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  1. Epstein-Barr virus BGLF4 kinase retards cellular S-phase progression and induces chromosomal abnormality.

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    Yu-Hsin Chang

    Full Text Available Epstein-Barr virus (EBV induces an uncoordinated S-phase-like cellular environment coupled with multiple prophase-like events in cells replicating the virus. The EBV encoded Ser/Thr kinase BGLF4 has been shown to induce premature chromosome condensation through activation of condensin and topoisomerase II and reorganization of the nuclear lamina to facilitate the nuclear egress of nucleocapsids in a pathway mimicking Cdk1. However, the observation that RB is hyperphosphorylated in the presence of BGLF4 raised the possibility that BGLF4 may have a Cdk2-like activity to promote S-phase progression. Here, we investigated the regulatory effects of BGLF4 on cell cycle progression and found that S-phase progression and DNA synthesis were interrupted by BGLF4 in mammalian cells. Expression of BGLF4 did not compensate Cdk1 defects for DNA replication in S. cerevisiae. Using time-lapse microscopy, we found the fate of individual HeLa cells was determined by the expression level of BGLF4. In addition to slight cell growth retardation, BGLF4 elicits abnormal chromosomal structure and micronucleus formation in 293 and NCP-TW01 cells. In Saos-2 cells, BGLF4 induced the hyperphosphorylation of co-transfected RB, while E2F1 was not released from RB-E2F1 complexes. The E2F1 regulated activities of the cyclin D1 and ZBRK1 promoters were suppressed by BGLF4 in a dose dependent manner. Detection with phosphoamino acid specific antibodies revealed that, in addition to Ser780, phosphorylation of the DNA damage-responsive Ser612 on RB was enhanced by BGLF4. Taken together, our study indicates that BGLF4 may directly or indirectly induce a DNA damage signal that eventually interferes with host DNA synthesis and delays S-phase progression.

  2. A Quantitative bgl Operon Model for E. coli Requires BglF Conformational Change for Sugar Transport

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    Chopra, Paras; Bender, Andreas

    The bgl operon is responsible for the metabolism of β-glucoside sugars such as salicin or arbutin in E. coli. Its regulatory system involves both positive and negative feedback mechanisms and it can be assumed to be more complex than that of the more closely studied lac and trp operons. We have developed a quantitative model for the regulation of the bgl operon which is subject to in silico experiments investigating its behavior under different hypothetical conditions. Upon administration of 5mM salicin as an inducer our model shows 80-fold induction, which compares well with the 60-fold induction measured experimentally. Under practical conditions 5-10mM inducer are employed, which is in line with the minimum inducer concentration of 1mM required by our model. The necessity of BglF conformational change for sugar transport has been hypothesized previously, and in line with those hypotheses our model shows only minor induction if conformational change is not allowed. Overall, this first quantitative model for the bgl operon gives reasonable predictions that are close to experimental results (where measured). It will be further refined as values of the parameters are determined experimentally. The model was developed in Systems Biology Markup Language (SBML) and it is available from the authors and from the Biomodels repository [www.ebi.ac.uk/biomodels].

  3. Maribavir Inhibits Epstein-Barr Virus Transcription through the EBV Protein Kinase

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    Whitehurst, Christopher B.; Sanders, Marcia K.; Law, Mankit; Wang, Fu-Zhang; Xiong, Jie; Dittmer, Dirk P.

    2013-01-01

    Maribavir (MBV) inhibits Epstein-Barr virus (EBV) replication and the enzymatic activity of the viral protein kinase BGLF4. MBV also inhibits expression of multiple EBV transcripts during EBV lytic infection. Here we demonstrate, with the use of a BGLF4 knockout virus, that effects of MBV on transcription take place primarily through inhibition of BGLF4. MBV inhibits viral genome copy numbers and infectivity to levels similar to and exceeding levels produced by BGLF4 knockout virus. PMID:23449792

  4. RECQL5 Suppresses Oncogenic JAK2-Induced Replication Stress and Genomic Instability

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    Edwin Chen

    2015-12-01

    Full Text Available JAK2V617F is the most common oncogenic lesion in patients with myeloproliferative neoplasms (MPNs. Despite the ability of JAK2V617F to instigate DNA damage in vitro, MPNs are nevertheless characterized by genomic stability. In this study, we address this paradox by identifying the DNA helicase RECQL5 as a suppressor of genomic instability in MPNs. We report increased RECQL5 expression in JAK2V617F-expressing cells and demonstrate that RECQL5 is required to counteract JAK2V617F-induced replication stress. Moreover, RECQL5 depletion sensitizes JAK2V617F mutant cells to hydroxyurea (HU, a pharmacological inducer of replication stress and the most common treatment for MPNs. Using single-fiber chromosome combing, we show that RECQL5 depletion in JAK2V617F mutant cells impairs replication dynamics following HU treatment, resulting in increased double-stranded breaks and apoptosis. Cumulatively, these findings identify RECQL5 as a critical regulator of genome stability in MPNs and demonstrate that replication stress-associated cytotoxicity can be amplified specifically in JAK2V617F mutant cells through RECQL5-targeted synthetic lethality.

  5. Purification and enzymatic characterization of secretory glycoside hydrolase family 3 (GH3) aryl β-glucosidases screened from Aspergillus oryzae genome.

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    Kudo, Kanako; Watanabe, Akira; Ujiie, Seiryu; Shintani, Takahiro; Gomi, Katsuya

    2015-12-01

    By a global search of the genome database of Aspergillus oryzae, we found 23 genes encoding putative β-glucosidases, among which 10 genes with a signal peptide belonging to glycoside hydrolase family 3 (GH3) were overexpressed in A. oryzae using the improved glaA gene promoter. Consequently, crude enzyme preparations from three strains, each harboring the genes AO090038000223 (bglA), AO090103000127 (bglF), and AO090003001511 (bglJ), showed a substrate preference toward p-nitrophenyl-β-d-glucopyranoside (pNPGlc) and thus were purified to homogeneity and enzymatically characterized. All the purified enzymes (BglA, BglF, and BglJ) preferentially hydrolyzed aryl β-glycosides, including pNPGlc, rather than cellobiose, and these enzymes were proven to be aryl β-glucosidases. Although the specific activity of BglF toward all the substrates tested was significantly low, BglA and BglJ showed appreciably high activities toward pNPGlc and arbutin. The kinetic parameters of BglA and BglJ for pNPGlc suggested that both the enzymes had relatively higher hydrolytic activity toward pNPGlc among the fungal β-glucosidases reported. The thermal and pH stabilities of BglA were higher than those of BglJ, and BglA was particularly stable in a wide pH range (pH 4.5-10). In contrast, BglJ was the most heat- and alkaline-labile among the three β-glucosidases. Furthermore, BglA was more tolerant to ethanol than BglJ; as a result, it showed much higher hydrolytic activity toward isoflavone glycosides in the presence of ethanol than BglJ. This study suggested that the mining of novel β-glucosidases exhibiting higher activity from microbial genome sequences is of great use for the production of beneficial compounds such as isoflavone aglycones. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. Efficient production of infectious viruses requires enzymatic activity of Epstein-Barr virus protein kinase.

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    Murata, Takayuki; Isomura, Hiroki; Yamashita, Yoriko; Toyama, Shigenori; Sato, Yoshitaka; Nakayama, Sanae; Kudoh, Ayumi; Iwahori, Satoko; Kanda, Teru; Tsurumi, Tatsuya

    2009-06-20

    The Epstein-Barr virus (EBV) BGLF4 gene product is the only protein kinase encoded by the virus genome. In order to elucidate its physiological roles in viral productive replication, we here established a BGLF4-knockout mutant and a revertant virus. While the levels of viral DNA replication of the deficient mutant were equivalent to those of the wild-type and the revertant, virus production was significantly impaired. Expression of the BGLF4 protein in trans fully complemented the low yield of the mutant virus, while expression of a kinase-dead (K102I) form of the protein failed to restore the virus titer. These results demonstrate that BGLF4 plays a significant role in production of infectious viruses and that the kinase activity is crucial.

  7. Characterization of Camptothecin-induced Genomic Changes in the Camptothecin-resistant T-ALL-derived Cell Line CPT-K5

    DEFF Research Database (Denmark)

    Kjeldsen, Eigil; Nielsen, Christine J F; Roy, Amit

    2018-01-01

    -K5 and its parental cell line. We identified copy number alterations affecting genes important for maintaining genome integrity and reducing CPT-induced DNA damage. We show for the first time that short tandem repeats are targets for TOP1 cleavage, that can be differentially stimulated by CPT.......Acquisition of resistance to topoisomerase I (TOP1)-targeting camptothecin (CPT) derivatives is a major clinical problem. Little is known about the underlying chromosomal and genomic mechanisms. We characterized the CPT-K5 cell line expressing mutant CPT-resistant TOP1 and its parental T......-cell derived acute lymphoblastic leukemia CPT-sensitive RPMI-8402 cell line by karyotyping and molecular genetic methods, including subtractive oligo-based array comparative genomic hybridization (soaCGH) analysis. Karyotyping revealed that CPT-K5 cells had acquired additional structural aberrations...

  8. Identification of herpesvirus proteins that contribute to G1/S arrest.

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    Paladino, Patrick; Marcon, Edyta; Greenblatt, Jack; Frappier, Lori

    2014-04-01

    Lytic infection by herpesviruses induces cell cycle arrest at the G1/S transition. This appears to be a function of multiple herpesvirus proteins, but only a minority of herpesvirus proteins have been examined for cell cycle effects. To gain a more comprehensive understanding of the viral proteins that contribute to G1/S arrest, we screened a library of over 200 proteins from herpes simplex virus type 1, human cytomegalovirus, and Epstein-Barr virus (EBV) for effects on the G1/S interface, using HeLa fluorescent, ubiquitination-based cell cycle indicator (Fucci) cells in which G1/S can be detected colorimetrically. Proteins from each virus were identified that induce accumulation of G1/S cells, predominantly tegument, early, and capsid proteins. The identification of several capsid proteins in this screen suggests that incoming viral capsids may function to modulate cellular processes. The cell cycle effects of selected EBV proteins were further verified and examined for effects on p53 and p21 as regulators of the G1/S transition. Two EBV replication proteins (BORF2 and BMRF1) were found to induce p53 but not p21, while a previously uncharacterized tegument protein (BGLF2) was found to induce p21 protein levels in a p53-independent manner. Proteomic analyses of BGLF2-interacting proteins identified interactions with the NIMA-related protein kinase (NEK9) and GEM-interacting protein (GMIP). Silencing of either NEK9 or GMIP induced p21 without affecting p53 and abrogated the ability of BGLF2 to further induce p21. Collectively, these results suggest multiple viral proteins contribute to G1/S arrest, including BGLF2, which induces p21 levels likely by interfering with the functions of NEK9 and GMIP. Most people are infected with multiple herpesviruses, whose proteins alter the infected cells in several ways. During lytic infection, the viral proteins block cell proliferation just before the cellular DNA replicates. We used a novel screening method to identify proteins

  9. Radiation-induced instability of human genome

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    Ryabchenko, N.N.; Demina, Eh.A.

    2014-01-01

    A brief review is dedicated to the phenomenon of radiation-induced genomic instability where the increased level of genomic changes in the offspring of irradiated cells is characteristic. Particular attention is paid to the problems of genomic instability induced by the low-dose radiation, role of the bystander effect in formation of radiation-induced instability, and its relationship with individual radiosensitivity. We believe that in accordance with the paradigm of modern radiobiology the increased human individual radiosensitivity can be formed due to the genome instability onset and is a significant risk factor for radiation-induced cancer

  10. AID/APOBEC cytosine deaminase induces genome-wide kataegis

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    Lada Artem G

    2012-12-01

    Full Text Available Abstract Clusters of localized hypermutation in human breast cancer genomes, named “kataegis” (from the Greek for thunderstorm, are hypothesized to result from multiple cytosine deaminations catalyzed by AID/APOBEC proteins. However, a direct link between APOBECs and kataegis is still lacking. We have sequenced the genomes of yeast mutants induced in diploids by expression of the gene for PmCDA1, a hypermutagenic deaminase from sea lamprey. Analysis of the distribution of 5,138 induced mutations revealed localized clusters very similar to those found in tumors. Our data provide evidence that unleashed cytosine deaminase activity is an evolutionary conserved, prominent source of genome-wide kataegis events. Reviewers This article was reviewed by: Professor Sandor Pongor, Professor Shamil R. Sunyaev, and Dr Vladimir Kuznetsov.

  11. Nuclear translocation and regulation of intranuclear distribution of cytoplasmic poly(A-binding protein are distinct processes mediated by two Epstein Barr virus proteins.

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    Richard Park

    Full Text Available Many viruses target cytoplasmic polyA binding protein (PABPC to effect widespread inhibition of host gene expression, a process termed viral host-shutoff (vhs. During lytic replication of Epstein Barr Virus (EBV we observed that PABPC was efficiently translocated from the cytoplasm to the nucleus. Translocated PABPC was diffusely distributed but was excluded from viral replication compartments. Vhs during EBV infection is regulated by the viral alkaline nuclease, BGLF5. Transfection of BGLF5 alone into BGLF5-KO cells or uninfected 293 cells promoted translocation of PAPBC that was distributed in clumps in the nucleus. ZEBRA, a viral bZIP protein, performs essential functions in the lytic program of EBV, including activation or repression of downstream viral genes. ZEBRA is also an essential replication protein that binds to viral oriLyt and interacts with other viral replication proteins. We report that ZEBRA also functions as a regulator of vhs. ZEBRA translocated PABPC to the nucleus, controlled the intranuclear distribution of PABPC, and caused global shutoff of host gene expression. Transfection of ZEBRA alone into 293 cells caused nuclear translocation of PABPC in the majority of cells in which ZEBRA was expressed. Co-transfection of ZEBRA with BGLF5 into BGLF5-KO cells or uninfected 293 cells rescued the diffuse intranuclear pattern of PABPC seen during lytic replication. ZEBRA mutants defective for DNA-binding were capable of regulating the intranuclear distribution of PABPC, and caused PABPC to co-localize with ZEBRA. One ZEBRA mutant, Z(S186E, was deficient in translocation yet was capable of altering the intranuclear distribution of PABPC. Therefore ZEBRA-mediated nuclear translocation of PABPC and regulation of intranuclear PABPC distribution are distinct events. Using a click chemistry-based assay for new protein synthesis, we show that ZEBRA and BGLF5 each function as viral host shutoff factors.

  12. Mechanisms of cadmium induced genomic instability

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    Filipic, Metka, E-mail: metka.filipic@nib.si [National Institute of Biology, Department for Genetic Toxicology and Cancer Biology, Ljubljana (Slovenia)

    2012-05-01

    Cadmium is an ubiquitous environmental contaminant that represents hazard to humans and wildlife. It is found in the air, soil and water and, due to its extremely long half-life, accumulates in plants and animals. The main source of cadmium exposure for non-smoking human population is food. Cadmium is primarily toxic to the kidney, but has been also classified as carcinogenic to humans by several regulatory agencies. Current evidence suggests that exposure to cadmium induces genomic instability through complex and multifactorial mechanisms. Cadmium dose not induce direct DNA damage, however it induces increase in reactive oxygen species (ROS) formation, which in turn induce DNA damage and can also interfere with cell signalling. More important seems to be cadmium interaction with DNA repair mechanisms, cell cycle checkpoints and apoptosis as well as with epigenetic mechanisms of gene expression control. Cadmium mediated inhibition of DNA repair mechanisms and apoptosis leads to accumulation of cells with unrepaired DNA damage, which in turn increases the mutation rate and thus genomic instability. This increases the probability of developing not only cancer but also other diseases associated with genomic instability. In the in vitro experiments cadmium induced effects leading to genomic instability have been observed at low concentrations that were comparable to those observed in target organs and tissues of humans that were non-occupationally exposed to cadmium. Therefore, further studies aiming to clarify the relevance of these observations for human health risks due to cadmium exposure are needed.

  13. Mechanisms of cadmium induced genomic instability

    International Nuclear Information System (INIS)

    Filipič, Metka

    2012-01-01

    Cadmium is an ubiquitous environmental contaminant that represents hazard to humans and wildlife. It is found in the air, soil and water and, due to its extremely long half-life, accumulates in plants and animals. The main source of cadmium exposure for non-smoking human population is food. Cadmium is primarily toxic to the kidney, but has been also classified as carcinogenic to humans by several regulatory agencies. Current evidence suggests that exposure to cadmium induces genomic instability through complex and multifactorial mechanisms. Cadmium dose not induce direct DNA damage, however it induces increase in reactive oxygen species (ROS) formation, which in turn induce DNA damage and can also interfere with cell signalling. More important seems to be cadmium interaction with DNA repair mechanisms, cell cycle checkpoints and apoptosis as well as with epigenetic mechanisms of gene expression control. Cadmium mediated inhibition of DNA repair mechanisms and apoptosis leads to accumulation of cells with unrepaired DNA damage, which in turn increases the mutation rate and thus genomic instability. This increases the probability of developing not only cancer but also other diseases associated with genomic instability. In the in vitro experiments cadmium induced effects leading to genomic instability have been observed at low concentrations that were comparable to those observed in target organs and tissues of humans that were non-occupationally exposed to cadmium. Therefore, further studies aiming to clarify the relevance of these observations for human health risks due to cadmium exposure are needed.

  14. Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing

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    Plant Ramona N

    2006-08-01

    Full Text Available Abstract Background Whole genome amplification is an increasingly common technique through which minute amounts of DNA can be multiplied to generate quantities suitable for genetic testing and analysis. Questions of amplification-induced error and template bias generated by these methods have previously been addressed through either small scale (SNPs or large scale (CGH array, FISH methodologies. Here we utilized whole genome sequencing to assess amplification-induced bias in both coding and non-coding regions of two bacterial genomes. Halobacterium species NRC-1 DNA and Campylobacter jejuni were amplified by several common, commercially available protocols: multiple displacement amplification, primer extension pre-amplification and degenerate oligonucleotide primed PCR. The amplification-induced bias of each method was assessed by sequencing both genomes in their entirety using the 454 Sequencing System technology and comparing the results with those obtained from unamplified controls. Results All amplification methodologies induced statistically significant bias relative to the unamplified control. For the Halobacterium species NRC-1 genome, assessed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 119 times greater than those from unamplified material, 164.0 times greater for Repli-G, 165.0 times greater for PEP-PCR and 252.0 times greater than the unamplified controls for DOP-PCR. For Campylobacter jejuni, also analyzed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 15 times greater than those from unamplified material, 19.8 times greater for Repli-G, 61.8 times greater for PEP-PCR and 220.5 times greater than the unamplified controls for DOP-PCR. Conclusion Of the amplification methodologies examined in this paper, the multiple displacement amplification products generated the least bias, and produced significantly higher yields of amplified DNA.

  15. DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA.

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    Wang'ondu, Ruth; Teal, Stuart; Park, Richard; Heston, Lee; Delecluse, Henri; Miller, George

    2015-01-01

    Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.

  16. Bystander effects in UV-induced genomic instability: Antioxidants inhibit delayed mutagenesis induced by ultraviolet A and B radiation

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    Dahle Jostein

    2005-01-01

    Full Text Available Abstract Background Genomic instability is characteristic of many types of human cancer. Recently, we reported that ultraviolet radiation induced elevated mutation rates and chromosomal instability for many cell generations after ultraviolet irradiation. The increased mutation rates of unstable cells may allow them to accumulate aberrations that subsequently lead to cancer. Ultraviolet A radiation, which primarily acts by oxidative stress, and ultraviolet B radiation, which initially acts by absorption in DNA and direct damage to DNA, both produced genomically unstable cell clones. In this study, we have determined the effect of antioxidants on induction of delayed mutations by ultraviolet radiation. Delayed mutations are indicative of genomic instability. Methods Delayed mutations in the hypoxanthine phosphoribosyl transferase (hprt gene were detected by incubating the cells in medium selectively killing hprt mutants for 8 days after irradiation, followed by a 5 day period in normal medium before determining mutation frequencies. Results The UVB-induced delayed hprt mutations were strongly inhibited by the antioxidants catalase, reduced glutathione and superoxide dismutase, while only reduced glutathione had a significant effect on UVA-induced delayed mutations. Treatment with antioxidants had only minor effects on early mutation frequenies, except that reduced glutathione decreased the UVB-induced early mutation frequency by 24 %. Incubation with reduced glutathione was shown to significantly increase the intracellular amount of reduced glutathione. Conclusion The strong effects of these antioxidants indicate that genomic instability, which is induced by the fundamentally different ultraviolet A and ultraviolet B radiation, is mediated by reactive oxygen species, including hydrogen peroxide and downstream products. However, cells take up neither catalase nor SOD, while incubation with glutathione resulted in increased intracellular levels of

  17. Genomic rearrangement in radiation-induced murine myeloid leukemia

    International Nuclear Information System (INIS)

    Ishihara, Hiroshi

    1994-01-01

    After whole body irradiation of 3Gy X ray to C3H/He male mice, acute myeloid leukemia is induced at an incidence of 20 to 30% within 2 years. We have studied the mechanism of occurrence of this radiation-induced murine myeloid leukemia. Detection and isolation of genomic structural aberration which may be accumulated accompanied with leukemogenesis are helpful in analyzing the complicated molecular process from radiation damage to leukemogenesis. So, our research work was done in three phases. First, structures of previously characterized oncogenes and cytokine-related genes were analyzed, and abnormal structures of fms(protooncogene encoding M-CSF receptor gene)-related and myc-related genes were found in several leukemia cells. Additionally, genomic structural aberration of IL-3 gene was observed in some leukemia cells, so that construction of genomic libraries and cloning of the abnormal IL-3 genomic DNAs were performed to characterize the structure. Secondly, because the breakage of chromosome 2 that is frequently observed in myeloid leukemia locates in proximal position of IL-1 gene cluster in some cases, the copy number of IL-1 gene was determined and the gene was cloned. Lastly, the abnormal genome of leukemia cell was cloned by in-gel competence reassociation method. We discussed these findings and evaluated the analysis of the molecular process of leukemogenesis using these cloned genomic fragments. (author)

  18. From Genomics to Gene Therapy: Induced Pluripotent Stem Cells Meet Genome Editing.

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    Hotta, Akitsu; Yamanaka, Shinya

    2015-01-01

    The advent of induced pluripotent stem (iPS) cells has opened up numerous avenues of opportunity for cell therapy, including the initiation in September 2014 of the first human clinical trial to treat dry age-related macular degeneration. In parallel, advances in genome-editing technologies by site-specific nucleases have dramatically improved our ability to edit endogenous genomic sequences at targeted sites of interest. In fact, clinical trials have already begun to implement this technology to control HIV infection. Genome editing in iPS cells is a powerful tool and enables researchers to investigate the intricacies of the human genome in a dish. In the near future, the groundwork laid by such an approach may expand the possibilities of gene therapy for treating congenital disorders. In this review, we summarize the exciting progress being made in the utilization of genomic editing technologies in pluripotent stem cells and discuss remaining challenges toward gene therapy applications.

  19. Ionizing radiation induced genomic instability and its relation to radiation carcinogenesis

    International Nuclear Information System (INIS)

    Wang Zhongwen

    2000-01-01

    There are widespread testimonies that the genomic instability induced by ionizing irradiation exits in mammal and its vitro cells. Genomic instability can enhance the frequency of genetic changes among the progeny of the original irradiated cells. In the radiation-leukemogenesis, there is no significant difference between controls and CBA/H mouses of PPI (preconception patent irradiation), but the offsprings of the PPI recipients show a different character (shorter latent period and higher incidence) after an extra γ-radiation. The radiation-induced genomic instability may get the genome on the verge of mutation and lead to carcinogens following mutation of some critical genes. The genomic instability, as the early event of initiation of carcinomas, may be play a specific or unique role

  20. Radiation-induced hyperproliferation of intestinal crypts results in elevated genome instability with inactive p53-related genomic surveillance.

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    Zhou, Xin; Ma, Xiaofei; Wang, Zhenhua; Sun, Chao; Wang, Yupei; He, Yang; Zhang, Hong

    2015-12-15

    Radiation-induced hyperproliferation of intestinal crypts is well documented, but its potential tumorigenic effects remain elusive. Here we aim to determine the genomic surveillance process during crypt hyperproliferation, and its consequential outcome after ionizing radiation. Crypt regeneration in the intestine was induced by a single dose of 12Gy abdominal irradiation. γ-H2AX, 53BP1 and DNA-PKcs were used as DNA repair surrogates to investigate the inherent ability of intestinal crypt cells to recognize and repair double-strand breaks. Ki67 staining and the 5-bromo-2'-deoxyuridine incorporation assay were used to study patterns of cell proliferation in regenerating crypts. Staining for ATM, p53, Chk1 and Chk2 was performed to study checkpoint activation and release. Apoptosis was evaluated through H&E staining and terminal deoxynucleotidyl transferase (dUTP) nick-end labeling. The ATM-p53 pathway was immediately activated after irradiation. A second wave of DSBs in crypt cells was observed in regenerating crypts, accompanied with significantly increased chromosomal bridges. The p53-related genomic surveillance pathway was not active during the regeneration phase despite DSBs and chromosomal bridges in the cells of regenerating crypts. Non-homologous end joining (NHEJ) DSBs repair was involved in the DSBs repair process, as indicated by p-DNA-PKcs staining. Intestinal crypt cells retained hyperproliferation with inactive p53-related genomic surveillance system. NHEJ was involved in the resultant genomic instability during hyperproliferation. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Complete genome sequences and comparative genome analysis of Lactobacillus plantarum strain 5-2 isolated from fermented soybean.

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    Liu, Chen-Jian; Wang, Rui; Gong, Fu-Ming; Liu, Xiao-Feng; Zheng, Hua-Jun; Luo, Yi-Yong; Li, Xiao-Ran

    2015-12-01

    Lactobacillus plantarum is an important probiotic and is mostly isolated from fermented foods. We sequenced the genome of L. plantarum strain 5-2, which was derived from fermented soybean isolated from Yunnan province, China. The strain was determined to contain 3114 genes. Fourteen complete insertion sequence (IS) elements were found in 5-2 chromosome. There were 24 DNA replication proteins and 76 DNA repair proteins in the 5-2 genome. Consistent with the classification of L. plantarum as a facultative heterofermentative lactobacillus, the 5-2 genome encodes key enzymes required for the EMP (Embden-Meyerhof-Parnas) and phosphoketolase (PK) pathways. Several components of the secretion machinery are found in the 5-2 genome, which was compared with L. plantarum ST-III, JDM1 and WCFS1. Most of the specific proteins in the four genomes appeared to be related to their prophage elements. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Non-genomic actions of retinoic acid induce pi3k signaling pathway and phosphorylation of nuclear proteins

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    Laserna Mendieta, Emilio J.; Masiá, Susana; Barettino, Domingo

    2007-01-01

    Retinoic acid (RA), the active form of vitamin A, induces neuroblastoma cells SH-SY5Y to differentiate. In addition to its classical transcriptional actions regulating the expression of specific genes, RA acts in an extra-genomic way, modulating the activity of relevant signalling cascades. In particular, RA treatment of SH-SY5Y neuroblastoma cells results in activation of phosphatidyl-inositol-3-kinase (PI3K) signaling pathway, and this activation is required for RA-induced differentiation (...

  3. Use of γ-ray-induced mutations in the genome era in rice

    International Nuclear Information System (INIS)

    Kusaba, Makoto

    2007-01-01

    Ionizing radiation has been used for inducing mutations and improving crops since the discovery by STADLER (1928) that X-rays could induce mutations in barley. At the end of 2004, the whole genome sequence of rice was determined (INTERNATIONAL RICE GENOME SEQUENCING PROJECT, 2005). What can γ-ray-induced mutations contribute now that this has been achieved? One answer could be the elucidation of the functions of the numerous genes revealed by the complete sequence of the rice genome. This includes identification of mutants through reverse genetics and the isolation of genes containing mutations through forward genetics using molecular markers and sequence information. Another answer could be mutation breeding using reverse genetics. But first we must know what kind of DNA lesions are caused by γ-rays. In this article, I describe the production of DNA lesions, and then discuss how γ-ray-induced mutations can contribute to the elucidation of gene function and to mutation breeding. (author)

  4. Dioxin induces genomic instability in mouse embryonic fibroblasts.

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    Merja Korkalainen

    Full Text Available Ionizing radiation and certain other exposures have been shown to induce genomic instability (GI, i.e., delayed genetic damage observed many cell generations later in the progeny of the exposed cells. The aim of this study was to investigate induction of GI by a nongenotoxic carcinogen, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD. Mouse embryonic fibroblasts (C3H10T1/2 were exposed to 1, 10 or 100 nM TCDD for 2 days. Micronuclei (MN and expression of selected cancer-related genes were assayed both immediately and at a delayed point in time (8 days. For comparison, similar experiments were done with cadmium, a known genotoxic agent. TCDD treatment induced an elevated frequency of MN at 8 days, but not directly after the exposure. TCDD-induced alterations in gene expression were also mostly delayed, with more changes observed at 8 days than at 2 days. Exposure to cadmium produced an opposite pattern of responses, with pronounced effects immediately after exposure but no increase in MN and few gene expression changes at 8 days. Although all responses to TCDD alone were delayed, menadione-induced DNA damage (measured by the Comet assay, was found to be increased directly after a 2-day TCDD exposure, indicating that the stability of the genome was compromised already at this time point. The results suggested a flat dose-response relationship consistent with dose-response data reported for radiation-induced GI. These findings indicate that TCDD, although not directly genotoxic, induces GI, which is associated with impaired DNA damage response.

  5. Simulated space radiation-induced mutants in the mouse kidney display widespread genomic change.

    Directory of Open Access Journals (Sweden)

    Mitchell S Turker

    Full Text Available Exposure to a small number of high-energy heavy charged particles (HZE ions, as found in the deep space environment, could significantly affect astronaut health following prolonged periods of space travel if these ions induce mutations and related cancers. In this study, we used an in vivo mutagenesis assay to define the mutagenic effects of accelerated 56Fe ions (1 GeV/amu, 151 keV/μm in the mouse kidney epithelium exposed to doses ranging from 0.25 to 2.0 Gy. These doses represent fluences ranging from 1 to 8 particle traversals per cell nucleus. The Aprt locus, located on chromosome 8, was used to select induced and spontaneous mutants. To fully define the mutagenic effects, we used multiple endpoints including mutant frequencies, mutation spectrum for chromosome 8, translocations involving chromosome 8, and mutations affecting non-selected chromosomes. The results demonstrate mutagenic effects that often affect multiple chromosomes for all Fe ion doses tested. For comparison with the most abundant sparsely ionizing particle found in space, we also examined the mutagenic effects of high-energy protons (1 GeV, 0.24 keV/μm at 0.5 and 1.0 Gy. Similar doses of protons were not as mutagenic as Fe ions for many assays, though genomic effects were detected in Aprt mutants at these doses. Considered as a whole, the data demonstrate that Fe ions are highly mutagenic at the low doses and fluences of relevance to human spaceflight, and that cells with considerable genomic mutations are readily induced by these exposures and persist in the kidney epithelium. The level of genomic change produced by low fluence exposure to heavy ions is reminiscent of the extensive rearrangements seen in tumor genomes suggesting a potential initiation step in radiation carcinogenesis.

  6. Simulated space radiation-induced mutants in the mouse kidney display widespread genomic change.

    Science.gov (United States)

    Turker, Mitchell S; Grygoryev, Dmytro; Lasarev, Michael; Ohlrich, Anna; Rwatambuga, Furaha A; Johnson, Sorrel; Dan, Cristian; Eckelmann, Bradley; Hryciw, Gwen; Mao, Jian-Hua; Snijders, Antoine M; Gauny, Stacey; Kronenberg, Amy

    2017-01-01

    Exposure to a small number of high-energy heavy charged particles (HZE ions), as found in the deep space environment, could significantly affect astronaut health following prolonged periods of space travel if these ions induce mutations and related cancers. In this study, we used an in vivo mutagenesis assay to define the mutagenic effects of accelerated 56Fe ions (1 GeV/amu, 151 keV/μm) in the mouse kidney epithelium exposed to doses ranging from 0.25 to 2.0 Gy. These doses represent fluences ranging from 1 to 8 particle traversals per cell nucleus. The Aprt locus, located on chromosome 8, was used to select induced and spontaneous mutants. To fully define the mutagenic effects, we used multiple endpoints including mutant frequencies, mutation spectrum for chromosome 8, translocations involving chromosome 8, and mutations affecting non-selected chromosomes. The results demonstrate mutagenic effects that often affect multiple chromosomes for all Fe ion doses tested. For comparison with the most abundant sparsely ionizing particle found in space, we also examined the mutagenic effects of high-energy protons (1 GeV, 0.24 keV/μm) at 0.5 and 1.0 Gy. Similar doses of protons were not as mutagenic as Fe ions for many assays, though genomic effects were detected in Aprt mutants at these doses. Considered as a whole, the data demonstrate that Fe ions are highly mutagenic at the low doses and fluences of relevance to human spaceflight, and that cells with considerable genomic mutations are readily induced by these exposures and persist in the kidney epithelium. The level of genomic change produced by low fluence exposure to heavy ions is reminiscent of the extensive rearrangements seen in tumor genomes suggesting a potential initiation step in radiation carcinogenesis.

  7. Attenuation of the cyproterone acetate-induced testicular hypofunction by a novel nutraceutical lycopene: a genomic approach.

    Science.gov (United States)

    Tripathy, A; Ghosh, A; Dey, A; Pakhira, B P; Ghosh, D

    2017-10-01

    This study was designed to explore the cyproterone acetate (CPA)-induced andrological hypofunction and its correction by oral administration of lycopene. In this concern, spermatogenic, biochemical, histological and genomic profiles were studied. Cyproterone acetate administration for 1 month helped to develop infertile model rats. A significant recovery was noted in sperm motility, sperm count, sperm viability, hypo-osmotic swelling tail-coiled spermatozoa; activities of testicular ∆ 5 , 3β-hydroxysteroid dehydrogenase (HSD), 17β-HSD, catalase (CAT) and superoxide dismutase (SOD); and levels of conjugated diene (CD), malondialdehyde (MDA), testicular cholesterol and serum testosterone after the administration of lycopene at 1.5 mg/0.5 ml Tween-80/100 g body weight/day for last 1 month to infertile model rats. Simultaneously, qRT-PCR study of Bax, Bcl-2, caspase-3, ∆ 5 , 3β-HSD and 17β-HSD genes in testicular tissue showed a significant rectification towards the control in CPA-pre-treated cum CPA-lycopene-cotreated rats. Side-by-side histological and histometric studies showed a significant correction in qualitative analysis of spermatogenesis and seminiferous tubular diameter (STD) in CPA-pre-treated cum CPA-lycopene-cotreated rats. Lycopene showed outstanding efficacy in the management of CPA-induced testicular hypofunction with special reference to correction in oxidative stress-induced testicular apoptosis at genomic level. © 2016 Blackwell Verlag GmbH.

  8. Complete Genome Sequence of Escherichia coli Strain WG5

    DEFF Research Database (Denmark)

    Imamovic, Lejla; Misiakou, Maria-Anna; van der Helm, Eric

    2018-01-01

    Escherichia coli strain WG5 is a widely used host for phage detection, including somatic coliphages employed as standard ISO method 10705-1 (2000). Here, we present the complete genome sequence of a commercial E. coli WG5 strain.......Escherichia coli strain WG5 is a widely used host for phage detection, including somatic coliphages employed as standard ISO method 10705-1 (2000). Here, we present the complete genome sequence of a commercial E. coli WG5 strain....

  9. IMA Genome-F 5G

    OpenAIRE

    Wingfield, Brenda D.; Barnes, Irene; Wilhelm de Beer, Z.; De Vos, Lieschen; Duong, Tuan A.; Kanzi, Aquillah M.; Naidoo, Kershney; Nguyen, Hai D.T.; Santana, Quentin C.; Sayari, Mohammad; Seifert, Keith A.; Steenkamp, Emma T.; Trollip, Conrad; van der Merwe, Nicolaas A.; van der Nest, Magriet A.

    2015-01-01

    The genomes of Ceratocystis eucalypticola, Chrysoporthe cubensis, Chrysoporthe deuterocubensis, Davidsoniella virescens, Fusarium temperatum, Graphilbum fragrans, Penicillium nordicum and Thielaviopsis musarum are presented in this genome announcement. These seven genomes are from plant pathogens and otherwise economically important fungal species. The genome sizes range from 28 Mb in the case of T. musarum to 45 Mb for Fusarium temperatum. These genomes include the first reports of genomes f...

  10. Genomic mutation study for long-term cells induced by carbon ions

    International Nuclear Information System (INIS)

    Wang, X.; Furusawa, Y.; Suzuki, M.; Hirayama, R.; Matsumoto, Y.; Qin, Y.

    2007-01-01

    samples at the HPRT locus was detected to measure 6-thioguanine-resistant colonies. Results: The survival fraction of the cells irradiated by carbon ions was lower than sparsely ionizing (low LET) radiation, and it was also lower in high dose rate (∼1.5Gy/min) than in low dose rate (∼0.008Gy/min) irradiated at the same dose. Furthermore, high dose (D10 dose) induced high mutation fraction. Low dose and low dose rate had the low mutation fractions and different dose rate induced different mutation fraction even at the same dose. Mutation fraction had lower value before 6 days, and the higher level between 12 to 24 days after irradiated, then value went down. Conclusion: The results suggest that high LET radiation may cause higher mutation induction dependent with irradiated dose. Different dose rate also may induce different mutation affection. The heavy ion radiation may cause the long-term genomic mutation and instability of cells. HPRT mutant detection system can be application evaluating the carcinogenic effects of space environmental radiation and heavy ion radiation therapy.

  11. Multimode drug inducible CRISPR/Cas9 devices for transcriptional activation and genome editing

    Science.gov (United States)

    Lu, Jia; Zhao, Chen; Zhao, Yingze; Zhang, Jingfang; Zhang, Yue; Chen, Li; Han, Qiyuan; Ying, Yue; Peng, Shuai; Ai, Runna; Wang, Yu

    2018-01-01

    Abstract Precise investigation and manipulation of dynamic biological processes often requires molecular modulation in a controlled inducible manner. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) has emerged as a versatile tool for targeted gene editing and transcriptional programming. Here, we designed and vigorously optimized a series of Hybrid drug Inducible CRISPR/Cas9 Technologies (HIT) for transcriptional activation by grafting a mutated human estrogen receptor (ERT2) to multiple CRISPR/Cas9 systems, which renders them 4-hydroxytamoxifen (4-OHT) inducible for the access of genome. Further, extra functionality of simultaneous genome editing was achieved with one device we named HIT2. Optimized terminal devices herein delivered advantageous performances in comparison with several existing designs. They exerted selective, titratable, rapid and reversible response to drug induction. In addition, these designs were successfully adapted to an orthogonal Cas9. HIT systems developed in this study can be applied for controlled modulation of potentially any genomic loci in multiple modes. PMID:29237052

  12. Synergistic Interactions with PI3K Inhibition that Induce Apoptosis. | Office of Cancer Genomics

    Science.gov (United States)

    Activating mutations involving the PI3K pathway occur frequently in human cancers. However, PI3K inhibitors primarily induce cell cycle arrest, leaving a significant reservoir of tumor cells that may acquire or exhibit resistance. We searched for genes that are required for the survival of PI3K mutant cancer cells in the presence of PI3K inhibition by conducting a genome scale shRNA-based apoptosis screen in a PIK3CA mutant human breast cancer cell. We identified 5 genes (PIM2, ZAK, TACC1, ZFR, ZNF565) whose suppression induced cell death upon PI3K inhibition.

  13. Sequence-Based Mapping and Genome Editing Reveal Mutations in Stickleback Hps5 Cause Oculocutaneous Albinism and the casper Phenotype

    Directory of Open Access Journals (Sweden)

    James C. Hart

    2017-09-01

    Full Text Available Here, we present and characterize the spontaneous X-linked recessive mutation casper, which causes oculocutaneous albinism in threespine sticklebacks (Gasterosteus aculeatus. In humans, Hermansky-Pudlak syndrome results in pigmentation defects due to disrupted formation of the melanin-containing lysosomal-related organelle (LRO, the melanosome. casper mutants display not only reduced pigmentation of melanosomes in melanophores, but also reductions in the iridescent silver color from iridophores, while the yellow pigmentation from xanthophores appears unaffected. We mapped casper using high-throughput sequencing of genomic DNA from bulked casper mutants to a region of the stickleback X chromosome (chromosome 19 near the stickleback ortholog of Hermansky-Pudlak syndrome 5 (Hps5. casper mutants have an insertion of a single nucleotide in the sixth exon of Hps5, predicted to generate an early frameshift. Genome editing using CRISPR/Cas9 induced lesions in Hps5 and phenocopied the casper mutation. Injecting single or paired Hps5 guide RNAs revealed higher incidences of genomic deletions from paired guide RNAs compared to single gRNAs. Stickleback Hps5 provides a genetic system where a hemizygous locus in XY males and a diploid locus in XX females can be used to generate an easily scored visible phenotype, facilitating quantitative studies of different genome editing approaches. Lastly, we show the ability to better visualize patterns of fluorescent transgenic reporters in Hps5 mutant fish. Thus, Hps5 mutations present an opportunity to study pigmented LROs in the emerging stickleback model system, as well as a tool to aid in assaying genome editing and visualizing enhancer activity in transgenic fish.

  14. Comprehensive evaluation of genome-wide 5-hydroxymethylcytosine profiling approaches in human DNA.

    Science.gov (United States)

    Skvortsova, Ksenia; Zotenko, Elena; Luu, Phuc-Loi; Gould, Cathryn M; Nair, Shalima S; Clark, Susan J; Stirzaker, Clare

    2017-01-01

    The discovery that 5-methylcytosine (5mC) can be oxidized to 5-hydroxymethylcytosine (5hmC) by the ten-eleven translocation (TET) proteins has prompted wide interest in the potential role of 5hmC in reshaping the mammalian DNA methylation landscape. The gold-standard bisulphite conversion technologies to study DNA methylation do not distinguish between 5mC and 5hmC. However, new approaches to mapping 5hmC genome-wide have advanced rapidly, although it is unclear how the different methods compare in accurately calling 5hmC. In this study, we provide a comparative analysis on brain DNA using three 5hmC genome-wide approaches, namely whole-genome bisulphite/oxidative bisulphite sequencing (WG Bis/OxBis-seq), Infinium HumanMethylation450 BeadChip arrays coupled with oxidative bisulphite (HM450K Bis/OxBis) and antibody-based immunoprecipitation and sequencing of hydroxymethylated DNA (hMeDIP-seq). We also perform loci-specific TET-assisted bisulphite sequencing (TAB-seq) for validation of candidate regions. We show that whole-genome single-base resolution approaches are advantaged in providing precise 5hmC values but require high sequencing depth to accurately measure 5hmC, as this modification is commonly in low abundance in mammalian cells. HM450K arrays coupled with oxidative bisulphite provide a cost-effective representation of 5hmC distribution, at CpG sites with 5hmC levels >~10%. However, 5hmC analysis is restricted to the genomic location of the probes, which is an important consideration as 5hmC modification is commonly enriched at enhancer elements. Finally, we show that the widely used hMeDIP-seq method provides an efficient genome-wide profile of 5hmC and shows high correlation with WG Bis/OxBis-seq 5hmC distribution in brain DNA. However, in cell line DNA with low levels of 5hmC, hMeDIP-seq-enriched regions are not detected by WG Bis/OxBis or HM450K, either suggesting misinterpretation of 5hmC calls by hMeDIP or lack of sensitivity of the latter methods. We

  15. Efficient Genome Editing in Induced Pluripotent Stem Cells with Engineered Nucleases In Vitro.

    Science.gov (United States)

    Termglinchan, Vittavat; Seeger, Timon; Chen, Caressa; Wu, Joseph C; Karakikes, Ioannis

    2017-01-01

    Precision genome engineering is rapidly advancing the application of the induced pluripotent stem cells (iPSCs) technology for in vitro disease modeling of cardiovascular diseases. Targeted genome editing using engineered nucleases is a powerful tool that allows for reverse genetics, genome engineering, and targeted transgene integration experiments to be performed in a precise and predictable manner. However, nuclease-mediated homologous recombination is an inefficient process. Herein, we describe the development of an optimized method combining site-specific nucleases and the piggyBac transposon system for "seamless" genome editing in pluripotent stem cells with high efficiency and fidelity in vitro.

  16. Acetone utilization by sulfate-reducing bacteria: draft genome sequence of Desulfococcus biacutus and a proteomic survey of acetone-inducible proteins.

    Science.gov (United States)

    Gutiérrez Acosta, Olga B; Schleheck, David; Schink, Bernhard

    2014-07-11

    The sulfate-reducing bacterium Desulfococcus biacutus is able to utilize acetone for growth by an inducible degradation pathway that involves a novel activation reaction for acetone with CO as a co-substrate. The mechanism, enzyme(s) and gene(s) involved in this acetone activation reaction are of great interest because they represent a novel and yet undefined type of activation reaction under strictly anoxic conditions. In this study, a draft genome sequence of D. biacutus was established. Sequencing, assembly and annotation resulted in 159 contigs with 5,242,029 base pairs and 4773 predicted genes; 4708 were predicted protein-encoding genes, and 3520 of these had a functional prediction. Proteins and genes were identified that are specifically induced during growth with acetone. A thiamine diphosphate-requiring enzyme appeared to be highly induced during growth with acetone and is probably involved in the activation reaction. Moreover, a coenzyme B12- dependent enzyme and proteins that are involved in redox reactions were also induced during growth with acetone. We present for the first time the genome of a sulfate reducer that is able to grow with acetone. The genome information of this organism represents an important tool for the elucidation of a novel reaction mechanism that is employed by a sulfate reducer in acetone activation.

  17. Radiation and chemotherapy bystander effects induce early genomic instability events: telomere shortening and bridge formation coupled with mitochondrial dysfunction.

    LENUS (Irish Health Repository)

    Gorman, Sheeona

    2012-02-01

    The bridge breakage fusion cycle is a chromosomal instability mechanism responsible for genomic changes. Radiation bystander effects induce genomic instability; however, the mechanism driving this instability is unknown. We examined if radiation and chemotherapy bystander effects induce early genomic instability events such as telomere shortening and bridge formation using a human colon cancer explant model. We assessed telomere lengths, bridge formations, mitochondrial membrane potential and levels of reactive oxygen species in bystander cells exposed to medium from irradiated and chemotherapy-treated explant tissues. Bystander cells exposed to media from 2Gy, 5Gy, FOLFOX treated tumor and matching normal tissue showed a significant reduction in telomere lengths (all p values <0.018) and an increase in bridge formations (all p values <0.017) compared to bystander cells treated with media from unirradiated tissue (0Gy) at 24h. There was no significant difference between 2Gy and 5Gy treatments, or between effects elicited by tumor versus matched normal tissue. Bystander cells exposed to media from 2Gy irradiated tumor tissue showed significant depolarisation of the mitochondrial membrane potential (p=0.012) and an increase in reactive oxygen species levels. We also used bystander cells overexpressing a mitochondrial antioxidant manganese superoxide dismutase (MnSOD) to examine if this antioxidant could rescue the mitochondrial changes and subsequently influence nuclear instability events. In MnSOD cells, ROS levels were reduced (p=0.02) and mitochondrial membrane potential increased (p=0.04). These events were coupled with a decrease in percentage of cells with anaphase bridges and a decrease in the number of cells undergoing telomere length shortening (p values 0.01 and 0.028 respectively). We demonstrate that radiation and chemotherapy bystander responses induce early genomic instability coupled with defects in mitochondrial function. Restoring mitochondrial

  18. Widespread occurrence of organelle genome-encoded 5S rRNAs including permuted molecules.

    Science.gov (United States)

    Valach, Matus; Burger, Gertraud; Gray, Michael W; Lang, B Franz

    2014-12-16

    5S Ribosomal RNA (5S rRNA) is a universal component of ribosomes, and the corresponding gene is easily identified in archaeal, bacterial and nuclear genome sequences. However, organelle gene homologs (rrn5) appear to be absent from most mitochondrial and several chloroplast genomes. Here, we re-examine the distribution of organelle rrn5 by building mitochondrion- and plastid-specific covariance models (CMs) with which we screened organelle genome sequences. We not only recover all organelle rrn5 genes annotated in GenBank records, but also identify more than 50 previously unrecognized homologs in mitochondrial genomes of various stramenopiles, red algae, cryptomonads, malawimonads and apusozoans, and surprisingly, in the apicoplast (highly derived plastid) genomes of the coccidian pathogens Toxoplasma gondii and Eimeria tenella. Comparative modeling of RNA secondary structure reveals that mitochondrial 5S rRNAs from brown algae adopt a permuted triskelion shape that has not been seen elsewhere. Expression of the newly predicted rrn5 genes is confirmed experimentally in 10 instances, based on our own and published RNA-Seq data. This study establishes that particularly mitochondrial 5S rRNA has a much broader taxonomic distribution and a much larger structural variability than previously thought. The newly developed CMs will be made available via the Rfam database and the MFannot organelle genome annotator. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Transposable elements as stress adaptive capacitors induce genomic instability in fungal pathogen Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Sonia Chadha

    Full Text Available A fundamental problem in fungal pathogenesis is to elucidate the evolutionary forces responsible for genomic rearrangements leading to races with fitter genotypes. Understanding the adaptive evolutionary mechanisms requires identification of genomic components and environmental factors reshaping the genome of fungal pathogens to adapt. Herein, Magnaporthe oryzae, a model fungal plant pathogen is used to demonstrate the impact of environmental cues on transposable elements (TE based genome dynamics. For heat shock and copper stress exposed samples, eight TEs belonging to class I and II family were employed to obtain DNA profiles. Stress induced mutant bands showed a positive correlation with dose/duration of stress and provided evidences of TEs role in stress adaptiveness. Further, we demonstrate that genome dynamics differ for the type/family of TEs upon stress exposition and previous reports of stress induced MAGGY transposition has underestimated the role of TEs in M. oryzae. Here, we identified Pyret, MAGGY, Pot3, MINE, Mg-SINE, Grasshopper and MGLR3 as contributors of high genomic instability in M. oryzae in respective order. Sequencing of mutated bands led to the identification of LTR-retrotransposon sequences within regulatory regions of psuedogenes. DNA transposon Pot3 was identified in the coding regions of chromatin remodelling protein containing tyrosinase copper-binding and PWWP domains. LTR-retrotransposons Pyret and MAGGY are identified as key components responsible for the high genomic instability and perhaps these TEs are utilized by M. oryzae for its acclimatization to adverse environmental conditions. Our results demonstrate how common field stresses change genome dynamics of pathogen and provide perspective to explore the role of TEs in genome adaptability, signalling network and its impact on the virulence of fungal pathogens.

  20. Complete genome sequence of Paenibacillus riograndensis SBR5(T), a Gram-positive diazotrophic rhizobacterium.

    Science.gov (United States)

    Brito, Luciana Fernandes; Bach, Evelise; Kalinowski, Jörn; Rückert, Christian; Wibberg, Daniel; Passaglia, Luciane M; Wendisch, Volker F

    2015-08-10

    Paenibacillus riograndensis is a Gram-positive rhizobacterium which exhibits plant growth promoting activities. It was isolated from the rhizosphere of wheat grown in the state of Rio Grande do Sul, Brazil. Here we announce the complete genome sequence of P. riograndensis strain SBR5(T). The genome of P. riograndensis SBR5(T) consists of a circular chromosome of 7,893,056bps. The genome was finished and fully annotated, containing 6705 protein coding genes, 87 tRNAs and 27 rRNAs. The knowledge of the complete genome helped to explain why P. riograndensis SBR5(T) can grow with the carbon sources arabinose and mannitol, but not myo-inositol, and to explain physiological features such as biotin auxotrophy and antibiotic resistances. The genome sequence will be valuable for functional genomics and ecological studies as well as for application of P. riograndensis SBR5(T) as plant growth-promoting rhizobacterium. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Gene expression profile and genomic alterations in colonic tumours induced by 1,2-dimethylhydrazine (DMH) in rats

    International Nuclear Information System (INIS)

    Femia, Angelo Pietro; Luceri, Cristina; Toti, Simona; Giannini, Augusto; Dolara, Piero; Caderni, Giovanna

    2010-01-01

    Azoxymethane (AOM) or 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats shares many phenotypical similarities with human sporadic colon cancer and is a reliable model for identifying chemopreventive agents. Genetic mutations relevant to human colon cancer have been described in this model, but comprehensive gene expression and genomic analysis have not been reported so far. Therefore, we applied genome-wide technologies to study variations in gene expression and genomic alterations in DMH-induced colon cancer in F344 rats. For gene expression analysis, 9 tumours (TUM) and their paired normal mucosa (NM) were hybridized on 4 × 44K Whole rat arrays (Agilent) and selected genes were validated by semi-quantitative RT-PCR. Functional analysis on microarray data was performed by GenMAPP/MappFinder analysis. Array-comparative genomic hybridization (a-CGH) was performed on 10 paired TUM-NM samples hybridized on Rat genome arrays 2 × 105K (Agilent) and the results were analyzed by CGH Analytics (Agilent). Microarray gene expression analysis showed that Defcr4, Igfbp5, Mmp7, Nos2, S100A8 and S100A9 were among the most up-regulated genes in tumours (Fold Change (FC) compared with NM: 183, 48, 39, 38, 36 and 32, respectively), while Slc26a3, Mptx, Retlna and Muc2 were strongly down-regulated (FC: -500; -376, -167, -79, respectively). Functional analysis showed that pathways controlling cell cycle, protein synthesis, matrix metalloproteinases, TNFα/NFkB, and inflammatory responses were up-regulated in tumours, while Krebs cycle, the electron transport chain, and fatty acid beta oxidation were down-regulated. a-CGH analysis showed that four TUM out of ten had one or two chromosomal aberrations. Importantly, one sample showed a deletion on chromosome 18 including Apc. The results showed complex gene expression alterations in adenocarcinomas encompassing many altered pathways. While a-CGH analysis showed a low degree of genomic imbalance, it is interesting to

  2. Novel genomes and genome constitutions identified by GISH and 5S rDNA and knotted1 genomic sequences in the genus Setaria.

    Science.gov (United States)

    Zhao, Meicheng; Zhi, Hui; Doust, Andrew N; Li, Wei; Wang, Yongfang; Li, Haiquan; Jia, Guanqing; Wang, Yongqiang; Zhang, Ning; Diao, Xianmin

    2013-04-11

    The Setaria genus is increasingly of interest to researchers, as its two species, S. viridis and S. italica, are being developed as models for understanding C4 photosynthesis and plant functional genomics. The genome constitution of Setaria species has been studied in the diploid species S. viridis, S. adhaerans and S. grisebachii, where three genomes A, B and C were identified respectively. Two allotetraploid species, S. verticillata and S. faberi, were found to have AABB genomes, and one autotetraploid species, S. queenslandica, with an AAAA genome, has also been identified. The genomes and genome constitutions of most other species remain unknown, even though it was thought there are approximately 125 species in the genus distributed world-wide. GISH was performed to detect the genome constitutions of Eurasia species of S. glauca, S. plicata, and S. arenaria, with the known A, B and C genomes as probes. No or very poor hybridization signal was detected indicating that their genomes are different from those already described. GISH was also performed reciprocally between S. glauca, S. plicata, and S. arenaria genomes, but no hybridization signals between each other were found. The two sets of chromosomes of S. lachnea both hybridized strong signals with only the known C genome of S. grisebachii. Chromosomes of Qing 9, an accession formerly considered as S. viridis, hybridized strong signal only to B genome of S. adherans. Phylogenetic trees constructed with 5S rDNA and knotted1 markers, clearly classify the samples in this study into six clusters, matching the GISH results, and suggesting that the F genome of S. arenaria is basal in the genus. Three novel genomes in the Setaria genus were identified and designated as genome D (S. glauca), E (S. plicata) and F (S. arenaria) respectively. The genome constitution of tetraploid S. lachnea is putatively CCC'C'. Qing 9 is a B genome species indigenous to China and is hypothesized to be a newly identified species. The

  3. Genome-wide high-resolution mapping of UV-induced mitotic recombination events in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Yi Yin

    2013-10-01

    Full Text Available In the yeast Saccharomyces cerevisiae and most other eukaryotes, mitotic recombination is important for the repair of double-stranded DNA breaks (DSBs. Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH. In this study, LOH events induced by ultraviolet (UV light are mapped throughout the genome to a resolution of about 1 kb using single-nucleotide polymorphism (SNP microarrays. UV doses that have little effect on the viability of diploid cells stimulate crossovers more than 1000-fold in wild-type cells. In addition, UV stimulates recombination in G1-synchronized cells about 10-fold more efficiently than in G2-synchronized cells. Importantly, at high doses of UV, most conversion events reflect the repair of two sister chromatids that are broken at approximately the same position whereas at low doses, most conversion events reflect the repair of a single broken chromatid. Genome-wide mapping of about 380 unselected crossovers, break-induced replication (BIR events, and gene conversions shows that UV-induced recombination events occur throughout the genome without pronounced hotspots, although the ribosomal RNA gene cluster has a significantly lower frequency of crossovers.

  4. Pomegranate Intake Protects Against Genomic Instability Induced by Medical X-rays In Vivo in Mice.

    Science.gov (United States)

    Nallanthighal, Sameera; Shirode, Amit B; Judd, Julius A; Reliene, Ramune

    2016-01-01

    Ionizing radiation (IR) is a well-documented human carcinogen. The increased use of IR in medical procedures has doubled the annual radiation dose and may increase cancer risk. Genomic instability is an intermediate lesion in IR-induced cancer. We examined whether pomegranate extract (PE) suppresses genomic instability induced by x-rays. Mice were treated orally with PE and exposed to an x-ray dose of 2 Gy. PE intake suppressed x-ray-induced DNA double-strand breaks (DSBs) in peripheral blood and chromosomal damage in bone marrow. We hypothesized that PE-mediated protection against x-ray-induced damage may be due to the upregulation of DSB repair and antioxidant enzymes and/or increase in glutathione (GSH) levels. We found that expression of DSB repair genes was not altered (Nbs1 and Rad50) or was reduced (Mre11, DNA-PKcs, Ku80, Rad51, Rad52 and Brca2) in the liver of PE-treated mice. Likewise, mRNA levels of antioxidant enzymes were reduced (Gpx1, Cat, and Sod2) or were not altered (HO-1 and Sod1) as a function of PE treatment. In contrast, PE-treated mice with and without IR exposure displayed higher hepatic GSH concentrations than controls. Thus, ingestion of pomegranate polyphenols is associated with inhibition of x-ray-induced genomic instability and elevated GSH, which may reduce cancer risk.

  5. Bystander effects, adaptive response and genomic instability induced by prenatal irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Streffer, Christian [Institute for Science and Ethics, University Duisburg-Essen, Auf dem Sutan 12, D-45239 Essen (Germany)]. E-mail: streffer.essen@t-online.de

    2004-12-02

    The developing human embryo and fetus undergo very radiosensitive stages during the prenatal development. It is likely that the induction of low dose related effects such as bystander effects, the adaptive response, and genomic instability would have profound effects on embryonic and fetal development. In this paper, I review what has been reported on the induction of these three phenomena in exposed embryos and fetuses. All three phenomena have been shown to occur in murine embryonic or fetal cells and structures, although the induction of an adaptive response (and also likely the induction of bystander effects) are limited in terms of when during development they can be induced and the dose or dose-rate used to treat animals in utero. In contrast, genomic instability can be induced throughout development, and the effects of radiation exposure on genome instability can be observed for long times after irradiation including through pre- and postnatal development and into the next generation of mice. There are clearly strain-specific differences in the induction of these phenomena and all three can lead to long-term detrimental effects. This is true for the adaptive response as well. While induction of an adaptive response can make fetuses more resistant to some gross developmental defects induced by a subsequent high dose challenge with ionizing radiation, the long-term effects of this low dose exposure are detrimental. The negative effects of all three phenomena reflect the complexity of fetal development, a process where even small changes in the timing of gene expression or suppression can have dramatic effects on the pattern of biological events and the subsequent development of the mammalian organism.

  6. Terminal structures of West Nile virus genomic RNA and their interactions with viral NS5 protein

    International Nuclear Information System (INIS)

    Dong Hongping; Zhang Bo; Shi Peiyong

    2008-01-01

    Genome cyclization is essential for flavivirus replication. We used RNases to probe the structures formed by the 5'-terminal 190 nucleotides and the 3'-terminal 111 nucleotides of the West Nile virus (WNV) genomic RNA. When analyzed individually, the two RNAs adopt stem-loop structures as predicted by the thermodynamic-folding program. However, when mixed together, the two RNAs form a duplex that is mediated through base-pairings of two sets of RNA elements (5'CS/3'CSI and 5'UAR/3'UAR). Formation of the RNA duplex facilitates a conformational change that leaves the 3'-terminal nucleotides of the genome (position - 8 to - 16) to be single-stranded. Viral NS5 binds specifically to the 5'-terminal stem-loop (SL1) of the genomic RNA. The 5'SL1 RNA structure is essential for WNV replication. The study has provided further evidence to suggest that flavivirus genome cyclization and NS5/5'SL1 RNA interaction facilitate NS5 binding to the 3' end of the genome for the initiation of viral minus-strand RNA synthesis

  7. Analysis of radiation-induced genome alterations in Vigna unguiculata

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    van der Vyver C

    2011-09-01

    Full Text Available Christell van der Vyver1, B Juan Vorster2, Karl J Kunert3, Christopher A Cullis41Institute for Plant Biotechnology, Department of Genetics, University of Stellenbosch, Stellenbosch, South Africa; 2Department of Plant Production and Soil Science, and 3Department of Plant Science, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, South Africa; 4Case Western Reserve University, Department of Biology, Cleveland, OH, USAAbstract: Seeds from an inbred Vigna unguiculata (cowpea cultivar were gamma-irradiated with a dose of 180 Gy in order to identify and characterize possible mutations. Three techniques, ie, random amplified polymorphic DNA, microsatellites, and representational difference analysis, were used to characterize possible DNA variation among the mutants and nonirradiated control plants both immediately after irradiation and in subsequent generations. A large portion of putative radiation-induced genome changes had significant similarities to chloroplast sequences. The frequency of mutation at three of these isolated polymorphic regions with chloroplast similarity was further determined by polymerase chain reaction screening using a large number of individual parental, M1, and M2 plants. Analysis of these sequences indicated that the rate at which various regions of the genome is mutated in irradiation experiments differs significantly and also that mutations have variable “repair” rates. Furthermore, regions of the nuclear DNA derived from the chloroplast genome are highly susceptible to modification by radiation treatment. Overall, data have provided detailed information on the effects of gamma irradiation on the cowpea genome and about the ability of the plant to repair these genome changes in subsequent plant generations.Keywords: mutation breeding, gamma radiation, genetic mutations, cowpea, representational difference analysis

  8. Fine organization of genomic regions tagged to the 5S rDNA locus of the bread wheat 5B chromosome.

    Science.gov (United States)

    Sergeeva, Ekaterina M; Shcherban, Andrey B; Adonina, Irina G; Nesterov, Michail A; Beletsky, Alexey V; Rakitin, Andrey L; Mardanov, Andrey V; Ravin, Nikolai V; Salina, Elena A

    2017-11-14

    The multigene family encoding the 5S rRNA, one of the most important structurally-functional part of the large ribosomal subunit, is an obligate component of all eukaryotic genomes. 5S rDNA has long been a favored target for cytological and phylogenetic studies due to the inherent peculiarities of its structural organization, such as the tandem arrays of repetitive units and their high interspecific divergence. The complex polyploid nature of the genome of bread wheat, Triticum aestivum, and the technically difficult task of sequencing clusters of tandem repeats mean that the detailed organization of extended genomic regions containing 5S rRNA genes remains unclear. This is despite the recent progress made in wheat genomic sequencing. Using pyrosequencing of BAC clones, in this work we studied the organization of two distinct 5S rDNA-tagged regions of the 5BS chromosome of bread wheat. Three BAC-clones containing 5S rDNA were identified in the 5BS chromosome-specific BAC-library of Triticum aestivum. Using the results of pyrosequencing and assembling, we obtained six 5S rDNA- containing contigs with a total length of 140,417 bp, and two sets (pools) of individual 5S rDNA sequences belonging to separate, but closely located genomic regions on the 5BS chromosome. Both regions are characterized by the presence of approximately 70-80 copies of 5S rDNA, however, they are completely different in their structural organization. The first region contained highly diverged short-type 5S rDNA units that were disrupted by multiple insertions of transposable elements. The second region contained the more conserved long-type 5S rDNA, organized as a single tandem array. FISH using probes specific to both 5S rDNA unit types showed differences in the distribution and intensity of signals on the chromosomes of polyploid wheat species and their diploid progenitors. A detailed structural organization of two closely located 5S rDNA-tagged genomic regions on the 5BS chromosome of bread

  9. The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine.

    Science.gov (United States)

    Huang, Yun; Pastor, William A; Zepeda-Martínez, Jorge A; Rao, Anjana

    2012-10-01

    5-Hydroxymethylcytosine (5hmC) is a recently discovered base in the mammalian genome, produced upon oxidation of 5-methylcytosine (5mC) in a process catalyzed by TET proteins. The biological functions of 5hmC and further oxidation products of 5mC are under intense investigation, as they are likely intermediates in DNA demethylation pathways. Here we describe a novel protocol to profile 5hmC at a genome-wide scale. This approach is based on sodium bisulfite-mediated conversion of 5hmC to cytosine-5-methylenesulfonate (CMS); CMS-containing DNA fragments are then immunoprecipitated using a CMS-specific antiserum. The anti-CMS technique is highly specific with a low background, and is much less dependent on 5hmC density than anti-5hmC immunoprecipitation (IP). Moreover, it does not enrich for CA and CT repeats, as noted for 5hmC DNA IP using antibodies to 5hmC. The anti-CMS protocol takes 3 d to complete.

  10. Genetic and epigenetic alterations induced by different levels of rye genome integration in wheat recipient.

    Science.gov (United States)

    Zheng, X L; Zhou, J P; Zang, L L; Tang, A T; Liu, D Q; Deng, K J; Zhang, Y

    2016-06-17

    The narrow genetic variation present in common wheat (Triticum aestivum) varieties has greatly restricted the improvement of crop yield in modern breeding systems. Alien addition lines have proven to be an effective means to broaden the genetic diversity of common wheat. Wheat-rye addition lines, which are the direct bridge materials for wheat improvement, have been wildly used to produce new wheat cultivars carrying alien rye germplasm. In this study, we investigated the genetic and epigenetic alterations in two sets of wheat-rye disomic addition lines (1R-7R) and the corresponding triticales. We used expressed sequence tag-simple sequence repeat, amplified fragment length polymorphism, and methylation-sensitive amplification polymorphism analyses to analyze the effects of the introduction of alien chromosomes (either the entire genome or sub-genome) to wheat genetic background. We found obvious and diversiform variations in the genomic primary structure, as well as alterations in the extent and pattern of the genomic DNA methylation of the recipient. Meanwhile, these results also showed that introduction of different rye chromosomes could induce different genetic and epigenetic alterations in its recipient, and the genetic background of the parents is an important factor for genomic and epigenetic variation induced by alien chromosome addition.

  11. Experimental Induction of Genome Chaos.

    Science.gov (United States)

    Ye, Christine J; Liu, Guo; Heng, Henry H

    2018-01-01

    Genome chaos, or karyotype chaos, represents a powerful survival strategy for somatic cells under high levels of stress/selection. Since the genome context, not the gene content, encodes the genomic blueprint of the cell, stress-induced rapid and massive reorganization of genome topology functions as a very important mechanism for genome (karyotype) evolution. In recent years, the phenomenon of genome chaos has been confirmed by various sequencing efforts, and many different terms have been coined to describe different subtypes of the chaotic genome including "chromothripsis," "chromoplexy," and "structural mutations." To advance this exciting field, we need an effective experimental system to induce and characterize the karyotype reorganization process. In this chapter, an experimental protocol to induce chaotic genomes is described, following a brief discussion of the mechanism and implication of genome chaos in cancer evolution.

  12. CRISPR-Cas9-Based Genome Editing of Human Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Giacalone, Joseph C; Sharma, Tasneem P; Burnight, Erin R; Fingert, John F; Mullins, Robert F; Stone, Edwin M; Tucker, Budd A

    2018-02-28

    Human induced pluripotent stem cells (hiPSCs) are the ideal cell source for autologous cell replacement. However, for patients with Mendelian diseases, genetic correction of the original disease-causing mutation is likely required prior to cellular differentiation and transplantation. The emergence of the CRISPR-Cas9 system has revolutionized the field of genome editing. By introducing inexpensive reagents that are relatively straightforward to design and validate, it is now possible to correct genetic variants or insert desired sequences at any location within the genome. CRISPR-based genome editing of patient-specific iPSCs shows great promise for future autologous cell replacement therapies. One caveat, however, is that hiPSCs are notoriously difficult to transfect, and optimized experimental design considerations are often necessary. This unit describes design strategies and methods for efficient CRISPR-based genome editing of patient- specific iPSCs. Additionally, it details a flexible approach that utilizes positive selection to generate clones with a desired genomic modification, Cre-lox recombination to remove the integrated selection cassette, and negative selection to eliminate residual hiPSCs with intact selection cassettes. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

  13. Radiation-induced genomic instability, and the cloning and functional analysis of its related gene

    International Nuclear Information System (INIS)

    Muto, Masahiro; Kanari, Yasuyoshi; Kubo, Eiko; Yamada, Yutaka

    2000-01-01

    Exposure to ionizing radiation produces a number of biological consequences including gene mutations, chromosome aberrations, cellular transformation and cell death. The classical view has been that mutations occur at the sites of DNA damage, that is, damage produced by radiation is converted into a mutation during subsequent DNA replication or as a consequence of enzymatic repair processes. However, many investigators have presented evidence for an alternative mechanism to explain these biological effects. This evidence suggests that radiation may induce a process of genomic instability that is transmissible over many generations of cell replication and that serves to enhance the probability of the occurrence of such genetic effects among the progeny of the irradiated cell after many generations of cell replication. If such a process exists in vivo, it could have significant implications for mechanisms of carcinogenesis. Exposure of B10 mice to fractionated X-irradiation induces a high incidence of thymic lymphomas, whereas the incidence in STS/A mice is very low. Such strain differences are presumably determined genetically, and various genetic factors have been reported to be involved in radiation-induced lymphomagenesis. The mechanism of radiation-induced lymphomagenesis appears to develop through a complex and multistep process. Using this experimental system, we characterized the prelymphoma cells induced by radiation, and identified the genetic changes preceding the development of thymic lymphomas by comparing the oncogenic alterations with the pattern of T cell receptor (TCR) γ rearrangements. In these studies, the latent expression of some chromosomal aberrations and p53 mutations in irradiated progeny has been interpreted to be a manifestation of genomic instability. In the present report we review the results of in vivo studies conducted in our laboratory that support the hypothesis of genomic instability induced by radiation, and we describe the

  14. RecQL5 promotes genome stabilization through two parallel mechanisms--interacting with RNA polymerase II and acting as a helicase.

    Science.gov (United States)

    Islam, M Nurul; Fox, David; Guo, Rong; Enomoto, Takemi; Wang, Weidong

    2010-05-01

    The RecQL5 helicase is essential for maintaining genome stability and reducing cancer risk. To elucidate its mechanism of action, we purified a RecQL5-associated complex and identified its major component as RNA polymerase II (Pol II). Bioinformatics and structural modeling-guided mutagenesis revealed two conserved regions in RecQL5 as KIX and SRI domains, already known in transcriptional regulators for Pol II. The RecQL5-KIX domain binds both initiation (Pol IIa) and elongation (Pol IIo) forms of the polymerase, whereas the RecQL5-SRI domain interacts only with the elongation form. Fully functional RecQL5 requires both helicase activity and associations with the initiation polymerase, because mutants lacking either activity are partially defective in the suppression of sister chromatid exchange and resistance to camptothecin-induced DNA damage, and mutants lacking both activities are completely defective. We propose that RecQL5 promotes genome stabilization through two parallel mechanisms: by participation in homologous recombination-dependent DNA repair as a RecQ helicase and by regulating the initiation of Pol II to reduce transcription-associated replication impairment and recombination.

  15. Fluorescent strategy based on cationic conjugated polymer fluorescence resonance energy transfer for the quantification of 5-(hydroxymethyl)cytosine in genomic DNA.

    Science.gov (United States)

    Hong, Tingting; Wang, Tianlu; Guo, Pu; Xing, Xiwen; Ding, Fei; Chen, Yuqi; Wu, Jinjun; Ma, Jingwei; Wu, Fan; Zhou, Xiang

    2013-11-19

    DNA methylation is dynamically reprogrammed during early embryonic development in mammals. It can be explained partially by the discovery of 5-(hydroxymethyl)cytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC), which are identified as key players involved in both active and passive demethylation pathways. As one of the ten-eleven translocation oxidation products, 5-hmC was found relatively abundant in neuron cells and embryonic stem cells. Herein we report a new method for 5-hmC quantification in genomic DNA based on CCP-FRET (cationic conjugated polymers act as the energy donor and induce fluorescence resonance energy transfer) assay combined with KRuO4 oxidation. 5-hmC in genomic DNA can be selectively transformed into 5-fC by the oxidation of KRuO4 and then labeled with hydroxylamine-BODIPY (BODIPY = 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore through the reaction between 5-fC and hydroxylamine-BODIPY. After the fluorescently labeled DNA was captured by CCP through electrostatic interactions, a significant FRET between CCP and hydroxylamine-BODIPY fluorophore was observed. This CCP-FRET-based assay benefits from light-harvesting, large Stokes shift, and optical signal amplification properties of the CCP. Furthermore, this CCP-FRET-based assay was quite successfully demonstrated for the 5-hmC quantification in three types of cells (mESc, HeLa, HEK 293T), providing a much more convenient choice for 5-hmC quantification in genomic DNA.

  16. Co-diversification of Enterococcus faecium core genomes and PBP5: evidences of pbp5 horizontal transfer

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    Carla Novais

    2016-10-01

    Full Text Available Ampicillin resistance has greatly contributed to the recent dramatic increase of a cluster of human adapted Enterococcus faecium lineages (ST17, ST18 and ST78 in hospital-based infections. Changes in the chromosomal pbp5 gene have been associated with different levels of ampicillin susceptibility, leading to protein variants (designated as PBP5 C-types to keep the nomenclature used in previous works with diverse degrees of reduction in penicillin affinity. Our goal was to use a comparative genomics approach to evaluate the relationship between the diversity of PBP5 among E. faecium isolates of different phylogenomic groups as well as to assess the pbp5 transferability among isolates of disparate clonal lineages. The analyses of 78 selected E. faecium strains as well as published E. faecium genomes, suggested that the diversity of pbp5 mirrors the phylogenomic diversification of E. faecium. The presence of identical PBP5 C-types as well as similar pbp5 genetic environments in different E. faecium lineages and clones from quite different geographical and environmental origin was also documented and would indicate their horizontal gene transfer among E. faecium populations. This was supported by experimental assays showing transfer of large (≈180-280 kb chromosomal genetic platforms containing pbp5 alleles, ponA (transglycosilase and other metabolic and adaptive features, from E. faecium donor isolates to suitable E. faecium recipient strains. Mutation profile analysis of PBP5 from available genomes and strains from this study suggests that the spread of PBP5 C-types might have occurred even in the absence of a significant ampicillin resistance phenotype. In summary, genetic platforms containing pbp5 sequences were stably maintained in particular E. faecium lineages, but were also able to be transferred among E. faecium clones of different origins, emphasizing the growing risk of further spread of ampicillin resistance in this nosocomial pathogen.

  17. Genomic instability induced by 60Co γ ray radiation in normal human liver cells

    International Nuclear Information System (INIS)

    Gen Xiaohua; Guo Xianhua; Zuo Yahui; Wang Xiaoli; Wang Zhongwen

    2007-01-01

    Objective: To explore the genomic instability induced by 60 Co γ rays. Methods: The cloning efficiency and micronucleus efficiency of normal human liver cell irradiated by 60 Co γ rays were detected, and the method of single cell gel electrophoresis (SCGE) was carried out to measure DNA chains damage. The fast-growing cells were divided into different dose-groups and then irradiated by 60 Co γ rays. After 40 populations doubling, the progenies were secondly irradiated with 2 Gy 60 Co γ rays. Results: The cloning efficiency decreased with the increase of doses after the initial irradiation. After the survival cells were given second irradiation, both results of SCGE and micronucleus frequency showed that the second damage was correlated with the original irradiation doses. Conclusions: 60 Co γ rays can not only induce the immediate biological effects in liver cells, but also lead to the genomic instability in the descendants that leads to an enhanced frequency of genetic changes occurring among the progeny of the original irradiated cell. The expanding effect of second event helps to study the genomic instability. (authors)

  18. The JAK-STAT transcriptional regulator, STAT-5, activates the ATM DNA damage pathway to induce HPV 31 genome amplification upon epithelial differentiation.

    Directory of Open Access Journals (Sweden)

    Shiyuan Hong

    Full Text Available High-risk human papillomavirus (HPV must evade innate immune surveillance to establish persistent infections and to amplify viral genomes upon differentiation. Members of the JAK-STAT family are important regulators of the innate immune response and HPV proteins downregulate expression of STAT-1 to allow for stable maintenance of viral episomes. STAT-5 is another member of this pathway that modulates the inflammatory response and plays an important role in controlling cell cycle progression in response to cytokines and growth factors. Our studies show that HPV E7 activates STAT-5 phosphorylation without altering total protein levels. Inhibition of STAT-5 phosphorylation by the drug pimozide abolishes viral genome amplification and late gene expression in differentiating keratinocytes. In contrast, treatment of undifferentiated cells that stably maintain episomes has no effect on viral replication. Knockdown studies show that the STAT-5β isoform is mainly responsible for this activity and that this is mediated through the ATM DNA damage response. A downstream target of STAT-5, the peroxisome proliferator-activated receptor γ (PPARγ contributes to the effects on members of the ATM pathway. Overall, these findings identify an important new regulatory mechanism by which the innate immune regulator, STAT-5, promotes HPV viral replication through activation of the ATM DNA damage response.

  19. Radiation-induced genomic instability: Are epigenetic mechanisms the missing link?

    Energy Technology Data Exchange (ETDEWEB)

    Aypar, Umut; Morgan, William F.; Baulch, Janet E.

    2011-02-01

    Purpose: This review examines the evidence for the hypothesis that epigenetics are involved in the initiation and perpetuation of radiation-induced genomic instability (RIGI). Conclusion: In addition to the extensively studied targeted effects of radiation, it is now apparent that non-targeted delayed effects such as RIGI are also important post-irradiation outcomes. In RIGI, unirradiated progeny cells display phenotypic changes at delayed times after radiation of the parental cell. RIGI is thought to be important in the process of carcinogenesis, however, the mechanism by which this occurs remains to be elucidated. In the genomically unstable clones developed by Morgan and colleagues, radiation-induced mutations, double-strand breaks, or changes in mRNA levels alone could not account for the initiation or perpetuation of RIGI. Since changes in the DNA sequence could not fully explain the mechanism of RIGI, inherited epigenetic changes may be involved. Epigenetics are known to play an important role in many cellular processes and epigenetic aberrations can lead to carcinogenesis. Recent studies in the field of radiation biology suggest that the changes in methylation patterns may be involved in RIGI. Together these clues have led us to hypothesize that epigenetics may be the missing link in understanding the mechanism behind RIGI.

  20. The genome of Chelonid herpesvirus 5 harbors atypical genes

    Science.gov (United States)

    Ackermann, Mathias; Koriabine, Maxim; Hartmann-Fritsch, Fabienne; de Jong, Pieter J.; Lewis, Teresa D.; Schetle, Nelli; Work, Thierry M.; Dagenais, Julie; Balazs, George H.; Leong, Jo-Ann C.

    2012-01-01

    The Chelonid fibropapilloma-associated herpesvirus (CFPHV; ChHV5) is believed to be the causative agent of fibropapillomatosis (FP), a neoplastic disease of marine turtles. While clinical signs and pathology of FP are well known, research on ChHV5 has been impeded because no cell culture system for its propagation exists. We have cloned a BAC containing ChHV5 in pTARBAC2.1 and determined its nucleotide sequence. Accordingly, ChHV5 has a type D genome and its predominant gene order is typical for the varicellovirus genus within thealphaherpesvirinae. However, at least four genes that are atypical for an alphaherpesvirus genome were also detected, i.e. two members of the C-type lectin-like domain superfamily (F-lec1, F-lec2), an orthologue to the mouse cytomegalovirus M04 (F-M04) and a viral sialyltransferase (F-sial). Four lines of evidence suggest that these atypical genes are truly part of the ChHV5 genome: (1) the pTARBAC insertion interrupted the UL52 ORF, leaving parts of the gene to either side of the insertion and suggesting that an intact molecule had been cloned. (2) Using FP-associated UL52 (F-UL52) as an anchor and the BAC-derived sequences as a means to generate primers, overlapping PCR was performed with tumor-derived DNA as template, which confirmed the presence of the same stretch of “atypical” DNA in independent FP cases. (3) Pyrosequencing of DNA from independent tumors did not reveal previously undetected viral sequences, suggesting that no apparent loss of viral sequence had happened due to the cloning strategy. (4) The simultaneous presence of previously known ChHV5 sequences and F-sial as well as F-M04 sequences was also confirmed in geographically distinct Australian cases of FP. Finally, transcripts of F-sial and F-M04 but not transcripts of lytic viral genes were detected in tumors from Hawaiian FP-cases. Therefore, we suggest that F-sial and F-M04 may play a role in FP pathogenesis

  1. Upregulation of FOXM1 induces genomic instability in human epidermal keratinocytes

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    Philpott Michael P

    2010-02-01

    Full Text Available Abstract Background The human cell cycle transcription factor FOXM1 is known to play a key role in regulating timely mitotic progression and accurate chromosomal segregation during cell division. Deregulation of FOXM1 has been linked to a majority of human cancers. We previously showed that FOXM1 was upregulated in basal cell carcinoma and recently reported that upregulation of FOXM1 precedes malignancy in a number of solid human cancer types including oral, oesophagus, lung, breast, kidney, bladder and uterus. This indicates that upregulation of FOXM1 may be an early molecular signal required for aberrant cell cycle and cancer initiation. Results The present study investigated the putative early mechanism of UVB and FOXM1 in skin cancer initiation. We have demonstrated that UVB dose-dependently increased FOXM1 protein levels through protein stabilisation and accumulation rather than de novo mRNA expression in human epidermal keratinocytes. FOXM1 upregulation in primary human keratinocytes triggered pro-apoptotic/DNA-damage checkpoint response genes such as p21, p38 MAPK, p53 and PARP, however, without causing significant cell cycle arrest or cell death. Using a high-resolution Affymetrix genome-wide single nucleotide polymorphism (SNP mapping technique, we provided the evidence that FOXM1 upregulation in epidermal keratinocytes is sufficient to induce genomic instability, in the form of loss of heterozygosity (LOH and copy number variations (CNV. FOXM1-induced genomic instability was significantly enhanced and accumulated with increasing cell passage and this instability was increased even further upon exposure to UVB resulting in whole chromosomal gain (7p21.3-7q36.3 and segmental LOH (6q25.1-6q25.3. Conclusion We hypothesise that prolonged and repeated UVB exposure selects for skin cells bearing stable FOXM1 protein causes aberrant cell cycle checkpoint thereby allowing ectopic cell cycle entry and subsequent genomic instability. The aberrant

  2. Genomic and non-genomic effects of androgens in the cardiovascular system: clinical implications.

    Science.gov (United States)

    Lucas-Herald, Angela K; Alves-Lopes, Rheure; Montezano, Augusto C; Ahmed, S Faisal; Touyz, Rhian M

    2017-07-01

    The principle steroidal androgens are testosterone and its metabolite 5α-dihydrotestosterone (DHT), which is converted from testosterone by the enzyme 5α-reductase. Through the classic pathway with androgens crossing the plasma membrane and binding to the androgen receptor (AR) or via mechanisms independent of the ligand-dependent transactivation function of nuclear receptors, testosterone induces genomic and non-genomic effects respectively. AR is widely distributed in several tissues, including vascular endothelial and smooth muscle cells. Androgens are essential for many developmental and physiological processes, especially in male reproductive tissues. It is now clear that androgens have multiple actions besides sex differentiation and sexual maturation and that many physiological systems are influenced by androgens, including regulation of cardiovascular function [nitric oxide (NO) release, Ca 2+ mobilization, vascular apoptosis, hypertrophy, calcification, senescence and reactive oxygen species (ROS) generation]. This review focuses on evidence indicating that interplay between genomic and non-genomic actions of testosterone may influence cardiovascular function. © 2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  3. DNA Oncogenic Virus-Induced Oxidative Stress, Genomic Damage, and Aberrant Epigenetic Alterations

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    Mankgopo Magdeline Kgatle

    2017-01-01

    Full Text Available Approximately 20% of human cancers is attributable to DNA oncogenic viruses such as human papillomavirus (HPV, hepatitis B virus (HBV, and Epstein-Barr virus (EBV. Unrepaired DNA damage is the most common and overlapping feature of these DNA oncogenic viruses and a source of genomic instability and tumour development. Sustained DNA damage results from unceasing production of reactive oxygen species and activation of inflammasome cascades that trigger genomic changes and increased propensity of epigenetic alterations. Accumulation of epigenetic alterations may interfere with genome-wide cellular signalling machineries and promote malignant transformation leading to cancer development. Untangling and understanding the underlying mechanisms that promote these detrimental effects remain the major objectives for ongoing research and hope for effective virus-induced cancer therapy. Here, we review current literature with an emphasis on how DNA damage influences HPV, HVB, and EBV replication and epigenetic alterations that are associated with carcinogenesis.

  4. Genome wide analysis of drug-induced torsades de pointes: lack of common variants with large effect sizes.

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    Elijah R Behr

    Full Text Available Marked prolongation of the QT interval on the electrocardiogram associated with the polymorphic ventricular tachycardia Torsades de Pointes is a serious adverse event during treatment with antiarrhythmic drugs and other culprit medications, and is a common cause for drug relabeling and withdrawal. Although clinical risk factors have been identified, the syndrome remains unpredictable in an individual patient. Here we used genome-wide association analysis to search for common predisposing genetic variants. Cases of drug-induced Torsades de Pointes (diTdP, treatment tolerant controls, and general population controls were ascertained across multiple sites using common definitions, and genotyped on the Illumina 610k or 1M-Duo BeadChips. Principal Components Analysis was used to select 216 Northwestern European diTdP cases and 771 ancestry-matched controls, including treatment-tolerant and general population subjects. With these sample sizes, there is 80% power to detect a variant at genome-wide significance with minor allele frequency of 10% and conferring an odds ratio of ≥2.7. Tests of association were carried out for each single nucleotide polymorphism (SNP by logistic regression adjusting for gender and population structure. No SNP reached genome wide-significance; the variant with the lowest P value was rs2276314, a non-synonymous coding variant in C18orf21 (p  =  3×10(-7, odds ratio = 2, 95% confidence intervals: 1.5-2.6. The haplotype formed by rs2276314 and a second SNP, rs767531, was significantly more frequent in controls than cases (p  =  3×10(-9. Expanding the number of controls and a gene-based analysis did not yield significant associations. This study argues that common genomic variants do not contribute importantly to risk for drug-induced Torsades de Pointes across multiple drugs.

  5. The Role of DNA Methylation Changes in Radiation-Induced Transgenerational Genomic Instability and Bystander Effects in cranial irradiated Mice

    Science.gov (United States)

    Zhang, Meng; Sun, Yeqing; Gao, Yinglong; Zhang, Baodong

    Heavy-ion radiation could lead to genome instability in the germline, and therefore to transgenerational genome and epigenome instability in offspring of exposed males. The exact mechanisms of radiation-induced genome instability in directly exposed and in bystander organ remain obscure, yet accumulating evidence points to the role of DNA methylation changes in genome instability development. The potential of localized body-part exposures to affect the germline and thus induce genome and epigenome changes in the progeny has not been studied. To investigate whether or not the paternal cranial irradiation can exert deleterious changes in the protected germline and the offsprings, we studied the alteration of DNA methylation in the shielded testes tissue. Here we report that the localized paternal cranial irradiation results in a significant altered DNA methylation in sperm cells and leads to a profound epigenetic dysregulation in the unexposed progeny conceived 3 months after paternal exposure. The possible molecular mechanisms and biological consequences of the observed changes are discussed. Keywords: Heavy-ion radiation; Transgenerational effect; Genomic Instability Bystander Effects; DNA methylation.

  6. Comprehensive genomic analysis of a plant growth-promoting rhizobacterium Pantoea agglomerans strain P5.

    Science.gov (United States)

    Shariati J, Vahid; Malboobi, Mohammad Ali; Tabrizi, Zeinab; Tavakol, Elahe; Owilia, Parviz; Safari, Maryam

    2017-11-15

    In this study, we provide a comparative genomic analysis of Pantoea agglomerans strain P5 and 10 closely related strains based on phylogenetic analyses. A next-generation shotgun strategy was implemented using the Illumina HiSeq 2500 technology followed by core- and pan-genome analysis. The genome of P. agglomerans strain P5 contains an assembly size of 5082485 bp with 55.4% G + C content. P. agglomerans consists of 2981 core and 3159 accessory genes for Coding DNA Sequences (CDSs) based on the pan-genome analysis. Strain P5 can be grouped closely with strains PG734 and 299 R using pan and core genes, respectively. All the predicted and annotated gene sequences were allocated to KEGG pathways. Accordingly,  genes involved in plant growth-promoting (PGP) ability, including phosphate solubilization, IAA and siderophore production, acetoin and 2,3-butanediol synthesis and bacterial secretion, were assigned. This study provides an in-depth view of the PGP characteristics of strain P5, highlighting its potential use in agriculture as a biofertilizer.

  7. A plant-based chemical genomics screen for the identification of flowering inducers.

    Science.gov (United States)

    Fiers, Martijn; Hoogenboom, Jorin; Brunazzi, Alice; Wennekes, Tom; Angenent, Gerco C; Immink, Richard G H

    2017-01-01

    Floral timing is a carefully regulated process, in which the plant determines the optimal moment to switch from the vegetative to reproductive phase. While there are numerous genes known that control flowering time, little information is available on chemical compounds that are able to influence this process. We aimed to discover novel compounds that are able to induce flowering in the model plant Arabidopsis. For this purpose we developed a plant-based screening platform that can be used in a chemical genomics study. Here we describe the set-up of the screening platform and various issues and pitfalls that need to be addressed in order to perform a chemical genomics screening on Arabidopsis plantlets. We describe the choice for a molecular marker, in combination with a sensitive reporter that's active in plants and is sufficiently sensitive for detection. In this particular screen, the firefly Luciferase marker was used, fused to the regulatory sequences of the floral meristem identity gene APETALA1 (AP1) , which is an early marker for flowering. Using this screening platform almost 9000 compounds were screened, in triplicate, in 96-well plates at a concentration of 25 µM. One of the identified potential flowering inducing compounds was studied in more detail and named Flowering1 (F1). F1 turned out to be an analogue of the plant hormone Salicylic acid (SA) and appeared to be more potent than SA in the induction of flowering. The effect could be confirmed by watering Arabidopsis plants with SA or F1, in which F1 gave a significant reduction in time to flowering in comparison to SA treatment or the control. In this study a chemical genomics screening platform was developed to discover compounds that can induce flowering in Arabidopsis. This platform was used successfully, to identify a compound that can speed-up flowering in Arabidopsis.

  8. The Genomes OnLine Database (GOLD) v.5: a metadata management system based on a four level (meta)genome project classification

    Science.gov (United States)

    Reddy, T.B.K.; Thomas, Alex D.; Stamatis, Dimitri; Bertsch, Jon; Isbandi, Michelle; Jansson, Jakob; Mallajosyula, Jyothi; Pagani, Ioanna; Lobos, Elizabeth A.; Kyrpides, Nikos C.

    2015-01-01

    The Genomes OnLine Database (GOLD; http://www.genomesonline.org) is a comprehensive online resource to catalog and monitor genetic studies worldwide. GOLD provides up-to-date status on complete and ongoing sequencing projects along with a broad array of curated metadata. Here we report version 5 (v.5) of the database. The newly designed database schema and web user interface supports several new features including the implementation of a four level (meta)genome project classification system and a simplified intuitive web interface to access reports and launch search tools. The database currently hosts information for about 19 200 studies, 56 000 Biosamples, 56 000 sequencing projects and 39 400 analysis projects. More than just a catalog of worldwide genome projects, GOLD is a manually curated, quality-controlled metadata warehouse. The problems encountered in integrating disparate and varying quality data into GOLD are briefly highlighted. GOLD fully supports and follows the Genomic Standards Consortium (GSC) Minimum Information standards. PMID:25348402

  9. The Genomes OnLine Database (GOLD) v.5: a metadata management system based on a four level (meta)genome project classification

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, Tatiparthi B. K. [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Thomas, Alex D. [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Stamatis, Dimitri [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Bertsch, Jon [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Isbandi, Michelle [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Jansson, Jakob [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Mallajosyula, Jyothi [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Pagani, Ioanna [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Lobos, Elizabeth A. [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Kyrpides, Nikos C. [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); King Abdulaziz Univ., Jeddah (Saudi Arabia)

    2014-10-27

    The Genomes OnLine Database (GOLD; http://www.genomesonline.org) is a comprehensive online resource to catalog and monitor genetic studies worldwide. GOLD provides up-to-date status on complete and ongoing sequencing projects along with a broad array of curated metadata. Within this paper, we report version 5 (v.5) of the database. The newly designed database schema and web user interface supports several new features including the implementation of a four level (meta)genome project classification system and a simplified intuitive web interface to access reports and launch search tools. The database currently hosts information for about 19 200 studies, 56 000 Biosamples, 56 000 sequencing projects and 39 400 analysis projects. More than just a catalog of worldwide genome projects, GOLD is a manually curated, quality-controlled metadata warehouse. The problems encountered in integrating disparate and varying quality data into GOLD are briefly highlighted. Lastly, GOLD fully supports and follows the Genomic Standards Consortium (GSC) Minimum Information standards.

  10. Draft Genome Sequence of Lactobacillus delbrueckii subsp. bulgaricus LBB.B5

    NARCIS (Netherlands)

    Urshev, Z.; Hajo, K.; Lenoci, L.; Bron, P.A.; Dijkstra, A.; Alkema, W.; Wels, M.; Siezen, R.J.; Minkova, S.; Hijum, S.A. van

    2016-01-01

    Lactobacillus delbrueckii subsp. bulgaricus LBB.B5 originates from homemade Bulgarian yogurt and was selected for its ability to form a strong association with Streptococcus thermophilus The genome sequence will facilitate elucidating the genetic background behind the contribution of LBB.B5 to the

  11. The 5′ and 3′ Untranslated Regions of the Flaviviral Genome

    Directory of Open Access Journals (Sweden)

    Wy Ching Ng

    2017-06-01

    Full Text Available Flaviviruses are enveloped arthropod-borne viruses with a single-stranded, positive-sense RNA genome that can cause serious illness in humans and animals. The 11 kb 5′ capped RNA genome consists of a single open reading frame (ORF, and is flanked by 5′ and 3′ untranslated regions (UTR. The ORF is a polyprotein that is processed into three structural and seven non-structural proteins. The UTRs have been shown to be important for viral replication and immune modulation. Both of these regions consist of elements that are essential for genome cyclization, resulting in initiation of RNA synthesis. Genome mutation studies have been employed to investigate each component of the essential elements to show the necessity of each component and its role in viral RNA replication and growth. Furthermore, the highly structured 3′UTR is responsible for the generation of subgenomic flavivirus RNA (sfRNA that helps the virus evade host immune response, thereby affecting viral pathogenesis. In addition, changes within the 3′UTR have been shown to affect transmissibility between vector and host, which can influence the development of vaccines.

  12. A genome-wide association study of heparin-induced thrombocytopenia using an electronic medical record

    DEFF Research Database (Denmark)

    Karnes, Jason H; Cronin, Robert M; Rollin, Jerome

    2015-01-01

    Heparin-induced thrombocytopenia (HIT) is an unpredictable, potentially catastrophic adverse effect of heparin treatment resulting from an immune response to platelet factor 4 (PF4)/heparin complexes. No genome-wide evaluations have been performed to identify potential genetic influences on HIT. ...

  13. Genomic instability following irradiation

    International Nuclear Information System (INIS)

    Hacker-Klom, U.B.; Goehde, W.

    2001-01-01

    Ionising irradiation may induce genomic instability. The broad spectrum of stress reactions in eukaryontic cells to irradiation complicates the discovery of cellular targets and pathways inducing genomic instability. Irradiation may initiate genomic instability by deletion of genes controlling stability, by induction of genes stimulating instability and/or by activating endogeneous cellular viruses. Alternatively or additionally it is discussed that the initiation of genomic instability may be a consequence of radiation or other agents independently of DNA damage implying non nuclear targets, e.g. signal cascades. As a further mechanism possibly involved our own results may suggest radiation-induced changes in chromatin structure. Once initiated the process of genomic instability probably is perpetuated by endogeneous processes necessary for proliferation. Genomic instability may be a cause or a consequence of the neoplastic phenotype. As a conclusion from the data available up to now a new interpretation of low level radiation effects for radiation protection and in radiotherapy appears useful. The detection of the molecular mechanisms of genomic instability will be important in this context and may contribute to a better understanding of phenomenons occurring at low doses <10 cSv which are not well understood up to now. (orig.)

  14. Genome-Wide Mapping of 5mC and 5hmC Identified Differentially Modified Genomic Regions in Late-Onset Severe Preeclampsia: A Pilot Study.

    Directory of Open Access Journals (Sweden)

    Lisha Zhu

    Full Text Available Preeclampsia (PE is a leading cause of perinatal morbidity and mortality. However, as a common form of PE, the etiology of late-onset PE is elusive. We analyzed 5-methylcytosine (5mC and 5-hydroxymethylcytosine (5hmC levels in the placentas of late-onset severe PE patients (n = 4 and normal controls (n = 4 using a (hydroxymethylated DNA immunoprecipitation approach combined with deep sequencing ([h]MeDIP-seq, and the results were verified by (hMeDIP-qPCR. The most significant differentially methylated regions (DMRs were verified by MassARRAY EppiTYPER in an enlarged sample size (n = 20. Bioinformatics analysis identified 714 peaks of 5mC that were associated with 403 genes and 119 peaks of 5hmC that were associated with 61 genes, thus showing significant differences between the PE patients and the controls (>2-fold, p<0.05. Further, only one gene, PTPRN2, had both 5mC and 5hmC changes in patients. The ErbB signaling pathway was enriched in those 403 genes that had significantly different 5mC level between the groups. This genome-wide mapping of 5mC and 5hmC in late-onset severe PE and normal controls demonstrates that both 5mC and 5hmC play epigenetic roles in the regulation of the disease, but work independently. We reveal the genome-wide mapping of DNA methylation and DNA hydroxymethylation in late-onset PE placentas for the first time, and the identified ErbB signaling pathway and the gene PTPRN2 may be relevant to the epigenetic pathogenesis of late-onset PE.

  15. Drosophila Sld5 is essential for normal cell cycle progression and maintenance of genomic integrity

    Energy Technology Data Exchange (ETDEWEB)

    Gouge, Catherine A. [Department of Biology, East Carolina University East Carolina University, Greenville, NC 27858 (United States); Christensen, Tim W., E-mail: christensent@ecu.edu [Department of Biology, East Carolina University East Carolina University, Greenville, NC 27858 (United States)

    2010-09-10

    Research highlights: {yields} Drosophila Sld5 interacts with Psf1, PPsf2, and Mcm10. {yields} Haploinsufficiency of Sld5 leads to M-phase delay and genomic instability. {yields} Sld5 is also required for normal S phase progression. -- Abstract: Essential for the normal functioning of a cell is the maintenance of genomic integrity. Failure in this process is often catastrophic for the organism, leading to cell death or mis-proliferation. Central to genomic integrity is the faithful replication of DNA during S phase. The GINS complex has recently come to light as a critical player in DNA replication through stabilization of MCM2-7 and Cdc45 as a member of the CMG complex which is likely responsible for the processivity of helicase activity during S phase. The GINS complex is made up of 4 members in a 1:1:1:1 ratio: Psf1, Psf2, Psf3, And Sld5. Here we present the first analysis of the function of the Sld5 subunit in a multicellular organism. We show that Drosophila Sld5 interacts with Psf1, Psf2, and Mcm10 and that mutations in Sld5 lead to M and S phase delays with chromosomes exhibiting hallmarks of genomic instability.

  16. Radiation-induced genomic instability and bystander effects: related inflammatory-type responses to radiation-induced stress and injury? A review.

    Science.gov (United States)

    Lorimore, S A; Wright, E G

    2003-01-01

    To review studies of radiation responses in the haemopoietic system in the context of radiation-induced genomic instability, bystander effects and inflammatory-type processes. There is considerable evidence that cells that themselves are not exposed to ionizing radiation but are the progeny of cells irradiated many cell divisions previously may express a high frequency of gene mutations, chromosomal aberrations and cell death. These effects are collectively known as radiation-induced genomic instability. A second untargeted effect results in non-irradiated cells exhibiting responses typically associated with direct radiation exposure but occurs as a consequence of contact with irradiated cells or by receiving soluble signals from irradiated cells. These effects are collectively known as radiation-induced bystander effects. Reported effects include increases or decreases in damage-inducible and stress-related proteins; increases or decreases in reactive oxygen species, cell death or cell proliferation, and induction of mutations and chromosome aberrations. This array of responses is reminiscent of effects mediated by cytokines and other similar regulatory factors that may involve, but do not necessarily require, gap junction-mediated transfer, have multiple inducers and a variety of context-dependent consequences in different cell systems. That chromosomal instability in haemopoietic cells can be induced by an indirect bystander-type mechanism both in vitro and in vivo provides a potential link between these two untargeted effects and there are radiation responses in vivo consistent with the microenvironment contributing secondary cell damage as a consequence of an inflammatory-type response to radiation-induced injury. Intercellular signalling, production of cytokines and free radicals are features of inflammatory responses that have the potential for both bystander-mediated and persisting damage as well as for conferring a predisposition to malignancy. The

  17. Genome Sequences of Subcluster K5 Mycobacteriophages AlleyCat, Edugator, and Guillsminger.

    Science.gov (United States)

    King, Rodney A; Slowan-Pomeroy, Tina M; Thomas, Jodi E; Ahmed, Tithe; Alexander, Katie L; Biddle, James M; Daniels, Makenzie K; Rowlett, Jenna R; Senay, Taylor E; Rinehart, Claire A; Staples, Amanda K; Rowland, Naomi S; Gaffney, Bobby L; Emmons, Christine B; Hauk, Maya D; Nguyen, Rebecca L; Naegele, Leonard; Strickland, Summer S; Briggs, Laura A; Rush, Alexander N; Saha, Sanghamitra; Sadana, Rachna; Cresawn, Steven G; Russell, Daniel A; Garlena, Rebecca A; Pope, Welkin H; Jacobs-Sera, Deborah; Hatfull, Graham F

    2017-11-09

    Bacteriophages AlleyCat, Edugator, and Guillsminger were isolated on Mycobacterium smegmatis mc 2 155 from enriched soil samples. All are members of mycobacteriophage subcluster K5, with genomes of 62,112 to 63,344 bp. Each genome contains 92 to 99 predicted protein-coding genes and one tRNA. Guillsminger is the first mycobacteriophage to carry an IS 1380 family transposon. Copyright © 2017 King et al.

  18. Genomic instability in rat: Breakpoints induced by ionising radiation and interstitial telomeric-like sequences

    International Nuclear Information System (INIS)

    Camats, Nuria; Ruiz-Herrera, Aurora; Parrilla, Juan Jose; Acien, Maribel; Paya, Pilar; Giulotto, Elena; Egozcue, Josep; Garcia, Francisca; Garcia, Montserrat

    2006-01-01

    The Norwegian rat (Rattus norvegicus) is the most widely studied experimental species in biomedical research although little is known about its chromosomal structure. The characterisation of possible unstable regions of the karyotype of this species would contribute to the better understanding of its genomic architecture. The cytogenetic effects of ionising radiation have been widely used for the study of genomic instability, and the importance of interstitial telomeric-like sequences (ITSs) in instability of the genome has also been reported in previous studies in vertebrates. In order to describe the unstable chromosomal regions of R. norvegicus, the distribution of breakpoints induced by X-irradiation and ITSs in its karyotype were analysed in this work. For the X-irradiation analysis, 52 foetuses (from 14 irradiated rats) were studied, 4803 metaphases were analysed, and a total of 456 breakpoints induced by X-rays were detected, located in 114 chromosomal bands, with 25 of them significantly affected by X-irradiation (hot spots). For the analysis of ITSs, three foetuses (from three rats) were studied, 305 metaphases were analysed and 121 ITSs were detected, widely distributed in the karyotype of this species. Seventy-six percent of all hot spots analysed in this study were co-localised with ITSs

  19. Genomic instability in rat: Breakpoints induced by ionising radiation and interstitial telomeric-like sequences

    Energy Technology Data Exchange (ETDEWEB)

    Camats, Nuria [Institut de Biotecnologia i Biomedicina (IBB), Universitat Autonoma de Barcelona, 08193 Barcelona (Spain); Departament de Biologia Cel.lular, Fisiologia i Immunologia Universitat Autonoma de Barcelona, 08193 Barcelona (Spain); Ruiz-Herrera, Aurora [Departament de Biologia Cel.lular, Fisiologia i Immunologia Universitat Autonoma de Barcelona, 08193 Barcelona (Spain); Parrilla, Juan Jose [Servicio de Ginecologia y Obstetricia, Hospital Universitario Virgen de la Arrixaca, Ctra, Madrid-Cartagena, s/n, El Palmar, 30120 Murcia (Spain); Acien, Maribel [Servicio de Ginecologia y Obstetricia, Hospital Universitario Virgen de la Arrixaca, Ctra, Madrid-Cartagena, s/n, El Palmar, 30120 Murcia (Spain); Paya, Pilar [Servicio de Ginecologia y Obstetricia, Hospital Universitario Virgen de la Arrixaca, Ctra, Madrid-Cartagena, s/n, El Palmar, 30120 Murcia (Spain); Giulotto, Elena [Dipartimento di Genetica e Microbiologia Adriano Buzzati Traverso, Universita degli Studi di Pavia, 27100 Pavia (Italy); Egozcue, Josep [Departament de Biologia Cel.lular, Fisiologia i Immunologia Universitat Autonoma de Barcelona, 08193 Barcelona (Spain); Garcia, Francisca [Institut de Biotecnologia i Biomedicina (IBB), Universitat Autonoma de Barcelona, 08193 Barcelona (Spain); Garcia, Montserrat [Institut de Biotecnologia i Biomedicina (IBB), Universitat Autonoma de Barcelona, 08193 Barcelona (Spain) and Departament de Biologia Cellular, Fisiologia i Immunologia Universitat Autonoma de Barcelona, 08193 Barcelona (Spain)]. E-mail: Montserrat.Garcia.Caldes@uab.es

    2006-03-20

    The Norwegian rat (Rattus norvegicus) is the most widely studied experimental species in biomedical research although little is known about its chromosomal structure. The characterisation of possible unstable regions of the karyotype of this species would contribute to the better understanding of its genomic architecture. The cytogenetic effects of ionising radiation have been widely used for the study of genomic instability, and the importance of interstitial telomeric-like sequences (ITSs) in instability of the genome has also been reported in previous studies in vertebrates. In order to describe the unstable chromosomal regions of R. norvegicus, the distribution of breakpoints induced by X-irradiation and ITSs in its karyotype were analysed in this work. For the X-irradiation analysis, 52 foetuses (from 14 irradiated rats) were studied, 4803 metaphases were analysed, and a total of 456 breakpoints induced by X-rays were detected, located in 114 chromosomal bands, with 25 of them significantly affected by X-irradiation (hot spots). For the analysis of ITSs, three foetuses (from three rats) were studied, 305 metaphases were analysed and 121 ITSs were detected, widely distributed in the karyotype of this species. Seventy-six percent of all hot spots analysed in this study were co-localised with ITSs.

  20. Development of radiation-induced mutation techniques and functional genomics studies

    International Nuclear Information System (INIS)

    Kim, Dong Sub; Kang, Si Yong; Kim, Jin Baek

    2012-01-01

    This project has been performed to develop plant genetic resources using radiation (gamma-rays, ion-beam, space environments), to conduct functional genomics studies with mutant resources, and to develop new radiation plant breeding techniques using various radiation sources during 3 years. In the first section, we developed flower genetic resources, functional crop resources, and bio-industrial plant resources. In the second section, we cloned several mutated genes and studied mechanisms of gene expression and genetic diversity of mutations induced by gamma-rays. In the third section, we developed new plant breeding techniques using gamma-phytotron, heavy ion-beam, and space environments. Based on these results, a total of 8 cultivars containing Chrysanthemum, Hibiscus, kenaf, rice, and soybean were applied for plant variety protection (PVP) and a total of 4 cultivars were registered for PVP. Also, license agreement for the dwarf type Hibiscus mutant 'Ggoma' was conducted with Supro co. and the manufacturing technology for natural antioxidant pear-grape vinegar was transferred into Enzenic co. Also, 8 gene sequences, such as F3'H and LDOX genes associated with flower color in Chrysanthemum and EPSPS gene from Korean lawn grass, were registered in the database of National Center for Biotechnology Information (NCBI). In the future study, we will develop new radiation mutation breeding techniques through the mutation spectrum induced by various radiation sources, the studies for mechanism of the cellular response to radiation, and the comparative·structural·functional genomics studies for useful traits

  1. RNA interactions in the 5' region of the HIV-1 genome

    DEFF Research Database (Denmark)

    Damgaard, Christian Kroun; Andersen, Ebbe Sloth; Knudsen, Bjarne

    2004-01-01

    The untranslated leader of the dimeric HIV-1 RNA genome is folded into a complex structure that plays multiple and essential roles in the viral replication cycle. Here, we have investigated secondary and tertiary structural elements within the 5' 744 nucleotides of the HIV-1 genome using...... a combination of bioinformatics, enzymatic probing, native gel electrophoresis, and UV-crosslinking experiments. We used a recently developed RNA folding algorithm (Pfold) to predict the common secondary structure of an alignment of 20 divergent HIV-1 sequences. Combining this analysis with biochemical data, we...

  2. The genome structure of Arachis hypogaea (Linnaeus, 1753 and an induced Arachis allotetraploid revealed by molecular cytogenetics

    Directory of Open Access Journals (Sweden)

    Eliza F. de M. B. do Nascimento

    2018-03-01

    Full Text Available Peanut, Arachis hypogaea (Linnaeus, 1753 is an allotetraploid cultivated plant with two subgenomes derived from the hybridization between two diploid wild species, A. duranensis (Krapovickas & W. C. Gregory, 1994 and A. ipaensis (Krapovickas & W. C. Gregory, 1994, followed by spontaneous chromosomal duplication. To understand genome changes following polyploidy, the chromosomes of A. hypogaea, IpaDur1, an induced allotetraploid (A. ipaensis × A. duranensis4x and the diploid progenitor species were cytogenetically compared. The karyotypes of the allotetraploids share the number and general morphology of chromosomes; DAPI+ bands pattern and number of 5S rDNA loci. However, one 5S rDNA locus presents a heteromorphic FISH signal in both allotetraploids, relative to corresponding progenitor. Whilst for A. hypogaea the number of 45S rDNA loci was equivalent to the sum of those present in the diploid species, in IpaDur1, two loci have not been detected. Overall distribution of repetitive DNA sequences was similar in both allotetraploids, although A. hypogaea had additional CMA3+ bands and few slight differences in the LTR-retrotransposons distribution compared to IpaDur1. GISH showed that the chromosomes of both allotetraploids had preferential hybridization to their corresponding diploid genomes. Nevertheless, at least one pair of IpaDur1 chromosomes had a clear mosaic hybridization pattern indicating recombination between the subgenomes, clear evidence that the genome of IpaDur1 shows some instability comparing to the genome of A. hypogaea that shows no mosaic of subgenomes, although both allotetraploids derive from the same progenitor species. For some reasons, the chromosome structure of A. hypogaea is inherently more stable, or, it has been at least, partially stabilized through genetic changes and selection.

  3. Noncontiguous finished genome sequence and description of Paenibacillus ihumii sp. nov. strain AT5

    Directory of Open Access Journals (Sweden)

    A.H. Togo

    2016-03-01

    Full Text Available Paenibacillus ihumii sp. nov. strain AT5 (= CSUR 1981 = DSM 100664 is the type strain of P. ihumii. This bacterium was isolated from a stool sample from a morbidly obese French patient using the culturomics approach. The genome of this Gram-negative, facultative anaerobic, motile and spore-forming bacillus is 5 924 686 bp long. Genomic analysis identified 253 (5% of 3812 genes as ORFans and at least 2599 (50.03% of 5194 orthologous proteins not shared with the closest phylogenetic species.

  4. Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA.

    Science.gov (United States)

    Shahal, Tamar; Gilat, Noa; Michaeli, Yael; Redy-Keisar, Orit; Shabat, Doron; Ebenstein, Yuval

    2014-08-19

    5-Hydroxymethylcytosine (5hmC), a modified form of the DNA base cytosine, is an important epigenetic mark linked to regulation of gene expression in development, and tumorigenesis. We have developed a spectroscopic method for a global quantification of 5hmC in genomic DNA. The assay is performed within a multiwell plate, which allows simultaneous recording of up to 350 samples. Our quantification procedure of 5hmC is direct, simple, and rapid. It relies on a two-step protocol that consists of enzymatic glucosylation of 5hmC with an azide-modified glucose, followed by a "click reaction" with an alkyne-fluorescent tag. The fluorescence intensity recorded from the DNA sample is proportional to its 5hmC content and can be quantified by a simple plate reader measurement. This labeling technique is specific and highly sensitive, allowing detection of 5hmC down to 0.002% of the total nucleotides. Our results reveal significant variations in the 5hmC content obtained from different mouse tissues, in agreement with previously reported data.

  5. Genome-wide mapping for clinically relevant predictors of lamotrigine- and phenytoin-induced hypersensitivity reactions.

    LENUS (Irish Health Repository)

    McCormack, Mark

    2012-03-01

    An association between carbamazepine-induced hypersensitivity and HLA-A*3101 has been reported in populations of both European and Asian descent. We aimed to investigate HLA-A*3101 and other common variants across the genome as markers for cutaneous adverse drug reactions (cADRs) attributed to lamotrigine and phenytoin.

  6. The 5'UTR-specific mutation in VEEV TC-83 genome has a strong effect on RNA replication and subgenomic RNA synthesis, but not on translation of the encoded proteins.

    Science.gov (United States)

    Kulasegaran-Shylini, Raghavendran; Thiviyanathan, Varatharasa; Gorenstein, David G; Frolov, Ilya

    2009-04-25

    Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Viruses in the VEEV serocomplex continuously circulate in the Central and South America. The only currently available attenuated strain VEEV TC-83 is being used only for vaccination of at-risk laboratory workers and military personnel. Its attenuated phenotype was shown to rely only on two point mutations, one of which, G3A, was found in the 5' untranslated region (5'UTR) of the viral genome. Our data demonstrate that the G3A mutation strongly affects the secondary structure of VEEV 5'UTR, but has only a minor effect on translation. The indicated mutation increases replication of the viral genome, downregulates transcription of the subgenomic RNA, and, thus, affects the ratio of genomic and subgenomic RNA synthesis. These findings and the previously reported G3A-induced, higher sensitivity of VEEV TC-83 to IFN-alpha/beta suggest a plausible explanation for its attenuated phenotype.

  7. Replication and virus-induced transcriptome of HAdV-5 in normal host cells versus cancer cells--differences of relevance for adenoviral oncolysis.

    Directory of Open Access Journals (Sweden)

    Dominik E Dorer

    Full Text Available Adenoviruses (Ads, especially HAdV-5, have been genetically equipped with tumor-restricted replication potential to enable applications in oncolytic cancer therapy. Such oncolytic adenoviruses have been well tolerated in cancer patients, but their anti-tumor efficacy needs to be enhanced. In this regard, it should be considered that cancer cells, dependent on their tissue of origin, can differ substantially from the normal host cells to which Ads are adapted by complex virus-host interactions. Consequently, viral replication efficiency, a key determinant of oncolytic activity, might be suboptimal in cancer cells. Therefore, we have analyzed both the replication kinetics of HAdV-5 and the virus-induced transcriptome in human bronchial epithelial cells (HBEC in comparison to cancer cells. This is the first report on genome-wide expression profiling of Ads in their native host cells. We found that E1A expression and onset of viral genome replication are most rapid in HBEC and considerably delayed in melanoma cells. In squamous cell lung carcinoma cells, we observed intermediate HAdV-5 replication kinetics. Infectious particle production, viral spread and lytic activity of HAdV-5 were attenuated in melanoma cells versus HBEC. Expression profiling at the onset of viral genome replication revealed that HAdV-5 induced the strongest changes in the cellular transcriptome in HBEC, followed by lung cancer and melanoma cells. We identified prominent regulation of genes involved in cell cycle and DNA metabolism, replication and packaging in HBEC, which is in accord with the necessity to induce S phase for viral replication. Strikingly, in melanoma cells HAdV-5 triggered opposing regulation of said genes and, in contrast to lung cancer cells, no weak S phase induction was detected when using the E2F promoter as reporter. Our results provide a rationale for improving oncolytic adenoviruses either by adaptation of viral infection to target tumor cells or by

  8. Fenton reaction induced cancer in wild type rats recapitulates genomic alterations observed in human cancer.

    Directory of Open Access Journals (Sweden)

    Shinya Akatsuka

    Full Text Available Iron overload has been associated with carcinogenesis in humans. Intraperitoneal administration of ferric nitrilotriacetate initiates a Fenton reaction in renal proximal tubules of rodents that ultimately leads to a high incidence of renal cell carcinoma (RCC after repeated treatments. We performed high-resolution microarray comparative genomic hybridization to identify characteristics in the genomic profiles of this oxidative stress-induced rat RCCs. The results revealed extensive large-scale genomic alterations with a preference for deletions. Deletions and amplifications were numerous and sometimes fragmented, demonstrating that a Fenton reaction is a cause of such genomic alterations in vivo. Frequency plotting indicated that two of the most commonly altered loci corresponded to a Cdkn2a/2b deletion and a Met amplification. Tumor sizes were proportionally associated with Met expression and/or amplification, and clustering analysis confirmed our results. Furthermore, we developed a procedure to compare whole genomic patterns of the copy number alterations among different species based on chromosomal syntenic relationship. Patterns of the rat RCCs showed the strongest similarity to the human RCCs among five types of human cancers, followed by human malignant mesothelioma, an iron overload-associated cancer. Therefore, an iron-dependent Fenton chemical reaction causes large-scale genomic alterations during carcinogenesis, which may result in distinct genomic profiles. Based on the characteristics of extensive genome alterations in human cancer, our results suggest that this chemical reaction may play a major role during human carcinogenesis.

  9. Development of radiation-induced mutation techniques and functional genomics studies

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dong Sub; Kang, Si Yong; Kim, Jin Baek [KAERI, Daejeon (Korea, Republic of); and others

    2012-01-15

    This project has been performed to develop plant genetic resources using radiation (gamma-rays, ion-beam, space environments), to conduct functional genomics studies with mutant resources, and to develop new radiation plant breeding techniques using various radiation sources during 3 years. In the first section, we developed flower genetic resources, functional crop resources, and bio-industrial plant resources. In the second section, we cloned several mutated genes and studied mechanisms of gene expression and genetic diversity of mutations induced by gamma-rays. In the third section, we developed new plant breeding techniques using gamma-phytotron, heavy ion-beam, and space environments. Based on these results, a total of 8 cultivars containing Chrysanthemum, Hibiscus, kenaf, rice, and soybean were applied for plant variety protection (PVP) and a total of 4 cultivars were registered for PVP. Also, license agreement for the dwarf type Hibiscus mutant 'Ggoma' was conducted with Supro co. and the manufacturing technology for natural antioxidant pear-grape vinegar was transferred into Enzenic co. Also, 8 gene sequences, such as F3'H and LDOX genes associated with flower color in Chrysanthemum and EPSPS gene from Korean lawn grass, were registered in the database of National Center for Biotechnology Information (NCBI). In the future study, we will develop new radiation mutation breeding techniques through the mutation spectrum induced by various radiation sources, the studies for mechanism of the cellular response to radiation, and the comparative{center_dot}structural{center_dot}functional genomics studies for useful traits.

  10. Draft Genome Sequence of Lactobacillus delbrueckii subsp. bulgaricus LBB.B5.

    Science.gov (United States)

    Urshev, Zoltan; Hajo, Karima; Lenoci, Leonardo; Bron, Peter A; Dijkstra, Annereinou; Alkema, Wynand; Wels, Michiel; Siezen, Roland J; Minkova, Svetlana; van Hijum, Sacha A F T

    2016-10-06

    Lactobacillus delbrueckii subsp. bulgaricus LBB.B5 originates from homemade Bulgarian yogurt and was selected for its ability to form a strong association with Streptococcus thermophilus The genome sequence will facilitate elucidating the genetic background behind the contribution of LBB.B5 to the taste and aroma of yogurt and its exceptional protocooperation with S. thermophilus. Copyright © 2016 Urshev et al.

  11. Comparative genome analysis of three eukaryotic parasites with differing abilities to transform leukocytes reveals key mediators of theileria-induced leukocyte transformation

    KAUST Repository

    Hayashida, Kyoko

    2012-09-04

    We sequenced the genome of Theileria orientalis, a tick-borne apicomplexan protozoan parasite of cattle. The focus of this study was a comparative genome analysis of T. orientalis relative to other highly pathogenic Theileria species, T. parva and T. annulata. T. parva and T. annulata induce transformation of infected cells of lymphocyte or macrophage/monocyte lineages; in contrast, T. orientalis does not induce uncontrolled proliferation of infected leukocytes and multiplies predominantly within infected erythrocytes. While synteny across homologous chromosomes of the three Theileria species was found to be well conserved overall, subtelomeric structures were found to differ substantially, as T. orientalis lacks the large tandemly arrayed subtelomere-encoded variable secreted protein-encoding gene family. Moreover, expansion of particular gene families by gene duplication was found in the genomes of the two transforming Theileria species, most notably, the TashAT/TpHN and Tar/Tpr gene families. Gene families that are present only in T. parva and T. annulata and not in T. orientalis, Babesia bovis, or Plasmo-dium were also identified. Identification of differences between the genome sequences of Theileria species with different abilities to transform and immortalize bovine leukocytes will provide insight into proteins and mechanisms that have evolved to induce and regulate this process. The T. orientalis genome database is available at http://totdb.czc.hokudai.ac.jp/. 2012 Hayashida et al. T.

  12. Phenotypic and genomic analysis of serotype 3 Sabin poliovirus vaccine produced in MRC-5 cell substrate.

    Science.gov (United States)

    Alirezaie, Behnam; Taqavian, Mohammad; Aghaiypour, Khosrow; Esna-Ashari, Fatemeh; Shafyi, Abbas

    2011-05-01

    The cell substrate has a pivotal role in live virus vaccines production. It is necessary to evaluate the effects of the cell substrate on the properties of the propagated viruses, especially in the case of viruses which are unstable genetically such as polioviruses, by monitoring the molecular and phenotypical characteristics of harvested viruses. To investigate the presence/absence of mutation(s), the near full-length genomic sequence of different harvests of the type 3 Sabin strain of poliovirus propagated in MRC-5 cells were determined. The sequences were compared with genomic sequences of different virus seeds, vaccines, and OPV-like isolates. Nearly complete genomic sequencing results, however, revealed no detectable mutations throughout the genome RNA-plaque purified (RSO)-derived monopool of type 3 OPVs manufactured in MRC-5. Thirty-six years of experience in OPV production, trend analysis, and vaccine surveillance also suggest that: (i) different monopools of serotype 3 OPV produced in MRC-5 retained their phenotypic characteristics (temperature sensitivity and neuroattenuation), (ii) MRC-5 cells support the production of acceptable virus yields, (iii) OPV replicated in the MRC-5 cell substrate is a highly efficient and safe vaccine. These results confirm previous reports that MRC-5 is a desirable cell substrate for the production of OPV. Copyright © 2011 Wiley-Liss, Inc.

  13. Herpesvirus Genome Recognition Induced Acetylation of Nuclear IFI16 Is Essential for Its Cytoplasmic Translocation, Inflammasome and IFN-β Responses.

    Directory of Open Access Journals (Sweden)

    Mairaj Ahmed Ansari

    2015-07-01

    Full Text Available The IL-1β and type I interferon-β (IFN-β molecules are important inflammatory cytokines elicited by the eukaryotic host as innate immune responses against invading pathogens and danger signals. Recently, a predominantly nuclear gamma-interferon-inducible protein 16 (IFI16 involved in transcriptional regulation has emerged as an innate DNA sensor which induced IL-1β and IFN-β production through inflammasome and STING activation, respectively. Herpesvirus (KSHV, EBV, and HSV-1 episomal dsDNA genome recognition by IFI16 leads to IFI16-ASC-procaspase-1 inflammasome association, cytoplasmic translocation and IL-1β production. Independent of ASC, HSV-1 genome recognition results in IFI16 interaction with STING in the cytoplasm to induce interferon-β production. However, the mechanisms of IFI16-inflammasome formation, cytoplasmic redistribution and STING activation are not known. Our studies here demonstrate that recognition of herpesvirus genomes in the nucleus by IFI16 leads into its interaction with histone acetyltransferase p300 and IFI16 acetylation resulting in IFI16-ASC interaction, inflammasome assembly, increased interaction with Ran-GTPase, cytoplasmic redistribution, caspase-1 activation, IL-1β production, and interaction with STING which results in IRF-3 phosphorylation, nuclear pIRF-3 localization and interferon-β production. ASC and STING knockdowns did not affect IFI16 acetylation indicating that this modification is upstream of inflammasome-assembly and STING-activation. Vaccinia virus replicating in the cytoplasm did not induce nuclear IFI16 acetylation and cytoplasmic translocation. IFI16 physically associates with KSHV and HSV-1 genomes as revealed by proximity ligation microscopy and chromatin-immunoprecipitation studies which is not hampered by the inhibition of acetylation, thus suggesting that acetylation of IFI16 is not required for its innate sensing of nuclear viral genomes. Collectively, these studies identify the

  14. Lack of specificity of chromosome breaks resulting from radiation-induced genomic instability in Chinese hamster cells

    International Nuclear Information System (INIS)

    Trott, K.-R.; Teibe, A.

    1998-01-01

    In V79 Chinese hamster cells, radiation-induced genomic instability results in a persistently increased frequency of micronuclei, dicentric chromosomes and apoptosis and in decreased colony-forming ability. These manifestations of radiation-induced genomic instability may be attributed to an increased rate of chromosome breakage events many generations after irradiation. This chromosomal instability does not seem to be a property which has been inflicted on individual chromosomes at the time of irradiation. Rather, it appears to be secondary to an increased level of non-specific clastogenic factors in the progeny of most if not all irradiated cells. This conclusion is drawn from the observations presented here, that all the chromosomes in surviving V79 cells are involved in the formation of dicentric chromosome aberrations 1 or 2 weeks after irradiation with about equal probability if corrections are made for chromosome length. (orig.)

  15. Complete genome sequence of Shigella flexneri 5b and comparison with Shigella flexneri 2a

    Directory of Open Access Journals (Sweden)

    Xue Ying

    2006-07-01

    Full Text Available Abstract Background Shigella bacteria cause dysentery, which remains a significant threat to public health. Shigella flexneri is the most common species in both developing and developed countries. Five Shigella genomes have been sequenced, revealing dynamic and diverse features. To investigate the intra-species diversity of S. flexneri genomes further, we have sequenced the complete genome of S. flexneri 5b strain 8401 (abbreviated Sf8401 and compared it with S. flexneri 2a (Sf301. Results The Sf8401 chromosome is 4.5-Mb in size, a little smaller than that of Sf301, mainly because the former lacks the SHI-1 pathogenicity island (PAI. Compared with Sf301, there are 6 inversions and one translocation in Sf8401, which are probably mediated by insertion sequences (IS. There are clear differences in the known PAIs between these two genomes. The bacteriophage SfV segment remaining in SHI-O of Sf8401 is clearly larger than the remnants of bacteriophage SfII in Sf301. SHI-1 is absent from Sf8401 but a specific related protein is found next to the pheV locus. SHI-2 is involved in one intra-replichore inversion near the origin of replication, which may change the expression of iut/iuc genes. Moreover, genes related to the glycine-betaine biosynthesis pathway are present only in Sf8401 among the known Shigella genomes. Conclusion Our data show that the two S. flexneri genomes are very similar, which suggests a high level of structural and functional conservation between the two serotypes. The differences reflect different selection pressures during evolution. The ancestor of S. flexneri probably acquired SHI-1 and SHI-2 before SHI-O was integrated and the serotypes diverged. SHI-1 was subsequently deleted from the S. flexneri 5b genome by recombination, but stabilized in the S. flexneri 2a genome. These events may have contributed to the differences in pathogenicity and epidemicity between the two serotypes of S. flexneri.

  16. Genome-Wide Locations of Potential Epimutations Associated with Environmentally Induced Epigenetic Transgenerational Inheritance of Disease Using a Sequential Machine Learning Prediction Approach.

    Science.gov (United States)

    Haque, M Muksitul; Holder, Lawrence B; Skinner, Michael K

    2015-01-01

    Environmentally induced epigenetic transgenerational inheritance of disease and phenotypic variation involves germline transmitted epimutations. The primary epimutations identified involve altered differential DNA methylation regions (DMRs). Different environmental toxicants have been shown to promote exposure (i.e., toxicant) specific signatures of germline epimutations. Analysis of genomic features associated with these epimutations identified low-density CpG regions (machine learning computational approach to predict all potential epimutations in the genome. A number of previously identified sperm epimutations were used as training sets. A novel machine learning approach using a sequential combination of Active Learning and Imbalance Class Learner analysis was developed. The transgenerational sperm epimutation analysis identified approximately 50K individual sites with a 1 kb mean size and 3,233 regions that had a minimum of three adjacent sites with a mean size of 3.5 kb. A select number of the most relevant genomic features were identified with the low density CpG deserts being a critical genomic feature of the features selected. A similar independent analysis with transgenerational somatic cell epimutation training sets identified a smaller number of 1,503 regions of genome-wide predicted sites and differences in genomic feature contributions. The predicted genome-wide germline (sperm) epimutations were found to be distinct from the predicted somatic cell epimutations. Validation of the genome-wide germline predicted sites used two recently identified transgenerational sperm epimutation signature sets from the pesticides dichlorodiphenyltrichloroethane (DDT) and methoxychlor (MXC) exposure lineage F3 generation. Analysis of this positive validation data set showed a 100% prediction accuracy for all the DDT-MXC sperm epimutations. Observations further elucidate the genomic features associated with transgenerational germline epimutations and identify a genome

  17. Enhanced micronucleus formation in the descendants of {gamma}-ray-irradiated tobacco cells: Evidence for radiation-induced genomic instability in plant cells

    Energy Technology Data Exchange (ETDEWEB)

    Yokota, Yuichiro, E-mail: yokota.yuichiro@jaea.go.jp [Life Science and Biotechnology Division, Quantum Beam Science Directorate, Japan Atomic Energy Agency, 1233 Watanuki-machi, Takasaki, Gunma 370-1292 (Japan); Funayama, Tomoo; Hase, Yoshihiro [Life Science and Biotechnology Division, Quantum Beam Science Directorate, Japan Atomic Energy Agency, 1233 Watanuki-machi, Takasaki, Gunma 370-1292 (Japan); Hamada, Nobuyuki [Radiation Safety Research Center, Nuclear Technology Research Laboratory, Central Research Institute of Electric Power Industry, 2-11-1 Iwado-kita, Komae, Tokyo 201-8511 (Japan); Kobayashi, Yasuhiko; Tanaka, Atsushi; Narumi, Issay [Life Science and Biotechnology Division, Quantum Beam Science Directorate, Japan Atomic Energy Agency, 1233 Watanuki-machi, Takasaki, Gunma 370-1292 (Japan)

    2010-09-10

    Ionizing radiation-induced genomic instability has been documented in various end points such as chromosomal aberrations and mutations, which arises in the descendants of irradiated mammalian or yeast cells many generations after the initial insult. This study aimed at addressing radiation-induced genomic instability in higher plant tobacco cells. We thus investigated micronucleus (MN) formation and cell proliferation in tobacco cells irradiated with {gamma}-rays and their descendants. In {gamma}-irradiated cells, cell cycle was arrested at G{sub 2}/M phase at around 24 h post-irradiation but released afterward. In contrast, MN frequency peaked at 48 h post-irradiation. Almost half of 40 Gy-irradiated cells had MN at 48 h post-irradiation, but proliferated as actively as sham-irradiated cells up to 120 h post-irradiation. Moreover, the descendants that have undergone at least 22 generations after irradiation still showed a two-fold MN frequency compared to sham-irradiated cells. This is the direct evidence for radiation-induced genomic instability in tobacco cells.

  18. A subset of herpes simplex virus replication genes induces DNA amplification within the host cell genome

    Energy Technology Data Exchange (ETDEWEB)

    Heilbronn, R.; zur Hausen, H. (Deutsches Krebsforschungszentrum, Heidelberg (West Germany))

    1989-09-01

    Herpes simplex virus (HSV) induces DNA amplification of target genes within the host cell chromosome. To characterize the HSV genes that mediate the amplification effect, combinations of cloned DNA fragments covering the entire HSV genome were transiently transfected into simian virus 40 (SV40)-transformed hamster cells. This led to amplification of the integrated SV40 DNA sequences to a degree comparable to that observed after transfection of intact virion DNA. Transfection of combinations of subclones and of human cytomegalovirus immediate-early promoter-driven expression constructs for individual open reading frames led to the identification of sic HSV genes which together were necessary and sufficient for the induction of DNA amplification: UL30 (DNA polymerase), UL29 (major DNA-binding protein), UL5, UL8, UL42, and UL52. All of these genes encode proteins necessary for HSV DNA replication. However, an additional gene coding for an HSV origin-binding protein (UL9) was required for origin-dependent HSV DNA replication but was dispensable for SV40 DNA amplification. The results show that a subset of HSV replication genes is sufficient for the induction of DNA amplification. This opens the possibility that HSV expresses functions sufficient for DNA amplification but separate from those responsible for lytic viral growth. HSV infection may thereby induce DNA amplification within the host cell genome without killing the host by lytic viral growth. This may lead to persistence of a cell with a new genetic phenotype, which would have implications for the pathogenicity of the virus in vivo.

  19. Path from schizophrenia genomics to biology: gene regulation and perturbation in neurons derived from induced pluripotent stem cells and genome editing.

    Science.gov (United States)

    Duan, Jubao

    2015-02-01

    Schizophrenia (SZ) is a devastating mental disorder afflicting 1% of the population. Recent genome-wide association studies (GWASs) of SZ have identified >100 risk loci. However, the causal variants/genes and the causal mechanisms remain largely unknown, which hinders the translation of GWAS findings into disease biology and drug targets. Most risk variants are noncoding, thus likely regulate gene expression. A major mechanism of transcriptional regulation is chromatin remodeling, and open chromatin is a versatile predictor of regulatory sequences. MicroRNA-mediated post-transcriptional regulation plays an important role in SZ pathogenesis. Neurons differentiated from patient-specific induced pluripotent stem cells (iPSCs) provide an experimental model to characterize the genetic perturbation of regulatory variants that are often specific to cell type and/or developmental stage. The emerging genome-editing technology enables the creation of isogenic iPSCs and neurons to efficiently characterize the effects of SZ-associated regulatory variants on SZ-relevant molecular and cellular phenotypes involving dopaminergic, glutamatergic, and GABAergic neurotransmissions. SZ GWAS findings equipped with the emerging functional genomics approaches provide an unprecedented opportunity for understanding new disease biology and identifying novel drug targets.

  20. Protection of vanillin derivative VND3207 on genome damage and apoptosis of human lymphoblastoid cells induced by γ-ray irradiation

    International Nuclear Information System (INIS)

    Huang Rui; Huang Bo; He Xingpeng; Xu Qinzhi; Wang Yu; Zhou Pingkun

    2009-01-01

    To determine the protective effect of vanillin derivative VND3207 on the genome damage and apoptosis of human lymphoblastoid AHH-1 cells induced by γ-ray irradiation, the techniques of single-cell gel electrophoresis, micronucleus test, Annexin V-FACS assay, and the double-fluorescein staining and fluorescent microscope observation were used. Neutral single-cell gel electrophoresis showed that the initial DNA double-strand breaks caused by 2 Gy 60 Co γ-ray was significantly decreased by VND3207 in the range of 540 μmol/L. This significant phenomenonwas demonstrated by the fact that the comet tail-moment was significantly shortened and the DNA content in the comet tail was reduced when the cells were protected with VND3207, and the radio-protective effect increases along with the increasing of drug concentration. Similarly, the yield of micronucleus was reduced by 540 μmol/L of VND3207 in a concentration-dependency in AHH-1 cells irradiated with 0.5 Gy, 1.0 Gy and 2.0 Gy 60 Co γ-rays. 40 μmol/L VND3207 resulted in 40% reduction in the yield of micronucleus induced by 2.0 Gy. The occurrence of apoptosis enhanced along with the time from 8 h to 48 h post 4 Gy irradiation, and 40 μmol/L of VND3207 significantly decreased the induction of apoptosis. This work has further demonstrated a good protection of VND3207 on γ-ray-induced cell genome damage and apoptosis. (authors)

  1. Cytoplasmic ATR Activation Promotes Vaccinia Virus Genome Replication

    Directory of Open Access Journals (Sweden)

    Antonio Postigo

    2017-05-01

    Full Text Available In contrast to most DNA viruses, poxviruses replicate their genomes in the cytoplasm without host involvement. We find that vaccinia virus induces cytoplasmic activation of ATR early during infection, before genome uncoating, which is unexpected because ATR plays a fundamental nuclear role in maintaining host genome integrity. ATR, RPA, INTS7, and Chk1 are recruited to cytoplasmic DNA viral factories, suggesting canonical ATR pathway activation. Consistent with this, pharmacological and RNAi-mediated inhibition of canonical ATR signaling suppresses genome replication. RPA and the sliding clamp PCNA interact with the viral polymerase E9 and are required for DNA replication. Moreover, the ATR activator TOPBP1 promotes genome replication and associates with the viral replisome component H5. Our study suggests that, in contrast to long-held beliefs, vaccinia recruits conserved components of the eukaryote DNA replication and repair machinery to amplify its genome in the host cytoplasm.

  2. TIA-1 and TIAR interact with 5'-UTR of enterovirus 71 genome and facilitate viral replication.

    Science.gov (United States)

    Wang, Xiaohui; Wang, Huanru; Li, Yixuan; Jin, Yu; Chu, Ying; Su, Airong; Wu, Zhiwei

    2015-10-16

    Enterovirus 71 is one of the major causative pathogens of HFMD in children. Upon infection, the viral RNA is translated in an IRES-dependent manner and requires several host factors for effective replication. Here, we found that T-cell-restricted intracellular antigen 1 (TIA-1), and TIA-1 related protein (TIAR) were translocated from nucleus to cytoplasm after EV71 infection and localized to the sites of viral replication. We found that TIA-1 and TIAR can facilitate EV71 replication by enhancing the viral genome synthesis in host cells. We demonstrated that both proteins bound to the stem-loop I of 5'-UTR of viral genome and improved the stability of viral genomic RNA. Our results suggest that TIA-1 and TIAR are two new host factors that interact with 5-UTR of EV71 genome and positively regulate viral replication. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. WARBURG EFFECT AND TRANSLOCATION-INDUCED GENOMIC INSTABILITY: TWO YEAST MODELS FOR CANCER CELLS

    Directory of Open Access Journals (Sweden)

    Valentina eTosato

    2013-01-01

    Full Text Available Yeast has been established as an efficient model system to study biological principles underpinning human health. In this review we focus on yeast models covering two aspects of cancer formation and progression i the activity of pyruvate kinase (PK, which recapitulates metabolic features of cancer cells, including the Warburg effect, and ii Bridge-Induced chromosome Translocation (BIT mimicking genome instability in cancer. Saccharomyces cerevisiae is an excellent model to study cancer cell metabolism, as exponentially growing yeast cells exhibit many metabolic similarities with rapidly proliferating cancer cells. The metabolic reconfiguration includes an increase in glucose uptake and fermentation, at the expense of respiration and oxidative phosphorylation (the Warburg effect, and involves a broad reconfiguration of nucleotide and amino acid metabolism. Both in yeast and humans, the regulation of this process seems to have a central player, pyruvate kinase, which is up-regulated in cancer, and to occur mostly on a post-transcriptional and posttranslational basis. Furthermore, BIT allows to generate selectable translocation-derived recombinants (translocants, between any two desired chromosomal locations, in wild-type yeast strains transformed with a linear DNA cassette carrying a selectable marker flanked by two DNA sequences homologous to different chromosomes. Using the Bridge-Induced Translocation system, targeted non-reciprocal translocations in mitosis are easily inducible. An extensive collection of different yeast translocants exhibiting genome instability and aberrant phenotypes similar to cancer cells has been produced and subjected to analysis. In this review, we hence provide an overview upon two yeast cancer models, and extrapolate general principles for mimicking human disease mechanisms in yeast.

  4. Genome-Wide Locations of Potential Epimutations Associated with Environmentally Induced Epigenetic Transgenerational Inheritance of Disease Using a Sequential Machine Learning Prediction Approach.

    Directory of Open Access Journals (Sweden)

    M Muksitul Haque

    Full Text Available Environmentally induced epigenetic transgenerational inheritance of disease and phenotypic variation involves germline transmitted epimutations. The primary epimutations identified involve altered differential DNA methylation regions (DMRs. Different environmental toxicants have been shown to promote exposure (i.e., toxicant specific signatures of germline epimutations. Analysis of genomic features associated with these epimutations identified low-density CpG regions (<3 CpG / 100bp termed CpG deserts and a number of unique DNA sequence motifs. The rat genome was annotated for these and additional relevant features. The objective of the current study was to use a machine learning computational approach to predict all potential epimutations in the genome. A number of previously identified sperm epimutations were used as training sets. A novel machine learning approach using a sequential combination of Active Learning and Imbalance Class Learner analysis was developed. The transgenerational sperm epimutation analysis identified approximately 50K individual sites with a 1 kb mean size and 3,233 regions that had a minimum of three adjacent sites with a mean size of 3.5 kb. A select number of the most relevant genomic features were identified with the low density CpG deserts being a critical genomic feature of the features selected. A similar independent analysis with transgenerational somatic cell epimutation training sets identified a smaller number of 1,503 regions of genome-wide predicted sites and differences in genomic feature contributions. The predicted genome-wide germline (sperm epimutations were found to be distinct from the predicted somatic cell epimutations. Validation of the genome-wide germline predicted sites used two recently identified transgenerational sperm epimutation signature sets from the pesticides dichlorodiphenyltrichloroethane (DDT and methoxychlor (MXC exposure lineage F3 generation. Analysis of this positive validation

  5. Reciprocal activation of α5-nAChR and STAT3 in nicotine-induced human lung cancer cell proliferation.

    Science.gov (United States)

    Zhang, Yao; Jia, Yanfei; Li, Ping; Li, Huanjie; Xiao, Dongjie; Wang, Yunshan; Ma, Xiaoli

    2017-07-20

    Cigarette smoking is the top environmental risk factor for lung cancer. Nicotine, the addictive component of cigarettes, induces lung cancer cell proliferation, invasion and migration via the activation of nicotinic acetylcholine receptors (nAChRs). Genome-wide association studies (GWAS) show that CHRNA5 gene encoding α5-nAChR is especially relevant to lung cancer. However, the mechanism of this subunit in lung cancer is not clear. In the present study, we demonstrate that the expression of α5-nAChR is correlated with phosphorylated STAT3 (pSTAT3) expression, smoking history and lower survival of non-small cell lung cancer (NSCLC) samples. Nicotine increased the levels of α5-nAChR mRNA and protein in NSCLC cell lines and activated the JAK2/STAT3 signaling cascade. Nicotine-induced activation of JAK2/STAT3 signaling was inhibited by the silencing of α5-nAChR. Characterization of the CHRNA5 promoter revealed four STAT3-response elements. ChIP assays confirmed that the CHRNA5 promoter contains STAT3 binding sites. By silencing STAT3 expression, nicotine-induced upregulation of α5-nAChR was suppressed. Downregulation of α5-nAChR and/or STAT3 expression inhibited nicotine-induced lung cancer cell proliferation. These results suggest that there is a feedback loop between α5-nAChR and STAT3 that contributes to the nicotine-induced tumor cell proliferation, which indicates that α5-nAChR is an important therapeutic target involved in tobacco-associated lung carcinogenesis. Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  6. Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv. Makino

    Directory of Open Access Journals (Sweden)

    Ri Gao

    2016-10-01

    Full Text Available Autopolyploidy is widespread in higher plants and plays an important role in the process of evolution. The present study successfully induced autotetraploidys from Chrysanthemum lavandulifolium by colchicine. The plant morphology, genomic, transcriptomic, and epigenetic changes between tetraploid and diploid plants were investigated. Ligulate flower, tubular flower and leaves of tetraploid plants were greater than those of the diploid plants. Compared with diploid plants, the genome changed as a consequence of polyploidization in tetraploid plants, namely, 1.1% lost fragments and 1.6% novel fragments occurred. In addition, DNA methylation increased after genome doubling in tetraploid plants. Among 485 common transcript-derived fragments (TDFs, which existed in tetraploid and diploid progenitors, 62 fragments were detected as differentially expressed TDFs, 6.8% of TDFs exhibited up-regulated gene expression in the tetraploid plants and 6.0% exhibited down-regulation. The present study provides a reference for further studying the autopolyploidization role in the evolution of C. lavandulifolium. In conclusion, the autopolyploid C. lavandulifolium showed a global change in morphology, genome and gene expression compared with corresponding diploid.

  7. Features of 5'-splice-site efficiency derived from disease-causing mutations and comparative genomics

    DEFF Research Database (Denmark)

    Roca, Xavier; Olson, Andrew J; Rao, Atmakuri R

    2008-01-01

    Many human diseases, including Fanconi anemia, hemophilia B, neurofibromatosis, and phenylketonuria, can be caused by 5'-splice-site (5'ss) mutations that are not predicted to disrupt splicing, according to position weight matrices. By using comparative genomics, we identify pairwise dependencies...

  8. Perspectives on the role of bystander effect and genomic instability on therapy-induced secondary malignancy

    International Nuclear Information System (INIS)

    Perumal, Venkatachalam; Raavi, Venkateswarlu; Kanagaraj, Karthik; Shangamithra, V.; Paul, Solomon F.D.; Chinnadurai, M.

    2017-01-01

    Deviation from the orchestra of regulated cell division into unregulated and then result into the formation of tumor, is known as carcinogenesis. While causes and hallmarks of many cancer types are well established, newer concepts on tumor cell response to treatment, challenges established therapeutic regime and drives into alternative toward the better management. The phenomena of therapeutics induced bystander response, and genomic instability on late effects of cancer therapy is emerging as a newer challenge. Bystander response is defined as the manifestation of radiation/chemotherapy drug signatures on the unexposed cells which are in the closer vicinity of the directly exposed; on the other hand, genomic instability is defined as the expression of radiation/chemotherapy drug signatures in the progeny of exposed cells. Unequivocally, existence of those phenomena has been demonstrated with many cell types (both in vitro and in vivo) followed by radiation and widely used chemotherapeutic drugs. Nevertheless, it is also revealed that the effects are variable and depend on dose, type of radiation/chemicals agents, experimental model, type of donor and recipient cells, and biomarkers adopted; moreover, to observe those effects, reactive oxygen species has been reported as leading mediators of those responses when compared to other molecules such as interleukins, cytokines, and inflammatory markers. Available data on those phenomena and our findings suggest that a role of therapeutic drugs induced bystander effects, and genomic instability on the development of secondary malignancy cannot be ruled out completely. (author)

  9. SMC5/6: Shaping, protecting and preparing the genome for safe reproduction

    NARCIS (Netherlands)

    Verver, D.E.

    2015-01-01

    SMC5/6 is one of the three structural maintenance of chromosomes (SMC) protein complexes and is involved in numerous processes involving DNA damage repair and genomic integrity maintenance. Although such DNA damage control mechanisms, together with highly dynamic changes in chromatin composition and

  10. Genomic evolution, recombination, and inter-strain diversity of chelonid alphaherpesvirus 5 from Florida and Hawaii green sea turtles with fibropapillomatosis

    Science.gov (United States)

    Morrison, Cheryl L.; Iwanowicz, Luke R.; Work, Thierry M.; Fahsbender, Elizabeth; Breitbart, Mya; Adams, Cynthia; Iwanowicz, Deborah; Sanders, Lakyn; Ackermann, Mathias; Cornman, Robert S.

    2018-01-01

    Chelonid alphaherpesvirus 5 (ChHV5) is a herpesvirus associated with fibropapillomatosis (FP) in sea turtles worldwide. Single-locus typing has previously shown differentiation between Atlantic and Pacific strains of this virus, with low variation within each geographic clade. However, a lack of multi-locus genomic sequence data hinders understanding of the rate and mechanisms of ChHV5 evolutionary divergence, as well as how these genomic changes may contribute to differences in disease manifestation. To assess genomic variation in ChHV5 among five Hawaii and three Florida green sea turtles, we used high-throughput short-read sequencing of long-range PCR products amplified from tumor tissue using primers designed from the single available ChHV5 reference genome from a Hawaii green sea turtle. This strategy recovered sequence data from both geographic regions for approximately 75% of the predicted ChHV5 coding sequences. The average nucleotide divergence between geographic populations was 1.5%; most of the substitutions were fixed differences between regions. Protein divergence was generally low (average 0.08%), and ranged between 0 and 5.3%. Several atypical genes originally identified and annotated in the reference genome were confirmed in ChHV5 genomes from both geographic locations. Unambiguous recombination events between geographic regions were identified, and clustering of private alleles suggests the prevalence of recombination in the evolutionary history of ChHV5. This study significantly increased the amount of sequence data available from ChHV5 strains, enabling informed selection of loci for future population genetic and natural history studies, and suggesting the (possibly latent) co-infection of individuals by well-differentiated geographic variants.

  11. Genomic evolution, recombination, and inter-strain diversity of chelonid alphaherpesvirus 5 from Florida and Hawaii green sea turtles with fibropapillomatosis.

    Science.gov (United States)

    Morrison, Cheryl L; Iwanowicz, Luke; Work, Thierry M; Fahsbender, Elizabeth; Breitbart, Mya; Adams, Cynthia; Iwanowicz, Deb; Sanders, Lakyn; Ackermann, Mathias; Cornman, Robert S

    2018-01-01

    Chelonid alphaherpesvirus 5 (ChHV5) is a herpesvirus associated with fibropapillomatosis (FP) in sea turtles worldwide. Single-locus typing has previously shown differentiation between Atlantic and Pacific strains of this virus, with low variation within each geographic clade. However, a lack of multi-locus genomic sequence data hinders understanding of the rate and mechanisms of ChHV5 evolutionary divergence, as well as how these genomic changes may contribute to differences in disease manifestation. To assess genomic variation in ChHV5 among five Hawaii and three Florida green sea turtles, we used high-throughput short-read sequencing of long-range PCR products amplified from tumor tissue using primers designed from the single available ChHV5 reference genome from a Hawaii green sea turtle. This strategy recovered sequence data from both geographic regions for approximately 75% of the predicted ChHV5 coding sequences. The average nucleotide divergence between geographic populations was 1.5%; most of the substitutions were fixed differences between regions. Protein divergence was generally low (average 0.08%), and ranged between 0 and 5.3%. Several atypical genes originally identified and annotated in the reference genome were confirmed in ChHV5 genomes from both geographic locations. Unambiguous recombination events between geographic regions were identified, and clustering of private alleles suggests the prevalence of recombination in the evolutionary history of ChHV5. This study significantly increased the amount of sequence data available from ChHV5 strains, enabling informed selection of loci for future population genetic and natural history studies, and suggesting the (possibly latent) co-infection of individuals by well-differentiated geographic variants.

  12. Genomic evolution, recombination, and inter-strain diversity of chelonid alphaherpesvirus 5 from Florida and Hawaii green sea turtles with fibropapillomatosis

    Directory of Open Access Journals (Sweden)

    Cheryl L. Morrison

    2018-02-01

    Full Text Available Chelonid alphaherpesvirus 5 (ChHV5 is a herpesvirus associated with fibropapillomatosis (FP in sea turtles worldwide. Single-locus typing has previously shown differentiation between Atlantic and Pacific strains of this virus, with low variation within each geographic clade. However, a lack of multi-locus genomic sequence data hinders understanding of the rate and mechanisms of ChHV5 evolutionary divergence, as well as how these genomic changes may contribute to differences in disease manifestation. To assess genomic variation in ChHV5 among five Hawaii and three Florida green sea turtles, we used high-throughput short-read sequencing of long-range PCR products amplified from tumor tissue using primers designed from the single available ChHV5 reference genome from a Hawaii green sea turtle. This strategy recovered sequence data from both geographic regions for approximately 75% of the predicted ChHV5 coding sequences. The average nucleotide divergence between geographic populations was 1.5%; most of the substitutions were fixed differences between regions. Protein divergence was generally low (average 0.08%, and ranged between 0 and 5.3%. Several atypical genes originally identified and annotated in the reference genome were confirmed in ChHV5 genomes from both geographic locations. Unambiguous recombination events between geographic regions were identified, and clustering of private alleles suggests the prevalence of recombination in the evolutionary history of ChHV5. This study significantly increased the amount of sequence data available from ChHV5 strains, enabling informed selection of loci for future population genetic and natural history studies, and suggesting the (possibly latent co-infection of individuals by well-differentiated geographic variants.

  13. Causes of genome instability

    DEFF Research Database (Denmark)

    Langie, Sabine A S; Koppen, Gudrun; Desaulniers, Daniel

    2015-01-01

    function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make......Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genome's integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus...

  14. Whole Genome Sequence of the Heterozygous Clinical Isolate Candida krusei 81-B-5

    Directory of Open Access Journals (Sweden)

    Christina A. Cuomo

    2017-09-01

    Full Text Available Candida krusei is a diploid, heterozygous yeast that is an opportunistic fungal pathogen in immunocompromised patients. This species also is utilized for fermenting cocoa beans during chocolate production. One major concern in the clinical setting is the innate resistance of this species to the most commonly used antifungal drug fluconazole. Here, we report a high-quality genome sequence and assembly for the first clinical isolate of C. krusei, strain 81-B-5, into 11 scaffolds generated with PacBio sequencing technology. Gene annotation and comparative analysis revealed a unique profile of transporters that could play a role in drug resistance or adaptation to different environments. In addition, we show that, while 82% of the genome is highly heterozygous, a 2.0 Mb region of the largest scaffold has undergone loss of heterozygosity. This genome will serve as a reference for further genetic studies of this pathogen.

  15. Genome Sequence of Selenium-Solubilizing Bacterium Caulobacter vibrioides T5M6

    DEFF Research Database (Denmark)

    Wang, Yihua; Qin, Yanan; Kot, Witold

    2016-01-01

    Caulobacter vibrioides T5M6 is a Gram-negative strain that strongly solubilizes selenium (Se) mineral into Se(IV) and was isolated from a selenium mining area in Enshi, southwest China. This strain produces the phytohormone IAA and promotes plant growth. Here we present the genome of this strain...

  16. The genomes and comparative genomics of Lactobacillus delbrueckii phages.

    Science.gov (United States)

    Riipinen, Katja-Anneli; Forsman, Päivi; Alatossava, Tapani

    2011-07-01

    Lactobacillus delbrueckii phages are a great source of genetic diversity. Here, the genome sequences of Lb. delbrueckii phages LL-Ku, c5 and JCL1032 were analyzed in detail, and the genetic diversity of Lb. delbrueckii phages belonging to different taxonomic groups was explored. The lytic isometric group b phages LL-Ku (31,080 bp) and c5 (31,841 bp) showed a minimum nucleotide sequence identity of 90% over about three-fourths of their genomes. The genomic locations of their lysis modules were unique, and the genomes featured several putative overlapping transcription units of genes. LL-Ku and c5 virions displayed peptidoglycan hydrolytic activity associated with a ~36-kDa protein similar in size to the endolysin. Unexpectedly, the 49,433-bp genome of the prolate phage JCL1032 (temperate, group c) revealed a conserved gene order within its structural genes. Lb. delbrueckii phages representing groups a (a phage LL-H), b and c possessed only limited protein sequence homology. Genomic comparison of LL-Ku and c5 suggested that diversification of Lb. delbrueckii phages is mainly due to insertions, deletions and recombination. For the first time, the complete genome sequences of group b and c Lb. delbrueckii phages are reported.

  17. Naftopidil inhibits 5-hydroxytryptamine-induced bladder contraction in rats.

    Science.gov (United States)

    Sakai, Takumi; Kasahara, Ken-ichi; Tomita, Ken-ichi; Ikegaki, Ichiro; Kuriyama, Hiroshi

    2013-01-30

    Naftopidil is an α(1D) and α(1A) subtype-selective α(1)-adrenoceptor antagonist that has been used to treat lower urinary tract symptoms of benign prostatic hyperplasia. In this study, we investigated the effects of naftopidil on 5-hydroxytryptamine (5-HT)-induced rat bladder contraction (10(-8)-10(-4) M). Naftopidil (0.3, 1, and 3 μM) inhibited 5-HT-induced bladder contraction in a concentration-dependent manner. On the other hand, other α(1)-adrenoceptor antagonists, tamsulosin, silodosin or prazosin, did not inhibit 5-HT-induced bladder contraction. The 5-HT-induced bladder contraction was inhibited by both ketanserin and 4-(4-fluoronaphthalen-1-yl)-6-propan-2-ylpyrimidin-2-amine (RS127445), serotonin 5-HT(2A) and 5-HT(2B) receptor antagonists, respectively. In addition, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and α-methyl-5-HT, 5-HT(2A) and 5-HT(2) receptor agonists, respectively, induced bladder contraction. The 5-HT-induced bladder contraction was not inhibited by N-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-N-pyridin-2-yl-cyclohexanecarboxamide (WAY-100635), [1-[2[(methylsulfonyl)amino]ethyl]-4-piperidinyl]methyl-1-methyl-1H-indole-3-carboxylate (GR113808) or (R)-3-[2-[2-(4-methylpiperidin-1-yl)ethyl]pyrrolidine-1-sulphonyl]phenol (SB269970), 5-HT(1A), 5-HT(4) and 5-HT(7) receptor antagonists, respectively. Naftopidil inhibited both the 5-HT(2A) and 5-HT(2) receptor agonists-induced bladder contractions. Naftopidil binds to the human 5-HT(2A) and 5-HT(2B) receptors with pKi values of 6.55 and 7.82, respectively. These results suggest that naftopidil inhibits 5-HT-induced bladder contraction via blockade of the 5-HT(2A) and 5-HT(2B) receptors in rats. Furthermore, 5-HT-induced bladder contraction was enhanced in bladder strips obtained from bladder outlet obstructed rats, with this contraction inhibited by naftopidil. The beneficial effects of naftopidil on storage symptoms such as urinary frequency and nocturia in patients with benign

  18. Non-homologous end-joining genes are not inactivated in human radiation-induced sarcomas with genomic instability

    International Nuclear Information System (INIS)

    Lefevre, S.H.; Coquelle, A.; Gonin-Laurent, N.

    2005-01-01

    DNA double-strand break (DSB) repair pathways are implicated in the maintenance of genomic stability. However the alterations of these pathways, as may occur in human tumor cells with strong genomic instability, remain poorly characterized. We analyzed the loss of heterozygosity (LOH) and the presence of mutations for a series of genes implicated in DSB repair by non-homologous end-joining in five radiation-induced sarcomas devoid of both active Tp53 and Rb1. LOH was recurrently observed for 8 of the 9 studied genes (KU70, KU80, XRCC4, LIG4, Artemis, MRE11, RAD50, NBS1) but not for DNA-PKcs. No mutation was found in the remaining allele of the genes with LOH and the mRNA expression did not correlate with the allelic status. Our findings suggest that non-homologous end-joining repair pathway alteration is unlikely to be involved in the high genomic instability observed in these tumors. (author)

  19. Genome-wide pharmacogenomic study of citalopram-induced side effects in STAR*D.

    Science.gov (United States)

    Adkins, D E; Clark, S L; Åberg, K; Hettema, J M; Bukszár, J; McClay, J L; Souza, R P; van den Oord, E J C G

    2012-07-03

    Affecting about 1 in 12 Americans annually, depression is a leading cause of the global disease burden. While a range of effective antidepressants are now available, failure and relapse rates remain substantial, with intolerable side effect burden the most commonly cited reason for discontinuation. Thus, understanding individual differences in susceptibility to antidepressant therapy side effects will be essential to optimize depression treatment. Here we perform genome-wide association studies (GWAS) to identify genetic variation influencing susceptibility to citalopram-induced side effects. The analysis sample consisted of 1762 depression patients, successfully genotyped for 421K single-nucleotide polymorphisms (SNPs), from the Sequenced Treatment Alternatives to Relieve Depression (STAR(*)D) study. Outcomes included five indicators of citalopram side effects: general side effect burden, overall tolerability, sexual side effects, dizziness and vision/hearing side effects. Two SNPs met our genome-wide significance criterion (qeffects of citalopram on vision/hearing side effects (P=3.27 × 10(-8), q=0.026). The second genome-wide significant finding, representing a haplotype spanning ∼30 kb and eight genotyped SNPs in a gene desert on chromosome 13, was associated with general side effect burden (P=3.22 × 10(-7), q=0.096). Suggestive findings were also found for SNPs at LAMA1, AOX2P, EGFLAM, FHIT and RTP2. Although our findings require replication and functional validation, this study demonstrates the potential of GWAS to discover genes and pathways that potentially mediate adverse effects of antidepressant medications.

  20. Genome-wide transcriptional profiling of skin and dorsal root ganglia after ultraviolet-B-induced inflammation.

    Directory of Open Access Journals (Sweden)

    John M Dawes

    Full Text Available Ultraviolet-B (UVB-induced inflammation produces a dose-dependent mechanical and thermal hyperalgesia in both humans and rats, most likely via inflammatory mediators acting at the site of injury. Previous work has shown that the gene expression of cytokines and chemokines is positively correlated between species and that these factors can contribute to UVB-induced pain. In order to investigate other potential pain mediators in this model we used RNA-seq to perform genome-wide transcriptional profiling in both human and rat skin at the peak of hyperalgesia. In addition we have also measured transcriptional changes in the L4 and L5 DRG of the rat model. Our data show that UVB irradiation produces a large number of transcriptional changes in the skin: 2186 and 3888 genes are significantly dysregulated in human and rat skin, respectively. The most highly up-regulated genes in human skin feature those encoding cytokines (IL6 and IL24, chemokines (CCL3, CCL20, CXCL1, CXCL2, CXCL3 and CXCL5, the prostanoid synthesising enzyme COX-2 and members of the keratin gene family. Overall there was a strong positive and significant correlation in gene expression between the human and rat (R = 0.8022. In contrast to the skin, only 39 genes were significantly dysregulated in the rat L4 and L5 DRGs, the majority of which had small fold change values. Amongst the most up-regulated genes in DRG were REG3B, CCL2 and VGF. Overall, our data shows that numerous genes were up-regulated in UVB irradiated skin at the peak of hyperalgesia in both human and rats. Many of the top up-regulated genes were cytokines and chemokines, highlighting again their potential as pain mediators. However many other genes were also up-regulated and might play a role in UVB-induced hyperalgesia. In addition, the strong gene expression correlation between species re-emphasises the value of the UVB model as translational tool to study inflammatory pain.

  1. The Role of Abcb5 Alleles in Susceptibility to Haloperidol-Induced Toxicity in Mice and Humans

    Science.gov (United States)

    Zheng, Ming; Zhang, Haili; Dill, David L.; Clark, J. David; Tu, Susan; Yablonovitch, Arielle L.; Tan, Meng How; Zhang, Rui; Rujescu, Dan; Wu, Manhong; Tessarollo, Lino; Vieira, Wilfred; Gottesman, Michael M.; Deng, Suhua; Eberlin, Livia S.; Zare, Richard N.; Billard, Jean-Martin; Gillet, Jean-Pierre; Li, Jin Billy; Peltz, Gary

    2015-01-01

    Background We know very little about the genetic factors affecting susceptibility to drug-induced central nervous system (CNS) toxicities, and this has limited our ability to optimally utilize existing drugs or to develop new drugs for CNS disorders. For example, haloperidol is a potent dopamine antagonist that is used to treat psychotic disorders, but 50% of treated patients develop characteristic extrapyramidal symptoms caused by haloperidol-induced toxicity (HIT), which limits its clinical utility. We do not have any information about the genetic factors affecting this drug-induced toxicity. HIT in humans is directly mirrored in a murine genetic model, where inbred mouse strains are differentially susceptible to HIT. Therefore, we genetically analyzed this murine model and performed a translational human genetic association study. Methods and Findings A whole genome SNP database and computational genetic mapping were used to analyze the murine genetic model of HIT. Guided by the mouse genetic analysis, we demonstrate that genetic variation within an ABC-drug efflux transporter (Abcb5) affected susceptibility to HIT. In situ hybridization results reveal that Abcb5 is expressed in brain capillaries, and by cerebellar Purkinje cells. We also analyzed chromosome substitution strains, imaged haloperidol abundance in brain tissue sections and directly measured haloperidol (and its metabolite) levels in brain, and characterized Abcb5 knockout mice. Our results demonstrate that Abcb5 is part of the blood-brain barrier; it affects susceptibility to HIT by altering the brain concentration of haloperidol. Moreover, a genetic association study in a haloperidol-treated human cohort indicates that human ABCB5 alleles had a time-dependent effect on susceptibility to individual and combined measures of HIT. Abcb5 alleles are pharmacogenetic factors that affect susceptibility to HIT, but it is likely that additional pharmacogenetic susceptibility factors will be discovered

  2. The role of Abcb5 alleles in susceptibility to haloperidol-induced toxicity in mice and humans.

    KAUST Repository

    Zheng, Ming

    2015-02-03

    We know very little about the genetic factors affecting susceptibility to drug-induced central nervous system (CNS) toxicities, and this has limited our ability to optimally utilize existing drugs or to develop new drugs for CNS disorders. For example, haloperidol is a potent dopamine antagonist that is used to treat psychotic disorders, but 50% of treated patients develop characteristic extrapyramidal symptoms caused by haloperidol-induced toxicity (HIT), which limits its clinical utility. We do not have any information about the genetic factors affecting this drug-induced toxicity. HIT in humans is directly mirrored in a murine genetic model, where inbred mouse strains are differentially susceptible to HIT. Therefore, we genetically analyzed this murine model and performed a translational human genetic association study.A whole genome SNP database and computational genetic mapping were used to analyze the murine genetic model of HIT. Guided by the mouse genetic analysis, we demonstrate that genetic variation within an ABC-drug efflux transporter (Abcb5) affected susceptibility to HIT. In situ hybridization results reveal that Abcb5 is expressed in brain capillaries, and by cerebellar Purkinje cells. We also analyzed chromosome substitution strains, imaged haloperidol abundance in brain tissue sections and directly measured haloperidol (and its metabolite) levels in brain, and characterized Abcb5 knockout mice. Our results demonstrate that Abcb5 is part of the blood-brain barrier; it affects susceptibility to HIT by altering the brain concentration of haloperidol. Moreover, a genetic association study in a haloperidol-treated human cohort indicates that human ABCB5 alleles had a time-dependent effect on susceptibility to individual and combined measures of HIT. Abcb5 alleles are pharmacogenetic factors that affect susceptibility to HIT, but it is likely that additional pharmacogenetic susceptibility factors will be discovered.ABCB5 alleles alter susceptibility to

  3. Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription.

    Science.gov (United States)

    Cifuentes-Muñoz, Nicolás; Branttie, Jean; Slaughter, Kerri Beth; Dutch, Rebecca Ellis

    2017-12-15

    induce the formation of large cytoplasmic granules, named inclusion bodies, for genome replication and transcription. Unlike other cytoplasmic structures, such as stress granules and processing bodies, inclusion bodies are exclusively present in infected cells and contain HMPV RNA and proteins to more efficiently transcribe and replicate the viral genome. Though inclusion body formation is nuanced, it corresponds to a more generalized strategy used by different viruses, including filoviruses and rhabdoviruses, for genome transcription and replication. Thus, an understanding of inclusion body formation is crucial for the discovery of innovative therapeutic targets. Copyright © 2017 American Society for Microbiology.

  4. Genomic Analysis Reveals Contrasting PIFq Contribution to Diurnal Rhythmic Gene Expression in PIF-Induced and -Repressed Genes.

    Science.gov (United States)

    Martin, Guiomar; Soy, Judit; Monte, Elena

    2016-01-01

    Members of the PIF quartet (PIFq; PIF1, PIF3, PIF4, and PIF5) collectively contribute to induce growth in Arabidopsis seedlings under short day (SD) conditions, specifically promoting elongation at dawn. Their action involves the direct regulation of growth-related and hormone-associated genes. However, a comprehensive definition of the PIFq-regulated transcriptome under SD is still lacking. We have recently shown that SD and free-running (LL) conditions correspond to "growth" and "no growth" conditions, respectively, correlating with greater abundance of PIF protein in SD. Here, we present a genomic analysis whereby we first define SD-regulated genes at dawn compared to LL in the wild type, followed by identification of those SD-regulated genes whose expression depends on the presence of PIFq. By using this sequential strategy, we have identified 349 PIF/SD-regulated genes, approximately 55% induced and 42% repressed by both SD and PIFq. Comparison with available databases indicates that PIF/SD-induced and PIF/SD-repressed sets are differently phased at dawn and mid-morning, respectively. In addition, we found that whereas rhythmicity of the PIF/SD-induced gene set is lost in LL, most PIF/SD-repressed genes keep their rhythmicity in LL, suggesting differential regulation of both gene sets by the circadian clock. Moreover, we also uncovered distinct overrepresented functions in the induced and repressed gene sets, in accord with previous studies in other examined PIF-regulated processes. Interestingly, promoter analyses showed that, whereas PIF/SD-induced genes are enriched in direct PIF targets, PIF/SD-repressed genes are mostly indirectly regulated by the PIFs and might be more enriched in ABA-regulated genes.

  5. FANTOM5 CAGE profiles of human and mouse reprocessed for GRCh38 and GRCm38 genome assemblies.

    Science.gov (United States)

    Abugessaisa, Imad; Noguchi, Shuhei; Hasegawa, Akira; Harshbarger, Jayson; Kondo, Atsushi; Lizio, Marina; Severin, Jessica; Carninci, Piero; Kawaji, Hideya; Kasukawa, Takeya

    2017-08-29

    The FANTOM5 consortium described the promoter-level expression atlas of human and mouse by using CAGE (Cap Analysis of Gene Expression) with single molecule sequencing. In the original publications, GRCh37/hg19 and NCBI37/mm9 assemblies were used as the reference genomes of human and mouse respectively; later, the Genome Reference Consortium released newer genome assemblies GRCh38/hg38 and GRCm38/mm10. To increase the utility of the atlas in forthcoming researches, we reprocessed the data to make them available on the recent genome assemblies. The data include observed frequencies of transcription starting sites (TSSs) based on the realignment of CAGE reads, and TSS peaks that are converted from those based on the previous reference. Annotations of the peak names were also updated based on the latest public databases. The reprocessed results enable us to examine frequencies of transcription initiations on the recent genome assemblies and to refer promoters with updated information across the genome assemblies consistently.

  6. The 5S rDNA family evolves through concerted and birth-and-death evolution in fish genomes: an example from freshwater stingrays

    Science.gov (United States)

    2011-01-01

    Background Ribosomal 5S genes are well known for the critical role they play in ribosome folding and functionality. These genes are thought to evolve in a concerted fashion, with high rates of homogenization of gene copies. However, the majority of previous analyses regarding the evolutionary process of rDNA repeats were conducted in invertebrates and plants. Studies have also been conducted on vertebrates, but these analyses were usually restricted to the 18S, 5.8S and 28S rRNA genes. The recent identification of divergent 5S rRNA gene paralogs in the genomes of elasmobranches and teleost fishes indicate that the eukaryotic 5S rRNA gene family has a more complex genomic organization than previously thought. The availability of new sequence data from lower vertebrates such as teleosts and elasmobranches enables an enhanced evolutionary characterization of 5S rDNA among vertebrates. Results We identified two variant classes of 5S rDNA sequences in the genomes of Potamotrygonidae stingrays, similar to the genomes of other vertebrates. One class of 5S rRNA genes was shared only by elasmobranches. A broad comparative survey among 100 vertebrate species suggests that the 5S rRNA gene variants in fishes originated from rounds of genome duplication. These variants were then maintained or eliminated by birth-and-death mechanisms, under intense purifying selection. Clustered multiple copies of 5S rDNA variants could have arisen due to unequal crossing over mechanisms. Simultaneously, the distinct genome clusters were independently homogenized, resulting in the maintenance of clusters of highly similar repeats through concerted evolution. Conclusions We believe that 5S rDNA molecular evolution in fish genomes is driven by a mixed mechanism that integrates birth-and-death and concerted evolution. PMID:21627815

  7. Double-strand break repair-adox: Restoration of suppressed double-strand break repair during mitosis induces genomic instability.

    Science.gov (United States)

    Terasawa, Masahiro; Shinohara, Akira; Shinohara, Miki

    2014-12-01

    Double-strand breaks (DSBs) are one of the severest types of DNA damage. Unrepaired DSBs easily induce cell death and chromosome aberrations. To maintain genomic stability, cells have checkpoint and DSB repair systems to respond to DNA damage throughout most of the cell cycle. The failure of this process often results in apoptosis or genomic instability, such as aneuploidy, deletion, or translocation. Therefore, DSB repair is essential for maintenance of genomic stability. During mitosis, however, cells seem to suppress the DNA damage response and proceed to the next G1 phase, even if there are unrepaired DSBs. The biological significance of this suppression is not known. In this review, we summarize recent studies of mitotic DSB repair and discuss the mechanisms of suppression of DSB repair during mitosis. DSB repair, which maintains genomic integrity in other phases of the cell cycle, is rather toxic to cells during mitosis, often resulting in chromosome missegregation and aberration. Cells have multiple safeguards to prevent genomic instability during mitosis: inhibition of 53BP1 or BRCA1 localization to DSB sites, which is important to promote non-homologous end joining or homologous recombination, respectively, and also modulation of the non-homologous end joining core complex to inhibit DSB repair. We discuss how DSBs during mitosis are toxic and the multiple safeguard systems that suppress genomic instability. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  8. Characterization of 5-hydroxytryptamine-induced contraction and acetylcholine-induced relaxation in isolated chicken basilar artery.

    Science.gov (United States)

    Matsumoto, F; Watanabe, Y; Obi, T; Islam, M Z; Yamazaki-Himeno, E; Shiraishi, M; Miyamoto, A

    2012-05-01

    The aim of the present study was to clarify the responsiveness of the chicken basilar artery to 5-hydroxytryptamine (5-HT) and acetylcholine (ACh) and to characterize the related receptor subtypes in vitro. Basilar arteries were obtained from freshly slaughtered broiler chickens. The 5-HT induced concentration-dependent contraction of the arteries. The concentration-response curves for 5-HT were shifted 30-fold to the right by methiothepin (a 5-HT(1) and 5-HT(2) receptor antagonist) and 3-fold to the right by ketanserin (a 5-HT(2) receptor antagonist). In the presence of ketanserin, the concentration-response curve for 5-HT was shifted 10-fold to the right by methiothepin. The pA(2) value for methiothepin was 8.26. The ACh induced concentration-dependent relaxation under conditions of precontraction by 5-HT. The concentration-response curve for ACh was shifted to the right by atropine [a nonselective muscarinic (M) receptor antagonist] and hexahydro-sila-difenidol hydrochloride, a p-fluoroanalog (pFHHSiD, an M(3) receptor antagonist), but not by pirenzepine (an M(1) receptor antagonist) or methoctramine (an M(2) receptor antagonist). The pA(2) value for pFHHSiD was 7.55. Nω-Nitro-l-arginine (a nitric oxide synthase inhibitor) inhibited ACh-induced relaxation by approximately 50%. These results suggest that 5-HT induces contraction via activation of 5-HT(1) and 5-HT(2) receptors and that ACh induces relaxation via activation of the M(3) receptor. The 5-HT(1) receptor might play a dominant role in 5-HT-induced contraction. One of the factors involved in ACh-induced relaxation is probably nitric oxide released from endothelial cells.

  9. Radiation-induced genomic instability is associated with DNA methylation changes in cultured human keratinocytes

    International Nuclear Information System (INIS)

    Kaup, Sahana; Grandjean, Valerie; Mukherjee, Rajarshi; Kapoor, Aparna; Keyes, Edward; Seymour, Colin B.; Mothersill, Carmel E.; Schofield, Paul N.

    2006-01-01

    The mechanism by which radiation-induced genomic instability is initiated, propagated and effected is currently under intense scrutiny. We have investigated the potential role of altered genomic methylation patterns in the cellular response to irradiation and have found evidence for widespread dysregulation of CpG methylation persisting up to 20 population doublings post-irradiation. Similar effects are seen with cells treated with medium from irradiated cells (the 'bystander effect') rather than subjected to direct irradiation. Using an arbitrarily primed methylation sensitive PCR screening method we have demonstrated that irradiation causes reproducible alterations in the methylation profile of a human keratinocyte cell line, HPV-G, and have further characterised one of these sequences as being a member of a retrotransposon element derived sequence family on chromosome 7; MLT1A. Multiple changes were also detected in the screen, which indicate that although the response of cells is predominantly hypermethylation, specific hypomethylation occurs as well. Sequence specific changes are also reported in the methylation of the pericentromeric SAT2 satellite sequence. This is the first demonstration that irradiation results in the induction of heritable methylation changes in mammalian cells, and provides a link between the various non-radiological instigators of genomic instability, the perpetuation of the unstable state and several of its manifestations

  10. Coordinated Changes in Mutation and Growth Rates Induced by Genome Reduction

    Directory of Open Access Journals (Sweden)

    Issei Nishimura

    2017-07-01

    Full Text Available Genome size is determined during evolution, but it can also be altered by genetic engineering in laboratories. The systematic characterization of reduced genomes provides valuable insights into the cellular properties that are quantitatively described by the global parameters related to the dynamics of growth and mutation. In the present study, we analyzed a small collection of W3110 Escherichia coli derivatives containing either the wild-type genome or reduced genomes of various lengths to examine whether the mutation rate, a global parameter representing genomic plasticity, was affected by genome reduction. We found that the mutation rates of these cells increased with genome reduction. The correlation between genome length and mutation rate, which has been reported for the evolution of bacteria, was also identified, intriguingly, for genome reduction. Gene function enrichment analysis indicated that the deletion of many of the genes encoding membrane and transport proteins play a role in the mutation rate changes mediated by genome reduction. Furthermore, the increase in the mutation rate with genome reduction was highly associated with a decrease in the growth rate in a nutrition-dependent manner; thus, poorer media showed a larger change that was of higher significance. This negative correlation was strongly supported by experimental evidence that the serial transfer of the reduced genome improved the growth rate and reduced the mutation rate to a large extent. Taken together, the global parameters corresponding to the genome, growth, and mutation showed a coordinated relationship, which might be an essential working principle for balancing the cellular dynamics appropriate to the environment.

  11. Efficient, footprint-free human iPSC genome editing by consolidation of Cas9/CRISPR and piggyBac technologies.

    Science.gov (United States)

    Wang, Gang; Yang, Luhan; Grishin, Dennis; Rios, Xavier; Ye, Lillian Y; Hu, Yong; Li, Kai; Zhang, Donghui; Church, George M; Pu, William T

    2017-01-01

    Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA), donor DNA and piggyBac transposase resulted in efficient, targeted genome editing and concurrent scarless transgene excision. Using this approach, in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40-50% and homology-directed repair (HDR) frequencies of 10-20%.

  12. 5-Lipoxygenase Deficiency Reduces Acetaminophen-Induced Hepatotoxicity and Lethality

    Directory of Open Access Journals (Sweden)

    Miriam S. N. Hohmann

    2013-01-01

    Full Text Available 5-Lipoxygenase (5-LO converts arachidonic acid into leukotrienes (LTs and is involved in inflammation. At present, the participation of 5-LO in acetaminophen (APAP-induced hepatotoxicity and liver damage has not been addressed. 5-LO deficient (5-LO-/- mice and background wild type mice were challenged with APAP (0.3–6 g/kg or saline. The lethality, liver damage, neutrophil and macrophage recruitment, LTB4, cytokine production, and oxidative stress were assessed. APAP induced a dose-dependent mortality, and the dose of 3 g/kg was selected for next experiments. APAP induced LTB4 production in the liver, the primary target organ in APAP toxicity. Histopathological analysis revealed that 5-LO-/- mice presented reduced APAP-induced liver necrosis and inflammation compared with WT mice. APAP-induced lethality, increase of plasma levels of aspartate aminotransferase and alanine aminotransferase, liver cytokine (IL-1β, TNF-α, IFN-γ, and IL-10, superoxide anion, and thiobarbituric acid reactive substances production, myeloperoxidase and N-acetyl-β-D-glucosaminidase activity, Nrf2 and gp91phox mRNA expression, and decrease of reduced glutathione and antioxidant capacity measured by 2,2′-azinobis(3-ethylbenzothiazoline 6-sulfonate assay were prevented in 5-LO-/- mice compared to WT mice. Therefore, 5-LO deficiency resulted in reduced mortality due to reduced liver inflammatory and oxidative damage, suggesting 5-LO is a promising target to reduce APAP-induced lethality and liver inflammatory/oxidative damage.

  13. Reference quality assembly of the 3.5 Gb genome of Capsicum annuum form a single linked-read library

    Science.gov (United States)

    Linked-Read sequencing technology has recently been employed successfully for de novo assembly of multiple human genomes, however the utility of this technology for complex plant genomes is unproven. We evaluated the technology for this purpose by sequencing the 3.5 gigabase (Gb) diploid pepper (Cap...

  14. 17β-estradiol-induced regulation of the novel 5-HT1A-related transcription factors NUDR and Freud-1 in SH SY5Y cells.

    Science.gov (United States)

    Adeosun, Samuel O; Albert, Paul R; Austin, Mark C; Iyo, Abiye H

    2012-05-01

    Nuclear deformed epidermal autoregulatory factor-1 (NUDR/Deaf-1) and five prime repressor element under dual repression (Freud-1) are novel transcriptional regulators of the 5-HT(1A) receptor, a receptor that has been implicated in the pathophysiology of various psychiatric illnesses. The antidepressant effect of 17β-Estradiol (17βE(2)) is purported to involve the downregulation of this receptor. We investigated the possible role of NUDR and Freud-1 in 17βE(2)-induced downregulation of the 5-HT(1A) receptor in the neuroblastoma cell line SH SY5Y. Cells were treated with 10 nM of 17βE(2) for 3 or 48 h, followed by a 24-h withdrawal period. Proteins were isolated and analyzed by western blotting. 17βE(2) treatment increased NUDR immunoreactivity while Freud-1 and the 5-HT(1A) receptor showed significant decreases. Upon withdrawal of 17βE(2), protein expression returned to control levels, except for NUDR, which remained significantly elevated in the 3-h treatment. Taken together, these data support a non-genomic downregulation of 5-HT(1A) receptor protein by 17βE(2), which does not involve NUDR and Freud-1. Rather, changes in both transcription factors seem to be compensatory/homeostatic responses to changes in 5-HT(1A) receptor induced by 17βE(2). These observations further highlight the importance of NUDR and Freud-1 in regulating 5-HT(1A) receptor expression.

  15. CRISPR/Cas9 Genome Editing: A Promising Tool for Therapeutic Applications of Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Zhang, Yanli; Sastre, Danuta; Wang, Feng

    2018-01-01

    Induced pluripotent stem cells hold tremendous potential for biological and therapeutic applications. The development of efficient technologies for targeted genome alteration of stem cells in disease models is a prerequisite for utilizing stem cells to their full potential. The revolutionary technology for genome editing known as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) system is recently recognized as a powerful tool for editing DNA at specific loci. The ease of use of the CRISPR-Cas9 technology will allow us to improve our understanding of genomic variation in disease processes via cellular and animal models. More recently, this system was modified to repress (CRISPR interference, CRISPRi) or activate (CRISPR activation, CRISPRa) gene expression without alterations in the DNA, which amplified the scope of applications of CRISPR systems for stem cell biology. Here, we highlight latest advances of CRISPR-associated applications in human pluripotent stem cells. The challenges and future prospects of CRISPR-based systems for human research are also discussed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. Transmissible gastroenteritis coronavirus genome packaging signal is located at the 5' end of the genome and promotes viral RNA incorporation into virions in a replication-independent process.

    Science.gov (United States)

    Morales, Lucia; Mateos-Gomez, Pedro A; Capiscol, Carmen; del Palacio, Lorena; Enjuanes, Luis; Sola, Isabel

    2013-11-01

    Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5' end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. The integrity of the entire sequence domain was necessary because deletion of any of the five structural motifs defined within this region abrogated specific packaging of this viral RNA. One of these RNA motifs was the stem-loop SL5, a highly conserved motif in coronaviruses located at nucleotide positions 106 to 136. Partial deletion or point mutations within this motif also abrogated packaging. Using TGEV-derived defective minigenomes replicated in trans by a helper virus, we have shown that TGEV RNA packaging is a replication-independent process. Furthermore, the last 494 nt of the genomic 3' end were not essential for packaging, although this region increased packaging efficiency. TGEV RNA sequences identified as necessary for viral genome packaging were not sufficient to direct packaging of a heterologous sequence derived from the green fluorescent protein gene. These results indicated that TGEV genome packaging is a complex process involving many factors in addition to the identified RNA packaging signal. The identification of well-defined RNA motifs within the TGEV RNA genome that are essential for packaging will be useful for designing packaging-deficient biosafe coronavirus-derived vectors and providing new targets for antiviral therapies.

  17. Xp22.3 genomic deletions involving the CDKL5 gene in girls with early onset epileptic encephalopathy.

    Science.gov (United States)

    Mei, Davide; Marini, Carla; Novara, Francesca; Bernardina, Bernardo D; Granata, Tiziana; Fontana, Elena; Parrini, Elena; Ferrari, Anna R; Murgia, Alessandra; Zuffardi, Orsetta; Guerrini, Renzo

    2010-04-01

    Mutations of the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) cause an X-linked encephalopathy with early onset intractable epilepsy, including infantile spasms and other seizure types, and a Rett syndrome (RTT)-like phenotype. Very limited information is available on the frequency and phenotypic spectrum associated with CDKL5 deletions/duplications. We investigated the role of CDKL5 deletions/duplications in causing early onset intractable epilepsy of unknown etiology in girls. We studied 49 girls with early onset intractable epilepsy, with or without infantile spasms, and developmental impairment, for whom no etiologic factors were obvious after clinical examination, brain magnetic resonance imaging (MRI) and expanded screening for inborn errors of metabolism. We performed CDKL5 gene mutation analysis in all and multiplex ligation dependent probe amplification assay (MLPA) in those who were mutation negative. Custom Array-comparative genomic hybridization (CGH), breakpoint polymerase chain reaction (PCR) analysis, and X-inactivation studies were performed in patients in whom MLPA uncovered a genomic alteration. We found CDKL5 mutations in 8.2% (4 of 49) of patients and genomic deletions in 8.2% (4 of 49). Overall, abnormalities of the CDKL5 gene accounted for 16.3% (8 of 49) of patients. CDKL5 gene deletions are an under-ascertained cause of early onset intractable epilepsy in girls. Genetic testing of CDKL5, including both mutation and deletion/duplication analysis, should be considered in this clinical subgroup.

  18. Antioxidant Supplementation Reduces Genomic Aberrations in Human Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Junfeng Ji

    2014-01-01

    Full Text Available Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs using oncogenic transcription factors. However, this method leads to genetic aberrations in iPSCs via unknown mechanisms, which may limit their clinical use. Here, we demonstrate that the supplementation of growth media with antioxidants reduces the genome instability of cells transduced with the reprogramming factors. Antioxidant supplementation did not affect transgene expression level or silencing kinetics. Importantly, iPSCs made with antioxidants had significantly fewer de novo copy number variations, but not fewer coding point mutations, than iPSCs made without antioxidants. Our results suggest that the quality and safety of human iPSCs might be enhanced by using antioxidants in the growth media during the generation and maintenance of iPSCs.

  19. Genomic and proteomic analyses of Prdm5 reveal interactions with insulator binding proteins in embryonic stem cells

    DEFF Research Database (Denmark)

    Galli, Giorgio Giacomo; Carrara, Matteo; Francavilla, Chiara

    2013-01-01

    PRDM proteins belong to the SET- domain protein family involved in the regulation of gene expression. Although few PRDM members possess histone methyltransferase activity, the molecular mechanisms by which the other members exert transcriptional regulation remain to be delineated. In this study, we...... find that Prdm5 is highly expressed in mouse embryonic stem cells (mES) and exploit this cellular system to characterize molecular functions of Prdm5. By combining proteomics and next generation sequencing technologies we identify Prdm5 interaction partners and genomic occupancy. We demonstrate that......, despite Prdm5 is dispensable for mES cell maintenance, it directly targets genomic regions involved in early embryonic development and affects the expression of a subset of developmental regulators during cell differentiation. Importantly, Prdm5 interacts with Ctcf, Cohesin and TFIIIC and co...

  20. SUMO E3 ligase Mms21 prevents spontaneous DNA damage induced genome rearrangements.

    Directory of Open Access Journals (Sweden)

    Jason Liang

    2018-03-01

    Full Text Available Mms21, a subunit of the Smc5/6 complex, possesses an E3 ligase activity for the Small Ubiquitin-like MOdifier (SUMO. Here we show that the mms21-CH mutation, which inactivates Mms21 ligase activity, causes increased accumulation of gross chromosomal rearrangements (GCRs selected in the dGCR assay. These dGCRs are formed by non-allelic homologous recombination between divergent DNA sequences mediated by Rad52-, Rrm3- and Pol32-dependent break-induced replication. Combining mms21-CH with sgs1Δ caused a synergistic increase in GCRs rates, indicating the distinct roles of Mms21 and Sgs1 in suppressing GCRs. The mms21-CH mutation also caused increased rates of accumulating uGCRs mediated by breakpoints in unique sequences as revealed by whole genome sequencing. Consistent with the accumulation of endogenous DNA lesions, mms21-CH mutants accumulate increased levels of spontaneous Rad52 and Ddc2 foci and had a hyper-activated DNA damage checkpoint. Together, these findings support that Mms21 prevents the accumulation of spontaneous DNA lesions that cause diverse GCRs.

  1. The role of free radicals and stress signalling in persistent genomic instability induced by long wavelength UV light

    International Nuclear Information System (INIS)

    Phillipson, R.; McMillan, T.J.

    2003-01-01

    Induction of persistent genomic instability has commonly been investigated with ionising radiation. It has been characterised as a decrease in plating efficiency, and an increase in chromosomal aberrations and mutation frequency in the progeny of cells that survive the initial irradiation. We now present data demonstrating the phenomenon following exposure to long-wavelength solar UV-A (320-400nm) radiation at environmentally relevant doses. Using the spontaneously immortalised human skin keratinocyte line, HaCaT, we observed a significant decrease in plating efficiency (77 +/- 2% of control), and increase in micronuclei (2.5 fold) and mutation frequency (2 fold), 7 days after the initial radiation insult. Modification of UV-A-induced instability by incubation with exogenous catalase implicated reactive oxygen species (ROS), in-particular hydrogen peroxide, in the production and/or maintenance of the phenomenon. Assessment of anti-oxidant enzymes revealed a significant increase in glutathione-s-transferase activity (158 +/- 4% of control) at day 7 in the irradiated cell population, which was inhibited by incubation with exogenous catalase (97 +/- 3%), providing further evidence for an ROS-mediated pathway. Furthermore, inhibition of UV-A-induced micronuclei at day 7 by the flavonoid-containing-protein inhibitor diphenyleneiodonium (DPI) indicates that the NADPH oxidase family of enzymes may be involved in this phenomenon. Measurement of superoxide production by the cytochrome c reduction assay revealed that the irradiated cell population produce 50% more superoxide than the unirradiated controls, and that incubation with DPI led to a preferential reduction in superoxide production in the UV-A treated population at day 7. Finally, NADPH oxidase activity is increased significantly over controls in UV-A-treated cells. These data demonstrate that oxidative stress, analogous to that produced by ionising radiation, induces persistent genomic instability through a

  2. Warburg effect and translocation-induced genomic instability: two yeast models for cancer cells

    International Nuclear Information System (INIS)

    Tosato, Valentina; Grüning, Nana-Maria; Breitenbach, Michael; Arnak, Remigiusz; Ralser, Markus; Bruschi, Carlo V.

    2013-01-01

    Yeast has been established as an efficient model system to study biological principles underpinning human health. In this review we focus on yeast models covering two aspects of cancer formation and progression (i) the activity of pyruvate kinase (PK), which recapitulates metabolic features of cancer cells, including the Warburg effect, and (ii) chromosome bridge-induced translocation (BIT) mimiking genome instability in cancer. Saccharomyces cerevisiae is an excellent model to study cancer cell metabolism, as exponentially growing yeast cells exhibit many metabolic similarities with rapidly proliferating cancer cells. The metabolic reconfiguration includes an increase in glucose uptake and fermentation, at the expense of respiration and oxidative phosphorylation (the Warburg effect), and involves a broad reconfiguration of nucleotide and amino acid metabolism. Both in yeast and humans, the regulation of this process seems to have a central player, PK, which is up-regulated in cancer, and to occur mostly on a post-transcriptional and post-translational basis. Furthermore, BIT allows to generate selectable translocation-derived recombinants (“translocants”), between any two desired chromosomal locations, in wild-type yeast strains transformed with a linear DNA cassette carrying a selectable marker flanked by two DNA sequences homologous to different chromosomes. Using the BIT system, targeted non-reciprocal translocations in mitosis are easily inducible. An extensive collection of different yeast translocants exhibiting genome instability and aberrant phenotypes similar to cancer cells has been produced and subjected to analysis. In this review, we hence provide an overview upon two yeast cancer models, and extrapolate general principles for mimicking human disease mechanisms in yeast.

  3. Warburg effect and translocation-induced genomic instability: two yeast models for cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Tosato, Valentina [International Centre for Genetic Engineering and Biotechnology, Trieste (Italy); Grüning, Nana-Maria [Cambridge System Biology Center, Department of Biochemistry, University of Cambridge, Cambridge (United Kingdom); Breitenbach, Michael [Division of Genetics, Department of Cell Biology, University of Salzburg, Salzburg (Austria); Arnak, Remigiusz [International Centre for Genetic Engineering and Biotechnology, Trieste (Italy); Ralser, Markus [Cambridge System Biology Center, Department of Biochemistry, University of Cambridge, Cambridge (United Kingdom); Bruschi, Carlo V., E-mail: bruschi@icgeb.org [International Centre for Genetic Engineering and Biotechnology, Trieste (Italy)

    2013-01-18

    Yeast has been established as an efficient model system to study biological principles underpinning human health. In this review we focus on yeast models covering two aspects of cancer formation and progression (i) the activity of pyruvate kinase (PK), which recapitulates metabolic features of cancer cells, including the Warburg effect, and (ii) chromosome bridge-induced translocation (BIT) mimiking genome instability in cancer. Saccharomyces cerevisiae is an excellent model to study cancer cell metabolism, as exponentially growing yeast cells exhibit many metabolic similarities with rapidly proliferating cancer cells. The metabolic reconfiguration includes an increase in glucose uptake and fermentation, at the expense of respiration and oxidative phosphorylation (the Warburg effect), and involves a broad reconfiguration of nucleotide and amino acid metabolism. Both in yeast and humans, the regulation of this process seems to have a central player, PK, which is up-regulated in cancer, and to occur mostly on a post-transcriptional and post-translational basis. Furthermore, BIT allows to generate selectable translocation-derived recombinants (“translocants”), between any two desired chromosomal locations, in wild-type yeast strains transformed with a linear DNA cassette carrying a selectable marker flanked by two DNA sequences homologous to different chromosomes. Using the BIT system, targeted non-reciprocal translocations in mitosis are easily inducible. An extensive collection of different yeast translocants exhibiting genome instability and aberrant phenotypes similar to cancer cells has been produced and subjected to analysis. In this review, we hence provide an overview upon two yeast cancer models, and extrapolate general principles for mimicking human disease mechanisms in yeast.

  4. The problem of induced genomic instability in the child organism under conditions of long-term effect of small radiation doses

    International Nuclear Information System (INIS)

    Suskov, I.I.; Kuz'mina, N.S.

    2001-01-01

    The phenomenological aspects of the genomic instability induced in the descendants of the multi-divided cells having been exposed to the radiation are examined. It is demonstrated that the regularity of the genomic instability induction do not correspond to the classical conception of the radiation genetics (hit principle and target theory). The mechanisms and the biological significance of this new genetic phenomenon in the child organism under conditions of low-intensive effect of small-dose radiation and its connection with the state of health are discussed [ru

  5. Array-based assay detects genome-wide 5-mC and 5-hmC in the brains of humans, non-human primates, and mice.

    Science.gov (United States)

    Chopra, Pankaj; Papale, Ligia A; White, Andrew T J; Hatch, Andrea; Brown, Ryan M; Garthwaite, Mark A; Roseboom, Patrick H; Golos, Thaddeus G; Warren, Stephen T; Alisch, Reid S

    2014-02-13

    Methylation on the fifth position of cytosine (5-mC) is an essential epigenetic mark that is linked to both normal neurodevelopment and neurological diseases. The recent identification of another modified form of cytosine, 5-hydroxymethylcytosine (5-hmC), in both stem cells and post-mitotic neurons, raises new questions as to the role of this base in mediating epigenetic effects. Genomic studies of these marks using model systems are limited, particularly with array-based tools, because the standard method of detecting DNA methylation cannot distinguish between 5-mC and 5-hmC and most methods have been developed to only survey the human genome. We show that non-human data generated using the optimization of a widely used human DNA methylation array, designed only to detect 5-mC, reproducibly distinguishes tissue types within and between chimpanzee, rhesus, and mouse, with correlations near the human DNA level (R(2) > 0.99). Genome-wide methylation analysis, using this approach, reveals 6,102 differentially methylated loci between rhesus placental and fetal tissues with pathways analysis significantly overrepresented for developmental processes. Restricting the analysis to oncogenes and tumor suppressor genes finds 76 differentially methylated loci, suggesting that rhesus placental tissue carries a cancer epigenetic signature. Similarly, adapting the assay to detect 5-hmC finds highly reproducible 5-hmC levels within human, rhesus, and mouse brain tissue that is species-specific with a hierarchical abundance among the three species (human > rhesus > mouse). Annotation of 5-hmC with respect to gene structure reveals a significant prevalence in the 3'UTR and an association with chromatin-related ontological terms, suggesting an epigenetic feedback loop mechanism for 5-hmC. Together, these data show that this array-based methylation assay is generalizable to all mammals for the detection of both 5-mC and 5-hmC, greatly improving the utility of mammalian model systems

  6. Complete genome sequencing of Agrobacterium sp. H13-3, the former Rhizobium lupini H13-3, reveals a tripartite genome consisting of a circular and a linear chromosome and an accessory plasmid but lacking a tumor-inducing Ti-plasmid.

    Science.gov (United States)

    Wibberg, Daniel; Blom, Jochen; Jaenicke, Sebastian; Kollin, Florian; Rupp, Oliver; Scharf, Birgit; Schneiker-Bekel, Susanne; Sczcepanowski, Rafael; Goesmann, Alexander; Setubal, Joao Carlos; Schmitt, Rüdiger; Pühler, Alfred; Schlüter, Andreas

    2011-08-20

    Agrobacterium sp. H13-3, formerly known as Rhizobium lupini H13-3, is a soil bacterium that was isolated from the rhizosphere of Lupinus luteus. The isolate has been established as a model system for studying novel features of flagellum structure, motility and chemotaxis within the family Rhizobiaceae. The complete genome sequence of Agrobacterium sp. H13-3 has been established and the genome structure and phylogenetic assignment of the organism was analysed. For de novo sequencing of the Agrobacterium sp. H13-3 genome, a combined strategy comprising 454-pyrosequencing on the Genome Sequencer FLX platform and PCR-based amplicon sequencing for gap closure was applied. The finished genome consists of three replicons and comprises 5,573,770 bases. Based on phylogenetic analyses, the isolate could be assigned to the genus Agrobacterium biovar I and represents a genomic species G1 strain within this biovariety. The highly conserved circular chromosome (2.82 Mb) of Agrobacterium sp. H13-3 mainly encodes housekeeping functions characteristic for an aerobic, heterotrophic bacterium. Agrobacterium sp. H13-3 is a motile bacterium driven by the rotation of several complex flagella. Its behaviour towards external stimuli is regulated by a large chemotaxis regulon and a total of 17 chemoreceptors. Comparable to the genome of Agrobacterium tumefaciens C58, Agrobacterium sp. H13-3 possesses a linear chromosome (2.15 Mb) that is related to its reference replicon and features chromosomal and plasmid-like properties. The accessory plasmid pAspH13-3a (0.6 Mb) is only distantly related to the plasmid pAtC58 of A. tumefaciens C58 and shows a mosaic structure. A tumor-inducing Ti-plasmid is missing in the sequenced strain H13-3 indicating that it is a non-virulent isolate. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. PCR artifact in testing for homologous recombination in genomic editing in zebrafish.

    Directory of Open Access Journals (Sweden)

    Minho Won

    Full Text Available We report a PCR-induced artifact in testing for homologous recombination in zebrafish. We attempted to replace the lnx2a gene with a donor cassette, mediated by a TALEN induced double stranded cut. The donor construct was flanked with homology arms of about 1 kb at the 5' and 3' ends. Injected embryos (G0 were raised and outcrossed to wild type fish. A fraction of the progeny appeared to have undergone the desired homologous recombination, as tested by PCR using primer pairs extending from genomic DNA outside the homology region to a site within the donor cassette. However, Southern blots revealed that no recombination had taken place. We conclude that recombination happened during PCR in vitro between the donor integrated elsewhere in the genome and the lnx2a locus. We conclude that PCR alone may be insufficient to verify homologous recombination in genome editing experiments in zebrafish.

  8. [Genome loses all 5-methylcytosine a life span. How is this connected with accumulation of mutations during aging?].

    Science.gov (United States)

    Mazin, A L

    1993-01-01

    The 5-methylcytosine (5mC) content in liver DNA has been determined for rats of different age. The rate of the 5mC loss from DNA is maximal in pre- and neonatal rats, 1.28% of reduction of the 5mC content per day, then it decreases to 0.33% and becomes minimal and constant in adult rats, 0.028% per day. During pregnancy and the first 15 days of postnatal development rat genome loses 49% of all 5mC. Within the next 45 days 15% of 5mC disappears, and during maximal rat life span, about four years, 39% of the genomic 5mC may be lost. Thus, it has been found for the first time that the animal genome loses practically all 5mC residues during the life span. Analysis of the literature data shows that for embryos the rate of the 5mC loss from DNA proves to be higher than that for adult animals by 96 times for mice, 69-for rats and 28-for cows. The rate of embryonal DNA hypomethylation may be inversely proportional to the pregnancy duration of species. In adult animals the rate inversely correlates with their maximal life span and accounts for the 5mC loss from DNA of a mouse by 0.028%, of a rat by 0.024%, of a hamster by 0.007%, of a cow by 0.004% and of a human being by 0.0005% per day. During the entire ontogenesis, the genome of a mouse loses 93% of all 5mC residues, that of a rat-101% and of a cow-88%. The age-dependent loss of 5mC from DNA is also typical for cell lines aging in vitro. It is constant, as a rule, and correlates with the number of cell population doublings (PD). The removal of all 5mC from DNA corresponds to 70-130 PD for human, 40-60 PD-for hamster and 6 PD- for mouse cells. In immortal lines the level of DNA methylation is stable or grows with age. A possible mechanism of an age-related 5mC loss from DNA is discussed. DNA hypomethylation may result from 5mC deamination directly at the moment of replicative DNA methylation and subsequent reparation of the G.T mispairs which leads to accumulation of the 5mC-->T+C substitutions in the genome with each

  9. Genomic instability--an evolving hallmark of cancer.

    Science.gov (United States)

    Negrini, Simona; Gorgoulis, Vassilis G; Halazonetis, Thanos D

    2010-03-01

    Genomic instability is a characteristic of most cancers. In hereditary cancers, genomic instability results from mutations in DNA repair genes and drives cancer development, as predicted by the mutator hypothesis. In sporadic (non-hereditary) cancers the molecular basis of genomic instability remains unclear, but recent high-throughput sequencing studies suggest that mutations in DNA repair genes are infrequent before therapy, arguing against the mutator hypothesis for these cancers. Instead, the mutation patterns of the tumour suppressor TP53 (which encodes p53), ataxia telangiectasia mutated (ATM) and cyclin-dependent kinase inhibitor 2A (CDKN2A; which encodes p16INK4A and p14ARF) support the oncogene-induced DNA replication stress model, which attributes genomic instability and TP53 and ATM mutations to oncogene-induced DNA damage.

  10. Genome-wide identification and comparative analysis of cytosine-5 DNA methyltransferases and demethylase families in wild and cultivated peanut

    Directory of Open Access Journals (Sweden)

    Pengfei eWang

    2016-02-01

    Full Text Available AbstractDNA methylation plays important roles in genome protection, regulation of gene expression and was associated with plants development. Plant DNA methylation pattern was mediated by cytosine-5 DNA methyltransferases and demethylase. Although the genomes of AA and BB wild peanuts have been fully sequence, these two gene families have not been studied. In this study we report the identification and analysis of putative cytosine-5 DNA methyltransferases (C5-MTases and demethylase in AA and BB wild peanuts. Cytosine-5 DNA methyltransferases in AA and BB wild peanuts could be classified in known MET, CMT and DRM2 groups based on their domain organization. This result was supported by the gene and protein structural characteristics and phylogenetic analysis. We found that some wild peanut DRM2 numbers didn’t contain UBA domain which was different from other plants such as Arabidopsis, maize, soybean. Five DNA demethylase were found in AA genome and five in BB genome. The selective pressure analysis showed that wild peanut C5-MTases gene mainly underwent purifying selection but many positive selection sites can be detected. Conversely, DNA demethylase genes mainly underwent positive selection during evolution. Additionally, the expression dynamic of cytosine-5 DNA methyltransferases and demethylase genes in different cultivated peanut tissues were analyzed. Expression result showed that cold, heat or drought stress could influence the expression level of C5-MTases and DNA demethylase genes in cultivated peanut. These results are useful for better understanding the complexity of these two gene families, and will facilitate epigenetic studies in peanut.

  11. DNA repair efficiency in germ cells and early mouse embryos and consequences for radiation-induced transgenerational genomic damage

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Wyrobek, Andrew J.

    2009-01-18

    Exposure to ionizing radiation and other environmental agents can affect the genomic integrity of germ cells and induce adverse health effects in the progeny. Efficient DNA repair during gametogenesis and the early embryonic cycles after fertilization is critical for preventing transmission of DNA damage to the progeny and relies on maternal factors stored in the egg before fertilization. The ability of the maternal repair machinery to repair DNA damage in both parental genomes in the fertilizing egg is especially crucial for the fertilizing male genome that has not experienced a DNA repair-competent cellular environment for several weeks prior to fertilization. During the DNA repair-deficient period of spermatogenesis, DNA lesions may accumulate in sperm and be carried into the egg where, if not properly repaired, could result in the formation of heritable chromosomal aberrations or mutations and associated birth defects. Studies with female mice deficient in specific DNA repair genes have shown that: (i) cell cycle checkpoints are activated in the fertilized egg by DNA damage carried by the sperm; and (ii) the maternal genotype plays a major role in determining the efficiency of repairing genomic lesions in the fertilizing sperm and directly affect the risk for abnormal reproductive outcomes. There is also growing evidence that implicates DNA damage carried by the fertilizing gamete as a mediator of postfertilization processes that contribute to genomic instability in subsequent generations. Transgenerational genomic instability most likely involves epigenetic mechanisms or error-prone DNA repair processes in the early embryo. Maternal and embryonic DNA repair processes during the early phases of mammalian embryonic development can have far reaching consequences for the genomic integrity and health of subsequent generations.

  12. A re-sequencing based assessment of genomic heterogeneity and fast neutron-induced deletions in a common bean cultivar

    Directory of Open Access Journals (Sweden)

    Jamie A. O'Rourke

    2013-06-01

    Full Text Available A small fast neutron mutant population has been established from Phaseolus vulgaris cv. Red Hawk. We leveraged the available P. vulgaris genome sequence and high throughput next generation DNA sequencing to examine the genomic structure of five Phaseolus vulgaris cv. Red Hawk fast neutron mutants with striking visual phenotypes. Analysis of these genomes identified three classes of structural variation; between cultivar variation, natural variation within the fast neutron mutant population, and fast neutron induced mutagenesis. Our analyses focused on the latter two classes. We identified 23 large deletions (>40 bp common to multiple individuals, illustrating residual heterogeneity and regions of structural variation within the common bean cv. Red Hawk. An additional 18 large deletions were identified in individual mutant plants. These deletions, ranging in size from 40 bp to 43,000 bp, are potentially the result of fast neutron mutagenesis. Six of the 18 deletions lie near or within gene coding regions, identifying potential candidate genes causing the mutant phenotype.

  13. Scribl: an HTML5 Canvas-based graphics library for visualizing genomic data over the web.

    Science.gov (United States)

    Miller, Chase A; Anthony, Jon; Meyer, Michelle M; Marth, Gabor

    2013-02-01

    High-throughput biological research requires simultaneous visualization as well as analysis of genomic data, e.g. read alignments, variant calls and genomic annotations. Traditionally, such integrative analysis required desktop applications operating on locally stored data. Many current terabyte-size datasets generated by large public consortia projects, however, are already only feasibly stored at specialist genome analysis centers. As even small laboratories can afford very large datasets, local storage and analysis are becoming increasingly limiting, and it is likely that most such datasets will soon be stored remotely, e.g. in the cloud. These developments will require web-based tools that enable users to access, analyze and view vast remotely stored data with a level of sophistication and interactivity that approximates desktop applications. As rapidly dropping cost enables researchers to collect data intended to answer questions in very specialized contexts, developers must also provide software libraries that empower users to implement customized data analyses and data views for their particular application. Such specialized, yet lightweight, applications would empower scientists to better answer specific biological questions than possible with general-purpose genome browsers currently available. Using recent advances in core web technologies (HTML5), we developed Scribl, a flexible genomic visualization library specifically targeting coordinate-based data such as genomic features, DNA sequence and genetic variants. Scribl simplifies the development of sophisticated web-based graphical tools that approach the dynamism and interactivity of desktop applications. Software is freely available online at http://chmille4.github.com/Scribl/ and is implemented in JavaScript with all modern browsers supported.

  14. Generation of an ICF syndrome model by efficient genome editing of human induced pluripotent stem cells using the CRISPR system.

    Science.gov (United States)

    Horii, Takuro; Tamura, Daiki; Morita, Sumiyo; Kimura, Mika; Hatada, Izuho

    2013-09-30

    Genome manipulation of human induced pluripotent stem (iPS) cells is essential to achieve their full potential as tools for regenerative medicine. To date, however, gene targeting in human pluripotent stem cells (hPSCs) has proven to be extremely difficult. Recently, an efficient genome manipulation technology using the RNA-guided DNase Cas9, the clustered regularly interspaced short palindromic repeats (CRISPR) system, has been developed. Here we report the efficient generation of an iPS cell model for immunodeficiency, centromeric region instability, facial anomalies syndrome (ICF) syndrome using the CRISPR system. We obtained iPS cells with mutations in both alleles of DNA methyltransferase 3B (DNMT3B) in 63% of transfected clones. Our data suggest that the CRISPR system is highly efficient and useful for genome engineering of human iPS cells.

  15. Transmissible Gastroenteritis Coronavirus Genome Packaging Signal Is Located at the 5′ End of the Genome and Promotes Viral RNA Incorporation into Virions in a Replication-Independent Process

    Science.gov (United States)

    Morales, Lucia; Mateos-Gomez, Pedro A.; Capiscol, Carmen; del Palacio, Lorena; Sola, Isabel

    2013-01-01

    Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5′ end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. The integrity of the entire sequence domain was necessary because deletion of any of the five structural motifs defined within this region abrogated specific packaging of this viral RNA. One of these RNA motifs was the stem-loop SL5, a highly conserved motif in coronaviruses located at nucleotide positions 106 to 136. Partial deletion or point mutations within this motif also abrogated packaging. Using TGEV-derived defective minigenomes replicated in trans by a helper virus, we have shown that TGEV RNA packaging is a replication-independent process. Furthermore, the last 494 nt of the genomic 3′ end were not essential for packaging, although this region increased packaging efficiency. TGEV RNA sequences identified as necessary for viral genome packaging were not sufficient to direct packaging of a heterologous sequence derived from the green fluorescent protein gene. These results indicated that TGEV genome packaging is a complex process involving many factors in addition to the identified RNA packaging signal. The identification of well-defined RNA motifs within the TGEV RNA genome that are essential for packaging will be useful for designing packaging-deficient biosafe coronavirus-derived vectors and providing new targets for antiviral therapies. PMID:23966403

  16. Demethylation by 5-aza-2'-deoxycytidine in colorectal cancer cells targets genomic DNA whilst promoter CpG island methylation persists

    International Nuclear Information System (INIS)

    Mossman, David; Kim, Kyu-Tae; Scott, Rodney J

    2010-01-01

    DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. Aberrant epigenetic gene silencing in tumours is a frequent event, yet the factors which dictate which genes are targeted for inactivation are unknown. DNA methylation and histone acetylation can be modified with the chemical agents 5-aza-2'-deoxycytidine (5-aza-dC) and Trichostatin A (TSA) respectively. The aim of this study was to analyse de-methylation and re-methylation and its affect on gene expression in colorectal cancer cell lines treated with 5-aza-dC alone and in combination with TSA. We also sought to identify methylation patterns associated with long term reactivation of previously silenced genes. Colorectal cancer cell lines were treated with 5-aza-dC, with and without TSA, to analyse global methylation decreases by High Performance Liquid Chromatography (HPLC). Re-methylation was observed with removal of drug treatments. Expression arrays identified silenced genes with differing patterns of expression after treatment, such as short term reactivation or long term reactivation. Sodium bisulfite sequencing was performed on the CpG island associated with these genes and expression was verified with real time PCR. Treatment with 5-aza-dC was found to affect genomic methylation and to a lesser extent gene specific methylation. Reactivated genes which remained expressed 10 days post 5-aza-dC treatment featured hypomethylated CpG sites adjacent to the transcription start site (TSS). In contrast, genes with uniformly hypermethylated CpG islands were only temporarily reactivated. These results imply that 5-aza-dC induces strong de-methylation of the genome and initiates reactivation of transcriptionally inactive genes, but this does not require gene associated CpG island de-methylation to occur. In addition, for three of our selected genes, hypomethylation at the TSS of an epigenetically silenced gene is associated with the long term reversion of

  17. Draft genome sequence of a GES-5-producing Serratia marcescens isolated in southern Brazil

    Directory of Open Access Journals (Sweden)

    Carolina Silva Nodari

    Full Text Available Abstract Serratia marcescens is a Gram-negative rod intrinsically resistant to polymyxins and usually associated with wound, respiratory and urinary tract infections. The whole genome of the first GES-5-producing S. marcescens isolated from a Brazilian patient was sequenced using Ion Torrent PGM System. Besides blaGES-5, we were able to identify genes encoding for other β-lactamases, for aminoglycoside modifying enzymes and for an efflux pump to tetracyclines.

  18. Sequence analysis of the PIP5K locus in Eimeria maxima provides further evidence for eimerian genome plasticity and segmental organization.

    Science.gov (United States)

    Song, B K; Pan, M Z; Lau, Y L; Wan, K L

    2014-07-29

    Commercial flocks infected by Eimeria species parasites, including Eimeria maxima, have an increased risk of developing clinical or subclinical coccidiosis; an intestinal enteritis associated with increased mortality rates in poultry. Currently, infection control is largely based on chemotherapy or live vaccines; however, drug resistance is common and vaccines are relatively expensive. The development of new cost-effective intervention measures will benefit from unraveling the complex genetic mechanisms that underlie host-parasite interactions, including the identification and characterization of genes encoding proteins such as phosphatidylinositol 4-phosphate 5-kinase (PIP5K). We previously identified a PIP5K coding sequence within the E. maxima genome. In this study, we analyzed two bacterial artificial chromosome clones presenting a ~145-kb E. maxima (Weybridge strain) genomic region spanning the PIP5K gene locus. Sequence analysis revealed that ~95% of the simple sequence repeats detected were located within regions comparable to the previously described feature-rich segments of the Eimeria tenella genome. Comparative sequence analysis with the orthologous E. maxima (Houghton strain) region revealed a moderate level of conserved synteny. Unique segmental organizations and telomere-like repeats were also observed in both genomes. A number of incomplete transposable elements were detected and further scrutiny of these elements in both orthologous segments revealed interesting nesting events, which may play a role in facilitating genome plasticity in E. maxima. The current analysis provides more detailed information about the genome organization of E. maxima and may help to reveal genotypic differences that are important for expression of traits related to pathogenicity and virulence.

  19. First complete genome sequence of parainfluenza virus 5 isolated from lesser panda.

    Science.gov (United States)

    Zhai, Jun-Qiong; Zhai, Shao-Lun; Lin, Tao; Liu, Jian-Kui; Wang, He-Xing; Li, Bing; Zhang, He; Zou, Shu-Zhan; Zhou, Xia; Wu, Meng-Fan; Chen, Wu; Luo, Man-Lin

    2017-05-01

    Parainfluenza virus 5 (PIV5) is widespread in mammals and humans. Up to now, there is little information about PIV5 infection in lesser pandas. In this study, a PIV5 variant (named ZJQ-221) was isolated from a lesser panda with respiratory disease in Guangzhou zoo in Guangdong province, southern China. The full-length genome of ZJQ-221 was found to be 15,246 nucleotides and consisted of seven non-overlapping genes encoding eight proteins (i.e., NP, V, P, M, F, SH, HN and L). Sequence alignment and genetic analysis revealed that ZJQ-221 shared a close relationship with a PIV5 strain of canine-origin (1168-1) from South Korea. The findings of this study confirm the presence of PIV5 in lesser panda and indicate this mammal as a possible natural reservoir. Furthermore they highlight the urgent need to strengthen viral surveillance and control of PIV5 in zoo animals.

  20. "After the Genome 5, Conference to be held October 6-10, 1999, Jackson Hole, Wyoming"

    Energy Technology Data Exchange (ETDEWEB)

    Brent, Roger [Molecular Sciences Inst., Milpitas, CA (United States)

    1999-10-06

    become cognizant of the issues raised by this future, and, in response, the organizers intend to distinguish this meeting from other "postgenomic" meetings by bringing together intellectuals from subject fields far outside of conventional biology with the expectation that this will help focus thinking beyond the immediate future. To this end, After the Genome 5 will bring together industrial and university researchers, including: 1) Physicists, chemists, and engineers who are devising and using new data gathering techniques, such as microarrays, protein mass spectrometry, and single molecule measurements 2) Computer scientists from fields as diverse as geology and wargames, who have experience moving from broad knowledge of systems to analysis that results in models and simulations 3) Neurobiologists and computer scientists who combine physiological experimentation and computer modeling to understand single cells and small networks of cells 4) Biologists who are trying to model genetic networks 5) All- around visionary thinkers 7) policy makers, to suggest how to convey any good ideas to organizations that can commit resources to them.

  1. Genome Maps, a new generation genome browser.

    Science.gov (United States)

    Medina, Ignacio; Salavert, Francisco; Sanchez, Rubén; de Maria, Alejandro; Alonso, Roberto; Escobar, Pablo; Bleda, Marta; Dopazo, Joaquín

    2013-07-01

    Genome browsers have gained importance as more genomes and related genomic information become available. However, the increase of information brought about by new generation sequencing technologies is, at the same time, causing a subtle but continuous decrease in the efficiency of conventional genome browsers. Here, we present Genome Maps, a genome browser that implements an innovative model of data transfer and management. The program uses highly efficient technologies from the new HTML5 standard, such as scalable vector graphics, that optimize workloads at both server and client sides and ensure future scalability. Thus, data management and representation are entirely carried out by the browser, without the need of any Java Applet, Flash or other plug-in technology installation. Relevant biological data on genes, transcripts, exons, regulatory features, single-nucleotide polymorphisms, karyotype and so forth, are imported from web services and are available as tracks. In addition, several DAS servers are already included in Genome Maps. As a novelty, this web-based genome browser allows the local upload of huge genomic data files (e.g. VCF or BAM) that can be dynamically visualized in real time at the client side, thus facilitating the management of medical data affected by privacy restrictions. Finally, Genome Maps can easily be integrated in any web application by including only a few lines of code. Genome Maps is an open source collaborative initiative available in the GitHub repository (https://github.com/compbio-bigdata-viz/genome-maps). Genome Maps is available at: http://www.genomemaps.org.

  2. Transmissible Gastroenteritis Coronavirus Genome Packaging Signal Is Located at the 5′ End of the Genome and Promotes Viral RNA Incorporation into Virions in a Replication-Independent Process

    OpenAIRE

    Morales, Lucia; Mateos-Gomez, Pedro A.; Capiscol, Carmen; del Palacio, Lorena; Enjuanes, Luis; Sola, Isabel

    2013-01-01

    Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5′ end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. Th...

  3. Combining Induced Pluripotent Stem Cells and Genome Editing Technologies for Clinical Applications.

    Science.gov (United States)

    Chang, Chia-Yu; Ting, Hsiao-Chien; Su, Hong-Lin; Jeng, Jing-Ren

    2018-01-01

    In this review, we introduce current developments in induced pluripotent stem cells (iPSCs), site-specific nuclease (SSN)-mediated genome editing tools, and the combined application of these two novel technologies in biomedical research and therapeutic trials. The sustainable pluripotent property of iPSCs in vitro not only provides unlimited cell sources for basic research but also benefits precision medicines for human diseases. In addition, rapidly evolving SSN tools efficiently tailor genetic manipulations for exploring gene functions and can be utilized to correct genetic defects of congenital diseases in the near future. Combining iPSC and SSN technologies will create new reliable human disease models with isogenic backgrounds in vitro and provide new solutions for cell replacement and precise therapies.

  4. Src Kinase Dependent Rapid Non-genomic Modulation of Hippocampal Spinogenesis Induced by Androgen and Estrogen

    Directory of Open Access Journals (Sweden)

    Mika Soma

    2018-05-01

    Full Text Available Dendritic spine is a small membranous protrusion from a neuron's dendrite that typically receives input from an axon terminal at the synapse. Memories are stored in synapses which consist of spines and presynapses. Rapid modulations of dendritic spines induced by hippocampal sex steroids, including dihydrotestosterone (DHT, testosterone (T, and estradiol (E2, are essential for synaptic plasticity. Molecular mechanisms underlying the rapid non-genomic modulation through synaptic receptors of androgen (AR and estrogen (ER as well as its downstream kinase signaling, however, have not been well understood. We investigated the possible involvement of Src tyrosine kinase in rapid changes of dendritic spines in response to androgen and estrogen, including DHT, T, and E2, using hippocampal slices from adult male rats. We found that the treatments with DHT (10 nM, T (10 nM, and E2 (1 nM increased the total density of spines by ~1.22 to 1.26-fold within 2 h using super resolution confocal imaging of Lucifer Yellow-injected CA1 pyramidal neurons. We examined also morphological changes of spines in order to clarify differences between three sex steroids. From spine head diameter analysis, DHT increased middle- and large-head spines, whereas T increased small- and middle-head spines, and E2 increased small-head spines. Upon application of Src tyrosine kinase inhibitor, the spine increases induced through DHT, T, and E2 treatments were completely blocked. These results imply that Src kinase is essentially involved in sex steroid-induced non-genomic modulation of the spine density and morphology. These results also suggest that rapid effects of exogenously applied androgen and estrogen can occur in steroid-depleted conditions, including “acute” hippocampal slices and the hippocampus of gonadectomized animals.

  5. Dramatic improvement in genome assembly achieved using doubled-haploid genomes.

    Science.gov (United States)

    Zhang, Hong; Tan, Engkong; Suzuki, Yutaka; Hirose, Yusuke; Kinoshita, Shigeharu; Okano, Hideyuki; Kudoh, Jun; Shimizu, Atsushi; Saito, Kazuyoshi; Watabe, Shugo; Asakawa, Shuichi

    2014-10-27

    Improvement in de novo assembly of large genomes is still to be desired. Here, we improved draft genome sequence quality by employing doubled-haploid individuals. We sequenced wildtype and doubled-haploid Takifugu rubripes genomes, under the same conditions, using the Illumina platform and assembled contigs with SOAPdenovo2. We observed 5.4-fold and 2.6-fold improvement in the sizes of the N50 contig and scaffold of doubled-haploid individuals, respectively, compared to the wildtype, indicating that the use of a doubled-haploid genome aids in accurate genome analysis.

  6. Genome-Wide Association Studies Suggest Limited Immune Gene Enrichment in Schizophrenia Compared to 5 Autoimmune Diseases

    DEFF Research Database (Denmark)

    Pouget, Jennie G; Gonçalves, Vanessa F; Spain, Sarah L

    2016-01-01

    There has been intense debate over the immunological basis of schizophrenia, and the potential utility of adjunct immunotherapies. The major histocompatibility complex is consistently the most powerful region of association in genome-wide association studies (GWASs) of schizophrenia and has been...... in immune genes contributes to schizophrenia. We show that there is no enrichment of immune loci outside of the MHC region in the largest genetic study of schizophrenia conducted to date, in contrast to 5 diseases of known immune origin. Among 108 regions of the genome previously associated...

  7. Generation of an ICF Syndrome Model by Efficient Genome Editing of Human Induced Pluripotent Stem Cells Using the CRISPR System

    Directory of Open Access Journals (Sweden)

    Izuho Hatada

    2013-09-01

    Full Text Available Genome manipulation of human induced pluripotent stem (iPS cells is essential to achieve their full potential as tools for regenerative medicine. To date, however, gene targeting in human pluripotent stem cells (hPSCs has proven to be extremely difficult. Recently, an efficient genome manipulation technology using the RNA-guided DNase Cas9, the clustered regularly interspaced short palindromic repeats (CRISPR system, has been developed. Here we report the efficient generation of an iPS cell model for immunodeficiency, centromeric region instability, facial anomalies syndrome (ICF syndrome using the CRISPR system. We obtained iPS cells with mutations in both alleles of DNA methyltransferase 3B (DNMT3B in 63% of transfected clones. Our data suggest that the CRISPR system is highly efficient and useful for genome engineering of human iPS cells.

  8. Loss of Heterozygosity at an Unlinked Genomic Locus Is Responsible for the Phenotype of a Candida albicans sap4Δ sap5Δ sap6Δ Mutant ▿

    OpenAIRE

    Dunkel, Nico; Morschhäuser, Joachim

    2011-01-01

    The diploid genome of the pathogenic yeast Candida albicans exhibits a high degree of heterozygosity. Genomic alterations that result in a loss of heterozygosity at specific loci may affect phenotypes and confer a selective advantage under certain conditions. Such genomic rearrangements can also occur during the construction of C. albicans mutants and remain undetected. The SAP2 gene on chromosome R encodes a secreted aspartic protease that is induced and required for growth of C. albicans wh...

  9. Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering.

    Science.gov (United States)

    Nuñez, James K; Harrington, Lucas B; Doudna, Jennifer A

    2016-03-18

    The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems.

  10. Genomics-enabled analysis of the emergent disease cotton bacterial blight.

    Directory of Open Access Journals (Sweden)

    Anne Z Phillips

    2017-09-01

    Full Text Available Cotton bacterial blight (CBB, an important disease of (Gossypium hirsutum in the early 20th century, had been controlled by resistant germplasm for over half a century. Recently, CBB re-emerged as an agronomic problem in the United States. Here, we report analysis of cotton variety planting statistics that indicate a steady increase in the percentage of susceptible cotton varieties grown each year since 2009. Phylogenetic analysis revealed that strains from the current outbreak cluster with race 18 Xanthomonas citri pv. malvacearum (Xcm strains. Illumina based draft genomes were generated for thirteen Xcm isolates and analyzed along with 4 previously published Xcm genomes. These genomes encode 24 conserved and nine variable type three effectors. Strains in the race 18 clade contain 3 to 5 more effectors than other Xcm strains. SMRT sequencing of two geographically and temporally diverse strains of Xcm yielded circular chromosomes and accompanying plasmids. These genomes encode eight and thirteen distinct transcription activator-like effector genes. RNA-sequencing revealed 52 genes induced within two cotton cultivars by both tested Xcm strains. This gene list includes a homeologous pair of genes, with homology to the known susceptibility gene, MLO. In contrast, the two strains of Xcm induce different clade III SWEET sugar transporters. Subsequent genome wide analysis revealed patterns in the overall expression of homeologous gene pairs in cotton after inoculation by Xcm. These data reveal important insights into the Xcm-G. hirsutum disease complex and strategies for future development of resistant cultivars.

  11. Construction and Analysis of Siberian Tiger Bacterial Artificial Chromosome Library with Approximately 6.5-Fold Genome Equivalent Coverage

    Science.gov (United States)

    Liu, Changqing; Bai, Chunyu; Guo, Yu; Liu, Dan; Lu, Taofeng; Li, Xiangchen; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun

    2014-01-01

    Bacterial artificial chromosome (BAC) libraries are extremely valuable for the genome-wide genetic dissection of complex organisms. The Siberian tiger, one of the most well-known wild primitive carnivores in China, is an endangered animal. In order to promote research on its genome, a high-redundancy BAC library of the Siberian tiger was constructed and characterized. The library is divided into two sub-libraries prepared from blood cells and two sub-libraries prepared from fibroblasts. This BAC library contains 153,600 individually archived clones; for PCR-based screening of the library, BACs were placed into 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 116.5 kb, representing approximately 6.46 genome equivalents of the haploid genome and affording a 98.86% statistical probability of obtaining at least one clone containing a unique DNA sequence. Screening the library with 19 microsatellite markers and a SRY sequence revealed that each of these markers were present in the library; the average number of positive clones per marker was 6.74 (range 2 to 12), consistent with 6.46 coverage of the tiger genome. Additionally, we identified 72 microsatellite markers that could potentially be used as genetic markers. This BAC library will serve as a valuable resource for physical mapping, comparative genomic study and large-scale genome sequencing in the tiger. PMID:24608928

  12. Two low coverage bird genomes and a comparison of reference-guided versus de novo genome assemblies.

    Science.gov (United States)

    Card, Daren C; Schield, Drew R; Reyes-Velasco, Jacobo; Fujita, Matthew K; Andrew, Audra L; Oyler-McCance, Sara J; Fike, Jennifer A; Tomback, Diana F; Ruggiero, Robert P; Castoe, Todd A

    2014-01-01

    As a greater number and diversity of high-quality vertebrate reference genomes become available, it is increasingly feasible to use these references to guide new draft assemblies for related species. Reference-guided assembly approaches may substantially increase the contiguity and completeness of a new genome using only low levels of genome coverage that might otherwise be insufficient for de novo genome assembly. We used low-coverage (∼3.5-5.5x) Illumina paired-end sequencing to assemble draft genomes of two bird species (the Gunnison Sage-Grouse, Centrocercus minimus, and the Clark's Nutcracker, Nucifraga columbiana). We used these data to estimate de novo genome assemblies and reference-guided assemblies, and compared the information content and completeness of these assemblies by comparing CEGMA gene set representation, repeat element content, simple sequence repeat content, and GC isochore structure among assemblies. Our results demonstrate that even lower-coverage genome sequencing projects are capable of producing informative and useful genomic resources, particularly through the use of reference-guided assemblies.

  13. Two low coverage bird genomes and a comparison of reference-guided versus de novo genome assemblies

    Science.gov (United States)

    Card, Daren C.; Schield, Drew R.; Reyes-Velasco, Jacobo; Fujita, Matthre K.; Andrew, Audra L.; Oyler-McCance, Sara J.; Fike, Jennifer A.; Tomback, Diana F.; Ruggiero, Robert P.; Castoe, Todd A.

    2014-01-01

    As a greater number and diversity of high-quality vertebrate reference genomes become available, it is increasingly feasible to use these references to guide new draft assemblies for related species. Reference-guided assembly approaches may substantially increase the contiguity and completeness of a new genome using only low levels of genome coverage that might otherwise be insufficient for de novo genome assembly. We used low-coverage (~3.55.5x) Illumina paired-end sequencing to assemble draft genomes of two bird species (the Gunnison Sage-Grouse, Centrocercus minimus, and the Clark's Nutcracker, Nucifraga columbiana). We used these data to estimate de novo genome assemblies and reference-guided assemblies, and compared the information content and completeness of these assemblies by comparing CEGMA gene set representation, repeat element content, simple sequence repeat content, and GC isochore structure among assemblies. Our results demonstrate that even lower-coverage genome sequencing projects are capable of producing informative and useful genomic resources, particularly through the use of reference-guided assemblies.

  14. Phosphate steering by Flap Endonuclease 1 promotes 5′-flap specificity and incision to prevent genome instability

    KAUST Repository

    Tsutakawa, Susan E.

    2017-06-27

    DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5\\'-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5\\'-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 5\\'polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via phosphate steering\\', basic residues energetically steer an inverted ss 5\\'-flap through a gateway over FEN1\\'s active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA) repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 5\\'-flap specificity and catalysis, preventing genomic instability.

  15. Cloning of Bovine herpesvirus type 1 and type 5 as infectious bacterial artifical chromosomes

    Directory of Open Access Journals (Sweden)

    Ackermann Mathias

    2009-10-01

    Full Text Available Abstract Background Bovine herpesviruses type 1 (BoHV1 and type 5 (BoHV5 are two closely related pathogens of cattle. The identity of the two viruses on the amino acid level averages 82%. Despite their high antigenetic similarities the two pathogens induce distinctive clinical signs. BoHV1 causes respiratory and genital tract infections while BoHV5 leads to severe encephalitis in calves. Findings The viral genomes of BoHV1 and BoHV5 were cloned as infectious bacterial artificial chromosomes (BACs. First, recombinant viruses carrying the genetic elements for propagation in bacteria were generated. Second, DNA from these recombinant viruses were transferred into prokaryotic cells. Third, DNA from these bacteria were transferred into eukaryotic cells. Progeny viruses from BAC transfections showed similar kinetics as their corresponding wild types. Conclusion The two viral genomes of BoHV1 and BoHV5 cloned as BACs are accessible to the tools of bacterial genetics. The ability to easily manipulate the viral genomes on a molecular level in future experiments will lead to a better understanding of the difference in pathogenesis induced by these two closely related bovine herpesviruses.

  16. Genomic instability and radiation

    Energy Technology Data Exchange (ETDEWEB)

    Little, John B [Harvard School of Public Health, Boston, MA 02115 (United States)

    2003-06-01

    Genomic instability is a hallmark of cancer cells, and is thought to be involved in the process of carcinogenesis. Indeed, a number of rare genetic disorders associated with a predisposition to cancer are characterised by genomic instability occurring in somatic cells. Of particular interest is the observation that transmissible instability can be induced in somatic cells from normal individuals by exposure to ionising radiation, leading to a persistent enhancement in the rate at which mutations and chromosomal aberrations arise in the progeny of the irradiated cells after many generations of replication. If such induced instability is involved in radiation carcinogenesis, it would imply that the initial carcinogenic event may not be a rare mutation occurring in a specific gene or set of genes. Rather, radiation may induce a process of instability in many cells in a population, enhancing the rate at which the multiple gene mutations necessary for the development of cancer may arise in a given cell lineage. Furthermore, radiation could act at any stage in the development of cancer by facilitating the accumulation of the remaining genetic events required to produce a fully malignant tumour. The experimental evidence for such induced instability is reviewed. (review)

  17. Genomic instability and radiation

    International Nuclear Information System (INIS)

    Little, John B

    2003-01-01

    Genomic instability is a hallmark of cancer cells, and is thought to be involved in the process of carcinogenesis. Indeed, a number of rare genetic disorders associated with a predisposition to cancer are characterised by genomic instability occurring in somatic cells. Of particular interest is the observation that transmissible instability can be induced in somatic cells from normal individuals by exposure to ionising radiation, leading to a persistent enhancement in the rate at which mutations and chromosomal aberrations arise in the progeny of the irradiated cells after many generations of replication. If such induced instability is involved in radiation carcinogenesis, it would imply that the initial carcinogenic event may not be a rare mutation occurring in a specific gene or set of genes. Rather, radiation may induce a process of instability in many cells in a population, enhancing the rate at which the multiple gene mutations necessary for the development of cancer may arise in a given cell lineage. Furthermore, radiation could act at any stage in the development of cancer by facilitating the accumulation of the remaining genetic events required to produce a fully malignant tumour. The experimental evidence for such induced instability is reviewed. (review)

  18. Draft genome sequence of a GES-5-producing Serratia marcescens isolated in southern Brazil.

    Science.gov (United States)

    Nodari, Carolina Silva; Siebert, Marina; Matte, Ursula da Silveira; Barth, Afonso Luís

    Serratia marcescens is a Gram-negative rod intrinsically resistant to polymyxins and usually associated with wound, respiratory and urinary tract infections. The whole genome of the first GES-5-producing S. marcescens isolated from a Brazilian patient was sequenced using Ion Torrent PGM System. Besides bla GES-5 , we were able to identify genes encoding for other β-lactamases, for aminoglycoside modifying enzymes and for an efflux pump to tetracyclines. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  19. Inhibition of cisplatin-induced vomiting by selective 5-hydroxytryptamine M-receptor antagonism.

    OpenAIRE

    Miner, W. D.; Sanger, G. J.

    1986-01-01

    MDL 72222, the selective 5-hydroxytryptamine (5-HT) M-receptor antagonist, prevented or reduced cisplatin-induced emesis in ferrets. It is suggested that cisplatin-induced, and possibly other cytotoxic drug-induced vomiting may involve a 5-HT M-receptor mechanism.

  20. Cell-type-specific genome editing with a microRNA-responsive CRISPR–Cas9 switch

    Science.gov (United States)

    Hirosawa, Moe; Fujita, Yoshihiko; Parr, Callum J. C.; Hayashi, Karin; Kashida, Shunnichi; Hotta, Akitsu; Woltjen, Knut

    2017-01-01

    Abstract The CRISPR–Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR–Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information. PMID:28525578

  1. Suicidal function of DNA methylation in age-related genome disintegration.

    Science.gov (United States)

    Mazin, Alexander L

    2009-10-01

    This article is dedicated to the 60th anniversary of 5-methylcytosine discovery in DNA. Cytosine methylation can affect genetic and epigenetic processes, works as a part of the genome-defense system and has mutagenic activity; however, the biological functions of this enzymatic modification are not well understood. This review will put forward the hypothesis that the host-defense role of DNA methylation in silencing and mutational destroying of retroviruses and other intragenomic parasites was extended during evolution to most host genes that have to be inactivated in differentiated somatic cells, where it acquired a new function in age-related self-destruction of the genome. The proposed model considers DNA methylation as the generator of 5mC>T transitions that induce 40-70% of all spontaneous somatic mutations of the multiple classes at CpG and CpNpG sites and flanking nucleotides in the p53, FIX, hprt, gpt human genes and some transgenes. The accumulation of 5mC-dependent mutations explains: global changes in the structure of the vertebrate genome throughout evolution; the loss of most 5mC from the DNA of various species over their lifespan and the Hayflick limit of normal cells; the polymorphism of methylation sites, including asymmetric mCpNpN sites; cyclical changes of methylation and demethylation in genes. The suicidal function of methylation may be a special genetic mechanism for increasing DNA damage and the programmed genome disintegration responsible for cell apoptosis and organism aging and death.

  2. Sequencing and Analysis of the Pseudomonas fluorescens GcM5-1A Genome: A Pathogen Living in the Surface Coat of Bursaphelenchus xylophilus.

    Directory of Open Access Journals (Sweden)

    Kai Feng

    Full Text Available It is known that several bacteria are adherent to the surface coat of pine wood nematode (Bursaphelenchus xylophilus, but their function and role in the pathogenesis of pine wilt disease remains debatable. The Pseudomonas fluorescens GcM5-1A is a bacterium isolated from the surface coat of pine wood nematodes. In previous studies, GcM5-1A was evident in connection with the pathogenicity of pine wilt disease. In this study, we report the de novo sequencing of the GcM5-1A genome. A 600-Mb collection of high-quality reads was obtained and assembled into sequence contigs spanning a 6.01-Mb length. Sequence annotation predicted 5,413 open reading frames, of which 2,988 were homologous to genes in the other four sequenced P. fluorescens isolates (SBW25, WH6, Pf0-1 and Pf-5 and 1,137 were unique to GcM5-1A. Phylogenetic studies and genome comparison revealed that GcM5-1A is more closely related to SBW25 and WH6 isolates than to Pf0-1 and Pf-5 isolates. Towards study of pathogenesis, we identified 79 candidate virulence factors in the genome of GcM5-1A, including the Alg, Fl, Waa gene families, and genes coding the major pathogenic protein fliC. In addition, genes for a complete T3SS system were identified in the genome of GcM5-1A. Such systems have proved to play a critical role in subverting and colonizing the host organisms of many gram-negative pathogenic bacteria. Although the functions of the candidate virulence factors need yet to be deciphered experimentally, the availability of this genome provides a basic platform to obtain informative clues to be addressed in future studies by the pine wilt disease research community.

  3. Figure 5 from Integrative Genomics Viewer: Visualizing Big Data | Office of Cancer Genomics

    Science.gov (United States)

    Split-Screen View. The split-screen view is useful for exploring relationships of genomic features that are independent of chromosomal location. Color is used here to indicate mate pairs that map to different chromosomes, chromosomes 1 and 6, suggesting a translocation event. Adapted from Figure 8; Thorvaldsdottir H et al. 2012

  4. Efficient Multiple Genome Modifications Induced by the crRNAs, tracrRNA and Cas9 Protein Complex in Zebrafish

    Science.gov (United States)

    Ohga, Rie; Ota, Satoshi; Kawahara, Atsuo

    2015-01-01

    The type II clustered regularly interspaced short palindromic repeats (CRISPR) associated with Cas9 endonuclease (CRISPR/Cas9) has become a powerful genetic tool for understanding the function of a gene of interest. In zebrafish, the injection of Cas9 mRNA and guide-RNA (gRNA), which are prepared using an in vitro transcription system, efficiently induce DNA double-strand breaks (DSBs) at the targeted genomic locus. Because gRNA was originally constructed by fusing two short RNAs CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), we examined the effect of synthetic crRNAs and tracrRNA with Cas9 mRNA or Cas9 protein on the genome editing activity. We previously reported that the disruption of tyrosinase (tyr) by tyr-gRNA/Cas9 mRNA causes a retinal pigment defect, whereas the disruption of spns2 by spns2-gRNA1/Cas9 mRNA leads to a cardiac progenitor migration defect in zebrafish. Here, we found that the injection of spns2-crRNA1, tyr-crRNA and tracrRNA with Cas9 mRNA or Cas9 protein simultaneously caused a migration defect in cardiac progenitors and a pigment defect in retinal epithelial cells. A time course analysis demonstrated that the injection of crRNAs and tracrRNA with Cas9 protein rapidly induced genome modifications compared with the injection of crRNAs and tracrRNA with Cas9 mRNA. We further show that the crRNA-tracrRNA-Cas9 protein complex is functional for the visualization of endogenous gene expression; therefore, this is a very powerful, ready-to-use system in zebrafish. PMID:26010089

  5. Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using boric acid-functionalized nano-microsphere fluorescent probes.

    Science.gov (United States)

    Chen, Hua-Yan; Wei, Jing-Ru; Pan, Jiong-Xiu; Zhang, Wei; Dang, Fu-Quan; Zhang, Zhi-Qi; Zhang, Jing

    2017-05-15

    5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 β-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared with other fluorescence detection methods, this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Construction and Analysis of Siberian Tiger Bacterial Artificial Chromosome Library with Approximately 6.5-Fold Genome Equivalent Coverage

    Directory of Open Access Journals (Sweden)

    Changqing Liu

    2014-03-01

    Full Text Available Bacterial artificial chromosome (BAC libraries are extremely valuable for the genome-wide genetic dissection of complex organisms. The Siberian tiger, one of the most well-known wild primitive carnivores in China, is an endangered animal. In order to promote research on its genome, a high-redundancy BAC library of the Siberian tiger was constructed and characterized. The library is divided into two sub-libraries prepared from blood cells and two sub-libraries prepared from fibroblasts. This BAC library contains 153,600 individually archived clones; for PCR-based screening of the library, BACs were placed into 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 116.5 kb, representing approximately 6.46 genome equivalents of the haploid genome and affording a 98.86% statistical probability of obtaining at least one clone containing a unique DNA sequence. Screening the library with 19 microsatellite markers and a SRY sequence revealed that each of these markers were present in the library; the average number of positive clones per marker was 6.74 (range 2 to 12, consistent with 6.46 coverage of the tiger genome. Additionally, we identified 72 microsatellite markers that could potentially be used as genetic markers. This BAC library will serve as a valuable resource for physical mapping, comparative genomic study and large-scale genome sequencing in the tiger.

  7. Cadmium-induced genomic instability in Arabidopsis: Molecular toxicological biomarkers for early diagnosis of cadmium stress.

    Science.gov (United States)

    Wang, Hetong; He, Lei; Song, Jie; Cui, Weina; Zhang, Yanzhao; Jia, Chunyun; Francis, Dennis; Rogers, Hilary J; Sun, Lizong; Tai, Peidong; Hui, Xiujuan; Yang, Yuesuo; Liu, Wan

    2016-05-01

    Microsatellite instability (MSI) analysis, random-amplified polymorphic DNA (RAPD), and methylation-sensitive arbitrarily primed PCR (MSAP-PCR) are methods to evaluate the toxicity of environmental pollutants in stress-treated plants and human cancer cells. Here, we evaluate these techniques to screen for genetic and epigenetic alterations of Arabidopsis plantlets exposed to 0-5.0 mg L(-1) cadmium (Cd) for 15 d. There was a substantial increase in RAPD polymorphism of 24.5, and in genomic methylation polymorphism of 30.5-34.5 at CpG and of 14.5-20 at CHG sites under Cd stress of 5.0 mg L(-1) by RAPD and of 0.25-5.0 mg L(-1) by MSAP-PCR, respectively. However, only a tiny increase of 1.5 loci by RAPD occurred under Cd stress of 4.0 mg L(-1), and an additional high dose (8.0 mg L(-1)) resulted in one repeat by MSI analysis. MSAP-PCR detected the most significant epigenetic modifications in plantlets exposed to Cd stress, and the patterns of hypermethylation and polymorphisms were consistent with inverted U-shaped dose responses. The presence of genomic methylation polymorphism in Cd-treated seedlings, prior to the onset of RAPD polymorphism, MSI and obvious growth effects, suggests that these altered DNA methylation loci are the most sensitive biomarkers for early diagnosis and risk assessment of genotoxic effects of Cd pollution in ecotoxicology. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Site-specific genome editing for correction of induced pluripotent stem cells derived from dominant dystrophic epidermolysis bullosa.

    Science.gov (United States)

    Shinkuma, Satoru; Guo, Zongyou; Christiano, Angela M

    2016-05-17

    Genome editing with engineered site-specific endonucleases involves nonhomologous end-joining, leading to reading frame disruption. The approach is applicable to dominant negative disorders, which can be treated simply by knocking out the mutant allele, while leaving the normal allele intact. We applied this strategy to dominant dystrophic epidermolysis bullosa (DDEB), which is caused by a dominant negative mutation in the COL7A1 gene encoding type VII collagen (COL7). We performed genome editing with TALENs and CRISPR/Cas9 targeting the mutation, c.8068_8084delinsGA. We then cotransfected Cas9 and guide RNA expression vectors expressed with GFP and DsRed, respectively, into induced pluripotent stem cells (iPSCs) generated from DDEB fibroblasts. After sorting, 90% of the iPSCs were edited, and we selected four gene-edited iPSC lines for further study. These iPSCs were differentiated into keratinocytes and fibroblasts secreting COL7. RT-PCR and Western blot analyses revealed gene-edited COL7 with frameshift mutations degraded at the protein level. In addition, we confirmed that the gene-edited truncated COL7 could neither associate with normal COL7 nor undergo triple helix formation. Our data establish the feasibility of mutation site-specific genome editing in dominant negative disorders.

  9. Genome-wide screen of DNA methylation changes induced by low dose X-ray radiation in mice.

    Directory of Open Access Journals (Sweden)

    Jingzi Wang

    Full Text Available Epigenetic mechanisms play a key role in non-targeted effects of radiation. The purpose of this study was to investigate global hypomethylation and promoter hypermethylation of particular genes induced by low dose radiation (LDR. Thirty male BALB/c mice were divided into 3 groups: control, acutely exposed (0.5 Gy X-rays, and chronic exposure for 10 days (0.05Gy/d×10d. High-performance liquid chromatography (HPLC and MeDIP-quantitative polymerase chain reaction (qPCR were used to study methylation profiles. DNMT1 and MBD2 expression was determined by qPCR and western blot assays. Methylation and expression of Rad23b and Ddit3 were determined by bisulfate sequencing primers (BSP and qPCR, respectively. The results show that LDR induced genomic hypomethylation in blood 2 h postirraditaion, but was not retained at 1-month. DNMT1 and MBD2 were downregulated in a tissue-specific manner but did not persist. Specific hypermethylation was observed for 811 regions in the group receiving chronic exposure, which covered almost all key biological processes as indicated by GO and KEGG pathway analysis. Eight hypermethylated genes (Rad23b, Tdg, Ccnd1, Ddit3, Llgl1, Rasl11a, Tbx2, Scl6a15 were verified by MeDIP-qPCR. Among them, Rad23b and Ddit3 gene displayed tissue-specific methylation and downregulation, which persisted for 1-month postirradiation. Thus, LDR induced global hypomethylation and tissue-specific promoter hypermethylation of particular genes. Promoter hypermethylation, rather than global hypomethylation, was relatively stable. Dysregulation of methylation might be correlated with down-regulation of DNMT1 and MBD2, but much better understanding the molecular mechanisms involved in this process will require further study.

  10. Study of genomic instability induced by low dose ionizing radiation

    International Nuclear Information System (INIS)

    Seoane, A.; Crudeli, C.; Dulout, F.

    2006-01-01

    The crews of commercial flights and services staff of radiology and radiotherapy from hospitals are exposed to low doses of ionizing radiation. Genomic instability includes those adverse effects observed in cells, several generations after the exposure occurred. The purpose of this study was to analyze the occurrence of genomic instability by very low doses of ionizing radiation [es

  11. Genome Analysis of Fimbriiglobus ruber SP5T, a Planctomycete with Confirmed Chitinolytic Capability.

    Science.gov (United States)

    Ravin, Nikolai V; Rakitin, Andrey L; Ivanova, Anastasia A; Beletsky, Alexey V; Kulichevskaya, Irina S; Mardanov, Andrey V; Dedysh, Svetlana N

    2018-04-01

    Members of the bacterial order Planctomycetales have often been observed in associations with Crustacea. The ability to degrade chitin, however, has never been reported for any of the cultured planctomycetes although utilization of N -acetylglucosamine (GlcNAc) as a sole carbon and nitrogen source is well recognized for these bacteria. Here, we demonstrate the chitinolytic capability of a member of the family Gemmataceae , Fimbriiglobus ruber SP5 T , which was isolated from a peat bog. As revealed by metatranscriptomic analysis of chitin-amended peat, the pool of 16S rRNA reads from F. ruber increased in response to chitin availability. Strain SP5 T displayed only weak growth on amorphous chitin as a sole source of carbon but grew well with chitin as a source of nitrogen. The genome of F. ruber SP5 T is 12.364 Mb in size and is the largest among all currently determined planctomycete genomes. It encodes several enzymes putatively involved in chitin degradation, including two chitinases affiliated with the glycoside hydrolase (GH) family GH18, GH20 family β- N -acetylglucosaminidase, and the complete set of enzymes required for utilization of GlcNAc. The gene encoding one of the predicted chitinases was expressed in Escherichia coli , and the endochitinase activity of the recombinant enzyme was confirmed. The genome also contains genes required for the assembly of type IV pili, which may be used to adhere to chitin and possibly other biopolymers. The ability to use chitin as a source of nitrogen is of special importance for planctomycetes that inhabit N-depleted ombrotrophic wetlands. IMPORTANCE Planctomycetes represent an important part of the microbial community in Sphagnum -dominated peatlands, but their potential functions in these ecosystems remain poorly understood. This study reports the presence of chitinolytic potential in one of the recently described peat-inhabiting members of the family Gemmataceae , Fimbriiglobus ruber SP5 T This planctomycete uses

  12. Molecular characterization of DnaJ 5 homologs in silkworm Bombyx mori and its expression during egg diapause.

    Science.gov (United States)

    Sirigineedi, Sasibhushan; Vijayagowri, Esvaran; Murthy, Geetha N; Rao, Guruprasada; Ponnuvel, Kangayam M

    2014-12-01

    A comparison of the cDNA sequences (1 056 bp) of Bombyx mori DnaJ 5 homolog with B. mori genome revealed that unlike in other Hsps, it has an intron of 234 bp. The DnaJ 5 homolog contains 351 amino acids, of which 70 contain the conserved DnaJ domain at the N-terminal end. This homolog of B. mori has all desirable functional domains similar to other insects, and the 13 different DnaJ homologs identified in B. mori genome were distributed on different chromosomes. The expressed sequence tag database analysis of Hsp40 gene expression revealed higher expression in wing disc followed by diapause-induced eggs. Microarray analysis revealed higher expression of DnaJ 5 homolog at 18th h after oviposition in diapause-induced eggs. Further validation of DnaJ 5 expression through qPCR in diapause-induced and nondiapause eggs at different time intervals revealed higher expression in diapause eggs at 18 and 24 h after oviposition, which coincided with the expression of Hsp70 as the Hsp 40 is its co-chaperone. This study thus provides an outline of the genome organization of Hsp40 gene, and its role in egg diapause induction in B. mori. © 2013 Institute of Zoology, Chinese Academy of Sciences.

  13. A Simple Method to Decode the Complete 18-5.8-28S rRNA Repeated Units of Green Algae by Genome Skimming.

    Science.gov (United States)

    Lin, Geng-Ming; Lai, Yu-Heng; Audira, Gilbert; Hsiao, Chung-Der

    2017-11-06

    Green algae, Chlorella ellipsoidea , Haematococcus pluvialis and Aegagropila linnaei (Phylum Chlorophyta) were simultaneously decoded by a genomic skimming approach within 18-5.8-28S rRNA region. Whole genomic DNAs were isolated from green algae and directly subjected to low coverage genome skimming sequencing. After de novo assembly and mapping, the size of complete 18-5.8-28S rRNA repeated units for three green algae were ranged from 5785 to 6028 bp, which showed high nucleotide diversity (π is around 0.5-0.6) within ITS1 and ITS2 (Internal Transcribed Spacer) regions. Previously, the evolutional diversity of algae has been difficult to decode due to the inability design universal primers that amplify specific marker genes across diverse algal species. In this study, our method provided a rapid and universal approach to decode the 18-5.8-28S rRNA repeat unit in three green algal species. In addition, the completely sequenced 18-5.8-28S rRNA repeated units provided a solid nuclear marker for phylogenetic and evolutionary analysis for green algae for the first time.

  14. Trans-generational radiation-induced chromosomal instability in the female enhances the action of chemical mutagens

    International Nuclear Information System (INIS)

    Camats, Nuria; Garcia, Francisca; Parrilla, Juan Jose; Calaf, Joaquim; Martin, Miguel; Caldes, Montserrat Garcia

    2008-01-01

    Genomic instability can be produced by ionising radiation, so-called radiation-induced genomic instability, and chemical mutagens. Radiation-induced genomic instability occurs in both germinal and somatic cells and also in the offspring of irradiated individuals, and it is characterised by genetic changes including chromosomal rearrangements. The majority of studies of trans-generational, radiation-induced genomic instability have been described in the male germ line, whereas the authors who have chosen the female as a model are scarce. The aim of this work is to find out the radiation-induced effects in the foetal offspring of X-ray-treated female rats and, at the same time, the possible impact of this radiation-induced genomic instability on the action of a chemical mutagen. In order to achieve both goals, the quantity and quality of chromosomal damage were analysed. In order to detect trans-generational genomic instability, a total of 4806 metaphases from foetal tissues from the foetal offspring of X-irradiated female rats (5 Gy, acute dose) were analysed. The study's results showed that there is radiation-induced genomic instability: the number of aberrant metaphases and the breaks per total metaphases studied increased and were found to be statistically significant (p ≤ 0.05), with regard to the control group. In order to identify how this trans-generational, radiation-induced chromosomal instability could influence the chromosomal behaviour of the offspring of irradiated rat females in front of a chemical agent (aphidicolin), a total of 2481 metaphases were studied. The observed results showed that there is an enhancement of the action of the chemical agent: chromosomal breaks per aberrant metaphases show significant differences (p ≤ 0.05) in the X-ray- and aphidicolin-treated group as regards the aphidicolin-treated group. In conclusion, our findings indicate that there is trans-generational, radiation-induced chromosomal instability in the foetal cells

  15. Trans-generational radiation-induced chromosomal instability in the female enhances the action of chemical mutagens

    Energy Technology Data Exchange (ETDEWEB)

    Camats, Nuria [Institut de Biotecnologia i Biomedicina (IBB), Universitat Autonoma de Barcelona, 08193 Barcelona (Spain); Departament de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, 08193 Barcelona (Spain); Garcia, Francisca [Institut de Biotecnologia i Biomedicina (IBB), Universitat Autonoma de Barcelona, 08193 Barcelona (Spain); Parrilla, Juan Jose [Servicio de Ginecologia y Obstetricia, Hospital Universitario Virgen de la Arrixaca, 30120 El Palmar, Murcia (Spain); Calaf, Joaquim [Servei de Ginecologia i Obstetricia, Hospital Universitari de la Santa Creu i Sant Pau, 08025 Barcelona (Spain); Martin, Miguel [Departament de Pediatria, d' Obstetricia i Ginecologia i de Medicina Preventiva, Universitat Autonoma de Barcelona, 08193 Barcelona (Spain); Caldes, Montserrat Garcia [Institut de Biotecnologia i Biomedicina (IBB), Universitat Autonoma de Barcelona, 08193 Barcelona (Spain); Departament de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, 08193 Barcelona (Spain)], E-mail: Montserrat.Garcia.Caldes@uab.es

    2008-04-02

    Genomic instability can be produced by ionising radiation, so-called radiation-induced genomic instability, and chemical mutagens. Radiation-induced genomic instability occurs in both germinal and somatic cells and also in the offspring of irradiated individuals, and it is characterised by genetic changes including chromosomal rearrangements. The majority of studies of trans-generational, radiation-induced genomic instability have been described in the male germ line, whereas the authors who have chosen the female as a model are scarce. The aim of this work is to find out the radiation-induced effects in the foetal offspring of X-ray-treated female rats and, at the same time, the possible impact of this radiation-induced genomic instability on the action of a chemical mutagen. In order to achieve both goals, the quantity and quality of chromosomal damage were analysed. In order to detect trans-generational genomic instability, a total of 4806 metaphases from foetal tissues from the foetal offspring of X-irradiated female rats (5 Gy, acute dose) were analysed. The study's results showed that there is radiation-induced genomic instability: the number of aberrant metaphases and the breaks per total metaphases studied increased and were found to be statistically significant (p {<=} 0.05), with regard to the control group. In order to identify how this trans-generational, radiation-induced chromosomal instability could influence the chromosomal behaviour of the offspring of irradiated rat females in front of a chemical agent (aphidicolin), a total of 2481 metaphases were studied. The observed results showed that there is an enhancement of the action of the chemical agent: chromosomal breaks per aberrant metaphases show significant differences (p {<=} 0.05) in the X-ray- and aphidicolin-treated group as regards the aphidicolin-treated group. In conclusion, our findings indicate that there is trans-generational, radiation-induced chromosomal instability in the foetal

  16. Phytozome Comparative Plant Genomics Portal

    Energy Technology Data Exchange (ETDEWEB)

    Goodstein, David; Batra, Sajeev; Carlson, Joseph; Hayes, Richard; Phillips, Jeremy; Shu, Shengqiang; Schmutz, Jeremy; Rokhsar, Daniel

    2014-09-09

    The Dept. of Energy Joint Genome Institute is a genomics user facility supporting DOE mission science in the areas of Bioenergy, Carbon Cycling, and Biogeochemistry. The Plant Program at the JGI applies genomic, analytical, computational and informatics platforms and methods to: 1. Understand and accelerate the improvement (domestication) of bioenergy crops 2. Characterize and moderate plant response to climate change 3. Use comparative genomics to identify constrained elements and infer gene function 4. Build high quality genomic resource platforms of JGI Plant Flagship genomes for functional and experimental work 5. Expand functional genomic resources for Plant Flagship genomes

  17. The role of radiation types and dose in induced genomic instability

    International Nuclear Information System (INIS)

    Kadhim Munira, A.

    2007-01-01

    Complete text of publication follows. Genomic Instability (GI) is defined as long-term alterations induced by low-dose exposure to a variety of genotoxic agents in mammalian cells that act to increase the 'apparent' spontaneous mutation frequency.GI is a hallmark of tumorigenic progression and is observed in the progeny of irradiated and bystander cells as the delayed and stochastic appearance of de novo chromosomal aberrations, gene mutations and delayed lethal mutations both in vitro and in vivo. It occurs at a frequency several orders of magnitude greater than would be expected for mutation in a single gene, implying that GI is a multigenic phenomenon. The expression of GI can be influenced by genotype, cell type and radiation quality; however the underlying mechanisms are not fully understood. While several studies have demonstrated GI induction by high and low LET radiation, our work on human and mouse primary cell systems has shown significant differences in the capacity to induce GI and the spectrum of alterations depending on LET. These differences might be attributed to differences in radiation track structure, radiation dose and radiation exposure regime (distribution of hit and un hit cells). In this presentation I shall review the role of radiation quality; describe the possible mechanisms underlining the observed differences between radiation type and present results of experiments demonstrating that the dose of low LET radiation might be the most significant factor in determining the role of radiation type in the induction of GI.

  18. A Portrait of Ribosomal DNA Contacts with Hi-C Reveals 5S and 45S rDNA Anchoring Points in the Folded Human Genome.

    Science.gov (United States)

    Yu, Shoukai; Lemos, Bernardo

    2016-12-31

    Ribosomal RNAs (rRNAs) account for >60% of all RNAs in eukaryotic cells and are encoded in the ribosomal DNA (rDNA) arrays. The rRNAs are produced from two sets of loci: the 5S rDNA array resides exclusively on human chromosome 1, whereas the 45S rDNA array resides on the short arm of five human acrocentric chromosomes. The 45S rDNA gives origin to the nucleolus, the nuclear organelle that is the site of ribosome biogenesis. Intriguingly, 5S and 45S rDNA arrays exhibit correlated copy number variation in lymphoblastoid cells (LCLs). Here we examined the genomic architecture and repeat content of the 5S and 45S rDNA arrays in multiple human genome assemblies (including PacBio MHAP assembly) and ascertained contacts between the rDNA arrays and the rest of the genome using Hi-C datasets from two human cell lines (erythroleukemia K562 and lymphoblastoid cells). Our analyses revealed that 5S and 45S arrays each have thousands of contacts in the folded genome, with rDNA-associated regions and genes dispersed across all chromosomes. The rDNA contact map displayed conserved and disparate features between two cell lines, and pointed to specific chromosomes, genomic regions, and genes with evidence of spatial proximity to the rDNA arrays; the data also showed a lack of direct physical interaction between the 5S and 45S rDNA arrays. Finally, the analysis identified an intriguing organization in the 5S array with Alu and 5S elements adjacent to one another and organized in opposite orientation along the array. Portraits of genome folding centered on the ribosomal DNA array could help understand the emergence of concerted variation, the control of 5S and 45S expression, as well as provide insights into an organelle that contributes to the spatial localization of human chromosomes during interphase. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  19. Mixtures of xenoestrogens disrupt estradiol-induced non-genomic signaling and downstream functions in pituitary cells.

    Science.gov (United States)

    Viñas, René; Watson, Cheryl S

    2013-03-26

    Our study examines the effects of xenoestrogen mixtures on estradiol-induced non-genomic signaling and associated functional responses. Bisphenol-A, used to manufacture plastic consumer products, and nonylphenol, a surfactant, are estrogenic by a variety of assays, including altering many intracellular signaling pathways; bisphenol-S is now used as a bisphenol-A substitute. All three compounds contaminate the environment globally. We previously showed that bisphenol-S, bisphenol-A, and nonylphenol alone rapidly activated several kinases at very low concentrations in the GH3/B6/F10 rat pituitary cell line. For each assay we compared the response of individual xenoestrogens at environmentally relevant concentrations (10-15 -10-7 M), to their mixture effects on 10-9 M estradiol-induced responses. We used a medium-throughput plate immunoassay to quantify phosphorylations of extracellular signal-regulated kinases (ERKs) and c-Jun-N-terminal kinases (JNKs). Cell numbers were assessed by crystal violet assay to compare the proliferative effects. Apoptosis was assessed by measuring caspase 8 and 9 activities via the release of the fluorescent product 7-amino-4-trifluoromethylcoumarin. Prolactin release was measured by radio-immunoassay after a 1 min exposure to all individual and combinations of estrogens. Individual xenoestrogens elicited phospho-activation of ERK in a non-monotonic dose- (fM-nM) and mostly oscillating time-dependent (2.5-60 min) manner. When multiple xenoestrogens were combined with nM estradiol, the physiologic estrogen's response was attenuated. Individual bisphenol compounds did not activate JNK, while nonylphenol did; however, the combination of two or three xenoestrogens with estradiol generated an enhanced non-monotonic JNK dose-response. Estradiol and all xenoestrogen compounds induced cell proliferation individually, while the mixtures of these compounds with estradiol suppressed proliferation below that of the vehicle control, suggesting a

  20. Genome chaos: survival strategy during crisis.

    Science.gov (United States)

    Liu, Guo; Stevens, Joshua B; Horne, Steven D; Abdallah, Batoul Y; Ye, Karen J; Bremer, Steven W; Ye, Christine J; Chen, David J; Heng, Henry H

    2014-01-01

    Genome chaos, a process of complex, rapid genome re-organization, results in the formation of chaotic genomes, which is followed by the potential to establish stable genomes. It was initially detected through cytogenetic analyses, and recently confirmed by whole-genome sequencing efforts which identified multiple subtypes including "chromothripsis", "chromoplexy", "chromoanasynthesis", and "chromoanagenesis". Although genome chaos occurs commonly in tumors, both the mechanism and detailed aspects of the process are unknown due to the inability of observing its evolution over time in clinical samples. Here, an experimental system to monitor the evolutionary process of genome chaos was developed to elucidate its mechanisms. Genome chaos occurs following exposure to chemotherapeutics with different mechanisms, which act collectively as stressors. Characterization of the karyotype and its dynamic changes prior to, during, and after induction of genome chaos demonstrates that chromosome fragmentation (C-Frag) occurs just prior to chaotic genome formation. Chaotic genomes seem to form by random rejoining of chromosomal fragments, in part through non-homologous end joining (NHEJ). Stress induced genome chaos results in increased karyotypic heterogeneity. Such increased evolutionary potential is demonstrated by the identification of increased transcriptome dynamics associated with high levels of karyotypic variance. In contrast to impacting on a limited number of cancer genes, re-organized genomes lead to new system dynamics essential for cancer evolution. Genome chaos acts as a mechanism of rapid, adaptive, genome-based evolution that plays an essential role in promoting rapid macroevolution of new genome-defined systems during crisis, which may explain some unwanted consequences of cancer treatment.

  1. Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9

    OpenAIRE

    Feng, Yan; Chen, Cheng; Han, Yuxiang; Chen, Zelin; Lu, Xiaochan; Liang, Fang; Li, Song; Qin, Wei; Lin, Shuo

    2016-01-01

    The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5′-NGG-3′ protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutation...

  2. Autophagy regulates chlorpyrifos-induced apoptosis in SH-SY5Y cells

    International Nuclear Information System (INIS)

    Park, Jae Hyeon; Lee, Jeong Eun; Shin, In Chul; Koh, Hyun Chul

    2013-01-01

    Recent studies have shown that up-regulation of autophagy may be a tractable therapeutic intervention for clearing disease-causing proteins, including α-synuclein, ubiquitin, and other misfolded or aggregated proteins in pesticide-induced neurodegeneration. In a previous study, we reported that chlorpyrifos (CPF)-induced mitochondria-dependent apoptosis is mediated through reactive oxygen species in SH-SY5Y cells. In this study, we explored a novel pharmacotherapeutic approach to prevent CPF neurotoxicity involving the regulation of autophagy. We investigated the modulation of CPF-induced apoptosis according to autophagy regulation. We found that CPF induced apoptosis in SH-SY5Y cells, as demonstrated by the activation of caspase-3 and nuclear condensation. In addition, we observed that cells treated with CPF underwent autophagic cell death by monitoring the expression of LC3-II and p62. Pretreatment with the autophagy inducer rapamycin significantly enhanced the cell viability of CPF-exposed cells, and the enhancement of cell viability was partially due to alleviation of CPF-induced apoptosis via a decrease in levels of cleaved caspase-3. Specifically, rapamycin pretreatment decreased Bax and increased Bcl-2 expression in mitochondria. In addition, rapamycin significantly decreased cytochrome c release in from mitochondria into the cytosol. However, pretreatment of cells with the autophagy inhibitor, 3-methyladenine (3MA), remarkably increased CPF toxicity in these cells; this with correlated with increased expression of Bax and decreased expression of Bcl-2 in mitochondria. Our results suggest that CPF-induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against CPF-induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders. - Highlights: ► Chlorpyrifos (CPF) is cytotoxic to SH-SY5Y cells ► CPF-induced cytotoxicity is mediated by

  3. Autophagy regulates chlorpyrifos-induced apoptosis in SH-SY5Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jae Hyeon [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of); Lee, Jeong Eun [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Shin, In Chul [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Koh, Hyun Chul, E-mail: hckoh@hanyang.ac.kr [Department of Pharmacology, College of Medicine, Hanyang University (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of)

    2013-04-01

    Recent studies have shown that up-regulation of autophagy may be a tractable therapeutic intervention for clearing disease-causing proteins, including α-synuclein, ubiquitin, and other misfolded or aggregated proteins in pesticide-induced neurodegeneration. In a previous study, we reported that chlorpyrifos (CPF)-induced mitochondria-dependent apoptosis is mediated through reactive oxygen species in SH-SY5Y cells. In this study, we explored a novel pharmacotherapeutic approach to prevent CPF neurotoxicity involving the regulation of autophagy. We investigated the modulation of CPF-induced apoptosis according to autophagy regulation. We found that CPF induced apoptosis in SH-SY5Y cells, as demonstrated by the activation of caspase-3 and nuclear condensation. In addition, we observed that cells treated with CPF underwent autophagic cell death by monitoring the expression of LC3-II and p62. Pretreatment with the autophagy inducer rapamycin significantly enhanced the cell viability of CPF-exposed cells, and the enhancement of cell viability was partially due to alleviation of CPF-induced apoptosis via a decrease in levels of cleaved caspase-3. Specifically, rapamycin pretreatment decreased Bax and increased Bcl-2 expression in mitochondria. In addition, rapamycin significantly decreased cytochrome c release in from mitochondria into the cytosol. However, pretreatment of cells with the autophagy inhibitor, 3-methyladenine (3MA), remarkably increased CPF toxicity in these cells; this with correlated with increased expression of Bax and decreased expression of Bcl-2 in mitochondria. Our results suggest that CPF-induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against CPF-induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders. - Highlights: ► Chlorpyrifos (CPF) is cytotoxic to SH-SY5Y cells ► CPF-induced cytotoxicity is mediated by

  4. Genomic and proteomic analysis of soybean heritable variations induced by space flight

    Institute of Scientific and Technical Information of China (English)

    HE Jie; GAO Yong; SUN Ye-qing

    2009-01-01

    To analyze the biological effects of space environment, the diversity of genomic DNA between the space flight soybean 194(4126) with phenotype of good yield and good fruit quality induced by space flight and the soybean with ground control was studied by amplified fragment length polymorphism (AFLP) method, and the polymorphism of space flight soybean 194(4126) was 3.56%. The differences of protein expression of seeds and leaves between the two kinds of soybeans were analysed by two-dimensional electrophoresis, PDQuest software and MALDI-TOF mass spectrometry. Results show that the loss and decrease of protein expression in 194(4126) soybean are subjected to the space fight of seeds, and three special proteins including Dehydrin, MAT1 and ceQORH are identified. It is concluded that the space environment changes the phenotype and geno-type of soybeans due to the space flight of seeds.

  5. Determination of the number of copies of genes coding for 5s-rRNA and tRNA in the genomes of 43 species of wheat and Aegilops

    International Nuclear Information System (INIS)

    Vakhitov, V.A.; Gimalov, F.R.; Nikonorov, Yu.M.

    1986-01-01

    The number of 5s-rRNA and tRNA genes has been studied in 43 species of wheat and Aegilops differing in ploidy level, genomic composition and origin. It has been demonstrated that the repeatability of the 5s-rRNA and tRNA genes increases in wheat with increasing ploidy level, but not in proportion to the genome size. In Aegilops, in distinction from wheat, the relative as well as absolute number of 5s-RNA genes increases with increasing ploidy level. The proportion of the sequences coding for tRNA in the dipoloid and polyploid Aegilops species is practically similar, while the number of tRNA genes increases almost 2-3 times with increasing ploidy level. Large variability has been recorded between the species with similar genomic composition and ploidy level in respect of the number of the 5s-rRNA and tRNA genes. It has been demonstrated that integration of the initial genomes of the amphidiploids is accompanied by elimination of a particular part of these genomes. It has been concluded that the mechanisms of establishment and evolution of genomes in the intra- and intergeneric allopolyploids are not identical

  6. Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch.

    Science.gov (United States)

    Hirosawa, Moe; Fujita, Yoshihiko; Parr, Callum J C; Hayashi, Karin; Kashida, Shunnichi; Hotta, Akitsu; Woltjen, Knut; Saito, Hirohide

    2017-07-27

    The CRISPR-Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR-Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Biogeography and environmental genomics of the Roseobacter-affiliated pelagic CHAB-I-5 lineage

    DEFF Research Database (Denmark)

    Billerbeck, Sara; Wemheuer, Bernd; Voget, Sonja

    2016-01-01

    The identification and functional characterization of microbial communities remains a prevailing topic in microbial oceanography as information on environmentally relevant pelagic prokaryotes is still limited. The Roseobacter group, an abundant lineage of marine Alphaproteobacteria, can constitute...... large proportions of the bacterioplankton. Roseobacters also occur associated with eukaryotic organisms and possess streamlined as well as larger genomes from 2.2 to >5 Mpb. Here, we show that one pelagic cluster of this group, CHAB-I-5, occurs globally from tropical to polar regions and accounts for up...

  8. Genome-wide Analyses Identify KIF5A as a Novel ALS Gene.

    Science.gov (United States)

    Nicolas, Aude; Kenna, Kevin P; Renton, Alan E; Ticozzi, Nicola; Faghri, Faraz; Chia, Ruth; Dominov, Janice A; Kenna, Brendan J; Nalls, Mike A; Keagle, Pamela; Rivera, Alberto M; van Rheenen, Wouter; Murphy, Natalie A; van Vugt, Joke J F A; Geiger, Joshua T; Van der Spek, Rick A; Pliner, Hannah A; Shankaracharya; Smith, Bradley N; Marangi, Giuseppe; Topp, Simon D; Abramzon, Yevgeniya; Gkazi, Athina Soragia; Eicher, John D; Kenna, Aoife; Mora, Gabriele; Calvo, Andrea; Mazzini, Letizia; Riva, Nilo; Mandrioli, Jessica; Caponnetto, Claudia; Battistini, Stefania; Volanti, Paolo; La Bella, Vincenzo; Conforti, Francesca L; Borghero, Giuseppe; Messina, Sonia; Simone, Isabella L; Trojsi, Francesca; Salvi, Fabrizio; Logullo, Francesco O; D'Alfonso, Sandra; Corrado, Lucia; Capasso, Margherita; Ferrucci, Luigi; Moreno, Cristiane de Araujo Martins; Kamalakaran, Sitharthan; Goldstein, David B; Gitler, Aaron D; Harris, Tim; Myers, Richard M; Phatnani, Hemali; Musunuri, Rajeeva Lochan; Evani, Uday Shankar; Abhyankar, Avinash; Zody, Michael C; Kaye, Julia; Finkbeiner, Steven; Wyman, Stacia K; LeNail, Alex; Lima, Leandro; Fraenkel, Ernest; Svendsen, Clive N; Thompson, Leslie M; Van Eyk, Jennifer E; Berry, James D; Miller, Timothy M; Kolb, Stephen J; Cudkowicz, Merit; Baxi, Emily; Benatar, Michael; Taylor, J Paul; Rampersaud, Evadnie; Wu, Gang; Wuu, Joanne; Lauria, Giuseppe; Verde, Federico; Fogh, Isabella; Tiloca, Cinzia; Comi, Giacomo P; Sorarù, Gianni; Cereda, Cristina; Corcia, Philippe; Laaksovirta, Hannu; Myllykangas, Liisa; Jansson, Lilja; Valori, Miko; Ealing, John; Hamdalla, Hisham; Rollinson, Sara; Pickering-Brown, Stuart; Orrell, Richard W; Sidle, Katie C; Malaspina, Andrea; Hardy, John; Singleton, Andrew B; Johnson, Janel O; Arepalli, Sampath; Sapp, Peter C; McKenna-Yasek, Diane; Polak, Meraida; Asress, Seneshaw; Al-Sarraj, Safa; King, Andrew; Troakes, Claire; Vance, Caroline; de Belleroche, Jacqueline; Baas, Frank; Ten Asbroek, Anneloor L M A; Muñoz-Blanco, José Luis; Hernandez, Dena G; Ding, Jinhui; Gibbs, J Raphael; Scholz, Sonja W; Floeter, Mary Kay; Campbell, Roy H; Landi, Francesco; Bowser, Robert; Pulst, Stefan M; Ravits, John M; MacGowan, Daniel J L; Kirby, Janine; Pioro, Erik P; Pamphlett, Roger; Broach, James; Gerhard, Glenn; Dunckley, Travis L; Brady, Christopher B; Kowall, Neil W; Troncoso, Juan C; Le Ber, Isabelle; Mouzat, Kevin; Lumbroso, Serge; Heiman-Patterson, Terry D; Kamel, Freya; Van Den Bosch, Ludo; Baloh, Robert H; Strom, Tim M; Meitinger, Thomas; Shatunov, Aleksey; Van Eijk, Kristel R; de Carvalho, Mamede; Kooyman, Maarten; Middelkoop, Bas; Moisse, Matthieu; McLaughlin, Russell L; Van Es, Michael A; Weber, Markus; Boylan, Kevin B; Van Blitterswijk, Marka; Rademakers, Rosa; Morrison, Karen E; Basak, A Nazli; Mora, Jesús S; Drory, Vivian E; Shaw, Pamela J; Turner, Martin R; Talbot, Kevin; Hardiman, Orla; Williams, Kelly L; Fifita, Jennifer A; Nicholson, Garth A; Blair, Ian P; Rouleau, Guy A; Esteban-Pérez, Jesús; García-Redondo, Alberto; Al-Chalabi, Ammar; Rogaeva, Ekaterina; Zinman, Lorne; Ostrow, Lyle W; Maragakis, Nicholas J; Rothstein, Jeffrey D; Simmons, Zachary; Cooper-Knock, Johnathan; Brice, Alexis; Goutman, Stephen A; Feldman, Eva L; Gibson, Summer B; Taroni, Franco; Ratti, Antonia; Gellera, Cinzia; Van Damme, Philip; Robberecht, Wim; Fratta, Pietro; Sabatelli, Mario; Lunetta, Christian; Ludolph, Albert C; Andersen, Peter M; Weishaupt, Jochen H; Camu, William; Trojanowski, John Q; Van Deerlin, Vivianna M; Brown, Robert H; van den Berg, Leonard H; Veldink, Jan H; Harms, Matthew B; Glass, Jonathan D; Stone, David J; Tienari, Pentti; Silani, Vincenzo; Chiò, Adriano; Shaw, Christopher E; Traynor, Bryan J; Landers, John E

    2018-03-21

    To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Pre-exposure to 50 Hz magnetic fields modifies menadione-induced genotoxic effects in human SH-SY5Y neuroblastoma cells.

    Directory of Open Access Journals (Sweden)

    Jukka Luukkonen

    Full Text Available BACKGROUND: Extremely low frequency (ELF magnetic fields (MF are generated by power lines and various electric appliances. They have been classified as possibly carcinogenic by the International Agency for Research on Cancer, but a mechanistic explanation for carcinogenic effects is lacking. A previous study in our laboratory showed that pre-exposure to ELF MF altered cancer-relevant cellular responses (cell cycle arrest, apoptosis to menadione-induced DNA damage, but it did not include endpoints measuring actual genetic damage. In the present study, we examined whether pre-exposure to ELF MF affects chemically induced DNA damage level, DNA repair rate, or micronucleus frequency in human SH-SY5Y neuroblastoma cells. METHODOLOGY/PRINCIPAL FINDINGS: Exposure to 50 Hz MF was conducted at 100 µT for 24 hours, followed by chemical exposure for 3 hours. The chemicals used for inducing DNA damage and subsequent micronucleus formation were menadione and methyl methanesulphonate (MMS. Pre-treatment with MF enhanced menadione-induced DNA damage, DNA repair rate, and micronucleus formation in human SH-SY5Y neuroblastoma cells. Although the results with MMS indicated similar effects, the differences were not statistically significant. No effects were observed after MF exposure alone. CONCLUSIONS: The results confirm our previous findings showing that pre-exposure to MFs as low as 100 µT alters cellular responses to menadione, and show that increased genotoxicity results from such interaction. The present findings also indicate that complementary data at several chronological points may be critical for understanding the MF effects on DNA damage, repair, and post-repair integrity of the genome.

  10. Pre-exposure to 50 Hz magnetic fields modifies menadione-induced genotoxic effects in human SH-SY5Y neuroblastoma cells.

    Science.gov (United States)

    Luukkonen, Jukka; Liimatainen, Anu; Höytö, Anne; Juutilainen, Jukka; Naarala, Jonne

    2011-03-23

    Extremely low frequency (ELF) magnetic fields (MF) are generated by power lines and various electric appliances. They have been classified as possibly carcinogenic by the International Agency for Research on Cancer, but a mechanistic explanation for carcinogenic effects is lacking. A previous study in our laboratory showed that pre-exposure to ELF MF altered cancer-relevant cellular responses (cell cycle arrest, apoptosis) to menadione-induced DNA damage, but it did not include endpoints measuring actual genetic damage. In the present study, we examined whether pre-exposure to ELF MF affects chemically induced DNA damage level, DNA repair rate, or micronucleus frequency in human SH-SY5Y neuroblastoma cells. Exposure to 50 Hz MF was conducted at 100 µT for 24 hours, followed by chemical exposure for 3 hours. The chemicals used for inducing DNA damage and subsequent micronucleus formation were menadione and methyl methanesulphonate (MMS). Pre-treatment with MF enhanced menadione-induced DNA damage, DNA repair rate, and micronucleus formation in human SH-SY5Y neuroblastoma cells. Although the results with MMS indicated similar effects, the differences were not statistically significant. No effects were observed after MF exposure alone. The results confirm our previous findings showing that pre-exposure to MFs as low as 100 µT alters cellular responses to menadione, and show that increased genotoxicity results from such interaction. The present findings also indicate that complementary data at several chronological points may be critical for understanding the MF effects on DNA damage, repair, and post-repair integrity of the genome.

  11. Genetic and epigenetic variations induced by wheat-rye 2R and 5R monosomic addition lines.

    Science.gov (United States)

    Fu, Shulan; Sun, Chuanfei; Yang, Manyu; Fei, Yunyan; Tan, Feiqun; Yan, Benju; Ren, Zhenglong; Tang, Zongxiang

    2013-01-01

    Monosomic alien addition lines (MAALs) can easily induce structural variation of chromosomes and have been used in crop breeding; however, it is unclear whether MAALs will induce drastic genetic and epigenetic alterations. In the present study, wheat-rye 2R and 5R MAALs together with their selfed progeny and parental common wheat were investigated through amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP) analyses. The MAALs in different generations displayed different genetic variations. Some progeny that only contained 42 wheat chromosomes showed great genetic/epigenetic alterations. Cryptic rye chromatin has introgressed into the wheat genome. However, one of the progeny that contained cryptic rye chromatin did not display outstanding genetic/epigenetic variation. 78 and 49 sequences were cloned from changed AFLP and MSAP bands, respectively. Blastn search indicated that almost half of them showed no significant similarity to known sequences. Retrotransposons were mainly involved in genetic and epigenetic variations. Genetic variations basically affected Gypsy-like retrotransposons, whereas epigenetic alterations affected Copia-like and Gypsy-like retrotransposons equally. Genetic and epigenetic variations seldom affected low-copy coding DNA sequences. The results in the present study provided direct evidence to illustrate that monosomic wheat-rye addition lines could induce different and drastic genetic/epigenetic variations and these variations might not be caused by introgression of rye chromatins into wheat. Therefore, MAALs may be directly used as an effective means to broaden the genetic diversity of common wheat.

  12. Genomic research perspectives in Kazakhstan

    Directory of Open Access Journals (Sweden)

    Ainur Akilzhanova

    2014-01-01

    Full Text Available Introduction: Technological advancements rapidly propel the field of genome research. Advances in genetics and genomics such as the sequence of the human genome, the human haplotype map, open access databases, cheaper genotyping and chemical genomics, have transformed basic and translational biomedical research. Several projects in the field of genomic and personalized medicine have been conducted at the Center for Life Sciences in Nazarbayev University. The prioritized areas of research include: genomics of multifactorial diseases, cancer genomics, bioinformatics, genetics of infectious diseases and population genomics. At present, DNA-based risk assessment for common complex diseases, application of molecular signatures for cancer diagnosis and prognosis, genome-guided therapy, and dose selection of therapeutic drugs are the important issues in personalized medicine. Results: To further develop genomic and biomedical projects at Center for Life Sciences, the development of bioinformatics research and infrastructure and the establishment of new collaborations in the field are essential. Widespread use of genetic tools will allow the identification of diseases before the onset of clinical symptoms, the individualization of drug treatment, and could induce individual behavioral changes on the basis of calculated disease risk. However, many challenges remain for the successful translation of genomic knowledge and technologies into health advances, such as medicines and diagnostics. It is important to integrate research and education in the fields of genomics, personalized medicine, and bioinformatics, which will be possible with opening of the new Medical Faculty at Nazarbayev University. People in practice and training need to be educated about the key concepts of genomics and engaged so they can effectively apply their knowledge in a matter that will bring the era of genomic medicine to patient care. This requires the development of well

  13. Genome-derived vaccines.

    Science.gov (United States)

    De Groot, Anne S; Rappuoli, Rino

    2004-02-01

    Vaccine research entered a new era when the complete genome of a pathogenic bacterium was published in 1995. Since then, more than 97 bacterial pathogens have been sequenced and at least 110 additional projects are now in progress. Genome sequencing has also dramatically accelerated: high-throughput facilities can draft the sequence of an entire microbe (two to four megabases) in 1 to 2 days. Vaccine developers are using microarrays, immunoinformatics, proteomics and high-throughput immunology assays to reduce the truly unmanageable volume of information available in genome databases to a manageable size. Vaccines composed by novel antigens discovered from genome mining are already in clinical trials. Within 5 years we can expect to see a novel class of vaccines composed by genome-predicted, assembled and engineered T- and Bcell epitopes. This article addresses the convergence of three forces--microbial genome sequencing, computational immunology and new vaccine technologies--that are shifting genome mining for vaccines onto the forefront of immunology research.

  14. Complex DNA Damage: A Route to Radiation-Induced Genomic Instability and Carcinogenesis

    Directory of Open Access Journals (Sweden)

    Ifigeneia V. Mavragani

    2017-07-01

    Full Text Available Cellular effects of ionizing radiation (IR are of great variety and level, but they are mainly damaging since radiation can perturb all important components of the cell, from the membrane to the nucleus, due to alteration of different biological molecules ranging from lipids to proteins or DNA. Regarding DNA damage, which is the main focus of this review, as well as its repair, all current knowledge indicates that IR-induced DNA damage is always more complex than the corresponding endogenous damage resulting from endogenous oxidative stress. Specifically, it is expected that IR will create clusters of damage comprised of a diversity of DNA lesions like double strand breaks (DSBs, single strand breaks (SSBs and base lesions within a short DNA region of up to 15–20 bp. Recent data from our groups and others support two main notions, that these damaged clusters are: (1 repair resistant, increasing genomic instability (GI and malignant transformation and (2 can be considered as persistent “danger” signals promoting chronic inflammation and immune response, causing detrimental effects to the organism (like radiation toxicity. Last but not least, the paradigm shift for the role of radiation-induced systemic effects is also incorporated in this picture of IR-effects and consequences of complex DNA damage induction and its erroneous repair.

  15. Building a model: developing genomic resources for common milkweed (Asclepias syriaca) with low coverage genome sequencing.

    Science.gov (United States)

    Straub, Shannon C K; Fishbein, Mark; Livshultz, Tatyana; Foster, Zachary; Parks, Matthew; Weitemier, Kevin; Cronn, Richard C; Liston, Aaron

    2011-05-04

    Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed. The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first

  16. Genomic instability and radiation effects

    International Nuclear Information System (INIS)

    Christian Streffer

    2007-01-01

    Complete text of publication follows. Cancer, genetic mutations and developmental abnormalities are apparently associated with an increased genomic instability. Such phenomena have been frequently shown in human cancer cells in vitro and in situ. It is also well-known that individuals with a genetic predisposition for cancer proneness, such as ataxia telangiectesia, Fanconi anaemia etc. demonstrate a general high genomic instability e.g. in peripheral lymphocytes before a cancer has developed. Analogous data have been found in mice which develop a specific congenital malformation which has a genetic background. Under these aspects it is of high interest that ionising radiation can increase the genomic instability of mammalian cells after exposures in vitro an in vivo. This phenomenon is expressed 20 to 40 cell cycles after the exposure e.g. by de novo chromosomal aberrations. Such effects have been observed with high and low LET radiation, high LET radiation is more efficient. With low LET radiation a good dose response is observed in the dose range 0.2 to 2.0 Gy, Recently it has been reported that senescence and genomic instability was induced in human fibroblasts after 1 mGy carbon ions (1 in 18 cells are hit), apparently bystander effects also occurred under these conditions. The instability has been shown with DNA damage, chromosomal aberrations, gene mutation and cell death. It is also transferred to the next generation of mice with respect to gene mutations, chromosomal aberrations and congenital malformations. Several mechanisms have been discussed. The involvement of telomeres has gained interest. Genomic instability seems to be induced by a general lesion to the whole genome. The transmission of one chromosome from an irradiated cell to an non-irradiated cell leads to genomic instability in the untreated cells. Genomic instability increases mutation rates in the affected cells in general. As radiation late effects (cancer, gene mutations and congenital

  17. The genome of the Hi5 germ cell line from Trichoplusia ni, an agricultural pest and novel model for small RNA biology.

    Science.gov (United States)

    Fu, Yu; Yang, Yujing; Zhang, Han; Farley, Gwen; Wang, Junling; Quarles, Kaycee A; Weng, Zhiping; Zamore, Phillip D

    2018-01-29

    We report a draft assembly of the genome of Hi5 cells from the lepidopteran insect pest, Trichoplusia ni , assigning 90.6% of bases to one of 28 chromosomes and predicting 14,037 protein-coding genes. Chemoreception and detoxification gene families reveal T. ni -specific gene expansions that may explain its widespread distribution and rapid adaptation to insecticides. Transcriptome and small RNA data from thorax, ovary, testis, and the germline-derived Hi5 cell line show distinct expression profiles for 295 microRNA- and >393 piRNA-producing loci, as well as 39 genes encoding small RNA pathway proteins. Nearly all of the W chromosome is devoted to piRNA production, and T. ni siRNAs are not 2´- O -methylated. To enable use of Hi5 cells as a model system, we have established genome editing and single-cell cloning protocols. The T. ni genome provides insights into pest control and allows Hi5 cells to become a new tool for studying small RNAs ex vivo. © 2018, Fu et al.

  18. TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants

    DEFF Research Database (Denmark)

    Wendt, Toni; Holm, Preben Bach; Starker, Colby G

    2013-01-01

    , and their broad targeting range. Here we report the assembly of several TALENs for a specific genomic locus in barley. The cleavage activity of individual TALENs was first tested in vivo using a yeast-based, single-strand annealing assay. The most efficient TALEN was then selected for barley transformation....... Analysis of the resulting transformants showed that TALEN-induced double strand breaks led to the introduction of short deletions at the target site. Additional analysis revealed that each barley transformant contained a range of different mutations, indicating that mutations occurred independently...

  19. Life-cycle and genome of OtV5, a large DNA virus of the pelagic marine unicellular green alga Ostreococcus tauri.

    Directory of Open Access Journals (Sweden)

    Evelyne Derelle

    Full Text Available Large DNA viruses are ubiquitous, infecting diverse organisms ranging from algae to man, and have probably evolved from an ancient common ancestor. In aquatic environments, such algal viruses control blooms and shape the evolution of biodiversity in phytoplankton, but little is known about their biological functions. We show that Ostreococcus tauri, the smallest known marine photosynthetic eukaryote, whose genome is completely characterized, is a host for large DNA viruses, and present an analysis of the life-cycle and 186,234 bp long linear genome of OtV5. OtV5 is a lytic phycodnavirus which unexpectedly does not degrade its host chromosomes before the host cell bursts. Analysis of its complete genome sequence confirmed that it lacks expected site-specific endonucleases, and revealed the presence of 16 genes whose predicted functions are novel to this group of viruses. OtV5 carries at least one predicted gene whose protein closely resembles its host counterpart and several other host-like sequences, suggesting that horizontal gene transfers between host and viral genomes may occur frequently on an evolutionary scale. Fifty seven percent of the 268 predicted proteins present no similarities with any known protein in Genbank, underlining the wealth of undiscovered biological diversity present in oceanic viruses, which are estimated to harbour 200Mt of carbon.

  20. Genome size analyses of Pucciniales reveal the largest fungal genomes.

    Science.gov (United States)

    Tavares, Sílvia; Ramos, Ana Paula; Pires, Ana Sofia; Azinheira, Helena G; Caldeirinha, Patrícia; Link, Tobias; Abranches, Rita; Silva, Maria do Céu; Voegele, Ralf T; Loureiro, João; Talhinhas, Pedro

    2014-01-01

    Rust fungi (Basidiomycota, Pucciniales) are biotrophic plant pathogens which exhibit diverse complexities in their life cycles and host ranges. The completion of genome sequencing of a few rust fungi has revealed the occurrence of large genomes. Sequencing efforts for other rust fungi have been hampered by uncertainty concerning their genome sizes. Flow cytometry was recently applied to estimate the genome size of a few rust fungi, and confirmed the occurrence of large genomes in this order (averaging 225.3 Mbp, while the average for Basidiomycota was 49.9 Mbp and was 37.7 Mbp for all fungi). In this work, we have used an innovative and simple approach to simultaneously isolate nuclei from the rust and its host plant in order to estimate the genome size of 30 rust species by flow cytometry. Genome sizes varied over 10-fold, from 70 to 893 Mbp, with an average genome size value of 380.2 Mbp. Compared to the genome sizes of over 1800 fungi, Gymnosporangium confusum possesses the largest fungal genome ever reported (893.2 Mbp). Moreover, even the smallest rust genome determined in this study is larger than the vast majority of fungal genomes (94%). The average genome size of the Pucciniales is now of 305.5 Mbp, while the average Basidiomycota genome size has shifted to 70.4 Mbp and the average for all fungi reached 44.2 Mbp. Despite the fact that no correlation could be drawn between the genome sizes, the phylogenomics or the life cycle of rust fungi, it is interesting to note that rusts with Fabaceae hosts present genomes clearly larger than those with Poaceae hosts. Although this study comprises only a small fraction of the more than 7000 rust species described, it seems already evident that the Pucciniales represent a group where genome size expansion could be a common characteristic. This is in sharp contrast to sister taxa, placing this order in a relevant position in fungal genomics research.

  1. Neuropeptide Y Y5 receptor antagonism attenuates cocaine-induced effects in mice

    DEFF Research Database (Denmark)

    Sørensen, Gunnar; Jensen, Morten; Weikop, Pia

    2012-01-01

    Rationale Several studies suggest a role for neuropeptide Y (NPY) in addiction to drugs of abuse, including cocaine. However, the NPY receptors mediating addiction-related effects remain to be determined. Objectives To explore the potential role of Y5 NPY receptors in cocaine-induced behavioural...... effects. Methods The Y5 antagonist L-152,804 and Y5-knockout (Y5-KO) mice were tested in two models of cocaine addiction-related behaviour: acute self-administration and cocaine-induced hyperactivity. We also studied effects of Y5 receptor antagonism on cocaine-induced c-fos expression and extracellular...... effects, suggesting that Y5 receptors could be a potential therapeutic target in cocaine addiction....

  2. Non-targeted and delayed effects of exposure to ionizing radiation: I. Radiation-induced genomic instability and bystander effects in vitro

    Science.gov (United States)

    Morgan, William F.

    2003-01-01

    A long-standing dogma in the radiation sciences is that energy from radiation must be deposited in the cell nucleus to elicit a biological effect. A number of non-targeted, delayed effects of ionizing radiation have been described that challenge this dogma and pose new challenges to evaluating potential hazards associated with radiation exposure. These effects include induced genomic instability and non-targeted bystander effects. The in vitro evidence for non-targeted effects in radiation biology will be reviewed, but the question as to how one extrapolates from these in vitro observations to the risk of radiation-induced adverse health effects such as cancer remains open.

  3. An integrated CRISPR Bombyx mori genome editing system with improved efficiency and expanded target sites.

    Science.gov (United States)

    Ma, Sanyuan; Liu, Yue; Liu, Yuanyuan; Chang, Jiasong; Zhang, Tong; Wang, Xiaogang; Shi, Run; Lu, Wei; Xia, Xiaojuan; Zhao, Ping; Xia, Qingyou

    2017-04-01

    Genome editing enabled unprecedented new opportunities for targeted genomic engineering of a wide variety of organisms ranging from microbes, plants, animals and even human embryos. The serial establishing and rapid applications of genome editing tools significantly accelerated Bombyx mori (B. mori) research during the past years. However, the only CRISPR system in B. mori was the commonly used SpCas9, which only recognize target sites containing NGG PAM sequence. In the present study, we first improve the efficiency of our previous established SpCas9 system by 3.5 folds. The improved high efficiency was also observed at several loci in both BmNs cells and B. mori embryos. Then to expand the target sites, we showed that two newly discovered CRISPR system, SaCas9 and AsCpf1, could also induce highly efficient site-specific genome editing in BmNs cells, and constructed an integrated CRISPR system. Genome-wide analysis of targetable sites was further conducted and showed that the integrated system cover 69,144,399 sites in B. mori genome, and one site could be found in every 6.5 bp. The efficiency and resolution of this CRISPR platform will probably accelerate both fundamental researches and applicable studies in B. mori, and perhaps other insects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Genome Engineering for Personalized Arthritis Therapeutics.

    Science.gov (United States)

    Adkar, Shaunak S; Brunger, Jonathan M; Willard, Vincent P; Wu, Chia-Lung; Gersbach, Charles A; Guilak, Farshid

    2017-10-01

    Arthritis represents a family of complex joint pathologies responsible for the majority of musculoskeletal conditions. Nearly all diseases within this family, including osteoarthritis, rheumatoid arthritis, and juvenile idiopathic arthritis, are chronic conditions with few or no disease-modifying therapeutics available. Advances in genome engineering technology, most recently with CRISPR-Cas9, have revolutionized our ability to interrogate and validate genetic and epigenetic elements associated with chronic diseases such as arthritis. These technologies, together with cell reprogramming methods, including the use of induced pluripotent stem cells, provide a platform for human disease modeling. We summarize new evidence from genome-wide association studies and genomics that substantiates a genetic basis for arthritis pathogenesis. We also review the potential contributions of genome engineering in the development of new arthritis therapeutics. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. The resurrection genome of Boea hygrometrica: A blueprint for survival of dehydration

    Science.gov (United States)

    Xiao, Lihong; Yang, Ge; Zhang, Liechi; Yang, Xinhua; Zhao, Shuang; Ji, Zhongzhong; Zhou, Qing; Hu, Min; Wang, Yu; Chen, Ming; Xu, Yu; Jin, Haijing; Xiao, Xuan; Hu, Guipeng; Bao, Fang; Hu, Yong; Wan, Ping; Li, Legong; Deng, Xin; Kuang, Tingyun; Xiang, Chengbin; Zhu, Jian-Kang; Oliver, Melvin J.; He, Yikun

    2015-01-01

    “Drying without dying” is an essential trait in land plant evolution. Unraveling how a unique group of angiosperms, the Resurrection Plants, survive desiccation of their leaves and roots has been hampered by the lack of a foundational genome perspective. Here we report the ∼1,691-Mb sequenced genome of Boea hygrometrica, an important resurrection plant model. The sequence revealed evidence for two historical genome-wide duplication events, a compliment of 49,374 protein-coding genes, 29.15% of which are unique (orphan) to Boea and 20% of which (9,888) significantly respond to desiccation at the transcript level. Expansion of early light-inducible protein (ELIP) and 5S rRNA genes highlights the importance of the protection of the photosynthetic apparatus during drying and the rapid resumption of protein synthesis in the resurrection capability of Boea. Transcriptome analysis reveals extensive alternative splicing of transcripts and a focus on cellular protection strategies. The lack of desiccation tolerance-specific genome organizational features suggests the resurrection phenotype evolved mainly by an alteration in the control of dehydration response genes. PMID:25902549

  6. Genomics and physiology of a marine flavobacterium encoding a proteorhodopsin and a xanthorhodopsin-like protein.

    Directory of Open Access Journals (Sweden)

    Thomas Riedel

    Full Text Available Proteorhodopsin (PR photoheterotrophy in the marine flavobacterium Dokdonia sp. PRO95 has previously been investigated, showing no growth stimulation in the light at intermediate carbon concentrations. Here we report the genome sequence of strain PRO95 and compare it to two other PR encoding Dokdonia genomes: that of strain 4H-3-7-5 which shows the most similar genome, and that of strain MED134 which grows better in the light under oligotrophic conditions. Our genome analysis revealed that the PRO95 genome as well as the 4H-3-7-5 genome encode a protein related to xanthorhodopsins. The genomic environment and phylogenetic distribution of this gene suggest that it may have frequently been recruited by lateral gene transfer. Expression analyses by RT-PCR and direct mRNA-sequencing showed that both rhodopsins and the complete β-carotene pathway necessary for retinal production are transcribed in PRO95. Proton translocation measurements showed enhanced proton pump activity in response to light, supporting that one or both rhodopsins are functional. Genomic information and carbon source respiration data were used to develop a defined cultivation medium for PRO95, but reproducible growth always required small amounts of yeast extract. Although PRO95 contains and expresses two rhodopsin genes, light did not stimulate its growth as determined by cell numbers in a nutrient poor seawater medium that mimics its natural environment, confirming previous experiments at intermediate carbon concentrations. Starvation or stress conditions might be needed to observe the physiological effect of light induced energy acquisition.

  7. HIV-1 vaccine-induced T-cell responses cluster in epitope hotspots that differ from those induced in natural infection with HIV-1.

    Science.gov (United States)

    Hertz, Tomer; Ahmed, Hasan; Friedrich, David P; Casimiro, Danilo R; Self, Steven G; Corey, Lawrence; McElrath, M Juliana; Buchbinder, Susan; Horton, Helen; Frahm, Nicole; Robertson, Michael N; Graham, Barney S; Gilbert, Peter

    2013-01-01

    Several recent large clinical trials evaluated HIV vaccine candidates that were based on recombinant adenovirus serotype 5 (rAd-5) vectors expressing HIV-derived antigens. These vaccines primarily elicited T-cell responses, which are known to be critical for controlling HIV infection. In the current study, we present a meta-analysis of epitope mapping data from 177 participants in three clinical trials that tested two different HIV vaccines: MRKAd-5 HIV and VRC-HIVAD014-00VP. We characterized the population-level epitope responses in these trials by generating population-based epitope maps, and also designed such maps using a large cohort of 372 naturally infected individuals. We used these maps to address several questions: (1) Are vaccine-induced responses randomly distributed across vaccine inserts, or do they cluster into immunodominant epitope hotspots? (2) Are the immunodominance patterns observed for these two vaccines in three vaccine trials different from one another? (3) Do vaccine-induced hotspots overlap with epitope hotspots induced by chronic natural infection with HIV-1? (4) Do immunodominant hotspots target evolutionarily conserved regions of the HIV genome? (5) Can epitope prediction methods be used to identify these hotspots? We found that vaccine responses clustered into epitope hotspots in all three vaccine trials and some of these hotspots were not observed in chronic natural infection. We also found significant differences between the immunodominance patterns generated in each trial, even comparing two trials that tested the same vaccine in different populations. Some of the vaccine-induced immunodominant hotspots were located in highly variable regions of the HIV genome, and this was more evident for the MRKAd-5 HIV vaccine. Finally, we found that epitope prediction methods can partially predict the location of vaccine-induced epitope hotspots. Our findings have implications for vaccine design and suggest a framework by which different

  8. Punctuated evolution of prostate cancer genomes.

    Science.gov (United States)

    Baca, Sylvan C; Prandi, Davide; Lawrence, Michael S; Mosquera, Juan Miguel; Romanel, Alessandro; Drier, Yotam; Park, Kyung; Kitabayashi, Naoki; MacDonald, Theresa Y; Ghandi, Mahmoud; Van Allen, Eliezer; Kryukov, Gregory V; Sboner, Andrea; Theurillat, Jean-Philippe; Soong, T David; Nickerson, Elizabeth; Auclair, Daniel; Tewari, Ashutosh; Beltran, Himisha; Onofrio, Robert C; Boysen, Gunther; Guiducci, Candace; Barbieri, Christopher E; Cibulskis, Kristian; Sivachenko, Andrey; Carter, Scott L; Saksena, Gordon; Voet, Douglas; Ramos, Alex H; Winckler, Wendy; Cipicchio, Michelle; Ardlie, Kristin; Kantoff, Philip W; Berger, Michael F; Gabriel, Stacey B; Golub, Todd R; Meyerson, Matthew; Lander, Eric S; Elemento, Olivier; Getz, Gad; Demichelis, Francesca; Rubin, Mark A; Garraway, Levi A

    2013-04-25

    The analysis of exonic DNA from prostate cancers has identified recurrently mutated genes, but the spectrum of genome-wide alterations has not been profiled extensively in this disease. We sequenced the genomes of 57 prostate tumors and matched normal tissues to characterize somatic alterations and to study how they accumulate during oncogenesis and progression. By modeling the genesis of genomic rearrangements, we identified abundant DNA translocations and deletions that arise in a highly interdependent manner. This phenomenon, which we term "chromoplexy," frequently accounts for the dysregulation of prostate cancer genes and appears to disrupt multiple cancer genes coordinately. Our modeling suggests that chromoplexy may induce considerable genomic derangement over relatively few events in prostate cancer and other neoplasms, supporting a model of punctuated cancer evolution. By characterizing the clonal hierarchy of genomic lesions in prostate tumors, we charted a path of oncogenic events along which chromoplexy may drive prostate carcinogenesis. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Genome Stability Pathways in Head and Neck Cancers

    Directory of Open Access Journals (Sweden)

    Glenn Jenkins

    2013-01-01

    Full Text Available Genomic instability underlies the transformation of host cells toward malignancy, promotes development of invasion and metastasis and shapes the response of established cancer to treatment. In this review, we discuss recent advances in our understanding of genomic stability in squamous cell carcinoma of the head and neck (HNSCC, with an emphasis on DNA repair pathways. HNSCC is characterized by distinct profiles in genome stability between similarly staged cancers that are reflected in risk, treatment response and outcomes. Defective DNA repair generates chromosomal derangement that can cause subsequent alterations in gene expression, and is a hallmark of progression toward carcinoma. Variable functionality of an increasing spectrum of repair gene polymorphisms is associated with increased cancer risk, while aetiological factors such as human papillomavirus, tobacco and alcohol induce significantly different behaviour in induced malignancy, underpinned by differences in genomic stability. Targeted inhibition of signalling receptors has proven to be a clinically-validated therapy, and protein expression of other DNA repair and signalling molecules associated with cancer behaviour could potentially provide a more refined clinical model for prognosis and treatment prediction. Development and expansion of current genomic stability models is furthering our understanding of HNSCC pathophysiology and uncovering new, promising treatment strategies.

  10. Genome Stability Pathways in Head and Neck Cancers

    Science.gov (United States)

    O'Byrne, Kenneth J.; Panizza, Benedict; Richard, Derek J.

    2013-01-01

    Genomic instability underlies the transformation of host cells toward malignancy, promotes development of invasion and metastasis and shapes the response of established cancer to treatment. In this review, we discuss recent advances in our understanding of genomic stability in squamous cell carcinoma of the head and neck (HNSCC), with an emphasis on DNA repair pathways. HNSCC is characterized by distinct profiles in genome stability between similarly staged cancers that are reflected in risk, treatment response and outcomes. Defective DNA repair generates chromosomal derangement that can cause subsequent alterations in gene expression, and is a hallmark of progression toward carcinoma. Variable functionality of an increasing spectrum of repair gene polymorphisms is associated with increased cancer risk, while aetiological factors such as human papillomavirus, tobacco and alcohol induce significantly different behaviour in induced malignancy, underpinned by differences in genomic stability. Targeted inhibition of signalling receptors has proven to be a clinically-validated therapy, and protein expression of other DNA repair and signalling molecules associated with cancer behaviour could potentially provide a more refined clinical model for prognosis and treatment prediction. Development and expansion of current genomic stability models is furthering our understanding of HNSCC pathophysiology and uncovering new, promising treatment strategies. PMID:24364026

  11. Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line

    International Nuclear Information System (INIS)

    Araújo, E.S.S. de; Vasques, L.R.; Stabellini, R.; Krepischi, A.C.V.; Pereira, L.V.

    2014-01-01

    DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting that XIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2′-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance of XIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A

  12. Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line

    Energy Technology Data Exchange (ETDEWEB)

    Araújo, E.S.S. de [Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Centro Internacional de Pesquisa, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Vasques, L.R. [Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Stabellini, R.; Krepischi, A.C.V. [Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Centro Internacional de Pesquisa, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Pereira, L.V. [Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil)

    2014-10-17

    DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting that XIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2′-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance of XIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A.

  13. Building a model: developing genomic resources for common milkweed (Asclepias syriaca with low coverage genome sequencing

    Directory of Open Access Journals (Sweden)

    Weitemier Kevin

    2011-05-01

    Full Text Available Abstract Background Milkweeds (Asclepias L. have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L. could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. Results A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp and 5S rDNA (120 bp sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp, with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae unigenes (median coverage of 0.29× and 66% of single copy orthologs (COSII in asterids (median coverage of 0.14×. From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites and phylogenetics (low-copy nuclear genes studies were developed. Conclusions The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species

  14. Deleterious mutation accumulation in organelle genomes.

    Science.gov (United States)

    Lynch, M; Blanchard, J L

    1998-01-01

    It is well established on theoretical grounds that the accumulation of mildly deleterious mutations in nonrecombining genomes is a major extinction risk in obligately asexual populations. Sexual populations can also incur mutational deterioration in genomic regions that experience little or no recombination, i.e., autosomal regions near centromeres, Y chromosomes, and organelle genomes. Our results suggest, for a wide array of genes (transfer RNAs, ribosomal RNAs, and proteins) in a diverse collection of species (animals, plants, and fungi), an almost universal increase in the fixation probabilities of mildly deleterious mutations arising in mitochondrial and chloroplast genomes relative to those arising in the recombining nuclear genome. This enhanced width of the selective sieve in organelle genomes does not appear to be a consequence of relaxed selection, but can be explained by the decline in the efficiency of selection that results from the reduction of effective population size induced by uniparental inheritance. Because of the very low mutation rates of organelle genomes (on the order of 10(-4) per genome per year), the reduction in fitness resulting from mutation accumulation in such genomes is a very long-term process, not likely to imperil many species on time scales of less than a million years, but perhaps playing some role in phylogenetic lineage sorting on time scales of 10 to 100 million years.

  15. Genome-Wide Locations of Potential Epimutations Associated with Environmentally Induced Epigenetic Transgenerational Inheritance of Disease Using a Sequential Machine Learning Prediction Approach

    OpenAIRE

    Haque, M. Muksitul; Holder, Lawrence B.; Skinner, Michael K.

    2015-01-01

    Environmentally induced epigenetic transgenerational inheritance of disease and phenotypic variation involves germline transmitted epimutations. The primary epimutations identified involve altered differential DNA methylation regions (DMRs). Different environmental toxicants have been shown to promote exposure (i.e., toxicant) specific signatures of germline epimutations. Analysis of genomic features associated with these epimutations identified low-density CpG regions (

  16. Draft Genome Sequences of the Fish Pathogen Vibrio harveyi Strains VH2 and VH5

    DEFF Research Database (Denmark)

    Castillo, Daniel; D'Alvise, Paul; Middelboe, Mathias

    2015-01-01

    Vibrio harveyi is an important marine pathogen that is responsible for vibriosis outbreaks in cultured fish and invertebrates worldwide. Here, we announce the draft genome sequences of V. harveyi strains VH2 and VH5, isolated from farmed juvenile Seriola dumerili during outbreaks of vibriosis...... in Crete, Greece....

  17. Harnessing CRISPR-Cas systems for bacterial genome editing.

    Science.gov (United States)

    Selle, Kurt; Barrangou, Rodolphe

    2015-04-01

    Manipulation of genomic sequences facilitates the identification and characterization of key genetic determinants in the investigation of biological processes. Genome editing via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) constitutes a next-generation method for programmable and high-throughput functional genomics. CRISPR-Cas systems are readily reprogrammed to induce sequence-specific DNA breaks at target loci, resulting in fixed mutations via host-dependent DNA repair mechanisms. Although bacterial genome editing is a relatively unexplored and underrepresented application of CRISPR-Cas systems, recent studies provide valuable insights for the widespread future implementation of this technology. This review summarizes recent progress in bacterial genome editing and identifies fundamental genetic and phenotypic outcomes of CRISPR targeting in bacteria, in the context of tool development, genome homeostasis, and DNA repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Genome-wide local ancestry approach identifies genes and variants associated with chemotherapeutic susceptibility in African Americans.

    Directory of Open Access Journals (Sweden)

    Heather E Wheeler

    Full Text Available Chemotherapeutic agents are used in the treatment of many cancers, yet variable resistance and toxicities among individuals limit successful outcomes. Several studies have indicated outcome differences associated with ancestry among patients with various cancer types. Using both traditional SNP-based and newly developed gene-based genome-wide approaches, we investigated the genetics of chemotherapeutic susceptibility in lymphoblastoid cell lines derived from 83 African Americans, a population for which there is a disparity in the number of genome-wide studies performed. To account for population structure in this admixed population, we incorporated local ancestry information into our association model. We tested over 2 million SNPs and identified 325, 176, 240, and 190 SNPs that were suggestively associated with cytarabine-, 5'-deoxyfluorouridine (5'-DFUR-, carboplatin-, and cisplatin-induced cytotoxicity, respectively (p≤10(-4. Importantly, some of these variants are found only in populations of African descent. We also show that cisplatin-susceptibility SNPs are enriched for carboplatin-susceptibility SNPs. Using a gene-based genome-wide association approach, we identified 26, 11, 20, and 41 suggestive candidate genes for association with cytarabine-, 5'-DFUR-, carboplatin-, and cisplatin-induced cytotoxicity, respectively (p≤10(-3. Fourteen of these genes showed evidence of association with their respective chemotherapeutic phenotypes in the Yoruba from Ibadan, Nigeria (p<0.05, including TP53I11, COPS5 and GAS8, which are known to be involved in tumorigenesis. Although our results require further study, we have identified variants and genes associated with chemotherapeutic susceptibility in African Americans by using an approach that incorporates local ancestry information.

  19. Genomic stability during cellular reprogramming: Mission impossible?

    Energy Technology Data Exchange (ETDEWEB)

    Joest, Mathieu von; Búa Aguín, Sabela; Li, Han, E-mail: han.li@pasteur.fr

    2016-06-15

    The generation of induced pluripotent stem cells (iPSCs) from adult somatic cells is one of the most exciting discoveries in recent biomedical research. It holds tremendous potential in drug discovery and regenerative medicine. However, a series of reports highlighting genomic instability in iPSCs raises concerns about their clinical application. Although the mechanisms cause genomic instability during cellular reprogramming are largely unknown, several potential sources have been suggested. This review summarizes current knowledge on this active research field and discusses the latest efforts to alleviate the genomic insults during cellular reprogramming to generate iPSCs with enhanced quality and safety.

  20. Draft genome sequence of Halorubrum tropicale strain V5, a novel halophilic archaeon isolated from the solar salterns of Cabo Rojo, Puerto Rico.

    Science.gov (United States)

    Sánchez-Nieves, Rubén; Facciotti, Marc T; Saavedra-Collado, Sofía; Dávila-Santiago, Lizbeth; Rodríguez-Carrero, Roy; Montalvo-Rodríguez, Rafael

    2016-03-01

    The genus Halorubrum is a member of the family Halobacteriaceae which currently has the highest number of described species (31) of all the haloarchaea. Here we report the draft genome sequence of strain V5, a new species within this genus that was isolated from the solar salterns of Cabo Rojo, Puerto Rico. Assembly was performed and rendered the genome into 17 contigs (N50 = 515,834 bp), the largest of which contains 1,031,026 bp. The genome consists of 3.57 MB in length with G + C content of 67.6%. In general, the genome includes 4 rRNAs, 52 tRNAs, and 3246 protein-coding sequences. The NCBI accession number for this genome is LIST00000000 and the strain deposit number is CECT9000.

  1. The zebrafish genome: a review and msx gene case study.

    Science.gov (United States)

    Postlethwait, J H

    2006-01-01

    Zebrafish is one of several important teleost models for understanding principles of vertebrate developmental, molecular, organismal, genetic, evolutionary, and genomic biology. Efficient investigation of the molecular genetic basis of induced mutations depends on knowledge of the zebrafish genome. Principles of zebrafish genomic analysis, including gene mapping, ortholog identification, conservation of syntenies, genome duplication, and evolution of duplicate gene function are discussed here using as a case study the zebrafish msxa, msxb, msxc, msxd, and msxe genes, which together constitute zebrafish orthologs of tetrapod Msx1, Msx2, and Msx3. Genomic analysis suggests orthologs for this difficult to understand group of paralogs.

  2. Isolation and characterization of 5S rDNA sequences in catfishes genome (Heptapteridae and Pseudopimelodidae): perspectives for rDNA studies in fish by C0t method.

    Science.gov (United States)

    Gouveia, Juceli Gonzalez; Wolf, Ivan Rodrigo; de Moraes-Manécolo, Vivian Patrícia Oliveira; Bardella, Vanessa Belline; Ferracin, Lara Munique; Giuliano-Caetano, Lucia; da Rosa, Renata; Dias, Ana Lúcia

    2016-12-01

    Sequences of 5S ribosomal RNA (rRNA) are extensively used in fish cytogenomic studies, once they have a flexible organization at the chromosomal level, showing inter- and intra-specific variation in number and position in karyotypes. Sequences from the genome of Imparfinis schubarti (Heptapteridae) were isolated, aiming to understand the organization of 5S rDNA families in the fish genome. The isolation of 5S rDNA from the genome of I. schubarti was carried out by reassociation kinetics (C 0 t) and PCR amplification. The obtained sequences were cloned for the construction of a micro-library. The obtained clones were sequenced and hybridized in I. schubarti and Microglanis cottoides (Pseudopimelodidae) for chromosome mapping. An analysis of the sequence alignments with other fish groups was accomplished. Both methods were effective when using 5S rDNA for hybridization in I. schubarti genome. However, the C 0 t method enabled the use of a complete 5S rRNA gene, which was also successful in the hybridization of M. cottoides. Nevertheless, this gene was obtained only partially by PCR. The hybridization results and sequence analyses showed that intact 5S regions are more appropriate for the probe operation, due to conserved structure and motifs. This study contributes to a better understanding of the organization of multigene families in catfish's genomes.

  3. Genome editing reveals a role for OCT4 in human embryogenesis.

    Science.gov (United States)

    Fogarty, Norah M E; McCarthy, Afshan; Snijders, Kirsten E; Powell, Benjamin E; Kubikova, Nada; Blakeley, Paul; Lea, Rebecca; Elder, Kay; Wamaitha, Sissy E; Kim, Daesik; Maciulyte, Valdone; Kleinjung, Jens; Kim, Jin-Soo; Wells, Dagan; Vallier, Ludovic; Bertero, Alessandro; Turner, James M A; Niakan, Kathy K

    2017-10-05

    Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

  4. Sleep-inducing N-alkyl-5-[m-(trifluoromethyl)phenyl]-5-hydroxy-2-pyrrolidinones and N-alkyl-3-(trifluoromethyl)cinnamamides.

    Science.gov (United States)

    Houlihan, W J; Gogerty, J H; Ryan, E A; Schmitt, G

    1985-01-01

    A series of N-alkyl-3-[m-(trifluoromethyl)phenyl]-5-hydroxy-2-pyrrolidinones and N-alkyl-3-(trifluoromethyl)-cinnamamides were prepared and screened in a series of tests designed to detect potential sleep inducers. The more active members of the series were evaluated for their ability to induce sleep in Cebus monkeys. The most active compound, N-methyl-5-[m-(trifluoromethyl)phenyl]-5-hydroxy-2-pyrrolidinone, was equal to methaqualone.

  5. Genetic and epigenetic variations induced by wheat-rye 2R and 5R monosomic addition lines.

    Directory of Open Access Journals (Sweden)

    Shulan Fu

    Full Text Available BACKGROUND: Monosomic alien addition lines (MAALs can easily induce structural variation of chromosomes and have been used in crop breeding; however, it is unclear whether MAALs will induce drastic genetic and epigenetic alterations. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, wheat-rye 2R and 5R MAALs together with their selfed progeny and parental common wheat were investigated through amplified fragment length polymorphism (AFLP and methylation-sensitive amplification polymorphism (MSAP analyses. The MAALs in different generations displayed different genetic variations. Some progeny that only contained 42 wheat chromosomes showed great genetic/epigenetic alterations. Cryptic rye chromatin has introgressed into the wheat genome. However, one of the progeny that contained cryptic rye chromatin did not display outstanding genetic/epigenetic variation. 78 and 49 sequences were cloned from changed AFLP and MSAP bands, respectively. Blastn search indicated that almost half of them showed no significant similarity to known sequences. Retrotransposons were mainly involved in genetic and epigenetic variations. Genetic variations basically affected Gypsy-like retrotransposons, whereas epigenetic alterations affected Copia-like and Gypsy-like retrotransposons equally. Genetic and epigenetic variations seldom affected low-copy coding DNA sequences. CONCLUSIONS/SIGNIFICANCE: The results in the present study provided direct evidence to illustrate that monosomic wheat-rye addition lines could induce different and drastic genetic/epigenetic variations and these variations might not be caused by introgression of rye chromatins into wheat. Therefore, MAALs may be directly used as an effective means to broaden the genetic diversity of common wheat.

  6. Mutations in Cytosine-5 tRNA Methyltransferases Impact Mobile Element Expression and Genome Stability at Specific DNA Repeats

    Directory of Open Access Journals (Sweden)

    Bianca Genenncher

    2018-02-01

    Full Text Available The maintenance of eukaryotic genome stability is ensured by the interplay of transcriptional as well as post-transcriptional mechanisms that control recombination of repeat regions and the expression and mobility of transposable elements. We report here that mutations in two (cytosine-5 RNA methyltransferases, Dnmt2 and NSun2, impact the accumulation of mobile element-derived sequences and DNA repeat integrity in Drosophila. Loss of Dnmt2 function caused moderate effects under standard conditions, while heat shock exacerbated these effects. In contrast, NSun2 function affected mobile element expression and genome integrity in a heat shock-independent fashion. Reduced tRNA stability in both RCMT mutants indicated that tRNA-dependent processes affected mobile element expression and DNA repeat stability. Importantly, further experiments indicated that complex formation with RNA could also contribute to the impact of RCMT function on gene expression control. These results thus uncover a link between tRNA modification enzymes, the expression of repeat DNA, and genomic integrity.

  7. The Genomic Evolution of Prostate Cancer

    Science.gov (United States)

    2017-06-01

    the proposed project : 1. To continue to acquire a comprehensive understanding of prostate cancer genomics . 2. To develop an understanding of... Genetics I • ECEV 35901 Evolutionary Genomics • Fundamentals of Clinical Research • HGEN 47400 Introduction to Probability and Statistics for Geneticists...Marc Gillard,2 David M. Hatcher,5 Westin R. Tom,5 Walter M. Stadler2 and Kevin P. White1,2,3 1Institute for Genomics and Systems Biology , Departments of

  8. Novel cross-strand three-purine stack of the highly conserved 5'-GA/AAG-5' internal loop at the 3'-end termini of Parvovirus Genomes

    International Nuclear Information System (INIS)

    Chou, S.-H.; Chin, K.-H.

    2001-01-01

    We have used two-dimensional nuclear magnetic resonance (2D-NMR), distance geometry (DG) and molecular dynamics / energy minimization (MD/EM) methods to study a 2x3 asymmetric internal loop structure of the highly conserved '5'-(GA)/(AAG)-5' bubble' present at the 3'-end hairpin of the single-stranded DNA genome of parvoviruses. This motif contains an unpaired adenosine stacked between two bracketed sheared G·A pairs. However, the phenomenal cross-strand G-G and A-A stacking in the tandem sheared G·A pairs has undergone considerable change. A novel three-purine stacking pattern is observed instead; the inserted A18 base is completely un-stacked from its neighboring G17 and A19 bases, but well stacked with the cross-strand A4 and G3 bases to form a novel A4/A18/G3 stack that is different from the double G/G, A/A or quadruple G/G/G/G stack present in the 5'-(GA)/(AG)-5' or 5'-(GGA)/(AGG)-5' motifs. Unlike the bulged purine residue that usually causes about 20 degree kink in the helical axis of the parent helix when bracketed by canonical G·C or A·T base pairs, no significant kink is observed in the present helix containing a bulged-adenine that is bracketed by sheared G ·A pairs. The phosphodiesters connecting G3-A4 and G17-A18 residues adopt unusual ζ torsional angles close to the trans domain, yet that connecting A18-A19 residues resumes the normal ζ(g - ) value. The well structured '5'-(GAA)/(AG)-5'' internal loop in the parvovirus genomes explains its resistance to single-strand specific endonuclease susceptibility

  9. How do students react to analyzing their own genomes in a whole-genome sequencing course?: outcomes of a longitudinal cohort study.

    Science.gov (United States)

    Sanderson, Saskia C; Linderman, Michael D; Zinberg, Randi; Bashir, Ali; Kasarskis, Andrew; Zweig, Micol; Suckiel, Sabrina; Shah, Hardik; Mahajan, Milind; Diaz, George A; Schadt, Eric E

    2015-11-01

    Health-care professionals need to be trained to work with whole-genome sequencing (WGS) in their practice. Our aim was to explore how students responded to a novel genome analysis course that included the option to analyze their own genomes. This was an observational cohort study. Questionnaires were administered before (T3) and after the genome analysis course (T4), as well as 6 months later (T5). In-depth interviews were conducted at T5. All students (n = 19) opted to analyze their own genomes. At T5, 12 of 15 students stated that analyzing their own genomes had been useful. Ten reported they had applied their knowledge in the workplace. Technical WGS knowledge increased (mean of 63.8% at T3, mean of 72.5% at T4; P = 0.005). In-depth interviews suggested that analyzing their own genomes may increase students' motivation to learn and their understanding of the patient experience. Most (but not all) of the students reported low levels of WGS results-related distress and low levels of regret about their decision to analyze their own genomes. Giving students the option of analyzing their own genomes may increase motivation to learn, but some students may experience personal WGS results-related distress and regret. Additional evidence is required before considering incorporating optional personal genome analysis into medical education on a large scale.

  10. Epstein-Barr Virus BKRF4 Gene Product Is Required for Efficient Progeny Production.

    Science.gov (United States)

    Masud, H M Abdullah Al; Watanabe, Takahiro; Yoshida, Masahiro; Sato, Yoshitaka; Goshima, Fumi; Kimura, Hiroshi; Murata, Takayuki

    2017-12-01

    Epstein-Barr virus (EBV), a member of human gammaherpesvirus, infects mainly B cells. EBV has two alternative life cycles, latent and lytic, and is reactivated occasionally from the latent stage to the lytic cycle. To combat EBV-associated disorders, understanding the molecular mechanisms of the EBV lytic replication cycle is also important. Here, we focused on an EBV lytic gene, BKRF4. Using our anti-BKRF4 antibody, we revealed that the BKRF4 gene product is expressed during the lytic cycle with late kinetics. To characterize the role of BKRF4, we constructed BKRF4-knockout mutants using the bacterial artificial chromosome (BAC) and CRISPR/Cas9 systems. Although disruption of the BKRF4 gene had almost no effect on viral protein expression and DNA synthesis, it significantly decreased progeny virion levels in HEK293 and Akata cells. Furthermore, we show that BKRF4 is involved not only in production of progeny virions but also in increasing the infectivity of the virus particles. Immunoprecipitation assays revealed that BKRF4 interacted with a virion protein, BGLF2. We showed that the C-terminal region of BKRF4 was critical for this interaction and for efficient progeny production. Immunofluorescence analysis revealed that BKRF4 partially colocalized with BGLF2 in the nucleus and perinuclear region. Finally, we showed that BKRF4 is a phosphorylated, possible tegument protein and that the EBV protein kinase BGLF4 may be important for this phosphorylation. Taken together, our data suggest that BKRF4 is involved in the production of infectious virions. IMPORTANCE Although the latent genes of EBV have been studied extensively, the lytic genes are less well characterized. This study focused on one such lytic gene, BKRF4, which is conserved only among gammaherpesviruses (ORF45 of Kaposi's sarcoma-associated herpesvirus or murine herpesvirus 68). After preparing the BKRF4 knockout virus using B95-8 EBV-BAC, we demonstrated that the BKRF4 gene was involved in infectious

  11. Comparative genomics of the marine bacterial genus Glaciecola reveals the high degree of genomic diversity and genomic characteristic for cold adaptation.

    Science.gov (United States)

    Qin, Qi-Long; Xie, Bin-Bin; Yu, Yong; Shu, Yan-Li; Rong, Jin-Cheng; Zhang, Yan-Jiao; Zhao, Dian-Li; Chen, Xiu-Lan; Zhang, Xi-Ying; Chen, Bo; Zhou, Bai-Cheng; Zhang, Yu-Zhong

    2014-06-01

    To what extent the genomes of different species belonging to one genus can be diverse and the relationship between genomic differentiation and environmental factor remain unclear for oceanic bacteria. With many new bacterial genera and species being isolated from marine environments, this question warrants attention. In this study, we sequenced all the type strains of the published species of Glaciecola, a recently defined cold-adapted genus with species from diverse marine locations, to study the genomic diversity and cold-adaptation strategy in this genus.The genome size diverged widely from 3.08 to 5.96 Mb, which can be explained by massive gene gain and loss events. Horizontal gene transfer and new gene emergence contributed substantially to the genome size expansion. The genus Glaciecola had an open pan-genome. Comparative genomic research indicated that species of the genus Glaciecola had high diversity in genome size, gene content and genetic relatedness. This may be prevalent in marine bacterial genera considering the dynamic and complex environments of the ocean. Species of Glaciecola had some common genomic features related to cold adaptation, which enable them to thrive and play a role in biogeochemical cycle in the cold marine environments.

  12. The cacao Criollo genome v2.0: an improved version of the genome for genetic and functional genomic studies.

    Science.gov (United States)

    Argout, X; Martin, G; Droc, G; Fouet, O; Labadie, K; Rivals, E; Aury, J M; Lanaud, C

    2017-09-15

    Theobroma cacao L., native to the Amazonian basin of South America, is an economically important fruit tree crop for tropical countries as a source of chocolate. The first draft genome of the species, from a Criollo cultivar, was published in 2011. Although a useful resource, some improvements are possible, including identifying misassemblies, reducing the number of scaffolds and gaps, and anchoring un-anchored sequences to the 10 chromosomes. We used a NGS-based approach to significantly improve the assembly of the Belizian Criollo B97-61/B2 genome. We combined four Illumina large insert size mate paired libraries with 52x of Pacific Biosciences long reads to correct misassembled regions and reduced the number of scaffolds. We then used genotyping by sequencing (GBS) methods to increase the proportion of the assembly anchored to chromosomes. The scaffold number decreased from 4,792 in assembly V1 to 554 in V2 while the scaffold N50 size has increased from 0.47 Mb in V1 to 6.5 Mb in V2. A total of 96.7% of the assembly was anchored to the 10 chromosomes compared to 66.8% in the previous version. Unknown sites (Ns) were reduced from 10.8% to 5.7%. In addition, we updated the functional annotations and performed a new RefSeq structural annotation based on RNAseq evidence. Theobroma cacao Criollo genome version 2 will be a valuable resource for the investigation of complex traits at the genomic level and for future comparative genomics and genetics studies in cacao tree. New functional tools and annotations are available on the Cocoa Genome Hub ( http://cocoa-genome-hub.southgreen.fr ).

  13. Merkel cell polyomavirus small T antigen induces genome instability by E3 ubiquitin ligase targeting.

    Science.gov (United States)

    Kwun, H J; Wendzicki, J A; Shuda, Y; Moore, P S; Chang, Y

    2017-12-07

    The formation of a bipolar mitotic spindle is an essential process for the equal segregation of duplicated DNA into two daughter cells during mitosis. As a result of deregulated cellular signaling pathways, cancer cells often suffer a loss of genome integrity that might etiologically contribute to carcinogenesis. Merkel cell polyomavirus (MCV) small T (sT) oncoprotein induces centrosome overduplication, aneuploidy, chromosome breakage and the formation of micronuclei by targeting cellular ligases through a sT domain that also inhibits MCV large T oncoprotein turnover. These results provide important insight as to how centrosome number and chromosomal stability can be affected by the E3 ligase targeting capacity of viral oncoproteins such as MCV sT, which may contribute to Merkel cell carcinogenesis.

  14. Genome wide transcriptional response of Saccharomyces cerevisiae to stress-induced perturbations

    Directory of Open Access Journals (Sweden)

    Hilal eTaymaz-Nikerel

    2016-02-01

    Full Text Available Cells respond to environmental and/or genetic perturbations in order to survive and proliferate. Characterization of the changes after various stimuli at different -omics levels is crucial to comprehend the adaptation of cells to changing conditions. Genome wide quantification and analysis of transcript levels, the genes affected by perturbations, extends our understanding of cellular metabolism by pointing out the mechanisms that play role in sensing the stress caused by those perturbations and related signaling pathways, and in this way guides us to achieve endeavors such as rational engineering of cells or interpretation of disease mechanisms. Saccharomyces cerevisiae as a model system has been studied in response to different perturbations and corresponding transcriptional profiles were followed either statically or/and dynamically, short- and long- term. This review focuses on response of yeast cells to diverse stress inducing perturbations including nutritional changes, ionic stress, salt stress, oxidative stress, osmotic shock, as well as to genetic interventions such as deletion and over-expression of genes. It is aimed to conclude on common regulatory phenomena that allow yeast to organize its transcriptomic response after any perturbation under different external conditions.

  15. Analyses of charophyte chloroplast genomes help characterize the ancestral chloroplast genome of land plants.

    Science.gov (United States)

    Civaň, Peter; Foster, Peter G; Embley, Martin T; Séneca, Ana; Cox, Cymon J

    2014-04-01

    Despite the significance of the relationships between embryophytes and their charophyte algal ancestors in deciphering the origin and evolutionary success of land plants, few chloroplast genomes of the charophyte algae have been reconstructed to date. Here, we present new data for three chloroplast genomes of the freshwater charophytes Klebsormidium flaccidum (Klebsormidiophyceae), Mesotaenium endlicherianum (Zygnematophyceae), and Roya anglica (Zygnematophyceae). The chloroplast genome of Klebsormidium has a quadripartite organization with exceptionally large inverted repeat (IR) regions and, uniquely among streptophytes, has lost the rrn5 and rrn4.5 genes from the ribosomal RNA (rRNA) gene cluster operon. The chloroplast genome of Roya differs from other zygnematophycean chloroplasts, including the newly sequenced Mesotaenium, by having a quadripartite structure that is typical of other streptophytes. On the basis of the improbability of the novel gain of IR regions, we infer that the quadripartite structure has likely been lost independently in at least three zygnematophycean lineages, although the absence of the usual rRNA operonic synteny in the IR regions of Roya may indicate their de novo origin. Significantly, all zygnematophycean chloroplast genomes have undergone substantial genomic rearrangement, which may be the result of ancient retroelement activity evidenced by the presence of integrase-like and reverse transcriptase-like elements in the Roya chloroplast genome. Our results corroborate the close phylogenetic relationship between Zygnematophyceae and land plants and identify 89 protein-coding genes and 22 introns present in the chloroplast genome at the time of the evolutionary transition of plants to land, all of which can be found in the chloroplast genomes of extant charophytes.

  16. Epigenetic dysregulation underlies radiation-induced transgenerational genome instability in vivo

    International Nuclear Information System (INIS)

    Koturbash, Igor; Baker, Mike; Loree, Jonathan; Kutanzi, Kristy; Hudson, Darryl; Pogribny, Igor; Sedelnikova, Olga; Bonner, William; Kovalchuk, Olga

    2006-01-01

    Purpose: Although modern cancer radiation therapy has led to increased patient survival rates, the risk of radiation treatment-related complications is becoming a growing problem. Among various complications, radiation also poses a threat to the progeny of exposed parents. It causes transgenerational genome instability that is linked to transgenerational carcinogenesis. Although the occurrence of transgenerational genome instability, which manifests as elevated delayed and nontargeted mutation, has been well documented, the mechanisms by which it arises remain obscure. We hypothesized that epigenetic alterations may play a pivotal role in the molecular etiology of transgenerational genome instability. Methods and Materials: We studied the levels of cytosine DNA methylation in somatic tissues of unexposed offspring upon maternal, paternal, or combined parental exposure. Results: We observed a significant loss of global cytosine DNA methylation in the thymus tissue of the offspring upon combined parental exposure. The loss of DNA methylation was paralleled by a significant decrease in the levels of maintenance (DNMT1) and de novo methyltransferases DNMT3a and 3b and methyl-CpG-binding protein MeCP2. Along with profound changes in DNA methylation, we noted a significant accumulation of DNA strand breaks in thymus, which is a radiation carcinogenesis target organ. Conclusions: The observed changes were indicative of a profound epigenetic dysregulation in the offspring, which in turn could lead to genome destabilization and possibly could serve as precursor for transgenerational carcinogenesis. Future studies are clearly needed to address the cellular and carcinogenic repercussions of those changes

  17. Cambridge Healthtech Institute's 5th Annual Conference: impact of genomics on medicine.

    Science.gov (United States)

    Zanders, E D

    2001-08-01

    The recent publications in Nature and Science by the Human Genome Consortium and Celera Genomics, respectively, while being landmark achievements in themselves, have also given pause for thought. A definitive catalogue of human genes is still not available but the broad picture of how humans compare with lower organisms at the genomic level is becoming clearer. The full impact of these findings on the practice of medicine is hard to predict, but research being conducted now, in the early years of the 21st century, will form the basis of future advances in the diagnosis and treatment of disease. Exactly what this will entail is the subject of intense debate, but there are some common starting points that were discussed at this meeting in Munich. The main theme to emerge was the need to move beyond the human genome sequence towards an understanding of proteins and their interactions in complex biological pathways, thereby increasing opportunities for drug discovery through the identification of new targets. The majority of the talks were therefore devoted to the description of technological advances in the analysis of gene and protein expression (and interaction) and in the use of various methods of gene deletion in order to validate individual proteins as drug targets. Perhaps it will still be a few years before it will be possible to report on the application of genomic analyses to routine medical practice at the first point of care for patients but when that happens, the research efforts described here will have been worthwhile.

  18. Genome Architecture and Its Roles in Human Copy Number Variation

    Directory of Open Access Journals (Sweden)

    Lu Chen

    2014-12-01

    Full Text Available Besides single-nucleotide variants in the human genome, large-scale genomic variants, such as copy number variations (CNVs, are being increasingly discovered as a genetic source of human diversity and the pathogenic factors of diseases. Recent experimental findings have shed light on the links between different genome architectures and CNV mutagenesis. In this review, we summarize various genomic features and discuss their contributions to CNV formation. Genomic repeats, including both low-copy and high-copy repeats, play important roles in CNV instability, which was initially known as DNA recombination events. Furthermore, it has been found that human genomic repeats can also induce DNA replication errors and consequently result in CNV mutations. Some recent studies showed that DNA replication timing, which reflects the high-order information of genomic organization, is involved in human CNV mutations. Our review highlights that genome architecture, from DNA sequence to high-order genomic organization, is an important molecular factor in CNV mutagenesis and human genomic instability.

  19. Predictive genomics: a cancer hallmark network framework for predicting tumor clinical phenotypes using genome sequencing data.

    Science.gov (United States)

    Wang, Edwin; Zaman, Naif; Mcgee, Shauna; Milanese, Jean-Sébastien; Masoudi-Nejad, Ali; O'Connor-McCourt, Maureen

    2015-02-01

    Tumor genome sequencing leads to documenting thousands of DNA mutations and other genomic alterations. At present, these data cannot be analyzed adequately to aid in the understanding of tumorigenesis and its evolution. Moreover, we have little insight into how to use these data to predict clinical phenotypes and tumor progression to better design patient treatment. To meet these challenges, we discuss a cancer hallmark network framework for modeling genome sequencing data to predict cancer clonal evolution and associated clinical phenotypes. The framework includes: (1) cancer hallmarks that can be represented by a few molecular/signaling networks. 'Network operational signatures' which represent gene regulatory logics/strengths enable to quantify state transitions and measures of hallmark traits. Thus, sets of genomic alterations which are associated with network operational signatures could be linked to the state/measure of hallmark traits. The network operational signature transforms genotypic data (i.e., genomic alterations) to regulatory phenotypic profiles (i.e., regulatory logics/strengths), to cellular phenotypic profiles (i.e., hallmark traits) which lead to clinical phenotypic profiles (i.e., a collection of hallmark traits). Furthermore, the framework considers regulatory logics of the hallmark networks under tumor evolutionary dynamics and therefore also includes: (2) a self-promoting positive feedback loop that is dominated by a genomic instability network and a cell survival/proliferation network is the main driver of tumor clonal evolution. Surrounding tumor stroma and its host immune systems shape the evolutionary paths; (3) cell motility initiating metastasis is a byproduct of the above self-promoting loop activity during tumorigenesis; (4) an emerging hallmark network which triggers genome duplication dominates a feed-forward loop which in turn could act as a rate-limiting step for tumor formation; (5) mutations and other genomic alterations have

  20. γ-Tocotrienol prevents 5-FU-induced reactive oxygen species production in human oral keratinocytes through the stabilization of 5-FU-induced activation of Nrf2.

    Science.gov (United States)

    Takano, Hideyuki; Momota, Yukihiro; Kani, Kouichi; Aota, Keiko; Yamamura, Yoshiko; Yamanoi, Tomoko; Azuma, Masayuki

    2015-04-01

    Chemotherapy-induced oral mucositis is a common adverse event in patients with oral squamous cell carcinoma, and is initiated through a variety of mechanisms, including the generation of reactive oxygen species (ROS). In this study, we examined the preventive effect of γ-tocotrienol on the 5-FU-induced ROS production in human oral keratinocytes (RT7). We treated RT7 cells with 5-FU and γ-tocotrienol at concentrations of 10 µg/ml and 10 nM, respectively. When cells were treated with 5-FU alone, significant growth inhibition was observed as compared to untreated cells. This inhibition was, in part, due to the ROS gene-rated by 5-FU treatment, because N-acetyl cysteine (NAC), a ROS scavenger, significantly ameliorated the growth of RT7 cells. γ-tocotrienol showed no cytotoxic effect on the growth of RT7 cells. Simultaneous treatment of cells with these agents resulted in the significant recovery of cell growth, owing to the suppression of ROS generation by γ-tocotrienol. Whereas 5-FU stimulated the expression of NF-E2-related factor 2 (Nrf2) protein in the nucleus up to 12 h after treatment of RT7 cells, γ-tocotrienol had no obvious effect on the expression of nuclear Nrf2 protein. Of note, the combined treatment with both agents stabilized the 5-FU-induced nuclear Nrf2 protein expression until 24 h after treatment. In addition, expression of Nrf2-dependent antioxidant genes, such as heme oxygenase-1 (HO-1) and quinone oxidoreductase-1 (NQO-1), was significantly augmented by treatment of cells with both agents. These findings suggest that γ-tocotrienol could prevent 5-FU-induced ROS generation by stabilizing Nrf2 activation, thereby leading to ROS detoxification and cell survival in human oral keratinocytes.

  1. The Whole Genome Assembly and Comparative Genomic Research of Thellungiella parvula (Extremophile Crucifer Mitochondrion

    Directory of Open Access Journals (Sweden)

    Xuelin Wang

    2016-01-01

    Full Text Available The complete nucleotide sequences of the mitochondrial (mt genome of an extremophile species Thellungiella parvula (T. parvula have been determined with the lengths of 255,773 bp. T. parvula mt genome is a circular sequence and contains 32 protein-coding genes, 19 tRNA genes, and three ribosomal RNA genes with a 11.5% coding sequence. The base composition of 27.5% A, 27.5% T, 22.7% C, and 22.3% G in descending order shows a slight bias of 55% AT. Fifty-three repeats were identified in the mitochondrial genome of T. parvula, including 24 direct repeats, 28 tandem repeats (TRs, and one palindromic repeat. Furthermore, a total of 199 perfect microsatellites have been mined with a high A/T content (83.1% through simple sequence repeat (SSR analysis and they were distributed unevenly within this mitochondrial genome. We also analyzed other plant mitochondrial genomes’ evolution in general, providing clues for the understanding of the evolution of organelles genomes in plants. Comparing with other Brassicaceae species, T. parvula is related to Arabidopsis thaliana whose characters of low temperature resistance have been well documented. This study will provide important genetic tools for other Brassicaceae species research and improve yields of economically important plants.

  2. Genome sequence of a 5,310-year-old maize cob provides insights into the early stages of maize domestication

    DEFF Research Database (Denmark)

    Ramos Madrigal, Jazmin; Smith, Bruce D.; Moreno Mayar, José Victor

    2016-01-01

    The complex evolutionary history of maize (Zea mays L. ssp. mays) has been clarified with genomic-level data from modern landraces and wild teosinte grasses [1, 2], augmenting archaeological findings that suggest domestication occurred between 10,000 and 6,250 years ago in southern Mexico [3, 4......]. Maize rapidly evolved under human selection, leading to conspicuous phenotypic transformations, as well as adaptations to varied environments [5]. Still, many questions about the domestication process remain unanswered because modern specimens do not represent the full range of past diversity due...... to abandonment of unproductive lineages, genetic drift, on-going natural selection, and recent breeding activity. To more fully understand the history and spread of maize, we characterized the draft genome of a 5,310-year-old archaeological cob excavated in the Tehuacan Valley of Mexico. We compare this ancient...

  3. Acetone utilization by sulfate-reducing bacteria: draft genome sequence of Desulfococcus biacutus and a proteomic survey of acetone-inducible proteins

    OpenAIRE

    Gutiérrez Acosta, Olga B; Schleheck, David; Schink, Bernhard

    2014-01-01

    Background The sulfate-reducing bacterium Desulfococcus biacutus is able to utilize acetone for growth by an inducible degradation pathway that involves a novel activation reaction for acetone with CO as a co-substrate. The mechanism, enzyme(s) and gene(s) involved in this acetone activation reaction are of great interest because they represent a novel and yet undefined type of activation reaction under strictly anoxic conditions. Results In this study, a draft genome sequence of D. biacutus ...

  4. Complete genome sequence of Pseudomonas rhizosphaerae IH5T (=DSM 16299T), a phosphate-solubilizing rhizobacterium for bacterial biofertilizer.

    Science.gov (United States)

    Kwak, Yunyoung; Jung, Byung Kwon; Shin, Jae-Ho

    2015-01-10

    Pseudomonas rhizosphaerae IH5(T) (=DSM 16299(T)), isolated from the rhizospheric soil of grass growing in Spain, has been reported as a novel species of the genus Pseudomonas harboring insoluble phosphorus solubilizing activity. To understanding the multifunctional biofertilizer better, we report the complete genome sequence of P. rhizosphaerae IH5(T). Copyright © 2014 Elsevier B.V. All rights reserved.

  5. The Genome of the Chicken DT40 Bursal Lymphoma Cell Line

    DEFF Research Database (Denmark)

    Molnar, Janos; Poti, Adam; Pipek, Orsolya

    2014-01-01

    The chicken DT40 cell line is a widely used model system in the study of multiple cellular processes due to the efficiency of homologous gene targeting. The cell line was derived from a bursal lymphoma induced by avian leukosis virus infection. In this study we characterized the genome of the cell...... chicken genomes and the Gallus gallus reference genome, we found no unique mutational processes shaping the DT40 genome except for a mild increase in insertion and deletion events, particularly deletions at tandem repeats. We mapped coding sequence mutations that are unique to the DT40 genome; mutations...

  6. Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection.

    Science.gov (United States)

    Ye, Lin; Wang, Jiaming; Beyer, Ashley I; Teque, Fernando; Cradick, Thomas J; Qi, Zhongxia; Chang, Judy C; Bao, Gang; Muench, Marcus O; Yu, Jingwei; Levy, Jay A; Kan, Yuet Wai

    2014-07-01

    Individuals homozygous for the C-C chemokine receptor type 5 gene with 32-bp deletions (CCR5Δ32) are resistant to HIV-1 infection. In this study, we generated induced pluripotent stem cells (iPSCs) homozygous for the naturally occurring CCR5Δ32 mutation through genome editing of wild-type iPSCs using a combination of transcription activator-like effector nucleases (TALENs) or RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 together with the piggyBac technology. Remarkably, TALENs or CRISPR-Cas9-mediated double-strand DNA breaks resulted in up to 100% targeting of the colonies on one allele of which biallelic targeting occurred at an average of 14% with TALENs and 33% with CRISPR. Excision of the piggyBac using transposase seamlessly reproduced exactly the naturally occurring CCR5Δ32 mutation without detectable exogenous sequences. We differentiated these modified iPSCs into monocytes/macrophages and demonstrated their resistance to HIV-1 challenge. We propose that this strategy may provide an approach toward a functional cure of HIV-1 infection.

  7. Intrinsically disordered region of influenza A NP regulates viral genome packaging via interactions with viral RNA and host PI(4,5)P2.

    Science.gov (United States)

    Kakisaka, Michinori; Yamada, Kazunori; Yamaji-Hasegawa, Akiko; Kobayashi, Toshihide; Aida, Yoko

    2016-09-01

    To be incorporated into progeny virions, the viral genome must be transported to the inner leaflet of the plasma membrane (PM) and accumulate there. Some viruses utilize lipid components to assemble at the PM. For example, simian virus 40 (SV40) targets the ganglioside GM1 and human immunodeficiency virus type 1 (HIV-1) utilizes phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2]. Recent studies clearly indicate that Rab11-mediated recycling endosomes are required for influenza A virus (IAV) trafficking of vRNPs to the PM but it remains unclear how IAV vRNP localized or accumulate underneath the PM for viral genome incorporation into progeny virions. In this study, we found that the second intrinsically disordered region (IDR2) of NP regulates two binding steps involved in viral genome packaging. First, IDR2 facilitates NP oligomer binding to viral RNA to form vRNP. Secondly, vRNP assemble by interacting with PI(4,5)P2 at the PM via IDR2. These findings suggest that PI(4,5)P2 functions as the determinant of vRNP accumulation at the PM. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Possible radioprotective effect of folic acid supplementation on low dose ionizing radiation-induced genomic instability in vitro.

    Science.gov (United States)

    Padula, Gisel; Ponzinibbio, María Virginia; Seoane, Analia I

    2016-08-01

    Ionizing radiation (IR) induces DNA damage through production of single and double-strand breaks and reactive oxygen species (ROS). Folic acid (FA) prevents radiation-induced DNA damage by modification of DNA synthesis and/or repair and as a radical scavenger. We hypothesized that in vitro supplementation with FA will decrease the sensitivity of cells to genetic damage induced by low dose of ionizing radiation. Annexin V, comet and micronucleus assays were performed in cultured CHO cells. After 7 days of pre-treatment with 0, 100, 200 or 300 nM FA, cultures were exposed to radiation (100 mSv). Two un-irradiated controls were executed (0 and 100 nM FA). Data were statistically analyzed with X2-test and linear regression analysis (P 0.05). We observed a significantly decreased frequency of apoptotic cells with the increasing FA concentration (P <0.05). The same trend was observed when analyzing DNA damage and chromosomal instability (P <0.05 for 300 nM). Only micronuclei frequencies showed significant differences for linear regression analysis (R2=94.04; P <0.01). Our results have demonstrated the radioprotective effect of folic acid supplementation on low dose ionizing radiation-induced genomic instability in vitro; folate status should be taken into account when studying the effect of low dose radiation in environmental or occupational exposure.

  9. Draft genome sequences of four uropathogenic escherichia coli 04:H5 isolates (ATCC 700414,700415,700416 and 700417)

    Science.gov (United States)

    Uropathogenic Escherichia coli O4: H5 isolates ATCC 700414, 700415, 700416, and 700417 were recovered from women with first-time urinary tract infections. Here, we report the draft genome sequences for these four E. coli isolates, which are currently being used to validate food safety processing tec...

  10. An Atypical Human Induced Pluripotent Stem Cell Line With a Complex, Stable, and Balanced Genomic Rearrangement Including a Large De Novo 1q Uniparental Disomy

    Science.gov (United States)

    Steichen, Clara; Maluenda, Jérôme; Tosca, Lucie; Luce, Eléanor; Pineau, Dominique; Dianat, Noushin; Hannoun, Zara; Tachdjian, Gérard; Melki, Judith

    2015-01-01

    Human induced pluripotent stem cells (hiPSCs) hold great promise for cell therapy through their use as vital tools for regenerative and personalized medicine. However, the genomic integrity of hiPSCs still raises some concern and is one of the barriers limiting their use in clinical applications. Numerous articles have reported the occurrence of aneuploidies, copy number variations, or single point mutations in hiPSCs, and nonintegrative reprogramming strategies have been developed to minimize the impact of the reprogramming process on the hiPSC genome. Here, we report the characterization of an hiPSC line generated by daily transfections of modified messenger RNAs, displaying several genomic abnormalities. Karyotype analysis showed a complex genomic rearrangement, which remained stable during long-term culture. Fluorescent in situ hybridization analyses were performed on the hiPSC line showing that this karyotype is balanced. Interestingly, single-nucleotide polymorphism analysis revealed the presence of a large 1q region of uniparental disomy (UPD), demonstrating for the first time that UPD can occur in a noncompensatory context during nonintegrative reprogramming of normal fibroblasts. PMID:25650439

  11. The plasticizer butyl benzyl phthalate induces genomic changes in rat mammary gland after neonatal/prepubertal exposure

    Directory of Open Access Journals (Sweden)

    Lamartiniere Coral A

    2007-12-01

    Full Text Available Abstract Background Phthalate esters like n-butyl benzyl phthalate (BBP are widely used plasticizers. BBP has shown endocrine-disrupting properties, thus having a potential effect on hormone-sensitive tissues. The aim of this study is to determine the effect of neonatal/prepubertal exposure (post-natal days 2–20 to BBP on maturation parameters and on the morphology, proliferative index and genomic signature of the rat mammary gland at different ages of development (21, 35, 50 and 100 days. Results Here we show that exposure to BBP increased the uterine weight/body weight ratio at 21 days and decreased the body weight at time of vaginal opening. BBP did not induce significant changes on the morphology of the mammary gland, but increased proliferative index in terminal end buds at 35 days and in lobules 1 at several ages. Moreover, BBP had an effect on the genomic profile of the mammary gland mainly at the end of the exposure (21 days, becoming less prominent thereafter. By this age a significant number of genes related to proliferation and differentiation, communication and signal transduction were up-regulated in the glands of the exposed animals. Conclusion These results suggest that BBP has an effect in the gene expression profile of the mammary gland.

  12. Metagenome-Assembled Genome Sequences of Acetobacterium sp. Strain MES1 and Desulfovibrio sp. Strain MES5 from a Cathode-Associated Acetogenic Microbial Community.

    Science.gov (United States)

    Ross, Daniel E; Marshall, Christopher W; May, Harold D; Norman, R Sean

    2017-09-07

    Draft genome sequences of Acetobacterium sp. strain MES1 and Desulfovibrio sp. strain MES5 were obtained from the metagenome of a cathode-associated community enriched within a microbial electrosynthesis system (MES). The draft genome sequences provide insight into the functional potential of these microorganisms within an MES and a foundation for future comparative analyses. Copyright © 2017 Ross et al.

  13. A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status

    Science.gov (United States)

    Friso, Simonetta; Choi, Sang-Woon; Girelli, Domenico; Mason, Joel B.; Dolnikowski, Gregory G.; Bagley, Pamela J.; Olivieri, Oliviero; Jacques, Paul F.; Rosenberg, Irwin H.; Corrocher, Roberto; Selhub, Jacob

    2002-01-01

    DNA methylation, an essential epigenetic feature of DNA that modulates gene expression and genomic integrity, is catalyzed by methyltransferases that use the universal methyl donor S-adenosyl-l-methionine. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate (5-methylTHF), the methyl donor for synthesis of methionine from homocysteine and precursor of S-adenosyl-l-methionine. In the present study we sought to determine the effect of folate status on genomic DNA methylation with an emphasis on the interaction with the common C677T mutation in the MTHFR gene. A liquid chromatography/MS method for the analysis of nucleotide bases was used to assess genomic DNA methylation in peripheral blood mononuclear cell DNA from 105 subjects homozygous for this mutation (T/T) and 187 homozygous for the wild-type (C/C) MTHFR genotype. The results show that genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine (tHcy) levels (P < 0.01). T/T genotypes had a diminished level of DNA methylation compared with those with the C/C wild-type (32.23 vs.62.24 ng 5-methylcytosine/μg DNA, P < 0.0001). When analyzed according to folate status, however, only the T/T subjects with low levels of folate accounted for the diminished DNA methylation (P < 0.0001). Moreover, in T/T subjects DNA methylation status correlated with the methylated proportion of red blood cell folate and was inversely related to the formylated proportion of red blood cell folates (P < 0.03) that is known to be solely represented in those individuals. These results indicate that the MTHFR C677T polymorphism influences DNA methylation status through an interaction with folate status. PMID:11929966

  14. Strains of bacterial species induce a greatly varied acute adaptive immune response: The contribution of the accessory genome.

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    Uri Sela

    2018-01-01

    Full Text Available A fundamental question in human susceptibility to bacterial infections is to what extent variability is a function of differences in the pathogen species or in individual humans. To focus on the pathogen species, we compared in the same individual the human adaptive T and B cell immune response to multiple strains of two major human pathogens, Staphylococcus aureus and Streptococcus pyogenes. We found wide variability in the acute adaptive immune response induced by various strains of a species, with a unique combination of activation within the two arms of the adaptive response. Further, this was also accompanied by a dramatic difference in the intensity of the specific protective T helper (Th response. Importantly, the same immune response differences induced by the individual strains were maintained across multiple healthy human donors. A comparison of isogenic phage KO strains, demonstrated that of the pangenome, prophages were the major contributor to inter-strain immune heterogeneity, as the T cell response to the remaining "core genome" was noticeably blunted. Therefore, these findings extend and modify the notion of an adaptive response to a pathogenic bacterium, by implying that the adaptive immune response signature of a bacterial species should be defined either per strain or alternatively to the species' 'core genome', common to all of its strains. Further, our results demonstrate that the acquired immune response variation is as wide among different strains within a single pathogenic species as it is among different humans, and therefore may explain in part the clinical heterogeneity observed in patients infected with the same species.

  15. Annealing-induced near-surface ordering in disordered Ga0.5In0.5P

    International Nuclear Information System (INIS)

    Luo, J.S.; Olson, J.M.; Wu, M.

    1995-01-01

    Most samples of Ga 0.5 In 0.5 P grown by metalorganic chemical vapor deposition (MOCVD) on (001)-like surfaces are partially ordered and exhibit distinctive reflectance difference spectral (RDS) features associated with the anisotropic properties of the ordered bulk structure. It is known that the ordering is not a ground-state property of the bulk but is surface-induced during growth. On the other hand, Ga 0.5 In 0.5 P grown by liquid-phase epitaxy (LPE) is completely disordered, and it has been shown that its RD spectrum is essentially featureless. In this article, we present a study of the effects of annealing (in a PH 3 /H 2 atmosphere) on LPE-grown Ga 0.5 In 0.5 P using ex situ and in situ RDS. The annealing temperatures and times used in this study (650 degree C and tens of minutes) have virtually no effect on the bulk optical or structural properties of MOCVD-grown Ga 0.5 In 0.5 P. For LPE-grown Ga 0.5 In 0.5 P, we find that annealing induces bulk-like RDS features at both E 0 and E 1 with line shapes similar to those observed for MOCVD-grown ordered Ga 0.5 In 0.5 P. These bulk-like spectral features are, however, due to near-surface reconstruction of Ga and In because they are effectively quenched by exposure to air. Also, the E 0 feature becomes sharper and both the E 0 and the E 1 features red-shift as the annealing process is prolonged. This indicates that this reconstruction is kinetically limited, presumably by the slow interdiffusion of Ga and In necessary to achieve the ordered bulk-like structure. copyright 1995 American Vacuum SocietyGa 0.5 In 0.5 P

  16. Bipyrimidine Signatures as a Photoprotective Genome Strategy in G + C-rich Halophilic Archaea.

    Science.gov (United States)

    Jones, Daniel L; Baxter, Bonnie K

    2016-09-02

    Halophilic archaea experience high levels of ultraviolet (UV) light in their environments and demonstrate resistance to UV irradiation. DNA repair systems and carotenoids provide UV protection but do not account for the high resistance observed. Herein, we consider genomic signatures as an additional photoprotective strategy. The predominant forms of UV-induced DNA damage are cyclobutane pyrimidine dimers, most notoriously thymine dimers (T^Ts), which form at adjacent Ts. We tested whether the high G + C content seen in halophilic archaea serves a photoprotective function through limiting T nucleotides, and thus T^T lesions. However, this speculation overlooks the other bipyrimidine sequences, all of which capable of forming photolesions to varying degrees. Therefore, we designed a program to determine the frequencies of the four bipyrimidine pairs (5' to 3': TT, TC, CT, and CC) within genomes of halophilic archaea and four other randomized sample groups for comparison. The outputs for each sampled genome were weighted by the intrinsic photoreactivities of each dinucleotide pair. Statistical methods were employed to investigate intergroup differences. Our findings indicate that the UV-resistance seen in halophilic archaea can be attributed in part to a genomic strategy: high G + C content and the resulting bipyrimidine signature reduces the genomic photoreactivity.

  17. Genomic instability: potential contributions to tumour and normal tissue response, and second tumours, after radiotherapy

    International Nuclear Information System (INIS)

    Hendry, Jolyon H.

    2001-01-01

    Purpose: Induced genomic instability generally refers to a type of damage which is transmissible down cell generations, and which results in a persistently enhanced frequency of de novo mutations, chromosomal abnormalities or lethality in a significant fraction of the descendant cell population. The potential contribution of induced genomic instability to tumour and normal tissue response, and second tumours, after radiotherapy, is explored. Results: The phenomenon of spontaneous genomic instability is well known in some rare genetic diseases (e.g. Gorlin's syndrome), and there is evidence in such cases that it can lead to a greater propensity for carcinogenesis (with shortened latency) which is enhanced after irradiation. It is unclear what role induced genomic instability plays in the response of normal individuals, but persistent chromosomal instability has been detected in vivo in lymphocytes and keratinocytes from irradiated normal individuals. Such induced genomic instability might play some role in tumour response in a subset of tumours with specific defects in damage response genes, but again its contribution to radiocurability in the majority of cancer patients is unclear. In normal tissues, genomic instability induced in wild-type cells leading to delayed cell death might contribute to more severe or prolonged early reactions as a consequence of increased cell loss, a longer time required for recovery, and greater residual injury. In tumours, induced genomic instability reflected in delayed reductions in clonogenic capacity might contribute to the radiosensitivity of primary tumours, and also to a lower incidence, longer latency and slower growth rate of recurrences and metastases. Conclusions: The evidence which is reviewed shows that there is little information at present to support these propositions, but what exists is consistent with their expectations. Also, it is not yet clear to what extent mutations associated with genomic instability

  18. Efficient genome editing in hematopoietic stem cells with helper-dependent Ad5/35 vectors expressing site-specific endonucleases under microRNA regulation

    Directory of Open Access Journals (Sweden)

    Kamola Saydaminova

    Full Text Available Genome editing with site-specific endonucleases has implications for basic biomedical research as well as for gene therapy. We generated helper-dependent, capsid-modified adenovirus (HD-Ad5/35 vectors for zinc-finger nuclease (ZFN– or transcription activator-like effector nuclease (TALEN–mediated genome editing in human CD34+ hematopoietic stem cells (HSCs from mobilized adult donors. The production of these vectors required that ZFN and TALEN expression in HD-Ad5/35 producer 293-Cre cells was suppressed. To do this, we developed a microRNA (miRNA-based system for regulation of gene expression based on miRNA expression profiling of 293-Cre and CD34+ cells. Using miR-183-5p and miR-218-5p based regulation of transgene gene expression, we first produced an HD-Ad5/35 vector expressing a ZFN specific to the HIV coreceptor gene ccr5. We demonstrated that HD-Ad5/35.ZFNmiR vector conferred ccr5 knock out in primitive HSC (i.e., long-term culture initiating cells and NOD/SCID repopulating cells. The ccr5 gene disruption frequency achieved in engrafted HSCs found in the bone marrow of transplanted mice is clinically relevant for HIV therapy considering that these cells can give rise to multiple lineages, including all the lineages that represent targets and reservoirs for HIV. We produced a second HD-Ad5/35 vector expressing a TALEN targeting the DNase hypersensitivity region 2 (HS2 within the globin locus control region. This vector has potential for targeted gene correction in hemoglobinopathies. The miRNA regulated HD-Ad5/35 vector platform for expression of site-specific endonucleases has numerous advantages over currently used vectors as a tool for genome engineering of HSCs for therapeutic purposes.

  19. Structural dynamics of retroviral genome and the packaging.

    Science.gov (United States)

    Miyazaki, Yasuyuki; Miyake, Ariko; Nomaguchi, Masako; Adachi, Akio

    2011-01-01

    Retroviruses can cause diseases such as AIDS, leukemia, and tumors, but are also used as vectors for human gene therapy. All retroviruses, except foamy viruses, package two copies of unspliced genomic RNA into their progeny viruses. Understanding the molecular mechanisms of retroviral genome packaging will aid the design of new anti-retroviral drugs targeting the packaging process and improve the efficacy of retroviral vectors. Retroviral genomes have to be specifically recognized by the cognate nucleocapsid domain of the Gag polyprotein from among an excess of cellular and spliced viral mRNA. Extensive virological and structural studies have revealed how retroviral genomic RNA is selectively packaged into the viral particles. The genomic area responsible for the packaging is generally located in the 5' untranslated region (5' UTR), and contains dimerization site(s). Recent studies have shown that retroviral genome packaging is modulated by structural changes of RNA at the 5' UTR accompanied by the dimerization. In this review, we focus on three representative retroviruses, Moloney murine leukemia virus, human immunodeficiency virus type 1 and 2, and describe the molecular mechanism of retroviral genome packaging.

  20. The RNA 5 of Prunus necrotic ringspot virus is a biologically inactive copy of the 3'-UTR of the genomic RNA 3.

    Science.gov (United States)

    Di Terlizzi, B; Skrzeczkowski, L J; Mink, G I; Scott, S W; Zimmerman, M T

    2001-01-01

    In addition to the four RNAs known to be encapsidated by Prunus necrotic ringspot virus (PNRSV) and Apple mosaic virus (ApMV), an additional small RNA (RNA 5) was present in purified preparations of several isolates of both viruses. RNA 5 was always produced following infection of a susceptible host by an artificial mixture of RNAs 1, 2, 3, and 4 indicating that it was a product of viral replication. RNA 5 does not activate the infectivity of mixtures that contain the three genomic RNAs (RNA 1 + RNA 2 + RNA 3) nor does it appear to modify symptom expression. Results from hybridization studies suggested that RNA 5 had partial sequence homology with RNAs 1, 2, 3, and 4. Cloning and sequencing the RNA 5 of isolate CH 57/1-M of PNRSV, and the 3' termini of the RNA 1, RNA 2 and RNA 3 of this isolate indicated that it was a copy of the 3' untranslated terminal region (3'-UTR) of the genomic RNA 3.

  1. Air pollution and genomic instability: The role of particulate matter in lung carcinogenesis

    International Nuclear Information System (INIS)

    Santibáñez-Andrade, Miguel; Quezada-Maldonado, Ericka Marel; Osornio-Vargas, Álvaro; Sánchez-Pérez, Yesennia; García-Cuellar, Claudia M.

    2017-01-01

    In this review, we summarize and discuss the evidence regarding the interaction between air pollution, especially particulate matter (PM), and genomic instability. PM has been widely studied in the context of several diseases, and its role in lung carcinogenesis gained relevance due to an increase in cancer cases for which smoking does not seem to represent the main risk factor. According to epidemiological and toxicological evidence, PM acts as a carcinogenic factor in humans, inducing high rates of genomic alterations. Here, we discuss not only how PM is capable of inducing genomic instability during the carcinogenic process but also how our genetic background influences the response to the sources of damage. - Highlights: • Air pollution represents a worldwide problem with impact on human health. • Particulate matter (PM) has a recognized carcinogenic potential in humans. • Lung cancer susceptibility depends on gene-environment interactions. • Epidemiological and experimental evidence links PM exposure to genomic instability. • PM and genomic instability are co-dependent factors during cancer continuum. - We summarize the association between particulate matter (a component of air pollution) and genomic instability as well as discuss how new strategies to study the impact of air pollution on genomic instability and lung-cancer development could improve our understanding of the lung-cancer genome.

  2. [Loss of total 5-methylcytosine from the genome during cell culture aging coincides with the Hayflick limit].

    Science.gov (United States)

    Mazin, A L

    1993-01-01

    Analyzing the data about the age-related 5-methylcytosine (5mC) loss from DNA of cell cultures, the following conclusions have been made: 1. The rate of 5mC loss from DNA does not depend on the cell donor age; it remains constant during the logarithmic phase of cell growth, and may vary significantly in different cell lines. 2. The rate is inversely proportional to their Hayflick limit and to the species lifespan of cell donors. 3. In immortal cell lines the 5mC content in DNA is stable or increases with aging. 4. Hayflick limit estimations coincide with or are lower than the number of cell population doublings that corresponds to all 5mC loss from cell genome. A simple and fast method has been proposed for Hayflick limit prognostication by analysis of the rate of DNA hypomethylation. It may be used for early diagnosis of precrisis and immortal cell lines. Evidence has been obtained that age-dependent 5mC loss from DNA is the result of accumulating 5mC-->T+C substitutions that occur during DNA methylation in every cell division. The loss of all genomic 5mC residues during the lifespan may correspond to accumulation of about 3 x 10(6) 5mC-->T transitions or, on average, one mutation per gene. This may be one of the main reasons of the "catastrophe of errors" and cessation of cell proliferation. It is calculated that the rate of 5mC-->T transitions in normal cells may be 2.3 x 10(-5) per site in each cell doubling in human, 6 x 10(-5) in hamster, and 4.6 x 10(-4) in mouse. DNA methylation as a generator of mutations may be a "counter" of cell divisions and thus be one of the molecular mechanisms of the Hayflick phenomenon. The conclusion is made that the DNA methylation system may be considered as a genetically programmed mechanism for accumulating mutations during cell aging.

  3. Complete genome sequence of Pseudomonas citronellolis P3B5, a candidate for microbial phyllo-remediation of hydrocarbon-contaminated sites.

    Science.gov (United States)

    Remus-Emsermann, Mitja N P; Schmid, Michael; Gekenidis, Maria-Theresia; Pelludat, Cosima; Frey, Jürg E; Ahrens, Christian H; Drissner, David

    2016-01-01

    Pseudomonas citronellolis is a Gram negative, motile gammaproteobacterium belonging to the order Pseudomonadales and the family Pseudomonadaceae . We isolated strain P3B5 from the phyllosphere of basil plants ( Ocimum basilicum L.). Here we describe the physiology of this microorganism, its full genome sequence, and detailed annotation. The 6.95 Mbp genome contains 6071 predicted protein coding sequences and 96 RNA coding sequences. P. citronellolis has been the subject of many studies including the investigation of long-chain aliphatic compounds and terpene degradation. Plant leaves are covered by long-chain aliphates making up a waxy layer that is associated with the leaf cuticle. In addition, basil leaves are known to contain high amounts of terpenoid substances, hinting to a potential nutrient niche that might be exploited by P. citronellolis . Furthermore, the isolated strain exhibited resistance to several antibiotics. To evaluate the potential of this strain as source of transferable antibiotic resistance genes on raw consumed herbs we therefore investigated if those resistances are encoded on mobile genetic elements. The availability of the genome will be helpful for comparative genomics of the phylogenetically broad pseudomonads, in particular with the sequence of the P. citronellolis type strain PRJDB205 not yet publicly available. The genome is discussed with respect to a phyllosphere related lifestyle, aliphate and terpenoid degradation, and antibiotic resistance.

  4. Collateral Effects of Antibiotics: Carbadox and Metronidazole Induce VSH-1 and Facilitate Gene Transfer among Brachyspira hyodysenteriae Strains▿

    Science.gov (United States)

    Stanton, Thaddeus B.; Humphrey, Samuel B.; Sharma, Vijay K.; Zuerner, Richard L.

    2008-01-01

    Brachyspira hyodysenteriae is an anaerobic spirochete and the etiologic agent of swine dysentery. The genome of this spirochete contains a mitomycin C-inducible, prophage-like gene transfer agent designated VSH-1. VSH-1 particles package random 7.5-kb fragments of the B. hyodysenteriae genome and transfer genes between B. hyodysenteriae cells. The chemicals and conditions inducing VSH-1 production are largely unknown. Antibiotics used in swine management and stressors inducing traditional prophages might induce VSH-1 and thereby stimulate lateral gene transfer between B. hyodysenteriae cells. In these studies, VSH-1 induction was initially detected by a quantitative real-time reverse transcriptase PCR assay evaluating increased transcription of hvp38 (VSH-1 head protein gene). VSH-1 induction was confirmed by detecting VSH-1-associated 7.5-kb DNA and VSH-1 particles in B. hyodysenteriae cultures. Nine antibiotics (chlortetracycline, lincomycin, tylosin, tiamulin, virginiamycin, ampicillin, ceftriaxone, vancomycin, and florfenicol) at concentrations affecting B. hyodysenteriae growth did not induce VSH-1 production. By contrast, VSH-1 was detected in B. hyodysenteriae cultures treated with mitomycin C (10 μg/ml), carbadox (0.5 μg/ml), metronidazole (0.5 μg/ml), and H2O2 (300 μM). Carbadox- and metronidazole-induced VSH-1 particles transmitted tylosin and chloramphenicol resistance determinants between B. hyodysenteriae strains. The results of these studies suggest that certain antibiotics may induce the production of prophage or prophage-like elements by intestinal bacteria and thereby impact intestinal microbial ecology. PMID:18359835

  5. Plum pox virus (PPV) genome expression in genetically engineered RNAi plants

    Science.gov (United States)

    An important approach to controlling sharka disease caused by Plum pox virus (PPV) is the development of PPV resistant plants using small interfering RNAs (siRNA) technology. In order to evaluate siRNA induced gene silencing, we studied, based on knowledge of the PPV genome sequence, virus genome t...

  6. Bcl-2 associated athanogene 5 (Bag5) is overexpressed in prostate cancer and inhibits ER-stress induced apoptosis

    International Nuclear Information System (INIS)

    Bruchmann, Anja; Roller, Corinna; Walther, Tamara Vanessa; Schäfer, Georg; Lehmusvaara, Sara; Visakorpi, Tapio; Klocker, Helmut; Cato, Andrew C B; Maddalo, Danilo

    2013-01-01

    The Bag (Bcl-2 associated athanogene) family of proteins consists of 6 members sharing a common, single-copied Bag domain through which they interact with the molecular chaperone Hsp70. Bag5 represents an exception in the Bag family since it consists of 5 Bag domains covering the whole protein. Bag proteins like Bag1 and Bag3 have been implicated in tumor growth and survival but it is not known whether Bag5 also exhibits this function. Bag5 mRNA and protein expression levels were investigated in prostate cancer patient samples using real-time PCR and immunoblot analyses. In addition immunohistological studies were carried out to determine the expression of Bag5 in tissue arrays. Analysis of Bag5 gene expression was carried out using one-way ANOVA and Bonferroni’s Multiple Comparison test. The mean values of the Bag5 stained cells in the tissue array was analyzed by Mann-Whitney test. Functional studies of the role of Bag5 in prostate cancer cell lines was performed using overexpression and RNA interference analyses. Our results show that Bag5 is overexpressed in malignant prostate tissue compared to benign samples. In addition we could show that Bag5 levels are increased following endoplasmic reticulum (ER)-stress induction, and Bag5 relocates from the cytoplasm to the ER during this process. We also demonstrate that Bag5 interacts with the ER-resident chaperone GRP78/BiP and enhances its ATPase activity. Bag5 overexpression in 22Rv.1 prostate cancer cells inhibited ER-stress induced apoptosis in the unfolded protein response by suppressing PERK-eIF2-ATF4 activity while enhancing the IRE1-Xbp1 axis of this pathway. Cells expressing high levels of Bag5 showed reduced sensitivity to apoptosis induced by different agents while Bag5 downregulation resulted in increased stress-induced cell death. We have therefore shown that Bag5 is overexpressed in prostate cancer and plays a role in ER-stress induced apoptosis. Furthermore we have identified GRP78/BiP as a novel

  7. Whole-Genome Sequencing and iPLEX MassARRAY Genotyping Map an EMS-Induced Mutation Affecting Cell Competition in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Chang-Hyun Lee

    2016-10-01

    Full Text Available Cell competition, the conditional loss of viable genotypes only when surrounded by other cells, is a phenomenon observed in certain genetic mosaic conditions. We conducted a chemical mutagenesis and screen to recover new mutations that affect cell competition between wild-type and RpS3 heterozygous cells. Mutations were identified by whole-genome sequencing, making use of software tools that greatly facilitate the distinction between newly induced mutations and other sources of apparent sequence polymorphism, thereby reducing false-positive and false-negative identification rates. In addition, we utilized iPLEX MassARRAY for genotyping recombinant chromosomes. These approaches permitted the mapping of a new mutation affecting cell competition when only a single allele existed, with a phenotype assessed only in genetic mosaics, without the benefit of complementation with existing mutations, deletions, or duplications. These techniques expand the utility of chemical mutagenesis and whole-genome sequencing for mutant identification. We discuss mutations in the Atm and Xrp1 genes identified in this screen.

  8. Genome Editing in Induced Pluripotent Stem Cells using CRISPR/Cas9.

    Science.gov (United States)

    Ben Jehuda, Ronen; Shemer, Yuval; Binah, Ofer

    2018-06-01

    The development of the reprogramming technology led to generation of induced Pluripotent Stem Cells (iPSC) from a variety of somatic cells. Ever since, fast growing knowledge of different efficient protocols enabled the differentiation of these iPSCs into different cells types utilized for disease modeling. Indeed, iPSC-derived cells have been increasingly used for investigating molecular and cellular pathophysiological mechanisms underlying inherited diseases. However, a major barrier in the field of iPSC-based disease modeling relies on discriminating between the effects of the causative mutation and the genetic background of these cells. In the past decade, researchers have made great improvement in genome editing techniques, with one of the latest being CRISPR/Cas9. Using a single non-sequence specific protein combined with a small guiding RNA molecule, this state-of-the-art approach enables modifications of genes with high efficiency and accuracy. By so doing, this technique enables the generation of isogenic controls or isogenic mutated cell lines in order to focus on the pathologies caused by a specific mutation. In this article, we review the latest studies combining iPSC and CRISPR/Cas9 technologies for the investigation of the molecular and cellular mechanisms underlying inherited diseases including immunological, metabolic, hematological, neurodegenerative and cardiac diseases.

  9. Task 1.5 Genomic Shift and Drift Trends of Emerging Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    Borucki, M

    2010-01-05

    The Lawrence Livermore National Laboratory (LLNL) Bioinformatics group has recently taken on a role in DTRA's Transformation Medical Technologies Initiative (TMTI). The high-level goal of TMTI is to accelerate the development of broad-spectrum countermeasures. To achieve those goals, TMTI has a near term need to conduct analyses of genomic shift and drift trends of emerging pathogens, with a focused eye on select agent pathogens, as well as antibiotic and virulence markers. Most emerging human pathogens are zoonotic viruses with a genome composed of RNA. The high mutation rate of the replication enzymes of RNA viruses contributes to sequence drift and provides one mechanism for these viruses to adapt to diverse hosts (interspecies transmission events) and cause new human and zoonotic diseases. Additionally, new viral pathogens frequently emerge due to genetic shift (recombination and segment reassortment) which allows for dramatic genotypic and phenotypic changes to occur rapidly. Bacterial pathogens also evolve via genetic drift and shift, although sequence drift generally occurs at a much slower rate for bacteria as compared to RNA viruses. However, genetic shift such as lateral gene transfer and inter- and intragenomic recombination enables bacteria to rapidly acquire new mechanisms of survival and antibiotic resistance. New technologies such as rapid whole genome sequencing of bacterial genomes, ultra-deep sequencing of RNA virus populations, metagenomic studies of environments rich in antibiotic resistance genes, and the use of microarrays for the detection and characterization of emerging pathogens provide mechanisms to address the challenges posed by the rapid emergence of pathogens. Bioinformatic algorithms that enable efficient analysis of the massive amounts of data generated by these technologies as well computational modeling of protein structures and evolutionary processes need to be developed to allow the technology to fulfill its potential.

  10. Error-free versus mutagenic processing of genomic uracil--relevance to cancer.

    Science.gov (United States)

    Krokan, Hans E; Sætrom, Pål; Aas, Per Arne; Pettersen, Henrik Sahlin; Kavli, Bodil; Slupphaug, Geir

    2014-07-01

    Genomic uracil is normally processed essentially error-free by base excision repair (BER), with mismatch repair (MMR) as an apparent backup for U:G mismatches. Nuclear uracil-DNA glycosylase UNG2 is the major enzyme initiating BER of uracil of U:A pairs as well as U:G mismatches. Deficiency in UNG2 results in several-fold increases in genomic uracil in mammalian cells. Thus, the alternative uracil-removing glycosylases, SMUG1, TDG and MBD4 cannot efficiently complement UNG2-deficiency. A major function of SMUG1 is probably to remove 5-hydroxymethyluracil from DNA with general back-up for UNG2 as a minor function. TDG and MBD4 remove deamination products U or T mismatched to G in CpG/mCpG contexts, but may have equally or more important functions in development, epigenetics and gene regulation. Genomic uracil was previously thought to arise only from spontaneous cytosine deamination and incorporation of dUMP, generating U:G mismatches and U:A pairs, respectively. However, the identification of activation-induced cytidine deaminase (AID) and other APOBEC family members as DNA-cytosine deaminases has spurred renewed interest in the processing of genomic uracil. Importantly, AID triggers the adaptive immune response involving error-prone processing of U:G mismatches, but also contributes to B-cell lymphomagenesis. Furthermore, mutational signatures in a substantial fraction of other human cancers are consistent with APOBEC-induced mutagenesis, with U:G mismatches as prime suspects. Mutations can be caused by replicative polymerases copying uracil in U:G mismatches, or by translesion polymerases that insert incorrect bases opposite abasic sites after uracil-removal. In addition, kataegis, localized hypermutations in one strand in the vicinity of genomic rearrangements, requires APOBEC protein, UNG2 and translesion polymerase REV1. What mechanisms govern error-free versus error prone processing of uracil in DNA remains unclear. In conclusion, genomic uracil is an

  11. Evidence that personal genome testing enhances student learning in a course on genomics and personalized medicine.

    Directory of Open Access Journals (Sweden)

    Keyan Salari

    Full Text Available An emerging debate in academic medical centers is not about the need for providing trainees with fundamental education on genomics, but rather the most effective educational models that should be deployed. At Stanford School of Medicine, a novel hands-on genomics course was developed in 2010 that provided students the option to undergo personal genome testing as part of the course curriculum. We hypothesized that use of personal genome testing in the classroom would enhance the learning experience of students. No data currently exist on how such methods impact student learning; thus, we surveyed students before and after the course to determine its impact. We analyzed responses using paired statistics from the 31 medical and graduate students who completed both pre-course and post-course surveys. Participants were stratified by those who did (N = 23 or did not (N = 8 undergo personal genome testing. In reflecting on the experience, 83% of students who underwent testing stated that they were pleased with their decision compared to 12.5% of students who decided against testing (P = 0.00058. Seventy percent of those who underwent personal genome testing self-reported a better understanding of human genetics on the basis of having undergone testing. Further, students who underwent personal genome testing demonstrated an average 31% increase in pre- to post-course scores on knowledge questions (P = 3.5×10(-6; this was significantly higher (P = 0.003 than students who did not undergo testing, who showed a non-significant improvement. Undergoing personal genome testing and using personal genotype data in the classroom enhanced students' self-reported and assessed knowledge of genomics, and did not appear to cause significant anxiety. At least for self-selected students, the incorporation of personal genome testing can be an effective educational tool to teach important concepts of clinical genomic testing.

  12. BAC CGH-array identified specific small-scale genomic imbalances in diploid DMBA-induced rat mammary tumors

    International Nuclear Information System (INIS)

    Samuelson, Emma; Karlsson, Sara; Partheen, Karolina; Nilsson, Staffan; Szpirer, Claude; Behboudi, Afrouz

    2012-01-01

    Development of breast cancer is a multistage process influenced by hormonal and environmental factors as well as by genetic background. The search for genes underlying this malignancy has recently been highly productive, but the etiology behind this complex disease is still not understood. In studies using animal cancer models, heterogeneity of the genetic background and environmental factors is reduced and thus analysis and identification of genetic aberrations in tumors may become easier. To identify chromosomal regions potentially involved in the initiation and progression of mammary cancer, in the present work we subjected a subset of experimental mammary tumors to cytogenetic and molecular genetic analysis. Mammary tumors were induced with DMBA (7,12-dimethylbenz[a]anthrazene) in female rats from the susceptible SPRD-Cu3 strain and from crosses and backcrosses between this strain and the resistant WKY strain. We first produced a general overview of chromosomal aberrations in the tumors using conventional kartyotyping (G-banding) and Comparative Genome Hybridization (CGH) analyses. Particular chromosomal changes were then analyzed in more details using an in-house developed BAC (bacterial artificial chromosome) CGH-array platform. Tumors appeared to be diploid by conventional karyotyping, however several sub-microscopic chromosome gains or losses in the tumor material were identified by BAC CGH-array analysis. An oncogenetic tree analysis based on the BAC CGH-array data suggested gain of rat chromosome (RNO) band 12q11, loss of RNO5q32 or RNO6q21 as the earliest events in the development of these mammary tumors. Some of the identified changes appear to be more specific for DMBA-induced mammary tumors and some are similar to those previously reported in ACI rat model for estradiol-induced mammary tumors. The later group of changes is more interesting, since they may represent anomalies that involve genes with a critical role in mammary tumor development. Genetic

  13. Super-resolution imaging of a 2.5 kb non-repetitive DNA in situ in the nuclear genome using molecular beacon probes

    Science.gov (United States)

    Ni, Yanxiang; Cao, Bo; Ma, Tszshan; Niu, Gang; Huo, Yingdong; Huang, Jiandong; Chen, Danni; Liu, Yi; Yu, Bin; Zhang, Michael Q; Niu, Hanben

    2017-01-01

    High-resolution visualization of short non-repetitive DNA in situ in the nuclear genome is essential for studying looping interactions and chromatin organization in single cells. Recent advances in fluorescence in situ hybridization (FISH) using Oligopaint probes have enabled super-resolution imaging of genomic domains with a resolution limit of 4.9 kb. To target shorter elements, we developed a simple FISH method that uses molecular beacon (MB) probes to facilitate the probe-target binding, while minimizing non-specific fluorescence. We used three-dimensional stochastic optical reconstruction microscopy (3D-STORM) with optimized imaging conditions to efficiently distinguish sparsely distributed Alexa-647 from background cellular autofluorescence. Utilizing 3D-STORM and only 29–34 individual MB probes, we observed 3D fine-scale nanostructures of 2.5 kb integrated or endogenous unique DNA in situ in human or mouse genome, respectively. We demonstrated our MB-based FISH method was capable of visualizing the so far shortest non-repetitive genomic sequence in 3D at super-resolution. DOI: http://dx.doi.org/10.7554/eLife.21660.001 PMID:28485713

  14. Genomics of aerobic cellulose utilization systems in actinobacteria.

    Directory of Open Access Journals (Sweden)

    Iain Anderson

    Full Text Available Cellulose degrading enzymes have important functions in the biotechnology industry, including the production of biofuels from lignocellulosic biomass. Anaerobes including Clostridium species organize cellulases and other glycosyl hydrolases into large complexes known as cellulosomes. In contrast, aerobic actinobacteria utilize systems comprised of independently acting enzymes, often with carbohydrate binding domains. Numerous actinobacterial genomes have become available through the Genomic Encyclopedia of Bacteria and Archaea (GEBA project. We identified putative cellulose-degrading enzymes belonging to families GH5, GH6, GH8, GH9, GH12, GH48, and GH51 in the genomes of eleven members of the actinobacteria. The eleven organisms were tested in several assays for cellulose degradation, and eight of the organisms showed evidence of cellulase activity. The three with the highest cellulase activity were Actinosynnema mirum, Cellulomonas flavigena, and Xylanimonas cellulosilytica. Cellobiose is known to induce cellulolytic enzymes in the model organism Thermobifida fusca, but only Nocardiopsis dassonvillei showed higher cellulolytic activity in the presence of cellobiose. In T. fusca, cellulases and a putative cellobiose ABC transporter are regulated by the transcriptional regulator CelR. Nine organisms appear to use the CelR site or a closely related binding site to regulate an ABC transporter. In some, CelR also regulates cellulases, while cellulases are controlled by different regulatory sites in three organisms. Mining of genome data for cellulose degradative enzymes followed by experimental verification successfully identified several actinobacteria species which were not previously known to degrade cellulose as cellulolytic organisms.

  15. MicroRNA-486-5p suppresses TGF-ß2-induced proliferation ...

    Indian Academy of Sciences (India)

    Bei Liu

    2017-09-27

    486-5p) on TGF-b2-induced proliferation, invasion ... The human lens epithelial cell line was purchased from ... MiR-486-5p mimics and negative control mimics were ... specific binding was blocked using 5% non-fat milk for 1.

  16. MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells | Office of Cancer Genomics

    Science.gov (United States)

    The discovery of cancer dependencies has the potential to inform therapeutic strategies and to identify putative drug targets. Integrating data from comprehensive genomic profiling of cancer cell lines and from functional characterization of cancer cell dependencies, we discovered that loss of the enzyme methylthioadenosine phosphorylase (MTAP) confers a selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor suppressor gene, CDKN2A.

  17. ''After the Genome 5 Conference'' to be held October 6-10, 1999 in Jackson Hole, Wyoming

    Energy Technology Data Exchange (ETDEWEB)

    Roger Brent

    1999-10-06

    entire organisms become available.Since then, many more biologists have become cognizant of the issues raised by this future, and, in response, the organizers intend to distinguish this meeting from other ''postgenomic'' meetings by bringing together intellectuals from subject fields far outside of conventional biology with the expectation that this will help focus thinking beyond the immediate future. To this end, After the Genome 5 will bring together industrial and university researchers, including: (1) Physicists, chemists, and engineers who are devising and using new data gathering techniques, such as microarrays, protein mass spectrometry, and single molecule measurements (2) Computer scientists from fields as diverse as geology and wargames, who have experience moving from broad knowledge of systems to analysis that results in models and simulations (3) Neurobiologists and computer scientists who combine physiological experimentation and computer modeling to understand single cells and small networks of cells (4) Biologists who are trying to model genetic networks (5) All-around visionary thinkers (6) policy makers, to suggest how to convey any good ideas to organizations that can commit resources to them.

  18. Structural dynamics of retroviral genome and the packaging

    Directory of Open Access Journals (Sweden)

    Yasuyuki eMiyazaki

    2011-12-01

    Full Text Available Retroviruses can cause diseases such as AIDS, leukemia and tumors, but are also used as vectors for human gene therapy. All retroviruses, except foamy viruses, package two copies of unspliced genomic RNA into their progeny viruses. Understanding the molecular mechanisms of retroviral genome packaging will aid the design of new anti-retroviral drugs targeting the packaging process and improve the efficacy of retroviral vectors. Retroviral genomes have to be specifically recognized by the cognate nucleocapsid (NC domain of the Gag polyprotein from among an excess of cellular and spliced viral mRNA. Extensive virological and structural studies have revealed how retroviral genomic RNA is selectively packaged into the viral particles. The genomic area responsible for the packaging is generally located in the 5’ untranslated region (5’ UTR, and contains dimerization site(s. Recent studies have shown that retroviral genome packaging is modulated by structural changes of RNA at the 5’ UTR accompanied by the dimerization. In this review, we focus on three representative retroviruses, Moloney murine leukemia virus (MoMLV, human immunodeficiency virus type 1 (HIV-1 and 2 (HIV-2, and describe the molecular mechanism of retroviral genome packaging.

  19. Genome Sequences of Marine Shrimp Exopalaemon carinicauda Holthuis Provide Insights into Genome Size Evolution of Caridea.

    Science.gov (United States)

    Yuan, Jianbo; Gao, Yi; Zhang, Xiaojun; Wei, Jiankai; Liu, Chengzhang; Li, Fuhua; Xiang, Jianhai

    2017-07-05

    Crustacea, particularly Decapoda, contains many economically important species, such as shrimps and crabs. Crustaceans exhibit enormous (nearly 500-fold) variability in genome size. However, limited genome resources are available for investigating these species. Exopalaemon carinicauda Holthuis, an economical caridean shrimp, is a potential ideal experimental animal for research on crustaceans. In this study, we performed low-coverage sequencing and de novo assembly of the E. carinicauda genome. The assembly covers more than 95% of coding regions. E. carinicauda possesses a large complex genome (5.73 Gb), with size twice higher than those of many decapod shrimps. As such, comparative genomic analyses were implied to investigate factors affecting genome size evolution of decapods. However, clues associated with genome duplication were not identified, and few horizontally transferred sequences were detected. Ultimately, the burst of transposable elements, especially retrotransposons, was determined as the major factor influencing genome expansion. A total of 2 Gb repeats were identified, and RTE-BovB, Jockey, Gypsy, and DIRS were the four major retrotransposons that significantly expanded. Both recent (Jockey and Gypsy) and ancestral (DIRS) originated retrotransposons responsible for the genome evolution. The E. carinicauda genome also exhibited potential for the genomic and experimental research of shrimps.

  20. Detection of γ-ray-induced DNA damages in malformed dominant lethal embryos of the Japanese medaka (Oryzias latipes) using AP-PCR fingerprinting

    International Nuclear Information System (INIS)

    Kubota, Yoshiko; Shimada, Atsuko; Shima, Akihiro

    1992-01-01

    Adult male fish of the medaka HNI strain exposed to 9.5 Gy or 19 Gy (0.95 Gy/min) of γ-rays were mated with non-irradiated female fish of the Hd-rR strain. Genomic DNA was prepared from malformed individual embryos which were expected to be dominant lethal and used for AP-PCR fingerprinting. By the use of a part of the T3 promoter sequence (20 mer), which is not found in the medaka genome as an arbitrary primer, polymorphisms were found in genomic fingerprints which could distinguish the parental strains. On the other hand, fingerprints of F1 hybrids were found to be the sum of those of their parents. Based on these findings, the fingerprints of genomic DNA of each severely malformed embryo were analyzed, because it was expected that radiation-induced genomic damages resulting in severe malformation and eventually in dominant lethals should be detected as changes in paternal fingerprints of F1 hybrids. Indeed, changes were found in genomic DNA as loss of some paternal bands in fingerprints of malformed embryos. One of 10 malformed embryos obtained from 9.5 Gy γ-irradiated males had lost 5 bands. These results indicated a possibility that quantitative as well as qualitative estimation of γ-ray-induced DNA damages can be made by this method which does not require the functional selection based on a specific target gene. (author). 16 refs., 3 figs., 1 tab

  1. Gene activation by induced DNA rearrangements

    International Nuclear Information System (INIS)

    Schnipper, L.E.; Chan, V.; Sedivy, J.; Jat, P.; Sharp, P.A.

    1989-01-01

    A murine cell line (EN/NIH) containing the retroviral vector ZIPNeoSV(x)1 that was modified by deletion of the enhancer elements in the viral long terminal repeats has been used as an assay system to detect induced DNA rearrangements that result in activation of a transcriptionally silent reporter gene encoded by the viral genome. The spontaneous frequency of G418 resistance is less than 10(-7), whereas exposure to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or the combination of UV irradiation plus TPA resulted in the emergence of drug resistant cell lines at a frequency of 5 per 10(6) and 67 per 10(6) cells, respectively. In several of the cell lines that were analyzed a low level of amplification of one of the two parental retroviral integrants was observed, whereas in others no alteration in the region of the viral genome was detected. To determine the effect of the SV40 large T antigen on induced DNA rearrangements, EN/NIH cells were transfected with a temperature sensitive (ts) mutant of SV40 T. Transfectants were maintained at the permissive temperature (33 degrees C) for varying periods of time (1-5 days) in order to vary SV40 T antigen exposure, after which they were shifted to 39.5 degrees C for selection in G418. The frequency of emergence of drug resistant cell clones increased with duration of exposure to large T antigen (9-52 per 10(6) cells over 1-5 days, respectively), and all cell lines analyzed demonstrated DNA rearrangements in the region of the neo gene. A novel 18-kilobase pair XbaI fragment was cloned from one cell line which revealed the presence of a 2.0-kilobase pair EcoRI segment containing an inverted duplication which hybridized to neo sequences. It is likely that the observed rearrangement was initiated by the specific binding of large T antigen to the SV40 origin of replication encoded within the viral genome

  2. A systematic review of the pathophysiology of 5-fluorouracil-induced cardiotoxicity

    DEFF Research Database (Denmark)

    Polk, Anne; Vistisen, Kirsten; Vaage-Nilsen, Merete

    2014-01-01

    BACKGROUND: Cardiotoxicity is a serious side effect to treatment with 5-fluorouracil (5-FU), but the underlying mechanisms are not fully understood. The objective of this systematic review was to evaluate the pathophysiology of 5-FU- induced cardiotoxicity. METHODS: We systematically searched Pub...

  3. Cooperation between Epstein-Barr Virus Immune Evasion Proteins Spreads Protection from CD8+ T Cell Recognition across All Three Phases of the Lytic Cycle

    Science.gov (United States)

    Quinn, Laura L.; Zuo, Jianmin; Abbott, Rachel J. M.; Shannon-Lowe, Claire; Tierney, Rosemary J.; Hislop, Andrew D.; Rowe, Martin

    2014-01-01

    CD8+ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L), interference by BILF1 increases with progression through lytic cycle (IEevasion functions are actually relevant in the context of lytic virus replication, and secondly identify lytic-cycle phase-specific effects that provide mechanistic insight into the immunodominance pattern seen for CD8+ T cell responses to EBV lytic antigens. PMID:25144360

  4. DNA rearrangements from γ-irradiated normal human fibroblasts preferentially occur in transcribed regions of the genome

    International Nuclear Information System (INIS)

    Forrester, H.B.; Radford, I.R.

    2003-01-01

    Full text: DNA rearrangement events leading to chromosomal aberrations are central to ionizing radiation-induced cell death. Although DNA double-strand breaks are probably the lesion that initiates formation of chromosomal aberrations, little is understood about the molecular mechanisms that generate and modulate DNA rearrangement. Examination of the sequences that flank sites of DNA rearrangement may provide information regarding the processes and enzymes involved in rearrangement events. Accordingly, we developed a method using inverse PCR that allows the detection and sequencing of putative radiation-induced DNA rearrangements in defined regions of the human genome. The method can detect single copies of a rearrangement event that has occurred in a particular region of the genome and, therefore, DNA rearrangement detection does not require survival and continued multiplication of the affected cell. Ionizing radiation-induced DNA rearrangements were detected in several different regions of the genome of human fibroblast cells that were exposed to 30 Gy of γ-irradiation and then incubated for 24 hours at 37 deg C. There was a 3- to 5-fold increase in the number of products amplified from irradiated as compared with control cells in the target regions 5' to the C-MYC, CDKN1A, RB1, and FGFR2 genes. Sequences were examined from 121 DNA rearrangements. Approximately half of the PCR products were derived from possible inter-chromosomal rearrangements and the remainder were from intra-chromosomal events. A high proportion of the sequences that rearranged with target regions were located in genes, suggesting that rearrangements may occur preferentially in transcribed regions. Eighty-four percent of the sequences examined by reverse transcriptase PCR were from transcribed sequences in IMR-90 cells. The distribution of DNA rearrangements within the target regions is non-random and homology occurs between the sequences involved in rearrangements in some cases but is not

  5. Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming

    NARCIS (Netherlands)

    Krijger, Peter Hugo Lodewijk; Di Stefano, Bruno; de Wit, Elzo; Limone, Francesco; van Oevelen, Chris; de Laat, Wouter; Graf, Thomas

    2016-01-01

    Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated

  6. Genome Defense Mechanisms in Neurospora and Associated Specialized Proteins

    Directory of Open Access Journals (Sweden)

    Ranjan Tamuli

    2010-06-01

    Full Text Available Neurospora crassa, the filamentous fungus possesses widest array of genome defense mechanisms known to any eukaryotic organism, including a process called repeat-induced point mutation (RIP. RIP is a genome defense mechanism that hypermutates repetitive DNA sequences; analogous to genomic imprinting in mammals. As an impact of RIP, Neurospora possesses many fewer genes in multigene families than expected. A DNA methyltransferase homologue, RID was shown to be essential for RIP. Recently, a variant catalytic subunit of translesion DNA polymerase zeta (Pol zeta has been found to be essential for dominant RIP suppressor phenotype. Meiotic silencing and quelling are two other genome defense mechanisms in Neurospora, and proteins required for these two processes have been identified through genetic screens.

  7. Draft Genome Sequence of Pseudomonas sp. Strain In5 Isolated from a Greenlandic Disease Suppressive Soil with Potent Antimicrobial Activity

    DEFF Research Database (Denmark)

    Hennessy, Rosanna C.; Glaring, Mikkel Andreas; Frydenlund Michelsen, Charlotte

    2015-01-01

    Pseudomonas sp. In5 is an isolate of disease suppressive soil with potent activity against pathogens. Its antifungal activity has been linked to a gene cluster encoding nonribosomal peptide synthetases producing the peptides nunamycin and nunapeptin. The genome sequence will provide insight into ...

  8. Distinct Mechanisms of Nuclease-Directed DNA-Structure-Induced Genetic Instability in Cancer Genomes.

    Science.gov (United States)

    Zhao, Junhua; Wang, Guliang; Del Mundo, Imee M; McKinney, Jennifer A; Lu, Xiuli; Bacolla, Albino; Boulware, Stephen B; Zhang, Changsheng; Zhang, Haihua; Ren, Pengyu; Freudenreich, Catherine H; Vasquez, Karen M

    2018-01-30

    Sequences with the capacity to adopt alternative DNA structures have been implicated in cancer etiology; however, the mechanisms are unclear. For example, H-DNA-forming sequences within oncogenes have been shown to stimulate genetic instability in mammals. Here, we report that H-DNA-forming sequences are enriched at translocation breakpoints in human cancer genomes, further implicating them in cancer etiology. H-DNA-induced mutations were suppressed in human cells deficient in the nucleotide excision repair nucleases, ERCC1-XPF and XPG, but were stimulated in cells deficient in FEN1, a replication-related endonuclease. Further, we found that these nucleases cleaved H-DNA conformations, and the interactions of modeled H-DNA with ERCC1-XPF, XPG, and FEN1 proteins were explored at the sub-molecular level. The results suggest mechanisms of genetic instability triggered by H-DNA through distinct structure-specific, cleavage-based replication-independent and replication-dependent pathways, providing critical evidence for a role of the DNA structure itself in the etiology of cancer and other human diseases. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  9. A linear mitochondrial genome of Cyclospora cayetanensis (Eimeriidae, Eucoccidiorida, Coccidiasina, Apicomplexa) suggests the ancestral start position within mitochondrial genomes of eimeriid coccidia.

    Science.gov (United States)

    Ogedengbe, Mosun E; Qvarnstrom, Yvonne; da Silva, Alexandre J; Arrowood, Michael J; Barta, John R

    2015-05-01

    The near complete mitochondrial genome for Cyclospora cayetanensis is 6184 bp in length with three protein-coding genes (Cox1, Cox3, CytB) and numerous lsrDNA and ssrDNA fragments. Gene arrangements were conserved with other coccidia in the Eimeriidae, but the C. cayetanensis mitochondrial genome is not circular-mapping. Terminal transferase tailing and nested PCR completed the 5'-terminus of the genome starting with a 21 bp A/T-only region that forms a potential stem-loop. Regions homologous to the C. cayetanensis mitochondrial genome 5'-terminus are found in all eimeriid mitochondrial genomes available and suggest this may be the ancestral start of eimeriid mitochondrial genomes. Copyright © 2015 Australian Society for Parasitology Inc. All rights reserved.

  10. Common genomic signaling among initial DNA damage and radiation-induced apoptosis in peripheral blood lymphocytes from locally advanced breast cancer patients

    DEFF Research Database (Denmark)

    Henríquez-Hernández, Luis Alberto; Pinar, Beatriz; Carmona-Vigo, Ruth

    2013-01-01

    PURPOSE: To investigate the genomic signaling that defines sensitive lymphocytes to radiation and if such molecular profiles are consistent with clinical toxicity; trying to disclose the radiobiology mechanisms behind these cellular processes. PATIENTS AND METHODS: Twelve consecutive patients...... suffering from locally advanced breast cancer and treated with high-dose hyperfractionated radiotherapy were recruited. Initial DNA damage was measured by pulsed-field gel electrophoresis and radiation-induced apoptosis was measured by flow cytometry. Gene expression was assessed by DNA microarray. RESULTS...

  11. Whole-genome sequence of the Tibetan frog Nanorana parkeri and the comparative evolution of tetrapod genomes.

    Science.gov (United States)

    Sun, Yan-Bo; Xiong, Zi-Jun; Xiang, Xue-Yan; Liu, Shi-Ping; Zhou, Wei-Wei; Tu, Xiao-Long; Zhong, Li; Wang, Lu; Wu, Dong-Dong; Zhang, Bao-Lin; Zhu, Chun-Ling; Yang, Min-Min; Chen, Hong-Man; Li, Fang; Zhou, Long; Feng, Shao-Hong; Huang, Chao; Zhang, Guo-Jie; Irwin, David; Hillis, David M; Murphy, Robert W; Yang, Huan-Ming; Che, Jing; Wang, Jun; Zhang, Ya-Ping

    2015-03-17

    The development of efficient sequencing techniques has resulted in large numbers of genomes being available for evolutionary studies. However, only one genome is available for all amphibians, that of Xenopus tropicalis, which is distantly related from the majority of frogs. More than 96% of frogs belong to the Neobatrachia, and no genome exists for this group. This dearth of amphibian genomes greatly restricts genomic studies of amphibians and, more generally, our understanding of tetrapod genome evolution. To fill this gap, we provide the de novo genome of a Tibetan Plateau frog, Nanorana parkeri, and compare it to that of X. tropicalis and other vertebrates. This genome encodes more than 20,000 protein-coding genes, a number similar to that of Xenopus. Although the genome size of Nanorana is considerably larger than that of Xenopus (2.3 vs. 1.5 Gb), most of the difference is due to the respective number of transposable elements in the two genomes. The two frogs exhibit considerable conserved whole-genome synteny despite having diverged approximately 266 Ma, indicating a slow rate of DNA structural evolution in anurans. Multigenome synteny blocks further show that amphibians have fewer interchromosomal rearrangements than mammals but have a comparable rate of intrachromosomal rearrangements. Our analysis also identifies 11 Mb of anuran-specific highly conserved elements that will be useful for comparative genomic analyses of frogs. The Nanorana genome offers an improved understanding of evolution of tetrapod genomes and also provides a genomic reference for other evolutionary studies.

  12. Wild tobacco genomes reveal the evolution of nicotine biosynthesis.

    Science.gov (United States)

    Xu, Shuqing; Brockmöller, Thomas; Navarro-Quezada, Aura; Kuhl, Heiner; Gase, Klaus; Ling, Zhihao; Zhou, Wenwu; Kreitzer, Christoph; Stanke, Mario; Tang, Haibao; Lyons, Eric; Pandey, Priyanka; Pandey, Shree P; Timmermann, Bernd; Gaquerel, Emmanuel; Baldwin, Ian T

    2017-06-06

    Nicotine, the signature alkaloid of Nicotiana species responsible for the addictive properties of human tobacco smoking, functions as a defensive neurotoxin against attacking herbivores. However, the evolution of the genetic features that contributed to the assembly of the nicotine biosynthetic pathway remains unknown. We sequenced and assembled genomes of two wild tobaccos, Nicotiana attenuata (2.5 Gb) and Nicotiana obtusifolia (1.5 Gb), two ecological models for investigating adaptive traits in nature. We show that after the Solanaceae whole-genome triplication event, a repertoire of rapidly expanding transposable elements (TEs) bloated these Nicotiana genomes, promoted expression divergences among duplicated genes, and contributed to the evolution of herbivory-induced signaling and defenses, including nicotine biosynthesis. The biosynthetic machinery that allows for nicotine synthesis in the roots evolved from the stepwise duplications of two ancient primary metabolic pathways: the polyamine and nicotinamide adenine dinucleotide (NAD) pathways. In contrast to the duplication of the polyamine pathway that is shared among several solanaceous genera producing polyamine-derived tropane alkaloids, we found that lineage-specific duplications within the NAD pathway and the evolution of root-specific expression of the duplicated Solanaceae-specific ethylene response factor that activates the expression of all nicotine biosynthetic genes resulted in the innovative and efficient production of nicotine in the genus Nicotiana Transcription factor binding motifs derived from TEs may have contributed to the coexpression of nicotine biosynthetic pathway genes and coordinated the metabolic flux. Together, these results provide evidence that TEs and gene duplications facilitated the emergence of a key metabolic innovation relevant to plant fitness.

  13. Carcinogen susceptibility is regulated by genome architecture and predicts cancer mutagenesis.

    Science.gov (United States)

    García-Nieto, Pablo E; Schwartz, Erin K; King, Devin A; Paulsen, Jonas; Collas, Philippe; Herrera, Rafael E; Morrison, Ashby J

    2017-10-02

    The development of many sporadic cancers is directly initiated by carcinogen exposure. Carcinogens induce malignancies by creating DNA lesions (i.e., adducts) that can result in mutations if left unrepaired. Despite this knowledge, there has been remarkably little investigation into the regulation of susceptibility to acquire DNA lesions. In this study, we present the first quantitative human genome-wide map of DNA lesions induced by ultraviolet (UV) radiation, the ubiquitous carcinogen in sunlight that causes skin cancer. Remarkably, the pattern of carcinogen susceptibility across the genome of primary cells significantly reflects mutation frequency in malignant melanoma. Surprisingly, DNase-accessible euchromatin is protected from UV, while lamina-associated heterochromatin at the nuclear periphery is vulnerable. Many cancer driver genes have an intrinsic increase in carcinogen susceptibility, including the BRAF oncogene that has the highest mutation frequency in melanoma. These findings provide a genome-wide snapshot of DNA injuries at the earliest stage of carcinogenesis. Furthermore, they identify carcinogen susceptibility as an origin of genome instability that is regulated by nuclear architecture and mirrors mutagenesis in cancer. © 2017 The Authors.

  14. A RecET-assisted CRISPR-Cas9 genome editing in Corynebacterium glutamicum.

    Science.gov (United States)

    Wang, Bo; Hu, Qitiao; Zhang, Yu; Shi, Ruilin; Chai, Xin; Liu, Zhe; Shang, Xiuling; Zhang, Yun; Wen, Tingyi

    2018-04-23

    Extensive modification of genome is an efficient manner to regulate the metabolic network for producing target metabolites or non-native products using Corynebacterium glutamicum as a cell factory. Genome editing approaches by means of homologous recombination and counter-selection markers are laborious and time consuming due to multiple round manipulations and low editing efficiencies. The current two-plasmid-based CRISPR-Cas9 editing methods generate false positives due to the potential instability of Cas9 on the plasmid, and require a high transformation efficiency for co-occurrence of two plasmids transformation. Here, we developed a RecET-assisted CRISPR-Cas9 genome editing method using a chromosome-borne Cas9-RecET and a single plasmid harboring sgRNA and repair templates. The inducible expression of chromosomal RecET promoted the frequencies of homologous recombination, and increased the efficiency for gene deletion. Due to the high transformation efficiency of a single plasmid, this method enabled 10- and 20-kb region deletion, 2.5-, 5.7- and 7.5-kb expression cassette insertion and precise site-specific mutation, suggesting a versatility of this method. Deletion of argR and farR regulators as well as site-directed mutation of argB and pgi genes generated the mutant capable of accumulating L-arginine, indicating the stability of chromosome-borne Cas9 for iterative genome editing. Using this method, the model-predicted target genes were modified to redirect metabolic flux towards 1,2-propanediol biosynthetic pathway. The final engineered strain produced 6.75 ± 0.46 g/L of 1,2-propanediol that is the highest titer reported in C. glutamicum. Furthermore, this method is available for Corynebacterium pekinense 1.563, suggesting its universal applicability in other Corynebacterium species. The RecET-assisted CRISPR-Cas9 genome editing method will facilitate engineering of metabolic networks for the synthesis of interested bio-based products from renewable

  15. Gene conversion in the rice genome

    DEFF Research Database (Denmark)

    Xu, Shuqing; Clark, Terry; Zheng, Hongkun

    2008-01-01

    -chromosomal conversions distributed between chromosome 1 and 5, 2 and 6, and 3 and 5 are more frequent than genome average (Z-test, P ... is not tightly linked to natural selection in the rice genome. To assess the contribution of segmental duplication on gene conversion statistics, we determined locations of conversion partners with respect to inter-chromosomal segment duplication. The number of conversions associated with segmentation is less...... involved in conversion events. CONCLUSION: The evolution of gene families in the rice genome may have been accelerated by conversion with pseudogenes. Our analysis suggests a possible role for gene conversion in the evolution of pathogen-response genes....

  16. Understanding and exploiting the genomic response to hypoxia

    International Nuclear Information System (INIS)

    Giaccia, A.J.

    2003-01-01

    The tumor microenvironment influences both therapeutic outcome and malignant progression. Of the many factors that may be altered in the tumor microenvironment, changes in tumor oxygenation have been strongly associated with a lower probability of local tumor control and survival. In vitro studies indicate that cells exposed to a low oxygen environment exhibit multiple phenotypes, including cell-cycle arrest, increased expression of pro-angiogenic genes, increased invasive capacity, increased apoptosis, increased anaerobic metabolism and altered differentiation programs. While the mechanistic basis of hypoxia as an impediment to radiotherapy and chemotherapy is well understood, it is unclear what changes in the cellular phenotype are important in understanding how hypoxia modifies malignant progression. One insight into how hypoxia modulates malignant progression comes from understanding the critical transcriptional regulators of gene expression under hypoxic conditions such as hypoxia inducible factor 1 (HIF-1) as well as changes in gene expression in untransformed and transformed cells. Overall, about 1.5% of the genome is found to be transcriptionally responsive to changes in oxygenation. Most importantly, the coordinated changes in gene expression under hypoxic conditions underscore the physiologic basis for altering gene expression in response to a low oxygen environment. In addition, some hypoxia-induced genes exhibit increased expression after reoxygenation, suggesting that they are regulated both by hypoxia and oxidative stress. Analysis of the genomic response to hypoxia has several therapeutic uses. First, it allows one to ask the question of what the cellular consequences are to inhibition of the transcriptional response to hypoxia such as by targeting the HIF-1 transcription factor. While the effect of loss of HIF-1 in tumors leads to inhibition of tumor growth, it does not eliminate tumors. In fact, studies indicate that inhibition of HIF-1 leads to a

  17. Complete genome sequence of DSM 30083(T), the type strain (U5/41(T)) of Escherichia coli, and a proposal for delineating subspecies in microbial taxonomy.

    OpenAIRE

    Meier-Kolthoff, Jan P; Hahnke, Richard L; Petersen, Jörn; Scheuner, Carmen; Michael, Victoria; Fiebig, Anne; Rohde, Christine; Rohde, Manfred; Fartmann, Berthold; Goodwin, Lynne A; Chertkov, Olga; Reddy, Tbk; Pati, Amrita; Ivanova, Natalia N; Markowitz, Victor

    2014-01-01

    Although Escherichia coli is the most widely studied bacterial model organism and often considered to be the model bacterium per se, its type strain was until now forgotten from microbial genomics. As a part of the G enomic E ncyclopedia of B acteria and A rchaea project, we here describe the features of E. coli DSM 30083T together with its genome sequence and annotation as well as novel aspects of its phenotype. The 5,038,133 bp containing genome sequence includes 4,762 protein-coding genes ...

  18. Bufei Huoxue Capsule Attenuates PM2.5-Induced Pulmonary Inflammation in Mice

    Directory of Open Access Journals (Sweden)

    Yue Jing

    2017-01-01

    Full Text Available Atmospheric fine particulate matter 2.5 (PM 2.5 may carry many toxic substances on its surface and this may pose a public health threat. Epidemiological research indicates that cumulative ambient PM2.5 is correlated to morbidity and mortality due to pulmonary and cardiovascular diseases and cancer. Mitigating the toxic effects of PM2.5 is therefore highly desired. Bufei Huoxue (BFHX capsules have been used in China to treat pulmonary heart disease (cor pulmonale. Thus, we assessed the effects of BFHX capsules on PM2.5-induced pulmonary inflammation and the underlying mechanisms of action. Using Polysearch and Cytoscape 3.2.1 software, pharmacological targets of BFHX capsules in atmospheric PM2.5-related respiratory disorders were predicted and found to be related to biological pathways of inflammation and immune function. In a mouse model of PM2.5-induced inflammation established with intranasal instillation of PM2.5 suspension, BFHX significantly reduced pathological response and inflammatory mediators including IL-4, IL-6, IL-10, IL-8, TNF-α, and IL-1β. BFHX also reduced keratinocyte growth factor (KGF, secretory immunoglobulin A (sIgA, and collagen fibers deposition in lung and improved lung function. Thus, BFHX reduced pathological responses induced by PM2.5, possibly via regulation of inflammatory mediators in mouse lungs.

  19. A Gene Gravity Model for the Evolution of Cancer Genomes: A Study of 3,000 Cancer Genomes across 9 Cancer Types

    Science.gov (United States)

    Lin, Chen-Ching; Zhao, Junfei; Jia, Peilin; Li, Wen-Hsiung; Zhao, Zhongming

    2015-01-01

    Cancer development and progression result from somatic evolution by an accumulation of genomic alterations. The effects of those alterations on the fitness of somatic cells lead to evolutionary adaptations such as increased cell proliferation, angiogenesis, and altered anticancer drug responses. However, there are few general mathematical models to quantitatively examine how perturbations of a single gene shape subsequent evolution of the cancer genome. In this study, we proposed the gene gravity model to study the evolution of cancer genomes by incorporating the genome-wide transcription and somatic mutation profiles of ~3,000 tumors across 9 cancer types from The Cancer Genome Atlas into a broad gene network. We found that somatic mutations of a cancer driver gene may drive cancer genome evolution by inducing mutations in other genes. This functional consequence is often generated by the combined effect of genetic and epigenetic (e.g., chromatin regulation) alterations. By quantifying cancer genome evolution using the gene gravity model, we identified six putative cancer genes (AHNAK, COL11A1, DDX3X, FAT4, STAG2, and SYNE1). The tumor genomes harboring the nonsynonymous somatic mutations in these genes had a higher mutation density at the genome level compared to the wild-type groups. Furthermore, we provided statistical evidence that hypermutation of cancer driver genes on inactive X chromosomes is a general feature in female cancer genomes. In summary, this study sheds light on the functional consequences and evolutionary characteristics of somatic mutations during tumorigenesis by propelling adaptive cancer genome evolution, which would provide new perspectives for cancer research and therapeutics. PMID:26352260

  20. Analysis of gene order data supports vertical inheritance of the leukotoxin operon and genome rearrangements in the 5' flanking region in genus Mannheimia

    DEFF Research Database (Denmark)

    Larsen, Jesper; Kuhnert, Peter; Frey, Joachim

    2007-01-01

    subclades, thus reaffirming the hypothesis of vertical inheritance of the leukotoxin operon. The presence of individual 5' flanking regions in M. haemolytica + M. glucosida and M. granulomatis reflects later genome rearrangements within each subclade. The evolution of the novel 5' flanking region in M...

  1. Novel cross-strand three-purine stack of the highly conserved 5'-GA/AAG-5' internal loop at the 3'-end termini of Parvovirus Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Chou, S.-H.; Chin, K.-H. [National Chung-Hsing University, Institute of Biochemistry (China)

    2001-12-15

    We have used two-dimensional nuclear magnetic resonance (2D-NMR), distance geometry (DG) and molecular dynamics / energy minimization (MD/EM) methods to study a 2x3 asymmetric internal loop structure of the highly conserved '5'-(GA)/(AAG)-5' bubble' present at the 3'-end hairpin of the single-stranded DNA genome of parvoviruses. This motif contains an unpaired adenosine stacked between two bracketed sheared G{center_dot}A pairs. However, the phenomenal cross-strand G-G and A-A stacking in the tandem sheared G{center_dot}A pairs has undergone considerable change. A novel three-purine stacking pattern is observed instead; the inserted A18 base is completely un-stacked from its neighboring G17 and A19 bases, but well stacked with the cross-strand A4 and G3 bases to form a novel A4/A18/G3 stack that is different from the double G/G, A/A or quadruple G/G/G/G stack present in the 5'-(GA)/(AG)-5' or 5'-(GGA)/(AGG)-5' motifs. Unlike the bulged purine residue that usually causes about 20 degree kink in the helical axis of the parent helix when bracketed by canonical G{center_dot}C or A{center_dot}T base pairs, no significant kink is observed in the present helix containing a bulged-adenine that is bracketed by sheared G {center_dot}A pairs. The phosphodiesters connecting G3-A4 and G17-A18 residues adopt unusual {zeta} torsional angles close to the trans domain, yet that connecting A18-A19 residues resumes the normal {zeta}(g{sup -}) value. The well structured '5'-(GAA)/(AG)-5'' internal loop in the parvovirus genomes explains its resistance to single-strand specific endonuclease susceptibility.

  2. Telomerase activation by genomic rearrangements in high-risk neuroblastoma

    Science.gov (United States)

    Peifer, Martin; Hertwig, Falk; Roels, Frederik; Dreidax, Daniel; Gartlgruber, Moritz; Menon, Roopika; Krämer, Andrea; Roncaioli, Justin L.; Sand, Frederik; Heuckmann, Johannes M.; Ikram, Fakhera; Schmidt, Rene; Ackermann, Sandra; Engesser, Anne; Kahlert, Yvonne; Vogel, Wenzel; Altmüller, Janine; Nürnberg, Peter; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Mariappan, Aruljothi; Heynck, Stefanie; Mariotti, Erika; Henrich, Kai-Oliver; Glöckner, Christian; Bosco, Graziella; Leuschner, Ivo; Schweiger, Michal R.; Savelyeva, Larissa; Watkins, Simon C.; Shao, Chunxuan; Bell, Emma; Höfer, Thomas; Achter, Viktor; Lang, Ulrich; Theissen, Jessica; Volland, Ruth; Saadati, Maral; Eggert, Angelika; de Wilde, Bram; Berthold, Frank; Peng, Zhiyu; Zhao, Chen; Shi, Leming; Ortmann, Monika; Büttner, Reinhard; Perner, Sven; Hero, Barbara; Schramm, Alexander; Schulte, Johannes H.; Herrmann, Carl; O’Sullivan, Roderick J.; Westermann, Frank; Thomas, Roman K.; Fischer, Matthias

    2016-01-01

    Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system1. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive2–4. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type1,2,5. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours. PMID:26466568

  3. Genome-Wide Mutational Signature of the Chemotherapeutic Agent Mitomycin C in Caenorhabditis elegans.

    Science.gov (United States)

    Tam, Annie S; Chu, Jeffrey S C; Rose, Ann M

    2015-11-12

    Cancer therapy largely depends on chemotherapeutic agents that generate DNA lesions. However, our understanding of the nature of the resulting lesions as well as the mutational profiles of these chemotherapeutic agents is limited. Among these lesions, DNA interstrand crosslinks are among the more toxic types of DNA damage. Here, we have characterized the mutational spectrum of the commonly used DNA interstrand crosslinking agent mitomycin C (MMC). Using a combination of genetic mapping, whole genome sequencing, and genomic analysis, we have identified and confirmed several genomic lesions linked to MMC-induced DNA damage in Caenorhabditis elegans. Our data indicate that MMC predominantly causes deletions, with a 5'-CpG-3' sequence context prevalent in the deleted regions of DNA. Furthermore, we identified microhomology flanking the deletion junctions, indicative of DNA repair via nonhomologous end joining. Based on these results, we propose a general repair mechanism that is likely to be involved in the biological response to this highly toxic agent. In conclusion, the systematic study we have described provides insight into potential sequence specificity of MMC with DNA. Copyright © 2016 Tam et al.

  4. Genome-Wide Mutational Signature of the Chemotherapeutic Agent Mitomycin C in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Annie S. Tam

    2016-01-01

    Full Text Available Cancer therapy largely depends on chemotherapeutic agents that generate DNA lesions. However, our understanding of the nature of the resulting lesions as well as the mutational profiles of these chemotherapeutic agents is limited. Among these lesions, DNA interstrand crosslinks are among the more toxic types of DNA damage. Here, we have characterized the mutational spectrum of the commonly used DNA interstrand crosslinking agent mitomycin C (MMC. Using a combination of genetic mapping, whole genome sequencing, and genomic analysis, we have identified and confirmed several genomic lesions linked to MMC-induced DNA damage in Caenorhabditis elegans. Our data indicate that MMC predominantly causes deletions, with a 5′-CpG-3′ sequence context prevalent in the deleted regions of DNA. Furthermore, we identified microhomology flanking the deletion junctions, indicative of DNA repair via nonhomologous end joining. Based on these results, we propose a general repair mechanism that is likely to be involved in the biological response to this highly toxic agent. In conclusion, the systematic study we have described provides insight into potential sequence specificity of MMC with DNA.

  5. GPBAR1/TGR5 mediates bile acid-induced cytokine expression in murine Kupffer cells.

    Directory of Open Access Journals (Sweden)

    Guiyu Lou

    Full Text Available GPBAR1/TGR5 is a novel plasma membrane-bound G protein-coupled bile acid (BA receptor. BAs are known to induce the expression of inflammatory cytokines in the liver with unknown mechanism. Here we show that without other external stimuli, TGR5 activation alone induced the expression of interleukin 1β (IL-1β and tumor necrosis factor-α (TNF-α in murine macrophage cell line RAW264.7 or murine Kupffer cells. The TGR5-mediated increase of pro-inflammatory cytokine expression was suppressed by JNK inhibition. Moreover, the induced pro-inflammatory cytokine expression in mouse liver by 1% cholic acid (CA diet was blunted in JNK-/- mice. TGR5 activation by its ligands enhanced the phosphorylation levels, DNA-binding and trans-activities of c-Jun and ATF2 transcription factors. Finally, the induced pro-inflammatory cytokine expression in Kupffer cells by TGR5 activation correlated with the suppression of Cholesterol 7α-hydroxylase (Cyp7a1 expression in murine hepatocytes. These results suggest that TGR5 mediates the BA-induced pro-inflammatory cytokine production in murine Kupffer cells through JNK-dependent pathway. This novel role of TGR5 may correlate to the suppression of Cyp7a1 expression in hepatocytes and contribute to the delicate BA feedback regulation.

  6. Adenovirus type 5 induces progression of quiescent rat cells into S phase without polyamine accumulation.

    Science.gov (United States)

    Cheetham, B F; Shaw, D C; Bellett, A J

    1982-01-01

    Adenovirus type 5 induces cellular DNA synthesis and thymidine kinase in quiescent rat cells but does not induce ornithine decarboxylase. We now show that unlike serum, adenovirus type 5 fails to induce S-adenosylmethionine decarboxylase or polyamine accumulation. The inhibition by methylglyoxal bis(guanylhydrazone) of the induction of thymidine kinase by adenovirus type 5 is probably unrelated to its effects on polyamine biosynthesis. Thus, induction of cellular thymidine kinase and DNA replication by adenovirus type 5 is uncoupled from polyamine accumulation. PMID:7177112

  7. Transcription facilitated genome-wide recruitment of topoisomerase I and DNA gyrase.

    Science.gov (United States)

    Ahmed, Wareed; Sala, Claudia; Hegde, Shubhada R; Jha, Rajiv Kumar; Cole, Stewart T; Nagaraja, Valakunja

    2017-05-01

    Movement of the transcription machinery along a template alters DNA topology resulting in the accumulation of supercoils in DNA. The positive supercoils generated ahead of transcribing RNA polymerase (RNAP) and the negative supercoils accumulating behind impose severe topological constraints impeding transcription process. Previous studies have implied the role of topoisomerases in the removal of torsional stress and the maintenance of template topology but the in vivo interaction of functionally distinct topoisomerases with heterogeneous chromosomal territories is not deciphered. Moreover, how the transcription-induced supercoils influence the genome-wide recruitment of DNA topoisomerases remains to be explored in bacteria. Using ChIP-Seq, we show the genome-wide occupancy profile of both topoisomerase I and DNA gyrase in conjunction with RNAP in Mycobacterium tuberculosis taking advantage of minimal topoisomerase representation in the organism. The study unveils the first in vivo genome-wide interaction of both the topoisomerases with the genomic regions and establishes that transcription-induced supercoils govern their recruitment at genomic sites. Distribution profiles revealed co-localization of RNAP and the two topoisomerases on the active transcriptional units (TUs). At a given locus, topoisomerase I and DNA gyrase were localized behind and ahead of RNAP, respectively, correlating with the twin-supercoiled domains generated. The recruitment of topoisomerases was higher at the genomic loci with higher transcriptional activity and/or at regions under high torsional stress compared to silent genomic loci. Importantly, the occupancy of DNA gyrase, sole type II topoisomerase in Mtb, near the Ter domain of the Mtb chromosome validates its function as a decatenase.

  8. Complete genome sequence of Ikoma lyssavirus.

    Science.gov (United States)

    Marston, Denise A; Ellis, Richard J; Horton, Daniel L; Kuzmin, Ivan V; Wise, Emma L; McElhinney, Lorraine M; Banyard, Ashley C; Ngeleja, Chanasa; Keyyu, Julius; Cleaveland, Sarah; Lembo, Tiziana; Rupprecht, Charles E; Fooks, Anthony R

    2012-09-01

    Lyssaviruses (family Rhabdoviridae) constitute one of the most important groups of viral zoonoses globally. All lyssaviruses cause the disease rabies, an acute progressive encephalitis for which, once symptoms occur, there is no effective cure. Currently available vaccines are highly protective against the predominantly circulating lyssavirus species. Using next-generation sequencing technologies, we have obtained the whole-genome sequence for a novel lyssavirus, Ikoma lyssavirus (IKOV), isolated from an African civet in Tanzania displaying clinical signs of rabies. Genetically, this virus is the most divergent within the genus Lyssavirus. Characterization of the genome will help to improve our understanding of lyssavirus diversity and enable investigation into vaccine-induced immunity and protection.

  9. Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System

    Directory of Open Access Journals (Sweden)

    Yichun Bai

    2016-04-01

    Full Text Available In recent years, genome engineering technology has provided unprecedented opportunities for site-specific modification of biological genomes. Clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated (Cas 9 is one such means that can target a specific genome locus. It has been applied in human cells and many other organisms. Meanwhile, to efficiently enrich targeted cells, several surrogate systems have also been developed. However, very limited information exists on the application of CRISPR/Cas9 in chickens. In this study, we employed the CRISPR/Cas9 system to induce mutations in the peroxisome proliferator-activated receptor-γ (PPAR-γ, ATP synthase epsilon subunit (ATP5E, and ovalbumin (OVA genes in chicken DF-1 cells. The results of T7E1 assays showed that the mutation rate at the three different loci was 0.75%, 0.5%, and 3.0%, respectively. In order to improve the mutation efficiency, we used the PuroR gene for efficient enrichment of genetically modified cells with the surrogate reporter system. The mutation rate, as assessed via the T7E1 assay, increased to 60.7%, 61.3%, and 47.3%, and subsequent sequence analysis showed that the mutation efficiency increased to 94.7%, 95%, and 95%, respectively. In addition, there were no detectable off-target mutations in three potential off-target sites using the T7E1 assay. As noted above, the CRISPR/Cas9 system is a robust tool for chicken genome editing.

  10. Whole-Genome Sequencing and iPLEX MassARRAY Genotyping Map an EMS-Induced Mutation Affecting Cell Competition in Drosophila melanogaster.

    Science.gov (United States)

    Lee, Chang-Hyun; Rimesso, Gerard; Reynolds, David M; Cai, Jinlu; Baker, Nicholas E

    2016-10-13

    Cell competition, the conditional loss of viable genotypes only when surrounded by other cells, is a phenomenon observed in certain genetic mosaic conditions. We conducted a chemical mutagenesis and screen to recover new mutations that affect cell competition between wild-type and RpS3 heterozygous cells. Mutations were identified by whole-genome sequencing, making use of software tools that greatly facilitate the distinction between newly induced mutations and other sources of apparent sequence polymorphism, thereby reducing false-positive and false-negative identification rates. In addition, we utilized iPLEX MassARRAY for genotyping recombinant chromosomes. These approaches permitted the mapping of a new mutation affecting cell competition when only a single allele existed, with a phenotype assessed only in genetic mosaics, without the benefit of complementation with existing mutations, deletions, or duplications. These techniques expand the utility of chemical mutagenesis and whole-genome sequencing for mutant identification. We discuss mutations in the Atm and Xrp1 genes identified in this screen. Copyright © 2016 Lee et al.

  11. Protective effect of dexamethasone on 5-FU-induced oral mucositis in hamsters.

    Science.gov (United States)

    Ribeiro, Susana Barbosa; de Araújo, Aurigena Antunes; Araújo Júnior, Raimundo Fernandes de; Brito, Gerly Anne de Castro; Leitão, Renata Carvalho; Barbosa, Maisie Mitchele; Garcia, Vinicius Barreto; Medeiros, Aldo Cunha; Medeiros, Caroline Addison Carvalho Xavier de

    2017-01-01

    Oral mucositis (OM) is an important side effect of cancer treatment, characterized by ulcerative lesions in the mucosa of patients undergoing radiotherapy or chemotherapy, which has marked effects on patient quality of life and cancer therapy continuity. Considering that few protocols have demonstrated efficacy in preventing this side effect, the aim of this study was to examine the effect of dexamethasone (DEX) on OM induced by 5-fluorouracil (5-FU) in hamsters by studying signaling pathways. OM was induced in hamsters by 5-FU followed by mechanical trauma (MT) on day 4. On day 10, the animals were euthanized. The experimental groups included saline, MT, 5-FU, and DEX (0.25, 0.5, or 1 mg/kg). Macroscopic, histopathological, and immunohistochemical analyses as well as immunofluorescence experiments were performed on the oral mucosa of the animals. The oral mucosal samples were analyzed by enzyme-linked immunosorbent assays, and quantitative real-time polymerase chain reaction (qPCR). DEX (0.5 or 1 mg/kg) reduced inflammation and ulceration of the oral mucosa of hamsters. In addition, DEX (1 mg/kg) reduced the cytokine levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and macrophage migration inhibitory factor (MIF). DEX (1 mg/kg) also reduced the immunoexpression of cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-2, transforming growth factor (TGF)-β, MIF, Smad 2/3, Smad 2/3 phosphorylated and NFκB p65 in the jugal mucosa. Finally, DEX (1 mg/kg) increased interleukin-1 receptor-associated kinase 3 (IRAK-M), glucocorticoid-induced leucine zipper (GILZ), and mitogen-activated protein kinase (MKP1) gene expression and reduced NFκB p65 and serine threonine kinase (AKt) gene expression, relative to the 5-FU group. Thus, DEX improved OM induced by 5-FU in hamsters.

  12. Protective effect of dexamethasone on 5-FU-induced oral mucositis in hamsters.

    Directory of Open Access Journals (Sweden)

    Susana Barbosa Ribeiro

    Full Text Available Oral mucositis (OM is an important side effect of cancer treatment, characterized by ulcerative lesions in the mucosa of patients undergoing radiotherapy or chemotherapy, which has marked effects on patient quality of life and cancer therapy continuity. Considering that few protocols have demonstrated efficacy in preventing this side effect, the aim of this study was to examine the effect of dexamethasone (DEX on OM induced by 5-fluorouracil (5-FU in hamsters by studying signaling pathways. OM was induced in hamsters by 5-FU followed by mechanical trauma (MT on day 4. On day 10, the animals were euthanized. The experimental groups included saline, MT, 5-FU, and DEX (0.25, 0.5, or 1 mg/kg. Macroscopic, histopathological, and immunohistochemical analyses as well as immunofluorescence experiments were performed on the oral mucosa of the animals. The oral mucosal samples were analyzed by enzyme-linked immunosorbent assays, and quantitative real-time polymerase chain reaction (qPCR. DEX (0.5 or 1 mg/kg reduced inflammation and ulceration of the oral mucosa of hamsters. In addition, DEX (1 mg/kg reduced the cytokine levels of tumor necrosis factor (TNF-α, interleukin (IL-1β, and macrophage migration inhibitory factor (MIF. DEX (1 mg/kg also reduced the immunoexpression of cyclooxygenase (COX-2, matrix metalloproteinase (MMP-2, transforming growth factor (TGF-β, MIF, Smad 2/3, Smad 2/3 phosphorylated and NFκB p65 in the jugal mucosa. Finally, DEX (1 mg/kg increased interleukin-1 receptor-associated kinase 3 (IRAK-M, glucocorticoid-induced leucine zipper (GILZ, and mitogen-activated protein kinase (MKP1 gene expression and reduced NFκB p65 and serine threonine kinase (AKt gene expression, relative to the 5-FU group. Thus, DEX improved OM induced by 5-FU in hamsters.

  13. Genomic characterization of Haemophilus parasuis SH0165, a highly virulent strain of serovar 5 prevalent in China.

    Directory of Open Access Journals (Sweden)

    Zhuofei Xu

    Full Text Available Haemophilus parasuis can be either a commensal bacterium of the porcine respiratory tract or an opportunistic pathogen causing Glässer's disease, a severe systemic disease that has led to significant economical losses in the pig industry worldwide. We determined the complete genomic sequence of H. parasuis SH0165, a highly virulent strain of serovar 5, which was isolated from a hog pen in North China. The single circular chromosome was 2,269,156 base pairs in length and contained 2,031 protein-coding genes. Together with the full spectrum of genes detected by the analysis of metabolic pathways, we confirmed that H. parasuis generates ATP via both fermentation and respiration, and possesses an intact TCA cycle for anabolism. In addition to possessing the complete pathway essential for the biosynthesis of heme, this pathogen was also found to be well-equipped with different iron acquisition systems, such as the TonB system and ABC-type transport complexes, to overcome iron limitation during infection and persistence. We identified a number of genes encoding potential virulence factors, such as type IV fimbriae and surface polysaccharides. Analysis of the genome confirmed that H. parasuis is naturally competent, as genes related to DNA uptake are present. A nine-mer DNA uptake signal sequence (ACAAGCGGT, identical to that found in Actinobacillus pleuropneumoniae and Mannheimia haemolytica, followed by similar downstream motifs, was identified in the SH0165 genome. Genomic and phylogenetic comparisons with other Pasteurellaceae species further indicated that H. parasuis was closely related to another swine pathogenic bacteria A. pleuropneumoniae. The comprehensive genetic analysis presented here provides a foundation for future research on the metabolism, natural competence and virulence of H. parasuis.

  14. Complete genome sequence of Acidihalobacter prosperus strain F5, an extremely acidophilic, iron- and sulfur-oxidizing halophile with potential industrial applicability in saline water bioleaching of chalcopyrite.

    Science.gov (United States)

    Khaleque, Himel N; Corbett, Melissa K; Ramsay, Joshua P; Kaksonen, Anna H; Boxall, Naomi J; Watkin, Elizabeth L J

    2017-11-20

    Successful process development for the bioleaching of mineral ores, particularly the refractory copper sulfide ore chalcopyrite, remains a challenge in regions where freshwater is scarce and source water contains high concentrations of chloride ion. In this study, a pure isolate of Acidihalobacter prosperus strain F5 was characterized for its ability to leach base metals from sulfide ores (pyrite, chalcopyrite and pentlandite) at increasing chloride ion concentrations. F5 successfully released base metals from ores including pyrite and pentlandite at up to 30gL -1 chloride ion and chalcopyrite up to 18gL -1 chloride ion. In order to understand the genetic mechanisms of tolerance to high acid, saline and heavy metal stress the genome of F5 was sequenced and analysed. As well as being the first strain of Ac. prosperus to be isolated from Australia it is also the first complete genome of the Ac. prosperus species to be sequenced. The F5 genome contains genes involved in the biosynthesis of compatible solutes and genes encoding monovalent cation/proton antiporters and heavy metal transporters which could explain its abilities to tolerate high salinity, acidity and heavy metal stress. Genome analysis also confirmed the presence of genes involved in copper tolerance. The study demonstrates the potential biotechnological applicability of Ac. prosperus strain F5 for saline water bioleaching of mineral ores. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

    KAUST Repository

    Rashid, Fahad

    2017-02-23

    Human flap endonuclease 1 (FEN1) and related structure-specific 5\\'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5\\'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually \\'locks\\' protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.

  16. Mitochondrial genome sequences and comparative genomics ofPhytophthora ramorum and P. sojae

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Frank N.; Douda, Bensasson; Tyler, Brett M.; Boore,Jeffrey L.

    2007-01-01

    The complete sequences of the mitochondrial genomes of theoomycetes of Phytophthora ramorum and P. sojae were determined during thecourse of their complete nuclear genome sequencing (Tyler, et al. 2006).Both are circular, with sizes of 39,314 bp for P. ramorum and 42,975 bpfor P. sojae. Each contains a total of 37 identifiable protein-encodinggenes, 25 or 26 tRNAs (P. sojae and P. ramorum, respectively)specifying19 amino acids, and a variable number of ORFs (7 for P. ramorum and 12for P. sojae) which are potentially additional functional genes.Non-coding regions comprise approximately 11.5 percent and 18.4 percentof the genomes of P. ramorum and P. sojae, respectively. Relative to P.sojae, there is an inverted repeat of 1,150 bp in P. ramorum thatincludes an unassigned unique ORF, a tRNA gene, and adjacent non-codingsequences, but otherwise the gene order in both species is identical.Comparisons of these genomes with published sequences of the P. infestansmitochondrial genome reveals a number of similarities, but the gene orderin P. infestans differs in two adjacent locations due to inversions.Sequence alignments of the three genomes indicated sequence conservationranging from 75 to 85 percent and that specific regions were morevariable than others.

  17. A Comprehensive Toolbox for Genome Editing in Cultured Drosophila melanogaster Cells

    Directory of Open Access Journals (Sweden)

    Stefan Kunzelmann

    2016-06-01

    Full Text Available Custom genome editing has become an essential element of molecular biology. In particular, the generation of fusion constructs with epitope tags or fluorescent proteins at the genomic locus facilitates the analysis of protein expression, localization, and interaction partners at physiologic levels. Following up on our initial publication, we now describe a considerably simplified, more efficient, and readily scalable experimental workflow for PCR-based genome editing in cultured Drosophila melanogaster cells. Our analysis at the act5C locus suggests that PCR-based homology arms of 60 bp are sufficient to reach targeting efficiencies of up to 80% after selection; extension to 80 bp (PCR or 500 bp (targeting vector did not further improve the yield. We have expanded our targeting system to N-terminal epitope tags; this also allows the generation of cell populations with heterologous expression control of the tagged locus via the copper-inducible mtnDE promoter. We present detailed, quantitative data on editing efficiencies for several genomic loci that may serve as positive controls or benchmarks in other laboratories. While our first PCR-based editing approach offered only blasticidin-resistance for selection, we now introduce puromycin-resistance as a second, independent selection marker; it is thus possible to edit two loci (e.g., for coimmunoprecipitation without marker removal. Finally, we describe a modified FLP recombinase expression plasmid that improves the efficiency of marker cassette FLP-out. In summary, our technique and reagents enable a flexible, robust, and cloning-free genome editing approach that can be parallelized for scale-up.

  18. Minimal Contribution of APOBEC3-Induced G-to-A Hypermutation to HIV-1 Recombination and Genetic Variation.

    Science.gov (United States)

    Delviks-Frankenberry, Krista A; Nikolaitchik, Olga A; Burdick, Ryan C; Gorelick, Robert J; Keele, Brandon F; Hu, Wei-Shau; Pathak, Vinay K

    2016-05-01

    Although the predominant effect of host restriction APOBEC3 proteins on HIV-1 infection is to block viral replication, they might inadvertently increase retroviral genetic variation by inducing G-to-A hypermutation. Numerous studies have disagreed on the contribution of hypermutation to viral genetic diversity and evolution. Confounding factors contributing to the debate include the extent of lethal (stop codon) and sublethal hypermutation induced by different APOBEC3 proteins, the inability to distinguish between G-to-A mutations induced by APOBEC3 proteins and error-prone viral replication, the potential impact of hypermutation on the frequency of retroviral recombination, and the extent to which viral recombination occurs in vivo, which can reassort mutations in hypermutated genomes. Here, we determined the effects of hypermutation on the HIV-1 recombination rate and its contribution to genetic variation through recombination to generate progeny genomes containing portions of hypermutated genomes without lethal mutations. We found that hypermutation did not significantly affect the rate of recombination, and recombination between hypermutated and wild-type genomes only increased the viral mutation rate by 3.9 × 10-5 mutations/bp/replication cycle in heterozygous virions, which is similar to the HIV-1 mutation rate. Since copackaging of hypermutated and wild-type genomes occurs very rarely in vivo, recombination between hypermutated and wild-type genomes does not significantly contribute to the genetic variation of replicating HIV-1. We also analyzed previously reported hypermutated sequences from infected patients and determined that the frequency of sublethal mutagenesis for A3G and A3F is negligible (4 × 10-21 and1 × 10-11, respectively) and its contribution to viral mutations is far below mutations generated during error-prone reverse transcription. Taken together, we conclude that the contribution of APOBEC3-induced hypermutation to HIV-1 genetic

  19. Loss of 5‐lipoxygenase activity protects mice against paracetamol‐induced liver toxicity

    Science.gov (United States)

    Pu, Shiyun; Ren, Lin; Liu, Qinhui; Kuang, Jiangying; Shen, Jing; Cheng, Shihai; Zhang, Yuwei; Jiang, Wei; Zhang, Zhiyong; Jiang, Changtao

    2015-01-01

    Background and Purpose Paracetamol (acetaminophen) is the most widely used over‐the‐counter analgesic and overdosing with paracetamol is the leading cause of hospital admission for acute liver failure. 5‐Lipoxygenase (5‐LO) catalyses arachidonic acid to form LTs, which lead to inflammation and oxidative stress. In this study, we examined whether deletion or pharmacological inhibition of 5‐LO could protect mice against paracetamol‐induced hepatic toxicity. Experimental Approach Both genetic deletion and pharmacological inhibition of 5‐LO in C57BL/6J mice were used to study the role of this enzyme in paracetamol induced liver toxicity. Serum and tissue biochemistry, H&E staining, and real‐time PCR were used to assess liver toxicity. Key Results Deletion or pharmacological inhibition of 5‐LO in mice markedly ameliorated paracetamol‐induced hepatic injury, as shown by decreased serum alanine transaminase and aspartate aminotransferase levels and hepatic centrilobular necrosis. The hepatoprotective effect of 5‐LO inhibition was associated with induction of the antitoxic phase II conjugating enzyme, sulfotransferase2a1, suppression of the pro‐toxic phase I CYP3A11 and reduction of the hepatic transporter MRP3. In 5‐LO−/− mice, levels of GSH were increased, and oxidative stress decreased. In addition, PPAR α, a nuclear receptor that confers resistance to paracetamol toxicity, was activated in 5‐LO−/− mice. Conclusions and Implications The activity of 5‐LO may play a critical role in paracetamol‐induced hepatic toxicity by regulating paracetamol metabolism and oxidative stress. PMID:26398229

  20. Epigenetic control of mobile DNA as an interface between experience and genome change

    Directory of Open Access Journals (Sweden)

    James A. Shapiro

    2014-04-01

    Full Text Available Mobile DNA in the genome is subject to RNA-targeted epigenetic control. This control regulates the activity of transposons, retrotransposons and genomic proviruses. Many different life history experiences alter the activities of mobile DNA and the expression of genetic loci regulated by nearby insertions. The same experiences induce alterations in epigenetic formatting and lead to trans-generational modifications of genome expression and stability. These observations lead to the hypothesis that epigenetic formatting directed by non-coding RNA provides a molecular interface between life history events and genome alteration.

  1. Why size really matters when sequencing plant genomes

    Czech Academy of Sciences Publication Activity Database

    Kelly, L.J.; Leitch, A.R.; Fay, M. F.; Renny-Byfield, S.; Pellicer, J.; Macas, Jiří; Leitch, I.J.

    2012-01-01

    Roč. 5, č. 4 (2012), s. 415-425 ISSN 1755-0874 Institutional research plan: CEZ:AV0Z50510513 Institutional support: RVO:60077344 Keywords : C-value * genome assembly * genome size evolution * genome sequencing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.924, year: 2012

  2. Ornithine decarboxylase antizyme induces hypomethylation of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2 in human oral cancer cell line.

    Directory of Open Access Journals (Sweden)

    Daisuke Yamamoto

    2010-09-01

    Full Text Available Methylation of CpG islands of genome DNA and lysine residues of histone H3 and H4 tails regulates gene transcription. Inhibition of polyamine synthesis by ornithine decarboxylase antizyme-1 (OAZ in human oral cancer cell line resulted in accumulation of decarboxylated S-adenosylmethionine (dcSAM, which acts as a competitive inhibitor of methylation reactions. We anticipated that accumulation of dcSAM impaired methylation reactions and resulted in hypomethylation of genome DNA and histone tails.Global methylation state of genome DNA and lysine residues of histone H3 and H4 tails were assayed by Methylation by Isoschizomers (MIAMI method and western blotting, respectively, in the presence or absence of OAZ expression. Ectopic expression of OAZ mediated hypomethylation of CpG islands of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2. Protein level of DNA methyltransferase 3B (DNMT3B and histone H3K9me specific methyltransferase G9a were down-regulated in OAZ transfectant.OAZ induced hypomethylation of CpG islands of global genome DNA and H3K9me2 by down-regulating DNMT3B and G9a protein level. Hypomethylation of CpG islands of genome DNA and histone H3K9me2 is a potent mechanism of induction of the genes related to tumor suppression and DNA double strand break repair.

  3. [Efficient genome editing in human pluripotent stem cells through CRISPR/Cas9].

    Science.gov (United States)

    Liu, Gai-gai; Li, Shuang; Wei, Yu-da; Zhang, Yong-xian; Ding, Qiu-rong

    2015-11-01

    The RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has offered a new platform for genome editing with high efficiency. Here, we report the use of CRISPR/Cas9 technology to target a specific genomic region in human pluripotent stem cells. We show that CRISPR/Cas9 can be used to disrupt a gene by introducing frameshift mutations to gene coding region; to knock in specific sequences (e.g. FLAG tag DNA sequence) to targeted genomic locus via homology directed repair; to induce large genomic deletion through dual-guide multiplex. Our results demonstrate the versatile application of CRISPR/Cas9 in stem cell genome editing, which can be widely utilized for functional studies of genes or genome loci in human pluripotent stem cells.

  4. An Inhibitory Motif on the 5’UTR of Several Rotavirus Genome Segments Affects Protein Expression and Reverse Genetics Strategies

    Science.gov (United States)

    Papa, Guido; Eichwald, Catherine; Burrone, Oscar R.

    2016-01-01

    Rotavirus genome consists of eleven segments of dsRNA, each encoding one single protein. Viral mRNAs contain an open reading frame (ORF) flanked by relatively short untranslated regions (UTRs), whose role in the viral cycle remains elusive. Here we investigated the role of 5’UTRs in T7 polymerase-driven cDNAs expression in uninfected cells. The 5’UTRs of eight genome segments (gs3, gs5-6, gs7-11) of the simian SA11 strain showed a strong inhibitory effect on the expression of viral proteins. Decreased protein expression was due to both compromised transcription and translation and was independent of the ORF and the 3’UTR sequences. Analysis of several mutants of the 21-nucleotide long 5’UTR of gs 11 defined an inhibitory motif (IM) represented by its primary sequence rather than its secondary structure. IM was mapped to the 5’ terminal 6-nucleotide long pyrimidine-rich tract 5’-GGY(U/A)UY-3’. The 5’ terminal position within the mRNA was shown to be essentially required, as inhibitory activity was lost when IM was moved to an internal position. We identified two mutations (insertion of a G upstream the 5’UTR and the U to A mutation of the fifth nucleotide of IM) that render IM non-functional and increase the transcription and translation rate to levels that could considerably improve the efficiency of virus helper-free reverse genetics strategies. PMID:27846320

  5. A microculture system for the measurement of antigen-induced murine lymphocyte proliferation: advantages of 5% horse serum and 5 X 10(-5) M mercaptoethanol.

    Science.gov (United States)

    Brummer, E; Vris, T W; Lawrence, H S

    1977-01-01

    Short term microculture systems which measure murine lymphocyte proliferative responses to mitogens are well established. We demonstrate here that these microculture methods are not suitable for antigen-induced responses because of the high levels of murine lymphocyte proliferation in control cultures associated with the use of fetal calf serum or human serum. We also show that this problem can be eliminated with the use of a combination of 5% horse serum and 5 X 10(-5) M mercaptoethanol. We describe an antigen-induced murine lymphocyte proliferation microculture system in which good stimulation indices are achieved and the lymphocyte proliferation in control cultures remain at a low level throughout the 7 day culture period.

  6. Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs

    Science.gov (United States)

    Flather, Dylan; Cathcart, Andrea L.; Cruz, Casey; Baggs, Eric; Ngo, Tuan; Gershon, Paul D.; Semler, Bert L.

    2016-01-01

    Despite being intensely studied for more than 50 years, a complete understanding of the enterovirus replication cycle remains elusive. Specifically, only a handful of cellular proteins have been shown to be involved in the RNA replication cycle of these viruses. In an effort to isolate and identify additional cellular proteins that function in enteroviral RNA replication, we have generated multiple recombinant polioviruses containing RNA affinity tags within the 3′ or 5′ noncoding region of the genome. These recombinant viruses retained RNA affinity sequences within the genome while remaining viable and infectious over multiple passages in cell culture. Further characterization of these viruses demonstrated that viral protein production and growth kinetics were unchanged or only slightly altered relative to wild type poliovirus. However, attempts to isolate these genetically-tagged viral genomes from infected cells have been hindered by high levels of co-purification of nonspecific proteins and the limited matrix-binding efficiency of RNA affinity sequences. Regardless, these recombinant viruses represent a step toward more thorough characterization of enterovirus ribonucleoprotein complexes involved in RNA replication. PMID:26861382

  7. The human genome project

    International Nuclear Information System (INIS)

    Worton, R.

    1996-01-01

    The Human Genome Project is a massive international research project, costing 3 to 5 billion dollars and expected to take 15 years, which will identify the all the genes in the human genome - i.e. the complete sequence of bases in human DNA. The prize will be the ability to identify genes causing or predisposing to disease, and in some cases the development of gene therapy, but this new knowledge will raise important ethical issues

  8. Preliminary Genomic Characterization of Ten Hardwood Tree Species from Multiplexed Low Coverage Whole Genome Sequencing.

    Directory of Open Access Journals (Sweden)

    Margaret Staton

    Full Text Available Forest health issues are on the rise in the United States, resulting from introduction of alien pests and diseases, coupled with abiotic stresses related to climate change. Increasingly, forest scientists are finding genetic/genomic resources valuable in addressing forest health issues. For a set of ten ecologically and economically important native hardwood tree species representing a broad phylogenetic spectrum, we used low coverage whole genome sequencing from multiplex Illumina paired ends to economically profile their genomic content. For six species, the genome content was further analyzed by flow cytometry in order to determine the nuclear genome size. Sequencing yielded a depth of 0.8X to 7.5X, from which in silico analysis yielded preliminary estimates of gene and repetitive sequence content in the genome for each species. Thousands of genomic SSRs were identified, with a clear predisposition toward dinucleotide repeats and AT-rich repeat motifs. Flanking primers were designed for SSR loci for all ten species, ranging from 891 loci in sugar maple to 18,167 in redbay. In summary, we have demonstrated that useful preliminary genome information including repeat content, gene content and useful SSR markers can be obtained at low cost and time input from a single lane of Illumina multiplex sequence.

  9. Genome editing: a robust technology for human stem cells.

    Science.gov (United States)

    Chandrasekaran, Arun Pandian; Song, Minjung; Ramakrishna, Suresh

    2017-09-01

    Human pluripotent stem cells comprise induced pluripotent and embryonic stem cells, which have tremendous potential for biological and therapeutic applications. The development of efficient technologies for the targeted genome alteration of stem cells in disease models is a prerequisite for utilizing stem cells to their full potential. Genome editing of stem cells is possible with the help of synthetic nucleases that facilitate site-specific modification of a gene of interest. Recent advances in genome editing techniques have improved the efficiency and speed of the development of stem cells for human disease models. Zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system are powerful tools for editing DNA at specific loci. Here, we discuss recent technological advances in genome editing with site-specific nucleases in human stem cells.

  10. Fluoxetine-induced inhibition of synaptosomal [3H]5-HT release: Possible Ca2+-channel inhibition

    International Nuclear Information System (INIS)

    Stauderman, K.A.; Gandhi, V.C.; Jones, D.J.

    1992-01-01

    Fluoxetine, a selective 5-Ht uptake inhibitor, inhibited 15 mM K + -induced [ 3 H]5-HT release from rat spinal cord and cortical synaptosomes at concentrations > 0.5 uM. This effect reflected a property shared by another selective 5-HT uptake inhibitor paroxetine but not by less selective uptake inhibitors such as amitriptyline, desipramine, imipramine or nortriptyline. Inhibition of release by fluoxetine was inversely related to both the concentration of K + used to depolarize the synaptosomes and the concentration of external Ca 2+ . Experiments aimed at determining a mechanism of action revealed that fluoxetine did not inhibit voltage-independent release of [ 3 H]5-HT release induced by the Ca 2+ -ionophore A 23187 or Ca 2+ -independent release induced by fenfluramine. Moreover the 5-HT autoreceptor antagonist methiothepin did not reverse the inhibitory actions of fluoxetine on K + -induced release. Further studies examined the effects of fluoxetine on voltage-dependent Ca 2+ channels and Ca 2+ entry

  11. Ensembl Genomes 2016: more genomes, more complexity.

    Science.gov (United States)

    Kersey, Paul Julian; Allen, James E; Armean, Irina; Boddu, Sanjay; Bolt, Bruce J; Carvalho-Silva, Denise; Christensen, Mikkel; Davis, Paul; Falin, Lee J; Grabmueller, Christoph; Humphrey, Jay; Kerhornou, Arnaud; Khobova, Julia; Aranganathan, Naveen K; Langridge, Nicholas; Lowy, Ernesto; McDowall, Mark D; Maheswari, Uma; Nuhn, Michael; Ong, Chuang Kee; Overduin, Bert; Paulini, Michael; Pedro, Helder; Perry, Emily; Spudich, Giulietta; Tapanari, Electra; Walts, Brandon; Williams, Gareth; Tello-Ruiz, Marcela; Stein, Joshua; Wei, Sharon; Ware, Doreen; Bolser, Daniel M; Howe, Kevin L; Kulesha, Eugene; Lawson, Daniel; Maslen, Gareth; Staines, Daniel M

    2016-01-04

    Ensembl Genomes (http://www.ensemblgenomes.org) is an integrating resource for genome-scale data from non-vertebrate species, complementing the resources for vertebrate genomics developed in the context of the Ensembl project (http://www.ensembl.org). Together, the two resources provide a consistent set of programmatic and interactive interfaces to a rich range of data including reference sequence, gene models, transcriptional data, genetic variation and comparative analysis. This paper provides an update to the previous publications about the resource, with a focus on recent developments. These include the development of new analyses and views to represent polyploid genomes (of which bread wheat is the primary exemplar); and the continued up-scaling of the resource, which now includes over 23 000 bacterial genomes, 400 fungal genomes and 100 protist genomes, in addition to 55 genomes from invertebrate metazoa and 39 genomes from plants. This dramatic increase in the number of included genomes is one part of a broader effort to automate the integration of archival data (genome sequence, but also associated RNA sequence data and variant calls) within the context of reference genomes and make it available through the Ensembl user interfaces. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Acute food deprivation reverses morphine-induced locomotion deficits in M5 muscarinic receptor knockout mice.

    Science.gov (United States)

    Steidl, Stephan; Lee, Esther; Wasserman, David; Yeomans, John S

    2013-09-01

    Lesions of the pedunculopontine tegmental nucleus (PPT), one of two sources of cholinergic input to the ventral tegmental area (VTA), block conditioned place preference (CPP) for morphine in drug-naïve rats. M5 muscarinic cholinergic receptors, expressed by midbrain dopamine neurons, are critical for the ability of morphine to increase nucleus accumbens dopamine levels and locomotion, and for morphine CPP. This suggests that M5-mediated PPT cholinergic inputs to VTA dopamine neurons critically contribute to morphine-induced dopamine activation, reward and locomotion. In the current study we tested whether food deprivation, which reduces PPT contribution to morphine CPP in rats, could also reduce M5 contributions to morphine-induced locomotion in mice. Acute 18-h food deprivation reversed the phenotypic differences usually seen between non-deprived wild-type and M5 knockout mice. That is, food deprivation increased morphine-induced locomotion in M5 knockout mice but reduced morphine-induced locomotion in wild-type mice. Food deprivation increased saline-induced locomotion equally in wild-type and M5 knockout mice. Based on these findings, we suggest that food deprivation reduces the contribution of M5-mediated PPT cholinergic inputs to the VTA in morphine-induced locomotion and increases the contribution of a PPT-independent pathway. The contributions of cholinergic, dopaminergic and GABAergic neurons to the effects of acute food deprivation are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Genomic dark matter: the reliability of short read mapping illustrated by the genome mappability score.

    Science.gov (United States)

    Lee, Hayan; Schatz, Michael C

    2012-08-15

    Genome resequencing and short read mapping are two of the primary tools of genomics and are used for many important applications. The current state-of-the-art in mapping uses the quality values and mapping quality scores to evaluate the reliability of the mapping. These attributes, however, are assigned to individual reads and do not directly measure the problematic repeats across the genome. Here, we present the Genome Mappability Score (GMS) as a novel measure of the complexity of resequencing a genome. The GMS is a weighted probability that any read could be unambiguously mapped to a given position and thus measures the overall composition of the genome itself. We have developed the Genome Mappability Analyzer to compute the GMS of every position in a genome. It leverages the parallelism of cloud computing to analyze large genomes, and enabled us to identify the 5-14% of the human, mouse, fly and yeast genomes that are difficult to analyze with short reads. We examined the accuracy of the widely used BWA/SAMtools polymorphism discovery pipeline in the context of the GMS, and found discovery errors are dominated by false negatives, especially in regions with poor GMS. These errors are fundamental to the mapping process and cannot be overcome by increasing coverage. As such, the GMS should be considered in every resequencing project to pinpoint the 'dark matter' of the genome, including of known clinically relevant variations in these regions. The source code and profiles of several model organisms are available at http://gma-bio.sourceforge.net

  14. Molecular analysis of point mutations in a barley genome exposed to MNU and gamma rays

    Energy Technology Data Exchange (ETDEWEB)

    Kurowska, Marzena, E-mail: mkurowsk@us.edu.pl [Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Jagiellonska 28, 40-032 Katowice (Poland); Labocha-Pawlowska, Anna; Gnizda, Dominika; Maluszynski, Miroslaw; Szarejko, Iwona [Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Jagiellonska 28, 40-032 Katowice (Poland)

    2012-10-15

    We present studies aimed at determining the types and frequencies of mutations induced in the barley genome after treatment with chemical (N-methyl-N-nitrosourea, MNU) and physical (gamma rays) mutagens. We created M{sub 2} populations of a doubled haploid line and used them for the analysis of mutations in targeted DNA sequences and over an entire barley genome using TILLING (Targeting Induced Local Lesions in Genomes) and AFLP (Amplified Fragment Length Polymorphism) technique, respectively. Based on the TILLING analysis of the total DNA sequence of 4,537,117 bp in the MNU population, the average mutation density was estimated as 1/504 kb. Only one nucleotide change was found after an analysis of 3,207,444 bp derived from the highest dose of gamma rays applied. MNU was clearly a more efficient mutagen than gamma rays in inducing point mutations in barley. The majority (63.6%) of the MNU-induced nucleotide changes were transitions, with a similar number of G > A and C > T substitutions. The similar share of G > A and C > T transitions indicates a lack of bias in the repair of O{sup 6}-methylguanine lesions between DNA strands. There was, however, a strong specificity of the nucleotide surrounding the O{sup 6}-meG at the -1 position. Purines formed 81% of nucleotides observed at the -1 site. Scanning the barley genome with AFLP markers revealed ca. a three times higher level of AFLP polymorphism in MNU-treated as compared to the gamma-irradiated population. In order to check whether AFLP markers can really scan the whole barley genome for mutagen-induced polymorphism, 114 different AFLP products, were cloned and sequenced. 94% of bands were heterogenic, with some bands containing up to 8 different amplicons. The polymorphic AFLP products were characterised in terms of their similarity to the records deposited in a GenBank database. The types of sequences present in the polymorphic bands reflected the organisation of the barley genome.

  15. Two Inducible Prophages of an Antarctic Pseudomonas sp. ANT_H14 Use the Same Capsid for Packaging Their Genomes - Characterization of a Novel Phage Helper-Satellite System.

    Directory of Open Access Journals (Sweden)

    Lukasz Dziewit

    Full Text Available Two novel prophages ФAH14a and ФAH14b of a psychrotolerant Antarctic bacterium Pseudomonas sp. ANT_H14 have been characterized. They were simultaneously induced with mitomycin C and packed into capsids of the same size and protein composition. The genome sequences of ФAH14a and ФAH14b have been determined. ФAH14b, the phage with a smaller genome (16,812 bp seems to parasitize ФAH14a (55,060 bp and utilizes its capsids, as only the latter encodes a complete set of structural proteins. Both viruses probably constitute a phage helper-satellite system, analogous to the P2-P4 duo. This study describes the architecture and function of the ФAH14a and ФAH14b genomes. Moreover, a functional analysis of a ФAH14a-encoded lytic enzyme and a DNA methyltransferase was performed. In silico analysis revealed the presence of the homologs of ФAH14a and ФAH14b in other Pseudomonas genomes, which may suggest that helper-satellite systems related to the one described in this work are common in pseudomonads.

  16. Full-Genome Analysis of Avian Influenza A(H5N1) Virus from a Human, North America, 2013

    Science.gov (United States)

    Pabbaraju, Kanti; Tellier, Raymond; Wong, Sallene; Li, Yan; Bastien, Nathalie; Tang, Julian W.; Drews, Steven J.; Jang, Yunho; Davis, C. Todd; Tipples, Graham A.

    2014-01-01

    Full-genome analysis was conducted on the first isolate of a highly pathogenic avian influenza A(H5N1) virus from a human in North America. The virus has a hemagglutinin gene of clade 2.3.2.1c and is a reassortant with an H9N2 subtype lineage polymerase basic 2 gene. No mutations conferring resistance to adamantanes or neuraminidase inhibitors were found. PMID:24755439

  17. The dynamic genome of Hydra

    Science.gov (United States)

    Chapman, Jarrod A.; Kirkness, Ewen F.; Simakov, Oleg; Hampson, Steven E.; Mitros, Therese; Weinmaier, Therese; Rattei, Thomas; Balasubramanian, Prakash G.; Borman, Jon; Busam, Dana; Disbennett, Kathryn; Pfannkoch, Cynthia; Sumin, Nadezhda; Sutton, Granger G.; Viswanathan, Lakshmi Devi; Walenz, Brian; Goodstein, David M.; Hellsten, Uffe; Kawashima, Takeshi; Prochnik, Simon E.; Putnam, Nicholas H.; Shu, Shengquiang; Blumberg, Bruce; Dana, Catherine E.; Gee, Lydia; Kibler, Dennis F.; Law, Lee; Lindgens, Dirk; Martinez, Daniel E.; Peng, Jisong; Wigge, Philip A.; Bertulat, Bianca; Guder, Corina; Nakamura, Yukio; Ozbek, Suat; Watanabe, Hiroshi; Khalturin, Konstantin; Hemmrich, Georg; Franke, André; Augustin, René; Fraune, Sebastian; Hayakawa, Eisuke; Hayakawa, Shiho; Hirose, Mamiko; Hwang, Jung Shan; Ikeo, Kazuho; Nishimiya-Fujisawa, Chiemi; Ogura, Atshushi; Takahashi, Toshio; Steinmetz, Patrick R. H.; Zhang, Xiaoming; Aufschnaiter, Roland; Eder, Marie-Kristin; Gorny, Anne-Kathrin; Salvenmoser, Willi; Heimberg, Alysha M.; Wheeler, Benjamin M.; Peterson, Kevin J.; Böttger, Angelika; Tischler, Patrick; Wolf, Alexander; Gojobori, Takashi; Remington, Karin A.; Strausberg, Robert L.; Venter, J. Craig; Technau, Ulrich; Hobmayer, Bert; Bosch, Thomas C. G.; Holstein, Thomas W.; Fujisawa, Toshitaka; Bode, Hans R.; David, Charles N.; Rokhsar, Daniel S.; Steele, Robert E.

    2015-01-01

    The freshwater cnidarian Hydra was first described in 17021 and has been the object of study for 300 years. Experimental studies of Hydra between 1736 and 1744 culminated in the discovery of asexual reproduction of an animal by budding, the first description of regeneration in an animal, and successful transplantation of tissue between animals2. Today, Hydra is an important model for studies of axial patterning3, stem cell biology4 and regeneration5. Here we report the genome of Hydra magnipapillata and compare it to the genomes of the anthozoan Nematostella vectensis6 and other animals. The Hydra genome has been shaped by bursts of transposable element expansion, horizontal gene transfer, trans-splicing, and simplification of gene structure and gene content that parallel simplification of the Hydra life cycle. We also report the sequence of the genome of a novel bacterium stably associated with H. magnipapillata. Comparisons of the Hydra genome to the genomes of other animals shed light on the evolution of epithelia, contractile tissues, developmentally regulated transcription factors, the Spemann–Mangold organizer, pluripotency genes and the neuromuscular junction. PMID:20228792

  18. Spirochaetes as intestinal pathogens: Lessons from a Brachyspira genome

    Directory of Open Access Journals (Sweden)

    Hampson David J

    2009-05-01

    Full Text Available Abstract Anaerobic spirochaetes of the genus Brachyspira have long been known as important gut pathogens of pigs, but increasingly they are recognised as causing disease in birds and other animal species, including human beings. The genome sequence of the major swine pathogen Brachyspira hyodysenteriae was recently published, and this revealed extensive genome optimisation that leads to adaptation to the complex environment of the colon. The genome sequences of other pathogenic and non-pathogenic Brachyspira species are becoming available, and this data will help to reveal how these species have evolved and adapted to varied lifestyles in the large intestines of different species, and why some but not others can induce colitis and diarrhoea.

  19. tigaR: integrative significance analysis of temporal differential gene expression induced by genomic abnormalities

    NARCIS (Netherlands)

    Miok, V.; Wilting, S.M.; van de Wiel, M.A.; Jaspers, A.; van Noort, P.I.; Brakenhoff, R.H.; Snijders, P.J.F.; Steenbergen, R.D.M.; van Wieringen, W.N.

    2014-01-01

    Background: To determine which changes in the host cell genome are crucial for cervical carcinogenesis, a longitudinal in vitro model system of HPV-transformed keratinocytes was profiled in a genome-wide manner. Four cell lines affected with either HPV16 or HPV18 were assayed at 8 sequential time

  20. phiGENOME: an integrative navigation throughout bacteriophage genomes.

    Science.gov (United States)

    Stano, Matej; Klucar, Lubos

    2011-11-01

    phiGENOME is a web-based genome browser generating dynamic and interactive graphical representation of phage genomes stored in the phiSITE, database of gene regulation in bacteriophages. phiGENOME is an integral part of the phiSITE web portal (http://www.phisite.org/phigenome) and it was optimised for visualisation of phage genomes with the emphasis on the gene regulatory elements. phiGENOME consists of three components: (i) genome map viewer built using Adobe Flash technology, providing dynamic and interactive graphical display of phage genomes; (ii) sequence browser based on precisely formatted HTML tags, providing detailed exploration of genome features on the sequence level and (iii) regulation illustrator, based on Scalable Vector Graphics (SVG) and designed for graphical representation of gene regulations. Bringing 542 complete genome sequences accompanied with their rich annotations and references, makes phiGENOME a unique information resource in the field of phage genomics. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. IL-8 induces miR-424-5p expression and modulates SOCS2/STAT5 signaling pathway in oral squamous cell carcinoma.

    Science.gov (United States)

    Peng, Hsuan-Yu; Jiang, Shih-Sheng; Hsiao, Jenn-Ren; Hsiao, Michael; Hsu, Yuan-Ming; Wu, Guan-Hsun; Chang, Wei-Min; Chang, Jang-Yang; Jin, Shiow-Lian Catherine; Shiah, Shine-Gwo

    2016-06-01

    Suppressor of cytokine signaling (SOCS) proteins are negative feedback regulators of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Dysregulation of SOCS protein expression in cancers can be one of the mechanisms that maintain STAT activation, but this mechanism is still poorly understood in oral squamous cell carcinoma (OSCC). Here, we report that SOCS2 protein is significantly downregulated in OSCC patients and its levels are inversely correlated with miR-424-5p expression. We identified the SOCS2 protein, which modulates STAT5 activity, as a direct target of miR-424-5p. The miR-424-5p-induced STAT5 phosphorylation, matrix metalloproteinases (MMPs) expression, and cell migration and invasion were blocked by SOCS2 restoration, suggesting that miR-424-5p exhibits its oncogenic activity through negatively regulating SOCS2 levels. Furthermore, miR-424-5p expression could be induced by the cytokine IL-8 primarily through enhancing STAT5 transcriptional activity rather than NF-κB signaling. Antagomir-mediated inactivation of miR-424-5p prevented the IL-8-induced cell migration and invasion, indicating that miR-424-5p is required for IL-8-induced cellular invasiveness. Taken together, these data indicate that STAT5-dependent expression of miR-424-5p plays an important role in mediating IL-8/STAT5/SOCS2 feedback loop, and scavenging miR-424-5p function using antagomir may have therapeutic potential for the treatment of OSCC. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  2. Causes of genome instability: the effect of low dose chemical exposures in modern society

    Science.gov (United States)

    Langie, Sabine A.S.; Koppen, Gudrun; Desaulniers, Daniel; Al-Mulla, Fahd; Al-Temaimi, Rabeah; Amedei, Amedeo; Azqueta, Amaya; Bisson, William H.; Brown, Dustin; Brunborg, Gunnar; Charles, Amelia K.; Chen, Tao; Colacci, Annamaria; Darroudi, Firouz; Forte, Stefano; Gonzalez, Laetitia; Hamid, Roslida A.; Knudsen, Lisbeth E.; Leyns, Luc; Lopez de Cerain Salsamendi, Adela; Memeo, Lorenzo; Mondello, Chiara; Mothersill, Carmel; Olsen, Ann-Karin; Pavanello, Sofia; Raju, Jayadev; Rojas, Emilio; Roy, Rabindra; Ryan, Elizabeth; Ostrosky-Wegman, Patricia; Salem, Hosni K.; Scovassi, Ivana; Singh, Neetu; Vaccari, Monica; Van Schooten, Frederik J.; Valverde, Mahara; Woodrick, Jordan; Zhang, Luoping; van Larebeke, Nik; Kirsch-Volders, Micheline; Collins, Andrew R.

    2015-01-01

    Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genome’s integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus, genome instability can be defined as an enhanced tendency for the genome to acquire mutations; ranging from changes to the nucleotide sequence to chromosomal gain, rearrangements or loss. This review raises the hypothesis that in addition to known human carcinogens, exposure to low dose of other chemicals present in our modern society could contribute to carcinogenesis by indirectly affecting genome stability. The selected chemicals with their mechanisms of action proposed to indirectly contribute to genome instability are: heavy metals (DNA repair, epigenetic modification, DNA damage signaling, telomere length), acrylamide (DNA repair, chromosome segregation), bisphenol A (epigenetic modification, DNA damage signaling, mitochondrial function, chromosome segregation), benomyl (chromosome segregation), quinones (epigenetic modification) and nano-sized particles (epigenetic pathways, mitochondrial function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make scientists aware of the increasing need to unravel the underlying mechanisms via which chemicals at low doses can induce genome instability and thus promote carcinogenesis. PMID:26106144

  3. The inhibition of cholera toxin-induced 5-HT release by the 5-HT3 receptor antagonist, granisetron, in the rat

    Science.gov (United States)

    Turvill, J L; Connor, P; Farthing, M J G

    2000-01-01

    The secretagogue 5-hydroxytryptamine (5-HT) is implicated in the pathophysiology of cholera. 5-HT released from enterochromaffin cells after cholera toxin exposure is thought to activate non-neuronally (5-HT2 dependent) and neuronally (5-HT3 dependent) mediated water and electrolyte secretion. CT-secretion can be reduced by preventing the release of 5-HT. Enterochromaffin cells possess numerous receptors that, under basal conditions, modulate 5-HT release. These include basolateral 5-HT3 receptors, the activation of which is known to enhance 5-HT release. Until now, 5-HT3 receptor antagonists (e.g. granisetron) have been thought to inhibit cholera toxin-induced fluid secretion by blockading 5-HT3 receptors on secretory enteric neurones. Instead we postulated that they act by inhibiting cholera toxin-induced enterochromaffin cell degranulation. Isolated intestinal segments in anaesthetized male Wistar rats, pre-treated with granisetron 75 μg kg−1, lidoocaine 6 mg kg−1 or saline, were instilled with a supramaximal dose of cholera toxin or saline. Net fluid movement was determined by small intestinal perfusion or gravimetry and small intestinal and luminal fluid 5-HT levels were determined by HPLC with fluorimetric detection. Intraluminal 5-HT release was proportional to the reduction in tissue 5-HT levels and to the onset of water and electrolyte secretion, suggesting that luminal 5-HT levels reflect enterochromaffin cell activity. Both lidocaine and granisetron inhibited fluid secretion. However, granisetron alone, and proportionately, reduced 5-HT release. The simultaneous inhibition of 5-HT release and fluid secretion by granisetron suggests that 5-HT release from enterochromaffin cells is potentiated by endogenous 5-HT3 receptors. The accentuated 5-HT release promotes cholera toxin-induced fluid secretion. PMID:10882387

  4. A tailing genome walking method suitable for genomes with high local GC content.

    Science.gov (United States)

    Liu, Taian; Fang, Yongxiang; Yao, Wenjuan; Guan, Qisai; Bai, Gang; Jing, Zhizhong

    2013-10-15

    The tailing genome walking strategies are simple and efficient. However, they sometimes can be restricted due to the low stringency of homo-oligomeric primers. Here we modified their conventional tailing step by adding polythymidine and polyguanine to the target single-stranded DNA (ssDNA). The tailed ssDNA was then amplified exponentially with a specific primer in the known region and a primer comprising 5' polycytosine and 3' polyadenosine. The successful application of this novel method for identifying integration sites mediated by φC31 integrase in goat genome indicates that the method is more suitable for genomes with high complexity and local GC content. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Nitrogen gas plasma treatment of bacterial spores induces oxidative stress that damages the genomic DNA.

    Science.gov (United States)

    Sakudo, Akikazu; Toyokawa, Yoichi; Nakamura, Tetsuji; Yagyu, Yoshihito; Imanishi, Yuichiro

    2017-01-01

    Gas plasma, produced by a short high‑voltage pulse generated from a static induction thyristor power supply [1.5 kilo pulse/sec (kpps)], was demonstrated to inactivate Geobacillus stearothermophilus spores (decimal reduction time at 15 min, 2.48 min). Quantitative polymerase chain reaction and enzyme‑linked immunosorbent assays further indicated that nitrogen gas plasma treatment for 15 min decreased the level of intact genomic DNA and increased the level of 8-hydroxy-2'-deoxyguanosine, a major product of DNA oxidation. Three potential inactivation factors were generated during operation of the gas plasma instrument: Heat, longwave ultraviolet-A and oxidative stress (production of hydrogen peroxide, nitrite and nitrate). Treatment of the spores with hydrogen peroxide (3x2‑4%) effectively inactivated the bacteria, whereas heat treatment (100˚C), exposure to UV-A (75‑142 mJ/cm2) and 4.92 mM peroxynitrite (•ONOO‑), which is decomposed into nitrite and nitrate, did not. The results of the present study suggest the gas plasma treatment inactivates bacterial spores primarily by generating hydrogen peroxide, which contributes to the oxidation of the host genomic DNA.

  6. Social isolation stress induces ATF-7 phosphorylation and impairs silencing of the 5-HT 5B receptor gene

    Science.gov (United States)

    Maekawa, Toshio; Kim, Seungjoon; Nakai, Daisuke; Makino, Chieko; Takagi, Tsuyoshi; Ogura, Hiroo; Yamada, Kazuyuki; Chatton, Bruno; Ishii, Shunsuke

    2010-01-01

    Many symptoms induced by isolation rearing of rodents may be relevant to neuropsychiatric disorders, including depression. However, identities of transcription factors that regulate gene expression in response to chronic social isolation stress remain elusive. The transcription factor ATF-7 is structurally related to ATF-2, which is activated by various stresses, including inflammatory cytokines. Here, we report that Atf-7-deficient mice exhibit abnormal behaviours and increased 5-HT receptor 5B (Htr5b) mRNA levels in the dorsal raphe nuclei. ATF-7 silences the transcription of Htr5B by directly binding to its 5′-regulatory region, and mediates histone H3-K9 trimethylation via interaction with the ESET histone methyltransferase. Isolation-reared wild-type (WT) mice exhibit abnormal behaviours that resemble those of Atf-7-deficient mice. Upon social isolation stress, ATF-7 in the dorsal raphe nucleus is phosphorylated via p38 and is released from the Htr5b promoter, leading to the upregulation of Htr5b. Thus, ATF-7 may have a critical role in gene expression induced by social isolation stress. PMID:19893493

  7. The genome of the pear (Pyrus bretschneideri Rehd.).

    Science.gov (United States)

    Wu, Jun; Wang, Zhiwen; Shi, Zebin; Zhang, Shu; Ming, Ray; Zhu, Shilin; Khan, M Awais; Tao, Shutian; Korban, Schuyler S; Wang, Hao; Chen, Nancy J; Nishio, Takeshi; Xu, Xun; Cong, Lin; Qi, Kaijie; Huang, Xiaosan; Wang, Yingtao; Zhao, Xiang; Wu, Juyou; Deng, Cao; Gou, Caiyun; Zhou, Weili; Yin, Hao; Qin, Gaihua; Sha, Yuhui; Tao, Ye; Chen, Hui; Yang, Yanan; Song, Yue; Zhan, Dongliang; Wang, Juan; Li, Leiting; Dai, Meisong; Gu, Chao; Wang, Yuezhi; Shi, Daihu; Wang, Xiaowei; Zhang, Huping; Zeng, Liang; Zheng, Danman; Wang, Chunlei; Chen, Maoshan; Wang, Guangbiao; Xie, Lin; Sovero, Valpuri; Sha, Shoufeng; Huang, Wenjiang; Zhang, Shujun; Zhang, Mingyue; Sun, Jiangmei; Xu, Linlin; Li, Yuan; Liu, Xing; Li, Qingsong; Shen, Jiahui; Wang, Junyi; Paull, Robert E; Bennetzen, Jeffrey L; Wang, Jun; Zhang, Shaoling

    2013-02-01

    The draft genome of the pear (Pyrus bretschneideri) using a combination of BAC-by-BAC and next-generation sequencing is reported. A 512.0-Mb sequence corresponding to 97.1% of the estimated genome size of this highly heterozygous species is assembled with 194× coverage. High-density genetic maps comprising 2005 SNP markers anchored 75.5% of the sequence to all 17 chromosomes. The pear genome encodes 42,812 protein-coding genes, and of these, ~28.5% encode multiple isoforms. Repetitive sequences of 271.9 Mb in length, accounting for 53.1% of the pear genome, are identified. Simulation of eudicots to the ancestor of Rosaceae has reconstructed nine ancestral chromosomes. Pear and apple diverged from each other ~5.4-21.5 million years ago, and a recent whole-genome duplication (WGD) event must have occurred 30-45 MYA prior to their divergence, but following divergence from strawberry. When compared with the apple genome sequence, size differences between the apple and pear genomes are confirmed mainly due to the presence of repetitive sequences predominantly contributed by transposable elements (TEs), while genic regions are similar in both species. Genes critical for self-incompatibility, lignified stone cells (a unique feature of pear fruit), sorbitol metabolism, and volatile compounds of fruit have also been identified. Multiple candidate SFB genes appear as tandem repeats in the S-locus region of pear; while lignin synthesis-related gene family expansion and highly expressed gene families of HCT, C3'H, and CCOMT contribute to high accumulation of both G-lignin and S-lignin. Moreover, alpha-linolenic acid metabolism is a key pathway for aroma in pear fruit.

  8. Amino acid limitation induces down-regulation of WNT5a at transcriptional level

    International Nuclear Information System (INIS)

    Wang Zuguang; Chen Hong

    2009-01-01

    An aberrant WNT signaling contributes to the development and progression of multiple cancers. WNT5a is one of the WNT signaling molecules. This study was designed to test the hypothesis that amino acid deprivation induces changes in the WNT signaling pathway in colon cancer cells. Results showed that targets of the amino acid response pathway, ATF3 and p21, were induced in the human colon cancer cell line SW480 during amino acid limitation. There was a significant decrease in the WNT5a mRNA level following amino acid deprivation. The down-regulation of WNT5a mRNA by amino acid deprivation is not due to mRNA destabilization. There is a reduction of nuclear β-catenin protein level by amino acid limitation. Under amino acid limitation, phosphorylation of ERK1/2 was increased and the blockage of ERK1/2 by the inhibitor U0126 partially restored WNT5a mRNA level. In conclusion, amino acid limitation in colon cancer cells induces phosphorylation of ERK1/2, which then down-regulates WNT5a expression.

  9. The genome sequence of Caenorhabditis briggsae: a platform for comparative genomics.

    Directory of Open Access Journals (Sweden)

    Lincoln D Stein

    2003-11-01

    Full Text Available The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp and C. elegans (100.3 Mbp genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C

  10. A 'new lease of life': FnCpf1 possesses DNA cleavage activity for genome editing in human cells.

    Science.gov (United States)

    Tu, Mengjun; Lin, Li; Cheng, Yilu; He, Xiubin; Sun, Huihui; Xie, Haihua; Fu, Junhao; Liu, Changbao; Li, Jin; Chen, Ding; Xi, Haitao; Xue, Dongyu; Liu, Qi; Zhao, Junzhao; Gao, Caixia; Song, Zongming; Qu, Jia; Gu, Feng

    2017-11-02

    Cpf1 nucleases were recently reported to be highly specific and programmable nucleases with efficiencies comparable to those of SpCas9. AsCpf1 and LbCpf1 require a single crRNA and recognize a 5'-TTTN-3' protospacer adjacent motif (PAM) at the 5' end of the protospacer for genome editing. For widespread application in precision site-specific human genome editing, the range of sequences that AsCpf1 and LbCpf1 can recognize is limited due to the size of this PAM. To address this limitation, we sought to identify a novel Cpf1 nuclease with simpler PAM requirements. Specifically, here we sought to test and engineer FnCpf1, one reported Cpf1 nuclease (FnCpf1) only requires 5'-TTN-3' as a PAM but does not exhibit detectable levels of nuclease-induced indels at certain locus in human cells. Surprisingly, we found that FnCpf1 possesses DNA cleavage activity in human cells at multiple loci. We also comprehensively and quantitatively examined various FnCpf1 parameters in human cells, including spacer sequence, direct repeat sequence and the PAM sequence. Our study identifies FnCpf1 as a new member of the Cpf1 family for human genome editing with distinctive characteristics, which shows promise as a genome editing tool with the potential for both research and therapeutic applications. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. HPCE quantification of 5-methyl-2'-deoxycytidine in genomic DNA: methodological optimization for chestnut and other woody species.

    Science.gov (United States)

    Hasbún, Rodrigo; Valledor, Luís; Rodríguez, José L; Santamaria, Estrella; Ríos, Darcy; Sanchez, Manuel; Cañal, María J; Rodríguez, Roberto

    2008-01-01

    Quantification of deoxynucleosides using micellar high-performance capillary electrophoresis (HPCE) is an efficient, fast and inexpensive evaluation method of genomic DNA methylation. This approach has been demonstrated to be more sensitive and specific than other methods for the quantification of DNA methylation content. However, effective detection and quantification of 5-methyl-2'-deoxycytidine depend of the sample characteristics. Previous works have revealed that in most woody species, the quality and quantity of RNA-free DNA extracted that is suitable for analysis by means of HPCE varies among species of the same gender, among tissues taken from the same tree, and vary in the same tissue depending on the different seasons of the year. The aim of this work is to establish a quantification method of genomic DNA methylation that lends itself to use in different Castanea sativa Mill. materials, and in other angiosperm and gymnosperm woody species. Using a DNA extraction kit based in silica membrane has increased the resolutive capacity of the method. Under these conditions, it can be analyzed different organs or tissues of angiosperms and gymnosperms, regardless of their state of development. We emphasized the importance of samples free of nucleosides, although, in the contrary case, the method ensures the effective separation of deoxynucleosides and identification of 5-methyl-2'-deoxycytidine.

  12. Evolutionary force of AT-rich repeats to trap genomic and episomal DNAs into the rice genome: lessons from endogenous pararetrovirus.

    Science.gov (United States)

    Liu, Ruifang; Koyanagi, Kanako O; Chen, Sunlu; Kishima, Yuji

    2012-12-01

    In plant genomes, the incorporation of DNA segments is not a common method of artificial gene transfer. Nevertheless, various segments of pararetroviruses have been found in plant genomes in recent decades. The rice genome contains a number of segments of endogenous rice tungro bacilliform virus-like sequences (ERTBVs), many of which are present between AT dinucleotide repeats (ATrs). Comparison of genomic sequences between two closely related rice subspecies, japonica and indica, allowed us to verify the preferential insertion of ERTBVs into ATrs. In addition to ERTBVs, the comparative analyses showed that ATrs occasionally incorporate repeat sequences including transposable elements, and a wide range of other sequences. Besides the known genomic sequences, the insertion sequences also represented DNAs of unclear origins together with ERTBVs, suggesting that ATrs have integrated episomal DNAs that would have been suspended in the nucleus. Such insertion DNAs might be trapped by ATrs in the genome in a host-dependent manner. Conversely, other simple mono- and dinucleotide sequence repeats (SSR) were less frequently involved in insertion events relative to ATrs. Therefore, ATrs could be regarded as hot spots of double-strand breaks that induce non-homologous end joining. The insertions within ATrs occasionally generated new gene-related sequences or involved structural modifications of existing genes. Likewise, in a comparison between Arabidopsis thaliana and Arabidopsis lyrata, the insertions preferred ATrs to other SSRs. Therefore ATrs in plant genomes could be considered as genomic dumping sites that have trapped various DNA molecules and may have exerted a powerful evolutionary force. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  13. Use of reiterative primer extension methodology to map UV-induced photoproducts at the nucleotide level in the laci gene from genomic DNA

    International Nuclear Information System (INIS)

    Chandrasekhar, D.; Houten, B. Van

    1994-01-01

    A newly developed reiterative primer extension assay has been employed to examine photoproduct formation and repair at the nucleotide level. Analysis of UV-induced DNA photoproduct hotspots in the first 184 base pairs of the laci genes of genomic E. coli DNA has revealed that photoproducts are formed linearly with dose and display a sequence-dependent increase. Generally, pyrimdine dimers were twice as frequent as all other UV-induced photoproducts. However, specific sites showed differing distributions. A post-irradiation recovery period revealed differences in the repair efficiency at individual nucleotides. Repair of photoproducts on the transcribed strand was generally twice as efficient as repair of photoproducts on the nontranscribed strand, indicating that strand-specific DNA repair occurs in the constitutively transcribed laci gene of E. coli. The UV-induced DNA photoproduct distribution following repair was well correlated with an established UV-induced mutation spectrum for wild-type E. coli cells. This analysis revealed that photoproduct hotspots on the efficiently repaired transcribed strand did not correlate with mutagenic hotspots. These data strongly support the hypothesis that mutations arise at inefficiently repaired sites on the nontranscribed strand

  14. GenomeRNAi: a database for cell-based RNAi phenotypes.

    Science.gov (United States)

    Horn, Thomas; Arziman, Zeynep; Berger, Juerg; Boutros, Michael

    2007-01-01

    RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at http://rnai.dkfz.de.

  15. Compactness of viral genomes: effect of disperse and localized random mutations

    Science.gov (United States)

    Lošdorfer Božič, Anže; Micheletti, Cristian; Podgornik, Rudolf; Tubiana, Luca

    2018-02-01

    Genomes of single-stranded RNA viruses have evolved to optimize several concurrent properties. One of them is the architecture of their genomic folds, which must not only feature precise structural elements at specific positions, but also allow for overall spatial compactness. The latter was shown to be disrupted by random synonymous mutations, a disruption which can consequently negatively affect genome encapsidation. In this study, we use three mutation schemes with different degrees of locality to mutate the genomes of phage MS2 and Brome Mosaic virus in order to understand the observed sensitivity of the global compactness of their folds. We find that mutating local stretches of their genomes’ sequence or structure is less disruptive to their compactness compared to inducing randomly-distributed mutations. Our findings are indicative of a mechanism for the conservation of compactness acting on a global scale of the genomes, and have several implications for understanding the interplay between local and global architecture of viral RNA genomes.

  16. Statistical analyses of conserved features of genomic islands in bacteria.

    Science.gov (United States)

    Guo, F-B; Xia, Z-K; Wei, W; Zhao, H-L

    2014-03-17

    We performed statistical analyses of five conserved features of genomic islands of bacteria. Analyses were made based on 104 known genomic islands, which were identified by comparative methods. Four of these features include sequence size, abnormal G+C content, flanking tRNA gene, and embedded mobility gene, which are frequently investigated. One relatively new feature, G+C homogeneity, was also investigated. Among the 104 known genomic islands, 88.5% were found to fall in the typical length of 10-200 kb and 80.8% had G+C deviations with absolute values larger than 2%. For the 88 genomic islands whose hosts have been sequenced and annotated, 52.3% of them were found to have flanking tRNA genes and 64.7% had embedded mobility genes. For the homogeneity feature, 85% had an h homogeneity index less than 0.1, indicating that their G+C content is relatively uniform. Taking all the five features into account, 87.5% of 88 genomic islands had three of them. Only one genomic island had only one conserved feature and none of the genomic islands had zero features. These statistical results should help to understand the general structure of known genomic islands. We found that larger genomic islands tend to have relatively small G+C deviations relative to absolute values. For example, the absolute G+C deviations of 9 genomic islands longer than 100,000 bp were all less than 5%. This is a novel but reasonable result given that larger genomic islands should have greater restrictions in their G+C contents, in order to maintain the stable G+C content of the recipient genome.

  17. Evidence for the involvement of 5-lipoxygenase products in ethanol-induced intestinal plasma protein loss

    International Nuclear Information System (INIS)

    Beck, I.T.; Boyd, A.J.; Dinda, P.K.

    1988-01-01

    In this study the authors investigated whether the products of 5-lipoxygenase (5-LO) were involved in the jejunal microvascular injury induced by intraluminal ethanol (ETH). A group of rabbits was given orally a selective inhibitor of 5-LO in two 10-mg doses, 24, and 2 h before the experiments. A jejunal segment was perfused with a control solution (control segment) and an adjacent segment with an ETH-containing solution (ETH-perfused segment). In a series of experiments, they measured 5-LO activity of the jejunal segments of both groups using the generation of leukotriene B 4 (LTB 4 ) as an index. In a second series of experiments, they determined the ETH-induced intraluminal protein loss, which was taken as a measure of mucosal microvascular damage. The ETH-induced increase in protein loss was significantly lower in the treated than in the untreated group. These findings suggest that products of 5-LO are involved in the ETH-induced jejunal microvascular injury

  18. REARRANGEMENT IN THE B-GENOME FROM DIPLOID PROGENITOR TO WHEAT ALLOPOLYPOLID

    Directory of Open Access Journals (Sweden)

    Salina E.A.

    2012-08-01

    Full Text Available Three key periods that were accompanied by considerable rearrangements in the B genome of wheat and its progenitor can be considered. The first period covers the period from the divergence of diploid Triticum and Aegilops species from their common progenitor (2.5–6 million years ago to formation of the tetraploid T. diccocoides (about 500 thousand years ago. Significant genomic rearrangements in the diploid progenitor of the B genome, Ae. speltoides (SS genome, involved a considerable amplification of repeated DNA sequences, which led to an increase in the number of heterochromatin blocks on chromosomes relative to other diploid Aegilops and Triticum species. Our analysis has demonstrated that during this period the Spelt1 repeats intensively amplified as well as several mobile elements proliferated, in particular, the genome-specific gypsy LTR-retrotransposon Fatima and CACTA DNA-transposon Caspar. The second period in the B-genome evolution was associated with the emergence of tetraploid (BBAA genome and its subsequent evolution. The third most important event leading to the next rearrangement of the B genome took place relatively recently, 7000–9500 years ago, being associated with the emergence of hexaploid wheat with the genomic formula BBAADD. The evolution of the B/S genome involved intergenomic and intragenomic translocations and chromosome inversions. So far, five rearrangements in the B-genome chromosomes of polyploid wheats has been observed and described; the majority of them took place during the formation and evolution of tetraploid species. The mapping of the S-genome chromosomes and comparison with the B-genome chromosome maps have demonstrated that individual rearrangements pre-existed in Ae. speltoides; moreover, Ae. speltoides is polymorphic for these rearrangements.Chromosome 5B is nearly 870 Mbp (5BL = 580 Mbp and 5BS = 290 Mbp and is known to carry important genes controlling the key aspects of wheat biology, in

  19. DNA lesions induced by replication stress trigger mitotic aberration and tetraploidy development.

    Directory of Open Access Journals (Sweden)

    Yosuke Ichijima

    Full Text Available During tumorigenesis, cells acquire immortality in association with the development of genomic instability. However, it is still elusive how genomic instability spontaneously generates during the process of tumorigenesis. Here, we show that precancerous DNA lesions induced by oncogene acceleration, which induce situations identical to the initial stages of cancer development, trigger tetraploidy/aneuploidy generation in association with mitotic aberration. Although oncogene acceleration primarily induces DNA replication stress and the resulting lesions in the S phase, these lesions are carried over into the M phase and cause cytokinesis failure and genomic instability. Unlike directly induced DNA double-strand breaks, DNA replication stress-associated lesions are cryptogenic and pass through cell-cycle checkpoints due to limited and ineffective activation of checkpoint factors. Furthermore, since damaged M-phase cells still progress in mitotic steps, these cells result in chromosomal mis-segregation, cytokinesis failure and the resulting tetraploidy generation. Thus, our results reveal a process of genomic instability generation triggered by precancerous DNA replication stress.

  20. Dynamic maps of UV damage formation and repair for the human genome.

    Science.gov (United States)

    Hu, Jinchuan; Adebali, Ogun; Adar, Sheera; Sancar, Aziz

    2017-06-27

    Formation and repair of UV-induced DNA damage in human cells are affected by cellular context. To study factors influencing damage formation and repair genome-wide, we developed a highly sensitive single-nucleotide resolution damage mapping method [high-sensitivity damage sequencing (HS-Damage-seq)]. Damage maps of both cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] from UV-irradiated cellular and naked DNA revealed that the effect of transcription factor binding on bulky adducts formation varies, depending on the specific transcription factor, damage type, and strand. We also generated time-resolved UV damage maps of both CPDs and (6-4)PPs by HS-Damage-seq and compared them to the complementary repair maps of the human genome obtained by excision repair sequencing to gain insight into factors that affect UV-induced DNA damage and repair and ultimately UV carcinogenesis. The combination of the two methods revealed that, whereas UV-induced damage is virtually uniform throughout the genome, repair is affected by chromatin states, transcription, and transcription factor binding, in a manner that depends on the type of DNA damage.

  1. DNA damage checkpoint kinase ATM regulates germination and maintains genome stability in seeds.

    Science.gov (United States)

    Waterworth, Wanda M; Footitt, Steven; Bray, Clifford M; Finch-Savage, William E; West, Christopher E

    2016-08-23

    Genome integrity is crucial for cellular survival and the faithful transmission of genetic information. The eukaryotic cellular response to DNA damage is orchestrated by the DNA damage checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR). Here we identify important physiological roles for these sensor kinases in control of seed germination. We demonstrate that double-strand breaks (DSBs) are rate-limiting for germination. We identify that desiccation tolerant seeds exhibit a striking transcriptional DSB damage response during germination, indicative of high levels of genotoxic stress, which is induced following maturation drying and quiescence. Mutant atr and atm seeds are highly resistant to aging, establishing ATM and ATR as determinants of seed viability. In response to aging, ATM delays germination, whereas atm mutant seeds germinate with extensive chromosomal abnormalities. This identifies ATM as a major factor that controls germination in aged seeds, integrating progression through germination with surveillance of genome integrity. Mechanistically, ATM functions through control of DNA replication in imbibing seeds. ATM signaling is mediated by transcriptional control of the cell cycle inhibitor SIAMESE-RELATED 5, an essential factor required for the aging-induced delay to germination. In the soil seed bank, seeds exhibit increased transcript levels of ATM and ATR, with changes in dormancy and germination potential modulated by environmental signals, including temperature and soil moisture. Collectively, our findings reveal physiological functions for these sensor kinases in linking genome integrity to germination, thereby influencing seed quality, crucial for plant survival in the natural environment and sustainable crop production.

  2. Genetic and epigenetic variation in 5S ribosomal RNA genes reveals genome dynamics in Arabidopsis thaliana.

    Science.gov (United States)

    Simon, Lauriane; Rabanal, Fernando A; Dubos, Tristan; Oliver, Cecilia; Lauber, Damien; Poulet, Axel; Vogt, Alexander; Mandlbauer, Ariane; Le Goff, Samuel; Sommer, Andreas; Duborjal, Hervé; Tatout, Christophe; Probst, Aline V

    2018-04-06

    Organized in tandem repeat arrays in most eukaryotes and transcribed by RNA polymerase III, expression of 5S rRNA genes is under epigenetic control. To unveil mechanisms of transcriptional regulation, we obtained here in depth sequence information on 5S rRNA genes from the Arabidopsis thaliana genome and identified differential enrichment in epigenetic marks between the three 5S rDNA loci situated on chromosomes 3, 4 and 5. We reveal the chromosome 5 locus as the major source of an atypical, long 5S rRNA transcript characteristic of an open chromatin structure. 5S rRNA genes from this locus translocated in the Landsberg erecta ecotype as shown by linkage mapping and chromosome-specific FISH analysis. These variations in 5S rDNA locus organization cause changes in the spatial arrangement of chromosomes in the nucleus. Furthermore, 5S rRNA gene arrangements are highly dynamic with alterations in chromosomal positions through translocations in certain mutants of the RNA-directed DNA methylation pathway and important copy number variations among ecotypes. Finally, variations in 5S rRNA gene sequence, chromatin organization and transcripts indicate differential usage of 5S rDNA loci in distinct ecotypes. We suggest that both the usage of existing and new 5S rDNA loci resulting from translocations may impact neighboring chromatin organization.

  3. FR-like EBNA1 binding repeats in the human genome

    International Nuclear Information System (INIS)

    D'Herouel, Aymeric Fouquier; Birgersdotter, Anna; Werner, Maria

    2010-01-01

    Epstein-Barr virus (EBV) is widely spread in the human population. EBV nuclear antigen 1 (EBNA1) is a transcription factor that activates viral genes and is necessary for viral replication and partitioning, which binds the EBV genome cooperatively. We identify similar EBNA1 repeat binding sites in the human genome using a nearest-neighbor positional weight matrix. Previously experimentally verified EBNA1 sites in the human genome are successfully recovered by our approach. Most importantly, 40 novel regions are identified in the human genome, constituted of tandemly repeated binding sites for EBNA1. Genes located in the vicinity of these regions are presented as possible targets for EBNA1-mediated regulation. Among these, four are discussed in more detail: IQCB1, IMPG1, IRF2BP2 and TPO. Incorporating the cooperative actions of EBNA1 is essential when identifying regulatory regions in the human genome and we believe the findings presented here are highly valuable for the understanding of EBV-induced phenotypic changes.

  4. Whole-genome sequencing of a laboratory-evolved yeast strain

    Directory of Open Access Journals (Sweden)

    Dunham Maitreya J

    2010-02-01

    Full Text Available Abstract Background Experimental evolution of microbial populations provides a unique opportunity to study evolutionary adaptation in response to controlled selective pressures. However, until recently it has been difficult to identify the precise genetic changes underlying adaptation at a genome-wide scale. New DNA sequencing technologies now allow the genome of parental and evolved strains of microorganisms to be rapidly determined. Results We sequenced >93.5% of the genome of a laboratory-evolved strain of the yeast Saccharomyces cerevisiae and its ancestor at >28× depth. Both single nucleotide polymorphisms and copy number amplifications were found, with specific gains over array-based methodologies previously used to analyze these genomes. Applying a segmentation algorithm to quantify structural changes, we determined the approximate genomic boundaries of a 5× gene amplification. These boundaries guided the recovery of breakpoint sequences, which provide insights into the nature of a complex genomic rearrangement. Conclusions This study suggests that whole-genome sequencing can provide a rapid approach to uncover the genetic basis of evolutionary adaptations, with further applications in the study of laboratory selections and mutagenesis screens. In addition, we show how single-end, short read sequencing data can provide detailed information about structural rearrangements, and generate predictions about the genomic features and processes that underlie genome plasticity.

  5. Chronic hypoxia promotes pulmonary artery endothelial cell proliferation through H2O2-induced 5-lipoxygenase.

    Directory of Open Access Journals (Sweden)

    Kristi M Porter

    Full Text Available Pulmonary Hypertension (PH is a progressive disorder characterized by endothelial dysfunction and proliferation. Hypoxia induces PH by increasing vascular remodeling. A potential mediator in hypoxia-induced PH development is arachidonate 5-Lipoxygenase (ALOX5. While ALOX5 metabolites have been shown to promote pulmonary vasoconstriction and endothelial cell proliferation, the contribution of ALOX5 to hypoxia-induced proliferation remains unknown. We hypothesize that hypoxia exposure stimulates HPAEC proliferation by increasing ALOX5 expression and activity. To test this, human pulmonary artery endothelial cells (HPAEC were cultured under normoxic (21% O2 or hypoxic (1% O2 conditions for 24-, 48-, or 72 hours. In a subset of cells, the ALOX5 inhibitor, zileuton, or the 5-lipoxygenase activating protein inhibitor, MK-886, was administered during hypoxia exposure. ALOX5 expression was measured by qRT-PCR and western blot and HPAEC proliferation was assessed. Our results demonstrate that 24 and 48 hours of hypoxia exposure have no effect on HPAEC proliferation or ALOX5 expression. Seventy two hours of hypoxia significantly increases HPAEC ALOX5 expression, hydrogen peroxide (H2O2 release, and HPAEC proliferation. We also demonstrate that targeted ALOX5 gene silencing or inhibition of the ALOX5 pathway by pharmacological blockade attenuates hypoxia-induced HPAEC proliferation. Furthermore, our findings indicate that hypoxia-induced increases in cell proliferation and ALOX5 expression are dependent on H2O2 production, as administration of the antioxidant PEG-catalase blocks these effects and addition of H2O2 to HPAEC promotes proliferation. Overall, these studies indicate that hypoxia exposure induces HPAEC proliferation by activating the ALOX5 pathway via the generation of H2O2.

  6. Identification and analysis of genome-wide SNPs provide insight into signatures of selection and domestication in channel catfish (Ictalurus punctatus.

    Directory of Open Access Journals (Sweden)

    Luyang Sun

    Full Text Available Domestication and selection for important performance traits can impact the genome, which is most often reflected by reduced heterozygosity in and surrounding genes related to traits affected by selection. In this study, analysis of the genomic impact caused by domestication and artificial selection was conducted by investigating the signatures of selection using single nucleotide polymorphisms (SNPs in channel catfish (Ictalurus punctatus. A total of 8.4 million candidate SNPs were identified by using next generation sequencing. On average, the channel catfish genome harbors one SNP per 116 bp. Approximately 6.6 million, 5.3 million, 4.9 million, 7.1 million and 6.7 million SNPs were detected in the Marion, Thompson, USDA103, Hatchery strain, and wild population, respectively. The allele frequencies of 407,861 SNPs differed significantly between the domestic and wild populations. With these SNPs, 23 genomic regions with putative selective sweeps were identified that included 11 genes. Although the function for the majority of the genes remain unknown in catfish, several genes with known function related to aquaculture performance traits were included in the regions with selective sweeps. These included hypoxia-inducible factor 1β. HIFιβ.. and the transporter gene ATP-binding cassette sub-family B member 5 (ABCB5. HIF1β. is important for response to hypoxia and tolerance to low oxygen levels is a critical aquaculture trait. The large numbers of SNPs identified from this study are valuable for the development of high-density SNP arrays for genetic and genomic studies of performance traits in catfish.

  7. Two Inducible Prophages of an Antarctic Pseudomonas sp. ANT_H14 Use the Same Capsid for Packaging Their Genomes – Characterization of a Novel Phage Helper-Satellite System

    Science.gov (United States)

    Dziewit, Lukasz; Radlinska, Monika

    2016-01-01

    Two novel prophages ФAH14a and ФAH14b of a psychrotolerant Antarctic bacterium Pseudomonas sp. ANT_H14 have been characterized. They were simultaneously induced with mitomycin C and packed into capsids of the same size and protein composition. The genome sequences of ФAH14a and ФAH14b have been determined. ФAH14b, the phage with a smaller genome (16,812 bp) seems to parasitize ФAH14a (55,060 bp) and utilizes its capsids, as only the latter encodes a complete set of structural proteins. Both viruses probably constitute a phage helper-satellite system, analogous to the P2-P4 duo. This study describes the architecture and function of the ФAH14a and ФAH14b genomes. Moreover, a functional analysis of a ФAH14a-encoded lytic enzyme and a DNA methyltransferase was performed. In silico analysis revealed the presence of the homologs of ФAH14a and ФAH14b in other Pseudomonas genomes, which may suggest that helper-satellite systems related to the one described in this work are common in pseudomonads. PMID:27387973

  8. Induced Genome-Wide Binding of Three Arabidopsis WRKY Transcription Factors during Early MAMP-Triggered Immunity.

    Science.gov (United States)

    Birkenbihl, Rainer P; Kracher, Barbara; Somssich, Imre E

    2017-01-01

    During microbial-associated molecular pattern-triggered immunity (MTI), molecules derived from microbes are perceived by cell surface receptors and upon signaling to the nucleus initiate a massive transcriptional reprogramming critical to mount an appropriate host defense response. WRKY transcription factors play an important role in regulating these transcriptional processes. Here, we determined on a genome-wide scale the flg22-induced in vivo DNA binding dynamics of three of the most prominent WRKY factors, WRKY18, WRKY40, and WRKY33. The three WRKY factors each bound to more than 1000 gene loci predominantly at W-box elements, the known WRKY binding motif. Binding occurred mainly in the 500-bp promoter regions of these genes. Many of the targeted genes are involved in signal perception and transduction not only during MTI but also upon damage-associated molecular pattern-triggered immunity, providing a mechanistic link between these functionally interconnected basal defense pathways. Among the additional targets were genes involved in the production of indolic secondary metabolites and in modulating distinct plant hormone pathways. Importantly, among the targeted genes were numerous transcription factors, encoding predominantly ethylene response factors, active during early MTI, and WRKY factors, supporting the previously hypothesized existence of a WRKY subregulatory network. Transcriptional analysis revealed that WRKY18 and WRKY40 function redundantly as negative regulators of flg22-induced genes often to prevent exaggerated defense responses. © 2016 American Society of Plant Biologists. All rights reserved.

  9. Limonene inhibits methamphetamine-induced locomotor activity via regulation of 5-HT neuronal function and dopamine release.

    Science.gov (United States)

    Yun, Jaesuk

    2014-05-15

    Methamphetamine is a psychomotor stimulant that produces hyperlocomotion in rodents. Limonene (a cyclic terpene from citrus essential oils) has been reported to induce sedative effects. In this study, we demonstrated that limonene administration significantly inhibited serotonin (5-hydroxytryptamine, 5-HT)-induced head twitch response in mice. In rats, pretreatment with limonene decreased hyperlocomotion induced by methamphetamine injection. In addition, limonene reversed the increase in dopamine levels in the nucleus accumbens of rats given methamphetamine. These results suggest that limonene may inhibit stimulant-induced behavioral changes via regulating dopamine levels and 5-HT receptor function. Copyright © 2013 Elsevier GmbH. All rights reserved.

  10. The Role of Hippocampal 5HT3 Receptors in Harmaline-Induced Memory Deficit

    Directory of Open Access Journals (Sweden)

    Mohammad Nasehi

    2015-07-01

    Full Text Available Introduction: The plethora of studies indicated that there is a cross talk relationship between harmaline and serotonergic (5-HT system on cognitive and non-cognitive behaviors. Thus, the purpose of this study is to assess the effects of hippocampal 5-HT4 receptor on memory acquisition deficit induced by harmaline.  Methods: Harmaline was injected peritoneally, while 5-HT4 receptor agonist (RS67333 and antagonist (RS23597-190 were injected intra-hippocampal. A single-trial step-down passive avoidance, open field and tail flick tasks were used for measurement of memory, locomotor activity and pain responses, respectively.  Results: The data revealed that pre-training injection of higher dose of harmaline (1 mg/kg, RS67333 (0.5 ng/mouse and RS23597-190 (0.5 ng/mouse decreased memory acquisition process in the adult mice. Moreover, concurrent pre-training administration of subthreshold dose of RS67333 (0.005 ng/mouse or RS23597-190 (0.005 ng/mouse with subthreshold dose of harmaline (0.5 mg/kg, i.p. intensify impairment of memory acquisition. All above interventions did not change locomotion and tail flick behaviors.  Discussion: The results demonstrated that the synergistic effect between both hippocampal 5-HT4 receptor agonist and antagonist with impairment of memory acquisition induced by harmaline, indicating a modulatory effect for hippocampal 5HT4 receptor on Harmaline induced amnesia.

  11. Genomics using the Assembly of the Mink Genome

    DEFF Research Database (Denmark)

    Guldbrandtsen, Bernt; Cai, Zexi; Sahana, Goutam

    2018-01-01

    The American Mink’s (Neovison vison) genome has recently been sequenced. This opens numerous avenues of research both for studying the basic genetics and physiology of the mink as well as genetic improvement in mink. Using genotyping-by-sequencing (GBS) generated marker data for 2,352 Danish farm...... mink runs of homozygosity (ROH) were detect in mink genomes. Detectable ROH made up on average 1.7% of the genome indicating the presence of at most a moderate level of genomic inbreeding. The fraction of genome regions found in ROH varied. Ten percent of the included regions were never found in ROH....... The ability to detect ROH in the mink genome also demonstrates the general reliability of the new mink genome assembly. Keywords: american mink, run of homozygosity, genome, selection, genomic inbreeding...

  12. Nucleotide Excision Repair in Cellular Chromatin: Studies with Yeast from Nucleotide to Gene to Genome

    Directory of Open Access Journals (Sweden)

    Simon Reed

    2012-09-01

    Full Text Available Here we review our development of, and results with, high resolution studies on global genome nucleotide excision repair (GGNER in Saccharomyces cerevisiae. We have focused on how GGNER relates to histone acetylation for its functioning and we have identified the histone acetyl tranferase Gcn5 and acetylation at lysines 9/14 of histone H3 as a major factor in enabling efficient repair. We consider results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences. In the latter case we also see a role for acetylation at histone H4. We then go on to outline the development of a high resolution genome-wide approach that enables one to examine correlations between histone modifications and the nucleotide excision repair (NER of UV-induced cyclobutane pyrimidine dimers throughout entire genomes. This is an approach that will enable rapid advances in understanding the complexities of how compacted chromatin in chromosomes is processed to access DNA damage and then returned to its pre-damaged status to maintain epigenetic codes.

  13. 2′-deoxy-5,6-dihydro-5-azacytidine—a less toxic alternative of 2′-deoxy-5-azacytidine

    Science.gov (United States)

    Matoušová, Marika; Votruba, Ivan; Otmar, Miroslav; Tloušťová, Eva; Günterová, Jana

    2011-01-01

    Restoration of transcriptionally silenced genes by means of methyltransferases inhibitors plays a crucial role in the current therapy of myelodysplastic syndromes and certain types of leukemias. A comparative study of hypomethylating activities of a series of 5-azacytidine nucleosides: 5-azacytidine (AC), 2′-deoxy-5-azacytidine (DAC) and its α-anomer (α-DAC), 5,6-dihydro-5-azacytidine (DHAC), 2′-deoxy-5,6-dihydro-5-azacytidine (DHDAC, KP-1212) and its α-anomer (α-DHDAC), and of a 2-pyrimidone ribonucleoside (zebularine) was conducted. Methylation-specific PCR was employed to detect the efficiency of individual agents on cyclin-dependent kinase inhibitor 2B and thrombospondin-1 hypermethylated gene loci. Overall changes in DNA methylation level were quantified by direct estimation of 5-methyl-2′-deoxycytidine-5′-monophosphate by HPLC using digested genomic DNA. Flow cytometric analysis of cell cycle progression and apoptotic markers was used to determine cytotoxicity of the compounds. mRNA expression was measured using qRT-PCR. 2′-deoxy-5,6-dihydro-5-azacytidine was found to be less cytotoxic and more stable than 2′-deoxy-5-azacytidine at the doses that induce comparable DNA hypomethylation and gene reactivation. This makes it a valuable tool for epigenetic research and worth further investigations to elucidate its possible therapeutic potential. PMID:21566456

  14. 5-HT(1A) receptor antagonism reverses and prevents fluoxetine-induced sexual dysfunction in rats.

    Science.gov (United States)

    Sukoff Rizzo, Stacey J; Pulicicchio, Claudine; Malberg, Jessica E; Andree, Terrance H; Stack, Gary P; Hughes, Zoë A; Schechter, Lee E; Rosenzweig-Lipson, Sharon

    2009-09-01

    Sexual dysfunction associated with antidepressant treatment continues to be a major compliance issue for antidepressant therapies. 5-HT(1A) antagonists have been suggested as beneficial adjunctive treatment in respect of antidepressant efficacy; however, the effects of 5-HT(1A) antagonism on antidepressant-induced side-effects has not been fully examined. The present study was conducted to evaluate the ability of acute or chronic treatment with 5-HT(1A) antagonists to alter chronic fluoxetine-induced impairments in sexual function. Chronic 14-d treatment with fluoxetine resulted in a marked reduction in the number of non-contact penile erections in sexually experienced male rats, relative to vehicle-treated controls. Acute administration of the 5-HT(1A) antagonist WAY-101405 resulted in a complete reversal of chronic fluoxetine-induced deficits on non-contact penile erections at doses that did not significantly alter baselines. Chronic co-administration of the 5-HT(1A) antagonists WAY-100635 or WAY-101405 with fluoxetine prevented fluoxetine-induced deficits in non-contact penile erections in sexually experienced male rats. Moreover, withdrawal of WAY-100635 from co-treatment with chonic fluoxetine, resulted in a time-dependent reinstatement of chronic fluoxetine-induced deficits in non-contact penile erections. Additionally, chronic administration of SSA-426, a molecule with dual activity as both a SSRI and 5-HT(1A) antagonist, did not produce deficits in non-contact penile erections at doses demonstrated to have antidepressant-like activity in the olfactory bulbectomy model. Taken together, these data suggest that 5-HT(1A) antagonist treatment may have utility for the management of SSRI-induced sexual dysfunction.

  15. Visualization for genomics: the Microbial Genome Viewer.

    NARCIS (Netherlands)

    Kerkhoven, R.; Enckevort, F.H.J. van; Boekhorst, J.; Molenaar, D; Siezen, R.J.

    2004-01-01

    SUMMARY: A Web-based visualization tool, the Microbial Genome Viewer, is presented that allows the user to combine complex genomic data in a highly interactive way. This Web tool enables the interactive generation of chromosome wheels and linear genome maps from genome annotation data stored in a

  16. Genome resource banking of biomedically important laboratory animals.

    Science.gov (United States)

    Agca, Yuksel

    2012-11-01

    Genome resource banking is the systematic collection, storage, and redistribution of biomaterials in an organized, logistical, and secure manner. Genome cryobanks usually contain biomaterials and associated genomic information essential for progression of biomedicine, human health, and research. In that regard, appropriate genome cryobanks could provide essential biomaterials for both current and future research projects in the form of various cell types and tissues, including sperm, oocytes, embryos, embryonic or adult stem cells, induced pluripotent stem cells, and gonadal tissues. In addition to cryobanked germplasm, cryobanking of DNA, serum, blood products, and tissues from scientifically, economically, and ecologically important species has become a common practice. For revitalization of the whole organism, cryopreserved germplasm in conjunction with assisted reproductive technologies, offer a powerful approach for research model management, as well as assisting in animal production for agriculture, conservation, and human reproductive medicine. Recently, many developed and developing countries have allocated substantial resources to establish genome resources banks which are responsible for safeguarding scientifically, economically, and ecologically important wild type, mutant, and transgenic plants, fish, and local livestock breeds, as well as wildlife species. This review is dedicated to the memory of Dr. John K. Critser, who has made profound contributions to the science of cryobiology and establishment of genome research and resources centers for mice, rats, and swine. Emphasis will be given to application of genome resource banks to species with substantial contributions to the advancement of biomedicine and human health. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells

    Science.gov (United States)

    Tu, Mengjun; Lin, Li; Cheng, Yilu; He, Xiubin; Sun, Huihui; Xie, Haihua; Fu, Junhao; Liu, Changbao; Li, Jin; Chen, Ding; Xi, Haitao; Xue, Dongyu; Liu, Qi; Zhao, Junzhao; Gao, Caixia; Song, Zongming; Qu, Jia

    2017-01-01

    Abstract Cpf1 nucleases were recently reported to be highly specific and programmable nucleases with efficiencies comparable to those of SpCas9. AsCpf1 and LbCpf1 require a single crRNA and recognize a 5′-TTTN-3′ protospacer adjacent motif (PAM) at the 5′ end of the protospacer for genome editing. For widespread application in precision site-specific human genome editing, the range of sequences that AsCpf1 and LbCpf1 can recognize is limited due to the size of this PAM. To address this limitation, we sought to identify a novel Cpf1 nuclease with simpler PAM requirements. Specifically, here we sought to test and engineer FnCpf1, one reported Cpf1 nuclease (FnCpf1) only requires 5′-TTN-3′ as a PAM but does not exhibit detectable levels of nuclease-induced indels at certain locus in human cells. Surprisingly, we found that FnCpf1 possesses DNA cleavage activity in human cells at multiple loci. We also comprehensively and quantitatively examined various FnCpf1 parameters in human cells, including spacer sequence, direct repeat sequence and the PAM sequence. Our study identifies FnCpf1 as a new member of the Cpf1 family for human genome editing with distinctive characteristics, which shows promise as a genome editing tool with the potential for both research and therapeutic applications. PMID:28977650

  18. Integration of genome-scale metabolic networks into whole-body PBPK models shows phenotype-specific cases of drug-induced metabolic perturbation.

    Science.gov (United States)

    Cordes, Henrik; Thiel, Christoph; Baier, Vanessa; Blank, Lars M; Kuepfer, Lars

    2018-01-01

    Drug-induced perturbations of the endogenous metabolic network are a potential root cause of cellular toxicity. A mechanistic understanding of such unwanted side effects during drug therapy is therefore vital for patient safety. The comprehensive assessment of such drug-induced injuries requires the simultaneous consideration of both drug exposure at the whole-body and resulting biochemical responses at the cellular level. We here present a computational multi-scale workflow that combines whole-body physiologically based pharmacokinetic (PBPK) models and organ-specific genome-scale metabolic network (GSMN) models through shared reactions of the xenobiotic metabolism. The applicability of the proposed workflow is illustrated for isoniazid, a first-line antibacterial agent against Mycobacterium tuberculosis , which is known to cause idiosyncratic drug-induced liver injuries (DILI). We combined GSMN models of a human liver with N-acetyl transferase 2 (NAT2)-phenotype-specific PBPK models of isoniazid. The combined PBPK-GSMN models quantitatively describe isoniazid pharmacokinetics, as well as intracellular responses, and changes in the exometabolome in a human liver following isoniazid administration. Notably, intracellular and extracellular responses identified with the PBPK-GSMN models are in line with experimental and clinical findings. Moreover, the drug-induced metabolic perturbations are distributed and attenuated in the metabolic network in a phenotype-dependent manner. Our simulation results show that a simultaneous consideration of both drug pharmacokinetics at the whole-body and metabolism at the cellular level is mandatory to explain drug-induced injuries at the patient level. The proposed workflow extends our mechanistic understanding of the biochemistry underlying adverse events and may be used to prevent drug-induced injuries in the future.

  19. TALENs: customizable molecular DNA scissors for genome engineering of plants.

    Science.gov (United States)

    Chen, Kunling; Gao, Caixia

    2013-06-20

    Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology. Transcription activator-like effector nucleases (TALENs), consisting of an engineered specific (TALE) DNA binding domain and a Fok I cleavage domain, are newly developed versatile reagents for genome engineering in different organisms. Because of the simplicity of the DNA recognition code and their modular assembly, TALENs can act as customizable molecular DNA scissors inducing double-strand breaks (DSBs) at given genomic location. Thus, they provide a valuable approach to targeted genome modifications such as mutations, insertions, replacements or chromosome rearrangements. In this article, we review the development of TALENs, and summarize the principles and tools for TALEN-mediated gene targeting in plant cells, as well as current and potential strategies for use in plant research and crop improvement. Copyright © 2013. Published by Elsevier Ltd.

  20. Investigation of intercellular salicylic acid accumulation during compatible and incompatible Arabidopsis-pseudomonas syringae interactions using a fast neutron-generated mutant allele of EDS5 identified by genetic mapping and whole-genome sequencing.

    Directory of Open Access Journals (Sweden)

    Jessie L Carviel

    Full Text Available A whole-genome sequencing technique developed to identify fast neutron-induced deletion mutations revealed that iap1-1 is a new allele of EDS5 (eds5-5. RPS2-AvrRpt2-initiated effector-triggered immunity (ETI was compromised in iap1-1/eds5-5 with respect to in planta bacterial levels and the hypersensitive response, while intra- and intercellular free salicylic acid (SA accumulation was greatly reduced, suggesting that SA contributes as both an intracellular signaling molecule and an antimicrobial agent in the intercellular space during ETI. During the compatible interaction between wild-type Col-0 and virulent Pseudomonas syringae pv. tomato (Pst, little intercellular free SA accumulated, which led to the hypothesis that Pst suppresses intercellular SA accumulation. When Col-0 was inoculated with a coronatine-deficient strain of Pst, high levels of intercellular SA accumulation were observed, suggesting that Pst suppresses intercellular SA accumulation using its phytotoxin coronatine. This work suggests that accumulation of SA in the intercellular space is an important component of basal/PAMP-triggered immunity as well as ETI to pathogens that colonize the intercellular space.

  1. IGF-1 induces the epithelial-mesenchymal transition via Stat5 in hepatocellular carcinoma.

    Science.gov (United States)

    Zhao, Chuanzong; Wang, Qian; Wang, Ben; Sun, Qi; He, Zhaobin; Hong, Jianguo; Kuehn, Florian; Liu, Enyu; Zhang, Zongli

    2017-12-19

    It has been reported that the epithelial-mesenchymal transition (EMT) plays an important role in hepatocellular carcinoma (HCC). However, the relationship between the insulin-like growth factor-1 (IGF-1) and EMT of HCC was not fully elucidated. In the present work, we found that the expression of N-cadherin, Vimentin, Snail1, Snail2, and Twist1 was positively associated with IGF-1R expression, while E-cadherin expression was negatively associated with IGF-1 expression in human HCC samples. Furthermore, we observed that IGF-1 up-regulated the expression of N-cadherin, Vimentin, Snail1, Snail2 and Twist1, and down-regulated the expression of E-cadherin. In addition, Stat5 was induced in IGF-1-treated HepG2 and Hep3B cells, and Stat5 inhibition or siRNA significantly affected IGF-1-induced EMT in HepG2 and Hep3B cells. In conclusion, IGF-1 induces EMT of HCC via Stat5 signaling pathway. Thus, IGF-1/Stat5 can be recommended as a potential and novel therapeutic strategy for HCC patients.

  2. HCV RNA traffic and association with NS5A in living cells

    Energy Technology Data Exchange (ETDEWEB)

    Fiches, Guillaume N.; Eyre, Nicholas S.; Aloia, Amanda L.; Van Der Hoek, Kylie [Department of Molecular and Cellular Biology, Research Centre for Infectious Diseases, University of Adelaide, Adelaide and Centre for Cancer Biology, SA Pathology, Adelaide, SA (Australia); Betz-Stablein, Brigit; Luciani, Fabio [Systems Immunology, School of Medical Sciences, University of New South Wales, Sydney, NSW (Australia); Chopra, Abha [Institute for Immunology and infectious diseases (IIID), Murdoch University, Perth, WA (Australia); Beard, Michael R., E-mail: michael.beard@adelaide.edu.au [Department of Molecular and Cellular Biology, Research Centre for Infectious Diseases, University of Adelaide, Adelaide and Centre for Cancer Biology, SA Pathology, Adelaide, SA (Australia)

    2016-06-15

    The spatiotemporal dynamics of Hepatitis C Virus (HCV) RNA localisation are poorly understood. To address this we engineered HCV genomes harbouring MS2 bacteriophage RNA stem-loops within the 3′-untranslated region to allow tracking of HCV RNA via specific interaction with a MS2-Coat-mCherry fusion protein. Despite the impact of these insertions on viral fitness, live imaging revealed that replication of tagged-HCV genomes induced specific redistribution of the mCherry-tagged-MS2-Coat protein to motile and static foci. Further analysis showed that HCV RNA was associated with NS5A in both static and motile structures while a subset of motile NS5A structures was devoid of HCV RNA. Further investigation of viral RNA traffic with respect to lipid droplets (LDs) revealed HCV RNA-positive structures in close association with LDs. These studies provide new insights into the dynamics of HCV RNA traffic with NS5A and LDs and provide a platform for future investigations of HCV replication and assembly. - Highlights: • HCV can tolerate can bacteriophage MS2 stem-loop insertions within the 3′ UTR. • MS2 stem-loop containing HCV genomes allow for real-time imaging of HCV RNA. • HCV RNA is both static and motile and associates with NS5A and lipid droplets.

  3. HCV RNA traffic and association with NS5A in living cells

    International Nuclear Information System (INIS)

    Fiches, Guillaume N.; Eyre, Nicholas S.; Aloia, Amanda L.; Van Der Hoek, Kylie; Betz-Stablein, Brigit; Luciani, Fabio; Chopra, Abha; Beard, Michael R.

    2016-01-01

    The spatiotemporal dynamics of Hepatitis C Virus (HCV) RNA localisation are poorly understood. To address this we engineered HCV genomes harbouring MS2 bacteriophage RNA stem-loops within the 3′-untranslated region to allow tracking of HCV RNA via specific interaction with a MS2-Coat-mCherry fusion protein. Despite the impact of these insertions on viral fitness, live imaging revealed that replication of tagged-HCV genomes induced specific redistribution of the mCherry-tagged-MS2-Coat protein to motile and static foci. Further analysis showed that HCV RNA was associated with NS5A in both static and motile structures while a subset of motile NS5A structures was devoid of HCV RNA. Further investigation of viral RNA traffic with respect to lipid droplets (LDs) revealed HCV RNA-positive structures in close association with LDs. These studies provide new insights into the dynamics of HCV RNA traffic with NS5A and LDs and provide a platform for future investigations of HCV replication and assembly. - Highlights: • HCV can tolerate can bacteriophage MS2 stem-loop insertions within the 3′ UTR. • MS2 stem-loop containing HCV genomes allow for real-time imaging of HCV RNA. • HCV RNA is both static and motile and associates with NS5A and lipid droplets.

  4. A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants.

    Science.gov (United States)

    Pilkington, Sarah M; Crowhurst, Ross; Hilario, Elena; Nardozza, Simona; Fraser, Lena; Peng, Yongyan; Gunaseelan, Kularajathevan; Simpson, Robert; Tahir, Jibran; Deroles, Simon C; Templeton, Kerry; Luo, Zhiwei; Davy, Marcus; Cheng, Canhong; McNeilage, Mark; Scaglione, Davide; Liu, Yifei; Zhang, Qiong; Datson, Paul; De Silva, Nihal; Gardiner, Susan E; Bassett, Heather; Chagné, David; McCallum, John; Dzierzon, Helge; Deng, Cecilia; Wang, Yen-Yi; Barron, Lorna; Manako, Kelvina; Bowen, Judith; Foster, Toshi M; Erridge, Zoe A; Tiffin, Heather; Waite, Chethi N; Davies, Kevin M; Grierson, Ella P; Laing, William A; Kirk, Rebecca; Chen, Xiuyin; Wood, Marion; Montefiori, Mirco; Brummell, David A; Schwinn, Kathy E; Catanach, Andrew; Fullerton, Christina; Li, Dawei; Meiyalaghan, Sathiyamoorthy; Nieuwenhuizen, Niels; Read, Nicola; Prakash, Roneel; Hunter, Don; Zhang, Huaibi; McKenzie, Marian; Knäbel, Mareike; Harris, Alastair; Allan, Andrew C; Gleave, Andrew; Chen, Angela; Janssen, Bart J; Plunkett, Blue; Ampomah-Dwamena, Charles; Voogd, Charlotte; Leif, Davin; Lafferty, Declan; Souleyre, Edwige J F; Varkonyi-Gasic, Erika; Gambi, Francesco; Hanley, Jenny; Yao, Jia-Long; Cheung, Joey; David, Karine M; Warren, Ben; Marsh, Ken; Snowden, Kimberley C; Lin-Wang, Kui; Brian, Lara; Martinez-Sanchez, Marcela; Wang, Mindy; Ileperuma, Nadeesha; Macnee, Nikolai; Campin, Robert; McAtee, Peter; Drummond, Revel S M; Espley, Richard V; Ireland, Hilary S; Wu, Rongmei; Atkinson, Ross G; Karunairetnam, Sakuntala; Bulley, Sean; Chunkath, Shayhan; Hanley, Zac; Storey, Roy; Thrimawithana, Amali H; Thomson, Susan; David, Charles; Testolin, Raffaele; Huang, Hongwen; Hellens, Roger P; Schaffer, Robert J

    2018-04-16

    Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) 'Hongyang' draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models. A second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within 'Hongyang' The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned 'Hort16A' cDNAs and comparing with the predicted protein models for Red5 and both the original 'Hongyang' assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised 'Hongyang' annotation, respectively, compared with 90.9% to the Red5 models. Our study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and

  5. Reference genome-independent assessment of mutation density using restriction enzyme-phased sequencing

    Directory of Open Access Journals (Sweden)

    Monson-Miller Jennifer

    2012-02-01

    Full Text Available Abstract Background The availability of low cost sequencing has spurred its application to discovery and typing of variation, including variation induced by mutagenesis. Mutation discovery is challenging as it requires a substantial amount of sequencing and analysis to detect very rare changes and distinguish them from noise. Also challenging are the cases when the organism of interest has not been sequenced or is highly divergent from the reference. Results We describe the development of a simple method for reduced representation sequencing. Input DNA was digested with a single restriction enzyme and ligated to Y adapters modified to contain a sequence barcode and to provide a compatible overhang for ligation. We demonstrated the efficiency of this method at SNP discovery using rice and arabidopsis. To test its suitability for the discovery of very rare SNP, one control and three mutagenized rice individuals (1, 5 and 10 mM sodium azide were used to prepare genomic libraries for Illumina sequencers by ligating barcoded adapters to NlaIII restriction sites. For genome-dependent discovery 15-30 million of 80 base reads per individual were aligned to the reference sequence achieving individual sequencing coverage from 7 to 15×. We identified high-confidence base changes by comparing sequences across individuals and identified instances consistent with mutations, i.e. changes that were found in a single treated individual and were solely GC to AT transitions. For genome-independent discovery 70-mers were extracted from the sequence of the control individual and single-copy sequence was identified by comparing the 70-mers across samples to evaluate copy number and variation. This de novo "genome" was used to align the reads and identify mutations as above. Covering approximately 1/5 of the 380 Mb genome of rice we detected mutation densities ranging from 0.6 to 4 per Mb of diploid DNA depending on the mutagenic treatment. Conclusions The

  6. The functional genomic studies of curcumin.

    Science.gov (United States)

    Huminiecki, Lukasz; Horbańczuk, Jarosław; Atanasov, Atanas G

    2017-10-01

    Curcumin is a natural plant-derived compound that has attracted a lot of attention for its anti-cancer activities. Curcumin can slow proliferation of and induce apoptosis in cancer cell lines, but the precise mechanisms of these effects are not fully understood. However, many lines of evidence suggested that curcumin has a potent impact on gene expression profiles; thus, functional genomics should be the key to understanding how curcumin exerts its anti-cancer activities. Here, we review the published functional genomic studies of curcumin focusing on cancer. Typically, a cancer cell line or a grafted tumor were exposed to curcumin and profiled with microarrays, methylation assays, or RNA-seq. Crucially, these studies are in agreement that curcumin has a powerful effect on gene expression. In the majority of the studies, among differentially expressed genes we found genes involved in cell signaling, apoptosis, and the control of cell cycle. Curcumin can also induce specific methylation changes, and is a powerful regulator of the expression of microRNAs which control oncogenesis. We also reflect on how the broader technological progress in transcriptomics has been reflected on the field of curcumin. We conclude by discussing the areas where more functional genomic studies are highly desirable. Integrated OMICS approaches will clearly be the key to understanding curcumin's anticancer and chemopreventive effects. Such strategies may become a template for elucidating the mode of action of other natural products; many natural products have pleiotropic effects that are well suited for a systems-level analysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Prevalence, Host Range, and Comparative Genomic Analysis of Temperate Ochrobactrum Phages

    Directory of Open Access Journals (Sweden)

    Claudia Jäckel

    2017-06-01

    Full Text Available Ochrobactrum and Brucella are closely related bacteria that populate different habitats and differ in their pathogenic properties. Only little is known about mobile genetic elements in these genera which might be important for survival and virulence. Previous studies on Brucella lysogeny indicated that active phages are rare in this genus. To gain insight into the presence and nature of prophages in Ochrobactrum, temperate phages were isolated from various species and characterized in detail. In silico analyses disclosed numerous prophages in published Ochrobactrum genomes. Induction experiments showed that Ochrobactrum prophages can be induced by various stress factors and that some strains released phage particles even under non-induced conditions. Sixty percent of lysates prepared from 125 strains revealed lytic activity. The host range and DNA similarities of 19 phages belonging to the families Myoviridae, Siphoviridae, or Podoviridae were determined suggesting that they are highly diverse. Some phages showed relationship to the temperate Brucella inopinata phage BiPB01. The genomic sequences of the myovirus POA1180 (41,655 bp and podovirus POI1126 (60,065 bp were analyzed. Phage POA1180 is very similar to a prophage recently identified in a Brucella strain isolated from an exotic frog. The POA1180 genome contains genes which may confer resistance to chromate and the ability to take up sulfate. Phage POI1126 is related to podoviruses of Sinorhizobium meliloti (PCB5, Erwinia pyrifoliae (Pep14, and Burkholderia cenocepacia (BcepIL02 and almost identical to an unnamed plasmid of the Ochrobactrum intermedium strain LMG 3301. Further experiments revealed that the POI1126 prophage indeed replicates as an extrachromosomal element. The data demonstrate for the first time that active prophages are common in Ochrobactrum and suggest that atypical brucellae also may be a reservoir for temperate phages.

  8. Full Genome Sequence and sfRNA Interferon Antagonist Activity of Zika Virus from Recife, Brazil.

    Directory of Open Access Journals (Sweden)

    Claire L Donald

    2016-10-01

    Full Text Available The outbreak of Zika virus (ZIKV in the Americas has transformed a previously obscure mosquito-transmitted arbovirus of the Flaviviridae family into a major public health concern. Little is currently known about the evolution and biology of ZIKV and the factors that contribute to the associated pathogenesis. Determining genomic sequences of clinical viral isolates and characterization of elements within these are an important prerequisite to advance our understanding of viral replicative processes and virus-host interactions.We obtained a ZIKV isolate from a patient who presented with classical ZIKV-associated symptoms, and used high throughput sequencing and other molecular biology approaches to determine its full genome sequence, including non-coding regions. Genome regions were characterized and compared to the sequences of other isolates where available. Furthermore, we identified a subgenomic flavivirus RNA (sfRNA in ZIKV-infected cells that has antagonist activity against RIG-I induced type I interferon induction, with a lesser effect on MDA-5 mediated action.The full-length genome sequence including non-coding regions of a South American ZIKV isolate from a patient with classical symptoms will support efforts to develop genetic tools for this virus. Detection of sfRNA that counteracts interferon responses is likely to be important for further understanding of pathogenesis and virus-host interactions.

  9. Early nongenomic events in aldosterone action in renal collecting duct cells: PKCalpha activation, mineralocorticoid receptor phosphorylation, and cross-talk with the genomic response.

    Science.gov (United States)

    Le Moëllic, Cathy; Ouvrard-Pascaud, Antoine; Capurro, Claudia; Cluzeaud, Francoise; Fay, Michel; Jaisser, Frederic; Farman, Nicolette; Blot-Chabaud, Marcel

    2004-05-01

    Effects of aldosterone on its target cells have long been considered to be mediated exclusively through the genomic pathway; however, evidence has been provided for rapid effects of the hormone that may involve nongenomic mechanisms. Whether an interaction exists between these two signaling pathways is not yet established. In this study, the authors show that aldosterone triggers both early nongenomic and late genomic increase in sodium transport in the RCCD(2) rat cortical collecting duct cell line. In these cells, the early (up to 2.5 h) aldosterone-induced increase in short-circuit current (Isc) is not blocked by the mineralocorticoid receptor (MR) antagonist RU26752, it does not require mRNA or protein synthesis, and it involves the PKCalpha signaling pathway. In addition, this early response is reproduced by aldosterone-BSA, which acts at the cell surface and presumably does not enter the cells (aldo-BSA is unable to trigger the late response). The authors also show that MR is rapidly phosphorylated on serine and threonine residues by aldosterone or aldosterone-BSA. In contrast, the late (4 to 24 h) aldosterone-induced increase in ion transport occurs through activation of the MR and requires mRNA and protein synthesis. Interestingly, nongenomic and genomic aldosterone actions appear to be interdependent. Blocking the PKCalpha pathway results in the inhibition of the late genomic response to aldosterone, as demonstrated by the suppression of aldosterone-induced increase in MR transactivation activity, alpha1 Na(+)/K(+)/ATPase mRNA, and Isc. These data suggest cross-talk between the nongenomic and genomic responses to aldosterone in renal cells and suggest that the aldosterone-MR mediated increase in mRNA/protein synthesis and ion transport depends, at least in part, upon PKCalpha activation. E-mail: marcel.blot-chabaud@pharmacie.univ-mrs.fr

  10. [Current advances and future prospects of genome editing technology in the field of biomedicine.

    Science.gov (United States)

    Sakuma, Tetsushi

    Genome editing technology can alter the genomic sequence at will, contributing the creation of cellular and animal models of human diseases including hereditary disorders and cancers, and the generation of the mutation-corrected human induced pluripotent stem cells for ex vivo regenerative medicine. In addition, novel approaches such as drug development using genome-wide CRISPR screening and cancer suppression using epigenome editing technology, which can change the epigenetic modifications in a site-specific manner, have also been conducted. In this article, I summarize the current advances and future prospects of genome editing technology in the field of biomedicine.

  11. The Glyphosate-Based Herbicide Roundup Does not Elevate Genome-Wide Mutagenesis of Escherichia coli.

    Science.gov (United States)

    Tincher, Clayton; Long, Hongan; Behringer, Megan; Walker, Noah; Lynch, Michael

    2017-10-05

    Mutations induced by pollutants may promote pathogen evolution, for example by accelerating mutations conferring antibiotic resistance. Generally, evaluating the genome-wide mutagenic effects of long-term sublethal pollutant exposure at single-nucleotide resolution is extremely difficult. To overcome this technical barrier, we use the mutation accumulation/whole-genome sequencing (MA/WGS) method as a mutagenicity test, to quantitatively evaluate genome-wide mutagenesis of Escherichia coli after long-term exposure to a wide gradient of the glyphosate-based herbicide (GBH) Roundup Concentrate Plus. The genome-wide mutation rate decreases as GBH concentration increases, suggesting that even long-term GBH exposure does not compromise the genome stability of bacteria. Copyright © 2017 Tincher et al.

  12. An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system.

    Science.gov (United States)

    Zhu, Xiaoxiao; Xu, Yajie; Yu, Shanshan; Lu, Lu; Ding, Mingqin; Cheng, Jing; Song, Guoxu; Gao, Xing; Yao, Liangming; Fan, Dongdong; Meng, Shu; Zhang, Xuewen; Hu, Shengdi; Tian, Yong

    2014-09-19

    The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo. So far, the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here, we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. We developed 6 strains of genome-modified mice using CRISPR/Cas9 system, and utilized this approach to genotype mice from F0 to F2 generation, which included single and multiplexed genome-modified mice. We also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%. We further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus, PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN.

  13. Transliterating transmission of genome damage in rats

    International Nuclear Information System (INIS)

    Slovinska, L.; Sanova, S.; Misurova, E.

    2004-01-01

    We studied the influence of gamma radiation (3 Gy) on slowly proliferating liver tissue of male rats and their progeny considering to induction and duration of latent damage. The irradiation caused latent cytogenetic damage in the liver in irradiated males of the F 0 generation manifesting itself during induced proliferation of hepatocytes (after partial hepatectomy) by reduced proliferating activity, a higher frequency of chromosomal aberrations and higher proportion of cells with apoptotic DNA fragments. In the progeny of irradiated males (F 1 and F 2 generation), the latent genome damage manifested itself during liver regeneration after partial hepatectomy by similar, but less pronounced changes compared with irradiated males of the parental generation. This finding indicates the transfer of the part of radiation-induced genome damage from parents to their progeny. Irradiation of F 1 and F 2 progeny of irradiated males (their total radiation load was 3+3 Gy, 3+0+3 Gy respectively) caused less changes as irradiation of progeny of non-irradiated control males (their total radiation load was 0+3 Gy, 0+0+3 Gy respectively). (authors)

  14. The perennial ryegrass GenomeZipper: targeted use of genome resources for comparative grass genomics.

    Science.gov (United States)

    Pfeifer, Matthias; Martis, Mihaela; Asp, Torben; Mayer, Klaus F X; Lübberstedt, Thomas; Byrne, Stephen; Frei, Ursula; Studer, Bruno

    2013-02-01

    Whole-genome sequences established for model and major crop species constitute a key resource for advanced genomic research. For outbreeding forage and turf grass species like ryegrasses (Lolium spp.), such resources have yet to be developed. Here, we present a model of the perennial ryegrass (Lolium perenne) genome on the basis of conserved synteny to barley (Hordeum vulgare) and the model grass genome Brachypodium (Brachypodium distachyon) as well as rice (Oryza sativa) and sorghum (Sorghum bicolor). A transcriptome-based genetic linkage map of perennial ryegrass served as a scaffold to establish the chromosomal arrangement of syntenic genes from model grass species. This scaffold revealed a high degree of synteny and macrocollinearity and was then utilized to anchor a collection of perennial ryegrass genes in silico to their predicted genome positions. This resulted in the unambiguous assignment of 3,315 out of 8,876 previously unmapped genes to the respective chromosomes. In total, the GenomeZipper incorporates 4,035 conserved grass gene loci, which were used for the first genome-wide sequence divergence analysis between perennial ryegrass, barley, Brachypodium, rice, and sorghum. The perennial ryegrass GenomeZipper is an ordered, information-rich genome scaffold, facilitating map-based cloning and genome assembly in perennial ryegrass and closely related Poaceae species. It also represents a milestone in describing synteny between perennial ryegrass and fully sequenced model grass genomes, thereby increasing our understanding of genome organization and evolution in the most important temperate forage and turf grass species.

  15. Genome-wide mutagenesis and multi-drug resistance in American trypanosomes induced by the front-line drug benznidazole

    KAUST Repository

    Campos, Mônica C.

    2017-10-25

    Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and affects 5–8 million people in Latin America. Although the nitroheterocyclic compound benznidazole has been the front-line drug for several decades, treatment failures are common. Benznidazole is a pro-drug and is bio-activated within the parasite by the mitochondrial nitroreductase TcNTR-1, leading to the generation of reactive metabolites that have trypanocidal activity. To better assess drug action and resistance, we sequenced the genomes of T. cruzi Y strain (35.5 Mb) and three benznidazole-resistant clones derived from a single drug-selected population. This revealed the genome-wide accumulation of mutations in the resistant parasites, in addition to variations in DNA copy-number. We observed mutations in DNA repair genes, linked with increased susceptibility to DNA alkylating and inter-strand cross-linking agents. Stop-codon-generating mutations in TcNTR-1 were associated with cross-resistance to other nitroheterocyclic drugs. Unexpectedly, the clones were also highly resistant to the ergosterol biosynthesis inhibitor posaconazole, a drug proposed for use against T. cruzi infections, in combination with benznidazole. Our findings therefore identify the highly mutagenic activity of benznidazole metabolites in T. cruzi, demonstrate that this can result in multi-drug resistance, and indicate that vigilance will be required if benznidazole is used in combination therapy.

  16. Loss of Kdm5c Causes Spurious Transcription and Prevents the Fine-Tuning of Activity-Regulated Enhancers in Neurons

    Directory of Open Access Journals (Sweden)

    Marilyn Scandaglia

    2017-10-01

    Full Text Available During development, chromatin-modifying enzymes regulate both the timely establishment of cell-type-specific gene programs and the coordinated repression of alternative cell fates. To dissect the role of one such enzyme, the intellectual-disability-linked lysine demethylase 5C (Kdm5c, in the developing and adult brain, we conducted parallel behavioral, transcriptomic, and epigenomic studies in Kdm5c-null and forebrain-restricted inducible knockout mice. Together, genomic analyses and functional assays demonstrate that Kdm5c plays a critical role as a repressor responsible for the developmental silencing of germline genes during cellular differentiation and in fine-tuning activity-regulated enhancers during neuronal maturation. Although the importance of these functions declines after birth, Kdm5c retains an important genome surveillance role preventing the incorrect activation of non-neuronal and cryptic promoters in adult neurons.

  17. Family genome browser: visualizing genomes with pedigree information.

    Science.gov (United States)

    Juan, Liran; Liu, Yongzhuang; Wang, Yongtian; Teng, Mingxiang; Zang, Tianyi; Wang, Yadong

    2015-07-15

    Families with inherited diseases are widely used in Mendelian/complex disease studies. Owing to the advances in high-throughput sequencing technologies, family genome sequencing becomes more and more prevalent. Visualizing family genomes can greatly facilitate human genetics studies and personalized medicine. However, due to the complex genetic relationships and high similarities among genomes of consanguineous family members, family genomes are difficult to be visualized in traditional genome visualization framework. How to visualize the family genome variants and their functions with integrated pedigree information remains a critical challenge. We developed the Family Genome Browser (FGB) to provide comprehensive analysis and visualization for family genomes. The FGB can visualize family genomes in both individual level and variant level effectively, through integrating genome data with pedigree information. Family genome analysis, including determination of parental origin of the variants, detection of de novo mutations, identification of potential recombination events and identical-by-decent segments, etc., can be performed flexibly. Diverse annotations for the family genome variants, such as dbSNP memberships, linkage disequilibriums, genes, variant effects, potential phenotypes, etc., are illustrated as well. Moreover, the FGB can automatically search de novo mutations and compound heterozygous variants for a selected individual, and guide investigators to find high-risk genes with flexible navigation options. These features enable users to investigate and understand family genomes intuitively and systematically. The FGB is available at http://mlg.hit.edu.cn/FGB/. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  18. Characterization of the genome of a phylogenetically distinct tospovirus and its interactions with the local lesion-induced host Chenopodium quinoa by whole-transcriptome analyses.

    Science.gov (United States)

    Chou, Wan-Chen; Lin, Shih-Shun; Yeh, Shyi-Dong; Li, Siang-Ling; Peng, Ying-Che; Fan, Ya-Hsu; Chen, Tsung-Chi

    2017-01-01

    Chenopodium quinoa is a natural local lesion host of numerous plant viruses, including tospoviruses (family Bunyaviridae). Groundnut chlorotic fan-spot tospovirus (GCFSV) has been shown to consistently induce local lesions on the leaves of C. quinoa 4 days post-inoculation (dpi). To reveal the whole genome of GCFSV and its interactions with C. quinoa, RNA-seq was performed to determine the transcriptome profiles of C. quinoa leaves. The high-throughput reads from infected C. quinoa leaves were used to identify the whole genome sequence of GCFSV and its single nucleotide polymorphisms. Our results indicated that GCFSV is a phylogenetically distinct tospovirus. Moreover, 27,170 coding and 29,563 non-coding sequences of C. quinoa were identified through de novo assembly, mixing reads from mock and infected samples. Several key genes involved in the modulation of hypersensitive response (HR) were identified. The expression levels of 4,893 deduced complete genes annotated using the Arabidopsis genome indicated that several HR-related orthologues of pathogenesis-related proteins, transcription factors, mitogen-activated protein kinases, and defense proteins were significantly expressed in leaves that formed local lesions. Here, we also provide new insights into the replication progression of a tospovirus and the molecular regulation of the C. quinoa response to virus infection.

  19. Pressure-induced ferroelectric to antiferroelectric phase transformation in porous PZT95/5 ceramics

    International Nuclear Information System (INIS)

    Zeng, T.; Dong, X.L.; Chen, X.F.; Yao, C.H.; He, H.L.

    2007-01-01

    The hydrostatic pressure-induced ferroelectric to antiferroelectric (FE-AFE) phase transformation of PZT95/5 ceramics was investigated as a function of porosity, pore shape and pore size. FE-AFE phase transformations were more diffuse and occurred at lower hydrostatic pressures with increasing porosity. The porous PZT95/5 ceramics with spherical pores exhibited higher transformation pressures than those with irregular pores. Moreover, FE-AFE phase transformations of porous PZT95/5 ceramics with polydisperse irregular pores were more diffuse than those of porous PZT95/5 ceramics with monodisperse irregular pores. The relation between pore structure and hydrostatic pressure-induced FE-AFE transformation was established according to stress concentration theory. (copyright 2007 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  20. Genome sequencing reveals the potential of an indigenous arsenotrophic bacterium; Achromobacter sp. KAs 3-5 for sub-surface arsenic mobilization and strategies for bioremediation

    Directory of Open Access Journals (Sweden)

    Balaram Mohapatra

    2017-12-01

    Full Text Available Prevalence of toxic arsenic (As oxyanion species in oligotrophic groundwater of south-east Asiatic regions (India and Bangladesh has threatened the health of millions of people. As-transforming bacteria alter the mobility, speciation and bioavailability of As in the aquifer ecosystem, hence play important roles in the biogeochemical cycling of As. Till date, only 19 cultivable As-transforming bacterial strains have been reported but with no description on their detail genomic and physiological perspective of As homeostasis. In this study, the draft genome (5.7 Mbp of an As-transforming, aromatic hydrocarbon utilizing and iron disproportioning indigenous groundwater bacterium KAs 3-5 has been obtained by Ion-Torrent sequencing revealed 65% genomic GC content, 5100 protein coding genes, and taxonomic affiliation to the members of genus Achromobacter, with >85% of genomic completeness. Phylogenomic signatures like MLST of 10 house-keeping genes, cut-off of <95% of average nucleotide/amino acid identity (ANI/OrthoANI/AAI, <0.99 of tetra-nucleotide correlations, and <70% value of DNA-DNA homology with nearest phylogenetic neighbors exhibited its species distinctiveness among all the described Achromobacter sp. members. Pan-genomic analysis confirmed the strain’s potential to adapt wide array of environmental stresses with a higher abundance of unique genes for metabolism of amino acids, polyketide, xenobiotics, nitroso compounds, aromatic hydrocarbons and most necessarily complete operon cluster for As-resistance/transformation/detoxification, as well as genes for transport, and signal transduction mechanisms. The genome analysis also highlighted its genetic determinants for loss of functions for antibiotic resistance, pathogenicity regulations, and gain of new/acquired functions for Fe-transport, fatty acids uptake-metabolism, motility, heavy metal (Cu-Zn-Co metabolism and several putative/hypothetical proteins owing to its capacity to acquired

  1. Radiation induced genomic instability

    International Nuclear Information System (INIS)

    Morgan, W.

    2003-01-01

    This presentation will focus on delayed genetic effects occurring in the progeny of cells after exposure to ionizing radiation. We have developed a model system for investigating those genetic effects occurring multiple generations after radiation exposure. The presentation will describe some of the delayed effects observed after radiation exposures including delayed chromosomal rearrangements, and recombination events as determined by a plasmid based assay system. We will present new data on how changes in gene expression as measured by differential display and DNA microarray analysis provides a mechanism by which cells display a memory of irradiation, and introduce candidate genes that may play a role in initiating and perpetuation the unstable phenotype. These results will be discussed in terms of the recently described non-targeted Death Inducing Effect (DIE) where by secreted factors from clones of unstable cells can elicit effects in non irradiated cells and may serve to perpetuate the unstable phenotype in cells that themselves were not irradiated

  2. Herpes simplex virus types 1 and 2 induce shutoff of host protein synthesis by different mechanisms in Friend erythroleukemia cells

    International Nuclear Information System (INIS)

    Hill, T.M.; Sinden, R.R.; Sadler, J.R.

    1983-01-01

    Herpes simplex virus type 1 (HSV-1) and HSV-2 disrupt host protein synthesis after viral infection. We have treated both viral types with agents which prevent transcription of the viral genome and used these treated viruses to infect induced Friend erythroleukemia cells. By measuring the changes in globin synthesis after infection, we have determined whether expression of the viral genome precedes the shutoff of host protein synthesis or whether the inhibitor molecule enters the cells as part of the virion. HSV-2-induced shutoff of host protein synthesis was insensitive to the effects of shortwave (254-nm) UV light and actinomycin D. Both of the treatments inhibited HSV-1-induced host protein shutoff. Likewise, treatment of HSV-1 with the cross-linking agent 4,5',8-trimethylpsoralen and longwave (360-nm) UV light prevented HSV-1 from inhibiting cellular protein synthesis. Treatment of HSV-2 with 4,5',8-trimethylpsoralen did not affect the ability of the virus to interfere with host protein synthesis, except at the highest doses of longwave UV light. It was determined that the highest longwave UV dosage damaged the HSV-2 virion as well as cross-linking the viral DNA. The results suggest that HSV-2 uses a virion-associated component to inhibit host protein synthesis and that HSV-1 requires the expression of the viral genome to cause cellular protein synthesis shutoff

  3. Genomic insight into the common carp (Cyprinus carpio genome by sequencing analysis of BAC-end sequences

    Directory of Open Access Journals (Sweden)

    Wang Jintu

    2011-04-01

    Full Text Available Abstract Background Common carp is one of the most important aquaculture teleost fish in the world. Common carp and other closely related Cyprinidae species provide over 30% aquaculture production in the world. However, common carp genomic resources are still relatively underdeveloped. BAC end sequences (BES are important resources for genome research on BAC-anchored genetic marker development, linkage map and physical map integration, and whole genome sequence assembling and scaffolding. Result To develop such valuable resources in common carp (Cyprinus carpio, a total of 40,224 BAC clones were sequenced on both ends, generating 65,720 clean BES with an average read length of 647 bp after sequence processing, representing 42,522,168 bp or 2.5% of common carp genome. The first survey of common carp genome was conducted with various bioinformatics tools. The common carp genome contains over 17.3% of repetitive elements with GC content of 36.8% and 518 transposon ORFs. To identify and develop BAC-anchored microsatellite markers, a total of 13,581 microsatellites were detected from 10,355 BES. The coding region of 7,127 genes were recognized from 9,443 BES on 7,453 BACs, with 1,990 BACs have genes on both ends. To evaluate the similarity to the genome of closely related zebrafish, BES of common carp were aligned against zebrafish genome. A total of 39,335 BES of common carp have conserved homologs on zebrafish genome which demonstrated the high similarity between zebrafish and common carp genomes, indicating the feasibility of comparative mapping between zebrafish and common carp once we have physical map of common carp. Conclusion BAC end sequences are great resources for the first genome wide survey of common carp. The repetitive DNA was estimated to be approximate 28% of common carp genome, indicating the higher complexity of the genome. Comparative analysis had mapped around 40,000 BES to zebrafish genome and established over 3

  4. Genomic insight into the common carp (Cyprinus carpio) genome by sequencing analysis of BAC-end sequences

    Science.gov (United States)

    2011-01-01

    Background Common carp is one of the most important aquaculture teleost fish in the world. Common carp and other closely related Cyprinidae species provide over 30% aquaculture production in the world. However, common carp genomic resources are still relatively underdeveloped. BAC end sequences (BES) are important resources for genome research on BAC-anchored genetic marker development, linkage map and physical map integration, and whole genome sequence assembling and scaffolding. Result To develop such valuable resources in common carp (Cyprinus carpio), a total of 40,224 BAC clones were sequenced on both ends, generating 65,720 clean BES with an average read length of 647 bp after sequence processing, representing 42,522,168 bp or 2.5% of common carp genome. The first survey of common carp genome was conducted with various bioinformatics tools. The common carp genome contains over 17.3% of repetitive elements with GC content of 36.8% and 518 transposon ORFs. To identify and develop BAC-anchored microsatellite markers, a total of 13,581 microsatellites were detected from 10,355 BES. The coding region of 7,127 genes were recognized from 9,443 BES on 7,453 BACs, with 1,990 BACs have genes on both ends. To evaluate the similarity to the genome of closely related zebrafish, BES of common carp were aligned against zebrafish genome. A total of 39,335 BES of common carp have conserved homologs on zebrafish genome which demonstrated the high similarity between zebrafish and common carp genomes, indicating the feasibility of comparative mapping between zebrafish and common carp once we have physical map of common carp. Conclusion BAC end sequences are great resources for the first genome wide survey of common carp. The repetitive DNA was estimated to be approximate 28% of common carp genome, indicating the higher complexity of the genome. Comparative analysis had mapped around 40,000 BES to zebrafish genome and established over 3,100 microsyntenies, covering over 50% of

  5. eGenomics: Cataloguing Our Complete Genome Collection III

    Directory of Open Access Journals (Sweden)

    Dawn Field

    2007-01-01

    Full Text Available This meeting report summarizes the proceedings of the “eGenomics: Cataloguing our Complete Genome Collection III” workshop held September 11–13, 2006, at the National Institute for Environmental eScience (NIEeS, Cambridge, United Kingdom. This 3rd workshop of the Genomic Standards Consortium was divided into two parts. The first half of the three-day workshop was dedicated to reviewing the genomic diversity of our current and future genome and metagenome collection, and exploring linkages to a series of existing projects through formal presentations. The second half was dedicated to strategic discussions. Outcomes of the workshop include a revised “Minimum Information about a Genome Sequence” (MIGS specification (v1.1, consensus on a variety of features to be added to the Genome Catalogue (GCat, agreement by several researchers to adopt MIGS for imminent genome publications, and an agreement by the EBI and NCBI to input their genome collections into GCat for the purpose of quantifying the amount of optional data already available (e.g., for geographic location coordinates and working towards a single, global list of all public genomes and metagenomes.

  6. Genomic imprinting of IGF2 in marsupials is methylation dependent

    Directory of Open Access Journals (Sweden)

    Imumorin Ikhide

    2008-05-01

    Full Text Available Abstract Background- Parent-specific methylation of specific CpG residues is critical to imprinting in eutherian mammals, but its importance to imprinting in marsupials and, thus, the evolutionary origins of the imprinting mechanism have been the subject of controversy. This has been particularly true for the imprinted Insulin-like Growth Factor II (IGF2, a key regulator of embryonic growth in vertebrates and a focal point of the selective forces leading to genomic imprinting. The presence of the essential imprinting effector, DNMT3L, in marsupial genomes and the demonstration of a differentially methylated region (DMR in the retrotransposon-derived imprinted gene, PEG10, in tammar wallaby argue for a role for methylation in imprinting, but several studies have found no evidence of parent-specific methylation at other imprinted loci in marsupials. Results- We performed the most extensive search to date for allele-specific patterns of CpG methylation within CpG isochores or CpG enriched segments across a 22 kilobase region surrounding the IGF2 gene in the South American opossum Monodelphis domestica. We identified a previously unknown 5'-untranslated exon for opossum IGF2, which is flanked by sequences defining a putative neonatal promoter, a DMR and an active Matrix Attachment Region (MAR. Demethylation of this DMR in opossum neonatal fibroblasts results in abherrant biallelic expression of IGF2. Conclusion- The demonstration of a DMR and an active MAR in the 5' flank of opossum IGF2 mirrors the regulatory features of the 5' flank of Igf2 in mice. However, demethylation induced activation of the maternal allele of IGF2 in opossum differs from the demethylation induced repression of the paternal Igf2 allele in mice. While it can now be concluded that parent-specific DNA methylation is an epigentic mark common to Marsupialia and Eutheria, the molecular mechanisms of transcriptional silencing at imprinted loci have clearly evolved along independent

  7. Evolution of genome size and complexity in the rhabdoviridae.

    Directory of Open Access Journals (Sweden)

    Peter J Walker

    2015-02-01

    Full Text Available RNA viruses exhibit substantial structural, ecological and genomic diversity. However, genome size in RNA viruses is likely limited by a high mutation rate, resulting in the evolution of various mechanisms to increase complexity while minimising genome expansion. Here we conduct a large-scale analysis of the genome sequences of 99 animal rhabdoviruses, including 45 genomes which we determined de novo, to identify patterns of genome expansion and the evolution of genome complexity. All but seven of the rhabdoviruses clustered into 17 well-supported monophyletic groups, of which eight corresponded to established genera, seven were assigned as new genera, and two were taxonomically ambiguous. We show that the acquisition and loss of new genes appears to have been a central theme of rhabdovirus evolution, and has been associated with the appearance of alternative, overlapping and consecutive ORFs within the major structural protein genes, and the insertion and loss of additional ORFs in each gene junction in a clade-specific manner. Changes in the lengths of gene junctions accounted for as much as 48.5% of the variation in genome size from the smallest to the largest genome, and the frequency with which new ORFs were observed increased in the 3' to 5' direction along the genome. We also identify several new families of accessory genes encoded in these regions, and show that non-canonical expression strategies involving TURBS-like termination-reinitiation, ribosomal frame-shifts and leaky ribosomal scanning appear to be common. We conclude that rhabdoviruses have an unusual capacity for genomic plasticity that may be linked to their discontinuous transcription strategy from the negative-sense single-stranded RNA genome, and propose a model that accounts for the regular occurrence of genome expansion and contraction throughout the evolution of the Rhabdoviridae.

  8. Evolution of genome size and complexity in the rhabdoviridae.

    Science.gov (United States)

    Walker, Peter J; Firth, Cadhla; Widen, Steven G; Blasdell, Kim R; Guzman, Hilda; Wood, Thomas G; Paradkar, Prasad N; Holmes, Edward C; Tesh, Robert B; Vasilakis, Nikos

    2015-02-01

    RNA viruses exhibit substantial structural, ecological and genomic diversity. However, genome size in RNA viruses is likely limited by a high mutation rate, resulting in the evolution of various mechanisms to increase complexity while minimising genome expansion. Here we conduct a large-scale analysis of the genome sequences of 99 animal rhabdoviruses, including 45 genomes which we determined de novo, to identify patterns of genome expansion and the evolution of genome complexity. All but seven of the rhabdoviruses clustered into 17 well-supported monophyletic groups, of which eight corresponded to established genera, seven were assigned as new genera, and two were taxonomically ambiguous. We show that the acquisition and loss of new genes appears to have been a central theme of rhabdovirus evolution, and has been associated with the appearance of alternative, overlapping and consecutive ORFs within the major structural protein genes, and the insertion and loss of additional ORFs in each gene junction in a clade-specific manner. Changes in the lengths of gene junctions accounted for as much as 48.5% of the variation in genome size from the smallest to the largest genome, and the frequency with which new ORFs were observed increased in the 3' to 5' direction along the genome. We also identify several new families of accessory genes encoded in these regions, and show that non-canonical expression strategies involving TURBS-like termination-reinitiation, ribosomal frame-shifts and leaky ribosomal scanning appear to be common. We conclude that rhabdoviruses have an unusual capacity for genomic plasticity that may be linked to their discontinuous transcription strategy from the negative-sense single-stranded RNA genome, and propose a model that accounts for the regular occurrence of genome expansion and contraction throughout the evolution of the Rhabdoviridae.

  9. Evolution of Genome Size and Complexity in the Rhabdoviridae

    Science.gov (United States)

    Walker, Peter J.; Firth, Cadhla; Widen, Steven G.; Blasdell, Kim R.; Guzman, Hilda; Wood, Thomas G.; Paradkar, Prasad N.; Holmes, Edward C.; Tesh, Robert B.; Vasilakis, Nikos

    2015-01-01

    RNA viruses exhibit substantial structural, ecological and genomic diversity. However, genome size in RNA viruses is likely limited by a high mutation rate, resulting in the evolution of various mechanisms to increase complexity while minimising genome expansion. Here we conduct a large-scale analysis of the genome sequences of 99 animal rhabdoviruses, including 45 genomes which we determined de novo, to identify patterns of genome expansion and the evolution of genome complexity. All but seven of the rhabdoviruses clustered into 17 well-supported monophyletic groups, of which eight corresponded to established genera, seven were assigned as new genera, and two were taxonomically ambiguous. We show that the acquisition and loss of new genes appears to have been a central theme of rhabdovirus evolution, and has been associated with the appearance of alternative, overlapping and consecutive ORFs within the major structural protein genes, and the insertion and loss of additional ORFs in each gene junction in a clade-specific manner. Changes in the lengths of gene junctions accounted for as much as 48.5% of the variation in genome size from the smallest to the largest genome, and the frequency with which new ORFs were observed increased in the 3’ to 5’ direction along the genome. We also identify several new families of accessory genes encoded in these regions, and show that non-canonical expression strategies involving TURBS-like termination-reinitiation, ribosomal frame-shifts and leaky ribosomal scanning appear to be common. We conclude that rhabdoviruses have an unusual capacity for genomic plasticity that may be linked to their discontinuous transcription strategy from the negative-sense single-stranded RNA genome, and propose a model that accounts for the regular occurrence of genome expansion and contraction throughout the evolution of the Rhabdoviridae. PMID:25679389

  10. Integrated Genomic Analysis of Diverse Induced Pluripotent Stem Cells from the Progenitor Cell Biology Consortium.

    Science.gov (United States)

    Salomonis, Nathan; Dexheimer, Phillip J; Omberg, Larsson; Schroll, Robin; Bush, Stacy; Huo, Jeffrey; Schriml, Lynn; Ho Sui, Shannan; Keddache, Mehdi; Mayhew, Christopher; Shanmukhappa, Shiva Kumar; Wells, James; Daily, Kenneth; Hubler, Shane; Wang, Yuliang; Zambidis, Elias; Margolin, Adam; Hide, Winston; Hatzopoulos, Antonis K; Malik, Punam; Cancelas, Jose A; Aronow, Bruce J; Lutzko, Carolyn

    2016-07-12

    The rigorous characterization of distinct induced pluripotent stem cells (iPSC) derived from multiple reprogramming technologies, somatic sources, and donors is required to understand potential sources of variability and downstream potential. To achieve this goal, the Progenitor Cell Biology Consortium performed comprehensive experimental and genomic analyses of 58 iPSC from ten laboratories generated using a variety of reprogramming genes, vectors, and cells. Associated global molecular characterization studies identified functionally informative correlations in gene expression, DNA methylation, and/or copy-number variation among key developmental and oncogenic regulators as a result of donor, sex, line stability, reprogramming technology, and cell of origin. Furthermore, X-chromosome inactivation in PSC produced highly correlated differences in teratoma-lineage staining and regulator expression upon differentiation. All experimental results, and raw, processed, and metadata from these analyses, including powerful tools, are interactively accessible from a new online portal at https://www.synapse.org to serve as a reusable resource for the stem cell community. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Genomic Prediction from Whole Genome Sequence in Livestock: The 1000 Bull Genomes Project

    DEFF Research Database (Denmark)

    Hayes, Benjamin J; MacLeod, Iona M; Daetwyler, Hans D

    Advantages of using whole genome sequence data to predict genomic estimated breeding values (GEBV) include better persistence of accuracy of GEBV across generations and more accurate GEBV across breeds. The 1000 Bull Genomes Project provides a database of whole genome sequenced key ancestor bulls....... In a dairy data set, predictions using BayesRC and imputed sequence data from 1000 Bull Genomes were 2% more accurate than with 800k data. We could demonstrate the method identified causal mutations in some cases. Further improvements will come from more accurate imputation of sequence variant genotypes...

  12. Fisetin Induces Apoptosis Through p53-Mediated Up-Regulation of DR5 Expression in Human Renal Carcinoma Caki Cells.

    Science.gov (United States)

    Min, Kyoung-Jin; Nam, Ju-Ock; Kwon, Taeg Kyu

    2017-08-02

    Fisetin is a natural compound found in fruits and vegetables such as strawberries, apples, cucumbers, and onions. Since fisetin can elicit anti-cancer effects, including anti-proliferation and anti-migration, we investigated whether fisetin induced apoptosis in human renal carcinoma (Caki) cells. Fisetin markedly induced sub-G1 population and cleavage of poly (ADP-ribose) polymerase (PARP), which is a marker of apoptosis, and increased caspase activation. We found that pan-caspase inhibitor (z-VAD-fmk) inhibited fisetin-induced apoptosis. In addition, fisetin induced death receptor 5 (DR5) expression at the transcriptional level, and down-regulation of DR5 by siRNA blocked fisetin-induced apoptosis. Furthermore, fisetin induced p53 protein expression through up-regulation of protein stability, whereas down-regulation of p53 by siRNA markedly inhibited fisetin-induced DR5 expression. In contrast, fisetin induced up-regulation of CHOP expression and reactive oxygen species production, which had no effect on fisetin-induced apoptosis. Taken together, our study demonstrates that fisetin induced apoptosis through p53 mediated up-regulation of DR5 expression at the transcriptional level.

  13. Contrasting patterns of evolution of 45S and 5S rDNA families uncover new aspects in the genome constitution of the agronomically important grass Thinopyrum intermedium (Triticeae).

    Science.gov (United States)

    Mahelka, Václav; Kopecky, David; Baum, Bernard R

    2013-09-01

    We employed sequencing of clones and in situ hybridization (genomic and fluorescent in situ hybridization [GISH and rDNA-FISH]) to characterize both the sequence variation and genomic organization of 45S (herein ITS1-5.8S-ITS2 region) and 5S (5S gene + nontranscribed spacer) ribosomal DNA (rDNA) families in the allohexaploid grass Thinopyrum intermedium. Both rDNA families are organized within several rDNA loci within all three subgenomes of the allohexaploid species. Both families have undergone different patterns of evolution. The 45S rDNA family has evolved in a concerted manner: internal transcribed spacer (ITS) sequences residing within the arrays of two subgenomes out of three got homogenized toward one major ribotype, whereas the third subgenome contained a minor proportion of distinct unhomogenized copies. Homogenization mechanisms such as unequal crossover and/or gene conversion were coupled with the loss of certain 45S rDNA loci. Unlike in the 45S family, the data suggest that neither interlocus homogenization among homeologous chromosomes nor locus loss occurred in 5S rDNA. Consistently with other Triticeae, the 5S rDNA family in intermediate wheatgrass comprised two distinct array types-the long- and short-spacer unit classes. Within the long and short units, we distinguished five and three different types, respectively, likely representing homeologous unit classes donated by putative parental species. Although the major ITS ribotype corresponds in our phylogenetic analysis to the E-genome species, the minor ribotype corresponds to Dasypyrum. 5S sequences suggested the contributions from Pseudoroegneria, Dasypyrum, and Aegilops. The contribution from Aegilops to the intermediate wheatgrass' genome is a new finding with implications in wheat improvement. We discuss rDNA evolution and potential origin of intermediate wheatgrass.

  14. Genomic Selection Improves Heat Tolerance in Dairy Cattle

    Science.gov (United States)

    Garner, J. B.; Douglas, M. L.; Williams, S. R. O; Wales, W. J.; Marett, L. C.; Nguyen, T. T. T.; Reich, C. M.; Hayes, B. J.

    2016-01-01

    Dairy products are a key source of valuable proteins and fats for many millions of people worldwide. Dairy cattle are highly susceptible to heat-stress induced decline in milk production, and as the frequency and duration of heat-stress events increases, the long term security of nutrition from dairy products is threatened. Identification of dairy cattle more tolerant of heat stress conditions would be an important progression towards breeding better adapted dairy herds to future climates. Breeding for heat tolerance could be accelerated with genomic selection, using genome wide DNA markers that predict tolerance to heat stress. Here we demonstrate the value of genomic predictions for heat tolerance in cohorts of Holstein cows predicted to be heat tolerant and heat susceptible using controlled-climate chambers simulating a moderate heatwave event. Not only was the heat challenge stimulated decline in milk production less in cows genomically predicted to be heat-tolerant, physiological indicators such as rectal and intra-vaginal temperatures had reduced increases over the 4 day heat challenge. This demonstrates that genomic selection for heat tolerance in dairy cattle is a step towards securing a valuable source of nutrition and improving animal welfare facing a future with predicted increases in heat stress events. PMID:27682591

  15. Role of annexin A5 in cisplatin-induced toxicity in renal cells: molecular mechanism of apoptosis.

    Science.gov (United States)

    Jeong, Jin-Joo; Park, Nahee; Kwon, Yeo-Jung; Ye, Dong-Jin; Moon, Aree; Chun, Young-Jin

    2014-01-24

    Annexin A5 belongs to a large family of calcium-binding and phospholipid-binding proteins and may act as an endogenous regulator of various pathophysiological processes. There is increasing evidence that annexin A5 is related to cytotoxicity, but the precise function of this protein has yet to be elucidated. In this study, we aimed to verify the function of annexin A5 in the apoptosis of renal epithelial cells. Real-time PCR and Western blot analysis, together with immunofluorescence analysis, showed that the expression of annexin A5 significantly increased in the presence of cisplatin in both human and rat renal epithelial cells. With regard to the mechanism of cisplatin-induced apoptosis, apoptosis-inducing factor (AIF) release into the cytosol was observed, and the underlying mechanism was identified as voltage-dependent anion channel (VDAC) oligomerization. Mitochondrial membrane potential (Δψm) was found to be greatly disrupted in cisplatin-treated cells. Moreover, cisplatin strongly induced translocation of annexin A5 into mitochondria. To understand the functional significance of annexin A5 in renal cell death, we used a siRNA-mediated approach to knock down annexin A5. Annexin A5 depletion by siRNA led to decreased annexin A5 translocation into mitochondria and significantly reduced VDAC oligomerization and AIF release. Annexin A5 siRNA also increased cell viability compared with the control. Moreover, expression of annexin A5 was induced by other nephrotoxicants such as CdCl2 and bacitracin. Taken together, our data suggest that annexin A5 may play a crucial role in cisplatin-induced toxicity by mediating the mitochondrial apoptotic pathway via the induction and oligomerization of VDAC.

  16. De novo assembly of a haplotype-resolved human genome.

    Science.gov (United States)

    Cao, Hongzhi; Wu, Honglong; Luo, Ruibang; Huang, Shujia; Sun, Yuhui; Tong, Xin; Xie, Yinlong; Liu, Binghang; Yang, Hailong; Zheng, Hancheng; Li, Jian; Li, Bo; Wang, Yu; Yang, Fang; Sun, Peng; Liu, Siyang; Gao, Peng; Huang, Haodong; Sun, Jing; Chen, Dan; He, Guangzhu; Huang, Weihua; Huang, Zheng; Li, Yue; Tellier, Laurent C A M; Liu, Xiao; Feng, Qiang; Xu, Xun; Zhang, Xiuqing; Bolund, Lars; Krogh, Anders; Kristiansen, Karsten; Drmanac, Radoje; Drmanac, Snezana; Nielsen, Rasmus; Li, Songgang; Wang, Jian; Yang, Huanming; Li, Yingrui; Wong, Gane Ka-Shu; Wang, Jun

    2015-06-01

    The human genome is diploid, and knowledge of the variants on each chromosome is important for the interpretation of genomic information. Here we report the assembly of a haplotype-resolved diploid genome without using a reference genome. Our pipeline relies on fosmid pooling together with whole-genome shotgun strategies, based solely on next-generation sequencing and hierarchical assembly methods. We applied our sequencing method to the genome of an Asian individual and generated a 5.15-Gb assembled genome with a haplotype N50 of 484 kb. Our analysis identified previously undetected indels and 7.49 Mb of novel coding sequences that could not be aligned to the human reference genome, which include at least six predicted genes. This haplotype-resolved genome represents the most complete de novo human genome assembly to date. Application of our approach to identify individual haplotype differences should aid in translating genotypes to phenotypes for the development of personalized medicine.

  17. Origins of the Xylella fastidiosa prophage-like regions and their impact in genome differentiation.

    Directory of Open Access Journals (Sweden)

    Alessandro de Mello Varani

    Full Text Available Xylella fastidiosa is a Gram negative plant pathogen causing many economically important diseases, and analyses of completely sequenced X. fastidiosa genome strains allowed the identification of many prophage-like elements and possibly phage remnants, accounting for up to 15% of the genome composition. To better evaluate the recent evolution of the X. fastidiosa chromosome backbone among distinct pathovars, the number and location of prophage-like regions on two finished genomes (9a5c and Temecula1, and in two candidate molecules (Ann1 and Dixon were assessed. Based on comparative best bidirectional hit analyses, the majority (51% of the predicted genes in the X. fastidiosa prophage-like regions are related to structural phage genes belonging to the Siphoviridae family. Electron micrograph reveals the existence of putative viral particles with similar morphology to lambda phages in the bacterial cell in planta. Moreover, analysis of microarray data indicates that 9a5c strain cultivated under stress conditions presents enhanced expression of phage anti-repressor genes, suggesting switches from lysogenic to lytic cycle of phages under stress-induced situations. Furthermore, virulence-associated proteins and toxins are found within these prophage-like elements, thus suggesting an important role in host adaptation. Finally, clustering analyses of phage integrase genes based on multiple alignment patterns reveal they group in five lineages, all possessing a tyrosine recombinase catalytic domain, and phylogenetically close to other integrases found in phages that are genetic mosaics and able to perform generalized and specialized transduction. Integration sites and tRNA association is also evidenced. In summary, we present comparative and experimental evidence supporting the association and contribution of phage activity on the differentiation of Xylella genomes.

  18. Evolution of the Largest Mammalian Genome.

    Science.gov (United States)

    Evans, Ben J; Upham, Nathan S; Golding, Goeffrey B; Ojeda, Ricardo A; Ojeda, Agustina A

    2017-06-01

    The genome of the red vizcacha rat (Rodentia, Octodontidae, Tympanoctomys barrerae) is the largest of all mammals, and about double the size of their close relative, the mountain vizcacha rat Octomys mimax, even though the lineages that gave rise to these species diverged from each other only about 5 Ma. The mechanism for this rapid genome expansion is controversial, and hypothesized to be a consequence of whole genome duplication or accumulation of repetitive elements. To test these alternative but nonexclusive hypotheses, we gathered and evaluated evidence from whole transcriptome and whole genome sequences of T. barrerae and O. mimax. We recovered support for genome expansion due to accumulation of a diverse assemblage of repetitive elements, which represent about one half and one fifth of the genomes of T. barrerae and O. mimax, respectively, but we found no strong signal of whole genome duplication. In both species, repetitive sequences were rare in transcribed regions as compared with the rest of the genome, and mostly had no close match to annotated repetitive sequences from other rodents. These findings raise new questions about the genomic dynamics of these repetitive elements, their connection to widespread chromosomal fissions that occurred in the T. barrerae ancestor, and their fitness effects-including during the evolution of hypersaline dietary tolerance in T. barrerae. ©The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  19. VISTA - computational tools for comparative genomics

    Energy Technology Data Exchange (ETDEWEB)

    Frazer, Kelly A.; Pachter, Lior; Poliakov, Alexander; Rubin,Edward M.; Dubchak, Inna

    2004-01-01

    Comparison of DNA sequences from different species is a fundamental method for identifying functional elements in genomes. Here we describe the VISTA family of tools created to assist biologists in carrying out this task. Our first VISTA server at http://www-gsd.lbl.gov/VISTA/ was launched in the summer of 2000 and was designed to align long genomic sequences and visualize these alignments with associated functional annotations. Currently the VISTA site includes multiple comparative genomics tools and provides users with rich capabilities to browse pre-computed whole-genome alignments of large vertebrate genomes and other groups of organisms with VISTA Browser, submit their own sequences of interest to several VISTA servers for various types of comparative analysis, and obtain detailed comparative analysis results for a set of cardiovascular genes. We illustrate capabilities of the VISTA site by the analysis of a 180 kilobase (kb) interval on human chromosome 5 that encodes for the kinesin family member3A (KIF3A) protein.

  20. Identification of a large genomic region in UV-irradiated human cells which has fewer cyclobutane pyrimidine dimers than most genomic regions

    International Nuclear Information System (INIS)

    Kantor, G.J.; Deiss-Tolbert, D.M.

    1995-01-01

    Size separation after UV-endonuclease digestion of DNA from UV-irradiated human cells using denaturing conditions fractionates the genome based on cyclobutane pyrimidine dimer content. We have examined the largest molecules available (50-80 kb; about 5% of the DNA) after fractionation and those of average size (5-15 kb) for content of some specific genes. We find that the largest molecules are not a representative sampling of the genome. Three contiguous genes located in a G+C-rich isochore (tyrosine hydroxylase, insulin, insulin-like growth factor II) have concentrations two to three times greater in the largest molecules. This shows that this genomic region has fewer pyrimidine dimers than most other genomic regions. In contrast, the β-actin genomic region, which has a similar G+C content, has an equal concentration in both fractions as do the p53 and β-globin genomic regions, which are A+T-rich. These data show that DNA damage in the form of cyclobutane pyrimidine dimers occurs with different probabilities in specific isochores. Part of the reason may be the relative G-C content, but other factors must play a significant role. We also report that the transcriptionally inactive insulin region is repaired at the genome-overall rate in normal cells and is not repaired in xeroderma pigmentosum complementation group C cells. (author)

  1. Genomic localization, sequence analysis, and transcription of the putative human cytomegalovirus DNA polymerase gene

    International Nuclear Information System (INIS)

    Heilbronn, T.; Jahn, G.; Buerkle, A.; Freese, U.K.; Fleckenstein, B.; Zur Hausen, H.

    1987-01-01

    The human cytomegalovirus (HCMV)-induced DNA polymerase has been well characterized biochemically and functionally, but its genomic location has not yet been assigned. To identify the coding sequence, cross-hybridization with the herpes simplex virus type 1 (HSV-1) polymerase gene was used, as suggested by the close similarity of the herpes group virus-induced DNA polymerases to the HCMV DNA polymerase. A cosmid and plasmid library of the entire HCMV genome was screened with the BamHI Q fragment of HSF-1 at different stringency conditions. One PstI-HincII restriction fragment of 850 base pairs mapping within the EcoRI M fragment of HCMV cross-hybridized at T/sub m/ - 25/degrees/C. Sequence analysis revealed one open reading frame spanning the entire sequence. The amino acid sequence showed a highly conserved domain of 133 amino acids shared with the HSV and putative Esptein-Barr virus polymerase sequences. This domain maps within the C-terminal part of the HSV polymerase gene, which has been suggested to contain part of the catalytic center of the enzyme. Transcription analysis revealed one 5.4-kilobase early transcript in the sense orientation with respect to the open reading frame identified. This transcript appears to code for the 140-kilodalton HCMV polymerase protein

  2. Genome sequence of a Proteus mirabilis strain isolated from the salivary glands of larval Lucilia sericata

    Science.gov (United States)

    We announced a draft genome sequence of a Proteus mirabilis strain derived from Lucilia sericata salivary glands. This strain is demonstrated to attract and induce oviposition by L. sericata, a common blow fly important to medicine, agriculture, and forensics. The genome will help to dissect inter...

  3. Nuclear envelope and genome interactions in cell fate

    Science.gov (United States)

    Talamas, Jessica A.; Capelson, Maya

    2015-01-01

    The eukaryotic cell nucleus houses an organism’s genome and is the location within the cell where all signaling induced and development-driven gene expression programs are ultimately specified. The genome is enclosed and separated from the cytoplasm by the nuclear envelope (NE), a double-lipid membrane bilayer, which contains a large variety of trans-membrane and associated protein complexes. In recent years, research regarding multiple aspects of the cell nucleus points to a highly dynamic and coordinated concert of efforts between chromatin and the NE in regulation of gene expression. Details of how this concert is orchestrated and how it directs cell differentiation and disease are coming to light at a rapid pace. Here we review existing and emerging concepts of how interactions between the genome and the NE may contribute to tissue specific gene expression programs to determine cell fate. PMID:25852741

  4. Iron and genome stability: An update

    International Nuclear Information System (INIS)

    Prá, Daniel; Franke, Silvia Isabel Rech; Henriques, João Antonio Pêgas; Fenech, Michael

    2012-01-01

    Iron is an essential micronutrient which is required in a relatively narrow range for maintaining metabolic homeostasis and genome stability. Iron participates in oxygen transport and mitochondrial respiration as well as in antioxidant and nucleic acid metabolism. Iron deficiency impairs these biological pathways, leading to oxidative stress and possibly carcinogenesis. Iron overload has been linked to genome instability as well as to cancer risk increase, as seen in hereditary hemochromatosis. Iron is an extremely reactive transition metal that can interact with hydrogen peroxide to generate hydroxyl radicals that form the 8-hydroxy-guanine adduct, cause point mutations as well as DNA single and double strand breaks. Iron overload also induces DNA hypermethylation and can reduce telomere length. The current Recommended Dietary Allowances (RDA) for iron, according with Institute of Medicine Dietary Reference Intake (DRI), is based in the concept of preventing anemia, and ranges from 7 mg/day to 18 mg/day depending on life stage and gender. Pregnant women need 27 mg/day. The maximum safety level for iron intake, the Upper Level (UL), is 40–45 mg/day, based on the prevention of gastrointestinal distress associated to high iron intakes. Preliminary evidence indicates that 20 mg/day iron, an intake slightly higher than the RDA, may reduce the risk of gastrointestinal cancer in the elderly as well as increasing genome stability in lymphocytes of children and adolescents. Current dietary recommendations do not consider the concept of genome stability which is of concern because damage to the genome has been linked to the origin and progression of many diseases and is the most fundamental pathology. Given the importance of iron for homeostasis and its potential influence over genome stability and cancer it is recommended to conduct further studies that conclusively define these relationships.

  5. Iron and genome stability: An update

    Energy Technology Data Exchange (ETDEWEB)

    Pra, Daniel, E-mail: daniel_pra@yahoo.com [PPG em Promocao da Saude, Universidade de Santa Cruz do Sul (UNISC), Santa Cruz do Sul, RS (Brazil); PPG em Saude e Comportamento, Universidade Catolica de Pelotas, Pelotas, RS (Brazil); Franke, Silvia Isabel Rech [PPG em Promocao da Saude, Universidade de Santa Cruz do Sul (UNISC), Santa Cruz do Sul, RS (Brazil); Henriques, Joao Antonio Pegas [Instituto de Biotecnologia, Universidade de Caxias do Sul, Caxias do Sul, RS (Brazil); Fenech, Michael [CSIRO Food and Nutritional Sciences, Adelaide, SA (Australia)

    2012-05-01

    Iron is an essential micronutrient which is required in a relatively narrow range for maintaining metabolic homeostasis and genome stability. Iron participates in oxygen transport and mitochondrial respiration as well as in antioxidant and nucleic acid metabolism. Iron deficiency impairs these biological pathways, leading to oxidative stress and possibly carcinogenesis. Iron overload has been linked to genome instability as well as to cancer risk increase, as seen in hereditary hemochromatosis. Iron is an extremely reactive transition metal that can interact with hydrogen peroxide to generate hydroxyl radicals that form the 8-hydroxy-guanine adduct, cause point mutations as well as DNA single and double strand breaks. Iron overload also induces DNA hypermethylation and can reduce telomere length. The current Recommended Dietary Allowances (RDA) for iron, according with Institute of Medicine Dietary Reference Intake (DRI), is based in the concept of preventing anemia, and ranges from 7 mg/day to 18 mg/day depending on life stage and gender. Pregnant women need 27 mg/day. The maximum safety level for iron intake, the Upper Level (UL), is 40-45 mg/day, based on the prevention of gastrointestinal distress associated to high iron intakes. Preliminary evidence indicates that 20 mg/day iron, an intake slightly higher than the RDA, may reduce the risk of gastrointestinal cancer in the elderly as well as increasing genome stability in lymphocytes of children and adolescents. Current dietary recommendations do not consider the concept of genome stability which is of concern because damage to the genome has been linked to the origin and progression of many diseases and is the most fundamental pathology. Given the importance of iron for homeostasis and its potential influence over genome stability and cancer it is recommended to conduct further studies that conclusively define these relationships.

  6. Rapid sequencing of the bamboo mitochondrial genome using Illumina technology and parallel episodic evolution of organelle genomes in grasses.

    Science.gov (United States)

    Ma, Peng-Fei; Guo, Zhen-Hua; Li, De-Zhu

    2012-01-01

    Compared to their counterparts in animals, the mitochondrial (mt) genomes of angiosperms exhibit a number of unique features. However, unravelling their evolution is hindered by the few completed genomes, of which are essentially Sanger sequenced. While next-generation sequencing technologies have revolutionized chloroplast genome sequencing, they are just beginning to be applied to angiosperm mt genomes. Chloroplast genomes of grasses (Poaceae) have undergone episodic evolution and the evolutionary rate was suggested to be correlated between chloroplast and mt genomes in Poaceae. It is interesting to investigate whether correlated rate change also occurred in grass mt genomes as expected under lineage effects. A time-calibrated phylogenetic tree is needed to examine rate change. We determined a largely completed mt genome from a bamboo, Ferrocalamus rimosivaginus (Poaceae), through Illumina sequencing of total DNA. With combination of de novo and reference-guided assembly, 39.5-fold coverage Illumina reads were finally assembled into scaffolds totalling 432,839 bp. The assembled genome contains nearly the same genes as the completed mt genomes in Poaceae. For examining evolutionary rate in grass mt genomes, we reconstructed a phylogenetic tree including 22 taxa based on 31 mt genes. The topology of the well-resolved tree was almost identical to that inferred from chloroplast genome with only minor difference. The inconsistency possibly derived from long branch attraction in mtDNA tree. By calculating absolute substitution rates, we found significant rate change (∼4-fold) in mt genome before and after the diversification of Poaceae both in synonymous and nonsynonymous terms. Furthermore, the rate change was correlated with that of chloroplast genomes in grasses. Our result demonstrates that it is a rapid and efficient approach to obtain angiosperm mt genome sequences using Illumina sequencing technology. The parallel episodic evolution of mt and chloroplast

  7. The diploid genome sequence of an individual human.

    Directory of Open Access Journals (Sweden)

    Samuel Levy

    2007-09-01

    Full Text Available Presented here is a genome sequence of an individual human. It was produced from approximately 32 million random DNA fragments, sequenced by Sanger dideoxy technology and assembled into 4,528 scaffolds, comprising 2,810 million bases (Mb of contiguous sequence with approximately 7.5-fold coverage for any given region. We developed a modified version of the Celera assembler to facilitate the identification and comparison of alternate alleles within this individual diploid genome. Comparison of this genome and the National Center for Biotechnology Information human reference assembly revealed more than 4.1 million DNA variants, encompassing 12.3 Mb. These variants (of which 1,288,319 were novel included 3,213,401 single nucleotide polymorphisms (SNPs, 53,823 block substitutions (2-206 bp, 292,102 heterozygous insertion/deletion events (indels(1-571 bp, 559,473 homozygous indels (1-82,711 bp, 90 inversions, as well as numerous segmental duplications and copy number variation regions. Non-SNP DNA variation accounts for 22% of all events identified in the donor, however they involve 74% of all variant bases. This suggests an important role for non-SNP genetic alterations in defining the diploid genome structure. Moreover, 44% of genes were heterozygous for one or more variants. Using a novel haplotype assembly strategy, we were able to span 1.5 Gb of genome sequence in segments >200 kb, providing further precision to the diploid nature of the genome. These data depict a definitive molecular portrait of a diploid human genome that provides a starting point for future genome comparisons and enables an era of individualized genomic information.

  8. Analysis of human HPRT- deletion mutants by the microarray-CGH (comparative genomic hybridization)

    International Nuclear Information System (INIS)

    Kodaira, M.; Sasaki, K.; Tagawa, H.; Omine, H.; Kushiro, J.; Takahashi, N.; Katayama, H.

    2003-01-01

    We are trying to evaluate genetic effects of radiation on human using mutation frequency as an indicator. For the efficient detection of mutations, it is important to understand the mechanism and the characteristics of radiation-induced mutations. We have started the analysis of hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutants induced by X-ray in order to clarify the deletion size and the mutation-distribution. We analyzed 39 human X-ray induced HPRT-deletion mutants by using the microarray-CGH. The array for this analysis contains 57 BAC clones covering as much as possible of the 4Mb of the 5' side and 10Mb of the 3' side of the HPRT gene based on the NCBI genome database. DNA from parent strain and each HPRT-mutant strain are labeled with Cy5 and Cy3 respectively, and were mixed and hybridized on the array. Fluorescent intensity ratio of the obtained spots was analyzed using software we developed to identify clones corresponding to the deletion region. The deletion in these strains ranged up to 3.5 Mb on the 5' side and 6 Mb on the 3' side of the HPRT gene. Deletions in 13 strains ended around BAC clones located at about 3 Mb on the 5' side. On the 3' side, deletions extended up to the specific clones located at 1.5 Mb in 11 strains. The mutations seem to be complex on the 3' end of deletion; some accompanied duplications with deletions and others could not be explained by one mutation event. We need to confirm these results, taking into account the experimental reproducibility and the accuracy of the published genetic map. The results of the research using the microarray-CGH help us to search the regions where deletions are easily induced and to identify the factors affecting the range of deletions

  9. The complete nucleotide sequences of the 5 genetically distinct plastid genomes of Oenothera, subsection Oenothera: II. A microevolutionary view using bioinformatics and formal genetic data.

    Science.gov (United States)

    Greiner, Stephan; Wang, Xi; Herrmann, Reinhold G; Rauwolf, Uwe; Mayer, Klaus; Haberer, Georg; Meurer, Jörg

    2008-09-01

    A unique combination of genetic features and a rich stock of information make the flowering plant genus Oenothera an appealing model to explore the molecular basis of speciation processes including nucleus-organelle coevolution. From representative species, we have recently reported complete nucleotide sequences of the 5 basic and genetically distinguishable plastid chromosomes of subsection Oenothera (I-V). In nature, Oenothera plastid genomes are associated with 6 distinct, either homozygous or heterozygous, diploid nuclear genotypes of the 3 basic genomes A, B, or C. Artificially produced plastome-genome combinations that do not occur naturally often display interspecific plastome-genome incompatibility (PGI). In this study, we compare formal genetic data available from all 30 plastome-genome combinations with sequence differences between the plastomes to uncover potential determinants for interspecific PGI. Consistent with an active role in speciation, a remarkable number of genes have high Ka/Ks ratios. Different from the Solanacean cybrid model Atropa/tobacco, RNA editing seems not to be relevant for PGIs in Oenothera. However, predominantly sequence polymorphisms in intergenic segments are proposed as possible sources for PGI. A single locus, the bidirectional promoter region between psbB and clpP, is suggested to contribute to compartmental PGI in the interspecific AB hybrid containing plastome I (AB-I), consistent with its perturbed photosystem II activity.

  10. Induction of genomic instability and activation of autophagy in artificial human aneuploid cells

    Energy Technology Data Exchange (ETDEWEB)

    Ariyoshi, Kentaro [Hirosaki University, Institute of Radiation Emergency Medicine, 66-1 Hon-cho, Hirosaki 036-8564 (Japan); Miura, Tomisato; Kasai, Kosuke; Fujishima, Yohei [Department of Biomedical Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki 036-8564 (Japan); Oshimura, Mitsuo [Chromosome Engineering Research Center (CERC), Tottori University, Nishicho 86, Yonago, Tottori 683-8503 (Japan); Yoshida, Mitsuaki A., E-mail: ariyoshi@hirosaki-u.ac.jp [Hirosaki University, Institute of Radiation Emergency Medicine, 66-1 Hon-cho, Hirosaki 036-8564 (Japan)

    2016-08-15

    Highlights: • Clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability. • Increased autophagy was observed in the artificially aneuploid clones. • Inhibition of autophagy resulted in increased genomic instability and DNA damage. • Intracellular levels of reactive oxygen species were up-regulated in the artificially aneuploid clones. - Abstract: Chromosome missegregation can lead to a change in chromosome number known as aneuploidy. Although aneuploidy is a known hallmark of cancer cells, the various mechanisms by which altered gene and/or DNA copy number facilitate tumorigenesis remain unclear. To understand the effect of aneuploidy occurring in non-tumorigenic human breast epithelial cells, we generated clones harboring artificial aneuploidy using microcell-mediated chromosome transfer. Our results demonstrate that clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability. Also, the increased autophagy was observed in the artificially aneuploidy clones, and inhibition of autophagy resulted in increased genomic instability and DNA damage. In addition, the intracellular levels of reactive oxygen species were up-regulated in the artificially aneuploid clones, and inhibition of autophagy further increased the production of reactive oxygen species. Together, these results suggest that even a single extraneous chromosome can induce genomic instability, and that autophagy triggered by aneuploidy-induced stress is a mechanism to protect cells bearing abnormal chromosome number.

  11. Induction of genomic instability and activation of autophagy in artificial human aneuploid cells

    International Nuclear Information System (INIS)

    Ariyoshi, Kentaro; Miura, Tomisato; Kasai, Kosuke; Fujishima, Yohei; Oshimura, Mitsuo; Yoshida, Mitsuaki A.

    2016-01-01

    Highlights: • Clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability. • Increased autophagy was observed in the artificially aneuploid clones. • Inhibition of autophagy resulted in increased genomic instability and DNA damage. • Intracellular levels of reactive oxygen species were up-regulated in the artificially aneuploid clones. - Abstract: Chromosome missegregation can lead to a change in chromosome number known as aneuploidy. Although aneuploidy is a known hallmark of cancer cells, the various mechanisms by which altered gene and/or DNA copy number facilitate tumorigenesis remain unclear. To understand the effect of aneuploidy occurring in non-tumorigenic human breast epithelial cells, we generated clones harboring artificial aneuploidy using microcell-mediated chromosome transfer. Our results demonstrate that clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability. Also, the increased autophagy was observed in the artificially aneuploidy clones, and inhibition of autophagy resulted in increased genomic instability and DNA damage. In addition, the intracellular levels of reactive oxygen species were up-regulated in the artificially aneuploid clones, and inhibition of autophagy further increased the production of reactive oxygen species. Together, these results suggest that even a single extraneous chromosome can induce genomic instability, and that autophagy triggered by aneuploidy-induced stress is a mechanism to protect cells bearing abnormal chromosome number.

  12. Non-targeted and delayed effects of exposure to ionizing radiation: II. Radiation-induced genomic instability and bystander effects in vivo, clastogenic factors and transgenerational effects

    Science.gov (United States)

    Morgan, William F.

    2003-01-01

    The goal of this review is to summarize the evidence for non-targeted and delayed effects of exposure to ionizing radiation in vivo. Currently, human health risks associated with radiation exposures are based primarily on the assumption that the detrimental effects of radiation occur in irradiated cells. Over the years a number of non-targeted effects of radiation exposure in vivo have been described that challenge this concept. These include radiation-induced genomic instability, bystander effects, clastogenic factors produced in plasma from irradiated individuals that can cause chromosomal damage when cultured with nonirradiated cells, and transgenerational effects of parental irradiation that can manifest in the progeny. These effects pose new challenges to evaluating the risk(s) associated with radiation exposure and understanding radiation-induced carcinogenesis.

  13. Functional analysis of the stem-loop structures at the 5' end of the Aichi virus genome

    International Nuclear Information System (INIS)

    Nagashima, Shigeo; Sasaki, Jun; Taniguchi, Koki

    2003-01-01

    Aichi virus is a member of the family Picornaviridae. Computer-assisted secondary structure prediction suggested the formation of three stem-loop structures (SL-A, SL-B, and SL-C from the 5' end) within the 5'-end 120 nucleotides of the genome. We have already shown that the most 5'-end stem-loop, SL-A, is critical for viral RNA replication. Here, using an infectious cDNA clone and a replicon harboring a luciferase gene, we revealed that formation of SL-B and SL-C on the positive strand is essential for viral RNA replication. In addition, the specific nucleotide sequence of the loop segment of SL-B was also shown to be critical for viral RNA replication. Mutations of the upper and lower stems of SL-C that do not disrupt the base-pairings hardly affected RNA replication, but decreased the yields of viable viruses significantly compared with for the wild-type. This suggests that SL-C plays a role at some step besides RNA replication during virus infection

  14. Genome-to-genome analysis highlights the effect of the human innate and adaptive immune systems on the hepatitis C virus.

    Science.gov (United States)

    Ansari, M Azim; Pedergnana, Vincent; L C Ip, Camilla; Magri, Andrea; Von Delft, Annette; Bonsall, David; Chaturvedi, Nimisha; Bartha, Istvan; Smith, David; Nicholson, George; McVean, Gilean; Trebes, Amy; Piazza, Paolo; Fellay, Jacques; Cooke, Graham; Foster, Graham R; Hudson, Emma; McLauchlan, John; Simmonds, Peter; Bowden, Rory; Klenerman, Paul; Barnes, Eleanor; Spencer, Chris C A

    2017-05-01

    Outcomes of hepatitis C virus (HCV) infection and treatment depend on viral and host genetic factors. Here we use human genome-wide genotyping arrays and new whole-genome HCV viral sequencing technologies to perform a systematic genome-to-genome study of 542 individuals who were chronically infected with HCV, predominantly genotype 3. We show that both alleles of genes encoding human leukocyte antigen molecules and genes encoding components of the interferon lambda innate immune system drive viral polymorphism. Additionally, we show that IFNL4 genotypes determine HCV viral load through a mechanism dependent on a specific amino acid residue in the HCV NS5A protein. These findings highlight the interplay between the innate immune system and the viral genome in HCV control.

  15. Genome U-Plot: a whole genome visualization.

    Science.gov (United States)

    Gaitatzes, Athanasios; Johnson, Sarah H; Smadbeck, James B; Vasmatzis, George

    2018-05-15

    The ability to produce and analyze whole genome sequencing (WGS) data from samples with structural variations (SV) generated the need to visualize such abnormalities in simplified plots. Conventional two-dimensional representations of WGS data frequently use either circular or linear layouts. There are several diverse advantages regarding both these representations, but their major disadvantage is that they do not use the two-dimensional space very efficiently. We propose a layout, termed the Genome U-Plot, which spreads the chromosomes on a two-dimensional surface and essentially quadruples the spatial resolution. We present the Genome U-Plot for producing clear and intuitive graphs that allows researchers to generate novel insights and hypotheses by visualizing SVs such as deletions, amplifications, and chromoanagenesis events. The main features of the Genome U-Plot are its layered layout, its high spatial resolution and its improved aesthetic qualities. We compare conventional visualization schemas with the Genome U-Plot using visualization metrics such as number of line crossings and crossing angle resolution measures. Based on our metrics, we improve the readability of the resulting graph by at least 2-fold, making apparent important features and making it easy to identify important genomic changes. A whole genome visualization tool with high spatial resolution and improved aesthetic qualities. An implementation and documentation of the Genome U-Plot is publicly available at https://github.com/gaitat/GenomeUPlot. vasmatzis.george@mayo.edu. Supplementary data are available at Bioinformatics online.

  16. The Switchgrass Genome: Tools and Strategies

    Directory of Open Access Journals (Sweden)

    Michael D. Casler

    2011-11-01

    Full Text Available Switchgrass ( L. is a perennial grass species receiving significant focus as a potential bioenergy crop. In the last 5 yr the switchgrass research community has produced a genetic linkage map, an expressed sequence tag (EST database, a set of single nucleotide polymorphism (SNP markers that are distributed across the 18 linkage groups, 4x sampling of the AP13 genome in 400-bp reads, and bacterial artificial chromosome (BAC libraries containing over 200,000 clones. These studies have revealed close collinearity of the switchgrass genome with those of sorghum [ (L. Moench], rice ( L., and (L. P. Beauv. Switchgrass researchers have also developed several microarray technologies for gene expression studies. Switchgrass genomic resources will accelerate the ability of plant breeders to enhance productivity, pest resistance, and nutritional quality. Because switchgrass is a relative newcomer to the genomics world, many secrets of the switchgrass genome have yet to be revealed. To continue to efficiently explore basic and applied topics in switchgrass, it will be critical to capture and exploit the knowledge of plant geneticists and breeders on the next logical steps in the development and utilization of genomic resources for this species. To this end, the community has established a switchgrass genomics executive committee and work group ( [verified 28 Oct. 2011].

  17. MicroRNA-34a promotes genomic instability by a broad suppression of genome maintenance mechanisms downstream of the oncogene KSHV-vGPCR.

    Science.gov (United States)

    Krause, Claudia J; Popp, Oliver; Thirunarayanan, Nanthakumar; Dittmar, Gunnar; Lipp, Martin; Müller, Gerd

    2016-03-01

    The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded chemokine receptor vGPCR acts as an oncogene in Kaposi's sarcomagenesis. Until now, the molecular mechanisms by which the vGPCR contributes to tumor development remain incompletely understood. Here, we show that the KSHV-vGPCR contributes to tumor progression through microRNA (miR)-34a-mediated induction of genomic instability. Large-scale analyses on the DNA, gene and protein level of cell lines derived from a mouse model of vGPCR-driven tumorigenesis revealed that a vGPCR-induced upregulation of miR-34a resulted in a broad suppression of genome maintenance genes. A knockdown of either the vGPCR or miR-34a largely restored the expression of these genes and confirmed miR-34a as a downstream effector of the KSHV-vGPCR that compromises genome maintenance mechanisms. This novel, protumorigenic role of miR-34a questions the use of miR-34a mimetics in cancer therapy as they could impair genome stability.

  18. Fisetin Induces Apoptosis Through p53-Mediated Up-Regulation of DR5 Expression in Human Renal Carcinoma Caki Cells

    Directory of Open Access Journals (Sweden)

    Kyoung-jin Min

    2017-08-01

    Full Text Available Fisetin is a natural compound found in fruits and vegetables such as strawberries, apples, cucumbers, and onions. Since fisetin can elicit anti-cancer effects, including anti-proliferation and anti-migration, we investigated whether fisetin induced apoptosis in human renal carcinoma (Caki cells. Fisetin markedly induced sub-G1 population and cleavage of poly (ADP-ribose polymerase (PARP, which is a marker of apoptosis, and increased caspase activation. We found that pan-caspase inhibitor (z-VAD-fmk inhibited fisetin-induced apoptosis. In addition, fisetin induced death receptor 5 (DR5 expression at the transcriptional level, and down-regulation of DR5 by siRNA blocked fisetin-induced apoptosis. Furthermore, fisetin induced p53 protein expression through up-regulation of protein stability, whereas down-regulation of p53 by siRNA markedly inhibited fisetin-induced DR5 expression. In contrast, fisetin induced up-regulation of CHOP expression and reactive oxygen species production, which had no effect on fisetin-induced apoptosis. Taken together, our study demonstrates that fisetin induced apoptosis through p53 mediated up-regulation of DR5 expression at the transcriptional level.

  19. New sequence-based data on the relative DNA contents of chromosomes in the normal male and female human diploid genomes for radiation molecular cytogenetics

    Directory of Open Access Journals (Sweden)

    Repin Mikhail V

    2009-06-01

    Full Text Available Abstract Background The objective of this work is to obtain the correct relative DNA contents of chromosomes in the normal male and female human diploid genomes for the use at FISH analysis of radiation-induced chromosome aberrations. Results The relative DNA contents of chromosomes in the male and female human diploid genomes have been calculated from the publicly available international Human Genome Project data. New sequence-based data on the relative DNA contents of human chromosomes were compared with the data recommended by the International Atomic Energy Agency in 2001. The differences in the values of the relative DNA contents of chromosomes obtained by using different approaches for 15 human chromosomes, mainly for large chromosomes, were below 2%. For the chromosomes 13, 17, 20 and 22 the differences were above 5%. Conclusion New sequence-based data on the relative DNA contents of chromosomes in the normal male and female human diploid genomes were obtained. This approach, based on the genome sequence, can be recommended for the use in radiation molecular cytogenetics.

  20. Exchange bias induced at a Co2FeAl0.5Si0.5/Cr interface

    International Nuclear Information System (INIS)

    Yu, C N T; Vick, A J; Inami, N; Ono, K; Frost, W; Hirohata, A

    2017-01-01

    In order to engineer the strength of an exchange bias in a cubic Heusler alloy layer, crystalline strain has been induced at a ferromagnet/antiferromagnet interface by their lattice mismatch in addition to the conventional interfacial exchange coupling between them. Such interfaces have been formed in (Co 2 FeAl 0.5 Si 0.5 (CFAS)/Cr) 3 structures grown by ultrahigh vacuum molecular beam epitaxy. The magnetic and structural properties have been characterised to investigate the exchange interactions at the CFAS/Cr interfaces. Due to the interfacial lattice mismatch of 1.4%, the maximum offset of 18 Oe in a magnetisation curve has been measured for the case of a CFAS (2 nm)/Cr (0.9 nm) interface at 193 K. The half-metallic property of CFAS has been observed to remain unchanged, which agrees with the theoretical prediction by Culbert et al (2008 J. Appl. Phys . 103 07D707). Such a strain-induced exchange bias may provide insight of the interfacial interactions and may offer a wide flexibility in spintronic device design. (paper)

  1. The Jujube Genome Provides Insights into Genome Evolution and the Domestication of Sweetness/Acidity Taste in Fruit Trees.

    Science.gov (United States)

    Huang, Jian; Zhang, Chunmei; Zhao, Xing; Fei, Zhangjun; Wan, KangKang; Zhang, Zhong; Pang, Xiaoming; Yin, Xiao; Bai, Yang; Sun, Xiaoqing; Gao, Lizhi; Li, Ruiqiang; Zhang, Jinbo; Li, Xingang

    2016-12-01

    Jujube (Ziziphus jujuba Mill.) belongs to the Rhamnaceae family and is a popular fruit tree species with immense economic and nutritional value. Here, we report a draft genome of the dry jujube cultivar 'Junzao' and the genome resequencing of 31 geographically diverse accessions of cultivated and wild jujubes (Ziziphus jujuba var. spinosa). Comparative analysis revealed that the genome of 'Dongzao', a fresh jujube, was ~86.5 Mb larger than that of the 'Junzao', partially due to the recent insertions of transposable elements in the 'Dongzao' genome. We constructed eight proto-chromosomes of the common ancestor of Rhamnaceae and Rosaceae, two sister families in the order Rosales, and elucidated the evolutionary processes that have shaped the genome structures of modern jujubes. Population structure analysis revealed the complex genetic background of jujubes resulting from extensive hybridizations between jujube and its wild relatives. Notably, several key genes that control fruit organic acid metabolism and sugar content were identified in the selective sweep regions. We also identified S-locus genes controlling gametophytic self-incompatibility and investigated haplotype patterns of the S locus in the jujube genomes, which would provide a guideline for parent selection for jujube crossbreeding. This study provides valuable genomic resources for jujube improvement, and offers insights into jujube genome evolution and its population structure and domestication.

  2. A Genome-Wide Breast Cancer Scan in African Americans

    Science.gov (United States)

    2010-06-01

    SNPs from the African American breast cancer scan to COGs , a European collaborative study which is has designed a SNP array with that will be genotyped...Award Number: W81XWH-08-1-0383 TITLE: A Genome-wide Breast Cancer Scan in African Americans PRINCIPAL INVESTIGATOR: Christopher A...SUBTITLE A Genome-wide Breast Cancer Scan in African Americans 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-08-1-0383 5c. PROGRAM

  3. A Thousand Fly Genomes: An Expanded Drosophila Genome Nexus.

    Science.gov (United States)

    Lack, Justin B; Lange, Jeremy D; Tang, Alison D; Corbett-Detig, Russell B; Pool, John E

    2016-12-01

    The Drosophila Genome Nexus is a population genomic resource that provides D. melanogaster genomes from multiple sources. To facilitate comparisons across data sets, genomes are aligned using a common reference alignment pipeline which involves two rounds of mapping. Regions of residual heterozygosity, identity-by-descent, and recent population admixture are annotated to enable data filtering based on the user's needs. Here, we present a significant expansion of the Drosophila Genome Nexus, which brings the current data object to a total of 1,121 wild-derived genomes. New additions include 305 previously unpublished genomes from inbred lines representing six population samples in Egypt, Ethiopia, France, and South Africa, along with another 193 genomes added from recently-published data sets. We also provide an aligned D. simulans genome to facilitate divergence comparisons. This improved resource will broaden the range of population genomic questions that can addressed from multi-population allele frequencies and haplotypes in this model species. The larger set of genomes will also enhance the discovery of functionally relevant natural variation that exists within and between populations. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  4. Visualization for genomics: the Microbial Genome Viewer.

    Science.gov (United States)

    Kerkhoven, Robert; van Enckevort, Frank H J; Boekhorst, Jos; Molenaar, Douwe; Siezen, Roland J

    2004-07-22

    A Web-based visualization tool, the Microbial Genome Viewer, is presented that allows the user to combine complex genomic data in a highly interactive way. This Web tool enables the interactive generation of chromosome wheels and linear genome maps from genome annotation data stored in a MySQL database. The generated images are in scalable vector graphics (SVG) format, which is suitable for creating high-quality scalable images and dynamic Web representations. Gene-related data such as transcriptome and time-course microarray experiments can be superimposed on the maps for visual inspection. The Microbial Genome Viewer 1.0 is freely available at http://www.cmbi.kun.nl/MGV

  5. Capsaicin sensitizes TRAIL-induced apoptosis through Sp1-mediated DR5 up-regulation: Involvement of Ca2+ influx

    International Nuclear Information System (INIS)

    Moon, Dong-Oh; Kang, Chang-Hee; Kang, Sang-Hyuck; Choi, Yung-Hyun; Hyun, Jin-Won; Chang, Weon-Young; Kang, Hee-Kyoung; Koh, Young-Sang; Maeng, Young-Hee; Kim, Young-Ree; Kim, Gi-Young

    2012-01-01

    Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various malignant cells, several cancers including human hepatocellular carcinoma (HCC) exhibit potent resistance to TRAIL-induced cell death. The aim of this study is to evaluate the anti-cancer potential of capsaicin in TRAIL-induced cancer cell death. As indicated by assays that measure phosphatidylserine exposure, mitochondrial activity and activation of caspases, capsaicin potentiated TRAIL-resistant cells to lead to cell death. In addition, we found that capsaicin induces the cell surface expression of TRAIL receptor DR5, but not DR4 through the activation Sp1 on its promoter region. Furthermore, we investigated that capsaicin-induced DR5 expression and apoptosis are inhibited by calcium chelator or inhibitors for calmodulin-dependent protein kinase. Taken together, our data suggest that capsaicin sensitizes TRAIL-mediated HCC cell apoptosis by DR5 up-regulation via calcium influx-dependent Sp1 activation. Highlights: ► Capsaicin sensitizes TRAIL-induced apoptosis through activation of caspases. ► Capsaicin induces expression of DR5 through Sp1 activation. ► Capsaicin activates calcium signaling pathway.

  6. Sex-specific hippocampal 5-hydroxymethylcytosine is disrupted in response to acute stress.

    Science.gov (United States)

    Papale, Ligia A; Li, Sisi; Madrid, Andy; Zhang, Qi; Chen, Li; Chopra, Pankaj; Jin, Peng; Keleş, Sündüz; Alisch, Reid S

    2016-12-01

    Environmental stress is among the most important contributors to increased susceptibility to develop psychiatric disorders. While it is well known that acute environmental stress alters gene expression, the molecular mechanisms underlying these changes remain largely unknown. 5-hydroxymethylcytosine (5hmC) is a novel environmentally sensitive epigenetic modification that is highly enriched in neurons and is associated with active neuronal transcription. Recently, we reported a genome-wide disruption of hippocampal 5hmC in male mice following acute stress that was correlated to altered transcript levels of genes in known stress related pathways. Since sex-specific endocrine mechanisms respond to environmental stimulus by altering the neuronal epigenome, we examined the genome-wide profile of hippocampal 5hmC in female mice following exposure to acute stress and identified 363 differentially hydroxymethylated regions (DhMRs) linked to known (e.g., Nr3c1 and Ntrk2) and potentially novel genes associated with stress response and psychiatric disorders. Integration of hippocampal expression data from the same female mice found stress-related hydroxymethylation correlated to altered transcript levels. Finally, characterization of stress-induced sex-specific 5hmC profiles in the hippocampus revealed 778 sex-specific acute stress-induced DhMRs some of which were correlated to altered transcript levels that produce sex-specific isoforms in response to stress. Together, the alterations in 5hmC presented here provide a possible molecular mechanism for the adaptive sex-specific response to stress that may augment the design of novel therapeutic agents that will have optimal effectiveness in each sex. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Cytogenetic and molecular characterization of human radio-induced tumours

    International Nuclear Information System (INIS)

    Lefevre, S.

    2002-09-01

    After a brief recall of some fundamentals regarding radiobiology, this research thesis discusses some epidemiological aspects of radio carcinogenesis, based on epidemiological studies performed on people having survived to Hiroshima, Nagasaki and Chernobyl, but also performed on people submitted to domestic or professional exposures to radon, or to medicine-related exposures. The author highlights some predispositions to radio-induced cancers. Then, she discusses the genetic mechanisms of radio-induced carcinogenesis and the genetic alterations observed in human radio-induced tumours. She discusses and comments the genomic instability, its mechanisms and some models observed on mice, and describes the various forms of radio-induced genomic instability. After a discussion of all these aspects, the author draws some perspectives for future research works

  8. Hyperthyroidism enhances 5-HT-induced contraction of the rat pulmonary artery: role of calcium-activated chloride channel activation.

    Science.gov (United States)

    Oriowo, Mabayoje A; Oommen, Elsie; Khan, Islam

    2011-11-01

    Experimentally-induced hyperthyroidism in rodents is associated with signs and symptoms of pulmonary hypertension. The main objective of the present study was to investigate the effect of thyroxine-induced pulmonary hypertension on the contractile response of the pulmonary artery to 5-HT and the possible underlying signaling pathway. 5-HT concentration-dependently contracted artery segments from control and thyroxine-treated rats with pD(2) values of 5.04 ± 0.19 and 5.34 ± 0.14, respectively. The maximum response was significantly greater in artery segments from thyroxine-treated rats. Neither BW 723C86 (5-HT(2B)-receptor agonist) nor CP 93129 (5-HT(1B)-receptor agonist) contracted ring segments of the pulmonary artery from control and thyroxine-treated rats at concentrations up to 10(-4)M. There was no significant difference in the level of expression of 5-HT(2A)-receptor protein between the two groups. Ketanserin (3 × 10(-8)M) produced a rightward shift of the concentration-response curve to 5-HT in both groups with equal potency (-logK(B) values were 8.1 ± 0.2 and 7.9 ± 0.1 in control and thyroxine-treated rats, respectively). Nifedipine (10(-6)M) inhibited 5-HT-induced contractions in artery segments from control and thyroxine-treated rats and was more effective against 5-HT-induced contraction in artery segments for thyroxine-treated rats. The calcium-activated chloride channel blocker, niflumic acid (10(-4)M) also inhibited 5-HT-induced contractions in artery segments from control and thyroxine-treated rats and was more effective against 5-HT-induced contraction in artery segments for thyroxine-treated rats. It was concluded that hyperthyroidism enhanced 5-HT-induced contractions of the rat pulmonary artery by a mechanism involving increased activity of calcium-activated chloride channels. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. RECG maintains plastid and mitochondrial genome stability by suppressing extensive recombination between short dispersed repeats.

    Directory of Open Access Journals (Sweden)

    Masaki Odahara

    2015-03-01

    Full Text Available Maintenance of plastid and mitochondrial genome stability is crucial for photosynthesis and respiration, respectively. Recently, we have reported that RECA1 maintains mitochondrial genome stability by suppressing gross rearrangements induced by aberrant recombination between short dispersed repeats in the moss Physcomitrella patens. In this study, we studied a newly identified P. patens homolog of bacterial RecG helicase, RECG, some of which is localized in both plastid and mitochondrial nucleoids. RECG partially complements recG deficiency in Escherichia coli cells. A knockout (KO mutation of RECG caused characteristic phenotypes including growth delay and developmental and mitochondrial defects, which are similar to those of the RECA1 KO mutant. The RECG KO cells showed heterogeneity in these phenotypes. Analyses of RECG KO plants showed that mitochondrial genome was destabilized due to a recombination between 8-79 bp repeats and the pattern of the recombination partly differed from that observed in the RECA1 KO mutants. The mitochondrial DNA (mtDNA instability was greater in severe phenotypic RECG KO cells than that in mild phenotypic ones. This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants. Some of the induced recombination caused efficient genomic rearrangements in RECG KO mitochondria. Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci. In addition, the RECG KO mutation caused remarkable plastid abnormalities and induced recombination between short repeats (12-63 bp in the plastid DNA. These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.

  10. Cooperation between Epstein-Barr virus immune evasion proteins spreads protection from CD8+ T cell recognition across all three phases of the lytic cycle.

    Directory of Open Access Journals (Sweden)

    Laura L Quinn

    2014-08-01

    Full Text Available CD8+ T cell responses to Epstein-Barr virus (EBV lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE and some early (E antigens are more frequently observed than responses to epitopes of late (L expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE- and early (E-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L, interference by BILF1 increases with progression through lytic cycle (IE

  11. 2´-deoxy-5,6-dihydro-5-azacytidine - a less toxic alternative of 2´-deoxy-5-azacytidine: a comparative study of hypomethylating potential.

    Science.gov (United States)

    Matoušová, Marika; Votruba, Ivan; Otmar, Miroslav; Tloušťová, Eva; Günterová, Jana; Mertlíková-Kaiserová, Helena

    2011-06-01

    Restoration of transcriptionally silenced genes by means of methyltransferases inhibitors plays a crucial role in the current therapy of myelodysplastic syndromes and certain types of leukemias. A comparative study of hypomethylating activities of a series of 5-azacytidine nucleosides: 5-azacytidine (AC), 2'-deoxy-5-azacytidine (DAC) and its α-anomer (α-DAC), 5,6-dihydro-5-azacytidine (DHAC), 2'-deoxy-5,6-dihydro-5-azacytidine (DHDAC, KP-1212) and its α-anomer (α-DHDAC), and of a 2-pyrimidone ribonucleoside (zebularine) was conducted. Methylation-specific PCR was employed to detect the efficiency of individual agents on cyclin-dependent kinase inhibitor 2B and thrombospondin-1 hypermethylated gene loci. Overall changes in DNA methylation level were quantified by direct estimation of 5-methyl-2'-deoxycytidine-5'-monophosphate by HPLC using digested genomic DNA. Flow cytometric analysis of cell cycle progression and apoptotic markers was used to determine cytotoxicity of the compounds. mRNA expression was measured using qRT-PCR. 2'-deoxy-5,6-dihydro-5-azacytidine was found to be less cytotoxic and more stable than 2'-deoxy-5-azacytidine at the doses that induce comparable DNA hypomethylation and gene reactivation. This makes it a valuable tool for epigenetic research and worth further investigations to elucidate its possible therapeutic potential.

  12. Genome-wide Analyses Identify KIF5A as a Novel ALS Gene

    NARCIS (Netherlands)

    Nicolas, Aude; Kenna, Kevin P.; Renton, Alan E.; Ticozzi, Nicola; Faghri, Faraz; Chia, Ruth; Dominov, Janice A.; Kenna, Brendan J.; Nalls, Mike A.; Keagle, Pamela; Rivera, Alberto M.; van Rheenen, Wouter; Murphy, Natalie A.; van Vugt, Joke J.F.A.; Geiger, Joshua T.; van der Spek, Rick; Pliner, Hannah A.; Smith, Bradley N.; Marangi, Giuseppe; Topp, Simon D.; Abramzon, Yevgeniya; Gkazi, Athina Soragia; Eicher, John D.; Kenna, Aoife; Logullo, Francesco O.; Simone, Isabella L.; Logroscino, Giancarlo; Salvi, Fabrizio; Bartolomei, Ilaria; Borghero, Giuseppe; Murru, Maria Rita; Costantino, Emanuela; Pani, Carla; Puddu, Roberta; Caredda, Carla; Piras, Valeria; Tranquilli, Stefania; Cuccu, Stefania; Corongiu, Daniela; Melis, Maurizio; Milia, Antonio; Marrosu, Francesco; Marrosu, Maria Giovanna; Floris, Gianluca; Cannas, Antonino; Capasso, Margherita; Caponnetto, Claudia; Mancardi, Gianluigi; Origone, Paola; Mandich, Paola; Conforti, Francesca L.; Cavallaro, Sebastiano; Mora, Gabriele; Marinou, Kalliopi; Sideri, Riccardo; Penco, Silvana; Mosca, Lorena; Lunetta, Christian; Pinter, Giuseppe Lauria; Corbo, Massimo; Riva, Nilo; Carrera, Paola; Volanti, Paolo; Mandrioli, Jessica; Fini, Nicola; Fasano, Antonio; Tremolizzo, Lucio; Arosio, Alessandro; Ferrarese, Carlo; Trojsi, Francesca; Tedeschi, Gioacchino; Monsurrò, Maria Rosaria; Piccirillo, Giovanni; Femiano, Cinzia; Ticca, Anna; Ortu, Enzo; La Bella, Vincenzo; Spataro, Rossella; Colletti, Tiziana; Sabatelli, Mario; Zollino, Marcella; Conte, Amelia; Luigetti, Marco; Lattante, Serena; Marangi, Giuseppe; Santarelli, Marialuisa; Petrucci, Antonio; Pugliatti, Maura; Pirisi, Angelo; Parish, Leslie D.; Occhineri, Patrizia; Giannini, Fabio; Battistini, Stefania; Ricci, Claudia; Benigni, Michele; Cau, Tea B.; Loi, Daniela; Calvo, Andrea; Moglia, Cristina; Brunetti, Maura; Barberis, Marco; Restagno, Gabriella; Casale, Federico; Marrali, Giuseppe; Fuda, Giuseppe; Ossola, Irene; Cammarosano, Stefania; Canosa, Antonio; Ilardi, Antonio; Manera, Umberto; Grassano, Maurizio; Tanel, Raffaella; Pisano, Fabrizio; Mora, Gabriele; Calvo, Andrea; Mazzini, Letizia; Riva, Nilo; Mandrioli, Jessica; Caponnetto, Claudia; Battistini, Stefania; Volanti, Paolo; La Bella, Vincenzo; Conforti, Francesca L.; Borghero, Giuseppe; Messina, Sonia; Simone, Isabella L.; Trojsi, Francesca; Salvi, Fabrizio; Logullo, Francesco O.; D'Alfonso, Sandra; Corrado, Lucia; Capasso, Margherita; Ferrucci, Luigi; Harms, Matthew B.; Goldstein, David B.; Shneider, Neil A.; Goutman, Stephen A.; Simmons, Zachary; Miller, Timothy M.; Chandran, Siddharthan; Pal, Suvankar; Manousakis, George; Appel, Stanley H.; Simpson, Ericka; Wang, Leo; Baloh, Robert H.; Gibson, Summer B.; Bedlack, Richard; Lacomis, David; Sareen, Dhruv; Sherman, Alexander; Bruijn, Lucie; Penny, Michelle; Moreno, Cristiane de Araujo Martins; Kamalakaran, Sitharthan; Goldstein, David B.; Allen, Andrew S.; Appel, Stanley; Baloh, Robert H.; Bedlack, Richard S.; Boone, Braden E.; Brown, Robert; Carulli, John P.; Chesi, Alessandra; Chung, Wendy K.; Cirulli, Elizabeth T.; Cooper, Gregory M.; Couthouis, Julien; Day-Williams, Aaron G.; Dion, Patrick A.; Gibson, Summer B.; Gitler, Aaron D.; Glass, Jonathan D.; Goldstein, David B.; Han, Yujun; Harms, Matthew B.; Harris, Tim; Hayes, Sebastian D.; Jones, Angela L.; Keebler, Jonathan; Krueger, Brian J.; Lasseigne, Brittany N.; Levy, Shawn E.; Lu, Yi Fan; Maniatis, Tom; McKenna-Yasek, Diane; Miller, Timothy M.; Myers, Richard M.; Petrovski, Slavé; Pulst, Stefan M.; Raphael, Alya R.; Ravits, John M.; Ren, Zhong; Rouleau, Guy A.; Sapp, Peter C.; Shneider, Neil A.; Simpson, Ericka; Sims, Katherine B.; Staropoli, John F.; Waite, Lindsay L.; Wang, Quanli; Wimbish, Jack R.; Xin, Winnie W.; Gitler, Aaron D.; Harris, Tim; Myers, Richard M.; Phatnani, Hemali; Kwan, Justin; Sareen, Dhruv; Broach, James R.; Simmons, Zachary; Arcila-Londono, Ximena; Lee, Edward B.; Van Deerlin, Vivianna M.; Shneider, Neil A.; Fraenkel, Ernest; Ostrow, Lyle W.; Baas, Frank; Zaitlen, Noah; Berry, James D.; Malaspina, Andrea; Fratta, Pietro; Cox, Gregory A.; Thompson, Leslie M.; Finkbeiner, Steve; Dardiotis, Efthimios; Miller, Timothy M.; Chandran, Siddharthan; Pal, Suvankar; Hornstein, Eran; MacGowan, Daniel J.L.; Heiman-Patterson, Terry D.; Hammell, Molly G.; Patsopoulos, Nikolaos A.; Dubnau, Joshua; Nath, Avindra; Phatnani, Hemali; Musunuri, Rajeeva Lochan; Evani, Uday Shankar; Abhyankar, Avinash; Zody, Michael C.; Kaye, Julia; Finkbeiner, Steven; Wyman, Stacia K.; LeNail, Alexander; Lima, Leandro; Fraenkel, Ernest; Rothstein, Jeffrey D.; Svendsen, Clive N.; Thompson, Leslie M.; Van Eyk, Jenny; Maragakis, Nicholas J.; Berry, James D.; Glass, Jonathan D.; Miller, Timothy M.; Kolb, Stephen J.; Baloh, Robert H.; Cudkowicz, Merit; Baxi, Emily; Kaye, Julia; Finkbeiner, Steven; Wyman, Stacia K.; Finkbeiner, Steven; LeNail, Alex; Lima, Leandro; Fraenkel, Ernest; Fraenkel, Ernest; Svendsen, Clive N.; Svendsen, Clive N.; Thompson, Leslie M.; Thompson, Leslie M.; Van Eyk, Jennifer E.; Berry, James D.; Berry, James D.; Miller, Timothy M.; Kolb, Stephen J.; Cudkowicz, Merit; Cudkowicz, Merit; Baxi, Emily; Benatar, Michael; Taylor, J. Paul; Wu, Gang; Rampersaud, Evadnie; Wuu, Joanne; Rademakers, Rosa; Züchner, Stephan; Schule, Rebecca; McCauley, Jacob; Hussain, Sumaira; Cooley, Anne; Wallace, Marielle; Clayman, Christine; Barohn, Richard; Statland, Jeffrey; Ravits, John M.; Swenson, Andrea; Jackson, Carlayne; Trivedi, Jaya; Khan, Shaida; Katz, Jonathan; Jenkins, Liberty; Burns, Ted; Gwathmey, Kelly; Caress, James; McMillan, Corey; Elman, Lauren; Pioro, Erik P.; Heckmann, Jeannine; So, Yuen; Walk, David; Maiser, Samuel; Zhang, Jinghui; Benatar, Michael; Taylor, J. Paul; Taylor, J. Paul; Rampersaud, Evadnie; Wu, Gang; Wuu, Joanne; Silani, Vincenzo; Ticozzi, Nicola; Gellera, Cinzia; Ratti, Antonia; Taroni, Franco; Lauria, Giuseppe; Verde, Federico; Fogh, Isabella; Tiloca, Cinzia; Comi, Giacomo P.; Sorarù, Gianni; Cereda, Cristina; D'Alfonso, Sandra; Corrado, Lucia; De Marchi, Fabiola; Corti, Stefania; Ceroni, Mauro; Mazzini, Letizia; Siciliano, Gabriele; Filosto, Massimiliano; Inghilleri, Maurizio; Peverelli, Silvia; Colombrita, Claudia; Poletti, Barbara; Maderna, Luca; Del Bo, Roberto; Gagliardi, Stella; Querin, Giorgia; Bertolin, Cinzia; Pensato, Viviana; Castellotti, Barbara; Lauria, Giuseppe; Verde, Federico; Fogh, Isabella; Tiloca, Cinzia; Fogh, Isabella; Comi, Giacomo P.; Sorarù, Gianni; Cereda, Cristina; Camu, William; Mouzat, Kevin; Lumbroso, Serge; Corcia, Philippe; Meininger, Vincent; Besson, Gérard; Lagrange, Emmeline; Clavelou, Pierre; Guy, Nathalie; Couratier, Philippe; Vourch, Patrick; Danel, Véronique; Bernard, Emilien; Lemasson, Gwendal; Corcia, Philippe; Laaksovirta, Hannu; Myllykangas, Liisa; Jansson, Lilja; Valori, Miko; Ealing, John; Hamdalla, Hisham; Rollinson, Sara; Pickering-Brown, Stuart; Orrell, Richard W.; Sidle, Katie C.; Malaspina, Andrea; Hardy, John; Singleton, Andrew B.; Johnson, Janel O.; Arepalli, Sampath; Sapp, Peter C.; McKenna-Yasek, Diane; Polak, Meraida; Asress, Seneshaw; Al-Sarraj, Safa; King, Andrew; Troakes, Claire; Vance, Caroline; de Belleroche, Jacqueline; Baas, Frank; ten Asbroek, Anneloor L.M.A.; Muñoz-Blanco, José Luis; Hernandez, Dena G.; Ding, Jinhui; Gibbs, J. Raphael; Scholz, Sonja W.; Scholz, Sonja W.; Floeter, Mary Kay; Campbell, Roy H.; Landi, Francesco; Bowser, Robert; Pulst, Stefan M.; Ravits, John M.; MacGowan, Daniel J.L.; Kirby, Janine; Pioro, Erik P.; Pamphlett, Roger; Broach, James; Gerhard, Glenn; Dunckley, Travis L.; Brady, Christopher B.; Brady, Christopher B.; Kowall, Neil W.; Troncoso, Juan C.; Le Ber, Isabelle; Mouzat, Kevin; Lumbroso, Serge; Mouzat, Kevin; Lumbroso, Serge; Heiman-Patterson, Terry D.; Heiman-Patterson, Terry D.; Kamel, Freya; Van Den Bosch, Ludo; Van Den Bosch, Ludo; Baloh, Robert H.; Strom, Tim M.; Meitinger, Thomas; Strom, Tim M.; Shatunov, Aleksey; Van Eijk, Kristel R.; de Carvalho, Mamede; de Carvalho, Mamede; Kooyman, Maarten; Middelkoop, Bas; Moisse, Matthieu; McLaughlin, Russell; Van Es, Michael A.; Weber, Markus; Boylan, Kevin B.; Van Blitterswijk, Marka; Rademakers, Rosa; Morrison, Karen; Basak, A. Nazli; Mora, Jesús S.; Drory, Vivian; Shaw, Pamela; Turner, Martin R.; Talbot, Kevin; Hardiman, Orla; Williams, Kelly L.; Fifita, Jennifer A.; Nicholson, Garth A.; Blair, Ian P.; Nicholson, Garth A.; Rouleau, Guy A.; Esteban-Pérez, Jesús; García-Redondo, Alberto; Al-Chalabi, Ammar; Al Kheifat, Ahmad; Al-Chalabi, Ammar; Andersen, Peter M.; Basak, A. Nazli; Blair, Ian P.; Chio, Adriano; Cooper-Knock, Jonathan; Corcia, Philippe; Couratier, Philippe; de Carvalho, Mamede; Dekker, Annelot; Drory, Vivian; Redondo, Alberto Garcia; Gotkine, Marc; Hardiman, Orla; Hide, Winston; Iacoangeli, Alfredo; Glass, Jonathan D.; Kenna, Kevin P.; Kiernan, Matthew; Kooyman, Maarten; Landers, John E.; McLaughlin, Russell; Middelkoop, Bas; Mill, Jonathan; Neto, Miguel Mitne; Moisse, Matthieu; Pardina, Jesus Mora; Morrison, Karen; Newhouse, Stephen; Pinto, Susana; Pulit, Sara; Robberecht, Wim; Shatunov, Aleksey; Shaw, Pamela; Shaw, Chris; Silani, Vincenzo; Sproviero, William; Tazelaar, Gijs; Ticozzi, Nicola; Van Damme, Philip; van den Berg, Leonard; van der Spek, Rick; Van Eijk, Kristel R.; Van Es, Michael A.; van Rheenen, Wouter; van Vugt, Joke J.F.A.; Veldink, Jan H.; Weber, Markus; Williams, Kelly L.; Van Damme, Philip; Robberecht, Wim; Zatz, Mayana; Robberecht, Wim; Bauer, Denis C.; Twine, Natalie A.; Rogaeva, Ekaterina; Zinman, Lorne; Ostrow, Lyle W.; Maragakis, Nicholas J.; Rothstein, Jeffrey D.; Simmons, Zachary; Cooper-Knock, Johnathan; Brice, Alexis; Goutman, Stephen A.; Feldman, Eva L.; Gibson, Summer B.; Taroni, Franco; Ratti, Antonia; Ratti, Antonia; Gellera, Cinzia; Van Damme, Philip; Robberecht, Wim; Fratta, Pietro; Sabatelli, Mario; Lunetta, Christian; Ludolph, Albert C.; Andersen, Peter M.; Weishaupt, Jochen H.; Camu, William; Trojanowski, John Q.; Van Deerlin, Vivianna M.; Brown, Robert H.; van den Berg, Leonard; Veldink, Jan H.; Harms, Matthew B.; Glass, Jonathan D.; Stone, David J.; Tienari, Pentti; Silani, Vincenzo; Silani, Vincenzo; Chiò, Adriano; Shaw, Christopher E.; Chiò, Adriano; Traynor, Bryan J.; Landers, John E.; Traynor, Bryan J.

    2018-01-01

    To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494

  13. The RAB2B-GARIL5 Complex Promotes Cytosolic DNA-Induced Innate Immune Responses.

    Science.gov (United States)

    Takahama, Michihiro; Fukuda, Mitsunori; Ohbayashi, Norihiko; Kozaki, Tatsuya; Misawa, Takuma; Okamoto, Toru; Matsuura, Yoshiharu; Akira, Shizuo; Saitoh, Tatsuya

    2017-09-19

    Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces the IFN antiviral response. However, the regulatory mechanisms that mediate cGAS-triggered signaling have not been fully explored. Here, we show the involvement of a small GTPase, RAB2B, and its effector protein, Golgi-associated RAB2B interactor-like 5 (GARIL5), in the cGAS-mediated IFN response. RAB2B-deficiency affects the IFN response induced by cytosolic DNA. Consistent with this, RAB2B deficiency enhances replication of vaccinia virus, a DNA virus. After DNA stimulation, RAB2B colocalizes with stimulator of interferon genes (STING), the downstream signal mediator of cGAS, on the Golgi apparatus. The GTP-binding activity of RAB2B is required for its localization on the Golgi apparatus and for recruitment of GARIL5. GARIL5 deficiency also affects the IFN response induced by cytosolic DNA and enhances replication of vaccinia virus. These findings indicate that the RAB2B-GARIL5 complex promotes IFN responses against DNA viruses by regulating the cGAS-STING signaling axis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  14. The RAB2B-GARIL5 Complex Promotes Cytosolic DNA-Induced Innate Immune Responses

    Directory of Open Access Journals (Sweden)

    Michihiro Takahama

    2017-09-01

    Full Text Available Cyclic GMP-AMP synthase (cGAS is a cytosolic DNA sensor that induces the IFN antiviral response. However, the regulatory mechanisms that mediate cGAS-triggered signaling have not been fully explored. Here, we show the involvement of a small GTPase, RAB2B, and its effector protein, Golgi-associated RAB2B interactor-like 5 (GARIL5, in the cGAS-mediated IFN response. RAB2B-deficiency affects the IFN response induced by cytosolic DNA. Consistent with this, RAB2B deficiency enhances replication of vaccinia virus, a DNA virus. After DNA stimulation, RAB2B colocalizes with stimulator of interferon genes (STING, the downstream signal mediator of cGAS, on the Golgi apparatus. The GTP-binding activity of RAB2B is required for its localization on the Golgi apparatus and for recruitment of GARIL5. GARIL5 deficiency also affects the IFN response induced by cytosolic DNA and enhances replication of vaccinia virus. These findings indicate that the RAB2B-GARIL5 complex promotes IFN responses against DNA viruses by regulating the cGAS-STING signaling axis.

  15. CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo.

    Directory of Open Access Journals (Sweden)

    Nadia T Sebastian

    2017-07-01

    Full Text Available Latent HIV infection of long-lived cells is a barrier to viral clearance. Hematopoietic stem and progenitor cells are a heterogeneous population of cells, some of which are long-lived. CXCR4-tropic HIVs infect a broad range of HSPC subtypes, including hematopoietic stem cells, which are multi-potent and long-lived. However, CCR5-tropic HIV infection is limited to more differentiated progenitor cells with life spans that are less well understood. Consistent with emerging data that restricted progenitor cells can be long-lived, we detected persistent HIV in restricted HSPC populations from optimally treated people. Further, genotypic and phenotypic analysis of amplified env alleles from donor samples indicated that both CXCR4- and CCR5-tropic viruses persisted in HSPCs. RNA profiling confirmed expression of HIV receptor RNA in a pattern that was consistent with in vitro and in vivo results. In addition, we characterized a CD4high HSPC sub-population that was preferentially targeted by a variety of CXCR4- and CCR5-tropic HIVs in vitro. Finally, we present strong evidence that HIV proviral genomes of both tropisms can be transmitted to CD4-negative daughter cells of multiple lineages in vivo. In some cases, the transmitted proviral genomes contained signature deletions that inactivated the virus, eliminating the possibility that coincidental infection explains the results. These data support a model in which both stem and non-stem cell progenitors serve as persistent reservoirs for CXCR4- and CCR5-tropic HIV proviral genomes that can be passed to daughter cells.

  16. CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo.

    Science.gov (United States)

    Sebastian, Nadia T; Zaikos, Thomas D; Terry, Valeri; Taschuk, Frances; McNamara, Lucy A; Onafuwa-Nuga, Adewunmi; Yucha, Ryan; Signer, Robert A J; Riddell, James; Bixby, Dale; Markowitz, Norman; Morrison, Sean J; Collins, Kathleen L

    2017-07-01

    Latent HIV infection of long-lived cells is a barrier to viral clearance. Hematopoietic stem and progenitor cells are a heterogeneous population of cells, some of which are long-lived. CXCR4-tropic HIVs infect a broad range of HSPC subtypes, including hematopoietic stem cells, which are multi-potent and long-lived. However, CCR5-tropic HIV infection is limited to more differentiated progenitor cells with life spans that are less well understood. Consistent with emerging data that restricted progenitor cells can be long-lived, we detected persistent HIV in restricted HSPC populations from optimally treated people. Further, genotypic and phenotypic analysis of amplified env alleles from donor samples indicated that both CXCR4- and CCR5-tropic viruses persisted in HSPCs. RNA profiling confirmed expression of HIV receptor RNA in a pattern that was consistent with in vitro and in vivo results. In addition, we characterized a CD4high HSPC sub-population that was preferentially targeted by a variety of CXCR4- and CCR5-tropic HIVs in vitro. Finally, we present strong evidence that HIV proviral genomes of both tropisms can be transmitted to CD4-negative daughter cells of multiple lineages in vivo. In some cases, the transmitted proviral genomes contained signature deletions that inactivated the virus, eliminating the possibility that coincidental infection explains the results. These data support a model in which both stem and non-stem cell progenitors serve as persistent reservoirs for CXCR4- and CCR5-tropic HIV proviral genomes that can be passed to daughter cells.

  17. Hydrogen Peroxide Toxicity Induces Ras Signaling in Human Neuroblastoma SH-SY5Y Cultured Cells

    Directory of Open Access Journals (Sweden)

    Jirapa Chetsawang

    2010-01-01

    Full Text Available It has been reported that overproduction of reactive oxygen species occurs after brain injury and mediates neuronal cells degeneration. In the present study, we examined the role of Ras signaling on hydrogen peroxide-induced neuronal cells degeneration in dopaminergic neuroblastoma SH-SY5Y cells. Hydrogen peroxide significantly reduced cell viability in SH-SY5Y cultured cells. An inhibitor of the enzyme that catalyzes the farnesylation of Ras proteins, FTI-277, and a competitive inhibitor of GTP-binding proteins, GDP-beta-S significantly decreased hydrogen peroxide-induced reduction in cell viability in SH-SY5Y cultured cells. The results of this study might indicate that a Ras-dependent signaling pathway plays a role in hydrogen peroxide-induced toxicity in neuronal cells.

  18. The First Complete Chloroplast Genome Sequences in Actinidiaceae: Genome Structure and Comparative Analysis.

    Science.gov (United States)

    Yao, Xiaohong; Tang, Ping; Li, Zuozhou; Li, Dawei; Liu, Yifei; Huang, Hongwen

    2015-01-01

    Actinidia chinensis is an important economic plant belonging to the basal lineage of the asterids. Availability of a complete Actinidia chloroplast genome sequence is crucial to understanding phylogenetic relationships among major lineages of angiosperms and facilitates kiwifruit genetic improvement. We report here the complete nucleotide sequences of the chloroplast genomes for Actinidia chinensis and A. chinensis var deliciosa obtained through de novo assembly of Illumina paired-end reads produced by total DNA sequencing. The total genome size ranges from 155,446 to 157,557 bp, with an inverted repeat (IR) of 24,013 to 24,391 bp, a large single copy region (LSC) of 87,984 to 88,337 bp and a small single copy region (SSC) of 20,332 to 20,336 bp. The genome encodes 113 different genes, including 79 unique protein-coding genes, 30 tRNA genes and 4 ribosomal RNA genes, with 16 duplicated in the inverted repeats, and a tRNA gene (trnfM-CAU) duplicated once in the LSC region. Comparisons of IR boundaries among four asterid species showed that IR/LSC borders were extended into the 5' portion of the psbA gene and IR contraction occurred in Actinidia. The clap gene has been lost from the chloroplast genome in Actinidia, and may have been transferred to the nucleus during chloroplast evolution. Twenty-seven polymorphic simple sequence repeat (SSR) loci were identified in the Actinidia chloroplast genome. Maximum parsimony analyses of a 72-gene, 16 taxa angiosperm dataset strongly support the placement of Actinidiaceae in Ericales within the basal asterids.

  19. Genome-wide analysis of mutations in mutant lineages selected following fast-neutron irradiation mutagenesis of Arabidopsis thaliana

    KAUST Repository

    Belfield, E.J.; Gan, X.; Mithani, A.; Brown, C.; Jiang, C.; Franklin, K.; Alvey, E.; Wibowo, A.; Jung, M.; Bailey, K.; Kalwani, S.; Ragoussis, J.; Mott, R.; Harberd, N.P.

    2012-01-01

    Ionizing radiation has long been known to induce heritable mutagenic change in DNA sequence. However, the genome-wide effect of radiation is not well understood. Here we report the molecular properties and frequency of mutations in phenotypically selected mutant lines isolated following exposure of the genetic model flowering plant Arabidopsis thaliana to fast neutrons (FNs). Previous studies suggested that FNs predominantly induce deletions longer than a kilobase in A. thaliana. However, we found a higher frequency of single base substitution than deletion mutations. While the overall frequency and molecular spectrum of fast-neutron (FN)-induced single base substitutions differed substantially from those of "background" mutations arising spontaneously in laboratory-grown plants, G:C>A:T transitions were favored in both. We found that FN-induced G:C>A:T transitions were concentrated at pyrimidine dinucleotide sites, suggesting that FNs promote the formation of mutational covalent linkages between adjacent pyrimidine residues. In addition, we found that FNs induced more single base than large deletions, and that these single base deletions were possibly caused by replication slippage. Our observations provide an initial picture of the genome-wide molecular profile of mutations induced in A. thaliana by FN irradiation and are particularly informative of the nature and extent of genome-wide mutation in lines selected on the basis of mutant phenotypes from FN-mutagenized A. thaliana populations.

  20. Genome-wide analysis of mutations in mutant lineages selected following fast-neutron irradiation mutagenesis of Arabidopsis thaliana

    KAUST Repository

    Belfield, E.J.

    2012-04-12

    Ionizing radiation has long been known to induce heritable mutagenic change in DNA sequence. However, the genome-wide effect of radiation is not well understood. Here we report the molecular properties and frequency of mutations in phenotypically selected mutant lines isolated following exposure of the genetic model flowering plant Arabidopsis thaliana to fast neutrons (FNs). Previous studies suggested that FNs predominantly induce deletions longer than a kilobase in A. thaliana. However, we found a higher frequency of single base substitution than deletion mutations. While the overall frequency and molecular spectrum of fast-neutron (FN)-induced single base substitutions differed substantially from those of "background" mutations arising spontaneously in laboratory-grown plants, G:C>A:T transitions were favored in both. We found that FN-induced G:C>A:T transitions were concentrated at pyrimidine dinucleotide sites, suggesting that FNs promote the formation of mutational covalent linkages between adjacent pyrimidine residues. In addition, we found that FNs induced more single base than large deletions, and that these single base deletions were possibly caused by replication slippage. Our observations provide an initial picture of the genome-wide molecular profile of mutations induced in A. thaliana by FN irradiation and are particularly informative of the nature and extent of genome-wide mutation in lines selected on the basis of mutant phenotypes from FN-mutagenized A. thaliana populations.

  1. The Sequenced Angiosperm Genomes and Genome Databases.

    Science.gov (United States)

    Chen, Fei; Dong, Wei; Zhang, Jiawei; Guo, Xinyue; Chen, Junhao; Wang, Zhengjia; Lin, Zhenguo; Tang, Haibao; Zhang, Liangsheng

    2018-01-01

    Angiosperms, the flowering plants, provide the essential resources for human life, such as food, energy, oxygen, and materials. They also promoted the evolution of human, animals, and the planet earth. Despite the numerous advances in genome reports or sequencing technologies, no review covers all the released angiosperm genomes and the genome databases for data sharing. Based on the rapid advances and innovations in the database reconstruction in the last few years, here we provide a comprehensive review for three major types of angiosperm genome databases, including databases for a single species, for a specific angiosperm clade, and for multiple angiosperm species. The scope, tools, and data of each type of databases and their features are concisely discussed. The genome databases for a single species or a clade of species are especially popular for specific group of researchers, while a timely-updated comprehensive database is more powerful for address of major scientific mysteries at the genome scale. Considering the low coverage of flowering plants in any available database, we propose construction of a comprehensive database to facilitate large-scale comparative studies of angiosperm genomes and to promote the collaborative studies of important questions in plant biology.

  2. The genome and transcriptome of Phalaenopsis yield insights into floral organ development and flowering regulation

    Directory of Open Access Journals (Sweden)

    Jian-Zhi Huang

    2016-05-01

    Full Text Available The Phalaenopsis orchid is an important potted flower of high economic value around the world. We report the 3.1 Gb draft genome assembly of an important winter flowering Phalaenopsis ‘KHM190’ cultivar. We generated 89.5 Gb RNA-seq and 113 million sRNA-seq reads to use these data to identify 41,153 protein-coding genes and 188 miRNA families. We also generated a draft genome for Phalaenopsis pulcherrima ‘B8802,’ a summer flowering species, via resequencing. Comparison of genome data between the two Phalaenopsis cultivars allowed the identification of 691,532 single-nucleotide polymorphisms. In this study, we reveal that the key role of PhAGL6b in the regulation of labellum organ development involves alternative splicing in the big lip mutant. Petal or sepal overexpressing PhAGL6b leads to the conversion into a lip-like structure. We also discovered that the gibberellin pathway that regulates the expression of flowering time genes during the reproductive phase change is induced by cool temperature. Our work thus depicted a valuable resource for the flowering control, flower architecture development, and breeding of the Phalaenopsis orchids.

  3. Radiation-induced genomic instability and bystander effects: inter-related inflammatory-type non-targeted effects of exposure to ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Wright, E.G. (Molecular and Cellular Pathology Laboratories, Division of Pathology and Neuroscience, Ninewells Hospital and Medical School, Dundee, Scotland (United Kingdom))

    2008-12-15

    The dogma that genetic alterations are restricted to directly irradiated cells has been challenged by observations in which effects of ionizing radiation, characteristically associated with the consequences of energy deposition in the cell nucleus, arise in non-irradiated cells. These, so called, untargeted effects are demonstrated in cells that are the descendants of irradiated cells (radiation-induced genomic instability) or in cells that have communicated with neighbouring irradiated cells (radiation-induced bystander effects). There are also reports of long-range signals in vivo, known as clastogenic factors, with the capacity to induce damage in unirradiated cells. Clastogenic factors may be related to the inflammatory responses that have been implicated in some of the pathological consequences of radiation exposures. The phenotypic expression of untargeted effects reflects a balance between the type of signals produced and the responses of cell populations to such signals, both of which may be significantly influenced by cell type and genotype. There is accumulating evidence that untargeted effects in vitro involve inter-cellular signalling, production of cytokines and free radical generation. These are also features of inflammatory responses in vivo that are known to have the potential for both bystander-mediated and persisting damage as well as for conferring a predisposition to malignancy. At present it is far from clear how untargeted effects contribute to overall cellular radiation responses and in vivo consequences but it is possible that the various untargeted effects may reflect inter-related aspects of a non-specific inflammatory-type response to radiation-induced stress and injury and be involved in a variety of the pathological consequences of radiation exposures. (orig.)

  4. Radiation-induced genomic instability and bystander effects: inter-related inflammatory-type non-targeted effects of exposure to ionizing radiation

    International Nuclear Information System (INIS)

    Wright, E.G.

    2008-01-01

    The dogma that genetic alterations are restricted to directly irradiated cells has been challenged by observations in which effects of ionizing radiation, characteristically associated with the consequences of energy deposition in the cell nucleus, arise in non-irradiated cells. These, so called, untargeted effects are demonstrated in cells that are the descendants of irradiated cells (radiation-induced genomic instability) or in cells that have communicated with neighbouring irradiated cells (radiation-induced bystander effects). There are also reports of long-range signals in vivo, known as clastogenic factors, with the capacity to induce damage in unirradiated cells. Clastogenic factors may be related to the inflammatory responses that have been implicated in some of the pathological consequences of radiation exposures. The phenotypic expression of untargeted effects reflects a balance between the type of signals produced and the responses of cell populations to such signals, both of which may be significantly influenced by cell type and genotype. There is accumulating evidence that untargeted effects in vitro involve inter-cellular signalling, production of cytokines and free radical generation. These are also features of inflammatory responses in vivo that are known to have the potential for both bystander-mediated and persisting damage as well as for conferring a predisposition to malignancy. At present it is far from clear how untargeted effects contribute to overall cellular radiation responses and in vivo consequences but it is possible that the various untargeted effects may reflect inter-related aspects of a non-specific inflammatory-type response to radiation-induced stress and injury and be involved in a variety of the pathological consequences of radiation exposures. (orig.)

  5. Genome Analysis of Staphylococcus agnetis, an Agent of Lameness in Broiler Chickens.

    Science.gov (United States)

    Al-Rubaye, Adnan A K; Couger, M Brian; Ojha, Sohita; Pummill, Jeff F; Koon, Joseph A; Wideman, Robert F; Rhoads, Douglas D

    2015-01-01

    Lameness in broiler chickens is a significant animal welfare and financial issue. Lameness can be enhanced by rearing young broilers on wire flooring. We have identified Staphylococcus agnetis as significantly involved in bacterial chondronecrosis with osteomyelitis (BCO) in proximal tibia and femorae, leading to lameness in broiler chickens in the wire floor system. Administration of S. agnetis in water induces lameness. Previously reported in some cases of cattle mastitis, this is the first report of this poorly described pathogen in chickens. We used long and short read next generation sequencing to assemble single finished contigs for the genome and a large plasmid from the chicken pathogen. Comparison of the S. agnetis genome to those of other pathogenic Staphylococci shows that S.agnetis contains a distinct repertoire of virulence determinants. Additionally, the S. agnetis genome has several regions that differ substantially from the genomes of other pathogenic Staphylococci. Comparison of our finished genome to a recent draft genome for a cattle mastitis isolate suggests that future investigations focus on the evolutionary epidemiology of this emerging pathogen of domestic animals.

  6. Genome Analysis of Staphylococcus agnetis, an Agent of Lameness in Broiler Chickens.

    Directory of Open Access Journals (Sweden)

    Adnan A K Al-Rubaye

    Full Text Available Lameness in broiler chickens is a significant animal welfare and financial issue. Lameness can be enhanced by rearing young broilers on wire flooring. We have identified Staphylococcus agnetis as significantly involved in bacterial chondronecrosis with osteomyelitis (BCO in proximal tibia and femorae, leading to lameness in broiler chickens in the wire floor system. Administration of S. agnetis in water induces lameness. Previously reported in some cases of cattle mastitis, this is the first report of this poorly described pathogen in chickens. We used long and short read next generation sequencing to assemble single finished contigs for the genome and a large plasmid from the chicken pathogen. Comparison of the S. agnetis genome to those of other pathogenic Staphylococci shows that S.agnetis contains a distinct repertoire of virulence determinants. Additionally, the S. agnetis genome has several regions that differ substantially from the genomes of other pathogenic Staphylococci. Comparison of our finished genome to a recent draft genome for a cattle mastitis isolate suggests that future investigations focus on the evolutionary epidemiology of this emerging pathogen of domestic animals.

  7. Big Data Analysis of Human Genome Variations

    KAUST Repository

    Gojobori, Takashi

    2016-01-25

    Since the human genome draft sequence was in public for the first time in 2000, genomic analyses have been intensively extended to the population level. The following three international projects are good examples for large-scale studies of human genome variations: 1) HapMap Data (1,417 individuals) (http://hapmap.ncbi.nlm.nih.gov/downloads/genotypes/2010-08_phaseII+III/forward/), 2) HGDP (Human Genome Diversity Project) Data (940 individuals) (http://www.hagsc.org/hgdp/files.html), 3) 1000 genomes Data (2,504 individuals) http://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/ If we can integrate all three data into a single volume of data, we should be able to conduct a more detailed analysis of human genome variations for a total number of 4,861 individuals (= 1,417+940+2,504 individuals). In fact, we successfully integrated these three data sets by use of information on the reference human genome sequence, and we conducted the big data analysis. In particular, we constructed a phylogenetic tree of about 5,000 human individuals at the genome level. As a result, we were able to identify clusters of ethnic groups, with detectable admixture, that were not possible by an analysis of each of the three data sets. Here, we report the outcome of this kind of big data analyses and discuss evolutionary significance of human genomic variations. Note that the present study was conducted in collaboration with Katsuhiko Mineta and Kosuke Goto at KAUST.

  8. GenColors-based comparative genome databases for small eukaryotic genomes.

    Science.gov (United States)

    Felder, Marius; Romualdi, Alessandro; Petzold, Andreas; Platzer, Matthias; Sühnel, Jürgen; Glöckner, Gernot

    2013-01-01

    Many sequence data repositories can give a quick and easily accessible overview on genomes and their annotations. Less widespread is the possibility to compare related genomes with each other in a common database environment. We have previously described the GenColors database system (http://gencolors.fli-leibniz.de) and its applications to a number of bacterial genomes such as Borrelia, Legionella, Leptospira and Treponema. This system has an emphasis on genome comparison. It combines data from related genomes and provides the user with an extensive set of visualization and analysis tools. Eukaryote genomes are normally larger than prokaryote genomes and thus pose additional challenges for such a system. We have, therefore, adapted GenColors to also handle larger datasets of small eukaryotic genomes and to display eukaryotic gene structures. Further recent developments include whole genome views, genome list options and, for bacterial genome browsers, the display of horizontal gene transfer predictions. Two new GenColors-based databases for two fungal species (http://fgb.fli-leibniz.de) and for four social amoebas (http://sacgb.fli-leibniz.de) were set up. Both new resources open up a single entry point for related genomes for the amoebozoa and fungal research communities and other interested users. Comparative genomics approaches are greatly facilitated by these resources.

  9. Implementing genomics and pharmacogenomics in the clinic: The National Human Genome Research Institute's genomic medicine portfolio.

    Science.gov (United States)

    Manolio, Teri A

    2016-10-01

    Increasing knowledge about the influence of genetic variation on human health and growing availability of reliable, cost-effective genetic testing have spurred the implementation of genomic medicine in the clinic. As defined by the National Human Genome Research Institute (NHGRI), genomic medicine uses an individual's genetic information in his or her clinical care, and has begun to be applied effectively in areas such as cancer genomics, pharmacogenomics, and rare and undiagnosed diseases. In 2011 NHGRI published its strategic vision for the future of genomic research, including an ambitious research agenda to facilitate and promote the implementation of genomic medicine. To realize this agenda, NHGRI is consulting and facilitating collaborations with the external research community through a series of "Genomic Medicine Meetings," under the guidance and leadership of the National Advisory Council on Human Genome Research. These meetings have identified and begun to address significant obstacles to implementation, such as lack of evidence of efficacy, limited availability of genomics expertise and testing, lack of standards, and difficulties in integrating genomic results into electronic medical records. The six research and dissemination initiatives comprising NHGRI's genomic research portfolio are designed to speed the evaluation and incorporation, where appropriate, of genomic technologies and findings into routine clinical care. Actual adoption of successful approaches in clinical care will depend upon the willingness, interest, and energy of professional societies, practitioners, patients, and payers to promote their responsible use and share their experiences in doing so. Published by Elsevier Ireland Ltd.

  10. Molecular characterisation of the full-length genome of olive latent virus 1 isolated from tomato.

    Science.gov (United States)

    Hasiów-Jaroszewska, Beata; Borodynko, Natasza; Pospieszny, Henryk

    2011-05-01

    Olive latent virus 1 (OLV-1) is a species of the Necrovirus genus. So far, it has been reported to infect olive, citrus tree and tulip. Here, we determined and analysed the complete genomic sequence of an isolate designated as CM1, which was collected from tomato plant in the Wielkopolska region of Poland and represents the prevalent isolate of OLV-1. The CM1 genome consists of monopartite single-stranded positive-sense RNA genome sized 3,699 nt with five open reading frames (ORFs) and small inter-cistronic regions. ORF1 encodes a polypeptide with a molecular weight of 23 kDa and the read-through (RT) of its amber stop codon results in ORF1 RT that encodes the virus RNA-dependent RNA polymerase. ORF2 and ORF3 encode two peptides, with 8 kDa and 6 kDa, respectively, which appear to be involved in cell-to-cell movement. ORF4 is located in the 3' terminal and encodes a protein with 30 kDa identified as the viral coat protein (CP). The differences in CP region of four OLV-1 isolates whose sequences have been deposited in GenBank were observed. Nucleotide sequence identities of the CP of tomato CM1 isolate with those of olive, citrus and tulip isolates were 91.8%, 89.5% and 92.5%, respectively. In contrast to other OLV-1 isolates, CM1 induced necrotic spots on tomato plants and elicited necrotic local lesions on Nicotiana benthamiana, followed by systemic infection. This is the third complete genomic sequence of OLV-1 reported and the first one from tomato.

  11. Coconut genome size determined by flow cytometry: Tall versus Dwarf types.

    Science.gov (United States)

    Freitas Neto, M; Pereira, T N S; Geronimo, I G C; Azevedo, A O N; Ramos, S R R; Pereira, M G

    2016-02-11

    Coconuts (Cocos nucifera L.) are tropical palm trees that are classified into Tall and Dwarf types based on height, and both types are diploid (2n = 2x = 32 chromosomes). The reproduction mode is autogamous for Dwarf types and allogamous for Tall types. One hypothesis for the origin of the Dwarf coconut suggests that it is a Tall variant that resulted from either mutation or inbreeding, and differences in genome size between the two types would support this hypothesis. In this study, we estimated the genome sizes of 14 coconut accessions (eight Tall and six Dwarf types) using flow cytometry. Nuclei were extracted from leaf discs and stained with propidium iodide, and Pisum sativum (2C = 9.07 pg DNA) was used as an internal standard. Histograms with good resolution and low coefficients of variation (2.5 to 3.2%) were obtained. The 2C DNA content ranged from 5.72 to 5.48 pg for Tall accessions and from 5.58 to 5.52 pg for Dwarf accessions. The mean genome sizes for Tall and Dwarf specimens were 5.59 and 5.55 pg, respectively. Among all accessions, Rennel Island Tall had the highest mean DNA content (5.72 pg), whereas West African Tall had the lowest (5.48 pg). The mean coconut genome size (2C = 5.57 pg, corresponding to 2723.73 Mbp/haploid set) was classified as small. Only small differences in genome size existed among the coconut accessions, suggesting that the Dwarf type did not evolve from the Tall type.

  12. Comparative Genomics Reveals High Genomic Diversity in the Genus Photobacterium.

    Science.gov (United States)

    Machado, Henrique; Gram, Lone

    2017-01-01

    Vibrionaceae is a large marine bacterial family, which can constitute up to 50% of the prokaryotic population in marine waters. Photobacterium is the second largest genus in the family and we used comparative genomics on 35 strains representing 16 of the 28 species described so far, to understand the genomic diversity present in the Photobacterium genus. Such understanding is important for ecophysiology studies of the genus. We used whole genome sequences to evaluate phylogenetic relationships using several analyses (16S rRNA, MLSA, fur , amino-acid usage, ANI), which allowed us to identify two misidentified strains. Genome analyses also revealed occurrence of higher and lower GC content clades, correlating with phylogenetic clusters. Pan- and core-genome analysis revealed the conservation of 25% of the genome throughout the genus, with a large and open pan-genome. The major source of genomic diversity could be traced to the smaller chromosome and plasmids. Several of the physiological traits studied in the genus did not correlate with phylogenetic data. Since horizontal gene transfer (HGT) is often suggested as a source of genetic diversity and a potential driver of genomic evolution in bacterial species, we looked into evidence of such in Photobacterium genomes. Genomic islands were the source of genomic differences between strains of the same species. Also, we found transposase genes and CRISPR arrays that suggest multiple encounters with foreign DNA. Presence of genomic exchange traits was widespread and abundant in the genus, suggesting a role in genomic evolution. The high genetic variability and indications of genetic exchange make it difficult to elucidate genome evolutionary paths and raise the awareness of the roles of foreign DNA in the genomic evolution of environmental organisms.

  13. Rodent malaria parasites : genome organization & comparative genomics

    NARCIS (Netherlands)

    Kooij, Taco W.A.

    2006-01-01

    The aim of the studies described in this thesis was to investigate the genome organization of rodent malaria parasites (RMPs) and compare the organization and gene content of the genomes of RMPs and the human malaria parasite P. falciparum. The release of the complete genome sequence of P.

  14. Radiation-induced genomic instability driven by de novo chromosomal rearrangement hot spots

    International Nuclear Information System (INIS)

    Grosovsky, A.J.; Allen, R.N.; Moore, S.R.

    2003-01-01

    Genomic instability has become generally recognized as a critical contributor to tumor progression by generating the necessary number of genetic alterations required for expression of a clinically significant malignancy. Our study of chromosomal instability investigates the hypothesis that chromosomal rearrangements can generate novel breakage-prone sites, resulting in instability acting predominantly in cis. Here we present an analysis of the karyotypic distribution of instability associated chromosomal rearrangements in TK6 and derivative human lymphoblasts. Karyotypic analysis performed on a total of 455 independent clones included 183 rearrangements distributed among 100 separate unstable clones. The results demonstrate that the breakpoints of chromosomal rearrangements in unstable clones are non-randomly distributed throughout the genome. This pattern is statistically significant, and incompatible with expectations for random breakage associated with loss or alteration of a trans-acting factor. Furthermore, specific chromosomal breakage hot spots associated with instability have been identified; these occur in several independent unstable clones and are often repeatedly broken and rejoined during the outgrowth of an individual clone. In complimentary studies, genomic instability was generated without any exposure to a DNA-damaging agent, but rather by transfection with alpha heterochromatin DNA. In a prospective analysis, human-hamster hybrid AL cells containing a single human chromosome 11 were transfected with heterochromatic alpha DNA repeats and clones were analyzed by chromosome 11 painting. Transfection with alpha DNA was associated with karyotypic heterogeneity in 40% of clones examined; control transfections with plasmid alone did not lead to karyotypic heterogeneity

  15. Biosynthesis of ribose-5-phosphate and erythrose-4-phosphate in archaea: a phylogenetic analysis of archaeal genomes

    Directory of Open Access Journals (Sweden)

    Tim Soderberg

    2005-01-01

    Full Text Available A phylogenetic analysis of the genes encoding enzymes in the pentose phosphate pathway (PPP, the ribulose monophosphate (RuMP pathway, and the chorismate pathway of aromatic amino acid biosynthesis, employing data from 13 complete archaeal genomes, provides a potential explanation for the enigmatic phylogenetic patterns of the PPP genes in archaea. Genomic and biochemical evidence suggests that three archaeal species (Methanocaldococcus jannaschii, Thermoplasma acidophilum and Thermoplasma volcanium produce ribose-5-phosphate via the nonoxidative PPP (NOPPP, whereas nine species apparently lack an NOPPP but may employ a reverse RuMP pathway for pentose synthesis. One species (Halobacterium sp. NRC-1 lacks both the NOPPP and the RuMP pathway but may possess a modified oxidative PPP (OPPP, the details of which are not yet known. The presence of transketolase in several archaeal species that are missing the other two NOPPP genes can be explained by the existence of differing requirements for erythrose-4-phosphate (E4P among archaea: six species use transketolase to make E4P as a precursor to aromatic amino acids, six species apparently have an alternate biosynthetic pathway and may not require the ability to make E4P, and one species (Pyrococcus horikoshii probably does not synthesize aromatic amino acids at all.

  16. A photoactivatable Cre-loxP recombination system for optogenetic genome engineering.

    Science.gov (United States)

    Kawano, Fuun; Okazaki, Risako; Yazawa, Masayuki; Sato, Moritoshi

    2016-12-01

    Genome engineering techniques represented by the Cre-loxP recombination system have been used extensively for biomedical research. However, powerful and useful techniques for genome engineering that have high spatiotemporal precision remain elusive. Here we develop a highly efficient photoactivatable Cre recombinase (PA-Cre) to optogenetically control genome engineering in vivo. PA-Cre is based on the reassembly of split Cre fragments by light-inducible dimerization of the Magnet system. PA-Cre enables sharp induction (up to 320-fold) of DNA recombination and is efficiently activated even by low-intensity illumination (∼0.04 W m -2 ) or short periods of pulsed illumination (∼30 s). We demonstrate that PA-Cre allows for efficient DNA recombination in an internal organ of living mice through noninvasive external illumination using a LED light source. The present PA-Cre provides a powerful tool to greatly facilitate optogenetic genome engineering in vivo.

  17. miR-339-5p inhibits alcohol-induced brain inflammation through regulating NF-κB pathway

    International Nuclear Information System (INIS)

    Zhang, Yu; Wei, Guangkuan; Di, Zhiyong; Zhao, Qingjie

    2014-01-01

    Graphical abstract: - Highlights: • Alcohol upregulates miR-339-5p expression. • miR-339-5p inhibits the NF-kB pathway. • miR-339-5p interacts with and blocks activity of IKK-beat and IKK-epsilon. • miR-339-5p modulates IL-1β, IL-6 and TNF-α. - Abstract: Alcohol-induced neuroinflammation is mediated by the innate immunesystem. Pro-inflammatory responses to alcohol are modulated by miRNAs. The miRNA miR-339-5p has previously been found to be upregulated in alcohol-induced neuroinflammation. However, little has been elucidated on the regulatory functions of this miRNA in alcohol-induced neuroinflammation. We investigated the function of miR-339-5p in alcohol exposed brain tissue and isolated microglial cells using ex vivo and in vitro techniques. Our results show that alcohol induces transcription of miR 339-5p, IL-6, IL-1β and TNF-α in mouse brain tissue and isolated microglial cells by activating NF-κB. Alcohol activation of NF-κB allows for nuclear translocation of the NF-κB subunit p65 and expression of pro-inflammatory mediators. miR-339-5p inhibited expression of these pro-inflammatory factors through the NF-κB pathway by abolishing IKK-β and IKK-ε activity

  18. miR-339-5p inhibits alcohol-induced brain inflammation through regulating NF-κB pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yu [Department of Neurology, The First Affiliated School of Harbin Medical University, Harbin 150001 (China); Wei, Guangkuan [Department of Neurology, Heilongjiang Provincial Hospital, Harbin 150036 (China); Di, Zhiyong [Department of Laboratory, Heilongjiang Provincial Hospital, Harbin 150036 (China); Zhao, Qingjie, E-mail: zhaoqingjie2013@163.com [Department of Neurology, The First Affiliated School of Harbin Medical University, Harbin 150001 (China)

    2014-09-26

    Graphical abstract: - Highlights: • Alcohol upregulates miR-339-5p expression. • miR-339-5p inhibits the NF-kB pathway. • miR-339-5p interacts with and blocks activity of IKK-beat and IKK-epsilon. • miR-339-5p modulates IL-1β, IL-6 and TNF-α. - Abstract: Alcohol-induced neuroinflammation is mediated by the innate immunesystem. Pro-inflammatory responses to alcohol are modulated by miRNAs. The miRNA miR-339-5p has previously been found to be upregulated in alcohol-induced neuroinflammation. However, little has been elucidated on the regulatory functions of this miRNA in alcohol-induced neuroinflammation. We investigated the function of miR-339-5p in alcohol exposed brain tissue and isolated microglial cells using ex vivo and in vitro techniques. Our results show that alcohol induces transcription of miR 339-5p, IL-6, IL-1β and TNF-α in mouse brain tissue and isolated microglial cells by activating NF-κB. Alcohol activation of NF-κB allows for nuclear translocation of the NF-κB subunit p65 and expression of pro-inflammatory mediators. miR-339-5p inhibited expression of these pro-inflammatory factors through the NF-κB pathway by abolishing IKK-β and IKK-ε activity.

  19. Folic acid functionalized surface highlights 5-methylcytosine-genomic content within circulating tumor cells

    KAUST Repository

    Malara, Natalia; Coluccio, Maria Laura; Limongi, Tania; Asande, Monica; Trunzo, Valentina; Cojoc, Gheorghe; Raso, Cinzia; Candeloro, Patrizio; Perozziello, Gerardo; Raimondo, Raffaella; De Vitis, Stefania; Roveda, Laura; Renne, Maria; Prati, Ubaldo; Mollace, Vincenzo; Di Fabrizio, Enzo M.

    2014-01-01

    Although the detection of methylated cell free DNA represents one of the most promising approaches for relapse risk assessment in cancer patients, the low concentration of cell-free circulating DNA constitutes the biggest obstacle in the development of DNA methylation-based biomarkers from blood. This paper describes a method for the measurement of genomic methylation content directly on circulating tumor cells (CTC), which could be used to deceive the aforementioned problem. Since CTC are disease related blood-based biomarkers, they result essential to monitor tumor's stadiation, therapy, and early relapsing lesions. Within surface's bio-functionalization and cell's isolation procedure standardization, the presented approach reveals a singular ability to detect high 5-methylcytosine CTC-subset content in the whole CTC compound, by choosing folic acid (FA) as transducer molecule. Sensitivity and specificity, calculated for FA functionalized surface (FA-surface), result respectively on about 83% and 60%. FA-surface, allowing the detection and characterization of early metastatic dissemination, provides a unique advance in the comprehension of tumors progression and dissemination confirming the presence of CTC and its association with high risk of relapse. This functionalized surface identifying and quantifying high 5-methylcytosine CTC-subset content into the patient's blood lead significant progress in cancer risk assessment, also providing a novel therapeutic strategy.© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Folic acid functionalized surface highlights 5-methylcytosine-genomic content within circulating tumor cells

    KAUST Repository

    Malara, Natalia

    2014-07-01

    Although the detection of methylated cell free DNA represents one of the most promising approaches for relapse risk assessment in cancer patients, the low concentration of cell-free circulating DNA constitutes the biggest obstacle in the development of DNA methylation-based biomarkers from blood. This paper describes a method for the measurement of genomic methylation content directly on circulating tumor cells (CTC), which could be used to deceive the aforementioned problem. Since CTC are disease related blood-based biomarkers, they result essential to monitor tumor\\'s stadiation, therapy, and early relapsing lesions. Within surface\\'s bio-functionalization and cell\\'s isolation procedure standardization, the presented approach reveals a singular ability to detect high 5-methylcytosine CTC-subset content in the whole CTC compound, by choosing folic acid (FA) as transducer molecule. Sensitivity and specificity, calculated for FA functionalized surface (FA-surface), result respectively on about 83% and 60%. FA-surface, allowing the detection and characterization of early metastatic dissemination, provides a unique advance in the comprehension of tumors progression and dissemination confirming the presence of CTC and its association with high risk of relapse. This functionalized surface identifying and quantifying high 5-methylcytosine CTC-subset content into the patient\\'s blood lead significant progress in cancer risk assessment, also providing a novel therapeutic strategy.© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.