Identification of apoptosis-related PLZF target genes

International Nuclear Information System (INIS)

Bernardo, Maria Victoria; Yelo, Estefania; Gimeno, Lourdes; Campillo, Jose Antonio; Parrado, Antonio

2007-01-01

The PLZF gene encodes a BTB/POZ-zinc finger-type transcription factor, involved in physiological development, proliferation, differentiation, and apoptosis. In this paper, we investigate proliferation, survival, and gene expression regulation in stable clones from the human haematopoietic K562, DG75, and Jurkat cell lines with inducible expression of PLZF. In Jurkat cells, but not in K562 and DG75 cells, PLZF induced growth suppression and apoptosis in a cell density-dependent manner. Deletion of the BTB/POZ domain of PLZF abrogated growth suppression and apoptosis. PLZF was expressed with a nuclear speckled pattern distinctively in the full-length PLZF-expressing Jurkat clones, suggesting that the nuclear speckled localization is required for PLZF-induced apoptosis. By microarray analysis, we identified that the apoptosis-inducer TP53INP1, ID1, and ID3 genes were upregulated, and the apoptosis-inhibitor TERT gene was downregulated. The identification of apoptosis-related PLZF target genes may have biological and clinical relevance in cancer typified by altered PLZF expression

Genes Involved in Human Ribosome Biogenesis areTranscriptionally Upregulated in Colorectal Cancer

DEFF Research Database (Denmark)

Mansilla, Francisco; Lamy, Philippe; Ørntoft, Torben Falck

2009-01-01

Microarray gene expression profiling comprising 168 colorectal adenocarcinomas and 10 normal mucosas showed that over 79% of the genes involved in human ribosome biogenesis are significantly upregulated (log2>0.5, p<10-3) when compared to normal mucosa. Overexpression was independent of microsate......Microarray gene expression profiling comprising 168 colorectal adenocarcinomas and 10 normal mucosas showed that over 79% of the genes involved in human ribosome biogenesis are significantly upregulated (log2>0.5, p... of microsatellite status. The promoters of the genes studied showed a significant enrichment for several transcription factor binding sites. There was a significant correlation between the number of binding site targets for these transcription factors and the observed gene transcript upregulation. The upregulation...

Targeting the human lysozyme gene on bovine αs1- casein gene ...

African Journals Online (AJOL)

Targeting an exogenous gene into a favorable gene locus and for expression under endogenous regulators is an ideal method in mammary gland bioreactor research. For this purpose, a gene targeting vector was constructed to targeting the human lysozyme gene on bovine αs1-casein gene locus. In this case, the ...

Identification and characterization of nuclear genes involved in photosynthesis in Populus

Science.gov (United States)

2014-01-01

Background The gap between the real and potential photosynthetic rate under field conditions suggests that photosynthesis could potentially be improved. Nuclear genes provide possible targets for improving photosynthetic efficiency. Hence, genome-wide identification and characterization of the nuclear genes affecting photosynthetic traits in woody plants would provide key insights on genetic regulation of photosynthesis and identify candidate processes for improvement of photosynthesis. Results Using microarray and bulked segregant analysis strategies, we identified differentially expressed nuclear genes for photosynthesis traits in a segregating population of poplar. We identified 515 differentially expressed genes in this population (FC ≥ 2 or FC ≤ 0.5, P photosynthesis by the nuclear genome mainly involves transport, metabolism and response to stimulus functions. Conclusions This study provides new genome-scale strategies for the discovery of potential candidate genes affecting photosynthesis in Populus, and for identification of the functions of genes involved in regulation of photosynthesis. This work also suggests that improving photosynthetic efficiency under field conditions will require the consideration of multiple factors, such as stress responses. PMID:24673936

Identification of gene targets against dormant phase Mycobacterium tuberculosis infections

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Murphy Dennis J

2007-07-01

Full Text Available Abstract Background Mycobacterium tuberculosis, the causative agent of tuberculosis (TB, infects approximately 2 billion people worldwide and is the leading cause of mortality due to infectious disease. Current TB therapy involves a regimen of four antibiotics taken over a six month period. Patient compliance, cost of drugs and increasing incidence of drug resistant M. tuberculosis strains have added urgency to the development of novel TB therapies. Eradication of TB is affected by the ability of the bacterium to survive up to decades in a dormant state primarily in hypoxic granulomas in the lung and to cause recurrent infections. Methods The availability of M. tuberculosis genome-wide DNA microarrays has lead to the publication of several gene expression studies under simulated dormancy conditions. However, no single model best replicates the conditions of human pathogenicity. In order to identify novel TB drug targets, we performed a meta-analysis of multiple published datasets from gene expression DNA microarray experiments that modeled infection leading to and including the dormant state, along with data from genome-wide insertional mutagenesis that examined gene essentiality. Results Based on the analysis of these data sets following normalization, several genome wide trends were identified and used to guide the selection of targets for therapeutic development. The trends included the significant up-regulation of genes controlled by devR, down-regulation of protein and ATP synthesis, and the adaptation of two-carbon metabolism to the hypoxic and nutrient limited environment of the granuloma. Promising targets for drug discovery were several regulatory elements (devR/devS, relA, mprAB, enzymes involved in redox balance and respiration, sulfur transport and fixation, pantothenate, isoprene, and NAD biosynthesis. The advantages and liabilities of each target are discussed in the context of enzymology, bacterial pathways, target tractability

Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

Science.gov (United States)

Osato, Naoki

2018-01-19

Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional

Targeted gene insertion for molecular medicine.

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Voigt, Katrin; Izsvák, Zsuzsanna; Ivics, Zoltán

2008-11-01

Genomic insertion of a functional gene together with suitable transcriptional regulatory elements is often required for long-term therapeutical benefit in gene therapy for several genetic diseases. A variety of integrating vectors for gene delivery exist. Some of them exhibit random genomic integration, whereas others have integration preferences based on attributes of the targeted site, such as primary DNA sequence and physical structure of the DNA, or through tethering to certain DNA sequences by host-encoded cellular factors. Uncontrolled genomic insertion bears the risk of the transgene being silenced due to chromosomal position effects, and can lead to genotoxic effects due to mutagenesis of cellular genes. None of the vector systems currently used in either preclinical experiments or clinical trials displays sufficient preferences for target DNA sequences that would ensure appropriate and reliable expression of the transgene and simultaneously prevent hazardous side effects. We review in this paper the advantages and disadvantages of both viral and non-viral gene delivery technologies, discuss mechanisms of target site selection of integrating genetic elements (viruses and transposons), and suggest distinct molecular strategies for targeted gene delivery.

Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells.

Science.gov (United States)

Byrne, Susan M; Ortiz, Luis; Mali, Prashant; Aach, John; Church, George M

2015-02-18

Sequence-specific nucleases such as TALEN and the CRISPR/Cas9 system have so far been used to disrupt, correct or insert transgenes at precise locations in mammalian genomes. We demonstrate efficient 'knock-in' targeted replacement of multi-kilobase genes in human induced pluripotent stem cells (iPSC). Using a model system replacing endogenous human genes with their mouse counterpart, we performed a comprehensive study of targeting vector design parameters for homologous recombination. A 2.7 kilobase (kb) homozygous gene replacement was achieved in up to 11% of iPSC without selection. The optimal homology arm length was around 2 kb, with homology length being especially critical on the arm not adjacent to the cut site. Homologous sequence inside the cut sites was detrimental to targeting efficiency, consistent with a synthesis-dependent strand annealing (SDSA) mechanism. Using two nuclease sites, we observed a high degree of gene excisions and inversions, which sometimes occurred more frequently than indel mutations. While homozygous deletions of 86 kb were achieved with up to 8% frequency, deletion frequencies were not solely a function of nuclease activity and deletion size. Our results analyzing the optimal parameters for targeting vector design will inform future gene targeting efforts involving multi-kilobase gene segments, particularly in human iPSC. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Tumor-targeted inhibition by a novel strategy - mimoretrovirus expressing siRNA targeting the Pokemon gene.

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Tian, Zhiqiang; Wang, Huaizhi; Jia, Zhengcai; Shi, Jinglei; Tang, Jun; Mao, Liwei; Liu, Hongli; Deng, Yijing; He, Yangdong; Ruan, Zhihua; Li, Jintao; Wu, Yuzhang; Ni, Bing

2010-12-01

Pokemon gene has crucial but versatile functions in cell differentiation, proliferation and tumorigenesis. It is a master regulator of the ARF-HDM2-p53 and Rb-E2F pathways. The facts that the expression of Pokemon is essential for tumor formation and many kinds of tumors over-express the Pokemon gene make it an attractive target for therapeutic intervention for cancer treatment. In this study, we used an RNAi strategy to silence the Pokemon gene in a cervical cancer model. To address the issues involving tumor specific delivery and durable expression of siRNA, we applied the Arg-Gly-Asp (RGD) peptide ligand and polylysine (K(18)) fusion peptide to encapsulate a recombinant retrovirus plasmid expressing a siRNA targeting the Pokemon gene and produced the 'mimoretrovirus'. At charge ratio 2.0 of fusion peptide/plasmid, the mimoretrovirus formed stable and homogenous nanoparticles, and provided complete DNase I protection and complete gel retardation. This nanoparticle inhibited SiHa cell proliferation and invasion, while it promoted SiHa cell apoptosis. The binding of the nanoparticle to SiHa cells was mediated via the RGD-integrin α(v)β(3) interaction, as evidenced by the finding that unconjugated RGD peptide inhibited this binding significantly. This tumor-targeting mimoretrovirus exhibited excellent anti-tumor capacity in vivo in a nude mouse model. Moreover, the mimoretrovirus inhibited tumor growth with a much higher efficiency than recombinant retrovirus expressing siRNA or the K(18)/P4 nanoparticle lacking the RGD peptide. Results suggest that the RNAi/RGD-based mimoretrovirus developed in this study represents a novel anti-tumor strategy that may be applicable to most research involving cancer therapy and, thus, has promising potential as a cervical cancer treatment.

TBC2target: A Resource of Predicted Target Genes of Tea Bioactive Compounds

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Shihua Zhang

2018-02-01

Full Text Available Tea is one of the most popular non-alcoholic beverages consumed worldwide. Numerous bioactive constituents of tea were confirmed to possess healthy benefits via the mechanisms of regulating gene expressions or protein activities. However, a complete interacting profile between tea bioactive compounds (TBCs and their target genes is lacking, which put an obstacle in the study of healthy function of tea. To fill this gap, we developed a database of target genes of TBCs (TBC2target, http://camellia.ahau.edu.cn/TBC2target based on a pharmacophore mapping approach. In TBC2target, 6,226 interactions between 240 TBCs and 673 target genes were documented. TBC2target contains detailed information about each interacting entry, such as TBC, CAS number, PubChem CID, source of compound (e.g., green, black, compound type, target gene(s of TBC, gene symbol, gene ID, ENSEMBL ID, PDB ID, TBC bioactivity and the reference. Using the TBC-target associations, we constructed a bipartite network and provided users the global network and local sub-network visualization and topological analyses. The entire database is free for online browsing, searching and downloading. In addition, TBC2target provides a BLAST search function to facilitate use of the database. The particular strengths of TBC2target are the inclusion of the comprehensive TBC-target interactions, and the capacity to visualize and analyze the interacting networks, which may help uncovering the beneficial effects of tea on human health as a central resource in tea health community.

TBC2target: A Resource of Predicted Target Genes of Tea Bioactive Compounds.

Science.gov (United States)

Zhang, Shihua; Zhang, Liang; Wang, Yijun; Yang, Jian; Liao, Mingzhi; Bi, Shoudong; Xie, Zhongwen; Ho, Chi-Tang; Wan, Xiaochun

2018-01-01

Tea is one of the most popular non-alcoholic beverages consumed worldwide. Numerous bioactive constituents of tea were confirmed to possess healthy benefits via the mechanisms of regulating gene expressions or protein activities. However, a complete interacting profile between tea bioactive compounds (TBCs) and their target genes is lacking, which put an obstacle in the study of healthy function of tea. To fill this gap, we developed a database of target genes of TBCs (TBC2target, http://camellia.ahau.edu.cn/TBC2target) based on a pharmacophore mapping approach. In TBC2target, 6,226 interactions between 240 TBCs and 673 target genes were documented. TBC2target contains detailed information about each interacting entry, such as TBC, CAS number, PubChem CID, source of compound (e.g., green, black), compound type, target gene(s) of TBC, gene symbol, gene ID, ENSEMBL ID, PDB ID, TBC bioactivity and the reference. Using the TBC-target associations, we constructed a bipartite network and provided users the global network and local sub-network visualization and topological analyses. The entire database is free for online browsing, searching and downloading. In addition, TBC2target provides a BLAST search function to facilitate use of the database. The particular strengths of TBC2target are the inclusion of the comprehensive TBC-target interactions, and the capacity to visualize and analyze the interacting networks, which may help uncovering the beneficial effects of tea on human health as a central resource in tea health community.

Mipu1, a novel direct target gene, is involved in hypoxia inducible factor 1-mediated cytoprotection.

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Kangkai Wang

Full Text Available Mipu1 (myocardial ischemic preconditioning up-regulated protein 1, recently identified in our lab, is a novel zinc-finger transcription factor which is up-regulated during ischemic preconditioning. However, it is not clear what transcription factor contributes to its inducible expression. In the present study, we reported that HIF-1 regulates the inducible expression of Mipu1 which is involved in the cytoprotection of HIF-1α against oxidative stress by inhibiting Bax expression. Our results showed that the inducible expression of Mipu1 was associated with the expression and activation of transcription factor HIF-1 as indicated by cobalt chloride (CoCl2 treatment, HIF-1α overexpression and knockdown assays. EMSA and luciferase reporter gene assays showed that HIF-1α bound to the hypoxia response element (HRE within Mipu1 promoter region and promoted its transcription. Moreover, our results revealed that Mipu1 inhibited the expression of Bax, an important pro-apoptosis protein associated with the intrinsic pathway of apoptosis, elevating the cytoprotection of HIF-1 against hydrogen peroxide (H2O2-mediated injury in H9C2 cells. Our findings implied that Bax may be a potential target gene of transcription factor Mipu1, and provided a novel insight for understanding the cytoprotection of HIF-1 and new clues for further elucidating the mechanisms by which Mipu1 protects cell against pathological stress.

Novel Hematopoietic Target Genes in the NRF2-Mediated Transcriptional Pathway

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Michelle R. Campbell

2013-01-01

Full Text Available Nuclear factor- (erythroid-derived 2 like 2 (NFE2L2, NRF2 is a key transcriptional activator of the antioxidant response pathway and is closely related to erythroid transcription factor NFE2. Under oxidative stress, NRF2 heterodimerizes with small Maf proteins and binds cis-acting enhancer sequences found near oxidative stress response genes. Using the dietary isothiocyanate sulforaphane (SFN to activate NRF2, chromatin immunoprecipitation sequencing (ChIP-seq identified several hundred novel NRF2-mediated targets beyond its role in oxidative stress. Activated NRF2 bound the antioxidant response element (ARE in promoters of several known and novel target genes involved in iron homeostasis and heme metabolism, including known targets FTL and FTH1, as well as novel binding in the globin locus control region. Five novel NRF2 target genes were chosen for followup: AMBP, ABCB6, FECH, HRG-1 (SLC48A1, and TBXAS1. SFN-induced gene expression in erythroid K562 and lymphoid cells were compared for each target gene. NRF2 silencing showed reduced expression in lymphoid, lung, and hepatic cells. Furthermore, stable knockdown of NRF2 negative regulator KEAP1 in K562 cells resulted in increased NQO1, AMBP, and TBXAS1 expression. NFE2 binding sites in K562 cells revealed similar binding profiles as lymphoid NRF2 sites in all potential NRF2 candidates supporting a role for NRF2 in heme metabolism and erythropoiesis.

Gene transfer technology and genetic radioisotope targeting therapy

International Nuclear Information System (INIS)

Wang Jiaqiong; Wang Zizheng

2004-01-01

With deeper cognition about mechanisms of disease at the cellular and molecular level, gene therapy has become one of the most important research fields in medical molecular biology at present. Gene transfer technology plays an important role during the course of gene therapy, and further improvement should be made about vectors carrying target gene sequences. Also, gene survey is needed during gene therapy, and gene imaging is the most effective method. The combination of gene therapy and targeted radiotherapy, that is, 'Genetic Radioisotope Targeting Therapy', will be a novel approach to tumor gene therapy

Turmeric (Curcuma longa): miRNAs and their regulating targets are involved in development and secondary metabolite pathways.

Science.gov (United States)

Singh, Noopur; Sharma, Ashok

Turmeric has been used as a therapeutic herb over centuries in traditional medicinal systems due to the presence of several secondary metabolite compounds. microRNAs are known to regulate gene expression at the post-transcriptional level by transcriptional cleavage or translation repression. miRNAs have been demonstrated to play an active role in secondary metabolism regulation. The present work was focused on the identification of the miRNAs involved in the regulation of secondary metabolite and development process of turmeric. Eighteen miRNA families were identified for turmeric. Sixteen miRNA families were observed to regulate 238 target transcripts. LncRNAs targets of the putative miRNA candidates were also predicted. Our results indicated their role in binding, reproduction, stress, and other developmental processes. Gene annotation and pathway analysis illustrated the biological function of the targets regulated by the putative miRNAs. The miRNA-mediated gene regulatory network also revealed co-regulated targets that were regulated by two or more miRNA families. miR156 and miR5015 were observed to be involved in rhizome development. miR5021 showed regulation for terpenoid backbone biosynthesis and isoquinoline alkaloid biosynthesis pathways. The flavonoid biosynthesis pathway was observed to be regulated by miR2919. The analysis revealed the probable involvement of three miRNAs (miR1168.2, miR156b and miR1858) in curcumin biosynthesis. Other miRNAs were found to be involved in the growth and developmental process of turmeric. Phylogenetic analysis of selective miRNAs was also performed. Copyright © 2017 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

The mechanism of gene targeting in human somatic cells.

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Yinan Kan

2014-04-01

Full Text Available Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB repair known as homologous recombination (HR. The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells.

Biallelic targeting of expressed genes in mouse embryonic stem cells using the Cas9 system

NARCIS (Netherlands)

Zhang, Yu; Vanoli, Fabio; LaRocque, Jeannine R.; Krawczyk, Przemek M.; Jasin, Maria

2014-01-01

Gene targeting - homologous recombination between transfected DNA and a chromosomal locus - is greatly stimulated by a DNA break in the target locus. Recently, the RNA-guided Cas9 endonuclease, involved in bacterial adaptive immunity, has been modified to function in mammalian cells. Unlike other

Transcriptome profiling of the Plutella xylostella (Lepidoptera: Plutellidae) ovary reveals genes involved in oogenesis.

Science.gov (United States)

Peng, Lu; Wang, Lei; Yang, Yi-Fan; Zou, Ming-Min; He, Wei-Yi; Wang, Yue; Wang, Qing; Vasseur, Liette; You, Min-Sheng

2017-12-30

As a specialized organ, the insect ovary performs valuable functions by ensuring fecundity and population survival. Oogenesis is the complex physiological process resulting in the production of mature eggs, which are involved in epigenetic programming, germ cell behavior, cell cycle regulation, etc. Identification of the genes involved in ovary development and oogenesis is critical to better understand the reproductive biology and screening for the potential molecular targets in Plutella xylostella, a worldwide destructive pest of economically major crops. Based on transcriptome sequencing, a total of 7.88Gb clean nucleotides was obtained, with 19,934 genes and 1861 new transcripts being identified. Expression profiling indicated that 61.7% of the genes were expressed (FPKM≥1) in the P. xylostella ovary. GO annotation showed that the pathways of multicellular organism reproduction and multicellular organism reproduction process, as well as gamete generation and chorion were significantly enriched. Processes that were most likely relevant to reproduction included the spliceosome, ubiquitin mediated proteolysis, endocytosis, PI3K-Akt signaling pathway, insulin signaling pathway, cAMP signaling pathway, and focal adhesion were identified in the top 20 'highly represented' KEGG pathways. Functional genes involved in oogenesis were further analyzed and validated by qRT-PCR to show their potential predominant roles in P. xylostella reproduction. Our newly developed P. xylostella ovary transcriptome provides an overview of the gene expression profiling in this specialized tissue and the functional gene network closely related to the ovary development and oogenesis. This is the first genome-wide transcriptome dataset of P. xylostella ovary that includes a subset of functionally activated genes. This global approach will be the basis for further studies on molecular mechanisms of P. xylostella reproduction aimed at screening potential molecular targets for integrated pest

Targeted integration of genes in Xenopus tropicalis

DEFF Research Database (Denmark)

Shi, Zhaoying; Tian, Dandan; Xin, Huhu

2017-01-01

With the successful establishment of both targeted gene disruption and integration methods in the true diploid frog Xenopus tropicalis, this excellent vertebrate genetic model now is making a unique contribution to modelling human diseases. Here, we summarize our efforts on establishing homologous...... recombination-mediated targeted integration in Xenopus tropicalis, the usefulness, and limitation of targeted integration via the homology-independent strategy, and future directions on how to further improve targeted gene integration in Xenopus tropicalis....

MicroRNA-target gene responses to lead-induced stress in cotton (Gossypium hirsutum L.).

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He, Qiuling; Zhu, Shuijin; Zhang, Baohong

2014-09-01

MicroRNAs (miRNAs) play key roles in plant responses to various metal stresses. To investigate the miRNA-mediated plant response to heavy metals, cotton (Gossypium hirsutum L.), the most important fiber crop in the world, was exposed to different concentrations (0, 25, 50, 100, and 200 µM) of lead (Pb) and then the toxicological effects were investigated. The expression patterns of 16 stress-responsive miRNAs and 10 target genes were monitored in cotton leaves and roots by quantitative real-time PCR (qRT-PCR); of these selected genes, several miRNAs and their target genes are involved in root development. The results show a reciprocal regulation of cotton response to lead stress by miRNAs. The characterization of the miRNAs and the associated target genes in response to lead exposure would help in defining the potential roles of miRNAs in plant adaptation to heavy metal stress and further understanding miRNA regulation in response to abiotic stress.

Identification of hookworm DAF-16/FOXO response elements and direct gene targets.

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Xin Gao

2010-08-01

Full Text Available The infective stage of the parasitic nematode hookworm is developmentally arrested in the environment and needs to infect a specific host to complete its life cycle. The canine hookworm (Ancylostoma caninum is an excellent model for investigating human hookworm infections. The transcription factor of A. caninum, Ac-DAF-16, which has a characteristic fork head or "winged helix" DNA binding domain (DBD, has been implicated in the resumption of hookworm development in the host. However, the precise roles of Ac-DAF-16 in hookworm parasitism and its downstream targets are unknown. In the present study, we combined molecular techniques and bioinformatics to identify a group of Ac-DAF-16 binding sites and target genes.The DNA binding domain of Ac-DAF-16 was used to select genomic fragments by in vitro genomic selection. Twenty four bound genomic fragments were analyzed for the presence of the DAF-16 family binding element (DBE and possible alternative Ac-DAF-16 bind motifs. The 22 genes linked to these genomic fragments were identified using bioinformatics tools and defined as candidate direct gene targets of Ac-DAF-16. Their developmental stage-specific expression patterns were examined. Also, a new putative DAF-16 binding element was identified.Our results show that Ac-DAF-16 is involved in diverse biological processes throughout hookworm development. Further investigation of these target genes will provide insights into the molecular basis by which Ac-DAF-16 regulates its downstream gene network in hookworm infection.

A Morpholino-based screen to identify novel genes involved in craniofacial morphogenesis

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Melvin, Vida Senkus; Feng, Weiguo; Hernandez-Lagunas, Laura; Artinger, Kristin Bruk; Williams, Trevor

2014-01-01

BACKGROUND The regulatory mechanisms underpinning facial development are conserved between diverse species. Therefore, results from model systems provide insight into the genetic causes of human craniofacial defects. Previously, we generated a comprehensive dataset examining gene expression during development and fusion of the mouse facial prominences. Here, we used this resource to identify genes that have dynamic expression patterns in the facial prominences, but for which only limited information exists concerning developmental function. RESULTS This set of ~80 genes was used for a high throughput functional analysis in the zebrafish system using Morpholino gene knockdown technology. This screen revealed three classes of cranial cartilage phenotypes depending upon whether knockdown of the gene affected the neurocranium, viscerocranium, or both. The targeted genes that produced consistent phenotypes encoded proteins linked to transcription (meis1, meis2a, tshz2, vgll4l), signaling (pkdcc, vlk, macc1, wu:fb16h09), and extracellular matrix function (smoc2). The majority of these phenotypes were not altered by reduction of p53 levels, demonstrating that both p53 dependent and independent mechanisms were involved in the craniofacial abnormalities. CONCLUSIONS This Morpholino-based screen highlights new genes involved in development of the zebrafish craniofacial skeleton with wider relevance to formation of the face in other species, particularly mouse and human. PMID:23559552

Changes in gene expression in PBMCs profiles of PPARα target genes in obese and non-obese individuals during fasting.

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Felicidade, Ingrid; Marcarini, Juliana Cristina; Carreira, Clísia Mara; Amarante, Marla Karine; Afman, Lydia A; Mantovani, Mário Sérgio; Ribeiro, Lúcia Regina

2015-01-01

The prevalence of obesity has risen dramatically and the World Health Organization estimates that 700 million people will be obese worldwide by 2015. Approximately, 50% of the Brazilian population above 20 years of age is overweight, and 16% is obese. This study aimed to evaluate the differences in the expression of PPARα target genes in human peripheral blood mononuclear cells (PBMCs) and free fatty acids (FFA) in obese and non-obese individuals after 24 h of fasting. We first presented evidence that Brazilian people exhibit expression changes in PPARα target genes in PBMCs under fasting conditions. Q-PCR was utilized to assess the mRNA expression levels of target genes. In both groups, the FFA concentrations increased significantly after 24 h of fasting. The basal FFA mean concentration was two-fold higher in the obese group compared with the non-obese group. After fasting, all genes evaluated in this study showed increased expression levels compared with basal expression in both groups. However, our results reveal no differences in gene expression between the obese and non-obese, more studies are necessary to precisely delineate the associated mechanisms, particularly those that include groups with different degrees of obesity and patients with diabetes mellitus type 2 because the expression of the main genes that are involved in β-oxidation and glucose level maintenance are affected by these factors. © 2014 S. Karger AG, Basel.

Protein targeting in the analysis of learning and memory: a potential alternative to gene targeting.

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Gerlai, R; Williams, S P; Cairns, B; Van Bruggen, N; Moran, P; Shih, A; Caras, I; Sauer, H; Phillips, H S; Winslow, J W

1998-11-01

Gene targeting using homologous recombination in embryonic stem (ES) cells offers unprecedented precision with which one may manipulate single genes and investigate the in vivo effects of defined mutations in the mouse. Geneticists argue that this technique abrogates the lack of highly specific pharmacological tools in the study of brain function and behavior. However, by now it has become clear that gene targeting has some limitations too. One problem is spatial and temporal specificity of the generated mutation, which may appear in multiple brain regions or even in other organs and may also be present throughout development, giving rise to complex, secondary phenotypical alterations. This may be a disadvantage in the functional analysis of a number of genes associated with learning and memory processes. For example, several proteins, including neurotrophins--cell-adhesion molecules--and protein kinases, that play a significant developmental role have recently been suggested to be also involved in neural and behavioral plasticity. Knocking out genes of such proteins may lead to developmental alterations or even embryonic lethality in the mouse, making it difficult to study their function in neural plasticity, learning, and memory. Therefore, alternative strategies to gene targeting may be needed. Here, we suggest a potentially useful in vivo strategy based on systemic application of immunoadhesins, genetically engineered fusion proteins possessing the Fc portion of the human IgG molecule and, for example, a binding domain of a receptor of interest. These proteins are stable in vivo and exhibit high binding specificity and affinity for the endogenous ligand of the receptor, but lack the ability to signal. Thus, if delivered to the brain, immunoadhesins may specifically block signalling of the receptor of interest. Using osmotic minipumps, the protein can be infused in a localized region of the brain for a specified period of time (days or weeks). Thus, the location

A new electrospray method for targeted gene delivery.

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Boehringer, Stephan; Ruzgys, Paulius; Tamò, Luca; Šatkauskas, Saulius; Geiser, Thomas; Gazdhar, Amiq; Hradetzky, David

2018-03-05

A challenge for gene therapy is absence of safe and efficient local delivery of therapeutic genetic material. An efficient and reproducible physical method of electrospray for localized and targeted gene delivery is presented. Electrospray works on the principle of coulombs repulsion, under influence of electric field the liquid carrying genetic material is dispersed into micro droplets and is accelerated towards the targeted tissue, acting as a counter electrode. The accelerated droplets penetrate the targeted cells thus facilitating the transfer of genetic material into the cell. The work described here presents the principle of electrospray for gene delivery, the basic instrument design, and the various optimized parameters to enhance gene transfer in vitro. We estimate a transfection efficiency of up to 60% was achieved. We describe an efficient gene transfer method and a potential electrospray-mediated gene transfer mechanism.

Problem-Solving Test: Targeted Gene Disruption

Science.gov (United States)

Szeberenyi, Jozsef

2008-01-01

Mutational inactivation of a specific gene is the most powerful technique to analyze the biological function of the gene. This approach has been used for a long time in viruses, bacteria, yeast, and fruit fly, but looked quite hopeless in more complex organisms. Targeted inactivation of specific genes (also known as knock-out mutation) in mice is…

Apolipoprotein gene involved in lipid metabolism

Science.gov (United States)

Rubin, Edward; Pennacchio, Len A.

2007-07-03

Methods and materials for studying the effects of a newly identified human gene, APOAV, and the corresponding mouse gene apoAV. The sequences of the genes are given, and transgenic animals which either contain the gene or have the endogenous gene knocked out are described. In addition, single nucleotide polymorphisms (SNPs) in the gene are described and characterized. It is demonstrated that certain SNPs are associated with diseases involving lipids and triglycerides and other metabolic diseases. These SNPs may be used alone or with SNPs from other genes to study individual risk factors. Methods for intervention in lipid diseases, including the screening of drugs to treat lipid-related or diabetic diseases are also disclosed.

Identification of Plagl1/Zac1 binding sites and target genes establishes its role in the regulation of extracellular matrix genes and the imprinted gene network.

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Varrault, Annie; Dantec, Christelle; Le Digarcher, Anne; Chotard, Laëtitia; Bilanges, Benoit; Parrinello, Hugues; Dubois, Emeric; Rialle, Stéphanie; Severac, Dany; Bouschet, Tristan; Journot, Laurent

2017-10-13

PLAGL1/ZAC1 undergoes parental genomic imprinting, is paternally expressed, and is a member of the imprinted gene network (IGN). It encodes a zinc finger transcription factor with anti-proliferative activity and is a candidate tumor suppressor gene on 6q24 whose expression is frequently lost in various neoplasms. Conversely, gain of PLAGL1 function is responsible for transient neonatal diabetes mellitus, a rare genetic disease that results from defective pancreas development. In the present work, we showed that Plagl1 up-regulation was not associated with DNA damage-induced cell cycle arrest. It was rather associated with physiological cell cycle exit that occurred with contact inhibition, growth factor withdrawal, or cell differentiation. To gain insights into Plagl1 mechanism of action, we identified Plagl1 target genes by combining chromatin immunoprecipitation and genome-wide transcriptomics in transfected cell lines. Plagl1-elicited gene regulation correlated with multiple binding to the proximal promoter region through a GC-rich motif. Plagl1 target genes included numerous genes involved in signaling, cell adhesion, and extracellular matrix composition, including collagens. Plagl1 targets also included 22% of the 409 genes that make up the IGN. Altogether, this work identified Plagl1 as a transcription factor that coordinated the regulation of a subset of IGN genes and controlled extracellular matrix composition. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Characterization of differentially expressed genes involved in pathways associated with gastric cancer.

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Hao Li

Full Text Available To explore the patterns of gene expression in gastric cancer, a total of 26 paired gastric cancer and noncancerous tissues from patients were enrolled for gene expression microarray analyses. Limma methods were applied to analyze the data, and genes were considered to be significantly differentially expressed if the False Discovery Rate (FDR value was 2. Subsequently, Gene Ontology (GO categories were used to analyze the main functions of the differentially expressed genes. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG database, we found pathways significantly associated with the differential genes. Gene-Act network and co-expression network were built respectively based on the relationships among the genes, proteins and compounds in the database. 2371 mRNAs and 350 lncRNAs considered as significantly differentially expressed genes were selected for the further analysis. The GO categories, pathway analyses and the Gene-Act network showed a consistent result that up-regulated genes were responsible for tumorigenesis, migration, angiogenesis and microenvironment formation, while down-regulated genes were involved in metabolism. These results of this study provide some novel findings on coding RNAs, lncRNAs, pathways and the co-expression network in gastric cancer which will be useful to guide further investigation and target therapy for this disease.

Gene targeting in adult rhesus macaque fibroblasts

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Wolf Don P

2008-03-01

Full Text Available Abstract Background Gene targeting in nonhuman primates has the potential to produce critical animal models for translational studies related to human diseases. Successful gene targeting in fibroblasts followed by somatic cell nuclear transfer (SCNT has been achieved in several species of large mammals but not yet in primates. Our goal was to establish the protocols necessary to achieve gene targeting in primary culture of adult rhesus macaque fibroblasts as a first step in creating nonhuman primate models of genetic disease using nuclear transfer technology. Results A primary culture of adult male fibroblasts was transfected with hTERT to overcome senescence and allow long term in vitro manipulations. Successful gene targeting of the HPRT locus in rhesus macaques was achieved by electroporating S-phase synchronized cells with a construct containing a SV40 enhancer. Conclusion The cell lines reported here could be used for the production of null mutant rhesus macaque models of human genetic disease using SCNT technology. In addition, given the close evolutionary relationship and biological similarity between rhesus macaques and humans, the protocols described here may prove useful in the genetic engineering of human somatic cells.

Identification of target genes of transcription factor activator protein 2 gamma in breast cancer cells

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Ailan, He; Shuanglin, Xiang; Xiangwen, Xiao; Daolong, Ren; Lu, Gan; Xiaofeng, Ding; Xi, Qiao; Xingwang, Hu; Rushi, Liu; Jian, Zhang

2009-01-01

Activator protein 2 gamma (AP-2γ) is a member of the transcription factor activator protein-2 (AP-2) family, which is developmentally regulated and plays a role in human neoplasia. AP-2γ has been found to be overexpressed in most breast cancers, and have a dual role to inhibit tumor initiation and promote tumor progression afterwards during mammary tumorigensis. To identify the gene targets that mediate its effects, we performed chromatin immunoprecipitation (ChIP) to isolate AP-2γ binding sites on genomic DNA from human breast cancer cell line MDA-MB-453. 20 novel DNA fragments proximal to potential AP-2γ targets were obtained. They are categorized into functional groups of carcinogenesis, metabolism and others. A combination of sequence analysis, reporter gene assays, quantitative real-time PCR, electrophoretic gel mobility shift assays and immunoblot analysis further confirmed the four AP-2γ target genes in carcinogenesis group: ErbB2, CDH2, HPSE and IGSF11. Our results were consistent with the previous reports that ErbB2 was the target gene of AP-2γ. Decreased expression and overexpression of AP-2γ in human breast cancer cells significantly altered the expression of these four genes, indicating that AP-2γ directly regulates them. This suggested that AP-2γ can coordinate the expression of a network of genes, involving in carcinogenesis, especially in breast cancer. They could serve as therapeutic targets against breast cancers in the future

Molecular Subtyping of Primary Prostate Cancer Reveals Specific and Shared Target Genes of Different ETS Rearrangements

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Paula Paulo

2012-07-01

Full Text Available This work aimed to evaluate whether ETS transcription factors frequently involved in rearrangements in prostate carcinomas (PCa, namely ERG and ETV1, regulate specific or shared target genes. We performed differential expression analysis on nine normal prostate tissues and 50 PCa enriched for different ETS rearrangements using exon-level expression microarrays, followed by in vitro validation using cell line models. We found specific deregulation of 57 genes in ERG-positive PCa and 15 genes in ETV1-positive PCa, whereas deregulation of 27 genes was shared in both tumor subtypes. We further showed that the expression of seven tumor-associated ERG target genes (PLA1A, CACNA1D, ATP8A2, HLA-DMB, PDE3B, TDRD1, and TMBIM1 and two tumor-associated ETV1 target genes (FKBP10 and GLYATL2 was significantly affected by specific ETS silencing in VCaP and LNCaP cell line models, respectively, whereas the expression of three candidate ERG and ETV1 shared targets (GRPR, KCNH8, and TMEM45B was significantly affected by silencing of either ETS. Interestingly, we demonstrate that the expression of TDRD1, the topmost overexpressed gene of our list of ERG-specific candidate targets, is inversely correlated with the methylation levels of a CpG island found at -66 bp of the transcription start site in PCa and that TDRD1 expression is regulated by direct binding of ERG to the CpG island in VCaP cells. We conclude that ETS transcription factors regulate specific and shared target genes and that TDRD1, FKBP10, and GRPR are promising therapeutic targets and can serve as diagnostic markers for molecular subtypes of PCa harboring specific fusion gene rearrangements.

Targeting Gene-Viro-Therapy with AFP driving Apoptin gene shows potent antitumor effect in hepatocarcinoma

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Zhang Kang-Jian

2012-02-01

Full Text Available Abstract Background Gene therapy and viral therapy are used for cancer therapy for many years, but the results are less than satisfactory. Our aim was to construct a new recombinant adenovirus which is more efficient to kill hepatocarcinoma cells but more safe to normal cells. Methods By using the Cancer Targeting Gene-Viro-Therapy strategy, Apoptin, a promising cancer therapeutic gene was inserted into the double-regulated oncolytic adenovirus AD55 in which E1A gene was driven by alpha fetoprotein promoter along with a 55 kDa deletion in E1B gene to form AD55-Apoptin. The anti-tumor effects and safety were examined by western blotting, virus yield assay, real time polymerase chain reaction, 3-(4,5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide assay, Hoechst33342 staining, Fluorescence-activated cell sorting, xenograft tumor model, Immunohistochemical assay, liver function analysis and Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling assay. Results The recombinant virus AD55-Apoptin has more significant antitumor effect for hepatocelluar carcinoma cell lines (in vitro than that of AD55 and even ONYX-015 but no or little impair on normal cell lines. Furthermore, it also shows an obvious in vivo antitumor effect on the Huh-7 liver carcinoma xenograft in nude mice with bigger beginning tumor volume till about 425 mm3 but has no any damage on the function of liver. The induction of apoptosis is involved in AD55-Apoptin induced antitumor effects. Conclusion The AD55-Apoptin can be a potential anti-hepatoma agent with remarkable antitumor efficacy as well as higher safety in cancer targeting gene-viro-therapy system.

Dnmts and Tet target memory-associated genes after appetitive olfactory training in honey bees.

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Biergans, Stephanie D; Giovanni Galizia, C; Reinhard, Judith; Claudianos, Charles

2015-11-04

DNA methylation and demethylation are epigenetic mechanisms involved in memory formation. In honey bees DNA methyltransferase (Dnmt) function is necessary for long-term memory to be stimulus specific (i.e. to reduce generalization). So far, however, it remains elusive which genes are targeted and what the time-course of DNA methylation is during memory formation. Here, we analyse how DNA methylation affects memory retention, gene expression, and differential methylation in stimulus-specific olfactory long-term memory formation. Out of 30 memory-associated genes investigated here, 9 were upregulated following Dnmt inhibition in trained bees. These included Dnmt3 suggesting a negative feedback loop for DNA methylation. Within these genes also the DNA methylation pattern changed during the first 24 hours after training. Interestingly, this was accompanied by sequential activation of the DNA methylation machinery (i.e. Dnmts and Tet). In sum, memory formation involves a temporally complex epigenetic regulation of memory-associated genes that facilitates stimulus specific long-term memory in the honey bee.

Multi-targeted priming for genome-wide gene expression assays

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Adomas Aleksandra B

2010-08-01

Full Text Available Abstract Background Complementary approaches to assaying global gene expression are needed to assess gene expression in regions that are poorly assayed by current methodologies. A key component of nearly all gene expression assays is the reverse transcription of transcribed sequences that has traditionally been performed by priming the poly-A tails on many of the transcribed genes in eukaryotes with oligo-dT, or by priming RNA indiscriminately with random hexamers. We designed an algorithm to find common sequence motifs that were present within most protein-coding genes of Saccharomyces cerevisiae and of Neurospora crassa, but that were not present within their ribosomal RNA or transfer RNA genes. We then experimentally tested whether degenerately priming these motifs with multi-targeted primers improved the accuracy and completeness of transcriptomic assays. Results We discovered two multi-targeted primers that would prime a preponderance of genes in the genomes of Saccharomyces cerevisiae and Neurospora crassa while avoiding priming ribosomal RNA or transfer RNA. Examining the response of Saccharomyces cerevisiae to nitrogen deficiency and profiling Neurospora crassa early sexual development, we demonstrated that using multi-targeted primers in reverse transcription led to superior performance of microarray profiling and next-generation RNA tag sequencing. Priming with multi-targeted primers in addition to oligo-dT resulted in higher sensitivity, a larger number of well-measured genes and greater power to detect differences in gene expression. Conclusions Our results provide the most complete and detailed expression profiles of the yeast nitrogen starvation response and N. crassa early sexual development to date. Furthermore, our multi-targeting priming methodology for genome-wide gene expression assays provides selective targeting of multiple sequences and counter-selection against undesirable sequences, facilitating a more complete and

The effect of COMT gene on the target precision of the athlete movement

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E. V. Mikhailova

2014-01-01

Full Text Available The aim of the study was to find correlation between COMT gene alleles and the target precision of the athlete movement. 68 Russian competing athletes involved in boxing and volleyball, participated in the study. We found interrelation between COMT Met allele and a tall stature in the volleyball players.

The landscape of human genes involved in the immune response to parasitic worms

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Fumagalli Matteo

2010-08-01

Full Text Available Abstract Background More than 2 billion individuals worldwide suffer from helminth infections. The highest parasite burdens occur in children and helminth infection during pregnancy is a risk factor for preterm delivery and reduced birth weight. Therefore, helminth infections can be regarded as a strong selective pressure. Results Here we propose that candidate susceptibility genes for parasitic worm infections can be identified by searching for SNPs that display a strong correlation with the diversity of helminth species/genera transmitted in different geographic areas. By a genome-wide search we identified 3478 variants that correlate with helminth diversity. These SNPs map to 810 distinct human genes including loci involved in regulatory T cell function and in macrophage activation, as well as leukocyte integrins and co-inhibitory molecules. Analysis of functional relationships among these genes identified complex interaction networks centred around Th2 cytokines. Finally, several genes carrying candidate targets for helminth-driven selective pressure also harbour susceptibility alleles for asthma/allergy or are involved in airway hyper-responsiveness, therefore expanding the known parallelism between these conditions and parasitic infections. Conclusions Our data provide a landscape of human genes that modulate susceptibility to helminths and indicate parasitic worms as one of the major selective forces in humans.

The SET1 Complex Selects Actively Transcribed Target Genes via Multivalent Interaction with CpG Island Chromatin

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David A. Brown

2017-09-01

Full Text Available Chromatin modifications and the promoter-associated epigenome are important for the regulation of gene expression. However, the mechanisms by which chromatin-modifying complexes are targeted to the appropriate gene promoters in vertebrates and how they influence gene expression have remained poorly defined. Here, using a combination of live-cell imaging and functional genomics, we discover that the vertebrate SET1 complex is targeted to actively transcribed gene promoters through CFP1, which engages in a form of multivalent chromatin reading that involves recognition of non-methylated DNA and histone H3 lysine 4 trimethylation (H3K4me3. CFP1 defines SET1 complex occupancy on chromatin, and its multivalent interactions are required for the SET1 complex to place H3K4me3. In the absence of CFP1, gene expression is perturbed, suggesting that normal targeting and function of the SET1 complex are central to creating an appropriately functioning vertebrate promoter-associated epigenome.

Advances in sarcoma gene mutations and therapeutic targets.

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Gao, Peng; Seebacher, Nicole A; Hornicek, Francis; Guo, Zheng; Duan, Zhenfeng

2018-01-01

Sarcomas are rare and complex malignancies that have been associated with a poor prognostic outcome. Over the last few decades, traditional treatment with surgery and/or chemotherapy has not significantly improved outcomes for most types of sarcomas. In recent years, there have been significant advances in the understanding of specific gene mutations that are important in driving the pathogenesis and progression of sarcomas. Identification of these new gene mutations, using next-generation sequencing and advanced molecular techniques, has revealed a range of potential therapeutic targets. This, in turn, may lead to the development of novel agents targeted to different sarcoma subtypes. In this review, we highlight the advances made in identifying sarcoma gene mutations, including those of p53, RB, PI3K and IDH genes, as well as novel therapeutic strategies aimed at utilizing these mutant genes. In addition, we discuss a number of preclinical studies and ongoing early clinical trials in sarcoma targeting therapies, as well as gene editing technology, which may provide a better choice for sarcoma patient management. Published by Elsevier Ltd.

Polymorphisms in miRNA genes and their involvement in autoimmune diseases susceptibility.

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Latini, Andrea; Ciccacci, Cinzia; Novelli, Giuseppe; Borgiani, Paola

2017-08-01

MicroRNAs (miRNAs) are small non-coding RNA molecules that negatively regulate the expression of multiple protein-encoding genes at the post-transcriptional level. MicroRNAs are involved in different pathways, such as cellular proliferation and differentiation, signal transduction and inflammation, and play crucial roles in the development of several diseases, such as cancer, diabetes, and cardiovascular diseases. They have recently been recognized to play a role also in the pathogenesis of autoimmune diseases. Although the majority of studies are focused on miRNA expression profiles investigation, a growing number of studies have been investigating the role of polymorphisms in miRNA genes in the autoimmune diseases development. Indeed, polymorphisms affecting the miRNA genes can modify the set of targets they regulate or the maturation efficiency. This review is aimed to give an overview about the available studies that have investigated the association of miRNA gene polymorphisms with the susceptibility to various autoimmune diseases and to their clinical phenotypes.

Identification of transcription coactivator OCA-B-dependent genes involved in antigen-dependent B cell differentiation by cDNA array analyses.

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Kim, Unkyu; Siegel, Rachael; Ren, Xiaodi; Gunther, Cary S; Gaasterland, Terry; Roeder, Robert G

2003-07-22

The tissue-specific transcriptional coactivator OCA-B is required for antigen-dependent B cell differentiation events, including germinal center formation. However, the identity of OCA-B target genes involved in this process is unknown. This study has used large-scale cDNA arrays to monitor changes in gene expression patterns that accompany mature B cell differentiation. B cell receptor ligation alone induces many genes involved in B cell expansion, whereas B cell receptor and helper T cell costimulation induce genes associated with B cell effector function. OCA-B expression is induced by both B cell receptor ligation alone and helper T cell costimulation, suggesting that OCA-B is involved in B cell expansion as well as B cell function. Accordingly, several genes involved in cell proliferation and signaling, such as Lck, Kcnn4, Cdc37, cyclin D3, B4galt1, and Ms4a11, have been identified as OCA-B-dependent genes. Further studies on the roles played by these genes in B cells will contribute to an understanding of B cell differentiation.

Alternative epigenetic chromatin states of polycomb target genes.

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Yuri B Schwartz

2010-01-01

Full Text Available Polycomb (PcG regulation has been thought to produce stable long-term gene silencing. Genomic analyses in Drosophila and mammals, however, have shown that it targets many genes, which can switch state during development. Genetic evidence indicates that critical for the active state of PcG target genes are the histone methyltransferases Trithorax (TRX and ASH1. Here we analyze the repertoire of alternative states in which PcG target genes are found in different Drosophila cell lines and the role of PcG proteins TRX and ASH1 in controlling these states. Using extensive genome-wide chromatin immunoprecipitation analysis, RNAi knockdowns, and quantitative RT-PCR, we show that, in addition to the known repressed state, PcG targets can reside in a transcriptionally active state characterized by formation of an extended domain enriched in ASH1, the N-terminal, but not C-terminal moiety of TRX and H3K27ac. ASH1/TRX N-ter domains and transcription are not incompatible with repressive marks, sometimes resulting in a "balanced" state modulated by both repressors and activators. Often however, loss of PcG repression results instead in a "void" state, lacking transcription, H3K27ac, or binding of TRX or ASH1. We conclude that PcG repression is dynamic, not static, and that the propensity of a target gene to switch states depends on relative levels of PcG, TRX, and activators. N-ter TRX plays a remarkable role that antagonizes PcG repression and preempts H3K27 methylation by acetylation. This role is distinct from that usually attributed to TRX/MLL proteins at the promoter. These results have important implications for Polycomb gene regulation, the "bivalent" chromatin state of embryonic stem cells, and gene expression in development.

An animal model for Norrie disease (ND): gene targeting of the mouse ND gene.

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Berger, W; van de Pol, D; Bächner, D; Oerlemans, F; Winkens, H; Hameister, H; Wieringa, B; Hendriks, W; Ropers, H H

1996-01-01

In order to elucidate the cellular and molecular processes which are involved in Norrie disease (ND), we have used gene targeting technology to generate ND mutant mice. The murine homologue of the ND gene was cloned and shown to encode a polypeptide that shares 94% of the amino acid sequence with its human counterpart. RNA in situ hybridization revealed expression in retina, brain and the olfactory bulb and epithelium of 2 week old mice. Hemizygous mice carrying a replacement mutation in exon 2 of the ND gene developed retrolental structures in the vitreous body and showed an overall disorganization of the retinal ganglion cell layer. The outer plexiform layer disappears occasionally, resulting in a juxtaposed inner and outer nuclear layer. At the same regions, the outer segments of the photoreceptor cell layer are no longer present. These ocular findings are consistent with observations in ND patients and the generated mouse line provides a faithful model for study of early pathogenic events in this severe X-linked recessive neurological disorder.

TargetMine, an integrated data warehouse for candidate gene prioritisation and target discovery.

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Yi-An Chen

Full Text Available Prioritising candidate genes for further experimental characterisation is a non-trivial challenge in drug discovery and biomedical research in general. An integrated approach that combines results from multiple data types is best suited for optimal target selection. We developed TargetMine, a data warehouse for efficient target prioritisation. TargetMine utilises the InterMine framework, with new data models such as protein-DNA interactions integrated in a novel way. It enables complicated searches that are difficult to perform with existing tools and it also offers integration of custom annotations and in-house experimental data. We proposed an objective protocol for target prioritisation using TargetMine and set up a benchmarking procedure to evaluate its performance. The results show that the protocol can identify known disease-associated genes with high precision and coverage. A demonstration version of TargetMine is available at http://targetmine.nibio.go.jp/.

Transcriptome analysis identifies genes involved in ethanol response of Saccharomyces cerevisiae in Agave tequilana juice.

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Ramirez-Córdova, Jesús; Drnevich, Jenny; Madrigal-Pulido, Jaime Alberto; Arrizon, Javier; Allen, Kirk; Martínez-Velázquez, Moisés; Alvarez-Maya, Ikuri

2012-08-01

During ethanol fermentation, yeast cells are exposed to stress due to the accumulation of ethanol, cell growth is altered and the output of the target product is reduced. For Agave beverages, like tequila, no reports have been published on the global gene expression under ethanol stress. In this work, we used microarray analysis to identify Saccharomyces cerevisiae genes involved in the ethanol response. Gene expression of a tequila yeast strain of S. cerevisiae (AR5) was explored by comparing global gene expression with that of laboratory strain S288C, both after ethanol exposure. Additionally, we used two different culture conditions, cells grown in Agave tequilana juice as a natural fermentation media or grown in yeast-extract peptone dextrose as artificial media. Of the 6368 S. cerevisiae genes in the microarray, 657 genes were identified that had different expression responses to ethanol stress due to strain and/or media. A cluster of 28 genes was found over-expressed specifically in the AR5 tequila strain that could be involved in the adaptation to tequila yeast fermentation, 14 of which are unknown such as yor343c, ylr162w, ygr182c, ymr265c, yer053c-a or ydr415c. These could be the most suitable genes for transforming tequila yeast to increase ethanol tolerance in the tequila fermentation process. Other genes involved in response to stress (RFC4, TSA1, MLH1, PAU3, RAD53) or transport (CYB2, TIP20, QCR9) were expressed in the same cluster. Unknown genes could be good candidates for the development of recombinant yeasts with ethanol tolerance for use in industrial tequila fermentation.

CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation.

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Miki, Daisuke; Zhang, Wenxin; Zeng, Wenjie; Feng, Zhengyan; Zhu, Jian-Kang

2018-05-17

Homologous recombination-based gene targeting is a powerful tool for precise genome modification and has been widely used in organisms ranging from yeast to higher organisms such as Drosophila and mouse. However, gene targeting in higher plants, including the most widely used model plant Arabidopsis thaliana, remains challenging. Here we report a sequential transformation method for gene targeting in Arabidopsis. We find that parental lines expressing the bacterial endonuclease Cas9 from the egg cell- and early embryo-specific DD45 gene promoter can improve the frequency of single-guide RNA-targeted gene knock-ins and sequence replacements via homologous recombination at several endogenous sites in the Arabidopsis genome. These heritable gene targeting can be identified by regular PCR. Our approach enables routine and fine manipulation of the Arabidopsis genome.

Target genes of myostatin loss-of-function in muscles of late bovine fetuses

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Hocquette Jean-François

2007-03-01

Full Text Available Abstract Background Myostatin, a muscle-specific member of the Transforming Growth Factor beta family, negatively regulates muscle development. Double-muscled (DM cattle have a loss-of-function mutation in their myostatin gene responsible for the hypermuscular phenotype. Thus, these animals are a good model for understanding the mechanisms underpinning muscular hypertrophy. In order to identify individual genes or networks that may be myostatin targets, we looked for genes that were differentially expressed between DM and normal (NM animals (n = 3 per group in the semitendinosus muscle (hypertrophied in DM animals at 260 days of fetal development (when the biochemical differentiation of muscle is intensive. A heterologous microarray (human and murine oligonucleotide sequences of around 6,000 genes expressed in muscle was used. Results Many genes were found to be differentially expressed according to genetic type (some with a more than 5-fold change, and according to the presence of one or two functional myostatin allele(s. They belonged to various functional categories. The genes down-regulated in DM fetuses were mainly those encoding extracellular matrix proteins, slow contractile proteins and ribosomal proteins. The genes up-regulated in DM fetuses were mainly involved in the regulation of transcription, cell cycle/apoptosis, translation or DNA metabolism. These data highlight features indicating that DM muscle is shifted towards a more glycolytic metabolism, and has an altered extracellular matrix composition (e.g. down-regulation of COL1A1 and COL1A2, and up-regulation of COL4A2 and decreased adipocyte differentiation (down-regulation of C1QTNF3. The altered gene expression in the three major muscle compartments (fibers, connective tissue and intramuscular adipose tissue is consistent with the well-known characteristics of DM cattle. In addition, novel potential targets of the myostatin gene were identified (MB, PLN, troponins, ZFHX1B

Insulators target active genes to transcription factories and polycomb-repressed genes to polycomb bodies.

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Hua-Bing Li

2013-04-01

Full Text Available Polycomb bodies are foci of Polycomb proteins in which different Polycomb target genes are thought to co-localize in the nucleus, looping out from their chromosomal context. We have shown previously that insulators, not Polycomb response elements (PREs, mediate associations among Polycomb Group (PcG targets to form Polycomb bodies. Here we use live imaging and 3C interactions to show that transgenes containing PREs and endogenous PcG-regulated genes are targeted by insulator proteins to different nuclear structures depending on their state of activity. When two genes are repressed, they co-localize in Polycomb bodies. When both are active, they are targeted to transcription factories in a fashion dependent on Trithorax and enhancer specificity as well as the insulator protein CTCF. In the absence of CTCF, assembly of Polycomb bodies is essentially reduced to those representing genomic clusters of Polycomb target genes. The critical role of Trithorax suggests that stable association with a specialized transcription factory underlies the cellular memory of the active state.

Targeted gene therapy and cell reprogramming in Fanconi anemia

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Rio, Paula; Baños, Rocio; Lombardo, Angelo; Quintana-Bustamante, Oscar; Alvarez, Lara; Garate, Zita; Genovese, Pietro; Almarza, Elena; Valeri, Antonio; Díez, Begoña; Navarro, Susana; Torres, Yaima; Trujillo, Juan P; Murillas, Rodolfo; Segovia, Jose C; Samper, Enrique; Surralles, Jordi; Gregory, Philip D; Holmes, Michael C; Naldini, Luigi; Bueren, Juan A

2014-01-01

Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. PMID:24859981

Exploring genes and pathways involved in migraine

NARCIS (Netherlands)

Eising, E.

2017-01-01

The research in this thesis was aimed at identifying genes and molecular pathways involved in migraine. To this end, two gene expression analyses were performed in brain tissue obtained from transgenic mouse models for familial hemiplegic migraine (FHM), a monogenic subtype of migraine with aura.

Targeted Gene Therapy of Cancer: Second Amendment toward Holistic Therapy.

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Barar, Jaleh; Omidi, Yadollah

2013-01-01

It seems solid tumors are developing smart organs with specialized cells creating specified bio-territory, the so called "tumor microenvironment (TME)", in which there is reciprocal crosstalk among cancer cells, immune system cells and stromal cells. TME as an intricate milieu also consists of cancer stem cells (CSCs) that can resist against chemotherapies. In solid tumors, metabolism and vascularization appears to be aberrant and tumor interstitial fluid (TIF) functions as physiologic barrier. Thus, chemotherapy, immunotherapy and gene therapy often fail to provide cogent clinical outcomes. It looms that it is the time to accept the fact that initiation of cancer could be generation of another form of life that involves a cluster of thousands of genes, while we have failed to observe all aspects of it. Hence, the current treatment modalities need to be re-visited to cover all key aspects of disease using combination therapy based on the condition of patients. Perhaps personalized cluster of genes need to be simultaneously targeted.

Targeted Gene Therapy of Cancer: Second Amendment toward Holistic Therapy

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Jaleh Barar

2013-02-01

Full Text Available It seems solid tumors are developing smart organs with specialized cells creating specified bio-territory, the so called “tumor microenvironment (TME”, in which there is reciprocal crosstalk among cancer cells, immune system cells and stromal cells. TME as an intricate milieu also consists of cancer stem cells (CSCs that can resist against chemotherapies. In solid tumors, metabolism and vascularization appears to be aberrant and tumor interstitial fluid (TIF functions as physiologic barrier. Thus, chemotherapy, immunotherapy and gene therapy often fail to provide cogent clinical outcomes. It looms that it is the time to accept the fact that initiation of cancer could be generation of another form of life that involves a cluster of thousands of genes, while we have failed to observe all aspects of it. Hence, the current treatment modalities need to be re-visited to cover all key aspects of disease using combination therapy based on the condition of patients. Perhaps personalized cluster of genes need to be simultaneously targeted.

Host genes involved in Agrobacterium-mediated transformation

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Soltani, Jalal

2009-01-01

Agrobacterium is the nature’s genetic engineer that can transfer genes across the kingdom barriers to both prokaryotic and eukaryotic host cells. The host genes which are involved in Agrobacterium-mediated transformatiom (AMT) are not well known. Here, I studied in a systematic way to identify the

The SET1 Complex Selects Actively Transcribed Target Genes via Multivalent Interaction with CpG Island Chromatin.

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Brown, David A; Di Cerbo, Vincenzo; Feldmann, Angelika; Ahn, Jaewoo; Ito, Shinsuke; Blackledge, Neil P; Nakayama, Manabu; McClellan, Michael; Dimitrova, Emilia; Turberfield, Anne H; Long, Hannah K; King, Hamish W; Kriaucionis, Skirmantas; Schermelleh, Lothar; Kutateladze, Tatiana G; Koseki, Haruhiko; Klose, Robert J

2017-09-05

Chromatin modifications and the promoter-associated epigenome are important for the regulation of gene expression. However, the mechanisms by which chromatin-modifying complexes are targeted to the appropriate gene promoters in vertebrates and how they influence gene expression have remained poorly defined. Here, using a combination of live-cell imaging and functional genomics, we discover that the vertebrate SET1 complex is targeted to actively transcribed gene promoters through CFP1, which engages in a form of multivalent chromatin reading that involves recognition of non-methylated DNA and histone H3 lysine 4 trimethylation (H3K4me3). CFP1 defines SET1 complex occupancy on chromatin, and its multivalent interactions are required for the SET1 complex to place H3K4me3. In the absence of CFP1, gene expression is perturbed, suggesting that normal targeting and function of the SET1 complex are central to creating an appropriately functioning vertebrate promoter-associated epigenome. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Targeting the human lysozyme gene on bovine αs1- casein gene ...

African Journals Online (AJOL)

ajl yemi

2011-11-28

Nov 28, 2011 ... Targeting an exogenous gene into a favorable gene locus and for expression under endogenous regulators is ... case, the expression of human lysozyme could be regulated by the endogenous cis-element of αs1- casein gene in .... Mouse mammary epithelial C127 cells (Cell Bank, Chinese. Academy of ...

Co-factors necessary for PPAR mediated transactivation of endogenous target genes

DEFF Research Database (Denmark)

Grøntved, Lars; Nielsen, Ronni; Stunnenberg, Henk

of endogenous target gene in different cell types are elusive. To mutually compare the ability of the PPAR subtypes to activate endogenous target genes in a given cell, PPARa, PPARb/d and PPARg2 were HA tagged and rapidly, equally and synchronously expressed using adenoviral delivery. Within a few hours after...... subtype specific activation of target genes. Accumulating evidence suggests that transcriptional co-factors can function as master regulators for nuclear receptors and impose promoter selectivity. To study co-factor necessity for PPAR mediated transactivation of endogenous target genes, specific co...

Targeted gene therapy and cell reprogramming in Fanconi anemia.

Science.gov (United States)

Rio, Paula; Baños, Rocio; Lombardo, Angelo; Quintana-Bustamante, Oscar; Alvarez, Lara; Garate, Zita; Genovese, Pietro; Almarza, Elena; Valeri, Antonio; Díez, Begoña; Navarro, Susana; Torres, Yaima; Trujillo, Juan P; Murillas, Rodolfo; Segovia, Jose C; Samper, Enrique; Surralles, Jordi; Gregory, Philip D; Holmes, Michael C; Naldini, Luigi; Bueren, Juan A

2014-06-01

Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

Specific genetic modifications of domestic animals by gene targeting and animal cloning

Directory of Open Access Journals (Sweden)

Zhou Jiangfeng

2003-11-01

Full Text Available Abstract The technology of gene targeting through homologous recombination has been extremely useful for elucidating gene functions in mice. The application of this technology was thought impossible in the large livestock species until the successful creation of the first mammalian clone "Dolly" the sheep. The combination of the technologies for gene targeting of somatic cells with those of animal cloning made it possible to introduce specific genetic mutations into domestic animals. In this review, the principles of gene targeting in somatic cells and the challenges of nuclear transfer using gene-targeted cells are discussed. The relevance of gene targeting in domestic animals for applications in bio-medicine and agriculture are also examined.

Stimulation of autophagy by the p53 target gene Sestrin2.

Science.gov (United States)

Maiuri, Maria Chiara; Malik, Shoaib Ahmad; Morselli, Eugenia; Kepp, Oliver; Criollo, Alfredo; Mouchel, Pierre-Luc; Carnuccio, Rosa; Kroemer, Guido

2009-05-15

The oncosuppressor protein p53 regulates autophagy in a dual fashion. The pool of cytoplasmic p53 protein represses autophagy in a transcription-independent fashion, while the pool of nuclear p53 stimulates autophagy through the transactivation of specific genes. Here we report the discovery that Sestrin2, a novel p53 target gene, is involved in the induction of autophagy. Depletion of Sestrin2 by RNA interference reduced the level of autophagy in a panel of p53-sufficient human cancer cell lines responding to distinct autophagy inducers. In quantitative terms, Sestrin2 depletion was as efficient in preventing autophagy induction as was the depletion of Dram, another p53 target gene. Knockout of either Sestrin2 or Dram reduced autophagy elicited by nutrient depletion, rapamycin, lithium or thapsigargin. Moreover, autophagy induction by nutrient depletion or pharmacological stimuli led to an increase in Sestrin2 expression levels in p53-proficient cells. In strict contrast, the depletion of Sestrin2 or Dram failed to affect autophagy in p53-deficient cells and did not modulate the inhibition of baseline autophagy by a cytoplasmic p53 mutant that was reintroduced into p53-deficient cells. We conclude that Sestrin2 acts as a positive regulator of autophagy in p53-proficient cells.

Target genes prediction and functional analysis of microRNAs differentially expressed in gastric cancer stem cells MKN-45

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Zohreh Salehi

2017-01-01

Conclusions: Bioinformatics analysis such as DAVID database, GO biological process, GO molecular function, Kyoto encyclopedia of genes and genomes pathways, BioCarta pathway, Panther pathway, and Reactome pathway revealed that target genes of differentially expressed miRNAs in gastric CSCs were connected to pivotal biological pathways that involved in cell cycle regulation, stemness properties, and differentiation.

Positive-negative-selection-mediated gene targeting in rice

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Zenpei eShimatani

2015-01-01

Full Text Available Gene targeting (GT refers to the designed modification of genomic sequence(s through homologous recombination (HR. GT is a powerful tool both for the study of gene function and for molecular breeding. However, in transformation of higher plants, non-homologous end joining (NHEJ occurs overwhelmingly in somatic cells, masking HR-mediated GT. Positive-negative selection (PNS is an approach for finding HR-mediated GT events because it can eliminate NHEJ effectively by expression of a negative-selection marker gene. In rice—a major crop worldwide—reproducible PNS-mediated GT of endogenous genes has now been successfully achieved. The procedure is based on strong PNS using diphtheria toxin A-fragment as a negative marker, and has succeeded in the directed modification of several endogenous rice genes in various ways. In addition to gene knock-outs and knock-ins, a nucleotide substitution in a target gene was also achieved recently. This review presents a summary of the development of the rice PNS system, highlighting its advantages. Different types of gene modification and gene editing aimed at developing new plant breeding technology (NPBT based on PNS are discussed.

Regulation, overexpression, and target gene identification of Potato Homeobox 15 (POTH15) – a class-I KNOX gene in potato

Science.gov (United States)

Mahajan, Ameya S.; Kondhare, Kirtikumar R.; Rajabhoj, Mohit P.; Kumar, Amit; Ghate, Tejashree; Ravindran, Nevedha; Habib, Farhat; Siddappa, Sundaresha; Banerjee, Anjan K.

2016-01-01

Potato Homeobox 15 (POTH15) is a KNOX-I (Knotted1-like homeobox) family gene in potato that is orthologous to Shoot Meristemless (STM) in Arabidopsis. Despite numerous reports on KNOX genes from different species, studies in potato are limited. Here, we describe photoperiodic regulation of POTH15, its overexpression phenotype, and identification of its potential targets in potato (Solanum tuberosum ssp. andigena). qRT-PCR analysis showed a higher abundance of POTH15 mRNA in shoot tips and stolons under tuber-inducing short-day conditions. POTH15 promoter activity was detected in apical and axillary meristems, stolon tips, tuber eyes, and meristems of tuber sprouts, indicating its role in meristem maintenance and leaf development. POTH15 overexpression altered multiple morphological traits including leaf and stem development, leaflet number, and number of nodes and branches. In particular, the rachis of the leaf was completely reduced and leaves appeared as a bouquet of leaflets. Comparative transcriptomic analysis of 35S::GUS and two POTH15 overexpression lines identified more than 6000 differentially expressed genes, including 2014 common genes between the two overexpression lines. Functional analysis of these genes revealed their involvement in responses to hormones, biotic/abiotic stresses, transcription regulation, and signal transduction. qRT-PCR of selected candidate target genes validated their differential expression in both overexpression lines. Out of 200 randomly chosen POTH15 targets, 173 were found to have at least one tandem TGAC core motif, characteristic of KNOX interaction, within 3.0kb in the upstream sequence of the transcription start site. Overall, this study provides insights to the role of POTH15 in controlling diverse developmental processes in potato. PMID:27217546

Cancer gene therapy with targeted adenoviruses.

Science.gov (United States)

Bachtarzi, Houria; Stevenson, Mark; Fisher, Kerry

2008-11-01

Clinical experience with adenovirus vectors has highlighted the need for improved delivery and targeting. This manuscript aims to provide an overview of the techniques currently under development for improving adenovirus delivery to malignant cells in vivo. Primary research articles reporting improvements in adenoviral gene delivery are described. Strategies include genetic modification of viral coat proteins, non-genetic modifications including polymer encapsulation approaches and pharmacological interventions. Reprogramming adenovirus tropism in vitro has been convincingly demonstrated using a range of genetic and physical strategies. These studies have provided new insights into our understanding of virology and the field is progressing. However, there are still some limitations that need special consideration before adenovirus-targeted cancer gene therapy emerges as a routine treatment in the clinical setting.

Bullying involvement and empathy: Child and target characteristics

NARCIS (Netherlands)

Noorden, T.H.J. van; Cillessen, A.H.N.; Haselager, G.J.T.; Lansu, T.A.M.; Bukowski, W.M.

2017-01-01

This study investigated how the bullying involvement of a child and a target peer are related to empathy. The role of gender was also considered. We hypothesized that empathy primarily varies depending on the bullying role of the target peer. Participants were 264 7-12-year-old children

Uncovering Direct Targets of MiR-19a Involved in Lung Cancer Progression.

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Kumiko Yamamoto

Full Text Available Micro RNAs (miRNAs regulate the expression of target genes posttranscriptionally by pairing incompletely with mRNA in a sequence-specific manner. About 30% of human genes are regulated by miRNAs, and a single miRNA is capable of reducing the production of hundreds of proteins by means of incomplete pairing upon miRNA-mRNA binding. Lately, evidence implicating miRNAs in the development of lung cancers has been emerging. In particular, miR-19a, which is highly expressed in malignant lung cancer cells, is considered the key miRNA for tumorigenesis. However, its direct targets remain underreported. In the present study, we focused on six potential miR-19a target genes selected by miRNA target prediction software. To evaluate these genes as direct miR-19a target genes, we performed luciferase, pull-down, and western blot assays. The luciferase activity of plasmids with each miR-19a-binding site was observed to decrease, while increased luciferase activity was observed in the presence of anti-miR-19a locked nucleic acid (LNA. The pull-down assay showed biotinylated miR-19a to bind to AGO2 protein and to four of six potential target mRNAs. Western blot analysis showed that the expression levels of the four genes changed depending on treatment with miR-19a mimic or anti-miR-19a-LNA. Finally, FOXP1, TP53INP1, TNFAIP3, and TUSC2 were identified as miR-19a targets. To examine the function of these four target genes in lung cancer cells, LK79 (which has high miR-19a expression and A549 (which has low miR-19a expression were used. The expression of the four target proteins was higher in A549 than in LK79 cells. The four miR-19a target cDNA expression vectors suppressed cell viability, colony formation, migration, and invasion of A549 and LK79 cells, but LK79 cells transfected with FOXP1 and TP53INP1 cDNAs showed no difference compared to the control cells in the invasion assay.

Targeted gene deletion of miRNAs in mice by TALEN system.

Science.gov (United States)

Takada, Shuji; Sato, Tempei; Ito, Yoshiaki; Yamashita, Satoshi; Kato, Tomoko; Kawasumi, Miyuri; Kanai-Azuma, Masami; Igarashi, Arisa; Kato, Tomomi; Tamano, Moe; Asahara, Hiroshi

2013-01-01

Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs) and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A) tail worked better than that of with poly(A) tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.

Targeted gene therapy of xeroderma pigmentosum cells using meganuclease and TALEN™.

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Aurélie Dupuy

Full Text Available Xeroderma pigmentosum group C (XP-C is a rare human syndrome characterized by hypersensitivity to UV light and a dramatic predisposition to skin neoplasms. XP-C cells are deficient in the nucleotide excision repair (NER pathway, a complex process involved in the recognition and removal of DNA lesions. Several XPC mutations have been described, including a founder mutation in North African patients involving the deletion of a TG dinucleotide (ΔTG located in the middle of exon 9. This deletion leads to the expression of an inactive truncated XPC protein, normally involved in the first step of NER. New approaches used for gene correction are based on the ability of engineered nucleases such as Meganucleases, Zinc-Finger nucleases or TALE nucleases to accurately generate a double strand break at a specific locus and promote correction by homologous recombination through the insertion of an exogenous DNA repair matrix. Here, we describe the targeted correction of the ΔTG mutation in XP-C cells using engineered meganuclease and TALEN™. The methylated status of the XPC locus, known to inhibit both of these nuclease activities, led us to adapt our experimental design to optimize their in vivo efficacies. We show that demethylating treatment as well as the use of TALEN™ insensitive to CpG methylation enable successful correction of the ΔTG mutation. Such genetic correction leads to re-expression of the full-length XPC protein and to the recovery of NER capacity, attested by UV-C resistance of the corrected cells. Overall, we demonstrate that nuclease-based targeted approaches offer reliable and efficient strategies for gene correction.

Metabolic modeling to identify engineering targets for Komagataella phaffii: The effect of biomass composition on gene target identification.

Science.gov (United States)

Cankorur-Cetinkaya, Ayca; Dikicioglu, Duygu; Oliver, Stephen G

2017-11-01

Genome-scale metabolic models are valuable tools for the design of novel strains of industrial microorganisms, such as Komagataella phaffii (syn. Pichia pastoris). However, as is the case for many industrial microbes, there is no executable metabolic model for K. phaffiii that confirms to current standards by providing the metabolite and reactions IDs, to facilitate model extension and reuse, and gene-reaction associations to enable identification of targets for genetic manipulation. In order to remedy this deficiency, we decided to reconstruct the genome-scale metabolic model of K. phaffii by reconciling the extant models and performing extensive manual curation in order to construct an executable model (Kp.1.0) that conforms to current standards. We then used this model to study the effect of biomass composition on the predictive success of the model. Twelve different biomass compositions obtained from published empirical data obtained under a range of growth conditions were employed in this investigation. We found that the success of Kp1.0 in predicting both gene essentiality and growth characteristics was relatively unaffected by biomass composition. However, we found that biomass composition had a profound effect on the distribution of the fluxes involved in lipid, DNA, and steroid biosynthetic processes, cellular alcohol metabolic process, and oxidation-reduction process. Furthermore, we investigated the effect of biomass composition on the identification of suitable target genes for strain development. The analyses revealed that around 40% of the predictions of the effect of gene overexpression or deletion changed depending on the representation of biomass composition in the model. Considering the robustness of the in silico flux distributions to the changing biomass representations enables better interpretation of experimental results, reduces the risk of wrong target identification, and so both speeds and improves the process of directed strain development

Targeting Herpetic Keratitis by Gene Therapy

Directory of Open Access Journals (Sweden)

Hossein Mostafa Elbadawy

2012-01-01

Full Text Available Ocular gene therapy is rapidly becoming a reality. By November 2012, approximately 28 clinical trials were approved to assess novel gene therapy agents. Viral infections such as herpetic keratitis caused by herpes simplex virus 1 (HSV-1 can cause serious complications that may lead to blindness. Recurrence of the disease is likely and cornea transplantation, therefore, might not be the ideal therapeutic solution. This paper will focus on the current situation of ocular gene therapy research against herpetic keratitis, including the use of viral and nonviral vectors, routes of delivery of therapeutic genes, new techniques, and key research strategies. Whereas the correction of inherited diseases was the initial goal of the field of gene therapy, here we discuss transgene expression, gene replacement, silencing, or clipping. Gene therapy of herpetic keratitis previously reported in the literature is screened emphasizing candidate gene therapy targets. Commonly adopted strategies are discussed to assess the relative advantages of the protective therapy using antiviral drugs and the common gene therapy against long-term HSV-1 ocular infections signs, inflammation and neovascularization. Successful gene therapy can provide innovative physiological and pharmaceutical solutions against herpetic keratitis.

Targeted gene deletion of miRNAs in mice by TALEN system.

Directory of Open Access Journals (Sweden)

Shuji Takada

Full Text Available Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A tail worked better than that of with poly(A tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.

Ultrasound-targeted microbubble destruction improves the low density lipoprotein receptor gene expression in HepG2 cells

International Nuclear Information System (INIS)

Guo Dongping; Li Xiaoyu; Sun, Ping; Tang Yibo; Chen Xiuying; Chen Qi; Fan Leming; Zang Bin; Shao Lizheng; Li Xiaorong

2006-01-01

Ultrasound-targeted microbubble destruction had been employed in gene delivery and promised great potential. Liver has unique features that make it attractive for gene therapy. However, it poses formidable obstacles to hepatocyte-specific gene delivery. This study was designed to test the efficiency of therapeutic gene transfer and expression mediated by ultrasound/microbubble strategy in HepG 2 cell line. Air-filled albumin microbubbles were prepared and mixed with plasmid DNA encoding low density lipoprotein receptor (LDLR) and green fluorescent protein. The mixture of the DNA and microbubbles was administer to cultured HepG 2 cells under variable ultrasound conditions. Transfection rate of the transferred gene and cell viability were assessed by FACS analysis, confocal laser scanning microscopy, Western blot analysis and Trypan blue staining. The result demonstrated that microbubbles with ultrasound irradiation can significantly elevate exogenous LDLR gene expression and the expressed LDLRs were functional and active to uptake their ligands. We conclude that ultrasound-targeted microbubble destruction has the potential to promote safe and efficient LDLR gene transfer into hepatocytes. With further refinement, it may represent an effective nonviral avenue of gene therapy for liver-involved genetic diseases

De-repressing LncRNA-Targeted Genes to Upregulate Gene Expression: Focus on Small Molecule Therapeutics

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Roya Pedram Fatemi

2014-01-01

Full Text Available Non-protein coding RNAs (ncRNAs make up the overwhelming majority of transcripts in the genome and have recently gained attention for their complex regulatory role in cells, including the regulation of protein-coding genes. Furthermore, ncRNAs play an important role in normal development and their expression levels are dysregulated in several diseases. Recently, several long noncoding RNAs (lncRNAs have been shown to alter the epigenetic status of genomic loci and suppress the expression of target genes. This review will present examples of such a mechanism and focus on the potential to target lncRNAs for achieving therapeutic gene upregulation by de-repressing genes that are epigenetically silenced in various diseases. Finally, the potential to target lncRNAs, through their interactions with epigenetic enzymes, using various tools, such as small molecules, viral vectors and antisense oligonucleotides, will be discussed. We suggest that small molecule modulators of a novel class of drug targets, lncRNA-protein interactions, have great potential to treat some cancers, cardiovascular disease, and neurological disorders.

Gene targeting approaches to complex genetic diseases: atherosclerosis and essential hypertension.

OpenAIRE

Smithies, O; Maeda, N

1995-01-01

Gene targeting allows precise, predetermined changes to be made in a chosen gene in the mouse genome. To date, targeting has been used most often for generation of animals completely lacking the product of a gene of interest. The resulting "knockout" mice have confirmed some hypotheses, have upset others, but have rarely been uninformative. Models of several human genetic diseases have been produced by targeting--including Gaucher disease, cystic fibrosis, and the fragile X syndrome. These di...

Stably Expressed Genes Involved in Basic Cellular Functions.

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Kejian Wang

Full Text Available Stably Expressed Genes (SEGs whose expression varies within a narrow range may be involved in core cellular processes necessary for basic functions. To identify such genes, we re-analyzed existing RNA-Seq gene expression profiles across 11 organs at 4 developmental stages (from immature to old age in both sexes of F344 rats (n = 4/group; 320 samples. Expression changes (calculated as the maximum expression / minimum expression for each gene of >19000 genes across organs, ages, and sexes ranged from 2.35 to >109-fold, with a median of 165-fold. The expression of 278 SEGs was found to vary ≤4-fold and these genes were significantly involved in protein catabolism (proteasome and ubiquitination, RNA transport, protein processing, and the spliceosome. Such stability of expression was further validated in human samples where the expression variability of the homologous human SEGs was significantly lower than that of other genes in the human genome. It was also found that the homologous human SEGs were generally less subject to non-synonymous mutation than other genes, as would be expected of stably expressed genes. We also found that knockout of SEG homologs in mouse models was more likely to cause complete preweaning lethality than non-SEG homologs, corroborating the fundamental roles played by SEGs in biological development. Such stably expressed genes and pathways across life-stages suggest that tight control of these processes is important in basic cellular functions and that perturbation by endogenous (e.g., genetics or exogenous agents (e.g., drugs, environmental factors may cause serious adverse effects.

An ensemble method to predict target genes and pathways in uveal melanoma

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Wei Chao

2018-04-01

Full Text Available This work proposes to predict target genes and pathways for uveal melanoma (UM based on an ensemble method and pathway analyses. Methods: The ensemble method integrated a correlation method (Pearson correlation coefficient, PCC, a causal inference method (IDA and a regression method (Lasso utilizing the Borda count election method. Subsequently, to validate the performance of PIL method, comparisons between confirmed database and predicted miRNA targets were performed. Ultimately, pathway enrichment analysis was conducted on target genes in top 1000 miRNA-mRNA interactions to identify target pathways for UM patients. Results: Thirty eight of the predicted interactions were matched with the confirmed interactions, indicating that the ensemble method was a suitable and feasible approach to predict miRNA targets. We obtained 50 seed miRNA-mRNA interactions of UM patients and extracted target genes from these interactions, such as ASPG, BSDC1 and C4BP. The 601 target genes in top 1,000 miRNA-mRNA interactions were enriched in 12 target pathways, of which Phototransduction was the most significant one. Conclusion: The target genes and pathways might provide a new way to reveal the molecular mechanism of UM and give hand for target treatments and preventions of this malignant tumor.

Mannan-Modified PLGA Nanoparticles for Targeted Gene Delivery

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Fansheng Kong

2012-01-01

Full Text Available The studies of targeted gene delivery nanocarriers have gained increasing attention during the past decades. In this study, mannan modified DNA loaded bioadhesive PLGA nanoparticles (MAN-DNA-NPs were investigated for targeted gene delivery to the Kupffer cells (KCs. Bioadhesive PLGA nanoparticles were prepared and subsequently bound with pEGFP. Following the coupling of the mannan-based PE-grafted ligands (MAN-PE with the DNA-NPs, the MAN-DNA-NPs were delivered intravenously to rats. The transfection efficiency was determined from the isolated KCs and flow cytometry was applied for the quantitation of gene expression after 48 h post transfection. The size of the MAN-DNA-NPs was found to be around 190 nm and the Zeta potential was determined to be −15.46mV. The pEGFP binding capacity of MAN-DNA-NPs was (88.9±5.8% and the in vitro release profiles of the MAN-DNA-NPs follow the Higuchi model. When compared with non-modified DNA-NPs and Lipofectamine 2000-DNA, MAN-DNA-NPs produced the highest gene expressions, especially in vivo. The in vivo data from flow cytometry analysis showed that MAN-DNA-NPs displayed a remarkably higher transfection efficiency (39% than non-modified DNA-NPs (25% and Lipofectamine 2000-DNA (23% in KCs. The results illustrate that MAN-DNA-NPs have the ability to target liver KCs and could function as promising active targeting drug delivery vectors.

Lmx1b-targeted cis-regulatory modules involved in limb dorsalization.

Science.gov (United States)

Haro, Endika; Watson, Billy A; Feenstra, Jennifer M; Tegeler, Luke; Pira, Charmaine U; Mohan, Subburaman; Oberg, Kerby C

2017-06-01

Lmx1b is a homeodomain transcription factor responsible for limb dorsalization. Despite striking double-ventral (loss-of-function) and double-dorsal (gain-of-function) limb phenotypes, no direct gene targets in the limb have been confirmed. To determine direct targets, we performed a chromatin immunoprecipitation against Lmx1b in mouse limbs at embryonic day 12.5 followed by next-generation sequencing (ChIP-seq). Nearly 84% ( n =617) of the Lmx1b-bound genomic intervals (LBIs) identified overlap with chromatin regulatory marks indicative of potential cis -regulatory modules (PCRMs). In addition, 73 LBIs mapped to CRMs that are known to be active during limb development. We compared Lmx1b-bound PCRMs with genes regulated by Lmx1b and found 292 PCRMs within 1 Mb of 254 Lmx1b-regulated genes. Gene ontological analysis suggests that Lmx1b targets extracellular matrix production, bone/joint formation, axonal guidance, vascular development, cell proliferation and cell movement. We validated the functional activity of a PCRM associated with joint-related Gdf5 that provides a mechanism for Lmx1b-mediated joint modification and a PCRM associated with Lmx1b that suggests a role in autoregulation. This is the first report to describe genome-wide Lmx1b binding during limb development, directly linking Lmx1b to targets that accomplish limb dorsalization. © 2017. Published by The Company of Biologists Ltd.

Deduction of Novel Genes Potentially Involved in Osteoblasts of Rheumatoid Arthritis Using Next-Generation Sequencing and Bioinformatic Approaches

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Yi-Jen Chen

2017-11-01

Full Text Available The role of osteoblasts in peri-articular bone loss and bone erosion in rheumatoid arthritis (RA has gained much attention, and microRNAs are hypothesized to play critical roles in the regulation of osteoblast function in RA. The aim of this study is to explore novel microRNAs differentially expressed in RA osteoblasts and to identify genes potentially involved in the dysregulated bone homeostasis in RA. RNAs were extracted from cultured normal and RA osteoblasts for sequencing. Using the next generation sequencing and bioinformatics approaches, we identified 35 differentially expressed microRNAs and 13 differentially expressed genes with potential microRNA–mRNA interactions in RA osteoblasts. The 13 candidate genes were involved mainly in cell–matrix adhesion, as classified by the Gene Ontology. Two genes of interest identified from RA osteoblasts, A-kinase anchoring protein 12 (AKAP12 and leucin rich repeat containing 15 (LRRC15, were found to express more consistently in the related RA synovial tissue arrays in the Gene Expression Omnibus database, with the predicted interactions with miR-183-5p and miR-146a-5p, respectively. The Ingenuity Pathway Analysis identified AKAP12 as one of the genes involved in protein kinase A signaling and the function of chemotaxis, interconnecting with molecules related to neovascularization. The findings indicate new candidate genes as the potential indicators in evaluating therapies targeting chemotaxis and neovascularization to control joint destruction in RA.

Expression profiles of genes involved in tanshinone biosynthesis of ...

Indian Academy of Sciences (India)

Expression profiles of genes involved in tanshinone biosynthesis of two. Salvia miltiorrhiza genotypes with different tanshinone contents. Zhenqiao Song, Jianhua Wang and Xingfeng Li. J. Genet. 95, 433–439. Table 1. S. miltiorrhiza genes and primer pairs used for qRT-PCR. Gene. GenBank accession. Primer name.

Tumor Microenvironment Gene Signature as a Prognostic Classifier and Therapeutic Target

Science.gov (United States)

2016-06-01

AWARD NUMBER: W81XWH-14-1-0107 TITLE: Tumor Microenvironment Gene Signature as a Prognostic Classifier and Therapeutic Target PRINCIPAL...AND SUBTITLE Tumor Microenvironment Gene Signature as a 5a. CONTRACT NUMBER W81XWH-14-1-0107 Prognostic Classifier and Therapeutic Target 5b...gene signature that correlates with poor survival in ovarian cancer patients. We are refining this gene signature to develop biomarkers for the

Selective Inhibition of Histone Deacetylation in Melanoma Increases Targeted Gene Delivery by a Bacteriophage Viral Vector

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Samuel Campbell

2018-04-01

Full Text Available The previously developed adeno-associated virus/phage (AAVP vector, a hybrid between M13 bacteriophage (phage viruses that infect bacteria only and human Adeno-Associated Virus (AAV, is a promising tool in targeted gene therapy against cancer. AAVP can be administered systemically and made tissue specific through the use of ligand-directed targeting. Cancer cells and tumor-associated blood vessels overexpress the αν integrin receptors, which are involved in tumor angiogenesis and tumor invasion. AAVP is targeted to these integrins via a double cyclic RGD4C ligand displayed on the phage capsid. Nevertheless, there remain significant host-defense hurdles to the use of AAVP in targeted gene delivery and subsequently in gene therapy. We previously reported that histone deacetylation in cancer constitutes a barrier to AAVP. Herein, to improve AAVP-mediated gene delivery to cancer cells, we combined the vector with selective adjuvant chemicals that inhibit specific histone deacetylases (HDAC. We examined the effects of the HDAC inhibitor C1A that mainly targets HDAC6 and compared this to sodium butyrate, a pan-HDAC inhibitor with broad spectrum HDAC inhibition. We tested the effects on melanoma, known for HDAC6 up-regulation, and compared this side by side with a normal human kidney HEK293 cell line. Varying concentrations were tested to determine cytotoxic levels as well as effects on AAVP gene delivery. We report that the HDAC inhibitor C1A increased AAVP-mediated transgene expression by up to ~9-fold. These findings indicate that selective HDAC inhibition is a promising adjuvant treatment for increasing the therapeutic value of AAVP.

Coverage analysis of lists of genes involved in heterogeneous ...

Indian Academy of Sciences (India)

Genes involved in myopathies: 82 genes, based on the disease groups ... 605517 Muscular dystrophy-dystroglycanopathy (congenital with brain and eye ..... Epilepsy, X-linked, with variable learning disabilities and behavior disorders. 300491.

Gene-carried hepatoma targeting complex induced high gene transfection efficiency with low toxicity and significant antitumor activity

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Zhao QQ

2012-06-01

Full Text Available Qing-Qing Zhao,1,2 Yu-Lan Hu,1 Yang Zhou,3 Ni Li,1 Min Han,1 Gu-Ping Tang,4 Feng Qiu,2 Yasuhiko Tabata,5 Jian-Qing Gao,11Institute of Pharmaceutics, Zhejiang University, Hangzhou, China; 2Department of Pharmacy, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China; 3Institute of Biochemistry, Iowa State University, Ames, IA, USA; 4Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Hangzhou, China; 5Institute for Frontier Medical Sciences, Kyoto University, Kyoto, JapanBackground: The success of gene transfection is largely dependent on the development of a vehicle or vector that can efficiently deliver a gene to cells with minimal toxicity.Methods: A liver cancer-targeted specific peptide (FQHPSF sequence was successfully synthesized and linked with chitosan-linked polyethylenimine (CP to form a new targeted gene delivery vector called CPT (CP/peptide. The structure of CPT was confirmed by 1H nuclear magnetic resonance spectroscopy and ultraviolet spectrophotometry. The particle size of CPT/DNA complexes was measured using laser diffraction spectrometry and the cytotoxicity of the copolymer was evaluated by methylthiazol tetrazolium method. The transfection efficiency evaluation of the CP copolymer was performed using luciferase activity assay. Cellular internalization of the CP/DNA complex was observed under confocal laser scanning microscopy. The targeting specificity of the polymer coupled to peptide was measured by competitive inhibition transfection study. The liver targeting specificity of the CPT copolymer in vivo was demonstrated by combining the copolymer with a therapeutic gene, interleukin-12, and assessed by its abilities in suppressing the growth of ascites tumor in mouse model.Results: The results showed that the liver cancer-targeted specific peptide was successfully synthesized and linked with CP to form a new targeted gene delivery vector called CPT. The composition of CPT

microRNAs targeting DEAD-box helicases are involved in salinity stress response in rice (Oryza sativa L.

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Macovei Anca

2012-10-01

Full Text Available Abstract Background Rice (Oryza sativa L., one of the most important food crop in the world, is considered to be a salt-sensitive crop. Excess levels of salt adversely affect all the major metabolic activities, including cell wall damage, cytoplasmic lysis and genomic stability. In order to cope with salt stress, plants have evolved high degrees of developmental plasticity, including adaptation via cascades of molecular networks and changes in gene expression profiles. Posttranscriptional regulation, through the activity of microRNAs, also plays an important role in the plant response to salinity conditions. MicroRNAs are small endogenous RNAs that modulate gene expression and are involved in the most essential physiological processes, including plant development and adaptation to environmental changes. Results In the present study, we investigated the expression profiles of osa-MIR414, osa-MIR408 and osa-MIR164e along with their targeted genes, under salinity stress conditions in wild type and transgenic rice plants ectopically expressing the PDH45 (Pea DNA Helicase gene. The present miRNAs were predicted to target the OsABP (ATP-Binding Protein, OsDSHCT (DOB1/SK12/helY-like DEAD-box Helicase and OsDBH (DEAD-Box Helicase genes, included in the DEAD-box helicase family. An in silico characterization of the proteins was performed and the miRNAs predicted targets were validated by RLM-5′RACE. The qRT-PCR analysis showed that the OsABP, OsDBH and OsDSHCT genes were up-regulated in response to 100 and 200 mM NaCl treatments. The present study also highlighted an increased accumulation of the gene transcripts in wild type plants, with the exception of the OsABP mRNA which showed the highest level (15.1-fold change compared to control in the transgenic plants treated with 200 mM NaCl. Salinity treatments also affected the expression of osa-MIR414, osa-MIR164e and osa-MIR408, found to be significantly down-regulated, although the changes in mi

Identification and Regulation of c-Myb Target Genes in MCF-7 Cells

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O'Rourke John P

2011-01-01

Full Text Available Abstract Background The c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells. Although oncogenic versions of c-Myb were first associated with leukemias, over expression or rearrangement of the c-myb gene is common in several types of solid tumors, including breast cancers. Expression of the c-myb gene in human breast cancer cells is dependent on estrogen stimulation, but little is known about the activities of the c-Myb protein or what genes it regulates in estrogen-stimulated cells. Methods We used chromatin immunoprecipitation coupled with whole genome promoter tiling microarrays to identify endogenous c-Myb target genes in human MCF-7 breast cancer cells and characterized the activity of c-Myb at a panel of target genes during different stages of estrogen deprivation and stimulation. Results By using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs. Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1. By studying a panel of these targets to validate the results, we found that estradiol stimulation triggered the association of c-Myb with promoters and that association correlated with increased target gene expression. We studied one target gene, CXCR4, in detail, showing that c-Myb associated with the CXCR4 gene promoter and activated a CXCR4 reporter gene in transfection assays. Conclusions Our results show that c-Myb associates with a surprisingly large number of promoters in human cells. The results also suggest that estradiol stimulation leads to large-scale, genome-wide changes in c-Myb activity and subsequent changes in gene expression in human breast cancer

Identification and Regulation of c-Myb Target Genes in MCF-7 Cells

International Nuclear Information System (INIS)

Quintana, Anita M; Liu, Fan; O'Rourke, John P; Ness, Scott A

2011-01-01

The c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells. Although oncogenic versions of c-Myb were first associated with leukemias, over expression or rearrangement of the c-myb gene is common in several types of solid tumors, including breast cancers. Expression of the c-myb gene in human breast cancer cells is dependent on estrogen stimulation, but little is known about the activities of the c-Myb protein or what genes it regulates in estrogen-stimulated cells. We used chromatin immunoprecipitation coupled with whole genome promoter tiling microarrays to identify endogenous c-Myb target genes in human MCF-7 breast cancer cells and characterized the activity of c-Myb at a panel of target genes during different stages of estrogen deprivation and stimulation. By using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs. Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1. By studying a panel of these targets to validate the results, we found that estradiol stimulation triggered the association of c-Myb with promoters and that association correlated with increased target gene expression. We studied one target gene, CXCR4, in detail, showing that c-Myb associated with the CXCR4 gene promoter and activated a CXCR4 reporter gene in transfection assays. Our results show that c-Myb associates with a surprisingly large number of promoters in human cells. The results also suggest that estradiol stimulation leads to large-scale, genome-wide changes in c-Myb activity and subsequent changes in gene expression in human breast cancer cells

RNAi-Based Identification of Gene-Specific Nuclear Cofactor Networks Regulating Interleukin-1 Target Genes

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Johanna Meier-Soelch

2018-04-01

Full Text Available The potent proinflammatory cytokine interleukin (IL-1 triggers gene expression through the NF-κB signaling pathway. Here, we investigated the cofactor requirements of strongly regulated IL-1 target genes whose expression is impaired in p65 NF-κB-deficient murine embryonic fibroblasts. By two independent small-hairpin (shRNA screens, we examined 170 genes annotated to encode nuclear cofactors for their role in Cxcl2 mRNA expression and identified 22 factors that modulated basal or IL-1-inducible Cxcl2 levels. The functions of 16 of these factors were validated for Cxcl2 and further analyzed for their role in regulation of 10 additional IL-1 target genes by RT-qPCR. These data reveal that each inducible gene has its own (quantitative requirement of cofactors to maintain basal levels and to respond to IL-1. Twelve factors (Epc1, H2afz, Kdm2b, Kdm6a, Mbd3, Mta2, Phf21a, Ruvbl1, Sin3b, Suv420h1, Taf1, and Ube3a have not been previously implicated in inflammatory cytokine functions. Bioinformatics analysis indicates that they are components of complex nuclear protein networks that regulate chromatin functions and gene transcription. Collectively, these data suggest that downstream from the essential NF-κB signal each cytokine-inducible target gene has further subtle requirements for individual sets of nuclear cofactors that shape its transcriptional activation profile.

Identification of genes potentially involved in solute stress response in Sphingomonas wittichii RW1 by transposon mutant recovery

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Edith eCoronado

2014-11-01

Full Text Available The term water stress refers to the effects of low water availability on microbial growth and physiology. Water availability has been proposed as a major constraint for the use of microorganisms in contaminated sites with the purpose of bioremediation. Sphingomonas wittichii RW1 is a bacterium capable of degrading the xenobiotic compounds dibenzofuran and dibenzo-p-dioxin, and has potential to be used for targeted bioremediation. The aim of the current work was to identify genes implicated in water stress in RW1 by means of transposon mutagenesis and mutant growth experiments. Conditions of low water potential were mimicked by adding NaCl to the growth media. Three different mutant selection or separation method were tested, which, however recovered different mutants. Recovered transposon mutants with poorer growth under salt-induced water stress carried insertions in genes involved in proline and glutamate biosynthesis, and further in a gene putatively involved in aromatic compound catabolism. Transposon mutants growing poorer on medium with lowered water potential also included ones that had insertions in genes involved in more general functions such as transcriptional regulation, elongation factor, cell division protein, RNA polymerase β or an aconitase.

Genomewide Analysis of Aryl Hydrocarbon Receptor Binding Targets Reveals an Extensive Array of Gene Clusters that Control Morphogenetic and Developmental Programs

Science.gov (United States)

Sartor, Maureen A.; Schnekenburger, Michael; Marlowe, Jennifer L.; Reichard, John F.; Wang, Ying; Fan, Yunxia; Ma, Ci; Karyala, Saikumar; Halbleib, Danielle; Liu, Xiangdong; Medvedovic, Mario; Puga, Alvaro

2009-01-01

Background The vertebrate aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates cellular responses to environmental polycyclic and halogenated compounds. The naive receptor is believed to reside in an inactive cytosolic complex that translocates to the nucleus and induces transcription of xenobiotic detoxification genes after activation by ligand. Objectives We conducted an integrative genomewide analysis of AHR gene targets in mouse hepatoma cells and determined whether AHR regulatory functions may take place in the absence of an exogenous ligand. Methods The network of AHR-binding targets in the mouse genome was mapped through a multipronged approach involving chromatin immunoprecipitation/chip and global gene expression signatures. The findings were integrated into a prior functional knowledge base from Gene Ontology, interaction networks, Kyoto Encyclopedia of Genes and Genomes pathways, sequence motif analysis, and literature molecular concepts. Results We found the naive receptor in unstimulated cells bound to an extensive array of gene clusters with functions in regulation of gene expression, differentiation, and pattern specification, connecting multiple morphogenetic and developmental programs. Activation by the ligand displaced the receptor from some of these targets toward sites in the promoters of xenobiotic metabolism genes. Conclusions The vertebrate AHR appears to possess unsuspected regulatory functions that may be potential targets of environmental injury. PMID:19654925

Non-viral gene therapy that targets motor neurons in vivo

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Mary-Louise eRogers

2014-10-01

Full Text Available A major challenge in neurological gene therapy is safe delivery of transgenes to sufficient cell numbers from the circulation or periphery. This is particularly difficult for diseases involving spinal cord motor neurons such as amyotrophic lateral sclerosis (ALS. We have examined the feasibility of non-viral gene delivery to spinal motor neurons from intraperitoneal injections of plasmids carried by ‘immunogene’ nanoparticles targeted for axonal retrograde transport using antibodies. PEGylated polyethylenimine (PEI-PEG12 as DNA carrier was conjugated to an antibody (MLR2 to the neurotrophin receptor p75 (p75NTR. We used a plasmid (pVIVO2 designed for in vivo gene delivery that produces minimal immune responses, has improved nuclear entry into post mitotic cells and also expresses green fluorescent protein (GFP. MLR2-PEI-PEG12 carried pVIVO2 and was specific for mouse motor neurons in mixed cultures containing astrocytes. While only 8% of motor neurons expressed GFP 72 h post transfection in vitro, when the immunogene was given intraperitonealy to neonatal C57BL/6J mice GFP specific motor neuron expression was observed in 25.4% of lumbar, 18.3% of thoracic and 17.0 % of cervical motor neurons, 72 h post transfection. PEI-PEG12 carrying pVIVO2 by itself did not transfect motor neurons in vivo, demonstrating the need for specificity via the p75NTR antibody MLR2. This is the first time that specific transfection of spinal motor neurons has been achieved from peripheral delivery of plasmid DNA as part of a non-viral gene delivery agent. These results stress the specificity and feasibility of immunogene delivery targeted for p75NTR expressing motor neurons, but suggests that further improvements are required to increase the transfection efficiency of motor neurons in vivo.

A Genome-Wide Identification of the WRKY Family Genes and a Survey of Potential WRKY Target Genes in Dendrobium officinale.

Science.gov (United States)

He, Chunmei; Teixeira da Silva, Jaime A; Tan, Jianwen; Zhang, Jianxia; Pan, Xiaoping; Li, Mingzhi; Luo, Jianping; Duan, Jun

2017-08-23

The WRKY family, one of the largest families of transcription factors, plays important roles in the regulation of various biological processes, including growth, development and stress responses in plants. In the present study, 63 DoWRKY genes were identified from the Dendrobium officinale genome. These were classified into groups I, II, III and a non-group, each with 14, 28, 10 and 11 members, respectively. ABA-responsive, sulfur-responsive and low temperature-responsive elements were identified in the 1-k upstream regulatory region of DoWRKY genes. Subsequently, the expression of the 63 DoWRKY genes under cold stress was assessed, and the expression profiles of a large number of these genes were regulated by low temperature in roots and stems. To further understand the regulatory mechanism of DoWRKY genes in biological processes, potential WRKY target genes were investigated. Among them, most stress-related genes contained multiple W-box elements in their promoters. In addition, the genes involved in polysaccharide synthesis and hydrolysis contained W-box elements in their 1-k upstream regulatory regions, suggesting that DoWRKY genes may play a role in polysaccharide metabolism. These results provide a basis for investigating the function of WRKY genes and help to understand the downstream regulation network in plants within the Orchidaceae.

A targeted resequencing gene panel for focal epilepsy.

Science.gov (United States)

Hildebrand, Michael S; Myers, Candace T; Carvill, Gemma L; Regan, Brigid M; Damiano, John A; Mullen, Saul A; Newton, Mark R; Nair, Umesh; Gazina, Elena V; Milligan, Carol J; Reid, Christopher A; Petrou, Steven; Scheffer, Ingrid E; Berkovic, Samuel F; Mefford, Heather C

2016-04-26

We report development of a targeted resequencing gene panel for focal epilepsy, the most prevalent phenotypic group of the epilepsies. The targeted resequencing gene panel was designed using molecular inversion probe (MIP) capture technology and sequenced using massively parallel Illumina sequencing. We demonstrated proof of principle that mutations can be detected in 4 previously genotyped focal epilepsy cases. We searched for both germline and somatic mutations in 251 patients with unsolved sporadic or familial focal epilepsy and identified 11 novel or very rare missense variants in 5 different genes: CHRNA4, GRIN2B, KCNT1, PCDH19, and SCN1A. Of these, 2 were predicted to be pathogenic or likely pathogenic, explaining ∼0.8% of the cohort, and 8 were of uncertain significance based on available data. We have developed and validated a targeted resequencing panel for focal epilepsies, the most important clinical class of epilepsies, accounting for about 60% of all cases. Our application of MIP technology is an innovative approach that will be advantageous in the clinical setting because it is highly sensitive, efficient, and cost-effective for screening large patient cohorts. Our findings indicate that mutations in known genes likely explain only a small proportion of focal epilepsy cases. This is not surprising given the established clinical and genetic heterogeneity of these disorders and underscores the importance of further gene discovery studies in this complex syndrome. © 2016 American Academy of Neurology.

Anti-EGFR immunonanoparticles containing IL12 and salmosin genes for targeted cancer gene therapy.

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Kim, Jung Seok; Kang, Seong Jae; Jeong, Hwa Yeon; Kim, Min Woo; Park, Sang Il; Lee, Yeon Kyung; Kim, Hong Sung; Kim, Keun Sik; Park, Yong Serk

2016-09-01

Tumor-directed gene delivery is of major interest in the field of cancer gene therapy. Varied functionalizations of non-viral vectors have been suggested to enhance tumor targetability. In the present study, we prepared two different types of anti-EGF receptor (EGFR) immunonanoparticles containing pDNA, neutrally charged liposomes and cationic lipoplexes, for tumor-directed transfection of cancer therapeutic genes. Even though both anti-EGFR immunonanoparticles had a high binding affinity to the EGFR-positive cancer cells, the anti-EGFR immunolipoplex formulation exhibited approximately 100-fold higher transfection to the target cells than anti-EGFR immunoliposomes. The lipoplex formulation also showed a higher transfection to SK-OV-3 tumor xenografts in mice. Thus, IL12 and/or salmosin genes were loaded in the anti-EGFR immunolipoplexes and intravenously administered to mice carrying SK-OV-3 tumors. Co-transfection of IL12 and salmosin genes using anti-EGFR immunolipoplexes significantly reduced tumor growth and pulmonary metastasis. Furthermore, combinatorial treatment with doxorubicin synergistically inhibited tumor growth. These results suggest that anti-EGFR immunolipoplexes containing pDNA encoding therapeutic genes could be utilized as a gene-transfer modality for cancer gene therapy.

Immuno-Oncology-The Translational Runway for Gene Therapy: Gene Therapeutics to Address Multiple Immune Targets.

Science.gov (United States)

Weß, Ludger; Schnieders, Frank

2017-12-01

Cancer therapy is once again experiencing a paradigm shift. This shift is based on extensive clinical experience demonstrating that cancer cannot be successfully fought by addressing only single targets or pathways. Even the combination of several neo-antigens in cancer vaccines is not sufficient for successful, lasting tumor eradication. The focus has therefore shifted to the immune system's role in cancer and the striking abilities of cancer cells to manipulate and/or deactivate the immune system. Researchers and pharma companies have started to target the processes and cells known to support immune surveillance and the elimination of tumor cells. Immune processes, however, require novel concepts beyond the traditional "single-target-single drug" paradigm and need parallel targeting of diverse cells and mechanisms. This review gives a perspective on the role of gene therapy technologies in the evolving immuno-oncology space and identifies gene therapy as a major driver in the development and regulation of effective cancer immunotherapy. Present challenges and breakthroughs ranging from chimeric antigen receptor T-cell therapy, gene-modified oncolytic viruses, combination cancer vaccines, to RNA therapeutics are spotlighted. Gene therapy is recognized as the most prominent technology enabling effective immuno-oncology strategies.

In silico identification of miRNAs and their target genes and analysis of gene co-expression network in saffron (Crocus sativus L.) stigma

Science.gov (United States)

Zinati, Zahra; Shamloo-Dashtpagerdi, Roohollah; Behpouri, Ali

2016-01-01

As an aromatic and colorful plant of substantive taste, saffron (Crocus sativus L.) owes such properties of matter to growing class of the secondary metabolites derived from the carotenoids, apocarotenoids. Regarding the critical role of microRNAs in secondary metabolic synthesis and the limited number of identified miRNAs in C. sativus, on the other hand, one may see the point how the characterization of miRNAs along with the corresponding target genes in C. sativus might expand our perspectives on the roles of miRNAs in carotenoid/apocarotenoid biosynthetic pathway. A computational analysis was used to identify miRNAs and their targets using EST (Expressed Sequence Tag) library from mature saffron stigmas. Then, a gene co- expression network was constructed to identify genes which are potentially involved in carotenoid/apocarotenoid biosynthetic pathways. EST analysis led to the identification of two putative miRNAs (miR414 and miR837-5p) along with the corresponding stem- looped precursors. To our knowledge, this is the first report on miR414 and miR837-5p in C. sativus. Co-expression network analysis indicated that miR414 and miR837-5p may play roles in C. sativus metabolic pathways and led to identification of candidate genes including six transcription factors and one protein kinase probably involved in carotenoid/apocarotenoid biosynthetic pathway. Presence of transcription factors, miRNAs and protein kinase in the network indicated multiple layers of regulation in saffron stigma. The candidate genes from this study may help unraveling regulatory networks underlying the carotenoid/apocarotenoid biosynthesis in saffron and designing metabolic engineering for enhanced secondary metabolites. PMID:28261627

Re-Factoring Glycolytic Genes for Targeted Engineering of Catabolism in Gram-Negative Bacteria

DEFF Research Database (Denmark)

Sánchez-Pascuala, Alberto; Nikel, Pablo I.; de Lorenzo, Víctor

2018-01-01

the potential applications of such a portable tool for targeted pathway engineering, in the present protocol we describe how the genes encoding all the enzymes of the linear EMP route have been individually recruited from the genome of E. coli K-12, edited in silico to remove their endogenous regulatory signals......The Embden-Meyerhof-Parnas (EMP) pathway is widely accepted to be the biochemical standard of glucose catabolism. The well-characterized glycolytic route of Escherichia coli, based on the EMP catabolism, is an example of an intricate pathway in terms of genomic organization of the genes involved...... and patterns of gene expression and regulation. This intrinsic genetic and metabolic complexity renders it difficult to engineer glycolytic activities and transfer them onto other microbial cell factories, thus limiting the biotechnological potential of bacterial hosts that lack the route. Taking into account...

Targeted gene panels and microbiota analysis provide insight into the effects of effects of alternative production diet formulations on channel catfish nutritional physiology

Science.gov (United States)

The present research evaluated targeted gene panels and microbiota analysis to provide greater insight into the effects of alternatively-sourced dietary ingredients on production indices, gut health, changes in the gut microbiota and genes involved in the regulation of appetite, growth, metabolism, ...

Inducement of radionuclides targeting therapy by gene transfection

International Nuclear Information System (INIS)

Luo Quanyong

2001-01-01

The author presents an overview of gene transfection methods to genetically induce tumor cells to express enhanced levels of cell surface antigens and receptors to intake radiolabeled antibody and peptide targeting and thus increase their therapeutic effect in radiotherapy. The current research include inducement of radioimmunotherapy through CEA gene transfection, inducement of iodine-131 therapy by sodium iodide symporter gene transfection and inducement of MIBG therapy by noradrenaline transporter gene transfection. These studies raise the prospect that gene-therapy techniques could be used to enable the treatment of a wide range of tumors with radiopharmaceuticals of established clinical acceptability

STAT3 Target Genes Relevant to Human Cancers

International Nuclear Information System (INIS)

Carpenter, Richard L.; Lo, Hui-Wen

2014-01-01

Since its discovery, the STAT3 transcription factor has been extensively studied for its function as a transcriptional regulator and its role as a mediator of development, normal physiology, and pathology of many diseases, including cancers. These efforts have uncovered an array of genes that can be positively and negatively regulated by STAT3, alone and in cooperation with other transcription factors. Through regulating gene expression, STAT3 has been demonstrated to play a pivotal role in many cellular processes including oncogenesis, tumor growth and progression, and stemness. Interestingly, recent studies suggest that STAT3 may behave as a tumor suppressor by activating expression of genes known to inhibit tumorigenesis. Additional evidence suggested that STAT3 may elicit opposing effects depending on cellular context and tumor types. These mixed results signify the need for a deeper understanding of STAT3, including its upstream regulators, parallel transcription co-regulators, and downstream target genes. To help facilitate fulfilling this unmet need, this review will be primarily focused on STAT3 downstream target genes that have been validated to associate with tumorigenesis and/or malignant biology of human cancers

Cloning and Characterizing Genes Involved in Monoterpene Induced Mammary Tumor Regression.

Science.gov (United States)

1996-10-01

AD GRANT NUMBER DAMDI7-94-J-4041 TITLE: Cloning and Characterizing Genes Involved in Monoterpene Induced Mammary Tumor Regression PRINCIPAL...October 1996 Annual (1 Sep 95 - 31 Aug 96) 4. TITLE AND SUBTITLE 5. FUNDING NUMBERS Cloning and Characterizing Genes Involved in Monoterpene Induced... Monoterpene -induced/repressed genes were identified in regressing rat mammary carcinomas treated with dietary limonene using a newly developed method

Magnetic nanoparticles for targeted therapeutic gene delivery and magnetic-inducing heating on hepatoma

International Nuclear Information System (INIS)

Yuan, Chenyan; Zhang, Jia; Li, Hongbo; Zhang, Hao; Wang, Ling; Zhang, Dongsheng; An, Yanli

2014-01-01

Gene therapy holds great promise for treating cancers, but their clinical applications are being hampered due to uncontrolled gene delivery and expression. To develop a targeted, safe and efficient tumor therapy system, we constructed a tissue-specific suicide gene delivery system by using magnetic nanoparticles (MNPs) as carriers for the combination of gene therapy and hyperthermia on hepatoma. The suicide gene was hepatoma-targeted and hypoxia-enhanced, and the MNPs possessed the ability to elevate temperature to the effective range for tumor hyperthermia as imposed on an alternating magnetic field (AMF). The tumoricidal effects of targeted gene therapy associated with hyperthermia were evaluated in vitro and in vivo. The experiment demonstrated that hyperthermia combined with a targeted gene therapy system proffer an effective tool for tumor therapy with high selectivity and the synergistic effect of hepatoma suppression. (paper)

Cloning, characterization and targeting of the mouse HEXA gene

Energy Technology Data Exchange (ETDEWEB)

Wakamatsu, N.; Trasler, J.M.; Gravel, R.A. [McGill Univ., Quebec (Canada)] [and others

1994-09-01

The HEXA gene, encoding the {alpha} subunit of {beta}-hexosaminidase A, is essential for the metabolism of ganglioside G{sub M2}, and defects in this gene cause Tay-Sachs disease in humans. To elucidate the role of the gene in the nervous system of the mouse and to establish a mouse model of Tay-Sachs disease, we have cloned and characterized the HEXA gene and targeted a disruption of the gene in mouse ES cells. The mouse HEXA gene spans {approximately}26 kb and consists of 14 exons, similar to the human gene. A heterogeneous transcription initiation site was identified 21-42 bp 5{prime} of the initiator ATG, with two of the sites fitting the consensus CTCA (A = start) as seen for some weak initiator systems. Promoter analysis showed that the first 150 bp 5{prime} of the ATG contained 85% of promoter activity observed in constructs containing up to 1050 bp of 5{prime} sequence. The active region contained a sequence matching that of the adenovirus major late promoter upstream element factor. A survey of mouse tissues showed that the highest mRNA levels were in (max to min): testis (5.5 x brain cortex), adrenal, epididymis, heart, brain, lung, kidney, and liver (0.3 x brain cortex). A 12 kb BstI/SalI fragment containing nine exons was disrupted with the insertion of the bacterial neo{sup r} gene in exon 11 and was targeted into 129/Sv ES cells by homologous recombination. Nine of 153 G418 resistant clones were correctly targeted as confirmed by Southern blotting. The heterozygous ES cells were microinjected into mouse blastocysts and implanted into pseudo-pregnant mice. Nine male chimeric mice, showing that 40-95% chimerism for the 129/Sv agouti coat color marker, are being bred in an effort to generate germline transmission of the disrupted HEXA gene.

Gene therapy and radionuclides targeting therapy in mammary carcinoma

International Nuclear Information System (INIS)

Song Jinhua

2003-01-01

Breast carcinoma's gene therapy is a hotspot in study of the tumor's therapy in the recent years. Currently the major therapy methods that in the experimentative and primary clinical application phases include immunological gene therapy, multidrug resistance gene therapy, antisense oligonucleotide therapy and suicide gene therapy. The gene targeting brachytherapy, which is combined with gene therapy and radiotherapy has enhanced the killer effects of the suicide gene and nuclide in tumor cells. That has break a new path in tumor's gene therapy. The further study in this field will step up it's space to the clinical application

Genes involved in Beauveria bassiana infection to Galleria mellonella.

Science.gov (United States)

Chen, Anhui; Wang, Yulong; Shao, Ying; Zhou, Qiumei; Chen, Shanglong; Wu, Yonghua; Chen, Hongwei; Liu, Enqi

2018-05-01

The ascomycete fungus Beauveria bassiana is a natural pathogen of hundreds of insect species and is commercially produced as an environmentally friendly mycoinsecticide. Many genes involved in fungal insecticide infection have been identified but few have been further explored. In this study, we constructed three transcriptomes of B. bassiana at 24, 48 and 72 h post infection of insect pests (BbI) or control (BbC). There were 3148, 3613 and 4922 genes differentially expressed at 24, 48 and 72 h post BbI/BbC infection, respectively. A large number of genes and pathways involved in infection were identified. To further analyze those genes, expression patterns across different infection stages (0, 12, 24, 36, 48, 60, 72 and 84 h) were studied using quantitative RT-PCR. This analysis showed that the infection-related genes could be divided into four patterns: highly expressed throughout the whole infection process (thioredoxin 1); highly expressed during early stages of infection but lowly expressed after the insect death (adhesin protein Mad1); lowly expressed during early infection but highly expressed after insect death (cation transporter, OpS13); or lowly expressed across the entire infection process (catalase protein). The data provide novel insights into the insect-pathogen interaction and help to uncover the molecular mechanisms involved in fungal infection of insect pests.

Identification of novel target genes involved in Indian Fanconi anemia patients using microarray.

Science.gov (United States)

Shyamsunder, Pavithra; Ganesh, Kripa S; Vidyasekar, Prasanna; Mohan, Sheila; Verma, Rama Shanker

2013-12-01

Fanconi anemia (FA) is a genetic disorder characterized by progressive bone marrow failure and a predisposition to cancers. Mutations have been documented in 15 FA genes that participate in the FA-BRCA DNA repair pathway, a fundamental pathway in the development of the disease and the presentation of its characteristic symptoms. Certain symptoms such as oxygen sensitivity, hematological abnormalities and impaired immunity suggest that FA proteins could participate in or independently control other pathways as well. In this study, we identified 9 DNA repair genes that were down regulated in a genome wide analysis of 6 Indian Fanconi anemia patients. Functional clustering of a total of 233 dysregulated genes identified key biological processes that included regulation of transcription, DNA repair, cell cycle and chromosomal organization. Microarray data revealed the down regulation of ATXN3, ARID4A and ETS-1, which were validated by RTPCR in a subsequent sample set of 9 Indian FA patients. Here we report for the first time a gene expression profile of Fanconi anemia patients from the Indian population and a pool of genes that might aid in the acquisition and progression of the FA phenotype. © 2013 Elsevier B.V. All rights reserved.

Expression profiling identifies genes involved in emphysema severity

Directory of Open Access Journals (Sweden)

Bowman Rayleen V

2009-09-01

Full Text Available Abstract Chronic obstructive pulmonary disease (COPD is a major public health problem. The aim of this study was to identify genes involved in emphysema severity in COPD patients. Gene expression profiling was performed on total RNA extracted from non-tumor lung tissue from 30 smokers with emphysema. Class comparison analysis based on gas transfer measurement was performed to identify differentially expressed genes. Genes were then selected for technical validation by quantitative reverse transcriptase-PCR (qRT-PCR if also represented on microarray platforms used in previously published emphysema studies. Genes technically validated advanced to tests of biological replication by qRT-PCR using an independent test set of 62 lung samples. Class comparison identified 98 differentially expressed genes (p p Gene expression profiling of lung from emphysema patients identified seven candidate genes associated with emphysema severity including COL6A3, SERPINF1, ZNHIT6, NEDD4, CDKN2A, NRN1 and GSTM3.

Identification of the genes involved in odorant reception and detection in the palm weevil Rhynchophorus ferrugineus, an important quarantine pest, by antennal transcriptome analysis

KAUST Repository

Antony, Binu; Soffan, Alan; Jakše, Jernej; Abdelazim, Mahmoud M.; Aldosari, Saleh A.; Aldawood, Abdulrahman S.; Pain, Arnab

2016-01-01

Our study presents the first comprehensive catalogue of olfactory gene families involved in pheromone and general odorant detection in R. ferrugineus, which are potential novel targets for pest control strategies.

Cis-regulatory element based targeted gene finding: genome-wide identification of abscisic acid- and abiotic stress-responsive genes in Arabidopsis thaliana.

Science.gov (United States)

Zhang, Weixiong; Ruan, Jianhua; Ho, Tuan-Hua David; You, Youngsook; Yu, Taotao; Quatrano, Ralph S

2005-07-15

A fundamental problem of computational genomics is identifying the genes that respond to certain endogenous cues and environmental stimuli. This problem can be referred to as targeted gene finding. Since gene regulation is mainly determined by the binding of transcription factors and cis-regulatory DNA sequences, most existing gene annotation methods, which exploit the conservation of open reading frames, are not effective in finding target genes. A viable approach to targeted gene finding is to exploit the cis-regulatory elements that are known to be responsible for the transcription of target genes. Given such cis-elements, putative target genes whose promoters contain the elements can be identified. As a case study, we apply the above approach to predict the genes in model plant Arabidopsis thaliana which are inducible by a phytohormone, abscisic acid (ABA), and abiotic stress, such as drought, cold and salinity. We first construct and analyze two ABA specific cis-elements, ABA-responsive element (ABRE) and its coupling element (CE), in A.thaliana, based on their conservation in rice and other cereal plants. We then use the ABRE-CE module to identify putative ABA-responsive genes in A.thaliana. Based on RT-PCR verification and the results from literature, this method has an accuracy rate of 67.5% for the top 40 predictions. The cis-element based targeted gene finding approach is expected to be widely applicable since a large number of cis-elements in many species are available.

Plant Genes Involved in Symbiotic Sinal Perception/Signal Transduction

DEFF Research Database (Denmark)

Binder, A; Soyano, T; Hayashi, H

2014-01-01

to nodule primordia formation, and the infection thread initiation in the root hairs guiding bacteria towards dividing cortical cells. This chapter focuses on the plant genes involved in the recognition of the symbiotic signal produced by rhizobia, and the downstream genes, which are part of a complex...... symbiotic signalling pathway that leads to the generation of calcium spiking in the nuclear regions and activation of transcription factors controlling symbiotic genes induction...

Identification and profiling of microRNAs and their target genes from developing caprine skeletal Muscle.

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Yanhong Wang

Full Text Available Goat is an important agricultural animal for meat production. Functional studies have demonstrated that microRNAs (miRNAs regulate gene expression at the post-transcriptional level and play an important role in various biological processes. Although studies on miRNAs expression profiles have been performed in various animals, relatively limited information about goat muscle miRNAs has been reported. To investigate the miRNAs involved in regulating different periods of skeletal muscle development, we herein performed a comprehensive research for expression profiles of caprine miRNAs during two developmental stages of skeletal muscles: fetal stage and six month-old stage. As a result, 15,627,457 and 15,593,721 clean reads were obtained from the fetal goat library (FC and the six month old goat library (SMC, respectively. 464 known miRNAs and 83 novel miRNA candidates were identified. Furthermore, by comparing the miRNA profile, 336 differentially expressed miRNAs were identified and then the potential targets of the differentially expressed miRNAs were predicted. To understand the regulatory network of miRNAs during muscle development, the mRNA expression profiles for the two development stages were characterized and 7322 differentially expressed genes (DEGs were identified. Then the potential targets of miRNAs were compared to the DEGs, the intersection of the two gene sets were screened out and called differentially expressed targets (DE-targets, which were involved in 231 pathways. Ten of the 231 pathways that have smallest P-value were shown as network figures. Based on the analysis of pathways and networks, we found that miR-424-5p and miR-29a might have important regulatory effect on muscle development, which needed to be further studied. This study provided the first global view of the miRNAs in caprine muscle tissues. Our results help elucidation of complex regulatory networks between miRNAs and mRNAs and for the study of muscle

Gene silencing in Tribolium castaneum as a tool for the targeted identification of candidate RNAi targets in crop pests.

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Knorr, Eileen; Fishilevich, Elane; Tenbusch, Linda; Frey, Meghan L F; Rangasamy, Murugesan; Billion, Andre; Worden, Sarah E; Gandra, Premchand; Arora, Kanika; Lo, Wendy; Schulenberg, Greg; Valverde-Garcia, Pablo; Vilcinskas, Andreas; Narva, Kenneth E

2018-02-01

RNAi shows potential as an agricultural technology for insect control, yet, a relatively low number of robust lethal RNAi targets have been demonstrated to control insects of agricultural interest. In the current study, a selection of lethal RNAi target genes from the iBeetle (Tribolium castaneum) screen were used to demonstrate efficacy of orthologous targets in the economically important coleopteran pests Diabrotica virgifera virgifera and Meligethes aeneus. Transcript orthologs of 50 selected genes were analyzed in D. v. virgifera diet-based RNAi bioassays; 21 of these RNAi targets showed mortality and 36 showed growth inhibition. Low dose injection- and diet-based dsRNA assays in T. castaneum and D. v. virgifera, respectively, enabled the identification of the four highly potent RNAi target genes: Rop, dre4, ncm, and RpII140. Maize was genetically engineered to express dsRNA directed against these prioritized candidate target genes. T 0 plants expressing Rop, dre4, or RpII140 RNA hairpins showed protection from D. v. virgifera larval feeding damage. dsRNA targeting Rop, dre4, ncm, and RpII140 in M. aeneus also caused high levels of mortality both by injection and feeding. In summary, high throughput systems for model organisms can be successfully used to identify potent RNA targets for difficult-to-work with agricultural insect pests.

Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands.

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Luo, Yan; Wang, Yongsheng; Liu, Jun; Cui, Chenchen; Wu, Yongyan; Lan, Hui; Chen, Qi; Liu, Xu; Quan, Fusheng; Guo, Zekun; Zhang, Yong

2016-02-08

Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription-activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock.

Temporal expression of genes involved in the biosynthesis of ...

African Journals Online (AJOL)

Gibberellins (GAs) are a large family of endogenous plant growth regulators. Bioactive GAs influence nearly all processes during plant growth and development. In the present study, we cloned and identified 10 unique genes that are potentially involved in the biosynthesis of GAs, including one BpGGDP gene, two BpCPS ...

Comparative genomics identification of a novel set of temporally regulated hedgehog target genes in the retina.

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McNeill, Brian; Perez-Iratxeta, Carol; Mazerolle, Chantal; Furimsky, Marosh; Mishina, Yuji; Andrade-Navarro, Miguel A; Wallace, Valerie A

2012-03-01

The hedgehog (Hh) signaling pathway is involved in numerous developmental and adult processes with many links to cancer. In vertebrates, the activity of the Hh pathway is mediated primarily through three Gli transcription factors (Gli1, 2 and 3) that can serve as transcriptional activators or repressors. The identification of Gli target genes is essential for the understanding of the Hh-mediated processes. We used a comparative genomics approach using the mouse and human genomes to identify 390 genes that contained conserved Gli binding sites. RT-qPCR validation of 46 target genes in E14.5 and P0.5 retinal explants revealed that Hh pathway activation resulted in the modulation of 30 of these targets, 25 of which demonstrated a temporal regulation. Further validation revealed that the expression of Bok, FoxA1, Sox8 and Wnt7a was dependent upon Sonic Hh (Shh) signaling in the retina and their regulation is under positive and negative controls by Gli2 and Gli3, respectively. We also show using chromatin immunoprecipitation that Gli2 binds to the Sox8 promoter, suggesting that Sox8 is an Hh-dependent direct target of Gli2. Finally, we demonstrate that the Hh pathway also modulates the expression of Sox9 and Sox10, which together with Sox8 make up the SoxE group. Previously, it has been shown that Hh and SoxE group genes promote Müller glial cell development in the retina. Our data are consistent with the possibility for a role of SoxE group genes downstream of Hh signaling on Müller cell development. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

Self-focusing therapeutic gene delivery with intelligent gene vector swarms: intra-swarm signalling through receptor transgene expression in targeted cells.

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Tolmachov, Oleg E

2015-01-01

Gene delivery in vivo that is tightly focused on the intended target cells is essential to maximize the benefits of gene therapy and to reduce unwanted side-effects. Cell surface markers are immediately available for probing by therapeutic gene vectors and are often used to direct gene transfer with these vectors to specific target cell populations. However, it is not unusual for the choice of available extra-cellular markers to be too scarce to provide a reliable definition of the desired therapeutically relevant set of target cells. Therefore, interrogation of intra-cellular determinants of cell-specificity, such as tissue-specific transcription factors, can be vital in order to provide detailed cell-guiding information to gene vector particles. An important improvement in cell-specific gene delivery can be achieved through auto-buildup in vector homing efficiency using intelligent 'self-focusing' of swarms of vector particles on target cells. Vector self-focusing was previously suggested to rely on the release of diffusible chemo-attractants after a successful target-specific hit by 'scout' vector particles. I hypothesize that intelligent self-focusing behaviour of swarms of cell-targeted therapeutic gene vectors can be accomplished without the employment of difficult-to-use diffusible chemo-attractants, instead relying on the intra-swarm signalling through cells expressing a non-diffusible extra-cellular receptor for the gene vectors. In the proposed model, cell-guiding information is gathered by the 'scout' gene vector particles, which: (1) attach to a variety of cells via a weakly binding (low affinity) receptor; (2) successfully facilitate gene transfer into these cells; (3) query intra-cellular determinants of cell-specificity with their transgene expression control elements and (4) direct the cell-specific biosynthesis of a vector-encoded strongly binding (high affinity) cell-surface receptor. Free members of the vector swarm loaded with therapeutic cargo

Cancer gene therapy targeting angiogenesis: An updated Review

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Liu, Ching-Chiu; Shen, Zan; Kung, Hsiang-Fu; Lin, Marie CM

2006-01-01

Since the relationship between angiogenesis and tumor growth was established by Folkman in 1971, scientists have made efforts exploring the possibilities in treating cancer by targeting angiogenesis. Inhibition of angiogenesis growth factors and administration of angiogenesis inhibitors are the basics of anti-angiogenesis therapy. Transfer of anti-angiogenesis genes has received attention recently not only because of the advancement of recombinant vectors, but also because of the localized and sustained expression of therapeutic gene product inside the tumor after gene transfer. This review provides the up-to-date information about the strategies and the vectors studied in the field of anti-angiogenesis cancer gene therapy. PMID:17109514

New gene targets for glucagon-like peptide-1 during embryonic development and in undifferentiated pluripotent cells.

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Sanz, Carmen; Blázquez, Enrique

2011-09-01

In humans, glucagon-like peptide (GLP-1) functions during adult life as an incretin hormone with anorexigenic and antidiabetogenic properties. Also, the therapeutic potential of GLP-1 in preventing the adipocyte hyperplasia associated with obesity and in bolstering the maintenance of human mesenchymal stem cell (hMSC) stores by promoting the proliferation and cytoprotection of hMSC seems to be relevant. Since these observations suggest a role for GLP-1 during developmental processes, the aim of the present work was to characterize GLP-1 in early development as well as its gene targets in mouse embryonic stem (mES) cells. Mouse embryos E6, E8, and E10.5 and pluripotent mES were used for the inmunodetection of GLP-1 and GLP-1 receptor. Quantitative real-time PCR was used to determine the expression levels of GLP-1R in several tissues from E12.5 mouse embryos. Additionally, GLP-1 gene targets were studied in mES by multiple gene expression analyses. GLP-1 and its receptors were identified in mES and during embryonic development. In pluripotent mES, GLP-1 modified the expression of endodermal, ectodermal, and mesodermal gene markers as well as sonic hedgehog, noggin, members of the fibroblast and hepatic growth factor families, and others involved in pancreatic development. Additionally, GLP-1 promoted the expression of the antiapoptotic gene bcl2 and at the same time reduced proapoptotic caspase genes. Our results indicate that apart from the effects and therapeutic benefits of GLP-1 in adulthood, it may have additional gene targets in mES cells during embryonic life. Furthermore, the pathophysiological implications of GLP-1 imbalance in adulthood may have a counterpart during development.

EBF factors drive expression of multiple classes of target genes governing neuronal development.

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Green, Yangsook S; Vetter, Monica L

2011-04-30

Early B cell factor (EBF) family members are transcription factors known to have important roles in several aspects of vertebrate neurogenesis, including commitment, migration and differentiation. Knowledge of how EBF family members contribute to neurogenesis is limited by a lack of detailed understanding of genes that are transcriptionally regulated by these factors. We performed a microarray screen in Xenopus animal caps to search for targets of EBF transcriptional activity, and identified candidate targets with multiple roles, including transcription factors of several classes. We determined that, among the most upregulated candidate genes with expected neuronal functions, most require EBF activity for some or all of their expression, and most have overlapping expression with ebf genes. We also found that the candidate target genes that had the most strongly overlapping expression patterns with ebf genes were predicted to be direct transcriptional targets of EBF transcriptional activity. The identification of candidate targets that are transcription factor genes, including nscl-1, emx1 and aml1, improves our understanding of how EBF proteins participate in the hierarchy of transcription control during neuronal development, and suggests novel mechanisms by which EBF activity promotes migration and differentiation. Other candidate targets, including pcdh8 and kcnk5, expand our knowledge of the types of terminal differentiated neuronal functions that EBF proteins regulate.

Mitochondrial targeted neuron focused genes in hippocampus of rats with traumatic brain injury.

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Sharma, Pushpa; Su, Yan A; Barry, Erin S; Grunberg, Neil E; Lei, Zhang

2012-09-01

Mild traumatic brain injury (mTBI) represents a major health problem in civilian populations as well as among the military service members due to (1) lack of effective treatments, and (2) our incomplete understanding about the progression of secondary cell injury cascades resulting in neuronal cell death due to deficient cellular energy metabolism and damaged mitochondria. The aim of this study was to identify and delineate the mitochondrial targeted genes responsible for altered brain energy metabolism in the injured brain. Rats were either grouped into naïve controls or received lateral fluid percussion brain injury (2-2.5 atm) and followed up for 7 days. Rats were either grouped into naïve controls or received lateral fluid percussion brain injury (2-2.5 atm) and followed for 7 days. The severity of brain injury was evaluated by the neurological severity scale-revised (NSS-R) at 3 and 5 days post TBI and immunohistochemical analyses at 7 days post TBI. The expression profiles of mitochondrial-targeted genes across the hippocampus from TBI and naïe rats were also examined by oligo-DNA microarrays. NSS-R scores of TBI rats (5.4 ± 0.5) in comparison to naïe rats (3.9 ± 0.5) and H and E staining of brain sections suggested a mild brain injury. Bioinformatics and systems biology analyses showed 31 dysregulated genes, 10 affected canonical molecular pathways including a number of genes involved in mitochondrial enzymes for oxidative phosphorylation, mitogen-activated protein Kinase (MAP), peroxisome proliferator-activated protein (PPAP), apoptosis signaling, and genes responsible for long-term potentiation of Alzheimer's and Parkinson's diseases. Our results suggest that dysregulated mitochondrial-focused genes in injured brains may have a clinical utility for the development of future therapeutic strategies aimed at the treatment of TBI.

Discovery of a novel gene involved in autolysis of Clostridium cells.

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Yang, Liejian; Bao, Guanhui; Zhu, Yan; Dong, Hongjun; Zhang, Yanping; Li, Yin

2013-06-01

Cell autolysis plays important physiological roles in the life cycle of clostridial cells. Understanding the genetic basis of the autolysis phenomenon of pathogenic Clostridium or solvent producing Clostridium cells might provide new insights into this important species. Genes that might be involved in autolysis of Clostridium acetobutylicum, a model clostridial species, were investigated in this study. Twelve putative autolysin genes were predicted in C. acetobutylicum DSM 1731 genome through bioinformatics analysis. Of these 12 genes, gene SMB_G3117 was selected for testing the in tracellular autolysin activity, growth profile, viable cell numbers, and cellular morphology. We found that overexpression of SMB_G3117 gene led to earlier ceased growth, significantly increased number of dead cells, and clear electrolucent cavities, while disruption of SMB_G3117 gene exhibited remarkably reduced intracellular autolysin activity. These results indicate that SMB_G3117 is a novel gene involved in cellular autolysis of C. acetobutylicum.

Gene Dosage Analysis in a Clinical Environment: Gene-Targeted Microarrays as the Platform-of-Choice

Directory of Open Access Journals (Sweden)

Donald R. Love

2013-03-01

Full Text Available The role of gene deletion and duplication in the aetiology of disease has become increasingly evident over the last decade. In addition to the classical deletion/duplication disorders diagnosed using molecular techniques, such as Duchenne Muscular Dystrophy and Charcot-Marie-Tooth Neuropathy Type 1A, the significance of partial or whole gene deletions in the pathogenesis of a large number single-gene disorders is becoming more apparent. A variety of dosage analysis methods are available to the diagnostic laboratory but the widespread application of many of these techniques is limited by the expense of the kits/reagents and restrictive targeting to a particular gene or portion of a gene. These limitations are particularly important in the context of a small diagnostic laboratory with modest sample throughput. We have developed a gene-targeted, custom-designed comparative genomic hybridisation (CGH array that allows twelve clinical samples to be interrogated simultaneously for exonic deletions/duplications within any gene (or panel of genes on the array. We report here on the use of the array in the analysis of a series of clinical samples processed by our laboratory over a twelve-month period. The array has proven itself to be robust, flexible and highly suited to the diagnostic environment.

Regulatory network analysis of Epstein-Barr virus identifies functional modules and hub genes involved in infectious mononucleosis.

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Poorebrahim, Mansour; Salarian, Ali; Najafi, Saeideh; Abazari, Mohammad Foad; Aleagha, Maryam Nouri; Dadras, Mohammad Nasr; Jazayeri, Seyed Mohammad; Ataei, Atousa; Poortahmasebi, Vahdat

2017-05-01

Epstein-Barr virus (EBV) is the most common cause of infectious mononucleosis (IM) and establishes lifetime infection associated with a variety of cancers and autoimmune diseases. The aim of this study was to develop an integrative gene regulatory network (GRN) approach and overlying gene expression data to identify the representative subnetworks for IM and EBV latent infection (LI). After identifying differentially expressed genes (DEGs) in both IM and LI gene expression profiles, functional annotations were applied using gene ontology (GO) and BiNGO tools, and construction of GRNs, topological analysis and identification of modules were carried out using several plugins of Cytoscape. In parallel, a human-EBV GRN was generated using the Hu-Vir database for further analyses. Our analysis revealed that the majority of DEGs in both IM and LI were involved in cell-cycle and DNA repair processes. However, these genes showed a significant negative correlation in the IM and LI states. Furthermore, cyclin-dependent kinase 2 (CDK2) - a hub gene with the highest centrality score - appeared to be the key player in cell cycle regulation in IM disease. The most significant functional modules in the IM and LI states were involved in the regulation of the cell cycle and apoptosis, respectively. Human-EBV network analysis revealed several direct targets of EBV proteins during IM disease. Our study provides an important first report on the response to IM/LI EBV infection in humans. An important aspect of our data was the upregulation of genes associated with cell cycle progression and proliferation.

Mapping of HNF4alpha target genes in intestinal epithelial cells

DEFF Research Database (Denmark)

Boyd, Mette; Bressendorff, Simon; Moller, Jette

2009-01-01

ABSTRACT: BACKGROUND: The role of HNF4alpha has been extensively studied in hepatocytes and pancreatic beta-cells, and HNF4alpha is also regarded as key regulator of intestinal epithelial cell differentiation as well. The aim of the present work is to identify novel HNF4alpha target genes....... The HNF4alpha ChIP-chip data was matched with gene expression and histone H3 acetylation status of the promoters in order to identify HNF4alpha binding to actively transcribed genes with an open chromatin structure. RESULTS: 1,541 genes were identified as potential HNF4alpha targets, many of which have...

Towards prostate cancer gene therapy: Development of a chlorotoxin-targeted nanovector for toxic (melittin) gene delivery.

Science.gov (United States)

Tarokh, Zahra; Naderi-Manesh, Hossein; Nazari, Mahboobeh

2017-03-01

Prostate cancer is the second leading cause of death due to cancer in men. Owing to shortcomings in the current treatments, other therapies are being considered. Toxic gene delivery is one of the most effective methods for cancer therapy. Cationic polymers are able to form stable nanoparticles via interaction with nucleic acids electrostatically. Branched polyethylenimine that contains amine groups has notable buffering capacity and the ability to escape from endosome through the proton sponge effect. However, the cytotoxicity of this polymer is high, and modification is one of the applicable strategies to overcome this problem. In this study, PEI was targeted with chlorotoxin (CTX) via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) cross-linker. CTX can bind specifically to matrix metalloproteinase-2 that is overexpressed in certain cancers. Melittin as the major component of bee venom has been reported to have anti-cancer activity. This was thus selected to deliver to PC3 cell line. Flow cytometry analysis revealed that transfection efficiency of targeted nanoparticles is significantly higher compared to non-targeted nanoparticles. Targeted nanoparticles carrying the melittin gene also decreased cell viability of PC3 cells significantly while no toxic effects were observed on NIH3T3 cell line. Therefore, CTX-targeted nanoparticles carrying the melittin gene could serve as an appropriate gene delivery system for prostate and other MMP-2 positive cancer cells. Copyright © 2016 Elsevier B.V. All rights reserved.

CCDB: a curated database of genes involved in cervix cancer.

Science.gov (United States)

Agarwal, Subhash M; Raghav, Dhwani; Singh, Harinder; Raghava, G P S

2011-01-01

The Cervical Cancer gene DataBase (CCDB, http://crdd.osdd.net/raghava/ccdb) is a manually curated catalog of experimentally validated genes that are thought, or are known to be involved in the different stages of cervical carcinogenesis. In spite of the large women population that is presently affected from this malignancy still at present, no database exists that catalogs information on genes associated with cervical cancer. Therefore, we have compiled 537 genes in CCDB that are linked with cervical cancer causation processes such as methylation, gene amplification, mutation, polymorphism and change in expression level, as evident from published literature. Each record contains details related to gene like architecture (exon-intron structure), location, function, sequences (mRNA/CDS/protein), ontology, interacting partners, homology to other eukaryotic genomes, structure and links to other public databases, thus augmenting CCDB with external data. Also, manually curated literature references have been provided to support the inclusion of the gene in the database and establish its association with cervix cancer. In addition, CCDB provides information on microRNA altered in cervical cancer as well as search facility for querying, several browse options and an online tool for sequence similarity search, thereby providing researchers with easy access to the latest information on genes involved in cervix cancer.

Identification of genes involved in the biology of atypical teratoid/rhabdoid tumours using Drosophila melanogaster

Science.gov (United States)

Jeibmann, Astrid; Eikmeier, Kristin; Linge, Anna; Kool, Marcel; Koos, Björn; Schulz, Jacqueline; Albrecht, Stefanie; Bartelheim, Kerstin; Frühwald, Michael C.; Pfister, Stefan M.; Paulus, Werner; Hasselblatt, Martin

2014-06-01

Atypical teratoid/rhabdoid tumours (AT/RT) are malignant brain tumours. Unlike most other human brain tumours, AT/RT are characterized by inactivation of one single gene, SMARCB1. SMARCB1 is a member of the evolutionarily conserved SWI/SNF chromatin remodelling complex, which has an important role in the control of cell differentiation and proliferation. Little is known, however, about the pathways involved in the oncogenic effects of SMARCB1 inactivation, which might also represent targets for treatment. Here we report a comprehensive genetic screen in the fruit fly that revealed several genes not yet associated with loss of snr1, the Drosophila homologue of SMARCB1. We confirm the functional role of identified genes (including merlin, kibra and expanded, known to regulate hippo signalling pathway activity) in human rhabdoid tumour cell lines and AT/RT tumour samples. These results demonstrate that fly models can be employed for the identification of clinically relevant pathways in human cancer.

Antitumor bystander effect induced by radiation-inducible target gene therapy combined with α particle irradiation

International Nuclear Information System (INIS)

Liu Hui; Jin Chufeng; Wu Yican; Ge Shenfang; Wu Lijun; FDS Team

2012-01-01

In this work, we investigated the bystander effect of the tumor and normal cells surrounding the target region caused by radiation-inducible target gene therapy combined with α-particle irradiation. The receptor tumor cell A549 and normal cell MRC-5 were co-cultured with the donor cells irradiated to 0.5 Gy or the non-irradiated donor cells, and their survival and apoptosis fractions were evaluated. The results showed that the combined treatment of Ad-ET and particle irradiation could induce synergistic antitumor effect on A549 tumor cell, and the survival fraction of receptor cells co-cultured with the irradiated cells decreased by 6%, compared with receptor cells co-cultured with non-irradiated cells, and the apoptosis fraction increased in the same circumstance, but no difference was observed with the normal cells. This study demonstrates that Ad-ET combined with α-particle irradiation can significantly cause the bystander effect on neighboring tumor cells by inhibiting cell growth and inducing apoptosis, without obvious toxicity to normal cells. This suggests that combining radiation-inducible TRAIL gene therapy and irradiation may improve tumor treatment efficacy by specifically targeting tumor cells and even involving the neighboring tumor cells. (authors)

On revealing the gene targets of Ebola virus microRNAs involved in the human skin microbiome

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Pei-Chun Hsu

2018-01-01

Full Text Available Ebola virus, a negative-sense single-stranded RNA virus, causes severe viral hemorrhagic fever and has a high mortality rate. Histopathological and immunopathological analyses of Ebola virus have revealed that histopathological changes in skin tissue are associated with various degrees of endothelial cell swelling and necrosis. The interactions of microbes within or on a host are a crucial for the skin immune shield. The discovery of microRNAs (miRNAs in Ebola virus implies that immune escape, endothelial cell rupture, and tissue dissolution during Ebola virus infection are a result of the effects of Ebola virus miRNAs. Keratinocytes obtained from normal skin can attach and spread through expression of the thrombospondin family of proteins, playing a role in initiation of cell-mediated immune responses in the skin. Several miRNAs have been shown to bind the 3′ untranslated region of thrombospondin mRNA, thereby controlling its stability and translational activity. In this study, we discovered short RNA sequences that may act as miRNAs from Propionibacterium acnes using a practical workflow of bioinformatics methods. Subsequently, we deciphered the common target gene. These RNA sequences tended to bind to the same thrombospondin protein, THSD4, emphasizing the potential importance of the synergistic binding of miRNAs from Ebola virus, Propionibacterium acnes, and humans to the target. These results provide important insights into the molecular mechanisms of thrombospondin proteins and miRNAs in Ebola virus infection.

Involvement of Chromatin Remodeling Genes and the Rho GTPases RhoB and CDC42 in Ovarian Clear Cell Carcinoma

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Nicolai Skovbjerg Arildsen

2017-05-01

Full Text Available ObjectiveOvarian clear cell carcinomas (OCCCs constitute a rare ovarian cancer subtype with distinct clinical features, but may nonetheless be difficult to distinguish morphologically from other subtypes. There is limited knowledge of genetic events driving OCCC tumorigenesis beyond ARID1A, which is reportedly mutated in 30–50% of OCCCs. We aimed to further characterize OCCCs by combined global transcriptional profiling and targeted deep sequencing of a panel of well-established cancer genes. Increased knowledge of OCCC-specific genetic aberrations may help in guiding development of targeted treatments and ultimately improve patient outcome.MethodsGene expression profiling of formalin-fixed, paraffin-embedded (FFPE tissue from a cohort of the major ovarian cancer subtypes (cohort 1; n = 67 was performed using whole-genome cDNA-mediated Annealing, Selection, extension and Ligation (WG-DASL bead arrays, followed by pathway, gene module score, and gene ontology analyses, respectively. A second FFPE cohort of 10 primary OCCCs was analyzed by targeted DNA sequencing of a panel of 60 cancer-related genes (cohort 2. Non-synonymous and non-sense variants affecting single-nucleotide variations and insertions or deletions were further analyzed. A tissue microarray of 43 OCCCs (cohort 3 was used for validation by immunohistochemistry and chromogenic in situ hybridization.ResultsGene expression analyses revealed a distinct OCCC profile compared to other histological subtypes, with, e.g., ERBB2, TFAP2A, and genes related to cytoskeletal actin regulation being overexpressed in OCCC. ERBB2 was, however, not overexpressed on the protein level and ERBB2 amplification was rare in the validation cohort. Targeted deep sequencing revealed non-synonymous variants or insertions/deletions in 11/60 cancer-related genes. Genes involved in chromatin remodeling, including ARID1A, SPOP, and KMT2D were frequently mutated across OCCC tumors.ConclusionOCCCs appear

Inhibition of human esophageal squamous cell carcinomas by targeted silencing of tumor enhancer genes: an overview

International Nuclear Information System (INIS)

Islamian, Jalil Pirayesh; Mohammadi, Mohsen; Baradaran, Behzad

2014-01-01

Esophageal cancer has been reported as the ninth most common malignancy and ranks as the sixth most frequent cause of death worldwide. Esophageal cancer treatment involves surgery, chemotherapy, radiation therapy, or combination therapy. Novel strategies are needed to boost the oncologic outcome. Recent advances in the molecular biology of esophageal cancer have documented the role of genetic alterations in tumorigenesis. Oncogenes serve a pivotal function in tumorigenesis. Targeted therapies are directed at the unique molecular signature of cancer cells for enhanced efficacy with low toxicity. RNA interference (RNAi) technology is a powerful tool for silencing endogenous or exogenous genes in mammalian cells. Related results have shown that targeting oncogenes with siRNAs, specifically the mRNA, effectively reduces tumor cell proliferation and induces apoptotic cell death. This article will briefly review studies on silencing tumor enhancer genes related to the induction of esophageal cancer

EBF factors drive expression of multiple classes of target genes governing neuronal development

Directory of Open Access Journals (Sweden)

Vetter Monica L

2011-04-01

Full Text Available Abstract Background Early B cell factor (EBF family members are transcription factors known to have important roles in several aspects of vertebrate neurogenesis, including commitment, migration and differentiation. Knowledge of how EBF family members contribute to neurogenesis is limited by a lack of detailed understanding of genes that are transcriptionally regulated by these factors. Results We performed a microarray screen in Xenopus animal caps to search for targets of EBF transcriptional activity, and identified candidate targets with multiple roles, including transcription factors of several classes. We determined that, among the most upregulated candidate genes with expected neuronal functions, most require EBF activity for some or all of their expression, and most have overlapping expression with ebf genes. We also found that the candidate target genes that had the most strongly overlapping expression patterns with ebf genes were predicted to be direct transcriptional targets of EBF transcriptional activity. Conclusions The identification of candidate targets that are transcription factor genes, including nscl-1, emx1 and aml1, improves our understanding of how EBF proteins participate in the hierarchy of transcription control during neuronal development, and suggests novel mechanisms by which EBF activity promotes migration and differentiation. Other candidate targets, including pcdh8 and kcnk5, expand our knowledge of the types of terminal differentiated neuronal functions that EBF proteins regulate.

Identification of downstream metastasis-associated target genes regulated by LSD1 in colon cancer cells.

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Chen, Jiang; Ding, Jie; Wang, Ziwei; Zhu, Jian; Wang, Xuejian; Du, Jiyi

2017-03-21

This study aims to identify downstream target genes regulated by lysine-specific demethylase 1 (LSD1) in colon cancer cells and investigate the molecular mechanisms of LSD1 influencing invasion and metastasis of colon cancer. We obtained the expression changes of downstream target genes regulated by small-interfering RNA-LSD1 and LSD1-overexpression via gene expression profiling in two human colon cancer cell lines. An Affymetrix Human Transcriptome Array 2.0 was used to identify differentially expressed genes (DEGs). We screened out LSD1-target gene associated with proliferation, metastasis, and invasion from DEGs via Gene Ontology and Pathway Studio. Subsequently, four key genes (CABYR, FOXF2, TLE4, and CDH1) were computationally predicted as metastasis-related LSD1-target genes. ChIp-PCR was applied after RT-PCR and Western blot validations to detect the occupancy of LSD1-target gene promoter-bound LSD1. A total of 3633 DEGs were significantly upregulated, and 4642 DEGs were downregulated in LSD1-silenced SW620 cells. A total of 4047 DEGs and 4240 DEGs were upregulated and downregulated in LSD1-overexpressed HT-29 cells, respectively. RT-PCR and Western blot validated the microarray analysis results. ChIP assay results demonstrated that LSD1 might be negative regulators for target genes CABYR and CDH1. The expression level of LSD1 is negatively correlated with mono- and dimethylation of histone H3 lysine4(H3K4) at LSD1- target gene promoter region. No significant mono-methylation and dimethylation of H3 lysine9 methylation was detected at the promoter region of CABYR and CDH1. LSD1- depletion contributed to the upregulation of CABYR and CDH1 through enhancing the dimethylation of H3K4 at the LSD1-target genes promoter. LSD1- overexpression mediated the downregulation of CABYR and CDH1expression through decreasing the mono- and dimethylation of H3K4 at LSD1-target gene promoter in colon cancer cells. CABYR and CDH1 might be potential LSD1-target genes in colon

Gene targeting using homologous recombination in embryonic stem cells: The future for behavior genetics?

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Robert eGerlai

2016-04-01

Full Text Available Gene targeting with homologous recombination in embryonic stem cells created a revolution in the analysis of the function of genes in behavioral brain research. The technology allowed unprecedented precision with which one could manipulate genes and study the effect of this manipulation on the central nervous system. With gene targeting, the uncertainty inherent in psychopharmacology regarding whether a particular compound would act only through a specific target was removed. Thus, gene targeting became highly popular. However, with this popularity came the realization that like other methods, gene targeting also suffered from some technical and principal problems. For example, two decades ago, issues about compensatory changes and about genetic linkage were raised. Since then, the technology developed, and its utility has been better delineated. This review will discuss the pros and cons of the technique along with these advancements from the perspective of the neuroscientist user. It will also compare and contrast methods that may represent novel alternatives to the homologous recombination based gene targeting approach, including the TALEN and the CRISPR/Cas9 systems. The goal of the review is not to provide detailed recipes, but to attempt to present a short summary of these approaches a behavioral geneticist or neuroscientist may consider for the analysis of brain function and behavior.

MicroRNA profiling reveals dysregulated microRNAs and their target gene regulatory networks in cemento-ossifying fibroma.

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Pereira, Thaís Dos Santos Fontes; Brito, João Artur Ricieri; Guimarães, André Luiz Sena; Gomes, Carolina Cavaliéri; de Lacerda, Júlio Cesar Tanos; de Castro, Wagner Henriques; Coimbra, Roney Santos; Diniz, Marina Gonçalves; Gomez, Ricardo Santiago

2018-01-01

Cemento-ossifying fibroma (COF) is a benign fibro-osseous neoplasm of uncertain pathogenesis, and its treatment results in morbidity. MicroRNAs (miRNA) are small non-coding RNAs that regulate gene expression and may represent therapeutic targets. The purpose of the study was to generate a comprehensive miRNA profile of COF compared to normal bone. Additionally, the most relevant pathways and target genes of differentially expressed miRNA were investigated by in silico analysis. Nine COF and ten normal bone samples were included in the study. miRNA profiling was carried out by using TaqMan® OpenArray® Human microRNA panel containing 754 validated human miRNAs. We identified the most relevant miRNAs target genes through the leader gene approach, using STRING and Cytoscape software. Pathways enrichment analysis was performed using DIANA-miRPath. Eleven miRNAs were downregulated (hsa-miR-95-3p, hsa-miR-141-3p, hsa-miR-205-5p, hsa-miR-223-3p, hsa-miR-31-5p, hsa-miR-944, hsa-miR-200b-3p, hsa-miR-135b-5p, hsa-miR-31-3p, hsa-miR-223-5p and hsa-miR-200c-3p), and five were upregulated (hsa-miR-181a-5p, hsa-miR-181c-5p, hsa-miR-149-5p, hsa-miR-138-5p and hsa-miR-199a-3p) in COF compared to normal bone. Eighteen common target genes were predicted, and the leader genes approach identified the following genes involved in human COF: EZH2, XIAP, MET and TGFBR1. According to the biology of bone and COF, the most relevant KEGG pathways revealed by enrichment analysis were proteoglycans in cancer, miRNAs in cancer, pathways in cancer, p53-, PI3K-Akt-, FoxO- and TGF-beta signalling pathways, which were previously found to be differentially regulated in bone neoplasms, odontogenic tumours and osteogenesis. miRNA dysregulation occurs in COF, and EZH2, XIAP, MET and TGFBR1 are potential targets for functional analysis validation. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Gene targeting by the vitamin D response element binding protein reveals a role for vitamin D in osteoblast mTOR signaling.

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Lisse, Thomas S; Liu, Ting; Irmler, Martin; Beckers, Johannes; Chen, Hong; Adams, John S; Hewison, Martin

2011-03-01

Transcriptional regulation by hormonal 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] involves occupancy of vitamin D response elements (VDREs) by the VDRE binding protein (VDRE-BP) or 1,25(OH)(2)D(3)-bound vitamin D receptor (VDR). This relationship is disrupted by elevated VDRE-BP, causing a form of hereditary vitamin D-resistant rickets (HVDRR). DNA array analysis showed that of 114 genes regulated by 1,25(OH)(2)D(3) in control cells, almost all (113) were rendered insensitive to the hormone in VDRE-BP-overexpressing HVDRR cells. Among these was the gene for DNA-damage-inducible transcript 4 (DDIT4), an inhibitor of mammalian target of rapamycin (mTOR) signaling. Chromatin immunoprecipitation PCR using 1,25(OH)(2)D(3)-treated osteoblasts confirmed that VDR and VDRE-BP compete for binding to the DDIT4 gene promoter. Expression of DDIT4 mRNA in these cells was induced (1.6-6 fold) by 1,25(OH)(2)D(3) (10-100 nM), and Western blot and flow cytometry analysis showed that this response involved suppression of phosphorylated S6K1(T389) (a downstream target of mTOR) similar to rapamycin treatment. siRNA knockdown of DDIT4 completely abrogated antiproliferative responses to 1,25(OH)(2)D(3), whereas overexpression of VDRE-BP exerted a dominant-negative effect on transcription of 1,25(OH)(2)D(3)-target genes. DDIT4, an inhibitor of mTOR signaling, is a direct target for 1,25(OH)(2)D(3) and VDRE-BP, and functions to suppress cell proliferation in response to vitamin D.

Paired hormone response elements predict caveolin-1 as a glucocorticoid target gene.

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Marinus F van Batenburg

2010-01-01

Full Text Available Glucocorticoids act in part via glucocorticoid receptor binding to hormone response elements (HREs, but their direct target genes in vivo are still largely unknown. We developed the criterion that genomic occurrence of paired HREs at an inter-HRE distance less than 200 bp predicts hormone responsiveness, based on synergy of multiple HREs, and HRE information from known target genes. This criterion predicts a substantial number of novel responsive genes, when applied to genomic regions 10 kb upstream of genes. Multiple-tissue in situ hybridization showed that mRNA expression of 6 out of 10 selected genes was induced in a tissue-specific manner in mice treated with a single dose of corticosterone, with the spleen being the most responsive organ. Caveolin-1 was strongly responsive in several organs, and the HRE pair in its upstream region showed increased occupancy by glucocorticoid receptor in response to corticosterone. Our approach allowed for discovery of novel tissue specific glucocorticoid target genes, which may exemplify responses underlying the permissive actions of glucocorticoids.

Targeted disruption of the mouse Lipoma Preferred Partner gene

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Vervenne, Hilke B.V.K.; Crombez, Koen R.M.O.; Delvaux, Els L.; Janssens, Veerle; Ven, Wim J.M. van de; Petit, Marleen M.R.

2009-01-01

LPP (Lipoma Preferred Partner) is a zyxin-related cell adhesion protein that is involved in the regulation of cell migration. We generated mice with a targeted disruption of the Lpp gene and analysed the importance of Lpp for embryonic development and adult functions. Aberrant Mendelian inheritance in heterozygous crosses suggested partial embryonic lethality of Lpp -/- females. Fertility of Lpp -/- males was proven to be normal, however, females from Lpp -/- x Lpp -/- crosses produced a strongly reduced number of offspring, probably due to a combination of female embryonic lethality and aberrant pregnancies. Apart from these developmental and reproductive abnormalities, Lpp -/- mice that were born reached adulthood without displaying any additional macroscopic defects. On the other hand, Lpp -/- mouse embryonic fibroblasts exhibited reduced migration capacity, reduced viability, and reduced expression of some Lpp interaction partners. Finally, we discovered a short nuclear form of Lpp, expressed mainly in testis via an alternative promoter.

Trpac1, a pH response transcription regulator, is involved in cellulase gene expression in Trichoderma reesei.

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He, Ronglin; Ma, Lijuan; Li, Chen; Jia, Wendi; Li, Demao; Zhang, Dongyuan; Chen, Shulin

2014-12-01

Fungi grow over a relatively wide pH range and adapt to extracellular pH through a genetic regulatory system mediated by a key component PacC, which is a pH transcription regulator. The cellulase production of the filamentous fungi Trichoderma reesei is sensitive to ambient pH. To investigate the connection between cellulase expression regulation and ambient pH, an ortholog of Aspergillus nidulans pacC, Trpac1, was identified and functionally characterized using a target gene deletion strategy. Deleting Trpac1 dramatically increased the cellulase production and the transcription levels of the major cellulase genes at neutral pH, which suggested Trpac1 is involved in the regulation of cellulase production. It was further observed that the expression levels of transcription factors xyr1 and ace2 also increased in the ΔTrpac1 mutant at neutral pH. In addition, the ΔTrpac1 mutant exhibited conidiation defects under neutral and alkaline pH. These results implied that Trpac1 in involved in growth and development process and cellulase gene expression in T. reesei. Copyright © 2014 Elsevier Inc. All rights reserved.

Reproducible gene targeting in recalcitrant Escherichia coli isolates

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De Greve Henri

2011-06-01

Full Text Available Abstract Background A number of allele replacement methods can be used to mutate bacterial genes. For instance, the Red recombinase system of phage Lambda has been used very efficiently to inactivate chromosomal genes in E. coli K-12, through recombination between regions of homology. However, this method does not work reproducibly in some clinical E. coli isolates. Findings The procedure was modified by using longer homologous regions (85 bp and 500-600 bp, to inactivate genes in the uropathogenic E. coli strain UTI89. An lrhA regulator mutant, and deletions of the lac operon as well as the complete type 1 fimbrial gene cluster, were obtained reproducibly. The modified method is also functional in other recalcitrant E. coli, like the avian pathogenic E. coli strain APEC1. The lrhA regulator and lac operon deletion mutants of APEC1 were successfully constructed in the same way as the UTI89 mutants. In other avian pathogenic E. coli strains (APEC3E, APEC11A and APEC16A it was very difficult or impossible to construct these mutants, with the original Red recombinase-based method, with a Red recombinase-based method using longer (85 bp homologous regions or with our modified protocol, using 500 - 600 bp homologous regions. Conclusions The method using 500-600 bp homologous regions can be used reliably in some clinical isolates, to delete single genes or entire operons by homologous recombination. However, it does not invariably show a greater efficiency in obtaining mutants, when compared to the original Red-mediated gene targeting method or to the gene targeting method with 85 bp homologous regions. Therefore the length of the homology regions is not the only limiting factor for the construction of mutants in these recalcitrant strains.

Identification of Target Genes Involved in Wound Healing Angiogenesis of Endothelial Cells with the Treatment of a Chinese 2-Herb Formula.

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Jacqueline Chor Wing Tam

Full Text Available Angiogenesis is vitally important in diabetic wound healing. We had previously demonstrated that a Chinese 2-herb formula (NF3 significantly stimulated angiogenesis of HUVEC in wound healing. However, the molecular mechanism has not yet been elucidated. In line with this, global expression profiling of NF3-treated HUVEC was performed so as to assess the regulatory role of NF3 involved in the underlying signaling pathways in wound healing angiogenesis. The microarray results illustrated that different panels of differentially expressed genes were strictly governed in NF3-treated HUVEC in a time-regulated manner. The microarray analysis followed by qRT-PCR and western blotting verification of NF3-treated HUVEC at 6 h revealed the involvement of various genes in diverse biological process, e.g., MAP3K14 in anti-inflammation; SLC5A8 in anti-tumorogenesis; DNAJB7 in protein translation; BIRC5, EPCAM, INSL4, MMP8 and NPR3 in cell proliferation; CXCR7, EPCAM, HAND1 and MMP8 in migration; CXCR7, EPCAM and MMP8 in tubular formation; and BIRC5, CXCR7, EPCAM, HAND1, MMP8 and UBD in angiogenesis. After 16 h incubation of NF3, other sets of genes were shown with differential expression in HUVEC, e.g., IL1RAPL2 and NR1H4 in anti-inflammation; miR28 in anti-tumorogenesis; GRIN1 and LCN1 in anti-oxidation; EPB41 in intracellular signal transduction; PRL and TFAP2A in cell proliferation; miR28, PRL and SCG2 in cell migration; PRL in tubular formation; and miR28, NR1H4 and PRL in angiogenesis. This study provided concrete scientific evidence in support of the regulatory role of NF3 on endothelial cells involved in wound healing angiogenesis.

Identification of Target Genes Involved in Wound Healing Angiogenesis of Endothelial Cells with the Treatment of a Chinese 2-Herb Formula.

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Tam, Jacqueline Chor Wing; Ko, Chun Hay; Koon, Chi Man; Cheng, Zhang; Lok, Wong Hing; Lau, Ching Po; Leung, Ping Chung; Fung, Kwok Pui; Chan, Wai Yee; Lau, Clara Bik San

2015-01-01

Angiogenesis is vitally important in diabetic wound healing. We had previously demonstrated that a Chinese 2-herb formula (NF3) significantly stimulated angiogenesis of HUVEC in wound healing. However, the molecular mechanism has not yet been elucidated. In line with this, global expression profiling of NF3-treated HUVEC was performed so as to assess the regulatory role of NF3 involved in the underlying signaling pathways in wound healing angiogenesis. The microarray results illustrated that different panels of differentially expressed genes were strictly governed in NF3-treated HUVEC in a time-regulated manner. The microarray analysis followed by qRT-PCR and western blotting verification of NF3-treated HUVEC at 6 h revealed the involvement of various genes in diverse biological process, e.g., MAP3K14 in anti-inflammation; SLC5A8 in anti-tumorogenesis; DNAJB7 in protein translation; BIRC5, EPCAM, INSL4, MMP8 and NPR3 in cell proliferation; CXCR7, EPCAM, HAND1 and MMP8 in migration; CXCR7, EPCAM and MMP8 in tubular formation; and BIRC5, CXCR7, EPCAM, HAND1, MMP8 and UBD in angiogenesis. After 16 h incubation of NF3, other sets of genes were shown with differential expression in HUVEC, e.g., IL1RAPL2 and NR1H4 in anti-inflammation; miR28 in anti-tumorogenesis; GRIN1 and LCN1 in anti-oxidation; EPB41 in intracellular signal transduction; PRL and TFAP2A in cell proliferation; miR28, PRL and SCG2 in cell migration; PRL in tubular formation; and miR28, NR1H4 and PRL in angiogenesis. This study provided concrete scientific evidence in support of the regulatory role of NF3 on endothelial cells involved in wound healing angiogenesis.

Metformin inhibits epithelial–mesenchymal transition in prostate cancer cells: Involvement of the tumor suppressor miR30a and its target gene SOX4

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Zhang, Jing; Shen, Chengwu; Wang, Lin; Ma, Quanping; Xia, Pingtian; Qi, Mei; Yang, Muyi; Han, Bo

2014-01-01

Highlights: • Metformin inhibits TGF-β-induced EMT in prostate cancer (PCa) cells. • Metformin upregulates tumor suppressor miR30a and downregulates SOX4 in PCa cells. • SOX4 is a target gene of miR30a. - Abstract: Tumor metastasis is the leading cause of mortality and morbidity of prostate cancer (PCa) patients. Epithelial–mesenchymal transition (EMT) plays a critical role in cancer progression and metastasis. Recent evidence suggested that diabetic patients treated with metformin have lower PCa risk and better prognosis. This study was aimed to investigate the effects of metformin on EMT in PCa cells and the possible microRNA (miRNA)-based mechanisms. MiRNAs have been shown to regulate various processes of cancer metastasis. We herein showed that metformin significantly inhibits proliferation of Vcap and PC-3 cells, induces G0/G1 cell cycle arrest and inhibits invasiveness and motility capacity of Vcap cells. Metformin could inhibit TGF-β-induced EMT in Vcap cells, as manifested by inhibition of the increase of N-cadherin (p = 0.013), Vimentin (p = 0.002) and the decrease of E-cadherin (p = 0.0023) and β-catenin (p = 0.034) at mRNA and protein levels. Notably, we demonstrated significant upregulation of miR30a levels by metformin (P < 0.05) and further experiments indicated that miR30a significantly inhibits proliferation and EMT process of Vcap cells. Interestingly, we identified that SOX4, a previously reported oncogenic transcriptional factor and modulator of EMT, is a direct target gene of miR30a. Finally, we screened the expression of miR30a and SOX4 in 84 PCa cases with radical prostatectomy. Of note, SOX4 overexpression is significantly associated with decreased levels of miR30a in PCa cases. In all, our study suggested that inhibition of EMT by metformin in PCa cells may involve upregulation of miR30a and downregulation of SOX4

Human microRNA target analysis and gene ontology clustering by GOmir, a novel stand-alone application.

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Roubelakis, Maria G; Zotos, Pantelis; Papachristoudis, Georgios; Michalopoulos, Ioannis; Pappa, Kalliopi I; Anagnou, Nicholas P; Kossida, Sophia

2009-06-16

microRNAs (miRNAs) are single-stranded RNA molecules of about 20-23 nucleotides length found in a wide variety of organisms. miRNAs regulate gene expression, by interacting with target mRNAs at specific sites in order to induce cleavage of the message or inhibit translation. Predicting or verifying mRNA targets of specific miRNAs is a difficult process of great importance. GOmir is a novel stand-alone application consisting of two separate tools: JTarget and TAGGO. JTarget integrates miRNA target prediction and functional analysis by combining the predicted target genes from TargetScan, miRanda, RNAhybrid and PicTar computational tools as well as the experimentally supported targets from TarBase and also providing a full gene description and functional analysis for each target gene. On the other hand, TAGGO application is designed to automatically group gene ontology annotations, taking advantage of the Gene Ontology (GO), in order to extract the main attributes of sets of proteins. GOmir represents a new tool incorporating two separate Java applications integrated into one stand-alone Java application. GOmir (by using up to five different databases) introduces miRNA predicted targets accompanied by (a) full gene description, (b) functional analysis and (c) detailed gene ontology clustering. Additionally, a reverse search initiated by a potential target can also be conducted. GOmir can freely be downloaded BRFAA.

Mining predicted essential genes of Brugia malayi for nematode drug targets.

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Sanjay Kumar

Full Text Available We report results from the first genome-wide application of a rational drug target selection methodology to a metazoan pathogen genome, the completed draft sequence of Brugia malayi, a parasitic nematode responsible for human lymphatic filariasis. More than 1.5 billion people worldwide are at risk of contracting lymphatic filariasis and onchocerciasis, a related filarial disease. Drug treatments for filariasis have not changed significantly in over 20 years, and with the risk of resistance rising, there is an urgent need for the development of new anti-filarial drug therapies. The recent publication of the draft genomic sequence for B. malayi enables a genome-wide search for new drug targets. However, there is no functional genomics data in B. malayi to guide the selection of potential drug targets. To circumvent this problem, we have utilized the free-living model nematode Caenorhabditis elegans as a surrogate for B. malayi. Sequence comparisons between the two genomes allow us to map C. elegans orthologs to B. malayi genes. Using these orthology mappings and by incorporating the extensive genomic and functional genomic data, including genome-wide RNAi screens, that already exist for C. elegans, we identify potentially essential genes in B. malayi. Further incorporation of human host genome sequence data and a custom algorithm for prioritization enables us to collect and rank nearly 600 drug target candidates. Previously identified potential drug targets cluster near the top of our prioritized list, lending credibility to our methodology. Over-represented Gene Ontology terms, predicted InterPro domains, and RNAi phenotypes of C. elegans orthologs associated with the potential target pool are identified. By virtue of the selection procedure, the potential B. malayi drug targets highlight components of key processes in nematode biology such as central metabolism, molting and regulation of gene expression.

Ndrg2 is a PGC-1α/ERRα target gene that controls protein synthesis and expression of contractile-type genes in C2C12 myotubes.

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Foletta, Victoria C; Brown, Erin L; Cho, Yoshitake; Snow, Rod J; Kralli, Anastasia; Russell, Aaron P

2013-12-01

The stress-responsive, tumor suppressor N-myc downstream-regulated gene 2 (Ndrg2) is highly expressed in striated muscle. In response to anabolic and catabolic signals, Ndrg2 is suppressed and induced, respectively, in mouse C2C12 myotubes. However, little is known about the mechanisms regulating Ndrg2 expression in muscle, as well as the biological role for Ndrg2 in differentiated myotubes. Here, we show that Ndrg2 is a target of a peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) and estrogen-related receptor alpha (ERRα) transcriptional program and is induced in response to endurance exercise, a physiological stress known also to increase PGC-1α/ERRα activity. Analyses of global gene and protein expression profiles in C2C12 myotubes with reduced levels of NDRG2, suggest that NDRG2 affects muscle growth, contractile properties, MAPK signaling, ion and vesicle transport and oxidative phosphorylation. Indeed, suppression of NDRG2 in myotubes increased protein synthesis and the expression of fast glycolytic myosin heavy chain isoforms, while reducing the expression of embryonic myosin Myh3, other contractile-associated genes and the MAPK p90 RSK1. Conversely, enhanced expression of NDRG2 reduced protein synthesis, and furthermore, partially blocked the increased protein synthesis rates elicited by a constitutively active form of ERRα. In contrast, suppressing or increasing levels of NDRG2 did not affect mRNA expression of genes involved in mitochondrial biogenesis that are regulated by PGC-1α or ERRα. This study shows that in C2C12 myotubes Ndrg2 is a novel PGC-1α/ERRα transcriptional target, which influences protein turnover and the regulation of genes involved in muscle contraction and function. © 2013 Elsevier B.V. All rights reserved.

Normal Collagen and Bone Production by Gene-targeted Human Osteogenesis Imperfecta iPSCs

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Deyle, David R; Khan, Iram F; Ren, Gaoying; Wang, Pei-Rong; Kho, Jordan; Schwarze, Ulrike; Russell, David W

2012-01-01

Osteogenesis imperfecta (OI) is caused by dominant mutations in the type I collagen genes. In principle, the skeletal abnormalities of OI could be treated by transplantation of patient-specific, bone-forming cells that no longer express the mutant gene. Here, we develop this approach by isolating mesenchymal cells from OI patients, inactivating their mutant collagen genes by adeno-associated virus (AAV)-mediated gene targeting, and deriving induced pluripotent stem cells (iPSCs) that were expanded and differentiated into mesenchymal stem cells (iMSCs). Gene-targeted iMSCs produced normal collagen and formed bone in vivo, but were less senescent and proliferated more than bone-derived MSCs. To generate iPSCs that would be more appropriate for clinical use, the reprogramming and selectable marker transgenes were removed by Cre recombinase. These results demonstrate that the combination of gene targeting and iPSC derivation can be used to produce potentially therapeutic cells from patients with genetic disease. PMID:22031238

Pancreatic Cancer Gene Therapy: From Molecular Targets to Delivery Systems

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Fillat, Cristina, E-mail: cristina.fillat@crg.es; Jose, Anabel; Ros, Xavier Bofill-De; Mato-Berciano, Ana; Maliandi, Maria Victoria; Sobrevals, Luciano [Programa Gens i Malaltia, Centre de Regulació Genòmica-CRG, UPF, Parc de Recerca Biomedica de Barcelona-PRBB and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona (Spain)

2011-01-18

The continuous identification of molecular changes deregulating critical pathways in pancreatic tumor cells provides us with a large number of novel candidates to engineer gene-targeted approaches for pancreatic cancer treatment. Targets—both protein coding and non-coding—are being exploited in gene therapy to influence the deregulated pathways to facilitate cytotoxicity, enhance the immune response or sensitize to current treatments. Delivery vehicles based on viral or non-viral systems as well as cellular vectors with tumor homing characteristics are a critical part of the design of gene therapy strategies. The different behavior of tumoral versus non-tumoral cells inspires vector engineering with the generation of tumor selective products that can prevent potential toxic-associated effects. In the current review, a detailed analysis of the different targets, the delivery vectors, the preclinical approaches and a descriptive update on the conducted clinical trials are presented. Moreover, future possibilities in pancreatic cancer treatment by gene therapy strategies are discussed.

TFII-I regulates target genes in the PI-3K and TGF-β signaling pathways through a novel DNA binding motif.

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Segura-Puimedon, Maria; Borralleras, Cristina; Pérez-Jurado, Luis A; Campuzano, Victoria

2013-09-25

General transcription factor (TFII-I) is a multi-functional protein involved in the transcriptional regulation of critical developmental genes, encoded by the GTF2I gene located on chromosome 7q11.23. Haploinsufficiency at GTF2I has been shown to play a major role in the neurodevelopmental features of Williams-Beuren syndrome (WBS). Identification of genes regulated by TFII-I is thus critical to detect molecular determinants of WBS as well as to identify potential new targets for specific pharmacological interventions, which are currently absent. We performed a microarray screening for transcriptional targets of TFII-I in cortex and embryonic cells from Gtf2i mutant and wild-type mice. Candidate genes with altered expression were verified using real-time PCR. A novel motif shared by deregulated genes was found and chromatin immunoprecipitation assays in embryonic fibroblasts were used to document in vitro TFII-I binding to this motif in the promoter regions of deregulated genes. Interestingly, the PI3K and TGFβ signaling pathways were over-represented among TFII-I-modulated genes. In this study we have found a highly conserved DNA element, common to a set of genes regulated by TFII-I, and identified and validated novel in vivo neuronal targets of this protein affecting the PI3K and TGFβ signaling pathways. Overall, our data further contribute to unravel the complexity and variability of the different genetic programs orchestrated by TFII-I. © 2013 Elsevier B.V. All rights reserved.

Analysis of Deregulated microRNAs and Their Target Genes in Gastric Cancer.

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Simonas Juzėnas

Full Text Available MicroRNAs (miRNAs are widely studied non-coding RNAs that modulate gene expression. MiRNAs are deregulated in different tumors including gastric cancer (GC and have potential diagnostic and prognostic implications. The aim of our study was to determine miRNA profile in GC tissues, followed by evaluation of deregulated miRNAs in plasma of GC patients. Using available databases and bioinformatics methods we also aimed to evaluate potential target genes of confirmed differentially expressed miRNA and validate these findings in GC tissues.The study included 51 GC patients and 51 controls. Initially, we screened miRNA expression profile in 13 tissue samples of GC and 12 normal gastric tissues with TaqMan low density array (TLDA. In the second stage, differentially expressed miRNAs were validated in a replication cohort using qRT-PCR in tissue and plasma samples. Subsequently, we analyzed potential target genes of deregulated miRNAs using bioinformatics approach, determined their expression in GC tissues and performed correlation analysis with targeting miRNAs.Profiling with TLDA revealed 15 deregulated miRNAs in GC tissues compared to normal gastric mucosa. Replication analysis confirmed that miR-148a-3p, miR-204-5p, miR-223-3p and miR-375 were consistently deregulated in GC tissues. Analysis of GC patients' plasma samples showed significant down-regulation of miR-148a-3p, miR-375 and up-regulation of miR-223-3p compared to healthy subjects. Further, using bioinformatic tools we identified targets of replicated miRNAs and performed disease-associated gene enrichment analysis. Ultimately, we evaluated potential target gene BCL2 and DNMT3B expression by qRT-PCR in GC tissue, which correlated with targeting miRNA expression.Our study revealed miRNA profile in GC tissues and showed that miR-148a-3p, miR-223-3p and miR-375 are deregulated in GC plasma samples, but these circulating miRNAs showed relatively weak diagnostic performance as sole biomarkers

Bioinformatics Analysis Reveals Genes Involved in the Pathogenesis of Ameloblastoma and Keratocystic Odontogenic Tumor.

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Santos, Eliane Macedo Sobrinho; Santos, Hércules Otacílio; Dos Santos Dias, Ivoneth; Santos, Sérgio Henrique; Batista de Paula, Alfredo Maurício; Feltenberger, John David; Sena Guimarães, André Luiz; Farias, Lucyana Conceição

2016-01-01

Pathogenesis of odontogenic tumors is not well known. It is important to identify genetic deregulations and molecular alterations. This study aimed to investigate, through bioinformatic analysis, the possible genes involved in the pathogenesis of ameloblastoma (AM) and keratocystic odontogenic tumor (KCOT). Genes involved in the pathogenesis of AM and KCOT were identified in GeneCards. Gene list was expanded, and the gene interactions network was mapped using the STRING software. "Weighted number of links" (WNL) was calculated to identify "leader genes" (highest WNL). Genes were ranked by K-means method and Kruskal-Wallis test was used (Preview data was used to corroborate the bioinformatics data. CDK1 was identified as leader gene for AM. In KCOT group, results show PCNA and TP53 . Both tumors exhibit a power law behavior. Our topological analysis suggested leader genes possibly important in the pathogenesis of AM and KCOT, by clustering coefficient calculated for both odontogenic tumors (0.028 for AM, zero for KCOT). The results obtained in the scatter diagram suggest an important relationship of these genes with the molecular processes involved in AM and KCOT. Ontological analysis for both AM and KCOT demonstrated different mechanisms. Bioinformatics analyzes were confirmed through literature review. These results may suggest the involvement of promising genes for a better understanding of the pathogenesis of AM and KCOT.

A novel technology to target adenovirus vectors : application in cells involved in atherosclerosis

NARCIS (Netherlands)

Gras, Jan Cornelis Emile

2007-01-01

In this thesis a novel technology is described to target adenovirus vectors. Adenovirus vectors are powerful tools to modulate gene expression. The use of these vectors however, is hampered by the fact that many for gene therapy interesting cell types do not, or only at low levels express the CAR

Mechanisms involved in the transport of mercuric ions in target tissues

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Bridges, Christy C.; Zalups, Rudolfs K.

2016-01-01

Mercury exists in the environment in various forms, all of which pose a risk to human health. Despite guidelines regulating the industrial release of mercury into the environment, humans continue to be exposed regularly to various forms of this metal via inhalation or ingestion. Following exposure, mercuric ions are taken up by and accumulate in numerous organs, including brain, intestine, kidney, liver, and placenta. In order to understand the toxicological effects of exposure to mercury, a thorough understanding of the mechanisms that facilitate entry of mercuric ions into target cells must first be obtained. A number of mechanisms for the transport of mercuric ions into target cells and organs have been proposed in recent years. However, the ability of these mechanisms to transport mercuric ions and the regulatory features of these carriers have not been characterized completely. The purpose of this review is to summarize the current findings related to the mechanisms that may be involved in the transport of inorganic and organic forms of mercury in target tissues and organs. This review will describe mechanisms known to be involved in the transport of mercury and will also propose additional mechanisms that may potentially be involved in the transport of mercuric ions into target cells. PMID:27422290

Preparation and characterization of magnetic gene vectors for targeting gene delivery

Energy Technology Data Exchange (ETDEWEB)

Zheng, S.W.; Liu, G. [College of Chemistry, Chemical Engineering and Materials Science and Key Laboratory of Organic Synthesis of Jiangsu Province, Soochow University, SIP, Suzhou 215123 (China); Hong, R.Y., E-mail: rhong@suda.edu.cn [College of Chemistry, Chemical Engineering and Materials Science and Key Laboratory of Organic Synthesis of Jiangsu Province, Soochow University, SIP, Suzhou 215123 (China); State Key Laboratory of Multi-phase Complex Systems, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080 (China); Li, H.Z. [State Key Laboratory of Multi-phase Complex Systems, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080 (China); Li, Y.G., E-mail: ilguoliang@sohu.com [Department of radiology, the First Affiliated Hospital of Soochow University, Suzhou 215007 (China); Wei, D.G., E-mail: dougwei@deas.harvard.edu [Center for Nanoscale Systems, School of Engineering and Applied Science, Harvard University, 11 Oxford Street, Cambridge, MA 02139 (United States)

2012-10-15

Highlights: Black-Right-Pointing-Pointer PEI is ideal candidate polymer for the design of gene delivery systems. Black-Right-Pointing-Pointer PEI-CMD-MNPs exhibited a typical superparamagnetic behavior. Black-Right-Pointing-Pointer PEI-CMD-MNPs were well stable over the entire range of pH and NaCl concentration. Black-Right-Pointing-Pointer DNA-PEI-CMD-MNPs transfected cells by a magnet have higher transfection efficiency and gene expression efficiency. - Abstract: The PEI-CMD-MNPs were successfully prepared by the surface modification of magnetic Fe{sub 3}O{sub 4} nanoparticles with carboxymethyl dextran (CMD) and polyethyleneimine (PEI). The PEI-CMD-MNPs polyplexes exhibited a typical superparamagnetic behavior and were well stable over the entire range of pH and NaCl concentration. These PEI-CMD-MNPs were used as magnetic gene vectors for targeting gene delivery. The prepared MNPs at different surface modification stages were characterized using Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA), field emissions canning electron microscopy (FE-SEM), powder X-ray diffraction (XRD) and dynamic laser light scattering (DLS) analysis. The magnetic properties were studied by vibrating sample magnetometer (VSM). To evaluate the performance of the magnetic nanoparticles as gene transfer vector, the PEI-CMD-MNPs were used to delivery green fluorescent protein (GFP) gene into BHK21 cells. The expression of GFP gene was detected by fluorescence microscope. DNA-PEI-CMD-MNPs polyplexes absorbed by the cells were also monitored by Magnetic resonance imaging (MRI). The transfection efficiency and gene expression efficiency of that transfected with a magnet were much higher than that of standard transfection.

Potential downstream target genes of aberrant ETS transcription factors are differentially affected in Ewing's sarcoma and prostate carcinoma.

Directory of Open Access Journals (Sweden)

Maria J Camões

Full Text Available FLI1 and ERG, the major ETS transcription factors involved in rearrangements in the Ewing's sarcoma family of tumors (ESFT and in prostate carcinomas (PCa, respectively, belong to the same subfamily, having 98% sequence identity in the DNA binding domain. We therefore decided to investigate whether the aberrant transcription factors in both malignancies have some common downstream targets. We crossed a publicly available list of all putative EWSR1-FLI1 target genes in ESFT with our microarray expression data on 24 PCa and 6 non-malignant prostate tissues (NPT and choose four genes among the top-most differentially expressed between PCa with (PCa ERG+ and without (PCa ETS- ETS fusion genes (HIST1H4L, KCNN2, ECRG4 and LDOC1, as well as four well-validated direct targets of the EWSR1-FLI1 chimeric protein in ESFT (NR0B1, CAV1, IGFBP3 and TGFBR2. Using quantitative expression analysis in 16 ESFT and seven alveolar rhabdomyosarcomas (ARMS, we were able to validate the four genes previously described as direct targets of the EWSR1-FLI1 oncoprotein, showing overexpression of CAV1 and NR0B1 and underexpression of IGFBP3 and TGFBR2 in ESFT as compared to ARMS. Although none of these four genes showed significant expression differences between PCa ERG+ and PCa ETS-, CAV1, IGFBP3 and TGFBR2 were less expressed in PCa in an independent series of 56 PCa and 15 NPT, as also observed for ECRG4 and LDOC1, suggesting a role in prostate carcinogenesis in general. On the other hand, we demonstrate for the first time that both HIST1H4L and KCNN2 are significantly overexpressed in PCa ERG+ and that ERG binds to the promoter of these genes. Conversely, KCNN2 was found underexpressed in ESFT relative to ARMS, suggesting that the EWSR1-ETS oncoprotein may have the opposite effect of ERG rearrangements in PCa. We conclude that aberrant ETS transcription factors modulate target genes differently in ESFT and PCa.

Identification of target genes of synovial sarcoma-associated fusion oncoprotein using human pluripotent stem cells

Energy Technology Data Exchange (ETDEWEB)

Hayakawa, Kazuo [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Ikeya, Makoto [Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Fukuta, Makoto [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Woltjen, Knut [Department of Reprogramming Sciences, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Tamaki, Sakura; Takahara, Naoko; Kato, Tomohisa; Sato, Shingo [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Otsuka, Takanobu [Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Toguchida, Junya, E-mail: togjun@frontier.kyoto-u.ac.jp [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto (Japan)

2013-03-22

Highlights: ► We tried to identify targets of synovial sarcoma (SS)-associated SYT–SSX fusion gene. ► We established pluripotent stem cell (PSC) lines with inducible SYT–SSX gene. ► SYT–SSX responsive genes were identified by the induction of SYT–SSX in PSC. ► SS-related genes were selected from database by in silico analyses. ► 51 genes were finally identified among SS-related genes as targets of SYT–SSX in PSC. -- Abstract: Synovial sarcoma (SS) is a malignant soft tissue tumor harboring chromosomal translocation t(X; 18)(p11.2; q11.2), which produces SS-specific fusion gene, SYT–SSX. Although precise function of SYT–SSX remains to be investigated, accumulating evidences suggest its role in gene regulation via epigenetic mechanisms, and the product of SYT–SSX target genes may serve as biomarkers of SS. Lack of knowledge about the cell-of-origin of SS, however, has placed obstacle in the way of target identification. Here we report a novel approach to identify SYT–SSX2 target genes using human pluripotent stem cells (hPSCs) containing a doxycycline-inducible SYT–SSX2 gene. SYT–SSX2 was efficiently induced both at mRNA and protein levels within three hours after doxycycline administration, while no morphological change of hPSCs was observed until 24 h. Serial microarray analyses identified genes of which the expression level changed more than twofold within 24 h. Surprisingly, the majority (297/312, 95.2%) were up-regulated genes and a result inconsistent with the current concept of SYT–SSX as a transcriptional repressor. Comparing these genes with SS-related genes which were selected by a series of in silico analyses, 49 and 2 genes were finally identified as candidates of up- and down-regulated target of SYT–SSX, respectively. Association of these genes with SYT–SSX in SS cells was confirmed by knockdown experiments. Expression profiles of SS-related genes in hPSCs and human mesenchymal stem cells (hMSCs) were strikingly

Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection.

Science.gov (United States)

Saito, Shinta; Ura, Kiyoe; Kodama, Miho; Adachi, Noritaka

2015-06-30

Targeted gene modification by homologous recombination provides a powerful tool for studying gene function in cells and animals. In higher eukaryotes, non-homologous integration of targeting vectors occurs several orders of magnitude more frequently than does targeted integration, making the gene-targeting technology highly inefficient. For this reason, negative-selection strategies have been employed to reduce the number of drug-resistant clones associated with non-homologous vector integration, particularly when artificial nucleases to introduce a DNA break at the target site are unavailable or undesirable. As such, an exon-trap strategy using a promoterless drug-resistance marker gene provides an effective way to counterselect non-homologous integrants. However, constructing exon-trapping targeting vectors has been a time-consuming and complicated process. By virtue of highly efficient att-mediated recombination, we successfully developed a simple and rapid method to construct plasmid-based vectors that allow for exon-trapping gene targeting. These exon-trap vectors were useful in obtaining correctly targeted clones in mouse embryonic stem cells and human HT1080 cells. Most importantly, with the use of a conditionally cytotoxic gene, we further developed a novel strategy for negative selection, thereby enhancing the efficiency of counterselection for non-homologous integration of exon-trap vectors. Our methods will greatly facilitate exon-trapping gene-targeting technologies in mammalian cells, particularly when combined with the novel negative selection strategy.

Microarray-Based Identification of Transcription Factor Target Genes

NARCIS (Netherlands)

Gorte, M.; Horstman, A.; Page, R.B.; Heidstra, R.; Stromberg, A.; Boutilier, K.A.

2011-01-01

Microarray analysis is widely used to identify transcriptional changes associated with genetic perturbation or signaling events. Here we describe its application in the identification of plant transcription factor target genes with emphasis on the design of suitable DNA constructs for controlling TF

Epigenetic Editing: targeted rewriting of epigenetic marks to modulate expression of selected target genes.

NARCIS (Netherlands)

de Groote, M.L.; Verschure, P.J.; Rots, M.G.

2012-01-01

Despite significant advances made in epigenetic research in recent decades, many questions remain unresolved, especially concerning cause and consequence of epigenetic marks with respect to gene expression modulation (GEM). Technologies allowing the targeting of epigenetic enzymes to predetermined

Epigenetic Editing : targeted rewriting of epigenetic marks to modulate expression of selected target genes

NARCIS (Netherlands)

de Groote, Marloes L.; Verschure, Pernette J.; Rots, Marianne G.

2012-01-01

Despite significant advances made in epigenetic research in recent decades, many questions remain unresolved, especially concerning cause and consequence of epigenetic marks with respect to gene expression modulation (GEM). Technologies allowing the targeting of epigenetic enzymes to predetermined

Cadmium-mediated disruption of cortisol biosynthesis involves suppression of corticosteroidogenic genes in rainbow trout

International Nuclear Information System (INIS)

Sandhu, Navdeep; Vijayan, Mathilakath M.

2011-01-01

Cadmium is widely distributed in the aquatic environment and is toxic to fish even at sublethal concentrations. This metal is an endocrine disruptor, and one well established role in teleosts is the suppression of adrenocorticotrophic hormone (ACTH)-stimulated cortisol biosynthesis by the interrenal tissue. However the mechanism(s) leading to this steroid suppression is poorly understood. We tested the hypothesis that cadmium targets genes encoding proteins critical for corticosteroid biosynthesis, including melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (P450scc), in rainbow trout (Oncorhynchus mykiss). To test this, head kidney slices (containing the interrenal tissues) were incubated in vitro with cadmium chloride (0, 10, 100 and 1000 nM) for 4 h either in the presence or absence of ACTH (0.5 IU/mL). In the unstimulated head kidney slices, cadmium exposure did not affect basal cortisol secretion and the mRNA levels of MC2R and P450scc, while StAR gene expression was significantly reduced. Cadmium exposure significantly suppressed ACTH-stimulated cortisol production in a dose-related fashion. This cadmium-mediated suppression in corticosteroidogenesis corresponded with a significant reduction in MC2R, StAR and P450scc mRNA levels in trout head kidney slices. The inhibition of ACTH-stimulated cortisol production and suppression of genes involved in corticosteroidogenesis by cadmium were completely abolished in the presence of 8-Bromo-cAMP (a cAMP analog). Overall, cadmium disrupts the expression of genes critical for corticosteroid biosynthesis in rainbow trout head kidney slices. However, the rescue of cortisol production as well as StAR and P450scc gene expressions by cAMP analog suggests that cadmium impact occurs upstream of cAMP production. We propose that MC2R signaling, the primary step in ACTH-induced cortocosteroidogenesis, is a key target for cadmium-mediated disruption of

Cadmium-mediated disruption of cortisol biosynthesis involves suppression of corticosteroidogenic genes in rainbow trout

Energy Technology Data Exchange (ETDEWEB)

Sandhu, Navdeep [Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1 (Canada); Vijayan, Mathilakath M., E-mail: mvijayan@uwaterloo.ca [Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1 (Canada)

2011-05-15

Cadmium is widely distributed in the aquatic environment and is toxic to fish even at sublethal concentrations. This metal is an endocrine disruptor, and one well established role in teleosts is the suppression of adrenocorticotrophic hormone (ACTH)-stimulated cortisol biosynthesis by the interrenal tissue. However the mechanism(s) leading to this steroid suppression is poorly understood. We tested the hypothesis that cadmium targets genes encoding proteins critical for corticosteroid biosynthesis, including melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (P450scc), in rainbow trout (Oncorhynchus mykiss). To test this, head kidney slices (containing the interrenal tissues) were incubated in vitro with cadmium chloride (0, 10, 100 and 1000 nM) for 4 h either in the presence or absence of ACTH (0.5 IU/mL). In the unstimulated head kidney slices, cadmium exposure did not affect basal cortisol secretion and the mRNA levels of MC2R and P450scc, while StAR gene expression was significantly reduced. Cadmium exposure significantly suppressed ACTH-stimulated cortisol production in a dose-related fashion. This cadmium-mediated suppression in corticosteroidogenesis corresponded with a significant reduction in MC2R, StAR and P450scc mRNA levels in trout head kidney slices. The inhibition of ACTH-stimulated cortisol production and suppression of genes involved in corticosteroidogenesis by cadmium were completely abolished in the presence of 8-Bromo-cAMP (a cAMP analog). Overall, cadmium disrupts the expression of genes critical for corticosteroid biosynthesis in rainbow trout head kidney slices. However, the rescue of cortisol production as well as StAR and P450scc gene expressions by cAMP analog suggests that cadmium impact occurs upstream of cAMP production. We propose that MC2R signaling, the primary step in ACTH-induced cortocosteroidogenesis, is a key target for cadmium-mediated disruption of

Retinoschisislike alterations in the mouse eye caused by gene targeting of the Norrie disease gene.

Science.gov (United States)

Ruether, K; van de Pol, D; Jaissle, G; Berger, W; Tornow, R P; Zrenner, E

1997-03-01

To investigate the retinal function and morphology of mice carrying a replacement mutation in exon 2 of the Norrie disease gene. Recently, Norrie disease mutant mice have been generated using gene targeting technology. The mutation removes the 56 N-terminal amino acids of the Norrie gene product. Ganzfeld electroretinograms (ERGs) were obtained in five animals hemizygous or homozygous for the mutant gene and in three female animals heterozygous for the mutant gene. As controls, three males carrying the wild-type gene were examined. Electroretinogram testing included rod a- and b-wave V-log I functions, oscillatory potentials, and cone responses. The fundus morphology has been visualized by scanning laser ophthalmoscopy. Rod and cone ERG responses and fundus morphology were not significantly different among female heterozygotes and wild-type mice. In contrast, the hemizygous mice displayed a severe loss of ERG b-wave, leading to a negatively shaped scotopic ERG and a marked reduction of oscillatory potentials. The a-wave was normal at low intensities, and only with brighter flashes was there a moderate amplitude loss. Cone amplitudes were barely recordable in the gene-targeted males. Ophthalmoscopy revealed snowflakelike vitreal changes, retinoschisis, and pigment epithelium irregularities in hemizygotes and homozygotes, but no changes in female heterozygotes. The negatively shaped scotopic ERG in male mice with a Norrie disease gene mutation probably was caused by retinoschisis. Pigment epithelial changes and degenerations of the outer retina are relatively mild. These findings may be a clue to the embryonal retinoschisislike pathogenesis of Norrie disease in humans or it may indicate a different expression of the Norrie disease gene defect in mice compared to that in humans.

Genome-wide identification of polycomb target genes reveals a functional association of Pho with Scm in Bombyx mori.

Science.gov (United States)

Li, Zhiqing; Cheng, Daojun; Mon, Hiroaki; Tatsuke, Tsuneyuki; Zhu, Li; Xu, Jian; Lee, Jae Man; Xia, Qingyou; Kusakabe, Takahiro

2012-01-01

Polycomb group (PcG) proteins are evolutionarily conserved chromatin modifiers and act together in three multimeric complexes, Polycomb repressive complex 1 (PRC1), Polycomb repressive complex 2 (PRC2), and Pleiohomeotic repressive complex (PhoRC), to repress transcription of the target genes. Here, we identified Polycomb target genes in Bombyx mori with holocentric centromere using genome-wide expression screening based on the knockdown of BmSCE, BmESC, BmPHO, or BmSCM gene, which represent the distinct complexes. As a result, the expressions of 29 genes were up-regulated after knocking down 4 PcG genes. Particularly, there is a significant overlap between targets of BmPho (331 out of 524) and BmScm (331 out of 532), and among these, 190 genes function as regulator factors playing important roles in development. We also found that BmPho, as well as BmScm, can interact with other Polycomb components examined in this study. Further detailed analysis revealed that the C-terminus of BmPho containing zinc finger domain is involved in the interaction between BmPho and BmScm. Moreover, the zinc finger domain in BmPho contributes to its inhibitory function and ectopic overexpression of BmScm is able to promote transcriptional repression by Gal4-Pho fusions including BmScm-interacting domain. Loss of BmPho expression causes relocalization of BmScm into the cytoplasm. Collectively, we provide evidence of a functional link between BmPho and BmScm, and propose two Polycomb-related repression mechanisms requiring only BmPho associated with BmScm or a whole set of PcG complexes.

Genome-wide identification of polycomb target genes reveals a functional association of Pho with Scm in Bombyx mori.

Directory of Open Access Journals (Sweden)

Zhiqing Li

Full Text Available Polycomb group (PcG proteins are evolutionarily conserved chromatin modifiers and act together in three multimeric complexes, Polycomb repressive complex 1 (PRC1, Polycomb repressive complex 2 (PRC2, and Pleiohomeotic repressive complex (PhoRC, to repress transcription of the target genes. Here, we identified Polycomb target genes in Bombyx mori with holocentric centromere using genome-wide expression screening based on the knockdown of BmSCE, BmESC, BmPHO, or BmSCM gene, which represent the distinct complexes. As a result, the expressions of 29 genes were up-regulated after knocking down 4 PcG genes. Particularly, there is a significant overlap between targets of BmPho (331 out of 524 and BmScm (331 out of 532, and among these, 190 genes function as regulator factors playing important roles in development. We also found that BmPho, as well as BmScm, can interact with other Polycomb components examined in this study. Further detailed analysis revealed that the C-terminus of BmPho containing zinc finger domain is involved in the interaction between BmPho and BmScm. Moreover, the zinc finger domain in BmPho contributes to its inhibitory function and ectopic overexpression of BmScm is able to promote transcriptional repression by Gal4-Pho fusions including BmScm-interacting domain. Loss of BmPho expression causes relocalization of BmScm into the cytoplasm. Collectively, we provide evidence of a functional link between BmPho and BmScm, and propose two Polycomb-related repression mechanisms requiring only BmPho associated with BmScm or a whole set of PcG complexes.

Characterization of Zur-dependent genes and direct Zur targets in Yersinia pestis

Directory of Open Access Journals (Sweden)

Wang Xiaoyi

2009-06-01

Full Text Available Abstract Background The zinc uptake regulator Zur is a Zn2+-sensing metalloregulatory protein involved in the maintenance of bacterial zinc homeostasis. Up to now, regulation of zinc homeostasis by Zur is poorly understood in Y. pestis. Results We constructed a zur null mutant of Y. pestis biovar microtus strain 201. Microarray expression analysis disclosed a set of 154 Zur-dependent genes of Y. pestis upon exposure to zinc rich condition. Real-time reverse transcription (RT-PCR was subsequently used to validate the microarray data. Based on the 154 Zur-dependent genes, predicted regulatory Zur motifs were used to screen for potential direct Zur targets including three putative operons znuA, znuCB and ykgM-RpmJ2. The LacZ reporter fusion analysis verified that Zur greatly repressed the promoter activity of the above three operons. The subsequent electrophoretic mobility shift assay (EMSA demonstrated that a purified Zur protein was able to bind to the promoter regions of the above three operons. The DNase I footprinting was used to identify the Zur binding sites for the above three operons, verifying the Zur box sequence as predicted previously in γ-Proteobacteria. The primer extension assay was further used to determine the transcription start sites for the above three operons and to localize the -10 and -35 elements. Zur binding sites overlapped the -10 sequence of its target promoters, which was consistent with the previous observation that Zur binding would block the entry of the RNA polymerase to repress the transcription of its target genes. Conclusion Zur as a repressor directly controls the transcription of znuA, znuCB and ykgM-RpmJ2 in Y. pestis by employing a conserved mechanism of Zur-promoter DNA association as observed in γ-Proteobacteria. Zur contributes to zinc homeostasis in Y. pestis likely through transcriptional repression of the high-affinity zinc uptake system ZnuACB and two alternative ribosomal proteins YkgM and RpmJ2.

Genes and genetic variations involved in the development of hypertension: focusing on a Greek patient cohort.

Science.gov (United States)

Kouremenos, Nikolaos; Zacharopoulou, Ioanna V; Triantafyllidi, Helen; Zacharopoulos, Georgios V; Mornos, Cristian; Filippatos, Gerasimos; Lekakis, John; Kremastinos, Dimitrios; Manolis, Athanasios I; Gavras, Haralambos

2014-01-01

Essential hypertension (HTN) is a multifactorial disease involving environmental, genetic and other factors. Over the past years, genetic studies of essential HTN have increased dramatically but the molecular mechanisms involved are still unknown. As part of a research program coordinated by Boston university (USA), we studied the role of various genes and single nucleotide polymorphisms (SNPs) in the inheritance or the onset of HTN in African-American, Caucasian-American and Greek families. Among 128 Greek families with a history of HTN, we studied 1474 people. Of the total examined, 273 men and 286 women were hypertensive. Based on 410 DNA samples from the hypertensive subjects, different SNPs were examined. An overall meta-analysis of the results from the Greek families, as well as a comparison with the 2 other groups (African-Americans and Caucasian-Americans), was performed. We report SNPs that are associated with the inheritance of HTN and are located either at the promoters of N-methyltransferase and catalase genes, or within the coding region of NEDD4L ubiquitin ligase gene, or SNPs in mitochondrial DNA of hypertensive probands. Furthermore, we clarified the role of hereditary predisposition in the development of HTN, showing that the presence of maternal HTN was significantly higher in African-Americans and Greeks compared to Caucasian-Americans (81.7%, 84.8%, and 65%), while the paternal HTN showed no such difference (50%, 48.3% and 44.9%), respectively. Although genetic factors that were correlated with HTN were identified, it was not possible to identify a single gene that should be targeted for the treatment of HTN. Nevertheless, the important role of the maternal hereditary predisposition to HTN in the Greek patients and the responsible genetic factors involved should be further examined.

The light gene of Drosophila melanogaster encodes a homologue of VPS41, a yeast gene involved in cellular-protein trafficking.

Science.gov (United States)

Warner, T S; Sinclair, D A; Fitzpatrick, K A; Singh, M; Devlin, R H; Honda, B M

1998-04-01

Mutations in a number of genes affect eye colour in Drosophila melanogaster; some of these "eye-colour" genes have been shown to be involved in various aspects of cellular transport processes. In addition, combinations of viable mutant alleles of some of these genes, such as carnation (car) combined with either light (lt) or deep-orange (dor) mutants, show lethal interactions. Recently, dor was shown to be homologous to the yeast gene PEP3 (VPS18), which is known to be involved in intracellular trafficking. We have undertaken to extend our earlier work on the lt gene, in order to examine in more detail its expression pattern and to characterize its gene product via sequencing of a cloned cDNA. The gene appears to be expressed at relatively high levels in all stages and tissues examined, and shows strong homology to VPS41, a gene involved in cellular-protein trafficking in yeast and higher eukaryotes. Further genetic experiments also point to a role for lt in transport processes: we describe lethal interactions between viable alleles of lt and dor, as well as phenotypic interactions (reductions in eye pigment) between allels of lt and another eye-colour gene, garnet (g), whose gene product has close homology to a subunit of the human adaptor complex, AP-3.

GEM2Net: from gene expression modeling to -omics networks, a new CATdb module to investigate Arabidopsis thaliana genes involved in stress response.

Science.gov (United States)

Zaag, Rim; Tamby, Jean Philippe; Guichard, Cécile; Tariq, Zakia; Rigaill, Guillem; Delannoy, Etienne; Renou, Jean-Pierre; Balzergue, Sandrine; Mary-Huard, Tristan; Aubourg, Sébastien; Martin-Magniette, Marie-Laure; Brunaud, Véronique

2015-01-01

CATdb (http://urgv.evry.inra.fr/CATdb) is a database providing a public access to a large collection of transcriptomic data, mainly for Arabidopsis but also for other plants. This resource has the rare advantage to contain several thousands of microarray experiments obtained with the same technical protocol and analyzed by the same statistical pipelines. In this paper, we present GEM2Net, a new module of CATdb that takes advantage of this homogeneous dataset to mine co-expression units and decipher Arabidopsis gene functions. GEM2Net explores 387 stress conditions organized into 18 biotic and abiotic stress categories. For each one, a model-based clustering is applied on expression differences to identify clusters of co-expressed genes. To characterize functions associated with these clusters, various resources are analyzed and integrated: Gene Ontology, subcellular localization of proteins, Hormone Families, Transcription Factor Families and a refined stress-related gene list associated to publications. Exploiting protein-protein interactions and transcription factors-targets interactions enables to display gene networks. GEM2Net presents the analysis of the 18 stress categories, in which 17,264 genes are involved and organized within 681 co-expression clusters. The meta-data analyses were stored and organized to compose a dynamic Web resource. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Functional characterization of endogenous siRNA target genes in Caenorhabditis elegans

Directory of Open Access Journals (Sweden)

Heikkinen Liisa

2008-06-01

Full Text Available Abstract Background Small interfering RNA (siRNA molecules mediate sequence specific silencing in RNA interference (RNAi, a gene regulatory phenomenon observed in almost all organisms. Large scale sequencing of small RNA libraries obtained from C. elegans has revealed that a broad spectrum of siRNAs is endogenously transcribed from genomic sequences. The biological role and molecular diversity of C. elegans endogenous siRNA (endo-siRNA molecules, nonetheless, remain poorly understood. In order to gain insight into their biological function, we annotated two large libraries of endo-siRNA sequences, identified their cognate targets, and performed gene ontology analysis to identify enriched functional categories. Results Systematic trends in categorization of target genes according to the specific length of siRNA sequences were observed: 18- to 22-mer siRNAs were associated with genes required for embryonic development; 23-mers were associated uniquely with post-embryonic development; 24–26-mers were associated with phosphorus metabolism or protein modification. Moreover, we observe that some argonaute related genes associate with siRNAs with multiple reads. Sequence frequency graphs suggest that different lengths of siRNAs share similarities in overall sequence structure: the 5' end begins with G, while the body predominates with U and C. Conclusion These results suggest that the lengths of endogenous siRNA molecules are consequential to their biological functions since the gene ontology categories for their cognate mRNA targets vary depending upon their lengths.

E2F target genes: unraveling the biology

DEFF Research Database (Denmark)

Bracken, Adrian P; Ciro, Marco; Cocito, Andrea

2004-01-01

The E2F transcription factors are downstream effectors of the retinoblastoma protein (pRB) pathway and are required for the timely regulation of numerous genes essential for DNA replication and cell cycle progression. Several laboratories have used genome-wide approaches to discover novel target...

Crispr-mediated Gene Targeting of Human Induced Pluripotent Stem Cells.

Science.gov (United States)

Byrne, Susan M; Church, George M

2015-01-01

CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem or induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, we describe an optimized protocol for genome engineering of human iPSCs using a simple transient transfection of plasmids and/or single-stranded oligonucleotides. With this protocol, we achieve transfection efficiencies greater than 60%, with gene disruption efficiencies from 1-25% and gene insertion/replacement efficiencies from 0.5-10% without any further selection or enrichment steps. We also describe how to design and assess optimal sgRNA target sites and donor targeting vectors; cloning individual iPSC by single cell FACS sorting, and genotyping successfully edited cells.

Construction of a mouse model of factor VIII deficiency by gene targeting

Energy Technology Data Exchange (ETDEWEB)

Bi, L.; Lawler, A.; Gearhart, J. [Univ. of Pennsylvania School of Medicine, Philadelphia, PA (United States)] [and others

1994-09-01

To develop a small animal model of hemophilia A for gene therapy experiments, we set out to construct a mouse model for factor VIII deficiency by gene targeting. First, we screened a mouse liver cDNA library using a human FVIII cDNA probe. We cloned a 2.6 Kb partial mouse factor VIII cDNA which extends from 800 base pairs of the 3{prime} end of exon 14 to the 5{prime} end of exon 26. A mouse genomic library made from strain 129 was then screened to obtain genomic fragments covering the exons desired for homologous recombination. Two genomic clones were obtained, and one covering exon 15 through 22 was used for gene targeting. To make gene targeting constructs, a 5.8 Kb genomic DNA fragment covering exons 15 to 19 of the mouse FVIII gene was subcloned, and the neo expression cassette was inserted into exons 16 and 17 separately by different strategies. These two constructs were named MFVIIIC-16 and MFVIIIC-17. The constructs were linearized and transfected into strain 129 mouse ES cells by electroporation. Factor VIII gene-knockout ES cell lines were selected by G-418 and screened by genomic Southern blots. Eight exon 16 targeted cell lines and five exon 17 targeted cell lines were obtained. Three cell lines from each construct were injected into blastocysts and surgically transferred into foster mothers. Multiple chimeric mice with 70-90% hair color derived from the ES-cell genotype were seen with both constructs. Germ line transmission of the ES-cell genotype has been obtained for the MFVIIIC-16 construct, and multiple hemophilia A carrier females have been identified. Factor VIII-deficient males will be conceived soon.

Context dependent regulatory patterns of the androgen receptor and androgen receptor target genes

International Nuclear Information System (INIS)

Olsen, Jan Roger; Azeem, Waqas; Hellem, Margrete Reime; Marvyin, Kristo; Hua, Yaping; Qu, Yi; Li, Lisha; Lin, Biaoyang; Ke, XI- Song; Øyan, Anne Margrete; Kalland, Karl- Henning

2016-01-01

Expression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells, thus androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation. A clearer understanding of the context dependent activation of the AR and its target genes is therefore desirable. Immortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts. A variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen- and AR-dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial cells, but did not exert a positive feedback on endogenous AR expression. Treatment of basal prostate epithelial cells with inhibitors of epigenetic gene silencing was not efficient in

Identification of Phytophthora sojae genes involved in asexual ...

Indian Academy of Sciences (India)

ual sporulation or germination. But molecular details about asexual spore development in P. sojae are limited (Tyler et al. 2006). In the present study, to understand the molecular basis of asexual spore development in P. sojae, we investigated gene expression changes involved in asexual sporulation after ul- traviolet (UV) ...

Genes and Gut Bacteria Involved in Luminal Butyrate Reduction Caused by Diet and Loperamide.

Science.gov (United States)

Hwang, Nakwon; Eom, Taekil; Gupta, Sachin K; Jeong, Seong-Yeop; Jeong, Do-Youn; Kim, Yong Sung; Lee, Ji-Hoon; Sadowsky, Michael J; Unno, Tatsuya

2017-11-28

Unbalanced dietary habits and gut dysmotility are causative factors in metabolic and functional gut disorders, including obesity, diabetes, and constipation. Reduction in luminal butyrate synthesis is known to be associated with gut dysbioses, and studies have suggested that restoring butyrate formation in the colon may improve gut health. In contrast, shifts in different types of gut microbiota may inhibit luminal butyrate synthesis, requiring different treatments to restore colonic bacterial butyrate synthesis. We investigated the influence of high-fat diets (HFD) and low-fiber diets (LFD), and loperamide (LPM) administration, on key bacteria and genes involved in reduction of butyrate synthesis in mice. MiSeq-based microbiota analysis and HiSeq-based differential gene analysis indicated that different types of bacteria and genes were involved in butyrate metabolism in each treatment. Dietary modulation depleted butyrate kinase and phosphate butyryl transferase by decreasing members of the Bacteroidales and Parabacteroides . The HFD also depleted genes involved in succinate synthesis by decreasing Lactobacillus . The LFD and LPM treatments depleted genes involved in crotonoyl-CoA synthesis by decreasing Roseburia and Oscilllibacter . Taken together, our results suggest that different types of bacteria and genes were involved in gut dysbiosis, and that selected treatments may be needed depending on the cause of gut dysfunction.

Engineering liposomal nanoparticles for targeted gene therapy.

Science.gov (United States)

Zylberberg, C; Gaskill, K; Pasley, S; Matosevic, S

2017-08-01

Recent mechanistic studies have attempted to deepen our understanding of the process by which liposome-mediated delivery of genetic material occurs. Understanding the interactions between lipid nanoparticles and cells is still largely elusive. Liposome-mediated delivery of genetic material faces systemic obstacles alongside entry into the cell, endosomal escape, lysosomal degradation and nuclear uptake. Rational design approaches for targeted delivery have been developed to reduce off-target effects and enhance transfection. These strategies, which have included the modification of lipid nanoparticles with target-specific ligands to enhance intracellular uptake, have shown significant promise at the proof-of-concept stage. Control of physical and chemical specifications of liposome composition, which includes lipid-to-DNA charge, size, presence of ester bonds, chain length and nature of ligand complexation, is integral to the performance of targeted liposomes as genetic delivery agents. Clinical advances are expected to rely on such systems in the therapeutic application of liposome nanoparticle-based gene therapy. Here, we discuss the latest breakthroughs in the development of targeted liposome-based agents for the delivery of genetic material, paying particular attention to new ligand and cationic lipid design as well as recent in vivo advances.

The transcriptional repressor DREAM is involved in thyroid gene expression

International Nuclear Information System (INIS)

D'Andrea, Barbara; Di Palma, Tina; Mascia, Anna; Motti, Maria Letizia; Viglietto, Giuseppe; Nitsch, Lucio; Zannini, Mariastella

2005-01-01

Downstream regulatory element antagonistic modulator (DREAM) was originally identified in neuroendocrine cells as a calcium-binding protein that specifically binds to downstream regulatory elements (DRE) on DNA, and represses transcription of its target genes. To explore the possibility that DREAM may regulate the endocrine activity of the thyroid gland, we analyzed its mRNA expression in undifferentiated and differentiated thyroid cells. We demonstrated that DREAM is expressed in the normal thyroid tissue as well as in differentiated thyroid cells in culture while it is absent in FRT poorly differentiated cells. In the present work, we also show that DREAM specifically binds to DRE sites identified in the 5' untranslated region (UTR) of the thyroid-specific transcription factors Pax8 and TTF-2/FoxE1 in a calcium-dependent manner. By gel retardation assays we demonstrated that thapsigargin treatment increases the binding of DREAM to the DRE sequences present in Pax8 and TTF-2/Foxe1 5' UTRs, and this correlates with a significant reduction of the expression of these genes. Interestingly, in poorly differentiated thyroid cells overexpression of exogenous DREAM strongly inhibits Pax8 expression. Moreover, we provide evidence that a mutated form of DREAM unable to bind Ca 2+ interferes with thyroid cell proliferation. Therefore, we propose that in thyroid cells DREAM is a mediator of the calcium-signaling pathway and it is involved in the regulation of thyroid cell function

Non-canonical PRC1.1 Targets Active Genes Independent of H3K27me3 and Is Essential for Leukemogenesis

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Vincent van den Boom

2016-01-01

Full Text Available Polycomb proteins are classical regulators of stem cell self-renewal and cell lineage commitment and are frequently deregulated in cancer. Here, we find that the non-canonical PRC1.1 complex, as identified by mass-spectrometry-based proteomics, is critically important for human leukemic stem cells. Downmodulation of PRC1.1 complex members, like the DNA-binding subunit KDM2B, strongly reduces cell proliferation in vitro and delays or even abrogates leukemogenesis in vivo in humanized xenograft models. PRC1.1 components are significantly overexpressed in primary AML CD34+ cells. Besides a set of genes that is targeted by PRC1 and PRC2, ChIP-seq studies show that PRC1.1 also binds a distinct set of genes that are devoid of H3K27me3, suggesting a gene-regulatory role independent of PRC2. This set encompasses genes involved in metabolism, which have transcriptionally active chromatin profiles. These data indicate that PRC1.1 controls specific genes involved in unique cell biological processes required for leukemic cell viability.

Dual CRISPR-Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica.

Science.gov (United States)

Gao, Difeng; Smith, Spencer; Spagnuolo, Michael; Rodriguez, Gabriel; Blenner, Mark

2018-05-29

CRISPR-Cas9 technology has been successfully applied in Yarrowia lipolytica for targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest. The targeted regions for excision vary in length from 0.3 kb up to 3.5 kb and contain both non-coding and coding regions. The majority of the gene excisions are repaired by perfect nonhomologous end-joining without indel. Based on this dual cleavage system, two targeted markerless integration methods are developed by providing repair templates. While both strategies are effective, homology mediated end joining (HMEJ) based method are twice as efficient as homology recombination (HR) based method. In both cases, dual cleavage leads to similar or improved gene integration efficiencies compared to gene excision without integration. This dual cleavage strategy will be useful for not only generating more predictable and robust gene knockout, but also for efficient targeted markerless integration, and simultaneous knockout and integration in Y. lipolytica. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Construction of RNAi lentiviral vector targeting mouse Islet-1 gene

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Shen-shen ZHI

2011-02-01

Full Text Available Objective To construct and select RNAi lentiviral vectors that can silence mouse Islet-1 gene effectively.Methods Three groups of RNAi-target of mouse Islet-1 gene were designed,and corresponding shRNA oligo(sh1,sh2 and sh3 were synthesized,and then they were respectively inserted to the PLVTHM vector that had been digested by endonuclease.Agarose gel electrophoresis and sequencing were used to select and indentify the positive clones.The positive clones were extracted and then mixed with E.coli to amplify positive clones.The amplified clones were then infected into 293T along with the other 3 helper plasmids to produce lentiviral vector.After the construction of the lentiviral vector,plaque formation test was performed to determine the titer of lentiviral vector.The lentiviral vectors were then infected into C3H10T1/2 cells.The transfect efficiency of the lentiviral vectors was determined with flow cytometry with detection of green fluorescent protein(GFP.Q-PCR was employed to detect the RNAi efficiency of the lentiviral vectors.Results Agarose gel electrophoresis analysis showed that the clones with right gene at the target size were successfully established;gene sequencing showed that the right DNA fragments had been inserted;plaque formation test showed that the titer of the virus solution was 3.87×108TU/ml;the transfect efficiency of the lentiviral vector infected into C3H10T1/2 cells was 90.36%.All the 3 groups of shRNA targets(sh1,sh2 and sh3 showed an inhibitory effect on Islet-1 gene,and the sh1 showed the highest inhibitory effect(76.8%,as compared with that of normal cells(P < 0.05.Conclusion The RNAi lentiviral vector that can effectively silence the mouse Islet-1 gene has been constructed successfully,which may lay a foundation for further investigation of Islet-1 gene.

Nonviral Gene Targeting at rDNA Locus of Human Mesenchymal Stem Cells

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Youjin Hu

2013-01-01

Full Text Available Background. Genetic modification, such as the addition of exogenous genes to the MSC genome, is crucial to their use as cellular vehicles. Due to the risks associated with viral vectors such as insertional mutagenesis, the safer nonviral vectors have drawn a great deal of attention. Methods. VEGF, bFGF, vitamin C, and insulin-transferrin-selenium-X were supplemented in the MSC culture medium. The cells’ proliferation and survival capacity was measured by MTT, determination of the cumulative number of cells, and a colony-forming efficiency assay. The plasmid pHr2-NL was constructed and nucleofected into MSCs. The recombinants were selected using G418 and characterized using PCR and Southern blotting. Results. BFGF is critical to MSC growth and it acted synergistically with vitamin C, VEGF, and ITS-X, causing the cells to expand significantly. The neomycin gene was targeted to the rDNA locus of human MSCs using a nonviral human ribosomal targeting vector. The recombinant MSCs retained multipotential differentiation capacity, typical levels of hMSC surface marker expression, and a normal karyotype, and none were tumorigenic in nude mice. Conclusions. Exogenous genes can be targeted to the rDNA locus of human MSCs while maintaining the characteristics of MSCs. This is the first nonviral gene targeting of hMSCs.

A model of gene-gene and gene-environment interactions and its implications for targeting environmental interventions by genotype

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Wallace Helen M

2006-10-01

Full Text Available Abstract Background The potential public health benefits of targeting environmental interventions by genotype depend on the environmental and genetic contributions to the variance of common diseases, and the magnitude of any gene-environment interaction. In the absence of prior knowledge of all risk factors, twin, family and environmental data may help to define the potential limits of these benefits in a given population. However, a general methodology to analyze twin data is required because of the potential importance of gene-gene interactions (epistasis, gene-environment interactions, and conditions that break the 'equal environments' assumption for monozygotic and dizygotic twins. Method A new model for gene-gene and gene-environment interactions is developed that abandons the assumptions of the classical twin study, including Fisher's (1918 assumption that genes act as risk factors for common traits in a manner necessarily dominated by an additive polygenic term. Provided there are no confounders, the model can be used to implement a top-down approach to quantifying the potential utility of genetic prediction and prevention, using twin, family and environmental data. The results describe a solution space for each disease or trait, which may or may not include the classical twin study result. Each point in the solution space corresponds to a different model of genotypic risk and gene-environment interaction. Conclusion The results show that the potential for reducing the incidence of common diseases using environmental interventions targeted by genotype may be limited, except in special cases. The model also confirms that the importance of an individual's genotype in determining their risk of complex diseases tends to be exaggerated by the classical twin studies method, owing to the 'equal environments' assumption and the assumption of no gene-environment interaction. In addition, if phenotypes are genetically robust, because of epistasis

Identification of genes involved in DNA replication of the Autographa californica baculovirus

NARCIS (Netherlands)

Kool, M.; Ahrens, C. H.; Goldbach, R. W.; Rohrmann, G. F.; Vlak, J. M.

1994-01-01

By use of a transient replication assay, nine genes involved in DNA replication were identified in the genome of the Autographa californica baculovirus. Six genes encoding helicase, DNA polymerase, IE-1, LEF-1, LEF-2, and LEF-3 are essential for DNA replication while three genes encoding P35, IE-2,

Genome-wide identification of structural variants in genes encoding drug targets

DEFF Research Database (Denmark)

Rasmussen, Henrik Berg; Dahmcke, Christina Mackeprang

2012-01-01

The objective of the present study was to identify structural variants of drug target-encoding genes on a genome-wide scale. We also aimed at identifying drugs that are potentially amenable for individualization of treatments based on knowledge about structural variation in the genes encoding...

Transcriptomic Analysis Using Olive Varieties and Breeding Progenies Identifies Candidate Genes Involved in Plant Architecture.

Science.gov (United States)

González-Plaza, Juan J; Ortiz-Martín, Inmaculada; Muñoz-Mérida, Antonio; García-López, Carmen; Sánchez-Sevilla, José F; Luque, Francisco; Trelles, Oswaldo; Bejarano, Eduardo R; De La Rosa, Raúl; Valpuesta, Victoriano; Beuzón, Carmen R

2016-01-01

Plant architecture is a critical trait in fruit crops that can significantly influence yield, pruning, planting density and harvesting. Little is known about how plant architecture is genetically determined in olive, were most of the existing varieties are traditional with an architecture poorly suited for modern growing and harvesting systems. In the present study, we have carried out microarray analysis of meristematic tissue to compare expression profiles of olive varieties displaying differences in architecture, as well as seedlings from their cross pooled on the basis of their sharing architecture-related phenotypes. The microarray used, previously developed by our group has already been applied to identify candidates genes involved in regulating juvenile to adult transition in the shoot apex of seedlings. Varieties with distinct architecture phenotypes and individuals from segregating progenies displaying opposite architecture features were used to link phenotype to expression. Here, we identify 2252 differentially expressed genes (DEGs) associated to differences in plant architecture. Microarray results were validated by quantitative RT-PCR carried out on genes with functional annotation likely related to plant architecture. Twelve of these genes were further analyzed in individual seedlings of the corresponding pool. We also examined Arabidopsis mutants in putative orthologs of these targeted candidate genes, finding altered architecture for most of them. This supports a functional conservation between species and potential biological relevance of the candidate genes identified. This study is the first to identify genes associated to plant architecture in olive, and the results obtained could be of great help in future programs aimed at selecting phenotypes adapted to modern cultivation practices in this species.

Identification of novel target genes for safer and more specific control of root-knot nematodes from a pan-genome mining.

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Etienne G J Danchin

2013-10-01

Full Text Available Root-knot nematodes are globally the most aggressive and damaging plant-parasitic nematodes. Chemical nematicides have so far constituted the most efficient control measures against these agricultural pests. Because of their toxicity for the environment and danger for human health, these nematicides have now been banned from use. Consequently, new and more specific control means, safe for the environment and human health, are urgently needed to avoid worldwide proliferation of these devastating plant-parasites. Mining the genomes of root-knot nematodes through an evolutionary and comparative genomics approach, we identified and analyzed 15,952 nematode genes conserved in genomes of plant-damaging species but absent from non target genomes of chordates, plants, annelids, insect pollinators and mollusks. Functional annotation of the corresponding proteins revealed a relative abundance of putative transcription factors in this parasite-specific set compared to whole proteomes of root-knot nematodes. This may point to important and specific regulators of genes involved in parasitism. Because these nematodes are known to secrete effector proteins in planta, essential for parasitism, we searched and identified 993 such effector-like proteins absent from non-target species. Aiming at identifying novel targets for the development of future control methods, we biologically tested the effect of inactivation of the corresponding genes through RNA interference. A total of 15 novel effector-like proteins and one putative transcription factor compatible with the design of siRNAs were present as non-redundant genes and had transcriptional support in the model root-knot nematode Meloidogyne incognita. Infestation assays with siRNA-treated M. incognita on tomato plants showed significant and reproducible reduction of the infestation for 12 of the 16 tested genes compared to control nematodes. These 12 novel genes, showing efficient reduction of parasitism when

Id-1 gene and gene products as therapeutic targets for treatment of breast cancer and other types of carcinoma

Science.gov (United States)

Desprez, Pierre-Yves; Campisi, Judith

2014-08-19

A method for treatment of breast cancer and other types of cancer. The method comprises targeting and modulating Id-1 gene expression, if any, for the Id-1 gene, or gene products in breast or other epithelial cancers in a patient by delivering products that modulate Id-1 gene expression. When expressed, Id-1 gene is a prognostic indicator that cancer cells are invasive and metastatic.

Bioinformatic analysis of patient-derived ASPS gene expressions and ASPL-TFE3 fusion transcript levels identify potential therapeutic targets.

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David G Covell

Full Text Available Gene expression data, collected from ASPS tumors of seven different patients and from one immortalized ASPS cell line (ASPS-1, was analyzed jointly with patient ASPL-TFE3 (t(X;17(p11;q25 fusion transcript data to identify disease-specific pathways and their component genes. Data analysis of the pooled patient and ASPS-1 gene expression data, using conventional clustering methods, revealed a relatively small set of pathways and genes characterizing the biology of ASPS. These results could be largely recapitulated using only the gene expression data collected from patient tumor samples. The concordance between expression measures derived from ASPS-1 and both pooled and individual patient tumor data provided a rationale for extending the analysis to include patient ASPL-TFE3 fusion transcript data. A novel linear model was exploited to link gene expressions to fusion transcript data and used to identify a small set of ASPS-specific pathways and their gene expression. Cellular pathways that appear aberrantly regulated in response to the t(X;17(p11;q25 translocation include the cell cycle and cell adhesion. The identification of pathways and gene subsets characteristic of ASPS support current therapeutic strategies that target the FLT1 and MET, while also proposing additional targeting of genes found in pathways involved in the cell cycle (CHK1, cell adhesion (ARHGD1A, cell division (CDC6, control of meiosis (RAD51L3 and mitosis (BIRC5, and chemokine-related protein tyrosine kinase activity (CCL4.

Sexually dimorphic gene regulation in brain as a target for endocrine disrupters: Developmental exposure of rats to 4-methylbenzylidene camphor

International Nuclear Information System (INIS)

Maerkel, Kirsten; Durrer, Stefan; Henseler, Manuel; Schlumpf, Margret; Lichtensteiger, Walter

2007-01-01

The developing neuroendocrine brain represents a potential target for endocrine active chemicals. The UV filter 4-methylbenzylidene camphor (4-MBC) exhibits estrogenic activity, but also interferes with the thyroid axis. We investigated effects of pre- and postnatal exposure to 4-MBC in the same rat offspring at brain and reproductive organ levels. 4-MBC (7, 24, 47 mg/kg/day) was administered in chow to the parent generation before mating, during gestation and lactation, and to the offspring until adulthood. mRNA of estrogen target genes involved in control of sexual behavior and gonadal functions was measured by real-time RT-PCR in ventromedial hypothalamic nucleus (VMH) and medial preoptic area (MPO) of adult offspring. 4-MBC exposure affected mRNA levels of ER alpha, progesterone receptor (PR), preproenkephalin (PPE) and insulin-like growth factor-I (IGF-I) in a sex- and region-specific manner. In order to assess possible changes in sensitivity of target genes to estrogens, offspring were gonadectomized on day 70, injected with estradiol (E2, 10 or 50 μg/kg s.c.) or vehicle on day 84, and sacrificed 6 h later. The acute induction of PR mRNA, and repression (at 6 h) of PPE mRNA by E2 was enhanced by 4-MBC in male and female VMH and female MPO, whereas male MPO exhibited reduced responsiveness of both genes. Steroid receptor coactivator SRC-1 mRNA levels were increased in female VMH and MPO. The data indicate profound sex- and region-specific alterations in the regulation of estrogen target genes at brain level. Effect patterns in baseline and E2-induced gene expression differ from those in uterus and prostate

Attribution of human characteristics and bullying involvement in childhood: Distinguishing between targets

NARCIS (Netherlands)

Noorden, T.H.J. van; Haselager, G.J.T.; Lansu, T.A.M.; Cillessen, A.H.N.; Bukowski, W.M.

2016-01-01

This investigation researched the association between the attribution of human characteristics and bullying involvement in children by distinguishing between targets. Study 1 focused on the attribution of human characteristics by bullies, victims, bully/victims, and non-involved children toward

PHOX2B-mediated regulation of ALK expression: in vitro identification of a functional relationship between two genes involved in neuroblastoma.

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Tiziana Bachetti

Full Text Available BACKGROUND: Neuroblastoma (NB is a severe pediatric tumor originating from neural crest derivatives and accounting for 15% of childhood cancer mortality. The heterogeneous and complex genetic etiology has been confirmed with the identification of mutations in two genes, encoding for the receptor tyrosine kinase Anaplastic Lymphoma Kinase (ALK and the transcription factor Paired-like Homeobox 2B (PHOX2B, in a limited proportion of NB patients. Interestingly, these two genes are overexpressed in the great majority of primary NB samples and cell lines. These observations led us to test the hypothesis of a regulatory or functional relationship between ALK and PHOX2B underlying NB pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Following this possibility, we first confirmed a striking correlation between the transcription levels of ALK, PHOX2B and its direct target PHOX2A in a panel of NB cell lines. Then, we manipulated their expression in NB cell lines by siRNA-mediated knock-down and forced over-expression of each gene under analysis. Surprisingly, PHOX2B- and PHOX2A-directed siRNAs efficiently downregulated each other as well as ALK gene and, consistently, the enhanced expression of PHOX2B in NB cells yielded an increment of ALK protein. We finally demonstrated that PHOX2B drives ALK gene transcription by directly binding its promoter, which therefore represents a novel PHOX2B target. CONCLUSIONS/SIGNIFICANCE: These findings provide a compelling explanation of the concurrent involvement of these two genes in NB pathogenesis and are going to foster a better understanding of molecular interactions at the base of the disease. Moreover, this work opens new perspectives for NBs refractory to conventional therapies that may benefit from the design of novel therapeutic RNAi-based approaches for multiple gene targets.

Haloperidol induces pharmacoepigenetic response by modulating miRNA expression, global DNA methylation and expression profiles of methylation maintenance genes and genes involved in neurotransmission in neuronal cells.

Science.gov (United States)

Swathy, Babu; Banerjee, Moinak

2017-01-01

Haloperidol has been extensively used in various psychiatric conditions. It has also been reported to induce severe side effects. We aimed to evaluate whether haloperidol can influence host methylome, and if so what are the possible mechanisms for it in neuronal cells. Impact on host methylome and miRNAs can have wide spread alterations in gene expression, which might possibly help in understanding how haloperidol may impact treatment response or induce side effects. SK-N-SH, a neuroblasoma cell line was treated with haloperidol at 10μm concentration for 24 hours and global DNA methylation was evaluated. Methylation at global level is maintained by methylation maintenance machinery and certain miRNAs. Therefore, the expression of methylation maintenance genes and their putative miRNA expression profiles were assessed. These global methylation alterations could result in gene expression changes. Therefore genes expressions for neurotransmitter receptors, regulators, ion channels and transporters were determined. Subsequently, we were also keen to identify a strong candidate miRNA based on biological and in-silico approach which can reflect on the pharmacoepigenetic trait of haloperidol and can also target the altered neuroscience panel of genes used in the study. Haloperidol induced increase in global DNA methylation which was found to be associated with corresponding increase in expression of various epigenetic modifiers that include DNMT1, DNMT3A, DNMT3B and MBD2. The expression of miR-29b that is known to putatively regulate the global methylation by modulating the expression of epigenetic modifiers was observed to be down regulated by haloperidol. In addition to miR-29b, miR-22 was also found to be downregulated by haloperidol treatment. Both these miRNA are known to putatively target several genes associated with various epigenetic modifiers, pharmacogenes and neurotransmission. Interestingly some of these putative target genes involved in neurotransmission

Haloperidol induces pharmacoepigenetic response by modulating miRNA expression, global DNA methylation and expression profiles of methylation maintenance genes and genes involved in neurotransmission in neuronal cells.

Directory of Open Access Journals (Sweden)

Babu Swathy

Full Text Available Haloperidol has been extensively used in various psychiatric conditions. It has also been reported to induce severe side effects. We aimed to evaluate whether haloperidol can influence host methylome, and if so what are the possible mechanisms for it in neuronal cells. Impact on host methylome and miRNAs can have wide spread alterations in gene expression, which might possibly help in understanding how haloperidol may impact treatment response or induce side effects.SK-N-SH, a neuroblasoma cell line was treated with haloperidol at 10μm concentration for 24 hours and global DNA methylation was evaluated. Methylation at global level is maintained by methylation maintenance machinery and certain miRNAs. Therefore, the expression of methylation maintenance genes and their putative miRNA expression profiles were assessed. These global methylation alterations could result in gene expression changes. Therefore genes expressions for neurotransmitter receptors, regulators, ion channels and transporters were determined. Subsequently, we were also keen to identify a strong candidate miRNA based on biological and in-silico approach which can reflect on the pharmacoepigenetic trait of haloperidol and can also target the altered neuroscience panel of genes used in the study.Haloperidol induced increase in global DNA methylation which was found to be associated with corresponding increase in expression of various epigenetic modifiers that include DNMT1, DNMT3A, DNMT3B and MBD2. The expression of miR-29b that is known to putatively regulate the global methylation by modulating the expression of epigenetic modifiers was observed to be down regulated by haloperidol. In addition to miR-29b, miR-22 was also found to be downregulated by haloperidol treatment. Both these miRNA are known to putatively target several genes associated with various epigenetic modifiers, pharmacogenes and neurotransmission. Interestingly some of these putative target genes involved in

Prediction of target genes for miR-140-5p in pulmonary arterial hypertension using bioinformatics methods.

Science.gov (United States)

Li, Fangwei; Shi, Wenhua; Wan, Yixin; Wang, Qingting; Feng, Wei; Yan, Xin; Wang, Jian; Chai, Limin; Zhang, Qianqian; Li, Manxiang

2017-12-01

The expression of microRNA (miR)-140-5p is known to be reduced in both pulmonary arterial hypertension (PAH) patients and monocrotaline-induced PAH models in rat. Identification of target genes for miR-140-5p with bioinformatics analysis may reveal new pathways and connections in PAH. This study aimed to explore downstream target genes and relevant signaling pathways regulated by miR-140-5p to provide theoretical evidences for further researches on role of miR-140-5p in PAH. Multiple downstream target genes and upstream transcription factors (TFs) of miR-140-5p were predicted in the analysis. Gene ontology (GO) enrichment analysis indicated that downstream target genes of miR-140-5p were enriched in many biological processes, such as biological regulation, signal transduction, response to chemical stimulus, stem cell proliferation, cell surface receptor signaling pathways. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis found that downstream target genes were mainly located in Notch, TGF-beta, PI3K/Akt, and Hippo signaling pathway. According to TF-miRNA-mRNA network, the important downstream target genes of miR-140-5p were PPI, TGF-betaR1, smad4, JAG1, ADAM10, FGF9, PDGFRA, VEGFA, LAMC1, TLR4, and CREB. After thoroughly reviewing published literature, we found that 23 target genes and seven signaling pathways were truly inhibited by miR-140-5p in various tissues or cells; most of these verified targets were in accordance with our present prediction. Other predicted targets still need further verification in vivo and in vitro .

Identification of novel androgen receptor target genes in prostate cancer

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Gerald William L

2007-06-01

Full Text Available Abstract Background The androgen receptor (AR plays critical roles in both androgen-dependent and castrate-resistant prostate cancer (PCa. However, little is known about AR target genes that mediate the receptor's roles in disease progression. Results Using Chromatin Immunoprecipitation (ChIP Display, we discovered 19 novel loci occupied by the AR in castrate resistant C4-2B PCa cells. Only four of the 19 AR-occupied regions were within 10-kb 5'-flanking regulatory sequences. Three were located up to 4-kb 3' of the nearest gene, eight were intragenic and four were in gene deserts. Whereas the AR occupied the same loci in C4-2B (castrate resistant and LNCaP (androgen-dependent PCa cells, differences between the two cell lines were observed in the response of nearby genes to androgens. Among the genes strongly stimulated by DHT in C4-2B cells – D-dopachrome tautomerase (DDT, Protein kinase C delta (PRKCD, Glutathione S- transferase theta 2 (GSTT2, Transient receptor potential cation channel subfamily V member 3 (TRPV3, and Pyrroline-5-carboxylate reductase 1 (PYCR1 – most were less strongly or hardly stimulated in LNCaP cells. Another AR target gene, ornithine aminotransferase (OAT, was AR-stimulated in a ligand-independent manner, since it was repressed by AR siRNA knockdown, but not stimulated by DHT. We also present evidence for in vivo AR-mediated regulation of several genes identified by ChIP Display. For example, PRKCD and PYCR1, which may contribute to PCa cell growth and survival, are expressed in PCa biopsies from primary tumors before and after ablation and in metastatic lesions in a manner consistent with AR-mediated stimulation. Conclusion AR genomic occupancy is similar between LNCaP and C4-2B cells and is not biased towards 5' gene flanking sequences. The AR transcriptionally regulates less than half the genes nearby AR-occupied regions, usually but not always, in a ligand-dependent manner. Most are stimulated and a few are

Preservation of genes involved in sterol metabolism in cholesterol auxotrophs: facts and hypotheses.

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Giovanna Vinci

Full Text Available BACKGROUND: It is known that primary sequences of enzymes involved in sterol biosynthesis are well conserved in organisms that produce sterols de novo. However, we provide evidence for a preservation of the corresponding genes in two animals unable to synthesize cholesterol (auxotrophs: Drosophila melanogaster and Caenorhabditis elegans. PRINCIPAL FINDINGS: We have been able to detect bona fide orthologs of several ERG genes in both organisms using a series of complementary approaches. We have detected strong sequence divergence between the orthologs of the nematode and of the fruitfly; they are also very divergent with respect to the orthologs in organisms able to synthesize sterols de novo (prototrophs. Interestingly, the orthologs in both the nematode and the fruitfly are still under selective pressure. It is possible that these genes, which are not involved in cholesterol synthesis anymore, have been recruited to perform different new functions. We propose a more parsimonious way to explain their accelerated evolution and subsequent stabilization. The products of ERG genes in prototrophs might be involved in several biological roles, in addition to sterol synthesis. In the case of the nematode and the fruitfly, the relevant genes would have lost their ancestral function in cholesterogenesis but would have retained the other function(s, which keep them under pressure. CONCLUSIONS: By exploiting microarray data we have noticed a strong expressional correlation between the orthologs of ERG24 and ERG25 in D. melanogaster and genes encoding factors involved in intracellular protein trafficking and folding and with Start1 involved in ecdysteroid synthesis. These potential functional connections are worth being explored not only in Drosophila, but also in Caenorhabditis as well as in sterol prototrophs.

Identification of Spt5 target genes in zebrafish development reveals its dual activity in vivo.

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Keerthi Krishnan

Full Text Available Spt5 is a conserved essential protein that represses or stimulates transcription elongation in vitro. Immunolocalization studies on Drosophila polytene chromosomes suggest that Spt5 is associated with many loci throughout the genome. However, little is known about the prevalence and identity of Spt5 target genes in vivo during development. Here, we identify direct target genes of Spt5 using fog(sk8 zebrafish mutant, which disrupts the foggy/spt5 gene. We identified that fog(sk8 and their wildtype siblings differentially express less than 5% of genes examined. These genes participate in diverse biological processes from stress response to cell fate specification. Up-regulated genes exhibit shorter overall gene length compared to all genes examined. Through chromatin immunoprecipitation in zebrafish embryos, we identified a subset of developmentally critical genes that are bound by both Spt5 and RNA polymerase II. The protein occupancy patterns on these genes are characteristic of both repressive and stimulatory elongation regulation. Together our findings establish Spt5 as a dual regulator of transcription elongation in vivo and identify a small but diverse set of target genes critically dependent on Spt5 during development.

Ultrasound-mediated vascular gene transfection by cavitation of endothelial-targeted cationic microbubbles.

Science.gov (United States)

Xie, Aris; Belcik, Todd; Qi, Yue; Morgan, Terry K; Champaneri, Shivam A; Taylor, Sarah; Davidson, Brian P; Zhao, Yan; Klibanov, Alexander L; Kuliszewski, Michael A; Leong-Poi, Howard; Ammi, Azzdine; Lindner, Jonathan R

2012-12-01

Ultrasound-mediated gene delivery can be amplified by acoustic disruption of microbubble carriers that undergo cavitation. We hypothesized that endothelial targeting of microbubbles bearing cDNA is feasible and, through optimizing proximity to the vessel wall, increases the efficacy of gene transfection. Contrast ultrasound-mediated gene delivery is a promising approach for site-specific gene therapy, although there are concerns with the reproducibility of this technique and the safety when using high-power ultrasound. Cationic lipid-shelled decafluorobutane microbubbles bearing a targeting moiety were prepared and compared with nontargeted microbubbles. Microbubble targeting efficiency to endothelial adhesion molecules (P-selectin or intercellular adhesion molecule [ICAM]-1) was tested using in vitro flow chamber studies, intravital microscopy of tumor necrosis factor-alpha (TNF-α)-stimulated murine cremaster muscle, and targeted contrast ultrasound imaging of P-selectin in a model of murine limb ischemia. Ultrasound-mediated transfection of luciferase reporter plasmid charge coupled to microbubbles in the post-ischemic hindlimb muscle was assessed by in vivo optical imaging. Charge coupling of cDNA to the microbubble surface was not influenced by the presence of targeting ligand, and did not alter the cavitation properties of cationic microbubbles. In flow chamber studies, surface conjugation of cDNA did not affect attachment of targeted microbubbles at microvascular shear stresses (0.6 and 1.5 dyne/cm(2)). Attachment in vivo was also not affected by cDNA according to intravital microscopy observations of venular adhesion of ICAM-1-targeted microbubbles and by ultrasound molecular imaging of P-selectin-targeted microbubbles in the post-ischemic hindlimb in mice. Transfection at the site of high acoustic pressures (1.0 and 1.8 MPa) was similar for control and P-selectin-targeted microbubbles but was associated with vascular rupture and hemorrhage. At 0.6 MPa

Tris(1,3-dichloro-2-propyl) phosphate perturbs the expression of genes involved in immune response and lipid and steroid metabolism in chicken embryos

International Nuclear Information System (INIS)

Farhat, Amani; Buick, Julie K.; Williams, Andrew; Yauk, Carole L.; O'Brien, Jason M.; Crump, Doug; Williams, Kim L.; Chiu, Suzanne; Kennedy, Sean W.

2014-01-01

We previously demonstrated that in ovo exposure to the flame retardant tris(1,3-dichloro-2-propyl) phosphate (TDCPP) decreased plasma thyroxine levels, reduced growth parameters, and decreased gallbladder size in chicken embryos. In the current study DNA microarrays were used to evaluate global mRNA expression in liver tissue of male chicken embryos that exhibited the above mentioned effects. Injected doses were dimethyl sulfoxide vehicle control, 7.6 or 45 μg TDCPP/g egg. TDCPP caused significant changes in the expression of five genes at the low dose and 47 genes at the high dose (False Discovery Rate p ≤ 0.1, fold change ≥ 1.5). The gene expression analysis suggested a compromised immune function, a state of cholestatic liver/biliary fibrosis, and disrupted lipid and steroid metabolism. Circulating bile acid levels were elevated, which is an indication of liver dysfunction, and plasma cholesterol levels were reduced; however, hepatic bile acid and cholesterol levels were unaltered. Interactome analyses identified apolipoprotein E, hepatocyte nuclear factor 4 alpha, and peroxisome proliferator-activated receptor alpha as key regulatory molecules involved in the effects of TDCPP. Our results demonstrate a targeted effect of TDCPP toxicity on lipid metabolism, including cholesterol, that helps explain the aforementioned phenotypic effects, as chicken embryos are highly dependent on yolk lipids for growth and maintenance throughout development. Finally, our results are in concordance with the literature that describes TDCPP as a cancer-causing agent, since the majority of dysregulated genes were involved in cancer pathways. - Highlights: • TDCPP dysregulates genes involved in immune function and lipid metabolism. • A targeted effect of TDCPP toxicity on cholesterol metabolism is apparent. • A state of cholestatic liver fibrosis is suggested by the expression profile. • Elevated plasma bile acids suggest that TDCPP causes liver dysfunction

Tris(1,3-dichloro-2-propyl) phosphate perturbs the expression of genes involved in immune response and lipid and steroid metabolism in chicken embryos

Energy Technology Data Exchange (ETDEWEB)

Farhat, Amani [Department of Biology, University of Ottawa, Ottawa, ON K1N 6N5 (Canada); National Wildlife Research Centre, Environment Canada, Ottawa, ON K1A 0H3 (Canada); Buick, Julie K.; Williams, Andrew; Yauk, Carole L.; O' Brien, Jason M. [Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON K1A 0K9 (Canada); Crump, Doug; Williams, Kim L.; Chiu, Suzanne [National Wildlife Research Centre, Environment Canada, Ottawa, ON K1A 0H3 (Canada); Kennedy, Sean W., E-mail: sean.kennedy@ec.gc.ca [Department of Biology, University of Ottawa, Ottawa, ON K1N 6N5 (Canada); National Wildlife Research Centre, Environment Canada, Ottawa, ON K1A 0H3 (Canada)

2014-03-01

We previously demonstrated that in ovo exposure to the flame retardant tris(1,3-dichloro-2-propyl) phosphate (TDCPP) decreased plasma thyroxine levels, reduced growth parameters, and decreased gallbladder size in chicken embryos. In the current study DNA microarrays were used to evaluate global mRNA expression in liver tissue of male chicken embryos that exhibited the above mentioned effects. Injected doses were dimethyl sulfoxide vehicle control, 7.6 or 45 μg TDCPP/g egg. TDCPP caused significant changes in the expression of five genes at the low dose and 47 genes at the high dose (False Discovery Rate p ≤ 0.1, fold change ≥ 1.5). The gene expression analysis suggested a compromised immune function, a state of cholestatic liver/biliary fibrosis, and disrupted lipid and steroid metabolism. Circulating bile acid levels were elevated, which is an indication of liver dysfunction, and plasma cholesterol levels were reduced; however, hepatic bile acid and cholesterol levels were unaltered. Interactome analyses identified apolipoprotein E, hepatocyte nuclear factor 4 alpha, and peroxisome proliferator-activated receptor alpha as key regulatory molecules involved in the effects of TDCPP. Our results demonstrate a targeted effect of TDCPP toxicity on lipid metabolism, including cholesterol, that helps explain the aforementioned phenotypic effects, as chicken embryos are highly dependent on yolk lipids for growth and maintenance throughout development. Finally, our results are in concordance with the literature that describes TDCPP as a cancer-causing agent, since the majority of dysregulated genes were involved in cancer pathways. - Highlights: • TDCPP dysregulates genes involved in immune function and lipid metabolism. • A targeted effect of TDCPP toxicity on cholesterol metabolism is apparent. • A state of cholestatic liver fibrosis is suggested by the expression profile. • Elevated plasma bile acids suggest that TDCPP causes liver dysfunction.

Target genes discovery through copy number alteration analysis in human hepatocellular carcinoma.

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Gu, De-Leung; Chen, Yen-Hsieh; Shih, Jou-Ho; Lin, Chi-Hung; Jou, Yuh-Shan; Chen, Chian-Feng

2013-12-21

High-throughput short-read sequencing of exomes and whole cancer genomes in multiple human hepatocellular carcinoma (HCC) cohorts confirmed previously identified frequently mutated somatic genes, such as TP53, CTNNB1 and AXIN1, and identified several novel genes with moderate mutation frequencies, including ARID1A, ARID2, MLL, MLL2, MLL3, MLL4, IRF2, ATM, CDKN2A, FGF19, PIK3CA, RPS6KA3, JAK1, KEAP1, NFE2L2, C16orf62, LEPR, RAC2, and IL6ST. Functional classification of these mutated genes suggested that alterations in pathways participating in chromatin remodeling, Wnt/β-catenin signaling, JAK/STAT signaling, and oxidative stress play critical roles in HCC tumorigenesis. Nevertheless, because there are few druggable genes used in HCC therapy, the identification of new therapeutic targets through integrated genomic approaches remains an important task. Because a large amount of HCC genomic data genotyped by high density single nucleotide polymorphism arrays is deposited in the public domain, copy number alteration (CNA) analyses of these arrays is a cost-effective way to reveal target genes through profiling of recurrent and overlapping amplicons, homozygous deletions and potentially unbalanced chromosomal translocations accumulated during HCC progression. Moreover, integration of CNAs with other high-throughput genomic data, such as aberrantly coding transcriptomes and non-coding gene expression in human HCC tissues and rodent HCC models, provides lines of evidence that can be used to facilitate the identification of novel HCC target genes with the potential of improving the survival of HCC patients.

Identification of highly effective target genes for RNAi-mediated control of emerald ash borer, Agrilus planipennis.

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Rodrigues, Thais B; Duan, Jian J; Palli, Subba R; Rieske, Lynne K

2018-03-22

Recent study has shown that RNA interference (RNAi) is efficient in emerald ash borer (EAB), Agrilus planipennis, and that ingestion of double-stranded RNA (dsRNA) targeting specific genes causes gene silencing and mortality in neonates. Here, we report on the identification of highly effective target genes for RNAi-mediated control of EAB. We screened 13 candidate genes in neonate larvae and selected the most effective target genes for further investigation, including their effect on EAB adults and on a non-target organism, Tribolium castaneum. The two most efficient target genes selected, hsp (heat shock 70-kDa protein cognate 3) and shi (shibire), caused up to 90% mortality of larvae and adults. In EAB eggs, larvae, and adults, the hsp is expressed at higher levels when compared to that of shi. Ingestion of dsHSP and dsSHI caused mortality in both neonate larvae and adults. Administration of a mixture of both dsRNAs worked better than either dsRNA by itself. In contrast, injection of EAB.dsHSP and EAB.dsSHI did not cause mortality in T. castaneum. Thus, the two genes identified cause high mortality in the EAB with no apparent phenotype effects in a non-target organism, the red flour beetle, and could be used in RNAi-mediated control of this invasive pest.

Advances in plant gene-targeted and functional markers: a review

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Poczai Péter

2013-02-01

Full Text Available Abstract Public genomic databases have provided new directions for molecular marker development and initiated a shift in the types of PCR-based techniques commonly used in plant science. Alongside commonly used arbitrarily amplified DNA markers, other methods have been developed. Targeted fingerprinting marker techniques are based on the well-established practices of arbitrarily amplified DNA methods, but employ novel methodological innovations such as the incorporation of gene or promoter elements in the primers. These markers provide good reproducibility and increased resolution by the concurrent incidence of dominant and co-dominant bands. Despite their promising features, these semi-random markers suffer from possible problems of collision and non-homology analogous to those found with randomly generated fingerprints. Transposable elements, present in abundance in plant genomes, may also be used to generate fingerprints. These markers provide increased genomic coverage by utilizing specific targeted sites and produce bands that mostly seem to be homologous. The biggest drawback with most of these techniques is that prior genomic information about retrotransposons is needed for primer design, prohibiting universal applications. Another class of recently developed methods exploits length polymorphism present in arrays of multi-copy gene families such as cytochrome P450 and β-tubulin genes to provide cross-species amplification and transferability. A specific class of marker makes use of common features of plant resistance genes to generate bands linked to a given phenotype, or to reveal genetic diversity. Conserved DNA-based strategies have limited genome coverage and may fail to reveal genetic diversity, while resistance genes may be under specific evolutionary selection. Markers may also be generated from functional and/or transcribed regions of the genome using different gene-targeting approaches coupled with the use of RNA information

Advances in plant gene-targeted and functional markers: a review

Science.gov (United States)

2013-01-01

Public genomic databases have provided new directions for molecular marker development and initiated a shift in the types of PCR-based techniques commonly used in plant science. Alongside commonly used arbitrarily amplified DNA markers, other methods have been developed. Targeted fingerprinting marker techniques are based on the well-established practices of arbitrarily amplified DNA methods, but employ novel methodological innovations such as the incorporation of gene or promoter elements in the primers. These markers provide good reproducibility and increased resolution by the concurrent incidence of dominant and co-dominant bands. Despite their promising features, these semi-random markers suffer from possible problems of collision and non-homology analogous to those found with randomly generated fingerprints. Transposable elements, present in abundance in plant genomes, may also be used to generate fingerprints. These markers provide increased genomic coverage by utilizing specific targeted sites and produce bands that mostly seem to be homologous. The biggest drawback with most of these techniques is that prior genomic information about retrotransposons is needed for primer design, prohibiting universal applications. Another class of recently developed methods exploits length polymorphism present in arrays of multi-copy gene families such as cytochrome P450 and β-tubulin genes to provide cross-species amplification and transferability. A specific class of marker makes use of common features of plant resistance genes to generate bands linked to a given phenotype, or to reveal genetic diversity. Conserved DNA-based strategies have limited genome coverage and may fail to reveal genetic diversity, while resistance genes may be under specific evolutionary selection. Markers may also be generated from functional and/or transcribed regions of the genome using different gene-targeting approaches coupled with the use of RNA information. Such techniques have the

Molecular evolution of pentatricopeptide repeat genes reveals truncation in species lacking an editing target and structural domains under distinct selective pressures

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Hayes Michael L

2012-05-01

selection even in the absence of an editing site target suggests that unrecognized function(s might exist for this PPR protein. PPR gene sequences that encode helix A are under strong selection, and could be involved in RNA substrate recognition.

Molecular evolution of pentatricopeptide repeat genes reveals truncation in species lacking an editing target and structural domains under distinct selective pressures.

Science.gov (United States)

Hayes, Michael L; Giang, Karolyn; Mulligan, R Michael

2012-05-14

Pentatricopeptide repeat (PPR) proteins are required for numerous RNA processing events in plant organelles including C-to-U editing, splicing, stabilization, and cleavage. Fifteen PPR proteins are known to be required for RNA editing at 21 sites in Arabidopsis chloroplasts, and belong to the PLS class of PPR proteins. In this study, we investigate the co-evolution of four PPR genes (CRR4, CRR21, CLB19, and OTP82) and their six editing targets in Brassicaceae species. PPR genes are composed of approximately 10 to 20 tandem repeats and each repeat has two α-helical regions, helix A and helix B, that are separated by short coil regions. Each repeat and structural feature was examined to determine the selective pressures on these regions. All of the PPR genes examined are under strong negative selection. Multiple independent losses of editing site targets are observed for both CRR21 and OTP82. In several species lacking the known editing target for CRR21, PPR genes are truncated near the 17th PPR repeat. The coding sequences of the truncated CRR21 genes are maintained under strong negative selection; however, the 3' UTR sequences beyond the truncation site have substantially diverged. Phylogenetic analyses of four PPR genes show that sequences corresponding to helix A are high compared to helix B sequences. Differential evolutionary selection of helix A versus helix B is observed in both plant and mammalian PPR genes. PPR genes and their cognate editing sites are mutually constrained in evolution. Editing sites are frequently lost by replacement of an edited C with a genomic T. After the loss of an editing site, the PPR genes are observed with three outcomes: first, few changes are detected in some cases; second, the PPR gene is present as a pseudogene; and third, the PPR gene is present but truncated in the C-terminal region. The retention of truncated forms of CRR21 that are maintained under strong negative selection even in the absence of an editing site target

BDNF gene delivery mediated by neuron-targeted nanoparticles is neuroprotective in peripheral nerve injury

OpenAIRE

Lopes, CDF; Gonçalves, NP; Gomes, CP; Saraiva, MJ; Pêgo, AP

2017-01-01

Neuron-targeted gene delivery is a promising strategy to treat peripheral neuropathies. Here we propose the use of polymeric nanoparticles based on thiolated trimethyl chitosan (TMCSH) to mediate targeted gene delivery to peripheral neurons upon a peripheral and minimally invasive intramuscular administration. Nanoparticles were grafted with the non-toxic carboxylic fragment of the tetanus neurotoxin (HC) to allow neuron targeting and were explored to deliver a plasmid DNA encoding for the br...

Examination of Signatures of Recent Positive Selection on Genes Involved in Human Sialic Acid Biology.

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Moon, Jiyun M; Aronoff, David M; Capra, John A; Abbot, Patrick; Rokas, Antonis

2018-03-28

Sialic acids are nine carbon sugars ubiquitously found on the surfaces of vertebrate cells and are involved in various immune response-related processes. In humans, at least 58 genes spanning diverse functions, from biosynthesis and activation to recycling and degradation, are involved in sialic acid biology. Because of their role in immunity, sialic acid biology genes have been hypothesized to exhibit elevated rates of evolutionary change. Consistent with this hypothesis, several genes involved in sialic acid biology have experienced higher rates of non-synonymous substitutions in the human lineage than their counterparts in other great apes, perhaps in response to ancient pathogens that infected hominins millions of years ago (paleopathogens). To test whether sialic acid biology genes have also experienced more recent positive selection during the evolution of the modern human lineage, reflecting adaptation to contemporary cosmopolitan or geographically-restricted pathogens, we examined whether their protein-coding regions showed evidence of recent hard and soft selective sweeps. This examination involved the calculation of four measures that quantify changes in allele frequency spectra, extent of population differentiation, and haplotype homozygosity caused by recent hard and soft selective sweeps for 55 sialic acid biology genes using publicly available whole genome sequencing data from 1,668 humans from three ethnic groups. To disentangle evidence for selection from confounding demographic effects, we compared the observed patterns in sialic acid biology genes to simulated sequences of the same length under a model of neutral evolution that takes into account human demographic history. We found that the patterns of genetic variation of most sialic acid biology genes did not significantly deviate from neutral expectations and were not significantly different among genes belonging to different functional categories. Those few sialic acid biology genes that

Engineering nucleases for gene targeting: safety and regulatory considerations.

Science.gov (United States)

Pauwels, Katia; Podevin, Nancy; Breyer, Didier; Carroll, Dana; Herman, Philippe

2014-01-25

Nuclease-based gene targeting (NBGT) represents a significant breakthrough in targeted genome editing since it is applicable from single-celled protozoa to human, including several species of economic importance. Along with the fast progress in NBGT and the increasing availability of customized nucleases, more data are available about off-target effects associated with the use of this approach. We discuss how NBGT may offer a new perspective for genetic modification, we address some aspects crucial for a safety improvement of the corresponding techniques and we also briefly relate the use of NBGT applications and products to the regulatory oversight. Copyright © 2013 Elsevier B.V. All rights reserved.

Effects of insulin and exercise training on FGF21, its receptors and target genes in obesity and type 2 diabetes

DEFF Research Database (Denmark)

Sørensen, Rikke Kruse; Vienberg, Sara Gry; Vind, Birgitte F

2017-01-01

obesity with and without type 2 diabetes led to reduced expression of KLB, but increased FGFR1c expression. However, the expression of most FGF21 target genes was unaltered except for reduced CIDEA expression in individuals with type 2 diabetes. CONCLUSIONS....../INTERPRETATION: Insulin-induced expression of muscle FGF21 correlates strongly with a rise in serum FGF21, and this response appears intact in overweight/obesity and type 2 diabetes. FGF21 resistance may involve reduced KLB expression in WAT. However, increased FGFR1c expression or other mechanisms seem to ensure...... that insulin and exercise increase FGF21 in plasma. Obesity and type 2 diabetes are potentially FGF21-resistant states, but to what extent FGF21 responses to insulin and exercise training are preserved, and whether FGF21, its receptors and target genes are altered, remains to be established. METHODS...

Two potential hookworm DAF-16 target genes, SNR-3 and LPP-1: gene structure, expression profile, and implications of a cis-regulatory element in the regulation of gene expression.

Science.gov (United States)

Gao, Xin; Goggin, Kevin; Dowling, Camille; Qian, Jason; Hawdon, John M

2015-01-08

Hookworms infect nearly 700 million people, causing anemia and developmental stunting in heavy infections. Little is known about the genomic structure or gene regulation in hookworms, although recent publication of draft genome assemblies has allowed the first investigations of these topics to be undertaken. The transcription factor DAF-16 mediates multiple developmental pathways in the free living nematode Caenorhabditis elegans, and is involved in the recovery from the developmentally arrested L3 in hookworms. Identification of downstream targets of DAF-16 will provide a better understanding of the molecular mechanism of hookworm infection. Genomic Fragment 2.23 containing a DAF-16 binding element (DBE) was used to identify overlapping complementary expressed sequence tags (ESTs). These sequences were used to search a draft assembly of the Ancylostoma caninum genome, and identified two neighboring genes, snr-3 and lpp-1, in a tail-to-tail orientation. Expression patterns of both genes during parasitic development were determined by qRT-PCR. DAF-16 dependent cis-regulatory activity of fragment 2.23 was investigated using an in vitro reporter system. The snr-3 gene spans approximately 5.6 kb in the genome and contains 3 exons and 2 introns, and contains the DBE in its 3' untranslated region. Downstream from snr-3 in a tail-to-tail arrangement is the gene lpp-1. The lpp-1 gene spans more than 6 kb and contains 10 exons and 9 introns. The A. caninum genome contains 2 apparent splice variants, but there are 7 splice variants in the A. ceylanicum genome. While the gene order is similar, the gene structures of the hookworm genes differ from their C. elegans orthologs. Both genes show peak expression in the late L4 stage. Using a cell culture based expression system, fragment 2.23 was found to have both DAF-16-dependent promoter and enhancer activity that required an intact DBE. Two putative DAF-16 targets were identified by genome wide screening for DAF-16 binding

Genus beta human papillomavirus E6 proteins vary in their effects on the transactivation of p53 target genes.

Science.gov (United States)

White, Elizabeth A; Walther, Johanna; Javanbakht, Hassan; Howley, Peter M

2014-08-01

The genus beta human papillomaviruses (beta HPVs) cause cutaneous lesions and are thought to be involved in the initiation of some nonmelanoma skin cancers (NMSCs), particularly in patients with the genetic disorder epidermodysplasia verruciformis (EV). We have previously reported that at least two of the genus beta HPV E6 proteins bind to and/or increase the steady-state levels of p53 in squamous epithelial cells. This is in contrast to a well-characterized ability of the E6 proteins of cancer-associated HPVs of genus alpha HPV, which inactivate p53 by targeting its ubiquitin-mediated proteolysis. In this study, we have investigated the ability of genus beta E6 proteins from eight different HPV types to block the transactivation of p53 target genes following DNA damage. We find that the E6 proteins from diverse beta HPV species and types vary in their capacity to block the induction of MDM2, p21, and proapoptotic genes after genotoxic stress. We conclude that some genus beta HPV E6 proteins inhibit at least some p53 target genes, although perhaps not by the same mechanism or to the same degree as the high-risk genus alpha HPV E6 proteins. This study addresses the ability of various human papillomavirus E6 proteins to block the activation of p53-responsive cellular genes following DNA damage in human keratinocytes, the normal host cell for HPVs. The E6 proteins encoded by the high-risk, cancer-associated HPV types of genus alpha HPV have a well-established activity to target p53 degradation and thereby inhibit the response to DNA damage. In this study, we have investigated the ability of genus beta HPV E6 proteins from eight different HPV types to block the ability of p53 to transactivate downstream genes following DNA damage. We find that some, but not all, genus beta HPV E6 proteins can block the transactivation of some p53 target genes. This differential response to DNA damage furthers the understanding of cutaneous HPV biology and may help to explain the

Thioridazine affects transcription of genes involved in cell wall biosynthesis in methicillin-resistant Staphylococcus aureus

DEFF Research Database (Denmark)

Bonde, Mette; Højland, Dorte Heidi; Kolmos, Hans Jørn

2011-01-01

have previously shown that the expression of some resistance genes is abolished after treatment with thioridazine and oxacillin. To further understand the mechanism underlying the reversal of resistance, we tested the expression of genes involved in antibiotic resistance and cell wall biosynthesis...... in response to thioridazine in combination with oxacillin. We observed that the oxacillin-induced expression of genes belonging to the VraSR regulon is reduced by the addition of thioridazine. The exclusion of such key factors involved in cell wall biosynthesis will most likely lead to a weakened cell wall...... reversal of resistance by thioridazine relies on decreased expression of specific genes involved in cell wall biosynthesis....

Combined serial analysis of gene expression and transcription factor binding site prediction identifies novel-candidate-target genes of Nr2e1 in neocortex development.

Science.gov (United States)

Schmouth, Jean-François; Arenillas, David; Corso-Díaz, Ximena; Xie, Yuan-Yun; Bohacec, Slavita; Banks, Kathleen G; Bonaguro, Russell J; Wong, Siaw H; Jones, Steven J M; Marra, Marco A; Simpson, Elizabeth M; Wasserman, Wyeth W

2015-07-24

Nr2e1 (nuclear receptor subfamily 2, group e, member 1) encodes a transcription factor important in neocortex development. Previous work has shown that nuclear receptors can have hundreds of target genes, and bind more than 300 co-interacting proteins. However, recognition of the critical role of Nr2e1 in neural stem cells and neocortex development is relatively recent, thus the molecular mechanisms involved for this nuclear receptor are only beginning to be understood. Serial analysis of gene expression (SAGE), has given researchers both qualitative and quantitative information pertaining to biological processes. Thus, in this work, six LongSAGE mouse libraries were generated from laser microdissected tissue samples of dorsal VZ/SVZ (ventricular zone and subventricular zone) from the telencephalon of wild-type (Wt) and Nr2e1-null embryos at the critical development ages E13.5, E15.5, and E17.5. We then used a novel approach, implementing multiple computational methods followed by biological validation to further our understanding of Nr2e1 in neocortex development. In this work, we have generated a list of 1279 genes that are differentially expressed in response to altered Nr2e1 expression during in vivo neocortex development. We have refined this list to 64 candidate direct-targets of NR2E1. Our data suggested distinct roles for Nr2e1 during different neocortex developmental stages. Most importantly, our results suggest a possible novel pathway by which Nr2e1 regulates neurogenesis, which includes Lhx2 as one of the candidate direct-target genes, and SOX9 as a co-interactor. In conclusion, we have provided new candidate interacting partners and numerous well-developed testable hypotheses for understanding the pathways by which Nr2e1 functions to regulate neocortex development.

[Discovery of the target genes inhibited by formic acid in Candida shehatae].

Science.gov (United States)

Cai, Peng; Xiong, Xujie; Xu, Yong; Yong, Qiang; Zhu, Junjun; Shiyuan, Yu

2014-01-04

At transcriptional level, the inhibitory effects of formic acid was investigated on Candida shehatae, a model yeast strain capable of fermenting xylose to ethanol. Thereby, the target genes were regulated by formic acid and the transcript profiles were discovered. On the basis of the transcriptome data of C. shehatae metabolizing glucose and xylose, the genes responsible for ethanol fermentation were chosen as candidates by the combined method of yeast metabolic pathway analysis and manual gene BLAST search. These candidates were then quantitatively detected by RQ-PCR technique to find the regulating genes under gradient doses of formic acid. By quantitative analysis of 42 candidate genes, we finally identified 10 and 5 genes as markedly down-regulated and up-regulated targets by formic acid, respectively. With regard to gene transcripts regulated by formic acid in C. shehatae, the markedly down-regulated genes ranking declines as follows: xylitol dehydrogenase (XYL2), acetyl-CoA synthetase (ACS), ribose-5-phosphate isomerase (RKI), transaldolase (TAL), phosphogluconate dehydrogenase (GND1), transketolase (TKL), glucose-6-phosphate dehydrogenase (ZWF1), xylose reductase (XYL1), pyruvate dehydrogenase (PDH) and pyruvate decarboxylase (PDC); and a declining rank for up-regulated gens as follows: fructose-bisphosphate aldolase (ALD), glucokinase (GLK), malate dehydrogenase (MDH), 6-phosphofructokinase (PFK) and alcohol dehydrogenase (ADH).

Potential impact of miR-137 and its targets in schizophrenia

Directory of Open Access Journals (Sweden)

Carrie eWright

2013-04-01

Full Text Available The significant impact of microRNAs (miRNAs on disease pathology is becoming increasingly evident. These small non-coding RNAs have the ability to post-transcriptionally silence the expression of thousands of genes. Therefore, dysregulation of even a single miRNA could confer a large polygenic effect. Schizophrenia is a genetically complex illness thought to involve multiple genes each contributing a small risk. Large genome-wide association studies identified miR-137, a miRNA shown to be involved in neuronal maturation, as one of the top risk genes. To assess the potential mechanism of impact of miR-137 in this disorder and identify its targets, we used a combination of literature searches, Ingenuity Pathway Analysis (IPA, and freely accessible bioinformatics resources. Using TargetScan and the Schizophrenia Gene Resource (SZGR database, we found that in addition to CSMD1, C10orf26, CACNA1C, TCF4, and ZNF804A, five schizophrenia risk genes whose transcripts are also validated miR-137 targets, there are other schizophrenia-associated genes that may be targets of miR-137, including ERBB4, GABRA1, GRIN2A, GRM5, GSK3B, NRG2 and HTR2C. IPA analyses of all the potential targets identified several nervous system functions as the top canonical pathways including synaptic long-term potentiation, a process implicated in learning and memory mechanisms and recently shown to be altered in patients with schizophrenia. Among the subset of targets involved in nervous system development and function, the top scoring pathways were ephrin receptor signaling and axonal guidance, processes that are critical for proper circuitry formation and were shown to be disrupted in schizophrenia. These results suggest that miR-137 may indeed play a substantial role in the genetic etiology of schizophrenia by regulating networks involved in neural development and brain function.

Deep comparative genomics among Chlamydia trachomatis lymphogranuloma venereum isolates highlights genes potentially involved in pathoadaptation.

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Borges, Vítor; Gomes, João Paulo

2015-06-01

Lymphogranuloma venereum (LGV) is a human sexually transmitted disease caused by the obligate intracellular bacterium Chlamydia trachomatis (serovars L1-L3). LGV clinical manifestations range from severe ulcerative proctitis (anorectal syndrome), primarily caused by the epidemic L2b strains, to painful inguinal lymphadenopathy (the typical LGV bubonic form). Besides potential host-related factors, the differential disease severity and tissue tropism among LGV strains is likely a function of the genetic backbone of the strains. We aimed to characterize the genetic variability among LGV strains as strain- or serovar-specific mutations may underlie phenotypic signatures, and to investigate the mutational events that occurred throughout the pathoadaptation of the epidemic L2b lineage. By analyzing 20 previously published genomes from L1, L2, L2b and L3 strains and two new genomes from L2b strains, we detected 1497 variant sites and about 100 indels, affecting 453 genes and 144 intergenic regions, with 34 genes displaying a clear overrepresentation of nonsynonymous mutations. Effectors and/or type III secretion substrates (almost all of those described in the literature) and inclusion membrane proteins showed amino acid changes that were about fivefold more frequent than silent changes. More than 120 variant sites occurred in plasmid-regulated virulence genes, and 66% yielded amino acid changes. The identified serovar-specific variant sites revealed that the L2b-specific mutations are likely associated with higher fitness and pointed out potential targets for future highly discriminatory diagnostic/typing tests. By evaluating the evolutionary pathway beyond the L2b clonal radiation, we observed that 90.2% of the intra-L2b variant sites occurring in coding regions involve nonsynonymous mutations, where CT456/tarp has been the main target. Considering the progress on C. trachomatis genetic manipulation, this study may constitute an important contribution for prioritizing

Genome-Wide Identification of the Target Genes of AP2-O, a Plasmodium AP2-Family Transcription Factor.

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Izumi Kaneko

2015-05-01

Full Text Available Stage-specific transcription is a fundamental biological process in the life cycle of the Plasmodium parasite. Proteins containing the AP2 DNA-binding domain are responsible for stage-specific transcriptional regulation and belong to the only known family of transcription factors in Plasmodium parasites. Comprehensive identification of their target genes will advance our understanding of the molecular basis of stage-specific transcriptional regulation and stage-specific parasite development. AP2-O is an AP2 family transcription factor that is expressed in the mosquito midgut-invading stage, called the ookinete, and is essential for normal morphogenesis of this stage. In this study, we identified the genome-wide target genes of AP2-O by chromatin immunoprecipitation-sequencing and elucidate how this AP2 family transcription factor contributes to the formation of this motile stage. The analysis revealed that AP2-O binds specifically to the upstream genomic regions of more than 500 genes, suggesting that approximately 10% of the parasite genome is directly regulated by AP2-O. These genes are involved in distinct biological processes such as morphogenesis, locomotion, midgut penetration, protection against mosquito immunity and preparation for subsequent oocyst development. This direct and global regulation by AP2-O provides a model for gene regulation in Plasmodium parasites and may explain how these parasites manage to control their complex life cycle using a small number of sequence-specific AP2 transcription factors.

Evaluating Transcription Factor Activity Changes by Scoring Unexplained Target Genes in Expression Data.

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Evi Berchtold

Full Text Available Several methods predict activity changes of transcription factors (TFs from a given regulatory network and measured expression data. But available gene regulatory networks are incomplete and contain many condition-dependent regulations that are not relevant for the specific expression measurement. It is not known which combination of active TFs is needed to cause a change in the expression of a target gene. A method to systematically evaluate the inferred activity changes is missing. We present such an evaluation strategy that indicates for how many target genes the observed expression changes can be explained by a given set of active TFs. To overcome the problem that the exact combination of active TFs needed to activate a gene is typically not known, we assume a gene to be explained if there exists any combination for which the predicted active TFs can possibly explain the observed change of the gene. We introduce the i-score (inconsistency score, which quantifies how many genes could not be explained by the set of activity changes of TFs. We observe that, even for these minimal requirements, published methods yield many unexplained target genes, i.e. large i-scores. This holds for all methods and all expression datasets we evaluated. We provide new optimization methods to calculate the best possible (minimal i-score given the network and measured expression data. The evaluation of this optimized i-score on a large data compendium yields many unexplained target genes for almost every case. This indicates that currently available regulatory networks are still far from being complete. Both the presented Act-SAT and Act-A* methods produce optimal sets of TF activity changes, which can be used to investigate the difficult interplay of expression and network data. A web server and a command line tool to calculate our i-score and to find the active TFs associated with the minimal i-score is available from https://services.bio.ifi.lmu.de/i-score.

Horizontal transfer of a eukaryotic plastid-targeted protein gene to cyanobacteria

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Keeling Patrick J

2007-06-01

Full Text Available Abstract Background Horizontal or lateral transfer of genetic material between distantly related prokaryotes has been shown to play a major role in the evolution of bacterial and archaeal genomes, but exchange of genes between prokaryotes and eukaryotes is not as well understood. In particular, gene flow from eukaryotes to prokaryotes is rarely documented with strong support, which is unusual since prokaryotic genomes appear to readily accept foreign genes. Results Here, we show that abundant marine cyanobacteria in the related genera Synechococcus and Prochlorococcus acquired a key Calvin cycle/glycolytic enzyme from a eukaryote. Two non-homologous forms of fructose bisphosphate aldolase (FBA are characteristic of eukaryotes and prokaryotes respectively. However, a eukaryotic gene has been inserted immediately upstream of the ancestral prokaryotic gene in several strains (ecotypes of Synechococcus and Prochlorococcus. In one lineage this new gene has replaced the ancestral gene altogether. The eukaryotic gene is most closely related to the plastid-targeted FBA from red algae. This eukaryotic-type FBA once replaced the plastid/cyanobacterial type in photosynthetic eukaryotes, hinting at a possible functional advantage in Calvin cycle reactions. The strains that now possess this eukaryotic FBA are scattered across the tree of Synechococcus and Prochlorococcus, perhaps because the gene has been transferred multiple times among cyanobacteria, or more likely because it has been selectively retained only in certain lineages. Conclusion A gene for plastid-targeted FBA has been transferred from red algae to cyanobacteria, where it has inserted itself beside its non-homologous, functional analogue. Its current distribution in Prochlorococcus and Synechococcus is punctate, suggesting a complex history since its introduction to this group.

A Convenient Cas9-based Conditional Knockout Strategy for Simultaneously Targeting Multiple Genes in Mouse.

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Chen, Jiang; Du, Yinan; He, Xueyan; Huang, Xingxu; Shi, Yun S

2017-03-31

The most powerful way to probe protein function is to characterize the consequence of its deletion. Compared to conventional gene knockout (KO), conditional knockout (cKO) provides an advanced gene targeting strategy with which gene deletion can be performed in a spatially and temporally restricted manner. However, for most species that are amphiploid, the widely used Cre-flox conditional KO (cKO) system would need targeting loci in both alleles to be loxP flanked, which in practice, requires time and labor consuming breeding. This is considerably significant when one is dealing with multiple genes. CRISPR/Cas9 genome modulation system is advantaged in its capability in targeting multiple sites simultaneously. Here we propose a strategy that could achieve conditional KO of multiple genes in mouse with Cre recombinase dependent Cas9 expression. By transgenic construction of loxP-stop-loxP (LSL) controlled Cas9 (LSL-Cas9) together with sgRNAs targeting EGFP, we showed that the fluorescence molecule could be eliminated in a Cre-dependent manner. We further verified the efficacy of this novel strategy to target multiple sites by deleting c-Maf and MafB simultaneously in macrophages specifically. Compared to the traditional Cre-flox cKO strategy, this sgRNAs-LSL-Cas9 cKO system is simpler and faster, and would make conditional manipulation of multiple genes feasible.

Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.

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Tatiana Flisikowska

Full Text Available Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+ and IgG(+ B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.

Identification of key genes involved in polysaccharide bioflocculant synthesis in Bacillus licheniformis.

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Chen, Zhen; Liu, Peize; Li, Zhipeng; Yu, Wencheng; Wang, Zhi; Yao, Haosheng; Wang, Yuanpeng; Li, Qingbiao; Deng, Xu; He, Ning

2017-03-01

The present study reports the sequenced genome of Bacillus licheniformis CGMCC 2876, which is composed of a 4,284,461 bp chromosome that contains 4,188 protein-coding genes, 72 tRNA genes, and 21 rRNA genes. Additional analysis revealed an eps gene cluster with 16 open reading frames. Conserved Domains Database analysis combined with qPCR experiments indicated that all genes in this cluster were involved in polysaccharide bioflocculant synthesis. Phosphoglucomutase and UDP-glucose pyrophosphorylase were supposed to be key enzymes in polysaccharide secretion in B. licheniformis. A biosynthesis pathway for the production of polysaccharide bioflocculant involving the integration of individual genes was proposed based on functional analysis. Overexpression of epsDEF from the eps gene cluster in B. licheniformis CGMCC 2876 increased the flocculating activity of the recombinant strain by 90% compared to the original strain. Similarly, the crude yield of polysaccharide bioflocculant was enhanced by 27.8%. Overexpression of the UDP-glucose pyrophosphorylase gene not only increased the flocculating activity by 71% but also increased bioflocculant yield by 13.3%. Independent of UDP-N-acetyl-D-mannosamine dehydrogenase gene, flocculating activity, and polysaccharide yield were negatively impacted by overexpression of the UDP-N-acetylglucosamine 2-epimerase gene. Overall, epsDEF and gtaB2 were identified as key genes for polysaccharide bioflocculant synthesis in B. licheniformis. These results will be useful for further engineering of B. licheniformis for industrial bioflocculant production. Biotechnol. Bioeng. 2017;114: 645-655. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

DNA demethylases target promoter transposable elements to positively regulate stress responsive genes in Arabidopsis.

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Le, Tuan-Ngoc; Schumann, Ulrike; Smith, Neil A; Tiwari, Sameer; Au, Phil Chi Khang; Zhu, Qian-Hao; Taylor, Jennifer M; Kazan, Kemal; Llewellyn, Danny J; Zhang, Ren; Dennis, Elizabeth S; Wang, Ming-Bo

2014-09-17

DNA demethylases regulate DNA methylation levels in eukaryotes. Arabidopsis encodes four DNA demethylases, DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 (DML2), and DML3. While DME is involved in maternal specific gene expression during seed development, the biological function of the remaining DNA demethylases remains unclear. We show that ROS1, DML2, and DML3 play a role in fungal disease resistance in Arabidopsis. A triple DNA demethylase mutant, rdd (ros1 dml2 dml3), shows increased susceptibility to the fungal pathogen Fusarium oxysporum. We identify 348 genes differentially expressed in rdd relative to wild type, and a significant proportion of these genes are downregulated in rdd and have functions in stress response, suggesting that DNA demethylases maintain or positively regulate the expression of stress response genes required for F. oxysporum resistance. The rdd-downregulated stress response genes are enriched for short transposable element sequences in their promoters. Many of these transposable elements and their surrounding sequences show localized DNA methylation changes in rdd, and a general reduction in CHH methylation, suggesting that RNA-directed DNA methylation (RdDM), responsible for CHH methylation, may participate in DNA demethylase-mediated regulation of stress response genes. Many of the rdd-downregulated stress response genes are downregulated in the RdDM mutants nrpd1 and nrpe1, and the RdDM mutants nrpe1 and ago4 show enhanced susceptibility to F. oxysporum infection. Our results suggest that a primary function of DNA demethylases in plants is to regulate the expression of stress response genes by targeting promoter transposable element sequences.

In silico prediction of novel therapeutic targets using gene-disease association data.

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Ferrero, Enrico; Dunham, Ian; Sanseau, Philippe

2017-08-29

Target identification and validation is a pressing challenge in the pharmaceutical industry, with many of the programmes that fail for efficacy reasons showing poor association between the drug target and the disease. Computational prediction of successful targets could have a considerable impact on attrition rates in the drug discovery pipeline by significantly reducing the initial search space. Here, we explore whether gene-disease association data from the Open Targets platform is sufficient to predict therapeutic targets that are actively being pursued by pharmaceutical companies or are already on the market. To test our hypothesis, we train four different classifiers (a random forest, a support vector machine, a neural network and a gradient boosting machine) on partially labelled data and evaluate their performance using nested cross-validation and testing on an independent set. We then select the best performing model and use it to make predictions on more than 15,000 genes. Finally, we validate our predictions by mining the scientific literature for proposed therapeutic targets. We observe that the data types with the best predictive power are animal models showing a disease-relevant phenotype, differential expression in diseased tissue and genetic association with the disease under investigation. On a test set, the neural network classifier achieves over 71% accuracy with an AUC of 0.76 when predicting therapeutic targets in a semi-supervised learning setting. We use this model to gain insights into current and failed programmes and to predict 1431 novel targets, of which a highly significant proportion has been independently proposed in the literature. Our in silico approach shows that data linking genes and diseases is sufficient to predict novel therapeutic targets effectively and confirms that this type of evidence is essential for formulating or strengthening hypotheses in the target discovery process. Ultimately, more rapid and automated target

New inter-correlated genes targeted by diatom-derived polyunsaturated aldehydes in the sea urchin Paracentrotus lividus.

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Ruocco, Nadia; Maria Fedele, Anna; Costantini, Susan; Romano, Giovanna; Ianora, Adrianna; Costantini, Maria

2017-08-01

The marine environment is continually subjected to the action of stressors (including natural toxins), which represent a constant danger for benthic communities. In the present work using network analysis we identified ten genes on the basis of associated functions (FOXA, FoxG, GFI-1, nodal, JNK, OneCut/Hnf6, TAK1, tcf4, TCF7, VEGF) in the sea urchin Paracentrotus lividus, having key roles in different processes, such as embryonic development and asymmetry, cell fate specification, cell differentiation and morphogenesis, and skeletogenesis. These genes are correlated with three HUB genes, Foxo, Jun and HIF1A. Real Time qPCR revealed that during sea urchin embryonic development the expression levels of these genes were modulated by three diatom-derived polyunsaturated aldehydes (PUAs), decadienal, heptadienal and octadienal. Our findings show how changes in gene expression levels may be used as an early indicator of stressful conditions in the marine environment. The identification of key genes and the molecular pathways in which they are involved represents a fundamental tool in understanding how marine organisms try to afford protection against toxicants, to avoid deleterious consequences and irreversible damages. The genes identified in this work as targets for PUAs can be considered as possible biomarkers to detect exposure to different environmental pollutants. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Screening key candidate genes and pathways involved in insulinoma by microarray analysis.

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Zhou, Wuhua; Gong, Li; Li, Xuefeng; Wan, Yunyan; Wang, Xiangfei; Li, Huili; Jiang, Bin

2018-06-01

Insulinoma is a rare type tumor and its genetic features remain largely unknown. This study aimed to search for potential key genes and relevant enriched pathways of insulinoma.The gene expression data from GSE73338 were downloaded from Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified between insulinoma tissues and normal pancreas tissues, followed by pathway enrichment analysis, protein-protein interaction (PPI) network construction, and module analysis. The expressions of candidate key genes were validated by quantitative real-time polymerase chain reaction (RT-PCR) in insulinoma tissues.A total of 1632 DEGs were obtained, including 1117 upregulated genes and 514 downregulated genes. Pathway enrichment results showed that upregulated DEGs were significantly implicated in insulin secretion, and downregulated DEGs were mainly enriched in pancreatic secretion. PPI network analysis revealed 7 hub genes with degrees more than 10, including GCG (glucagon), GCGR (glucagon receptor), PLCB1 (phospholipase C, beta 1), CASR (calcium sensing receptor), F2R (coagulation factor II thrombin receptor), GRM1 (glutamate metabotropic receptor 1), and GRM5 (glutamate metabotropic receptor 5). DEGs involved in the significant modules were enriched in calcium signaling pathway, protein ubiquitination, and platelet degranulation. Quantitative RT-PCR data confirmed that the expression trends of these hub genes were similar to the results of bioinformatic analysis.The present study demonstrated that candidate DEGs and enriched pathways were the potential critical molecule events involved in the development of insulinoma, and these findings were useful for better understanding of insulinoma genesis.

TCDD dysregulation of 13 AHR-target genes in rat liver

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Watson, John D.; Prokopec, Stephenie D.; Smith, Ashley B.; Okey, Allan B.; Pohjanvirta, Raimo; Boutros, Paul C.

2014-01-01

Despite several decades of research, the complete mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other xenobiotic agonists of the aryl hydrocarbon receptor (AHR) cause toxicity remains unclear. While it has been shown that the AHR is required for all major manifestations of toxicity, the specific downstream changes involved in the development of toxic phenotypes remain unknown. Here we examine a panel of 13 genes that are AHR-regulated in many species and tissues. We profiled their hepatic mRNA abundances in two rat strains with very different sensitivities to TCDD: the TCDD-sensitive Long–Evans (Turku/AB; L–E) and the TCDD-resistant Han/Wistar (Kuopio; H/W). We evaluated doses ranging from 0 to 3000 μg/kg at 19 h after TCDD exposure and time points ranging from 1.5 to 384 h after exposure to 100 μg/kg TCDD. Twelve of 13 genes responded to TCDD in at least one strain, and seven of these showed statistically significant inter-strain differences in the time course analysis (Aldh3a1, Cyp1a2, Cyp1b1, Cyp2a1, Fmo1, Nfe2l2 and Nqo1). Cyp2s1 did not respond to TCDD in either rat strain. Five genes exhibited biphasic responses to TCDD insult (Ahrr, Aldh3a1, Cyp1b1, Nfe2l2 and Nqo1), suggesting a secondary event, such as association with additional transcriptional modulators. Of the 12 genes that responded to TCDD during the dose–response analysis, none had an ED 50 equivalent to that of Cyp1a1, the most sensitive gene in this study, while nine genes responded to doses at least 10–100 fold higher, in at least one strain (Ahrr (L–E), Aldh3a1 (both), Cyp1a2 (both), Cyp1b1 (both), Cyp2a1 (L–E), Inmt (both), Nfe2l2 (L–E), Nqo1 (L–E) and Tiparp (both)). These data shed new light on the association of the AHR target genes with TCDD toxicity, and in particular the seven genes exhibiting strain-specific differences represent strong candidate mediators of Type-II toxicities. - Highlights: • NanoString measured hepatic mRNA molecules following

TCDD dysregulation of 13 AHR-target genes in rat liver

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Watson, John D., E-mail: john.watson@oicr.on.ca [Ontario Institute for Cancer Research, Department of Informatics and Bio-computing Program, Toronto (Canada); Prokopec, Stephenie D., E-mail: stephenie.prokopec@oicr.on.ca [Ontario Institute for Cancer Research, Department of Informatics and Bio-computing Program, Toronto (Canada); Smith, Ashley B., E-mail: ashleyblaines@gmail.com [Ontario Institute for Cancer Research, Department of Informatics and Bio-computing Program, Toronto (Canada); Okey, Allan B., E-mail: allan.okey@utoronto.ca [Department of Pharmacology and Toxicology, University of Toronto, Toronto (Canada); Pohjanvirta, Raimo, E-mail: raimo.pohjanvirta@helsinki.fi [Laboratory of Toxicology, National Institute for Health and Welfare, Kuopio (Finland); Department of Food Hygiene and Environmental Health, University of Helsinki, Helsinki (Finland); Boutros, Paul C., E-mail: paul.boutros@oicr.on.ca [Ontario Institute for Cancer Research, Department of Informatics and Bio-computing Program, Toronto (Canada); Department of Pharmacology and Toxicology, University of Toronto, Toronto (Canada); Department of Medical Biophysics, University of Toronto, Toronto (Canada)

2014-02-01

Despite several decades of research, the complete mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other xenobiotic agonists of the aryl hydrocarbon receptor (AHR) cause toxicity remains unclear. While it has been shown that the AHR is required for all major manifestations of toxicity, the specific downstream changes involved in the development of toxic phenotypes remain unknown. Here we examine a panel of 13 genes that are AHR-regulated in many species and tissues. We profiled their hepatic mRNA abundances in two rat strains with very different sensitivities to TCDD: the TCDD-sensitive Long–Evans (Turku/AB; L–E) and the TCDD-resistant Han/Wistar (Kuopio; H/W). We evaluated doses ranging from 0 to 3000 μg/kg at 19 h after TCDD exposure and time points ranging from 1.5 to 384 h after exposure to 100 μg/kg TCDD. Twelve of 13 genes responded to TCDD in at least one strain, and seven of these showed statistically significant inter-strain differences in the time course analysis (Aldh3a1, Cyp1a2, Cyp1b1, Cyp2a1, Fmo1, Nfe2l2 and Nqo1). Cyp2s1 did not respond to TCDD in either rat strain. Five genes exhibited biphasic responses to TCDD insult (Ahrr, Aldh3a1, Cyp1b1, Nfe2l2 and Nqo1), suggesting a secondary event, such as association with additional transcriptional modulators. Of the 12 genes that responded to TCDD during the dose–response analysis, none had an ED{sub 50} equivalent to that of Cyp1a1, the most sensitive gene in this study, while nine genes responded to doses at least 10–100 fold higher, in at least one strain (Ahrr (L–E), Aldh3a1 (both), Cyp1a2 (both), Cyp1b1 (both), Cyp2a1 (L–E), Inmt (both), Nfe2l2 (L–E), Nqo1 (L–E) and Tiparp (both)). These data shed new light on the association of the AHR target genes with TCDD toxicity, and in particular the seven genes exhibiting strain-specific differences represent strong candidate mediators of Type-II toxicities. - Highlights: • NanoString measured hepatic mRNA molecules

Genes involved in cell division in mycoplasmas

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Frank Alarcón

2007-01-01

Full Text Available Bacterial cell division has been studied mainly in model systems such as Escherichia coli and Bacillus subtilis, where it is described as a complex process with the participation of a group of proteins which assemble into a multiprotein complex called the septal ring. Mycoplasmas are cell wall-less bacteria presenting a reduced genome. Thus, it was important to compare their genomes to analyze putative genes involved in cell division processes. The division and cell wall (dcw cluster, which in E. coli and B. subtilis is composed of 16 and 17 genes, respectively, is represented by only three to four genes in mycoplasmas. Even the most conserved protein, FtsZ, is not present in all mycoplasma genomes analyzed so far. A model for the FtsZ protein from Mycoplasma hyopneumoniae and Mycoplasma synoviae has been constructed. The conserved residues, essential for GTP/GDP binding, are present in FtsZ from both species. A strong conservation of hydrophobic amino acid patterns is observed, and is probably necessary for the structural stability of the protein when active. M. synoviae FtsZ presents an extended amino acid sequence at the C-terminal portion of the protein, which may participate in interactions with other still unknown proteins crucial for the cell division process.

Oligopeptide complex for targeted non-viral gene delivery to adipocytes

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Won, Young-Wook; Adhikary, Partho Protim; Lim, Kwang Suk; Kim, Hyung Jin; Kim, Jang Kyoung; Kim, Yong-Hee

2014-12-01

Commercial anti-obesity drugs acting in the gastrointestinal tract or the central nervous system have been shown to have limited efficacy and severe side effects. Anti-obesity drug development is thus focusing on targeting adipocytes that store excess fat. Here, we show that an adipocyte-targeting fusion-oligopeptide gene carrier consisting of an adipocyte-targeting sequence and 9-arginine (ATS-9R) selectively transfects mature adipocytes by binding to prohibitin. Injection of ATS-9R into obese mice confirmed specific binding of ATS-9R to fat vasculature, internalization and gene expression in adipocytes. We also constructed a short-hairpin RNA (shRNA) for silencing fatty-acid-binding protein 4 (shFABP4), a key lipid chaperone in fatty-acid uptake and lipid storage in adipocytes. Treatment of obese mice with ATS-9R/shFABP4 led to metabolic recovery and body-weight reduction (>20%). The ATS-9R/shFABP4 oligopeptide complex could prove to be a safe therapeutic approach to regress and treat obesity as well as obesity-induced metabolic syndromes.

A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum.

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Khang, Chang Hyun; Park, Sook-Young; Lee, Yong-Hwan; Kang, Seogchan

2005-06-01

Rapid progress in fungal genome sequencing presents many new opportunities for functional genomic analysis of fungal biology through the systematic mutagenesis of the genes identified through sequencing. However, the lack of efficient tools for targeted gene replacement is a limiting factor for fungal functional genomics, as it often necessitates the screening of a large number of transformants to identify the desired mutant. We developed an efficient method of gene replacement and evaluated factors affecting the efficiency of this method using two plant pathogenic fungi, Magnaporthe grisea and Fusarium oxysporum. This method is based on Agrobacterium tumefaciens-mediated transformation with a mutant allele of the target gene flanked by the herpes simplex virus thymidine kinase (HSVtk) gene as a conditional negative selection marker against ectopic transformants. The HSVtk gene product converts 5-fluoro-2'-deoxyuridine to a compound toxic to diverse fungi. Because ectopic transformants express HSVtk, while gene replacement mutants lack HSVtk, growing transformants on a medium amended with 5-fluoro-2'-deoxyuridine facilitates the identification of targeted mutants by counter-selecting against ectopic transformants. In addition to M. grisea and F. oxysporum, the method and associated vectors are likely to be applicable to manipulating genes in a broad spectrum of fungi, thus potentially serving as an efficient, universal functional genomic tool for harnessing the growing body of fungal genome sequence data to study fungal biology.

Genes related to antioxidant metabolism are involved in Methylobacterium mesophilicum-soybean interaction.

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Araújo, Welington Luiz; Santos, Daiene Souza; Dini-Andreote, Francisco; Salgueiro-Londoño, Jennifer Katherine; Camargo-Neves, Aline Aparecida; Andreote, Fernando Dini; Dourado, Manuella Nóbrega

2015-10-01

The genus Methylobacterium is composed of pink-pigmented methylotrophic bacterial species that are widespread in natural environments, such as soils, stream water and plants. When in association with plants, this genus colonizes the host plant epiphytically and/or endophytically. This association is known to promote plant growth, induce plant systemic resistance and inhibit plant infection by phytopathogens. In the present study, we focused on evaluating the colonization of soybean seedling-roots by Methylobacterium mesophilicum strain SR1.6/6. We focused on the identification of the key genes involved in the initial step of soybean colonization by methylotrophic bacteria, which includes the plant exudate recognition and adaptation by planktonic bacteria. Visualization by scanning electron microscopy revealed that M. mesophilicum SR1.6/6 colonizes soybean roots surface effectively at 48 h after inoculation, suggesting a mechanism for root recognition and adaptation before this period. The colonization proceeds by the development of a mature biofilm on roots at 96 h after inoculation. Transcriptomic analysis of the planktonic bacteria (with plant) revealed the expression of several genes involved in membrane transport, thus confirming an initial metabolic activation of bacterial responses when in the presence of plant root exudates. Moreover, antioxidant genes were mostly expressed during the interaction with the plant exudates. Further evaluation of stress- and methylotrophic-related genes expression by qPCR showed that glutathione peroxidase and glutathione synthetase genes were up-regulated during the Methylobacterium-soybean interaction. These findings support that glutathione (GSH) is potentially a key molecule involved in cellular detoxification during plant root colonization. In addition to methylotrophic metabolism, antioxidant genes, mainly glutathione-related genes, play a key role during soybean exudate recognition and adaptation, the first step in

Dysregulated homeostasis of target tissues or autoantigens - A novel principle in autoimmunity.

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Petersen, Frank; Yue, Xiaoyang; Riemekasten, Gabriela; Yu, Xinhua

2017-06-01

Monogenic autoimmune disorders provide a powerful tool for our understanding of the principles of autoimmunity due to the obvious impact of a single gene on the disease. So far, approximately 100 single gene defects causing murine monogenic autoimmune disorders have been reported and the functional characterization of these genes will provide significant progress in understanding the nature of autoimmunity. According to their function, genes leading to monogenic autoimmune disorders can be categorized into two groups. An expectable first group contains genes involved in the homeostasis of the immune system, including homeostasis of immune organs and immune cells. Intriguingly, the second group consists of genes functionally involved in the homeostasis of target tissues or autoantigens. According to our novel hypothesis, we propose that autoimmunity represents a consequence of a dysregulated homeostasis of the immune system and/or its targets including autoantigens and target tissues. In this review we refer to both aspects of homeostasis in autoimmunity with a highlight on the role of the homeostasis of target tissues and autoantigens. Copyright © 2017 Elsevier B.V. All rights reserved.

Integrative analysis of RUNX1 downstream pathways and target genes

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Liu Marjorie

2008-07-01

Full Text Available Abstract Background The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML. The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. Results Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1 cell lines with RUNX1 mutations from FPD-AML patients, 2 over-expression of RUNX1 and CBFβ, and 3 Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFβ. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. Conclusion This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease

Comparative Proteomic Analysis Provides insight into the Key Proteins as Possible Targets Involved in Aspirin Inhibiting Biofilm Formation of Staphylococcus xylosus

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Chang-Geng Xu

2017-08-01

Full Text Available Staphylococcus xylosus is an opportunistic pathogen that causes infection in humans and cow mastitis. And S. xylosus possesses a strong ability to form biofilms in vitro. As biofilm formation facilitates resistance to antimicrobial agents, the discovery of new medicinal properties for classic drugs is highly desired. Aspirin, which is the most common active component of non-steroidal anti-inflammatory compounds, affects the biofilm-forming capacity of various bacterial species. We have found that aspirin effectively inhibits biofilm formation of S. xylosus by Crystal violet (CV staining and scanning electron microscopy analyses. The present study sought to elucidate possible targets of aspirin in suppressing S. xylosus biofilm formation. Based on an isobaric tag for relative and absolute quantitation (iTRAQ fold-change of >1.2 or <0.8 (P-value < 0.05, 178 differentially expressed proteins, 111 down-regulated and 67 up-regulated, were identified after application of aspirin to cells at a 1/2 minimal inhibitory concentration. Gene ontology analysis indicated enrichment in metabolic processes for the majority of the differentially expressed proteins. We then used the Kyoto Encyclopedia of Genes and Genomes (KEGG pathway database to analyze a large number of differentially expressed proteins and identified genes involved in biosynthesis of amino acids pathway, carbon metabolism (pentose phosphate and glycolytic pathways, tricarboxylic acid cycle and nitrogen metabolism (histidine metabolism. These novel proteins represent candidate targets in aspirin-mediated inhibition of S. xylosus biofilm formation at sub-MIC levels. The findings lay the foundation for further studies to identify potential aspirin targets.

Immunotherapy Targets Common Cancer Mutation

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In a study of an immune therapy for colorectal cancer that involved a single patient, researchers identified a method for targeting the cancer-causing protein produced by a mutant form of the KRAS gene.

Optimizations of siRNA design for the activation of gene transcription by targeting the TATA-box motif.

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Miaomiao Fan

Full Text Available Small interfering RNAs (siRNAs are widely used to repress gene expression by targeting mRNAs. Some reports reveal that siRNAs can also activate or inhibit gene expression through targeting the gene promoters. Our group has found that microRNAs (miRNAs could activate gene transcription via interaction with the TATA-box motif in gene promoters. To investigate whether siRNA targeting the same region could upregulate the promoter activity, we test the activating efficiency of siRNAs targeting the TATA-box motif of 16 genes and perform a systematic analysis to identify the common features of the functional siRNAs for effective activation of gene promoters. Further, we try various modifications to improve the activating efficiency of siRNAs and find that it is quite useful to design the promoter-targeting activating siRNA by following several rules such as (a complementary to the TATA-box-centered region; (b UA usage at the first two bases of the antisense strand; (c twenty-three nucleotides (nts in length; (d 2'-O-Methyl (2'-OMe modification at the 3' terminus of the antisense strand; (e avoiding mismatches at the 3' end of the antisense strand. The optimized activating siRNAs potently enhance the expression of interleukin-2 (IL-2 gene in human and mouse primary CD4+ T cells with a long-time effect. Taken together, our study provides a guideline for rational design the promoter-targeting siRNA to sequence-specifically enhance gene expression.

Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system.

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Dahan-Meir, Tal; Filler-Hayut, Shdema; Melamed-Bessudo, Cathy; Bocobza, Samuel; Czosnek, Henryk; Aharoni, Asaph; Levy, Avraham A

2018-04-18

Current breeding relies mostly on random mutagenesis and recombination to generate novel genetic variation. However, targeted genome editing is becoming an increasingly important tool for precise plant breeding. Using the CRISPR-Cas system combined with the bean yellow dwarf virus rolling circle replicon we optimized a method for targeted mutagenesis and gene replacement in tomato. The carotenoid isomerase (CRTISO) and phytoene synthase 1 (PSY1) genes from the carotenoid biosynthesis pathway were chosen as targets due to their easily detectable change of phenotype. We took advantage of the geminiviral replicon amplification as a mean to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double strand break (DSB) in the target gene, via homologous recombination. Mutagenesis experiments, performed in the Micro-Tom variety achieved precise modification of the CRTISO and PSY1 loci at an efficiency of up to 90%. In the gene targeting experiments, our target was a fast-neutron-induced crtiso allele that contained a 281bp deletion. This deletion was repaired with the wildtype sequence through homologous recombination between the CRISPR-Cas-induced DSB in the crtiso target and the amplified donor in 25% of the plants transformed. This shows that efficient gene targeting can be achieved in the absence of selection markers or reporters using a single and modular construct that is adaptable to other tomato targets and other crops. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Integration of TP53, DREAM, MMB-FOXM1 and RB-E2F target gene analyses identifies cell cycle gene regulatory networks.

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Fischer, Martin; Grossmann, Patrick; Padi, Megha; DeCaprio, James A

2016-07-27

Cell cycle (CC) and TP53 regulatory networks are frequently deregulated in cancer. While numerous genome-wide studies of TP53 and CC-regulated genes have been performed, significant variation between studies has made it difficult to assess regulation of any given gene of interest. To overcome the limitation of individual studies, we developed a meta-analysis approach to identify high confidence target genes that reflect their frequency of identification in independent datasets. Gene regulatory networks were generated by comparing differential expression of TP53 and CC-regulated genes with chromatin immunoprecipitation studies for TP53, RB1, E2F, DREAM, B-MYB, FOXM1 and MuvB. RNA-seq data from p21-null cells revealed that gene downregulation by TP53 generally requires p21 (CDKN1A). Genes downregulated by TP53 were also identified as CC genes bound by the DREAM complex. The transcription factors RB, E2F1 and E2F7 bind to a subset of DREAM target genes that function in G1/S of the CC while B-MYB, FOXM1 and MuvB control G2/M gene expression. Our approach yields high confidence ranked target gene maps for TP53, DREAM, MMB-FOXM1 and RB-E2F and enables prediction and distinction of CC regulation. A web-based atlas at www.targetgenereg.org enables assessing the regulation of any human gene of interest. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Quantification of differential gene expression by multiplexed targeted resequencing of cDNA

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Arts, Peer; van der Raadt, Jori; van Gestel, Sebastianus H.C.; Steehouwer, Marloes; Shendure, Jay; Hoischen, Alexander; Albers, Cornelis A.

2017-01-01

Whole-transcriptome or RNA sequencing (RNA-Seq) is a powerful and versatile tool for functional analysis of different types of RNA molecules, but sample reagent and sequencing cost can be prohibitive for hypothesis-driven studies where the aim is to quantify differential expression of a limited number of genes. Here we present an approach for quantification of differential mRNA expression by targeted resequencing of complementary DNA using single-molecule molecular inversion probes (cDNA-smMIPs) that enable highly multiplexed resequencing of cDNA target regions of ∼100 nucleotides and counting of individual molecules. We show that accurate estimates of differential expression can be obtained from molecule counts for hundreds of smMIPs per reaction and that smMIPs are also suitable for quantification of relative gene expression and allele-specific expression. Compared with low-coverage RNA-Seq and a hybridization-based targeted RNA-Seq method, cDNA-smMIPs are a cost-effective high-throughput tool for hypothesis-driven expression analysis in large numbers of genes (10 to 500) and samples (hundreds to thousands). PMID:28474677

Huntington's disease and its therapeutic target genes: a global functional profile based on the HD Research Crossroads database.

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Kalathur, Ravi Kiran Reddy; Hernández-Prieto, Miguel A; Futschik, Matthias E

2012-06-28

Huntington's disease (HD) is a fatal progressive neurodegenerative disorder caused by the expansion of the polyglutamine repeat region in the huntingtin gene. Although the disease is triggered by the mutation of a single gene, intensive research has linked numerous other genes to its pathogenesis. To obtain a systematic overview of these genes, which may serve as therapeutic targets, CHDI Foundation has recently established the HD Research Crossroads database. With currently over 800 cataloged genes, this web-based resource constitutes the most extensive curation of genes relevant to HD. It provides us with an unprecedented opportunity to survey molecular mechanisms involved in HD in a holistic manner. To gain a synoptic view of therapeutic targets for HD, we have carried out a variety of bioinformatical and statistical analyses to scrutinize the functional association of genes curated in the HD Research Crossroads database. In particular, enrichment analyses were performed with respect to Gene Ontology categories, KEGG signaling pathways, and Pfam protein families. For selected processes, we also analyzed differential expression, using published microarray data. Additionally, we generated a candidate set of novel genetic modifiers of HD by combining information from the HD Research Crossroads database with previous genome-wide linkage studies. Our analyses led to a comprehensive identification of molecular mechanisms associated with HD. Remarkably, we not only recovered processes and pathways, which have frequently been linked to HD (such as cytotoxicity, apoptosis, and calcium signaling), but also found strong indications for other potentially disease-relevant mechanisms that have been less intensively studied in the context of HD (such as the cell cycle and RNA splicing, as well as Wnt and ErbB signaling). For follow-up studies, we provide a regularly updated compendium of molecular mechanism, that are associated with HD, at http://hdtt.sysbiolab.eu Additionally

Huntington's Disease and its therapeutic target genes: a global functional profile based on the HD Research Crossroads database

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Kalathur Ravi Kiran

2012-06-01

Full Text Available Abstract Background Huntington’s disease (HD is a fatal progressive neurodegenerative disorder caused by the expansion of the polyglutamine repeat region in the huntingtin gene. Although the disease is triggered by the mutation of a single gene, intensive research has linked numerous other genes to its pathogenesis. To obtain a systematic overview of these genes, which may serve as therapeutic targets, CHDI Foundation has recently established the HD Research Crossroads database. With currently over 800 cataloged genes, this web-based resource constitutes the most extensive curation of genes relevant to HD. It provides us with an unprecedented opportunity to survey molecular mechanisms involved in HD in a holistic manner. Methods To gain a synoptic view of therapeutic targets for HD, we have carried out a variety of bioinformatical and statistical analyses to scrutinize the functional association of genes curated in the HD Research Crossroads database. In particular, enrichment analyses were performed with respect to Gene Ontology categories, KEGG signaling pathways, and Pfam protein families. For selected processes, we also analyzed differential expression, using published microarray data. Additionally, we generated a candidate set of novel genetic modifiers of HD by combining information from the HD Research Crossroads database with previous genome-wide linkage studies. Results Our analyses led to a comprehensive identification of molecular mechanisms associated with HD. Remarkably, we not only recovered processes and pathways, which have frequently been linked to HD (such as cytotoxicity, apoptosis, and calcium signaling, but also found strong indications for other potentially disease-relevant mechanisms that have been less intensively studied in the context of HD (such as the cell cycle and RNA splicing, as well as Wnt and ErbB signaling. For follow-up studies, we provide a regularly updated compendium of molecular mechanism, that are

Identification of the Drosophila skpA gene as a novel target of the transcription factor DREF

International Nuclear Information System (INIS)

Dang Thi Phuong Thao; Ida, Hiroyuki; Yoshida, Hideki; Yamaguchi, Masamitsu

2006-01-01

SKPa is component of a Drosophila SCF complex that functions in combination with the ubiquitin-conjugating enzyme UbcD1. skpA null mutation results in centrosome overduplication, unusual chromatin condensation, defective endoreduplication and cell-cycle progression. While the molecular mechanisms that regulate expression of the skpA gene are poorly understood, the DNA replication-related element (DRE) and the DRE-binding factor (DREF) play important roles in regulating proliferation-related genes in Drosophila and DRE (5'-TATCGATA) and DRE-like (5'-CATCGATT) sequences were here found to be involved in skpA promoter activity. Thus both luciferase transient expression assays in cultured Drosophila S2 cells using skpA promoter-luciferase fusion plasmids and anti-lacZ immunostaining of various tissues from transgenic third instar larvae carrying the skpA promoter-lacZ fusion genes provided supportive evidence. Furthermore, anti-SKPa immunostaining of eye imaginal discs from flies overexpressing DREF showed ectopic expression of protein in the region posterior to the morphogenetic furrow where DREF is overexpressed. Knockdown of DREF in some tissues where SKPa distribution is well known almost completely abrogated the skpA gene expression. These findings, taken together, indicate that the Drosophila skpA gene is a novel target of the transcription factor DREF

Identification of miRNAs and their target genes in developing soybean seeds by deep sequencing

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Chen Shou-Yi

2011-01-01

Full Text Available Abstract Background MicroRNAs (miRNAs regulate gene expression by mediating gene silencing at transcriptional and post-transcriptional levels in higher plants. miRNAs and related target genes have been widely studied in model plants such as Arabidopsis and rice; however, the number of identified miRNAs in soybean (Glycine max is limited, and global identification of the related miRNA targets has not been reported in previous research. Results In our study, a small RNA library and a degradome library were constructed from developing soybean seeds for deep sequencing. We identified 26 new miRNAs in soybean by bioinformatic analysis and further confirmed their expression by stem-loop RT-PCR. The miRNA star sequences of 38 known miRNAs and 8 new miRNAs were also discovered, providing additional evidence for the existence of miRNAs. Through degradome sequencing, 145 and 25 genes were identified as targets of annotated miRNAs and new miRNAs, respectively. GO analysis indicated that many of the identified miRNA targets may function in soybean seed development. Additionally, a soybean homolog of Arabidopsis SUPPRESSOR OF GENE SLIENCING 3 (AtSGS3 was detected as a target of the newly identified miRNA Soy_25, suggesting the presence of feedback control of miRNA biogenesis. Conclusions We have identified large numbers of miRNAs and their related target genes through deep sequencing of a small RNA library and a degradome library. Our study provides more information about the regulatory network of miRNAs in soybean and advances our understanding of miRNA functions during seed development.

[Identification of Clonorchis sinensis metacercariae based on PCR targeting ribosomal DNA ITS regions and COX1 gene].

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Yang, Qing-Li; Shen, Ji-Qing; Jiang, Zhi-Hua; Yang, Yi-Chao; Li, Hong-Mei; Chen, Ying-Dan; Zhou, Xiao-Nong

2014-06-01

To identify Clonorchis sinensis metacercariae using PCR targeting ribosomal DNA ITS region and COX1 gene. Pseudorasbora parva were collected from Hengxian County of Guangxi at the end of May 2013. Single metacercaria of C. sinensis and other trematodes were separated from muscle tissue of P. parva by digestion method. Primers targeting ribosomal DNA ITS region and COX1 gene of C. sinensis were designed for PCR and the universal primers were used as control. The sensitivity and specificity of the PCR detection were analyzed. C. sinensis metacercariae at different stages were identified by PCR. DNA from single C. sinensis metacercaria was detected by PCR targeting ribosomal DNA ITS region and COX1 gene. The specific amplicans have sizes of 437/549, 156/249 and 195/166 bp, respectively. The ratio of the two positive numbers in PCR with universal primers and specific primers targeting C. sinensis ribosomal DNA ITS1 and ITS2 regions was 0.905 and 0.952, respectively. The target gene fragments were amplified by PCR using COX1 gene-specific primers. The PCR with specific primers did not show any non-specific amplification. However, the PCR with universal primers targeting ribosomal DNA ITS regions performed serious non-specific amplification. C. sinensis metacercariae at different stages are identified by morphological observation and PCR method. Species-specific primers targeting ribosomal DNA ITS region show higher sensitivity and specificity than the universal primers. PCR targeting COX1 gene shows similar sensitivity and specificity to PCR with specific primers targeting ribosomal DNA ITS regions.

ESTs analysis reveals putative genes involved in symbiotic seed germination in Dendrobium officinale.

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Zhao, Ming-Ming; Zhang, Gang; Zhang, Da-Wei; Hsiao, Yu-Yun; Guo, Shun-Xing

2013-01-01

Dendrobiumofficinale (Orchidaceae) is one of the world's most endangered plants with great medicinal value. In nature, D. officinale seeds must establish symbiotic relationships with fungi to germinate. However, the molecular events involved in the interaction between fungus and plant during this process are poorly understood. To isolate the genes involved in symbiotic germination, a suppression subtractive hybridization (SSH) cDNA library of symbiotically germinated D. officinale seeds was constructed. From this library, 1437 expressed sequence tags (ESTs) were clustered to 1074 Unigenes (including 902 singletons and 172 contigs), which were searched against the NCBI non-redundant (NR) protein database (E-value cutoff, e(-5)). Based on sequence similarity with known proteins, 579 differentially expressed genes in D. officinale were identified and classified into different functional categories by Gene Ontology (GO), Clusters of orthologous Groups of proteins (COGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expression levels of 15 selected genes emblematic of symbiotic germination were confirmed via real-time quantitative PCR. These genes were classified into various categories, including defense and stress response, metabolism, transcriptional regulation, transport process and signal transduction pathways. All transcripts were upregulated in the symbiotically germinated seeds (SGS). The functions of these genes in symbiotic germination were predicted. Furthermore, two fungus-induced calcium-dependent protein kinases (CDPKs), which were upregulated 6.76- and 26.69-fold in SGS compared with un-germinated seeds (UGS), were cloned from D. officinale and characterized for the first time. This study provides the first global overview of genes putatively involved in D. officinale symbiotic seed germination and provides a foundation for further functional research regarding symbiotic relationships in orchids.

ESTs analysis reveals putative genes involved in symbiotic seed germination in Dendrobium officinale.

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Ming-Ming Zhao

Full Text Available Dendrobiumofficinale (Orchidaceae is one of the world's most endangered plants with great medicinal value. In nature, D. officinale seeds must establish symbiotic relationships with fungi to germinate. However, the molecular events involved in the interaction between fungus and plant during this process are poorly understood. To isolate the genes involved in symbiotic germination, a suppression subtractive hybridization (SSH cDNA library of symbiotically germinated D. officinale seeds was constructed. From this library, 1437 expressed sequence tags (ESTs were clustered to 1074 Unigenes (including 902 singletons and 172 contigs, which were searched against the NCBI non-redundant (NR protein database (E-value cutoff, e(-5. Based on sequence similarity with known proteins, 579 differentially expressed genes in D. officinale were identified and classified into different functional categories by Gene Ontology (GO, Clusters of orthologous Groups of proteins (COGs and Kyoto Encyclopedia of Genes and Genomes (KEGG pathways. The expression levels of 15 selected genes emblematic of symbiotic germination were confirmed via real-time quantitative PCR. These genes were classified into various categories, including defense and stress response, metabolism, transcriptional regulation, transport process and signal transduction pathways. All transcripts were upregulated in the symbiotically germinated seeds (SGS. The functions of these genes in symbiotic germination were predicted. Furthermore, two fungus-induced calcium-dependent protein kinases (CDPKs, which were upregulated 6.76- and 26.69-fold in SGS compared with un-germinated seeds (UGS, were cloned from D. officinale and characterized for the first time. This study provides the first global overview of genes putatively involved in D. officinale symbiotic seed germination and provides a foundation for further functional research regarding symbiotic relationships in orchids.

ESTs Analysis Reveals Putative Genes Involved in Symbiotic Seed Germination in Dendrobium officinale

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Zhao, Ming-Ming; Zhang, Gang; Zhang, Da-Wei; Hsiao, Yu-Yun; Guo, Shun-Xing

2013-01-01

Dendrobium officinale (Orchidaceae) is one of the world’s most endangered plants with great medicinal value. In nature, D . officinale seeds must establish symbiotic relationships with fungi to germinate. However, the molecular events involved in the interaction between fungus and plant during this process are poorly understood. To isolate the genes involved in symbiotic germination, a suppression subtractive hybridization (SSH) cDNA library of symbiotically germinated D . officinale seeds was constructed. From this library, 1437 expressed sequence tags (ESTs) were clustered to 1074 Unigenes (including 902 singletons and 172 contigs), which were searched against the NCBI non-redundant (NR) protein database (E-value cutoff, e-5). Based on sequence similarity with known proteins, 579 differentially expressed genes in D . officinale were identified and classified into different functional categories by Gene Ontology (GO), Clusters of orthologous Groups of proteins (COGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expression levels of 15 selected genes emblematic of symbiotic germination were confirmed via real-time quantitative PCR. These genes were classified into various categories, including defense and stress response, metabolism, transcriptional regulation, transport process and signal transduction pathways. All transcripts were upregulated in the symbiotically germinated seeds (SGS). The functions of these genes in symbiotic germination were predicted. Furthermore, two fungus-induced calcium-dependent protein kinases (CDPKs), which were upregulated 6.76- and 26.69-fold in SGS compared with un-germinated seeds (UGS), were cloned from D . officinale and characterized for the first time. This study provides the first global overview of genes putatively involved in D . officinale symbiotic seed germination and provides a foundation for further functional research regarding symbiotic relationships in orchids. PMID:23967335

MicroRNA-31 controls phenotypic modulation of human vascular smooth muscle cells by regulating its target gene cellular repressor of E1A-stimulated genes

International Nuclear Information System (INIS)

Wang, Jie; Yan, Cheng-Hui; Li, Yang; Xu, Kai; Tian, Xiao-Xiang; Peng, Cheng-Fei; Tao, Jie; Sun, Ming-Yu; Han, Ya-Ling

2013-01-01

Phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of a variety of proliferative vascular diseases. The cellular repressor of E1A-stimulated genes (CREG) has been shown to play an important role in phenotypic modulation of VSMCs. However, the mechanism regulating CREG upstream signaling remains unclear. MicroRNAs (miRNAs) have recently been found to play a critical role in cell differentiation via target-gene regulation. This study aimed to identify a miRNA that binds directly to CREG, and may thus be involved in CREG-mediated VSMC phenotypic modulation. Computational analysis indicated that miR-31 bound to the CREG mRNA 3′ untranslated region (3′-UTR). miR-31 was upregulated in quiescent differentiated VSMCs and downregulated in proliferative cells stimulated by platelet-derived growth factor and serum starvation, demonstrating a negative relationship with the VSMC differentiation marker genes, smooth muscle α-actin, calponin and CREG. Using gain-of-function and loss-of-function approaches, CREG and VSMC differentiation marker gene expression levels were shown to be suppressed by a miR-31 mimic, but increased by a miR-31 inhibitor at both protein and mRNA levels. Notably, miR-31 overexpression or inhibition affected luciferase expression driven by the CREG 3′-UTR containing the miR-31 binding site. Furthermore, miR-31-mediated VSMC phenotypic modulation was inhibited in CREG-knockdown human VSMCs. We also determined miR-31 levels in the serum of patients with coronary artery disease (CAD), with or without in stent restenosis and in healthy controls. miR-31 levels were higher in the serum of CAD patients with restenosis compared to CAD patients without restenosis and in healthy controls. In summary, these data demonstrate that miR-31 not only directly binds to its target gene CREG and modulates the VSMC phenotype through this interaction, but also can be an important biomarker in diseases involving VSMC

Gene therapy prospects--intranasal delivery of therapeutic genes.

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Podolska, Karolina; Stachurska, Anna; Hajdukiewicz, Karolina; Małecki, Maciej

2012-01-01

Gene therapy is recognized to be a novel method for the treatment of various disorders. Gene therapy strategies involve gene manipulation on broad biological processes responsible for the spreading of diseases. Cancer, monogenic diseases, vascular and infectious diseases are the main targets of gene therapy. In order to obtain valuable experimental and clinical results, sufficient gene transfer methods are required. Therapeutic genes can be administered into target tissues via gene carriers commonly defined as vectors. The retroviral, adenoviral and adeno-associated virus based vectors are most frequently used in the clinic. So far, gene preparations may be administered directly into target organs or by intravenous, intramuscular, intratumor or intranasal injections. It is common knowledge that the number of gene therapy clinical trials has rapidly increased. However, some limitations such as transfection efficiency and stable and long-term gene expression are still not resolved. Consequently, great effort is focused on the evaluation of new strategies of gene delivery. There are many expectations associated with intranasal delivery of gene preparations for the treatment of diseases. Intranasal delivery of therapeutic genes is regarded as one of the most promising forms of pulmonary gene therapy research. Gene therapy based on inhalation of gene preparations offers an alternative way for the treatment of patients suffering from such lung diseases as cystic fibrosis, alpha-1-antitrypsin defect, or cancer. Experimental and first clinical trials based on plasmid vectors or recombinant viruses have revealed that gene preparations can effectively deliver therapeutic or marker genes to the cells of the respiratory tract. The noninvasive intranasal delivery of gene preparations or conventional drugs seems to be very encouraging, although basic scientific research still has to continue.

Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.

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Lu, Sha; Yin, Xiaoyan; Spollen, William; Zhang, Ning; Xu, Dong; Schoelz, James; Bilyeu, Kristin; Zhang, Zhanyuan J

2015-01-01

In the past decade, RNA silencing has gained significant attention because of its success in genomic scale research and also in the genetic improvement of crop plants. However, little is known about the molecular basis of siRNA processing in association with its target transcript. To reveal this process for improving hpRNA-mediated gene silencing in crop plants, the soybean GmFAD3 gene family was chosen as a test model. We analyzed RNAi mutant soybean lines in which three members of the GmFAD3 gene family were silenced. The silencing levels of FAD3A, FAD3B and FAD3C were correlated with the degrees of sequence homology between the inverted repeat of hpRNA and the GmFAD3 transcripts in the RNAi lines. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs corresponding to the loop portion of the hairpin transcript were detected while much lower levels of siRNAs were found outside of the target region. siRNAs generated from the 318-bp inverted repeat were found to be diced much more frequently at stem sequences close to the loop and associated with the inferred cleavage sites on the target transcripts, manifesting "hot spots". The top candidate hpRNA-derived siRNA share certain sequence features with mature miRNA. This is the first comprehensive and detailed study revealing the siRNA-mediated gene silencing mechanism in crop plants using gene family GmFAD3 as a test model.

An ABA-responsive DRE-binding protein gene from Setaria italica, SiARDP, the target gene of SiAREB, plays a critical role under drought stress.

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Li, Cong; Yue, Jing; Wu, Xiaowei; Xu, Cong; Yu, Jingjuan

2014-10-01

The DREB (dehydration-responsive element binding)-type transcription factors regulate the expression of stress-inducible genes by binding the DRE/CRT cis-elements in promoter regions. The upstream transcription factors that regulate the transcription of DREB transcription factors have not been clearly defined, although the function of DREB transcription factors in abiotic stress is known. In this study, an abscisic acid (ABA)-responsive DREB-binding protein gene (SiARDP) was cloned from foxtail millet (Setaria italica). The transcript level of SiARDP increased not only after drought, high salt, and low temperature stresses, but also after an ABA treatment in foxtail millet seedlings. Two ABA-responsive elements (ABRE1: ACGTGTC; ABRE2: ACGTGGC) exist in the promoter of SiARDP. Further analyses showed that two ABA-responsive element binding (AREB)-type transcription factors, SiAREB1 and SiAREB2, could physically bind to the ABRE core element in vitro and in vivo. The constitutive expression of SiARDP in Arabidopsis thaliana enhanced drought and salt tolerance during seed germination and seedling development, and overexpression of SiARDP in foxtail millet improved drought tolerance. The expression levels of target genes of SiARDP were upregulated in transgenic Arabidopsis and foxtail millet. These results reveal that SiARDP, one of the target genes of SiAREB, is involved in ABA-dependent signal pathways and plays a critical role in the abiotic stress response in plants. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Ancient Origin of the U2 Small Nuclear RNA Gene-Targeting Non-LTR Retrotransposons Utopia.

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Kojima, Kenji K; Jurka, Jerzy

2015-01-01

Most non-long terminal repeat (non-LTR) retrotransposons encoding a restriction-like endonuclease show target-specific integration into repetitive sequences such as ribosomal RNA genes and microsatellites. However, only a few target-specific lineages of non-LTR retrotransposons are distributed widely and no lineage is found across the eukaryotic kingdoms. Here we report the most widely distributed lineage of target sequence-specific non-LTR retrotransposons, designated Utopia. Utopia is found in three supergroups of eukaryotes: Amoebozoa, SAR, and Opisthokonta. Utopia is inserted into a specific site of U2 small nuclear RNA genes with different strength of specificity for each family. Utopia families from oomycetes and wasps show strong target specificity while only a small number of Utopia copies from reptiles are flanked with U2 snRNA genes. Oomycete Utopia families contain an "archaeal" RNase H domain upstream of reverse transcriptase (RT), which likely originated from a plant RNase H gene. Analysis of Utopia from oomycetes indicates that multiple lineages of Utopia have been maintained inside of U2 genes with few copy numbers. Phylogenetic analysis of RT suggests the monophyly of Utopia, and it likely dates back to the early evolution of eukaryotes.

Genome-wide target profiling of piggyBac and Tol2 in HEK 293: pros and cons for gene discovery and gene therapy

Science.gov (United States)

2011-01-01

Background DNA transposons have emerged as indispensible tools for manipulating vertebrate genomes with applications ranging from insertional mutagenesis and transgenesis to gene therapy. To fully explore the potential of two highly active DNA transposons, piggyBac and Tol2, as mammalian genetic tools, we have conducted a side-by-side comparison of the two transposon systems in the same setting to evaluate their advantages and disadvantages for use in gene therapy and gene discovery. Results We have observed that (1) the Tol2 transposase (but not piggyBac) is highly sensitive to molecular engineering; (2) the piggyBac donor with only the 40 bp 3'-and 67 bp 5'-terminal repeat domain is sufficient for effective transposition; and (3) a small amount of piggyBac transposases results in robust transposition suggesting the piggyBac transpospase is highly active. Performing genome-wide target profiling on data sets obtained by retrieving chromosomal targeting sequences from individual clones, we have identified several piggyBac and Tol2 hotspots and observed that (4) piggyBac and Tol2 display a clear difference in targeting preferences in the human genome. Finally, we have observed that (5) only sites with a particular sequence context can be targeted by either piggyBac or Tol2. Conclusions The non-overlapping targeting preference of piggyBac and Tol2 makes them complementary research tools for manipulating mammalian genomes. PiggyBac is the most promising transposon-based vector system for achieving site-specific targeting of therapeutic genes due to the flexibility of its transposase for being molecularly engineered. Insights from this study will provide a basis for engineering piggyBac transposases to achieve site-specific therapeutic gene targeting. PMID:21447194

Transcriptome analysis reveals key differentially expressed genes involved in wheat grain development

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Yonglong Yu

2016-04-01

Full Text Available Wheat seed development is an important physiological process of seed maturation and directly affects wheat yield and quality. In this study, we performed dynamic transcriptome microarray analysis of an elite Chinese bread wheat cultivar (Jimai 20 during grain development using the GeneChip Wheat Genome Array. Grain morphology and scanning electron microscope observations showed that the period of 11–15 days post-anthesis (DPA was a key stage for the synthesis and accumulation of seed starch. Genome-wide transcriptional profiling and significance analysis of microarrays revealed that the period from 11 to 15 DPA was more important than the 15–20 DPA stage for the synthesis and accumulation of nutritive reserves. Series test of cluster analysis of differential genes revealed five statistically significant gene expression profiles. Gene ontology annotation and enrichment analysis gave further information about differentially expressed genes, and MapMan analysis revealed expression changes within functional groups during seed development. Metabolic pathway network analysis showed that major and minor metabolic pathways regulate one another to ensure regular seed development and nutritive reserve accumulation. We performed gene co-expression network analysis to identify genes that play vital roles in seed development and identified several key genes involved in important metabolic pathways. The transcriptional expression of eight key genes involved in starch and protein synthesis and stress defense was further validated by qRT-PCR. Our results provide new insight into the molecular mechanisms of wheat seed development and the determinants of yield and quality.

Gene Therapy Targeting HIV Entry

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Chuka Didigu

2014-03-01

Full Text Available Despite the unquestionable success of antiretroviral therapy (ART in the treatment of HIV infection, the cost, need for daily adherence, and HIV-associated morbidities that persist despite ART all underscore the need to develop a cure for HIV. The cure achieved following an allogeneic hematopoietic stem cell transplant (HSCT using HIV-resistant cells, and more recently, the report of short-term but sustained, ART-free control of HIV replication following allogeneic HSCT, using HIV susceptible cells, have served to both reignite interest in HIV cure research, and suggest potential mechanisms for a cure. In this review, we highlight some of the obstacles facing HIV cure research today, and explore the roles of gene therapy targeting HIV entry, and allogeneic stem cell transplantation in the development of strategies to cure HIV infection.

Gene targeting and cloning in pigs using fetal liver derived cells.

Science.gov (United States)

Waghmare, Sanjeev K; Estrada, Jose; Reyes, Luz; Li, Ping; Ivary, Bess; Sidner, Richard A; Burlak, Chris; Tector, A Joseph

2011-12-01

Since there are no pig embryonic stem cells, pig genetic engineering is done in fetal fibroblasts that remain totipotent for only 3 to 5 wk. Nuclear donor cells that remain totipotent for longer periods of time would facilitate complicated genetic engineering in pigs. The goal of this study was to test the feasibility of using fetal liver-derived cells (FLDC) to perform gene targeting, and create a genetic knockout pig. FLDC were isolated and processed using a human liver stem cell protocol. Single copy α-1,3-galactosyl transferase knockout (GTKO) FLDCs were created using electroporation and neomycin resistant colonies were screened using PCR. Homozygous GTKO cells were created through loss of heterozygosity mutations in single GTKO FLDCs. Double GTKO FLDCs were used in somatic cell nuclear transfer (SCNT) to create GTKO pigs. FLDCs grew for more than 80 population doublings, maintaining normal karyotype. Gene targeting and loss of heterozygosity mutations produced homozygous GTKO FLDCs. FLDCs used in SCNT gave rise to homozygous GTKO pigs. FDLCs can be used in gene targeting and SCNT to produce genetically modified pigs. The increased life span in culture compared to fetal fibroblasts may facilitate genetic engineering in the pig. Copyright © 2011 Elsevier Inc. All rights reserved.

Identification of the Drosophila Mes4 gene as a novel target of the transcription factor DREF

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Suyari, Osamu; Ida, Hiroyuki [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Yoshioka, Yasuhide; Kato, Yasuko; Hashimoto, Reina [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Venture Laboratory, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Yamaguchi, Masamitsu, E-mail: myamaguc@kit.ac.jp [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan)

2009-05-01

The Mes4 gene has been identified as one of the maternal Dorsal target genes in Drosophila. In the present study, we found a DNA replication-related element (DRE, 5'-TATCGATA) in the Mes4 promoter recognized by the DRE-binding factor (DREF). Luciferase transient expression assays in S2 cells using Mes4 promoter-luciferase fusion plasmids revealed that the DRE sequence is essential for Mes4 promoter activity. Requirement of DRE for Mes4 promoter activity was further confirmed by anti-{beta}-galactosidase antibody-staining of various tissues from transgenic flies carrying Mes4 promoter-lacZ fusion genes. Furthermore, wild type Mes4 promoter activity was decreased by 40% in DREF-depleted S2 cells. These results indicate that DREF positively regulates Mes4 gene expression. Band mobility shift analyses using Kc cell nuclear extracts further indicated that the DRE sequence in the Mes4 promoter is especially important for binding to DREF. Moreover, specific binding of DREF to the involved genomic region could be demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. These results, taken together, indicate that the DRE/DREF system activates transcription of the Mes4 gene. In addition, knockdown of the Mes4 gene in wing imaginal discs using the GAL4-UAS system caused an atrophied wing phenotype, suggesting that Mes4 is required for wing morphogenesis.

Identification of the Drosophila Mes4 gene as a novel target of the transcription factor DREF

International Nuclear Information System (INIS)

Suyari, Osamu; Ida, Hiroyuki; Yoshioka, Yasuhide; Kato, Yasuko; Hashimoto, Reina; Yamaguchi, Masamitsu

2009-01-01

The Mes4 gene has been identified as one of the maternal Dorsal target genes in Drosophila. In the present study, we found a DNA replication-related element (DRE, 5'-TATCGATA) in the Mes4 promoter recognized by the DRE-binding factor (DREF). Luciferase transient expression assays in S2 cells using Mes4 promoter-luciferase fusion plasmids revealed that the DRE sequence is essential for Mes4 promoter activity. Requirement of DRE for Mes4 promoter activity was further confirmed by anti-β-galactosidase antibody-staining of various tissues from transgenic flies carrying Mes4 promoter-lacZ fusion genes. Furthermore, wild type Mes4 promoter activity was decreased by 40% in DREF-depleted S2 cells. These results indicate that DREF positively regulates Mes4 gene expression. Band mobility shift analyses using Kc cell nuclear extracts further indicated that the DRE sequence in the Mes4 promoter is especially important for binding to DREF. Moreover, specific binding of DREF to the involved genomic region could be demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. These results, taken together, indicate that the DRE/DREF system activates transcription of the Mes4 gene. In addition, knockdown of the Mes4 gene in wing imaginal discs using the GAL4-UAS system caused an atrophied wing phenotype, suggesting that Mes4 is required for wing morphogenesis.

A transcriptomic scan for potential candidate genes involved in osmoregulation in an obligate freshwater palaemonid prawn (Macrobrachium australiense

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Azam Moshtaghi

2016-10-01

Full Text Available Background Understanding the genomic basis of osmoregulation (candidate genes and/or molecular mechanisms controlling the phenotype addresses one of the fundamental questions in evolutionary ecology. Species distributions and adaptive radiations are thought to be controlled by environmental salinity levels, and efficient osmoregulatory (ionic balance ability is the main mechanism to overcome the problems related to environmental salinity gradients. Methods To better understand how osmoregulatory performance in freshwater (FW crustaceans allow individuals to acclimate and adapt to raised salinity conditions, here we (i, reviewed the literature on genes that have been identified to be associated with osmoregulation in FW crustaceans, and (ii, performed a transcriptomic analysis using cDNA libraries developed from mRNA isolated from three important osmoregulatory tissues (gill, antennal gland, hepatopancreas and total mRNA from post larvae taken from the freshwater prawn, Macrobrachium australiense using Illumina deep sequencing technology. This species was targeted because it can complete its life cycle totally in freshwater but, like many Macrobrachium sp., can also tolerate brackish water conditions and hence should have genes associated with tolerance of both FW and saline conditions. Results We obtained between 55.4 and 65.2 million Illumina read pairs from four cDNA libraries. Overall, paired end sequences assembled into a total of 125,196 non-redundant contigs (≥200 bp with an N50 length of 2,282 bp and an average contig length of 968 bp. Transcriptomic analysis of M. australiense identified 32 different gene families that were potentially involved with osmoregulatory capacity. A total of 32,597 transcripts were specified with gene ontology (GO terms identified on the basis of GO categories. Abundance estimation of expressed genes based on TPM (transcript per million ≥20 showed 1625 transcripts commonly expressed in all four libraries

Identifying genes and gene networks involved in chromium metabolism and detoxification in Crambe abyssinica

International Nuclear Information System (INIS)

Zulfiqar, Asma; Paulose, Bibin; Chhikara, Sudesh; Dhankher, Om Parkash

2011-01-01

Chromium pollution is a serious environmental problem with few cost-effective remediation strategies available. Crambe abyssinica (a member of Brassicaseae), a non-food, fast growing high biomass crop, is an ideal candidate for phytoremediation of heavy metals contaminated soils. The present study used a PCR-Select Suppression Subtraction Hybridization approach in C. abyssinica to isolate differentially expressed genes in response to Cr exposure. A total of 72 differentially expressed subtracted cDNAs were sequenced and found to represent 43 genes. The subtracted cDNAs suggest that Cr stress significantly affects pathways related to stress/defense, ion transporters, sulfur assimilation, cell signaling, protein degradation, photosynthesis and cell metabolism. The regulation of these genes in response to Cr exposure was further confirmed by semi-quantitative RT-PCR. Characterization of these differentially expressed genes may enable the engineering of non-food, high-biomass plants, including C. abyssinica, for phytoremediation of Cr-contaminated soils and sediments. - Highlights: → Molecular mechanism of Cr uptake and detoxification in plants is not well known. → We identified differentially regulated genes upon Cr exposure in Crambe abyssinica. → 72 Cr-induced subtracted cDNAs were sequenced and found to represent 43 genes. → Pathways linked to stress, ion transport, and sulfur assimilation were affected. → This is the first Cr transcriptome study in a crop with phytoremediation potential. - This study describes the identification and isolation of differentially expressed genes involved in chromium metabolism and detoxification in a non-food industrial oil crop Crambe abyssinica.

Identifying genes and gene networks involved in chromium metabolism and detoxification in Crambe abyssinica

Energy Technology Data Exchange (ETDEWEB)

Zulfiqar, Asma, E-mail: asmazulfiqar08@yahoo.com [Department of Plant, Soil, and Insect Sciences, 270 Stockbridge Road, University of Massachusetts Amherst, MA 01003 (United States); Paulose, Bibin, E-mail: bpaulose@psis.umass.edu [Department of Plant, Soil, and Insect Sciences, 270 Stockbridge Road, University of Massachusetts Amherst, MA 01003 (United States); Chhikara, Sudesh, E-mail: sudesh@psis.umass.edu [Department of Plant, Soil, and Insect Sciences, 270 Stockbridge Road, University of Massachusetts Amherst, MA 01003 (United States); Dhankher, Om Parkash, E-mail: parkash@psis.umass.edu [Department of Plant, Soil, and Insect Sciences, 270 Stockbridge Road, University of Massachusetts Amherst, MA 01003 (United States)

2011-10-15

Chromium pollution is a serious environmental problem with few cost-effective remediation strategies available. Crambe abyssinica (a member of Brassicaseae), a non-food, fast growing high biomass crop, is an ideal candidate for phytoremediation of heavy metals contaminated soils. The present study used a PCR-Select Suppression Subtraction Hybridization approach in C. abyssinica to isolate differentially expressed genes in response to Cr exposure. A total of 72 differentially expressed subtracted cDNAs were sequenced and found to represent 43 genes. The subtracted cDNAs suggest that Cr stress significantly affects pathways related to stress/defense, ion transporters, sulfur assimilation, cell signaling, protein degradation, photosynthesis and cell metabolism. The regulation of these genes in response to Cr exposure was further confirmed by semi-quantitative RT-PCR. Characterization of these differentially expressed genes may enable the engineering of non-food, high-biomass plants, including C. abyssinica, for phytoremediation of Cr-contaminated soils and sediments. - Highlights: > Molecular mechanism of Cr uptake and detoxification in plants is not well known. > We identified differentially regulated genes upon Cr exposure in Crambe abyssinica. > 72 Cr-induced subtracted cDNAs were sequenced and found to represent 43 genes. > Pathways linked to stress, ion transport, and sulfur assimilation were affected. > This is the first Cr transcriptome study in a crop with phytoremediation potential. - This study describes the identification and isolation of differentially expressed genes involved in chromium metabolism and detoxification in a non-food industrial oil crop Crambe abyssinica.

Single-step generation of rabbits carrying a targeted allele of the tyrosinase gene using CRISPR/Cas9.

Science.gov (United States)

Honda, Arata; Hirose, Michiko; Sankai, Tadashi; Yasmin, Lubna; Yuzawa, Kazuaki; Honsho, Kimiko; Izu, Haruna; Iguchi, Atsushi; Ikawa, Masahito; Ogura, Atsuo

2015-01-01

Targeted genome editing of nonrodent mammalian species has provided the potential for highly accurate interventions into gene function in humans and the generation of useful animal models of human diseases. Here we show successful clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas)-mediated gene targeting via circular plasmid injection in rabbits. The rabbit tyrosinase gene (TYR) was effectively disrupted, and we confirmed germline transmission by pronuclear injection of a circular plasmid expressing humanized Cas9 (hCas9) and single-guide RNA. Direct injection into pronuclear stage zygotes was possible following an in vitro validation assay. Neither off-target mutagenesis nor hCas9 transgenesis was detected in any of the genetically targeted pups and embryos examined. Gene targeting with this rapid and simplified strategy will help accelerate the development of translational research using other nonrodent mammalian species.

Vitamin D Pathway Status and the Identification of Target Genes in the Mouse Mammary Gland

Science.gov (United States)

2014-11-01

breast cancer stem cells with oncolytic herpes simplex virus. Cancer Gene Therapy 2012;19(10):707-14. June 21, 2012 – Poster Presentation – Presented...AD_________________ Award Number: W81XWH-11-1-0152 TITLE: Vitamin D Pathway Status and the Identification of Target Genes in the Mouse Mammary... Identification of Target Genes in the 5b. GRANT NUMBER W81XWH-11-1-0152 Mouse Mammary Gland 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT

Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

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Alok Arun

Full Text Available Real-time quantitative reverse transcription PCR (qRT-PCR is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae, two developmental stages (pupal and adult and two sexes (male and female, all of which were subjected to two food treatments (food stress and control feeding ad libitum. The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the

Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

Science.gov (United States)

Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M

2015-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression

The MSX1 homeoprotein recruits G9a methyltransferase to repressed target genes in myoblast cells.

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Jingqiang Wang

Full Text Available Although the significance of lysine modifications of core histones for regulating gene expression is widely appreciated, the mechanisms by which these modifications are incorporated at specific regulatory elements during cellular differentiation remains largely unknown. In our previous studies, we have shown that in developing myoblasts the Msx1 homeoprotein represses gene expression by influencing the modification status of chromatin at its target genes. We now show that genomic binding by Msx1 promotes enrichment of the H3K9me2 mark on repressed target genes via recruitment of G9a histone methyltransferase, the enzyme responsible for catalyzing this histone mark. Interaction of Msx1 with G9a is mediated via the homeodomain and is required for transcriptional repression and regulation of cellular differentiation, as well as enrichment of the H3K9me2 mark in proximity to Msx1 binding sites on repressed target genes in myoblast cells as well as the developing limb. We propose that regulation of chromatin status by Msx1 recruitment of G9a and other histone modifying enzymes to regulatory regions of target genes represents an important means of regulating the gene expression during development.

Identification of the human ApoAV gene as a novel RORα target gene

International Nuclear Information System (INIS)

Lind, Ulrika; Nilsson, Tina; McPheat, Jane; Stroemstedt, Per-Erik; Bamberg, Krister; Balendran, Clare; Kang, Daiwu

2005-01-01

Retinoic acid receptor-related orphan receptor-α (RORα) (NR1F1) is an orphan nuclear receptor with a potential role in metabolism. Previous studies have shown that RORα regulates transcription of the murine Apolipoprotein AI gene and human Apolipoprotein CIII genes. In the present study, we present evidence that RORα also induces transcription of the human Apolipoprotein AV gene, a recently identified apolipoprotein associated with triglyceride levels. Adenovirus-mediated overexpression of RORα increased the endogenous expression of ApoAV in HepG2 cells and RORα also enhanced the activity of an ApoAV promoter construct in transiently transfected HepG2 cells. Deletion and mutation studies identified three AGGTCA motifs in the ApoAV promoter that mediate RORα transactivation, one of which overlaps with a previously identified binding site for PPARα. Together, these results suggest a novel mechanism whereby RORα modulates lipid metabolism and implies RORα as a potential target for the treatment of dyslipidemia and atherosclerosis

HisB as novel selection marker for gene targeting approaches in Aspergillus niger.

Science.gov (United States)

Fiedler, Markus R M; Gensheimer, Tarek; Kubisch, Christin; Meyer, Vera

2017-03-08

For Aspergillus niger, a broad set of auxotrophic and dominant resistance markers is available. However, only few offer targeted modification of a gene of interest into or at a genomic locus of choice, which hampers functional genomics studies. We thus aimed to extend the available set by generating a histidine auxotrophic strain with a characterized hisB locus for targeted gene integration and deletion in A. niger. A histidine-auxotrophic strain was established via disruption of the A. niger hisB gene by using the counterselectable pyrG marker. After curing, a hisB - , pyrG - strain was obtained, which served as recipient strain for further studies. We show here that both hisB orthologs from A. nidulans and A. niger can be used to reestablish histidine prototrophy in this recipient strain. Whereas the hisB gene from A. nidulans was suitable for efficient gene targeting at different loci in A. niger, the hisB gene from A. niger allowed efficient integration of a Tet-on driven luciferase reporter construct at the endogenous non-functional hisB locus. Subsequent analysis of the luciferase activity revealed that the hisB locus is tight under non-inducing conditions and allows even higher luciferase expression levels compared to the pyrG integration locus. Taken together, we provide here an alternative selection marker for A. niger, hisB, which allows efficient homologous integration rates as well as high expression levels which compare favorably to the well-established pyrG selection marker.

Enrichment of putative PAX8 target genes at serous epithelial ovarian cancer susceptibility loci

DEFF Research Database (Denmark)

Kar, Siddhartha P; Adler, Emily; Tyrer, Jonathan

2017-01-01

BACKGROUND: Genome-wide association studies (GWAS) have identified 18 loci associated with serous ovarian cancer (SOC) susceptibility but the biological mechanisms driving these findings remain poorly characterised. Germline cancer risk loci may be enriched for target genes of transcription factors...... (TFs) critical to somatic tumorigenesis. METHODS: All 615 TF-target sets from the Molecular Signatures Database were evaluated using gene set enrichment analysis (GSEA) and three GWAS for SOC risk: discovery (2196 cases/4396 controls), replication (7035 cases/21 693 controls; independent from discovery...... to interact with PAX8 in the literature to the PAX8-target set and applying an alternative to GSEA, interval enrichment, further confirmed this association (P=0.006). Fifteen of the 157 genes from this expanded PAX8 pathway were near eight loci associated with SOC risk at P

A targeted next-generation sequencing assay for the molecular diagnosis of genetic disorders with orodental involvement

Science.gov (United States)

Prasad, Megana K; Geoffroy, Véronique; Vicaire, Serge; Jost, Bernard; Dumas, Michael; Le Gras, Stéphanie; Switala, Marzena; Gasse, Barbara; Laugel-Haushalter, Virginie; Paschaki, Marie; Leheup, Bruno; Droz, Dominique; Dalstein, Amelie; Loing, Adeline; Grollemund, Bruno; Muller-Bolla, Michèle; Lopez-Cazaux, Séréna; Minoux, Maryline; Jung, Sophie; Obry, Frédéric; Vogt, Vincent; Davideau, Jean-Luc; Davit-Beal, Tiphaine; Kaiser, Anne-Sophie; Moog, Ute; Richard, Béatrice; Morrier, Jean-Jacques; Duprez, Jean-Pierre; Odent, Sylvie; Bailleul-Forestier, Isabelle; Rousset, Monique Marie; Merametdijan, Laure; Toutain, Annick; Joseph, Clara; Giuliano, Fabienne; Dahlet, Jean-Christophe; Courval, Aymeric; El Alloussi, Mustapha; Laouina, Samir; Soskin, Sylvie; Guffon, Nathalie; Dieux, Anne; Doray, Bérénice; Feierabend, Stephanie; Ginglinger, Emmanuelle; Fournier, Benjamin; de la Dure Molla, Muriel; Alembik, Yves; Tardieu, Corinne; Clauss, François; Berdal, Ariane; Stoetzel, Corinne; Manière, Marie Cécile; Dollfus, Hélène; Bloch-Zupan, Agnès

2016-01-01

Background Orodental diseases include several clinically and genetically heterogeneous disorders that can present in isolation or as part of a genetic syndrome. Due to the vast number of genes implicated in these disorders, establishing a molecular diagnosis can be challenging. We aimed to develop a targeted next-generation sequencing (NGS) assay to diagnose mutations and potentially identify novel genes mutated in this group of disorders. Methods We designed an NGS gene panel that targets 585 known and candidate genes in orodental disease. We screened a cohort of 101 unrelated patients without a molecular diagnosis referred to the Reference Centre for Oro-Dental Manifestations of Rare Diseases, Strasbourg, France, for a variety of orodental disorders including isolated and syndromic amelogenesis imperfecta (AI), isolated and syndromic selective tooth agenesis (STHAG), isolated and syndromic dentinogenesis imperfecta, isolated dentin dysplasia, otodental dysplasia and primary failure of tooth eruption. Results We discovered 21 novel pathogenic variants and identified the causative mutation in 39 unrelated patients in known genes (overall diagnostic rate: 39%). Among the largest subcohorts of patients with isolated AI (50 unrelated patients) and isolated STHAG (21 unrelated patients), we had a definitive diagnosis in 14 (27%) and 15 cases (71%), respectively. Surprisingly, COL17A1 mutations accounted for the majority of autosomal-dominant AI cases. Conclusions We have developed a novel targeted NGS assay for the efficient molecular diagnosis of a wide variety of orodental diseases. Furthermore, our panel will contribute to better understanding the contribution of these genes to orodental disease. Trial registration numbers NCT01746121 and NCT02397824. PMID:26502894

Polymorphisms of genes involved in polycyclic aromatic hydrocarbons’ biotransformation and atherosclerosis

Science.gov (United States)

Marinković, Natalija; Pašalić, Daria; Potočki, Slavica

2013-01-01

Polycyclic aromatic hydrocarbons (PAHs) are among the most prevalent environmental pollutants and result from the incomplete combustion of hydrocarbons (coal and gasoline, fossil fuel combustion, byproducts of industrial processing, natural emission, cigarette smoking, etc.). The first phase of xenobiotic biotransformation in the PAH metabolism includes activities of cytochrome P450 from the CYP1 family and microsomal epoxide hydrolase. The products of this biotransformation are reactive oxygen species that are transformed in the second phase through the formation of conjugates with glutathione, glucuronate or sulphates. PAH exposure may lead to PAH-DNA adduct formation or induce an inflammatory atherosclerotic plaque phenotype. Several genetic polymorphisms of genes encoded for enzymes involved in PAH biotransformation have been proven to lead to the development of diseases. Enzyme CYP P450 1A1, which is encoded by the CYP1A1 gene, is vital in the monooxygenation of lipofilic substrates, while GSTM1 and GSTT1 are the most abundant isophorms that conjugate and neutralize oxygen products. Some single nucleotide polymorphisms of the CYP1A1 gene as well as the deletion polymorphisms of GSTT1 and GSTM1 may alter the final specific cellular inflammatory respond. Occupational exposure or conditions from the living environment can contribute to the production of PAH metabolites with adverse effects on human health. The aim of this study was to obtain data on biotransformation and atherosclerosis, as well as data on the gene polymorphisms involved in biotransformation, in order to better study gene expression and further elucidate the interaction between genes and the environment. PMID:24266295

Fast and sensitive detection of indels induced by precise gene targeting

DEFF Research Database (Denmark)

Yang, Zhang; Steentoft, Catharina; Hauge, Camilla

2015-01-01

The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies. A major limitation in application of precise gene editing lies in lack of sensitive and fast methods to detect...... and characterize the induced DNA changes. Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site. These indels are often small and difficult and laborious to detect...

Genome-wide analysis of murine renal distal convoluted tubular cells for the target genes of mineralocorticoid receptor

Energy Technology Data Exchange (ETDEWEB)

Ueda, Kohei [Department of Nephrology and Endocrinology, The University of Tokyo, Tokyo (Japan); Fujiki, Katsunori; Shirahige, Katsuhiko [Research Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo (Japan); Gomez-Sanchez, Celso E. [Endocrine Section, G.V. (Sonny) Montgomery VA Medical Center, MS (United States); Endocrinology, University of Mississippi Medical Center, MS (United States); Fujita, Toshiro [Division of Clinical Epigenetics, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo (Japan); Nangaku, Masaomi [Department of Nephrology and Endocrinology, The University of Tokyo, Tokyo (Japan); Nagase, Miki, E-mail: mnagase-tky@umin.ac.jp [Department of Nephrology and Endocrinology, The University of Tokyo, Tokyo (Japan); Department of Anatomy and Life Structure, School of Medicine Juntendo University, Tokyo (Japan)

2014-02-28

Highlights: • We define a target gene of MR as that with MR-binding to the adjacent region of DNA. • We use ChIP-seq analysis in combination with microarray. • We, for the first time, explore the genome-wide binding profile of MR. • We reveal 5 genes as the direct target genes of MR in the renal epithelial cell-line. - Abstract: Background and objective: Mineralocorticoid receptor (MR) is a member of nuclear receptor family proteins and contributes to fluid homeostasis in the kidney. Although aldosterone-MR pathway induces several gene expressions in the kidney, it is often unclear whether the gene expressions are accompanied by direct regulations of MR through its binding to the regulatory region of each gene. The purpose of this study is to identify the direct target genes of MR in a murine distal convoluted tubular epithelial cell-line (mDCT). Methods: We analyzed the DNA samples of mDCT cells overexpressing 3xFLAG-hMR after treatment with 10{sup −7} M aldosterone for 1 h by chromatin immunoprecipitation with deep-sequence (ChIP-seq) and mRNA of the cell-line with treatment of 10{sup −7} M aldosterone for 3 h by microarray. Results: 3xFLAG-hMR overexpressed in mDCT cells accumulated in the nucleus in response to 10{sup −9} M aldosterone. Twenty-five genes were indicated as the candidate target genes of MR by ChIP-seq and microarray analyses. Five genes, Sgk1, Fkbp5, Rasl12, Tns1 and Tsc22d3 (Gilz), were validated as the direct target genes of MR by quantitative RT-qPCR and ChIP-qPCR. MR binding regions adjacent to Ctgf and Serpine1 were also validated. Conclusions: We, for the first time, captured the genome-wide distribution of MR in mDCT cells and, furthermore, identified five MR target genes in the cell-line. These results will contribute to further studies on the mechanisms of kidney diseases.

Effect of Chemical Prevention Drugs-based MicroRNAs and Their Target Genes 
on Tumor Inhibition

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Yanhui JIANG

2015-04-01

Full Text Available Chemopreventive drugs including natural chemopreventive drugs and synthetic chemopreventive drugs, it not only can prevent cancer, can also play a role in tumor treatment. MicroRNAs (miRNAs is a kind of short chains of non-coding RNA, regulating the expression of many genes through the way of degradation of mRNA or inhibitting mRNA translation. In recent years, more and more studies have shown that chemopreventive drugs through influence the expression of miRNAs and their target genes play a role in the prevention and treatment in a variety of tumors, and chemopreventive drugs on the experimental study of miRNAs and their target genes in tumor have demonstrated a good safety and efficacy. Effect on chemopreventive drugs-based microRNAs and their target genes into cancer cells will be expected as a new starting point for cancer research. The thesis expounds and analyzes between the natural chemopreventive drugs and synthetic chemopreventive drugs and miRNAs and their target genes in tumor research progress.

Efficient and Heritable Gene Targeting in Tilapia by CRISPR/Cas9

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Li, Minghui; Yang, Huihui; Zhao, Jiue; Fang, Lingling; Shi, Hongjuan; Li, Mengru; Sun, Yunlv; Zhang, Xianbo; Jiang, Dongneng; Zhou, Linyan; Wang, Deshou

2014-01-01

Studies of gene function in non-model animals have been limited by the approaches available for eliminating gene function. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated) system has recently become a powerful tool for targeted genome editing. Here, we report the use of the CRISPR/Cas9 system to disrupt selected genes, including nanos2, nanos3, dmrt1, and foxl2, with efficiencies as high as 95%. In addition, mutations in dmrt1 and foxl2 induced by CRISPR/Cas9 were efficiently transmitted through the germline to F1. Obvious phenotypes were observed in the G0 generation after mutation of germ cell or somatic cell-specific genes. For example, loss of Nanos2 and Nanos3 in XY and XX fish resulted in germ cell-deficient gonads as demonstrated by GFP labeling and Vasa staining, respectively, while masculinization of somatic cells in both XY and XX gonads was demonstrated by Dmrt1 and Cyp11b2 immunohistochemistry and by up-regulation of serum androgen levels. Our data demonstrate that targeted, heritable gene editing can be achieved in tilapia, providing a convenient and effective approach for generating loss-of-function mutants. Furthermore, our study shows the utility of the CRISPR/Cas9 system for genetic engineering in non-model species like tilapia and potentially in many other teleost species. PMID:24709635

Reconstruction of the gene regulatory network involved in the sonic hedgehog pathway with a potential role in early development of the mouse brain.

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Jinhua Liu

2014-10-01

Full Text Available The Sonic hedgehog (Shh signaling pathway is crucial for pattern formation in early central nervous system development. By systematically analyzing high-throughput in situ hybridization data of E11.5 mouse brain, we found that Shh and its receptor Ptch1 define two adjacent mutually exclusive gene expression domains: Shh+Ptch1- and Shh-Ptch1+. These two domains are associated respectively with Foxa2 and Gata3, two transcription factors that play key roles in specifying them. Gata3 ChIP-seq experiments and RNA-seq assays on Gata3-knockdown cells revealed that Gata3 up-regulates the genes that are enriched in the Shh-Ptch1+ domain. Important Gata3 targets include Slit2 and Slit3, which are involved in the process of axon guidance, as well as Slc18a1, Th and Qdpr, which are associated with neurotransmitter synthesis and release. By contrast, Foxa2 both up-regulates the genes expressed in the Shh+Ptch1- domain and down-regulates the genes characteristic of the Shh-Ptch1+ domain. From these and other data, we were able to reconstruct a gene regulatory network governing both domains. Our work provides the first genome-wide characterization of the gene regulatory network involved in the Shh pathway that underlies pattern formation in the early mouse brain.

The feasibility of a targeted ultrasound contrast agent carrying genes and cell-penetrating peptides to hypoxic HUVEC

International Nuclear Information System (INIS)

Tian Ju; Wang Zhigang; Ren Jianli; Zhang Qingfeng; Liu Li

2012-01-01

Objective: To prepare an anti-P-selectin targeted ultrasound contrast agent carrying genes and cell-penetrating peptides (CPP) and to investigate its feasibility of delivery to hypoxic human umbilical vein endothelial cells (HUVEC). Methods: Anti-P-selectin targeted ultrasound contrast agent carrying a green fluorescent protein gene (pEGFP-N1) and CPP was prepared by mechanical vibration and carbodiimide techniques. The appearance, distribution, concentration and diameter of the ultrasound contrast agent were measured. The gene and CPP distribution on the agent was investigated using confocal laser scanning microscopy (CLSM). The efficiency of the ultrasound contrast agent to carry the gene and CPP was investigated by fluorospectrophotometry. HUVEC were cultured in vitro and hypoxic HUVEC were prepared using hydrogen peroxide (H 2 O 2 ). Hypoxic HUVEC were randomly assigned targeted ultrasound contrast agents and non-targeted ultrasound contrast agents for transfection. The transfection effect of green fluorescent protein in the two groups was observed using fluorescence microscopy and flow cytometry. T-test and linear correlation analysis were used for statistical analysis. Results: The average diameter of anti-P-selectin targeted ultrasound contrast agents carrying gene and CPP was (2.15 ±0.36) μm and the concentration was (1.58 ± 0.23) × 10 7 /ml.The results of CLSM showed that gene and CPP were distributed on the shell of the agent. The gene encapsulation efficiency was 28% (y=0.932x-0.09, r=0.993, P<0.05), and the CPP encapsulation efficiency was 25% (y=5.875x-0.81, r=0.987, P<0.05). EGFP expression was observed using fluorescence microscopy in targeted ultrasound contrast agents and non-targeted ultrasound contrast agents. The average transfection efficiencies of targeted ultrasound contrast agents and non-targeted ultrasound contrast agents were (18.74 ± 0.47) % and (15.34 ± 0.22) % after 24 h (t=10.923, P<0.001). Conclusions: The in vitro studies

Calcineurin Targets Involved in Stress Survival and Fungal Virulence.

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Hee-Soo Park

2016-09-01

Full Text Available Calcineurin governs stress survival, sexual differentiation, and virulence of the human fungal pathogen Cryptococcus neoformans. Calcineurin is activated by increased Ca2+ levels caused by stress, and transduces signals by dephosphorylating protein substrates. Herein, we identified and characterized calcineurin substrates in C. neoformans by employing phosphoproteomic TiO2 enrichment and quantitative mass spectrometry. The identified targets include the transactivator Crz1 as well as novel substrates whose functions are linked to P-bodies/stress granules (PBs/SGs and mRNA translation and decay, such as Pbp1 and Puf4. We show that Crz1 is a bona fide calcineurin substrate, and Crz1 localization and transcriptional activity are controlled by calcineurin. We previously demonstrated that thermal and other stresses trigger calcineurin localization to PBs/SGs. Several calcineurin targets localized to PBs/SGs, including Puf4 and Pbp1, contribute to stress resistance and virulence individually or in conjunction with Crz1. Moreover, Pbp1 is also required for sexual development. Genetic epistasis analysis revealed that Crz1 and the novel targets Lhp1, Puf4, and Pbp1 function in a branched calcineurin pathway that orchestrates stress survival and virulence. These findings support a model whereby calcineurin controls stress and virulence, at the transcriptional level via Crz1, and post-transcriptionally by localizing to PBs/SGs and acting on targets involved in mRNA metabolism. The calcineurin targets identified in this study share little overlap with known calcineurin substrates, with the exception of Crz1. In particular, the mRNA binding proteins and PBs/SGs residents comprise a cohort of novel calcineurin targets that have not been previously linked to calcineurin in mammals or in Saccharomyces cerevisiae. This study suggests either extensive evolutionary rewiring of the calcineurin pathway, or alternatively that these novel calcineurin targets have yet

Genome-wide identification of direct HBx genomic targets

KAUST Repository

Guerrieri, Francesca

2017-02-17

Background The Hepatitis B Virus (HBV) HBx regulatory protein is required for HBV replication and involved in HBV-related carcinogenesis. HBx interacts with chromatin modifying enzymes and transcription factors to modulate histone post-translational modifications and to regulate viral cccDNA transcription and cellular gene expression. Aiming to identify genes and non-coding RNAs (ncRNAs) directly targeted by HBx, we performed a chromatin immunoprecipitation sequencing (ChIP-Seq) to analyse HBV recruitment on host cell chromatin in cells replicating HBV. Results ChIP-Seq high throughput sequencing of HBx-bound fragments was used to obtain a high-resolution, unbiased, mapping of HBx binding sites across the genome in HBV replicating cells. Protein-coding genes and ncRNAs involved in cell metabolism, chromatin dynamics and cancer were enriched among HBx targets together with genes/ncRNAs known to modulate HBV replication. The direct transcriptional activation of genes/miRNAs that potentiate endocytosis (Ras-related in brain (RAB) GTPase family) and autophagy (autophagy related (ATG) genes, beclin-1, miR-33a) and the transcriptional repression of microRNAs (miR-138, miR-224, miR-576, miR-596) that directly target the HBV pgRNA and would inhibit HBV replication, contribute to HBx-mediated increase of HBV replication. Conclusions Our ChIP-Seq analysis of HBx genome wide chromatin recruitment defined the repertoire of genes and ncRNAs directly targeted by HBx and led to the identification of new mechanisms by which HBx positively regulates cccDNA transcription and HBV replication.

Re-Factoring Glycolytic Genes for Targeted Engineering of Catabolism in Gram-Negative Bacteria.

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Sánchez-Pascuala, Alberto; Nikel, Pablo I; de Lorenzo, Víctor

2018-01-01

The Embden-Meyerhof-Parnas (EMP) pathway is widely accepted to be the biochemical standard of glucose catabolism. The well-characterized glycolytic route of Escherichia coli, based on the EMP catabolism, is an example of an intricate pathway in terms of genomic organization of the genes involved and patterns of gene expression and regulation. This intrinsic genetic and metabolic complexity renders it difficult to engineer glycolytic activities and transfer them onto other microbial cell factories, thus limiting the biotechnological potential of bacterial hosts that lack the route. Taking into account the potential applications of such a portable tool for targeted pathway engineering, in the present protocol we describe how the genes encoding all the enzymes of the linear EMP route have been individually recruited from the genome of E. coli K-12, edited in silico to remove their endogenous regulatory signals, and synthesized de novo following a standard (i.e., GlucoBrick) that facilitates their grouping in the form of functional modules that can be combined at the user's will. This novel genetic tool allows for the à la carte implementation or boosting of EMP pathway activities into different Gram-negative bacteria. The potential of the GlucoBrick platform is further illustrated by engineering novel glycolytic activities in the most representative members of the Pseudomonas genus (Pseudomonas putida and Pseudomonas aeruginosa).

Identification of new developmentally regulated genes involved in Streptomyces coelicolor sporulation.

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Salerno, Paola; Persson, Jessica; Bucca, Giselda; Laing, Emma; Ausmees, Nora; Smith, Colin P; Flärdh, Klas

2013-12-05

The sporulation of aerial hyphae of Streptomyces coelicolor is a complex developmental process. Only a limited number of the genes involved in this intriguing morphological differentiation programme are known, including some key regulatory genes. The aim of this study was to expand our knowledge of the gene repertoire involved in S. coelicolor sporulation. We report a DNA microarray-based investigation of developmentally controlled gene expression in S. coelicolor. By comparing global transcription patterns of the wild-type parent and two mutants lacking key regulators of aerial hyphal sporulation, we found a total of 114 genes that had significantly different expression in at least one of the two mutants compared to the wild-type during sporulation. A whiA mutant showed the largest effects on gene expression, while only a few genes were specifically affected by whiH mutation. Seven new sporulation loci were investigated in more detail with respect to expression patterns and mutant phenotypes. These included SCO7449-7451 that affect spore pigment biogenesis; SCO1773-1774 that encode an L-alanine dehydrogenase and a regulator-like protein and are required for maturation of spores; SCO3857 that encodes a protein highly similar to a nosiheptide resistance regulator and affects spore maturation; and four additional loci (SCO4421, SCO4157, SCO0934, SCO1195) that show developmental regulation but no overt mutant phenotype. Furthermore, we describe a new promoter-probe vector that takes advantage of the red fluorescent protein mCherry as a reporter of cell type-specific promoter activity. Aerial hyphal sporulation in S. coelicolor is a technically challenging process for global transcriptomic investigations since it occurs only as a small fraction of the colony biomass and is not highly synchronized. Here we show that by comparing a wild-type to mutants lacking regulators that are specifically affecting processes in aerial hypha, it is possible to identify previously

An Efficient Method for Identifying Gene Fusions by Targeted RNA Sequencing from Fresh Frozen and FFPE Samples.

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Jonathan A Scolnick

Full Text Available Fusion genes are known to be key drivers of tumor growth in several types of cancer. Traditionally, detecting fusion genes has been a difficult task based on fluorescent in situ hybridization to detect chromosomal abnormalities. More recently, RNA sequencing has enabled an increased pace of fusion gene identification. However, RNA-Seq is inefficient for the identification of fusion genes due to the high number of sequencing reads needed to detect the small number of fusion transcripts present in cells of interest. Here we describe a method, Single Primer Enrichment Technology (SPET, for targeted RNA sequencing that is customizable to any target genes, is simple to use, and efficiently detects gene fusions. Using SPET to target 5701 exons of 401 known cancer fusion genes for sequencing, we were able to identify known and previously unreported gene fusions from both fresh-frozen and formalin-fixed paraffin-embedded (FFPE tissue RNA in both normal tissue and cancer cells.

Genes involved in translation of Mycoplasma hyopneumoniae and Mycoplasma synoviae

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Mônica de Oliveira Santos

2007-01-01

Full Text Available This is a report on the analysis of genes involved in translation of the complete genomes of Mycoplasma hyopneumoniae strain J and 7448 and Mycoplasma synoviae. In both genomes 31 ORFs encoding large ribosomal subunit proteins and 19 ORFs encoding small ribosomal subunit proteins were found. Ten ribosomal protein gene clusters encoding 42 ribosomal proteins were found in M. synoviae, while 8 clusters encoding 39 ribosomal proteins were found in both M. hyopneumoniae strains. The L33 gene of the M. hyopneumoniae strain 7448 presented two copies in different locations. The genes encoding initiation factors (IF-1, IF-2 and IF-3, elongation factors (EF-G, EF-Tu, EF-Ts and EF-P, and the genes encoding the ribosome recycling factor (frr and one polypeptide release factor (prfA were present in the genomes of M. hyopneumoniae and M. synoviae. Nineteen aminoacyl-tRNA synthases had been previously identified in both mycoplasmas. In the two strains of M. hyopneumoniae, J and 7448, only one set of 5S, 16S and 23S rRNAs had been identified. Two sets of 16S and 23S rRNA genes and three sets of 5S rRNA genes had been identified in the M. synoviae genome.

Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.

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Sha Lu

Full Text Available In the past decade, RNA silencing has gained significant attention because of its success in genomic scale research and also in the genetic improvement of crop plants. However, little is known about the molecular basis of siRNA processing in association with its target transcript. To reveal this process for improving hpRNA-mediated gene silencing in crop plants, the soybean GmFAD3 gene family was chosen as a test model. We analyzed RNAi mutant soybean lines in which three members of the GmFAD3 gene family were silenced. The silencing levels of FAD3A, FAD3B and FAD3C were correlated with the degrees of sequence homology between the inverted repeat of hpRNA and the GmFAD3 transcripts in the RNAi lines. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs corresponding to the loop portion of the hairpin transcript were detected while much lower levels of siRNAs were found outside of the target region. siRNAs generated from the 318-bp inverted repeat were found to be diced much more frequently at stem sequences close to the loop and associated with the inferred cleavage sites on the target transcripts, manifesting "hot spots". The top candidate hpRNA-derived siRNA share certain sequence features with mature miRNA. This is the first comprehensive and detailed study revealing the siRNA-mediated gene silencing mechanism in crop plants using gene family GmFAD3 as a test model.

Association analysis of schizophrenia on 18 genes involved in neuronal migration

DEFF Research Database (Denmark)

Kähler, Anna K; Djurovic, Srdjan; Kulle, Bettina

2008-01-01

neuronal function, morphology, and formation of synaptic connections. We have investigated the putative association between SZ and gene variants engaged in the neuronal migration process, by performing an association study on 839 cases and 1,473 controls of Scandinavian origin. Using a gene-wide approach......Several lines of evidence support the theory of schizophrenia (SZ) being a neurodevelopmental disorder. The structural, cytoarchitectural and functional brain abnormalities reported in patients with SZ, might be due to aberrant neuronal migration, since the final position of neurons affects......, tagSNPs in 18 candidate genes have been genotyped, with gene products involved in the neuron-to-glial cell adhesion, interactions with the DISC1 protein and/or rearrangements of the cytoskeleton. Of the 289 markers tested, 19 markers located in genes MDGA1, RELN, ITGA3, DLX1, SPARCL1, and ASTN1...

Twin target self-amplification-based DNA machine for highly sensitive detection of cancer-related gene.

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Xu, Huo; Jiang, Yifan; Liu, Dengyou; Liu, Kai; Zhang, Yafeng; Yu, Suhong; Shen, Zhifa; Wu, Zai-Sheng

2018-06-29

The sensitive detection of cancer-related genes is of great significance for early diagnosis and treatment of human cancers, and previous isothermal amplification sensing systems were often based on the reuse of target DNA, the amplification of enzymatic products and the accumulation of reporting probes. However, no reporting probes are able to be transformed into target species and in turn initiate the signal of other probes. Herein we reported a simple, isothermal and highly sensitive homogeneous assay system for tumor suppressor p53 gene detection based on a new autonomous DNA machine, where the signaling probe, molecular beacon (MB), was able to execute the function similar to target DNA besides providing the common signal. In the presence of target p53 gene, the operation of DNA machine can be initiated, and cyclical nucleic acid strand-displacement polymerization (CNDP) and nicking/polymerization cyclical amplification (NPCA) occur, during which the MB was opened by target species and cleaved by restriction endonuclease. In turn, the cleaved fragments could activate the next signaling process as target DNA did. According to the functional similarity, the cleaved fragment was called twin target, and the corresponding fashion to amplify the signal was named twin target self-amplification. Utilizing this newly-proposed DNA machine, the target DNA could be detected down to 0.1 pM with a wide dynamic range (6 orders of magnitude) and single-base mismatched targets were discriminated, indicating a very high assay sensitivity and good specificity. In addition, the DNA machine was not only used to screen the p53 gene in complex biological matrix but also was capable of practically detecting genomic DNA p53 extracted from A549 cell line. This indicates that the proposed DNA machine holds the potential application in biomedical research and early clinical diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

The Immature Fiber Mutant Phenotype of Cotton (Gossypium hirsutum Is Linked to a 22-bp Frame-Shift Deletion in a Mitochondria Targeted Pentatricopeptide Repeat Gene

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Gregory N. Thyssen

2016-06-01

Full Text Available Cotton seed trichomes are the most important source of natural fibers globally. The major fiber thickness properties influence the price of the raw material, and the quality of the finished product. The recessive immature fiber (im gene reduces the degree of fiber cell wall thickening by a process that was previously shown to involve mitochondrial function in allotetraploid Gossypium hirsutum. Here, we present the fine genetic mapping of the im locus, gene expression analysis of annotated proteins near the locus, and association analysis of the linked markers. Mapping-by-sequencing identified a 22-bp deletion in a pentatricopeptide repeat (PPR gene that is completely linked to the immature fiber phenotype in 2837 F2 plants, and is absent from all 163 cultivated varieties tested, although other closely linked marker polymorphisms are prevalent in the diversity panel. This frame-shift mutation results in a transcript with two long open reading frames: one containing the N-terminal transit peptide that targets mitochondria, the other containing only the RNA-binding PPR domains, suggesting that a functional PPR protein cannot be targeted to mitochondria in the im mutant. Taken together, these results suggest that PPR gene Gh_A03G0489 is involved in the cotton fiber wall thickening process, and is a promising candidate gene at the im locus. Our findings expand our understanding of the molecular mechanisms that modulate cotton fiber fineness and maturity, and may facilitate the development of cotton varieties with superior fiber attributes.

MicroRNA-429 induces tumorigenesis of human non-small cell lung cancer cells and targets multiple tumor suppressor genes

Energy Technology Data Exchange (ETDEWEB)

Lang, Yaoguo; Xu, Shidong; Ma, Jianqun; Wu, Jun [Department of Thoracic Surgery, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, Heilongjiang 150081 (China); Jin, Shi; Cao, Shoubo [Department of Medical Oncology, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, Heilongjiang 150081 (China); Yu, Yan, E-mail: yuyan@hrbmu.edu.cn [Department of Medical Oncology, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, Heilongjiang 150081 (China)

2014-07-18

Highlights: • MiR-429 expression is upregulated in non-small cell lung cancer (NSCLC). • MiR-429 inhibits PTEN, RASSF8 and TIMP2 expression. • MiR-429 promotes metastasis and proliferation. • We report important regulatory mechanisms involved in NSCLC progression. • MiR-429 is a potential therapeutic target and diagnostic marker. - Abstract: Lung cancer is the major cause of cancer death globally. MicroRNAs are evolutionally conserved small noncoding RNAs that are critical for the regulation of gene expression. Aberrant expression of microRNA (miRNA) has been implicated in cancer initiation and progression. In this study, we demonstrated that the expression of miR-429 are often upregulated in non-small cell lung cancer (NSCLC) compared with normal lung tissues, and its expression level is also increased in NSCLC cell lines compared with normal lung cells. Overexpression of miR-429 in A549 NSCLC cells significantly promoted cell proliferation, migration and invasion, whereas inhibition of miR-429 inhibits these effects. Furthermore, we demonstrated that miR-429 down-regulates PTEN, RASSF8 and TIMP2 expression by directly targeting the 3′-untranslated region of these target genes. Taken together, our results suggest that miR-429 plays an important role in promoting the proliferation and metastasis of NSCLC cells and is a potential target for NSCLC therapy.

CRISPR-Cas Targeting of Host Genes as an Antiviral Strategy.

Science.gov (United States)

Chen, Shuliang; Yu, Xiao; Guo, Deyin

2018-01-16

Currently, a new gene editing tool-the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) system-is becoming a promising approach for genetic manipulation at the genomic level. This simple method, originating from the adaptive immune defense system in prokaryotes, has been developed and applied to antiviral research in humans. Based on the characteristics of virus-host interactions and the basic rules of nucleic acid cleavage or gene activation of the CRISPR-Cas system, it can be used to target both the virus genome and host factors to clear viral reservoirs and prohibit virus infection or replication. Here, we summarize recent progress of the CRISPR-Cas technology in editing host genes as an antiviral strategy.

The hnRNP 2H9 gene, which is involved in the splicing reaction, is a multiply spliced gene

DEFF Research Database (Denmark)

Honoré, B

2000-01-01

The hnRNP 2H9 gene products are involved in the splicing process and participate in early heat shock-induced splicing arrest. By combining low/high stringency hybridisation, database search, Northern and Western blotting it is shown that the gene is alternatively spliced into at least six...

Genome-wide identification and characterization of microRNA genes and their targets in flax (Linum usitatissimum): Characterization of flax miRNA genes.

Science.gov (United States)

Barvkar, Vitthal T; Pardeshi, Varsha C; Kale, Sandip M; Qiu, Shuqing; Rollins, Meaghen; Datla, Raju; Gupta, Vidya S; Kadoo, Narendra Y

2013-04-01

MicroRNAs (miRNAs) are small (20-24 nucleotide long) endogenous regulatory RNAs that play important roles in plant growth and development. They regulate gene expression at the post-transcriptional level by translational repression or target degradation and gene silencing. In this study, we identified 116 conserved miRNAs belonging to 23 families from the flax (Linum usitatissimum L.) genome using a computational approach. The precursor miRNAs varied in length; while most of the mature miRNAs were 21 nucleotide long, intergenic and showed conserved signatures of RNA polymerase II transcripts in their upstream regions. Promoter region analysis of the flax miRNA genes indicated prevalence of MYB transcription factor binding sites. Four miRNA gene clusters containing members of three phylogenetic groups were identified. Further, 142 target genes were predicted for these miRNAs and most of these represent transcriptional regulators. The miRNA encoding genes were expressed in diverse tissues as determined by digital expression analysis as well as real-time PCR. The expression of fourteen miRNAs and nine target genes was independently validated using the quantitative reverse transcription PCR (qRT-PCR). This study suggests that a large number of conserved plant miRNAs are also found in flax and these may play important roles in growth and development of flax.

A Novel PCR Assay for Listeria welshimeri Targeting Transcriptional Regulator Gene lwe1801

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Transcriptional regulator genes encode a group of specialized molecules that play essential roles in microbial responses to changing external conditions. These genes have been shown to possess species or group specificity and are useful as detection targets for diagnostic application. The present st...

Systems Biology-Based Investigation of Cellular Antiviral Drug Targets Identified by Gene-Trap Insertional Mutagenesis.

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Feixiong Cheng

2016-09-01

Full Text Available Viruses require host cellular factors for successful replication. A comprehensive systems-level investigation of the virus-host interactome is critical for understanding the roles of host factors with the end goal of discovering new druggable antiviral targets. Gene-trap insertional mutagenesis is a high-throughput forward genetics approach to randomly disrupt (trap host genes and discover host genes that are essential for viral replication, but not for host cell survival. In this study, we used libraries of randomly mutagenized cells to discover cellular genes that are essential for the replication of 10 distinct cytotoxic mammalian viruses, 1 gram-negative bacterium, and 5 toxins. We herein reported 712 candidate cellular genes, characterizing distinct topological network and evolutionary signatures, and occupying central hubs in the human interactome. Cell cycle phase-specific network analysis showed that host cell cycle programs played critical roles during viral replication (e.g. MYC and TAF4 regulating G0/1 phase. Moreover, the viral perturbation of host cellular networks reflected disease etiology in that host genes (e.g. CTCF, RHOA, and CDKN1B identified were frequently essential and significantly associated with Mendelian and orphan diseases, or somatic mutations in cancer. Computational drug repositioning framework via incorporating drug-gene signatures from the Connectivity Map into the virus-host interactome identified 110 putative druggable antiviral targets and prioritized several existing drugs (e.g. ajmaline that may be potential for antiviral indication (e.g. anti-Ebola. In summary, this work provides a powerful methodology with a tight integration of gene-trap insertional mutagenesis testing and systems biology to identify new antiviral targets and drugs for the development of broadly acting and targeted clinical antiviral therapeutics.

Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated, homology-independent knock-in system.

Science.gov (United States)

Katoh, Yohei; Michisaka, Saki; Nozaki, Shohei; Funabashi, Teruki; Hirano, Tomoaki; Takei, Ryota; Nakayama, Kazuhisa

2017-04-01

The CRISPR/Cas9 system has revolutionized genome editing in virtually all organisms. Although the CRISPR/Cas9 system enables the targeted cleavage of genomic DNA, its use for gene knock-in remains challenging because levels of homologous recombination activity vary among various cells. In contrast, the efficiency of homology-independent DNA repair is relatively high in most cell types. Therefore the use of a homology-independent repair mechanism is a possible alternative for efficient genome editing. Here we constructed a donor knock-in vector optimized for the CRISPR/Cas9 system and developed a practical system that enables efficient disruption of target genes by exploiting homology-independent repair. Using this practical knock-in system, we successfully disrupted genes encoding proteins involved in ciliary protein trafficking, including IFT88 and IFT20, in hTERT-RPE1 cells, which have low homologous recombination activity. The most critical concern using the CRISPR/Cas9 system is off-target cleavage. To reduce the off-target cleavage frequency and increase the versatility of our knock-in system, we constructed a universal donor vector and an expression vector containing Cas9 with enhanced specificity and tandem sgRNA expression cassettes. We demonstrated that the second version of our system has improved usability. © 2017 Katoh et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Spontaneous autoimmunity in 129 and C57BL/6 mice-implications for autoimmunity described in gene-targeted mice.

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Anne E Bygrave

2004-08-01

Full Text Available Systemic lupus erythematosus (SLE is a multisystem autoimmune disorder in which complex genetic factors play an important role. Several strains of gene-targeted mice have been reported to develop SLE, implicating the null genes in the causation of disease. However, hybrid strains between 129 and C57BL/6 mice, widely used in the generation of gene-targeted mice, develop spontaneous autoimmunity. Furthermore, the genetic background markedly influences the autoimmune phenotype of SLE in gene-targeted mice. This suggests an important role in the expression of autoimmunity of as-yet-uncharacterised background genes originating from these parental mouse strains. Using genome-wide linkage analysis, we identified several susceptibility loci, derived from 129 and C57BL/6 mice, mapped in the lupus-prone hybrid (129 x C57BL/6 model. By creating a C57BL/6 congenic strain carrying a 129-derived Chromosome 1 segment, we found that this 129 interval was sufficient to mediate the loss of tolerance to nuclear antigens, which had previously been attributed to a disrupted gene. These results demonstrate important epistatic modifiers of autoimmunity in 129 and C57BL/6 mouse strains, widely used in gene targeting. These background gene influences may account for some, or even all, of the autoimmune traits described in some gene-targeted models of SLE.

Global Developmental Gene Programing Involves a Nuclear Form of Fibroblast Growth Factor Receptor-1 (FGFR1.

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Christopher Terranova

Full Text Available Genetic studies have placed the Fgfr1 gene at the top of major ontogenic pathways that enable gastrulation, tissue development and organogenesis. Using genome-wide sequencing and loss and gain of function experiments the present investigation reveals a mechanism that underlies global and direct gene regulation by the nuclear form of FGFR1, ensuring that pluripotent Embryonic Stem Cells differentiate into Neuronal Cells in response to Retinoic Acid. Nuclear FGFR1, both alone and with its partner nuclear receptors RXR and Nur77, targets thousands of active genes and controls the expression of pluripotency, homeobox, neuronal and mesodermal genes. Nuclear FGFR1 targets genes in developmental pathways represented by Wnt/β-catenin, CREB, BMP, the cell cycle and cancer-related TP53 pathway, neuroectodermal and mesodermal programing networks, axonal growth and synaptic plasticity pathways. Nuclear FGFR1 targets the consensus sequences of transcription factors known to engage CREB-binding protein, a common coregulator of transcription and established binding partner of nuclear FGFR1. This investigation reveals the role of nuclear FGFR1 as a global genomic programmer of cell, neural and muscle development.

BDNF gene delivery mediated by neuron-targeted nanoparticles is neuroprotective in peripheral nerve injury.

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Lopes, Cátia D F; Gonçalves, Nádia P; Gomes, Carla P; Saraiva, Maria J; Pêgo, Ana P

2017-03-01

Neuron-targeted gene delivery is a promising strategy to treat peripheral neuropathies. Here we propose the use of polymeric nanoparticles based on thiolated trimethyl chitosan (TMCSH) to mediate targeted gene delivery to peripheral neurons upon a peripheral and minimally invasive intramuscular administration. Nanoparticles were grafted with the non-toxic carboxylic fragment of the tetanus neurotoxin (HC) to allow neuron targeting and were explored to deliver a plasmid DNA encoding for the brain-derived neurotrophic factor (BDNF) in a peripheral nerve injury model. The TMCSH-HC/BDNF nanoparticle treatment promoted the release and significant expression of BDNF in neural tissues, which resulted in an enhanced functional recovery after injury as compared to control treatments (vehicle and non-targeted nanoparticles), associated with an improvement in key pro-regenerative events, namely, the increased expression of neurofilament and growth-associated protein GAP-43 in the injured nerves. Moreover, the targeted nanoparticle treatment was correlated with a significantly higher density of myelinated axons in the distal stump of injured nerves, as well as with preservation of unmyelinated axon density as compared with controls and a protective role in injury-denervated muscles, preventing them from denervation. These results highlight the potential of TMCSH-HC nanoparticles as non-viral gene carriers to deliver therapeutic genes into the peripheral neurons and thus, pave the way for their use as an effective therapeutic intervention for peripheral neuropathies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cell cycle and anti-estrogen effects synergize to regulate cell proliferation and ER target gene expression.

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Mathieu Dalvai

Full Text Available Antiestrogens are designed to antagonize hormone induced proliferation and ERalpha target gene expression in mammary tumor cells. Commonly used drugs such as OH-Tamoxifen and ICI 182780 (Fulvestrant block cell cycle progression in G0/G1. Inversely, the effect of cell cycle stage on ER regulated gene expression has not been tested directly. We show that in ERalpha-positive breast cancer cells (MCF-7 the estrogen receptor gene and downstream target genes are cell cycle regulated with expression levels varying as much as three-fold between phases of the cell cycle. Steroid free culture conditions commonly used to assess the effect of hormones or antiestrogens on gene expression also block MCF-7 cells in G1-phase when several ERalpha target genes are overexpressed. Thus, cell cycle effects have to be taken into account when analyzing the impact of hormonal treatments on gene transcription. We found that antiestrogens repress transcription of several ERalpha target genes specifically in S phase. This observation corroborates the more rapid and strong impact of antiestrogen treatments on cell proliferation in thymidine, hydroxyurea or aphidicolin arrested cells and correlates with an increase of apoptosis compared to similar treatments in lovastatin or nocodazol treated cells. Hence, cell cycle effects synergize with the action of antiestrogens. An interesting therapeutic perspective could be to enhance the action of anti-estrogens by associating hormone-therapy with specific cell cycle drugs.

Genes Involved in Initial Follicle Recruitment May Be Associated with Age at Menopause

NARCIS (Netherlands)

Voorhuis, Marlies; Broekmans, Frank J.; Fauser, Bart C. J. M.; Onland-Moret, N. Charlotte; van der Schouw, Yvonne T.

Context: Timing of menopause is largely influenced by genetic factors. Because menopause occurs when the follicle pool in the ovaries has become exhausted, genes involved in primordial follicle recruitment can be considered as candidate genes for timing of menopause. Objective: The aim was to study

The distribution of BRAF gene fusions in solid tumors and response to targeted therapy.

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Ross, Jeffrey S; Wang, Kai; Chmielecki, Juliann; Gay, Laurie; Johnson, Adrienne; Chudnovsky, Jacob; Yelensky, Roman; Lipson, Doron; Ali, Siraj M; Elvin, Julia A; Vergilio, Jo-Anne; Roels, Steven; Miller, Vincent A; Nakamura, Brooke N; Gray, Adam; Wong, Michael K; Stephens, Philip J

2016-02-15

Although the BRAF V600E base substitution is an approved target for the BRAF inhibitors in melanoma, BRAF gene fusions have not been investigated as anticancer drug targets. In our study, a wide variety of tumors underwent comprehensive genomic profiling for hundreds of known cancer genes using the FoundationOne™ or FoundationOne Heme™ comprehensive genomic profiling assays. BRAF fusions involving the intact in-frame BRAF kinase domain were observed in 55 (0.3%) of 20,573 tumors, across 12 distinct tumor types, including 20 novel BRAF fusions. These comprised 29 unique 5' fusion partners, of which 31% (9) were known and 69% (20) were novel. BRAF fusions included 3% (14/531) of melanomas; 2% (15/701) of gliomas; 1.0% (3/294) of thyroid cancers; 0.3% (3/1,062) pancreatic carcinomas; 0.2% (8/4,013) nonsmall-cell lung cancers and 0.2% (4/2,154) of colorectal cancers, and were enriched in pilocytic (30%) vs. nonpilocytic gliomas (1%; p < 0.0001), Spitzoid (75%) vs. nonSpitzoid melanomas (1%; p = 0.0001), acinar (67%) vs. nonacinar pancreatic cancers (<1%; p < 0.0001) and papillary (3%) vs. nonpapillary thyroid cancers (0%; p < 0.03). Clinical responses to trametinib and sorafenib are presented. In conclusion, BRAF fusions are rare driver alterations in a wide variety of malignant neoplasms, but enriched in Spitzoid melanoma, pilocytic astrocytomas, pancreatic acinar and papillary thyroid cancers. © 2015 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.

Gene Targeting and Expression Modulation by Peptide Nucleic Acids (PNA)

DEFF Research Database (Denmark)

Nielsen, Peter E

2010-01-01

Peptide nucleic acids (PNA) are artificial structural mimics of nucleic acids capable of sequence specific hybridization to both RNA and DNA. Thus they have obvious potential as gene targeting agents for drug discovery approaches. An overview with emphasis on recent progress on RNA "interference...

Identification and expression analyses of WRKY genes reveal their involvement in growth and abiotic stress response in watermelon (Citrullus lanatus).

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Yang, Xiaozhen; Li, Hao; Yang, Yongchao; Wang, Yongqi; Mo, Yanling; Zhang, Ruimin; Zhang, Yong; Ma, Jianxiang; Wei, Chunhua; Zhang, Xian

2018-01-01

Despite identification of WRKY family genes in numerous plant species, a little is known about WRKY genes in watermelon, one of the most economically important fruit crops around the world. Here, we identified a total of 63 putative WRKY genes in watermelon and classified them into three major groups (I-III) and five subgroups (IIa-IIe) in group II. The structure analysis indicated that ClWRKYs with different WRKY domains or motifs may play different roles by regulating respective target genes. The expressions of ClWRKYs in different tissues indicate that they are involved in various tissue growth and development. Furthermore, the diverse responses of ClWRKYs to drought, salt, or cold stress suggest that they positively or negatively affect plant tolerance to various abiotic stresses. In addition, the altered expression patterns of ClWRKYs in response to phytohormones such as, ABA, SA, MeJA, and ETH, imply the occurrence of complex cross-talks between ClWRKYs and plant hormone signals in regulating plant physiological and biological processes. Taken together, our findings provide valuable clues to further explore the function and regulatory mechanisms of ClWRKY genes in watermelon growth, development, and adaption to environmental stresses.

Identification and expression analyses of WRKY genes reveal their involvement in growth and abiotic stress response in watermelon (Citrullus lanatus.

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Xiaozhen Yang

Full Text Available Despite identification of WRKY family genes in numerous plant species, a little is known about WRKY genes in watermelon, one of the most economically important fruit crops around the world. Here, we identified a total of 63 putative WRKY genes in watermelon and classified them into three major groups (I-III and five subgroups (IIa-IIe in group II. The structure analysis indicated that ClWRKYs with different WRKY domains or motifs may play different roles by regulating respective target genes. The expressions of ClWRKYs in different tissues indicate that they are involved in various tissue growth and development. Furthermore, the diverse responses of ClWRKYs to drought, salt, or cold stress suggest that they positively or negatively affect plant tolerance to various abiotic stresses. In addition, the altered expression patterns of ClWRKYs in response to phytohormones such as, ABA, SA, MeJA, and ETH, imply the occurrence of complex cross-talks between ClWRKYs and plant hormone signals in regulating plant physiological and biological processes. Taken together, our findings provide valuable clues to further explore the function and regulatory mechanisms of ClWRKY genes in watermelon growth, development, and adaption to environmental stresses.

sugE: A gene involved in tributyltin (TBT) resistance of Aeromonas molluscorum Av27.

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Cruz, Andreia; Micaelo, Nuno; Félix, Vitor; Song, Jun-Young; Kitamura, Shin-Ichi; Suzuki, Satoru; Mendo, Sónia

2013-01-01

The mechanism of bacterial resistance to tributyltin (TBT) is still unclear. The results herein presented contribute to clarify that mechanism in the TBT-resistant bacterium Aeromonas molluscorum Av27. We have identified and cloned a new gene that is involved in TBT resistance in this strain. The gene is highly homologous (84%) to the Aeromonas hydrophila-sugE gene belonging to the small multidrug resistance gene family (SMR), which includes genes involved in the transport of lipophilic drugs. In Av27, expression of the Av27-sugE was observed at the early logarithmic growth phase in the presence of a high TBT concentration (500 μM), thus suggesting the contribution of this gene for TBT resistance. E. coli cells transformed with Av27-sugE become resistant to ethidium bromide (EtBr), chloramphenicol (CP) and tetracycline (TE), besides TBT. According to the Moriguchi logP (miLogP) values, EtBr, CP and TE have similar properties and are substrates for the sugE-efflux system. Despite the different miLogP of TBT, E. coli cells transformed with Av27-sugE become resistant to this compound. So it seems that TBT is also a substrate for the SugE protein. The modelling studies performed also support this hypothesis. The data herein presented clearly indicate that sugE is involved in TBT resistance of this bacterium.

Genes involved in long-chain alkene biosynthesis in Micrococcus luteus

Energy Technology Data Exchange (ETDEWEB)

Beller, Harry R.; Goh, Ee-Been; Keasling, Jay D.

2010-01-07

Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which four decades ago was reported to biosynthesize iso- and anteiso branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty-acid overproducing E. coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-CoA produced the same C27 monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or -ACP) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (?-ketoacyl-ACP synthase III), which catalyzes decarboxylative Claisen condensation during

Targeted, homology-driven gene insertion in stem cells by ZFN-loaded 'all-in-one' lentiviral vectors

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Cai, Yujia; Laustsen, Anders; Zhou, Yan

2016-01-01

-driven mechanism into safe loci. This insertion mechanism is driven by time-restricted exposure of treated cells to ZFNs. We show targeted gene integration in human stem cells, including CD34+ hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted insertions are identified in 89......% of transduced iPSCs. Our findings demonstrate the applicability of nuclease-loaded 'all-in-one' IDLVs for site-directed gene insertion in stem cell based gene therapies....

Genome-wide identification of Bcl11b gene targets reveals role in brain-derived neurotrophic factor signaling.

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Bin Tang

Full Text Available B-cell leukemia/lymphoma 11B (Bcl11b is a transcription factor showing predominant expression in the striatum. To date, there are no known gene targets of Bcl11b in the nervous system. Here, we define targets for Bcl11b in striatal cells by performing chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq in combination with genome-wide expression profiling. Transcriptome-wide analysis revealed that 694 genes were significantly altered in striatal cells over-expressing Bcl11b, including genes showing striatal-enriched expression similar to Bcl11b. ChIP-seq analysis demonstrated that Bcl11b bound a mixture of coding and non-coding sequences that were within 10 kb of the transcription start site of an annotated gene. Integrating all ChIP-seq hits with the microarray expression data, 248 direct targets of Bcl11b were identified. Functional analysis on the integrated gene target list identified several zinc-finger encoding genes as Bcl11b targets, and further revealed a significant association of Bcl11b to brain-derived neurotrophic factor/neurotrophin signaling. Analysis of ChIP-seq binding regions revealed significant consensus DNA binding motifs for Bcl11b. These data implicate Bcl11b as a novel regulator of the BDNF signaling pathway, which is disrupted in many neurological disorders. Specific targeting of the Bcl11b-DNA interaction could represent a novel therapeutic approach to lowering BDNF signaling specifically in striatal cells.

Gene-environment interaction involving recently identified colorectal cancer susceptibility loci

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Kantor, Elizabeth D.; Hutter, Carolyn M.; Minnier, Jessica; Berndt, Sonja I.; Brenner, Hermann; Caan, Bette J.; Campbell, Peter T.; Carlson, Christopher S.; Casey, Graham; Chan, Andrew T.; Chang-Claude, Jenny; Chanock, Stephen J.; Cotterchio, Michelle; Du, Mengmeng; Duggan, David; Fuchs, Charles S.; Giovannucci, Edward L.; Gong, Jian; Harrison, Tabitha A.; Hayes, Richard B.; Henderson, Brian E.; Hoffmeister, Michael; Hopper, John L.; Jenkins, Mark A.; Jiao, Shuo; Kolonel, Laurence N.; Le Marchand, Loic; Lemire, Mathieu; Ma, Jing; Newcomb, Polly A.; Ochs-Balcom, Heather M.; Pflugeisen, Bethann M.; Potter, John D.; Rudolph, Anja; Schoen, Robert E.; Seminara, Daniela; Slattery, Martha L.; Stelling, Deanna L.; Thomas, Fridtjof; Thornquist, Mark; Ulrich, Cornelia M.; Warnick, Greg S.; Zanke, Brent W.; Peters, Ulrike; Hsu, Li; White, Emily

2014-01-01

BACKGROUND Genome-wide association studies have identified several single nucleotide polymorphisms (SNPs) that are associated with risk of colorectal cancer (CRC). Prior research has evaluated the presence of gene-environment interaction involving the first 10 identified susceptibility loci, but little work has been conducted on interaction involving SNPs at recently identified susceptibility loci, including: rs10911251, rs6691170, rs6687758, rs11903757, rs10936599, rs647161, rs1321311, rs719725, rs1665650, rs3824999, rs7136702, rs11169552, rs59336, rs3217810, rs4925386, and rs2423279. METHODS Data on 9160 cases and 9280 controls from the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) and Colon Cancer Family Registry (CCFR) were used to evaluate the presence of interaction involving the above-listed SNPs and sex, body mass index (BMI), alcohol consumption, smoking, aspirin use, post-menopausal hormone (PMH) use, as well as intake of dietary calcium, dietary fiber, dietary folate, red meat, processed meat, fruit, and vegetables. Interaction was evaluated using a fixed-effects meta-analysis of an efficient Empirical Bayes estimator, and permutation was used to account for multiple comparisons. RESULTS None of the permutation-adjusted p-values reached statistical significance. CONCLUSIONS The associations between recently identified genetic susceptibility loci and CRC are not strongly modified by sex, BMI, alcohol, smoking, aspirin, PMH use, and various dietary factors. IMPACT Results suggest no evidence of strong gene-environment interactions involving the recently identified 16 susceptibility loci for CRC taken one at a time. PMID:24994789

Tumor targeted gene therapy

International Nuclear Information System (INIS)

Kang, Joo Hyun

2006-01-01

Knowledge of molecular mechanisms governing malignant transformation brings new opportunities for therapeutic intervention against cancer using novel approaches. One of them is gene therapy based on the transfer of genetic material to an organism with the aim of correcting a disease. The application of gene therapy to the cancer treatment had led to the development of new experimental approaches such as suicidal gene therapy, inhibition of oncogenes and restoration of tumor-suppressor genes. Suicidal gene therapy is based on the expression in tumor cells of a gene encoding an enzyme that converts a prodrug into a toxic product. Representative suicidal genes are Herpes simplex virus type 1 thymidine kinase (HSV1-tk) and cytosine deaminase (CD). Especially, physicians and scientists of nuclear medicine field take an interest in suicidal gene therapy because they can monitor the location and magnitude, and duration of expression of HSV1-tk and CD by PET scanner

Use of an activated beta-catenin to identify Wnt pathway target genes in caenorhabditis elegans, including a subset of collagen genes expressed in late larval development.

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Jackson, Belinda M; Abete-Luzi, Patricia; Krause, Michael W; Eisenmann, David M

2014-04-16

The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin-dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1 col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle.

Targeted deficiency of the transcriptional activator Hnf1alpha alters subnuclear positioning of its genomic targets.

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Reini F Luco

2008-05-01

Full Text Available DNA binding transcriptional activators play a central role in gene-selective regulation. In part, this is mediated by targeting local covalent modifications of histone tails. Transcriptional regulation has also been associated with the positioning of genes within the nucleus. We have now examined the role of a transcriptional activator in regulating the positioning of target genes. This was carried out with primary beta-cells and hepatocytes freshly isolated from mice lacking Hnf1alpha, an activator encoded by the most frequently mutated gene in human monogenic diabetes (MODY3. We show that in Hnf1a-/- cells inactive endogenous Hnf1alpha-target genes exhibit increased trimethylated histone H3-Lys27 and reduced methylated H3-Lys4. Inactive Hnf1alpha-targets in Hnf1a-/- cells are also preferentially located in peripheral subnuclear domains enriched in trimethylated H3-Lys27, whereas active targets in wild-type cells are positioned in more central domains enriched in methylated H3-Lys4 and RNA polymerase II. We demonstrate that this differential positioning involves the decondensation of target chromatin, and show that it is spatially restricted rather than a reflection of non-specific changes in the nuclear organization of Hnf1a-deficient cells. This study, therefore, provides genetic evidence that a single transcriptional activator can influence the subnuclear location of its endogenous genomic targets in primary cells, and links activator-dependent changes in local chromatin structure to the spatial organization of the genome. We have also revealed a defect in subnuclear gene positioning in a model of a human transcription factor disease.

Targeted Adenoviral Vector Demonstrates Enhanced Efficacy for In Vivo Gene Therapy of Uterine Leiomyoma.

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Abdelaziz, Mohamed; Sherif, Lotfy; ElKhiary, Mostafa; Nair, Sanjeeta; Shalaby, Shahinaz; Mohamed, Sara; Eziba, Noura; El-Lakany, Mohamed; Curiel, David; Ismail, Nahed; Diamond, Michael P; Al-Hendy, Ayman

2016-04-01

Gene therapy is a potentially effective non-surgical approach for the treatment of uterine leiomyoma. We demonstrated that targeted adenovirus vector, Ad-SSTR-RGD-TK/GCV, was highly effective in selectively inducing apoptosis and inhibiting proliferation of human leiomyoma cells in vitro while sparing normal myometrial cells. An in-vivo study, to compare efficacy and safety of modified adenovirus vector Ad-SSTR-RGD-TK/GCV versus untargeted vector for treatment of leiomyoma. Female nude mice were implanted with rat leiomyoma cells subcutaneously. Then mice were randomized into three groups. Group 1 received Ad-LacZ (marker gene), Group 2 received untargeted Ad-TK, and Group 3 received the targeted Ad-SSTR-RGD-TK. Tumors were measured weekly for 4 weeks. Then mice were sacrificed and tissue samples were collected. Evaluation of markers of apoptosis, proliferation, extracellular matrix, and angiogenesis was performed using Western Blot & Immunohistochemistry. Statistical analysis was done using ANOVA. Dissemination of adenovirus was assessed by PCR. In comparison with the untargeted vector, the targeted adenoviral vector significantly shrank leiomyoma size (P leiomyoma lesions with both targeted and untargeted adenovirus. Targeted adenovirus, effectively reduces tumor size in leiomyoma without dissemination to other organs. Further evaluation of this localized targeted strategy for gene therapy is needed in appropriate preclinical humanoid animal models in preparation for a future pilot human trial. © The Author(s) 2016.

A study of the evolution of human microRNAs by their apparent repression effectiveness on target genes.

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Yong Huang

Full Text Available BACKGROUND: Even though the genomes of many model species have already been sequenced, our knowledge of gene regulation in evolution is still very limited. One big obstacle is that it is hard to predict the target genes of transcriptional factors accurately from sequences. In this respect, microRNAs (miRNAs are different from transcriptional factors, as target genes of miRNAs can be readily predicted from sequences. This feature of miRNAs offers an unprecedented vantage point for evolutionary analysis of gene regulation. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we analyzed a particular aspect of miRNA evolution, the differences in the "apparent repression effectiveness (ARE" between human miRNAs of different conservational levels. ARE is a measure we designed to evaluate the repression effect of miRNAs on target genes based on publicly available gene expression data in normal tissues and miRNA targeting and expression data. We found that ARE values of more conserved miRNAs are significantly higher than those of less conserved miRNAs in general. We also found the gain in expression abundance and broadness of miRNAs in evolution contributed to the gain in ARE. CONCLUSIONS/SIGNIFICANCE: The ARE measure quantifies the repressive effects of miRNAs and enables us to study the influences of many factors on miRNA-mediated repression, such as conservational levels and expression levels of miRNAs. The gain in ARE can be explained by the existence of a trend of miRNAs in evolution to effectively control more target genes, which is beneficial to the miRNAs but not necessarily to the organism at all times. Our results from miRNAs gave us an insight of the complex interplay between regulators and target genes in evolution.

A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets.

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Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

2014-12-05

Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways.

Capturing the target genes of BldD in Saccharopolyspora erythraea using improved genomic SELEX method.

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Wu, Hang; Mao, Yongrong; Chen, Meng; Pan, Hui; Huang, Xunduan; Ren, Min; Wu, Hao; Li, Jiali; Xu, Zhongdong; Yuan, Hualing; Geng, Ming; Weaver, David T; Zhang, Lixin; Zhang, Buchang

2015-03-01

BldD (SACE_2077), a key developmental regulator in actinomycetes, is the first identified transcriptional factor in Saccharopolyspora erythraea positively regulating erythromycin production and morphological differentiation. Although the BldD of S. erythraea binds to the promoters of erythromycin biosynthetic genes, the interaction affinities are relatively low, implying the existence of its other target genes in S. erythraea. Through the genomic systematic evolution of ligands by exponential enrichment (SELEX) method that we herein improved, four DNA sequences of S. erythraea A226, corresponding to the promoter regions of SACE_0306 (beta-galactosidase), SACE_0811 (50S ribosomal protein L25), SACE_3410 (fumarylacetoacetate hydrolase), and SACE_6014 (aldehyde dehydrogenase), were captured with all three BldD concentrations of 0.5, 1, and 2 μM, while the previously identified intergenic regions of eryBIV-eryAI and ermE-eryCI plus the promoter region of SACE_7115, the amfC homolog for aerial mycelium formation, could be captured only when the BldD's concentration reached 2 μM. Electrophoretic mobility shift assay (EMSA) analysis indicated that BldD specifically bound to above seven DNA sequences, and quantitative real-time PCR (qRT-PCR) assay showed that the transcriptional levels of the abovementioned target genes decreased when bldD was disrupted in A226. Furthermore, SACE_7115 and SACE_0306 in A226 were individually inactivated, showing that SACE_7115 was predominantly involved in aerial mycelium formation, while SACE_0306 mainly controlled erythromycin production. This study provides valuable information for better understanding of the pleiotropic regulator BldD in S. erythraea, and the improved method may be useful for uncovering regulatory networks of other transcriptional factors.

Insights into the regulation of human CNV-miRNAs from the view of their target genes

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Wu Xudong

2012-12-01

Full Text Available Abstract Background microRNAs (miRNAs represent a class of small (typically 22 nucleotides in length non-coding RNAs that can degrade their target mRNAs or block their translation. Recent research showed that copy number alterations of miRNAs and their target genes are highly prevalent in cancers; however, the evolutionary and biological functions of naturally existing copy number variable miRNAs (CNV-miRNAs among individuals have not been studied extensively throughout the genome. Results In this study, we comprehensively analyzed the properties of genes regulated by CNV-miRNAs, and found that CNV-miRNAs tend to target a higher average number of genes and prefer to synergistically regulate the same genes; further, the targets of CNV-miRNAs tend to have higher variability of expression within and between populations. Finally, we found the targets of CNV-miRNAs are more likely to be differentially expressed among tissues and developmental stages, and participate in a wide range of cellular responses. Conclusions Our analyses of CNV-miRNAs provide new insights into the impact of copy number variations on miRNA-mediated post-transcriptional networks. The deeper interpretation of patterns of gene expression variation and the functional characterization of CNV-miRNAs will help to broaden the current understanding of the molecular basis of human phenotypic diversity.

RNA-seq based identification and mutant validation of gene targets related to ethanol resistance in cyanobacterial Synechocystis sp. PCC 6803

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Wang Jiangxin

2012-12-01

Full Text Available Abstract Background Fermentation production of biofuel ethanol consumes agricultural crops, which will compete directly with the food supply. As an alternative, photosynthetic cyanobacteria have been proposed as microbial factories to produce ethanol directly from solar energy and CO2. However, the ethanol productivity from photoautotrophic cyanobacteria is still very low, mostly due to the low tolerance of cyanobacterial systems to ethanol stress. Results To build a foundation necessary to engineer robust ethanol-producing cyanobacterial hosts, in this study we applied a quantitative transcriptomics approach with a next-generation sequencing technology, combined with quantitative reverse-transcript PCR (RT-PCR analysis, to reveal the global metabolic responses to ethanol in model cyanobacterial Synechocystis sp. PCC 6803. The results showed that ethanol exposure induced genes involved in common stress responses, transporting and cell envelope modification. In addition, the cells can also utilize enhanced polyhydroxyalkanoates (PHA accumulation and glyoxalase detoxication pathway as means against ethanol stress. The up-regulation of photosynthesis by ethanol was also further confirmed at transcriptional level. Finally, we used gene knockout strains to validate the potential target genes related to ethanol tolerance. Conclusion RNA-Seq based global transcriptomic analysis provided a comprehensive view of cellular response to ethanol exposure. The analysis provided a list of gene targets for engineering ethanol tolerance in cyanobacterium Synechocystis.

Differentiation and injury-repair signals modulate the interaction of E2F and pRB proteins with novel target genes in keratinocytes.

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Chang, Wing Y; Andrews, Joseph; Carter, David E; Dagnino, Lina

2006-08-01

E2F transcription factors are central to epidermal morphogenesis and regeneration after injury. The precise nature of E2F target genes involved in epidermal formation and repair has yet to be determined. Identification of these genes is essential to understand how E2F proteins regulate fundamental aspects of epidermal homeostasis and transformation. We have conducted a genome-wide screen using CpG island microarray analysis to identify novel promoters bound by E2F3 and E2F5 in human keratinocytes. We further characterized several of these genes, and determined that multiple E2F and retinoblastoma (pRb) family proteins associate with them in exponentially proliferating cells. We also assessed the effect on E2F and pRb binding to those genes in response to differentiation induced by bone morphogenetic protein-6 (BMP-6), or to activation of repair mechanisms induced by transforming growth factor-beta (TGF-beta). These studies demonstrate promoter- and cytokine-specific changes in binding profiles of E2F and/or pRb family proteins. For example, E2F1, 3, 4 and p107 were recruited to the N-myc promoter in cells treated with BMP-6, whereas E2F1, 3, 4, 5, p107 and p130 were bound to this promoter in the presence of TGF-beta. Functionally, these different interactions resulted in transcriptional repression by BMP-6 and TGF-beta of the N-myc gene, via mechanisms that involved E2F binding to the promoter and association with pRb-family proteins. Thus, multiple combinations of E2F and pRb family proteins may associate with and transcriptionally regulate a given target promoter in response to differentiation and injury-repair stimuli in epidermal keratinocytes.

Growth rate regulated genes and their wide involvement in the Lactococcus lactis stress responses

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Redon Emma

2008-07-01

Full Text Available Abstract Background The development of transcriptomic tools has allowed exhaustive description of stress responses. These responses always superimpose a general response associated to growth rate decrease and a specific one corresponding to the stress. The exclusive growth rate response can be achieved through chemostat cultivation, enabling all parameters to remain constant except the growth rate. Results We analysed metabolic and transcriptomic responses of Lactococcus lactis in continuous cultures at different growth rates ranging from 0.09 to 0.47 h-1. Growth rate was conditioned by isoleucine supply. Although carbon metabolism was constant and homolactic, a widespread transcriptomic response involving 30% of the genome was observed. The expression of genes encoding physiological functions associated with biogenesis increased with growth rate (transcription, translation, fatty acid and phospholipids metabolism. Many phages, prophages and transposon related genes were down regulated as growth rate increased. The growth rate response was compared to carbon and amino-acid starvation transcriptomic responses, revealing constant and significant involvement of growth rate regulations in these two stressful conditions (overlap 27%. Two regulators potentially involved in the growth rate regulations, llrE and yabB, have been identified. Moreover it was established that genes positively regulated by growth rate are preferentially located in the vicinity of replication origin while those negatively regulated are mainly encountered at the opposite, thus indicating the relationship between genes expression and their location on chromosome. Although stringent response mechanism is considered as the one governing growth deceleration in bacteria, the rigorous comparison of the two transcriptomic responses clearly indicated the mechanisms are distinct. Conclusion This work of integrative biology was performed at the global level using transcriptomic analysis

AAV-Mediated Gene Targeting Is Significantly Enhanced by Transient Inhibition of Nonhomologous End Joining or the Proteasome In Vivo

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Paulk, Nicole K.; Loza, Laura Marquez; Finegold, Milton J.

2012-01-01

Abstract Recombinant adeno-associated virus (rAAV) vectors have clear potential for use in gene targeting but low correction efficiencies remain the primary drawback. One approach to enhancing efficiency is a block of undesired repair pathways like nonhomologous end joining (NHEJ) to promote the use of homologous recombination. The natural product vanillin acts as a potent inhibitor of NHEJ by inhibiting DNA-dependent protein kinase (DNA-PK). Using a homology containing rAAV vector, we previously demonstrated in vivo gene repair frequencies of up to 0.1% in a model of liver disease hereditary tyrosinemia type I. To increase targeting frequencies, we administered vanillin in combination with rAAV. Gene targeting frequencies increased up to 10-fold over AAV alone, approaching 1%. Fah−/−Ku70−/− double knockout mice also had increased gene repair frequencies, genetically confirming the beneficial effects of blocking NHEJ. A second strategy, transient proteasomal inhibition, also increased gene-targeting frequencies but was not additive to NHEJ inhibition. This study establishes the benefit of transient NHEJ inhibition with vanillin, or proteasome blockage with bortezomib, for increasing hepatic gene targeting with rAAV. Functional metabolic correction of a clinically relevant disease model was demonstrated and provided evidence for the feasibility of gene targeting as a therapeutic strategy. PMID:22486314

ESKIMO1 is a key gene involved in water economy as well as cold acclimation and salt tolerance

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Yu Agnes

2008-12-01

Full Text Available Abstract Background Drought is a major social and economic problem resulting in huge yield reduction in the field. Today's challenge is to develop plants with reduced water requirements and stable yields in fluctuating environmental conditions. Arabidopsis thaliana is an excellent model for identifying potential targets for plant breeding. Drought tolerance in the field was successfully conferred to crops by transferring genes from this model species. While involved in a plant genomics programme, which aims to identify new genes responsible for plant response to abiotic stress, we identified ESKIMO1 as a key gene involved in plant water economy as well as cold acclimation and salt tolerance. Results All esk1 mutants were more tolerant to freezing, after acclimation, than their wild type counterpart. esk1 mutants also showed increased tolerance to mild water deficit for all traits measured. The mutant's improved tolerance to reduced water supply may be explained by its lower transpiration rate and better water use efficiency (WUE, which was assessed by carbon isotope discrimination and gas exchange measurements. esk1 alleles were also shown to be more tolerant to salt stress. Transcriptomic analysis of one mutant line and its wild-type background was carried out. Under control watering conditions a number of genes were differentially expressed between the mutant and the wild type whereas under mild drought stress this list of genes was reduced. Among the genes that were differentially expressed between the wild type and mutant, two functional categories related to the response to stress or biotic and abiotic stimulus were over-represented. Under salt stress conditions, all gene functional categories were represented equally in both the mutant and wild type. Based on this transcriptome analysis we hypothesise that in control conditions the esk1 mutant behaves as if it was exposed to drought stress. Conclusion Overall our findings suggest that the

Disruption of a cystine transporter downregulates expression of genes involved in sulfur regulation and cellular respiration

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Jessica A. Simpkins

2016-06-01

Full Text Available Cystine and cysteine are important molecules for pathways such as redox signaling and regulation, and thus identifying cellular deficits upon deletion of the Saccharomyces cerevisiae cystine transporter Ers1p allows for a further understanding of cystine homeostasis. Previous complementation studies using the human ortholog suggest yeast Ers1p is a cystine transporter. Human CTNS encodes the protein Cystinosin, a cystine transporter that is embedded in the lysosomal membrane and facilitates the export of cystine from the lysosome. When CTNS is mutated, cystine transport is disrupted, leading to cystine accumulation, the diagnostic hallmark of the lysosomal storage disorder cystinosis. Here, we provide biochemical evidence for Ers1p-dependent cystine transport. However, the accumulation of intracellular cystine is not observed when the ERS1 gene is deleted from ers1-Δ yeast, supporting the existence of modifier genes that provide a mechanism in ers1-Δ yeast that prevents or corrects cystine accumulation. Upon comparison of the transcriptomes of isogenic ERS1+ and ers1-Δ strains of S. cerevisiae by DNA microarray followed by targeted qPCR, sixteen genes were identified as being differentially expressed between the two genotypes. Genes that encode proteins functioning in sulfur regulation, cellular respiration, and general transport were enriched in our screen, demonstrating pleiotropic effects of ers1-Δ. These results give insight into yeast cystine regulation and the multiple, seemingly distal, pathways that involve proper cystine recycling.

Small changes in gene expression of targeted osmoregulatory genes when exposing marine and freshwater threespine stickleback (Gasterosteus aculeatus to abrupt salinity transfers.

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Annette Taugbøl

Full Text Available Salinity is one of the key factors that affects metabolism, survival and distribution of fish species, as all fish osmoregulate and euryhaline fish maintain osmotic differences between their extracellular fluid and either freshwater or seawater. The threespine stickleback (Gasterosteus aculeatus is a euryhaline species with populations in both marine and freshwater environments, where the physiological and genomic basis for salinity tolerance adaptation is not fully understood. Therefore, our main objective in this study was to investigate gene expression of three targeted osmoregulatory genes (Na+/K+-ATPase (ATPA13, cystic fibrosis transmembrane regulator (CFTR and a voltage gated potassium channel gene (KCNH4 and one stress related heat shock protein gene (HSP70 in gill tissue from marine and freshwater populations when exposed to non-native salinity for periods ranging from five minutes to three weeks. Overall, the targeted genes showed highly plastic expression profiles, in addition the expression of ATP1A3 was slightly higher in saltwater adapted fish and KCNH4 and HSP70 had slightly higher expression in freshwater. As no pronounced changes were observed in the expression profiles of the targeted genes, this indicates that the osmoregulatory apparatuses of both the marine and landlocked freshwater stickleback population have not been environmentally canalized, but are able to respond plastically to abrupt salinity challenges.

Small changes in gene expression of targeted osmoregulatory genes when exposing marine and freshwater threespine stickleback (Gasterosteus aculeatus) to abrupt salinity transfers.

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Taugbøl, Annette; Arntsen, Tina; Ostbye, Kjartan; Vøllestad, Leif Asbjørn

2014-01-01

Salinity is one of the key factors that affects metabolism, survival and distribution of fish species, as all fish osmoregulate and euryhaline fish maintain osmotic differences between their extracellular fluid and either freshwater or seawater. The threespine stickleback (Gasterosteus aculeatus) is a euryhaline species with populations in both marine and freshwater environments, where the physiological and genomic basis for salinity tolerance adaptation is not fully understood. Therefore, our main objective in this study was to investigate gene expression of three targeted osmoregulatory genes (Na+/K+-ATPase (ATPA13), cystic fibrosis transmembrane regulator (CFTR) and a voltage gated potassium channel gene (KCNH4) and one stress related heat shock protein gene (HSP70)) in gill tissue from marine and freshwater populations when exposed to non-native salinity for periods ranging from five minutes to three weeks. Overall, the targeted genes showed highly plastic expression profiles, in addition the expression of ATP1A3 was slightly higher in saltwater adapted fish and KCNH4 and HSP70 had slightly higher expression in freshwater. As no pronounced changes were observed in the expression profiles of the targeted genes, this indicates that the osmoregulatory apparatuses of both the marine and landlocked freshwater stickleback population have not been environmentally canalized, but are able to respond plastically to abrupt salinity challenges.

Genome-wide targeted prediction of ABA responsive genes in rice based on over-represented cis-motif in co-expressed genes.

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Lenka, Sangram K; Lohia, Bikash; Kumar, Abhay; Chinnusamy, Viswanathan; Bansal, Kailash C

2009-02-01

Abscisic acid (ABA), the popular plant stress hormone, plays a key role in regulation of sub-set of stress responsive genes. These genes respond to ABA through specific transcription factors which bind to cis-regulatory elements present in their promoters. We discovered the ABA Responsive Element (ABRE) core (ACGT) containing CGMCACGTGB motif as over-represented motif among the promoters of ABA responsive co-expressed genes in rice. Targeted gene prediction strategy using this motif led to the identification of 402 protein coding genes potentially regulated by ABA-dependent molecular genetic network. RT-PCR analysis of arbitrarily chosen 45 genes from the predicted 402 genes confirmed 80% accuracy of our prediction. Plant Gene Ontology (GO) analysis of ABA responsive genes showed enrichment of signal transduction and stress related genes among diverse functional categories.

Homologous gene targeting of a carotenoids biosynthetic gene in Rhodosporidium toruloides by Agrobacterium-mediated transformation.

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Sun, Wenyi; Yang, Xiaobing; Wang, Xueying; Lin, Xinping; Wang, Yanan; Zhang, Sufang; Luan, Yushi; Zhao, Zongbao K

2017-07-01

To target a carotenoid biosynthetic gene in the oleaginous yeast Rhodosporidium toruloides by using the Agrobacterium-mediated transformation (AMT) method. The RHTO_04602 locus of R. toruloides NP11, previously assigned to code the carotenoid biosynthetic gene CRTI, was amplified from genomic DNA and cloned into the binary plasmid pZPK-mcs, resulting in pZPK-CRT. A HYG-expression cassette was inserted into the CRTI sequence of pZPK-CRT by utilizing the restriction-free clone strategy. The resulted plasmid was used to transform R. toruloides cells according to the AMT method, leading to a few white transformants. Sequencing analysis of those transformants confirmed homologous recombination and insertional inactivation of CRTI. When the white variants were transformed with a CRTI-expression cassette, cells became red and produced carotenoids as did the wild-type strain NP11. Successful homologous targeting of the CrtI locus confirmed the function of RHTO_04602 in carotenoids biosynthesis in R. toruloides. It provided valuable information for metabolic engineering of this non-model yeast species.

Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

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Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong; Kim, Min Ju; Park, Sang-Min; Ryu, Junghyun; Ahn, Jin Seop; Heo, Soon Young; Kang, Jee Hyun; Choi, You Jung [Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan (Korea, Republic of); Choi, Seong-Jun [Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of); Shim, Hosup, E-mail: shim@dku.edu [Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan (Korea, Republic of); Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of); Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of)

2014-10-03

Highlights: • ATM gene-targeted pigs were produced by somatic cell nuclear transfer. • A novel large animal model for ataxia telangiectasia was developed. • The new model may provide an alternative to the mouse model. - Abstract: Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.

Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

International Nuclear Information System (INIS)

Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong; Kim, Min Ju; Park, Sang-Min; Ryu, Junghyun; Ahn, Jin Seop; Heo, Soon Young; Kang, Jee Hyun; Choi, You Jung; Choi, Seong-Jun; Shim, Hosup

2014-01-01

Highlights: • ATM gene-targeted pigs were produced by somatic cell nuclear transfer. • A novel large animal model for ataxia telangiectasia was developed. • The new model may provide an alternative to the mouse model. - Abstract: Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies

MicroRNA-424/503 cluster members regulate bovine granulosa cell proliferation and cell cycle progression by targeting SMAD7 gene through activin signalling pathway.

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Pande, Hari Om; Tesfaye, Dawit; Hoelker, Michael; Gebremedhn, Samuel; Held, Eva; Neuhoff, Christiane; Tholen, Ernst; Schellander, Karl; Wondim, Dessie Salilew

2018-05-01

The granulosa cells are indispensable for follicular development and its function is orchestrated by several genes, which in turn posttranscriptionally regulated by microRNAs (miRNA). In our previous study, the miRRNA-424/503 cluster was found to be highly abundant in bovine granulosa cells (bGCs) of preovulatory dominant follicle compared to subordinate counterpart at day 19 of the bovine estrous cycle. Other study also indicated the involvement of miR-424/503 cluster in tumour cell resistance to apoptosis suggesting this miRNA cluster may involve in cell survival. However, the role of miR-424/503 cluster in granulosa cell function remains elusive Therefore, this study aimed to investigate the role of miRNA-424/503 cluster in bGCs function using microRNA gain- and loss-of-function approaches. The role of miR-424/503 cluster members in granulosa cell function was investigated by overexpressing or inhibiting its activity in vitro cultured granulosa cells using miR-424/503 mimic or inhibitor, respectively. Luciferase reporter assay showed that SMAD7 and ACVR2A are the direct targets of the miRNA-424/503 cluster members. In line with this, overexpression of miRNA-424/503 cluster members using its mimic and inhibition of its activity by its inhibitor reduced and increased, respectively the expression of SMAD7 and ACVR2A. Furthermore, flow cytometric analysis indicated that overexpression of miRNA-424/503 cluster members enhanced bGCs proliferation by promoting G1- to S- phase cell cycle transition. Modulation of miRNA-424/503 cluster members tended to increase phosphorylation of SMAD2/3 in the Activin signalling pathway. Moreover, sequence specific knockdown of SMAD7, the target gene of miRNA-424/503 cluster members, using small interfering RNA also revealed similar phenotypic and molecular alterations observed when miRNA-424/503 cluster members were overexpressed. Similarly, to get more insight about the role of miRNA-424/503 cluster members in activin signalling

Barriers to Liposomal Gene Delivery: from Application Site to the Target.

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Saffari, Mostafa; Moghimi, Hamid Reza; Dass, Crispin R

2016-01-01

Gene therapy is a therapeutic approach to deliver genetic material into cells to alter their function in entire organism. One promising form of gene delivery system (DDS) is liposomes. The success of liposome-mediated gene delivery is a multifactorial issue and well-designed liposomal systems might lead to optimized gene transfection particularly in vivo. Liposomal gene delivery systems face different barriers from their site of application to their target, which is inside the cells. These barriers include presystemic obstacles (epithelial barriers), systemic barriers in blood circulation and cellular barriers. Epithelial barriers differ depending on the route of administration. Systemic barriers include enzymatic degradation, binding and opsonisation. Both of these barriers can act as limiting hurdles that genetic material and their vector should overcome before reaching the cells. Finally liposomes should overcome cellular barriers that include cell entrance, endosomal escape and nuclear uptake. These barriers and their impact on liposomal gene delivery will be discussed in this review.

Recent advances in dendrimer-based nanovectors for tumor-targeted drug and gene delivery

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Kesharwani, Prashant; Iyer, Arun K.

2015-01-01

Advances in the application of nanotechnology in medicine have given rise to multifunctional smart nanocarriers that can be engineered with tunable physicochemical characteristics to deliver one or more therapeutic agent(s) safely and selectively to cancer cells, including intracellular organelle-specific targeting. Dendrimers having properties resembling biomolecules, with well-defined 3D nanopolymeric architectures, are emerging as a highly attractive class of drug and gene delivery vector. The presence of numerous peripheral functional groups on hyperbranched dendrimers affords efficient conjugation of targeting ligands and biomarkers that can recognize and bind to receptors overexpressed on cancer cells for tumor-cell-specific delivery. The present review compiles the recent advances in dendrimer-mediated drug and gene delivery to tumors by passive and active targeting principles with illustrative examples. PMID:25555748

Identification of estrogen target genes during zebrafish embryonic development through transcriptomic analysis.

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Ruixin Hao

Full Text Available Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 µM 17β-estradiol (E2 or vehicle from 3 hours to 4 days post fertilization (dpf, harvested at 1, 2, 3 and 4 dpf, and subjected to RNA extraction for transcriptome analysis using microarrays. Differentially expressed genes by E2-treatment were analyzed with hierarchical clustering followed by biological process and tissue enrichment analysis. Markedly distinct sets of genes were up and down-regulated by E2 at the four different time points. Among these genes, only the well-known estrogenic marker vtg1 was co-regulated at all time points. Despite this, the biological functional categories targeted by E2 were relatively similar throughout zebrafish development. According to knowledge-based tissue enrichment, estrogen responsive genes were clustered mainly in the liver, pancreas and brain. This was in line with the developmental dynamics of estrogen-target tissues that were visualized using transgenic zebrafish containing estrogen responsive elements driving the expression of GFP (Tg(5xERE:GFP. Finally, the identified embryonic estrogen-responsive genes were compared to already published estrogen-responsive genes identified in male adult zebrafish (Gene Expression Omnibus database. The expressions of a few genes were co-regulated by E2 in both embryonic and adult zebrafish. These could potentially be used as estrogenic biomarkers for exposure to estrogens or estrogenic endocrine disruptors in zebrafish. In conclusion, our data suggests that estrogen effects on early embryonic zebrafish development are stage- and tissue- specific.

Gene therapy: An overview

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Sudip Indu

2013-01-01

Full Text Available Gene therapy "the use of genes as medicine" involves the transfer of a therapeutic or working copy of a gene into specific cells of an individual in order to repair a faulty gene copy. The technique may be used to replace a faulty gene, or to introduce a new gene whose function is to cure or to favorably modify the clinical course of a condition. The objective of gene therapy is to introduce new genetic material into target cells while causing no damage to the surrounding healthy cells and tissues, hence the treatment related morbidity is decreased. The delivery system includes a vector that delivers a therapeutic gene into the patient′s target cell. Functional proteins are created from the therapeutic gene causing the cell to return to a normal stage. The vectors used in gene therapy can be viral and non-viral. Gene therapy, an emerging field of biomedicine, is still at infancy and much research remains to be done before this approach to the treatment of condition will realize its full potential.