Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes

International Nuclear Information System (INIS)

Zhang, H.; Wang, J.C.; Liu, L.F.

1988-01-01

Treatment of HeLa cells with a DNA topoisomerase I-specific inhibitor, camptothecin, results in rapid cessation of the synthesis of the 45S rRNA precursor. The inhibition of rRNA synthesis is reversible following drug removal and correlates with the presence of camptothecin-trapped topoisomerase I-DNA abortive complexes, which can be detected as topoisomerase I-linked DNA breaks upon lysis with sodium dodecyl sulfate. These breaks were found to be concentrated within the transcribed region of human rRNA genes. No such sites can be detected in the inactive human rRNA genes in mouse-human hybrid cells, suggesting a preferential association of topoisomerase I with actively transcribed genes. The distribution of RNA polymerase molecules along the transcription unit of human rRNA genes in camptothecin-treated HeLa cells, as assayed by nuclear run-on transcription, shows a graded decrease of the RNA polymerase density toward the 3' end of the transcription unit; the density is minimally affected near the 5' start of the transcription unit. These results suggest that DNA topoisomerase I is normally involved in the elongation step of transcription, especially when the transcripts are long, and that camptothecin interferes with this role

Human DNA repair and recombination genes

International Nuclear Information System (INIS)

Thompson, L.H.; Weber, C.A.; Jones, N.J.

1988-09-01

Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs

DNA-mediated gene transfer into ataxia-telangiectasia cells

International Nuclear Information System (INIS)

Crescenzi, M.; Pulciani, S.; Carbonari, M.; Tedesco, L.; Russo, G.; Gaetano, C.; Fiorilli, M.

1986-01-01

The complete description of the genetic lesion(s) underlying the AT mutation might, therefore, highlight not only a DNA-repair pathwa, but also an important aspect of the physiology of lymphocytes. DNA-mediated gene transfer into eukaryotic cells has proved a powerful tool for the molecular cloning of certain mammalian genes. The possibility to clone a given gene using this technology depends, basically, on the availability of a selectable marker associated with the expression of the transfected gene in the recipient cell. Recently, a human DNA repair gene has been cloned in CHO mutant cells by taking advantage of the increased resistance to ultraviolet radiation of the transformants. As a preliminary step toward the molecular cloning of the AT gene(s), the authors have attempted to confer radioresistance to AT cells by transfection with normal human DNA

Molecular genetic analysis of a vaccinia virus gene with an essential role in DNA replication

International Nuclear Information System (INIS)

Evans, E.V.A.

1989-01-01

The poxvirus, vaccinia, is large DNA virus which replicates in the cytoplasma of the host cell. The virus is believed to encode most or all of the functions required for the temporally regulated transcription and replication of its 186 kilobase genome. Physical and genetic autonomy from the host make vaccinia a useful eukaryotic organism in which to study replication genes and proteins, using a combination of biochemical and genetic techniques. Essential viral functions for replication are identified by conditional lethal mutants that fail to synthesize DNA at the non-permissive temperatures. One such group contains the non-complementing alleles ts17, ts24, ts69 (WR strain). Studies were undertaken to define the phenotype of ts mutants, and to identify and characterize the affected gene and protein. Mutant infection was essentially normal at 32 degree C, but at 39 degree C the mutants did not incorporate 3 H-thymidine into nascent viral DNA or synthesize late viral proteins. If mutant cultures were shifted to non-permissive conditions at the height of replication, DNA synthesis was halted rapidly, implying that the mutants are defective in DNA elongation. The gene affected in the WR mutants and in ts6389, a DNA-minus mutant of the IHD strain, was mapped by marker rescue and corresponds to open reading frame 5 (orfD5) of the viral HindIII D fragment

Identification of Bicarbonate as a Trigger and Genes Involved with Extracellular DNA Export in Mycobacterial Biofilms

Directory of Open Access Journals (Sweden)

Sasha J. Rose

2016-12-01

Full Text Available Extracellular DNA (eDNA is an integral biofilm matrix component of numerous pathogens, including nontuberculous mycobacteria (NTM. Cell lysis is the source of eDNA in certain bacteria, but the source of eDNA remains unidentified for NTM, as well as for other eDNA-containing bacterial species. In this study, conditions affecting eDNA export were examined, and genes involved with the eDNA export mechanism were identified. After a method for monitoring eDNA in real time in undisturbed biofilms was established, different conditions affecting eDNA were investigated. Bicarbonate positively influenced eDNA export in a pH-independent manner in Mycobacterium avium, M. abscessus, and M. chelonae. The surface-exposed proteome of M. avium in eDNA-containing biofilms revealed abundant carbonic anhydrases. Chemical inhibition of carbonic anhydrases with ethoxzolamide significantly reduced eDNA export. An unbiased transposon mutant library screen for eDNA export in M. avium identified many severely eDNA-attenuated mutants, including one not expressing a unique FtsK/SpoIIIE-like DNA-transporting pore, two with inactivation of carbonic anhydrases, and nine with inactivation of genes belonging to a unique genomic region, as well as numerous mutants involved in metabolism and energy production. Complementation of nine mutants that included the FtsK/SpoIIIE and carbonic anhydrase significantly restored eDNA export. Interestingly, several attenuated eDNA mutants have mutations in genes encoding proteins that were found with the surface proteomics, and many more mutations are localized in operons potentially encoding surface proteins. Collectively, our data strengthen the evidence of eDNA export being an active mechanism that is activated by the bacterium responding to bicarbonate.

Variation of DNA methylation patterns associated with gene expression in rice (Oryza sativa) exposed to cadmium.

Science.gov (United States)

Feng, Sheng Jun; Liu, Xue Song; Tao, Hua; Tan, Shang Kun; Chu, Shan Shan; Oono, Youko; Zhang, Xian Duo; Chen, Jian; Yang, Zhi Min

2016-12-01

We report genome-wide single-base resolution maps of methylated cytosines and transcriptome change in Cd-exposed rice. Widespread differences were identified in CG and non-CG methylation marks between Cd-exposed and Cd-free rice genomes. There are 2320 non-redundant differentially methylated regions detected in the genome. RNA sequencing revealed 2092 DNA methylation-modified genes differentially expressed under Cd exposure. More genes were found hypermethylated than those hypomethylated in CG, CHH and CHG (where H is A, C or T) contexts in upstream, gene body and downstream regions. Many of the genes were involved in stress response, metal transport and transcription factors. Most of the DNA methylation-modified genes were transcriptionally altered under Cd stress. A subset of loss of function mutants defective in DNA methylation and histone modification activities was used to identify transcript abundance of selected genes. Compared with wide type, mutation of MET1 and DRM2 resulted in general lower transcript levels of the genes under Cd stress. Transcripts of OsIRO2, OsPR1b and Os09g02214 in drm2 were significantly reduced. A commonly used DNA methylation inhibitor 5-azacytidine was employed to investigate whether DNA demethylation affected physiological consequences. 5-azacytidine provision decreased general DNA methylation levels of selected genes, but promoted growth of rice seedlings and Cd accumulation in rice plant. © 2016 John Wiley & Sons Ltd.

Isolation and characterization of the dnaA gene of Rickettsia prowazekii

International Nuclear Information System (INIS)

Waite, R.T.; Shaw, E.I.; Winkler, H.H.; Wood, D.G.

1998-01-01

The dnaA gene encoding the initiator protein of DNA replication was isolated from the obligate intracellular bacterium, Rickettsia prowazekii. Comparison of the deduced amino acid sequence of R. prowazekii DnaA with other bacterial DnaA proteins revealed extensive similarity. However, the rickettsial sequence is unique in the number of basic lysine residues found within a highly conserved portion of the putative DNA binding region, suggesting that the rickettsial protein may recognize a DNA sequence that differs from the consensus DnaA box sequence identified in other bacteria. Consensus DnaA box sequences, found upstream of many bacterial dnaA genes, were not identified upstream of rickettsial dnaA gene. In addition, gene organization within this region differed from that of other bacteria. The putative start of transcription of the rickettsial dnaA gene was localized to a site 522 nucleotides upstream of the DnaA start codon. Key words: Rickettsia prowazekii; dnaA gene; initiator protein (authors)

DNA Methylation in Inflammatory Genes among Children with Obstructive Sleep Apnea

OpenAIRE

Kim, Jinkwan; Bhattacharjee, Rakesh; Khalyfa, Abdelnaby; Kheirandish-Gozal, Leila; Capdevila, Oscar Sans; Wang, Yang; Gozal, David

2012-01-01

Background: Pediatric obstructive sleep apnea (OSA) leads to multiple end-organ morbidities that are mediated by the cumulative burden of oxidative stress and inflammation. Because not all children with OSA exhibit increased systemic inflammation, genetic and environmental factors may be affecting patterns of DNA methylation in genes subserving inflammatory functions.

DNA methylation in a Scottish family multiply affected by bipolar disorder and major depressive disorder.

Science.gov (United States)

Walker, Rosie May; Christoforou, Andrea Nikie; McCartney, Daniel L; Morris, Stewart W; Kennedy, Nicholas A; Morten, Peter; Anderson, Susan Maguire; Torrance, Helen Scott; Macdonald, Alix; Sussmann, Jessika Elizabeth; Whalley, Heather Clare; Blackwood, Douglas H R; McIntosh, Andrew Mark; Porteous, David John; Evans, Kathryn Louise

2016-01-01

Bipolar disorder (BD) is a severe, familial psychiatric condition. Progress in understanding the aetiology of BD has been hampered by substantial phenotypic and genetic heterogeneity. We sought to mitigate these confounders by studying a multi-generational family multiply affected by BD and major depressive disorder (MDD), who carry an illness-linked haplotype on chromosome 4p. Within a family, aetiological heterogeneity is likely to be reduced, thus conferring greater power to detect illness-related changes. As accumulating evidence suggests that altered DNA methylation confers risk for BD and MDD, we compared genome-wide methylation between (i) affected carriers of the linked haplotype (ALH) and married-in controls (MIs), (ii) well unaffected haplotype carriers (ULH) and MI, (iii) ALH and ULH and (iv) all haplotype carriers (LH) and MI. Nominally significant differences in DNA methylation were observed in all comparisons, with differences withstanding correction for multiple testing when the ALH or LH group was compared to the MIs. In both comparisons, we observed increased methylation at a locus in FANCI, which was accompanied by increased FANCI expression in the ALH group. FANCI is part of the Fanconi anaemia complementation (FANC) gene family, which are mutated in Fanconi anaemia and participate in DNA repair. Interestingly, several FANC genes have been implicated in psychiatric disorders. Regional analyses of methylation differences identified loci implicated in psychiatric illness by genome-wide association studies, including CACNB2 and the major histocompatibility complex. Gene ontology analysis revealed enrichment for methylation differences in neurologically relevant genes. Our results highlight altered DNA methylation as a potential mechanism by which the linked haplotype might confer risk for mood disorders. Differences in the phenotypic outcome of haplotype carriers might, in part, arise from additional changes in DNA methylation that converge on

Divergence of gene body DNA methylation and evolution of plant duplicate genes.

Directory of Open Access Journals (Sweden)

Jun Wang

Full Text Available It has been shown that gene body DNA methylation is associated with gene expression. However, whether and how deviation of gene body DNA methylation between duplicate genes can influence their divergence remains largely unexplored. Here, we aim to elucidate the potential role of gene body DNA methylation in the fate of duplicate genes. We identified paralogous gene pairs from Arabidopsis and rice (Oryza sativa ssp. japonica genomes and reprocessed their single-base resolution methylome data. We show that methylation in paralogous genes nonlinearly correlates with several gene properties including exon number/gene length, expression level and mutation rate. Further, we demonstrated that divergence of methylation level and pattern in paralogs indeed positively correlate with their sequence and expression divergences. This result held even after controlling for other confounding factors known to influence the divergence of paralogs. We observed that methylation level divergence might be more relevant to the expression divergence of paralogs than methylation pattern divergence. Finally, we explored the mechanisms that might give rise to the divergence of gene body methylation in paralogs. We found that exonic methylation divergence more closely correlates with expression divergence than intronic methylation divergence. We show that genomic environments (e.g., flanked by transposable elements and repetitive sequences of paralogs generated by various duplication mechanisms are associated with the methylation divergence of paralogs. Overall, our results suggest that the changes in gene body DNA methylation could provide another avenue for duplicate genes to develop differential expression patterns and undergo different evolutionary fates in plant genomes.

The phytochemical 3,3'-diindolylmethane decreases expression of AR-controlled DNA damage repair genes through repressive chromatin modifications and is associated with DNA damage in prostate cancer cells.

Science.gov (United States)

Palomera-Sanchez, Zoraya; Watson, Gregory W; Wong, Carmen P; Beaver, Laura M; Williams, David E; Dashwood, Roderick H; Ho, Emily

2017-09-01

Androgen receptor (AR) is a transcription factor involved in normal prostate physiology and prostate cancer (PCa) development. 3,3'-Diindolylmethane (DIM) is a promising phytochemical agent against PCa that affects AR activity and epigenetic regulators in PCa cells. However, whether DIM suppresses PCa via epigenetic regulation of AR target genes is unknown. We assessed epigenetic regulation of AR target genes in LNCaP PCa cells and showed that DIM treatment led to epigenetic suppression of AR target genes involved in DNA repair (PARP1, MRE11, DNA-PK). Decreased expression of these genes was accompanied by an increase in repressive chromatin marks, loss of AR occupancy and EZH2 recruitment to their regulatory regions. Decreased DNA repair gene expression was associated with an increase in DNA damage (γH2Ax) and up-regulation of genomic repeat elements LINE1 and α-satellite. Our results suggest that DIM suppresses AR-dependent gene transcription through epigenetic modulation, leading to DNA damage and genome instability in PCa cells. Published by Elsevier Inc.

DNA methylation in inflammatory genes among children with obstructive sleep apnea.

Science.gov (United States)

Kim, Jinkwan; Bhattacharjee, Rakesh; Khalyfa, Abdelnaby; Kheirandish-Gozal, Leila; Capdevila, Oscar Sans; Wang, Yang; Gozal, David

2012-02-01

Pediatric obstructive sleep apnea (OSA) leads to multiple end-organ morbidities that are mediated by the cumulative burden of oxidative stress and inflammation. Because not all children with OSA exhibit increased systemic inflammation, genetic and environmental factors may be affecting patterns of DNA methylation in genes subserving inflammatory functions. DNA from matched children with OSA with and without high levels of high-sensitivity C-reactive protein (hsCRP) were assessed for DNA methylation levels of 24 inflammatory-related genes. Primer-based polymerase chain reaction assays in a case-control setting involving 47 OSA cases and 31 control subjects were conducted to confirm the findings; hsCRP and myeloid-related protein (MRP) 8/14 levels were also assayed. Forkhead box P3 (FOXP3) and interferon regulatory factor 1 (IRF1) showed higher methylation in six children with OSA and high hsCRP levels compared with matched children with OSA and low hsCRP levels (P DNA methylation levels compared with children with OSA and low CRP levels and control subjects. IRF1 did not exhibit significant differences. FOXP3 DNA methylation levels correlated with hsCRP and MRP 8/14 levels and with apnea-hypopnea index (AHI), BMI z score, and apolipoprotein B levels. A stepwise multiple regression model showed that AHI was independently associated with FOXP3 DNA methylation levels (P gene, which regulates expression of T regulatory lymphocytes, is more likely to display increased methylation among children with OSA who exhibit increased systemic inflammatory responses. Thus, epigenetic modifications may constitute an important determinant of inflammatory phenotype in OSA, and FOXP3 DNA methylation levels may provide a potential biomarker for end-organ vulnerability.

Gene assembly via one-pot chemical ligation of DNA promoted by DNA nanostructures

DEFF Research Database (Denmark)

Manuguerra, Ilenia; Croce, Stefano; El-Sagheer, Afaf H.

2018-01-01

Current gene synthesis methods are driven by enzymatic reactions. Here we report the one-pot synthesis of a chemically-ligated gene from 14 oligonucleotides. The chemical ligation benefits from the highly efficient click chemistry approach templated by DNA nanostructures, and produces modified DNA...

Polymorphisms in metabolism and repair genes affects DNA damage caused by open-cast coal mining exposure.

Science.gov (United States)

Espitia-Pérez, Lyda; Sosa, Milton Quintana; Salcedo-Arteaga, Shirley; León-Mejía, Grethel; Hoyos-Giraldo, Luz Stella; Brango, Hugo; Kvitko, Katia; da Silva, Juliana; Henriques, João A P

2016-09-15

Increasing evidence suggest that occupational exposure to open-cast coal mining residues like dust particles, heavy metals and Polycyclic Aromatic Hydrocarbons (PAHs) may cause a wide range of DNA damage and genomic instability that could be associated to initial steps in cancer development and other work-related diseases. The aim of our study was to evaluate if key polymorphisms in metabolism genes CYP1A1Msp1, GSTM1Null, GSTT1Null and DNA repair genes XRCC1Arg194Trp and hOGG1Ser326Cys could modify individual susceptibility to adverse coal exposure effects, considering the DNA damage (Comet assay) and micronucleus formation in lymphocytes (CBMN) and buccal mucosa cells (BMNCyt) as endpoints for genotoxicity. The study population is comprised of 200 healthy male subjects, 100 open-cast coal-mining workers from "El Cerrejón" (world's largest open-cast coal mine located in Guajira - Colombia) and 100 non-exposed referents from general population. The data revealed a significant increase of CBMN frequency in peripheral lymphocytes of occupationally exposed workers carrying the wild-type variant of GSTT1 (+) gene. Exposed subjects carrying GSTT1null polymorphism showed a lower micronucleus frequency compared with their positive counterparts (FR: 0.83; P=0.04), while BMNCyt, frequency and Comet assay parameters in lymphocytes: Damage Index (DI) and percentage of DNA in the tail (Tail % DNA) were significantly higher in exposed workers with the GSTM1Null polymorphism. Other exfoliated buccal mucosa abnormalities related to cell death (Karyorrhexis and Karyolysis) were increased in GSTT/M1Null carriers. Nuclear buds were significantly higher in workers carrying the CYP1A1Msp1 (m1/m2, m2/m2) allele. Moreover, BMNCyt frequency and Comet assay parameters were significantly lower in exposed carriers of XRCC1Arg194Trp (Arg/Trp, Trp/Trp) and hOGG1Ser326Cys (Ser/Cys, Cys/Cys), thereby providing new data to the increasing evidence about the protective role of these polymorphisms

Gene transcription profiles, global DNA methylation and potential transgenerational epigenetic effects related to Zn exposure history in Daphnia magna

International Nuclear Information System (INIS)

Vandegehuchte, Michiel B.; De Coninck, Dieter; Vandenbrouck, Tine; De Coen, Wim M.; Janssen, Colin R.

2010-01-01

A reduced level of DNA methylation has recently been described in both Zn-exposed and non-exposed offspring of Daphnia magna exposed to Zn. The hypothesis examined in this study is that DNA hypomethylation has an effect on gene transcription. A second hypothesis is that accumulative epigenetic effects can affect gene transcription in non-exposed offspring from parents with an exposure history of more than one generation. Transcriptional gene regulation was studied with a cDNA microarray. In the exposed and non-exposed hypomethylated daphnids, a large proportion of common genes were similarly up- or down-regulated, indicating a possible effect of the DNA hypomethylation. Two of these genes can be mechanistically involved in DNA methylation reduction. The similar transcriptional regulation of two and three genes in the F 0 and F 1 exposed daphnids on one hand and their non-exposed offspring on the other hand, could be the result of a one-generation temporary transgenerational epigenetic effect, which was not accumulative. - Zn-induced DNA hypomethylation is related to gene transcription in Daphnia magna and Zn exposure potentially induced limited temporary transgenerational effects on gene transcription.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1).

Science.gov (United States)

Nwaobi, Sinifunanya E; Olsen, Michelle L

2015-09-26

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

DNA sequence responsible for the amplification of adjacent genes.

Science.gov (United States)

Pasion, S G; Hartigan, J A; Kumar, V; Biswas, D K

1987-10-01

A 10.3-kb DNA fragment in the 5'-flanking region of the rat prolactin (rPRL) gene was isolated from F1BGH(1)2C1, a strain of rat pituitary tumor cells (GH cells) that produces prolactin in response to 5-bromodeoxyuridine (BrdU). Following transfection and integration into genomic DNA of recipient mouse L cells, this DNA induced amplification of the adjacent thymidine kinase gene from Herpes simplex virus type 1 (HSV1TK). We confirmed the ability of this "Amplicon" sequence to induce amplification of other linked or unlinked genes in DNA-mediated gene transfer studies. When transferred into the mouse L cells with the 10.3-5'rPRL gene sequence of BrdU-responsive cells, both the human growth hormone and the HSV1TK genes are amplified in response to 5-bromodeoxyuridine. This observation is substantiated by BrdU-induced amplification of the cotransferred bacterial Neo gene. Cotransfection studies reveal that the BrdU-induced amplification capability is associated with a 4-kb DNA sequence in the 5'-flanking region of the rPRL gene of BrdU-responsive cells. These results demonstrate that genes of heterologous origin, linked or unlinked, and selected or unselected, can be coamplified when located within the amplification boundary of the Amplicon sequence.

Maternal folate depletion and high-fat feeding from weaning affects DNA methylation and DNA repair in brain of adult offspring.

Science.gov (United States)

Langie, Sabine A S; Achterfeldt, Sebastian; Gorniak, Joanna P; Halley-Hogg, Kirstin J A; Oxley, David; van Schooten, Frederik J; Godschalk, Roger W L; McKay, Jill A; Mathers, John C

2013-08-01

The mechanisms through which environmental and dietary factors modulate DNA repair are still unclear but may include dysregulation of gene expression due to altered epigenetic markings. In a mouse model, we investigated the effect of maternal folate depletion during pregnancy and lactation, and high-fat feeding from weaning, on base excision repair (BER) and DNA methylation and expression of selected BER-related genes in the brain of adult offspring. While folate depletion did not affect BER activity of the mothers, BER increased in the offspring at weaning (P=0.052). In the long term, as observed in 6-mo-old offspring, the double insult, i.e., maternal low-folate supply and high-fat feeding from weaning, decreased BER activity significantly in the cortex, cerebellum, hippocampus, and subcortical regions (P≤0.017). This fall in BER activity was associated with small changes in methylation or expression of BER-related genes. Maternal folate depletion led to slightly increased oxidative DNA damage levels in subcortical regions of adult offspring, which may increase sensitivity to oxidative stress and predispose to neurological disorders. In summary, our data suggest that low-folate supply during early life may leave an epigenetic mark that can predispose the offspring to further dietary insults, causing adverse effects during adult life.

Epigenetic changes of DNA repair genes in cancer.

Science.gov (United States)

Lahtz, Christoph; Pfeifer, Gerd P

2011-02-01

'Every Hour Hurts, The Last One Kills'. That is an old saying about getting old. Every day, thousands of DNA damaging events take place in each cell of our body, but efficient DNA repair systems have evolved to prevent that. However, our DNA repair system and that of most other organisms are not as perfect as that of Deinococcus radiodurans, for example, which is able to repair massive amounts of DNA damage at one time. In many instances, accumulation of DNA damage has been linked to cancer, and genetic deficiencies in specific DNA repair genes are associated with tumor-prone phenotypes. In addition to mutations, which can be either inherited or somatically acquired, epigenetic silencing of DNA repair genes may promote tumorigenesis. This review will summarize current knowledge of the epigenetic inactivation of different DNA repair components in human cancer.

The impact of endurance exercise on global and AMPK gene-specific DNA methylation

Energy Technology Data Exchange (ETDEWEB)

King-Himmelreich, Tanya S.; Schramm, Stefanie; Wolters, Miriam C.; Schmetzer, Julia; Möser, Christine V.; Knothe, Claudia [pharmazentrum frankfurt/ZAFES, Institut für Klinische Pharmakologie, Klinikum der Goethe-Universität Frankfurt, Theodor Stern Kai 7, 60590, Frankfurt am Main (Germany); Resch, Eduard [Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project Group for Translational Medicine & Pharmacology (TMP), 60596, Frankfurt/Main (Germany); Peil, Johannes [Sports Clinic, Bad Nauheim, MCI GmbH, In der Aue 30-32, 61231, Bad Nauheim (Germany); Geisslinger, Gerd [pharmazentrum frankfurt/ZAFES, Institut für Klinische Pharmakologie, Klinikum der Goethe-Universität Frankfurt, Theodor Stern Kai 7, 60590, Frankfurt am Main (Germany); Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project Group for Translational Medicine & Pharmacology (TMP), 60596, Frankfurt/Main (Germany); Niederberger, Ellen, E-mail: e.niederberger@em.uni-frankfurt.de [pharmazentrum frankfurt/ZAFES, Institut für Klinische Pharmakologie, Klinikum der Goethe-Universität Frankfurt, Theodor Stern Kai 7, 60590, Frankfurt am Main (Germany)

2016-05-27

Alterations in gene expression as a consequence of physical exercise are frequently described. The mechanism of these regulations might depend on epigenetic changes in global or gene-specific DNA methylation levels. The AMP-activated protein kinase (AMPK) plays a key role in maintenance of energy homeostasis and is activated by increases in the AMP/ATP ratio as occurring in skeletal muscles after sporting activity. To analyze whether exercise has an impact on the methylation status of the AMPK promoter, we determined the AMPK methylation status in human blood samples from patients before and after sporting activity in the context of rehabilitation as well as in skeletal muscles of trained and untrained mice. Further, we examined long interspersed nuclear element 1 (LINE-1) as indicator of global DNA methylation changes. Our results revealed that light sporting activity in mice and humans does not alter global DNA methylation but has an effect on methylation of specific CpG sites in the AMPKα2 gene. These regulations were associated with a reduced AMPKα2 mRNA and protein expression in muscle tissue, pointing at a contribution of the methylation status to AMPK expression. Taken together, these results suggest that exercise influences AMPKα2 gene methylation in human blood and eminently in the skeletal muscle of mice and therefore might repress AMPKα2 gene expression. -- Highlights: •AMPK gene methylation increases after moderate endurance exercise in humans and mice. •AMPKα mRNA and protein decrease after moderate endurance exercise in mice. •Global DNA methylation is not affected under the same conditions.

The impact of endurance exercise on global and AMPK gene-specific DNA methylation

International Nuclear Information System (INIS)

King-Himmelreich, Tanya S.; Schramm, Stefanie; Wolters, Miriam C.; Schmetzer, Julia; Möser, Christine V.; Knothe, Claudia; Resch, Eduard; Peil, Johannes; Geisslinger, Gerd; Niederberger, Ellen

2016-01-01

Alterations in gene expression as a consequence of physical exercise are frequently described. The mechanism of these regulations might depend on epigenetic changes in global or gene-specific DNA methylation levels. The AMP-activated protein kinase (AMPK) plays a key role in maintenance of energy homeostasis and is activated by increases in the AMP/ATP ratio as occurring in skeletal muscles after sporting activity. To analyze whether exercise has an impact on the methylation status of the AMPK promoter, we determined the AMPK methylation status in human blood samples from patients before and after sporting activity in the context of rehabilitation as well as in skeletal muscles of trained and untrained mice. Further, we examined long interspersed nuclear element 1 (LINE-1) as indicator of global DNA methylation changes. Our results revealed that light sporting activity in mice and humans does not alter global DNA methylation but has an effect on methylation of specific CpG sites in the AMPKα2 gene. These regulations were associated with a reduced AMPKα2 mRNA and protein expression in muscle tissue, pointing at a contribution of the methylation status to AMPK expression. Taken together, these results suggest that exercise influences AMPKα2 gene methylation in human blood and eminently in the skeletal muscle of mice and therefore might repress AMPKα2 gene expression. -- Highlights: •AMPK gene methylation increases after moderate endurance exercise in humans and mice. •AMPKα mRNA and protein decrease after moderate endurance exercise in mice. •Global DNA methylation is not affected under the same conditions.

Single gene retrieval from thermally degraded DNA

Indian Academy of Sciences (India)

To simulate single gene retrieval from ancient DNA, several related factors have been investigated. By monitoring a 889 bp polymerase chain reaction (PCR) product and genomic DNA degradation, we find that heat and oxygen (especially heat) are both crucial factors influencing DNA degradation. The heat influence ...

DNA Array-Based Gene Profiling

Science.gov (United States)

Mocellin, Simone; Provenzano, Maurizio; Rossi, Carlo Riccardo; Pilati, Pierluigi; Nitti, Donato; Lise, Mario

2005-01-01

Cancer is a heterogeneous disease in most respects, including its cellularity, different genetic alterations, and diverse clinical behaviors. Traditional molecular analyses are reductionist, assessing only 1 or a few genes at a time, thus working with a biologic model too specific and limited to confront a process whose clinical outcome is likely to be governed by the combined influence of many genes. The potential of functional genomics is enormous, because for each experiment, thousands of relevant observations can be made simultaneously. Accordingly, DNA array, like other high-throughput technologies, might catalyze and ultimately accelerate the development of knowledge in tumor cell biology. Although in its infancy, the implementation of DNA array technology in cancer research has already provided investigators with novel data and intriguing new hypotheses on the molecular cascade leading to carcinogenesis, tumor aggressiveness, and sensitivity to antiblastic agents. Given the revolutionary implications that the use of this technology might have in the clinical management of patients with cancer, principles of DNA array-based tumor gene profiling need to be clearly understood for the data to be correctly interpreted and appreciated. In the present work, we discuss the technical features characterizing this powerful laboratory tool and review the applications so far described in the field of oncology. PMID:15621987

Gene organization in rice revealed by full-length cDNA mapping and gene expression analysis through microarray.

Directory of Open Access Journals (Sweden)

Kouji Satoh

Full Text Available Rice (Oryza sativa L. is a model organism for the functional genomics of monocotyledonous plants since the genome size is considerably smaller than those of other monocotyledonous plants. Although highly accurate genome sequences of indica and japonica rice are available, additional resources such as full-length complementary DNA (FL-cDNA sequences are also indispensable for comprehensive analyses of gene structure and function. We cross-referenced 28.5K individual loci in the rice genome defined by mapping of 578K FL-cDNA clones with the 56K loci predicted in the TIGR genome assembly. Based on the annotation status and the presence of corresponding cDNA clones, genes were classified into 23K annotated expressed (AE genes, 33K annotated non-expressed (ANE genes, and 5.5K non-annotated expressed (NAE genes. We developed a 60mer oligo-array for analysis of gene expression from each locus. Analysis of gene structures and expression levels revealed that the general features of gene structure and expression of NAE and ANE genes were considerably different from those of AE genes. The results also suggested that the cloning efficiency of rice FL-cDNA is associated with the transcription activity of the corresponding genetic locus, although other factors may also have an effect. Comparison of the coverage of FL-cDNA among gene families suggested that FL-cDNA from genes encoding rice- or eukaryote-specific domains, and those involved in regulatory functions were difficult to produce in bacterial cells. Collectively, these results indicate that rice genes can be divided into distinct groups based on transcription activity and gene structure, and that the coverage bias of FL-cDNA clones exists due to the incompatibility of certain eukaryotic genes in bacteria.

Comparing the DNA hypermethylome with gene mutations in human colorectal cancer.

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Kornel E Schuebel

2007-09-01

Full Text Available We have developed a transcriptome-wide approach to identify genes affected by promoter CpG island DNA hypermethylation and transcriptional silencing in colorectal cancer. By screening cell lines and validating tumor-specific hypermethylation in a panel of primary human colorectal cancer samples, we estimate that nearly 5% or more of all known genes may be promoter methylated in an individual tumor. When directly compared to gene mutations, we find larger numbers of genes hypermethylated in individual tumors, and a higher frequency of hypermethylation within individual genes harboring either genetic or epigenetic changes. Thus, to enumerate the full spectrum of alterations in the human cancer genome, and to facilitate the most efficacious grouping of tumors to identify cancer biomarkers and tailor therapeutic approaches, both genetic and epigenetic screens should be undertaken.

A comparison of synthetic oligodeoxynucleotides, DNA fragments and AAV-1 for targeted episomal and chromosomal gene repair

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Leclerc Xavier

2009-04-01

Full Text Available Abstract Background Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. An attractive alternative to these approaches would be to correct the endogenous mutated gene in the affected individual. This study presents a quantitative comparison of the repair efficiency using different forms of donor nucleic acids, including synthetic DNA oligonucleotides, double stranded DNA fragments with sizes ranging from 200 to 2200 bp and sequences carried by a recombinant adeno-associated virus (rAAV-1. Evaluation of each gene repair strategy was carried out using two different reporter systems, a mutated eGFP gene or a dual construct with a functional eGFP and an inactive luciferase gene, in several different cell systems. Gene targeting events were scored either following transient co-transfection of reporter plasmids and donor DNAs, or in a system where a reporter construct was stably integrated into the chromosome. Results In both episomal and chromosomal assays, DNA fragments were more efficient at gene repair than oligonucleotides or rAAV-1. Furthermore, the gene targeting frequency could be significantly increased by using DNA repair stimulating drugs such as doxorubicin and phleomycin. Conclusion Our results show that it is possible to obtain repair frequencies of 1% of the transfected cell population under optimized transfection protocols when cells were pretreated with phleomycin using rAAV-1 and dsDNA fragments.

Dietary selenomethionine increases exon-specific DNA methylation of the p53 gene in rat liver and colon mucosa.

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Zeng, Huawei; Yan, Lin; Cheng, Wen-Hsing; Uthus, Eric O

2011-08-01

The regulation of site-specific DNA methylation of tumor suppressor genes has been considered as a leading mechanism by which certain nutrients exert their anticancer property. This study was to investigate whether selenium (Se) affects the methylation of globe genomic DNA and the exon-specific p53 gene. Three groups of rats (n = 6-7/group) were fed the AIN-93G basal diet supplemented with 0 [Se deficient (D)], 0.15 [Se adequate (A)], or 4 mg [Se supranutritional (S)] (Se as l-selenomethionine)/kg diet for 104 d, respectively. Rats fed the A or S diet had greater plasma and liver glutathione peroxidase activity, liver thioredoxin reductase activity, and plasma homocysteine concentration than those fed the D diet. However, compared with the A diet, rats fed the S diet did not further increase these Se-dependent enzyme activities or homocysteine concentration. In contrast, Se concentrations in kidney, liver, gastrocnemius muscle, and plasma were increased in a Se-dose-dependent manner. Interestingly, rats fed the S diet had significantly less global liver genomic DNA methylation than those fed the D diet. However, the S diet significantly increased the methylation of the p53 gene (exons 5-8) but not the β-actin gene (exons 2-3) DNA in liver and colon mucosa compared with those fed the D diet. Taken together, long-term Se consumption not only affects selenoprotein enzyme activities, homocysteine, tissue Se concentrations, and global genomic DNA methylation but also increases exon-specific DNA methylation of the p53 gene in a Se-dose-dependent manner in rat liver and colon mucosa.

DNA-dependent protein kinase inhibits AID-induced antibody gene conversion.

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Adam J L Cook

2007-04-01

Full Text Available Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID-dependent hypermutation of Ig V(DJ rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(DJ regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(DJ hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs. Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion.

The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli.

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Burgers, P M; Kornberg, A; Sakakibara, Y

1981-09-01

An Escherichia coli mutant, dnaN59, stops DNA synthesis promptly upon a shift to a high temperature; the wild-type dnaN gene carried in a transducing phage encodes a polypeptide of about 41,000 daltons [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553; Yuasa, S. & Sakakibara, Y. (1980) Mol. Gen. Genet. 180, 267-273]. We now find that the product of dnaN gene is the beta subunit of DNA polymerase III holoenzyme, the principal DNA synthetic multipolypeptide complex in E. coli. The conclusion is based on the following observations: (i) Extracts from dnaN59 cells were defective in phage phi X174 and G4 DNA synthesis after the mutant cells had been exposed to the increased temperature. (ii) The enzymatic defect was overcome by addition of purified beta subunit but not by other subunits of DNA polymerase III holoenzyme or by other replication proteins required for phi X174 DNA synthesis. (iii) Partially purified beta subunit from the dnaN mutant, unlike that from the wild type, was inactive in reconstituting the holoenzyme when mixed with the other purified subunits. (iv) Increased dosage of the dnaN gene provided by a plasmid carrying the gene raised cellular levels of the beta subunit 5- to 6-fold.

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

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

2002-01-01

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

Aberrant DNA methylation of WNT pathway genes in the development and progression of CIMP-negative colorectal cancer.

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Galamb, Orsolya; Kalmár, Alexandra; Péterfia, Bálint; Csabai, István; Bodor, András; Ribli, Dezső; Krenács, Tibor; Patai, Árpád V; Wichmann, Barnabás; Barták, Barbara Kinga; Tóth, Kinga; Valcz, Gábor; Spisák, Sándor; Tulassay, Zsolt; Molnár, Béla

2016-08-02

The WNT signaling pathway has an essential role in colorectal carcinogenesis and progression, which involves a cascade of genetic and epigenetic changes. We aimed to analyze DNA methylation affecting the WNT pathway genes in colorectal carcinogenesis in promoter and gene body regions using whole methylome analysis in 9 colorectal cancer, 15 adenoma, and 6 normal tumor adjacent tissue (NAT) samples by methyl capture sequencing. Functional methylation was confirmed on 5-aza-2'-deoxycytidine-treated colorectal cancer cell line datasets. In parallel with the DNA methylation analysis, mutations of WNT pathway genes (APC, β-catenin/CTNNB1) were analyzed by 454 sequencing on GS Junior platform. Most differentially methylated CpG sites were localized in gene body regions (95% of WNT pathway genes). In the promoter regions, 33 of the 160 analyzed WNT pathway genes were differentially methylated in colorectal cancer vs. normal, including hypermethylated AXIN2, CHP1, PRICKLE1, SFRP1, SFRP2, SOX17, and hypomethylated CACYBP, CTNNB1, MYC; 44 genes in adenoma vs. NAT; and 41 genes in colorectal cancer vs. adenoma comparisons. Hypermethylation of AXIN2, DKK1, VANGL1, and WNT5A gene promoters was higher, while those of SOX17, PRICKLE1, DAAM2, and MYC was lower in colon carcinoma compared to adenoma. Inverse correlation between expression and methylation was confirmed in 23 genes, including APC, CHP1, PRICKLE1, PSEN1, and SFRP1. Differential methylation affected both canonical and noncanonical WNT pathway genes in colorectal normal-adenoma-carcinoma sequence. Aberrant DNA methylation appears already in adenomas as an early event of colorectal carcinogenesis.

DNA methylation of amino acid transporter genes in the human placenta.

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Simner, C; Novakovic, B; Lillycrop, K A; Bell, C G; Harvey, N C; Cooper, C; Saffery, R; Lewis, R M; Cleal, J K

2017-12-01

Placental transfer of amino acids via amino acid transporters is essential for fetal growth. Little is known about the epigenetic regulation of amino acid transporters in placenta. This study investigates the DNA methylation status of amino acid transporters and their expression across gestation in human placenta. BeWo cells were treated with 5-aza-2'-deoxycytidine to inhibit methylation and assess the effects on amino acid transporter gene expression. The DNA methylation levels of amino acid transporter genes in human placenta were determined across gestation using DNA methylation array data. Placental amino acid transporter gene expression across gestation was also analysed using data from publically available Gene Expression Omnibus data sets. The expression levels of these transporters at term were established using RNA sequencing data. Inhibition of DNA methylation in BeWo cells demonstrated that expression of specific amino acid transporters can be inversely associated with DNA methylation. Amino acid transporters expressed in term placenta generally showed low levels of promoter DNA methylation. Transporters with little or no expression in term placenta tended to be more highly methylated at gene promoter regions. The transporter genes SLC1A2, SLC1A3, SLC1A4, SLC7A5, SLC7A11 and SLC7A10 had significant changes in enhancer DNA methylation across gestation, as well as gene expression changes across gestation. This study implicates DNA methylation in the regulation of amino acid transporter gene expression. However, in human placenta, DNA methylation of these genes remains low across gestation and does not always play an obvious role in regulating gene expression, despite clear evidence for differential expression as gestation proceeds. Copyright © 2017. Published by Elsevier Ltd.

Isolating human DNA repair genes using rodent-cell mutants

International Nuclear Information System (INIS)

Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

1987-01-01

The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab

DNA Nanotechnology for Precise Control over Drug Delivery and Gene Therapy.

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Angell, Chava; Xie, Sibai; Zhang, Liangfang; Chen, Yi

2016-03-02

Nanomedicine has been growing exponentially due to its enhanced drug targeting and reduced drug toxicity. It uses the interactions where nanotechnological components and biological systems communicate with each other to facilitate the delivery performance. At this scale, the physiochemical properties of delivery systems strongly affect their capacities. Among current delivery systems, DNA nanotechnology shows many advantages because of its unprecedented engineering abilities. Through molecular recognition, DNA nanotechnology can be used to construct a variety of nanostructures with precisely controllable size, shape, and surface chemistry, which can be appreciated in the delivery process. In this review, different approaches that are currently used for the construction of DNA nanostructures are reported. Further, the utilization of these DNA nanostructures with the well-defined parameters for the precise control in drug delivery and gene therapy is discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

nfi-1 affects behavior and life-span in C. elegans but is not essential for DNA replication or survival

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Hirono Keiko

2005-10-01

Full Text Available Abstract Background The Nuclear Factor I (one (NFI family of transcription/replication factors plays essential roles in mammalian gene expression and development and in adenovirus DNA replication. Because of its role in viral DNA replication NFI has long been suspected to function in host DNA synthesis. Determining the requirement for NFI proteins in mammalian DNA replication is complicated by the presence of 4 NFI genes in mice and humans. Loss of individual NFI genes in mice cause defects in brain, lung and tooth development, but the presence of 4 homologous NFI genes raises the issue of redundant roles for NFI genes in DNA replication. No NFI genes are present in bacteria, fungi or plants. However single NFI genes are present in several simple animals including Drosophila and C. elegans, making it possible to test for a requirement for NFI in multicellular eukaryotic DNA replication and development. Here we assess the functions of the single nfi-1 gene in C. elegans. Results C. elegans NFI protein (CeNFI binds specifically to the same NFI-binding site recognized by vertebrate NFIs. nfi-1 encodes alternatively-spliced, maternally-inherited transcripts that are expressed at the single cell stage, during embryogenesis, and in adult muscles, neurons and gut cells. Worms lacking nfi-1 survive but have defects in movement, pharyngeal pumping and egg-laying and have a reduced life-span. Expression of the muscle gene Ce titin is decreased in nfi-1 mutant worms. Conclusion NFI gene function is not needed for survival in C. elegans and thus NFI is likely not essential for DNA replication in multi-cellular eukaryotes. The multiple defects in motility, egg-laying, pharyngeal pumping, and reduced lifespan indicate that NFI is important for these processes. Reduction in Ce titin expression could affect muscle function in multiple tissues. The phenotype of nfi-1 null worms indicates that NFI functions in multiple developmental and behavioral systems in C

DNA dynamics play a role as a basal transcription factor in the positioning and regulation of gene transcription initiation

OpenAIRE

Alexandrov, Boian S.; Gelev, Vladimir; Yoo, Sang Wook; Alexandrov, Ludmil B.; Fukuyo, Yayoi; Bishop, Alan R.; Rasmussen, Kim ?.; Usheva, Anny

2009-01-01

We assess the role of DNA breathing dynamics as a determinant of promoter strength and transcription start site (TSS) location. We compare DNA Langevin dynamic profiles of representative gene promoters, calculated with the extended non-linear PBD model of DNA with experimental data on transcription factor binding and transcriptional activity. Our results demonstrate that DNA dynamic activity at the TSS can be suppressed by mutations that do not affect basal transcription factor binding–DNA co...

Multiple controls affect arsenite oxidase gene expression in Herminiimonas arsenicoxydans

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Coppée Jean-Yves

2010-02-01

Full Text Available Abstract Background Both the speciation and toxicity of arsenic are affected by bacterial transformations, i.e. oxidation, reduction or methylation. These transformations have a major impact on environmental contamination and more particularly on arsenic contamination of drinking water. Herminiimonas arsenicoxydans has been isolated from an arsenic- contaminated environment and has developed various mechanisms for coping with arsenic, including the oxidation of As(III to As(V as a detoxification mechanism. Results In the present study, a differential transcriptome analysis was used to identify genes, including arsenite oxidase encoding genes, involved in the response of H. arsenicoxydans to As(III. To get insight into the molecular mechanisms of this enzyme activity, a Tn5 transposon mutagenesis was performed. Transposon insertions resulting in a lack of arsenite oxidase activity disrupted aoxR and aoxS genes, showing that the aox operon transcription is regulated by the AoxRS two-component system. Remarkably, transposon insertions were also identified in rpoN coding for the alternative N sigma factor (σ54 of RNA polymerase and in dnaJ coding for the Hsp70 co-chaperone. Western blotting with anti-AoxB antibodies and quantitative RT-PCR experiments allowed us to demonstrate that the rpoN and dnaJ gene products are involved in the control of arsenite oxidase gene expression. Finally, the transcriptional start site of the aoxAB operon was determined using rapid amplification of cDNA ends (RACE and a putative -12/-24 σ54-dependent promoter motif was identified upstream of aoxAB coding sequences. Conclusion These results reveal the existence of novel molecular regulatory processes governing arsenite oxidase expression in H. arsenicoxydans. These data are summarized in a model that functionally integrates arsenite oxidation in the adaptive response to As(III in this microorganism.

Whole genome DNA methylation: beyond genes silencing

OpenAIRE

Tirado-Magallanes, Roberto; Rebbani, Khadija; Lim, Ricky; Pradhan, Sriharsa; Benoukraf, Touati

2016-01-01

The combination of DNA bisulfite treatment with high-throughput sequencing technologies has enabled investigation of genome-wide DNA methylation at near base pair level resolution, far beyond that of the kilobase-long canonical CpG islands that initially revealed the biological relevance of this covalent DNA modification. The latest high-resolution studies have revealed a role for very punctual DNA methylation in chromatin plasticity, gene regulation and splicing. Here, we aim to outline the ...

Chronic exposure to microcystin-LR affected mitochondrial DNA maintenance and caused pathological changes of lung tissue in mice

International Nuclear Information System (INIS)

Li, Xinxiu; Xu, Lizhi; Zhou, Wei; Zhao, Qingya; Wang, Yaping

2016-01-01

Microcystin-LR (MC-LR), an important variant of cyanotoxin family, was frequently encountered in the contaminated aquatic environment and taken as a potent hepatotoxin. However, a little was known on the association between the long-term MC-LR exposure and lung damage. In this study, we investigated the changes of the pulmonary histopathology, mitochondrial DNA (mtDNA) integrity and the expression of mtDNA encoded genes in the mice with chronic exposed to MC-LR at different concentrations (1, 5, 10, 20 and 40 μg/L) for 12 months. Our results showed that the long-term and persistent exposure to MC-LR disturbed the balance of redox system, influenced mtDNA stability, changed the expression of mitochondrial genes in the lung cells. Notably, MC-LR exposure influenced the level of inflammatory cytokines and resulted in thickening of the alveolar septa. In conclusion, chronic exposure to MC-LR affected mtDNA maintenance, and caused lung impairment in mice. - Highlights: • A simulated natural exposure to MC-LR caused the lung pathological changes. • The chronic exposure disturbed the redox system balance of lung tissue cells. • The chronic exposure impaired the mtDNA stability and mitochondria function. • The lung was one of the vulnerable organs to MC-LR exposure in mice. - Long-term exposure to MC-LR in drinking water disturbed the balance of redox system, affected mitochondrial DNA maintenance and caused lung impairment in mice.

Effect of specific enzyme inhibitors on replication, total genome DNA repair and on gene-specific DNA repair after UV irradiation in CHO cells

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Jones, J.C.; Stevsner, Tinna; Bohr, Vilhelm A. (National Cancer Institute, NIH, Bethesda, MD (USA). Division of Cancer Treatment, Laboratory of Molecular Pharmacology); Mattern, M.R. (Smith Kline Beecham Pharmaceuticals, King of Prussia, PA (USA). Department of Biomolecular Discovery)

1991-09-01

The effects were studied of some specific enzyme inhibitors on DNA repair and replication after UV damage in Chinese hamster ovary cells. The DNA repair was studied at the level of the average, overall genome and also in the active dihydrofolate reductase gene. Replication was measured in the overall genome. The inhibitors were tested of DNA poly-merase {alpha} and {delta} (aphidicolin), of poly(ADPr) polymerase (3-aminobenzamide), of ribonucleotide reductase (hydroxyurea), of topo-isomerase I (camptothecin), and of topoisomerase II (merbarone, VP-16). In addition, the effects were tested of the potential topoisomerase I activator, {beta}-lapachone. All of these compounds inhibited genome replication and all topoisomerase inhibitors affected the overall genome repair; {beta}-lapachone stimulated it. None of these compounds had any effect on the gene-specific repair. (author). 36 refs.; 3 figs.; 2 tabs.

Genetic polymorphisms in homologous recombination repair genes in healthy Slovenian population and their influence on DNA damage

International Nuclear Information System (INIS)

Goricar, Katja; Erculj, Nina; Zadel, Maja; Dolzan, Vita

2012-01-01

Homologous recombination (HR) repair is an important mechanism involved in repairing double-strand breaks in DNA and for maintaining genomic stability. Polymorphisms in genes coding for enzymes involved in this pathway may influence the capacity for DNA repair. The aim of this study was to select tag single nucleotide polymorphisms (SNPs) in specific genes involved in HR repair, to determine their allele frequencies in a healthy Slovenian population and their influence on DNA damage detected with comet assay. In total 373 individuals were genotyped for nine tag SNPs in three genes: XRCC3 722C>T, XRCC3 -316A>G, RAD51 -98G>C, RAD51 -61G>T, RAD51 1522T>G, NBS1 553G>C, NBS1 1197A>G, NBS1 37117C>T and NBS1 3474A>C using competitive allele-specific amplification (KASPar assay). Comet assay was performed in a subgroup of 26 individuals to determine the influence of selected SNPs on DNA damage. We observed that age significantly affected genotype frequencies distribution of XRCC3 -316A>G (P = 0.039) in healthy male blood donors. XRCC3 722C>T (P = 0.005), RAD51 -61G>T (P = 0.023) and NBS1 553G>C (P = 0.008) had a statistically significant influence on DNA damage. XRCC3 722C>T, RAD51 -61G>T and NBS1 553G>C polymorphisms significantly affect the repair of damaged DNA and may be of clinical importance as they are common in Slovenian population

Quantitative DNA methylation analysis of candidate genes in cervical cancer.

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Erin M Siegel

Full Text Available Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2. A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97-1.00, p-value = 0.003. Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated.

DNA methyltransferase 3A gene polymorphism contributes to daily life stress susceptibility

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Barliana MI

2017-12-01

Full Text Available Melisa I Barliana,1,2 Shintya N Amalya,1 Ivan S Pradipta,3 Sofa D Alfian,3 Arif SW Kusuma,1,2 Tiana Milanda,1,4 Rizky Abdulah3,4 1Department of Biological Pharmacy, Biotechnology Pharmacy Laboratory, 2Pharmacy Services Development Research Center, 3Department of Pharmacology and Clinical Pharmacy, Clinical Pharmacy Laboratory, 4Center for Drug Discovery and Product Development, Faculty of Pharmacy, Universitas Padjadjaran, Jatinangor, West Java, Indonesia Abstract: Daily life stress markedly affects the response toward stressful stimuli. DNA methy­lation is one of the factors that regulate this response, and is a normal mechanism of somatic cell growth, but its regulatory gene variations may cause alterations in the stress response. The aim of the present study was to investigate genotypic variants of the DNA methyltransferase 3A (DNMT3A gene in 129 healthy subjects and evaluate its association with daily life stress. Blood samples were collected, and genomic DNA was isolated. DNA was amplified using specific tetra primers for DNMT3A (C/T rs11683424 and visualized following 2% agarose gel electrophoresis. The association of DNMT3A genetic variants with daily life stress was analyzed using the Kessler Psychological Distress Scale (K10. We observed that the distribution of subjects with genotype CC (wild type, CT (heteromutant, and TT (homomutant was 13.95%, 81.4%, and 4.65%, respectively. Genetic variations significantly affected the daily life stress condition (p=0.04 in Indonesian healthy subjects, but most of the subjects with the CT phenotype were classified in a stress condition. Keywords: daily life stressor, DNA methylation, epigenetic, Kessler Psychological Distress Scale (K10, rs11683424, DNMT3A

Expression of protein-coding genes embedded in ribosomal DNA

DEFF Research Database (Denmark)

Johansen, Steinar D; Haugen, Peik; Nielsen, Henrik

2007-01-01

Ribosomal DNA (rDNA) is a specialised chromosomal location that is dedicated to high-level transcription of ribosomal RNA genes. Interestingly, rDNAs are frequently interrupted by parasitic elements, some of which carry protein genes. These are non-LTR retrotransposons and group II introns that e...... in the nucleolus....

Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

Institute of Scientific and Technical Information of China (English)

2008-01-01

High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.

Molecular cloning and analysis of DNA repair gene from the radioresistant bacterium deinococcus radiodurans

International Nuclear Information System (INIS)

Du Zeji; Wang Mingsuo

1998-12-01

Deinococcus radiodurans (Dr) possesses a prominent ability to repair DNA injury induced by various DNA-damaging agents including mitomycin C (MC), ultraviolet light (UV) and ionizing radiation. A DNA repair mutant Dr KH3111 is a streptomycin resistant (Sm R ) derivative of KH311 which is generated by treatment with nitrosoguanidine and is sensitive to MC, 8-trimethyl-psoralen, UV and γ-ray irradiation. Gene affected by a mutation in the mutant is identified and its nucleotide sequence is determined. A complete open reading frame (ORF) which encompassed the KH3111 mutation region is found and tentatively designated as orf144b. The deduced amino acid (aa) sequence of orf144b consists of 284 aa and has no significant homology to other known proteins. The exact KH3111 mutation site is one nucleotide altered (G to A) in the sequence of orf144b in the mutant. The KH3111 mutation causes the substitution of Gly for Glu at aa position 149 of Orf144b. Survival measurements of a revertant KH3112 which was produced by transforming with DNA containing a part of the orf144b gene of KD8301 showed that the resistances to MC, UV and γ-ray in the revertant were fully restored at a level equal to the wild type. Thus, the orf144b gene required for the multiple-DNA-damaging agent resistance of Dr was designated with the name of pprA (Pleiotropic gene promoting DNA repair). This new gene can express in E. coli at very high level, and make the host E. coli resistant to MC, UV and γ-ray. The pprA gene does not express in normal Dr, but it can be induced to express by treatment with MC, UV and γ-ray. It was thought that the PprA polypeptide is a cytoplasmic protein because of the absence of characteristics found in the aa sequence of membrane proteins

DnaB gene product-independence of DNA polymerase III-directed repair synthesis in Escherichia coli K-12

International Nuclear Information System (INIS)

Billen, D.; Hellermann, G.R.

1977-01-01

An investigation has been carried out into the role of dnaB gene product in X-ray-induced repair synthesis carried out by DNA polymerase III in toluene-treated Escherichia coli K-12. A polAl polBlOO dnaB mutant deficient in both DNA polymerase I and II activities was used, and it was shown that the level of X-ray-induced, ATP-dependent, non-conservative DNA synthesis was, unlike semi-conservative DNA synthesis, unaffected by a temperature shift from 30 0 to 42 0 C. The dnaB gene product was not therefore necessary for DNA polymerase III-directed repair synthesis, which occurred in the absence of replicative synthesis. (U.K.)

Factors affecting expression of the recF gene of Escherichia coli K-12.

Science.gov (United States)

Sandler, S J; Clark, A J

1990-01-31

This report describes four factors which affect expression of the recF gene from strong upstream lambda promoters under temperature-sensitive cIAt2-encoded repressor control. The first factor was the long mRNA leader sequence consisting of the Escherichia coli dnaN gene and 95% of the dnaA gene and lambda bet, N (double amber) and 40% of the exo gene. When most of this DNA was deleted, RecF became detectable in maxicells. The second factor was the vector, pBEU28, a runaway replication plasmid. When we substituted pUC118 for pBEU28, RecF became detectable in whole cells by the Coomassie blue staining technique. The third factor was the efficiency of initiation of translation. We used site-directed mutagenesis to change the mRNA leader, ribosome-binding site and the 3 bp before and after the translational start codon. Monitoring the effect of these mutational changes by translational fusion to lacZ, we discovered that the efficiency of initiation of translation was increased 30-fold. Only an estimated two- or threefold increase in accumulated levels of RecF occurred, however. This led us to discover the fourth factor, namely sequences in the recF gene itself. These sequences reduce expression of the recF-lacZ fusion genes 100-fold. The sequences responsible for this decrease in expression occur in four regions in the N-terminal half of recF. Expression is reduced by some sequences at the transcriptional level and by others at the translational level.

Interactive effects of ultraviolet-B radiation and pesticide exposure on DNA photo-adduct accumulation and expression of DNA damage and repair genes in Xenopus laevis embryos

International Nuclear Information System (INIS)

Yu, Shuangying; Tang, Song; Mayer, Gregory D.; Cobb, George P.; Maul, Jonathan D.

2015-01-01

Highlights: • Interactive effects of UVB radiation-pesticide co-exposures were examined in frogs. • Responses included induction of DNA photo-adducts and DNA damage and repair genes. • Elevated DNA adduct levels occurred for co-exposures compared to UVB alone. • One mechanism is that pesticides may alter nuclear excision repair gene expression. - Abstract: Pesticide use and ultraviolet-B (UVB) radiation have both been suggested to adversely affect amphibians; however, little is known about their interactive effects. One potential adverse interaction could involve pesticide-induced dysregulation of DNA repair pathways, resulting in greater numbers of DNA photo-adducts from UVB exposure. In the present study, we investigated the interactive effects of UVB radiation and two common pesticides (endosulfan and α-cypermethrin) on induction of DNA photo-adducts and expression of DNA damage and repair related genes in African clawed frog (Xenopus laevis) embryos. We examined 13 genes that are, collectively, involved in stress defense, cell cycle arrest, nucleotide excision repair (NER), base excision repair, mismatch repair, DNA repair regulation, and apoptosis. We exposed X. laevis embryos to 0, 25, and 50 μg/L endosulfan or 0, 2.5, and 5.0 μg/L α-cypermethrin for 96 h, with environmentally relevant exposures of UVB radiation during the last 7 h of the 96 h exposure. We measured the amount of cyclobutane pyrimidine dimers (CPDs) and mRNA abundance of the 13 genes among treatments including control, pesticide only, UVB only, and UVB and pesticide co-exposures. Each of the co-exposure scenarios resulted in elevated CPD levels compared to UVB exposure alone, suggesting an inhibitory effect of endosulfan and α-cypermethrin on CPD repair. This is attributed to results indicating that α-cypermethrin and endosulfan reduced mRNA abundance of XPA and HR23B, respectively, to levels that may affect the initial recognition of DNA lesions. In contrast, both pesticides

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,

Identification of DNA repair genes in the human genome

International Nuclear Information System (INIS)

Hoeijmakers, J.H.J.; van Duin, M.; Westerveld, A.; Yasui, A.; Bootsma, D.

1986-01-01

To identify human DNA repair genes we have transfected human genomic DNA ligated to a dominant marker to excision repair deficient xeroderma pigmentosum (XP) and CHO cells. This resulted in the cloning of a human gene, ERCC-1, that complements the defect of a UV- and mitomycin-C sensitive CHO mutant 43-3B. The ERCC-1 gene has a size of 15 kb, consists of 10 exons and is located in the region 19q13.2-q13.3. Its primary transcript is processed into two mRNAs by alternative splicing of an internal coding exon. One of these transcripts encodes a polypeptide of 297 aminoacids. A putative DNA binding protein domain and nuclear location signal could be identified. Significant AA-homology is found between ERCC-1 and the yeast excision repair gene RAD10. 58 references, 6 figures, 1 table

When gene medication is also genetic modification--regulating DNA treatment.

Science.gov (United States)

Foss, Grethe S; Rogne, Sissel

2007-07-26

The molecular methods used in DNA vaccination and gene therapy resemble in many ways the methods applied in genetic modification of organisms. In some regulatory regimes, this creates an overlap between 'gene medication' and genetic modification. In Norway, an animal injected with plasmid DNA, in the form of DNA vaccine or gene therapy, currently is viewed as being genetically modified for as long as the added DNA is present in the animal. However, regulating a DNA-vaccinated animal as genetically modified creates both regulatory and practical challenges. It is also counter-intuitive to many biologists. Since immune responses can be elicited also to alter traits, the borderline between vaccination and the modification of properties is no longer distinct. In this paper, we discuss the background for the Norwegian interpretation and ways in which the regulatory challenge can be handled.

The application of DNA microarrays in gene expression analysis.

Science.gov (United States)

van Hal, N L; Vorst, O; van Houwelingen, A M; Kok, E J; Peijnenburg, A; Aharoni, A; van Tunen, A J; Keijer, J

2000-03-31

DNA microarray technology is a new and powerful technology that will substantially increase the speed of molecular biological research. This paper gives a survey of DNA microarray technology and its use in gene expression studies. The technical aspects and their potential improvements are discussed. These comprise array manufacturing and design, array hybridisation, scanning, and data handling. Furthermore, it is discussed how DNA microarrays can be applied in the working fields of: safety, functionality and health of food and gene discovery and pathway engineering in plants.

DNA context represents transcription regulation of the gene in mouse embryonic stem cells

Science.gov (United States)

Ha, Misook; Hong, Soondo

2016-04-01

Understanding gene regulatory information in DNA remains a significant challenge in biomedical research. This study presents a computational approach to infer gene regulatory programs from primary DNA sequences. Using DNA around transcription start sites as attributes, our model predicts gene regulation in the gene. We find that H3K27ac around TSS is an informative descriptor of the transcription program in mouse embryonic stem cells. We build a computational model inferring the cell-type-specific H3K27ac signatures in the DNA around TSS. A comparison of embryonic stem cell and liver cell-specific H3K27ac signatures in DNA shows that the H3K27ac signatures in DNA around TSS efficiently distinguish the cell-type specific H3K27ac peaks and the gene regulation. The arrangement of the H3K27ac signatures inferred from the DNA represents the transcription regulation of the gene in mESC. We show that the DNA around transcription start sites is associated with the gene regulatory program by specific interaction with H3K27ac.

Amphibian antimicrobial peptide fallaxin analogue FL9 affects virulence gene expression and DNA replication in Staphylococcus aureus

DEFF Research Database (Denmark)

Gottschalk, Sanne; Gottlieb, Caroline Trebbien; Vestergaard, Martin

2015-01-01

antimicrobials. In the present study, the analogue FL9, based on the amphibian AMP fallaxin, was studied to elucidate its mode of action and antibacterial activity against the human pathogen Staphylococcus aureus. Our data showed that FL9 may have a dual mode of action against S. aureus. At concentrations around...... and at alkaline pH, while it was compromised by acidic pH and exposure to serum. Furthermore, at subinhibitory concentrations of FL9, S. aureus responded by increasing the expression of two major virulence factor genes, namely the regulatory rnaIII and hla, encoding α-haemolysin. In addition, the S. aureus...... the MIC, FL9 bound DNA, inhibited DNA synthesis and induced the SOS DNA damage response, whereas at concentrations above the MIC the interaction between S. aureus and FL9 led to membrane disruption. The antibacterial activity of the peptide was maintained over a wide range of NaCl and MgCl2 concentrations...

Transfer of Chinese hamster DNA repair gene(s) into repair-deficient human cells (Xeroderma pigmentosum)

International Nuclear Information System (INIS)

Karentz, D.; Cleaver, J.E.

1985-01-01

Transfer of repair genes by DNA transfection into repair-deficient Xeroderma pigmentosum (XP) cells has thus far been unsuccessful, presenting an obstacle to cloning XP genes. The authors chose an indirect route to transfer repair genes in chromosome fragments. DNA repair-competent (UV resistant) hybrid cell lines were established by PEG-mediated fusions of DNA repair-deficient (UV sensitive) human fibroblasts (XP12RO) with wild type Chinese hamster (CHO) cells (AA8). CHO cells were exposed to 5 Krad X-rays prior to fusions, predisposing hybrid cells to lose CHO chromosome fragments preferentially. Repair-competent hybrids were selected by periodic exposures to UV light. Secondary and tertiary hybrid cell lines were developed by fusion of X-irradiated hybrids to XP12RO. The hybrid cell lines exhibit resistance to UV that is comparable to that of CHO cells and they are proficient at repair replication after UV exposure. Whole cell DNA-DNA hybridizations indicate that the hybrids have greater homology to CHO DNA than is evident between XP12RO and CHO. These observations indicate that CHO DNA sequences which can function in repair of UV-damaged DNA in human cells have been transferred into the genome of the repair-deficient XP12RO cells

A mechanism of gene amplification driven by small DNA fragments.

Directory of Open Access Journals (Sweden)

Kuntal Mukherjee

Full Text Available DNA amplification is a molecular process that increases the copy number of a chromosomal tract and often causes elevated expression of the amplified gene(s. Although gene amplification is frequently observed in cancer and other degenerative disorders, the molecular mechanisms involved in the process of DNA copy number increase remain largely unknown. We hypothesized that small DNA fragments could be the trigger of DNA amplification events. Following our findings that small fragments of DNA in the form of DNA oligonucleotides can be highly recombinogenic, we have developed a system in the yeast Saccharomyces cerevisiae to capture events of chromosomal DNA amplification initiated by small DNA fragments. Here we demonstrate that small DNAs can amplify a chromosomal region, generating either tandem duplications or acentric extrachromosomal DNA circles. Small fragment-driven DNA amplification (SFDA occurs with a frequency that increases with the length of homology between the small DNAs and the target chromosomal regions. SFDA events are triggered even by small single-stranded molecules with as little as 20-nt homology with the genomic target. A double-strand break (DSB external to the chromosomal amplicon region stimulates the amplification event up to a factor of 20 and favors formation of extrachromosomal circles. SFDA is dependent on Rad52 and Rad59, partially dependent on Rad1, Rad10, and Pol32, and independent of Rad51, suggesting a single-strand annealing mechanism. Our results reveal a novel molecular model for gene amplification, in which small DNA fragments drive DNA amplification and define the boundaries of the amplicon region. As DNA fragments are frequently found both inside cells and in the extracellular environment, such as the serum of patients with cancer or other degenerative disorders, we propose that SFDA may be a common mechanism for DNA amplification in cancer cells, as well as a more general cause of DNA copy number variation

PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

International Nuclear Information System (INIS)

Binkova, Blanka; Chvatalova, Irena; Lnenickova, Zdena; Milcova, Alena; Tulupova, Elena; Farmer, Peter B.; Sram, Radim J.

2007-01-01

Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22-50 years) working outdoors in the downtown area of Prague and in matched 'unexposed' controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by 32 P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32-55 μg/m 3 , PM2.5 27-38 μg/m 3 , c-PAHs 18-22 ng/m 3 ; personal exposure to c-PAHs: 9.7 ng/m 3 versus 5.8 ng/m 3 (P 8 nucleotides versus 0.82 ± 0.23 adducts/10 8 nucleotides, P = 0.065), whereas the level of the B[a]P-'like' adduct was significantly higher in exposed group (0.122 ± 0.036 adducts/10 8 nucleotides versus 0.099 ± 0.035 adducts/10 8 nucleotides, P = 0.003). A significant difference in both the total (P < 0.05) and the B[a]P-'like' DNA adducts (P < 0.01) between smokers and nonsmokers within both groups was observed. A significant positive association between DNA adduct and cotinine

Gene Transfer into the Lung by Nanoparticle Dextran-Spermine/Plasmid DNA Complexes

Directory of Open Access Journals (Sweden)

Syahril Abdullah

2010-01-01

Full Text Available A novel cationic polymer, dextran-spermine (D-SPM, has been found to mediate gene expression in a wide variety of cell lines and in vivo through systemic delivery. Here, we extended the observations by determining the optimal conditions for gene expression of D-SPM/plasmid DNA (D-SPM/pDNA in cell lines and in the lungs of BALB/c mice via instillation delivery. In vitro studies showed that D-SPM could partially protect pDNA from degradation by nuclease and exhibited optimal gene transfer efficiency at D-SPM to pDNA weight-mixing ratio of 12. In the lungs of mice, the levels of gene expression generated by D-SPM/pDNA are highly dependent on the weight-mixing ratio of D-SPM to pDNA, amount of pDNA in the complex, and the assay time postdelivery. Readministration of the complex at day 1 following the first dosing showed no significant effect on the retention and duration of gene expression. The study also showed that there was a clear trend of increasing size of the complexes as the amount of pDNA was increased, where the sizes of the D-SPM/pDNA complexes were within the nanometer range.

A multiplex branched DNA assay for parallel quantitative gene expression profiling.

Science.gov (United States)

Flagella, Michael; Bui, Son; Zheng, Zhi; Nguyen, Cung Tuong; Zhang, Aiguo; Pastor, Larry; Ma, Yunqing; Yang, Wen; Crawford, Kimberly L; McMaster, Gary K; Witney, Frank; Luo, Yuling

2006-05-01

We describe a novel method to quantitatively measure messenger RNA (mRNA) expression of multiple genes directly from crude cell lysates and tissue homogenates without the need for RNA purification or target amplification. The multiplex branched DNA (bDNA) assay adapts the bDNA technology to the Luminex fluorescent bead-based platform through the use of cooperative hybridization, which ensures an exceptionally high degree of assay specificity. Using in vitro transcribed RNA as reference standards, we demonstrated that the assay is highly specific, with cross-reactivity less than 0.2%. We also determined that the assay detection sensitivity is 25,000 RNA transcripts with intra- and interplate coefficients of variance of less than 10% and less than 15%, respectively. Using three 10-gene panels designed to measure proinflammatory and apoptosis responses, we demonstrated sensitive and specific multiplex gene expression profiling directly from cell lysates. The gene expression change data demonstrate a high correlation coefficient (R(2)=0.94) compared with measurements obtained using the single-plex bDNA assay. Thus, the multiplex bDNA assay provides a powerful means to quantify the gene expression profile of a defined set of target genes in large sample populations.

Alteration of Gene Expression, DNA Methylation, and Histone Methylation in Free Radical Scavenging Networks in Adult Mouse Hippocampus following Fetal Alcohol Exposure.

Directory of Open Access Journals (Sweden)

Eric J Chater-Diehl

Full Text Available The molecular basis of Fetal Alcohol Spectrum Disorders (FASD is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse's lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as "Free radical scavenging". We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was "Peroxisome biogenesis"; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD.

Alteration of Gene Expression, DNA Methylation, and Histone Methylation in Free Radical Scavenging Networks in Adult Mouse Hippocampus following Fetal Alcohol Exposure.

Science.gov (United States)

Chater-Diehl, Eric J; Laufer, Benjamin I; Castellani, Christina A; Alberry, Bonnie L; Singh, Shiva M

2016-01-01

The molecular basis of Fetal Alcohol Spectrum Disorders (FASD) is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse's lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as "Free radical scavenging". We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was "Peroxisome biogenesis"; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD.

Gene Therapy for Chronic HBV-Can We Eliminate cccDNA?

Science.gov (United States)

Bloom, Kristie; Maepa, Mohube Betty; Ely, Abdullah; Arbuthnot, Patrick

2018-04-12

Chronic infection with the hepatitis B virus (HBV) is a global health concern and accounts for approximately 1 million deaths annually. Amongst other limitations of current anti-HBV treatment, failure to eliminate the viral covalently closed circular DNA (cccDNA) and emergence of resistance remain the most worrisome. Viral rebound from latent episomal cccDNA reservoirs occurs following cessation of therapy, patient non-compliance, or the development of escape mutants. Simultaneous viral co-infections, such as by HIV-1, further complicate therapeutic interventions. These challenges have prompted development of novel targeted hepatitis B therapies. Given the ease with which highly specific and potent nucleic acid therapeutics can be rationally designed, gene therapy has generated interest for antiviral application. Gene therapy strategies developed for HBV include gene silencing by harnessing RNA interference, transcriptional inhibition through epigenetic modification of target DNA, genome editing by designer nucleases, and immune modulation with cytokines. DNA-binding domains and effectors based on the zinc finger (ZF), transcription activator-like effector (TALE), and clustered regularly interspaced short palindromic repeat (CRISPR) systems are remarkably well suited to targeting episomal cccDNA. This review discusses recent developments and challenges facing the field of anti-HBV gene therapy, its potential curative significance and the progress towards clinical application.

Isolation and characterization of the human uracil DNA glycosylase gene

International Nuclear Information System (INIS)

Vollberg, T.M.; Siegler, K.M.; Cool, B.L.; Sirover, M.A.

1989-01-01

A series of anti-human placental uracil DNA glycosylase monoclonal antibodies was used to screen a human placental cDNA library in phage λgt11. Twenty-seven immunopositive plaques were detected and purified. One clone containing a 1.2-kilobase (kb) human cDNA insert was chosen for further study by insertion into pUC8. The resultant recombinant plasmid selected by hybridization a human placental mRNA that encoded a 37-kDa polypeptide. This protein was immunoprecipitated specifically by an anti-human placenta uracil DNA glycosylase monoclonal antibody. RNA blot-hybridization (Northern) analysis using placental poly(A) + RNA or total RNA from four different human fibroblast cell strains revealed a single 1.6-kb transcript. Genomic blots using DNA from each cell strain digested with either EcoRI or PstI revealed a complex pattern of cDNA-hydridizing restriction fragments. The genomic analysis for each enzyme was highly similar in all four human cell strains. In contrast, a single band was observed when genomic analysis was performed with the identical DNA digests with an actin gene probe. During cell proliferation there was an increase in the level of glycosylase mRNA that paralleled the increase in uracil DNA glycosylase enzyme activity. The isolation of the human uracil DNA glycosylase gene permits an examination of the structure, organization, and expression of a human DNA repair gene

Visually Relating Gene Expression and in vivo DNA Binding Data

Energy Technology Data Exchange (ETDEWEB)

Huang, Min-Yu; Mackey, Lester; Ker?,; nen, Soile V. E.; Weber, Gunther H.; Jordan, Michael I.; Knowles, David W.; Biggin, Mark D.; Hamann, Bernd

2011-09-20

Gene expression and in vivo DNA binding data provide important information for understanding gene regulatory networks: in vivo DNA binding data indicate genomic regions where transcription factors are bound, and expression data show the output resulting from this binding. Thus, there must be functional relationships between these two types of data. While visualization and data analysis tools exist for each data type alone, there is a lack of tools that can easily explore the relationship between them. We propose an approach that uses the average expression driven by multiple of ciscontrol regions to visually relate gene expression and in vivo DNA binding data. We demonstrate the utility of this tool with examples from the network controlling early Drosophila development. The results obtained support the idea that the level of occupancy of a transcription factor on DNA strongly determines the degree to which the factor regulates a target gene, and in some cases also controls whether the regulation is positive or negative.

Sorting through the chaff, nDNA gene trees for phylogenetic inference and hybrid identification of annual sunflowers (Helianthus sect. Helianthus).

Science.gov (United States)

Moody, Michael L; Rieseberg, Loren H

2012-07-01

The annual sunflowers (Helianthus sect. Helianthus) present a formidable challenge for phylogenetic inference because of ancient hybrid speciation, recent introgression, and suspected issues with deep coalescence. Here we analyze sequence data from 11 nuclear DNA (nDNA) genes for multiple genotypes of species within the section to (1) reconstruct the phylogeny of this group, (2) explore the utility of nDNA gene trees for detecting hybrid speciation and introgression; and (3) test an empirical method of hybrid identification based on the phylogenetic congruence of nDNA gene trees from tightly linked genes. We uncovered considerable topological heterogeneity among gene trees with or without three previously identified hybrid species included in the analyses, as well as a general lack of reciprocal monophyly of species. Nonetheless, partitioned Bayesian analyses provided strong support for the reciprocal monophyly of all species except H. annuus (0.89 PP), the most widespread and abundant annual sunflower. Previous hypotheses of relationships among taxa were generally strongly supported (1.0 PP), except among taxa typically associated with H. annuus, apparently due to the paraphyly of the latter in all gene trees. While the individual nDNA gene trees provided a useful means for detecting recent hybridization, identification of ancient hybridization was problematic for all ancient hybrid species, even when linkage was considered. We discuss biological factors that affect the efficacy of phylogenetic methods for hybrid identification.

AKT phosphorylates H3-threonine 45 to facilitate termination of gene transcription in response to DNA damage

OpenAIRE

Lee, Jong-Hyuk; Kang, Byung-Hee; Jang, Hyonchol; Kim, Tae Wan; Choi, Jinmi; Kwak, Sojung; Han, Jungwon; Cho, Eun-Jung; Youn, Hong-Duk

2015-01-01

Post-translational modifications of core histones affect various cellular processes, primarily through transcription. However, their relationship with the termination of transcription has remained largely unknown. In this study, we show that DNA damage-activated AKT phosphorylates threonine 45 of core histone H3 (H3-T45). By genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis, H3-T45 phosphorylation was distributed throughout DNA damage-responsive gene loci, particularly ...

Soil DNA extraction procedure influences protist 18S rRNA gene community profiling outcome

DEFF Research Database (Denmark)

Santos, Susana S.; Nunes, Ines Marques; Nielsen, Tue K.

2017-01-01

Advances in sequencing technologies allow deeper studies of the soil protist diversity and function. However, little attention has been given to the impact of the chosen soil DNA extraction procedure to the overall results. We examined the effect of three acknowledged DNA recovery methods, two...... manual methods (ISOm-11063, GnS-GII) and one commercial kit (MoBio), on soil protist community structures obtained from different sites with different land uses. Results from 18S rRNA gene amplicon sequencing suggest that DNA extraction method significantly affect the replicate homogeneity, the total...... number of operational taxonomic units (OTUs) recovered and the overall taxonomic structure and diversity of soil protist communities. However, DNA extraction effects did not overwhelm the natural variation among samples, as the community data still strongly grouped by geographical location...

A genome-wide study of DNA methylation patterns and gene expression levels in multiple human and chimpanzee tissues.

Directory of Open Access Journals (Sweden)

Athma A Pai

2011-02-01

Full Text Available The modification of DNA by methylation is an important epigenetic mechanism that affects the spatial and temporal regulation of gene expression. Methylation patterns have been described in many contexts within and across a range of species. However, the extent to which changes in methylation might underlie inter-species differences in gene regulation, in particular between humans and other primates, has not yet been studied. To this end, we studied DNA methylation patterns in livers, hearts, and kidneys from multiple humans and chimpanzees, using tissue samples for which genome-wide gene expression data were also available. Using the multi-species gene expression and methylation data for 7,723 genes, we were able to study the role of promoter DNA methylation in the evolution of gene regulation across tissues and species. We found that inter-tissue methylation patterns are often conserved between humans and chimpanzees. However, we also found a large number of gene expression differences between species that might be explained, at least in part, by corresponding differences in methylation levels. In particular, we estimate that, in the tissues we studied, inter-species differences in promoter methylation might underlie as much as 12%-18% of differences in gene expression levels between humans and chimpanzees.

Candidate genes for cross-resistance against DNA-damaging drugs

DEFF Research Database (Denmark)

Wittig, Rainer; Nessling, Michelle; Will, Rainer D

2002-01-01

Drug resistance of tumor cells leads to major drawbacks in the treatment of cancer. To identify candidate genes for drug resistance, we compared the expression patterns of the drug-sensitive human malignant melanoma cell line MeWo and three derived sublines with acquired resistance to the DNA...... as several apoptosis-related genes, in particular STK17A and CRYAB. As MPP1 and CRYAB are also among the 14 genes differentially expressed in all three of the drug-resistant sublines, they represent the strongest candidates for resistance against DNA-damaging drugs....

Cytogenetic responses to ionizing radiation exposure of human fibroblasts with knocked-down expressions of various DNA damage signaling genes

Science.gov (United States)

Zhang, Ye; Rohde, Larry; Wu, Honglu

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have demonstrated that genes with up-regulated expression induced by IR may play important roles in DNA damage sensing, cell cycle checkpoint and chromosomal repair, the relationship between the regulation of gene expression by IR and its impact on cytogenetic responses to ionizing radiation has not been systematically studied. Here, the expression of 25 genes selected based on their transcriptional changes in response to IR or from their known DNA repair roles were individually knocked down by siRNA transfection in human fibroblast cells. Chromosome aberrations (CA) and micronuclei (MN) formation were measured as the cytogenetic endpoints. Our results showed that the yields of MN and/or CA formation were significantly increased by suppressed expression of some of the selected genes in DSB and other DNA repair pathways. Knocked-down expression of other genes showed significant impact on cell cycle progression, possibly because of severe impairment of DNA damage repair. Of these 11 genes that affected the cytogenetic response, 9 were up-regulated in the cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulating the biological consequences after IR. Failure to express these IR-responsive genes, such as by gene mutation, could seriously change the outcome of the post IR scenario and lead to carcinogenesis.

Gene conversion of ribosomal DNA in Nicotiana tabacum is associated with undermethylated, decondensed and probably active gene units.

Science.gov (United States)

Lim, K Y; Kovarik, A; Matýăsek, R; Bezdĕk, M; Lichtenstein, C P; Leitch, A R

2000-06-01

We examined the structure, intranuclear distribution and activity of ribosomal DNA (rDNA) in Nicotiana sylvestris (2n = 2x = 24) and N. tomentosiformis (2n = 2x = 24) and compared these with patterns in N. tabacum (tobacco, 2n = 4x = 48). We also examined a long-established N. tabacum culture, TBY-2. Nicotiana tabacum is an allotetraploid thought to be derived from ancestors of N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). Nicotiana sylvestris has three rDNA loci, one locus each on chromosomes 10, 11, and 12. In root-tip meristematic interphase cells, the site on chromosome 12 remains condensed and inactive, while the sites on chromosomes 10 and 11 show activity at the proximal end of the locus only. Nicotiana tomentosiformis has one major locus on chromosome 3 showing activity and a minor, inactive locus on chromosome 11. In N. tabacum cv. 095-55, there are four rDNA loci on T3, S10, S11/t and S12 (S11/t carries a small T-genome translocation). The locus on S12 remains condensed and inactive in root-tip meristematic cells while the others show activity, including decondensation at interphase and secondary constrictions at metaphase. Nicotiana tabacum DNA digested with methylcytosine-sensitive enzymes revealed a hybridisation pattern for rDNA that resembled that of N. tomentosiformis and not N. sylvestris. The data indicate that active, undermethylated genes are of the N. tomentosiformis type. Since S-genome chromosomes of N. tabacum show rDNA expression, the result indicates rDNA gene conversion of the active rDNA units on these chromosomes. Gene conversion in N. tabacum is consistent with the results of previous work. However, using primers specific for the S-genome rDNA intergenic sequences (IGS) in the polymerase chain reaction (PCR) show that rDNA gene conversion has not gone to completion in N. tabacum. Furthermore, using methylation-insensitive restriction enzymes we demonstrate that about 8% of the rDNA units remain of the N

Rearrangement of Rag-1 recombinase gene in DNA-repair deficient/immunodeficient ``wasted`` mice

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Woloschak, G.E.; Weaver, P.; Churchill, M.; Chang-Liu, C-M. [Argonne National Lab., IL (United States); Libertin, C.R. [Loyola Univ., Maywood, IL (United States)

1992-11-01

Mice recessive for the autosomal gene ``wasted`` (wst) display a disease pattern which includes increased sensitivity to the killing effects of ionizing radiation, immunodeficiency, and neurologic dysfunction. The recent cloning and characterization of recombinase genes (Rag-l/Rag-2) expressed in lymphoid and possibly central nervous system tissues prompted us to examine expression of these genes in DNA repair-deficient/immunodeficient wasted mice. Our results revealed that in thymus tissue, a small Rag-I transcript (1.0 kb) was detected in wst/wst mice that was not evident in thymus from control mice. In wst/{sm_bullet} mice, a two-fold increase in Rag-1 mRNA was evident in thymus tissue. Rag-2 mRNA could only be detected in thymus tissue from wst/{sm_bullet} and not from wst/wst or parental control BCF, mice. Southern blots revealed a rearrangement or deletion within the Rag-1 gene of affected wasted mice that was not evident in known strain-specific parental or littermate controls. These results support the idea that the Rag-1 gene may map at or near the locus for the wasted mutation. In addition, they suggest the importance of recombinase function in normal immune and central nervous system development as well as the potential contribution of this gene family to the normal repair of radiation-induced DNA damage.

Myopathic mtDNA Depletion Syndrome Due to Mutation in TK2 Gene.

Science.gov (United States)

Martín-Hernández, Elena; García-Silva, María Teresa; Quijada-Fraile, Pilar; Rodríguez-García, María Elena; Rivera, Henry; Hernández-Laín, Aurelio; Coca-Robinot, David; Fernández-Toral, Joaquín; Arenas, Joaquín; Martín, Miguel A; Martínez-Azorín, Francisco

2017-01-01

Whole-exome sequencing was used to identify the disease gene(s) in a Spanish girl with failure to thrive, muscle weakness, mild facial weakness, elevated creatine kinase, deficiency of mitochondrial complex III and depletion of mtDNA. With whole-exome sequencing data, it was possible to get the whole mtDNA sequencing and discard any pathogenic variant in this genome. The analysis of whole exome uncovered a homozygous pathogenic mutation in thymidine kinase 2 gene ( TK2; NM_004614.4:c.323 C>T, p.T108M). TK2 mutations have been identified mainly in patients with the myopathic form of mtDNA depletion syndromes. This patient presents an atypical TK2-related myopathic form of mtDNA depletion syndromes, because despite having a very low content of mtDNA (TK2 gene in mtDNA depletion syndromes and expanded the phenotypic spectrum.

SigG Does Not Control Gene Expression in Response to DNA Damage in Mycobacterium tuberculosis H37Rv ▿ §

OpenAIRE

Smollett, Katherine L.; Dawson, Lisa F.; Davis, Elaine O.

2010-01-01

Expression of the Mycobacterium tuberculosis sigG sigma factor was induced by a variety of DNA-damaging agents, but inactivation of sigG did not affect induction of gene expression or bacterial survival under these conditions. Therefore, SigG does not control the DNA repair response of M. tuberculosis H37Rv.

Sleep loss reduces the DNA-binding of BMAL1, CLOCK, and NPAS2 to specific clock genes in the mouse cerebral cortex.

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Valérie Mongrain

Full Text Available We have previously demonstrated that clock genes contribute to the homeostatic aspect of sleep regulation. Indeed, mutations in some clock genes modify the markers of sleep homeostasis and an increase in homeostatic sleep drive alters clock gene expression in the forebrain. Here, we investigate a possible mechanism by which sleep deprivation (SD could alter clock gene expression by quantifying DNA-binding of the core-clock transcription factors CLOCK, NPAS2, and BMAL1 to the cis-regulatory sequences of target clock genes in mice. Using chromatin immunoprecipitation (ChIP, we first showed that, as reported for the liver, DNA-binding of CLOCK and BMAL1 to target clock genes changes in function of time-of-day in the cerebral cortex. Tissue extracts were collected at ZT0 (light onset, -6, -12, and -18, and DNA enrichment of E-box or E'-box containing sequences was measured by qPCR. CLOCK and BMAL1 binding to Cry1, Dbp, Per1, and Per2 depended on time-of-day, with maximum values reached at around ZT6. We then observed that SD, performed between ZT0 and -6, significantly decreased DNA-binding of CLOCK and BMAL1 to Dbp, consistent with the observed decrease in Dbp mRNA levels after SD. The DNA-binding of NPAS2 and BMAL1 to Per2 was also decreased by SD, although SD is known to increase Per2 expression in the cortex. DNA-binding to Per1 and Cry1 was not affected by SD. Our results show that the sleep-wake history can affect the clock molecular machinery directly at the level of chromatin binding thereby altering the cortical expression of Dbp and Per2 and likely other targets. Although the precise dynamics of the relationship between DNA-binding and mRNA expression, especially for Per2, remains elusive, the results also suggest that part of the reported circadian changes in DNA-binding of core clock components in tissues peripheral to the suprachiasmatic nuclei could, in fact, be sleep-wake driven.

Sleep loss reduces the DNA-binding of BMAL1, CLOCK, and NPAS2 to specific clock genes in the mouse cerebral cortex.

Science.gov (United States)

Mongrain, Valérie; La Spada, Francesco; Curie, Thomas; Franken, Paul

2011-01-01

We have previously demonstrated that clock genes contribute to the homeostatic aspect of sleep regulation. Indeed, mutations in some clock genes modify the markers of sleep homeostasis and an increase in homeostatic sleep drive alters clock gene expression in the forebrain. Here, we investigate a possible mechanism by which sleep deprivation (SD) could alter clock gene expression by quantifying DNA-binding of the core-clock transcription factors CLOCK, NPAS2, and BMAL1 to the cis-regulatory sequences of target clock genes in mice. Using chromatin immunoprecipitation (ChIP), we first showed that, as reported for the liver, DNA-binding of CLOCK and BMAL1 to target clock genes changes in function of time-of-day in the cerebral cortex. Tissue extracts were collected at ZT0 (light onset), -6, -12, and -18, and DNA enrichment of E-box or E'-box containing sequences was measured by qPCR. CLOCK and BMAL1 binding to Cry1, Dbp, Per1, and Per2 depended on time-of-day, with maximum values reached at around ZT6. We then observed that SD, performed between ZT0 and -6, significantly decreased DNA-binding of CLOCK and BMAL1 to Dbp, consistent with the observed decrease in Dbp mRNA levels after SD. The DNA-binding of NPAS2 and BMAL1 to Per2 was also decreased by SD, although SD is known to increase Per2 expression in the cortex. DNA-binding to Per1 and Cry1 was not affected by SD. Our results show that the sleep-wake history can affect the clock molecular machinery directly at the level of chromatin binding thereby altering the cortical expression of Dbp and Per2 and likely other targets. Although the precise dynamics of the relationship between DNA-binding and mRNA expression, especially for Per2, remains elusive, the results also suggest that part of the reported circadian changes in DNA-binding of core clock components in tissues peripheral to the suprachiasmatic nuclei could, in fact, be sleep-wake driven.

Vitrification affects nuclear maturation and gene expression of immature human oocytes

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Abbas Shahedi

2017-02-01

Full Text Available Background: Vitrification of oocytes is a fast-freezing technique, which may affect the quality of the human oocyte, and consequently affects the embryo development, pregnancy and birth. The aim of the current study was to investigate the consequence of in-vitro vitrification on maturation status of immature human oocytes, additionally, expression levels of stress, and apoptosis related genes. Materials and Methods: The total of 213 human immature oocytes which routinely discarded from assisted reproduction clinics were collected and divided into two groups including: (I fresh germinal vesicle (GV oocytes (n=106 (matured in-vitro  (fIVM , and  (II GV oocytes (n=107 that initially vitrified, then matured in  in-vitro (vIVM. After 36 hours of incubation, the oocytes were evaluated for nuclear maturation and expression level of DNA methyltransferase (DNMT1, stress related genes (Sod1 and Hsp70, and apoptotic related genes (Bax and Bcl-2 by quantitative Real-Time PCR. Results: Oocyte maturation rates were reduced in vIVM compared to fIVM oocytes (P=0.001. The expression of stress (Sod1 and Hsp70, and apoptotic-related genes (Bax and Bcl-2 in vIVM were significantly higher compared to the fIVM group. Additionally, pro-apoptotic gene up-regulated 4.3 times more than anti-apoptotic gene in vIVM oocyte. However, DNMT1 gene expression was reduced in vIVM oocyte (P = 0.047. Conclusions: The low survival rate of vitrified In-vitro matured GV oocytes could definitely be explained by the alterations of their gene expression profile. 

PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

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Binkova, Blanka [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Chvatalova, Irena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Lnenickova, Zdena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Milcova, Alena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Tulupova, Elena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester (United Kingdom); Farmer, Peter B. [Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester (United Kingdom); Sram, Radim J. [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic)]. E-mail: sram@biomed.cas.cz

2007-07-01

Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22-50 years) working outdoors in the downtown area of Prague and in matched 'unexposed' controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by {sup 32}P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32-55 {mu}g/m{sup 3}, PM2.5 27-38 {mu}g/m{sup 3}, c-PAHs 18-22 ng/m{sup 3}; personal exposure to c-PAHs: 9.7 ng/m{sup 3} versus 5.8 ng/m{sup 3} (P < 0.01) for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92 {+-} 0.28 adducts/10{sup 8} nucleotides versus 0.82 {+-} 0.23 adducts/10{sup 8} nucleotides, P = 0.065), whereas the level of the B[a]P-'like' adduct was significantly higher in exposed group (0.122 {+-} 0.036 adducts/10{sup 8} nucleotides versus 0.099 {+-} 0.035 adducts/10{sup 8} nucleotides, P = 0

Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene

International Nuclear Information System (INIS)

Kouzarides, T.; Bankier, A.T.; Satchwell, S.C.; Weston, K.; Tomlinson, P.; Barrell, B.G.

1987-01-01

DNA sequence analysis has revealed that the gene coding for the human cytomegalovirus (HCMV) DNA polymerase is present within the long unique region of the virus genome. Identification is based on extensive amino acid homology between the predicted HCMV open reading frame HFLF2 and the DNA polymerase of herpes simplex virus type 1. The authors present here a 5280 base-pair DNA sequence containing the HCMV pol gene, along with the analysis of transcripts encoded within this region. Since HCMV pol also shows homology to the predicted Epstein-Barr virus pol, they were able to analyze the extent of homology between the DNA polymerases of three distantly related herpes viruses, HCMV, Epstein-Barr virus, and herpes simplex virus. The comparison shows that these DNA polymerases exhibit considerable amino acid homology and highlights a number of highly conserved regions; two such regions show homology to sequences within the adenovirus type 2 DNA polymerase. The HCMV pol gene is flanked by open reading frames with homology to those of other herpes viruses; upstream, there is a reading frame homologous to the glycoprotein B gene of herpes simplex virus type I and Epstein-Barr virus, and downstream there is a reading frame homologous to BFLF2 of Epstein-Barr virus

Chromosomal location of the human gene for DNA polymerase β

International Nuclear Information System (INIS)

McBride, O.W.; Zmudzka, B.Z.; Wilson, S.H.

1987-01-01

Inhibition studies indicate that DNA polymerase β has a synthetic role in DNA repair after exposure of mammalian cells to some types of DNA-damaging agents. The primary structure of the enzyme is highly conserved in vertebrates, and nearly full-length cDNAs for the enzyme were recently cloned from mammalian cDNA libraries. Southern blot analysis of DNA from a panel of human-rodent somatic cell hybrids, using portions of the cDNA as probe, indicates that the gene for human DNA polymerase β is single copy and located on the short arm or proximal long arm of chromosome 8 (8pter-8q22). A restriction fragment length polymorphism (RFLP) was detected in normal individuals by using a probe from the 5' end of the cDNA, and this RFLP probably is due to an insertion or duplication of DNA in 20-25% of the population. This restriction site can be used as one marker for chromosome 8 genetic linkage studies and for family studies of traits potentially involving this DNA repair gene

[Analysis of USH2A gene mutation in a Chinese family affected with Usher syndrome].

Science.gov (United States)

Li, Pengcheng; Liu, Fei; Zhang, Mingchang; Wang, Qiufen; Liu, Mugen

2015-08-01

To investigate the disease-causing mutation in a Chinese family affected with Usher syndrome type II. All of the 11 members from the family underwent comprehensive ophthalmologic examination and hearing test, and their genomic DNA were isolated from venous leukocytes. PCR and direct sequencing of USH2A gene were performed for the proband. Wild type and mutant type minigene vectors containing exon 42, intron 42 and exon 43 of the USH2A gene were constructed and transfected into Hela cells by lipofectamine reagent. Reverse transcription (RT)-PCR was carried out to verify the splicing of the minigenes. Pedigree analysis and clinical diagnosis indicated that the patients have suffered from autosomal recessive Usher syndrome type II. DNA sequencing has detected a homozygous c.8559-2A>G mutation of the USH2A gene in the proband, which has co-segregated with the disease in the family. The mutation has affected a conserved splice site in intron 42, which has led to inactivation of the splice site. Minigene experiment has confirmed the retaining of intron 42 in mature mRNA. The c.8559-2A>G mutation in the USH2A gene probably underlies the Usher syndrome type II in this family. The splice site mutation has resulted in abnormal splicing of USH2A pre-mRNA.

DNMT1-interacting RNAs block gene specific DNA methylation

Science.gov (United States)

Di Ruscio, Annalisa; Ebralidze, Alexander K.; Benoukraf, Touati; Amabile, Giovanni; Goff, Loyal A.; Terragni, Joylon; Figueroa, Maria Eugenia; De Figureido Pontes, Lorena Lobo; Alberich-Jorda, Meritxell; Zhang, Pu; Wu, Mengchu; D’Alò, Francesco; Melnick, Ari; Leone, Giuseppe; Ebralidze, Konstantin K.; Pradhan, Sriharsa; Rinn, John L.; Tenen, Daniel G.

2013-01-01

Summary DNA methylation was described almost a century ago. However, the rules governing its establishment and maintenance remain elusive. Here, we present data demonstrating that active transcription regulates levels of genomic methylation. We identified a novel RNA arising from the CEBPA gene locus critical in regulating the local DNA methylation profile. This RNA binds to DNMT1 and prevents CEBPA gene locus methylation. Deep sequencing of transcripts associated with DNMT1 combined with genome-scale methylation and expression profiling extended the generality of this finding to numerous gene loci. Collectively, these results delineate the nature of DNMT1-RNA interactions and suggest strategies for gene selective demethylation of therapeutic targets in disease. PMID:24107992

DNA repair synthesis dependent on the uvrA,B gene products

International Nuclear Information System (INIS)

Moses, R.E.; Moody, E.E.M.

1975-01-01

Ultraviolet irradiation of toluene-treated Escherichia coli causes an inhibition of replicative DNA synthesis. This is followed by the appearance of nonconservative DNA repair synthesis which does not require either the polymerase or 5' → 3' exonucleolytic activities of DNA polymerase I. The repair synthesis may be catalyzed by DNA polymerase III activity but does not require a functional DNA polymerase II. The ultraviolet-induced synthesis requires ATP and is dependent on a functional uvrA and uvrB gene product. However, other uvr gene products are not required for the synthesis. The recB function is also not required

To What Extent Does DNA Methylation Affect Phenotypic Variation in Cattle?

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Stephanie McKAY

2015-07-01

Full Text Available DNA methylation is an environmentally influenced epigenetic modification that regulates gene transcription and has the potential to influence variation in economically important phenotypes in agricultural species. We have utilized a novel approach to evaluate the relationship between genetic and epigenetic variation and downstream phenotypes. To begin with, we have integrated RNA-Seq and methyl binding domain sequencing (MBD-Seq data in order to determine the extent to which DNA methylation affects phenotypic variation in economically important traits of cattle. MBD-Seq is a technique that involves the sample enrichment of methylated genomic regions followed by their next-generation sequencing. This study utilized Illumina next generation sequencing technology to perform both RNA-Seq and MBD-Seq. NextGENe software (SoftGenetics, State College, PA was employed for quality trimming and aligning the sequence reads to the UMD3.1 bovine reference genome, generating counts of matched reads and methylated peak identification. Subsequently, we identified and quantified genome-wide methylated regions and characterized the extent of differential methylation and differential expression between two groups of animals with extreme phenotypes. The program edgeR from the R software package (version 3.0.1 was employed for identifying differentially methylated regions and regions of differential expression. Finally, Partial Correlation with Information Theory (PCIT was performed to identify transcripts and methylation events that exhibit differential hubbing. A differential hub is defined as a gene network hub that is more highly connected in one treatment group than the other. This analysis produced every possible pair-wise interaction that subsequently enabled us to look at network interactions of how methylation affects expression. (co-expression, co-methylation, methylation x expression. Genomic regions of interest derived from this analysis were then aligned

Variation in the DNA methylation pattern of expressed and nonexpressed genes in chicken.

Science.gov (United States)

Cooper, D N; Errington, L H; Clayton, R M

1983-01-01

Using methyl-sensitive and -insensitive restriction enzymes, Hpa II and Msp I, the methylation status of various chicken genes was examined in different tissues and developmental stages. Tissue-specific differences in methylation were found for the delta-crystallin, beta-tubulin, G3PDH, rDNA, and actin genes but not for the histone genes. Developmental decreases in methylation were noted for the delta-crystallin and actin genes in chicken kidney between embryo and adult. Since most of the sequences examined were housekeeping genes, transcriptional differences are apparently not a necessary accompaniment to changes in DNA methylation at the CpG sites examined. The only exception is sperm DNA where the delta-crystallin, beta-tubulin, and actin genes are highly methylated and almost certainly not transcribed. However the G3PDH genes are no more highly methylated in sperm than in other somatic tissues. Many sequences homologous to the rDNA and histone probes used are unmethylated in all tissues examined including sperm, but a methylated rDNA subfraction is more heavily methylated in sperm than in other tissues. We speculate as to the significance of these differences in sperm DNA methylation in the light of possible requirements for early gene activation and the probable deleterious mutagenic effects of heavy methylation within coding sequences.

Evolutionary Transition of Promoter and Gene Body DNA Methylation across Invertebrate-Vertebrate Boundary.

Science.gov (United States)

Keller, Thomas E; Han, Priscilla; Yi, Soojin V

2016-04-01

Genomes of invertebrates and vertebrates exhibit highly divergent patterns of DNA methylation. Invertebrate genomes tend to be sparsely methylated, and DNA methylation is mostly targeted to a subset of transcription units (gene bodies). In a drastic contrast, vertebrate genomes are generally globally and heavily methylated, punctuated by the limited local hypo-methylation of putative regulatory regions such as promoters. These genomic differences also translate into functional differences in DNA methylation and gene regulation. Although promoter DNA methylation is an important regulatory component of vertebrate gene expression, its role in invertebrate gene regulation has been little explored. Instead, gene body DNA methylation is associated with expression of invertebrate genes. However, the evolutionary steps leading to the differentiation of invertebrate and vertebrate genomic DNA methylation remain unresolved. Here we analyzed experimentally determined DNA methylation maps of several species across the invertebrate-vertebrate boundary, to elucidate how vertebrate gene methylation has evolved. We show that, in contrast to the prevailing idea, a substantial number of promoters in an invertebrate basal chordate Ciona intestinalis are methylated. Moreover, gene expression data indicate significant, epigenomic context-dependent associations between promoter methylation and expression in C. intestinalis. However, there is no evidence that promoter methylation in invertebrate chordate has been evolutionarily maintained across the invertebrate-vertebrate boundary. Rather, body-methylated invertebrate genes preferentially obtain hypo-methylated promoters among vertebrates. Conversely, promoter methylation is preferentially found in lineage- and tissue-specific vertebrate genes. These results provide important insights into the evolutionary origin of epigenetic regulation of vertebrate gene expression. © The Author(s) 2015. Published by Oxford University Press on behalf

Repair of DNA damage in the human metallothionein gene family

International Nuclear Information System (INIS)

Leadon, S.A.; Snowden, M.M.

1987-01-01

In order to distinguish enhanced repair of a sequence due to its transcriptional activity from enhanced repair due to chromatin alterations brought about by integration of a sequence into the genome, we have investigated the repair of damage both in endogenous genes and in cell lines that contain an integrated gene with an inducible promoter. The endogenous genes we are studying are the metallothioneins (MTs), a multigene family in man consisting of about 10-12 members. Cultured cells were exposed to 10-J/m 2 uv light and allowed to repair in the presence of bromodeoxyuridine. The DNA was then isolated, digested with Eco RI, and fully hybrid density DNA made by semiconservative synthesis was separated from unreplicated DNA by centrifugation in CsCl density gradients. Unreplicated, parental-density DNA was then reacted with a monoclonal antibody against bromouracil. 1 ref., 1 fig., 1 tab

Rearrangement of RAG-1 recombinase gene in DNA-repair deficient ``wasted`` mice

Energy Technology Data Exchange (ETDEWEB)

Woloschak, G.E.; Libertin, C.R.; Weaver, P. [Loyola Univ., Chicago, IL (United States); Churchill, M.; Chang-Liu, C.M. [Argonne National Lab., IL (United States)

1993-11-01

Mice recessive for the autosomal gene ``wasted`` wst display a disease pattern which includes increased sensitivity to the killing effects of ionizing radiation, immunodeficiency, and neurologic dysfunction. The recent cloning and characterization of recombinase genes (RAG-l/RAG-2) expressed in lymphoid and possibly central nervous system tissues prompted us to examine expression of these genes in DNA repair-deficient/immunodeficient wasted mice. Our results revealed expression of RAG-1 mRNA in spinal cord (but not brain) of control mice; no expression of RAG-1 mRNA was detected in spinal cord or brain from wst/wst mice or their normal littermates (wst/{center_dot}mice). In thymus tissue, a small RAG-1 transcript (1.0 kb) was detected in wst/wst mice that was not evident in thymus from control mice. In wst/{center_dot}mice, a two-fold increase in RAG-1 mRNA was evident in thymus tissue. RAG-2 mRNA could only be detected in thymus tissue from wst/{center_dot} and not from wst/wst or parental control BCF{sub 1} mice. Southern blots revealed a rearrangement/deletion within the RAG-1 gene of affected wasted mice, not evident in known strain-specific parental or littermate controls. These results support the idea that the RAG-1 gene may map at or near the locus for the wasted mutation. In addition, they suggest the importance of recombinase function in normal immune and central nervous system development as well as the potential contribution of this gene family to the normal repair of radiation-induced DNA damage.

MethylMix 2.0: an R package for identifying DNA methylation genes.

Science.gov (United States)

Cedoz, Pierre-Louis; Prunello, Marcos; Brennan, Kevin; Gevaert, Olivier

2018-04-14

DNA methylation is an important mechanism regulating gene transcription, and its role in carcinogenesis has been extensively studied. Hyper and hypomethylation of genes is a major mechanism of gene expression deregulation in a wide range of diseases. At the same time, high-throughput DNA methylation assays have been developed generating vast amounts of genome wide DNA methylation measurements. We developed MethylMix, an algorithm implemented in R to identify disease specific hyper and hypomethylated genes. Here we present a new version of MethylMix that automates the construction of DNA-methylation and gene expression datasets from The Cancer Genome Atlas (TCGA). More precisely, MethylMix 2.0 incorporates two major updates: the automated downloading of DNA methylation and gene expression datasets from TCGA and the automated preprocessing of such datasets: value imputation, batch correction and CpG sites clustering within each gene. The resulting datasets can subsequently be analyzed with MethylMix to identify transcriptionally predictive methylation states. We show that the Differential Methylation Values created by MethylMix can be used for cancer subtyping. olivier.gevaert@stanford.edu. https://bioconductor.org/packages/release/bioc/manuals/MethylMix/man/MethylMix.pdf. MethylMix 2.0 was implemented as an R package and is available in bioconductor.

Differences in the ovine HSP90AA1 gene expression rates caused by two linked polymorphisms at its promoter affect rams sperm DNA fragmentation under environmental heat stress conditions.

Science.gov (United States)

Salces-Ortiz, Judit; Ramón, Manuel; González, Carmen; Pérez-Guzmán, M Dolores; Garde, J Julián; García-Álvarez, Olga; Maroto-Morales, Alejandro; Calvo, Jorge H; Serrano, M Magdalena

2015-01-01

Heat shock (HS) is one of the best-studied exogenous cellular stresses. Almost all tissues, cell types, metabolic pathways and biochemical reactions are affected in greater or lesser extent by HS. However, there are some especially thermo sensible cellular types such as the mammalian male germ cells. The present study examined the role of three INDELs in conjunction with the -660G/C polymorphism located at the HSP90AA1 promoter region over the gene expression rate under HS. Specially, the -668insC INDEL, which is very close to the -660G/C transversion, is a good candidate to be implied in the transcriptional regulation of the gene by itself or in a cooperative way with this SNP. Animals carrying the genotype II-668 showed higher transcription rates than those with ID-668 (FC = 3.07) and DD-668 (FC = 3.40) genotypes for samples collected under HS. A linkage between gene expression and sperm DNA fragmentation was also found. When HS conditions were present along or in some stages of the spermatogenesis, alternative genotypes of the -668insC and -660G/C mutations are involved in the effect of HS over sperm DNA fragmentation. Thus, unfavorable genotypes in terms of gene expression induction (ID-668GC-660 and DD-668GG-660) do not produce enough mRNA (stored as messenger ribonucleoprotein particles) and Hsp90α protein to cope with future thermal stress which might occur in posterior stages when transcriptional activity is reduced and cell types and molecular processes are more sensible to heat (spermatocytes in pachytene and spermatids protamination). This would result in the impairment of DNA packaging and the consequent commitment of the events occurring shortly after fertilization and during embryonic development. In the short-term, the assessment of the relationship between sperm DNA fragmentation sensitivity and ram's fertility will be of interest to a better understanding of the mechanisms of response to HS and its consequences on animal production and

Application of DNA Machineries for the Barcode Patterned Detection of Genes or Proteins.

Science.gov (United States)

Zhou, Zhixin; Luo, Guofeng; Wulf, Verena; Willner, Itamar

2018-06-05

The study introduces an analytical platform for the detection of genes or aptamer-ligand complexes by nucleic acid barcode patterns generated by DNA machineries. The DNA machineries consist of nucleic acid scaffolds that include specific recognition sites for the different genes or aptamer-ligand analytes. The binding of the analytes to the scaffolds initiate, in the presence of the nucleotide mixture, a cyclic polymerization/nicking machinery that yields displaced strands of variable lengths. The electrophoretic separation of the resulting strands provides barcode patterns for the specific detection of the different analytes. Mixtures of DNA machineries that yield, upon sensing of different genes (or aptamer ligands), one-, two-, or three-band barcode patterns are described. The combination of nucleic acid scaffolds acting, in the presence of polymerase/nicking enzyme and nucleotide mixture, as DNA machineries, that generate multiband barcode patterns provide an analytical platform for the detection of an individual gene out of many possible genes. The diversity of genes (or other analytes) that can be analyzed by the DNA machineries and the barcode patterned imaging is given by the Pascal's triangle. As a proof-of-concept, the detection of one of six genes, that is, TP53, Werner syndrome, Tay-Sachs normal gene, BRCA1, Tay-Sachs mutant gene, and cystic fibrosis disorder gene by six two-band barcode patterns is demonstrated. The advantages and limitations of the detection of analytes by polymerase/nicking DNA machineries that yield barcode patterns as imaging readout signals are discussed.

Germline stem cell gene PIWIL2 mediates DNA repair through relaxation of chromatin.

Directory of Open Access Journals (Sweden)

De-Tao Yin

Full Text Available DNA damage response (DDR is an intrinsic barrier of cell to tumorigenesis initiated by genotoxic agents. However, the mechanisms underlying the DDR are not completely understood despite of extensive investigation. Recently, we have reported that ectopic expression of germline stem cell gene PIWIL2 is associated with tumor stem cell development, although the underlying mechanisms are largely unknown. Here we show that PIWIL2 is required for the repair of DNA-damage induced by various types of genotoxic agents. Upon ultraviolet (UV irradiation, silenced PIWIL2 gene in normal human fibroblasts was transiently activated after treatment with UV light. This activation was associated with DNA repair, because Piwil2-deficienct mouse embryonic fibroblasts (mili(-/- MEFs were defective in cyclobutane pyrimidine dimers (CPD repair after UV treatment. As a result, the UV-treated mili(-/- MEFs were more susceptible to apoptosis, as characterized by increased levels of DNA damage-associated apoptotic proteins, such as active caspase-3, cleaved Poly (ADP-ribose polymerase (PARP and Bik. The impaired DNA repair in the mili(-/- MEFs was associated with the reductions of histone H3 acetylation and chromatin relaxation, although the DDR pathway downstream chromatin relaxation appeared not to be directly affected by Piwil2. Moreover, guanine-guanine (Pt-[GG] and double strand break (DSB repair were also defective in the mili(-/- MEFs treated by genotoxic chemicals Cisplatin and ionizing radiation (IR, respectively. The results indicate that Piwil2 can mediate DNA repair through an axis of Piwil2 → histone acetylation → chromatin relaxation upstream DDR pathways. The findings reveal a new role for Piwil2 in DNA repair and suggest that Piwil2 may act as a gatekeeper against DNA damage-mediated tumorigenesis.

GeneBreak: detection of recurrent DNA copy number aberration-associated chromosomal breakpoints within genes [version 2; referees: 2 approved

Directory of Open Access Journals (Sweden)

Evert van den Broek

2017-07-01

Full Text Available Development of cancer is driven by somatic alterations, including numerical and structural chromosomal aberrations. Currently, several computational methods are available and are widely applied to detect numerical copy number aberrations (CNAs of chromosomal segments in tumor genomes. However, there is lack of computational methods that systematically detect structural chromosomal aberrations by virtue of the genomic location of CNA-associated chromosomal breaks and identify genes that appear non-randomly affected by chromosomal breakpoints across (large series of tumor samples. ‘GeneBreak’ is developed to systematically identify genes recurrently affected by the genomic location of chromosomal CNA-associated breaks by a genome-wide approach, which can be applied to DNA copy number data obtained by array-Comparative Genomic Hybridization (CGH or by (low-pass whole genome sequencing (WGS. First, ‘GeneBreak’ collects the genomic locations of chromosomal CNA-associated breaks that were previously pinpointed by the segmentation algorithm that was applied to obtain CNA profiles. Next, a tailored annotation approach for breakpoint-to-gene mapping is implemented. Finally, dedicated cohort-based statistics is incorporated with correction for covariates that influence the probability to be a breakpoint gene. In addition, multiple testing correction is integrated to reveal recurrent breakpoint events. This easy-to-use algorithm, ‘GeneBreak’, is implemented in R (www.cran.r-project.org and is available from Bioconductor (www.bioconductor.org/packages/release/bioc/html/GeneBreak.html.

Uropathogenic Escherichia coli virulence genes: invaluable approaches for designing DNA microarray probes.

Science.gov (United States)

Jahandeh, Nadia; Ranjbar, Reza; Behzadi, Payam; Behzadi, Elham

2015-01-01

The pathotypes of uropathogenic Escherichia coli (UPEC) cause different types of urinary tract infections (UTIs). The presence of a wide range of virulence genes in UPEC enables us to design appropriate DNA microarray probes. These probes, which are used in DNA microarray technology, provide us with an accurate and rapid diagnosis and definitive treatment in association with UTIs caused by UPEC pathotypes. The main goal of this article is to introduce the UPEC virulence genes as invaluable approaches for designing DNA microarray probes. Main search engines such as Google Scholar and databases like NCBI were searched to find and study several original pieces of literature, review articles, and DNA gene sequences. In parallel with in silico studies, the experiences of the authors were helpful for selecting appropriate sources and writing this review article. There is a significant variety of virulence genes among UPEC strains. The DNA sequences of virulence genes are fabulous patterns for designing microarray probes. The location of virulence genes and their sequence lengths influence the quality of probes. The use of selected virulence genes for designing microarray probes gives us a wide range of choices from which the best probe candidates can be chosen. DNA microarray technology provides us with an accurate, rapid, cost-effective, sensitive, and specific molecular diagnostic method which is facilitated by designing microarray probes. Via these tools, we are able to have an accurate diagnosis and a definitive treatment regarding UTIs caused by UPEC pathotypes.

The gene expressions of DNA methylation/demethylation enzymes ...

African Journals Online (AJOL)

A decrease in mRNA levels for cytochrome c oxidase (COX) subunits was observed in skeletal muscle of hypothyroid rats. However, the precise expression mechanisms of the related genes in hypothyroid state still remain unclear. This study investigated gene expressions of DNA methyltransferases (Dnmts), DNA ...

Maternal high-fat diet associated with altered gene expression, DNA methylation, and obesity risk in mouse offspring

Science.gov (United States)

Zaidi, Rabab; Shah, Shyam; Oakley, M. Elsa; Pavlatos, Cassondra; El Idrissi, Samir; Xing, Xiaoyun; Li, Daofeng; Wang, Ting; Cheverud, James M.

2018-01-01

We investigated maternal obesity in inbred SM/J mice by assigning females to a high-fat diet or a low-fat diet at weaning, mating them to low-fat-fed males, cross-fostering the offspring to low-fat-fed SM/J nurses at birth, and weaning the offspring onto a high-fat or low-fat diet. A maternal high-fat diet exacerbated obesity in the high-fat-fed daughters, causing them to weigh more, have more fat, and have higher serum levels of leptin as adults, accompanied by dozens of gene expression changes and thousands of DNA methylation changes in their livers and hearts. Maternal diet particularly affected genes involved in RNA processing, immune response, and mitochondria. Between one-quarter and one-third of differentially expressed genes contained a differentially methylated region associated with maternal diet. An offspring high-fat diet reduced overall variation in DNA methylation, increased body weight and organ weights, increased long bone lengths and weights, decreased insulin sensitivity, and changed the expression of 3,908 genes in the liver. Although the offspring were more affected by their own diet, their maternal diet had epigenetic effects lasting through adulthood, and in the daughters these effects were accompanied by phenotypic changes relevant to obesity and diabetes. PMID:29447215

Maternal high-fat diet associated with altered gene expression, DNA methylation, and obesity risk in mouse offspring.

Science.gov (United States)

Keleher, Madeline Rose; Zaidi, Rabab; Shah, Shyam; Oakley, M Elsa; Pavlatos, Cassondra; El Idrissi, Samir; Xing, Xiaoyun; Li, Daofeng; Wang, Ting; Cheverud, James M

2018-01-01

We investigated maternal obesity in inbred SM/J mice by assigning females to a high-fat diet or a low-fat diet at weaning, mating them to low-fat-fed males, cross-fostering the offspring to low-fat-fed SM/J nurses at birth, and weaning the offspring onto a high-fat or low-fat diet. A maternal high-fat diet exacerbated obesity in the high-fat-fed daughters, causing them to weigh more, have more fat, and have higher serum levels of leptin as adults, accompanied by dozens of gene expression changes and thousands of DNA methylation changes in their livers and hearts. Maternal diet particularly affected genes involved in RNA processing, immune response, and mitochondria. Between one-quarter and one-third of differentially expressed genes contained a differentially methylated region associated with maternal diet. An offspring high-fat diet reduced overall variation in DNA methylation, increased body weight and organ weights, increased long bone lengths and weights, decreased insulin sensitivity, and changed the expression of 3,908 genes in the liver. Although the offspring were more affected by their own diet, their maternal diet had epigenetic effects lasting through adulthood, and in the daughters these effects were accompanied by phenotypic changes relevant to obesity and diabetes.

The gene expressions of DNA methylation/demethylation enzymes ...

African Journals Online (AJOL)

user

2011-01-31

Jan 31, 2011 ... A decrease in mRNA levels for cytochrome c oxidase (COX) subunits was observed in skeletal muscle of hypothyroid rats. However, the precise expression mechanisms of the related genes in hypothyroid state still remain unclear. This study investigated gene expressions of DNA methyltransferases.

GenePublisher: automated analysis of DNA microarray data

DEFF Research Database (Denmark)

Knudsen, Steen; Workman, Christopher; Sicheritz-Ponten, T.

2003-01-01

GenePublisher, a system for automatic analysis of data from DNA microarray experiments, has been implemented with a web interface at http://www.cbs.dtu.dk/services/GenePublisher. Raw data are uploaded to the server together with aspecification of the data. The server performs normalization...

Complete gene expression profiling of Saccharopolyspora erythraea using GeneChip DNA microarrays

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Bordoni Roberta

2007-11-01

Full Text Available Abstract Background The Saccharopolyspora erythraea genome sequence, recently published, presents considerable divergence from those of streptomycetes in gene organization and function, confirming the remarkable potential of S. erythraea for producing many other secondary metabolites in addition to erythromycin. In order to investigate, at whole transcriptome level, how S. erythraea genes are modulated, a DNA microarray was specifically designed and constructed on the S. erythraea strain NRRL 2338 genome sequence, and the expression profiles of 6494 ORFs were monitored during growth in complex liquid medium. Results The transcriptional analysis identified a set of 404 genes, whose transcriptional signals vary during growth and characterize three distinct phases: a rapid growth until 32 h (Phase A; a growth slowdown until 52 h (Phase B; and another rapid growth phase from 56 h to 72 h (Phase C before the cells enter the stationary phase. A non-parametric statistical method, that identifies chromosomal regions with transcriptional imbalances, determined regional organization of transcription along the chromosome, highlighting differences between core and non-core regions, and strand specific patterns of expression. Microarray data were used to characterize the temporal behaviour of major functional classes and of all the gene clusters for secondary metabolism. The results confirmed that the ery cluster is up-regulated during Phase A and identified six additional clusters (for terpenes and non-ribosomal peptides that are clearly regulated in later phases. Conclusion The use of a S. erythraea DNA microarray improved specificity and sensitivity of gene expression analysis, allowing a global and at the same time detailed picture of how S. erythraea genes are modulated. This work underlines the importance of using DNA microarrays, coupled with an exhaustive statistical and bioinformatic analysis of the results, to understand the transcriptional

DNA repair and cancer

International Nuclear Information System (INIS)

Rathore, Shakuntla; Joshi, Pankaj Kumar; Gaur, Sudha

2012-01-01

DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecule that encode it's genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many one million individual molecular lesions per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions include potentially harmful mutation in cell's genome which affect the survival of it's daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. Inherited mutation that affect DNA repair genes are strongly associated with high cancer risks in humans. Hereditary non polyposis colorectal cancer (HNPCC) is strongly associated with specific mutation in the DNA mismatch repair pathway. BRCA1, BRCA2 two famous mutation conferring a hugely increased risk of breast cancer on carrier, are both associated with a large number of DNA repair pathway, especially NHEJ and homologous recombination. Cancer therapy procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell to repair DNA damage, resulting in cell death. Cells that are most rapidly dividing most typically cancer cells are preferentially affected. The side effect is that other non-cancerous but rapidly dividing cells such as stem cells in the bone marrow are also affected. Modern cancer treatment attempt to localize the DNA damage to cells and tissue only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor) or by biochemical means (exploiting a feature unique to cancer cells in the body). (author)

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.

A Genome-Wide mQTL Analysis in Human Adipose Tissue Identifies Genetic Variants Associated with DNA Methylation, Gene Expression and Metabolic Traits

DEFF Research Database (Denmark)

Volkov, Petr; Olsson, Anders H; Gillberg, Linn

2016-01-01

Little is known about the extent to which interactions between genetics and epigenetics may affect the risk of complex metabolic diseases and/or their intermediary phenotypes. We performed a genome-wide DNA methylation quantitative trait locus (mQTL) analysis in human adipose tissue of 119 men, w...... and epigenetic variation in both cis and trans positions influencing gene expression in adipose tissue and in vivo (dys)metabolic traits associated with the development of obesity and diabetes.......Little is known about the extent to which interactions between genetics and epigenetics may affect the risk of complex metabolic diseases and/or their intermediary phenotypes. We performed a genome-wide DNA methylation quantitative trait locus (mQTL) analysis in human adipose tissue of 119 men......, where 592,794 single nucleotide polymorphisms (SNPs) were related to DNA methylation of 477,891 CpG sites, covering 99% of RefSeq genes. SNPs in significant mQTLs were further related to gene expression in adipose tissue and obesity related traits. We found 101,911 SNP-CpG pairs (mQTLs) in cis and 5...

Decreased expression level of BER genes in Alzheimer's disease patients is not derivative of their DNA methylation status.

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Sliwinska, Agnieszka; Sitarek, Przemysław; Toma, Monika; Czarny, Piotr; Synowiec, Ewelina; Krupa, Renata; Wigner, Paulina; Bialek, Katarzyna; Kwiatkowski, Dominik; Korycinska, Anna; Majsterek, Ireneusz; Szemraj, Janusz; Galecki, Piotr; Sliwinski, Tomasz

2017-10-03

Neurodegeneration in Alzheimer's disease can be caused by accumulation of oxidative DNA damage resulting from altered expression of genes involved in the base excision repair system (BER). Promoter methylation can affect the profile of BER genes expression. Decreased expression of BER genes was observed in the brains of AD patients. The aim of our study was to compare the expression and methylation profiles of six genes coding for proteins involved in BER, namely: hOGG1, APE1, MUTYH, NEIL1, PARP1 and XRCC1, in the peripheral blood cells of AD patients and healthy volunteers. The study consisted of 100 persons diagnosed with Alzheimer's disease according to DSM-IV criteria, and 110 healthy volunteers. DNA and total RNA were isolated from venous blood cells. Promoter methylation profiles were obtained by High Resolution Melting (HRM) analysis of bisulfide converted DNA samples. Real-time PCR with TaqMan probes was employed for gene expression analysis. APE1, hOGG1, MUTYH, PARP1 and NEIL1 were significantly (pgenes. The methylation status of promoters is not associated with downregulation of BER genes. Our results show that downregulation of BER genes detected in peripheral blood samples could reflect the changes occurring in the brain of patients with AD, and may be a useful biomarker of this disease. Copyright © 2017 Elsevier Inc. All rights reserved.

Atypical DNA methylation of genes encoding cysteine-rich peptides in Arabidopsis thaliana

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You Wanhui

2012-04-01

Full Text Available Abstract Background In plants, transposons and non-protein-coding repeats are epigenetically silenced by CG and non-CG methylation. This pattern of methylation is mediated in part by small RNAs and two specialized RNA polymerases, termed Pol IV and Pol V, in a process called RNA-directed DNA methylation. By contrast, many protein-coding genes transcribed by Pol II contain in their gene bodies exclusively CG methylation that is independent of small RNAs and Pol IV/Pol V activities. It is unclear how the different methylation machineries distinguish between transposons and genes. Here we report on a group of atypical genes that display in their coding region a transposon-like methylation pattern, which is associated with gene silencing in sporophytic tissues. Results We performed a methylation-sensitive amplification polymorphism analysis to search for targets of RNA-directed DNA methylation in Arabidopsis thaliana and identified several members of a gene family encoding cysteine-rich peptides (CRPs. In leaves, the CRP genes are silent and their coding regions contain dense, transposon-like methylation in CG, CHG and CHH contexts, which depends partly on the Pol IV/Pol V pathway and small RNAs. Methylation in the coding region is reduced, however, in the synergid cells of the female gametophyte, where the CRP genes are specifically expressed. Further demonstrating that expressed CRP genes lack gene body methylation, a CRP4-GFP fusion gene under the control of the constitutive 35 S promoter remains unmethylated in leaves and is transcribed to produce a translatable mRNA. By contrast, a CRP4-GFP fusion gene under the control of a CRP4 promoter fragment acquires CG and non-CG methylation in the CRP coding region in leaves similar to the silent endogenous CRP4 gene. Conclusions Unlike CG methylation in gene bodies, which does not dramatically affect Pol II transcription, combined CG and non-CG methylation in CRP coding regions is likely to

Hepatitis B virus DNA integration and transactivation of cellular genes

Directory of Open Access Journals (Sweden)

Vijay Kumar

2007-02-01

Full Text Available

Chronic hepatitis B virus (HBV infection is etiologically related to human hepatocellular carcinoma (HCC. Most HCCs contain integrated HBV DNA in hepatocyte, suggesting that the integration may be involved in carcinogenesis. Available data on the integrants from human hepatocellular carcinomas seem to represent primary integrants as well as the products of secondary rearrangements. By means of structural analyses of the possible primary integrants, it has been observed that the replication intermediates of the viral genome are the preferred substrates for integration. The integrated HBV DNA and the target cellular DNA are invariably associated with deletions, possibly reflecting the substrate for, and the mechanism of, the integration reaction. The host DNA sequences as well as the target site of integration in chromosomes are selected randomly suggesting that HBV DNA integration should bring about random mutagenic effects. Analysis of the samples recovered from hepatocellular carcinomas show that the integrated HBV DNA can mediate secondary rearrangements of chromosomes, such as translocations, inversions, deletions and (possibly amplifications. The integration of HBV DNA into the host genome occurs at early steps of clonal tumor expansion. The integration has been shown in a number of cases to affect a variety of cancer-related genes and to exert insertional mutagenesis. However, in contrast to the woodchuck model, in which specific HBV-DNA integration is detectable in most cases, insertional activation or inactivation of cellular genes appears to be a rare event in man. The discovery of transactivating functions exerted by HBx and truncated HBs(urface proteins supports the notion that these could be relevant to hepatocarcinogenesis as these transactivator sequences have been found in a large number of HCC tumors or hepatoma-derived cell lines. The HBx

Conidiogenesis-related DNA photolyase gene in Beauveria bassiana.

Science.gov (United States)

Lee, Se Jin; Lee, Mi Rong; Kim, Sihyeon; Kim, Jong Cheol; Park, So Eun; Shin, Tae Young; Kim, Jae Su

2018-03-01

Beauveria bassiana is an entomopathogenic fungi used in environmentally mindful pest management. Its main active ingredient, conidia, is commercially available as a fungal biopesticide. Many studies of conidia production have focused on how to optimize culture conditions for maximum productivity and stability against unfavorable abiotic factors. However, understanding of how conidiogenesis-related genes provide improved conidial production remains unclear. In this study, we focus on identifying conidiogenesis-related genes in B. bassiana ERL1170 using a random mutagenesis technique. Transformation of ERL1170 using restriction enzyme-mediated integration generated one morphologically different transformant, ERL1170-pABeG #163. The transformant was confirmed to represent B. bassiana, and the binary vector was successfully integrated into the genome of ERL1170. Compared to the wild type, transformant #163 showed very slow hyphal growth and within 6 days only produced bassiana exhibits thread-like hyphae and conidiophore structures and circular conidia. To determine the location of the randomly inserted DNA, we conducted thermal asymmetric interlaced (TAIL) PCR and Escherichia coli cloning to clearly sequence the disrupted region. We identified one colony (colony No. 7) with an insertion site identified as DNA photolyase. This was confirmed through a gene knock-out study. It is possible the gene that encodes for DNA photolyase was disrupted during the insertion process and might be involved in fungal conidiogenesis. This work serves as a platform for exploring the function of a variety of B. bassiana genes involved in pest management and their downstream processing. Copyright © 2018 Elsevier Inc. All rights reserved.

The application of DNA microarrays in gene expression analysis

NARCIS (Netherlands)

Hal, van N.L.W.; Vorst, O.; Houwelingen, van A.M.M.L.; Kok, E.J.; Peijnenburg, A.A.C.M.; Aharoni, A.; Tunen, van A.J.; Keijer, J.

2000-01-01

DNA microarray technology is a new and powerful technology that will substantially increase the speed of molecular biological research. This paper gives a survey of DNA microarray technology and its use in gene expression studies. The technical aspects and their potential improvements are discussed.

DNA-transporting nanoparticles : design and in vitro evaluation of DNA and formulation for non-viral gene delivery

NARCIS (Netherlands)

van Gaal, E.V.B.

2010-01-01

The aim of gene therapy is to treat, cure or prevent a disease by replacing defective genes, introducing new genes or changing the expression of a person’s genes. Success of gene therapy is dependent on successful delivery of DNA from the site of administration into cell nuclei. Naturally occurring

Optimizing hyaluronidase dose and plasmid DNA delivery greatly improves gene electrotransfer efficiency in rat skeletal muscle

DEFF Research Database (Denmark)

Åkerström, Thorbjörn; Vedel, Kenneth; Needham Andersen, Josefine

2015-01-01

Transfection of rat skeletal muscle in vivo is a widely used research model. However, gene electrotransfer protocols have been developed for mice and yield variable results in rats. We investigated whether changes in hyaluronidase pre-treatment and plasmid DNA delivery can improve transfection...... with a homogenous distribution. We also show that transfection was stable over five weeks of regular exercise or inactivity. Our findings show that species-specific plasmid DNA delivery and hyaluronidase pre-treatment greatly improves transfection efficiency in rat skeletal muscle....... efficiency in rat skeletal muscle. We found that pre-treating the muscle with a hyaluronidase dose suitable for rats (0.56. U/g b.w.) prior to plasmid DNA injection increased transfection efficiency by >200% whereas timing of the pre-treatment did not affect efficiency. Uniformly distributing plasmid DNA...

Chitosan-Graft-Polyethylenimine/DNA Nanoparticles as Novel Non-Viral Gene Delivery Vectors Targeting Osteoarthritis

Science.gov (United States)

Lv, Lulu; Zhao, Huiqing

2014-01-01

The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes. PMID:24392152

Tagging of blast resistance gene(s) to DNA markers and marker-assisted selection (MAS) in rice improvement

International Nuclear Information System (INIS)

Zhuang, J.Y.; Lu, J.; Qian, H.R.; Lin, H.X.; Zheng, K.L.

1998-01-01

This paper reports progress made on the tagging of blast resistance gene(s) to DNA markers and on the initiation of marker-assisted selection (MAS) for blast resistance in rice improvement. A pair of near isogenic lines, K8OR and K79S, were developed using a Chinese landrace Hong-jiao-zhan as the resistance donor. Ten putatively positive markers were identified by screening 177 mapped DNA markers. Using the F 2 population of 143 plants and the derived F 3 lines, three Restriction Fragment Length Polymorphism (RFLP) markers (RG81, RG869 and RZ397) on chromosome 12 of rice were identified to be closely linked to the blast resistance gene Pi-12(t). The genetic distance between Pi-12(t) and the closest marker RG869 was 5.1 cM. By employing the bulk segregant analysis (BSA) procedure, six of 199 arbitrary primers were found to produce positive Randomly Amplified Polymorphic DNA (RAPD) bands. Tight linkage between Pi-12(t) and three RAPD bands, each from a different primer, was confirmed after amplification of DNA of all F 2 individuals. Two fragments were cloned and sequenced, and two sequence characterised amplified re-ion (SCAR) markers were established. In two other F 3 populations, Xian-feng I/Tetep and Xian-feng, 1/Hong-jiao-zhan, the blast resistance was found to be controlled by interactions of two or more genes. One resistance gene was located in the vicinity of RG81 in both populations. Work to identify other gene(s) is currently under way. Marker assisted selection for blast resistance was initiated. Crosses were made between elite varieties and blast resistance donors to develop populations for DNA marker-assisted selection of blast resistance. In addition, 48 varieties widely used in current rice breeding programs were provided by rice breeders. DNA marker-based polymorphism among, these varieties and resistance donors were analysed to produce a database for future MAS program. (author)

[Analysis of SOX10 gene mutation in a family affected with Waardenburg syndrome type II].

Science.gov (United States)

Zheng, Lei; Yan, Yousheng; Chen, Xue; Zhang, Chuan; Zhang, Qinghua; Feng, Xuan; Hao, Shen

2018-02-10

OBJECTIVE To detect potential mutation of SOX10 gene in a pedigree affected with Warrdenburg syndrome type II. METHODS Genomic DNA was extracted from peripheral blood samples of the proband and his family members. Exons and flanking sequences of MITF, PAX3, SOX10, SNAI2, END3 and ENDRB genes were analyzed by chip capturing and high throughput sequencing. Suspected mutations were verified with Sanger sequencing. RESULTS A c.127C>T (p.R43X) mutation of the SOX10 gene was detected in the proband, for which both parents showed a wild-type genotype. CONCLUSION The c.127C>T (p.R43X) mutation of SOX10 gene probably underlies the ocular symptoms and hearing loss of the proband.

Non-electrostatic complexes with DNA: towards novel synthetic gene delivery systems.

Science.gov (United States)

Soto, J; Bessodes, M; Pitard, B; Mailhe, P; Scherman, D; Byk, G

2000-05-01

We have developed new DNA complexing amphiphile based on Hoechst 33258 interaction with DNA grooves. The synthesis and physicochemical characterisation of lipid/DNA complexes, as compared to that of classical lipopolyamine for gene delivery, are described and discussed.

Mutations affecting RNA polymerase I-stimulated exchange and rDNA recombination in yeast

International Nuclear Information System (INIS)

Lin, Y.H.; Keil, R.L.

1991-01-01

HOT1 is a cis-acting recombination-stimulatory sequence isolated from the rDNA repeat unit of yeast. The ability of HOT1 to stimulate mitotic exchange appears to depend on its ability to promote high levels of RNA polymerase I transcription. A qualitative colony color sectoring assay was developed to screen for trans-acting mutations that alter the activity of HOT1. Both hypo-recombination and hyper-recombination mutants were isolated. Genetic analysis of seven HOT1 recombination mutants (hrm) that decrease HOT1 activity shows that they behave as recessive nuclear mutations and belong to five linkage groups. Three of these mutations, hrm1, hrm2, and hrm3, also decrease rDNA exchange but do not alter recombination in the absence of HOT1. Another mutation, hrm4, decreases HOT1-stimulated recombination but does not affect rDNA recombination or exchange in the absence of HOT1. Two new alleles of RAD52 were also isolated using this screen. With regard to HOT1 activity, rad52 is epistatic to all four hrm mutations indicating that the products of the HRM genes and of RAD52 mediate steps in the same recombination pathway. Finding mutations that decrease both the activity of HOT1 and exchange in the rDNA supports the hypothesis that HOT1 plays a role in rDNA recombination

The Human Tyrosyl-DNA Phosphodiesterase 1 (hTdp1) Inhibitor NSC120686 as an Exploratory Tool to Investigate Plant Tdp1 Genes.

Science.gov (United States)

Macovei, Anca; Pagano, Andrea; Sabatini, Maria Elisa; Grandi, Sofia; Balestrazzi, Alma

2018-03-28

The hTdp1 (human tyrosyl-DNA phosphodiesterase 1) inhibitor NSC120686 has been used, along with topoisomerase inhibitors, as a pharmacophoric model to restrain the Tdp1 activity as part of a synergistic treatment for cancer. While this compound has an end-point application in medical research, in plants, its application has not been considered so far. The originality of our study consists in the use of hTdp1 inhibitor in Medicago truncatula cells, which, unlike human cells, contain two Tdp1 genes. Hence, the purpose of this study was to test the hTdp1 inhibitor NSC120686 as an exploratory tool to investigate the plant Tdp1 genes, since their characterization is still in incipient phases. To do so, M. truncatula calli were exposed to increasing (75, 150, 300 μM) concentrations of NSC120686. The levels of cell mortality and DNA damage, measured via diffusion assay and comet assay, respectively, were significantly increased when the highest doses were used, indicative of a cytotoxic and genotoxic threshold. In addition, the NSC120686-treated calli and untreated MtTdp1α -depleted calli shared a similar response in terms of programmed cell death (PCD)/necrosis and DNA damage. Interestingly, the expression profiles of MtTdp1α and MtTdp1β genes were differently affected by the NSC120686 treatment, as MtTdp1α was upregulated while MtTdp1β was downregulated. The NSC120686 treatment affected not only the MtTdp1 genes but also other genes with roles in alternative DNA repair pathways. Since the expression patterns of these genes were different than what was observed in the MtTdp1α -depleted plants, it could be hypothesized that the NSC120686 treatment exerts a different influence compared to that resulting from the lack of the MtTdp1α gene function.

Rearrangement of Rag-1 recombinase gene in DNA-repair deficient/immunodeficient wasted'' mice

Energy Technology Data Exchange (ETDEWEB)

Woloschak, G.E.; Weaver, P.; Churchill, M.; Chang-Liu, C-M. (Argonne National Lab., IL (United States)); Libertin, C.R. (Loyola Univ., Maywood, IL (United States))

1992-01-01

Mice recessive for the autosomal gene wasted'' (wst) display a disease pattern which includes increased sensitivity to the killing effects of ionizing radiation, immunodeficiency, and neurologic dysfunction. The recent cloning and characterization of recombinase genes (Rag-l/Rag-2) expressed in lymphoid and possibly central nervous system tissues prompted us to examine expression of these genes in DNA repair-deficient/immunodeficient wasted mice. Our results revealed that in thymus tissue, a small Rag-I transcript (1.0 kb) was detected in wst/wst mice that was not evident in thymus from control mice. In wst/[sm bullet] mice, a two-fold increase in Rag-1 mRNA was evident in thymus tissue. Rag-2 mRNA could only be detected in thymus tissue from wst/[sm bullet] and not from wst/wst or parental control BCF, mice. Southern blots revealed a rearrangement or deletion within the Rag-1 gene of affected wasted mice that was not evident in known strain-specific parental or littermate controls. These results support the idea that the Rag-1 gene may map at or near the locus for the wasted mutation. In addition, they suggest the importance of recombinase function in normal immune and central nervous system development as well as the potential contribution of this gene family to the normal repair of radiation-induced DNA damage.

Selective Gene Delivery for Integrating Exogenous DNA into Plastid and Mitochondrial Genomes Using Peptide-DNA Complexes.

Science.gov (United States)

Yoshizumi, Takeshi; Oikawa, Kazusato; Chuah, Jo-Ann; Kodama, Yutaka; Numata, Keiji

2018-05-14

Selective gene delivery into organellar genomes (mitochondrial and plastid genomes) has been limited because of a lack of appropriate platform technology, even though these organelles are essential for metabolite and energy production. Techniques for selective organellar modification are needed to functionally improve organelles and produce transplastomic/transmitochondrial plants. However, no method for mitochondrial genome modification has yet been established for multicellular organisms including plants. Likewise, modification of plastid genomes has been limited to a few plant species and algae. In the present study, we developed ionic complexes of fusion peptides containing organellar targeting signal and plasmid DNA for selective delivery of exogenous DNA into the plastid and mitochondrial genomes of intact plants. This is the first report of exogenous DNA being integrated into the mitochondrial genomes of not only plants, but also multicellular organisms in general. This fusion peptide-mediated gene delivery system is a breakthrough platform for both plant organellar biotechnology and gene therapy for mitochondrial diseases in animals.

DNA repair-related genes in sugarcane expressed sequence tags (ESTs

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R.M.A. Costa

2001-12-01

Full Text Available There is much interest in the identification and characterization of genes involved in DNA repair because of their importance in the maintenance of the genome integrity. The high level of conservation of DNA repair genes means that these genetic elements may be used in phylogenetic studies as a source of information on the genetic origin and evolution of species. The mechanisms by which damaged DNA is repaired are well understood in bacteria, yeast and mammals, but much remains to be learned as regards plants. We identified genes involved in DNA repair mechanisms in sugarcane using a similarity search of the Brazilian Sugarcane Expressed Sequence Tag (SUCEST database against known sequences deposited in other public databases (National Center of Biotechnology Information (NCBI database and the Munich Information Center for Protein Sequences (MIPS Arabidopsis thaliana database. This search revealed that most of the various proteins involved in DNA repair in sugarcane are similar to those found in other eukaryotes. However, we also identified certain intriguing features found only in plants, probably due to the independent evolution of this kingdom. The DNA repair mechanisms investigated include photoreactivation, base excision repair, nucleotide excision repair, mismatch repair, non-homologous end joining, homologous recombination repair and DNA lesion tolerance. We report the main differences found in the DNA repair machinery in plant cells as compared to other organisms. These differences point to potentially different strategies plants employ to deal with DNA damage, that deserve further investigation.A identificação e caracterização de genes envolvidos com reparo de DNA são de grande interesse, dada a sua importância na manutenção da integridade genômica. Além disso, a alta conservação dos genes de reparo de DNA faz com que possam ser utilizados como fonte de informação no que diz respeito à origem e evolução das esp

Ionizing and ultraviolet radiation enhances the efficiency of DNA mediated gene transfer in vitro

International Nuclear Information System (INIS)

Perez, C.F.

1984-08-01

The enhancement effects of ionizing and non-ionizing radiation on the efficiency of DNA mediated gene transfer were studied. Confluent Rat-2 cells were transfected with purified SV40 viral DNA, irradiated with either X-rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were non-linear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X-rays or 330 MeV/amu argon particles at the Berkeley Bevalac showed a higher frequency of HAT + colonies/survivor than unirradiated transfected cells. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK-transformation by both X-rays and ultraviolet radiation. The results demonstrate that radiation enhancement of the efficiency of DNA mediated gene transfer is not explained by increased nuclear uptake of the transfected DNA. Radiation increases the competence of the transfected cell population for genetic transformation. Three models for this increased competence are presented. The targeted integration model, the inducible recombination model, the partition model, and the utilization of DNA mediated gene transfer for DNA repair studies are discussed. 465 references

Ionizing and ultraviolet radiation enhances the efficiency of DNA mediated gene transfer in vitro

Energy Technology Data Exchange (ETDEWEB)

Perez, C.F.

1984-08-01

The enhancement effects of ionizing and non-ionizing radiation on the efficiency of DNA mediated gene transfer were studied. Confluent Rat-2 cells were transfected with purified SV40 viral DNA, irradiated with either X-rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were non-linear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X-rays or 330 MeV/amu argon particles at the Berkeley Bevalac showed a higher frequency of HAT/sup +/ colonies/survivor than unirradiated transfected cells. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK-transformation by both X-rays and ultraviolet radiation. The results demonstrate that radiation enhancement of the efficiency of DNA mediated gene transfer is not explained by increased nuclear uptake of the transfected DNA. Radiation increases the competence of the transfected cell population for genetic transformation. Three models for this increased competence are presented. The targeted integration model, the inducible recombination model, the partition model, and the utilization of DNA mediated gene transfer for DNA repair studies are discussed. 465 references.

DR-Integrator: a new analytic tool for integrating DNA copy number and gene expression data.

Science.gov (United States)

Salari, Keyan; Tibshirani, Robert; Pollack, Jonathan R

2010-02-01

DNA copy number alterations (CNA) frequently underlie gene expression changes by increasing or decreasing gene dosage. However, only a subset of genes with altered dosage exhibit concordant changes in gene expression. This subset is likely to be enriched for oncogenes and tumor suppressor genes, and can be identified by integrating these two layers of genome-scale data. We introduce DNA/RNA-Integrator (DR-Integrator), a statistical software tool to perform integrative analyses on paired DNA copy number and gene expression data. DR-Integrator identifies genes with significant correlations between DNA copy number and gene expression, and implements a supervised analysis that captures genes with significant alterations in both DNA copy number and gene expression between two sample classes. DR-Integrator is freely available for non-commercial use from the Pollack Lab at http://pollacklab.stanford.edu/ and can be downloaded as a plug-in application to Microsoft Excel and as a package for the R statistical computing environment. The R package is available under the name 'DRI' at http://cran.r-project.org/. An example analysis using DR-Integrator is included as supplemental material. Supplementary data are available at Bioinformatics online.

The evolution of CHROMOMETHYLASES and gene body DNA methylation in plants.

Science.gov (United States)

Bewick, Adam J; Niederhuth, Chad E; Ji, Lexiang; Rohr, Nicholas A; Griffin, Patrick T; Leebens-Mack, Jim; Schmitz, Robert J

2017-05-01

The evolution of gene body methylation (gbM), its origins, and its functional consequences are poorly understood. By pairing the largest collection of transcriptomes (>1000) and methylomes (77) across Viridiplantae, we provide novel insights into the evolution of gbM and its relationship to CHROMOMETHYLASE (CMT) proteins. CMTs are evolutionary conserved DNA methyltransferases in Viridiplantae. Duplication events gave rise to what are now referred to as CMT1, 2 and 3. Independent losses of CMT1, 2, and 3 in eudicots, CMT2 and ZMET in monocots and monocots/commelinids, variation in copy number, and non-neutral evolution suggests overlapping or fluid functional evolution of this gene family. DNA methylation within genes is widespread and is found in all major taxonomic groups of Viridiplantae investigated. Genes enriched with methylated CGs (mCG) were also identified in species sister to angiosperms. The proportion of genes and DNA methylation patterns associated with gbM are restricted to angiosperms with a functional CMT3 or ortholog. However, mCG-enriched genes in the gymnosperm Pinus taeda shared some similarities with gbM genes in Amborella trichopoda. Additionally, gymnosperms and ferns share a CMT homolog closely related to CMT2 and 3. Hence, the dependency of gbM on a CMT most likely extends to all angiosperms and possibly gymnosperms and ferns. The resulting gene family phylogeny of CMT transcripts from the most diverse sampling of plants to date redefines our understanding of CMT evolution and its evolutionary consequences on DNA methylation. Future, functional tests of homologous and paralogous CMTs will uncover novel roles and consequences to the epigenome.

DNA methyltransferase 1 mutations and mitochondrial pathology: is mtDNA methylated?

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Alessandra eMaresca

2015-03-01

Full Text Available Autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN and Hereditary sensory neuropathy with dementia and hearing loss (HSN1E are two rare, overlapping neurodegenerative syndromes that have been recently linked to allelic dominant pathogenic mutations in the DNMT1 gene, coding for DNA (cytosine-5-methyltransferase 1. DNMT1 is the enzyme responsible for maintaining the nuclear genome methylation patterns during the DNA replication and repair, thus regulating gene expression. The mutations responsible for ADCA-DN and HSN1E affect the replication foci targeting sequence domain, which regulates DNMT1 binding to chromatin. DNMT1 dysfunction is anticipated to lead to a global alteration of the DNA methylation pattern with predictable downstream consequences on gene expression. Interestingly, ADCA-DN and HSN1E phenotypes share some clinical features typical of mitochondrial diseases, such as optic atrophy, peripheral neuropathy and deafness, and some biochemical evidence of mitochondrial dysfunction. The recent discovery of a mitochondrial isoform of DNMT1 and its proposed role in methylating mitochondrial DNA (mtDNA suggests that DNMT1 mutations may directly affect mtDNA and mitochondrial physiology. On the basis of this latter finding the link between DNMT1 abnormal activity and mitochondrial dysfunction in ADCA-DN and HSN1E appears intuitive, however mtDNA methylation remains highly debated. In the last years several groups demonstrated the presence of 5-methylcytosine in mtDNA by different approaches, but, on the other end, the opposite evidence that mtDNA is not methylated has also been published. Since over 1500 mitochondrial proteins are encoded by the nuclear genome, the altered methylation of these genes may well have a critical role in leading to the mitochondrial impairment observed in ADCA-DN and HSN1E. Thus, many open questions still remain unanswered, such as why mtDNA should be methylated, and how this process is

Cumulative Impact of Polychlorinated Biphenyl and Large Chromosomal Duplications on DNA Methylation, Chromatin, and Expression of Autism Candidate Genes.

Science.gov (United States)

Dunaway, Keith W; Islam, M Saharul; Coulson, Rochelle L; Lopez, S Jesse; Vogel Ciernia, Annie; Chu, Roy G; Yasui, Dag H; Pessah, Isaac N; Lott, Paul; Mordaunt, Charles; Meguro-Horike, Makiko; Horike, Shin-Ichi; Korf, Ian; LaSalle, Janine M

2016-12-13

Rare variants enriched for functions in chromatin regulation and neuronal synapses have been linked to autism. How chromatin and DNA methylation interact with environmental exposures at synaptic genes in autism etiologies is currently unclear. Using whole-genome bisulfite sequencing in brain tissue and a neuronal cell culture model carrying a 15q11.2-q13.3 maternal duplication, we find that significant global DNA hypomethylation is enriched over autism candidate genes and affects gene expression. The cumulative effect of multiple chromosomal duplications and exposure to the pervasive persistent organic pollutant PCB 95 altered methylation of more than 1,000 genes. Hypomethylated genes were enriched for H2A.Z, increased maternal UBE3A in Dup15q corresponded to reduced levels of RING1B, and bivalently modified H2A.Z was altered by PCB 95 and duplication. These results demonstrate the compounding effects of genetic and environmental insults on the neuronal methylome that converge upon dysregulation of chromatin and synaptic genes. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Genomic survey and expression analysis of DNA repair genes in the genus Leptospira.

Science.gov (United States)

Martins-Pinheiro, Marinalva; Schons-Fonseca, Luciane; da Silva, Josefa B; Domingos, Renan H; Momo, Leonardo Hiroyuki Santos; Simões, Ana Carolina Quirino; Ho, Paulo Lee; da Costa, Renata M A

2016-04-01

Leptospirosis is an emerging zoonosis with important economic and public health consequences and is caused by pathogenic leptospires. The genus Leptospira belongs to the order Spirochaetales and comprises saprophytic (L. biflexa), pathogenic (L. interrogans) and host-dependent (L. borgpetersenii) members. Here, we present an in silico search for DNA repair pathways in Leptospira spp. The relevance of such DNA repair pathways was assessed through the identification of mRNA levels of some genes during infection in animal model and after exposition to spleen cells. The search was performed by comparison of available Leptospira spp. genomes in public databases with known DNA repair-related genes. Leptospires exhibit some distinct and unexpected characteristics, for instance the existence of a redundant mechanism for repairing a chemically diverse spectrum of alkylated nucleobases, a new mutS-like gene and a new shorter version of uvrD. Leptospira spp. shares some characteristics from Gram-positive, as the presence of PcrA, two RecQ paralogs and two SSB proteins; the latter is considered a feature shared by naturally competent bacteria. We did not find a significant reduction in the number of DNA repair-related genes in both pathogenic and host-dependent species. Pathogenic leptospires were enriched for genes dedicated to base excision repair and non-homologous end joining. Their evolutionary history reveals a remarkable importance of lateral gene transfer events for the evolution of the genus. Up-regulation of specific DNA repair genes, including components of SOS regulon, during infection in animal model validates the critical role of DNA repair mechanisms for the complex interplay between host/pathogen.

Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

Science.gov (United States)

Tsabar, Michael; Mason, Jennifer M.; Chan, Yuen-Ling; Bishop, Douglas K.; Haber, James E.

2015-01-01

Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1ATR/Tel1ATM-dependent DNA damage response or caffeine's inhibition of 5′ to 3′ resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments. PMID:26019181

Asynchronous DNA replication within the human β-globin gene locus

International Nuclear Information System (INIS)

Epner, E.; Forrester, W.C.; Groudine, M.

1988-01-01

The timing of DNA replication of the human β-globin gene locus has been studied by blot hybridization of newly synthesized BrdUrd-substituted DNA from cells in different stages of the S phase. Using probes that span >120 kilobases across the human β-globin gene locus, the authors show that the majority of this domain replicates in early S phase in the human erythroleukemia cell line K562 and in middle-to-late S phase in the lymphoid cell line Manca. However, in K562 cells three small regions display a strikingly different replication pattern than adjacent sequences. These islands, located in the inter-γ-globin gene region and approximately 20 kilobases 5' to the ε-globin gene and 20 kilobases 3' to the β-globin gene, replicate later and throughout S phase. A similar area is also present in the α-globin gene region in K562 cells. They suggest that these regions may represent sites of termination of replication forks

Observation of intermittency in gene expression on cDNA microarrays

CERN Document Server

Peterson, L E

2002-01-01

We used scaled factorial moments to search for intermittency in the log expression ratios (LERs) for thousands of genes spotted on cDNA microarrays (gene chips). Results indicate varying levels of intermittency in gene expression. The observation of intermittency in the data analyzed provides a complimentary handle on moderately expressed genes, generally not tackled by conventional techniques.

A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs

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Gong Shiaochin

2009-03-01

Full Text Available Abstract Background Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. Results We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1 subclone genes of interest into BAC linking vectors, (2 insert desired reporter genes into respective genes and (3 link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. Conclusion The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs.

Science.gov (United States)

Maye, Peter; Stover, Mary Louise; Liu, Yaling; Rowe, David W; Gong, Shiaochin; Lichtler, Alexander C

2009-03-13

Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP) reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1) subclone genes of interest into BAC linking vectors, (2) insert desired reporter genes into respective genes and (3) link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

An SGS3-like protein functions in RNA-directed DNA methylation and transcriptional gene silencing in Arabidopsis

KAUST Repository

Zheng, Zhimin

2010-01-06

RNA-directed DNA methylation (RdDM) is an important epigenetic mechanism for silencing transgenes and endogenous repetitive sequences such as transposons. The RD29A promoter-driven LUCIFERASE transgene and its corresponding endogenous RD29A gene are hypermethylated and silenced in the Arabidopsis DNA demethylase mutant ros1. By screening for second-site suppressors of ros1, we identified the RDM12 locus. The rdm12 mutation releases the silencing of the RD29A-LUC transgene and the endogenous RD29A gene by reducing the promoter DNA methylation. The rdm12 mutation also reduces DNA methylation at endogenous RdDM target loci, including transposons and other repetitive sequences. In addition, the rdm12 mutation affects the levels of small interfering RNAs (siRNAs) from some of the RdDM target loci. RDM12 encodes a protein with XS and coiled-coil domains, and is similar to SGS3, which is a partner protein of RDR6 and can bind to double-stranded RNAs with a 5′ overhang, and is required for several post-transcriptional gene silencing pathways. Our results show that RDM12 is a component of the RdDM pathway, and suggest that RdDM may involve double-stranded RNAs with a 5′ overhang and the partnering between RDM12 and RDR2. © 2010 Blackwell Publishing Ltd.

Intrinsically bent DNA in replication origins and gene promoters.

Science.gov (United States)

Gimenes, F; Takeda, K I; Fiorini, A; Gouveia, F S; Fernandez, M A

2008-06-24

Intrinsically bent DNA is an alternative conformation of the DNA molecule caused by the presence of dA/dT tracts, 2 to 6 bp long, in a helical turn phase DNA or with multiple intervals of 10 to 11 bp. Other than flexibility, intrinsic bending sites induce DNA curvature in particular chromosome regions such as replication origins and promoters. Intrinsically bent DNA sites are important in initiating DNA replication, and are sometimes found near to regions associated with the nuclear matrix. Many methods have been developed to localize bent sites, for example, circular permutation, computational analysis, and atomic force microscopy. This review discusses intrinsically bent DNA sites associated with replication origins and gene promoter regions in prokaryote and eukaryote cells. We also describe methods for identifying bent DNA sites for circular permutation and computational analysis.

Cloning, DNA sequence, and expression of the Rhodobacter sphaeroides cytochrome c/sub 2/ gene

Energy Technology Data Exchange (ETDEWEB)

Donohue, T.J.; McEwan, A.G.; Kaplan, S.

1986-11-01

The Rhodobacter sphaeroides cytochrome c/sub 2/ functions as a mobile electron carrier in both aerobic and photosynthetic electron transport chains. Synthetic deoxyoligonucleotide probes, based on the known amino acid sequence of this protein (M/sub r/ 14,000), were used to identify and clone the cytochrome c/sub 2/ structural gene (cycA). DNA sequence analysis of the cycA gene indicated the presence of a typical procaryotic 21-residue signal sequence, suggesting that this periplasmic protein is synthesized in vivo as a precursor. Synthesis of an immunoreactive cytochrome c/sub 2/ precursor protein (M/sub r/ 15,500) was observed in vitro when plasmids containing the cycA gene were used as templates in an R. sphaeroides coupled transcription-translation system. Approximately 500 base pairs of DNA upstream of the cycA gene was sufficient to allow expression of this gene product in vitro. Northern blot analysis with an internal cycA-specific probe identified at least two possibly monocistronic transcripts present in both different cellular levels and relative stoichiometries in steady-state cells grown under different physiological conditions. The ratio of the small (740-mucleotide) and large (920-nucleotide) cycA-specific mRNA species was dependent on cultural conditions but was not affected by light intensity under photosynthetic conditions. These results suggest that the increase in the cellular level of the cytochrome c/sub 2/ protein found in photosynthetic cells was due, in part, to increased transcription of the single-copy cyc operon.

Use of electroporation for high-molecular-weight DNA-mediated gene transfer.

Science.gov (United States)

Jastreboff, M M; Ito, E; Bertino, J R; Narayanan, R

1987-08-01

Electroporation was used to introduce high-molecular-weight DNA into murine hematopoietic cells and NIH3T3 cells. CCRF-CEM cells were stably transfected with SV2NEO plasmid and the genomic DNA from G-418-resistant clones (greater than 65 kb) was introduced into mouse bone marrow and NIH3T3 cells by electroporation. NEO sequences and expression were detected in the hematopoietic tissues of lethally irradiated mice, with 24% of individual spleen colonies expressing NEO. The frequency of genomic DNA transfer into NIH3T3 cells was 0.25 X 10(-3). Electroporation thus offers a powerful mode of gene transfer not only of cloned genes but also of high-molecular-weight DNA into cells.

DNA Methylation of Regulatory Regions of Imprinted Genes at Birth and Its Relation to Infant Temperament

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Bernard F. Fuemmeler

2016-01-01

Full Text Available BACKGROUND DNA methylation of the differentially methylated regions (DMRs of imprinted genes is relevant to neurodevelopment. METHODS DNA methylation status of the DMRs of nine imprinted genes in umbilical cord blood leukocytes was analyzed in relation to infant behaviors and temperament (n = 158. RESULTS MEG3 DMR levels were positively associated with internalizing ( β = 0.15, P = 0.044 and surgency ( β = 0.19, P = 0.018 behaviors, after adjusting for birth weight, gender, gestational age at birth, maternal age at delivery, race/ethnicity, education level, smoking status, parity, and a history of anxiety or depression. Higher methylation levels at the intergenic MEG3-IG methylation regions were associated with surgency ( β = 0.28, P = 0.0003 and PEG3 was positively related to externalizing ( β = 0.20, P = 0.01 and negative affectivity ( β = 0.18, P = 0.02. CONCLUSION While the small sample size limits inference, these pilot data support gene-specific associations between epigenetic differences in regulatory regions of imprinted domains at birth and later infant temperament.

Essential and non-essential DNA replication genes in the model halophilic Archaeon, Halobacterium sp. NRC-1

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DasSarma Shiladitya

2007-06-01

Full Text Available Abstract Background Information transfer systems in Archaea, including many components of the DNA replication machinery, are similar to those found in eukaryotes. Functional assignments of archaeal DNA replication genes have been primarily based upon sequence homology and biochemical studies of replisome components, but few genetic studies have been conducted thus far. We have developed a tractable genetic system for knockout analysis of genes in the model halophilic archaeon, Halobacterium sp. NRC-1, and used it to determine which DNA replication genes are essential. Results Using a directed in-frame gene knockout method in Halobacterium sp. NRC-1, we examined nineteen genes predicted to be involved in DNA replication. Preliminary bioinformatic analysis of the large haloarchaeal Orc/Cdc6 family, related to eukaryotic Orc1 and Cdc6, showed five distinct clades of Orc/Cdc6 proteins conserved in all sequenced haloarchaea. Of ten orc/cdc6 genes in Halobacterium sp. NRC-1, only two were found to be essential, orc10, on the large chromosome, and orc2, on the minichromosome, pNRC200. Of the three replicative-type DNA polymerase genes, two were essential: the chromosomally encoded B family, polB1, and the chromosomally encoded euryarchaeal-specific D family, polD1/D2 (formerly called polA1/polA2 in the Halobacterium sp. NRC-1 genome sequence. The pNRC200-encoded B family polymerase, polB2, was non-essential. Accessory genes for DNA replication initiation and elongation factors, including the putative replicative helicase, mcm, the eukaryotic-type DNA primase, pri1/pri2, the DNA polymerase sliding clamp, pcn, and the flap endonuclease, rad2, were all essential. Targeted genes were classified as non-essential if knockouts were obtained and essential based on statistical analysis and/or by demonstrating the inability to isolate chromosomal knockouts except in the presence of a complementing plasmid copy of the gene. Conclusion The results showed that ten

Histone H3.3 promotes IgV gene diversification by enhancing formation of AID-accessible single-stranded DNA.

Science.gov (United States)

Romanello, Marina; Schiavone, Davide; Frey, Alexander; Sale, Julian E

2016-07-01

Immunoglobulin diversification is driven by activation-induced deaminase (AID), which converts cytidine to uracil within the Ig variable (IgV) regions. Central to the recruitment of AID to the IgV genes are factors that regulate the generation of single-stranded DNA (ssDNA), the enzymatic substrate of AID Here, we report that chicken DT40 cells lacking variant histone H3.3 exhibit reduced IgV sequence diversification. We show that this results from impairment of the ability of AID to access the IgV genes due to reduced formation of ssDNA during IgV transcription. Loss of H3.3 also diminishes IgV R-loop formation. However, reducing IgV R-loops by RNase HI overexpression in wild-type cells does not affect IgV diversification, showing that these structures are not necessary intermediates for AID access. Importantly, the reduction in the formation of AID-accessible ssDNA in cells lacking H3.3 is independent of any effect on the level of transcription or the kinetics of RNAPII elongation, suggesting the presence of H3.3 in the nucleosomes of the IgV genes increases the chances of the IgV DNA becoming single-stranded, thereby creating an effective AID substrate. © 2016 MRC Laboratory of Molecular Biology. Published under the terms of the CC BY 4.0 license.

Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22

International Nuclear Information System (INIS)

Tsai-Pflugfelder, M.; Liu, L.F.; Liu, A.A.; Tewey, K.M.; Whang-Peng, J.; Knutsen, T.; Huebner, K.; Croce, C.M.; Wang, J.C.

1988-01-01

Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage λ was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a λgt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes

Hepatitis B virus DNA polymerase gene polymorphism based ...

African Journals Online (AJOL)

Hepatitis B virus DNA polymerase gene polymorphism based prediction of genotypes in chronic HBV patients from Western India. Yashwant G. Chavan, Sharad R. Pawar, Minal Wani, Amol D. Raut, Rabindra N. Misra ...

Satellite DNA-based artificial chromosomes for use in gene therapy.

Science.gov (United States)

Hadlaczky, G

2001-04-01

Satellite DNA-based artificial chromosomes (SATACs) can be made by induced de novo chromosome formation in cells of different mammalian species. These artificially generated accessory chromosomes are composed of predictable DNA sequences and they contain defined genetic information. Prototype human SATACs have been successfully constructed in different cell types from 'neutral' endogenous DNA sequences from the short arm of the human chromosome 15. SATACs have already passed a number of hurdles crucial to their further development as gene therapy vectors, including: large-scale purification; transfer of purified artificial chromosomes into different cells and embryos; generation of transgenic animals and germline transmission with purified SATACs; and the tissue-specific expression of a therapeutic gene from an artificial chromosome in the milk of transgenic animals.

Differentially expressed genes in Populus simonii x P. nigra in respnse to NaCl stress using cDNA-AFLP

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Salinity is an important environmental factor limiting growth and productivity of plants, and affects almost every aspect of the plant physiology and biochemistry. The objective of this study was to apply cDNA-AFLP and to identify differentially expressed genes in response to NaCl stress vs. no-stre...

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

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

1989-09-01

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

Horizontal gene transfer and nucleotide compositional anomaly in large DNA viruses

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Ogata Hiroyuki

2007-12-01

Full Text Available Abstract Background DNA viruses have a wide range of genome sizes (5 kb up to 1.2 Mb, compared to 0.16 Mb to 1.5 Mb for obligate parasitic bacteria that do not correlate with their virulence or the taxonomic distribution of their hosts. The reasons for such large variation are unclear. According to the traditional view of viruses as gifted "gene pickpockets", large viral genome sizes could originate from numerous gene acquisitions from their hosts. We investigated this hypothesis by studying 67 large DNA viruses with genome sizes larger than 150 kb, including the recently characterized giant mimivirus. Given that horizontally transferred DNA often have anomalous nucleotide compositions differing from the rest of the genome, we conducted a detailed analysis of the inter- and intra-genome compositional properties of these viruses. We then interpreted their compositional heterogeneity in terms of possible causes, including strand asymmetry, gene function/expression, and horizontal transfer. Results We first show that the global nucleotide composition and nucleotide word usage of viral genomes are species-specific and distinct from those of their hosts. Next, we identified compositionally anomalous (cA genes in viral genomes, using a method based on Bayesian inference. The proportion of cA genes is highly variable across viruses and does not exhibit a significant correlation with genome size. The vast majority of the cA genes were of unknown function, lacking homologs in the databases. For genes with known homologs, we found a substantial enrichment of cA genes in specific functional classes for some of the viruses. No significant association was found between cA genes and compositional strand asymmetry. A possible exogenous origin for a small fraction of the cA genes could be confirmed by phylogenetic reconstruction. Conclusion At odds with the traditional dogma, our results argue against frequent genetic transfers to large DNA viruses from their

Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity.

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Zhang, Jin; Ruhlman, Tracey A; Sabir, Jamal S M; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K

2016-02-17

Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear-plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Yeast DNA-repair gene RAD14 encodes a zinc metalloprotein with affinity for ultraviolet-damaged DNA

International Nuclear Information System (INIS)

Guzder, S.N.; Sung, P.; Prakash, S.; Prakash, L.

1993-01-01

Xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers due to a defect in excision repair of UV light-damaged DNA. Of the seven XP complementation groups, A--G, group A represents a severe and frequent form of the disease. The Saccharomyces cerevisiae RAD14 gene is a homolog of the XP-A correcting (XPAC) gene. Like XP-A cells, rad14-null mutants are defective in the incision step of excision repair of UV-damaged DNA. The authors have purified RAD14 protein to homogeneity from extract of a yeast strain genetically tailored to overexpress RAD14. As determined by atomic emission spectroscopy, RAD14 contains one zinc atom. They also show in vitro that RAD14 binds zinc but does not bind other divalent metal ions. In DNA mobility-shift assays, RAD14 binds specifically to UV-damaged DNA. Removal of cyclobutane pyrimidine dimers from damaged DNA by enzymatic photoreactivation has no effect on binding, strongly suggesting that RAD14 recognizes pyrimidine(6-4)pyrimidone photoproduct sites. These findings indicate that RAD14 functions in damage recognition during excision repair. 37 refs., 4 figs

Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB

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Munir, Ahsan; Waseem, Hassan; Williams, Maggie R.; Stedtfeld, Robert D.; Gulari, Erdogan; Tiedje, James M.; Hashsham, Syed A.

2017-01-01

Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics) to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs). Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R2 = 0.8131). PMID:28555058

Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB

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Ahsan Munir

2017-05-01

Full Text Available Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs. Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R2 = 0.8131.

Periodic expression of nuclear and mitochondrial DNA replication genes during the trypanosomatid cell cycle.

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Pasion, S G; Brown, G W; Brown, L M; Ray, D S

1994-12-01

In trypanosomatids, DNA replication in the nucleus and in the single mitochondrion (or kinetoplast) initiates nearly simultaneously, suggesting that the DNA synthesis (S) phases of the nucleus and the mitochondrion are coordinately regulated. To investigate the basis for the temporal link between nuclear and mitochondrial DNA synthesis phases the expression of the genes encoding DNA ligase I, the 51 and 28 kDa subunits of replication protein A, dihydrofolate reductase and the mitochondrial type II topoisomerase were analyzed during the cell cycle progression of synchronous cultures of Crithidia fasciculata. These DNA replication genes were all expressed periodically, with peak mRNA levels occurring just prior to or at the peak of DNA synthesis in the synchronized cultures. A plasmid clone (pdN-1) in which TOP2, the gene encoding the mitochondrial topoisomerase, was disrupted by the insertion of a NEO drug-resistance cassette was found to express both a truncated TOP2 mRNA and a truncated topoisomerase polypeptide. The truncated mRNA was also expressed periodically coordinate with the expression of the endogenous TOP2 mRNA indicating that cis elements necessary for periodic expression are contained within cloned sequences. The expression of both TOP2 and nuclear DNA replication genes at the G1/S boundary suggests that regulated expression of these genes may play a role in coordinating nuclear and mitochondrial S phases in trypanosomatids.

Molecular genetic and biochemical analyses of a DNA repair gene from Serratia marcescens

International Nuclear Information System (INIS)

Murphy, K.E.

1989-01-01

In Escherichia coli, the SOS response and two 3-methyladenine DNA glycosylases (TagI and TagII) are required for repair of DNA damaged by alkylating agents such as methyl methanesulfonate (MMS). Mutations of the recA gene eliminate the SOS response. TagI and TagII are encoded by the tag and alkA genes, respectively. A gene (rpr) encoding 3-methyladenine DNA glycosylase activity was isolated from the Gram-negative bacterium Serratia marcescens. The gene, localized to a 1.5-kilobase pair SmaI-HindIII restriction fragment, was cloned into plasmid pUC18. The clone complemented E. coli tag alkA and recA mutations for MMS resistance. The rpr gene did not, however, complement recA mutations for resistance to ultraviolet light or the ability to perform homologous recombination reactions, nor did it complement E. coli ada or alkB mutations. Two proteins of molecular weights 42,000 and 16,000 were produced from the rpr locus. Analysis of deletion and insertion mutants of rpr suggested that the 42kD molecule is the active protein. The 16kD protein may either be a breakdown product of the 42kD species or may be encoded by another gene overlapping the reading frame of the rpr gene. Biochemical assays showed that the rpr gene product (Rpr) possesses 3-methyladenine DNA glycosylase activity

Apparent polyploidization after gamma irradiation: pitfalls in the use of quantitative polymerase chain reaction (qPCR) for the estimation of mitochondrial and nuclear DNA gene copy numbers.

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Kam, Winnie W Y; Lake, Vanessa; Banos, Connie; Davies, Justin; Banati, Richard

2013-05-30

Quantitative polymerase chain reaction (qPCR) has been widely used to quantify changes in gene copy numbers after radiation exposure. Here, we show that gamma irradiation ranging from 10 to 100 Gy of cells and cell-free DNA samples significantly affects the measured qPCR yield, due to radiation-induced fragmentation of the DNA template and, therefore, introduces errors into the estimation of gene copy numbers. The radiation-induced DNA fragmentation and, thus, measured qPCR yield varies with temperature not only in living cells, but also in isolated DNA irradiated under cell-free conditions. In summary, the variability in measured qPCR yield from irradiated samples introduces a significant error into the estimation of both mitochondrial and nuclear gene copy numbers and may give spurious evidence for polyploidization.

Modulation of DNA binding by gene-specific transcription factors.

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Schleif, Robert F

2013-10-01

The transcription of many genes, particularly in prokaryotes, is controlled by transcription factors whose activity can be modulated by controlling their DNA binding affinity. Understanding the molecular mechanisms by which DNA binding affinity is regulated is important, but because forming definitive conclusions usually requires detailed structural information in combination with data from extensive biophysical, biochemical, and sometimes genetic experiments, little is truly understood about this topic. This review describes the biological requirements placed upon DNA binding transcription factors and their consequent properties, particularly the ways that DNA binding affinity can be modulated and methods for its study. What is known and not known about the mechanisms modulating the DNA binding affinity of a number of prokaryotic transcription factors, including CAP and lac repressor, is provided.

Expression of DNA repair genes in ovarian cancer samples: biological and clinical considerations.

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Ganzinelli, M; Mariani, P; Cattaneo, D; Fossati, R; Fruscio, R; Corso, S; Ricci, F; Broggini, M; Damia, G

2011-05-01

The purpose of this study was to investigate retrospectively the mRNA expression of genes involved in different DNA repair pathways implicated in processing platinum-induced damage in 171 chemotherapy-naïve ovarian tumours and correlate the expression of the different genes with clinical parameters. The expression of genes involved in DNA repair pathways (PARP1, ERCC1, XPA, XPF, XPG, BRCA1, FANCA, FANCC, FANCD2, FANCF and PolEta), and in DNA damage transduction (Chk1 and Claspin) was measured by RT-PCR in 13 stage I borderline and 77 stage I and 88 III ovarian carcinomas. ERCC1, XPA, XPF and XPG genes were significantly less expressed in stage III than in stage I carcinoma; BRCA1, FANCA, FANCC, FANCD2 gene expressions were low in borderline tumours, higher in stage I carcinomas and lower in stage III samples. High levels of ERCC1, XPA, FANCC, XPG and PolEta correlated with an increase in Overall Survival (OS) and Progression Free Survival (PFS), whilst high BRCA1 levels were associated with PFS on univariate analysis. With multivariate analyses no genes retained an association when adjusted by stage, grade and residual tumour. A tendency towards a better PFS was observed in patients with the highest level of ERCC1 and BRCA1 after platinum-based therapy than those given both platinum and taxol. The expression of DNA repair genes differed in borderline stage I, stage I and stage III ovarian carcinomas. The role of DNA repair genes in predicting the response in ovarian cancer patients seems far from being established. Copyright © 2010 Elsevier Ltd. All rights reserved.

A functional polymorphism in the reduced folate carrier gene and DNA hypomethylation in mothers of children with autism.

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James, S Jill; Melnyk, Stepan; Jernigan, Stefanie; Pavliv, Oleksandra; Trusty, Timothy; Lehman, Sara; Seidel, Lisa; Gaylor, David W; Cleves, Mario A

2010-09-01

The biologic basis of autism is complex and is thought to involve multiple and variable gene-environment interactions. While the logical focus has been on the affected child, the impact of maternal genetics on intrauterine microenvironment during pivotal developmental windows could be substantial. Folate-dependent one carbon metabolism is a highly polymorphic pathway that regulates the distribution of one-carbon derivatives between DNA synthesis (proliferation) and DNA methylation (cell-specific gene expression and differentiation). These pathways are essential to support the programmed shifts between proliferation and differentiation during embryogenesis and organogenesis. Maternal genetic variants that compromise intrauterine availability of folate derivatives could alter fetal cell trajectories and disrupt normal neurodevelopment. In this investigation, the frequency of common functional polymorphisms in the folate pathway was investigated in a large population-based sample of autism case-parent triads. In case-control analysis, a significant increase in the reduced folate carrier (RFC1) G allele frequency was found among case mothers, but not among fathers or affected children. Subsequent log linear analysis of the RFC1 A80G genotype within family trios revealed that the maternal G allele was associated with a significant increase in risk of autism whereas the inherited genotype of the child was not. Further, maternal DNA from the autism mothers was found to be significantly hypomethylated relative to reference control DNA. Metabolic profiling indicated that plasma homocysteine, adenosine, and S-adenosylhomocyteine were significantly elevated among autism mothers consistent with reduced methylation capacity and DNA hypomethylation. Together, these results suggest that the maternal genetics/epigenetics may influence fetal predisposition to autism. (c) 2010 Wiley-Liss, Inc.

DNA repair genes RAD52 and SRS2, a cell wall synthesis regulator gene SMI1, and the membrane sterol synthesis scaffold gene ERG28 are important in efficient Agrobacterium-mediated yeast transformation with chromosomal T-DNA.

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Ohmine, Yuta; Satoh, Yukari; Kiyokawa, Kazuya; Yamamoto, Shinji; Moriguchi, Kazuki; Suzuki, Katsunori

2016-04-02

Plant pathogenic Agrobacterium strains can transfer T-DNA regions of their Ti plasmids to a broad range of eukaryotic hosts, including fungi, in vitro. In the recent decade, the yeast Saccharomyces cerevisiae is used as a model host to reveal important host proteins for the Agrobacterium-mediated transformation (AMT). Further investigation is required to understand the fundamental mechanism of AMT, including interaction at the cell surface, to expand the host range, and to develop new tools. In this study, we screened a yeast mutant library for low AMT mutant strains by advantage of a chromosome type T-DNA, which transfer is efficient and independent on integration into host chromosome. By the mutant screening, we identified four mutant strains (srs2Δ, rad52Δ, smi1Δ and erg28Δ), which showed considerably low AMT efficiency. Structural analysis of T-DNA product replicons in AMT colonies of mutants lacking each of the two DNA repair genes, SRS2 and RAD52, suggested that the genes act soon after T-DNA entry for modification of the chromosomal T-DNA to stably maintain them as linear replicons and to circularize certain T-DNA simultaneously. The cell wall synthesis regulator SMI1 might have a role in the cell surface interaction between the donor and recipient cells, but the smi1Δ mutant exhibited pleiotropic effect, i.e. low effector protein transport as well as low AMT for the chromosomal T-DNA, but relatively high AMT for integrative T-DNAs. The ergosterol synthesis regulator/enzyme-scaffold gene ERG28 probably contributes by sensing a congested environment, because growth of erg28Δ strain was unaffected by the presence of donor bacterial cells, while the growth of the wild-type and other mutant yeast strains was suppressed by their presence. RAD52 and the DNA helicase/anti-recombinase gene SRS2 are necessary to form and maintain artificial chromosomes through the AMT of chromosomal T-DNA. A sterol synthesis scaffold gene ERG28 is important in the high

Distinct co-evolution patterns of genes associated to DNA polymerase III DnaE and PolC

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Engelen Stefan

2012-02-01

Full Text Available Abstract Background Bacterial genomes displaying a strong bias between the leading and the lagging strand of DNA replication encode two DNA polymerases III, DnaE and PolC, rather than a single one. Replication is a highly unsymmetrical process, and the presence of two polymerases is therefore not unexpected. Using comparative genomics, we explored whether other processes have evolved in parallel with each polymerase. Results Extending previous in silico heuristics for the analysis of gene co-evolution, we analyzed the function of genes clustering with dnaE and polC. Clusters were highly informative. DnaE co-evolves with the ribosome, the transcription machinery, the core of intermediary metabolism enzymes. It is also connected to the energy-saving enzyme necessary for RNA degradation, polynucleotide phosphorylase. Most of the proteins of this co-evolving set belong to the persistent set in bacterial proteomes, that is fairly ubiquitously distributed. In contrast, PolC co-evolves with RNA degradation enzymes that are present only in the A+T-rich Firmicutes clade, suggesting at least two origins for the degradosome. Conclusion DNA replication involves two machineries, DnaE and PolC. DnaE co-evolves with the core functions of bacterial life. In contrast PolC co-evolves with a set of RNA degradation enzymes that does not derive from the degradosome identified in gamma-Proteobacteria. This suggests that at least two independent RNA degradation pathways existed in the progenote community at the end of the RNA genome world.

pH-Dependent DNA Distortion and Repression of Gene Expression by Pectobacterium atrosepticum PecS.

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Deochand, Dinesh K; Meariman, Jacob K; Grove, Anne

2016-07-15

Transcriptional activity is exquisitely sensitive to changes in promoter DNA topology. Transcription factors may therefore control gene activity by modulating the relative positioning of -10 and -35 promoter elements. The plant pathogen Pectobacterium atrosepticum, which causes soft rot in potatoes, must alter gene expression patterns to ensure growth in planta. In the related soft-rot enterobacterium Dickeya dadantii, PecS functions as a master regulator of virulence gene expression. Here, we report that P. atrosepticum PecS controls gene activity by altering promoter DNA topology in response to pH. While PecS binds the pecS promoter with high affinity regardless of pH, it induces significant DNA distortion only at neutral pH, the pH at which the pecS promoter is repressed in vivo. At pH ∼8, DNA distortions are attenuated, and PecS no longer represses the pecS promoter. A specific histidine (H142) located in a crevice between the dimerization- and DNA-binding regions is required for pH-dependent changes in DNA distortion and repression of gene activity, and mutation of this histidine renders the mutant protein incapable of repressing the pecS promoter. We propose that protonated PecS induces a DNA conformation at neutral pH in which -10 and -35 promoter elements are suboptimally positioned for RNA polymerase binding; on deprotonation of PecS, binding is no longer associated with significant changes in DNA conformation, allowing gene expression. We suggest that this mode of gene regulation leads to differential expression of the PecS regulon in response to alkalinization of the plant apoplast.

Neutral and Non-Neutral Evolution of Duplicated Genes with Gene Conversion

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Jeffrey A. Fawcett

2011-02-01

Full Text Available Gene conversion is one of the major mutational mechanisms involved in the DNA sequence evolution of duplicated genes. It contributes to create unique patters of DNA polymorphism within species and divergence between species. A typical pattern is so-called concerted evolution, in which the divergence between duplicates is maintained low for a long time because of frequent exchanges of DNA fragments. In addition, gene conversion affects the DNA evolution of duplicates in various ways especially when selection operates. Here, we review theoretical models to understand the evolution of duplicates in both neutral and non-neutral cases. We also explain how these theories contribute to interpreting real polymorphism and divergence data by using some intriguing examples.

Radioactive cDNA microarrys for gene expression profiles in antidepressant therapy

International Nuclear Information System (INIS)

Lee, M. S.; Han, B. J.; Cha, J. H.; Ryu, Y. M.; Shin, E. K.; Park, J. H.; Park, Y. H.; Kim, M. K.

2002-01-01

Using radioactive cDNA microarray, we investigated a pattern of gene regulation under treatment of antidepressant on patients of depressive disoder. Basic microarray technology was performed as previously described in our research. The bioinformatic selection of human cDNAs, which is specifically designed for psychiatry, neurology, and signal transduction, were arrayed on nylon membranes. Using with 33P-labeled probes, this method provided highly sensitive gene expression profiles of our interest including brain receptors, drug metabolism, and cellular signalings. Gene expression profiles were also classified into several categories in accordance with the gene-regulation of antidepressant. The gene profiles of our interest were significantly up- (16 genes, >2.0 of Z-ratio) or down- (24 genes, <-2.0 of Z ratio) regulated when compared the good responsed group with the bad-responsed one. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology

Exercise-associated DNA methylation change in skeletal muscle and the importance of imprinted genes: a bioinformatics meta-analysis.

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Brown, William M

2015-12-01

Epigenetics is the study of processes--beyond DNA sequence alteration--producing heritable characteristics. For example, DNA methylation modifies gene expression without altering the nucleotide sequence. A well-studied DNA methylation-based phenomenon is genomic imprinting (ie, genotype-independent parent-of-origin effects). We aimed to elucidate: (1) the effect of exercise on DNA methylation and (2) the role of imprinted genes in skeletal muscle gene networks (ie, gene group functional profiling analyses). Gene ontology (ie, gene product elucidation)/meta-analysis. 26 skeletal muscle and 86 imprinted genes were subjected to g:Profiler ontology analysis. Meta-analysis assessed exercise-associated DNA methylation change. g:Profiler found four muscle gene networks with imprinted loci. Meta-analysis identified 16 articles (387 genes/1580 individuals) associated with exercise. Age, method, sample size, sex and tissue variation could elevate effect size bias. Only skeletal muscle gene networks including imprinted genes were reported. Exercise-associated effect sizes were calculated by gene. Age, method, sample size, sex and tissue variation were moderators. Six imprinted loci (RB1, MEG3, UBE3A, PLAGL1, SGCE, INS) were important for muscle gene networks, while meta-analysis uncovered five exercise-associated imprinted loci (KCNQ1, MEG3, GRB10, L3MBTL1, PLAGL1). DNA methylation decreased with exercise (60% of loci). Exercise-associated DNA methylation change was stronger among older people (ie, age accounted for 30% of the variation). Among older people, genes exhibiting DNA methylation decreases were part of a microRNA-regulated gene network functioning to suppress cancer. Imprinted genes were identified in skeletal muscle gene networks and exercise-associated DNA methylation change. Exercise-associated DNA methylation modification could rewind the 'epigenetic clock' as we age. CRD42014009800. Published by the BMJ Publishing Group Limited. For permission to use (where

Genome-Wide DNA Methylation Indicates Silencing of Tumor Suppressor Genes in Uterine Leiomyoma

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Navarro, Antonia; Yin, Ping; Monsivais, Diana; Lin, Simon M.; Du, Pan; Wei, Jian-Jun; Bulun, Serdar E.

2012-01-01

Background Uterine leiomyomas, or fibroids, represent the most common benign tumor of the female reproductive tract. Fibroids become symptomatic in 30% of all women and up to 70% of African American women of reproductive age. Epigenetic dysregulation of individual genes has been demonstrated in leiomyoma cells; however, the in vivo genome-wide distribution of such epigenetic abnormalities remains unknown. Principal Findings We characterized and compared genome-wide DNA methylation and mRNA expression profiles in uterine leiomyoma and matched adjacent normal myometrial tissues from 18 African American women. We found 55 genes with differential promoter methylation and concominant differences in mRNA expression in uterine leiomyoma versus normal myometrium. Eighty percent of the identified genes showed an inverse relationship between DNA methylation status and mRNA expression in uterine leiomyoma tissues, and the majority of genes (62%) displayed hypermethylation associated with gene silencing. We selected three genes, the known tumor suppressors KLF11, DLEC1, and KRT19 and verified promoter hypermethylation, mRNA repression and protein expression using bisulfite sequencing, real-time PCR and western blot. Incubation of primary leiomyoma smooth muscle cells with a DNA methyltransferase inhibitor restored KLF11, DLEC1 and KRT19 mRNA levels. Conclusions These results suggest a possible functional role of promoter DNA methylation-mediated gene silencing in the pathogenesis of uterine leiomyoma in African American women. PMID:22428009

Neuronal DNA Methyltransferases: Epigenetic Mediators between Synaptic Activity and Gene Expression?

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Bayraktar, Gonca; Kreutz, Michael R

2018-04-01

DNMT3A and 3B are the main de novo DNA methyltransferases (DNMTs) in the brain that introduce new methylation marks to non-methylated DNA in postmitotic neurons. DNA methylation is a key epigenetic mark that is known to regulate important cellular processes in neuronal development and brain plasticity. Accumulating evidence disclosed rapid and dynamic changes in DNA methylation of plasticity-relevant genes that are important for learning and memory formation. To understand how DNMTs contribute to brain function and how they are regulated by neuronal activity is a prerequisite for a deeper appreciation of activity-dependent gene expression in health and disease. This review discusses the functional role of de novo methyltransferases and in particular DNMT3A1 in the adult brain with special emphasis on synaptic plasticity, memory formation, and brain disorders.

Diversification of DnaA dependency for DNA replication in cyanobacterial evolution.

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Ohbayashi, Ryudo; Watanabe, Satoru; Ehira, Shigeki; Kanesaki, Yu; Chibazakura, Taku; Yoshikawa, Hirofumi

2016-05-01

Regulating DNA replication is essential for all living cells. The DNA replication initiation factor DnaA is highly conserved in prokaryotes and is required for accurate initiation of chromosomal replication at oriC. DnaA-independent free-living bacteria have not been identified. The dnaA gene is absent in plastids and some symbiotic bacteria, although it is not known when or how DnaA-independent mechanisms were acquired. Here, we show that the degree of dependency of DNA replication on DnaA varies among cyanobacterial species. Deletion of the dnaA gene in Synechococcus elongatus PCC 7942 shifted DNA replication from oriC to a different site as a result of the integration of an episomal plasmid. Moreover, viability during the stationary phase was higher in dnaA disruptants than in wild-type cells. Deletion of dnaA did not affect DNA replication or cell growth in Synechocystis sp. PCC 6803 or Anabaena sp. PCC 7120, indicating that functional dependency on DnaA was already lost in some nonsymbiotic cyanobacterial lineages during diversification. Therefore, we proposed that cyanobacteria acquired DnaA-independent replication mechanisms before symbiosis and such an ancestral cyanobacterium was the sole primary endosymbiont to form a plastid precursor.

Polymorphisms of Selected DNA Repair Genes and Lung Cancer in Chromium Exposure.

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Halasova, E; Matakova, T; Skerenova, M; Krutakova, M; Slovakova, P; Dzian, A; Javorkova, S; Pec, M; Kypusova, K; Hamzik, J

2016-01-01

Chromium is a well-known mutagen and carcinogen involved in lung cancer development. DNA repair genes play an important role in the elimination of genetic changes caused by chromium exposure. In the present study, we investigated the polymorphisms of the following DNA repair genes: XRCC3, participating in the homologous recombination repair, and hMLH1 and hMSH2, functioning in the mismatch repair. We focused on the risk the polymorphisms present in the development of lung cancer regarding the exposure to chromium. We analyzed 106 individuals; 45 patients exposed to chromium with diagnosed lung cancer and 61 healthy controls. Genotypes were determined by a PCR-RFLP method. We unravelled a potential for increased risk of lung cancer development in the hMLH1 (rs1800734) AA genotype in the recessive model. In conclusion, gene polymorphisms in the DNA repair genes underscores the risk of lung cancer development in chromium exposed individuals.

DNA tagging of blast resistant gene(s in three Brazilian rice cultivars

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S.S. Sandhu

2003-12-01

Full Text Available Rice blast is the most important fungal disease of rice and is caused by Pyricularia oryzae Sacc. (Telomorph Magnoporthe grisea Barr.. Seven randomly amplified polymorphic DNA (RAPD markers OPA5, OPG17, OPG18, OPG19, OPF9, OPF17 and OPF19 showed very clear polymorphism in resistant cultivar lines which differed from susceptible lines. By comparing different susceptible lines, nine DNA amplifications of seven primers (OPA5(1000, OPA5(1200, OPG17(700, OPG18(850, OPG19(500, OPG19(600, OPF9(600, OPF17(1200 and OPF19(600 were identified as dominant markers for the blast resistant gene in resistant cultivar lines. These loci facilitate the indirect scoring of blast resistant and blast susceptible genotypes. The codomine RAPDs markers will facilitate marker-assisted selection of the blast resistant gene in two blast resistant genotypes of rice (Labelle and Line 11 and will be useful in rice breeding programs.

A DNA methylation microarray-based study identifies ERG as a gene commonly methylated in prostate cancer.

Science.gov (United States)

Schwartzman, Jacob; Mongoue-Tchokote, Solange; Gibbs, Angela; Gao, Lina; Corless, Christopher L; Jin, Jennifer; Zarour, Luai; Higano, Celestia; True, Lawrence D; Vessella, Robert L; Wilmot, Beth; Bottomly, Daniel; McWeeney, Shannon K; Bova, G Steven; Partin, Alan W; Mori, Motomi; Alumkal, Joshi

2011-10-01

DNA methylation of promoter regions is a common event in prostate cancer, one of the most common cancers in men worldwide. Because prior reports demonstrating that DNA methylation is important in prostate cancer studied a limited number of genes, we systematically quantified the DNA methylation status of 1505 CpG dinucleotides for 807 genes in 78 paraffin-embedded prostate cancer samples and three normal prostate samples. The ERG gene, commonly repressed in prostate cells in the absence of an oncogenic fusion to the TMPRSS2 gene, was one of the most commonly methylated genes, occurring in 74% of prostate cancer specimens. In an independent group of patient samples, we confirmed that ERG DNA methylation was common, occurring in 57% of specimens, and cancer-specific. The ERG promoter is marked by repressive chromatin marks mediated by polycomb proteins in both normal prostate cells and prostate cancer cells, which may explain ERG's predisposition to DNA methylation and the fact that tumors with ERG DNA methylation were more methylated, in general. These results demonstrate that bead arrays offer a high-throughput method to discover novel genes with promoter DNA methylation such as ERG, whose measurement may improve our ability to more accurately detect prostate cancer.

Sperm DNA fragmentation affects epigenetic feature in human male pronucleus.

Science.gov (United States)

Rajabi, H; Mohseni-Kouchesfehani, H; Eslami-Arshaghi, T; Salehi, M

2018-02-01

To evaluate whether the sperm DNA fragmentation affects male pronucleus epigenetic factors, semen analysis was performed and DNA fragmentation was assessed by the method of sperm chromatin structure assay (SCSA). Human-mouse interspecies fertilisation was used to create human male pronucleus. Male pronucleus DNA methylation and H4K12 acetylation were evaluated by immunostaining. Results showed a significant positive correlation between the level of sperm DNA fragmentation and DNA methylation in male pronuclei. In other words, an increase in DNA damage caused an upsurge in DNA methylation. In the case of H4K12 acetylation, no correlation was detected between DNA damage and the level of histone acetylation in the normal group, but results for the group in which male pronuclei were derived from sperm cells with DNA fragmentation, increased DNA damage led to a decreased acetylation level. Sperm DNA fragmentation interferes with the active demethylation process and disrupts the insertion of histones into the male chromatin in the male pronucleus, following fertilisation. © 2017 Blackwell Verlag GmbH.

DNA demethylation activates genes in seed maternal integument development in rice (Oryza sativa L.).

Science.gov (United States)

Wang, Yifeng; Lin, Haiyan; Tong, Xiaohong; Hou, Yuxuan; Chang, Yuxiao; Zhang, Jian

2017-11-01

DNA methylation is an important epigenetic modification that regulates various plant developmental processes. Rice seed integument determines the seed size. However, the role of DNA methylation in its development remains largely unknown. Here, we report the first dynamic DNA methylomic profiling of rice maternal integument before and after pollination by using a whole-genome bisulfite deep sequencing approach. Analysis of DNA methylation patterns identified 4238 differentially methylated regions underpin 4112 differentially methylated genes, including GW2, DEP1, RGB1 and numerous other regulators participated in maternal integument development. Bisulfite sanger sequencing and qRT-PCR of six differentially methylated genes revealed extensive occurrence of DNA hypomethylation triggered by double fertilization at IAP compared with IBP, suggesting that DNA demethylation might be a key mechanism to activate numerous maternal controlling genes. These results presented here not only greatly expanded the rice methylome dataset, but also shed novel insight into the regulatory roles of DNA methylation in rice seed maternal integument development. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

DNA-mediated gene transfer into human diploid fibroblasts derived from normal and ataxia-telangiectasia donors: parameters for DNA transfer and properties of DNA transformants

International Nuclear Information System (INIS)

Debenham, P.G.; Webb, M.B.T.; Masson, W.K.; Cox, R.

1984-01-01

An investigation was made of the feasibility of DNA-mediated gene transfer into human diploid fibroblasts derived from patients with the radiation sensitive syndrome ataxia-telangiectasia (A-T) and from a normal donor. Although they are markedly different in their growth characteristics, both normal and A-T strains give similar frequencies for DNA transfer in a model system using the recombinant plasmid pSV2-gpt. pSV2-gpt DNA transformants arise with a frequency between 10 -5 and 10 -4 per viable cell. Analysis of such transformants, although possible, is severely handicapped by the limited clonal life span of diploid human cells. Despite these problems it may be concluded that diploid human fibroblasts are competent recipients for DNA-mediated gene transfer and the putative repair deficiency of A-T does not markedly effect the efficiency of this process. (author)

A data mining approach for classifying DNA repair genes into ageing-related or non-ageing-related

Directory of Open Access Journals (Sweden)

Vasieva Olga

2011-01-01

Full Text Available Abstract Background The ageing of the worldwide population means there is a growing need for research on the biology of ageing. DNA damage is likely a key contributor to the ageing process and elucidating the role of different DNA repair systems in ageing is of great interest. In this paper we propose a data mining approach, based on classification methods (decision trees and Naive Bayes, for analysing data about human DNA repair genes. The goal is to build classification models that allow us to discriminate between ageing-related and non-ageing-related DNA repair genes, in order to better understand their different properties. Results The main patterns discovered by the classification methods are as follows: (a the number of protein-protein interactions was a predictor of DNA repair proteins being ageing-related; (b the use of predictor attributes based on protein-protein interactions considerably increased predictive accuracy of attributes based on Gene Ontology (GO annotations; (c GO terms related to "response to stimulus" seem reasonably good predictors of ageing-relatedness for DNA repair genes; (d interaction with the XRCC5 (Ku80 protein is a strong predictor of ageing-relatedness for DNA repair genes; and (e DNA repair genes with a high expression in T lymphocytes are more likely to be ageing-related. Conclusions The above patterns are broadly integrated in an analysis discussing relations between Ku, the non-homologous end joining DNA repair pathway, ageing and lymphocyte development. These patterns and their analysis support non-homologous end joining double strand break repair as central to the ageing-relatedness of DNA repair genes. Our work also showcases the use of protein interaction partners to improve accuracy in data mining methods and our approach could be applied to other ageing-related pathways.

Alteration of gene expression and DNA methylation in drug-resistant gastric cancer.

Science.gov (United States)

Maeda, Osamu; Ando, Takafumi; Ohmiya, Naoki; Ishiguro, Kazuhiro; Watanabe, Osamu; Miyahara, Ryoji; Hibi, Yoko; Nagai, Taku; Yamada, Kiyofumi; Goto, Hidemi

2014-04-01

The mechanisms of drug resistance in cancer are not fully elucidated. To study the drug resistance of gastric cancer, we analyzed gene expression and DNA methylation profiles of 5-fluorouracil (5-FU)- and cisplatin (CDDP)-resistant gastric cancer cells and biopsy specimens. Drug-resistant gastric cancer cells were established with culture for >10 months in a medium containing 5-FU or CDDP. Endoscopic biopsy specimens were obtained from gastric cancer patients who underwent chemotherapy with oral fluoropyrimidine S-1 and CDDP. Gene expression and DNA methylation analyses were performed using microarray, and validated using real-time PCR and pyrosequencing, respectively. Out of 17,933 genes, 541 genes commonly increased and 569 genes decreased in both 5-FU- and CDDP-resistant AGS cells. Genes with expression changed by drugs were related to GO term 'extracellular region' and 'p53 signaling pathway' in both 5-FU- and CDDP-treated cells. Expression of 15 genes including KLK13 increased and 12 genes including ETV7 decreased, in both drug-resistant cells and biopsy specimens of two patients after chemotherapy. Out of 10,365 genes evaluated with both expression microarray and methylation microarray, 74 genes were hypermethylated and downregulated, or hypomethylated and upregulated in either 5-FU-resistant or CDDP-resistant cells. Of these genes, expression of 21 genes including FSCN1, CPT1C and NOTCH3, increased from treatment with a demethylating agent. There are alterations of gene expression and DNA methylation in drug-resistant gastric cancer; they may be related to mechanisms of drug resistance and may be useful as biomarkers of gastric cancer drug sensitivity.

Sequence homology and expression profile of genes associated with DNA repair pathways in Mycobacterium leprae.

Science.gov (United States)

Sharma, Mukul; Vedithi, Sundeep Chaitanya; Das, Madhusmita; Roy, Anindya; Ebenezer, Mannam

2017-01-01

Survival of Mycobacterium leprae, the causative bacteria for leprosy, in the human host is dependent to an extent on the ways in which its genome integrity is retained. DNA repair mechanisms protect bacterial DNA from damage induced by various stress factors. The current study is aimed at understanding the sequence and functional annotation of DNA repair genes in M. leprae. T he genome of M. leprae was annotated using sequence alignment tools to identify DNA repair genes that have homologs in Mycobacterium tuberculosis and Escherichia coli. A set of 96 genes known to be involved in DNA repair mechanisms in E. coli and Mycobacteriaceae were chosen as a reference. Among these, 61 were identified in M. leprae based on sequence similarity and domain architecture. The 61 were classified into 36 characterized gene products (59%), 11 hypothetical proteins (18%), and 14 pseudogenes (23%). All these genes have homologs in M. tuberculosis and 49 (80.32%) in E. coli. A set of 12 genes which are absent in E. coli were present in M. leprae and in Mycobacteriaceae. These 61 genes were further investigated for their expression profiles in the whole transcriptome microarray data of M. leprae which was obtained from the signal intensities of 60bp probes, tiling the entire genome with 10bp overlaps. It was noted that transcripts corresponding to all the 61 genes were identified in the transcriptome data with varying expression levels ranging from 0.18 to 2.47 fold (normalized with 16SrRNA). The mRNA expression levels of a representative set of seven genes ( four annotated and three hypothetical protein coding genes) were analyzed using quantitative Polymerase Chain Reaction (qPCR) assays with RNA extracted from skin biopsies of 10 newly diagnosed, untreated leprosy cases. It was noted that RNA expression levels were higher for genes involved in homologous recombination whereas the genes with a low level of expression are involved in the direct repair pathway. This study provided

DNA methylation and gene expression of TXNIP in adult offspring of women with diabetes in pregnancy.

Directory of Open Access Journals (Sweden)

Azadeh Houshmand-Oeregaard

Full Text Available Fetal exposure to maternal diabetes increases the risk of type 2 diabetes (T2DM, possibly mediated by epigenetic mechanisms. Low blood TXNIP DNA methylation has been associated with elevated glucose levels and risk of T2DM, and increased skeletal muscle TXNIP gene expression was reported in subjects with impaired glucose metabolism or T2DM. Subcutaneous adipose tissue (SAT and skeletal muscle play a key role in the control of whole body glucose metabolism and insulin action. The extent to which TXNIP DNA methylation levels are decreased and/or gene expression levels increased in SAT or skeletal muscle of a developmentally programmed at-risk population is unknown.The objective of this study was to investigate TXNIP DNA methylation and gene expression in SAT and skeletal muscle, and DNA methylation in blood, from adult offspring of women with gestational diabetes (O-GDM, n = 82 or type 1 diabetes (O-T1DM, n = 67 in pregnancy compared with offspring of women from the background population (O-BP, n = 57.SAT TXNIP DNA methylation was increased (p = 0.032 and gene expression decreased (p = 0.001 in O-GDM, but these differences were attenuated after adjustment for confounders. Neither blood/muscle TXNIP DNA methylation nor muscle gene expression differed between groups.We found no evidence of decreased TXNIP DNA methylation or increased gene expression in metabolic target tissues of offspring exposed to maternal diabetes. Further studies are needed to confirm and understand the paradoxical SAT TXNIP DNA methylation and gene expression changes in O-GDM subjects.

Junk DNA enhances pEI-based non-viral gene delivery

NARCIS (Netherlands)

Gaal, E.V.B. van; Oosting, R.S.; Hennink, W.E.; Crommelin, D.J.A.; Mastrobattista, E.

Gene therapy aims at delivering exogenous DNA into the nuclei of target cells to establish expression of a therapeutic protein. Non-viral gene delivery is examined as a safer alternative to viral approaches, but is presently characterized by a low efficiency. In the past years several non-viral

Amphibian antimicrobial peptide fallaxin analogue FL9 affects virulence gene expression and DNA replication in Staphylococcus aureus.

Science.gov (United States)

Gottschalk, Sanne; Gottlieb, Caroline T; Vestergaard, Martin; Hansen, Paul R; Gram, Lone; Ingmer, Hanne; Thomsen, Line E

2015-12-01

The rapid rise in antibiotic-resistant pathogens is causing increased health concerns, and consequently there is an urgent need for novel antimicrobial agents. Antimicrobial peptides (AMPs), which have been isolated from a wide range of organisms, represent a very promising class of novel antimicrobials. In the present study, the analogue FL9, based on the amphibian AMP fallaxin, was studied to elucidate its mode of action and antibacterial activity against the human pathogen Staphylococcus aureus. Our data showed that FL9 may have a dual mode of action against S. aureus. At concentrations around the MIC, FL9 bound DNA, inhibited DNA synthesis and induced the SOS DNA damage response, whereas at concentrations above the MIC the interaction between S. aureus and FL9 led to membrane disruption. The antibacterial activity of the peptide was maintained over a wide range of NaCl and MgCl(2) concentrations and at alkaline pH, while it was compromised by acidic pH and exposure to serum. Furthermore, at subinhibitory concentrations of FL9, S. aureus responded by increasing the expression of two major virulence factor genes, namely the regulatory rnaIII and hla, encoding α-haemolysin. In addition, the S. aureus-encoded natural tolerance mechanisms included peptide cleavage and the addition of positive charge to the cell surface, both of which minimized the antimicrobial activity of FL9. Our results add new information about FL9 and its effect on S. aureus, which may aid in the future development of analogues with improved therapeutic potential.

Identification and localization of a gene that specifies production of Escherichia coli DNA topoisomerase I

International Nuclear Information System (INIS)

Trucksis, M.; Depew, R.E.

1981-01-01

A gene that specifies production of Escherichia coli DNA topoisomerase I (ω protein) was identified with the aid of a radioimmunoassay for this protein. E. coli DNA topoisomerase I was produced by Salmonella typhimurium merodiploids that harbored E. coli plasmid F' 123, but not by strains that lost this plasmid. Analysis of strains with spontaneous deletions of F' 123 showed that the gene, topA, required for production of the E. coli ω protein was between the trp operon and the cysB gene. Deletions that eliminated topA also eliminated the supX gene. We suggest that topA is the structural gene of E. coli DNA topoisomerase I and that topA is identical to supX

DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines.

Science.gov (United States)

Yamagishi, J; Isobe, R; Takebuchi, T; Bando, H

2003-03-01

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef- 6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.

Transgelin gene is frequently downregulated by promoter DNA hypermethylation in breast cancer.

Science.gov (United States)

Sayar, Nilufer; Karahan, Gurbet; Konu, Ozlen; Bozkurt, Betul; Bozdogan, Onder; Yulug, Isik G

2015-01-01

CpG hypermethylation in gene promoters is a frequent mechanism of tumor suppressor gene silencing in various types of cancers. It usually occurs at early steps of cancer progression and can be detected easily, giving rise to development of promising biomarkers for both detection and progression of cancer, including breast cancer. 5-aza-2'-deoxycytidine (AZA) is a DNA demethylating and anti-cancer agent resulting in induction of genes suppressed via DNA hypermethylation. Using microarray expression profiling of AZA- or DMSO-treated breast cancer and non-tumorigenic breast (NTB) cells, we identified for the first time TAGLN gene as a target of DNA hypermethylation in breast cancer. TAGLN expression was significantly and frequently downregulated via promoter DNA hypermethylation in breast cancer cells compared to NTB cells, and also in 13/21 (61.9 %) of breast tumors compared to matched normal tissues. Analyses of public microarray methylation data showed that TAGLN was also hypermethylated in 63.02 % of tumors compared to normal tissues; relapse-free survival of patients was worse with higher TAGLN methylation; and methylation levels could discriminate between tumors and healthy tissues with 83.14 % sensitivity and 100 % specificity. Additionally, qRT-PCR and immunohistochemistry experiments showed that TAGLN expression was significantly downregulated in two more independent sets of breast tumors compared to normal tissues and was lower in tumors with poor prognosis. Colony formation was increased in TAGLN silenced NTB cells, while decreased in overexpressing BC cells. TAGLN gene is frequently downregulated by DNA hypermethylation, and TAGLN promoter methylation profiles could serve as a future diagnostic biomarker, with possible clinical impact regarding the prognosis in breast cancer.

Varicella-zoster virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription.

Science.gov (United States)

Khalil, Mohamed I; Sommer, Marvin H; Hay, John; Ruyechan, William T; Arvin, Ann M

2015-07-01

The VZV genome has two origins of DNA replication (oriS), each of which consists of an AT-rich sequence and three origin binding protein (OBP) sites called Box A, C and B. In these experiments, the mutation in the core sequence CGC of the Box A and C not only inhibited DNA replication but also inhibited both ORF62 and ORF63 expression in reporter gene assays. In contrast the Box B mutation did not influence DNA replication or flanking gene transcription. These results suggest that efficient DNA replication enhances ORF62 and ORF63 transcription. Recombinant viruses carrying these mutations in both sites and one with a deletion of the whole oriS were constructed. Surprisingly, the recombinant virus lacking both copies of oriS retained the capacity to replicate in melanoma and HELF cells suggesting that VZV has another origin of DNA replication. Copyright © 2015 Elsevier Inc. All rights reserved.

Stress-induced gene expression and behavior are controlled by DNA methylation and methyl donor availability in the dentate gyrus

Science.gov (United States)

Saunderson, Emily A.; Spiers, Helen; Gutierrez-Mecinas, Maria; Trollope, Alexandra F.; Shaikh, Abeera; Mill, Jonathan; Reul, Johannes M. H. M.

2016-01-01

Stressful events evoke long-term changes in behavioral responses; however, the underlying mechanisms in the brain are not well understood. Previous work has shown that epigenetic changes and immediate-early gene (IEG) induction in stress-activated dentate gyrus (DG) granule neurons play a crucial role in these behavioral responses. Here, we show that an acute stressful challenge [i.e., forced swimming (FS)] results in DNA demethylation at specific CpG (5′-cytosine–phosphate–guanine-3′) sites close to the c-Fos (FBJ murine osteosarcoma viral oncogene homolog) transcriptional start site and within the gene promoter region of Egr-1 (early growth response protein 1) specifically in the DG. Administration of the (endogenous) methyl donor S-adenosyl methionine (SAM) did not affect CpG methylation and IEG gene expression at baseline. However, administration of SAM before the FS challenge resulted in an enhanced CpG methylation at the IEG loci and suppression of IEG induction specifically in the DG and an impaired behavioral immobility response 24 h later. The stressor also specifically increased the expression of the de novo DNA methyltransferase Dnmt3a [DNA (cytosine-5-)-methyltransferase 3 alpha] in this hippocampus region. Moreover, stress resulted in an increased association of Dnmt3a enzyme with the affected CpG loci within the IEG genes. No effects of SAM were observed on stress-evoked histone modifications, including H3S10p-K14ac (histone H3, phosphorylated serine 10 and acetylated lysine-14), H3K4me3 (histone H3, trimethylated lysine-4), H3K9me3 (histone H3, trimethylated lysine-9), and H3K27me3 (histone H3, trimethylated lysine-27). We conclude that the DNA methylation status of IEGs plays a crucial role in FS-induced IEG induction in DG granule neurons and associated behavioral responses. In addition, the concentration of available methyl donor, possibly in conjunction with Dnmt3a, is critical for the responsiveness of dentate neurons to environmental

Quantification of differential gene expression by multiplexed targeted resequencing of cDNA

Science.gov (United States)

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

Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

Energy Technology Data Exchange (ETDEWEB)

Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon [College of Medicine, Univ. of Korea, Seoul (Korea, Republic of)

2003-07-01

Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology.

Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

International Nuclear Information System (INIS)

Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon

2003-01-01

Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology

cDNA fingerprinting of osteoprogenitor cells to isolate differentiation stage-specific genes.

OpenAIRE

Candeliere, G A; Rao, Y; Floh, A; Sandler, S D; Aubin, J E

1999-01-01

A cDNA fingerprinting strategy was developed to identify genes based on their differential expression pattern during osteoblast development. Preliminary biological and molecular staging of cDNA pools prepared by global amplification PCR allowed discrim-inating choices to be made in selection of expressed sequence tags (ESTs) to be isolated. Sequencing of selected ESTs confirmed that both known and novel genes can be isolated from any developmental stage of interest, e.g. from primitive progen...

Species-level para- and polyphyly in DNA barcode gene trees

DEFF Research Database (Denmark)

Mutanen, Marko; Kivelä, Sami M.; Vos, Rutger A.

2016-01-01

was paid to accurate species identification to ensure data integrity. We investigated the effects of tree-building method, sampling effort, and other methodological issues, all of which can influence estimates of non-monophyly. We found a 12% incidence of non-monophyly, a value significantly lower than...... between species and gene genealogies, as indicated by situations where conspecific individuals do not form a monophyletic cluster in a gene tree. In two previous reviews, non-monophyly has been reported as being common in mitochondrial DNA gene trees. We developed a novel web service "Monophylizer......" to detect non-monophyly in phylogenetic trees and used it to ascertain the incidence of species non-monophyly in COI (a.k.a. cox1) barcode sequence data from 4977 species and 41,583 specimens of European Lepidoptera, the largest data set of DNA barcodes analyzed from this regard. Particular attention...

Gene-gun DNA vaccination aggravates respiratory syncytial virus-induced pneumonitis

DEFF Research Database (Denmark)

Bartholdy, Christina; Olszewska, Wieslawa; Stryhn, Anette

2004-01-01

elicited with recombinant vaccinia virus expressing the complete RSV M2 protein, but stronger than those induced by a similar DNA construct without the beta2m gene. DNA vaccination led to enhanced pulmonary disease after RSV challenge, with increased weight loss and cell recruitment to the lung. Depletion......A CD8+ T-cell memory response to respiratory syncytial virus (RSV) was generated by using a DNA vaccine construct encoding the dominant Kd-restricted epitope from the viral transcription anti-terminator protein M2 (M2(82-90)), linked covalently to human beta2-microglobulin (beta2m). Cutaneous gene...... of CD8+ T cells reduced, but did not abolish, enhancement of disease. Mice vaccinated with a construct encoding a class I-restricted lymphocytic choriomeningitis virus epitope and beta2m suffered more severe weight loss after RSV infection than unvaccinated RSV-infected mice, although RSV-specific CD8...

Unwinding after high salinity stress: Pea DNA helicase 45 over- expression in tobacco confers high salinity tolerance without affecting yield (abstract)

International Nuclear Information System (INIS)

Tuteja, N.

2005-01-01

Soil salinity is an increasing threat for agriculture and is a major factor in reducing plant productivity; therefore, it is necessary to obtain salinity-tolerant varieties. A typical characteristic of soil salinity is the induction of multiple stress- inducible genes. Some of the genes encoding osmolytes, ion channels or enzymes are able to confer salinity-tolerant phenotypes when transferred to sensitive plants. As salinity stress affects the cellular gene-expression machinery, it is evident that molecules involved in nucleic acid processing including helicases, are likely to be affected as well. DNA helicases unwind duplex DNA and are involved in replication, repair, recombination and transcription while RNA helicases unfold the secondary structures in RNA and are involved in transcription, ribosome biogenesis and translation initiation. We have earlier reported the isolation of a pea DNA helicase 45 (PDH45) that exhibits striking homology with eIF-4A (Plant J. 24:219-230,2000). Here we report that PDH45 mRNA is induced in pea seedlings in response to high salt and its over- expression driven by a constitutive CAMV-355-promoter in tobacco plants confers salinity tolerance, thus suggesting a new pathway for manipulating stress tolerance in crop plants. The T0 transgenic plants showed high-levels of PDH45 protein in normal and stress conditions, as compared to wild type (WT) plants. The T0 transgenics also showed tolerance to high salinity as tested by a leaf disc senescence assay. The T1 transgenics were able to grow to maturity and set normal viable seeds under continuous salinity stress, without any reduction in plant yield, in terms of seed weight. Measurement of Na/sup +/ ions in different parts of the plant showed higher accumulation in the old leaves and negligible in seeds of T1 transgenic lines as compared with the WT plants. The possible mechanism of salinity tolerance will be discussed. Over-expression of PDH45 provides a possible example of the

Molecular and clinical characterization of the myopathic form of mitochondrial DNA depletion syndrome caused by mutations in the thymidine kinase (TK2) gene.

Science.gov (United States)

Chanprasert, Sirisak; Wang, Jing; Weng, Shao-Wen; Enns, Gregory M; Boué, Daniel R; Wong, Brenda L; Mendell, Jerry R; Perry, Deborah A; Sahenk, Zarife; Craigen, William J; Alcala, Francisco J Climent; Pascual, Juan M; Melancon, Serge; Zhang, Victor Wei; Scaglia, Fernando; Wong, Lee-Jun C

2013-01-01

Mitochondrial DNA (mtDNA) depletion syndromes (MDSs) are a clinically and molecularly heterogeneous group of mitochondrial cytopathies characterized by severe mtDNA copy number reduction in affected tissues. Clinically, MDSs are mainly categorized as myopathic, encephalomyopathic, hepatocerebral, or multi-systemic forms. To date, the myopathic form of MDS is mainly caused by mutations in the TK2 gene, which encodes thymidine kinase 2, the first and rate limiting step enzyme in the phosphorylation of pyrimidine nucleosides. We analyzed 9 unrelated families with 11 affected subjects exhibiting the myopathic form of MDS, by sequencing the TK2 gene. Twelve mutations including 4 novel mutations were detected in 9 families. Skeletal muscle specimens were available from 7 out of 11 subjects. Respiratory chain enzymatic activities in skeletal muscle were measured in 6 subjects, and enzymatic activities were reduced in 3 subjects. Quantitative analysis of mtDNA content in skeletal muscle was performed in 5 subjects, and marked mtDNA content reduction was observed in each. In addition, we outline the molecular and clinical characteristics of this syndrome in a total of 52 patients including those previously reported, and a total of 36 TK2 mutations are summarized. Clinically, hypotonia and proximal muscle weakness are the major phenotypes present in all subjects. In summary, our study expands the molecular and clinical spectrum associated with TK2 deficiency. © 2013.

AKT phosphorylates H3-threonine 45 to facilitate termination of gene transcription in response to DNA damage.

Science.gov (United States)

Lee, Jong-Hyuk; Kang, Byung-Hee; Jang, Hyonchol; Kim, Tae Wan; Choi, Jinmi; Kwak, Sojung; Han, Jungwon; Cho, Eun-Jung; Youn, Hong-Duk

2015-05-19

Post-translational modifications of core histones affect various cellular processes, primarily through transcription. However, their relationship with the termination of transcription has remained largely unknown. In this study, we show that DNA damage-activated AKT phosphorylates threonine 45 of core histone H3 (H3-T45). By genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis, H3-T45 phosphorylation was distributed throughout DNA damage-responsive gene loci, particularly immediately after the transcription termination site. H3-T45 phosphorylation pattern showed close-resemblance to that of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation, which establishes the transcription termination signal. AKT1 was more effective than AKT2 in phosphorylating H3-T45. Blocking H3-T45 phosphorylation by inhibiting AKT or through amino acid substitution limited RNA decay downstream of mRNA cleavage sites and decreased RNA polymerase II release from chromatin. Our findings suggest that AKT-mediated phosphorylation of H3-T45 regulates the processing of the 3' end of DNA damage-activated genes to facilitate transcriptional termination. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genetic manipulation in Sulfolobus islandicus and functional analysis of DNA repair genes

DEFF Research Database (Denmark)

Zhang, Changyi; Tian, Bin; Li, Suming

2013-01-01

Recently, a novel gene-deletion method was developed for the crenarchaeal model Sulfolobus islandicus, which is a suitable tool for addressing gene essentiality in depth. Using this technique, we have investigated functions of putative DNA repair genes by constructing deletion mutants and studying...

Densely ionizing radiation affects DNA methylation of selective LINE-1 elements

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Prior, Sara; Miousse, Isabelle R. [Department of Environmental and Occupational Health, Fay W. Boozman College of Public Health, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Nzabarushimana, Etienne [Department of Environmental and Occupational Health, Fay W. Boozman College of Public Health, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Department of Bioinformatics, School of Informatics and Computing, Indiana University, Bloomington, IN 47405 (United States); Pathak, Rupak [Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Skinner, Charles; Kutanzi, Kristy R. [Department of Environmental and Occupational Health, Fay W. Boozman College of Public Health, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Allen, Antiño R. [Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Raber, Jacob [Departments of Behavioral Neuroscience, Neurology, and Radiation Medicine, Division of Neuroscience, ONPRC, Oregon Health & Science University, Portland, OR 97239 (United States); Tackett, Alan J. [Department of Biochemistry, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Hauer-Jensen, Martin [Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Nelson, Gregory A. [Department of Basic Sciences, Division of Radiation Research, Loma Linda University, Loma Linda, CA 92350 (United States); and others

2016-10-15

Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2′-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promoter type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5′-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR. - Highlights: • DNA methylation of LINE-1 elements is dependent on their evolutionary age. • Densely ionizing radiation affects DNA methylation of selective LINE-1 elements. • Radiation-induced reactivation of LINE-1 is DNA methylation-independent. • Histone modifications dictate the transcriptional activity of LINE-1.

Densely ionizing radiation affects DNA methylation of selective LINE-1 elements

International Nuclear Information System (INIS)

Prior, Sara; Miousse, Isabelle R.; Nzabarushimana, Etienne; Pathak, Rupak; Skinner, Charles; Kutanzi, Kristy R.; Allen, Antiño R.; Raber, Jacob; Tackett, Alan J.; Hauer-Jensen, Martin; Nelson, Gregory A.

2016-01-01

Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2′-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promoter type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5′-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR. - Highlights: • DNA methylation of LINE-1 elements is dependent on their evolutionary age. • Densely ionizing radiation affects DNA methylation of selective LINE-1 elements. • Radiation-induced reactivation of LINE-1 is DNA methylation-independent. • Histone modifications dictate the transcriptional activity of LINE-1.

Hda-mediated inactivation of the DnaA protein and dnaA gene autoregulation act in concert to ensure homeostatic maintenance of the Escherichia coli chromosome.

Science.gov (United States)

Riber, Leise; Olsson, Jan A; Jensen, Rasmus B; Skovgaard, Ole; Dasgupta, Santanu; Marinus, Martin G; Løbner-Olesen, Anders

2006-08-01

Initiation of DNA replication in Eschericia coli requires the ATP-bound form of the DnaA protein. The conversion of DnaA-ATP to DnaA-ADP is facilitated by a complex of DnaA, Hda (homologous to DnaA), and DNA-loaded beta-clamp proteins in a process termed RIDA (regulatory inactivation of DnaA). Hda-deficient cells initiate replication at each origin mainly once per cell cycle, and the rare reinitiation events never coincide with the end of the origin sequestration period. Therefore, RIDA is not the predominant mechanism to prevent immediate reinitiation from oriC. The cellular level of Hda correlated directly with dnaA gene expression such that Hda deficiency led to reduced dnaA gene expression, and overproduction of Hda led to DnaA overproduction. Hda-deficient cells were very sensitive to variations in the cellular level of DnaA, and DnaA overproduction led to uncontrolled initiation of replication from oriC, causing severe growth retardation or cell death. Based on these observations, we propose that both RIDA and dnaA gene autoregulation are required as homeostatic mechanisms to ensure that initiation of replication occurs at the same time relative to cell mass in each cell cycle.

Effect of ionizing radiation on DNA-mediated gene transfer efficiency

International Nuclear Information System (INIS)

Rubin, J.S.; Hall, E.J.; Hei, T.K.

1986-01-01

Ionizing radiation causes a number of molecular changes in cells including DNA damage and gene amplification. In this study the authors examined whether radiation can effect the efficiency of integration and expression of exogenous DNA sequences. They examined both 137 Cs γ rays and various monoenergetic neutron beams. This enabled them to test whether the LET or RBE of the radiation had any effect. Rat2 cells were transfected with various amounts of the bacterial plasmid pSV2-GPT along with carrier DNA for 24 hours

The FRIABLE1 gene product affects cell adhesion in Arabidopsis.

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Lutz Neumetzler

Full Text Available Cell adhesion in plants is mediated predominantly by pectins, a group of complex cell wall associated polysaccharides. An Arabidopsis mutant, friable1 (frb1, was identified through a screen of T-DNA insertion lines that exhibited defective cell adhesion. Interestingly, the frb1 plants displayed both cell and organ dissociations and also ectopic defects in organ separation. The FRB1 gene encodes a Golgi-localized, plant specific protein with only weak sequence similarities to known proteins (DUF246. Unlike other cell adhesion deficient mutants, frb1 mutants do not have reduced levels of adhesion related cell wall polymers, such as pectins. Instead, FRB1 affects the abundance of galactose- and arabinose-containing oligosaccharides in the Golgi. Furthermore, frb1 mutants displayed alteration in pectin methylesterification, cell wall associated extensins and xyloglucan microstructure. We propose that abnormal FRB1 action has pleiotropic consequences on wall architecture, affecting both the extensin and pectin matrices, with consequent changes to the biomechanical properties of the wall and middle lamella, thereby influencing cell-cell adhesion.

A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1

International Nuclear Information System (INIS)

Sebastian, J.; Sancar, G.B.

1991-01-01

The PHR1 gene of Saccharomyces cerevisiae encodes the DNA repair enzyme photolyase. Transcription of PHR1 increases in response to treatment of cells with 254-nm radiation and chemical agents that damage DNA. The authors here the identification of a damage-responsive DNA binding protein, termed photolyase regulatory protein (PRP), and its cognate binding site, termed the PHR1 transcription after DNA damage. PRP activity, monitored by electrophoretic-mobility-shift assay, was detected in cells during normal growth but disappeared within 30 min after irradiation. Copper-phenanthroline footprinting of PRP-DNA complexes revealed that PRP protects a 39-base-pair region of PHR1 5' flanking sequence beginning 40 base pairs upstream from the coding sequence. Thus these observations establish that PRP is a damage-responsive repressor of PHR1 transcription

Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes

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Fu Juanjuan

2011-07-01

Full Text Available Abstract To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP or Gaussia luciferase (G.luc were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

Horizontal gene transfer from macrophages to ischemic muscles upon delivery of naked DNA with Pluronic block copolymers.

Science.gov (United States)

Mahajan, Vivek; Gaymalov, Zagit; Alakhova, Daria; Gupta, Richa; Zucker, Irving H; Kabanov, Alexander V

2016-01-01

Intramuscular administration of plasmid DNA (pDNA) with non-ionic Pluronic block copolymers increases gene expression in injected muscles and lymphoid organs. We studied the role of immune cells in muscle transfection upon inflammation. Local inflammation in murine hind limb ischemia model (MHLIM) drastically increased DNA, RNA and expressed protein levels in ischemic muscles injected with pDNA/Pluronic. The systemic inflammation (MHLIM or peritonitis) also increased expression of pDNA/Pluronic in the muscles. When pDNA/Pluronic was injected in ischemic muscles the reporter gene, Green Fluorescent Protein (GFP) co-localized with desmin(+) muscle fibers and CD11b(+) macrophages (MØs), suggesting transfection of MØs along with the muscle cells. P85 enhanced (∼ 4 orders) transfection of MØs with pDNA in vitro. Moreover, adoptively transferred MØs were shown to pass the transgene to inflamed muscle cells in MHLIM. Using a co-culture of myotubes (MTs) and transfected MØs expressing a reporter gene under constitutive (cmv-luciferase) or muscle specific (desmin-luciferase) promoter we demonstrated that P85 enhances horizontal gene transfer from MØ to MTs. Therefore, MØs can play an important role in muscle transfection with pDNA/Pluronic during inflammation, with both inflammation and Pluronic contributing to the increased gene expression. pDNA/Pluronic has potential for therapeutic gene delivery in muscle pathologies that involve inflammation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cloning and characterization of human DNA repair genes

International Nuclear Information System (INIS)

Thompson, L.H.; Brookman, K.W.; Weber, C.A.; Salazar, E.P.; Stewart, S.A.; Carrano, A.V.

1987-01-01

The isolation of two addition human genes that give efficient restoration of the repair defects in other CHO mutant lines is reported. The gene designated ERCC2 (Excision Repair Complementing Chinese hamster) corrects mutant UV5 from complementation group 1. They recently cloned this gene by first constructing a secondary transformant in which the human gene was shown to have become physically linked to the bacterial gpt dominant-marker gene by cotransfer in calcium phosphate precipitates in the primary transfection. Transformants expressing both genes were recovered by selecting for resistance to both UV radiation and mycophenolic acid. Using similar methods, the human gene that corrects CHO mutant EM9 was isolated in cosmids and named XRCC1 (X-ray Repair Complementing Chinese hamster). In this case, transformants were recovered by selecting for resistance to CldUrd, which kills EM9 very efficiently. In both genomic and cosmid transformants, the XRCC1 gene restored resistance to the normal range. DNA repair was studied using the kinetics of strand-break rejoining, which was measured after exposure to 137 Cs γ-rays

Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

International Nuclear Information System (INIS)

Gustafsson, Ann-Sofie; Abramenkovs, Andris; Stenerlöw, Bo

2014-01-01

Highlights: • We reduced the level of DNA-PKcs with siRNA and examined cells after γ-irradiation. • Low DNA-PKcs levels lead to radiosensitivity but did not affect repair of DSB. • Low DNA-PKcs levels may block progression of mitosis. • DNA-PKcs role in mitotic progression is independent of its role in DSB repair. • We suggest different mechanisms by which loss of DNA-PKcs function sensitize cells. - Abstract: Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure

Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

Energy Technology Data Exchange (ETDEWEB)

Gustafsson, Ann-Sofie, E-mail: ann-sofie.gustafsson@bms.uu.se; Abramenkovs, Andris; Stenerlöw, Bo

2014-11-15

Highlights: • We reduced the level of DNA-PKcs with siRNA and examined cells after γ-irradiation. • Low DNA-PKcs levels lead to radiosensitivity but did not affect repair of DSB. • Low DNA-PKcs levels may block progression of mitosis. • DNA-PKcs role in mitotic progression is independent of its role in DSB repair. • We suggest different mechanisms by which loss of DNA-PKcs function sensitize cells. - Abstract: Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure

Sequence homology and expression profile of genes associated with dna repair pathways in Mycobacterium leprae

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Mukul Sharma

2017-01-01

Full Text Available Background: Survival of Mycobacterium leprae, the causative bacteria for leprosy, in the human host is dependent to an extent on the ways in which its genome integrity is retained. DNA repair mechanisms protect bacterial DNA from damage induced by various stress factors. The current study is aimed at understanding the sequence and functional annotation of DNA repair genes in M. leprae. Methods: T he genome of M. leprae was annotated using sequence alignment tools to identify DNA repair genes that have homologs in Mycobacterium tuberculosis and Escherichia coli. A set of 96 genes known to be involved in DNA repair mechanisms in E. coli and Mycobacteriaceae were chosen as a reference. Among these, 61 were identified in M. leprae based on sequence similarity and domain architecture. The 61 were classified into 36 characterized gene products (59%, 11 hypothetical proteins (18%, and 14 pseudogenes (23%. All these genes have homologs in M. tuberculosis and 49 (80.32% in E. coli. A set of 12 genes which are absent in E. coli were present in M. leprae and in Mycobacteriaceae. These 61 genes were further investigated for their expression profiles in the whole transcriptome microarray data of M. leprae which was obtained from the signal intensities of 60bp probes, tiling the entire genome with 10bp overlaps. Results: It was noted that transcripts corresponding to all the 61 genes were identified in the transcriptome data with varying expression levels ranging from 0.18 to 2.47 fold (normalized with 16SrRNA. The mRNA expression levels of a representative set of seven genes ( four annotated and three hypothetical protein coding genes were analyzed using quantitative Polymerase Chain Reaction (qPCR assays with RNA extracted from skin biopsies of 10 newly diagnosed, untreated leprosy cases. It was noted that RNA expression levels were higher for genes involved in homologous recombination whereas the genes with a low level of expression are involved in the

Homology of genes for exopolysaccharide synthesis in Rhizobium leguminosarum and effect of cloned exo genes on nodule formation

International Nuclear Information System (INIS)

Skorupska, A.; Derylo, M.

1993-01-01

A 5.4 kb BamHI fragment of Rhizobium leguminosarum (R.l.) bv. trifolii TA1 was found to carry genes involved in exopolysaccharide synthesis (exo genes). This fragment was strongly hybridized to the total DNA from DNA from R.l. bv. viciae and bv. phaseoli digested with EcoRI. No homology was found with total DNA of R. melilotic and Rhizobium sp. NGR 234. The exo genes R.l. bv. trifolii TA1 conjugally introduced into R.l. bv. viciae 1302 considerably affected the symbiosis: the nodules induced on vetch were abortive and did not fix nitrogen. On the other hand, Phaseolus beans infected with R.l. bv. trifolii exo genes formed the nitrogen-fixing nodules. It can be conduced that additional copies of exo genes introduced into wild type R.l. strains can disturb the synthesis of acidic exopolysaccharides and affect symbiosis of the plants forming indeterminate nodules, but do not affect symbiosis of the plants forming the determinate nodules. (author). 29 refs, 2 figs, 2 tabs

Silencing of PINK1 expression affects mitochondrial DNA and oxidative phosphorylation in dopaminergic cells.

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Matthew E Gegg

Full Text Available Mitochondrial dysfunction has been implicated in the pathogenesis of Parkinson's disease (PD. Impairment of the mitochondrial electron transport chain (ETC and an increased frequency in deletions of mitochondrial DNA (mtDNA, which encodes some of the subunits of the ETC, have been reported in the substantia nigra of PD brains. The identification of mutations in the PINK1 gene, which cause an autosomal recessive form of PD, has supported mitochondrial involvement in PD. The PINK1 protein is a serine/threonine kinase localized in mitochondria and the cytosol. Its precise function is unknown, but it is involved in neuroprotection against a variety of stress signalling pathways.In this report we have investigated the effect of silencing PINK1 expression in human dopaminergic SH-SY5Y cells by siRNA on mtDNA synthesis and ETC function. Loss of PINK1 expression resulted in a decrease in mtDNA levels and mtDNA synthesis. We also report a concomitant loss of mitochondrial membrane potential and decreased mitochondrial ATP synthesis, with the activity of complex IV of the ETC most affected. This mitochondrial dysfunction resulted in increased markers of oxidative stress under basal conditions and increased cell death following treatment with the free radical generator paraquat.This report highlights a novel function of PINK1 in mitochondrial biogenesis and a role in maintaining mitochondrial ETC activity. Dysfunction of both has been implicated in sporadic forms of PD suggesting that these may be key pathways in the development of the disease.

MOLECULAR CLONING OF OVINE cDNA LEPTIN GENE

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CLAUDIA TEREZIA SOCOL

2008-05-01

Full Text Available An efficient bacterial transformation system suitable for cloning the coding sequence of the ovine leptin gene in E. coli DH5α host cells using the pGEMT easy vector it is described in this paper. The necessity of producing leptin is based on the fact that the role of this molecule in the animal and human organism is still unknown, leptin not existing as commercial product on the Romanian market. The results obtained in the bacterial transformation, cloning, recombinant clones selection, control of the insertion experiments and DNA computational analysis represent the first steps in further genetic engineering experiments such as production of DNA libraries, DNA sequencing, protein expression, etc., for a further contribution in elucidating the role of leptin in the animal and human organism.

Optimal control of gene mutation in DNA replication.

Science.gov (United States)

Yu, Juanyi; Li, Jr-Shin; Tarn, Tzyh-Jong

2012-01-01

We propose a molecular-level control system view of the gene mutations in DNA replication from the finite field concept. By treating DNA sequences as state variables, chemical mutagens and radiation as control inputs, one cell cycle as a step increment, and the measurements of the resulting DNA sequence as outputs, we derive system equations for both deterministic and stochastic discrete-time, finite-state systems of different scales. Defining the cost function as a summation of the costs of applying mutagens and the off-trajectory penalty, we solve the deterministic and stochastic optimal control problems by dynamic programming algorithm. In addition, given that the system is completely controllable, we find that the global optimum of both base-to-base and codon-to-codon deterministic mutations can always be achieved within a finite number of steps.

The majority of inducible DNA repair genes in Mycobacterium tuberculosis are induced independently of RecA.

Science.gov (United States)

Rand, Lucinda; Hinds, Jason; Springer, Burkhard; Sander, Peter; Buxton, Roger S; Davis, Elaine O

2003-11-01

In many species of bacteria most inducible DNA repair genes are regulated by LexA homologues and are dependent on RecA for induction. We have shown previously by analysing the induction of recA that two mechanisms for the induction of gene expression following DNA damage exist in Mycobacterium tuberculosis. Whereas one of these depends on RecA and LexA in the classical way, the other mechanism is independent of both of these proteins and induction occurs in the absence of RecA. Here we investigate the generality of each of these mechanisms by analysing the global response to DNA damage in both wild-type M. tuberculosis and a recA deletion strain of M. tuberculosis using microarrays. This revealed that the majority of the genes that were induced remained inducible in the recA mutant stain. Of particular note most of the inducible genes with known or predicted functions in DNA repair did not depend on recA for induction. Amongst these are genes involved in nucleotide excision repair, base excision repair, damage reversal and recombination. Thus, it appears that this novel mechanism of gene regulation is important for DNA repair in M. tuberculosis.

Genome-wide DNA methylation profiles reveal novel candidate genes associated with meat quality at different age stages in hens.

Science.gov (United States)

Zhang, Meng; Yan, Feng-Bin; Li, Fang; Jiang, Ke-Ren; Li, Dong-Hua; Han, Rui-Li; Li, Zhuan-Jan; Jiang, Rui-Rui; Liu, Xiao-Jun; Kang, Xiang-Tao; Sun, Gui-Rong

2017-04-05

Poultry meat quality is associated with breed, age, tissue and other factors. Many previous studies have focused on distinct breeds; however, little is known regarding the epigenetic regulatory mechanisms in different age stages, such as DNA methylation. Here, we compared the global DNA methylation profiles between juvenile (20 weeks old) and later laying-period (55 weeks old) hens and identified candidate genes related to the development and meat quality of breast muscle using whole-genome bisulfite sequencing. The results showed that the later laying-period hens, which had a higher intramuscular fat (IMF) deposition capacity and water holding capacity (WHC) and less tenderness, exhibited higher global DNA methylation levels than the juvenile hens. A total of 2,714 differentially methylated regions were identified in the present study, which corresponded to 378 differentially methylated genes, mainly affecting muscle development, lipid metabolism, and the ageing process. Hypermethylation of the promoters of the genes ABCA1, COL6A1 and GSTT1L and the resulting transcriptional down-regulation in the later laying-period hens may be the reason for the significant difference in the meat quality between the juvenile and later laying-period hens. These findings contribute to a better understanding of epigenetic regulation in the skeletal muscle development and meat quality of chicken.

DNA polymorphism of HLA class II genes in primary biliary cirrhosis

DEFF Research Database (Denmark)

Morling, Niels; Dalhoff, K; Fugger, L

1992-01-01

We investigated the DNA restriction fragment length polymorphism of the major histocompatibility complex class II genes: HLA-DRB, -DQA, -DQB, DPA, -DPB, the serologically defined HLA-A, B, C, DR antigens, and the primed lymphocyte typing defined HLA-DP antigens in 23 Danish patients with primary...... than 0.05, 'corrected' P greater than 0.05). No DNA fragments specific for DRB1*0301 (DR3) could be identified. The frequencies in PBC of other genetic markers including DRw8, DRB1*08, HLA-DP antigens, DPA, and DPB genes did not differ significantly from those in controls. The associations between PBC...

Multiple and variable NHEJ-like genes are involved in resistance to DNA damage in Streptomyces ambofaciens

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Grégory Hoff

2016-11-01

Full Text Available Non homologous end-joining (NHEJ is a double strand break (DSB repair pathway which does not require any homologous template and can ligate two DNA ends together. The basic bacterial NHEJ machinery involves two partners: the Ku protein, a DNA end binding protein for DSB recognition and the multifunctional LigD protein composed a ligase, a nuclease and a polymerase domain, for end processing and ligation of the broken ends. In silico analyses performed in the 38 sequenced genomes of Streptomyces species revealed the existence of a large panel of NHEJ-like genes. Indeed, ku genes or ligD domain homologues are scattered throughout the genome in multiple copies and can be distinguished in two categories: the core NHEJ gene set constituted of conserved loci and the variable NHEJ gene set constituted of NHEJ-like genes present in only a part of the species. In Streptomyces ambofaciens ATCC 23877, not only the deletion of core genes but also that of variable genes led to an increased sensitivity to DNA damage induced by electron beam irradiation. Multiple mutants of ku, ligase or polymerase encoding genes showed an aggravated phenotype compared to single mutants. Biochemical assays revealed the ability of Ku-like proteins to protect and to stimulate ligation of DNA ends. RT-qPCR and GFP fusion experiments suggested that ku-like genes show a growth phase dependent expression profile consistent with their involvement in DNA repair during spores formation and/or germination.

Role of gene 59 of bacteriophage T4 in repair of uv-irradiated and alkylated DNA in vivo

International Nuclear Information System (INIS)

Wu, R.; Wu, J.L.; Yeh, Y.C.

1975-01-01

Nonsense mutants in gene 59 (amC5, am HL628) were used to study the role of this gene in the repair of uv-damaged and alkylated DNA of bacteriophage T4 in vivo. The higher sensitivity to uv irradiation and alkylation of gene 59 mutants after exposure to these agents was established by a comparison of the survival fractions with wild type. Zonal centrifugal analysis of both parental and nascent mutant intracellular DNA molecules after uv irradiation showed that immediately after exposure the size of single-stranded DNA fragments was the same as the wild-type intracellular DNA. However, the capability of rejoining fragmented intracellular DNA was greatly reduced in the mutant. In contrast, the wild-type-infected cells under the same condition resumed DNA replication and repaired its DNA to normal size. Methyl methanesulfonate induced more randomly fragmented intracellular DNA, when compared to uv irradiation. The rate of rejoining under these conditions as judged from their sedimentation profiles was also greatly reduced in mutant-infected cells. Further evidence is presented that uv repair is not a simple consequence of arrested DNA replication, which is a phenotype of the mutant when infected in a nonpermissive host, Escherichia coli B(su - ), but rather that the DNA repair function of gene 59 is independent of the replication function. These and other data presented indicate that a product(s) of gene 59 is essential for both repair of uv lesions and repair of alkylation damage of DNA in vivo. It is suggested that gene 59 may have two functions during viral development: DNA replication and replication repair of DNA molecules

Roles of genes and Alu repeats in nonlinear correlations of HUMHBB DNA sequence

International Nuclear Information System (INIS)

Xiao Yi; Huang Yanzhao

2004-01-01

DNA sequences of different species and different portion of the DNA of the same species may have completely different correlation properties, but the origin of these correlations is still not very clear and is currently being investigated, especially in different particular cases. We report here a study of the DNA sequence of human beta globin region (HUMHBB) which has strong linear and nonlinear correlations. We studied the roles of two of the typical elements of DNA sequence, genes and Alu repeats, in the nonlinear correlations of HUMHBB. We find that there exist strong nonlinear correlations between the exons or introns in different genes and between the Alu repeats. They may be one of the major sources of the nonlinear correlations in HUMBHB

Novel PVA-DNA nanoparticles prepared by ultra high pressure technology for gene delivery

International Nuclear Information System (INIS)

Kimura, Tsuyoshi; Okuno, Akira; Miyazaki, Kozo; Furuzono, Tsutomu; Ohya, Yuichi; Ouchi, Tatsuro; Mutsuo, Shingo; Yoshizawa, Hidekazu; Kitamura, Yoshiro; Fujisato, Toshiyta; Kishida, Akio

2004-01-01

Polyvinyl alcohol (PVA)-DNA nanoparticles have been developed by ultra high pressure (UHP) technology. Mixture solutions of DNA and PVA having various molecular weights (Mw) and degree of saponifications (DS) were treated under 10,000 atmospheres (981 MPa) condition at 40 deg. C for 10 min. Agarose gel electrophoresis and scanning electron microscope observation revealed that the PVA-DNA nanoparticles with average diameter of about 200 nm were formed. Using PVA of higher Mw and degree of saponifications, the amount of nanoparticles formed increased. The driving force of nanoparticle formation was the hydrogen bonding between DNA and PVA. In order to apply the PVA-DNA nanoparticles for gene delivery, the cytotoxicity and the cellular uptake of them were investigated using Raw264 cell lines. The cell viability was not influenced whether the presence of the PVA-DNA nanoparticles. Further, the nanoparticles internalized into cells were observed by fluorescent microscope. These results indicates that the PVA-DNA nanoparticles prepared by UHP technology showed be useful as drug carrier, especially for gene delivery

Triple negative breast cancers have a reduced expression of DNA repair genes.

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Enilze Ribeiro

Full Text Available DNA repair is a key determinant in the cellular response to therapy and tumor repair status could play an important role in tailoring patient therapy. Our goal was to evaluate the mRNA of 13 genes involved in different DNA repair pathways (base excision, nucleotide excision, homologous recombination, and Fanconi anemia in paraffin embedded samples of triple negative breast cancer (TNBC compared to luminal A breast cancer (LABC. Most of the genes involved in nucleotide excision repair and Fanconi Anemia pathways, and CHK1 gene were significantly less expressed in TNBC than in LABC. PARP1 levels were higher in TNBC than in LABC. In univariate analysis high level of FANCA correlated with an increased overall survival and event free survival in TNBC; however multivariate analyses using Cox regression did not confirm FANCA as independent prognostic factor. These data support the evidence that TNBCs compared to LABCs harbour DNA repair defects.

DNA entropy reveals a significant difference in complexity between housekeeping and tissue specific gene promoters.

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Thomas, David; Finan, Chris; Newport, Melanie J; Jones, Susan

2015-10-01

The complexity of DNA can be quantified using estimates of entropy. Variation in DNA complexity is expected between the promoters of genes with different transcriptional mechanisms; namely housekeeping (HK) and tissue specific (TS). The former are transcribed constitutively to maintain general cellular functions, and the latter are transcribed in restricted tissue and cells types for specific molecular events. It is known that promoter features in the human genome are related to tissue specificity, but this has been difficult to quantify on a genomic scale. If entropy effectively quantifies DNA complexity, calculating the entropies of HK and TS gene promoters as profiles may reveal significant differences. Entropy profiles were calculated for a total dataset of 12,003 human gene promoters and for 501 housekeeping (HK) and 587 tissue specific (TS) human gene promoters. The mean profiles show the TS promoters have a significantly lower entropy (pentropy distributions for the 3 datasets show that promoter entropies could be used to identify novel HK genes. Functional features comprise DNA sequence patterns that are non-random and hence they have lower entropies. The lower entropy of TS gene promoters can be explained by a higher density of positive and negative regulatory elements, required for genes with complex spatial and temporary expression. Copyright © 2015 Elsevier Ltd. All rights reserved.

Regulation of UGT1A1 and HNF1 transcription factor gene expression by DNA methylation in colon cancer cells

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Harvey Mario

2010-01-01

Full Text Available Abstract Background UDP-glucuronosyltransferase 1A1 (UGT1A1 is a pivotal enzyme involved in metabolism of SN-38, the active metabolite of irinotecan commonly used to treat metastatic colorectal cancer. We previously demonstrated aberrant methylation of specific CpG dinucleotides in UGT1A1-negative cells, and revealed that methylation state of the UGT1A1 5'-flanking sequence is negatively correlated with gene transcription. Interestingly, one of these CpG dinucleotides (CpG -4 is found close to a HNF1 response element (HRE, known to be involved in activation of UGT1A1 gene expression, and within an upstream stimulating factor (USF binding site. Results Gel retardation assays revealed that methylation of CpG-4 directly affect the interaction of USF1/2 with its cognate sequence without altering the binding for HNF1-alpha. Luciferase assays sustained a role for USF1/2 and HNF1-alpha in UGT1A1 regulation in colon cancer cells. Based on the differential expression profiles of HNF1A gene in colon cell lines, we also assessed whether methylation affects its expression. In agreement with the presence of CpG islands in the HNF1A promoter, treatments of UGT1A1-negative HCT116 colon cancer cells with a DNA methyltransferase inhibitor restore HNF1A gene expression, as observed for UGT1A1. Conclusions This study reveals that basal UGT1A1 expression in colon cells is positively regulated by HNF1-alpha and USF, and negatively regulated by DNA methylation. Besides, DNA methylation of HNF1A could also play an important role in regulating additional cellular drug metabolism and transporter pathways. This process may contribute to determine local inactivation of drugs such as the anticancer agent SN-38 by glucuronidation and define tumoral response.

Comparative analysis of DNA nanoparticles and AAVs for ocular gene delivery.

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Zongchao Han

Full Text Available Gene therapy is a critical tool for the treatment of monogenic retinal diseases. However, the limited vector capacity of the current benchmark delivery strategy, adeno-associated virus (AAV, makes development of larger capacity alternatives, such as compacted DNA nanoparticles (NPs, critical. Here we conduct a side-by-side comparison of self-complementary AAV and CK30PEG NPs using matched ITR plasmids. We report that although AAVs are more efficient per vector genome (vg than NPs, NPs can drive gene expression on a comparable scale and longevity to AAV. We show that subretinally injected NPs do not leave the eye while some of the AAV-injected animals exhibited vector DNA and GFP expression in the visual pathways of the brain from PI-60 onward. As a result, these NPs have the potential to become a successful alternative for ocular gene therapy, especially for the multitude of genes too large for AAV vectors.

Functional role of DNA mismatch repair gene PMS2 in prostate cancer cells.

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Fukuhara, Shinichiro; Chang, Inik; Mitsui, Yozo; Chiyomaru, Takeshi; Yamamura, Soichiro; Majid, Shahana; Saini, Sharanjot; Deng, Guoren; Gill, Ankurpreet; Wong, Darryn K; Shiina, Hiroaki; Nonomura, Norio; Lau, Yun-Fai C; Dahiya, Rajvir; Tanaka, Yuichiro

2015-06-30

DNA mismatch repair (MMR) enzymes act as proofreading complexes that maintains genomic integrity and MMR-deficient cells show an increased mutation rate. MMR has also been shown to influence cell signaling and the regulation of tumor development. MMR consists of various genes and includes post-meiotic segregation (PMS) 2 which is a vital component of mutL-alpha. In prostate, the functional role of this gene has never been reported and in this study, our aim was to investigate the effect of PMS2 on growth properties of prostate cancer (PCa) cells. Previous studies have shown PMS2 to be deficient in DU145 cells and this lack of expression was confirmed by Western blotting whereas normal prostatic PWR-1E and RWPE-1 cells expressed this gene. PMS2 effects on various growth properties of DU145 were then determined by creating stable gene transfectants. Interestingly, PMS2 caused decreased cell proliferation, migration, invasion, and in vivo growth; and increased apoptosis as compared to vector control. We further analyzed genes affected by PMS2 expression and observe the apoptosis-related TMS1 gene to be significantly upregulated whereas anti-apoptotic BCL2A1 was downregulated. These results demonstrate a functional role for PMS2 to protect against PCa progression by enhancing apoptosis of PCa cells.

Correlation of SHOX2 Gene Amplification and DNA Methylation in Lung Cancer Tumors

International Nuclear Information System (INIS)

Schneider, Katja U; Liebenberg, Volker; Kneip, Christoph; Seegebarth, Anke; Erdogan, Fikret; Rappold, Gudrun; Schmidt, Bernd; Dietrich, Dimo; Fleischhacker, Michael; Leschber, Gunda; Merk, Johannes; Schäper, Frank; Stapert, Henk R; Vossenaar, Erik R; Weickmann, Sabine

2011-01-01

DNA methylation in the SHOX2 locus was previously used to reliably detect lung cancer in a group of critical controls, including 'cytologically negative' samples with no visible tumor cell content, at a high specificity based on the analysis of bronchial lavage samples. This study aimed to investigate, if the methylation correlates with SHOX2 gene expression and/or copy number alterations. An amplification of the SHOX2 gene locus together with the observed tumor-specific hypermethylation might explain the good performance of this marker in bronchial lavage samples. SHOX2 expression, gene copy number and DNA methylation were determined in lung tumor tissues and matched morphologically normal adjacent tissues (NAT) from 55 lung cancer patients. Quantitative HeavyMethyl (HM) real-time PCR was used to detect SHOX2 DNA methylation levels. SHOX2 expression was assayed with quantitative real-time PCR, and copy numbers alterations were measured with conventional real-time PCR and array CGH. A hypermethylation of the SHOX2 locus in tumor tissue as compared to the matched NAT from the same patient was detected in 96% of tumors from a group of 55 lung cancer patients. This correlated highly significantly with the frequent occurrence of copy number amplification (p < 0.0001), while the expression of the SHOX2 gene showed no difference. Frequent gene amplification correlated with hypermethylation of the SHOX2 gene locus. This concerted effect qualifies SHOX2 DNA methylation as a biomarker for lung cancer diagnosis, especially when sensitive detection is needed, i.e. in bronchial lavage or blood samples

Mitochondrial DNA depletion syndrome due to mutations in the RRM2B gene.

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Bornstein, Belén; Area, Estela; Flanigan, Kevin M; Ganesh, Jaya; Jayakar, Parul; Swoboda, Kathryn J; Coku, Jorida; Naini, Ali; Shanske, Sara; Tanji, Kurenai; Hirano, Michio; DiMauro, Salvatore

2008-06-01

Mitochondrial DNA depletion syndrome (MDS) is characterized by a reduction in mtDNA copy number and has been associated with mutations in eight nuclear genes, including enzymes involved in mitochondrial nucleotide metabolism (POLG, TK2, DGUOK, SUCLA2, SUCLG1, PEO1) and MPV17. Recently, mutations in the RRM2B gene, encoding the p53-controlled ribonucleotide reductase subunit, have been described in seven infants from four families, who presented with various combinations of hypotonia, tubulopathy, seizures, respiratory distress, diarrhea, and lactic acidosis. All children died before 4 months of age. We sequenced the RRM2B gene in three unrelated cases with unexplained severe mtDNA depletion. The first patient developed intractable diarrhea, profound weakness, respiratory distress, and died at 3 months. The other two unrelated patients had a much milder phenotype and are still alive at ages 27 and 36 months. All three patients had lactic acidosis and severe depletion of mtDNA in muscle. Muscle histochemistry showed RRF and COX deficiency. Sequencing the RRM2B gene revealed three missense mutations and two single nucleotide deletions in exons 6, 8, and 9, confirming that RRM2B mutations are important causes of MDS and that the clinical phenotype is heterogeneous and not invariably fatal in infancy.

Patterns of prokaryotic lateral gene transfers affecting parasitic microbial eukaryotes

DEFF Research Database (Denmark)

Alsmark, Cecilia; Foster, Peter G; Sicheritz-Pontén, Thomas

2013-01-01

BACKGROUND: The influence of lateral gene transfer on gene origins and biology in eukaryotes is poorly understood compared with those of prokaryotes. A number of independent investigations focusing on specific genes, individual genomes, or specific functional categories from various eukaryotes have...... approach to systematically investigate lateral gene transfer affecting the proteomes of thirteen, mainly parasitic, microbial eukaryotes, representing four of the six eukaryotic super-groups. All of the genomes investigated have been significantly affected by prokaryote-to-eukaryote lateral gene transfers...... indicated that lateral gene transfer does indeed affect eukaryotic genomes. However, the lack of common methodology and criteria in these studies makes it difficult to assess the general importance and influence of lateral gene transfer on eukaryotic genome evolution. RESULTS: We used a phylogenomic...

Comprehensive Pathway-Based Association Study of DNA Repair Gene Variants and the Risk of Nasopharyngeal Carcinoma

Science.gov (United States)

Qin, Hai-De; Shugart, Yin Yao; Bei, Jin-Xin; Pan, Qing-Hua; Chen, Lina; Feng, Qi-Sheng; Chen, Li-Zhen; Huang, Wei; Liu, Jian Jun; Jorgensen, Timothy J.; Zeng, Yi-Xin; Jia, Wei-Hua

2011-01-01

DNA repair plays a central role in protecting against environmental carcinogenesis, and genetic variants of DNA repair genes have been reported to be associated with several human malignancies. To assess whether DNA gene variants were associated with nasopharyngeal carcinoma (NPC) risk, a candidate gene association study was conducted among the Cantonese population within the Guangdong Province, China --the ethnic group with the highest risk for NPC. A two-stage study design was utilized. In the discovery stage, 676 tagging SNPs covering 88 DNA repair genes were genotyped in a matched case-control study (cases/controls = 755/755). Eleven SNPs with Ptrend Cantonese population (cases/controls = 1,568/1,297). Two of the SNPs (rs927220 and rs11158728) – both in RAD51L1 – remained strongly associated with NPC. The SNP rs927220 had a significant Pcombined of 5.55 × 10−5, with OR = 1.20 (95%CI = 1.10 to 1.30), Bonferroni corrected P = 0.0381. The other SNP (rs11158728), which is in strong LD with rs927220 (r2 = 0.7), had a significant Pcombined of 2.0 × 10−4, Bonferroni corrected P = 0.1372. Gene-environment interaction analysis suggested that the exposures of salted-fish consumption and cigarette smoking had potential interactions with DNA repair gene variations, but need to be further investigated. Our findings support the notion that DNA repair genes, in particular RAD51L1, play a role in NPC etiology and development. PMID:21368091

Potential efficacy of mitochondrial genes for animal DNA barcoding: a case study using eutherian mammals.

Science.gov (United States)

Luo, Arong; Zhang, Aibing; Ho, Simon Yw; Xu, Weijun; Zhang, Yanzhou; Shi, Weifeng; Cameron, Stephen L; Zhu, Chaodong

2011-01-28

A well-informed choice of genetic locus is central to the efficacy of DNA barcoding. Current DNA barcoding in animals involves the use of the 5' half of the mitochondrial cytochrome oxidase 1 gene (CO1) to diagnose and delimit species. However, there is no compelling a priori reason for the exclusive focus on this region, and it has been shown that it performs poorly for certain animal groups. To explore alternative mitochondrial barcoding regions, we compared the efficacy of the universal CO1 barcoding region with the other mitochondrial protein-coding genes in eutherian mammals. Four criteria were used for this comparison: the number of recovered species, sequence variability within and between species, resolution to taxonomic levels above that of species, and the degree of mutational saturation. Based on 1,179 mitochondrial genomes of eutherians, we found that the universal CO1 barcoding region is a good representative of mitochondrial genes as a whole because the high species-recovery rate (> 90%) was similar to that of other mitochondrial genes, and there were no significant differences in intra- or interspecific variability among genes. However, an overlap between intra- and interspecific variability was still problematic for all mitochondrial genes. Our results also demonstrated that any choice of mitochondrial gene for DNA barcoding failed to offer significant resolution at higher taxonomic levels. We suggest that the CO1 barcoding region, the universal DNA barcode, is preferred among the mitochondrial protein-coding genes as a molecular diagnostic at least for eutherian species identification. Nevertheless, DNA barcoding with this marker may still be problematic for certain eutherian taxa and our approach can be used to test potential barcoding loci for such groups.

DNA polymorphism of HLA class II genes in pauciarticular juvenile rheumatoid arthritis

DEFF Research Database (Denmark)

Morling, N; Friis, J; Fugger, L

1991-01-01

We investigated the DNA restriction fragment length polymorphism (RFLP) of the major histocompatibility complex (MHC) class II genes: HLA-DRB, -DQA, -DQB, DPA, and -DPB in 54 patients with pauciarticular juvenile rheumatoid arthritis (PJRA) and in healthy Danes. The frequencies of DNA fragments a...

Presence of a consensus DNA motif at nearby DNA sequence of the mutation susceptible CG nucleotides.

Science.gov (United States)

Chowdhury, Kaushik; Kumar, Suresh; Sharma, Tanu; Sharma, Ankit; Bhagat, Meenakshi; Kamai, Asangla; Ford, Bridget M; Asthana, Shailendra; Mandal, Chandi C

2018-01-10

Complexity in tissues affected by cancer arises from somatic mutations and epigenetic modifications in the genome. The mutation susceptible hotspots present within the genome indicate a non-random nature and/or a position specific selection of mutation. An association exists between the occurrence of mutations and epigenetic DNA methylation. This study is primarily aimed at determining mutation status, and identifying a signature for predicting mutation prone zones of tumor suppressor (TS) genes. Nearby sequences from the top five positions having a higher mutation frequency in each gene of 42 TS genes were selected from a cosmic database and were considered as mutation prone zones. The conserved motifs present in the mutation prone DNA fragments were identified. Molecular docking studies were done to determine putative interactions between the identified conserved motifs and enzyme methyltransferase DNMT1. Collective analysis of 42 TS genes found GC as the most commonly replaced and AT as the most commonly formed residues after mutation. Analysis of the top 5 mutated positions of each gene (210 DNA segments for 42 TS genes) identified that CG nucleotides of the amino acid codons (e.g., Arginine) are most susceptible to mutation, and found a consensus DNA "T/AGC/GAGGA/TG" sequence present in these mutation prone DNA segments. Similar to TS genes, analysis of 54 oncogenes not only found CG nucleotides of the amino acid Arg as the most susceptible to mutation, but also identified the presence of similar consensus DNA motifs in the mutation prone DNA fragments (270 DNA segments for 54 oncogenes) of oncogenes. Docking studies depicted that, upon binding of DNMT1 methylates to this consensus DNA motif (C residues of CpG islands), mutation was likely to occur. Thus, this study proposes that DNMT1 mediated methylation in chromosomal DNA may decrease if a foreign DNA segment containing this consensus sequence along with CG nucleotides is exogenously introduced to dividing

In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma.

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Bekim Sadikovic

Full Text Available Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.

TLR9 agonists oppositely modulate DNA repair genes in tumor versus immune cells and enhance chemotherapy effects.

Science.gov (United States)

Sommariva, Michele; De Cecco, Loris; De Cesare, Michelandrea; Sfondrini, Lucia; Ménard, Sylvie; Melani, Cecilia; Delia, Domenico; Zaffaroni, Nadia; Pratesi, Graziella; Uva, Valentina; Tagliabue, Elda; Balsari, Andrea

2011-10-15

Synthetic oligodeoxynucleotides expressing CpG motifs (CpG-ODN) are a Toll-like receptor 9 (TLR9) agonist that can enhance the antitumor activity of DNA-damaging chemotherapy and radiation therapy in preclinical mouse models. We hypothesized that the success of these combinations is related to the ability of CpG-ODN to modulate genes involved in DNA repair. We conducted an in silico analysis of genes implicated in DNA repair in data sets obtained from murine colon carcinoma cells in mice injected intratumorally with CpG-ODN and from splenocytes in mice treated intraperitoneally with CpG-ODN. CpG-ODN treatment caused downregulation of DNA repair genes in tumors. Microarray analyses of human IGROV-1 ovarian carcinoma xenografts in mice treated intraperitoneally with CpG-ODN confirmed in silico findings. When combined with the DNA-damaging drug cisplatin, CpG-ODN significantly increased the life span of mice compared with individual treatments. In contrast, CpG-ODN led to an upregulation of genes involved in DNA repair in immune cells. Cisplatin-treated patients with ovarian carcinoma as well as anthracycline-treated patients with breast cancer who are classified as "CpG-like" for the level of expression of CpG-ODN modulated DNA repair genes have a better outcome than patients classified as "CpG-untreated-like," indicating the relevance of these genes in the tumor cell response to DNA-damaging drugs. Taken together, the findings provide evidence that the tumor microenvironment can sensitize cancer cells to DNA-damaging chemotherapy, thereby expanding the benefits of CpG-ODN therapy beyond induction of a strong immune response.

Candidate luminal B breast cancer genes identified by genome, gene expression and DNA methylation profiling.

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Stéphanie Cornen

Full Text Available Breast cancers (BCs of the luminal B subtype are estrogen receptor-positive (ER+, highly proliferative, resistant to standard therapies and have a poor prognosis. To better understand this subtype we compared DNA copy number aberrations (CNAs, DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes, in 32 luminal B tumors with those observed in 156 BCs of the other molecular subtypes. Frequent CNAs included 8p11-p12 and 11q13.1-q13.2 amplifications, 7q11.22-q34, 8q21.12-q24.23, 12p12.3-p13.1, 12q13.11-q24.11, 14q21.1-q23.1, 17q11.1-q25.1, 20q11.23-q13.33 gains and 6q14.1-q24.2, 9p21.3-p24,3, 9q21.2, 18p11.31-p11.32 losses. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs presented a deregulated expression in relation with their CNAs, including 11 genes previously reported associated with endocrine resistance. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q, and seven candidate oncogenes are potential therapeutic targets. A total of 100 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 of these was associated with poor survival in luminal tumors. Twenty-four genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a meta-analysis of next-generation sequencing data in 875 BCs, KCNB2 mutations were associated with luminal B cases while candidate TSGs MDN1 (6q15 and UTRN (6q24, were mutated in this subtype. In conclusion, we have reported luminal B candidate genes that may play a role in the development and/or hormone resistance of this aggressive subtype.

Fructose-Rich Diet Affects Mitochondrial DNA Damage and Repair in Rats.

Science.gov (United States)

Cioffi, Federica; Senese, Rosalba; Lasala, Pasquale; Ziello, Angela; Mazzoli, Arianna; Crescenzo, Raffaella; Liverini, Giovanna; Lanni, Antonia; Goglia, Fernando; Iossa, Susanna

2017-03-24

Evidence indicates that many forms of fructose-induced metabolic disturbance are associated with oxidative stress and mitochondrial dysfunction. Mitochondria are prominent targets of oxidative damage; however, it is not clear whether mitochondrial DNA (mtDNA) damage and/or its lack of repair are events involved in metabolic disease resulting from a fructose-rich diet. In the present study, we evaluated the degree of oxidative damage to liver mtDNA and its repair, in addition to the state of oxidative stress and antioxidant defense in the liver of rats fed a high-fructose diet. We used male rats feeding on a high-fructose or control diet for eight weeks. Our results showed an increase in mtDNA damage in the liver of rats fed a high-fructose diet and this damage, as evaluated by the expression of DNA polymerase γ, was not repaired; in addition, the mtDNA copy number was found to be significantly reduced. A reduction in the mtDNA copy number is indicative of impaired mitochondrial biogenesis, as is the finding of a reduction in the expression of genes involved in mitochondrial biogenesis. In conclusion, a fructose-rich diet leads to mitochondrial and mtDNA damage, which consequently may have a role in liver dysfunction and metabolic diseases.

Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription.

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Xie, Qiu; Li, Caihua; Song, Xiaozhen; Wu, Lihua; Jiang, Qian; Qiu, Zhiyong; Cao, Haiyan; Yu, Kaihui; Wan, Chunlei; Li, Jianting; Yang, Feng; Huang, Zebing; Niu, Bo; Jiang, Zhengwen; Zhang, Ting

2017-03-17

The biogenesis of ribosomes in vivo is an essential process for cellular functions. Transcription of ribosomal RNA (rRNA) genes is the rate-limiting step in ribosome biogenesis controlled by environmental conditions. Here, we investigated the role of folate antagonist on changes of DNA double-strand breaks (DSBs) landscape in mouse embryonic stem cells. A significant DSB enhancement was detected in the genome of these cells and a large majority of these DSBs were found in rRNA genes. Furthermore, spontaneous DSBs in cells under folate deficiency conditions were located exclusively within the rRNA gene units, representing a H3K4me1 hallmark. Enrichment H3K4me1 at the hot spots of DSB regions enhanced the recruitment of upstream binding factor (UBF) to rRNA genes, resulting in the increment of rRNA genes transcription. Supplement of folate resulted in a restored UBF binding across DNA breakage sites of rRNA genes, and normal rRNA gene transcription. In samples from neural tube defects (NTDs) with low folate level, up-regulation of rRNA gene transcription was observed, along with aberrant UBF level. Our results present a new view by which alterations in folate levels affects DNA breakage through epigenetic control leading to the regulation of rRNA gene transcription during the early stage of development. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

Science.gov (United States)

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.

The co-repressor SMRT delays DNA damage-induced caspase activation by repressing pro-apoptotic genes and modulating the dynamics of checkpoint kinase 2 activation.

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Claudio Scafoglio

Full Text Available Checkpoint kinase 2 (Chk2 is a major regulator of DNA damage response and can induce alternative cellular responses: cell cycle arrest and DNA repair or programmed cell death. Here, we report the identification of a new role of Chk2 in transcriptional regulation that also contributes to modulating the balance between survival and apoptosis following DNA damage. We found that Chk2 interacts with members of the NCoR/SMRT transcriptional co-regulator complexes and serves as a functional component of the repressor complex, being required for recruitment of SMRT on the promoter of pro-apoptotic genes upon DNA damage. Thus, the co-repressor SMRT exerts a critical protective action against genotoxic stress-induced caspase activation, repressing a functionally important cohort of pro-apoptotic genes. Amongst them, SMRT is responsible for basal repression of Wip1, a phosphatase that de-phosphorylates and inactivates Chk2, thus affecting a feedback loop responsible for licensing the correct timing of Chk2 activation and the proper execution of the DNA repair process.

Examination of gene expression in mice exposed to low dose radiation using affymetrix cDNA microarrays

Energy Technology Data Exchange (ETDEWEB)

Morris, D.; Knox, D.; Lavoie, J.; Lemon, J.; Boreham, D. [McMaster Univ., Hamilton, Ontario (Canada)

2005-07-01

'Full text:' Gamma radiation acts via the indirect effect to damage cells by producing reactive oxygen species (ROS). These ROS are capable damaging macromolecules and, altering signal pathways and gene transcription. Cells have evolved enzymes and mechanisms to scavenge ROS and repair oxidative damage. Microarrays allow the survey of the gene transcription activity of thousands of genes simultaneously. Messenger RNA is extracted from cells, hybridized with the complementary DNA (cDNA) of a microarray chip, and examined with a chip reader. Affymetrix microarray chips have been produced by the CSCHAH in Winnipeg containing 26000 murine genes. Groups of female mice have been exposed to low dose whole body chronic gamma radiation exposures of 0,50,100, and 120 mGy, corresponding to 15,30,60, and 75 weeks, respectively. MRNA from mice brain tissue has been extracted, isolated, converted to cDNA and labeled. Gene expression in each irradiated mouse was compared to the pooled expression of the control mice. Analysis of gene expression levels are performed with microarray analytical software, Array Pro by Media Cybernetics, and powerful statistical software, BRB microarray tools. Differences in gene expressions, focusing on genes for cytokines, DNA repair mechanisms, immuno-modulators, apoptosis pathways, and enzymatic anti-oxidant systems, are being examined and will be reported. (author)

Nonparametric testing for DNA copy number induced differential mRNA gene expression

NARCIS (Netherlands)

van Wieringen, W.N.; van de Wiel, M.A.

2009-01-01

The central dogma of molecular biology relates DNA with mRNA. Array CGH measures DNA copy number and gene expression microarrays measure the amount of mRNA. Methods that integrate data from these two platforms may uncover meaningful biological relationships that further our understanding of cancer.

Rapid assessment of repair of ultraviolet DNA damage with a modified host-cell reactivation assay using a luciferase reporter gene and correlation with polymorphisms of DNA repair genes in normal human lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Qiao Yawei; Spitz, Margaret R.; Guo Zhaozheng; Hadeyati, Mohammad; Grossman, Lawrence; Kraemer, Kenneth H.; Wei Qingyi

2002-11-30

As DNA repair plays an important role in genetic susceptibility to cancer, assessment of the DNA repair phenotype is critical for molecular epidemiological studies of cancer. In this report, we compared use of the luciferase (luc) reporter gene in a host-cell reactivation (HCR) (LUC) assay of repair of ultraviolet (UV) damage to DNA to use of the chloramphenicol (cat) gene-based HCR (CAT) assay we used previously for case-control studies. We performed both the assays on cryopreserved lymphocytes from 102 healthy non-Hispanic white subjects. There was a close correlation between DNA repair capacity (DRC) as measured by the LUC and CAT assays. Although these two assays had similar variation, the LUC assay was faster and more sensitive. We also analyzed the relationship between DRC and the subjects' previously determined genotypes for four polymorphisms of two nucleotide-excision repair (NER) genes (in intron 9 of xeroderma pigmentosum (XP) C and exons 6, 10 and 23 of XPD) and one polymorphism of a base-excision repair gene in exon 10 of X-ray complementing group 1 (XRCC1). The DRC was significantly lower in subjects homozygous for one or more polymorphisms of the two NER genes than in subjects with other genotypes (P=0.010). In contrast, the polymorphic XRCC1 allele had no significant effect on DRC. These results suggest that the post-UV LUC assay measures NER phenotype and that polymorphisms of XPC and XPD genes modulate DRC. For population studies of the DNA repair phenotype, many samples need to be evaluated, and so the LUC assay has several advantages over the CAT assay: the LUC assay was more sensitive, had less variation, was not radioactive, was easier to perform, and required fewer cryopreserved cells. These features make the LUC-based HCR assay suitable for molecular epidemiological studies.

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

Science.gov (United States)

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.

DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Kenyon, C.J.; Walker, G.C.

1988-05-01

Operon fusions in Escherichia coli were obtained that showed increased beta-galactosidase expression in response to treatment with the DNA-damaging agent mitomycin C. These fusions were generated by using the Mud(ApR, lac) vector to insert the lactose structural genes randomly into the bacterial chromosome. Induction of beta-galactosidase in these strains, which carried fusions of lac to these din (damage-inducible) loci, was (i) triggered by UV light as well as by mitomycin C and (ii) abolished by either a recA- or a lexA- mutation. Similar characteristics of induction were observed when the lactose genes were fused to a prophage lambda promoter by using Mud(ApR, lac). These results indicate that E. coli contains a set of genes that, like prophage lambda genes, are expressed in response to DNA-damaging agents and regulated by the recA and lexA gene products. These din genes map at five bacterial loci. One din::Mud(ApR, lac) insertion results in a UV-sensitive phenotype and may be within the uvrA transcriptional unit.

The DNA damage response of C. elegans affected by gravity sensing and radiosensitivity during the Shenzhou-8 spaceflight

Energy Technology Data Exchange (ETDEWEB)

Gao, Ying [Center of Medical Physics and Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Shushanhu Road 350, Hefei 230031 (China); Cancer Hospital, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Shushanhu Road 350, Hefei 230031 (China); Xu, Dan; Zhao, Lei [Institute of Environmental Systems Biology, College of Environmental Science and Engineering, Dalian Maritime University, Linghai Road 1, Dalian 116026 (China); Sun, Yeqing, E-mail: yqsun@dlmu.edu.cn [Institute of Environmental Systems Biology, College of Environmental Science and Engineering, Dalian Maritime University, Linghai Road 1, Dalian 116026 (China)

2017-01-15

Highlights: • Extrinsic condition and intrinsic sensitivity both affect responses to spaceflight. • Protein phosphorylation/dephosphorylation is sensitive to μg and space radiation. • Microgravity affects transcription depending on dystrophin gene dys-1 in C.elegans. • Loss-function of apoptotic gene ced-1 leads protective responses to space radiation. - Abstract: Space radiation and microgravity are recognized as primary and inevitable risk factors for humans traveling in space, but the reports regarding their synergistic effects remain inconclusive and vary across studies due to differences in the environmental conditions and intrinsic biological sensitivity. Thus, we studied the synergistic effects on transcriptional changes in the global genome and DNA damage response (DDR) by using dys-1 mutant and ced-1 mutant of C. elegans, which respectively presented microgravity-insensitivity and radiosensitivity when exposure to spaceflight condition (SF) and space radiation (SR). The dys-1 mutation induced similar transcriptional changes under both conditions, including the transcriptional distribution and function of altered genes. The majority of alterations were related to metabolic shift under both conditions, including transmembrane transport, lipid metabolic processes and proteolysis. Under SF and SR conditions, 12/14 and 10/13 altered pathways, respectively, were both grouped in the metabolism category. Out of the 778 genes involved in DDR, except eya-1 and ceh-34, 28 altered genes in dys-1 mutant showed no predicted protein interactions, or anti-correlated miRNAs during spaceflight. The ced-1 mutation induced similar changes under SF and SR; however, these effects were stronger than those of the dys-1 mutant. The additional genes identified were related to phosphorous/phosphate metabolic processes and growth rather than, metabolism, especially for environmental information processing under SR. Although the DDR profiles were significantly changed under

The DNA damage response of C. elegans affected by gravity sensing and radiosensitivity during the Shenzhou-8 spaceflight

International Nuclear Information System (INIS)

Gao, Ying; Xu, Dan; Zhao, Lei; Sun, Yeqing

2017-01-01

Highlights: • Extrinsic condition and intrinsic sensitivity both affect responses to spaceflight. • Protein phosphorylation/dephosphorylation is sensitive to μg and space radiation. • Microgravity affects transcription depending on dystrophin gene dys-1 in C.elegans. • Loss-function of apoptotic gene ced-1 leads protective responses to space radiation. - Abstract: Space radiation and microgravity are recognized as primary and inevitable risk factors for humans traveling in space, but the reports regarding their synergistic effects remain inconclusive and vary across studies due to differences in the environmental conditions and intrinsic biological sensitivity. Thus, we studied the synergistic effects on transcriptional changes in the global genome and DNA damage response (DDR) by using dys-1 mutant and ced-1 mutant of C. elegans, which respectively presented microgravity-insensitivity and radiosensitivity when exposure to spaceflight condition (SF) and space radiation (SR). The dys-1 mutation induced similar transcriptional changes under both conditions, including the transcriptional distribution and function of altered genes. The majority of alterations were related to metabolic shift under both conditions, including transmembrane transport, lipid metabolic processes and proteolysis. Under SF and SR conditions, 12/14 and 10/13 altered pathways, respectively, were both grouped in the metabolism category. Out of the 778 genes involved in DDR, except eya-1 and ceh-34, 28 altered genes in dys-1 mutant showed no predicted protein interactions, or anti-correlated miRNAs during spaceflight. The ced-1 mutation induced similar changes under SF and SR; however, these effects were stronger than those of the dys-1 mutant. The additional genes identified were related to phosphorous/phosphate metabolic processes and growth rather than, metabolism, especially for environmental information processing under SR. Although the DDR profiles were significantly changed under

Deoxynucleoside salvage enzymes and tissue specific mitochondrial DNA depletion.

Science.gov (United States)

Wang, L

2010-06-01

Adequate mitochondrial DNA (mtDNA) copies are required for normal mitochondria function and reductions in mtDNA copy number due to genetic alterations cause tissue-specific mtDNA depletion syndrome (MDS). There are eight nuclear genes, directly or indirectly involved in mtDNA replication and mtDNA precursor synthesis, which have been identified as the cause of MDS. However, the tissue specific pathology of these nuclear gene mutations is not well understood. Here, mtDNA synthesis, mtDNA copy number control, and mtDNA turnover, as well as the synthesis of mtDNA precursors in relation to the levels of salvage enzymes are discussed. The question why MDS caused by TK2 and p53R2 mutations are predominantly muscle specific while dGK deficiency affected mainly liver will be addressed.

DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations

Energy Technology Data Exchange (ETDEWEB)

Fortes, F.P. [CIPE, Laboratrio de Oncogentica Molecular, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Kuasne, H. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Urologia, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP (Brazil); Marchi, F.A. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Programa Inter-Institucional em Bioinformtica, Instituto de Matemtica e Estatstica, Universidade So Paulo, So Paulo, SP (Brazil); Miranda, P.M. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Rogatto, S.R. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Urologia, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP (Brazil); Achatz, M.I. [CIPE, Laboratrio de Oncogentica Molecular, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Oncogentica, A.C. Camargo Cancer Center, So Paulo, SP (Brazil)

2015-04-28

Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG) pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17) in 30 patients with germline TP53mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR) with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results.

A mutation in the aroE gene affects pigment production, virulence, and chemotaxis in Xanthomonas oryzae pv. oryzae.

Science.gov (United States)

Kim, Hong-Il; Noh, Tae-Hwan; Lee, Chang-Soo; Park, Young-Jin

2015-01-01

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB) in rice. To study its function, a random insertion mutation library of Xoo was constructed using the Tn5 transposon. A mutant strain with decreased virulence against the susceptible rice cultivar IR24 was isolated from the library (aroE mutant), which also had extremely low pigment production. Thermal asymmetric interlaced-polymerase chain reaction (TAIL-PCR) and sequence analysis of the mutant revealed that the transposon was inserted into the aroE gene (encoding shikimate dehydrogenase). To investigate gene expression changes in the pigment- and virulence-deficient mutant, DNA microarray analysis was performed, which showed downregulation of 20 genes involved in the chemotaxis of Xoo. Our findings reveal that mutation of the aroE gene affects virulence and pigment production, as well as expression of genes involved in Xoo chemotaxis. Copyright © 2014 Elsevier GmbH. All rights reserved.

Exposure to 3,3',5-triiodothyronine affects histone and RNA polymerase II modifications, but not DNA methylation status, in the regulatory region of the Xenopus laevis thyroid hormone receptor βΑ gene.

Science.gov (United States)

Kasai, Kentaro; Nishiyama, Norihito; Izumi, Yushi; Otsuka, Shunsuke; Ishihara, Akinori; Yamauchi, Kiyoshi

2015-11-06

Thyroid hormones (THs) play a critical role in amphibian metamorphosis, during which the TH receptor (TR) gene, thrb, is upregulated in a tissue-specific manner. The Xenopus laevis thrb gene has 3 TH response elements (TREs) in the 5' flanking regulatory region and 1 TRE in the exon b region, around which CpG sites are highly distributed. To clarify whether exposure to 3,3',5-triiodothyronine (T3) affects histone and RNA polymerase II (RNAPII) modifications and the level of DNA methylation in the 5' regulatory region, we conducted reverse transcription-quantitative polymerase chain reaction, bisulfite sequencing and chromatin immunoprecipitation assay using X. laevis cultured cells and premetamorphic tadpoles treated with or without 2 nM T3. Exposure to T3 increased the amount of the thrb transcript, in parallel with enhanced histone H4 acetylation and RNAPII recruitment, and probably phosphorylation of RNAPII at serine 5, in the 5' regulatory and exon b regions. However, the 5' regulatory region remained hypermethylated even with exposure to T3, and there was no significant difference in the methylation status between DNAs from T3-untreated and -treated cultured cells or tadpole tissues. Our results demonstrate that exposure to T3 induced euchromatin-associated epigenetic marks by enhancing histone acetylation and RNAPII recruitment, but not by decreasing the level of DNA methylation, in the 5' regulatory region of the X. laevis thrb gene. Copyright © 2015 Elsevier Inc. All rights reserved.

Epigenetic Alteration by DNA Promoter Hypermethylation of Genes Related to Transforming Growth Factor-β (TGF-β) Signaling in Cancer

Energy Technology Data Exchange (ETDEWEB)

Khin, Sann Sanda [Kobe University Graduate School of Medicine, Division of Diagnostic Molecular Pathology, Kobe 650-0017 (Japan); Pathology Research Unit, Department of Medical Research (Central Myanmar), Naypyitaw, Union of (Myanmar); Kitazawa, Riko [Kobe University Graduate School of Medicine, Division of Diagnostic Molecular Pathology, Kobe 650-0017 (Japan); Ehime University Graduate School of Medicine, Toon 791-0295, Ehime (Japan); Kondo, Takeshi; Idei, Yuka; Fujimoto, Masayo [Kobe University Graduate School of Medicine, Division of Diagnostic Molecular Pathology, Kobe 650-0017 (Japan); Haraguchi, Ryuma [Ehime University Graduate School of Medicine, Toon 791-0295, Ehime (Japan); Mori, Kiyoshi [Kobe University Graduate School of Medicine, Division of Diagnostic Molecular Pathology, Kobe 650-0017 (Japan); Kitazawa, Sohei, E-mail: kitazawa@m.ehime-u.ac.jp [Kobe University Graduate School of Medicine, Division of Diagnostic Molecular Pathology, Kobe 650-0017 (Japan); Ehime University Graduate School of Medicine, Toon 791-0295, Ehime (Japan)

2011-03-03

Epigenetic alterations in cancer, especially DNA methylation and histone modification, exert a significant effect on the deregulated expression of cancer-related genes and lay an epigenetic pathway to carcinogenesis and tumor progression. Global hypomethylation and local hypermethylation of CpG islands in the promoter region, which result in silencing tumor suppressor genes, constitute general and major epigenetic modification, the hallmark of the neoplastic epigenome. Additionally, methylation-induced gene silencing commonly affects a number of genes and increases with cancer progression. Indeed, cancers with a high degree of methylation (CpG island methylator phenotype/CIMP) do exist and represent a distinct subset of certain cancers including colorectal, bladder and kidney. On the other hand, signals from the microenvironment, especially those from transforming growth factor-β (TGF-β), induce targeted de novo epigenetic alterations of cancer-related genes. While TGF-β signaling has been implicated in two opposite roles in cancer, namely tumor suppression and tumor promotion, its deregulation is also partly induced by epigenetic alteration itself. Although the epigenetic pathway to carcinogenesis and cancer progression has such reciprocal complexity, the important issue is to identify genes or signaling pathways that are commonly silenced in various cancers in order to find early diagnostic and therapeutic targets. In this review, we focus on the epigenetic alteration by DNA methylation and its role in molecular modulations of the TGF-β signaling pathway that cause or underlie altered cancer-related gene expression in both phases of early carcinogenesis and late cancer progression.

Epigenetic Alteration by DNA Promoter Hypermethylation of Genes Related to Transforming Growth Factor-β (TGF-β) Signaling in Cancer

International Nuclear Information System (INIS)

Khin, Sann Sanda; Kitazawa, Riko; Kondo, Takeshi; Idei, Yuka; Fujimoto, Masayo; Haraguchi, Ryuma; Mori, Kiyoshi; Kitazawa, Sohei

2011-01-01

Epigenetic alterations in cancer, especially DNA methylation and histone modification, exert a significant effect on the deregulated expression of cancer-related genes and lay an epigenetic pathway to carcinogenesis and tumor progression. Global hypomethylation and local hypermethylation of CpG islands in the promoter region, which result in silencing tumor suppressor genes, constitute general and major epigenetic modification, the hallmark of the neoplastic epigenome. Additionally, methylation-induced gene silencing commonly affects a number of genes and increases with cancer progression. Indeed, cancers with a high degree of methylation (CpG island methylator phenotype/CIMP) do exist and represent a distinct subset of certain cancers including colorectal, bladder and kidney. On the other hand, signals from the microenvironment, especially those from transforming growth factor-β (TGF-β), induce targeted de novo epigenetic alterations of cancer-related genes. While TGF-β signaling has been implicated in two opposite roles in cancer, namely tumor suppression and tumor promotion, its deregulation is also partly induced by epigenetic alteration itself. Although the epigenetic pathway to carcinogenesis and cancer progression has such reciprocal complexity, the important issue is to identify genes or signaling pathways that are commonly silenced in various cancers in order to find early diagnostic and therapeutic targets. In this review, we focus on the epigenetic alteration by DNA methylation and its role in molecular modulations of the TGF-β signaling pathway that cause or underlie altered cancer-related gene expression in both phases of early carcinogenesis and late cancer progression

Virus-specific DNA sequences present in cells which carry the herpes simplex virus thymidine kinase gene.

Science.gov (United States)

Minson, A C; Darby, G K; Wildy, P

1979-11-01

Two independently derived cell lines which carry the herpes simplex type 2 thymidine kinase gene have been examined for the presence of HSV-2-specific DNA sequences. Both cell lines contained 1 to 3 copies per cell of a sequence lying within map co-ordinates 0.2 to 0.4 of the HSV-2 genome. Revertant cells, which contained no detectable thymidine kinase, did not contain this DNA sequence. The failure of EcoR1-restricted HSV-2 DNA to act as a donor of the thymidine kinase gene in transformation experiments suggests that the gene lies close to the EcoR1 restriction site within this sequence at a map position of approx. 0.3. The HSV-2 kinase gene is therefore approximately co-linear with the HSV-1 gene.

Laboratory of Mutagenesis and DNA Repair

International Nuclear Information System (INIS)

2000-01-01

Full text: Two main lines of research were continued: the first one concerned the mechanisms controlling the fidelity of DNA replication in Escherichia coli; the second concerned cellular responses of Saccharomyces cerevisiae to DNA damaging agents. We have been investigating the question whether during chromosomal DNA replication in Escherichia coli the two DNA strands may be replicated with differential accuracy. To address this question we set up a new system that allows the examination of mutagenesis either of the leading strand or the lagging strand. Our results suggest that the lagging strand replication of the E. coli chromosome may be more accurate than leading strand replication. More recently, we studied mutagenesis of the two strands in recA730 strains which exhibit constitutive expression of the SOS system. Our results clearly indicate that in recA730 strains there is a significant difference in the fidelity of replication between the two replicating strands. Based on our data we propose a model describing a possible mechanism of SOS mutagenesis. To get more insight into cellular responses to DNA damage we have isolated several novel genes of S. cerevisiae, the transcription of which is induced by DNA lesions. Main effort was concentrated on the characterization of the DIN7 gene. We found that Din7p specifically affects the metabolism of mitochondrial DNA (mtDNA). The elevated level of Din7p results in an increased frequency of mitochondrial petite mutants, as well as in a higher frequency of mitochondrial point mutations. Din7p affects also the stability of microsatellite sequences present in the mitochondrial genome. As expected, Din7p was found to be located in mitochondria. In another project, we found that the DIN8 gene isolated in our laboratory is identical with the UMP1 gene encoding a chaperone-like protein involved in 20S proteasome maturation. Interestingly, induction of UMP1 expression in response to DNA damage is subject to regulation

Land use type significantly affects microbial gene transcription in soil.

Science.gov (United States)

Nacke, Heiko; Fischer, Christiane; Thürmer, Andrea; Meinicke, Peter; Daniel, Rolf

2014-05-01

Soil microorganisms play an essential role in sustaining biogeochemical processes and cycling of nutrients across different land use types. To gain insights into microbial gene transcription in forest and grassland soil, we isolated mRNA from 32 sampling sites. After sequencing of generated complementary DNA (cDNA), a total of 5,824,229 sequences could be further analyzed. We were able to assign nonribosomal cDNA sequences to all three domains of life. A dominance of bacterial sequences, which were affiliated to 25 different phyla, was found. Bacterial groups capable of aromatic compound degradation such as Phenylobacterium and Burkholderia were detected in significantly higher relative abundance in forest soil than in grassland soil. Accordingly, KEGG pathway categories related to degradation of aromatic ring-containing molecules (e.g., benzoate degradation) were identified in high abundance within forest soil-derived metatranscriptomic datasets. The impact of land use type forest on community composition and activity is evidently to a high degree caused by the presence of wood breakdown products. Correspondingly, bacterial groups known to be involved in lignin degradation and containing ligninolytic genes such as Burkholderia, Bradyrhizobium, and Azospirillum exhibited increased transcriptional activity in forest soil. Higher solar radiation in grassland presumably induced increased transcription of photosynthesis-related genes within this land use type. This is in accordance with high abundance of photosynthetic organisms and plant-infecting viruses in grassland.

DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids

Science.gov (United States)

Canovas, Sebastian; Ivanova, Elena; Romar, Raquel; García-Martínez, Soledad; Soriano-Úbeda, Cristina; García-Vázquez, Francisco A; Saadeh, Heba; Andrews, Simon; Kelsey, Gavin; Coy, Pilar

2017-01-01

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT. DOI: http://dx.doi.org/10.7554/eLife.23670.001 PMID:28134613

Actin and myosin contribute to mammalian mitochondrial DNA maintenance

Science.gov (United States)

Reyes, A.; He, J.; Mao, C. C.; Bailey, L. J.; Di Re, M.; Sembongi, H.; Kazak, L.; Dzionek, K.; Holmes, J. B.; Cluett, T. J.; Harbour, M. E.; Fearnley, I. M.; Crouch, R. J.; Conti, M. A.; Adelstein, R. S.; Walker, J. E.; Holt, I. J.

2011-01-01

Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and β-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of β-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some β-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance. PMID:21398640

Gene activation by induced DNA rearrangements

International Nuclear Information System (INIS)

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

1989-01-01

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

The (PrS/HGF-pDNA) multilayer films for gene-eluting stent coating: Gene-protecting, anticoagulation, antibacterial properties, and in vivo antirestenosis evaluation.

Science.gov (United States)

Chang, Hao; Ren, Ke-feng; Zhang, He; Wang, Jin-lei; Wang, Bai-liang; Ji, Jian

2015-02-01

Vascular gene-eluting stents (GES) is a promising strategy for treatment of cardiovascular disease. Very recently, we have proved that the (protamine sulfate/plasmid DNA encoding hepatocyte growth factor) (PrS/HGF-pDNA) multilayer can serve as a powerful tool for enhancing competitiveness of endothelial cell over smooth muscle cell, which opens perspectives for the regulation of intercellular competitiveness in the field of interventional therapy. However, before the gene multilayer films could be used in vascular stents for real clinical application, the preservation of gene bioactivity during the industrial sterilization and the hemocompatibility of film should be taken into account. Actually, both are long been ignored issues in the field of gene coating for GES. In this study, we demonstrate that the (PrS/HGF-pDNA) multilayer film exhibits the good gene-protecting abilities, which is confirmed by using the industrial sterilizations (gamma irradiation and ethylene oxide) and a routine storage condition (dry state at 4°C for 30 days). Furthermore, hemocompatible measurements (such as platelet adhesion and whole blood coagulation) and antibacterial assays (bacteria adhesion and growth inhibition) indicate the good anticoagulation and antibacterial properties of the (PrS/HGF-pDNA) multilayer film. The in vivo preliminary data of angiography and histological analysis suggest that the (PrS/HGF-pDNA) multilayer coated stent can reduce the in-stent restenosis. This work reveals that the (PrS/HGF-pDNA) multilayer film could be a promising candidate as coating for GES, which is of great potential in future clinic application. © 2014 Wiley Periodicals, Inc.

Size effect on transfection and cytotoxicity of nanoscale plasmid DNA/polyethyleneimine complexes for aerosol gene delivery

Energy Technology Data Exchange (ETDEWEB)

Hoon Byeon, Jeong, E-mail: jbyeon@purdue.edu [Department of Chemistry, Purdue University, West Lafayette, Indiana 47907 (United States); Kim, Jang-Woo, E-mail: jwkim@hoseo.edu [Department of Digital Display Engineering, Hoseo University, Asan 336-795 (Korea, Republic of)

2014-02-03

Nanoscale plasmid DNA (pDNA)/polyethyleneimine (PEI) complexes were fabricated in the aerosol state using a nebulization system consisting of a collison atomizer and a cool-walled diffusion dryer. The aerosol fabricated nanoscale complexes were collected and employed to determine fundamental properties of the complexes, such as size, structure, surface charge, and in vitro gene transfection efficiency and cytotoxicity. The results showed that mass ratio between pDNA and PEI should be optimized to enhance gene transfection efficiency without a significant loss of cell viability. These findings may support practical advancements in the field of nonviral gene delivery.

LNA effects on DNA binding and conformation

DEFF Research Database (Denmark)

Pabon-Martinez, Y Vladimir; Xu, You; Villa, Alessandra

2017-01-01

-substitution in the duplex pyrimidine strand alters the double helix structure, affecting x-displacement, slide and twist favoring triplex formation through enhanced TFO major groove accommodation. Collectively, these findings should facilitate the design of potent anti-gene ONs.......The anti-gene strategy is based on sequence-specific recognition of double-strand DNA by triplex forming (TFOs) or DNA strand invading oligonucleotides to modulate gene expression. To be efficient, the oligonucleotides (ONs) should target DNA selectively, with high affinity. Here we combined...... hybridization analysis and electrophoretic mobility shift assay with molecular dynamics (MD) simulations to better understand the underlying structural features of modified ONs in stabilizing duplex- and triplex structures. Particularly, we investigated the role played by the position and number of locked...

Common variants in mismatch repair genes associated with increased risk of sperm DNA damage and male infertility

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Ji Guixiang

2012-05-01

Full Text Available Abstract Background The mismatch repair (MMR pathway plays an important role in the maintenance of the genome integrity, meiotic recombination and gametogenesis. This study investigated whether genetic variations in MMR genes are associated with an increased risk of sperm DNA damage and male infertility. Methods We selected and genotyped 21 tagging single nucleotide polymorphisms (SNPs in five MMR genes (MLH1, MLH3, PMS2, MSH4 and MSH5 using the SNPstream 12-plex platform in a case-control study of 1,292 idiopathic infertility patients and 480 fertile controls in a Chinese population. Sperm DNA damage levels were detected with the Tdt-mediated dUTP nick end labelling (TUNEL assay in 450 cases. Fluorescence resonance energy transfer (FRET and co-immunoprecipitation techniques were employed to determine the effects of functional variants. Results One intronic SNP in MLH1 (rs4647269 and two non-synonymous SNPs in PMS2 (rs1059060, Ser775Asn and MSH5 (rs2075789, Pro29Ser seem to be risk factors for the development of azoospermia or oligozoospermia. Meanwhile, we also identified a possible contribution of PMS2 rs1059060 to the risk of male infertility with normal sperm count. Among patients with normal sperm count, MLH1 rs4647269 and PMS2 rs1059060 were associated with increased sperm DNA damage. Functional analysis revealed that the PMS2 rs1059060 can affect the interactions between MLH1 and PMS2. Conclusions Our results provide evidence supporting the involvement of genetic polymorphisms in MMR genes in the aetiology of male infertility.

Methamphetamine and HIV-Tat alter murine cardiac DNA methylation and gene expression

Energy Technology Data Exchange (ETDEWEB)

Koczor, Christopher A., E-mail: ckoczor@emory.edu; Fields, Earl; Jedrzejczak, Mark J.; Jiao, Zhe; Ludaway, Tomika; Russ, Rodney; Shang, Joan; Torres, Rebecca A.; Lewis, William

2015-11-01

This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10 d, 3 mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change > 1.5, p < 0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides for calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects. - Highlights: • HIV-1 Tat and methamphetamine (METH) alter cardiac gene expression and epigenetics. • METH impacts gene expression or epigenetics more significantly than Tat expression. • METH alters cardiac mitochondrial function and calcium signaling independent of Tat. • METH alters DNA methylation, expression, and protein abundance of

Methamphetamine and HIV-Tat alter murine cardiac DNA methylation and gene expression

International Nuclear Information System (INIS)

Koczor, Christopher A.; Fields, Earl; Jedrzejczak, Mark J.; Jiao, Zhe; Ludaway, Tomika; Russ, Rodney; Shang, Joan; Torres, Rebecca A.; Lewis, William

2015-01-01

This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10 d, 3 mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change > 1.5, p < 0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides for calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects. - Highlights: • HIV-1 Tat and methamphetamine (METH) alter cardiac gene expression and epigenetics. • METH impacts gene expression or epigenetics more significantly than Tat expression. • METH alters cardiac mitochondrial function and calcium signaling independent of Tat. • METH alters DNA methylation, expression, and protein abundance of

Mapping of gene transcripts by nuclease protection assays and cDNA primer extension

International Nuclear Information System (INIS)

Calzone, F.J.; Britten, R.J.; Davidson, E.J.

1987-01-01

An important problem often faced in the molecular characterization of genes is the precise mapping of those genomic sequences transcribed into RNA. This requires identification of the genomic site initiating gene transcription, the location of genomic sequences removed from the primary gene transcript during RNA processing, and knowledge of sequences terminating the processed gene transcript. The objective of the protocols described here is the generation of transcription maps utilizing relatively uncharacterized gene fragments. The basic approach is hybridization of a single-stranded DNA probe with cellular RNA, followed by treatment with a single-strand-specific nuclease that does not attack DNA-RNA hybrids, in order to destroy any unreacted probe sequences. Thus the probe sequences included in the hybrid duplexes are protected from nuclease digestion. The sizes of the protected probe fragments determined by gel electrophoresis correspond to the lengths of the hybridized sequence elements

DNA demethylation by 5-aza-2-deoxycytidine treatment abrogates 17 beta-estradiol-induced cell growth and restores expression of DNA repair genes in human breast cancer cells.

Science.gov (United States)

Singh, Kamaleshwar P; Treas, Justin; Tyagi, Tulika; Gao, Weimin

2012-03-01

Prolonged exposure to elevated levels of estrogen is a risk factor for breast cancer. Though increased cell growth and loss of DNA repair capacity is one of the proposed mechanisms for estrogen-induced cancers, the mechanism through which estrogen induces cell growth and decreases DNA repair capacity is not clear. DNA hypermethylation is known to inactivate DNA repair genes and apoptotic response in cancer cells. Therefore, the objective of this study was to determine the role of DNA hypermethylation in estrogen-induced cell growth and regulation of DNA repair genes expression in breast cancer cells. To achieve this objective, the estrogen-responsive MCF-7 cells either pretreated with 5-aza-2-deoxycytidine (5-aza-dC) or untreated (as control) were exposed to 17 beta-estradiol (E2), and its effect on cell growth and expression of DNA repair genes were measured. The result revealed that 5-aza-dC abrogates the E2-induced growth in MCF-7 cells. An increased expression of OGG1, MSH4, and MLH1 by 5-aza-dC treatment alone, suggest the DNA hypermethylation as a potential cause for decreased expression of these genes in MCF-7 cells. The decreased expression of ERCC1, XPC, OGG1, and MLH1 by E2 alone and its restoration by co-treatment with 5-aza-dC further suggest that E2 reduces the expression of these DNA repair genes potentially through promoter hypermethylation. Reactivation of mismatch repair (MMR) gene MLH1 and abrogation of E2-induced cell growth by 5-aza-dC treatment suggest that estrogen causes increased growth in breast cancer cells potentially through the inhibition of MMR-mediated apoptotic response. In summary, this study suggests that estrogen increases cell growth and decreases the DNA repair capacity in breast cancer cells, at least in part, through epigenetic mechanism. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Assembly of a biocompatible triazole-linked gene by one-pot click-DNA ligation

Science.gov (United States)

Kukwikila, Mikiembo; Gale, Nittaya; El-Sagheer, Afaf H.; Brown, Tom; Tavassoli, Ali

2017-11-01

The chemical synthesis of oligonucleotides and their enzyme-mediated assembly into genes and genomes has significantly advanced multiple scientific disciplines. However, these approaches are not without their shortcomings; enzymatic amplification and ligation of oligonucleotides into genes and genomes makes automation challenging, and site-specific incorporation of epigenetic information and/or modified bases into large constructs is not feasible. Here we present a fully chemical one-pot method for the assembly of oligonucleotides into a gene by click-DNA ligation. We synthesize the 335 base-pair gene that encodes the green fluorescent protein iLOV from ten functionalized oligonucleotides that contain 5ʹ-azide and 3ʹ-alkyne units. The resulting click-linked iLOV gene contains eight triazoles at the sites of chemical ligation, and yet is fully biocompatible; it is replicated by DNA polymerases in vitro and encodes a functional iLOV protein in Escherichia coli. We demonstrate the power and potential of our one-pot gene-assembly method by preparing an epigenetically modified variant of the iLOV gene.

Transcription profiles of mitochondrial genes correlate with mitochondrial DNA haplotypes in a natural population of Silene vulgaris

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Olson Matthew S

2010-01-01

Full Text Available Abstract Background Although rapid changes in copy number and gene order are common within plant mitochondrial genomes, associated patterns of gene transcription are underinvestigated. Previous studies have shown that the gynodioecious plant species Silene vulgaris exhibits high mitochondrial diversity and occasional paternal inheritance of mitochondrial markers. Here we address whether variation in DNA molecular markers is correlated with variation in transcription of mitochondrial genes in S. vulgaris collected from natural populations. Results We analyzed RFLP variation in two mitochondrial genes, cox1 and atp1, in offspring of ten plants from a natural population of S. vulgaris in Central Europe. We also investigated transcription profiles of the atp1 and cox1 genes. Most DNA haplotypes and transcription profiles were maternally inherited; for these, transcription profiles were associated with specific mitochondrial DNA haplotypes. One individual exhibited a pattern consistent with paternal inheritance of mitochondrial DNA; this individual exhibited a transcription profile suggestive of paternal but inconsistent with maternal inheritance. We found no associations between gender and transcript profiles. Conclusions Specific transcription profiles of mitochondrial genes were associated with specific mitochondrial DNA haplotypes in a natural population of a gynodioecious species S. vulgaris. Our findings suggest the potential for a causal association between rearrangements in the plant mt genome and transcription product variation.

DNA replication factor C1 mediates genomic stability and transcriptional gene silencing in Arabidopsis

KAUST Repository

Liu, Qian; Wang, Junguo; Miki, Daisuke; Xia, Ran; Yu, Wenxiang; He, Junna; Zheng, Zhimin; Zhu, Jian-Kang; Gonga, Zhizhong

2010-01-01

Genetic screening identified a suppressor of ros1-1, a mutant of REPRESSOR OF SILENCING1 (ROS1; encoding a DNA demethylation protein). The suppressor is a mutation in the gene encoding the largest subunit of replication factor C (RFC1). This mutation of RFC1 reactivates the unlinked 35S-NPTII transgene, which is silenced in ros1 and also increases expression of the pericentromeric Athila retrotransposons named transcriptional silent information in a DNA methylationindependent manner. rfc1 is more sensitive than the wild type to the DNA-damaging agent methylmethane sulphonate and to the DNA inter- and intra- cross-linking agent cisplatin. The rfc1 mutant constitutively expresses the G2/M-specific cyclin CycB1;1 and other DNA repair-related genes. Treatment with DNA-damaging agents mimics the rfc1 mutation in releasing the silenced 35S-NPTII, suggesting that spontaneously induced genomic instability caused by the rfc1 mutation might partially contribute to the released transcriptional gene silencing (TGS). The frequency of somatic homologous recombination is significantly increased in the rfc1 mutant. Interestingly, ros1 mutants show increased telomere length, but rfc1 mutants show decreased telomere length and reduced expression of telomerase. Our results suggest that RFC1 helps mediate genomic stability and TGS in Arabidopsis thaliana. © 2010 American Society of Plant Biologists.

DNA replication factor C1 mediates genomic stability and transcriptional gene silencing in Arabidopsis

KAUST Repository

Liu, Qian

2010-07-01

Genetic screening identified a suppressor of ros1-1, a mutant of REPRESSOR OF SILENCING1 (ROS1; encoding a DNA demethylation protein). The suppressor is a mutation in the gene encoding the largest subunit of replication factor C (RFC1). This mutation of RFC1 reactivates the unlinked 35S-NPTII transgene, which is silenced in ros1 and also increases expression of the pericentromeric Athila retrotransposons named transcriptional silent information in a DNA methylationindependent manner. rfc1 is more sensitive than the wild type to the DNA-damaging agent methylmethane sulphonate and to the DNA inter- and intra- cross-linking agent cisplatin. The rfc1 mutant constitutively expresses the G2/M-specific cyclin CycB1;1 and other DNA repair-related genes. Treatment with DNA-damaging agents mimics the rfc1 mutation in releasing the silenced 35S-NPTII, suggesting that spontaneously induced genomic instability caused by the rfc1 mutation might partially contribute to the released transcriptional gene silencing (TGS). The frequency of somatic homologous recombination is significantly increased in the rfc1 mutant. Interestingly, ros1 mutants show increased telomere length, but rfc1 mutants show decreased telomere length and reduced expression of telomerase. Our results suggest that RFC1 helps mediate genomic stability and TGS in Arabidopsis thaliana. © 2010 American Society of Plant Biologists.

Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes.

Science.gov (United States)

Metts, J; West, J; Doares, S H; Matthysse, A G

1991-02-01

Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis. Two of the mutants were avirulent on all hosts tested. The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N. debneyi, N. glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato). That the mutant phenotype was due to the transposon insertion was determined by cloning the DNA containing the transposon insertion and using the cloned DNA to replace the wild-type DNA in the parent bacterial strain by marker exchange. The transposon insertions in the three mutants mapped at three widely separated locations on the bacterial chromosome. The effects of the mutations on various steps in tumor formation were examined. All three mutants showed no alteration in binding to carrot cells. However, none of the mutants showed any induction of vir genes by acetosyringone under conditions in which the parent strain showed vir gene induction. When the mutant bacteria were examined for changes in surface components, it was found that all three of the mutants showed a similar alteration in lipopolysaccharide (LPS). LPS from the mutants was larger in size and more heavily saccharide substituted than LPS from the parent strain. Two of the mutants showed no detectable alteration in outer membrane and periplasmic space proteins. The third mutant, Ivr-225, was missing a 79-kDa surface peptide. The reason(s) for the failure of vir gene induction in these mutants and its relationship, if any, to the observed alteration in LPS are unknown.

DNA mutation motifs in the genes associated with inherited diseases.

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Michal Růžička

Full Text Available Mutations in human genes can be responsible for inherited genetic disorders and cancer. Mutations can arise due to environmental factors or spontaneously. It has been shown that certain DNA sequences are more prone to mutate. These sites are termed hotspots and exhibit a higher mutation frequency than expected by chance. In contrast, DNA sequences with lower mutation frequencies than expected by chance are termed coldspots. Mutation hotspots are usually derived from a mutation spectrum, which reflects particular population where an effect of a common ancestor plays a role. To detect coldspots/hotspots unaffected by population bias, we analysed the presence of germline mutations obtained from HGMD database in the 5-nucleotide segments repeatedly occurring in genes associated with common inherited disorders, in particular, the PAH, LDLR, CFTR, F8, and F9 genes. Statistically significant sequences (mutational motifs rarely associated with mutations (coldspots and frequently associated with mutations (hotspots exhibited characteristic sequence patterns, e.g. coldspots contained purine tract while hotspots showed alternating purine-pyrimidine bases, often with the presence of CpG dinucleotide. Using molecular dynamics simulations and free energy calculations, we analysed the global bending properties of two selected coldspots and two hotspots with a G/T mismatch. We observed that the coldspots were inherently more flexible than the hotspots. We assume that this property might be critical for effective mismatch repair as DNA with a mutation recognized by MutSα protein is noticeably bent.

Non-equilibrium repressor binding kinetics link DNA damage dose to transcriptional timing within the SOS gene network.

Science.gov (United States)

Culyba, Matthew J; Kubiak, Jeffrey M; Mo, Charlie Y; Goulian, Mark; Kohli, Rahul M

2018-06-01

Biochemical pathways are often genetically encoded as simple transcription regulation networks, where one transcription factor regulates the expression of multiple genes in a pathway. The relative timing of each promoter's activation and shut-off within the network can impact physiology. In the DNA damage repair pathway (known as the SOS response) of Escherichia coli, approximately 40 genes are regulated by the LexA repressor. After a DNA damaging event, LexA degradation triggers SOS gene transcription, which is temporally separated into subsets of 'early', 'middle', and 'late' genes. Although this feature plays an important role in regulating the SOS response, both the range of this separation and its underlying mechanism are not experimentally defined. Here we show that, at low doses of DNA damage, the timing of promoter activities is not separated. Instead, timing differences only emerge at higher levels of DNA damage and increase as a function of DNA damage dose. To understand mechanism, we derived a series of synthetic SOS gene promoters which vary in LexA-operator binding kinetics, but are otherwise identical, and then studied their activity over a large dose-range of DNA damage. In distinction to established models based on rapid equilibrium assumptions, the data best fit a kinetic model of repressor occupancy at promoters, where the drop in cellular LexA levels associated with higher doses of DNA damage leads to non-equilibrium binding kinetics of LexA at operators. Operators with slow LexA binding kinetics achieve their minimal occupancy state at later times than operators with fast binding kinetics, resulting in a time separation of peak promoter activity between genes. These data provide insight into this remarkable feature of the SOS pathway by demonstrating how a single transcription factor can be employed to control the relative timing of each gene's transcription as a function of stimulus dose.

A parasitic selfish gene that affects host promiscuity.

Science.gov (United States)

Giraldo-Perez, Paulina; Goddard, Matthew R

2013-11-07

Selfish genes demonstrate transmission bias and invade sexual populations despite conferring no benefit to their hosts. While the molecular genetics and evolutionary dynamics of selfish genes are reasonably well characterized, their effects on hosts are not. Homing endonuclease genes (HEGs) are one well-studied family of selfish genes that are assumed to be benign. However, we show that carrying HEGs is costly for Saccharomyces cerevisiae, demonstrating that these genetic elements are not necessarily benign but maybe parasitic. We estimate a selective load of approximately 1-2% in 'natural' niches. The second aspect we examine is the ability of HEGs to affect hosts' sexual behaviour. As all selfish genes critically rely on sex for spread, then any selfish gene correlated with increased host sexuality will enjoy a transmission advantage. While classic parasites are known to manipulate host behaviour, we are not aware of any evidence showing a selfish gene is capable of affecting host promiscuity. The data presented here show a selfish element may increase the propensity of its eukaryote host to undergo sex and along with increased rates of non-Mendelian inheritance, this may counterbalance mitotic selective load and promote spread. Demonstration that selfish genes are correlated with increased promiscuity in eukaryotes connects with ideas suggesting that selfish genes promoted the evolution of sex initially.

Obesity is associated with depot-specific alterations in adipocyte DNA methylation and gene expression

DEFF Research Database (Denmark)

Sonne, Si Brask; Yadav, Rachita; Yin, Guangliang

2017-01-01

The present study aimed to identify genes exhibiting concomitant obesity-dependent changes in DNA methylation and gene expression in adipose tissues in the mouse using diet-induced obese (DIO) C57BL/6J and genetically obese ob/ob mice as models. Mature adipocytes were isolated from epididymal...... and inguinal adipose tissues of ob/ob and DIO C57BL/6J mice. DNA methylation was analyzed by MeDIP-sequencing and gene expression by microarray analysis. The majority of differentially methylated regions (DMRs) were hypomethylated in obese mice. Global methylation of long interspersed elements indicated......57BL/6J mice occurred primarily in exons, whereas inguinal adipocytes of ob/ob mice exhibited a higher enrichment of DMRs in promoter regions than in other regions of the genome, suggesting an influence of leptin on DNA methylation in inguinal adipocytes. We observed altered methylation...

Gain of DNA methylation is enhanced in the absence of CTCF at the human retinoblastoma gene promoter

International Nuclear Information System (INIS)

Dávalos-Salas, Mercedes; Furlan-Magaril, Mayra; González-Buendía, Edgar; Valdes-Quezada, Christian; Ayala-Ortega, Erandi; Recillas-Targa, Félix

2011-01-01

Long-term gene silencing throughout cell division is generally achieved by DNA methylation and other epigenetic processes. Aberrant DNA methylation is now widely recognized to be associated with cancer and other human diseases. Here we addressed the contribution of the multifunctional nuclear factor CTCF to the epigenetic regulation of the human retinoblastoma (Rb) gene promoter in different tumoral cell lines. To assess the DNA methylation status of the Rb promoter, genomic DNA from stably transfected human erythroleukemic K562 cells expressing a GFP reporter transgene was transformed with sodium bisulfite, and then PCR-amplified with modified primers and sequenced. Single- and multi-copy integrants with the CTCF binding site mutated were isolated and characterized by Southern blotting. Silenced transgenes were reactivated using 5-aza-2'-deoxycytidine and Trichostatin-A, and their expression was monitored by fluorescent cytometry. Rb gene expression and protein abundance were assessed by RT-PCR and Western blotting in three different glioma cell lines, and DNA methylation of the promoter region was determined by sodium bisulfite sequencing, together with CTCF dissociation and methyl-CpG-binding protein incorporation by chromatin immunoprecipitation assays. We found that the inability of CTCF to bind to the Rb promoter causes a dramatic loss of gene expression and a progressive gain of DNA methylation. This study indicates that CTCF plays an important role in maintaining the Rb promoter in an optimal chromatin configuration. The absence of CTCF induces a rapid epigenetic silencing through a progressive gain of DNA methylation. Consequently, CTCF can now be seen as one of the epigenetic components that allows the proper configuration of tumor suppressor gene promoters. Its aberrant dissociation can then predispose key genes in cancer cells to acquire DNA methylation and epigenetic silencing

Production of DNA microarray and expression analysis of genes from Xylella fastidiosa in different culture media

Directory of Open Access Journals (Sweden)

Regiane de Fátima Travensolo

2009-06-01

Full Text Available DNA Microarray was developed to monitor the expression of many genes from Xylella fastidiosa, allowing the side by-side comparison of two situations in a single experiment. The experiments were performed using X. fastidiosa cells grown in two culture media: BCYE and XDM2. The primers were synthesized, spotted onto glass slides and the array was hybridized against fluorescently labeled cDNAs. The emitted signals were quantified, normalized and the data were statistically analyzed to verify the differentially expressed genes. According to the data, 104 genes were differentially expressed in XDM2 and 30 genes in BCYE media. The present study showed that DNA microarray technique efficiently differentiate the expressed genes under different conditions.DNA Microarray foi desenvolvida para monitorar a expressão de muitos genes de Xylella fastidiosa, permitindo a comparação de duas situações distintas em um único experimento. Os experimentos foram feitos utilizando células de X. fastidiosa cultivada em dois meios de cultura: BCYE e XDM2. Pares de oligonucleotídeos iniciadores foram sintetizados, depositados em lâminas de vidro e o arranjo foi hibridizado contra cDNAs marcados fluorescentemente. Os sinais emitidos foram quantificados, normalizados e os dados foram estatisticamente analisados para verificar os genes diferencialmente expressos. De acordo com nossos dados, 104 genes foram diferencialmente expressos para o meio de cultura XDM2 e 30 genes para o BCYE. No presente estudo, nós demonstramos que a técnica de DNA microarrays eficientemente diferencia genes expressos sob diferentes condições de cultivo.

Development of library preparation method able to correct gene expression levels in rice anther and isolate a trace expression gene mediated in cold-resistance

Energy Technology Data Exchange (ETDEWEB)

Yamaguchi, Tomoya; Koike, Setsuo [Tohoku National Agricultural Experiment Station, Morioka (Japan)

2000-02-01

When cDNA library is prepared by a previously developed method, genes of which expression level is high are apt to be cloned at a high frequency, whereas genes of which expression level are low, are difficult to be cloned. A low-expression gene has been cloned at very low frequency. Therefore, the gene encoding the key enzyme that is involved in growth disturbance of rice pollen has not been identified. In this study, development of a library preparing method able to correct the expression level was attempted using highly sensitive detection method with radioisotope and some genes related to cold-resistance of rice were isolated. Double strand DNAs were synthesized using mRNA extract from rice anthers and annealed following heat-denaturation. It has been known that single strand DNA molecules abundantly existing in DNA solution can easily aggregate to form double strand DNA, but single stranded DNA molecules poor in the solution are apt to still remain as single strand after annealing. Thus, the amount of single strand DNA would be balanced in the solution between abundant DNA and poor DNA species. The authors succeeded to prepare a gene library including low and high expression genes at similar proportions. Moreover, spin trap method that allows RI labeling of DNA bound to latex particle, was developed to detect with high sensitivity, especially for genes that are expressed at low level. The present method could be used for recovery, detection and quantitative analysis of radiolabeled single strand DNA. Thus, it was demonstrated that the stage from tetrad sperm to small sperm might be easily affected by cold stress. The present results suggest that the expressions of {beta}-1 and {beta}-3 glucanase, which are involved in the release of small sperms following meiosis in the pollen formation, might be easily affected by cold stress. (M.N.)

Development of library preparation method able to correct gene expression levels in rice anther and isolate a trace expression gene mediated in cold-resistance

International Nuclear Information System (INIS)

Yamaguchi, Tomoya; Koike, Setsuo

2000-01-01

When cDNA library is prepared by a previously developed method, genes of which expression level is high are apt to be cloned at a high frequency, whereas genes of which expression level are low, are difficult to be cloned. A low-expression gene has been cloned at very low frequency. Therefore, the gene encoding the key enzyme that is involved in growth disturbance of rice pollen has not been identified. In this study, development of a library preparing method able to correct the expression level was attempted using highly sensitive detection method with radioisotope and some genes related to cold-resistance of rice were isolated. Double strand DNAs were synthesized using mRNA extract from rice anthers and annealed following heat-denaturation. It has been known that single strand DNA molecules abundantly existing in DNA solution can easily aggregate to form double strand DNA, but single stranded DNA molecules poor in the solution are apt to still remain as single strand after annealing. Thus, the amount of single strand DNA would be balanced in the solution between abundant DNA and poor DNA species. The authors succeeded to prepare a gene library including low and high expression genes at similar proportions. Moreover, spin trap method that allows RI labeling of DNA bound to latex particle, was developed to detect with high sensitivity, especially for genes that are expressed at low level. The present method could be used for recovery, detection and quantitative analysis of radiolabeled single strand DNA. Thus, it was demonstrated that the stage from tetrad sperm to small sperm might be easily affected by cold stress. The present results suggest that the expressions of β-1 and β-3 glucanase, which are involved in the release of small sperms following meiosis in the pollen formation, might be easily affected by cold stress. (M.N.)

A defect in the thymidine kinase 2 gene causing isolated mitochondrial myopathy without mtDNA depletion.

Science.gov (United States)

Leshinsky-Silver, E; Michelson, M; Cohen, S; Ginsberg, M; Sadeh, M; Barash, V; Lerman-Sagie, T; Lev, D

2008-07-01

Isolated mitochondrial myopathies (IMM) are either due to primary defects in mtDNA, in nuclear genes that control mtDNA abundance and structure such as thymidine kinase 2 (TK2), or due to CoQ deficiency. Defects in the TK2 gene have been found to be associated with mtDNA depletion attributed to a depleted mitochondrial dNTP pool in non-dividing cells. We report an unusual case of IMM, homozygous for the H90N mutation in the TK2 gene but unlike other cases with the same mutation, does not demonstrate mtDNA depletion. The patient's clinical course is relatively mild and a muscle biopsy showed ragged red muscle fibers with a mild decrease in complexes I and an increase in complexes IV and II activities. This report extends the phenotypic expression of TK2 defects and suggests that all patients who present with an IMM even with normal quantities of mtDNA should be screened for TK2 mutations.

GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells.

Science.gov (United States)

Kostyuk, Svetlana; Smirnova, Tatiana; Kameneva, Larisa; Porokhovnik, Lev; Speranskij, Anatolij; Ershova, Elizaveta; Stukalov, Sergey; Izevskaya, Vera; Veiko, Natalia

2015-01-01

Cell free DNA (cfDNA) circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA) and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci). As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR), PCNA (FACS)) and antiapoptotic genes (BCL2 (RT-PCR and FACS), BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR)). Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs). Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR), in the level of fatty acid binding protein FABP4 (FACS analysis) and in the level of fat (Oil Red O). GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose-derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis.

DNA repair gene polymorphisms in relation to chromosome aberration frequencies in retired radiation workers

International Nuclear Information System (INIS)

Wilding, Craig S.; Relton, Caroline L.; Rees, Gwen S.; Tarone, Robert E.; Whitehouse, Caroline A.; Tawn, E. Janet

2005-01-01

Polymorphic variation in DNA repair genes was examined in a group of retired workers from the British Nuclear Fuels plc facility at Sellafield in relation to previously determined translocation frequencies in peripheral blood lymphocytes. Variation at seven polymorphisms in four genes involved in the base excision repair (XRCC1 R194W, R399Q and a [AC] n microsatellite in the 3' UTR) and double strand break repair (XRCC3 T241M and a [AC] n microsatellite in intron 3 of XRCC3, XRCC4 I134T, and a GACTAn microsatellite located 120kb 5' of XRCC5) pathways was determined for 291 retired radiation workers who had received cumulative occupational external radiation doses of between 0 and 1873mSv. When the interaction between radiation dose and each DNA repair gene polymorphism was examined in relation to translocation frequency there was no evidence for any of the polymorphisms studied influencing the response to occupational exposure. A positive interaction observed between genotype (individuals with at least one allele >=20 repeat units) at a microsatellite locus in the XRCC3 gene and smoking status should be interpreted cautiously because interactions were investigated for seven polymorphisms and two exposures. Nonetheless, further research is warranted to examine whether this DNA repair gene variant might be associated with a sub-optimal repair response to smoking-induced DNA damage and hence an increased frequency of translocations

Corruption of the intra-gene DNA methylation architecture is a hallmark of cancer.

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Bartlett, Thomas E; Zaikin, Alexey; Olhede, Sofia C; West, James; Teschendorff, Andrew E; Widschwendter, Martin

2013-01-01

Epigenetic processes--including DNA methylation--are increasingly seen as having a fundamental role in chronic diseases like cancer. It is well known that methylation levels at particular genes or loci differ between normal and diseased tissue. Here we investigate whether the intra-gene methylation architecture is corrupted in cancer and whether the variability of levels of methylation of individual CpGs within a defined gene is able to discriminate cancerous from normal tissue, and is associated with heterogeneous tumour phenotype, as defined by gene expression. We analysed 270985 CpGs annotated to 18272 genes, in 3284 cancerous and 681 normal samples, corresponding to 14 different cancer types. In doing so, we found novel differences in intra-gene methylation pattern across phenotypes, particularly in those genes which are crucial for stem cell biology; our measures of intra-gene methylation architecture are a better determinant of phenotype than measures based on mean methylation level alone (K-S test [Formula: see text] in all 14 diseases tested). These per-gene methylation measures also represent a considerable reduction in complexity, compared to conventional per-CpG beta-values. Our findings strongly support the view that intra-gene methylation architecture has great clinical potential for the development of DNA-based cancer biomarkers.

HMGB1-mediated DNA bending: Distinct roles in increasing p53 binding to DNA and the transactivation of p53-responsive gene promoters.

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Štros, Michal; Kučírek, Martin; Sani, Soodabeh Abbasi; Polanská, Eva

2018-03-01

HMGB1 is a chromatin-associated protein that has been implicated in many important biological processes such as transcription, recombination, DNA repair, and genome stability. These functions include the enhancement of binding of a number of transcription factors, including the tumor suppressor protein p53, to their specific DNA-binding sites. HMGB1 is composed of two highly conserved HMG boxes, linked to an intrinsically disordered acidic C-terminal tail. Previous reports have suggested that the ability of HMGB1 to bend DNA may explain the in vitro HMGB1-mediated increase in sequence-specific DNA binding by p53. The aim of this study was to reinvestigate the importance of HMGB1-induced DNA bending in relationship to the ability of the protein to promote the specific binding of p53 to short DNA duplexes in vitro, and to transactivate two major p53-regulated human genes: Mdm2 and p21/WAF1. Using a number of HMGB1 mutants, we report that the HMGB1-mediated increase in sequence-specific p53 binding to DNA duplexes in vitro depends very little on HMGB1-mediated DNA bending. The presence of the acidic C-terminal tail of HMGB1 and/or the oxidation of the protein can reduce the HMGB1-mediated p53 binding. Interestingly, the induction of transactivation of p53-responsive gene promoters by HMGB1 requires both the ability of the protein to bend DNA and the acidic C-terminal tail, and is promoter-specific. We propose that the efficient transactivation of p53-responsive gene promoters by HMGB1 depends on complex events, rather than solely on the promotion of p53 binding to its DNA cognate sites. Copyright © 2018 Elsevier B.V. All rights reserved.

Differential structural status of the RNA counterpart of an undecamer quasi-palindromic DNA sequence present in LCR of human β-globin gene cluster.

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Kaushik, Mahima; Kukreti, Shrikant

2015-01-01

Our previous work on structural polymorphism shown at a single nucleotide polymorphism (SNP) (A → G) site located on HS4 region of locus control region (LCR) of β-globin gene has established a hairpin → duplex equilibrium corresponding to A → B like DNA transition (Kaushik M, Kukreti, R., Grover, D., Brahmachari, S.K. and Kukreti S. Nucleic Acids Res. 2003; Kaushik M, Kukreti S. Nucleic Acids Res. 2006). The G-allele of A → G SNP has been shown to be significantly associated with the occurrence of β-thalassemia. Considering the significance of this 11-nt long quasi-palindromic sequence [5'-TGGGG(G/A)CCCCA; HP(G/A)11] of β-globin gene LCR, we further explored the differential behavior of the same DNA sequence with its RNA counterpart, using various biophysical and biochemical techniques. In contrast to its DNA counterpart exhibiting a A → B structural transition and an equilibrium between duplex and hairpin forms, the studied RNA oligonucleotide sequence [5'-UGGGG(G/A)CCCCA; RHP(G/A)11] existed only in duplex form (A-conformation) and did not form hairpin. The single residue difference from A to G led to the unusual thermal stability of the RNA structure formed by the studied sequence. Since, naturally occurring mutations and various SNP sites may stabilize or destabilize the local DNA/RNA secondary structures, these structural transitions may affect the gene expression by a change in the protein-DNA recognition patterns.

Coordinate regulation of stromelysin and collagenase genes determined with cDNA probes

International Nuclear Information System (INIS)

Frisch, S.M.; Clark, E.J.; Werb, Z.

1987-01-01

Secreted proteinases are required for tumor metastasis, angiogenesis, and tissue remodeling during wound healing and embryonic growth. Thus, the regulation of the genes of secreted proteinases may serve as an interesting model for growth-controlled genes in general. The authors studied the genes of the secreted proteinases stromelysin and collagenase by using molecularly cloned cDNAs from each proteinase. Stromelysin cDNA was cloned by differential screening of a total cDNA library from rabbit synovial cells treated with phorbol 12-myristate 13-acetate, which yielded a clone of 1.2 kilobase pairs; collagenase cDNA was obtained by cloning reverse transcripts of anti-collagenase-immunoadsorbed polysomal mRNA, which yielded a clone of 0.8 kilobase pairs. Stromelysin and collagenase mRNA species of 2.2 and 2.4 kilobases, respectively, were detected on hybridization blots of RNA from phorbol 12-myristate 13-acetate-treated but not untreated rabbit synovial cells. Expression of stromelysin mRNA was also induced in rabbit alveolar macrophages and rabbit brain capillary endothelial cells treated with phorbol 12-myristate 13-acetate. Stromelysin and collagenase mRNA were both induced by phorbol 12-myristate 13-acetate and cytochalasin B at a constant ratio of the two gene products; this suggest coordinate regulation. The fact that induction was blocked after inhibition of protein synthesis by cycloheximide implicates an indirect signal transduction pathway that requires new protein synthesis

A novel Listeria monocytogenes-based DNA delivery system for cancer gene therapy.

LENUS (Irish Health Repository)

van Pijkeren, Jan Peter

2012-01-31

Bacteria-mediated transfer of plasmid DNA to mammalian cells (bactofection) has been shown to have significant potential as an approach to express heterologous proteins in various cell types. This is achieved through entry of the entire bacterium into cells, followed by release of plasmid DNA. In a murine model, we show that Listeria monocytogenes can invade and spread in tumors, and establish the use of Listeria to deliver genes to tumors in vivo. A novel approach to vector lysis and release of plasmid DNA through antibiotic administration was developed. Ampicillin administration facilitated both plasmid transfer and safety control of vector. To further improve on the gene delivery system, we selected a Listeria monocytogenes derivative that is more sensitive to ampicillin, and less pathogenic than the wild-type strain. Incorporation of a eukaryotic-transcribed lysin cassette in the plasmid further increased bacterial lysis. Successful gene delivery of firefly luciferase to growing tumors in murine models and to patient breast tumor samples ex vivo was achieved. The model described encompasses a three-phase treatment regimen, involving (1) intratumoral administration of vector followed by a period of vector spread, (2) systemic ampicillin administration to induce vector lysis and plasmid transfer, and (3) systemic administration of combined moxifloxacin and ampicillin to eliminate systemic vector. For the first time, our results reveal the potential of Listeria monocytogenes for in vivo gene delivery.

Digital quantification of gene methylation in stool DNA by emulsion-PCR coupled with hydrogel immobilized bead-array.

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Liu, Yunlong; Wu, Haiping; Zhou, Qiang; Song, Qinxin; Rui, Jianzhong; Zou, Bingjie; Zhou, Guohua

2017-06-15

Aberrations of gene methylation in stool DNA (sDNA) is an effective biomarker for non-invasive colorectal cancer diagnosis. However, it is challenging to accurately quantitate the gene methylation levels in sDNA due to the low abundance and degradation of sDNA. In this study, a digital quantification strategy was proposed by combining emulsion PCR (emPCR) with hydrogel immobilized bead-array. The assay includes following steps: bisulfite conversion of sDNA, pre-amplification by PCR with specific primers containing 5' universal sequences, emPCR of pre-amplicons with beaded primers to achieve single-molecular amplification and identification of hydrogel embedding beads coated with amplicons. The sensitivity and the specificity of the method are high enough to pick up 0.05% methylated targets from unmethylated DNA background. The successful detection of hypermethylated vimentin gene in clinical stool samples suggests that the proposed method should be a potential tool for non-invasive colorectal cancer screening. Copyright © 2016 Elsevier B.V. All rights reserved.

A Small-Molecule Inducible Synthetic Circuit for Control of the SOS Gene Network without DNA Damage.

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Kubiak, Jeffrey M; Culyba, Matthew J; Liu, Monica Yun; Mo, Charlie Y; Goulian, Mark; Kohli, Rahul M

2017-11-17

The bacterial SOS stress-response pathway is a pro-mutagenic DNA repair system that mediates bacterial survival and adaptation to genotoxic stressors, including antibiotics and UV light. The SOS pathway is composed of a network of genes under the control of the transcriptional repressor, LexA. Activation of the pathway involves linked but distinct events: an initial DNA damage event leads to activation of RecA, which promotes autoproteolysis of LexA, abrogating its repressor function and leading to induction of the SOS gene network. These linked events can each independently contribute to DNA repair and mutagenesis, making it difficult to separate the contributions of the different events to observed phenotypes. We therefore devised a novel synthetic circuit to unlink these events and permit induction of the SOS gene network in the absence of DNA damage or RecA activation via orthogonal cleavage of LexA. Strains engineered with the synthetic SOS circuit demonstrate small-molecule inducible expression of SOS genes as well as the associated resistance to UV light. Exploiting our ability to activate SOS genes independently of upstream events, we further demonstrate that the majority of SOS-mediated mutagenesis on the chromosome does not readily occur with orthogonal pathway induction alone, but instead requires DNA damage. More generally, our approach provides an exemplar for using synthetic circuit design to separate an environmental stressor from its associated stress-response pathway.

DNA probe labeling with digoxigenin-dUTP and its application in gene diagnosis

International Nuclear Information System (INIS)

Liu Guoyang

1992-01-01

DNA probe labeling by the randomly primed incorporation of digoxigenin-dUTP is reported. The sensitivity of color reaction and hybridization were 32 fg and 200 fg, respectively, and both were specific for the target. Single-copy and multi-copy gene fragments among 2 μg human genomic DNA were detected by β IVS II, Fr 3-42 and 3'HVR labeled with digoxigenin-dUTP. The results were consistent with a radioactive control assay. This method has been successfully used in the gene diagnosis of adult polycystic kidney disease

CDKL5 is a brain MeCP2 target gene regulated by DNA methylation.

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Carouge, Delphine; Host, Lionel; Aunis, Dominique; Zwiller, Jean; Anglard, Patrick

2010-06-01

Rett syndrome and its "early-onset seizure" variant are severe neurodevelopmental disorders associated with mutations within the MECP2 and the CDKL5 genes. Antidepressants and drugs of abuse induce the expression of the epigenetic factor MeCP2, thereby influencing chromatin remodeling. We show that increased MeCP2 levels resulted in the repression of Cdkl5 in rat brain structures in response to cocaine, as well as in cells exposed to serotonin, or overexpressing MeCP2. In contrast, Cdkl5 was induced by siRNA-mediated knockdown of Mecp2 and by DNA-methyltransferase inhibitors, demonstrating its regulation by MeCP2 and by DNA methylation. Cdkl5 gene methylation and its methylation-dependent binding to MeCP2 were increased in the striatum of cocaine-treated rats. Our data demonstrate that Cdkl5 is a MeCP2-repressed target gene providing a link between genes the mutation of which generates overlapping symptoms. They highlight DNA methylation changes as a potential mechanism participating in the long-term plasticity triggered by pharmacological agents.

Epigenetic Alteration by DNA Promoter Hypermethylation of Genes Related to Transforming Growth Factor-β (TGF-β Signaling in Cancer

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Kiyoshi Mori

2011-03-01

Full Text Available Epigenetic alterations in cancer, especially DNA methylation and histone modification, exert a significant effect on the deregulated expression of cancer-related genes and lay an epigenetic pathway to carcinogenesis and tumor progression. Global hypomethylation and local hypermethylation of CpG islands in the promoter region, which result in silencing tumor suppressor genes, constitute general and major epigenetic modification, the hallmark of the neoplastic epigenome. Additionally, methylation-induced gene silencing commonly affects a number of genes and increases with cancer progression. Indeed, cancers with a high degree of methylation (CpG island methylator phenotype/CIMP do exist and represent a distinct subset of certain cancers including colorectal, bladder and kidney. On the other hand, signals from the microenvironment, especially those from transforming growth factor-β (TGF-β, induce targeted de novo epigenetic alterations of cancer-related genes. While TGF-β signaling has been implicated in two opposite roles in cancer, namely tumor suppression and tumor promotion, its deregulation is also partly induced by epigenetic alteration itself. Although the epigenetic pathway to carcinogenesis and cancer progression has such reciprocal complexity, the important issue is to identify genes or signaling pathways that are commonly silenced in various cancers in order to find early diagnostic and therapeutic targets. In this review, we focus on the epigenetic alteration by DNA methylation and its role in molecular modulations of the TGF-β signaling pathway that cause or underlie altered cancer-related gene expression in both phases of early carcinogenesis and late cancer progression.

Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation

Energy Technology Data Exchange (ETDEWEB)

Banks, G.R.; Taylor, S.Y. (National Institute for Medical Research, London (England))

1988-12-01

The Ustilago maydis PYR3 gene encoding dihydroorotase activity was cloned by direct complementation of Escherichia coli pyrC mutations. PYR3 transformants of E. coli pyrC mutants expressed homologous transcripts of a variety of sizes and regained dihydroorotase activity. PYR3 also complemented Saccharomyces cerevisiae ura4 mutations, and again multiple transcripts were expressed in transformants, and enzyme activity was regained. A 1.25-kilobase poly(rA)+ PYR3 transcript was detected in U. maydis itself. Linear DNA carrying the PYR3 gene transformed a U. maydis pyr3-1 pyrimidine auxotroph to prototrophy. Hybridization analysis revealed that three different types of transformants could be generated, depending on the structure of the transforming DNA used. The first type involved exchange of chromosomal mutant gene sequences with the cloned wild-type plasmid sequences. A second type had integrated linear transforming DNA at the chromosomal PYR3 locus, probably via a single crossover event. The third type had integrated transforming DNA sequences at multiple sites in the U. maydis genome. In the last two types, tandemly reiterated copies of the transforming DNA were found to have been integrated. All three types had lost the sensitivity of the parental pyr3-1 mutant to UV irradiation. They had also regained dihydroorotase activity, although its level did not correlate with the PYR3 gene copy number.

Development and validation of an integrated DNA walking strategy to detect GMO expressing cry genes.

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Fraiture, Marie-Alice; Vandamme, Julie; Herman, Philippe; Roosens, Nancy H C

2018-06-27

Recently, an integrated DNA walking strategy has been proposed to prove the presence of GMO via the characterisation of sequences of interest, including their transgene flanking regions and the unnatural associations of elements in their transgenic cassettes. To this end, the p35S, tNOS and t35S pCAMBIA elements have been selected as key targets, allowing the coverage of most of GMO, EU authorized or not. In the present study, a bidirectional DNA walking method anchored on the CryAb/c genes is proposed with the aim to cover additional GMO and additional sequences of interest. The performance of the proposed bidirectional DNA walking method anchored on the CryAb/c genes has been evaluated in a first time for its feasibility using several GM events possessing these CryAb/c genes. Afterwards, its sensitivity has been investigated through low concentrations of targets (as low as 20 HGE). In addition, to illustrate its applicability, the entire workflow has been tested on a sample mimicking food/feed matrices analysed in GMO routine analysis. Given the successful assessment of its performance, the present bidirectional DNA walking method anchored on the CryAb/c genes can easily be implemented in GMO routine analysis by the enforcement laboratories and allows completing the entire DNA walking strategy in targeting an additional transgenic element frequently found in GMO.

Extraction of Total DNA and RNA from Marine Filter Samples and Generation of a cDNA as Universal Template for Marker Gene Studies.

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Schneider, Dominik; Wemheuer, Franziska; Pfeiffer, Birgit; Wemheuer, Bernd

2017-01-01

Microbial communities play an important role in marine ecosystem processes. Although the number of studies targeting marker genes such as the 16S rRNA gene has been increased in the last few years, the vast majority of marine diversity is rather unexplored. Moreover, most studies focused on the entire bacterial community and thus disregarded active microbial community players. Here, we describe a detailed protocol for the simultaneous extraction of DNA and RNA from marine water samples and for the generation of cDNA from the isolated RNA which can be used as a universal template in various marker gene studies.

Alterations in radioresistance of eucaryotic cells after the transfer of genomic wildtype DNA and metallothionein genes

International Nuclear Information System (INIS)

Lohrer, H.

1987-01-01

The presented paper describes experiments concerning the alteration of radiosensitivity of eucaryotic cells after gene transfer. Ionizing radiation (γ- or X-ray) induces DNA single- or double strand breaks, which are religated by an unknown repair system. Repair deficient cells are highly sensitive to ionizing radiation. In the experiments described, cells from a patient with the heritable disease Ataxia telangiectasia were used as well as two X-ray sensitive CHO mutant cell lines. After gene transfer of an intact human DNA repair gene or a metallothionein gene the cells should regain radioresistance. (orig.) [de

Anchoring of self-assembled plasmid DNA/ anti-DNA antibody/cationic lipid micelles on bisphosphonate-modified stent for cardiovascular gene delivery

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Ma G

2013-03-01

Full Text Available Guilei Ma,1,# Yong Wang,1,# Ilia Fishbein,2 Mei Yu,1 Linhua Zhang,1 Ivan S Alferiev,2 Jing Yang,1 Cunxian Song,1 Robert J Levy2 1Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China; 2Children's Hospital of Philadelphia, Abramson Research Building, Philadelphia, PA, USA #These authors contributed equally to this work Purpose: To investigate the anchoring of plasmid DNA/anti-DNA antibody/cationic lipid tri-complex (DAC micelles onto bisphosphonate-modified 316 L coronary stents for cardiovascular site-specific gene delivery. Methods: Stents were first modified with polyallylamine bisphosphonate (PAA-BP, thereby enabling the retention of a PAA-BP molecular monolayer that permits the anchoring (via vector-binding molecules of DAC micelles. DAC micelles were then chemically linked onto the PAA-BP-modified stents by using N-succinimidyl-3-(2-pyridyldithiol-propionate (SPDP as a crosslinker. Rhodamine-labeled DNA was used to assess the anchoring of DAC micelles, and radioactive-labeled antibody was used to evaluate binding capacity and stability. DAC micelles (encoding green fluorescent protein were tethered onto the PAA-BP-modified stents, which were assessed in cell culture. The presence of a PAA-BP molecular monolayer on the steel surface was confirmed by X-ray photoelectron spectroscopy and atomic force microscope analysis. Results: The anchoring of DAC micelles was generally uniform and devoid of large-scale patches of defects. Isotopic quantification confirmed that the amount of antibody chemically linked on the stents was 17-fold higher than that of the physical adsorbed control stents and its retention time was also significantly longer. In cell culture, numerous green fluorescent protein-positive cells were found on the PAA-BP modified stents, which demonstrated high localization and efficiency of gene delivery. Conclusion: The DAC micelle

Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction.

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Birla, Bhagyashree S; Chou, Hui-Hsien

2015-01-01

Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple double-stranded DNA fragments. Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor. In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number dsDNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the Gibson Assembly protocol, we have assembled 3 different genes from up to 45 dsDNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to most gene sequences, and can improve both the efficiency and cost of gene assembly.

Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction.

Directory of Open Access Journals (Sweden)

Bhagyashree S Birla

Full Text Available Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple double-stranded DNA fragments. Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor. In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number dsDNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the Gibson Assembly protocol, we have assembled 3 different genes from up to 45 dsDNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to most gene sequences, and can improve both the efficiency and cost of gene assembly.

Dietary Berries and Ellagic Acid Prevent Oxidative DNA Damage and Modulate Expression of DNA Repair Genes

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Ramesh C. Gupta

2008-03-01

Full Text Available DNA damage is a pre-requisite for the initiation of cancer and agents that reduce this damage are useful in cancer prevention. In this study, we evaluated the ability of whole berries and berry phytochemical, ellagic acid to reduce endogenous oxidative DNA damage. Ellagic acid was selected based on > 95% inhibition of 8-oxodeoxyguosine (8-oxodG and other unidentified oxidative DNA adducts induced by 4-hydroxy-17B;-estradiol and CuCl2 in vitro. Inhibition of the latter occurred at lower concentrations (10 u(microM than that for 8-oxodG (100 u(microM. In the in vivo study, female CD-1 mice (n=6 were fed either a control diet or diet supplemented with ellagic acid (400 ppm and dehydrated berries (5% w/w with varying ellagic acid contents -- blueberry (low, strawberry (medium and red raspberry (high, for 3 weeks. Blueberry and strawberry diets showed moderate reductions in endogenous DNA adducts (25%. However, both red raspberry and ellagic acid diets showed a significant reduction of 59% (p < 0.001 and 48% (p < 0.01, respectively. Both diets also resulted in a 3-8 fold over-expression of genes involved in DNA repair such as xeroderma pigmentosum group A complementing protein (XPA, DNA excision repair protein (ERCC5 and DNA ligase III (DNL3. These results suggest that red raspberry and ellagic acid reduce endogenous oxidative DNA damage by mechanisms which may involve increase in DNA repair.

Genomics and radical mediated DNA damage: major differences between ionizing radiation and DNA-cleaving enediynes

International Nuclear Information System (INIS)

Cosgrove, J.P.; Begley, T.J.; Samson, L.D.; Dedon, P.C.

2003-01-01

While the evidence is strong for radical-mediated oxidative processes in the pathophysiology of cancer and aging, the mechanisms by which cells respond to oxidative stress have eluded definition. To this end, we have undertaken genomic studies comparing the response of S. cerevisiae to DNA-specific oxidizing agents, the enediynes calicheamicin (CAL), esperamicin (ESP), and neocarzinostatin (NCS), and the non-specific gamma-radiation (RAD). While RAD results in relatively indiscriminate oxidation of cellular molecules, the enediynes are highly specific to DNA and produce damage by a common mechanism involving radical-mediated oxidation of deoxyribose. Transcriptional profiling in response to these agents (80% survival; 15 min exposure; Affymetrix) revealed unexpected differences between RAD and the enediynes and among the three enediynes. Only 2 genes responded in common to all agents, while 9 genes were regulated in common for the 3 enediynes (no DNA repair genes altered in common). The limited common gene expression changes for the 3 enediynes may result from differences in deoxyribose oxidation chemistry, DNA and chromatin targets or the proportions of single- and double-strand DNA lesions. RAD produced a more robust response than the enediynes, altering expression of 195 and 52 genes by more than 2- and 5-fold, respectively, compared to 16-44 and *2 genes, respectively, for the enediynes. This suggests that the transcriptional response varies in intensity according to the number of cellular features affected by the toxin. Genes showing the strongest up-regulation with RAD: ribonucleotide reductase, multidrug resistance, DS break repair/RAD51, GSH transferase; strongly reduced gene expression: TEL1 (damage signaling), NAT2 (acetyltransferase). Genomic phenotyping studies, using a subset of the Research Genetics deletion library, revealed that loss of apn1, the major AP endonuclease, caused resistance to NCS, possibly due to reduced formation of protein-DNA cross

Application of DNA chips in the analysis of gene mutation in HBV

International Nuclear Information System (INIS)

Wang Yongzhong; Ruan Lihua; Zhou Guoping; Wu Guoxiang; Chen Min

2005-01-01

Objective: To investigate the clinical applicability of DNA chips for analysis of gene mutation in HBV. Methods: Serum HBV DNA from 47 patients with viral hepatitis type B was amplified with PCR. Possible gene mutations were searched for in site 1896 of pre-C section, sites 1762,1764 of BCP section and sites 528, 552 of P section with DNA chip method based upon membrane coloration. Results: In the 32 patients without lamivudine treatment, the results were as follows: (1) 6 specimens with HBsAg + , HBeAg + , HBeAb - , no mutations observed. (2) 13 specimens with HBsAg + , HBeAg - , HBeAb + , mutations at site 1896, pre- C 4 cases, mutations at sites 1762,1764, BCP 11 cases. (3) 13 specimens with HBsAg + , HBeAg + , HBeAb + , mutations at site 1896 pre -C 4 cases, mutations at sites 1762,1764 BCP 13 cases. In the 15 patients after 48 weeks treatment with lamivudine but remained HBV DNA positive, mutations were observed at: site 1896 pre-C, 5 cases, sites 1762,1764 BCP, 6 cases, site 528 P section, 2 cases, site 552 P section, YVDD 4 cases, YIDD 7 cases. Conclusion: Mutations at sites 1896, 1762,1764 were more frequent in patients with HBeAb + and were related to the negative expression of HBeAg, Mutations at 1762,1764 BCP were closely related to the changes of HBeAg/HBeAb. P section mutations were only observed after lamivadine treatment and were related to resistance against the drug. DNA chip method based upon membrane coloration for detection of gene mutation was expedient and specific and worth popularization. (authors)

Dynamic DNA cytosine methylation in the Populus trichocarpa genome: tissue-level variation and relationship to gene expression

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Vining Kelly J

2012-01-01

Full Text Available Abstract Background DNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes. However, large-scale epigenomic studies have been applied to very few plant species, and variability in methylation among specialized tissues and its relationship to gene expression is poorly understood. Results We surveyed DNA methylation from seven distinct tissue types (vegetative bud, male inflorescence [catkin], female catkin, leaf, root, xylem, phloem in the reference tree species black cottonwood (Populus trichocarpa. Using 5-methyl-cytosine DNA immunoprecipitation followed by Illumina sequencing (MeDIP-seq, we mapped a total of 129,360,151 36- or 32-mer reads to the P. trichocarpa reference genome. We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data. Qualitative DNA methylation differences among tissues were obvious on a chromosome scale. Methylated genes had lower expression than unmethylated genes, but genes with methylation in transcribed regions ("gene body methylation" had even lower expression than genes with promoter methylation. Promoter methylation was more frequent than gene body methylation in all tissues except male catkins. Male catkins differed in demethylation of particular transposable element categories, in level of gene body methylation, and in expression range of genes with methylated transcribed regions. Tissue-specific gene expression patterns were correlated with both gene body and promoter methylation. Conclusions We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined. In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation.

The Tyrosyl-DNA Phosphodiesterase 1β (Tdp1β Gene Discloses an Early Response to Abiotic Stresses

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Maria Elisa Sabatini

2017-11-01

Full Text Available Tyrosyl-DNA phosphodiesterase 1 (Tdp1 is involved in DNA repair pathways as it mends the topoisomerase I—DNA covalent complexes. In plants, a small Tdp1 gene family, composed by Tdp1α and Tdp1β genes, was identified, but the roles of these genes in abiotic stress responses are not fully understood. To investigate their specific stress response patterns, the present study made use of bioinformatic and molecular tools to look into the Tdp1β gene function, so far described only in the plant kingdom, and compare it with Tdp1α gene coding for the canonical, highly conserved α isoform. The expression profiles of Tdp1α and Tdp1β genes were examined under abiotic stress conditions (cold, heat, high osmolarity, salt, and UV-B in two model species, Arabidopsis thaliana and Medicago truncatula. The two isoforms of topoisomerase I (TOP1α and TOP1β were also taken into consideration in view of their known roles in DNA metabolism and cell proliferation. Data relative to gene expression in Arabidopsis were retrieved from the AtGenExpress microarray dataset, while quantitative Real-Time PCR was carried out to evaluate the stress response in M. truncatula cell cultures. These analyses revealed that Tdp1β gene expression was enhanced during the first hour of treatment, whereas Tdp1α enhanced expression succeeded at subsequent timepoints. In agreement with the gene-specific responses to abiotic stress conditions, the promoter regions of Tdp1α and Tdp1β genes are well equipped with stress-related cis-elements. An in-depth bioinformatic characterization of the HIRAN motif, a distinctive feature of the Tdp1β protein, showed its wide distribution in chromatin remodeling and DNA repair proteins. The reported data suggests that Tdp1β functions in the early response to abiotic stresses.

Immunohistochemical loss of 5-hydroxymethylcytosine expression in acute myeloid leukaemia: relationship to somatic gene mutations affecting epigenetic pathways.

Science.gov (United States)

Magotra, Minoti; Sakhdari, Ali; Lee, Paul J; Tomaszewicz, Keith; Dresser, Karen; Hutchinson, Lloyd M; Woda, Bruce A; Chen, Benjamin J

2016-12-01

Genes affecting epigenetic pathways are frequently mutated in myeloid malignancies, including acute myeloid leukaemia (AML). The genes encoding TET2, IDH1 and IDH2 are among the most commonly mutated genes, and cause defective conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5hmC), impairing demethylation of DNA, and presumably serving as driver mutations in leukaemogenesis. The aim of this study was to correlate 5hmC immunohistochemical loss with the mutation status of genes involved in epigenetic pathways in AML. Immunohistochemical staining with an anti-5hmC antibody was performed on 41 decalcified, formalin-fixed paraffin-embedded (FFPE) bone marrow biopsies from patients with AML. Archived DNA was subjected to next-generation sequencing for analysis of a panel of genes, including TET2, IDH1, IDH2, WT1 and DNMT3A. TET2, IDH1, IDH2, WT1 and DNMT3A mutations were found in 46% (19/41) of the cases. Ten of 15 cases (67%) with TET2, IDH1, IDH2 or WT1 mutations showed deficient 5hmC staining, whereas nine of 26 cases (35%) without a mutation in these genes showed loss of 5hmC. It is of note that all four cases with TET2 mutations showed deficient 5hmC staining. Overall, somatic mutations in TET2, IDH1, IDH2, WT1 and DNMT3A were common in our cohort of AML cases. Immunohistochemical staining for 5hmC was lost in the majority of cases harbouring mutations in these genes, reflecting the proposed relationship between dysfunctional epigenetic pathways and leukaemogenesis. © 2016 John Wiley & Sons Ltd.

DNA Methylation and Gene Expression Profiling of Ewing Sarcoma Primary Tumors Reveal Genes That Are Potential Targets of Epigenetic Inactivation

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Nikul Patel

2012-01-01

Full Text Available The role of aberrant DNA methylation in Ewing sarcoma is not completely understood. The methylation status of 503 genes in 52 formalin-fixed paraffin-embedded EWS tumors and 3 EWS cell lines was compared to human mesenchymal stem cell primary cultures (hMSCs using bead chip methylation analysis. Relative expression of methylated genes was assessed in 5-Aza-2-deoxycytidine-(5-AZA-treated EWS cell lines and in a cohort of primary EWS samples and hMSCs by gene expression and quantitative RT-PCR. 129 genes demonstrated statistically significant hypermethylation in EWS tumors compared to hMSCs. Thirty-six genes were profoundly methylated in EWS and unmethylated in hMSCs. 5-AZA treatment of EWS cell lines resulted in upregulation of expression of hundreds of genes including 162 that were increased by at least 2-fold. The expression of 19 of 36 candidate hypermethylated genes was increased following 5-AZA. Analysis of gene expression from an independent cohort of tumors confirmed decreased expression of six of nineteen hypermethylated genes (AXL, COL1A1, CYP1B1, LYN, SERPINE1, and VCAN. Comparing gene expression and DNA methylation analyses proved to be an effective way to identify genes epigenetically regulated in EWS. Further investigation is ongoing to elucidate the role of these epigenetic alterations in EWS pathogenesis.

A comparison of digital gene expression profiling and methyl DNA immunoprecipitation as methods for gene discovery in honeybee (Apis mellifera behavioural genomic analyses.

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Cui Guan

Full Text Available The honey bee has a well-organized system of division of labour among workers. Workers typically progress through a series of discrete behavioural castes as they age, and this has become an important case study for exploring how dynamic changes in gene expression can influence behaviour. Here we applied both digital gene expression analysis and methyl DNA immunoprecipitation analysis to nurse, forager and reverted nurse bees (nurses that have returned to the nursing state after a period spent foraging from the same colony in order to compare the outcomes of these different forms of genomic analysis. A total of 874 and 710 significantly differentially expressed genes were identified in forager/nurse and reverted nurse/forager comparisons respectively. Of these, 229 genes exhibited reversed directions of gene expression differences between the forager/nurse and reverted nurse/forager comparisons. Using methyl-DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq we identified 366 and 442 significantly differentially methylated genes in forager/nurse and reverted nurse/forager comparisons respectively. Of these, 165 genes were identified as differentially methylated in both comparisons. However, very few genes were identified as both differentially expressed and differentially methylated in our comparisons of nurses and foragers. These findings confirm that changes in both gene expression and DNA methylation are involved in the nurse and forager behavioural castes, but the different analytical methods reveal quite distinct sets of candidate genes.

Functional analyses of PtRDM1 gene overexpression in poplars and evaluation of its effect on DNA methylation and response to salt stress.

Science.gov (United States)

Movahedi, Ali; Zhang, Jiaxin; Sun, Weibo; Mohammadi, Kourosh; Almasi Zadeh Yaghuti, Amir; Wei, Hui; Wu, Xiaolong; Yin, Tongming; Zhuge, Qiang

2018-06-01

Epigenetic modification by DNA methylation is necessary for all cellular processes, including genetic expression events, DNA repair, genomic imprinting and regulation of tissue development. It occurs almost exclusively at the C5 position of symmetric CpG and asymmetric CpHpG and CpHpH sites in genomic DNA. The RNA-directed DNA methylation (RDM1) gene is crucial for heterochromatin and DNA methylation. We overexpressed PtRDM1 gene from Populus trichocarpa to amplify transcripts of orthologous RDM1 in 'Nanlin895' (P. deltoides × P. euramericana 'Nanlin895'). This overexpression resulted in increasing RDM1 transcript levels: by ∼150% at 0 mM NaCl treatment and by ∼300% at 60 mM NaCl treatment compared to WT (control) poplars. Genomic cytosine methylation was monitored within 5.8S rDNA and histone H3 loci by bisulfite sequencing. In total, transgenic poplars revealed more DNA methylation than WT plants. In our results, roots revealed more methylated CG contexts than stems and leaves whereas, histone H3 presented more DNA methylation than 5.8S rDNA in both WT and transgenic poplars. The NaCl stresses enhanced more DNA methylation in transgenic poplars than WT plants through histone H3 and 5.8 rDNA loci. Also, the overexpression of PtRDM1 resulted in hyper-methylation, which affected plant phenotype. Transgenic poplars revealed significantly more regeneration of roots than WT poplars via NaCl treatments. Our results proved that RDM1 protein enhanced the DNA methylation by chromatin remodeling (e.g. histone H3) more than repetitive DNA sequences (e.g. 5.8S rDNA). Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Development of Gene Expression Fingerprints for Identification of Environmental Contaminants Using cDNA Arrays

National Research Council Canada - National Science Library

Inouye, L

2004-01-01

...) to develop cDNA array-based assays that map gene expression from contaminant exposures. Results substantiate that distinct gene expression profiles exist for major contaminant classes such as PARs, PCBs, and PCDD/Fs...

Ternary polyplex micelles with PEG shells and intermediate barrier to complexed DNA cores for efficient systemic gene delivery.

Science.gov (United States)

Li, Junjie; Chen, Qixian; Zha, Zengshi; Li, Hui; Toh, Kazuko; Dirisala, Anjaneyulu; Matsumoto, Yu; Osada, Kensuke; Kataoka, Kazunori; Ge, Zhishen

2015-07-10

Simultaneous achievement of prolonged retention in blood circulation and efficient gene transfection activity in target tissues has always been a major challenge hindering in vivo applications of nonviral gene vectors via systemic administration. Herein, we constructed novel rod-shaped ternary polyplex micelles (TPMs) via complexation between the mixed block copolymers of poly(ethylene glycol)-b-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-b-PAsp(DET)) and poly(N-isopropylacrylamide)-b-PAsp(DET) (PNIPAM-b-PAsp(DET)) and plasmid DNA (pDNA) at room temperature, exhibiting distinct temperature-responsive formation of a hydrophobic intermediate layer between PEG shells and pDNA cores through facile temperature increase from room temperature to body temperature (~37 °C). As compared with binary polyplex micelles of PEG-b-PAsp(DET) (BPMs), TPMs were confirmed to condense pDNA into a more compact structure, which achieved enhanced tolerability to nuclease digestion and strong counter polyanion exchange. In vitro gene transfection results demonstrated TPMs exhibiting enhanced gene transfection efficiency due to efficient cellular uptake and endosomal escape. Moreover, in vivo performance evaluation after intravenous injection confirmed that TPMs achieved significantly prolonged blood circulation, high tumor accumulation, and promoted gene expression in tumor tissue. Moreover, TPMs loading therapeutic pDNA encoding an anti-angiogenic protein remarkably suppressed tumor growth following intravenous injection into H22 tumor-bearing mice. These results suggest TPMs with PEG shells and facilely engineered intermediate barrier to inner complexed pDNA have great potentials as systemic nonviral gene vectors for cancer gene therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Study of design parameters affecting the motion of DNA for nanoinjection

International Nuclear Information System (INIS)

David, Regis A; Jensen, Brian D; Howell, Larry L; Black, Justin L; Burnett, Sandra H

2012-01-01

This paper reports the effects of various parameters on the attraction and repulsion of DNA to and from a silicon lance. An understanding of DNA motion is crucial for a new approach to insert DNA, or other foreign microscopic matter, into a living cell. The approach, called nanoinjection, uses electrical forces to attract and repel the desired substance to a micromachined lance designed to pierce the cell membranes. We have developed mathematical models to predict the trajectory of DNA. The mathematical model allows investigation of the attraction/repulsion process by varying specific parameters. We find that the ground electrode placement, lance orientation and lance penetration significantly affect attraction or repulsion efficiency, while the gap, lance direction, lance tip width, lance tip half-angle and lance tip height do not. (paper)

StpA and Hha stimulate pausing by RNA polymerase by promoting DNA-DNA bridging of H-NS filaments.

Science.gov (United States)

Boudreau, Beth A; Hron, Daniel R; Qin, Liang; van der Valk, Ramon A; Kotlajich, Matthew V; Dame, Remus T; Landick, Robert

2018-06-20

In enterobacteria, AT-rich horizontally acquired genes, including virulence genes, are silenced through the actions of at least three nucleoid-associated proteins (NAPs): H-NS, StpA and Hha. These proteins form gene-silencing nucleoprotein filaments through direct DNA binding by H-NS and StpA homodimers or heterodimers. Both linear and bridged filaments, in which NAPs bind one or two DNA segments, respectively, have been observed. Hha can interact with H-NS or StpA filaments, but itself lacks a DNA-binding domain. Filaments composed of H-NS alone can inhibit transcription initiation and, in the bridged conformation, slow elongating RNA polymerase (RNAP) by promoting backtracking at pause sites. How the other NAPs modulate these effects of H-NS is unknown, despite evidence that they help regulate subsets of silenced genes in vivo (e.g. in pathogenicity islands). Here we report that Hha and StpA greatly enhance H-NS-stimulated pausing by RNAP at 20°C. StpA:H-NS or StpA-only filaments also stimulate pausing at 37°C, a temperature at which Hha:H-NS or H-NS-only filaments have much less effect. In addition, we report that both Hha and StpA greatly stimulate DNA-DNA bridging by H-NS filaments. Together, these observations indicate that Hha and StpA can affect H-NS-mediated gene regulation by stimulating bridging of H-NS/DNA filaments.

Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia

Directory of Open Access Journals (Sweden)

C.B. Sacramento

2010-08-01

Full Text Available The main objective of the present study was to find suitable DNA-targeting sequences (DTS for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS and hypoxia-responsive element (HRE sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF. The rate of plasmid nuclear transport and consequent gene expression under normoxia (20% O2 and hypoxia (less than 5% O2 were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50% lower under hypoxia, while the HRE plasmid was about 50% higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.

The DnaJ Gene Family in Pepper (Capsicum annuum L.: Comprehensive Identification, Characterization and Expression Profiles

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Yunyan Kang

2017-05-01

Full Text Available The DnaJ proteins which function as molecular chaperone played critical roles in plant growth and development and response to heat stress (HS and also called heat shock protein 40 based on molecular weight. However, little was reported on this gene family in pepper. Recently, the release of the whole pepper genome provided an opportunity for identifying putative DnaJ homologous. In this study, a total of 76 putative pepper DnaJ genes (CaDnaJ01 to CaDnaJ76 were identified using bioinformatics methods and classified into five groups by the presence of the complete three domains (J-domain, zinc finger domain, and C-terminal domain. Chromosome mapping suggested that segmental duplication and tandem duplication were occurred in evolution. The multiple stress-related cis-elements were found in the promoter region of these CaDnaJ genes, which indicated that the CaDnaJs might be involved in the process of responding to complex stress conditions. In addition, expression profiles based on RNA-seq showed that the 47 CaDnaJs were expressed in at least one tissue tested. The result implied that they could be involved in the process of pepper growth and development. qRT-PCR analysis found that 80.60% (54/67 CaDnaJs were induced by HS, indicated that they could participated in pepper response to high temperature treatments. In conclusion, all these results would provide a comprehensive basis for further analyzing the function of CaDnaJ members and be also significant for elucidating the evolutionary relationship in pepper.

The effect of co-administration of DNA carrying chicken interferon-gamma gene on protection of chickens against infectious bursal disease by DNA-mediated vaccination.

Science.gov (United States)

Hsieh, Ming Kun; Wu, Ching Ching; Lin, Tsang Long

2006-11-17

The purpose of the present study was to determine whether DNA vaccination by co-administration of DNA coding for chicken interferon-gamma (IFN-gamma) gene and DNA encoding for the VP243 gene of IBDV could enhance immune response and protection efficacy of chickens against challenge by IBDV. Plasmids carrying VP243 gene of IBDV strain variant E (VE) (P/VP243/E) and chicken IFN-gamma gene (P/cIFN-gamma) were constructed, respectively. One-day-old chickens were intramuscularly injected with P/VP243/E, or P/cIFN-gamma, or both once, twice, or three times into the thigh muscle of one leg or the thigh muscles of two separate legs at weekly intervals. Chickens were orally challenged with IBDV strain VE at 3 weeks of age and observed for 10 days. Chickens receiving two plasmids in the same site two times had significantly higher (Pprotection and those receiving two plasmids in the same sites did not have any protection against IBD. The enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) titers to IBDV of chickens in the groups with three doses of P/VP243/E were significantly higher (Pprotected by DNA vaccination did not have detectable IBDV antigen in the bursae as determined by immunofluorescent antibody assay (IFA). The results indicated that co-administration of plasmid encoding chicken IFN-gamma gene with plasmid encoding a large segment gene of the IBDV did not enhance immune response and protection against challenge by IBDV.

Analysis of variants in DNA damage signalling genes in bladder cancer

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Bishop D Timothy

2008-07-01

Full Text Available Abstract Background Chemicals from occupational exposure and components of cigarette smoke can cause DNA damage in bladder urothelium. Failure to repair DNA damage by DNA repair proteins may result in mutations leading to genetic instability and the development of bladder cancer. Immunohistochemistry studies have shown DNA damage signal activation in precancerous bladder lesions which is lost on progression, suggesting that the damage signalling mechanism acts as a brake to further tumorigenesis. Single nucleotide polymorphisms (SNPs in DSB signalling genes may alter protein function. We hypothesized that SNPs in DSB signalling genes may modulate predisposition to bladder cancer and influence the effects of environmental exposures. Methods We recruited 771 cases and 800 controls (573 hospital-based and 227 population-based from a previous case-control study and interviewed them regarding their smoking habits and occupational history. DNA was extracted from a peripheral blood sample and genotyping of 24 SNPs in MRE11, NBS1, RAD50, H2AX and ATM was undertaken using an allelic discrimination method (Taqman. Results Smoking and occupational dye exposure were strongly associated with bladder cancer risk. Using logistic regression adjusting for age, sex, smoking and occupational dye exposure, there was a marginal increase in risk of bladder cancer for an MRE11 3'UTR SNP (rs2155209, adjusted odds ratio 1.54 95% CI (1.13–2.08, p = 0.01 for individuals homozygous for the rare allele compared to those carrying the common homozygous or heterozygous genotype. However, in the hospital-based controls, the genotype distribution for this SNP deviated from Hardy-Weinberg equilibrium. None of the other SNPs showed an association with bladder cancer and we did not find any significant interaction between any of these polymorphisms and exposure to smoking or dye exposure. Conclusion Apart from a possible effect for one MRE11 3'UTR SNP, our study does not support

Fiscal 2000 pioneering research report on the basic technology for novel DNA drug creation using anti-gene engineering; 2000 nendo anti gene kogaku ni yoru shinki DNA drug soshutsu kiban gijutsu chosa kenkyu hokokusho

Energy Technology Data Exchange (ETDEWEB)

NONE

2001-03-01

Research is conducted concerning the possibility of anti-gene engineering basic to the application of DNA (deoxyribonucleic acid) drugs capable of serving as functional DNAs to the control of the expression of industrially useful substance producing genes or of anomalous genes and concerning the possibility of novel industry creation on the strength of the said engineering. The specific research items are described below. Technical seeds are investigated relating to the tissue- and cell-specific drug delivery system for the expression of the molecular device function of the DNA drug. Concerning molecular target technologies such as the anti-sense method, possibilities are studied of utilizing the currently available technical seeds for the eventual creation of novel industries. Concerning novel designing methods utilizing genome information such as SNPs (single nucleotide polymorphisms), investigations are conducted to determine if they would help novel technology development and novel material development. The domestic state is surveyed in relation to DNA drugs, and possibilities are investigated of novel substance production and novel medicine creation with the aid of anti-gene engineering. (NEDO)

Gene-specific DNA methylation association with serum levels of C-reactive protein in African Americans.

Directory of Open Access Journals (Sweden)

Yan V Sun

Full Text Available A more thorough understanding of the differences in DNA methylation (DNAm profiles in populations may hold promise for identifying molecular mechanisms through which genetic and environmental factors jointly contribute to human diseases. Inflammation is a key molecular mechanism underlying several chronic diseases including cardiovascular disease, and it affects DNAm profile on both global and locus-specific levels. To understand the impact of inflammation on the DNAm of the human genome, we investigated DNAm profiles of peripheral blood leukocytes from 966 African American participants in the Genetic Epidemiology Network of Arteriopathy (GENOA study. By testing the association of DNAm sites on CpG islands of over 14,000 genes with C-reactive protein (CRP, an inflammatory biomarker of cardiovascular disease, we identified 257 DNAm sites in 240 genes significantly associated with serum levels of CRP adjusted for age, sex, body mass index and smoking status, and corrected for multiple testing. Of the significantly associated DNAm sites, 80.5% were hypomethylated with higher CRP levels. The most significant Gene Ontology terms enriched in the genes associated with the CRP levels were immune system process, immune response, defense response, response to stimulus, and response to stress, which are all linked to the functions of leukocytes. While the CRP-associated DNAm may be cell-type specific, understanding the DNAm association with CRP in peripheral blood leukocytes of multi-ethnic populations can assist in unveiling the molecular mechanism of how the process of inflammation affects the risks of developing common disease through epigenetic modifications.

GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells

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Svetlana Kostyuk

2015-01-01

Full Text Available Background. Cell free DNA (cfDNA circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Principal Findings. Exposure of haMSCs to GC-DNA induces short-term oxidative stress (determined with H2DCFH-DA and results in both single- and double-strand DNA breaks (comet assay and γH2AX, foci. As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR, PCNA (FACS and antiapoptotic genes (BCL2 (RT-PCR and FACS, BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR. Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs. Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR, in the level of fatty acid binding protein FABP4 (FACS analysis and in the level of fat (Oil Red O. Conclusions. GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stem cells differentiation in human adipose—derived mesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis.

DNA methylation in obesity

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Małgorzata Pokrywka

2014-11-01

Full Text Available The number of overweight and obese people is increasing at an alarming rate, especially in the developed and developing countries. Obesity is a major risk factor for diabetes, cardiovascular disease, and cancer, and in consequence for premature death. The development of obesity results from the interplay of both genetic and environmental factors, which include sedentary life style and abnormal eating habits. In the past few years a number of events accompanying obesity, affecting expression of genes which are not directly connected with the DNA base sequence (e.g. epigenetic changes, have been described. Epigenetic processes include DNA methylation, histone modifications such as acetylation, methylation, phosphorylation, ubiquitination, and sumoylation, as well as non-coding micro-RNA (miRNA synthesis. In this review, the known changes in the profile of DNA methylation as a factor affecting obesity and its complications are described.

An innovative strategy to clone positive modifier genes of defects caused by mtDNA mutations: MRPS18C as suppressor gene of m.3946G>A mutation in MT-ND1 gene.

Science.gov (United States)

Rodríguez-García, María Elena; Cotrina-Vinagre, Francisco Javier; Carnicero-Rodríguez, Patricia; Martínez-Azorín, Francisco

2017-07-01

We have developed a new functional complementation approach to clone modifier genes which overexpression is able to suppress the biochemical defects caused by mtDNA mutations (suppressor genes). This strategy consists in transferring human genes into respiratory chain-deficient fibroblasts, followed by a metabolic selection in a highly selective medium. We used a normalized expression cDNA library in an episomal vector (pREP4) to transfect the fibroblasts, and a medium with glutamine and devoid of any carbohydrate source to select metabolically. Growing the patient's fibroblasts in this selective medium, the deficient cells rapidly disappear unless they are rescued by the cDNA of a suppressor gene. The use of an episomal vector allows us to carry out several rounds of transfection/selection (cyclical phenotypic rescue) to enrich the rescue with true clones of suppressor genes. Using fibroblasts from a patient with epileptic encephalopathy with the m.3946G>A (p.E214K) mutation in the MT-ND1 gene, several candidate genes were identified and one of them was characterized functionally. Thus, overexpression of MRPS18C gene (that encode for bS18m protein) suppressed the molecular defects produced by this mtDNA mutation, recovering the complex I activity and reducing the ROS produced by this complex to normal levels. We suggest that modulation of bS18m expression may be an effective therapeutic strategy for the patients with this mutation.

Radiosensitivity and genes

Energy Technology Data Exchange (ETDEWEB)

Qiyue, Hu; Mingyue, Lun [Suzhou Medical Coll., JS (China)

1995-07-01

Reported effects of some oncogenes, tumour suppressor genes and DNA repair genes on sensitivity of cells to ionizing radiation are reviewed. The role of oncogenes in cellular response to irradiation is discussed, especially the extensively studied oncogenes such as the ras gene family. For tumour suppressor genes, mainly the p53, which is increasingly implicated as a gene affecting radiosensitivity, is reviewed. It is considered that there is a cell cycle checkpoint determinant which is postulated to be able to arrest the irradiated cells in G{sub 1} phase to allow them to repair damage before they undergo DNA synthesis. So far there are six DNA repair genes which have been cloned in mammalian cells, but only one, XRCC1, appears to be involved in repair of human X-ray damage. XRCC1 can correct high sisterchromatid exchange levels when transferred into EM{sub 9} cells, but its expression seems to have no correlation with radiosensitivity of human neck and head tumour cells. Radiosensitivity is an intricate issue which may involve many factors. A scheme of cellular reactions after exposure to irradiation is proposed to indicate a possible sequence of events initiated by ionizing radiation.

Radiosensitivity and genes

International Nuclear Information System (INIS)

Hu Qiyue; Lun Mingyue

1995-07-01

Reported effects of some oncogenes, tumour suppressor genes and DNA repair genes on sensitivity of cells to ionizing radiation are reviewed. The role of oncogenes in cellular response to irradiation is discussed, especially the extensively studied oncogenes such as the ras gene family. For tumour suppressor genes, mainly the p53, which is increasingly implicated as a gene affecting radiosensitivity, is reviewed. It is considered that there is a cell cycle checkpoint determinant which is postulated to be able to arrest the irradiated cells in G 1 phase to allow them to repair damage before they undergo DNA synthesis. So far there are six DNA repair genes which have been cloned in mammalian cells, but only one, XRCC1, appears to be involved in repair of human X-ray damage. XRCC1 can correct high sisterchromatid exchange levels when transferred into EM 9 cells, but its expression seems to have no correlation with radiosensitivity of human neck and head tumour cells. Radiosensitivity is an intricate issue which may involve many factors. A scheme of cellular reactions after exposure to irradiation is proposed to indicate a possible sequence of events initiated by ionizing radiation

Review: Clinical aspects of hereditary DNA Mismatch repair gene mutations

NARCIS (Netherlands)

Sijmons, Rolf H.; Hofstra, Robert M. W.

Inherited mutations of the DNA Mismatch repair genes MLH1, MSH2, MSH6 and PMS2 can result in two hereditary tumor syndromes: the adult-onset autosomal dominant Lynch syndrome, previously referred to as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and the childhood-onset autosomal recessive

Polymorphisms in human DNA repair genes and head and neck ...

Indian Academy of Sciences (India)

Abstract. Genetic polymorphisms in some DNA repair proteins are associated with a number of malignant transformations like head and ... Such studies may benefit from analysis of multiple genes or polymorphisms and from the ... low survival and high morbidity when diagnosed in advanced ...... racial and/or ethnic cohort.

Immunohistochemical and DNA sequencing analysis on human mismatch repair gene MLH1 in cervical squamous cell carcinoma with LOH of this gene

NARCIS (Netherlands)

Hu, X.; Guo, Z.; Pang, T.; Li, Q.; Afink, G.; Pontén, J.

2000-01-01

BACKGROUND: The human MLH1 gene (hMLH1) is one of the DNA mismatch repair genes. Defects in these genes are believed to be the underlying cause of microsatellite instability (MSI). MSI has been demonstrated in many human cancers such as colon cancer and some female-specific tumors. The hMLH1 gene