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Sample records for dna affects gene

  1. Factors affecting SFHR gene correction efficiency with single-stranded DNA fragment

    SciTech Connect

    Tsuchiya, Hiroyuki; Harashima, Hideyoshi; Kamiya, Hiroyuki . E-mail: hirokam@pharm.hokudai.ac.jp

    2005-11-04

    A 606-nt single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, improves the gene correction efficiency by 12-fold as compared with a PCR fragment, which is the conventional type of fragment used in the small fragment homologous replacement method [H. Tsuchiya, H. Harashima, H. Kamiya, Increased SFHR gene correction efficiency with sense single-stranded DNA, J. Gene Med. 7 (2005) 486-493]. To reveal the characteristic features of this gene correction with the ss DNA fragment, the effects on the gene correction in CHO-K1 cells of the chain length, 5'-phosphate, adenine methylation, and transcription were studied. Moreover, the possibility that the ss DNA fragment is integrated into the target DNA was examined with a radioactively labeled ss DNA fragment. The presence of methylated adenine, but not the 5'-phosphate, enhanced the gene correction efficiency, and the optimal length of the ss DNA fragment ({approx}600 nt) was determined. Transcription of the target gene did not affect the gene correction efficiency. In addition, the target DNA recovered from the transfected CHO-K1 cells was radioactive. The results obtained in this study indicate that length and adenine methylation were important factors affecting the gene correction efficiency, and that the ss DNA fragment was integrated into the double-stranded target DNA.

  2. Low intensity infrared laser affects expression of oxidative DNA repair genes in mitochondria and nucleus

    NASA Astrophysics Data System (ADS)

    Fonseca, A. S.; Magalhães, L. A. G.; Mencalha, A. L.; Geller, M.; Paoli, F.

    2014-11-01

    Practical properties and physical characteristics of low intensity lasers have made possible their application to treat soft tissue diseases. Excitation of intracellular chromophores by red and infrared radiation at low energy fluences with increase of mitochondrial metabolism is the basis of the biostimulation effect but free radicals can be produced. DNA lesions induced by free radicals are repaired by the base excision repair pathway. In this work, we evaluate the expression of POLγ and APEX2 genes related to repair of mitochondrial and nuclear DNA, respectively. Skin and muscle tissue of Wistar rats were exposed to low intensity infrared laser at different fluences. One hour and 24 hours after laser exposure, tissue samples were withdrawn for total RNA extraction, cDNA synthesis, and evaluation of POLγ and APEX2 mRNA expression by real time quantitative polymerase chain reaction. Skin and muscle tissue of Wistar rats exposed to laser radiation show different expression of POLγ and APEX2 mRNA depending of the fluence and time after exposure. Our study suggests that a low intensity infrared laser affects expression of genes involved in repair of oxidative lesions in mitochondrial and nuclear DNA.

  3. Extremely low-frequency electromagnetic fields do not affect DNA damage and gene expression profiles of yeast and human lymphocytes.

    PubMed

    Luceri, Cristina; De Filippo, Carlotta; Giovannelli, Lisa; Blangiardo, Marta; Cavalieri, Duccio; Aglietti, Filippo; Pampaloni, Monica; Andreuccetti, Daniele; Pieri, Lapo; Bambi, Franco; Biggeri, Annibale; Dolara, Piero

    2005-09-01

    We studied the effects of extremely low-frequency (50 Hz) electromagnetic fields (EMFs) on peripheral human blood lymphocytes and DBY747 Saccharomyces cerevisiae. Graded exposure to 50 Hz magnetic flux density was obtained with a Helmholtz coil system set at 1, 10 or 100 microT for 18 h. The effects of EMFs on DNA damage were studied with the single-cell gel electrophoresis assay (comet assay) in lymphocytes. Gene expression profiles of EMF-exposed human and yeast cells were evaluated with DNA microarrays containing 13,971 and 6,212 oligonucleotides, respectively. After exposure to the EMF, we did not observe an increase in the amount of strand breaks or oxidated DNA bases relative to controls or a variation in gene expression profiles. The results suggest that extremely low-frequency EMFs do not induce DNA damage or affect gene expression in these two different eukaryotic cell systems.

  4. Prenatal tobacco smoke exposure affects global and gene-specific DNA methylation.

    PubMed

    Breton, Carrie V; Byun, Hyang-Min; Wenten, Made; Pan, Fei; Yang, Allen; Gilliland, Frank D

    2009-09-01

    Prenatal exposure to tobacco smoke increases the risk for diseases later in the child's life that may be mediated through alterations in DNA methylation. To demonstrate that differences in DNA methylation patterns occur in children exposed to tobacco smoke and that variation in detoxification genes may alter these associations. Methylation of DNA repetitive elements, LINE1 and AluYb8, was measured using bisulfite conversion and pyrosequencing in buccal cells of 348 children participating in the Children's Health Study. Gene-specific CpG methylation differences associated with smoke exposure were screened in 272 participants in the Children's Health Study children using an Illumina GoldenGate panel. CpG loci that demonstrated a statistically significant difference in methylation were validated by pyrosequencing. Estimates were standardized across loci using a Z score to enable cross-comparison of results. DNA methylation patterns were associated with in utero exposure to maternal smoking. Exposed children had significantly lower methylation of AluYb8 (beta, -0.31; P = 0.03). Differences in smoking-related effects on LINE1 methylation were observed in children with the common GSTM1 null genotype. Differential methylation of CpG loci in eight genes was identified through the screen. Two genes, AXL and PTPRO, were validated by pyrosequencing and showed significant increases in methylation of 0.37 (P = 0.005) and 0.34 (P = 0.02) in exposed children. The associations with maternal smoking varied by a common GSTP1 haplotype. Life-long effects of in utero exposures may be mediated through alterations in DNA methylation. Variants in detoxification genes may modulate the effects of in utero exposure through epigenetic mechanisms.

  5. Genetic mapping of nth, a gene affecting endonuclease III (thymine glycol-DNA glycosylase) in Escherichia coli K-12.

    PubMed Central

    Weiss, B; Cunningham, R P

    1985-01-01

    The nth gene of Escherichia coli affects the production of endonuclease III, a glycosylase-endonuclease that attacks DNA damaged by oxidizing agents or by ionizing radiation. An nth insertion mutant and a deletion mutant were studied. nth is located between add and tyrS on the linkage map of E. coli K-12 and was 97% linked to tyrS in a transduction with phage P1. PMID:3886628

  6. Physical Factors Affecting Plasmid DNA Compaction in Stearylamine-Containing Nanoemulsions Intended for Gene Delivery

    PubMed Central

    Silva, André Leandro; Júnior, Francisco Alexandrino; Verissimo, Lourena Mafra; Agnez-Lima, Lucymara Fassarella; Egito, Lucila Carmem Monte; de Oliveira, Anselmo Gomes; do Egito, Eryvaldo Socrates Tabosa

    2012-01-01

    Cationic lipids have been used in the development of non-viral gene delivery systems as lipoplexes. Stearylamine, a cationic lipid that presents a primary amine group when in solution, is able to compact genetic material by electrostatic interactions. In dispersed systems such as nanoemulsions this lipid anchors on the oil/water interface confering a positive charge to them. The aim of this work was to evaluate factors that influence DNA compaction in cationic nanoemulsions containing stearylamine. The influence of the stearylamine incorporation phase (water or oil), time of complexation, and different incubation temperatures were studied. The complexation rate was assessed by electrophoresis migration on agarose gel 0.7%, and nanoemulsion and lipoplex characterization was done by Dynamic Light Scattering (DLS). The results demonstrate that the best DNA compaction process occurs after 120 min of complexation, at low temperature (4 ± 1 °C), and after incorporation of the cationic lipid into the aqueous phase. Although the zeta potential of lipoplexes was lower than the results found for basic nanoemulsions, the granulometry did not change. Moreover, it was demonstrated that lipoplexes are suitable vehicles for gene delivery. PMID:24281666

  7. Physical factors affecting plasmid DNA compaction in stearylamine-containing nanoemulsions intended for gene delivery.

    PubMed

    Silva, André Leandro; Alexandrino, Francisco; Verissimo, Lourena Mafra; Agnez-Lima, Lucymara Fassarella; Egito, Lucila Carmem Monte; de Oliveira, Anselmo Gomes; do Egito, Eryvaldo Socrates Tabosa

    2012-06-18

    Cationic lipids have been used in the development of non-viral gene delivery systems as lipoplexes. Stearylamine, a cationic lipid that presents a primary amine group when in solution, is able to compact genetic material by electrostatic interactions. In dispersed systems such as nanoemulsions this lipid anchors on the oil/water interface confering a positive charge to them. The aim of this work was to evaluate factors that influence DNA compaction in cationic nanoemulsions containing stearylamine. The influence of the stearylamine incorporation phase (water or oil), time of complexation, and different incubation temperatures were studied. The complexation rate was assessed by electrophoresis migration on agarose gel 0.7%, and nanoemulsion and lipoplex characterization was done by Dynamic Light Scattering (DLS). The results demonstrate that the best DNA compaction process occurs after 120 min of complexation, at low temperature (4 ± 1 °C), and after incorporation of the cationic lipid into the aqueous phase. Although the zeta potential of lipoplexes was lower than the results found for basic nanoemulsions, the granulometry did not change. Moreover, it was demonstrated that lipoplexes are suitable vehicles for gene delivery.

  8. DNA Methylation of Lipid-Related Genes Affects Blood Lipid Levels

    PubMed Central

    Pfeiffer, Liliane; Wahl, Simone; Pilling, Luke C.; Reischl, Eva; Sandling, Johanna K.; Kunze, Sonja; Holdt, Lesca M.; Kretschmer, Anja; Schramm, Katharina; Adamski, Jerzy; Klopp, Norman; Illig, Thomas; Hedman, Åsa K.; Roden, Michael; Hernandez, Dena G.; Singleton, Andrew B.; Thasler, Wolfgang E.; Grallert, Harald; Gieger, Christian; Herder, Christian; Teupser, Daniel; Meisinger, Christa; Spector, Timothy D.; Kronenberg, Florian; Prokisch, Holger; Melzer, David; Peters, Annette; Deloukas, Panos; Ferrucci, Luigi; Waldenberger, Melanie

    2016-01-01

    Background Epigenetic mechanisms might be involved in the regulation of interindividual lipid level variability and thus may contribute to the cardiovascular risk profile. The aim of this study was to investigate the association between genome-wide DNA methylation and blood lipid levels high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and total cholesterol. Observed DNA methylation changes were also further analyzed to examine their relationship with previous hospitalized myocardial infarction. Methods and Results Genome-wide DNA methylation patterns were determined in whole blood samples of 1776 subjects of the Cooperative Health Research in the Region of Augsburg F4 cohort using the Infinium HumanMethylation450 BeadChip (Illumina). Ten novel lipid-related CpG sites annotated to various genes including ABCG1, MIR33B/SREBF1, and TNIP1 were identified. CpG cg06500161, located in ABCG1, was associated in opposite directions with both high-density lipoprotein cholesterol (β coefficient=−0.049; P=8.26E-17) and triglyceride levels (β=0.070; P=1.21E-27). Eight associations were confirmed by replication in the Cooperative Health Research in the Region of Augsburg F3 study (n=499) and in the Invecchiare in Chianti, Aging in the Chianti Area study (n=472). Associations between triglyceride levels and SREBF1 and ABCG1 were also found in adipose tissue of the Multiple Tissue Human Expression Resource cohort (n=634). Expression analysis revealed an association between ABCG1 methylation and lipid levels that might be partly mediated by ABCG1 expression. DNA methylation of ABCG1 might also play a role in previous hospitalized myocardial infarction (odds ratio, 1.15; 95% confidence interval=1.06–1.25). Conclusions Epigenetic modifications of the newly identified loci might regulate disturbed blood lipid levels and thus contribute to the development of complex lipid-related diseases. PMID:25583993

  9. Extended in vitro maturation affects gene expression and DNA methylation in bovine oocytes.

    PubMed

    Heinzmann, Julia; Mattern, Felix; Aldag, Patrick; Bernal-Ulloa, Sandra Milena; Schneider, Tamara; Haaf, Thomas; Niemann, Heiner

    2015-10-01

    To mimic post-ovulatory ageing, we have extended the in vitro maturation (IVM) phase to 48 h and examined effects on (i) developmental potential, (ii) expression of a panel of developmentally important genes and (iii) gene-specific epigenetic marks. Results were compared with the 24 h IVM protocol (control) usually employed for bovine oocytes. Cleavage rates and blastocyst yields were significantly reduced in oocytes after extended IVM. No significant differences were observed in the methylation of entire alleles in oocytes for the genes bH19, bSNRPN, bZAR1, bOct4 and bDNMT3A. However, we found differentially methylated CpG sites in the bDNMT3Ls locus in oocytes after extended IVM and in embryos derived from them compared with controls. Moreover, embryos derived from the 48 h matured oocyte group were significantly less methylated at CpG5 and CpG7 compared with the 24 h group. CpG7 was significantly hypermethylated in embryos produced from the control oocytes, but not in oocytes matured for 48 h. Furthermore, methylation for CpG5-CpG8 of bDNMT3Ls was significantly lower in oocytes of the 24 h group compared with embryos derived therefrom, whereas no such difference was found for oocytes and embryos of the in vitro aged group. Expression of most of the selected genes was not affected by duration of IVM. However, transcript abundance for the imprinted gene bIGF2R was significantly reduced in oocytes analyzed after extended IVM compared with control oocytes. Transcript levels for bPRDX1, bDNMT3A and bBCLXL were significantly reduced in 4- to 8-cell embryos derived from in vitro aged oocytes. These results indicate that extended IVM leads to ageing-like alterations and demonstrate that epigenetic mechanisms are critically involved in ageing of bovine oocytes, which warrants further studies into epigenetic mechanisms involved in ageing of female germ cells, including humans.

  10. Disruption of a DNA topoisomerase I gene affects morphogenesis in Arabidopsis.

    PubMed

    Takahashi, Taku; Matsuhara, Shio; Abe, Mitsutomo; Komeda, Yoshibumi

    2002-09-01

    The genesis of phyllotaxis, which often is associated with the Fibonacci series of numbers, is an old unsolved puzzle in plant morphogenesis. Here, we show that disruption of an Arabidopsis topoisomerase (topo) I gene named TOP1alpha affects phyllotaxis and plant architecture. The divergence angles and internode lengths between two successive flowers were more random in the top1alpha mutant than in the wild type. The top1alpha plants sporadically produced multiple flowers from one node, and the number of floral organ primordia often was different. The mutation also caused the twisting of inflorescences and individual flowers and the serration of leaf margins. These morphological abnormalities indicate that TOP1alpha may play a critical role in the maintenance of a regular pattern of organ initiation. The top1alpha mutant transformed with the RNA interference construct for TOP1beta, another topo I gene arrayed tandemly with TOP1alpha, was found to be lethal at young seedling stages, suggesting that topo I activity is essential in plants.

  11. Early life adversity and serotonin transporter gene variation interact to affect DNA methylation of the corticotropin-releasing factor gene promoter region in the adult rat brain.

    PubMed

    van der Doelen, Rick H A; Arnoldussen, Ilse A; Ghareh, Hussein; van Och, Liselot; Homberg, Judith R; Kozicz, Tamás

    2015-02-01

    The interaction between childhood maltreatment and the serotonin transporter (5-HTT) gene linked polymorphic region has been associated with increased risk to develop major depression. This Gene × Environment interaction has furthermore been linked with increased levels of anxiety and glucocorticoid release upon exposure to stress. Both endophenotypes are regulated by the neuropeptide corticotropin-releasing factor (CRF) or hormone, which is expressed by the paraventricular nucleus of the hypothalamus, the bed nucleus of the stria terminalis, and the central amygdala (CeA). Therefore, we hypothesized that altered regulation of the expression of CRF in these areas represents a major neurobiological mechanism underlying the interaction of early life stress and 5-HTT gene variation. The programming of gene transcription by Gene × Environment interactions has been proposed to involve epigenetic mechanisms such as DNA methylation. In this study, we report that early life stress and 5-HTT genotype interact to affect DNA methylation of the Crf gene promoter in the CeA of adult male rats. Furthermore, we found that DNA methylation of a specific site in the Crf promoter significantly correlated with CRF mRNA levels in the CeA. Moreover, CeA CRF mRNA levels correlated with stress coping behavior in a learned helplessness paradigm. Together, our findings warrant further investigation of the link of Crf promoter methylation and CRF expression in the CeA with behavioral changes that are relevant for psychopathology.

  12. DNA polymerase beta promoter mutations affect gene transcription, translation and the sensitivity of esophageal cancer cells to cisplatin treatment.

    PubMed

    Wang, Tao; Zang, Wenqiao; Ma, Yunyun; Li, Min; Xuan, Xiaoyan; Wang, Na; Wu, Rui; Li, Yuebai; Dong, Ziming; Zhao, Guoqiang

    2013-02-01

    The ability of a promoter to initiate transcription is important for the control of gene expression. Mutations in the DNA polymerase beta (po1β) promoter may affect the transcription of this gene; however, the relationship between these mutations and the upregulation of the expression of po1β remains unclear. Therefore, in the present study, three po1β promoter mutants (M1, -37 C→A; M2, -114 G→A, -37 C→A; M3, -194 T→C) were generated to examine the effect of promoter mutations on polβ gene expression and sensitivity to cisplatin. We found that the M1 and M2 mutant polβ promoter constructs showed higher RLA than the wild-type polβ promoter (P < 0.01), whereas the activity of the M3 polβ promoter did not differ significantly from that of the wild-type polβ promoter (P > 0.05). The expression levels of polβ mRNA and protein were significantly higher (P < 0.01) and the sensitivity to cisplatin was significantly lower (P < 0.05) in Eca9706(-/-)-M1 and Eca9706(-/-)-M2 cells than in Eca9706(-/-)-W. The expression levels of polβ mRNA and protein and the sensitivity to cisplatin were not significantly different between Eca9706(-/-)-M3 and Eca9706(-/-)-W cells (P > 0.05).These results revealed that specific mutations of the polymerase beta gene promoter significantly enhanced the gene's transcriptional activity. These mutations correspondingly increased the gene's mRNA and protein product, at the same time reduced the esophageal cancer cells' sensitivity to cisplatin.

  13. Melting profiles may affect detection of residual HPV L1 gene DNA fragments in Gardasil®.

    PubMed

    Lee, Sin Hang

    2014-03-01

    Gardasil® is a quadrivalent human papillomavirus (HPV) protein-based vaccine containing genotype-specific L1 capsid proteins of HPV-16, HPV-18, HPV-6 and HPV-11 in the form of virus-like-particles (VLPs) as the active ingredient. The VLPs are produced by a DNA recombinant technology. It is uncertain if the residual HPV L1 gene DNA fragments in the vaccine products are considered contaminants or excipients of the Gardasil® vaccine. Because naked viral DNA fragments, if present in the vaccine, may bind to the insoluble amorphous aluminum hydroxyphosphate sulfate (AAHS) adjuvant which may help deliver the foreign DNA into macrophages, causing unintended pathophysiologic effects, experiments were undertaken to develop tests for HPV L1 gene DNA fragments in the final products of Gardasil® by polymerase chain reaction (PCR) and direct DNA sequencing. The results showed that while the HPV-11 and HPV-18 L1 gene DNA fragments in Gardasil® were readily amplified by the common GP6/MY11 degenerate consensus primers, the HPV-16 L1 gene DNA may need specially designed non-degenerate PCR primers for amplification at different regions of the L1 gene and different stringency conditions for detection. These variable melting profiles of HPV DNA in the insoluble fraction of the Gardasil® vaccine suggest that the HPV DNA fragments are firmly bound to the aluminum AAHS adjuvant. All methods developed for detecting residual HPV DNA in the vaccine Gardasil® for quality assurance must take into consideration the variable melting profiles of the DNA to avoid false negative results.

  14. Variation in DNA repair gene XRCC3 affects susceptibility to astrocytomas and glioblastomas.

    PubMed

    Custódio, A C; Almeida, L O; Pinto, G R; Santos, M J; Almeida, J R W; Clara, C A; Rey, J A; Casartelli, C

    2012-02-10

    The gene XRCC3 (X-ray cross complementing group 3) has the task of repairing damage that occurs when there is recombination between homologous chromosomes. Repair of recombination between homologous chromosomes plays an important role in maintaining genome integrity, although it is known that double-strand breaks are the main inducers of chromosomal aberrations. Changes in the XRCC3 protein lead to an increase in errors in chromosome segregation due to defects in centrosomes, resulting in aneuploidy and other chromosomal aberrations, such as small increases in telomeres. We examined XRCC3 Thr241Met polymorphism using PCR-RFLP in 80 astrocytoma and glioblastoma samples. The individuals of the control group (N = 100) were selected from the general population of the São Paulo State. Odds ratio and 95%CI were calculated using a logistic regression model. Patients who had the allele Met of the XRCC3 Thr241Met polymorphism had a significantly increased risk of tumor development (odds ratio = 3.13; 95% confidence interval = 1.50-6.50). There were no significant differences in overall survival of patients. We suggest that XRCC3 Thr241Met polymorphism is involved in susceptibility for developing astrocytomas and glioblastomas.

  15. Two novel pathogenic mitochondrial DNA mutations affecting organelle number and protein synthesis. Is the tRNA(Leu(UUR)) gene an etiologic hot spot?

    PubMed Central

    Moraes, C T; Ciacci, F; Bonilla, E; Jansen, C; Hirano, M; Rao, N; Lovelace, R E; Rowland, L P; Schon, E A; DiMauro, S

    1993-01-01

    We identified two patients with pathogenic single nucleotide changes in two different mitochondrial tRNA genes: the first mutation in the tRNA(Asn) gene, and the ninth known mutation in the tRNA(Leu(UUR)) gene. The mutation in tRNA(Asn) was associated with isolated ophthalmoplegia, whereas the mutation in tRNA(Leu(UUR)) caused a neurological syndrome resembling MERRF (myoclonus epilepsy and ragged-red fibers) plus optic neuropathy, retinopathy, and diabetes. Both mutations were heteroplasmic, with higher percentages of mutant mtDNA in affected tissues, and undetectable levels in maternal relatives. Analysis of single muscle fibers indicated that morphological and biochemical alterations appeared only when the proportions of mutant mtDNA exceeded 90% of the total cellular mtDNA pool. The high incidence of mutations in the tRNA(Leu(UUR)) gene suggests that this region is an "etiologic hot spot" in mitochondrial disease. Images PMID:8254046

  16. The transcriptional coregulator MAML1 affects DNA methylation and gene expression patterns in human embryonic kidney cells.

    PubMed

    Putnik, Milica; Brodin, David; Wojdacz, Tomasz K; Fagerström-Billai, Fredrik; Dahlman-Wright, Karin; Wallberg, Annika E

    2016-03-01

    Mastermind-like 1 (MAML1) is a transcriptional coregulator that has been associated with early development of many systems such as neuronal, muscular and urogenital. The present study aimed to explore the genome wide effects of MAML1 on DNA methylation and RNA expression in human embryonic kidney cells. Infinium HumanMethylation450 BeadChip Illumina array, methylation-sensitive high-resolution melt technique, Chip Analysis Methylation Pipeline and RNA profiling approaches were used to study MAML1 effects on the epigenome. We found that 11802 CpG sites were differentially methylated in MAML1-expressing cells while only 225 genes were differentially expressed. MAML1 overexpression induced more global differential hypermethylation than hypomethylation changes. In addition, the differentially methylated regions were mapped predominantly to 3'untranslated regions, intragenic regions and gene bodies and to a lesser extent to gene regulatory sequences. Gene ontology analysis revealed that the differentially changed genes (including HOXC11, HTATIP2, SLFN12 and SOX11) are involved in the regulation of urogenital system development, cell adhesion and embryogenesis. This study is the first report that shows the global effect of a single coregulator on DNA methylation and gene expression. Our results stress and support the effects of transcriptional coregulators on the cell methylome.

  17. Global and gene specific DNA methylation in breast cancer cells was not affected during epithelial-to-mesenchymal transition in vitro.

    PubMed

    Smolkova, B; Miklikova, S; Kajabova, V Horvathova; Babelova, A; Yamani, N El; Zduriencikova, M; Fridrichova, I; Zmetakova, I; Krivulcik, T; Kalinkova, L; Matuskova, M; Kucerova, L; Dusinska, M

    2016-01-01

    Epithelial-to-mesenchymal transition (EMT) significantly affects the risk of metastasising in breast cancer. Plasticity and reversibility of EMT suggest that epigenetic mechanisms could be the key drivers of these processes, but little is known about the dynamics of EMT-related epigenetic alterations. We hypothesised that EMT, mediated by autocrine and paracrine signals, will be accompanied by changes in DNA methylation profiles. Therefore, conditioned medium from adipose tissue-derived mesenchymal stromal cells was used for induction of EMT in human breast cancer SK-BR-3 cell line. EMT-related morphological alterations and changes in gene expression of EMT-associated markers were assessed. To reverse EMT, 20 nm size gold nanoparticles (AuNPs) synthesized by the citrate reduction method were applied. Finally, DNA methylation of LINE-1 sequences and promoter methylation of TIMP3, ADAM23 and BRMS1 genes were quantitatively evaluated by pyrosequencing. Despite the presence of EMT-associated morphological and gene expression changes in tumour cells, EMT induced by adipose tissue-derived mesenchymal stromal cells had almost no effect on LINE-1 and gene-specific DNA methylation patterns of TIMP3, ADAM23 and BRMS1 genes. Although treatment for 24, 48 or 72 hours with 20 nm AuNPs at a concentration of 3 µg/ml slightly decreased gene expression of EMT-associated markers in SK-BR-3 cells, it did not alter global or gene-specific DNA methylation. Our results suggest that changes in DNA methylation are not detectable in vitro in early phases of EMT. Previously published positive findings could represent rather the sustained presence of potent EMT-inducing signals or the synergistic effect of various epigenetic mechanisms. Treatment with AuNPs slightly attenuated EMT, and their therapeutic potential needs to be further investigated.

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

    PubMed

    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

  19. Selective ploidy ablation, a high-throughput plasmid transfer protocol, identifies new genes affecting topoisomerase I–induced DNA damage

    PubMed Central

    Reid, Robert J.D.; González-Barrera, Sergio; Sunjevaric, Ivana; Alvaro, David; Ciccone, Samantha; Wagner, Marisa; Rothstein, Rodney

    2011-01-01

    We have streamlined the process of transferring plasmids into any yeast strain library by developing a novel mating-based, high-throughput method called selective ploidy ablation (SPA). SPA uses a universal plasmid donor strain that contains conditional centromeres on every chromosome. The plasmid-bearing donor is mated to a recipient, followed by removal of all donor-strain chromosomes, producing a haploid strain containing the transferred plasmid. As proof of principle, we used SPA to transfer plasmids containing wild-type and mutant alleles of DNA topoisomerase I (TOP1) into the haploid yeast gene-disruption library. Overexpression of Top1 identified only one sensitive mutation, rpa34, while overexpression of top1-T722A allele, a camptothecin mimetic, identified 190 sensitive gene-disruption strains along with rpa34. In addition to known camptothecin-sensitive strains, this set contained mutations in genes involved in the Rpd3 histone deacetylase complex, the kinetochore, and vesicle trafficking. We further show that mutations in several ESCRT vesicle trafficking components increase Top1 levels, which is dependent on SUMO modification. These findings demonstrate the utility of the SPA technique to introduce plasmids into the haploid gene-disruption library to discover new interacting pathways. PMID:21173034

  20. Fasting and refeeding affect the expression of the Inhibitor of DNA Binding (ID) genes in rainbow trout (Oncorhynchus mykiss) muscle.

    PubMed

    Gahr, Scott A; Weber, Gregory M; Rexroad, Caird E

    2006-08-01

    The Inhibitor of DNA Binding/Differentiation (ID) proteins are a family of dominant negative regulators of the basic helix-loop-helix (bHLH) transcription factors, shown in mammals to delay cell differentiation and prolong proliferation. In the current study we used real-time PCR to investigate the effects of fasting and refeeding on the expression of ID genes in rainbow trout muscle. Fry shortly following yolk-sac absorption (approximately 250 mg) were used in a pair of experiments. In the first experiment, the treatment groups included fish fed or fasted throughout the duration of the experiment, and fish fasted for 14 days followed by feeding for the remainder of the experiment. The second experiment consisted of the same treatment groups; however the fish were only fasted for 7 days prior to refeeding. In both experiments, ID gene expression in the muscle of fasted fish was significantly lower than the fed samples after 7 days. Refeeding for 3 or 7 days returned the ID expression to levels similar to the fed fish. The reduction of ID expression during a fast and the subsequent return to fed levels with refeeding suggests the ID proteins participate in the regulation of muscle growth in the rainbow trout.

  1. Dehydroepiandrosterone affects the expression of multiple genes in rat liver including 11 beta-hydroxysteroid dehydrogenase type 1: a cDNA array analysis.

    PubMed

    Gu, Shi; Ripp, Sharon L; Prough, Russell A; Geoghegan, Thomas E

    2003-03-01

    Dehydroepiandrosterone (DHEA) is a C-19 adrenal steroid precursor to the gonadal steroids. In humans, circulating levels of DHEA, as its sulfated conjugate, are high at puberty and throughout early adulthood but decline with age. Dietary supplementation to maintain high levels of DHEA purportedly has beneficial effects on cognitive memory, the immune system, and fat and carbohydrate metabolism. In rodents, DHEA is a peroxisome proliferator that induces genes for the classical peroxisomal and microsomal enzymes associated with this response. These effects are mediated through activation of peroxisome proliferator-activated receptor alpha (PPAR alpha). However, DHEA can affect the expression of genes independently of PPAR alpha, including the gene for the major inducible drug and xenobiotic metabolizing enzyme, cytochrome P450 3A23. To elucidate the biochemistry associated with DHEA treatment, we employed a cDNA gene expression array using liver RNA from rats treated with DHEA or the classic peroxisome proliferator nafenopin. Principal components analysis identified 30 to 35 genes whose expression was affected by DHEA and/or nafenopin. Some were genes previously identified as PPAR-responsive genes. Changes in expression of several affected genes were verified by quantitative reverse transcriptase-polymerase chain reaction. These included aquaporin 3, which was induced by DHEA and to a lesser extent nafenopin, nuclear tyrosine phosphatase, which was induced by both agents, and 11 beta-hydroxysteroid dehydrogenase 1, which was decreased by treatment with DHEA in a dose-dependent fashion. Regulation of 11 beta-hydroxysteroid dehydrogenase 1 expression is important since the enzyme is believed to amplify local glucocorticoid signaling, and its repression may cause some of the metabolic effects associated with DHEA.

  2. Post-weaning selenium and folate supplementation affects gene and protein expression and global DNA methylation in mice fed high-fat diets.

    PubMed

    Bermingham, Emma N; Bassett, Shalome A; Young, Wayne; Roy, Nicole C; McNabb, Warren C; Cooney, Janine M; Brewster, Di T; Laing, William A; Barnett, Matthew P G

    2013-03-05

    Consumption of high-fat diets has negative impacts on health and well-being, some of which may be epigenetically regulated. Selenium and folate are two compounds which influence epigenetic mechanisms. We investigated the hypothesis that post-weaning supplementation with adequate levels of selenium and folate in offspring of female mice fed a high-fat, low selenium and folate diet during gestation and lactation will lead to epigenetic changes of potential importance for long-term health. Female offspring of mothers fed the experimental diet were either maintained on this diet (HF-low-low), or weaned onto a high-fat diet with sufficient levels of selenium and folate (HF-low-suf), for 8 weeks. Gene and protein expression, DNA methylation, and histone modifications were measured in colon and liver of female offspring. Adequate levels of selenium and folate post-weaning affected gene expression in colon and liver of offspring, including decreasing Slc2a4 gene expression. Protein expression was only altered in the liver. There was no effect of adequate levels of selenium and folate on global histone modifications in the liver. Global liver DNA methylation was decreased in mice switched to adequate levels of selenium and folate, but there was no effect on methylation of specific CpG sites within the Slc2a4 gene in liver. Post-weaning supplementation with adequate levels of selenium and folate in female offspring of mice fed high-fat diets inadequate in selenium and folate during gestation and lactation can alter global DNA methylation in liver. This may be one factor through which the negative effects of a poor diet during early life can be ameliorated. Further research is required to establish what role epigenetic changes play in mediating observed changes in gene and protein expression, and the relevance of these changes to health.

  3. Feeding conditions and breed affect the level of DNA methylation of the mitochondrial uncoupling protein 3 gene in chicken breast muscle.

    PubMed

    Gao, G L; Wang, H W; Zhao, X Z; Li, Qin; Li, J; Li, Q R; Wang, Q G

    2015-04-01

    To investigate the effects of feed condition and breed on the level of DNA methylation for the uncoupling protein 3 (UCP3) gene, which is an important candidate gene for regulating intramuscular fat (IMF) content in chicken breast muscle, breast muscle of Daninghe (DNH) and Qingjiaoma (QJM) chickens under scatter-feed and captivity-feed conditions was analyzed. Using RNA sequencing, 47 and 113 candidate genes were determined to be related to feed conditions and breed, respectively, and 7 differentially expressed genes were confirmed by real-time quantitative PCR, including UCP3. The mRNA levels of UCP3 were significantly different between the 2 feed conditions. The DNA region from bp +1700 to +2459 of the UCP3 gene was studied using the bisulfite sequencing method and contained 46 methylation sites and 3 CpG islands. The results showed that the methylation level of this UCP3 region was lower in DNH chickens (0.77% to 0.88%, P = 0.012) and QJM chickens (0.88% to 0.91%, P = 0.20) under scatter-feed conditions than under captivity-feed conditions. The mean methylation level of UCP3 in DNH chickens was lower than that in QJM chickens under scatter-feed conditions (DNH to QJM, 0.77% to 0.88%, P = 0.007), which suggests that breed affects the mean methylation level of UCP3 under scatter-feed conditions. In summary, our findings suggest that feed condition and breed affect the methylation of UCP3 in chicken breast muscle.

  4. Coordinate expression of Escherichia coli dnaA and dnaN genes.

    PubMed

    Sako, T; Sakakibara, Y

    1980-01-01

    The defects of temperature-sensitive dnaA and dnaN mutants of Escherichia coli are complemented by a recombinant lambda phage, which carries the bacterial DNA segment composed of two EcoRI segments of 1.0 and 3.3 kilobases. Derivatives of the phage, which have an insertion segment of Tn3 in the dnaA gene, are much less active in expressing the dnaN gene function than the parent phage. The dnaN gene activity was determined as the efficiency of superinfecting phage to suppress loss of the viability of lambda lysogenic dnaN59 cells at the non-permissive temperature. Deletions that include the end of the dnaA gene distal to the dnaN gene also reduce the expression of the dnaN gene function. Deletion and insertion in the dnaN gene do not affect the expression of the dnaA gene function. The expression of the dnaN gene function by the dnaA- dnaN+ phages remains weak upon simultaneous infection with dnaA+ dnaN- phages. Thus the insertion and deletion of the dnaA gene influence in cis the expresion of the dnaN gene. We propose that the dnaA and dnaN genes constitute an operon, where the former is upstream to the latter.

  5. Homologous and homeologous intermolecular gene conversion are not differentially affected by mutations in the DNA damage or the mismatch repair genes RAD1, RAD50, RAD51, RAD52, RAD54, PMS1 and MSH2

    SciTech Connect

    Porter, G.; Westmoreland, J.; Priebe, S.

    1996-06-01

    Mismatch repair (MMR) genes or genes involved in both DNA damage repair and homologous recombination might affect homeologous vs. homologous recombination differentially. Spontaneous mitotic gene conversion between a chromosome and a homologous or homeologous donor sequence (14% diverged) on a single copy plasmid was examined in wild-type Saccharomyces cerevisiae strains and in MMR or DNA damage repair mutants. Homologous recombination in rad51, rad52 and rad54 mutants was considerably reduced, while there was little effect of rad1, rad50, pms1 and msh2 null mutations. DNA divergence resulted in no differential effect on recombination rates in the wild type or the mutants; there was only a five- to 10-fold reduction in homeologous relative to homologous recombination regardless of background. Since DNA divergence is known to affect recombination in some systems, we propose that differences in the role of MMR depends on the mode of recombination and/or the level of divergence. Based on analysis of the recombination breakpoints, there is a minimum of three homologous bases required at a recombination junction. A comparison of Rad{sup +} vs. rad52 strains revealed that while all conversion tracts are continuous, elimination of RAD52 leads to the appearance of a novel class of very short conversion tracts. 67 refs., 5 figs., 4 tabs.

  6. Homologous and Homeologous Intermolecular Gene Conversion Are Not Differentially Affected by Mutations in the DNA Damage or the Mismatch Repair Genes Rad1, Rad50, Rad51, Rad52, Rad54, Pms1 and Msh2

    PubMed Central

    Porter, G.; Westmoreland, J.; Priebe, S.; Resnick, M. A.

    1996-01-01

    Mismatch repair (MMR) genes or genes involved in both DNA damage repair and homologous recombination might affect homeologous vs. homologous recombination differentially. Spontaneous mitotic gene conversion between a chromosome and a homologous or homeologous donor sequence (14% diverged) on a single copy plasmid was examined in wild-type Saccharomyces cerevisiae strains and in MMR or DNA damage repair mutants. Homologous recombination in rad51, rad52 and rad54 mutants was considerably reduced, while there was little effect of rad1, rad50, pms1 and msh2 null mutations. DNA divergence resulted in no differential effect on recombination rates in the wild type or the mutants; there was only a five- to 10-fold reduction in homeologous relative to homologous recombination regardless of background. Since DNA divergence is known to affect recombination in some systems, we propose that differences in the role of MMR depends on the mode of recombination and/or the level of divergence. Based on analysis of the recombination breakpoints, there is a minimum of three homologous bases required at a recombination junction. A comparison of Rad(+) vs. rad52 strains revealed that while all conversion tracts are continuous, elimination of RAD52 leads to the appearance of a novel class of very short conversion tracts. PMID:8725224

  7. Maternal age and ovarian stimulation independently affect oocyte mtDNA copy number and cumulus cell gene expression in bovine clones.

    PubMed

    Cree, Lynsey M; Hammond, Elizabeth R; Shelling, Andrew N; Berg, Martin C; Peek, John C; Green, Mark P

    2015-06-01

    rates, numbers and diameters were monitored by ultrasonography and aspirated when the lead follicles were >14 mm in diameter. Follicle characteristics were analysed using a mixed model procedure. Quantitative PCR (qPCR) was used to determine mtDNA copy number and reverse transcriptase-qPCR (RT-qPCR) was used to measure gene expression in oocytes and cumulus cells. Method of ovarian stimulation (P = 0.04), but not maternal age (P > 0.1), was associated with a lower mtDNA copy number in oocytes. Neither factor affected mtDNA copy number in cumulus cells. In oocytes, maternal age had no effect on gene expression; however, ovarian stimulation in older females increased the expression of GRP78 (P = 0.02), a gene involved in ER stress. In cumulus cells, increasing maternal age was associated with the higher expression of genes involved in mitochondrial maintenance (TXN2 P = 0.008 and TFAM P = 0.03), whereas ovarian stimulation decreased the expression of genes involved in mitochondrial oxidative stress and apoptosis (TXN2 P = 0.002, PRDX3 P = 0.03 and BAX P = 0.03). The low number of oocyte and cumulus cell samples collected from the unstimulated cycles limited the analysis. Fertilization and developmental potential of the oocytes was not assessed because these were used for mtDNA and gene expression quantification. Delineation of the independent effects of maternal age and ovarian stimulation regimen on mtDNA copy number gene expression in oocytes and cumulus cells was enabled by the removal of genetic and environmental variability in this bovine model for human IVF. Therefore, these extend upon previous knowledge and findings provide relevant insights that are applicable for improving human ovarian stimulation regimens. Funding was provided by Fertility Associates and the University of Auckland. J.C.P. is a shareholder of Fertility Associates and M.P.G. received a fellowship from Fertility Associates. The other authors of this manuscript declare no conflict of interest that

  8. Testing the Oncogenic Relevance of Cell Adhesion and Cytosketal Genes Affected by DNA Deletions in Breast Cancer

    DTIC Science & Technology

    2010-07-01

    and hair follicle derived cells as targets for the v-rasHa oncogene in mouse skin carcinogenesis. Carcinogenesis 12, 1119–1124. Wicki, A., Lehembre, F...potential oncogenic significance of genes directly involved in cell adhesion and the cytoskeleton. The aim of this study was therefore to directly test ...expression of candidate cancer genes belonging to the cytoskeletal/cell adhesion category, (2) use these tools to test the oncogenic significance of

  9. DNA Microarray and Gene Ontology Enrichment Analysis Reveals That a Mutation in opsX Affects Virulence and Chemotaxis in Xanthomonas oryzae pv. oryzae.

    PubMed

    Kim, Hong-Il; Park, Young-Jin

    2016-06-01

    Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight (BLB) in rice (Oryza sativa L.). In this study, we investigated the effect of a mutation in opsX (XOO1056), which encodes a saccharide biosynthesis regulatory protein, on the virulence and bacterial chemotaxis of Xoo. We performed DNA microarray analysis, which showed that 63 of 2,678 genes, including genes related to bacterial motility (flagellar and chemotaxis proteins) were significantly downregulated (<-2 log2 fold changes) by the mutation in opsX. Indeed, motility assays showed that the mutant strain was nonmotile on semisolid agar swarm plates. In addition, a mutant strain (opsX::Tn5) showed decreased virulence against the susceptible rice cultivar, IR24. Quantitative real-time RT-PCR reaction was performed to confirm the expression levels of these genes, including those related to flagella and chemotaxis, in the opsX mutant. Our findings revealed that mutation of opsX affects both virulence and bacterial motility. These results will help to improve our understanding of Xoo and provide insight into Xoo-rice interactions.

  10. Fibronectin affects transient MMP2 gene expression through DNA demethylation changes in non-invasive breast cancer cell lines.

    PubMed

    Pereira, Isabela T; Ramos, Edneia A S; Costa, Erico T; Camargo, Anamaria A; Manica, Graciele C M; Klassen, Liliane M B; Chequin, Andressa; Braun-Prado, Karin; Pedrosa, Fábio de O; Souza, Emanuel M; Costa, Fabricio F; Klassen, Giseli

    2014-01-01

    Metastasis accounts for more than 90% of cancer deaths. Cells from primary solid tumors may invade adjacent tissues and migrate to distant sites where they establish new colonies. The tumor microenvironment is now recognized as an important participant in the signaling that induces cancer cell migration. An essential process for metastasis is extracellular matrix (ECM) degradation by metalloproteases (MMPs), which allows tumor cells to invade local tissues and to reach blood vessels. The members of this protein family include gelatinase A, or MMP-2, which is responsible for the degradation of type IV collagen, the most abundant component of the basal membrane, that separates epithelial cells in the stroma. It is known that fibronectin is capable of promoting the expression of MMP-2 in MCF7 breast cancer cells in culture. In addition, it was already shown that the MMP2 gene expression is regulated by epigenetic mechanisms. In this work, we showed that fibronectin was able to induce MMP2 expression by 30% decrease in its promoter methylation. In addition, a histone marker for an open chromatin conformation was significantly increased. These results indicate a new role for fibronectin in the communication between cancer cells and the ECM, promoting epigenetic modifications.

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

    PubMed Central

    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

  12. Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes

    PubMed Central

    Li, Fuyang; Papworth, Monika; Minczuk, Michal; Rohde, Christian; Zhang, Yingying; Ragozin, Sergei; Jeltsch, Albert

    2007-01-01

    Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation. PMID:17151075

  13. How the Neanderthal in Your Genes Affects Your Health

    MedlinePlus

    ... medlineplus.gov/news/fullstory_163749.html How the Neanderthal in Your Genes Affects Your Health The DNA ... 23, 2017 THURSDAY, Feb. 23, 2017 (HealthDay News) -- Neanderthals were wiped out about 40,000 years ago, ...

  14. A Sequence-Specific Interaction between the Saccharomyces cerevisiae rRNA Gene Repeats and a Locus Encoding an RNA Polymerase I Subunit Affects Ribosomal DNA Stability

    PubMed Central

    Cahyani, Inswasti; Cridge, Andrew G.; Engelke, David R.; Ganley, Austen R. D.

    2014-01-01

    The spatial organization of eukaryotic genomes is linked to their functions. However, how individual features of the global spatial structure contribute to nuclear function remains largely unknown. We previously identified a high-frequency interchromosomal interaction within the Saccharomyces cerevisiae genome that occurs between the intergenic spacer of the ribosomal DNA (rDNA) repeats and the intergenic sequence between the locus encoding the second largest RNA polymerase I subunit and a lysine tRNA gene [i.e., RPA135-tK(CUU)P]. Here, we used quantitative chromosome conformation capture in combination with replacement mapping to identify a 75-bp sequence within the RPA135-tK(CUU)P intergenic region that is involved in the interaction. We demonstrate that the RPA135-IGS1 interaction is dependent on the rDNA copy number and the Msn2 protein. Surprisingly, we found that the interaction does not govern RPA135 transcription. Instead, replacement of a 605-bp region within the RPA135-tK(CUU)P intergenic region results in a reduction in the RPA135-IGS1 interaction level and fluctuations in rDNA copy number. We conclude that the chromosomal interaction that occurs between the RPA135-tK(CUU)P and rDNA IGS1 loci stabilizes rDNA repeat number and contributes to the maintenance of nucleolar stability. Our results provide evidence that the DNA loci involved in chromosomal interactions are composite elements, sections of which function in stabilizing the interaction or mediating a functional outcome. PMID:25421713

  15. Specific DNA replication mutations affect telomere length in Saccharomyces cerevisiae.

    PubMed Central

    Adams, A K; Holm, C

    1996-01-01

    To investigate the relationship between the DNA replication apparatus and the control of telomere length, we examined the effects of several DNA replication mutations on telomere length in Saccharomyces cerevisiae. We report that a mutation in the structural gene for the large subunit of DNA replication factor C (cdc44/rfc1) causes striking increases in telomere length. A similar effect is seen with mutations in only one other DNA replication gene: the structural gene for DNA polymerase alpha (cdc17/pol1) (M.J. Carson and L. Hartwell, Cell 42:249-257, 1985). For both genes, the telomere elongation phenotype is allele specific and appears to correlate with the penetrance of the mutations. Furthermore, fluorescence-activated cell sorter analysis reveals that those alleles that cause elongation also exhibit a slowing of DNA replication. To determine whether elongation is mediated by telomerase or by slippage of the DNA polymerase, we created cdc17-1 mutants carrying deletions of the gene encoding the RNA component of telomerase (TLC1). cdc17-1 strains that would normally undergo telomere elongation failed to do so in the absence of telomerase activity. This result implies that telomere elongation in cdc17-1 mutants is mediated by the action of telomerase. Since DNA replication involves transfer of the nascent strand from polymerase alpha to replication factor C (T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1950-1960, 1991; T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1961-1968, 1991; S. Waga and B. Stillman, Nature [London] 369:207-212, 1994), one possibility is that this step affects the regulation of telomere length. PMID:8756617

  16. Novel Functional Residues in the Core Domain of Histone H2B Regulate Yeast Gene Expression and Silencing and Affect the Response to DNA Damage ▿

    PubMed Central

    Kyriss, McKenna N. M.; Jin, Yi; Gallegos, Isaura J.; Sanford, James A.; Wyrick, John J.

    2010-01-01

    Previous studies have identified novel modifications in the core fold domain of histone H2B, but relatively little is known about the function of these putative histone modification sites. We have mutated core modifiable residues that are conserved in Saccharomyces cerevisiae histone H2B and characterized the effects of the mutants on yeast silencing, gene expression, and the DNA damage response. We identified three histone H2B core modifiable residues as functionally important. We find that mutating H2B K49 in yeast confers a UV sensitivity phenotype, and we confirm that the homologous residue in human histone H2B is acetylated and methylated in human cells. Our results also indicate that mutating H2B K111 impairs the response to methyl methanesulfonate (MMS)-induced DNA lesions and disrupts telomeric silencing and Sir4 binding. In contrast, mutating H2B R102 enhances silencing at yeast telomeres and the HML silent mating loci and increases Sir4 binding to these regions. The H2B R102A mutant also represses the expression of endogenous genes adjacent to yeast telomeres, which is likely due to the ectopic spreading of the Sir complex in this mutant strain. We propose a structural model by which H2B R102 and K111 regulate the binding of the Sir complex to the nucleosome. PMID:20479120

  17. Metagenomic DNA fragments that affect Escherichia coli mutational pathways.

    PubMed

    Yang, Hanjing; To, Kam H; Aguila, Sharon J; Miller, Jeffrey H

    2006-08-01

    A multicopy cloning approach was used to search for metagenomic DNA fragments that affect Escherichia coli mutational pathways. Soil metagenomic expression libraries were constructed with DNA samples prepared directly from soil samples collected from the UCLA Botanical Garden. Using frameshift mutator screening, we obtained a total of 26 unique metagenomic fragments that stimulate frameshift rates in an E. coli wild-type host. Mutational enhancer strains such as an ndk-deficient strain and a temperature sensitive mutS strain (mutS60) were used to further verify the mutator phenotype. We found that the presence of multiple copies of certain types of metagenomic DNA sequence repeats cause general genome instability in the wild-type E. coli host and the effect can be suppressed by overproducing a DNA mismatch component MutL. In addition, we identified nine metagenomic mutator genes (designated as smu genes) that encode proteins that have not been linked to mutator phenotypes prior to this study including a putative RNA methyltransferase Smu10A. The strain overproducing Smu10A displays one prominent base substitution hotspot in the rpoB gene, which coincides with the base substitution hotspot we have observed in cells that are partially deficient in the proofreading function carried out by the DNA polymerase III epsilon subunit. Based on the structural conservation of DNA replication/recombination/repair machineries among microorganisms, this approach would allow us to both identify new mutational pathways in E. coli and to find genes involved in DNA replication, recombination or DNA repair from vast unculturable microbes.

  18. Whole genome DNA methylation: beyond genes silencing

    PubMed Central

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

    2017-01-01

    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 major biological consequences of DNA methylation recently discovered. We also discuss the necessity of tuning DNA methylation resolution into an adequate scale to ease the integration of the methylome information with other chromatin features and transcription events such as gene expression, nucleosome positioning, transcription factors binding dynamic, gene splicing and genomic imprinting. Finally, our review sheds light on DNA methylation heterogeneity in cell population and the different approaches used for its assessment, including the contribution of single cell DNA analysis technology. PMID:27895318

  19. The A1555G mutation in the 12S rRNA gene of human mtDNA: recurrent origins and founder events in families affected by sensorineural deafness.

    PubMed

    Torroni, A; Cruciani, F; Rengo, C; Sellitto, D; López-Bigas, N; Rabionet, R; Govea, N; López De Munain, A; Sarduy, M; Romero, L; Villamar, M; del Castillo, I; Moreno, F; Estivill, X; Scozzari, R

    1999-11-01

    The mtDNA variation of 50 Spanish and 4 Cuban families affected by nonsyndromic sensorineural deafness due to the A1555G mutation in the 12S rRNA gene was studied by high-resolution RFLP analysis and sequencing of the control region. Phylogenetic analyses of haplotypes and detailed survey of population controls revealed that the A1555G mutation can be attributed to >/=30 independent mutational events among the 50 Spanish families and that it occurs on mtDNA haplogroups that are common in all European populations. This indicates that the relatively high detection rate of this mutation in Spain is not due to sampling biases or to a single major founder event. Moreover, the distribution of these mutational events on different haplogroups is compatible with a random occurrence of the A1555G mutation and tends to support the conclusion that mtDNA backgrounds do not play a significant role in the expression of the mutation. Overall, these findings appear to indicate that the rare detection of this mutation in other populations is most likely due to inadequacy in patient ascertainment and molecular screening. This probable lack of identification of the A1555G mutation in subjects affected by sensorineural hearing loss implies that their maternally related relatives are not benefiting from presymptomatic detection and information concerning their increased risk of ototoxicity due to aminoglycoside treatments.

  20. The A1555G Mutation in the 12S rRNA Gene of Human mtDNA: Recurrent Origins and Founder Events in Families Affected by Sensorineural Deafness

    PubMed Central

    Torroni, Antonio; Cruciani, Fulvio; Rengo, Chiara; Sellitto, Daniele; López-Bigas, Núria; Rabionet, Raquel; Govea, Nancy; López de Munain, Adolfo; Sarduy, Maritza; Romero, Lourdes; Villamar, Manuela; del Castillo, Ignacio; Moreno, Felipe; Estivill, Xavier; Scozzari, Rosaria

    1999-01-01

    Summary The mtDNA variation of 50 Spanish and 4 Cuban families affected by nonsyndromic sensorineural deafness due to the A1555G mutation in the 12S rRNA gene was studied by high-resolution RFLP analysis and sequencing of the control region. Phylogenetic analyses of haplotypes and detailed survey of population controls revealed that the A1555G mutation can be attributed to ⩾30 independent mutational events among the 50 Spanish families and that it occurs on mtDNA haplogroups that are common in all European populations. This indicates that the relatively high detection rate of this mutation in Spain is not due to sampling biases or to a single major founder event. Moreover, the distribution of these mutational events on different haplogroups is compatible with a random occurrence of the A1555G mutation and tends to support the conclusion that mtDNA backgrounds do not play a significant role in the expression of the mutation. Overall, these findings appear to indicate that the rare detection of this mutation in other populations is most likely due to inadequacy in patient ascertainment and molecular screening. This probable lack of identification of the A1555G mutation in subjects affected by sensorineural hearing loss implies that their maternally related relatives are not benefiting from presymptomatic detection and information concerning their increased risk of ototoxicity due to aminoglycoside treatments. PMID:10521300

  1. 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.

    PubMed

    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.

  2. Experimental factors affecting the robustness of DNA methylation analysis

    PubMed Central

    Pharo, Heidi D.; Honne, Hilde; Vedeld, Hege M.; Dahl, Christina; Andresen, Kim; Liestøl, Knut; Jeanmougin, Marine; Guldberg, Per; Lind, Guro E.

    2016-01-01

    Diverging methylation frequencies are often reported for the same locus in the same disease, underscoring the need for limiting technical variability in DNA methylation analyses. We have investigated seven likely sources of variability at different steps of bisulfite PCR-based DNA methylation analyses using a fully automated quantitative methylation-specific PCR setup of six gene promoters across 20 colon cancer cell lines. Based on >15,000 individual PCRs, all tested parameters affected the normalized percent of methylated reference (PMR) differences, with a fourfold varying magnitude. Additionally, large variations were observed across the six genes analyzed. The highest variation was seen using single-copy genes as reference for normalization, followed by different amounts of template in the PCR, different amounts of DNA in the bisulfite reaction, and storage of bisulfite converted samples. Finally, when a highly standardized pipeline was repeated, the difference in PMR value for the same assay in the same cell line was on average limited to five (on a 0–100 scale). In conclusion, a standardized pipeline is essential for consistent methylation results, where parameters are kept constant for all samples. Nevertheless, a certain level of variation in methylation values must be expected, underscoring the need for careful interpretation of data. PMID:27671843

  3. Regulation of mammalian horizontal gene transfer by apoptotic DNA fragmentation

    PubMed Central

    Yan, B; Wang, H; Li, F; Li, C-Y

    2006-01-01

    Previously it was shown that horizontal DNA transfer between mammalian cells can occur through the uptake of apoptotic bodies, where genes from the apoptotic cells were transferred to neighbouring cells phagocytosing the apoptotic bodies. The regulation of this process is poorly understood. It was shown that the ability of cells as recipient of horizontally transferred DNA was enhanced by deficiency of p53 or p21. However, little is known with regard to the regulation of DNA from donor apoptotic cells. Here we report that the DNA fragmentation factor/caspase-activated DNase (DFF/CAD), which is the endonuclease responsible for DNA fragmentation during apoptosis, plays a significant role in regulation of horizontal DNA transfer. Cells with inhibited DFF/CAD function are poor donors for horizontal gene transfer (HGT) while their ability of being recipients of HGT is not affected. PMID:17146478

  4. Does inbreeding affect gene expression in birds?

    PubMed

    Hansson, Bengt; Naurin, Sara; Hasselquist, Dennis

    2014-09-01

    Inbreeding increases homozygosity, exposes genome-wide recessive deleterious alleles and often reduces fitness. The physiological and reproductive consequences of inbreeding may be manifested already during gene regulation, but the degree to which inbreeding influences gene expression is unknown in most organisms, including in birds. To evaluate the pattern of inbreeding-affected gene expression over the genome and in relation to sex, we performed a transcriptome-wide gene expression (10 695 genes) study of brain tissue of 10-day-old inbred and outbred, male and female zebra finches. We found significantly lower gene expression in females compared with males at Z-linked genes, confirming that dosage compensation is incomplete in female birds. However, inbreeding did not affect gene expression at autosomal or sex-linked genes, neither in males nor in females. Analyses of single genes again found a clear sex-biased expression at Z-linked genes, whereas only a single gene was significantly affected by inbreeding. The weak effect of inbreeding on gene expression in zebra finches contrasts to the situation, for example, in Drosophila where inbreeding has been found to influence gene expression more generally and at stress-related genes in particular.

  5. 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.

    PubMed

    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

  6. 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

    PubMed Central

    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

  7. DNA repair genes of mammalian cells

    SciTech Connect

    Thompson, L.H.; Brookman, K.W.; Salazar, E.P.; Fuscoe, J.C.; Weber, C.A.

    1985-09-27

    In the CHO cell line various mutations affecting DNA repair have been obtained. Mutants that belong to five genetic complementation groups for UV sensitivity and resemble the cells from individuals having the cancer-prone genetic disorder xeroderma pigmentosum were previously identified. Each mutant is defective in the incision step of nucleotide excision repair and hypersensitive to bulky DNA lesions. A sixth genetic complementation group for UV sensitivity has now been identified with UV27-1. These UV mutants can be divided into two subgroups; only Groups 2 and 4 are extremely sensitive to mitomycin C and other DNA cross-linking agents. The clear-cut phenotypes of the CHO mutants have allowed us to construct hybrid cells by fusion with human lymphocytes and thereby identify which human chromosomes carry genes that correct the CHO mutations. The first two mutants analyzed, UV20 (excision-repair deficient; UV Group 2) and EM9, which has very high SCE, are both corrected by chromosome 19. 46 refs., 3 figs.

  8. Different mating-type-regulated genes affect the DNA repair defects of Saccharomyces RAD51, RAD52 and RAD55 mutants.

    PubMed

    Valencia-Burton, Maria; Oki, Masaya; Johnson, Jean; Seier, Tracey A; Kamakaka, Rohinton; Haber, James E

    2006-09-01

    Saccharomyces cerevisiae cells expressing both a- and alpha-mating-type (MAT) genes (termed mating-type heterozygosity) exhibit higher rates of spontaneous recombination and greater radiation resistance than cells expressing only MATa or MATalpha. MAT heterozygosity suppresses recombination defects of four mutations involved in homologous recombination: complete deletions of RAD55 or RAD57, an ATPase-defective Rad51 mutation (rad51-K191R), and a C-terminal truncation of Rad52, rad52-Delta327. We investigated the genetic basis of MAT-dependent suppression of these mutants by deleting genes whose expression is controlled by the Mata1-Matalpha2 repressor and scoring resistance to both campothecin (CPT) and phleomycin. Haploid rad55Delta strains became more damage resistant after deleting genes required for nonhomologous end-joining (NHEJ), a process that is repressed in MATa/MATalpha cells. Surprisingly, NHEJ mutations do not suppress CPT sensitivity of rad51-K191R or rad52-Delta327. However, rad51-K191R is uniquely suppressed by deleting the RME1 gene encoding a repressor of meiosis or its coregulator SIN4; this effect is independent of the meiosis-specific homolog, Dmc1. Sensitivity of rad52-Delta327 to CPT was unexpectedly increased by the MATa/MATalpha-repressed gene YGL193C, emphasizing the complex ways in which MAT regulates homologous recombination. The rad52-Delta327 mutation is suppressed by deleting the prolyl isomerase Fpr3, which is not MAT regulated. rad55Delta is also suppressed by deletion of PST2 and/or YBR052C (RFS1, rad55 suppressor), two members of a three-gene family of flavodoxin-fold proteins that associate in a nonrandom fashion with chromatin. All three recombination-defective mutations are made more sensitive by deletions of Rad6 and of the histone deacetylases Rpd3 and Ume6, although these mutations are not themselves CPT or phleomycin sensitive.

  9. Major genes affecting ovulation rate in sheep.

    PubMed

    Davis, George Henry

    2005-01-01

    Research conducted since 1980 in relation to inheritance patterns and DNA testing of major genes for prolificacy has shown that major genes have the potential to significantly increase the reproductive performance of sheep flocks throughout the world. Mutations that increase ovulation rate have been discovered in the BMPR-1B, BMP15 and GDF9 genes, and others are known to exist from the expressed inheritance patterns although the mutations have not yet been located. In the case of BMP15, four different mutations have been discovered but each produces the same phenotype. The modes of inheritance of the different prolificacy genes include autosomal dominant genes with additive effects on ovulation rate (BMPR-1B; Lacaune), autosomal over-dominant genes with infertility in homozygous females (GDF9), X-linked over-dominant genes with infertility in homozygous females (BMP15), and X-linked maternally imprinted genes (FecX2). The size of the effect of one copy of a mutation on ovulation rate ranges from an extra 0.4 ovulations per oestrus for the FecX2 mutation to an extra 1.5 ovulations per oestrus for the BMPR-1B mutation. A commercial DNA testing service enables some of these mutations to be used in genetic improvement programmes based on marker assisted selection.

  10. Major genes affecting ovulation rate in sheep

    PubMed Central

    2005-01-01

    Research conducted since 1980 in relation to inheritance patterns and DNA testing of major genes for prolificacy has shown that major genes have the potential to significantly increase the reproductive performance of sheep flocks throughout the world. Mutations that increase ovulation rate have been discovered in the BMPR-1B, BMP15 and GDF9 genes, and others are known to exist from the expressed inheritance patterns although the mutations have not yet been located. In the case of BMP15, four different mutations have been discovered but each produces the same phenotype. The modes of inheritance of the different prolificacy genes include autosomal dominant genes with additive effects on ovulation rate (BMPR-1B; Lacaune), autosomal over-dominant genes with infertility in homozygous females (GDF9), X-linked over-dominant genes with infertility in homozygous females (BMP15), and X-linked maternally imprinted genes (FecX2). The size of the effect of one copy of a mutation on ovulation rate ranges from an extra 0.4 ovulations per oestrus for the FecX2 mutation to an extra 1.5 ovulations per oestrus for the BMPR-1B mutation. A commercial DNA testing service enables some of these mutations to be used in genetic improvement programmes based on marker assisted selection. PMID:15601592

  11. Changes in liver cell DNA methylation status in diabetic mice affect its FT-IR characteristics.

    PubMed

    Vidal, Benedicto de Campos; Ghiraldini, Flávia Gerelli; Mello, Maria Luiza S

    2014-01-01

    Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD) mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR) profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile. The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO) and adequate software. Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including νas -CH3 stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of -CH3 groups. Other spectral differences were found at 1700-1500 cm(-1) and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice.

  12. AQUATIC PLANT SPECIATION AFFECTED BY DIVERSIFYING SELECTION OF ORGANELLE DNA REGIONS(1).

    PubMed

    Kato, Syou; Misawa, Kazuharu; Takahashi, Fumio; Sakayama, Hidetoshi; Sano, Satomi; Kosuge, Keiko; Kasai, Fumie; Watanabe, Makoto M; Tanaka, Jiro; Nozaki, Hisayoshi

    2011-10-01

    Many of the genes that control photosynthesis are carried in the chloroplast. These genes differ among species. However, evidence has yet to be reported revealing the involvement of organelle genes in the initial stages of plant speciation. To elucidate the molecular basis of aquatic plant speciation, we focused on the unique plant species Chara braunii C. C. Gmel. that inhabits both shallow and deep freshwater habitats and exhibits habitat-based dimorphism of chloroplast DNA (cpDNA). Here, we examined the "shallow" and "deep" subpopulations of C. braunii using two nuclear DNA (nDNA) markers and cpDNA. Genetic differentiation between the two subpopulations was measured in both nDNA and cpDNA regions, although phylogenetic analyses suggested nuclear gene flow between subpopulations. Neutrality tests based on Tajima's D demonstrated diversifying selection acting on organelle DNA regions. Furthermore, both "shallow" and "deep" haplotypes of cpDNA detected in cultures originating from bottom soils of three deep environments suggested that migration of oospores (dormant zygotes) between the two habitats occurs irrespective of the complete habitat-based dimorphism of cpDNA from field-collected vegetative thalli. Therefore, the two subpopulations are highly selected by their different aquatic habitats and show prezygotic isolation, which represents an initial process of speciation affected by ecologically based divergent selection of organelle genes.

  13. DNA methylation of retrotransposons, DNA transposons and genes in sugar beet (Beta vulgaris L.).

    PubMed

    Zakrzewski, Falk; Schmidt, Martin; Van Lijsebettens, Mieke; Schmidt, Thomas

    2017-03-03

    The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses activity of transposable elements (TEs), affects gene expression, and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of the worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%) in particular, satellite DNA, retrotransposons, and DNA transposons. Genome-wide cytosine methylation in the sugar beet genome was studied in leaves and leaf-derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H=A, C, and T), and CHH sites, whereas the TE pattern differed, depending on the classes 1 (retrotransposons) and 2 (DNA transposons), respectively. Along genes and TEs, the CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing toward a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome-wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared to leaves. This article is protected by copyright. All rights reserved.

  14. Human DNA repair and recombination genes

    SciTech Connect

    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.

  15. Identification of the dnaA and dnaN gene products of Escherichia coli.

    PubMed

    Yuasa, S; Sakakibara, Y

    1980-01-01

    A specialized transducing lambda phage carrying the dnaN genes of Escherichia coli specifies two proteins of about 41 and 48 kilodaltons (kd). The temperature-sensitive mutations, dnaN59 and dnaA167, were found to result in altered isoelectric points of the 41 and 48 kd proteins, respectively. Thus the dnaN gene product was identified as a weakly acidic 41 and 48 kd protein. The synthesis of the dnaN gene product is greatly reduced by insertion of a transposon Tn3 in the dnaA gene and by deletion in the gene at the distal end to the dnaN gene. Temperature-sensitive dnaA mutations, on the dnaN gene product. These results indicate that the synthesis of the dnaN gene product is dependent on the structural integrity of the dnaA gene.

  16. DNA repair genes in the Megavirales pangenome.

    PubMed

    Blanc-Mathieu, Romain; Ogata, Hiroyuki

    2016-06-01

    The order 'Megavirales' represents a group of eukaryotic viruses with a large genome encoding a few hundred up to two thousand five hundred genes. Several members of Megavirales possess genes involved in major DNA repair pathways. Some of these genes were likely inherited from an ancient virus world and some others were derived from the genomes of their hosts. Here we examine molecular phylogenies of key DNA repair enzymes in light of recent hypotheses on the origin of Megavirales, and propose that the last common ancestors of the individual families of the order Megavirales already possessed DNA repair functions to achieve and maintain a moderately large genome and that this repair capacity gradually increased, in a family-dependent manner, during their recent evolution.

  17. Controlling gene expression by DNA mechanics: emerging insights and challenges.

    PubMed

    Levens, David; Baranello, Laura; Kouzine, Fedor

    2016-09-01

    Transcription initiation is a major control point for the precise regulation of gene expression. Our knowledge of this process has been mainly derived from protein-centric studies wherein cis-regulatory DNA sequences play a passive role, mainly in arranging the protein machinery to coalesce at the transcription start sites of genes in a spatial and temporal-specific manner. However, this is a highly dynamic process in which molecular motors such as RNA polymerase II (RNAPII), helicases, and other transcription factors, alter the level of mechanical force in DNA, rather than simply a set of static DNA-protein interactions. The double helix is a fiber that responds to flexural and torsional stress, which if accumulated, can affect promoter output as well as change DNA and chromatin structure. The relationship between DNA mechanics and the control of early transcription initiation events has been under-investigated. Genomic techniques to display topological stress and conformational variation in DNA across the mammalian genome provide an exciting new insight on the role of DNA mechanics in the early stages of the transcription cycle. Without understanding how torsional and flexural stresses are generated, transmitted, and dissipated, no model of transcription will be complete and accurate.

  18. Controlling gene expression by DNA mechanics: emerging insights and challenges.

    PubMed

    Levens, David; Baranello, Laura; Kouzine, Fedor

    2016-11-01

    Transcription initiation is a major control point for the precise regulation of gene expression. Our knowledge of this process has been mainly derived from protein-centric studies wherein cis-regulatory DNA sequences play a passive role, mainly in arranging the protein machinery to coalesce at the transcription start sites of genes in a spatial and temporal-specific manner. However, this is a highly dynamic process in which molecular motors such as RNA polymerase II (RNAPII), helicases, and other transcription factors, alter the level of mechanical force in DNA, rather than simply a set of static DNA-protein interactions. The double helix is a fiber that responds to flexural and torsional stress, which if accumulated, can affect promoter output as well as change DNA and chromatin structure. The relationship between DNA mechanics and the control of early transcription initiation events has been under-investigated. Genomic techniques to display topological stress and conformational variation in DNA across the mammalian genome provide an exciting new insight on the role of DNA mechanics in the early stages of the transcription cycle. Without understanding how torsional and flexural stresses are generated, transmitted, and dissipated, no model of transcription will be complete and accurate.

  19. Ipr gene control of the anti-DNA antibody response.

    PubMed

    Pisetsky, D S; Caster, S A; Roths, J B; Murphy, E D

    1982-05-01

    The influence of the Ipr gene on the anti-DNA antibody response was investigated in MRL and B6 Ipr/Ipr inbred mice, MRL +/+ mice less than a yr of age produced low levels of anti-DNA antibody, whereas older animals of this strain demonstrated levels in some instances comparable to those of the more severely affected MRL Ipr/Ipr mice. This result indicates a tendency to autoreactivity in MRL mice independent of the Ipr gene. To determine whether other mice bearing the Ipr gene would also express autoantibodies, the anti-DNA antibody responses of B6 Ipr/Ipr mice were studied. This strain was development by matings to transfer the Ipr gene into another inbred background and allow evaluation of the action independent of other disturbances of the MRL mice. Mice of this strain produced antibodies to DNA, with female animals displaying significantly higher levels than males. This result demonstrates that the Ipr gene can stimulate autoantibody production in mice other than the MRL strain and does not require abnormalities unique to this background to potentiate autoreactivity.

  20. Naked DNA for liver gene transfer.

    PubMed

    Liu, Feng; Tyagi, Pradeep

    2005-01-01

    The majority of acquired and inherited genetic disorders, including most inborn errors of metabolism, are manifested in the liver. Therefore, it is hardly any surprise to see a large number of Medline reports describing gene therapy efforts in preclinical settings directed toward this organ (Inoue et al., 2004; Oka and Chen, 2004). Of late, non-viral vectors have garnered a lot of attention from the biomedical research community engaged in liver gene therapy (Gupta et al., 2004). However, the first initiative toward gene transfer to the liver using a non-viral approach was taken by Hickman et al. (1994), who applied the technique of naked DNA injection pioneered by Wolff (1990) for skeletal muscle. Direct injection of naked DNA resulted in low, variable and localized gene expression in the rat liver. Consequently, several developments reported in the literature since then aimed to improve hepatic gene expression by employing both surgical and nonsurgical methods. These developments include the exploitation of the unique vasculature of liver as well as the use of electric and mechanical force as an adjunct to the systemic administration of the naked plasmid gene. This chapter focuses on these developments reported from various laboratories, including ours. In addition, the underlying mechanism responsible for the dramatic increase in gene expression using these latest approaches for non-viral gene transfer to the liver is also discussed.

  1. Sperm DNA fragmentation affects epigenetic feature in human male pronucleus.

    PubMed

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

    2017-03-06

    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.

  2. DNA Methylation of BDNF Gene in Schizophrenia

    PubMed Central

    Çöpoğlu, Ümit Sertan; İğci, Mehri; Bozgeyik, Esra; Kokaçya, M. Hanifi; İğci, Yusuf Ziya; Dokuyucu, Recep; Arı, Mustafa; Savaş, Haluk A.

    2016-01-01

    Background Although genetic factors are risk factors for schizophrenia, some environmental factors are thought to be required for the manifestation of disease. Epigenetic mechanisms regulate gene functions without causing a change in the nucleotide sequence of DNA. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that regulates synaptic transmission and plasticity. It has been suggested that BDNF may play a role in the pathophysiology of schizophrenia. It is established that methylation status of the BDNF gene is associated with fear learning, memory, and stressful social interactions. In this study, we aimed to investigate the DNA methylation status of BDNF gene in patients with schizophrenia. Material/Methods The study included 49 patients (33 male and 16 female) with schizophrenia and 65 unrelated healthy controls (46 male and 19 female). Determination of methylation pattern of CpG islands was based on the principle that bisulfite treatment of DNA results in conversion of unmethylated cytosine residues into uracil, whereas methylated cytosine residues remain unmodified. Methylation-specific PCR was performed with primers specific for either methylated or unmethylated DNA. Results There was no significant difference in methylated or un-methylated status for BDNF promoters between schizophrenia patients and controls. The mean duration of illness was significantly lower in the hemi-methylated group compared to the non-methylated group for BDNF gene CpG island-1 in schizophrenia patients. Conclusions Although there were no differences in BDNF gene methylation status between schizophrenia patients and healthy controls, there was an association between duration of illness and DNA methylation. PMID:26851233

  3. DNA Methylation of BDNF Gene in Schizophrenia.

    PubMed

    Çöpoğlu, Ümit Sertan; Igci, Mehri; Bozgeyik, Esra; Kokaçya, M Hanifi; İğci, Yusuf Ziya; Dokuyucu, Recep; Ari, Mustafa; Savaş, Haluk A

    2016-02-06

    BACKGROUND Although genetic factors are risk factors for schizophrenia, some environmental factors are thought to be required for the manifestation of disease. Epigenetic mechanisms regulate gene functions without causing a change in the nucleotide sequence of DNA. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that regulates synaptic transmission and plasticity. It has been suggested that BDNF may play a role in the pathophysiology of schizophrenia. It is established that methylation status of the BDNF gene is associated with fear learning, memory, and stressful social interactions. In this study, we aimed to investigate the DNA methylation status of BDNF gene in patients with schizophrenia. MATERIAL AND METHODS The study included 49 patients (33 male and 16 female) with schizophrenia and 65 unrelated healthy controls (46 male and 19 female). Determination of methylation pattern of CpG islands was based on the principle that bisulfite treatment of DNA results in conversion of unmethylated cytosine residues into uracil, whereas methylated cytosine residues remain unmodified. Methylation-specific PCR was performed with primers specific for either methylated or unmethylated DNA. RESULTS There was no significant difference in methylated or un-methylated status for BDNF promoters between schizophrenia patients and controls. The mean duration of illness was significantly lower in the hemi-methylated group compared to the non-methylated group for BDNF gene CpG island-1 in schizophrenia patients. CONCLUSIONS Although there were no differences in BDNF gene methylation status between schizophrenia patients and healthy controls, there was an association between duration of illness and DNA methylation.

  4. Preferential DNA repair in expressed genes

    SciTech Connect

    Hanawalt, P.C.

    1987-12-01

    Potentially deleterious alterations to DNA occur nonrandomly within the mammalian genome. These alterations include the adducts produced by many chemical carcinogens, but not the UV-induced cyclobutane pyrimidine dimer, which may be an exception. Recent studies in our laboratory have shown that the excision repair of pyrimidine dimers and certain other lesions is nonrandom in the mammalian genome, exhibiting a distinct preference for actively transcribed DNA sequences. An important consequence of this fact is that mutagenesis and carcinogenesis may be determined in part by the activities of the relevant genes. Repair may also be processive, and a model is proposed in which excision repair is coupled to transcription at the nuclear matrix. Similar but freely diffusing repair complexes may account for the lower overall repair efficiencies in the silent domains of the genome. Risk assessment in relations to chemical carcinogenesis requires assays that determine effective levels of DNA damage for producing malignancy. The existence of nonrandom repair in the genome casts into doubt the reliability of overall indicators of DNA binding and lesion repair for such determinations. Furthermore, some apparent differences between the intragenomic repair heterogeneity in rodent cells and that in human cells mandate a reevaluation of rodent test systems for human risk assessment. Tissue-specific and cell-specific differences in the coordinate regulation of gene expression and DNA repair may account for corresponding differences in the carcinogenic response.

  5. SERS gene probe for DNA diagnostics

    NASA Astrophysics Data System (ADS)

    Stokes, David L.; Allain, Leonardo R.; Isola, Narayana R.; Vo-Dinh, Tuan

    2003-07-01

    We describe the development of a surface-enhanced Raman scattering gene (SERGen) probe technology for rapid screening for diseases and pathogens through DNA hybridization assays. The technology combines the use of gene probes labeled with SERS-active markers, and nanostructured metallic platforms for inducing the SERS effect. As a result, SERGen-based methods can offer the spectral selectivity and sensitivity of SERS as well as the molecular specificity of DNA sequence hybridization. Furthermore, these new probe s preclude the use of radioactive labels. As illustrated herein, SERGen probes have been used as primers in polymerase chain reaction (PCR) amplifications of specific DNA sequences, hence further boosting the sensitivity of the technology. We also describe several approaches to developing SERS-active DNA assay platforms, addressing the challenges of making the SERGen technology accessible and practical for clinical settings. The usefulness of the SERGen approach has been demonstrated in the detection of HIV, BRCA1 breast cancer, and BAX genes. There is great potential for the use of numerous SERGen probes for multiplexed detection of multiple biological targets.

  6. Changes in Liver Cell DNA Methylation Status in Diabetic Mice Affect Its FT-IR Characteristics

    PubMed Central

    Vidal, Benedicto de Campos; Ghiraldini, Flávia Gerelli; Mello, Maria Luiza S.

    2014-01-01

    Background Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD) mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR) profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile. Methodology/Principal Findings The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO) and adequate software. Conclusions/Significance Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including νas –CH3 stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of –CH3 groups. Other spectral differences were found at 1700–1500 cm−1 and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice. PMID:25019512

  7. Brief Guide to Genomics: DNA, Genes and Genomes

    MedlinePlus

    ... guía de genómica A Brief Guide to Genomics DNA, Genes and Genomes Deoxyribonucleic acid (DNA) is the ... and lead to a disease such as cancer. DNA Sequencing Sequencing simply means determining the exact order ...

  8. Organization and transcription of the dnaA and dnaN genes of Escherichia coli.

    PubMed

    Sakakibara, Y; Tsukano, H; Sako, T

    1981-01-01

    The locations of the linked dnaA and dnaN genes of Escherichia coli in a specialized transducing lambda phage genome have been determined by electron microscopic heteroduplex analysis, using phages with deletions or insertions in the dnaA or dnaN gene. The transcription initiation sites for the dna genes were also localized by electron microscopic analysis of DNA-RBA heteroduplex molecules formed between the E. coli DNA fragment of the phage genome and the in vitro transcription products of the fragment. The dnaN gene was found to be transcribed in the same direction as the dnaA gene, and predominantly from the promoter of the dnaA gene.

  9. A Limited Number of Globin Genes in Human DNA

    PubMed Central

    Gambino, Roberto; Kacian, Daniel; O'Donnell, Joyce; Ramirez, Francesco; Marks, Paul A.; Bank, Arthur

    1974-01-01

    The number of globin genes in human cells was determined by hybridizing DNA from human spleens to 3H-labeled DNA complementary to human globin mRNA. Assuming the rates of reannealing of complementary DNA and cellular DNA are similar, the extent of hybridization of complementary DNA at various ratios of cellular DNA to complementary DNA indicate that there are fewer than 10 globin gene copies per haploid human genome. An alternative analysis of the data, which introduces no assumptions concerning the relative rates of reaction of complementary DNA and cellular DNA, indicates fewer than 20 globin gene copies are present. DNA isolated from the spleen of a patient with β+ thalassemia contained a number of globin gene copies similar to that of normal DNA. PMID:4530276

  10. A DNA element in the slo gene modulates ethanol tolerance.

    PubMed

    Krishnan, Harish R; Li, Xiaolei; Ghezzi, Alfredo; Atkinson, Nigel S

    2016-03-01

    In Drosophila, the slo gene encodes BK-type Ca(2+)-activated K(+) channels and is involved in producing rapid functional tolerance to sedation with ethanol. Drosophila are ideal for the study of functional ethanol tolerance because the adult does not acquire metabolic ethanol tolerance (Scholz, Ramond, Singh, & Heberlein, 2000). It has been shown that mutations in slo block the capacity to acquire tolerance, that sedation with ethanol vapor induces slo gene expression in the nervous system, and that transgenic induction of slo can phenocopy tolerance (Cowmeadow, Krishnan, & Atkinson, 2005; Cowmeadow et al., 2006). Here we use ethanol-induced histone acetylation to map a DNA regulatory element in the slo transcriptional control region and functionally test the element for a role in producing ethanol tolerance. Histone acetylation is commonly associated with activating transcription factors. We used the chromatin immunoprecipitation assay to map histone acetylation changes following ethanol sedation to identify an ethanol-responsive DNA element. Ethanol sedation induced an increase in histone acetylation over a 60 n DNA element called 6b, which is situated between the two ethanol-responsive neural promoters of the slo gene. Removal of the 6b element from the endogenous slo gene affected the production of functional ethanol tolerance as assayed in an ethanol-vapor recovery from sedation assay. Removal of element 6b extended the period of functional ethanol tolerance from ∼10 days to more than 21 days after a single ethanol-vapor sedation. This study demonstrates that mapping the position of ethanol-induced histone acetylation is an effective way to identify DNA regulatory elements that help to mediate the response of a gene to ethanol. Using this approach, we identified a DNA element, which is conserved among Drosophila species, and which is important for producing a behaviorally relevant ethanol response.

  11. Cloning of Bacteroides fragilis plasmid genes affecting metronidazole resistance and ultraviolet survival in Escherichia coli

    SciTech Connect

    Wehnert, G.U.; Abratt, V.R.; Goodman, H.J.; Woods, D.R. )

    1990-03-01

    Since reduced metronidazole causes DNA damage, resistance to metronidazole was used as a selection method for the cloning of Bacteroides fragilis genes affecting DNA repair mechanisms in Escherichia coli. Genes from B. fragilis Bf-2 were cloned on a recombinant plasmid pMT100 which made E. coli AB1157 and uvrA, B, and C mutant strains more resistant to metronidazole, but more sensitive to far uv irradiation under aerobic conditions. The loci affecting metronidazole resistance and uv sensitivity were linked and located on a 5-kb DNA fragment which originated from the small 6-kb cryptic plasmid pBFC1 present in B. fragilis Bf-2 cells.

  12. Nucleosome-free DNA regions differentially affect distant communication in chromatin.

    PubMed

    Nizovtseva, Ekaterina V; Clauvelin, Nicolas; Todolli, Stefjord; Polikanov, Yury S; Kulaeva, Olga I; Wengrzynek, Scott; Olson, Wilma K; Studitsky, Vasily M

    2017-04-07

    Communication between distantly spaced genomic regions is one of the key features of gene regulation in eukaryotes. Chromatin per se can stimulate efficient enhancer-promoter communication (EPC); however, the role of chromatin structure and dynamics in this process remains poorly understood. Here we show that nucleosome spacing and the presence of nucleosome-free DNA regions can modulate chromatin structure/dynamics and, in turn, affect the rate of EPC in vitro and in silico. Increasing the length of internucleosomal linker DNA from 25 to 60 bp results in more efficient EPC. The presence of longer nucleosome-free DNA regions can positively or negatively affect the rate of EPC, depending upon the length and location of the DNA region within the chromatin fiber. Thus the presence of histone-free DNA regions can differentially affect the efficiency of EPC, suggesting that gene regulation over a distance could be modulated by changes in the length of internucleosomal DNA spacers. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Characterization of the mammalian DNA polymerase gene(s) and enzyme(s). Annual progress report

    SciTech Connect

    Mishra, N.C.

    1995-01-01

    Two Genes for DNA polymerase delta were identified from the wild type Chinese hamster ovary cells. These genes were cloned via RT-PCR from mRNA prepared the Chinese hamster ovary cells using primers specific to conserved sequences of the DNA polymerase {delta} gene. The first gene encodes a PCNA dependent DNA polymerase {delta} gene whereas the second gene encodes a PCNA independent DNA polymerase {delta} gene. Methods were developed to clone these genes in expression vector and host systems. The role of the two genes in DNA replication and repair was determined.

  14. Therapeutic option of plasmid-DNA based gene transfer.

    PubMed

    Taniyama, Yoshiaki; Azuma, Junya; Kunugiza, Yasuo; Iekushi, Kazuma; Rakugi, Hiromi; Morishita, Ryuichi

    2012-01-01

    Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common method in clinical cases because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid deoxyribonucleic acid (DNA)-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a phase III clinical trial did not show sufficient efficiency. In this situation, more efficient plasmid DNA gene transfer is needed all over the world. This review focuses on plasmid DNA gene transfer and its enhancement, including ultrasound with microbubbles, electroporation, hydrodynamic method, gene gun, jet injection, cationic lipids and cationic polymers.

  15. TP53 codon 72 polymorphism affects accumulation of mtDNA damage in human cells

    PubMed Central

    Altilia, Serena; Santoro, Aurelia; Malagoli, Davide; Lanzarini, Catia; Álvarez, Josué Adolfo Ballesteros; Galazzo, Gianluca; Porter, Donald Carl; Crocco, Paolina; Rose, Giuseppina; Passarino, Giuseppe; Roninson, Igor Boris; Franceschi, Claudio; Salvioli, Stefano

    2012-01-01

    Human TP53 gene is characterised by a polymorphism at codon 72 leading to an Arginine-to-Proline (R/P) substitution. The two resulting p53 isoforms have a different subcellular localisation after stress (more nuclear or more mitochondrial for the P or R isoform, respectively). p53P72 variant is more efficient than p53R72 in inducing the expression of genes involved in nuclear DNA repair. Since p53 is involved also in mitochondrial DNA (mtDNA) maintenance, we wondered whether these p53 isoforms are associated with different accumulation of mtDNA damage. We observed that cells bearing p53R72 accumulate lower amount of mtDNA damage upon rotenone stress with respect to cells bearing p53P72, and that p53R72 co-localises with polymerase gamma more than p53P72. We also analysed the in vivo accumulation of heteroplasmy in a 300 bp fragment of mtDNA D-loop of 425 aged subjects. We observed that subjects with heteroplasmy higher than 5% are significantly less than expected in the p53R72/R72 group. On the whole, these data suggest that the polymorphism of TP53 at codon 72 affects the accumulation of mtDNA mutations, likely through the different ability of the two p53 isoforms to bind to polymerase gamma, and may contribute to in vivo accumulation of mtDNA mutations. PMID:22289634

  16. Structural analysis of the dnaA and dnaN genes of Escherichia coli.

    PubMed

    Ohmori, H; Kimura, M; Nagata, T; Sakakibara, Y

    1984-05-01

    The nucleotide sequence of the entire region containing the Escherichia coli dnaA and dnaN genes was determined. Base substitutions by such mutations as dnaA46, dnaA167, dnaN59, and dnaN806 were also identified. Analyses of coding frames, the mutational base substitutions, and other data indicate that dnaN follows dnaA, both have the same orientation, and are separated by only 4 bp. The deduced amino acid sequence specifies Mrs and isoelectric points consistent with those of the previously identified gene products. The transcriptional initiation site of the dnaA gene was assigned by analysis of in vitro RNA products. Examination of the intercistronic sequence and analysis of in vitro transcription supported the notion that the dnaA and dnaN genes constitute a single operon.

  17. Discoordinate gene expression in the dnaA-dnaN operon of Escherichia coli.

    PubMed

    Quiñones, A; Messer, W

    1988-07-01

    The dnaN gene of Escherichia coli encodes the beta-subunit of the DNA polymerase III holoenzyme. Previous work has established that dnaN lies immediately downstream of dnaA and that both genes may be cotranscribed from the dnaA promoters; no promoter for dnaN has been described. We investigated the in vivo regulation of transcription of the dnaN gene by transcriptional fusions to the galK gene, translational fusion to the lacZ gene and S1 mapping analysis. We found that there are at least three dnaN promoters residing entirely in the reading frame of the preceding dnaA gene, and that transcription from these promoters can occur independently of dnaA transcription which, however, extends at least up to dnaN. Furthermore, we found evidence for the inducibility of the dnaN promoters in a dam background under conditions of simultaneously reduced dnaA transcription. These results are consistent with the hypothesis that although dnaA and dnaN are organized in an operon considerable discoordinate transcription can occur, thus uncoupling dnaN and dnaA regulation, when needed.

  18. The first determination of DNA sequence of a specific gene.

    PubMed

    Inouye, Masayori

    2016-05-10

    How and when the first DNA sequence of a gene was determined? In 1977, F. Sanger came up with an innovative technology to sequence DNA by using chain terminators, and determined the entire DNA sequence of the 5375-base genome of bacteriophage φX 174 (Sanger et al., 1977). While this Sanger's achievement has been recognized as the first DNA sequencing of genes, we had determined DNA sequence of a gene, albeit a partial sequence, 11 years before the Sanger's DNA sequence (Okada et al., 1966).

  19. Nanoscale structure of protamine/DNA complexes for gene delivery

    NASA Astrophysics Data System (ADS)

    Motta, Simona; Brocca, Paola; Del Favero, Elena; Rondelli, Valeria; Cantù, Laura; Amici, Augusto; Pozzi, Daniela; Caracciolo, Giulio

    2013-02-01

    Understanding the internal packing of gene carriers is a key-factor to realize both gene protection during transport and de-complexation at the delivery site. Here, we investigate the structure of complexes formed by DNA fragments and protamine, applied in gene delivery. We found that complexes are charge- and size-tunable aggregates, depending on the protamine/DNA ratio, hundred nanometers in size. Their compactness and fractal structure depend on the length of the DNA fragments. Accordingly, on the local scale, the sites of protamine/DNA complexation assume different morphologies, seemingly displaying clumping ability for the DNA network only for shorter DNA fragments.

  20. Transcriptional organization of the dnaN and recF genes of Escherichia coli K-12.

    PubMed

    Armengod, M E; García-Sogo, M; Lambíes, E

    1988-08-25

    The dnaN gene of Escherichia coli determines the beta subunit of DNA polymerase III, a multisubunit enzyme responsible for most of the replicative DNA synthesis. The dnaN gene maps between the dnaA and recF genes. We have characterized the regulatory region of the dnaN gene by screening DNA restriction fragments for promoter activity, S1 mapping of mRNAs, deletion analysis, and in vivo dnaN complementation tests. There are at least three dnaN promoters located in the second half of the dnaA coding region. The one closest to the dnaN structural gene is the weakest, but it provides sufficient dnaN expression for complementation when the gene is present on a multicopy plasmid. Deletion of sequences needed for initiation of dnaN translation or introduction of nonsense codons into dnaN causes reduction of recF expression. However, a deletion inactivating dnaN without changing the reading frame of the gene does not affect expression of the recF gene. These results indicate that the dnaN and recF genes are organized in an operon. We have previously shown the presence of termination signals within the dnaN coding region (Armengod, M.E., and Lambíes, E. (1986) Gene (Amst.) 43, 183-196). Therefore, we propose that the polarity produced by nonsense mutations in dnaN is primarily transcriptional. The uncoupling of transcription and translation of the dnaN gene (when translation is interrupted by premature nonsense codons or by other mechanisms) probably results in transcription termination at termination signals in dnaN.

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

    PubMed Central

    Rose, Sasha J.

    2016-01-01

    ABSTRACT 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. PMID:27923918

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

    PubMed

    Rose, Sasha J; Bermudez, Luiz E

    2016-12-06

    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.

  3. Altered gene expression correlates with DNA structure.

    PubMed

    Kohwi, Y; Kohwi-Shigematsu, T

    1991-12-01

    We examined the participation of triplex DNA structure in gene regulation using a poly(dG)-poly(dC) sequence as a model. We show that a poly(dG)-poly(dC) sequence, which can adopt an intramolecular dG.dG.dC triplex under superhelical strain, strongly augments gene expression when placed 5' to a promoter. The activity of this sequence exhibits a striking length dependency: dG tracts of 27-30 bp augment the expression of a reporter gene to a level comparable to that observed with the polyoma enhancer in mouse LTK- cells, whereas tracts of 35 bp and longer have virtually no effect. A supercoiled plasmid containing a dG tract of 30 bp competes in vivo for a trans-acting factor as revealed by reduction in the reporter gene transcription driven by the (dG)29/promoter of the test plasmid, while dGs of 35 bp and longer in the competition plasmid failed to compete. In purified supercoiled plasmid DNA at a superhelical density of -0.05, dG tracts of 32 bp and longer form a triplex, whereas those of 30 bp and shorter remain double-stranded under a PBS solution. These results suggest that a localized superhelical strain can exist, at least transiently, in mouse LTK- cells, and before being relaxed by topoisomerases this rapidly induces dG tracts of 35 bp and longer to adopt a triplex preventing the factor from binding. Thus, these data suggest that a poly(dG)-poly(dC) sequence can function as a negative regulator by adopting an intramolecular triple helix structure in vivo.

  4. Methods for isolation of cell-free plasma DNA strongly affect DNA yield.

    PubMed

    Fleischhacker, Michael; Schmidt, Bernd; Weickmann, Sabine; Fersching, Debora M I; Leszinski, Gloria S; Siegele, Barbara; Stötzer, Oliver J; Nagel, Dorothea; Holdenrieder, Stefan

    2011-11-20

    Extracellular nucleic acids are present in plasma, serum, and other body fluids and their analysis has gained increasing attention during recent years. Because of the small quantity and highly fragmented nature of cell-free DNA in plasma and serum, a fast, efficient, and reliable isolation method is still a problem and so far there is no agreement on a standardized method. We used spin columns from commercial suppliers (QIAamp DNA Blood Midi Kit from Qiagen; NucleoSpin Kit from Macherey-Nagel; MagNA Pure isolation system from Roche Diagnostics) to isolate DNA from 44 plasma samples in parallel at laboratories in Berlin and Munich. DNA in all samples was quantified by real-time PCR on a LightCycler 480 using three different targets (GAPDH, ß-globin, ERV). The quantities of cell-free DNA for the different isolation methods and genes varied between medians of 1.6 ng/mL and 28.1 ng/mL. This considerable variation of absolute DNA values was mainly caused by the use of different isolation methods (p<0.0001). Comparable results were achieved by the use of the genes GAPDH and ERV while higher values were obtained by use of ß-globin. The laboratory site had only minor influence on DNA yield when manual extraction methods were used.

  5. Marker gene tethering by nucleoporins affects gene expression in plants.

    PubMed

    Smith, Sarah; Galinha, Carla; Desset, Sophie; Tolmie, Frances; Evans, David; Tatout, Christophe; Graumann, Katja

    2015-01-01

    In non-plant systems, chromatin association with the nuclear periphery affects gene expression, where interactions with nuclear envelope proteins can repress and interactions with nucleoporins can enhance transcription. In plants, both hetero- and euchromatin can localize at the nuclear periphery, but the effect of proximity to the nuclear periphery on gene expression remains largely unknown. This study explores the putative function of Seh1 and Nup50a nucleoporins on gene expression by using the Lac Operator / Lac Repressor (LacI-LacO) system adapted to Arabidopsis thaliana. We used LacO fused to the luciferase reporter gene (LacO:Luc) to investigate whether binding of the LacO:Luc transgene to nucleoporin:LacI protein fusions alters luciferase expression. Two separate nucleoporin-LacI-YFP fusions were introduced into single insert, homozygous LacO:Luc Arabidopsis plants. Homozygous plants carrying LacO:Luc and a single insert of either Seh1-LacI-YFP or Nup50a-LacI-YFP were tested for luciferase activity and compared to plants containing LacO:Luc only. Seh1-LacI-YFP increased, while Nup50a-LacI-YFP decreased luciferase activity. Seh1-LacI-YFP accumulated at the nuclear periphery as expected, while Nup50a-LacI-YFP was nucleoplasmic and was not selected for further study. Protein and RNA levels of luciferase were quantified by western blotting and RT-qPCR, respectively. Increased luciferase activity in LacO:Luc+Seh1-LacI-YFP plants was correlated with increased luciferase protein and RNA levels. This change of luciferase expression was abolished by disruption of LacI-LacO binding by treating with IPTG in young seedlings, rosette leaves and inflorescences. This study suggests that association with the nuclear periphery is involved in the regulation of gene expression in plants.

  6. A nuclear-mitochondrial DNA interaction affecting hearing impairment in mice

    PubMed Central

    Johnson, Kenneth R.; Zheng, Qing Yin; Bykhovskaya, Yelena; Spirina, Olga; Fischel-Ghodsian, Nathan

    2010-01-01

    The pathophysiologic pathways and clinical expression of mitochondrial DNA (mtDNA) mutations are not well understood. This is mainly the result of the heteroplasmic nature of most pathogenic mtDNA mutations and of the absence of clinically relevant animal models with mtDNA mutations. mtDNA mutations predisposing to hearing impairment in humans are generally homoplasmic, yet some individuals with these mutations have severe hearing loss, whereas their maternal relatives with the identical mtDNA mutation have normal hearing1,2. Epidemiologic, biochemical and genetic data indicate that nuclear genes are often the main determinants of these differences in phenotype3–5. To identify a mouse model for maternally inherited hearing loss, we screened reciprocal backcrosses of three inbred mouse strains, A/J, NOD/LtJ and SKH2/J, with age-related hearing loss (AHL). In the (A/J×CAST/Ei)×A/J backcross, mtDNA derived from the A/J strain exerted a significant detrimental effect on hearing when compared with mtDNA from the CAST/Ei strain. This effect was not seen in the (NOD/LtJ × CAST/Ei)×NOD/LtJ and (SKH2/J×CAST/Ei)×SKH2/J backcrosses. Genotyping revealed that this effect was seen only in mice homozygous for the A/J allele at the Ahl locus on mouse chromosome 10. Sequencing of the mitochondrial genome in the three inbred strains revealed a single nucleotide insertion in the tRNA-Arg gene (mt-Tr) as the probable mediator of the mitochondrial effect. This is the first mouse model with a naturally occurring mtDNA mutation affecting a clinical phenotype, and it provides an experimental model to dissect the pathophysiologic processes connecting mtDNA mutations to hearing loss. PMID:11175788

  7. DNA Topoisomerase Iα Affects the Floral Transition1[OPEN

    PubMed Central

    Gong, Ximing; Shen, Lisha; Peng, Ya Zhi; Gan, Yinbo

    2017-01-01

    DNA topoisomerases modulate DNA topology to maintain chromosome superstructure and genome integrity, which is indispensable for DNA replication and RNA transcription. Their function in plant development still remains largely unknown. Here, we report a hitherto unidentified role of Topoisomerase Iα (TOP1α) in controlling flowering time in Arabidopsis (Arabidopsis thaliana). Loss of function of TOP1α results in early flowering under both long and short days. This is attributed mainly to a decrease in the expression of a central flowering repressor, FLOWERING LOCUS C (FLC), and its close homologs, MADS AFFECTING FLOWERING4 (MAF4) and MAF5, during the floral transition. TOP1α physically binds to the genomic regions of FLC, MAF4, and MAF5 and promotes the association of RNA polymerase II complexes to their transcriptional start sites. These correlate with the changes in histone modifications but do not directly affect nucleosome occupancy at these loci. Our results suggest that TOP1α mediates DNA topology to facilitate the recruitment of RNA polymerase II at FLC, MAF4, and MAF5 in conjunction with histone modifications, thus facilitating the expression of these key flowering repressors to prevent precocious flowering in Arabidopsis. PMID:27837087

  8. New differentially expressed genes and differential DNA methylation underlying refractory epilepsy

    PubMed Central

    Xu, Tao; Liu, Shiyong; Yuan, Jinxian; Huang, Hao; Qin, Lu; Yang, Hui; Chen, Lifen; Tan, Xinjie; Chen, Yangmei

    2016-01-01

    Epigenetics underlying refractory epilepsy is poorly understood, especially in patients without distinctive genetic alterations. DNA methylation may affect gene expression in epilepsy without affecting DNA sequences. Herein, we analyzed genome-wide DNA methylation and gene expression in brain tissues of 10 patients with refractory epilepsy using methylated DNA immunoprecipitation linked with sequencing and mRNA Sequencing. Diverse distribution of differentially methylated genes was found in X chromosome, while differentially methylated genes appeared rarely in Y chromosome. 62 differentially expressed genes, such as MMP19, AZGP1, DES, and LGR6 were correlated with refractory epilepsy for the first time. Although general trends of differentially enriched gene ontology terms and Kyoto Encyclopedia of Genes and Genome pathways in this study are consistent with previous researches, differences also exist in many specific gene ontology terms and Kyoto Encyclopedia of Genes and Genome pathways. These findings provide a new genome-wide profiling of DNA methylation and gene expression in brain tissues of patients with refractory epilepsy, which may provide a basis for further study on the etiology and mechanisms of refractory epilepsy. PMID:27903967

  9. GeneBreak: detection of recurrent DNA copy number aberration-associated chromosomal breakpoints within genes.

    PubMed

    van den Broek, Evert; van Lieshout, Stef; Rausch, Christian; Ylstra, Bauke; van de Wiel, Mark A; Meijer, Gerrit A; Fijneman, Remond J A; Abeln, Sanne

    2016-01-01

    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).

  10. Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.

    PubMed Central

    Singh, K K; Samson, L

    1995-01-01

    Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7761422

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

    PubMed

    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

  12. [Bioinformatics analysis of DNA demethylase genes in Lonicera japonica Thunb].

    PubMed

    Qi, Lin-jie; Yuan, Yuan; Wu, Chong; Huang, Lu-qi; Chen, Ping

    2015-03-01

    The DNA demethylase genes are widespread in plants. Four DNA demethylase genes (LJDME1, LJDME2, LJDME3 and LJDME4) were obtained from transcriptome dataset of Lonicera japonica Thunb by using bioinformatics methods and the proteins' physicochemical properties they encoded were predicted. The phylogenetic tree showed that the four DNA demethylase genes and Arabidopsis thaliana DME had a close relationship. The result of gene expression model showed that four DNA demethylase genes were different between species. The expression levels of LJDME1 and LJDME2 were even more higher in Lonicera japonica var. chinensis than those in L. japonica. LJDME] and LJDME2 maybe regulate the active compounds of L. japonica. This study aims to lay a foundation for further understanding of the function of DNA demethylase genes in L. japonica.

  13. Induction of DNA damage by deguelin is mediated through reducing DNA repair genes in human non-small cell lung cancer NCI-H460 cells.

    PubMed

    Ji, Bin-Chuan; Yu, Chien-Chih; Yang, Su-Tso; Hsia, Te-Chun; Yang, Jai-Sing; Lai, Kuang-Chi; Ko, Yang-Ching; Lin, Jen-Jyh; Lai, Tung-Yuan; Chung, Jing-Gung

    2012-04-01

    It has been shown that deguelin, one of the compounds of rotenoids from flavonoid family, induced cytotoxic effects through induction of cell cycle arrest and apoptosis in many types of human cancer cell lines, but deguelin-affected DNA damage and repair gene expression (mRNA) are not clarified yet. We investigated the effects of deguelin on DNA damage and associated gene expression in human lung cancer NCI-H460 cells in vitro. DNA damage was assayed by using the comet assay and DNA gel electrophoresis and the results indicated that NCI-H460 cells treated with 0, 50, 250 and 500 nM deguelin led to a longer DNA migration smear based on the single cell electrophoresis and DNA fragmentation occurred based on the examination of DNA gel electrophoresis. DNA damage and repair gene expression (mRNA) were evaluated by using real-time PCR assay and the results indicated that 50 and 250 nM deguelin for a 24-h exposure in NCI-H460 cells, decreased the gene levels of breast cancer 1, early onset (BRCA1), DNA-dependent serine/threonine protein kinase (DNA-PK), O6-methylguanine-DNA methyltransferase (MGMT), p53, ataxia telangiectasia mutated (ATM) and ataxia-telangiectasia and Rad3-related (ATR) mRNA expressions. Collectively, the present study showed that deguelin caused DNA damage and inhibited DNA damage and repair gene expressions, which might be due to deguelin-inhibited cell growth in vitro.

  14. Induction of dnaN and dnaQ gene expression in Escherichia coli by alkylation damage to DNA.

    PubMed Central

    Quiñones, A; Kaasch, J; Kaasch, M; Messer, W

    1989-01-01

    The dnaN and dnaQ genes encode the beta-subunit and the epsilon-subunit of the DNA polymerase III holoenzyme. By transcriptional fusions to the galK gene, translational fusions to lacZ and comparative S1 mapping analysis, we investigated the in-vivo regulation of dnaN and dnaQ. We found that DNA damage caused by the alkylating agent methyl methanesulphonate (MMS) leads to a significant induction in dnaN and dnaQ gene expression suggesting a requirement of increased amounts of at least some DNA polymerase III holoenzyme subunits for recovery from DNA damage caused by MMS. These results are first evidences that subunits of the DNA polymerase III holoenzyme are DNA damage inducible. This MMS induction of dnaN and dnaQ gene expression is unrelated to the adaptive response. It was not observed in lexA and recA mutants which abolish the induction of the SOS response. Images PMID:2656258

  15. Induction of dnaN and dnaQ gene expression in Escherichia coli by alkylation damage to DNA.

    PubMed

    Quiñones, A; Kaasch, J; Kaasch, M; Messer, W

    1989-02-01

    The dnaN and dnaQ genes encode the beta-subunit and the epsilon-subunit of the DNA polymerase III holoenzyme. By transcriptional fusions to the galK gene, translational fusions to lacZ and comparative S1 mapping analysis, we investigated the in-vivo regulation of dnaN and dnaQ. We found that DNA damage caused by the alkylating agent methyl methanesulphonate (MMS) leads to a significant induction in dnaN and dnaQ gene expression suggesting a requirement of increased amounts of at least some DNA polymerase III holoenzyme subunits for recovery from DNA damage caused by MMS. These results are first evidences that subunits of the DNA polymerase III holoenzyme are DNA damage inducible. This MMS induction of dnaN and dnaQ gene expression is unrelated to the adaptive response. It was not observed in lexA and recA mutants which abolish the induction of the SOS response.

  16. Wilson disease: changes in methionine metabolism and inflammation affect global DNA methylation in early liver disease

    PubMed Central

    Medici, Valentina; Shibata, Noreene M.; Kharbanda, Kusum K.; LaSalle, Janine M.; Woods, Rima; Liu, Sarah; Engelberg, Jesse A.; Devaraj, Sridevi; Török, Natalie J.; Jiang, Joy X.; Havel, Peter J.; Lönnerdal, Bo; Kim, Kyoungmi; Halsted, Charles H.

    2012-01-01

    Hepatic methionine metabolism may play an essential role in regulating methylation status and liver injury in Wilson disease (WD) through the inhibition of S-adenosylhomocysteine hydrolase (SAHH) by copper (Cu) and the consequent accumulation of S-adenosylhomocysteine (SAH). We studied the transcript levels of selected genes related to liver injury, levels of SAHH, SAH, DNA methyltransferases genes (Dnmt1, Dnmt3a, Dnmt3b) and global DNA methylation in the tx-j mouse (tx-j), an animal model of WD. Findings were compared to those in control C3H mice, and in response to Cu chelation by penicillamine (PCA) and dietary supplementation of the methyl donor betaine to modulate inflammatory and methylation status. Transcript levels of selected genes related to endoplasmic reticulum stress, lipid synthesis, and fatty acid oxidation were down-regulated at baseline in tx-j mice, further down-regulated in response to PCA, and showed little to no response to betaine. Hepatic Sahh transcript and protein levels were reduced in tx-j mice with consequent increase of SAH levels. Hepatic Cu accumulation was associated with inflammation, as indicated by histopathology and elevated serum ALT and liver tumor necrosis factor alpha (Tnf-α) levels. Dnmt3b was down-regulated in tx-j mice together with global DNA hypomethylation. PCA treatment of tx-j mice reduced Tnf-α and ALT levels, betaine treatment increased S-adenosylmethionine and up-regulated Dnmt3b levels, and both treatments restored global DNA methylation levels. Conclusion: reduced hepatic Sahh expression was associated with increased liver SAH levels in the tx-j model of WD, with consequent global DNA hypomethylation. Increased global DNA methylation was achieved by reducing inflammation by Cu chelation or by providing methyl groups. We propose that increased SAH levels and inflammation affect widespread epigenetic regulation of gene expression in WD. PMID:22945834

  17. Wilson's disease: changes in methionine metabolism and inflammation affect global DNA methylation in early liver disease.

    PubMed

    Medici, Valentina; Shibata, Noreene M; Kharbanda, Kusum K; LaSalle, Janine M; Woods, Rima; Liu, Sarah; Engelberg, Jesse A; Devaraj, Sridevi; Török, Natalie J; Jiang, Joy X; Havel, Peter J; Lönnerdal, Bo; Kim, Kyoungmi; Halsted, Charles H

    2013-02-01

    Hepatic methionine metabolism may play an essential role in regulating methylation status and liver injury in Wilson's disease (WD) through the inhibition of S-adenosylhomocysteine hydrolase (SAHH) by copper (Cu) and the consequent accumulation of S-adenosylhomocysteine (SAH). We studied the transcript levels of selected genes related to liver injury, levels of SAHH, SAH, DNA methyltransferases genes (Dnmt1, Dnmt3a, Dnmt3b), and global DNA methylation in the tx-j mouse (tx-j), an animal model of WD. Findings were compared to those in control C3H mice, and in response to Cu chelation by penicillamine (PCA) and dietary supplementation of the methyl donor betaine to modulate inflammatory and methylation status. Transcript levels of selected genes related to endoplasmic reticulum stress, lipid synthesis, and fatty acid oxidation were down-regulated at baseline in tx-j mice, further down-regulated in response to PCA, and showed little to no response to betaine. Hepatic Sahh transcript and protein levels were reduced in tx-j mice with consequent increase of SAH levels. Hepatic Cu accumulation was associated with inflammation, as indicated by histopathology and elevated serum alanine aminotransferase (ALT) and liver tumor necrosis factor alpha (Tnf-α) levels. Dnmt3b was down-regulated in tx-j mice together with global DNA hypomethylation. PCA treatment of tx-j mice reduced Tnf-α and ALT levels, betaine treatment increased S-adenosylmethionine and up-regulated Dnmt3b levels, and both treatments restored global DNA methylation levels. Reduced hepatic Sahh expression was associated with increased liver SAH levels in the tx-j model of WD, with consequent global DNA hypomethylation. Increased global DNA methylation was achieved by reducing inflammation by Cu chelation or by providing methyl groups. We propose that increased SAH levels and inflammation affect widespread epigenetic regulation of gene expression in WD. Copyright © 2012 American Association for the Study of

  18. Estrogen and progesterone receptor status affect genome-wide DNA methylation profile in breast cancer.

    PubMed

    Li, Lian; Lee, Kyoung-Mu; Han, Wonshik; Choi, Ji-Yeob; Lee, Ji-Young; Kang, Gyeong Hoon; Park, Sue Kyung; Noh, Dong-Young; Yoo, Keun-Young; Kang, Daehee

    2010-11-01

    DNA methylation is the main epigenetic modification that occurs at the early stages of carcinogenesis. We performed a genome-wide DNA methylation profiling to evaluate whether the DNA methylation state is different in the estrogen receptor (ER) and progesterone receptor (PR) status of breast cancer. Twelve ER+/PR+ and 12 ER-/PR- breast cancer tissues were selected from the biorepository of the Seoul Breast Cancer Study for Infinium Methylation Assay. The difference of the DNA methylation state of 27 578 methylation sites in 14 000 genes between two groups was evaluated by Student's t-test. False discovery rate (FDR) was estimated to evaluate the probability of false positive associations. Of the 27 578 sites, 148 sites (0.54%) were significantly different between ER+/PR+ and ER-/PR- breast cancers (P < 0.001); 93 hypermethylated and 55 hypomethylated. Five genes, FAM124B (P = 7.26 × 10(-7)), MANEAL (P = 3.38 × 10(-7)), ST6GALNAC1 (P = 2.85 × 10(-6)), NAV1 (P = 5.94 × 10(-6)) and PER1 (P = 6.45 × 10(-6)) remained significant after correction for multiple tests (FDR < 0.05). In a subsequent replication study for five genes, four of the five genes were validated; FAM124B and ST6GALNAC1 were significantly hypermethylated, and NAV1 and PER1 were significantly hypomethylated in ER+/PR+ breast cancers (P < 0.05). In the first genome-wide DNA methylation profiling according to the receptor status of breast cancer, we found that ER/PR status affects the DNA methylation state of FAM124B, ST6GALNAC1, NAV1 and PER1 in breast cancer.

  19. Isolating human DNA repair genes using rodent-cell mutants

    SciTech Connect

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

    1987-03-23

    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.

  20. Lineage-Specific Genes Are Prominent DNA Damage Hotspots during Leukemic Transformation of B Cell Precursors.

    PubMed

    Boulianne, Bryant; Robinson, Mark E; May, Philippa C; Castellano, Leandro; Blighe, Kevin; Thomas, Jennifer; Reid, Alistair; Müschen, Markus; Apperley, Jane F; Stebbing, Justin; Feldhahn, Niklas

    2017-02-14

    In human leukemia, lineage-specific genes represent predominant targets of deletion, with lymphoid-specific genes frequently affected in lymphoid leukemia and myeloid-specific genes in myeloid leukemia. To investigate the basis of lineage-specific alterations, we analyzed global DNA damage in primary B cell precursors expressing leukemia-inducing oncogenes by ChIP-seq. We identified more than 1,000 sensitive regions, of which B lineage-specific genes constitute the most prominent targets. Identified hotspots at B lineage genes relate to DNA-DSBs, affect genes that harbor genomic lesions in human leukemia, and associate with ectopic deletion in successfully transformed cells. Furthermore, we show that most identified regions overlap with gene bodies of highly expressed genes and that induction of a myeloid lineage phenotype in transformed B cell precursors promotes de novo DNA damage at myeloid loci. Hence, we demonstrate that lineage-specific transcription predisposes lineage-specific genes in transformed B cell precursors to DNA damage, which is likely to promote the frequent alteration of lineage-specific genes in human leukemia. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. Glutamate racemase from Mycobacterium tuberculosis inhibits DNA gyrase by affecting its DNA-binding

    PubMed Central

    Sengupta, Sugopa; Shah, Meera; Nagaraja, Valakunja

    2006-01-01

    Glutamate racemase (MurI) catalyses the conversion of l-glutamate to d-glutamate, an important component of the bacterial cell wall. MurI from Escherichia coli inhibits DNA gyrase in presence of the peptidoglycan precursor. Amongst the two-glutamate racemases found in Bacillus subtilis, only one inhibits gyrase, in absence of the precursor. Mycobacterium tuberculosis has a single gene encoding glutamate racemase. Action of M.tuberculosis MurI on DNA gyrase activity has been examined and its mode of action elucidated. We demonstrate that mycobacterial MurI inhibits DNA gyrase activity, in addition to its precursor independent racemization function. The inhibition is not species-specific as E.coli gyrase is also inhibited but is enzyme-specific as topoisomerase I activity remains unaltered. The mechanism of inhibition is different from other well-known gyrase inhibitors. MurI binds to GyrA subunit of the enzyme leading to a decrease in DNA-binding of the holoenzyme. The sequestration of the gyrase by MurI results in inhibition of all reactions catalysed by DNA gyrase. MurI is thus not a typical potent inhibitor of DNA gyrase and instead its role could be in modulation of the gyrase activity. PMID:17020913

  2. Yeast DNA sequences initiating gene expression in Escherichia coli.

    PubMed

    Lewin, Astrid; Tran, Thi Tuyen; Jacob, Daniela; Mayer, Martin; Freytag, Barbara; Appel, Bernd

    2004-01-01

    DNA transfer between pro- and eukaryotes occurs either during natural horizontal gene transfer or as a result of the employment of gene technology. We analysed the capacity of DNA sequences from a eukaryotic donor organism (Saccharomyces cerevisiae) to serve as promoter region in a prokaryotic recipient (Escherichia coli) by creating fusions between promoterless luxAB genes from Vibrio harveyi and random DNA sequences from S. cerevisiae and measuring the luminescence of transformed E. coli. Fifty-four out of 100 randomly analysed S. cerevisiae DNA sequences caused considerable gene expression in E. coli. Determination of transcription start sites within six selected yeast sequences in E. coli confirmed the existence of bacterial -10 and -35 consensus sequences at appropriate distances upstream from transcription initiation sites. Our results demonstrate that the probability of transcription of transferred eukaryotic DNA in bacteria is extremely high and does not require the insertion of the transferred DNA behind a promoter of the recipient genome.

  3. DNA-templated semiconductor nanocrystal growth for controlled DNA packing and gene delivery.

    PubMed

    Gao, Li; Ma, Nan

    2012-01-24

    DNA-templated semiconductor nanocrystal (SNC) growth represents a facile means to generate bioactive hybrid nanostructures by directly integrating DNA molecules and luminescent SNCs together via a one-step synthesis, which has been applied to biosensing and cell imaging. In this study we for the first time demonstrated that DNA-templated CdS SNC growth could also be used to rationally tune the structures and activities of large DNA molecules. We explored the synergistic effects of nanocrystal growth on the sizes and charges of DNA molecules and demonstrate that the CdS growth-induced DNA packing could be used as a smart gene delivery system. Herein we used DNA plasmids encoding intact enhanced green fluorescence protein (EGFP) genes as templates to grow CdS SNCs and found that the stepwise growth of CdS nanocrystals can spontaneously induce DNA condensation and negative charge shielding in a synergistic manner. The condensed DNA plasmids exhibited efficient cellular uptake and a relative gene transfection efficiency of 32%. The transfection efficiency can be further doubled in the presence of chloroquine. We elucidated that the gene transfection and expression is controlled by reversible DNA packing, where ligand exchange of DNA with intracellular glutathione molecules plays a critical role in the recovery of DNA plasmids for gene expression. © 2011 American Chemical Society

  4. Plasmid DNA-based gene transfer with ultrasound and microbubbles.

    PubMed

    Taniyama, Yoshiaki; Azuma, Junya; Rakugi, Hiromi; Morishita, Ryuichi

    2011-12-01

    Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common option in the real world because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid DNA-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a Phase III clinical trial did not show sufficient efficiency. Recently, a Phase III clinical trial of hepatocyte growth factor gene therapy in peripheral artery disease (PAD) showed improvement of ischemic ulcers, but it could not salvage limbs from amputation. In addition, a Phase I/II clinical study of fibroblast growth factor gene therapy in PAD extended amputation-free survival, but it seemed to fail in Phase III. In this situation, we and others have developed plasmid DNA-based gene transfer using ultrasound with microbubbles to enhance its efficiency while maintaining safety. Ultrasound-mediated gene transfer has been reported to augment the gene transfer efficiency and select the target organ using cationic microbubble phospholipids which bind negatively charged DNA. Ultrasound with microbubblesis likely to create new therapeutic options inavariety of diseases.

  5. Comprehensive investigation of DNA methylation and gene expression in trisomy 21 placenta.

    PubMed

    Lim, Ji Hyae; Kim, Shin Young; Han, Jung Yeol; Kim, Moon Young; Park, So Yeon; Ryu, Hyun Mee

    2016-06-01

    Trisomy 21 (T21) is the most common aneuploidy affecting humans and is caused by an extra copy of all or part of chromosome 21 (chr21). DNA methylation is an epigenetic event that plays an important role in human diseases via regulation of gene expression. However, the integrative association between DNA methylation and gene expression in T21 fetal placenta has yet to be determined. We profiled expression of 207 genes on chr21 and their DNA methylation patterns in placenta samples from normal and DS fetuses using microarray analysis and predicted the functions of differentially expressed genes using bioinformatics tools. We found 47 genes with significantly increased expression in the T21 placenta compared to the normal placenta. Hypomethylation of the 47 genes was observed in the T21 placenta. Most of hypomethylated DNA positions were intragenic regions, i.e. regions inside a gene. Moreover, gene expression and hypomethylated DNA position showed significantly positive associations. By analyzing the properties of the gene-disease network, we found that increased genes in the T21 placenta were significantly associated with T21 and T21 complications such as mental retardation, neurobehavioral manifestations, and congenital abnormalities. To our knowledge, this is the first study to comprehensively survey the association between gene expression and DNA methylation in chr21 of the T21 fetal placenta. Our findings provide a broad overview of the relationships between gene expression and DNA methylation in the placentas of fetuses with T21 and could contribute to future research efforts concerning genes involvement in disease pathogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Characterization of DNA methyltransferase and demethylase genes in Fragaria vesca.

    PubMed

    Gu, Tingting; Ren, Shuai; Wang, Yuanhua; Han, Yuhui; Li, Yi

    2016-06-01

    DNA methylation is an epigenetic modification essential for gene regulations in plants, but understanding on how it is involved in fruit development, especially in non-climacteric fleshy fruit, is limited. The diploid woodland strawberry (Fragaria vesca) is an important model for non-climacteric fruit crops. In this study, we identified DNA methyltransferase genes and demethylase genes in Fragaria vesca and other angiosperm species. In accordance with previous studies, our phylogenetic analyses of those DNA methylation modifiers support the clustering of those genes into several classes. Our data indicate that whole-genome duplications and tandem duplications contributed to the expansion of those DNA methylation modifiers in angiosperms. We have further demonstrated that some DNA methylase and demethylase genes reach their highest expression levels in strawberry fleshy fruits when turning from white to red, suggesting that DNA methylation might undergo a dramatic change at the onset of fleshy fruit-ripening process. In addition, we have observed that expression of some DNA demethylase genes increases in response to various abiotic stresses including heat, cold, drought and salinity. Collectively, our study indicates a regulatory role of DNA methylation in the turning stage of non-climacteric fleshy fruit and responses to environment stimuli, and would facilitate functional studies of DNA methylation in the growth and development of non-climacteric fruits.

  7. Intranuclear DNA density affects chromosome condensation in metazoans

    PubMed Central

    Hara, Yuki; Iwabuchi, Mari; Ohsumi, Keita; Kimura, Akatsuki

    2013-01-01

    Chromosome condensation is critical for accurate inheritance of genetic information. The degree of condensation, which is reflected in the size of the condensed chromosomes during mitosis, is not constant. It is differentially regulated in embryonic and somatic cells. In addition to the developmentally programmed regulation of chromosome condensation, there may be adaptive regulation based on spatial parameters such as genomic length or cell size. We propose that chromosome condensation is affected by a spatial parameter called the chromosome amount per nuclear space, or “intranuclear DNA density.” Using Caenorhabditis elegans embryos, we show that condensed chromosome sizes vary during early embryogenesis. Of importance, changing DNA content to haploid or polyploid changes the condensed chromosome size, even at the same developmental stage. Condensed chromosome size correlates with interphase nuclear size. Finally, a reduction in nuclear size in a cell-free system from Xenopus laevis eggs resulted in reduced condensed chromosome sizes. These data support the hypothesis that intranuclear DNA density regulates chromosome condensation. This suggests an adaptive mode of chromosome condensation regulation in metazoans. PMID:23783035

  8. Intranuclear DNA density affects chromosome condensation in metazoans.

    PubMed

    Hara, Yuki; Iwabuchi, Mari; Ohsumi, Keita; Kimura, Akatsuki

    2013-08-01

    Chromosome condensation is critical for accurate inheritance of genetic information. The degree of condensation, which is reflected in the size of the condensed chromosomes during mitosis, is not constant. It is differentially regulated in embryonic and somatic cells. In addition to the developmentally programmed regulation of chromosome condensation, there may be adaptive regulation based on spatial parameters such as genomic length or cell size. We propose that chromosome condensation is affected by a spatial parameter called the chromosome amount per nuclear space, or "intranuclear DNA density." Using Caenorhabditis elegans embryos, we show that condensed chromosome sizes vary during early embryogenesis. Of importance, changing DNA content to haploid or polyploid changes the condensed chromosome size, even at the same developmental stage. Condensed chromosome size correlates with interphase nuclear size. Finally, a reduction in nuclear size in a cell-free system from Xenopus laevis eggs resulted in reduced condensed chromosome sizes. These data support the hypothesis that intranuclear DNA density regulates chromosome condensation. This suggests an adaptive mode of chromosome condensation regulation in metazoans.

  9. Mitochondrial DNA Damage and its Consequences for Mitochondrial Gene Expression

    PubMed Central

    Cline, Susan D.

    2012-01-01

    How mitochondria process DNA damage and whether a change in the steady-state level of mitochondrial DNA damage (mtDNA) contributes to mitochondrial dysfunction are questions that fuel burgeoning areas of research into aging and disease pathogenesis. Over the past decade, researchers have identified and measured various forms of endogenous and environmental mtDNA damage and have elucidated mtDNA repair pathways. Interestingly, mitochondria do not appear to contain the full range of DNA repair mechanisms that operate in the nucleus, although mtDNA contains types of damage that are targets of each nuclear DNA repair pathway. The reduced repair capacity may, in part, explain the high mutation frequency of the mitochondrial chromosome. Since mtDNA replication is dependent on transcription, mtDNA damage may alter mitochondrial gene expression at three levels: by causing DNA polymerase γ nucleotide incorporation errors leading to mutations, by interfering with the priming of mtDNA replication by the mitochondrial RNA polymerase, or by inducing transcriptional mutagenesis or premature transcript termination. This review summarizes our current knowledge of mtDNA damage, its repair, and its effects on mtDNA integrity and gene expression. PMID:22728831

  10. Integrative analysis of DNA copy number, DNA methylation and gene expression in multiple myeloma reveals alterations related to relapse

    PubMed Central

    Krzeminski, Patryk; Corchete, Luis A.; García, Juan L.; López-Corral, Lucía; Fermiñán, Encarna; García, Eva M.; Martín, Ana A.; Hernández-Rivas, Jesús M.; García-Sanz, Ramón; Miguel, Jesús F. San; Gutiérrez, Norma C.

    2016-01-01

    Multiple myeloma (MM) remains incurable despite the introduction of novel agents, and a relapsing course is observed in most patients. Although the development of genomic technologies has greatly improved our understanding of MM pathogenesis, the mechanisms underlying relapse have been less thoroughly investigated. In this study, an integrative analysis of DNA copy number, DNA methylation and gene expression was conducted in matched diagnosis and relapse samples from MM patients. Overall, the acquisition of abnormalities at relapse was much more frequent than the loss of lesions present at diagnosis, and DNA losses were significantly more frequent in relapse than in diagnosis samples. Interestingly, copy number abnormalities involving more than 100 Mb of DNA at relapse significantly affect the gene expression of these samples, provoking a particular deregulation of the IL-8 pathway. On the other hand, no significant modifications of gene expression were observed in those samples with less than 100 Mb affected by chromosomal changes. Although several statistical approaches were used to identify genes whose abnormal expression at relapse was regulated by methylation, only two genes that were significantly deregulated in relapse samples (SORL1 and GLT1D1) showed a negative correlation between methylation and expression. Further analysis revealed that DNA methylation was involved in regulating SORL1 expression in MM. Finally, relevant changes in gene expression observed in relapse samples, such us downregulation of CD27 and P2RY8, were most likely not preceded by alterations in the corresponding DNA. Taken together, these results suggest that the genomic heterogeneity described at diagnosis remains at relapse. PMID:27811368

  11. Integrative analysis of DNA copy number, DNA methylation and gene expression in multiple myeloma reveals alterations related to relapse.

    PubMed

    Krzeminski, Patryk; Corchete, Luis A; García, Juan L; López-Corral, Lucía; Fermiñán, Encarna; García, Eva M; Martín, Ana A; Hernández-Rivas, Jesús M; García-Sanz, Ramón; San Miguel, Jesús F; Gutiérrez, Norma C

    2016-12-06

    Multiple myeloma (MM) remains incurable despite the introduction of novel agents, and a relapsing course is observed in most patients. Although the development of genomic technologies has greatly improved our understanding of MM pathogenesis, the mechanisms underlying relapse have been less thoroughly investigated. In this study, an integrative analysis of DNA copy number, DNA methylation and gene expression was conducted in matched diagnosis and relapse samples from MM patients. Overall, the acquisition of abnormalities at relapse was much more frequent than the loss of lesions present at diagnosis, and DNA losses were significantly more frequent in relapse than in diagnosis samples. Interestingly, copy number abnormalities involving more than 100 Mb of DNA at relapse significantly affect the gene expression of these samples, provoking a particular deregulation of the IL-8 pathway. On the other hand, no significant modifications of gene expression were observed in those samples with less than 100 Mb affected by chromosomal changes. Although several statistical approaches were used to identify genes whose abnormal expression at relapse was regulated by methylation, only two genes that were significantly deregulated in relapse samples (SORL1 and GLT1D1) showed a negative correlation between methylation and expression. Further analysis revealed that DNA methylation was involved in regulating SORL1 expression in MM. Finally, relevant changes in gene expression observed in relapse samples, such us downregulation of CD27 and P2RY8, were most likely not preceded by alterations in the corresponding DNA. Taken together, these results suggest that the genomic heterogeneity described at diagnosis remains at relapse.

  12. The novel quinolone CHM-1 induces DNA damage and inhibits DNA repair gene expressions in a human osterogenic sarcoma cell line.

    PubMed

    Chen, Hung-Yi; Lu, Hsu-Feng; Yang, Jai-Sing; Kuo, Sheng-Chu; Lo, Chyi; Yang, Mei-Due; Chiu, Tsan-Hung; Chueh, Fu-Shin; Ho, Heng-Chien; Ko, Yang-Ching; Chung, Jing-Gung

    2010-10-01

    20-Fluoro-6,7-methylenedioxy-2-phenyl-4-quino-lone (CHM-1) has been reported to induce cell cycle arrest and apoptosis in many types of cancer cells. However, there is no available information to show CHM-1 affecting DNA damage and expression of associated repair genes. Herein, we investigated whether or not CHM-1 induced DNA damage and affected DNA repair gene expression in U-2 OS human osterogenic sarcoma cells. The comet assay showed that incubation of U-2 OS cells with 0, 0.75, 1.5, 3 and 6 μM of CHM-1 led to a longer DNA migration smear (comet tail). DNA gel electrophoresis showed that 3 μM of CHM-1 for 24 and 48 h treatment induced DNA fragmentation in U-2 OS cells. Real-time PCR analysis showed that treatment with 3 μM of CHM-1 for 24 h reduced the mRNA expression levels of ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR), breast cancer 1, early onset (BRCA1), 14-3-3sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK) and O(6)-methylguanine-DNA methyltransferase (MGMT) genes in a time-dependent manner. Taken together, the results indicate that CHM-1 caused DNA damage and reduced DNA repair genes in U-2 OS cells, which may be the mechanism for CHM-1-inhibited cell growth and induction of apoptosis.

  13. DNA replication-dependent induction of gene proximity by androgen.

    PubMed

    Coll-Bastus, Nuria; Mao, Xueying; Young, Bryan D; Sheer, Denise; Lu, Yong-Jie

    2015-02-15

    The male hormone androgen, working through the androgen receptor (AR), plays a major role in physiological process and disease development. Previous studies of AR mainly focus on its transcriptional activity. Here, we found that androgen-induced TMPRSS2 and ERG gene proximity is mediated by AR control of DNA replication rather than gene transcription. We demonstrate that, in both AR transactivation-positive and -negative prostate cells, androgen regulates DNA replication and androgen-induced gene proximity relies on both DNA replication-licensing and actual DNA replication activity. Androgen stimulation advances DNA replication timing of certain genomic regions, which may potentially increase gene proximity through sharing the same replication factory at a similar time. Therefore, we have revealed novel mechanisms of AR biological function, which will stimulate new research directions. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  14. Listeria monocytogenes DNA Glycosylase AdlP Affects Flagellar Motility, Biofilm Formation, Virulence, and Stress Responses

    PubMed Central

    Zhang, Ting; Bae, Dongryeoul

    2016-01-01

    ABSTRACT The temperature-dependent alteration of flagellar motility gene expression is critical for the foodborne pathogen Listeria monocytogenes to respond to a changing environment. In this study, a genetic determinant, L. monocytogenes f2365_0220 (lmof2365_0220), encoding a putative protein that is structurally similar to the Bacillus cereus alkyl base DNA glycosylase (AlkD), was identified. This determinant was involved in the transcriptional repression of flagellar motility genes and was named adlP (encoding an AlkD-like protein [AdlP]). Deletion of adlP activated the expression of flagellar motility genes at 37°C and disrupted the temperature-dependent inhibition of L. monocytogenes motility. The adlP null strains demonstrated decreased survival in murine macrophage-like RAW264.7 cells and less virulence in mice. Furthermore, the deletion of adlP significantly decreased biofilm formation and impaired the survival of bacteria under several stress conditions, including the presence of a DNA alkylation compound (methyl methanesulfonate), an oxidative agent (H2O2), and aminoglycoside antibiotics. Our findings strongly suggest that adlP may encode a bifunctional protein that transcriptionally represses the expression of flagellar motility genes and influences stress responses through its DNA glycosylase activity. IMPORTANCE We discovered a novel protein that we named AlkD-like protein (AdlP). This protein affected flagellar motility, biofilm formation, and virulence. Our data suggest that AdlP may be a bifunctional protein that represses flagellar motility genes and influences stress responses through its DNA glycosylase activity. PMID:27316964

  15. The structure and duplex context of DNA interstrand crosslinks affects the activity of DNA polymerase η

    PubMed Central

    Roy, Upasana; Mukherjee, Shivam; Sharma, Anjali; Frank, Ekaterina G.; Schärer, Orlando D.

    2016-01-01

    Several important anti-tumor agents form DNA interstrand crosslinks (ICLs), but their clinical efficiency is counteracted by multiple complex DNA repair pathways. All of these pathways require unhooking of the ICL from one strand of a DNA duplex by nucleases, followed by bypass of the unhooked ICL by translesion synthesis (TLS) polymerases. The structures of the unhooked ICLs remain unknown, yet the position of incisions and processing of the unhooked ICLs significantly influence the efficiency and fidelity of bypass by TLS polymerases. We have synthesized a panel of model unhooked nitrogen mustard ICLs to systematically investigate how the state of an unhooked ICL affects pol η activity. We find that duplex distortion induced by a crosslink plays a crucial role in translesion synthesis, and length of the duplex surrounding an unhooked ICL critically affects polymerase efficiency. We report the synthesis of a putative ICL repair intermediate that mimics the complete processing of an unhooked ICL to a single crosslinked nucleotide, and find that it provides only a minimal obstacle for DNA polymerases. Our results raise the possibility that, depending on the structure and extent of processing of an ICL, its bypass may not absolutely require TLS polymerases. PMID:27257072

  16. Identification of Novel Gene Targets and Functions of p21-Activated Kinase 1 during DNA Damage by Gene Expression Profiling

    PubMed Central

    Motwani, Mona; Li, Da-Qiang; Horvath, Anelia; Kumar, Rakesh

    2013-01-01

    P21-activated kinase 1 (PAK1), a serine/threonine protein kinase, modulates many cellular processes by phosphorylating its downstream substrates. In addition to its role in the cytoplasm, PAK1 also affects gene transcription due to its nuclear localization and association with chromatin. It is now recognized that PAK1 kinase activity and its nuclear translocation are rapidly stimulated by ionizing radiation (IR), and that PAK1 activation is a component of the DNA damage response. Owing to the role of PAK1 in the cell survival, its association with the chromatin, and now, stimulation by ionizing radiation, we hypothesize that PAK1 may be contributing to modulation of genes with roles in cellular processes that might be important in the DNA damage response. The purpose of this study was to identify new PAK1 targets in response to ionizing radiation with putative role in the DNA damage response. We examined the effect of IR on the gene expression patterns in the murine embryonic fibroblasts with or without Pak1 using microarray technology. Differentially expressed transcripts were identified using Gene Spring GX 10.0.2. Pathway, network, functional analyses and gene family classification were carried out using Kyoto Encyclopedia of Genes and Genomes (KEGG), Ingenuity Pathway, Gene Ontology and PANTHER respectively. Selective targets of PAK1 were validated by RT-qPCR. For the first time, we provide a genome-wide analysis of PAK1 and identify its targets with potential roles in the DNA damage response. Gene Ontology analysis identified genes in the IR-stimulated cells that were involved in cell cycle arrest and cell death. Pathway analysis revealed p53 pathway being most influenced by IR responsive, PAK1 targets. Gene family of transcription factors was over represented and gene networks involved in DNA replication, repair and cellular signaling were identified. In brief, this study identifies novel PAK1 dependent IR responsive genes which reveal new aspects of PAK1

  17. NADH dehydrogenase subunit genes in the mitochondrial DNA of yeasts.

    PubMed Central

    Nosek, J; Fukuhara, H

    1994-01-01

    The genes encoding the NADH dehydrogenase subunits of respiratory complex I have not been identified so far in the mitochondrial DNA (mtDNA) of yeasts. In the linear mtDNA of Candida parapsilosis, we found six new open reading frames whose sequences were unambiguously homologous to those of the genes known to code for NADH dehydrogenase subunit proteins of different organisms, i.e., ND1, ND2, ND3, ND4L, ND5, and ND6. The gene for ND4 also appears to be present, as judged from hybridization experiments with a Podospora gene probe. Specific transcripts from these open reading frames (ND genes) could be detected in the mitochondria. Hybridization experiments using C. parapsilosis genes as probes suggested that ND genes are present in the mtDNAs of a wide range of yeast species including Candida catenulata, Pichia guilliermondii, Clavispora lusitaniae, Debaryomyces hansenii, Hansenula polymorpha, and others. Images PMID:7521869

  18. Two affected siblings with nuclear cataract associated with a novel missense mutation in the CRYGD gene.

    PubMed

    Messina-Baas, Olga Maud; Gonzalez-Huerta, Luz Maria; Cuevas-Covarrubias, Sergio Alberto

    2006-08-24

    To identify the disease locus for nuclear congenital cataract in a nonconsanguineous family with two affected members. One family with two affected members with congenital cataract and 170 normal controls were examined. DNA from leukocytes and bucal swabs was isolated to analyze the CRYGA-D cluster genes and microsatellite markers D2S325, D2S2382, and D2S126, and to discard paternity through gene scan with several highly polymorphic markers. DNA sequencing analysis of the CRYGA-D cluster genes of the two affected members showed a novel heterozygous missense mutation c.320A > C within exon 3 of the CRYGD gene. This transversion mutation resulted in the substitution of glutamic acid 107 by an alanine (E107A). Analysis of the two unaffected members of the family and the normal parents showed a normal sequence of the CRYGA-D cluster genes. This mutation was not found in a group of 170 unrelated controls. We consider that it is unlikely that this abnormal allele represents a rare polymorphism. DNA analysis showed no evidence for non-paternity while genotyping indicated that the haplotype of the mother co-segregated with the disease. In this study we describe the mutation c.320A > C (E107A) in the CRYGD gene associated with nuclear congenital cataract. Haplotype analysis strongly suggests that the origin of the mutation was transmitted through the mother.

  19. Functional cooperation of the dnaE and dnaN gene products in Escherichia coli.

    PubMed Central

    Kuwabara, N; Uchida, H

    1981-01-01

    A system was designed to isolate second-site intergenic suppressors of a thermosensitive mutation of the dnaE gene of Escherichia coli. The dnaE gene codes for the alpha subunit of DNA polymerase III [McHenry, C. S. & Crow, W. (1979) J. Biol. Chem. 254, 1748-1753]. One such suppressor, named sueA77, was finely mapped and found to be located at 82 min on the E. coli chromosome, between dnaA and recF, and within the dnaN gene [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553]. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme [Burgers, P. M. J., Kornberg, A. & Sakakibara, Y. (1981) Proc. Natl. Acad. Sci. USA 78, 5391-5395]. The sueA77 mutation was trans-dominant over its wild-type allele, and it suppressed different thermosensitive mutations of dnaE with different maximal permissive temperature. These properties were interpreted as providing genetic evidence for interaction of the dnaE and dnaN gene products in E. coli. Images PMID:6458043

  20. Functional cooperation of the dnaE and dnaN gene products in Escherichia coli.

    PubMed

    Kuwabara, N; Uchida, H

    1981-09-01

    A system was designed to isolate second-site intergenic suppressors of a thermosensitive mutation of the dnaE gene of Escherichia coli. The dnaE gene codes for the alpha subunit of DNA polymerase III [McHenry, C. S. & Crow, W. (1979) J. Biol. Chem. 254, 1748-1753]. One such suppressor, named sueA77, was finely mapped and found to be located at 82 min on the E. coli chromosome, between dnaA and recF, and within the dnaN gene [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553]. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme [Burgers, P. M. J., Kornberg, A. & Sakakibara, Y. (1981) Proc. Natl. Acad. Sci. USA 78, 5391-5395]. The sueA77 mutation was trans-dominant over its wild-type allele, and it suppressed different thermosensitive mutations of dnaE with different maximal permissive temperature. These properties were interpreted as providing genetic evidence for interaction of the dnaE and dnaN gene products in E. coli.

  1. DNA Methylation is Developmentally Regulated for Genes Essential for Cardiogenesis

    PubMed Central

    Chamberlain, Alyssa A.; Lin, Mingyan; Lister, Rolanda L.; Maslov, Alex A.; Wang, Yidong; Suzuki, Masako; Wu, Bingruo; Greally, John M.; Zheng, Deyou; Zhou, Bin

    2014-01-01

    Background DNA methylation is a major epigenetic mechanism altering gene expression in development and disease. However, its role in the regulation of gene expression during heart development is incompletely understood. The aim of this study is to reveal DNA methylation in mouse embryonic hearts and its role in regulating gene expression during heart development. Methods and Results We performed the genome‐wide DNA methylation profiling of mouse embryonic hearts using methyl‐sensitive, tiny fragment enrichment/massively parallel sequencing to determine methylation levels at ACGT sites. The results showed that while global methylation of 1.64 million ACGT sites in developing hearts remains stable between embryonic day (E) 11.5 and E14.5, a small fraction (2901) of them exhibit differential methylation. Gene Ontology analysis revealed that these sites are enriched at genes involved in heart development. Quantitative real‐time PCR analysis of 350 genes with differential DNA methylation showed that the expression of 181 genes is developmentally regulated, and 79 genes have correlative changes between methylation and expression, including hyaluronan synthase 2 (Has2). Required for heart valve formation, Has2 expression in the developing heart valves is downregulated at E14.5, accompanied with increased DNA methylation in its enhancer. Genetic knockout further showed that the downregulation of Has2 expression is dependent on DNA methyltransferase 3b, which is co‐expressed with Has2 in the forming heart valve region, indicating that the DNA methylation change may contribute to the Has2 enhancer's regulating function. Conclusions DNA methylation is developmentally regulated for genes essential to heart development, and abnormal DNA methylation may contribute to congenital heart disease. PMID:24947998

  2. Mutagens manufactured in fungal culture may affect DNA/RNA of producing fungi.

    PubMed

    Paterson, R R M; Lima, N

    2009-04-01

    Self-produced mutagens in culture by fungi may affect DNA analysis of the same fungi. This has not been considered previously. Many fungi produce numerous mutagenic secondary metabolites (SM) in culture. There is a paradox of growing fungi in media to produce representative DNA which also support mutagenic SM. This is a crucial issue in developing diagnostic and phylogenetic methods, especially for closely-related fungi. For example, idh gene analysis of the patulin metabolic pathway in fungi can be interpreted as producing some false negative and positive results in terms of possession, or nonpossession, of the gene from mutated strains. The most obvious mycotoxins and fungi to consider in this regard are aflatoxins and Aspergillus, as aflatoxins are the most mutagenic natural compounds. Many other fungi and SM are relevant. Conditions to grow fungi have not been selected to inhibit SM production although relevant data exist. In fact, fungi repair damaged nucleic acid (NA) and are capable of removing toxins by employing transporter proteins. These and NA repair mechanisms could be inhibited by secondary metabolites. Mutagenic effects may involve inhibition of DNA stabilizing enzymes. There may be an equivalent situation for bacteria. Researchers need to devise methods to reduce SM for valid protocols. More work on how mutagens affect the NA of producing fungus in vitro is required. The current review assesses the potential seriousness of the situation with selected papers.

  3. Predicting preferential DNA vector insertion sites: implications for functional genomics and gene therapy

    PubMed Central

    Hackett, Christopher S; Geurts, Aron M; Hackett, Perry B

    2007-01-01

    Viral and transposon vectors have been employed in gene therapy as well as functional genomics studies. However, the goals of gene therapy and functional genomics are entirely different; gene therapists hope to avoid altering endogenous gene expression (especially the activation of oncogenes), whereas geneticists do want to alter expression of chromosomal genes. The odds of either outcome depend on a vector's preference to integrate into genes or control regions, and these preferences vary between vectors. Here we discuss the relative strengths of DNA vectors over viral vectors, and review methods to overcome barriers to delivery inherent to DNA vectors. We also review the tendencies of several classes of retroviral and transposon vectors to target DNA sequences, genes, and genetic elements with respect to the balance between insertion preferences and oncogenic selection. Theoretically, knowing the variables that affect integration for various vectors will allow researchers to choose the vector with the most utility for their specific purposes. The three principle benefits from elucidating factors that affect preferences in integration are as follows: in gene therapy, it allows assessment of the overall risks for activating an oncogene or inactivating a tumor suppressor gene that could lead to severe adverse effects years after treatment; in genomic studies, it allows one to discern random from selected integration events; and in gene therapy as well as functional genomics, it facilitates design of vectors that are better targeted to specific sequences, which would be a significant advance in the art of transgenesis. PMID:18047689

  4. Genome-wide gene expression and DNA methylation differences in abnormally cloned and normally natural mating piglets.

    PubMed

    Zou, C; Fu, Y; Li, C; Liu, H; Li, G; Li, J; Zhang, H; Wu, Y; Li, C

    2016-08-01

    Many studies have proved that DNA methylation can regulate gene expression and further affect skeletal muscle growth and development of pig, whereas the mechanisms of how DNA methylation or gene expression alteration ultimately lead to phenotypical differences between the cloned and natural mating pigs remain elusive. This study aimed to investigate genome-wide gene expression and DNA methylation differences between abnormally cloned and normally natural mating piglets and identify molecular markers related to skeletal muscle growth and development in pig. The DNA methylation and genome-wide gene expression in the two groups of piglets were analysed through methylated DNA immunoprecipitation binding high-throughput sequencing and RNA sequencing respectively. We detected 1493 differentially expressed genes between the two groups, of which 382 genes were also differentially methylated. The results of the integrative analysis between DNA methylation and gene expression revealed that the DNA methylation levels showed a significantly negative and monotonic correlation with gene expression levels around the transcription start site of genes. By contrast, no notable monotonic correlation was observed in other regions. Furthermore, we identified some interesting genes and signalling pathways (e.g. myosin, heavy chain 7 and mammalian target of rapamycin) which possibly play essential roles in skeletal muscle growth and development. The results of this study provide insights into the relationship of DNA methylation with gene expression in newborn piglets and into the mechanisms in abnormally cloned animals through somatic cell nuclear transfer.

  5. Monitoring of dnaK gene expression in Porphyromonas gingivalis by oxygen stress using DNA microarray.

    PubMed

    Araki, Makoto; Hiratsuka, Koichi; Kiyama-Kishikawa, Michiko; Abiko, Yoshimitsu

    2004-06-01

    Porphyromonas gingivalis, a Gram-negative anaerobe associated with adult periodontitis, expresses numerous potential virulence factors. dnaK, a member of the heat shock protein family, functions as a molecular chaperone and plays a role in microbial pathogenicity. However, little is known regarding its gene expression caused by oxygen stress in P. gingivalis. In the present study, a custom-made DNA microarray was designed and used to monitor dnaK gene expression in P. gingivalis caused by oxygen stress. The results demonstrated that dnaK mRNA was up-regulated in a short time, and the DNA microarray results were confirmed by real-time polymerase chain reaction analysis. These findings suggest that oxygen stress stimulates gene expression of dnaK and may have a relationship to the aerotolerance activity of this organism as well as its expression of pathogenesis.

  6. DNA affects the composition of lipoplex protein corona: a proteomics approach.

    PubMed

    Capriotti, Anna L; Caracciolo, Giulio; Caruso, Giuseppe; Foglia, Patrizia; Pozzi, Daniela; Samperi, Roberto; Laganà, Aldo

    2011-08-01

    The distribution of drug delivery systems into the body is affected by plasma proteins adsorbed onto their surface. Furthermore, an exact understanding of the structure and morphology of drug carriers is fundamental to understand their role as gene delivery systems. In this work, the adsorption of human plasma proteins bound to cationic liposomes and to their relative DNA lipoplexes was compared. A shotgun proteomics approach based on HPLC coupled to high resolution MS was used for an efficient identification of proteins adsorbed onto liposome and lipoplex surfaces. The distinct pattern of proteins adsorbed helps to better understand the DNA compaction process. The experimental evidence leads us to hypothesize that polyanionic DNA is associated to the lipoplex surface and can interact with basic plasma proteins. Such a finding is in agreement with recent results showing that lipoplexes are multilamellar DNA/lipid domains partially decorated with DNA at their surface. Proteomics experiments showed that the lipoplex corona is rich of biologically relevant proteins such as fibronectin, histones and complement proteins. Our results provide novel insights to understand how lipoplexes activate the immune system and why they are rapidly cleared from the blood stream. The differences in the protein adsorption data detected in the presented experiments could be the basis for the establishment of a correlation between protein adsorption pattern and in vivo fate of intravenously administered nanoparticles and will require some consideration in the future.

  7. Ternary nanoparticles composed of cationic solid lipid nanoparticles, protamine, and DNA for gene delivery

    PubMed Central

    He, Sai-Nan; Li, Yun-Long; Yan, Jing-Jing; Zhang, Wei; Du, Yong-Zhong; Yu, He-Yong; Hu, Fu-Qiang; Yuan, Hong

    2013-01-01

    Background The objective of this research was to design an effective gene delivery system composed of cationic solid lipid nanoparticles (SLNs), protamine, and Deoxyribonucleic acid DNA. Methods Cationic SLNs were prepared using an aqueous solvent diffusion method with octadecylamine as the cationic lipid material. First, protamine was combined with DNA to form binary protamine/DNA nanoparticles, and the ternary nanoparticle gene delivery system was then obtained by combining binary protamine/DNA nanoparticles with cationic SLNs. The size, zeta potential, and ability of the binary and ternary nanoparticles to compact and protect DNA were characterized. The effect of octadecylamine content in SLNs and the SLNS/DNA ratios on transfection efficiency, cellular uptake and cytotoxicity of the ternary nanoparticles were also assessed using HEK293 cells. Results When the weight ratio of protamine to DNA reached 1.5:1, the plasmid DNA could be effectively compacted and protected. The average hydrodynamic diameter of the ternary nanoparticles when combined with protamine increased from 188.50 ± 0.26 nm to 259.33 ± 3.44 nm, and the zeta potential increased from 25.50 ± 3.30 mV to 33.40 ± 2.80 mV when the weight ratio of SLNs to DNA increased from 16/3 to 80/3. The ternary nanoparticles showed high gene transfection efficiency compared with Lipofectamine™ 2000/DNA nanoparticles. Several factors that might affect gene transfection efficiency, such as content and composition of SLNs, post-transfection time, and serum were examined. The ternary nanoparticles composed of SLNs with 15 wt% octadecylamine (50/3 weight ratio of SLNs to DNA) showed the best transfection efficiency (26.13% ± 5.22%) in the presence of serum. It was also found that cellular uptake of the ternary nanoparticles was better than that of the SLN/DNA and binary protamine/DNA nanoparticle systems, and DNA could be transported to the nucleus. Conclusion SLNs enhanced entry of binary protamine/DNA

  8. Modelling Robust Feedback Control Mechanisms That Ensure Reliable Coordination of Histone Gene Expression with DNA Replication

    PubMed Central

    Corrigall, Holly; Ebenhöh, Oliver; Müller, Berndt

    2016-01-01

    Histone proteins are key elements in the packing of eukaryotic DNA into chromosomes. A little understood control system ensures that histone gene expression is balanced with DNA replication so that histone proteins are produced in appropriate amounts. Disturbing or disrupting this system affects genome stability and gene expression, and has detrimental consequences for human development and health. It has been proposed that feedback control involving histone proteins contributes to this regulation and there is evidence implicating cell cycle checkpoint molecules activated when DNA synthesis is impaired in this control. We have developed mathematical models that incorporate these control modes in the form of inhibitory feedback of histone gene expression from free histone proteins, and alternatively a direct link that couples histone RNA synthesis to DNA synthesis. Using our experimental evidence and related published data we provide a simplified description of histone protein synthesis during S phase. Both models reproduce the coordination of histone gene expression with DNA replication during S phase and the down-regulation of histone RNA when DNA synthesis is interrupted, but only the model incorporating histone protein feedback control was able to effectively simulate the coordinate expression of a simplified histone gene family. Our combined theoretical and experimental approach supports the hypothesis that the regulation of histone gene expression involves feedback control. PMID:27798685

  9. [Analysis of AVPR2 gene mutation in a pedigree affected with congenital nephrogenic diabetes insipidus].

    PubMed

    Dai, Zhijuan; Ruan, Luya; Jin, Jian; Qian, Yanying; Wang, Liang; Shi, Zhen; Wu, Chaoming

    2016-10-01

    To detect potential mutation in a pedigree affected with congenital nephrogenic diabetes insipidus (NDI). Clinical data of a male patient affected with NDI was collected. Genomic DNA was extracted from peripheral blood samples from the patient and five family members. The whole coding region of the arginine vasopressin receptor 2 (AVPR2) gene was amplified by PCR and directly sequenced. The patient presented polyuria and polydipsia postnatally. Computerized tomography revealed bilateral hydronephrosis and hydroureter. The patient was responsive to hydrochlorothiazide but not to desmopressin. DNA analysis identified a hemizygous missence mutation c.295 T>C in exon 2 of the AVPR2 gene in the proband. His mother and grandmother were both heterozygous for the same mutation. The congenital NDI in the patient was probably due to mutation of the AVPR2 gene.

  10. Genes with stable DNA methylation levels show higher evolutionary conservation than genes with fluctuant DNA methylation levels.

    PubMed

    Zhang, Ruijie; Lv, Wenhua; Luan, Meiwei; Zheng, Jiajia; Shi, Miao; Zhu, Hongjie; Li, Jin; Lv, Hongchao; Zhang, Mingming; Shang, Zhenwei; Duan, Lian; Jiang, Yongshuai

    2015-11-24

    Different human genes often exhibit different degrees of stability in their DNA methylation levels between tissues, samples or cell types. This may be related to the evolution of human genome. Thus, we compared the evolutionary conservation between two types of genes: genes with stable DNA methylation levels (SM genes) and genes with fluctuant DNA methylation levels (FM genes). For long-term evolutionary characteristics between species, we compared the percentage of the orthologous genes, evolutionary rate dn/ds and protein sequence identity. We found that the SM genes had greater percentages of the orthologous genes, lower dn/ds, and higher protein sequence identities in all the 21 species. These results indicated that the SM genes were more evolutionarily conserved than the FM genes. For short-term evolutionary characteristics among human populations, we compared the single nucleotide polymorphism (SNP) density, and the linkage disequilibrium (LD) degree in HapMap populations and 1000 genomes project populations. We observed that the SM genes had lower SNP densities, and higher degrees of LD in all the 11 HapMap populations and 13 1000 genomes project populations. These results mean that the SM genes had more stable chromosome genetic structures, and were more conserved than the FM genes.

  11. Visually Relating Gene Expression and in vivo DNA Binding Data

    SciTech Connect

    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.

  12. Higher plant mitochondrial DNA: Genomes, genes, mutants, transcription, translation

    SciTech Connect

    Not Available

    1986-01-01

    This volume contains brief summaries of 63 presentations given at the International Workshop on Higher Plant Mitochondrial DNA. The presentations are organized into topical discussions addressing plant genomes, mitochondrial genes, cytoplasmic male sterility, transcription, translation, plasmids and tissue culture. (DT)

  13. Hyperglycemia Differentially Affects Maternal and Fetal DNA Integrity and DNA Damage Response

    PubMed Central

    Moreli, Jusciele B.; Santos, Janine H.; Lorenzon-Ojea, Aline Rodrigues; Corrêa-Silva, Simone; Fortunato, Rodrigo S.; Rocha, Clarissa Ribeiro; Rudge, Marilza V.; Damasceno, Débora C.; Bevilacqua, Estela; Calderon, Iracema M.

    2016-01-01

    Objective: Investigate the DNA damage and its cellular response in blood samples from both mother and the umbilical cord of pregnancies complicated by hyperglycemia. Methods: A total of 144 subjects were divided into 4 groups: normoglycemia (ND; 46 cases), mild gestational hyperglycemia (MGH; 30 cases), gestational diabetes mellitus (GDM; 45 cases) and type-2 diabetes mellitus (DM2; 23 cases). Peripheral blood mononuclear cell (PBMC) isolation and/or leukocytes from whole maternal and umbilical cord blood were obtained from all groups at delivery. Nuclear and mitochondrial DNA damage were measured by gene-specific quantitative PCR, and the expression of mRNA and proteins involved in the base excision repair (BER) pathway were assessed by real-time qPCR and Western blot, respectively. Apoptosis was measured in vitro experiments by caspase 3/7 activity and ATP levels. Results: GDM and DM2 groups were characterized by an increase in oxidative stress biomarkers, an increase in nuclear and mitochondrial DNA damage, and decreased expression of mRNA (APE1, POLβ and FEN1) and proteins (hOGG1, APE1) involved in BER. The levels of hyperglycemia were associated with the in vitro apoptosis pathway. Blood levels of DNA damage in umbilical cord were similar among the groups. Newborns of diabetic mothers had increased expression of BER mRNA (APE1, POLβ and FEN1) and proteins (hOGG1, APE1, POLβ and FEN1). A diabetes-like environment was unable to induce apoptosis in the umbilical cord blood cells. Conclusions: Our data show relevant asymmetry between maternal and fetal blood cell susceptibility to DNA damage and apoptosis induction. Maternal cells seem to be more predisposed to changes in an adverse glucose environment. This may be due to differential ability in upregulating multiple genes involved in the activation of DNA repair response, especially the BER mechanism. However if this study shows a more effective adaptive response by the fetal organism, it also calls for

  14. [Scientific ethics of gene therapy for individuals. The urgency for DNA gene surgery].

    PubMed

    Valenzuela, Carlos Y

    2003-10-01

    Gene therapy for individuals is mainly directed to somatic or germ cells. The present technology aims to insert a DNA segment in the recipient cells. This therapy is useful in Mendelian recessive diseases. There is an ethical moratorium to perform insertion gene therapy in germ cells, because this procedure increases the human genome. Somatic cell gene therapy cures individuals but increases the gene frequency of genetic diseases in the population. This occurs because the descendants of the cured patient should carry his or her "ill" genes. We denots by "DNA gene surgery" the procedure that replaces "ill" nucleotide(s) by healthy one(s) conserving the genome size and the gene context of expression and regulation. Several procedures for gene surgery have been applied to cells and animals. Those based on DNA repair as Chimeric RNA/DNA, one stranded oligonucleotides and tristranded DNA. Those based on DNA recombination with oligo DNA or one stranded DNA, and transposable DNA segments. Gene surgery can be applied to germ cell gene therapy without ethical contraindications. It can cure Mendelian dominant diseases and it can be applied to heterozygotes. It preserves the regulation and expression gene context. If a technical safe procedure is available, the entire mankind could be treated and cured of all the Mendelian diseases, in one generation. Susceptibilities for all diseases could also be treated. The moratorium for research on germ cell gene therapy by gene surgery should be interrupted. Safe gene surgery is a moral imperative for gene therapy of patients and their descendants, for the treatment of dominant genetic diseases and for heterozygous carriers of recessive disorders.

  15. DNA methylation and gene expression in Mimulus guttatus.

    PubMed

    Colicchio, Jack M; Miura, Fumihito; Kelly, John K; Ito, Takashi; Hileman, Lena C

    2015-07-07

    The presence of methyl groups on cytosine nucleotides across an organism's genome (methylation) is a major regulator of genome stability, crossing over, and gene regulation. The capacity for DNA methylation to be altered by environmental conditions, and potentially passed between generations, makes it a prime candidate for transgenerational epigenetic inheritance. Here we conduct the first analysis of the Mimulus guttatus methylome, with a focus on the relationship between DNA methylation and gene expression. We present a whole genome methylome for the inbred line Iron Mountain 62 (IM62). DNA methylation varies across chromosomes, genomic regions, and genes. We develop a model that predicts gene expression based on DNA methylation (R(2) = 0.2). Post hoc analysis of this model confirms prior relationships, and identifies novel relationships between methylation and gene expression. Additionally, we find that DNA methylation is significantly depleted near gene transcriptional start sites, which may explain the recently discovered elevated rate of recombination in these same regions. The establishment here of a reference methylome will be a useful resource for the continued advancement of M. guttatus as a model system. Using a model-based approach, we demonstrate that methylation patterns are an important predictor of variation in gene expression. This model provides a novel approach for differential methylation analysis that generates distinct and testable hypotheses regarding gene expression.

  16. Global and gene specific DNA methylation changes during zebrafish development

    USDA-ARS?s Scientific Manuscript database

    DNA methylation is dynamic through the life of an organism. In this study, we measured the global and gene specific DNA methylation changes in zebrafish at different developmental stages. We found that the methylation percentage of cytosines was 11.75 ± 0.96% in 3.3 hour post fertilization (hpf) zeb...

  17. Methods of Combinatorial Optimization to Reveal Factors Affecting Gene Length

    PubMed Central

    Bolshoy, Alexander; Tatarinova, Tatiana

    2012-01-01

    In this paper we present a novel method for genome ranking according to gene lengths. The main outcomes described in this paper are the following: the formulation of the genome ranking problem, presentation of relevant approaches to solve it, and the demonstration of preliminary results from prokaryotic genomes ordering. Using a subset of prokaryotic genomes, we attempted to uncover factors affecting gene length. We have demonstrated that hyperthermophilic species have shorter genes as compared with mesophilic organisms, which probably means that environmental factors affect gene length. Moreover, these preliminary results show that environmental factors group together in ranking evolutionary distant species. PMID:23300345

  18. Methods of combinatorial optimization to reveal factors affecting gene length.

    PubMed

    Bolshoy, Alexander; Tatarinova, Tatiana

    2012-01-01

    In this paper we present a novel method for genome ranking according to gene lengths. The main outcomes described in this paper are the following: the formulation of the genome ranking problem, presentation of relevant approaches to solve it, and the demonstration of preliminary results from prokaryotic genomes ordering. Using a subset of prokaryotic genomes, we attempted to uncover factors affecting gene length. We have demonstrated that hyperthermophilic species have shorter genes as compared with mesophilic organisms, which probably means that environmental factors affect gene length. Moreover, these preliminary results show that environmental factors group together in ranking evolutionary distant species.

  19. Plasmid DNA gene therapy by electroporation: principles and recent advances.

    PubMed

    Murakami, Tatsufumi; Sunada, Yoshihide

    2011-12-01

    Simple plasmid DNA injection is a safe and feasible gene transfer method, but it confers low transfection efficiency and transgene expression. This non-viral gene transfer method is enhanced by physical delivery methods, such as electroporation and the use of a gene gun. In vivo electroporation has been rapidly developed over the last two decades to deliver DNA to various tissues or organs. It is generally considered that membrane permeabilization and DNA electrophoresis play important roles in electro-gene transfer. Skeletal muscle is a well characterized target tissue for electroporation, because it is accessible and allows for long-lasting gene expression ( > one year). Skin is also a target tissue because of its accessibility and immunogenicity. Numerous studies have been performed using in vivo electroporation in animal models of disease. Clinical trials of DNA vaccines and immunotherapy for cancer treatment using in vivo electroporation have been initiated in patients with melanoma and prostate cancer. Furthermore, electroporation has been applied to DNA vaccines for infectious diseases to enhance immunogenicity, and the relevant clinical trials have been initiated. The gene gun approach is also being applied for the delivery of DNA vaccines against infectious diseases to the skin. Here, we review recent advances in the mechanism of in vivo electroporation, and summarize the findings of recent preclinical and clinical studies using this technology.

  20. Silencing of the pentose phosphate pathway genes influences DNA replication in human fibroblasts.

    PubMed

    Fornalewicz, Karolina; Wieczorek, Aneta; Węgrzyn, Grzegorz; Łyżeń, Robert

    2017-09-05

    Previous reports and our recently published data indicated that some enzymes of glycolysis and the tricarboxylic acid cycle can affect the genome replication process by changing either the efficiency or timing of DNA synthesis in human normal cells. Both these pathways are connected with the pentose phosphate pathway (PPP pathway). The PPP pathway supports cell growth by generating energy and precursors for nucleotides and amino acids. Therefore, we asked if silencing of genes coding for enzymes involved in the pentose phosphate pathway may also affect the control of DNA replication in human fibroblasts. Particular genes coding for PPP pathway enzymes were partially silenced with specific siRNAs. Such cells remained viable. We found that silencing of the H6PD, PRPS1, RPE genes caused less efficient enterance to the S phase and decrease in efficiency of DNA synthesis. On the other hand, in cells treated with siRNA against G6PD, RBKS and TALDO genes, the fraction of cells entering the S phase was increased. However, only in the case of G6PD and TALDO, the ratio of BrdU incorporation to DNA was significantly changed. The presented results together with our previously published studies illustrate the complexity of the influence of genes coding for central carbon metabolism on the control of DNA replication in human fibroblasts, and indicate which of them are especially important in this process. Copyright © 2017. Published by Elsevier B.V.

  1. Characterization of the DNA polymerase gene of human herpesvirus 6.

    PubMed Central

    Teo, I A; Griffin, B E; Jones, M D

    1991-01-01

    The construction of a recombinant bacteriophage lambda library containing overlapping clones covering 155 kbp of the 161-kbp genome of the Ugandan U1102 isolate of human herpesvirus 6 (HHV-6) is described. The use of degenerate-primer polymerase chain reaction allowed the isolation of a DNA probe for the DNA polymerase gene of HHV-6, which was subsequently used to isolate and position the pol gene on the physical map of the viral genome. A 4.4-kbp EcoRI DNA restriction fragment containing the pol gene was isolated and sequenced. The open reading frames flanking the pol gene code for the HHV-6 glycoprotein B gene and the human cytomegalovirus UL53 homolog. This arrangement is different from that seen in the alpha and gamma herpesvirus families, lending further support to the notion that HHV-6 is a member of the beta herpesvirus group. Images PMID:1651403

  2. Quantitative DNA Methylation Analysis of Candidate Genes in Cervical Cancer

    PubMed Central

    Siegel, Erin M.; Riggs, Bridget M.; Delmas, Amber L.; Koch, Abby; Hakam, Ardeshir; Brown, Kevin D.

    2015-01-01

    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. PMID:25826459

  3. A DNA Bubble-Mediated Gene Regulation System Based on Thrombin-Bound DNA Aptamers.

    PubMed

    Wang, Jing; Yang, Le; Cui, Xun; Zhang, Zhe; Dong, Lichun; Guan, Ningzi

    2017-05-19

    We describe here a novel approach to enhance the transcription of a target gene in cell-free systems by symmetrically introducing duplex aptamers upstream to a T7 promoter in both the sense and antisense strands of double-stranded plasmids, which leads to the formation of a DNA bubble due to the none-complementary state of the ssDNA region harboring the aptamer sequences. With the presence of thrombins, the DNA bubble would be enlarged due to the binding of aptamers with thrombins. Consequently, the recognition region of the promoter contained in the DNA bubble can be more easily recognized and bound by RNA polymerases, and the separation efficiency of the unwinding region can also be significantly improved, leading to the enhanced expression of the target gene at the transcriptional level. The effectiveness of the proposed gene regulation system was demonstrated by enhancing the expression of gfp and ecaA genes in cell-free systems.

  4. DNA-water interactions distinguish messenger RNA genes from transfer RNA genes.

    PubMed

    Khandelwal, Garima; Jayaram, B

    2012-05-30

    Physicochemical properties of DNA sequences as a guide to developing insights into genome organization has received little attention. Here, we utilize the energetics of DNA to further advance the knowledge on its language at a molecular level. Specifically, we ask the question whether physicochemical properties of different functional units on genomes differ. We extract intramolecular and solvation energies of different DNA base pair steps from a comprehensive set of molecular dynamics simulations. We then investigate the solvation behavior of DNA sequences coding for mRNAs and tRNAs. Distinguishing mRNA genes from tRNA genes is a tricky problem in genome annotation without assumptions on length of DNA and secondary structure of the product of transcription. We find that solvation energetics of DNA behaves as an extremely efficient property in discriminating 2,063,537 genes coding for mRNAs from 56,251 genes coding for tRNAs in all (~1500) completely sequenced prokaryotic genomes.

  5. How the gene-patenting race is affecting science

    SciTech Connect

    Wuethrich, B.

    1993-09-04

    Since the National Institutes of Health first filed for patents on thousand fragments of human genes in 1992, many researchers are confronting difficult problems arising at the intersection of science, private enterprise, and the law. At present scientists understand the function of fewer than 1,500 human genes. Decoding all these genes in the goal of the Human Genome Project, sponsored by NIH and DOE. This paper discusses the complex practical, political, ethical, and economic issues involved in describing portions of DNA sequences and the patenting (and ownership) of those sequences.

  6. Effects of folylpolyglutamate synthase modulation on global and gene-specific DNA methylation and gene expression in human colon and breast cancer cells.

    PubMed

    Kim, Sung-Eun; Hinoue, Toshinori; Kim, Michael S; Sohn, Kyoung-Jin; Cho, Robert C; Weisenberger, Daniel J; Laird, Peter W; Kim, Young-In

    2016-03-01

    Folylpolyglutamate synthase (FPGS) plays a critical role in intracellular folate homeostasis. FPGS-induced polyglutamylated folates are better substrates for several enzymes involved in the generation of S-adenosylmethionine, the primary methyl group donor, and hence FPGS modulation may affect DNA methylation. DNA methylation is an important epigenetic determinant in gene expression and aberrant DNA methylation is mechanistically linked cancer development. We investigated whether FPGS modulation would affect global and gene-specific promoter DNA methylation with consequent functional effects on gene expression profiles in HCT116 colon and MDA-MB-435 breast cancer cells. Although FPGS modulation altered global DNA methylation and DNA methyltransferases (DNMT) activity, the effects of FPGS modulation on global DNA methylation and DNMT activity could not be solely explained by intracellular folate concentrations and content of long-chain folylpolyglutamates, and it may be cell-specific. FPGS modulation influenced differential gene expression and promoter cytosine-guanine dinucleotide sequences (CpG) DNA methylation involved in cellular development, cell cycle, cell death and molecular transport. Some of the altered gene expression was associated with promoter CpG DNA methylation changes. In both the FPGS-overexpressed HCT116 and MDA-MB-435 cell lines, we identified several differentially expressed genes involved in folate biosynthesis and one-carbon metabolism, which might in part have contributed to the observed increased efficacy of 5-fluorouracil in response to FPGS overexpression. Our data suggest that FPGS modulation affects global and promoter CpG DNA methylation and expression of several genes involved in important biological pathways. The potential role of FPGS modulation in DNA methylation and its associated downstream functional effects warrants further studies. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Nutri-epigenomic Studies Related to Neural Tube Defects: Does Folate Affect Neural Tube Closure Via Changes in DNA Methylation?

    PubMed

    Rochtus, Anne; Jansen, Katrien; Van Geet, Chris; Freson, Kathleen

    2015-01-01

    Neural tube defects (NTDs), affecting 1-2 per 1000 pregnancies, are severe congenital malformations that arise from the failure of neurulation during early embryonic development. The methylation hypothesis suggests that folate prevents NTDs by stimulating cellular methylation reactions. Folate is central to the one-carbon metabolism that produces pyrimidines and purines for DNA synthesis and for the generation of the methyldonor S-adenosyl-methionine. This review focuses on the relation between the folate-mediated one-carbon metabolism, DNA methylation and NTDs. Studies will be discussed that investigated global or locus-specific DNA methylation differences in patients with NTDs. Folate deficiency may increase NTD risk by decreasing DNA methylation, but to date, human studies vary widely in study design in terms of analyzing different clinical subtypes of NTDs, using different methylation quantification assays and using DNA isolated from diverse types of tissues. Some studies have focused mainly on global DNA methylation differences while others have quantified specific methylation differences for imprinted genes, transposable elements and DNA repair enzymes. Findings of global DNA hypomethylation and LINE-1 hypomethylation suggest that epigenetic alterations may disrupt neural tube closure. However, current research does not support a linear relation between red blood cell folate concentration and DNA methylation. Further studies are required to better understand the interaction between folate, DNA methylation changes and NTDs.

  8. The Slx5-Slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination.

    PubMed

    Burgess, Rebecca C; Rahman, Sadia; Lisby, Michael; Rothstein, Rodney; Zhao, Xiaolan

    2007-09-01

    Recombination is important for repairing DNA lesions, yet it can also lead to genomic rearrangements. This process must be regulated, and recently, sumoylation-mediated mechanisms were found to inhibit Rad51-dependent recombination. Here, we report that the absence of the Slx5-Slx8 complex, a newly identified player in the SUMO (small ubiquitin-like modifier) pathway, led to increased Rad51-dependent and Rad51-independent recombination. The increases were most striking during S phase, suggesting an accumulation of DNA lesions during replication. Consistent with this view, Slx8 protein localized to replication centers. In addition, like SUMO E2 mutants, slx8Delta mutants exhibited clonal lethality, which was due to the overamplification of 2 microm, an extrachromosomal plasmid. Interestingly, in both SUMO E2 and slx8Delta mutants, clonal lethality was rescued by deleting genes required for Rad51-independent recombination but not those involved in Rad51-dependent events. These results suggest that sumoylation negatively regulates Rad51-independent recombination, and indeed, the Slx5-Slx8 complex affected the sumoylation of several enzymes involved in early steps of Rad51-independent recombination. We propose that, during replication, the Slx5-Slx8 complex helps prevent DNA lesions that are acted upon by recombination. In addition, the complex inhibits Rad51-independent recombination via modulating the sumoylation of DNA repair proteins.

  9. DNA methylation differences in monozygotic twin pairs discordant for schizophrenia identifies psychosis related genes and networks.

    PubMed

    Castellani, Christina A; Laufer, Benjamin I; Melka, Melkaye G; Diehl, Eric J; O'Reilly, Richard L; Singh, Shiva M

    2015-05-06

    Despite their singular origin, monozygotic twin pairs often display discordance for complex disorders including schizophrenia. It is a common (1%) and often familial disease with a discordance rate of ~50% in monozygotic twins. This high discordance is often explained by the role of yet unknown environmental, random, and epigenetic factors. The involvement of DNA methylation in this disease appears logical, but remains to be established. We have used blood DNA from two pairs of monozygotic twins discordant for schizophrenia and their parents in order to assess genome-wide methylation using a NimbleGen Methylation Promoter Microarray. The genome-wide results show that differentially methylated regions (DMRs) exist between members representing discordant monozygotic twins. Some DMRs are shared with parent(s) and others appear to be de novo. We found twenty-seven genes affected by DMR changes that were shared in the affected member of two discordant monozygotic pairs from unrelated families. Interestingly, the genes affected by pair specific DMRs share specific networks. Specifically, this study has identified two networks; "cell death and survival" and a "cellular movement and immune cell trafficking". These two networks and the genes affected have been previously implicated in the aetiology of schizophrenia. The results are compatible with the suggestion that DNA methylation may contribute to the discordance of monozygotic twins for schizophrenia. Also, this may be accomplished by the direct effect of gene specific methylation changes on specific biological networks rather than individual genes. It supports the extensive genetic, epigenetic and phenotypic heterogeneity implicated in schizophrenia.

  10. Gene expression profiles in varicose veins using complementary DNA microarray.

    PubMed

    Lee, Seokjong; Lee, Wonchae; Choe, Yoonseok; Kim, Dowon; Na, Gunyeon; Im, Sanguk; Kim, Jinoh; Kim, Moonkyu; Kim, Jungchul; Cho, Joonyong

    2005-04-01

    There has been little information reported about the genetic event concerning the pathophysiology of varicose vein (VV). The purpose of this study was to examine the differentiation of gene expression in the wall of VV using complementary deoxyribonucleic acid (cDNA) microarrays. The study was performed with four pairs of VVs and control veins (CVs). cDNA specimens of VVs were prepared from the ribonucleic acid-isolated VVs of patients who underwent venous obliteration, using radiofrequency, as well as from CVs of those who underwent aortocoronary bypass grafting. Each set of VVs and CVs was hybridized with high-density microarray containing 3,063 human cDNAs. The finding of microarray hybridization were scanned, analyzed, and classified with the cluster program. Among 3,063 cDNA clones, 82 genes were up-regulated in VVs, and some of the up-regulated genes, which were detected by cDNA microarray, including transforming growth factor 3-induced gene (BIGH3), tubulin, lumican, actinin, collagen type I, versican, actin, and tropomyosin, belonged to extracelluar matrix molecules, cytoskeletal proteins, or myofibroblasts. Many up-regulated genes were found in Ws by applying cDNA microarray. These gene profiles suggested a pathway associated with fibrosis and that wound healing might be related to the pathophysiology of VVs.

  11. Spermatozoal cell death-inducing DNA fragmentation factor-α-like effector A (CIDEA) gene expression and DNA fragmentation in infertile men with metabolic syndrome and normal seminogram.

    PubMed

    Elsamanoudy, Ayman Z; Abdalla, Hussein Abdelaziz; Hassanien, Mohammed; Gaballah, Mohammad A

    2016-01-01

    This is the first study to investigate spermatozoal cell death-inducing DNA fragmentation factor-α-like effector A (CIDEA) gene expression and DNA fragmentations in the spermatozoa of men diagnosed with metabolic syndrome (MS) who have normal seminograms with unexplained infertility, and to correlate these parameters with seminal glucose concentration. This study included 120 participants: 75 male subjects with MS (38 fertile and 37 infertile), and a control group of 45 fertile males without MS. HOMA-IR, semen analysis, and biochemical measurement of seminal plasma insulin and glucose levels were carried out. Spermatozoal insulin gene and CIDEA gene expressions were performed by the RT-PCR method. The percentage of spermatozoal DNA fragmentation was also estimated. The spermatozoal insulin and CIDEA gene expression, as well as the DNA fragmentation, were significantly higher in the infertile MS group than in the fertile MS group, and significantly higher in both the MS groups than in the control group. Seminal glucose concentration showed significant positive correlations with seminal insulin level, spermatozoa insulin, CIDEA gene expression, and DNA fragmentation. Moreover, there was a positive correlation between spermatozoa CIDEA gene expression and DNA fragmentation. It can be concluded that MS may affect male fertility at the molecular level, through its possible inducing effect of spermatozoa CIDEA and insulin gene expression, DNA fragmentation, and increased seminal glucose.

  12. Aberrant DNA methylation impacts gene expression and prognosis in breast cancer subtypes.

    PubMed

    Győrffy, Balázs; Bottai, Giulia; Fleischer, Thomas; Munkácsy, Gyöngyi; Budczies, Jan; Paladini, Laura; Børresen-Dale, Anne-Lise; Kristensen, Vessela N; Santarpia, Libero

    2016-01-01

    DNA methylation has a substantial impact on gene expression, affecting the prognosis of breast cancer (BC) patients dependent on molecular subtypes. In this study, we investigated the prognostic relevance of the expression of genes reported as aberrantly methylated, and the link between gene expression and DNA methylation in BC subtypes. The prognostic value of the expression of 144 aberrantly methylated genes was evaluated in ER+/HER2-, HER2+, and ER-/HER2- molecular BC subtypes, in a meta-analysis of two large transcriptomic cohorts of BC patients (n = 1,938 and n = 1,640). The correlation between gene expression and DNA methylation in distinct gene regions was also investigated in an independent dataset of 104 BCs. Survival and Pearson correlation analyses were computed for each gene separately. The expression of 48 genes was significantly associated with BC prognosis (p < 0.05), and 32 of these prognostic genes exhibited a direct expression-methylation correlation. The expression of several immune-related genes, including CD3D and HLA-A, was associated with both relapse-free survival (HR = 0.42, p = 3.5E-06; HR = 0.35, p = 1.7E-08) and overall survival (HR = 0.50, p = 5.5E-04; HR = 0.68, p = 4.5E-02) in ER-/HER2- BCs. On the overall, the distribution of both positive and negative expression-methylation correlation in distinct gene regions have different effects on gene expression and prognosis in BC subtypes. This large-scale meta-analysis allowed the identification of several genes consistently associated with prognosis, whose DNA methylation could represent a promising biomarker for prognostication and clinical stratification of patients with distinct BC subtypes.

  13. CG gene body DNA methylation changes and evolution of duplicated genes in cassava

    PubMed Central

    Wang, Haifeng; Beyene, Getu; Zhai, Jixian; Feng, Suhua; Fahlgren, Noah; Taylor, Nigel J.; Bart, Rebecca; Carrington, James C.; Jacobsen, Steven E.; Ausin, Israel

    2015-01-01

    DNA methylation is important for the regulation of gene expression and the silencing of transposons in plants. Here we present genome-wide methylation patterns at single-base pair resolution for cassava (Manihot esculenta, cultivar TME 7), a crop with a substantial impact in the agriculture of subtropical and tropical regions. On average, DNA methylation levels were higher in all three DNA sequence contexts (CG, CHG, and CHH, where H equals A, T, or C) than those of the most well-studied model plant Arabidopsis thaliana. As in other plants, DNA methylation was found both on transposons and in the transcribed regions (bodies) of many genes. Consistent with these patterns, at least one cassava gene copy of all of the known components of Arabidopsis DNA methylation pathways was identified. Methylation of LTR transposons (GYPSY and COPIA) was found to be unusually high compared with other types of transposons, suggesting that the control of the activity of these two types of transposons may be especially important. Analysis of duplicated gene pairs resulting from whole-genome duplication showed that gene body DNA methylation and gene expression levels have coevolved over short evolutionary time scales, reinforcing the positive relationship between gene body methylation and high levels of gene expression. Duplicated genes with the most divergent gene body methylation and expression patterns were found to have distinct biological functions and may have been under natural or human selection for cassava traits. PMID:26483493

  14. CG gene body DNA methylation changes and evolution of duplicated genes in cassava.

    PubMed

    Wang, Haifeng; Beyene, Getu; Zhai, Jixian; Feng, Suhua; Fahlgren, Noah; Taylor, Nigel J; Bart, Rebecca; Carrington, James C; Jacobsen, Steven E; Ausin, Israel

    2015-11-03

    DNA methylation is important for the regulation of gene expression and the silencing of transposons in plants. Here we present genome-wide methylation patterns at single-base pair resolution for cassava (Manihot esculenta, cultivar TME 7), a crop with a substantial impact in the agriculture of subtropical and tropical regions. On average, DNA methylation levels were higher in all three DNA sequence contexts (CG, CHG, and CHH, where H equals A, T, or C) than those of the most well-studied model plant Arabidopsis thaliana. As in other plants, DNA methylation was found both on transposons and in the transcribed regions (bodies) of many genes. Consistent with these patterns, at least one cassava gene copy of all of the known components of Arabidopsis DNA methylation pathways was identified. Methylation of LTR transposons (GYPSY and COPIA) was found to be unusually high compared with other types of transposons, suggesting that the control of the activity of these two types of transposons may be especially important. Analysis of duplicated gene pairs resulting from whole-genome duplication showed that gene body DNA methylation and gene expression levels have coevolved over short evolutionary time scales, reinforcing the positive relationship between gene body methylation and high levels of gene expression. Duplicated genes with the most divergent gene body methylation and expression patterns were found to have distinct biological functions and may have been under natural or human selection for cassava traits.

  15. Integrative analysis of DNA methylation and gene expression in butyrate-treated CHO cells.

    PubMed

    Wippermann, Anna; Rupp, Oliver; Brinkrolf, Karina; Hoffrogge, Raimund; Noll, Thomas

    2016-11-24

    The cellular mechanisms responsible for the versatile properties of CHO cells as the major production cell line for biopharmaceutical molecules are not entirely understood yet, although several 'omics' data facilitate the understanding of CHO cells and their reactions to environmental conditions. However, genome-wide studies of epigenetic processes such as DNA methylation are still limited. To prove the applicability and usefulness of integrating DNA methylation and gene expression data in a biotechnological context, we exemplarily analyzed the time course of cellular reactions upon butyrate addition in antibody-producing CHO cells by whole-genome bisulfite sequencing and CHO-specific cDNA microarrays. Gene expression and DNA methylation analyses showed that pathways known to be affected by butyrate, including cell cycle and apoptosis, as well as pathways potentially involved in butyrate-induced hyperproductivity such as central energy metabolism and protein biosynthesis were affected. Differentially methylated regions were furthermore found to contain binding-site motifs of specific transcription factors and were hypothesized to represent regulatory regions closely connected to the cellular response to butyrate. Generally, our experiment underlines the benefit of integrating DNA methylation and gene expression data, as it provided potential novel candidate genes for rational cell line development and allowed for new insights into the butyrate effect on CHO cells.

  16. Cloning, sequencing and analysis of dnaK -dnaJ gene cluster of Bacillus megaterium.

    PubMed

    Bao, Fangming; Gong, Lei; Shao, Weilan

    2008-12-01

    The DNA fragment of heat shock genes (hrcA-grpE-dnaK-dnaJ) containing complete hrcA-grpE-dnaK operon and the transcription unit of dnaJ was cloned, sequensed and analyzed from Bacillus megaterium RF5. The sequence of hrcA, grpE and dnaJ were first time reported, and their coding products exibit 60%, 63% and 81% of identities to the homologs of B. subtilis. A sigmaA-type promoter of Gram-positive bacteria (PA1) and a terminator were located upstream of the hrcA and downstream of dnaK, and a Controlling inverted repeat of chaperone expression element (CIRCE) was identified between PA1 and hrcA. Another sigmaA-type promoter (PA2) and a terminator were found upstream and downstream of dnaJ, indicating B. megaterium has a transcription unit containing a single gene dnaJ. The structure of dnaJ transcription unit is more similar to that of Listeria monocytogenes than other species of Bacillus. A partial protein-based phylogenetic tree, derived from Gram-positive bacteria using HrcA sequence, indicated a closer phylogenetic relationship between B. megaterium and Geobacillus species than other two Bacillus species.

  17. Analysis of thirteen trinucleotide repeat loci as candidate genes for Schizophrenia and bipolar affective disorder

    SciTech Connect

    Jain, S.; Leggo, J.; Ferguson-Smith, M.A.; Rubinsztein, D.C.

    1996-04-09

    A group of diseases are due to abnormal expansions of trinucleotide repeats. These diseases all affect the nervous system. In addition, they manifest the phenomenon of anticipation, in which the disease tends to present at an earlier age or with greater severity in successive generations. Many additional genes with trinucleotide repeats are believed to be expressed in the human brain. As anticipation has been reported in schizophrenia and bipolar affective disorder, we have examined allele distributions of 13 trinucleotide repeat-containing genes, many novel and all expressed in the brain, in genomic DNA from schizophrenic (n = 20-97) and bipolar affective disorder patients (23-30) and controls (n = 43-146). No evidence was obtained to implicate expanded alleles in these 13 genes as causal factors in these diseases. 26 refs., 1 fig., 2 tabs.

  18. Quantifying the Effect of DNA Packaging on Gene Expression Level

    NASA Astrophysics Data System (ADS)

    Kim, Harold

    2010-10-01

    Gene expression, the process by which the genetic code comes alive in the form of proteins, is one of the most important biological processes in living cells, and begins when transcription factors bind to specific DNA sequences in the promoter region upstream of a gene. The relationship between gene expression output and transcription factor input which is termed the gene regulation function is specific to each promoter, and predicting this gene regulation function from the locations of transcription factor binding sites is one of the challenges in biology. In eukaryotic organisms (for example, animals, plants, fungi etc), DNA is highly compacted into nucleosomes, 147-bp segments of DNA tightly wrapped around histone protein core, and therefore, the accessibility of transcription factor binding sites depends on their locations with respect to nucleosomes - sites inside nucleosomes are less accessible than those outside nucleosomes. To understand how transcription factor binding sites contribute to gene expression in a quantitative manner, we obtain gene regulation functions of promoters with various configurations of transcription factor binding sites by using fluorescent protein reporters to measure transcription factor input and gene expression output in single yeast cells. In this talk, I will show that the affinity of a transcription factor binding site inside and outside the nucleosome controls different aspects of the gene regulation function, and explain this finding based on a mass-action kinetic model that includes competition between nucleosomes and transcription factors.

  19. Divergence of Gene Body DNA Methylation and Evolution of Plant Duplicate Genes

    PubMed Central

    Wang, Jun; Marowsky, Nicholas C.; Fan, Chuanzhu

    2014-01-01

    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. PMID:25310342

  20. Nanoscopic structure of DNA condensed for gene delivery.

    PubMed Central

    Dunlap, D D; Maggi, A; Soria, M R; Monaco, L

    1997-01-01

    Scanning force microscopy was used to examine DNA condensates prepared with varying stoichiometries of lipospermine or polyethylenimine in physiological solution. For the first time, individual DNA strands were clearly visualized in incomplete condensates without drying. Using lipospermine at sub-saturating concentrations, discrete nuclei of condensation were observed often surrounded by folded loops of DNA. Similar packing of DNA loops occurred for polyethylenimine-induced condensation. Increasing the amount of the condensing agent led to the progressive coalescence or aggregation of initial condensation nuclei through folding rather than winding the DNA. At over-saturating charge ratios of the cationic lipid or polymer to DNA, condensates had sizes smaller than or equal to those measured previously in electron micrographs. Polyethylenimine condensates were more compact than lipospermine condensates and both produced more homogeneously compacted plasmids when used in a 2-4-fold charge excess. The size and morphology of the condensates may affect their efficiency in transfection. PMID:9224610

  1. Multiplex cDNA quantification method that facilitates the standardization of gene expression data

    PubMed Central

    Gotoh, Osamu; Murakami, Yasufumi; Suyama, Akira

    2011-01-01

    Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h. PMID:21415008

  2. DNA replication timing influences gene expression level

    PubMed Central

    2017-01-01

    Eukaryotic genomes are replicated in a reproducible temporal order; however, the physiological significance is poorly understood. We compared replication timing in divergent yeast species and identified genomic features with conserved replication times. Histone genes were among the earliest replicating loci in all species. We specifically delayed the replication of HTA1-HTB1 and discovered that this halved the expression of these histone genes. Finally, we showed that histone and cell cycle genes in general are exempt from Rtt109-dependent dosage compensation, suggesting the existence of pathways excluding specific loci from dosage compensation mechanisms. Thus, we have uncovered one of the first physiological requirements for regulated replication time and demonstrated a direct link between replication timing and gene expression. PMID:28539386

  3. Differences in DNA Binding Specificity of Floral Homeotic Protein Complexes Predict Organ-Specific Target Genes.

    PubMed

    Smaczniak, Cezary; Muiño, Jose M; Chen, Dijun; Angenent, Gerco C; Kaufmann, Kerstin

    2017-08-01

    Floral organ identities in plants are specified by the combinatorial action of homeotic master regulatory transcription factors. However, how these factors achieve their regulatory specificities is still largely unclear. Genome-wide in vivo DNA binding data show that homeotic MADS domain proteins recognize partly distinct genomic regions, suggesting that DNA binding specificity contributes to functional differences of homeotic protein complexes. We used in vitro systematic evolution of ligands by exponential enrichment followed by high-throughput DNA sequencing (SELEX-seq) on several floral MADS domain protein homo- and heterodimers to measure their DNA binding specificities. We show that specification of reproductive organs is associated with distinct binding preferences of a complex formed by SEPALLATA3 and AGAMOUS. Binding specificity is further modulated by different binding site spacing preferences. Combination of SELEX-seq and genome-wide DNA binding data allows differentiation between targets in specification of reproductive versus perianth organs in the flower. We validate the importance of DNA binding specificity for organ-specific gene regulation by modulating promoter activity through targeted mutagenesis. Our study shows that intrafamily protein interactions affect DNA binding specificity of floral MADS domain proteins. Differential DNA binding of MADS domain protein complexes plays a role in the specificity of target gene regulation. © 2017 American Society of Plant Biologists. All rights reserved.

  4. Expression of petite mitochondrial DNA in vivo: zygotic gene rescue.

    PubMed

    Strausberg, R L; Butow, R A

    1977-07-01

    A protocol is introduced for probing the organization and regulation of expression of the yeast mitochondrial genome, termed "zygotic gene rescue." The procedure is based on the notion that genes retained on mitochondrial DNA of on the notion that genes retained on mitochondrial DNA of petites can be expressed in zygotes of a cross between petite and wild type. To test the validity of this notion, we have taken advantage of our ability to discriminate, by mobility differences on sodium dodecyl sulfate/polyacrylamide gels, different forms of the product of alleles of the mitochondrial gene, varI. In petite strains that have retained the varI gene, its characteristic product appears in zygotes 4-5 hr after mating; no product is observed in petite strains deleted in the varI locus. Our studies indicate that (i) expression in the zygote of the varI gene in the petite genome is not exclusively the result of recombination with mitochondrial DNA of the wild-type tester, and (ii) the varI gene is probably reiterated in the petite mitochondrial genome. The strength of the technique of zygotic gene rescue in the analysis of the mitochondrial genome is discussed.

  5. A parasitic selfish gene that affects host promiscuity.

    PubMed

    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.

  6. Genetic variants of the DNA repair genes from Exome Aggregation Consortium (EXAC) database: significance in cancer.

    PubMed

    Das, Raima; Ghosh, Sankar Kumar

    2017-04-01

    DNA repair pathway is a primary defense system that eliminates wide varieties of DNA damage. Any deficiencies in them are likely to cause the chromosomal instability that leads to cell malfunctioning and tumorigenesis. Genetic polymorphisms in DNA repair genes have demonstrated a significant association with cancer risk. Our study attempts to give a glimpse of the overall scenario of the germline polymorphisms in the DNA repair genes by taking into account of the Exome Aggregation Consortium (ExAC) database as well as the Human Gene Mutation Database (HGMD) for evaluating the disease link, particularly in cancer. It has been found that ExAC DNA repair dataset (which consists of 228 DNA repair genes) comprises 30.4% missense, 12.5% dbSNP reported and 3.2% ClinVar significant variants. 27% of all the missense variants has the deleterious SIFT score of 0.00 and 6% variants carrying the most damaging Polyphen-2 score of 1.00, thus affecting the protein structure and function. However, as per HGMD, only a fraction (1.2%) of ExAC DNA repair variants was found to be cancer-related, indicating remaining variants reported in both the databases to be further analyzed. This, in turn, may provide an increased spectrum of the reported cancer linked variants in the DNA repair genes present in ExAC database. Moreover, further in silico functional assay of the identified vital cancer-associated variants, which is essential to get their actual biological significance, may shed some lights in the field of targeted drug development in near future. Copyright © 2017. Published by Elsevier B.V.

  7. Noncatalytic, N-terminal Domains of DNA Polymerase Lambda Affect Its Cellular Localization and DNA Damage Response.

    PubMed

    Stephenson, Anthony A; Taggart, David J; Suo, Zucai

    2017-04-13

    Specialized DNA polymerases, such as DNA polymerase lambda (Polλ), are important players in DNA damage tolerance and repair pathways. Knowing how DNA polymerases are regulated and recruited to sites of DNA damage is imperative to understanding these pathways. Recent work has suggested that Polλ plays a role in several distinct DNA damage tolerance and repair pathways. In this paper, we report previously unknown roles of the N-terminal domains of human Polλ for modulating its involvement in DNA damage tolerance and repair. By using Western blot analysis, fluorescence microscopy, and cell survival assays, we found that the BRCA1 C-terminal (BRCT) and proline/serine-rich (PSR) domains of Polλ affect its cellular localization and DNA damage responses. The nuclear localization signal (NLS) of Polλ was necessary to overcome the impediment of its nuclear localization caused by its BRCT and PSR domains. Induction of DNA damage resulted in recruitment of Polλ to chromatin, which was controlled by its BRCT and PSR domains. In addition, the presence of both domains was required for Polλ-mediated tolerance of oxidative DNA damage but not DNA methylation damage. These findings suggest that the N-terminal domains of Polλ are important for regulating its responses to DNA damage.

  8. Haploinsufficiency of DNA Damage Response Genes and their Potential Influence in Human Genomic Disorders

    PubMed Central

    O’Driscoll, Mark

    2008-01-01

    Genomic disorders are a clinically diverse group of conditions caused by gain, loss or re-orientation of a genomic region containing dosage-sensitive genes. One class of genomic disorder is caused by hemizygous deletions resulting in haploinsufficiency of a single or, more usually, several genes. For example, the heterozygous contiguous gene deletion on chromosome 22q11.2 causing DiGeorge syndrome involves at least 20-30 genes. Determining how the copy number variation (CNV) affects human variation and contributes to the aetiology and progression of various genomic disorders represents important questions for the future. Here, I will discuss the functional significance of one form of CNV, haploinsufficiency (i.e. loss of a gene copy), of DNA damage response components and its association with certain genomic disorders. There is increasing evidence that haploinsufficiency for certain genes encoding key players in the cells response to DNA damage, particularly those of the Ataxia Telangiectasia and Rad3-related (ATR)-pathway, has a functional impact. I will review this evidence and present examples of some well known clinically similar genomic disorders that have recently been shown to be defective in the ATR-dependent DNA damage response. Finally, I will discuss the potential implications of a haploinsufficiency-induced defective DNA damage response for the clinical management of certain human genomic disorders. PMID:19440510

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

    PubMed

    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.

  10. Oligonucleotide-mediated gene repair at DNA level: the potential applications for gene therapy.

    PubMed

    Liu, Chang-Mei; Liu, De-Pei; Liang, Chih-Chuan

    2002-10-01

    Mutations in gene sequence can cause many genetic disorders, and researchers have attempted to develop treatments or cures at the DNA level for these diseases. Several strategies including triple-helix-forming oligonucleotides (TFOs), chimeric RNA/DNA oligonucleotide (RDO), and short single-stranded oligodeoxynucleotide (ODN) have been used to correct the dysfunctional genes in situ in the chromosome. Experimental data from cells and animal models suggest that all these strategies can repair the mutations in situ at DNA level. More effective structures of oligonucleotide, efficient delivery systems, and gene correction efficiency should be improved. Development of these strategies holds great potentials for treatments of genetic defects and other disorders.

  11. Expression of the dnaN and dnaQ genes of Escherichia coli is inducible by mitomycin C.

    PubMed

    Kaasch, M; Kaasch, J; Quiñones, A

    1989-10-01

    The dnaN and dnaQ genes encode the beta subunit and the epsilon subunit of the DNA polymerase III holoenzyme. Using translational fusions to lacZ we found that DNA damage caused by mitomycin C induces expression of the dnaA and dnaQ genes. This induction was not observed in lexA and recA mutants which block the induction of the SOS response, suggesting a relationship between the mechanism(s) of genetic control of DNA polymerase III holoenzyme and the SOS regulatory network. Nevertheless, there is evidence that the mitomycin C induction of dnaN and dnaQ is not a simple lexA-regulated process, because nalidixic acid (an excellent SOS inducer) does not increase dnaN and dnaQ gene expression, and the time course of induction is abnormally slow.

  12. Reference gene selection for in vitro cell-free DNA analysis and gene expression profiling.

    PubMed

    Bronkhorst, Abel Jacobus; Aucamp, Janine; Wentzel, Johannes F; Pretorius, Piet J

    2016-05-01

    (i) To optimize cell-free DNA (cfDNA) and mRNA quantification using eight housekeeping genes (HKGs), (ii) to determine if there is a difference in the occurrence of HKGs in the cfDNA and mRNA of normal cells and cancer cells, and (iii) to investigate whether there is some selectivity involved in the release of cfDNA. cfDNA was isolated directly from the growth medium of 3 cultured cancer cell lines and one non-malignant, primary cell line. At the same time interval, mRNA was isolated from these cells and cDNA was synthesized. CfDNA and cDNA were then amplified with real-time PCR utilizing eight different HKGs. For all cell lines tested, Beta-actin (ACTB) is the most appropriate HKG to use as a control for cfDNA and mRNA quantification. There was no clear difference in the occurrence of HKGs between cancer cells and healthy cells. Lastly, there is a consistent and distinct difference between the mRNA expression and cfDNA of all cell lines. This study reveals a new candidate HKG for a robust control in cfDNA analysis and gene expression profiling, and should be considered for optimal analysis. Furthermore, results indicate that cfDNA is selectively released from cells into culture medium. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  13. Loss of Caenorhabditis elegans UNG-1 uracil-DNA glycosylase affects apoptosis in response to DNA damaging agents.

    PubMed

    Skjeldam, Hanne K; Kassahun, Henok; Fensgård, Oyvind; SenGupta, Tanima; Babaie, Eshrat; Lindvall, Jessica M; Arczewska, Katarzyna; Nilsen, Hilde

    2010-08-05

    The nematode Caenorhabditis elegans has been used extensively to study responses to DNA damage. In contrast, little is known about DNA repair in this organism. C. elegans is unusual in that it encodes few DNA glycosylases and the uracil-DNA glycosylase (UDG) encoded by the ung-1 gene is the only known UDG. C. elegans could therefore become a valuable model organism for studies of the genetic interaction networks involving base excision repair (BER). As a first step towards characterization of BER in C. elegans, we show that the UNG-1 protein is an active uracil-DNA glycosylase. We demonstrate that an ung-1 mutant has reduced ability to repair uracil-containing DNA but that an alternative Ugi-inhibited activity is present in ung-1 nuclear extracts. Finally, we demonstrate that ung-1 mutants show altered levels of apoptotic cell corpses formed in response to DNA damaging agents. Increased apoptosis in the ung-1 mutant in response to ionizing radiation (IR) suggests that UNG-1 contributes to repair of IR-induced DNA base damage in vivo. Following treatment with paraquat however, the apoptotic corpse-formation was reduced. Gene expression profiling suggests that this phenotype is a consequence of compensatory transcriptomic shifts that modulate oxidative stress responses in the mutant and not an effect of reduced DNA damage signaling. 2010 Elsevier B.V. All rights reserved.

  14. Modulation of DNA binding by gene-specific transcription factors.

    PubMed

    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.

  15. Gene Specific Impedimetric Bacterial DNA Sensor for Rheumatic Heart Disease.

    PubMed

    Singh, Swati; Kaushal, Ankur; Gupta, Sunil; Kumar, Ashok

    2017-03-01

    An impedimetric mga gene specific DNA sensor was developed by immobilization of single stranded DNA probe onto the screen printed modified gold-dendrimer nanohybrid composite electrode for early and rapid detection of S. pyogenes in human throat swab samples causing rheumatic heart disease. Electrochemical impedance response was measured after hybridization with bacterial single stranded genomic DNA (ssG-DNA) with probe. The sensor was found highly specific to S. pyogenes and can detect as low as 0.01 ng ssDNA in 6 µL sample only in 30 min. The nanohybrid sensor was also tested with non-specific pathogens and characterized by FTIR. An early detection of the pathogen S. pyogenes in human can save damage of mitral and aortic heart valves (rheumatic heart disease) by proper medical care.

  16. Land use type significantly affects microbial gene transcription in soil.

    PubMed

    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.

  17. Tudor Nuclease Genes and Programmed DNA Rearrangements in Tetrahymena thermophila▿

    PubMed Central

    Howard-Till, Rachel A.; Yao, Meng-Chao

    2007-01-01

    Proteins containing a Tudor domain and domains homologous to staphylococcal nucleases are found in a number of eukaryotes. These “Tudor nucleases” have been found to be associated with the RNA-induced silencing complex (A. A. Caudy, R. F. Ketting, S. M. Hammond, A. M. Denli, A. M. Bathoorn, B. B. Tops, J. M. Silva, M. M. Myers, G. J. Hannon, and R. H. Plasterk, Nature 425:411-414, 2003). We have identified two Tudor nuclease gene homologs, TTN1 and TTN2, in the ciliate Tetrahymena thermophila, which has two distinct small-RNA pathways. Characterization of single and double KOs of TTN1 and TTN2 shows that neither of these genes is essential for growth or sexual reproduction. Progeny of TTN2 KOs and double knockouts occasionally show minor defects in the small-RNA-guided process of DNA deletion but appear to be normal in hairpin RNA-induced gene silencing, suggesting that Tudor nucleases play only a minor role in RNA interference in Tetrahymena. Previous studies of Tetrahymena have shown that inserted copies of the neo gene from Escherichia coli are often deleted from the developing macronucleus during sexual reproduction (Y. Liu, X. Song, M. A. Gorovsky, and K. M. Karrer, Eukaryot. Cell 4:421-431, 2005; M. C. Yao, P. Fuller, and X. Xi, Science 300:1581-1584, 2003). This transgene deletion phenomenon is hypothesized to be a form of genome defense. Analysis of the Tudor nuclease mutants revealed exceptionally high rates of deletion of the neo transgene at the TTN2 locus but no deletion at the TTN1 locus. When present in the same genome, however, the neo gene is deleted at high rates even at the TTN1 locus, further supporting a role for trans-acting RNA in this process. This deletion is not affected by the presence of the same sequence in the macronucleus, thus providing a counterargument for the role of the macronuclear genome in specifying all sequences for deletion. PMID:17715366

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

    PubMed

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

    2014-11-01

    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 which is uncoupled from its essential function in DSB repair. This could have implications for the development of therapeutic strategies aiming to radiosensitize tumors by affecting the DNA-PKcs function.

  19. Emodin, aloe-emodin and rhein induced DNA damage and inhibited DNA repair gene expression in SCC-4 human tongue cancer cells.

    PubMed

    Chen, Ya-Yin; Chiang, Su-Yin; Lin, Jaung-Geng; Yang, Jai-Sing; Ma, Yi-Shih; Liao, Ching-Lung; Lai, Tung-Yuan; Tang, Nou-Ying; Chung, Jing-Gung

    2010-03-01

    In our primary studies, we have shown that emodin, aloe-emodin and rhein induced cytotoxic effects, including cell cycle arrest and apoptosis in SCC-4 human tongue cancer cells. However, details regarding their effects on DNA damage and repair gene expression in SCC-4 cells are not clear. We investigated whether or not emodin, aloe-emodin and rhein induced DNA damage and inhibited DNA repair gene expression in SCC-4 cells. Comet assay (single cell electrophoresis) indicated that incubation of SCC-4 cells with 0, 20, 30 and 40 microM of emodin, 0, 25, 50 and 100 microM of aloe-emodin or rhein led to a longer DNA migration smear (comet tail). This means that all examined agents induced DNA damage in SCC-4 cells and these effects are dose-dependent but emodin is stronger than that of aloe-emodin or rhein. The results from real-time PCR assay demonstrated that 30 microM of emodin or aloe-emodin used for 24 and 48 h treatment in SCC-4 cells significantly inhibited expression of genes associated with DNA damage and repair [ataxia telangiectasia mutated (ATM); ataxia-telangiectasia and Rad3-related (ATR); 14-3-3sigma (14-3-3sigma); breast cancer 1, early onset (BRCA1); and DNA-dependent serine/threonine protein kinase (DNA-PK)]; only rhein suppressed the expression of O(6)-methylguanine-DNA methyltransferase (MGMT) mRNA with 48 h treatment, but had no effect on ATM expression. On 24 h treatment, only aloe-emodin significantly affected ATM expression. These effects may be the vital factors for emodin, aloe-emodin and rhein induction of DNA damage in vitro. In conclusion, these agents induced DNA damage followed by the inhibition of DNA repair-associated gene expressions, including ATM, ATR, 14-3-3sigma, BRCA1, DNA-PK and MGMT in SCC-4 human tongue cancer cells.

  20. Leptin gene promoter DNA methylation in WNIN obese mutant rats.

    PubMed

    Kalashikam, Rajender Rao; Inagadapa, Padmavathi J N; Thomas, Anju Elizabeth; Jeyapal, Sugeetha; Giridharan, Nappan Veettil; Raghunath, Manchala

    2014-02-05

    Obesity has become an epidemic in worldwide population. Leptin gene defect could be one of the causes for obesity. Two mutant obese rats WNIN/Ob and WNIN/GROb, isolated at National Centre for Laboratory Animal Sciences (NCLAS), Hyderabad, India, were found to be leptin resistant. The present study aims to understand the regulatory mechanisms underlying the resistance by promoter DNA methylation of leptin gene in these mutant obese rats. Male obese mutant homozygous, carrier and heterozygous rats of WNIN/Ob and WNIN/GROb strain of 6 months old were studied to check the leptin gene expression (RT-PCR) and promoter DNA methylation (MassARRAY Compact system, SEQUENOM) of leptin gene by invivo and insilico approach. Homozygous WNIN/Ob and WNIN/GROb showed significantly higher leptin gene expression compared to carrier and lean counterparts. Leptin gene promoter DNA sequence region was analyzed ranging from transcription start site (TSS) to-550 bp length and found four CpGs in this sequence among them only three CpG loci (-309, -481, -502) were methylated in these WNIN mutant rat phenotypes. The increased percentage of methylation in WNIN mutant lean and carrier phenotypes is positively correlated with transcription levels. Thus genetic variation may have effect on methylation percentages and subsequently on the regulation of leptin gene expression which may lead to obesity in these obese mutant rat strains.

  1. Electric field-mediated gene transfer into K562 cells: optimization of parameters affecting efficiency.

    PubMed

    Croaker, G M; Wass, E J; Iland, H J

    1990-07-01

    Since hemopoietic cells are refractory to transfection by conventional chemical means, we have developed a reliable and efficient gene transfer system for K562 cells which uses electric field-mediated gene transfer (EFMGT). EFMGT involves the exposure of cells in suspension to an electric field which transiently allows the entry of DNA into the cell and its subsequent integration and expression. Plasmids bearing the neo gene were used to identify and select transfected clonogenic cells manifested by geneticin resistance in semisolid medium. Transfection efficiency is significantly affected by the following variables: voltage, capacitance, time constant, number of pulses, buffer type and temperature, DNA concentration, configuration, and promoter type. Cell cycle status also appears to be critical as shown in studies employing aphidicolin synchronization. Using optimal conditions, we have consistently achieved a transfection efficiency of 0.3-0.4% of clonogenic cells per microgram DNA. Stability of neo gene expression was also demonstrated after 4 months in nonselective culture conditions. This level of efficiency compares favorably with other reports of gene transfer into human hemopoietic progenitor cells.

  2. Parameters affecting organization and transfection efficiency of amphiphilic copolymers/DNA carriers.

    PubMed

    Roques, Caroline; Bouchemal, Kawthar; Ponchel, Gilles; Fromes, Yves; Fattal, Elias

    2009-08-19

    Amphiphilic block copolymers are attracting increasing interest in the field of gene therapy, especially for transfection of striated muscles. However, little is known about the parameters affecting their transfection efficiency in vivo. These copolymers can self-assemble as micelles in certain conditions. Since micellization strongly depends on the temperature and ionic content of the preparation medium, the present paper aimed at investigating the influence of these parameters in the context of gene delivery. We first assessed the micellization of pluronic L64 and tetronic 304 at various temperatures in water, saline or Tyrode's salts solution. Pluronic L64 can form micelles at temperatures above 37 degrees C in water or at 37 degrees C in the Tyrode's salts solution, in the range of concentration investigated. For tetronic 304, CMC was found to be far below the concentrations used to transfer DNA. Pluronic L64 interacted with DNA only in the presence of micelles. Moreover, in vivo evaluation demonstrated that significantly improved transfection efficiency was obtained at 37 degrees C in Tyrode's salts solution for pluronic L64 based formulations, compared to 4 degrees C and 20 degrees C. Such differences were not recorded with tetronic 304. Finally, optimized formulations of both tetronic 304 and pluronic L64 were able to mediate efficient transfection in dystrophic muscles.

  3. Mitochondrial DNA haplotypes induce differential patterns of DNA methylation that result in differential chromosomal gene expression patterns

    PubMed Central

    Lee, William T; Sun, Xin; Tsai, Te-Sha; Johnson, Jacqueline L; Gould, Jodee A; Garama, Daniel J; Gough, Daniel J; McKenzie, Matthew; Trounce, Ian A; St. John, Justin C

    2017-01-01

    Mitochondrial DNA copy number is strictly regulated during development as naive cells differentiate into mature cells to ensure that specific cell types have sufficient copies of mitochondrial DNA to perform their specialised functions. Mitochondrial DNA haplotypes are defined as specific regions of mitochondrial DNA that cluster with other mitochondrial sequences to show the phylogenetic origins of maternal lineages. Mitochondrial DNA haplotypes are associated with a range of phenotypes and disease. To understand how mitochondrial DNA haplotypes induce these characteristics, we used four embryonic stem cell lines that have the same set of chromosomes but possess different mitochondrial DNA haplotypes. We show that mitochondrial DNA haplotypes influence changes in chromosomal gene expression and affinity for nuclear-encoded mitochondrial DNA replication factors to modulate mitochondrial DNA copy number, two events that act synchronously during differentiation. Global DNA methylation analysis showed that each haplotype induces distinct DNA methylation patterns, which, when modulated by DNA demethylation agents, resulted in skewed gene expression patterns that highlight the effectiveness of the new DNA methylation patterns established by each haplotype. The haplotypes differentially regulate α-ketoglutarate, a metabolite from the TCA cycle that modulates the TET family of proteins, which catalyse the transition from 5-methylcytosine, indicative of DNA methylation, to 5-hydroxymethylcytosine, indicative of DNA demethylation. Our outcomes show that mitochondrial DNA haplotypes differentially modulate chromosomal gene expression patterns of naive and differentiating cells by establishing mitochondrial DNA haplotype-specific DNA methylation patterns. PMID:28900542

  4. The nucleotide sequence of the dnaA gene and the first part of the dnaN gene of Escherichia coli K-12.

    PubMed Central

    Hansen, E B; Hansen, F G; von Meyenburg, K

    1982-01-01

    The nucleotide sequence of the dnaA gene and the first 10% of the dnaN gene was determined. From the nucleotide sequence the amino acid sequence of the dnaA gene product was derived. It is a basic protein of 467 amino acid residues with a molecular weight of 52.5 kD. The expression of the dnaA gene is in the counterclockwise direction like the one of the dnaN gene, for which potential startsites were found. PMID:6296774

  5. The nucleotide sequence of the dnaA gene and the first part of the dnaN gene of Escherichia coli K-12.

    PubMed

    Hansen, E B; Hansen, F G; von Meyenburg, K

    1982-11-25

    The nucleotide sequence of the dnaA gene and the first 10% of the dnaN gene was determined. From the nucleotide sequence the amino acid sequence of the dnaA gene product was derived. It is a basic protein of 467 amino acid residues with a molecular weight of 52.5 kD. The expression of the dnaA gene is in the counterclockwise direction like the one of the dnaN gene, for which potential startsites were found.

  6. DNA uptake sequences in Neisseria gonorrhoeae as intrinsic transcriptional terminators and markers of horizontal gene transfer

    PubMed Central

    Gurung, Neesha

    2016-01-01

    DNA uptake sequences are widespread throughout the Neisseria gonorrhoeae genome. These short, conserved sequences facilitate the exchange of endogenous DNA between members of the genus Neisseria. Often the DNA uptake sequences are present as inverted repeats that are able to form hairpin structures. It has been suggested previously that DNA uptake sequence inverted repeats present 3′ of genes play a role in rho-independent termination and attenuation. However, there is conflicting experimental evidence to support this role. The aim of this study was to determine the role of DNA uptake sequences in transcriptional termination. Both bioinformatics predictions, conducted using TransTermHP, and experimental evidence, from RNA-seq data, were used to determine which inverted repeat DNA uptake sequences are transcriptional terminators and in which direction. Here we show that DNA uptake sequences in the inverted repeat configuration occur in N. gonorrhoeae both where the DNA uptake sequence precedes the inverted version of the sequence and also, albeit less frequently, in reverse order. Due to their symmetrical configuration, inverted repeat DNA uptake sequences can potentially act as bi-directional terminators, therefore affecting transcription on both DNA strands. This work also provides evidence that gaps in DNA uptake sequence density in the gonococcal genome coincide with areas of DNA that are foreign in origin, such as prophage. This study differentiates for the first time, to our knowledge, between DNA uptake sequences that form intrinsic transcriptional terminators and those that do not, providing characteristic features within the flanking inverted repeat that can be identified. PMID:28348864

  7. DNMT1-interacting RNAs block gene specific DNA methylation

    PubMed Central

    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

  8. DNA Methylation Occurred around Lowly Expressed Genes of Plastid DNA during Tomato Fruit Development.

    PubMed

    Ngernprasirtsiri, J; Kobayashi, H; Akazawa, T

    1988-09-01

    We have analyzed DNA methylation of plastid DNA from fully ripened red fruits, green mature fruits, and green leaves of tomato (Lycopersicon esculentum var. Firstmore). Essentially identical restriction profiles were obtained between chromoplast and chloroplast DNAs by EcoRI digestion. BstNI/EcoRII and HpaII/MspI are pairs of isoschizomers that can discriminate between methylated and unmethylated DNAs. These endonucleases produced different restriction patterns of plastid DNAs from tomato fruits compared to tomato leaves. Moreover, we have found from Southern blots that methylation was not detected in DNA fragments containing certain genes that are actively expressed in chromoplasts, whereas DNA fragments bearing genes that are barely transcribed in chromoplasts are methylated.

  9. Proteasome inhibition enhances resistance to DNA damage via upregulation of Rpn4-dependent DNA repair genes.

    PubMed

    Karpov, Dmitry S; Spasskaya, Daria S; Tutyaeva, Vera V; Mironov, Alexander S; Karpov, Vadim L

    2013-09-17

    The 26S proteasome is an ATP-dependent multi-subunit protease complex and the major regulator of intracellular protein turnover and quality control. However, its role in the DNA damage response is controversial. We addressed this question in yeast by disrupting the transcriptional regulation of the PRE1 proteasomal gene. The mutant strain has decreased proteasome activity and is hyper-resistant to various DNA-damaging agents. We found that Rpn4-target genes MAG1, RAD23, and RAD52 are overexpressed in this strain due to Rpn4 stabilisation. These genes represent three different pathways of base excision, nucleotide excision and double strand break repair by homologous recombination (DSB-HR). Consistently, the proteasome mutant displays increased DSB-HR activity. Our data imply that the proteasome may have a negative role in DNA damage response.

  10. Towards understanding the breast cancer epigenome: a comparison of genome-wide DNA methylation and gene expression data

    PubMed Central

    Michiels, Stefan; Metzger-Filho, Otto; Saini, Kamal S.

    2016-01-01

    Until recently, an elevated disease risk has been ascribed to a genetic predisposition, however, exciting progress over the past years has discovered alternate elements of inheritance that involve epigenetic regulation. Epigenetic changes are heritably stable alterations that include DNA methylation, histone modifications and RNA-mediated silencing. Aberrant DNA methylation is a common molecular basis for a number of important human diseases, including breast cancer. Changes in DNA methylation profoundly affect global gene expression patterns. What is emerging is a more dynamic and complex association between DNA methylation and gene expression than previously believed. Although many tools have already been developed for analyzing genome-wide gene expression data, tools for analyzing genome-wide DNA methylation have not yet reached the same level of refinement. Here we provide an in-depth analysis of DNA methylation in parallel with gene expression data characteristics and describe the particularities of low-level and high-level analyses of DNA methylation data. Low-level analysis refers to pre-processing of methylation data (i.e. normalization, transformation and filtering), whereas high-level analysis is focused on illustrating the application of the widely used class comparison, class prediction and class discovery methods to DNA methylation data. Furthermore, we investigate the influence of DNA methylation on gene expression by measuring the correlation between the degree of CpG methylation and the level of expression and to explore the pattern of methylation as a function of the promoter region. PMID:26657508

  11. Towards understanding the breast cancer epigenome: a comparison of genome-wide DNA methylation and gene expression data.

    PubMed

    Singhal, Sandeep K; Usmani, Nawaid; Michiels, Stefan; Metzger-Filho, Otto; Saini, Kamal S; Kovalchuk, Olga; Parliament, Matthew

    2016-01-19

    Until recently, an elevated disease risk has been ascribed to a genetic predisposition, however, exciting progress over the past years has discovered alternate elements of inheritance that involve epigenetic regulation. Epigenetic changes are heritably stable alterations that include DNA methylation, histone modifications and RNA-mediated silencing. Aberrant DNA methylation is a common molecular basis for a number of important human diseases, including breast cancer. Changes in DNA methylation profoundly affect global gene expression patterns. What is emerging is a more dynamic and complex association between DNA methylation and gene expression than previously believed. Although many tools have already been developed for analyzing genome-wide gene expression data, tools for analyzing genome-wide DNA methylation have not yet reached the same level of refinement. Here we provide an in-depth analysis of DNA methylation in parallel with gene expression data characteristics and describe the particularities of low-level and high-level analyses of DNA methylation data. Low-level analysis refers to pre-processing of methylation data (i.e. normalization, transformation and filtering), whereas high-level analysis is focused on illustrating the application of the widely used class comparison, class prediction and class discovery methods to DNA methylation data. Furthermore, we investigate the influence of DNA methylation on gene expression by measuring the correlation between the degree of CpG methylation and the level of expression and to explore the pattern of methylation as a function of the promoter region.

  12. Diaphanous gene mutation affects spiral cleavage and chirality in snails

    PubMed Central

    Kuroda, Reiko; Fujikura, Kohei; Abe, Masanori; Hosoiri, Yuji; Asakawa, Shuichi; Shimizu, Miho; Umeda, Shin; Ichikawa, Futaba; Takahashi, Hiromi

    2016-01-01

    L-R (left and right) symmetry breaking during embryogenesis and the establishment of asymmetric body plan are key issues in developmental biology, but the onset including the handedness-determining gene locus still remains unknown. Using pure dextral (DD) and sinistral (dd) strains of the pond snail Lymnaea stagnalis as well as its F2 through to F10 backcrossed lines, the single handedness-determining-gene locus was mapped by genetic linkage analysis, BAC cloning and chromosome walking. We have identified the actin-related diaphanous gene Lsdia1 as the strongest candidate. Although the cDNA and derived amino acid sequences of the tandemly duplicated Lsdia1 and Lsdia2 genes are very similar, we could discriminate the two genes/proteins in our molecular biology experiments. The Lsdia1 gene of the sinistral strain carries a frameshift mutation that abrogates full-length LsDia1 protein expression. In the dextral strain, it is already translated prior to oviposition. Expression of Lsdia1 (only in the dextral strain) and Lsdia2 (in both chirality) decreases after the 1-cell stage, with no asymmetric localization throughout. The evolutionary relationships among body handedness, SD/SI (spiral deformation/spindle inclination) at the third cleavage, and expression of diaphanous proteins are discussed in comparison with three other pond snails (L. peregra, Physa acuta and Indoplanorbis exustus). PMID:27708420

  13. A superfamily of DNA transposons targeting multicopy small RNA genes.

    PubMed

    Kojima, Kenji K; Jurka, Jerzy

    2013-01-01

    Target-specific integration of transposable elements for multicopy genes, such as ribosomal RNA and small nuclear RNA (snRNA) genes, is of great interest because of the relatively harmless nature, stable inheritance and possible application for targeted gene delivery of target-specific transposable elements. To date, such strict target specificity has been observed only among non-LTR retrotransposons. We here report a new superfamily of sequence-specific DNA transposons, designated Dada. Dada encodes a DDE-type transposase that shows a distant similarity to transposases encoded by eukaryotic MuDR, hAT, P and Kolobok transposons, as well as the prokaryotic IS256 insertion element. Dada generates 6-7 bp target site duplications upon insertion. One family of Dada DNA transposons targets a specific site inside the U6 snRNA genes and are found in various fish species, water flea, oyster and polycheate worm. Other target sequences of the Dada transposons are U1 snRNA genes and different tRNA genes. The targets are well conserved in multicopy genes, indicating that copy number and sequence conservation are the primary constraints on the target choice of Dada transposons. Dada also opens a new frontier for target-specific gene delivery application.

  14. Gene-specific DNA methylation of DNMT3B and MTHFR and colorectal adenoma risk.

    PubMed

    Ho, Vikki; Ashbury, Janet E; Taylor, Sherryl; Vanner, Stephen; King, Will D

    2015-12-01

    DNA methyltransferase 3B (DNMT3B) and methylenetetrahydrofolate reductase (MTHFR) are genes which encode enzymes critical to one-carbon metabolism. Polymorphisms in these genes have been implicated in colorectal cancer etiology; however, epigenetic modifications such as gene-specific DNA methylation also affect gene expression. DNA methylation of DNMT3B and MTHFR was quantified in blood leukocytes using Sequenom EpiTYPER® among 272 participants undergoing a screening colonoscopy. DNA methylation was quantified in 66 and 28CpG sites of DNMT3B and MTHFR respectively, and conceptualized using two approaches. First, measures representing average methylation across all CpG sites were created. Second, unsupervised principal component (PC) analysis was used to identify summary variables representing methylation around the transcription start site and in the gene-coding area for both DNMT3B and MTHFR. Logistic regression was used to compare methylation levels between participants diagnosed with colorectal adenoma(s) versus those with a normal colonoscopy via the estimation of odds ratios (ORs) and 95% confidence intervals (95% CIs) for the risk of colorectal adenomas. No association was observed between average DNA methylation of either DNMT3B or MTHFR and colorectal adenoma risk. For DNMT3B, increasing DNA methylation of CpG sites in the gene-coding area was associated with a higher risk of colorectal adenomas (OR=1.34; 95% CI: 1.01-1.79 per SD). This research provides preliminary evidence that methylation of DNMT3B may have functional significance with respect to colorectal adenomas, precursors to the vast majority of colorectal cancers.

  15. Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

    PubMed

    Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

    2015-11-01

    Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1.

  16. DNA sequence variation and regulation of genes involved in pathogenesis of pulmonary tuberculosis.

    PubMed

    Qidwai, T; Jamal, F; Khan, M Y

    2012-06-01

    DNA sequence variations [copy number variations, single nucleotide polymorphisms (SNPs) and microsatellite repeats] play an important role in susceptibility/resistance to tuberculosis and other infectious diseases like malaria and HIV. Different population exhibit variable associations with tuberculosis susceptibility and severity because of DNA sequence variations in both host and parasite. A number of genes and their polymorphisms have been identified that appear to be important in tuberculosis. In this article, several case-control studies of tuberculosis including a number of genes in different population have been explored. Furthermore, this review summarizes the current studies of host polymorphisms and their association with tuberculosis in different population. We have computationally predicted 275 SNPs which occur in transcription factor binding sites for transcription factors in 19 genes involved in pathogenesis of tuberculosis. Some common SNPs are rs1327474, rs755622, rs1801274, rs396991, rs5030737, rs1800451, rs1800450, rs3763313 rs3763313, rs9268494 and rs9268492 that have been found to play a role in disease. Presence of non-synonimous polymorphisms in coding region might affect the structure of protein, whereas polymorphisms in promoter region affect the level of gene products, consequently altering the susceptibility/resistance to disease. Based on this prediction, we hypothesize that these genes play an important role in susceptibility to tuberculosis through an altered expression of gene product via the modification of transcriptional regulation of gene. © 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd.

  17. Gene Specific DNA Sensors for Diagnosis of Pathogenic Infections.

    PubMed

    Datta, Manali; Desai, Dignya; Kumar, Ashok

    2017-06-01

    Gene specific DNA based sensors have potential applications for rapid and real time monitoring of hybridization signal with the target nucleic acid of pathogens. Different types of DNA based sensors and their applications have been studied for rapid and accurate detection of pathogens causing human diseases. These sensors are based on surface plasmon resonance, quantum-dots, molecular beacons, piezoelectric and electrochemical etc. Curbing epidemics at an early stage is one of the massive challenges in healthcare systems. Timely detection of the causative organism may provide a solution to restrain mortality caused by the disease. With the advent of interdisciplinary sciences, bioelectronics has emerged as an effective alternative for disease diagnostics. Gene specific DNA sensors present themselves as cost-effective, sensitive and specific platforms for detection of disease causing pathogens. The mini review explores different transducer based sensors and their potential in diagnosis of acute and chronic diseases.

  18. Humans and chimpanzees differ in their cellular response to DNA damage and non-coding sequence elements of DNA repair-associated genes.

    PubMed

    Weis, E; Galetzka, D; Herlyn, H; Schneider, E; Haaf, T

    2008-01-01

    both species. Genetic differences in non-coding sequence elements may affect gene regulation in the DNA repair network and thus contribute to species differences in DNA repair and cancer susceptibility.

  19. Genetic analysis of DNA methylation and gene expression levels in whole blood of healthy human subjects

    PubMed Central

    2012-01-01

    Background The predominant model for regulation of gene expression through DNA methylation is an inverse association in which increased methylation results in decreased gene expression levels. However, recent studies suggest that the relationship between genetic variation, DNA methylation and expression is more complex. Results Systems genetic approaches for examining relationships between gene expression and methylation array data were used to find both negative and positive associations between these levels. A weighted correlation network analysis revealed that i) both transcriptome and methylome are organized in modules, ii) co-expression modules are generally not preserved in the methylation data and vice-versa, and iii) highly significant correlations exist between co-expression and co-methylation modules, suggesting the existence of factors that affect expression and methylation of different modules (i.e., trans effects at the level of modules). We observed that methylation probes associated with expression in cis were more likely to be located outside CpG islands, whereas specificity for CpG island shores was present when methylation, associated with expression, was under local genetic control. A structural equation model based analysis found strong support in particular for a traditional causal model in which gene expression is regulated by genetic variation via DNA methylation instead of gene expression affecting DNA methylation levels. Conclusions Our results provide new insights into the complex mechanisms between genetic markers, epigenetic mechanisms and gene expression. We find strong support for the classical model of genetic variants regulating methylation, which in turn regulates gene expression. Moreover we show that, although the methylation and expression modules differ, they are highly correlated. PMID:23157493

  20. Reduced rDNA copy number does not affect "competitive" chromosome pairing in XYY males of Drosophila melanogaster.

    PubMed

    Maggert, Keith A

    2014-03-20

    The ribosomal DNA (rDNA) arrays are causal agents in X-Y chromosome pairing in meiosis I of Drosophila males. Despite broad variation in X-linked and Y-linked rDNA copy number, polymorphisms in regulatory/spacer sequences between rRNA genes, and variance in copy number of interrupting R1 and R2 retrotransposable elements, there is little evidence that different rDNA arrays affect pairing efficacy. I investigated whether induced rDNA copy number polymorphisms affect chromosome pairing in a "competitive" situation in which complex pairing configurations were possible using males with XYY constitution. Using a common normal X chromosome, one of two different full-length Y chromosomes, and a third chromosome from a series of otherwise-isogenic rDNA deletions, I detected no differences in X-Y or Y-Y pairing or chromosome segregation frequencies that could not be attributed to random variation alone. This work was performed in the context of an undergraduate teaching program at Texas A&M University, and I discuss the pedagogical utility of this and other such experiments.

  1. Reduced rDNA Copy Number Does Not Affect “Competitive” Chromosome Pairing in XYY Males of Drosophila melanogaster

    PubMed Central

    Maggert, Keith A.

    2014-01-01

    The ribosomal DNA (rDNA) arrays are causal agents in X-Y chromosome pairing in meiosis I of Drosophila males. Despite broad variation in X-linked and Y-linked rDNA copy number, polymorphisms in regulatory/spacer sequences between rRNA genes, and variance in copy number of interrupting R1 and R2 retrotransposable elements, there is little evidence that different rDNA arrays affect pairing efficacy. I investigated whether induced rDNA copy number polymorphisms affect chromosome pairing in a “competitive” situation in which complex pairing configurations were possible using males with XYY constitution. Using a common normal X chromosome, one of two different full-length Y chromosomes, and a third chromosome from a series of otherwise-isogenic rDNA deletions, I detected no differences in X-Y or Y-Y pairing or chromosome segregation frequencies that could not be attributed to random variation alone. This work was performed in the context of an undergraduate teaching program at Texas A&M University, and I discuss the pedagogical utility of this and other such experiments. PMID:24449686

  2. DNA cleavage and Trp53 differentially affect SINE transcription.

    PubMed

    Hagan, Christy R; Rudin, Charles M

    2007-03-01

    Among the cellular responses observed following treatment with DNA-damaging agents is the activation of Short Interspersed Elements (SINEs; retrotransposable genetic elements that comprise over 10% of the human genome). By placing a human SINE (the Alu element) into murine cells, we have previously shown that DNA-damaging agents such as etoposide can induce both upregulation of SINE transcript levels and SINE retrotransposition. A similarly cytotoxic (but not genotoxic) exposure to vincristine was not associated with SINE activation. Here we demonstrate that multiple other genotoxic exposures are associated with upregulation of SINE transcript levels. By comparing the effects of similarly cytotoxic doses of the topoisomerase II inhibitors etoposide and merbarone, we confirm that DNA strand breakage is specifically associated with SINE induction. By evaluating transcription rate and RNA stability, we demonstrate that SINE induction by genotoxic exposure is associated with transcriptional induction and not with transcript stabilization. Finally we demonstrate that SINE induction by genotoxic stress is mediated by a Trp53-independent pathway, and in fact that Trp53 plays an inhibitory role in attenuating the transcriptional induction of SINE elements following exposure to a genotoxic agent. Together these data support a model in which initial DNA damage can trigger genomic instability due to SINE activation, a response which may be amplified in cancer cells lacking functional TP53. Copyright (c) 2006 Wiley-Liss, Inc.

  3. Hormonal induction of transfected genes depends on DNA topology.

    PubMed Central

    Piña, B; Haché, R J; Arnemann, J; Chalepakis, G; Slater, E P; Beato, M

    1990-01-01

    Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells. In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA. Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility. This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids. In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexamethasone. A similar result was obtained with fibroblasts that contain endogenous glucocorticoid receptors. When the distance between receptor-binding sites or between the binding sites and the promoter was increased, the dependence of progestin induction on DNA topology was more pronounced. In contrast to the original plasmid, these constructs also revealed a similar topological dependence for induction by glucocorticoids. The differential influence of DNA topology is not due to differences in the affinity of the two hormone receptors for DNA of various topologies, but probably reflects an influence of DNA topology on the interaction between different DNA-bound receptor molecules and between receptors and other transcription factors. Images PMID:2153920

  4. Bone mineral density-affecting genes in Africans.

    PubMed Central

    Gong, Gordon; Haynatzki, Gleb; Haynatzka, Vera; Howell, Ryan; Kosoko-Lasaki, Sade; Fu, Yun-Xin; Yu, Fei; Gallagher, John C.; Wilson, M. Roy

    2006-01-01

    BACKGROUND: We have recently reported the role of environmental exposure in the ethnic diversity of bone mineral density (BMD). Potential genetic difference has not been adequately assessed. PURPOSE: To determine allele frequencies of BMD-affecting genes and their association with BMD in Africans. METHODS: Allele frequencies at 18 polymorphic sites in 13 genes that affect BMD in Asians and/or Caucasians were determined in 143 recent immigrants (55 men and 88 women, 18-51 years of age) from sub-Saharan Sudan to the United States. Genetic association studies were performed. RESULTS: Among the 14 single-nucleotide polymorphisms (SNPs), 10 were significantly different in allele frequency between Sudanese and Asians, and 10 between Sudanese and Caucasians. Only the osteocalcin gene was not significantly different in allele frequency among Sudanese, Asians and Caucasians. Allele frequencies in the TGFB, COL1A1 and CSR genes were extremely low (<0.04) in the Sudanese. Frequencies of microsatellite alleles in four genes were significantly different among Sudanese, Asians and Caucasians. SNPs in the VDR and ERalpha genes were associated with BMD and/or BMC (bone mineral content) at several bone sites. CONCLUSIONS: Genetic difference may play a role in the ethnic diversity in BMD and/or BMC. PMID:16895279

  5. DNA methylation patterns of protein coding genes and long noncoding RNAs in female schizophrenic patients.

    PubMed

    Liao, Qi; Wang, Yunliang; Cheng, Jia; Dai, Dongjun; Zhou, Xingyu; Zhang, Yuzheng; Gao, Shugui; Duan, Shiwei

    2015-02-01

    Schizophrenia (SCZ) is a complex mental disorder contributed by both genetic and epigenetic factors. Long noncoding RNAs (lncRNAs) was recently found playing an important regulatory role in mental disorders. However, little was known about the DNA methylation of lncRNAs, although numerous SCZ studies have been performed on genetic polymorphisms or epigenetic marks in protein coding genes. We presented a comprehensive genome wide DNA methylation study of both protein coding genes and lncRNAs in female patients with paranoid and undifferentiated SCZ. Using the methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq), 8,163 and 764 peaks were identified in paranoid and undifferentiated SCZ, respectively (p < 1 × 10-5). Gene ontology analysis showed that the hypermethylated regions were enriched in the genes related to neuron system and brain for both paranoid and undifferentiated SCZ (p < 0.05). Among these peaks, 121 peaks were located in gene promoter regions that might affect gene expression and influence the SCZ related pathways. Interestingly, DNA methylation of 136 and 23 known lncRNAs in Refseq database were identified in paranoid and undifferentiated SCZ, respectively. In addition, ∼20% of intergenic peaks annotated based on Refseq genes were overlapped with lncRNAs in UCSC and gencode databases. In order to show the results well for most biological researchers, we created an online database to display and visualize the information of DNA methyation peaks in both types of SCZ (http://www.bioinfo.org/scz/scz.htm). Our results showed that the aberrant DNA methylation of lncRNAs might be another important epigenetic factor for SCZ.

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

    PubMed

    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.

  7. DNA radiolysis. Mapping of the gene regulation domains

    NASA Astrophysics Data System (ADS)

    Spotheim-Maurizot, Mélanie; Franchet-Beuzit, Jenny; Isabelle, Valerie; Tartier, Laurence; Charlier, Michel

    1995-11-01

    Ionizing radiations induce strand breaks and modifications of nucleotides (base and/or sugar) in DNA. In aerated solution, the damages are mainly due to the attack of DNA by the hydroxyl radicals (OH ·) issued from the radiolysis of water. The lesions occur at every nucleotides along the DNA molecule. For γ-rays, β-rays and fast neutrons, we have determined at each nucleotide the probability of strand breakage at neutral pH and the probability of base and sugar modification leading also to strand breakage after an alkaline treatment. The method of sequencing gel electrophoresis was used for this purpose. We have shown that the probability of getting a radiation induced damage at a given nucleotide is modulated by: i) the chemical nature of this nucleotide, ii) the local conformation determined by the sequence of nucleotides and by DNA strandedness (single or double stranded), iii) the type of structure to which the nucleotide belongs (right-handed B- or left-handed Z-DNA) and iv) the presence of proteins specifically (e.g. repressors) or nonspecifically (histones or histone-like) bound to DNA. Therefore, radiolysis may be a convenient tool for mapping gene regulation domains in which DNA is often in non-canonical B forms or is in interaction with regulatory proteins.

  8. groE genes affect SOS repair in Escherichia coli

    SciTech Connect

    Liu, S.K.; Tessman, I. )

    1990-10-01

    Repair of UV-irradiated bacteriophage in Escherichia coli by Weigle reactivation requires functional recA+ and umuD+C+ genes. When the cells were UV irradiated, the groE heat shock gene products, GroES and GroEL, were needed for at least 50% of the Weigle reactivation of the single-stranded DNA phage S13. Because of repression of the umuDC and recA genes, Weigle reactivation is normally blocked by the lexA3(Ind-) mutation (which creates a noncleavable LexA protein), but it was restored by a combination of a high-copy-number umuD+C+ plasmid and a UV dose that increases groE expression. Maximal reactivation was achieved by elevated amounts of the Umu proteins, which was accomplished in part by UV-induced expression of the groE genes. By increasing the number of copies of the umuD+C+ genes, up to 50% of the normal amount of reactivation of S13 was achieved in an unirradiated recA+ host.

  9. Aberrant DNA methylation of microRNA genes in human breast cancer - a critical appraisal.

    PubMed

    Lehmann, Ulrich

    2014-06-01

    Aberrant DNA methylation of regulatory sequences is a well-documented mechanism of functional deletion of genes with anti-tumourigenic properties including microRNAs. This review discusses the publications describing aberrant methylation of microRNA genes in human breast cancer cells. Among the anti-tumourigenic properties of epigenetically inactivated microRNA genes, the inhibition of proliferation and of epithelial-to-mesenchymal transition (EMT) are the best studied. Several studies are conceptually very interesting and present a comprehensive functional characterization of anti-tumorigenic microRNAs. The link between microRNA expression and gene methylation is not addressed directly by all studies and a number of studies are limited in their strength by not including primary breast cancer specimens or by analysing very small sets of primary human specimens. The publications cover a wide range of DNA methylation detection techniques, often making direct comparison of results challenging. Despite the identification and thorough characterization of many interesting candidates and functionally important microRNA genes affected by DNA methylation, the translation of microRNA gene methylation as a new biomarker into the daily routine practice has not yet worked out.

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

    PubMed

    Liu, Jinghe; Liang, Xingwei; Zhu, Jiaqiao; Wei, Liang; Hou, Yi; Chen, Da-Yuan; Sun, Qing-Yuan

    2008-09-01

    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, Dnmt1 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 the 5' regions of Dnmt1 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.

  11. Characterization of the mammalian DNA polymerase gene(s) and enzyme(s). Annual progress report

    SciTech Connect

    Mishra, N.C.

    1994-01-01

    Consistent with the long term goal of our research to understand the nature of the key enzymes in eukaryotic DNA replication we have characterized the properties of the wild type DNA polymerases of the {alpha}-family and their mutants. We have also provided evidence for the role of aphidicolin in the elongation process of the in vivo DNA replication in eukaryotic cells. We also developed a technology for planned prep from a large numbers of clones for direct screening by size or restriction digestion in order to facilitate our goals to clone the DNA polymerase gene.

  12. On the origin and evolutionary consequences of gene body DNA methylation

    PubMed Central

    Bewick, Adam J.; Ji, Lexiang; Niederhuth, Chad E.; Willing, Eva-Maria; Hofmeister, Brigitte T.; Shi, Xiuling; Wang, Li; Lu, Zefu; Rohr, Nicholas A.; Hartwig, Benjamin; Kiefer, Christiane; Deal, Roger B.; Schmutz, Jeremy; Grimwood, Jane; Stroud, Hume; Jacobsen, Steven E.; Schneeberger, Korbinian; Zhang, Xiaoyu; Schmitz, Robert J.

    2016-01-01

    In plants, CG DNA methylation is prevalent in the transcribed regions of many constitutively expressed genes (gene body methylation; gbM), but the origin and function of gbM remain unknown. Here we report the discovery that Eutrema salsugineum has lost gbM from its genome, to our knowledge the first instance for an angiosperm. Of all known DNA methyltransferases, only CHROMOMETHYLASE 3 (CMT3) is missing from E. salsugineum. Identification of an additional angiosperm, Conringia planisiliqua, which independently lost CMT3 and gbM, supports that CMT3 is required for the establishment of gbM. Detailed analyses of gene expression, the histone variant H2A.Z, and various histone modifications in E. salsugineum and in Arabidopsis thaliana epigenetic recombinant inbred lines found no evidence in support of any role for gbM in regulating transcription or affecting the composition and modification of chromatin over evolutionary timescales. PMID:27457936

  13. Reference-based gene model prediction on DNA contigs

    SciTech Connect

    Xu, Y.; Uberbacher, E.C.

    1997-01-01

    This paper presents an algorithm for constructing multiple gene models on a set of contigs of a large genomic clone. The algorithm first uses pattern recognition-based methods to locate exons or partial exons in each contig, and then applies protein homology or EST information from the databases, as reference models, to parse the predicted exons into gene models. In the phase of gene model construction, the algorithm uses a unified framework for genes ranging from situation with homologous proteins/ESTs to no homologous protein/EST in the database. By exploiting protein homology or EST information, the algorithm is able to (1) parse exons into multiple gene models over a set of DNA contigs (possibly unoriented and unordered); (2) remove falsely predicted exons; and (3) identify and locate exons missed by the initial exon prediction.

  14. DEVELOPMENT OF A 950-GENE DNA ARRAY FOR EXAMINING GENE EXPRESSION PATTERNS IN MOUSE TESTIS

    EPA Science Inventory

    Development of a 950-gene DNA array for examining gene expression patterns in mouse testis.

    Rockett JC, Christopher Luft J, Brian Garges J, Krawetz SA, Hughes MR, Hee Kirn K, Oudes AJ, Dix DJ.

    Reproductive Toxicology Division, National Health and Environmental Effec...

  15. Does varicocelectomy affect DNA fragmentation in infertile patients?

    PubMed Central

    Telli, Onur; Sarici, Hasmet; Kabar, Mucahit; Ozgur, Berat Cem; Resorlu, Berkan; Bozkurt, Selen

    2015-01-01

    Introduction: The aims of this study were to investigate the effect of varicocelectomy on DNA fragmentation index and semen parameters in infertile patients before and after surgical repair of varicocele. Materials and Methods: In this prospective study, 72 men with at least 1-year history of infertility, varicocele and oligospermia were examined. Varicocele sperm samples were classified as normal or pathological according to the 2010 World Health Organization guidelines. The acridine orange test was used to assess the DNA fragmentation index (DFI) preoperatively and postoperatively. Results: DFI decreased significantly after varicocelectomy from 34.5% to 28.2% (P = 0.024). In addition all sperm parameters such as mean sperm count, sperm concentration, progressive motility and sperm morphology significantly increased from 19.5 × 106 to 30.7 × 106, 5.4 × 106/ml to 14.3 × 106/ml, and 19.9% to 31.2% (P < 0.001) and 2.6% to 3.1% (P = 0.017). The study was limited by the loss to follow-up of some patients and unrecorded pregnancy outcome due to short follow-up. Conclusion: Varicocele causes DNA-damage in spermatozoa. We suggest that varicocelectomy improves sperm parameters and decreases DFI. PMID:25878412

  16. Heterogeneous staining: a tool for studies of how fluorescent dyes affect the physical properties of DNA.

    PubMed

    Nyberg, Lena; Persson, Fredrik; Akerman, Björn; Westerlund, Fredrik

    2013-10-01

    The commonly used fluorescent dye YOYO-1 (YOYO) has, using bulk techniques, been demonstrated to stain DNA heterogeneously at substoichiometric concentrations. We here, using nanofluidic channels and fluorescence microscopy, investigate the heterogeneous staining on the single DNA molecule level and demonstrate that the dye distribution is continuous. The equilibration of YOYO on DNA is extremely slow but can be accelerated by increasing the ionic strength and/or the temperature. Furthermore, we demonstrate how to use the heterogeneous staining as a tool for detailed and time-efficient studies of how fluorescent dyes affect the physical properties of DNA. We show that the relative increase in extension of DNA with increasing amount of YOYO bound is higher at low ionic strengths and also extrapolate the extension of native DNA. Our study reveals important information on how YOYO affects the physical properties of DNA, but it also has broader applications. First, it reveals how cationic intercalators, such as potential DNA drugs, affect DNA under strong confinement. Second, the strategy of using heterogeneous staining is of general use for single molecule studies of DNA interacting with proteins or ligands.

  17. Identification of smut-responsive genes in sugarcane using cDNA-SRAP.

    PubMed

    Huang, N; Zhang, Y Y; Xiao, X H; Huang, L; Wu, Q B; Que, Y X; Xu, L P

    2015-06-18

    Sugarcane smut, caused by the fungus Sporisorium scitamineum, is one of the main diseases that affect sugarcane worldwide. In the present study, the cDNA-SRAP technique was used to identify genes that are likely to be involved in the response of sugarcane to S. scitamineum infection. In total, 21 bands with significant differential expression during cDNA-SRAP analysis were cloned and sequenced. Real-time qPCR confirmation demonstrated that expression of 19 of these 21 differential bands was consistent with the expression observed during cDNA-SRAP analysis, with a deduced false positive rate of 9.5%. Sequence alignment indicated that 18 of 19 differentially expressed genes showed homologies from 19% to 100% to certain genes in GenBank, including the following genes: topoisomerase (EU048780), ethylene insensitive (EU048778), and tetraspanin (EU048770). A real-time qPCR assay showed that during 0-72 h after pathogen infection, expression of the topoisomerase and the ethylene insensitive genes was upregulated, whereas expression of the tetraspanin gene was downregulated, identical to the expression patterns observed under salicylic acid treatment. Therefore, all three genes are thought to play a role during S. scitamineum challenge, but with different functions. To our knowledge, this is the first report on the application of cDNA-SRAP in differential gene expression analysis of sugarcane during a sugarcane-S. scitamineum interaction. The results obtained also contribute to a better understanding of the molecular mechanisms associated with sugarcane-S. scitamineum interactions.

  18. Multivalent dendrimer vectors with DNA intercalation motifs for gene delivery.

    PubMed

    Wong, Pamela T; Tang, Kenny; Coulter, Alexa; Tang, Shengzhuang; Baker, James R; Choi, Seok Ki

    2014-11-10

    Poly(amido amine) (PAMAM) dendrimers constitute an important class of nonviral, cationic vectors in gene delivery. Here we report on a new concept for dendrimer vector design based on the incorporation of dual binding motifs: DNA intercalation, and receptor recognition for targeted delivery. We prepared a series of dendrimer conjugates derived from a fifth generation (G5) PAMAM dendrimer, each conjugated with multiple folate (FA) or riboflavin (RF) ligands for cell receptor targeting, and with 3,8-diamino-6-phenylphenanthridinium ("DAPP")-derived ligands for anchoring a DNA payload. Polyplexes of each dendrimer with calf thymus dsDNA were made and characterized by surface plasmon resonance (SPR) spectroscopy, dynamic light scattering (DLS) and zeta potential measurement. These studies provided evidence supporting polyplex formation based on the observation of tight DNA-dendrimer adhesion, and changes in particle size and surface charge upon coincubation. Further SPR studies to investigate the adhesion of the polyplex to a model surface immobilized with folate binding protein (FBP), demonstrated that the DNA payload has only a minimal effect on the receptor binding activity of the polyplex: KD = 0.22 nM for G5(FA)(DAPP) versus 0.98 nM for its polyplex. Finally, we performed in vitro transfection assays to determine the efficiency of conjugate mediated delivery of a luciferase-encoding plasmid into the KB cancer cell line and showed that RF-conjugated dendrimers were 1 to 2 orders of magnitude more effective in enhancing luciferase gene transfection than a plasmid only control. In summary, this study serves as a proof of concept for DNA-ligand intercalation as a motif in the design of multivalent dendrimer vectors for targeted gene delivery.

  19. Effect of loss of T-DNA genes on MIA biosynthetic pathway gene regulation and alkaloid accumulation in Catharanthus roseus hairy roots.

    PubMed

    Taneja, Jyoti; Jaggi, Monika; Wankhede, Dhammaprakash Pandhari; Sinha, Alok Krishna

    2010-10-01

    Hairy roots are generated by integration of T-DNA in host plant genome from root inducing (Ri) plasmid of Agrobacterium rhizogenes and have been utilized for production of secondary metabolites in different plant systems. In Catharanthus roseus, hairy roots are known to show different morphologies, growth patterns, and alkaloid contents. It is also known that during transformation, there is a differential loss of a few T-DNA genes. To decipher the effect of loss of T-DNA genes on the various aspects of hairy roots, ten hairy root clones were analyzed for the presence or absence of T-DNA genes and its implications. It was found that the loss of a few ORFs drastically affects the growth and morphological patterns of hairy roots. The absence of T(R)-DNA from hairy roots revealed increased transcript accumulation and higher alkaloid concentrations, whereas callusing among hairy root lines led to decreased transcript and alkaloid accumulation. Significantly higher expression of MIA biosynthetic pathway genes and low abundance of regulator transcripts in hairy root clones in comparison with non-transformed control roots were also observed. This study indicates that it is not only the integration of T-DNA at certain region of host plant genome but also the presence or absence of important ORFs that affects the expression patterns of MIA biosynthetic pathway genes, regulators, and accumulation of specific alkaloids.

  20. Polymorphisms in DNA repair genes and associations with cancer risk.

    PubMed

    Goode, Ellen L; Ulrich, Cornelia M; Potter, John D

    2002-12-01

    Common polymorphisms in DNA repair genes may alter protein function and an individual's capacity to repair damaged DNA; deficits in repair capacity may lead to genetic instability and carcinogenesis. To establish our overall understanding of possible in vivo relationships between DNA repair polymorphisms and the development of cancer, we performed a literature review of epidemiological studies that assessed associations between such polymorphisms and risk of cancer. Thirty studies of polymorphisms in OGG1, XRCC1, ERCC1, XPC, XPD, XPF, BRCA2, and XRCC3 were identified in the April 30, 2002 MEDLINE database (National Center for Biotechnology Information. PubMed Database: http://www.ncbi.nlm.nih.gov/entrez). These studies focused on adult glioma, bladder cancer, breast cancer, esophageal cancer, lung cancer, prostate cancer, skin cancer (melanoma and nonmelanoma), squamous cell carcinoma of the head and neck, and stomach cancer. We found that a small proportion of the published studies were large and population-based. Nonetheless, published data were consistent with associations between: (a) the OGG1 S326C variant and increased risk of various types of cancer; (b) the XRCC1 R194W variant and reduced risk of various types of cancer; and (c) the BRCA2 N372H variant and increased risk of breast cancer. Suggestive results were seen for polymorphisms in other genes; however, small sample sizes may have contributed to false-positive or false-negative findings. We conclude that large, well-designed studies of common polymorphisms in DNA repair genes are needed. Such studies may benefit from analysis of multiple genes or polymorphisms and from the consideration of relevant exposures that may influence the likelihood of cancer in the presence of reduced DNA repair capacity.

  1. DNA methylation and differential gene regulation in photoreceptor cell death.

    PubMed

    Farinelli, P; Perera, A; Arango-Gonzalez, B; Trifunovic, D; Wagner, M; Carell, T; Biel, M; Zrenner, E; Michalakis, S; Paquet-Durand, F; Ekström, P A R

    2014-12-04

    Retinitis pigmentosa (RP) defines a group of inherited degenerative retinal diseases causing progressive loss of photoreceptors. To this day, RP is still untreatable and rational treatment development will require a thorough understanding of the underlying cell death mechanisms. Methylation of the DNA base cytosine by DNA methyltransferases (DNMTs) is an important epigenetic factor regulating gene expression, cell differentiation, cell death, and survival. Previous studies suggested an involvement of epigenetic mechanisms in RP, and in this study, increased cytosine methylation was detected in dying photoreceptors in the rd1, rd2, P23H, and S334ter rodent models for RP. Ultrastructural analysis of photoreceptor nuclear morphology in the rd1 mouse model for RP revealed a severely altered chromatin structure during retinal degeneration that coincided with an increased expression of the DNMT isozyme DNMT3a. To identify disease-specific differentially methylated DNA regions (DMRs) on a genomic level, we immunoprecipitated methylated DNA fragments and subsequently analyzed them with a targeted microarray. Genome-wide comparison of DMRs between rd1 and wild-type retina revealed hypermethylation of genes involved in cell death and survival as well as cell morphology and nervous system development. When correlating DMRs with gene expression data, we found that hypermethylation occurred alongside transcriptional repression. Consistently, motif analysis showed that binding sites of several important transcription factors for retinal physiology were hypermethylated in the mutant model, which also correlated with transcriptional silencing of their respective target genes. Finally, inhibition of DNMTs in rd1 organotypic retinal explants using decitabine resulted in a substantial reduction of photoreceptor cell death, suggesting inhibition of DNA methylation as a potential novel treatment in RP.

  2. Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis.

    PubMed

    Bahl, Martin Iain; Bergström, Anders; Licht, Tine Rask

    2012-04-01

    Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly or following freeze storage of three homogenized human fecal samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples; however, differences were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six different primer pairs targeting 16S rRNA genes of significant bacterial groups, and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven of nine cases, the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly higher in fecal samples that had been frozen compared to identical samples that had not. This effect was further supported by qPCR analysis of bacterial groups within these two phyla. The results demonstrate that storage conditions of fecal samples may adversely affect the determined Firmicutes to Bacteroidetes ratio, which is a frequently used biomarker in gut microbiology. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  3. Persistence of DNA in carcasses, slime and avian feces may affect interpretation of environmental DNA data.

    PubMed

    Merkes, Christopher M; McCalla, S Grace; Jensen, Nathan R; Gaikowski, Mark P; Amberg, Jon J

    2014-01-01

    The prevention of non-indigenous aquatic invasive species spreading into new areas is a goal of many resource managers. New techniques have been developed to survey for species that are difficult to capture with conventional gears that involve the detection of their DNA in water samples (eDNA). This technique is currently used to track the invasion of bigheaded carps (silver carp and bighead carp; Hypophthalmichthys molitrix and H. nobilis) in the Chicago Area Waterway System and Upper Mississippi River. In both systems DNA has been detected from silver carp without the capture of a live fish, which has led to some uncertainty about the source of the DNA. The potential contribution to eDNA by vectors and fomites has not been explored. Because barges move from areas with a high abundance of bigheaded carps to areas monitored for the potential presence of silver carp, we used juvenile silver carp to simulate the barge transport of dead bigheaded carp carcasses, slime residue, and predator feces to determine the potential of these sources to supply DNA to uninhabited waters where it could be detected and misinterpreted as indicative of the presence of live bigheaded carp. Our results indicate that all three vectors are feasible sources of detectable eDNA for at least one month after their deposition. This suggests that current monitoring programs must consider alternative vectors of DNA in the environment and consider alternative strategies to minimize the detection of DNA not directly released from live bigheaded carps.

  4. Persistence of DNA in Carcasses, Slime and Avian Feces May Affect Interpretation of Environmental DNA Data

    PubMed Central

    Merkes, Christopher M.; McCalla, S. Grace; Jensen, Nathan R.; Gaikowski, Mark P.; Amberg, Jon J.

    2014-01-01

    The prevention of non-indigenous aquatic invasive species spreading into new areas is a goal of many resource managers. New techniques have been developed to survey for species that are difficult to capture with conventional gears that involve the detection of their DNA in water samples (eDNA). This technique is currently used to track the invasion of bigheaded carps (silver carp and bighead carp; Hypophthalmichthys molitrix and H. nobilis) in the Chicago Area Waterway System and Upper Mississippi River. In both systems DNA has been detected from silver carp without the capture of a live fish, which has led to some uncertainty about the source of the DNA. The potential contribution to eDNA by vectors and fomites has not been explored. Because barges move from areas with a high abundance of bigheaded carps to areas monitored for the potential presence of silver carp, we used juvenile silver carp to simulate the barge transport of dead bigheaded carp carcasses, slime residue, and predator feces to determine the potential of these sources to supply DNA to uninhabited waters where it could be detected and misinterpreted as indicative of the presence of live bigheaded carp. Our results indicate that all three vectors are feasible sources of detectable eDNA for at least one month after their deposition. This suggests that current monitoring programs must consider alternative vectors of DNA in the environment and consider alternative strategies to minimize the detection of DNA not directly released from live bigheaded carps. PMID:25402206

  5. Posttranslational arginylation enzyme Ate1 affects DNA mutagenesis by regulating stress response

    PubMed Central

    Kumar, Akhilesh; Birnbaum, Michael D; Patel, Devang M; Morgan, William M; Singh, Jayanti; Barrientos, Antoni; Zhang, Fangliang

    2016-01-01

    Arginyltransferase 1 (Ate1) mediates protein arginylation, a poorly understood protein posttranslational modification (PTM) in eukaryotic cells. Previous evidence suggest a potential involvement of arginylation in stress response and this PTM was traditionally considered anti-apoptotic based on the studies of individual substrates. However, here we found that arginylation promotes cell death and/or growth arrest, depending on the nature and intensity of the stressing factor. Specifically, in yeast, mouse and human cells, deletion or downregulation of the ATE1 gene disrupts typical stress responses by bypassing growth arrest and suppressing cell death events in the presence of disease-related stressing factors, including oxidative, heat, and osmotic stresses, as well as the exposure to heavy metals or radiation. Conversely, in wild-type cells responding to stress, there is an increase of cellular Ate1 protein level and arginylation activity. Furthermore, the increase of Ate1 protein directly promotes cell death in a manner dependent on its arginylation activity. Finally, we found Ate1 to be required to suppress mutation frequency in yeast and mammalian cells during DNA-damaging conditions such as ultraviolet irradiation. Our study clarifies the role of Ate1/arginylation in stress response and provides a new mechanism to explain the link between Ate1 and a variety of diseases including cancer. This is also the first example that the modulation of the global level of a PTM is capable of affecting DNA mutagenesis. PMID:27685622

  6. Development of safe and effective nonviral gene therapy by eliminating CpG motifs from plasmid DNA vector.

    PubMed

    Takahashi, Yuki; Nishikawa, Makiya; Takakura, Yoshinobu

    2012-01-01

    Nonviral gene therapy is expected to become a regular treatment for a variety of difficult-to-treat diseases, such as cancer and virus infection. Plasmid DNA, which is used in most nonviral gene delivery systems, usually contains, unmethylated cytosine-guanine dinucleotides, so called CpG motifs. CpG motifs are recognized by immune cells as a danger signal, leading to an inflammatory response. Such inflammatory responses could affect the safety and effectiveness of nonviral gene therapy. Therefore, reducing the number of CpG motifs in plasmid DNA has been used to increase the potency of plasmid DNA-based gene therapy. Previous studies have demonstrated that CpG reduction can extend the time period of transgene expression from plasmid DNA after in vivo gene transfer. In this review, the biological functions of the CpG motif are briefly summarized. Then, safety issues of nonviral gene therapy are discussed from the viewpoint of the inflammatory response to the CpG motif in plasmid DNA, and the effects of the CpG motif in plasmid DNA on the transgene expression profile of nonviral gene transfer are reviewed.

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

    PubMed

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

    2010-04-01

    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 contacts. We use this effect to establish the separate contributions of transcription factor binding and DNA dynamics to transcriptional activity. Our results argue against a purely 'transcription factor-centric' view of transcription initiation, suggesting that both DNA dynamics and transcription factor binding are necessary conditions for transcription initiation.

  8. Toxic Diatom Aldehydes Affect Defence Gene Networks in Sea Urchins.

    PubMed

    Varrella, Stefano; Romano, Giovanna; Costantini, Susan; Ruocco, Nadia; Ianora, Adrianna; Bentley, Matt G; Costantini, Maria

    2016-01-01

    Marine organisms possess a series of cellular strategies to counteract the negative effects of toxic compounds, including the massive reorganization of gene expression networks. Here we report the modulated dose-dependent response of activated genes by diatom polyunsaturated aldehydes (PUAs) in the sea urchin Paracentrotus lividus. PUAs are secondary metabolites deriving from the oxidation of fatty acids, inducing deleterious effects on the reproduction and development of planktonic and benthic organisms that feed on these unicellular algae and with anti-cancer activity. Our previous results showed that PUAs target several genes, implicated in different functional processes in this sea urchin. Using interactomic Ingenuity Pathway Analysis we now show that the genes targeted by PUAs are correlated with four HUB genes, NF-κB, p53, δ-2-catenin and HIF1A, which have not been previously reported for P. lividus. We propose a working model describing hypothetical pathways potentially involved in toxic aldehyde stress response in sea urchins. This represents the first report on gene networks affected by PUAs, opening new perspectives in understanding the cellular mechanisms underlying the response of benthic organisms to diatom exposure.

  9. Toxic Diatom Aldehydes Affect Defence Gene Networks in Sea Urchins

    PubMed Central

    Varrella, Stefano; Ruocco, Nadia; Ianora, Adrianna; Bentley, Matt G.; Costantini, Maria

    2016-01-01

    Marine organisms possess a series of cellular strategies to counteract the negative effects of toxic compounds, including the massive reorganization of gene expression networks. Here we report the modulated dose-dependent response of activated genes by diatom polyunsaturated aldehydes (PUAs) in the sea urchin Paracentrotus lividus. PUAs are secondary metabolites deriving from the oxidation of fatty acids, inducing deleterious effects on the reproduction and development of planktonic and benthic organisms that feed on these unicellular algae and with anti-cancer activity. Our previous results showed that PUAs target several genes, implicated in different functional processes in this sea urchin. Using interactomic Ingenuity Pathway Analysis we now show that the genes targeted by PUAs are correlated with four HUB genes, NF-κB, p53, δ-2-catenin and HIF1A, which have not been previously reported for P. lividus. We propose a working model describing hypothetical pathways potentially involved in toxic aldehyde stress response in sea urchins. This represents the first report on gene networks affected by PUAs, opening new perspectives in understanding the cellular mechanisms underlying the response of benthic organisms to diatom exposure. PMID:26914213

  10. The study of the relation of DNA repair pathway genes SNPs and the sensitivity to radiotherapy and chemotherapy of NSCLC

    PubMed Central

    Wang, Chunbo; Nie, Huan; Li, Yiqun; Liu, Guiyou; Wang, Xu; Xing, Shijie; Zhang, Liping; Chen, Xin; Chen, Yue; Li, Yu

    2016-01-01

    To analyze the relation between SNPs in DNA repair pathway-related genes and sensitivity of tumor radio-chemotherapy, 26 SNPs in 20 DNA repair genes were genotyped on 176 patients of NSCLC undertaking radio-chemotherapy treatment. In squamous cell carcinoma (SCC), as the rs2228000, rs2228001 (XPC), rs2273953 (TP73), rs2279744 (MDM2), rs2299939 (PTEN) and rs8178085, rs12334811 (DNA-PKcs) affected the sensitivity to chemotherapy, so did the rs8178085, rs12334811 to radiotherapy. Moreover rs344781, rs2273953 and rs12334811 were related with the survival time of SCC. In general, the “good” genotype GG (rs12334811) showed greater efficacy of radio-chemotherapy and MSF (24 months) on SCC. In adenocarcinoma, as the rs2699887 (PIK3), rs12334811 (DNA-PKcs) influenced the sensitivity to chemotherapy, so did the rs2299939, rs2735343 (PTEN) to radiotherapy. And rs402710, rs80270, rs2279744 and rs2909430 impacted the survival time of the adenocarcinoma patients. Both GG (rs2279744) and AG (rs2909430) showed a shorter survival time (MFS = 6). Additionally, some SNPs such as rs2228000, rs2228001 and rs344781 were found to regulate the expression of DNA repair pathway genes through eQTLs dataset analysis. These results indicate that SNPs in DNA repair pathway genes might regulate the expression and affect the DNA damage repair, and thereby impact the efficacy of radio-chemotherapy and the survival time of NSCLC. PMID:27246533

  11. Independent component analysis of Alzheimer's DNA microarray gene expression data

    PubMed Central

    Kong, Wei; Mou, Xiaoyang; Liu, Qingzhong; Chen, Zhongxue; Vanderburg, Charles R; Rogers, Jack T; Huang, Xudong

    2009-01-01

    Background Gene microarray technology is an effective tool to investigate the simultaneous activity of multiple cellular pathways from hundreds to thousands of genes. However, because data in the colossal amounts generated by DNA microarray technology are usually complex, noisy, high-dimensional, and often hindered by low statistical power, their exploitation is difficult. To overcome these problems, two kinds of unsupervised analysis methods for microarray data: principal component analysis (PCA) and independent component analysis (ICA) have been developed to accomplish the task. PCA projects the data into a new space spanned by the principal components that are mutually orthonormal to each other. The constraint of mutual orthogonality and second-order statistics technique within PCA algorithms, however, may not be applied to the biological systems studied. Extracting and characterizing the most informative features of the biological signals, however, require higher-order statistics. Results ICA is one of the unsupervised algorithms that can extract higher-order statistical structures from data and has been applied to DNA microarray gene expression data analysis. We performed FastICA method on DNA microarray gene expression data from Alzheimer's disease (AD) hippocampal tissue samples and consequential gene clustering. Experimental results showed that the ICA method can improve the clustering results of AD samples and identify significant genes. More than 50 significant genes with high expression levels in severe AD were extracted, representing immunity-related protein, metal-related protein, membrane protein, lipoprotein, neuropeptide, cytoskeleton protein, cellular binding protein, and ribosomal protein. Within the aforementioned categories, our method also found 37 significant genes with low expression levels. Moreover, it is worth noting that some oncogenes and phosphorylation-related proteins are expressed in low levels. In comparison to the PCA and support

  12. Relationship of eukaryotic DNA replication to committed gene expression: general theory for gene control.

    PubMed Central

    Villarreal, L P

    1991-01-01

    The historic arguments for the participation of eukaryotic DNA replication in the control of gene expression are reconsidered along with more recent evidence. An earlier view in which gene commitment was achieved with stable chromatin structures which required DNA replication to reset expression potential (D. D. Brown, Cell 37:359-365, 1984) is further considered. The participation of nonspecific stable repressor of gene activity (histones and other chromatin proteins), as previously proposed, is reexamined. The possible function of positive trans-acting factors is now further developed by considering evidence from DNA virus models. It is proposed that these positive factors act to control the initiation of replicon-specific DNA synthesis in the S phase (early or late replication timing). Stable chromatin assembles during replication into potentially active (early S) or inactive (late S) states with prevailing trans-acting factors (early) or repressing factors (late) and may asymmetrically commit daughter templates. This suggests logical schemes for programming differentiation based on replicons and trans-acting initiators. This proposal requires that DNA replication precede major changes in gene commitment. Prior evidence against a role for DNA replication during terminal differentiation is reexamined along with other results from terminal differentiation of lower eukaryotes. This leads to a proposal that DNA replication may yet underlie terminal gene commitment, but that for it to do so there must exist two distinct modes of replication control. In one mode (mitotic replication) replicon initiation is tightly linked to the cell cycle, whereas the other mode (terminal replication) initiation is not cell cycle restricted, is replicon specific, and can lead to a terminally differentiated state. Aberrant control of mitotic and terminal modes of DNA replication may underlie the transformed state. Implications of a replicon basis for chromatin structure-function and

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

    PubMed

    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.

  14. Benzo[a]pyrene decreases global and gene specific DNA methylation during zebrafish development

    PubMed Central

    Fang, Xiefan; Thornton, Cammi; Scheffler, Brian E.; Willett, Kristine L.

    2013-01-01

    DNA methylation is important for gene regulation and is vulnerable to early-life exposure to environmental contaminants. We found that direct waterborne benzo[a]pyrene (BaP) exposure at 24 μg/L from 2.5 to 96 hours post fertilization (hpf) to zebrafish embryos significantly decreased global cytosine methylation by 44.8% and promoter methylation in vasa by 17%. Consequently, vasa expression was significantly increased by 33%. In contrast, BaP exposure at environmentally relevant concentrations did not change CpG island methylation or gene expression in cancer genes such as ras-association domain family member 1 (rassf1), telomerase reverse transcriptase (tert), c-jun, and c-myca. Similarly, BaP did not change gene expression of DNA methyltransferase 1 (dnmt1) and glycine N-methyltransferase (gnmt). While total DNMT activity was not affected, GNMT enzyme activity was moderately increased. In summary, BaP is an epigenetic modifier for global and gene specific DNA methylation status in zebrafish larvae. PMID:23542452

  15. Analysis of Mycobacterium leprae gene expression using DNA microarray.

    PubMed

    Akama, Takeshi; Tanigawa, Kazunari; Kawashima, Akira; Wu, Huhehasi; Ishii, Norihisa; Suzuki, Koichi

    2010-10-01

    Mycobacterium leprae, the causative agent of leprosy, does not grow under in vitro condition, making molecular analysis of this bacterium difficult. For this reason, bacteriological information regarding M. leprae gene function is limited compared with other mycobacterium species. In this study, we performed DNA microarray analysis to clarify the RNA expression profile of the Thai53 strain of M. leprae grown in footpads of hypertensive nude rats (SHR/NCrj-rnu). Of 1605 M. leprae genes, 315 showed signal intensity twofold higher than the median. These genes include Acyl-CoA metabolic enzymes and drug metabolic enzymes, which might be related to the virulence of M. leprae. In addition, consecutive RNA expression profile and in silico analyses enabled identification of possible operons within the M. leprae genome. The present results will shed light on M. leprae gene function and further our understanding of the pathogenesis of leprosy.

  16. Immunomodulation by mucosal gene transfer using TGF-beta DNA.

    PubMed Central

    Kuklin, N A; Daheshia, M; Chun, S; Rouse, B T

    1998-01-01

    This report evaluates the efficacy of DNA encoding TGF-beta administered mucosally to suppress immunity and modulate the immunoinflammatory response to herpes simplex virus (HSV) infection. A single intranasal administration of an eukaryotic expression vector encoding TGF-beta1 led to expression in the lung and lymphoid tissue. T cell-mediated immune responses to HSV infection were suppressed with this effect persisting as measured by the delayed-type hypersensitivity reaction for at least 7 wk. Treated animals were more susceptible to systemic infection with HSV. Multiple prophylactic mucosal administrations of TGF-beta DNA also suppressed the severity of ocular lesions caused by HSV infection, although no effects on this immunoinflammatory response were evident after therapeutic treatment with TGF-beta DNA. Our results demonstrate that the direct mucosal gene transfer of immunomodulatory cytokines provides a convenient means of modulating immunity and influencing the expression of inflammatory disorders. PMID:9664086

  17. Alternative splicing of DNA damage response genes and gastrointestinal cancers.

    PubMed

    Rahmutulla, Bahityar; Matsushita, Kazuyuki; Nomura, Fumio

    2014-12-14

    Alternative splicing, which is a common phenomenon in mammalian genomes, is a fundamental process of gene regulation and contributes to great protein diversity. Alternative splicing events not only occur in the normal gene regulation process but are also closely related to certain diseases including cancer. In this review, we briefly demonstrate the concept of alternative splicing and DNA damage and describe the association of alternative splicing and cancer pathogenesis, focusing on the potential relationship of alternative splicing, DNA damage, and gastrointestinal cancers. We will also discuss whether alternative splicing leads to genetic instability, which is considered to be a driving force for tumorigenesis. Better understanding of the role and mechanism of alternative splicing in tumorigenesis may provide new directions for future cancer studies.

  18. Advances in Non-Viral DNA Vectors for Gene Therapy

    PubMed Central

    Hardee, Cinnamon L.; Arévalo-Soliz, Lirio Milenka; Hornstein, Benjamin D.; Zechiedrich, Lynn

    2017-01-01

    Uses of viral vectors have thus far eclipsed uses of non-viral vectors for gene therapy delivery in the clinic. Viral vectors, however, have certain issues involving genome integration, the inability to be delivered repeatedly, and possible host rejection. Fortunately, development of non-viral DNA vectors has progressed steadily, especially in plasmid vector length reduction, now allowing these tools to fill in specifically where viral or other non-viral vectors may not be the best options. In this review, we examine the improvements made to non-viral DNA gene therapy vectors, highlight opportunities for their further development, address therapeutic needs for which their use is the logical choice, and discuss their future expansion into the clinic. PMID:28208635

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

    NASA Astrophysics Data System (ADS)

    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.

  20. Evolving DNA motifs to predict GeneChip probe performance

    PubMed Central

    Langdon, WB; Harrison, AP

    2009-01-01

    Background Affymetrix High Density Oligonuclotide Arrays (HDONA) simultaneously measure expression of thousands of genes using millions of probes. We use correlations between measurements for the same gene across 6685 human tissue samples from NCBI's GEO database to indicated the quality of individual HG-U133A probes. Low correlation indicates a poor probe. Results Regular expressions can be automatically created from a Backus-Naur form (BNF) context-free grammar using strongly typed genetic programming. Conclusion The automatically produced motif is better at predicting poor DNA sequences than an existing human generated RE, suggesting runs of Cytosine and Guanine and mixtures should all be avoided. PMID:19298675

  1. Adenovirus with DNA Packaging Gene Mutations Increased Virus Release

    PubMed Central

    Wechman, Stephen L.; Rao, Xiao-Mei; McMasters, Kelly M.; Zhou, Heshan Sam

    2016-01-01

    Adenoviruses (Ads) have been extensively manipulated for the development of cancer selective replication, leading to cancer cell death or oncolysis. Clinical studies using E1-modified oncolytic Ads have shown that this therapeutic platform was safe, but with limited efficacy, indicating the necessity of targeting other viral genes for manipulation. To improve the therapeutic efficacy of oncolytic Ads, we treated the entire Ad genome repeatedly with UV-light and have isolated AdUV which efficiently lyses cancer cells as reported previously (Wechman, S. L. et al. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection. Viruses 2016, 8, 6). In this report, we show that no mutations were observed in the early genes (E1 or E4) of AdUV while several mutations were observed within the Ad late genes which have structural or viral DNA packaging functions. This study also reported the increased release of AdUV from cancer cells. In this study, we found that AdUV inhibits tumor growth following intratumoral injection. These results indicate the potentially significant role of the viral late genes, in particular the DNA packaging genes, to enhance Ad oncolysis. PMID:27999391

  2. DNA interference: DNA-induced gene silencing in the appendicularian Oikopleura dioica.

    PubMed

    Omotezako, Tatsuya; Onuma, Takeshi A; Nishida, Hiroki

    2015-05-22

    RNA interference is widely employed as a gene-silencing system in eukaryotes for host defence against invading nucleic acids. In response to invading double-stranded RNA (dsRNA), mRNA is degraded in sequence-specific manner. So far, however, DNA interference (DNAi) has been reported only in plants, ciliates and archaea, and has not been explored in Metazoa. Here, we demonstrate that linear double-stranded DNA promotes both sequence-specific transcription blocking and mRNA degradation in developing embryos of the appendicularian Oikopleura dioica. Introduced polymerase chain reaction (PCR) products or linearized plasmids encoding Brachyury induced tail malformation and mRNA degradation. This malformation was also promoted by DNA fragments of the putative 5'-flanking region and intron without the coding region. PCR products encoding Zic-like1 and acetylcholine esterase also induced loss of sensory organ and muscle acetylcholinesterase activity, respectively. Co-injection of mRNA encoding EGFP and mCherry, and PCR products encoding these fluorescent proteins, induced sequence-specific decrease in the green or red fluorescence, respectively. These results suggest that O. dioica possesses a defence system against exogenous DNA and RNA, and that DNA fragment-induced gene silencing would be mediated through transcription blocking as well as mRNA degradation. This is the first report of DNAi in Metazoa. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

  3. FISH comets show that the salvage enzyme TK1 contributes to gene-specific DNA repair

    PubMed Central

    McAllister, Katherine A.; Yasseen, Akeel A.; McKerr, George; Downes, C. S.; McKelvey-Martin, Valerie J.

    2014-01-01

    Thymidine kinase 1 (TK1) is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP. TK1 deficiency has for a long time been known to cause increased cellular sensitivity to DNA damage. We have examined preferential strand break repair of DNA domains in TK1+ and TK1- clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumor suppressor (TP53) and human telomerase reverse transcriptase (hTERT) gene regions, over 1 h after 5Gy γ-irradiation. Results showed that repair of the TP53 and hTERT gene regions was more efficient in TK1+ compared to TK1- cells, a trend also reflected to a lesser degree in genomic DNA repair between the cell-lines. The targeted gene-specific repair in TK+ cells occurred rapidly, mainly over the first 15 min repair-period. Therefore, TK1 is needed for preferential repair of actively transcribed regions, through a previously unsuspected mechanism. In principle, TK1 could exert its protective effects through supply of a supplementary dTTP pool for accurate repair of damaged genes; but Raji TK1+ cells in thymidine free media still show preferential repair of transcribed regions. TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents. PMID:25152750

  4. Getting the most from gene delivery by repeated DNA transfections

    NASA Astrophysics Data System (ADS)

    Montani, Maura; Marchini, Cristina; Badillo Pazmay, Gretta Veronica; Andreani, Cristina; Bartolacci, Caterina; Amici, Augusto; Pozzi, Daniela; Caracciolo, Giulio

    2015-06-01

    Intracellular delivery of reporter genes causes cells to be luminescent or fluorescent, this condition being of tremendous relevance in applied physics research. Potential applications range from the study of spatial distribution and dynamics of plasma membrane and cytosolic proteins up to the rational design of nanocarriers for gene therapy. Since efficiency of gene delivery is the main limit in most biophysical studies, versatile methods that can maximize gene expression are urgently needed. Here, we describe a robust methodology based on repeated gene delivery in mammalian cells. We find this procedure to be much more efficient than the more traditional route of gene delivery making it possible to get high-quality data without affecting cell viability. Implications for biophysical investigations are discussed.

  5. ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism

    PubMed Central

    Desai, Radha; Frazier, Ann E.; Durigon, Romina; Patel, Harshil; Jones, Aleck W.; Dalla Rosa, Ilaria; Lake, Nicole J.; Compton, Alison G.; Mountford, Hayley S.; Tucker, Elena J.; Mitchell, Alice L. R.; Jackson, Deborah; Sesay, Abdul; Di Re, Miriam; van den Heuvel, Lambert P.; Burke, Derek; Lunke, Sebastian; McGillivray, George; Mandelstam, Simone; Mochel, Fanny; Keren, Boris; Jardel, Claude; Turner, Anne M.; Ian Andrews, P.; Smeitink, Jan; Spelbrink, Johannes N.; Heales, Simon J.; Kohda, Masakazu; Ohtake, Akira; Murayama, Kei; Okazaki, Yasushi; Lombès, Anne; Holt, Ian J.; Thorburn, David R.; Spinazzola, Antonella

    2017-01-01

    Abstract Although mitochondrial disorders are clinically heterogeneous, they frequently involve the central nervous system and are among the most common neurogenetic disorders. Identifying the causal genes has benefited enormously from advances in high-throughput sequencing technologies; however, once the defect is known, researchers face the challenge of deciphering the underlying disease mechanism. Here we characterize large biallelic deletions in the region encoding the ATAD3C, ATAD3B and ATAD3A genes. Although high homology complicates genomic analysis of the ATAD3 defects, they can be identified by targeted analysis of standard single nucleotide polymorphism array and whole exome sequencing data. We report deletions that generate chimeric ATAD3B/ATAD3A fusion genes in individuals from four unrelated families with fatal congenital pontocerebellar hypoplasia, whereas a case with genomic rearrangements affecting the ATAD3C/ATAD3B genes on one allele and ATAD3B/ATAD3A genes on the other displays later-onset encephalopathy with cerebellar atrophy, ataxia and dystonia. Fibroblasts from affected individuals display mitochondrial DNA abnormalities, associated with multiple indicators of altered cholesterol metabolism. Moreover, drug-induced perturbations of cholesterol homeostasis cause mitochondrial DNA disorganization in control cells, while mitochondrial DNA aggregation in the genetic cholesterol trafficking disorder Niemann-Pick type C disease further corroborates the interdependence of mitochondrial DNA organization and cholesterol. These data demonstrate the integration of mitochondria in cellular cholesterol homeostasis, in which ATAD3 plays a critical role. The dual problem of perturbed cholesterol metabolism and mitochondrial dysfunction could be widespread in neurological and neurodegenerative diseases. PMID:28549128

  6. DNA Repair in Drosophila: Mutagens, Models, and Missing Genes.

    PubMed

    Sekelsky, Jeff

    2017-02-01

    The numerous processes that damage DNA are counterbalanced by a complex network of repair pathways that, collectively, can mend diverse types of damage. Insights into these pathways have come from studies in many different organisms, including Drosophila melanogaster Indeed, the first ideas about chromosome and gene repair grew out of Drosophila research on the properties of mutations produced by ionizing radiation and mustard gas. Numerous methods have been developed to take advantage of Drosophila genetic tools to elucidate repair processes in whole animals, organs, tissues, and cells. These studies have led to the discovery of key DNA repair pathways, including synthesis-dependent strand annealing, and DNA polymerase theta-mediated end joining. Drosophila appear to utilize other major repair pathways as well, such as base excision repair, nucleotide excision repair, mismatch repair, and interstrand crosslink repair. In a surprising number of cases, however, DNA repair genes whose products play important roles in these pathways in other organisms are missing from the Drosophila genome, raising interesting questions for continued investigations. Copyright © 2017 by the Genetics Society of America.

  7. DNA methylation profiling identifies CG methylation clusters in Arabidopsis genes.

    PubMed

    Tran, Robert K; Henikoff, Jorja G; Zilberman, Daniel; Ditt, Renata F; Jacobsen, Steven E; Henikoff, Steven

    2005-01-26

    Cytosine DNA methylation in vertebrates is widespread, but methylation in plants is found almost exclusively at transposable elements and repetitive DNA. Within regions of methylation, methylcytosines are typically found in CG, CNG, and asymmetric contexts. CG sites are maintained by a plant homolog of mammalian Dnmt1 acting on hemi-methylated DNA after replication. Methylation of CNG and asymmetric sites appears to be maintained at each cell cycle by other mechanisms. We report a new type of DNA methylation in Arabidopsis, dense CG methylation clusters found at scattered sites throughout the genome. These clusters lack non-CG methylation and are preferentially found in genes, although they are relatively deficient toward the 5' end. CG methylation clusters are present in lines derived from different accessions and in mutants that eliminate de novo methylation, indicating that CG methylation clusters are stably maintained at specific sites. Because 5-methylcytosine is mutagenic, the appearance of CG methylation clusters over evolutionary time predicts a genome-wide deficiency of CG dinucleotides and an excess of C(A/T)G trinucleotides within transcribed regions. This is exactly what we find, implying that CG methylation clusters have contributed profoundly to plant gene evolution. We suggest that CG methylation clusters silence cryptic promoters that arise sporadically within transcription units.

  8. Transcriptional Activators of Human Genes with Programmable DNA-Specificity

    PubMed Central

    Hahn, Simone; Streubel, Jana; Bonas, Ulla; Behrens, Sven-Erik; Boch, Jens

    2011-01-01

    TAL (transcription activator-like) effectors are translocated by Xanthomonas bacteria into plant cells where they activate transcription of target genes. DNA target sequence recognition occurs in a unique mode involving a central domain of tandem repeats. Each repeat recognizes a single base pair in a contiguous DNA sequence and a pair of adjacent hypervariable amino acid residues per repeat specifies which base is bound. Rearranging the repeats allows the design of novel TAL proteins with predictable DNA-recognition specificities. TAL protein-based transcriptional activation in plant cells is mediated by a C-terminal activation domain (AD). Here, we created synthetic TAL proteins with designed repeat compositions using a novel modular cloning strategy termed “Golden TAL Technology”. Newly programmed TAL proteins were not only functional in plant cells, but also in human cells and activated targeted expression of exogenous as well as endogenous genes. Transcriptional activation in different human cell lines was markedly improved by replacing the TAL-AD with the VP16-AD of herpes simplex virus. The creation of TAL proteins with potentially any desired DNA-recognition specificity allows their versatile use in biotechnology. PMID:21625585

  9. Persistence of DNA in carcasses, slime and avian feces may affect interpretation of environmental DNA data

    USGS Publications Warehouse

    Merkes, Christopher M.; McCalla, S. Grace; Jensen, Nathan R.; Gaikowski, Mark P.; Amberg, Jon J.

    2014-01-01

    The prevention of non-indigenous aquatic invasive species spreading into new areas is a goal of many resource managers. New techniques have been developed to survey for species that are difficult to capture with conventional gears that involve the detection of their DNA in water samples (eDNA). This technique is currently used to track the invasion of bigheaded carps (silver carp and bighead carp; Hypophthalmichthys molitrix and H. nobilis) in the Chicago Area Waterway System and Upper Mississippi River. In both systems DNA has been detected from silver carp without the capture of a live fish, which has led to some uncertainty about the source of the DNA. The potential contribution to eDNA by vectors and fomites has not been explored. Because barges move from areas with a high abundance of bigheaded carps to areas monitored for the potential presence of silver carp, we used juvenile silver carp to simulate the barge transport of dead bigheaded carp carcasses, slime residue, and predator feces to determine the potential of these sources to supply DNA to uninhabited waters where it could be detected and misinterpreted as indicative of the presence of live bigheaded carp. Our results indicate that all three vectors are feasible sources of detectable eDNA for at least one month after their deposition. This suggests that current monitoring programs must consider alternative vectors of DNA in the environment and consider alternative strategies to minimize the detection of DNA not directly released from live bigheaded carps.

  10. Both DNA strands of antibody genes are hypermutation targets

    PubMed Central

    Milstein, Cesar; Neuberger, Michael S.; Staden, Rodger

    1998-01-01

    During the maturation of the immune response, antibody genes are subjected to localized hypermutation. Mutations are not evenly distributed along the V gene; intrinsic hot spots exist that are correlated with primary sequence motifs. Although the mechanism of hypermutation remains unknown, it has been proposed to exhibit DNA strand polarity because purine residues on the coding strand are more frequently targeted for mutation than pyrimidines. However, this polarity may not be an intrinsic property of the hypermutation mechanism but a consequence of evolutionary-selected peculiarities of V gene sequences. Furthermore, the possibility that both strands are hypermutation targets has received little attention. To discriminate between these possibilities, we have analyzed the average frequency of mutations of each of the three bases of all nucleotide triplets by using large databases taken from both V and non-V mutation targets. We also have reassessed the sequence motifs associated with hot spots. We find that even in non-Ig sequences, A mutates more than T, consistent with a strand-dependent component to targeting. However, the mutation biases of triplets and of their inverted complements are correlated, demonstrating that there is a sequence-specific but strand-independent component to mutational targeting. Thus, there are two aspects of the hypermutation process that are sensitive to local DNA sequences, one that is DNA strand-dependent and the other that is not. PMID:9671757

  11. Local DNA hypomethylation activates genes in rice endosperm

    PubMed Central

    Zemach, Assaf; Kim, M. Yvonne; Silva, Pedro; Rodrigues, Jessica A.; Dotson, Bradley; Brooks, Matthew D.; Zilberman, Daniel

    2010-01-01

    Cytosine methylation silences transposable elements in plants, vertebrates, and fungi but also regulates gene expression. Plant methylation is catalyzed by three families of enzymes, each with a preferred sequence context: CG, CHG (H = A, C, or T), and CHH, with CHH methylation targeted by the RNAi pathway. Arabidopsis thaliana endosperm, a placenta-like tissue that nourishes the embryo, is globally hypomethylated in the CG context while retaining high non-CG methylation. Global methylation dynamics in seeds of cereal crops that provide the bulk of human nutrition remain unknown. Here, we show that rice endosperm DNA is hypomethylated in all sequence contexts. Non-CG methylation is reduced evenly across the genome, whereas CG hypomethylation is localized. CHH methylation of small transposable elements is increased in embryos, suggesting that endosperm demethylation enhances transposon silencing. Genes preferentially expressed in endosperm, including those coding for major storage proteins and starch synthesizing enzymes, are frequently hypomethylated in endosperm, indicating that DNA methylation is a crucial regulator of rice endosperm biogenesis. Our data show that genome-wide reshaping of seed DNA methylation is conserved among angiosperms and has a profound effect on gene expression in cereal crops. PMID:20937895

  12. DNA hydroxymethylation controls cardiomyocyte gene expression in development and hypertrophy

    PubMed Central

    Greco, Carolina M.; Kunderfranco, Paolo; Rubino, Marcello; Larcher, Veronica; Carullo, Pierluigi; Anselmo, Achille; Kurz, Kerstin; Carell, Thomas; Angius, Andrea; Latronico, Michael V. G.; Papait, Roberto; Condorelli, Gianluigi

    2016-01-01

    Methylation at 5-cytosine (5-mC) is a fundamental epigenetic DNA modification associated recently with cardiac disease. In contrast, the role of 5-hydroxymethylcytosine (5-hmC)—5-mC's oxidation product—in cardiac biology and disease is unknown. Here we assess the hydroxymethylome in embryonic, neonatal, adult and hypertrophic mouse cardiomyocytes, showing that dynamic modulation of hydroxymethylated DNA is associated with specific transcriptional networks during heart development and failure. DNA hydroxymethylation marks the body of highly expressed genes as well as distal regulatory regions with enhanced activity. Moreover, pathological hypertrophy is characterized by a shift towards a neonatal 5-hmC distribution pattern. We also show that the ten-eleven translocation 2 (TET2) enzyme regulates the expression of key cardiac genes, such as Myh7, through 5-hmC deposition on the gene body and at enhancers. Thus, we provide a genome-wide analysis of 5-hmC in the cardiomyocyte and suggest a role for this epigenetic modification in heart development and disease. PMID:27489048

  13. Transient Silencing of DNA Repair Genes Improves Targeted Gene Integration in the Filamentous Fungus Trichoderma reesei.

    PubMed

    Chum, Pak Yang; Schmidt, Georg; Saloheimo, Markku; Landowski, Christopher P

    2017-08-01

    Trichoderma reesei is a filamentous fungus that is used worldwide to produce industrial enzymes. Industrial strains have traditionally been created though systematic strain improvement using mutagenesis and screening approaches. It is also desirable to specifically manipulate the genes of the organism to further improve and to modify the strain. Targeted integration in filamentous fungi is typically hampered by very low frequencies of homologous recombination. To address this limitation, we have developed a simple transient method for silencing genes in T. reesei Using gene-specific small interfering RNAs (siRNAs) targeted to mus53, we could achieve up to 90% knockdown of mus53 mRNA. As a practical example, we demonstrated that transient silencing of DNA repair genes significantly improved homologous integration of DNA at a specific locus in a standard protoplast transformation. The best transient silencing of mus53 with siRNAs in protoplasts could achieve up to 59% marker gene integration.IMPORTANCE The previous solution for improving targeted integration efficiency has been deleting nonhomologous end joining (NHEJ) DNA repair genes. However, deleting these important repair genes may lead to unintended consequences for genomic stability and could lead to the accumulation of spontaneous mutations. Our method of transiently silencing NHEJ repair pathway genes allows recovery of their important repair functions. Here we report a silencing approach for improving targeted DNA integration in filamentous fungi. Furthermore, our transient silencing method is a truly flexible approach that is capable of knocking down the expression of a target gene in growing mycelial cultures, which could facilitate the broad study of gene functions in T. reesei. Copyright © 2017 American Society for Microbiology.

  14. Benzo(a)pyrene induces similar gene expression changes in testis of DNA repair proficient and deficient mice

    PubMed Central

    2010-01-01

    Background Benzo [a]pyrene (B[a]P) exposure induces DNA adducts at all stages of spermatogenesis and in testis, and removal of these lesions is less efficient in nucleotide excision repair deficient Xpc-/- mice than in wild type mice. In this study, we investigated by using microarray technology whether compromised DNA repair in Xpc-/- mice may lead to a transcriptional reaction of the testis to cope with increased levels of B[a]P induced DNA damage. Results Two-Way ANOVA revealed only 4 genes differentially expressed between wild type and Xpc-/- mice, and 984 genes between testes of B[a]P treated and untreated mice irrespective of the mouse genotype. However, the level in which these B[a]P regulated genes are expressed differs between Wt and Xpc-/- mice (p = 0.000000141), and were predominantly involved in the regulation of cell cycle, translation, chromatin structure and spermatogenesis, indicating a general stress response. In addition, analysis of cell cycle phase dependent gene expression revealed that expression of genes involved in G1-S and G2-M phase arrest was increased after B[a]P exposure in both genotypes. A slightly higher induction of average gene expression was observed at the G2-M checkpoint in Xpc-/- mice, but this did not reach statistical significance (P = 0.086). Other processes that were expected to have changed by exposure, like apoptosis and DNA repair, were not found to be modulated at the level of gene expression. Conclusion Gene expression in testis of untreated Xpc-/- and wild type mice were very similar, with only 4 genes differentially expressed. Exposure to benzo(a)pyrene affected the expression of genes that are involved in cell cycle regulation in both genotypes, indicating that the presence of unrepaired DNA damage in testis blocks cell proliferation to protect DNA integrity in both DNA repair proficient and deficient animals. PMID:20504355

  15. Associations between DNA methylation and schizophrenia-related intermediate phenotypes - a gene set enrichment analysis.

    PubMed

    Hass, Johanna; Walton, Esther; Wright, Carrie; Beyer, Andreas; Scholz, Markus; Turner, Jessica; Liu, Jingyu; Smolka, Michael N; Roessner, Veit; Sponheim, Scott R; Gollub, Randy L; Calhoun, Vince D; Ehrlich, Stefan

    2015-06-03

    Multiple genetic approaches have identified microRNAs as key effectors in psychiatric disorders as they post-transcriptionally regulate expression of thousands of target genes. However, their role in specific psychiatric diseases remains poorly understood. In addition, epigenetic mechanisms such as DNA methylation, which affect the expression of both microRNAs and coding genes, are critical for our understanding of molecular mechanisms in schizophrenia. Using clinical, imaging, genetic, and epigenetic data of 103 patients with schizophrenia and 111 healthy controls of the Mind Clinical Imaging Consortium (MCIC) study of schizophrenia, we conducted gene set enrichment analysis to identify markers for schizophrenia-associated intermediate phenotypes. Genes were ranked based on the correlation between DNA methylation patterns and each phenotype, and then searched for enrichment in 221 predicted microRNA target gene sets. We found the predicted hsa-miR-219a-5p target gene set to be significantly enriched for genes (EPHA4, PKNOX1, ESR1, among others) whose methylation status is correlated with hippocampal volume independent of disease status. Our results were strengthened by significant associations between hsa-miR-219a-5p target gene methylation patterns and hippocampus-related neuropsychological variables. IPA pathway analysis of the respective predicted hsa-miR-219a-5p target genes revealed associated network functions in behavior and developmental disorders. Altered methylation patterns of predicted hsa-miR-219a-5p target genes are associated with a structural aberration of the brain that has been proposed as a possible biomarker for schizophrenia. The (dys)regulation of microRNA target genes by epigenetic mechanisms may confer additional risk for developing psychiatric symptoms. Further study is needed to understand possible interactions between microRNAs and epigenetic changes and their impact on risk for brain-based disorders such as schizophrenia.

  16. Associations between DNA methylation and schizophrenia-related intermediate phenotypes a gene set enrichment analysis

    PubMed Central

    Hass, Johanna; Walton, Esther; Wright, Carrie; Beyer, Andreas; Scholz, Markus; Turner, Jessica; Liu, Jingyu; Smolka, Michael N.; Roessner, Veit; Sponheim, Scott R.; Gollub, Randy L.; Calhoun, Vince D.; Ehrlich, Stefan

    2015-01-01

    Multiple genetic approaches have identified microRNAs as key effectors in psychiatric disorders as they post-transcriptionally regulate expression of thousands of target genes. However, their role in specific psychiatric diseases remains poorly understood. In addition, epigenetic mechanisms such as DNA methylation, which affect the expression of both microRNAs and coding genes, are critical for our understanding of molecular mechanisms in schizophrenia. Using clinical, imaging, genetic, and epigenetic data of 103 patients with schizophrenia and 111 healthy controls of the Mind Clinical Imaging Consortium (MCIC) study of schizophrenia, we conducted gene set enrichment analysis to identify markers for schizophrenia-associated intermediate phenotypes. Genes were ranked based on the correlation between DNA methylation patterns and each phenotype, and then searched for enrichment in 221 predicted microRNA target gene sets. We found the predicted hsa-miR-219a-5p target gene set to be significantly enriched for genes (EPHA4, PKNOX1, ESR1, amongst others) whose methylation status is correlated with hippocampal volume independent of disease status. Our results were strengthened by significant associations between hsa-miR-219a-5p target gene methylation patterns and hippocampus-related neuropsychological variables. IPA pathway analysis of the respective predicted hsa-miR-219a-5p target genes revealed associated network functions in behaviour and developmental disorders. Altered methylation patterns of predicted hsa-miR-219a-5p target genes are associated with a structural aberration of the brain that has been proposed as a possible biomarker for schizophrenia. The (dys)regulation of microRNA target genes by epigenetic mechanisms may confer additional risk for developing psychiatric symptoms. Further study is needed to understand possible interactions between microRNAs and epigenetic changes and their impact on risk for brain-based disorders such as schizophrenia. PMID

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

    PubMed

    Bartholdy, Christina; Olszewska, Wieslawa; Stryhn, Anette; Thomsen, Allan Randrup; Openshaw, Peter J M

    2004-10-01

    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-gun immunization of BALB/c mice with this construct induced an antigen-specific CD8+ T-cell memory. After intranasal RSV challenge, accelerated CD8+ T-cell responses were observed in pulmonary lymph nodes and virus clearance from the lungs was enhanced. The construct induced weaker CD8+ T-cell responses than those 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 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+ T-cell responses were not induced. Thus, in addition to specific CD8+ T cell-mediated immunopathology, gene-gun DNA vaccination causes non-specific enhancement of RSV disease without affecting virus clearance.

  18. How hormone receptor-DNA binding affects nucleosomal DNA: the role of symmetry.

    PubMed Central

    Bishop, T C; Kosztin, D; Schulten, K

    1997-01-01

    Molecular dynamics simulations have been employed to determine the optimal conformation of an estrogen receptor DNA binding domain dimer bound to a consensus response element, ds(AGGTCACAGTGACCT), and to a nonconsensus response element, ds(AGAACACAGTGACCT). The structures simulated were derived from a crystallographic structure and solvated by a sphere (45-A radius) of explicit water and counterions. Long-range electrostatic interactions were accounted for during 100-ps simulations by means of a fast multipole expansion algorithm combined with a multiple time-step scheme in the molecular dynamics package NAMD. The simulations demonstrate that the dimer induces a bent and underwound (10.7 bp/turn) conformation in the DNA. The bending reflects the dyad symmetry of the receptor dimer and can be described as an S-shaped curve in the helical axis of DNA when projected onto a plane. A similar bent and underwound conformation is observed for nucleosomal DNA near the nucleosome's dyad axis that reflects the symmetry of the histone octamer. We propose that when a receptor dimer binds to a nucleosome, the most favorable dimer-DNA and histone-DNA interactions are achieved if the respective symmetry axes are aligned. Such positioning of a receptor dimer over the dyad of nucleosome B in the mouse mammary tumor virus promoter is in agreement with experiment. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 9 FIGURE 11 PMID:9129808

  19. A multistep genomic screen identifies new genes required for repair of DNA double-strand breaks in Saccharomyces cerevisiae.

    PubMed

    McKinney, Jennifer Summers; Sethi, Sunaina; Tripp, Jennifer DeMars; Nguyen, Thuy N; Sanderson, Brian A; Westmoreland, James W; Resnick, Michael A; Lewis, L Kevin

    2013-04-15

    Efficient mechanisms for rejoining of DNA double-strand breaks (DSBs) are vital because misrepair of such lesions leads to mutation, aneuploidy and loss of cell viability. DSB repair is mediated by proteins acting in two major pathways, called homologous recombination and nonhomologous end-joining. Repair efficiency is also modulated by other processes such as sister chromatid cohesion, nucleosome remodeling and DNA damage checkpoints. The total number of genes influencing DSB repair efficiency is unknown. To identify new yeast genes affecting DSB repair, genes linked to gamma radiation resistance in previous genome-wide surveys were tested for their impact on repair of site-specific DSBs generated by in vivo expression of EcoRI endonuclease. Eight members of the RAD52 group of DNA repair genes (RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, MRE11 and XRS2) and 73 additional genes were found to be required for efficient repair of EcoRI-induced DSBs in screens utilizing both MATa and MATα deletion strain libraries. Most mutants were also sensitive to the clastogenic chemicals MMS and bleomycin. Several of the non-RAD52 group genes have previously been linked to DNA repair and over half of the genes affect nuclear processes. Many proteins encoded by the protective genes have previously been shown to associate physically with each other and with known DNA repair proteins in high-throughput proteomics studies. A majority of the proteins (64%) share sequence similarity with human proteins, suggesting that they serve similar functions. We have used a genetic screening approach to detect new genes required for efficient repair of DSBs in Saccharomyces cerevisiae. The findings have spotlighted new genes that are critical for maintenance of genome integrity and are therefore of greatest concern for their potential impact when the corresponding gene orthologs and homologs are inactivated or polymorphic in human cells.

  20. Quantitative expression of candidate genes affecting eggshell color.

    PubMed

    Zheng, Chuanwei; Li, Zesheng; Yang, Ning; Ning, Zhonghua

    2014-05-01

    There are three pigments that affect the color of an eggshell: protoporphyrin, biliverdin and biliverdin-zinc chelate. Protoporphyrin is the main pigment in brown and light-brown eggshells, whereas very little protoporphyrin is found in white eggshells. Eggshell protoporphyrin is derived from the heme formation in birds. Coproporphyrinogen III oxidase (CPOX) and ferrochelatase (FECH) represent rate-limiting enzymes for the heme-biosynthetic pathway. Breast cancer resistance protein (BCRP), feline leukemia virus receptor (FLVCR), and heme-responsive gene-1 (HRG1) serve as primary transporters for both protoporphyrinogen and heme. Finally, four organic anion transporting polypeptide family members (including solute carrier organic anion transporter family, SLCO1C1, SLCO1A2, SLCO1B3 and LOC418189) may affect pigment transport within eggshells. Here we measured gene expression levels in key tissues of egg-producing hens. We analyzed three different types of hens that generated distinct eggshell colors: white, pink or brown. Our data revealed three ways in which eggshell color was genetically influenced. First, high-level expression of CPOX generated more protoporphyrinogen and a brown eggshell color. In contrast, high expression of FECH likely converted more protoporphyrinogen into heme, reduced protoporphyrinogen levels within the eggshell and generated a light color. Second, heme transporters also affected eggshell color. High-level expression of BCRP, HRG1 and FLVCR were associated with brown, white and generally lighter eggshell colors, respectively. Finally, protoporphyrin precipitation also affected eggshell color, as high expression of both SLCO1A2 and SLCO1C1 were associated with brown eggshell color. As such, we have identified seven genes in which expression levels in different tissues were associated with eggshell color. © 2014 Japanese Society of Animal Science.

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

    PubMed

    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.

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

    PubMed Central

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

    1981-01-01

    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. PMID:6458041

  3. Gestational diabetes mellitus epigenetically affects genes predominantly involved in metabolic diseases.

    PubMed

    Ruchat, Stephanie-May; Houde, Andrée-Anne; Voisin, Grégory; St-Pierre, Julie; Perron, Patrice; Baillargeon, Jean-Patrice; Gaudet, Daniel; Hivert, Marie-France; Brisson, Diane; Bouchard, Luigi

    2013-09-01

    Offspring exposed to gestational diabetes mellitus (GDM) have an increased risk for chronic diseases, and one promising mechanism for fetal metabolic programming is epigenetics. Therefore, we postulated that GDM exposure impacts the offspring's methylome and used an epigenomic approach to explore this hypothesis. Placenta and cord blood samples were obtained from 44 newborns, including 30 exposed to GDM. Women were recruited at first trimester of pregnancy and followed until delivery. GDM was assessed after a 75-g oral glucose tolerance test at 24-28 weeks of pregnancy. DNA methylation was measured at>485,000 CpG sites (Infinium HumanMethylation450 BeadChips). Ingenuity Pathway Analysis was conducted to identify metabolic pathways epigenetically affected by GDM. Our results showed that 3,271 and 3,758 genes in placenta and cord blood, respectively, were potentially differentially methylated between samples exposed or not to GDM (p-values down to 1 × 10(-06); none reached the genome-wide significance levels), with more than 25% (n = 1,029) being common to both tissues. Mean DNA methylation differences between groups were 5.7 ± 3.2% and 3.4 ± 1.9% for placenta and cord blood, respectively. These genes were likely involved in the metabolic diseases pathway (up to 115 genes (11%), p-values for pathways = 1.9 × 10(-13)genes, 326 in placenta and 117 in cord blood were also associated with newborn weight. Our results therefore suggest that GDM has epigenetic effects on genes preferentially involved in the metabolic diseases pathway, with consequences on fetal growth and development, and provide supportive evidence that DNA methylation is involved in fetal metabolic programming.

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

    SciTech Connect

    King-Himmelreich, Tanya S.; Schramm, Stefanie; Wolters, Miriam C.; Schmetzer, Julia; Möser, Christine V.; Knothe, Claudia; Geisslinger, Gerd

    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.

  5. LexA-independent DNA damage-mediated induction of gene expression in Myxococcus xanthus.

    PubMed

    Campoy, Susana; Fontes, Marta; Padmanabhan, S; Cortés, Pilar; Llagostera, Montserrat; Barbé, Jordi

    2003-08-01

    Myxococcus xanthus, a member of the Proteobacteria delta-class, has two independent recA genes, recA1 and recA2, but only recA2 is DNA damage-inducible. The lexA gene has been isolated from M. xanthus by PCR amplification with oligonucleotides designed after sequence identification by tblastn analysis of its genome at the Cereon Microbial Sequence Database. The M. xanthus purified LexA protein is shown to bind specifically to the consensus sequence CTRHAMRYBYGTTCAGS present upstream of lexA and recA2. A degenerate copy of this motif but with important differences can be identified in the region upstream of the recA1 gene. A knock-out lexA(Def) mutant that has been generated does not differ significantly from wild type in morphology, growth rate, light-induced carotenogenesis or development. Using transcriptional lacZ fusions and quantitative RT-PCR analysis, it has been demonstrated that expression of both lexA and recA2 genes is constitutive in the lexA(Def) mutant, whereas the transcription of the DNA damage non-inducible recA1 gene is not affected in this strain. recN and ssb, whose expression in Escherichia coli are LexA-regulated, are induced by DNA damage in the M. xanthus lexA(Def) mutant. These data reveal the existence of different regulatory mechanisms for DNA damage-inducible genes in bacteria belonging to different phyla.

  6. Hydrophobin gene expression affects hyphal wall composition in Schizophyllum commune.

    PubMed

    van Wetter, M A; Wösten, H A; Sietsma, J H; Wessels, J G

    2000-11-01

    Disruption of the SC3 hydrophobin gene of Schizophyllum commune (DeltaSC3 strain) affected the composition of the cell wall. Compared to a wild-type strain the amount of mucilage (i.e., water-soluble (1-3)beta-glucan with single glucose residues attached by (1-6)beta-linkages) increased considerably, while the amount of alkali-resistant glucan (linked to chitin) decreased. Reintroduction of the SC3 gene or other hydrophobins genes expressed behind the SC3 promotor restored wild-type cell wall composition. However, addition of purified SC3 protein to the medium or growing the DeltaSC3 strain in spent medium of the wild-type strain had no effect. In young cultures of wild-type strains of S.commune, not yet expressing SC3, the amount of mucilage was also relatively high. These data show that hydrophobins not only function at hydrophilic/hydrophobic interfaces, as shown previously, but also affect wall composition.

  7. A Bacillus subtilis dnaG mutant harbours a mutation in a gene homologous to the dnaN gene of Escherichia coli.

    PubMed

    Ogasawara, N; Moriya, S; Mazza, G; Yoshikawa, H

    1986-01-01

    A dnaG mutation of Bacillus subtilis, dnaG5, was found to be linked closely to recF. We have reported previously that two putative dna genes, 'dnaA' and 'dnaN', highly homologous to Escherichia coli's dnaA and dnaN, respectively, were located adjacent to recF [Ogasawara et al., EMBO J., 4 (1985) 3345-3350]. Transformation by various fragments cloned from the 'dnaA'-recF region of the wild-type cell revealed that a 532-bp AluI fragment containing 5'-portion of the 'dnaN' gene could transform the dnaG5 mutation. The nucleotide (nt) sequence of the same fragment cloned from the mutant cell shows a single nt change in the ORF of 'dnaN' which in turn causes a single amino acid alteration from Gly to Arg. The 'dnaN' gene is now proven to be a dna gene, mutations in which result in instant arrest of chromosomal replication.

  8. African swine fever virus multigene family 360 and 530 genes affect host interferon response.

    PubMed

    Afonso, C L; Piccone, M E; Zaffuto, K M; Neilan, J; Kutish, G F; Lu, Z; Balinsky, C A; Gibb, T R; Bean, T J; Zsak, L; Rock, D L

    2004-02-01

    African swine fever virus (ASFV) multigene family 360 and 530 (MGF360/530) genes affect viral growth in macrophage cell cultures and virulence in pigs (L. Zsak, Z. Lu, T. G. Burrage, J. G. Neilan, G. F. Kutish, D. M. Moore, and D. L. Rock, J. Virol. 75:3066-3076, 2001). The mechanism by which these novel genes affect virus-host interactions is unknown. To define MGF360/530 gene function, we compared macrophage transcriptional responses following infection with parental ASFV (Pr4) and an MGF360/530 deletion mutant (Pr4 Delta 35). A swine cDNA microarray containing 7,712 macrophage cDNA clones was used to compare the transcriptional profiles of swine macrophages infected with Pr4 and Pr4 Delta 35 at 3 and 6 h postinfection (hpi). While at 3 hpi most (7,564) of the genes had similar expression levels in cells infected with either virus, 38 genes had significantly increased (>2.0-fold, P < 0.05) mRNA levels in Pr4 Delta 35-infected macrophages. Similar up-regulation of these genes was observed at 6 hpi. Viral infection was required for this induced transcriptional response. Most Pr Delta 35 up-regulated genes were part of a type I interferon (IFN) response or were genes that are normally induced by double-stranded RNA and/or viral infection. These included monocyte chemoattractant protein, transmembrane protein 3, tetratricopeptide repeat protein 1, a ubiquitin-like 17-kDa protein, ubiquitin-specific protease ISG43, an RNA helicase DEAD box protein, GTP-binding MX protein, the cytokine IP-10, and the PKR activator PACT. Differential expression of IFN early-response genes in Pr4 Delta 35 relative to Pr4 was confirmed by Northern blot analysis and real-time PCR. Analysis of IFN-alpha mRNA and secreted IFN-alpha levels at 3, 8, and 24 hpi revealed undetectable IFN-alpha in mock- and Pr4-infected macrophages but significant IFN-alpha levels at 24 hpi in Pr4 Delta 35-infected macrophages. The absence of IFN-alpha in Pr4-infected macrophages suggests that MGF360/530 genes

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

    SciTech Connect

    Woloschak, G.E.; Weaver, P.; Churchill, M.; Chang-Liu, C-M.; Libertin, C.R.

    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.

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

    SciTech Connect

    Woloschak, G.E.; Weaver, P.; Churchill, M.; Chang-Liu, C-M. ); Libertin, C.R. )

    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.

  11. Mapping of bionic array electric field focusing in plasmid DNA-based gene electrotransfer.

    PubMed

    Browne, C J; Pinyon, J L; Housley, D M; Crawford, E N; Lovell, N H; Klugmann, M; Housley, G D

    2016-04-01

    Molecular medicine through gene therapy is challenged to achieve targeted action. This is now possible utilizing bionic electrode arrays for focal delivery of naked (plasmid) DNA via gene electrotransfer. Here, we establish the properties of array-based electroporation affecting targeted gene delivery. An array with eight 300 μm platinum ring electrodes configured as a cochlear implant bionic interface was used to transduce HEK293 cell monolayers with a plasmid-DNA green fluorescent protein (GFP) reporter gene construct. Electroporation parameters were pulse intensity, number, duration, separation and electrode configuration. The latter determined the shape of the electric fields, which were mapped using a voltage probe. Electrode array-based electroporation was found to require ~100 × lower applied voltages for cell transduction than conventional electroporation. This was found to be due to compression of the field lines orthogonal to the array. A circular area of GFP-positive cells was created when the electrodes were ganged together as four adjacent anodes and four cathodes, whereas alternating electrode polarity created a linear area of GFP-positive cells. The refinement of gene delivery parameters was validated in vivo in the guinea pig cochlea. These findings have significant clinical ramifications, where spatiotemporal control of gene expression can be predicted by manipulation of the electric field via current steering at a cellular level.

  12. Mapping of bionic array electric field focusing in plasmid DNA-based gene electrotransfer

    PubMed Central

    Browne, C J; Pinyon, J L; Housley, D M; Crawford, E N; Lovell, N H; Klugmann, M; Housley, G D

    2016-01-01

    Molecular medicine through gene therapy is challenged to achieve targeted action. This is now possible utilizing bionic electrode arrays for focal delivery of naked (plasmid) DNA via gene electrotransfer. Here, we establish the properties of array-based electroporation affecting targeted gene delivery. An array with eight 300 μm platinum ring electrodes configured as a cochlear implant bionic interface was used to transduce HEK293 cell monolayers with a plasmid-DNA green fluorescent protein (GFP) reporter gene construct. Electroporation parameters were pulse intensity, number, duration, separation and electrode configuration. The latter determined the shape of the electric fields, which were mapped using a voltage probe. Electrode array-based electroporation was found to require ~100 × lower applied voltages for cell transduction than conventional electroporation. This was found to be due to compression of the field lines orthogonal to the array. A circular area of GFP-positive cells was created when the electrodes were ganged together as four adjacent anodes and four cathodes, whereas alternating electrode polarity created a linear area of GFP-positive cells. The refinement of gene delivery parameters was validated in vivo in the guinea pig cochlea. These findings have significant clinical ramifications, where spatiotemporal control of gene expression can be predicted by manipulation of the electric field via current steering at a cellular level. PMID:26826485

  13. Search for genes essential for pneumococcal transformation: the RADA DNA repair protein plays a role in genomic recombination of donor DNA.

    PubMed

    Burghout, Peter; Bootsma, Hester J; Kloosterman, Tomas G; Bijlsma, Jetta J E; de Jongh, Christa E; Kuipers, Oscar P; Hermans, Peter W M

    2007-09-01

    We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counterselection of mutants in 21 different genes during the challenge. Eight of the counterselected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants of seven of the eight remaining genes, i.e., spr0459-spr0460, spr0777, spr0838, spr1259-spr1260, and spr1357, resulted in reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation with chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and in DNA damage repair in S. pneumoniae.

  14. Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus

    PubMed Central

    Monier, Adam; Pagarete, António; de Vargas, Colomban; Allen, Michael J.; Read, Betsy; Claverie, Jean-Michel; Ogata, Hiroyuki

    2009-01-01

    Interactions between viruses and phytoplankton, the main primary producers in the oceans, affect global biogeochemical cycles and climate. Recent studies are increasingly revealing possible cases of gene transfers between cyanobacteria and phages, which might have played significant roles in the evolution of cyanobacteria/phage systems. However, little has been documented about the occurrence of horizontal gene transfer in eukaryotic phytoplankton/virus systems. Here we report phylogenetic evidence for the transfer of seven genes involved in the sphingolipid biosynthesis pathway between the cosmopolitan eukaryotic microalga Emiliania huxleyi and its large DNA virus EhV. PCR assays indicate that these genes are prevalent in E. huxleyi and EhV strains isolated from different geographic locations. Patterns of protein and gene sequence conservation support that these genes are functional in both E. huxleyi and EhV. This is the first clear case of horizontal gene transfer of multiple functionally linked enzymes in a eukaryotic phytoplankton–virus system. We examine arguments for the possible direction of the gene transfer. The virus-to-host direction suggests the existence of ancient viruses that controlled the complex metabolic pathway in order to infect primitive eukaryotic cells. In contrast, the host-to-virus direction suggests that the serial acquisition of genes involved in the same metabolic pathway might have been a strategy for the ancestor of EhVs to stay ahead of their closest relatives in the great evolutionary race for survival. PMID:19451591

  15. Hemoglobin Pasadena: identification of the gene mutant by DNA analysis using synthetic DNA probes.

    PubMed

    Rahbar, S; Rosen, R; Nozari, G; Lee, T D; Asmerom, Y; Wallace, R B

    1988-03-01

    Hemoglobin Pasadena [beta 75(E19)Leu----Arg] was found in a boy who had an acute episode of anemia and rapid splenic enlargement. His father was the only other member of a large family with this hemoglobinopathy. We have used gene mapping techniques for direct identification of the beta-globin gene mutation. To correlate the DNA findings with the structural identification of this variant, we have also performed globin chain separation and analysis of the tryptic peptides using high performance liquid chromatography and secondary ion mass spectral analysis.

  16. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes

    PubMed Central

    Barber-Zucker, Shiran; Gordân, Raluca; Lukatsky, David B.

    2015-01-01

    Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using

  17. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes.

    PubMed

    Afek, Ariel; Cohen, Hila; Barber-Zucker, Shiran; Gordân, Raluca; Lukatsky, David B

    2015-08-01

    Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using

  18. Histone deacetylase inhibition modulates histone acetylation at gene promoter regions and affects genome-wide gene transcription in Schistosoma mansoni

    PubMed Central

    Anderson, Letícia; Gomes, Monete Rajão; daSilva, Lucas Ferreira; Pereira, Adriana da Silva Andrade; Mourão, Marina M.; Romier, Christophe; Pierce, Raymond

    2017-01-01

    Background Schistosomiasis is a parasitic disease infecting hundreds of millions of people worldwide. Treatment depends on a single drug, praziquantel, which kills the Schistosoma spp. parasite only at the adult stage. HDAC inhibitors (HDACi) such as Trichostatin A (TSA) induce parasite mortality in vitro (schistosomula and adult worms), however the downstream effects of histone hyperacetylation on the parasite are not known. Methodology/Principal findings TSA treatment of adult worms in vitro increased histone acetylation at H3K9ac and H3K14ac, which are transcription activation marks, not affecting the unrelated transcription repression mark H3K27me3. We investigated the effect of TSA HDACi on schistosomula gene expression at three different time points, finding a marked genome-wide change in the transcriptome profile. Gene transcription activity was correlated with changes on the chromatin acetylation mark at gene promoter regions. Moreover, combining expression data with ChIP-Seq public data for schistosomula, we found that differentially expressed genes having the H3K4me3 mark at their promoter region in general showed transcription activation upon HDACi treatment, compared with those without the mark, which showed transcription down-regulation. Affected genes are enriched for DNA replication processes, most of them being up-regulated. Twenty out of 22 genes encoding proteins involved in reducing reactive oxygen species accumulation were down-regulated. Dozens of genes encoding proteins with histone reader motifs were changed, including SmEED from the PRC2 complex. We targeted SmEZH2 methyltransferase PRC2 component with a new EZH2 inhibitor (GSK343) and showed a synergistic effect with TSA, significantly increasing schistosomula mortality. Conclusions/Significance Genome-wide gene expression analyses have identified important pathways and cellular functions that were affected and may explain the schistosomicidal effect of TSA HDACi. The change in expression

  19. Histone deacetylase inhibition modulates histone acetylation at gene promoter regions and affects genome-wide gene transcription in Schistosoma mansoni.

    PubMed

    Anderson, Letícia; Gomes, Monete Rajão; daSilva, Lucas Ferreira; Pereira, Adriana da Silva Andrade; Mourão, Marina M; Romier, Christophe; Pierce, Raymond; Verjovski-Almeida, Sergio

    2017-04-01

    Schistosomiasis is a parasitic disease infecting hundreds of millions of people worldwide. Treatment depends on a single drug, praziquantel, which kills the Schistosoma spp. parasite only at the adult stage. HDAC inhibitors (HDACi) such as Trichostatin A (TSA) induce parasite mortality in vitro (schistosomula and adult worms), however the downstream effects of histone hyperacetylation on the parasite are not known. TSA treatment of adult worms in vitro increased histone acetylation at H3K9ac and H3K14ac, which are transcription activation marks, not affecting the unrelated transcription repression mark H3K27me3. We investigated the effect of TSA HDACi on schistosomula gene expression at three different time points, finding a marked genome-wide change in the transcriptome profile. Gene transcription activity was correlated with changes on the chromatin acetylation mark at gene promoter regions. Moreover, combining expression data with ChIP-Seq public data for schistosomula, we found that differentially expressed genes having the H3K4me3 mark at their promoter region in general showed transcription activation upon HDACi treatment, compared with those without the mark, which showed transcription down-regulation. Affected genes are enriched for DNA replication processes, most of them being up-regulated. Twenty out of 22 genes encoding proteins involved in reducing reactive oxygen species accumulation were down-regulated. Dozens of genes encoding proteins with histone reader motifs were changed, including SmEED from the PRC2 complex. We targeted SmEZH2 methyltransferase PRC2 component with a new EZH2 inhibitor (GSK343) and showed a synergistic effect with TSA, significantly increasing schistosomula mortality. Genome-wide gene expression analyses have identified important pathways and cellular functions that were affected and may explain the schistosomicidal effect of TSA HDACi. The change in expression of dozens of histone reader genes involved in regulation of the

  20. Approaches to diagnose DNA mismatch repair gene defects in cancer.

    PubMed

    Peña-Diaz, Javier; Rasmussen, Lene Juel

    2016-02-01

    The DNA repair pathway mismatch repair (MMR) is responsible for the recognition and correction of DNA biosynthetic errors caused by inaccurate nucleotide incorporation during replication. Faulty MMR leads to failure to address the mispairs or insertion deletion loops (IDLs) left behind by the replicative polymerases and results in increased mutation load at the genome. The realization that defective MMR leads to a hypermutation phenotype and increased risk of tumorigenesis highlights the relevance of this pathway for human disease. The association of MMR defects with increased risk of cancer development was first observed in colorectal cancer patients that carried inactivating germline mutations in MMR genes and the disease was named as hereditary non-polyposis colorectal cancer (HNPCC). Currently, a growing list of cancers is found to be MMR defective and HNPCC has been renamed Lynch syndrome (LS) partly to include the associated risk of developing extra-colonic cancers. In addition, a number of non-hereditary, mostly epigenetic, alterations of MMR genes have been described in sporadic tumors. Besides conferring a strong cancer predisposition, genetic or epigenetic inactivation of MMR genes also renders cells resistant to some chemotherapeutic agents. Therefore, diagnosis of MMR deficiency has important implications for the management of the patients, the surveillance of their relatives in the case of LS and for the choice of treatment. Some of the alterations found in MMR genes have already been well defined and their pathogenicity assessed. Despite this substantial wealth of knowledge, the effects of a large number of alterations remain uncharacterized (variants of uncertain significance, VUSs). The advent of personalized genomics is likely to increase the list of VUSs found in MMR genes and anticipates the need of diagnostic tools for rapid assessment of their pathogenicity. This review describes current tools and future strategies for addressing the relevance

  1. Requirement of the Escherichia coli dnaA gene product for plasmid F maintenance.

    PubMed Central

    Kline, B C; Kogoma, T; Tam, J E; Shields, M S

    1986-01-01

    There are DnaA protein-binding sites in at least one F origin of replication, and only potentially leaky dnaA(Ts) mutations had ever been used in previous studies indicating that F replication was independent of the dnaA gene product. Here we show that an Escherichia coli dnaA::Tn10 host which does not make a dnaA gene product cannot sustain autonomous or integrated F plasmid maintenance. PMID:3020005

  2. Soil DNA Extraction Procedure Influences Protist 18S rRNA Gene Community Profiling Outcome.

    PubMed

    Santos, Susana S; Nunes, Inês; Nielsen, Tue K; Jacquiod, Samuel; Hansen, Lars H; Winding, Anne

    2017-07-01

    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. The commercial DNA extraction kit was associated with the highest diversity estimates and with a corresponding higher retrieval of Excavata, Cercozoa and Amoebozoa-related taxa. Overall, our findings indicate that this extraction offers a compromise between rare and dominant taxa representation, while providing high replication reproducibility. A comprehensive understanding of the DNA extraction techniques impact on soil protist diversity can enable more accurate diversity assays. Copyright © 2017 Elsevier GmbH. All rights reserved.

  3. Characterization of How DNA Modifications Affect DNA Binding by C2H2 Zinc Finger Proteins

    PubMed Central

    Patel, A.; Hashimoto, H.; Zhang, X.; Cheng, X.

    2016-01-01

    Much is known about vertebrate DNA methylation and oxidation; however, much less is known about how modified cytosine residues within particular sequences are recognized. Among the known methylated DNA-binding domains, the Cys2-His2 zinc finger (ZnF) protein superfamily is the largest with hundreds of members, each containing tandem ZnFs ranging from 3 to >30 fingers. We have begun to biochemically and structurally characterize these ZnFs not only on their sequence specificity but also on their sensitivity to various DNA modifications. Rather than following published methods of refolding insoluble ZnF arrays, we have expressed and purified soluble forms of ZnFs, ranging in size from a tandem array of two to six ZnFs, from seven different proteins. We also describe a fluorescence polarization assay to measure ZnFs affinity with oligonucleotides containing various modifications and our approaches for cocrystallization of ZnFs with oligonucleotides. PMID:27372763

  4. Common risk genes for affective and schizophrenic psychoses.

    PubMed

    Maier, Wolfgang

    2008-06-01

    The familial-genetic relationship between affective and schizophrenic disorders is receiving a re-emergence of interest. The reasons are a series of cross-diagnostic molecular-genetic discoveries: specific alleles in the genes for dysbindin (DTNBP1), neuregulin (NRG1) and DAOA (G72/G30) reveal associations for each of both groups of disorders in the same direction in some but not all reported studies. These findings cannot just be false positives because of confirming metaanalyses. Furthermore there is some pathophysiological support: the mentioned genes are involved in biochemical pathways, which are contributing to both disorders partly in a similar and partly in a different manner. The new levels of evidence enrich the classical continuity/discontinuity debate on the relationship between both groups of disorders.

  5. Gene replacement and expression of foreign DNA in mycobacteria.

    PubMed Central

    Husson, R N; James, B E; Young, R A

    1990-01-01

    A system that permits molecular genetic manipulation of mycobacteria was developed on the basis of the yeast paradigm of gene replacement by homologous recombination. A shuttle vector that can replicate autonomously at a high copy number in Escherichia coli but must integrate into homologous DNA for survival in Mycobacterium smegmatis was constructed. The vector contains a ColE1 origin of replication, antibiotic resistance markers for ampicillin and kanamycin, a nutritional marker (pyrF) that allows both positive and negative selection in E. coli and M. smegmatis, and unique restriction sites that permit insertion of foreign DNA. Transformation of mycobacteria with this vector results in integration of its DNA into the genomic pyrF locus by either a single or a double homologous recombination event. With this system, the 65-kilodalton Mycobacterium leprae stress protein antigen was inserted into the M. smegmatis genome and expressed. This gene replacement technology, together with a uniquely useful pyrF marker, should be valuable for investigating mycobacterial pathobiology, for the development of candidate mycobacterial vaccine vehicles, and as a model for the development of molecular genetic systems in other pathogenic microorganisms. Images FIG. 2 FIG. 3 PMID:2153655

  6. Activation of endocrine-related gene expression in placental choriocarcinoma cell lines following DNA methylation knock-down.

    PubMed

    Hogg, K; Robinson, W P; Beristain, A G

    2014-07-01

    Increasingly, placental DNA methylation is assessed as a factor in pregnancy-related complications, yet the transcriptional impact of such findings is not always clear. Using a proliferative in vitro placental model, the effect of DNA methylation loss on gene activation was evaluated at a number of genes selected for being differentially methylated in pre-eclampsia-associated placentae in vivo. We aimed to determine whether reduced DNA methylation at specific loci was associated with transcriptional changes at the corresponding gene, thus providing mechanistic underpinnings for previous clinical findings and to assess the degree of transcriptional response amongst our candidate genes. BeWo and JEG3 choriocarcinoma cells were exposed to 1 μM 5-Aza-2'-deoxycytidine (5-Aza-CdR) or vehicle control for 48 h, and re-plated and cultured for a further 72 h in normal media before cells were harvested for RNA and DNA. Bisulphite pyrosequencing confirmed that DNA methylation was reduced by ∼30-50% points at the selected loci studied in both cell lines. Gene activation, measured by qRT-PCR, was highly variable and transcript specific, indicating differential sensitivity to DNA methylation. Most notably, loss of DNA methylation at the leptin (LEP) promoter corresponded to a 200-fold and 40-fold increase in LEP expression in BeWo and JEG3 cells, respectively (P < 0.01). Transcripts of steroidogenic pathway enzymes CYP11A1 and HSD3B1 were up-regulated ∼40-fold in response to 5-Aza-CdR exposure in BeWo cells (P < 0.01). Other transcripts, including aromatase (CYP19), HSD11B2, inhibin (INHBA) and glucocorticoid receptor (NR3C1) were more moderately, although significantly, affected by loss of associated DNA methylation. These data present a mixed effect of DNA methylation changes at selected loci supporting cautionary interpretation of DNA methylation results in the absence of functional data.

  7. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  8. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  9. Deletion of PLCB1 gene in schizophrenia-affected patients.

    PubMed

    Lo Vasco, Vincenza Rita; Cardinale, Giuseppina; Polonia, Patrizia

    2012-04-01

    A prevalence of 1% in the general population and approximately 50% concordance rate in monozygotic twins was reported for schizophrenia, suggesting that genetic predisposition affecting neurodevelopmental processes might combine with environmental risk factors. A multitude of pathways seems to be involved in the aetiology and/or pathogenesis of schizophrenia, including dopaminergic, serotoninergic, muscarinic and glutamatergic signalling. The phosphoinositide signal transduction system and related phosphoinositide-specific phospholipase C (PI-PLC) enzymes seem to represent a point of convergence in these networking pathways during the development of selected brain regions. The existence of a susceptibility locus on the short arm of chromosome 20 moved us to analyse PLCB1, the gene codifying for PI-PLC β1 enzyme, which maps on 20p12. By using interphase fluorescent in situ hybridization methodology, we found deletions of PLCB1 in orbito-frontal cortex samples of schizophrenia-affected patients.

  10. Deletion of PLCB1 gene in schizophrenia-affected patients

    PubMed Central

    Vasco, Vincenza Rita Lo; Cardinale, Giuseppina; Polonia, Patrizia

    2012-01-01

    Abstract A prevalence of 1% in the general population and approximately 50% concordance rate in monozygotic twins was reported for schizophrenia, suggesting that genetic predisposition affecting neurodevelopmental processes might combine with environmental risk factors. A multitude of pathways seems to be involved in the aetiology and/or pathogenesis of schizophrenia, including dopaminergic, serotoninergic, muscarinic and glutamatergic signalling. The phosphoinositide signal transduction system and related phosphoinositide-specific phospholipase C (PI-PLC) enzymes seem to represent a point of convergence in these networking pathways during the development of selected brain regions. The existence of a susceptibility locus on the short arm of chromosome 20 moved us to analyse PLCB1, the gene codifying for PI-PLC β1 enzyme, which maps on 20p12. By using interphase fluorescent in situ hybridization methodology, we found deletions of PLCB1 in orbito-frontal cortex samples of schizophrenia-affected patients. PMID:22507702

  11. Agrobacterium T-DNA integration in Arabidopsis is correlated with DNA sequence compositions that occur frequently in gene promoter regions.

    PubMed

    Schneeberger, Richard G; Zhang, Ke; Tatarinova, Tatiana; Troukhan, Max; Kwok, Shing F; Drais, Josh; Klinger, Kevin; Orejudos, Francis; Macy, Kimberly; Bhakta, Amit; Burns, James; Subramanian, Gopal; Donson, Jonathan; Flavell, Richard; Feldmann, Kenneth A

    2005-10-01

    Mobile insertion elements such as transposons and T-DNA generate useful genetic variation and are important tools for functional genomics studies in plants and animals. The spectrum of mutations obtained in different systems can be highly influenced by target site preferences inherent in the mechanism of DNA integration. We investigated the target site preferences of Agrobacterium T-DNA insertions in the chromosomes of the model plant Arabidopsis thaliana. The relative frequencies of insertions in genic and intergenic regions of the genome were calculated and DNA composition features associated with the insertion site flanking sequences were identified. Insertion frequencies across the genome indicate that T-strand integration is suppressed near centromeres and rDNA loci, progressively increases towards telomeres, and is highly correlated with gene density. At the gene level, T-DNA integration events show a statistically significant preference for insertion in the 5' and 3' flanking regions of protein coding sequences as well as the promoter region of RNA polymerase I transcribed rRNA gene repeats. The increased insertion frequencies in 5' upstream regions compared to coding sequences are positively correlated with gene expression activity and DNA sequence composition. Analysis of the relationship between DNA sequence composition and gene activity further demonstrates that DNA sequences with high CG-skew ratios are consistently correlated with T-DNA insertion site preference and high gene expression. The results demonstrate genomic and gene-specific preferences for T-strand integration and suggest that DNA sequences with a pronounced transition in CG- and AT-skew ratios are preferred targets for T-DNA integration.

  12. A mRad51-GFP antimorphic allele affects homologous recombination and DNA damage sensitivity.

    PubMed

    Uringa, Evert-Jan; Baldeyron, Céline; Odijk, Hanny; Wassenaar, Evelyne; van Cappellen, Wiggert A; Maas, Alex; Hoeijmakers, Jan H J; Baarends, Willy M; Kanaar, Roland; Essers, Jeroen

    2015-01-01

    Accurate DNA double-strand break repair through homologous recombination is essential for preserving genome integrity. Disruption of the gene encoding RAD51, the protein that catalyzes DNA strand exchange during homologous recombination, results in lethality of mammalian cells. Proteins required for homologous recombination, also play an important role during DNA replication. To explore the role of RAD51 in DNA replication and DSB repair, we used a knock-in strategy to express a carboxy-terminal fusion of green fluorescent protein to mouse RAD51 (mRAD51-GFP) in mouse embryonic stem cells. Compared to wild-type cells, heterozygous mRad51(+/wt-GFP) embryonic stem cells showed increased sensitivity to DNA damage induced by ionizing radiation and mitomycin C. Moreover, gene targeting was found to be severely impaired in mRad51(+/wt-GFP) embryonic stem cells. Furthermore, we found that mRAD51-GFP foci were not stably associated with chromatin. From these experiments we conclude that this mRad51-GFP allele is an antimorphic allele. When this allele is present in a heterozygous condition over wild-type mRad51, embryonic stem cells are proficient in DNA replication but display defects in homologous recombination and DNA damage repair. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Sister chromatid exchange, DNA repair, and single-gene mutation

    SciTech Connect

    Carrano, A.V.; Thompson, L.H.

    1982-01-01

    Sister chromatid exchange (SCE) has been studied in cultured mammalian cells with regard to the nature of the inducing lesion, mutation induction, and factors that modify the observed frequency following mutagen exposure, SCEs can be induced by a wide spectrum of DNA lesions and, for nine agents examined, the frequency of induced SCE is linearly related to induced single-gene mutation. Further, a deficiency in DNA repair may alter the expression of both SCE and mutation in a qualitatively similar manner. The frequency of SCE induced by mitomycin-C is suppressed in heterochromatic relative to euchromatin and, in nondividing lymphocytes, the lesions leading to the formation of SCEs may persist for several months.

  14. Polymorphism of the DNA Base Excision Repair Genes in Keratoconus

    PubMed Central

    Wojcik, Katarzyna A.; Synowiec, Ewelina; Sobierajczyk, Katarzyna; Izdebska, Justyna; Blasiak, Janusz; Szaflik, Jerzy; Szaflik, Jacek P.

    2014-01-01

    Keratoconus (KC) is a degenerative corneal disorder for which the exact pathogenesis is not yet known. Oxidative stress is reported to be associated with this disease. The stress may damage corneal biomolecules, including DNA, and such damage is primarily removed by base excision repair (BER). Variation in genes encoding BER components may influence the effectiveness of corneal cells to cope with oxidative stress. In the present work we genotyped 5 polymorphisms of 4 BER genes in 284 patients and 353 controls. The A/A genotype of the c.–1370T>A polymorphism of the DNA polymerase γ (POLG) gene was associated with increased occurrence of KC, while the A/T genotype was associated with decreased occurrence of KC. The A/G genotype and the A allele of the c.1196A>G polymorphism of the X-ray repair cross-complementing group 1 (XRCC1) were associated with increased, and the G/G genotype and the G allele, with decreased KC occurrence. Also, the C/T and T as well as C/C genotypes and alleles of the c.580C>T polymorphism of the same gene displayed relationship with KC occurrence. Neither the g.46438521G>C polymorphism of the Nei endonuclease VIII-like 1 (NEIL1) nor the c.2285T>C polymorphism of the poly(ADP-ribose) polymerase-1 (PARP-1) was associated with KC. In conclusion, the variability of the XRCC1 and POLG genes may play a role in KC pathogenesis and determine the risk of this disease. PMID:25356504

  15. Intrauterine growth restriction affects hippocampal dual specificity phosphatase 5 gene expression and epigenetic characteristics

    PubMed Central

    Ke, Xingrao; McKnight, Robert A.; Caprau, Diana; O'Grady, Shannon; Fu, Qi; Yu, Xing; Callaway, Christopher W.; Albertine, Kurt H.

    2011-01-01

    Intrauterine growth retardation (IUGR) predisposes humans toward hippocampal morbidities, such as impaired learning and memory. Hippocampal dual specificity phosphatase 5 (DUSP5) may be involved in these morbidities because DUSP5 regulates extracellular signal-regulated kinase phosphorylation (Erk). In the rat, IUGR causes postnatal changes in hippocampal gene expression and epigenetic characteristics. However, the impact of IUGR upon hippocampal DUSP5 expression and epigenetic characteristics is not known. We therefore hypothesized that IUGR affects hippocampal 1) DUSP5 expression, DNA CpG methylation, and histone code, and 2) erk1/2 phosphorylation in a well-characterized rat model of IUGR. We found that IUGR significantly decreased DUSP5 expression in the day of life (DOL) 0 and 21 male rat, while decreasing only DUSP5 protein levels in the DOL21 female rat. Fluorescent in situ hybridization and immunohistochemistry analyses localized the changes in DUSP5 mRNA and protein, many of which occurred in the dentate gyrus. IUGR also caused sex-specific differences in DNA CpG methylation and histone code in two sites of the hippocampal DUSP5 gene, a 5′-flanking specificity protein-1 (SP1) site and exon 2. Finally, when IUGR decreased DUSP5 protein levels, Erk phosphorylation increased. We conclude that IUGR affects hippocampal DUSP5 expression and epigenetic characteristics in a sex-specific manner. PMID:21828247

  16. Transient gene expression in epidermal cells of plant leaves by biolistic DNA delivery.

    PubMed

    Ueki, Shoko; Magori, Shimpei; Lacroix, Benoît; Citovsky, Vitaly

    2013-01-01

    Transient gene expression is a useful approach for studying the functions of gene products. In the case of plants, Agrobacterium infiltration is a method of choice for transient introduction of genes for many species. However, this technique does not work efficiently in some species, such as Arabidopsis thaliana. Moreover, the infection of Agrobacterium is known to induce dynamic changes in gene expression patterns in the host plants, possibly affecting the function and localization of the proteins to be tested. These problems can be circumvented by biolistic delivery of the genes of interest. Here, we present an optimized protocol for biolistic delivery of plasmid DNA into epidermal cells of plant leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol allows efficient and reproducible transient expression of diverse genes in Arabidopsis, Nicotiana benthamiana and N. tabacum, and is suitable for studies of the biological function and subcellular localization of the gene products directly in planta. The protocol also can be easily adapted to other species by optimizing the delivery gas pressure.

  17. Differential elimination of rDNA genes in bobbed mutants of Drosophila melanogaster.

    PubMed Central

    Terracol, R; Prud'homme, N

    1986-01-01

    In Drosophila melanogaster, the multiply repeated genes encoding 18S and 28S rRNA are located on the X and Y chromosomes. A large percentage of these repeats are interrupted in the 28S region by insertions of two types. We compared the restriction patterns from a subcloned wild-type Oregon R strain to those of spontaneous and ethyl methanesulfonate-induced bobbed mutants. Bobbed mutations were found to be deficiencies that modified the organization of the rDNA locus. Genes without insertions were deleted about twice as often as genes with type I insertions. Type II insertion genes were not decreased in number, except in the mutant having the most bobbed phenotype. Reversion to wild type was associated with an increase in gene copy number, affecting exclusively genes without insertions. One hypothesis which explains these results is the partial clustering of genes by type. The initial deletion could then be due either to an unequal crossover or to loss of material without exchange. Some of our findings indicated that deletion may be associated with an amplification phenomenon, the magnitude of which would be dependent on the amount of clustering of specific gene types at the locus. Images PMID:3023865

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

    PubMed Central

    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, and PE38 stimulate DNA replication. No stimulation by the AcMNPV pcna gene, encoding a protein with sequence homology to proliferating-cell nuclear antigen, was observed. A pattern of amino acids found in a number of single-stranded-DNA-binding proteins was identified in the carboxyl-terminal region of IE-1. Images PMID:7972036

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

    PubMed

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

    2010-10-01

    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 F0 and F1 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. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  20. How-to-Do-It: Recombinant DNA Made Easy II. Gene, Gene, Who's Got the Gene?

    ERIC Educational Resources Information Center

    Thomson, Robert G.

    1989-01-01

    Described is an activity in which students are able to determine that DNA can be transferred between bacteria and should be able to predict the type of DNA transferred. Methods, materials, and results are discussed. (CW)

  1. OS052. Preeclampsia candidate genes differentially methylated in maternal leukocyte DNA.

    PubMed

    White, W; Brost, B; O'Brien, J; Rose, C; Davies, N; Sun, Z; Turner, S; Garovic, V

    2012-07-01

    Altered gene expression in biomarkers associated with preeclampsia/ eclampsia (PE) could be explained in part by epigenetic phenomena such as variable methylation We sought to characterize the methylation profiles of candidate genes known to be associated with the preeclampsia phenotype in maternal leukocyte DNA in preeclamptic cases and normotensive controls at the time of delivery. Methylation profiles of maternal leukocyte DNA were evaluated in 14 PE cases and 14 normotensive controls. Subjects were nulliparous, non-smokers, age and BMI matched. Genomic DNA was run on a commercially available beadchip human methylation assay. Mean methylation at sites in genes from a well-defined preeclampsia gene set present on our platform were compared using a t-test. QC confirmed high correlation of replicates and detection p values >95%. Of the 39 genes in the "preeclampsia gene set", 34 were present on our platform with 73 CpG sites. Seven out of 34 tested in this gene set had differential methylation with p value <0.05. Two genes were found to be less methylated in PE which may result in more expression. AGT (-3%;p= 0.027), angiotensin, is a potent vasoconstrictor with exaggerated effect in PE. DDAH1 (-6%;p=0.031) is involved in nitric oxide generation, via asymmetric dimethylarginine (ADMA), levels of which are known to be altered in PE. Five genes were more methylated and therefore may correlate with reduced transcription. CALCA (+4%;p=0.001) forms calcitonin-gene related peptide, a potent vasodilator decreased in the PE . F5 (+1%;p=0.016), coagulation Factor V, is a target of activated protein C, and increased resistance related to genetic variants (Factor V Leiden) or pregnancy have been associated with PE. MTHFR (+3%;p=0.041) regulates homocysteine; high levels are associated with a 20X increase in risk for PE. POMC (+4%;p=0.014) produces beta endorphin and through ACTH stimulates aldosterone, both decreased in PE. PTGS2 (+3%;p=0.03) is part of the COX 2

  2. DNA demethylation induced by 5-azacytidine does not affect fragile X expression.

    PubMed Central

    Glover, T W; Coyle-Morris, J; Pearce-Birge, L; Berger, C; Gemmill, R M

    1986-01-01

    Experiments were performed to determine the role of DNA demethylation in fragile X expression. Fragile X positive lymphoblastoid cells were treated with 5-azacytidine and harvested for analysis of fragile X expression both directly following treatment and after a recovery period in the absence of the drug. The effectiveness of 5-azacytidine treatment in inducing DNA demethylation was concurrently monitored by analysis of methylation changes at random autosomal loci in isolated DNA from treated cells. Under conditions where 5-azacytidine was found to inhibit fragile X expression, no DNA demethylation was observed. At the time when demethylation did occur, fragile X expression was not affected. These results strongly indicate that DNA demethylation is not involved in fragile X expression. Images Fig. 1 PMID:2420174

  3. Organization of gene and non-gene sequences in micronuclear DNA of Oxytricha nova.

    PubMed Central

    Boswell, R E; Jahn, C L; Greslin, A F; Prescott, D M

    1983-01-01

    In order to study the derivation of the macronuclear genome from the micronuclear genome in Oxytricha nova micronuclear DNA was partially digested with EcoRI, size fractionated, and then cloned in the lambda phage Charon 8. Clones were selected a) at random b) by hybridization with macronuclear DNA or c) by hybridization with clones of macronuclear DNA. One group of these clones contains only unique sequence DNA, and all of these had sequences that were homologous to macronuclear sequences. The number of macronuclear genes with sequences homologous to these micronuclear clones indicates that macronuclear sequences are clustered in the micronuclear genome. Many micronuclear clones contain repetitive DNA sequences and hybridize to numerous EcoRI fragments of total micronuclear DNA, yielding similar but non-identical patterns. Some micronuclear clones containing these repetitive sequences also contained unique sequence DNA that hybridized to a macronuclear sequence. These clones define a major interspersed repetitive sequence family in the micronuclear genome that is eliminated during formation of the macronuclear genome. Images PMID:6304639

  4. Identification of target genes conferring ethanol stress tolerance to Saccharomyces cerevisiae based on DNA microarray data analysis.

    PubMed

    Hirasawa, Takashi; Yoshikawa, Katsunori; Nakakura, Yuki; Nagahisa, Keisuke; Furusawa, Chikara; Katakura, Yoshio; Shimizu, Hiroshi; Shioya, Suteaki

    2007-08-01

    During industrial production process using yeast, cells are exposed to the stress due to the accumulation of ethanol, which affects the cell growth activity and productivity of target products, thus, the ethanol stress-tolerant yeast strains are highly desired. To identify the target gene(s) for constructing ethanol stress tolerant yeast strains, we obtained the gene expression profiles of two strains of Saccharomyces cerevisiae, namely, a laboratory strain and a strain used for brewing Japanese rice wine (sake), in the presence of 5% (v/v) ethanol, using DNA microarray. For the selection of target genes for breeding ethanol stress tolerant strains, clustering of DNA microarray data was performed. For further selection, the ethanol sensitivity of the knockout mutants in each of which the gene selected by DNA microarray analysis is deleted, was also investigated. The integration of the DNA microarray data and the ethanol sensitivity data of knockout strains suggests that the enhancement of expression of genes related to tryptophan biosynthesis might confer the ethanol stress tolerance to yeast cells. Indeed, the strains overexpressing tryptophan biosynthesis genes showed a stress tolerance to 5% ethanol. Moreover, the addition of tryptophan to the culture medium and overexpression of tryptophan permease gene conferred ethanol stress tolerance to yeast cells. These results indicate that overexpression of the genes for trypophan biosynthesis increases the ethanol stress tolerance. Tryptophan supplementation to culture and overexpression of the tryptophan permease gene are also effective for the increase in ethanol stress tolerance. Our methodology for the selection of target genes for constructing ethanol stress tolerant strains, based on the data of DNA microarray analysis and phenotypes of knockout mutants, was validated.

  5. Iron nanoparticles significantly affect the in vitro and in vivo expression of Id genes.

    PubMed

    Zou, Jinglu; Wang, Xin; Zhang, Ling; Wang, Jinke

    2015-03-16

    In recent DNA microarray studies, we found that the transcription of the Id3 gene was significantly down-regulated in five cell lines (RAW264.7, Hepa1-6, THP-1, HepG2, and HL7702) treated with two doses (50 and 100 μg/mL) of a DMSA-coated magnetite nanoparticle. Given the regulatory roles of Id genes in the cell cycle, growth, and differentiation, we wanted to do more investigations on the effect of the nanoparticle upon the Id genes. This study detected the expression of Id genes in six cell lines (the above cell lines plus HeLa) treated with the nanoparticle at the same doses using quantitative PCR. The results revealed that the expression of Id genes was significantly affected by the nanoparticle in these cell lines. Under each treatment, the Id3 gene was significantly (p < 0.01) down-regulated in all cell lines, the Id1 gene was significantly down-regulated in all cell lines except the RAW264.7 cells, and the Id2 gene was significantly down-regulated in the HepG2, HL7702, and HeLa cells. Because the Id1, Id2, and Id3 genes were significantly down-regulated in three liver-derived cell lines (Hepa1-6, HepG2, and HL7702) in both microarray and PCR detections, this study then detected the expression of Id genes in the liver tissues of mice that were intravenously injected with the nanoparticle at two doses (2 and 5 mg/kg body weight). The results revealed that the expression of Id1, Id2, and Id3 genes was also significantly down-regulated in the liver tissues under each treatment. Another Id gene, Id4, was also significantly regulated in some cells or liver tissues treated with the nanoparticle. These results reveal that the nanoparticle exerts a significant effect on the in vitro and in vivo expression of Id genes. This study thus provides new insights into the Id-related nanotoxicity of the nanoparticle and the close relationship between the regulation of Id genes and iron.

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

    PubMed

    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.

  7. Mutation in fucose synthesis gene of Klebsiella pneumoniae affects capsule composition and virulence in mice.

    PubMed

    Pan, Po-Chang; Chen, Hui-Wen; Wu, Po-Kuan; Wu, Yu-Yang; Lin, Chun-Hung; Wu, June H

    2011-02-01

    The emerging pathogenicity of Klebsiella pneumoniae (KP) is evident by the increasing number of clinical cases of liver abscess (LA) due to KP infection. A unique property of KP is its thick mucoid capsule. The bacterial capsule has been found to contain fucose in KP strains causing LA but not in those causing urinary tract infections. The products of the gmd and wcaG genes are responsible for converting mannose to fucose in KP. A KP strain, KpL1, which is known to have a high death rate in infected mice, was mutated by inserting an apramycin-resistance gene into the gmd. The mutant expressed genes upstream and downstream of gmd, but not gmd itself, as determined by reverse transcriptase polymerase chain reaction. The DNA mapping confirmed the disruption of the gmd gene. This mutant decreased its ability to kill infected mice and showed decreased virulence in infected HepG2 cells. Compared with wild-type KpL1, the gmd mutant lost fucose in capsular polysaccharides, increased biofilm formation and interacted more readily with macrophages. The mutant displayed morphological changes with long filament forms and less uniform sizes. The mutation also converted the serotype from K1 of wild-type to K2 and weak K3. The results indicate that disruption of the fucose synthesis gene affected the pathophysiology of this bacterium and may be related to the virulence of this KpL1 strain.

  8. DNA Methylation and Genome Evolution in Honeybee: Gene Length, Expression, Functional Enrichment Covary with the Evolutionary Signature of DNA Methylation

    PubMed Central

    Zeng, Jia; Yi, Soojin V.

    2010-01-01

    A growing body of evidence suggests that DNA methylation is functionally divergent among different taxa. The recently discovered functional methylation system in the honeybee Apis mellifera presents an attractive invertebrate model system to study evolution and function of DNA methylation. In the honeybee, DNA methylation is mostly targeted toward transcription units (gene bodies) of a subset of genes. Here, we report an intriguing covariation of length and epigenetic status of honeybee genes. Hypermethylated and hypomethylated genes in honeybee are dramatically different in their lengths for both exons and introns. By analyzing orthologs in Drosophila melanogaster, Acyrthosiphon pisum, and Ciona intestinalis, we show genes that were short and long in the past are now preferentially situated in hyper- and hypomethylated classes respectively, in the honeybee. Moreover, we demonstrate that a subset of high-CpG genes are conspicuously longer than expected under the evolutionary relationship alone and that they are enriched in specific functional categories. We suggest that gene length evolution in the honeybee is partially driven by evolutionary forces related to regulation of gene expression, which in turn is associated with DNA methylation. However, lineage-specific patterns of gene length evolution suggest that there may exist additional forces underlying the observed interaction between DNA methylation and gene lengths in the honeybee. PMID:20924039

  9. Biodegradable DNA Nanoparticles that Provide Widespread Gene Delivery in the Brain

    PubMed Central

    Mastorakos, Panagiotis; Song, Eric; Zhang, Clark; Berry, Sneha; Park, Hee Won; Kim, Young Eun; Park, Jong Sung; Lee, Seulki; Suk, Jung Soo; Hanes, Justin

    2016-01-01

    Successful gene therapy of neurological disorders is predicated on achieving widespread and uniform transgene expression throughout the affected disease area in the brain. However, conventional gene vectors preferentially travel through low-resistance perivascular spaces and/or are confined to the administration site even with the aid of a pressure-driven flow provided by convection-enhanced delivery. Biodegradable DNA nanoparticles offer a safe gene delivery platform devoid of adverse effects associated with virus-based or synthetic non-biodegradable systems. Using a state-of-the-art biodegradable polymer, poly(β-amino ester), we engineered colloidally stable sub-100 nm DNA nanoparticles coated with a non-adhesive polyethylene glycol corona that are able to avoid the adhesive and steric hindrances imposed by the extracellular matrix. Following convection enhanced delivery, these brain-penetrating nanoparticles were able to homogeneously distribute throughout the rodent striatum and mediate widespread and high-level transgene expression. These nanoparticles provide a biodegradable DNA nanoparticle platform enabling uniform transgene expression patterns in vivo and hold promise for the treatment of neurological diseases. PMID:26680637

  10. Conservative inheritance of newly synthesized DNA in double-strand break-induced gene conversion.

    PubMed

    Ira, Grzegorz; Satory, Dominik; Haber, James E

    2006-12-01

    To distinguish among possible mechanisms of repair of a double-strand break (DSB) by gene conversion in budding yeast, Saccharomyces cerevisiae, we employed isotope density transfer to analyze budding yeast mating type (MAT) gene switching in G2/M-arrested cells. Both of the newly synthesized DNA strands created during gene conversion are found at the repaired locus, leaving the donor unchanged. These results support suggestions that mitotic DSBs are primarily repaired by a synthesis-dependent strand-annealing mechanism. We also show that the proportion of crossing-over associated with DSB-induced ectopic recombination is not affected by the presence of nonhomologous sequences at one or both ends of the DSB or the presence of additional sequences that must be copied from the donor.

  11. DNA Sequence Heterogeneity of Campylobacter jejuni CJIE4 Prophages and Expression of Prophage Genes

    PubMed Central

    Clark, Clifford G.; Chong, Patrick M.; McCorrister, Stuart J.; Mabon, Philip; Walker, Matthew; Westmacott, Garrett R.

    2014-01-01

    Campylobacter jejuni carry temperate bacteriophages that can affect the biology or virulence of the host bacterium. Known effects include genomic rearrangements and resistance to DNA transformation. C. jejuni prophage CJIE1 shows sequence variability and variability in the content of morons. Homologs of the CJIE1 prophage enhance both adherence and invasion to cells in culture and increase the expression of a specific subset of bacterial genes. Other C. jejuni temperate phages have so far not been well characterized. In this study we describe investigations into the DNA sequence variability and protein expression in a second prophage, CJIE4. CJIE4 sequences were obtained de novo from DNA sequencing of five C. jejuni isolates, as well as from whole genome sequences submitted to GenBank by other research groups. These CJIE4 DNA sequences were heterogenous, with several different insertions/deletions (indels) in different parts of the prophage genome. Two variants of a 3–4 kb region inserted within CJIE4 had different gene content that distinguished two major conserved CJIE4 prophage families. Additional indels were detected throughout the prophage. Detection of proteins in the five isolates characterized in our laboratory in isobaric Tags for Relative and Absolute Quantitation (iTRAQ) experiments indicated that prophage proteins within each of the two large indel variants were expressed during growth of the bacteria on Mueller Hinton agar plates. These proteins included the extracellular DNase associated with resistance to DNA transformation and prophage repressor proteins. Other proteins associated with known or suspected roles in prophage biology were also expressed from CJIE4, including capsid protein, the phage integrase, and MazF, a type II toxin-antitoxin system protein. Together with the results previously obtained for the CJIE1 prophage these results demonstrate that sequence variability and expression of moron genes are both general properties of temperate

  12. Optimal salt concentration of vehicle for plasmid DNA enhances gene transfer mediated by electroporation.

    PubMed

    Lee, Min-Jae; Cho, Soon-Shin; Jang, Hyung-Suk; Lim, Young Shin; You, Ji-Ran; Park, Jangwon; Suh, Hearan; Kim, Jeong-a; Park, Jong-Sang; Kim, Duk-Kyung

    2002-09-30

    In vivo electroporation has emerged as a leading technology for developing nonviral gene therapies, and the various technical parameters governing electroporation efficiency have been optimized by both theoretical and experimental analysis. However, most electroporation parameters focused on the electric conditions and the preferred vehicle for plasmid DNA injections has been normal saline. We hypothesized that salts in vehicle for plasmid DNA must affect the efficiency of DNA transfer because cations would alter ionic atmosphere, ionic strength, and conductivity of their medium. Here, we show that half saline (71 mM) is an optimal vehicle for in vivo electroporation of naked DNA in skeletal muscle. With various salt concentrations, two reporter genes, luciferase and beta-galactosidase were injected intramuscularly under our optimal electric condition (125 V/cm, 4 pulses x 2 times, 50 ms, 1 Hz). Exact salt concentrations of DNA vehicle were measured by the inductively coupled plasma-atomic emission spectrometer (ICP-AES) and the conductivity change in the tissue induced by the salt in the medium was measured by Low-Frequency (LF) Impedance Analyzer. Luciferase expression increased as cation concentration of vehicle decreased and this result can be visualized by X-Gal staining. However, at lower salt concentration, transfection efficiency was diminished because the hypoosmotic stress and electrical injury by low conductivity induced myofiber damage. At optimal salt concentration (71 mM), we observed a 3-fold average increase in luciferase expression in comparison with the normal saline condition (p < 0.01). These results provide a valuable experimental parameter for in vivo gene therapy mediated by electroporation.

  13. In-depth cDNA library sequencing provides quantitative gene expression profiling in cancer biomarker discovery.

    PubMed

    Yang, Wanling; Ying, Dingge; Lau, Yu-Lung

    2009-06-01

    Quantitative gene expression analysis plays an important role in identifying differentially expressed genes in various pathological states, gene expression regulation and co-regulation, shedding light on gene functions. Although microarray is widely used as a powerful tool in this regard, it is suboptimal quantitatively and unable to detect unknown gene variants. Here we demonstrated effective detection of differential expression and co-regulation of certain genes by expressed sequence tag analysis using a selected subset of cDNA libraries. We discussed the issues of sequencing depth and library preparation, and propose that increased sequencing depth and improved preparation procedures may allow detection of many expression features for less abundant gene variants. With the reduction of sequencing cost and the emerging of new generation sequencing technology, in-depth sequencing of cDNA pools or libraries may represent a better and powerful tool in gene expression profiling and cancer biomarker detection. We also propose using sequence-specific subtraction to remove hundreds of the most abundant housekeeping genes to increase sequencing depth without affecting relative expression ratio of other genes, as transcripts from as few as 300 most abundantly expressed genes constitute about 20% of the total transcriptome. In-depth sequencing also represents a unique advantage of detecting unknown forms of transcripts, such as alternative splicing variants, fusion genes, and regulatory RNAs, as well as detecting mutations and polymorphisms that may play important roles in disease pathogenesis.

  14. Paternal benzo[a]pyrene exposure affects gene expression in the early developing mouse embryo.

    PubMed

    Brevik, Asgeir; Lindeman, Birgitte; Rusnakova, Vendula; Olsen, Ann-Karin; Brunborg, Gunnar; Duale, Nur

    2012-09-01

    The health of the offspring depends on the genetic constitution of the parental germ cells. The paternal genome appears to be important; e.g., de novo mutations in some genes seem to arise mostly from the father, whereas epigenetic modifications of DNA and histones are frequent in the paternal gonads. Environmental contaminants which may affect the integrity of the germ cells comprise the polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P). B[a]P has received much attention due to its ubiquitous distribution, its carcinogenic and mutagenic potential, and also effects on reproduction. We conducted an in vitro fertilization (IVF) experiment using sperm cells from B[a]P-exposed male mice to study effects of paternal B[a]P exposure on early gene expression in the developing mouse embryo. Male mice were exposed to a single acute dose of B[a]P (150 mg/kg, ip) 4 days prior to isolation of cauda sperm, followed by IVF of oocytes from unexposed superovulated mice. Gene expression in fertilized zygotes/embryos was determined using reverse transcription-qPCR at the 1-, 2-, 4-, 8-, and blastocyst cell stages of embryo development. We found that paternal B[a]P exposure altered the expression of numerous genes in the developing embryo especially at the blastocyst stage. Some genes were also affected at earlier developmental stages. Embryonic gene expression studies seem useful to identify perturbations of signaling pathways resulting from exposure to contaminants, and can be used to address mechanisms of paternal effects on embryo development.

  15. Gluten affects epithelial differentiation-associated genes in small intestinal mucosa of coeliac patients.

    PubMed

    Juuti-Uusitalo, K; Mäki, M; Kainulainen, H; Isola, J; Kaukinen, K

    2007-11-01

    In coeliac disease gluten induces an immunological reaction in genetically susceptible patients, and influences on epithelial cell proliferation and differentiation in the small-bowel mucosa. Our aim was to find novel genes which operate similarly in epithelial proliferation and differentiation in an epithelial cell differentiation model and in coeliac disease patient small-bowel mucosal biopsy samples. The combination of cDNA microarray data originating from a three-dimensional T84 epithelial cell differentiation model and small-bowel mucosal biopsy samples from untreated and treated coeliac disease patients and healthy controls resulted in 30 genes whose mRNA expression was similarly affected. Nine of 30 were located directly or indirectly in the receptor tyrosine kinase pathway starting from the epithelial growth factor receptor. Removal of gluten from the diet resulted in a reversion in the expression of 29 of the 30 genes in the small-bowel mucosal biopsy samples. Further characterization by blotting and labelling revealed increased epidermal growth factor receptor and beta-catenin protein expression in the small-bowel mucosal epithelium in untreated coeliac disease patients compared to healthy controls and treated coeliac patients. We found 30 genes whose mRNA expression was affected similarly in the epithelial cell differentiation model and in the coeliac disease patient small-bowel mucosal biopsy samples. In particular, those genes involved in the epithelial growth factor-mediated signalling pathways may be involved in epithelial cell differentiation and coeliac disease pathogenesis. The epithelial cell differentiation model is a useful tool for studying gene expression changes in the crypt-villus axis.

  16. Brief isoflurane anaesthesia affects differential gene expression, gene ontology and gene networks in rat brain.

    PubMed

    Lowes, Damon A; Galley, Helen F; Moura, Alessandro P S; Webster, Nigel R

    2017-01-15

    Much is still unknown about the mechanisms of effects of even brief anaesthesia on the brain and previous studies have simply compared differential expression profiles with and without anaesthesia. We hypothesised that network analysis, in addition to the traditional differential gene expression and ontology analysis, would enable identification of the effects of anaesthesia on interactions between genes. Rats (n=10 per group) were randomised to anaesthesia with isoflurane in oxygen or oxygen only for 15min, and 6h later brains were removed. Differential gene expression and gene ontology analysis of microarray data was performed. Standard clustering techniques and principal component analysis with Bayesian rules were used along with social network analysis methods, to quantitatively model and describe the gene networks. Anaesthesia had marked effects on genes in the brain with differential regulation of 416 probe sets by at least 2 fold. Gene ontology analysis showed 23 genes were functionally related to the anaesthesia and of these, 12 were involved with neurotransmitter release, transport and secretion. Gene network analysis revealed much greater connectivity in genes from brains from anaesthetised rats compared to controls. Other importance measures were also altered after anaesthesia; median [range] closeness centrality (shortest path) was lower in anaesthetized animals (0.07 [0-0.30]) than controls (0.39 [0.30-0.53], p<0.0001) and betweenness centrality was higher (53.85 [32.56-70.00]% compared to 5.93 [0-30.65]%, p<0.0001). Simply studying the actions of individual components does not fully describe dynamic and complex systems. Network analysis allows insight into the interactions between genes after anaesthesia and suggests future targets for investigation. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    PubMed

    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; Koturbash, Igor

    2016-10-01

    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. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. How Does Guanine-Cytosine Base Pair Affect Excess-Electron Transfer in DNA?

    PubMed

    Lin, Shih-Hsun; Fujitsuka, Mamoru; Majima, Tetsuro

    2015-06-25

    Charge transfer and proton transfer in DNA have attracted wide attention due to their relevance in biological processes and so on. Especially, excess-electron transfer (EET) in DNA has strong relation to DNA repair. However, our understanding on EET in DNA still remains limited. Herein, by using a strongly electron-donating photosensitizer, trimer of 3,4-ethylenedioxythiophene (3E), and an electron acceptor, diphenylacetylene (DPA), two series of functionalized DNA oligomers were synthesized for investigation of EET dynamics in DNA. The transient absorption measurements during femtosecond laser flash photolysis showed that guanine:cytosine (G:C) base pair affects EET dynamics in DNA by two possible mechanisms: the excess-electron quenching by proton transfer with the complementary G after formation of C(•-) and the EET hindrance by inserting a G:C base pair as a potential barrier in consecutive thymines (T's). In the present paper, we provided useful information based on the direct kinetic measurements, which allowed us to discuss EET through oligonucleotides for the investigation of DNA damage/repair.

  19. GRAIL seeks out genes buried in DNA sequence

    SciTech Connect

    Roberts, L.

    1991-11-08

    When the Human Genome Project achieves its ultimate goal, supposedly around 2005, biologists will have in hand the exact sequence of all 3 billion nucleotides arrayed along the human chromosomes. But they have never been entirely sure how they will read the language of the long string of As, Gs, Ts, and Cs. How will they even be able to pick out the genes, which account for a mere 5% of the genome, from the mass of letters in between Now Edward Ubergacher, a biophysicist-turned-computational-biologist at Oak Ridge National Laboratory, has come one step toward providing an answer: a new artificial intelligence program, called GRAIL, that can pick out the coding regions of genes in a long stretch of sequence data. So far, the Oak Ridge team has analyzed 5 million bases of DNA. One year ago, even 6 months ago, it was virtually impossible to go into human genomic sequence and find genes by computer with any reliability. Now we can go in and find 90% of the genes very quickly. GRAIL can be used on a PC, not a supercomputer, and it provides an answer almost instantly.

  20. DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing

    PubMed Central

    Nishina, Kazutaka; Piao, Wenying; Yoshida-Tanaka, Kie; Sujino, Yumiko; Nishina, Tomoko; Yamamoto, Tsuyoshi; Nitta, Keiko; Yoshioka, Kotaro; Kuwahara, Hiroya; Yasuhara, Hidenori; Baba, Takeshi; Ono, Fumiko; Miyata, Kanjiro; Miyake, Koichi; Seth, Punit P.; Low, Audrey; Yoshida, Masayuki; Bennett, C. Frank; Kataoka, Kazunori; Mizusawa, Hidehiro; Obika, Satoshi; Yokota, Takanori

    2015-01-01

    Antisense oligonucleotides (ASOs) are recognized therapeutic agents for the modulation of specific genes at the post-transcriptional level. Similar to any medical drugs, there are opportunities to improve their efficacy and safety. Here we develop a short DNA/RNA heteroduplex oligonucleotide (HDO) with a structure different from double-stranded RNA used for short interfering RNA and single-stranded DNA used for ASO. A DNA/locked nucleotide acid gapmer duplex with an α-tocopherol-conjugated complementary RNA (Toc-HDO) is significantly more potent at reducing the expression of the targeted mRNA in liver compared with the parent single-stranded gapmer ASO. Toc-HDO also improves the phenotype in disease models more effectively. In addition, the high potency of Toc-HDO results in a reduction of liver dysfunction observed in the parent ASO at a similar silencing effect. HDO technology offers a novel concept of therapeutic oligonucleotides, and the development of this molecular design opens a new therapeutic field. PMID:26258894

  1. Ivory identification by DNA profiling of cytochrome b gene.

    PubMed

    Lee, James Chun-I; Hsieh, Hsing-Mei; Huang, Li-Hung; Kuo, Yi-Chen; Wu, Jane-Hong; Chin, Shih-Chien; Lee, An-Hsing; Linacre, Adrian; Tsai, Li-Chin

    2009-03-01

    Ivory can be visually identified in its native form as coming from an elephant species; however, determining from which of the three extant elephant species a section of ivory originates is more problematic. We report on a method that will identify and distinguish the protected and endangered elephant species, Elephas maximus or Loxodonta sp. To identify the species of elephant from ivory products, we developed three groups of nested PCR amplifications within the cytochrome b gene that generate amplification products using highly degraded DNA isolated from confiscated ivory samples dating from 1995. DNA from a total of 382 out of 453 ivory samples were successfully isolated and amplified leading to species identification. All sequences were searched against GenBank and found to match with E. maximus and Loxodonta sp. with at least 99% similarity. The samples that were tested came from eight Asian elephants, 14 African forest elephants (Loxodonta cyclotis), and 360 African savannah elephants (Loxodonta africana). This study demonstrates a high success rate in species identification of ivory by a nested PCR approach within the cytochrome b gene which provides the necessary information for the protection of endangered species conservation.

  2. DNA methylation of stress-related genes and LINE-1 repetitive elements across the healthy human placenta

    PubMed Central

    Non, Amy L.; Binder, Alexandra M.; Barault, Ludovic; Rancourt, Rebecca C.; Kubzansky, Laura D.; Michels, Karin B.

    2012-01-01

    Objectives DNA methylation is known to play a critical role in regulating development of placental morphology and physiology. The methylation of genes mediated by glucocorticoid hormones may be particularly vulnerable to intrauterine stress in the placenta. However little is known about DNA methylation of stress-related genes within a healthy placenta, and particularly whether methylation occurs uniformly across different regions of the placenta, which is a critical question for researchers seeking to analyze methylation patterns. We examined DNA methylation across four regions of the placenta to evaluate methylation levels of stress-related genes within a healthy placenta, and to evaluate whether methylation patterns vary by sampling location. Study Design We evaluated levels of DNA methylation of three stress-related genes: NR3C1, BDNF, and 11B-HSD2 and of the repetitive element, LINE-1, in four different sample locations of 20 healthy placentas. Main Outcome Measures Pyrosequencing was used to quantify levels of methylation at CpG sites within the promoter regions of each of the three stress-related genes, and global methylation of LINE-1. Results Very low levels of methylation were found across all three stress-related genes; no gene showed a median methylation level greater than 4.20% across placental regions. Variation in methylation between placental regions for stress-related genes and for LINE-1 was minimal. Conclusions Our data suggest that these frequently studied stress-related genes have low levels of methylation in healthy placenta tissue. Minimal variation between sites suggests that sampling location does not affect DNA methylation analyses of these genes or of LINE-1 repetitive elements. PMID:22222044

  3. DNA methylation of stress-related genes and LINE-1 repetitive elements across the healthy human placenta.

    PubMed

    Non, A L; Binder, A M; Barault, L; Rancourt, R C; Kubzansky, L D; Michels, K B

    2012-03-01

    DNA methylation is known to play a critical role in regulating development of placental morphology and physiology. The methylation of genes mediated by glucocorticoid hormones may be particularly vulnerable to intrauterine stress in the placenta. However little is known about DNA methylation of stress-related genes within a healthy placenta, and particularly whether methylation occurs uniformly across different regions of the placenta, which is a critical question for researchers seeking to analyze methylation patterns. We examined DNA methylation across four regions of the placenta to evaluate methylation levels of stress-related genes within a healthy placenta, and to evaluate whether methylation patterns vary by sampling location. We evaluated levels of DNA methylation of three stress-related genes: NR3C1, BDNF, and 11B-HSD2 and of the repetitive element, LINE-1, in four different sample locations of 20 healthy placentas. Pyrosequencing was used to quantify levels of methylation at CpG sites within the promoter regions of each of the three stress-related genes, and global methylation of LINE-1. Very low levels of methylation were found across all three stress-related genes; no gene showed a median methylation level greater than 4.20% across placental regions. Variation in methylation between placental regions for stress-related genes and for LINE-1 was minimal. Our data suggest that these frequently studied stress-related genes have low levels of methylation in healthy placenta tissue. Minimal variation between sites suggests that sampling location does not affect DNA methylation analyses of these genes or of LINE-1 repetitive elements. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Non-small cell lung cancer exhibits transcript overexpression of genes associated with homologous recombination and DNA replication pathways.

    PubMed

    Saviozzi, Silvia; Ceppi, Paolo; Novello, Silvia; Ghio, Paolo; Lo Iacono, Marco; Borasio, Piero; Cambieri, Alberto; Volante, Marco; Papotti, Mauro; Calogero, Raffaele A; Scagliotti, Giorgio V

    2009-04-15

    Genes involved in DNA repair and replication have been recently investigated as predictive markers of response to chemotherapy in non-small cell lung cancer (NSCLC). However, few data on the expression of these genes in tumor compared with corresponding normal lung are available. The aim of this study was to evaluate differential mRNA levels of 22 DNA repair genes of five different DNA repair pathways: direct, base excision, nucleotide excision (NER), double-strand break (DSBR), and postreplicative repair. In addition, six genes involved in DNA replication (REP) and three telomere maintenance genes were investigated. Total RNAs extracted from fresh-frozen tumors and corresponding normal tissues of 50 consecutive chemo-naïve resected NSCLC patients were analyzed. Transcript levels were quantified by real-time PCR. A significant overexpression was detected in 20 of 30 (67%) genes, mostly belonging to DSBR pathways, whereas others (XPA, XPC, and UBE2N; 10%) were significantly underexpressed. For 7 of 30 (23%) genes, mostly belonging to NER pathway, no significant difference between paired tumor and normal samples was observed. Transcript overexpression of DSBR and REP genes was significantly higher in poorly differentiated carcinomas and DSBR levels were higher in men compared with women. The transcriptional overexpression of four genes (XRCC5, TOP3B, TYMS, and UNG) showed significant correlation with a shorter patients' outcome at the univariate, whereas only stage of disease appeared as an independent factor affecting prognosis, as assessed by multivariate analysis. In conclusion, genes belonging to DNA repair/replication pathways are overexpressed in NSCLC and are associated with a more aggressive phenotype.

  5. Genes associated with genotype-specific DNA methylation in squamous cell carcinoma as candidate drug targets

    PubMed Central

    2014-01-01

    Background Aberrant DNA methylation is often associated with cancers. Thus, screening genes with cancer-associated aberrant DNA methylation is a useful method to identify candidate cancer-causing genes. Aberrant DNA methylation is also genotype dependent. Thus, the selection of genes with genotype-specific aberrant DNA methylation in cancers is potentially important for tailor-made medicine. The selected genes are important candidate drug targets. Results The recently proposed principal component analysis based selection of genes with aberrant DNA methylation was applied to genotype and DNA methylation patterns in squamous cell carcinoma measured using single nucleotide polymorphism (SNP) arrays. SNPs that are frequently found in cancers are usually highly methylated, and the genes that were selected using this method were reported previously to be related to cancers. Thus, genes with genotype-specific DNA methylation patterns will be good therapeutic candidates. The tertiary structures of the proteins encoded by the selected genes were successfully inferred using two profile-based protein structure servers, FAMS and Phyre2. Candidate drugs for three of these proteins, tyrosine kinase receptor (ALK), EGLN3 protein, and NUAK family SNF1-like kinase 1 (NUAK1), were identified by ChooseLD. Conclusions We detected genes with genotype-specific DNA methylation in squamous cell carcinoma that are candidate drug targets. Using in silico drug discovery, we successfully identified several candidate drugs for the ALK, EGLN3 and NUAK1 genes that displayed genotype-specific DNA methylation. PMID:24565165

  6. Longer resistance of some DNA traits from BT176 maize to gastric juice from gastrointestinal affected patients.

    PubMed

    Ferrini, A M; Mannoni, V; Pontieri, E; Pourshaban, M

    2007-01-01

    The presence of antibiotic resistance marker genes in genetically engineered plants is one of the most controversial issues related to Genetically Modified Organism (GMO)-containing food, raising concern about the possibility that these markers could increase the pool of antibiotic resistance genes. This study investigates the in vitro survival of genes bla and cryIA(b) of maize Bt176 in human gastric juice samples. Five samples of gastric juice were collected from patients affected by gastro-esophageal reflux or celiac disease and three additional samples were obtained by pH modification with NaHCO3. DNA was extracted from maize Bt176 and incubated with samples of gastric juices at different times. The survival of the target traits (bla gene, whole 1914 bp gene cry1A(b), and its 211 bp fragment) was determined using PCR. The stability of the target genes was an inverse function of their lengths in all the samples. Survival in samples from untreated subjects was below the normal physiological time of gastric digestion. On the contrary, survival time in samples from patients under anti-acid drug treatment or in samples whose pH was modified, resulted strongly increased. Our data indicate the possibility that in particular cases the survival time could be so delayed that, as a consequence, some traits of DNA could reach the intestine. In general, this aspect must be considered for vulnerable consumers (people suffering from gastrointestinal diseases related to altered digestive functionality, physiological problems or drug side-effects) in the risk analysis usually referred to healthy subjects.

  7. Intrinsic features of Aedes aegypti genes affect transcriptional responsiveness of mosquito genes to dengue virus infection.

    PubMed

    Behura, Susanta K; Severson, David W

    2012-10-01

    Dengue virus infection causes significant morbidity and mortality in humans world-wide. The Aedes aegypti mosquito is the major vector that spreads dengue virus to humans. Interaction between dengue viruses and A. aegypti is a multi-factorial phenomena that is determined by both virus and mosquito genotypes. Although, studies have suggested significant association of mosquito vectorial capacity with population variation of dengue virus, specifications of the vector factors that may influence vector-virus compatibility are very limited in the literature. Recently, we have shown that a large number of genes are differentially expressed between MOYO-S (susceptible) and MOYO-R (refractory) A. aegypti strains upon infection with dengue virus (JAM-1409 genotype). In the current study, we show that specific intrinsic features of A. aegypti genes are significantly associated with 'responsiveness' of mosquito genes to dengue infection. Binomial logistic regression analysis further reveals differential marginal effects of these features on gene responsiveness of mosquitoes to the viral infection. Thus, our result shows that intrinsic features of genes significantly affect differential expression of A. aegypti genes to dengue infection. The information will benefit further investigations on evolution of genes among natural populations of A. aegypti conferring differential susceptibility to dengue virus. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Sublethal gamma irradiation affects reproductive impairment and elevates antioxidant enzyme and DNA repair activities in the monogonont rotifer Brachionus koreanus.

    PubMed

    Han, Jeonghoon; Won, Eun-Ji; Kim, Il-Chan; Yim, Joung Han; Lee, Su-Jae; Lee, Jae-Seong

    2014-10-01

    To examine the effects of gamma radiation on marine organisms, we irradiated several doses of gamma ray to the microzooplankton Brachionus koreanus, and measured in vivo and in vitro endpoints including the survival rate, lifespan, fecundity, population growth, gamma ray-induced oxidative stress, and modulated patterns of enzyme activities and gene expressions after DNA damage. After gamma radiation, no individuals showed any mortality within 96 h even at a high intensity (1200 Gy). However, a reduced fecundity (e.g. cumulated number of offspring) of B. koreanus at over 150 Gy was observed along with a slight decrease in lifespan. At 150 Gy and 200 Gy, the reduced fecundity of the rotifers led to a significant decrease in population growth, although in the second generation the population growth pattern was not affected even at 200 Gy when compared to the control group. At sub-lethal doses, reactive oxygen species (ROS) levels dose-dependently increased with GST enzyme activity. In addition, up-regulations of the antioxidant and chaperoning genes in response to gamma radiation were able to recover cellular damages, and life table parameters were significantly influenced, particularly with regard to fecundity. DNA repair-associated genes showed significantly up-regulated expression patterns in response to sublethal doses (150 and 200 Gy), as shown in the expression of the gamma-irradiated B. koreanus p53 gene, suggesting that these sublethal doses were not significantly fatal to B. koreanus but induced DNA damages leading to a decrease of the population size. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Analysis of gene transcription in cells lacking DNA-PK activity.

    PubMed

    Bryntesson, F; Regan, J C; Jeggo, P A; Taccioli, G E; Hubank, M

    2001-08-01

    The DNA-dependent protein kinase (DNA-PK), comprised of the Ku70/Ku80 (now known as G22p1/Xrcc5) heterodimer and the catalytic subunit DNA-PKcs (now known as Prkdc), is required for the nonhomologous end joining (NHEJ) pathway of DNA double-strand break repair. The mechanism of action of DNA-PK remains unclear. We have investigated whether DNA-PK regulates gene transcription in vivo after DNA damage using the subtractive hybridization technique of cDNA representational difference analysis (cDNA RDA). Differential transcription, both radiation-dependent and independent, was detected and confirmed in primary mouse embryo fibroblasts from DNA-PKcs(-/-) and DNA-PKcs(+/+) mice. We present evidence that transcription of the extracellular matrix gene laminin alpha 4 (Lama4) is regulated by DNA-PK in a radiation-independent manner. However, screening of both primary and immortalized DNA-PKcs-deficient cell lines demonstrates that the majority of differences were not consistently dependent on DNA-PK status. Similar results were obtained in experiments using KU mutant hamster cell lines, indicating heterogeneity of transcription between closely related cell lines. Our results suggest that while DNA-PK may be involved in limited gene-specific transcription, it does not play a major role in the transcriptional response to DNA damage.

  10. l-Ornithine affects peripheral clock gene expression in mice.

    PubMed

    Fukuda, Takafumi; Haraguchi, Atsushi; Kuwahara, Mari; Nakamura, Kaai; Hamaguchi, Yutaro; Ikeda, Yuko; Ishida, Yuko; Wang, Guanying; Shirakawa, Chise; Tanihata, Yoko; Ohara, Kazuaki; Shibata, Shigenobu

    2016-10-05

    The peripheral circadian clock is entrained by factors in the external environment such as scheduled feeding, exercise, and mental and physical stresses. In addition, recent studies in mice demonstrated that some food components have the potential to control the peripheral circadian clock during scheduled feeding, although information about these components remains limited. l-Ornithine is a type of non-protein amino acid that is present in foods and has been reported to have various physiological functions. In human trials, for example, l-ornithine intake improved a subjective index of sleep quality. Here we demonstrate, using an in vivo monitoring system, that repeated oral administration of l-ornithine at an early inactive period in mice induced a phase advance in the rhythm of PER2 expression. By contrast, l-ornithine administration to mouse embryonic fibroblasts did not affect the expression of PER2, indicating that l-ornithine indirectly alters the phase of PER2. l-Ornithine also increased plasma levels of insulin, glucose and glucagon-like peptide-1 alongside mPer2 expression, suggesting that it exerts its effects probably via insulin secretion. Collectively, these findings demonstrate that l-ornithine affects peripheral clock gene expression and may expand the possibilities of L-ornithine as a health food.

  11. l-Ornithine affects peripheral clock gene expression in mice

    PubMed Central

    Fukuda, Takafumi; Haraguchi, Atsushi; Kuwahara, Mari; Nakamura, Kaai; Hamaguchi, Yutaro; Ikeda, Yuko; Ishida, Yuko; Wang, Guanying; Shirakawa, Chise; Tanihata, Yoko; Ohara, Kazuaki; Shibata, Shigenobu

    2016-01-01

    The peripheral circadian clock is entrained by factors in the external environment such as scheduled feeding, exercise, and mental and physical stresses. In addition, recent studies in mice demonstrated that some food components have the potential to control the peripheral circadian clock during scheduled feeding, although information about these components remains limited. l-Ornithine is a type of non-protein amino acid that is present in foods and has been reported to have various physiological functions. In human trials, for example, l-ornithine intake improved a subjective index of sleep quality. Here we demonstrate, using an in vivo monitoring system, that repeated oral administration of l-ornithine at an early inactive period in mice induced a phase advance in the rhythm of PER2 expression. By contrast, l-ornithine administration to mouse embryonic fibroblasts did not affect the expression of PER2, indicating that l-ornithine indirectly alters the phase of PER2. l-Ornithine also increased plasma levels of insulin, glucose and glucagon-like peptide-1 alongside mPer2 expression, suggesting that it exerts its effects probably via insulin secretion. Collectively, these findings demonstrate that l-ornithine affects peripheral clock gene expression and may expand the possibilities of L-ornithine as a health food. PMID:27703199

  12. How nanochannel confinement affects the DNA melting transition within the Poland-Scheraga model

    NASA Astrophysics Data System (ADS)

    Reiter-Schad, Michaela; Werner, Erik; Tegenfeldt, Jonas O.; Mehlig, Bernhard; Ambjörnsson, Tobias

    2015-09-01

    When double-stranded DNA molecules are heated, or exposed to denaturing agents, the two strands are separated. The statistical physics of this process has a long history and is commonly described in terms of the Poland-Scheraga (PS) model. Crucial to this model is the configurational entropy for a melted region (compared to the entropy of an intact region of the same size), quantified by the loop factor. In this study, we investigate how confinement affects the DNA melting transition, by using the loop factor for an ideal Gaussian chain. By subsequent numerical solutions of the PS model, we demonstrate that the melting temperature depends on the persistence lengths of single-stranded and double-stranded DNA. For realistic values of the persistence lengths, the melting temperature is predicted to decrease with decreasing channel diameter. We also demonstrate that confinement broadens the melting transition. These general findings hold for the three scenarios investigated: 1. homo-DNA, i.e., identical basepairs along the DNA molecule, 2. random sequence DNA, and 3. "real" DNA, here T4 phage DNA. We show that cases 2 and 3 in general give rise to broader transitions than case 1. Case 3 exhibits a similar phase transition as case 2 provided the random sequence DNA has the same ratio of AT to GC basepairs (A - adenine, T - thymine, G - guanine, C - cytosine). A simple analytical estimate for the shift in melting temperature is provided as a function of nanochannel diameter. For homo-DNA, we also present an analytical prediction of the melting probability as a function of temperature.

  13. [Gene expression of AAV-ITR ssDNA mini vector in skeletal muscle of mice].

    PubMed

    Zhu, Dongqin; Zhang, Yun; Liu, Xiaomei; Zhang, Chun

    2014-11-01

    AAV-ITR single strand DNA mini vector (AAV-ITR ssDNA mini vector) is a novel gene expression vector based on AAV-ITR. We have shown efficient gene expression of AAV-ITR ssDNA mini vector in HEK 293T. Here, we studied the efficacy of gene expression of AAV-ITR ssDNA mini vector in vivo. We injected the skeletal muscle of ICR mice separately with equal molars of AAV-ITR ssDNA mini vector, ITR mutated AAV-ITR single strand DNA mini vector (AAV-ITRmm ssDNA mutant vector), AAV-ITR dsDNA and pUC57-minivector-GFP, combined with TurboFect. Florescence microscope analysis of skeletal muscle section shows that AAV-ITR ssDNA mini vector had higher expression efficiency and longer expression period. We extracted DNA from the muscle three months after injection and quantified three vectors by Real-time PCR. RT-PCR analysis shows that there were highest copy numbers of AAV-ITR ssDNA mini vector existing in muscle. Stable existing of AAV- TR ssDNA mini vector in muscle could be the molecular basis of long term gene expression of the vector. The results suggest that AAV-ITR ssDNA mini vector might be a promising vector for gene therapy.

  14. DNA Binding Region” of BRCA1 Affects Genetic Stability through modulating the Intra-S-Phase Checkpoint

    PubMed Central

    Masuda, Takaaki; Xu, Xiaoling; Dimitriadis, Emilios K.; Lahusen, Tyler; Deng, Chu-Xia

    2016-01-01

    The breast cancer associated gene 1 (BRCA1) contains 3 domains: an N-terminal RING domain with ubiquitin E3 ligase activity, C-terminal BRCT protein interaction domain and a central region. RING and BRCT domains are well characterized, yet the function of the central region remains unclear. In this study, we identified an essential DNA binding region (DBR: 421-701 amino acids) within the central region of human BRCA1, and found that BRCA1 brings DNA together and preferably binds to splayed-arm DNA in a sequence-independent manner. To investigate the biological role of the DBR, we generated mouse ES cells, which lack the DBR (ΔDBR) by using the TALEN method. The ΔDBR cells exhibited decreased survival as compared to the wild type (WT) cells treated with a PARP inhibitor, however they have an intact ability to conduct DNA repair mediated by homologous recombination (HR). The ΔDBR cells continued to incorporate more EdU in the presence of hydroxyurea (HU), which causes replication stress and exhibited reduced viability than the WT cells. Moreover, phosphorylation of CHK1, which regulates the intra-S phase checkpoint, was moderately decreased in ΔDBR cells. These data suggest that DNA binding by BRCA1 affects the stability of DNA replication folks, resulting in weakened intra-S-phase checkpoint control in the ΔDBR cells. The ΔDBR cells also exhibited an increased number of abnormal chromosome structures as compared with WT cells, indicating that the ΔDBR cells have increased genetic instability. Thus, we demonstrated that the DBR of BRCA1 modulates genetic stability through the intra-S-phase checkpoint activated by replication stress. PMID:26884712

  15. "DNA Binding Region" of BRCA1 Affects Genetic Stability through modulating the Intra-S-Phase Checkpoint.

    PubMed

    Masuda, Takaaki; Xu, Xiaoling; Dimitriadis, Emilios K; Lahusen, Tyler; Deng, Chu-Xia

    2016-01-01

    The breast cancer associated gene 1 (BRCA1) contains 3 domains: an N-terminal RING domain with ubiquitin E3 ligase activity, C-terminal BRCT protein interaction domain and a central region. RING and BRCT domains are well characterized, yet the function of the central region remains unclear. In this study, we identified an essential DNA binding region (DBR: 421-701 amino acids) within the central region of human BRCA1, and found that BRCA1 brings DNA together and preferably binds to splayed-arm DNA in a sequence-independent manner. To investigate the biological role of the DBR, we generated mouse ES cells, which lack the DBR (ΔDBR) by using the TALEN method. The ΔDBR cells exhibited decreased survival as compared to the wild type (WT) cells treated with a PARP inhibitor, however they have an intact ability to conduct DNA repair mediated by homologous recombination (HR). The ΔDBR cells continued to incorporate more EdU in the presence of hydroxyurea (HU), which causes replication stress and exhibited reduced viability than the WT cells. Moreover, phosphorylation of CHK1, which regulates the intra-S phase checkpoint, was moderately decreased in ΔDBR cells. These data suggest that DNA binding by BRCA1 affects the stability of DNA replication folks, resulting in weakened intra-S-phase checkpoint control in the ΔDBR cells. The ΔDBR cells also exhibited an increased number of abnormal chromosome structures as compared with WT cells, indicating that the ΔDBR cells have increased genetic instability. Thus, we demonstrated that the DBR of BRCA1 modulates genetic stability through the intra-S-phase checkpoint activated by replication stress.

  16. mtDNA depletion confers specific gene expression profiles in human cells grown in culture and in xenograft

    PubMed Central

    Magda, Darren; Lecane, Philip; Prescott, Julia; Thiemann, Patricia; Ma, Xuan; Dranchak, Patricia K; Toleno, Donna M; Ramaswamy, Krishna; Siegmund, Kimberly D; Hacia, Joseph G

    2008-01-01

    Background Interactions between the gene products encoded by the mitochondrial and nuclear genomes play critical roles in eukaryotic cellular function. However, the effects mitochondrial DNA (mtDNA) levels have on the nuclear transcriptome have not been defined under physiological conditions. In order to address this issue, we characterized the gene expression profiles of A549 lung cancer cells and their mtDNA-depleted ρ0 counterparts grown in culture and as tumor xenografts in immune-deficient mice. Results Cultured A549 ρ0 cells were respiration-deficient and showed enhanced levels of transcripts relevant to metal homeostasis, initiation of the epithelial-mesenchymal transition, and glucuronidation pathways. Several well-established HIF-regulated transcripts showed increased or decreased abundance relative to the parental cell line. Furthermore, growth in culture versus xenograft has a significantly greater influence on expression profiles, including transcripts involved in mitochondrial structure and both aerobic and anaerobic energy metabolism. However, both in vitro and in vivo, mtDNA levels explained the majority of the variance observed in the expression of transcripts in glucuronidation, tRNA synthetase, and immune surveillance related pathways. mtDNA levels in A549 xenografts also affected the expression of genes, such as AMACR and PHYH, involved in peroxisomal lipid metabolic pathways. Conclusion We have identified mtDNA-dependent gene expression profiles that are shared in cultured cells and in xenografts. These profiles indicate that mtDNA-depleted cells could provide informative model systems for the testing the efficacy of select classes of therapeutics, such as anti-angiogenesis agents. Furthermore, mtDNA-depleted cells grown culture and in xenografts provide a powerful means to investigate possible relationships between mitochondrial activity and gene expression profiles in normal and pathological cells. PMID:18980691

  17. Study of design parameters affecting the motion of DNA for nanoinjection

    NASA Astrophysics Data System (ADS)

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

    2012-05-01

    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.

  18. Complete gene expression profiling of Saccharopolyspora erythraea using GeneChip DNA microarrays

    PubMed Central

    Peano, Clelia; Bicciato, Silvio; Corti, Giorgio; Ferrari, Francesco; Rizzi, Ermanno; Bonnal, Raoul JP; Bordoni, Roberta; Albertini, Alberto; Bernardi, Luigi Rossi; Donadio, Stefano; De Bellis, Gianluca

    2007-01-01

    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 organization of the chromosomes

  19. A recent evolutionary change affects a regulatory element in the human FOXP2 gene.

    PubMed

    Maricic, Tomislav; Günther, Viola; Georgiev, Oleg; Gehre, Sabine; Curlin, Marija; Schreiweis, Christiane; Naumann, Ronald; Burbano, Hernán A; Meyer, Matthias; Lalueza-Fox, Carles; de la Rasilla, Marco; Rosas, Antonio; Gajovic, Srecko; Kelso, Janet; Enard, Wolfgang; Schaffner, Walter; Pääbo, Svante

    2013-04-01

    The FOXP2 gene is required for normal development of speech and language. By isolating and sequencing FOXP2 genomic DNA fragments from a 49,000-year-old Iberian Neandertal and 50 present-day humans, we have identified substitutions in the gene shared by all or nearly all present-day humans but absent or polymorphic in Neandertals. One such substitution is localized in intron 8 and affects a binding site for the transcription factor POU3F2, which is highly conserved among vertebrates. We find that the derived allele of this site is less efficient than the ancestral allele in activating transcription from a reporter construct. The derived allele also binds less POU3F2 dimers than POU3F2 monomers compared with the ancestral allele. Because the substitution in the POU3F2 binding site is likely to alter the regulation of FOXP2 expression, and because it is localized in a region of the gene associated with a previously described signal of positive selection, it is a plausible candidate for having caused a recent selective sweep in the FOXP2 gene.

  20. Glycans affect DNA extraction and induce substantial differences in gut metagenomic studies

    PubMed Central

    Angelakis, Emmanouil; Bachar, Dipankar; Henrissat, Bernard; Armougom, Fabrice; Audoly, Gilles; Lagier, Jean-Christophe; Robert, Catherine; Raoult, Didier

    2016-01-01

    Exopolysaccharides produced by bacterial species and present in feces are extremely inhibitory to DNA restriction and can cause discrepancies in metagenomic studies. We determined the effects of different DNA extraction methods on the apparent composition of the gut microbiota using Illumina MiSeq deep sequencing technology. DNA was extracted from the stool from an obese female using 10 different methods and the choice of DNA extraction method affected the proportional abundance at the phylum level, species richness (Chao index, 227 to 2,714) and diversity (non parametric Shannon, 1.37 to 4.4). Moreover DNA was extracted from stools obtained from 83 different individuals by the fastest extraction assay and by an extraction assay that degradated exopolysaccharides. The fastest extraction method was able to detect 68% to 100% genera and 42% to 95% species whereas the glycan degradation extraction method was able to detect 56% to 93% genera and 25% to 87% species. To allow a good liberation of DNA from exopolysaccharides commonly presented in stools, we recommend the mechanical lysis of stools plus glycan degradation, used here for the first time. Caution must be taken in the interpretation of current metagenomic studies, as the efficiency of DNA extraction varies widely among stool samples. PMID:27188959

  1. Association of DNA methylation and monoamine oxidase A gene expression in the brains of different dog breeds.

    PubMed

    Eo, JungWoo; Lee, Hee-Eun; Nam, Gyu-Hwi; Kwon, Yun-Jeong; Choi, Yuri; Choi, Bong-Hwan; Huh, Jae-Won; Kim, Minkyu; Lee, Sang-Eun; Seo, Bohyun; Kim, Heui-Soo

    2016-04-15

    The monoamine oxidase A (MAOA) gene is an important candidate gene for human behavior that encodes an enzyme regulating the metabolism of key neurotransmitters. The regulatory mechanisms of the MAOA gene in dogs are yet to be elucidated. We measured MAOA gene transcription and analyzed the VNTR genotype and methylation status of the gene promoter region in different dog breeds to determine whether MAOA expression is correlated with the MAOA genotype or epigenetic modification in dogs. We found brain-specific expression of the MAOA gene and different transcription levels in different dog breeds including Beagle, Sapsaree, and German shepherd, and also a robust association of the DNA methylation of the gene promoter with mRNA levels. However, the 90 bp tandem repeats that we observed near the transcription start site were not variable, indicating no correlation with canine MAOA activity. These results show that differential DNA methylation in the MAOA promoter region may affect gene expression by modulating promoter activity. Moreover, the distinctive patterns of MAOA expression and DNA methylation may be involved in breed-specific or individual behavioral characteristics, such as aggression, because behavioral phenotypes are related to different physiological and neuroendocrine responses. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    PubMed

    Pai, Athma A; Bell, Jordana T; Marioni, John C; Pritchard, Jonathan K; Gilad, Yoav

    2011-02-01

    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.

  3. DNA methylation affected by male sterile cytoplasm in rice (Oryza sativa L.)

    USDA-ARS?s Scientific Manuscript database

    Male sterile cytoplasm plays an important role in hybrid rice and cytoplasmic effects are sufficiently documented. However, no reports are available on DNA methylation affected by male sterile cytoplasm in hybrid rice. We used a methylation sensitive amplified polymorphism (MSAP) technique to charac...

  4. Comprehensive gene expression profiling following DNA vaccination of rainbow trout against infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Purcell, Maureen K.; Nichols, Krista M.; Winton, James R.; Kurath, Gael; Thorgaard, Gary H.; Wheeler, Paul; Hansen, John D.; Herwig, Russell P.; Park, Linda K.

    2006-01-01

    The DNA vaccine based on the glycoprotein gene of Infectious hematopoietic necrosis virus induces a non-specific anti-viral immune response and long-term specific immunity against IHNV. This study characterized gene expression responses associated with the early anti-viral response. Homozygous rainbow trout were injected intra-muscularly (I.M.) with vector DNA or the IHNV DNA vaccine. Gene expression in muscle tissue (I.M. site) was evaluated using a 16,008 feature salmon cDNA microarray. Eighty different genes were significantly modulated in the vector DNA group while 910 genes were modulated in the IHNV DNA vaccinate group relative to control group. Quantitative reverse-transcriptase PCR was used to examine expression of selected immune genes at the I.M. site and in other secondary tissues. In the localized response (I.M. site), the magnitudes of gene expression changes were much greater in the vaccinate group relative to the vector DNA group for the majority of genes analyzed. At secondary systemic sites (e.g. gill, kidney and spleen), type I IFN-related genes were up-regulated in only the IHNV DNA vaccinated group. The results presented here suggest that the IHNV DNA vaccine induces up-regulation of the type I IFN system across multiple tissues, which is the functional basis of early anti-viral immunity.

  5. Gypenosides causes DNA damage and inhibits expression of DNA repair genes of human oral cancer SAS cells.

    PubMed

    Lu, Kung-Wen; Chen, Jung-Chou; Lai, Tung-Yuan; Yang, Jai-Sing; Weng, Shu-Wen; Ma, Yi-Shih; Tang, Nou-Ying; Lu, Pei-Jung; Weng, Jing-Ru; Chung, Jing-Gung

    2010-01-01

    Gypenosides (Gyp) are the major components of Gynostemma pentaphyllum Makino, a Chinese medical plant. Recently, Gyp has been shown to induce cell cycle arrest and apoptosis in many human cancer cell lines. However, there is no available information to address the effects of Gyp on DNA damage and DNA repair-associated gene expression in human oral cancer cells. Therefore, we investigated whether Gyp induced DNA damage and DNA repair gene expression in human oral cancer SAS cells. The results from flow cytometric assay indicated that Gyp-induced cytotoxic effects led to a decrease in the percentage of viable SAS cells. The results from comet assay revealed that the incubation of SAS cells with Gyp led to a longer DNA migration smear (comet tail) when compared with control and this effect was dose-dependent. The results from real-time PCR analysis indicated that treatment of SAS cells with 180 mug/ml of Gyp for 24 h led to a decrease in 14-3-3sigma, DNA-dependent serine/threonine protein kinase (DNAPK), p53, ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and breast cancer gene 1 (BRCA1) mRNA expression. These observations may explain the cell death caused by Gyp in SAS cells. Taken together, Gyp induced DNA damage and inhibited DNA repair-associated gene expressions in human oral cancer SAS cells in vitro.

  6. Tandem transcription termination sites in the dnaN gene of Escherichia coli.

    PubMed

    Armengod, M E; García-Sogo, M; Pérez-Roger, I; Macián, F; Navarro-Aviñó, J P

    1991-10-15

    The dnaN gene of Escherichia coli encodes the beta-subunit of DNA polymerase III and maps between the dnaA and recF genes. We demonstrated previously that dnaN and recF constitute a transcriptional unit under control of the dnaN promoters. However, the recF gene has its own promoter region located in the middle of the dnaN structural gene. In this report, we use S1 mapping of mRNAs, transcriptional and translational fusions to the galK and lacZ genes, and in vitro mutagenesis to identify and characterize three tandem transcription termination sites responsible for transcriptional polarity in the dnaN-recF operon. These sites are located in the dnaN gene, downstream from the recF promoter region. Cumulatively, they terminate about 80% of the untranslated transcripts started at the recF promoters. As expected, they do not reduce transcription coming from the dnaN promoters unless dnaN translation was prematurely disrupted by the presence of a nonsense codon. The particular arrangement of regulatory elements (promoters and terminators) in the dnaN-recF region provides an exceptional in vivo system to confirm the latent termination site model of transcriptional polarity. In addition, our results contribute to the understanding of the complex regulation of the dnaA, dnaN, and recF genes. We propose that these three genes constitute an operon and that the terminators described in this work could be used to reduce expression of the distal genes of the operon under circumstances in which the dnaN translation happens to be slowed down.

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

    PubMed

    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.

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

    PubMed

    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.

  9. Molecular cloning of a gene involved in methotrexate uptake by DNA-mediated gene transfer.

    PubMed

    Underhill, T M; Williams, F M; Murray, R C; Flintoff, W F

    1992-07-01

    A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from a wild-type Chinese hamster ovary genomic cosmid library. Primary and secondary transfectants, which contain a limited number of cosmid sequences, have been shown to regain methotrexate sensitivity and to take up methotrexate. Furthermore, the DNA from three cosmid clones, isolated from a primary methotrexate-sensitive transfectant, after transfection rescued the methotrexate-resistant phenotype at a high frequency. Restriction endonuclease analysis of the DNA of these cosmid clones indicated that they overlapped extensively and shared two regions of Chinese hamster ovary DNA of 6.6 kb and 20.6 kb. These observations indicate that a gene involved in methotrexate uptake is contained in its entirety within one of these regions. This is the first report of the functional molecular cloning of a gene involved in methotrexate uptake. A general strategy is also described for screening large cosmid libraries from primary transfectants.

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

    PubMed

    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.

  11. A High Phosphorus Diet Affects Lipid Metabolism in Rat Liver: A DNA Microarray Analysis

    PubMed Central

    Chun, Sunwoo; Bamba, Takeshi; Suyama, Tatsuya; Ishijima, Tomoko; Fukusaki, Eiichiro; Abe, Keiko; Nakai, Yuji

    2016-01-01

    A high phosphorus (HP) diet causes disorders of renal function, bone metabolism, and vascular function. We previously demonstrated that DNA microarray analysis is an appropriate method to comprehensively evaluate the effects of a HP diet on kidney dysfunction such as calcification, fibrillization, and inflammation. We reported that type IIb sodium-dependent phosphate transporter is significantly up-regulated in this context. In the present study, we performed DNA microarray analysis to investigate the effects of a HP diet on the liver, which plays a pivotal role in energy metabolism. DNA microarray analysis was performed with total RNA isolated from the livers of rats fed a control diet (containing 0.3% phosphorus) or a HP diet (containing 1.2% phosphorus). Gene Ontology analysis of differentially expressed genes (DEGs) revealed that the HP diet induced down-regulation of genes involved in hepatic amino acid catabolism and lipogenesis, while genes related to fatty acid β-oxidation process were up-regulated. Although genes related to fatty acid biosynthesis were down-regulated in HP diet-fed rats, genes important for the elongation and desaturation reactions of omega-3 and -6 fatty acids were up-regulated. Concentrations of hepatic arachidonic acid and eicosapentaenoic acid were increased in HP diet-fed rats. These essential fatty acids activate peroxisome proliferator-activated receptor alpha (PPARα), a transcription factor for fatty acid β-oxidation. Evaluation of the upstream regulators of DEGs using Ingenuity Pathway Analysis indicated that PPARα was activated in the livers of HP diet-fed rats. Furthermore, the serum concentration of fibroblast growth factor 21, a hormone secreted from the liver that promotes fatty acid utilization in adipose tissue as a PPARα target gene, was higher (p = 0.054) in HP diet-fed rats than in control diet-fed rats. These data suggest that a HP diet enhances energy expenditure through the utilization of free fatty acids

  12. A High Phosphorus Diet Affects Lipid Metabolism in Rat Liver: A DNA Microarray Analysis.

    PubMed

    Chun, Sunwoo; Bamba, Takeshi; Suyama, Tatsuya; Ishijima, Tomoko; Fukusaki, Eiichiro; Abe, Keiko; Nakai, Yuji

    2016-01-01

    A high phosphorus (HP) diet causes disorders of renal function, bone metabolism, and vascular function. We previously demonstrated that DNA microarray analysis is an appropriate method to comprehensively evaluate the effects of a HP diet on kidney dysfunction such as calcification, fibrillization, and inflammation. We reported that type IIb sodium-dependent phosphate transporter is significantly up-regulated in this context. In the present study, we performed DNA microarray analysis to investigate the effects of a HP diet on the liver, which plays a pivotal role in energy metabolism. DNA microarray analysis was performed with total RNA isolated from the livers of rats fed a control diet (containing 0.3% phosphorus) or a HP diet (containing 1.2% phosphorus). Gene Ontology analysis of differentially expressed genes (DEGs) revealed that the HP diet induced down-regulation of genes involved in hepatic amino acid catabolism and lipogenesis, while genes related to fatty acid β-oxidation process were up-regulated. Although genes related to fatty acid biosynthesis were down-regulated in HP diet-fed rats, genes important for the elongation and desaturation reactions of omega-3 and -6 fatty acids were up-regulated. Concentrations of hepatic arachidonic acid and eicosapentaenoic acid were increased in HP diet-fed rats. These essential fatty acids activate peroxisome proliferator-activated receptor alpha (PPARα), a transcription factor for fatty acid β-oxidation. Evaluation of the upstream regulators of DEGs using Ingenuity Pathway Analysis indicated that PPARα was activated in the livers of HP diet-fed rats. Furthermore, the serum concentration of fibroblast growth factor 21, a hormone secreted from the liver that promotes fatty acid utilization in adipose tissue as a PPARα target gene, was higher (p = 0.054) in HP diet-fed rats than in control diet-fed rats. These data suggest that a HP diet enhances energy expenditure through the utilization of free fatty acids

  13. Dietary Flavanols Modulate the Transcription of Genes Associated with Cardiovascular Pathology without Changes in Their DNA Methylation State

    PubMed Central

    Boby, Céline; Leroux, Christine; Declerck, Ken; Szarc vel Szic, Katarzyna; Heyninck, Karen; Laukens, Kris; Bizet, Martin; Defrance, Matthieu; Dedeurwaerder, Sarah; Calonne, Emilie; Fuks, Francois; Haegeman, Guy; Haenen, Guido R. M. M.; Bast, Aalt; Weseler, Antje R.

    2014-01-01

    Background In a recent intervention study, the daily supplementation with 200 mg monomeric and oligomeric flavanols (MOF) from grape seeds for 8 weeks revealed a vascular health benefit in male smokers. The objective of the present study was to determine the impact of MOF consumption on the gene expression profile of leukocytes and to assess changes in DNA methylation. Methodology/Principal Findings Gene expression profiles were determined using whole genome microarrays (Agilent) and DNA methylation was assessed using HumanMethylation450 BeadChips (Illumina). MOF significantly modulated the expression of 864 genes. The majority of the affected genes are involved in chemotaxis, cell adhesion, cell infiltration or cytoskeleton organisation, suggesting lower immune cell adhesion to endothelial cells. This was corroborated by in vitro experiments showing that MOF exposure of monocytes attenuates their adhesion to TNF-α-stimulated endothelial cells. Nuclear factor kappa B (NF-κB) reporter gene assays confirmed that MOF decrease the activity of NF-κB. Strong inter-individual variability in the leukocytes' DNA methylation was observed. As a consequence, on group level, changes due to MOF supplementation could not be found. Conclusion Our study revealed that an 8 week daily supplementation with 200 mg MOF modulates the expression of genes associated with cardiovascular disease pathways without major changes of their DNA methylation state. However, strong inter-individual variation in leukocyte DNA methylation may obscure the subtle epigenetic response to dietary flavanols. Despite the lack of significant changes in DNA methylation, the modulation of gene expression appears to contribute to the observed vascular health effect of MOF in humans. PMID:24763279

  14. Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

    PubMed Central

    Simon, J R; Moore, P D

    1987-01-01

    Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA. Images PMID:3302673

  15. Changes in mitochondrial DNA alter expression of nuclear encoded genes associated with tumorigenesis

    SciTech Connect

    Jandova, Jana; Janda, Jaroslav; Sligh, James E

    2012-10-15

    We previously reported the presence of a mtDNA mutation hotspot in UV-induced premalignant and malignant skin tumors in hairless mice. We have modeled this change (9821insA) in murine cybrid cells and demonstrated that this alteration in mtDNA associated with mtBALB haplotype can alter the biochemical characteristics of cybrids and subsequently can contribute to significant changes in their behavioral capabilities. This study shows that changes in mtDNA can produce differences in expression levels of specific nuclear-encoded genes, which are capable of triggering the phenotypes such as seen in malignant cells. From a potential list of differentially expressed genes discovered by microarray analysis, we selected MMP-9 and Col1a1 for further studies. Real-time PCR confirmed up-regulation of MMP-9 and down-regulation of Col1a1 in cybrids harboring the mtDNA associated with the skin tumors. These cybrids also showed significantly increased migration and invasion abilities compared to wild type. The non-specific MMP inhibitor, GM6001, was able to inhibit migratory and invasive abilities of the 9821insA cybrids confirming a critical role of MMPs in cellular motility. Nuclear factor-{kappa}B (NF-{kappa}B) is a key transcription factor for production of MMPs. An inhibitor of NF-{kappa}B activation, Bay 11-7082, was able to inhibit the expression of MMP-9 and ultimately decrease migration and invasion of mutant cybrids containing 9821insA. These studies confirm a role of NF-{kappa}B in the regulation of MMP-9 expression and through this regulation modulates the migratory and invasive capabilities of cybrids with mutant mtDNA. Enhanced migration and invasion abilities caused by up-regulated MMP-9 may contribute to the tumorigenic phenotypic characteristics of mutant cybrids. -- Highlights: Black-Right-Pointing-Pointer Cybrids are useful models to study the role of mtDNA changes in cancer development. Black-Right-Pointing-Pointer mtDNA changes affect the expression of nuclear

  16. The nucleotide sequence of the sheep MHC class II DNA gene

    SciTech Connect

    Wright, H.; Redmond, J.; Ballingall, K.T.; Wright, F.

    1995-01-11

    The human MHC class II DNA gene was identified and sequenced by Trowsdale and Kelly. When a molecular map of the HLA-D region became available, it was shown that the HLA-DNA gene was unusual in not having a B gene partner situated within a few kilobases (kb), the nearest B gene being HLA-DPB1. The nearest unpaired B gene is HLA-DOB which is approximately 160 kb telomeric of HLA-DNA. More recently, the mouse MHC class II genes H-20A and H-20B were shown to be equivalent to the HLA-DNA and HLA-DOB genes. Moreover, the mouse genes expressed an MHC class II protein whose tissue distribution was restricted to B cells and epithelial cell of the thymic medulla. No corresponding HLA-DN protein has been reported. 21 refs., 3 figs.

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

    PubMed

    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.

  18. Control of gene editing by manipulation of DNA repair mechanisms.

    PubMed

    Danner, Eric; Bashir, Sanum; Yumlu, Saniye; Wurst, Wolfgang; Wefers, Benedikt; Kühn, Ralf

    2017-04-03

    DNA double-strand breaks (DSBs) are produced intentionally by RNA-guided nucleases to achieve genome editing through DSB repair. These breaks are repaired by one of two main repair pathways, classic non-homologous end joining (c-NHEJ) and homology-directed repair (HDR), the latter being restricted to the S/G2 phases of the cell cycle and notably less frequent. Precise genome editing applications rely on HDR, with the abundant c-NHEJ formed mutations presenting a barrier to achieving high rates of precise sequence modifications. Here, we give an overview of HDR- and c-NHEJ-mediated DSB repair in gene editing and summarize the current efforts to promote HDR over c-NHEJ.

  19. From DNA Copy Number to Gene Expression: Local aberrations, Trisomies and Monosomies

    NASA Astrophysics Data System (ADS)

    Shay, Tal

    The goal of my PhD research was to study the effect of DNA copy number changes on gene expression. DNA copy number aberrations may be local, encompassing several genes, or on the level of an entire chromosome, such as trisomy and monosomy. The main dataset I studied was of Glioblastoma, obtained in the framework of a collaboration, but I worked also with public datasets of cancer and Down's Syndrome. The molecular basis of expression changes in Glioblastoma. Glioblastoma is the most common and aggressive type of primary brain tumors in adults. In collaboration with Prof. Hegi (CHUV, Switzerland), we analyzed a rich Glioblastoma dataset including clinical information, DNA copy number (array CGH) and expression profiles. We explored the correlation between DNA copy number and gene expression at the level of chromosomal arms and local genomic aberrations. We detected known amplification and over expression of oncogenes, as well as deletion and down-regulation of tumor suppressor genes. We exploited that information to map alterations of pathways that are known to be disrupted in Glioblastoma, and tried to characterize samples that have no known alteration in any of the studied pathways. Identifying local DNA aberrations of biological significance. Many types of tumors exhibit chromosomal losses or gains and local amplifications and deletions. A region that is aberrant in many tumors, or whose copy number change is stronger, is more likely to be clinically relevant, and not just a by-product of genetic instability. We developed a novel method that defines and prioritizes aberrations by formalizing these intuitions. The method scores each aberration by the fraction of patients harboring it, its length and its amplitude, and assesses the significance of the score by comparing it to a null distribution obtained by permutations. This approach detects genetic locations that are significantly aberrant, generating a 'genomic aberration profile' for each sample. The 'genomic

  20. Purkinje Cell Degeneration in pcd Mice Reveals Large Scale Chromatin Reorganization and Gene Silencing Linked to Defective DNA Repair*

    PubMed Central

    Baltanás, Fernando C.; Casafont, Iñigo; Lafarga, Vanesa; Weruaga, Eduardo; Alonso, José R.; Berciano, María T.; Lafarga, Miguel

    2011-01-01

    DNA repair protects neurons against spontaneous or disease-associated DNA damage. Dysfunctions of this mechanism underlie a growing list of neurodegenerative disorders. The Purkinje cell (PC) degeneration mutation causes the loss of nna1 expression and is associated with the postnatal degeneration of PCs. This PC degeneration dramatically affects nuclear architecture and provides an excellent model to elucidate the nuclear mechanisms involved in a whole array of neurodegenerative disorders. We used immunocytochemistry for histone variants and components of the DNA damage response, an in situ transcription assay, and in situ hybridization for telomeres to analyze changes in chromatin architecture and function. We demonstrate that the phosphorylation of H2AX, a DNA damage signal, and the trimethylation of the histone H4K20, a repressive mark, in extensive domains of genome are epigenetic hallmarks of chromatin in degenerating PCs. These histone modifications are associated with a large scale reorganization of chromatin, telomere clustering, and heterochromatin-induced gene silencing, all of them key factors in PC degeneration. Furthermore, ataxia telangiectasia mutated and 53BP1, two components of the DNA repair pathway, fail to be concentrated in the damaged chromatin compartments, even though the expression levels of their coding genes were slightly up-regulated. Although the mechanism by which Nna1 loss of function leads to PC neurodegeneration is undefined, the progressive accumulation of DNA damage in chromosome territories irreversibly compromises global gene transcription and seems to trigger PC degeneration and death. PMID:21700704

  1. The facioscapulohumeral muscular dystrophy (FSHD1) gene affects males more severely and more frequently than females.

    PubMed

    Zatz, M; Marie, S K; Cerqueira, A; Vainzof, M; Pavanello, R C; Passos-Bueno, M R

    1998-05-01

    We investigated 52 families of patients with facioscapulohumeral muscular dystrophy (FSHD1), including 172 patients (104 males and 68 females). Among 273 DNA samples which were analyzed with probe p13E-11, 131 (67 males and 64 females) were shown to carry an EcoRI fragment smaller than 35 kb; 114 among them were examined clinically and neurologically. Results of the present investigation showed that: a) there is no molecular evidence for autosomal or X-linked recessive inheritance of FSHD1; b) an excess of affected males, which is explained by a significantly greater proportion of females than males among asymptomatic cases and a significantly greater proportion of affected sons than daughters observed in the offspring of asymptomatic mothers; c) the penetrance of the FSHD1 gene until age 30 was estimated as 83% for both sexes but was significantly greater for males (95%) than for females (69%); d) new mutations occur significantly more frequently in females than males among somatic/germinal mosaic cases; and e) severely affected cases originated more often through new mutations or were transmitted through maternal than through paternal lines including somatic/germinal mothers. These observations have important implications for understanding the molecular mechanisms responsible for FSHD1 and for genetic and prognostic counseling according to the gender of the affected patient.

  2. DNA sequence of the Escherichia coli tonB gene.

    PubMed Central

    Postle, K; Good, R F

    1983-01-01

    The nucleotide sequence of a cloned section of the Escherichia coli chromosome containing the tonB gene has been determined. Transcription initiation and termination sites for tonB RNA have been determined by S1 nuclease mapping. The tonB promoter and terminator resemble other E. coli promoters and terminators; the sequence of the tonB terminator region suggests that it may function bidirectionally. The DNA sequence specifies an open translation reading frame between the 5' and 3' RNA termini whose location is consistent with the position of previously isolated tonB::IS1 mutations. The DNA sequence predicts a proline-rich protein with a calculated size of 26.1-26.6 kilodaltons (239-244 amino acids), depending on which of three potential initiation codons is utilized. The predicted NH2 terminus of tonB protein resembles the proteolytically cleaved signal sequences of E. coli periplasmic and outer membrane proteins; the overall hydrophilic character of the protein sequence suggests that the bulk of the tonB protein is not embedded within the inner or outer membrane. A significant discrepancy exists between the calculated size of tonB protein and the apparent size of 36 kilodaltons determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Images PMID:6310567

  3. Widespread DNA hypomethylation and differential gene expression in Turner syndrome

    PubMed Central

    Trolle, Christian; Nielsen, Morten Muhlig; Skakkebæk, Anne; Lamy, Philippe; Vang, Søren; Hedegaard, Jakob; Nordentoft, Iver; Ørntoft, Torben Falck; Pedersen, Jakob Skou; Gravholt, Claus Højbjerg

    2016-01-01

    Adults with 45,X monosomy (Turner syndrome) reflect a surviving minority since more than 99% of fetuses with 45,X monosomy die in utero. In adulthood 45,X monosomy is associated with increased morbidity and mortality, although strikingly heterogeneous with some individuals left untouched while others suffer from cardiovascular disease, autoimmune disease and infertility. The present study investigates the leukocyte DNAmethylation profile by using the 450K-Illumina Infinium assay and the leukocyte RNA-expression profile in 45,X monosomy compared with karyotypically normal female and male controls. We present results illustrating that genome wide X-chromosome RNA-expression profile, autosomal DNA-methylation profile, and the X-chromosome methylation profile clearly distinguish Turner syndrome from controls. Our results reveal genome wide hypomethylation with most differentially methylated positions showing a medium level of methylation. Contrary to previous studies, applying a single loci specific analysis at well-defined DNA loci, our results indicate that the hypomethylation extend to repetitive elements. We describe novel candidate genes that could be involved in comorbidity in TS and explain congenital urinary malformations (PRKX), premature ovarian failure (KDM6A), and aortic aneurysm formation (ZFYVE9 and TIMP1). PMID:27687697

  4. Polymorphisms in selected DNA repair genes and cell cycle regulating genes involved in the risk of papillary thyroid carcinoma.

    PubMed

    Halkova, Tereza; Dvorakova, Sarka; Sykorova, Vlasta; Vaclavikova, Eliska; Vcelak, Josef; Vlcek, Petr; Sykorova, Pavla; Kodetova, Daniela; Betka, Jan; Lastuvka, Petr; Bavor, Petr; Hoch, Jiri; Katra, Rami; Bendlova, Bela

    2016-06-07

    Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. In addition to causal somatic mutations in the BRAF gene and RET/PTC rearrangements, the contribution of single nucleotide polymorphisms (SNPs) in low-penetrance genes in the development of PTC has been proposed. Four SNPs in the XRCC1 (Arg399Gln, Arg280His, Arg194Trp and T-77C) and one SNP from each of three other genes participating in DNA repair pathways and/or cell cycle regulation (ATM Asp1853Asn, TP53 Arg72Pro, CDKN1B Val109Gly) were selected. The allelic and genotypic distributions of these variants as well as haplotypes of the XRCC1 were examined in 583 individuals comprising well-characterized cohorts of 209 PTC patients and 374 healthy volunteers. Correlations of polymorphism with clinical-pathological data and mutation status were performed. XRCC1 T-77C polymorphism affects the genetic susceptibility for PTC development in men, the specific combination of XRCC1 haplotypes correlates with RET/PTC incidence, CDKN1B Val109Gly significantly influences the risk of developing PTC regardless of gender and in PTC cases, selected genotypes of TP53 Arg72Pro and ATM Asp1853Asn were significantly associated with monitored tumour characteristics. It seems that SNPs in studied regulating genes contribute to the development of PTC and modify the tumour behaviour or characteristics.

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

    SciTech Connect

    Woloschak, G.E.; Libertin, C.R.; Weaver, P.; Churchill, M.; Chang-Liu, C.M.

    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.

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

    PubMed

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

    2017-05-29

    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).

  7. Periconceptional folate consumption is associated with neonatal DNA methylation modifications in neural crest regulatory and cancer development genes.

    PubMed

    Gonseth, Semira; Roy, Ritu; Houseman, E Andres; de Smith, Adam J; Zhou, Mi; Lee, Seung-Tae; Nusslé, Sébastien; Singer, Amanda W; Wrensch, Margaret R; Metayer, Catherine; Wiemels, Joseph L

    2015-01-01

    Folate deficiency during early embryonic development constitutes a risk factor for neural tube defects and potentially for childhood leukemia via unknown mechanisms. We tested whether folate consumption during the 12 months prior to conception induced DNA methylation modifications at birth in healthy neonates with a genome-wide and agnostic approach. We hypothesized that DNA methylation in genes involved in neural tube development and/or cancer susceptibility would be affected by folate exposure. We retrospectively assessed folate exposure at the time of conception by food-frequency questionnaires administered to the mothers of 343 healthy newborns. We measured genome-wide DNA methylation from neonatal blood spots. We implemented a method based on bootstrap resampling to decrease false-positive findings. Folate was inversely associated with DNA methylation throughout the genome. Among the top folate-associated genes that were replicated in an independent Gambian study were TFAP2A, a gene critical for neural crest development, STX11, a gene implicated in acute myeloid leukemia, and CYS1, a candidate gene for cystic kidney disease. Reduced periconceptional folate intake was associated with increased methylation and, in turn, decreased gene expression at these 3 loci. The top folate-sensitive genes defined by their associated CpG sites were enriched for numerous transcription factors by Gene Set Enrichment Analysis, including those implicated in cancer development (e.g., MYC-associated zinc finger protein). The influence of estimated periconceptional folate intake on neonatal DNA methylation levels provides potential mechanistic insights into the role of this vitamin in the development of neural tube defects and childhood cancers.

  8. Mitochondrial DNA Haplogroup Background Affects LHON, but Not Suspected LHON, in Chinese Patients

    PubMed Central

    Bi, Rui; Salas, Antonio; Li, Shiqiang; Xiao, Xueshan; Wang, Panfeng; Guo, Xiangming; Kong, Qing-Peng; Zhang, Qingjiong; Yao, Yong-Gang

    2011-01-01

    Recent studies have shown that mtDNA background could affect the clinical expression of Leber hereditary optic neuropathy (LHON). We analyzed the mitochondrial DNA (mtDNA) variation of 304 Chinese patients with m.11778G>A (sample #1) and of 843 suspected LHON patients who lack the three primary mutations (sample #2) to discern mtDNA haplogroup effect on disease onset. Haplogroup frequencies in the patient group was compared to frequencies in the general Han Chinese population (n = 1,689; sample #3). The overall matrilineal composition of the suspected LHON population resembles that of the general Han Chinese population, suggesting no association with mtDNA haplogroup. In contrast, analysis of these LHON patients confirms mtDNA haplogroup effect on LHON. Specifically, the LHON sample significantly differs from the general Han Chinese and suspected LHON populations by harboring an extremely lower frequency of haplogroup R9, in particular of its main sub-haplogroup F (#1 vs. #3, P-value = 1.46×10−17, OR = 0.051, 95% CI: 0.016–0.162; #1 vs. #2, P-value = 4.44×10−17, OR = 0.049, 95% CI: 0.015–0.154; in both cases, adjusted P-value <10−5) and higher frequencies of M7b (#1 vs. #3, adjusted P-value = 0.001 and #1 vs. #2, adjusted P-value = 0.004). Our result shows that mtDNA background affects LHON in Chinese patients with m.11778G>A but not suspected LHON. Haplogroup F has a protective effect against LHON, while M7b is a risk factor. PMID:22110754

  9. Mitochondrial DNA haplogroup background affects LHON, but not suspected LHON, in Chinese patients.

    PubMed

    Zhang, A-Mei; Jia, Xiaoyun; Bi, Rui; Salas, Antonio; Li, Shiqiang; Xiao, Xueshan; Wang, Panfeng; Guo, Xiangming; Kong, Qing-Peng; Zhang, Qingjiong; Yao, Yong-Gang

    2011-01-01

    Recent studies have shown that mtDNA background could affect the clinical expression of Leber hereditary optic neuropathy (LHON). We analyzed the mitochondrial DNA (mtDNA) variation of 304 Chinese patients with m.11778G>A (sample #1) and of 843 suspected LHON patients who lack the three primary mutations (sample #2) to discern mtDNA haplogroup effect on disease onset. Haplogroup frequencies in the patient group was compared to frequencies in the general Han Chinese population (n = 1,689; sample #3). The overall matrilineal composition of the suspected LHON population resembles that of the general Han Chinese population, suggesting no association with mtDNA haplogroup. In contrast, analysis of these LHON patients confirms mtDNA haplogroup effect on LHON. Specifically, the LHON sample significantly differs from the general Han Chinese and suspected LHON populations by harboring an extremely lower frequency of haplogroup R9, in particular of its main sub-haplogroup F (#1 vs. #3, P-value = 1.46×10(-17), OR = 0.051, 95% CI: 0.016-0.162; #1 vs. #2, P-value = 4.44×10(-17), OR = 0.049, 95% CI: 0.015-0.154; in both cases, adjusted P-value <10(-5)) and higher frequencies of M7b (#1 vs. #3, adjusted P-value = 0.001 and #1 vs. #2, adjusted P-value = 0.004). Our result shows that mtDNA background affects LHON in Chinese patients with m.11778G>A but not suspected LHON. Haplogroup F has a protective effect against LHON, while M7b is a risk factor.

  10. Determining the effect of DNA methylation on gene expression in cancer cells.

    PubMed

    Lee, Chai-Jin; Evans, Jared; Kim, Kwangsoo; Chae, Heejoon; Kim, Sun

    2014-01-01

    DNA methylation, a DNA modification by adding methyl group to cytosine, has an important role in the regulation of gene expression. DNA methylation is known to be associated with gene transcription by interfering with DNA-binding proteins, such as transcription factors. DNA methylation is closely related to tumorigenesis, and the methylation state of some genes can be used as a biomarker for tumorigenesis. Aberrant DNA methylation of genomic regions, including CpG islands, CpG shores, and first exons, is related to the altered gene expression pattern characteristics of all human cancers. Subheading 1 surveys recent developments on DNA methylation and gene expressions in cancer. Then we provide analysis of DNA methylation and gene expression in 30 breast cancer cell lines representing different tumor phenotypes. This study conducted an integrated analysis to identify the relationship between DNA methylation in various genomic regions and expression levels of downstream genes, using MethylCapseq data (affinity purification followed by next-generation sequencing of eluted DNA) and Affymetrix gene expression microarray data. The goal of this study was to assess genome-wide methylation profiles associated with different molecular subtypes of human breast cancer (luminal, basal A, and basal B) and to comprehensively investigate the effect of DNA methylation on gene expression in breast cancer phenotypes. This showed that methylation of genomic regions near transcription start sites, CpG island, CpG shore, and first exon was strongly associated with gene repression, and the effects of the regions on gene expression patterns were different for different molecular subtypes of breast cancer. The results further indicated that aberrant methylation of specific genomic regions was significantly associated with different breast cancer subtypes.

  11. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease.

    PubMed

    Dupuy, Aurélie; Sarasin, Alain

    2015-06-01

    Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients.

  12. DNA methylation affects nuclear organization, histone modifications, and linker histone binding but not chromatin compaction.

    PubMed

    Gilbert, Nick; Thomson, Inga; Boyle, Shelagh; Allan, James; Ramsahoye, Bernard; Bickmore, Wendy A

    2007-05-07

    DNA methylation has been implicated in chromatin condensation and nuclear organization, especially at sites of constitutive heterochromatin. How this is mediated has not been clear. In this study, using mutant mouse embryonic stem cells completely lacking in DNA methylation, we show that DNA methylation affects nuclear organization and nucleosome structure but not chromatin compaction. In the absence of DNA methylation, there is increased nuclear clustering of pericentric heterochromatin and extensive changes in primary chromatin structure. Global levels of histone H3 methylation and acetylation are altered, and there is a decrease in the mobility of linker histones. However, the compaction of both bulk chromatin and heterochromatin, as assayed by nuclease digestion and sucrose gradient sedimentation, is unaltered by the loss of DNA methylation. This study shows how the complete loss of a major epigenetic mark can have an impact on unexpected levels of chromatin structure and nuclear organization and provides evidence for a novel link between DNA methylation and linker histones in the regulation of chromatin structure.

  13. Cell-free DNA testing after combined test: factors affecting the uptake.

    PubMed

    Maiz, Nerea; Alzola, Irune; Murua, Emerson J; Rodríguez Santos, Javier

    2016-11-01

    First, to assess what was the uptake of cell free DNA (cfDNA) testing after a combined test and the maternal and fetal factors that influenced this decision, and second, to assess the uptake and factors that influence the choice of invasive testing. This observational retrospective study included 1083 singleton pregnancies who had a combined test for screening for Down syndrome between 11 (+) (0) and 13 (+) (6) weeks. Multivariate logistic regression analysis was used to determine which factors affected the uptake of cfDNA test and invasive testing among risk for trisomies 21, 18, and 13, maternal characteristics and fetal nuchal translucency (NT) thickness. Two-hundred fifty-seven (23.7%) women had a cfDNA test, 89 (8.2%) had an invasive test, and 737 (68.1%) had no further test. The uptake of cfDNA increased with the risk for trisomies (p < 0.001), maternal age (p = 0.013), and was higher in nulliparous women (p = 0.004). The uptake of invasive test increased with the risk for trisomies (p < 0.001) and NT thickness (p < 0.001). This study shows that the uptake of cfDNA testing increases with the risk for trisomies, maternal age, and is higher in nulliparous, whereas the uptake of invasive testing increases with the risk for trisomies and NT thickness.

  14. Structure and function of the DNA ligases encoded by the mammalian LIG3 gene.

    PubMed

    Tomkinson, Alan E; Sallmyr, Annahita

    2013-12-01

    Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA ligase, DNA ligase I. DNA ligase IIIα is a component of an alternative non-homologous end joining (NHEJ) pathway for DNA double-strand break (DSB) repair that is more active when the major DNA ligase IV-dependent pathway is defective. Unlike its other nuclear functions, the role of DNA ligase IIIα in alternative NHEJ is independent of its nuclear partner protein, X-ray repair cross-complementing protein 1 (XRCC1). DNA ligase IIIα is frequently overexpressed in cancer cells, acting as a biomarker for increased dependence upon alternative NHEJ for DSB repair and it is a promising novel therapeutic target. © 2013 Elsevier B.V. All rights reserved.

  15. Structure and function of the DNA ligases encoded by the mammalian LIG3 gene

    PubMed Central

    Tomkinson, Alan E.; Sallmyr, Annahita

    2013-01-01

    Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA ligase, DNA ligase I. DNA ligase IIIα is a component of an alternative non-homologous end joining (NHEJ) pathway for DNA double-strand break (DSB) repair that is more active when the major DNA ligase IV-dependent pathway is defective. Unlike its other nuclear functions, the role of DNA ligase IIIα in alternative NHEJ is independent of its nuclear partner protein, X-ray repair cross-complementing protein 1 (XRCC1). DNA ligase IIIα is frequently overexpressed in cancer cells, acting as a biomarker for increased dependence upon alternative NHEJ for DSB repair and it is a promising novel therapeutic target. PMID:24013086

  16. 16S rRNA gene probe quantitates residual host cell DNA in pharmaceutical-grade plasmid DNA.

    PubMed

    Wang, Kai-Yu; Guo, Ying-Jun; Sun, Shu-Han; Shi, Ke; Zhang, Shu; Wang, Kai-Hui; Yi-Zhang; Chen, Zu-Huan

    2006-03-24

    The development and widespread use of DNA-based vaccination against infectious pathogens have been a great triumph of medical science. Quality control of DNA vaccines as biopharmaceutical productions is a problem to solve. Residual genomic DNA of engineering bacteria has been identified as a potential risk factor, so whose level must be controlled under the regulatory standards. We report a dot-blot hybridization method to detect residual host cell DNA in purified DNA vaccines. The assay utilizes PCR amplified and digoxigenin-labeled Escherichia coli 16S rRNA gene as probe. The sensitivity of the dot-blot hybridization assay with E. coli 16S rRNA gene probe was evaluated in comparison with single copy UidR gene probe. The optimized dot-blot hybridization assay had both low background and a suitable sensitivity, detecting 10 pg of residual E. coli DNA. The method is suitable in the routine use of measuring the levels of residual E. coli DNA in the pharmaceutical-grade DNA vaccine.

  17. Beryllium chloride-induced oxidative DNA damage and alteration in the expression patterns of DNA repair-related genes.

    PubMed

    Attia, Sabry M; Harisa, Gamaleldin I; Hassan, Memy H; Bakheet, Saleh A

    2013-09-01

    Beryllium metal has physical properties that make its use essential for very specific applications, such as medical diagnostics, nuclear/fusion reactors and aerospace applications. Because of the widespread human exposure to beryllium metals and the discrepancy of the genotoxic results in the reported literature, detail assessments of the genetic damage of beryllium are warranted. Mice exposed to beryllium chloride at an oral dose of 23mg/kg for seven consecutive days exhibited a significant increase in the level of DNA-strand breaking and micronuclei formation as detected by a bone marrow standard comet assay and micronucleus test. Whereas slight beryllium chloride-induced oxidative DNA damage was detected following formamidopyrimidine DNA glycosylase digestion, digestion with endonuclease III resulted in considerable increases in oxidative DNA damage after the 11.5 and 23mg/kg/day treatment as detected by enzyme-modified comet assays. Increased 8-hydroxydeoxyguanosine was also directly correlated with increased bone marrow micronuclei formation and DNA strand breaks, which further confirm the involvement of oxidative stress in the induction of bone marrow genetic damage after exposure to beryllium chloride. Gene expression analysis on the bone marrow cells from beryllium chloride-exposed mice showed significant alterations in genes associated with DNA damage repair. Therefore, beryllium chloride may cause genetic damage to bone marrow cells due to the oxidative stress and the induced unrepaired DNA damage is probably due to the down-regulation in the expression of DNA repair genes, which may lead to genotoxicity and eventually cause carcinogenicity.

  18. Gene duplication and divergence affecting drug content in Cannabis sativa.

    PubMed

    Weiblen, George D; Wenger, Jonathan P; Craft, Kathleen J; ElSohly, Mahmoud A; Mehmedic, Zlatko; Treiber, Erin L; Marks, M David

    2015-12-01

    Cannabis sativa is an economically important source of durable fibers, nutritious seeds, and psychoactive drugs but few economic plants are so poorly understood genetically. Marijuana and hemp were crossed to evaluate competing models of cannabinoid inheritance and to explain the predominance of tetrahydrocannabinolic acid (THCA) in marijuana compared with cannabidiolic acid (CBDA) in hemp. Individuals in the resulting F2 population were assessed for differential expression of cannabinoid synthase genes and were used in linkage mapping. Genetic markers associated with divergent cannabinoid phenotypes were identified. Although phenotypic segregation and a major quantitative trait locus (QTL) for the THCA/CBDA ratio were consistent with a simple model of codominant alleles at a single locus, the diversity of THCA and CBDA synthase sequences observed in the mapping population, the position of enzyme coding loci on the map, and patterns of expression suggest multiple linked loci. Phylogenetic analysis further suggests a history of duplication and divergence affecting drug content. Marijuana is distinguished from hemp by a nonfunctional CBDA synthase that appears to have been positively selected to enhance psychoactivity. An unlinked QTL for cannabinoid quantity may also have played a role in the recent escalation of drug potency.

  19. Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

    2008-01-01

    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 shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

  20. Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

    2008-01-01

    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 shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

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

    PubMed Central

    Fuemmeler, Bernard F.; Lee, Chien-Ti; Soubry, Adelheid; Iversen, Edwin S.; Huang, Zhiqing; Murtha, Amy P.; Schildkraut, Joellen M.; Jirtle, Randy L.; Murphy, Susan K.; Hoyo, Cathrine

    2016-01-01

    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. PMID:27920589

  2. The HRDC domain of E. coli RecQ helicase controls single-stranded DNA translocation and double-stranded DNA unwinding rates without affecting mechanoenzymatic coupling.

    PubMed

    Harami, Gábor M; Nagy, Nikolett T; Martina, Máté; Neuman, Keir C; Kovács, Mihály

    2015-06-11

    DNA-restructuring activities of RecQ-family helicases play key roles in genome maintenance. These activities, driven by two tandem RecA-like core domains, are thought to be controlled by accessory DNA-binding elements including the helicase-and-RnaseD-C-terminal (HRDC) domain. The HRDC domain of human Bloom's syndrome (BLM) helicase was shown to interact with the RecA core, raising the possibility that it may affect the coupling between ATP hydrolysis, translocation along single-stranded (ss)DNA and/or unwinding of double-stranded (ds)DNA. Here, we determined how these activities are affected by the abolition of the ssDNA interaction of the HRDC domain or the deletion of the entire domain in E. coli RecQ helicase. Our data show that the HRDC domain suppresses the rate of DNA-activated ATPase activity in parallel with those of ssDNA translocation and dsDNA unwinding, regardless of the ssDNA binding capability of this domain. The HRDC domain does not affect either the processivity of ssDNA translocation or the tight coupling between the ATPase, translocation, and unwinding activities. Thus, the mechanochemical coupling of E. coli RecQ appears to be independent of HRDC-ssDNA and HRDC-RecA core interactions, which may play roles in more specialized functions of the enzyme.

  3. CHST11 gene expression and DNA methylation in breast cancer

    PubMed Central

    HERMAN, DAMIR; LEAKEY, TATIANA I.; BEHRENS, ALICE; YAO-BORENGASSER, AIWEI; COONEY, CRAIG A.; JOUSHEGHANY, FARIBA; PHANAVANH, BOUNLEUT; SIEGEL, ERIC R.; SAFAR, A. MAZIN; KOROURIAN, SOHEILA; KIEBER-EMMONS, THOMAS; MONZAVI-KARBASSI, BEHJATOLAH

    2015-01-01

    Our previously published data link P-selectin-reactive chondroitin sulfate structures on the surface of breast cancer cells to metastatic behavior of cells. We have shown that a particular sulfation pattern mediated by the expression of carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) correlates with P-selectin binding and aggressiveness of human breast cancer cell lines. The present study was performed to evaluate the prognostic value of CHST11 expression and determine whether aberrant DNA methylation controls CHST11 expression in breast cancer. Publicly available datasets were used to examine the association of CHST11 expression to aggressiveness and progression of breast cancer. Methylation status was analyzed using bisulfite genomic sequencing. 5-aza-2′-deoxycytidine (5AzadC) was used for DNA demethylation. Reduced representation bisulfite sequencing was performed in the CpG island of CHST11 with a minimum coverage of 10. Quantitative real-time RT-PCR was employed to confirm the expression profile of CHST11 in breast cancer cell lines. Flow cytometry was also used to confirm the expression of the CHST11 product, chondroitin sulfate A (CS-A). The expression of CHST11 was significantly higher in basal-like and Her2-amplified cell lines compared to luminal cell lines. CHST11 was also highly expressed in cancer tissues compared to normal tissues and the expression levels were significantly associated with tumor progression. We observed very low levels of DNA methylation in a CpG island of CHST11 in basal-like cells but very high levels in the same region in luminal cells. Treatment of MCF7 cells, a luminal cell line with very low expression of CHST11, with 5AzadC increased the expression of CHST11 and its immediate product, CS-A, in a dose-dependent manner. These results suggest that CHST11 may play a direct role in progression of breast cancer and that its expression is controlled by DNA methylation. Therefore, in addition to CHST11 mRNA levels, the

  4. CHST11 gene expression and DNA methylation in breast cancer.

    PubMed

    Herman, Damir; Leakey, Tatiana I; Behrens, Alice; Yao-Borengasser, Aiwei; Cooney, Craig A; Jousheghany, Fariba; Phanavanh, Bounleut; Siegel, Eric R; Safar, A Mazin; Korourian, Soheila; Kieber-Emmons, Thomas; Monzavi-Karbassi, Behjatolah

    2015-03-01

    Our previously published data link P-selectin-reactive chondroitin sulfate structures on the surface of breast cancer cells to metastatic behavior of cells. We have shown that a particular sulfation pattern mediated by the expression of carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) correlates with P-selectin binding and aggressiveness of human breast cancer cell lines. The present study was performed to evaluate the prognostic value of CHST11 expression and determine whether aberrant DNA methylation controls CHST11 expression in breast cancer. Publicly available datasets were used to examine the association of CHST11 expression to aggressiveness and progression of breast cancer. Methylation status was analyzed using bisulfite genomic sequencing. 5-aza-2'-deoxycytidine (5AzadC) was used for DNA demethylation. Reduced representation bisulfite sequencing was performed in the CpG island of CHST11 with a minimum coverage of 10. Quantitative real-time RT-PCR was employed to confirm the expression profile of CHST11 in breast cancer cell lines. Flow cytometry was also used to confirm the expression of the CHST11 product, chondroitin sulfate A (CS-A). The expression of CHST11 was significantly higher in basal-like and Her2-amplified cell lines compared to luminal cell lines. CHST11 was also highly expressed in cancer tissues compared to normal tissues and the expression levels were significantly associated with tumor progression. We observed very low levels of DNA methylation in a CpG island of CHST11 in basal-like cells but very high levels in the same region in luminal cells. Treatment of MCF7 cells, a luminal cell line with very low expression of CHST11, with 5AzadC increased the expression of CHST11 and its immediate product, CS-A, in a dose-dependent manner. These results suggest that CHST11 may play a direct role in progression of breast cancer and that its expression is controlled by DNA methylation. Therefore, in addition to CHST11 mRNA levels, the

  5. DNA damage response pathway and replication fork stress during oligonucleotide directed gene editing.

    PubMed

    Bonner, Melissa; Strouse, Bryan; Applegate, Mindy; Livingston, Paula; Kmiec, Eric B

    2012-04-03

    Single-stranded DNA oligonucleotides (ODNs) can be used to direct the exchange of nucleotides in the genome of mammalian cells in a process known as gene editing. Once refined, gene editing should become a viable option for gene therapy and molecular medicine. Gene editing is regulated by a number of DNA recombination and repair pathways whose natural activities often lead to single- and double-stranded DNA breaks. It has been previously shown that introduction of a phosphorotioated ODN, designed to direct a gene-editing event, into cells results in the activation of γH2AX, a well-recognized protein biomarker for double-stranded DNA breakage. Using a single copy, integrated mutant enhanced green fluorescent protein (eGFP) gene as our target, we now demonstrate that several types of ODNs, capable of directing gene editing, also activate the DNA damage response and the post-translational modification of proliferating cell nuclear antigen (PCNA), a signature modification of replication stress. We find that the gene editing reaction itself leads to transient DNA breakage, perhaps through replication fork collapse. Unmodified specific ODNs elicit a lesser degree of replication stress than their chemically modified counterparts, but are also less active in gene editing. Modified phosphothioate oligonucleotides (PTOs) are detrimental irrespective of the DNA sequence. Such collateral damage may prove problematic for proliferation of human cells genetically modified by gene editing.

  6. Dietary zinc restriction and repletion affects DNA integrity in healthy men123

    PubMed Central

    Song, Yang; Chung, Carolyn S; Bruno, Richard S; Traber, Maret G; Brown, Kenneth H; King, Janet C

    2009-01-01

    Background: Zinc plays an important role in antioxidant defense and the maintenance of cellular DNA integrity. However, no experimental human studies have been performed to examine the role of zinc status on DNA damage. Objective: We evaluated the effects of dietary zinc depletion and repletion on DNA strand breaks, oxidative stress, and antioxidant defenses in healthy men. Design: Nine healthy men with reported mean daily zinc intakes >11 mg/d were recruited. Subjects completed 3 consecutive dietary periods: baseline (days 1 to 13; 11 mg Zn/d), zinc depletion (days 14 to 55; 0.6 mg Zn/d for 1 wk and 4 mg Zn/d for 5 wk), and zinc repletion (days 56 to 83; 11 mg Zn/d for 4 wk with 20 mg supplemental Zn for first 7 d). Blood samples were collected on days 1, 13, 35, 55, and 83. DNA damage in peripheral blood cells, plasma oxidative stress, and antioxidant defense biomarkers were assessed. Results: Dietary zinc depletion (6 wk) was associated with increased DNA strand breaks in peripheral blood cells (day 13 compared with day 55; P < 0.05), changes that were ameliorated by zinc repletion (day 55 compared with day 83; P < 0.05). Plasma zinc concentrations were negatively correlated with DNA strand breaks (r = −0.60, P = 0.006) during the zinc-depletion period. Plasma α- and γ-tocopherol concentrations, plasma total antioxidant capacity, and erythrocyte superoxide dismutase activity did not change significantly, and plasma F2-isoprostanes were unaffected by dietary period. Conclusions: Changes in dietary zinc intake affected DNA single-strand breaks. Zinc appears to be a critical factor for maintaining DNA integrity in humans. PMID:19515738

  7. Do DNA Microarrays Tell the Story of Gene Expression?

    PubMed Central

    Rosenfeld, Simon

    2010-01-01

    Poor reproducibility of microarray measurements is a major obstacle to their application as an instrument for clinical diagnostics. In this paper, several aspects of poor reproducibility are analyzed. All of them belong to the category of interpretive weaknesses of DNA microarray technology. First, the attention is drawn to the fact that absence of the information regarding post-transcriptional mRNA stability makes it impossible to evaluate the level of gene activity from the relative mRNA abundances, the quantities available from microarray measurements. Second, irreducible intracellular variability with persistent patterns of stochasticity and burstiness put natural limits to reproducibility. Third, strong interactions within intracellular biomolecular networks make it highly problematic to build a bridge between transcription rates of individual genes and structural fidelity of their genetic codes. For these reasons, the microarray measurements of relative mRNA abundances are more appropriate in laboratory settings as a tool for scientific research, hypotheses generating and producing the leads for subsequent validation through more sophisticated technologies. As to clinical settings, where firm conclusive diagnoses, not the leads for further experimentation, are required, microarrays still have a long way to go until they become a reliable instrument in patient-related decision making. PMID:20628535

  8. Alphavirus vectors: applications for DNA vaccine production and gene expression.

    PubMed

    Lundstrom, K

    2000-01-01

    Replication-deficient alphavirus vectors have been developed for efficient high-level transgene expression. The broad host range of alphaviruses has allowed infection of a wide variety of mammalian cell lines and primary cultures. Particularly, G protein-coupled receptors have been expressed at high levels and subjected to binding and functional studies. Expression in suspension cultures has greatly facilitated production of large quantities of recombinant proteins for structural studies. Injection of recombinant alphavirus vectors into rodent brain resulted in local reporter gene expression. Highly neuron-specific expression was obtained in hippocampal slice cultures in vivo. Additionally, preliminary studies in animal models suggest that alphavirus vectors can be attractive candidates for gene therapy applications. Traditionally alphavirus vectors, either attenuated strains or replication-deficient particles, have been used to elicit efficient immune responses in animals. Recently, the application of alphaviruses has been extended to naked nucleic acids. Injection of DNA as well as RNA vectors has demonstrated efficient antigen production. In many cases, protection against lethal challenges has been obtained after immunization with alphavirus particles or nucleic acid vectors. Alphavirus vectors can therefore be considered as potentially promising vectors for vaccine production.

  9. Gene structure, DNA methylation, and imprinted expression of the human SNRPN gene

    SciTech Connect

    Glenn, C.C.; Jong, T.C.; Filbrandt, M.M.

    1996-02-01

    The human SNRPN (small nuclear ribonucleoprotein polypeptide N) gene is one of a gene family that encode proteins involved in pre-mRNA splicing and maps to the smallest deletion region involved in the Prader-Willi syndrome (PWS) within chromosome 15q11-q13. Paternal only expression of SNRPN has previously been demonstrated by use of cell lines from PWS patients (maternal allele only) and Angelman syndrome (AS) patients (paternal allele only). We have characterized two previously unidentified 5{prime} exons of the SNRPN gene and demonstrate that exons -1 and 0 are included in the full-length transcript. This gene is expressed in a wide range of somatic tissues and at high, approximately equal levels in all regions of the brain. Both the first exon of SNRPN (exon -1) and the putative transcription start site are embedded within a CpG island. This CpG island is extensively methylated on the repressed maternal allele and is unmethylated on the expressed paternal allele, in a wide range of fetal and adult somatic cells. This provides a quick and highly reliable diagnostic assay for PWS and AS, which is based on DNA-methylation analysis that has been tested on >100 patients in a variety of tissues. Conversely, several CpG sites {approximately}22 kb downstream of the transcription start site in intron 5 are preferentially methylated on the expressed paternal allele in somatic tissues and male germ cells, whereas these same sites are unmethylated in fetal oocytes. These findings are consistent with a key role for DNA methylation in the imprinted inheritance and subsequent gene expression of the human SNRPN gene. 59 refs., 9 figs., 1 tab.

  10. An increase in negative supercoiling in bacteria reveals topology-reacting gene clusters and a homeostatic response mediated by the DNA topoisomerase I gene.

    PubMed

    Ferrándiz, María-José; Martín-Galiano, Antonio J; Arnanz, Cristina; Camacho-Soguero, Isabel; Tirado-Vélez, José-Manuel; de la Campa, Adela G

    2016-09-06

    We studied the transcriptional response to an increase in DNA supercoiling in Streptococcus pneumoniae by using seconeolitsine, a new topoisomerase I inhibitor. A homeostatic response allowing recovery of supercoiling was observed in cells treated with subinhibitory seconeolitsine concentrations. Supercoiling increases of 40.7% (6 μM) and 72.9% (8 μM) were lowered to 8.5% and 44.1%, respectively. Likewise, drug removal facilitated the recovery of cell viability and DNA-supercoiling. Transcription of topoisomerase I depended on the supercoiling level. Also specific binding of topoisomerase I to the gyrase A gene promoter was detected by chromatin-immunoprecipitation. The transcriptomic response to 8 μM seconeolitsine had two stages. An early stage, associated to an increase in supercoiling, affected 10% of the genome. A late stage, manifested by supercoiling recovery, affected 2% of the genome. Nearly 25% of the early responsive genes formed 12 clusters with a coordinated transcription. Clusters were 6.7-31.4 kb in length and included 9-22 responsive genes. These clusters partially overlapped with those observed under DNA relaxation, suggesting that bacteria manage supercoiling stress using pathways with common components. This is the first report of a coordinated global transcriptomic response that is triggered by an increase in DNA supercoiling in bacteria. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. An increase in negative supercoiling in bacteria reveals topology-reacting gene clusters and a homeostatic response mediated by the DNA topoisomerase I gene

    PubMed Central

    Ferrándiz, María-José; Martín-Galiano, Antonio J.; Arnanz, Cristina; Camacho-Soguero, Isabel; Tirado-Vélez, José-Manuel; de la Campa, Adela G.

    2016-01-01

    We studied the transcriptional response to an increase in DNA supercoiling in Streptococcus pneumoniae by using seconeolitsine, a new topoisomerase I inhibitor. A homeostatic response allowing recovery of supercoiling was observed in cells treated with subinhibitory seconeolitsine concentrations. Supercoiling increases of 40.7% (6 μM) and 72.9% (8 μM) were lowered to 8.5% and 44.1%, respectively. Likewise, drug removal facilitated the recovery of cell viability and DNA-supercoiling. Transcription of topoisomerase I depended on the supercoiling level. Also specific binding of topoisomerase I to the gyrase A gene promoter was detected by chromatin-immunoprecipitation. The transcriptomic response to 8 μM seconeolitsine had two stages. An early stage, associated to an increase in supercoiling, affected 10% of the genome. A late stage, manifested by supercoiling recovery, affected 2% of the genome. Nearly 25% of the early responsive genes formed 12 clusters with a coordinated transcription. Clusters were 6.7–31.4 kb in length and included 9–22 responsive genes. These clusters partially overlapped with those observed under DNA relaxation, suggesting that bacteria manage supercoiling stress using pathways with common components. This is the first report of a coordinated global transcriptomic response that is triggered by an increase in DNA supercoiling in bacteria. PMID:27378778

  12. Cytogenetic and molecular localization of tipE: a gene affecting sodium channels in Drosophila melanogaster.

    PubMed

    Feng, G; Deák, P; Kasbekar, D P; Gil, D W; Hall, L M

    1995-04-01

    Voltage-sensitive sodium channels play a key role in nerve cells where they are responsible for the increase in sodium permeability during the rising phase of action potentials. In Drosophila melanogaster a subset of temperature-sensitive paralytic mutations affect sodium channel function. One such mutation is temperature-induced paralysis locus E (tipE), which has been shown by electrophysiology and ligand binding studies to reduce sodium channel numbers. Three new gamma-ray-induced tipE alleles associated with either visible deletions in 64AB or a translocation breakpoint within 64B2 provide landmarks for positional cloning of tipE. Beginning with the flanking cloned gene Ras2, a 140-kb walk across the translocation breakpoint was completed. Germline transformation using a 42-kb cosmid clone and successively smaller subclones localized the tipE gene within a 7.4-kb genomic DNA segment. Although this chromosome region is rich in transcripts, only three overlapping mRNAs (5.4, 4.4, and 1.7 kb) lie completely within the smallest rescuing construct. The small sizes of the rescuing construct and transcripts suggest that tipE does not encode a standard sodium channel alpha-subunit with four homologous repeats. Sequencing these transcripts will elucidate the role of the tipE gene product in sodium channel functional regulation.

  13. Cytogenetic and molecular localization of tipE: A gene affecting sodium channels in Drosophila melanogaster

    SciTech Connect

    Feng, G.; Deak, P.; Hall, L.M.

    1995-04-01

    Voltage-sensitive sodium channels play a key role in nerve cells where they are responsible for the increase in sodium permeability during the rising phase of action potentials. In Drosophila melanogaster a subset of temperature-sensitive paralytic mutations affect sodium channel function. One such mutation is temperature-induced paralysis locus E (tipE), which has been shown by electrophysiology and ligand binding studies to reduce sodium channel numbers. Three new {gamma}-ray-induced tipE alleles associated with either visible deletions in 64AB or a translocation breakpoint within 64B2 provide landmarks for positional cloning of tipE. Beginning with the flanking cloned gene Ras2, a 140-kb walk across the translocation breakpoint was completed. Germline transformation using a 42-kb cosmid clone and successively smaller subclones localized the tipE gene within a 7.4-kb genomic DNA segment. Although this chromosome region is rich in transcripts, only three overlapping mRNAs (5.4, 4.4, and 1.7 kb) lie completely within the smallest rescuing construct. The small sizes of the rescuing construct and transcripts suggests that tipE does not encode a standard sodium channel {alpha}-subunit with four homologous repeats. Sequencing these transcripts will elucidate the role of the tipE gene product in sodium channel functional regulation. 55 refs., 4 figs., 2 tabs.

  14. DNA adenine methylation of sams1 gene in symbiont-bearing Amoeba proteus.

    PubMed

    Jeon, Taeck J

    2008-10-01

    The expression of amoeba sams genes is switched from sams1 to sams2 when amoebae are infected with Legionella jeonii. To elucidate the mechanism for the inactivation of host sams1 gene by endosymbiotic bacteria, methylation states of the sams1 gene of D and xD amoebae was compared in this study. The sams1 gene of amoebae was methylated at an internal adenine residue of GATC site in symbiont-bearing xD amoebae but not in symbiont-free D amoebae, suggesting that the modification might have caused the inactivation of sams1 in xD amoebae. The sams1 gene of xD amoebae was inactivated at the transcriptional level. Analysis of DNA showed that adenine residues in L. jeonii sams were also methylated, implying that L. jeonii bacteria belong to a Dam methylase-positive strain. In addition, both SAM and Met appeared to act as negative regulators for the expression of sams1 whereas the expression of sams2 was not affected in amoebae.

  15. Candidate gene linkage approach to Identify DNA variants that predispose to preterm birth

    PubMed Central

    Bream, Elise N.A.; Leppellere, Cara R.; Cooper, Margaret E.; Dagle, John M.; Merrill, David C.; Christensen, Kaare; Simhan, Hyagriv N.; Fong, Chin-To; Hallman, Mikko; Muglia, Louis J.; Marazita, Mary L.; Murray, Jeffrey C.

    2013-01-01

    Background To identify genetic variants contributing to preterm birth using a linkage candidate gene approach. Methods We studied 99 single nucleotide polymorphisms for 33 genes in 257 families with preterm births segregating. Nonparametric and parametric analyses were used. Premature infants and mothers of premature infants were defined as affected cases in independent analyses. Results Analyses with the infant as the case identified two genes with evidence of linkage: CRHR1 (p=0.0012) and CYP2E1 (p=0.0011). Analyses with the mother as the case identified four genes with evidence of linkage: ENPP1 (p=0.003), IGFBP3 (p=0.006), DHCR7 (p=0.009), and TRAF2 (p=0.01). DNA sequence analysis of the coding exons and splice sites for CRHR1 and TRAF2 identified no new likely etiologic variants. Conclusion These findings suggest the involvement of six genes acting through the infant and/or the mother in the etiology of preterm birth. PMID:23168575

  16. Blocking transcription of the human rhodopsin gene by triplex-mediated DNA photocrosslinking.

    PubMed

    Intody, Z; Perkins, B D; Wilson, J H; Wensel, T G

    2000-11-01

    To explore the ability of triplex-forming oligodeoxyribonucleotides (TFOs) to inhibit genes responsible for dominant genetic disorders, we used two TFOs to block expression of the human rhodopsin gene, which encodes a G protein-coupled receptor involved in the blinding disorder autosomal dominant retinitis pigmentosa. Psoralen-modified TFOs and UVA irradiation were used to form photoadducts at two target sites in a plasmid expressing a rhodopsin-EGFP fusion, which was then transfected into HT1080 cells. Each TFO reduced rhodopsin-GFP expression by 70-80%, whereas treatment with both reduced expression by 90%. Expression levels of control genes on either the same plasmid or one co-transfected were not affected by the treatment. Mutations at one TFO target eliminated its effect on transcription, without diminishing inhibition by the other TFO. Northern blots indicated that TFO-directed psoralen photoadducts blocked progression of RNA polymerase, resulting in truncated transcripts. Inhibition of gene expression was not relieved over a 72 h period, suggesting that TFO-induced psoralen lesions are not repaired on this time scale. Irradiation of cells after transfection with plasmid and psoralen-TFOs produced photoadducts inside the cells and also inhibited expression of rhodopsin-EGFP. We conclude that directing DNA damage with psoralen-TFOs is an efficient and specific means for blocking transcription from the human rhodopsin gene.

  17. Blocking transcription of the human rhodopsin gene by triplex-mediated DNA photocrosslinking

    PubMed Central

    Intody, Zsofia; Perkins, Brian D.; Wilson, John H.; Wensel, Theodore G.

    2000-01-01

    To explore the ability of triplex-forming oligodeoxyribonucleotides (TFOs) to inhibit genes responsible for dominant genetic disorders, we used two TFOs to block expression of the human rhodopsin gene, which encodes a G protein-coupled receptor involved in the blinding disorder autosomal dominant retinitis pigmentosa. Psoralen-modified TFOs and UVA irradiation were used to form photoadducts at two target sites in a plasmid expressing a rhodopsin–EGFP fusion, which was then transfected into HT1080 cells. Each TFO reduced rhodopsin–GFP expression by 70–80%, whereas treatment with both reduced expression by 90%. Expression levels of control genes on either the same plasmid or one co-transfected were not affected by the treatment. Mutations at one TFO target eliminated its effect on transcription, without diminishing inhibition by the other TFO. Northern blots indicated that TFO-directed psoralen photoadducts blocked progression of RNA polymerase, resulting in truncated transcripts. Inhibition of gene expression was not relieved over a 72 h period, suggesting that TFO-induced psoralen lesions are not repaired on this time scale. Irradiation of cells after transfection with plasmid and psoralen–TFOs produced photoadducts inside the cells and also inhibited expression of rhodopsin–EGFP. We conclude that directing DNA damage with psoralen–TFOs is an efficient and specific means for blocking transcription from the human rhodopsin gene. PMID:11058128

  18. Reporter gene expression in dendritic cells after gene gun administration of plasmid DNA.

    PubMed

    Watkins, Craig; Hopkins, John; Harkiss, Gordon

    2005-07-21

    Dendritic cells (DC) play an integral role in plasmid DNA vaccination. However, the interaction between plasmid DNA and DC in vivo is incompletely understood. In this report, we utilise the sheep pseudoafferent cannulation model to examine the interaction between plasmid DNA encoding enhanced green fluorescent protein (pEGFP) and afferent lymph DC (ALDC) following gene gun administration. The results show that peaks of fluorescent ALDC tended to appear around days 1-4 and 9-13, then erratically thereafter for up to 2 months. Phenotypic analysis showed that EGFP+ ALDC expressed MHC class II, WC6, CD1b, and SIRPalpha markers. Plasmid, detected by PCR, was found in lymph cells and cell-free plasma on a daily basis, and was present variably for up to 2 months. Plasmid was also detected in purified CD1b+ ALDC, but the presence of plasmid did not correlate with EGFP expression by ALDC. Free EGFP in afferent lymph plasma was detectable by luminometry only after three administrations of the plasmid. The results show that gene gun administered pEGFP persisted for extended periods after a single administration, leeching out of skin on a daily basis. The plasmid was associated with both the cellular and fluid components of afferent lymph. EGFP protein appeared in afferent lymph in a pulsatile manner, but associated only with ALDC.

  19. Dimethylarsinic acid in drinking water changed the morphology of urinary bladder but not the expression of DNA repair genes of bladder transitional epithelium in F344 rats.

    PubMed

    Wang, Amy; Wolf, Douglas C; Sen, Banalata; Knapp, Geremy W; Holladay, Steven D; Huckle, William R; Caceci, Thomas; Robertson, John L

    2009-06-01

    Inorganic arsenic increases urinary bladder transitional cell carcinoma in humans. In F344 rats, dimethylarsinic acid (DMA[V]) increases transitional cell carcinoma. Arsenic-induced inhibition of DNA repair has been reported in cultured cell lines and in lymphocytes of arsenic-exposed humans, but it has not been studied in urinary bladder. Should inhibition of DNA damage repair in transitional epithelium occur, it may contribute to carcinogenesis or cocarcinogenesis. We investigated morphology and expression of DNA repair genes in F344 rat transitional cells following up to 100 ppm DMA(V) in drinking water for four weeks. Mitochondria were very sensitive to DMA(V), and swollen mitochondria appeared to be the main source of vacuoles in the transitional epithelium. Real-time reverse transcriptase polymerase chain reaction (Real-Time RT PCR) showed the mRNA levels of tested DNA repair genes, ataxia telangectasia mutant (ATM), X-ray repair cross-complementing group 1 (XRCC1), excision repair cross-complementing group 3/xeroderma pigmentosum B (ERCC3/XPB), and DNA polymerase beta (Polbeta), were not altered by DMA(V). These data suggested that either DMA(V) does not affect DNA repair in the bladder or DMA(V) affects DNA repair without affecting baseline mRNA levels of repair genes. The possibility remains that DMA(V) may lower damage-induced increases in repair gene expression or cause post-translational modification of repair enzymes.

  20. Clinical characteristics and prognosis of acute myeloid leukemia associated with DNA-methylation regulatory gene mutations

    PubMed Central

    Ryotokuji, Takeshi; Yamaguchi, Hiroki; Ueki, Toshimitsu; Usuki, Kensuke; Kurosawa, Saiko; Kobayashi, Yutaka; Kawata, Eri; Tajika, Kenji; Gomi, Seiji; Kanda, Junya; Kobayashi, Anna; Omori, Ikuko; Marumo, Atsushi; Fujiwara, Yusuke; Yui, Shunsuke; Terada, Kazuki; Fukunaga, Keiko; Hirakawa, Tsuneaki; Arai, Kunihito; Kitano, Tomoaki; Kosaka, Fumiko; Tamai, Hayato; Nakayama, Kazutaka; Wakita, Satoshi; Fukuda, Takahiro; Inokuchi, Koiti

    2016-01-01

    In recent years, it has been reported that the frequency of DNA-methylation regulatory gene mutations – mutations of the genes that regulate gene expression through DNA methylation – is high in acute myeloid leukemia. The objective of the present study was to elucidate the clinical characteristics and prognosis of acute myeloid leukemia with associated DNA-methylation regulatory gene mutation. We studied 308 patients with acute myeloid leukemia. DNA-methylation regulatory gene mutations were observed in 135 of the 308 cases (43.8%). Acute myeloid leukemia associated with a DNA-methylation regulatory gene mutation was more frequent in older patients (P<0.0001) and in patients with intermediate cytogenetic risk (P<0.0001) accompanied by a high white blood cell count (P=0.0032). DNA-methylation regulatory gene mutation was an unfavorable prognostic factor for overall survival in the whole cohort (P=0.0018), in patients aged ≤70 years, in patients with intermediate cytogenetic risk, and in FLT3-ITD-negative patients (P=0.0409). Among the patients with DNA-methylation regulatory gene mutations, 26.7% were found to have two or more such mutations and prognosis worsened with increasing number of mutations. In multivariate analysis DNA-methylation regulatory gene mutation was an independent unfavorable prognostic factor for overall survival (P=0.0424). However, patients with a DNA-methylation regulatory gene mutation who underwent allogeneic stem cell transplantation in first remission had a significantly better prognosis than those who did not undergo such transplantation (P=0.0254). Our study establishes that DNA-methylation regulatory gene mutation is an important unfavorable prognostic factor in acute myeloid leukemia. PMID:27247325

  1. A role for Rtt109 in buffering gene-dosage imbalance during DNA replication.

    PubMed

    Voichek, Yoav; Bar-Ziv, Raz; Barkai, Naama

    2016-07-03

    Chromatin can function as an integrator of DNA-related processes, allowing communication, for example, between DNA replication and gene transcription. Such communication is needed to overcome the gene-dosage imbalance introduced during DNA replication, when certain genes are replicated prior to others. Increased transcription of early replicating genes could alter regulatory balances. This does not occur, suggesting a mechanism that suppresses expression from newly replicated DNA. Critical to this buffering is Rtt109, which acetylates the internal K56 residue of newly synthesized histone H3 prior to incorporation onto DNA. H3K56ac distinguishes replicated from non-replicated DNA, communicating this information to the transcription machinery to ensure expression homeostasis during S phase.

  2. Candidate Luminal B Breast Cancer Genes Identified by Genome, Gene Expression and DNA Methylation Profiling

    PubMed Central

    Addou-Klouche, Lynda; Finetti, Pascal; Saade, Marie-Rose; Manai, Marwa; Carbuccia, Nadine; Bekhouche, Ismahane; Letessier, Anne; Charafe-Jauffret, Emmanuelle; Jacquemier, Jocelyne; Spicuglia, Salvatore; de The, Hugues; Viens, Patrice; Bertucci, François; Birnbaum, Daniel; Chaffanet, Max

    2014-01-01

    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. PMID:24416132

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

    PubMed

    Cornen, Stéphanie; Guille, Arnaud; Adélaïde, José; Addou-Klouche, Lynda; Finetti, Pascal; Saade, Marie-Rose; Manai, Marwa; Carbuccia, Nadine; Bekhouche, Ismahane; Letessier, Anne; Raynaud, Stéphane; Charafe-Jauffret, Emmanuelle; Jacquemier, Jocelyne; Spicuglia, Salvatore; de The, Hugues; Viens, Patrice; Bertucci, François; Birnbaum, Daniel; Chaffanet, Max

    2014-01-01

    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.

  4. Locus- and Site-Specific DNA Methylation of 19 kDa Zein Genes in Maize

    PubMed Central

    Li, Xinxin; Miclaus, Mihai; Messing, Joachim

    2016-01-01

    An interesting question in maize development is why only a single zein gene is highly expressed in each of the 19-kDa zein gene clusters (A and B types), z1A2-1 and z1B4, in the immature endosperm. For instance, epigenetic marks could provide a structural difference. Therefore, we investigated the DNA methylation of the arrays of gene copies in both promoter and gene body regions of leaf (non-expressing tissue as a control), normal endosperm, and cultured endosperm. Although we could show that expressed genes have much lower methylation levels in promoter regions than silent ones in both leaf and normal endosperm, there was surprisingly also a difference in the pattern of the z1A and z1B gene clusters. The expression of z1B gene is suppressed by increased DNA methylation and activated with reduced DNA methylation, whereas z1A gene expression is not. DNA methylation in gene coding regions is higher in leaf than in endosperm, whereas no significant difference is observed in gene bodies between expressed and non-expressed gene copies. A median CHG methylation (25–30%) appears to be optimal for gene expression. Moreover, tissue-cultured endosperm can reset the DNA methylation pattern and tissue-specific gene expression. These results reveal that DNA methylation changes of the 19-kDa zein genes is subject to plant development and tissue culture treatment, but varies in different chromosomal locations, indicating that DNA methylation changes do not apply to gene expression in a uniform fashion. Because tissue culture is used to produce transgenic plants, these studies provide new insights into variation of gene expression of integrated sequences. PMID:26741504

  5. Mutations that affect phosphorylation of the adenovirus DNA-binding protein alter its ability to enhance its own synthesis.

    PubMed Central

    Morin, N; Delsert, C; Klessig, D F

    1989-01-01

    The multifunctional adenovirus single-strand DNA-binding protein (DBP) is highly phosphorylated. Its phosphorylation sites are located in the amino-terminal domain of the protein, and its DNA- and RNA-binding activity resides in the carboxy-terminal half of the polypeptide. We have substituted cysteine or alanine for up to 10 of these potential phosphorylation sites by using oligonucleotide-directed mutagenesis. Alteration of one or a few of these sites had little effect on the viability of virus containing the mutated DBP. However, when eight or more sites were altered, viral growth decreased significantly. This suggests that the overall phosphorylation state of the protein was more important than whether any particular site was modified. The reduction in growth correlated with both depressed DNA replication and expression of late genes. This reduction was probably the result of lower DBP accumulation in mutant-infected cells. Interestingly, although the stability of the mutated DBP was not affected, DBP synthesis and the level of its mRNA were depressed 5- to 10-fold for the underphosphorylated protein. These results suggest that DBP enhances its own expression and imply that phosphorylation of the DBP may be important for this function. Similarities to several eucaryotic transcriptional activators, which are composed of negatively charged activating domains and separate binding domains, are discussed. Images PMID:2585602

  6. PcG Proteins, DNA Methylation, and Gene Repression by Chromatin Looping

    PubMed Central

    Tiwari, Vijay K; McGarvey, Kelly M; Licchesi, Julien D.F; Ohm, Joyce E; Herman, James G; Schübeler, Dirk; Baylin, Stephen B

    2008-01-01

    Many DNA hypermethylated and epigenetically silenced genes in adult cancers are Polycomb group (PcG) marked in embryonic stem (ES) cells. We show that a large region upstream (∼30 kb) of and extending ∼60 kb around one such gene, GATA-4, is organized—in Tera-2 undifferentiated embryonic carcinoma (EC) cells—in a topologically complex multi-loop conformation that is formed by multiple internal long-range contact regions near areas enriched for EZH2, other PcG proteins, and the signature PcG histone mark, H3K27me3. Small interfering RNA (siRNA)–mediated depletion of EZH2 in undifferentiated Tera-2 cells leads to a significant reduction in the frequency of long-range associations at the GATA-4 locus, seemingly dependent on affecting the H3K27me3 enrichments around those chromatin regions, accompanied by a modest increase in GATA-4 transcription. The chromatin loops completely dissolve, accompanied by loss of PcG proteins and H3K27me3 marks, when Tera-2 cells receive differentiation signals which induce a ∼60-fold increase in GATA-4 expression. In colon cancer cells, however, the frequency of the long-range interactions are increased in a setting where GATA-4 has no basal transcription and the loops encompass multiple, abnormally DNA hypermethylated CpG islands, and the methyl-cytosine binding protein MBD2 is localized to these CpG islands, including ones near the gene promoter. Removing DNA methylation through genetic disruption of DNA methyltransferases (DKO cells) leads to loss of MBD2 occupancy and to a decrease in the frequency of long-range contacts, such that these now more resemble those in undifferentiated Tera-2 cells. Our findings reveal unexpected similarities in higher order chromatin conformation between stem/precursor cells and adult cancers. We also provide novel insight that PcG-occupied and H3K27me3-enriched regions can form chromatin loops and physically interact in cis around a single gene in mammalian cells. The loops associate with a

  7. Chromatin remodelling and DNA repair genes are frequently mutated in endometrioid endometrial carcinoma.

    PubMed

    García-Sanz, Pablo; Triviño, Juan Carlos; Mota, Alba; Pérez López, María; Colás, Eva; Rojo-Sebastián, Alejandro; García, Ángel; Gatius, Sonia; Ruiz, María; Prat, Jaime; López-López, Rafael; Abal, Miguel; Gil-Moreno, Antonio; Reventós, Jaume; Matias-Guiu, Xavier; Moreno-Bueno, Gema

    2017-04-01

    In developed countries, endometrial carcinoma is the most common cancer that affects the female genital tract. Endometrial carcinoma is divided into two main histological types, type I or endometrioid and type II or non-endometrioid, each of which have characteristic, although not exclusive, molecular alterations and mutational profiles. Nevertheless, information about the implication and relevance of some of these genes in this disease is lacking. We sought here to identify new recurrently mutated genes in endometrioid cancers that play a role in tumourigenesis and that influence the clinical outcome. We focused on low-grade, non-ultramutated tumours as these tumours have a worse prognosis than the ultramutated POLE-positive endometrioid endometrial carcinomas (EECs). We performed exome-sequencing of 11 EECs with matched normal tissue and subsequently validated 15 candidate genes in 76 samples. For the first time, we show that mutations in chromatin remodelling-related genes (KMT2D, KMT2C, SETD1B and BCOR) and in DNA-repair-related genes (BRCA1, BRCA2, RAD50 and CHD4) are frequent in this subtype of endometrial cancer. The alterations to these genes occurred with frequencies ranging from 35.5% for KMT2D to 10.5% for BRCA1 and BCOR, with some showing a tendency toward co-occurrence (RAD50-KMT2D and RAD50-SETD1B). All these genes harboured specific mutational hotspots. In addition, the mutational status of KMT2C, KMT2D and SETD1B helps to predict the degree of myometrial invasion, a critical prognostic feature. These results highlight the possible implication of these genes in this disease, creating opportunities for new therapeutic approaches.

  8. The oli1 gene and flanking sequences in mitochondrial DNA of Saccharomyces cerevisiae: the complete nucleotide sequence of a 1.35 kilobase petite mitochondrial DNA genome covering the oli1 gene.

    PubMed

    Ooi, B G; Nagley, P

    1986-01-01

    As part of our genetic and molecular analysis of mutants of Saccharomyces cerevisiae affected in the oli1 gene (coding for mitochondrial ATPase subunit 9) we have determined the complete nucleotide sequence of the mtDNA genome of a petite (23-3) carrying this gene. Petite 23-3 (1,355 base pairs) retains a continuous segment of the relevant wild-type (J69-1B) mtDNA genome extending 983 nucleotides upstream, and 126 nucleotides downstream, of the 231 nucleotide oli1 coding region. There is a 15-nucleotide excision sequence in petite 23-3 mtDNA which occurs as a direct repeat in the wild-type mtDNA sequence flanking the unique petite mtDNA segment (interestingly, this excision sequence in petite 23-3 carries a single base substitution relative to the parental wild-type sequence). The putative replication origin of petite 23-3 is considered to be in its single G,C rich cluster, which differs in just one nucleotide from the standard oriS sequence. The DNA sequences in the intergenic regions flanking the oli1 gene of strain J69-1B (and its derivatives) have been systematically compared to those of the corresponding regions of mtDNA in strains derived from the D273-10B parent (sequences from the laboratory of A. Tzagoloff). The nature and distribution of the sequence divergences (base substitutions, base deletions or insertions, and more extensive rearrangements) are considered in the context of functions associated with mitochondrial gene expression which are ascribed to specialized sequences in the intergenic regions of the yeast mitochondrial genome.

  9. Gene expression profiling identifies eleven DNA repair genes down-regulated during mouse neural crest cell migration.

    PubMed

    Albino, Domenico; Brizzolara, Antonella; Moretti, Stefano; Falugi, Carla; Mirisola, Valentina; Scaruffi, Paola; Di Candia, Michele; Truini, Mauro; Coco, Simona; Bonassi, Stefano; Tonini, Gian Paolo

    2011-01-01

    Neural Crest Cells (NCCs) are transient multipotent migratory cells that derive from the embryonic neural crest which is itself derived from the margin of the neural tube. DNA repair genes are expressed in the early stages of mammalian development to reduce possible replication errors and genotoxic damage. Some birth defects and cancers are due to inappropriate or defective DNA repair machinery, indicating that the proper functioning of DNA repair genes in the early stages of fetal development is essential for maintaining DNA integrity. We performed a genome-wide expression analysis combining laser capture microdissection (LCM) and high-density oligo-microarray of murine NCCs at pre-migratory embryonic days 8.5 (E8.5), and at E13.5, as well as on neural crest-derived cells from the adrenal medulla at postnatal day 90. We found 11 genes involved in DNA repair activity (response to DNA damage stimulus, DNA damage checkpoint, base-excision repair, mismatch repair), over-expressed in the early stages of mouse embryo development. Expression of these 11 genes was very low or undetectable in the differentiated adrenal medulla of the adult mouse. Amongst the 11 genes, 6 had not been previously reported as being over-expressed during mouse embryonic development. High expression of DNA repair genes in enriched NCCs during early embryonic development may contribute to maintaining DNA integrity whilst failure of some of these genes may be associated with the onset of genetic disease and cancer. Our model of enriched murine NCCs and neural crest-derived cells can be used to elucidate the key roles of genes during normal embryonic development and in cancer pathogenesis.

  10. Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips.

    PubMed

    Kosuri, Sriram; Eroshenko, Nikolai; Leproust, Emily M; Super, Michael; Way, Jeffrey; Li, Jin Billy; Church, George M

    2010-12-01

    Development of cheap, high-throughput and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols and enzymatic error correction to develop a method for highly parallel gene synthesis. We tested our approach by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ∼35 kilobase pairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ∼2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.

  11. Human TOP3: a single-copy gene encoding DNA topoisomerase III.

    PubMed Central

    Hanai, R; Caron, P R; Wang, J C

    1996-01-01

    A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing. Expressing the cloned human cDNA in yeast (delta)top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA. On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12. Images Fig. 2 PMID:8622991

  12. Genome-wide profiling of DNA methylation and gene expression in Crassostrea gigas male gametes

    PubMed Central

    Olson, Claire E.; Roberts, Steven B.

    2014-01-01

    DNA methylation patterns and functions are variable across invertebrate taxa. In order to provide a better understanding of DNA methylation in the Pacific oyster (Crassostrea gigas), we characterized the genome-wide DNA methylation profile in male gamete cells using whole-genome bisulfite sequencing. RNA-Seq analysis was performed to examine the relationship between DNA methylation and transcript expression. Methylation status of over 7.6 million CpG dinucleotides was described with a majority of methylated regions occurring among intragenic regions. Overall, 15% of the CpG dinucleotides were determined to be methylated and the mitochondrial genome lacked DNA methylation. Integrative analysis of DNA methylation and RNA-Seq data revealed a positive association between methylation status, both in gene bodies and putative promoter regions, and expression. This study provides a comprehensive characterization of the distribution of DNA methylation in the oyster male gamete tissue and suggests that DNA methylation is involved in gene regulatory activity. PMID:24987376

  13. cDNA macroarray analysis of gene expression in synoviocytes stimulated with TNFalpha.

    PubMed

    Sugiyama, Tomoyasu; Ishii, Shizuko; Yamamoto, Jun ichi; Irie, Ryotaro; Saito, Kaoru; Otuki, Tetsuji; Wakamatsu, Ai; Suzuki, Yuzuru; Hio, Yuri; Ota, Toshio; Nishikawa, Tetsuo; Sugano, Sumio; Masuho, Yasuhiko; Isogai, Takao

    2002-04-24

    Gene expression of synoviocytes stimulated with tumor necrosis factor-alpha (TNFalpha) was studied by macroarray analysis to elucidate the cellular response and identify new biological functions of known and unknown genes. 10035 cDNA clones were used to make cDNA macroarrays of representative genes. Synoviocytes expressed large amounts of fibronectin and collagen mRNA. Statistical analysis of the macroarray data revealed 26 genes, including six new genes, which underwent significant alteration of gene expression in response to TNFalpha stimulation. These findings suggest that the synoviocyte response to TNFalpha stimulation forms the basis of development of various aspects of the pathophysiology of rheumatoid arthritis.

  14. Gene structure, expression, and DNA methylation characteristics of sea cucumber cyclin B gene during aestivation.

    PubMed

    Zhu, Aijun; Chen, Muyan; Zhang, Xiumei; Storey, Kenneth B

    2016-12-05

    The sea cucumber, Apostichopus japonicus, is a good model for studying environmentally-induced aestivation by a marine invertebrate. One of the central requirements of aestivation is the repression of energy-expensive cellular processes such as cell cycle progression. The present study identified the gene structure of the cell cycle regulator, cyclin B, and detected the expression levels of this gene over three stages of the annual aestivation-arousal cycle. Furthermore, the DNA methylation characteristics of cyclin B were analyzed in non-aestivation and deep-aestivation stages of sea cucumbers. We found that the cyclin B promoter contains a CpG island, three CCAAT-boxes and three cell cycle gene homology regions (CHRs). Application of qRT-PCR analysis showed significant downregulation of cyclin B transcript levels during deep-aestivation in comparison with non-aestivation in both intestine and longitudinal muscle, and these returned to basal levels after arousal from aestivation. Methylation analysis of the cyclin B core promoter revealed that its methylation level showed significant differences between non-aestivation and deep-aestivation stages (p<0.05) and interestingly, a positive correlation between Cyclin B transcripts expression and methylation levels of the core promoter was also observed. Our findings suggest that cell cycle progression may be reversibly arrested during aestivation as indicated by the changes in cyclin B expression levels and we propose that DNA methylation is one of the regulatory mechanisms involved in cyclin B transcriptional variation.

  15. Diethyl pyrocarbonate reaction with the lactose repressor protein affects both inducer and DNA binding

    SciTech Connect

    Sams, C.F.; Matthews, K.S.

    1988-04-05

    Modification of the lactose repressor protein of Escherichia coli with diethyl pyrocarbonate (DPC) results in decreased inducer binding as well as operator and nonspecific DNA binding. Spectrophotometric measurements indicated a maximum of three histidines per subunit was modified, and quantitation of lysine residues with trinitrobenzenesulfonate revealed the modification of one lysine residue. The loss of DNA binding, both operator and nonspecific, was correlated with histidine modification; removal of the carbethoxy groups from the histidines by hydroxylamine was accompanied by significant recovery of DNA binding function. The presence of inducing sugars during the DPC reaction had no effect on histidine modification or the loss of DNA binding activity. In contrast, inducer binding was not recovered upon reversal of the histidine modification. However, the presence of inducer during reaction protected lysine from reaction and also prevented the decrease in inducer binding; these results indicate that reaction of the lysine residue(s) may correlate to the loss of sugar binding activity. Since no difference in incorporation of radiolabeled carbethoxy was observed following reaction with diethyl pyrocarbonate in the presence or absence of inducer, the reagent appears to function as a catalyst in the modification of the lysine. The formation of an amide bond between the affected lysine and a nearby carboxylic acid moiety provides a possible mechanism for the activity loss. Reaction of the isolated NH2-terminal domain resulted in loss of DNA binding with modification of the single histidine at position 29. Results from the modification of core domain paralleled observations with intact repressor.

  16. The constant region affects antigen binding of antibodies to DNA by altering secondary structure.

    PubMed

    Xia, Yumin; Janda, Alena; Eryilmaz, Ertan; Casadevall, Arturo; Putterman, Chaim

    2013-11-01

    We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens.

  17. Preparation and incubation conditions affect the DNA integrity of ejaculated human spermatozoa

    PubMed Central

    Matsuura, Rieko; Takeuchi, Takumi; Yoshida, Atsumi

    2010-01-01

    Appropriate semen processing and assessment are critical for successful infertility treatment. We investigated whether laboratory procedures including semen preparation and incubation affect sperm DNA integrity. A total of 153 infertile men were involved. Conventional semen parameters and sperm chromatin structure assay (SCSA) parameters, that is, DNA fragmentation index (%DFI) and high DNA stainability (%HDS), were assessed on the fresh ejaculated semen samples, which were treated and incubated under different conditions. Negative correlations were identified between the %DFI and sperm concentration, motility, progressive motility and morphology. A lower percentage of DFI was detected in spermatozoa when density gradient centrifugation (DGC) was followed by swim-up treatment in comparison with DGC alone (P < 0.01). Although the %DFI increased in a time-dependent manner with incubation both at room temperature (RT) and at 37°C in air, the %DFI after 24 h at RT was significantly lower than that at 37°C (P < 0.05). Incubation with 5% CO2 was effective in maintaining sperm motility (P < 0.01); however, it induced further elevation of %DFI (P < 0.001). Thus, sperm DNA damage was associated with longer incubation periods. Interestingly, common culture conditions, such as maintaining pH and temperature, compromised the sperm DNA integrity. PMID:20562894

  18. Ancient Out-of-Africa Mitochondrial DNA Variants Associate with Distinct Mitochondrial Gene Expression Patterns

    PubMed Central

    Mishmar, Dan

    2016-01-01

    Mitochondrial DNA (mtDNA) variants have been traditionally used as markers to trace ancient population migrations. Although experiments relying on model organisms and cytoplasmic hybrids, as well as disease association studies, have served to underline the functionality of certain mtDNA SNPs, only little is known of the regulatory impact of ancient mtDNA variants, especially in terms of gene expression. By analyzing RNA-seq data of 454 lymphoblast cell lines from the 1000 Genomes Project, we found that mtDNA variants defining the most common African genetic background, the L haplogroup, exhibit a distinct overall mtDNA gene expression pattern, which was independent of mtDNA copy numbers. Secondly, intra-population analysis revealed subtle, yet significant, expression differences in four tRNA genes. Strikingly, the more prominent African mtDNA gene expression pattern best correlated with the expression of nuclear DNA-encoded RNA-binding proteins, and with SNPs within the mitochondrial RNA-binding proteins PTCD1 and MRPS7. Our results thus support the concept of an ancient regulatory transition of mtDNA-encoded genes as humans left Africa to populate the rest of the world. PMID:27812116

  19. DNA extraction from bovine mummified fetuses and detection of factor XI gene deficiency in the mummies.

    PubMed

    Ghanem, Mohamed Elshabrawy; Nishibori, Masahide; Nakao, Toshihiko; Moriyoshi, Masaharu

    2005-06-01

    Genomic DNA extracted from bovine mummified tissue is valuable material for detection of some genes that may contribute to fetal abnormalities. In this study bovine genomic DNA was extracted from the hardened tissue samples of ten bovine mummified fetuses. The amount of genomic DNA extracted from 2 g of the mummified tissues by the phenol/chloroform-ethanol method was low (less than 4 microg/ml) for all samples. The extracted DNA was then amplified by the GenomiPhi DNA amplification system. After amplification, the amount of DNA was increased to more than 100 microg/ml for all samples. This amplification system was shown to be a good tool for amplifying the genomic DNA of the mummified fetuses. The amplified genomic DNA was used for testing the mummies for Factor XI gene deficiency, an autosomal recessive deficiency involved in the early stages of the intrinsic blood coagulation pathway. Exon 12 of the Factor XI gene of the mummies was amplified by PCR. Two of the ten mummified fetuses were heterozygous for the Factor XI gene as indicated by the presence of two amplified DNA fragments of 320 bp and 244 bp. Factor XI deficiency has already been described in Holstein cattle. However, no report is available for bovine fetus. In this study, DNA was extracted and amplified from the bovine mummified fetuses, and the samples were successfully tested for Factor XI gene deficiency in the mummies.

  20. On the presence and role of human gene-body DNA methylation

    PubMed Central

    Jjingo, Daudi; Conley, Andrew B.; Yi, Soojin V.; Lunyak, Victoria V.; Jordan, I. King

    2012-01-01

    DNA methylation of promoter sequences is a repressive epigenetic mark that down-regulates gene expression. However, DNA methylation is more prevalent within gene-bodies than seen for promoters, and gene-body methylation has been observed to be positively correlated with gene expression levels. This paradox remains unexplained, and accordingly the role of DNA methylation in gene-bodies is poorly understood. We addressed the presence and role of human gene-body DNA methylation using a meta-analysis of human genome-wide methylation, expression and chromatin data sets. Methylation is associated with transcribed regions as genic sequences have higher levels of methylation than intergenic or promoter sequences. We also find that the relationship between gene-body DNA methylation and expression levels is non-monotonic and bell-shaped. Mid-level expressed genes have the highest levels of gene-body methylation, whereas the most lowly and highly expressed sets of genes both have low levels of methylation. While gene-body methylation can be seen to efficiently repress the initiation of intragenic transcription, the vast majority of methylated sites within genes are not associated with intragenic promoters. In fact, highly expressed genes initiate the most intragenic transcription, which is inconsistent with the previously held notion that gene-body methylation serves to repress spurious intragenic transcription to allow for efficient transcriptional elongation. These observations lead us to propose a model to explain the presence of human gene-body methylation. This model holds that the repression of intragenic transcription by gene-body methylation is largely epiphenomenal, and suggests that gene-body methylation levels are predominantly shaped via the accessibility of the DNA to methylating enzyme complexes. PMID:22577155

  1. Systematic Isolation and Characterization of Cadmium Tolerant Genes in Tobacco: A cDNA Library Construction and Screening Approach

    PubMed Central

    Zhang, Mei; Mo, Hui; Sun, Wen; Guo, Yan; Li, Jing

    2016-01-01

    Heavy metal pollution is a major limiting factor that severely affects plant growth worldwide, and the accumulation of heavy metal in the plant may be hazardous to human health. To identify the processes involved in cadmium detoxification, we constructed a cDNA library of tobacco roots acclimated to cadmium (Cd) stress. According to the results of functional screening cDNA library with a yeast Cd-sensitive mutant, ycf1Δ, we obtained a series of candidate genes that were involved in Cd response. Sequence analysis and yeast functional complementation of 24 positive cDNA clones revealed that, in addition to antioxidant genes, genes implicated in abiotic and biotic stress defenses, cellular metabolism, and signal transduction showed Cd detoxification effects in yeast. The real time RT-PCR analyses revealed that some Cd tolerance/ detoxification genes may be able to anticipate in other stresses such as biotic defense and water balance in tobacco. Taken together, our data suggest that plants’ acclimation to Cd stress is a highly complex process associated with broad gene functions. Moreover, our results provide insights into the Cd detoxification mechanisms along with the antioxidant system, defense gene induction, and calcium signal pathway. PMID:27579677

  2. Dnmt3L antagonizes DNA methylation at bivalent promoters and favors DNA methylation at gene bodies in ESCs.

    PubMed

    Neri, Francesco; Krepelova, Anna; Incarnato, Danny; Maldotti, Mara; Parlato, Caterina; Galvagni, Federico; Matarese, Filomena; Stunnenberg, Hendrik G; Oliviero, Salvatore

    2013-09-26

    The de novo DNA methyltransferase 3-like (Dnmt3L) is a catalytically inactive DNA methyltransferase that cooperates with Dnmt3a and Dnmt3b to methylate DNA. Dnmt3L is highly expressed in mouse embryonic stem cells (ESCs), but its function in these cells is unknown. Through genome-wide analysis of Dnmt3L knockdown in ESCs, we found that Dnmt3L is a positive regulator of methylation at the gene bodies of housekeeping genes and, more surprisingly, is also a negative regulator of methylation at promoters of bivalent genes. Dnmt3L is required for the differentiation of ESCs into primordial germ cells (PGCs) through the activation of the homeotic gene Rhox5. We demonstrate that Dnmt3L interacts with the Polycomb PRC2 complex in competition with the DNA methyltransferases Dnmt3a and Dnmt3b to maintain low methylation levels at the H3K27me3 regions. Thus, in ESCs, Dnmt3L counteracts the activity of de novo DNA methylases to maintain hypomethylation at promoters of bivalent developmental genes.

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

    PubMed

    Ha, Misook; Hong, Soondo

    2016-04-14

    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.

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

    NASA Astrophysics Data System (ADS)

    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 inferr