Dissertations / Theses on the topic 'Cytosine methylation'
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1
Currie, Graeme M. "DNA methylation at cytosine position 5." Thesis, Aston University, 1992. http://publications.aston.ac.uk/12603/.
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DNA methylation appears to be involved in the regulation of gene expression. Transcriptionally inactive (silenced) genes normally contain a high proportion of 5-methyl-2'-deoxycytosine residues whereas transcriptionally active genes show much reduced levels. There appears good reason to believe that chemical agents capable of methylating 2'-deoxycytosine might affect gene expression and as a result of hypermethylating promoter regions of cytosine-guanine rich oncogenic sequences, cancer related genes may be silenced. This thesis describes the synthesis of a number of `electrophilic' S-methylsulphonium compounds and assesses their ability to act as molecules capable of methylating cytosine at position 5 and also considers their potential as cytotoxic agents. DNA is methylated in vivo by DNA methyltransferase utilising S-adenoxylmethionine as the methyl donor. This thesis addresses the theory that S-adenoxylmethionine may be replaced as the methyl donor for DNA methytransferase by other sulphonium compounds. S-[3H-methyl]methionine sulphonium iodide was synthesised and experiments to assess the ability of this compounds to transfer methyl groups to cytosine in the presence of DNA methyltransferase were unsuccessful. A proline residue adjacent to a cysteine residue has been identified to a highly conserved feature of the active site region of a large number of prokaryotic DNA methyltransferases. The thesis examines the possibility that short peptides containing the Pro-Cys fragment may be able to facilitate the alkylation of cytosine position 5 by sulphonium compounds. Peptides were synthesised up to 9 amino acids in length but none were shown to exhibit significant activity. Molecular modelling techniques, including Chem-X, Quanta, BIPED and protein structure prediction programs were used to assess any structural similarities that may exist between short peptides containing a Pro-Cys fragment and similar sequences present in proteins. A number of similar structural features were observed.
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Al-Azzawi, Haneen. "Cytosine methylation and hydroxymethylation at the leptin promoter." Thesis, University of Nottingham, 2013. http://eprints.nottingham.ac.uk/13843/.
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Leptin is an important hormone well known for its role in regulating energy intake and expenditure. DNA methylation levels at the leptin promoter in adult tissues appear to correlate with environmental stresses experienced during early life. This suggests that, once established in early life, DNA methylation is stably transmitted over successive cell generations. The aim of the work presented in this thesis was to determine factors that contribute to the establishment and maintenance of this epigenetic mark at the leptin promoter and to investigate the individual roles of cytosine methylation and cytosine hydroxymethylation at this genomic locus. No effect of a high fat prenatal diet was observed on leptin promoter DNA methylation levels in the adipose tissue of pigs. However, this genomic region exhibited intermediate levels of DNA methylation, which is usually associated with gene silencing, even though adipose tissue is the primary site of leptin expression. Double stranded methylation data obtained from DNA methyltransferase (DNMT) mutant mouse embryonic stem cells (mESCs) was used to investigate the contributions of the three catalytically active DNMT enzymes to leptin promoter DNA methylation patterns. Depletion of DNMT3b resulted in increased methylation levels at the leptin promoter, consistent with preliminary data from mutant DNMT3b mouse tissues where similar increases in methylation levels were observed at specific CpG dinucleotides. Two mESC lines, either hypomethylated or hypermethylated at the leptin promoter, were tested for leptin mRNA expression and neither cell line expressed leptin mRNA, suggesting that some form of methylation may be required for leptin expression. To further investigate the relationship between DNA methylation and leptin expression, in vitro differentiated adipocytes were analysed. 3T3-L1 preadipocytes, which do not express leptin, exhibit high levels of DNA methylation and these high methylation levels are maintained after the cells differentiate into leptin-expressing adipocytes. Induction of cytosine hydroxymethylation at the leptin promoter was detected in differentiating and mature adipocytes and evidence is presented to suggest that cytosine hydroxymethylation at the leptin promoter correlates with leptin expression.
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Genger, Ruth Kathleen, and Ruth Genger@csiro au. "Cytosine methylation, methyltransferases and flowering time in Arabidopsis thaliana." The Australian National University. Faculty of Science, 2000. http://thesis.anu.edu.au./public/adt-ANU20011127.115231.
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Environmental signals such as photoperiod and temperature provide plants with seasonal information, allowing them to time flowering to occur in favourable conditions. Most ecotypes of the model plant Arabidopsis thaliana flower earlier in long photoperiods and after prolonged exposure to cold (vernalization). The vernalized state is stable through mitosis, but is not transmitted to progeny, suggesting that the vernalization signal may be transmitted via a modification of DNA such as cytosine methylation. The role of methylation in the vernalization response is investigated in this thesis.
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Arabidopsis plants transformed with an antisense construct to the cytosine methyltransferase METI (AMT) showed significant decreases in methylation. AMT plants flowered significantly earlier than unvernalized wildtype plants, and the promotion of flowering correlated with the extent of demethylation. The flowering time of mutants with decreased DNA methylation (ddm1) was promoted only in growth conditions in which wildtype plants showed a vernalization response, suggesting that the early flowering response to demethylation operated specifically through the vernalization pathway.
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The AMT construct was crossed into two late flowering mutants that differed in vernalization responsiveness. Demethylation promoted flowering of the vernalization responsive mutant fca, but not of the fe mutant, which has only a slight vernalization response. This supports the hypothesis that demethylation is a step in the vernalization pathway.
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The role of gibberellic acid (GA) in the early flowering response to demethylation was investigated by observing the effect of the gai mutation, which disrupts the GA signal transduction pathway, on flowering time in plants with demethylated DNA. The presence of a single gai allele delayed flowering, suggesting that the early flowering response to demethylation requires a functional GA signal transduction pathway, and that demethylation increases GA levels or responses, directly or indirectly.
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In most transgenic lines, AMT-mediated demethylation did not fully substitute for vernalization. This indicates that part of the response is not affected by METI-mediated methylation, and may involve a second methyltransferase or a factor other than methylation. A second Arabidopsis methyltransferase, METIIa, was characterized and compared to METI. The two genes are very similar throughout the coding region, and share the location of their eleven introns, indicating that they diverged relatively recently. Both are transcribed in all tissues and at all developmental stages assayed, but the level of expression of METI is significantly higher than that of METIIa. The possible functions of METI, METIIa, and other Arabidopsis cytosine methyltransferase genes recently identified are discussed.
4
Voss, Karl O. "Capillary electrophoresis for DNA sequencing and cytosine methylation analysis." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ29120.pdf.
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Spangler, Maribeth. "Cytosine Methylation of Phytophthora sojae by Methylated DNA Immunoprecipitation." Bowling Green State University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1339451917.
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Cull, Rebecca M. "Analysis of Cytosine Methylation in Soybean Pathogen Phytophthora sojae." Bowling Green State University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1404745644.
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Schmidt, Martin, Sarah Hense, André E. Minoche, Juliane C. Dohm, Heinz Himmelbauer, Thomas Schmidt, and Falk Zakrzewski. "Cytosine Methylation of an Ancient Satellite Family in the Wild Beet Beta procumbens." Karger, 2014. https://tud.qucosa.de/id/qucosa%3A70576.
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DNA methylation is an essential epigenetic feature for the regulation and maintenance of heterochromatin. Satellite DNA is a repetitive sequence component that often occurs in large arrays in heterochromatin of subtelomeric, intercalary and centromeric regions. Knowledge about the methylation status of satellite DNA is important for understanding the role of repetitive DNA in heterochromatization. In this study, we investigated the cytosine methylation of the ancient satellite family pEV in the wild beet Beta procumbens. The pEV satellite is widespread in species-specific pEV subfamilies in the genus Beta and most likely originated before the radiation of the Betoideae and Chenopodioideae. In B. procumbens , the pEV subfamily occurs abundantly and spans intercalary and centromeric regions. To uncover its cytosine methylation, we performed chromosome-wide immunostaining and bisulfite sequencing of pEV satellite repeats. We found that CG and CHG sites are highly methylated while CHH sites show only low levels of methylation. As a consequence of the low frequency of CG and CHG sites and the preferential occurrence of most cytosines in the CHH motif in pEV monomers, this satellite family displays only low levels of total cytosine methylation.
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Shock, Lisa. "Functional consequences of cytosine methylation in mitochondrial DNA catalyzed by DNA methyltransferase 1." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/271.
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Cytosine methylation of mitochondrial DNA (mtDNA) was first described several decades ago, but neither the mechanism generating this modification nor its functional significance was known. Because mitochondrial dysfunction is a hallmark characteristic of numerous human diseases, including neurological and cardiovascular disease, aging and cancer, this dissertation addressed whether epigenetic modification of mtDNA regulates mitochondrial function. We show that mtDNA contains not only 5-methylcytosine (5mC), but also 5-hydroxymethylcytosine (5hmC), suggesting that previous reports likely underestimated the degree of epigenetic modification within the mitochondrial genome. We questioned how these modifications were generated by looking for mitochondrial isoforms of the nuclear-encoded DNA methyltransferases. We found that an isoform of the most abundant mammalian methyltransferase, DNA methyltransferase 1 (DNMT1) translocates to mitochondria, driven by an in-frame mitochondrial targeting sequence (MTS) located upstream of the nuclear DNMT1 translational start site. This MTS is highly conserved across mammalian species, and directs a heterologous protein to the mitochondria. To investigate the function of mitochondrial DNMT1 (mtDNMT1), we created a cell line that carries a tandem-affinity purification (TAP) tag at the C-terminus of a single endogenous human DNMT1 allele. Using the DNMT1-TAP cell line, we showed that mtDNMT1 specifically binds mtDNA in a manner that is proportional to CpG density, proving its presence in the mitochondrial matrix. mtDNMT1 exhibits CpG-specific methyltransferase activity in vitro that is resistant to trypsin-treatment of intact mitochondria, but moderately susceptible to pharmacologic inhibition by the nucleoside analog 5-aza-2’-deoxycytidine (5-aza-dC). NRF1 and PGC1α, transcription factors that activate nuclear-encoded mitochondrial proteins in response to oxidative stress, were observed to up-regulate expression of mtDNMT1. Loss of p53, a tumor suppressor gene known to help control mitochondrial metabolism, also results in a striking increase in mtDNMT1 expression, and this up-regulation of mtDNMT1 appears to modify mitochondrial transcription in a gene-specific fashion. Our data suggests roles for mtDNMT1 in both the establishment and maintenance of cytosine methylation (from which 5hmC is presumably derived) and in the regulation of mitochondrial transcription. We propose that the enzymes responsible for epigenetic modification of mtDNA have potential as therapeutic targets, with relevance to a broad spectrum of human disorders.
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Nicolini, Francesco. "Investigating the role of DNA (cytosine-5)-methyltransferase 1 in mitochondrial DNA methylation." Thesis, King's College London (University of London), 2018. https://kclpure.kcl.ac.uk/portal/en/theses/investigating-the-role-of-dna-cytosine5methyltransferase-1-in-mitochondrial-dna-methylation(4036df6a-90c7-4259-83fe-9470104d29d9).html.
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Mitochondrial DNA (mtDNA) that escapes from autophagy-mediated degradation after hemodynamic stress can cause inflammation in the heart by activating TLR9 signalling pathway, in which unmethylated CpG motifs bind to TLR9. DNA methyltransferases (DNMTs) such as DNMT1 and DNMT3A are involved in nuclear DNA methylation. It has also been reported that two additional potential transcription initiation codons exist at 5’ region to the original DNMT1 and that this 1st-to-3rd ATG sequence fused to GFP cDNA can localise in mitochondria, suggesting that DNMT1 might affect mtDNA methylation patterns. The aim of this study is to elucidate the role of DNMT1 in mtDNA methylation. To achieve this aim, three different isoforms of mouse DNMT1 sequences (“nuclear DNMT1”, “whole DNMT1” and “mtDNMT1”) as well as a mouse DNMT3A isoform were generated through molecular cloning. Mitoprot II software analysis predicted a high chance of mitochondrial localization (>90%) for both whole DNMT1 and mtDNMT1 isoforms. Western blotting analysis confirmed overexpression of all DNMT1 constructs, and immunocytochemistry experiments confirmed the predicted localisation of DNMT1 isoforms. DNMT3A localisation was detected at nuclear level, but not at mitochondrial level. Adenoviral vectors for nuclear DNMT1 and mtDNMT1 were generated and the methylated-DNA immunoprecipitation/qPCR methylation analysis of the D-Loop showed a 3-to-5 fold increase in total methylation levels in mtDNMT1 overexpressed mouse cardiac endothelial cells. However, global percentage of methylation is low. CoIP-MS analysis after mtDNMT1 overexpression failed to highlight any candidates for mtDNMT1 co-interaction. These data suggest that the 1st-to-3rd ATG sequence contains the mitochondrial localisation signal for DNMT1, and that DNMT1 can localise to mitochondria through it and methylate mtDNA.
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Kan, Mun Seng. "Mutational analysis of M.HhaI to mimic #PSI#M.SpoI from Schizosaccharomyces pombe and Masc1 from Ascobolus immersus." Thesis, University of Sheffield, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310873.
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Tatra, Gurminder Singh. "Genomic cytosine methylation, its role in controlling stem elongation plasticity in Stellaria longipes (Caryophyllaceae)." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0017/MQ48047.pdf.
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CELIK, Selcen. "An Improved Antibody-based Method to Detect Whole Genome Cytosine Methylation in Mouse Embryonic Fibroblasts." Thesis, The University of Sydney, 2013. http://hdl.handle.net/2123/9500.
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Cytosine methylation (5meC) of the genome is an important epigenetic mark that acts as a regulator of gene expression and genome stability. The loss of methylation is reported to occur in the embryo following fertilisation. The immunological methods including antigenic retrieval with acid provide highly selective recognition of 5meC, and are commonly used to detect the changes in 5meC. However a recent study of the early embryo showed that a trypsin treatment after acid is required for increased antigenicity for 5meC epitope in mouse embryos. This thesis presents the conditions required for reliable immunological analysis of global patterns of 5meC. It used a somatic cell model (mouse embryonic fibroblasts) and examined the effects of various cell growth states and the consequences of exposure of cells to various genotoxic stresses on staining of 5meC. This thesis showed that antigenic unmasking of 5meC and MBD1 (methyl-binding-protein1) with tryptic digestion revealed a heterogeneity in nuclear arrangements of methylation in somatic cells. These are accompanied by the changes in heterochromatin during DNA damage. The results suggest a new model of DNA methylation within the mammalian genome. This represents multiple populations of genome which have different response to antigenic retrieval induced by some conditions, such cell growth and DNA damage.
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Royle, Jack W. "Functional characterisation of DNA methylation in the pest species Helicoverpa armigera." Thesis, Queensland University of Technology, 2020. https://eprints.qut.edu.au/201652/1/Jack_Royle_Thesis.pdf.
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This project investigated the role of DNA methylation in the cotton bollworm, Helicoverpa armigera. Through a combination of Next Generation Sequencing (NGS) and Liquid Chromatography Mass Spectrometry (LCMS) analyses, it was possible to quantify DNA methylation at different stages of the H. armigera life-cycle as well as uncover potential functional roles for this epigenetic modification in this economically devastating pest species. The results described provide further insights into the mechanisms of epigenetic systems in insects and provide a platform for future applied research.
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Golding, Michael Cameron. "Expression of the bovine DNA (cytosine 5) methyltransferase family during preimplantation development and aberrations induced by somatic cell nuclear transfer." Texas A&M University, 2003. http://hdl.handle.net/1969.1/1308.
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Bovine preimplantation embryos derived from nuclear transfer experiments exhibit a global state of genomic hypermethylation that likely account for the large number of developmental abnormalities observed to date. The central hypotheses of this work is that the genomic hypermethylation and improper epigenetic reprogramming reported in studies of bovine nuclear transfer, are in large part due to abnormal expression and regulation of the DNA methyltransferase proteins. Bovine Dnmt mRNAs display strong sequence homology to those of human and mouse and similar to other species, exist as multiple isoforms. Two of these splice variants, which have been termed Dnmt2γ and Dnmt3a4 represent previously unreported sequence combinations. Investigation of bovine DNA methyltransferase expression in the bovine oocyte and early preimplantation development has revealed an intricate system divergent from observations previously reported in the mouse. Specifically, the somatic version of Dnmt1 along with Dnmt2, 3a and 3b are all expressed during these initial stages of bovine development. Further, real time analyses of the Dnmt transcripts in cloned and in vitro produced embryos reveal significant differences in the mRNA expression levels of Dnmt1 and 2 but not Dnmt3a and 3b suggesting that the de novo methyltransferases may be functioning normally while Dnmt1 and Dnmt2 are aberrantly methylating the genome during a critical time when methylation levels should be receding. Real time PCR analysis of the Dnmt transcripts in fetal and adult tissues has revealed a developmental and tissue specific expression pattern suggesting that proper expression and function of these enzymes is a key element in the process of differentiation. These results are further supported by studies of Dnmt expression in aging bovine fibroblast cultures, which suggest that the Dnmts may play some as yet unidentified role in cellular senescence. Recently, it has been postulated that the cause of abnormal methylation observed in cloned embryos may be due in part to misexpression of the Dnmt1o isoform during preimplantation development. Work presented here raises new and significant hypotheses that must be considered both regarding the cadre of DNA methyltranferases that direct epigenetic programming during normal development and regarding the implication of abnormal DNMT expression in cloned embryos.
Bovine preimplantation embryos derived from nuclear transfer experiments exhibit a global state of genomic hypermethylation that likely account for the large number of developmental abnormalities observed to date. The central hypotheses of this work is that the genomic hypermethylation and improper epigenetic reprogramming reported in studies of bovine nuclear transfer, are in large part due to abnormal expression and regulation of the DNA methyltransferase proteins. Bovine Dnmt mRNAs display strong sequence homology to those of human and mouse and similar to other species, exist as multiple isoforms. Two of these splice variants, which have been termed Dnmt2γ and Dnmt3a4 represent previously unreported sequence combinations. Investigation of bovine DNA methyltransferase expression in the bovine oocyte and early preimplantation development has revealed an intricate system divergent from observations previously reported in the mouse. Specifically, the somatic version of Dnmt1 along with Dnmt2, 3a and 3b are all expressed during these initial stages of bovine development. Further, real time analyses of the Dnmt transcripts in cloned and in vitro produced embryos reveal significant differences in the mRNA expression levels of Dnmt1 and 2 but not Dnmt3a and 3b suggesting that the de novo methyltransferases may be functioning normally while Dnmt1 and Dnmt2 are aberrantly methylating the genome during a critical time when methylation levels should be receding. Real time PCR analysis of the Dnmt transcripts in fetal and adult tissues has revealed a developmental and tissue specific expression pattern suggesting that proper expression and function of these enzymes is a key element in the process of differentiation. These results are further supported by studies of Dnmt expression in aging bovine fibroblast cultures, which suggest that the Dnmts may play some as yet unidentified role in cellular senescence. Recently, it has been postulated that the cause of abnormal methylation observed in cloned embryos may be due in part to misexpression of the Dnmt1o isoform during preimplantation development. Work presented here raises new and significant hypotheses that must be considered both regarding the cadre of DNA methyltranferases that direct epigenetic programming during normal development and regarding the implication of abnormal DNMT expression in cloned embryos.
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Bienvenu, Carine. "Oxydations radicalaires de la 5-méthyl-2'-désoxycytidine." Université Joseph Fourier (Grenoble ; 1971-2015), 1997. http://www.theses.fr/1997GRE10039.
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Les reactions d'oxydation radicalaire des bases de l'adn peuvent etre initiees par differents agents du stress oxydant tels que les rayonnements du proche uv et ionisant. Ces dommages oxydants de l'adn sont susceptibles d'etre a l'origine de processus de cancerogenese et de mutagenese. La 5-methylcytosine est une base rare de l'adn impliquee dans de nombreuses fonctions biologiques cles pour la vie cellulaire. Cette base methylee est presente principalement au niveau des sequences repetitives cpg qui sont le siege de frequentes mutations. Nous nous sommes interesses a l'oxydation photosensibilisee de la 5-methyl-2'-desoxycytidine (m#5dcyd) en solution aqueuse aeree. Le cation radical pyrimidique initialement forme par un mecanisme de type i est implique dans deux voies competitives de transformation, a savoir un processus de deprotonation et une reaction d'hydratation. Les produits finals ont ete isoles par chromatographie liquide a haute performance et caracterises au moyen de diverses methodes spectroscopiques (uv, spectrometrie de masse, rmn du proton et du carbone, dichroisme circulaire). L'action du radical hydroxyle (oh) sur la 5-methyl-2'-desoxycytidine a egalement ete etudiee. Les resultats obtenus montrent que l'oxydation de ce nucleoside par le radical oh conduit, au moins partiellement, a la formation d'intermediaires radicalaires identiques a ceux produits par photosensibilisation. Des experiences d'irradiation uv et gamma de solutions aqueuses de m#5dcyd en presence d'oxygene 18 ont permis de proposer des mecanismes pour la formation des principaux produits d'oxydation de la m#5dcyd. Un autre volet de ce travail a concerne la mesure de produits d'oxydation de la m#5dcyd dans l'adn isole. Pour ce faire, une technique d'analyse sensible et specifique, la chromatographie gazeuse couplee a la spectrometrie de masse a ete mis en uvre. Ces mesures ont egalement necessite la synthese de molecules etalon enrichies avec des isotopes stables (#2h et #1#5n)
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Kgatle, Mankgopo Magdeline. "An investigation of genome-wide promoter region cytosine-phosphate-guanine (CpG) Island methylation profiles in patients with chronic hepatitis B virus infection." Doctoral thesis, University of Cape Town, 2014. http://hdl.handle.net/11427/8800.
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Includes bibliographical references.Hepatitis B virus (HBV) is oncogenic and a major cause of hepatocellular carcinoma (HCC) in the developing world. It integrates parts of its genome such as the HBx gene, core and surface antigens into the human genome. The integrated viral DNA disrupts gene function resulting in physiological changes that cause liver disease. The viral inserts are inactivated through methylation. This is a protective innate response driven by human DNA methyltransferases triggered by the presence of viral DNA inserts. This thesis investigates the hypothesis that during the innate response to methylate integrated HBV DNA, there is unintended methylation of genomic DNA around the intercalated viral DNA that could be adjacent host promoter Cytosine-phosphateGuanine (CpG) islands. This would activate or silence genes including tumour suppressors and result in the clinical disease phenotypes of hepatic inflammation, fibrosis and HCC that characterise chronic HBV infection. Genome-wide microarray analysis was used to investigate for the presence of promoter CpG island methylation in a cohort of patients with liver disease due to HBV infection, HCC, autoimmune hepatitis which is a non-viral liver disease and normal cases with no liver disease. The study identified hypermethylation in promoter regions, transcription start sites, gene exons and introns. Only sites in the promoter region and within 100bp upstream of a transcription start site were analysed for this thesis presentation. Using an extended cohort of patients with chronic HBV infection and normal controls, bisulfite DNA sequencing was used to validate and confirm the presence of DNA methylation in a selection of some of genes identified. HBV infected patients were shown to have hypermethylation in the promoter CpG island regions of several genes that regulate hepatic metabolism, tumour suppression, ribonucleic acid splicing, vitamin D receptor binding, protein ubiquitination and the cell cycle. Many of these genes have transcriptional binding factors that are known to be affected by the transcriptional transactivator HBx protein, suggesting that HBx protein is important in the pathogenesis of liver disease. Amongst the most hypermethylated core promoter regions identified were those for cyclin kinases genes such as Cyclin D3 (CCND3). CCND3 gene is important in liver regeneration and wound healing and its abnormal function has been linked to the development of liver fibrosis and HCC. Increased methylation of CCND3 gene was associated with HBV e antigen positive status and genotype D, supporting the hypothesis that increased methylation is associated with host and viral factors. Methylation induced alteration in the function of the identified gene promoters would affect cellular signalling with effects on cell growth, differentiation, proliferation and apoptosis. These changes would explain the development of hepatic inflammation, apoptosis, fibrosis and malignant transformation seen in chronic HBV infection. Further investigation of these genes will provide new insights on mechanisms of HBV induced liver disease and the development of new molecular diagnostic tools or therapeutic interventions.
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Panda, Kaushik Kant. "Genome-Wide Regulation of Both Canonical and Non-canonical RNA-directed DNA Methylation Mechanisms in Arabidopsis thaliana." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1512116266715136.
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Cramer, Jason. "EVOLUTION AND DIVERGENCE OF THE STRUCTURAL AND PHYSICAL PROPERTIES OF DNA BINDING BY METHYL-CYTOSINE BINDING DOMAIN FAMILY MEMBERS 2 AND 3." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3517.
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The studies presented in this dissertation, Evolution And Divergence Of The Structural And Physical Properties Of DNA Binding By Methyl-Cytosine Binding Domain Family Members 2 And 3, pertain primarily to two key epigenetic regulators involved with the biological interpretation of methylated DNA marks. We provide insights into the emergence and evolution of the MBD2 and MBD3 and how those molecular entities influence heritable changes in gene activity. We further provide details regarding the mystery surrounding MBD3 function and the MBD2-mediated capacity of primitive animals to carry out methylation-specific epigenetic mechanisms. In chapter two, we describe the DNA binding properties of MBD2 and MBD3. This study provides information regarding previously unidentified MBD3 binding properties and potential biological function. In chapter three, we show that sponges demonstrate a MBD2-mediated capacity for binding methylated DNA sites, recruit NuRD components in vitro, and knockdown of MBD2 in the freshwater desmosponge, Ephydatia muelleri, promotes an abnormal growth phenotype.
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Du, Toit Jean. "An analysis of the effect of transformation on global– and gene–specific DNA methylation in four cultured cell lines / Jean du Toit." Thesis, North-West University, 2010. http://hdl.handle.net/10394/4560.
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DNA methylation plays a role in several biological functions, such as gene expression regulation, and several endogenous and exogenous factors affect these DNA methylation patterns in the cell. One such alteration of a cell line's DNA methylation pattern is caused by the insertion of a vector into the cell line. Using the cytosine–extension assay and realtime methylation–specific PCR, alterations of DNA methylation levels on both global and gene–specific levels were investigated. In some cell lines the cellular transformation led to an increase in DNA methylation levels, and in others a decrease in DNA methylation amounts was observed. The same phenomenon was seen in the promoter regions of specific genes, showing that vector–insertion into a cell line caused DNA methylation alterations in many regions of the genome. These alterations in DNA methylation are investigated in this reduced representation study using enrichment of the methylated fraction of fragmented DNA and subsequent GS FLX Titanium sequencing of these methylated fragments. The results of sequence data analysis showed that methylated fragments are distributed over the whole genome, but could be related to only a few specific genes. These results have implications for cell culture work, biotechnological applications and uses in gene therapy.Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.
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Borgel, Julie. "Dynamique et mécanismes de ciblage de la méthylation de l’ADN au cours du développement précoce chez la souris." Thesis, Montpellier 2, 2011. http://www.theses.fr/2011MON20224.
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La méthylation de l'ADN est fortement reprogrammée pendant le développement chez les mammifères, où elle semble jouer un rôle essentiel dans la répression génique et le maintien de l'identité cellulaire. Néanmoins, les cibles de la méthylation de l'ADN, la cinétique d'acquisition et les mécanismes de ciblage au cours du développement sont mal connus. Le premier objectif de ma thèse a donc été d'identifier les cibles de la méthylation de l'ADN pendant l'embryogenèse chez la souris. Sachant que plusieurs études dans les cellules ES ont mis en évidence un lien entre l'histone méthyltransférase G9a et l'établissement de la méthylation de l'ADN, le deuxième objectif de ma thèse a été de tester le rôle de G9a dans l'établissement de la méthylation de l'ADN au cours de l'embryogenèse. Pour cela, j'ai développé une technique d'analyse de la méthylation à l'échelle génomique à partir d'un petit nombre de cellules. Nous avons observé que la méthylation est essentiellement catalysée par DNMT3B et est mise en place principalement pendant l'implantation de l'embryon entre le blastoscyste et l'épiblaste. Pendant cette période, la méthylation cible préférentiellement les gènes de la lignée germinale et est indispensable à leur répression. La méthylation cible aussi des gènes spécifiques de différentes lignées somatiques telles que la lignée hématopoïétique, et peut être effacée ultérieurement pendant la différentiation. De manière surprenante, nous avons identifié des promoteurs de gènes non soumis à l'empreinte qui semblent résister à la reprogrammation de la méthylation de l'ADN et hériter la méthylation des gamètes parentaux. Enfin, nous avons montré que, contrairement à ce que suggèrent les études dans les cellules ES, G9a ne semble pas être indispensable à l'acquisition et au maintien de la méthylation de l'ADN au niveau des promoteurs pendant le développement in vivoDNA methylation is an epigenetic mark extensively reprogrammed during mammalian development. It is believed to play essential functions in gene regulation and the maintenance of cellular identity. However, the target genes of DNA methylation and the mechanisms that recruit DNA methylation during development remain poorly understood. The first aim of my PhD project was to identify the target genes of DNA methylation during early mouse development in vivo. In addition, because several studies show that G9a is required for DNA methylation establishment and maintenance during ES cells differentiation, the second aim was to determine whether G9a is required for the establishment of promoter DNA methylation patterns during early development in vivo.To address these questions, I developped a genomics approach to map DNA methylation starting from very small amount of cells. .We observed a major epigenetic switch during implantation at the transition from the blastocyst to the postimplantation epiblast. During this period, DNA methylation is primarily targeted to repress the germline expression program. DNA methylation in the epiblast is also targeted to promoters of lineage-specific genes such as hematopoietic genes, which are subsequently demethylated during terminal differentiation. De novo methylation during early embryogenesis is catalyzed by Dnmt3b, and absence of DNA methylation leads to ectopic gene activation in the embryo. Surprisingly, we identify nonimprinted genes that escape post-fertilization DNA methylation reprogramming and seem to inherit promoter DNA methylation from parental gametes. Finally we show that, unlike what it was shown in ES cells, the absence of G9a in an in vivo context does not have a drastic effect on the maintenance and the establishment of promoter DNA methylation during early development
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Cribiu, Pauline. "Étude des effets inter et transgénérationnels de l’exposition parentale au stress chimique chez le crustacé amphipode Gammarus fossarum." Thesis, Lyon, 2020. http://www.theses.fr/2020LYSET002.
Full textAbstract:
À l’heure actuelle, les démarches écotoxicologiques (i.e. bioessais de laboratoire, approches biomarqueurs, tests in situ) évaluent les effets des contaminants majoritairement sur des temps de réponse courts, qui ne correspondent pas à l’échelle temporelle de la dynamique des populations. En plus de la toxicité manifestée au cours de l’exposition chimique, des effets peuvent pourtant s’observer plus tardivement au cours de la vie des organismes, voire de leur descendance. Ces effets différés peuvent avoir une influence importante sur la démographie, la résilience et l’acquisition de tolérance des populations ainsi que leur vulnérabilité face à de nouvelles perturbations environnementales. Leur étude constitue ainsi un véritable enjeu pour améliorer la compréhension des réponses des populations au stress chimique dans les écosystèmes. Dans ce contexte, l’objectif général de cette thèse a été d’explorer les effets intergénérationnels et transgénérationnels de l’exposition aux contaminants et leurs conséquences sur le fonctionnement des populations de l’espèce sentinelle Gammarus fossarum (Crustacé). Le parti-pris expérimental appliqué tout au long de ce travail combinant expérimentation multi-génération et modélisation en dynamique de population, a consisté à mettre en œuvre des expositions limitées à la génération parentale initiale (F0), puis à suivre le développement de trois générations successives en milieu non contaminé. Compte-tenu de l’implication potentiellement importante des mécanismes épigénétiques dans l’apparition d’effets différés, la méthylation globale des cytosines de l’ADN a été explorée pour la toute première fois chez cette espèce. Cette marque épigénétique s’est avérée être sensible au stress thermique, chimique avec le cadmium et à la privation alimentaire dans des conditions contrôlées au laboratoire. Une variabilité importante du niveau de base du biomarqueur entre populations naturelles de Gammarus fossarum a également été observée. Par ailleurs, suite à des expositions parentales de 3 semaines au cadmium et à la 3,4dichloroaniline, nous avons mis en évidence l’existence d’effets en cascade sur les traits d’histoire de vie de G. fossarum jusqu’à la troisième génération de descendants, après plus d’un an d’expérimentation. Nos travaux suggèrent également un rôle prépondérant des compromis entre les traits d’histoire de vie et entre les générations dans l’émergence des effets différés. Ces compromis se traduisent par un maintien de la capacité démographique de la population suite à l’exposition parentale au cadmium et sont ainsi certainement contraints par la stratégie d’histoire de vie de Gammarus fossarum. Au bilan, ces résultats soulignent l’intérêt de mener des études au-delà de la première génération de descendants et de généraliser les études à long terme sur les espèces environnementales non-modèles de laboratoire pour améliorer la compréhension des réponses populationnelles à la contamination et la pertinence écologique de l’évaluation actuelle des risquesMost of the current ecotoxicological approaches (i.e. laboratory bioassays, biomarker, in situ tests) assess the effects of contaminants at the individual level on short response time, that do not match the time scale of population dynamics. In addition to toxicity occurring during the chemical exposure of individuals, effects can arise later along the lifetime of organisms and of their progeny. Such delayed effects can lead to significant impact on population demography, resilience and tolerance, as well as on population vulnerability to new environmental disruptions. Studying these effects is a real challenge to improve the understanding of population response to chemical stress in ecosystems. In this context, the main purpose of this thesis was to explore intergenerational and transgenerational effects of parental contaminant exposure and their consequences on the functioning of the populations in the sentinel species Gammarus fossarum (Crustacea). To do so, challenging oneyear lab experiment together with population dynamics modelling were performed. The experimental statement was to only expose the parental generation (F0) and then to monitor the development of successive generations in an uncontaminated environment. Assuming a prevalent involvement of epigenetic mechanisms in the onset of delayed effects, this work explored for the first time the global genomic cytosine methylation level in Gammarus fossarum. The studied epigenetic mark was shown to be sensitive to heat stress, chemical stress (cadmium) and to food starvation in controlled laboratory conditions. A substantial variability in the basal level between several natural populations of Gammarus fossarum was also recorded. In the light of the multi-generational experiments, cascading effects were observed on G. fossarum life history traits until the third offspring generation after the parental exposure to cadmium or 3,4-dichloroaniline. In addition, a significant role of trade-offs between life-history traits and between generations can be suggested in the emergence of delayed effects. These trade-offs translate into the maintenance of demographic population capacity after the parental cadmium exposure and could be consequently constrained by life history strategy of Gammarus fossarum. Hence, these results highlight the interest of expanding the studied response time beyond the first offspring generation and of studying the long-term effects of chemical stress in non-target environmental species. Such approaches can be suggested to improve the understanding of natural population responses to contamination and to upgrade the ecological relevance of the current risk assessment
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Hubert, Benjamin. "La régulation épigénétique des éléments transposables dans les populations naturelles de Drosophila simulans." Phd thesis, Université Claude Bernard - Lyon I, 2010. http://tel.archives-ouvertes.fr/tel-00815956.
Full textAbstract:
La méthylation de l'ADN et les modifications post-traductionnelles des histones sont desmodifications épigénétiques qui interviennent dans la régulation des éléments transposables(ET) chez de nombreuses espèces. La proportion des ET dans les génomes varie selon lesespèces considérées et pose la question des mécanismes de régulation de ces ET. Au sein del'espèce Drosophila simulans, les populations naturelles présentent un polymorphisme uniquedans le nombre de copies des ET, ce qui en fait un excellent modèle pour étudier cettequestion. L'étude de la méthylation d'ADN et des modifications post-traductionnelles deshistones associées au rétrotransposon à LTR tirant dans la lignée germinale des populationsnaturelles a permis de montrer l'influence d'une copie d'ET sur la structure de la chromatineau site d'insertion. Dans un second volet, nous avons cherché à caractériser la méthylation del'ADN chez la drosophile, chez laquelle la fonction est encore mal connue. Nous avons, pardes approches spécifiques et globales, mesuré l'abondance de cette marque épigénétique chezla drosophile. Nous concluons que les taux de méthylation de l'ADN sont très faibles maisvariables entre espèces. Notre travail n'a pas permis de mettre en évidence un rôle de laméthylation de l'ADN dans le contrôle des ET, toutefois, nous ne pouvons pas exclure cesystème de régulation.
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"DNA cytosine methylation and DNA repair." Tulane University, 1987.
Find full textAbstract:
Thymine$\cdot$guanine (T$\cdot$G) and uracil$\cdot$guanine (U$\cdot$G) mismatched base pairs in DNA are formed by the spontaneous deamination of 5-methylcytosine (m$\sp5$C) and cytosine (C) residues, respectively. Covalently closed circular (CCC) heteroduplex M13mp18 replicative form (RF) DNAs with a T$\cdot$G or a U$\cdot$G mismatch in a unique restriction site were created by oligonucleotide-directed mutagenesis and were transfected into repair-proficient and repair-deficient E. coli. The CCC heteroduplex M13mp18 DNAs were unmethylated at all dam (5$\sp\prime$-GATC-3$\sp\prime$) sites in order to avoid methylation-directed and nick-directed biasing of repair. In an E. coli host containing uracil-DNA glycosylase ($ung\sp{+}$) and which is proficient in mismatch repair ($mut\sp{+}$), $\sim$97% of the U$\cdot$G-containing heteroduplex had the U residue excised by the uracil excision repair system. With the analogous T$\cdot$G mispair, mismatch repair operated on almost all the transfecting heteroduplexes to remove the T residue in 75% of the heteroduplexes when the T residue was on the minus-strand of the M13mp18 RF DNA. Similar preferential removal of the minus-strand's mismatched base was observed. In $ung\sp{-}$ hosts, which lack uracil-DNA glycosylase but have an intact Mut system, the U$\cdot$G mismatches are corrected by mismatch repair with the same directionality and to the same extent as were T$\cdot$G mispairs in $ung\sp{-}$ or $ung\sp{+}$ hosts. In E. coli hosts carrying insertions, deletions, or point mutations in the genes coding for the various components of the Mut system (mutL, mutS, MutH genes), the mismatch repair of T$\cdot$G mismatches is diminished but not abolished The ability of restriction endonucleases to hydrolyze their DNA recognition sites substituted with mismatched base pairs was investigated. The restriction enzymes studied included SalI, AccI, HincII, HindIII, SmaI, SstI, and KpnI. None of the restriction enzymes were able to completely cleave the mismatch-containing recognition sites under standard conditions. However, SmaI, SalI, and SstI, catalyzed partial digestion leading to an accumulation of DNA singly nicked at the mismatch-containing recognition site. Therefore, in some cases a transition-type substitution in only one strand of a recognition site inhibits restriction endonuclease-catalyzed digestion at that site although in others partial digestion occurs. Also this study yielded information on how different pyrimidine functional groups interacted with the restriction enzymes. (Abstract shortened with permission of author.)acase@tulane.edu
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Vargason, Jeffrey M. "The effect of cytosine methylation on DNA structure." Thesis, 2002. http://hdl.handle.net/1957/32388.
Full textAbstract:
DNA methylation is common in prokaryotes and eukaryotes and has been
implicated in various biological roles including gene silencing, X-chromosome
inactivation, and genomic imprinting. 5-methylcytosine the "fifth base" of the
genetic code comprises 1-3% of the human genome and is primarily found on
cytosines within the context of the CpG sequence. Although progress has been
made in understanding the biological roles of 5-methylcytosine, we are only
beginning to uncover how it changes the local structure and global conformation of
DNA. This thesis deals with the local perturbations in structure and hydration and
the global conformational changes induced by the presence of 5-methylcytosine in
DNA as determined by single crystal x-ray diffraction.
5-methylcytosine induces a novel conformation in the structure of duplex
DNA. This conformation has characteristics of both the A-DNA and B-DNA
conformations as well as some unique defining characteristics. This distinct duplex
provides a structural rationale for the increased rate of deamination in 5-methylcytosine relative to cytosine. In addition to this novel conformation, 5-methylcytosine stabilizes intermediates within the B-DNA to A-DNA transition pathway, thus providing a crystallographic map of the transition from B-DNA to A-DNA.
5-methylcytosine was also used as a tool to probe the stabilizing features of
the DNA four-way junction (known as the Holliday junction). The first crystal
structures of Holliday junctions were found serendipitously while studying duplex
DNA. The DNA four-way junction formation in these crystals was thought to be
stabilized by a network of sequence dependent hydrogen bonds at the junction
crossover. In this thesis, 5-methylcytosine was used to perturb these hydrogen
bonds; however, the junction persisted, suggesting that there is flexibility in the
types of sequences that can accommodate junction formation in the crystal, as well
as, flexibility in the global structure of the junction. Overall, this work describes
the effects of 5-methylcytosine on the local and global structure and hydration of
DNA structure, as well as raising some interesting questions regarding the
biological impact of methylation induced DNA structure.Graduation date: 2002
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Genger, Ruth Kathleen. "Cytosine methylation, methyltransferases and flowering time in Arabidopsis thaliana." Phd thesis, 2000. http://hdl.handle.net/1885/47082.
Full textAbstract:
Environmental signals such as photoperiod and temperature provide plants with seasonal information, allowing them to time flowering to occur in favourable conditions. Most ecotypes of the model plant Arabidopsis thaliana flower earlier in long photoperiods and after prolonged exposure to cold (vernalization). The vernalized state is stable through mitosis, but is not transmitted to progeny, suggesting that the vernalization signal may be transmitted via a modification of DNA such as cytosine methylation. The role of methylation in the vernalization response is investigated in this thesis. ¶ ...
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Pedersen, J. S., E. Valen, A. M. V. Velazquez, B. J. Parker, S. Lindgreen, B. Lilje, Desmond J. Tobin, et al. "Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome." 2014. http://hdl.handle.net/10454/7060.
Full textAbstract:
yesEpigenetic information is available from contemporary organisms, but is difficult to track back in evolutionary time. Here, we show that genome-wide epigenetic information can be gathered directly from next-generation sequence reads of DNA isolated from ancient remains. Using the genome sequence data generated from hair shafts of a 4000-yr-old Paleo- Eskimo belonging to the Saqqaq culture, we generate the first ancient nucleosome map coupled with a genome-wide survey of cytosine methylation levels. The validity of both nucleosome map and methylation levels were confirmed by the recovery of the expected signals at promoter regions, exon/intron boundaries, and CTCF sites. The top-scoring nucleosome calls revealed distinct DNA positioning biases, attesting to nucleotide-level accuracy. The ancient methylation levels exhibited high conservation over time, clustering closely with modern hair tissues. Using ancient methylation information, we estimated the age at death of the Saqqaq individual and illustrate how epigenetic information can be used to infer ancient gene expression. Similar epigenetic signatures were found in other fossil material, such as 110,000- to 130,000-yr-old bones, supporting the contention that ancient epigenomic information can be reconstructed from a deep past. Our findings lay the foundation for extracting epigenomic information from ancient samples, allowing shifts in epialleles to be tracked through evolutionary time, as well as providing an original window into modern epigenomics.
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Hudec, Michael. "Epigenetická regulace genů HLA asociovaných s celiakií." Master's thesis, 2017. http://www.nusl.cz/ntk/nusl-367819.
Full textAbstract:
Introduction: HLA class II system presents one of the most important mechanism in immune system, which is able to recognise pathogens and damaged cells. Some HLA class II alleles are associated with autoimmune diseases, for example celiac disease, which is typical by chronic inflammation of small intestine and other following symptoms. The risk HLA class II variants are DQ2 and DQ8. Epigenetic mechanisms that regulates gene expression, especially methylation of cytosine in promoter region of DQ2 and/or DQ8 alleles, could have influence on development of T lymphocytes in the thymus, where T-lymphocytes develop and pass a few stages in, and only the survival clones can be part of function immune system. Aim: The aim of this study is to compare methylation level of promoter regions of HLA DQ2 and DQ8 alleles between celiac patients and healthy controls. Another goal is to compare expression level of DQ2 and DQ8 variants between these two groups. Methods: DNA and RNA were isolated from full blood of two sets of donors. DNA was converted by bisulphite conversion and then amplified by Nested PCR. The PCR product was cloned to bacteria. Than positive colonies were selected. Subsequent methylation analysis was performed. RNA was converted to cDNA by Reverse transcription. Relative expression was analyzed...
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Zhang, Xiaolin. "Mutagenic mechanisms associated with DNA cytosine methylation, DNA base sequence context and DNA precursor pool asymmetry." Thesis, 1995. http://hdl.handle.net/1957/35148.
Full text
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"Functional analysis of the two subunits of DNA methyltransferase EcoHK311." Thesis, 2006. http://library.cuhk.edu.hk/record=b6074085.
Full textAbstract:
All mC5-MTases are monomeric enzymes, except M. EcoHK31I and M. AquI which are MTases composed of two poly peptides. M.EcoHK31I is a mC5-MTase which recognizes the sequence 5-YGGCCR-3' and consists of polypeptide alpha and beta, with the latter gene encoded in an alternative reading frame of the former. All of the conserved motifs in mC5-MTases can be found in polypeptide alpha, except motif IX, which is located in polypeptide beta. Both polypeptides are required for in vitro methylation.Methylation of cytosine residues in DNA occurs in diverse organisms from bacteria to humans. In higher eukaryotic organisms cytosine-C5 methyltransferase (mC5-MTase) is the only type of DNA MTase and it plays an important role in controlling a number of cellular processes including transcription genomic imprinting and DNA repair. In bacteria, there are three types of MTases, mC4-, mC5- and mAb-, classified according to the methylation site of the DNA. MTase and its cognate restriction endonuclease (ENase) form restriction-modification system. The role of MTase is to protect the host from its own ENase digestion while the ENase acts to degrade the invasion of foreign DNA. Sequence comparison of nearly 50 bacterial mC5-MTases has shown that these enzymes share an overall common protein architecture. Ten conserved motifs (I to X), each 10 to 20 amino acids in length, have been identified, five of which are highly conserved (I, IV, VI, VIII and X). In addition, all of these enzymes have a hypervariable region lying between motifs VIII and IX. It is called the target recognition domain (TRD), and is responsible for the specificity of DNA recognition and the choice of base to be methylated.
Since both of the polypeptides alpha and beta of M.EcoHK31I are sequenced and cloned into the expression vector separately, the role of DNA recognition and subunits interaction of individual polypeptides can be studied. By electromobility shift assay, we found that polypeptides alpha and beta complex recognize specific double strand oligos substrate. Polypeptide alpha-DNA formed aggregates and polypeptide beta alone did not bind DNA. Therefore, polypeptide beta assists the proper binding of polypeptide alpha to DNA substrate. Complex of polypeptide alpha and a polypeptide beta variant with N-terminal deletion of 41 amino acids showed a 16-fold reduction in methylation activity. Further deletion resulted in an inactive MTase. By surface plasmon resonance assay, the dissociation equilibrium constant (KD) of polypeptides alpha and beta complex was found to be 56.2nM and the KD for polypeptide alpha and DeltaN46-polypeptide beta complex was increased by about 95 folds, contributing by a drastic decrease in dissociate rate constant (kd) and an increase in association rate constant (ka). This indicated that the N-terminal region of polypeptide beta takes part in subunit interaction.
To pinpoint which amino acid residues located at the variable region of polypeptide alpha are important for DNA binding and subunits interaction, "charge-to-alanine scanning mutagenesis" were performed on 16 charge residues between Asp213 and Glu271 in the small domain. It was found that the five charge residues upstream of motif X are not required for activity. For other residues except K225, E240 and D245, the protein is active when the same charge is maintained.
Fung Wai To.
"March 2006."
Adviser: P. C. Shaw.
Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6376.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2006.
Includes bibliographical references (p. 180-201).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.
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LI, AHONG-FEN, and 李中芬. "Study of the relationship between DNA cytosine methylation and gene expression and characterization of arabidopsis DNA methylase genes." Thesis, 1993. http://ndltd.ncl.edu.tw/handle/18741613531694483392.
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"Effect of 5-methylation of cytosine residues on interactions of proteins with dna (mdbp, chromatin, m(5)c)." Tulane University, 1985.
Find full textAbstract:
Restriction enzyme Taq I was able to cleave four highly modified DNAs from bacteriophage T4, XP12, PBS1 and SP15. The relative ability of cohesive-ended TaqI fragments of these DNAs and of normal (lamda) DNA to be ligated by T4 DNA ligase was as follows: (lamda) DNA = XP12 DNA > T4 DNA = PBS1 DNA >> SP15 DNA. Extensive 5-methylation of cytosine residues as in XP12 DNA had no effect on ligation The interactions of DNA binding proteins with 5-methylcytosine (m('5)C)-rich DNA and with normal, cytosine (C)-rich DNA were studied by a nitrocellulose filter-binding assay. Little or no significant difference was seen in the binding of histone mixture. H1, H2b or H4 to m('5)C-rich DNA and normal DNA with a similar A+T content. High mobility group (HMG) proteins 14 and 17 showed a limited preference for normal C-containing DNA rather than m('5)C-rich DNA However, a mammalian protein which bound preferentially to m('5)C-rich DNA sequences has been identified. This methylated DNA-binding protein (MDBP) was extracted with 0.3 M NaCl from human placental nuclei and then partially purified by phosphocellulose, hydroxyapatite, DEAE-cellulose and Sephacryl S-200 chromatography. The effects of pH, the concentration of NaCl and divalent cations on the MDBP binding specificity have been determined. This protein strongly preferred to bind m('5)C-rich DNA only when the DNA was double-stranded and the extent of preferential binding of DNA by MDBP was correlated with its m('5)CpG doublet frequency. Also, DNA methylated by human DNA methyltransferase, which predominantly methylates CpG sites, bound better to MDBP than nick-translated DNA with similar m('5)C content but a random distribution of m('5)C residues. Therefore, MDBP appears to specifically recognize m('5)CpG containing sequencesacase@tulane.edu
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Tate, Courtney Marie. "Structure-function analysis of CXXC finger protein 1." Thesis, 2009. http://hdl.handle.net/1805/2044.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)This dissertation describes structure-function studies of CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, in order to determine the functional significance of Cfp1 protein domains and properties. Cfp1 is an important regulator of chromatin structure and is essential for mammalian development. Murine embryonic stem (ES) cells lacking Cfp1 (CXXC1-/-) are viable but demonstrate a variety of defects, including hypersensitivity to DNA damaging agents, reduced plating efficiency and growth, decreased global and gene-specific cytosine methylation, failure to achieve in vitro differentiation, aberrant histone methylation, and subnuclear mis-localization of Setd1A, the catalytic component of a histone H3K4 methyltransferase complex, and tri-methylated histone H3K4 (H3K4me3) with regions of heterochromatin. Expression of wild-type Cfp1 in CXXC1-/- ES cells rescues the observed defects, thereby providing a convenient method to assess structure-function relationships of Cfp1. Cfp1 cDNA expression constructs were stably transfected into CXXC1-/- ES cells to evaluate the ability of various Cfp1 fragments and mutations to rescue the CXXC1-/- ES cell phenotype. These experiments revealed that expression of either the amino half of Cfp1 (amino acids 1-367) or the carboxyl half of Cfp1 (amino acids 361-656) is sufficient to rescue the hypersensitivity to DNA damaging agents, plating efficiency, cytosine and histone methylation, and differentiation defects. These results reveal that Cfp1 contains redundant functional domains for appropriate regulation of cytosine methylation, histone methylation, and in vitro differentiation. Additional studies revealed that a point mutation (C169A) that abolishes DNA-binding activity of Cfp1 ablates the rescue activity of the 1-367 fragment, and a point mutation (C375A) that abolishes the interaction of Cfp1 with the Setd1A and Setd1B histone H3K4 methyltransferase complexes ablates the rescue activity of the 361-656 Cfp1 fragment. In addition, introduction of both point mutations (C169A and C375A) ablates the rescue activity of the full-length Cfp1 protein. These results indicate that retention of either DNA-binding or Setd1 association of Cfp1 is required to rescue hypersensitivity to DNA damaging agents, plating efficiency, cytosine and histone methylation, and in vitro differentiation. In contrast, confocal immunofluorescence analysis revealed that full-length Cfp1 is required to restrict Setd1A and histone H3K4me3 to euchromatic regions.
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Shapiro, Jonathan. "A Novel Approach to Identify Candidate Imprinted Genes in Humans." Thesis, 2012. http://hdl.handle.net/1807/32278.
Full textAbstract:
Many imprinted genes are necessary for normal human development. Approximately 70 imprinted genes have been identified in humans. I developed a novel approach to identify candidate imprinted genes in humans using the premise that imprinted genes are often associated with nearby parent-of-origin-specific DNA differentially methylated regions (DMRs). I identified parent-of-origin-specific DMRs using sodium bisulfite-based DNA (CpG) methylation profiling of uniparental tissues, mature cystic ovarian teratoma (MCT) and androgenetic complete hydatidiform mole (AnCHM), and biparental tissues, blood and placenta. In support of this approach, the CpG methylation profiling led to the identification of parent-of-origin-specific differentially methylated CpG sites (DMCpGs) in known parent-of-origin-specific DMRs. I found new DMRs for known imprinted genes NAP1L5 and ZNF597. Most importantly, I discovered many new DMCpGs, which were associated with nearby genes, i.e., candidate imprinted genes. Allelic expression analyses of one candidate imprinted gene, AXL, suggested polymorphic imprinting of AXL in human blood.