共查询到20条相似文献,搜索用时 15 毫秒
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N. W. Penn R. Suwalski C. O''Riley K. Bojanowski R. Yura 《The Biochemical journal》1972,126(4):781-790
A method is given for small-scale preparation of DNA from 1.0-1.5g of adult rat tissues. The product from brain or liver is characterized by base ratios and phosphorus content which accord with reported values for rat tissue. It is reasonably free of RNA, protein and glycogen. It contains 5-hydroxymethylcytosine at a content of about 15% of the total cytosine bases present. 5-Hydroxymethylcytosine is also demonstrable in mouse and frog brain DNA and in the crude cytidylic acid fractions obtained from RNA hydrolysates of rat brain and liver. 5-Hydroxymethylcytosine is identified by paper chromatography, u.v. spectra in acid and alkaline solutions and by its conversion into 5-hydroxymethyluracil. 相似文献
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S V Cannon A Cummings G W Teebor 《Biochemical and biophysical research communications》1988,151(3):1173-1179
The enzymatic release of 5-hydroxymethylcytosine from T2 bacteriophage DNA was effected by an extract of calf thymus. Like the previously described 5-hydroxymethyluracil DNA glycosylase, 5-hydroxymethylcytosine DNA glycosylase was not detectable in bacterial extracts. The phylogenetic distribution of these activities indicates that their primary function is the maintenance of methylcytosine residues in differentiated tissue. 相似文献
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蒋德伟 《中国生物化学与分子生物学报》2015,31(6):549-555
大脑的发育和神经系统疾病的发生发展是极其复杂的过程,涉及多种因素. 大量研究证实,表观遗传调控系统,如组蛋白甲基化、组蛋白乙酰化和DNA甲基化,是其中一类重要的调控因素. 近年来研究发现,DNA去甲基化中间产物5-羟甲基胞嘧啶(5hmC)是一种新的表观遗传标记形式,且在神经元内呈现非常高的水平. 这暗示5hmC可能在脑的生长发育以及中枢神经系统疾病的发生发展过程中有着重要的调控作用. 本文综述了近年来该领域的重要研究进展,并且提出一些今后的研究展望. 相似文献
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Active DNA demethylation performed by ten-eleven translocation (TET) enzymes produces 5-hydroxymethylcytosines, 5-formylcytosines, and 5-carboxylcytosines. Recent observations suggest that 5-hydroxymethylcytosine is a stable epigenetic mark rather than merely an intermediate of DNA demethylation. However, the clear functional role of this new epigenetic player is elusive. The contribution of 5-hydroxymethylation to DNA repair is being discussed currently. Recently, Jiang and colleagues have demonstrated that DNA damage response-activated ATR kinase phosphorylates TET3 in mammalian cells and promotes DNA demethylation and 5-hydroxymethylcytosine accumulation. Moreover, TET3 catalytic activity is important for proper DNA repair and cell survival. Here, we discuss recent studies on the potential role of 5-hydroxymethylation in DNA repair and genome integrity maintenance. 相似文献
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V G Ignashov 《Molekuliarnaia biologiia》1976,10(4):682-685
The treatment of denatured T4 phage DNA with antiserum for the DNA of this phage, containing antibodies against glucosylated 5-hydroxymethylcytosine, decreases the ability of DNA for renaturation. The greatest inhibiting activity is possessed by antiserum for T4 phage DNA irradiated with UV light, which contains antibodies not only against glucosylated 5-hydroxymethylcytosine, but also against the usual nitrogen bases. Antiserum against E. coli DNA, containing antibodies to the usual nitrogen bases, in equal dilutions with the antisera indicated above, shows less inhibitory activity on the renaturation of T4 phage DNA. 相似文献
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Uncovering the role of 5-hydroxymethylcytosine in the epigenome 总被引:1,自引:0,他引:1
Just over 2 years ago, TET1 was found to catalyse the oxidation of 5-methylcytosine, a well-known epigenetic mark, into 5-hydroxymethylcytosine in mammalian DNA. The exciting prospect of a novel epigenetic modification that may dynamically regulate DNA methylation has led to the rapid accumulation of publications from a wide array of fields, from biochemistry to stem cell biology. Although we have only started to scratch the surface, interesting clues on the role of 5-hydroxymethylcytosine are quickly emerging. 相似文献
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被称为"第六种碱基"的5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5hmC),广泛分布于多种哺乳动物的组织和细胞中,与胚胎发育,神经系统功能以及肿瘤研究高度相关.与5-甲基胞嘧啶(5-methylcytosine, 5mC)相比,5hmC在组织中含量更低,难以精确的检测.随着研究的深入,5hmC参与的重要生物学作用逐渐被人们发现,同时也促使着5hmC的检测和定量方法不断发展.为了区分5hmC与其他胞嘧啶衍生物,很多利用化学或者酶学修饰实现靶向检测或非靶向富集5hmC的方法应运而生.因此,选择并发展灵敏,准确,可靠的5hmC检测技术对于表观遗传研究至关重要.本文重点综述了近年来发展起来的5hmC检测和测序技术,通过比较分析各种方法的优缺点,为研究人员选择特定合适的方法开展相关研究提供重要的参考. 相似文献
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Brain development and aging are associated with alterations in multiple epigenetic systems,including DNA methylation and demethylation patterns.Here,we observed that the levels of the 5-hydroxymethylcytosine (5hmC) ten-eleven translocation (TET) enzyme-mediated active DNA demethylation products were dynamically changed and involved in postnatal brain development and aging in tree shrews (Tupaia belangeri chinensis).The levels of 5hmC in multiple anatomic structures showed a gradual increase throughout postnatal development,whereas a significant decrease in 5hmC was found in several brain regions in aged tree shrews,including in the prefrontal cortex and hippocampus,but not the cerebellum.Active changes in Tet mRNA levels indicated that TET2 and TET3 predominantly contributed to the changes in 5hmC levels.Our findings provide new insight into the dynamic changes in 5hmC levels in tree shrew brains during postnatal development and aging processes. 相似文献
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Junjie U Guo Yijing Su Chun Zhong Guo-li Ming Hongjun Song 《Cell cycle (Georgetown, Tex.)》2011,10(16):2662-2668
Cytosine methylation is the major epigenetic modification of metazoan DNA. Although there is strong evidence that active DNA demethylation occurs in animal cells, the molecular details of this process are unknown. The recent discovery of the TET protein family (TET1–3) 5-methylcytosine hydroxylases has provided a new entry point to reveal the identity of the long-sought DNA demethylase. Here, we review the recent progress in understanding the function of TET proteins and 5-hydroxymethylcytosine (5hmC) through various biochemical and genomic approaches, the current evidence for a role of 5hmC as an early intermediate in active DNA demethylation and the potential functions of TET proteins and 5hmC beyond active DNA demethylation. We also discuss how future studies can extend our knowledge of this novel epigenetic modification.Key words: TET1, 5-hydroxymethylcytosine, active DNA demethylation, epigenetic, DNA methylation, hippocampus, electroconvulsive stimulation, Gadd45b, BER 相似文献
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5-Hydroxymethylcytosine reacted with bisulfite and, instead of undergoing usual deamination process, gave cytosine 5-methylenesulfonate as the product. The conversion was rapid and quantitative, and the optimum pH was 4.5. The product was isolated as crystals and characterized. Cytosine 5-methylenesulfonate was only very slowly deaminated by treatment with bisulfite. 5-Hydroxymethyl-2'-deoxycytidine 5'-phosphate reacted with bisulfite in the same way as 5-hydroxymethylcytosine. Residues of 5-hydroxymethylcytosine in native as well as denatured T2 DNA were convertible to those of cytosine 5-methylenesulfonate by treatment of the DNA with bisulfite. While it is known that the 5-hydroxy-methyl groups of T-even bacteriophage DNA can be enzymatically glucosylated, this observation offers chemical evidence that the 5-hydrozymethyl groups in DNA are situated in such a way that they can readily react with external agents. 5-Hydroxymethyluracil gave uracil 5-methylenesulfonate on treatment with bisulfite. This reaction was much slower than that of 5-hydroxymethylcytosine, and the optimum pH was between 6 and 7. 相似文献
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Jonathan B. Coulter Cliona M. O'Driscoll Joseph P. Bressler 《The Journal of biological chemistry》2013,288(40):28792-28800
DNA methylation regulates gene expression throughout development and in a wide range of pathologies such as cancer and neurological disorders. Pathways controlling the dynamic levels and targets of methylation are known to be disrupted by chemicals and are therefore of great interest in both prevention and clinical contexts. Benzene and its metabolite hydroquinone have been shown to lead to decreased levels of DNA methylation, although the mechanism is not known. This study employs a cell culture model to investigate the mechanism of hydroquinone-mediated changes in DNA methylation. Exposures that do not affect HEK293 cell viability led to genomic and methylated reporter DNA demethylation. Hydroquinone caused reactivation of a methylated reporter plasmid that was prevented by the addition of N-acetylcysteine. Hydroquinone also caused an increase in Ten Eleven Translocation 1 activity and global levels of 5-hydroxymethylcytosine. 5-Hydroxymethylcytosine was found enriched at LINE-1 prior to a decrease in both 5-hydroxymethylcytosine and 5-methylcytosine. Ten Eleven Translocation-1 knockdown decreased 5-hydroxymethylcytosine formation following hydroquinone exposure as well as the induction of glutamate-cysteine ligase catalytic subunit and 14-3-3σ. Finally, Ten Eleven Translocation 1 knockdown decreased the percentage of cells accumulating in G2+M following hydroquinone exposure, indicating that it may have a role in cell cycle changes in response to toxicants. This work demonstrates that hydroquinone exposure leads to active and functional DNA demethylation in HEK293 cells in a mechanism involving reactive oxygen species and Ten Eleven Translocation 1 5-methylcytosine dioxygenase. 相似文献
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We describe strand-specific, base-resolution detection of 5-hydroxymethylcytosine (5-hmC) in genomic DNA with single-molecule sensitivity, combining a bioorthogonal, selective chemical labeling method of 5-hmC with single-molecule, real-time (SMRT) DNA sequencing. The chemical labeling not only allows affinity enrichment of 5-hmC-containing DNA fragments but also enhances the kinetic signal of 5-hmC during SMRT sequencing. We applied the approach to sequence 5-hmC in a genomic DNA sample with high confidence. 相似文献
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Robertson AB Dahl JA Vågbø CB Tripathi P Krokan HE Klungland A 《Nucleic acids research》2011,39(8):e55
Recently, 5-hydroxymethylcytosine (5hmC) was identified in mammalian genomic DNA. The biological role of this modification remains unclear; however, identifying the genomic location of this modified base will assist in elucidating its function. We describe a method for the rapid and inexpensive identification of genomic regions containing 5hmC. This method involves the selective glucosylation of 5hmC residues by the β-glucosyltransferase from T4 bacteriophage creating β-glucosyl-5-hydroxymethylcytosine (β-glu-5hmC). The β-glu-5hmC modification provides a target that can be efficiently and selectively pulled down by J-binding protein 1 coupled to magnetic beads. DNA that is precipitated is suitable for analysis by quantitative PCR, microarray or sequencing. Furthermore, we demonstrate that the J-binding protein 1 pull down assay identifies 5hmC at the promoters of developmentally regulated genes in human embryonic stem cells. The method described here will allow for a greater understanding of the temporal and spatial effects that 5hmC may have on epigenetic regulation at the single gene level. 相似文献
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DNA methylation at cytosines (5mC) is a major epigenetic modification involved in the regulation of multiple biological processes in mammals. How methylation is reversed was until recently poorly understood. The family of dioxygenases commonly known as Ten-eleven translocation (Tet) proteins are responsible for the oxidation of 5mC into three new forms, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Current models link Tet-mediated 5mC oxidation with active DNA demethylation. The higher oxidation products (5fC and 5caC) are recognized and excised by the DNA glycosylase TDG via the base excision repair pathway. Like DNA methyltransferases, Tet enzymes are important for embryonic development. We will examine the mechanism and biological significance of Tet-mediated 5mC oxidation in the context of pronuclear DNA demethylation in mouse early embryos. In contrast to its role in active demethylation in the germ cells and early embryo, a number of lines of evidence suggest that the intragenic 5hmC present in brain may act as a stable mark instead. This short review explores mechanistic aspects of TET oxidation activity, the impact Tet enzymes have on epigenome organization and their contribution to the regulation of early embryonic and neuronal development. [BMB Reports 2014; 47(11): 609-618] 相似文献
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《Epigenetics》2013,8(5):560-565
Recent studies reported the presence of 5-hydroxymethylcytosine (5 hmC) as an additional modification in mammalian genomic DNA. To date, 5 hmC has been detected only in mouse DNA isolated from embryonic stem cells, some adult tissues and in DNA from human bone marrow. Understanding its biological function will require the development of sensitive analytical methods that allow the detection and quantification of 5-hydroxymethylcytosine along with 5-methylcytosine and cytosine.Here we report the validation of a fast and sensitive method for the quantification of global 5-hydroxymethyl-2'-deoxycytidine (5 hmdC) in DNA. The method is based on a procedure consisting of fluorescence labeling of deoxyribonucleotides and analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). A double stranded DNA fragment containing a defined number of 5 hmdC residues was used for peak assignment, to establish separation conditions and to determine the limit of detection (LOD). The method yielded a LOD for 5 hmdC of 0.45 amol, which is equivalent to approximately to one 5 hmdC per 4,000 normal nucleotides (0.025%) using 1 μg of DNA as the matrix.By applying the calibrated assay to the analysis of various DNAs we show that 5 hmdC is present in human tissue and human cancer cell lines. We demonstrate that by using CE-LIF DNA can be analyzed in one run for both methylation and hydroxymethylation of cytosine with high sensitivity and accuracy. 相似文献
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《Bioorganic & medicinal chemistry letters》2014,24(1):294-297
Detection of DNA damage has been greatly improved following the development of equipment and techniques, however, discrimination between 5-hydroxymethylcytosine (5-hmC) and 5-methylcytosine (5-mC) is still a thorny problem. In the present study, an approach to oxidize and selective label (Ox-Labeling) 5-hmC in native DNA has been reported, which conveniently distinguishes 5-hmC from 5-mC using simple and effective processes. 相似文献
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Daniel Nettersheim Lukas C. Heukamp Florian Fronhoffs Marc J. Grewe Natalie Haas Anke Waha Friedemann Honecker Andreas Waha Glen Kristiansen Hubert Schorle 《PloS one》2013,8(12)
During mammalian development the fertilized zygote and primordial germ cells lose their DNA methylation within one cell cycle leading to the concept of active DNA demethylation. Recent studies identified the TET hydroxylases as key enzymes responsible for active DNA demethylation, catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. Further oxidation and activation of the base excision repair mechanism leads to replacement of a modified cytosine by an unmodified one. In this study, we analyzed the expression/activity of TET1-3 and screened for the presence of 5mC oxidation products in adult human testis and in germ cell cancers. By analyzing human testis sections, we show that levels of 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine are decreasing as spermatogenesis proceeds, while 5-methylcytosine levels remain constant. These data indicate that during spermatogenesis active DNA demethylation becomes downregulated leading to a conservation of the methylation marks in mature sperm. We demonstrate that all carcinoma in situ and the majority of seminomas are hypomethylated and hypohydroxymethylated compared to non-seminomas. Interestingly, 5-formylcytosine and 5-carboxylcytosine were detectable in all germ cell cancer entities analyzed, but levels did not correlate to the 5-methylcytosine or 5-hydroxymethylcytosine status. A meta-analysis of gene expression data of germ cell cancer tissues and corresponding cell lines demonstrates high expression of TET1 and the DNA glycosylase TDG, suggesting that germ cell cancers utilize the oxidation pathway for active DNA demethylation. During xenograft experiments, where seminoma-like TCam-2 cells transit to an embryonal carcinoma-like state DNMT3B and DNMT3L where strongly upregulated, which correlated to increasing 5-methylcytosine levels. Additionally, 5-hydroxymethylcytosine levels were elevated, demonstrating that de novo methylation and active demethylation accompanies this transition process. Finally, mutations of IDH1 (IDH1
R132) and IDH2 (IDH2
R172) leading to production of the TET inhibiting oncometabolite 2-hydroxyglutarate in germ cell cancer cell lines were not detected. 相似文献