Methylation of somatic vs germ cell DNAs analyzed by restriction endonuclease digestions

Kaput, James J.; Sneider, Thomas W.

doi:10.1093/nar/7.8.2303

Cited by 62 publications

(27 citation statements)

References 26 publications

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“…In conformity with a previous report (10) we found that thymus DNA contains about 1.5 times as much m5 Cyt as sperm DNA. However, at least for the MspI/HpaII sites tested, this difference is not due to hemimethylation.…”

Section: Introductionsupporting

confidence: 93%

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Distribution of 5-methylcytosine in the DNA of somatic and germline cells from bovine tissues

Sturm

Taylor

1981

Nucl Acids Res

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Genomic DNA of calf thymus contains 1.5 times as much 5-methylcytosine as similar sperm DNA, but the major EcoRI repeat fragment from satellite I of thymus contains ten times as much 5-methylcytosine as the corresponding fragment from sperm DNA. Restriction enzyme analyses of the total DNA and the satellite I fragment show that three HpaII sites in the fragment are completely unmethylated in sperm but fully methylated in thymus DNA. Undermethylation of many sites in the satellite DNAs can probably account for the lower level of methylation of sperm DNA rather than hemimethylation as previously suggested. These results are also discussed in relation to maintenance and de novo (initiation-type) methylases.

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Section: Introductionsupporting

confidence: 93%

“…With these ideas in mind we have examined the DNA of bovine thymus and sperm to test a hypothesis that the reduced methylation of some mammalian sperm DNAs might be explained by strand asynmnetric hemimethylation (10).…”

Section: Introductionmentioning

confidence: 99%

Distribution of 5-methylcytosine in the DNA of somatic and germline cells from bovine tissues

Sturm

Taylor

1981

Nucl Acids Res

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“…7 x 10'0 becquerels), (2)(3)(4)(5) ,g of DNA, and 1-2 units ofthe enzyme was incubated at 37°C for 1 hr. After further incubation in the presence of 7 ,g of protease K and 0.4% sodium dodecyl sulfate, the solution was made 0.4 M NaOH and incubated at 60°C for 30 min to destroy any RNAs that might possibly be methylated with contaminating RNA methyltransferase (5). The whole reaction mixture was then neutralized with HCl and extracted with phenol.…”

Section: Methodsmentioning

confidence: 99%

“…Evidence of tissue-specific methylation has been described (7)(8)(9), although the significance of the observed methylation patterns is unclear. A function in the regulation of gene expression is suggested especially by the effect of hypomethylation on globin synthesis in Friend cells (10) and the effect of azacytidine on differentiation (11,12).…”

mentioning

confidence: 99%

Differential activity of DNA methyltransferase in the life cycle of Chlamydomonas reinhardi.

Sano¹,

Grabowy²,

Sager³

1981

Proc. Natl. Acad. Sci. U.S.A.

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Two molecular weight. forms of DNA (cytosine-5-)-methyltransferase [S-adenosyl-L-methionine:DNA (cytosine-5-)-methyltransferase, EC 2.1.1.37], both active in assays in vitro, were isolated from the green alga Chlamydomonas reinhardi at various stages of the life cycle. The enzyme with Mr 60,000 was found in vegetative cells and gametes of both male (mtF) and female (mtt) mating types. The enzyme with Mr 200,000 was specific to gametic cells and zygotes, which are the only stages at which methylation of chloroplast DNA occurs in vivo. Chloroplast DNA from gametes was shown to be methylated on both strands at most if not all methylation sites.and the Mr 200,000 enzyme was shown to methylate both unmethylated. and hemimethylated sites, the latter at an elevated rate. Micrococcus luteus DNA showed the same nearest-neighbor frequencies of methylation after methylation by each molecular weight component. The data suggest strongly that the Mr 20o,ooo enzyme is the active multimeric form of the Mr 60,000 enzyme and that it acts as both initiation and maintenance methylase. It is proposed that methylation of chloroplast DNA in female. gametes and zygotes is regulated by assembly ofthe multimeric Mr 2oo,0o0 active enzyme, which in turn determines the maternal inheritance of-chloroplast DNA.Eukaryotic DNAs contain 5-methylcytosine as a minor component, at concentrations of 27% of total cytosine, depending upon the species (1, 2). The introduction ofmethyl residues into the 5 position ofcytosine occurs after replication and is catalyzed by a DNA methyltransferase (EC 2.1.1.37), which transfers the methyl group from S-adenosylmethionine to cytosine in doublestranded DNA (3). This class of enzymes, found in both plant and animal tissues (3), methylates only a small fraction of cytosines, mainly at C-G doublets (4). Very little evidence of site specificity of a eukaryotic methyltransferase has yet been reported, other than in our preliminary study (5).The physiological roles of methylation in eukaryotic cells are almost entirely unknown (6). Evidence of tissue-specific methylation has been described (7-9), although the significance of the observed methylation patterns is unclear. A function in the regulation of gene expression is suggested especially by the effect of hypomethylation on globin synthesis in Friend cells (10) and the effect of azacytidine on differentiation (11,12). A role for methylation and restriction in the inactivation or destruction of particular sequences of DNA or even whole chromosomes has been proposed (13). In a series ofstudies, we have demonstrated that a methylation and restriction system regulates the inheritance of chloroplast DNA in the sexual alga Chlamydomonas (14). In this organism, as well as in higher plants, chloroplast DNA from the female parent is preferentially transmitted to progeny, leading to the genetic phenomenon of maternal inheritance.In this paper, we describe the isolation and characterization of a DNA methyltransferase from Chlamydomonas. Methylating activity has been ...

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“…This requires efficient strategies for the detection of 5mC. Different concepts for discriminating between cytosine (C) and 5mC have been described which rely on endonuclease digestion,8 affinity enrichment,9 nanopore sequencing,10 different chemical behavior concerning redox reactivity,11 or selective deamination of C using sodium bisulfite 12…”

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confidence: 99%

Modified Nucleotides for Discrimination between Cytosine and the Epigenetic Marker 5‐Methylcytosine

2016

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Abstract5‐Methyl‐2′‐deoxycytosine, the most common epigenetic marker of DNA in eukaryotic cells, plays a key role in gene regulation and affects various cellular processes such as development and carcinogenesis. Therefore, the detection of 5mC can serve as an important biomarker for diagnostics. Here we describe that modified dGTP analogues as well as modified primers are able to sense the presence or absence of a single methylation of C, even though this modification does not interfere directly with Watson–Crick nucleobase pairing. By screening several modified nucleotide scaffolds, O6‐modified 2′‐deoxyguanosine analogues were identified as discriminating between C and 5mC. These modified nucleotides might find application in site‐specific 5mC detection, for example, through real‐time PCR approaches.

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Methylation of somatic vs germ cell DNAs analyzed by restriction endonuclease digestions

Cited by 62 publications

References 26 publications

Distribution of 5-methylcytosine in the DNA of somatic and germline cells from bovine tissues

Distribution of 5-methylcytosine in the DNA of somatic and germline cells from bovine tissues

Differential activity of DNA methyltransferase in the life cycle of Chlamydomonas reinhardi.

Modified Nucleotides for Discrimination between Cytosine and the Epigenetic Marker 5‐Methylcytosine

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