Nucleotide clusters in deoxyribonucleic acids XII. The distribution of 5-methylcytosine in pyrimidine oligonucleotides of mouse L-cell satellite DNA and main band DNA

Harbers, Klaus; Harbers, Barbara; Spencer, John H.

doi:10.1016/0006-291x(75)90572-0

Cited by 45 publications

(12 citation statements)

References 21 publications

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“…This question can only be resolved by analysis ofclones and subelones at widely different passage levels, which unfortunately was not possible within the limited replicative life-span of these diploid cell strains. Many highly repetitive sequences have levels of cytosine methylation that are several times higher than those observed for total or "main-band" DNA 415, 23,24), sometimes exceeding 40% of total cytosines (15). It thus was possible that the random drift in total DNA methylation might result from drift in such repetitive fractions.…”

Section: Discussionmentioning

confidence: 95%

Variability of DNA methylation patterns during serial passage of human diploid fibroblasts

Reis

Goldstein

1982

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

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We have examined DNA methylation in diploid human fibroblasts, early and late in their replicative life-span. The extent of methylation of -C-C-G-G-was measured by comparison of fragment sizes after digestion with methylation-specific restriction enzyme Hpa II or Msp I, or both. Methylation of -C-C-G-Gsites in total DNA, occurring predominantly at internal (3') cytosines, increased from 59% to 64% of sites in one cell strain at late passage, remained constant in another, and decreased in four other strains (54% to 48%, 58.5% to 48%, 55% to 51.5%, and 52% to 44.5%). Base composition analysis confirmed a substantial loss of total DNA 5-methylcytosine (mC) in one strain. Seven clonal isolates, examined at middle to late passage, ranged from 33% to 51% methylation of3' cytosines in -C-C-G-G-sites. Three discrete classes of highly repetitive DNA were found which contained Msp I sites at intervals of 45, 110, and 175 base pairs. These repeat families consistently had 70-80% of sites methylated at 3' cytosines, in all clones and in all strains examined both at early and at late passage. Thus, altered methylation of repetitive sequences is unlikely to account for the variable -C-C-G-G-methylation observed in total DNA. When DNA from one fibroblast strain and from eight pure clones isolated from that parental culture was digested with Msp I or Hpa H followed by EcoRI and probed for y-globin gene sequences, considerable interclonal and intraclonal heterogeneity was observed for methylation at four -C-C-G-Gsites in the y-globin codingregion ofDNA. Therefore, the pattern of methylation in endogenous gene regions appears to undergo random drift during replication of diploid fibroblasts.Cytosine methylation has been found to diminish at certain CpG sites in and around genes that have been expressed by a given tissue, relative to tissues that have never expressed those genes (1-7). It therefore has been proposed that differentiation involves a heritable loss of specific cytosine methylations, associated with-the initial expression ofnearby genes (3-5). Residual methylation at hypomethylated sites in expressing tissues has generally been attributed to nonexpressing cell types invariably present in whole tissues (1-5). However, the question of whether individual clonal isolates of a single diploid cell type also vary in methylation patterns has not been addressed.Recent studies of the heritability of site-specific DNA methylation have utilized exogenous transforming DNA molecules, either methylated in vitro or unmethylated (8,9). The results indicated only one instance of de novo methylation (8) and highly variable retention (15-70%) ofprior methylation over 25 cell generations (8,9). We now report that methylation of endogenous genes is also inherited unstably in clonal isolates of diploid human fibroblasts. MATERIALS AND METHODSHuman Diploid Fibroblast Strains. Fibroblast strains were established as described (10-12) from forearm biopsy specimens from normal donors and were grown in Eagle's medium with 15% fetal calf ...

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Section: Discussionmentioning

confidence: 95%

Variability of DNA methylation patterns during serial passage of human diploid fibroblasts

Reis

Goldstein

1982

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

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“…Since teratocarcinoma cells do undergo demethylation in response to specific inducers, and since these cells seem to mimic in many other respects the formation of primitive endoderm cells, it is a reasonable assumption that the yolk sac and placenta undermethylation reflect some form of a demethylation process. It should be kept in mind that as much as 50%o of the methyl moieties in mouse cells may be located in satellite sequences (35,36). Thus, changes either in the number of satellite copies or in their methylation state could account for some variations in the total methylation content.…”

Section: Discussionmentioning

confidence: 99%

Variations in DNA methylation during mouse cell differentiation in vivo and in vitro.

Razin¹,

Webb²,

Szyf³

et al. 1984

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

175

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Mouse teratocarcinoma cells induced to differentiate in vitro undergo a massive (30%) demethylation of DNA. A similar undermethylation is also observed in the mouse extraembryonic membranes, the yolk sac and placenta. In both cases, the decrease in methyl moieties occurs at a large number of CpG sites spread out over the entire genome, as indicated by a restriction enzyme analysis of several mouse genes including dhfr, 13-major globin, and the H-2K gene family. In contrast to this, the embryo itself appears to undergo methylation de novo during early stages of embryogenesis. Thus, as opposed to somatic cells, events during early mouse development are associated with wide variations in the level of DNA methylation. Although these changes in DNA methylation seem to be an integral part of the differentiation process, its relation to specific gene expression is still unclear.

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“…Paper and thin layer chromatographic methods have also been applied to digests of DNA which were labeled in vivo with radioactive precursors (8,9,10,11). This approach has high sensitivity b'ut quantitation of radiolabeled methylated bases using [3H] or [14C-methyl] methionine has had limited success due to the difficulty in determining the sizes of the appropriate methionine pools (12,13,14). More advanced chromatographic techniques including gas-liquid chromatography (15,16), cation-exchange chromatography (17,18,19,20,21,22,23,24,25), reversed-phase HPLC (26), paired-ion reversed-phase HPLC (27) and gas chromatographymass spectrometry (28) have also been applied to analysis of the major, minor or total base composition of DNAs.…”

Section: Introductionmentioning

confidence: 99%

Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA

et al. 1980

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We have developed a method to accurately determine (< 3% RSD) the complete major and modified base composition of a few micrograms of unlabeled DNA. The DNA samples were quantitatively hydrolyzed with DNase 1, Nuclease P1, and bacterial alkaline phosphatase. The resulting deoxyribonucleosides were directly separated in 70 min by reversed-phase high performance liquid chromatography with detection by ultraviolet absorption at 254 nm and 280 nm (RP-HPLC). The highly sensitive and selective dual wavelength quantitation greatly enhances the precision and accuracy of the chromatographic analysis. Contamination of DNA preparations with RNA does not interfere with the DNA analysis due to the high resolution of the chromatography. We have used this method for the quantitation of m5dCyd in 5 microgram of calf thymus and salmon sperm DNA in which the m5dCyd comprises only 1 to 2% of the total bases. This method should be a useful research tool in studies on various DNAs and DNA subfractions and should help to elucidate the functions of methylation of DNA.

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Nucleotide clusters in deoxyribonucleic acids XII. The distribution of 5-methylcytosine in pyrimidine oligonucleotides of mouse L-cell satellite DNA and main band DNA

Cited by 45 publications

References 21 publications

Variability of DNA methylation patterns during serial passage of human diploid fibroblasts

Variability of DNA methylation patterns during serial passage of human diploid fibroblasts

Variations in DNA methylation during mouse cell differentiation in vivo and in vitro.

Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA

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