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Biomedical subjects

A Razin

Publications and source records attributed to A Razin.

At least 73 records · Page 4Linked to original sources
PubMed1984

DNA methylation pattern is determined by the intracellular level of the methylase.

Extrachromosomal plasmid DNA is transiently undermethylated in Escherichia coli during amplification in the presence of chloramphenicol. In addition, undermethylation of phage lambda DNA was observed after thermal induction of a lambda c1857 lysogen while the integrated lambda phage DNA was found to be fully methylated. These methylation pattern changes occur under conditions (extensive replication) in which the intracellular methylase level becomes limiting. In an E. coli strain that harbors a plasmid that carries the dam methylase gene and therefore overproduces dam methylase, there is no undermethylation of dam sites in either of the extrachromosomal DNAs. The sites that are methylated by the mec methylase in both plasmid and lambda phage DNAs were undermethylated in the dam overproducer as well. These results indicate that the intracellular level of the E. coli methylase determines the DNA methylation pattern.

Bacteriophage lambda↗
PubMed1984

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

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, beta-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.

Animals↗
PubMed1983

Methylation of replicating and post-replicated mouse L-cell DNA.

We have introduced [alpha-32P]dGTP into permeabilized cells and measured the degree of methylation at CpG sites by nearest-neighbor analysis. This method reveals a lag of approximately 1 min between DNA synthesis and the modification event. When methylation is inhibited by the addition of S-adenosyl-L-homocysteine in the presence of continued DNA synthesis, the resulting hemimethylated sites are methylated immediately after the release of inhibition. The results suggest that the methylase activity in the cell allows immediate methylation but conditions at the replication fork bring about a short delay in the onset of the modification reaction.

Animals↗
PubMed1983

Pattern of methylation of two genes coding for housekeeping functions.

The distribution of sites that can be methylated was analyzed in the Chinese hamster adenine phosphoribosyl-transferase (aprt) gene and the patterns of methylation of this gene and the mouse dihydrofolate reductase (dhfr) gene were studied by using CpG restriction enzymes. Both genes were found to be unmethylated completely at their 5'-end region and methylated heavily throughout the rest of the gene. Because the hamster aprt gene can be inhibited by DNA methylation in vivo, the results suggest that 5' undermethylation of this gene may be a necessary condition for its expression. The pattern of methylation of each of these two genes was similar in sperm and all other somatic tissue DNAs. This is in contrast to many tissue-specific genes that were found to be highly methylated in sperm DNA and undermethylated in the tissue in which they are expressed. This result is consistent with the fact that both aprt and dhfr are key enzymes in the biosynthesis of nucleotides and therefore expected to be synthesized in all cells.

Adenine Phosphoribosyltransferase↗
PubMed1983

Sequence and substrate specificity of isolated DNA methylases from Escherichia coli C.

Two DNA methylase activities of Escherichia coli C, the mec (designates DNA-cytosine-methylase gene, which is also designated dcm) and dam gene products, were physically separated by DEAE-cellulose column chromatography. The sequence and substrate specificity of the two enzymes were studied in vitro. The experiments revealed that both enzymes show their expected sequence specificity under in vitro conditions, methylating symmetrically on both DNA strands. The mec enzyme methylates exclusively the internal cytosine residue of CCATGG sequences, and the dam enzyme methylates adenine residues at GATC sites. Substrate specificity experiments revealed that both enzymes methylate in vitro unmethylated duplex DNA as efficiently as hemimethylated DNA. The results of these experiments suggest that the methylation at a specific site takes place by two independent events. A methyl group in a site on one strand of the DNA does not facilitate the methylation of the same site on the opposite strand. With the dam methylase it was found that the enzyme is incapable of methylating GATC sites located at the ends of DNA molecules.

Base Sequence↗
PubMed1982

Studies on the biological role of DNA methylation: V. The pattern of E.coli DNA methylation.

The distribution of the methylatable sites GATC and CCATGG was studied by analyzing the molecular average size of restriction fragments of E. coli DNA. Both sites were found to be randomly distributed, reflecting a random pattern of methylation. The methylation pattern of specific sequences such as the origin of replication and rRNA genes has been studied in wild type E. coli and a methylation deficient (dam- dcm-) mutant. These sequences were found to be methylated in wild type cells and unmethylated in the mutant indicating that there is no effect of the state of methylation of these sequences on their expression. Analysis of the state of methylation of GATC sites in newly replicating DNA using the restriction enzyme Dpn I (cleaves only when both strands are methylated) revealed no detectable hemimethylated DNA suggesting that methylation occurs at the replication fork. Taking together the results presented here and previously published data (5), we arrive at the conclusion that the most likely function of E. coli DNA methylations is probably in preventing nuclease activity.

Base Composition↗
PubMed1982

Clonal inheritance of the pattern of DNA methylation in mouse cells.

DNA-mediated gene transfer was used to investigate the mode of inheritance of 5-methylcytosine in mouse L cells. Unmethylated phi X174 replicative form DNA remains unmethylated after its introduction and integration into these cells. On the other hand, phi X174 replicative form DNA that was methylated in vitro at its C-C-G-G residues retains these methylations as shown by restriction enzyme analysis with Hpa II and Msp I to detect methylation at this specific site. Although these unselected methylated vectors are prone to lose 30-40% of their methyl moieties upon transfection, this demethylation appears to be random. Once established, the resulting methylation pattern is stable for at least 100 cell generations. In order to examine the specificity of methylation inheritance, fully hemimethylated duplex phi X174 DNA was synthesized in vitro from primed single-strand phi X174 DNA by using 5-methyl deoxycytidine 5'-triphosphate. This molecule was inserted into mouse L cells by cotransformation and subsequently was analyzed by a series of restriction enzymes. Only methylations located at C-G residues were conserved after many generations of cell growth. The results suggest that the inheritance of the cellular DNA methylation pattern is based on a C-G-specific methylase that operates on newly replicated hemimethylated DNA.

5-Methylcytosine↗
PubMed1982

In vitro methylation of the hamster adenine phosphoribosyltransferase gene inhibits its expression in mouse L cells.

The effect of DNA methylation on the expression of the hamster adenine phosphoribosyltransferase (aprt) gene in mouse cells has been examined. This gene was methylated in vitro at all of its C-C-G-G sites by using Hpa II methylase and was inserted into mouse Ltk- aprt- L cells by cotransformation, with the herpes virus thymidine kinase gene as a selectable vector. Whereas clones carrying unmethylated aprt sequences were found to have an aprt+ phenotype as shown by their ability to grow in azaserine-containing medium, almost all clones carrying methylated aprt sequences were shown to be phenotypically aprt-. Blot hybridization analysis demonstrated that both the methylated and unmethylated aprt sequences were integrated into the cellular genome to the same extent and that the in vitro modification was stably maintained in these cells for many generations. When clones containing methylated aprt genes were exposed to conditions that select for the expression of the aprt gene, a low frequency of reversion to the aprt+ phenotype was observed. In all of these clones, this reversion was accompanied by reorganization and undermethylation of the aprt sequences. These results show that the expression of certain genes may be inhibited by site-specific methylation of these sequences and suggest that methylation may play a direct role in the regulation of gene expression.

5-Methylcytosine↗