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Biomedical subjects

M Szyf

Publications and source records attributed to M Szyf.

68 records · Page 4Linked to original sources
PubMed1986

Biological role of DNA methylation: sequence-specific single-strand breaks associated with hypomethylation of GATC sites in Escherichia coli DNA.

The effect of methylation of GATC sites in Escherichia coli DNA on the formation of single-strand breaks was studied with dam+, dam mutant, and Dam-overproducer strains. Single-strand breaks have been observed in dam mutant cells predominantly at TpT and, to a lesser extent, at CpC. In dam mutant cells harboring pTP166 (a plasmid containing the dam gene), no such nicks were observed.

Base Sequence↗
PubMed1985

Tissue-specific hypomethylation and expression of rat phosphoenolpyruvate carboxykinase gene induced by in vivo treatment of fetuses and neonates with 5-azacytidine.

Rat fetuses of 17-19-day gestation were injected in utero with 5-azacytidine (two to three daily injections of 40 micrograms/fetus). Neonates were injected with seven daily injections (1 mg/kg). DNA samples were isolated from the fetal and neonatal livers and neonatal spleen and subjected to analysis of their methylation status. Overall methylation was analyzed by the nearest-neighbor analysis (at CpG sites) and the pattern of methylation at CCGG sites by Southern blot analysis using phosphoenolpyruvate carboxykinase (PEPCK) sequences as probes. While DNAs from the liver and spleen undergo hypomethylation to the same extent in response to the 5-azacytidine treatment, the changes in the methylation patterns of the PEPCK gene in the two tissues are strikingly different. The changes observed indicate that a decrease in the methylase activity (inhibition by 5-azacytidine) results in site- and tissue-specific hypomethylation. The tissue-specific changes in the methylation pattern are associated with a tissue-specific expression of the PEPCK gene. Although the gene is hypomethylated by azacytidine in both liver and spleen, it is expressed only in the liver. The expression of already active genes (PEPCK in the kidney and albumin in the liver) is not further enhanced by the drug.

Animals↗
PubMed1985

Cell cycle-dependent regulation of eukaryotic DNA methylase level.

DNA methylase activity in the nuclei of somatic cells arrested at G0 increased markedly when the cells were subjected to a mitogenic stimulus. Treatment of mouse splenocytes with Concanavalin A resulted in about 20-fold increase in methylase activity within 20 h starting 12-15 h after Concanavalin A addition. The methylase level in rat liver was elevated approximately 3-fold at about 20-h posthepatectomy. A detailed time course of the increase in methylase activity with respect to the cell cycle revealed that the onset of this event coincided with the entry of the cells into S phase. In both systems, the extent of methylation in CpG sequences is not altered significantly even under conditions of active DNA synthesis which is induced by the mitogenic effect. These results suggest that the cell responds to the mitogenic stimulus by adjusting the DNA methylase activity to enable conservation of the methylation level in DNA.

Animals↗
PubMed1985

Cellular and viral DNA hypomethylation associated with induction of Epstein-Barr virus lytic cycle.

Epstein-Barr virus (EBV) producer and nonproducer cell lines have been treated with a combination of phorbol 12-myristate 13-acetate and n-butyrate (sodium salt). These inducers caused a massive hypomethylation of the EBV producer line P3HR-1 DNA (about 30%) at the time when DNA replication was inhibited. The viral DNA in these cells is heavily methylated as judged by digestion with Hpa II and probing with the Bam HI H fragment of EBV. However, upon induction with phorbol 12-myristate 13-acetate and n-butyrate, total hypomethylation of this viral DNA region was observed within 24 hr. This hypomethylation preceded EBV amplification, which became apparent only 32-36 hr after induction. When induction was carried out in the presence of retinoic acid, hypomethylation of cellular and viral DNA, viral DNA amplification, and production of the viral early antigen and viral capsid antigen were substantially inhibited. EBV DNA in another producer line (Jijoye nude) and in the nonproducer line Raji was hypomethylated and did not undergo further hypomethylation in response to induction. The observed hypomethylation of P3HR-1 and EBV DNA in the absence of DNA replication suggests that it is achieved by an active demethylation mechanism. This changes our perception of the DNA methylation phenomenon, since it has been generally accepted that hypomethylation of DNA takes place by a passive mechanism that involves DNA replication in the absence of methylation.

Butyrates↗
PubMed1985

Procaryotic and eucaryotic traits of DNA methylation in spiroplasmas (mycoplasmas).

Differences in the type of base methylated (cytosine or adenine) and in the extent of methylation were detected by high-pressure liquid chromatography in the DNAs of five spiroplasmas. Nearest neighbor analysis and digestion by restriction enzyme isoschizomers also revealed differences in methylation sequence specificity. Whereas in Spiroplasma floricola and Spiroplasma sp. strain PPS-1 5-methylcytosine was found on the 5' side of each of the four major bases, the cytosine in Spiroplasma apis DNA was methylated only when its 3' neighboring base was adenine or thymine. In Spiroplasma sp. strain MQ-1 over 95% of the methylated cytosine was in C-G sequences. Essentially all of the C-G sequences in the MQ-1 DNA were methylated. Partially purified extracts of S. apis and Spiroplasma sp. strain MQ-1 were used to study substrate and sequence specificity of the methylase activity. Methylation by the MQ-1 enzyme was exclusively at C-G sequences, resembling in this respect eucaryotic DNA methylases. However, the MQ-1 methylase differed from eucaryotic methylases by showing high activity on nonmethylated DNA duplexes, low activity with hemimethylated DNA duplexes, and no activity on single-stranded DNA.

Base Composition↗
PubMed1984

Methylation pattern of mouse mitochondrial DNA.

The pattern of methylation of mouse mitochondrial DNA (mtDNA) was studied using several techniques. By employing a sensitive analytical procedure it was possible to show that this DNA contains the modified base 5-methylcytosine (m5Cyt). This residue occurred exclusively at the dinucleotide sequence CpG at a frequency of 3 to 5%. The pattern of methylation was further investigated by determining the state of methylation of several MspI (HpaII) sites. Different sites were found to be methylated to a different extent, implying that methylation of mtDNA is nonrandom. Based on the known base composition and nucleotide sequence of mouse mtDNA, the dinucleotide sequence CpG was found to be underrepresented in this DNA. The features of mtDNA methylation (CpG methylation, partial methylation of specific sites and CpG underrepresentation) are also characteristic of vertebrate nuclear DNA. This resemblance may reflect functional relationship between the mitochondrial and nuclear genomes.

5-Methylcytosine↗
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↗
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↗
PubMedDate not supplied

De novo expression of the Muc2 gene in pancreas carcinoma cells is triggered by promoter demethylation.

It has been established that mucin-producing variants of different subtypes of pancreatic carcinomas, including the intraductal papillary and ductal mucinous tumors, have usually a more favorable prognosis. Intraductal papillary and ductal mucinous tumors have also been shown to ectopically express the intestinal mucin gene MUC2. The mechanism of the de novo expression of this gene in tumors may have potential implications for the modulation of its behavior. We studied, therefore, the mechanism of the de novo expression of MUC2 in pancreas carcinoma cells in vitro. The MUC2 gene promoter is methylated in the nonexpressing pancreatic cell line PANC-1 and is not methylated in the expressing cell line BxPC-3. The promoter is silenced by methylation as shown by reporter expression assays. De novo expression of MUC2 in PANC-1 cells is triggered by treating the cells with a pharmacological inhibitor of DNA methylation (5-aza-2'-deoxycytidine). There was no decrease or loss of expression of the methyltransferase DNMT1 in the MUC2-producing cells. These data show that the de novo expression of the MUC2 gene in pancreas carcinoma cells is associated with promoter demethylation. They warrant further investigations on the relationship between MUC2 promoter demethylation in pancreatic cancer and the prognosis of carcinoma patients.

Azacitidine↗
PubMedDate not supplied

Genomic structure of the human DNA methyltransferase gene.

We determined the genomic structure of the gene encoding human DNA methyltransferase (DNA MTase). Six overlapping human genomic DNA clones which include all of the known cDNA sequence were isolated. Analysis of these clones demonstrates that the human DNA MTase gene consists of at least 40 exons and 39 introns spanning a distance of 60 kilobases. Elucidation of the chromosomal organization of the human DNA MTase gene provides the template for future structure-function analysis of the properties of mammalian DNA MTase.

Cloning, Molecular↗