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Biomedical subjects

A Razin

Publications and source records attributed to A Razin.

At least 55 records · Page 3Linked to original sources
PubMed1991

DNA methylation and gene expression.

A large body of evidence demonstrates that DNA methylation plays a role in gene regulation in animal cells. Not only is there a correlation between gene transcription and undermethylation, but also transfection experiments clearly show that the presence of methyl moieties inhibits gene expression in vivo. Furthermore, gene activation can be induced by treatment of cells with 5-azacytidine, a potent demethylating agent. Methylation appears to influence gene expression by affecting the interactions with DNA of both chromatin proteins and specific transcription factors. Although methylation patterns are very stable in somatic cells, the early embryo is characterized by large alterations in DNA modification. New methodologies are now becoming available for studying methylation at this stage and in the germ line. During development, tissue-specific genes undergo demethylation in their tissue of expression. In tissue culture cells this process is highly specific and appears to involve an active mechanism which takes place in the absence of DNA replication. The X chromosome undergoes inactivation during development; this is accompanied by de novo methylation, which appears necessary to stably maintain its silent state. As opposed to the programmed changes in DNA methylation which occur in vivo, immortalized tissue culture cells demonstrate alterations in DNA modification which take place over a long time scale and which appear to be the result of selective pressures present during the growth of these cells in culture.

Animals↗
PubMed1990

Effect of CpG methylation on gene expression in transfected plant protoplasts.

Activity of the cat gene driven by the cauliflower mosaic virus 35S promoter has been assayed by transfecting petunia protoplasts with the pUC8CaMVCAT plasmid. In vitro methylation of this plasmid with M.HpaII (methylates C in CCGG sites) and M.HhaI (methylates GCGC sites) did not affect bacterial chloramphenicol acetyltransferase (CAT) activity. It should be noted, however, that no HpaII or HhaI sites are present in the promoter sequence. In contrast, in vitro methylation of the plasmid with the spiroplasma methylase M.SssI, which methylates all CpG sites, resulted in complete inhibition of CAT activity. The promoter sequence contains 16 CpG sites and 13 CpNpG sites that are known to be methylation sites in plant DNA. In the light of this fact, and considering the results of the experiments presented here, we conclude that methylation at all CpG sites leaving CpNpG sites unmethylated is sufficient to block gene activity in a plant cell. Methylation of CpNpG sites in plant cells may, therefore, play a role other than gene silencing.

Blotting, Southern↗
PubMed1990

Cloning, characterization, and expression in Escherichia coli of the gene coding for the CpG DNA methylase from Spiroplasma sp. strain MQ1(M.SssI).

We describe here the cloning, characterization and expression in E. coli of the gene coding for a DNA methylase from Spiroplasma sp. strain MQ1 (M.SssI). This enzyme methylates completely and exclusively CpG sequences. The Spiroplasma gene was transcribed in E. coli using its own promoter. Translation of the entire message required the use of an opal suppressor, suggesting that UGA triplets code for tryptophan in Spiroplasma. Sequence analysis of the gene revealed several UGA triplets, in a 1158 bp long open reading frame. The deduced amino acid sequence revealed in M.SssI all common domains characteristic of bacterial cytosine DNA methylases. The putative sequence recognition domain of M.SssI showed no obvious similarities with that of the mouse DNA methylase, in spite of their common sequence specificity. The cloned enzyme methylated exclusively CpG sequences both in vivo and in vitro. In contrast to the mammalian enzyme which is primarily a maintenance methylase, M.SssI displayed de novo methylase activity, characteristic of prokaryotic cytosine DNA methylases.

Amino Acid Sequence↗
PubMed1990

Demethylation of genes in animal cells.

Tissue-specific animal cell genes are usually fully methylated in the germ line and become demethylated in those cell types in which they are expressed. To investigate this process, we inserted a methylated IgG kappa gene into fibroblasts and lymphocytes at various stages of development. The results show that this gene undergoes demethylation only in the mature lymphocytes and therefore suggest that the ability to demethylate a gene is developmentally regulated. These studies were supported by similar experiments using the rat Insulin I gene, and in this case it appears that the cis-acting elements that control demethylation may be different from those responsible for gene activation. The ability to demethylate the housekeeping gene APRT is also under developmental control, because this occurs only in embryonic cells, both in tissue culture and in transgenic mice.

Adenine Phosphoribosyltransferase↗
PubMed1990

Tissue-specific methylation patterns and expression of the human apolipoprotein AI gene.

To better understand the tissue-specific expression of the human apolipoprotein (apo)AI gene, we performed a detailed analysis of the pattern of methylation of the gene in various human adult and embryonic tissues and in tissues of transgenic mice harboring the human apo-AI gene. In addition, the gene was analyzed also in liver and intestine-derived human cell lines (HepG2 and Caco2, respectively). Using methyl-sensitive restriction enzymes (HpaII, HhaI, and SmaI) and the appropriate radioactive probes, we were able to determine separately the status of methylation of the 5'-end, the body of the gene, and 3'-end flanking sequences. The apo-AI gene in tissues that express the gene was undermethylated at the 5'-end. However, the 5'-end of the gene in sperm and in all adult tissues that do not express the gene was heavily methylated. The body of the gene which contains a CpG island and the 3'-end flanking sequences were, in general, hypomethylated except for specific sites that showed partial methylation. In contrast, while the gene showed tissue-specific expression already in a 12-week-old embryo, the 5'-end was invariably hypomethylated in all tissues of the embryo. A human apo-AI transgene has recently been shown to be active exclusively in the liver, while the endogenous gene is expressed in both liver and intestine (6). We show here that the 5'-end of the apo-AI transgene was methylated in all tissues of the mouse (including intestine) except liver. The results presented here demonstrate a clear correlation between hypomethylation of the 5'-end and activity of the apo-AI gene. However, the observed methylation pattern of the gene in embryonic tissues suggests that tissue-specific expression precedes formation of the tissue-specific methylation pattern.

Animals↗
PubMed1988

Effect of in vitro DNA methylation on beta-globin gene expression.

When the human beta-globin gene was methylated at every cytosine residue and was inserted into mouse fibroblasts by DNA-mediated gene transfer, the transcription of the gene was strongly inhibited. This methylation also prevented expression and induction of the gene in mouse erythroleukemia cells. By using partially methylated hybrid molecules, it was shown that methylation-sensitive negative regulatory elements are located in both the 5' and 3' ends of the beta-globin gene but not in the 90-base-pair region usually associated with promoter activity. To further investigate the role of DNA methylation in the regulation of the beta-globin gene, 50-base-pair poly(dG-dC) tracts were introduced into various sites in a mouse-human hybrid gene, and these inserts were methylated by means of the Hha I methylase. Heavy methylation of these artificially added sites had no effect on either transcription initiation or elongation, suggesting that DNA modification operates through fixed endogenous sites in the gene domain.

Animals↗
PubMed1988

Relationship between transient DNA hypomethylation and erythroid differentiation of murine erythroleukemia cells.

The state of DNA methylation in mouse erythroleukemia (MEL) cells has been analyzed in relation to commitment to differentiation in response to treatment with hexamethylenebisacetamide (HMBA). Previous experiments have shown that induction by HMBA involves transient genome-wide hypomethylation of DNA that is achieved by replacement of 5-methylcytosine with cytosine residues. The experiments described in the present communication revealed that hypomethylation is a very early event in the process of differentiation. Exposure of the cells to 3-deazaadenosine, an adenosine analog, in combination with homocysteine, resulted in the intracellular accumulation of 3-deazaadenosylhomocysteine, which caused an inhibition of HMBA-induced hypomethylation that was correlated with a comparable inhibition of differentiation. While these experiments suggest that hypomethylation is a necessary step in the process of differentiation, other experiments reported here indicate that hypomethylation of DNA may be necessary but not sufficient to trigger the whole program of differentiation in MEL cells. We found, for example that exposure of the cells to cycloheximide during the first 24 hr of induction by HMBA resulted in complete inhibition of differentiation without significant effect on the HMBA-induced hypomethylation. This result also indicates that the enzymatic machinery required for the hypomethylation of DNA is present in uninduced cells.

Acetamides↗
PubMed1986

Characterization of a tumorigenic murine T-lymphoid-cell line spontaneously derived from an IL-2-dependent T-cell line.

The establishment of IL-2-independent T-cell lines spontaneously derived from long-term IL-2-dependent cytotoxic T-cell lines is described. Two lines (cloned and uncloned) studied in detail have shown the following characteristics: (1) Permanent loss of IL-2 dependence. (2) Partial or complete loss of both cytotoxic activity and the IL-2 receptor. (3) Increased expression of T-cell membrane markers (Thy1.2, Lyt1.2) compared with the parental line. (4) Lower level of DNA methylation than in freshly obtained lymphoid cells. (5) Different karyotypic pattern from the parental IL-2-dependent line, with a mean number of 39-40 chromosomes and a resemblance to T leukemic lines. (6) Leukemia caused in normal syngeneic C57BL/6 mice by the uncloned line, in contrast to the cloned IL-2-independent line or the parental dependent line. Unlike established leukemic lines, however, the independent line gave rise to tumors which regressed in some mice within a few days of their appearance. These findings suggest that T-cell lines maintained with IL-2 for prolonged periods of time (greater than 3 months) can undergo transformation and, therefore, should not be utilized for immunotherapeutic purposes.

Animals↗
PubMed1986

Replacement of 5-methylcytosine by cytosine: a possible mechanism for transient DNA demethylation during differentiation.

In an earlier study it was discovered that when Friend erythroleukemia cells (FELC) were exposed to a variety of chemical agents capable of inducing differentiation, their DNA underwent genome-wide transient demethylation. In an attempt to elucidate the biochemical mechanism responsible for this phenomenon we have induced FELC with 5 mM hexamethylenebisacetamide and labeled the DNA in vivo with a density label, 5-bromodeoxyuridine, and a radioactive label, deoxy[5-3H]cytidine. Newly replicated DNA (heavy-light) was separated from parental DNA (light-light) by isopycnic centrifugation. Incorporation of deoxy[5-3H]cytidine into light-light duplex DNA has been observed only in induced cells concomitantly with the demethylation of the DNA, whereas, in parallel experiments, deoxy[G-3H]adenosine was not incorporated into light-light DNA. It was also found that the labeling of light-light DNA with deoxy[5-3H]cytidine is transient since the 3H label was removed from the DNA during the period of de novo DNA methylation that follows the demethylation. These results, taken together, strongly suggest that the demethylation of the DNA during differentiation is achieved by an enzymatic mechanism whereby 5-methylcytosine is replaced by cytosine.

5-Methylcytosine↗
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

The "old age security hypothesis" reconsidered.

"That the introduction of a means for transferring present to future consumption other than children in a developing country will reduce the rate of population growth is shown to depend crucially on the assumption that parents do not care about the numbers or the welfare of the children they have. When parents do care, the conclusion no longer unambiguously follows because the new means for providing for parents' old age leads to a positive income effect."

Adolescent↗
PubMed1985

Sequential changes in DNA methylation patterns of the rat phosphoenolpyruvate carboxykinase gene during development.

The cytosolic phosphenolpyruvate carboxykinase [PEPCK; GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] gene was isolated from a rat genomic library, and a map of the methylatable sites C-C-G-G and G-C-G-C has been constructed. The extent of methylation of 18 sites in the PEPCK gene in adult liver, kidney, spleen, and heart muscle and in fetal liver has been analyzed using the 5-methylcytosine sensitive enzymes Hpa II and Hha I. This analysis revealed extensive undermethylation of the PEPCK gene in the adult liver and kidney (PEPCK-expressing tissue), whereas the gene in adult spleen and heart muscle as well as in fetal liver (PEPCK-nonexpressing tissues) was heavily methylated. However, unlike the gene in the adult nonexpressing tissues, a region in the middle of the gene was found to be partially hypomethylated in fetal liver. This hypomethylation correlates with the competence of the fetal liver gene to be expressed. Treatment of fetuses by in utero injection of 5-azacytidine causes a hypomethylation-associated activation of the PEPCK gene. Taken together, the present findings suggest a sequential loss of methyl groups during development. When related to PEPCK gene expression, the sequential loss of methyl groups demonstrates an early stage prior to transcription characterized by hypomethylation of discrete sites and a later developmental hypomethylation of all sites associated with the mature active PEPCK gene around the time of birth.

Age Factors↗
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↗