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Biomedical subjects

A Razin

Publications and source records attributed to A Razin.

At least 91 records · Page 5Linked to original sources
PubMed1981

Stable RNA synthesis and its control in Mycoplasma capricolum.

The synthesis of stable RNA in Mycoplasma capricolum was studied by [32P] labeling of cellular RNA of cells grown in a partially-defined medium in the presence or absence of an amino acid mixture supplement. The results indicate that M. capricolum employs the same stringent control mechanism used by E. coli cells, as judged by a decreased synthesis of stable RNA and accumulation of 5'-triphosphoguanosine-3'-diphosphate (pppGpp) and 5'-diphosphoguanosine-3'-diphosphate (ppGpp) in response to amino acid starvation. In addition, the results suggest that precursors of stable RNA accumulate and an intracellular pool of the precursors exists at all times under the growth conditions used by us. These findings may be interpreted to reflect a slow rate of RNA processing in M. capricolum.

Amino Acids↗
PubMed1981

Restriction enzyme digestion of hemimethylated DNA.

Hemimethylated duplex DNA of the bacteriophage phi X 174 was synthesized using primed repair synthesis is in vitro with E. coli DNA polymerase I followed by ligation to produce the covalently closed circular duplex (RFI). Single-stranded phi X DNA was used as a template, a synthetic oligonucleotide as primer and 5-methyldeoxycytidine-5'-triphosphate (5mdCTP) was used in place of dCTP. The hemimethylated product was used as substrate for cleavage by various restriction enzymes. Out of the 17 enzymes tested, only 5 (BstN I, Taq I, Hinc II, Hinf I and Hpa I) cleaved the hemimethylated DNA. Two enzymes (Msp I and Hae III) were able to produce nicks on the unmethylated strand of the cleavage site. Msp I, which is known to cleave at CCGG when the internal cytosine residue is methylated, does not cleave when both cytosines are methylated. Another enzyme, Apy I, cleaves at the sequence CCTAGG when the internal cytosine is methylated, but is inactive on hemimethylated DNA in which both cytosines are methylated. Hemimethylated molecules should be useful for studying DNA methylation both in vivo and in vitro.

Bacteriophage phi X 174↗
PubMed1980

DNA methylation and gene function.

In most higher organisms, DNA is modified after synthesis by the enzymatic conversion of many cytosine residues to 5-methylcytosine. For several years, control of gene activity by DNA methylation has been recognized as a logically attractive possibility, but experimental support has proved elusive. However, there is now reason to believe, from recent studies, that DNA methylation is a key element in the hierarchy of control mechanisms that govern vertebrate gene function and differentiation.

Animals↗
PubMed1980

Studies on the biological role of dna methylation; IV. Mode of methylation of DNA in E. coli cells.

Two pairs of restriction enzyme isoschizomers were used to study in vivo methylation of E. coli and extrachromosomal DNA. By use of the restriction enzymes MboI (which cleaves only the unmethylated GATC sequence) and its isoschizomer Sau3A (indifferent to methylated adenine at this sequence), we found that all the GATC sites in E. coli and in extrachromosomal DNAs are symmetrically methylated on both strands. The calculated number of GATC sites in E. coli DNA can account for all its m6Ade residues. Foreign DNA, like mouse mtDNA, which is not methylated at GATC sites became fully methylated at these sequences when introduced by transfection into E. coli cells. This experiment provides the first evidence for the operation of a de novo methylation mechanism for E. coli methylases not involved in restriction modification. When the two restriction enzyme isoschizomers, EcoRII and ApyI, were used to analyze the methylation pattern of CCTAGG sequences in E. coli C and phi X174 DNA, it was found that all these sites are methylated. The number of CCTAGG sites in E. coli C DNA does not account for all m5Cyt residues.

Base Sequence↗
PubMed1980

Methylated bases in mycoplasmal DNA.

The DNAs of four Mycoplasma and one Acholeplasma species were found to contain methylated bases. All of the five species contained 6-methyladenine (m6Ade), the methylated base characteristic of prokaryotic DNA. The extent of methylation of adenine residues in the mycoplasmal DNA ranged from 0.2% in Mycoplasma capricolum to about 2% in Mycoplasma arginini and Mycoplasma hyorhinis with intermediate methylation values for Mycoplasma orale and Acholeplasma laidlawii DNAs. About 5.8% of the cytosine residues in M. hyorhinis DNA were methylated also. Analysis of cell culture DNA for the presence of m6Ade as a means for detection of contamination by mycoplasmas, and the phylogenetic implications of the finding of methylated bases in mycoplasmal DNAs are discussed.

5-Methylcytosine↗
PubMed1980

Methylation of foreign DNA sequences in eukaryotic cells.

The herpesvirus thymidine kinase gene has been used to introduce foreign DNA sequences into mouse L cells by DNA-mediated gene transfer. These inserted genes were then assayed for methylation at the specific sequence C-C-G-G by using the restriction enzyme isoschizomers Hpa II and Msp I. Despite the fact that 70% of the cellular C-C-G-G sites are methylated, herpesvirus sequences, plasmid DNA, and growth hormone gene DNA were found to remain unmethylated in 90% of the clones that contain these genes. DNA that had been methylated in vitro with Hpa II methylase was also inserted into L cells. The presence of this modification in the vector DNA did not, however, guarantee that these sequences remained methylated in the recipient clones. Only 10% of all transformed clones were found to contain methylated C-C-G-G sequences in the vector DNA, and these modifications were stable for 25-50 generations. Hha I and Mbo I were used to probe for methyl groups at these restriction sites, but none of the inserted sequences acquired these modifications. These results are discussed in relation to various models put forth to explain the process of methylation in eukaryotic cells.

DNA↗
PubMed1979

Direct detection of methylated cytosine in DNA by use of the restriction enzyme MspI.

The extent of methylation of the internal C in the sequence CCGG in DNA from various eukaryotic sources has been determined using the restriction enzyme MspI known to be specific for this sequence. The methylation of the CCGG sequence is reflected in the restriction pattern obtained by DNA treated with MspI and its isoschizomer HpaII and analyzed by gel electrophoresis. A direct method for detection 5-methylcytosine in the sequence CCGG has been deviced. DNA fragments obtained with MspI were radioactively labeled at their 5' ends and subsequently degraded to the corresponding 5'-deoxyribonucleoside monophosphates. 5 methylcytidylic acid has been found in most of the 5' ends of MspI fragments of calf thymus DNA (about 90%) indicating heavy methylation of the sequence CCGG in calf thymus DNA. The results also reveal a symmetric methylation of both strands at this sequence in calf thymus DNA. In contrast, the CCGG sequence in other eukaryotic DNAs from organisms like Neurospora, Drosophila and Herpes virus proved to be undermethylated at this sequence.

Animals↗
PubMed1978

Efficient correction of a mutation by use of chemically synthesized DNA.

The mutated base in the am3 lysis-defective mutant of the bacteriophage phiX174 has been corrected by a combined in vitro enzymatic DNA synthesis and in vivo replication of the heteroduplex product. Chemically synthesized oligodeoxyribonucleotides carrying the wild-type sequence have been used to prime DNA synthesis with am3 phiX174 DNA serving as a template. The resultant semisynthetic heteroduplex composed of an am3(+) strand and a wild-type (-) strand, with one mismatched base pair at position 587 on the phiX174 DNA sequence, was used to infect spheroplasts. The progeny phage were analyzed by a parallel plaque assay on wild-type host, Escherichia coli C, to screen for wild-type phenotype, and on E. coli HF4714, an amber suppressor strain, to determine the total progeny phage. When a 23-base-long synthetic primer was used, about one-third of total progeny were found to be wild type. Shorter primers yielded lower percentages of wild type; they also had poorer priming activity.

Base Sequence↗
PubMed1977

Distribution of 5-methylcytosine in chromatin.

The content of 5-methylcytosine in eukaryotic DNA was measured by mass spectrometry. Almost equal amounts of methylated cytosine were found in the DNA of various tissues of the chicken. When chromatin or nuclei were digested with micrococcal nuclease, 50% of the DNA was found to be nuclease resistant. In contrast to this, over 75% of the 5-methylcytosine was protected from nuclease digestion by chromatin proteins. These results suggest that 5-methylcytosine is nonrandomly distributed with respect to the nucleoproteins.

Animals↗
PubMed1977

Studies on the biological role of DNA methylation: III Role in excision of one-genome long single-stranded phi X 174 DNA.

Accumulation of replicative intermediates of the bacteriophage phi X174 was observed in E. coli C infected cells when phage DNA methylation has been inhibited by nicotinamide or when cells were infected with a temperature-sensitive mutant in gene A. Analysis of the accumulating replicative intermediates by electron microscopy revealed that these molecules are composed of double-stranded DNA rings with multiple-genome length single-stranded "tails". These results suggest that the single 5-methylcytosine residue present in the phage DNA serves as a recognition site for the gene A protein mediating the excision of one-genome long phage DNA. This excision process is oligatory for the final maturation of the phage.

Coliphages↗
PubMed1976

Studies on the biological role of DNA methylation. II. Role of phiX174 DNA methylation in the process of viral progeny DNA synthesis.

In vivo inhibition of bacteriophage phiX174 DNA methylation by nicotinamide resulted in the accumulation of replicative intermediates with multiple-genome length single-stranded "tails". These abnormal replicative intermediates could not be chased into viral single-stranded circular DNA. The effect of nicotinamide on phage maturation and accumulation of abnormal replicative intermediates could be reversed by washing out the inhibitor. The results suggest that the single methyl group present in the viral DNA serves as a recognition site for a specific endonuclease, probably the gene A protein product, that is responsible for the excision of the single-stranded one-genome long viral DNA, before final maturation of the virus occurs.

Coliphages↗
PubMed1975

Studies on the biological role of DNA methylation: inhibition of methylation and maturation of the bacteriophage phichi174 by nicotinamide.

Nicotinamide was found to be a potent inhibitor of DNA methylation in vivo without interfering with protein or DNA synthesis. The inhibition of DNA methylation in a phage-infected cell resulted in a parallel decrease in the production of viable virus particles. In vitro experiments revealed that nicotinamide inhibits DNA methylase activity in a competitive fashion with respect to S-adenosylmethionine and non-competitively with respect to DNA. These results were interpreted to mean that DNA methylation is an essential step in the process of maturation of the bacteriophage phichi174.

Coliphages↗