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Biomedical subjects

D S Sarma

Publications and source records attributed to D S Sarma.

At least 127 records · Page 7Linked to original sources
PubMed1976

Nonrandom nature of in vivo methylation of dimethylnitrosamine and the subsequent removal of methylated products from rat liver chromatin DNA.

This investigation was designed to study whether methylation of liver chromatin DNA by dimethylnitrosamine (DMN) and the subsequent in vivo removal of DNA-bound methylated products are random. Liver chromatin DNA was fractionated into nuclease-digestible and nondigestible material 4 hr following the administration of [3H]DMN (0.5 mg/250 muCi/100 g body weight). Digestion of such methylated liver chromatin with pancreatic DNase I or micrococcal nuclease and analysis of nuclease-digested acid-soluble products revealed a discrepancy between the radioactivity released (72%) and the nucleotides released (50%) as measured by the absorbance at 260 nm. This discrepancy disappeared, and the rate and extent of release of both the radioactivity and the absorbance at 260 nm were identical when the total purified DNA isolated from methylated chromatin was used as the substrate instead of chromatin DNA in the nuclease reaction. These results, together with the fact that guanine contents of the DNA of the two fractions of the chromatin isolated by nuclease digestion were identical, suggest that methylation of the nuclease-accessible region of hepatic chromatin DNA is relatively greater than that of the inaccessible region. The study of the removal of methylated products in the accessible region of the chromatin DNA further reveals that, of the methylated products present at 4 hr, 62% is lost by 3 days, 87% is lost by 1 week and 94% is lost by 2 weeks. However, loss from the nuclease-inaccessible region of chromatin DNA is only 27% by 3 days, 49% by 1 week, and 86% by 2 weeks, thereby suggesting that the removal of methylated products from this region of chromatin DNA is relatively slower compared with that from the nuclease-accessible region of chromatin-DNA. The results of this study thus indicated (a) an increased methylation and faster rate of removal of DMN-induced methylated products in nuclease-accessible regions of chromatin DNA and (b) decreased methylation and slower rate of removal from the nuclease-inaccessible regions of chromatin DNA. It is concluded that the distribution and removal of DMN-induced methylated products in liver chromatin DNA is nonrandom as measured by this technique.

Animals↗
PubMed1975

Alkaline lysis of mammalian cells for sedimentation analysis of nuclear DNA. Conformation of released DNA as monitored by physical, electron microscopic and enzymological techniques.

The degree of single strandedness of the DNA released from rat liver nuclei by various alkaline lysing solutions (including some with sodium dodecyl sulfate) was determined both before and after sedimentation in alkaline sucrose gradients employing electron microscopy, melting profiles, circular dichroism measurements, and digestibility by S1 nuclease. Regardless of the technique employed, the results obtained following alkaline sucrose gradient centrifugation of the DNA are consistent. The DNA was completely single stranded as judged by electron microscopy, circular dichroism spectra, and digestibility by S1 nuclease, an enzyme that specifically hydrolyzes single-stranded DNA. This was not true if the DNA was analyzed following alkaline lysis of the nuclei but before centrifugation. Under conditions which gave a complete transition to the single-stranded state, as judged by melting profiles and circular dichroism spectra, only 10-15% of the DNA was hydrolyzed by S1 nuclease. An increase in the susceptibility of the released DNA to S1 nuclease was observed with increases in the pH of the lysing solution. In order to release DNA which was single stranded as judged by both physical and enzymological techniques, the rat liver nuclei were lysed for 30 min with a 0.3 M NaOH lysing solution containing 0.5% dodecyl sulfate, 0.3 M NaCl and 0.03 M EDTA.

Animals↗
PubMed1975

In vivo repair of rat liver DNA damaged by 3-hydroxyxanthine.

Using alkaline and neutral sucrose gradients, it was observed that administration of the hepatocarcinogen, 3-hydroxyxanthine, to rats, resulted in the slower sedimentation of liver DNA in alkaline and neutral sucrose gradients. Slower sedimentation of liver DNA in alkaline sucrose gradients was apparent within 4 h following the administration of the carcinogen at a dose of 50 mug/g body weight. The fragmentation of liver DNA was progressive with an increase in the dose. Such fragmentation caused by a dose of 100 mug/g was largely repaired by 24 h following the administration of the carcinogen. Fragmentation and repair of liver DNA with a dose of 100 mug or more/g body weight was also seen using neutral sucrose gradients. In neutral sucrose gradients significant repair of the fragmented DNA was seen by 24 h after carcinogen. Liver DNA from rats given 3-hydroxyxanthine (100 mug/g for 4 h) or calf thymus DNA incubated in vitro with this carcinogen did not show any change in their melting profile.

Animals↗
PubMed1975

Replication of hepatic DNA in rats treated with dimethylnitrosamine.

Replication of DNA containing unrepaired lesions such as depurinated sites, single-strand breaks or methylated bases such as O-6 and N-7 methylguanine was studied in the rat liver. Rat liver DNA was damaged by administering 10 mug dimethylnitrosamine (DMN)/g body wt i.p. 4 h prior to partial hepatectomy. The analysis of DNA on alkaline sucrose gradient revealed considerable damage to the parental strand at the time of and 48 h subsequent to partial hepatectomy. During this time interval, the synthesis of new strands was studied using labeled thymidine. In the control liver, radioactivity in DNA appeared as small fragments at 15 and 30 min following the administration of labeled thymidine which became bigger within 4 h. In the carcinogen-treated livers, the newly made DNA remained as small fragments for longer periods of time. Sometime between 4 and 24 h these became bigger in size than the parental damaged template DNA. Thus, with a delay, the newly made strands became eventually bigger, in spite of the fact that the parental template DNA strand was damaged. Such replication of DNA with unrepaired lesions (miscoding and/or non-coding) offers a mechanism by which the original damage to DNA caused by the carcinogen can be permanently imprinted on the newly made cell, a phenomenon that could account for the initiation of carcinogenesis under certain circumstances.

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