Methylation of satellite deoxyribonucleic acid in mouse neoplastic and non-neoplastic cell cultures

Summary Comparisons of nucleic acid methylation between paired neoplastic and non-neoplastic mouse cell lines have shown a striking difference in the deoxyribonucleic acid (DNA) peak eluted from methylated albumin-kieselguhr columns (R. Gantt and V. J. Evans, 1969, Cancer Res. 29: 536–541). Since mouse satellite DNA is relatively highly methylated, its 5-methylcytosine content was compared with mainband DNA in these two paired cell lines to determine whether this might account for the observed differences. The cell DNA was labeled with methyl-labeled methionine and isolated from the cells by repeated neutral cesium chloride isopycnic centrifugation. The satellite DNA strands were then separated in an alkaline cesium chloride gradient. Both the 5-methylcytosine content and the relative amounts of satellite DNA were indistinguishable in the paired cell lines. Further, the results showed that both strands of satellite DNA had virtually equal amounts of 5-methylcytosine, although the heavy strand contains 1.5 times more cytosine than the light strand.     

Involvement of deoxyribonucleic acid polymerase beta in nuclear deoxyribonucleic acid synthesis.

The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.     

Methylation of rat liver mitochondrial deoxyribonucleic acid by chemical carcinogens and associated alterations in physical properties.

The formation of 7-methylguanine in rat liver mitochondrial DNA following the administration of the powerful carcinogen, dimethylnitrosamine, and the weak carcinogen, methyl methanesulphonate was measured and compared to the alkylation of nuclear DNA by these agents. At all doses tested mitochondrial DNA was alkylated more extensively than nuclear DNA by dimethylnitrosamine but both types of cellular DNA were alkylated to about the same extent by methyl methanesulphonate. The physical structure of rat liver mitochondrial DNA isolated from animals treated with these agents was investigated by electrophoresis in agarose gels and by isopycnic centrifugation in CsCl gradients. These procedures carried out in the presence of ethidium bromide, an intercalating dye, separate closed circular forms of mitochondrial DNA from open circular molecules (containing a single-strand break) and linear molecules. Administration of dimethylnitrosamine produced a considerable decrease in the amount of mitochondrial DNA which could be isolated in the closed circular form and at higher doses of dimethylnitrosamine no closed circular mitochondrial DNA could be found. Methyl methanesulphonate was less effective at reducing the amount of closed circular mitochondrial DNA. One explantation of these results is that dimethylnitrosamine leads to strand breaks in mitochondrial DNA and the possible use of this system to investigate carcinogen-induced breaks in DNA is discussed.     

Structure of Caulobacter deoxyribonucleic acid.

The deoxyribonucleic acid of the dimorphic bacterium Caulobacter crescentus contains a component that renatures with rapid, unimolecular kinetics. This component was present in both swarmer and stalked cells and exhibited the sensitivity to endonuclease S1 expected for hairpin loops. Double-stranded side branches between 100 and 600 nucleotide pairs in length were visible in electron micrographs of rapidly reassociating deoxyribonucleic acid isolated by hydroxyapatite chromatography. No extrachromosomal elements were found in spite of systematic attempts to detect their presence. These results indicate that the rapidly reassociating fraction derives from inverted repeat sequences within the chromosome and not from cross-links or plasmids. We estimate that there are approximately 350 inverted repeat regions per Caulobacter genome. The kinetic complexity of Caulobacter deoxyribonucleic acid, however, is no greater than that of other bacteria.