Relationship Between Single-stranded DNA Isolated from Mouse Cells Transformed by Simian Virus 40 and Transcription of Cellular and Virus Genes

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Abstract
Summary A minor fraction of single-stranded DNA (ssDNA) was isolated by an improved method of hydroxylapatite chromatography (HAC) from the native nuclear DNA (nDNA) of SV-3T3 cells, non-productively transformed by SV40. Molecular hybridization, monitored by the use of S1 nuclease, HAC, isopycnic centrifugation and thermal melting showed that ssDNA from SV-3T3 cells (which amounts to 1.5 to 2% of the total nDNA) has the same characteristics as ssDNA previously isolated from other cell species. Only 27 to 28% of ssDNA can be self-hybridized but the greatest part can be reassociated to the non-repetitive portion of nDNA and up to 38% hybridized to homologous RNAs, as compared with 7 to 8% for bulk nDNA. Highly radioactive virus probes (SV40-3H-cRNA synthesized in a cell-free system and the separated ‘early’ and ‘late’ strands of SV40 DNA labelled with 125I) were annealed to different excess amounts of cellular DNA. Both the quantities of each probe hybridized at saturation levels and the various reaction kinetics indicated that ssDNA is greatly enriched for virus sequences, mainly originating from the ‘early’ DNA strand which is predominantly expressed in SV-3T3 cells. The mode of formation of ssDNA is discussed in the light of other findings on the effects of DNA untwisting proteins and susceptibility of active animal genes to selective enzymic attacks.