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

Abstract

SUMMARYA 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-3T 3 cells, non-productively transformed by SV4o. Molecular hybridization, monitored by the use of $1 nuclease, HAC, isopycnic centrifugation and thermal melting showed that ssDNA from SV-3T3 cells (which amounts to 1.5 to 2 ~o of the total nDNA) has the same characteristics as ssDNA previously isolated from other cell species. Only 27 to 28 ~/o of ssDNA can be selfhybridized 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 (SV4o-ZH-cRNA synthesized in a cell-free system and the separated 'early' and 'late' strands of SV4o DNA labelled with a25I) 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.

Manifold expression of new cellular genes in human lymphoid neoplasia.

“…1. RESULTS In all cases, ssDNA isolated from human lymphoid cells had the same characteristics as ssDNA isolated from other cell systems (7)(8)(9)(10)(11)(12)(13)(14). After two successive steps of hydroxylapatite chromatography purification, it amounted to 1.2-1.4% of the total nDNA and was 96-98% digested by S1 nuclease, whereas only 2-3% of bulk double-stranded nDNA was degraded under the same conditions.…”

“…The relationship between ssDNA and transcription was confirmed in certain cases of specific gene expression. In cells transformed by virus genomes integrated into the host genome, ssDNA was greatly enriched with virus-specific coding DNA sequences, as compared with nDNA, but contained few, if any, sequences from the noncoding DNA strand or from the nonexpressed virus genes (9)(10)(11). In rat myogenic cells (12) or mouse erythroleukemia cells (13) undergoing differentiation, the analysis of ssDNA reflected the changes in transcriptional activity.…”

“…Over the past few years, such a fraction has been isolated in our laboratory in the form of a minor single-stranded component (ssDNA). In all normal or malignant cell systems studied, from human, murine, or avian origin, ssDNA amounted to 1.5-2% of the total nuclear DNA (nDNA); most of the ssDNA could be reassociated with unique-copy nDNA and 30-35% hybridized with homologous cytoplasmic RNAs (7)(8)(9)(10)(11)(12)(13)(14). It was also shown that 70-0% of ssDNA from the various cell systems (9)(10)(11)(12)(13)(14) consists ofnon-selfreassociating DNA chains, which can be reassociated with nonrepetitive nDNA and contains nearly all the ssDNA sequences hybridizable with homologous mRNAs (10).…”

“…Aliquots were distributed in sealed tubes (covered with paraffin oil before sealing) and incubated in a water bath at 66°C. The fraction of DNA in hybrid form was usually determined by treating aliquots with SI nuclease and measuring the trichloroacetic acid-precipitable radioactivity (9)(10)(11)(12)(13)(14). The results were expressed as percentages of the input radioactivity rendered S1 nuclease resistant, after deduction ofcontrol values (time 0, "2I-labeled DNA incubated with yeast RNA), which usually did not exceed 2-3%.…”

“…The results were expressed as percentages of the input radioactivity rendered S1 nuclease resistant, after deduction ofcontrol values (time 0, "2I-labeled DNA incubated with yeast RNA), which usually did not exceed 2-3%. For iterative cross-hybridization assays, S1 nuclease treatment was omitted, DNARNA hybrids were treated with pancreatic ribonuclease (RNase A, 25 ,g/ml) and TI RNase (10 yg/ml, 30 min at 25°C), and DNARNA hybrids were separated by centrifuging to equilibrium in a CsS04 gradient as previously described (11)(12)(13) and as shown in Fig. 1.…”