Single-stranded DNA from oncornavirus-infected cells enriched in virus-specific DNA sequences

Abstract

We previously found that a minor fraction of single-stranded DNA (ss-DNA) isolated from native nuclear DNA of normal chicken embryonic cells and cells of other species hybridized with bulk nuclear DNA or cellular RNA in great excess. At least one-third of ss-DNA belonging to the nonrepetitious part of the cell genome could be hybridized to homologous RNAs. In the present work, similar results were obtained with ss-DNA from cells of chickens infected by avian myeloblastosis virus (AMV). To investigate whether this enrichment of ss-DNA in transcribed DNA sequences involves provirus DNA, radioactive AMV RNA and cDNA copies of AMV RNA were used. Most of the 70S AMV RNA hybridized much faster to ss-DNA from productively infected leukemic cells than to bulk DNA. cDNA, either double-stranded or single-stranded, made in the presence of actinomycin D hybridized to total nuclear DNA with similar kinetics. In contrast, about half of the double-stranded cDNA molecules hybridized 40-50 times faster to ss-DNA than to total DNA, indicating that only one of the provirus DNA strands seems to be present in ss-DNA. This was confirmed by the fact that relatively insignificant amounts of the ss-cDNA molecules made in the presence of actinomycin D could be annealed to ss-DNA as compared with bulk DNA. These results indicate that actively transcribed DNA sequences can be selectively distributed in the ss-DNA fraction, probably because of single strand breaks in the vicinity of transcription sites.The provirus theory of Temin (1), which postulates that the replication of RNA tumor viruses proceeds via DNA copies of viral RNA integrated in the host genome, has been substantiated by the discovery of successful transfection mediated by reverse transcriptase (2, 3), through an intermediate of viral DNA isolated from oncornavirus-transformed cells (4-6) and molecular hybridization between the viral genome and cell nucleic acids (7,8). In productively infected cells, the integrated provirus DNA appears to be more actively transcribed than most of the cellular genes (9).We have isolated, from the nDNA of various species, a minor fraction of single-stranded DNA (ss-DNA) and demonstrated that about one-third of the ss-DNA from cultured normal embryonic chicken cells (10) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be-hereby marked "advertsement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Relationship between Single‐Stranded DNA Isolated from Cultured Muscular Cells during Differentiation and the Transcription of Messenger RNA

“…consisting of single-stranded molecules, is related to synthesis of numerous RNA species [4--61. It was demonstrated that the ssDNA from chicken leukemic cells productively infected by avian myeloblastosis virus [5] or ssDNA from mouse cells non-productively transformed by simian virus (SV40) (Shaool, D. et al. unpublished results) are greatly enriched for DNA sequences encoding the viral RNA, but contain no or very few sequences from the non-transcribed DNA strand.…”

“…The amounts hybridized were determined by treating aliquots with S1 nuclease and measuring the acid precipitates collected on GF/B nitrocellulose Wathman filters, in a scintillation spectrometer as previously indicated [5] for 'H or in a Gamma 4000 Beckman counter at 70% efficiently for '"I.…”

“…Previous studies from our laboratory showed that a minor fraction of single-stranded DNA (ssDNA), usually amounting to 1.5 -2 of the total nuclear DNA, is related to the transcriptional activity, avian myeloblastosis virus [5] and ssDNA from mouse SV-3T3 cells (Shaool, D., Hanania, N., Harel, J . and May, E., unpublished results) non-productively transformed by simian virus 40 was found much enriched for transcribed virus-specific DNA sequences but contained no or very little DNA from the non-transcribed viral (or proviral) sequences.…”

“…Previous studies from our laboratory showed that a minor fraction of single-stranded DNA (ssDNA), usually amounting to 1.5 -2 of the total nuclear DNA, is related to the transcriptional activity, since 30 -35 (x of the ssDNA from chicken embryonic cells [4], chicken leukemic cells [5], human R.D. cells [6] and mouse SV-3T3 cells (Shaool, D., Hanania, N., Harel, J. and May, E., unpublished results), could be hybridized to total cytoplasmic RNA or purified polyadenylated RNA from homologous cells.…”

“…This relationship was confirmed in the case of tumor virus genes expressed in transformed cells. The ssDNA from chicken leukemic cells productively infected by avian myeloblastosis virus [5] and ssDNA from mouse SV-3T3 cells (Shaool, D., Hanania, N., Harel, J . and May, E., unpublished results) non-productively transformed by simian virus 40 was found much enriched for transcribed virus-specific DNA sequences but contained no or very little DNA from the non-transcribed viral (or proviral) sequences.…”