Integration of avian sarcoma virus DNA sequences in transformed mammalian cells.

Abstract

DNA from six avian sarcoma virus (ASV) transformed mammalian cell lines was digested with the restriction endonucleases EcoRI, Xho I, or Sal I, fractionated by agarose gel electrophoresis, transferred to nitrocellulose filter strips, and hybridized with specific ASV [32P]cDNA probes. DNA from al of the ASV-transformed cell lines yielded three common virus-specific DNA fragments (2.4, 1.8, and 1.3 X 106 daltons) upon cleavage with EcoRI. Xho I appeared to cleave at least once within the integrated provirus and yielded a common fragment of 3.3 X 166 daltons as well as a second virus-specific DNA fragment whose size varied from 4.0 to 5.0 X 106 daltons in the different transformed cell lines. Sal I did not cleave within the provirus and yielded a single major virus-specific fragment of about 11 X 106 daltons in all transformed lines examined. Using specific cDNA probes, we show that the 1.8 X 106-dalton EcoPd fragment contains sequences homologous to the 3' end of the viral RNA as well as to the src region of the viral genome. These studies clearly demonstrate that the same region on the ASV genome is utilized for provirus integration in different ASV-transformed cell lines. A central observation in the study of DNA and RNA tumor viruses is the persistence of viral genetic information in the host genome of the transformed cell (1, 2). Transformation of mammalian cells by the DNA tumor virus simian virus 40 (SV40) involves the covalent insertion of the viral DNA into the host cell genome (3). Recently, it has been shown that the arrangement of the integrated SV40 DNA sequences is different in independently isolated clones of SV40-transformed cells (4,5). These results imply that the site of SV40 DNA integration in the cellular genome is not unique and that the SV40 sequences involved in integration are not confined to a specific region of the viral genome (4, 5). Transformation of mammalian cells by RNA tumor viruses requires the formation of a DNA copy (provirus) of the viral RNA and the integration of the provirus into the host genome (2). Molecular hybridization studies have shown that avian sarcoma virus (ASV)-transformed mammalian cells contain one to two copies of covalently integrated provirus per cell and that normal mammalian cells contain little if any DNA sequences homologous to the ASV genome (6-8).To determine the arrangement of integrated proviral sequences in ASV-transformed mammalian cells, DNA from six ASV-transformed mammalian cell lines has been digested with restriction endonucleases EcoRI, Xho I or Sal I, fractionated by agarose gel electrophoresis, transferred to nitrocellulose filter strips, and hybridized to labeled cDNA probes complementary to either the entire ASV genome or to nucleotide sequences at the 3' or 5' end of the viral RNA.Comparison of the viral-specific DNA fragments generated by these restriction enzymes and the various [32P]cDNA probes (6).Restriction Enzymes, Agarose Gels, and Blotting Technique. EcoRI, Xho I, and Sal I restriction endonucleases were purchased from ...

Evidence for splicing of avian sarcoma virus 5′-terminal genomic sequences onto viral-specific RNA in infected cells

“…which contains only the sarc genes (26)(27)(28) (10), also hybridizes to all three species of RSV-specific mRNA from infected cells, these observations cannot be attributed to the redundant sequences adjacent to the poly(A) at the 3' end. These data are consistent with restriction endonuclease mapping studies of ASV proviral DNA which indicate that nucleotide sequences complementary to iDNA10o transcripts are not present on restriction fragments of ASV-specific DNA that code for the 28S and 21S viral-specific mRNA species (34). Therefore, the nucleotide sequences complementary to iDNA100 transcripts must be somehow attached or "spliced" onto the discrete classes of viral-specific mRNA identified in infected cells at some point during their synthesis or maturation.…”

Variety of Endogenous Proviruses in the Genomes of Chickens of Different Breeds

“…1) (Farashyan et al, 1979). These three fragments represent almost the whole genome of RSV (Collins & Parsons, 1977). Comparison Of the three bands on the autoradiograms corresponding to the three internal fragments of the provirus shows the quality of the 32p-labelled cDNA RAV-2 (RAV-2 and RSV genomes are very similar (Shoyab & Baluda, 1975).…”

Identification of transcriptional elements within the long terminal repeat of Rous sarcoma virus.

“…High-molecular-weight DNA was isolated from transfected ev-chicken embryo cells as previously described (6). The DNA was digested with either Pvul or EcoRI and electrophoresed in 0.75% agarose gels.…”