Introduction to virus-caused cancers

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 ...

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Collins¹,

Parsons²

1977

“…Avian RNA tumor viruses contain several species of RNA, the largest of which is believed to be the genetic material (1). From the sedimentation coefficient (60-70S) and the relative electrophoretic mobility the molecular weight of this RNA was estimated to be about 107 (2)(3)(4).…”

mentioning

confidence: 99%

The Nucleotide Sequence Complexity of Avian Tumor Virus RNA

Billeter¹,

Parsons²,

Coffin³

1974

121

The nucleotide sequence complexity of 60-70S RNA of Rous sarcoma virus was determined from the molar yields of 11 pure oligonucleotides of known chain length, obtained from [32P]RNA of Rous sarcoma virus by Ti RNase digestion and two-dimensional polyacrylamide gel electrophoresis. We calculate an apparent chain length of about 9800 nucleotides, corresponding to a molecular weight of 3.4 X 106. Assuming the molecular weight of 60-70S RNA of Rous sarcoma virus to be about 107, our data suggest that the virus genome consists of identical, or very similar, nucleotide sequences repeated three to four times.Avian RNA tumor viruses contain several species of RNA, the largest of which is believed to be the genetic material (1). From the sedimentation coefficient (60-70S) and the relative electrophoretic mobility the molecular weight of this RNA was estimated to be about 107 (2-4). Upon denaturation it is converted into RNA with an apparent molecular weight of 3 X 106 (3-5). These observations have led to the conclusion that the genome of avian tumor viruses consists of an aggregate of three to four subunits, each with a molecular weight of about 3 X 106 (5, 6). In order to determine the coding capacity of the viral genome, it is necessary to establish whether the subunits are similar or different with regard to their nucleotide sequence. From a study of hybridization kinetics of RNA of Rous sarcoma virus (RSV) to its complementary DNA, it was concluded that the subunits are different, and that the sum of their molecular weights is about 9.3 X 106 (7).In this paper we describe a chemical approach to the same problem. We have determined the molar yield of a number of well-characterized, pure oligonucleotides derived from 60-70S [32P]RNA of RSV (Schmidt-Ruppin strain), which, on the basis of their chain length and partial sequence data, are expected to occur only once in 107 daltons of RNA. From these data we calculate an apparent total chain length, which in this paper will be referred to as "nucleotide sequence complexity" or "complexity." The number obtained, 9,800, implies that the nucleotide sequences of 60-70S RNA of RSV are repeated three to four times. MATERIALS AND METHODSCells and Viruses. Chicken embryo fibroblast cultures were grown as described (8). Rous sarcoma virus (Schmidt-Ruppin strain, subgroup D) derived from a stock virus described by Altaner and Temin (9) was cloned (10) and passaged twice before use. Cell cultures infected with about 0.5 focus-forming unit/cell and transferred 3-5 days later (8) were used for labeling experiments.Preparation of 32P-labeled RNA. RSV [32P]RNA was prepared from 5 to 10 confluent cultures of RSV-infected chicken cells essentially as described (8) Two-dimensional Gel Electrophoresis of Labeled RNA. Unlabeled QB RNA was added to the sample to give a total of 100 jg. After precipitation with ethanol, the RNA was collected by centrifugation, dried, and dissolved in 200 ul of 0.1 M NaCl-50 mM Tris-HCl (pH 7.5)-5 mM EDTA. A small aliquot was removed for determination o...

“…Samples (1-20 ,g total RNA) were annealed with 0.05-0.1 ,ug of poly(dC)-DNA in 50 1 of 0.5 M NaCl-10 mM Tris-HC1 (pH 7.5) containing 1 Mg of oligo(C)&20. After 4 hr at 660, 2 Mg of poly(U) (Miles Laboratories, Elkhart, Ind.) were added and labeled hybrid was determined by poly(I)-Sephadex chromatography.…”

Section: Methodsmentioning

confidence: 93%

“…Progeny RNA would subsequently be produced by transcription of the DNA (1-3). While-the first part of the hypothesis is supported by a number of findings (1)(2)(3), the only evidence for DNA-dependent viral RNA synthesis is provided by the observations that actinomycin D inhibits virus formation (4,5) and that virus mutants are generated by exposure of infected cells to brornodeoxyuridine (6). Although the inhibition experiment demonstrates the requirement for a DNA-dependent step, it does not show that the inhibitor is acting directly on viral RNA synthesis, rather than, for instance, by preventing synthesis of protein(s) required for viral RNA replication.…”

mentioning

confidence: 94%

In Vitro Synthesis of Rous Sarcoma Virus-Specific RNA is Catalyzed by a DNA-Dependent RNA Polymerase

Rymo

Parsons

Coffin

et al. 1974

Synthesis of Rous sarcoma virus RNA was examined in vitro with a new assay for radioactive virus-specific RNA. Nuclei from infected and uninfected cells were incubated with ribonucleoside [a-$'2Ptriphos-phates, Mn++, Mg++ and (NH4)2SO4. Incorporation into total and viral RNA proceeded with similar kinetics for up to 25 min at 37°. About 0.5% of the RNA synthesized by the infected system was scored as virus-specific, compared to 0.03% of the RNA from the tuninfected system and 0.005% of the RNA synthesized by monkey kidney cell nuclei. Preincubation with DNase or actinomycin D completely suppressed total and virus-specific RNA synthesis. a-Amanitin, a specific inhibitor of eukaryotic RNA polymerase Il. completely inhibited virus-specific RNA synthesis, while reducing total RNA synthesis by only 50%. We conclude that tumor virus-specific RNA is synthesized on a DNA template, most probably by the host's RNA polymerase II.Temin's model for tumor virus RNA replication postulates that the infecting viral RNA is transcribed into DNA, which is then integrated into the host genome. Progeny RNA would subsequently be produced by transcription of the DNA (1-3). While-the first part of the hypothesis is supported by a number of findings (1-3), the only evidence for DNA-dependent viral RNA synthesis is provided by the observations that actinomycin D inhibits virus formation (4, 5) and that virus mutants are generated by exposure of infected cells to brornodeoxyuridine (6). Although the inhibition experiment demonstrates the requirement for a DNA-dependent step, it does not show that the inhibitor is acting directly on viral RNA synthesis, rather than, for instance, by preventing synthesis of protein(s) required for viral RNA replication. Failure to find tumor virus-specific minus strands in infected cells (J. Duffy, H. Diggelthann, and C. Weissmann, unpublished results; see however ref. (7) for an opposite result) argues against the alternative pathway of RNA-directed RNA replication.In order to clarify the nature of template and enzyme involved in Rous sarcoma virus RNA replication we developed a cell-free system of RNA synthesis in which about 0.5% of the RNA synthesized is virus-specific. Virus-specific RNA synthesis is as sensitive to actinomycin D and DNase as cellular RNA synthesis, supporting the view that the template involved is DNA. The high sensitivity of virus RNA synthesis to a-amanitin suggests that DNA-dependent RNA polymerase II is responsible for tufmor virus RNA synthesis. MATERIALS AND METHODSDNase I (electrophoretically purified, from Worthington Biochemical Corp., Freehold, N.J.) was treated with iodoacetate to eliminate RNase activity (8). RNase T1 was from Calbiochem; crude RNase T2 was prepared as described (9). a-Amanitin was a gift from Prof. T. Wieland. Ribonucleoside [a-32PItriphosphates were synthesized by a method based on published procedures (10, 11). All information regarding cells, cell lines, viruses, labeled viral nucleic acids, and other materials has been published (12,16). Chicke...