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. 1971 Nov;8(5):730–741. doi: 10.1128/jvi.8.5.730-741.1971

Deoxyribonucleic Acid Polymerase of Rous Sarcoma Virus: Reaction Conditions and Analysis of the Reaction Product Nucleic Acids

D H L Bishop 1,2, Ruth Ruprecht 1,2, R W Simpson 1,2, S Spiegelman 1,2
PMCID: PMC376254  PMID: 4332143

Abstract

Reaction conditions for Rous sarcoma virus ribonucleic acid (RNA)-instructed deoxyribonucleic acid (DNA) polymerase activity are described whereby the viral RNA is relatively protected from endogenous or added nuclease activity. Three analyses of reaction product nucleic acids (3H-RNA, 32P-DNA) were compared, namely, gel electrophoresis, Cs2SO4 gradient centrifugation, and hydroxyapatite column chromatography. It was found that hydroxyapatite analysis could be misleading unless the state of the template RNA was monitored concomitantly with the DNA analysis. Gel electrophoresis and Cs2SO4 gradient centrifugation gave comparable results. It was concluded that analyses of the product of reverse transcriptase reactions should not only refer to the template RNA and product DNA species, but also be performed with virus or viral RNA which do not have or obtain nicks in the 60S RNA. Otherwise, interpretation of the results would have the ambiguity of potential artifacts caused by those degraded RNA molecules.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aaslestad H. G., Clark H. F., Bishop D. H., Koprowski H. Comparison of the ribonucleic acid polymerases of two rhabdoviruses, Kern Canyon virus and vesicular stomatitis virus. J Virol. 1971 Jun;7(6):726–735. doi: 10.1128/jvi.7.6.726-735.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BONAR R. A., BEARD J. W. Virus of avian myeloblastosis. XII. Chemical constitution. J Natl Cancer Inst. 1959 Jul;23(1):183–197. [PubMed] [Google Scholar]
  3. Bader J. P., Steck T. L. Analysis of the ribonucleic acid of murine leukemia virus. J Virol. 1969 Oct;4(4):454–459. doi: 10.1128/jvi.4.4.454-459.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baltimore D. RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature. 1970 Jun 27;226(5252):1209–1211. doi: 10.1038/2261209a0. [DOI] [PubMed] [Google Scholar]
  5. Bishop D. H., Claybrook J. R., Spiegelman S. Electrophoretic separation of viral nucleic acids on polyacrylamide gels. J Mol Biol. 1967 Jun 28;26(3):373–387. doi: 10.1016/0022-2836(67)90310-5. [DOI] [PubMed] [Google Scholar]
  6. Bishop D. H. Complete transcription by the transcriptase of vesicular stomatitis virus. J Virol. 1971 Apr;7(4):486–490. doi: 10.1128/jvi.7.4.486-490.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bishop D. H., Obijeski J. F., Simpson R. W. Transcription of the influenza ribonucleic acid genome by a virion polymerase. I. Optimal conditions for in vitro activity of the ribonucleic acid-dependent ribonucleic acid polymerase. J Virol. 1971 Jul;8(1):66–73. doi: 10.1128/jvi.8.1.66-73.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bishop D. H., Obijeski J. F., Simpson R. W. Transcription of the influenza ribonucleic acid genome by a virion polymerase. II. Nature of the in vitro polymerase product. J Virol. 1971 Jul;8(1):74–80. doi: 10.1128/jvi.8.1.74-80.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bishop D. H., Roy P. Kinetics of RNA synthesis by vesicular stomatitis virus particles. J Mol Biol. 1971 May 14;57(3):513–527. doi: 10.1016/0022-2836(71)90106-9. [DOI] [PubMed] [Google Scholar]
  10. Bishop D. H., Roy P. Properties of the product synthesized by vesicular stomatitis virus particles. J Mol Biol. 1971 Jun 28;58(3):799–814. doi: 10.1016/0022-2836(71)90041-6. [DOI] [PubMed] [Google Scholar]
  11. Bishop J. M., Levinson W. E., Quintrell N., Sullivan D., Fanshier L., Jackson J. The low molecular weight RNAs of Rous sarcoma virus. I. The 4 S RNA. Virology. 1970 Sep;42(1):182–195. doi: 10.1016/0042-6822(70)90251-5. [DOI] [PubMed] [Google Scholar]
  12. Bishop J. M., Levinson W. E., Sullivan D., Fanshier L., Quintrell N., Jackson J. The low molecular weight RNAs of Rous sarcoma virus. II. The 7 S RNA. Virology. 1970 Dec;42(4):927–937. doi: 10.1016/0042-6822(70)90341-7. [DOI] [PubMed] [Google Scholar]
  13. Blair C. D., Duesberg P. H. Structure of Rauscher mouse leukaemia virus RNA. Nature. 1968 Oct 26;220(5165):396–399. doi: 10.1038/220396a0. [DOI] [PubMed] [Google Scholar]
  14. Bonar R. A., Sverak L., Bolognesi D. P., Langlois A. J., Beard D., Beard J. W. Ribonucleic acid components of BAI strain A (myeloblastosis) avian tumor virus. Cancer Res. 1967 Jun;27(6):1138–1157. [PubMed] [Google Scholar]
  15. CRAWFORD L. V., CRAWFORD E. M. The properties of Rous sarcoma virus purified by density gradient centrifugation. Virology. 1961 Feb;13:227–232. doi: 10.1016/0042-6822(61)90056-3. [DOI] [PubMed] [Google Scholar]
  16. Duesberg P. H., Canaani E. Complementarity between Rous sarcoma virus (RSV) RNA and the in vitro-synthesized DNA of the virus-associated DNA polymerase. Virology. 1970 Nov;42(3):783–788. doi: 10.1016/0042-6822(70)90325-9. [DOI] [PubMed] [Google Scholar]
  17. Duesberg P. H., Cardiff R. D. Structural relationships between the RNA of mammary tumor virus and those of other RNA tumor viruses. Virology. 1968 Dec;36(4):696–700. doi: 10.1016/0042-6822(68)90206-7. [DOI] [PubMed] [Google Scholar]
  18. Duesberg P. H. On the structure of RNA tumor viruses. Curr Top Microbiol Immunol. 1970;51:78–104. [PubMed] [Google Scholar]
  19. Duesberg P. H. Physical properties of Rous Sarcoma Virus RNA. Proc Natl Acad Sci U S A. 1968 Aug;60(4):1511–1518. doi: 10.1073/pnas.60.4.1511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Duesberg P. H., Vogt P. K. Differences between the ribonucleic acids of transforming and nontransforming avian tumor viruses. Proc Natl Acad Sci U S A. 1970 Dec;67(4):1673–1680. doi: 10.1073/pnas.67.4.1673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Erikson R. L. Studies on the RNA from avian myeloblastosis virus. Virology. 1969 Jan;37(1):124–131. doi: 10.1016/0042-6822(69)90313-4. [DOI] [PubMed] [Google Scholar]
  22. Fanshier L., Garapin A. C., McDonnell J., Faras A., Levinson W., Bishop J. M. Deoxyribonucleic acid polymerase associated with avian tumor viruses: secondary structure of the deoxyribonucleic acid product. J Virol. 1971 Jan;7(1):77–86. doi: 10.1128/jvi.7.1.77-86.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Faras A., Fanshier L., Garapin A. C., Levinson W., Bishop J. M. Deoxyribonucleic acid polymerase of Rous sarcoma virus: studies on the mechanism of double-stranded deoxyribonucleic acid synthesis. J Virol. 1971 May;7(5):539–548. doi: 10.1128/jvi.7.5.539-548.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Fujinaga K., Parsons J. T., Beard J. W., Beard D., Green M. Mechanism of carcinogenesis by RNA tumor viruses. 3. Formation of RNA, DNA complex and duplex DNA molecules by the DNA polymerase (s) of avian myeloblastosis virus. Proc Natl Acad Sci U S A. 1970 Nov;67(3):1432–1439. doi: 10.1073/pnas.67.3.1432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Garapin A. C., Fanshier L., Leong J. A., Jackson J., Levinson W., Bishop J. M. Deoxyribonucleic acid polymerases of Rous sarcoma virus: kinetics of deoxyribonucleic acid synthesis and specificity of the products. J Virol. 1971 Feb;7(2):227–232. doi: 10.1128/jvi.7.2.227-232.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Garapin A. C., McDonnell J. P., Levinson W., Quintrell N., Fanshier L., Bishop J. M. Deoxyribonucleic acid polymerase associated with Rous sarcoma virus and avian myeloblastosis virus: properties of the enzyme and its product. J Virol. 1970 Nov;6(5):589–598. doi: 10.1128/jvi.6.5.589-598.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hanafusa H. Rapid transformation of cells by Rous sarcoma virus. Proc Natl Acad Sci U S A. 1969 Jun;63(2):318–325. doi: 10.1073/pnas.63.2.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kacian D. L., Watson K. F., Burny A., Spiegelman S. Purification of the DNA polymerase of avian myeloblastosis virus. Biochim Biophys Acta. 1971 Sep 24;246(3):365–383. doi: 10.1016/0005-2787(71)90773-8. [DOI] [PubMed] [Google Scholar]
  29. Manly K. F., Smoler D. F., Bromfeld E., Baltimore D. Forms of deoxyribonucleic acid produced by virions of the ribonucleic acid tumor viruses. J Virol. 1971 Jan;7(1):106–111. doi: 10.1128/jvi.7.1.106-111.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McDonnell J. P., Garapin A. C., Levinson W. E., Quintrell N., Fanshier L., Bishop J. M. DNA polymerases of Rous sarcoma virus: delineation of two reactions with actinomycin. Nature. 1970 Oct 31;228(5270):433–435. doi: 10.1038/228433a0. [DOI] [PubMed] [Google Scholar]
  31. Montagnier L., Goldé A., Vigier P. A possible subunit structure of Rous sarcoma virus RNA. J Gen Virol. 1969 Apr;4(3):449–452. doi: 10.1099/0022-1317-4-3-449. [DOI] [PubMed] [Google Scholar]
  32. Quintrell N., Fanshier L., Evans B., Levinson W., Bishop J. M. Deoxyribonucleic acid polymerase(s) of Rous sarcoma virus: effects of virion-associated endonuclease on the enzymatic product. J Virol. 1971 Jul;8(1):17–27. doi: 10.1128/jvi.8.1.17-27.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rokutanda M., Rokutanda H., Green M., Fujinaga K., Ray R. K., Gurgo C. Formation of viral RNA-DNA hybrid molecules by the DNA polymerase of sarcoma-leukaemia viruses. Nature. 1970 Sep 5;227(5262):1026–1028. doi: 10.1038/2271026a0. [DOI] [PubMed] [Google Scholar]
  34. Spiegelman S., Burny A., Das M. R., Keydar J., Schlom J., Travnicek M., Watson K. Characterization of the products of DNA-directed DNA polymerases in oncogenic RNA viruses. Nature. 1970 Aug 8;227(5258):563–567. doi: 10.1038/227563a0. [DOI] [PubMed] [Google Scholar]
  35. Spiegelman S., Burny A., Das M. R., Keydar J., Schlom J., Travnicek M., Watson K. DNA-directed DNA polymerase activity in oncogenic RNA viruses. Nature. 1970 Sep 5;227(5262):1029–1031. doi: 10.1038/2271029a0. [DOI] [PubMed] [Google Scholar]
  36. Symons R. H. Modified procedure for the synthesis of 32P-labelled ribonucleoside 5'-monophosphates of high specific activity. Biochim Biophys Acta. 1968 Feb 26;155(2):609–610. doi: 10.1016/0005-2787(68)90205-0. [DOI] [PubMed] [Google Scholar]
  37. Temin H. M., Mizutani S. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature. 1970 Jun 27;226(5252):1211–1213. doi: 10.1038/2261211a0. [DOI] [PubMed] [Google Scholar]
  38. Trávnícek M., Ríman J. Chromatographic differences between lysyl-tRNA's from avian tumor virus BAI strain A and virus transformed cells. Biochim Biophys Acta. 1970 Jan 21;199(1):283–285. [PubMed] [Google Scholar]
  39. Trávnícek M. Some properties of amino acid-acceptor RNA isolated from avian tumour virus BAI strain A (avian myeloblastosis). Biochim Biophys Acta. 1969 Jun 17;182(2):427–439. [PubMed] [Google Scholar]
  40. Wollmann R. L., Kirsten W. H. Cellular origin of a mouse leukemia viral ribonucleic acid. J Virol. 1968 Nov;2(11):1241–1248. doi: 10.1128/jvi.2.11.1241-1248.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]

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