Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1982 May;42(2):538–546. doi: 10.1128/jvi.42.2.538-546.1982

Demonstration of biological activity and nucleotide sequence of an in vitro synthesized clone of the Moloney murine sarcoma virus mos gene.

D J Donoghue
PMCID: PMC256880  PMID: 7045395

Abstract

A clone of the Moloney murine sarcoma virus mos gene derived by in vitro reverse transcription was characterized. When assayed for focus formation by DNA transfection on NIH/3T3 cells, this clone was biologically inactive, presumably due to the absence of a long terminal repeat sequence. Therefore, a long terminal repeat was inserted into the clone by in vitro recombination, after which the most gene was able to transform NIH/3T3 cells efficiently. The nucleotide sequence encompassing the transforming region of this clone was determined. A single long open reading frame was observed, which potentially encodes a polypeptide of 41,000 daltons. This open reading frame initiates with the first five amino acids of the murine leukemia virus env gene, after which it enters the mos sequence, where it terminates. The nucleotide sequence described in this paper was compared with other sequences of mos in an effort to resolve discrepancies in the position of the long open reading frame. Although Moloney murine sarcoma virus retains the 3' splicing site of the murine leukemia virus env gene, a mos-specific mRNA which corresponds structurally to the murine leukemia virus env mRNA was not identified. The sequence described here revealed a single nucleotide change in the proposed env gene 3' splicing site which was retained in Moloney murine sarcoma virus. This deviation from the consensus 3' splicing sequence may underlie the observed absence of mos expression via the env gene splicing pathway.

Full text

PDF
538

Images in this article

539Image
on p.539
539Image
on p.539
542Image
on p.542
543Image
on p.543
544Image
on p.544

Selected References

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

  1. Andersson P., Goldfarb M. P., Weinberg R. A. A defined subgenomic fragment of in vitro synthesized Moloney sarcoma virus DNA can induce cell transformation upon transfection. Cell. 1979 Jan;16(1):63–75. doi: 10.1016/0092-8674(79)90188-0. [DOI] [PubMed] [Google Scholar]
  2. Ball J., McCarter J. A., Sunderland S. M. Evidence for helper independent murine sarcoma virus. I. Segregation of replication-defective and transformation-defective viruses. Virology. 1973 Nov;56(1):268–284. doi: 10.1016/0042-6822(73)90305-x. [DOI] [PubMed] [Google Scholar]
  3. Barbacid M., Lauver A. V., Devare S. G. Biochemical and immunological characterization of polyproteins coded for by the McDonough, Gardner-Arnstein, and Snyder-Theilen strains of feline sarcoma virus. J Virol. 1980 Jan;33(1):196–207. doi: 10.1128/jvi.33.1.196-207.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  5. Blair D. G., McClements W. L., Oskarsson M. K., Fischinger P. J., Vande Woude G. F. Biological activity of cloned Moloney sarcoma virus DNA: Terminally redundant sequences may enhance transformation efficiency. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3504–3508. doi: 10.1073/pnas.77.6.3504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blair D. G., Oskarsson M., Wood T. G., McClements W. L., Fischinger P. J., Vande Woude G. G. Activation of the transforming potential of a normal cell sequence: a molecular model for oncogenesis. Science. 1981 May 22;212(4497):941–943. doi: 10.1126/science.7233190. [DOI] [PubMed] [Google Scholar]
  7. Brown N. L., Smith M. The sequence of a region of bacteriophage phiX174 DNA coding for parts of genes A and B. J Mol Biol. 1977 Oct 15;116(1):1–28. doi: 10.1016/0022-2836(77)90115-2. [DOI] [PubMed] [Google Scholar]
  8. Canaani E., Robbins K. C., Aaronson S. A. The transforming gene of Moloney murine sarcoma virus. Nature. 1979 Nov 22;282(5737):378–383. doi: 10.1038/282378a0. [DOI] [PubMed] [Google Scholar]
  9. Chang E. H., Maryak J. M., Wei C. M., Shih T. Y., Shober R., Cheung H. L., Ellis R. W., Hager G. L., Scolnick E. M., Lowy D. R. Functional organization of the Harvey murine sarcoma virus genome. J Virol. 1980 Jul;35(1):76–92. doi: 10.1128/jvi.35.1.76-92.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cremer K., Reddy E. P., Aaronson S. A. Translational products of Moloney murine sarcoma virus RNA: identification of proteins encoded by the murine sarcoma virus src gene. J Virol. 1981 May;38(2):704–711. doi: 10.1128/jvi.38.2.704-711.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DeFeo D., Gonda M. A., Young H. A., Chang E. H., Lowy D. R., Scolnick E. M., Ellis R. W. Analysis of two divergent rat genomic clones homologous to the transforming gene of Harvey murine sarcoma virus. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3328–3332. doi: 10.1073/pnas.78.6.3328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dina D., Beemon K. Relationship between Moloney murine leukemia and sarcoma virus RNAs: purification and hybridization map of complementary DNAs from defined regions of Moloney murine sarcoma virus 124. J Virol. 1977 Sep;23(3):524–532. doi: 10.1128/jvi.23.3.524-532.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Donoghue D. J., Hunter T. Expression of transforming region of Moloney murine sarcoma virus in Escherichia coli as a fusion protein with small tumor antigen of polyoma virus. Proc Natl Acad Sci U S A. 1982 Feb;79(3):800–804. doi: 10.1073/pnas.79.3.800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Donoghue D. J., Sharp P. A., Weinberg R. A. An MSV-specific subgenomic mRNA in MSV-transformed G8-124 cells. Cell. 1979 May;17(1):53–63. doi: 10.1016/0092-8674(79)90294-0. [DOI] [PubMed] [Google Scholar]
  15. Donoghue D. J., Sharp P. A., Weinberg R. A. Comparative study of different isolates of murine sarcoma virus. J Virol. 1979 Dec;32(3):1015–1027. doi: 10.1128/jvi.32.3.1015-1027.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ferretti L., Sgaramella V. Temperature dependence of the joining by T4 DNA ligase of termini produced by type II restriction endonucleases. Nucleic Acids Res. 1981 Jan 10;9(1):85–93. doi: 10.1093/nar/9.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fiddes J. C. Nucleotide sequence of the intercistronic region between genes G and F in bacteriophage phiX174 DNA. J Mol Biol. 1976 Oct 15;107(1):1–24. doi: 10.1016/s0022-2836(76)80014-9. [DOI] [PubMed] [Google Scholar]
  18. Frankel A. E., Fischinger P. J. Nucleotide sequences in mouse DNA and RNA specific for Moloney sarcoma virus. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3705–3709. doi: 10.1073/pnas.73.10.3705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Goff S. P., Gilboa E., Witte O. N., Baltimore D. Structure of the Abelson murine leukemia virus genome and the homologous cellular gene: studies with cloned viral DNA. Cell. 1980 Dec;22(3):777–785. doi: 10.1016/0092-8674(80)90554-1. [DOI] [PubMed] [Google Scholar]
  20. Gopinathan K. P., Weymouth L. A., Kunkel T. A., Loeb L. A. Mutagenesis in vitro by DNA polymerase from an RNA tumour virus. Nature. 1979 Apr 26;278(5707):857–859. doi: 10.1038/278857a0. [DOI] [PubMed] [Google Scholar]
  21. Hu S., Davidson N. A heteroduplex study of the sequence relationships between the RNAs of M-MSV and M-MLV. Cell. 1977 Mar;10(3):469–477. doi: 10.1016/0092-8674(77)90034-4. [DOI] [PubMed] [Google Scholar]
  22. Kawai S., Yoshida M., Segawa K., Sugiyama H., Ishizaki R., Toyoshima K. Characterization of Y73, an avian sarcoma virus: a unique transforming gene and its product, a phosphopolyprotein with protein kinase activity. Proc Natl Acad Sci U S A. 1980 Oct;77(10):6199–6203. doi: 10.1073/pnas.77.10.6199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maat J., Smith A. J. A method for sequencing restriction fragments with dideoxynucleoside triphosphates. Nucleic Acids Res. 1978 Dec;5(12):4537–4545. doi: 10.1093/nar/5.12.4537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  25. Moloney J. B. A virus-induced rhabdomyosarcoma of mice. Natl Cancer Inst Monogr. 1966 Sep;22:139–142. [PubMed] [Google Scholar]
  26. Morrison D. A. Transformation and preservation of competent bacterial cells by freezing. Methods Enzymol. 1979;68:326–331. doi: 10.1016/0076-6879(79)68023-0. [DOI] [PubMed] [Google Scholar]
  27. Neil J. C., Breitman M. L., Vogt P. K. Characterization of a 105,000 molecular weight gag-related phosphoprotein from cells transformed by the defective avian sarcoma virus PRCII. Virology. 1981 Jan 15;108(1):98–110. doi: 10.1016/0042-6822(81)90530-4. [DOI] [PubMed] [Google Scholar]
  28. Oskarsson M., McClements W. L., Blair D. G., Maizel J. V., Vande Woude G. F. Properties of a normal mouse cell DNA sequence (sarc) homologous to the src sequence of Moloney sarcoma virus. Science. 1980 Mar 14;207(4436):1222–1224. doi: 10.1126/science.6243788. [DOI] [PubMed] [Google Scholar]
  29. Papkoff J., Hunter T., Beemon K. In vitro translation of virion RNA from Moloney murine sarcoma virus. Virology. 1980 Feb;101(1):91–103. doi: 10.1016/0042-6822(80)90486-9. [DOI] [PubMed] [Google Scholar]
  30. Papkoff J., Lai M. H., Hunter T., Verma I. M. Analysis of transforming gene products from Moloney murine sarcoma virus. Cell. 1981 Nov;27(1 Pt 2):109–119. doi: 10.1016/0092-8674(81)90365-2. [DOI] [PubMed] [Google Scholar]
  31. Reddy E. P., Smith M. J., Aaronson S. A. Complete nucleotide sequence and organization of the Moloney murine sarcoma virus genome. Science. 1981 Oct 23;214(4519):445–450. doi: 10.1126/science.6170110. [DOI] [PubMed] [Google Scholar]
  32. Rothenberg E., Donoghue D. J., Baltimore D. Analysis of a 5' leader sequence on murine leukemia virus 21S RNA: heteroduplex mapping with long reverse transcriptase products. Cell. 1978 Mar;13(3):435–451. doi: 10.1016/0092-8674(78)90318-5. [DOI] [PubMed] [Google Scholar]
  33. Sanger F., Coulson A. R. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol. 1975 May 25;94(3):441–448. doi: 10.1016/0022-2836(75)90213-2. [DOI] [PubMed] [Google Scholar]
  34. Scolnick E. M., Howk R. S., Anisowicz A., Peebles P. T., Scher C. D., Parks W. P. Separation of sarcoma virus-specific and leukemia virus-specific genetic sequences of Moloney sarcoma virus. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4650–4654. doi: 10.1073/pnas.72.11.4650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shalloway D., Zelenetz A. D., Cooper G. M. Molecular cloning and characterization of the chicken gene homologous to the transforming gene of Rous sarcoma virus. Cell. 1981 May;24(2):531–541. doi: 10.1016/0092-8674(81)90344-5. [DOI] [PubMed] [Google Scholar]
  36. Sharp P. A. Speculations on RNA splicing. Cell. 1981 Mar;23(3):643–646. doi: 10.1016/0092-8674(81)90425-6. [DOI] [PubMed] [Google Scholar]
  37. Shinnick T. M., Lerner R. A., Sutcliffe J. G. Nucleotide sequence of Moloney murine leukaemia virus. Nature. 1981 Oct 15;293(5833):543–548. doi: 10.1038/293543a0. [DOI] [PubMed] [Google Scholar]
  38. Thomas M., Cameron J. R., Davis R. W. Viable molecular hybrids of bacteriophage lambda and eukaryotic DNA. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4579–4583. doi: 10.1073/pnas.71.11.4579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tilghman S. M., Tiemeier D. C., Polsky F., Edgell M. H., Seidman J. G., Leder A., Enquist L. W., Norman B., Leder P. Cloning specific segments of the mammalian genome: bacteriophage lambda containing mouse globin and surrounding gene sequences. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4406–4410. doi: 10.1073/pnas.74.10.4406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tronick S. R., Robbins K. C., Canaani E., Devare S. G., Andersen P. R., Aaronson S. A. Molecular cloning of Moloney murine sarcoma virus: arrangement of virus-related sequences within the normal mouse genome. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6314–6318. doi: 10.1073/pnas.76.12.6314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Van Beveren C., Galleshaw J. A., Jonas V., Berns A. J., Doolittle R. F., Donoghue D. J., Verma I. M. Nucleotide sequence and formation of the transforming gene of a mouse sarcoma virus. Nature. 1981 Jan 22;289(5795):258–262. doi: 10.1038/289258a0. [DOI] [PubMed] [Google Scholar]
  42. Vande Woude G. F., Oskarsson M., Enquist L. W., Nomura S., Sullivan M., Fischinger P. J. Cloning of integrated Moloney sarcoma proviral DNA sequences in bacteriophage lambda. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4464–4468. doi: 10.1073/pnas.76.9.4464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Verma I. M., Lai M. H., Bosselman R. A., McKennett M. A., Fan H., Berns A. Molecular cloning of unintegrated Moloney mouse sarcoma virus DNA in bacteriophage lambda. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1773–1777. doi: 10.1073/pnas.77.4.1773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Vogelstein B., Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A. 1979 Feb;76(2):615–619. doi: 10.1073/pnas.76.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Witte O. N., Rosenberg N., Paskind M., Shields A., Baltimore D. Identification of an Abelson murine leukemia virus-encoded protein present in transformed fibroblast and lymphoid cells. Proc Natl Acad Sci U S A. 1978 May;75(5):2488–2492. doi: 10.1073/pnas.75.5.2488. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES