Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1985 Nov;5(11):3131–3138. doi: 10.1128/mcb.5.11.3131

Biologically active mutants with deletions in the v-mos oncogene assayed with retroviral vectors.

R J Bold, D J Donoghue
PMCID: PMC369128  PMID: 3018503

Abstract

We have constructed retroviral expression vectors by manipulation of the Moloney murine leukemia virus genome such that an exogenous DNA sequence may be inserted and subsequently expressed when introduced into mammalian cells. A series of N-terminal deletions of the v-mos oncogene was constructed and assayed for biological activity with these retroviral expression vectors. The results of the deletion analysis demonstrate that the region of p37mos coding region upstream of the third methionine codon is dispensable with respect to transformation. However, deletion mutants of v-mos which allow initiation of translation at the fourth methionine codon have lost the biological activity of the parental v-mos gene. Furthermore, experiments were also carried out to define the C-terminal limit of the active region of p37mos by the construction of premature termination mutants by the insertion of a termination oligonucleotide. Insertion of the oligonucleotide just 69 base pairs upstream from the wild-type termination site abolished the focus-forming ability of v-mos. Thus, we have shown the N-terminal limit of the active region of p37mos to be between the third and fourth methionines, while the C-terminal limit is within the last 23 amino acids of the protein.

Full text

PDF
3133

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. Bandyopadhyay P. K., Temin H. M. Expression of complete chicken thymidine kinase gene inserted in a retrovirus vector. Mol Cell Biol. 1984 Apr;4(4):749–754. doi: 10.1128/mcb.4.4.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barker W. C., Dayhoff M. O. Viral src gene products are related to the catalytic chain of mammalian cAMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1982 May;79(9):2836–2839. doi: 10.1073/pnas.79.9.2836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Brow M. A., Sen A., Sutcliffe J. G. Nucleotide sequence of the transforming gene of m1 murine sarcoma virus. J Virol. 1984 Feb;49(2):579–582. doi: 10.1128/jvi.49.2.579-582.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Canaani E., Dreazen O., Klar A., Rechavi G., Ram D., Cohen J. B., Givol D. Activation of the c-mos oncogene in a mouse plasmacytoma by insertion of an endogenous intracisternal A-particle genome. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7118–7122. doi: 10.1073/pnas.80.23.7118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cepko C. L., Roberts B. E., Mulligan R. C. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell. 1984 Jul;37(3):1053–1062. doi: 10.1016/0092-8674(84)90440-9. [DOI] [PubMed] [Google Scholar]
  9. Cohen J. B., Unger T., Rechavi G., Canaani E., Givol D. Rearrangement of the oncogene c-mos in mouse myeloma NSI and hybridomas. Nature. 1983 Dec 22;306(5945):797–799. doi: 10.1038/306797a0. [DOI] [PubMed] [Google Scholar]
  10. Cone R. D., Mulligan R. C. High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6349–6353. doi: 10.1073/pnas.81.20.6349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Donoghue D. J. Demonstration of biological activity and nucleotide sequence of an in vitro synthesized clone of the Moloney murine sarcoma virus mos gene. J Virol. 1982 May;42(2):538–546. doi: 10.1128/jvi.42.2.538-546.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Donoghue D. J., Hunter T. Recombinational junctions of variants of Moloney murine sarcoma virus: generation and divergence of a mammalian transforming gene. J Virol. 1983 Feb;45(2):607–617. doi: 10.1128/jvi.45.2.607-617.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Gattoni-Celli S., Hsiao W. L., Weinstein I. B. Rearranged c-mos locus in a MOPC 21 murine myeloma cell line and its persistence in hybridomas. Nature. 1983 Dec 22;306(5945):795–796. doi: 10.1038/306795a0. [DOI] [PubMed] [Google Scholar]
  17. Gilboa E., Kolbe M., Noonan K., Kucherlapati R. Construction of a mammalian transducing vector from the genome of Moloney murine leukemia virus. J Virol. 1982 Dec;44(3):845–851. doi: 10.1128/jvi.44.3.845-851.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  19. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hannink M., Donoghue D. J. Requirement for a signal sequence in biological expression of the v-sis oncogene. Science. 1984 Dec 7;226(4679):1197–1199. doi: 10.1126/science.6095451. [DOI] [PubMed] [Google Scholar]
  21. Hoffmann J. W., Steffen D., Gusella J., Tabin C., Bird S., Cowing D., Weinberg R. A. DNA methylation affecting the expression of murine leukemia proviruses. J Virol. 1982 Oct;44(1):144–157. doi: 10.1128/jvi.44.1.144-157.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Hwang L. H., Gilboa E. Expression of genes introduced into cells by retroviral infection is more efficient than that of genes introduced into cells by DNA transfection. J Virol. 1984 May;50(2):417–424. doi: 10.1128/jvi.50.2.417-424.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hwang L. S., Park J., Gilboa E. Role of intron-contained sequences in formation of moloney murine leukemia virus env mRNA. Mol Cell Biol. 1984 Nov;4(11):2289–2297. doi: 10.1128/mcb.4.11.2289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Joyner A. L., Bernstein A. Retrovirus transduction: generation of infectious retroviruses expressing dominant and selectable genes is associated with in vivo recombination and deletion events. Mol Cell Biol. 1983 Dec;3(12):2180–2190. doi: 10.1128/mcb.3.12.2180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kloetzer W. S., Maxwell S. A., Arlinghaus R. B. P85gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity. Proc Natl Acad Sci U S A. 1983 Jan;80(2):412–416. doi: 10.1073/pnas.80.2.412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Legerski R. J., Hodnett J. L., Gray H. B., Jr Extracellular nucleases of pseudomonas BAL 31. III. Use of the double-strand deoxyriboexonuclease activity as the basis of a convenient method for the mapping of fragments of DNA produced by cleavage with restriction enzymes. Nucleic Acids Res. 1978 May;5(5):1445–1464. doi: 10.1093/nar/5.5.1445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Miller A. D., Jolly D. J., Friedmann T., Verma I. M. A transmissible retrovirus expressing human hypoxanthine phosphoribosyltransferase (HPRT): gene transfer into cells obtained from humans deficient in HPRT. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4709–4713. doi: 10.1073/pnas.80.15.4709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Moloney J. B. A virus-induced rhabdomyosarcoma of mice. Natl Cancer Inst Monogr. 1966 Sep;22:139–142. [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Papkoff J., Nigg E. A., Hunter T. The transforming protein of Moloney murine sarcoma virus is a soluble cytoplasmic protein. Cell. 1983 May;33(1):161–172. doi: 10.1016/0092-8674(83)90345-8. [DOI] [PubMed] [Google Scholar]
  35. Papkoff J., Verma I. M., Hunter T. Detection of a transforming gene product in cells transformed by Moloney murine sarcoma virus. Cell. 1982 Jun;29(2):417–426. doi: 10.1016/0092-8674(82)90158-1. [DOI] [PubMed] [Google Scholar]
  36. Perkins A. S., Kirschmeier P. T., Gattoni-Celli S., Weinstein I. B. Design of a retrovirus-derived vector for expression and transduction of exogenous genes in mammalian cells. Mol Cell Biol. 1983 Jun;3(6):1123–1132. doi: 10.1128/mcb.3.6.1123. [DOI] [PMC free article] [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. Shtivelman E., Zakut R., Canaani E. Frequent generation of nonrescuable reorganized Moloney murine sarcoma viral genomes. Proc Natl Acad Sci U S A. 1984 Jan;81(2):294–298. doi: 10.1073/pnas.81.2.294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sorge J., Wright D., Erdman V. D., Cutting A. E. Amphotropic retrovirus vector system for human cell gene transfer. Mol Cell Biol. 1984 Sep;4(9):1730–1737. doi: 10.1128/mcb.4.9.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stacey A., Arbuthnott C., Kollek R., Coggins L., Ostertag W. Comparison of myeloproliferative sarcoma virus with Moloney murine sarcoma virus variants by nucleotide sequencing and heteroduplex analysis. J Virol. 1984 Jun;50(3):725–732. doi: 10.1128/jvi.50.3.725-732.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tabin C. J., Hoffmann J. W., Goff S. P., Weinberg R. A. Adaptation of a retrovirus as a eucaryotic vector transmitting the herpes simplex virus thymidine kinase gene. Mol Cell Biol. 1982 Apr;2(4):426–436. doi: 10.1128/mcb.2.4.426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Temin H. M. Function of the retrovirus long terminal repeat. Cell. 1982 Jan;28(1):3–5. doi: 10.1016/0092-8674(82)90367-1. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Van Beveren C., van Straaten F., Galleshaw J. A., Verma I. M. Nucleotide sequence of the genome of a murine sarcoma virus. Cell. 1981 Nov;27(1 Pt 2):97–108. doi: 10.1016/0092-8674(81)90364-0. [DOI] [PubMed] [Google Scholar]
  45. Vande Woude G. F., Oskarsson M., McClements W. L., Enquist L. W., Blair D. G., Fischinger P. J., Maizel J. V., Sullivan M. Characterization of integrated Moloney Sarcoma proviruses and flanking host sequences cloned in bacteriophage lambda. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 2):735–745. doi: 10.1101/sqb.1980.044.01.079. [DOI] [PubMed] [Google Scholar]
  46. Williams D. A., Lemischka I. R., Nathan D. G., Mulligan R. C. Introduction of new genetic material into pluripotent haematopoietic stem cells of the mouse. Nature. 1984 Aug 9;310(5977):476–480. doi: 10.1038/310476a0. [DOI] [PubMed] [Google Scholar]
  47. Wood T. G., McGeady M. L., Blair D. G., Vande Woude G. F. Long terminal repeat enhancement of v-mos transforming activity: identification of essential regions. J Virol. 1983 Jun;46(3):726–736. doi: 10.1128/jvi.46.3.726-736.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES