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. 1982 Jul;43(1):294–304. doi: 10.1128/jvi.43.1.294-304.1982

Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family.

M E Furth, L J Davis, B Fleurdelys, E M Scolnick
PMCID: PMC256120  PMID: 6287003

Abstract

We have isolated eight rat lymphocyte-myeloma hybrid cell lines producing monoclonal antibodies that react with the 21,000-dalton transforming protein (p21) encoded by the v-ras gene of Harvey murine sarcoma virus (Ha-MuSV). These antibodies specifically immunoprecipitate both phosphorylated and non-phosphorylated forms of p21 from lysates of cells transformed by Ha-MuSV. All eight react with the products of closely related ras genes expressed in cells transformed by two additional sarcoma viruses (rat sarcoma virus and BALB sarcoma virus) or by a cellular Harvey-ras gene placed under the control of a viral promoter. Three of the antibodies also react strongly with the p21 encoded by the v-ras gene of Kirsten MuSV. These same three antibodies immunoprecipitate the predominant p21 species synthesized normally in a variety of rodent cell lines, including the p21 produced at high levels in 416B murine hemopoietic cells. This suggests that an endogenous gene closely related to Kirsten-ras is expressed in these cells. The monoclonal antibodies have been used to confirm two properties associated with p21; localization at the inner surface of the membrane of Ha-MuSV-transformed cells, assayed by immunofluorescence microscopy, and binding of guanine nucleotides.

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Selected References

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

  1. Aaronson S. A., Barbacid M. Origin and biological properties of a new BALB/c mouse sarcoma virus. J Virol. 1978 Aug;27(2):366–373. doi: 10.1128/jvi.27.2.366-373.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andersen P. R., Devare S. G., Tronick S. R., Ellis R. W., Aaronson S. A., Scolnick E. M. Generation of BALB-MuSV and Ha-MuSC by type C virus transduction of homologous transforming genes from different species. Cell. 1981 Oct;26(1 Pt 1):129–134. doi: 10.1016/0092-8674(81)90041-6. [DOI] [PubMed] [Google Scholar]
  3. BLACK P. H., ROWE W. P., TURNER H. C., HUEBNER R. J. A SPECIFIC COMPLEMENT-FIXING ANTIGEN PRESENT IN SV40 TUMOR AND TRANSFORMED CELLS. Proc Natl Acad Sci U S A. 1963 Dec;50:1148–1156. doi: 10.1073/pnas.50.6.1148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Brugge J. S., Erikson R. L. Identification of a transformation-specific antigen induced by an avian sarcoma virus. Nature. 1977 Sep 22;269(5626):346–348. doi: 10.1038/269346a0. [DOI] [PubMed] [Google Scholar]
  6. Coffin J. M., Varmus H. E., Bishop J. M., Essex M., Hardy W. D., Jr, Martin G. S., Rosenberg N. E., Scolnick E. M., Weinberg R. A., Vogt P. K. Proposal for naming host cell-derived inserts in retrovirus genomes. J Virol. 1981 Dec;40(3):953–957. doi: 10.1128/jvi.40.3.953-957.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Collett M. S., Brugge J. S., Erikson R. L. Characterization of a normal avian cell protein related to the avian sarcoma virus transforming gene product. Cell. 1978 Dec;15(4):1363–1369. doi: 10.1016/0092-8674(78)90061-2. [DOI] [PubMed] [Google Scholar]
  8. Collett M. S., Erikson E., Purchio A. F., Brugge J. S., Erikson R. L. A normal cell protein similar in structure and function to the avian sarcoma virus transforming gene product. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3159–3163. doi: 10.1073/pnas.76.7.3159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Dexter T. M., Allen T. D., Scott D., Teich N. M. Isolation and characterisation of a bipotential haematopoietic cell line. Nature. 1979 Feb 8;277(5696):471–474. doi: 10.1038/277471a0. [DOI] [PubMed] [Google Scholar]
  11. Ellis R. W., DeFeo D., Maryak J. M., Young H. A., Shih T. Y., Chang E. H., Lowy D. R., Scolnick E. M. Dual evolutionary origin for the rat genetic sequences of Harvey murine sarcoma virus. J Virol. 1980 Nov;36(2):408–420. doi: 10.1128/jvi.36.2.408-420.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ellis R. W., Defeo D., Shih T. Y., Gonda M. A., Young H. A., Tsuchida N., Lowy D. R., Scolnick E. M. The p21 src genes of Harvey and Kirsten sarcoma viruses originate from divergent members of a family of normal vertebrate genes. Nature. 1981 Aug 6;292(5823):506–511. doi: 10.1038/292506a0. [DOI] [PubMed] [Google Scholar]
  13. Galfrè G., Milstein C., Wright B. Rat x rat hybrid myelomas and a monoclonal anti-Fd portion of mouse IgG. Nature. 1979 Jan 11;277(5692):131–133. doi: 10.1038/277131a0. [DOI] [PubMed] [Google Scholar]
  14. HARVEY J. J. AN UNIDENTIFIED VIRUS WHICH CAUSES THE RAPID PRODUCTION OF TUMOURS IN MICE. Nature. 1964 Dec 12;204:1104–1105. doi: 10.1038/2041104b0. [DOI] [PubMed] [Google Scholar]
  15. Huang A. L., Ostrowski M. C., Berard D., Hager G. L. Glucocorticoid regulation of the Ha-MuSV p21 gene conferred by sequences from mouse mammary tumor virus. Cell. 1981 Dec;27(2 Pt 1):245–255. doi: 10.1016/0092-8674(81)90408-6. [DOI] [PubMed] [Google Scholar]
  16. Huu Duc-Nguyen, Rosenblum E. N., Zeigel R. F. Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus. J Bacteriol. 1966 Oct;92(4):1133–1140. doi: 10.1128/jb.92.4.1133-1140.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Iscove N. N., Melchers F. Complete replacement of serum by albumin, transferrin, and soybean lipid in cultures of lipopolysaccharide-reactive B lymphocytes. J Exp Med. 1978 Mar 1;147(3):923–933. doi: 10.1084/jem.147.3.923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jainchill J. L., Aaronson S. A., Todaro G. J. Murine sarcoma and leukemia viruses: assay using clonal lines of contact-inhibited mouse cells. J Virol. 1969 Nov;4(5):549–553. doi: 10.1128/jvi.4.5.549-553.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  20. Khan A. S., Stephenson J. R. Feline leukemia virus: biochemical and immunological characterization of gag gene-coded structural proteins. J Virol. 1977 Sep;23(3):599–607. doi: 10.1128/jvi.23.3.599-607.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  22. Köhler G., Milstein C. Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur J Immunol. 1976 Jul;6(7):511–519. doi: 10.1002/eji.1830060713. [DOI] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Langbeheim H., Shih T. Y., Scolnick E. M. Identification of a normal vertebrate cell protein related to the p21 src of Harvey murine sarcoma virus. Virology. 1980 Oct 30;106(2):292–300. doi: 10.1016/0042-6822(80)90252-4. [DOI] [PubMed] [Google Scholar]
  25. Oppermann H., Levinson A. D., Varmus H. E., Levintow L., Bishop J. M. Uninfected vertebrate cells contain a protein that is closely related to the product of the avian sarcoma virus transforming gene (src). Proc Natl Acad Sci U S A. 1979 Apr;76(4):1804–1808. doi: 10.1073/pnas.76.4.1804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Peters R. L., Rabstein L. S., VanVleck R., Kelloff G. J., Huebner R. J. Naturally occurring sarcoma virus of the BALB/cCr mouse. J Natl Cancer Inst. 1974 Dec;53(6):1725–1729. [PubMed] [Google Scholar]
  27. Rasheed S., Gardner M. B., Huebner R. J. In vitro isolation of stable rat sarcoma viruses. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2972–2976. doi: 10.1073/pnas.75.6.2972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rohrschneider L. R., Eisenman R. N., Leitch C. R. Identification of a Rous sarcoma virus transformation-related protein in normal avian and mammalian cells. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4479–4483. doi: 10.1073/pnas.76.9.4479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ruscetti S. K., Turek L. P., Sherr C. J. Three independent isolates of feline sarcoma virus code for three distinct gag-x polyproteins. J Virol. 1980 Jul;35(1):259–264. doi: 10.1128/jvi.35.1.259-264.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Scolnick E. M., Papageorge A. G., Shih T. Y. Guanine nucleotide-binding activity as an assay for src protein of rat-derived murine sarcoma viruses. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5355–5359. doi: 10.1073/pnas.76.10.5355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Scolnick E. M., Shih T. Y., Maryak J., Ellis R., Chang E., Lowy D. Guanine nucleotide binding activity of the src gene product of rat-derived murine sarcoma viruses. Ann N Y Acad Sci. 1980;354:398–409. doi: 10.1111/j.1749-6632.1980.tb27981.x. [DOI] [PubMed] [Google Scholar]
  32. Scolnick E. M., Weeks M. O., Shih T. Y., Ruscetti S. K., Dexter T. M. Markedly elevated levels of an endogenous sarc protein in a hemopoietic precursor cell line. Mol Cell Biol. 1981 Jan;1(1):66–74. doi: 10.1128/mcb.1.1.66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Scolnick E. M., Williams D., Maryak J., Vass W., Goldberg R. J., Parks W. P. Type C particle-positive and type C particle-negative rat cell lines: characterization of the coding capacity of endogenous sarcoma virus-specific RNA. J Virol. 1976 Dec;20(3):570–582. doi: 10.1128/jvi.20.3.570-582.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Shih T. Y., Papageorge A. G., Stokes P. E., Weeks M. O., Scolnick E. M. Guanine nucleotide-binding and autophosphorylating activities associated with the p21src protein of Harvey murine sarcoma virus. Nature. 1980 Oct 23;287(5784):686–691. doi: 10.1038/287686a0. [DOI] [PubMed] [Google Scholar]
  35. Shih T. Y., Weeks M. O., Young H. A., Scholnick E. M. Identification of a sarcoma virus-coded phosphoprotein in nonproducer cells transformed by Kirsten or Harvey murine sarcoma virus. Virology. 1979 Jul 15;96(1):64–79. doi: 10.1016/0042-6822(79)90173-9. [DOI] [PubMed] [Google Scholar]
  36. Shih T. Y., Weeks M. O., Young H. A., Scolnick E. M. p21 of Kirsten murine sarcoma virus is thermolabile in a viral mutant temperature sensitive for the maintenance of transformation. J Virol. 1979 Aug;31(2):546–546. doi: 10.1128/jvi.31.2.546-546.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Willingham M. C., Pastan I., Shih T. Y., Scolnick E. M. Localization of the src gene product of the Harvey strain of MSV to plasma membrane of transformed cells by electron microscopic immunocytochemistry. Cell. 1980 Apr;19(4):1005–1014. doi: 10.1016/0092-8674(80)90091-4. [DOI] [PubMed] [Google Scholar]
  38. Witte O. N., Rosenberg N. E., Baltimore D. A normal cell protein cross-reactive to the major Abelson murine leukaemia virus gene product. Nature. 1979 Oct 4;281(5730):396–398. doi: 10.1038/281396a0. [DOI] [PubMed] [Google Scholar]
  39. Young H. A., Rasheed S., Sowder R., Benton C. V., Henderson L. E. Rat sarcoma virus: further analysis of individual viral isolates and the gene product. J Virol. 1981 Apr;38(1):286–293. doi: 10.1128/jvi.38.1.286-293.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Young H. A., Shih T. Y., Scolnick E. M., Rasheed S., Gardner M. B. Different rat-derived transforming retroviruses code for an immunologically related intracellular phosphoprotein. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3523–3527. doi: 10.1073/pnas.76.7.3523. [DOI] [PMC free article] [PubMed] [Google Scholar]

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