Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1987 Nov;84(22):8021–8025. doi: 10.1073/pnas.84.22.8021

Isolation of temperature-sensitive Abelson virus mutants by site-directed mutagenesis.

A Engelman 1, N Rosenberg 1
PMCID: PMC299468  PMID: 2825174

Abstract

Mutants of Abelson virus encoding temperature-sensitive protein-tyrosine kinase (EC 2.7.1.112) were created by site-directed mutagenesis using sequence information from temperature-sensitive mutants of the related v-src oncogene. Expression of these two independent mutations in Escherichia coli resulted in reduced phosphorylation of the mutant proteins at high temperature. Viruses containing one of the mutations induced conditional transformation of both NIH 3T3 and lymphoid cells when expressed in the context of a truncated transforming protein. These results underscore the functional homology between protein-tyrosine kinases and suggest that transfer of mutations within a related gene family may provide a rapid method to create mutants.

Full text

PDF
8023

Images in this article

Selected References

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

  1. Bargmann C. I., Hung M. C., Weinberg R. A. The neu oncogene encodes an epidermal growth factor receptor-related protein. Nature. 1986 Jan 16;319(6050):226–230. doi: 10.1038/319226a0. [DOI] [PubMed] [Google Scholar]
  2. Besmer P., Murphy J. E., George P. C., Qiu F. H., Bergold P. J., Lederman L., Snyder H. W., Jr, Brodeur D., Zuckerman E. E., Hardy W. D. A new acute transforming feline retrovirus and relationship of its oncogene v-kit with the protein kinase gene family. Nature. 1986 Apr 3;320(6061):415–421. doi: 10.1038/320415a0. [DOI] [PubMed] [Google Scholar]
  3. Beug H., Palmieri S., Freudenstein C., Zentgraf H., Graf T. Hormone-dependent terminal differentiation in vitro of chicken erythroleukemia cells transformed by ts mutants of avian erythroblastosis virus. Cell. 1982 Apr;28(4):907–919. doi: 10.1016/0092-8674(82)90070-8. [DOI] [PubMed] [Google Scholar]
  4. Bishop J. M. Cellular oncogenes and retroviruses. Annu Rev Biochem. 1983;52:301–354. doi: 10.1146/annurev.bi.52.070183.001505. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Dean M., Park M., Le Beau M. M., Robins T. S., Diaz M. O., Rowley J. D., Blair D. G., Vande Woude G. F. The human met oncogene is related to the tyrosine kinase oncogenes. 1985 Nov 28-Dec 4Nature. 318(6044):385–388. doi: 10.1038/318385a0. [DOI] [PubMed] [Google Scholar]
  7. Dorner A. J., Stoye J. P., Coffin J. M. Molecular basis of host range variation in avian retroviruses. J Virol. 1985 Jan;53(1):32–39. doi: 10.1128/jvi.53.1.32-39.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Drahos D., Szybalski W. Antitermination and termination functions of the cloned nutL, N, and tL1 modules of coliphage lambda. Gene. 1981 Dec;16(1-3):261–274. doi: 10.1016/0378-1119(81)90082-2. [DOI] [PubMed] [Google Scholar]
  9. Fincham V. J., Chiswell D. J., Wyke J. A. Mapping of nonconditional and conditional mutants in the src gene of Prague strain Rous sarcoma virus. Virology. 1982 Jan 15;116(1):72–83. doi: 10.1016/0042-6822(82)90404-4. [DOI] [PubMed] [Google Scholar]
  10. Fincham V. J., Wyke J. A. Localization of temperature-sensitive transformation mutations and back mutations in the Rous sarcoma virus src gene. J Virol. 1986 May;58(2):694–699. doi: 10.1128/jvi.58.2.694-699.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Goff S. P., Tabin C. J., Wang J. Y., Weinberg R., Baltimore D. Transfection of fibroblasts by cloned Abelson murine leukemia virus DNA and recovery of transmissible virus by recombination with helper virus. J Virol. 1982 Jan;41(1):271–285. doi: 10.1128/jvi.41.1.271-285.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hafen E., Basler K., Edstroem J. E., Rubin G. M. Sevenless, a cell-specific homeotic gene of Drosophila, encodes a putative transmembrane receptor with a tyrosine kinase domain. Science. 1987 Apr 3;236(4797):55–63. doi: 10.1126/science.2882603. [DOI] [PubMed] [Google Scholar]
  14. Kawai S., Hanafusa H. The effects of reciprocal changes in temperature on the transformed state of cells infected with a rous sarcoma virus mutant. Virology. 1971 Nov;46(2):470–479. doi: 10.1016/0042-6822(71)90047-x. [DOI] [PubMed] [Google Scholar]
  15. Kawakami T., Pennington C. Y., Robbins K. C. Isolation and oncogenic potential of a novel human src-like gene. Mol Cell Biol. 1986 Dec;6(12):4195–4201. doi: 10.1128/mcb.6.12.4195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kipreos E. T., Lee G. J., Wang J. Y. Isolation of temperature-sensitive tyrosine kinase mutants of v-abl oncogene by screening with antibodies for phosphotyrosine. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1345–1349. doi: 10.1073/pnas.84.5.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Latt S. A., Goff S. P., Tabin C. J., Paskind M., Wang J. Y., Baltimore D. Cloning and analysis of reverse transcript P160 genomes of Abelson murine leukemia virus. J Virol. 1983 Mar;45(3):1195–1199. doi: 10.1128/jvi.45.3.1195-1199.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Marth J. D., Peet R., Krebs E. G., Perlmutter R. M. A lymphocyte-specific protein-tyrosine kinase gene is rearranged and overexpressed in the murine T cell lymphoma LSTRA. Cell. 1985 Dec;43(2 Pt 1):393–404. doi: 10.1016/0092-8674(85)90169-2. [DOI] [PubMed] [Google Scholar]
  20. Martin-Zanca D., Hughes S. H., Barbacid M. A human oncogene formed by the fusion of truncated tropomyosin and protein tyrosine kinase sequences. 1986 Feb 27-Mar 5Nature. 319(6056):743–748. doi: 10.1038/319743a0. [DOI] [PubMed] [Google Scholar]
  21. Martin G. S. Rous sarcoma virus: a function required for the maintenance of the transformed state. Nature. 1970 Sep 5;227(5262):1021–1023. doi: 10.1038/2271021a0. [DOI] [PubMed] [Google Scholar]
  22. Mermer B., Malamy M., Coffin J. M. Rous sarcoma virus contains sequences which permit expression of the gag gene in Escherichia coli. Mol Cell Biol. 1983 Oct;3(10):1746–1758. doi: 10.1128/mcb.3.10.1746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Moscovici C., Moscovici M. G., Jimenez H., Lai M. M., Hayman M. J., Vogt P. K. Continuous tissue culture cell lines derived from chemically induced tumors of Japanese quail. Cell. 1977 May;11(1):95–103. doi: 10.1016/0092-8674(77)90320-8. [DOI] [PubMed] [Google Scholar]
  24. Moscovici M. G., Moscovici C. Isolation and characterization of a temperature-sensitive mutant of avian myeloblastosis virus. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1421–1425. doi: 10.1073/pnas.80.5.1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nishizawa M., Mayer B. J., Takeya T., Yamamoto T., Toyoshima K., Hanafusa H., Kawai S. Two independent mutations are required for temperature-sensitive cell transformation by a Rous sarcoma virus temperature-sensitive mutant. J Virol. 1985 Dec;56(3):743–749. doi: 10.1128/jvi.56.3.743-749.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pawson T., Guyden J., Kung T. H., Radke K., Gilmore T., Martin G. S. A strain of Fujinami sarcoma virus which is temperature-sensitive in protein phosphorylation and cellular transformation. Cell. 1980 Dec;22(3):767–775. doi: 10.1016/0092-8674(80)90553-x. [DOI] [PubMed] [Google Scholar]
  27. Prywes R., Foulkes J. G., Baltimore D. The minimum transforming region of v-abl is the segment encoding protein-tyrosine kinase. J Virol. 1985 Apr;54(1):114–122. doi: 10.1128/jvi.54.1.114-122.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Reddy E. P., Smith M. J., Srinivasan A. Nucleotide sequence of Abelson murine leukemia virus genome: structural similarity of its transforming gene product to other onc gene products with tyrosine-specific kinase activity. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3623–3627. doi: 10.1073/pnas.80.12.3623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Reed R. R. Transposon-mediated site-specific recombination: a defined in vitro system. Cell. 1981 Sep;25(3):713–719. doi: 10.1016/0092-8674(81)90178-1. [DOI] [PubMed] [Google Scholar]
  30. Reynolds F. H., Jr, Sacks T. L., Deobagkar D. N., Stephenson J. R. Cells nonproductively transformed by Abelson murine leukemia virus express a high molecular weight polyprotein containing structural and nonstructural components. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3974–3978. doi: 10.1073/pnas.75.8.3974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Reynolds F. H., Jr, Van de Ven W. J., Stephenson J. R. Abelson murine leukemia virus transformation-defective mutants with impaired P120-associated protein kinase activity. J Virol. 1980 Nov;36(2):374–386. doi: 10.1128/jvi.36.2.374-386.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rosenberg N. E., Clark D. R., Witte O. N. Abelson murine leukemia virus mutants deficient in kinase activity and lymphoid cell transformation. J Virol. 1980 Dec;36(3):766–774. doi: 10.1128/jvi.36.3.766-774.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schiff-Maker L., Rosenberg N. Gag-derived but not abl-derived determinants are exposed on the surface of Abelson virus-transformed cells. Virology. 1986 Oct 30;154(2):286–301. doi: 10.1016/0042-6822(86)90455-1. [DOI] [PubMed] [Google Scholar]
  35. Semba K., Nishizawa M., Miyajima N., Yoshida M. C., Sukegawa J., Yamanashi Y., Sasaki M., Yamamoto T., Toyoshima K. yes-related protooncogene, syn, belongs to the protein-tyrosine kinase family. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5459–5463. doi: 10.1073/pnas.83.15.5459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shibuya M., Hanafusa H. Nucleotide sequence of Fujinami sarcoma virus: evolutionary relationship of its transforming gene with transforming genes of other sarcoma viruses. Cell. 1982 Oct;30(3):787–795. doi: 10.1016/0092-8674(82)90283-5. [DOI] [PubMed] [Google Scholar]
  37. Shibuya M., Hanafusa H. Nucleotide sequence of Fujinami sarcoma virus: evolutionary relationship of its transforming gene with transforming genes of other sarcoma viruses. Cell. 1982 Oct;30(3):787–795. doi: 10.1016/0092-8674(82)90283-5. [DOI] [PubMed] [Google Scholar]
  38. Shoemaker C., Goff S., Gilboa E., Paskind M., Mitra S. W., Baltimore D. Structure of a cloned circular Moloney murine leukemia virus DNA molecule containing an inverted segment: implications for retrovirus integration. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3932–3936. doi: 10.1073/pnas.77.7.3932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Takahashi M., Cooper G. M. ret transforming gene encodes a fusion protein homologous to tyrosine kinases. Mol Cell Biol. 1987 Apr;7(4):1378–1385. doi: 10.1128/mcb.7.4.1378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Takemori T., Miyazoe I., Shirasawa T., Taniguchi M., Graf T. A temperature-sensitive mutant of Abelson murine leukemia virus confers inducibility of IgM expression to transformed lymphoid cells. EMBO J. 1987 Apr;6(4):951–956. doi: 10.1002/j.1460-2075.1987.tb04844.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Toyoshima K., Vogt P. K. Temperature sensitive mutants of an avian sarcoma virus. Virology. 1969 Dec;39(4):930–931. doi: 10.1016/0042-6822(69)90030-0. [DOI] [PubMed] [Google Scholar]
  42. Wang J. Y., Queen C., Baltimore D. Expression of an Abelson murine leukemia virus-encoded protein in Escherichia coli causes extensive phosphorylation of tyrosine residues. J Biol Chem. 1982 Nov 25;257(22):13181–13184. [PubMed] [Google Scholar]
  43. Watanabe S. M., Witte O. N. Site-directed deletions of Abelson murine leukemia virus define 3' sequences essential for transformation and lethality. J Virol. 1983 Mar;45(3):1028–1036. doi: 10.1128/jvi.45.3.1028-1036.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wigler M., Sweet R., Sim G. K., Wold B., Pellicer A., Lacy E., Maniatis T., Silverstein S., Axel R. Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell. 1979 Apr;16(4):777–785. doi: 10.1016/0092-8674(79)90093-x. [DOI] [PubMed] [Google Scholar]
  45. Witte O. N., Goff S., Rosenberg N., Baltimore D. A transformation-defective mutant of Abelson murine leukemia virus lacks protein kinase activity. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4993–4997. doi: 10.1073/pnas.77.8.4993. [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]
  47. Yamamoto T., Nishida T., Miyajima N., Kawai S., Ooi T., Toyoshima K. The erbB gene of avian erythroblastosis virus is a member of the src gene family. Cell. 1983 Nov;35(1):71–78. doi: 10.1016/0092-8674(83)90209-x. [DOI] [PubMed] [Google Scholar]
  48. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES