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. 1989 Nov;86(22):8669–8672. doi: 10.1073/pnas.86.22.8669

Molecular cloning and sequence of cDNA encoding polyoma medium tumor antigen-associated 61-kDa protein.

G Walter 1, F Ferre 1, O Espiritu 1, A Carbone-Wiley 1
PMCID: PMC298349  PMID: 2554323

Abstract

Polyoma virus medium tumor antigen forms specific complexes with several cellular proteins; among these is a protein of approximately 61 kDa. With antibodies directed against medium tumor antigen, the 61-kDa protein was purified from human 293 cells that were infected with a hybrid adenovirus and overexpressed medium tumor antigen. The purified 61-kDa protein was partially digested with protease V8, and one of the protease V8 fragments was isolated and partially sequenced. The amino acid sequence information was used to design mixed oligonucleotide probes for screening a cDNA library from human placenta. A clone was isolated that hybridized with two separate probes; the clone contained an insert with an open reading frame for 589 amino acids. By in vitro translation of the transcript from this insert, a protein was generated that had the same size and yielded the same pattern of protease V8 fragments as the original 61-kDa protein. Its amino acid sequence reveals 15 repeats, the majority of which are 39 amino acids long. This protein bears no resemblance to proteins in the data bank that was searched.

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Selected References

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  1. Bolen J. B., Thiele C. J., Israel M. A., Yonemoto W., Lipsich L. A., Brugge J. S. Enhancement of cellular src gene product associated tyrosyl kinase activity following polyoma virus infection and transformation. Cell. 1984 Oct;38(3):767–777. doi: 10.1016/0092-8674(84)90272-1. [DOI] [PubMed] [Google Scholar]
  2. Cheng S. H., Harvey R., Espino P. C., Semba K., Yamamoto T., Toyoshima K., Smith A. E. Peptide antibodies to the human c-fyn gene product demonstrate pp59c-fyn is capable of complex formation with the middle-T antigen of polyomavirus. EMBO J. 1988 Dec 1;7(12):3845–3855. doi: 10.1002/j.1460-2075.1988.tb03270.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  4. Courtneidge S. A. Activation of the pp60c-src kinase by middle T antigen binding or by dephosphorylation. EMBO J. 1985 Jun;4(6):1471–1477. doi: 10.1002/j.1460-2075.1985.tb03805.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Courtneidge S. A., Heber A. An 81 kd protein complexed with middle T antigen and pp60c-src: a possible phosphatidylinositol kinase. Cell. 1987 Sep 25;50(7):1031–1037. doi: 10.1016/0092-8674(87)90169-3. [DOI] [PubMed] [Google Scholar]
  6. Courtneidge S. A., Smith A. E. Polyoma virus transforming protein associates with the product of the c-src cellular gene. Nature. 1983 Jun 2;303(5916):435–439. doi: 10.1038/303435a0. [DOI] [PubMed] [Google Scholar]
  7. Garrels J. I. Two dimensional gel electrophoresis and computer analysis of proteins synthesized by clonal cell lines. J Biol Chem. 1979 Aug 25;254(16):7961–7977. [PubMed] [Google Scholar]
  8. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  9. Grussenmeyer T., Carbone-Wiley A., Scheidtmann K. H., Walter G. Interactions between polyomavirus medium T antigen and three cellular proteins of 88, 61, and 37 kilodaltons. J Virol. 1987 Dec;61(12):3902–3909. doi: 10.1128/jvi.61.12.3902-3909.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grussenmeyer T., Scheidtmann K. H., Hutchinson M. A., Eckhart W., Walter G. Complexes of polyoma virus medium T antigen and cellular proteins. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7952–7954. doi: 10.1073/pnas.82.23.7952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hanks S. K. Homology probing: identification of cDNA clones encoding members of the protein-serine kinase family. Proc Natl Acad Sci U S A. 1987 Jan;84(2):388–392. doi: 10.1073/pnas.84.2.388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jay E., Bambara R., Padmanabhan R., Wu R. DNA sequence analysis: a general, simple and rapid method for sequencing large oligodeoxyribonucleotide fragments by mapping. Nucleic Acids Res. 1974 Mar;1(3):331–353. doi: 10.1093/nar/1.3.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kaplan D. R., Whitman M., Schaffhausen B., Pallas D. C., White M., Cantley L., Roberts T. M. Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity. Cell. 1987 Sep 25;50(7):1021–1029. doi: 10.1016/0092-8674(87)90168-1. [DOI] [PubMed] [Google Scholar]
  14. Koch W., Carbone A., Walter G. Purified polyoma virus medium T antigen has tyrosine-specific protein kinase activity but no significant phosphatidylinositol kinase activity. Mol Cell Biol. 1986 Jun;6(6):1866–1874. doi: 10.1128/mcb.6.6.1866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kornbluth S., Sudol M., Hanafusa H. Association of the polyomavirus middle-T antigen with c-yes protein. Nature. 1987 Jan 8;325(7000):171–173. doi: 10.1038/325171a0. [DOI] [PubMed] [Google Scholar]
  16. Kypta R. M., Hemming A., Courtneidge S. A. Identification and characterization of p59fyn (a src-like protein tyrosine kinase) in normal and polyoma virus transformed cells. EMBO J. 1988 Dec 1;7(12):3837–3844. doi: 10.1002/j.1460-2075.1988.tb03269.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lathe R. Synthetic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations. J Mol Biol. 1985 May 5;183(1):1–12. doi: 10.1016/0022-2836(85)90276-1. [DOI] [PubMed] [Google Scholar]
  18. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  19. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Millán J. L. Molecular cloning and sequence analysis of human placental alkaline phosphatase. J Biol Chem. 1986 Mar 5;261(7):3112–3115. [PubMed] [Google Scholar]
  21. Pallas D. C., Cherington V., Morgan W., DeAnda J., Kaplan D., Schaffhausen B., Roberts T. M. Cellular proteins that associate with the middle and small T antigens of polyomavirus. J Virol. 1988 Nov;62(11):3934–3940. doi: 10.1128/jvi.62.11.3934-3940.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rundell K. Complete interaction of cellular 56,000- and 32,000-Mr proteins with simian virus 40 small-t antigen in productively infected cells. J Virol. 1987 Apr;61(4):1240–1243. doi: 10.1128/jvi.61.4.1240-1243.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rundell K., Major E. O., Lampert M. Association of cellular 56,000- and 32,000-molecular-weight protein with BK virus and polyoma virus t-antigens. J Virol. 1981 Mar;37(3):1090–1093. doi: 10.1128/jvi.37.3.1090-1093.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Schaffhausen B. S., Bockus B. J., Berkner K. L., Kaplan D., Roberts T. M. Characterization of middle T antigen expressed by using an adenovirus expression system. J Virol. 1987 Apr;61(4):1221–1225. doi: 10.1128/jvi.61.4.1221-1225.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sinha N. D., Biernat J., McManus J., Köster H. Polymer support oligonucleotide synthesis XVIII: use of beta-cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucleic Acids Res. 1984 Jun 11;12(11):4539–4557. doi: 10.1093/nar/12.11.4539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Walter G., Carbone-Wiley A., Joshi B., Rundell K. Homologous cellular proteins associated with simian virus 40 small T antigen and polyomavirus medium T antigen. J Virol. 1988 Dec;62(12):4760–4762. doi: 10.1128/jvi.62.12.4760-4762.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Walter G., Carbone A., Welch W. J. Medium tumor antigen of polyomavirus transformation-defective mutant NG59 is associated with 73-kilodalton heat shock protein. J Virol. 1987 Feb;61(2):405–410. doi: 10.1128/jvi.61.2.405-410.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Walter G. Production and use of antibodies against synthetic peptides. J Immunol Methods. 1986 Apr 17;88(2):149–161. doi: 10.1016/0022-1759(86)90001-3. [DOI] [PubMed] [Google Scholar]
  30. Wood W. I., Gitschier J., Lasky L. A., Lawn R. M. Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1585–1588. doi: 10.1073/pnas.82.6.1585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yang Y. C., Hearing P., Rundell K. Cellular proteins associated with simian virus 40 early gene products in newly infected cells. J Virol. 1979 Oct;32(1):147–154. doi: 10.1128/jvi.32.1.147-154.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]

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