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. 1985 Jan;5(1):127–132. doi: 10.1128/mcb.5.1.127

Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule.

D Wolf, N Harris, N Goldfinger, V Rotter
PMCID: PMC366686  PMID: 2580227

Abstract

Transfection of a cloned p53 gene into a p53 nonproducer Abelson murine leukemia virus-transformed cell line, L12, reconstituted p53 expression. The protein expressed in these cells was indistinguishable from that naturally expressed in p53 producer tumor cells. Conversely, p53 protein expressed in L12-derived clones that were established by transfection with a full-length p53 cDNA clone (pM8) exhibited a discrete immunological form. Immunoprecipitation of p53 with a panel of monoclonal anti-p53 antibodies showed that L12-derived clones that were transfected with the genomic p53 clone contained the same antigenic determinants as those found in the p53 protein expressed in tumor cells. These p53 proteins bound all monoclonal antibody types as well as the polyclonal anti-p53 tested. However, L12-derived clones established by transfection of the p53 cDNA clone (pM8) expressed a p53 protein that bound the RA3-2C2 and PAb200.47 anti-p53 monoclonal antibodies as well as polyclonal anti-p53 serum but totally lacked the antigenic receptor for the PAb122 and PAb421 monoclonal antibodies. The p53 proteins expressed by either genomic or cDNA p53 clones exhibited the same apparent molecular sizes and identical partial peptide maps. We suggest that transfection of the p53 gene induced expression of the entire group of the possible mRNA species, whereas cloned p53 cDNA (pM8) represented a single mRNA molecule that codes for a discrete species of p53 protein.

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Selected References

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

  1. Benchimol S., Pim D., Crawford L. Radioimmunoassay of the cellular protein p53 in mouse and human cell lines. EMBO J. 1982;1(9):1055–1062. doi: 10.1002/j.1460-2075.1982.tb01296.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Coffman R. L., Weissman I. L. A monoclonal antibody that recognizes B cells and B cell precursors in mice. J Exp Med. 1981 Feb 1;153(2):269–279. doi: 10.1084/jem.153.2.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Crawford L. V., Pim D. C., Gurney E. G., Goodfellow P., Taylor-Papadimitriou J. Detection of a common feature in several human tumor cell lines--a 53,000-dalton protein. Proc Natl Acad Sci U S A. 1981 Jan;78(1):41–45. doi: 10.1073/pnas.78.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Czosnek H. H., Bienz B., Givol D., Zakut-Houri R., Pravtcheva D. D., Ruddle F. H., Oren M. The gene and the pseudogene for mouse p53 cellular tumor antigen are located on different chromosomes. Mol Cell Biol. 1984 Aug;4(8):1638–1640. doi: 10.1128/mcb.4.8.1638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DeLeo A. B., Jay G., Appella E., Dubois G. C., Law L. W., Old L. J. Detection of a transformation-related antigen in chemically induced sarcomas and other transformed cells of the mouse. Proc Natl Acad Sci U S A. 1979 May;76(5):2420–2424. doi: 10.1073/pnas.76.5.2420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dippold W. G., Jay G., DeLeo A. B., Khoury G., Old L. J. p53 transformation-related protein: detection by monoclonal antibody in mouse and human cells. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1695–1699. doi: 10.1073/pnas.78.3.1695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  11. Gurney E. G., Harrison R. O., Fenno J. Monoclonal antibodies against simian virus 40 T antigens: evidence for distinct sublcasses of large T antigen and for similarities among nonviral T antigens. J Virol. 1980 Jun;34(3):752–763. doi: 10.1128/jvi.34.3.752-763.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Harlow E., Crawford L. V., Pim D. C., Williamson N. M. Monoclonal antibodies specific for simian virus 40 tumor antigens. J Virol. 1981 Sep;39(3):861–869. doi: 10.1128/jvi.39.3.861-869.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hoyt M. A., Knight D. M., Das A., Miller H. I., Echols H. Control of phage lambda development by stability and synthesis of cII protein: role of the viral cIII and host hflA, himA and himD genes. Cell. 1982 Dec;31(3 Pt 2):565–573. doi: 10.1016/0092-8674(82)90312-9. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Lane D. P., Crawford L. V. T antigen is bound to a host protein in SV40-transformed cells. Nature. 1979 Mar 15;278(5701):261–263. doi: 10.1038/278261a0. [DOI] [PubMed] [Google Scholar]
  17. Linzer D. I., Levine A. J. Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells. Cell. 1979 May;17(1):43–52. doi: 10.1016/0092-8674(79)90293-9. [DOI] [PubMed] [Google Scholar]
  18. Milner J. Different forms of p53 detected by monoclonal antibodies in non-dividing and dividing lymphocytes. Nature. 1984 Jul 12;310(5973):143–145. doi: 10.1038/310143a0. [DOI] [PubMed] [Google Scholar]
  19. Mulligan R. C., Berg P. Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2072–2076. doi: 10.1073/pnas.78.4.2072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Okayama H., Berg P. High-efficiency cloning of full-length cDNA. Mol Cell Biol. 1982 Feb;2(2):161–170. doi: 10.1128/mcb.2.2.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Oren M., Levine A. J. Molecular cloning of a cDNA specific for the murine p53 cellular tumor antigen. Proc Natl Acad Sci U S A. 1983 Jan;80(1):56–59. doi: 10.1073/pnas.80.1.56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rice D., Baltimore D. Regulated expression of an immunoglobulin kappa gene introduced into a mouse lymphoid cell line. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7862–7865. doi: 10.1073/pnas.79.24.7862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  24. Rotter V., Boss M. A., Baltimore D. Increased concentration of an apparently identical cellular protein in cells transformed by either Abelson murine leukemia virus or other transforming agents. J Virol. 1981 Apr;38(1):336–346. doi: 10.1128/jvi.38.1.336-346.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rotter V., Friedman H., Katz A., Zerivitz K., Wolf D. Variation in antigenic determinants of p53 transformation-related protein obtained from various species. J Immunol. 1983 Jul;131(1):329–333. [PubMed] [Google Scholar]
  26. Rotter V., Witte O. N., Coffman R., Baltimore D. Abelson murine leukemia virus-induced tumors elicit antibodies against a host cell protein, P50. J Virol. 1980 Nov;36(2):547–555. doi: 10.1128/jvi.36.2.547-555.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rotter V., Wolf D., Pravtcheva D., Ruddle F. H. Chromosomal assignment of the murine gene encoding the transformation-related protein p53. Mol Cell Biol. 1984 Feb;4(2):383–385. doi: 10.1128/mcb.4.2.383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rotter V. p53, a transformation-related cellular-encoded protein, can be used as a biochemical marker for the detection of primary mouse tumor cells. Proc Natl Acad Sci U S A. 1983 May;80(9):2613–2617. doi: 10.1073/pnas.80.9.2613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sarnow P., Ho Y. S., Williams J., Levine A. J. Adenovirus E1b-58kd tumor antigen and SV40 large tumor antigen are physically associated with the same 54 kd cellular protein in transformed cells. Cell. 1982 Feb;28(2):387–394. doi: 10.1016/0092-8674(82)90356-7. [DOI] [PubMed] [Google Scholar]
  30. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  31. Wolf D., Admon S., Oren M., Rotter V. Abelson murine leukemia virus-transformed cells that lack p53 protein synthesis express aberrant p53 mRNA species. Mol Cell Biol. 1984 Mar;4(3):552–558. doi: 10.1128/mcb.4.3.552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wolf D., Harris N., Rotter V. Reconstitution of p53 expression in a nonproducer Ab-MuLV-transformed cell line by transfection of a functional p53 gene. Cell. 1984 Aug;38(1):119–126. doi: 10.1016/0092-8674(84)90532-4. [DOI] [PubMed] [Google Scholar]
  33. Wolf D., Rotter V. Inactivation of p53 gene expression by an insertion of Moloney murine leukemia virus-like DNA sequences. Mol Cell Biol. 1984 Jul;4(7):1402–1410. doi: 10.1128/mcb.4.7.1402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zakut-Houri R., Oren M., Bienz B., Lavie V., Hazum S., Givol D. A single gene and a pseudogene for the cellular tumour antigen p53. Nature. 1983 Dec 8;306(5943):594–597. doi: 10.1038/306594a0. [DOI] [PubMed] [Google Scholar]

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