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. 1985 Mar;4(3):699–706. doi: 10.1002/j.1460-2075.1985.tb03686.x

Precise epitope mapping of the murine transformation-associated protein, p53.

A Wade-Evans, J R Jenkins
PMCID: PMC554245  PMID: 2408882

Abstract

Murine p53 cDNA sequences were cloned into an in vitro expression vector, Protem Hind. Four deletion libraries were generated using Bal31 double-stranded exonuclease; two being made from constructs encoding a fusion protein constructed from SV40 small t sequences and the p53 clone, p27.la; and two from the full length p53 clone, pp53-5. Both 5'- and 3'-terminal deletions of the p53 gene were made. Transcription of these constructs using Escherichia coli RNA polymerase holoenzyme, followed by translation in mRNA-dependent rabbit reticulocyte lysate, gave in vitro, truncated protein products which were immunoprecipitated by a panel of anti-p53 monoclonal antibodies. This approach enabled us to map accurately the binding sites of seven different monoclonal antibodies, demonstrating four distinct antigenic sites on p53. A synthetic peptide was constructed corresponding to the predicted amino acid sequence of one of these epitopes. This peptide competes with the epitope on the full length p53 protein for the relevant monoclonal antibodies and dissociates the corresponding p53/antibody complexes.

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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. Bienz B., Zakut-Houri R., Givol D., Oren M. Analysis of the gene coding for the murine cellular tumour antigen p53. EMBO J. 1984 Sep;3(9):2179–2183. doi: 10.1002/j.1460-2075.1984.tb02110.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chou P. Y., Fasman G. D. Prediction of protein conformation. Biochemistry. 1974 Jan 15;13(2):222–245. doi: 10.1021/bi00699a002. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. 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]
  10. 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]
  11. Jenkins J. R., Rudge K., Currie G. A. Cellular immortalization by a cDNA clone encoding the transformation-associated phosphoprotein p53. Nature. 1984 Dec 13;312(5995):651–654. doi: 10.1038/312651a0. [DOI] [PubMed] [Google Scholar]
  12. Jenkins J. R., Rudge K., Redmond S., Wade-Evans A. Cloning and expression analysis of full length mouse cDNA sequences encoding the transformation associated protein p53. Nucleic Acids Res. 1984 Jul 25;12(14):5609–5626. doi: 10.1093/nar/12.14.5609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  17. 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]
  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. Oren M., Maltzman W., Levine A. J. Post-translational regulation of the 54K cellular tumor antigen in normal and transformed cells. Mol Cell Biol. 1981 Feb;1(2):101–110. doi: 10.1128/mcb.1.2.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rotter V., Abutbul H., Ben-Ze'ev A. P53 transformation-related protein accumulates in the nucleus of transformed fibroblasts in association with the chromatin and is found in the cytoplasm of non-transformed fibroblasts. EMBO J. 1983;2(7):1041–1047. doi: 10.1002/j.1460-2075.1983.tb01543.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Ruscetti S. K., Scolnick E. M. Expression of a transformation-related protein (p53) in the malignant stage of Friend virus-induced diseases. J Virol. 1983 Jun;46(3):1022–1026. doi: 10.1128/jvi.46.3.1022-1026.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Tainer J. A., Getzoff E. D., Alexander H., Houghten R. A., Olson A. J., Lerner R. A., Hendrickson W. A. The reactivity of anti-peptide antibodies is a function of the atomic mobility of sites in a protein. Nature. 1984 Nov 8;312(5990):127–134. doi: 10.1038/312127a0. [DOI] [PubMed] [Google Scholar]
  26. Van Roy F., Fransen L., Fiers W. Phosphorylation patterns of tumour antigens in cells lytically infected or transformed by simian virus 40. J Virol. 1981 Oct;40(1):28–44. doi: 10.1128/jvi.40.1.28-44.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]

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