Advertisement

Purification of Messenger RNA by Polysome Isolation with Monoclonal Antibodies

  • Joseph P. Brown
  • Timothy M. Rose
  • Gregory D. Plowman

Abstract

In this chapter we discuss the use of monoclonal antibodies to enrich the mRNAs coding for low-abundance proteins by polysome immunopurification. In this technique polysomes (which consist of mRNA, ribosomes, and nascent polypeptide chains) are purified by immunoaffinity chromatography with antibodies that recognize antigenic determinants present on the nascent chains. While there have been numerous reports of the use of polyclonal antisera for this purpose, we are aware of only a few examples of the use of monoclonal antibodies, apart from our own work.1,2 Since the preceding chapter by Kraus addresses the use of polyclonal antisera, we shall focus on the differences between monoclonal antibodies and polyclonal antisera with respect to this application. Our own experience in cloning the mRNA for p97, a 97,000-dalton cell surface glycoprotein of human melanoma, which we identified by using monoclonal antibodies,3,5 will be described in some detail. The protein p97 is present in most human melanomas, but it is found in only trace amounts in normal adult tissues.6-8 It is representative of many other low-abundance cell-surface proteins in that it accounts for only 0.02% of total cell protein even in the cell line expressing the largest amounts of the antigen, and it is reasonable to assume that its mRNA comprises a similar proportion of the cellular mRNA.

Keywords

Polyclonal Antiserum Translation Product Immunoaffinity Chromatography Normal Adult Tissue Methyl Sulfonyl Fluoride 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Korman, A. J., Knudsen, P. J., Kaufman, J. F., and Strominger, J. L., 1982, cDNA clones for the heavy chain of HLA-DR antigens obtained after immunopurification of polysomes by monoclonal antibody, Proc. Natl. Acad. Sci. USA 79:1844–1848.PubMedCrossRefGoogle Scholar
  2. 2.
    Oren, M., and Levine, A. J., 1983, Molecular cloning of a cDNA specific for the murine p53 cellular tumor antigen, Proc. Natl. Acad. Sci. USA 80:56–59.PubMedCrossRefGoogle Scholar
  3. 3.
    Woodbury, R. G., Brown, J. P., Yeh, M.-Y., Hellstrom, I., and Hellstrom, K. E., 1980, Identification of a cell surface protein, p97, in human melanomas and certain other neoplasms, Proc. Natl. Acad. Sci. USA 77:2183–2187.PubMedCrossRefGoogle Scholar
  4. 4.
    Brown, J. P., Wright, P. W., Hart, C. E., Woodbury, R. G., Hellstrom, K. E., Hellstrom, I., and Wright, P., 1980, Protein antigens of normal and malignant human cells identified by immunoprecipitation with monoclonal antibodies, J. Biol. Chem. 255:4980–4983.PubMedGoogle Scholar
  5. 5.
    Dippold, W. G., Lloyd, K. O., Li, L. T., Ikeda, H., Oettgen, H. F., and Old, L. J., 1980, Cell surface antigens of human malignant melanoma: Definition of six antigenic systems with mouse monoclonal antibodies, Proc. Natl. Acad. Sci. USA 77:6114–6118.PubMedCrossRefGoogle Scholar
  6. 6.
    Brown, J. P., Nishiyama, K., Hellstrom, I., and Hellstrom, K. E., 1981, Structural characterization of human melanoma-associated antigen p97 using monoclonal antibodies, J. Immunol. 127:539–546.PubMedGoogle Scholar
  7. 7.
    Brown, J. P., Woodbury, R. G., Hart, C. E., Hellstrom, I., and Hellstrom, K. E., 1981, Quantitative analysis of melanoma-associated antigen p97 in normal and neoplastic tissues, Proc. Natl. Acad. Sci. USA 78:539–543.PubMedCrossRefGoogle Scholar
  8. 8.
    Garrigues, H. J., Tilgen, W., Hellstrom, I., Franke, W., and Hellstrom, K. E., 1982, Detection of a human melanoma-associated antigen, p97, in histological sections of primary human melanoma, Int. J. Cancer 29:511–515.PubMedCrossRefGoogle Scholar
  9. 9.
    Cundliffe, E., Cannon, M., and Davies, J., 1974, Mechanism of inhibition of eukaryotic protein synthesis by trichothecene fungal toxins, Proc. Natl. Acad. Sci. USA 71:30–34.PubMedCrossRefGoogle Scholar
  10. 10.
    Wei, C. M., Hansen, B. S., Vaughan, M. H., and McLaughlin, C. S., 1974, Mechanism of action of the mycotoxin trichodermin, a 12,13-epoxytrichothecene, Proc. Natl. Acad. Sci. USA 71:713–717.PubMedCrossRefGoogle Scholar
  11. 11.
    Pelham, H. R., and Jackson, R. J., 1976, An efficient mRNA-dependent translation system from reticulocyte lysates, Eur. J. Biochem. 67:247–256.PubMedCrossRefGoogle Scholar
  12. 12.
    Gurdon, J. B., 1974, The Control of Gene Expression in Animal Development, Harvard University Press, Cambridge.Google Scholar
  13. 13.
    Miskin, R., and Soreq, H., 1981, Microinjected Xenopus oocytes synthesize active human plasminogen activator, Nucleic Acids Res. 9:3355–3363.PubMedCrossRefGoogle Scholar
  14. 14.
    Plowman, G. D., Brown, J. P., Enns, C. A., Schroder, J., Nikinmaa, B., Sussman, H. H., Hellstrom, K. E., and Hellstrom, I., 1983, Assignment of the gene for human melanoma-associated antigen p97 to chromosome 3, Nature 303:70–72.PubMedCrossRefGoogle Scholar
  15. 15.
    Palacios, R., Palmiter, R. D., and Schimke, R. T., 1972, Identification and isolation of ovalbumin-synthesizing polysomes, J. Biol. Chem. 247:2316–2321.PubMedGoogle Scholar
  16. 16.
    Kraus, J. P., and Rosenberg, L. E., 1982, Purification of low-abundance messenger RNAs from rat liver by polysome immunoadsorption, Proc. Natl. Acad. Sci. USA 79:4015–4019.PubMedCrossRefGoogle Scholar
  17. 17.
    Shapiro, S. Z., and Young, J. R., 1981, An immunochemical method for mRNA purification, J. Biol Chem. 256:1495–1498.PubMedGoogle Scholar
  18. 18.
    Palmiter, R. D., 1974, Magnesium precipitation of ribonucleoprotein complexes. Expedient techniques for the isolation of undegraded polysomes and messenger ribonucleic acid, Biochemistry 13:3606–3615.PubMedCrossRefGoogle Scholar
  19. 19.
    Brown, J. P., Rose, T. M., Forstrom, J. W., Hellstrom, I., and Hellstrom, K. E., 1985, Isolation of a cDNA for a human melanoma-associated antigen in: Proceedings Eighth Nobel Conference of the Karolinska Institute, Raven Press, New York (in press).Google Scholar
  20. 20.
    Efstratiadis, A., and Villa-Komaroff, L., 1979, Cloning of double-stranded cDNA, in: Genetic Engineering (J. K. Stelow and A. Hollaender, eds.), Volume 1, Plenum Press, New York, pp. 15–36.CrossRefGoogle Scholar
  21. 21.
    Taub, F. and Thompson, B., 1982, An improved method for preparing large arrays of bacterial colonies containing plasmids for hybridization: in situ purification and stable binding of DNA on paper filters, Anal. Biochem. 126:222–230.PubMedCrossRefGoogle Scholar
  22. 22.
    Parnes, J. R., Velan, B., Felsenfeld, A., Ramanathan, L., Ferrini, U., Appella E., and Seidman, J. G., 1981, Mouse β2-microglobulin cDNA clones: A screening procedure for cDNA clones corresponding to rare mRNAs, Proc. Natl. Acad. Sci. USA 78:2253–2257.PubMedCrossRefGoogle Scholar
  23. 23.
    Brown, J. P., Hewick, R. M., Hellstrom, I., Hellstrom, K. E., Doolittle, R. F., and Dreyer, W. J., 1982, Human melanoma-associated p97 is structurally and functionally related to transferrin, Nature 296:171–173.PubMedCrossRefGoogle Scholar
  24. 24.
    Rotter, V., Witte, O. N., Coffman, R., and Baltimore, D., 1980, Abelson murine leukemia virus-induced tumors elicit antibodies against a host cell protein, p50, J. Virol. 36:547–555.PubMedGoogle Scholar

Copyright information

© Plenum Press, New York 1985

Authors and Affiliations

  • Joseph P. Brown
    • 1
  • Timothy M. Rose
    • 1
  • Gregory D. Plowman
    • 1
  1. 1.OncogenSeattleUSA

Personalised recommendations