Chromosoma

, Volume 80, Issue 3, pp 253–275 | Cite as

Monoclonal antibodies against chromosomal proteins of Drosophila melanogaster

Establishment of antibody producing cell lines and partial characterization of corresponding antigens
  • Harald Saumweber
  • Peter Symmons
  • Rainer Kabisch
  • Hans Will
  • F. Bonhoeffer
Article

Abstract

Total nuclear protein from the embryonic D. melanogaster cell line Kc and crude hydroxyapatite fractions thereof were used for immunization of mice. From the spleen cells of these mice we established 755 permanent lymphoid cell lines using the hybridoma technique originally developed by Köhler and Milstein (1975). Radioimmunoassay showed 455 of these cell lines secreted antibodies which bound to component(s) contained in the antigen mixtures used for immunization. Screening of 311 cell lines using indirect immunofluorescence revealed 58 lines whose antibodies showed a highly selective staining pattern on polytene chromosomes from the salivary glands of D. melanogaster third instar larvae. Eight of these cell lines were cloned and further characterized. We were able to order the staining patterns into three distinct classes based on the staining behaviour of the monoclonal antibodies: staining of active regions, staining of phase dark bands or staining of most interbands. The molecular weight of those antigens against which the monoclonal antibodies were directed was determined in SDS polyacrylamide gels.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Alfageme, C.R., Rudkin, G.T., Cohen, L.H.: Locations of chromosomal proteins in polytene chromosomes. Proc. nat. Acad. Sci.(Wash.) 73, 2038–2042 (1976)Google Scholar
  2. Ashburner, M.: Puffing patterns in Drosophila melanogaster and related species. In: Developmental studies on giant chromosomes (W. Beermann, ed.), pp. 101–151. New York: Springer Verlag 1972Google Scholar
  3. Ashburner, M.: Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. Develop. Biol. 39, 141–157 (1974)Google Scholar
  4. Benyajaiti, C., Worcel, A.: Isolation, characterization and structure of the folded interphase genome of Drosophila melanogaster. Cell 9, 393–407 (1976)Google Scholar
  5. Biessmann, H., Gjerset, R.A., Levy, W.B., McCarthy, J.B.: Fidelity of chromatin transcription in vitro. Biochemistry 15, 4356–4363 (1976)Google Scholar
  6. Bloom, K.S., Anderson, J.N.: Fractionation and characterization of chromosomal proteins by the hydroxyapatite dissociation method. J. biol. Chem. 253, 4446–4450 (1978)Google Scholar
  7. Bradford, M.N.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976)Google Scholar
  8. Claflin, L., Williams, K.: Mouse myeloma-spleen cell hybrids: enhanced hybridisation frequencies and rapid screening procedures. In: Current topics in microbiology and immunology 81, Lymphocyte hybridomas (F. Melchers, M. Potter and N. Warner, eds.), pp 107–109. Berlin, Heidelberg, New York: Springer 1978Google Scholar
  9. Dulbecco, R., Freeman, G.: Plaque production by the Polyoma virus. Virology 8, 396–397 (1959)Google Scholar
  10. Dulbecco, R., Vogt, M.: Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99, 167–182 (1954)Google Scholar
  11. Echalier, G., Ohanessian, A.: In vitro culture of Drosophila melanogaster embryonic cells. In Vitro 6, 162–172 (1970)Google Scholar
  12. Elgin, S.C.R., Bonner, J.: Partial Fractionation and chemical characterization of the major nonhistone chromosomal proteins. Biochemistry 11, 772–781 (1972)Google Scholar
  13. Elgin, S.C.R., Weintraub, H.: Chromosomal proteins and chromatin structure. Ann. Rev. Biochem. 44, 725–774 (1975)Google Scholar
  14. Finch, J.T., Klug, A.: Solenoidal model for superstructure in chromatin. Proc. nat. Acad. Sci. (Wash.) 73, 743–746 (1977)Google Scholar
  15. Gilbert, W., Müller-Hill, B.: Isolation of the Lac-repressor. Proc. nat. Acad. Sci. (Wash.) 56, 1891–1898 (1966)Google Scholar
  16. Greenleaf, A.L., Plagens, U., Jamrich, M., Bautz, E.K.F.: RNA polymerase B (or II) in heat induced puffs of Drosophila polytene chromosomes. Chromosoma (Berl.) 65, 127–136 (1978)Google Scholar
  17. Haemmerling, G.J.: T lymphocyte tissue culture lines produced by cell hybridization. Europ. J. Immunol. 7, 743–746 (1977)Google Scholar
  18. Hozier, J., Renz, M., Nehls, P.: Involvement of histone H1 in the organization of the chromosome fiber. Proc. nat. Acad. Sci. (Wash.) 74, 1879–1883 (1977)Google Scholar
  19. Jamrich, M., Greenleaf, A.L., Bautz, E.K.F.: Localization of RNA polymerase in polytene chromosomes of Drosophila melanogaster. Proc. nat. Acad. Sci. (Wash.) 74, 2079–2083 (1977)Google Scholar
  20. Kearney, J.F., Radbruch, A., Lisegang, B., Rajewsky, K.: A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J. Immunol. 123, 1548–1550 (1979)Google Scholar
  21. Klein, J.: Biology of the mouse histocompatibility-2 complex. Berlin, Heidelberg, New York: Springer 1975Google Scholar
  22. Köhler, G., Howe, S.C., Milstein, C.: Fusion between immunoglobulin secreting and non-secreting myeloma cell lines. Europ. J. Immunol. 6, 292–295 (1976)Google Scholar
  23. Köhler, G., Milstein, C.: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (Lond.) 256, 495–497 (1975)Google Scholar
  24. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227, 680–685 (1970)Google Scholar
  25. Lefevre, G. jr.: A photographic representation and interpretation of the polytene chromosomes of Drosophila melanogaster salivary glands. In: The genetics and biology of Drosophila, Vol. 1a (M. Ashburner and C. Novitski, eds.), pp. 31–66. New York, London: Academic Press 1976Google Scholar
  26. Lin, S., Riggs, R.D.: The general affinity of Lacrepressor for E. coli DNA: Implications for gene regulation in prokaryotes and eukaryotes. Cell 4, 107–111 (1973)Google Scholar
  27. Littlefield, J.W.: Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145, 709–710 (1964)Google Scholar
  28. Margulies, D.H., Knehl, W.M., Scharff, M.D.: Somatic cell hybridisation of mouse myeloma cells. Cell 8, 405–415 (1976)Google Scholar
  29. Mayfield, J.E., Serunian, L.A., Silver, L.M., Elgin, S.C.R.: A protein released by DNase I digestion of Drosophila nuclei is preferentially associated with puffs. Cell 14, 539–544 (1978)Google Scholar
  30. Melchers, F., Potter, M., Warner, N.L.: Preface. In: Current topics in microbiology and immunology 81, Lymphocyte hybridomas (F. Melchers, M. Potter and N. Warner, eds.), pp. IX–XVIII. Berlin, Heidelberg, New York: Springer 1978Google Scholar
  31. Ouchterlony, Ö., Nilsson, L.A.: Immunodiffusion and Immunoelectrophoresis. In: Handbook of experimental immunology (D.M. Weir, ed.), Vol. 1, Chapter, pp. 1–44. Oxford: Blackwell Scientific Publications 1978Google Scholar
  32. Parker, C.S., Roeder, R.G.: Selective and accurate transcription of the Xenopus laevis 5S RNA genes in isolated chromatin by purified RNA polymerase III. Proc. nat. Acad. Sci. (Wash.) 74, 44–48 (1977)Google Scholar
  33. Paulson, J.R., Laemmli, U.K.: The structure of histone-depleted metaphase chromosomes. Cell 12, 817–828 (1977)Google Scholar
  34. Peterson, J.L., McConkey, E.H.: Non-histone chromosomal proteins from HeLa cells. J. biol. Chem. 251, 548–554 (1976)Google Scholar
  35. Plagens, U., Greenleaf, A.L., Bautz, E.K.F.: Distribution of RNA polymerase on Drosophila polytene chromosomes as studied by indirect immunofluorescence. Chromosoma (Berl.) 59, 157–165 (1976)Google Scholar
  36. Pontecorvo, G.: Production of mammalian somatic cell hybrids by means of polyethylene glycol treatment. Somat. Cell Genet. 1, 397–400 (1975)Google Scholar
  37. Schäfer, W.: Der Mäuse-Inzuchtstamm STU. Entwicklung und Eigenschaften. Z. Naturforsch. 34c, 306–309 (1979)Google Scholar
  38. Scott, S.E.M., Sommerville, J.: Location of nuclear proteins on the chromosomes of newt oocytes. Nature (Lond.) 250, 680–682 (1974)Google Scholar
  39. Semeshin, V.F., Zhimulev, I.F., Belyaeva, E.S.: Electron microscope autoradiographic study on transcriptional activity of Drosophila melanogaster polytene chromosomes. Chromosoma (Berl.) 73, 163–177 (1979)Google Scholar
  40. Silver, L.M., Elgin, S.C.R.: A method for determination of the in situ distribution of chromosomal proteins. Proc. nat. Acad. Sci. (Wash.) 73, 423–427 (1976)Google Scholar
  41. Silver, L.M., Elgin, S.C.R.: Distribution patterns of three subfractions of Drosophila non-histone chromosomal proteins: Possible correlations with gene activity. Cell 11, 971–983 (1977)Google Scholar
  42. Silver, L.M., Elgin, S.C.R.: Production and characterization of antisera against three individual NHC proteins; a case of a generally distributed NHC protein. Chromosoma (Berl.) 68, 101–114 (1978a)Google Scholar
  43. Silver, L.M., Elgin, S.C.R.: Immunological analysis of protein distributions in Drosophila polytene chromosomes. In: The cell nucleus, Vol. V (H. Busch, ed.), pp. 215–262. New York: Academic Press 1978bGoogle Scholar
  44. Southern, E.M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. molec. Biol. 98, 503–517 (1975)Google Scholar
  45. Stollar, B.D.: Serological analyses of histones. In: Methods in cell biology, Vol. XVIII (G. Stein, J. Stein and L.J. Kleinsmith, eds.), pp. 105–122. New York: Academic Press 1978Google Scholar
  46. Weil, P.A., Luse, D.S., Segall, J., Roeder, R.G.: Selective and accurate initiation of transcription at the Ad2 major late promotor in a soluble system dependent on purified RNA polymerase II and DNA. Cell 18, 469–484 (1979a)Google Scholar
  47. Weil, P.A., Segall, J., Harris, B., Ng, S.-Y., Roeder, R.G.: Faithful transcription of eukaryotic genes by RNA polymerase III in systems reconstituted with purified DNA templates. J. biol. Chem. 254, 6163–6173 (1979b)Google Scholar
  48. Will, H.: Immunologische Charakterisierung von Nichthistonproteinen von Drosophila melanogaster. Ph. D. Thesis, Universität Heidelberg 1979Google Scholar
  49. Will, H., Bautz, E.K.F.: Immunological identification of a chromocenter associated protein in polytene chromosomes of Drosophila. Exp. Cell Res. 125, 401–410 (1980)Google Scholar
  50. Williams. A.F., Galfre, G., Milstein, C.: Analysis of cell surfaces by xenogenic myeloma-hybridoma antibodies: differentiation antigens of rat lymphocytes. Cell 12, 663–673 (1977)Google Scholar

Copyright information

© Springer-Verlag 1980

Authors and Affiliations

  • Harald Saumweber
    • 1
  • Peter Symmons
    • 1
  • Rainer Kabisch
    • 2
  • Hans Will
    • 2
  • F. Bonhoeffer
    • 1
  1. 1.Abteilung für Physikalische BiologieMax-Planck-Institut für VirusforschungTübingen 1
  2. 2.Molekulare Genetik, Universität HeidelbergHeidelbergFederal Republic of Germany

Personalised recommendations