Monoclonal antibodies prepared against the major Drosophila nuclear Matrix-pore complex-lamina glycoprotein bind specifically to the nuclear envelope in situ.
A high molecular weight glycoprotein found associated with a nuclear matrix-pore complex-lamina (NMPCL) preparation obtained from Drosophila melanogaster embryos has been shown by in vitro analyses to be largely confined to this subcellular fraction. In contrast with several of the NMPCL proteins, this glycoprotein remains completely insoluble after treatment with 5 M urea. It has, therefore, been possible to separate the glycoprotein from other NMPCL components by differential urea extraction. The glycoprotein in the 5 M urea-extracted pellet has been solubilized by boiling in sodium dodecyl sulfate and purified to near-homogeneity by sequential steps of chromatography on hydroxylapatite and Sephacryl S-300 (both run in the presence of 0.1% sodium dodecyl sulfate), followed by affinity chromatography on lentil lectin-Sepharose. Over 30 hybridoma cell lines producing antibodies against this glycoprotein have been obtained. Monoclonality has been established for two of these lines (designated AGP-26 and AGP-78), and the antibodies they secrete have been further characterized. Western blot analysis has shown both antibodies to be monospecific (with respect to other Drosophila embryo polypeptides) for the major NMPCL glycoprotein; in addition, antibody AGP-78 has been shown to be weakly cross-reactive with glycoproteins of similar or identical molecular weight found associated with isolated nuclear fractions obtained from Xenopus oocytes, as well as chicken, opossum, and rat livers. Finally, both antibodies AGP-26 and AGP-78 react exclusively with the Drosophila nuclear periphery (nuclear envelope) in situ as demonstrated by indirect immunofluorescence analysis of larval cryosections. Based on these results as well as upon those of biochemical studies reported previously (Berrios, M., Filson, A. J., Blobel, G, and Fisher, P. A. (1983) J. Biol. Chem. 258, 13384-13390), we conclude that the major Drosophila NMPCL glycoprotein is the specific homolog of the high molecular weight glycoprotein recently shown using immunoelectron microscopy to be a distinct component of the rat liver nuclear pore complex (Gerace, L., Ottaviano, Y., and Kondor-Koch, C. (1982) J. Cell Biol. 95, 826-837).
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002850:Chromatography, Gel; D004331:Drosophila melanogaster; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D008970:Molecular Weight; D009685:Nuclear Envelope / Q000276:immunology*
D000911:Antibodies, Monoclonal