Isolation and characterization of a proteinaceous subnuclear fraction composed of nuclear matrix, peripheral lamina, and nuclear pore complexes from embryos of Drosophila melanogaster.
Morphologically intact nuclei have been prepared from embryos of Drosophila melanogaster by a simple and rapid procedure. These nuclei have been further treated with high concentrations of DNase I and RNase A followed by sequential extraction with 2% Triton X-100 and 1 M NaCl to produce a structurally and biochemically distinct preparation designated Drosophila subnuclear fraction I (DSNF-I). As seen by phase-contrast microscopy, DSNF-I is composed of material which closely resembles unfractionated nuclei; residual internal nuclear structures including nucleolar remnants are clearly visible. By transmission electron microscopy, nuclear lamina, pore complexes, and a nuclear matrix are similarly identified. Biochemically, DSNF-I is composed almost entirely of protein (greater than 93%). SDS PAGE analysis reveals several major polypeptides; species at 174,000, 74,000, and 42,000 predominate. A polypeptide coincident with the Coomassie Blue-stainable 174-kdalton band has been shown by a novel technique of lectin affinity labeling to be a glycoprotein; a glycoprotein of similar or identical molecular weight has been found to be a component of nuclear envelope fractions isolated from the livers of rats, guinea pigs, opossums, and chickens. Antisera against several of the polypeptides in DSNF-I have been obtained from rabbits, and all of them show only little or no cross-reactivity with Drosophila cytoplasmic fractions. Initial results of immunocytochemical studies, while failing to positively localize either the 174- or 16-kdalton polypeptides, demonstrate a nuclear localization of the 74-kdalton antigen in all of several interphase cell types obtained from both Drosophila embryos and third-instar larvae.
D000818:Animals; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000032:analysis / Q000648:ultrastructure*; D004247:DNA / Q000032:analysis; D004331:Drosophila melanogaster / Q000196:embryology*; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis*; D006168:Guinea Pigs / Q000033:anatomy & histology; D007118:Immunoassay; D008970:Molecular Weight; D009685:Nuclear Envelope / Q000032:analysis; D009893:Opossums / Q000033:anatomy & histology; D011506:Proteins / Q000032:analysis*; D012313:RNA / Q000032:analysis; D013552:Swine / Q000033:anatomy & histology
D006023:Glycoproteins; D011506:Proteins; D012313:RNA; D004247:DNA