The 22 S cylinder particles of Xenopus laevis. II. Immunological characterization and localization of their proteins in tissues and cultured cells.
Cylinder-shaped particles of 10 nm diameter were isolated from nuclei of Xenopus laevis oocytes and purified by sucrose gradient centrifugation and DEAE-Sephacel chromatography. Antibodies to protein constituents of these isolated particles were elicited in guinea pigs and examined by immunoblotting and immunoprecipitation techniques as well as by immunofluorescence microscopy. The antibodies reacted with only two out of the 12 constituent polypeptides characteristic for these particles when examined in the denatured state by the immunoblotting technique, including the largest component of Mr 30 000, but were able to precipitate the whole ensemble of these polypeptides in immunoprecipitation experiments, in agreement with the notion that these proteins form the 22 S particle complex. The antibodies displayed a rather narrow range of interspecies cross-reactivity, showing reaction with cells of other amphibia but not with avian and mammalian cells. In oocytes as well as in transcriptionally active somatic cells the antigen was localized in the nucleoplasm, excluding nucleoli, as well as in the cytoplasm, usually suggesting a higher concentration in the nucleoplasm. During mitosis, the proteins were dispersed throughout the cytoplasm whereas the chromosomes were negative. Inactive cells such as mature erythrocytes, spermatids and spermatozoa were negative. These immunolocalization findings support our conclusion based on fractionation studies that the cylindershaped particles and their protein constituents occur both in the nucleoplasm and the cytoplasm of a broad range of cell types.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000032:analysis* / Q000276:immunology; D002478:Cells, Cultured; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007120:Immunochemistry; D008856:Microscopy, Fluorescence; D009865:Oocytes / Q000032:analysis / Q000276:immunology*; D011506:Proteins / Q000032:analysis* / Q000276:immunology; D014982:Xenopus laevis / Q000378:metabolism*
D011506:Proteins