Characterization of the 2A7 antigen as a 85-kDa human nucleocytoplasmic shuttling protein.
The murine monoclonal antibody 2A7 was found to react specifically with a 85-kDa human protein which is distributed throughout the nuclear interior in interphase and becomes associated with condensed chromosomes during mitosis. The 2A7 epitope was not detected in cells from other species. Two-dimensional immunoblotting analysis of HeLa cell homogenates further indicated that the 85-kDa polypeptide species recognized by the 2A7 antibody corresponds to an acidic protein which may be complexed in vivo within high-molecular-weight protein structures. Immunofluorescence monitoring of the 2A7 staining pattern during in situ preparation of nuclear matrices from HeLa cells demonstrated that the nucleoplasmic fraction of the antigen is readily solubilized by detergent and salts, whereas the nucleolar fraction resists detergent/salt extraction and DNase digestion, to be released only upon RNase activity. Mobility assays in human-mouse heterokaryons provided evidence that the 2A7 antigen is a nucleocytoplasmic shuttling protein. The nuclear distribution of this antigen remained unchanged upon drug-induced inhibition of RNA synthesis but was markedly altered by heat shock stress. All together, the data presented here suggest that the 2A7 antigen may have a function in RNA metabolism.
Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, K1N 6N5, Canada. mplev@science.uttawa.ca
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Paulin-Levasseur
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Julien
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000941:Antigens / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D018869:Heat-Shock Response; D006801:Humans; D006822:Hybrid Cells / Q000166:cytology / Q000378:metabolism; D007399:Interphase; D007526:Isoelectric Point; D051379:Mice; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D015530:Nuclear Matrix / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012995:Solubility; D013045:Species Specificity; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000941:Antigens; D011506:Proteins; D012333:RNA, Messenger; D003850:Deoxyribonuclease I; D012259:Ribonuclease, Pancreatic