Autoimmune serum from a patient with scleroderma was shown by indirect immunofluorescence to label nucleoli in a variety of cells tested including: rat kangaroo PtK2, Xenopus A6, 3T3, HeLa, and human peripheral blood lymphocytes. Immunoblot analysis of nucleolar proteins with the scleroderma antibody resulted in the labeling of a single protein band of 34 kD molecular weight with a pI of 8.5. Electron microscopic immunocytochemistry demonstrated that the protein recognized by the scleroderma antiserum was localized exclusively in the fibrillar region of the nucleolus which included both dense fibrillar and fibrillar center regions. Therefore, we have named this protein "fibrillarin". Fibrillarin was found on putative chromosomal nucleolar organizer regions (NORs) in metaphase and anaphase, and during telophase fibrillarin was found to be an early marker for the site of formation of the newly forming nucleolus. Double label indirect immunofluorescence and immunoelectron microscopy on normal, actinomycin D-segregated, and DRB-treated nucleoli showed that fibrillarin and nucleolar protein B23 were predominantly localized to the fibrillar and granular regions of the nucleolus, respectively. RNase A and DNase I digestion of cells in situ demonstrated that fibrillarin was partially removed by RNase and completely removed by DNase. These results suggest that fibrillarin is a widely occurring basic nonhistone nucleolar protein whose location and nuclease sensitivity may indicate some structural and/or functional role in the rDNA-containing dense fibrillar and fibrillar center regions of the nucleolus.
Monoclonal antibodies against nuclear antigens, such as DNA, histones, Sm, U1 RNP, and SS-B (La), which are targets of spontaneously occurring autoantibodies in systemic rheumatic diseases, have recently been produced by several investigators (1-5). The significance of using monoclonal antibodies against nuclear autoantigens lies in their monospecificity , as compared with human autoimmune sera, which frequently target more than 1 nuclear antigen (6). Therefore, monoclonal antinuclear antibodies should be important probes not only for standardization of clinical tests, but also for further dissecting the structure and function of reactive antigens. In a recent study, we showed that monoclonal anti-native DNA (anti-nDNA) antibodies also reacted with mitochondrial DNA in tissue culture cells (7). This observation facilitated studies to determine why certain human systemic lupus erythematosus sera that display high titers of anti-nDNA antibodies stain mitochondria in tissue culture cells (7).In the present study, we have shown that a monoclonal autoantibody derived from an autoimmune (New Zealand black X New Zealand white)F1 ([NZB/NZW]F1) mouse reacts with an M , 34,000 nucleolar protein. This protein was demonstrated to share an epitope which is also recognized by scleroderma antibodies reactive with an M , 34,000 nucleolar protein associated with the U3 RNP (8,9). MATERIALS AND METHODSMonoclonal antibodies. Monoclonal antibodies were produced by hybridoma technology as described previously (10). Splenocytes from a 6-month-old female, unmanipulated (NZBMZW)FI mouse were mixed, at a ratio of lO:l, with nonsecreting myeloma cells (P3x63Ag8.653) and fused with 35% polyethylene glycol. Hybrids were selected in hypoxanthine/aminopteridthymidine-containing medium, as described (10). After propagating hybridomas and subcloning
A time course study was conducted on nucleologenesis after release from a mitotic block in the presence and absence of actinomycin D to determine the composition and fate of prenucleolar bodies (PNBs). Prenucleolar bodies, whether naturally occurring or induced by actinomycin D treatment, stain with silver and contain phosphoproteins B23 and C23, two of the major proteins of the interphase nucleolus as determined by double label immunofluorescence with specific antibodies. The nucleolus is formed by fusion of PNBs, which subsequently "reorganize" and form internal fibrillar and peripheral granular regions. Actinomycin D prevents fusion of PNBs, which are then randomly dispersed throughout the nucleus but they still contain proteins B23 and C23. These results demonstrate that the nucleolus is formed by fusion of prenucleolar structures whose biochemical composition resembles the mature nucleolus, since PNBs contain at least two of the major nucleolar proteins.
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