Identification and localization of a novel nucleolar protein of high molecular weight by a monoclonal antibody

Abstract

A monoclonal murine antibody (No-I 14) is described which reacts specifically with a polypeptide of molecular weight (M,) 180000 present in low-speed nuclear pellets from oocytes and somatic cells of Xenopus laevis and X. borealis and in isolated amplified nucleoli. Two-dimensional gel electrophoresis has revealed the acidic nature of this polypeptide (isoelectric at pH of ca 4.2 in the presence of 9.5 M urea). A relatively large proportion of the protein is extracted at elevated ionic strength( i.e., at 0.4-0.5 M alkali salt) in a form sedimenting at approx. 7-8S , compatible with a monomeric state. It is also extracted by digestion with RNase but not with DNase. In immunofluorescence microscopy , antibody No-114 stains intensely nucleoli of oocytes and all somatic cells examined , including the residual nucleolar structure of Xenopus erythrocytes which are transcriptionally inactive. During mitosis the antigen does not remain associated with the nucleolar organizer regions (NOR) of chromosomes but is released and dispersed over the cytoplasm until telophase when it re-associates with the reforming interphase nucleoli. At higher resolution the immunofluorescent region is often resolved into a number of distinct subnucleolar components of varied size and shape. Immunoelectron microscopy using colloidal gold-coupled secondary antibodies reveals that the M, 180000 protein is confined to the dense fibrillar component of the nucleolus. This conclusion is also supported by its localization in the fibrillar part of segregated nucleoli of cells treated with actinomycin D. We conclude that nucleoli contain a prominent protein of M , 180000 which contributes to the general structure of the dense fibrillar component of the interphase nucleolus , independent of its specific transcriptional activity.In the past two decades considerable progress has been made in understanding the basic functional organization of the nucleolus. Combined morphological, biochemical, and auto radiographic data have established that the nucleolus is the site of the genes which code for pre-rRNAs, transcription of these genes, assembly of the pre-rRNA molecules with specific proteins into ribonucleoprotein complexes, and maturation of the preribosomal particles both at the RNA and protein level (for reviews see [1][2][3]). Based primarily on electron microscopic autoradiography of cells labeled with radioactive uridine, morphologically distinct components of nucleoli can be correlated with certain steps of the pathway of ribosome biogenesis [4][5][6][7][8][9]. Although nucleoli can display different shapes and arrangements of their compot)ept substructures, depending on the .specific cell type and metabolic condition of the cell, there is general agreement that primary transcripts containing pre-rRNA are located in the dense fibrillar part of the

A Novel Helicase-Type Protein in the Nucleolus: Protein NOH61

“…A very similar translocation has been reported for protein NO38/B23 after treatment with AMD and other cytotoxic drugs (Yung et al, 1985;Chan, 1992). When the same cells were counterstained with mAb No-114 directed against the 180-kDa nucleolar protein from X. laevis, also termed xNopp180 (Schmidt-Zachmann et al, 1984;Cairns and McStay, 1995), a specific labeling of the dense fibrillar component of segregated nucleoli was seen ( Figure 8CЉ). These experiments indicate that protein NOH61 binds to RNA and that its nucleolar accumulation depends on ongoing transcription.…”

Localization of nucleolar chromatin by immunocytochemistry and in situ hybridization at the electron microscopic level

“…This has been shown with antibodies reacting with various constituents of the dense fibrillar component as well as the granular component (e.g. Schmidt-Zachmann et al, 1984;Hiigle et al, 1985;Reimer et al, 1987a;Raska et al, 1989).…”

Nucleolar changes after microinjection of antibodies to RNA polymerase I into the nucleus of mammalian cells

“…The nucleoplasmic bodies present in microinjected Xenopus A6 cells contained, in addition, another specific DFC protein with an Mr of 180,000 (data not shown; cf. also Schmidt-Zachmann et al 1984). In contrast, precursor particles to the small ribosomal subunit as well as RNA polymerase I molecules could not be detected in these bodies.…”

“…In contrast, monoclonal antibody 72B9 reacting specifically with the DFC (Reimer et aL 1987b) stained one or two roundish and much larger nucleolar structures which could often be correlated with a region of the segregated nucleolus appearing light by phase contrast (Fig. la'; see also Schmidt-Zachmann et aL 1984;Hugle et aL 1985;Ochs et aL 1985). In the course of such AMD treatments, ribosomal protein SI, a marker for the GC (Hugle et aL 1985), progressively disappeared from the PtK 2 cell nucleoli (Fig.2a', b'), indicative of an AMDinduced clearance of precursor particles to the small ribosomal subunit from the nucleoli which in PtK 2 cells occurred much faster than in Xenopus cells of line A6 (Hugle et aL 1985).…”