Identification and localization of a novel nucleolar protein of high molecular weight by a monoclonal antibody.
A monoclonal murine antibody (No-114) is described which reacts specifically with a polypeptide of molecular weight (Mr) 180 000 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 Mr 180 000 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 Mr 180 000 which contributes to the general structure of the dense fibrillar component of the interphase nucleolus, independent of its specific transcriptional activity.
D000818:Animals; D000911:Antibodies, Monoclonal; D002466:Cell Nucleolus / Q000032:analysis*; D003429:Cross Reactions; D004586:Electrophoresis; D005260:Female; D007526:Isoelectric Point; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D009865:Oocytes / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D014981:Xenopus; D014982:Xenopus laevis
D000911:Antibodies, Monoclonal; D011506:Proteins