(oocyte nuclei, egg extracts, and somatic cells was analyzed by immunoblotting with NO29-specific antibodies. that NO29 might be involved in the nuclear and nucleolar accumulation of ribosomal proteins and the coordinated assembly of pre-ribosomal particles. The packaging of DNA and histones into nucleosomes as a first step in chromatin formation and the packaging of rRNA and ribosomal proteins (r-proteins) during ribosome formation are, at first glance, two basically different and for the most part spatially separated processes. The former occurs at chromatin borders in the nucleoplasm and the latter is confined to the nucleolus. However, in both processes, the eukaryotic cell has to overcome a common chemical problemi.e., the prevention of nonspecific associations and aggregations of these basic proteins with the numerous negatively charged nucleic acids and proteins abundant in the cytoplasm and in the nucleus. Biochemical studies have identified several proteins containing acidic regions that bind histones and can transfer them onto DNA to form nucleosome cores. Examples include major proteins of the amphibian oocyte such as nucleoplasmin and protein N1/N2 (1C5) as well as somatic cell proteins such as nucleoplasmin S (6), CAF-1 (7), and other related proteins (8C10). During ribosome assembly, AZD7507 the concerted interaction of r-proteins and rRNA is believed to involve certain nonribosomal nucleolar proteins (reviewed in 11C13). One of the best characterized among them is protein NO38, a major constituent of the granular component of nucleoli of (14), a homolog of mammalian protein B23 (15C19). Like nucleoplasmin (20), protein NO38/B23 tends to form stable oligomers (14, 21) and can shuttle between the nucleus and the cytoplasm (22). Because of its sequence homology to nucleoplasmin and its reported ability to bind to nucleic acids (23C25), protein NO38/B23 has been proposed to promote ribosome assembly and transport across the nuclear envelope (12). A direct association of protein NO38/B23 and r-proteins, however, has not yet been demonstrated. In addition, protein NO38/B23 has been reported to possess ribonuclease activity (26) and to associate with transcription factor YY1 (27), protein Rev of HIV-1 (28C30), and nucleolar protein p120 (31), suggesting that this protein has multiple functions. Although sequence elements required for the specific nucleolar accumulation of protein NO38/B23 have been described (32, 33), its direct and constitutive binding partners within the nucleolus remain to be identified. In immunoprecipitation experiments and affinity chromatography using cellular and nuclear extracts of cultured cells and antibodies to protein NO38 (14), we have recently isolated and cDNA-cloned a novel nucleolar, very acidic protein with an SDS/PAGE mobility corresponding to AZD7507 kidney epithelial cells (XLKE, line A6) and human hepatocellular carcinoma cells of line PLC have been described (34). Isolation and Fractionation of Oocyte Nuclei, Egg Extracts, and Ribosomal Subunits. Nuclei of mature (stages IVCVI) oocytes were isolated and fractionated (35) into low speed pellet (LSP), high speed pellet (HSP), and high speed supernatant (HSS). LSP fractions were cleared from yolk proteins by Freon extraction (36). Preparations of total egg extracts (37), ribosomes, and ribosomal subunits have been described (38). Immunoprecipitations from Cellular and AZD7507 Nuclear Extracts of A6 Cells. For l-[35S]methionine labeling, A6 cells were grown in methionine-reduced minimal essential medium, with 10% fetal calf serum and l-[35S]methionine, for 16 h before harvest. Cellular extracts were prepared as described (33). Nuclei from somatic cells were prepared according to Miake-Lye and Kirschner (39), resuspended in 5:1 buffer (10 mM Tris?HCl, pH 7.4/83 mM KCl/l7 mM NaCl/2 mM MgCl2), homogenized by sonication (Branson sonifier B-12; 5 20 sec, 75 W), and fractionated as described for oocyte nuclei. The resulting LSP was extracted in PBS, supplemented with 250 mM NaCl, for 20 min on ice. mAb 9E10 have been described (14, 33). Antibodies specific for the newly identified protein NO29 were obtained by immunization of guinea pigs with two synthesized peptides (40), EVTVPLANLK and SGFISSTAAQGPPSPAIE, coupled to keyhole limpet hemocyanin. While the first peptide sequence had originally been identified by direct amino acid sequencing of a polypeptide spot obtained after immunochromatography of cellular extracts with mAb No-185 and two-dimensional (2D) gel electrophoresis of the eluted proteins, the second one was deduced from the NO29 cDNA. All results shown have been obtained with antibodies affinity-purified on iodoacetyl-immobilized peptides (40). Gel Electrophoresis and Immunoblotting. SDS/PAGE and 2D-PAGE of proteins, using either isoelectric focusing (IEF) or nonequilibrium pH gradient electrophoresis (NEPHGE) in the first dimension separation were as described (14, 34, 35, 37). Immunoblotting was performed on Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore) as described (33), using protein NO29 antibodies, diluted 1:500 in AZD7507 Tris-buffered saline, and peroxidase-coupled secondary antibodies (Dianova, Hamburg, Germany) detected with the ECL system (Amersham). Isolation of cDNA Mouse monoclonal to MPS1 Clones. Total DNA from a Unizap cDNA library from kidney (Stratagene) was used for PCR with the library-specific reverse primer as sense primer and a degenerated antisense primer deduced from the amino acid sequence DEHNVVEVTAPN. An.