TY - CHAP A1 - Scheer, Ulrich A1 - Rose, Kathleen M. T1 - Localization of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immunocytochemistry N2 - Rabbit antibodies to RNA polymerase I from a rat hepatoma have been used to localize the enzyme in a variety of cells at the light and electron microscopic level. In interphase cells the immunofluorescence pattern indicated that polymerase I is contained exclusively within the nucleolus. That this fluorescence, which appeared punctated rather than uniform, represented transcriptional complexes of RNA polymerase I and rRNA genes was suggested by the observation that it was enhanced in regenerating liver and in a hepatoma and was markedly diminished in cells treated with actinomycin D. Electron microscopic immunolocalization using gold-coupled second antibodies showed that transcribed rRNA genes are located in, and probably confined to, the fibrillar centers of the nucleolus. In contrast, the surrounding dense fibrillar component, previously thought to be the site of nascent prerRNA, did not contain detectable amounts of polymerase I. During mitosis, polymerase I molecules were detected by immunofluorescence microscopy at the chromosomal nucleolus organizer region, indicating that a considerable quantity of the enzyme remains bound to the rRNA genes. From this we conclude that rRNA genes loaded with polymerase I molecules are transmitted from one cell generation to the next one and that factors other than the polymerase itself are involved in the modulation of transcription of DNA containing rRNA genes during the cell cycle. KW - nucleolus KW - nucleolus organizer KW - fibrillar centers KW - rRNA genes KW - anti-RNA polymerase I antibodies Y1 - 1984 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-33223 ER - TY - JOUR A1 - Scheer, Ulrich A1 - Lanfranchi, Gerolamo A1 - Rose, Kathleen M. A1 - Franke, Werner W. A1 - Ringertz, Nils R. T1 - Migration of rat RNA polymerase I into chick erythrocyte nuclei undergoing reactivation in chick-rat heterokaryons N2 - Transcriptionally inactive chick erythrocyte nudei were reactivated by Sendai virusinduced fusion of erythrocytes with rat L6j1 myoblasts. We used antibodies to trace the appearance of a specific protein engaged in transcription of a defined dass of genes, those coding for rRNA, during reactivation. Using immunofluorescence microscopy, we found increasing amounts of rat RNA polymerase I to appear, during a certain period of time after fusion, in the reforming nudeoli of the chick nudei. Amounts of rat RNA polymerase I sufficient to be detected by immunofluorescence microscopy had accumulated in the newly developed chick nudeoli 72- 190 h after fusion was initiated. This time interval coincides with the time when chick rRNA synthesis can first be detected. The results raise the possibility that during these stages of the reactivation process chick rRNA genes are transcribed by heterologous RNA polymerase I moleeules of rat origin. Y1 - 1983 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-33232 ER - TY - JOUR A1 - Scheer, Ulrich A1 - Hügle, Barbara A1 - Hazan, Rachel A1 - Rose, Kathleen M. T1 - Drug-induced dispersal of transcribed rRNA genes and transcriptional products: Immunolocalization and silver staining of different nucleolar components in rat cells treated with 5,6-dichloro-1-Beta-D-ribofuranosylbenzimidazole N2 - Upon incubation of cultured rat cells with the adenosine analogue 5,6-dichloro-l-β- D-ribofuranosylbenzimidazole (DRB), nucleoli reversibly dissociate into their substructures, disperse throughout the nuclear interior, and form nucleolar "necklaces". We have used this experimental system, which does not inhibit transcription of the rRNA genes, to study by immunocytochemistry the distribution of active rRNA genes and their transcriptional products during nucleolar dispersal and recovery to normal morphology. Antibodies to RNA polymerase I allow detection of template-engaged polymerase, and monoclonal antibodies to a ribosomal protein (S 1) of the small ribosomal subunit permit localization of nucleolar preribosomal particles. The results show that, under the action of DRB transcribed rRNA, genes spread throughout the nucleoplasm and finally appear in the form of several rows, each containing several (up to 30) granules positive for RNA polymerase land argyrophilic proteins. Nucleolar material containing preribosomal particles also appears in granular structures spread over the nucleoplasm but its distribution is distinct from that of rRNA gene-containing granules. We conclude that, although transcriptional units and preribosomal particles are both redistributed in response to DRB, these entities retain their individuality as functionally defined subunits. We further propose that each RNA polymerase-positive granular unit represents a single transcription unit and that each continuous array of granules ("string of nucleolar beads") reflects the linear distribution of rRNA genes along a nucleolar organizer region. Based on the total number of polymerase I-positive granules we estimate that a minimum of 60 rRNA genes are active during interphase of DRB-treated rat cells. Y1 - 1984 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-33216 ER - TY - JOUR A1 - Rose, Kathleen M. A1 - Szopa, Jan A1 - Han, Fu-Sheng A1 - Cheng, Yung-Chi A1 - Richter, Arndt A1 - Scheer, Ulrich T1 - Association of DNA topoisomerase I and RNA polymerase I: A possible role for topoisomerase I in ribosomal gene transcription N2 - RNA polymerase I preparations purified from a rat hepatoma contained DNA topoisomerase activity. The DNA topoisomerase associated with the polymerase had an Mr of 110000, required Mg2+ but not ATP, and was recognized by anti-topoisomerase I antibodies. When added to RNA polymerase I preparations containing topoisomerase activity, anti-topoisomerase I antibodies were able to inhibit the DNA relaxing activity of the preparation as well as RNA synthesis in vitro. RNA polymerase II prepared by analogous procedures did not contain topoisomerase activity and was not recognized by the antibodies. The topoisomerase I: polymerase I complex was reversibly dissociated by column chromatography on Sephacryl S200 in the presence of 0.25 M (NH4hS04. Topoisomerase I was immunolocalized in the transcriptionally active ribosomal gene complex containing RNA polymerase I in situ. These data indicate that topoisomerase I and RNA polymerase I are tightly complexed both in vivo and in vitro, and suggest a role for DNA topoisomerase I in the transcription of ribosomal genes. Y1 - 1988 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-33901 ER - TY - JOUR A1 - Reimer, Georg A1 - Rose, Kathleen M. A1 - Scheer, Ulrich A1 - Tan, Eng M. T1 - Autoantibody to RNA polymerase I in scleroderma sera N2 - Autoantibodies to components of the nucleolus are a unique serological feature of patients with scleroderma. There are autoantibodies of several specificities; one type produces a speckled pattern of nucleolar staining in immunofluorescence. In actinomycin D and 5,6-dichloro-{j-D-ribofuranosylbenzimidazoletreated Vero cells, staining was restricted to the fibrillar and not the granular regions. By double immunofluorescence, specific rabbit anti-RNA polymerase I antibodies stained the same fibrillar structures in drug-segregated nucleoli as scleroderma sera. Scleroderma sera immunoprecipitated 13 polypeptides from (35S)methionine-labeled HeLa cell extract with molecular weights ranging from 210,000 to 14,000. Similar polypeptides were precipitated by rabbit anti-RNA polymerase I antibodies, and their common identities were confirmed in immunoabsorption experiments. Microinjection of purified IgG from a patient with speckled nucleolar staining effectively inhibited ribosomal RNA transcription. Autoantibodies to RNA polymerase I were restricted to certain patients with scleroderma and were not found in other autoimmune diseases. Y1 - 1987 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-34294 ER - TY - JOUR A1 - Hadjiolova, Krassimira A1 - Rose, Kathleen M. A1 - Scheer, Ulrich T1 - Immunolocalization of nucleolar proteins after D-galactosamine-induced inhibition of transcription in rat hepatocytes N2 - No abstract available Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-33205 ER - TY - JOUR A1 - Benavente, Ricardo A1 - Rose, Kathleen M. A1 - Reimer, Georg A1 - Hügle-Dörr, Barbara A1 - Scheer, Ulrich T1 - Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells N2 - The formation of daughter nuclei and the reformation of nucleolar structures was studied after microinjection of antibodies to RNA polymerase I into dividing cultured cells (PtK2). The fate of several nucleolar proteins representing the three main structural subcomponents of the nucleolus was examined by immunofluorescence and electron microscopy. The results show that the RNA polymerase I antibodies do not interfere with normal mitotic progression or the early steps of nucleologenesis, i.e. , the aggregation of nucleolar material into prenucleolar bodies. However,they inhibit the telophasic coalescence of the prenucleolar bodies into the chromosomal nucleolar organizer regions, thus preventing the formation of new nucleoli. These prenucleolar bodies show a fibrillar organization that also compositionally resembles the dense fibrillar component of interphase nucleoli . We conclude that during normal nucleologenesis the dense fibrillar component forms from preformed entities around nucleolar organizer regions, and that this association seems to be dependent on the presence of an active form of RNA polymerase I. Y1 - 1987 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-33247 ER - TY - JOUR A1 - Benavente, Ricardo A1 - Reimer, Georg A1 - Rose, Kathleen M. A1 - Hügle-Dörr, Barbara A1 - Scheer, Ulrich T1 - Nucleolar changes after microinjection of antibodies to RNA polymerase I into the nucleus of mammalian cells N2 - After microinjection of antibodies against RNA polymerase I into the nuclei of cultured rat kangaroo (PtKz) and rat (RVF-SMC) cells alterations in nucleolar structure and composition were observed. These were detected by electron microscopy and double-label immunofluorescence microscopy using antibodies to proteins representative of the three major components of the nucleolus. The microinjected antibodies produced a progressive loss of the material of the dense fibrillar component (DFC) from the nucleoli which, at 4 h after injection, were transformed into bodies with purely granular component (GC) structure with attached fibrillar centers (FCs). Concomitantly, numerous extranucleolar aggregates appeared in the nucleoplasm which morphologically resembled fragments of the DFC and contained a protein (fibrillarin) diagnostic for this nucleolar structure. These observations indicate that the topological distribution of the material constituting the DFC can be experimentally influenced in interphase cells, apparently by modulating the transcriptional activity of the rRNA genes. These effects are different from nucleolar lesions induced by inhibitory drugs such as actinomycin D-dependent "nucleolar segregation". The structural alterations induced by antibodies to RNA polymerase I resemble, however, the initial events of nucleolar disintegration during mitotic prophase. Y1 - 1988 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-40666 ER -