A Drosophila anti-RNA polymerase II antibody recognizes a plant nucleolar antigen, RNA polymerase I, which is mostly localized in fibrillar centres

1991 ◽  
Vol 100 (1) ◽  
pp. 99-107 ◽  
Author(s):  
M. Martin ◽  
F.J. Medina
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Subunit ◽  
Rna Polymerases ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Dense Fibrillar Component ◽  
Transition Area ◽  
Fibrillar Component ◽  
Polymerase I

The distribution of nucleolar RNA polymerase in the nucleolus of onion root meristematic cells has been studied by means of an antibody originally raised against Drosophila RNA polymerase II. This antibody recognizes the homologous domains of the large subunit of the enzyme, which are highly conserved throughout evolution in the three classes of eucaryotic RNA polymerases. Given that RNA polymerase I is confined to the nucleolus, and that the onion cell nucleolus lacks digitations of extranucleolar chromatin, we conclude that the nucleolar enzyme localized is RNA polymerase I. A quantitative approach, independent of the existence of borderlines between nucleolar fibrillar centres and the dense fibrillar component, allowed us to show that the enzyme is localized in fibrillar centres and in the transition area between them and the dense fibrillar component, in parallel with the nucleolar DNA. These results, together with previous autoradiographic, cytochemical and immunocytochemical results, in this and other species, lead us to conclude that the activation of rDNA for transcription occurs in the fibrillar centres and pre-rRNA synthesis is expressed at the transition area between fibrillar centres and the dense fibrillar component. Fibrillar centres are connected to each other by extended RNA polymerase-bound DNA fibres, presumably active in transcription. This work provides evidence of the high evolutionary conservation of some domains of the large subunit of RNA polymerases and of the existence of fibrillar centres in the nucleolus of plant cells, totally homologous to those described in mammalian cells.

scholarly journals Assembly and Functional Organization of the Nucleolus: Ultrastructural Analysis ofSaccharomyces cerevisiaeMutants

2000 ◽  
Vol 11 (6) ◽  
pp. 2175-2189 ◽  
Author(s):  
Stéphanie Trumtel ◽  
Isabelle Léger-Silvestre ◽  
Pierre-Emmanuel Gleizes ◽  
Frédéric Teulières ◽  
Nicole Gas
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Functional Organization ◽  
Ultrastructural Localization ◽  
Rna Polymerase I ◽  
Wild Type ◽  
Rdna Transcription ◽  
Nucleolar Structure ◽  
Fibrillar Component ◽  
Polymerase I

Using Saccharomyces cerevisiae strains with genetically modified nucleoli, we show here that changing parameters as critical as the tandem organization of the ribosomal genes and the polymerase transcribing rDNA, although profoundly modifying the position and the shape of the nucleolus, only partially alter its functional subcompartmentation. High-resolution morphology achieved by cryofixation, together with ultrastructural localization of nucleolar proteins and rRNA, reveals that the nucleolar structure, arising upon transcription of rDNA from plasmids by RNA polymerase I, is still divided in functional subcompartments like the wild-type nucleolus. rRNA maturation is restricted to a fibrillar component, reminiscent of the dense fibrillar component in wild-type cells; a granular component is also present, whereas no fibrillar center can be distinguished, which directly links this latter substructure to rDNA chromosomal organization. Although morphologically different, the mininucleoli observed in cells transcribing rDNA with RNA polymerase II also contain a fibrillar subregion of analogous function, in addition to a dense core of unknown nature. Upon repression of rDNA transcription in this strain or in an RNA polymerase I thermosensitive mutant, the nucleolar structure falls apart (in a reversible manner), and nucleolar constituents partially relocate to the nucleoplasm, indicating that rRNA is a primary determinant for the assembly of the nucleolus.

scholarly journals Localisation of the Ki-67 antigen within the nucleolus. Evidence for a fibrillarin-deficient region of the dense fibrillar component

1996 ◽  
Vol 109 (6) ◽  
pp. 1253-1263 ◽  
Author(s):  
I.R. Kill
Keyword(s):  
Rna Polymerase ◽  
Indirect Immunofluorescence ◽  
Dermal Fibroblasts ◽  
Rna Polymerase I ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dense Fibrillar Component ◽  
Ki 67 ◽  
Fibrillar Component ◽  
Polymerase I

The Ki-67 antigen is detected in proliferating cells in all phases of the cell division cycle. Throughout most of interphase, the Ki-67 antigen is localised within the nucleous. To learn more about the relationship between the Ki-67 antigen and the nucleolus, we have compared the distribution of Ki-67 antibodies with that of a panel of antibodies reacting with nucleolar components by confocal laser scanning microscopy of normal human dermal fibroblasts in interphase stained in a double indirect immunofluorescence assay. During early G1, the Ki-67 antigen is detected at a large number of discrete foci throughout the nucleoplasm, extending to the nuclear envelope. During S-phase and G2, the antigen is located in the nucleolus. Double indirect immunofluorescence studies have revealed that during early to mid G1 the Ki-67 antigen is associated with reforming nucleoli within discrete domains which are distinct from domains containing two of the major nucleolar antigens fibrillarin and RNA polymerase I. Within mature nucleoli the Ki-67 antigen is absent from regions containing RNA polymerase I and displays only partial co-localisation within domains containing either fibrillarin or B23/nucleophosmin. Following disruption of nucleolar structure, induced by treatment of cells with the drug 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole or with actinomycin D, the Ki-67 antigen translocates to nucleoplasmic foci which are associated with neither fibrillarin nor RNA polymerase I. However, in treated cells the Ki-67 Ag remains associated with, but not co-localised to, regions containing B23/nucleophosmin. Our observations suggest that the Ki-67 antigen associates with a fibrillarin-deficient region of the dense fibrillar component of the nucleolus. Integrity of this region is lost following either nucleolar dispersal or nucleolar segregation.

The yeast alpha 2 protein can repress transcription by RNA polymerases I and II but not III

1993 ◽  
Vol 13 (7) ◽  
pp. 4029-4038
Author(s):  
B M Herschbach ◽  
A D Johnson
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Rna Polymerases ◽  
Rna Polymerase I ◽  
Cell Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cell Type Specific ◽  
Polymerase I ◽  
Rna Polymerase Ii Transcription

The alpha 2 protein of the yeast Saccharomyces cerevisiae normally represses a set of cell-type-specific genes (the a-specific genes) that are transcribed by RNA polymerase II. In this study, we determined whether alpha 2 can affect transcription by other RNA polymerases. We find that alpha 2 can repress transcription by RNA polymerase I but not by RNA polymerase III. Additional experiments indicate that alpha 2 represses RNA polymerase I transcription through the same pathway that it uses to repress RNA polymerase II transcription. These results implicate conserved components of the transcription machinery as mediators of alpha 2 repression and exclude several alternate models.

Three-dimensional organization of active rRNA genes within the nucleolus

2002 ◽  
Vol 115 (16) ◽  
pp. 3297-3307 ◽  
Author(s):  
Thierry Cheutin ◽  
Marie-Françoise O'Donohue ◽  
Adrien Beorchia ◽  
Marc Vandelaer ◽  
Hervé Kaplan ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Ultrastructural Level ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Fibrillar Component ◽  
Polymerase I

In this work, we have localized transcribing rRNA genes at the ultrastructural level and described their three-dimensional organization within the nucleolus by electron tomography. Isolated nucleoli, which exhibit a reduced transcriptional rate, were used to determine the sites of initial BrUTP incorporation (i.e. rRNA synthesis by the transcriptional machinery). Using pulse-chase experiments with BrUTP and an elongation inhibitor,cordycepin, it was possible to precisely localize the initial sites of BrUTP incorporation. Our data show that BrUTP incorporation initially takes place in the fibrillar centers and that elongating rRNAs rapidly enter the surrounding dense fibrillar component. Furthermore, we investigated the spatial arrangement of RNA polymerase I molecules within the whole volume of the fibrillar centers. Electron tomography was performed on thick sections of cells that had been labeled with anti-RNA polymerase I antibodies prior to embedding. Detailed tomographic analyses revealed that RNA polymerase I molecules are mainly localized within discrete clusters. In each of them, RNA polymerase I molecules were grouped as several coils, 60 nm in diameter. Overall, these findings have allowed us to propose a model for the three-dimensional organization of transcribing rDNA genes within the nucleolus.

scholarly journals Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells.

1987 ◽  
Vol 105 (4) ◽  
pp. 1483-1491 ◽  
Author(s):  
R Benavente ◽  
K M Rose ◽  
G Reimer ◽  
B Hügle-Dörr ◽  
U Scheer
Keyword(s):  
Rna Polymerase ◽  
Cultured Cells ◽  
Active Form ◽  
Nucleolar Organizer ◽  
Rna Polymerase I ◽  
Nucleolar Organizer Regions ◽  
Dense Fibrillar Component ◽  
Nucleolar Material ◽  
Fibrillar Component ◽  
Polymerase I

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.

Structural alterations of the nucleolus in mutants of Saccharomyces cerevisiae defective in RNA polymerase I

1993 ◽  
Vol 13 (4) ◽  
pp. 2441-2455
Author(s):  
M Oakes ◽  
Y Nogi ◽  
M W Clark ◽  
M Nomura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Rrna Processing ◽  
Structural Element ◽  
Nucleolar Structure ◽  
Pol I ◽  
Polymerase I

We have previously constructed mutants of Saccharomyces cerevisiae in which the gene for the second-largest subunit of RNA polymerase I (Pol I) is deleted. In these mutants, rRNA is synthesized by RNA polymerase II from a hybrid gene consisting of the 35S rRNA coding region fused to the GAL7 promoter on a plasmid. These strains thus grow in galactose but not glucose media. By immunofluorescence microscopy using antibodies against the known nucleolar proteins SSB1 and fibrillarin, we found that the intact crescent-shaped nucleolar structure is absent in these mutants; instead, several granules (called mininucleolar bodies [MNBs]) that stained with these antibodies were seen in the nucleus. Conversion of the intact nucleolar structure to MNBs was also observed in Pol I temperature-sensitive mutants at nonpermissive temperatures. These MNBs may structurally resemble prenucleolar bodies observed in higher eukaryotic cells and may represent a constituent of the normal nucleolus. Furthermore, cells under certain conditions that inhibit rRNA synthesis did not cause conversion of the nucleolus to MNBs. Thus, the role of Pol I in the maintenance of the intact nucleolar structure might include a role as a structural element in addition to (or instead of) a functional role to produce rRNA transcripts. Our study also shows that the intact nucleolar structure is not absolutely required for rRNA processing, ribosome assembly, or cell growth and that MNBs are possibly functional in rRNA processing in the Pol I deletion mutants.

scholarly journals Structural alterations of the nucleolus in mutants of Saccharomyces cerevisiae defective in RNA polymerase I.

1993 ◽  
Vol 13 (4) ◽  
pp. 2441-2455 ◽  
Author(s):  
M Oakes ◽  
Y Nogi ◽  
M W Clark ◽  
M Nomura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Rrna Processing ◽  
Structural Element ◽  
Nucleolar Structure ◽  
Pol I ◽  
Polymerase I

We have previously constructed mutants of Saccharomyces cerevisiae in which the gene for the second-largest subunit of RNA polymerase I (Pol I) is deleted. In these mutants, rRNA is synthesized by RNA polymerase II from a hybrid gene consisting of the 35S rRNA coding region fused to the GAL7 promoter on a plasmid. These strains thus grow in galactose but not glucose media. By immunofluorescence microscopy using antibodies against the known nucleolar proteins SSB1 and fibrillarin, we found that the intact crescent-shaped nucleolar structure is absent in these mutants; instead, several granules (called mininucleolar bodies [MNBs]) that stained with these antibodies were seen in the nucleus. Conversion of the intact nucleolar structure to MNBs was also observed in Pol I temperature-sensitive mutants at nonpermissive temperatures. These MNBs may structurally resemble prenucleolar bodies observed in higher eukaryotic cells and may represent a constituent of the normal nucleolus. Furthermore, cells under certain conditions that inhibit rRNA synthesis did not cause conversion of the nucleolus to MNBs. Thus, the role of Pol I in the maintenance of the intact nucleolar structure might include a role as a structural element in addition to (or instead of) a functional role to produce rRNA transcripts. Our study also shows that the intact nucleolar structure is not absolutely required for rRNA processing, ribosome assembly, or cell growth and that MNBs are possibly functional in rRNA processing in the Pol I deletion mutants.

scholarly journals The yeast alpha 2 protein can repress transcription by RNA polymerases I and II but not III.

1993 ◽  
Vol 13 (7) ◽  
pp. 4029-4038 ◽  
Author(s):  
B M Herschbach ◽  
A D Johnson
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Rna Polymerases ◽  
Rna Polymerase I ◽  
Cell Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cell Type Specific ◽  
Polymerase I ◽  
Rna Polymerase Ii Transcription

The alpha 2 protein of the yeast Saccharomyces cerevisiae normally represses a set of cell-type-specific genes (the a-specific genes) that are transcribed by RNA polymerase II. In this study, we determined whether alpha 2 can affect transcription by other RNA polymerases. We find that alpha 2 can repress transcription by RNA polymerase I but not by RNA polymerase III. Additional experiments indicate that alpha 2 represses RNA polymerase I transcription through the same pathway that it uses to repress RNA polymerase II transcription. These results implicate conserved components of the transcription machinery as mediators of alpha 2 repression and exclude several alternate models.

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