Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the present study immunofluorescence confocal laser scanning microscopy, autoradiography following (3)H-uridine incubation and transmission electron microscopy were used to evaluate the nucleolar protein localization, transcriptional activity, and nucleolar ultrastructure during genomic re-programming in bovine embryos reconstructed by nuclear transfer from granulosa cells into non-activated cytoplasts followed by activation. During the 1st cell cycle (1-cell embryos), no autoradiographic labelling was detected. Ultrastructurally, nucleoli devoid of a granular component were observed. During the 2nd cell cycle (2-cell embryos) autoradiographic labelling was also lacking and the embryos displayed varying degrees of nucleolar inactivation. During both the 3rd (4-cell embryos) and 4th (tentative 8-cell embryos), cell cycles autoradiographic labelling was lacking in some embryos, while others displayed labelling and associated formation of fibrillo-granular nucleoli. During the 5th cell cycle (tentative 16-cell embryos), all embryos displayed autoradiographic labelling and fibrillo-granular nucleoli. In some blastomeres, however, deviant nucleolar ultrastructure was observed. During the first cell cycle labelling of RNA polymerase I, fibrillarin, upstream binding factor (UBF) and nucleolin (C23) was localized to nuclear entities. During the 2nd cell cycle, only labelling of RNA polymerase I and fibrillarin persisted. During the 3rd and 4th cell cycle labelling of fibrillarin persisted, labelling of nucleophosmin (B23) appeared and that of nucleolin re-appeared. During the 5th cell cycle almost all embryos showed complete labelling of all proteins except for UBF, which lacked in more than half of the embryos. In conclusion, bovine granulosa cell nuclear transfer embryos showed re-modelling of the nucleoli to an inactive form followed by re-formation of fibrillo-granular nucleoli. The re-formation of fibrillo-granular nucleoli was initiated already during the 3rd cell cycle, which is one cell cycle earlier than in in vivo- and in vitro-derived bovine embryos. Moreover, in more than half of the embryos, UBF could not be immunocytochemically localized to the nucleolar compartment during the 5th cell cycle indicating lack of developmental potentials.
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PMID:Nucleolar protein allocation and ultrastructure in bovine embryos produced by nuclear transfer from granulosa cells. 1189 19

In the present study, immunofluorescence confocal laser scanning microscopy, autoradiography following (3)H-uridine incubation, and transmission electron microscopy were used to evaluate the nucleolar protein localization, transcriptional activity, and nucleolar ultrastructure during genomic reprogramming in bovine embryos reconstructed by nuclear transfer from in vitro-produced bovine morulae to activated cytoplasts. During the first cell cycle (one-cell embryos), no autoradiographic labelling was detected. Ultrastructurally, whorls consisting of densely packed fibrillar material were observed instead of nucleoli. During the second, third, and fourth cell cycle (two-, four-, and tentative eight-cell embryos), autoradiographically unlabelled nuclei contained vacuolated bodies consisting of densely packed fibrillar material. Also, during the fourth cell cycle, the first nucleoplasmic autoradiographic labelling was observed, but still without formation of fibrillo-granular nucleoli. During the fifth cell cycle (tentative 16-cell embryos), the nuclei displayed autoradiographic labelling over both nucleoplasm and presumptive nucleoli, and the formation of fibrillo-granular nucleoli was observed. In a certain proportion of blastomeres, however, abnormal patterns of nucleolar formation and apoptosis were noted. During the first two cell cycles, labelling of RNA polymerase I, fibrillarin, upstream binding factor (UBF), nucleolin (C23), and nucleophosmin (B23) was localized to nuclear entities. During the third cell cycle, labelling of topoisomerase I was observed in addition. During the fourth and fifth cell cycles, a substantial portion of the embryos presented blastomeres that lacked labelling of several of these nucleolar proteins. In conclusion, the nuclear transfer procedure was associated with remodelling of the nucleoli to an inactive form, followed by reformation of fibrillo-granular nucleoli during the fifth cell cycle. Moreover, a certain proportion of blastomeres failed to form functional nucleoli with respect to both ultrastructural organization and protein allocation.
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PMID:Nucleolar protein allocation and ultrastructure in bovine embryos produced by nuclear transfer from embryonic cells. 1190 Jun 41

The nucleolus is the site of ribosome biosynthesis, but is now known to have other functions as well. In the present study we have investigated how the distribution of signal recognition particle (SRP) RNA within the nucleolus relates to the known sites of ribosomal RNA synthesis, processing, and nascent ribosome assembly (i.e., the fibrillar centers, the dense fibrillar component (DFC), and the granular component). Very little SRP RNA was detected in fibrillar centers or the DFC of the nucleolus, as defined by the RNA polymerase I-specific upstream binding factor and the protein fibrillarin, respectively. Some SRP RNA was present in the granular component, as marked by the protein B23, indicating a possible interaction with ribosomal subunits at a later stage of maturation. However, a substantial portion of SRP RNA was also detected in regions of the nucleolus where neither B23, UBF, or fibrillarin were concentrated. Dual probe in situ hybridization experiments confirmed that a significant fraction of nucleolar SRP RNA was not spatially coincident with 28S ribosomal RNA. These results demonstrate that SRP RNA concentrates in an intranucleolar location other than the classical stations of ribosome biosynthesis, suggesting that there may be nucleolar regions that are specialized for other functions.
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PMID:Signal recognition particle RNA localization within the nucleolus differs from the classical sites of ribosome synthesis. 1242 65

After fertilization, ribosomal RNA synthesis is silenced during a period which depends on the species. Data concerning the reassembly of a functional nucleolus remain scarce. We have examined by immunocytochemistry, Western blots, and BrUTP microinjection the dynamics of major nucleolar proteins during the first cycles of mouse embryogenesis, in relation to rDNA transcription sites and coilin, a marker of Cajal bodies. We show that: (1) the reinitiation of rDNA transcription occurs at the two-cell stage, 44-45 h after hCG injection (hphCG), at the surface of the nucleolar precursor bodies (NPBs), where the RNA polymerase I (pol I) transcription complex is recruited 4-5 h before; (2) the NPBs are not equal in their ability to support recruitment of pol I and rDNA transcription; (3) maternally inherited fibrillarin undergoes a dynamic redistribution during the second cell stage, together with coilin, leading to the assembly of the Cajal body around 40 hphCG; and (4) the pol I complex is first recruited to the Cajal body before reaching its rDNA template. We also find that fibrillarin and B23 are both directly assembled around NPBs prior to ongoing pre-rRNA synthesis. Altogether, our results reveal a role of the Cajal bodies in the building of a functional nucleolus.
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PMID:The step-wise assembly of a functional nucleolus in preimplantation mouse embryos involves the cajal (coiled) body. 1249 Jan 98

Research on autoimmune diseases has revealed that autoimmunity can be induced by heavy metals such as mercury and gold. Following the introduction of platinum-containing catalytic converters in automobiles, the emission of platinum compounds constitutes an abundant environmental pollutant, however, potential immunological hazards resulting from platinum-containing emissions were not yet examined. In our previous studies on molecular mechanisms of heavy metal-induced autoimmunity, we showed a platinum-dependent subcellular redistribution of the autoantigen fibrillarin from the nucleolus to the nucleoplasm. Since H-2s mice constitute a valuable model to study the role of heavy metals in the development of systemic autoimmunity, we treated susceptible B10.S mice with hexachloroplatinate (Na2PtCl6, Pt4+) to examine whether platinum induces the production of autoantibodies. The present study shows for the first time that chronic administration of Pt4+ generated an autoimmune response in mice which targets distinct nucleoplasmic antigens. Dual-labeling revealed substantial colocalization of these nucleoplasmic autoantigens with (i) nascent RNA, (ii) the active, phosphorylated form of RNA polymerase II, and partial overlap with (iii) acetylated histone 4 protein, and (iv) 20S proteasomes in dendritic cells isolated from platinum-treated mice. The results suggest that platinum elicits antibodies against antigens associated with active sites of transcription which may be subject to proteasomal processing during heavy metal-induced autoimmunity.
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PMID:Platinum-induced autoantibodies target nucleoplasmic antigens related to active transcription. 1260 22

In the present study, ribosomal RNA (rRNA) gene activation, monitored through nucleolus development, was studied by autoradiography following (3)H-uridine incubation, transmission electron microscopy, and immunofluorescence confocal laser scanning microscopy of key nucleolar proteins involved in rRNA transcription (topoisomerase I, upstream binding factor, and RNA polymerase I) and processing (fibrillarin, nucleolin, and nucleophosmin) in in vivo developed, in vitro produced, and parthenogenetic bovine embryos. In general, in vivo developed embryos displayed formation of fibrillo-granular nucleoli during the 4th post-fertilization cell cycle. During the previous stages of development, nucleolus precursor bodies (NPBs) were observed. However, on some occasions the initial steps of nucleolus formation were observed already at the 2- and 4-cell stage in cases where such embryos were collected from superovulated animals together with later embryonic stages presenting nucleolar development and autoradiographic labeling. The in vitro produced embryos displayed very synchronous formation of fibrillo-granular nucleoli and autoradiographic labeling during the 4th cell cycle. In vivo developed and in vitro produced embryos displayed allocation of nucleolar proteins to fibrillar and granular compartments of the developing nucleoli during the 4th cell cycle. The parthenogenetic embryos typically displayed formation of fibrillo- granular nucleoli during the 5th cell cycle and autoradiographic labeling was not observed until the morula stage. Moreover, the 1-, 2-, and 4-cell parthenogenetic embryos practically lacked NPBs. On the other hand, parthenogenetic embryos displayed allocation of nucleoar proteins to nuclear entities during the 4th cell cycle. In conclusion, both in vivo developed and in vitro produced bovine embryos displayed activation of transcription and nucleolar development during the 4th cell cycle. However, in vivo developed embryos flushed together with later developmental stages displayed premature activation of these processes. Parthenogenetic bovine embryos, on the other hand, displayed a delayed activation.
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PMID:Nucleolar proteins and ultrastructure in bovine in vivo developed, in vitro produced, and parthenogenetic cleavage-stage embryos. 1265 36

Biogenesis of functional spliceosomal small nuclear RNAs (snRNAs) includes the post-transcriptional covalent modification of numerous internal nucleotides. We have recently demonstrated that synthesis of 2'-O-methylated nucleotides and pseudouridines in the RNA polymerase II-synthesized Sm snRNAs is directed by sequence-specific guide RNAs. Here, we provide evidence supporting the notion that modification of Sm snRNAs occurs in nucleoplasmic Cajal bodies (CBs), where modification guide RNAs accumulate. We show that short fragments of Sm snRNAs are correctly and efficiently modified when targeted to CBs, but not when these same fragments are targeted to the nucleolus. We also demonstrate that internal modification of the U2 snRNA occurs exclusively after nuclear import of the newly assembled Sm snRNP from the cytoplasm. Finally, we show that p80 coilin, the CB marker protein, is not required for snRNA modification. In coilin knockout cells, Sm snRNAs and their modification guide RNAs colocalize in residual CBs, which do not stockpile fibrillarin and fail to recruit the U3 small nucleolar RNA.
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PMID:Modification of Sm small nuclear RNAs occurs in the nucleoplasmic Cajal body following import from the cytoplasm. 1268 20

The expression of nucleolar-related proteins was studied as an indirect marker of the ribosomal RNA (rRNA) gene activation in porcine embryos up to the blastocyst stage produced in vivo and in vitro. A group of the in vivo-developed embryos were cultured with alpha-amanitin to block the de novo embryonic mRNA transcription. Localization of proteins involved in the rRNA transcription (upstream binding factor [UBF], topoisomerase I, RNA polymerase I [RNA Pol I], and the RNA Pol I-associated factor PAF53) and processing (fibrillarin, nucleophosmin, and nucleolin) was assessed by immunocytochemistry and confocal laser-scanning microscopy, and mRNA expression was determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). These findings were correlated with ultrastructural data and autoradiography following 20-min [3H]uridine incubation. Additionally, expression of the pocket proteins pRb and p130, which are involved in cell-cycle regulation, was assessed by semiquantitative RT-PCR up to the blastocyst stage. Toward the end of third cell cycle, the nuclei in non-alpha-amanitin-treated, in vivo-produced embryos displayed different stages of transformation of the nuclear precursor bodies (NPBs) into fibrillogranular nucleoli associated with autoradiographic labeling. However, on culture with alpha-amanitin, NPBs were not transformed into a fibrillogranular nucleolus during this cell cycle, demonstrating that embryonic nucleogenesis requires de novo mRNA transcription. Moreover, immunolocalization of RNA Pol I, but not of UBF, and the mRNA expression of PAF53 and UBF were significantly reduced or absent after culture with alpha-amanitin, indicating that RNA Pol I, PAF53, and presumably, UBF are derived from de novo embryonic transcription. Embryonic genomic activation was delayed in porcine embryos produced in vitro compared to the in vivo-derived counterparts with respect to mRNAs encoding PAF53 and UBF. Moreover, differences existed in the mRNA expression patterns of pRb between in vivo- and in vitro-developed embryos. These findings show, to our knowledge for the first time, a nucleolus-related gene expression in the preimplantation porcine embryo, and they highlight the differences in quality between in vivo and in vitro-produced embryos.
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PMID:Expression of nucleolar-related proteins in porcine preimplantation embryos produced in vivo and in vitro. 1458 13

The aim of this study was to describe the dynamic changes in the localization of the key nucleolar protein markers, fibrillarin, B23/nucleophosmin, C23/nucleolin, protein Nopp140, during the final stages of bovine oocyte growth. All these proteins were present in the large reticulated nucleoli of oocytes from the small-size category follicles (<1 mm). The entire nucleolus exhibited strong positivity for UBF (upstream binding factor, RNA polymerase I-specific transcription initiation factor), which displayed a dotted staining pattern. In contrast, protein p130 was diffusely distributed throughout the nucleus and excluded from nucleoli. In oocytes approaching the late period of growth (2-3-mm follicles), UBF localization shifted to the nucleolar periphery. Double staining of UBF-p130 revealed a gradual accumulation of p130 at the periphery shell around the nucleolus. In fully grown oocytes (>3-mm follicles), all studied nucleolar proteins were detected in the small compact nucleoli. The cap structure, attached to the compact nucleolus surface, was positive for UBF and PAF53 (subunit of RNA polymerase I). The UBF-positive cap showed a close structural association with p130. It is concluded that, during the process of oocyte nucleolus compaction, UBF and PAF53, proteins involved in the rDNA transcription, are segregated from fibrillarin and Nopp140, proteins essential for early steps of pre-rRNA processing. The observed changes may reflect the transition from pre-rRNA synthesis to pre-rRNA processing as an analysis of the relative abundance of the developmentally important gene transcripts confirmed. In addition, discovered structural association between UBF and p130 suggests a role for pocket proteins in ribosomal gene silencing in mammalian oocytes.
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PMID:Immunolocalization of upstream binding factor and pocket protein p130 during final stages of bovine oocyte growth. 1461 6

The nucleolus formation was studied as an indirect marker of the ribosomal RNA (rRNA) genes activation in porcine embryos following oocyte maturation, fertilization, and culture in vitro. Nucleologenesis was assessed by transmission electron microscopy (TEM), light microscopical autoradiography following 20 min of 3H-uridine incubation, and immunocytochemical localization of key nucleolar proteins involved in rRNA transcription (upstream binding factor (UBF), topoisomerase I, and RNA polymerase I) and processing (fibrillarin, nucleophosmin, nucleolin) by confocal laser scanning microscopy. During the first four post-fertilization cell cycles, TEM revealed spherical nucleolus precursor bodies (NPBs), consisting of densely packed fibrils, as the most prominent intra-nuclear entities of the blastomeres. Fibrillo-granular nucleoli were observed in some blastomeres in a single embryo during the 5th cell cycle, i.e., the tentative 16-cell stage, where formation of fibrillar centres (FC), a dense fibrillar component, and a granular component on the surface of the NPBs was seen. In this embryo, autoradiographic labeling was detected over the nucleoplasm and in particular over the nucleoli. Fibrillarin was immunocytochemically localized in the presumptive NPBs of the pronuclei. This protein was again localized to the presumptive NPBs together with nucleolin from late during the 3rd cell cycle, i.e., the four-cell stage in some embryos. UBF, RNA polymerase I, and nucleophosmin were localized to the presumptive NPBs in a proportion of the embryos at the 4th cell cycle, i.e., the tentative eight-cell stage and onwards. Toposiomerase I was not localized to intra-nuclear entities even during the 5th post-fertilization cell cycle. Moreover, a considerable proportion of the blastomere nuclei apparently did not show localization of other nucleolar proteins. In conclusion, porcine embryos produced in vitro display a substantial delay in or even lack of the development of functional nucleoli.
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PMID:Nucleolar ultrastructure and protein allocation in in vitro produced porcine embryos. 1511 26


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