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RNA Polymerase I (rna + polymerase_i)
Selected AbstractsMyosin Vb localises to nucleoli and associates with the RNA polymerase I transcription complexCYTOSKELETON, Issue 12 2009Andrew J. Lindsay Abstract It is becoming increasingly clear that the mammalian class V myosins are involved in a wide range of cellular processes such as receptor trafficking, mRNA transport, myelination in oligodendrocytes and cell division. Using paralog-specific antibodies, we observed significant nuclear localisation for both myosin Va and myosin Vb. Myosin Vb was present in nucleoli where it co-localises with RNA polymerase I, and newly synthesised ribosomal RNA (rRNA), indicating that it may play a role in transcription. Indeed, its nucleolar pattern was altered upon treatment with RNA polymerase I inhibitors. In contrast, myosin Va is largely excluded from nucleoli and is unaffected by these inhibitors. Myosin Vb was also found to physically associate with RNA polymerase I and actin in co-immunoprecipitation experiments. We propose that myosin Vb serves a role in rRNA transcription. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Modulation of oat arginine decarboxylase gene expression and genome organization in transgenic Trypanosoma cruzi epimastigotesFEBS JOURNAL, Issue 3 2006María P. Serra We have previously demonstrated that wild-type Trypanosoma cruzi epimastigotes lack arginine decarboxylase (ADC) enzymatic activity as well as its encoding gene. A foreign ADC has recently been expressed in T. cruzi after transformation with a recombinant plasmid containing the complete coding region of the oat ADC gene. In the present study, upon modulation of exogenous ADC expression, we found that ADC activity was detected early after transfection; subsequently it decreased to negligible levels between 2 and 3 weeks after electroporation and was again detected ,,4 weeks after electroporation. After this period, the ADC activity increased markedly and became expressed permanently. These changes of enzymatic activity showed a close correlation with the corresponding levels of ADC transcripts. To investigate whether the genome organization of the transgenic T. cruzi underwent any modification related to the expression of the heterologous gene, we performed PCR amplification assays, restriction mapping and pulse-field gel electrophoresis with DNA samples or chromosomes obtained from parasites collected at different time-points after transfection. The results indicated that the transforming plasmid remained as free episomes during the transient expression of the foreign gene. Afterwards, the free plasmid disappeared almost completely for several weeks and, finally, when the expression of the ADC gene became stable, two or more copies of the transforming plasmid arranged in tandem were integrated into a parasite chromosome (1.4 Mbp) bearing a ribosomal RNA locus. The sensitivity of transcription to ,-amanitin strongly suggests involvement of the protozoan RNA polymerase I in the transcription of the exogenous ADC gene. [source] Nuclear changes in necrotic HL-60 cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue S36 2001Roberta Bortul Abstract Cell death in eukaryotes can occur by either apoptosis or necrosis. Apoptosis is characterized by well-defined nuclear changes which are thought to be the consequence of both proteolysis and DNA fragmentation. On the other hand, the nuclear modifications that occur during necrosis are largely less known. Here, we have investigated whether or not nuclear modifications occur during ethanol-induced necrotic cell death of HL-60 cells. By means of immunofluorescence staining, we demonstrate that the patterns given by antibodies directed against some nuclear proteins (lamin B1, NuMA, topoisomerase II,, SC-35, B23/nucleophosmin) changed in necrotic cells. The changes in the spatial distribution of NuMA strongly resembled those described to occur during apoptosis. On the contrary, the fluorescent pattern characteristic for other nuclear proteins (C23/nucleolin, UBF, fibrillarin, RNA polymerase I) did not change during necrosis. By immunoblotting analysis, we observed that some nuclear proteins (SAF-A, SATB1, NuMA) were cleaved during necrosis, and in the case of SATB1, the apoptotic signature fragment of 70 kDa was also present to the same extent in necrotic samples. Caspase inhibitors did not prevent proteolytic cleavage of the aforementioned polypeptides during necrosis, while they were effective if apoptosis was induced. In contrast, lamin B1 and topoisomerase II, were uncleaved in necrotic cells, whereas they were proteolyzed during apoptosis. Transmission electron microscopy analysis revealed that slight morphological changes were present in the nuclear matrix fraction prepared from necrotic cells. However, these modifications (mainly consisting of a rarefaction of the inner fibrogranular network) were not as striking as those we have previously described in apoptotic HL-60 cells. Taken together, our results indicate that during necrosis marked biochemical and morphological changes do occur at the nuclear level. These alterations are quite distinct from those known to take place during apoptosis. Our results identify additional biochemical and morphological criteria that could be used to discriminate between the two types of cell death. J. Cell. Biochem. Suppl. 36: 19,31, 2001. © 2001 Wiley-Liss, Inc. [source] Rpc25, a conserved RNA polymerase III subunit, is critical for transcription initiationMOLECULAR MICROBIOLOGY, Issue 1 2005Cécile Zaros Summary Rpc25 is a strongly conserved subunit of RNA polymerase III with homology to Rpa43 in RNA polymerase I, Rpb7 in RNA polymerase II and the archaeal RpoE subunit. A central domain of Rpc25 can replaced the corresponding region of Rpb7 with little or no growth defect, underscoring the functional relatedness of these proteins. Rpc25 forms a heterodimer with Rpc17, another conserved component of RNA polymerase III. A conditional mutant (rpc25-S100P) impairs this interaction. rpc25-S100P and another conditional mutant obtained by complementation with the Schizosaccharomyces pombe subunit (rpc25-Sp) were investigated for the properties of their purified RNA polymerase III. The mutant enzymes were defective in the specific synthesis of pre-tRNA transcripts but acted at a wild-type level on poly[d(A-T)] templates. They were also indistinguishable from wild type in transcript elongation, cleavage and termination. These data indicate that Rpc25 is needed for transcription initiation but is not critical for the elongating properties of RNA polymerase III. [source] Overlapping sense and antisense transcription units in Trypanosoma bruceiMOLECULAR MICROBIOLOGY, Issue 4 2001Matthias Liniger Procyclins are the major surface glycoproteins of insect-form Trypanosoma brucei. The procyclin expression sites are polycistronic and are transcribed by an ,-amanitin-resistant polymerase, probably RNA polymerase I (Pol I). The expression sites are flanked by transcription units that are sensitive to ,-amanitin, which is a hallmark of Pol II-driven transcription. We have analysed a region of 9.5 kb connecting the EP/PAG2 expression site with the downstream transcription unit. The procyclin expression site is longer than was previously realized and contains an additional gene, procyclin-associated gene 4 (PAG4), and a region of unknown function, the T region, that gives rise to trans -spliced, polyadenylated RNAs containing small open reading frames (ORFs). Two new genes, GU1 and GU2, were identified in the downstream transcription unit on the opposite strand. Unexpectedly, the 3, untranslated region of GU2 and the complementary T transcripts overlap by several hundred base pairs. Replacement of GU2 by a unique tag confirmed that sense and antisense transcription occurred from a single chromosomal locus. Overlapping transcription is stage specific and may extend ,,10 kb in insect-form trypanosomes. The nucleotide composition of the T. brucei genome is such that antisense ORFs occur frequently. If stable mRNAs can be derived from both strands, the coding potential of the genome may be substantially larger than has previously been suspected. [source] The role of RNA polymerase I transcription and embryonic genome activation in nucleolar development in bovine preimplantation embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 7 2008O. Svarcova Abstract The aim of the present study was to investigate the role of RNA polymerase I (RPI) transcription in nucleolar development during major transcriptional activation (MTA) in cattle. Late eight-cell embryos were cultured in the absence (control group) or presence of actinomycin D (AD) (RPI inhibition, AD 0.2 µg/ml; total transcriptional inhibition, AD 2.0 µg/ml). Late four-cell embryos were cultured to late eight-cell stage in 0.2 µg/ml AD (MTA prevention, ADLT (long-term total transcriptional inhibition group). Embryos were processed for autoradiography, transmission electron microscopy, fluorescent in situ hybridization (ribosomal RNA, rRNA), silver staining (nucleolar proteins), and immunofluorescence (RPI). Control embryos displayed extranucleolar and nucleolar transcription, functional nucleoli, and distinct RPI localization. Nuclei (97%) showed large rRNA clusters, in 94.1% co-localized with nucleolar proteins deposits. In AD 0.2 group, only extranucleolar transcription was detected. Segregated dense-fibrillar and granular components, but no fibrillar centers, were observed. RPI was dispersed. Nuclei (55%) presented rRNA clusters, in 38.8% co-localized with silver-stained deposits. AD 2.0 and ADLT groups displayed no transcription and disintegrating nucleolar precursors. AD 2.0 (34%) and 14% (ADLT) of nuclei presented clusters of maternally inherited rRNA. In AD 2.0 group, RPI was dispersed, but 17.2% of nuclei showed colocalization of rRNA with nucleolar proteins. In ADLT group, RPI was lacking and clustering of nucleolar proteins was hampered. In conclusion, rDNA transcription is not required for targeting of rRNA processing proteins, rRNA is maternally inherited and target to rDNA independent of transcription, and de novo transcription is required for proper nucleologenesis in cattle. Mol. Reprod. Dev. 75: 1095,1103, 2008. © 2008 Wiley-Liss, Inc. [source] Close temporal relationship between onset of cancer and scleroderma in patients with RNA polymerase I/III antibodiesARTHRITIS & RHEUMATISM, Issue 9 2010Ami A. Shah Objective This study was undertaken to examine the temporal relationship between scleroderma development and malignancy, and to evaluate whether this differs by autoantibody status among affected patients. Methods Study participants had a diagnosis of scleroderma, a diagnosis of cancer, cancer, an available serum sample, and a cancer pathology specimen. Sera were tested for autoantibodies against topoisomerase I, centromere, and RNA polymerase I/III by immunoprecipitation and/or enzyme-linked immunosorbent assay. Clinical and demographic characteristics were compared across autoantibody categories. Expression of RNA polymerases I and III was evaluated by immunohistochemistry using cancerous tissue from patients with anti,RNA polymerase antibodies. Results Twenty-three patients were enrolled. Six patients tested positive for anti,RNA polymerase I/III, 5 for anti,topoisomerase I, and 8 for anticentromere, and 4 were not positive for any of these antigens. The median duration of scleroderma at cancer diagnosis differed significantly between groups (,1.2 years in the anti,RNA polymerase I/III group, +13.4 years in the anti,topoisomerase I group, +11.1 years in the anticentromere group, and +2.3 years in the group that was negative for all antigens tested) (P = 0.027). RNA polymerase III demonstrated a robust nucleolar staining pattern in 4 of 5 available tumors from patients with antibodies to RNA polymerase I/III. In contrast, nucleolar RNA polymerase III staining was not detected in any of 4 examined tumors from the RNA polymerase antibody,negative group (P = 0.048). Conclusion Our findings indicate that there is a close temporal relationship between the onset of cancer and scleroderma in patients with antibodies to RNA polymerase I/III, which is distinct from scleroderma patients with other autoantibody specificities. In this study, autoantibody response and tumor antigen expression are associated. We propose that malignancy may initiate the scleroderma-specific immune response and drive disease in a subset of scleroderma patients. [source] | ||||||||||