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)

The Bradyrhizobium japonicum NifA protein, the central regulator for nitrogen fixation gene expression, is encoded in the fixRnifA operon. This operon is activated during free-living anaerobic growth and in the symbiotic root nodule bacteroid state. In addition, it is expressed in aerobic conditions, albeit at a low level. Here, we report that this pattern of expression is due to the presence of two overlapping promoters: fixRp1, which is of the -24/-12 class recognized by the RNA polymerase sigma 54, and fixRp2, which shares homology with the -35 and -10 regions found in other putative B. japonicum housekeeping promoters. Primer extension analyses showed that fixRp1 directed the synthesis of a transcript, P1, that starts 12 nucleotides downstream of the -12 region. In addition to sigma 54, P1 was dependent on NifA and low oxygen tension. Transcripts originating from fixRp2 started at two sites: one coincided with P1, while the most abundant, P2 initiated just two nucleotides further downstream of P1. Expression from fixRp2 was dependent on the upstream -68 promoter region, a region known to bind a putative activator protein, but it was independent of sigma 54 and NifA. This promoter was expressed in aerobic and anaerobic conditions but was not expressed in 30-day-old bacteroids. Mutations in the conserved 12 region for the sigma 54 promoter did not show any transcript, because these mutations also disrupted the overlapping -10 region of the fixRp2 promoter. Conversely, mutations at the -24 region only affected the sigma 54-dependent P1 transcript, having no effect on the expression of P2. In the absence of omega(54), anaerobic expression from the fixRp(2) promoter was enhanced threefold, suggesting that in the wild-type strain, the two RNA polymerase holoenzymes must compete for binding to the same promoter region.
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PMID:Overlapping promoters for two different RNA polymerase holoenzymes control Bradyrhizobium japonicum nifA expression. 789 98

This study was designed to quantitate cardiac mRNA levels encoding components of the local renin-angiotensin system during the development of volume overload-induced cardiac hypertrophy. Changes in cardiac renin mRNA levels were measured in relation to renin activity in the left ventricle (LV) and in plasma after acute passive stretch of the heart caused by an aortovenocaval shunt in the rat. A quantitative reverse-transcriptase polymerase chain reaction method with competitive internal standards was used to measure mRNA levels in total RNA derived from cardiac tissues after shunt. Seven days after shunt surgery, LV weight was increased by 23%. Renin activities were elevated four- and twofold in plasma and LV, respectively. LV angiotensinogen mRNAs were not significantly increased by shunt surgery; they were twofold higher than phosphoglycerate kinase mRNA from the housekeeping gene PGK-1. By day 7, LV levels for renin mRNA were significantly increased from well below 0.25% to approximately 1% of PGK-1 mRNA. Identity between renin polymerase chain reaction products from kidney and heart cDNAs and absence of "reninlike" amplification products were supported by Southern blotting. Volume overload caused increased expression of the renin gene in the stretched myocardium. This finding is consistent with the concept of a myocardial renin-angiotensin system that can be activated by locally produced renin and contributes to the hypertrophy of cardiac muscle.
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PMID:Stretch-mediated activation of cardiac renin gene. 794 10

Two types of largest subunit RNA polymerase II (pol II) genes (pol IIA and pol IIB), differing in 3 amino acid substitutions, are encoded in the Trypanosoma brucei (stock 427-60) genome. As a result, the alpha-amanitin-resistant transcription of the procyclic acidic repetitive protein (PARP) and variant surface glycoprotein (VSG) genes was proposed to involve a modified, alpha-amanitin-resistant form of the largest subunit of pol II. Alternatively, pol I could transcribe the PARP and VSG genes. To discriminate between these two models, we deleted the N-terminal domain (about one-third of the polypeptide), which encodes the amino acid substitutions which discriminated the pol IIA and pol IIB genes, at both pol IIB alleles. The pol IIB- trypanosomes still transcribe the PARP genes and the VSG gene promoter region in insect-form trypanosomes by alpha-amanitin-resistant RNA polymerases, while control housekeeping genes are transcribed in an alpha-amanitin-sensitive manner, presumably by pol IIA. We conclude that the alpha-amanitin-resistant transcription of protein coding genes in T. brucei is not mediated by a diverged form of the largest subunit of pol II and that the presence of both the pol IIA and pol IIB genes is not essential for trypanosome viability. This conclusion was further supported by the finding that individual trypanosome variants exhibited allelic heterogeneity for the previously identified amino acid substitutions and that various permutations of the polymorphic amino acids generate at least four different types of largest subunit pol II genes. The expression of the PARP genes and the VSG gene promoter region by alpha-amanitin-resistant RNA polymerases in the pol IIB- trypanosomes provides evidence for transcription of these genes by pol I.
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PMID:Disruption of largest subunit RNA polymerase II genes in Trypanosoma brucei. 849 77

The Azorhizobium nifA promoter (PnifA) is positively regulated by two physiological signal transduction pathways, NtrBC, which signals anabolic N status, and FixLJK, which signals prevailing O2 status. Yet, PnifA response (gene product per unit time) to these two activating signals together is more than twice that of the summed, individual signals. In the absence of NIFA, a negative PnifA autoregulator, the fully induced PnifA response is more than 10-fold greater than that of summed, individual signals. Given this synergism, these two signal transduction pathways must interactively regulate PnifA activity. PnifA carries three cis-acting elements, an anaerobox, which presumably binds FIXK, a NIFAbox, which presumably binds NIFA itself, and a sigma 54 box, which presumably binds sigma 54 initiator, a subunit of RNA polymerase. For combinatorial analysis, single, double, and triple promoter mutations were constructed in these cis-acting elements, and PnifA activities were measured in six different trans-acting background, i.e., fixK, fixJ, nifA, ntrC, rpoF, and wild type. Under all physiological conditions studied, high-level PnifA activity required both FIXK in trans and the anaerobox element in cis. Surprisingly, because PnifA was hyperactive with a mutated sigma 54box, this cis-acting element mediates both negative and positive control. Because PnifA hyperactivity also required a wild-type upstream NIFAbox element, even in the absence of NIFA, a second upstream nifA transcription start superimposed on the NIFAbox element was hypothesized. When nifA mRNA 5' start points were mapped by primer extension, both a minor upstream transcript(s) starting 45 bp distal to the anaerobox and a major downstream transcript starting 10 bp distal to the sigma 54 box were observed. In Azorhizobium, RNA polymerase sigma 54 initiator subunits are encoded by a multigene family, which includes rpoF and rpoN genes. Because rpoF mutants show an Ntr+ phenotype, whereas rpoN mutants are Ntr-, multiple sigma 54 initiators are functionally distinct. Two independent rpoF mutants both show a tight Nif- phenotype. Moreover, rpoF product sigma 54F is absolutely required for high-level PnifA activity. In summary, the Azorhizobium nifA gene carries overlapping housekeeping-type and sigma 54-type promoters which interactively respond to different signals. Effectively, the upstream, housekeeping-type promoter responds to FIXK and positively regulates the downstream, sigma 54-type promoter. The downstream, sigma 54-type promoter responds to NTRC and negatively regulates the upstream, housekeeping-type promoter. In terms of transcript yield, the upstream, housekeeping-type promoter is therefore weak, and the downstream, sigma 54-type promoter is strong.
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PMID:Interactive regulation of Azorhizobium nifA transcription via overlapping promoters. 852 30

Early hematopoietic progenitors expressing the CD34+ phenotype can be harvested from the peripheral blood of normal individuals. We have optimized the liquid culture of human CD34+ peripheral blood progenitors (PBPs) to achieve differentiation into a population of cells consisting almost entirely of eosinophil progenitors and maturing eosinophils. Growth of CD34+ PBPs for 28 days in the presence of the combination of IL-3, granulocyte-macrophage colony-stimulating factor, and IL-5 resulted in an almost 250-fold increase in cell number, yielding a population that contained 83% maturing eosinophils. The residual population consisted of basophils and mast cells (3% by acidic toluidine blue staining, 15.2% by flow cytometric assay for binding to high-affinity IgE receptor) and immature cells. This provides an opportunity to examine the kinetics of the acquisition of specialized mature eosinophil characteristics during eosinophil differentiation. Several host-defense and bioactive proteins are found almost exclusively in eosinophil granules. In addition, stimulated eosinophils, like neutrophils, produce copious amounts of toxic oxygen radicals. We used our culture system and the sensitive technique of reverse-transcriptase polymerase chain reaction to analyze the kinetics of production of messenger RNA transcripts encoding several eosinophil proteins, including five eosinophil granule proteins and four subunit peptides of the superoxide-generating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in small numbers of differentiating eosinophils from peripheral blood CD34+ cells. Freshly isolated CD34+ PBPs contained transcripts for the ubiquitously present housekeeping protein phosphoglucokinase but contained no eosinophil granule protein transcripts and barely detectable amounts of some oxidase protein transcripts. On day 3 of culture, no cells recognizable by histochemical staining as eosinophils could be detected, but transcripts for all five eosinophil granule proteins were present. These transcripts increased several fold during the entire culture period. Similar kinetics were seen for all but one of the NADPH oxidase protein transcripts. However, transcripts for the p67phox NADPH oxidase protein were not detected until day 7, and functional oxidase activity did not appear until day 12. From that point, oxidase activity increased dramatically over the culture period. These studies demonstrate that commitment of CD34+ PBPs to the eosinophil lineage occurs very early, by day 3, but that further events in differentiation must take place before the appearance of histologically staining eosinophil granules and acquisition of functional oxidase capacity.
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PMID:Early commitment to the eosinophil lineage by cultured human peripheral blood CD34+ cells: messenger RNA analysis. 875 12

The human uracil-DNA glycosylase gene (UNG) spans approximately 13.5 kb including the promoter. UNG comprises 6 exons and 5 introns and was assigned to chromosome 12q23-q24.1 by radiation hybrid mapping. UNG exhibits typical features of housekeeping genes, including a 5' CpG island of 1.2 kb and a very GC-rich TATA-less promoter containing a number of elements involved in constitutive expression and cell cycle regulation. A smaller CpG island is located just downstream of the gene. Within the 15-kb sequence we identified 16 Alu retroposons, 2 of which contain putative competent RNA polymerase III promoters, 3 copies of medium reiteration frequency repeats, and 1 copy of a mammalian-wide interspersed repetitive element, as well as a 300-bp TA-dinucleotide repeat. In vitro methylation of the UNG promoter strongly reduced promoter activity, but methylation may not be involved in regulation of UNG in vivo since a narrow region of the 5' CpG island comprising the putative transcription factor binding region appears to be invariably methylation-free.
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PMID:Human uracil-DNA glycosylase gene: sequence organization, methylation pattern, and mapping to chromosome 12q23-q24.1. 888 63

We describe a new, simple and reliable semiautomated strategy for quantifying mRNA from archival specimens by using oligo(dT)25 paramagnetic beads and the reverse-transcriptase polymerase chain reaction (PCR) coupled with quantitative digital image analysis (Q-DIA). To evaluate the experimental conditions, we examined thymidylate synthase (TS) gene expression in mRNA isolated from both flash-frozen and formalin-fixed paraffin-embedded human biopsy samples using biopsy material obtained from 2 patients prior to chemotherapy with 5-fluorouracil. Following the electrophoretic separation of the PCR products through a 20% polyacrylamide gel, quantitation of the perimeters of the silver-nitrate-stained PCR products will be done by Q-DIA using a video frame-grabber board attached to a CCD camera using Image-Pro+ software. Validation of this approach will involve a comparison of the observed gene expression levels to TS protein levels obtained by tissue homogenization assays of TS, tetrahydrofolate, 5,10-methylenetetrahydrofolate, 2'-deoxyuridine-5'-monophosphate and 5-fluoro-2'-deoxyuridylate (FdUMP), by established [3H]FdUMP ligand-binding assays. The novelty of this method is that it offers a low-cost means whereby Q-DIA is performed directly from the gel to rapidly and accurately determine the level of TS gene expression, which is standardized against the beta-actin housekeeping gene. In the protocol described herein, gene expression studies can be done quickly and without the use of radioactive substances in both normal clinical samples shock frozen at the time of surgical excision and in formalin-fixed paraffin-embedded archival samples, which are commonly available in all hospital pathology departments. To demonstrate the utility of this method, mRNA was extracted from both nonpathological and tumor biopsies originating from both types of material from the same patients. TS gene expression in the flash-frozen and archival materials was compared to the level of TS intracellular enzyme activity in the same samples and a correlation of 89 and 80% between the shock-frozen and archival material relative to TS intracellular enzyme activity levels was observed. These findings suggest that routine semiautomated quantitative analysis of rare mRNA transcripts, e.g. TS, from archival material can be applied for retrospective studies.
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PMID:Detection of thymidylate synthase gene expression levels in formalin-fixed paraffin embedded tissue by semiquantitative, nonradioactive reverse transcriptase polymerase chain reaction. 898 25

It has been long known that neoplastic transformation is accompanied by a lowered requirement for extracellular Ca2+ for growth. The studies presented here demonstrate that human fibroblastic cell lines produce the two commonly found 'housekeeping' isoforms of the plasma membrane Ca(2+)-ATPase (PMCA), PMCA1b and 4b, and at the expression of both is demonstrably lower in cell lines neoplastically transformed by SV40 than in the corresponding parental cell lines. Western blot analyses of lysates from control (GM00037) and SV40-transformed (GM00637) skin fibroblasts revealed a 138 kDa PMCA whose level was significantly lower in the SV40-transformed cells relative to either total cellular protein or alpha-tubulin. Similar analyses of plasma membrane preparations from control WI-38) and SV40-transformed (WI-38VA13) lung fibroblasts revealed 3-4-fold lower levels of PMCA in the SV40-transformed cells. Competitive ELISAs performed on detergent solubilized plasma membrane preparations indicated at least 3-4-fold lower levels of PMCA in the SV40-transformed cell lines compared to controls. Reverse transcriptase coupled-PCR analyses showed that PMCA1b and PMCA4b were the only isoforms expressed in all four cell lines. The PMCA4b mRNA level detected by Northern analysis also was substantially lower in SV40 transformed skin fibroblasts than in non-transformed fibroblasts. Quantitative RT-PCR analyses showed levels of PMCA1b and 4b mRNAs to be 5 and 10-fold lower, respectively, in GM00637 than in GM00037 when the levels of PCR products were normalized to glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. These results demonstrate that the expression of these distinct PMCA genes is substantially lower in SV40 transformed human skin and lung fibroblasts and may be coordinately regulated in these cells.
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PMID:Analysis of plasma membrane Ca(2+)-ATPase expression in control and SV40-transformed human fibroblasts. 905 77

To analyze transcriptional control regions of Drosophila melanogaster housekeeping genes, we have characterized the promoter of the gene coding for the second-largest subunit of RNA polymerase II (DmRP140). Upstream of DmRP140 the genomic region harbors a gene which is transcribed in the opposite direction (DmRP140up). By determination of the transcription start sites of both genes we found a short non-transcribed intergenic region of 220 bp. Functional analysis of various promoter reportergene constructs by transient transfection of cultured cells revealed that sequences important for transcription of DmRP140 are located in the untranslated leader of the upstream gene. The onset of DmRP140 transcription during embryonic development was studied in transgenic flies using beta-galactosidase as reportergene. To distinguish between the maternally provided DmRP140 transcripts and the embryonically transcribed RNA the offspring of nontransformed females and male transformants was examined. The development of a sensitive detection assay based on a chemiluminescent substrate for beta-galactosidase allowed us to determine the onset of DmRP140 transcription to between 8-10 h after oviposition. Thus, DmRP140 transcription does not start following the transcriptional transition period between 2-3 h of development but occurs much later in embryogenesis coinciding with decreasing DNA synthesis and cell division rates.
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PMID:Transcription of DmRP140, the gene coding for the second-largest subunit of RNA polymerase II. 906 Oct 24

Cell differentiation in the Caulobacter crescentus cell cycle requires differential gene expression that is regulated primarily at the transcriptional level. Until now, however, a defined in vitro transcription system for the biochemical study of developmentally regulated transcription factors had not been available in this bacterium. We report here the purification of C. crescentus RNA polymerase holoenzymes and resolution of the core RNA polymerase from holoenzymes by chromatography on single-stranded DNA cellulose. The three RNA polymerase holoenzymes Esigma54, Esigma32, and Esigma73 were reconstituted exclusively from purified C. crescentus core and sigma factors. Reconstituted Esigma54 initiated transcription from the sigma54-dependent fljK promoter of C. crescentus in the presence of the transcription activator FlbD, and active Esigma32 specifically initiated transcription from the sigma32-dependent promoter of the C. crescentus heat-shock gene dnaK. For reconstitution of the Esigma73 holoenzyme, we overexpressed the C. crescentus rpoD gene in Escherichia coli and purified the full-length sigma73 protein. The reconstituted Esigma73 recognized the sigma70-dependent promoters of the E. coli lacUV5 and neo genes, as well as the sigma73-dependent housekeeping promoters of the C. crescentus pleC and rsaA genes. The ability of the C. crescentus Esigma73 RNA polymerase to recognize E. coli sigma70-dependent promoters is consistent with relaxed promoter specificity of this holoenzyme previously observed in vivo.
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PMID:Purification, characterization, and reconstitution of DNA-dependent RNA polymerases from Caulobacter crescentus. 926 Nov 76


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