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)

Nitric oxide (NO) is readily oxidized to nitrate and nitrite and NO activates guanylyl cyclase, increasing cyclic GMP levels. To determine if nitric oxide synthase (NOS) is present in urine collected daily from patients following renal transplantation, we evaluated NOS activity in the leukocyte-rich particulate fraction and measured nitrate, nitrite, and cyclic GMP levels in the supernatant fraction of the urine. Reverse transcriptase-PCR and cDNA sequencing confirmed the presence of inducible NOS (iNOS) in cells obtained from the urine of renal transplant patients with rejection. NOS activity was elevated significantly in renal transplant patients with rejection (6.40 +/- 1.47 pmol citrulline/min/mg protein) or with urinary tract infection (29.56 +/- 11.00 pmol citrulline/min/mg protein), when compared to post-renal transplantation patients without rejection or urinary tract infection (0.51 +/- 0.21 pmol citrulline/min/mg protein). Nitrate levels increased in renal transplant patients with rejection and nitrite levels increased in renal transplant patients with urinary tract infection (UTI). Cyclic GMP levels increased with both rejection and UTI. This study demonstrates the presence of NOS activity and inducible NOS-mRNA in cells isolated from the urine of patients undergoing renal allograft rejection.
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PMID:Nitric oxide synthase induction with renal transplant rejection or infection. 894 94

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

The recently identified P6A promoter of the anaerobically inducible focApfl operon of Escherichia coll overlaps the Fnr (fumarate-nitrate reduction regulator)-dependent P6 promoter. The Fnr-binding site of P6 and the -35 hexamer sequence of P6A are shared between the promoters. Inactivation of P6A, through introduction of a -10 hexamer mutation, resulted in enhanced anaerobic induction of operon expression. The dependence on the ArcA (aerobic respiration control regulator) and Fnr transcription factors for anaerobic induction was tested for several focA-lacZ and pfl-lacZ gene fusions. Anaerobic induction became more dependent on Fnr in derivatives lacking a functional P6A promoter compared with wild-type constructs. Moreover, aerobic expression of the focA gene was reduced by the p6A mutation, as was the dependence on ArcA for anaerobic induction. Inactivation of P6 severely reduced Fnr-dependent anaerobic induction, in accord with previous findings. Transcription analyses demonstrated that a mutation in the -10 hexamer sequence of either P6A or P6 did not adversely affect transcription from the remaining promoter. Taken together, these results indicate that the P6A promoter moderates the Fnr-dependent activation of P6 through competition for RNA polymerase binding.
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PMID:Overlapping promoters modulate Fnr- and ArcA-dependent anaerobic transcriptional activation of the focApfl operon in Escherichia coli. 908 61

Nitrate increases the transcription of the two Arabidopsis thaliana nitrate reductase genes. We demonstrated previously that 238 and 330 bp of the 5' flanking regions, designated as NP1 and NP2, of the two nitrate reductase genes NR1 and NR2, respectively, are sufficient for nitrate-dependent transcription (Y. Lin, C.-F. Hwang, J.B. Brown, C.-L. Cheng [1994] Plant Physiol 106: 477-484). Here we identify the cis-acting elements of NP1 and NP2 that are necessary for nitrate-dependent transcription by linker-scanning (LS) analysis. In transgenic plants one LS mutant of NP1 and two LS mutants of NP2 exhibited significantly lower nitrate-induced reporter gene chloramphenicol acetyltransferase activity. To distinguish which of these three mutants lost nitrate inducibility, competitive reverse-transcriptase polymerase chain reaction was used to measure the chloramphenicol acetyltransferase mRNA levels before and after nitrate induction. The single LS mutant in NP1 lost its response to nitrate, whereas the two LS mutants in NP2 partially lost their response to nitrate. A 12-bp sequence is conserved between the NP1 site and the two NP2 sites. This sequence motif is also conserved in the 5' flanking regions of other nitrate-inducible plant genes. Gel mobility shift experiments indicate that these three regions bind to similar proteins. The binding is constitutive with respect to nitrate treatment and was observed in both nonphotosynthetic suspension cells and green leaves.
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PMID:Sequences necessary for nitrate-dependent transcription of Arabidopsis nitrate reductase genes. 908 75

We have treated DBA/2-->C57BL/6 murine cardiac allograft recipients with anti-CD4 monoclonal antibody or with gallium nitrate to promote long-term (>60 days) allograft survival. Within this period, all grafts developed histologic evidence of ongoing vascular and parenchymal tissue remodeling, including interstitial fibrosis and neointimal hyperplasia, which are characteristic of chronic allograft rejection. To evaluate residual alloimmunity associated with the pharmacologic avoidance of acute graft rejection and the development of chronic tissue remodeling, we subjected these graft recipients to a battery of histologic and immunologic tests. Similar test results were obtained for graft recipients treated with either of the two immunosuppressive agents. All long-surviving allografts displayed histologic evidence of ongoing microvascular endothelial activation and interstitial leukocytic infiltration. Reverse transcriptase-polymerase chain reaction analyses demonstrated intragraft expression of mRNAs for interleukin (IL)-1, IL-2, IL-4, IL-6, tumor necrosis factor, interferon-gamma, and transforming growth factor-beta. All recipients had limiting dilution analysis-detectable, graft-reactive cytolytic T lymphocytes and helper T lymphocytes in their spleens and grafts, and all produced high titers of graft-reactive alloantibodies. In general, these observations indicate that (1) a similar immune status is achieved in long-surviving allografts and their recipients when either anti-CD4 monoclonal antibody or gallium nitrate was used for antirejection therapy, (2) this immune status is characterized by continuous, long-term inflammatory and immune processes that are qualitatively similar to those observed during acute allograft rejection, and (3) no specific immune responses developed selectively in long-term graft recipients to account for the avoidance of acute graft rejection or the development of chronic tissue remodeling in the graft.
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PMID:Prolonged murine cardiac allograft acceptance: characteristics of persistent active alloimmunity after treatment with gallium nitrate versus anti-CD4 monoclonal antibody. 913 72

We examined the effects of neutral salts and the non-ionic solute 2-methyl,-2,4-pentanediol (MPD) on transcript elongation by Escherichia coli RNA polymerase and on pausing induced by the multipartite his leader pause signal. All solutes tested slowed the overall rate of elongation, with anions showing the dominant effects in the order: (most inhibitory) HPO4(2-) > OAc- > SO4(2-) > ClO4- > I- approximately NO3- > Br approximately Cl- approximately MPD (least inhibitory). Although the protein structure-stabilizing anions HPO4(2-), OAc-, and SO4(2-) also increased the pause half-life at the his leader pause site, the remaining solutes accelerated escape from pause site in the order: (greatest acceleration) NO3- > ClO4- > I- > Br- > Cl- > MPD (least acceleration). Cl(-)-induced acceleration of escape from the pause site also occurred on mutant templates altered for the 3'-proximal region, RNA 3' end, or downstream DNA. The effect was eliminated, however, by base substitutions that destabilize the pause RNA hairpin or that extend it toward the 3' end. This "perfect hairpin" itself reduced the pause half-life by a factor of 3. We suggest that the pause RNA hairpin stabilizes a paused conformation of the transcription complex through an interaction with an easily disordered region of RNA polymerase. Extending the stem of the pause hairpin may disrupt the interaction by altering the position of the hairpin in the transcription complex. Anions may either compete for the interaction directly or disorder the site of hairpin interaction by chaotropic effects. We suggest that the negative effect of structure-stabilizing anions like OAc- and SO4(2-) may reflect passage of RNA polymerase through significantly different conformations during rapid elongation, some of which may expose hydrophobic surface.
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PMID:Effects of neutral salts on RNA chain elongation and pausing by Escherichia coli RNA polymerase. 914 40

The regulator of fumarate and nitrate reduction (FNR) protein of Escherichia coli is an oxygen-responsive transcription regulator that acts mainly to activate the transcription of genes associated with anaerobic energy generation during periods of oxygen starvation. The hlyX gene of the swine pathogen Actinobacillus pleuropneumoniae encodes an FNR homologue, HlyX, which can complement the anaerobic respiratory deficiencies of an fnr mutant. However, FNR and HlyX have distinct but overlapping regulons because during anaerobic incubation, hlyX-expressing E. coli K-12 strains produce an otherwise latent haemolysin. The gene encoding the 'latent' haemolysin has been designated hlyE and analysis of the promoter region by DNase I footprinting reveals the presence of an FNR- (HlyX-) binding site. Anaerobic expression of an hlyE::lacZ reporter was 6.5-fold higher in hlyX compared to fnr-expressing cells. Both FNR and HlyX recruited RNA polymerase to the hlyE promoter but formed different ternary complexes. One major transcript (tsp1) initiating at 78.5 bp downstream of the FNR-binding site and four minor transcripts initiating at 73.5 (tsp2), 71.5 (tsp3), 63.5 (tsp4) and 62.5 (tsp5) bp from the FNR site were detected. From the position of the FNR box relative to the transcript starts, hlyE is expressed from a Class I FNR-regulated promoter. Substitution of selected FNR amino acids with the residues found in the equivalent positions in HlyX indicated that Activating Region 1 (AR1) of FNR forms a surface encompassing beta to beta 11 and that the AR1 contact at Class I promoters is different to that at Class II promoters, although the same surface is involved. The FNR variant, FNR-A225T, combined the properties of FNR (good activation from Class II promoters) and HlyX (good activation of Class I promoters) and conferred the haemolytic phenotype.
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PMID:The molecular basis for the differential regulation of the hlyE-encoded haemolysin of Escherichia coli by FNR and HlyX lies in the improved activating region 1 contact of HlyX. 942 3

During tetrapyrrole biosynthesis 5-aminolevulinic acid dehydratase (ALAD) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA) to form one molecule of the pyrrole derivative porphobilinogen. In Escherichia coli, the enzyme is encoded by the gene hemB. The hemB gene was cloned from Pseudomonas aeruginosa by functional complementation of an E. coli hemB mutant. An open reading frame of 1011 bp encoding a protein of 336 amino acids (M(r) 37,008) was identified. The gene was mapped to SpeI fragment G and DpnI fragment G of the P. aeruginosa chromosome, corresponding to the 10 to 12 min region of the new map or 19 to 22 min interval of the old map. The 5' end of the hemB mRNA was determined and the -10 and -35 regions of a potential sigma 70-dependent promoter were localized. No obvious regulation of the hemB gene by oxygen, nitrate, heme or iron was detected. Alignment of the amino acid sequences deduced from hemB revealed a potential metal-binding site and indicated that the enzyme is Mg(2+)-dependent. P. aeruginosa hemB was overexpressed in an E. coli hemB mutant using the phage T7 RNA polymerase system and its Mg(2+)-dependent activity was directly demonstrated.
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PMID:Cloning, mapping and functional characterization of the hemB gene of Pseudomonas aeruginosa, which encodes a magnesium-dependent 5-aminolevulinic acid dehydratase. 952 30

The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, the napF operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the napF control region. In addition, the in vivo effects of Fnr-positive control mutations on napF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate napF operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro.
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PMID:Fnr, NarP, and NarL regulation of Escherichia coli K-12 napF (periplasmic nitrate reductase) operon transcription in vitro. 969 69

The recent outbreaks of Escherichia coli O157-associated food poisoning have focused attention on the virulence determinants of E. coli. Here, it is reported that single base substitutions in the fnr gene encoding the oxygen-responsive transcription regulator FNR (fumarate and nitrate reduction regulator) are sufficient to confer a hemolytic phenotype on E. coli K12, the widely used laboratory strain. The mechanism involves enhancing the expression of a normally dormant hemolysin gene (hlyE) located in the E. coli chromosome. The mutations direct single amino acid substitutions in the activating regions (AR1 and AR3) of FNR that contact RNA polymerase. It is concluded that altering a resident transcription regulator, or acquisition of a competent heterologous regulator, could generate a pool of hemolytic, and therefore more virulent, strains of E. coli in nature.
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PMID:Altering the anaerobic transcription factor FNR confers a hemolytic phenotype on Escherichia coli K12. 972 23


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