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)

Leptospiral protein antigens are of interest as potential virulence factors and as candidate serodiagnostic and immunoprotective reagents. We identified leptospiral protein antigens by screening a genomic expression library with serum from a rabbit hyperimmunized with formalin-killed, virulent Leptospira kirschneri serovar grippotyphosa. Genes expressing known outer membrane lipoproteins LipL32 and LipL41, the heat shock protein GroEL, and the alpha, beta, and beta' subunits of RNA polymerase were isolated from the library. In addition, a new leptospiral gene that in Escherichia coli expressed a 45-kDa antigen with an amino-terminal signal peptide followed by the spirochetal lipobox Val(-4)-Phe(-3)-Asn(-2)-Ala(-1) (downward arrow)Cys(+1) was isolated. We designated this putative lipoprotein LipL45. Immunoblot analysis of a panel of Leptospira strains probed with LipL45 antiserum demonstrated that many low-passage strains expressed LipL45. In contrast, LipL45 was not detected in high-passage, culture-attenuated strains, suggesting that LipL45 is a virulence-associated protein. In addition, all leptospiral strains tested, irrespective of culture passage, expressed a 31-kDa antigen that was recognized by LipL45 antiserum. Southern blot and peptide mapping studies indicated that this 31-kDa antigen was derived from the carboxy terminus of LipL45; therefore, it was designated P31(LipL45). Membrane fractionation studies demonstrated that P31(LipL45) is a peripheral membrane protein. Finally, we found that P31(LipL45) levels increased as Leptospira entered the stationary phase, indicating that P31(LipL45) levels were regulated. Hamsters infected with L. kirschneri formed an antibody response to LipL45, indicating that LipL45 was expressed during infection. Furthermore, the immunohistochemistry of kidneys from infected hamsters indicated that LipL45 was expressed by L. kirschneri that colonized the renal tubule. These observations suggest that expression of LipL45 responds to environmental cues, including those encountered during infection of a mammalian host.
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PMID:Novel 45-kilodalton leptospiral protein that is processed to a 31-kilodalton growth-phase-regulated peripheral membrane protein. 1174 98

Plasmablastic lymphoma is a relatively new entity that is considered to be a diffuse large B-cell lymphoma with an unique immunophenotype and a predilection for the oral cavity. We present a 50 year-old HIV-positive, bisexual, white male with a CD4 count 300/mm(3) and a viral HIV-RNA polymerase chain reaction (PCR) load of 237 copies/ml, who developed a painful, purple-red mass in the edentulous area of the maxillary right first molar. Erythematous gingival enlargements of the interdental papillae were seen in three of the dental quadrants. In addition, the patient was being managed with antiretroviral therapy and liposomal doxorubicin for recurrent cutaneous Kaposi's sarcoma (KS). Although oral KS was suspected, the gingival lesions were biopsied because they were refractory to chemotherapy and a lymphoma could not be excluded. Histopathologic examination revealed a lymphoid malignant neoplasm, consistent with a plasmablastic lymphoma. Immunoreactivity with vs38c, CD79a, kappa light chain, and IgG was readily identified in tumor cells; while only focal cells expressed CD20 and LCA (CD45RB). CD56, CD3, lambda light chain, and EMA were non-reactive. EBV was detected in the tumor by Southern hybridization, PCR amplification, in situ hybridization for EBER-1 DNA, and immunohistochemistry for latent membrane protein-1. The same tumor was negative for HHV-8 by PCR. Recognition of plasmablastic lymphoma is important, because it represents an HIV-associated malignancy that predominantly involves the oral cavity, may mimic KS and has a poor prognosis.
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PMID:Plasmablastic lymphoma: an HIV-associated entity with primary oral manifestations. 1175 27

The transcriptional activity and allele variation of the 28-kDa outer membrane protein gene (p28) of Ehrlichia chaffeensis were analyzed to determine the mechanism of the antigenic variation of the 28-kDa outer membrane proteins. Reverse transcriptase PCR amplification of mRNA indicated that 16 of the 22 members of the p28 multigene family were transcribed. Amino acid sequence analysis indicated that the p28-19 protein was produced in vitro in the Arkansas strain. The p28-19 gene and its promoter region were sequenced and compared in 12 clinical isolates of E. chaffeensis to determine allele variation. The variation of the p28-19 gene among the isolates is limited to three types represented by strains Arkansas, 91HE17, and Sapulpa, respectively. These results indicate that the majority of the p28 genes are active genes and that antigenic variation of the E. chaffeensis 28-kDa proteins may result from differential expression of the p28 gene family members rather than gene conversion.
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PMID:Antigenic variation of Ehrlichia chaffeensis resulting from differential expression of the 28-kilodalton protein gene family. 1189 44

In pathogenic Vibrio cholerae, the transmembrane DNA-binding protein ToxR co-ordinates the expression of over 20 genes, including those encoding important virulence factors such as cholera toxin and the toxin-co-regulated pilus. The outer membrane protein OmpT is the only member of the ToxR regulon known to be repressed by ToxR. In this study, we examined the environmental conditions that regulate OmpT expression and demonstrated that ompT transcription is upregulated 14-fold when the bacteria enter late log phase from early log phase. Deletion of the crp gene completely abolishes OmpT expression. Comparison of ompT transcription levels in the isogenic crp-, toxR- and crp-toxR- mutants revealed that (i) in the absence of ToxR, constitutive high-level ompT transcription is dependent on cAMP receptor protein (CRP); (ii) ToxR not only interferes with CRP-dependent ompT activation, but also abolishes the CRP-independent, basal level ompT transcription; thus, the mechanism by which ToxR represses ompT transcription involves both antiactivation and direct repression; (iii) both CRP and ToxR are required for the regulation of OmpT expression by growth phase. To provide further insights into the molecular mecha-nism of CRP-dependent activation of ompT transcription, we demonstrated that CRP-dependent activation requires a CRP binding site centred at -310 of the ompT promoter, without which the interaction of CRP with other CRP binding site(s) more proximal to the promoter results in repression. Mutations in two regions on CRP (AR1 and AR2) that directly contact RNA polymerase (RNAP) abolish activation, suggesting direct interaction of CRP with RNAP from -310 of the ompT promoter via DNA looping.
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PMID:ToxR interferes with CRP-dependent transcriptional activation of ompT in Vibrio cholerae. 1195 6

Phase variation of the outer membrane protein Ag43 in E. coli requires deoxyadenosine methylase (Dam) and OxyR. Previously, it was shown that OxyR is required for repression of the Ag43-encoding gene, agn43, and that Dam-dependent methylation of three GATC target sequences in the regulatory region abrogates OxyR binding. Here we report further characterization of agn43 transcription and its regulation. Transcription was initiated from a sigma(70)-dependent promoter at the G residue of the upstream GATC sequence. Template DNA and RNA polymerase were sufficient to obtain transcription in vitro, but DNA methylation enhanced the level of transcription. Analyses of transcription in vivo of agn'-lacZ with mutated Dam target sequences support this conclusion. Since methylation also abrogates OxyR binding, this indicates that methylation plays a dual role in facilitating agn43 transcription. In vitro transcription from an unmethylated template was repressed by OxyR(C199S), which resembles the reduced form of OxyR. Consistent with this and the role of Dam in OxyR binding, OxyR(C199S) protected from DNase I digestion the agn43 regulatory region from -16 to +42, which includes the three GATC sequences. Deletion analyses of the regulatory region showed that a 101-nucleotide region of the agn43 regulatory region containing the promoter and this OxyR binding region was sufficient for Dam- and OxyR-dependent phase variation
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PMID:Dam- and OxyR-dependent phase variation of agn43: essential elements and evidence for a new role of DNA methylation. 1202 51

Transcriptional regulation of the ferric citrate transport genes of Escherichia coli is initiated by the binding of ferric citrate to the outer membrane protein FecA. This binding elicits a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm, where the sigma factor FecI directs the RNA polymerase to the promoter upstream of the fecABCDE genes. An in vivo deletion analysis using a bacterial two-hybrid system assigned the interaction of the FecR and FecI proteins to the cytoplasmic portion of the FecR transmembrane protein and region 4 of FecI. Missense mutations randomly generated by PCR were localized to region 4 of FecI, and the mutants were impaired with regard to the interaction of FecR with FecI and fecB-lacZ transcription. The cloned region 4 of FecI interfered with fecB-lacZ transcription. Interaction of N-proximal regions of predicted FecR homologs with region 4 of predicted FecI homologs of Pseudomonas aeruginosa was demonstrated. The interaction was specific in that only cognate protein pairs interacted with each other; no interactions occurred between heterologous combinations of the P. aeruginosa proteins and between a P. aeruginosa FecI homolog and E. coli FecR. The results demonstrate that region 4 of FecI specifically binds FecR and that this binding is necessary for FecI to function as a sigma factor.
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PMID:Functional interaction of region 4 of the extracytoplasmic function sigma factor FecI with the cytoplasmic portion of the FecR transmembrane protein of the Escherichia coli ferric citrate transport system. 1205 67

In a speG-disrupted Escherichia coli mutant, which cannot metabolize spermidine to acetylspermidine, addition of spermidine to the medium caused a decrease in cell viability at the late stationary phase of growth. There were parallel decreases in the levels of ribosome modulation factor (RMF), the sigma(38) subunit of RNA polymerase, and the outer membrane protein C (OmpC). To clarify that these three proteins are strongly involved in cell viability, the rmf, rpoS (encoding sigma(38)), and ompC genes were disrupted. Viability of the triple mutant decreased to less than 1% of normal cells. The triple mutant had a reduced cell viability compared to any combination of double mutants, which also had a reduced cell viability. The single rmf and rpoS, but not ompC, mutant only slightly reduced cell viability. The results indicate that cooperative functions of these three proteins are necessary for cell viability at the late stationary phase. The triple mutant had a reduced level of ribosomes and of intracellular cations.
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PMID:Decrease in cell viability in an RMF, sigma(38), and OmpC triple mutant of Escherichia coli. 1243 78

In addition to being the major site of cerebrospinal fluid formation, the choroid plexus epithelium emerges as an important source of polypeptides in the brain. Physiologically regulated release of some polypeptides synthesized by the choroid plexus has been shown. The molecular mechanisms underlying this polypeptide secretion have not been characterized, however. In the present study, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein, two membrane fusion proteins playing a critical role in exocytosis in neurons and endocrine cells, were found to be expressed in the choroid plexus epithelium. It was also shown that in choroidal epithelium, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein stably interact. Two members of the vesicle-associated membrane protein family, vesicle-associated membrane protein-1 and vesicle-associated membrane protein-2, were expressed in the rat choroid plexus at the messenger RNA and protein level. However, their newly discovered isoforms, vesicle-associated membrane protein-1b and vesicle-associated membrane protein-2b, produced by alternative RNA splicing, were not detected in choroidal tissue. Immunohistochemistry demonstrated that vesicle-associated membrane protein is confined to the cytoplasm of choroidal epithelium, whereas synaptosome-associated protein of 25 kDa is associated with plasma membranes, albeit with a varied cellular distribution among species studied. Specifically, in the rat choroid plexus, synaptosome-associated protein of 25 kDa was localized to the basolateral membrane domain of choroidal epithelium and was expressed in small groups of cells. In comparison, in ovine and human choroidal tissues, apical staining for synaptosome-associated protein of 25 kDa was found in the majority of epithelial cells. These species-related differences in cellular synaptosome-associated protein of 25 kDa distribution suggested that the synaptosome-associated protein of 25 kDa homologue, synaptosome-associated protein of 23 kDa, is also expressed in the rat choroid plexus, which was confirmed by reverse-transcriptase polymerase chain reaction. Our findings suggest that synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein are involved in secretion of polypeptides from the choroid plexus epithelium. The presence of synaptosome-associated protein of 25 kDa and its homologue as well as multiple isoforms of vesicle-associated membrane protein in choroidal epithelium may play a role in the apical versus basolateral targeting of secretory vesicles.
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PMID:Expression of two membrane fusion proteins, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein, in choroid plexus epithelium. 1255 91

Illumination of dark-grown Myxococcus xanthus with blue light leads to the induction of carotenoid synthesis. Central to this response is the activation of the light-inducible promoter, PcarQRS, and the transcription of three downstream genes, carQ, carR and carS. Sequence analysis predicted that CarQ is a member of the ECF (extracytoplasmic function) subfamily of RNA polymerase sigma factors, and that CarR is an inner membrane protein. Genetic analysis strongly implied that CarR is an antisigma factor that sequesters CarQ in a transcriptionally inactive complex. Using in vitro transcription run-off assays, we present biochemical evidence that CarQ functions as a bacterial sigma factor and is responsible for transcription initiation at PcarQRS. Similar experiments using the crtI promoter failed to implicate CarQ in direct transcription of the crtI gene. Experiments using the yeast two-hybrid system demonstrated a protein-protein interaction between CarQ and CarR, providing evidence of a CarQ-CarR complex. The yeast two-hybrid system data also indicated that CarR is capable of oligomerization. Fractionation of M. xanthus membranes with the detergent sarkosyl showed that CarR was associated with the inner membrane. Furthermore, CarR was found to be unstable in illuminated stationary phase cells, providing a possible mechanism by which the CarR-CarQ complex is disrupted.
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PMID:Light-induced carotenogenesis in Myxococcus xanthus: functional characterization of the ECF sigma factor CarQ and antisigma factor CarR. 1265 58

Little is known about the stability of transcripts encoding membrane proteins in strong expression systems and its effect on membrane protein over-production. We have expressed all the genes encoding subunits of the membrane domain F(o) of the ATP synthase in a T7 RNA polymerase-based system. All of them but uncB (subunit a) were expressed separately at very high levels in the bacterial hosts Escherichia coli C41(DE3) and C43(DE3). However, expression of uncB was extremely toxic to the bacteria. Northern blot analysis showed that the level of accumulation of the mRNA from uncB was very low. Deletion of uncB in combination with gene fusion experiments demonstrated that the middle region of the gene, encoding amino acids 92-171, exhibited a dominant toxic phenotype associated with a very poor level of expression. Green fluorescent protein fusions with N- and C-ends of uncB helped to stabilize the mRNA and to obtain high yields of protein.
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PMID:Over-expression of Escherichia coli F1F(o)-ATPase subunit a is inhibited by instability of the uncB gene transcript. 1286 Mar 93


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