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)

Earlier, our laboratory reported the cloning of a chromosomally encoded cholera toxin (CT)-like enterotoxin gene from Salmonella typhimurium Q1 into pBR322. Cell lysates from the plasmid clone pC1, containing a 4.8 kb EcoR1 DNA fragment from Salmonella, caused elongation of Chinese hamster ovary (CHO) cells and this biologic activity was neutralized by anti-CT. However, this cloned gene product did not elicit fluid secretion in the rabbit intestinal loop (RIL) model, because of poor expression. We report here, subcloning of a 4.8 kb EcoRI and a 2.7 kb HindIII/Eco Rl fragment into a high expression T7 RNA polymerase/promoter system. Cell lysates from these clones elicited fluid secretion in the RIL model, caused firm induration in rabbit skin and elongated CHO cells. These biological activities were neutralized by anti-CT. SDS-PAGE and subsequent fluorographic analysis of Escherichia coli, harboring recombinant plasmids in a T7 expression system, revealed the presence of two prominent 35S-labeled polypeptides of 25 and 12 kDa, which were immunoprecipitated with anti-CT. The enterotoxin appeared to be 125 kDa in size, based on chromatography on a P-300 column, had a pl of 6.6 to 6.8, and was heat-labile (60 degrees C/5 min). Unlike cloned CT and heat-labile enterotoxin (LT-l), which were localized in the periplasm, the Salmonella enterotoxin was cytoplasmic in nature.
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PMID:Expression and characterization of the cloned Salmonella typhimurium enterotoxin. 145 24

The toxR gene of Vibrio cholerae encodes a transmembrane, DNA-binding protein that positively controls transcription of the genes for cholera toxin, TCP pili, and other proteins important in cholera pathogenesis. Nucleotide sequence analysis of the toxR upstream region has revealed that the heat shock gene htpG, encoding the bacterial homologue of the eukaryotic Hsp90 protein, was located immediately upstream and was divergently transcribed from toxR. Using lacZ transcriptional fusions, we have shown that neither toxR nor htpG expression was regulated by ToxR. However, the growth temperature had a coordinate but reciprocal effect on the expression from both the toxR and htpG promoters in V. cholerae; the decrease of toxR expression between 22 degrees C and 37 degrees C was proportional to the increase of htpG expression observed within that temperature range. A similar pattern of expression of the htpG and toxR promoters was observed in the heterologous host Escherichia coli, where this regulation was controlled by the level of the E. coli rpoH (htpR) gene product, sigma-32. Consistent with the temperature-regulated expression of the V. cholerae htpG promoter in E. coli, a sequence similar to the consensus sequence of the E. coli heat shock promoters was detected upstream from the V. cholerae htpG gene. We propose a model in which the regulation of toxR expression by temperature is controlled by the level of sigma-32 (RpoH) RNA polymerase.
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PMID:Expression of ToxR, the transcriptional activator of the virulence factors in Vibrio cholerae, is modulated by the heat shock response. 212 7

Colloidal gold is an electron-dense, lyophobic colloid that readily forms a stable electrostatic interaction with a variety of macromolecules. Monodispersed colloids ranging from 3-150 nm in diameter can be produced to provide the researcher with flexibility in selecting the optimally sized probe. Gold labeling of antibodies and lectins has been extensively used to study surface antigens and cell components. Recently, the use of gold labeling has been extended to study receptor-ligand binding, enzyme-substrate reactions, and transcellular pathways. Published applications include gold labeling of metabolites (low-density lipoproteins), enzymes (DNAase and RNAase, RNA polymerase, thrombin, collagenase, elastase), hormones (insulin, epidermal growth factor, glucagon), circulating plasma proteins (asialoglycoprotein, alpha 2-macroglobulin, factor VIII-von Willebrand factor), and endotoxins (tetanus toxin, cholera toxin). This broad spectrum of applications emphasizes the versatility and usefulness of colloidal gold as a probe in areas of cell biology related to receptors, endocytosis, transport, and functions of proteins.
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PMID:Colloidal gold: a pluripotent receptor probe. 635 33

Studies were performed to determine the mechanism underlying deficient arginine vasopressin (AVP)-stimulated adenylyl cyclase activity in chronic renal failure (CRF). As compared to control, principal cells cultured from the inner medullary collecting tubule of rats previously made uremic by 5/6 nephrectomy fail to accumulate cAMP when stimulated with AVP. CRF cells do respond normally to forskolin or cholera toxin and the defect in AVP responsiveness is not prevented by treatment with pertussis toxin, by cyclooxygenase inhibition, or by inhibition or down-regulation of protein kinase C. In contrast to their lack of responsiveness to AVP, CRF cells respond normally to other agonists of adenylyl cyclase such as isoproterenol or prostaglandin E2. Plasma membranes prepared from the inner medullae of CRF rats exhibit a marked decrease in apparent AVP receptor number but no change in the apparent number of beta adrenergic receptors. Reverse transcriptase PCR of total RNA from the inner medullae of CRF animals reveals virtual absence of V2 receptor mRNA; mRNA for alpha s is present in normal abundance. These studies indicate that AVP resistance in CRF is due, at least in part, to selective down-regulation of the V2 receptor as a consequence of decreased V2 receptor mRNA.
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PMID:Vasopressin resistance in chronic renal failure. Evidence for the role of decreased V2 receptor mRNA. 761 8

The nucleotide sequence of the Vibrio cholerae acfA gene (encoding an accessory colonization factor) has been determined. Sequence analysis revealed the presence of an open reading frame of 215 amino acids with a characteristic signal peptidase I (SPI) cleavage site at the N terminus. Electrophoretic analysis of proteins synthesized by Escherichia coli cells, following T7 promoter/RNA polymerase-directed expression of acfA, revealed a 23-kDa protein corresponding to the mature form of AcfA. The T7 expression system also showed that, in the presence of known SPI inhibitors, a 25-kDa unprocessed form of AcfA is produced.
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PMID:Isolation and characterization of the Vibrio cholerae acfA gene, required for efficient intestinal colonization. 773 17

The Yersinia enterocolitica surface antigen Myf is a fibrillar structure that resembles CS3 fimbriae. Gene myfA encodes the 21-kDa major subunit of the antigen, while genes myfB and myfC are required for the transport and assembly of pilin subunits at the bacterial cell surface. Here we show that the expression of Myf is regulated at the transcriptional level by temperature and pH. Gene myfA is transcribed at 37 degrees C and in acidic medium. The transcription start is preceded by a putative -10 box for the vegetative RNA polymerase as well as by sequences resembling the consensus sequence recognized by sigma 28. Thus, myfA could be transcribed either from a classical sigma 70 promoter or from a sigma 28 promoter. Transcription of myfA requires at least two genes, myfF and myfE, situated immediately upstream from myfA. The myfF product does not show similarity to any known regulatory protein. It is an 18.5-kDa protein with no typical helix-turn-helix motif and a unique hydrophobic domain in the NH2-terminal part. T7 expression, osmotic shock, fractionation experiments, and TnphoA fusion analyses carried out in Escherichia coli suggest that MyfF is associated with the inner membrane by means of its hydrophobic domain whereas the hydrophilic part protrudes in the periplasm. These features strikingly evoke ToxS, a protein involved in regulation of Tcp pilus production in Vibrio cholerae. MyfE resembles PsaE, a protein involved in regulation of pH6 antigen in Yersinia pestis. Genes myfF and myfE are presumably part of a whole regulatory network. MyfF could be an element of the signal transducing system.
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PMID:MyfF, an element of the network regulating the synthesis of fibrillae in Yersinia enterocolitica. 783 9

The Vibrio cholerae (Vc) gene (tagA) coding for the TagA lipoprotein has been isolated. Sequencing of tagA revealed the presence of an open reading frame (ORF) of 568 amino acids with a characteristic signal peptidase II cleavage site at the N terminus. Electrophoretic analysis of proteins synthesized by Escherichia coli (Ec) cells following T7 promoter/RNA polymerase-directed expression of tagA, revealed a closely migrating doublet of proteins corresponding to two species of TagA. Computer-generated alignment algorithms predict that a homology exists between Vc TagA and Ec K99 fimbriae biogenesis determinant FanD.
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PMID:Isolation and characterization of a Vibrio cholerae gene (tagA) that encodes a ToxR-regulated lipoprotein. 788 90

El Tor biotype Vibrio cholerae strains express a cell-associated mannose-sensitive hemagglutinin (MSHA) which is a putative attachment factor. Several MSHA-negative mutants from V. cholerae strain JBK70 were previously generated by Tn5 mutagenesis [Finn et al., Infect. Immun. 55 (1987) 942-946]. The chromosomal DNA regions containing the Tn5 insertions were isolated from eight strains for further analysis. Nucleotide sequencing of the insertional junctions and corresponding clones containing the intact chromosomal region from the parental strain revealed the presence of several contiguous ORFs. Only two ORFs of this region had received insertions, and these showed remarkable homology to genes involved in the general secretory pathway found in several Gram- bacterial species. Proteins corresponding to the observed ORFs were visualized with the T7 promoter/RNA polymerase expression system. Marker exchange mutagenesis was used to insert kanamycin-resistance cassettes and TnphoA insertions into different locations of this region in the chromosome of wild-type V. cholerae strains. The phenotypes of these mutants showed that this DNA region is involved in MSHA production, but is not required for general extracellular protein secretion.
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PMID:Genetic characterization of mannose-sensitive hemagglutinin (MSHA)-negative mutants of Vibrio cholerae derived by Tn5 mutagenesis. 795 47

In this study, we describe the molecular and antigenic characteristics of a cloned enterotoxin from Salmonella typhimurium strain Q1. The full length Salmonella enterotoxin gene (stn), localized on a 2.8 kb ClaI/PstI DNA fragment, was cloned from a genomic library of Salmonella. Based on nucleotide sequence analysis, the stn gene contained 749 bp that would encode a protein having a molecular size of 29,073. The most unusual feature of the stn gene was the presence of a rare initiation codon (TTG) in lieu of the typical ATG codon, which required site-directed mutagenesis to confirm the precise initiation site. The expression of the stn gene in a bacteriophage T7 RNA polymerase/promoter system was enhanced by introducing a typical ATG start codon and an optimal Shine-Dalgarno sequence upstream of the stn gene by site-directed mutagenesis. The stn gene was located opposite the hydHG operon that regulates labile hydrogenase activity in Salmonella species and Escherichia coli. The overall amino acid sequence of the enterotoxin was quite dissimilar to any other published sequence, including cholera toxin or other adenylate cyclase-activating proteins. However, an intriguing similarity in a small region of the amino acid sequence of Stn was observed with portions of the amino acid sequences from several other protein toxins known to ADP-ribosylate host cell proteins. This region of homology may indicate a conserved motif, within the active site, that is involved in the stimulation of adenylate cyclase activity.
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PMID:Molecular characterization of an enterotoxin from Salmonella typhimurium. 804 4

Biogenesis of the toxin-coregulated pilus (TCP) of Vibrio cholerae 01 is essential for successful bacterial colonization of the small intestine. Pilus assembly requires the products of at least seven genes located on the chromosome adjacent to the pilin-encoding gene, tcpA. Previously reported TnphoA insertions in the TCP-assembly-deficient V. cholerae strains, KP2.21 and KP4.2, were isolated from the chromosome for further analysis. Nucleotide sequencing of the tcpE::phoA and tcpF::phoA fusions and corresponding clones of the region containing the intact genes revealed the presence of two open reading frames (ORFs) of 340 and 338 amino acids, designated TcpE and TcpF, respectively. The partial sequence of an ORF downstream from the TcpF coding sequence was determined to correspond to the global virulence regulator, ToxT. Proteins corresponding to the observed ORFs were visualized with the T7 promoter/RNA polymerase expression system. Computer-generated alignment algorithms predict that a homology exists between TcpE and the Klebsiella pneumoniae pullulanase secretion proteins PulD and PulF, the Xanthomonas campestris extracellular enzyme secretion factor XpsF, the Bacillus subtilis DNA competence protein ComG-ORF2, and the Yersinia enterocolitica Yop secretion determinant YscC. These observations provide a model to investigate further the relationship between the secretion mechanisms utilized by these seemingly diverse virulence determinants. Additionally, an extreme C-terminal segment of TcpE shows striking homology to the transmembrane segment of the eukaryotic integrin beta-1 chain, which could imply a role for TcpE in not only TCP secretion, but also host cell interaction.
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PMID:Biogenesis and regulation of the Vibrio cholerae toxin-coregulated pilus: analogies to other virulence factor secretory systems. 809 77


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