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 rifampicin (Rif)-resistant RNA polymerase of phage T7 has proved invaluable for the exclusive over-expression, in Escherichia coli, of genes cloned downstream from the T7 phi 10 promoter [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 82 (1985) 1074-1078]. Here, we demonstrate that the system can be extended to Gram-negative bacteria other than E. coli, by the use of compatible wide host range plasmids. As an example, the Rif-resistant in vivo synthesis and specific radiolabelling of E. coli galactokinase in Pseudomonas ATCC19151, is demonstrated. The incidental observation that 30 min after treatment with Rif, two polypeptides continue to be synthesized in plasmid-free Pseudomonas ATCC19151, indicates that these proteins are produced by very stable mRNA species.
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PMID:Bacteriophage T7 RNA polymerase-controlled specific gene expression in Pseudomonas. 268 92

S1 nuclease mapping of RNA prepared from Pseudomonas amyloderamosa SB-15 suggested that the iam gene coding for isoamylase (glycogen 6-glucanohydrolase [EC 3.2.1.68]) is transcribed from two promoters. The transcription start site for the upstream promoter (termed P1) was located -182 base pairs from the first nucleotide of the initiation codon of iam, whereas the start site for the downstream promoter (termed P2) was 99 base pairs downstream of the P1 start site. Transcriptions from these promoters were induced by maltose and were not repressed by glucose. The promoter regions contained sequences homologous to the consensus sequence recognized by sigma 54 RNA polymerase of enteric bacteria and found in promoters of other Pseudomonas species. Northern (RNA) hybridization provided evidence that the iam gene is transcribed as monocistronic mRNAs with an approximate size of 2.6 kilobases.
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PMID:Transcription of the isoamylase gene (iam) in Pseudomonas amyloderamosa SB-15. 275 57

DNA-dependent RNA polymerase was purified from Pseudomonas aeruginosa. The subunit structure was typical of other eubacterial RNA polymerases in having beta' (157,000), beta (148,000), sigma (87,000), and alpha 2 (45,000) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was dependent on Mg2+, displaying optimal activity at 10 mM MgCl2. Ca2+ and Zn2+ could not replace MgCl2 in the assay system, while Mn2+, produced partial activity. KCl at concentrations greater than 10 mM inhibited enzyme activity. Optimal enzyme activity was observed at pH 8.5-9.0. The RNA polymerase was stable in 50% (w/v) glycerol at 4 degrees C for more than 3 months. Enzyme activity was inhibited in vitro by heparin, streptolydigin, streptovaracin, actinomycin D, and rifampicin.
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PMID:DNA-dependent RNA polymerase from Pseudomonas aeruginosa. 312 44

The general properties of the heat shock response in Pseudomonas aeruginosa were characterized. The transfer of cells from 30 to 45 degrees C repressed the synthesis of many cellular proteins and led to the enhanced production of 17 proteins. With antibodies raised against the Escherichia coli proteins, two polypeptides of P. aeruginosa with apparent molecular weights of 76,000 and 61,000 (76K and 61K proteins) were shown to be analogous to the DnaK and GroEL heat shock proteins of E. coli due to their immunologic cross-reactivity. The major sigma factor (sigma 87) of P. aeruginosa was shown to be a heat shock protein that was immunologically related to the sigma 70 of E. coli by using polyclonal antisera. A hybridoma was produced, and the monoclonal antibody MP-S-1 was specific for the sigma 87 and did not cross-react with sigma 70 of E. coli. A smaller 40K protein was immunoprecipitated with RNA polymerase antisera from cells that had been heat shocked. The 40K protein was also associated with RNA polymerase which had been purified from heat-shocked cells and may be the heat shock sigma factor of P. aeruginosa. Exposure to ethanol resulted in the production of seven new proteins, three of which appeared to be heat shock proteins.
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PMID:Heat shock response of Pseudomonas aeruginosa. 313 46

Bacteriophage phi 6 infects its host, the Gram-negative bacterium Pseudomonas syringae, by a protein-targeted fusion of the virus envelope with the host outer membrane. In this investigation we present results suggesting that the phage nucleocapsid penetrates the host cytoplasmic membrane via a membrane invagination and an intracellular vesicle. This indicates that the prokaryotic plasma membrane might be more dynamic and have more common features with eukaryotic membrane systems than previously expected. Most of the nucleocapsid surface lattice protein is degraded in the cell, and the nucleocapsid core particle containing the viral dsRNA segments and the proteins necessary for the viral RNA polymerase activity can be isolated from the infected cells. The penetration is dependent on the energized state of the host cytoplasmic membrane. About 25% of the entering core particles are re-used in the progeny viruses.
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PMID:The nucleocapsid of bacteriophage phi 6 penetrates the host cytoplasmic membrane. 316 5

We have determined the nucleotide sequence of the xylR gene for a transcriptional activator for the degradative pathway of aromatic hydrocarbons on the TOL plasmid from Pseudomonas putida. The 1698-bp sequence for a 566-amino acid (aa) protein (Mr 63741) was identified as the XylR-encoding sequence. Three regions in XylR show homology to Klebsiella pneumoniae NtrC and NifA, both of which are transcriptional activators for the ntr and nif genes involved in the nitrogen metabolism. The central region of XylR (aa 234-473) corresponds to the region that was proposed to interact with RNA polymerase having a sigma factor, NtrA [Drummond et al., EMBO J. 5 (1986) 441-447]. The C-terminal region (aa 515-558) has a putative DNA-binding structure. A short segment proximal to the central region (aa 211-229) is thought to be an interdomain linker. No amino acid homology was found in the N-terminal regions among these proteins. These findings suggest the interaction of XylR with an NtrA in the transcriptional activation of the degradative pathway.
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PMID:Nucleotide sequence of the regulatory gene xylR of the TOL plasmid from Pseudomonas putida. 316 74

A 2,598-base-pair (bp) SalI-HincII DNA fragment has been cloned which codes for vanillate demethylase, the enzyme responsible for the demethylation of vanillate (3-methoxy-4-hydroxybenzoate) to protocatechuate (3,4-dihydroxybenzoate). Complementation and insertional inactivation experiments have shown that this fragment carries two genes (vanA and vanB) which are predominantly cotranscribed from a promoter upstream of vanA. Nucleotide sequencing of the SalI-HincII fragment confirmed the genetic data: two open reading frames of 987 and 942 bp were present in the transcribed orientation. These had a very high G + C content in the third base of each codon, which is characteristic of Pseudomonas chromosomal genes. Expression of the genes in Escherichia coli with the T7 RNA polymerase-promoter system gave rise to two polypeptides of 36 and 33 kilodaltons which could be identified by deletion analysis as the products of vanA and vanB, respectively. A search of the protein sequence data bank indicated that the vanB gene product was related to the ferredoxin family.
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PMID:Cloning and sequencing of Pseudomonas genes encoding vanillate demethylase. 317 Apr 89

A 7 kb chromosomal DNA fragment from R. melilotii was cloned, which complemented temperature-sensitivity of an E. coli amber mutant in rpsA, the gene for ribosomal protein S1 (ES1). From complementation and maxicell analysis a 58 kd protein was identified as the homolog of protein S1 (RS1). DNA sequence analysis of the R. melilotii rpsA gene identified a protein of 568 amino acids, which showed 47% identical amino acid homology to protein S1 from E. coli. The RS1 protein lacked the two Cys residues which had been reported to play an important role for the function of ES1. Two repeats containing Shine-Dalgarno sequences were identified upstream of the structural gene. Binding studies with RNA polymerase from E. coli and Pseudomonas putida located one RNA-polymerase binding site close to the RS1 gene and another one several hundred basepairs upstream. One possible promoter was also identified by DNA sequence comparison with the corresponding E. coli promoter.
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PMID:Cloning and characterization of a gene from Rhizobium melilotii 2011 coding for ribosomal protein S1. 336 16

The Pseudomonas putida TOL plasmid pWWO carries an operon that specifies a meta-cleavage pathway for the catabolism of benzoate and toluates whose transcription is positively regulated by the xylS gene product. Stimulation of transcription of the operon is thought to result from activation of this protein by pathway substrates/effectors. In the present study, overexpression of the xylS gene has led to identification of the regulator as a 33 kDa protein. Overexpression of xylS also resulted in partially constitutive, i.e. effector-independent expression of the meta-cleavage operon. Determination of the polynucleotide sequence of the xylS gene revealed amino acid sequence homology with several DNA binding proteins, particularly with the araC products of Escherichia coli and Salmonella typhimurium and with the nifA and ntrC products of Klebsiella pneumoniae. Homologous sequences were mainly located in an alpha-helix-turn-alpha-helix domain of the polypeptide. Interestingly, amino acid sequence homology was also found with sigma factors of E. coli (ntrA and htpR products) and Bacillus subtilis (spoIIG and phage SPOI Gp34 products) and other RNA polymerase core-interacting proteins, such as the E. coli nusA product.
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PMID:The xylS gene positive regulator of TOL plasmid pWWO: identification, sequence analysis and overproduction leading to constitutive expression of meta cleavage operon. 347 26

Phage phi 6 has a genome consisting of three pieces of double-stranded RNA. Single-stranded RNA was prepared from phi 6 nucleocapsids by in vitro transcription with the phage RNA polymerase. These transcripts were polyadenylated and used as templates for the preparation of cDNA copies. The resulting DNA was cloned into the PstI restriction nuclease site of plasmid pBR322. Insert-bearing plasmids were annealed to phi 6 RNA to assign the inserts to their proper segments. In this way we identified inserts corresponding to the large, medium, and small segments. Two large overlapping inserts of the small segment constitute the complete complement of the segment as determined by the sequence analysis of the DNA. In vitro coupled transcription and translation showed that the small segment inserts were able to direct the synthesis of the four known genes in the small segment. Two overlapping inserts in the medium segment constitute the entire segment and were shown to direct the in vitro synthesis of two of the three known proteins of the medium segment. Several inserts bearing about one-third the complement of the large segment were also isolated, and one of these directed the synthesis of a peptide that resembles protein P1. Restriction endonuclease maps were prepared for the inserts, and by in vitro synthesis it was possible to refine the genetic map of phi 6. A chimeric plasmid was constructed that combines plasmids pUC8 and RSF1010. Inserts placed on this plasmid were transformed to Pseudomonas phaseolicola, the natural host of phage phi 6. It was possible to refine further the genetic map by complementation of nonsense mutants of phi 6 with the cDNA.
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PMID:cDNA cloning of portions of the bacteriophage phi 6 genome. 385 75


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