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)

Ribonucleic acid (RNA)-dependent RNA polymerase activity was demonstrated in the microsomal and ribosomal fraction from the spleen cells of immunized mice. The enzyme activity was solubilized by Triton X-100 from the fraction and partially purified by Biogel A 1.5 m column chromatography. The RNA-dependent RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RAN preparation (iotaRNA) as template made from the spleens of immunized mice but very low activity was found with an RNA preparation made from the spleens of normal mice. Incorporation of 3H-UTP markedly decreased in the presence of RNase but not in the presence of DNase. DNA preparations made from the spleens of immunized mice were inactive as template for this enzyme. The iotaRNA preparation was fractionated by sucrose density gradient centrifugation. A fraction corresponding to 12-13 S was most active as a template. It was followed by a fraction corresponding to 6-7 S. Sucrose gradient analysis of the 3H-UTP-labeled product was attempted. Some properties of this enzyme are described.
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PMID:Ribonucleic acid-dependent ribonucleic acid polymerase in the immune response. 123 May 9

In order to better understand the regulation of Pseudomonas aeruginosa flagellin expression we cloned the sigma factor of RNA polymerase used to transcribe the flagellin gene. It is a member of the sigma 28 class of alternative sigma factors described in several bacterial genera. Using the published sequence of the fliA gene encoding the sigma 28 from Salmonella typhimurium, we designed two oligonucleotides and, using the polymerase chain reaction, isolated the fliA gene from S. typhimurium chromosomal DNA. This heterologous probe was used in the DNA blot analysis of restriction digests of P. aeruginosa DNA. A 1.7 kb SalI-EcoRI fragment reacted with the probe and this fragment was cloned into the pBluescript vectors. The P. aeruginosa fliA gene was able to complement the motility defect of an Escherichia coli fliA mutant, but only when transcription was driven from the vector promoter. Insertional inactivation of the fliA gene with a gentamicin gene cassette rendered P. aeruginosa nonmotile and unable to express the flagellin gene. The 1.7 kb cloned fragment was sequenced and shown to contain the entire fliA gene. P. aeruginosa FliA shares 67% amino acid similarity with the homologous S. typhimurium sequence. Transcriptional analysis of the fliA gene showed that its expression was not dependent on RpoN, a sigma factor shown also to be required for flagellin synthesis. A reading frame downstream of fliA was found to encode the P. aeruginosa homologue of the enterobacterial cheY gene.
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PMID:The fliA (rpoF) gene of Pseudomonas aeruginosa encodes an alternative sigma factor required for flagellin synthesis. 156 Jul 74

The effects of mutations in the -10, -35, and Fnr box regions of the narGHJI promoter of Escherichia coli were determined by assaying the expression of beta-galactosidase from narG::lacZ fusion plasmids under aerobic and anaerobic conditions. A 1-base change in the -10 hexamer completely abolished expression, whereas a 3-base change to create the consensus TATAAT resulted in significant aerobic as well as anaerobic expression. A mutation in the putative -35 hexamer did not affect anaerobic expression but reduced aerobic expression from the construction with the -10 consensus sequence. A mutation in the Fnr box severely reduced anaerobic expression but did not affect aerobic expression. When the complete 5' region of the nar operon including the NarL box was present, nitrate stimulated both aerobic and anaerobic expression. Stimulation of expression by nitrate occurred in an fnr mutant but not in a narL mutant. We conclude that the rate of transcription of the nar operon is dependent on two distinct modes of transcription. One mode, which occurs at low levels, depends on the -10 and -35 hexamer sequences and is dramatically enhanced by changing the -10 sequence to the consensus TATAAT. The second depends on the -10 and Fnr box sequences but is independent of the -35 sequence. This second mode occurs at a very high level under anaerobic conditions when Fnr is activated and is also enhanced by changing the -10 sequence to the consensus TATAAT. NarL, activated by nitrate, stimulated both modes of transcription, indicating that it does not act through Fnr but that it directly affects the interaction of RNA polymerase with the promoter.
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PMID:Role of alternative promoter elements in transcription from the nar promoter of Escherichia coli. 173 6

The gene encoding rat fructose-1,6-bisphosphatase was isolated from a rat genomic Charon 4A library by screening with a cDNA to the rat liver enzyme. Southern blotting of rat genomic DNA showed that there is a single copy of the fructose-1,6-bisphosphatase gene. It extends over 23 kilobases and is composed of seven exons and six introns that range in size from 93 to 267 base pairs and from 1,400 to 11,300 base pairs, respectively. The intron/exon boundary sequences conform to consensus acceptor (GTn) and donor (nAG) sequences, and the exons in the gene appear to code for functional protein domains. The transcription start site, determined by 5'-extension sequencing of mRNA, was assigned to a guanine 119 bases 5' to the translation initiation AUG. The sequence of the gene upstream to the cap site contains characteristic RNA polymerase II promoter-binding sites: a putative TATA box at position -29 and a Sp 1 binding site (GGGGCGGAGA) at position -48. A 1,300-base pair fragment of 5'-flanking sequence containing these elements, ligated upstream from a firefly luciferase reporter gene and transfected into cultured normal rat kidney cells, demonstrated strong promoter activity. The accumulation of fructose-1,6-bisphosphatase mRNA in hepatocytes incubated with cAMP suggests that the gene may be cAMP-responsive, which is consistent with the presence of three consensus cAMP regulatory elements at positions -169, -282, and -698 in the 5'-flanking region of the gene. Expression of the fructose-1,6-bisphosphatase promoter-driven luciferaes gene was 2-3-fold activated by the cyclic nucleotide, suggesting that one or more of these elements may be functional. On the other hand, insulin decreased the expression of the endogenous gene in hepatocytes. Thus, expression of the fructose-1,6-bisphosphatase gene is regulated independently by both cAMP and insulin.
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PMID:The rat fructose-1,6-bisphosphatase gene. Structure and regulation of expression. 184 13

A gene encoding a carrier protein for glutamate and aspartate was cloned into Escherichia coli K-12 strain BK9MDG by using the high-copy-number plasmid pBR322. The gene (designated gltP) is probably identical to a gene recently cloned from E. coli B (Y. Deguchi, I. Yamato, and Y. Anraku, J. Bacteriol. 171:1314-1319). A 1.6-kilobase DNA fragment containing gltP was subcloned into the expression plasmids pT7-5 and pT7-6, and its product was identified by a phage T7 RNA polymerase-T7 promoter coupled system (S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. USA 82:1074-1078) as a polypeptide with an apparent mass of 38 kilodaltons. A portion of the gltP polypeptide was associated with the cytoplasmic membrane. The nucleotide sequence of the 1.6-kilobase fragment was determined. It contained an open reading frame capable of encoding a highly hydrophobic polypeptide of 395 amino acids, containing four possible transmembrane segments. Uptake of glutamate and aspartate was increased 5.5- and 4.5-fold, respectively, in strains containing gltP plasmids. Glutamate uptake was insensitive to the concentration of Na+ and was inhibited by L-cysteate and beta-hydroxyaspartate. These results suggest that gltP is a structural gene for a carrier protein of the Na(+)-independent, binding-protein-independent glutamate-aspartate transport system.
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PMID:Cloning and sequencing of a gene encoding a glutamate and aspartate carrier of Escherichia coli K-12. 197 22

We determined the DNA sequences of regions essential for bacteriophage P4 integration. A 20 base-pair core sequence in both phage (P4attP) and host (P4attB) attachment regions contains the recombination site. In P4attP this sequence is flanked by five repeated sequences. A 1.3 x 10(3) base open reading frame codes for P4 integrase. Two possible promoters are upstream from P4int. One would be recognized by Escherichia coli RNA polymerase and may be repressed by integrase protein. The second would be recognized by RNA polymerase modified after infection by a P4 helper phage, P2. The P4attB core sequence is the 3' end of a leucine tRNA gene. Downstream from this tRNA in E. coli K-12 is a region homologous to P4int that may be part of a cryptic prophage.
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PMID:Integration of satellite bacteriophage P4 in Escherichia coli. DNA sequences of the phage and host regions involved in site-specific recombination. 311 56

A 1,250 base pair Bacillus subtilis chromosomal HindIII restriction fragment (S fragment) has been cloned into the B. subtilis expression-probe plasmid pGR71. The S fragment induces the expression of the pGR71 chloramphenicol resistance gene shortly after the initiation of sporulation. The transcriptional promoter responsible for the expression of this temporally regulated genetic element has been identified and mapped in vitro. This promoter is recognized exclusively by the minor B. subtilis RNA polymerase that contains the 37,000 dalton sigma factor.
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PMID:A temporally regulated promoter from Bacillus subtilis is transcribed only by an RNA polymerase with a 37,000 dalton sigma factor. 631 73

RNA nucleotidyltransferase (EC 2.7.7.6) of Streptomyces granaticolor was purified by precipitation with polymin P and ammonium sulphate, affinity chromatography on DNA- cellulose and gell filtration on Biogel A 1.5 m. SDS-polyacrylamide gel electrophoresis revealed 8 protein bands of molar mass ranging from 37 to 130 kg/mol. Proteins of molar mass of 130 and 120 kg/mol were identified to be beta and beta subunits, respectively. The role of other subunits of the enzyme is discussed.
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PMID:Purification of DNA-dependent RNA polymerase from Streptomyces granaticolor. 664 21

The preparation and characterization of a protein from wheat germ showing strong affinity to amatoxins (ABP) but differing from RNA polymerase B (II) is described. The purification, traced by [3H]amatoxin (O-methyldehydroxymethyl-alpha-amanitin), comprises 4 chromatographic steps, on Biogel A 1.5, DEAE-Sephadex, phosphocellulose, and again Biogel A 1.5. The protein exhibited in dodecyl sulfate polyacrylamide gel electrophoresis one single band with a molecular mass of 29,000 Da. Its isoelectric point is 4.9. The dissociation constant of the complex ABP-[3H]amatoxin is KD20 = 5 X 10(-7)M as determined by equilibrium dialysis against alpha-amanitin. By cyanogen bromide the protein is split into two fragments with molecular masses of 22,000 and 7,000 Da, respectively whose amino acid analyses, on summation, give the amino acid composition of ABP.
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PMID:[Isolation and characterization of an amatoxin-binding protein from wheat germ (author's transl)]. 707 28

A 1.74-kb cDNA fragment containing the gp53 coding region has been cloned from bovine viral diarrhoea virus (BVDV) strain Singer by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis indicated that gp53 of BVDV strains Singer, NADL and SD-1 shared extensive sequence homology at both the RNA (85-94%) and protein (82-91%) levels. Nineteen cysteine residues and five potential N-linked glycosylation sites were identified within the sequenced region, all of which were conserved. These observations suggest that although the homology at the nucleotide sequence level may vary, there was strong structural conservation among bovine viral diarrhoea virus envelope proteins. Full-length gp53 was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST). The N-terminal half of gp53 was also synthesised in E. coli as a 28-KDa recombinant protein using the T7 RNA polymerase-directed expression system. Both recombinant proteins were expressed at high levels (approximately 30-50 mg/l). The recombinant proteins were recognised in ELISA and Western blot analyses by polyclonal serum raised against a mixture of BVDV and classical swine fever virus (CSFV). Rabbit antiserum raised against the 28-kDa recombinant protein reacted with different BVDV strains in ELISA and immunofluorescent antibody test, but not with CSFV in the same tests. These results demonstrated that the bacterial recombinant proteins have similar immunological properties to that of the native viral protein and, in conjunction with its homologous antisera, can be useful as diagnostic reagents.
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PMID:High level expression of the envelope glycoprotein (gp53) of bovine viral diarrhoea virus (Singer) and its potential use as diagnostic reagent. 785 9


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