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)

Adult diarrhea rotavirus (ADRV) has caused epidemics of diarrhea in China since 1982 and remains the only group B rotavirus associated with widespread disease in humans. We recently characterized the proteins of ADRV and have now proceeded to identify the gene segments encoding each protein. Viral RNA transcripts were synthesized in vitro with the endogenous viral RNA polymerase and separated by electrophoresis in agarose. The individual transcripts were translated in a cell-free system using nuclease-treated rabbit reticulocyte lysates. The translation products were compared with polypeptides found in purified virus and were characterized by SDS-PAGE, immunoprecipitation, and Western blot analysis using antisera to double- and single-shelled virions, virus cores, and monoclonal antibodies. Furthermore, individual RNA transcripts were hybridized to total dsRNA to determine their genomic origin. Based on this analysis, the core polypeptides VP1, VP2 and VP3 are encoded by segments 1, 2, and 3, respectively. The main polypeptides in the inner capsid, VP6, and the outer capsid, VP4 and VP7, are encoded by segments 6, 4, and 8 respectively. Segments 5, 7, and 9 code for 60, 45, and 30 kDa nonstructural polypeptides. Two other nonstructural polypeptides (24 and 25 kDa) are derived from gene segment 11. Gene segment 10 codes for a 26 kDa polypeptide that is precipitated with serum to ADRV and may be a structural protein VP9. With this exception, gene coding assignments of ADRV are comparable to those of the group A rotaviruses. Our results have clear implications for further work in cloning, sequencing, and expression genes of ADRV and can provide direction towards understanding the origin and the evolution of this virus.
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PMID:Coding assignments of the genome of adult diarrhea rotavirus. 132 59

Three open reading frames (ORFs) have been found in the region downstream of the luxG gene in the Photobacterium leiognathi lux operon. These genes (ORF I, II, and III) are not only closely linked to the lux operon and transcribed in the same direction but also show the same organization and code for proteins homologous in sequence to the gene products of ribB, ribA, and ribH of Bacillus subtilis, respectively. The Photobacterium leiognathi gene (ORF II) corresponding to ribA was expressed in Escherichia coli in the bacteriophage T7 promoter-RNA polymerase system and a 40 kDa 35S-labeled polypeptide has been detected on SDS-PAGE. Expression of DNA extending from luxBEG to ORF II inserted between a strong promoter and a reporter gene and transferred by conjugation into Vibrio harveyi did not affect the expression of the reporter gene. The results provide evidence that neither promoter nor terminator sites were present in the DNA between the luxG and ORF II indicating that these genes might be part of the lux operon.
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PMID:The lux genes in Photobacterium leiognathi are closely linked with genes corresponding in sequence to riboflavin synthesis genes. 133 74

The promoter region of the agarase gene (dagA) of Streptomyces coelicolor A3(2) is complex; it consists of four distinct promoters with different -10 and -35 regions. We report the isolation of a form of RNA polymerase that mediates transcription in vitro from the dagAp4 promoter. The core components of this RNA polymerase are associated with a polypeptide of c. 66 kDa; holoenzyme reconstitution experiments show that the 66 kDa polypeptide functions as a sigma factor that directs transcription from the dagAp4 and Bacillus subtilis veg promoters in vitro. Alignment of the DNA sequences of these two promoters shows that they have bases in common in the -10 and -35 regions and that these sequences are similar to those observed for the major RNA polymerases of other bacteria. N-terminal amino acid sequence analysis of the 66 kDa polypeptide revealed it to be the product of the hrdB gene. Previous experiments showed that the predicted amino acid sequence of the hrdB gene product is very similar to the major sigma factors of other bacteria and suggested that disruption of the hrdB gene is lethal. These observations together lead to the conclusion that we have isolated the major RNA polymerase of Streptomyces coelicolor A3(2). We have developed an improved protocol for the renaturation of sigma factors that have been isolated by preparative sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). This method involves renaturing the polypeptide in the presence of the bacterial chaperonin GroEL. We expect this protocol to find general application for renaturation of other polypeptides that have been subjected to SDS-PAGE.
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PMID:Isolation and characterization of the major vegetative RNA polymerase of Streptomyces coelicolor A3(2); renaturation of a sigma subunit using GroEL. 135 Mar 15

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 Saccharomyces cerevisiae RNA polymerase III transcription factor (TF)IIIB has been assembled from three components. An assembly pathway of these polypeptides, which specifies their interactions, has been determined. The TATA-binding protein, TBP, and the TFIIB-related BRF1 gene product BRF, together reconstitute the transcription factor activity and TFIIC-dependent DNA-binding activity of the B' component of TFIIIB. BRF alone weakly binds to a TFIIIC-tRNA gene complex; TBP greatly stabilizes this interaction. B" transcription factor activity is recovered with its previously identified 90 kd polypeptide from SDS-polyacrylamide gels. Incorporation of the 90 kd B" protein into the transcription complex requires TBP. The heparin-resistant TFIIIB-DNA complex retains all three of its constituent proteins, TBP, BRF, and B".
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PMID:The role of the TATA-binding protein in the assembly and function of the multisubunit yeast RNA polymerase III transcription factor, TFIIIB. 145 36

In the enteric bacterium, Escherichia coli, acyl coenzyme A synthetase (fatty acid:CoA ligase (AMP-forming) EC 6.2.1.3) activates exogenous long-chain fatty acids concomitant with their transport across the inner membrane into metabolically active CoA thioesters. These compounds serve as substrates for acyl-CoA dehydrogenase in the first step in the process of beta-oxidation. The acyl-CoA synthetase structural gene, fadD, has been identified on clone 6D1 of the Kohara E. coli gene library and by a process of subcloning and complementation analyses shown to be contained on a 2.2-kilobase NcoI-ClaI fragment of genomic DNA. The polypeptide encoded within this DNA fragment was identified following T7 RNA polymerase-dependent induction and estimated to be M(r) = 62,000 using SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of acyl-CoA synthetase was determined by automated sequencing to be Met-Lys-Lys-Val-Trp-Leu-Asn-Arg-Tyr-Pro. Sequence analysis of the 2.2-kilobase NcoI-ClaI fragment revealed a single open reading frame encoding these amino acids as the first 10 residues of a protein with a molecular weight of 62,028. The initiation codon for methionine was TTG. Primer extension of total in vivo mRNA from two fadD-specific oligonucleotides defined the transcriptional start at an adenine residue 60 base pairs upstream from the predicted translational start site. Two FadR operator sites of the fadD gene were identified at positions -13 to -29 (OD1) and positions -99 to -115 (OD2) by DNase I footprinting. Comparisons of the predicted amino acid sequence of the E. coli acyl-CoA synthetase to the deduced amino acid sequences of the rat and yeast acyl-CoA synthetases and the firefly luciferase demonstrated that these enzymes shared a significant degree of similarity. Based on the similar reaction mechanisms of these four enzymes, this similarity may define a region required for the same function.
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PMID:Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase. 146 45

Incubation of the E. coli RNA polymerase with a polypeptide factor from the protozoan Tetrahymena reduces the affinity of the holoenzyme for DNA. SDS-polyacrylamide gel electrophoresis of the peptide-treated RNA polymerase showed that the band pattern of the polymerase subunits was strongly altered. The three large subunits, beta', beta and sigma, disappear and a high number of rapidly migrating bands appeared. However, a brief heat treatment of the samples almost restored the original RNA polymerase subunit composition, and in addition a high molecular weight protein band approximately 240 kDa appeared. It is suggested that the Tetrahymena peptide specifically binds to the RNA polymerase and changes the structures of the large subunits.
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PMID:A peptide from Tetrahymena disrupts subunit organization of E. coli RNA polymerase. 147 89

The cleavage signal-1 protein (CS-1), a doublet antigen comprised of approx. 14-kDa and 18-kDa proteins has been shown to be present on the surface of sperm of various mammalian species including humans. Polyclonal antibodies to CS-1 inhibit the early cleavage of fertilized eggs without apparently affecting sperm penetration and pronuclear formation. We report here the cloning of the human CS-1 cDNA and its expression in vitro to obtain the recombinant protein (reCS-1) molecule. The CS-1 cDNA clone was isolated by immunological screening of a human testis lambda gt11 cDNA library with mono-specific polyclonal antibody against CS-1. The cDNA is 1828 bp long; the start codon assigned to the first ATG (bp 98-100) encodes a protein with 249 amino acid residues terminating at TAA (bp 845-847). The cDNA isolated has a 97-bp 5' and a 984-bp 3' untranslated region. The potential polyadenylation signal (5'-AATAAA) is at bp 1803-1808. An extensive computer search of the GenBank database did not indicate any extensive homology with any known sequence, indicating that CS-1 is a unique protein. The CS-1 cDNA was cloned in the transcription vector, pGEM-11Zf, to obtain high-level in vitro transcription by SP6 and T7 RNA polymerase. The transcribed CS-1 RNA was translated in a rabbit reticulocyte in vitro translation system and produced a 33-kDa reCS-1 protein, as assessed by migration in a SDS-polyacrylamide gel.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Human cleavage signal-1 protein; cDNA cloning, transcription and immunological analysis. 155 70

The protein-coding capacities of rat and human catechol O-methyltransferase (COMT) DNA clones were analysed by in vitro transcription and translation using bacteriophage RNA polymerase and rabbit reticulocyte lysate. Two types of clones corresponding to the structures of human placental cDNA clones were used. The shorter clones, containing the 663-residue open reading frame for the soluble COMT (S-COMT), produced 24-kDa (rat) and 26-kDa (human) polypeptides. Translation of the longer clones, containing 43 (rat) or 50 (human) amino acid amino-terminal extensions to the S-COMT polypeptides, yielded 28-kDa (rat) and 30-kDa (human) putative membrane-bound COMT (MB-COMT) polypeptides as the main products. These clones also yielded low amounts of the S-COMT polypeptides. Labelling time or ionic conditions during translation did not eliminate the shorter products, suggesting translation initiation from the second S-COMT AUG codon. In accordance with this postulation, the relative amount of S-COMT could be affected by changing the translation initiation contexts preceding the first AUG codon. The 28-kDa and 30-kDa products, but not the 24-kDa and 26-kDa products, associated with microsomal membranes cotranslationally, indicating that the amino-terminal extensions were functional signal sequences. However, the presence of membranes did not affect the mobilities of the proteins in SDS/polyacrylamide gels. The MB-COMT polypeptides could not be released from the microsomes by treatments with phospholipase C or alkali and were not protected by the microsomes against proteinase K digestion. These results indicate that MB-COMT synthesized in vitro is an integral membrane protein having an amino-terminal signal-anchor sequence.
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PMID:Cell-free synthesis of rat and human catechol O-methyltransferase. Insertion of the membrane-bound form into microsomal membranes in vitro. 176 63

We have shown by appropriate modification of the translational signals and using the strong T7 RNA polymerase promoter phi 10, that a cloned Saccharomyces cerevisiae pyruvate decarboxylase gene (pdc1) can be expressed in Escherichia coli. This protein, which migrated as a single band on SDS-polyacrylamide gels, was found to have a subunit molecular mass of approximately 62 kDa, similar to that of the enzyme produced by yeast. Polyclonal antibodies raised against purified yeast pyruvate decarboxylase recognized this bacterially produced protein. We found that this recombinant enzyme is active, indicating that the homotetramer encoded by the pdc1 gene is functional.
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PMID:Expression of active yeast pyruvate decarboxylase in Escherichia coli. 179 34


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