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)

To further investigate the role of p53 gene inactivation in gastric tumorigenesis, the mutational status of the p53 gene in primary human gastric cancer samples was examined. Reverse transcriptase polymerase chain reaction and subsequent direct sequencing of the p53 gene from gastric cancer samples revealed frequent point mutations of the p53 gene: some of these coincided with those previously identified in gastric cancer cell lines. In addition, both allelic deletion analysis using pYNZ 22 and polymerase chain reaction-restriction fragment length polymorphism analysis demonstrated an allelic deletion of the p53 gene in cancer tissue which contained a point mutation of the p53 gene in the remaining allele. Transfection of the wild-type or mutant p53 genes into gastric cancer cells showed that the wild-type but none of the mutated p53 genes suppressed the colony formation of gastric cancer cells. Furthermore, the incorporation of thymidine into DNA was reduced in cancer cells expressing the wild-type p53 gene. The glutathione S-transferase-wild type p53 fusion protein bound to simian virus 40 large T antigen in COS-1 cell lysate. None of the p53 fusion proteins containing mutations at codons 143, 175, 248, or 273 bound to simian virus 40 large T antigen. By contrast, two different mutant p53 fusion proteins containing mutations specifically observed in gastric cancer bound to simian virus 40 large T antigen. These results indicate that inactivation of the p53 gene through mutations and the allelic deletion may play an important role in gastric tumorigenesis. These mutations may cause a conformational change in the p53 protein resulting in the loss of the suppression by p53 of the growth of gastric cells, partly through disruption of the association of p53 protein with a cellular component.
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PMID:p53 gene mutations in human gastric cancer: wild-type p53 but not mutant p53 suppresses growth of human gastric cancer cells. 132 85

The Drosophila glutathione S-transferase D genes encode a family of isozymes. We have determined the amino acid sequence of a new member of this family by nucleotide sequence analysis of a genomic DNA clone. The open reading frame of this intronless gene should encode an isozyme subunit of 211 amino acids. This sequence has significant homology to the E. coli stringent starvation protein, SSP, which is also a protein of two identical 211 amino acid subunits. The two proteins have very similar overall amino acid composition as well. It is possible that SSP may be a glutathione S-transferase(s) in E. coli or is evolutionarily related to glutathione S-transferases. Because SSP is known to be tightly associated with the RNA polymerase holoenzyme during purification, it is conceivable that Drosophila glutathione S-transferase(s) may potentially interact with the transcription machinery in a fashion similar to SSP's interaction with E. coli RNA polymerase holoenzyme.
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PMID:Drosophila glutathione S-transferases have sequence homology to the stringent starvation protein of Escherichia coli. 173 92

Encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), which allowed easy purification of the fusion protein by affinity chromatography on immobilized glutathione. Inclusion of a thrombin cleavage site between the GST carrier and the viral enzyme facilitated the release of purified mature EMC virus RNA polymerase from the GST carrier by proteolysis with thrombin. The purified recombinant enzyme has a molecular mass of about 52 kDa and is recognized by polyclonal immune serum raised against a peptide sequence corresponding to the C-terminal region of the protein. The recombinant enzyme comigrates with immunoprecipitated EMC virus RNA polymerase from infected mouse L929 cell extracts when run in parallel lanes on a sodium dodecyl sulfate-polyacrylamide gel. The enzyme exhibits rifampin-resistant, poly(A)-dependent poly(U) polymerase activity and RNA polymerase activity, which are both oligo(U) dependent. Template-size products are synthesized in in vitro reactions with EMC virus genomic RNA or globin mRNA. The availability of recombinant EMC virus RNA polymerase in a purified form will allow biochemical analysis of its role in the replication of the virus as well as structure-function studies of this unique class of enzyme.
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PMID:Expression, purification, and properties of recombinant encephalomyocarditis virus RNA-dependent RNA polymerase. 185 68

Poliovirus protein 3B (also known as VPg) is covalently linked to the 5' ends of both genomic and antigenomic viral RNA. Genetic and biochemical studies have implicated protein 3AB, the membrane-bound precursor to VPg, in the initiation of genomic RNA synthesis. We have purified 3AB to near homogeneity following thrombin cleavage of purified glutathione S-transferase-3AB. When added to transcription reaction mixtures catalyzed by poliovirus RNA polymerase (3Dpol), 3AB stimulated RNA synthesis up to 75-fold with oligo(U)-primed virion RNA, globin mRNA, and unprimed synthetic, full-length minus-strand viral RNA as the templates. Synthetic VPg also stimulated RNA synthesis but was only 1 to 2% as effective as 3AB on a molar basis. The increased level of transcription was not the result of enhancing the elongation rate of the polymerase. No evidence was found for uridylylation of 3AB or for covalent linkage to RNA transcription products. 3AB sedimented as a multimer in glycerol gradients. In the presence of the polymerase, the sedimentation rate of both proteins increased, suggesting the formation of a complex. Detergent prevented both multimerization and complex formation. The polymerase also bound to immobilized glutathione S-transferase-3AB; this procedure was used to purify the polymerase to near homogeneity. These results suggest a mechanism for bringing together 3AB, 3Dpol (or its precursor 3CD), and viral RNA in host cell membranous vesicles in which all viral RNA synthesis occurs.
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PMID:Poliovirus protein 3AB forms a complex with and stimulates the activity of the viral RNA polymerase, 3Dpol. 747 38

We have generated a series of fusion proteins carrying portions of subunit IIc, the second largest subunit of Drosophila RNA polymerase I, and have used them in a domain interference assay to identify a fragment of the IIc subunit that carries the binding site for a basal transcription factor. Fusion proteins carrying a subunit IIc fragment spanning residues Ala519-Gly992 strongly inhibit promoter-driven transcription in both unfractionated nuclear extracts and in reconstituted systems. The same fusion proteins similarly inhibit dTFIIF stimulation of Pol II elongation on dC-tailed templates, suggesting that the IIc(A519-G992) fragment, which carries conserved regions D-H, interferes with transcription by binding to dTFIIF. Finally, dTFIIF can be specifically cross-linked to a GST-IIc(A519-G992) fusion protein or to subunit IIc in intact Pol II.
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PMID:Identifying a transcription factor interaction site on RNA polymerase II. 748 60

cDNA transcribed from bluetongue virus serotype 1 (Australia) dsRNA 5 coding for non-structural protein NS1 was amplified in a polymerase chain reaction and ligated downstream of the T7 RNA polymerase promoter in the bacterial expression plasmid pET-5b, as a fusion protein with glutathione S-transferase using the pGEX bacterial expression system or the metallothionein promoter in the yeast expression plasmid pYELC5. The linear epitopes bound by six monoclonal antibodies to NS1 were localised to two antigenic regions at the amino terminus by Western blots using a series of carboxy-terminal truncations of the NS1 protein overexpressed in Escherichia coli. Expression of truncated NS1 genes using the pGEX expression system in E. coli enabled a more detailed map of the two epitopes to be constructed. The first epitope is thought to lie between amino acid residues 40-59, while the second is defined by the peptide sequences flanking amino acid 96.
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PMID:Expression of the non-structural protein NS1 of bluetongue virus in bacteria and yeast: identification of two antigenic sites at the amino terminus. 751 25

We used direct RNA sequencing to determine the genomic organization of the region downstream from the G gene of viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus. This region contains a gene coding for a protein, identified as nonvirion protein (NV), and the gene coding for the RNA polymerase (L). Thus, VHSV genome organization was confirmed to be 3'-N-P-M-G-NV-L-5'. In both a virulent European (07-71) and an avirulent North American (Makah) strain, the NV gene is transcribed into a small mRNA that codes for a protein of 122 amino acids. It has no significant sequence similarity with the infectious hematopoietic necrosis virus NV protein nor with any other known protein. We expressed the NV protein as a fusion protein with the glutathione S-transferase of Schistosoma japonicum and used the purified fusion protein to immunize rabbits. The rabbit antiserum precipitated from infected cell extracts--and not from noninfected cells or purified virions--a protein of 14 kDa, well in accordance with the expected NV gene product size. The prediction that the NV protein is a nonstructural protein is supported by its absence from mature virions although it is present in infected cells.
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PMID:Distant strains of the fish rhabdovirus VHSV maintain a sixth functional cistron which codes for a nonstructural protein of unknown function. 757 46

An auxin-regulated gene, parA, comprises a gene family consisting of a handful genes which respond to various signals. Although Droog et al. (Plant Mol. Biol, 1993, 21, 965-972) postulated that the parA-related genes belong to the family of a cytoplasmic enzyme, glutathione S-transferase (GST), we detected a low level of GST activity in the parA products, whose value was below 1/30 of that of parB products encoding tobacco (Nicotiana tabacum L.) GST. Immunofluorescence studies using an antibody against parA protein revealed that the subcellular location of parA protein is the nucleus in cultured tobacco mesophyll protoplasts, while conventional GSTs' including the parB product were primarily located in the cytoplasm. Confocal laser scanning microscopy of tobacco BY-2 cells showed that the parA product was confined to the nucleus, but was excluded from the nucleolus. In addition, exon/intron organization of the parA family was appreciably different from that of conventional GSTs including parB. Furthermore, the parA protein is much more similar to a 24-kDa protein of Escherichia coli that is reported to bind to RNA polymerase. These different characteristics of parA compared with to the conventional GSTs, indicate that parA protein would have distinct functions, such as involvement in transcription, rather than functioning as a conventional GST. Transgenic tobacco plants that carried the parA promoter fused to a beta-glucuronidase gene were used to show that the parA gene is tissue-specific and also under developmental control.
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PMID:Expression of the auxin-regulated parA gene in transgenic tobacco and nuclear localization of its gene products. 776 32

Salmonella enterotoxin (Stn) is a virulence factor in S. typhimurium strain Q1 that causes both fluid secretion in ligated intestinal loops of rabbits and elongation of Chinese hamster ovary (CHO) cells. High-level expression systems are needed to provide Stn in soluble form for detailed study of the biological activity of Stn. To maximize the synthesis and solubility of Stn, we systematically compared the production of native Stn synthesized with a T7 RNA polymerase/promoter system to that of two fusion proteins: glutathione S-transferase::Stn (Gst::Stn) and thioredoxin A::Stn (TrxA::Stn). The latter fusion protein expression systems resulted in a 64-fold increase in Gst::Stn and TrxA::Stn antigen concentration, as measured by specific anti-peptide antibodies in an enzyme-linked immunosorbent assay (ELISA). Most of the toxin derived using these vector systems was insoluble; however, the solubility of the TrxA::Stn antigen increased by at least 50-fold, with a concomitant increase in CHO cell elongation activity. In addition, stn gene expression was enhanced more than 50-fold by addition of 0.2-0.4 M NaCl to Luria-Bertani medium. The biological activity of Stn also was increased in the high-osmolarity medium. Consequently, the expression of stn may be regulated by DNA supercoiling.
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PMID:Improved synthesis of Salmonella typhimurium enterotoxin using gene fusion expression systems. 802 62

The lipase gene from Pseudomonas aeruginosa TE3285 is followed by another gene, lipB. The lipase gene was expressed in Escherichia coli BL21(DE3)pLysS using the T7 RNA polymerase expression system. The mature lipase was accumulated as inclusion bodies at 42% of the total cell proteins. The inclusion bodies were solubilized with 8 M urea, but lipase activity was not detected in the solubilized preparation containing 85% lipase protein even after removing urea by dialysis. The lipB gene, positioned downstream of the lipase gene and thought to be necessary for the expression of the lipase gene, was expressed in Escherichia coli JM109 as a fusion with the glutathione transferase gene from Schistosoma japonicum. The fusion protein was partially purified on glutathione-agarose beads to 36% purity. Incubated with the fusion protein at a molar ratio of 1:1 at 4 degrees C for 24 h, the solubilized lipase showed lipase activity of about a tenth that of the purified lipase prepared from Pseudomonas aeruginosa TE3285. Magnesium ions and ATP were not essential but increased the activation. When the fusion protein was treated with thrombin to release the glutathione transferase part, it retained its activity. The lipase activation with lipB protein probably proceeds to form a 1:1 complex with the inactive, solubilized lipase protein but by a different mode from known chaperones.
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PMID:Lipase from Pseudomonas aeruginosa. Production in Escherichia coli and activation in vitro with a protein from the downstream gene. 834 92


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