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 gene, rpo 132, encoding the second-largest subunit of the vaccinia virus DNA-dependent RNA polymerase was identified and sequenced. Two complementary approaches, involving antiserum to purified vaccinia virus RNA polymerase, were used to locate the rpo 132 gene. One method involved the screening of a lambda gt11 library of vaccinia virus genome fragments and the other was based on the immunoprecipitation and polyacrylamide gel electrophoresis of the in vitro translation products of mRNA that hybridized to immobilized vaccinia virus DNA. The deduced open reading frame of the rpo 132 gene predicted a polypeptide of 1164 amino acid residues with sequence similarities to the second-largest RNA polymerase subunits of eubacteria, archaebacteria, and eukaryotes as well as to other poxviruses. Transcriptional analyses indicated that rpo 132 has both early and late RNA start sites and is expressed throughout infection.
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PMID:Identification, sequence, and expression of the gene encoding the second-largest subunit of the vaccinia virus DNA-dependent RNA polymerase. 182 7

An alternative approach to structure-function analysis of vesicular stomatitis virus (VSV) gene products and their interactions with one another during each phase of the viral life cycle is described. We showed previously by using the vaccinia virus-T7 RNA polymerase expression system that when cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with defective interfering (DI) particles, rapid and efficient replication and amplification of (DI) particle RNA occurred. Here, we demonstrate that all five VSV proteins can be expressed simultaneously when cells are contransfected with plasmids containing the matrix protein (M) gene and the glycoprotein (G) gene of VSV in addition to plasmids containing the genes for the N, NS, and L proteins. When cells coexpressing all five VSV proteins were superinfected with DI particles, which because of their defectiveness are unable to express any viral proteins or to replicate, DI particle replication, assembly, and budding were observed and infectious DI particles were released into the culture fluids. Omission of either the M or G protein expression resulted in no DI particle budding. The vector-supported DI particles were similar in size and morphology to the authentic DI particles generated from cells coinfected with DI particles and helper VSV and their infectivity could be blocked by anti-VSV or anti-G antiserum. The successful replication, assembly, and budding of DI particles from cells expressing all five VSV proteins from cloned cDNAs provide a powerful approach for detailed structure-function analysis of the VSV gene products in each step of the replicative cycle of the virus.
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PMID:Cells that express all five proteins of vesicular stomatitis virus from cloned cDNAs support replication, assembly, and budding of defective interfering particles. 184 19

The gene rpo35, encoding a subunit of the vaccinia virus DNA-dependent RNA polymerase, was identified, and its RNA and protein products were characterized. An Mr 35,000 polypeptide, which bound antibody to the purified RNA polymerase, was synthesized in reticulocyte lysates programmed with viral mRNA that hybridized to a 2,300-base pair segment of the viral genome. Determination of the sequence of the DNA segment revealed four potential protein coding regions, none of which had evident similarity to any described RNA polymerase subunit of prokaryotes or eukaryotes. One open reading frame that could encode a 35,400-Da protein was identified as rpo35 on the basis of mRNA hybridization, cell-free translation, and immunoprecipitation. The identification was confirmed by sequencing tryptic peptides of the authentic Mr 35,000 RNA polymerase subunit. Antiserum to the purified recombinant protein, expressed in bacteria, reacted specifically with a Mr 35,000 polypeptide that was detected starting 2 h after virus infection and that co-sedimented with RNA polymerase purified from virions. RNA analyses indicated that the 5'-end of an early transcript started 25 nucleotides upstream of rpo35, which is consistent with the location of an early promoter consensus sequence.
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PMID:Identification, sequence, and expression of the gene encoding a Mr 35,000 subunit of the vaccinia virus DNA-dependent RNA polymerase. 185 5

One of the most efficient systems for the expression of genes in the cytoplasm of animal cells utilizes a recombinant vaccinia virus encoding the bacteriophage T7 RNA polymerase. Cells infected with this virus are transfected with plasmid DNAs containing the gene to be expressed under T7 promoter control. The major limitation of this system is the efficiency with which DNA is introduced into the cell. Recently, a cationic liposome-mediated transfection reagent has yielded transfection frequencies of greater than 80%. To determine if commercially available cationic lipids could form liposomes that would yield similar transfection efficiencies, we tested liposomes prepared with five different cationic lipids. When used at appropriate concentrations in liposomes that also contained a neutral lipid, four of the five cationic lipids were effective in the transfection of HeLa cells. However, liposomes formed with the neutral lipid and one of the cationic lipids, dimethyldioctadecylammonium bromide (DDAB), gave transfection frequencies of greater than 95% and had a broad spectrum of effectiveness on a variety of cell lines. Liposomes containing DDAB are an inexpensive, highly efficient and reproducible alternative for the transfection of animal cells and are well suited for use with the vaccinia virus/T7 expression system.
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PMID:A new cationic liposome reagent mediating nearly quantitative transfection of animal cells. 186 62

Vaccinia virus RNA polymerase requires the heterodimeric protein, vaccinia early transcription factor (VETF), for transcription of early gene templates in vitro. We have analyzed the vaccinia growth factor promoter sequences interacting with VETF at the nucleotide level and provide evidence that the factor contacts the DNA at two separate sites. DNase I protection analysis showed that VETF was found to nucleotides -12 to -29 relative to the transcription initiation site, and also to nucleotides +8 to +10 downstream of the initiation site. The importance of both binding sites for stable complex formation was supported by methylation interference analysis. Using synthetic oligonucleotides encoding different parts of the vaccinia growth factor promoter, it was shown that nucleotides down-stream of the transcription initiation site are required for stable complex formation. Competition binding experiments demonstrated that only the upstream binding site contributes significantly to binding specificity. Binding to two separated DNA sequences results in a bend in the promoter DNA as demonstrated by electrophoretic mobility shift analysis of permuted DNA fragments. These findings suggest that VETF activates transcription by sequence specific binding and structural alteration of the promoter DNA helix.
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PMID:Promoter DNA contacts made by the vaccinia virus early transcription factor. 186 71

Vaccinia virus RNA polymerase requires the vaccinia early transcription factor, VETF, for the in vitro initiation of transcription at early gene promoters in a reaction requiring ATP hydrolysis. VETF binds specifically to early gene promoters and has an associated DNA-dependent ATPase activity. The effect of ATP on the interaction of VETF with the promoter for the vaccinia growth factor gene promoter has been examined. ATP had no marked effect on the steady-state level of promoter binding but dramatically affected the kinetics of dissociation of VETF from the promoter. The half-life of the VETF-promoter complex was greatly reduced in the presence of ATP. The destabilization of the complex was specific for ATP and dATP, consistent with the substrate specificity of the VETF-associated ATPase. ADP or the non-hydrolyzable ATP analog adenylyl-imidodiphosphate did not destabilize the complex suggesting that ATP hydrolysis is obligatory for dissociation. These findings provide a link between the promoter binding and ATPase activities associated with VETF and suggest that the ATP-dependent dissociation of the VETF-promoter complex is an important event in the transcription of vaccinia virus early genes.
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PMID:A role for ATP hydrolysis in vaccinia virus early gene transcription. Dissociation of the early transcription factor-promoter complex. 186 72

Transcription of vaccinia early genes by the viral RNA polymerase terminates downstream of a signal sequence TTTTTNT in the nontemplate DNA strand. Signal recognition occurs at the level of the sequence UUUUUNU in nascent RNA and depends on a virus-encoded termination factor (VTF). The presence of TTTTTNT elements within protein encoding regions of some early genes requires that these 5' proximal signals be ignored in order to achieve early expression of the full-sized proteins. In the case of the A18R gene, which contains a proximal terminator that is not utilized in vivo (Pacha et al., J. Virol. 64, 3853-3863 (1990)), the TTTTTNT sequence can be folded into a potential hairpin structure such that UUUUUNU would be part of a duplex stem in the nascent RNA. We find that the A18R putative hairpin is unable to promote factor-dependent termination in a purified in vitro transcription system. Sequence manipulations that abrogate the potential to form an RNA hairpin restore the activity of the TTTTTNT motif. The in vitro studies suggest that antitermination at the proximal site of the A18R gene may be mediated by secondary structure in the nascent RNA, and that early termination involves recognition by VTF and/or RNA polymerase of the UUUUUNU sequence in single-stranded form.
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PMID:Antitermination of vaccinia virus early transcription: possible role of RNA secondary structure. 192 85

Adenovirus (Ad) DNA polymerase (AdPol) and the preterminal protein (pTP) form a complex that is involved in the in vitro initiation of Ad DNA replication. Recombinant vaccinia viruses (vv) were constructed in which the genes encoding AdPol and pTP were cloned into a vaccinia/T7 hybrid expression-based vector downstream from the T7 promoter (pT7)/encephalomyocarditis virus (EMCV) 5'-untranslated region (UTR). HeLa cells infected with the recombinant vv-AdPol or vv-pTP or a mixture of both, together with the vv expressing T7 RNA polymerase produced significant levels of pTP and AdPol which were biologically active in the in vitro initiation of Ad DNA replication. These amounts of pTP and AdPol were only about two-fold less than the levels produced in insect cells infected with the recombinant baculovirus constructs expressing AdPol and pTP.
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PMID:Overproduction of adenovirus DNA polymerase and preterminal protein in HeLa cells. 193 14

The mutation in a vaccinia virus mutant resistant to inhibition by isatin-beta-thiosemicarbazone was mapped by marker rescue. DNA from the resistant mutant was cloned into cosmid and plasmid vectors and tested for its ability to convert wild-type vaccinia virus to IBT resistant virus in a helper-mediated marker rescue protocol. Resistance was mapped in this way to a 0.9-kb DNA fragment derived from the HindIII A fragment of vaccinia genome. Southern blot hybridization using this DNA as a probe demonstrated that the 0.9-kb fragment is contained within the DNA sequence encoding the second largest subunit of vaccinia RNA polymerase, rpo132. Thus, mutation of rpo132 can cause resistance to IBT in vaccinia virus.
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PMID:A vaccinia virus isatin-beta-thiosemicarbazone resistance mutation maps in the viral gene encoding the 132-kDa subunit of RNA polymerase. 196 56

The cellular isoform of the prion protein (PrPC) is a sialoglycoprotein bound almost exclusively on the external surface of the plasma membrane by a glycosyl phosphatidylinositol anchor. The deduced amino acid sequence of Syrian hamster PrPC identifies two potential sites for the addition of Asn-linked carbohydrates at amino acids 181-183 (Asn-Ile-Thr) and 197-199 (Asn-Phe-Thr). We have altered these sites by replacing the threonine residues with alanine and expressed the mutant proteins transiently in CV1 cells utilizing a mutagenesis vector with the T7 promoter located upstream from the PrP gene. The T7 RNA polymerase was supplied by infection with a recombinant vaccinia virus. The 3 mutant proteins (PrPAla183, PrPAla199 and PrPAla183/199) have a reduced relative molecular weight compared to wild-type (wt) PrP. Deglycosylation as well as synthesis in the presence of tunicamycin reduced the relative molecular weight of all the PrP species to that of the double mutant PrPAla183/199. Our results indicate that both single-site mutant prion proteins are glycosylated at non-mutated sites and they suggest that both potential sites for Asn-linked glycosylation are utilized in wt PrPC. Immunofluorescence studies demonstrate that while wt PrPC localizes to the cell surface, all the mutant PrP molecules accumulate intracellularly. The site of accumulation of PrPAla183 is probably prior to the mid-Golgi stack since this protein does not acquire resistance to endoglycosidase H. Whether the intracellular locations of the mutant PrPC species are the same as those identified for the scrapie isoform of the prion protein (PrPSc) remains to be established.
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PMID:Intracellular accumulation of the cellular prion protein after mutagenesis of its Asn-linked glycosylation sites. 198 82


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