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)

Using vaccinia virus as a selection and cloning vehicle, a thymidine kinase (TK) gene of fowlpox virus (FPV) has been identified. A plasmid, pF130, containing part of the HindIII-F region of vaccinia virus was used to shotgun clone EcoRI fragments of FPV DNA into TK- vaccinia virus and select for TK+ recombinants. The TK+ recombinant vaccinia virus contained a 5.5 kb EcoRI fragment of FPV. This FPV fragment was cloned into pUC9 and the presence of the TK gene in this fragment was confirmed by its ability to rescue TK+ vaccinia virus from TK- virus, when inserted into pF130. A recombinant vaccinia virus containing this FPV fragment induced TK enzyme activity in the cytoplasm of infected cells. The vaccinia virus RNA polymerase appeared able to recognize the FPV promoter sequences of the FPV TK gene since the fragment operated in the marker rescue, irrespective of its orientation to the vaccinia virus promoter in pF130. Using restriction enzyme analysis, insertion of subfragments of the 5.5 kb FPV fragment into pF130 and marker rescue, we were able to map the position of the TK gene in the 5.5 kb EcoRI fragment. This approach may facilitate identification and cloning of TK genes from other poxviruses.
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PMID:Identification and cloning of the fowlpox virus thymidine kinase gene using vaccinia virus. 301 54

A series of mutations, including 5' and 3' deletions, as well as insertions were introduced into the 5' flanking nucleotide sequence of a vaccinia virus late gene. This DNA has been shown previously to contain all the necessary elements for correct regulation of the gene most probably transcribed by the viral RNA polymerase. To facilitate the assays, the mutated DNA was fused to the chloramphenicol acetyltransferase gene and inserted into the genome of live vaccinia virus. The effects of the mutations on expression of the chimeric gene were studied by both enzyme assays and nuclease S1 analysis. The results showed that 5' deletions up to about 15 bp from the putative initiation site of transcription still yielded high levels of gene expression. All mutations, however, that deleted the authentic late mRNA start site, abolished promoter activity.
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PMID:Functional analysis of the 5' flanking sequence of a vaccinia virus late gene. 301 74

The sequence of the 8,600-base-pair HindIII H fragment, located at the center of the vaccinia virus genome, was determined to analyze several late genes. Seven major complete open reading frames (ORFs) and two that started from or continued into adjacent DNA segments were identified. ORFs were closely spaced and present on both DNA strands. Some adjacent ORFs had oppositely oriented overlapping termination codons or contiguous stop and start codons. Nucleotide compositional analysis indicated that the A-T frequency was consistently lowest in the first codon position. The sizes of the polypeptides predicted from the DNA sequence were compared with those determined by polyacrylamide gel electrophoresis of cell-free translation products of mRNAs selected by hybridization to cloned single-stranded DNA segments or synthesized in vitro by bacteriophage T7 RNA polymerase. Six transcripts that initiated within the HindIII H DNA fragment were detected, and of these, four were synthesized only at late times, one was synthesized only early, and one was synthesized early and late. The sites on the genome corresponding to the 5' ends of the transcripts were located by high-resolution nuclease S1 analysis. For late genes, the transcriptional and translational initiation sites mapped within a few nucleotides of each other, and in each case the sequence TAAATGG occurred at the start of the ORF. The extremely short leader and the absence of A or G in the -3 position, relative to the first nucleotide of the initiation codon, distinguishes the majority of vaccinia virus late genes from eucaryotic and vaccinia virus early genes.
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PMID:Conserved TAAATG sequence at the transcriptional and translational initiation sites of vaccinia virus late genes deduced by structural and functional analysis of the HindIII H genome fragment. 302 79

A high-molecular-weight protein complex that is capable of accurate transcription initiation and termination of vaccinia virus early genes without additional factors was demonstrated. The complex was solubilized by disruption of purified virions, freed of DNA by passage through a DEAE-cellulose column, and isolated by glycerol gradient sedimentation. All detectable RNA polymerase activity was associated with the transcription complex, whereas the majority of enzymes released from virus cores including mRNA (nucleoside-2'-O)methyltransferase, poly(A) polymerase, topoisomerase, nucleoside triphosphate phosphohydrolase II, protein kinase, and single-strand DNase sedimented more slowly. Activities corresponding to two enzymes, mRNA guanylyltransferase (capping enzyme) and nucleoside triphosphate phosphohydrolase I (DNA-dependent ATPase), partially sedimented with the complex. Silver-stained polyacrylamide gels, immunoblots, and autoradiographs confirmed the presence of subunits of vaccinia virus RNA polymerase, mRNA guanylyltransferase, and nucleoside triphosphate phosphohydrolase I, as well as additional unidentified polypeptides, in fractions with transcriptase activity. A possible role for the DNA-dependent ATPase was suggested by studies with ATP analogs with gamma-S or nonhydrolyzable beta-gamma-phosphodiester bonds. These analogs were used by vaccinia virus RNA polymerase to nonspecifically transcribe single-stranded DNA templates but did not support accurate transcription of early genes by the complex. Transcription also was sensitive to high concentrations of novobiocin; however, this effect could be attributed to inhibition of RNA polymerase or ATPase activities rather than topoisomerase.
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PMID:Sedimentation of an RNA polymerase complex from vaccinia virus that specifically initiates and terminates transcription. 303 83

Antiserum to a multisubunit DNA-dependent RNA polymerase from vaccinia virions was prepared to carry out genetic studies. This antiserum selectively inhibited the activity of the viral polymerase but had no effect on calf thymus RNA polymerase II. The specificity of the antiserum was further demonstrated by immunoprecipitation of RNA polymerase subunits from dissociated virus particles. The presence in vaccinia virus-infected cells of mRNA that encodes the polymerase subunits was determined by in vitro translation. Immunoprecipitable polypeptides with Mrs of about 135,000, 128,000, 36,000, 34,000, 31,000, 23,000, 21,000, 20,000, and 17,000 were made when early mRNA was added to reticulocyte extracts. The subunits were encoded within the vaccinia virus genome, as demonstrated by translation of early mRNA that hybridized to vaccinia virus DNA. The locations of the subunit genes were determined initially by hybridization of RNA to a series of overlapping 40-kilobase-pair DNA fragments that were cloned in a cosmid vector. Further mapping was achieved with cloned HindIII restriction fragments. Results of these studies indicated that RNA polymerase subunit genes are transcribed early in infection and are distributed within the highly conserved central portion of the poxvirus genome in HindIII fragments E, J, H, D, and A.
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PMID:DNA-dependent RNA polymerase subunits encoded within the vaccinia virus genome. 303 8

The M protein encoded by group A streptococci is a cell-wall polypeptide that has the property of enabling these organisms to evade the phagocytic cells of the human host. Therefore, the M protein plays a major role in the pathogenesis of streptococcal diseases. As an initial step toward the use of this protein as a target antigen for the production of protective anti-streptococcal immunity, a live vaccinia virus recombinant containing the M-protein gene has been constructed (VV:M6 delta). The bacterial M-protein DNA sequence is stable within this genetic context and is actively transcribed by viral RNA polymerase. Furthermore, high levels of immunoreactive M protein were detected in vivo when the VV:M6 delta recombinant was used to infect mammalian cells in culture. Thus, in addition to providing a powerful approach for dissecting the immunodominant domains of the M protein, the VV:M6 delta recombinant appears to be an excellent candidate vaccine for animal trials.
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PMID:Expression of streptococcal M protein in mammalian cells. 304 16

DNA coding for bacteriophage T7 RNA polymerase was ligated to a vaccinia virus transcriptional promoter and integrated within the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and stably expressed T7 RNA polymerase in mammalian cells. Target genes were constructed by inserting DNA segments that code for beta-galactosidase or chloramphenicol acetyltransferase into a plasmid with bacteriophage T7 promoter and terminator regions. When cells were infected with the recombinant vaccinia virus and transfected with plasmids containing the target genes, the latter were expressed at high levels. Chloramphenicol acetyltransferase activity was 400-600 times greater than that observed with conventional mammalian transient-expression systems regulated either by the enhancer and promoter regions of the Rous sarcoma virus long terminal repeat or by the simian virus 40 early region. The vaccinia/T7 hybrid virus forms the basis of a simple, rapid, widely applicable, and efficient mammalian expression system.
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PMID:Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. 309 28

A novel expression system based on coinfection of cells with two recombinant vaccinia viruses has been developed. One recombinant vaccinia virus contained the bacteriophage T7 RNA polymerase gene under control of a vaccinia virus promoter. The second recombinant vaccinia virus contained a target gene of choice flanked by bacteriophage T7 promoter and termination sequences. Maximum expression of the target gene occurred when cells were infected with 10 PFU of each recombinant virus. Although T7 RNA polymerase synthesis began shortly after infection, the target gene was not expressed until late times and was largely inhibited when DNA replication was blocked. Target gene transcripts were analyzed by agarose gel electrophoresis and had the predicted size. With this system, Escherichia coli beta-galactosidase, hepatitis B virus surface antigen, and human immunodeficiency virus envelope proteins were made. In each case, the level of synthesis was greater than had previously been obtained with the more conventional recombinant vaccinia virus expression system.
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PMID:Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes. 311 59

Vaccinia virus is an efficient expression vector with broad host range infectivity and large DNA capacity. This vector has been particularly useful for identifying target antigens for humoral and cell-mediated immunity. With increased levels of gene expression, obtained either with stronger vaccinia promoters or through incorporation of the bacteriophage T7 RNA polymerase gene into the vaccinia genome, proteins may be synthesized in mammalian cells for use as subunit vaccines. For use as a live recombinant vaccine, efforts are being made to attenuate vaccinia virus further, either by inactivating genes contributing to virulence or by introducing human lymphokine genes into the vaccinia genome.
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PMID:Roles of vaccinia virus in the development of new vaccines. 329 53

Despite the fact that cells infected with wild type vaccinia virus synthesize viral DNA and assemble progeny virus particles within the cytoplasm, the host cell nucleus is required for a productive infection. Recent evidence suggests that vaccinia virus selectively recruits components from the host cell nucleus into the cytoplasm for use by the developing virus. One of these components is the largest subunit of the cellular RNA polymerase II (Pol II).
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PMID:The role of the host cell nucleus in vaccinia virus morphogenesis. 331 10


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