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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The genome of infectious bursal disease virus (IBDV) consists of two segments of double-stranded (ds)RNA with molecular weights of 2.2 X 10(6) and 1.9 X 10(6) Da, respectively. After treatment of IBDV particles with proteinase K in the presence of sodium dodecyl sulfate (SDS), linear dsRNA molecules are released from the virus particles. However, after heating of virus particles at 100 degrees for 3 min in 1.5% SDS, without the protease, dsRNA-protein complexes can be seen under the electron microscope: Knob-like proteinaceous structures are linked to the ends of the dsRNA molecules of either size class which are circularized to form individual rings. A 90,000-Da IBDV structural polypeptide, the only protein encoded by the smaller genome segment, has been demonstrated to remain firmly linked to the IBDV genome under these conditions. No functional data exist about this circularizing protein; it is a probable candidate for an RNA-dependent RNA polymerase or an assembly protein for the two dsRNA segments. At high particle concentrations, or when the preparations are allowed to stand for several hours before spreading, these complexes tend to aggregate to form flower-like structures.
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PMID:The two segments of the infectious bursal disease virus genome are circularized by a 90,000-Da protein. 303 77

The cytoplasmic polyhedrosis virus of the silkworm, Bombyx mori, contains a ten-segmented, double-stranded RNA genome and five species of polypeptides, V1 (Mr 146,000), V2 (Mr 140,000), V3 (Mr 128,000)), V4 (Mr 62,000), and V5 (Mr 32,000). The virus contains an RNA-dependent RNA polymerase that transcribes the duplex genome RNA to form mRNA either in the infected animals or under appropriate conditions in vitro. We co-microinjected the virus, [alpha-32P]GTP and actinomycin D into oocytes of the frog, Xenopus laevis, and found that at least eight species of mRNA were formed in the oocytes. Virus-injected oocytes were labeled with [35S]methionine and cell extracts were treated with rabbit anti-cytoplasmic polyhedrosis virus immunoglobulin G. Analysis of the immunoprecipitates by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that V1, V2, and V3 were produced in the virus-injected oocytes whereas V4 and V5 were not. By injecting the separated double-stranded genome segments immediately after heat-denaturation into oocytes, it was found that V1 was coded for by segment 1 with a chain length of 4.2 kilobase pairs, V2 by segment 2 or 3, whose chain lengths are both 4.0 kilobase pairs, and V3 by segment 4 with a chain length of 3.2 kilobase pairs. These results demonstrate that the Xenopus oocyte is a very useful system for the coupled transcription-translation of double-stranded RNA viruses.
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PMID:Coupled transcription-translation of silkworm cytoplasmic polyhedrosis virus injected into oocytes of the frog, Xenopus laevis. 328 14

Exogenous polyribonucleotides stimulated the ribonucleic acid (RNA) transcriptase in Sendai virions. Added yeast RNA, polyadenylic acid, or polycytidylic acid increased incorporation of (3)H-guanosine monophosphate as much as fivefold. The products of stimulated reactions were virus-specific as determined by hybridization with Sendai virion RNA, but they sedimented more slowly (13s) than the product of an unstimulated reaction (16s). The stimulating activity was nondialyzable and heat stable, but was abolished by alkaline hydrolysis. Nucleoside monophosphates, individually or in combination, were ineffective, confirming the requirement for a polymer. Among other substances tested for effects on Sendai virion transcriptase, polyaspartic acid and polyglutamic acid stimulated the enzyme; polyinosinic acid, polyuridylic acid, and polyamines had no effect; and dextran sulfate and polyvinyl sulfate were inhibitory.
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PMID:Stimulation of Sendai virion transcriptase by polyanions. 434 27

The endogenous transcriptase present in purified vesicular stomatitis (VS) virions was solubilized with a Triton X-100 high-salt solution. The polymerase activity was purified on glycerol gradients and by phosphocellulose column chromatography; the viral proteins present in the active enzyme fractions were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was demonstrated that L protein, but not NS protein, was required for in vitro RNA synthesis on the VS viral nucleocapsid template. Solubilized L protein rebinds to the ribonucleoprotein template when the transcription complex is reconstituted, and the RNA synthesized in vitro by purified L protein hybridizes to virion RNA. Cyanogen bromide peptide fingerprints indicate that the large L protein is a unique polypeptide chain. It is concluded that the L protein functions as the transcriptase, and the nucleocapsid NS protein is not essential for in vitro RNA synthesis.
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PMID:L protein requirement for in vitro RNA synthesis by vesicular stomatitis virus. 435 10

Influenza B/LEE/40, B/Rome/1/67, B/Hong Kong/8/73, and B/Victoria/98926/70 viruses have a similar polypeptide composition as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These viruses are composed of six or seven polypeptides, depending on whether one or two high-molecular-weight polypeptides are resolved, ranging in molecular weights from 27,000 to 90,400. Three of these polypeptides, namely the heavy and light hemagglutinin chains and the neuraminidase, have attached carbohydrate. Highly purified influenza B/LEE/40 and B/Rome/1/67 virus preparations have RNA-dependent RNA polymerase activity equivalent to the incorporation of 100 and 30 pmol, respectively, of (3)H-UMP per mg of virus protein per h at 37 C, which is demonstrated only in detergent-treated virus suspensions. However, no RNA-dependent DNA polymerase enzyme activity was detected in the two viruses although virus suspensions were "activated" by heat, alpha-chymotrypsin, and detergents. Other enzymatic activities were associated with purified preparations of influenza B virus and were attributed to minor contamination of virus with host cell enzymes. Thus, nucleoside and deoxynucleoside phosphohydrolase enzymes were active in the absence of detergents and catalyzed the release of 1,200 and 1,800 nmol of P(i) per mg of virus protein in 30 min at 37 C from ATP and dATP substrates. Thin-layer chromatography indicated that the products of the phosphohydrolase enzymes of influenza B/LEE/40 were mainly nucleoside diphosphate and monophosphate. The latter enzymes were tightly bound to influenza B/LEE/40 virus and could not be removed completely by repeated centrifugation, including centrifugation of the virus to equilibrium in density gradients of 25 to 40% (wt/vol) cesium chloride. A low degree of RNase (approximately 0.01 mug% contamination) and phosphatase (10-30 nmol of P(i) released per mg of virus protein per 30 min) activity was detected in some, but not all, influenza B/LEE/40 virus preparations.
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PMID:Polypeptide composition of Influenza B viruses and enzymes associated with the purified virus particles. 435 55

The formation of reovirus double-stranded (ds) RNA and of oligo adenylic acid (oligo A) is inhibited by 5 mug of actinomycin D per ml added at the time of viral infection. Viral proteins are synthesized and assembled into dsRNA-deficient particles under these conditions. The addition of cycloheximide to infected cells during the mid-logarithmic phase of viral replication terminates protein and dsRNA synthesis, but allows continued oligo A synthesis for about 1 h. The (3)H-labeled oligo A formed in the presence of cycloheximide is incorporated into particles whose density in CsCl is identical to that of reovirions. Using the large particulate or virus factory-containing cytoplasmic fraction of infected L-cells, we have established an in vitro system for the synthesis of oligo A. The in vitro product migrates slightly faster in sodium dodecyl sulfate acrylamide gels than marker oligo A. Oligo A synthesis in vitro continues for about 1 h, requires, the presence of only one ribonucleoside triphosphate (ATP), is not inhibited by DNase or RNase, but is abruptly terminated by the addition of chymotrypsin to the reaction mixture. Oligo A formed both in vivo and in vitro is released from the factory fraction by chymotrypsin digestion. The enzymes which catalyze the synthesis of oligo A, dsRNA, and single-stranded RNA all exhibit a similar temperature dependence with an optimum of approximately 45 C. These results indicate that oligo A is formed within the core of the nascent virion after the completion of dsRNA synthesis; they suggest that the oligo A polymerase is an alternative activity of the virion-bound transcriptase and that it is regulated by outer capsomere proteins.
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PMID:Shythesis of reovirus oligo adenylic acid in vivo and in vitro. 485 7

Rotavirus gene products were examined, with the simian rotavirus SA11 as a model. The endogenous viral RNA-dependent RNA polymerase associated with single-shelled virus particles or with activated double-shelled particles was used to synthesize viral RNA transcripts. Sedimentation velocity sucrose gradient analysis of the RNA transcripts revealed four peaks at 9S, 12S, 14S, and 18S, whereas agarose gel electrophoresis under partially denaturing conditions revealed eight groups of RNA species ranging in molecular weight from 2 x 10(5) to 1.2 x 10(6). The transcripts synthesized in vitro were active in an mRNA-dependent cell-free translation system derived from rabbit reticulocytes. The transcripts directed the synthesis of 11 polypeptides that had molecular weights ranging from 125,000 to 20,000 when analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The products of in vitro translation were compared with polypeptides from purified virus and those synthesized in infected cells. Several of the polypeptides synthesized in vitro were designated as structural polypeptides by comparing the molecular weights determined by polyacrylamide gel electrophoresis analysis or by precipitation with hyperimmune serum prepared against purified virus. Three of the viral structural polypeptides (VP4, -5, and -5a) were not synthesized in vitro as primary gene products, demonstrating that processing must occur for the production of some structural polypeptides. Other in vitro-synthesized polypeptides were tentatively identified as either precursors to the viral glycoproteins or nonstructural polypeptides.
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PMID:In vitro transcription and translation of simian rotavirus SA11 gene products. 624 62

A template-dependent polyuridylic acid [poly(U)] polymerase has been isolated from BHK cells infected with foot-and-mouth disease virus (FMDV). Enzyme activity in a 20,000 x g supernatant of a cytoplasmic extract was concentrated by precipitation with 30 to 50% saturated ammonium sulfate. The poly(U) polymerase was freed of membranes by sodium dodecyl sulfate and 1,1,2-trichlorotrifluoroethane extraction, and RNA was removed by precipitation with 2 M LiCl. The solubilized poly(U) polymerase required polyadenylic acid as template complexed to an oligouridylic acid primer and Mg2+ for activity, but was inhibited by Mn2+. Antisera from animals infected with FMDV had previously been shown to inhibit the activity of FMDV RNA replicase complexed to the endogenous RNA template. The same antisera also inhibited the activity of poly(U) polymerase. Antisera depleted of antibody by absorption with the virus infection-associated antigen of FMDV no longer inhibited replicase and polymerase activities. The evidence suggests that FMDV RNA replicase, poly(U) polymerase, and the virus infection-associated antigen share a common protein.
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PMID:Isolation of a foot-and-mouth disease polyuridylic acid polymerase and its inhibition by antibody. 625 Dec 48

A poliovirus-specific RNA-dependent RNA polymerase was isolated from a cytoplasmic extract of infected HeLa cells and was shown to copurify with a single virus-specific protein. The polymerase was isolated from cells labeled with [35S]-methionine and was fractionated from other soluble cytoplasmic proteins by ammonium sulfate precipitation, phosphocellulose chromatography, gel filtration on Sephacryl S-200, and chromatography on hydroxylapatite. The activity of the enzyme was measured by using either polyadenylic acid or poliovirion RNA as a template in the presence of an oligouridylic acid primer. A single virus-specific protein that had an apparent molecular weight of 63,000 (p63) was found to copurify with this activity. Host-coded proteins were present in reduced molar amounts relative to p63. Noncapsid viral protein 2 (NCVP2) and other viral proteins were clearly separated from p63 by gel filtration on Sephacryl S-200. Polymerase activity coeluted from the column precisely with p63. NCVP2 was totally inactive as an RNA polymerase and did not stimulate the polymerase activity of p63. The purified enzyme sedimented at about 4S on a glycerol gradient and thus appeared to be a monomer of p63. Two-dimensional gel electrophoresis of the polymerase protein indicated that it had an isoelectric point of about 7.5. Thus, the viral polypeptide, p63, as defined by the above physical parameters, is an RNA-dependent RNA polymerase that can copy poliovirion RNA when oligouridylic acid is used as a primer.
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PMID:Identification of poliovirus polypeptide P63 as a soluble RNA-dependent RNA polymerase. 625 35

Mounting experimental evidence suggests that the TAT protein, released from human immunodeficiency virus-1 (HIV-1)-infected inflammatory cells, may genetically reprogram targeted cells within a localized environment to develop highly vascularized tumors of mesenchymal origin. The fibroblast growth factor (FGF) family of polypeptides has gained general acceptance as initiators of angiogenesis and functions as potent mitogens for mesoderm-derived cells. To evaluate a potential biological relationship between TAT and acidic FGF (FGF-1), primary murine embryonic fibroblasts either were transfected with the viral transactivator or were transduced (retrovirally mediated) with a secreted, chimeric form of the human polypeptide growth factor, human stomach tumor/Kaposi's sarcoma (hst/KS)FGF-1. Reverse transcriptase-polymerase chain reaction, Western blotting, in situ immunohistochemical, heparin affinity, DNA synthesis, and transient transfection techniques were used to confirm expression, localization, and functionality of the transgenes. Both transfected and transduced cells constitutively expressing either TAT or (hst/KS)FGF-1 adopted a transformed phenotype, maintained aggressive growth behavior, and demonstrated both induction of FGF-specific phosphotyrosyl proteins and nuclear association of FGF-1 and FGF-1 receptor. Increased levels of endogenous, murine FGF-1 mRNA (reverse transcriptase-polymerase chain reaction) and protein (immunoblot analysis) were apparent in both (hst/KS)FGF-1- and TAT-transformed cells. Medium conditioned by (hst/KS)FGF-1-transduced cells contained steady-state levels of biologically active FGF-1 which exhibited a representative molecular weight. Limited sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the conditioned medium from TAT-transformed cells demonstrated the appearance of FGF-1 as latent, high molecular weight complexes requiring reducing agents to activate full biological activity. Collectively, these results suggest that TAT induces the expression and secretion of FGF-1, which may be potentially relevant to the pathophysiological development of AIDS-Kaposi's sarcoma.
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PMID:The HIV-1 TAT protein induces the expression and extracellular appearance of acidic fibroblast growth factor. 754 39


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