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)

Translation of foreign mRNAs is enhanced by a cis-acting derivative (omega') of the 5'-leader sequence (omega) of tobacco mosaic virus RNA (vulgare strain). To explain this effect we have conducted several experiments in vitro. 1. The presence of various 5'-terminal sequences, including omega', did not significantly increase the half-lives of chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII) mRNAs in wheat-germ extract. Also, a long leader sequence, unrelated to omega', did not enhance expression of NPTII mRNA in vitro. 2. The ability of several leader sequences, including omega', to form multiple initiation complexes with 80S (wheat germ) ribosomes was examined using CAT or NPTII mRNAs incubated in the presence of sparsomycin. Formation of disome complexes was unrelated to the capacity of a 5'-leader sequence to enhance translation. 3. Expression of CAT mRNA in both wheat germ extract and messenger-dependent rabbit reticulocyte lysate was less susceptible to inhibition by increasing salt concentration when a 5'-proximal omega' sequence was present. This effect was less marked when the CAT mRNA was capped. Conversely at high salt concentrations, capping was less stimulatory for mRNA with a 5'-proximal omega' sequence. These data suggest that omega' and the cap enhance translation, at least in part, by a similar mechanism. We propose that both features reduce RNA secondary structure, thereby rendering the 5' terminus more accessible to scanning by 40S ribosomal subunits and/or interaction with associated initiation factors. This conclusion was supported by computer-based secondary-structure analyses of our SP6 RNA polymerase transcript sequences. The ability of 5' leader sequences from brome mosaic virus RNA 3, alfalfa mosaic virus RNA 4, and the genomic RNAs of turnip yellow mosaic virus, Rous sarcoma virus or tobacco mosaic virus (tomato strain) to enhance mRNA translation in eukaryotic systems may also be correlated with their respective secondary structures. A different mechanism probably accounts for the omega'-dependent enhancement of mRNA expression in Escherichia coli or in E. coli cell-free systems.
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PMID:Studies on the mechanism of translational enhancement by the 5'-leader sequence of tobacco mosaic virus RNA. 284 Nov 27

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 transient expression system in which chimeric genes are expressed in cells infected with vaccinia virus was developed. Recombinant plasmids containing the promoter regions of vaccinia virus genes ligated to the coding segment of the prokaryotic chloramphenicol acetyltransferase (CAT) gene were constructed. When the plasmids were introduced into vaccinia virus-infected cells by transfection, the chimeric gene was expressed and significant levels of CAT accumulated. CAT activity was not detected when the same recombinant plasmid was introduced into uninfected cells, nor was activity detected when the vaccinia virus promoter was absent from the plasmid or was replaced by simian virus 40 or Rous sarcoma virus promoters. This specificity indicated that expression is dependent on a cis-acting vaccinia virus promoter region within the recombinant plasmid and diffusible trans-acting transcription factors produced during virus infection. The lack of effect of a simian virus 40 enhancer element inserted upstream of the vaccinia virus promoter region also distinguished this system from systems dependent on RNA polymerase II. Although replication of the recombinant plasmid could not be detected in either uninfected or vaccinia virus-infected cells, an inhibitor of DNA synthesis significantly reduced CAT expression. This result, as well as the kinetics of CAT synthesis, suggests that replication of viral DNA templates can enhance transcription of chimeric genes in recombinant plasmids.
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PMID:Eukaryotic transient expression system dependent on transcription factors and regulatory DNA sequences of vaccinia virus. 385 41

Synthesis of Rous sarcoma virus RNA was examined in vitro with a new assay for radioactive virus-specific RNA. Nuclei from infected and uninfected cells were incubated with ribonucleoside [alpha-(32)P]triphosphates, Mn(++), Mg(++) and (NH(4))(2)SO(4). Incorporation into total and viral RNA proceeded with similar kinetics for up to 25 min at 37 degrees . About 0.5% of the RNA synthesized by the infected system was scored as virus-specific, compared to 0.03% of the RNA from the uninfected system and 0.005% of the RNA synthesized by monkey kidney cell nuclei. Preincubation with DNase or actinomycin D completely suppressed total and virus-specific RNA synthesis. alpha-Amanitin, a specific inhibitor of eukaryotic RNA polymerase II, completely inhibited virus-specific RNA synthesis, while reducing total RNA synthesis by only 50%. We conclude that tumor virus-specific RNA is synthesized on a DNA template, most probably by the host's RNA polymerase II.
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PMID:In vitro synthesis of Rous sarcoma virus-specific RNA is catalyzed by a DNA-dependent RNA polymerase. 436 1

Reverse transcriptase from avian retrovirus has a physically associated DNA endonuclease with novel substrate and cofactor requirements. A similar endonuclease activity copurifies with pp32, a protein from viral cores that has been identified with the non-alpha region of the beta subunit of reverse transcriptase. Several temperature-sensitive mutants of avian retrovirus with thermolabile DNA polymerase were tested for thermal sensitivity of their DNA endonuclease activity. Two pol mutants of Rous sarcoma virus, ts335 and ts337, had thermolabile DNA endonuclease; a temperature-resistant revertant of ts335 had a heat-stable DNA endonuclease. DNA endonuclease is therefore a product of the pol gene and an integral part of the reverse transcriptase. A second class of pol mutants, typified by ts568 and ts553, had thermolabile DNA polymerase, but heat-stable DNA endonuclease.
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PMID:Virus-coded DNA endonuclease from avian retrovirus. 616 35

Reverse transcriptase from Rous sarcoma virus and avian myeloblastosis virus was purified by a rapid two-step procedure using chromatography on phosphocellulose and heparin-Sepharose. The resulting enzyme was homogeneous, had a high specific activity and was free of contaminating nucleases. This procedure has been adapted to small-scale preparation of enzyme from mutant virus containing thermolabile reverse transcriptase, and is equally suitable for large-scale enzyme purification.
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PMID:Purification of reverse transcriptase from avian retroviruses using affinity chromatography on heparin-sepharose. 616 44

A temperature-sensitive mutant (LA83) of Rous sarcoma virus defective both in the transformation and replication function has been isolated and partially characterized. Temperature-shift experiments showed that the defects in both the focus-forming and replication functions were late and continuous. The mutant LA83 was complemented by avian leukosis viruses. Complementation of LA83 replication was also observed with the glycoprotein-deletion mutant, Brian high-titer RSV(-) suggesting that the env gene in LA83 was not defective. At the nonpermissive temperature LA83-infected cells produced noninfectious particles with a yield of about 30%. The noninfectious particles had only about 3% of reverse-transcriptase activity as the infectious LA83 produced at the permissive temperature. However, the LA83 virions were as thermolabile as the parent wild-type PR-B virions.
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PMID:Characterization of a replication-defective temperature-sensitive mutant of Rous sarcoma virus. 620 47

Specific initiation of transcription of Rous sarcoma virus by RNA polymerase II was obtained in a cell-free system using cloned RSV DNA as template. The site of initiation is located in the common "C" region of the long terminal repeat (LTR), 23 bp downstream from the promoter-like sequence TATTTAAG. This finding indicates that the basic information necessary for RSV transcription lies within the viral genome.
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PMID:Identification of a functional promoter in the long terminal repeat of Rous sarcoma virus. 625 99

We have used two methods to detect specific transcription of the chicken alpha 2 (type I) collagen gene in cell-free extracts derived from Rous sarcoma virus-transformed chicken embryo fibroblasts. The first method is a modification of the S1 nuclease mapping procedure which utilizes a DNA probe labeled with 32P at the 5' end of the HindIII linker originally used to clone the collagen promoter region into PBR322. The probe distinguishes newly made, specific RNA from endogenous RNA and nonspecific transcripts. Using this procedure we have found that chicken whole cell extracts support accurate initiation of transcription of the chicken alpha 2 (type I) collagen DNA template. Addition of either creatine phosphate, GTP, or UTP to concentrations of approximately 3 to 5 mM was found to stimulate RNA polymerase II transcription by 5- to 10-fold. The second method employs an avian myeloblastosis virus reverse transcriptase-catalyzed primary extension procedure, rendered in vitro-specific by use of a pBR322 fragment as primer. These two techniques should be useful for analyzing specific transcription in other types of cell-free extracts.
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PMID:Transcription of the chicken alpha 2 (Type I) collagen gene by homologous cell-free extracts. 628 36

A temperature-sensitive coordinate mutant tsLA83 of Prague (PR-B) strain of Rous sarcoma virus at the nonpermissive temperature (41 degrees) produces noninfectious virus particles (NI-LA83) which contained only 3% of the reverse-transcriptase activity present in infectious virions. Analyses of [35S]methionine-labeled NI-LA83 showed the presence of all of the viral proteins except reverse transcriptase. Pulse-chase analyses of the virus-specified proteins in cells infected with LA83 or PR-B showed that the gag and glycoprotein precursors, Pr76gag and gPr95env, respectively, were processed at both 35 and 41 degrees. The reverse-transcriptase precursor, Pr180gag-pol, however, was not processed in LA83-infected cells at 41 degrees. In contrast, cells infected with LA83 or PR-B at 35 degrees as well as with PR-B at 41 degrees showed normal cleavage of Pr180gag-pol. A shiftdown of LA83-infected cells at 41 degrees to the permissive temperature 35 degrees resulted in the normal processing of Pr180gag-pol and production of infectious virus containing reverse transcriptase. Electron microscopic analysis showed that at 41 degrees cells infected with LA83 showed a large number of budding structures but fewer released particles. A shiftdown from 41 to 35 degrees resulted in an increase of virus particles with a concomitant decrease in budding structures suggesting that the processing of reverse-transcriptase precursor is related to virion assembly.
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PMID:Impaired cleavage of the joint gag-pol polyprotein precursor and virion assembly in a temperature-sensitive mutant of Rous sarcoma virus. 633 Sep 81


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