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)

Wildtype and mutant v-Myc proteins were overexpressed in Escherichia coli using the T7 RNA polymerase system, and the in vitro DNA-binding activities of partially or highly purified proteins were analysed by native DNA-cellulose chromatography. For the construction of the expression plasmids, cloned proviral DNA from wildtype MC29 or from its spontaneous deletion mutant Q10C was used, the latter lacking internal v-myc sequences. Both the wildtype (p59) and the mutant (p42) recombinant protein contain at their amino termini 12 amino acids encoded by the vector, followed by 11 gag amino acids and 9 amino acids encoded by v-myc sequences derived from noncoding c-myc sequences. In addition, p59 contains 416 amino acids encoded by v-myc sequences derived from the complete chicken c-myc coding region, whereas p42 lacks 120 amino acids from the central region of the Myc protein including the highly acidic domain. Two additional proteins were engineered which contain the first 309 (p53) or the last 107 (p16) amino acids, respectively, of the Myc protein sequence in addition to vector-encoded amino acids. The p16 protein represents the carboxyl terminus of the Myc protein sequence containing both a muscle determination gene (MyoD1) homology region, including a basic motif and an amphipathic helix-loop-helix motif, and a leucine heptad repeat. All proteins, except p53 which lacks the carboxyl-terminal Myc protein sequences, bound to native DNA-cellulose and were eluted with 200-500 mM NaCl. Based on the DNA-binding activities of recombinant or spontaneous mutant v-Myc proteins extracted from bacterial or from transformed avian cells, we conclude that the DNA-binding domain of avian Myc proteins is confined within the last 86 carboxyl-terminal amino acids. The same region is also shown to be necessary and sufficient for Myc protein dimerization. This 86-amino acid region essentially encompasses a putative basic DNA contact surface and a tandem array of two presumed protein dimerization motifs, helix-loop-helix and leucine repeat.
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PMID:Myc protein structure: localization of DNA-binding and protein dimerization domains. 199 48

Pig-tailed macaques were vaccinated with a human T-cell lymphotropic virus type I (HTLV-I) subunit vaccine. Vaccinates and controls were challenged with simian T-cell lymphotropic virus type I (STLV-I)-infected cells. Vaccination yielded antibody responses to HTLV-I and STLV-I gag and env precursors. Controls developed HTLV-I and STLV-I antibody to gag and tax protein. Immunization produced syncytium inhibiting antibody and cellular cytotoxicity to virus-infected cells. Reverse transcriptase activity was present in control macaques only, implying that the subunit vaccine was protective against STLV-I infection.
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PMID:Evaluation of a HTLV-1 subunit vaccine in prevention of experimental STLV-I infection in Macaca nemestrina. 217 41

An enzyme immunoassay was developed to detect human immunodeficiency virus type 1 (HIV-1) DNA amplified by polymerase chain reaction (PCR-EIA). A set of primers (outer set) was used in PCR to amplify a segment of the HIV-1 gag gene from peripheral blood mononuclear cells. Hybrids between the amplified DNA and a RNA probe were measured in a microtiter plate immunoassay using a beta-D-galactosidase-conjugated monoclonal antibody to DNA-RNA hybrids and a fluorescent substrate. A second set of primers (nested set) located within the outer set was used in PCR with a known template to prepare the probe. One primer of the nested set included the T7 RNA polymerase promoter at its 5' end allowing transcription of a single-stranded RNA probe. Ten copies of HIV-1 DNA could be detected by PCR-EIA (42 fluorescent units with a background of 18 fluorescent units) compared with a detection limit of 1000 copies by ethidium bromide-stained agarose gel. HIV-1 DNA was detected by PCR-EIA in peripheral blood mononuclear cells from 32 of 33 seropositive patients (range 54-810 fluorescent units), and 0 of 25 seronegative patients (range 20-40 fluorescent units) (sensitivity 97%; specificity 100%). PCR-EIA offers a practical and nonisotopic method to objectively measure PCR-amplified HIV-1 DNA and has the potential for the measurement of other microbial pathogens in human body fluids.
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PMID:Enzyme immunoassay for detection of hybrids between PCR-amplified HIV-1 DNA and a RNA probe: PCR-EIA. 174 86

NIH 3T3 cells infected with Moloney murine leukemia virus (MoMLV) express high levels of virus-specific RNA. To inhibit replication of the virus, we stably introduced chimeric tRNA genes encoding antisense templates into NIH 3T3 cells via a retroviral vector. Efficient expression of hybrid tRNA-MoMLV antisense transcripts and inhibition of MoMLV replication were dependent on the use of a particular type of retroviral vector, the double-copy vector, in which the chimeric tRNA gene was inserted in the 3' long terminal repeat. MoMLV replication was inhibited up to 97% in cells expressing antisense RNA corresponding to the gag gene and less than twofold in cells expressing antisense RNA corresponding to the pol gene. RNA and protein analyses suggest that inhibition was exerted at the level of translation. These results suggest that RNA polymerase III-based antisense inhibition systems can be used to inhibit highly expressed viral genes and render cells resistant to viral replication via intracellular immunization strategies.
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PMID:Expression of chimeric tRNA-driven antisense transcripts renders NIH 3T3 cells highly resistant to Moloney murine leukemia virus replication. 224 70

The pol gene of Rous sarcoma virus is positioned downstream of the gag gene in a different, briefly overlapping reading frame; nevertheless, the primary translation product of pol is a gag-pol fusion protein. Two mechanisms, ribosomal frameshifting and RNA splicing, have been considered to explain this phenomenon. The frameshifting model is supported by synthesis of both gag protein and gag-pol fusion protein in a cell-free mammalian translation system programmed by a single RNA species that was synthesized from cloned viral DNA with a bacteriophage RNA polymerase. Under these conditions, the ratio of the gag protein to the fusion protein (about 20 to 1) is similar to that previously observed in infected cells, the frameshifting is specific for the gag-pol junction, and it is unaffected by large deletions in gag. In addition, synthesis of the fusion protein is ten times less efficient in an Escherichia coli cell-free translation system and cannot be explained by transcriptional errors or in vitro modification of the RNA. Ribosomal frameshifting may affect production of other proteins in higher eukaryotes, including proteins encoded by several retroviruses and transposable elements.
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PMID:Expression of the Rous sarcoma virus pol gene by ribosomal frameshifting. 241 54

Based on precedents from other retroviruses, the precursor of the human immunodeficiency virus (HIV-1) reverse transcriptase is predicted to be a polyprotein with a relative molecular mass (Mr) of 160,000 (160K) encoded by both the viral pol gene and the upstream gag gene. These two genes lie in different translational reading frames, with the 3' end of gag overlapping the 5' end of pol by 205 or 241 nucleotides. Thus, production of the gag-pol fusion protein would require either messenger RNA processing or translational frameshifting. The latter mechanism has been shown in the synthesis of the gag-pol proteins of two other retroviruses, Rous sarcoma virus (RSV) and mouse mammary tumour virus (MMTV). Here we report that translation of HIV-1 RNA synthesized in vitro by SP6 RNA polymerase yields significant amounts of a gag-pol fusion protein, indicating that efficient ribosomal frameshifting also occurs within the HIV-1 gag-pol overlap region. Site-directed mutagenesis and amino-acid sequencing localized the site of frameshifting to a UUA leucine codon near the 5' end of the overlap.
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PMID:Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. 244 6

The Saccharomyces cerevisiae viruses have a large viral double-stranded RNA which encodes the major viral capsid polypeptide. We have previously shown that this RNA (L1) also encodes a putative viral RNA-dependent RNA polymerase (D. F. Pietras, M. E. Diamond, and J. A. Bruenn, Nucleic Acids Res., 16:6226, 1988). The organization and expression of the viral genome is similar to that of the gag-pol region of the retroviruses. The complete sequence of L1 demonstrates two large open reading frames on the plus strand which overlap by 129 bases. The first is the gene for the capsid polypeptide, and the second is the gene for the putative RNA polymerase. One of the products of in vitro translation of the denatured viral double-stranded RNA is a polypeptide of the size expected of a capsid-polymerase fusion protein, resulting from a -1 frameshift within the overlapping region. A polypeptide of the size expected for a capsid-polymerase fusion product was found in virions, and it was recognized in Western blots (immunoblots) by antibodies to a synthetic peptide derived from the predicted polymerase sequence.
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PMID:Overlapping genes in a yeast double-stranded RNA virus. 266 62

An antiserum prepared against thymosin alpha 1, a hormone secreted by the thymus gland, effectively neutralized the AIDS-associated virus [HTLV-III/LAV (clone BH-10)] and blocked its replication in H9 cells. Reverse transcriptase activity and expression of the HTLV-III/LAV antigens p15 and p24 were inhibited by purified immunoglobulin G preparations of antisera to thymosin alpha 1. The antiviral activity of the antiserum was found to be due to a region of homology between thymosin alpha 1 and p17, a product of the gag gene of HTLV-III/LAV. Comparison of the primary sequences of thymosin alpha 1 and the gag protein revealed a 44% to 50% homology in an 18-amino acid region, between positions 11 and 28 on thymosin alpha 1 and 92 and 109 on the gag protein. The effectiveness of the thymosin alpha 1 antiserum and of immunoglobulin G-enriched preparations in blocking replication of HTLV-III(BH-10) in H9 cells suggests a novel approach to the development of an AIDS vaccine. A vaccine directed against the gag protein might overcome the problem of genetic drift in the envelope region of the virus and be useful against all genetic variants of HTLV-III/LAV.
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PMID:Neutralization of HTLV-III/LAV replication by antiserum to thymosin alpha 1. 301 Apr 64

Stable nonproducer (NP) cell lines transformed by the avian acute leukemia virus OK10 were established. All 13 studied NP quail cell lines released into the culture medium noninfectious, mostly reverse-transcriptase-negative particles containing the usual gag proteins. Infectious, transforming OK10 virus pseudotypes could be recovered by rescue with helper virus. p200, the putative transforming protein of OK10, was identified in in vitro translates of RNA from the noninfectious particles and in immunoprecipitates of cell extracts of the NP clones analyzed. Two NP clones, the reverse-transcriptase-negative B5 and -positive 9C cell lines, displayed striking differences in in vivo tumorigenicity. Although B5 induced no tumors in quails, 9C caused multiple tumors and cells derived from several tumors could be passaged in vitro. At no time during the in vivo passage or during more than 1 year of in vitro culture have the particles released by 9C cells acquired infectious properties. Possible reasons for the observed differences in tumorigenicity between the OK10 virus-transformed 9C and B5 NP cell lines are discussed.
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PMID:OK10-transformed cell lines: viral component generation and tumorigenicity. 620 53

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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