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)

We developed a stringently regulated expression system for mammalian cells that uses (i) the RNA polymerase, phi 10 promoter, and T phi transcriptional terminator of bacteriophage T7; (ii) the lac repressor, lac operator, rho-independent transcriptional terminators and the gpt gene of Escherichia coli; (iii) the RNA translational enhancer of encephalomyocarditis virus; and (iv) the genetic background of vaccinia virus. In cells infected with the recombinant vaccinia virus, reporter beta-galactosidase synthesis was not detected in the absence of inducer. An induction of at least 10,000- to 20,000-fold occurred upon addition of isopropyl beta-D-thiogalactopyranoside or by temperature elevation from 30 to 37 degrees C using a temperature-sensitive lac repressor. Regulated synthesis of the secreted and highly glycosylated human immunodeficiency virus 1 envelope protein gp120 was also demonstrated. Yields of both proteins were approximately 2 mg per 10(8) cells in 24 hr. Plasmid transfer vectors for cloning and expression of complete or incomplete open reading frames in recombinant vaccinia viruses are described.
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PMID:Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells. 762 18

Transcriptional elongation involves dynamic interactions among RNA polymerase and single-stranded and double-stranded nucleic acids in the ternary complex. In prokaryotes its regulation provides an important mechanism of genetic control. Analogous eukaryotic mechanisms are not well understood, but may control expression of proto-oncogenes and viruses, including the human immunodeficiency virus HIV-1 (ref. 8). The highly conserved eukaryotic transcriptional elongation factor TFIIS enables RNA polymerase II (RNAPII) to read though pause or termination sites, nucleosomes and sequence-specific DNA-binding proteins. Two distinct domains of human TFIIS, which bind RNAPII and nucleic acids, regulate read-through and possibly nascent transcript cleavage. Here we describe the three-dimensional NMR structure of a Cys4 nucleic-acid-binding domain from human TFIIS. Unlike previously characterized zinc modules, which contain an alpha-helix, this structure consists of a three-stranded beta-sheet. Analogous Cys4 structural motifs may occur in other proteins involved in DNA or RNA transactions, including RNAPII itself. This new structure, designated the Zn ribbon, extends the repertoire of Zn-mediated peptide architectures and highlights the growing recognition of the beta-sheet as a motif of nucleic-acid recognition.
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PMID:Structure of a new nucleic-acid-binding motif in eukaryotic transcriptional elongation factor TFIIS. 762 41

The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression in response to Tat is dependent on an element downstream of the HIV-1 transcriptional initiation site designated the trans-activating region (TAR). TAR forms a stable stem-loop RNA structure in which a 3-nt bulge structure and a 6-nt loop structure are important for Tat activation. In the absence of Tat, the HIV-1 promoter generates so-called short or nonprocessive transcripts terminating at +60, while in the presence of Tat the synthesis of these short transcripts is markedly decreased and transcripts that extend through the 9.0-kb HIV-1 genome are synthesized. Tat effects on transcriptional elongation are likely due to alterations in the elongation properties of RNA polymerase II. In this study we demonstrated that a set of cellular cofactors that modulate the binding of the cellular protein TRP-185 to the TAR RNA loop sequences also functioned to markedly stimulate the specific binding of hypophosphorylated (IIa) and hyperphosphorylated (IIo) RNA polymerase II to TAR RNA. The concentrations of RNA polymerase II required for this interaction with TAR RNA were similar to those required to initiate in vitro transcription from the HIV-1 long terminal repeat. RNA gel retardation analysis with wild-type and mutant TAR RNAs indicated that the TAR RNA loop and bulge sequences were critical for the binding of RNA polymerase II. The addition of wild-type but not mutant Tat protein to gel retardation analysis with TAR RNA and RNA polymerase II resulted in the loss of binding of RNA polymerase II binding to TAR RNA. These results suggest that Tat may function to alter RNA polymerase II, which is paused due to its binding to HIV-1 TAR RNA with resultant stimulation of its transcriptional elongation properties.
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PMID:Specific binding of RNA polymerase II to the human immunodeficiency virus trans-activating region RNA is regulated by cellular cofactors and Tat. 763 59

In an effort to facilitate the efficiency of human immunodeficiency virus type 1 (HIV-1) and/or human cytomegalovirus (HCMV) infection in primary monocyte/macrophages in vitro, the effect of low-speed centrifugation was studied. The infectivity of three strains (Bal, Ada-M, and IIIB) of HIV-1 tested was significantly enhanced by centrifugal inoculation at a force of 1500g for 60 min. Reverse transcriptase activity and HIV-1 p24 antigen in primary monocyte/macrophages infected by a centrifugal inoculation technique were detectable 3-7 days earlier and were more than 10-fold greater in magnitude (at an early stage of the infection) than those of control cells infected by the conventional inoculation technique. Examination of the cells by indirect immunofluorescence revealed higher expression of HIV-1 p24 protein in the monocyte/macrophages infected by the centrifugal inoculation technique. These differences were directly related to centrifugal inoculation and were evident up to 3 weeks after infection. Enhancement was not observed when centrifugation was carried out before or after HIV-1 infection. Centrifugal inoculation of HCMV also enhanced its immediate-early and early gene expression up to 30- to 50-fold, although neither late nuclear antigens and glycoproteins of HCMV nor infectious virus was detected in HCMV-infected monocyte/macrophage cultures. These results show that centrifugal inoculation is a useful technique for improving the efficiency of HCMV and HIV-1 infection in vitro.
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PMID:Centrifugal enhancement of human immunodeficiency virus type 1 infection and human cytomegalovirus gene expression in human primary monocyte/macrophages in vitro. 768 Mar 71

In vitro evaluation of a large chemical library of pharmacologically acceptable prototype compounds in a high-capacity, cellular-based screening system has led to the discovery of another family of human immunodeficiency virus type 1 (HIV-1) inhibitors. Through optimization of a lead compound, several alpha-anilinophenylacetamide (alpha-APA) derivatives have been identified that inhibit the replication of several HIV-1 strains (IIIB/LAI, RF, NDK, MN, HE) in a variety of host cell types at concentrations that are 10,000- to 100,000-fold lower than their cytotoxic concentrations. The IC50 of the alpha-APA derivative R 89439 for HIV-1 cytopathicity in MT-4 cells was 13 nM. The median 90% inhibitory concentration (IC90) in a variety of host cells was 50-100 nM. Although these alpha-APA derivatives are active against a tetrahydroimidazo [4,5,1-jk][1,4]benzodiazepin-2(1H)-thione-(TIBO)-resistant HIV-1 strain, they do not inhibit replication of HIV-2 (strains ROD and EHO) or simian immunodeficiency virus (strains Mac251, mndGB1, and agm3). An HIV-1 strain containing the Tyr181-->Cys mutation in the reverse transcriptase region displayed reduced sensitivity. alpha-APA derivative R 89439 inhibited virion and recombinant reverse transcriptase of HIV-1 but did not inhibit that of HIV-2. Reverse transcriptase inhibition depended upon the template/primer used. The relatively uncomplicated synthesis of R 89439, its potent anti-HIV-1 activity, and its favorable pharmacokinetic profile make R 89439 a good candidate for clinical studies.
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PMID:Potent and highly selective human immunodeficiency virus type 1 (HIV-1) inhibition by a series of alpha-anilinophenylacetamide derivatives targeted at HIV-1 reverse transcriptase. 768 Apr 76

The (-) enantiomers of 2',3'-dideoxy-5-fluoro-3'-thiacytidine [(-)-FTC] and 2',3'-dideoxy-3'-thiacytidine [(-)-BCH-189] were recently shown to inhibit selectively human immunodeficiency viruses (HIV) and hepatitis B virus in vitro. In the current study, the potential for HIV type 1 (HIV-1) resistance to these compounds was evaluated by serial passage of the virus in human peripheral blood mononuclear cells and MT-2 cells in the presence of increasing drug concentrations. Highly drug-resistant HIV-1 variants dominated the replicating virus population after two or more cycles of infection. The resistant variants were cross-resistant to (-)-FTC, (-)-BCH-189, and their (+) congeners but remained susceptible to 2',3'-dideoxycytidine, 3'-azido-3'-deoxythymidine, 3'-fluoro-3'-deoxythymidine, 2',3'-dideoxyinosine, phosphonoformate, the TIBO compound R82150, and the bis(heteroaryl)piperazine derivative U-87201E. Reverse transcriptase derived from drug-resistant viral particles was 15- to 50-fold less susceptible to the 5'-triphosphates of FTC and BCH-189 compared with enzyme from parental drug-susceptible virus. DNA sequence analysis of the reverse transcriptase gene amplified from resistant viruses consistently identified mutations at codon 184 from Met (ATG) to Val (GTG or GTA) or Ile (ATA). Sequence analysis of amplified reverse transcriptase from a patient who had received (-)-BCH-189 therapy for 4 months demonstrated a mixture of the Met-184-to-Val (GTG) mutation and the parental genotype, indicating that the Met-184 mutation can occur in vivo.
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PMID:Characterization of human immunodeficiency viruses resistant to oxathiolane-cytosine nucleosides. 768 16

Nystatin A was compared in vitro with amphotericin B, AZT, or foscarnet for their respective abilities to inhibit the replication of human immunodeficiency virus type 1 (HIV-1) in H9 cells. HIV-1-infected H9 cells were cultured for 7 days in the presence of each of these drugs, at various concentrations. Reverse transcriptase activity and p24 antigen production were quantitated. Untreated, HIV-1-infected H9 cells served as the control. Nystatin A inhibited viral replication most effectively at 10 micrograms/ml, a concentration that did not affect cell viability. Nystatin-A treatment inhibited RT activity by 85% and p24 production by 90%. These levels of inhibition were comparable to that mediated by amphotericin B, AZT, or foscarnet at 10, 25, and 50 micrograms/ml, respectively. Western blot analysis of the HIV-1-infected H9 cells treated with these drugs did not detect any expression of viral proteins. These findings were further corroborated by indirect immunofluorescence studies using monoclonal anti-gp120 FITC-conjugated antibodies and by polymerase chain reaction for proviral DNA analysis, using a 32P-labeled probe. These results suggest that Nystatin A merits attention as an antiviral drug for the treatment of HIV-1 infection. In vivo drug delivery by liposome encapsulation to overcome problems of bioavailability is currently under study.
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PMID:Inhibition of HIV-1 replication in H9 cells by nystatin-A compared with other antiviral agents. 768 87

The crystal structure of T7 RNA polymerase reveals a molecule organized around a cleft that can accommodate a double-stranded DNA template. A portion (approximately 45%) of the molecule displays extensive structural homology to the polymerase domain of Klenow fragment and more limited homology to the human immunodeficiency virus HIV-1 reverse transcriptase. A comparison of the structures and sequences of these polymerases identifies structural elements that may be responsible for discriminating between ribonucleotide and deoxyribonucleotide substrates, and RNA and DNA templates. The relative locations of the catalytic site and a specific promoter recognition residue allow the orientation of the polymerase on the template to be defined.
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PMID:Crystal structure of bacteriophage T7 RNA polymerase at 3.3 A resolution. 768 63

A virus causing Jembrana disease in Bali cattle (Bos javanicus) was demonstrated to have characteristics of a retrovirus. Reverse transcriptase activity was detected in virus purified by sucrose gradient centrifugation. Electron microscopic examination of tissue from the affected cattle indicated that the virus matured by C-type budding through the plasma membrane and into intracytoplasmic vacuoles of cells in lymphoid tissue, with the formation of circular enveloped virus particles ranging in diameter from 96 to 124 nm with an eccentric nucleoid. Western immunoblotting using sera from recovered animals demonstrated virus proteins of M(r) 100K, 45K, 42K, 33K, 26K, 16K and 14K. The 26K protein of Jembrana disease virus cross-reacted in Western blots with the 26K capsid protein of bovine immunodeficiency virus (BIV). The apparent morphogenesis, protein structure and antigenic relationship with BIV suggested the virus was a lentivirus.
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PMID:Characteristics of a retrovirus associated with Jembrana disease in Bali cattle. 769 Aug 40

Eukaryotic cellular mRNA is believed to be synthesized exclusively by RNA polymerase II (pol II), whereas pol I produces long rRNAs and pol III produces 5S rRNA, tRNA, and other small RNAs. To determine whether this functional differentiation is obligatory, we examined the translational potential of an artificial pol III transcript. The coding region of the human immunodeficiency virus type 1 tat gene was placed under the control of a strong pol III promoter from the adenovirus type 2 VA RNAI gene. The resultant chimera, pVA-Tat, was transcribed accurately in vivo and in vitro and gave rise to Tat protein, which transactivated a human immunodeficiency virus-driven chloramphenicol acetyltransferase reporter construct in transfected HeLa cells. pol III-specific mutations down-regulated VA-Tat RNA production in vivo and in vitro and dramatically reduced chloramphenicol acetyltransferase transactivation. As expected for a pol III transcript, VA-Tat RNA was not detectably capped at its 5' end or polyadenylated at its 3' end, but, like mRNA, it was associated with polysomes in a salt-stable manner. Mutational analysis of a short open reading frame upstream of the Tat-coding sequence implicates scanning in the initiation of VA-Tat RNA translation despite the absence of a cap. In comparison with tat mRNA generated by pol II, VA-Tat RNA was present on smaller polysomes and was apparently translated less efficiently, which is consistent with a relatively low initiation rate. Evidently, human cells are capable of utilizing pol III transcripts as functional mRNAs, and neither a cap nor a poly(A) tail is essential for translation, although they may be stimulatory. These findings raise the possibility that some cellular mRNAs are made by pol I or pol III.
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PMID:Functional mRNA can be generated by RNA polymerase III. 779 67


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