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)

Direct detection of foot-and-mouth disease (FMD) virus from infected bovine and porcine tissue was investigated using a modified polymerase chain reaction (PCR) technique. A high degree of conservation was found in the genomic region coding for the viral RNA polymerase among the seven FMD viral (FMDV) serotypes. An oligomeric primer pair and probe were constructed from consensus sequence data within this area. First strand cDNA was synthesized using random hexamers and Moloney MuLV reverse transcriptase. The oligomeric primers used for PCR of the random primed cDNA yielded a 454-base-pair target amplification product. The PCR product was sized by agarose gel electrophoresis and hybridized strongly with the consensus sequence oligomeric probe. The PCR product was further examined by digestion with NcoI, confirming the predicted internal restriction enzyme site. All seven serotypes of FMDV RNA were amplified in a few hours and the PCR product tested positive. The sensitivity of the enzymatic amplification for detection of FMDV was 10 TCID50 by gel electrophoresis and less than 1 TCID50 when combined with hybridization to a labeled probe. The technique was specific, as determined by examination of at least 12 other viruses, including enteroviruses and other agents of vesicular disease. In vitro enzymatic amplification of cDNA from FMDV RNA using the modified PCR technique is highly specific, rapid and at least as sensitive as presently used procedures for FMDV laboratory diagnosis.
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PMID:Rapid and sensitive detection of foot-and-mouth disease virus in tissues by enzymatic RNA amplification of the polymerase gene. 166 35

We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.
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PMID:Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography. 168 98

A polyacrylamide gel assay is used to measure the kinetics of adding a single deoxyribonucleotide onto either a correctly matched or mismatched primer 3' terminus (on M13 template) for all possible DNA base pairs and mispairs using Drosophila melanogaster DNA polymerase alpha (Pol alpha) and avian myeloblastosis virus reverse transcriptase. The reverse transcriptase catalyzes chain extension from transition mispairs (Pur.Pyr and Pyr.Pur, where Pur is purine and Pyr is pyrimidine) more efficiently than polymerase alpha. Reverse transcriptase extends G(primer).T almost 20% as efficiently as it extends A.T, while Pol alpha's G.T extension efficiency is less than 1%. For transversion mispairs (Pur.Pur and Pyr.Pyr), reverse transcriptase extends C.T and T.T with greater efficiency than polymerase alpha, while polymerase alpha is more efficient at extending A.G and G.G mispairs. Reverse transcriptase and polymerase alpha extend the G.G mispair at an efficiency of only 10(-6) and 10(-5), respectively, compared with G.C extension. The extension data for the two polymerases are compared with previously reported nucleotide misinsertion data for the same enzymes (Mendelman, L. V., Boosalis, M. S., Petruska, J., and Goodman, M. F. (1989) J. Biol. Chem. 264, 14415-14423). While the results obtained with reverse transcriptase and Pol alpha differ in detail, some general rules are indicated: (a) Pur.Pyr and Pyr.Pur mispairs, especially G.T and T.G, are easy to insert and even easier to extend; (b) Pyr.Pyr mispairs, especially C.C, are difficult to insert and slightly easier to extend; (c) Pur.Pur mispairs, notably G.G, are harder to extend than to insert. The comparison also shows that reverse transcriptase extends almost all mismatches more efficiently than it forms them, G.G being the only mismatch having a significantly lower efficiency of extension than insertion. Polymerase alpha inserts A.A mismatches most efficiently, but extends them inefficiently, thereby reducing the probability that such transversion mutations will occur in vivo. We show theoretically that when mispaired primers compete with properly matched primers for extension by polymerase, the relative velocities of extension depend on the concentration of the next correct dNTP substrate. The extension velocities depart from Michaelis-Menten kinetics by exhibiting positive cooperativity with respect to substrate concentration.
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PMID:Base mispair extension kinetics. Comparison of DNA polymerase alpha and reverse transcriptase. 168 52

Endoribonucleolytic cleavage by the ribonuclease H activity associated with HIV-1 reverse transcriptase was observed in vitro using substrates consisting of synthetic oligodeoxynucleotides hybridized to a 345 nucleotide T7 RNA polymerase transcript derived from the gag region of HIV-1. This observation suggests that a possible mechanism of action of antisense oligonucleotides in the inhibition of viral replication and expression may involve the selective "suicidal" ribonucleolytic cleavage of viral RNA by reverse transcriptase at the site of hybridization of the oligonucleotide.
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PMID:Endoribonucleolytic cleavage of RNA: oligodeoxynucleotide hybrids by the ribonuclease H activity of HIV-1 reverse transcriptase. 169 3

Reverse transcriptase has been purified from feline immunodeficiency virus (FIV) by DEAE-cellulose and phosphocellulose chromatography. The purified enzyme consists of a single protein with a Mr of 67,000. When proteolysis is not minimized during purification, a fragment of Mr 54,000 is also observed. This is similar to the reverse transcriptase from human immunodeficiency virus type 1 (HIV), which consists of a polypeptide of Mr 66,000; when proteolysis is not minimized during purification, a fragment of Mr 51,000 is also observed. In direct comparisons, the FIV reverse transcriptase is very similar to the HIV reverse transcriptase in template specificity and requirements for Mg2+. In contrast to these similarities, the FIV and HIV reverse transcriptases are substantially different in primary sequence, as determined by peptide mapping.
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PMID:Characterization of reverse transcriptase from feline immunodeficiency virus. 169 Jul 35

Reverse transcriptase required for the synthesis of msDNA.Ec67 in an Escherichia coli strain was purified as a large molecular weight complex with msDNA. The complex sedimented in a glycerol gradient at an s value greater than 19. The predominant protein species co-purifying with reverse transcriptase activity in the complex had a molecular weight estimated at 65,000 which is close to the expected size of 67,227 for the Ec67-reverse transcriptase. In addition, the large complex also contained msDNA.Ec67. The purified complex was able to synthesize cDNA using 5 S rRNA as a template (annealed to a synthetic DNA primer), and a double-stranded DNA using a synthetic DNA template (annealed to a synthetic DNA primer). When msDNA.Ec67 was used as a natural template:primer, the purified complex produced two major products: a 103-base single-stranded DNA by extending the 3' end of msDNA using msdRNA as a template, and a 60-base double-stranded DNA product resulting from the converse reaction in which the 3' end of msdRNA is extended using msDNA as a template. The results suggest that bacterial reverse transcriptase is capable of producing single-stranded cDNA and possibly double-stranded DNA as well. Possible implications of these findings on the biology of the msDNA-retron system are discussed.
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PMID:Reverse transcriptase from Escherichia coli exists as a complex with msDNA and is able to synthesize double-stranded DNA. 169 31

The two components of Camellia sinensis (tea plant) [i.e., (-)-epicatechin gallate and (-)-epigallocatechin gallate] were found to differentially inhibit the activities of reverse transcriptase and cellular DNA and RNA polymerases. Under the assay conditions optimized for each enzyme species, the strongest inhibition by these compounds was observed with reverse transcriptase. The concentrations of (-)-epicatechin gallate and (-)-epigallocatechin gallate required for 50% inhibition of the activity of human immunodeficiency virus (HIV) reverse transcriptase were in the range of 0.01-0.02 microgram/mL. On the other hand, neither (-)-epicatechin, (-)-epigallocatechin, nor gallic acid, the constituents of (-)-epicatechin gallate and (-)-epigallocatechin gallate, was inhibitory to the activity of HIV reverse transcriptase at concentrations up to 1 microgram/mL. The mode of inhibition of reverse transcriptase and other DNA polymerases by these compounds was competitive with respect to the template-primer, whereas the mode of inhibition of RNA polymerase was competitive with respect to the nucleotide substrate. The Ki values of HIV reverse transcriptase for (-)-epicatechin gallate and (-)-epigallocatechin gallate were determined to be 7.2 and 2.8 nM, respectively, which are smaller by 1-2 orders of magnitude than the Ki's of other DNA and RNA polymerases for these compounds.
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PMID:Differential inhibitory effects of some catechin derivatives on the activities of human immunodeficiency virus reverse transcriptase and cellular deoxyribonucleic and ribonucleic acid polymerases. 169 87

Preparations of recombinant reverse transcriptase RSV were isolated from E. coli HB101/pMF14 cell cultures. The enzyme purified to homogeneity was shown to be made up of two subunits with molecular masses of 97 +/- 4 and 61 +/- 3 kDa. A comparison of enzymatic properties of recombinant transcriptase to those of the enzyme isolated from the RSV (Rauss sarcoma) virus demonstrated that in the preparations under study the recombinant reverse transcriptase exists in a subunit form, alpha beta, and may acquire a relatively stable configuration, alpha 2.
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PMID:[Recombinant RNA-dependent DNA-polymerase from Rous sarcoma virus. Isolation and properties]. 169 35

A culture of rhesus monkey peripheral blood lymphocytes was divided into two parts; one was kept as an uninfected control, and the other was infected with a strain of simian immunodeficiency virus (SIVmac251) originally isolated from a rhesus monkey that died of a malignant lymphoma associated with acquired immune deficiency syndrome. Both cultures were sampled at successive intervals from 1 to 40 days postinfection. Each sample was subjected to in situ hybridization for detection of viral mRNA, immunocytochemical detection of viral core protein (p27), reverse transcriptase assay, electron microscopy, and immunophenotypic characterization of infected cells. These techniques were used to define viral growth kinetics of this novel lentivirus in peripheral blood lymphocytes. The first evidence of SIVmac251 replication was obtained by an in situ hybridization signal for viral mRNA at 2 days postinoculation. This was followed by detection of viral p27 core protein by immunocytochemistry on day 4. Reverse transcriptase activity above control values was not detected until day 8. Budding particles were not found in the infected cultures until 14 days postinfection. Results of in situ hybridization, immunocytochemistry, and reverse transcriptase assay indicated that two bursts of viral replication occurred during the course of this study. The first, at 3 weeks postinfection, was due to infection and subsequent depletion of CD4+ lymphocytes, while the second, 3 weeks later, resulted from a cycle of replication in CD8+ lymphocytes and the remaining CD4+ cells, culminating in the death of all cells on day 39 postinoculation.
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PMID:Study of long-term cultures of simian immunodeficiency virus (SIVmac 251)-infected peripheral blood lymphocytes. 169 33

The structural and enzymatic components of retroviral cores are formed by proteolytic cleavage of precursor polypeptides, mediated by the viral protease (PR). We constructed an active-site mutation, D37I, in the PR of avian leukosis virus. The D37I mutation was introduced into an infectious DNA clone, and quail cell lines expressing the mutant virus were established. These cell lines produce normal amounts of virus particles, the major internal protein components of which are the uncleaved gag and gag-pol precursors. As in other retroviral systems, the protease-defective virions are noninfectious and retain the "immature" type A morphology as determined by thin-section transmission electron microscopy. The virion cores are stable at nonionic detergent concentrations that completely disrupt wild-type cores. Digestion of mutant virions with exogenous PR in the presence of detergent leads to complete and correct cleavage of the gag precursor but incomplete cleavage of the gag-pol precursor. The protease-defective virions encapsidate normal amounts of genomic RNA and tRNA(Trp) that is properly annealed to the primer-binding site, but some of the genomic RNA remains monomeric. Results from UV cross-linking experiments show that the gag polyprotein of mutant virions interacts with viral RNA and that this interaction occurs through the nucleocapsid (NC) domain. However, within mutant virions the interaction of the NC domain with RNA differs from that of mature NC with RNA in wild-type virions. Reverse transcriptase (RT) activity associated with mutant virions is diminished but still detectable. Digestion of the virions with PR leads to a fivefold increase in activity, but this PR-mediated activation of RT is incomplete. Since in vitro cleavage of the gag-pol precursor is also incomplete, we hypothesize that amino acid sequences N terminal to the reverse transcriptase domain inhibit RT activity.
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PMID:Properties of avian retrovirus particles defective in viral protease. 169 12


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