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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Incorporation of 2'-deoxyribonucleotide 5'-triphosphate derivatives, chemically modified both in the base and at 3'-position, into DNA by four different DNA polymerases was investigated. It is shown that 3'-azido- and 3'-amino-2',3'-dideoxy-(E)-5-(2-bromovinyl)-uridine 5'-triphosphates effectively terminate DNA synthesis catalyzed by E. coli DNA polymerase I, rat liver DNA polymerase beta, and AMV reverse transcriptase. Calf thymus DNA polymerase alpha incorporates only the 3'-amino derivative. DNA polymerases I and beta catalyse DNA synthesis in the presence of beta-D-(2'-deoxyribofuranosyl)-1-benzimidazol 5'-triphosphate, inserting the corresponding monophosphate in place of -dGTP, whereas 3'-substituted analogues of this compound were inactive in the reactions.
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PMID:[Analogs of deoxyribonucleoside-5'-triphosphates modified in the base and sugar moieties: substrate properties in DNA biosynthesis in cell-free systems]. 343 59

Ten ribonucleic acid (RNA) tumor viruses grown in five different host cell species and three non-oncogenic viruses from three different virus groups have been examined for ribonuclease H content. Three different substrates were used to assay ribonuclease H: calf thymus [(3)H]RNA-deoxyribonucleic acid (DNA) hybrid prepared with denatured calf thymus DNA and Escherichia coli DNA-directed RNA polymerase, (3)H-polydenylic acid [(3)H-poly(A)] complexed to polydeoxythymidylic acid [poly(dT)], and (3)H-polyuridylic acid [(3)H-poly(U)] complexed to polydeoxyadenylic acid [poly(dA)]. All ten RNA tumor viruses contained ribonuclease H activity which degraded the RNA of both the calf thymus hybrid and poly(A)-poly(dT), whereas only the ribonuclease H in the Moloney strain of murine sarcoma-leukemia virus and in RD-feline leukemia virus hydrolyzed the RNA strand of poly(U)-poly(dA). No appreciable ribonuclease H activity was detected in influenza, Sendai, or vesicular stomatitis virus. The ribonuclease H and RNA-directed DNA polymerase activities in Moloney murine sarcoma-leukemia virus were inseparable by phosphocellulose chromatography or glycerol gradient centrifugation, but appeared to be partially separated by diethylaminoethyl-cellulose chromatography.
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PMID:Ribonuclease H: a ubiquitous activity in virions of ribonucleic acid tumor viruses. 411 67

Assays are described that permit one to distinguish the reverse transcriptase of RNA tumor viruses from known normal cellular DNA-instructed DNA polymerases. Template responses of purified reverse transcriptase were compared with those of similar preparations of the DNA polymerase I of Escherichia coli and of calf-thymus polymerase. All three enzymes responded well to the synthetic duplexes poly(dT).poly(A), poly(U).poly(A), and poly(dT).poly(dA). Hence, these duplexes can detect, but cannot distinguish reverse, transcriptase from the known normal DNA polymerases. However, certain oligomer-homopolymer complexes serve as excellent distinguishing agents. The reverse transcriptase responds very well to (dT)(10).poly(A) and very poorly to (dT)(10).poly(dA), whereas both cellular DNA polymerases do not exhibit this behavior.Purified single-stranded RNA also serves as a diagnostic device, since only reverse transcriptase gives a detectable response. To be definitive, a positive response to RNA must be accompanied by a demonstration via molecular hybridization that the DNA product is complementary to the RNA and not to some minor DNA contaminant.
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PMID:Distinguishing reverse transcriptase of an RNA tumor virus from other known DNA polymerases. 433 48

Polyethylene glycol enhances reverse transcription, augmenting both the rate and duration of polymerization. The effective mean molecular weight of polyethylene glycol is 6000 and the optimal concentration is 12% (w/w). Polyethylene glycol is effective on the reverse transcriptase reaction of all ten type B, C, and D viruses tested under a variety of exogenous, endogenous, and reconstitution assay systems, including the highly efficient conditions involving calf thymus DNA oligonucleotide primers. By three methods of synthesis, polyethylene glycol increased the yields of complementary [3H]DNA by a factor of 1.8--6.5. Polyethylene glycol does not alter the divalent cation requirements of the specificities of the enzyme. Complementary [3H]DNAs made in the presence of polyethylene glycol are indistinguishable in terms of size and sequence complementarity from those made in the absence of the polymer. The stimulatory effect was partly due to the ability of polyethylene glycol to stabilize reverse transcriptase. Preliminary tests indicate that polyethylene glycol also stimulates other nucleotide polymerases, such as the DNA-dependent DNA and RNA polymerases of Escherichia coli and the terminal transferase of calf thymus.
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PMID:Effects of polyethylene glycol on reverse transcriptase and other polymerase activities. 615 36

Mouse mammary tumor virus RNA was transcribed in vitro with avian myeloblastosis virus reverse transcriptase in the presence of calf thymus DNA oligomers. The yield of cDNA was markedly enchanced by increasing the concentration of the enzyme as well as primers in the reaction mixture. The average size of cDNA was approximately 200 residues, and it was not affected by the concentration of deoxynucleoside triphosphates when saturating concentration of enzyme was used. Nearly all of the viral sequences were represented in cDNA and it formed hybrids of high fidelity with viral RNA. Similar results were obtained when murine leukemia virus (AKR) RNA was transcribed. These observations will be useful for synthesizing cDNA of RNAs that are not easily available in sufficient quantities.
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PMID:Synthesis in high yield of complementary DNA of retroviral RNA. 615 40

The virus proteins, reverse transcriptase (RT) and p30, were found to increase with time in the subcellular fractions of lymphocytic tissue from either the thymus or spleen of AKR mice with spontaneous lymphocytic leukaemia. Significant levels of RT activity were first detected in the microsomal fractions of the two tissues at 15 and 20 weeks old, respectively. Although low amounts of p30 could be found in both tissues within the first week of life, the overall increase in the amount of p30 within each tissue followed much the same course as that shown by RT. In addition, a protein complex consisting of p30 and RT was first found in thymus and spleen lymphocytes of 15 and 20 week-old animals, respectively. The complex increased in amount in both organs as the animals aged, reaching a maximum level in 30 week-old mice. Repeated attempts to detect other virus proteins such as gp70 in association with the complex by immunological means were unsuccessful. The complex could not be found in lymphocytic tissue taken from younger animals or in 'non-target' organs, such as liver or kidney, of animals with leukaemia. In animals treated with antiviral IgG, which delayed the development of spontaneous leukaemia, the complex did not appear until much later in life (45 weeks) and then in considerably smaller amounts.
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PMID:Existence of a reverse transcriptase-p30 complex in AKR mice with a high incidence of spontaneous lymphocytic leukaemia. 616 45

N-Ethylmaleimide, a sulfhydryl-specific reagent, strongly inhibits AMV reverse transcriptase by specifically interfering with the template-binding site of the enzyme. However, the kinetics of inhibition differ widely with the composition and structure of the templates employed. The copying of templates with multiple 3'-hydroxyl termini appeared to be more susceptible to N-ethylmaleimide treatment, suggesting that the reagent may interfere with initiation of DNA synthesis. The ability of a template bound to enzyme prior to N-ethylmaleimide treatment to protect against inactivation of copying of other templates also, implies a common binding site for the different templates. Template exchange experiments demonstrated competition between activated calf thymus DNA and rAn . dT12--18 for binding to the enzyme. Thus, templates varying widely in composition and conformation appear to bind at a common site on reverse transcriptase. The experimental data also show suggestive evidence for small but finite differences in the requirements for optimal binding for templates of different structures.
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PMID:Template-binding site of AMV reverse transcriptase and inactivation of the enzyme by N-ethylmaleimide. 616 71

Human oocytes in different stages of maturation were obtained by follicular aspiration from women given Clomovid and Gonadex. Particles similar to type-C virus were observed in three out of 16 oocytes. The particles were irregularly distributed along the oocyte membrane. They were seen both in a state of budding and lying free in the perivitelline space. Reverse transcriptase activity was detected in three out of nine samples of follicular fluid obtained from women other than those donating the oocytes. The supernatants from bat lung cells and dog thymus cells cultivated with oocytes or follicular fluids were tested for reverse transcriptase. An increased activity was observed only transiently in one case. It is assumed that these findings indicate the expression of endogenous retroviruses in human oocytes.
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PMID:Morphological and microbiological signs of endogenous C-virus in human oocytes. 617 30

Model systems to study restricted primate retrovirus expression were established by de novo infection of canine foetal thymus cells (CF-2Th) and superinfection of HEL-12 cells with HEL-12 virus. In the resulting CF-2Th/HEL-12V cells and HEL-12/HEL-12V cells, four sequential stages of virus infection were defined by the production of reverse transcriptase (RT)-containing particles and expression of virus antigens as detected by radioimmunoassays. Stage 1 cells did not synthesize virus antigens or produce RT-containing particles. Stage 2 cells synthesized virus antigen but not RT-containing particles. Stage 3 cells synthesized antigen and produced RT-containing particles, and stage 4 cells synthesized virus antigens but no longer produced RT-containing particles. The duration of the four stage infection is 2 to 3 weeks in both cell types. Monospecific competition radioimmunoassays to detect HEL-12V p30 or gp70 antigen showed high levels of virus antigen throughout stages 2 to 4 of infection. Analysis of immunoprecipitates formed under conditions to detect either p30- or or gp70-containing proteins in cells pulsed and pulsed--chased with [3H]leucine showed the same spectrum of virus precursor polyproteins, intermediates and mature virion components in stage 2 to 4 cells in canine and human infections. Spent culture fluids collected from stage 3 and stage 4 CF-2Th/HEL-12V cells failed to reveal inhibitors of RT activity. Stage 4 CF-2Th/HEL-12V or HEL-12/HEL-12V cells labelled with [3H]uridine produced virions which incorporated [3H]uridine but did not have RT activity, suggesting that restricted infection is characterized by production of HEL-12V defective in RT activity.
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PMID:Restricted HEL-12 virus infections in de novo infected human and canine cells. 617 57

The effects of exogenously added spermine on activated (gapped) DNA-directed and poly(dC) . (dG)12-18-directed DNA synthesis were tested on the chromatographically separated DNA polymerase activities of Trypanosoma brucei brucei. Activated DNA-directed DNA synthesis by the Peak I (eluting from DNA-agarose at 0.15 M KCl) and Peak II (eluting at 0.3 M KCl) polymerase was consistently inhibited or stimulated, respectively, by exogenous spermine. Kinetic analysis revealed that inhibition of the Peak I enzyme with respect to template DNA occurred by a mixed mechanism, while a major factor in the stimulation of the Peak II enzyme by spermine appeared to be the polyamine-mediated reversal of "substrate inhibition' by DNA at concentrations above 10 micrograms/ml. The apparent Km values of Peak I and Peak II DNA polymerase for activated DNA were determined to be 5 and 0.5 microgram/ml, respectively. In contrast to the results observed with activated DNA, activation of Peak II-enzyme-catalyzed poly(dC)-directed DNA synthesis was similar at all template-primer concentrations. Peak I enzyme-catalyzed poly(dG) synthesis was either inhibited or slightly stimulated by spermine, depending upon the presence or absence of heteropolymeric DNA, respectively. Dose-dependent inhibition of DNA-directed DNA synthesis catalyzed by T. b. brucei DNA polymerases, murine thymus DNA polymerase alpha, and Rauscher murine leukemia virus reverse transcriptase by trypanocides was examined to determine a possible mechanism of selective toxicity by such agents. The drugs Antrycide (quinapyramine), pentamidine, imidocarb, Berenil (diminazene aceturate), WR-199-385-[2,5-bis(4-guanylphenyl)furan . 2HCl] and isometamidium inhibited DNA polymerases of the eucaryotic cells at approximately the same degree, and at similar concentrations. The presence of spermine in reaction mixtures did not spare any drug inhibition. Stimulation of reverse transcriptase activity was observed in the presence of Antrycide and imidocarb, however, this could be negated by stimulatory amounts of spermine present in the reaction mixture. The results, obtained using an activated DNA-directed assay system, suggest that trypanosomal DNA polymerases are not the selective target of trypanocidal drugs currently available.
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PMID:Effects of exogenous polyamine and trypanocides on the DNA polymerase activities from Trypanosoma brucei brucei, mouse thymus and murine leukemia virus. 617 72


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