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)

The 2'-azido analogs of poly(U) and poly(C), poly(dUz) [poly(2'-azido-2'-deoxyuridylic acid)], and poly-(dCz [poly(2'-azido-2'-deoxycytidylic acid)], were found to inhibit the RNA-directed DNA polymerase (reverse transcriptase) activity of murine leukemia (Moloney, Rauscher) and sarcoma (Moloney) virus, and feline leukemia (Theilen) and sarcoma (Gardner) virus, while under the same conditions the unsubstituted parent compounds failed to do so. In addition, poly(dUz) and poly(dCz) inhibited the replication of exogenous murine sarcoma virus (Moloney) in nontransformed cells (as assessed by an infectious center assay), but poly(dUz) failed to suppress the formation of endogenous sarcoma and leukemia viruses in transformed cell lines (MO-P, JLSV5). In these same cells, poly(dUz) failed to inhibit the multiplication of vesicular stomatitis virus. These data add further strength to the contention that reverse transcriptase is necessary for the productive infection and transformation of normal cells by oncornaviruses but is not essential maintenance of this transformed state and the continuous production of new viruses particles by these transformed cells.
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PMID:Inhibition of oncornavirus functions by 2'-azido polynucleotides. 4 74

The size of the DNA product synthesized by RNA-directed DNA polymerase (isolated from avian myeloblastosis virus) was found to be important for complementary DNA (cDNA)-mRNA hybridization reactions. Incomplete cDNA to rabbit reticulocyte globin mRNA formed poor hybrids and presumably lacked sequences needed for hybridization. The size of the cDNA synthesized was influenced by the reaction conditions used. The complementary DNA product contained 10 S material when synthesis was done at high deoxynucleoside triphosphate concentrations (greater than 50 muM) while the product was smaller than the template when synthesis was at lower concentrations. The concentration and size (oligo(dT)6 to (dT)10) of primer had little or no effect on the product size. Increasing the concentration of 10 S globin mRNA caused the cDNA product to contain more small material. The cDNA synthesized at high deoxynucleoside triphosphate concentrations was fractionated into heavy, medium, and light fractions by alkaline sucrose density centrifugation. All hybridized to globin mRNA. The larger cDNAs had a higher TM when hybridized to globin mRNA, a lower dTMP/dCMP ratio (indicating that the poly(dT) region constituted a smaller fraction of the molecule), and gave increased protection of 125I-labeled mRNA from nuclease digestion. The full size cDNA was especially useful for studying the RNA transcribed from chromatin by RNA polymerase. The complement of the 5' end of the mRNA is contained only in full size cDNA; the 5' end is the part of the mRNA first transcribed by the RNA polymerase assuming correct transcription. Thus, full size cDNA can hybridize more effectively to the short RNA transcripts that are obtained than partial cDNA. RNA transcribed from rabbit bone marrow chromatin by Escherichia coli RNA polymerase hybridized twice as efficiently to complete cDNA as it did to partial cDNA demonstrating the usefulness of full size cDNA.
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PMID:Importance of full size complementary DNA in nucleic acid hybridization. 5 64

Two approaches have been explored for the synthesis of double-stranded DNA from single-stranded DNA template complementary to rabbit 9S globin mRNA (cDNA). (i) cDNA was elongated with dCMP or dTMP homopolymeric tracts using terminal deoxynucleotidyltransferase (EC 2.7.7.31; nucleosidetriphosphate:DNA deoxynucleotidylexotransferase). cDNA-dC, in the presence of an oligo(dG)10 primer, was an efficient template with either DNA polymerase of Escherichia coli (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) or RNA-directed DNA polymerase of avian myeloblastosis virus. cDNA-dT [ with an oligo(dA)10 primer] functioned as template only with E. coli polymerase. (ii) cDNA, without homopolymeric tails, was also efficiently copied in the absence of oligonucleotide primer, by DNA polymerase of avian myeloblastosis virus or of E. coli. The product of the reaction consisted of long hairpin molecules which could be converted into DNA duplex (melting temperature, 93 degrees) by digestion with single-strand nuclease S1. The data indicate that a loop structure on the 3' end of cDNA allowed DNA synthesis to take place by a "self-priming" mechanism. Some of the double-stranded DNA synthesized corresponded to the entire sequence of the 9S mRNA template. The synthesis of full-length double-stranded DNA from mouse globin mRNA and immunoglobulin light chain mRNA is also discussed.
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PMID:Stepwise biosynthesis in vitro of globin genes from globin mRNA by DNA polymerase of avian myeloblastosis virus. 6 60

Details are presented of the in vitro synthesis of double-stranded DNA complementary to purified Xenopus globin messenger RNA, using a combination of reverse transcriptase, fragment 'A' of E. coli DNA polymerase 1 and S1 endonuclease. After selection of duplex DNA molecules approaching the length of Xenopus globin messenger RNA by sedimentation of the DNA through neutral sucrose gradients, the 3'-OH termini of the synthetic globin gene sequences were extended with short tracts of oligo dGMP using terminal transferase. This material was integrated into oligo dCMP-extended linear pCR1 plasmid DNA and amplified by transfection of E. coli. Plasmids carrying globin sequences were identified by hybridization of 32P-labelled globin mRNA to total cellular DNA in situ, by hybridization of purified plasmids to globin cDNA in solution, by analysis of recombinant DNA on polyacrylamide and agarose gels, and by heteroduplex mapping. The results show that extensive DNA copies of Xenopus globin mRNA have been integrated into recombinant plasmids.
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PMID:Recombinant plasmids containing Xenopus laevis globin structural genes derived from complementary DNA. 34 4

Deoxynucleoside analogs, AZT and/or ddN, are the therapeutic agents currently utilized to inhibit the human immunodeficiency virus (HIV) reverse transcriptase. The effects of their anabolic products, AZT-triphosphate (AZT-TP) and ddCTP on human cellular DNA metabolic processes were studied using highly purified, structurally and enzymatically defined forms of the two major human host DNA polymerases, alpha and beta, and compared to those of the reverse transcriptase purified from HIV viron. Human DNA polymerase alpha during processive DNA synthesis is able to incorporate AZT-monophosphate (AZT-MP) but not ddCMP into DNA, causing chain termination. During its initial encounter with a primer terminus, polymerase alpha is able to incorporate both AZT-MP and ddCMP into DNA chains. Polymerase beta is able to incorporate AZT-MP and ddCMP into DNA, causing chain termination in both modes of DNA synthesis. Steady state kinetic analyses demonstrate that polymerase alpha inserts one AZT-MP molecule into DNA for every 2500 dTMP molecules incorporated. Polymerase beta incorporates ddCMP with efficiency nearly equal to that of dCMP. HIV reverse transcriptase prefers to incorporate AZT-MP and ddCMP rather than dTMP and dCMP, respectively. The findings described here raise the concern that the capability of the two major host DNA polymerases to incorporate AZT-MP or ddCMP into DNA might cause adverse side effects on human DNA metabolism and mutation in the genomes of patients under long term continuous treatment with AZT and ddC.
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PMID:Human DNA polymerases alpha and beta are able to incorporate anti-HIV deoxynucleotides into DNA. 140 Apr 58

The in vitro fidelity of highly purified recombinant reverse transcriptase from simian immunodeficiency virus of African green monkeys (SIVagm) was determined. By using the phi X174am16 reversion assay an overall error rate of 1/19,000 was determined. This is 2.4-fold higher than the overall accuracy of purified recombinant HIV-1 reverse transcriptase, measured in parallel. The evaluation of error frequencies from nucleotide pool bias studies suggest an even higher accuracy for the SIVagm-derived reverse transcriptase. T:dGMP mismatches were formed most frequently with an error rate of 1/155,000, followed by G:dGMP (1/230,000), A:dGMP (1/315,000), G:dAMP (1/340,000), T:dCMP (1/540,000), T:dTMP (1/790,000), and A:dCMP (1/1,050,000) mispairs. Thus, according to pool bias effects and depending on the mismatch under consideration SIVagm reverse transcriptase appears to be 2 to 20-fold more accurate than the homologous enzyme from the human immunodeficiency virus type 1. This higher accuracy is not due to a co-purifying exonuclaease activity. Like the enzyme from HIV-1, the simian monkey-derived enzyme was found to be devoid of a proofreading 3' to 5' exonuclease.
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PMID:Fidelity of reverse transcriptase of the simian immunodeficiency virus from African green monkey. 170 65

The fidelity of DNA synthesis by reverse transcriptases from human immunodeficiency virus and other retroviruses was compared by measuring the rates of misincorporation of dCMP in the place of TMP in cell-free DNA synthesis with polyadenylic acid as the template. The fidelity of human immunodeficiency virus reverse transcriptase was found to be about one-third of that of the reverse transcriptases of other retroviruses.
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PMID:Low fidelity of cell-free DNA synthesis by reverse transcriptase of human immunodeficiency virus. 245 89

Reverse transcriptase from the human immunodeficiency virus type I (HIV-1) was expressed in E. coli and purified to near homogeneity. The enzyme was shown to contain reverse transcriptase, DNA polymerase and ribonuclease H activities. The DNA polymerase activity converted singly-primed phi X174 (+) DNA into the double-stranded form. Two third of the replication product is ligatable to covalently closed circular DNA (RFIV-form DNA) indicating that DNA synthesis by HIV reverse transcriptase can proceed until the enzyme matches the 5'-end of a pre-existing primer molecule. The in vitro accuracy of HIV reverse transcriptase was measured with the phi X174am16 reversion assay to be 1/7,400. Reversion rates for the individual mispairs were determined from pool bias studies to be 1/8,000 for the dGMP:T template mismatch, 1/35,000 for the dGMP:A template mismatch, 1/45,000 for the dAMP:G template mismatch, 1/73,000 for the dCMP:T template mispair, 1/140,000 for the dCMP:A template mispair, and 1/180,000 for the dGMP:G template mismatch. The dTMP:T template mispair was below the detection limit of the assay indicating a reversion rate of less than 1/300,000 for this particular mispair.
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PMID:Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural DNA. 246 38

The 2',3'-dideoxynucleoside triphosphates (ddNTPs) are potent substrate analog inhibitors of human immunodeficiency virus (HIV) reverse transcriptase and have clinical utility in the treatment of acquired immunodeficiency syndrome. Several issues regarding the interaction of these compounds with HIV reverse transcriptase were examined. The potency of unsubstituted ddNTPs and the 3'-azido analog of dTTP (AZTTP) was influenced by the choice of template. Both compounds were more potent with the complementary homopolymer templates than with gapped duplex DNA, although the Km for the competing dNTP was similar with different templates. The Ki for AZTTP was greater than for the unsubstituted ddNTPs with either a homopolymer or a gapped duplex DNA template. HIV reverse transcriptase incorporated ddCMP and AZTMP into primed phage m13 DNA at sites specified for insertion of dCMP and dTMP, respectively. ddCTP was more efficiently utilized as a substrate than was AZTTP. Primer elongation due to base misincorporation was observed in the absence of one dNTP. The combined effect of ddNTPs and the pyrophosphate analog phosphonoformate (PFA) on HIV reverse transcriptase was also examined, and inhibition by PFA in combination with ddTTP or AZTTP was mutually exclusive.
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PMID:Inhibition of human immunodeficiency virus reverse transcriptase by 2',3'-dideoxynucleoside triphosphates: template dependence, and combination with phosphonoformate. 247 97

Poly(2'-fluoro-2'-deoxyadenylic acid) (poly(dAfl)) and poly(2'-deoxycytidylic acid) (poly(dCfl)) were tested as templates in DNA synthesis reactions catalyzed by Xenopus laevis oocytes DNA polymerase alpha, mouse cell DNA polymerase gamma and avian myeloblastis virus (AMV) reverse transcriptase. Poly(dAfl).(dT)12 can fully substitute for poly(rA).(dT)12 as template with DNA polymerase gamma, to 50% with reverse transcriptase, but was poorly recognized by DNA polymerase alpha. DNA synthesis by reverse transcriptase with poly(dCfl).(dG)12 as template was 50% of that with poly(rC).(dG).(dG)12. The use of 2'-fluoropolymers as templates was more efficient at 37 degrees C than at 25 degrees C. No appreciable differences on the fidelity of DNA synthesis by reverse transcriptase were observed when dCMP misincorporation was measured with poly(dAfl).(dT)12 or poly(rA).(dT)12 as template primers. Poly(C) and poly-2'-O-methylcytidylic acid had no significant effect on the reaction catalyzed by DNA polymerase gamma and reverse transcriptase, independent of the synthetic polynucleotide complex utilized as template. On the other hand, poly(dCfl) was an inhibitor when poly(rA).(dT)12 or poly(dA).(dT)12 were used as templates, but not when poly(dAfl).(dT)12 was employed. Analogous results have been obtained with activated DNA and AMV 70 S RNA as templates in the reverse transcriptase reaction. The inhibition by poly(dCfl) was noncompetitive with regard to TTP, poly(dA) and poly(rA). Xenopus laevis oocytes DNA polymerase alpha was not inhibited by poly(dCfl).
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PMID:2'-Fluoro-2'-deoxypolynucleotides as templates and inhibitors for RNA- and DNA-dependent DNA polymerases. 257 20


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