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)

Terminal deoxynucleotidyl transferase (TdT) was used to prepare copolymers of dA and 1,N6-ethenodeoxyadenosine (epsilon dA). When used as templates for Escherichia coli DNA polymerase I (Pol I) and compared with poly (dA), normal dTTP incorporation was not significantly affected by the presence of 7% epsilon dA. dGTP misincorporation was only slightly increased and occurred about once for every 500 epsilon dA residues. The error-prone polymerase from avian myeloblastosis virus (AMV reverse transcriptase) increased this error rate 5- to 20-fold to a maximum of 1 dG/25 epsilon dA. No dCTP misincorporation was detected with either polymerase. In transcription with E. coli DNA-dependent RNA polymerase, no errors were revealed by nearest neighbor analysis. Poly (dA) treated with chloroacetaldehyde under conditions producing the same proportion of epsilon dA (without the hydrated form) as the synthesized template behaved in the same manner with a similar low level of misincorporation of dG. Such treatment of alternating poly d(A-T) caused structural changes indicative of crosslinks but did not alter its template properties. Increasing the amount of epsilon dA in either synthesized or modified polymers greatly decreased the template activity without increasing the error rate. It is suggested that epsilon dA generally does not prevent dT incorporation but behaves as a bulky lesion which is bypassed. In contrast to the low mutagenic efficiency of epsilon dA, O4-methyldeoxythymidine (m4dT), in copolymers with dA, directed the misincorporation of 1 dG/12 m4dT with Pol I and 1 dG/3 m4dT with reverse transcriptase. Nearest neighbor analysis of transcripts showed the incorporation of 1 dG/12 m4dT. These data are in agreement with the previous reported mutagenicity of m4dT in alternating poly d(A-T, m4T).
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PMID:Assessment of mutagenic efficiency of two carcinogen-modified nucleosides, 1,N6-ethenodeoxyadenosine and O4-methyldeoxythymidine, using polymerases of varying fidelity. 620 83

Poly(A)-RNA enriched for prothrombin was isolated by specific immunoprecipitation of bovine liver polysomes. Prothrombin consisted of about 8% of the cell-free translation products of this RNA. A double-stranded cDNA was synthesized by using reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) and made blunt-ended with nuclease S1. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved previously at the single Pst I site and similarly tailed with dGTP. The resulting plasmids were used to transform Escherichia coli strain RR1 under P3-EK1 conditions. Sixty-three tetracycline-resistant clones were obtained that hybridized to 32P-labeled cDNA synthesized from prothrombin-enriched mRNA. Recombinants containing cDNA to prothrombin mRNA sequences were screened by a solution hybridization assay with a [3H]cDNA synthesized from mRNA. This enriched mRNA was 50% prothrombin mRNA, as determined by a reticulocyte lysate translation assay. Three positive clones were identified by this assay; they contained bovine DNA inserts of 700, 500, and 400 base pairs. The DNA sequence of the 700-base-pair insert was then determined. This recombinant plasmid contained DNA coding for the carboxyl-terminal 160 residues of bovine prothrombin followed by a noncoding region of 119 base pairs and a poly(A) tail of 60 base pairs.
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PMID:Cloning and analysis of a cDNA coding for bovine prothrombin. 625 59

mRNA, isolated from the ligamentum nuchae of fetal sheep by guanidine HCl extraction and oligo(dT) cellulose chromatography, was used to synthesize blunt-ended cDNA molecules by the successive application of AMV reverse transcriptase, DNA polymerase and S1 nuclease. The cDNA was centrifuged on a 15-30% sucrose gradient and molecules greater than 700 bp were tailed with dCTP and cloned into the PstI site of pBR322 which had been tailed with dGTP. Ampicillin-sensitive and tetracycline-resistant colonies were screened by in situ hybridization with elastin-enriched mRNA that had been terminally labeled with 32p. Recombinant plasmids prepared from strongly hybridizing colonies were characterized by restriction mapping and the plasmid with the largest insert (1300 bp) thought to contain elastin sequences was characterized in more detail. The nick-translated cDNA hybridized to a single 3.5 kb mRNA species upon blot hybridization, a size identical to that previously identified for chick elastin mRNA (Burnett et al. (1982) J. Biol. Chem. 259, 1569-1572). Nucleotide sequencing of the 5' end of the cDNA demonstrated a sequence which was extremely GC rich and which corresponded to an amino acid sequence partially homologous to that previously identified in porcine tropoelastin (Foster et al. (1973) J. Biol. Chem. 248, 2876-2879). This is the first report of the identification of a plasmid containing sequences complementary to a translated region of elastin mRNA.
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PMID:Characterization of a sheep elastin cDNA clone containing translated sequences. 632 Aug 24

M13 DNA containing 20-30 apurinic/apyrimidinic (AP) sites per intact circular molecule was prepared by growing phage on an ung- dut- Escherichia coli mutant and treating the DNA with uracil N-glycosylase. AP sites obstruct in vitro DNA synthesis catalyzed by E. coli pol I. The position at which termination of synthesis occurs was determined for four enzymes. T4 DNA polymerase terminates one nucleotide before putative AP sites. DNA pol I, AMV reverse transcriptase, and DNA polymerase alpha terminate synthesis either before or at the site of an AP lesion depending on the particular sequence. We determined the identity of the nucleotide inserted opposite an AP site by synthesizing up to the lesion in a first-stage reaction using T4 DNA polymerase and then determining elongation in a second stage. Purines are inserted opposite AP sites more readily than pyrimidines, and dATP is more efficient than dGTP in promoting such elongation. The DNA-dependent conversion of dNTP to dNMP was determined in mixtures of all four dNTP's by using AP DNA. The production of dAMP from dATP occurs most readily. We conclude that there is an inherent specificity for the incorporation of adenine nucleotides opposite AP sites in this in vitro system. Insofar as the model system reflects in vivo mutational events, our data suggest that depurination should produce transversions and depyrimidination should produce transitions.
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PMID:Insertion of nucleotides opposite apurinic/apyrimidinic sites in deoxyribonucleic acid during in vitro synthesis: uniqueness of adenine nucleotides. 635 60

mRNA was isolated from the thoracic aortas of 16-day chick embryos and used to synthesize blunt-ended heteroduplex molecules consisting of one strand of mRNA and one of cDNA using AMV reverse transcriptase and S1 nuclease. The duplexes were tailed with dCTP and hybridized to the plasmid pBR322 which had been restricted with Pst I and tailed with dGTP. Recombinant plasmids were used to transform E. coli C600 and colonies containing elastin cDNA were selected by in situ hybridization with 32P labeled elastin mRNA and by hybrid selected translation using the nuclease-treated reticulocyte lysate system. mRNA recovered from hybridization to DNA of one clone, pWB1, markedly stimulated incorporation of [3H]valine into a protein which was immunoprecipitable with elastin-specific antibody and had a molecular weight of 72,000, characteristic of tropoelastin. The 230 bp insert of pWB1 was sequenced by the technique of Maxam and Gilbert and found to be derived from a nontranslated region of the 3' end of the mRNA. Nick-translated pWB1 was used to identify and to estimate the relative amounts of elastin mRNA in the developing chick embryo aorta by blot hybridization. A single mRNA species of 3.5 kb hybridized to the pWB1 probe and this species increased greatly in amount between day 7 and day 14. This increase was paralleled by an increase in translatable elastin mRNA and by the rate of elastin synthesis of aortas from various age embryos incubated in vivo. The injection of 150 microgram of hydrocortisone 21-phosphate into 8-day eggs produced a significant increase in both the relative rate of tropoelastin synthesized by the isolated aortas and the relative amount of elastin mRNA. These results suggest that the observed changes in elastin synthesis during development and after hydrocortisone administration are governed by the elastin mRNA content of the aortas.
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PMID:Control of elastin synthesis. 708 85

The finding of G-->A hypermutated retroviral genomes in which up to 40% of guanines may be substituted by adenines was proposed to result from the depletion of the intracellular dCTP concentration and suggested a means to hypermutagenize nucleic acids. Using a RNA/reverse transcriptase ratio of approximately 1:30, comparable to that within the retroviral replication complex, G-->A hypermutants were produced in a simple in vitro reaction using highly biased dNTP concentrations--i.e., a low ratio of [dCTP]/[dTTP]. Up to 38% of G residues could be substituted, the proportion being inversely proportional to the concentration of dCTP. As G-->A hypermutation resulted from elongation beyond multiple rG.dT mismatches, U-->C hypermutants resulting from multiple rU.dG mismatches were sought, and found, during cDNA synthesis using low [dATP] and high [dGTP]. Mixed G-->A and U-->C hypermutants could also be produced under conditions of low [dCTP] plus low [dATP] and high [dTTP] plus high [dGTP]. Hypermutagenesis should allow jumping through, and subsequent exploration of, sequence space to a greater degree than heretofore and, in conjunction with genetic screening, might be of use in the search of proteins or ribozymes with novel or enhanced properties.
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PMID:Hypermutagenesis of RNA using human immunodeficiency virus type 1 reverse transcriptase and biased dNTP concentrations. 752 43

Carbovir (CBV) [the (--)-enantiomer of the carbocyclic analog of 2',3'-dideoxy-2',3'-didehydroguanosine] is a potent inhibitor of human immunodeficiency virus type 1 (HIV) replication in vitro. We have characterized the metabolism of CBV and its effect on cellular metabolism in an effort to better understand its mechanism of action. CBV was primarily metabolized to the 5'-triphosphate of CBV (CBV-TP) to concentrations sufficient to inhibit HIV reverse transcriptase. Infection of CEM cells with HIV did not affect the metabolism of CBV. In CEM cells, there was no evidence of the degradation of CBV by purine nucleoside phosphorylase. The half-life of CBV-TP in CEM cells was 2.5 h, similar to that of the 5'-triphosphate of zidovudine (AZT). However, unlike the levels of the 5'-triphosphate of AZT, CBV-TP levels declined without evidence of a plateau. CBV did not affect the metabolism of AZT, and AZT did not affect the metabolism of CBV. A small amount of CBV was incorporated into DNA in intact CEM cells, and this incorporation was increased by incubation with mycophenolic acid, an inhibitor of IMP dehydrogenase. CBV specifically inhibited the incorporation of nucleic acid precursors into DNA but had no effect on the incorporation of radiolabeled precursors into RNA or protein. CBV did not decrease the level of TTP, dGTP, dCTP, or dATP. These results suggested that the cytotoxicity of CBV was due to the inhibition of DNA synthesis. Further studies are necessary to identify the target(s) responsible for growth inhibition.
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PMID:Metabolism of carbovir, a potent inhibitor of human immunodeficiency virus type 1, and its effects on cellular metabolism. 768 93

A new class of very potent and selective non-nucleoside inhibitors of HIV reverse transcriptase (RT) has recently been identified. The prototype compound trovirdine (LY 300046 HCl) and one analogue, MSC-127, have been studied with respect to inhibition of wild-type HIV-1 RT and RT with various mutations known to give rise to resistance to other non-nucleoside RT inhibitors, namely Leu100-->Ile (Ile100), Glu138-->Arg (Arg138), Tyr181-->Cys (Cys181) and Tyr188-->His (His188). The inhibition of HIV-1 RT by trovirdine and MSC-127 was reversible and template dependent. Trovirdine inhibited HIV-1 RT with an IC50 of 0.007 microM when employing heteropolymeric primer/template (oligo-DNA/ribosomal RNA) and dGTP as substrate. Enzyme kinetic studies showed that inhibition of RT by trovirdine was non-competitive with regard to deoxynucleoside triphosphates and uncompetitive with respect to varied primer/template under steady-state conditions. The amino acid changes Leu100, Tyr181 and Tyr188 gave rise to 25-, 147- and 12-fold decrease in inhibition by trovirdine. Enzyme-kinetic studies on trovirdine have been carried out using various RT mutants and compared to the properties of the earlier reported non-nucleoside RT inhibitors 9-Cl-TIBO, nevirapine and L-697,661.
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PMID:Inhibition of human immunodeficiency virus type 1 wild-type and mutant reverse transcriptases by the phenyl ethyl thiazolyl thiourea derivatives trovirdine and MSC-127. 866 92

In the search for effective antiviral agents, we have found 4-(2, 6-dichlorophenyl)-1, 2, 5-thiadiazol-3-yl-N-methyl, N-ethylcarbamate (RD4-2025) to be a highly potent and selective inhibitor of human immunodeficiency virus type 1 (HIV-1) in vitro. The 50% effective concentration of RD4-2025 for HIV-1-induced cytopathic effect in MT-4 cells was 37 nM, yet no antiviral activity was observed against HIV-2. In HIV-1 reverse transcriptase (RT) assays, RD4-2025 inhibited both RNA-dependent and DNA-dependent DNA polymerase activities of a recombinant HIV-1 RT with 50% inhibitory concentrations of 0.11 and 3.5 microM, respectively. However, the compound did not affect the activity of human DNA polymerase alpha. Kinetic studies revealed that the inhibition was noncompetitive with respect to dGTP as the substrate and poly(C)/(dG) 12-18 as the template/primer. These results were in accordance with those of nonnucleoside RT inhibitors (NNRTIs), such as R89439 (an alpha-anilinophenylacetamide derivative) and nevirapine, indicating that RD4-2025 also belongs to the family of NNRTIs.
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PMID:Inhibitory effect of 4-(2, 6-dichlorophenyl)-1, 2, 5-thiadiazol-3-yl-N-methyl, N-ethylcarbamate on replication of human immunodeficiency virus type 1 and the mechanism of action. 879 26

Nondenaturing gel electrophoresis was used to study the nucleotide substrate-induced conformational change in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). Dead-end complex was formed between HIV-1 RT, dideoxynucleotide chain-terminated primer, and DNA template in the presence of deoxynucleotide triphosphate (dNTP) complementary to the next position on the template. Complexes which form in the absence of the next complementary dNTP were disrupted by adding excess poly(rA)/oligo(dT) or heparin just prior to electrophoresis. Dead-end complex formation by noncomplementary dNTP's or ribonucleotides was at least 2000-fold less efficient than with the complementary nucleotide. When dA was the next nucleotide on the template, analogues of dTTP supported dead-end complex formation with increased apparent Kd (dTTP < dideoxy-TTP approximately alpha-thio-dTTP < dUTP < 3'-azidothymidine triphosphate). A similar relationship was observed for dGTP analogues across from dC on the template (dGTP < dideoxy-GTP < alpha-thio-dGTP << dITP < dideoxy-ITP). The optimal length of the primer/template duplex region for dead-end complex formation was between 20 and 32 base pairs. Primer-template with a mismatched primer terminus did not support dead-end complex formation, and primer terminated with 3'-azidothymidine formed dead-end complex with 25-fold elevated apparent Kd. By contrast, dead-end complex formation on primer terminated with dideoxy-IMP base paired with dC on the template was more efficient than on primer terminated with dideoxy-GMP. Implications for the mechanisms of discrimination between nucleotide analogues by HIV-1 RT are discussed.
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PMID:Nucleotide-induced stable complex formation by HIV-1 reverse transcriptase. 915 15


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