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)

We report that 2',3'-dideoxyadenosine and 2',3'-dideoxycytidine inhibit retroviral DNA synthesis and mRNA expression in T cells exposed to the virus that causes acquired immunodeficiency syndrome, and afford such cells long-term protection in vitro under conditions of substantial viral excess. Both 2',3'-dideoxyadenosine and 2',3'-dideoxycytidine appear to completely block reverse transcription from viral RNA to viral DNA. Viral mRNA expression is also not detected in cells protected by the drugs throughout 30 days of culture following exposure to the virus. Purine and pyrimidine analogues as 2',3'-dideoxynucleoside-5'-triphosphate serve as substrates for the human T-lymphotropic virus type III/lymphadenopathy-associated virus reverse transcriptase to elongate a DNA chain by one residue, after which the chain is terminated. Cloned normal helper/inducer T cells exposed to a cytopathic dose of the virus, but protected by the drugs, respond normally to specific antigen in vitro. These results suggest that the drugs could be promising agents for further studies in the experimental treatment of patients infected with retroviruses.
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PMID:Long-term inhibition of human T-lymphotropic virus type III/lymphadenopathy-associated virus (human immunodeficiency virus) DNA synthesis and RNA expression in T cells protected by 2',3'-dideoxynucleosides in vitro. 243 23

Mitsuya and Broder [Proc. Natl. Acad. Sci. USA 83:1911-1915 (1986)] demonstrated that every purine (adenosine, guanosine, and inosine) and pyrimidine (cytidine and thymidine) nucleoside containing the 2',3'-dideoxyribose configuration, when evaluated against human immunodeficiency virus (HIV) in vitro, significantly suppressed both the infectivity and the cytopathic effect of the virus, with 2',3'-dideoxycytidine (ddCyd) being the most potent of the series (total antiviral protection at 0.5-1.0 microM). We have compared three factors likely to be of significance in determining the pharmacological activity of these compounds, i.e., (i) their abilities to influence pool sizes of physiological deoxynucleoside-5'-triphosphates, (ii) their capacity to generate the corresponding 2',3'-dideoxynucleoside-5'-triphosphates, and (iii) the effectiveness of these nucleoside-5'-triphosphates as inhibitors of HIV reverse transcriptase. In MOLT-4 cells (a human T cell line), ddCyd was the compound most efficiently converted to its 5'-triphosphate, whereas 2',3'-dideoxyguanosine and 2',3'-dideoxythymidine were the compounds least efficiently converted, generating levels of their corresponding 5'-triphosphates less than 0.1% of that seen with ddCyd when these nucleosides were compared on an equimolar basis (5 microM). The 3'-azido analogue of 2',3'-dideoxythymidine fell intermediate between these two extremes. As inhibitors of HIV reverse transcriptase, however, all the 5'-triphosphates, with the exception of 2',3'-dideoxyinosine-5'-triphosphate, fell within a narrow range of activity (Ki, 0.10-0.26 microM), affinities some 40-60 fold greater than those of the corresponding physiological 2'-deoxynucleoside-5'-triphosphates. Significant alterations in pool sizes of physiological 2'-deoxynucleoside-5'-triphosphates were not observed at pharmacologically effective drug levels. The relative ability of 2',3'-dideoxynucleosides to generate 5'-triphosphates intracellularly thus correlates much more closely than do the other two factors examined, in capacity to block HIV replication. These studies support the conclusion that, for purposes of design of new compounds of this general class, factors influencing efficiency of nucleotide formation and degradation (e.g., membrane transport mechanisms, affinities for nucleoside kinases and for nucleotide kinases and phosphatases) may be of equal or even greater importance than differences in the relative abilities of the resultant 2',3'-dideoxynucleoside-5'-triphosphates to inhibit the viral reverse transcriptase.
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PMID:Factors determining the activity of 2',3'-dideoxynucleosides in suppressing human immunodeficiency virus in vitro. 245 90

The kinetics of forming all possible single base substitution errors are measured for Drosophila melanogaster DNA polymerase alpha and avian myeloblastosis virus reverse transcriptase. Seventeen sites along bacteriophage M13 DNA are investigated so that effects of nearest neighbor base stacking on misinsertion kinetics can be evaluated. Polymerase alpha appears to be more error prone than reverse transcriptase. Polymerase alpha forms transversion mispairs at rates comparable to transition mispairs with two exceptions; A.A and C.C are formed with significantly higher and lower efficiencies, respectively. Reverse transcriptase forms transversions with lower efficiencies than transitions, especially low being A.G, G.G, and C.C. For both enzymes, misinsertion frequencies vary typically by 10-fold for the same mispair in different locations. Misinsertion frequency can be expressed as a product of two components, one based on Km and the other on Vmax. DNA polymerase alpha appears to use primarily Km discrimination (100-5000-fold) to achieve insertion fidelity while reverse transcriptase shows a greater balance between Km and Vmax discrimination. Nearest-neighbor base stacking interactions appear to have opposite effects on the two discrimination components. The 5'-nearest neighbor influence on Km is greater for correct insertions than for incorrect, while the influence on Vmax is greater for the incorrect base. Target sites that have pyrimidine as the 5'-nearest neighbor to incoming nucleotides show a higher than average misinsertion component based on Km, but a lower than average component based on Vmax. Conversely, target sites with nearest neighbor purines have a higher than average Vmax component. These results imply that nucleotide misinsertion "hot spots" will occur next to pyrimidines when Km discrimination is dominant and next to purines when Vmax discrimination is dominant. When Vmax and Km discrimination components have similar magnitudes, nearest neighbor effects tend to cancel thereby reducing the effects of base stacking on insertion error rates.
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PMID:Nearest neighbor influences on DNA polymerase insertion fidelity. 247 45

The gene for rat cholecystokinin (CCK) was isolated from a rat genomic DNA library. The transcription unit spans 7 kilobases and is interrupted by two introns. The initiator methionine codon lies 2 bases into exon 2; therefore, exon 1 is a noncoding exon. The transcription initiation site was determined using avian myeloblastosis reverse transcriptase, a cDNA primer, and mRNA isolated from a rat medullary thyroid carcinoma. A "TATA"-like sequence precedes the transcription initiation site at position -34. The polyadenylation site for the gene was mapped by a nuclease protection assay using a cRNA generated by transcription of the exon 3 region of the CCK gene with SP6 bacteriophage RNA polymerase. The sequence AT-TAAA is found 22 bases 5' to the site determined to be the polyadenylation addition site. Two regions of simple repetitive DNA occur within the CCK lambda clone, one within intron 2 and the other 4 kilobases 3' to the gene. Sequence analysis of the repetitive element 3' distal to the gene revealed two copies of the sequence 5'-(AC)n-3', where n is 22 and 25. A 114-base pair sequence of predominantly repeating purine-pyrimidine nucleotides separates these two d(AC) repeats. Transcriptional control elements were investigated by fusing regions of the CCK gene to the structural gene encoding chloramphenicol acetyltransferase. Promoter activity was determined by transfecting COS-7 cells with plasmids containing the gene fusions, followed by determining chloramphenicol acetyltransferase activity in cellular extracts. The region necessary for expression of the CCK gene fusions in COS-7 cells is within 144 bases 5' to the initiation of transcription.
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PMID:A gene encoding rat cholecystokinin. Isolation, nucleotide sequence, and promoter activity. 298 40

N2,3-Ethenoguanine (N2,3-epsilon G) was recently identified in the liver of vinyl chloride-exposed rats. We have now synthesized the nucleoside and the 5'-diphosphate which was copolymerized with CDP. The deoxypolynucleotide complement, synthesized by AMV reverse transcriptase contained, in addition to dG, dC and dT. The total pyrimidine content was approximately equivalent to the N2,3-epsilon G content of the template. Incorporation of dC is neither lethal nor mutagenic, while dT incorporation represents a mutagenic event, occurring with approximately 20% frequency. N2,3-epsilon G X dT base pairs can have two hydrogen bonds with minimal helical distortion, as is also the case for N2,3-epsilon G X C base pairs. N2,3-epsilon G is the only derivative formed in vivo by the human carcinogen, vinyl chloride, that can be shown to have a high probability of causing transitions which could initiate malignant transformation.
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PMID:The vinyl chloride-derived nucleoside, N2,3-ethenoguanosine, is a highly efficient mutagen in transcription. 358 34

We previously reported that in the endogenous reaction of Rous sarcoma virus disrupted by melittin, plus-strand DNA initiates on a small oligonucleotide primer and that this initiation can be reconstructed in vitro in reactions containing purified minus-strand DNA as template, viral RNA as a source of primer, and reverse transcriptase (Smith et al., J. Virol. 49:200-204, 1984). Further studies on the specificity of initiation in the endogenous reaction have shown the following. (i) The primer was 12 nucleotides in length. Its sequence began with a 5' pyrimidine, followed by 11 purines, ending with rGrA-3'. This sequence was in agreement with the known plus-strand RNA sequence immediately upstream from the initiation site. Thus, the primer began one nucleotide 5' to the so-called polypurine tract that has been found on all retrovirus genomes. (ii) The transition point between RNA primer and DNA product was precisely located. It was before the end of the polypurine tract. Thus the polypurine tract, although essential for virus replication and probably a flag for the priming event, did not define the limits of the RNA primer. After primer removal, the DNA had a 5' phosphate, consistent with generation by the viral RNase H activity. The priming specificity in reconstructed reactions was also examined further, with the following observations. (i) When the source of RNA primer was prehybridized to the template viral DNA, the generation, utilization, and subsequent removal of primer were essentially the same as those observed in the endogenous reaction. In the absence of deliberate prehybridization, some specificity was lost. There were than additional locations for the 5' end of the primer as well as the transition point between RNA primer and DNA. (ii) Purine-rich oligoribonucleotides created by RNase A digestion of viral RNA could prime strong-stop plus DNA, but again with the loss of specificity relative to that in the endogenous reaction. (iii) The 5' end of the minus-strand DNA template was not required for initiation of strong-stop plus DNA. Therefore, the specificity of initiation did not depend upon an intramolecular interaction requiring the two inverted repeat sequences that flank the long terminal repeat.
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PMID:Specificity of initiation of plus-strand DNA by Rous sarcoma virus. 609 61

We have examined the effect of DNA lesions, which in vivo are potentially mutagenic, on in vitro DNA synthesis carried out by a number of purified DNA polymerases using a 0X174 template. Both acetyl aminofluorene (AAF) adducts and UV-induced pyrimidine dimers are blocks to elongation by DNA polymerases. On UV-irradiated DNA templates synthesis terminates one nucleotide before the sites of pyrimidine dimers with all of the enzymes tested: Pol I and Pol III holoenzyme from Escherichia coli, T4 DNA polymerase, avian myeloblastosis virus reverse transcriptase and a mammalian DNA polymerase alpha. With AAF, which reacts at the C-8 position of guanine, differences are observed between the above enzymes, with the latter two inserting a nucleotide opposite the site of the lesion. Substitution of Mn2+ for Mg2+ as the cation in the Pol I reactions causes changes in the termination pattern on both UV-irradiated and AAF-reacted templates. The significance of these results to the process of inducible error-prone repair and the possible bypass of lesions in the DNA is discussed.
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PMID:In vitro replication of mutagen-damaged DNA: sites of termination. 621 48

The complete 5'-terminal nucleotide sequence of the MRNA coding for the bovine common precursor of corticotropin and beta-lipotropin has been determined. The 5'-32P-labelled, 21-nucleotides-long, single-stranded DNA fragment complementary to a portion of the 5'-noncoding region of the mRNA was prepared from a cDNA clone and elongated by reverse transcriptase reaction with the mRNA as template. The DNA transcript formed was sequenced by the procedure of Maxam and Gilbert, and the resultant sequence was cross-checked by two-dimensional electrophoretic analysis of the partial alkaline digest of the 5'-32P-labelled mRNA. The 5'-terminal nucleotide residue was determined by two-dimensional thin-layer chromatography of the complete hydrolysis product of the 5'-32P-labelled mRNA. The nucleotide sequence determined, which partially overlaps the known sequence of the cloned cDNA, reveals the complete 5'-terminal sequence of the mRNA. This, in conjunction with our previous data, defines the complete primary structure of the mRNA. The mRNA is composed of 1098 nucleotides, including an unusually long 5'-noncoding sequence of 128 nucleotides. The presence of a 'cap' structure at the 5' terminus of the mRNA is suggested. The 5'-terminal 48 nucleotide residues of the mRNA are extremely purine-rich, having an A + G content of 83%, whereas all pyrimidine-rich segments are located downstream from there. Because the 5'-noncoding region of the mRNA contains three segments of potential secondary structure which partially overlap, it can exist in a number of alternative base-pairing configurations. However, its interaction with the 3'-terminal segment of 18-S rRNA at the site of maximal complementarity would fix the mRNA configuration in such a way as to bring the possible site of ribosome binding near the initiation codon.
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PMID:5'-terminal nucleotide sequence of the messenger RNA coding for bovine corticotropin/beta-lipotropin precursor. 626 Apr 86

The relative in vitro potency of nine human immunodeficiency virus (HIV) type 1 reverse transcriptase inhibitors was evaluated in a coculture assay which measures the frequencies of infectious primary cells from HIV-positive patients by the limiting dilution technique and measures their apparent reduction under increasing concentrations of drugs. An advantage of this assay is that it utilizes a variety of wild-type viruses not selected by in vitro propagation. Potency ranking placed the (-)-L-enantiomer of 2',3'-dideoxy-5-fluoro-3'-thiacytidine [(-)-FTC], an oxathiolane pyrimidine nucleoside analog (90% effective concentration = 55 nM), before 2',3'-dideoxycytidine (DDC) (74 nM), (-)-2',3'-dideoxy-3'-thiacytidine (3TC) (300 nM), 3'-azido-3'-deoxythymidine (AZT) (530 nM), TIBO R82913 (670 nM), and 2',3'-dideoxyinosine (DDI) (6,400 nM). HIV from AZT-naive patients' lymphocytes was more sensitive to the inhibitory effect of (-)-FTC, 3TC, or DDC than was highly AZT-resistant HIV obtained from AZT-treated patients' cells, indicating partial cross-resistance between thymidine and cytidine analogs. Combined inhibitory concentrations of AZT with (-)-FTC, 3TC, DDC, and DDI produced synergistic interactions as determined by the multiple-drug effect analysis. Synergistic interactions were demonstrable with AZT plus (-)-FTC or with AZT plus DDC with cells bearing AZT-resistant HIV. The inhibitory concentrations of AZT established by this cell-to-cell virus transmission assay are closer than those determined by the conventional assay system to the extracellular AZT concentrations required in patients' plasma to achieve comparable levels of HIV inhibition in vivo.
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PMID:Infectious amplification of wild-type human immunodeficiency virus from patients' lymphocytes and modulation by reverse transcriptase inhibitors in vitro. 750 8

The self-sustained sequence replication (3SR) reaction is an extremely efficient method for amplifying target DNA and RNA sequences that may be present in minute quantities. A serious problem often encountered in its practice is carryover contamination from products of previous 3SR reactions. A postamplification treatment of 3SR reaction products with the photoactive agent 4'-aminomethyl-4,5-dimethylisopsoralen (IP-10) was investigated as an approach for preventing carryover contamination by 3SR amplicons. Initially, inhibition of the amplification reaction by high concentrations of the reagent was observed. This problem was circumvented by developing a gel-based delivery of IP-10, and the method was found to provide highly efficient sterilization (approximately 10(6)-fold) of 3SR amplicons. Evaluation of this strategy on a number of 3SR targets has indicated that the degree of sterilization is dependent on the length of the amplified region and on the concentration of IP-10. It appears that the sterilization effect is caused by covalent modification of the pyrimidine bases of RNA and DNA, which renders them unusable as templates for the 3SR reaction. Modification of a purified RNA transcript with IP-10 was shown to prevent effectively reverse transcription by avian myeloblastosis virus reverse transcriptase (AMV RT). Similarly, treatment of a T7 RNA polymerase promoter-containing DNA template with IP-10 eliminated full-length transcription by T7 RNA polymerase. This isopsoralen method may be used to sterilize multiple 3SR reactions in a clinical assay with a convenient UV irradiation step.
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PMID:Photochemical sterilization of 3SR reactions. 750 79


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