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)

In an earlier study on minus-strand DNA synthesis catalyzed by murine leukemia virus reverse transcriptase, we described a prominent pause site near the polypurine tract (J. Guo, W. Wu, Z. Y. Yuan, K. Post, R. J. Crouch, and J. G . Levin, Biochemistry 34:5018-5029, 1995). We now report that pausing at this site is due to a stem-loop structure in the RNA template, formed by interaction of a number of bases in the polypurine tract, including the six G's, and a 3' sequence which includes four C's. Addition of human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein to reverse transcriptase reactions reduces pausing by approximately 8- to 10-fold and stimulates synthesis of full-length DNA. Thus, NC functions as an accessory protein during elongation of minus-strand DNA and increases the efficiency of DNA synthesis, in this case, by apparently destabilizing a region of secondary structure in the template. Since NC is associated with genomic RNA in the viral core and is likely to be part of a viral replication complex, these results suggest that NC may also promote efficient DNA synthesis during virus replication. Mutational analysis indicates that the features of HIV-1 NC which are important for reduction of pausing include the basic amino acids flanking the first zinc finger, the zinc fingers, and the cysteine and aromatic amino acids within the fingers. These findings suggest that reverse transcription might be targeted by drugs which inactivate the zinc fingers of HIV-1 NC.
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PMID:Human immunodeficiency virus type 1 nucleocapsid protein reduces reverse transcriptase pausing at a secondary structure near the murine leukemia virus polypurine tract. 879 60

From 1977 to 1993, 15,189 throat swab samples were received for isolation and identification of influenza virus in the Clinical Virology Laboratory, Veterans General Hospital-Taipei. Most of the samples came from the Pediatric Department. There were 634 identified strains of the influenza virus; the successful isolation rate was 4.17% in average/year. Among these isolates, 56.3% (357/634) were influenza B; 12.1% (77/634) were influenza A/H1N1 and 28.1% (178/634) were influenza A/H3N2. About 3.5% (22/634) were classified as flu-like agents because of no reaction with available monoclonal antibodies. In recent years, reverse transcriptase polymerase chain reaction (RT-PCR) was established here to re-evaluate these virus stocks. This method can provide rapid diagnosis method to identify influenza A/H1N1, A/H3N2 and B. Further, the RT-PCR method and sequencing of amplified DNA could be used to see the variation of virus isolates which were recirculated or which reappeared in the Taipei area.
Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi 1995 May
PMID:Isolation and identification of influenza viruses from clinical materials in 1977-1993 at Veterans General Hospital-Taipei. 977 90

HIV-1 replication is inhibited by the incorporation of chain-terminating nucleotides at the 3' end of the growing DNA chain. Here we show a nucleotide-dependent reaction catalyzed by HIV-1 reverse transcriptase that can efficiently remove the chain-terminating residue, yielding an extendible primer terminus. Radioactively labeled 3'-terminal residue from the primer can be transferred into a product that is resistant to calf intestinal alkaline phosphatase and sensitive to cleavage by snake venom phosphodiesterase. The products formed from different nucleotide substrates have unique electrophoretic migrations and have been identified as dinucleoside tri- or tetraphosphates. The reaction is inhibited by dNTPs that are complementary to the next position on the template (Ki approximately 5 microM), suggesting competition between dinucleoside polyphosphate synthesis and DNA polymerization. Dinucleoside polyphosphate synthesis was inhibited by an HIV-1 specific non-nucleoside inhibitor and was absent in mutant HIV-1 reverse transcriptase deficient in polymerase activity, indicating that this activity requires a functional polymerase active site. We suggest that dinucleoside polyphosphate synthesis occurs by transfer of the 3' nucleotide from the primer to the pyrophosphate moiety in the nucleoside di- or triphosphate substrate through a mechanism analogous to pyrophosphorolysis. Unlike pyrophosphorolysis, however, the reaction is nucleotide-dependent, is resistant to pyrophosphatase, and produces dinucleoside polyphosphates. Because it occurs at physiological concentrations of ribonucleoside triphosphates, this reaction may determine the in vivo activity of many nucleoside antiretroviral drugs.
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PMID:Unblocking of chain-terminated primer by HIV-1 reverse transcriptase through a nucleotide-dependent mechanism. 981 24

Stereochemical control of DNA biosynthesis was studied using several DNA-synthesizing complexes containing, in each case, a single substitution of a 2'-deoxy-D-nucleotide residue by an enantiomeric L-nucleotide residue in a DNA chain (either in the primer or in the template) as well as 2'-deoxy-L-ribonucleoside 5'-triphosphates (L-dNTPs) as substrates. Three template-dependent DNA polymerases were tested, Escherichia coli DNA polymerase I Klenow fragment, Thermus aquaticus DNA polymerase and avian myeloblastosis virus reverse transcriptase, as well as template-independent calf-thymus terminal deoxynucleotidyl transferase. Very stringent control of stereoselectivity was demonstrated for template-dependent DNA polymerases, whereas terminal deoxynucleotidyl transferase was less selective. DNA polymerase I and reverse transcriptase catalyzed formation of dinucleoside 5',5'-tetraphosphates when L-dTTP was used as substrate. Comparison between models of template-primer complexes, modified or not by a single L-nucleotide residue, revealed striking differences in their geometry.
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PMID:Stereochemical control of DNA biosynthesis. 1066 59

Molecular dynamics simulations of a ternary complex of HIV-1 reverse transcriptase (RT), double-stranded DNA, and bound dideoxynucleoside-5'-triphosphate (RT-DNA-ddNTP), utilizing the ddNTPs ddATP, betaFddATP, and alphaFddATP, explain the experimentally observed order of potency of these 5'-triphosphates as inhibitors of RT: ddATP > betaFddATP > alphaFddATP. On the basis of RT's known preference to bind the incoming dNTP (or ddNTP) with a north conformation at the polymerase site, alphaFddATP, which in solution prefers almost exclusively a north conformation, was predicted to be the most potent inhibitor. However, Tyr115, which appears to function as a steric gate to preclude the binding of ribonucleoside 5'-triphosphates, prevents the effective binding of alphaFddATP in its preferred north conformation. The south-biased betaFddATP, while able to bind to RT without hindrance by Tyr115, has to pay a high energy penalty to be flipped to the active north conformation at the polymerase site. Finally, the more flexible and less conformationally biased ddATP is able to switch to a north conformation at the RT site with a smaller energy penalty than betaFddATP. These results highlight the opposite conformational preferences of HIV-1 RT for alphaFddATP and betaFddATP and help establish conformational guidelines for optimal binding at the polymerase site of this enzyme.
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PMID:Interactions of conformationally biased north and south 2'-fluoro-2', 3'-dideoxynucleoside 5'-triphosphates with the active site of HIV-1 reverse transcriptase. 1098 66

Although peptide transport across the plasma membrane has been characterized well in the kidney and the intestine, the functional relevance of this transport in other organs has not been addressed. Here we report the cloning of a cDNA for a novel peptide/histidine transporter found in the rat (rPHT2), whose mRNA is expressed mainly in the lymphatic system. rPHT2 encodes a protein of 582 amino acids and showed 49% identity with the brain PHT (PHT1) [Yamashita, Shimada, Guo, Sato, Kohmura, Hayakawa, Takagi and Tohyama (1997) J. Biol. Chem. 272, 10205-10211]. rPHT2 mRNA was abundant in lung, spleen and thymus, and detected faintly in brain, liver, adrenal gland and heart by Northern-blot analysis and reverse transcriptase PCR (RT-PCR). Intense signals for the gene were found in immunocytes using in situ hybridization. Ectopic expression of rPHT2 protein in HEK-293T cells and BHK cells was not found on the cell surface, but was found on the lysosomal membrane using light- and electron-microscopic analysis. Recombinant rPHT2 protein reconstituted into liposomes showed proton-dependent transport activity with histidine and histidyl-leucine. These findings suggest that rPHT2 is involved in the protein catabolic pathway in the lymphatic system.
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PMID:Cloning of a lymphatic peptide/histidine transporter. 1133 35

Streptomyces coelicolor is the prototype for the investigation of antibiotic-producing and differentiating actinomycetes. As soil bacteria, streptomycetes can metabolize a wide variety of carbon sources and are hence vested with various specific permeases. Their activity and regulation substantially determine the nutritional state of the cell and, therefore, influence morphogenesis and antibiotic production. We have surveyed the genome of S. coelicolor A3(2) to provide a thorough description of the carbohydrate uptake systems. Among 81 ATP-binding cassette (ABC) permeases that are present in the genome, we found 45 to encode a putative solute binding protein, an essential feature for carbohydrate permease function. Similarity analysis allowed the prediction of putative ABC systems for transport of cellobiose and cellotriose, alpha-glucosides, lactose, maltose, maltodextrins, ribose, sugar alcohols, xylose, and beta-xylosides. A novel putative bifunctional protein composed of a substrate binding and a membrane-spanning moiety is likely to account for ribose or ribonucleoside uptake. Glucose may be incorporated by a proton-driven symporter of the major facilitator superfamily while a putative sodium-dependent permease of the solute-sodium symporter family may mediate uptake of galactose and a facilitator protein of the major intrinsic protein family may internalize glycerol. Of the predicted gene clusters, reverse transcriptase PCRs showed active gene expression in 8 of 11 systems. Together with the previously surveyed permeases of the phosphotransferase system that accounts for the uptake of fructose and N-acetylglucosamine, the genome of S. coelicolor encodes at least 53 potential carbohydrate uptake systems.
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PMID:In silico and transcriptional analysis of carbohydrate uptake systems of Streptomyces coelicolor A3(2). 1497 30

Oxidation of RNA precursors may disturb genetic information by mispair formations. In this study, effects of oxidized ribonucleoside triphosphates on in vitro transcription catalyzed by T7 RNA polymerase were examined. 8-Hydroxyguanosine 5'-triphosphate (8-OH-GTP) and 2-hydroxyadenosine 5'-triphosphate (2-OH-ATP) reduced amount of RNA. In addition, mRNA was converted to cDNA by reverse transcriptase, and the cDNA was then amplified by PCR. The PCR product was subsequently cloned into plasmid DNA and sequence of DNA was analyzed for each bacterial colony. The two oxidized ribonucleotides induced mutations in cDNA, suggesting disturbance of genetic information during transcription and/or reverse transcription. 8-OH-GTP induced T-->G plus T-->C mutations and 2-OH-ATP caused T-->C mutations. These results indicate that formation of these oxidized RNA precursors in cells affects transcription quantitatively and qualitatively. In addition, they are potential antiviral drugs that cause mutations in genomic RNA.
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PMID:Mutagenic properties of oxidized GTP and ATP in in vitro transcription-reverse transcription. 1715 Aug 36

Using cell lines and primary cells, it has been shown that translation control plays a key role regulating gene expression during physiological and pathological conditions. The relevance of this type of regulation in vivo (tissues, organs) remains to be elucidated, due to the lack of an efficient method for polysome-bound fractionation of solid tissue RNA samples. A simple and efficient method is described, in which tissue samples were pulverized in liquid nitrogen and lysed with NP40-lysis buffer in the presence of the RNAse inhibitors RNAsin and vanadyl-ribonucleoside complex. After cell lysis, the cytoplasmic extract was loaded into sucrose gradients, fractionated, and RNA prepared from each fraction. The obtained RNA was reverse transcribed with a low efficiency, a problem that was overcome by purifying polyA+ RNA. Aiming to use small quantities of solid tissue samples (10-20 mg/sample), polyA+ RNA purification was discarded, and the different components were individually screened for a negative effect on reverse transcription. The polysaccharide heparin, which is present as a nonspecific RNAse inhibitor, inhibits reverse transcriptase activity, and must be removed from RNA samples for an efficient reaction. Heparin was successfully removed by precipitation of the RNA with lithium chloride, as demonstrated by the reversal of the inhibition on RT-PCR reactions. In summary, we present a reliable method allowing us to prepare high-quality polysome-bound mRNA from small quantities of liquid-nitrogen-frozen solid tissue samples from both human and mouse origin, amenable for Northern blotting, RT-PCR reactions, and expression profiling analyses.
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PMID:Isolation of polysome-bound mRNA from solid tissues amenable for RT-PCR and profiling experiments. 1723 55

Oxidation of RNA precursors may disturb genetic information. In this study, the effects of oxidized RNA precursors on in vitro transcription were examined. Two oxidized ribonucleoside triphosphates, 8-hydroxyguanosine 5'-triphosphate (8-OH-GTP) and 2-hydroxyadenosine 5'-triphosphate (2-OH-ATP), were added to in vitro transcription reactions. The addition of 8-OH-GTP and 2-OH-ATP reduced the amount of RNA synthesized in vitro. Moreover, to examine qualitative alteration of the mRNA, it was converted to cDNA by reverse transcriptase, and the cDNA was then amplified by PCR. The PCR product was subsequently cloned into plasmid DNA, and the DNA sequence was analyzed for each bacterial colony. The two oxidized ribonucleotides induced mutations in cDNA, suggesting the disturbance of genetic information during transcription and/or reverse transcription. 8-OH-GTP induced T-->G plus T-->C mutations, and 2-OH-ATP caused T-->C mutations. These results indicate that the formation of these oxidized RNA precursors in cells affects transcription quantitatively and qualitatively.
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PMID:Effects of 8-hydroxy-GTP and 2-hydroxy-ATP on in vitro transcription. 1766 47


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