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)

Influenza B/LEE/40, B/Rome/1/67, B/Hong Kong/8/73, and B/Victoria/98926/70 viruses have a similar polypeptide composition as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These viruses are composed of six or seven polypeptides, depending on whether one or two high-molecular-weight polypeptides are resolved, ranging in molecular weights from 27,000 to 90,400. Three of these polypeptides, namely the heavy and light hemagglutinin chains and the neuraminidase, have attached carbohydrate. Highly purified influenza B/LEE/40 and B/Rome/1/67 virus preparations have RNA-dependent RNA polymerase activity equivalent to the incorporation of 100 and 30 pmol, respectively, of (3)H-UMP per mg of virus protein per h at 37 C, which is demonstrated only in detergent-treated virus suspensions. However, no RNA-dependent DNA polymerase enzyme activity was detected in the two viruses although virus suspensions were "activated" by heat, alpha-chymotrypsin, and detergents. Other enzymatic activities were associated with purified preparations of influenza B virus and were attributed to minor contamination of virus with host cell enzymes. Thus, nucleoside and deoxynucleoside phosphohydrolase enzymes were active in the absence of detergents and catalyzed the release of 1,200 and 1,800 nmol of P(i) per mg of virus protein in 30 min at 37 C from ATP and dATP substrates. Thin-layer chromatography indicated that the products of the phosphohydrolase enzymes of influenza B/LEE/40 were mainly nucleoside diphosphate and monophosphate. The latter enzymes were tightly bound to influenza B/LEE/40 virus and could not be removed completely by repeated centrifugation, including centrifugation of the virus to equilibrium in density gradients of 25 to 40% (wt/vol) cesium chloride. A low degree of RNase (approximately 0.01 mug% contamination) and phosphatase (10-30 nmol of P(i) released per mg of virus protein per 30 min) activity was detected in some, but not all, influenza B/LEE/40 virus preparations.
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PMID:Polypeptide composition of Influenza B viruses and enzymes associated with the purified virus particles. 435 55

The presence is reported of an RNA-instructed DNA polymerase in visna virus, the causative agent of a "slow" neurological disease in sheep. The product synthesized by the RNA-directed reaction has been shown to be a DNA heteropolymer by the following criteria: synthesis requires the presence of all four deoxyriboside triphosphates; the product is resistant to ribonuclease and alkali but is degraded by DNase; and the product has a density of 1.420 in Cs(2)SO(4) solution, characteristic of DNA.Visna virions, like those of the oncogenic RNA viruses, contain DNA polymerase activities that respond to a variety of double-stranded DNAs and to synthetic DNA.RNA hybrids.
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PMID:DNA polymerase activities in varions of visna virus, a causative agent of a "slow" neurological disease. 499 14

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

In this communication, we present data which describe optimum conditions for reverse transcription of large ribonucleic acid (RNA) templates into deoxyribonucleic acid (DNA) transcripts by the avian retrovirus reverse transcriptase in vitro. In contrast to previous studies, we have optimized all of the reaction components with respect to their influence on the size of DNA transcripts rather than the incorporation of radio-labeled deoxynucleoside triphosphates into acid-insoluble DNA product. The most dramatic effect on uninterrupted reverse transcription is the presence of physiological concentrations (i.e., 148 mM) of monovalent cation in the reaction mixture, although all of the components of the reaction influence the size of the DNA transcripts synthesized to some extent. The enzymatic conditions described herein for the uninterrupted reverse transcription of large RNA templates (greater than 1000--2000 nucleotides) are superior to those described previously because they are reproducible, do not require the presence of ribonuclease inhibitors, and do not result in the precipitation of components of the reaction mixture during incubation.
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PMID:Enzymatic synthesis of deoxyribonucleic acid by the avian retrovirus reverse transcriptase in vitro: optimum conditions required for transcription of large ribonucleic acid templates. 615 30

Polyspermine-ribonuclease (Mr approximately 17 000) and the enzyme transcriptase from Rauscher-leukaemia virus (Mr approximately 70 000) form a complex Mr approx. 160 000) such that the molar ratio of polyspermine-ribonuclease to reverse transcriptase is 5:1. The most favourable condition for complex-formation is in a solution consisting of 0.01 M-Tris/HCl buffer, pH 7.5, 0.25 M-KCl and 1 mM-Mn2+ at 37 degrees C. The association of the two enzymes retains full RNAase activity, but reverse-transcriptase activity is completely inhibited when ribonuclease-sensitive polymers such as (dG)12 x (rC)n or viral 70S RNA are used as primer templates.
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PMID:Complexing reverse transcriptase with polyspermine-ribonuclease. 616 6

The effect of the ribonuclease inhibitor polyvinyl sulfate on the activity of retroviral DNA polymerase and terminal c transferase was examined. This substance was found to be a potent inhibitor of these enzymes by virtue of competition between the sulfated sidechains of the molecule and the template primer for a site on the enzyme. The significance of these findings in relation to searching for reverse transcriptase is discussed.
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PMID:Inhibition of reverse transcriptase by polyvinyl sulfate (PVS). 616 65

Human thyroglobulin mRNA was isolated from Graves' goitres by size selection of total poly(A)-rich RNA in a sucrose gradient. It sedimented at 33 S, as in other mammalian species, and showed a single component of approximately 8500 bases by gel electrophoresis. cDNA was synthesized from the 33-S RNA by using reverse transcriptase in the presence of human placenta ribonuclease inhibitor and in conditions allowing the formation of long transcripts. The latter was made double-stranded using reverse transcriptase and 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 at the endonuclease PstI site and tailed with dGTP. The resulting plasmids were used to transform Escherichia coli C600 cells and four cloned recombinants were selected. Each plasmid DNA was shown to contain a sequence complementary to human thyroglobulin mRNA by hybridization with a labeled 33-S mRNA, visualization of cDNA . mRNA hybrids by electron microscopy and filter hybridization selection of mRNA directing the synthesis of immunologically related thyroglobulin peptides in the reticulocyte lysate. The four inserted DNA sequences were 1400 - 1800 base pairs long, two of them showing an homologous sequence of 1100 base pairs. Together, the four cloned DNA fragments represented 63% of the 8500 bases of human thyroglobulin mRNA.
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PMID:Cloning of four DNA fragments complementary to human thyroglobulin messenger RNA. 617 25

Partially purified human procollagen mRNAs have been copied using reverse transcriptase from avian myeloblastosis virus. Under standard conditions, using a high deoxynucleotide concentration, only incomplete cDNAs corresponding to one-fifth to one-half of the template could be synthesized. Addition of a ribonuclease inhibitor from human placenta in the reaction mixture allowed the synthesis of full-length cDNA copies from extremely long procollagen mRNA.
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PMID:Synthesis of full-length cDNAs from partially purified human procollagen mRNAs. 618 1

The single-stranded DNA containing the Moloney murine leukemia virus origin for plus-strand synthesis was cloned in M13mp2 and used as a template for avian myeloblastosis virus reverse transcriptase in the presence of Moloney RNA which had been treated with pancreatic RNase A. The RNA pieces containing the polypurine stretch near the plus-strand origin were processed, presumably by RNase H, to generate primers for DNA synthesis which initiated both at the correct origin site and at one nucleotide downstream from the correct site. Approximately 50% of the labeled DNA fragments synthesized under these conditions retained the priming RNA on their 5' ends. When the isolated fragments were hybridized back to the template DNA and again treated with the reverse transcriptase, all of the RNA was removed from the labeled DNA. By using 5'-end-labeled pancreatic RNase A-resistant fragments, it was possible to show that the RNA primers were removed intact. It appears from these results that the RNase H activity associated with the enzyme shows a preference for cutting at the junction between the RNA and DNA moieties of such complexes and therefore is ideally suited for removing RNA primers.
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PMID:Mechanism of RNA primer removal by the RNase H activity of avian myeloblastosis virus reverse transcriptase. 619 10

A 190-base-pair DNA-RNA hybrid containing the Moloney murine leukemia virus origin of plus-strand DNA synthesis was constructed and used as a source of template-primer for the reverse transcriptase in vitro. Synthesis was shown to initiate precisely at the known plus-strand origin. The observation that some of the origin fragments retained ribonucleotide residues on their 5' ends suggests that the primer for chain initiation is an RNA molecule left behind by RNase H during the degradation of the RNA moiety of the DNA-RNA hybrid. If the RNase H is responsible for creating the correct primer terminus, then it must possess a specific endonucleolytic activity capable of recognizing the sequence in the RNA where plus strands are initiated. The 16-base RNase A-resistant fragment which spans the plus-strand origin can also serve as a source of the specific plus-strand primer RNA. Evidence is presented that some of the plus-strand origin fragments synthesized in the endogenous reaction contain 5' ribonucleotides, suggesting that specific RNA primers for plus-strand initiation may be generated during reverse transcription in vivo as well.
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PMID:RNA-primed initiation of Moloney murine leukemia virus plus strands by reverse transcriptase in vitro. 620 82


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