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)

Simian sarcoma-associated virus type 1 propagated in human rhabdomyosarcoma cells exhibited characteristics typical of oncornaviruses but seemed to have several aberrant properties. It had a buoyant density of 1.14 g/cm3, had RNA-dependent DNA polymerase activity, seemed to be labile to high salt concentrations, and contained little 50 to 60S RNA but relatively large amounts of human ribosomal RNA. In addition to 50 to 60S RNA, purified virions contained smaller RNA molecules with sedimentation coefficients of 28 to 30S, 18 TO 20S, and 4 to 10S. Unlike the 50 to 60S RNA species, the smaller virion-associated RNAs lacked polyadenylic acid, and the 28 to 30S RNA had an average base composition similar to that of human ribosomal RNA. Upon heat denaturation, the native 50 to 60S RNA genome yielded polyadenylic acid-containing 28 to 30S subunits that degraded in to 18 to 20S molecules upon further heat treatment. The 50 to 60S viral RNA had a guanine plus cytosine content of 56%.
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PMID:RNA of simian sarcoma-associated virus type 1 produced in human tumor cells. 4 85

A RNA-dependent DNA polymerase was isolated from a human cell line derived from the bone marrow of a patient with polycythemia vera. The purification procedure included chromatography on phosphocellulose and oligo(dT)-cellulose, and glycerol gradient centrifugation. The enzyme could be distinguished from polymerase A by salt elution from phosphocellulose, utilization of poly(rC) - oligo(dG) and its molecular size of about 70000, as determined by centrifugation. Throughout the purification procedure ribonuclease H activity was co-purified. Upon dodecylsulfate-polyacrylamide electrophoresis on microgradient gels two main bands with molecular weights of 68000 and 66000 and three minor bands were detected. The enzyme preferentially used poly(rA) - oligo(dT) as template-primer compared with poly(dA) - oligo(dT). It incorporated dGMP into polymer on poly(rC) - oligo(dG).
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PMID:RNA-dependent DNA polymerase from a cell line derived from the bone marrow of a patient with polycythemia vera. 5 57

The synthesis of a complementary DNA copy (cDNA) of hen ovalbumin mRNA using AMV RNA-directed DNA polymerase was studied under different conditions of salt, deoxyribonucleotide concentrations, temperature, and time. It was observed that in the absence of monovalent cation at 46 degrees C a complete transcript of ovalbumin mRNA could be effected by the enzyme. The minimum deoxyribonucleotide requirement for complete synthesis was 35 muM for dATP, dGTP, and dCTP and 200 muM dTTP. By a number of different experimental criteria which included sedimentation on alkaline sucrose gradients and electrophoresis in polyacrylamide gels containing 98% formamide, direct electron microscope visualization, and protection of ovalbumin [25I]mRNA from nuclease digestion it could be demonstrated that a considerable fraction of a complete mRNA transcript was indeed synthesized. The cDNA/ovalbumin mRNA hybrid had a Tm on hydroxylapatite of 92 degrees C, indicating the synthesis of a RNA transcript with a high fidelity. When such a complete ovalbumin [3H]cDNA was synthesized with a specific activity of 10(8) cpm/mug and hyfridized to an excess of chick DNA, the kinetics of hybridization indicated that the cDNA was comprised of a nonrepetitive sequence.
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PMID:The synthesis and properties of the complete complementary DNA transcript of ovalbumin mRNA. 5 72

The alpha beta DNA polymerase of avian myeloblastosis virus was treated with dimethyl sulfoxide to dissociate the enzyme subunits. The dimethyl sulfoxide treated enzymes were passed over phosphocellulose to purify and characterize the dissociated subunits as well as to remove the dimethyl sulfoxide. RNA-directed DNA polymerase, RNase H, and nucleic acid-binding activity were monitored, as well as the subunit structure (on sodium dodecyl sulfate-polyacrylamide gels) of the various enzyme species obtained. With 30% dimethyl sulfoxide, the majority of DNA polymerase and RNase H activities as well as the alpha subunit were displaced from the alpha beta DNA polymerase position on phosphocellulose (0.23 M potassium phosphate) to the alpha DNA polymerase position (0.1 M). The association of DNA polymerase and RNase H activities with the alpha subunit suggests that alpha is the enzymatically active subunit in alpha beta. In addition to alpha DNA polymerase, a minor polymerase species eluted from phosphocellulose at 0.4 M potassium phosphate. The dissociated beta subunit eluted from phosphocellulose at a wide range of salt concentrations (0.28 to 0.5 M potassium phosphate). The dissociated beta subunit bound 3H-labeled murine leukemia virus RNA and [3H]poly(dT)-poly(dA) approximately 20-fold more avidly than alpha DNA polymerase alone. In contrast to the results with the alpha subunit, there was no correlation between DNA polymerase and RNase H activity profiles and the elution profile of the beta subunit from phosphocellulose. These observations suggest the beta subunit is either enzymatically inactive or possesses limited DNA polymerase and RNase H activity when compared with the alpha subunit.
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PMID:Dissociation of alpha beta DNA polymerase of avian myeloblastosis virus by dimethyl sulfoxide. 5 61

The synthesis of DNA on avian myeloblastosis virus (AMV) RNA as the primer-template using AMV reverse transcriptase in vitro has been examined as a function of the concentrations of these components, as well as a function of the ionic strenth of the assay medium. The results are consistent with the hypothesis that two types of sites exist on the AMV RNA: inactive "dead-end" sites that merely bind the enzyme, and active binding sites that lead to DNA synthesis. Velocity sedimentation studies of reverse transcriptase reveal that the enzyme becomes a dimer (or oligomer) at low salt concentrations and it is at these concentrations that the two types of sites are evident on the RNA. At high salt concentration the enzyme, which exists primarily as a monomer, is inactive with AMV RNA, although it is active when poly(rA)dT10 is used as the primer-template. We have shown that inactive sites are not due to binding of the reverse transcriptase to nicked regions or to partially denatured RNA molecules. We deduce that inactive sites are those containing incorrect 4S primer molecules. These results are discussed in terms of the mechanism of the interaction of the reverse transcriptase with AMV RNA.
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PMID:Mechanism of interaction of avian myeloblastosis virus reverse transcriptase with avian myeloblastosis virus RNA. 5 63

We have compared the relative merits of several procedures for the isolation of RNA-directed DNA polymerase (EC 2.7.7.7.) from cells using a reconsituted model system consisting of a mixture of woolly monkey (simian) sarcoma virus and a cultured human lymphoblastoid cell line, NC-37. When the cell-virus mixture was gently disrupted and fractionated by differential centrifugation, most of the added polymerase was recovered associated with a particulate fraction obtained from the post-mitochondrial supernatant. Purification of the polymerase was best achieved starting from this fraction. The particulate fraction itself can be purified by gel filtration through a Sepharose 2 B column. This procedure did not significantly alter the composition of viral and cellular DNA polymerases. Whereas as little as 7.5 - 10(5) viral particles were sufficient for the detection of RNA-directed DNA polymerase activity, a minimum of about 10(11) particles were necessary for the isolation and unequivocal characterization of the enzyme from the cell-virus mixture by subcellular fractionation and chromatographic separation from cellular DNA polymerases. Purified RNA-directed DNA polymerase had the same primer-template characteristics, sedimentation properties, and immunological cross reactivity as the enzyme purified from density gradient-banded virions of simian sarcoma virus. Methods involving total extraction of the cell-virus mixture either by repeated freezing and thawing followed by detergent treatment or by Dounce homogenization and treatment with high salt and detergent failed to provide RNA-directed DNA polymerase free of cellular DNA polymerases. Because of this, low levels of cellular RNA-directed DNA polymerase may be missed when these approaches are used.
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PMID:A comparative evaluation of methods for isolation of RNA-directed DNA polymerase from cells in a reconstituted system. 5 69

Rabbit lymphosarcoma tissues contain 70 S RNA and RNA-directed DNA polymerase encapsulated in particulate components that band in the density region of type C RNA viruses. RNA-directed DNA polymerase associated with the particles could be distinguished from cellular DNA polymerases by salt elution from phosphocellulose. The enzyme preferred the template primers poly(rA)-(dT)12-18 and poly(rC)-(dG)12-18 over other synthetic template primers and also utilized viral 70 S RNA as template; these properties are not observed with the known cellular DNA polymerases.
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PMID:Presence of a high-molecular-weight RNA and RNA-directed DNA polymerase in rabbit hereditary lymphosarcoma. 6 26

Using purified single-stranded ovalbumin complementary DNA (cDNAov) as a template for avian myeloblastosis (AM) virus reverse transcriptase, we have enzymatically synthesized a complete double-stranded cDNAov sequence. Our data suggests that many single-stranded cDNAov molecules contain short double-stranded sequences (hairpins) at their 3' termini capable of acting as primers for synthesis of complete double-stranded cDNAs. Optimum conditions for synthesis of the double-stranded cDNAov were found to be a high temperature (46 degrees) and a low salt concentration. Nevertheless, in all cases 40% of the initial single-stranded cDNAov molecules fail to prime for synthesis of a complementary double strand. Following synthesis, the second DNA strand is covalently linked to the first cDNAov strand as shown by analysis on alkaline sucrose gradients. The two strands have a high Tm on hydroxylapatite (89 degrees). These intact double-stranded cDNAov structures have a bouyant density in CsCl gradients of 1.700 g/cm3 and rapidly renature after heat denaturation with a C0t1/2 value of less than 2 X 10(-6) mol s liter(-1). All size classes of cDNAs, i.e. partial as well as complete transcripts of the mRNA, are capable of forming double-stranded structures. The closed loop of the double-stranded cDNAov could be opened with S1 nuclease. The denatured complementary strands of the cDNAov then renatured with the appropriate second order kinetics at a C0t1/2 value of 1.89 X 10(-3) mol s liter(-1). Using the enzyme terminal deoxyribonucleotidyltransferase to label to free 3'-terminal end of double-stranded [32P]cDNAov with 3H, we demonstrate a convenient procedure to study the site for restriction endonuclease cleavage within the ovalbumin gene.
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PMID:The ovalbumin gene. In vitro enzymatic synthesis and characterization. 6 59

The RNA-directed DNA polymerase of murine mammary tumor virus, a type B RNA tumor virus, was purified sequentially through DEAE-cellulose, phosphocellulose (step gradient), and phosphocellulose (linear salt gradient) chromatography followed by glycerol sedimentation centrifugation. During all stages of purification, coincident peaks of RNA-directed DNA polymerase activity, templated by polyribocytidylate-oligodeoxyguanidylate, and RNase H digestion of [3H]polyriboadenylate-polydeoxythymidylate were observed, and both enzymatic activities displayed a cation preference for magnesium. Under conditions that removed adventitiously associated nucleases, RNase H activity was found to co-purify with polymerase. The specificity of this nuclease was assayed with various prepared substrates, which indicated that the polymerase-associated RNase H activity was directed only against the RNA strand of an RNA-DNA hybrid. It is highly probable that RNase H (RNA-DNA hybrid: ribonucleotide-hydrolase, EC 3.1.4..34) and RNA-directed DNA polymerase of type B viruses are associated enzymatic activities analogous to those observed for avian and mammalian type C RNA tumor viruses.
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PMID:RNase H and RNA-directed DNA polymerase: associated enzymatic activities of murine mammary tumor virus. 6 21

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as a template for in vitro synthesis of 32P-labeled RNA and deoxysubstituted RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T1 and alkaline phosphatase digestion have been determined. In addition, the 3' nearest neighbor was determined for several fragments resulting from digestion with T1 ribonuclease. The utility of the deoxysubstitution technique was demonstrated by the ease with which the sequences of pyrimidine-rich fragments could be determined. Many sequences thus determined were long enough to fit uniquely with the alpha- or beta-globin amino acid sequences. The positions of these fits were found to be clustered, leading us to believe that only certain regions of the complementary DNA are transcribed by Escherichia coli RNA polymerase. Other unique characteristics of RNA synthesis from a complementary DNA template include a high yield of free poly(A) and the fact that one must use low rather than high salt buffers to obtain transcripts of high molecular weight.
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PMID:Rabbit globin mRNA: analysis of T1 RNAse digestion fragments. 6 35


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