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 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

Revertants of nonproducer human osteosarcoma (NP/KHOS) cells induced by Kirsten murine sarcoma virus were isolated after incubating at high temperature (40.5 degrees C) overnight and subcloning at 36 degrees C. The morphologic variants, from which murine sarcoma virus could no longer be rescued, had growth properties similar to those of the nontransformed, parent human osteosarcoma cells and did not release RNA-dependent DNA polymerase activity. These revertants were nontumorigenic in nude mice. The revertants supported leukemia virus growth and showed an enhanced sensitivity to murine sarcoma virus superinfection. Thus, the revertants were from human cells transformed by an oncogenic RNA virus.
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PMID:Revertants of human cells transformed by murine sarcoma virus. 6 97

An RNA-directed DNA polymerase associated with transformation-defective (td) segregant of Rous sarcoma virus (RSV) has been characterized. The enzyme required both a monovalent and a divalent cation, a sulfhydryl reducing agent, and all four deoxyribonucleoside triphosphates for the expression of maximal activity. Sensitivity of the endogenous RNA-directed DNA polymerase activity to a low concentration of pancreatic RNase indicated that the enzyme utilized the td virus endogenous RNA as template. Maximal DNA synthesis was observed in a reaction mixture of pH 8 - 8.5 at 45 C with a manganese concentration of 1 mM. The enzyme of the td virus responded to exogenous template-primers in a manner characteristic of DNA polymerase of RNA tumor viruses, and the response became substantially greater when noncomplementary precursors were omitted from the reaction mixture. The endogenous reaction kinetics were examined. Three phases of DNA synthesis could be distinguished. Evidence was obtained showing that during the third and slowest phase of DNA synthesis the reaction mixture was not depleted of precursors and that the enzyme was fully active to initiate DNA synthesis with newly-added viral or synthetic RNA templates. Comparison of TMP and dAMP incorporation kinetics suggested that at the initial phase the enzyme preferentially copies A-rich region(s) of viral RNA. A comparison was also made between the endogenous reaction of the td virus and that of its parent sarcoma virus. The pH optimum, metal ion requirements, effect of sulfhydryl agents, response to exogenous template-primers, and kinetics of DNA synthesis, were all compared. No significant difference between the reaction of the td virus and its sarcomatogenous counterpart could be demonstrated.
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PMID:Endogenous DNA polymerase of a transformation-defective rous sarcoma virus: characterization and comparison with the enzyme of the non-defective parent. 6 91

Various rat cell lines have been analyzed for expression of endogenous RNA homologous either to RT21C, a typical rat type C virus, or to Kirsten sarcoma virus. Cells have been found that express either (i) high levels of RNA homologous to RT21C rat type C virus and low levels of RNA homologous to Kirsten sarcoma virus (RT21Chigh,sarclow) or (ii) high levels of RNA homologous to Kirsten sarcoma virus and low levels of RNA homologous to typical rat type C virus (sarchigh, RT21Clow). The properties of these two classes of cell lines have been compared. Each type of cell contains an equal amount of the expressed RNA on polysomes. Cell lines that are RT21Chigh produce abundant rat p30 nad p12 structural proteins and release rat type C particles containing viral RNA and reverse transcriptase into supernatant fluids from these cultures. Cell lines that are sarchigh,RTC21Clow have no detectable rat viral p12 protein and no p30 protein immunoreactive in even broad interspecies radioimmunoassays, and do not release type C particles into the supernatant from the cultures. When the particle-negative cell lines are superinfected with heterologous mouse or wooly type C viruses or are producing typical rat type C virus particles, the endogenous sarcoma virus-specific RNA is secreted from these cells. The sarcoma virus-specific RNA can be transcribed in complementary DNA in the endogenous reverse transcriptase reactions carried out in vitro with such virus preparations. However, exposure of cells that are permissive to the helper virus with the particles containing sarcoma virus-specific RNA has not yet resulted in cell transformation or in the synthesis of these RNA sequences. The results suggest: (i) that the first step in the genesis of sarcoma viruses involves the packaging of this expressed sarcoma virus-specific RNA in helper viral particles; (ii) that efficient transmission of the sarcoma virus-specific RNA requires additional events; and (iii) that the formation of a stable sarcoma virus by recombination between the helper viral genome and part of the rescued sarcoma virus-specific RNA is much less common event than the rescue process itself.
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PMID:Type C particle-positive and type C particle-negative rat cell lines: characterization of the coding capacity of endogenous sarcoma virus-specific RNA. 6 49

Increasing evidence has accumulated that the direct assay of reverse transcriptase in human blood cells is of value in the diagnosis of leukaemia. The isolation and characterization of this enzyme has shown that it possesses remarkable similarities to the DNA-polymerase of the RNA-tumour virus of simian sarcoma. Hence, leukaemic cells in humans are thought to possess a virus-related gene, namely, reverse transcriptase. Various clinical reports have established the presence of this enzyme in blood cells, not only in the case of morphologically-proven malignant change, but also in cases classified as non-leukaemic from the morphological picture, such as acute leukaemia in remission and in the pre-leukaemic state. In confirmation and augmentation of earlier views we now report on the presence of reverse transcriptase in a patient with pancytopenia, who subsequently developed acute leukaemia i.e. isolation of the enzyme occurred in the pre-leukaemic state.
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PMID:[New polymerase gene in pancytopenia (author's transl)]. 6 82

Procedures were established for the isolation and partial purification of DNA polymerase, RNA polymerase and poly(A) polymerase activities from the cytoplasm and nuclei of NIH-Swiss mouse embryos. Based on the elution pattern of these enzyme activities from DEAE-cellulose and phosphocellulose columns in Tris-HCl buffer, pH 8.0, the apparent basicities of the enzymes can be arranged as follows: cytoplasmic(C) poly(A) polymerase greater than (C)DNA polymerase beta greater than (C)DNA polymerase alpha and nuclear(N) poly(A) polymerase greater than (N)DNA polymerase greater than (N)RNA polymerase I greater than (N)RNA polymerase II. Twenty rifamycins, including rifamycin B, rifamycin S, rifamycin SV, and rifamycin SV derivatives, were examined for their ability to inhibit the above mentioned nucleic acid polymerizing enzymes and Simian sarcoma virus type I (SSV-1) reverse transcriptase. Rifamycin SV 3'-formyldiphenylhydrazone, rifamycin SV 3'-formyl-n-octyloxime (AF/013) and rifamycin SV 3'-formyldiphenylmethyloxime (AF/05) inhibited all the tested enzyme activities. Rifamycin SV 3'-formylpropylphenyloxime (AF/015) inhibited cellular nucleic acid polymerase activities but not SSV-1 DNA polymerase activity. Rifamycin SV 3'-formyldinitrophenylhydrazone (AF/DNFL) strongly inhibited reverse transcriptase activity but did not inhibit cellular DNA polymerase activities. AF/DNFI slightly inhibited RNA and poly(A) polymerase activities. Rifamycin SV 3'-formyldipropylhydrazone (AF/DPI) and 2,6-dimethyl-4-N-benzyldemethyl-rifampicin (AF/ABDMP) slightly inhibited reverse transcriptase activity but did not inhibit cellular nucleic acid polymerase activities. Active rifamycin derivatives inhibited enzyme reactions by interacting with the enzyme proteins. Nascent polynucleotide chain elongation continued although at a reduced rate in the presence of inhibitor. The addition of increasing concentrations of nonionic detergent (Triton X-100) to rifamycin-inhibited enzyme reactions fully restored enzyme activities. The presence of highly lipophilic 3'-side chains on active rifamycins and the reversibility of enzyme inhibition by Triton X-100 suggest that the tested nucleic acid polymerizing enzymes may have hydrophobic regions with which inhibitory rifamycins interact.
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PMID:Interaction of rifamycins with mammalian nucleic acid polymerizing enzymes. 6 93

Murine Rauscher leukemia virus (MuRLV) from BALB/c plasma consisting of a mixture of an ecotropic and a xenotropic virus could be separated out by a selection process when propagated in human and simian cell cultures. This hypothesis is supported by obtaining consistently lower infectivity titers of human cell propagated RLV in human and simian cells as compared to MuRLV propagated in mouse cell cultures. Furthermore, RLV passaged in a simian cell culture failed to replicate in mouse cells, had a wide host range, was able to rescue Moloney sarcoma genome, possessed murine type C group-specific antigen, and was neutralized by anti-HRLV. Its reverse transcriptase was strongly inhibited by antiserum to MuRLV enzyme; however, antiserum to woolly monkey enzyme also inhibited (30%) its reverse transcriptase, suggesting some difference in antigenic properties. Inoculation of this virus in rhesus monkeys was inconclusive.
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PMID:Xenotropic properties of an isolate from murine Rauscher leukemia virus in primates. 6 54

Short term cultures of bovine leukemic lymphocytes release virus particles with biochemical properties of RNA oncogenic viruses. These particles, tentatively called Bovine Leukemia Virus (BLV) have a high molecular weight-reverse transcriptase complex and a density averaging 1.155 g/ml in sucrose solutions. Molecular hybridizations between BLV-3H cDNA and several viral RNAs show that BLV is not related to Mason-Pfizer Monkey Virus (MPMV) Simian Sarcoma Associated Virus (SSV-1) Feline Leukemia Virus (FeLV) or Avian Myeloblastosis Virus (AMV). Rauscher Leukemia Virus (RLV) exhibits a slight but reproducible relatednesse to BLV. The high preference of BLV reverse transcriptase for Mg++ as the divalent cation suggests that BLV might be an atypical mammalian leukemogenic type C virus. Hybridization studies using BLV 3H cDNA as a probe suggest that the DNA of bovine leukemic cells contains viral sequences that cannot be detected in normal bovine DNA.
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PMID:Bovine leukemia virus: an exogenous RNA oncogenic virus? 6 82

An antigen crossreacting with the 30,000-molecular-weight protein (p30) of the feline endogenous oncornavirus (RD114) was detected in a well-characterized human fibrosarcoma cell line, HT1080, by indirect immunofluorescence. Three antisera against RD114 p30 gave similar positive results, while two antisera prepared against simian sarcoma virus p30, one antiserum prepared against murine leukemia virus p30, and one antiserum prepared against feline leukemia virus p30 gave no immunofluorescence. The reactivity observed with the antiserum against RD114 p30 was detected in 10-40% of the cells at early passages and was no longer expressed by the forty-first subculture. The reactivity could be removed by adsorption of the antiserum with RD114-infected dog or human cells, but not by uninfected cells or by cells infected with an antigenically unrelated oncornavirus, feline leukemia virus. Neither complete virus particles nor reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) activity was detected in the culture. These experiments suggest that the fibrosarcoma cell line is expressing an antigen related to the p30 protein of RD114 baboon endogenous virus group of oncornaviruses without producing complete virions.
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PMID:Expression of antigenic crossreactivity to RD114 p 30 protein in a human fibrosarcoma cell line. 6 79

Sera from some leukemic cattle contain an antibody that inhibits the reverse transcriptase activity of the bovine leukemia virus. The antibody is not directed against the synthetic template or the major internal and envelope viral antigens. The antibody failed to inhibit the DNA polymerases of the murine leukemia virus, simian sarcoma-associated virus, avian myeloblastosis virus, or Escherichia coli. Conversely, the bovine leukemia virus enzyme was not inhibited by antibody against the reverse transcriptases of other C-type viruses. These findings agree with previous results showing that the major internal bovine leukemia virus protein lacks the known interspecies- and intraspecies-specific antigenic determinants indentified in the homologous proteins of other oncornaviruses.
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PMID:Inhibition of the reverse transcriptase of bovine leukemia virus by antibody in sera from leukemic cattle and immunological characterization of the enzyme. 6 83


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