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)

Reaction conditions for Rous sarcoma virus ribonucleic acid (RNA)-instructed deoxyribonucleic acid (DNA) polymerase activity are described whereby the viral RNA is relatively protected from endogenous or added nuclease activity. Three analyses of reaction product nucleic acids ((3)H-RNA, (32)P-DNA) were compared, namely, gel electrophoresis, Cs(2)SO(4) gradient centrifugation, and hydroxyapatite column chromatography. It was found that hydroxyapatite analysis could be misleading unless the state of the template RNA was monitored concomitantly with the DNA analysis. Gel electrophoresis and Cs(2)SO(4) gradient centrifugation gave comparable results. It was concluded that analyses of the product of reverse transcriptase reactions should not only refer to the template RNA and product DNA species, but also be performed with virus or viral RNA which do not have or obtain nicks in the 60S RNA. Otherwise, interpretation of the results would have the ambiguity of potential artifacts caused by those degraded RNA molecules.
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PMID:Deoxyribonucleic acid polymerase of Rous sarcoma virus: reaction conditions and analysis of the reaction product nucleic acids. 433 43

Antiserum to partially purified reverse transcriptase from the Schmidt-Ruppin strain of Rous sarcoma virus has been prepared and characterized. Antibody to the avian polymerase inhibited the reverse transcriptase activity of avian C-type viruses but had no effect on the polymerase activity from C-type viruses of other classes. The known mammalian C-type viral polymerases were significantly inhibited only by the antiserum to murine C-type viral polymerases; reverse transcriptases from four other mammalian viruses were immunologically distinct from both avian and mammalian C-type viral polymerases. Partially purified murine leukemia viral DNA polymerase activity was comparably reduced by specific antibody regardless of the template used for enzyme detection.
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PMID:Immunological relationships of reverse transcriptases from ribonucleic acid tumor viruses. 433 37

Early chicken embryos that are either positive or negative for group-specific antigens of avian leukosis viruses contained endogenous RNA-directed DNA polymerase activity. This endogenous DNA polymerase activity was not increased after mixture of soluble DNA polymerases isolated from chicken embryos with disrupted chicken embryo cells. The endogenous activity was resistant to treatment with deoxyribonuclease, and the initial rate of DNA synthesis was partially resistant to actinomycin D. In contrast, over 90% of the endogenous polymerase activity was destroyed by ribonuclease in medium with high salt concentration. The DNA product of the endogenous DNA polymerase activity from chicken embryos did not hybridize with RNA of Rous sarcoma virus or reticuloendotheliosis virus, whereas about 40% of this DNA product hybridized with the RNA from the same chicken-cell fraction. Antibody against DNA polymerase of avian myeloblastosis virus did not neutralize the chicken endogenous DNA polymerase activity. These results demonstrate that uninfected chicken embryo cells contain endogenous RNA-directed DNA polymerase activity that is not derived from avian leukosis or reticuloendotheliosis viruses.
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PMID:Endogenous RNA-directed DNA polymerase activity in uninfected chicken embryos. 433 97

Cells producing Rous sarcoma virus contain virus-specific ribonucleic acid (RNA) which can be identified by hybridization to single-stranded deoxyribonucleic acid (DNA) synthesized with RNA-directed DNA polymerase. Hybridization was detected by either fractionation on hydroxyapatite or hydrolysis with single strand-specific nucleases. Similar results were obtained with both procedures. The hybrids formed between enzymatically synthesized DNA and viral RNA have a high order of thermal stability, with only minor evidence of mismatched nucleotide sequences. Virus-specific RNA is present in both nuclei and cytoplasm of infected cells. This RNA is remarkably heterogeneous in size, including molecules which are probably restricted to the nucleus and which sediment in their native state more rapidly than the viral genome. The nature of the RNA found in cytoplasmic fractions varies from preparation to preparation, but heterogeneous RNA (ca. 4-50S), smaller than the viral genome, is always present in substantial amounts.
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PMID:Virus-specific ribonucleic acid in cells producing rous sarcoma virus: detection and characterization. 433 37

Stationary chicken embryo fibroblasts exposed to Rous sarcoma virus (RSV) remained stably infected for at least 5 days, but they did not release infectious virus or become transformed until after cell division. These infected stationary cells did not contain avian leukosis virus group-specific antigens or ribonucleic acid (RNA) hybridizable to deoxyribonucleic acid (DNA) made by the RSV endogenous RNA-directed DNA polymerase activity.
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PMID:Cell cycle-dependent activation of rous sarcoma virus-infected stationary chicken cells: avian leukosis virus group-specific antigens and ribonucleic acid. 433 98

The NH2-terminal amino acid sequence of the pp32 DNA binding protein has been determined, thus establishing its precise coding region in the polymerase gene of Rous sarcoma virus. Specific mutations were constructed in molecularly cloned Prague A DNA near the NH2- and COOH-termini of pp32 and the effects were assayed by transfection on chick embryo fibroblasts. Out-of-frame deletions at both sites and an in-frame deletion near the NH2 terminus rendered the DNA noninfectious and transformation negative. Single point mutations near the NH2 terminus reduced the transfection efficiency and the rate of virus replication. Biochemical studies indicated that the RNA-directed DNA polymerase and RNase H activities of the mutant viruses were not affected but the processing of the viral beta polypeptide was altered.
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PMID:Requirement of the avian retrovirus pp32 DNA binding protein domain for replication. 609 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

The precise src deletions in six transformation-defective (td) deletion mutants derived from the Schmidt-Ruppin strain of Rous sarcoma virus were determined by sequence analysis. Examination of the parental viral sequences neighboring the junctions of deletions in these td mutants revealed that these regions contained either directly repeated or inverted complementary sequences ranging from 9 to 28 nucleotides. Five td mutants were found to contain deletions flanked by directly repeated sequences, of which the 3' direct repeat was retained whereas the 5' direct repeat was deleted in the resulting td viral RNA. In the deletions of two td mutants where inverted complementary sequences were present at junctions of the deletions, both copies of the inverted complementary sequence were deleted in the td viral RNA. It is proposed from these observations that deletions of these mutants have been generated during the synthesis of minus-strand viral DNA by reverse transcriptase by jumping over a sequence flanked by direct repeats or by skipping a stem-and-loop structure formed via inverted complementary sequences on the viral RNA template. Data provide further information on the sequences in the td viral genome that are required for the generation of recovered sarcoma viruses (rASVs) by recombination with c-src. Sequence data of td viruses revealed that retaining as few as 82 nucleotides of the 3' src coding sequence is sufficient, whereas retaining as much as one-third of the 5' src but none of the 3' src coding sequences is not sufficient, for the generation of rASVs. Those that generate replication-competent rASVs retain, in addition to the 3' src region, a portion of the 5' src and/or its immediate upstream sequence that is homologous to exon 1 of the c-src DNA. These two sequence domains apparently provided 5' and 3' homologous regions for recombination between td viral genome and c-src DNA resulting in nondefective rASVs. Td109, which was shown previously to generate only replication-defective rASVs, retains 296 nucleotides of the 3' src sequence but lacks all the 5' src and 316 nucleotides of its immediate upstream region. It is concluded that the 5' src coding sequence and its immediate upstream region are not essential for the generation of rASVs. However, retaining a portion of those sequences is required for the generation of replication-competent rASVs.
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PMID:Mechanisms for the generation of src-deletion mutants and recovered sarcoma viruses: identification of viral sequences involved in src deletions and in recombination with c-src sequences. 609 66

The replication of transformation-defective mutants of the Prague strain of Rous sarcoma virus subgroup C was studied using roller cultures. Under such conditions, 10(5)--10(6) infectous units of virus per 0.2 ml were produced, as revealed in both the reverse transcriptase and 16Q complementation tests. A new td daPR-RSV-C mutant was isolated from duck-adapted PR-RSV-C. This mutant replicated in roller cultures with equal efficiency as the original td PR-RSV-C. It was verified that td daPR-RSV-C does not transform chicken fibroblasts, is not oncogenic for 3-week-old chickens and has subgroup C host-range specificity. Both td mutants replicate in duck cells and reach the same titres.
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PMID:Replication of transformation-defective mutants of the Prague strain of Rous sarcoma virus and isolation of a td mutant from duck-adapted PR-RSV-C. 615 95

Using cells transformed by Rous sarcoma virus (RSV), an RNA tumour virus whose genetic and structural composition is fully known, the virus-induced surface antigens acting as targets for antibodies and complement were studied. Among the virus structural proteins, only the envelope antigen gp85, but not the core group-specific proteins or reverse transcriptase, were able to mediate immune lysis in the 51Cr-release assay. The group-specific antigenic determinants of gp85 were predominantly involved. The virus-induced cell surface antigen (VCSA), specific for transformation, was the only other molecule effective. Since different cells express either of these antigens, further support is given to the non-identity of virus structural antigens and VCSA.
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PMID:Target antigens for antibodies and complement at the cell surface of RSV-transformed fibroblasts. 615 25


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