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)

Highly purified preparations of RNA-directed DNA polymerase from avian myeloblastosis virus (AMV) contain a Mn2+-activated endonuclease activity capable of nicking supercoiled DNA. This endonuclease activity co-sediments in glycerol gradients with the alphabeta form of AMV DNA polymerase, and co-chromatographs with DNA polymerase activity on DEAE-cellulose, phosphocellulose, and heparin-Sepharose. It is also present in AMV alphabeta-DNA polymerase purified by electrophoresis through nondenaturing polyacrylamide gels and subsequently chromatographed on poly(C)-agarose. alphabeta-associated endonuclease is co-immunoprecipitated with DNA polymerase activity by antiserum directed against alphabeta holoenzyme. The alpha form of AMV DNA polymerase lacks this activity. In its enzymatic properties, alphabeta-associated endonuclease resembles the endodeoxyribonuclease activity associated with the AMV p32 protein, which has been shown to be structurally related to the beta (but not the alpha) subunit of AMV DNA polymerase.
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PMID:Endonuclease activity of purified RNA-directed DNA polymerase from avian myeloblastosis virus. 8 98

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

The RNA-dependent DNA polymerase purified from B77 avian sarcoma virus exhibited two distinct DNA-processing activities. The alpha and beta 2 isoenzymes possessed an endodeoxyribonuclease activity capable of nicking simian virus 40 superhelical DNA, whereas the alpha beta isoenzyme performed as an untwisting topoisomerase. Both activities associated with the three molecular forms of the retroviral DNA polymerase were dependent on the presence of either Mn2+ or Mg2+ ions. From analysis of the denaturated DNA products, it is apparent that the alpha and beta 2 isoenzymes introduced two nicks, one per each strand in the superhelical simian virus 40 DNA molecules, whereas the alpha beta polymerase converted these supercoiled molecules to the relaxed covalently closed circular form. The notion that the DNA-processing activities are located on the DNA polymerase molecules was supported by the following: (i) the three isoenzymes were of a high purity; (ii) the activities cosedimented in glycerol gradients with the DNA polymerase activities of the alpha, beta 2, and alpha beta molecular forms; and (iii) immunoglobulin directed against the purified polymerase immunoprecipitated the DNA-processing activities. Chemical treatments of the DNA polymerase molecules (with pyridoxalphosphate, iodoacetamide, and sulfhydryl reagents), which inhibited the polymerase activity, also suppressed the endonucleolytic and topoisomerase activities, suggesting that cystein and amino groups play an important role in the active sites of the DNA-processing activities as well.
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PMID:DNA-processing activities associated with the purified alpha, beta 2, and alpha beta molecular forms of avian sarcoma virus RNA-dependent DNA polymerase. 617 42

Preparations of purified Rauscher murine leukemia virus were found to contain an endodeoxyribonuclease after disruption of the virus with nonionic detergents. The enzyme makes single-strand breaks in linear or covalently closed circular phage double-stranded DNA molecules. The enzyme was partially purified by ion-exchange chromatography on DEAE- and carboxymethyl-Sepharose columns followed by electrophoresis in DNA-containing polyacrylamide gels. The enzyme was separated from reverse transcriptase (p80pol), and the final endonuclease preparation contained no detectable reverse transcriptase activity. The DEAE-Sepharose column-purified endonuclease activity contained a polypeptide of about 40,000 Mr that we term p40. Peptide mapping experiments demonstrated that p40 shares methionine-labeled tryptic peptides with Pr200gag-pol and Pr135pol. Six major methionine-labeled tryptic peptides derived from p40 were found in Pr200gag-pol, but only five of these were detected in Pr135pol. The four core proteins (p30, p15, pp12, and p10) and p80pol plus p40 account for most, but not all, of the peptide sequences of Pr200gag-pol. The endonuclease-associated p40 is similar in size and precursor origin to the avian retrovirus-coded endonuclease (p32). In view of these similarities to the avian p32 endonuclease and its association with partially purified Rauscher murine leukemia virus-associated endonuclease preparations, we propose that p40 is the Rauscher murine leukemia virus-coded endonuclease.
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PMID:Endodeoxyribonuclease activity associated with Rauscher murine leukemia virus. 626 Sep 82

To evaluate the specificity and applicability to the study of human tumor cells of the reverse transcription (RT) in situ PCR and RT polymerase chain reaction (PCR) in situ hybridization techniques, we examined five melanoma cell lines and five nonmelanoma lines for tyrosinase mRNA using primers specific for tyrosinase. Each procedural step was optimized and minutely controlled, and results from the in situ techniques and solution-phase RT-PCR were compared. All melanoma lines showed a specific pattern of perinuclear cytoplasmic reaction not seen in nonmelanoma lines. There was exact agreement between the results from the RT in situ PCR and RT-PCR in situ hybridization techniques and those from solution-phase RT-PCR. Ribonuclease digestion abolished cytoplasmic staining, as did omission of the reverse transcriptase step. Nuclear staining was seen in melanoma and nonmelanoma lines, apparently as a result of DNA synthesis from repair-replication and mispriming or nonspecific amplification. Neither high concentrations of deoxyribonuclease nor long incubation periods abolished this effect completely. Demonstration of cytoplasmic mRNA by RT in situ PCR and RT-PCR in situ hybridization specifically identifies cells of melanocytic lineage.
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PMID:Demonstration of cytoplasmic tyrosinase mRNA in tissue-cultured cells by reverse transcription (RT) in situ polymerase chain reaction (PCR) and RT PCR in situ hybridization. 902 34

A P1 cloned insert of about 85.5 kilobases (kb) was isolated, containing four members of the human growth hormone/chorionic somatomammotropin (GH/CS) gene family and the thyroid hormone receptor interacting protein (TRIP-1) gene. The presence of the CS-like, CS-A, GH-variant and, most downstream, CS-B gene was confirmed by DNA blotting and sequence analysis. The TRIP-1 gene was detected 40 kb downstream of the CS-B gene and in the reverse transcriptional orientation to all the GH/CS genes. The TRIP-1 gene is highly homologous to the SUG-1 gene in yeast and is evolutionarily conserved among several species. Based on the common location of the GH and TRIP-1 (or homologue) genes on the same chromosome in the human, pig and rat genomes, we suggest that these loci are physically linked. Previously, it was reported that a muscle-specific sodium channel (SCN4A) gene is located immediately upstream of the pituitary growth hormone (GH-N) gene, and is linked to the GH gene locus in both humans and rats. This suggests a further linkage between the SCN4A, GH and TRIP-1 loci. Also, deoxyribonuclease hypersensitive sites have been reported in and around these loci and were associated with an important locus control region for the GH/CS genes. Unlike the GH/CS genes, we show, using reverse transcriptase-polymerase chain reaction that the TRIP-1 gene is expressed ubiquitously and, through RNA blotting, as a 1.4-kb transcript. This implies an open and active chromatin structure. The possible effect of this structure on the adjacent human GH/CS gene locus is discussed.
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PMID:Physical linkage of the human growth hormone gene family and the thyroid hormone receptor interacting protein-1 gene on chromosome 17. 966 65

The glycolipid galactosyldiacylglycerol (GDG), containing C16:0 and C18:1 fatty acids, was isolated from the sea alga Petalonia bingbamiae as a potent inhibitor of the activities of mammalian DNA polymerase alpha (pol. alpha). GDG, however, had no effect on pol. alpha from a fish or a higher plant. The inhibition of pol. alpha by GDG was dose-dependent with an IC50 value of 54 microM. The compound did not influence the activities of other replicative DNA polymerases such as mammalian pol. delta, or repair-related enzymes such as mammalian pol. beta. GDG also did not influence the activities of prokaryotic DNA polymerases such as the Klenow fragment of DNA polymerase I, T4 DNA polymerase, Taq DNA polymerase, DNA polymerases from the higher plant, cauliflower, or DNA metabolic enzymes such as calf thymus terminal deoxynucleotidyl transferase, human immunodeficiency virus type 1 reverse transcriptase and deoxyribonuclease 1. Kinetic analysis of the compound showed that pol. alpha was non-competitively inhibited with respect to both the DNA template and the nucleotide substrate. In this study, we demonstrated the structure-function relationship in the selective inhibition of pol. alpha by the glycolipid group.
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PMID:Galactosyldiacylglycerol, a mammalian DNA polymerase alpha-specific inhibitor from a sea alga, Petalonia bingbamiae. 1155 81

A deoxyribonuclease distinct from the previously isolated asparagus ribosome-inactivating proteins, possessing a molecular weight of 30 kDa and requiring a pH of 7.5 for optimum hydrolytic activity toward herring sperm DNA, was isolated from Asparagus officinalis seeds. The isolation procedure involved extraction with saline, (NH(4))(2)SO(4) precipitation, ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on CM-Sepharose, and FPLC gel filtration on Superdex 75. The doxyribonuclease was unadsorbed onto DEAE-cellulose and Affi-gel blue gel and adsorbed onto CM-Sepharose. It exhibited the novel N-terminal sequence, GIEVIKIREL. The deoxyribonuclease was purified to a specific activity of 1584 units/mg. It was devoid of ribonuclease, protease, and HIV-1 reverse transcriptase-inhibitory activities. However, it inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC(50) of 20 microM. It exhibited antifungal activity toward Botrytis cinerea but not toward Fusarium oxysporum and Mycosphaerella arachidicola.
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PMID:Isolation of a novel deoxyribonuclease with antifungal activity from Asparagus officinalis seeds. 1170 87

A protease designated pleureryn, with an N-terminal sequence dissimilar from previously reported mushroom metalloendopeptidases and showing only limited resemblance to aspartic proteinases, albeit considerable homology to DNA replication licensing factor, was isolated from fresh fruiting bodies of the edible mushroom Pleurotus eryngii. The purification protocol entailed ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel Blue gel, ion exchange chromatography on CM-Sepharose, and FPLC-gel filtration on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel Blue gel and CM-Sepharose. It demonstrated a single band with a molecular weight of 11.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pleureryn demonstrated a protease activity of 9364 U/mg toward casein. It exhibited a pH optimum of 5.0 and a temperature optimum of 45 degrees C, with substantial activity remaining at high temperatures and pH 4 and 12. The activity of the protease was adversely affected by pepstatin A, indicating that it is an aspartic protease. PMSF, trypsin inhibitor, and EDTA exerted no striking effect, suggesting that it is neither a serine protease nor a metalloprotease. It inhibited translation in a rabbit reticulocyte lysate system with an IC(50) of 20 nM. Pleureryn also exhibited some inhibitory activity against HIV-1 reverse transcriptase, reminiscent of a suppressive action of HIV-1 protease on its homologous reverse transcriptase but was devoid of ribonuclease, deoxyribonuclease, and antifungal activities.
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PMID:Pleureryn, a novel protease from fresh fruiting bodies of the edible mushroom Pleurotus eryngii. 1172 12

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