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)

A cell line (LB59Ly) derived from the leukocytes of a leukaemic cow was established as a monolayer, which spontaneously released large amounts of a retrovirus. This virus was found to be indistinguishable from the bovine leukaemia virus (BLV) produced by the commonly used high-producing heterologous cell line (FLK-BLV). Like the latter, its reverse transcriptase activity was greater in the presence of Mg2+ cations than in the presence of Mn2+ cations; its polyacrylamide gel electrophoresis pattern showed the presence of gp51, p24, p15, p12 and p10, and the antigenicity of the two major proteins completely cross-reacted with those of BLV from FLK-BLV cells. The virus was infectious and induced early and late polykaryocytosis, the specificity of which was demonstrated by use of specific anti-BLV sera.
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PMID:Establishment and propagation of a bovine leukaemia virus-producing cell line derived from the leukocyte of a leukaemic cow. 627 Feb 54

The RNA-directed DNA polymerase of the primate type-D retrovirus Mason-Pfizer virus was purified using ion-exchange and affinity chromatography, and molecular sieving. The enzyme was shown to have a molecular weight of approx. 80 000 as determined by sedimentation analysis, molecular sieving and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified RNA-directed DNA polymerase retained its ability to use a heteropolymeric RNA as a template. The Mason-Pfizer virus RNA-directed DNA polymerase was also characterized as to its divalent cation preference for several synthetic primertemplates and for heteropolymeric RNA. Mg2+ was preferred as its divalent cation for all primer-templates except oligo(dG).poly(rC)m for which it prefers Mn2+. The Mason-Pfizer virus enzyme was also shown to have a pH optimum of 8-8.5 and a temperature optimum of 37-40 degrees C. The stability of the Mason-Pfizer virus RNA-directed DNA polymerase was shown to differ when measured using different primer-templates.
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PMID:Purification and characterization of the RNA-directed DNA polymerase of a primate type-D retrovirus: Mason-Pfizer virus. 677 21

Exposure in vitro of various mammalian retroviruses to the chelating agents EDTA or EGTA in millimolar concentrations resulted in partial disintegration of viral membranes as measured by accessibility or even release of reverse transcriptase, an internal viral protein, without any other treatment usually required. Among the viruses responding to chelators were mammalian type C viruses, primate type D viruses and bovine leukemia virus. The effect was dose-dependent. The avian type C virus AMV, however, was found to be not susceptible to the agents. Rauscher mouse leukemia virus treated in vitro with EDTA or EGTA showed reduced infectivity in mice. The results are considered as evidence for some association of divalent cations with membranes of mammalian retroviruses. The disintegrating activity of EGTA suggests that Ca2+ is an integral constituent of viruses but Mg2+ may also be involved. These cations seem to be responsible for maintaining integrity of retroviral membranes which, after chelation of ions, are either disrupted or become permeable for the exogenous template of reverse transcriptase. In addition, the disintegrating activity of trifluoperazine may indicate that a calmodulin-like protein occurs in retroviral membranes.
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PMID:Disintegration of retroviruses by chelating agents. 681 93

Plant DNA polymerases and E. coli DNA polymerase I, but not animal DNA polymerases or avian reverse transcriptase, are strongly stimulated by ethidium bromide (EtdBr) when TMP incorporation is followed using a short oligo dT primer at 37 degrees C. The effect is observed with a poly A template in the presence of Mg2+ or Mn2+ and of poly dA template only in the presence of magnesium ions. When a longer primer like poly dT is used, EdtBr inhibited wheat DNA polymerase C activity. This result prompted us to study the effect of the incubation temperature on the drug mediated stimulation. With oligo dT primer the stimulation by EdtBr is not observed at a temperature of incubation lower than 35 degrees C. It is shown that the Tm of poly A-dT12 is around 35 degrees C and that EdtBr will clearly increase this value. The stimulation is lost when the enzyme is preincubated with the primer alone whereas it is not affected when the enzyme is preincubated with the template.
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PMID:Ethidium bromide stimulation of DNA polymerase activity by stabilization of the primer-template duplex. 682 Nov 57

Cell cultures established from the spleen of a Macaca nemestrina with enzootic retroperitoneal fibromatosis (ERF) spontaneously released a unique retrovirus. Throughout 14 serial passages, the spleen cell cultures remained fibroblastic and no cytopathic effect was evident. The virus incorporates [3H]uridine, contains an RNA-dependent DNA polymerase (RDDP), has a buoyant density of 1.15 g/cm3 in sucrose, and was designated MNV-1. Virion-associated reverse transcriptase showed no preference for either Mg2+ or Mn2+ in standard RDDP assays. Complementary DNA (cDNA) transcribed from polyadenylated MNV-1 RNA hybridized to genomic DNA and RNA extracted from diseased tissues but not to nucleic acids from normal tissues of a healthy Macaca nemestrina or a Macaca mulatta. MNV-1 is therefore exogenous to these species. MNV-1 had no detectable homology to the endogenous macaque virus isolates MAC-1 and MMC-1. Liquid hybridization of MNV-1 cDNA to viral RNA derived from exogenous and endogenous subhuman primate retroviruses (SiSV(SSAV), GALV-SF, BaEV-M7, and BILN) did not reveal any significant sequence homologies. In addition, MNV-1 does not share homology with bovine leukemia virus or Mason-Pfizer monkey virus as determined by Southern blot hybridization. We conclude that MNV-1 is a unique retrovirus which has not previously been described. As the ultrastructure of virions in in vitro cell cultures, as well as disease involved tissue, show some particles with type C morphology and others with type D morphology, MNV-1 may be comprised of more than one component.
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PMID:Isolation of a unique retrovirus, MNV-1, from Macaca nemestrina. 686 69

The RNase H activity of recombinant HIV-1 reverse transcriptase (RT) has been characterized with respect to inhibition by azidothymidylate (AZTMP) and N-ethylmaleimide (NEM) and to cleavage patterns using either poly(rA)/poly(dT) or poly(rG)/poly(dC) as model substrate and either Mg2+ or Mn2+ as divalent cation activator. The inhibitory potency of AZTMP and other nucleotide analogues was found to be dependent on both the composition of the substrate and the divalent cation. The enzyme was significantly more sensitive to AZTMP inhibition with poly(rG)/poly(dC) than with poly(rA)/poly(dT) as substrate and in Mn2+ than in Mg2+ with either substrate. Kinetic studies indicated that AZTMP is a competitive inhibitor with respect to the substrate in Mn2+ whereas it behaves as an uncompetitive inhibitor in Mg2+. These results suggest that the enzyme may exist in two distinct forms depending on whether Mg2+ or Mn2+ is the divalent cation activator. Consistent with this suggestion is the alteration in the mode of cleavage of the substrate upon substitution of Mg2+ with Mn2+. In Mg2+, hydrolysis of poly(rA)/poly(dT) appears to be solely endonucleolytic, whereas in Mn2+, hydrolysis is both endonucleolytic and exonucleolytic. With poly(rG)/poly(dC) as substrate, hydrolysis is both endonucleolytic and exonucleolytic in either Mg2+ or Mn2+. There is a positive correlation between sensitivity to AZTMP and production of mononucleotides, suggesting that the exonuclease activity of RNase H is preferentially inhibited by AZTMP. The sensitivity of RNase H to inhibition by N-ethylmaleimide was also found to be markedly influenced by the substrate composition and the divalent cation activator, being most sensitive under conditions in which endonucleolytic activity predominates.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Catalytically distinct conformations of the ribonuclease H of HIV-1 reverse transcriptase by substrate cleavage patterns and inhibition by azidothymidylate and N-ethylmaleimide. 750 46

Two ternary complexes of rat DNA polymerase beta (pol beta), a DNA template-primer, and dideoxycytidine triphosphate (ddCTP) have been determined at 2.9 A and 3.6 A resolution, respectively. ddCTP is the triphosphate of dideoxycytidine (ddC), a nucleoside analog that targets the reverse transcriptase of human immunodeficiency virus (HIV) and is at present used to treat AIDS. Although crystals of the two complexes belong to different space groups, the structures are similar, suggesting that the polymerase-DNA-ddCTP interactions are not affected by crystal packing forces. In the pol beta active site, the attacking 3'-OH of the elongating primer, the ddCTP phosphates, and two Mg2+ ions are all clustered around Asp190, Asp192, and Asp256. Two of these residues, Asp190 and Asp256, are present in the amino acid sequences of all polymerases so far studied and are also spatially similar in the four polymerases--the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, T7 RNA polymerase, and rat DNA pol beta--whose crystal structures are now known. A two-metal ion mechanism is described for the nucleotidyl transfer reaction and may apply to all polymerases. In the ternary complex structures analyzed, pol beta binds to the DNA template-primer in a different manner from that recently proposed for other polymerase-DNA models.
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PMID:Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP. 752 45

Cleavage specificity of RNase HI was examined on model Okazaki fragments, to determine the likely role of this nuclease in lagging strand DNA replication. Each substrate was prepared by annealing a short RNA primer, made by transcription in vitro, to a single-stranded synthetic DNA template, and subsequently extending the primer by DNA polymerization. The calf thymus RNase HI makes a structure-specific endonucleolytic cleavage in the RNA primer, releasing it intact, and leaving a mono-ribonucleotide at the 5' terminus of the RNA-DNA junction. This specific cleavage, one nucleotide upstream of the RNA-DNA junction, is RNA primer sequence- and length-independent. Cleavage specificity is lost if the RNA primer is not extended with DNA, or if the substrate has a nick at the RNA-DNA junction. In addition, the cleavage at a single site requires Mg2+. Cleavage in the presence of Mn2+ is less specific. Neither human immunodeficiency virus reverse transcriptase nor Escherichia coli RNases H perform such a structure-specific cleavage before an RNA-DNA junction. Our work indicates that calf RNase HI is designed to recognize Okazaki fragments. It has the specificity to remove their initiator RNA segments, except for one ribonucleotide, by a single endonucleolytic cleavage in vivo.
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PMID:Structure-specific cleavage of the RNA primer from Okazaki fragments by calf thymus RNase HI. 752 96

The stimulatory effect of Mg2+ and Mn2+ on the ribonuclease H (RNase H) functions of HIV-1 reverse transcriptase (RT) has been evaluated using a model 90-nt RNA template/36-nt DNA primer. Wild type enzyme exhibits similar endonuclease and directional processing activities in response to both cations, while RNase H activity (hydrolysis of double-stranded RNA) is only evident in the presence of Mn2+. Enzyme altered at the p66 residue Glu478 (Glu478-->Gln478), which participates in metal ion binding, is completely inactive in Mg2+. However, Mn2+ restores specifically its endoribonuclease activity. In the presence of Mn2+, mutant RT also catalyzes specific removal of the tRNA replication primer, eliminating the possibility of contaminating Escherichia coli RNase H in our recombinant enzyme. However, the efficiency with which mutant RT catalyzes transfer of nascent DNA between RNA templates (an event mandating RNase H activity) is severely reduced. These findings raise the possibility that directional processing activity is required to accelerate transfer of nascent DNA between templates during retroviral replication.
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PMID:Divalent cation modulation of the ribonuclease functions of human immunodeficiency virus reverse transcriptase. 754 83

A purified preparation of endogenous RNA-dependent DNA-polymerase (reverse transcriptase) earlier identified in rat brain (Ivanov, V.A., Pakhotin, P.I., Bobkova, N.V., and Ilyin, Yu.V. parallel Dokl. RAN (1992). V. 323, P. 173-177) has been obtained. A comparative analysis of the enzyme and recombinant reverse transcriptase coded by the mobile genome element jockey earlier expressed in a heterological cell system (Ivanov V.V., Melnikov A.A., Siunov A.V., Fodor I.I., Ilyin, Yu.V. parallel EMBO J. (1991), V. 10, P. 2489-2495) has been carried out. Like retroviral RNA-dependent DNA-polymerases, these enzymes show preference for polyribonucleotides and can use poly(rCm) as template. Besides they are inhibited by SH-reagents and require bivalent cations (Mg2+ or Mn2+) and detergent and/or KCl as ionic strength carrier. The enzymes differ drastically from retrovirus reverse transcriptases by a number of catalytic properties (low optima of concentration of requisite cations and ionic strength, strong preference for Mn2+, highly efficiency in using poly(rCm), lack of associated RNase H activity) but exhibit a high degree of similarity among themselves with regard to the above properties. It is suggested that endogenous reverse transcriptase from rat brain is a product of expression of the mobile genome element of the LINE family.
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PMID:[Endogenous cellular reverse transcriptase in the rat brain. A comparative analysis of the authentic enzyme and recombinant reverse transcriptase coded by a mobile genetic element of the LINE class]. 757 75


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