Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The functional organization of the murine leukemia virus reverse transcriptase was investigated by expressing a molecular clone containing AKR MuLV reverse transcriptase-coding sequences in Escherichia coli. A purified preparation of the expressed enzyme (pRT250 reverse transcriptase) consisted primarily of a 69-kilodalton protein that has normal levels of murine leukemia virus polymerase activity but 10-fold-reduced levels of RNase H compared with the viral enzyme. The deficit in RNase H activity was correlated with the absence of 60 to 65 amino acids normally present at the carboxyl end of murine leukemia virus reverse transcriptase. The results provide additional experimental evidence for the localization of polymerase and RNase H domains to the N- and C-terminal regions of reverse transcriptase, respectively.
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PMID:Functional organization of the murine leukemia virus reverse transcriptase: characterization of a bacterially expressed AKR DNA polymerase deficient in RNase H activity. 245 14

Selected conserved amino acids in the putative RNase H domain of reverse transcriptase (RT) were modified in a molecularly cloned infectious provirus and in a Moloney murine leukemia virus RT expression vector by site-directed mutagenesis. Substitution of either of two conserved aspartic acid residues in proviral DNA prevented production of infectious particles in transfected NIH 3T3 cells, and the same modifications depressed RT-associated RNase H activity by more than 25-fold with little or no effect on polymerase activity.
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PMID:Inhibition of RNase H activity and viral replication by single mutations in the 3' region of Moloney murine leukemia virus reverse transcriptase. 246 6

The region of the pol gene of the Moloney murine leukemia virus (M-MuLV) encoding the reverse transcriptase and RNase H activities was inserted in an eukaryotic expression vector and transiently expressed in human cultured cells. This results in the expression of high levels of reverse transcriptase activity. This enzyme, partially purified, also carries a RNase H activity, has the biochemical requirements of the viral enzyme and is recognized and inhibited by antibodies directed against a M-MuLV reverse transcriptase expressed in Escherichia coli.
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PMID:Expression of an enzymatically active murine retroviral reverse transcriptase in human cells. 246 4

The intrinsic properties of reverse transcriptase in reverse transcription were studied using a synthetic, partial ovalbumin mRNA with a synthetic DNA oligonucleotide annealed to the 3'-end of the RNA as a model substrate. With or without concomitant cDNA synthesis, the RNase H activity of avian myeloblastosis virus (AMV)-reverse transcriptase cleaved the substrate at a site which would leave a hybrid of between 7 and 14 base pairs between the 3' termini of the RNA and DNA oligonucleotide. Variability in the exact size of the hybrid probably reflects some weak base preference for cleavage by the enzyme. These short hybrids can be recognized as substrates by Escherichia coli RNase H and can be utilized by reverse transcriptase as sites for continuation of cDNA synthesis. Substrates with 5'-triphosphorylated termini, 3'-OH, 3'-phosphate, 3'-end hairpin structures and 20 base pair hybrids on the middle region of long RNA more than 300 bases or on circular RNA were all cleaved by AMV-reverse transcriptase-associated RNase H, indicating that the RNase H activity is essentially regarded as an endonuclease degrading RNA moiety in RNA-DNA hybrid. The modes of action of reverse transcriptase from murine leukemia virus and Rous-associated virus 2 were the same as that of AMV-reverse transcriptase, except that the size of the remaining hybrid and the specificity for cleavage depended on the reverse transcriptase. We propose a possible model to explain the mode of action of RNase H and RNA-dependent DNA polymerase activities in reverse transcription.
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PMID:Intrinsic properties of reverse transcriptase in reverse transcription. Associated RNase H is essentially regarded as an endonuclease. 247 53

Peripheral blood mononuclear cells (PBMNC) from 23 healthy subjects and 39 patients with B-cell chronic leukaemia (B-CLL) were assayed for ribonuclease H activity using as substrate the filter-immobilized synthetic homopolymer hybrid 3H-poly(rA):poly(dT). In 69% of the leukaemia patients examined enzyme activities were above those estimated in cells from the healthy controls. The mean enzyme levels for the normal and the leukaemic samples group were (per cent substrate hydrolysis): 8.1 +/- 8.9 and 58.7 +/- 40.8, respectively, their difference being statistically highly significant (P less than 0.0001). This result does not represent homogeneity within the cells but is due to a subclass of cells within the leukaemic clone containing the enzyme, thus contributing through pool size fluctuation to the wide variations of enzyme activity observed among the patients. These cells containing high activity could not be identified with either the prolymphocytes or the large lymphocytes. The activity of ribonuclease H in the examined CLL patients correlated with disease stage (Binet) (P = 0.011) and appeared to serve as a sensitive indicator of disease progression when compared with a number of other known prognostic parameters.
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PMID:Activity of ribonuclease H in cells of chronic B-lymphocytic leukaemia: correlation with clinical stage. 284 80

On the basis of earlier studies with both detergent-disrupted virions (the endogenous reaction) and an in vitro reconstructed reaction, the RNase H activity associated with Moloney murine leukemia virus reverse transcriptase has been implicated in the generation of plus-strand RNA primers during reverse transcription. Here we used an oligonucleotide extension assay to show that the RNA primers remaining bound to the plus DNA strands initiated at the normal origin in the in vitro reaction are heterogeneous in length. This result indicates that, although a precise cleavage generates the 3' end of the priming RNA, RNase H exhibits less specificity at other break sites. During the endogenous reaction, a kinetic analysis of the synthesis of plus strands corresponding to different regions of the genome suggested that additional sites for the initiation of plus-strand DNA existed upstream of the normal origin. Direct analysis of fragments produced in the endogenous reaction, as well as in the in vitro reaction, confirmed the existence of upstream plus-strand initiation sites. Several of these sites were mapped to the nucleotide level by the oligonucleotide extension method. A comparison of the nucleotide sequences surrounding the upstream initiation sites with the sequence at the normal plus-strand origin revealed common features, which suggests a mechanism for plus-strand priming based on sequence recognition by the RNase H/reverse transcriptase protein. Although primer removal by RNase H is highly efficient for DNA fragments initiated at the normal origin, the RNA primers were inefficiently removed from the fragments initiated at the upstream sites. This result suggests that primer removal, like primer generation, involves sequence recognition by the enzyme.
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PMID:The role of Moloney murine leukemia virus RNase H activity in the formation of plus-strand primers. 303 72

Ten ribonucleic acid (RNA) tumor viruses grown in five different host cell species and three non-oncogenic viruses from three different virus groups have been examined for ribonuclease H content. Three different substrates were used to assay ribonuclease H: calf thymus [(3)H]RNA-deoxyribonucleic acid (DNA) hybrid prepared with denatured calf thymus DNA and Escherichia coli DNA-directed RNA polymerase, (3)H-polydenylic acid [(3)H-poly(A)] complexed to polydeoxythymidylic acid [poly(dT)], and (3)H-polyuridylic acid [(3)H-poly(U)] complexed to polydeoxyadenylic acid [poly(dA)]. All ten RNA tumor viruses contained ribonuclease H activity which degraded the RNA of both the calf thymus hybrid and poly(A)-poly(dT), whereas only the ribonuclease H in the Moloney strain of murine sarcoma-leukemia virus and in RD-feline leukemia virus hydrolyzed the RNA strand of poly(U)-poly(dA). No appreciable ribonuclease H activity was detected in influenza, Sendai, or vesicular stomatitis virus. The ribonuclease H and RNA-directed DNA polymerase activities in Moloney murine sarcoma-leukemia virus were inseparable by phosphocellulose chromatography or glycerol gradient centrifugation, but appeared to be partially separated by diethylaminoethyl-cellulose chromatography.
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PMID:Ribonuclease H: a ubiquitous activity in virions of ribonucleic acid tumor viruses. 411 67

Infectious hamster leukemia virus (HaLV) contains a DNA polymerase different from those of murine and avian viruses. No endogenous reaction directed by the 60 to 70S RNA of HaLV could be demonstrated in detergenttreated HaLV virions, nor could the purified DNA polymerase copy added viral RNA. The virion RNA could, however, act as template for added avian myeloblastosis virus DNA polymerase and the HaLV DNA polymerase could efficiently utilize homopolymers as templates. The HaLV enzyme was like other reverse transcriptases in that certain ribohomopolymers were much better templates than the homologous deoxyribohomopolymers. No ribonuclease H activity could be shown in the HaLV enzyme, but neither could activity be found in the murine leukemia virus DNA polymerase. The hamster enzyme was unique in that poly(A) .oligo(dT) was a poor template, and globin mRNA primed with oligo(dT) was totally inactive as a template. Its uniqueness was also indicated by its subunit composition; electrophoresis of the HaLV DNA polymerase in sodium dodecyl sulfate-containing polyacrylamide gels revealed equimolar amounts of two polypeptides of molecular weight 68,000 and 53,000. The sedimentation rate of the enzyme in glycerol gradients was consistent with a structure containing one each of the two polypeptides. The enzyme thus appears to be structurally distinct from other known virion DNA polymerases. Its inability to carry out an endogenous reaction in vitro might result from an inability to utilize certain primers.
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PMID:Hamster leukemia virus: lack of endogenous DNA synthesis and unique structure of its DNA polymerase. 413 18

Ribonuclease H (RNA.DNA-hybrid ribonucleotidohydrolase, EC 3.1.4.34) has been reported to copurify with reverse transcriptase (RNA directed DNA polymerase) of RNA tumor viruses. In addition, viral specific ribonuclease H and reverse transcriptase of avian type-C viruses are thought to be part of the same polypeptide. In this report we show that a fraction of the ribonuclease H activity from Rauscher murine leukemia and Kirsten murine sarcoma viruses was separated from reverse transcriptase by anion exchange chromatography while the remaining portion co-purified with the viral polymerase. The amount of this co-purified nuclease activity was about 4- to 8-fold lower than the activity found in avian myeloblastosis virus (with respect to the ratio of ribonuclease H to reverse transcriptase) and this nuclease activity can only be detected by using labeled substrate of high specific radioactivity. However, a complete separation of ribonuclease H activity from reverse transcriptase was obtained by purifying core structures of the virus by sucrose density gradient centrifugation. While reverse transcriptase was present in the cores, there was no detectable ribonuclease H. Furthermore, a specific antibody against Rauscher leukemia virus reverse transcriptase did not inhibit any virion associated ribonuclease H activity. Our results suggest that in these virions these two enzyme activities reside in two separate molecules and probably in two different compartments of the virus. These findings emphasize a basic difference between the avian and murine type-C virus DNA polymerases.
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PMID:Separation of ribonuclease H and RNA directed DNA polymerase (reverse transcriptase) of murine type-C RNA tumor viruses. 413 16

Kirsten murine sarcoma-leukemia virus (Ki-MSV[MLV]) was found to contain less RNase H per unit of viral DNA polymerase than avian Rous sarcoma virus (RSV). Upon purification by chromatography on Sephadex G-200 and subsequent glycerol gradient sedimentation the avian DNA polymerase was obtained in association with a constant amount of RNase H. By contrast, equally purified DNA polymerase of Ki-MSV(MLV) and Moloney [Mo-MSV(MLV)] lacked detectable RNase H if assayed with two homopolymer and phage fd DNA-RNA hybrids as substrates. On the basis of picomoles of nucleotides turned over, the ratio of RNase H to purified avian DNA polymerase was 1:20 and that of RNase H to purified murine DNA polymerase ranged between <1:2,800 and 5,000. Based on the same activity with poly (A).oligo(dT) the activity of the murine DNA polymerase was 6 to 60 times lower than that of the avian enzyme with denatured salmon DNA template or with avian or murine viral RNA templates assayed under various conditions (native, heat-dissociated, with or without oligo(dT) and oligo(dC) and at different template enzyme ratios). The template activities of Ki-MSV(MLV) RNA and RSV RNA were enhanced uniformly by oligo(dT) but oligo(dC) was much less efficient in enhancing the activity of MSV(MLV) RNA than that of RSV RNA. It was concluded that the purified DNA polymerase of Ki-MSV(MLV) differs from that of Rous sarcoma virus in its lack of detectable RNase H and in its low capacity to transcribe viral RNA and denatured salmon DNA. Some aspects of these results are discussed.
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PMID:DNA polymerase of murine sarcoma-leukemia virus: lack of detectable RNase H and low activity with viral RNA and natural DNA templates. 435 18


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