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:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A RNA-dependent DNA polymerase was isolated from a human cell line derived from the bone marrow of a patient with polycythemia vera. The purification procedure included chromatography on phosphocellulose and oligo(dT)-cellulose, and glycerol gradient centrifugation. The enzyme could be distinguished from polymerase A by salt elution from phosphocellulose, utilization of poly(rC) - oligo(dG) and its molecular size of about 70000, as determined by centrifugation. Throughout the purification procedure ribonuclease H activity was co-purified. Upon dodecylsulfate-polyacrylamide electrophoresis on microgradient gels two main bands with molecular weights of 68000 and 66000 and three minor bands were detected. The enzyme preferentially used poly(rA) - oligo(dT) as template-primer compared with poly(dA) - oligo(dT). It incorporated dGMP into polymer on poly(rC) - oligo(dG).
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PMID:RNA-dependent DNA polymerase from a cell line derived from the bone marrow of a patient with polycythemia vera. 5 57

The influence of Rous sarcoma virus (RSV)-associated RNase H on the in vitro synthesis of DNA by the RSV RNA-directed DNA polymerase was determined under conditions whereby RNase H activity was selectively inhibited with NaF. Not only were we unable to detect any effect on the size, structure, or genetic complixity of the DNA product synthesized in the absence of RNase H activity, but the displacement of DNA from the 70S RNA:DNA hybrid structures was also unaffected. The suitability of 70S RNA:DNA hybrid structures synthesized in vitro to serve as a substrate for RNase H is discussed.
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PMID:In vitro transcription of 70S RNA by the RNA-directed DNA polymerase of Rouse sarcoma virus: lack of influence of RNase H. 5 43

The alpha beta DNA polymerase of avian myeloblastosis virus was treated with dimethyl sulfoxide to dissociate the enzyme subunits. The dimethyl sulfoxide treated enzymes were passed over phosphocellulose to purify and characterize the dissociated subunits as well as to remove the dimethyl sulfoxide. RNA-directed DNA polymerase, RNase H, and nucleic acid-binding activity were monitored, as well as the subunit structure (on sodium dodecyl sulfate-polyacrylamide gels) of the various enzyme species obtained. With 30% dimethyl sulfoxide, the majority of DNA polymerase and RNase H activities as well as the alpha subunit were displaced from the alpha beta DNA polymerase position on phosphocellulose (0.23 M potassium phosphate) to the alpha DNA polymerase position (0.1 M). The association of DNA polymerase and RNase H activities with the alpha subunit suggests that alpha is the enzymatically active subunit in alpha beta. In addition to alpha DNA polymerase, a minor polymerase species eluted from phosphocellulose at 0.4 M potassium phosphate. The dissociated beta subunit eluted from phosphocellulose at a wide range of salt concentrations (0.28 to 0.5 M potassium phosphate). The dissociated beta subunit bound 3H-labeled murine leukemia virus RNA and [3H]poly(dT)-poly(dA) approximately 20-fold more avidly than alpha DNA polymerase alone. In contrast to the results with the alpha subunit, there was no correlation between DNA polymerase and RNase H activity profiles and the elution profile of the beta subunit from phosphocellulose. These observations suggest the beta subunit is either enzymatically inactive or possesses limited DNA polymerase and RNase H activity when compared with the alpha subunit.
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PMID:Dissociation of alpha beta DNA polymerase of avian myeloblastosis virus by dimethyl sulfoxide. 5 61

A new instruction theory for antibody formation is presented. The reverse flow of information from the amino-acid sequences of small antigenic determinants to an antideterminant RNA (aRNA) seems feasible. Prerequisites are specific activating enzymes, tRNAs, ATP as well as some kind of membrane assembling the anticodons of tRNAs linearly, analogous to the linear primary structure of stretched polypeptides. Once synthesized, aRNA might be replicated, utilized as transfer factor and transcribed by means of Reverse Transcriptase into aDNA. Further steps would be the fusion of this aDNA with genetical performed DNA-molecules already coding for the basic strucures of different classes of immunoglobulins by means of a terminal deoxynucleotidyl-transferase. This could be a chromosomal or extrachromosomal integration. The second hypothesis concerns antigen-induced immunosuppression and the phenomenon of nonresponsiveness (tolerance). An overwhelming proteolysis might give rise to a degradation of antigens or receptor templates for antigenic determinants located on the surface of macrophages. On later exposure to a similar antigen proteolytic enzymes are already preformed abolishing rapidly antigenic information. The third hypothesis concerns antibody-induced immunosuppression and tolerance. Antideterminant information is integrated into the genome or established extra-chromosomally. The continuous presence of antibodies sets in motion a sequence of reactions causing an accumulation of all information intermediates including a complementary DNA strand to the aRNA. On exposure to the corresponding antigen aRNA is transcribed. However, translation might be inhibited by hybridisation with the complementary aDNA strand as well as specific RNA hydrolysis by RNase H. Concerning the immunogenity of antibodies, a proteolytical mechanism might also be possible. Taking this into account a tolerance could be suspended in the following way: 1. by influencing the overwhelming proteolytical degradation of antigenic determinants with simultaneous antigenic stimulation; 2. by substitution of aRNA to induce blocked antibody synthesis.
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PMID:[A new instruction theory: possibility of a reverse flow of information from polypeptide sequences to RNA particularly in antibody synthesis, and the mechanisms of tolerance induction and immunosuppression (author's transl)]. 5 2

The purified reverse transcriptase-RNase H complex from Friend murine leukemia virus consists of a single polypeptide of 84,000 molecular weight, which after mild protease treatment in vitro or after intentional degradation during the purification procedure allows the generation of several additional polypeptides. Degradation destroys the RNA-dependent DNA polymerase activity with native RNA templates and reduces RNase H but does not affect response to synthetic template primers such as poly (rA)-Oligo (dT). The properties of the intact murine enzyme consisting of a single polypeptide of 84,000 molecular weight are compared to those of the avian alpha subunit and the avian alpha beta enzyme complex. The intact murine enzyme resembles the avian beta-containing enzyme complex and is different from alpha in the following respects: (i) it binds to native RNA templates; (ii) it transcribes native RNA templates into DNA, a reaction which can be inhibited by actinomycin D; (iii) RNase H activity behaves like a processive exonuclease; and (iv) analysis of the RNase H digestion products reveals oligonucleotides approximately four bases in length.
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PMID:Further characterization of the Friend murine leukemia virus reverse transcriptase-RNase H complex. 5 72

Rauscher leukemia virus RNA-directed DNA polymerase has been purified to near homogeneity (greater than 90% pure) using affinity chromatography on polycytidylate-agarose with over 85% recovery of input enzymatic activity. The purified enzyme has a molecular weight of approximately 70,000 and appears to consist of a single polypeptide chain. The enzyme is free of DNase, but has RNase H activity. Analysis of the requirements for optimal rates of DNA synthesis by this enzyme using synthetic and natural template-primers has revealed template-specific variations in such requirements. During these studies it was observed that DNA synthesis catalyzed by Rauscher leukemia virus DNA polymerase is inhibited by the addition of inorganic phosphate. An analysis of the mechanism of phosphate inhibition was carried out using the synthetic template-primer poly(A)-(dT)10. It appears that by some mechanism, possibly involving the substrate binding site of the enzyme, phosphate ions inhibit DNA synthesis with a more acute effect on the rate of chain growth than on that of initiation. The extension of these studies to DNA synthesis catalyzed by a variety of mammalian type C viral reverse transcriptases revealed that low levels ( less than or equal to 2 mM) of inorganic phosphate strongly inhibited DNA synthesis. The susceptibility to phosphate inhibition appears unique to mammalian type C viral enzymes since the type B viral enzyme, Escherichia coli DNA polymerase I, avian myeloblastosis virus and Mason Pfizer monkey tumor virus reverse transcriptase and cellular DNA polymerases alpha and gamma are not inhibited by inorganic phosphate. This phenomenon of phosphate inhibition of various DNA polymerases, therefore, provides a new basis for the differentiation of the sources and nature of these enzymes.
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PMID:Purification and properties of Rauscher leukemia virus DNA polymerase and selective inhibition of mammalian viral reverse transcriptase by inorganic phosphate. 6 68

Three forms of the RNA-dependent DNA polymerase were isolated from highly purified avian sarcoma virus B77 grown in duck embryo fibroblasts, using sequential chromatography on DEAE-cellulose, phosphocellulose, and poly(U)-cellulose. One form, which sedimented with about 5.2 S, contained only one species of polypeptide, with a molecular weight of 63,000; a second sedimented with about 7.8 S and contained only one species of polypeptide with a molecular weight of 81,000; and a third form, which sedimented with about 7.3 S, contained two species of polypeptides with molecular weights of 63,000 and 81,000. The molecular constitution of the three enzyme forms were therefore alpha, beta2, and alphabeta. All three possessed almost the same specific activity with poly(rA)-oligo(dT) as the primer-template. Forms alpha and alphabeta of avian sarcoma virus DNA polymerase have already been described in the literature; form beta2 is a new form. All three forms possessed ribonuclease H activity, the relative specific activities of the alpha, beta2, and alphabeta forms being about 1:4:5. All three enzyme forms were inhibited by antiserum to the alphabeta form, but whereas the alpha and alphabeta forms could be inhibited about 95%, the maximum degree of inhibition of the beta2 form was about 80%. The three enzyme forms also differed with respect to heat stability at 46 degrees, the monomeric alpha form of the enzyme being only about one-half as stable as the two dimeric forms.
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PMID:RNA-dependent DNA polymerase of avian sarcoma virus B77. I. Isolation and partial characterization of the alpha, beta2, and alphabeta forms of the enzyme. 6 34

Based on the observation that in vitro transcription of Rous sarcoma virus (RSV) RNA by avian myeloblastosis virus DNA polymerase renders the RNA PROGRESSIVELY MORE SENSITIVE TO Escherichia coli RNase H digestion, a new procedure for the in situ analysis of this process has been developed. In vitro transcription products of 32P-labeled RSV RNA are first treated with RNase H, the resistant fraction is then digested to completion with RNase T1, and the oligonucleotides are analyzed by a fingerprint technique. By using the established order of these oligonucleotides along the RNA molecule, a comparison of the yields of each oligonucleotide, before and after transcription, allows qualitative and quantitative in situ analyses of the transcription process. Using this new procedure, we find that upon transcription of purified RSV RNA, DNA synthesis occurs mainly at three sites, one near the 5' end and two near the center of the subunit RNA molecule, and that most of these RNA molecules are competent templates for limited transcription at these specific sites. We also show that purified RSV 70S RNA contains a low-molecular-weight DNA hybridized to a nucleotide sequence near the center of the subunit molecule. Furthermore , we find that the low-molecular-weight nucleic acid fraction extracted from purified RSV virions contains DNA that can hybridize to RSV 70S RNA and that the virion DNA in such hybrids can function as a primer for an extensive in vitro reverse transcription.
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PMID:New procedure for the direct analysis of in vitro reverse transcription of Rous sarcoma virus RNA. 6 18

The RNA-directed DNA polymerase of murine mammary tumor virus, a type B RNA tumor virus, was purified sequentially through DEAE-cellulose, phosphocellulose (step gradient), and phosphocellulose (linear salt gradient) chromatography followed by glycerol sedimentation centrifugation. During all stages of purification, coincident peaks of RNA-directed DNA polymerase activity, templated by polyribocytidylate-oligodeoxyguanidylate, and RNase H digestion of [3H]polyriboadenylate-polydeoxythymidylate were observed, and both enzymatic activities displayed a cation preference for magnesium. Under conditions that removed adventitiously associated nucleases, RNase H activity was found to co-purify with polymerase. The specificity of this nuclease was assayed with various prepared substrates, which indicated that the polymerase-associated RNase H activity was directed only against the RNA strand of an RNA-DNA hybrid. It is highly probable that RNase H (RNA-DNA hybrid: ribonucleotide-hydrolase, EC 3.1.4..34) and RNA-directed DNA polymerase of type B viruses are associated enzymatic activities analogous to those observed for avian and mammalian type C RNA tumor viruses.
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PMID:RNase H and RNA-directed DNA polymerase: associated enzymatic activities of murine mammary tumor virus. 6 21

The RNase H activity associated with several RNA-directed DNA polymerases is inhibited by the addition of DNA, in contrast to RNase H activity from enzymes devoid of polymerizing activity. Kinetic investigations of the inhibitory effect of DNA, using purified Rauscher leukemia virus DNA polymerase as a test enzyme, revealed that the addition of DNA to an ongoing RNase H reaction causes an immediate cessation of RNase H activity. Concomitant initiation of DNA synthesis by inhibitory DNA can occur, provided that appropriate substrate and primer is available. Thus, in addition to providing a simple test for the distinction between polymerase-associated and polymerase-independent RNase H activity, this study strongly supports the concepts that (i) RNase H activity expressed by several mammalian oncoviral reverse transcriptases is an integral part of that molecule, and (ii) that the catalytic site of RNase H activity is also involuved in template-primer binding.
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PMID:Specific inhibition of DNA polymerase-associated RNase H by DNA. 6 22


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