RNA‐Dependent DNA polymerase and ribonuclease H from Friend virions

Weimann, B. J.; Schmidt, Jörg; Wolfrum, Dirk Ingo

doi:10.1016/0014-5793(74)81100-2

Cited by 13 publications

(6 citation statements)

References 17 publications

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“…However, since sodium dodecyl sulfate-gel analysis showed that all of our most highly purified MSV DNA polymerase preparations contained three polypeptides with mol wt of 82,000, 68,000, and 60,000, we cannot draw any definitive conclusions regarding the structural relationship of the two enzyme activities. Similar to our results, Weimann et al (26) found that the RNase H and DNA polymerase activities in Friend leukemia virus were not separable by standard fractionation procedures, their most highly purified enzyme preparation containing two components of mol wt 51,000 and 67,000 on sodium dodecyl sulfate-gels. The sedimentation coefficient (5.7S) determined for the Friend leukemia virus enzymes corresponded to a mol wt of 123,000 (26).…”

Section: Discussionsupporting

confidence: 91%

“…Similar to our results, Weimann et al (26) found that the RNase H and DNA polymerase activities in Friend leukemia virus were not separable by standard fractionation procedures, their most highly purified enzyme preparation containing two components of mol wt 51,000 and 67,000 on sodium dodecyl sulfate-gels. The sedimentation coefficient (5.7S) determined for the Friend leukemia virus enzymes corresponded to a mol wt of 123,000 (26). Wu et al (27) have also found multiple RNase H activities in murine RNA tumor viruses (Rauscher MLV and Kirsten MSV) separable by phosphocellulose chromatography.…”

Section: Discussionsupporting

confidence: 91%

“…It is clear from the results reported here as well as elsewhere (1,12,19,26,27) that Mn2+ is greatly preferred over Mg2+ as the divalent metal ion for assaying poly(A) (dT) 12-18-directed and 70S RNA-directed DNA polymerase activity and DNA polymerase-associated RNase H activity 1[HJpoly(A).poly(dT) degradation) of mammalian type C RNA tumor viruses. In fact, in our hands the 70S RNAdirected DNA polymerase activity and RNase H-I activity (DNA polymerase associated) of MSV could barely be detected in the presence of Mg2+ at a concentration that is optimal for AMV DNA polymerase-RNase H. Therefore, analyses (25) that attempt to compare the enzyme activities in avian versus mammalian RNA tumor viruses using Mg2+ as the only divalent metal ion for assay are inconclusive.…”

Section: Discussionsupporting

confidence: 57%

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Purification and characterization of the DNA polymerase and RNase H activities in Moloney murine sarcoma-leukemia virus

Gerard¹,

Grandgenett²

1975

J Virol

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Two RNase H (RNA-DNA hybrid ribonucleotidohydrolase, EC 3.1.4.34) activities separable by Sephadex G-100 gel filtration were identified in lysates of Moloney murine sarcoma-leukemia virus (MSV). The larger enzyme, which we have called RNase H-I, represented about 10% of the RNase H activity in the virion. RNase H-I (i) copurified with RNA-directed DNA polymerase from the virus, (ii) had a sedimentation coefficient of 4.4S (corresponds to an apparent mol wt of 70,000), (iii) required Mn2+ (2 mM optimum) for activity with a [3H ]poly(A) .poly(dT) substrate, (iv) eluted from phosphocellulose at 0.2 M KCl, and (v) degraded [3H]poly(A) .poly(dT) and [3H]poly(C) .poly(dG) at approximately equal rates. The smaller enzyme, designated RNase H-II, which represented the majority of the RNase H activity in the virus preparation, was shown to be different since it (i) had no detectable, associated DNA polymerase activity, (ii) had a sedimentation coefficient of 2.6S (corresponds to an apparent mol wt of 30,000), (iii) preferred Mg2+ (10 to 15 mM optimum) over Mn2+ (5 to 10 mM optimum) 2.5-fold for the degradation of [3H]poly(A) .poly(dT), and (iv) degraded [3H]poly(A) .poly(dT) 6 and 60 times faster than [3H]poly(C) .poly(dG) in the presence of Mn2+ and Mg2+, respectively. Moloney MSV DNA polymerase (RNase H-I), purified by Sephadex G-100 gel filtration followed by phosphocellulose, poly(A) oligo(dT)-cellulose, and DEAE-cellulose chromatography, transcribed heteropolymeric regions of avian myeloblastosis virus 70S RNA at a rate comparable to avian myeloblastosis virus DNA polymerase purified by the same procedure.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionsupporting

confidence: 91%

Section: Discussionsupporting

confidence: 57%

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Purification and characterization of the DNA polymerase and RNase H activities in Moloney murine sarcoma-leukemia virus

Gerard¹,

Grandgenett²

1975

J Virol

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“…Similar molecular weights have been reported for viral DNA polymerases of murine leukemia viruses and simian sarcoma virus [28,, for enzymes of human breast tumor cells [35] and of human acute leukemic blood cells [36]. Ribonuclease H activity, another unique property [41-451 and probably an integral part of viral RNA-dependent DNA polymerase [41,42] was found in the same range as the DNA polymerase after centrifugation.…”

Section: Discussionsupporting

confidence: 74%

RNA‐Dependent DNA Polymerase from a Cell Line Derived from the Bone Marrow of a Patient with Polycythemia vera

Weimann¹,

Schmidt²,

Kluge

et al. 1975

European Journal of Biochemistry

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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 70 000, as determined by centrifugation. Throughout the purification procedure ribonuclease H activity was co‐purified. Upon dodecyl‐sulfate‐polyacrylamide electrophoresis on microgradient gels two main bands with molecular weights of 68 000 and 66 000 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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“…The enzymes from Friend murine leukemia virus (Fr-MuLV) and from Moloney murine leukemia virus (Mo-MuLV) have been described as a single polypeptide of 80,000 to 84,000 molecular weight (8,12). In other reports the Fr-MuLV enzyme has been characterized as a two-polypeptide complex (15), and the Mo-MuLV enzyme has been shown to consist of three subunits (2). The two polypeptides observed for Fr-MuLV (15) resemble those of the hamster viral enzyme (13).…”

mentioning

confidence: 99%

Further characterization of the Friend murine leukemia virus reverse transcriptase-RNase H complex

Moelling¹

1976

J Virol

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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 a subunit and the avian a/3 enzyme complex. The intact murine enzyme resembles the avian /3-containing enzyme complex and is different from a 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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RNA‐Dependent DNA polymerase and ribonuclease H from Friend virions

Cited by 13 publications

References 17 publications

Purification and characterization of the DNA polymerase and RNase H activities in Moloney murine sarcoma-leukemia virus

Purification and characterization of the DNA polymerase and RNase H activities in Moloney murine sarcoma-leukemia virus

RNA‐Dependent DNA Polymerase from a Cell Line Derived from the Bone Marrow of a Patient with Polycythemia vera

Further characterization of the Friend murine leukemia virus reverse transcriptase-RNase H complex

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