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(Reverse Transcriptase) of Murine Type-C RNA Tumor Viruses (Anion Exchange Chromatography/Core Structure/Antibody to Reverse Transcriptase) ALAN M

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(Reverse Transcriptase) of Murine Type-C RNA Tumor Viruses (Anion Exchange Chromatography/Core Structure/Antibody to Reverse Transcriptase) ALAN M Proc. Nat. Acad. Sci. USA Vol. 71, No. 5, pp. 1871-1876, May 1974 Separation of Ribonuclease H and RNA Directed DNA Polymerase (Reverse Transcriptase) of Murine Type-C RNA Tumor Viruses (anion exchange chromatography/core structure/antibody to reverse transcriptase) ALAN M. WU*, M. G. SARNGADHARAN*, AND ROBERT C. GALLOt * Department of Molecular Biology, Bionetics Research Laboratory, and t Laboratory of Tumor Cell Biology, National Cancer Institute, Bethesda, Maryland 20014 Communicated by C. B. Anfinsen, January 21, 1974 ABSTRACT Ribonuclease H (RNA -DNA-hybrid ribo- was partially purified. Similar to the avian system, the RNase nucleotidohydrolase, EC 3.1.4.34) has been reported to H activity co-purified with reverse transcriptase activity, but copurify with reverse transcriptase (RNA directed DNA polymerase) of RNA tumor viruses. In addition, viral this could be accounted for by similar properties of two dis- specific ribonuclease H and reverse transcriptase of avian tinct enzymes. In this respect, it is important to note that the type-C viruses are thought to be part of the same poly- estimated molecular size of both mammalian cellular RNase peptide. In this report we show that a fraction of the ribo- H (7-9) and reverse transcriptase of type-C mammalian nuclease H activity from Rauscher murine leukemia and Kirsten murine sarcoma viruses was separated from reverse viruses (10, 11) are similar. transcriptase by anion exchange chromatography while the In this paper we report our studies on the relationship remaining portion co-purified with the viral polymerase. between RNase H and reverse transcriptase activities asso- The amount of this co-purified nuclease activity was about ciated with mouse type-C viruses. Our results indicate that at 4- to 8-fold lower than the activity found in avian myelo- least in murine type-C viruses, RNase H and reverse trans- blastosis virus (with respect to the ratio of ribonuclease H to reverse transcriptase) and this nuclease activity can only criptase not only reside on two separate polypeptides but are be detected by using labeled substrate ofhigh specific radio- probably located in two different "compartments" of the activity. However, a complete separation of ribonuclease H virus. activity from reverse transcriptase was obtained by purify- ing core structures of the virus by sucrose density gradient MATERIALS AND METHODS centrifugation. While reverse transcriptase was present in Viruses. Rauscher murine leukemia virus (R-MuLV) (1011 the cores, there was no detectable ribonuclease H. Further- more, a specific antibody against Rauscher leukemia virus particles per ml) grown in JLSV9 cells, was obtained through reverse transcriptase did not inhibit any virion associated the Virus Cancer Program, National Cancer Institute. Kirsten ribonuclease H activity. Our results suggest that in these murine sarcoma virus (Ki-MuSV) (1011 particles per ml) virions these two enzyme activities reside in two separate grown in normal rat-kidney cells was purchased from Electro- molecules and probably in two different compartments of the virus. These findings emphasize a basic difference Nucleonics. between the avian and murine type-C virus DNA polym- Preparation of Substrates. ['H](A). (dT)m was prepared by erases. incubating 0.24 ,umoles of [1H]ATP (20.9 Ci/mmole) and 5 units of poly(dT) with 330 of Escherichia coli RNA Ribonuclease H (RNase H) (RNA - DNA-hybrid ribonucleo- Amco ,ug tidohydrolase, EC 3.1.4.34) has been found in association polymerase in a reaction mixture of 2.5 ml containing 50 mM 5 mM 1 mM 5 mM di- with reverse transcriptase in type-C RNA tumor virus par- Tris HCl (pH 7.9), MgCl2, MnCl2, thiothreitol and at 370 for 30 min. The ticles (1-6), prompting some speculations on the mechanisms (DTT), 5% glycerol of integration of proviral DNA into host DNA (1, 3, 5). In reaction mixture was deproteinized with phenol-m-cresol-H20 avian myeloblastosis virus (AvMV), the virus-associated (50:7:5, w/v/v containing 0.1% 8-quinolinol) and the poly- after to 0.3 RNase H activity co-purified with reverse transcriptase meric product isolated by precipitation adjusting through cation and anion exchange chromatography and M NaCl with 2 volumes of absolute ethanol followed by a buffer con- sucrose or glycerol velocity gradient centrifugation (1, 3, 4, 6). Sephadex G-50 column chromatography using These two activities co-migrated during nondissociating gel taining 10 mM Tris - HCl (pH 7.4), 0.1 M NaCl, and 1 mM EDTA. - was as above that electrophoresis (6). It was concluded that two enzymatic ['2P](A)n (dT)m prepared except in activities reside on a single polypeptide (6). There is much less [a-'2P]ATP was used place of [3H]ATP. (A),. [3H1(dT)m information on RNase H of mammalian viruses. Grand- was prepared by the polymerization of [3H]deoxythymidine genett et al. (2) studied RNase H activity associated with triphosphate(TTP) (54 Ci/mmole) with R-MuLV reverse . various types of RNA tumor viruses. Among these, reverse transcriptase employing the template-primer, (dT)12_18 (A)n The are as described under the assay transcriptase from Moloney murine sarcoma virus (M-MuSV) details of the reaction system for reverse transcriptase (see below). The product was Abbreviations: RNase H, ribonuclease H; R-MuLV, Rauscher isolated and purified as described above. [3H](A)n was pre- murine leukemia virus; Ki-MuSV, Kirsten murine sarcoma virus; pared by a modified procedure of Singer and Guss (3, 12). The AvMV, avian myeloblastosis virus; M-MuSV, Moloney murine incubation mixture (150 ,A) contained 0.28 umole of [3H]ADP sarcoma virus; BSA, bovine serum albumin; PC, phosphocellu- (18 Ci/mmole), 0.1 M glycine buffer (pH 9.0), 10 mM MgC12, lose; DTT, dithiothreitol. 0.4 mM EDTA, 15,ug of bovine-serum albumin (BSA), and 1871 Downloaded by guest on September 30, 2021 1872 Microbiology: Wu et al. Proc. Nat. Acad. Sci. USA 71 (1974) TABLE 1. Reverse transcriptase and RNase H-like activities from Ki-MuSV and R-MuLV in the flow-through and 0.4 M KCl eluate from phosphocellulose column chromatography step* Reverse RNase transcriptase H-like activityt activityl 2-~ 4 Virus Fractions (pmoles/ml) (pmoles/ml) MuSV Flow-through 131 2675 ', ' (Kirsten) 0.4 M KCl fraction 9513 17 2 MuLV Flow-through 27 5234 0_C (Rauscher) 0.4 M KCl fraction 5119 20 -0.1 * For this study, fifty milliliters each of Ki-MuSV or R-MuLV 10 20 30 40 50 60 were used. See text for the details of enzyme purifications. The fraction number flow-through fractions from both Ki-MuSV and R-MuLV were 75 ml each and the 0.4 M KCl fractions 60 ml each. Peak fractions FIG. 1. DEAE-cellulose column chromatography of RNase were pooled and dialyzed before assay. H-like activities and reverse transcriptase activity from the flow- t Reverse transcriptase was assayed as follows. Reaction through fractions obtained from the PC column step. The details mixtures of 50 ,4 contained 50 mM Tris.HCl (pH 7.9), 1 mM of DEAE-cellulose column chromatography were described in Mn acetate, 60 mM KCl, 1 mM DTT, 0.2 mM EDTA, 10 ug/ml Materials and Methods. The sample applied was the flow-through (dT)1218-(A)., 5.6,AM [3H]TTP (13,000 cpm/pmole), and 20 fractions from the PC column chromatography of Ki-MuSV as ,l of enzyme fraction. The reactions were carried out for 45 min described in Table 1. Reverse transcriptase activities were at 300 and then the acid-insoluble radioactivity was determined. assayed with (dT)12,18. (A). as template-primer and RNase H t RNase H was assayed as follows. Reaction mixtures of 50 Ax activities with [3H](A).-(dT)m as substrate. The specific ac- contained 50 mM Tris HCl (pH 7.9), 1 mM Mn acetate, 1 mM tivity of [3H]TTP was 13,000 cpm/pmole and that of [3H] (A).-- DTT, and 10,000 cpm ['H] (A).* (dT)m. The reactions were carried (dT)m was 15,000 cpm/pmole. (The numbers on the outer left out at 370 for 45 min and terminated by adding 250 ,g of yeast and inner left ordinates have been multiplied by 10-2 and 10-', tRNA and 0.2 ml of 20% CCl1COOH containing 20mM Na pyro- respectively.) phosphate. After standing at 0° for 10 min, the-precipitate was removed by centrifugation at 3500 X g for 5 min. Aliquots of 0.65 units of polynucleotide phosphorylase (Miles Labora- 0.2 ml supernatant were added to 10 ml of Aquasol to determine tory). At the end of 3-hr incubation at 370, the reaction was the acid soluble radioactivity. The enzyme activities were con- terminated by adding sarkosyl to 0.3%. The product was verted to picomoles of labeled nucleotide incorporated or released The data show the activities of RNase purified by gel filtration as described above. qX-174 DNA- per 45 min/ml of enzyme. H and reverse transcriptase for the same aliquot of the pooled was of Stavri- ['H]RNA hybrid prepared using the procedure fractions. anopoulos et al. (13), substituting 4X-174 DNA in place of fi DNA as the template, and using (12.0 Ci/mmole) ['H]UTP activities were obtained in the flow-through and the 0.4 M the RNA strand. The final product was a 1:1 DNA to label KCl eluate. These were separately pooled, dialyzed against as determined analyses in a cesium sulfate RNA hybrid by 1, the enzyme activities further gradient. 100 volumes of buffer and density purified as described below. Enzyme Assays. The assays of purified and endogenous viral For DEAE-cellulose column chromatography, precycled reverse transcriptase (14, 15) and RNase H (3, 7) were per- DEAE-cellulose (DE 52, Whatman) was equilibrated with formed according to the procedures previously described.
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