Biochemical and immunological properties of the DNA polymerase and RNAase H activities of purified feline leukemia virus reverse transcriptase

Cancer Letters, 10 (1980) o Elsevier/North-Holland

207-221 Scientific Publishers Ltd.

207

BIOCHEMICAL AND IMMUNOLOGICAL PROPERTIES OF THE DNA POLYMERASE AND RNAase H ACTIVITIES OF PURIFIED FELINE LEUKEMIA VIRUS REVERSE TRANSCRIPTASE

HYUNE M. RHO and ROBERT C. GALL0 Labomtory of Tumor Cell Biology, National Health, Bethesda, MD 20205 (U.S.A.)

Cancer Institute,

National

Institutes

of

(Received 14 April 1980) (Revised version received 6 June 1980) (Accepted 9 June 1980)

SUMMARY

Feline leukemia virus DNA polymerase was purified by ion-exchange and nucleic acid affinity chromatographies. The enzyme consists of a single polypeptide chain of mol. wt. approx. 72,000 as determined by both glycerol density gradient centrifugation and SDS-polyacrylamide gel electrophoresis. The preferred divalent cation for DNA synthesis is Mn*’ on a variety of template-primers, and its optimum concentration appears to be significantly lower than reported results of other mammalian type-C viral enzymes. The purified enzyme also contained RNAase H activity. Both DNA polymerase and RNAase H activities appear to reside on the same molecule as demonstrated by the copurification of both activities through various purification steps. The divalent cation requirement for maximum activity of RNAase H is also similar to that of the DNA polymerase. RNAase H without detectable polymerase activity was generated by a limited chymotrypsin digestion of the purified reverse transcriptase. This RNAase H activity was inhibited equally effectively as RNAase H in the intact reverse transcriptase by antisera prepared against reverse transcriptase of feline leukemia virus. These results indicate that the RNAase H catalytic activity of reverse transcriptase is distinct from the polymerase portion of the molecule. Since the RNAase H activity appears to be more stable, the measurement of RNAase H activity with a proper antibody might be useful for assaying tumor cells for the presence of the viral enzyme.

INTRODUCTION

The biological function of reverse transcriptase in RNA tumor viruses is to catalyze synthesis of the DNA provirus the copy of the viral RNA

208

genome which is integrated into the host DNA. Most purified DNA polymerases from avian, rodent, feline, and simian RNA tumor viruses exhibit 2 enzyme activities: RNA-dependent DNA polymerase and RNAase H. The function of RNAase H during the synthesis of the proviral DNA is obscure. However, it has been proposed that during the synthesis of complementary DNA, RNAase H degrades the RNA template and provides a primer with 3’-OH to initiate the synthesis of the second strand of DNA [14,21]. Feline leukemia is a naturally occurring leukemia about which there is considerable information and evidence implicating feline leukemia virus ( FeLV) in its etiology [ 11. Detailed information on the reverse transcriptase of FeLV may be useful in following the transmission of the virus since antibodies to DNA polymerase can be a sensitive indicator of natural infection of cats by FeLV [4,11] and in controlling replication and transmission of the virus. In addition, there is evidence for the presence of immunoglobulins on the surface of some human myeloblastic leukemia cells [ 51. After their elution and isolation, these immunoglobulins sometimes interact with mammalian type-C viral DNA polymerases, especially that of FeLV [ 51. Detailed characterization of the reverse transcriptase might facilitate the characterization of the leukemic cell surface antigen complexed to the immunoglobulin which presumably should be structurally related in some aspects to reverse transcriptase. In this report, we describe purification and characterization of FeLV reverse transcriptase by biochemical and immunological techniques. Proteolytic digestion of the reverse transcriptase was able to generate RNAase H activity which was neutralized by antibody to the reverse transcriptase. MATERIALS

AND METHODS

Materials Theilen strain of FeLV was grown in a suspension culture of a cat embryo cell line (FL-74), and purified by sucrose density gradient. Antisera against purified reverse transcriptase of FeLV and simian sarcoma associated virus (SSAV) were prepared in goats as described [ 111. Other antisera were purchased from Cappel Lab. 3H-labeled nucleotides, and homopolymers were obtained from New England Nuclear Co., and oligomer-homopolymer and homopolymer-agarose from P.L. Biochemicals. E. coli DNA I polymerase was generously supplied by Dr. Loeb, Fox Chase Cancer Institute, Philadelphia, PA. Purification of FeLV reverse transcriptase FeLV (50 mg) was pelleted at 100, 000 X g for 90 min at 4°C. The resulting pellet was sonicated in 25 ml of Buffer A containing 50 mM Tris-HCl (pH 7.9), 500 mM KCl, 5 mM dithiothreitol (DTT), 20% glycerol, 0.5% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride (PMSF),

209

and chromatographed on a DEAE-cellulose column to remove nucleic acids. The flow-through was dialyzed overnight against Buffer A containing 20 mM NJ+bis[ 2-hydroxyethyll-2-aminoethane sulfonic acid (BES) (pH 6.5), 1 mM EDTA, 0.1% Triton X-100, 100 mM KCl, 20% glycerol, and 1 mM PMSF, and applied to a phosphocellulose (pll) column (1.5 X 10 cm) previously equilibrated with Buffer B. After it had been washed with the same buffer, the polymerase was eluted with a 100 ml of linear 0.1 to 1.0 M KC1 gradient in the buffer. The enzyme peak was pooled and dialyzed against Buffer C containing 50 mM Tris--HCl (pH ‘7.5), 100 mM KCl, 1 mM DTT, 5 mM MgC12,0.1% Triton X-100, 20% glycerol, and 1 mM PMSF, and further purified by a poly(C)-Sepharose column (1.0 X 5 cm) as described [3,11]. The column was washed with Buffer C and eluted with a total 80 ml of a linear gradient of 0.1 M to 1.0 M KC1 in the buffer. The enzyme was eluted between 0.2 and 0.30 M KC1 (Fig. 1B). The peak fractions were diluted with Buffer D containing 50 mM Tris-HCl (pH 7.4), 1 mM DTT, 0.01% Triton X-100, and 20% glycerol, and loaded on the poly(A) - oligo(dT)-cellulose column (1.5 X 10 cm) and washed with Buffer D containing 0.1 M KCl. The enzyme was eluted with a 100 ml of a linear gradient of 0.1 M-l.0 M KC1 in the buffer. The enzyme peak, eluted between 0.25 M and 0.32 M KC1 (Fig. lC), was concentrated by dialysis against Buffer E containing 100 mM Tris-HCl (pH 7.9), 100 mM KCl, 0.001% Triton X-100, 2 mM DTT, and 20% Ficoll. The concentrated enzyme was added to the equal volume of cold glycerol and stored at -20°C for a further characterization. DNA polymerase assays The standard reaction mixture for DNA polymerase activity in 0.1 ml contained 50 mM Tris-HCl (pH 7.9), 1 mM DTT, 0.05 mM MnCl,, 50 mM KCl, 0.01% Triton X-100, 20% glycerol, 10 tig BSA, 1 pg of poly(A) * oligo(dT), 15 PM of [3H]TTP (50 Ci/mmol). When AMV 70s RNA was used as template, 0.1 mM MnCl,, 15 r.lMof [3H]TTP, and 100 PM each of dATP, dCTP, dGTP were instead adopted in the standard reaction mixture. When poly( Cm) - oligo( dG) was used as template, 1 mM MnC12,and 15 PM of [3H]dGTP (30 Ci/mmol) were adopted in the standard reaction mixture. The reaction mixture was incubated at 37°C for 30 min and the synthesized polymer was measured on Whatman DEAE-cellulose (DE-81) paper disks as described [ 31. Synthesis of fd-DNA - [‘H]RNA hybrid The fd viral DNA is a circular single-stranded DNA isolated from the filamentous bacteriophage fd. The procedure to synthesize the fd-DNA C3H]RNA hybrid was as previously described from Sarngadharan et al. [ 131 with some modifications. Briefly, the reaction mixture (0.1 ml) contained 80 mM Tris-HCl (pH 8.0), 50 mM KCl, 10 mM DTT, 10 mM MnC13, 0.5 mM each of ATP, GTP, and CPP, 0.14 mM C3H]UTP (35 Ci/mmol),

210

2.0 0 r’ g

1.5

u) 9 !*

1.0

z -

0.5

0

C D.6

FRACTION NUMBER

Fig. 1. Purification of FeLV reverse transcriptase by ion exchange and sequential affinity chromatography. Fractions were assayed for RNA-directed DNA polymerase activity with poly(A) * oligo(dT) as described in the text. Salt concentrations were measured conductimetrically. (A) phosphocellulose chromatography; (B) poly( C)-Sepharose chromatography ; ( C) poly( A) * oligo( dT)-cellulose chromatography.

211

10 pg of fd-DNA (Miles Co.) and 3 pg of E. coli RNA polymerase (holoenzyme). After incubation at 37°C for 6 h, the hybrid was isolated by pronase and chloroform treatment followed by a Sephadex G-50 chromatography. The hybrid, with specific activity of 10,000 cpm/ng, was more than 90% resistant to RNAase A digestion in 50 mM Tris-HCl (pH 7.5), 0.3 M NaCl. RNAase H assay The standard reaction mixture for RNAase H assay contained in 0.1 ml: 50 mM Tris -HCl (pH 7.9), 1 mM DTT, 0.05 mM MnC12, 50 mM KCl, 10 ng of the fd-DNA - [‘H]RNA hybrid. The reaction mixture was incubated at 37°C for 15 min and the acid-soluble counts were measured by adding 40 pg of BSA and 4 ~1 of 100% trichloracetic acid. The mixture was cent& fuged and the supernatant was counted in liquid scintillation fluid. Glyceml gmdient FeLV DNA polymerase was layered on a lO-30% glycerol gradient containing 50 mM Tris-HCl (pH 7.4), 2 mM DTT, 0.3 M KCl, 0.05 mM MnCl,, 0.1% Triton X-100. The gradient was centrifuged for 18 h at 45,000 rev./min at 4°C in an SW 50.1 rotor of a Spinco ultracentrifuge. Fractions were collected from the bottom of the centrifuge tube, and aliquots from each fraction were assayed for DNA polymerase and RNAase H activities. Parallel gradients were run using E. coli DNA polymerase I and bovine serum albumin (BSA) as standard markers. E. cob DNA polymerase activity was measured with poly(dA) - oligo(dT) as template-primer. The position of BSA in the gradient was identified by a slab polyacrylamide gel electrophoresis followed by Coomassie blue staining. Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis in the presence of 0.1% SDS (SDS-PAGE) was performed according to the method of Laemmli [ 71. Polyacrylamide gels (10%) were stained with Coomassie blue and destained with 7.5% acetic acid. Immunologic assays The neutralization assay and double antibody immunoprecipitation were performed as previously described [ 161, with a minor modification. Briefly, FeLV reverse transcriptase was incubated with primary antibody (goat IgG) for 4 h at 4°C in a 50 ~1 reaction mixture containing 50 mM Tris-HCl (pH 7.8), 50 mM KCl, 0.05 mM MnCl,, 0.01% Triton X-100, and 1 mg/ml BSA. The proper amount of second antibody (rabbit IgG against goat) was then added to the reaction mixture to precipitate the primary antibody. After further incubation for 17 h at 4”C, the reaction mixture was diluted 4-fold. One series of reaction mixtures were centrifuged for 5 min in a Beckman microfuge B. The enzyme activity remaining in the super-

212

natant of the centrifuged samples and in an aliquot of the non-centrifuged samples were determined in the standard reaction mixture. The amount of enzyme was expressed as percent of control activity, based on the amount of polymerase activity remaining when the primary antibody consists solely of non-immune IgG. The amount of second antibody was properly titrated for a maximum precipitation. All antibodies and BSA were treated with 2 mM PMSF and cleared by centrifugation before use. Proteolysis

of FeLV

reverse

transcriptase

by chymotrypsin

FeLV reverse transcriptase (about 4 pg), purified by an additional affinity poly(G)-Sepharose column, was diluted with buffer containing 50 mM Tris-HCl (pH 7.4), 0.05 mM MnCl,, 0.3 M KCl, 5 mM DTT, 0.1% Triton X-100 and 20% glycerol. The enzyme was digested with 1 pg chymotrypsin at 37°C until the remaining polymerase activity was about 40% as described [ 81. The reaction was stopped by addition of 2 mM PMSF and adjusted to the final concentration of KC1 and DTT to 1.5 M and 10 mM, respectively. The reaction mixture was incubated at 4°C for 2 h with occasional shaking, and then diluted with a buffer containing 50 mM Tris-HCl (pH 7.4), 0.05 mM MnCl,, 5 mM DTT, 0.1% Triton X-100, and 20% glycerol to adjust the KC1 concentration to 20 mM. The diluted reaction mixture was then loaded on a poly(G)-agarose column (0.5 X 4 cm) previously equilibrated with the same buffer, and washed with the same buffer. The enzyme was eluted with a linear gradient of a 30 ml of O-O.8 M KC1 in the buffer. The polymerase and RNAase H were assayed through fractions as described above. RESULTS

AND DISCUSSION

Purification

of FeLV

reverse transcriptase

The FeLV enzyme extract was passed through a DEAE-cellulose column to remove nucleic acids. The enzyme was then chromatographed on a phosphocellulose at pH 6.5 (Fig. 1A). At this pH, reverse transcriptase was eluted at 0.4 M KCl. The major glycoprotein (gp70) was eluted between 0.1 and 0.2 M KC1 and major group specific internal protein (~30) between 0.2 and 0.3 M KC1 as reported [ 171. The phosphocellulose chromatography at pH 6.5 separated these 3 viral proteins with a minor cross-contamination. Subsequent chromatographies on poly( C)-Sepharose and the hybrid affinity column, poly( A) - oligo(dT)-cellulose, provided a relatively homogeneous reverse transcriptase (Figs. 1 and 3). However, additional Lentilelectinagarose chromatography gave a reverse transcriptase free of glycoproteins (data not shown). qetermination

of molecular

weight

An aliquot of the purified reverse transcriptase was adjusted to the same buffer strength as in the glycerol gradient and layered on lO--30% glycerol

16.

FRACTION NUMBER

Fig. 2. Glycerol gradient sedimentation of purified FeLV reverse transcriptase. The gradient was centrifuged and fractionated as described in the text. Aliquots of each fraction were assayed for DNA polymerase activity with poly( A) . oligo( dT) (0 ) and with . oligo(dG) (A). RNAase H activity (0) was analyzed with [3H]fd hybrid. PolY(W

gradient for a centrifugation. Figure 2 shows the sedimentation profile of polymerase activity with poly(A) * oligo(dT) and poly( Cm) * oligo(dG) as template-primer. RNAase H activity appeared at the same position as the polymerase activity in the gradient. Assuming the marker proteins behaved in a similar way in the sedimentation, FeLV reverse transcriptase sedimented at 72,000 daltons. This value is similar to the reported molecular weight estimates by gel filtration in 6 M guanidine-HCl [ 191. Figure 3 shows SDS-PAGE analysis of the purified FeLV reverse transcriptase, revealing a single polypeptide of mol. wt 72,000 relative to the marker protein bovine serum albumin. These results suggest that FeLV reverse transcriptase consists of a single polypeptide of 72,000 daltons.

Biochemical

properties

of FeLV reverse transcriptase

The DNA polymerase and RNAase H activities were copurified through several steps of purifications by ion-exchange and affinity chromatographies,

-

45,ooo

-

25,ooo

-DYE

Fig. 3. SDS-PAGE analysis of purified FeLV reverse transcriptase. Reverse transcriptase was subjected to electrophoresis on a 10% slab gel, and the gel was stained with Coomassie blue. The following proteins were used as markers: bovine serum albumin, mol. wt 68,000; ovalbumin, mol. wt 45,000; and chymotrypsin, mol. wt 25,000.

215 TABLE

1

EFFICIENCY OF DNA POLYMERASE

UTILIZATION

Template-Prime?

poly(Cm)

OF

VARIOUS

‘The amount of 0.58 fig/assay.

pmole incorporated

Mg” (5) Mn2+ (0.05)

TTP TTP dGTP dGTP TTP TTP TTP TTP

28.49 121.00 1.00 6.20 < 0.70 < 0.60 0.10 1.24

Mn2+ (10) ~g*+ iioj Mn*+ (0.1)

synthetic

FeLV

[ “HISubstrate

- olko(dGh 1y a

705 RNA

BY

Optimum divalent cation (mM)

Mg” (10) AMV

TEMPLATE-PRIMERS

template-primer

was

1 pg/assay and AMV

705 RNA

was

and glycerol gradient centrifugation. These results suggest that both enzyme activities reside on the same polypeptide molecule as reported for other retroviruses [ 14,211. The choice of divalent cation with various template-primers can markedly affect the transcription efficiency by FeLV DNA polymerase (Table 1). At the optimum concentration of Mn” (0.05--0.1 mM), poly(A) - oligo(dT)directed poly(dT) synthesis was 4 times more efficient than at the optimum Mg2+ concentration (5 mM). The cation requirement with modified template-primer [poly(Cm) - oligo(dG)] or natural template (AMV 70s RNA) was higher than with poly(A) - oligo(dT) (Table 1). In particular, the optimum concentration of Mn2’ (Fig. 4A) appeared to be lower with FeLV DNA polymerase than reported values with the enzymes from MuLV [ 2,201. This optimum concentration was not changed even though the concentration of salts (KC1 or NaCl) was changed (Fig. 4B). The optimum concentration of salts appeared to be between 50 mM and 80 mM (Fig. 4B), which is similar to the reported values for other mammalian and avian retroviruses [ 14,211. The optimum concentration of cation and salts for the RNAase activity of FeLV reverse transcriptase was similar to those of DNA polymerase (Figs. 5A, B). However, the RNAase H could use Mg2+ more effectively to catalyze the degradation of fd-DNA * [ 3H] RNA hybrid (Fig. 5A) as reported [ 201. Neutralization

and antibody

binding

assay

Antibodies prepared against DNA polymerase of disrupted virions have been shown to possess neutralization activities [15,18] and binding properties [6,10,11]. The FeLV DNA polymerase was tested for binding of hyperimmune IgG and for enzyme activity after its binding. Figure 6 shows

216

16 -

6-

0.025

0.1

0.4 DIVALANT

1

I

I

1

0

25

50

75

2

0.6

100

CATION

4

6

(mM)

,

I

1

I

/

125

150

175

200

225

SALT CONCENTRATION

6

c 251

(mMl

Fig. 4. Effects of cation and salt concentration on FeLV reverse transcriptase activity with poly( A) - oligo(dT). (A) Divalent cation preference of the enzyme in the presence of 50 mM KC1 was assayed with different concentration of Mn’+ (0) and Mg” (0). (B) Effects of KC1 ( l) and NaCl (A) on the enzyme activity were assayed in the presence of 0.05 mM Mn*+. Effects of KCl (0) and NaCl (A) were also investigated in the presence of 0.5 mM Mnl+, which is the known optimum concentration for mammalian retroviruses [ 12,201.

217

DIVALENT

CATION

0nM)

la

16

0

1 25

50

75

1 100

/ 125

I 150

SALT CONCENTRATION

I 175

1 200

1 225

I 250

(mM)

Fig. 5. Effects of cation and salt concentration on RNAase H activity with [ 3H]fd-hybrid. (A) Divalent cation preference of the RNAase H activity in the presence of 50 mM KCl was assayed with different concentrations of MnZ+ (0) and Mg*+ (0). (B) Effects of KCl ( l ) and NaCl (0 ) were assayed in the presence of 0.05 mM Mnl+.

218

loo

-,

so-

80 -

70 -

MI-

50 -

40 -

30 -

20 -

10 -

OL 0

I

0.0256

I

1

0.64

0.128 IgG

I

3.2

CONCENTRATION

I

1

I

16

80

400

[ug/ml]

Fig. 6. Enzyme neutralization by double antibody immunoprecipitation assay. The enzyme neutralization assays were carried out in duplicate as described in the text. The residual enzyme activities were measured before separation of the antibody-bound reverse transcriptase (0) and after separation by centrifugation ( l ).

that the residual polymerase activity was about lO--30% less when the immune complex was removed by centrifugation. Similar observations were reported with cellular DNA polymerase y [ 121. These results suggest that the binding of antibody to reverse transcriptase does not completely inhibit the DNA polymerase activity. It is possible that the immunogenic and catalytic sites are different. Genemtion of RNAase H by chymotrypsin FeLV reverse transcriptase consists of a single polypeptide with an estimated mol. wt of 72,000 and exhibits the properties of a viral reverse transcriptase containing both DNA polymerase and RNAase H activities.

219

To see if the 2 enzyme activities are located on different positions of the polypeptide molecule, the poly(G)-Sepharose purified FeLV reverse transcriptase was mildly digested with chymotrypsin and rechromatographed on poly(G)-Sepharose column. The chymotrypsin digestion caused degradation of the reverse transcriptase activities and gave rise to a new RNAase H peak which eluted at 0.1 M KC1 in the gradient (Fig. 7B). This peak did not show any significant DNA polymerase activity. Moreover, the RNAase H activity was completely inhibited by antisera prepared against FeLV DNA polymerase. This result suggests that the new RNAase H peak was originated from the FeLV reverse transcriptase by proteolytic cleavage. Final proof of a percursor-product relationship will await additional analysis. It is not known if DNA polymerase alone could be created by proteolytic

r

- 0.0 - 03

- 0..

-

_ .

0.)

- 0.2 B

P

OeCB

i

E g

-.m

f

0.0

f

I

8 0.s E -1

- 0.4

- 0-a

- 0.2

- 0.t

-a0

Fig. 7. Poly(G)Sepharose chromatography of FeLV reverse transcriptase before and after limited digestion with chymotrypsin. Aliquots from each fraction were assayed for DNA polymerase and RNAase H activities as described in the text. (A) control; (B) enzyme activities after digestion with chymotrypsin. DNA polymerase activity was assayed in the absence (0) and presence of antibodies (9); RNAase H activity was also assayed in the absence (0) and presence of antibodies (A). The amount of antiserum added was 10 rg IgG/reaction mixture.

6 Y

220

digestion. Since RNAase H activity is more stable than DNA polymerase as reported [ 8,9,20], assays for viral RNAase H activity with a proper antibody might be useful for assaying tumor cells for the presence of the viral enzyme. REFERENCES 1 Essex, M., Cotter, S., Stephenson, J., Aaronson, S. and Hardy, Jr., W. (1977) Leukemia, lymphoma, and fibrossrcoma of cats as models for similar diseases of man. In: Origin of Human Cancer, pp. 1197-1214. Editors: H.H. Hiatt, J.D. Watson and J.A. Winsten. Cold Spring Harbor Laboratory. 2 Gerad, G. and Grandgenett, D.P. (1975) Purification and characterization of the DNA polymerase and RNase H activities in Moloney sarcoma-leukemia virus. J. Virol., 15, 785-797. 3 Grandgenett, D.P. and Rho, H.M. (1975) Binding properties of avian myeloblastosis virus DNA polymerases to nucleic acid affinity columns. J. Virol., 15, 526-533. 4 Jacquemin, P.C., Saxinger, C., Gallo, R.C., Hardy, W.D. and Essex, M. (1978) Antibody response in cats to feline leukemia virus reverse transcriptase under natural conditions of exposure to the virus. Virology, 91, 472-476. 5 Jacquemin, P.C., Saxinger, C. and Gallo, R.C. (1978) Surface antibodies of human myelogenous leukemia leukocytes reactive with specific type-C viral reverse transcriptases. Nature, 276, 230-236. 6 Krakower, J., Barbacid, M. and Aaronson, S. (1977) Radioimmunoassay for mammalian type-C viral reverse transcriptase. J. Virol., 22, 331-339. 7 Laemmli, V.K. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. 8 Lai, M.H.T. and Verma, I.M. (1978) Reverse transcriptase of RNA tumor viruses. V. In vitro proteolysis of reverse transcriptase from avian myeloblastosis virus and isolation of a polypeptide manifesting only RNAase H activity. J. Virol., 25, 652663. 9 Moelling, K. (1976) Further characterization of the Friend murine leukemia virus reverse transcriptase-RNase H complex. J. Virol., l&418-425. 10 Panet, A., Baltimore, D. and Hanafusa, T. (1975) Quantitation of avian RNA tumor virus reverse transcriptase by radioimmunoassay. J. Virol., 16, 146-152. 11 Rho, H.M. and Gallo, R.C. (1979) Characterization of reverse transcriptase from feline leukemia virus by radioimmunoassay. Virology, 99, 192-196. 12 Robert-Guroff, M. and Gallo, R.C. (1974) Serological analysis of cellular and viral DNA polymerases by an antiserum to DNA polymerase -y of human lymphoblasts. Biochemistry, 16, 2874-2880. 13 Sarngadharan, M.G., Leis, J.P. and Gallo, R.C. (1975) Isolation and characterization of a ribonuclease from human leukemic blood cells specific for ribonucleic acid of ribonucleic acid-deoxyribonucleic acid hybrid molecules. J. Biol. Chem., 250, 365373. 14 Sarngadharan, M.G., Robert-Guroff, M. and Gallo, R.C. (1978) DNA polymerase of normal and neoplaatic mammalian cells. Biochim. Biophys. Acta, 516,419-487. 15 Scolnick, E.M., Parks, W.P. and Todaro, G.J. (1972) Reverse transcriptases of primate viruses as immunological markers. Science, 177, 1119-1121. 16 Smith, R.G., Abrell, J.W., Lewis, B.J. and Gallo, R.C. (1975) Serological analysis of human deoxyribonucleic acid polymerases. J. Biol. Chem., 250, 1702-1709. 17 Strand, M. and August, J.T. (1973) Structural proteins of oncogenic ribonucleic acid viruses. J. Biol. Chem., 248, 5627-5633. 18 Todaro, G.J. adn Gallo, R.C. (1973) Immunologic relationship of DNA polymerase

221 from human acute leukemia cells and primate and mouse leukemic virus reverse transcriptase. Nature, 244, 206209. 19 Tronick, S.R., Scolnick, E.M. and Parks, W.P. (1972) Reversible inactivation of the deoxyribonucleic acid polymerase of Rauscher leukemia virus. J. Virol., 10, 885888. 20 Verma, I.M. (1975) Studies on reverse transcriptase of RNA tumor viruses III. Properties of purified Moloney murine leukemia virus DNA polymerase and associated RNase H. J. Virol., 15, 843-854. 21 Verma, I.M. (1977) The reverse transcriptase. Biochim. Biophys. Acta, 473, l-38.