Proc. Nat. Acad. Sci. USA
Vol. 72, No. 10, pp. 4133-4136, October 1975 Medical Sciences
Purification of RNA-instructed DNA polymerase from human leukemic spleens (reverse transcriptase/leukemia)
S. S. WITKIN*, T. OHNO, AND S. SPIEGELMAN Institute of Cancer Research, College of Physicians & Surgeons, Columbia University, New York, N.Y. 10032
Contributed by S. Spiegelrman, June 23, 1975
ABSTRACT Particles possessing a density of 1.16 g/ml and encapsulating a 70S RNA and a RNA-instructed DNA polymerase (reverse transcriptase) have been prepared from the spleen of a patient with chronic lymphocytic leukemia. These particles have been converted to cores with a density of 1.26 g/ml and containing the enzyme-RNA complex, in complete analogy to the known RNA tumor viruses of avian and murine origin. The reverse transcriptase was purified from the cores by column chromatography to a stage showing a single major protein band of 70,000 daltons in a gel electrophoresis. The enzyme was capable of transcribing heteropolymeric RNA into DNA complements as demonstrated by specific back hybridization to template RNA. The leukemic spleen would a ppear to represent an important source of this enzyme, as well as other potentially important leukemiaspecific reagents.
The use of the "simultaneous detection test" (1) has permitted the identification of particles encapsulating a 70S RNA template with RNA-instructed DNA polymerase (reverse transcriptase) in human leukemic cells (2) as well as in a variety of other human neoplasias (3-6). The human leukemic reverse transcriptase has been partially purified and characterized from fresh leukemic cells (7, 8). The comparatively large amounts of peripheral leukemic white blood cells required for enzyme isolation have imposed severe restrictions on this type of research and confined it to the one or two laboratories that have access to the very limited supply of peripheral leukemic cells. One possible way out of this dilemma is to explore the use of spleens as a possible source of the leukemic enzyme. In addition to providing a possible solution of the logistic problem, such experiments would provide useful information on the relation between diseased tissue and the presence of the virus-like particles. Leukemias almost always involve the spleen and splenectomies are not infrequently performed therapeutically in instances of massive spleen enlargement or persistent platelet destruction. The technology of enzyme isolation from leukemic spleens was first worked out using the murine Rauscher leukemia model (Witkin and Spiegelman, in preparation). It was found that isopycnic separation of virus particles and their conversion to cores by non-ionic detergents (9, 10) provided material suitably enriched for enzyme purification. It is the purpose of the present paper to describe the use of this approach for the successful purification of a reverse transcriptase from a human leukemic spleen. MATERIALS AND METHODS Purification of Core Particles from Human Leukemic Spleen. Approximately 70 g of chronic lymphocytic leukeAbbreviations: AMV, avian myeloblastosis virus; RLV, Rauscher * Present address: Laboratory of Cell Biochemistry, Memorial Sloan-Kettering Cancer Center, New York, N.Y. 10021.
leukemia virus; BSA, bovine serum albumin.
4133
mic spleen were thawed, minced, suspended in 3 volumes of cold 5% sucrose (w/w) in TNM buffer (0.01 M Tris-HCl, pH 8.0, 0.15 M NaCl, 0.002 M MgCl2) and homogenized for approximately 2 min in a Silverson homogenizer (11). The mixture was then centrifuged at 4000 X g and 10,000 X g to remove nuclei and mitochondria, respectively. KCI (4 M) was added to the postmitochondrial supernatant to a final concentration of 0.8 M and the solution was centrifuged at 80,000 X g for 90 min. The high-speed supernatant was discarded and the pellet was resuspended in 5% sucrose in TNM buffer containing 0.8 M KCL. After an 8000 X g centrifugation to remove residual cell debris, the sample was layered in 10-ml aliquots on discontinuous sucrose gradients consisting of 5 ml of 50% sucrose and 10 ml of 25% sucrose, all in TNM buffer and 0.8 M KCI. The gradients were centrifuged at 176,000 X g for 120 min in a 60 Ti rotor. The material present in the 25-50% sucrose interface was removed, diluted about 5-fold, and solid (NH4)2SO4 was added with constant stirring over a 20-min period to a final concentration of 30%. The final pH of the solution was 7.0. After at least 60 min at 40, the solution was centrifuged at 12,000 X g for 30 min. The resulting pellet was drained and resuspended in 0.01 M Tris-HCI, pH 8.3, 0.005 M NaCI, 0.002 M EDTA (S buffer). An aliquot of this suspension was layered on a gradient of 30-65% sucrose in S buffer plus 0.005 M dithiothreitol. Centrifugation was for 16 hr at 98,000 X g in an SW-41 rotor. Eleven fractions of equal volume were collected from below and the density of each was determined by refractometry. Each fraction was then diluted with 0.01 M Tris'HCl, pH 8.0, centrifuged at 98,000 X g for 60 min, and the resulting pellets were resuspended in 100 ,l 0.01 M Tris-HCI, pH 8.0. Viral cores were prepared by procedures previously described (9, 10). An aliquot of the (NH4)2SO4 fraction in S buffer was adjusted to a final concentration of 1% Nonidet P-40 and 0.1 M dithiothreitol, incubated at 40 for 5 min and layered on a 30-65% sucrose gradient in S buffer and dithiothreitol. Centrifugation and core concentration were as described for viral purification. Disruption of Core Particles. The mildest conditions affording almost complete solubilization and recovery of the enzyme from the cores involved treatment with 1% Nonidet P-40 and 0.7 M KCL. Core pellets were resuspended in 0.01 M Tris-HCI (pH 8.0) containing 1% Nonidet P-40 and 0.7 M KCI, and were left at 40 for 20 min followed by dilution 50-fold in starting buffer (0.01 M potassium phosphate, pH 7.2, 10% glycerol, 0.001 M EDTA, 0.002 M dithiothreitol, 0.2% Nonidet P-40). DEAE-Cellulose Chromatography. The diluted disrupted cores were loaded on a 16.5 cm X 2.5 cm column of DEAE-cellulose (Whatman DE52) equilibrated with starting buffer. The column was then washed with 100 ml starting buffer and the enzyme activity was eluted by raising the
4134
Medical Sciences: Witkin et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
81 A
4
130
3
6
1.20 0
to
E
4
0
E EM
0
E
2 1.10
2
1.00 FRACTION NUMBER
FIG. 1. Isolation of virus and viral cores from human chronic lymphocytic leukemic spleen. A cytoplasmic particulate fraction was prepared from 20 g of infected spleen as described in Materials and Methods. Aliquots before (A) and after (B) treatment with Nonidet P-40 were layered on 30-65% sucrose gradients and centrifuged at 78,000 X g for 16 hr. Twelve equal fractions were collected from below, concentrated, and assayed for endogenous reverse transcriptase activity.
potassium phosphate to 0.40 M. Fractions of 3.2 ml were collected and 10 Mil aliquots of each were assayed for ability to incorporate [3H]deoxythymidine triphosphate (5 X 103 cpm/pmol) into acid-insoluble material using oligo(dT).poly(rA) as template. Phosphocellulose Chromatography. The pooled peak enzyme fractions from the DEAE-cellulose column were diluted 15-fold in starting buffer and loaded, at a constant rate of 20 ml/hr, on a 17 cm X 1.5 cm column of phosphocellulose (Whatman P11) equilibrated with starting buffer. The column was then washed with 60 ml starting buffer and the enzyme activity was eluted with 160 ml of a 0.01 M-0.5 M potassium phosphate gradient. The flow rate was maintained at 20 ml/hr and fractions of 1.6 ml were collected and assayed as described above. The enzyme activity in the pooled peak fractions was concentrated by rechromatography on a 4 cm X 0.9 cm phosphocellulose column and elution with 0.4 M potassium phosphate in starting buffer. Agarose Gel Filtration. The concentrated phosphocellulose activity was applied to a 50 cm X 0.9 cm agarose column (Bio-Gel A-0.5m) and equilibrated with 0.35 M potassium phosphate (pH 7.2) in starting buffer. The flow rate was about 4 ml/hr and 0.4 ml fractions were collected. Polyacrylamide Gel Electrophoresis. Polyacrylamide gel electrophoresis on 5% gels, in the presence of sodium dodecyl sulfate, was as reported previously (12). Polymerase Assays. The assay mixture for the synthetic templates reaction contained 5 Mumol of Tris*HCl (pH 8.0), 0.6 gmol of Mg9l2, 0.02 ,umol of unlabeled deoxyadenosine triphosphate, 0.003 M4mol of unlabeled deoxythymidine triphosphate, 0.1 Mimol of dithiothreitol, 0.4 Mg of oligo(dT)poly(rA) (Miles Research) and [3H]deoxythymidine triphosphate (New England Nuclear) to yield 5 X 103 cpm/pmol in a final volume of 100 ,ul. Incubation was for 10 min at 370 and was terminated by the addition of 5 volumes of cold trichloroacetic acid mix (10% trichloroacetic acid and 10% saturated sodium pyrophosphate in H20). After at least 10 min at 40, acid-precipitable radioactivity was collected on filters and counted as described (12). Assays using avian myeloblastosis virus (AMV) RNA or avian myeloblast DNA contained, in a final volume of 100 Mul, 5.0 Mimol of Tris-H1l (pH 8.0), 0.6 Mmol of MgCl2, 0.016 Mmol each of unlabeled deoxythymidine triphosphate, deoxyadenosine triphosphate, and deoxyguanosine triphos-
phate, 1.4 Mug of RNA or DNA, 0.1 pmol of dithiothreitol, and 0.002-0.016 gmol (6.4 X 102 to 2.0 X 104 cpm/pmol) of [3H]deoxycytidine triphosphate. In addition, the RNA-templated reactions contained 0.1 ;ig of oligo(dT) (Miles Research) and 10 gg of actinomycin D (Sigma). Reaction conditions and radioactivity determination were as described above. Purification of the reaction product, hybridization to AMV RNA or Rauscher leukemia virus (RLV) RNA, and analysis on Cs2SO4 equilibrium gradients were as previously described (11). RESULTS Reverse Transcriptase Activity in the Virus and Core Density Regions from Human Leukemia Spleens. The ammonium sulfate precipitates from the chronic lymphocytic leukemic spleens were treated as described in Materials and Methods. Eleven equal fractions were collected, concentrated, and assayed for endogenous activity (12). A sharp peak of enzyme activity was detected at a density of 1.17 g/ml (Fig. 1A). Detergent treatment (Fig. 1B) resulted in a shift of all of the endogenous enzyme activity to a density of 1.26-1.27 g/ml, characteristic of core-like particles. As had been done in our previous investigations (2, 3-6, 9, 10), it was established by ribonuclease or alkali sensitivity and Cs2SO4 gradient analysis (data not shown) that the products of the endogenous activities shown in Fig. 1A and B were complexes between the nascent DNA chain and a large RNA molecule. Chromatography of Spleen Reverse Transcriptase on DEAE Cellulose. Reverse transcriptase was solubilized from the core fraction in 1% Nonidet P-40 and 0.7 M KC1, diluted 50-fold to reduce the salt and detergent concentrations, and chromatographed on DEAE-cellulose as described in Materials and Methods. One main peak of oligo(dT)-poly(rA) activity was observed (Fig. 2A), containing 5% of the input protein. Greater than 90% of the activity was recovered, yielding a 19-fold enzyme enrichment. We have found that if the polymerase is associated with RNA, as in the core preparations, the enzyme activity will bind to DEAE-cellulose. In contrast, with preparations of enzyme dissociated from nucleic acid, the polymerase activity is detectable in the DEAE-cellulose flow-through. Chromatography on Phosphocellulose. The active frac-
Medical Sciences: Witkin et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
4135
UAt
A 0.4-
0.2E 20
10
2.0
B 0.5
I~~~I
If)
~~~~~~~~~~
x
a-
0 to to
30
O
I.0
.0.4 0.3 rji 0.2 9 0. I 0
8 Lg8.j
0.51
MIGRATION 20
5.0
40
60
80
BSA
C
100
COVALBUMIN
4.0
3.0
2.0 1.0
I 20
40
60
80
100
FRACTION NUMBER
FIG. 2. (A) DEAE-cellulose chromatography of leukemic spleen enzyme. Core-like particles isolated from a leukemic spleen were treated with 1% Nonidet P-40 and 0.7 M KCl and the solubilized enzyme activity was chromatographed on a 16.5 cm X 2.5 cm column. Elution was with 0.4 M potassium phosphate. Ten microliters of each fraction (3.2 ml) were assayed using an oligo(dT).poly(rA) template. (B) Phosphocellulose chromatography of leukemic spleen enzyme. The fractions from the pooled DEAE-cellulose peak activity were diluted and chromatographed on a 17 cm X 1.5 cm column. Elution was with 160 ml of an 0.01 M-0.5 M potassium phosphate gradient; 1.6 ml fractions were collected and 10 ,gl aliquots were assayed for oligo(dT)-poly(rA)-templated activity. (C) Agarose gel filtration of leukemic spleen enzyme. The peak of activity eluted from phosphocellulose was subjected to gel filtration on a 50 cm X 0.9 cm agarose column. The elution rate was 4 ml/hr and 0.4 ml fractions were collected. Four microliter aliquots were assayed for enzyme Activity with an oligo(dT).poly(rA) template. BSA = bovine serum albumin.
tions from the DEAE-cellulose column were pooled, diluted 15-fold, and chromatographed on phosphocellulose, using a 0.01 M-0.5 M potassium phosphate gradient. A sharp peak of oligo(dT)-poly(rA)-templated activity eluted at 0.27 M potassium phosphate (Fig. 2B). About 25% of the protein chromatographed on the column eluted within the peak activity with a recovery of 71% and a specific activity increased by 2.8-fold. The peak fractions were pooled and
concentrated by rechromatography on a small phosphocellulose column. A summary of the purification scheme is shown in Table 1. Agarose Gel Filtration. The concentrated phosphocellulose enzyme was filtered through a Bio-Gel AO.5m agarose column and each fraction was assayed for oligo(dT).poly(rA)templated activity. Routinely, two peaks of activity were detected (Fig. 2C). If the activity peak eluting first from the agarose column was rechromatographed on the same column, there was a shift of most of the activity to the position of the second peak. The first peak would appear to be an aggregate (possibly a dimer) of the enzyme. Sodium Dodecyl Sulfate-Acrylamide Gels. Portions of the two agarose peak fractions were electrophoresed on 5% acrylamide gels in the presence of 0.1% sodium dodecyl sulfate. Molecular weights of the separated polypeptides were determined using lysozyme, ovalbumin, bovine serum albumin, and aldolase as molecular weight markers. As shown by the gel photographs and scans of Fig. 3A and B, both agarose peaks yielded one major band, corresponding to a molecular weight of 70,000, supporting the conclusion that the first agarose peak is an aggregate of the second. The two agarose peaks were therefore pooled, glycerol was added to a final concentration of 50%, and the enzyme was stored for use at -20°. Table 2. Deoxynucleoside triphosphate and RNA requirements of leukemic RNA-dependent DNA polymerase Reaction*
Table 1. Purification of RNA-dependent DNA polymerase from a viral core fraction of human leukemic spleen
Fraction 1. Viral core region 2. DEAE-cellulose pool 3. Phosphocellulose pool
Total Total protein activity (mg) (pmol) 3.90 0.20 0.05
2011 1949 1400
>
FIG. 3. Sodium dodecyl sulfate-polyacrylamide gel agarose enzymes. Fractions 48(A) and 60(B) of Fig. 2C were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. After staining with Coomassie blue, the gels were scanned in a Gilford model 2400 spectrophotometer. The stained gel of each fraction is also shown.
Specific activity
(pmol/mg) 5.0 X 102 9.7 x 103 2.8 x 104
1. 2. 3. 4. 5. 6.
Complete -dATP -dGTP -dTTP -dATP, dGTP, dTTP -RNA
pmol [3H]dCMP polymerizedt 4.6
Vol. 72, No. 10, pp. 4133-4136, October 1975 Medical Sciences
Purification of RNA-instructed DNA polymerase from human leukemic spleens (reverse transcriptase/leukemia)
S. S. WITKIN*, T. OHNO, AND S. SPIEGELMAN Institute of Cancer Research, College of Physicians & Surgeons, Columbia University, New York, N.Y. 10032
Contributed by S. Spiegelrman, June 23, 1975
ABSTRACT Particles possessing a density of 1.16 g/ml and encapsulating a 70S RNA and a RNA-instructed DNA polymerase (reverse transcriptase) have been prepared from the spleen of a patient with chronic lymphocytic leukemia. These particles have been converted to cores with a density of 1.26 g/ml and containing the enzyme-RNA complex, in complete analogy to the known RNA tumor viruses of avian and murine origin. The reverse transcriptase was purified from the cores by column chromatography to a stage showing a single major protein band of 70,000 daltons in a gel electrophoresis. The enzyme was capable of transcribing heteropolymeric RNA into DNA complements as demonstrated by specific back hybridization to template RNA. The leukemic spleen would a ppear to represent an important source of this enzyme, as well as other potentially important leukemiaspecific reagents.
The use of the "simultaneous detection test" (1) has permitted the identification of particles encapsulating a 70S RNA template with RNA-instructed DNA polymerase (reverse transcriptase) in human leukemic cells (2) as well as in a variety of other human neoplasias (3-6). The human leukemic reverse transcriptase has been partially purified and characterized from fresh leukemic cells (7, 8). The comparatively large amounts of peripheral leukemic white blood cells required for enzyme isolation have imposed severe restrictions on this type of research and confined it to the one or two laboratories that have access to the very limited supply of peripheral leukemic cells. One possible way out of this dilemma is to explore the use of spleens as a possible source of the leukemic enzyme. In addition to providing a possible solution of the logistic problem, such experiments would provide useful information on the relation between diseased tissue and the presence of the virus-like particles. Leukemias almost always involve the spleen and splenectomies are not infrequently performed therapeutically in instances of massive spleen enlargement or persistent platelet destruction. The technology of enzyme isolation from leukemic spleens was first worked out using the murine Rauscher leukemia model (Witkin and Spiegelman, in preparation). It was found that isopycnic separation of virus particles and their conversion to cores by non-ionic detergents (9, 10) provided material suitably enriched for enzyme purification. It is the purpose of the present paper to describe the use of this approach for the successful purification of a reverse transcriptase from a human leukemic spleen. MATERIALS AND METHODS Purification of Core Particles from Human Leukemic Spleen. Approximately 70 g of chronic lymphocytic leukeAbbreviations: AMV, avian myeloblastosis virus; RLV, Rauscher * Present address: Laboratory of Cell Biochemistry, Memorial Sloan-Kettering Cancer Center, New York, N.Y. 10021.
leukemia virus; BSA, bovine serum albumin.
4133
mic spleen were thawed, minced, suspended in 3 volumes of cold 5% sucrose (w/w) in TNM buffer (0.01 M Tris-HCl, pH 8.0, 0.15 M NaCl, 0.002 M MgCl2) and homogenized for approximately 2 min in a Silverson homogenizer (11). The mixture was then centrifuged at 4000 X g and 10,000 X g to remove nuclei and mitochondria, respectively. KCI (4 M) was added to the postmitochondrial supernatant to a final concentration of 0.8 M and the solution was centrifuged at 80,000 X g for 90 min. The high-speed supernatant was discarded and the pellet was resuspended in 5% sucrose in TNM buffer containing 0.8 M KCL. After an 8000 X g centrifugation to remove residual cell debris, the sample was layered in 10-ml aliquots on discontinuous sucrose gradients consisting of 5 ml of 50% sucrose and 10 ml of 25% sucrose, all in TNM buffer and 0.8 M KCI. The gradients were centrifuged at 176,000 X g for 120 min in a 60 Ti rotor. The material present in the 25-50% sucrose interface was removed, diluted about 5-fold, and solid (NH4)2SO4 was added with constant stirring over a 20-min period to a final concentration of 30%. The final pH of the solution was 7.0. After at least 60 min at 40, the solution was centrifuged at 12,000 X g for 30 min. The resulting pellet was drained and resuspended in 0.01 M Tris-HCI, pH 8.3, 0.005 M NaCI, 0.002 M EDTA (S buffer). An aliquot of this suspension was layered on a gradient of 30-65% sucrose in S buffer plus 0.005 M dithiothreitol. Centrifugation was for 16 hr at 98,000 X g in an SW-41 rotor. Eleven fractions of equal volume were collected from below and the density of each was determined by refractometry. Each fraction was then diluted with 0.01 M Tris'HCl, pH 8.0, centrifuged at 98,000 X g for 60 min, and the resulting pellets were resuspended in 100 ,l 0.01 M Tris-HCI, pH 8.0. Viral cores were prepared by procedures previously described (9, 10). An aliquot of the (NH4)2SO4 fraction in S buffer was adjusted to a final concentration of 1% Nonidet P-40 and 0.1 M dithiothreitol, incubated at 40 for 5 min and layered on a 30-65% sucrose gradient in S buffer and dithiothreitol. Centrifugation and core concentration were as described for viral purification. Disruption of Core Particles. The mildest conditions affording almost complete solubilization and recovery of the enzyme from the cores involved treatment with 1% Nonidet P-40 and 0.7 M KCL. Core pellets were resuspended in 0.01 M Tris-HCI (pH 8.0) containing 1% Nonidet P-40 and 0.7 M KCI, and were left at 40 for 20 min followed by dilution 50-fold in starting buffer (0.01 M potassium phosphate, pH 7.2, 10% glycerol, 0.001 M EDTA, 0.002 M dithiothreitol, 0.2% Nonidet P-40). DEAE-Cellulose Chromatography. The diluted disrupted cores were loaded on a 16.5 cm X 2.5 cm column of DEAE-cellulose (Whatman DE52) equilibrated with starting buffer. The column was then washed with 100 ml starting buffer and the enzyme activity was eluted by raising the
4134
Medical Sciences: Witkin et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
81 A
4
130
3
6
1.20 0
to
E
4
0
E EM
0
E
2 1.10
2
1.00 FRACTION NUMBER
FIG. 1. Isolation of virus and viral cores from human chronic lymphocytic leukemic spleen. A cytoplasmic particulate fraction was prepared from 20 g of infected spleen as described in Materials and Methods. Aliquots before (A) and after (B) treatment with Nonidet P-40 were layered on 30-65% sucrose gradients and centrifuged at 78,000 X g for 16 hr. Twelve equal fractions were collected from below, concentrated, and assayed for endogenous reverse transcriptase activity.
potassium phosphate to 0.40 M. Fractions of 3.2 ml were collected and 10 Mil aliquots of each were assayed for ability to incorporate [3H]deoxythymidine triphosphate (5 X 103 cpm/pmol) into acid-insoluble material using oligo(dT).poly(rA) as template. Phosphocellulose Chromatography. The pooled peak enzyme fractions from the DEAE-cellulose column were diluted 15-fold in starting buffer and loaded, at a constant rate of 20 ml/hr, on a 17 cm X 1.5 cm column of phosphocellulose (Whatman P11) equilibrated with starting buffer. The column was then washed with 60 ml starting buffer and the enzyme activity was eluted with 160 ml of a 0.01 M-0.5 M potassium phosphate gradient. The flow rate was maintained at 20 ml/hr and fractions of 1.6 ml were collected and assayed as described above. The enzyme activity in the pooled peak fractions was concentrated by rechromatography on a 4 cm X 0.9 cm phosphocellulose column and elution with 0.4 M potassium phosphate in starting buffer. Agarose Gel Filtration. The concentrated phosphocellulose activity was applied to a 50 cm X 0.9 cm agarose column (Bio-Gel A-0.5m) and equilibrated with 0.35 M potassium phosphate (pH 7.2) in starting buffer. The flow rate was about 4 ml/hr and 0.4 ml fractions were collected. Polyacrylamide Gel Electrophoresis. Polyacrylamide gel electrophoresis on 5% gels, in the presence of sodium dodecyl sulfate, was as reported previously (12). Polymerase Assays. The assay mixture for the synthetic templates reaction contained 5 Mumol of Tris*HCl (pH 8.0), 0.6 gmol of Mg9l2, 0.02 ,umol of unlabeled deoxyadenosine triphosphate, 0.003 M4mol of unlabeled deoxythymidine triphosphate, 0.1 Mimol of dithiothreitol, 0.4 Mg of oligo(dT)poly(rA) (Miles Research) and [3H]deoxythymidine triphosphate (New England Nuclear) to yield 5 X 103 cpm/pmol in a final volume of 100 ,ul. Incubation was for 10 min at 370 and was terminated by the addition of 5 volumes of cold trichloroacetic acid mix (10% trichloroacetic acid and 10% saturated sodium pyrophosphate in H20). After at least 10 min at 40, acid-precipitable radioactivity was collected on filters and counted as described (12). Assays using avian myeloblastosis virus (AMV) RNA or avian myeloblast DNA contained, in a final volume of 100 Mul, 5.0 Mimol of Tris-H1l (pH 8.0), 0.6 Mmol of MgCl2, 0.016 Mmol each of unlabeled deoxythymidine triphosphate, deoxyadenosine triphosphate, and deoxyguanosine triphos-
phate, 1.4 Mug of RNA or DNA, 0.1 pmol of dithiothreitol, and 0.002-0.016 gmol (6.4 X 102 to 2.0 X 104 cpm/pmol) of [3H]deoxycytidine triphosphate. In addition, the RNA-templated reactions contained 0.1 ;ig of oligo(dT) (Miles Research) and 10 gg of actinomycin D (Sigma). Reaction conditions and radioactivity determination were as described above. Purification of the reaction product, hybridization to AMV RNA or Rauscher leukemia virus (RLV) RNA, and analysis on Cs2SO4 equilibrium gradients were as previously described (11). RESULTS Reverse Transcriptase Activity in the Virus and Core Density Regions from Human Leukemia Spleens. The ammonium sulfate precipitates from the chronic lymphocytic leukemic spleens were treated as described in Materials and Methods. Eleven equal fractions were collected, concentrated, and assayed for endogenous activity (12). A sharp peak of enzyme activity was detected at a density of 1.17 g/ml (Fig. 1A). Detergent treatment (Fig. 1B) resulted in a shift of all of the endogenous enzyme activity to a density of 1.26-1.27 g/ml, characteristic of core-like particles. As had been done in our previous investigations (2, 3-6, 9, 10), it was established by ribonuclease or alkali sensitivity and Cs2SO4 gradient analysis (data not shown) that the products of the endogenous activities shown in Fig. 1A and B were complexes between the nascent DNA chain and a large RNA molecule. Chromatography of Spleen Reverse Transcriptase on DEAE Cellulose. Reverse transcriptase was solubilized from the core fraction in 1% Nonidet P-40 and 0.7 M KC1, diluted 50-fold to reduce the salt and detergent concentrations, and chromatographed on DEAE-cellulose as described in Materials and Methods. One main peak of oligo(dT)-poly(rA) activity was observed (Fig. 2A), containing 5% of the input protein. Greater than 90% of the activity was recovered, yielding a 19-fold enzyme enrichment. We have found that if the polymerase is associated with RNA, as in the core preparations, the enzyme activity will bind to DEAE-cellulose. In contrast, with preparations of enzyme dissociated from nucleic acid, the polymerase activity is detectable in the DEAE-cellulose flow-through. Chromatography on Phosphocellulose. The active frac-
Medical Sciences: Witkin et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
4135
UAt
A 0.4-
0.2E 20
10
2.0
B 0.5
I~~~I
If)
~~~~~~~~~~
x
a-
0 to to
30
O
I.0
.0.4 0.3 rji 0.2 9 0. I 0
8 Lg8.j
0.51
MIGRATION 20
5.0
40
60
80
BSA
C
100
COVALBUMIN
4.0
3.0
2.0 1.0
I 20
40
60
80
100
FRACTION NUMBER
FIG. 2. (A) DEAE-cellulose chromatography of leukemic spleen enzyme. Core-like particles isolated from a leukemic spleen were treated with 1% Nonidet P-40 and 0.7 M KCl and the solubilized enzyme activity was chromatographed on a 16.5 cm X 2.5 cm column. Elution was with 0.4 M potassium phosphate. Ten microliters of each fraction (3.2 ml) were assayed using an oligo(dT).poly(rA) template. (B) Phosphocellulose chromatography of leukemic spleen enzyme. The fractions from the pooled DEAE-cellulose peak activity were diluted and chromatographed on a 17 cm X 1.5 cm column. Elution was with 160 ml of an 0.01 M-0.5 M potassium phosphate gradient; 1.6 ml fractions were collected and 10 ,gl aliquots were assayed for oligo(dT)-poly(rA)-templated activity. (C) Agarose gel filtration of leukemic spleen enzyme. The peak of activity eluted from phosphocellulose was subjected to gel filtration on a 50 cm X 0.9 cm agarose column. The elution rate was 4 ml/hr and 0.4 ml fractions were collected. Four microliter aliquots were assayed for enzyme Activity with an oligo(dT).poly(rA) template. BSA = bovine serum albumin.
tions from the DEAE-cellulose column were pooled, diluted 15-fold, and chromatographed on phosphocellulose, using a 0.01 M-0.5 M potassium phosphate gradient. A sharp peak of oligo(dT)-poly(rA)-templated activity eluted at 0.27 M potassium phosphate (Fig. 2B). About 25% of the protein chromatographed on the column eluted within the peak activity with a recovery of 71% and a specific activity increased by 2.8-fold. The peak fractions were pooled and
concentrated by rechromatography on a small phosphocellulose column. A summary of the purification scheme is shown in Table 1. Agarose Gel Filtration. The concentrated phosphocellulose enzyme was filtered through a Bio-Gel AO.5m agarose column and each fraction was assayed for oligo(dT).poly(rA)templated activity. Routinely, two peaks of activity were detected (Fig. 2C). If the activity peak eluting first from the agarose column was rechromatographed on the same column, there was a shift of most of the activity to the position of the second peak. The first peak would appear to be an aggregate (possibly a dimer) of the enzyme. Sodium Dodecyl Sulfate-Acrylamide Gels. Portions of the two agarose peak fractions were electrophoresed on 5% acrylamide gels in the presence of 0.1% sodium dodecyl sulfate. Molecular weights of the separated polypeptides were determined using lysozyme, ovalbumin, bovine serum albumin, and aldolase as molecular weight markers. As shown by the gel photographs and scans of Fig. 3A and B, both agarose peaks yielded one major band, corresponding to a molecular weight of 70,000, supporting the conclusion that the first agarose peak is an aggregate of the second. The two agarose peaks were therefore pooled, glycerol was added to a final concentration of 50%, and the enzyme was stored for use at -20°. Table 2. Deoxynucleoside triphosphate and RNA requirements of leukemic RNA-dependent DNA polymerase Reaction*
Table 1. Purification of RNA-dependent DNA polymerase from a viral core fraction of human leukemic spleen
Fraction 1. Viral core region 2. DEAE-cellulose pool 3. Phosphocellulose pool
Total Total protein activity (mg) (pmol) 3.90 0.20 0.05
2011 1949 1400
>
FIG. 3. Sodium dodecyl sulfate-polyacrylamide gel agarose enzymes. Fractions 48(A) and 60(B) of Fig. 2C were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. After staining with Coomassie blue, the gels were scanned in a Gilford model 2400 spectrophotometer. The stained gel of each fraction is also shown.
Specific activity
(pmol/mg) 5.0 X 102 9.7 x 103 2.8 x 104
1. 2. 3. 4. 5. 6.
Complete -dATP -dGTP -dTTP -dATP, dGTP, dTTP -RNA
pmol [3H]dCMP polymerizedt 4.6
