A simple purification of avian myeloblastosis virus reverse transcriptase for full-length transcription of 35 S RNA

ANALYTICAL

BIOCHEMISTRY

101,

88-96 (1980)

A Simple Purification of Avian Transcriptase for Full-Length JEANNE Instititute

F. RAMIREZ,~

C. MYERS,’ of Cancer Surgeons,

Research

and Department

Columbia

University,

Myeloblastosis Transcription

D. L. KACIAN,~ of Human 701

W.

M. FLOOD,

Genetics

168th

Virus Reverse of 35 S RNA

Street,

AND S. SPIEGELMAN~

and Development, New

York,

New

College York

of Physicians

&

10032

Received May 23, 1979 Complete transcription of large RNA templates by avian myeloblastosis virus reverse transcriptase requires a purified and concentrated enzyme. This report describes a simple 2-day procedure consisting of a DEAE column, a carboxymethyl-Sepharose column, and a concentration step. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the enzyme is free of contaminating protein and a series of rigorous assays reveal little if any exogenous ribonuclease or deoxyribonuclease activity. The reverse transcriptase purified by this method readily catalyzes synthesis of full-length complementary DNA from viral kNAs.

Recent developments in the technology of DNA manipulation have increased the need for convenient and reliable preparations of the relevant enzymes. In particular, the demonstration of eukaryotic transformation has made possible a host of experiments in molecular genetics. Purified reverse transcriptase which can completely copy large RNA molecules can contribute significantly to the success of this approach. We have also used the enzyme as an aid in sequencing cloned DNA fragments by a modification of the end-labeling procedure reported by Smith and Birnstiel (1). The DNA is excised by restriction endonucleases leaving an asymmetric overhang at the 5’ ends, and is then suitable for terminal labeling with deoxynucleoside [32P]triphosphates. DNA polymerases (pol I and T4)

commonly employed for this purpose are successful only if the DNA has not been internally nicked. We circumvented this difficulty by using reverse transcriptase devoid of deoxyribonuclease activity to exclusively label the 5’ termini of the thymidine kinase gene (S. Silverstein and J. Myers, unpublished results). We report here a simple and rapid procedure for isolating the avian myeloblastosis virus (AMV)” reverse transcriptase from small amounts of viral protein. The concentrated enzyme, free of ribonuclease and deoxyribonuclease activity, transcribes 35 S RNA into full-size cDNA. MATERIALS

Sources of materials used were as follows: Nonidet P-40, BDH Chemical Ltd.; sodium

I Present address: Department of Biochemistry, College of Medicine and Dentistry of New Jersey, Rutgers Medical School, Piscataway, N. J. 08854. * Present address: Department of Obstetrics & Gynecology, College of Medicine and Dentistry of New Jersey, Rutgers Medical School, Piscataway, N. J. 08854. s Present address: Department of Pathology, Barnes Hospital, St. Louis, MO. 63110. 4 To whom reprint requests should be addressed. 0003-2697/80/O 10088-09$02.00/O Copyright All rights

0 1980 by Academic Press, Inc. of reproduction in any form reserved.

rr Abbreviations used: AMV, avian myeloblastosis virus; CM, carboxymethyl; SDS, sodium dodecyl sulfate; DTT, dithiothreitol; NP-40, Nonidet P-40; TM, containing 0.25 M Tris-HCI, pH 8.3, 0.04 M MgCIZ; dXTP, containing 1 mM dATP, 1 mM r3H]dTTP, 275 cpm/pmol; TE, containing 0.05 M Tris-HCI, pH 8.3, I mM EDTA; TNE, containing 0.05 M Tris-HCI, pH 8.3, 0.1 M NaCI, 0.001 M EDTA; TCA. trichloroacetic acid. 88

AVIAN

MYELOBLASTOSIS

VIRUS

REVERSE

deoxycholate, Merck; glycerol (99.5+%, spectrophotometric grade, gold label), Aldrich Chemical Company, Inc.; dithiothreitol, poly(dT:rA), and unlabeled deoxynucleoside triphosphates, P-L Biochemicals; 13Hldeoxynucleoside triphosphates, Econofluor, and Protosol, New England Nuclear; diethyl pyroprocarbonate , Sigma; ammonium sulfate (enzyme grade), Schwarz-Mann; microgranular DEAE-cellulose (DE52, 10 meq/g dry wt), Whatman; CM-Sepharose CL-6B and Sephadex G-200, Pharmacia; Collodion bags (No. 100,75,000 M,), protein retention and associated apparatus, Schleicher and Schuell, Inc.: mixed bed resin (AG501-X8(D), 20-50 mesh, polyacrylamide, methylenebisacrylamide), Bio-Rad.

TRANSCRIPTASE

Preparation of AMV RNA, poliovirus RNA, and bacteriophage f 1 DNA has been previously described (2,3). Electrophoresis in 3.5% (w/w) polyacrylamide gels containing 98% (v/v) formamide was performed according to Duesberg and Vogt (4). SDSpolyacrylamide gel electrophoresis of purified enzyme has been detailed (5,6). To eliminate nuclease contamination, all glassware was baked 3-4 h at 200°C. Columns, column reservoirs, and tubing could not be baked. These were exposed to 0.1 N NaOH for at least 2 h and rinsed thoroughly with distilled water. Tubes for collecting column fractions were siliconized and baked. Preparation

of Reagents

Distilled water used for solutions was shaken at room temperature with 0.02% (v/v) diethyl pyrocarbonate for 1 h at room temperature. It was then heated in sealed bottles for 3 h in a boiling water bath to decompose the diethyl pyrocarbonate into ethanol and carbon dioxide. All potassium phosphate buffers were prepared from a 1 M solution, pH 7.2, by dilution. The pH was not further adjusted.

89

Dithiothrietol was frozen as a 1 M solution in convenient quantities and added to solutions immediately before use. Saturated ammonium sulfate was ajusted to pH 7.2 with ammonium hydroxide. A 10% (v/v) solution of Nonidet P-40 was deionized by stirring twice with mixed bed resin (5% of solution volume) for several hours. The solution was filtered twice through Whatman No. 3 paper, divided into 20-ml portions, and autoclaved for 30 min at 121°C. Detergent treated in this manner was used for all solutions. Buffers containing Nonidet and high concentrations of phosphate exhibit turbidity when warm. They are clear when cold. Preparation

METHODS

PURIFICATION

of DEAE-Cellulose

Whatman DE-52 (500 g) was suspended in 2 liters of 0.1 M potassium phosphate, pH 7.2. The cellulose was allowed to settle, and the supernatant containing fines was removed. This was done five times and then twice again using 0.01 M potassium phosphate. The mixture was poured into a Buchner funnel lined with nylon mesh and washed with 5 liters of 0.01 M potassium phosphate using gentle suction. The cellulose was then suspended in an equal volume of the same buffer containing 0.02% (w/v) sodium azide. It may be stored at 4°C for up to 2 years. Preparation

of CM-Sepharose

The beads were washed batchwise a minimum of five times with equal volumes of 0.1 M potassium phoshate, pH 7.2, 2 mM DTT, 10% (v/v) glycerol, 0.2% (v/v) NP-40. After the column was poured, one to two column volumes of the same buffer were passed through it to stabilize the bed. Enzyme Assays

One unit of enzyme activity converts 1 nmol of TTP to acid-insoluble form under

90

MYERS

the following conditions. The reaction mixture (100 ~1) contained 20 ~1 TM (0.25 M Tris-HCl, pH 8.3, 0.04 M MgC&); 4 ~1 poly(dT:rA) (100 pg/ml); 20 ~1 dXTP mixture (1 mM dATP, 1 mM [3H]dTTP, 275 cpm/ pmol); 36 ~1 water; and 20 ~1 enzyme and detergent-salt mix. Detergent-salt mix is prepared by mixing 2 ml TE (0.05 M Tris-HCl, pH 8.3, 1 mM EDTA), 0.2 ml 100% NP-40, 0.2 ml 10% (w/v) sodium deoxycholate, 0.6 ml 4 M KCl, 27 ml 0.01 M potassium phosphate, pH 7.2, 10% (v/v) glycerol, 0.2% (v/v) NP-40, and 2 mM DTT. It is thus equivalent to the crude extract applied to the DEAE column. The detergents and salts present in the crude extract significantly stimulate the enzyme activity; therefore, the assay mixture was adjusted with detergent-salt mix so that the enzyme yield could be determined throughout the procedure. Reactions were incubated for 10 min at 37°C and TCA-precipitable radioactivity was collected on nitrocellulose membrane filters. Purification

of AMV from

Plasma

The procedure has been detailed previously (7). Briefly, frozen plasma was thawed rapidly and placed into an ice slurry just before completely melted to prevent the temperature from rising above 5°C. One gram of kieselguhr was gently stirred into each 100 ml, and the plasma was centrifuged at 2000 rpm (SOOg) for 10 min at 4°C in the Sorvall GSA rotor. The supernatant was removed and filtered with gentle suction through a thin layer of kieselguhr atop Whatman No. 1 filter paper in a Buchner funnel. The filtrate was centrifuged in the Beckman Type 19 rotor at 18,000 rpm for 90 min at 4°C. The supernatant was gently decanted, and the pellets were resuspended in TNE (0.05 M Tris-HCl, pH 8.3, 0.1 M NaCl, 0.001 M EDTA), 10 ml per 100 ml original plasma, using a Dounce homogenizer with tight-fitting pestle. The suspension was

ET AL.

sonicated twice for 30 s (Branson Sonifier, model 185) at 100 W with a microtip while the suspension was cooled in an ice slurry. The virus was then layered over discontinuous sucrose gradients in Beckman SW 27 cellulose nitrate tubes consisting of 7 ml 50% (v/v) sucrose, 11 ml 35% sucrose, and 10 ml 20% sucrose in TNE, Ten milliliters of virus suspension was loaded on each, and they were spun at 27,000 rpm for 60 min at 4°C. The virus bands, found in the middle of the 35% layer, were removed, pooled, and diluted to three times their volume with TNE. The material was layered over discontinuous sucrose gradients consisting of 4.7 ml each of 60, 51, 42, 33, 24, and 15% (w/v) sucrose in TNE. They were centrifuged at 27,000 rpm in the SW 27 rotor overnight (or a minimum of 5 h) at 4°C. The virus band (p = 1.16 g/ml) was removed, and the yield was determined by reading the absorbance of a small portion in 1% sodium dodecyl sulfate and using the relationship 1.54 (A,,,) -0.76 (A,,,) = mg protein/ml (8). The virus was stored at -70°C in convenient portions, and it can be used for enzyme isolation for at least 2 years. Six hundred milliliters of plasma can be conveniently purified in one SW 27 rotor and should yield 80- 150 mg of virus protein. Purijcation

of Reverse

Transcriptase

A. Disruption of virus. The procedure is described for 15-20 mg viral protein. Virus from the equilibrium gradients was diluted four to five times with TE buffer (0.05 M Tris-HCl, pH 8.3, 0.001 M EDTA) and pelleted in the Beckman 50Ti rotor at 50,000 rpm at 2°C for 1 h. The pellets were drained and resuspended in TE buffer at a concentration of 10 mgiml. To the virus (2 ml) were added 0.2 ml 100% NP-40, 0.2 ml 10% (w/v) sodium deoxycholate, and 0.6 ml 4 M KCl. The solution was mixed until completely homogeneous and kept in an ice slurry for 15 min. The

AVIAN

MYELOBLASTOSIS

VIRUS REVERSE TRANSCRIPTASE

FRACTION

PURIFICATION

91

NUMBER

Frc. 1. Profiles of DEAE and CM-Sepharose columns. (A) Fractions of 0.35 ml were collected from a 1 x 5-cm DEAE column. A 1:lO dilution of each fraction was assayed as described under Methods. Fractions 8-12 were pooled. (B) Fractions of 0.5 ml were collected from a 0.9 X 60-cm CM-Sepharose column. A I:10 dilution of each fraction was assayed as described under Methods. Fractions 63-67 were pooled.

disrupted virus was centrifuged for 10 min at 4°C (10,000 rpm) in the Sorvall HB-4 rotor. The supernatant was removed, and the small pellet was discarded. B. DEAE column chromatogrphy. The supernatant was diluted to 10 times its volume with 10 mM potassium phosphate, pH 7.2, 2 mM DTT, 10% (v/v) glycerol, TABLE

1

PURIFICATION OF AMV REVERSE TRANSCRWTASE FROM 20 mg VIRAL PROTEIN Units of activity First day DEAE load Flow-through Wash Eluate pool Second day CM wash CM eluate pool NH$O, precipitation Second day (alternate) CM wash (with NP-40) CM eluate pool (with NP-40) Collodion bag concentration

Recovery (o/o)

4.7 x 103 1.4-3.7 x 102 0.94-1.9 x 102 4-4.5 x 103

85-95

1.4-1.9 x 103 1.4-1.9 x 103

30-40 30-40

-

100 3-8 2-4

iI


3.1-3.3 x 103

65-70

2.6-3.1

55-65

x 103

0.2% (v/v) NP-40. The solution (DEAE load) was then applied to a 1 X 5-cm column of DEAE-cellulose equilibrated with the same buffer, and the flow rate was maintained at 8- 10 ml per hour. The column was washed at 10 ml per hour with 20 ml of 50 mM potassium phosphate, pH 7.2, 2 mM DTT, 10% glycerol, and 0.2% NP-40. Elution was carried out at 4-6 ml per hour with 300 mM potassium phosphate, pH 7.2, 2 mM DTT, 10% glycerol, 0.2% NP-40. Fractions of 0.35 ml were collected. The peak fractions (fractions 8-12 in Fig. IA) were pooled and diluted to three times their volume with 10 mM potassium phosphate, pH 7.2, 2 mM DTT, 10% glycerol, 0.2% NP-40. The recovery at this stage was 85-95% (Table 1). C. CM-Sepharose column chromatography with concentration by ammonium suljute precipitution. The diluted DEAE p001 (5.5-7.5 ml) was loaded onto a 0.9 x 59.0-cm column of CM-Sepharose CL-6B equilibrated with 100 mM potassium phosphate, pH 7.2, 2 mM DTT, 10% glycerol, 0.2% NP-40. The flow rate was 6- 10 ml per hour. The column was then washed overnight with 40 ml of 100 mM potassium phosphate, pH 7.2, 2 mM DTT, 10% glycerol (NP-40 is omitted) at a flow rate of 3-4 ml per hour.

92

MYERS

ET AL.

Elution of the column was complete in 4-5 h and should be started early in the morning of the second day. The elution buffer was 300 mM potassium phosphate, pH 7.2,2 mM DTT, 10% glycerol (NP-40 is omitted), and fractions of 0.50-0.55 ml were collected. The enzyme elutes in a sharp peak (Fig. 1B) at about 0.8 column volumes. The peak 4-5 fractions (63-67 in Fig. 1B) were pooled and mixed with an equal volume of cold, saturated ammonium sulfate, pH 7.2. The mixture was kept in an ice slurry for 60-75 min and then centrifuged in the Beckman 50Ti rotor at 30,000 rpm at 2°C for 70 min. The supernatant was carefully decanted and the pellet (not visible) was resuspended in 200 mM potassium phosphate, pH 7.2, 10% glycerol, 4 mM DTT, 0.2% NP-40 to the desired concentration. Glycerol was added to give a final concentration of 50%, and the enzyme was stored at -20°C.

zo-

1

15IOPo5

15

18S16S I I

9 6

-

3

j

x z 1.5”

A 23s I

D. CM-Sepharose column chromatography with concentration by dialysis. CMSepharose column chromatography was performed as described above except that the wash and elution buffers contained 0.04% (v/v) NP-40. Peak fractions were pooled and placed in a Collodion bag mounted in its glass holder. The bag was placed into a tube filled with prechilled Sephadex G-200 up to the level of the solution and placed at 4°C. Every 15 min, the wet layer of beads adjacent to the bag was peeled away to expose dry material. With this procedure, 2.5 ml will concentrate over IO-fold in under 3 h. The enzyme was carefully removed, and the bag was rinsed with 25 ~1 of 0.01 M potassium phosphate, pH 7.2, 2 mM DTT, 10% glycerol, 0.2% NP-40. Glycerol was added to give a final concentration of 50%. The potassium phosphate concentration was adjusted to 0.1 M by determining the

,

1.2-

235 185165 I

I

B

2.0

I

1.6 1.2

10

20

30

40

50

60

70

10

20

FRACTION

30

40

50

60

70

5L IO

20

30

40

50

60

70

NUMBER

FIG. 2. Ribonuclease assay. Reaction mixtures (25 ~1) contained: 0.1 pg 28 S 13H]RNA (80,000 cpm/pg), 50 mM Tris-HCI, pH 8.3, 40 mM KCI, 8 mM MgCI? plus: (A) 3 ~1 salt-detergent buffer (16.5 mM Tris-HCI. 0.33 mM EDTA, 3.33% NP-40, 0.33% sodium deoxycholate, 0.4 M KCI, 50% glycerol); (B) 3 ~1 disrupted virus, 0.4 units/PI (in 50% glycerol); (C) 3 ~1 DEAE elution buffer diluted I: I with glycerol; (D) 3 ~1 DEAE peak fraction pool, 0.35 units/PI (in 50% glycerol); (E) 3 ~1 buffer (0.1 M potassium phosphate, 0.1% NP-40, 2 mM DTT, 50% glycerol): (F) 3 ~1 final enzyme, 2.3 units/PI in 50% glycerol. After incubation at 37°C for 30 min. IO ~1 of 0.1 M EDTA (pH 7.2) was added and the samples were lyophilized to dryness. To the lyophilized samples were added 30 PI formamide containing 2 mM phosphate buffer, 20 PI formamide:glycerol (I:l). and 0.5 ~1 0.04% bromphenol blue. Electrophoresis on 3.5% acrylamide formamide gels was for 16 h at 90 V (4). Gels were sliced into l.2-mm sections and dissolved in 10 ml Econofluor containing 3% Protosol for I6 h at 50°C.

AVIAN

MYELOBLASTOSIS

VIRUS

REVERSE

conductivity of a 1: 1000 dilution with standards of known molarity. NP-40 was brought to a final concentration of 0.1% by comparing the absorbance (a 1:40 dilution of enzyme solution) at 275 nm against known standards. The NP-40 concentration is reduced during the elution step because the detergent concentrates several-fold during dialysis, probably due to the formation of detergent micelles. RESULTS

The method described above uses only two columns and can be completed within 36 h, with much of the time free for other activities. Figure 1 shows typical enzyme activity profiles from the DEAE and CM-Sepharose columns. The reverse transcriptase elutes

FRACTION

TRANSCRIPTASE

PURIFICATION

93

sharply from both, and the fractions pooled are indicated. A relatively narrow region must be taken from the CM-Sepharose column to ensure removal of RNase, which elutes behind the reverse transcriptase. It is advisable to collect relatively small fractions, as described under Methods, to achieve good separation with optimal yield. Several attempts were made to widen the separation by gradient rather than step elution; however, no significant difference was noted (data not shown). Figure 2 shows the results of assays for ribonuclease activity in the finished enzyme as well as at various stages of the procedure. In these, fragmentation of 3Hlabeled 28 S ribosomal RNA was monitored on denaturing gels containing formamide using an RNA concentration fivefold less

NUMBER

FIG. 3. Deoxyribonuclease activity. Reaction mixtures (50 @I) contained 0’.3 pg [“H]f 1 DNA, 50 mM Tris-HCI, pH 8.3, 8 mM MgCI,, 40 mM KCI, plus: (A) 6 /II salt-detergent buffer (16.5 mM Tris-HCI, 0.33 mM EDTA, 3.33% NP-40,0.33% sodium deoxycholate, 0.4 M KCI, 50%glycerol); (B) 6pIdisrupted virus, 0.4 units/PI (in 50% glycerol): (C) 4 ~1 buffer (0. I M potassium phosphate, 0.1% NP-40, 2 mM DTT, 50% gIycero1); (D) 4 ~1 purified enzyme. 2.3 units/F1 (in 50% glycerol). Samples were incubated for I h at 37°C. terminated with 10 mM EDTA, and run on alkaline sucrosegradients (2). Fractions were collected directly into scintillation vials, neutralized, and counted in Aquasol.

94

MYERS

than that present in the usual synthetic reaction and an enzyme concentration twice as high. The crude extract (disrupted virus) contained considerable ribonuclease activity (Fig. 2B), and although DEAE column chromatography removes the bulk of the contaminating protein (over 90%, Refs. (6,9)), the amount of ribonuclease activity appears to be unchanged (Fig. 2D). The CM-Sepharose column effectively removes it, and the profiles of RNA incubated with enzyme (Fig. 2F) and without (Fig. 2E) are virtually indistinguishable. DNase activity was measured in disrupted virus preparations and the purified enzyme by observing the fragmentation of bacteriophage f 1 DNA on alkaline sucrose gradients. As seen in Fig. 3, the level of DNase activity in the disrupted virus purified by procedures described under Methods is very

ET AL.

low. There is an apparent widening of the peak, suggesting some degradation when the DNA was incubated with the crude extract (Fig. 3B). This is not seen with the purified enzyme (Fig. 3D), where the profile is virtually identical with its control. Although chemical purity of the final enzyme preparation was not a requirement of this work, it was of interest to examine the composition of the isolate by SDS-polyacrylamide gel electrophoresis. Figure 4 shows a scan of one such gel. The two subunits described previously (6) with molecular weights of 110,000 and 69,000 are present in the expected ratio of 1.6: 1. A few faint bands of higher molecular weight are also present. These constitute less than 1% of the material present in the reverse transcriptase bands. The suitability of the enzyme for synthetic

FIG. 4. Polyacrylamide gel electrophoresis of purified enzyme. Thirty microliters of enzyme (2.4 units/PI) was TCA precipitated and analyzed on 5% SDS-acrylamide gels (5,6). Electrophoresis was performed in 0.5 x IO-cm glass tubes at 12.5 mA/gel for 16 h. Gels were stained with Coomassie blue and scanned at wavelength 550 in the Gilford Model 2400 spectrophotometer. Enzymes purified by both procedures gave identical profiles.

AVIAN

MYELOBLASTOSIS

4 I ;

VIRUS

II

REVERSE

I

18

z 12

6

5

I

10

FRACTION

15

20

25

30

NUMBER

FIG. 5. Synthesis of poliovirus cDNA. Reverse transcriptase reactions contained 50 mM Tris-HCI (pH 8.3), 8 mM MgCI,, 0.4 mM dithiothreitol, 40 mM KCI, 0.2 mM dTTP. 0.2 mM dATP, 0.2 mM dGTP, 0.2 mM [3H]dCTP (5 Ci/mmol), 40 mM sodium pyrophosphate, 5 pg/ml of ohgo(dT),, 20 &ml of poliovirus RNA, and 80 units/ml of AMV reverse transcriptase. The enzyme was purified by CM-Sepharose (with NP-40) and concentrated in a Collodion bag as described under Methods. Reactions were incubated at 37°C for I h and terminated with EDTA. An aliquot was analyzed by alkaline sucrose gradient centrifugation (2). The position of [“H]f I DNA on a parallel gradient is shown.

purposes is seen in Fig. 5 and 6 which show alkaline sucrose gradient profiles of cDNAs synthesized from poliovirus and avian myeloblastosis virus RNAs using enzyme prepared as described. In each case, the major part of the DNA is equivalent in length to the template RNA: thus, this abbreviated purification method produces reverse transcriptase capable of synthesizing large cDNA. The enzyme concentrated with ammonium sulfate has yielded slightly more homogeneous DNA product than that prepared by the alternate method employing Collodion bags. DISCUSSION

The procedure described above replaces the cation column and glycerol gradient of

TRANSCRIPTASE

PURIFICATION

95

our previous procedure (10) with a single CM-Sepharose column. It is at least 1 day shorter, simpler to execute, and allows purification of small amounts of viral protein into a very concentrated enzyme. The reverse transcriptase preparation has very low levels of nuclease activity and is suitable for the synthesis of intact DNA copies of large, messenger RNAs. In approaching the synthesis of large cDNAs, several points require attention. First, the source, method of growth, harvesting, and purification of the virus are as important as the solubilization or column chromatography steps. We have always used plasma provided by Dr. J. Beard and purified the virus as described under Methods. Recently a similar enzyme isolation procedure from AMV plasma has also been reported by their group (9). Second, adequately purified enzyme is a necessary but not sufficient requirement for the synthesis of large cDNAs. The template RNA must be intact. A relatively small amount of fragmented material represents (fl

FRACTION

DNA

NUMBER

FIG. 6. Synthesis of AMV cDNA. cDNA was as described in the legend to that AMV RNA was present at 20 &ml transcriptase at 160 units/ml. The enzyme by CM-Sepharose (without NP-40) and by ammonium sulfate precipitation under Methods.

Synthesis of Fig. 5 except and reverse was purified concentrated as described

96

MYERS ET AL.

a much greater excess over intact template is not the case, either alone (11) or in when molar quantities are considered. The combination with sodium pyrophosphate quality of the template RNA should be (unpublished results). Indeed any change in verified by electrophoresis under stringent the reaction conditions which decrease net denaturing conditions. yield by weight of cDNA also results in a Third, RNase and DNase assays must be lower proportion of intact product. Since sufficiently sensitive to detect small amounts the addition of pyrophosphate limits the to the of activity. Test nucleic acids must be synthesis to DNA complementary roughly comparable in size to the template template RNA (2,3,11,12), addition of and expected product. Fragmentation meas- actinomycin D to the reaction is counterured by electrophoresis under denaturing productive. conditions, not loss of acid insolubility ACKNOWLEDGMENTS or other insensitive measures, must be monitored. We express our appreciation to Dr. Joseph Beard and Fourth, the optimal enzyme concentration Dr. Dorothy Beard for generously supplying the must be determined individually for each avian myeloblastosis virus used in these studies. We thank Drs. T. Ohno, D. Mills, and C. Dobkin for RNA template by maximizing the amount many helpful suggestions and discussions and Ms. of cDNA synthesized. For example, Susan LaFlamme for excellent technical assistance. poliovirus RNA requires about 60-80 This investigation was supported by Grant CA-02332 units per milliliter whereas AMV RNA and Contract NOI-CP-1016 awarded by the National needs 150-170 units per milliliter. The Cancer Institute. procedures described here permit a greater REFERENCES final enzyme concentration than previous I. Smith, H. 0.. and Birnstiel, M. L. (1976) Nucl. methods and are thus more suitable for Acids Res. 3, 2387-2389. large DNA synthesis by enabling transcrip2. Kacian, D. L., and Myers, J. C. (1976) Proc. tion of the RNA at maximal rate. Nat. Acad. Sri. USA 73, 2192-2195. Fifth, the addition of sodium pyrophos3. Myers, J. C., Spiegelman, S., and Kacian, D. L. (1977) Proc. Nor. Acad. Sci. USA 74, phate to the reaction, as described previously 2840-2843. (2,3), markedly enhances the yield of 4. Duesberg. P. H., and Vogt, P. K. (1973) ./. Viral. complementary DNA and inhibits anti12, 594-599. complementary DNA (11). We have shown 5. Shapiro, A. L., Vinuela, E., and Maize], J. V., elsewhere (12) that in the presence of Jr. (1967) Biochem. Biophys. Res. Commun. 28, 815-823. pyrophosphate the RNA:cDNA duplex 6. Kacian, D. L., Watson, K. F., Burny, A., and formed in the reaction is preserved, whereas Spiegelman, S. (1971) Biochem. Biophys. Acta in its absence the hybrid disappears. We 246, 365-383. believe that in the absence of pyrophos7. Kacian, D. L. (I977) in Methods in Virology phate the RNA strand is being degraded into (Maramorosch, K.. and Koprowski, H., eds.), fragments (by the ribonuclease H activity) Vol. 6, pp. l43- 184, Academic Press, New York. 8. Warburg, 0.. and Christian. W. (1942)Biochem. Z. that then serve as primers for the synthesis 310, 384-388. of DNA anticomplementary to the RNA 9. Houts, G. E., Miyagi, M., Ellis, C.. Beard. D., template (11,12). With enzyme molecules and Beard, J. W. (1979)J. Viral. 29, 517-522. occupied by these additional activities, the IO. Kacian, D. L., and Spiegelman, S. (1974) in rate of completion of the complementary Methods in Enzymology (Grossman, L., and Moldave, K., eds.). Vol. 29, pp. 150-173. DNA is decreased (12). Academic Press, New York. It might be expected that antinomycin D, II. Kacian, D. L., and Myers, J. C. (1976) Proc. which also inhibits DNA-dependent DNA Nat. Acad. Sci. USA 73, 3408-3412. synthesis by reverse transcriptase, would 12. Myers, J. C.. and Spiegelman, S. (1978) Proc. Nat. Acad. Sci. USA 73, 5329-5333. also enhance the yield of intact cDNA. This