Effects of polyethylene glycol on reverse transcriptase and other polymerase activities

353

Biochimica et Biophysica Acta, 606 (1980) 353--361

© Elsevier/North-Holland Biomedical Press

BBA 99603

EFFECTS OF P O L Y E T H Y L E N E GLYCOL ON R E V E R S E TRANSCRIPTASE AND O T H E R POLYMERASE ACTIVITIES

EMERSON W. CHAN *, PHYLIS J. DALE, ISABEL L. GRECO, JAMES G. ROSE and TIMOTHY E. O'CONNOR Division of Biological and Medical Research, Argonne National Laboratory, Bldg. 340, Argonne, IL 60439 (U.S.A.) (Received

May 17th,

1979)

Key words: Polyethylene glycol; Reverse transcriptase; DNA polymerase; RNA polymerase; Terminal transferase

Summary Polyethylene glycol enhances reverse transcription, augmenting both the rate and duration of polymerization. The effective mean molecular weight of polyethylene glycol is 6000 and the optimal concentration is 12% (w/w). Polyethylene glycol is effective on the reverse transcriptase reaction of all ten t y p e B, C, and D viruses tested under a variety of exogenous, endogenous, and reconstitution assay systems, including the highly efficient conditions involving calf t h y m u s DNA oligonucleotide primers. By three methods of synthesis, polyethylene glycol increased the yields of complementary [3H]DNA b y a factor of 1.8--6.5. Polyethylene glycol does not alter the divalent cation requirements or the specificities of the enzyme. Complementary [3H]DNAs made in the presence of polyethylene glycol are indistinguishable in terms of size and sequence complementarity from those made in the absence of the polymer. The stimulatory effect was partly due to the ability of polyethylene glycol to stabilize reverse transcriptase. Preliminary tests indicate that polyethylene glycol also stimulates other nucleotide polymerases, such as the DNAdependent DNA and R N A polymerases of Escherichia coli and the terminal transferase of calf thymus.

* To w h o m requests for reprints s h o u l d be sent. A b b r e v i a t i o n s : A M V , a v i a n m y e l o b l a s t o s i s virus; R - M U L V , R a u s c h e r m u r i n e l e u k e m i a virus: F B J - M U S V a n d F B R - M U S V a r e , r e s p e c t i v e l y , t h e F B J a n d F B R i s o l a t e s o f r o u t i n e o s t e o s a x c o m a viruses; R D l 1 4 , e n d o g e n o u s c a t virus; SSV, w o o l l y m o n k e y f i b r o s a r c o m a virus; G A L V , g i b b o n a p e l y m p h o m a virus; BV, b a b o o n e n d o g e n o u s virus: M M T V , r o u t i n e m a m m a r y t u m o r virus; M P M V , M a s o n - P f i z e r m o n k e y m a m m a r y t u m o r virus; [ 3 H ] c D N A , t r i t i a t e d c o m p l e m e n t a r y D N A ; C r o t , p r o d u c t o f initial R N A c o n c e n t r a t i o n a n d r e a c t i o n t i m e (M • s -1 ); SDS, s o d i u m d o d e c y l s u l f a t e .

354 Introduction Polyethylene glycol, a high molecular weight, water-soluble polymer, forms a colorless, neutral, and viscous solution in water. It is a versatile industrial and laboratory reagent. In using polyethylene glycol for concentration of retroviruses [1--6], we wished to determine whether polyethylene glycol had an effect on viral reverse transcriptase [7,8], an enzyme the activity of which is important for virus replication and detection and for the synthesis of radioactively labeled complementary DNA probes. We found that this polymer strongly stimulated reverse transcriptase reactions as well as those of DNAdependent DNA and R N A polymerases and terminal transferase. To our knowledge, the effects of this hydrophilic polymer on enzyme reactions have not been reported previously. Materials and Methods

Viruses. AMV, R-MULV, R D l 1 4 , SSV, GALV, BV, MMTV, and MPMV were obtained from the Division of Cancer Cause and Prevention, National Cancer Institute. FBJ and FBR-MUSV's were harvested from one-day-old spent media of chronically infected embroyonic fibroblast cell lines derived from BALB/c and SC-1 mice, respectively. Media were first clarified at 10 000 × g for 10 min and centrifuged at 100 000 × g for 1 h. The virus pellets-were resuspended in 0.01 M Tris-HC1 (pH 8.2), 0.15 M NaC1, and 0.002 M EDTA, layered onto a solution of 30% glycerol in the same buffer, and repelleted at 100 000 X g for 1 h. The final pellets were used for reverse transcriptase reactions and RNA extractions. Enzymes. Purified reverse transcriptase of AMV [9] was produced b y Life Sciences under a contract from the Division of Cancer Cause and Prevention, National Cancer Institute. Escherichia coli DNA polymerase I and R N A polymerase, bovine pancreatic DNAase, and terminal transferase were purchased from P.L. Biochemicals. Polyethylene glycol. Polyethylene glycols of mean molecular weights 600, 1000, 6000, and 20 000 were purchased from Fisher Scientific Company. The 6000 dalton polymer was used in all experiments except as indicated in Table II. Calf thymus deoxyoligonucleotide primers. Deoxyoligonucleotides were prepared by digesting calf thymus DNA (P.L. Biochemicals) according to the method of Taylor et al. [10]. Viral RNAs. RNAs were extracted from viruses by the SDS-phenol extraction procedure [11 ]. High molecular weight viral RNAs were fractionated from these preparations b y velocity glycerol density gradient centrifugation. R N A in the 60--70 S region was precipitated with ethanol and redissolved in 100 #l H20 and stored at --70°C. Exogenous reaction. The transcription of synthetic templates by detergentdisrupted virion enzymes was conducted as described b y Spiegelman et al. [12]. Synthetic template-primers, (rA)n "(dT)12, (rCm)n .(dG)12_1s, were obtained from P.L. Biochemicals. [Methyl-3H]dGTP and [methyl-3H]TTP were purchased from ICN Radiochemicals and used at 50 p m (1.2 • 103 cpm/pmol).

355 Reactions were carried o u t for 1 h at 37°C. Incorporations in control reactions in the absence of template were subtracted. Endogenous reaction. In this assay, detergent
Fig. 1 shows the effects of polyethylene glycol on the polymerization reactions of type C viral reverse transcriptases in four assay systems. Three crude systems with disrupted type C viruses and a reconstitution system with purified viral R N A and enzyme were tested. The crude systems included exogenous template assays, endogenous reactions, and simultaneous detection assays. In the exogenous template assay (Fig. 1A) the transcription of the synthetic template-primer (rC)n • (dG)l 2-18 b y detergent
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F i g . 1. E f f e c t s o f p o l y e t h y l e n e glycol on reverse transcriptase activity. The effects of polyethylene glycol on reverse transcriptase activity was tested under four different assay conditions. Parallel reactions were c a r r i e d o u t i n t h e a b s e n c e (©) o r p r e s e n c e (>() o f 1 2 % p o l y e t h y l e n e glycol. (A) Exogenous reaction. Approximately 5 p g o f A M V w e r e a s s a y e d a n d k i n e t i c s f o l l o w e d as d e s c r i b e d i n T a b l e I. ( B ) E n d o g e n o u s reaction. Standard reactions were carried out with 500 ~g of RDll4. (C) Simultaneous detection reaction. Standard assays were performed, each with approximately 300 pg of FBR virus. Ribonuclease controls (10 ~g/ml) of pancreatic ribonuclease A, P.L., Biochemicals) with and without polyethylene glycol a r e r e p r e s e n t e d b y (A). ( D ) P u r i f i e d r e c o n s t i t u t i o n r e a c t i o n . T e n u n i t s o f p u r i f i e d r e v e r s e t r a n s c r i p t a s e o f A M V w e r e u s e d t o t r a n s c r i b e 1 p g o f p u r i f i e d 6 0 - - 7 0 S R N A o f S S V . A t t i m e s i n d i c a t e d , 2-~ul a l i q u o t s were assayed for acid-precipitable radioactivity.

monitored. There was strong increase of both the rate and the duration of the reaction. The polymer was effective on the reverse transcriptase reactions of all type C, type B, and type D retroviruses tested (Table I). Equally good stimulation was observed with two other template-primers (rA), • (dT)12 and (rCm)n • (dG)]2-1s (data not shown). In the endogenous reaction (Fig. 1B), the detergent-disrupted RD114 virion enzyme was allowed to transcribe its associated RNA. Again, substantial stimulation by polyethylene glycol was evident. Comparable results were obtained with AMV, FBJ-MUSV, FBR-MUSV, and SSV (data not shown). In the simultaneous detection assay (Fig. 1C), the increase in the size of the peak, containing [3H]cDNA product complexed to 60--70 S RNA, clearly demonstrated the stimulation of the reaction by poly-

357

TABLE I EFFECTS OF P O L Y E T H Y L E N E G L Y C O L ON R E V E R S E T R A N S C R I P T A S E S D i s r u p t e d viruses w e r e t e s t e d in t h e a b s e n c e a n d p r e s e n c e o f 12% p o l y e t h y l e n e g l y c o l b y t h e e x o g e n o u s t e m p l a t e assay. K i n e t i c s w e r e f o l l o w e d b y s a m p l i n g 15 ~l o f t h e 100-/ll r e a c t i o n s for a c i d - p r e e i p i t a b l e r a d i o a c t i v i t y . T h e n u m b e r s given axe n e t r a d i o a c t i v i t y i n c o r p o r a t e d d u r i n g t h e first h o u r . A n i m a l origin

Chick Mouse

Virus

Type

AMV RLV FBJ FBR RDll4 SSV BV GALV MMTV MPMV

Cat Primate

Mouse Primate

I n c o r p o r a t i o n ( c p m X 10 - 3 )

C C C C C C C C B D

0%

12%

97.4 53.2 45.0 153.5 212.0 153.0 121.0 125.0 20.0 18.0

582.0 320.0 184.0 864.0 858.0 655.0 567.0 783.0 152.0 65.0

Enhancement (times)

5.4 6.0 4.0 5.6 4.0 4.3 4.7 6.3 7.6 3.6

ethylene glycol. By this assay, comparable stimulations were observed with different viruses that included FBJ-MUSV, FBR-MUSV, GALV, and SSV (data n o t shown). The fourth assay was a reconstitution system, (Fig. 1D), in which capacity of purified reverse transcriptase of AMV to copy purified 60--70 S R N A of SSV was found to be greatly augmented in the presence of the polymer. Comparable stimulations b y polyethylene glycol in such reconstitution reactions were observed in the transcription b y the same enzyme of other high molecular weight viral RNAs isolated from FBJ-MUSV, FBR-MUSV, AKR-MULV, R-MULV, and AMV (data not shown): For maximal stimulation, the effective polymer size was 6000 daltons (Table II) and the optimal concentration was 12% (w/w) (Fig. 2). Three lots of 6000 dalton polyethylene glycol from Fisher Scientific Company (including one autoclaved in H20) and one from Mallinckrodt Inc., were tested and found to be stimulatory. Reverse transcriptases showed similar requirements of divalent cations whether reactions were carried out in the absence or presence

TABLE EFFECTS

II OF POLYMER

SIZE ON

REVERSE

TRANSCRIPTASE

A predetermined a m o u n t of A M V was assayed b y the exogenous reaction in the absence and presence of 1 2 % polyethylene glycol having the indicated average molecular weights. Kinetics were followed as in Table I. Net incorporations within 1 h are shown.

Molecular weight (X 10 - 3 )

Incorporation ( c p m × 10 - 3 )

Enhancement (times)

None 0.6 1.0 6.0 20.0

2.0 3.2 12.4 16.4 2.9

-1.6 6.2 8.2 1.5

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Fig. 2. E f f e c t s o f p o l y e t h y l e n e glycol c o n c e n t r a t i o n on reverse t r a n s c r i p t a s e a c t i v i t y . T h e c f f e c t s of different concentrations of p o l y e t h y l e n e glycol on reverse transcriptase activity were tested u n d e r three assay c o n d i t i o n s : t h e e x o g e n o u s (o) a n d t h e e n d o g e n o u s (A) r e a c t i o n with d i s r u p t e d FBR virus a n d the r e c o n s t i t u t i o n (X) r e a c t i o n w i t h p u r i f i e d F B R R N A . T h e r e a c t i o n s w e r e i n c u b a t e d at 3 7 ° C for 2 h. T h e n e t i n c o r p o r a t i o n ( m i n u s z e r o t i m e ) in t h e p r e s e n c e of the p o l y m e r were c o m p a r e d w i t h t h o s e in the a b s e n c e o f t h e p o l y m e r a n d are e x p r e s s e d as r a t i o s o f s t i m u l a t i o n . Fig. 3. C h a r a c t e r i z a t i o n o f [ 3 H ] c D N A p r o d u c t s b y m o l e c u l a r h y b r i d i z a t i o n . SSV [ 3 H ] c D N A s m a d e in a 2 h e n d o g e n o u s r e a c t i o n in t h e a b s e n c e (o) a n d p r e s e n c e (X) of p o l y e t h y l e n e glycol w e r e a n n e a l e d to E. coil a n d SSV R N A s . H y b r i d s w e r e a n a l y z e d as d e s c r i b e d .

of polyethylene glycol (data not shown). The absolute dependence on and concentration optimum of either Mg 2÷ or Mn ~ were unaltered for GALV reverse transcriptase as tested in the exogenous reaction. Polyethylene glycol increased the yields of [3H]cDNA products 1.8--6.5 times for different viruses in differ-

T A B L E III E F F E C T ON [ 3 H ] c D N A YIELDS T h r e e t y p e s o f r e a c t i o n s w e r e used to s y n t h e s i z e v a r i o u s viral [ 3 H ] c D N A p r o d u c t s in t h e a b s e n c e a n d p r e s e n c e o f 12% p o l y e t h y l e n e glycol. T h e final yields of l a b e l e d D N A p r o d u c t for e a c h pair o f parallel r e a c t i o n s are given a n d c o m p a r e d . R e a c t i o n s w e r e p r i m e d w i t h e i t h e r calf t h y m u s d e o x y l i g o n u c l e o t i d e s (*) o r ( d T ) 1 2 - - 1 8 ( u n m a r k e d ) . Reactions

Viruses

P o l y e t h y l e n e glycol ( c p m × 10 - 3 ) 0%

Stimulation ( t i m es )

12%

Simultaneous detection

FBR

6.5

16.0

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FBR FBR

58.6 42.0

201.3 178.0

2.5 3.4 * 4.2 *

Reeonstitution (purified)

SSV AKR FBR FBR

86.8 50.4 6.6 360.0

157.7 90.3 11.8 640.0

1.8 1.8 1.8 * 1.8 *

FBR

55.0

355.0

6.5

359

ent synthetic reactions, including the highly efficient system using deoxyribonucleotides from calf thymus DNA as primers [10] (Table III). [SH]cDNAs synthesized in the absence and presence of the polymer, were characterized and compared by molecular hybridization (Fig. 3). Neither product made of SSV hybridized to E. coli RNA but both hybridized 90% to SSV RNA, forming very thermally stable DNA-RNA hybrid duplexes with midtransition temperatures of 85°C. Comparable results were obtained with parallel products of FBJMUSV and FBR-MUSV made by the reconstitution reaction. In addition, the above products made with or without the polymer were the same size, sedimenting as 4--6 S DNAs in alkaline velocity sucrose density gradients (data not shown). Polyethylene glycol has a stabilizing effect on reverse transcriptase (Fig. 4). When preincubated at 37°C in the absence of synthesis, purified AMV reverse transcriptase lost 80% of its activity in 1 h. When preincubated in the presence of 12% polyethylene glycol under otherwise identical conditions, the enzyme lost only 25% of its activity. In contrast, 30% glycerol had only minimal stabilizing effects. Time course of addition studies indicated that polyethylene glycol could prevent early cessation of the reaction (Fig. 5). When it was added at zero time, this early cessation did not occur. On addition of polyethylene glycol to a standard reaction that had essentially stopped at 1, 2, or 3 h after initiation, synthesis resumed at rates higher than the initial rate before the addition of

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Fig. 4. E f f e c t s o n t h e stability o f reverse transcriptase at 3 7 ° C . A l i q u o t s o f 6 . 4 units o f purified A M V reverse transcriptase w e r e p r e i n c u b a t e d at 3 7 ° C in 2 0 0 #1 of 0.01 M Tris ( p H 8.2), 0 . 1 5 M NaCI, and 5 m M d i t h i o t h r e i t o l either a l o n e ( o ) , w i t h 3 0 % g l y c e r o l ( ~ ) , or w i t h 12% p o l y e t h y l e n e g l y c o l (X). A t t h e t i m e s i n d i c a t e d , 5 #1 w e r e a s s a y e d b y the e x o g e n o u s r e a c t i o n for 1 h. I n c o r p o r a t i o n s are e x p r e s s e d as p r e c e n t a g e s o f initial a c t i v i t y a s s a y e d w i t h o u t p r e i n c u b a t i o n . Fig. 5. T i m e c o u r s e o f a d d i t i o n o f p o l y e t h y l e n e glycol. A large ( 3 0 0 #I) e x o g e n o u s r e a c t i o n w a s set up w i t h 50 # g o f A M V and a l i q u o t s o f 10 #I o f the r e a c t i o n w e r e t e r m i n a t e d at t i m e s i n d i c a t e d to o b t a i n the base-line k i n e t i c s o f t h e standard r e a c t i o n ( o ) . A l i q u o t s o f 50 ~l w e r e a s s a y e d further w i t h 50 #I o f p r e i n c u b a t i o n b u f f e r c o n t a i n i n g 24% p o l y e t h y l e n e g l y c o l . T h e s e r e a c t i o n s w e r e c o n t i n u e d at 3 7 ° C and their k i n e t i c s similarly d e t e r m i n e d . Kinetics o f the r e a c t i o n w i t h p o l y e t h y l e n e g l y c o l a d d i t i o n s at 0, 1, 2, and 3 h, are r e p r e s e n t e d b y e, ~, m a n d A r e s p e c t i v e l y ,

360 polyethylene glycol. The rate of reinitiated synthesis, however, declined as the time to reinitiation b y addition of polyethylene glycol was extended. Additional tests indicated that polyethylene glycol stimulated other nucleotide polymerases, including the DNA polymerase I and RNA polymerase from E. coli and the terminal transferase from calf thymus (data not shown). We have n o t a t t e m p t e d to optimize the assay systems or the stimulatory effects of polyethylene glycol for these enzymes. Discussion

These studies indicate a new application of polyethylene glycol as a laboratory reagent. Incorporation of the 6000 dalton polymer at 12% (w/w) resulted in strong stimulation o f reverse transcriptase reactions with an increase in both the rate and duration of the reaction. These effects were observed in both crude and purified assay systems. Moreover, polyethylene glycol was effective even in the highly efficient purified transcription system involving oligonucleotide primers of calf thymus DNA. In addition, polyethylene glycol was effective on all t y p e B, t y p e C, and t y p e D viral reverse transcriptases tested. Polyethylene glycol apparently does n o t alter the fundamental properties of reverse transcriptases in terms of divalent cation requirements and the size and specificity of the products made. Its primary effect appears to be the stabilization of reverse transcriptase. However, the fact that it can reinitiate synthesis of a standard reaction that has stopped indicates that enzyme stabilization is not its only effect. The mechanism(s) b y which polyethylene glycol enhances the overall synthetic reaction remains to be elucidated. The stimulatory effect of polyethylene glycol on polymerases has several potential practical applications. For molecular probing studies improved synthesis of specific nucleic acids by reverse transcriptase is attainable in the presence o f polyethylene glycol. Terminal deoxynucleotidyl transferase activity has been demonstrated in peripheral neoplastic cells in human lymphoblastic leukemia and l y m p h o m a [16--19] and for chronic myelogenous leukemia in blast crisis [18--20]. The determination of terminal transferase activity provides a biochemical marker helpful in the classification of a l y m p h o m a and in the selection of appropriate therapy. The observed enhancement of terminal transferase by polyethylene glycol should increase the sensitivity of detection for this marker. In addition, the exogenous assay for reverse transcriptase with a selected synthetic template is the most sensitive and convenient assay commonly used for the detection of R N A t u m o r viruses. In our hands, incorporation of 12% polyethylene glycol into the conventional reaction mixture provides a several-fold increase in sensitivity.

Acknowledgements This research was jointly supported by the U.S. Department of Energy under contract No. W-31-109-ENG-38 and the Division of Cancer Cause and Prevention, National Cancer Institute through an Interagency Agreement, contract No. Y01 CP 7-0504. We thank C.K. Lee, V.A. Pahnke, and M.F. Williams for the propagation, harvesting, and concentration of FBJ and FBR viruses. J.G.R.

361 is a summer undergraduate student. Present address: Department of Microbiology, University of Maine, Orono, ME 04473, U.S.A.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

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