Resolution and characterization of intracytoplasmic forms of reverse transcriptase from Rauscher leukemia virus-producing cells.

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0022-538X/78/0026-0001$02.00/0

Copyrighti 1978 AmericanSociety for Microbiology Printed in U.S.A.

Resolution

and

Characterization

of

Intracytoplasmic

Forms

of

Reverse

Transcriptase from Rauscher Leukemia

Virus-Producing

Cells

STUART L. MARCUS

MemorialSloan-Kettering Cancer Center, New York, New York10021

Received forpublication 9 November 1977

Themicrosomalsupernatantfraction obtained from a murinecellline

chroni-cally infected with and producing Rauscher leukemiavirus (JLSV-10)was found tocontaintwoforms of RNA-directed DNA polymerase (reverse transcriptase).

Thetwo enzyme forms, neither ofwhich is detectable in uninfected cells

(JLSV-9),wereinitiallypartiallypurified bypoly(C)-agarosechromatography, and their

separationwasachievedby phosphocellulose chromatography. Theenzymeform

elutingfirst fromphosphocellulose (0.3 MKCl),designated PCI, was found to be

identical in all parameters tested to that form isolated directly from purified

virions. The second enzymepeak, designated PC II, eluted from

phosphocellu-loseat0.5M KCI andwas notdetectable inpurified virions. The PC II enzyme

hasamolecular weight, determinedby velocitysedimentation, of approximately

109,000,ascomparedwith 70,000for the PC Ienzyme,andcouldnotbe further

dissociatedbyexposure tohighsaltornonionicdetergent. Mixing purifiedvirion

orPCI DNApolymerase with uninfected cells followed by fractionationdid not

produce the PCIIform, suggesting that it is neitheranartifactof purification nor

theresult of fortuitouscomplexingofreversetranscriptase with nornal cellular component(s). Both PCIandPCII enzymeforms appearedantigenically similar

tovirionDNApolymerase,demonstratedidentical divalent cationrequirements

forvarious template-primers, and were capableofcopying heteropolymeric

re-gions of rabbit globin mRNA. However, kinetic studies of heat inactivation

revealed that the PC II enzyme wasfarmore heat labile than the PC I form,

which appeared identical to the virion enzyme in this respect. Furthermore, whereas the PC I and virion-derived reverse transcriptase copied

poly(C)-(dG)12-18mostefficientlyat atemplate-to-primer molar nucleotide ratio of 25:1,

the PCII enzymepreferredaratio of5:1foroptimalratesofpoly(dG) synthesis.

Therefore, by these criteria, there appear to exist two intracellular fonms of

reversetranscriptaseinthe JLSV-10 Rauscher leukemiavirus-producingmurine

cell line.

RNA-directed DNA polymerase (reverse onthe

purification

ofreverse

transcriptase

from

transcriptase)has been showntoberequiredfor cells chronically infected with retrovirus (1, 4,

the productive infection and transformation of 8). Such studiesareneededto

identify

and

char-cellsbyavian retroviruses(17,31).Reversetran- acterize the form(s) of virion-related DNA

po-scriptase appears to be

required

for the initia- lymeraseinsuchcellsand also forthe

develop-tion,

although

notthe

maintenance,

ofthe vir- mentof

procedures

to

identify

theseenzymesin

ally transformed cell state (13, 29). Antibiotics human tumors, where retroviral involvement that inhibit the reverse transcriptase of has beensuggested (10).In thispaper,thepartial

Rauscher leukemia virus have been shown to purification and biochemical characterization of

preventtheleukemogenic activity of thatvirus twoforms ofreverse

transcriptase

fromthe

cy-(34), suggestingthat this enzymeplaysadirect toplasm ofRauscher leukemia

virus-producing

role in the induction of murine leukemia by cellsaredescribed.

exogenous virus. Anumberofreportsare

avail-ablein theliteratureconcerningthepurification MATERIALS AND METHODS

andbiochemical characterizationofmammalian Reagents.Allradioactivedeoxyribonucleoside tri-retroviralDNApolymerase (6,11, 12, 14, 23,26, phosphateswerepurchasedfrom Amersham/Searle.

30,32). Littleinformation, however, isavailable Unlabeled triphosphatesand template-primerswere 1

on November 10, 2019 by guest

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2

obtained from P-L Biochemicals. The molar ratio of volumes of ice-cold disruption buffer consisting of template to primer, unless otherwise indicated, was Tris-hydrochloride (pH 7.8), 1 mM dithiothreitol, 1 1:1 in the case ofpoly(A) (dT)12-,8and 20:1 in the case mMMgCl2,and 15%glycerol.Disruption was carried ofpoly(C) *(dG)12.18.Activated DNA was prepared as out with 40 strokes of a loose-fitting Dounce homog-describedbyAposhianandKornberg (2).Rabbitglo- enizer. Nuclei were removed by a 1,000 x g centrifu-bin mRNA was purchased from Amersham/Searle gation step carried out at4°C, and the resultingpellet and annealed tooligo(dT)12inthemolar ratio of 20:1 was discarded. The 1,000 x gsupernatant was sub-in 0.05 M Tris-hydrochloride, pH 7.8 (20). Starter jected to a10,000xgcentrifugationat40Cfor20min cultures of the non-virus-producing, continuous, to removemitochondria. Thesupernatantfrom that mouse bone marrow-derived fibroblastcelliine JLSV- step was then spun in an SW41 rotor for 1 h at 100,000 9 (3), as well as one chronically infected with and xg. Thepelletfraction thus obtained was suspended

actively producing Rauscher leukemia virus (JLSV- in1ml ofpoly(C)-agarosecolumnequilibrationbuffer,

10), were thegiftof Peter J.Gomatos of thisinstitute. and disruption buffer was added as previously de-Purified Rauscher leukemia virus and ahomogenous scribed forpurifiedRauscher leukemia virus(23). Pu-preparation ofEscherichia coli DNA polymerase I rification ofreverse transcriptase from this fraction

wereprovided byM. J.Modak of this institute. Puri- wasalsoaccomplishedaspreviouslydescribed(23)by

fied immunoglobulin G (IgG) preparations prepared using poly(C)-agarose affinity chromatography. The

frompreimmunized rabbit serum and fromantisera 100,000xgsupernatantfraction obtained from

JLSV-directedagainstpartiallypurifiedRauscherleukemia 9 or JLSV-10 cells was placed either directly onto

virus DNApolymerasewerethe kindgiftofCharles poly(C)-agaroseorafterthefollowingadditionswere

J. Sherr of the National Cancer Institute. DEAE- madetothat fraction: 0.05% (vol/vol)NonidetP-40, cellulose and phosphocellulose were obtained from 0.02%(wt/vol) sodiumdeoxycholate,and 0.04 MKCI. Whatman, Inc. Afterthe columnswerewashed with 10mil ofbuffer,

Preparationofpoly(C)-agarose.Poly(C)wasob- enzymewaseluted withalinear saltgradient(0to0.6

tained from P-L Biochemicals and was covalently M KCI)in wash buffer. Trasylol (Aprotinin), a

pro-coupledto agarosebeads (Sepharose 4B, Pharmacia tease inhibitor obtained fromMobay Chemical Co.,

Fine Chemicals, Inc.) througha modificationof the when usedwaspresent in the celldisruptionbufferat

original procedure (19).Cyanogenbromidewasadded aconcentration of 100U/ml. All columnswere run at

toSepharoseforactivation after firstbeingdissolved aflowrateof18ml/hat 4°C unlessotherwise

indi-inN,N-dimethylformamide (2ml ofsolvent for1g of cated. Fractions of 1-ml volumewerecollected.

BrCN).When thisprocedurerather than the addition Enzyme fractions that were obtained by elution

offinely dividedBrCN isused, the activationtime is from poly(C)-agarose weremade 0.5 M inKCI and

decreasedto 10min.All other stepsarethe same as passedthrough2-ml-bedvolumeDEAE-cellulose

col-previously described for coupling of poly(C)(19).After umns to remove any poly(C) that may have been

coupling,glycinewasadded to a final concentration of degraded during chromatography. The enzyme

frac-0.1 M tothepoly(C)-agarosesuspension,andstirring tions were thendiluted fivefold withpoly(C)-agarose wascontinued at4°Cfor2to4htoallow thecomplete column wash buffer andappliedto phosphocellulose substitution of activated groups thatmightbeleft on columns (0.9 by10cm) previously equilibrated with the agarose. Without suchblocking ofunsubstituted column wash buffercontaining0.1MKCI. Elution of

sites, someenzyme protein together with other pro- enzyme activity from phosphocellulose was

accom-teins may beinadvertentlycovalently coupledtothese plished (after columns were washedwith 10 ml of sitesduringthe course ofpurification, resultingin a buffer)with alinear saltgradient(0.1 M KCI to 0.8 M

loweringofyields. It isalso important to notethat KCI)inatotal volume of 60 ml. Peak enzyme fractions

during long periods of storage of poly(C)-agarose, wereusedimmediatelyorstored at0°Cinthe presence

traces of RNase may act to produce breaks in the of 100 ug ofbovineserum albumin monomer (Miles

polynucleotidestrands. Toremovefreepoly(C)from Laboratories, Inc.)per ml.

thematrix,columns should first be washed with 1M DNApolymerase assays.DNApolymerase

activ-KCI beforeequilibrationwith buffer.Poly(C)-agarose itywasassayedaspreviouslydescribed(23).Annealing columnswerepreparedwith1.5-to2-mlbedvolumes of varioustemplateandprimer combinations used in in Pasteurpipettesandequilibratedat4°C with buffer this study, both synthetic and natural in origin, was consisting of50 mM Tris-hydrochloride (pH 7.8), 1 carried out as described by Modak and Marcus (23).

mM dithiothreitol, and 10% (vol/vol) glycerol. We Molecular weight estimation. Sedimentation

haveshown thatpoly(C)-agaroseisaneffectiveaffin- coefficients were determined by centrifugation

ity matrix for the one-step purification of reverse through preformed 10 to 30%(vol/vol) linearglycerol

transcriptase from avian myeloblastosis virus (19) and gradienta in 0.05 MTris-hydrochloride buffer (pH 7.8) Rauscher leukemia virus(23) and for the partial pu- containing 1 mM dithiothreitol and 0.4 MKCI.DNA rification of murinemammary tumor virus DNA po- polymerase activity from peak poly(C)-agarose or lymerase (21). phosphocellulosecolumn fractions was diluted 1:1 in

Cell fractionation and enzyme purification. the same buffer used to prepare the glycerolgradients

CelLswere grown at 370C in plastic (Corning Glass and layered over5-ml gradients. Centrifugation was

Works) orglassrollerbottles in Eagle minimal essen- carried out in anSW50.1 rotor at 48,000 rpm for 16 h tial medium (5) supplemented with 10% fetal calf at4°C.Fractions were thencollectedfrom the bottom serum.Cellswereharvested inmid-logphase by scrap- of the tube and assayed for DNA polymerase activity ing and washed twice in phosphate-buffered saline. with poly(A) (dT)1218. Parallel gradients were run

Thecells(5 g[wetweight]) were then suspended in 5 with E. coli DNA polymerase I and purified

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derived Rauscher leukemia virus DNA polymeraseas 9 cells contained no poly(rCm)

(dG)12.18-di-molecular weight markers. The marker gradientswere rected synthesizing activity and were not

frac-assayedaspreviouslydescribed (19, 23). tionated

further

(data not

shown).

The relative Inhibition of DNA polymeraseactivitybyIgG recoveries of DNA polymerase activity from addition.Enzymefractions (10

pl)

wereincubatedat

viral

andcytoplasmicsources

are

listed

in

Table

00Cfor1h in atotal reaction volume of 35

pl

contain-ing 85 mM Tris-hydrochloride (pH 7.8), 100 mMKCI, 1. 1.7 mM dithiothreitol, 0.85 mM MnCl2, 0.085%

(vol/vol) Triton X-100, and the desired quantity of 5 immune or preimmuneIgG. Poly(A) *(dT)1218(0.5 ug) 40 _ A

and 5,uM[3H]dTTP (4,000 cpm/pmol) were added in

A30

0.6

a

25Spl

volume after preincubation, and polymerization I

was initiated by placing the reaction mixtures in a v 20 0.4

370Cbathfor 60 min. Thequantitationofincorporated 10-

'02

[3H]dTMP was carried out as described above for ____

DNApolymerase assays. x

RESULTS x

Purification of virion and microsomal 2 8

membrane pellet reverse transcriptase. Y?

Figure1shows theelutionprofilesof virion and e Y 6_ -0.6

JLSV-10 microsomal membrane pellet fraction , 0.5

DNA polymerase from poly(C)-agarose col- i 4 -0.4 T

umna. The template-primer poly(rCm).

(dG)12.

- 0.4

is,whichhas been reported specificforthe de- -> 0.3

tectionof reversetranscriptase activity (4),was 2 0.2

included in assays of column eluate fractions V

/._

from the cellular preparations. The

poly(A) *(dT)12.18-utilizingactivitycoeluted with 4 8 12 16 20 24 2 32 36 40

poly(rCm)*

(dG)12.i8-utilizing

activity from the 12 number

cytoplasmic pelletfraction at the same salt

con-Fracton

numbr

centration (0.24 M KCl) as did virion reverse FIG. 1. Elution from poly(C)-agarose columns of

transcrnptase. No terinal deoxynucleotidyl DNApolymeraseactivities from(A)

Rauscher

leuke-transferase activity was observed. F urther tud- mia

virions

and

(B)

the microsomalmembrane

peUet

ies (see below) revealed that the microsomal fraction derived from_{prepared and columns}

JLSV-10

_{were run}cell& Fractions_as _{described in}were

pellet DNA polymerase fromJLSV-10 cellspur-

the

text.Portions(20 fd)fromeach

fraction

were

as-ified in thismannerpossessed propertiesidenti- sayed forpoly(A)

(dT)1248-directed

(0)orpoly(rCm) calto those of the virion enzyme. Pellets pre-

(dG)i2-18directed

(0) or terminal

deoxynuclkotid-paredin a similar fashionfrom uninfectedJLSV- yltransferaseactivity(A)asdescribedinthe text. TABLE 1. Recovery ofpoly(A)*(dT)12i18andpoly(rCm) (dG)121s-directedDNAsyntheticactivityfromviral

and cellular sourcesafterpoly(C)-agarosechromatographya

Unitsof enzymeactivity'(x10-3)in:

Enzymesource Crude fractions Poly(C)-agarosecol- Poly(C)-agarosecol- Activityrecovered in

umnwash fractions umneluate fractions eluatefractions(%)

dTMP dGMP dTMP dGMP dTMP dGMP dTMP dGMP

Rauscher leukemia virus 160 10.1 15 0.9 115 7.5 72 75

(100Ag of viral protein)

JLSV-10 microsomal pellet 124 8.1 20.4 0.8 96 6.04 69 74

JLSV-10 microsomal super- 26.7 1.05 13.6 0.15 9.1 0.71 34 71

natant

JLSV-9 microsomal super- 6.0 NDC 5.6 ND 1.2 ND 20

natant

aApproximately5g(wetweight)ofeitherJLSV-9orJLSV-10celLswasharvested andprocessedas

described

inthe text, and the various fractionswerechromatographedonpoly(C)-agarose. Assaysfor DNAsynthetic activity directedbypoly(A)-(dT)12-1s (expressedasdTMPunits)orpoly(rCm)-(dG)12-18 (expressedasdGMP

unitsWcontained0.5mMMnCI2and50mMKCI,in additiontothe standardcomponentsdescribed in thetext. bOneunit ofactivityisdefinedasthat amountof enzymeincorporating1pmolof tritiated substrate into

acid-insolublematerialduring30 min of incubationat370C.

cND, Not detectable(<0.05pmolofdGMPincorporatedper

20-pd

portionunder assayconditions).

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4 MARCUS J. VIROL.

ResolutionofDNApolymerase activities phosphocellulose chromatograms of

poly(rC)-inJLSV-10high-speed cytoplasmic super- agarosecolumnpeaks corresponding to fractions

natant.Cellsweredisruptedasdescribed above 8to 12inclusive and15 to 19inclusive in Fig. 2A and in as gentleamanneras possible to avoid are shown in Fig. 3A and B, respectively. The

thepossibledisruption of enzyme complexes by secondarypeak of activity from poly(C)-agarose

detergent treatment(4). Nuclei were not used in (fractions 8 to 12) was resolved in this manner thefractionation procedure, since the supposed into two distinct peaks of roughly equivalent site of action andsynthesisof viral DNApolym- activity eluting from phosphocellulose at 0.3 M erase is thecytoplasm (12). In contrast to the and 0.5 M KCl. The primary peak of activity sharp, single-peak elution profiles shown for from poly(C)-agarose (fractions 15 to 19, Fig.

DNApolymerasefromRauscher leukemia virus 2A)elutedfromphosphocellulose as one major

and the JLSV-10 microsomal pellet

enzyme

(Fig. peak of activity at 0.3 M KCl (Fig. 3B), as did

1),elutionprofiles showing twopeaksof DNA the poly(C)-agarose peak reverse-transcriptase

polymerase activitywerereproduciblyobtained activityderived from either Rauscher leukemia

fromcytoplasmichigh-speedsupernatant chro- virus orJLSV-10 microsomal membranepellet

matographedonpoly(C)-agarose (Fig. 2A). The (data notshown). Therefore, the high-speed

cy-additionofdetergent(seeabove)toapproximate toplasmicsupernatantfrom Rauscher leukemia

conditionsused forchromatographyofdisrupted virus-producing cells appeared to contain two virions did notsignificantly alter the observed chromatographically separable forms of reverse elution profile. The primary peak of transcriptase, whereas the virion and

micro-poly(A)*(dT)12 18- andpoly(rCm)*(dG)12.18-uti- somalpelletfractionsappearedtocontain only

lizingactivityelutedat0.24 MKCI(asdoes the oneform of the enzyme. To determine whether

virion enzyme), whereas the secondary peak the two peaks of activity resolved by phospho-elutedat0.12 to 0.15MKCl. Chromatography cellulose chromatography were due to

artifac-ofJLSV-9 cytoplasmic high-speedsupernatant tual complexing of the virion enzyme with a

on poly(C)-agarose yielded the elution profile solublecytoplasmicconstituent, virion or

micro-shown inFig.2B. Smallamounts (seeTable1) somal pellet-derived enzyme was mixed with

ofpoly(A)*(dT)12.18-utilizing

activity

wereseen virus-freeJLSV-9 cellhomogenates,whichwere

toelute from 0.1 Mto0.22 MKCl,representing then fractionated in a manner identical to that

5 to10% of theactivityobserved in the JLSV-10 describedabove.Onlyonepeakof

reverse-tran-column eluates. No

poly(rCm)

*

(dG)12

18activity scriptase activity, eluting at 0.3 M KCI, was

wasobservedinJLSV-9column eluate fractions. resolved by phosphocellulose chromatography

The twopeaksofreverse-transcriptase activity in thismanner(datanotshown),suggesting that

obtainedfromvirus-producingcellswerepooled the0.5MKCIpeak activityfromJLSV-10cells

separately andfurther

analyzed by chromatog-

was indeedaform restricted tocellsproducing

raphy on

phosphocellulose

after passage virus. Further biochemical analysis of the two

through DEAE-cellulose to remove

possible

forms ofreversetranscriptase, designated PC I

traces of poly(C) (see above). Representative (0.3 M

KCl)

and PC II(0.5MKCl)

correspond-A B

50 C

0 6

40- -~~~~~~~~~~~~~~~~~~~~16v

e_o 30- 120 -0Q6

(J (~~~~~~~~~~1

~~~~~~~~~C

E

0-8 16 24 32 8 16 2 2

FractionNumber

FIG. 2. Elutionfrompoly(C)-agarose columns of DNApolymeraseactivityfrom high-speedmicrosomal

supernatantpreparationsderivedfrom (A)JLSV-1O cells and (B) JLSV-9 cells. High-speed cytoplasmic supernatantfractionswereprepared as described in the text.Portions(20

itl)

fromeach eluatefractionwere

assayed forpoly(A)J (dT)12.IS(0)-andpoly(rCm) (dG)1218(0)-utilizingactivity.

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VOL 26,1978 INTRACELLULAR R-MuLV DNA POLYMERASES 5

112

A B

o

OI

E -0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.8

IZS

T

EE

0. CL4

VO 2

2-~~~~~~~~~~~~~~~~~~~~~~

4 8 12 1610 24 28 23640 44 48 4 8 12 16 20 24 28 32 36 40 44 48

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FroctionNumber

FIG. 3. Chromatography of JLSV-10high-speedsupenatantpoly(rC)-agarosepooledeluatefractions8 to 12(A)and15to19(B) (seeFig. 2A)onphosphocellulose. Thepooled eluate fractionswereprocessedthrough

DEAE-cellulosecolumnsbefore phosphocelulose chromatography(see text). Phosphocellulosecolumneluate

fiactions

(2O-pdportions) upereassayedforpoIY(A)-(dT)12 8 (0)-andpoly(rCm)*(dG)1218 (0)-directedDNA synthesi8.

ing to their order of elution fromphosphocellu- ,

lose,

was carried out to detect

possible

differ- 18 +

encesin theirproperties. i

Analysis by

sedimentation

velocity.

The

16-molecularweights ofthe PCIandPC II forms ; 14

ofreverse transcriptase derived from the high- X 12

speed cytoplasmic supernatant fraction of °4

JLSV-10 cells were determined bysedimenta-

-tionthroughlinear 10 to30% (vol/vol) glycerol 8

-gradients in the presence of 0.4 MKCI(Fig.4).

Rauscher leukemia virus reverse transcriptase j 6 (70,000 daltons), purified by a single passage 4

-throughpoly(C)-agarose (23), and E. coli DNA 2

polymerase I (109,000 daltons) were used as

molecular weight markers. The PC I form of

reverse transcriptase (derivedfrommicrosomal B 2 4 6 8 10 12 14 16 18 20

pellet or supematant) cosedimented with the Fraction number T

virion-derived enzyme, indicating asedimenta-

FiG.

4. Glycerol gradient analysis of

JLSV-10

in-tion coefficientof4.2S corresponding to a mo- traceUularforms of reversetranscriptase.

Phospho-lecular weight of 70,000. The PC II enzyme ceUulose peakPC I(0) or PC II (A)fractions (150

consitently sedimented faster than the PC I id) werediluted 1:1 with 0.5M This-hydrochloride

form,at 5.5 to5.68, conrespondingto amolecular buffer, pH 7.8, containing 0.4 MKCI and I nM

weightof 109,000. The sedimentation patterns dithiothreitoland thenlayered on 10to 30% glycerol

were not alteredbythe useofNonidetP-40 at

gradients

prepared with thesame buffer.

Centrifu-0.2%(vol/vol)inaddition to 04 MKCI (datanot

gation

was

carried

out for 16h

with

an

SW5O.1

rotor.

shown. *Parallelgradients conaining

poly(C)-agarose-puri-shown). .

fled

Rauscher

lukemia

viru DNA polymerase

(0)

Template-primerutilization. Since the PC andE. coliDNA polymerase I (arrow indicates peak

I andPC II activities differed in apparent mo- fractionposition) were also runasmolecular

uwight

lecularweight, their respective abilitiestoutilize markers. FractionscoUectedfromthebottom of the

various template-primers (synthetic and natu-

cenrifuge

tubes were assayedfor DNApolymerase

ral)weretested andcomparedwiththose of the activitywithpoly(A)*(d-)-2-Ie

virion-derived

enzyme (23). Theresults of this

studyare shownin Table2. Both forms ofre- formswereableto copyheteropolymericregions

versetransciptasewereabletoutilize thesame ofrabbitglobin mRNA into DNA, thereby

con-spectrum of

template-primers

as has beenpre- firmingtheiridentityas truereverse

tanscrip-viouslydescribed for the Rauscher leukemia vi- tases.Optimal divalent cation concentrationsfor

rusenzyme(23), withnosignificantdifferences theuseofthevarioustemplate-primersfor both

observedbetween the PCIand PCI forms. It PCIand PC I enzymes werealso foundtobe

should be noted that both the PC I and PC II identical to those previously reported for the

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6 MARCUS J. VIROL.

TABLE 2. Utilizationofvarioustemplate-primersby PC I andPC IIJLSV-lO DNApolymerasesand Rauscher leukemia virus reverse transcrptase"

Substrate incorporationby:

Template-primer Divalent cation substrate PClen- PCIlen- Virion-de-substateP Ien PC I en-rived

en-zyme zyme zyme Synthetic

Poly(A) *(dT)1218 Mn2+ TTP 10.1 9.2 26

Poly(dA) (dT)1218 Mn2+ TTP <0.02 <0.02 <0.02

Poly(C) (dG)1218 Mg2+ dGTP 1.5 0.8 3.1

Poly(rCm) (dG)12-18 Mn2+ dGTP 0.8 0.7 1.0

Poly(dC) (dG)1218 Mg2+ dGTP 6.1 5.2 20.2

Natural

Activated calfthymus DNA Mg2+ dGTP 0.3 0.28 1.55

Globin mRNA+(dT)12 Mn2+ dGTP 0.15 0.13 0.21

GlobinmRNA+(dT)12 Mn2+-dATP and dCTP dGTP <0.01 <0.01 <0.01

(dT)12

Mg2e

orMn2+ dGTP <0.01 <0.01 <0.01

a

Assay

conditions and standard components used in reaction mixturesaregiveninthetext.In additionto

standardcomponentsandtemplate-primers,0.5mMMnCl2or2mMMgCl2was used whereverindicated,and

100 mMKCIwaspresent in all reactions.Approximately 5ngof RauscherleukemiavirusDNApolymerase enzymewasusedperreaction.Peakphosphocellulosefractions

(20-ptl

portions)ofPC I and PC IIenzymeswere

usedforeach reaction.Synthetictemplate-primer-directed reactionswereincubatedat37°Cfor30min.For

globin-mRNA-directedreactions,assaymixturesweredoubled insize,andthe incubation timewasincreasedto

60min.

Rauscher leukemia virus DNApolymerase (23).

optimal

ratio of

poly(C)

to

(dG)12.18

revealed

We have

recently

reported that Pi, at low differences between thetwointracellularforms concentrations(<10mM),effectivelyandselec- ofreverse

transcriptase (Fig.

5). The PC I

en-tively inhibits mammalian type C viral DNA zyme and the virion-derived reverse

transcrip-polymerases (23). The PCI, PCII, and virion- tasepreferred a template-to-primer ratio of 25:1, derivedreverse-transcriptase activitieswerein- whereas under identical conditions the PC II hibited80 to90%bytheaddition of2mMPito enzyme showed a marked preference for the reaction mixtures and to an equivalent degree 5:1ratio. Thisdifferencein therateof utiization

by 10mMN-ethylmaleimide (datanotshown). also serves to

explain

the

varying

responses

These results serve to identify the phosphate of the PC I and PC II enzymes to

sensitivity ofthe PC I and PC II enzymes and

poly(C)

-

(dG)12.18

showninTable

2,

inwhicha

also suggest that these reverse transcriptase 20:1template-to-primerratiowasused.

preparationsare notcontaminated with signifi- ApparentKmvalueswere also determined for

cantquantitiesof DNApolymerasesfOory,the substrate DNA precursors for thePC I,PC II,

formerbeingrelativelyresistanttoinhibitionby andvirion-derivedforms ofreversetranscriptase

N-ethylmaleimide (33)andthe latterbeingstim- with thesetwo

template-primers.

Allthreeforms

ulated rather than inhibitedbythe addition of ofreversetranscriptase yieldedanapparentKm Pitoreaction mixtures (16, 23). value for dTTP and dGTP of 20 ,uM. This value

In adetailed studyof thecatalytic properties did not change, regardless of the

template-to-of theRauscher leukemia virus DNApolymer- primer nucleotide ratio that was used. Apparent

ase,we have shownthatchangingthetemplate- Kmvaluesfor thesetwotemplate-primers for

all

to-primer nucleotide ratio can significantly af- threeforms ofreversetranscriptase were

iden-fect the rate of substrate polymerization (23). ticalat0.5 to0.7jug/ml.The pH optima for PC

The effect ofchanging the template-to-primer I, PC II, andthe virion-derived or microsomal

ratio on the rate of DNAsynthesis by PCI,PC pellet-derived reverse transcriptase were

deter-II,orthe Rauscherleukemiavirus-derived DNA mined by using Tris-hydrochloride buffers of polymerase wasdetermined, using afixed con- varying pH in the reaction mixture. For centration of

poly(A)looo

or poly(C)soo and an- poly(A).

(dT)12-1s-directed

synthesis, pH 7.8 to

nealing various amounts ofappropriate DNA 8.1 was the range found

optimal

for enzyme

oligomersasprimers (30).All three forms of the activity from all four sources. Therefore, al-enzymepreferredanequimolarratio ofpoly(A) thoughapparent kinetic constants for PC I and

to

(dT)o

nucleotides (datanotshown) for opti- PC II reverse transcriptase are identical with

mal rates of synthesis. Determination of the respect to substrateand template-primer

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[image:7.504.48.243.61.276.2]

VOL. 26, 1978 INTRACELLULAR R-MuLV DNA POLYMERASES 7

|1. 003' ,' \' z 2

~~~~~~~10

80

.E l 60 4 8 l 6 2 42

E~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Ig^G

o20_ _ ~~~~~~~~~~FIG.6. Inhibition of PCI1(O)and PCII1(0)

intra-C cellular JLSV-10 reverse transcriptase and JLSV-9

t;

~~~~~~~~~~DNA

polymerase

(A\)

by IgG directed against

X I I l I

~~~~~~~Rauscher

leukemia virus DNA polymerase. Enzyme

10 20 3 0 5 niiinsuiswr are out as described in

theextTheJLS-9 DAplymeaseactivityused Ratioottemplate to primer in this study was that derived frompoly(C)-agarose FIG. 5. Effect ofvarioustemplate-to-primernolar columneluatefractions (see Fig. 2) corresponding to

nucleotide ratiosofpoly(C) (dG)12.18 on theactivity differentareas of the elutionprofile, whichall gave

reversetranscrwptaseand Rauscher leukemia virus- (as picomoles offH]dTMP incorporated per hour) derived (0) reverse transcrptase. Poly(C) and~ were: PC I, 18; PC II, 13; JLSV-9 DNA polymerase,

(dO)12.18 were annealed in thedesiredratios as de- 4

scribed in the text. Enzyme activity inallcases was

KCI. Values corresponding to lOO'% activity in this DNAsynthesis by enzoymai acivtedluting8at

experiment

(aspicomoles of

pHJdGMP

ingcorporated

DAsnhssb nyai ciiyeuiga

per 30min)Jwere:PC Ipolymerase,2.5;-PClpolym- various salt concentrations after

chromatogra-erase, 2.0; Rauscherleukemia virus DNApolymnerase, phy of uninfected JLSV-9 extracts on

poly(C)-12.5. agarose (Fig. 2B) is also shown (Fig. 6). No

significantinhibitionof the normal cellular DNA

ties, as well as to pH optima, these enzymes may polymerase activity was observed. These results

bedistinguiishedby the template-to-primer nu- suggest that there are no significant antigenic

cleotide ratio forpoly(C)* (dG)1218requiredfor differences between the intracellular forms of

optimal rates of poly(dG) synthesis. reversetranscriptasethat wereresolvedby

phos-Susceptibility to IgG inhibition. The stud- phocellulose chromatography.

ies described above on the catalytic properties Enzyme stability and heat inactivation.

Of the PC I and PC II enzymes derived from Enzymefractions that were obtained by

phos-JLSV-10 Rauscher leukemia virus-producing phocellulose chromatography were either used

cells suggested that they were two forms of immediately or stored at000 after the addition

virion-coded reverse transcriptase. The immu- of bovineserumalbumin monomer (Miles

Lab-nological relatedness of the intracellular forms oratories, Inc.) to a final concentration of 100

of reverse transcriptase was tested by examining ,ug/ml. Long-term storage of PC I and PC II

the susceptibility of PC I and PC II toinhibition enzymefractionswas notattempted.During the

by IgG purified from rabbit antiserum directed course of the above studies, it was noted that,

againstpartiallypurifiedRauscherleukemiavi- even with the addition ofexogenous proteins,

rus DNApolymerase. The IgGpreparationthat

PCOII

activity declined at000more rapidly than

was used has been shown to be capable of de- did PC I activity. To determine whether this

tecting differences in mammalian type C viral decline in activity was intrinsic to a particular

DNA polymerases from various sources (28). enzymeform, the PC I and PC II enzyme

frac-The results of this study are shown in Fig. 6. tions, as well as poly(C)-agarose-purified

The ability of increasing quantities of anti- Rauscher leukemia virus DNA polymerase, were

Rauscher leukemia virusDNApolymeraseIgG heated for various periods of time at4500in the

to inhibit PC I and PC II enzyme forms is absence of

template-prmner,

substrate, or

diva-qualitatively

siilai

and equivalent to that de- lent cation (30). The enzymefractionswere then

termned withvirion-derived enzyme (data not cooled in an ice bath and tested for the loss of

shown). In addition to this study, the abilityof ability to direct DNA synthesis with

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8 MARCUS J. VIROL.

poly(rCm) (dG)1218as template-primer. There- identical to that found in the virion orin the sults of this study are shown in Fig. 7. The microsomal membranepellet fraction.

virion-purified reverse transcriptase and the in- DISCUSSION

tracellular PC I peak enzyme fraction

showed,

dIscUiON

underidenticalconditions, identical rates of heat Thisreport has described the presence of two inactivation. The PC II enzyme was far more forms ofRauscher leukemia virus DNA polym-sensitive to heatinactivation thanwasthe PCI erase in avirus-producingcell line.Fractionation enzyme or virion-derived polymerase. The ap- of the cytoplasmrevealed that the

majority

of proximate To.5 (one-half thermal inactivation reverse-transcriptase activity (>85%) in JLSV-time) for the PC I enzyme andRauscherleuke- 10 cells is found in the microsomal membrane mia virus-derived enzyme at this temperature, pellet fraction and that it is identical to the and measured with the template-primer, is 65

virion

form of the enzyme in such parameters as

man, whereas the T.6 for the PC II form Of molecular weight, optimal conditions for tem-reverse transcriptase is 11 mi. From this as well

plate-primer

utilization, and heat inactivation as the other data described above, the PC I

kinetics.

The high-speed cytoplasmic

superna-intracellular form of reverse transcriptase I tant fraction was found to contain only 10 to 20% of the total reverse-transcriptase activity,

100 andapproximately 20% of this soluble enzyme

90 activity was present in a form that differed from

the virion enzyme. The two forms that were

70 \ - t found in this fraction differ in their

position

of

70 _ elutionfromphosphocellulose columns. The PC

60 \ _ I

form

(eluting

first from

phosphocellulose)

ap-pears identicaltothevirion-derived enzyme in

so _ those

properties

thatwe havedescribed

previ-ously (23). The PC II enzyme form appears

40 higher in molecular weight (109,000 as opposed

to70,000[Fig.3]) than the PC I form, prefers a lowertemplate-to-primer ratio for optimal

copy-; \ming ofpoly(C) templates (Fig. 4), and is more

E

30 \ _ thermolabilethan PC I

(Fig.

7).

Both the PCI

and PC II forms ofreverse

transcriptase,

how-ever, appearantigenically similar in the degree

to which they are inhibited by IgG directed

20 _ agat the

partially purified

virion enzyme

(Fig.

6).

Although the quantity of PC II appears low compared with that of the PC Iformintotal cell fractions, it appears to be ofsignificant quantity since the majority of viral DNA polymerase

activityobserved is mostlikely due to the

pres-10.

sC

, ,

I0

I5

, ence

(in

the microsomal membrane

pellet)

of

20 30

budding

virus

particles

as

well

as"core"

parti-Minutes

[image:8.504.65.256.250.518.2]

o5

incubation

451

at cles. Because no treatment of cytoplasm was

FIG. 7. Kinetics ofheat inactivation of PCI(0) undertaken other than fractionation by

differ-andPCH(A) JLSV-10

intracelular

DNApolymer- ential centrifugation, the soluble enzyme frac-asesandRauscherleukemia virus reverse transcrip- tion should reflect the status of

reverse-tran-tase (0) at

450C.

Thepurified DNApolymerasesin scnptase formspriortopackaging in viral cores. buffer containing 0.05 MTris-hydrochloride, pH7.8, Processing ofthe uninfected cell line (JLSV-9) ImMdithiothreitol, 0.5MKCI, 10%glycerol, and 100 in anidentical manner produced no detectable pg of bovineserumalbumin monomer per ml, in 25- quantity ofreverse-transcriptaseactivity, as

de-ytl

portions, wereplaced in a45C waterbathfor termined by the ability of a DNA polymerase to

increasing period'softime,after which thete were copy themodified template poly(rCm)

(Fig.

2B).

cooled in anice bath.Ice-cold reaction mixtures were Experiments in which purified viral or

mi-then added to the enzyme fractions, and crosomal J n whic

purived

verse

tran-poly(rCm) (dG)128-directed synthesis wasinitiated crosomal JLSV-lO pellet-derived reverse

tran-byplacing the tubes in a 37°C water bath. Thepercent scriptase was mixed withJLSV-9 cells, which activity remaining was calculated byusing as con- werethen processedtodetect thepossible

for-trolsenzymefractions that had not been exposedto mation of PC II, yielded negative results (only

heat. the PC Iformwas observed). Therefore, PC II

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does not appear to arise from the fortuitous gent on the PC II intracellular form of reverse complexing of viral DNA polymerase with a transcriptase also failed to resolve high- and low-normal cellularcomponent or as a minor polym- molecular-weight components. Therefore, the erase ofuninfected cells. Theseexperiments also PC II enzyme

forn

that we have

described

does demonstrate for the first time the ability of not appear similar to the enzyme complex re-poly(C)-agarose to select for reverse transcrip- ported earlier.

tase in cytoplasmic preparations of virus-pro- The PC II enzyme form that we have de-ducing cells and to discriminate against cellular scribed appears similar in molecular weight to

polymerase. Such discrimination may be ob- anintracellular form of RD-114 virus DNA

po-served in the disproportionate loss of lymerase described by Gerwin et al. in infected

poly(A) (dT)128-directed DNA synthesis as cells (8). Although the high- and

low-molecular-compared with poly(rCm)-(dG)12.s-directed weight forms of the intracellular RD-114 virus

synthesis (specific forreverse transcriptase) in enzymes appeared identical with respect to

di-the JLSV-10 soluble cellular fraction poly(C)- valent cationoptima and apparent kinetic con-agarose column flow through (Table 1). The stants for substrates and template-primers,

identification of PC I and PC HIas"true" reverse other biochemical parameters, including the

transcriptases was made on the basis of their abilitytodirect naturalRNA-directed synthesis,

abilitytocopyheteropolymeric regions of rabbit were nottested (8). The increasedthermolability globin mRNA (Table 2) as well as on their of the PC II Rauscher leukemia virus intracel-responsetospecificantibodies (Fig.6). lular enzyme form, along with the increase in The existence of multiple forms of reverse apparent molecular weight, suggests an

altera-transcriptase withdiffering properties in intra- tion in structure as compared with the

virion-cellular particles obtained from cells infected derived form of reversetranscriptase. The

find-with gibbon ape leukemia virus has been re- ing that the PC II enzyme formcopiespoly(rC)

portedbyGillespieetal. (11). However, the two optimallyat atemplate-to-primer ratio different

forms studied by this group appeared to arise from that which is optimal for the virion enzyme via monomer-dimer interconversion mediated alsosuggests apossiblestructural difference

be-by salt and/or detergent concentrations. tweenthe twoenzymes. It istemptingto

spec-Rauscher leukemia virus DNApolymerasehas ulate, asothers have (8), that the

high-molecu-also beenreportedtoexistas aseries of aggre- lar-weight form serves as a precursor to the gate forms(26),althoughwehave shownthat in virion-packagedform inasystem similarto

that.

the presence of0.4MKCInosignificant degree of the/Bsubunit of avian retroviral reverse

tran-ofaggregationis observed(23). Bandyopadhyay scriptase(9). Our findings (i) that the PC II form

fractionatedand characterized the DNApolym- wasobservedonlyin soluble rather than

partic-erasesfrom JLSV-9(3)and JLSV-10(4)cellsby ulate fractions and (ii) thatmixing experiments

using classical purification procedures. He ob- failed togenerate the PC II form frompurified

servedauniqueDNApolymerasewithamolec- polymerase and uninfected cell components

ularweightof110,000in thecytoplasmofJLSV- would seem to lend support to that concept.

10cells,whichappearedtorepresentacomplex However,additionalpossibilitiesthat thePCII

formnedbetween allcytoplasmicviralpolymerase enzyme form(i)represents the DNApolymerase

and a 35,000-dalton cellular DNA polymerase. of a variant strain ofRauscher leukemia virus

Thecomplexcouldbeseparated by detergentor presentinsmall amounts or (ii) isproduced

by

phospholipasetreatmentandwasalsoobserved a related endogenous viral genome present in

by the author to be present in purified viral JLSV-9 cells that is activatedduring viral

infec-preparations. Attempts inourlaboratoryto re- tioncannot,atpresent, becompletelyruledout.

producetheseresultsby followingtheidentical Futurestudiesontheenzyme formPCIIwill

fractionationprocedurewereunsuccessful. How- concentrate onthequestionof whether it indeed

ever, it was found that the ammonium sulfate representsanenzymatically active intennediate

fractionationstep resulted in enrichment for the in thecleavage of the polyproteinprecursor to

PCI(virion) enzymeformn byprecipitatingpar- DNA

polymerase

(15) or a noncovalently but

ticle-bound

polymerase.

Furthermore, sedimen- tightlylinkedprotein-enzyme

complex.

tation velocity profiles of Rauscher leukemia

ACKNOWLEDGMENTS

virus reverse transcriptase from crude or puri- I thank Mukund J. Modak for helpful discussions and fiedenzymefractions,in thepresenceorabsence purified Rauscherleukemia virusand E.coliDNApolymerase ofdetergent, wereconsistentlyfree ofanysuch IandCharles J. Sherr foranti-reverse-tanscriptaseIgG.The 35,000-dalton polymerase activity (23)whenas- continuedinterestandencouragement of Nurul H. Sarkaris

sayedunderconditions reportedoptimalfor its appreciated._{for expert}_{technical assistance.}Thanksarealsoexpressed to Steven W.Smith

detection (4). Sedimentation velocity analysis Thisstudy wassupportedby Public Health Servicegrants

performed in thepresenceor absence of deter- CA-08748and CA-18369from the National CancerInstitute.

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LITERATURE CITED Virology53:258-273.

1.Allaudeen, H. S., M. G. Sarg haran, and R. C. 18. Marcus, S.L., and M. J.Modak.1976. Observations on Gallo. 1976. Acomparative evaluation of methods for the template specific conditions for DNA synthesisby isolation ofRNA-directedpolymerasein a reconstituted avianmyeloblastosis virus DNA polymerase. Nucleic system. Biochim.Biophys. Acta 435:45-62. AcidRes.3:1473-1486.

2. Aposhian, H. V., and A. Kornberg. 1972. Enzymatic 19. Marcus, S. L,M. J. Modak, and L F.Cavalieri.1974. synthesis ofdeoxyribonucleicacid. IX. Thepolymerase Purification of avianmyeloblastosisvirus DNA polym-formedafterT2bacteriophageinfectionofEscherichia eraseby affinity chromatography on polycytidylate-coli:a newenzyme.J. Biol. Chem.237:519-525. agarose. J.Virol.14:853-859.

3. Bandyopadhyay,A. K. 1975. Purificationand properties 20. Marcus,S.L, M. J.Modak,andL F.Cavalleri.1974. of nuclear and cytoplasmic DNA polymerases from Evidence fortemplate-specificsites on DNA polymer-JLS-V9cells. Arch.Biochem. Biophys.1":72-82. ases.Biochem.Biophys. Res.Commun.56:516-621. 4. Bandyopadhyay, A. K. 1975. Partialpurificationand 21. Marcus, S. L., N.H. Sarkar,and M. J. Modak. 1976.

properties of DNApolymerases from JLSV-10 cells. Purification andpropertiesofmurinemammary tumor Arch.Biochem.Biophys. 166:83-93. virus DNApolymerase.Virology71:242-254. 5. Eagle,H. 1959. Amino acidmetabolism in mammalian 22. Marcus,S.L,S. W.Smith,C. L.Jarowski,and M.J.

cell cultures. Science310:432-437. Modak. 1976. Terminaldeoxyribonucleotidyl transfer-6. Gerard, G. F.,and D. P.Grandgenett.1975.Purifica- an activity in acute undifferentiated leukemia.

Bio-tion and characterizaBio-tion of the DNA polymerase and chem.Biophys.Res. Commun. 70:37-44.

RNase HactivitiesinMoloneymurine sarcoma-leuke- 23. Modak, M. J., and S. L Marcus. 1977. Purificationand miavirus. J. Virol. 15:785-797. propertiesof Rauscher leukemia virus DNApolymerase 7. Gerard, G. F.,F.Rottman, andML Green.1974.Poly and selective inhibition ofmammalian virus reverse (2'TO-methylcytidylate) oligodeoxyguanylateas atem- transcriptase by inorganicphosphate. J.Biol. Chem. plateforthe ribonucleicacid directed deoxyribonucleic 252:11-19.

acidpolymeraseinribonucleic andtumorvirus particles 24. Modak,M.J.,S. LMarcus,and L F. Cavalieri. 1973. andaspecific probefor theribonucleic acid directed DNAcomplementaryto rabbitglobinmRNA madeby enzyme in transformed murine cells. Biochemistry E. coli DNApolymeraseI. Biochem. Biophys. Res.

13:1632-1640. Common.55:1-7.

8. Gerwin, B. I., S. G.Smith,and P. T.Peebles. 1975. 25. Moelling,K. 1974. Characterization ofreverse transcrip-Twoactive forms ofRD-114virus DNA polymerase in taseand RNase H from Friend murine leukemia virus. infected cells. Cell6:45-52. Virology62:46-59.

9.Gibson, W., and I. M. Verma. 1974.Studies on the 26. Nakajima,K.,K.Ohno,and Y. Ito. 1974. Interconver-reversetranscriptaseofRNAtumorviruses. I.Struc. sion of molecular size of the DNA polymerasefrom tural relatednessof the two subunits of avian RNA Rauscher leukemia virus.Intervirology3:332-341. tumor viruses. Proc. Natl. Acad. Sci. U.S.A. 27. Ritzi, E., D. S.Martin,R. LStolfi, andS.Spiegelman. 71:49914994. 1976. Plasma levels of a viral protein as adiagnostic 10.Gillespie, D., R. E.Gallagher, R.G. Smith, W. C. signalforthe presence of tumor: themurinemammary Saxinger,and R. C.Gallo.1975.On theevidencefor tumor model. Proc. Natl. Acad. Sci. U.S.A. Type C RNAtumor virusinformationand virus-related 73:4190-4194.

reversetranscriptaseinanimalsand inhuman leukemic 28. Sherr, C.F., M. M.Lieber,R. E.Benveniste,and G. cells,p. 1-27. In A.Gottlieb,0.J.Plescia,and D. H.L. J. Todaro. 1974. Endogenous baboon type C virus

Bishop (ed.), Fundamentalaspects ofneoplasia.Sprin- (M7):biochemical and immunologic characterization.

ger-Verlag,New York. Virology58:492-503.

11. Gillespie,D., W. C.Saxinger,andR.C. Gallo. 1975. 29. Ting,R.C.,S.S. Yang, and R. C. Gallo. 1972.Reverse Informationtransferincellsinfected by RNA tumor transcriptase RNA tumor virustransformationand de-virusesand extension to humanneoplasia. Prog. Nucleic rivativesofrifamycinSV.Nature (London) NewBiol. AcidRHs.Mol. Biol.15:1-108. 236:163-166.

12. Green,M., and G. F.Gerard.1974.RNA directed DNA 30. Verma,L.M. 1975. Studies onreversetranscriptase of polymerase-properties and functions in oncogenic RNA tumorviruses. IH.Properties of purified Moloney RNAviruses and cells. Prog. Nucleic AcidRes.Mol. murineleukemia virus DNA polymerase andassociated Biol. 14:187-334. RNase H.J. Virol. 15:843-854.

13. Hana ILH.,and T.Hanafusa. 1971. Non-infectious 31. Verma, I.M., G. F. Temple, H. Fan, and D. Baltimore. RSV deficient in DNA polymerase. Virology 1972. In vitro synthesis of DNA complementary to

43:313-320. rabbitreticulocyte RNA. Nature (London) NewBiol.

14. Hurwitz, J.,and J. P.Leis.1972.RNA-dependent DNA 235:163-167.

polymerase activityof RNA tumorviruses.I. Directing 32. Waters, L. C., and W.-K. Yang. 1974. Comparative influence of DNA in the reaction. J.Virol. 9:116-129. biochemicalproperties ofRNA-directed DNA polym-15.Jamjoom, G.A.,R.B. Naso, and R. B.Ariinghaus. erasesfrom Rauscher murine leukemiavirusandavian

1977.Furthercharacterization of intracellularprecursor myeloblastosisvirus. CancerRes.34:2585-2593. polypeptides of Rauscher leukemia virus. Virology 33. Weissbach, A. 1977. Eukaryotic DNA polymerases.

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16.Knopf, K.-W., MLYamada, and A.Weissbach.1976. 34. Wu, A. M., M. G. Sarugadharan, andR.C. Gallo. HeLacellDNApolymerasey,furtherpurification and 1974.Separation of ribonuclease H and RNA-directed propertiesof theenzyme. Biochemistry15:4540-4548. DNApolymerase (reverse transcriptase) of murine type 17. Linial,K,andW. S.Mason. 1973.Characterization of C RNAtumor viruses. Proc.Natl.Acad. Sci. U.S.A.

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