Simian Virus 40 Deoxyribonucleic Acid Synthesis: Analysis by Gel Electrophoresis

Copyright©1972 AmericanSociety for Microbiology

Simian Virus 40

Deoxyribonucleic

Acid

Synthesis:

Analysis by

Gel

Electrophoresis

PETER TEGTMEYER AND FRANCISCO MACASAET

Department ofPharmacology, Case Western Reserve University, Cleveland, Ohio, 44106

Received forpublication5July1972

An agarose-gel electrophoresis technique has been developed to study simian

virus 40

deoxyribonucleic

acid (DNA) synthesis. Superhelical DNA I, relaxed

DNA II, and replicative intermediate (RI) molecules were clearly resolved from

one anotherfor analytical purposes. Moreover, the RI molecules could be

identi-fied as early or late forms on the basis of theirelectrophoretic migration in

rela-tion to that ofDNA II. The techniquehas been utilizedto study the kinetics of

simian virus 40 DNA synthesis in pulse and in pulse-chase experiments. The

averagetimerequiredtocomplete the replication ofprelabeled RI moleculesandto convert them into DNA I was approximately 10min undertheexperimental

con-ditionsemployed.

Simian virus 40

(SV40) deoxyribonucleic

acid

(DNA) molecules have been studied by a

combi-nation oftechniques including dye

buoyant-den-sity

centrifugation,

velocity sedimentation, and

electronmicroscopy(1, 3, 8, 9, 12). Virion

parti-cles have been shown to contain superhelical

molecules (DNA I) which may be converted to

relaxed molecules (DNA II) by a single-strand

break (3). Cells infected by SV40 also contain a

viral

replicative intermediate

(RI) which has both

superhelical and relaxed regions (8, 12) and

polymeric forms of viralDNA (7). However,no

single technique has been utilized to clearly

dis-tinguish between the various forms of SV40 DNA

andtodetermine thedegree ofreplication of RI

molecules. Thorne has shown that the

super-helical and relaxed DNAof polyoma virus could

be resolved by electrophoresis through an agar

gel (14). Thepresentcommunication describes the

separation of the SV40DNAI, DNA II,and RI

moleculesatdifferent stagesofreplication witha

high degree ofresolution by a simple gel

elec-trophoresis technique. In addition, the kinetics of

the formation and processing of RI molecules

wereinvestigated.

MATERIALS AND METHODS

Cellculture. Africangreenmonkey kidney cell line AH (5)wascultivatedinF12medium containing2to

10%fetalcalfserum (FCS).

Virus.SV40strain WT (13), aclonedsmall-plaque

strain,wasusedfor allexperiments. Virus stockswere

prepared from cell lineAH monolayers inoculatedat

a multiplicity of 0.01 plaque-forming units (PFU)/ cell andplaque-assayed aspreviously described (13).

Infection of cells. AH monolayers grown to con-fluence in8-or32-o(45-or150-cm2 cellgrowing areas) prescription bottles were inoculated with 1.0 ml of

stock virusatamultiplicityof 10PFU/cell.After the

virus was allowed to adsorbto the cells for 2hr at

room temperature, the inoculum was replaced with

medium and the cells were incubated at 39 C.

Radioactive labeling of infected cells. After incuba-tion of the infected cultures for 48 hr, the mediumwas

aspirated, and4ml ofprewarmed medium containing

50to100,Ciof3H-thymidine (New England Nuclear Corp., Boston, Mass.; 15to20Ci7mmole) permlwas

addedtoeach culturebottle whichwasthen incubated underwaterattheappropriatetemperature.Atthe end of thepulse period, the culture bottles wererapidly

filled withaprechilled, 4 C solution of 0.137MNaCl,

0.005 M KCI, 0.01 M tris(hydroxymethyl)amino-methane (Tris) buffer, 0.001 M Na2HPO4, pH 7.4 (TBS). Thecell monolayer was further washedonce

with 10 ml of cold TBS before extraction. In

pulse-chase experiments, the medium containing isotope wasreplaced with prewarmed Hanks salt solution

con-taining 2% FCS andanexcessofunlabeled thymidine (100 Mg/ml). Under these conditions, the addition of 10 ,ug ofdeoxycytidine/ml tothechase medium (11)

did not alter the efficiency of the chase and was,

therefore, not utilized. After the appropriate time

period, the chase was rapidly terminated by the samemethoddescribed above fortermination of the

pulse.

Extraction of DNAfrom infected cells.Viral DNA

was selectively extracted from the infected cells as

describedbyHirt (6). Monolayers werelysed by 0.8 mlofasolution containing 0.01 MTris,0.01M

ethyl-enediaminetetraacetate (EDTA) and 0.6% sodium

dodecyl sulfate, pH 7.4, for 20minatroom tempera-ture. A 0.2-ml amountof5MNaClwas thenadded,

andthebottlewasgentlyinverted 10times.The mix-ture was poured intoa polyallomer centrifuge tube, 599

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stored at 4 Covernight, and thencentrifugedat4 Cfor 20min at 16,500rev/min in a Spinco SW65rotor.The supernatant fluid was dialyzed overnight in 0.01 M Tris and 0.01 M EDTA, pH 7.4. Thedialyzed super-natantfluid isreferred to as theHirtsupernatantfluid. Purification of SV40 virions and preparation of marker DNA. Stocksofvirus were grown in the pres-enceof 2MuCiof 14C-thymidine per ml. The virus in the medium was concentrated by polyethyleneglycoland isolated by isopycnic centrifugation in CsCl by the method ofFriedmann and Haas (4). DNA was ex-tractedfrom thepurifiedvirions by phenolcontaining 0.01 M Tris and 0.01M EDTA (pH 7.4). The aqueous phase wasdialyzed overnightat 4 Cagainst TBS.

Deoxyribonuclease treatment. Bovine pancreatic

deoxyribonuclease (electrophoreticallypure) was

ob-tained from Worthington Biochemical Corp. A 1-,ug amountof DNA extracted frompurified virions was

exposed for 10 min at 37 C to 0.02Kunitz units of

deoxyribonucleasein 0.2 ml of TBS (pH 7.4)

contain-ing0.001 M MgCl2 and 0.001 M

CaCI2.

The reaction

wasstopped byaddinganequal volumeof 4 C TBS

containing0.1 M EDTA.

Velocity sedimentation. Samples (0.1 ml) were

layeredonto 3mlof CsCl (p = 1.50 to 1.52g/cm3)in

0.01 MTris and 0.01 M EDTA, pH7.4, covered with 1mlof mineraloil,andcentrifugedat25Cfor 3.5 hr

at35,000rev/mininaSpinco SW65 rotor. Fractions (0.08 ml) weredrop collected, precipitated with 5%

trichloroaceticacid in the presence of 20

Mtg

ofcarrier

yeast ribonucleic acid, collected on nitrocellulose

filters,andassayedfor radioactivity inTriton-toluene

(10)byliquidscintillationcounting.

Dye buoyant-density centrifugation. Ethidium

bromide (EB)andCsClwereaddedtotheHirt

super-natantfluid to give a final concentration of 200

Mug

of

EB/ml and a final density of 1.560 g/cm3. Samples

(4 ml) werecentrifuged in a Spinco SW65 rotor at

40,000rev/minfor 65 hrat4 C. Fractions (0.06ml)

werecollectedfrom the bottom of the tubes. A10-Mliter

portionof eachfractionwasassayed forradioactivity

on Whatman 3-mm filter discs by the method of Bollum (2). The fractions ofinterest were dialyzed

overnightat4 Cagainst0.01 MTris and 0.01 M EDTA,

pH 7.4.

Preparation of agarose gels. The appropriate

con-centrations (weight/volume) of agarose (Seakem)

were autoclaved in electrophoresis buffer containing 0.036 MTris, 0.03 M NaH2PO4, and 0.001 M EDTA (pH7.5). The sterile agarose solutionwasdivided into

portionsandstoredatroomtemperatureforaslongas

1 yearwithout change inelectrophoresis characteris-tics. For use, the agarose wasliquified in a

boiling-waterbath, cooledto45C, and distributed to 8-cm

acrylictubes(0.6-cminternaldiameter). The gel tubes

werecooledfor30minatroomtemperature, and the top1-cmportionof the gel was removed with a scalpel blade. The tubes were then covered at the bottom

withwetdialysis tubingandimmediatelymountedin

theelectrophoresis apparatus (Model 70, Biox, West

Lafayette, Ind.) toprevent drying of the gels. Electrophoresis procedure. For electrophoresis, sodiumdodecylsulfate (SDS) was added to the elec-trophoresis buffer to a concentration of 0.2 g/100ml.

Thegeltubesweresubjectedtopre-electrophoresis in the SDS bufferatroomtemperature for 30 minat a

potential of 10v/tube (power source: model3-1014A, Buchler Instruments, Fort Lee, N.J.). Hirt super-natant fluid DNA samples (100Mlliters) were mixed with 50 Mliters of electrophoresis buffer containing 30% sucrose and 0.6% SDS and were warmed at 37 C for5 min.Samples (100,liters) wereloaded into the top of thegel tubes and subjectedtoelectrophoresisas

described in eachexperiment. The maximumquantity ofDNA added to agel tube in the experiments to be describedwas 10Mg.

Analysis of gels. After electrophoresis, the gelswere

eitheranalyzed immediately or stored at 4 C, foras

long as 1 week, with similar results.Coolingwasfound tofacilitateslicing of the gels. The gels were sliced into 2.5-mm discs which wereplaced into scintillation vials containing 10 ml of a Triton-toluene mixture (666 ml oftoluene, 333 ml of Triton X-100, 0.25 g of

dimethyl-1,4-bis-2-(5-phenyloxazolyl)benzene,and 8.25 g of

2,5-diphenyloxazole, Packard Instrument Co., Downers Grove, Ill.). The counting efficiency for 3H-labeled DNAsamples in the gel slices was less than 1% after 1 hr in the Triton-toluene mixture, but increased to stable levels of 15 to 20% after 2 days in the scintilla-tionfluid at room temperature. During this two-day period, the opaque gel slicesgradually became almost completely clear. More than 99% of 3H-labeled SV40 DNA remained within thegel slices after 1 month in thescintillation fluid as determined bytransferring the gel slice to a scintillation vial containing no Triton-toluene mixture and recounting the sample. The same procedureindicated that the scintillating events were actually occurring within the gel slice itself rather than in thetotalvolume of theTriton-toluene mixture. The counting efficiency of 3H-labeled DNA I samples in gelswhich had beendissolvedin 0.2mlof 30% H202 for 12 hrat70Cbefore mixture with 10 ml of Triton-toluene was approximately 20%. For

convenience,

samples were routinely assayed in intact gel slices.

Radioactivity of thesampleswas assayedinaliquid

scintillation spectrometer (series 720,Nuclear-Chicago Corp., Des Plaines, Ill.). Quench corrections were made by thechannels ratio method. Reconstruction experiments indicated thatsamplescounted in intact ordissolved gels fit similar quench correlation curves.

RESULTS

Electrophoresis of DNA extracted from virions.

SV40 DNA labeled with

'4C-thymidine

was

ex-tracted from purified virions as described in

Materials and Methods. DNAsamples, with and

without

deoxyribonuclease

treatment, were

sub-jectedto

electrophoresis

through 2% agarose

gels

as described in Fig. 1. Untreated virion-derived

DNA migrated as a large, sharp, leading peak

(DNA I) and as asmaller, trailing peak (DNA II).

No

heterogeneity

of either peak was observed

evenwhen thegels weresliced at 1-mm intervals.

No other DNA was observed on gels twice the

length illustrated here. Controlled

deoxyribo-nucleasetreatment

converted

DNA I into DNA

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12

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8

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4

2

C)

C.)

8

6

4

2

1

2

3

4

5

DISTANCE

MIGRATED

(CM)

FIG. 1. Gelelectrophoresisof SV40DNA IandII. DNA was extractedfrom purified SV40 virions and treated with deoxyribonuclease as described in Ma-terials and Methods. DNA samples suspended in loading buffercontaining 10% sucrose weresubjected

to electrophoresis for 2.5 hr through 2% agarose at

10 v/gel. Migration is from left (-) to right (+).

Thegelswere sliced andanalyzedasdescribed in Ma-terials and Methods. A, Untreated SV40 DNA; B, SV40 DNA treated with

deoxyribonuclease.

II. The untreated

'4C-labeled

DNA was

used

as

marker

DNAin the

remaining experiments.

Electrophoresis

of

SV40

RI.

To

examine

the

electrophoretic

behavior of SV40

RI,

AH

cells

were

labeled for 10

min

with

3H-thymidine

(100

,uCi/ml)

48

hr after

inoculation with SV40. The

viral DNA

was

selectively

extracted and

examined

by

a

variety of techniques

(Fig.

2). Velocity

sedi-mentation

analysis (Fig.

2A) showed

that mostof the

newly

synthesized viral

DNA

sedimented

asa

broad peak ahead of marker

DNA I

and II,

as

previously

described by others

(8, 9,

12).

Dye

buoyant-density centrifugation (Fig.

2B) showed

a

banding

of

viral

DNA at

heterogeneous

densi-ties

between the

densities

corresponding

to

marker

DNA Iand

II,

asalso

previously demonstrated

by

others

(8,

12). Gel electrophoresis

(Fig.

2C)

of the

same

sample clearly resolved

theDNA I

and

II

peaks.

Most

of

the

newly labeled viral

DNA

migrated

more

slowly

than DNA II. The

trailing

edge of this broad peak declined

sharply,

whereas the

leading

edge

gradually

extended through

the

DNA II

peak

to the base of the DNA I

peak.

Although

the DNA I,

II,

and

RI

peaks

overlapped

5

4

3

2

1U

03

'5

12

9

6

3

5

4

3

2

10

5

:

1

1~

4s,

3

₂

°

2

2

I

0

*0

5

4

3

2

DISTANCE MIGRATED

(CM)

FIG. 2. Acomparison of replicatingSV40 DNA as analyzed by velocity sedimentation, dye buoyant-density centrifugation, and gel electrophoresis. Cell line AH monolayers in 32-ounce(ca. 150cm2) bottles were inoculated with 10 PFU/cell, and 48 hours after infection at 39 C they were exposed to 100 ACi of 3H-thymidine per ml for 10 min. The Hirt supernatantfluidwasanalyzed by velocity

sedimenta-tion inneutralCsCl (A), dye buoyant-density centrifu-gation (B), andelectrophoresis through 1.5% agarose

for2hrat 10 v/gel (C).See Materials andMethods

for details of the techniques. Centrifugation fractions

werecollectedfrom the bottom of the tubesand num-beredintheorderof collection.

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tosome extent,an accurate estimateofthe relative

quantity ofisotope incorporated into each ofthe

three forms of viralDNAcouldeasilybe made on

the basis of this

single procedure.

In

parallel

ex-periments,

uninfected AH cultures

produced

no

detectable

DNA which

migrated

into thegels. To

further

correlate

the

behavior

of viral DNA in

dye

buoyant-density

centrifugation

and

gel

elec-trophoresis,

relevant

fractions from the

density

gradient

were

dialyzed

and

subjected

to

electro-phoresis

(Fig.

3).

Fraction a

(Fig.

2B),

cor-responding to

superhelical

DNA inthe EB-CsCl

gradient, comigrated throughanagarose

gel

with

marker DNA Ifor the most part.

However,

the

fraction also

contained

3H-DNA

which

migrated

somewhat more

slowly

than DNA I. This DNA

probably

represented

RI

molecules

early

in

the

replication

process

which

were not

completely

resolved from

DNA I in the

gradient

fraction

examined. Fraction

b

(Fig.

2B)

of

the

gradient,

corresponding

toRImolecules intermediate in the

replication

process

(8,

12),

migrated

to an

inter-mediate position

in the area

of

the agarose

gel

representing

RI

molecules. Fraction

c

(Fig.

2B)

of

the

gradient,

corresponding

to RI molecules

late in the process

of

replication

and to an

un-determined

portion of DNAII

(8, 12),

migrated

as a

small, sharp

peak

of DNA II anda

large

well-defined

peak of

RI

molecules

clearly behind

DNA

II on the

gel. Thus,

the

gel

technique separated

RI

molecules

in

various

stages of

replication.

In

addition,

DNA II

could

easily

be identifiedas a

small,

but

well-defined,

peak

superimposed

on a flat

portion of

the

broad,

RI

peak.

When

infected

cells

were

first

pulsed for

10 min with

3H-thymidine

and

then were

subsequently

chased

for

1 hr withan excess

of unlabeled

thymi-dine,

most of the

newly synthesized

DNA was

presentinformIDNAas

determined

by all three

methods. A

small

remaining

peak

corresponding

to RI DNA

could be identified

by

gel

electro-phoresis

analysis

(Fig. 4).

Studies

by

others have

shown that

3H-labeled

DNA I may reenter the

replicating pool

as the

parental

strands

of

repli-cating molecules

(8,

12).

Thus,

achase

would

not

beexpectedtobe

complete.

Replicative intermediate DNA was

also

examined

by

electrophoresis through

agarose

gels

of

varying

concentrations. Atanagarose

concen-tration

of

0.5%, all forms

of viral

DNA

migrated

as a

single peak.

Ata

1.0%

concentration,

formsI

and II were

poorly resolved

from each other but

could be

distinguished

from RI. Ata2%

concen-tration,

RI

did

not

completely

enterthe

gel

under the

conditions of electrophoresis,

but DNAIand IIwerewell

separated.

KineticsofviralRIsynthesis. Thehigh degree of

resolution of DNA 1,

11,

and RI by a single

2

I

%-O-%

x

CL)

30

2

4

2

5

4

3

2

v

5 '

1

4

N

3

so

2

a.)

I

0

5

4

3

2

1

2

3

4

5

[image:4.495.264.457.67.480.2]

DISTANCE MIGRATED (CM)

FIG. 3. Electrophoresis offractions a, b, and c from the dye buoyant-density centrifugation as indicated

inFig. 2B. Eachfraction was dialyzed against 0.01 M

Tris and 0.01 M ED TA, pH 7.4. A portion of the dialyzed fractions was subjected to electrophoresis through1.5%agarose at 10v/gelfor 2 hr.A, Fraction a from Fig. 2B; B, fraction b from Fig. 2B; and C, fractioncfrom Fig. 2B.

technique provided a means to measure

accu-rately

the rate of synthesis of RI. AH monolayers were

infected

at aninput multiplicity of 10

PFU/

cell and

labeled

with

3H-thymidine

for varying

periods of

time 48 hr after infection. The viral

DNA in the Hirt supernatant fluid was

examined

by

gel electrophoresis

(Fig. 5). With an increasing

duration of

thepulse period up to 40min, there

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6-

-~~~2

3-1 2 3 4 5

DISTANCE

MIGRATED

(CM)

FIG. 4. Gel electrophoresis ofSV40 DNA after a 3H-thymidine pulse and a 1-hr chase. Cell line AH monolayers in 32-ounce (ca 150 cm2) bottles were inoculated with 10PFU/cell,and 48 hrafterinfection

at39 C they were exposed to 100pCi of 3H-thymidine per ml for 10 min at 39 C and subsequently were chased with an excess ofunlabeled thymidine for 60 min. The Hirt supernatant fluid was subjected to electro-phoresisthrough 1.5%agarosefor2hrat10v/gel.

b

12

o

12

3

4

03

DISTANCE

MIGRATED (CM)

Fio. 5.Compositediagram of the gelelectrophoresis patterns of replicating SV40 DNA extracted from infected cells after exposure to 3H-thymidinefor

in-creasingperiods oftime. Cell line AHmonolayers in

8-ounce (ca 45 cm2) bottles were inoculated with 10

PFU/cellandincubatedat39 Cfor48 hr. Thecultures

were labeled with medium containing 50

pACi

of 3H-thymidine per mlforperiods of from 5 to 20 min.

Hirt supernatant fractions were subjected to electro-phoresis through 1.5% agarosefor 2 hrat 10 v/gel. The numbers in thefigure indicate the length ofthe

labeling periodinminutes.

was a

concomitant increase

in the

quantity

of

RI.

However,

the

shape

of the RI

peak,

asdetermined

by

gel

electrophoresis,

was

different

depending

on the

duration of

the

pulse period

(Fig. 5).

Aftera

5-min

pulse,

most of the

3H-labeled

DNA was

present in the

region

of

the

gel

which

was

pre-viously

shownto

correspond

tothe

location of

RI

molecules intermediate

inthe

replication

process. In

contrast, after

a

pulse

of 10minor

longer,

RI

molecules

late in the process of

replication

con-tained

most

of the

radiolabel. The shift

of

3H-DNA

tothe

late

region of the

RI peak

probably

coincided

with

the

average

time

period required

to

label

a

newly

replicated

strand

of

maximal

length.

The continued

increase

in

the

quantity of

3H-DNA in the RI region of the gel observed in

pulses longer

than 10 min

could be

explained,

in

part,

as a

reutilization of

recently

labeled

3H-DNA

I

molecules

as

the

parental strands of

newly

initiated

RI

molecules

and

possibly

asaresult of

a

depletion

of

endogenous thymidine

pools.

Processing

of RI molecules.

Pulse-chase

experi-mentswere

utilized

to

study

the rate at

which RI

molecules

were

completed

and

converted

into

DNA I.

Infected

AHcells were pulse-labeled

with

3H-thymidine

for

5

min.

The

incorporated label

was then

chased

with an excess

of

unlabeled

thymidine for varying

periods

of

time (Fig. 6). RI

disappeared

rapidly during

the

first

10min

of

the

chase witha

reciprocal

increase

in

DNA

I.

There-after,

the conversion of RI

into DNA

I

appeared

tooccurat amuch

slower

rate.

When the

length

of the

initial

pulse

period

was

varied,

the

efficiency

ofthe

chase, but

not the

kinetics

ofthe

chase,

depended

on the

length of

the

preceding

pulse

(Fig. 7).

The rapid phase of the chase has

been

interpreted

to

parallel the completion

of

replica-tion

of

thenewprogenystrands of RI

molecules,

whereas

the slow

phase

of the chase

reflected,

in

part, the

continued

reutilization

of

prelabeled

DNA Iasthe parentalstrands of newly

initiated

3

10

so

x go

2

TOTAL

DNA

I

IXR

IAI

DN

I

,~~~~~~~~~~~~

10

20

30

40

CHASE

TIME

(MIN)

FIG. 6. Kinetics of processing of 3H-thymidine-labeled SV40 DNA during chase periodsoffrom 0 to

40 minutes.Cell line AH cultures in 8-ounce(ca 45 cm') bottles were infected andpulse labeledfor 5 min as

described in Fig.5. At the end of thepulse, the medium containing 3H-thymidinewasreplaced with medium

con-taining an excess ofunlabeled thymidine. DNA was

extractedfrom the cultures and analyzed by gel electro-phoresisasdescribed in Fig.5. The total counts/min of

the S V40 DNAcomponentswereplotted above.

603

I

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100A

60

-J

l 40

k--40

-d

20-5

15 40

CHASE TIME (MIN)

FIG. 7. Effect ofthe lenigth ofthepulseperiodon

the efficientcy anidkinetics ofthe chase. Cell line AH

monolayersin 8-ounce (ca 45 cm2) bottleswere inocu-lated with 10 PFU/cell, anzd 48 hrafter infection at

39 C they were pulse labeled with 3H-thymidine for periods of from 5 to 40miili. Atthe entdofthepulse, themedium conitaininlg50IuCi of 3H-thymidineperml

was replaced with medium conitaininlg ani excess of unlabeled thymidinie. DNA was extracted from the cultures after 15- anid 40-mimi chase periodsand

ana-lyzed by gel electrophoresis as described in Fig. 5. The numbers in thefigureindicate the durationofthe initialpulseperiodinminiutes.

RI molecules. Although the factors responsible

forthe slowphaseofthe chase have not beenfully

investigated,

pulse-chase studiesprovide a useful meanstomeasuretherateofsynthesisof

replica-tive intermediatesand theirconversion intoDNA

Iunder standard conditions.

DISCUSSION

A new and simple technique utilizing

electro-phoresis through SDS-agarose gels has been

developedtoobtainahigh degreeofresolution of

the various formsofSV40 DNA. Accurate

esti-mates of the relative quantities of radiolabeled

DNA1,11,and RI couldeasily bemade, andRI

molecules at different stages in the replication

process could be identified. In the presence of

SDS, the migration ofDNA molecules through

gelswouldbeexpectedtodepend primarilyonthe

sizeandshape of the molecules. Previousstudies

by others utilizing density gradient and electron

microscopy techniques have shown that the RI

molecules, early in the process of replication,

remain predominantly superhelical in

configura-tion(8, 12). Thesemolecules would be expectedto

migrate through gels more slowly than DNA I

becauseof theirslightly larger size and less

super-helical configuration. RI molecules, late in the

processofreplication, havebeenshown by others

tobeuptotwice the size of DNAIandIIandto

be

predominantly

or

completely

relaxed in

con-figuration

(8,

12).

These

molecules would be

ex-pected

to

migrate

more

slowly

than DNA II.

Some RI

molecules,

intermediate in theprocessof

replication,

would

migrate

like DNA II. The

behavior of fractions derived from

dye

buoyant-density centrifugation

on

subsequent gel

electro-phoresis

strongly supported these

interpretations.

In contrast,

dye

buoyant-density

centrifugation

cannot be used to quantitate DNA II in the

presence

of

late RI

molecules in

a

relaxed

con-figuration. Velocity sedimentation failstoseparate

early

and late RImolecules

(12),

because the con-current increase in molecular weight and the

relaxation of replicating molecules affect the

sedimentation of the molecules in opposing

direc-tions.

Thus, gel electrophoresis

provides

favorable

resolutionfor monitoring SV40 DNA synthesis

by

asingle and simpletechnique.

ACKNOWLEDGMENTS

This investigation wassupported bya pilotgrant from the Cuyahoga Unit, Ohio Division, American Cancer Society.

WeareindebtedtoD.Caston for his adviceonthegel tech-niques uLsedin these studies.

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