Identification of the herpes simplex virus DNA sequences present in six herpes simplex virus thymidine kinase-transformed mouse cell lines.

0022-538X/80/01-0272/14$02.00/0

Identification of the Herpes Simplex Virus DNA Sequences

Present in

Six Herpes Simplex Virus Thymidine

Kinase-Transformed

Mouse Cell Lines

JEFFREY M.

LEIDEN,'

NIZAFRENKEL,'* ANDFREDRAPP2

Department of Biology, The University of Chicago, Chicago, Illinois60637,1 and Department of Microbiology and Specialized Cancer Research Center, The Milton S. Hershey Medical Center, The

Pennsylvania State University College of Medicine, Hershey, Pennsylvania170332

We

have

used

a

novel filter

hybridization approach to detect and map the

herpes

simplex virus

(HSV) DNA

sequences which are present

in four HSV

thymidine kinase (HSVtk+)-transformed cell lines which

were

derived by

exposure

of

thymidine kinase negative (tk-)

mouse cells to UV light-irradiated HSV type 2

(HSV-2).

Inaddition, we have mapped the

HSV-1

DNAsequences which are present

in

two

HSV-ltk+-transformed

cell lines

produced by

transfection of

tk-mouse

cells with sheared HSV-1 DNA. The results of these studies

can

be

summarized

as

follows.

(i)

The

only

HSV DNA

sequences

which

werecommon to

all HSVtk+-transformed

cells

were

those

located between

map

coordinates

0.28

and

0.32.

Thus, this region contains

all

of the viral

DNAsequences

which

are necessary

for the expression

of HSV-mediated tk transformation. (ii)

Many

of the

cell lines also contained variable

amounts

of non-tk

gene

viral DNA

sequences

located between

map

coordinates

0.11 to 0.57

and

0.82 to 1.00,

suggesting that

incorporation of the viral DNA

sequences

located between these

map

coordinates

is

a

relatively random

event.

(iii) The viral DNA

sequences

located between

map

coordinates

0 to 0.11

and

0.57 to 0.82 were

uniformly absent from all of the

HSVtk+ cell lines

tested,

suggesting that there is

a strong

negative

selective

pressure

against incorporation of these viral DNA

sequences.

During their

lytic cycle, herpes simplex

vi-ruses

have been

shown

toprogram

the

synthesis

of

a

virus-specific thymidine

kinase.

(tk)

enzyme

(6, 15). This tk has been shown

to

be distinct

from

both the nuclear and mitochondrial

mouse

and hamster cell

enzymes

with

respect to a

number

of biochemical and

immunological

prop-erties, including thennal

stability,

electropho-retic

mobility,

and

antigenic specificity

(16, 26).

The

enzyme

has

been shown

to

belong

to

the

,B

class

(11) of viral

proteins,

in that its

synthesis

requires

the

presence

of

functional

early

(a)

viral

polypeptides and is turned off late in infection

by late

(y)

viral

polypeptides (7,

18; R. Honess

and B.

Roizman,

unpublished observations).

Munyon

et

al.

(27)

were

the

first

to

show that

a

small

proportion

of

tk-negative (tk-)

mouse

cells could

be

stably

transformed

tothe

tk-pos-itive

(tk+)

phenotype

after exposure

of

the

tk-cells

to

UV

light-irradiated herpes

simplex

virus type 1

(HSV-1).

The

tk

enzyme

made in

such

biochemically

transformed

cells has been

shown

topossess the

biochemical

and

immunological

characteristics of

the

tk

produced during lytic

HSV infections

(4, 17, 26,

34).

More

recently,

transformation of tk- mouse

cells

to the

tk+

phenotype has been

accom-plished by transfecting the tk-

cells

with either

sheared HSV

DNA,

or

isolated restriction

en-zyme

fragments of HSV DNA (1,

21, 23,37). By

using these

approaches, Wigler

et

al. (37) have

shown

that

a

3.4-kilobase

fragment

of HSV-1 (F)

DNA is sufficient

to

stably

transform tk- cells

to

the

HSV

tk-positive (HSVtk+)

phenotype.

Furthernore, Maitland and McDougall

(21)

have

reported that the purified restriction

en-zyme

fragments

of

HSV-2 (333) DNA which

contain the viral DNA

sequences

located

be-tween map

coordinates

0.53

and

0.65 are

suffi-cient

to

transform tk-

mouse

cells

to

the

HSVtk+

phenotype.

This

map

location

for the HSV-2

tk-transforming

genewas

surprising

in

light

of re-cent

studies of HSV-1

x

HSV-2 recombinants

which

have

revealed

that most,

if

not

all,

of

the genes

of these

two

viruses

are

colinear

(24,

25,

29),

and

which have

mapped

the

HSV-1 tk

gene

between coordinates

0.27and 0.35

(25).

The studies

described

in

this paper were

de-signed

toboth

unambiguously

map

the HSV-1

and

HSV-2

tk genes andto

study

the

patterns

of

incorporation

of non-tk gene

viral

DNA

se-quences in

HSVtk+

transformants

produced by

infection of tk-mouse cells with UV

light-irra-diatedHSVor

by

transfection of thesecells with

272

on November 10, 2019 by guest

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HSV DNA

SEQUENCES

IN HSVtk+-TRANSFORMED 273

sheared viral DNA. Toward these endswehave

developed

a novel

hybridization mapping

ap-proach

which has allowed the detection and identification of the HSV DNA sequences which are present in a number of

HSVtk+

cell lines. Our results have revealed that

(i)

the

only region

of the HSV genome

which

is present in

all

HSVtk+-transformed cells is located between

map

coordinates 0.28 and

0.32, and (ii)

a

wide

variety

of non-tk gene viral DNA sequences can be

stably incorporated

into the

HSVtk+

cells.

However, specific regions

of the

HSV

genome appear to be

uniformly

absent from such

HSVtk+

transformants.

MATERIALS AND METHODS

Virus. HSV-2 strains 333 and 324 (both isolated frompenile lesions) and Silow (isolated from a vaginal lesion)wereobtained from W. Rawls (McMaster Uni-versity,Hamilton, Ontario,Canada). HSV-1 (1023) is arecombinant betweenHSV-1 (HFEM) and HSV-1 (MP) (32).HSV-2 strain G, HSV-1 strains F and MP, and HSV-1 (1023) were obtained from B. Roizman

(UniversityofChicago,Chicago,Ill.). HSV-1 (Justin) wasobtained from A.Sabin.

HSVtk -transformed mouse cells. HSVtk+-transformed cell lines 33A+, 39A+,

59W,

and Silow wereobtainedby the biochemical (tk)transformation method ofMunyon etal. (27), as modified by Rapp and Turner (30). NclAcllO (tk-) cells, inbred Swiss mouse cells lacking tk activity, were originally ob-tained from R. Goldberg (National Institutes of

Health, Bethesda, Md.). The stocks of HSV-2 to be used fortransfornationwereUV-irradiated for5min

at 46 ergs/s per

mm2.

This UV-irradiated viruswas

then usedtoinfect the NclAcllOcells insuspension

(6x 105cellsper 100-mmplate) at a multiplicity of 2 to 4PFU/cell (as calculated before UV irradiation). After 72 h,selectivemediumcontaining MTAGG (0.28 ygofmethotrexate sodium perml,4,ugof thymidine perml, 13

jug

of adenosine perml, 14,ugofguanosine perml, and7.5,ugofglycineperml) replaced nonse-lectivemedium(27).Three weeks afterinfection,foci of tk+-transformed cells appeared on the infected plates and werepicked witha Pasteurpipette. Cell

lines were grown and continuously propagated in

MTAGG-containingmedium.HSVtk+ cell lines33A+ and39A+weretransformedbyHSV-2(333), cellline

59W

wastransformedbyHSV-2 (324), andcellline SilowwastransformedbyHSV-2(Silow).Cellswere

passagedatleast50 to 100times (every4to 5days) before being used in the studies described in this paper. Cell lines 8N and 5Awere obtained from D.

Polacek and B. Roizman. These celllines were

pro-duced after transfection of L tk- CllD cells with sheared HSV-1 (1023) DNA, by using the calcium-phosphateprecipitationmethodof Grahametal. (8). After transfection, the cellswereswitchedto Little-field's HAT medium (0.01 M hypoxanthine,4.4,IM methotrexate, and 16,uMthymidine) (20). Surviving colonieswerepicked by using small glass cylinders and werefurtherpropagated inHAT-containing medium. Passage15of each of thesecell lineswasused inthe experiments describedbelow.

Assay of transformed-cell tk activity. HSV-2tk+ cells between passages50 to 100 wereassayedfor tk activityby using the method of Lin andMunyon

(19) asmodifiedby Rapp and Turner(30). Both non-infected and infected (18 h with HSV-2 (333), at 2 PFU/cell) controlcultures,composedofnormalNclA cllO(tk-)and NIH Swissmouse(tk+) cells,werealso assayed.

Toselectively inactivate the viral tk activity,cell

extracts wereincubatedat40°C for 30 min (28).

Purification of viral DNA. Purification of viral DNAwas performed asfollows. Vero cells were in-fected with 5 PFU/cell of HSV in 199-V medium containing 1.5

juCi

of [3H]thymidine (Amersham Corp.) per ml. After 20 hat 37°C,the infected cells werescraped into the medium andpelleted. The cell pelletwasrinsed twice with coldphosphate-buffered saline andresuspended in lysisbuffer (0.1 M NaCl,

0.01 MTris-hydrochloride(pH8.0),0.01MEDTA;13 ml/109cells). Sodiumdodecylsulfate(SDS) and pro-nase (Sigma Chemical Co.), pretreated according to Hotta andBassel(12),wereaddedtofinal concentra-tions of 0.6% and1mg/ml,respectively,and the

mix-ture was incubated at 37°C for 4to 6h. The lysis

mixturewasthen dilutedto afinal volume of150ml with 0.01 M Tris-hydrochloride (pH 8.0)-0.001 M EDTA(TE)and addedto195gof solid CsCl(Eastern Chemical).The refractive index of theresulting solu-tion was adjusted to 1.4005, and the mixture was

centrifugedat40,000rpmfor 20 hat20°Cina Beck-manVTi5OrotorinanL-5centrifuge.Fractions con-taining viral DNAwerecombined. Therefractive in-dex of this pooled peak material wasreadjusted to 1.4005withaCsClsolution,and theresulting mixture wasrecentrifugedtoequilibrium asdescribed above. The viralpeak material from these secondgradients

waspooled anddialyzed against TE buffer.

Mapping approach.Themapping approachwhich has been usedto identify the viral DNA sequences presentin theHSVtk+celllines isschematically rep-resented in Fig. 1. Basically, this approach involves threesequential steps: (i) aninitial density gradient

centrifugation step of the transformed-cell DNA,to enrichfor transformed cell-associated viral DNA se-quences, (ii)invitrolabelingof thegradient-selected

DNA followedbyapreselectionhybridizationtoHSV DNA immobilizedon nitrocellulosefilterstofurther enrich for the cell-associated viral DNA sequences, and(iii) hybridization of thepreselected transformed-cell DNA to blots containing restriction enzyme

frag-mentsof HSV DNA.

(i) Extraction and CsCl density gradient

cen-trifugationoftransformed-cell DNA. DNA from either1.25x 108or2.5x108cellswaslabeled in vivo by growing the cells for2daysin the presenceof 0.5 ,uCiof[3H]thymidine (Amersham Corp.) perml.The cellswereharvestedby rinsing the monolayers three times with ice-cold phosphate-buffered saline, fol-lowedby lysis in25mlofamixturecontaining 0.01 M

Tris-hydrochloride (pH 8.0),0.01 M EDTA, 0.01 M NaCl, 0.5% SDS, and1mgof Proteinase K (Merck &

Co., Inc.) per ml. The lysis mixture was incubated overnightat37°C and sheared on icefivetimes (30s/

time) at20,000 rpm inaVirTis45homogenizer. The

sheared DNA wasextractedwith 1volumeofphenol

and1volume ofchloroformandwasdialyzedat room VOL. 33,1980

on November 10, 2019 by guest

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TRANSFORMED CELL DN SEAREDWITH _ ENOLCHLOROFORM

DNM

EXrRATDwim VIRTIS ZVGENIZER DIALYSIS

PROTEINASE KAm

SDS

CSCLDENSITYGRADIENT

CENTRIFFUGATION

4

UG

GRADIENT

SELECiTE

PRESELECTION

HYBRIDIZATION DN LABELLED INVITRO TOSMA-LLNITROCELLULOSE

-WITH

32P

NUCLEOTIDES FILTERCONTAINING 1.5 uG

DENATUED HSVDNA

EhLTION OFHYBRIDIZED PRESELECTED DNA

-_-HYBRIDIZATIONOF ELUTED

DMA

TOSOUMERN TYPE BLOTS CONTAINING

RESTRICTION ENZYMEFRAGMENTS

[image:3.504.60.458.52.241.2]

OF HSV

DNA

FIG. 1. Diagrammatic representation of the hybridization approach. The individual steps of this approach aredescribed in detail in the text.

temperatureforapproximately12h against TEbuffer. The DNA mixture was then resheared five times as above, adjustedto atotal volume of 25 ml, and added to 32.5 gof solid CsCl (Eastern Chemical) to yield a final refractive index of 1.4005. Equilibrium density centrifugationwasdone inaVTi5O Beckman rotor at 40,000 rpmat20°Cfor 20 h.Sixty

600-pl

fractions were collected per gradient. The fractions between densities 1.710and 1.735g/cm3 (asdetermined from refractive indices) were pooled and dialyzed against several changes of TE bufferat4°C for3days. The DNA was then precipitated with ethanol. Figure 2 shows the results of a reconstruction experiment in which a mixture ofpurified 32P-labeled HSV-2 DNA and 3H-labeled tk- cell DNA wassheared andsubjected to

equilibrium densitycentrifugation as described above. As shown in Fig. 2, the sheared viral and cell DNAs sedimented in discrete bands. The fractions between densities 1.710and 1.735g/cm3contained 75% of the

32P-viral DNA label and only 2% of the 3H-labeled

cellularDNA, representinga40-fold enrichment.

(ii) Invitro labeling of DNA. Viral DNA and CsCl gradient-selected transformed-cell DNAswere labeled in vitro with [a-32P]dGTP and[a-32P]dCTP

(350 Ci/mmol; Amersham Corp.) with Escherichia coli DNA polymerase I, essentially as described by

Maniatis et al. (22). The specific activities of DNA labeledbythisprocedure rangedbetween 7x 107and 1.6x 108

cpm/4g.

(iii) Preparation of nitrocellulose filters

con-taining HSVDNA. The small nitrocellulose filters used in thepreselectionhybridizationstepwere pre-paredasfollows. A10-to15-,ugamountofHSV-1 or HSV-2 DNAwassuspendedin0.9mloflxSSC(lx

SSC=0.15MNaClplus0.015Msodiumcitrate)and

sheared fivetimesthrougha26-gauge needle. A

0.1-ml amountof3NNaOHwasadded,and the mixture

wasincubated for10minat roomtemperature.This

mixture was then added to 9 ml of 28x SSC and

filteredtwotimesthroughaHAWP02500 nitrocellu-lose membrane filter (Millipore Corp.), which had

1250-2

0

0.

NL

1000- 750-500

250

N

1-I\'

~~~~

.1~~~~~~~

N-'

-1.74

-1.70

11.66 r400

300

200

100

z 0

s

2

8 16 24 32 40 48 56

FractionNumber

FIG. 2. CsClequilibrium densitycentrifugationof amixtureofshearedtk-cell DNA andHSV-1DNA. A mixtureof

32P-labeled

HSV-1DNA and

3H-labeled

CIIDtk- cellularDNA wasshearedto an average molecularweight of5x 106and sedimentedto

equi-librium inaCsCldensity gradientasdescribed in the text.

been prewashed with 28x SSC. After filtration, the filterwaswashed with10ml of28xSSC, dried over-nightat roomtemperature, and bakedat80°C for5h. Eight replicate 2-mm-diameter filterswere punched out of each 25-mm-diameter filter, with a leather punch. It wasfoundthat filtration in alkalias

origi-nallydescribedby Baker (2) along with theuseof very high salt(28x SSC) resultedinthe best retention of HSV DNA.

Nitrocellulose "blots" containing various restriction enzyme fragments of HSV DNA were prepared as follows. HSV DNA wasdigested with restriction en-donucleases(NewEngland Biolabs),andsubjectedto

r

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[image:3.504.266.457.283.443.2]

electrophoresisat50 V for 20 h inahorizontalagarose gel (10 by30cm;0.4%),asdescribed byHayward (10).

A 10-to15-,ugamountof HSV DNAwasloadedonto 4-cm-wide wells. DNA wastransferred from the gel

ontoasheet ofBA 85 nitrocellulose filter(Schleicher &Schuell Co.), essentially asdescribedbySouthern

(33). Afterblotting, replicate blotswereprepared by cutting the nitrocellulose filter into 3-mm vertical stripswhichwere allowedto dry overnight atroom

temperatureandthenbakedat80°Cfor 5 h.

(iv)Preselectionhybridizations. Nitrocellulose

ifiters(2-mm diameter), preparedasdescribed above

andcontaining1.5

tLg

of immobilizeddenatured HSV DNA,werepreincubated for6 h at65°Cinasolution

containing6x SSC and 0.02% bovineserum albumin

(BSA), 0.02% polyvinylpyrrolidone, and0.02% Ficoll (5).

Invitro-labeled transformed-cell DNA (4ug) was

precipitated with ethanol ina1.5-ml microfuge tube

andresuspended by heatingat65°C for30minin 20

pl

ofhybridization solution containing6xSSC,0.01 M EDTA, 30% formamide, and 1 mg of E. coli DNA (Miles Laboratories, Inc.) per ml. The resuspended

DNAwas thensealed in acapillary pipette and

de-natured for 7minat117°C.Theresultingdenatured

DNAwasplacedintoaBeemcapsule (Beem Corp.),

and 2

pl

of 1Ox Denhardt solution (lx Denhardt

solution is 0.02%BSA,0.02%Ficoll, 0.02%

polyvinyl-pyrrolidone)inwaterwasadded. Apreincubated

ni-trocellulosefilter (2-mm diameter) containing

immo-bilizedHSVDNAwasblotteddryandsubmergedin

thehybridizationsolutionatthe bottomof the Beem capsule. The filter and hybridization solution were

overlaid with mineraloil,andhybridizationwas

car-ried outat65°Cfor 16 h. Afterhybridizationthe small

nitrocellulose filterwaswashed threetimesatroom

temperature with 2x SSC and twice for 1 h at65°Cin

10 ml of 6x SSC-30% formamide-lx Denhardt

solu-tion-0.5% SDS. After these washes, the hybridized

DNAwaselutedfrom the filtersbyincubation of each

filterin 100

pl

of elution buffer (0.1MNaOH,0.01 M EDTA, 0.1%SDS) at45°Cfor 2h,asoriginally

de-scribedby Gronebergetal.(9).Thesolution of eluted

DNAwasneutralized with 1 NHCI.Itshould be noted

that the efficiency of the preselection hybridization

stepwasgreatly improved by minimizingthe volume

of the hybridization solution. For convenience, we

choseafinalvolume of 20 tdperfilter,yieldinga50%

hybridization efficiencyforpurifiedviral DNA anda

background of0.02 to 0.04% for cellular DNA and, therefore, representing a 1,200- to 2,500-fold

enrich-mentfor viral DNAsequences.

(v)Blothybridizations.Nitrocellulose blotswere

preincubatedfor6 hat65°CinalxDenhardt solution

in 6x SSC.Thesepreincubatedfilterstripswerethen

hybridized inaglassscintillation vialcontaining the

elutedDNA fromthepreselection hybridizationstep

in1ml ofasolutioncontaining6xSSC,lxDenhardt

solution, 30%formamide, 10% dextransulfate

(Phar-macia FineChemicals, Inc.),0.5%SDS, and200ytg of

denatured E. coli DNA per ml. Hybridization was

carried out at 65°C under mineraloilwith vigorous

shakingfor 20h.It should benotedthatthe inclusion

of 10% dextran sulfate(36)intotheblothybridization

mixtures was found to result in a two- to fourfold

increase in thehybridization efficiency.Inaddition, it wasfound that theincorporationofexcessunlabeled E. coli DNA into the hybridization solutions resulted insignificantlyreduced levels ofbackground,as com-paredtohybridizationsperformedinthe presence of

excessunlabeled salmon sperm DNA. After

hybridi-zation, the nitrocelulose strips were washed three timesat roomtemperaturewith2xSSC and twice for 2 h with 6x SSC-lx Denhardt solution-30% form-amide-0.5% SDS at 650C. Finally, thenitroceUulose strips were dried andautoradiogrammed onCronex 2c medical X-ray film (Du PontCo.) either with(at -70°C),orwithout(atroomtemperature)high speed

intensifyingscreens(Du PontCo.). Whenapplicable,

the relativeintensities ofautoradiographicbandswere determined by scanning the autoradiograms with a Transidyne2955scanningdensitometerequipped with anautomaticintegrator.

Theoreticaltreatmentof the fractional recov-eryofintegratedviral DNA from the CsCl

equi-librium density centrifugation step. Consider a piece of HSV DNA of average viralbuoyant density,

Pv (=

1.729

g/cm3)

(14),

and size

V,

which is

integrated

intoceUular DNA sequences of average buoyant den-sity

pc

(= 1.690

g/cm3).

After randomshearing of the transformed-ceU DNA to double-stranded fragments

of sizeL, anyintegrated sequence of viral DNA

wil

be present in anassortmentofpieces of sheared DNA, containing various amounts of viral and ceUular se-quences. In order for a piece of this sheared trans-formedcellDNAtoberecoveredfrom the CsCl

equi-librium density centrifugation step (i.e., to display a

buoyantdensitygreaterthanorequaltop=1.710g/

cm3),

itmustcontainaminimumfraction of viral DNA sequences. This minimum fraction

(t)

canbe easily

calculated from thefollowing equation:p=

(t) pv

+

(1 - f)pc (equation 1). Itfollows that for apiece of

transformed-cellDNAoflength L,Lfrepresentsthe minimum amountof viral DNA whichmust be con-tained in thatpieceifit istobe recoveredfrom the

densitygradient preselectionstep.

Assumingthat

pv

= 1.729

g/cm3,

Pc = 1.690

g/cm3,

p=1.710

g/cm3,

and L=6 x106(i.e., the average size

of the sheared transformed-cell

DN4),

then from

equation1f=0.5andLf=3x106.

Thds,

any

piece

of integrated viral DNA of molecularweightVless than 3x

10'

willnotberecovered from the CsCl gradient purification step. On the other hand, when the size of anintegrated piece of viral DNA is greater than 3x

106, allofthe viral DNA sequences contained in this piece of integratedDNA wiU be recovered tosome

extentduring the CsCl purification step. Theexact

fractional recovery ofagivenviral DNA sequence will

dependuponitspositionXalongthe stretch of

inte-gratedviral DNA.Therelationshipbetween the frac-tional recovery F and the position X, for the viral DNA sequences contained in variouspieces of inte-grated viralDNAof sizes V>Lf is shown in Fig. 3. As seeninthatfigure, those viral DNA sequences located at both edges of the piece of integrated viral DNA (i.e.,at X=0andX= V)willberecovered from the equilibrium density step with anefficiency of F= 1

-fwhich is independent ofboth L, the size of the

shearedtransformed-cellDNA, and V, the size of the integrated viral DNA sequence. As described above

on November 10, 2019 by guest

http://jvi.asm.org/

276

LEIDEN, FRENKEL, AND RAPP

1.0

1.0

0 V1-fu U VI V2 V3U V3

X(positionalongV)

FIG. 3. Diagrammatic representation of the frac-tionalrecoveryofintegrated viral DNAsequences as afunction of the position of the integrated viral DNA sequences. L = average length of sheared

trans-formedcellDNA,X=distanceofagiven viral DNA sequencefromoneend of the integrated piece of viral

DNA, F=fractionalrecoveryfrom CsClequilibrium

density gradient ofagiven viral DNAsequence

lo-catedatpositionX,f=minimumfraction ofapiece

of shearedtransformed-cell DNA whichmustbe

com-posed of viral DNAsequencesif thatpiece of

trans-formed cell DNA is to be recoveredfrom the CsCl equilibrium density centrifugationstep,and V=the

sizeofan integrated stretch of viral DNA. As

de-scribed in thetext, agivenviralDNAsequencewill

be included inan assortment of fragments of

ran-domly sheared transformed-cell DNA. Fwas

calcu-latedasthefraction ofthese fragments which contain

atleast Lfamountof viral DNA sequences. Fwas

calculatedfor threecasesdiffering withrespect tothe size Vof theintegrated viral DNAasdepicted in the upperright handcornerofthefigure.

usingourexperimentalconditionsf=0.5.Therefore,

those viral DNAsequenceslocated atboth edgesof

thepiece ofintegratedviral DNAwillbe recovered

with an efficiency F = 0.5. Viral DNA sequences

locatedatinternalpositionsof theintegratedstretch

of viral DNA (V> X> 0) will be recovered with

efficiencieswhichvarylinearlybetween1-f(= 0.5)

and 1.0. Insummary,by usingourexperimental

con-ditions this theoretical treatmentpredictsthat all of

the viral DNAsequencespresentinintegrated pieces

ofviral DNA withmolecular weightsgreaterthan 3

x

10'

willbe recoveredwitha minimum efficiencyof

50%.

RESULTS

Biochemical characterization ofHSVtk+ transformed celllines.

HSV-2tk+

transform-antswereobtainedasdescribedabove after ex-posureofNclA cllO(tk-)mousecells toseveral

strains of UV

light-irradiated

HSV-2

(30).

The

resultanttk+ colonieswerecontinuously

propa-gatedinMTAGG selective medium.The various

celllineswereassayedfortkactivityafter 50to

100passages.

These

tests

(Table

1)

have

shown

that

all of the cell lines

express a

tk

activity

which

displays

the

characteristic thermal

labil-ity of the

HSV-2

enzyme

(28).

HSV-ltk+ transformants

were produced by

transfecting

L tk-

C11D

mouse cells with HSV-1

(1023)

DNA

which

was

randomly sheared

to

fragments of molecular weight

20 x

106

to 30 x

106

(D.

Polacek and B.

Roizman,

unpublished

data).

After

24

h, the cells

were

placed in

Little-field's

HAT

medium

(20)

to

select for

tk+

cells.

Surviving colonies

were

picked

after 2 to 3 weeks

and

grown

continuously for

15 to 20 passages in

HAT-containing medium.

Characterization of the

mapping

ap-proach.

The

mapping approach which

we

de-veloped

(Fig. 1) involves three sequential

steps.

In the

first

step,

transformed-cell

DNA

is

ran-domly sheared

to

fragments

of molecular weight 5x 10 to 7x

106

and subjected to

CsCl

equilib-rium density

centrifugation.

Because HSV and

cellular

DNAs

display widely different buoyant

densities (1.726 and

1.729

g/cm3

for HSV-1

and

HSV-2 DNAs,

respectively, [14] and

1.69 to 1.70

g/cm3

for

cellular DNA), this

step

results in the

partial purification of

any

viral

DNA sequences

which

are present

in

the transformed cells.

In

the second

step,

the DNA

banding

at

densities

greater

than

or

equal

to 1.710

g/cm3

is labeled,

in

vitro, with

a-32P-nucleotides

and further

en-riched for viral DNA

sequences

by hybridization

to a

small nitrocellulose

filter

containing

dena-tured unlabeled HSV DNA.

Finally,

the

hybrid-ized, labeled transformed

cell

DNA is

eluted

from the

filters and

rehybridized

to

Southem

type

blots

containing

unlabeled restriction

en-zyme

fragments

of

HSV DNA

(33). The resulting

bands

are

visualized

by

autoradiography and

are

TABLE 1. Viral tkactivity in biochemically

transformedmouse cells nmolof[3H]TdR

in-corporatedper mgof

protein in 45 mina

Cellextract Residual

Preincu- activity

Noprein.

bation

at

cubation 4oocb

NclA cllO(tk-) 0.029 0.043 148.3 NclA cllO(tk-)+ 0.30 0.081 27.0

HSV-2(333)

NIH Swissmouse(tk-) 0.20 0.18 90.0 NIHSwissmouse(tk-) 0.81 0.15 18.5

+HSV-2(333)

33A+cells 0.27 0.039 14.4

39A+cells 0.23 0.066 28.7

59D+cells 0.48 0.11 22.9

Silow cells 0.35 0.057 16.3

The assay forthymidine kinase wasperformed as

de-scribedin thetext.TdR, Thymidine.

bPreincubationwas

performed

for30min.

J.

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identified

by

comparison

to

autoradiograms

of

replicate

control blots which

were

hybridized

to

purified, in vitro-labeled HSV DNA.

Specificity and

sensitivity

ofthe

mapping

approach.

To

assess

the

specificity

of the

map-ping

approach which is

described

above, DNAs

from

uninfected

Vero

and

L

tk-

CllD cells,

as

well

as

calf

thymus DNA

were

processed

through the

preselection

steps

described above

and

hybridized

to

blots

containing restriction

enzyme

fragments

of viral DNA.

Autoradi-ograms

of these blots

were

devoid of

identifiable

bands

(data

not

shown).

In

anattempt to

determine the

sensitivity

of

this

approach

as

regards

unintegrated

viral DNA

sequences,

either

3 x

10-2

or 3 x 10-l

jig

of

purified viral DNA

(representing

1.0

and

10

cop-ies

per

cell,

respectively)

were

added

to

lysates

of

2 x 108

CllD

tk-

cells, and the resultant

mixtures

were

sequentially

processed

as

de-scribed above.

The band

patterns of these

recon-struction

hybridizations

were

compared

to

those

of

control

hybridizations in which

replicate

ni-trocellulose blots

were

hybridized

to

purified, in

vitro-labeled HSV DNA. The results of

these

hybridizations

are

shown

in

Fig.

4

and

5

and

revealed the

following.

(i)

As seenin

Fig.

4,

the

mapping

approach is

sufficiently

sensitive

to

detect

an

unintegrated

piece of HSV DNA of

size

106

(e.g.,

KpnI

fragment

S,

1.1 x

106

in

molecular

weight),

present in anaverage

abun-dance

of

one copy per

cell.

(ii) In Fig.

5, we

compared

the relative molarities of the

corre-sponding

bands from the

reconstruction and

control

hybridizations.

As seen in

that

figure,

there

was no

significant difference between the

molarities of the control and reconstruction

bands,

indicating

that the

preselection

steps

did

not

result

in

the loss of

specific subsets of viral

DNA

sequences.

(iii) To determine whether the

mapping

approach

described above would

yield

information

concerning

the

number of

copies

per

cell of the viral DNA

sequences present

in

a

given

transformed cell

line,

we

compared the

absolute intensities of corresponding bands from

autoradiograms

of the

1

and

10 copy per

cell

reconstruction

experiments. These ratios ranged

from

7.9to21.9

(Table

2).

Thus, the technique

is

quantitative within limits of

approximately

twofolderror.

Because

the

buoyant density

of

cell-associated

HSV

DNA sequences

depends

upon

whether

these sequences are

integrated into

the

host

genome, it

was

also necessary

to

consider the

sensitivity

of

the

mapping approach

for

inte-grated

transformed-cell associated HSV

DNA sequences. Whethera

specific

sequence of

inte-grated viral

DNA will be recovered

from

the

Kpn I

C

AB

p

C ,

D 0

E

s

F t..

G o

H "

I

J

K LM

N

0

Id 5d 5d

ld

10 10 1

C

10

II

9DE3-I~I

I I LmS

#N

p

0

RS

PQ

~~~~T

R

FIG. 4. Hybridization reconstruction experiment.

Either 3x10-2or3x101pgofpurifiedHSV-2DNA

(representing 1 and 10copiespercell, respectively),

weremixedwithalysateof2x 108CIIDtk- cells.

The resultant mixtureswerepreselectedtoenrich for

viral DNAsequencesandhybridizedtonitrocellulose

blotscontainingtheKpnIorEcoRI/HpaIrestriction

enzymefragments of HSV-2(G)DNAasdescribed in

the text. C = control: replicate blots which were

hybridizedtopurifiedinvitro-labeled HSV-2DNA,

10=invitro-labeled DNAfromthe 10copy percell

reconstruction mixture, 1 = in vitro-labeled DNA

fromtheIcopy per cellreconstructionmixture,Id= 1day of autoradiographicexposure,and 5d=5days

of autoradiographicexposure.

CsCl

density gradient purificationstep (i.e., will be includedinpiecesofsheared

transformed-cell

DNA with buoyant densities greater than or

equalto1.710g/cm3) will dependupontheexact

size and density of the integrated viral DNA

sequence, as well as upon the density of the surrounding cell DNA sequences. All of these parameters will, mostprobably, vary from cell

linetocellline,andit istherefore

impossible

to

EcoRI/Hpa I

5d

5d

10

1

I

i..

O.

.Y.

0.

.

on November 10, 2019 by guest

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[image:6.504.250.445.75.409.2]

278 LEIDEN, FRENKEL, AND RAPP

FIG. 5. Relative abundances of hybridized bands from autoradiograms of control and reconstruction

hybridizations. Top:densitometric scansoftheautoradiogramsshown in Fig. 4.Bottom: relativemolarities

ofthe various bands. These molarities were calculated relative to the molarities of the corresponding

fragments in virion DNA. See reference 31 for a review of the relevantstructural features of HSV DNA. The relativemolaritieswere obtainedbydividing the fractional area under each peak of thedensitometricscan by theexpectedfractionalarea, asdeterminedfrom the sizesand knownmolaritiesofthe fragments contained in eachpeak,-i.e., for1Mfragments ofsize M (daltons), molarity= (fractional area)

(M/108)-';

for0.5 M

fragments, molarity=2(fractionalarea)

(M/108)

-1;for 0.25 Mfragments,molarity=4(fractionalarea)(MI

108)

-1.Eachtripletofbarsrepresents,from lefttoright,therelativemolarities of the bands

from

the 10 copy per cellreconstruction,thecontrol,andthe1copy per cell reconstructionexperiments.*, Agroup offragments

including either a 0.5 M or a 0.25 Mfragment, i.e.,containing sequences

from

theinvertedrepeats of HSV DNA(ab and ac). It is noteworthy that these groups of fragments exhibit relative molarities greater than 1.0. Thisfindingmostlikely reflectsthefactthat the ab and ac repeatedsequencesarepresentingreater than unitmolarity in the labeledprobe DNA and, therefore, confirms the

hypothesis

that thehybridization is

sensitive to the amountsoflabeledprobeDNApresent in thehybridization solution.

experimentally

or

theoretically

determine the

absolute

sensitivity

of the

mapping approach

for

all

cases

of

integrated

viral

DNA sequences.

However, in the theoretical

treatment

presented

above,

we

have calculated the fractional

recov-ery

(from

the

CsCl

density gradient)

of the viral

DNA

sequences

contained in

pieces

of

viral

DNA

of average

buoyant density,

which

are

integrated into cellular DNA of

average

buoyant

density.

Thesecalculations

predict

that,

by

using

our

experimental

conditions,

any HSV

DNA sequence which is present ina

piece

of

integrated

viral DNA of

molecular

weight

greater than 3 x 10

will be recovered from

the

CsCl

gradient

preselection

step

with

a

minimum

efficiency

of 50%.

Mapping

of the HSV-2

DNA sequences present in

HSV-2tk+

33A+

cells. Cell

line

33A+

was

produced

by infection

of

tk-

mouse

cells

with

UV

light-irradiated

HSV-2

(333).

DNA extracted

from

passage 50 of

this cell line

was

preselected and

hybridized,

as described

above,

to

blots

containing

either the

KpnI

or

EcoRI/HpaI

restriction

enzyme

fragments

of

HSV-2

(G)

DNA. Theresults of

these

hybridi-zations

(Fig. 6)

can

be

summarized

as

follows.

(i) The

maps

generated from the

two

different

restriction

enzyme

cleavages used in these

ex-periments

were

in

agreement

and

indicated that

this

cell line contains

a

contiguous

set

of

HSV-2

(G)

DNA

sequences

located between map

coor-dinates

0.14

and

0.57.

(ii)

A

comparison

of the

intensities of the

bands

produced by 33A+

DNA with those

produced by

the 1 copy per cell reconstruction

mixture,

which was

hybridized

to

replicate

nitrocellulose

blots, revealed

that

33A+

cells

contain between 1

and

5

copies

per cell

of

these

HSV-2 DNA sequences

(Fig.

6). (iii)

To

assess

the

reproducibility

of the

mapping

ap-proach,

two

additional

preparations of 33A+

DNA

(passages

60-70)

were

separately

prese-lected and

hybridized

to

different

setsof

filters

and

blots

containing

HSV-2

DNA.

The

three

preparations of transformed-cell DNA

yielded

identical

hybridization patterns

(Fig.

6).

Mapping of the viral DNA

sequences present in

three

additional

HSV-2tk+

trans-formants.

In an attempt to

further delineate

J. VIROL.

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[image:8.504.50.240.81.583.2]

TABLE 2.

Comparison of the

intensities of the

ditional

HSV-2tk+-transformed

cell lines:

39A+

bandsfrom the1and 10copy per-cell reconstruction cells, produced

by

exposure

of tk-

cells

to

UV

experinents

light-irradiated HSV-2 (333) and

59D+

and

Silow

Restriction endonu-

Fra

t

Intensity 10/inten-

cells, produced by

infection of

tk- cells with UV

clease

agmen

sity 1"

light-irradiated HSV-2 (324) and HSV-2 (Silow),

KpnI

A 8.3

respectively.

The results of these

mapping

ex-B

periments

(Fig. 7) revealed the

following:

39A+

c

cells

contained

a

contiguous

set

of

viral

DNA

D

16.44

sequences located between map coordinates 0.14

and

0.42.

Silow cells contained

a

contiguous

E

10.08

stretch of viral DNA sequences mapping

be-tween

coordinates

0.21 and 0.32.

Finally, 59D+

F

8.43

cells

contained

a

contiguous

set

of

viral

se-G

12.3

quences

spanning

coordinates 0.28 to 0.42.

H

In addition to the

viral DNA sequences

de-I827

scribed

above,

cell lines

39A+,

59D+,

and

Silow

I*8.27

all

contained

two sets

of viral DNA sequences

J

8.86

which

were

present

in

significantly

lower

abun-dances (less than 0.1 copy per cell). These

low-L

8.03

abundance

viral DNA sequences have been

ten-M

tatively mapped in

two

separate

regions

of the

viral genome located

between map coordinates

N

7.89

0.06 to 0.20 and 0.82

to 0.93.

o

A

comparison of the map

positions of the viral

p

12.2

DNA sequences present in the four

HSV-2tk+

transformants

(Fig.

6

and

7) reveals that the

only region of the HSV-2 genome which is

com-EcoRI/HpaI

A 7.99

mon to all four cell

lines

is located between map

B

coordinates

0.28

and 0.32.

Thus, this region of

C

the

HSV-2 genome

must

contain the HSV-2 tk

D 9.03 gene.

E

Mapping

of the HSV-1 DNA sequences

F

9.94

present

in

two

HSV-ltk'-transformed

cell

lines. Cell lines 5A and 8N

were

produced

by

G

13.91

transfection

of L tk-

CllD

mouse

cells

with

HSV-1

(1023) DNA which was

randomly

H

8.54

sheared to fragments of molecular weight 20 x

I 10.55

106 to 30

x

106. DNAs from

passage 15 of these

J

cell lines

were

preselected

and

hybridized,

as

described

above,

to

nitrocellulose

blots

contain-L

9.27

ing various restriction enzyme

fragments

of

M

HSV-1 DNA.

Autoradiograms

of the

hybridized

blots from these

experiments (Fig. 8 and 9)

re-N

8.84

vealed that (i)

cell

line 5A contains a contiguous

O

9.45

set

of HSV-1

DNA

sequences

mapping between

coordinates

0.27

and

0.41, and

(ii) cell

line

8N

P

20.74

contained four sets of

noncontiguous L region

viral

DNA

sequences

mapping between

coordi-Q

21.96

nates

0.11

to

0.17,

0.29 to

0.32,

0.34

to 0.40, and

R

10.0

0.52 to 0.56. In

addition,

as seen

in

Fig. 9,

8N

s DNA

hybridized

to the

BglII/HindIII

fragment

'Ratios of theintensities of the

corresponding bands (as

Q of HSV-1

(Justin)

DNA,

and to the

BglII/

determiined bydensitometric scanning as described in the HindIII

fragment R,

but not to the

Bglll/

text)of the 10 and 1copy per cell reconstruction experiments HindIH fragment P, of HSV-1 (F) DNA.

More-shown inFig. 1. The5-day autoradiographic exposures were

over,

it hybridized tothe0.5 M fragmentsfrom

used for the densitometric

scanning,

the

right

side

of the S

region of HSV-1

DNA more

efficiently than to those from the left side

the

map

position

of

the tk

gene

of

various strains

of the S region.

Therefore, we have concluded

of

HSV-2,

we

mapped

the viral

DNA

sequences

that this

cell

contains a contiguous set of S

which are

present

in

late

passages

of

three

ad-

region DNA

sequences, composed of the

U.

se-33,

on November 10, 2019 by guest

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LEIDEN, J. VIROL.

Kpn I

LcoKI

/IHpaI

A B c A P

;r:'A + :5, C R33A4C

fE*<w <

.oR9b0.:*k

DPt~~~~~~~~~~~~~~~~~

F* w .. Z~~DDE g t s v *w

**@4tW~ ~ ~ ~ ~ J

N

K *

N X 4

F:.~ ~ ~ ~ ~ .

6 7 .

33A

HSV-2(G)EcoRIfipal _LIN 4FT+ -- t M+--

-G FTSJ B M.

HSV-2(G)KpnI - 4+ Ht it ---

-F K LPO H NOJ M 0 D0

MW , -I-.,---r--- --.

0 10 20 30 40 5 50 *'A%

ft4hC,

FIG. 6. Mapping ofthe HSV-2 DNA sequences present in the

HSV-2tkt

cellline 33A . Top: control DNA

(purifiedin vitro-labeled HSV-2[G] DNA) (C)orin vitro-labeled33A+ DNA(33A+) whichwasprocessed

through thepreselection steps washybridized tonitrocellulose strips containing either the KpnI, orthe

EcoRI/HpaI restriction enzyme fragments of HSV-2(G) DNA. A, B, and C, Autoradiograms from three separatehybridizationexperiments,eachdone withaseparatepreparationof 33A + DNA (passages50-70of thecellline)andaseparatesetofnitrocellulose blots. InexperimentCa1copy percellreconstruction mixture (seelegendtoFig. 4)washybridizedto areplicate nitrocellulose strip (R).Inexperiment A,autoradiography

wasperformed for either 5 days

(panels

2 and 10) or 3 weeks (panels 3 and 11). Bottom: schematic representation ofthe regions of homology between 33A+ DNA andthe KpnI, or EcoRI/HpaIrestriction enzymefragments ofHSV-2(G)DNA. The restrictionenzyme mapsweretakenfrom G. S.Hayward, T. G. Buchman, andB.Roizman(unpublisheddata).The dotted line represents aregionof uncertain homology, duetothecomigration oftheKpnIfragmentsLand M.

quences

mapping

between

coordinates

0.91and present in six

HSVtk+-transformed cell lines.

0.96

and

a

portion of

theacsequences.

Whereas

this

method

requires

relatively

small

DISCUSSION

amounts of

transformed-cell

DNA,

itpossesses

sufficient

sensitivity

to

detect

a small piece of In the

studies

reported

in

this

paper, we

have

HSV

DNA which ispresent in an average

abun-described

a

novel

mapping

approach

which has

dance

of1 copy per

cell.

Thus, this

procedure

allowed

the

simultaneous detection

and

identi-

could

be

applied

in

studies

of

cells

transformed

fication

of

the

HSV

DNA sequences which are

by

other viruses

containing

genomes which

on November 10, 2019 by guest

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[image:9.504.104.408.70.465.2]

EcoRI/

Hpal

Kpnil

C Silow 39A+ 59D+ R R C Siow39A4 C59D R R BA

F F

F 4 H

H1 2 4301G 21

K

LM'a'

N K

O LM *

P~ ~ ~ ~ ~ ~ ~~P

T R~~~~~~~~~~~~

1 2 3 4 5 6 7 8 9 10 11 1213

-ASIow

39A+

590+

EcoRI/lpal

i- i Hi -i--- i i i H

"s ^ C rCe I a u I D e I u D^O

KpnlI

L N G r- IS J C MV IMr In11UR Q

-Sibw

39A4

- D59D+

F K LPO H NQJ M G D I S C 1R1 A R2

,.. -- ---_,-

--~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

0 10 20 30 40 50 60 70 80 90 100

(daetonX10o6)

FIG. 7. Mapping ofthe HSV-2 DNAsequences present in theHSV-2tk+ celllines Silow,

39At,

and

59Dt.

Top: control[purifiedin vitro-labeledHSV-2(G)DNA](panels 1, 5, and 8)orin vitro-labeledSilow (panels 2and6), 39A+(panels3and7), and 59D+(panels4and9)transformed-cellDNAs, whichwerepreselected to enrichforviral DNA sequences,werehybridizedtonitrocellulose blotscontaining either the KpnIor

EcoRII

HpaIrestriction enzymefragments of HSV-2(G)DNA.Autoradiographywascarriedoutfor 4 days. Bottom: schematicrepresentationoftheregionsof homologybetweenSilow, 39A+,and59D+ DNAs and the KpnIor

EcoRI/HpaIrestriction enzymefragmentsofHSV-2(G) DNA. The restriction enzyme mapsweretakenfrom G. S. Hayward, T. G. Buchman, and B. Roizman (unpublished data). Dotted lines representaregion of uncertainhomology,duetocomigration of the KpnIfragmentsLand M. It should be noted that as described in thetextallthreeHSVtk+cellDNAsshowed lowintensityhybridizationtofragmentslocated between map coordinates 0.06 to0.20and0.82 to 0.93.Becauseof their extremely low abundances, these regions are not displayed in the bottom partofthefigure.

on November 10, 2019 by guest

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[image:10.504.104.396.70.541.2]

282 LEIDEN, FRENKEL, AND RAPP

A

BgIII/HindIII

Bgl

11

B

Bglll

Bgll/Hlirlclll

--+I-- t4$t-s--- i---- t - t

J K 0PN M D G F H

H J TUOS 0 G F L K M D IN P R

0 10 20 30 40 50 60 70 830 90 100C

(dialtonsX

FIG. 8. Mapping ofthe HSV-1 DNA sequencespresent in theHSV-ltk+ cell line 5A. A, Control DNA

(purifiedin vitro-labeled HSV-1(F)DNA(C)orin vitro-labeled 5ADNA, whichwaspreselectedasdescribed

in thetext, washybridized toreplicate nitrocellulose blotscontainingeither theBglIIrestriction enzyme

fragments of HSV-1(MP) DNA, orthe BglII/HindIII restriction enzymefragments of HSV-I(F) DNA. B,

Schematic representation ofthe regions ofhomology between 5A DNA andtheBglII and BglII/HindIII

restrictionenzymefragments ofHSV-I DNA. The restrictionenzyme maps weretaken from G. S.Hayward,

T. G.Buchman,and B. Roizman(unpublished data).

play buoyant densities different from those of

host cellular DNA. On the other hand, this

approach suffers from two distinct disadvan-tages. First, ityieldsnoinformationconcerning theintegrationorarrangementof the viral DNA sequenceswithin the transformedcells.Second, theincorporationof the

CsCl

gradient preselec-tion step could result in the failure to detect small stretches of integrated viral DNA se-quences. However, theoretical considerations

have indicated that theshearing and

centrifu-gation conditionsused in the studiesreportedin

thispaperwould haveallowed the detectionand

mappingofpiecesofintegratedviral DNA with

molecularweights greaterthan 3 x 106 (i.e.,3%

of the HSVgenome).

Mappingof theHSV-2 tkgene. Themaps

shown in

Fig.

10 indicate that the sequences

located betweenmapcoordinates 0.28and 0.32 are the only viral DNA sequences which are common to all of the HSV-2tk+-transformed

cells.Similarmappingstudies of the HSVDNA sequences which are present in six

HSV-ltk+

transformants (including the cell lines 5A and

8N discussedin thispaper,andfour additional HSV-ltk+-transformed cell lines which will be discussed elsewhere) have shown that the se-quenceslocated between mapcoordinates0.28

and 0.32arealso theonly viral DNAsequences common to all HSV-ltk+-transformed cells.

Therefore, both the HSV-1 and HSV-2 tkgenes arelocated between map coordinates 0.28 and

0.32, and this location does not vary between

viral strains. These results are in accord with

thefindings of Morseetal. (25). However, they

areindisagreement with the results of Maitland andMcDougall (21).

Non-tk gene viral DNA sequences

pres-ent in HSVtk+ cells. Inaddition to the viral DNA sequences located between map coordi-nates 0.28 and 0.32, all of the

HSVtk+-trans-formed cell lineswhichwehave tested contain a substantial, but variable, amount of non-tk

geneviral DNA. Severalpointsarenoteworthy

concerning the possible expression and

func-tion(s) of these viral DNA sequences. First, if

C 5A

AB

ID

F

KL MN

p RS

TU

C 5A A

F t

GH l

J 4

.: 4

K6.

L f

*::

NJd oh P -i

J. VIROL.

on November 10, 2019 by guest

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[image:11.504.67.462.62.354.2]

F Jus BglII/Hindill BglII/Hirdill

CBN

| A . I

I

Gf .s.A<

1-

N1

[image:12.504.50.444.70.328.2] [image:12.504.47.239.463.574.2]

-4 0o* <

|<ST st*

U

1 2 3 4 5 6

Jus

KmI

C SN

7 8

B

(MP)Bgl11

(F) BglII/Hindill

(Jus)BglIl/Hindill

(Jus)Kpn

J K OPNM D G F H L

-8tt -144t4}tt1---l-i---F-1$ H JTUOSO G F L K M D IN PR

_.j-^P4 --*-.

-+-H U J STPRVO G F M L N E 0 K

RUG Z E aWH MSNPv.X B LC' A YT J OUR: F K AA

0 10 20 30 40 50 60 70 so 90 10(0o

FIG. 9. Mapping ofthe HSV-1 DNA sequencespresent in the

HSV-ltk'

cell line8N. A, ControlDNA

(purifiedinvitro-labeled HSV-1DNA)orinvitro-labeled 8NDNA,whichwaspreselectedto enrichforviral DNA sequences, was hybridized to replicate nitrocellulose blots containing various restriction enzyme

fragmentsofDNAfromHSV-1 strainsMP,F,and Justin(Jus). KpnIfragmentsR1 andR2areboth derived

fromthe endsofthe Lregionandreflecttheheterogeneity ofthe viral DNAsequencesin thisregion ofthe

HSV-1genome(35;H.Locker andN.Frenkel, manuscriptinpreparation). B,Schematicrepresentation ofthe

regions ofhomologybetween 8N DNAandtheBglII, BglII/HindIII,andKpnIrestrictionenzymefragments ofHSV-1 DNA. TheBglIIandBgIII/HindIIIrestrictionenzymemapsweretakenfromG.S.Hayward,T.G. Buchman,andB. Roizman(unpublished data). TheKpnIrestrictionenzyme mapwastakenfromH.Locker

and N. Frenkel(manuscriptinpreparation).Dotted linesrepresentregions ofuncertainhomology,whichare

presumablyduetothepresenceofthe invertedrepeatacsequencesin8N DNA(seetext).

Sequencearrangwnwit

1-a L_

s-J-ab Lk dAikhc

0 0.1 CQ2 Q3 0.4 Q5 06 0.7 08 Q.9 1.0

r . , . . .

0 Q W 0.4 0I 0I 0. OI Q ID

I~~~~~~~~~~~~~~~~~~~

0.1

liQ

0 S 6 0. S 9 1

FIG. 10. Summary ofthemapping data oftheHSV DNA sequences present in the six HSVtk+-trans-formedcel lines.Thedottedlinesrepresentaregion

ofuncertainhomology (see legendtoFig. 6).

thesesequences encodeanyoftheknownlytic

functions ofHSV, the expression of these lytic functionsmustbesuppressed inthe

transfonned

cells. Second, it has been previously suggested

that some of these non-tk gene viral DNA se-quences may exert a positiveregulatory effect on viral tk gene expression in these cells and may,

therefore,

bepositively selectedforduring propagation of the cells inHAT medium (17). Thispossibility currentlyseemsunlikely in light

of the fact that none ofthe

HSVtk+

cell lines tested in this study contain the viral DNA se-quences which are known to encode the early (a)viralgenes.

The results shown in Fig. 10, when taken together with the mapping data of four addi-tional

HSV-ltk+

transformantstobepresented

elsewhere,

havealso revealedthat therearetwo

regionsoftheviralgenomewhichappeartobe uniformly absent frommost,ifnotall, HSVtk+ transformants. These regions are located be-tween map coordinates 0 to 0.06, and 0.57 to

0.82. One can envision at leasttwo alternative explanations for the uniform absence of these

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F G

Hi

A L MN

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R

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TV v wx yi

(daltonsX10-6)

on November 10, 2019 by guest

http://jvi.asm.org/

284

LEIDEN, FRENKEL, AND RAPP

viral

DNA sequences. First, these sequences may

contain

nonsuppressible

viral lytic

func-tions. Thus,

any

cell

originally containing

them

would be unable

to

survive. In

this

light it

is

interesting

to note

that

Morse et

al.

(25) have

mapped

a

host

shutoff function

between

coor-dinates

0.52

and

0.59 on the HSV-2 genome.

Alternatively, the

sequences which are

uni-formly deleted from the transformed cells

could

contain

early (a) viral functions

which are

needed

to turn on

the expression of lytic

viral

functions

located elsewhere

on

the

genome (11).

Thus the absence of these regions

could assure

the lack ofexpression of late viral lytic

functions.

This

possibility is especially attractive in

view of recent

studies which have indicated that

some

of

the

early viral

genes are

located within the

inverted

repeat

regions of the L

component

of

HSV DNA

as

well

as

between

map

coordinates

0.56

and

0.79

(3,13,29;

L.

Morse and B.

Roizman,

unpublished data).

Patterns

of incorporation of HSV DNA

during

HSV-mediated tk

transformation.

Our

mapping studies have revealed that

someof

the

HSV-2tk+ cell lines

whichwe

have examined

(cell lines 39A+, 59D+, and

Silow)

contain viral

DNA

sequences

which

werepresent

in

very

low

abundance. One

can

envision

at

least

two

under-lying mechanisms which could

generate

these

observed abundance

pattems.

First,

it is

possible

that

each of these cell lines

represents

the

prod-uct

of

several

independent

transformation

events

(of

several different

cells),

all

of which

resulted in the

incorporation of the tk

gene,

but

each of which involved the

incorporation

of

a

different

set

of

contiguous

non-tk

gene

viral

DNA

sequences.

This is

possible given

that the

HSV-2tk+ cell lines used

in

these studies

were

not

extensively

cloned.

Alternatively,

it is

pos-sible that these

cell

lines

may

have been the

product of

a

single

transformation

event,

but

that

following

the

initial

cloning

in

HAT

me-dium there

was a

period

of loss of non-tk

gene

viral DNA

sequences

from selected cells within

the

originally

uniform

population.

These

possi-bilities

are

currently

being

differentiated

by

mapping

studies of the DNA from subclones of

late

passages of these

cell

lines.

Finally,

it

should also

be

noted that

most, if

not

all,

of

the

UV

light-irradiated

HSV-produced

cell lines

appearto

contain

a

single

contiguous

sequence

of HSV

DNA,

whereas 8N

cells which

were

produced

by transfection of tk- cells

with

sheared

HSV DNA

clearly

containa

large

num-ber of

noncontiguous

HSV

DNAsequences. This difference

might reflect

the fact that DNA

trans-fections result

in the

incorporation

of

multiple

DNA

fragments

into thesame

cell.

Moreover,

the

finding

of the

multiple

noncontiguous

setsof

HSV

DNA sequences in

8N

cells

indicates that

the

incorporation of viral

DNA

fragments during

HSV-mediated tk transformation is

a relatively

random

process,

i.e., that

any piece

of viral

DNA

which does

not

encode

lytic HSV functions

can

be

incorporated into the cells during

HSV-me-diated tk transformation.

ACKNOWLEDGMENTS

We thank B. Burckart forskillfullygrowing thecells and viruses used inthese studies, H. Locker and B. Roizman for useful discussions, N.Turnerforperformingthe tk assays, and D.Polacek and B. Roizman for providing the 8N and5A cell lines.

The work performed at the University of Chicago was

supported byPublic HealthServiceresearch grants AI-15488

and CA-19264 from the National Institutes of Health, and by National Science Foundation grant PCM78-16298. J.M.L. is a

predoctoral fellow supportedby Public Health Service grant

5-T32 HD-07009-03.

The workperformedattheHershey Medical Center was supported by contractNO1CP 53516 within the Virus Cancer Program of the National Cancer Institute and grant CA 18450 awarded by the National Cancer Institute. A preliminary report of thesedata waspresented in the Herpesvirus Work-shop,Cambridge,England, August 1978.

LITERATURE CITED

1. Bacchetti, S., and F. Graham. 1977. Transfer of the gene forthymidinekinase tothymidinekinase-deficient human celis by purified herpes simplex viral DNA. Proc.Natl. Acad. Sci.U.S.A.74:1590-1594.

2. Baker,R. F. 1977.Bindingof DNA tocellulose nitrate

filtersunder denaturingconditions. Analyt. Biochem. 78:569-571.

3. Clements,J.B., R. J. Watson, and N. M.Wilkie.1977.

Temporal regulation of herpes simplexvirus type 1

transcription: location of transcripts on the viral ge-nome.Cell 12:275-285.

4. Davidson, R., S. Adelstein, and M. Oxman. 1973. Herpessimplexvirusasa sourceofthymidine kinase

forthymidinekinasedeficient mouse cells: suppression

and reactivation of the viral enzyme. Proc. Natl. Acad. Sci. U.S.A. 70:1912-1916.

5. Denhardt,D.T. 1966.Amembrane filter technique for

the detection of complementary DNA. Biochem.

Bio-phys.Res.Comm.23:641-646.

6. Dubbs,D.R.,andS. Kit.1964.Mutant strains ofherpes

simplexdeficient inthymidine kinase-inducing activity.

Virology22:493-502.

7. Ga.rfinkle, B., and B. McAuslan. 1974.Regulation of

herpessimplex virus induced thymidine kinase.

Bio-chem.Biophys. Res.Commun.58:822-829.

8. Graham,F.L.,A. J. Van derEb,and H.L.Heijneker. 1974. Size and location of the transforming region in human adenovirustype5 DNA.Nature251:687-691.

9. Groneberg,J., Y.Chardonnet,and W.Doerfler. 1977.

Integrated viral sequences in adenovirus type 12 trans-formed hamster cells. Cell 10:101-111.

10. Hayward,G.1974.Uniquedouble-strandedfragmentsof

bacteriophage T5 DNA resulting from preferential

shearinducedbreakage ofnicks.Proc.Natl.Acad. Sci. U.S.A.39:679-701.

11.Honess, R. W., and B. Roizman. 1975. Regulation of herpesvirus macromolecular synthesis. Sequential tran-sition ofpolypeptidesynthesis requires frunctional viral polypeptides. Proc. Natl. Acad. Sci. U.S.A. 72:1276-1295.

12.Hotta, Y., and A. Bassel. 1965. Molecular size and

circularity of DNA in cells ofmammals and higher

plants. Proc. Natl. Acad. Sci. U.S.A. 53:356-362.

on November 10, 2019 by guest

http://jvi.asm.org/

13. Jones,P.C.,G.S. Hayward,and B.Roizman. 1977. Anatomyof herpes simplex virus DNA. VII. aRNA is homologous to non-contiguous sites in both L and S componentsof viral DNA. J. Virol. 21:268-276.

14. Kieff,E.D., S.Bachenheimer, andB.Roizman. 1971.

Size, composition and structure of the DNA of subtypes 1and 2herpes simplex virus. J. Virol. 8:125-132. 15. Kit,S.,and D. R. Dubbs.1963.Acquisitionofthymidine

kinaseactivity by herpes simplex infected mouse fibro-blastcells. Biochem. Biophys. Res. Commun. 11:55-59.

16. Klemperer,H.G., G. R. Haynes, W. I. H. Shedden,

and D. H. Watson. 1967. A virus specific thymidine kinase in BHK21 cells infected with herpes simplex virus.Virology 31:120-128.

17.Leiden, J.,R. Buttyan, and P. G. Spear. 1976. Herpes

simplex virus gene expression in transformedcells. I.

Regulation of viral thymidine kinase gene in trans-formed Lcells by products of superinfecting virus. J. Virol. 20:413-424.

18. Leung, W. C. 1978. Evidence for a herpes simplex virus-specific factorcontrolling the transcription of deoxy-pyrimidinekinase. J. Virol. 27:269-274.

19. Lin, S.-S. and W. Munyon. 1974. Expression of the viral thymidine kinase gene in herpes simplex virus trans-formed L cells. J. Virol. 14:1199-1208.

20.Littlefield, J. 1964. Selection of hybrids frommatingsof fibroblasts in vitro and their presumed recombinants. Science 145:709-710.

21. Maitland, N. J., and J. K. McDougall. 1977. Biochem-ical transformation of mouse cells by fragments of herpes simplex virus DNA.Cell11:233-241.

22. Maniatis, T., A. Jeffrey, and D. G. Kleid. 1975. Nu-cleotide sequences of the rightward operator of phage

A.Proc. Natl. Acad.Sci. U.S.A. 72:1184-1188. 23. Minson, A.C.,P.Wildy, A. Buchan, and G. Darby.

1978.Introduction of the herpessimplexvirus thymi-dine kinase gene into mouse cells using virus DNA or transformedcellDNA.Cell13:581-587.

24. Morse,L.S.,T.G.Buchman, B. Roizman, and P. A.

Schaffer. 1977. Anatomy of herpes simplex virus DNA. IX. Apparentexclusion of some parental DNA arrange-mentsin the generation ofintertypic (HSV-1 x HSV-2) recombinants. J. Virol. 24:231-248.

25. Morse,L.S.,L.Pereira, B. Roizman, and P. A.

Schaf-fer.1978.Anatomy of HSV DNA. X. Mapping of viral genesby analysis of polypeptides and functions

speci-fiedby HSV-1xHSV-2 recombinants. J. Virol. 26:389-410.

26. Munyon, W.,R.Buchsbaum,E.Paoletti,J.Mann,E.

Kraiselburd, and D. Davis. 1972.Electrophoresis of thymidine kinase activity synthesized by cells trans-formedbyherpes simplex virus.Virology 49:683-689.

27. Munyon, W.,E.Kraiselburd,D.Davis,and J. Mann.

1971. Transferofthymidinekinasetothymidine kinase-less L cells by infection with ultraviolet-irradiated herpessimplex virus. J. Virol. 7:813-820.

28. Ogino, T., R. Shiman, and F. Rapp. 1973. Deoxythy-midine kinase from rabbitkidney cellsinfected with herpessimplex virus types1and2.Intervirology 1:80-95.

29.Preston, V. G., A. J.Davison, H. S.Marsden,M. C. Timbury, J. H.Subak-Sharpe, and N. M. Wilkie. 1978.Recombinants betweenherpessimplexvirustypes 1and 2:analyses of genomestructuresandexpression of immediateearly polypeptides.J.Virol. 28:499-517.

30. Rapp, F., and N. Turner. 1978. Biochemical

transfor-mation ofmousecellsby herpessimplexvirus types1

and2:comparisonof different methods for inactivation

of viruses. Arch. Virol. 56:77-87.

31. Roizman, B. 1979. The structure and isomerization of

herpessimplex virus genomes.Cell 16:481-494.

32. Ruyechan,W.T., L.S.Morse,D. M.Knipe,and B.

Roizman.1979.Moleculargenetics ofherpes simplex

virus.II. Mapping of the major viralglycoproteinsand ofthe genetic loci specifying the social behavior of infected cells. J. Virol. 29:677-697.

33. Southern, E. M. 1975. Detection ofspecificsequences among DNAfragmentsseparatedby gel

electrophore-sis. J.Mol. Biol.98:503-517.

34. Thouless, M. and P.Wildy.1975.Deoxypyrimidine ki-nasesof herpessimplexvirusestype1and2:comparison ofserological and structural properties. J. Gen. Virol. 26:159-170.

35. Wagner, M. J., and W. C. Summers.1978.Structure of thejoint S region and the termini oftheDNA ofherpes

simplexvirustype1.J. Virol. 27:374-387.

36. Wetmur, J. G. 1975.Acceleration of DNArenaturation rates.Biopolymers14:2517-2524.

37. Wigler, M., S. Silverstein, L. Lee, A. Pellicer, Y.

Cheng,and R. Axel.1977.Transfer ofpurified herpes

virusthymidine kinase gene tocultured mousecells. Cell11:223-232.

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http://jvi.asm.org/