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DNA of Epstein-Barr Virus. II. Comparison of the Molecular Weights of Restriction Endonuclease Fragments of the DNA of Epstein-Barr Virus Strains and Identification of End Fragments of the B95-8 Strain


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JOURNAL Aug. 1977,

Copyright t© 1977 American SocietyforMicrobiology Printed inU.S.A.

DNA of Epstein-Barr Virus


Comparison of the Molecular Weights of Restriction Endonuclease

Fragments of the DNA of Epstein-Barr Virus Strains and Identification

of End Fragments of the B95-8 Strain


Departments of Medicine and Microbiology, The Committee on Virology, The UniversityofChicago, Chicago, Illinois 60637

Received for publication 4 March 1977

Incubation ofthe DNA of the B95-8 strain ofEpstein-Barrvirus EBV (B95-8)

DNA] with EcoRI,Hsu I,SalI, orKpnIrestriction endonucleaseyielded 8 to 15

fragments separableon 0.4%agarose gelsand ranginginmolecularweight from

less than 1to morethan30 x 106. Bam I and Bgl IIyieldedfragments smaller than 11 x 106. Preincubation of EBV (B95-8) DNA with lambda exonuclease

resultedinadecreaseintheHsu I Aand Sal I A and D fragments, indicating

that these fragmentsarepositionednear termini. Theelectrophoreticprofiles of

the fragmentsproduced by cleavage of the DNA of the B95-8, HR-1, andJijoye strainsofEBV wereeach distinctive. The molecularweights of some EcoRI, Hsu

I, andSal I fragments from the DNA of theHR-1 strain ofEBV IEBV (HR-1) DNA] and of EcoRI fragments of the DNA of the Jijoye strain of EBV were

identical to that offragments produced by cleavage of EBV (B95-8) DNA with

the same enzyme, whereas others were unique to each strain. Some Hsu I,

EcoRI, and Sal I fragments of EBV (HR-1) DNA and Kpn I fragments of EBV

(B95-8) DNA were present inhalf-molar abundance relative to the majority of

the fragments. In these instances, the sum of the molecular weights of the

fragmentswas inexcessof108, theknownmolecularweight of EBV (HR-1) and

(B95-8) DNA. The simplest interpretation ofthis finding is that each EBV

(HR-1), andpossibly also (B95-8), DNApreparationcontainstwopopulationsof

DNAmolecules thatdiffer in thearrangementofDNAsequences abouta


point, suchas has beendescribed for

herpes simplex

virus DNA. Minor

frag-mentscould also beobservediftherewere morethanonedifference in


structureof the DNAs. The data do not exclude more extensiveheterogeneity in

primary structure of the DNA of the HR-1 strain. However, the observation

thattherelative molarabundance ofmajorand minorfragmentsof EBV


DNAdidnotvarybetweenpreparations from cultures thathadbeenmaintained


forseveral yearsfavors the former


overthe latter.

Current knowledge of the DNAs of human Epstein-Barrviruses(EBV)canbe summarized

asfollows. (i) VirionDNA


from HR-1

(11) and B95-8 (18) continuous




EBV(HR-1) DNA and

EBV(B95-8)DNA,respectively]is alinear dou-ble-stranded DNA of 100 x 10" daltons (23). Most DNAstrands have asizeless than 50 x

10" daltons in alkaline sucrose gradients (23).

(ii) The buoyant density ofEBV (HR-1) and

(B95-8) DNAs is 1.718 g/cm3inneutralcesium

chloride, suggestingabasecomposition of57 to 58guanine-plus-cytosine molespercent (12, 19,

23, 24, 30, 31). Fragments ofEBV DNAs

pro-ducedby mechanical shear haveadistribution ofbuoyant density in neutral cesium chloride

ranging from 1.716 to 1.721 g/cm3, suggesting

a narrow range of variation in proportional

contentof guanineandcytosineversusadenine



(23). EBV


DNA is rela-tively enriched for sequences with a slightly higher thanaveragebuoyantdensity (23). (iii)

Kinetic and absorptive reciprocal

hybridiza-tions with


viral DNAs indicated that

EBV (B95-8) and (HR-1) DNAs are

approxi-mately 85 to92% homologous (23, 27) and that

EBV(B95-8) DNAlacksapproximately8 to 15%

of the sequencesofEBV (HR-1)DNA (23). (iv) Covalently closed circularEBVDNA hasbeen demonstrated within some infected cells (15).

From thisobservation, itmay be inferredthat

sequences nearboth termini oflinearmolecules

possess homology.

Two analyses of the fragmentsproduced by 421

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cleavage of EBV DNA with restrictionenzymes

have been reported. Some of the fragments

pro-duced by cleavage of EBV (B95-8) and (HR-1)

DNAs were found to have similar molecular weights, whereas others were found to differ

(10, 27). In one ofthese previous reports (27),

avian myeloblastosis virus reverse

transcrip-tase was used to label EBV DNA in vitro. The

amountofradioactivity incorporated into

frag-mentsof DNA was variable and was not

propor-tional to the molecular weight of the DNA

frag-ment. High-molecular-weight fragments were

underrepresented, even assuming that the la-beling of fragments would be irrespective of

size. In the other report (10) the DNA was

labeled in vivo, but thequantitation of the label

in each fragment wasnot presented.

In this paper, we report the results of an

analysis of the fragments produced by cleavage

of EBV (B95-8) DNA with six restriction

en-zymes, the comparative analysis of the

frag-ments of EBV (HR-1) DNA, and preliminary

resultsof identification of the end fragments of

EBV (B95-8) DNA using lambda

5'-exonucle-ase. The long-term goal of these studies is to

correlate biological function with base

se-quences and their organization in EBV DNA.

The organization of viral DNA is likely to be important in control of transcription and may

berelated to thedifferential processing of

spe-cificviral RNA species in restringently infected

cells (9, 21).


Cell cultures. Cultures of HR-1 (11) (obtained from G. Klein, Karolinska Institute, Stockholm, Sweden, and from R. Glaser, Pennsylvania State University College ofMedicine, Hershey),B95-8(18) (obtained from G. Miller, YaleUniversity, New Ha-ven, Conn.), andJijoye (obtained from P. Gerber, Division of Biological Standards, National

Insti-tutesof Health, Bethesda, Md.) cellsweregrownat

350CinRPMI1640mediumsupplementedwith 10%

heat-inactivated (560C for 30 min) fetal calfserum

(both from GIBCO, Grand Island, N.Y.). Cultures

weregrownfor 3-month intervalsinmedia contain-ingtylocine (60


(GIBCO) and spectinomycin



(The UpjohnCo.,Kalamazoo, Mich.)or

inmedia without antibiotics.

Restriction enzymesandmarker DNAs. Restric-tionendonuclease SalI (fromStreptomyces albus), Kpn I (from Klebsiellapneumoniae), Bam I (from Bacillusamyloliquefaciens), andBglII(from

Bacil-lusglobiggi) and 32P-labeled herpessimplex type 1

(MP strain) [HSV-1(MP)]DNA wereobtainedfrom G. Haywardinthelaboratory of B. Roizman, Uni-versity of Chicago. EcoRI endonuclease was

pur-chased fromNew England Biolabs, Beverly, Mass.

HsuIendonucleasewasprepared from Haemophilus

suis (Hsu I and Hin III are isoschizomers) by a procedure devised byR. Robertsand G. Hayward. Allsteps werecarriedout at4°C.Briefly, 10gof H.


suiscellswassuspendedin abufferconsistingof50 mM NaCl, 10 mM 2-mercaptoethanol, and 20 mM

Tris-hydrochloride (pH 7.6), sonically treated 25 times for 30 s at 300 W(Artek Corp., Farmingdale,

N.Y.), and centrifuged at 100,000 x gfor 90 min.

One milliliter of 10% streptomycin sulfate was

added for each 1,500 unitsofabsorbencyat260 nm,

andthe precipitate wasremovedby centrifugation

at 10,000 x g for 30 min. A 50 to 70% saturated

ammonium sulfate fraction of the supernatant was

resuspended inabufferconsistingof1 MNaCl, 10 mM2-mercaptoethanol, and20mM

Tris-hydrochlo-ride (pH 7.6),dialyzedagainstthesamebuffer,and passed throughacolumn(2.5by50cm) of Bio-Gel A.

The fractions containing the cleanest activity

against T5 DNA were pooled, dialyzed againstPC

buffer(10%glycerol,50mMKCl,0.1mMEDTA,10 mM2-mercaptoethanol, and10mM potassium

phos-phate, pH 7.9),and loaded onto aphosphocellulose

(P. H. WhatmanCorp.) column (25by1.6cm).

Frac-tions were eluted with a linear 0.05 to 1 M KCl gradient and tested for activity against T5 DNA. Fractions containing the desired activity were

pooled, dialyzed against50%glycerolin 0.3M NaCl and20 mMTris-hydrochloride (pH 7.6), and stored

at -20°C.

T5bl phagewas grownanditsDNAwaspurified asdescribedpreviously(7).

Preparationof labeledDNA.Eight liters of HR-1

orB95-8 cultures or12 liters ofJijoye cultureswas

concentrated fivefold by aspiration of the superna-tantmedia and incubated for2 days with [3H]thy-midine (60Ci/mmol; New England Nuclear Corp., Boston, Mass.) or [3H]cytosine (50 Ci/mmol; New

England Nuclear Corp., Boston, Mass.) at a final

concentration of3 ,Ci/ml. For those experiments in which DNA was labeled with [32P]orthophos-phate, cellswereconcentrated 10-fold, reincubated

inmedia consistingof50% old and50% new phos-phate-free RPMI 1640 (obtained from GIBCO) for

24 h, and then resuspended for 24 h in 50 ,Ci of [32P]orthophosphatepermlinphosphate-free RPMI

1640 supplemented with 10% dialyzed calfserum

and0.05 MHEPES buffer(N-2-hydroxyethyl piper-azine-N'-2-ethanesulfonic acid), pH 7.4. Virus was

purified fromtheculture mediumaspreviously de-scribed (2), and the radiolabeled viral DNA was

isolatedbygentle phenol extraction(23). Sedimenta-tion of the labeled EBV (B95-8) and (HR-1) DNA

preparationsin sucrosevelocitygradientsusing T4

DNA as amarker(23)demonstrated thatmorethan

95% of the labeled EBV (HR-1) and (B95-8) DNA preparationssedimentedwith thevelocity expected formoleculesof 100 x 106daltons.

Exonuclease digestion. To identify those

frag-mentslocatedattheends ofEBVDNAmolecules,a

brief lambda exonuclease digestion wasperformed

before incubation with restriction enzymes. The amountoflambda exonuclease (giftofG. Hayward

inthe laboratory ofB. Roizman) requiredto

elimi-nate terminal fragments from the gel profileswas determinedby usingT5bl DNA. Each reaction mix-turecontained3.5 ggof EBV DNA perml, 67mM glycine phosphate (pH 9.6), 50 ,g of bovine serum albumin per ml, 1 mM 2-mercaptoethanol, and 3

mMMgCl2(28). The reaction mixturewasincubated

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at250C for5min, quenchedin icewith the addition of 1/10 volume of 0.1 M EDTA, extracted with phenol, and dialyzed extensively against 0.15 M NaCi, 20 mM Tris (pH 7.6), and 1 mMEDTA, fol-lowed by 20 mM NaCl, 20 mM Tris (pH 7.6), and

1 mM EDTA.

Restrictionenzyme analyses. EBVDNA(0.5to2 ,ug) was incubated for 2.5 hat370C in 0.3 ml ofa

solution containing20mMMgCl2,20mMNaCl, and 20 mM Tris-hydrochloride (pH 7.6) for digestion withHsu I,Sal I, Kpn I, Bam I, andBgl II orina

solution containing20mMMgCl2,50mMNaCl, and 150 mM Tris-hydrochloride (pH 7.6) for digestion with EcoRI. The volumeofrestrictionenzymeused in each reaction mixture was two- tofivefold greater than the volume neededtocompletelydigest3 ,ug of T5bl DNA. Twoto3ggofunlabeled T5b1 DNA was included in each incubation mixture to ascertain that the enzyme activity was adequate toyield limit digests (4). The enzyme reaction was terminated by the addition of20mM EDTA. In those experiments used to measure the sizeof the fragments of EBV DNA, fragmentsofT5bl and HSV-1 (MP)DNA were

used as internalmarkers (6). An HsuIdigest (6) of 32P-labeled HSV-1 (MP) DNA wasaddedtothe

sam-ple before electrophoresis.

DNA samples were layered onto 0.4% (wt/vol) cylindrical agarosegels (1 by28cm)and electropho-resed at40 Vfor36h at4°C (5). Fragments of T5bl DNA and EBV DNA were visualized and photo-graphed underUVlight after stainingfor 2 to4h in asolution containing0.5,gof ethidiumbromide per ml. The relative position and absorbance of EBV and T5bl DNAfragmentsweredeterminedby scan-ningtransparencyphotographs of theethidium bro-mide-stained gels in an Acta II spectrophotometer equipped with gel scanner. The position of frag-ments of 3H-labeled EBV and 32P-labeled HSV-1 DNAs was determined by cutting the gels

trans-versely into 1-mm slices andmeasuring the radioac-tivity presentineach slice (6). Relative molarratios werecalculatedbydividing theamountof radioac-tivityineachEBVDNAfragmentby the molecular weight of thefragmentasdetermined from its elec-trophoretic migration relative to T5bl and HSV-1 (MP) markers. Between85and90%of the radioac-tively labeledEBV (B95-8) or (HR-1) DNA applied

tothe gel wasroutinely recoveredinspecific frag-ments. The relative molar ratios listed in Table2

represent theresults ofanalyses of three [3H]thy-midine-labeled DNA preparations. Average relative molar ratios of between0.4and0.6wereconsidered

tobe 0.5; those between0.8and1.2wereconsidered

to be 1. To obtain molar ratios of low-molecular-weight fragments, gels wererunfor shorter

inter-vals than those shown in Fig. 1and2.


Comparison of the DNAs of the B95-8 and

HR-1 strains of EBV. Between 4 and 8 ,ug of

DNAwas regularly recovered from virus

puri-fied from 8 liters of B95-8 and HR-1 cultures.

Thespecificactivity of the purified DNA varied

between 2 x 104 and 105 3Hcpm/,ug when

cul-tures were incubated in [3H]thymidine or


[3H]cytosine and was approximately 104 cpm/

,ug when cultures were incubated with [32P]_


Initially, six restriction endonucleases(Hsu

I, Sal I,EcoRI,Kpn I,Bgl II, and BamI)were tested to determine which would yield a small

number offragments of EBV (B95-8) DNA that

could be resolved on agarose gels (Fig. 1). The

molecular weights of EBV DNA fragments

wereestimated by comparison of their

electro-phoretic mobility with that of fragments of T5bl and HSV (MP) DNA. The relative molar

ratios were calculated as described inMaterials

and Methods. The results were as follows: (i)

EcoRI, Hsu I, andSal I each yielded up to 15

fragments, ranging in molecular weight from

0.6 x 106to 32 x 106, whereasBam I andBgl II

eachgave more than 20fragmentssmallerthan

11 x 106 (Fig. 1, Table 1). (ii) Almost all

frag-ments produced by digestion of EBV (B95-8)

DNA with EcoRI, Sal I, and Hsu I contained

an amount of [3H]thymidine (Fig. 1, Table 1)

proportional to their molecular weight. One

EcoRI fragment (fragment D) contained

be-tweenone-quarterand one-halfof the expected

[3H]thymidine label (Fig. 1 and Table 1). Two

minorfragments(Fig. 1, Hsu Ifractions168to

171 and EcoRI fractions 149 to 151) were

con-sistently observed, butwere notassignedletter

designations because they were present in

molarratios of less than 0.25. Two other

frag-ments, the Gfragment ofEcoRIandIfragment

of Hsu I, contained more label than would be

expected ifthese bands contained single

frag-mentsof theirestimatedmolecularweight(Fig.

1and Table 1). Analysis of the Sal I, Hsu I, and

EcoRIfragments of EBV (B95-8) DNAlabeled

with [32P]orthophosphate andofSalIand Hsu I

fragments labeledwith [3H]cytosine confirmed the relative molar ratios obtained with

[3H]-thymidine-labeled DNA. The only discrepancy observed was that the Hsu I C fragment

con-tained slightly more [3H]thymidine label and

the Bfragment slightly less thanwasexpected

forfragmentsoftheirsize(Fig. 1),whereasthe

reverse result wasobtained when the DNA was

labeledwith[3H]cytosine(data notshown). (iii)

Thesums ofthe molecular weights of the

frag-ments produced by EcoRI, Sal I, and Hsu I

digestion (Table 1) were close to the reported molecular weight for EBV (B95-8) DNA (23).

(iv)Treatment of EBV (B95-8) DNAwith Kpn I

yielded at least 15 separable fragments. The

Kpn I C and E fragments probably consisted of

two components, since these fragments

con-tained approximately twice as much label as

expected for fragments with their estimated molecular weight (Table 1). In addition, three

KpnIfragments (B, L, and M) werepresent in

approximately half-molar amounts. The sum of

on November 10, 2019 by guest








50 100 150 200 250 300


. 5.

E4 I3.


_-50 00 i50 200 250



370 6.5 6.0







50 loo 150 200 250

Bom I 750


FIG. 1. Distribution oftritium label inagarosegel electropherograms of fragments ofEBV(B95-8)DNA

produced bytreatmentwith HsuI, SalI, BglII,EcoRI,Kpn I,andBamIrestrictionenzymes.Theprocedures

used in labeling andpurifying EBVDNA, in treating the DNA with restriction enzymes, in separating fragments ofthe DNA in 0.4%agarosegels,and indeterminingthe distributionofradioactivityin thegelsare

described in Materials and Methods. The direction ofelectrophoresisisfrom lefttoright.

themolecular weights of the Kpn Ifragmentsof

EBV(B95-8) DNAwasapproximately 1.3 x 108 (Table 1). Intact T5bl DNA mixed with the EBV(B95-8)DNAbefore incubationwith Kpn I

enzyme wasfully digested.

Theresultsof analyseson agarosegelsof the

fragmentsproduced by digestionof EBV(HR-1)

DNAwith EcoRl, Hsu I, orSalI (Fig. 2)

indi-catedthe following. (i) Ineach instance, some

of the fragments were identical in molecular

weighttoafragment produced by treatingEBV

(B95-8) DNA with the same enzyme (Fig. 4).

Other fragments in each EBV DNA

prepara-tionwereunique (Fig. 4). (ii) Some fragments

of EBV(HR-1)DNAproducedby EcoRI, Hsu I,

andSal I contained halfas much label as

ex-pected for afragment of their size (Fig. 2 and

Table 1). Each enzyme produced at least two

suchfragments presentinhalf-molar amounts

(Fig. 2 andTable 1). Thefinding of half-molar

fragmentswasconfirmedby analysesinwhich

the amount ofDNA in eachfragment was

de-termined from the relative optical density of

the fragment in ethidium bromide-stainedgels.

Threeminorcomponents(HsuI fractions93 to

98, 136to 149, and 157to159)wereconsistently

present, but were notassignedletter

designa-tions becausetheywerepresentinmolar ratios

of less than 0.25. (iii)Thesumsof the molecular

weights ofthe EcoRI, Hsu I, and Sal I

frag-mentswere132 x 10", 160 x 10",and 130 x


respectively (Table 1). (iv) Identical results

were obtained using EBV purified from HR-1

culturesoriginallyobtained from G. Kleinand

passagedfor3 years inourlaboratoryorHR-1


TheHR-1 cell linewasoriginallyderived(11)

fromacontinuous cultureoflymphoblasts



4 3




2-Bg E 75

a70. °65.

60--.I .o d4--*-Ik 150 -1260- 250



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TABLE 1. EBV restriction enzyme fragments

B-95 HR-1

Frag- EcoRI Hsu I Sal I KpnI EcoRI Hsu I Sal I


SizeaMolarity Size Molarity Size Molarity Size Molarity Size Molarity Size Molarity Size Molarity

A 32.5 1 28b 1 32b 1 29 1 26 1 35 0.5-1 27.5 0.5

B 21 1 20 1 27.5 1 22 0.5 21 1 27 0.5 22.5 0.5

C 11.61 1 17.6 1 19.5 1 11 2 20 1 17.6 1 12.8 1

D 8.2 0.25-0.5 13.8 1 11.0b 1 8.4 1 14.5 1 16.0 0.5 11.0 2

E 7.5 1 7.0 1 8.3 1 6.9 2 12.5 0.25 11.3 2 9.2 1

F 5.4 1 4.0 1 5.0 1 6.6 1 10 0.5 9.6 1 8.3 1

G 4.1 1.5-2 3.5 1 4.0 1 6.2 1 8.0 1 8.0 1 7.5 1

H 3.6 1 3.1 1 2.0 NDc 4.1 1 5.4 1 4.4 1 4.8 1

I 2.8 1 2.2 2 4.0 1 4.2 0.5 4.0 1 4.0 0.5

J 2.2 1 2.1 1 2.6 1 3.5 0.5 3.5 1 3.4 0.5

K 1.5 1 1.7 1 2.5 1 2.8 1-2 3.1 1 2.5 2

L 1.0 1 1.3 1 2.4 0.5 2.1 NDc 2.2 NDc 2.0 2

M 0.6 NDc 1.0 |NDc 2.1 0.5 1.5 NDc 2.1 NDc 1.3 3

1.8 ND~c 1.6 NI~c

1.5 ND~c 1.5 ND~c

Total 105x 106 105x 106 109 x 106 129 x106 132 x 106 158 x 106 130 x 106 mol


aMolecularweightestimated from position of marker DNAs as described in Materials and Methods. bEndfragment.

eND,Molarities not calculated. The total number of counts per minute in these fragments was less than two times the totalbackground.

dInthose instances where the relative molar ratio was clearly in excess of 1, the fragment was considered to have two

componentsof equal molecularweight.

joye)established from aBurkitt tumor


Comparisonof the DNA ofEBVfrom Jijoye and HR-1 cultures would therefore be of interest. However, the production ofEBV from Jijoye cultures is


and lessconstantthan from the HR-1 cell line(4).To obtain sufficientEBV

(Jijoye) DNA for analysis, it was necessary to

take a broad region of the dextran


This DNA was not run on sucrose


before treatmentwith restrictionenzyme. The

summary data of


of EBV (Jijoye)

DNA (Fig. 4) were derived from






and unlabeled EBV




3) run in parallel with



frag-ments of EBV (HR-1) DNA. The data indicate

that the EcoRI A


of EBV (Jijoye) DNA is


than the




HR-1 DNA and that there isno




tothe D


of EBV (HR-1) DNA. The differences between

frag-ments of EBV (Jijoye) DNA and EBV


DNA were confinedto EBV (HR-1) DNA

frag-ments that were not present in EBV (B95-8)

DNA. However, the molecular weights of the



DNA fragments which differed from those ofEBV (HR-1) DNAdidnot



offragments of

EBV (B95-8) DNA. The backgroundin the re-gion of lower-molecular-weight fragments of

EBV (Jijoye) DNA was much higher than in


comparable analyses

of EBV (HR-1) or

(B95-8) DNA, likely as aresult of

contamina-tion and random degradation of the EBV (Ji-joye) DNA.Therefore, the relative molar abun-dance of


of EBV (Jijoye) DNAcould



fromthese data.

Identification of the end fragments ofEBV

(B95-8) DNA. The distribution of [3H]thymi-dine label inSal I orHsu I restriction endo-nuclease fragments of [3H]thymidine-labeled

EBV (B95-8) DNA was compared with that

of DNA which had been preincubated with

lambda exonucleaseinordertoidentify the end fragments. Lambda exonuclease isaprocessive

enzyme that attacks 5'-phosphoryl termini of

native DNA preferentially (1), although not

exclusively (26). After lambda exonuclease

treatment and


with restriction

en-zyme, fragments positioned at ends of native

EBV DNA would be expectedto consistof

na-tive DNA witha



tail. The migration of DNA that contains single-and


segments is aberrant on

agarosegels (8).Incontrol experiments

under-taken to determine the proper condition for lambda exonuclease treatment, terminal

frag-ments of


DNAof7.7 x 10i and8.6 x 106

daltons were more than 90% reduced without

any decrease in the



The results of lambda exonuclease pretreat-mentofEBV (B95-8)DNAillustratedin



and 6 indicate the


(i) The Hsu I A

fragmentwasremovedby lambda exonuclease


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So I




'-50 10. 150

FIG. 2. Distributionoftritium11 electropherograms of fragments of beledEBV(HR-1)DNAproduced Hsu I, SalI, and EcoRI restrict

procedures used are described

Methods. The directionof electropi


digestion (Fig. 5)and is theref

(within 8 x 10" daltons) an (

(B95-8) DNA molecule. No chi

ent in the amount offragment

and presumably the other ens

smaller than J. (ii) Sal I A a

weresignificantly decreasedb;

cleasepretreatment(Fig. 6)an

identified asterminal fragmei

fragmentdiffered from other So


packed, bands, all of which

lambda exonuclease treatment. The simplest



of this


is that there ex-ists slight length variabilityattheD-fragment

terminusof theEBV(B95-8) DNAmolecule.


These studies were undertaken to begin to

determine the arrangement of sequences in

EBV DNA. EcoRI, Hsu I, Sal I, and Kpn I

14t K LM N

I J K LM restrictionenzymescleaveEBVDNAinto

frag-AA ,,_Aid mentsthat are separable on 0.4% agarose gels,

whereas Bam I and Bgl II yield fragments

whosesize is too close to beseparable. On the


basis of their susceptibility to degradation by

lambda exonuclease, the Hsu I A and Sal I A

and D fragments of EBV (B95-8) DNA have

been identified as terminalfragments.

The analyses reported here of the size and

relative molar abundancy of restriction enzyme

fragments of the DNA of EBV strains indicate

thatthere aredifferencesin base sequence

be-tween strainsof EBV and between DNA

mole-K ' cules of thesame strainofEBV. Several lines of



evidence indicate thatthere is



the DNA molecules of at least one strain of EBV.Thus, some Hsu I and Sal I fragments of

EBV (HR-1) DNA are present in equal molar

abundance; other fragments are present in half-molar abundance; andthe sum ofthe molecular

weight of the fragmentsproduced by each

en-zyme is in excess of the known molecular

weight ofEBV(HR-1) DNA (23).Inevaluating the significance of these observations, the fol-lowing points should be considered. (i) The

presence of minor bands is unlikely to be an

artifact of incomplete digestion of the DNA.

IJ^ T5bl DNA was incubated with EBV DNA in

each enzyme reaction mixture and was itself

completely digested. Moreover, theratioof

mi-abelinagarose gel nor to major bands was constant in repeated


experiments and was not affected by increasing

'by treatment with the concentration of restriction enzymes. (ii)


enzymes. The The EBV (HR-1) DNA used inthese analyses

In Materials and


is from left

sedimented in

sucrose velocity gradients as a


band of


100 x 106daltons.

(iii) EBV (HR-1) DNA containsonly EBV

ho-mologous sequences. Thus, DNAfrom cells

in-Fore locatednear fected withEBVdrives denatured EBV


end of the EBV DNAmorethan90% into


whereas DNA

range wasappar- from uninfected cells hasnoeffectonthe

rena-lts B through J, turation ofEBV







d is a fragment (iv)


on agarose


of the restriction

nd Dfragments enzyme





ylambdaexonu- HR-1 culturesthathavebeenmaintained




for several years



re-nts. The Sal I D sults.



in Taken


these datasuggest that EBV

crete, but


(HR-1) DNA containsatleasttwopopulations

decreased after ofmolecules with a

high degree




i c

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\ T5bl






50 100 150 200

FIG. 3. Electropherogram of fragments of I3H]thymidine-labeledEBV


DNAproduced by treat-ment withEcoRIrestriction enzyme. Thephotograph ofan agarosegel stained with ethidium bromide is

positioned aboveagraph of the distribution oftritiumlabelinthatgel. Theproceduresusedaredescribedin

Materials and Methods.The direction ofelectrophoresis isfromlefttoright.

and with unique and common restriction

en-zyme fragments. The simplestexplanation for

theseobservations is that they may be due to

alternative molecular arrangements of

identi-cal DNA sequences, possibly in association

with invertedrepeats in the DNA. Precedence

for this hypothesis has been established from

studies of thesequenceofherpessimplexDNA

(25, 29). Another possibility is that the HR-1

cultures containastablemixtureoftwoclosely

related strains of EBV. This possibility has

beensuggestedby studies thathavefoundtwo

distincttypes ofEBV-related nuclearantigen,

EBNA, in lymphocytes infected with EBV

(HR-1) (3). Comparative analysis of the DNA

in other strains of EBV could help to

distin-guish between these alternatives. Theanalyses

reported here of EBV (B95-8) DNAare

ambigu-ousinthis regardinthat submolar fragments

andatotal molecularweight for all fragments

in excess of 100 x 106 were clearly obtained

in only one instance, i.e., digestion of EBV

(B95-8) DNA with Kpn Irestriction nuclease.

Withregardto thedifferences reportedhere

between the DNA of strains ofEBV,the

follow-ing points shouldbe made. (i) EBV (HR-1) is

producedfromthe HR-1 clone(11) ofa continu-ous lymphoblast culture (Jijoye) established

from a Burkitt tumor biopsy. EBV (B95-8) is

produced from a clone of marmoset cells

in-fectedinvitrowith EBVderivedfroma

contin-uousculture oflymphoblastsestablished froma

patient with infectious mononucleosis (18).

EBV (B95-8) has the ability to transform but

cannotinduce early antigenonsuperinfection,

whereas EBV (HR-1)caninduce early antigen

butlacks the abilityto transform (16, 17). The

enhanced ability of EBV (B95-8) to transform

could result from the presence of DNA

se-quences required for transformation or from

the absence of DNA sequences that might

in-hibit transformation. (ii) Some ofthe fragments

produced by treating EBV (B95-8) or (HR-1)

DNA withEcoRI, Hsu I, and Sal I restriction

enzymes have identical molecular weights in

agarosegels. Since EBV (B95-8) DNA contains approximately 85% of the sequences of EBV

(HR-1) DNA (23, 27) and EBV (HR-1) DNA








VOL. 1977


on November 10, 2019 by guest




Eco RI

895 Jijoye HR




10-0 a


HsuI 895 HR-I


SalI B95 HR-I

B95/Sal I A




3-- m

- =

-- _


B95(X exo) /Sal I

FIG. 4. Comparison of the molecular weights of fragments of EBV (B95-8), (Jipoye), and (HR-1) DNAs treated with EcoRI, Hsu I, or Sal I restriction enzyme. Fragments present in molar ratios of less than 1 are indicated by narrow bands.

B95/ Hsu I








2-50 100 i50

FIG. 5.Agarosegelelectropherogr striction enzymefragmentsofI"H]


EBV(B95-8) DNA(upperpanel) an dine-labeled EBV (B95-8) DNA w treated with lambda exonuclease (1 digest5'endsof theDNA. The dire phoresis isfromlefttoright. The bo agarosegel (to therightoffragment lower panel contracted during the The small band oflabeled DNA l rapidly than fragment Ginthe lower therefore, tobefragmentH.

contains more than 95% of the

EBV (B95-8) DNA (23), it is Hi

fragments which are of the sa: weighthave similar base sequence

uniquetoeach strainare




50 0,0 150 200

FIG. 6. Agcirose gelelectropherogram of Sal I re-strictionenzymefragments of13H]thymidine-labeled EBV(B95-8) DNA (upperpanel) and of [3H]thymi-dine-labeled EBV (B95-8) DNA which had been treated with lambda exonuclease to digest 5' ends of the DNA. Thedirection ofelectrophoresis isfrom left to right.

in one or more fragments whose molecular

weight is distinctive. Hybridization of

frag-F H ments with DNA will permitidentification of

sequencesunique to EBV(HR-1)DNAandmay

permit the identification of sequences unique

to EBV (B95-8) DNA. (iii) EBV (Jijoye) is not

as amenable to biochemical analyses as its


HR-1 clone. Neverthe-less, it is clear from these studies

that the

treat-mentofEBV (Jijoye)DNAwith EcoRI

restric-tionenzymeyieldsfragments thatareidentical

F G H I i andothers thatdiffer inmolecular



fragments ofEBV (HR-1) DNA. As might be



from the common








d of


between their DNA


than between

which had been those of EBV (HR-1) and (B95-8) DNAs. That

lower panel) to the size ofmany of the


common to

ctionofelectro- EBV




is also common to

ttom endofthe EBV(B95-8)DNA and that all of thefragments

G) showninthe which differ also differ from those ofEBV

(B95-electrophoresis. 8)DNA suggestthat there aresequences


more erentially conservedinEBV DNA.

-nanilias likely

sequences of

kely that the me molecular




We wish to acknowledge the contributions of G. Hay-ward forhelpfuldiscussions;W.King forassistance in the

preparationof T5DNAand HsuI restrictionenzyme;and

P.Gerberforsuggestingstudiesof EBV(Jijoye) andfor the

gift of Jijoye cells.

This work was supported by Public Health Service











A 8C



on November 10, 2019 by guest



grants 1-P01 CA-19264-01 and5-R01CA-17281-02fromthe National Cancer Institute and by AmericanCancerSociety grantVC-113B.


Currentstudies using "blots" offragments of viral

DNA (N. Traub,R. Pritchett, and E. Kieff, manu-script submitted for publication) have identified thoseEcoRIand Hsu Ifragments of the DNA of the HR-1 strain ofEBV that contain DNA sequences

missing from the DNA of the B95-8 strain. These sequencesarecomponents of the EcoRI C and D and

the HsuIE andNfragmentsof EBV(HR-1) DNA. The EcoRI K (and possibly A) and Hsu I B

frag-mentsof theDNA of the B95-8 strain of EBVcontain sequences that aremissingfrom EBV(HR-1) DNA.


1. Carter, M. D., and G. M. Radding. 1971. The roleof exonuclease and protein of phagexin genetic recom-bination.II. Its substrate specificity and the mode of action ofXexonuclease. J. Biol. Chem. 246:2502-2510.

2. Dolyniuk, M., R. Pritchett, and E. Kieff. 1976. Pro-teins of Epstein-Barr virus. I. Analysis of the poly-peptides of purified enveloped Epstein-Barr virus. J. Virol. 17:935-949.

3. Fresen, K. O., and H. zur Hausen. 1976. Establishment ofEBNA-expressing cell lines by infection of Epstein-Barr virus (EBV)-genome negative human lym-phoma cells with different EBV strains. Int. J.


4. Gabain,A., G. Hayward, and H. Bujard. 1976. Mapping ofthe Hind III, EcoR1, SalI and SmaIrestriction endonuclease cleavage fragments from bacteriophage T5 DNA. Mol. Gen. Genet. 143:279-290.

5. Hayward, G. S. 1974. Unique double-stranded frag-ments of bacteriophage T5 DNA resulting from pref-erential shearinduced breakage of nicks. Proc. Natl. Acad. Sci. U.S.A. 71:2108-2112.

6. Hayward, G., N. Frenkel, and B. Roizman. 1975. Anat-omy of herpes simplex virus DNA. I. Strain

differ-encesandheterogeneityinthe locations of restriction endonuclease cleavage sites. Proc. Natl. Acad. Sci. U.S.A. 72:1768-1772.

7. Hayward, G. S., and M. G. Smith. 1972.The chromo-someofbacteriophage T5. I. Analysis of the single-stranded interruptions by agarose gel electrophore-sis. J.Mol. Biol. 63:383-395.

8. Hayward, G. S., and M. G. Smith. 1972. The chromo-someofbacteriophage T5. II. Arrangement of single strandfragmentsinT5+andT5st(0)DNAmolecules. J. Mol. Biol. 63:397-407.

9. Hayward, S. D., and E. D. Kieff. 1976. Epstein-Barr virus-specific RNA. I. Analysis of viral RNA in cellu-lar extracts and in the polyribosomal fraction of per-missiveand nonpermissive lymphoblastoid cell lines. J. Virol. 18:518-525.

10. Hayward, D., R. Pritchett, T. Orellana, W. King, and E.Kieff. 1976. The DNA ofEpstein-Barrvirus.

Frag-ments produced by restriction enzymes: homologous

DNAand RNAin lymphoblastoid cells, p. 619-639. In D.Baltimore, A. Huang, and C. F. Fox (ed.),Animal virology, vol. 4. Academic Press Inc., New York.

11. Hinuma, Y., M. Konn, J. Yamaguchi, D. Wudarski, J. Blakeslee, and J. Grace. 1967. Immunofluorescence

andherpes-type virus particlesintheP3HR-1Burkitt lymphoma cell line. J. Virol. 1:1045-1051.

12. Jehn, U., T. Lindahl, and G. Klein. 1972. Fate of virus

DNAintheabortive infection of human lymphoid cell lines by Epstein-Barr virus. J. Gen. Virol.


13. Kawai,Y.,M.Nonoyama,andJ.Pagano.1973.

Reasso-ciation kinetics for EBV DNA: nonhomology to

mammalian DNA and homology of viral DNA in

variousdiseases. J. Virol. 12:1006-1012.

14. Kieff, E., andJ.Levine. 1974.Homology between

Burk-ittherpes viral DNA and DNAin continuous lympho-blastoidcells from patients with infectious

mononu-cleosis. Proc. Natl. Acad. Sci. U.S.A.71:355-358. 15. Lindahl, T., A. Adams, G. Bjursell, G. W.Bornkamm,

C. Kaschka-Dierich, andU.Jehn. 1976.Covalently closed circularduplex DNA of Epstein-Barr virus ina

human lymphoid cell line. J. Mol. Biol. 102:511-530.

16. Menezes, J., W. Leibold, and G. Klein. 1975. Biological differences between different Epstein-Barr virus (EBV) strains with regardtolymphocyte transform-ing ability. Exp. Cell Res. 92:478-484.

17. Miller, G., and M. Lipman. 1973. Release of infectious Epstein-Barr virus by transformed marmoset

leuko-cytes.Proc. Natl.Acad. Sci. U.S.A. 70:190-194.

18. Miller, G., T. Shope,H.Lisco,D.Still, andM.Lipman. 1972.Epstein-Barr virus:transformation, cytopathic changes, and viral antigens in squirrel monkey and marmosetleukocytes. Proc. Natl. Acad. Sci. U.S.A. 69:383-387.

19. Nonoyama, M., and J. Pagano. 1972. Separation of Epstein-Barr virus DNA from large chromosomal DNA in non-virus producingcells. Nature (London) New Biol. 238:169-171.

20. Nonoyama, M., and J. S. Pagano. 1973. Homology be-tweenEpstein-Barr virus DNA and viral DNA from Burkitt'slymphoma and nasopharyngealcarcinoma

determined by DNA-DNA reassociationkinetics.

Na-ture(London) 242:44-47.

21. Orellana, T., and E. Kieff. 1977. Epptein-Barr virus-specific RNA. II. Analysis ofpoly~denylated viral RNAinrestringent,abortive, and productive infec-tion. J. Virol.22:321-330.

22. Pritchett, R., M. Pedersen, and E. Kieff. 1976.

Com-plexityof EBVhomologousDNAincontinuous lym-phoblastoid cell lines. Virology 74:227-231.

23. Pritchett, R. F., S. D. Hayward, and E. D. Kieff. 1975.

DNAofEpstein-Barr virus. I. Comparative studies of the DNA ofEpstein-Barr virus from HR-1 and B95-8 cells: size, structure, and relatedness. J. Virol.


24. Schulte-Holthausen,H., andH. zurHausen.1970.

Par-tialpurificationof theEpstein-Barrvirus and some

propertiesof its DNA.Virology40:776-779. 25. Sheldrick, P., andN.Berthelot. 1974.Inverted

repeti-tions in thechromosome ofherpessimplexvirus.Cold Spring Harbor Symp. Quant. Biol.39:667-678. 26. Sriprakash,K. S., N. Lundh, M. D. Huh, and C. M.

Radding.1975. Thespecificityof lambdaexonuclease: interactions with single stranded DNA. J. Biol.

Chem. 250:5438-5445.

27. Sugden, B., W. C. Summers, and G. Klein. 1976. Nu-cleic acid renaturationand restrictionendonuclease

cleavage analysesshow that the DNAs of a

trans-forming and a nontransforming strain of Epstein-Barr virusshareapproximately90%of their

nucleo-tidesequences. J. Virol. 18:765-775.

28. Wadsworth, S., G. Hayward,and B. Roizman. 1976. Anatomyofherpes simplex virusDNA. V.

Termi-nallyrepetitive sequences.J.Virol. 17:503-512. 29. Wadsworth, S., R. J. Jacob, and B. Roizman. 1975.

Anatomyofherpessimplex virus. II. Size,

composi-tion, and arrangement ofinverted terminal repeti-tions.J.Virol. 15:1487-1497.

30. Wagner,E.K.,B.Roizman,T.Savage,P. G.Spear, M.

Mizell, F.Darr,and D. Sypowicz.1970. Characteri-zationof the DNA of theherpesvirusassociated with Luckeadenocarcinoma of thefrogand Burkitt lym-phomaof man.Virology42:257-261.

31. Weinberg, A.,and Y. Becker.1969.StudiesonEBvirus

ofBurkitt'slymphoma. Virology39:312-321.

on November 10, 2019 by guest



FIG.1.producedfragmentsdescribedused Distribution of tritium label in agarose gel electropherograms offragments ofEBV (B95-8) DNA by treatment with Hsu I, Sal I, Bgl II, EcoRI, Kpn I, and Bam I restriction enzymes
TABLE 1. EBV restriction enzyme fragments
FIG. 2.electropherograms Distribution oftritium 11 offragments ofabel
FIG. 3.positionedMaterialsment Electropherogram of fragments of I3H]thymidine-labeled EBV (Jijoye) DNA produced by treat- with EcoRI restriction enzyme


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