DNA of Epstein-Barr virus. VI. Mapping of the internal tandem reiteration.

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0022-538X/79/08-0315/10$02.00/0

DNA

of Epstein-Barr Virus

VI. Mapping

of the

Internal

Tandem Reiteration

DOUGLASS GIVENANDELLIOTTKIEFF*

SectionofInfectious Disease, Departmentof Medicine,andDepartments of MicrobiologyandVirology,

Kovler

Viral

Oncology

Laboratories, The

University of Chicago, Chicago,

Illinois60637

Received forpublication 15 January 1979

Epstein-Barr virus (B95-8) DNA consists of short (10 x 106) and long (87 x

106) unique DNA sequencesjoinedby10tandem reiterations of a 1.85x 106DNA

segment. The reiterated sequence contains BamI and BglII sites separated by 4

X 105. The 4.5 x 105 and 14.0 x 105 segments generated by cleavage of the

reiterated DNA with BamI andBglIIcontainsequences whichhybridize to each

other,suggestingthat theinternaltandemlyreiterated sequence has a direct or

inverted repeat within it. Theoppositeends of thelinear, nicked, double-stranded

DNAmolecule(R.F.Pritchett,S. D.Hayward,and E. D.Kieff,J.Virol.

15:556-569, 1975) consist of from1to 12direct repeatsof another3 x 105sequence (D.

Given and E. Kieff,J.Virol. 28:524-542, 1978; D. Given, D. Yee,K.Griem, and

E. Kieff,J. Virol.30:852-862, 1979).There isnohomology between the internal

reiterated sequence and either terminus. However, part of theinternal reiteration

(lessthan 5x105)is reiteratedat twoseparate locations in thelong unique region.

The internal reiterations are a source of variation within EBV (B95-8) DNA

preparations. Thus,althoughthemajority of molecules contain 10tandem reit-erations, some molecules have 9, 8, 7, 6, 5, 4, or fewer tandem reiterations. A

consequence of thisvariabilityis that theKpnIAfragmentand theEcoRI/HsuI

Afragmentconsist ofafamilyofsevenor morefragments differinginthe number

of tandem internal reiterations. The EcoRI/HsuI A fragment of EBV (W91)

DNA is approximately 6 x 106 smaller than the largest and dominant

EcoRI/

HsuIAfragmentof EBV(B95-8)DNA. EBV (W91)DNAalso differs from EBV

(B95-8) DNAbyanadditional7x 106to 8x106of DNA in thelongunique DNA

region (D. Given and E. Kieff, J. Virol. 28:524-542, 1978; N. Raab-Traub, R.

Pritchett, and E.

Kieff,

J. Virol. 27:388-398, 1978). These data suggest the possibilitythat thesmaller number of internal reiterations in EBV (W91) DNA may be a consequence of the additional unique DNA and a restriction in the overall size of EBV DNA.

Epstein-Barr virus (EBV) DNA is a linear, nicked, double-stranded molecule of 105 x

10'

(15). Thebuoyantdensity (11, 13, 15, 18, 24,25)

andmelting temperature (11, 15) of the DNA

are compatible with a guanine-plus-cytosine

content of57 to 58%. The DNA of the B95-8

isolate of EBV has beenaprototypeforstudies

of theorganizationof EBV DNA(for review,see

reference 10).Both endsof the DNA consist of

avariable number of directrepeatsof thesame

sequence of400to500basepairs (6, 7).The size

(8) and order (6) of theHsuI, Sall, and EcoRI

restriction endonuclease fragments of EBV

(B95-8) DNA areshown in Fig. 1. These data

and the analysis of partially denatured

mole-cules(4) indicatethat,with theexceptionof the

variability inthe number ofcopies ofthe

reit-erationateach endof theDNA,theremainder

ofthe DNAisorganizedin asinglearrangement.

The results of treatment of EBV DNA with

KpnI restrictionendonucleasearediscordant(8)

as KpnIproduces a 20 x 106 fragment in

sub-molar amounts, and the sum of the molecular

weightofallfragments,includingthesubmolar fragment, isapproximately 20 x

106

more than themolecularweight of EBV DNA.

Analysis of the fragments produced by

cleav-age of the DNA with BamI andBglII reveals

thepresenceofa2 x 106fragment inexcessive

amounts (8, 17). Complementary RNA made

fromthe 2x 106 BamIfragment, whichis

pres-entin a 10-fold molarexcess, hybridizestothe

HsuIAand EcoRIAfragments, indicatingthat

there areapproximately 10tandemreiterations

of the2 x10' sequence in theHsuIAand EcoRI

Afragments(17).Partof this reiteratedDNA is transcribed in Burkitt tumor tissue (3) and in

restringently infectedBurkitt tumorcellsgrown

315

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EcoRI IKhet I J

HsuI

SalI Hhet

NmH-EcoRI/Hsu I

A

A G G F K- B

a JG H E SI

G F

G E C F.H

E H C Dhet

C KL F D FGHhet

B Dhet

A a (H) (F) (C) (E) J (D) (G)(HIhet)

l _ I I I I

[image:2.504.78.408.54.143.2]

0 10 20 30 40 50 60 70 80 90 100 110

FIG. 1. EcoRI(6),SailI (6),HsuI(6),andEcoRI/HsuI restriction endonucleasecleavage sites in EB V (B95-8)DNA.Fragmentsaredesignated by capitalletters(6).Acapitalletter enclosedby parenthesesabove the EcoRI/HsuIfragmentsindicatestentativeassignment ofmaplocation based on correspondence between the sizes ofthefragments (Fig. 3)andthe sizes ofthe EcoRI and HsuIoverlap regions. The positions of the EcoRI/HsuIA,B, and Jfragmentsweredetermined byhybridization ofblotsofagarose gelscontaining

EcoRI/HsuIdigest ofEBV(B95-8)DNAtolabeled HsuI AorBorEcoRI Cfragment ofEBV(B95-8) DNA.

in culture (14). The objective of these

experi-ments was todeterminethe location of the 2 x

106reiteration within the EcoRI A andHsuI A

fragments. In thecourseof this work itbecame

apparentthat the discordantfindingwithKpnI isduetothepresence of a minorpopulationof

molecules which contain fewer copies of the

internal reiteration butare otherwise identical

tothe dominantpopulation.

MATERIALS AND METHODS Cellculture, viruspurification, and prepara-tion of viral DNA. Viral DNA was obtained by

sodiumdodecylsulfate-phenolextraction(12)of virus

purifiedfromtheextracellularfluid of B95-8 and W91 cell cultures. InitialculturesofB95-8and W91cells were obtained from G.Miller,Yale University,New

Haven, Conn.Theproceduresused tomaintain cul-turesforviruspurificationand thepurificationof virus havebeen describedpreviously (5).

Separation and determinationof the molecu-larweight ofrestrictionendonucleasefragments

of EBV. EBV DNA was incubated with atwo- to

fivefold excess ofKpnI (NewEnglandBiolabs,

Bev-erly, Mass.), EcoRI, HsuI (8) (or its isoschizomer,

HindIII), Sall, XbaI, BamI,orBglII(BRL, Bethesda, Md.)for 2.5 hat37°C (6). For doubledigestionwith

EcoRI,HsuI, BamI, orBglII,2to 4yig of the DNA wasincubated at37°Cwithafivefoldexcessof enzyme in asolutionconsistingof 50 mMNaCl,7mM

MgC92,

2mM

,8-mercaptoethanol,

and 20 mM Tris-hydrochlo-ride,pH 7.4.EcoRI, HsuI,SalI, XbaI,KpnI,EcoRI/

HsuI, BamI, andBglII DNA fragmentswere sepa-ratedbyelectrophoresisat40Cin 0.3 to 0.4%(wt/vol)

agarose gels incylinders (1 by 28 cm) or inslabs(0.5

by23by 27cm) (6, 8). BamI, BglII, BamI/EcoRI,

BamI/HsuI,BglII/HsuI,andBamI/BglIIfragments

were also separated in 0.8% (wt/vol) agarose gels.

DNAfragmentswere stainedwithethidium bromide andphotographed (type57film,PolaroidCorp.,Oak

Brook,Ill.) under UVillumination (6,8). DNA

frag-mentstobecleaved with asecondenzymeorto be labeled in vitro were cut from the gelunder direct visualization.Theagarosewasdissolved in 5 volumes of5 M sodium perchlorate at 450C, the DNA was separated by chromatography on hydroxylapatite

(HTP, Bio-RadLaboratories, Richmond,Calif.),and the ethidium bromidewasremovedbydialysis against

Dowex 50 resin in the H form (6). The molecular

weights of the EcoRI/HsuI fragments were deter-mined from a log linear plot of the electrophoretic

mobility of thefragments in a 0.3% (wt/vol) agarose slab gel relative tolambda DNA (BRL) (22), EcoRI

fragments oflambda DNA (22), HsuI fragments of EBV(B95-8)and (HR-1)DNAs (8), and EcoRI frag-mentsof EBV(B95-8)DNA (8) as reference DNAs in adjacent wells on the same slab. The molecular

weights of BamI or BglII fragmentswere similarly determined from the comparativeelectrophoretic mo-bility of thefragments in 0.3% (wt/vol) agarose gels with the EcoRIfragmentsof EBV(HR-1)DNA(8)as described above or in 0.8% (wt/vol) agarose slab gels relative to EcoRIfragmentsoflambda DNA (22). The

sizesof the BamI orBglIIfragments of theseparated

HsuI A orEcoRI Afragmentsand of theBamI/HsuI,

BamI/EcoRI, BglII/HsuI, BglII/EcoRI, or BamI/

BglIIfragmentsweredeterminedby electrophoresis

in 0.8 to 1% agarosegels relative to theEcoRI frag-ments of lambda DNA(22).Themolecular weight of the smallest EcoRI fragmentof lambda DNA is 2.1 x 108(22).Therefore, determinationof themolecular weightoffragmentssmaller than106from their

elec-trophoreticmobility relativetothe EcoRIfragments

oflambda DNA issubjecttoerror, even in 1% agarose gels.

Labeling ofDNA in vitro. EBV DNA or

frag-ments of EBV DNA were labeled in vitro by nick

translation (6, 9) using DNApolymerase Iof Esche-richiacoli (Boehringer MannheimCorp., New York) and a-3P-labeled dCTP (300 Ci/mmol; Amersham

Corp.,ArlingtonHeights,Ill.). The specific activity of thelabeled DNAwas0.5x l08 to1 x108cpm/,ug.

HybridizationoflabeledDNA toblots of frag-mentsof EBV DNA. Blots (20) ofseparated frag-ments of EBV DNA wereincubatedat680Cin 1mlof asolution consisting of 104 to 105 cpm of denatured, labeled EBV DNA, 1 mg ofdenatured calf thymus DNA,0.01 MEDTA, 1.25 M NaCl, and 0.05 M

Tris-hydrochloride, pH7.4.After18hthe filterwaswashed for4h in4xSSC (lx SSC is0.15MNaCl plus 0.015 Msodiumcitrate) at550Canddried at600Cfor1h. ThefilterwasthenexposedtoX-rayfilm(SB5,Kodak

Corp., Rochester, N.Y.), with an intensifying screen

(Cronex Lightning Plus, Du Pont Co., Wilmington,

Del.)at-70°C.

RESULTS

KpnIfragmentsofEBV(B95-8) DNA.The

orderofarrangement of theKpnIfragmentsin

J.

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EPSTEIN-BARR 317

EBV(B95-8) DNA wasdeterminedby

hybridi-zation of blots of

KpnI

fragmentsof EBV DNA to labeled EcoRI,

HsuI, SalI,

or BamI

frag-ments whose map positions have been

previ-ously determined (6). The results (Fig. 2)

indi-cate that each ofthe KpnI fragments of EBV DNA, withthe exception of the minor fragments slightly smaller than A and the B fragment, mapped to a specific and unique location. The B (previously C [8]) fragment was 2 M relative to

A EBV (B95-8)DNAKpn

fragment

linkage

Kpn profile: EcoRIfragrrents

A B F G H I

B-o

C-s; E

-OF - 69 B-10'5X2 -;C

84

26 M

6

__ 2I2

H-24

26

K 24

0H

Hsu fragments Sc

E-4

GK I G-if .,

net a*

.*

I

O

p

t

I~~~~

I~~~~~

Ca fragments

B C D A B C E

B- B

0K-F

:# ~~~~~N-

M-N

EcoRl/Hsul Bam

A v

t

A A f

B

Linkage ofKpnl fragmentsof EBV(B95-8) DNA

EBV(B95-8)DNA probe

IJ A G FK B

Atzt/>_\\-t---\r ErEcoRi fragment

GKi

DE:

KhetG A O B J C WMI)1B D E &_N$i)F her

AEC C' B

r--- F- -C--I-I -- Sc! fragment

0 10 20 30 4 5r 6c 7 40Cc0cOC

FIG. 2. Mappingof KpnIfragmentsofEBV(B95-8)DNAby hybridizationtolabeledfragmentsofknown mapposition(Fig.1). (A) Photographofanethidiumbromide-stained agarosegelofanelectrophoretically separatedKpnI digest ofEBV(B95-8) DNA is shown in the upperleft. Thesizes ofthefragments were determined from their electrophoretic mobility in 0.3% (wt/vol) and 0.4% (wt/vol) agarose gels (6, 8).

Radiofluorogramsofblotsof KpnIrestriction endonucleasefragmentsofEBV(B95-8)DNAwhich had been

hybridizedtotheseparated32P-labeledEcoRI,HsuI, SalI, EcoRI/HsuI,orBamIfragments ofEBV(B95-8)

DNA. (B) Summary of linkagedatafor KpnIfragments. Thearrowsdrawn indicate hybridization ofthe labeledEcoRI,HsuI,orSalIfragmenttoKpnIfragmentsonblots.The orderoftheKpnI0 andH,M andI,

andNandKfragmentswithin the map distances indicatedby parentheseshasnotbeendetermined.

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[image:3.504.93.409.128.587.2]

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318

otherfragments (8), could be resolved intotwo

discrete bands in 0.3%agarosegels, and mapped

to two distinct locations, 37 x 106 to48 x 106 and66 x 106to 77 x 106.The minorfragments

smaller thanAranged in size from 16 x 106to

27 x 106, hybridized to all labeled fragments

whichhybridized totheAfragment, and failed

tohybridizetootherlabeled fragmentsof EBV

DNA. These data suggest the possibility that

theminorfragments resulted from thepresence

ofa minorpopulation ofEBV DNA molecules

which differ from the majorpopulation only in

the length of the KpnI A fragment. In several

instances, a labeled fragmentsuch asEcoRI G

orK orHsuI B hybridized to asmall extent to

a KpnI fragment (E, K, and B, respectively)

which mapped by other dataat a distant site.

Thiswaslikelyto be a consequence of

contam-ination ofthelabeled fragmentwith an adjacent

fragment from the gel in which the fragments

were separated before labeling in vitro. Thus,

the labeled EcoRI G fragment hybridizes

pri-marilytoKpnI fragments B, C, and J,but also

toKpnIfragment E. The hybridization to KpnI

fragmentE is, however, likelyto be an artifact

of contamination ofthe EcoRI G fragmentwith

a smallamount of theEcoRI H fragment since

thelabeled EcoRIfragmentHhybridizes

exclu-sively to the KpnI fragment E. Similarly, the

; .og MoIecula We!gh' (daltc

I. .!.s

!

,J/

v

/

/'

/

/ ..

EcoRI fragment K is contaminated withasmall

amountof EcoRIhet, and the HsuI fragment B

iscontaminated with the HsuIfragment C.

EcoRI/HsuI A fragments of EBV (B95-8)

and(W91) DNA. TheKpnIenzyme is difficult

towork with because of itslability (6). Digestion

of EBV DNA withacombination of EcoRI and

HsuIrestriction endonucleasesproducesalarge

fragment, EcoRI/HsuI A,which containsmost

of the DNA of theKpnIAfragment (Fig. 1and

2).Thefragmentsproduced bydouble digestion

of EBV(B95-8)or(W91)DNA withboth EcoRI

and HsuI are shown in Fig. 3. The molecular

weight of theAfragmentof EBV(B95-8) DNA

is estimated from itselectrophoretic mobility in

0.3%agarose gelstobeapproximately28 x 106. Minorcomponentswhichtogether compriseless

than 10% of theAfragmentareevidentat26x

106, 24 x 106, 22 x 106, and 20 x 106 (Fig. 3A).

The major andminorcomponentsof the

EcoRI/

HsuIAfragmentwereseparated,labeled in

vi-tro,andhybridizedtoblotscontainingallof the

EcoRI/HsuIfragments.The labeledmajor

com-ponenthybridizedtothemajorand minor

com-ponents of the EcoRI/HsuI A fragments and

alsohybridized totheEcoRI/HsuI Jfragment

to a much lesser extent (Fig. 3B; see Fig. 8,

column5). The results obtained with the labeled

minor components were identical to those

ob-ns1 B

W91 8

I-4?z L

_m

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I

FIG. 3. (A) EBV (B95-8) DNAwasincubated with a combination of EcoRI and HsuI restriction endonu-cleases, subjectedtoelectrophoresis ina0.35%(wt/vol)agarose slab gel,stained with ethidium bromide, and photographed under UV illumination. The sizes of theEcoRI/HsuIfragments were determined from a log linearplotof the electrophoretic mobility of theEcoRI/HsuIfragments relativetoEcoRIorHsuI fragments

ofEBV(B95-8) DNAorHsuI fragments ofEBV(HR-I)DNA (8). The calibration curve shownabovewas

constructedusing HsuI (B through L)fragmentsof EBV (B95-8)DNA and intact lambda DNA. (B)EcoRII

HsuIfragmentsofEBV(W91orB95-8)DNAwereseparated by electrophoresisinadjacentwellsofa0.35% agarose slabgel.Photograph of the ethidium bromide-stained gel with the fragments of W91 (column 2) and B95-8(column 3) DNA. Autoradiograms of blots of theEcoRI/HsuI fragmentsof EBV (W91orB95-8) DNA whichwerehybridizedtothe32P-labeledEcoRI/HsuIAfragmentareshowntotheleft (column 1)and tothe right (column 4), respectively.

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taimedwith themajor component. As many as

10 minor components differing in molecular

weight byapproximately2x 106 could be

distin-guished influorogramsofblotsof EBV (B95-8)

DNA hybridized to thelabeledEcoRI/HsuI A

fragment (Fig. 3B). These data suggested the

possibility that the difference in sizes of the

EcoRI/HsuIA fragmentswasdueto variation

inthe number ofcopiesof the 2x106reiteration

(6, 17) and that there was homology between

theEcoRI/HsuIAfragmentand another region

of the EBV genome (EcoRI/HsuI J fragment;

seebelowandFig.8,column5).The size of the

EcoRI/Hsu I A fragment of EBV (W91) DNA

is 22 x 106. Minor components which together

constitutedapproximately10%of the total DNA were visible at20 x 106, 18 x 106, 16 x 106, 14

X 106, 12 x 106,and 10 X 106influorograms of

blots of EBV (W91) DNA hybridized to the

labeledEcoRI/HsuIAfragment (Fig.3B).

Mapping within theEcoRI/HsuI A

frag-ment. BamI and

BglfI

are known to cleave

within the tandem reiteration in the

EcoRI/

HsuIAfragment (6, 17). Therefore, aseriesof

experiments was undertaken using BamI and

BglIItoestablish the arrangement of sequences withinthe EcoRI/HsuI Afragment. One objec-tiveofthis approachwastodemonstrate directly

that the difference inthe sizesof themajorand

minor EcoRI/HsuI A fragments was due to a

difference in the number of copies of the

reiter-ated DNA and not to adifferenceinthe sizesof

theuniquesequence componentsof theEcoRI/

HsuI Afragment. A loglinear plot of the size

and electrophoretic mobility of the BamI and

BglIl fragments of EBV (B95-8) DNA in 0.8%

(wt/vol) agarose

gels

is shown in Fig. 4. The molecular weight ofthe reiterated DNA

frag-ment,BamI V,previously termedBamI S (17)

or

BglIl

R, was determined to be 1.85 x

10"

relative to the molecularweightofEcoRI

frag-mentsof lambdaDNA.

TheBglIIAfragmentelectrophoresedin 0.8%

gelsasabroad band and in 0.3%gelsas

multiple

discrete bands differing by approximately 3 x

105 in molecular weight, indicating that the

BglIIA

fragment

extendstoincludeoneend of

the DNA(6, 7).Threeapproacheswereusedto

map the BamI and

BglII

cut sites within the

EcoRI/HsuIAfragment. Inthefirstapproach,

the BamI and BglII fragments which contain

sequences homologous to the EcoRI/HsuI A

fragmentwereidentifiedbyhybridizationof the

labeledEcoRI/HsuI AfragmentDNA toblots

ofseparatedBamI andBglIIfragmentsof EBV

DNA(Fig. 5A andB).The secondapproachwas

totentativelyidentifythe BamI andBglII

frag-ments which contain EcoRI or HsuI sites

by

hybridization ofthelabeledEcoRI/HsuIA

frag-Log Moleculor Weight (doilons)

6 65 7

C.D05.5¢.

m E49

_ 45

L 34

N.l2 5:c5

P2z20

: -x rv

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FIG. 4. Log linearplotofsize andelectrophoretic

mobility ofBamI(photographofethidium

bromide-stainedgel on the left) and BglII (photograph of

ethidiumbromide-stainedgelontheright) fragments

ofEBV(B95-8)DNA in 0.8% agarosegelsrelativeto

EcoRIfragmentsoflambda DNA(22).Themolecular weights of the tuo largest fragments (BamI and

BglII)weredetermined in 0.4% agarosegelsrelative to HsuI (MP) DNA (8). The broken line indicates regionsoftheloglinearplotdrawnby extrapolation.

TheestimateofthemolecularweightsofBamIand BglII fragmentsfromtheseregions istherefore sub-jecttogreater error.

ment to blots of EBV DNA doubly cut with

BglIIandHsuI(Fig. 5A), with BamI and EcoRI (Fig. 5B),orwithBamI and HsuI (Fig. 5B). The

thirdapproachwas to recuttheisolated EcoRI

AorHsuIAfragmentwithBamIorBglIIand

todetermine the size of the resultantfragments

(Fig. 5C). The results are summarized in Fig. 6

andwereasfollows.

(i) The EcoRI/HsuI A fragment hybridizes primarily tothe

Bglll

A, C, or D and R

frag-ments(Fig. 5A,column2)andtotheBamI Bor

C, G, V, and Xfragments (Fig. 5B, column 2). (ii)TheBglIIAfragmentextendstotheend

ofthe DNA and is 10 x 106 to 13 x 106. This

fragment must therefore contain the short,

unique region. TheBglIICDfragment mustlie

totheright of the reiterated BglIIRfragment

and include the first HsuI cut site, since it is

reduced in size from5.8 x 106 to 4.0X 106when

theDNAiscutwith both HsuI and

Bglfl

(Fig. 5A, column 4) and contains sequences homolo-goustotheEcoRI/HsuIN Bfragment (Fig. 5A,

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el

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4.

3!v

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.il,

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FIG. 5. Radiofluorogramsofblotsof BglIIorBglII/HsuIfragments (A)orBamI,BamI/HsuI,orBamIl

EcoRIfragments (B) ofEBV(B95-8)DNA orofblotsofisolated EcoRI AorHsuI Afragmentrecutwith BamI orBglII (C),which had beenhybridizedtolabeled EBV(B95-8)DNA(designatedT.P.)ortolabeled

restriction enzymefragments ofEBV(B95-8) DNA (designatedbythe letters at the topofeach blot). The molecularsizeofeachfragmentwasdeterminedbycomparison ofelectrophoretic mobilitytoEcoRIfragments ofphagelambda DNAcoelectrophoresedinadjacentwells.

Eco RI IKhlt II A S.I ICV V VV V V V VIVV G IXI

Ugh A R R R R R R R R RR C,0

HsuI A |

o .0 20 30 40

FIG. 6. Mapofthe BamI sitesfrom4.2x 106to 33 x10'and oftheBglIIcut sites from 0 to 34.3 x10'in EBV(B95-8)DNA.

column 3). Thearrangement of theBglII

frag-mentsand of the EcoRI and HsuI

cleavage

sites

(6) from 0 to 36 x 106 is shown in Fig. 6. The

arrangement was confirmed by the results of

recleavage of the purified HsuIA or EcoRI A fragment with BglII (Fig. 5C). As expected,

cleavage of the HsuI A fragment with BglII

resulted in three fragments: the BglII A

frag-ment, the reiteratedBglII R fragment, and a

third fragment, BglII CD, whichis reducedin

size because it extendsbeyondtheHsuI

cleavage

site (Fig. 5C, column 1, andFig. 6).

Cleavage

of

the isolated EcoRI A fragment withBglII

re-sultedinfull-sizeBglIIR andCDfragmentsand

aBglII Afragment reducedin size because of

cleavare

by EcoRIatthe EcoRI siteslocatedat

1x 10,3.8x

106,

and6x

106

from theleft end

oftheBglIIAfragment(Fig.5C,column2,and

Fig. 6).

(iii) Similarly, the BamI BC fragment

con-tains sequences homologous to the EcoRI J

fragment (Fig. 5B, column 3) and is reduced in

sizefrom5.8 x 106to 4.1 x 106when the DNA

is cut with EcoRI andBamI (Fig. 5B and Fig.

5C, column 4), indicating that this fragment

mustlietotheleft of thereiteration.The BamI

XfragmentmustcrosstheHsuIcutsite since it

is reduced from1 x 106 (Fig. 5B,columns2and

4)to0.4x 106 (Fig. 5B,column 5,and Fig. 5C,

column3) when the DNA iscleavedwith BamI

and HsuI.Furthermore,asexpected,thelabeled

EcoRI/HsuIBfragment hybridizestothe BamI

X fragment (data not shown). The BamI G

fragment must extend from the reiteration to the BamI Xfragment since it ishomologousto

the EcoRI/HsuI A fragment and is not

de-creased in sizeby cleavagewith HsuI (Fig. 5B,

column 5) or EcoRI (Fig. 5B, column 4). The

mapof the BamIsites from4.2x 106to33x 106

is shown inFig.6.Theseresultswereconfirmed

by analysis ofthefragments producedwhenthe

purified HsuI A orEcoRI Afragment was cut

with BamI and identifiedonblots with the

la-beledEcoRI/HsuIAfragment (Fig. 5C, columns

3and4). Asexpected, the BamIBC, G, andV

fragmentswere cleaved from theHsuI A

frag-mentandwereidenticalin sizetothefragments

cutfromintact EBV(B95-8)DNA, whereasthe

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X fragment was reduced to approximately 4 x

105, confirming that the size of the component

of the X fragment to the left of the first HsuI

cut site isapproximately 4 x 105.Furthermore,

the BamI BC fragment is smaller when cleaved

from the EcoRI Afragment since the EcoRI site

at6 x 106isin the BamI BC fragment (Fig. 5C,

column 4, and Fig. 6).

(iv) An unexpected finding when blots of

BamIfragmentswerehybridizedtothe labeled

BglIIRorBamI Vfragmentwasthat the

radi-ofluorogramappearedidenticaltothat shown in

Fig. 5B, column 2. All four BamI fragments,

including X, hybridized tothe labeled Bgll R

orBamI Vfragment.These data indicatedthat

the BamI Xfragmentwhich isseparated from

the tandem reiterations by the length of the

BamI G fragment,contains sequences

homolo-gous to BglIl R and BamI V fragments. The

sequences homologoustoBamI V and

BglII

R

fragments mustlie to the left of the HsuI cut

site in the BamI X fragment shown in Fig. 6,

since the labeled EcoRI/HsuI Afragment

hy-bridized to the0.4x 106fragmentof X andnot

to the0.6 x 106fragmentof BamI X (Fig. 5B,

column 5). Thehomologybetween theinternal

reiteration and the BamI Xfragmentisfurther

defined bystudies with labeled components of

thereiterated DNA below.

Toconfirm that themajorand minor

compo-nents of the EcoRI/HsuI A fragment do not

differin thecontentofuniquesequences,a mi-nor band was separated from a

gel,

incubated withBamI,andre-electrophoresed. The sizesof thefragmentsproduced by

cleavage

of themajor

and minorEcoRI/HsuIAfragmentcomponents

withBamIwereidentical (datanotshown).

Arrangement of BamI and BglH sites

within the internal tandem reiterations.

The sizes of the components of BglII A and

BamIBCtotherightof the EcoRI J-A siteare

4.5x

10"

(Fig.5C, column2) and4.1 x 106 (Fig.

5C, column 4), respectively. The sizes of the

componentsofBglII CD and BamI G and Xto

the left of the HsuI A-B siteare 4 x 106 (Fig.

5A, column 4) and4.1 x 106and0.4x 106 (Fig.

5B, column 5),respectively. As summarized in

Fig. 6, theseobservations,that thedistance from

the EcoRI J-Acutsitetothe BgIIIsite within

the first intemal reiteration is 4 x i05 longer

than the distance to the first BamI site and that

the distance from the BglII site in the last

reiterationtotheHsuIA-Bsiteis 5x 105shorter

thanthecorrespondingBamIdistance,both

sug-gest thatthe BamI site is 4 x 105to5 x 105to

the leftof theBglII sitein the reiteration. To

confirm the distance between the BamI and

BglIIsites within the reiteratedDNA,thesizes of the BamI/BglII double-digest fragments

weredetermined by electrophoresis in a 1%

aga-rose gel with EcoRI fragments of lambda DNA

in adjacent wells. As expected, the reiterated

DNA is cleaved into two components (Fig. 7,

column 2). The electrophoretic mobility of the

large component of the reiterated DNA, BglII

RI,

is slightly faster than the smallest EcoRI fragment of lambda, indicating a size of 1.4 x 106. The rapid mobility of the smaller

compo-nent,BglIIRK, placesit in the nonlinear part of

thegelbut suggests a size of approximately 4 x

105to 5 x i05.

The data, which are summarized in Fig. 6, indicate that theBamI site in the internal

reit-eration is approximately 4 x 105 to the left of

theBglIIsite and therefore in the leftward 1.4

x

10'

of the reiterated DNA. If the BamI site is

near the left end of the reiteration, BamI BC

wouldcontainonly the left end of the reiterated

sequence and BamI G would contain most of

one complete reiteration, i.e., all of the DNA

betweentheBamIandBglII sites,BglIIR and

most ofthe rest of the reiteration, BglII

RI.

If

there are no reiterations within the reiterated

1.85 x 106sequence, the extent ofhybridization

oflabeledBglII

R,

toBamI BC as opposed to

BamI G should be proportional to the amount

A orobe T. BgiR8: R BgIRIBgIRs

BrmnC58 U * i

8rnG

36)-Bgi Rl

9)-BaiRI (i40) I*

SamTX ii0)- 0

Sg;R, .45)-

I

I6

B p Bg RIBgi

A- I I I L- I i

S S

S

R-I

9

Diot BiOrn 1/g egSv

FIG. 7. Radiofluorogramsofblotsoffragmentsof

EBV(B95-8)DNAproduced by cleavagewitha com-binationofBamI andBglII(BamI/BglII) (A)orby cleavage withBglII (B)whichwerehybridizedtothe

32P-labeledEBV(B95-8)DNA (T.P., column1)orto

labeledBglIIRfragmentortothe large (BglRd or

small(Bgl-RJfragmentobtained whenBglIIR iscut into two fragments byBamI. Thefragments which hybridizeareidentified byname,whereknown,and by sizeontheleft.

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322 GIVEN AND KIEFF

ofBglII

Ri

inBamI BCasopposedtoBamIG

and to the distance from the left end of the

reiteratedsequence tothe BamI siteasopposed

tothe distance from theBglllsitetotheright

end of the reiteration.

Furthermore, Bglll

R.

should hybridizeto BglII A andBamI G, but

not toBamI BC(Fig. 6).To estimate the relative

amountofBglII

RI

inBamIBCversusBamI G

and to further check the data shown inFig. 6,

BglIIR,, BglIIR., andBglIIR were isolated

from gels, labeled in vitro, and hybridized to

blots of BglII and BamI/BglIH fragments of

EBV DNA. The resultsare as follows. (i)

La-beled BglII R1 hybridizes extensively to the

BamI BC

fragment (Fig.

7A, column 2) and

hardlyatall totheBamI Gfragment (Fig. 7A, column 2) from which BglII R. had been

re-movedby digestion withBglII and BamI. An

overlong exposureof the blot (Fig. 7A,column

3)demonstrated that thehyrbridizationtoBamI

G was barely above background. These data

indicate that the BamI site must be near the

right endof the left 1.40x106 component of the

reiterated sequence. (ii) Asexpected, BgIII

R.

hybridized extensivelytoBglIIA (Fig. 7B,

col-umn3) andhardlyatalltoBamI BC (Fig. 7A,

column 5). (iii) A surprising finding was that BglII

R,

hybridizedslightlytoBglII

R.

(Fig.7A, column4) and thatBglII

R.

hybridizedtoBglII

R,

(Fig. 7A,column5).These data suggest that

there ispartialhomologybetween the short and

longcomponentsof the reiteration suchaswould

occuriftherewere adirectorinvertedrepeatof

partof the short segment within thelong

seg-ment. (iv)Thepartialcopy of thereiteration in

the left 4 x 105 component of BamI X has

extensivehomologyto

BglII

R. (Fig. 7A,column 5)andonlytracehomologytoBglII R, (Fig. 7A, column 4). (v) In long exposure,

radiofluoro-grams of blots hybridized to labeled BglII

R,

(Fig. 7A, column 4, and Fig. 7B, column 2),

additionalfragments,BglIIIJ andBglII L,with homology to the labeled BglII

R,

preparation

could beseen.BglII IJ isalso apparent inlong

exposures ofBglII blots hybridized to labeled

BglII R, BamI V, or EcoRI/HsuI Afragment

(see Fig. 8) and probably contains DNA with

homology to BglII

RI. BglII

L, however, was

not apparent in long exposures ofBglII blots

hybridized to labeled BglII R, BamI V, or

EcoRI/HsuIAandisprobablyidentifiedinFig. 7B, column2, because of contamination of the

labeled

BglII

RH

preparationwithanother

frag-ment of similarsize.

Homology between the internal

reitera-tion and DNA in the long, unique region.

Long-exposure radiofluorograms of blots of

EcoRI,HsuI, EcoRI/HsuI,orBglII fragments

which had been hybridized to labeled EcoRI/

HsuIA, BamI V, or BgIII R fragments revealed

homology between the internal reiteration and

the HsuI F (Fig. 8, column 2),EcoRIC (Fig. 8,

column 3),EcoRI/HsuI J (Fig. 8, column 5), and

BglII IJ (Fig. 8, column 6) fragments. The BglII IJfragmentis part of EcoRI C (Fig. 8, column

7).Theseresultsindicate that within the region

ofoverlapofEcoRI C and HsuI F, i.e., between

94 x

106

and 97 x 106 from the left end of EBV

DNA (Fig. 1), there exists a sequence with

ho-mology to theinternal reiteration. The extent of

hybridization ofthe labeledEcoRI/HsuI A or

BamI V fragment to EcoRI C,HsuIF,BglII IJ,

orEcoRI/HsuI Jfragmentwasdetermined from

scans ofautoradiograms tobe less than 3% of

the extent ofhybridizationtoEcoRIA,HsuI A,

BglIIR, orEcoRI/HsuIAfragment,indicating

thatthere is less than 1 complete copy of the

reiteration in the EcoRI C, HsuI F, BglII IJ,

and EcoRI/HsuI J fragments. The BglII IJ

fragment has homology both to the large, BglII RH (Fig. 7B,colunm 2), andsmall, BglIIRK (Fig. 7B, column 3), component of the reiteration.

DISCUSSION

The results of the mapping of the BamI and BglIIrestriction enzymecleavagesites near the internal reiteration whichjoins the short (10 x

106)andlong (87x 106)unique regions of EBV

(B95-8) DNA are summarized in Fig. 6 and 9. The existence of short and long unique DNA

sequences joined by reiterated DNA and

boundedatthe termini by direct repeats is

sim-FIG. 8. Radiofluorograms of blots of the Hsu,

EcoRI,EcoRI/HsuI,orBglIIfr-agmentofEBVDNA whichwashybridizedtothe labeledEcoRI/HsuI A

fr-agment (E/HA),labeled EcoRIfr-agmentC(EcoRI

C),orlabeled EBV DNA(T,P).

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EPSTEIN-BARR 323

EBV(B95-8) DNA

TR IR IR IR TR

0 10 20 30 40 50 60 70 s0 90 100 110xlol

TR Teminol repetition IR Internol repOeition L Loo lorgelyuniquo region

S Shortlorgelyuniquo region Siteofinsertionofodditionol DNA

inW91,JijoyeondHR-I isolates

FIG. 9. Diagram oforganization ofEBV(B95-8)

DNA.

ilartothe molecularorganization of herpes

sim-plex virus (19, 23), bovine mammillitis virus (2),

and pseudorabies virus (21) DNAs. However,

EBVDNAdiffersfrom these otherherpesviral

DNAs in that there aretandem directinternal

reiterations in EBV DNA and there is no

ho-mology between the internal reiteration and

either terminus. Moreover, the short and long

uniqueregionsof EBV DNA donotinvert

rela-tive to each other during DNA replication as

has been observed with herpes simplex (23),

bovinemammillitis(2),andpseudorabies viruses

(21).

The minor KpnI (8) and EcoRI/HsuI

frag-mentsarise throughthepresencein each EBV

(B95-8) DNA preparation of molecules with a

smaller number of internal reiterations in the DNA.Thus,althoughthemajorityofmolecules

of EBV (B95-8) DNA contain 10 to 11 tandem

reiterations,somemoleculeshave 9,8,7,6, 5, or

4 tandem reiterations. We do not asyet know

whether such variantmoleculesarealways

gen-eratedduring thecourse of viral DNA

replica-tion. The findings that cells infected with the

B95-8 isolate and the cell line from which the

B95-8viruswasisolated contained circular DNA

molecules 6 x 106 smaller than EBV (B95-8)

DNA (1) support the hypothesis that shorter

DNAmolecules containing fewer copies of the

internal reiteration maybe infectious andmay

yieldprogenywithalargerorsmaller number of

internal tandem reiterations. Furthermore, the

finding that theEcoRI/HsuIAfragment of EBV

(W91) and (AG876) (N. Raab-Traub and E.

Kieff, manuscript in preparation) DNAs is

smaller than the EcoRI/HsuI A fragment of

EBV (B95-8) DNA suggeststhat these Burkitt

tumor isolates ofEBV, which have 7 x 106 of

additional DNAinsertednearthe distal end of

the long, unique region (6, 16), may have a

smallernumber ofinternaltandem

reiterations,

possibly as a consequence of the additional

uniqueDNA.

Previous estimates of the size of the HsuI A

andEcoRIAfragmentswerebasedonthe

elec-trophoretic mobilityof thesefragmentsin 0.4%

agarose gels relative to the HsuI fragments of

herpes simplex virus (MPstrain) DNA, the

larg-estfragment of which is 27.5 x

106,

orthe HsuI

fragments of T5blDNA, thelargestfragment of

whichis34 x106(8). Thelog linear relationship

between molecular weight and electrophoretic

mobility in 0.4% agarose gels is parabolic for

fragments inexcessof23 x 106 to 25 x 106(8).

The HsuI A and EcoRIAfragmentsareslightly

larger than thelargest standards, and previous

estimates of the sizes of these fragments are

therefore subject to error. Three lines of

evi-dence indicate that the size of theEcoRI/HsuI

Afragment is28 x 106 and, therefore, that the

truesizes ofthe HsuIAandEcoRIAfragments

are 34 x 106 and42 x 106daltons, respectively.

First, theelectrophoreticmobility of theEcoRI/

HsuI A fragment relative to the HsuI B

frag-mentof EBV (B95-8) DNA (20 x 106 [8]), the

HsuI Bfragmentof EBV(HR-1)DNA(27x106

[8]), and intact lambda DNA (32 x 106 [22]) is

compatible withasize of28 x 106. Second, six

minorbands, eachdifferingby approximately2

x 10 in molecular weight, are clearly

distin-guishable above the EcoRI/HsuI B fragment in

blots of theEcoRI/HsuI fragments whichwere

hybridized to the labeled EcoRI/HsuI A

frag-ment.The molecularweight of theEcoRI/HsuI

B fragment is14 x 106.The size ofthe

EcoRII

HsuIAfragment,which islargerthan thelargest

minorEcoRI/HsuI Afragment, musttherefore

be28x 106. Third, the molecular weight of the

unique-sequencecomponentoftheEcoRI/HsuI Afragmentis8.5x106.The size of the reiterated

component, BamI VorBglIIRfragment,is1.85

x

101;

to 1.9 x 106. TheBamI Vfragment com-ponent is present in at least 10-fold excess of

other BamIfragments(17).

The revisedestimates of the sizes of the HsuI

A and EcoRI Afragments suggested by these

databringthesum of themolecularweightsof

the EcoRI or HsuI fragmentsof EBV (B95-8)

DNAto 116x 106, which isapproximately 10%

largerthan estimates basedon ameasurement

of thelengthofEBV DNA (1,7, 15). Although

the latter estimates of the molecularweight of

EBV (B95-8) DNAarebasedultimatelyonthe

length of 4X174 DNA, a molecule of known

molecular weight, there are several possible

sources of error in the determination of the

molecular weight of EBV DNA from its length,

the most significant of which is the possibility that thehigherguanine-plus-cytosinecontentof EBV DNA results in a higher mass per unit

lengththan thatof4X174 replicative-formIIor

PM2 DNA.

The significance of theinternaltandem

reit-eration, of the homology between the

compo-nents of the reiteration which is likely to be a consequence of a reiteration within the

reitera-VOL. 31,1979

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tion,and of thehomologousDNAsequences in

theBamIX andHsulFfragmentsof thelong,

unique region is uncertain. From the relative

hybridization of labeled BamI V orBglII R to

theHsuIFand HsuI Afragments,thelengthof

the regionofhomology between the reiterated

DNA and HsuI F fragmentis estimated to be

less than 5x 105,whereas the component of the

BamI Xfragment homologous to the internal reiterationiscontained in the4 x 105 region of

BamIX fragment to the left of the HsuI A-B

site. TheHsuI F sequences homologous to the

internalreiteration havemorehomology tothe BglII

RI

componentthantotheBglII

R.

com-ponent,whereas thereverseistruefor BamI X.

Itis not known whether the DNA in BamI X

and HsuIF,which ishomologoustotheinternal

reiteration, is a continuous sequence or is

sepa-ratedby regionsofnonhomologyorwhetherthe

homologoussequenceswithin the reiterationor

in BamI X and HsuI F are direct or inverted

repeats.Regionsofhomologyin the DNA could

play aroleinDNAor RNA

synthesis

or could

beaconsequenceof gene

duplication.

Pertinent

tothe latter is theobservation that the internal

reiteration encodesstable

polyadenylated

RNA

inBurkitttumortissueand themostabundant

RNA in

restringently

infected Burkitt tumor

cellsgrowninculture

(3,

14).

ACKNOWLEDGMENTS

NancyRaab-Traub,WalterKing,and MarkHeller collab-oratedon someof theseexperiments. MaryHawke andNancy

Treptowprovidedexcellentassistance.

This research was supported by Public Health Service grantsCA 19264-03andCA 17281-04 from the National Cancer Institute and grant VC 113D from the American Cancer Society. D.G. isa predoctoraltrainee supported byPublic Health Service grant HD 07009-03.

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