Isolation and characterization of recombinant DNA clones of avian retroviruses: size heterogeneity and instability of the direct repeat.

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0022-538X/80/03-1026/08$02.00/0

Isolation and

Characterization of Recombinant DNA Clones of

Avian

Retroviruses:

Size

Heterogeneity and Instability of the

Direct Repeat

GRACEJU,LAWRENCE BOONE,ANDA. M.SKALKA*

DepartmentofCellBiology, RocheInstitute of Molecular Biology, Nutley, New Jersey 07110

Unintegrated proviral DNA of Schmidt-Ruppin B Rous sarcoma virus was

cloned in the bacteriophage A vector Charon 21A. A total of 12 independent

recombinantXSRBtdclones which were derived from the

transformation-defec-tive componentinthe viral preparationwereanalyzed with restriction

endonu-cleases and molecular hybridization techniques. Three classes of clones were

observed.Type Iclonescontained a5.0-megadaltoninsert of viral DNA, type II

clones contained phage withtwosizeclassesof inserts (5.0 and 5.2megadaltons),

andonetype IIIclone containedonly a5.2-megadaltoninsert. Thesmallerinsert

present in type II clones appeared to be derived by deletion of one copy of a

directlyrepeatedsequencewhichwaspresent in the larger insert. Mapping data

indicated that thedeletion includes all orpart of the terminalrepeat found in

lineardouble-stranded proviralDNA.Similarresults were obtained fromXRAV2

recombinant clones derived from Rous-associatedvirus type 2.Analysis of DNA

fromtype IIandtype IIIclones ofASRBtdandXRAV2revealed limited

hetero-geneityinthe size ofthedirectrepeat.

Replication of the retroviruses involves the

production of proviral DNA intermediates

be-fore integration into the host cell genome. At

least three types of intermediates have been

identifiedincells afterexogenousinfection:

lin-eardouble-stranded DNAandtwo sizes of

co-valently closed circular DNA (10). For the avian retroviruses, the linear double-stranded DNA

has been shown to contain a direct repeat of

approximately300basepairs (bp) (0.2

megadal-ton

[Md])

atitstermini.Thisduplication is also

present in the largerof the two circular

inter-mediates (16, 25), which may be formed from

the linearDNAbyintramolecularjoiningatthe

termini. Restrictionmappingdata indicatethat

thesmaller circular specieshasonlyonecopyof

the terminal sequence (25). The circular forms

arefoundexclusivelyinthenucleus(26),and,as

withbacteriophage lambda(9), acircular

inter-mediate of the provirus is thought to be the substrate for integrationinto the host genome (10, 13).Integrated provirushas also been found tocontain terminal redundancies (17, 24),

sug-gestingthattherepeatedsequencesareinvolved

intheintegrationreaction. Thelimitedquantity

of viral DNA found in infected cellshas made

analysis of these intermediates difficult.

How-ever,larger amountsofpurifiedDNAcan now

be obtainedbyusing recombinant DNA

meth-odology.

Unintegrated circular proviral DNA from

chicken embryo fibroblasts infected with

Schmidt-Ruppin B Rous sarcoma virus

(SR-RSV-B)wasisolated and cloned in the

bacterio-phageX vectorCharon21A(Ch21A)by usingan

enzymeknowntocuttheproviralDNA atonly

one site. Three types ofXSRBtd recombinant

DNA clones derived from the

transformation-defective (td) componentof the viruswere

iso-lated.OnetypecontainedaviralDNAinsert of

5.0Md,asecond containedalargerviralinsert

of 5.2 Md, and athird contained recombinant

molecules with either of thesetwosize classes of

inserts. Experimentsusing cloneswith twosize

classes indicated that the smaller insert could

arise by deletion of one copy of the repeated

sequence from thelarger insert during growth

within procaryotic host cells. Similar results

wereobtained with recombinant clonesprepared

fromunintegrated proviralDNAof

Rous-asso-ciated virustype2(RAV-2). Furthermore,in six

independent clones examined, the size of the

repeated sequences was found to be

heteroge-neous. The implications of these results for

mechanisms ofviralreplicationin vivoare

dis-cussed.

MATERIALS AND METHODS

Celis

and viruses. Chickenembryofibroblasts

(gs-chf; SPAFAS, Norwich, Conn.) wereprepared and propagated as described previously (20). The SR-RSV-B usedin theseexperimentswasarecombinant

betweenNY68,atemperature-sensitivemutantof

SR-RSV-A (subgroup A), and RAV-2 (subgroup B). It 1026

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contained the temperature-sensitive mutation of NY68 in the transformation gene (src) and the sub-group B specificity of RAV-2 (18). The virus was obtainedas acloned stock from H.Hanafusa, Rocke-feller University. The A vector Ch21A has been de-scribedpreviously (4). Escherichia coli K-12 strains

employedweretheEK1 hosts ED8767(recArk-mk') (22) and C600 (recA-rk-mk), which was obtained fromR.Davis, Stanford University, and the EK2 host DP50supF(recA+).

DNAisolation. To prepareproviral DNA,25petri

dishes(150mm2) of chicken embryofibroblastswere infectedathighmultiplicity (multiplicity of infection, >1 focus-forming unit per cell). After48h, infected cellswerelysed with detergent, and high-molecular-weightnuclear DNAwaspelleted by the method of Hirt(15). The Hirtsupernatant fractionwastreated withproteinase K (50,Ig/ml) at37°C for 2.5 h, ex-tracted with phenol, and precipitated with ethanoL Theethanolprecipitatewas nextdissolved,extracted with chloroform-isoamyl alcohol (100:1), and again concentratedbyethanolprecipitation.The DNAwas then dissolved in TEN buffer (0.01 M Tris,pH 7.4,

0.001MEDTA,0.01MNaCl),treated withRNase A

(50,ug/ml)at37°Cfor1h, extracted with phenol and

chloroform-isoamyl alcohol,and then ethanol precip-itated. The precipitate wasdissolved and covalently closed circular viral DNAwasseparatedfrom relaxed circles and linear moleculesby isopycnic centrifuga-tion inacesium chloride-propidium diiodide density gradient (27).

Recombinant DNAtechniques. All cloning ex-perimentswereconducted under the P2-EK2 or P3-EK1 conditionsspecified by the December 1978 re-visedNational Institutes of Health guidelines.

A portion of the covalently closed proviral DNA

preparation of SR-RSV-B was digested with Sail, ligatedtoSalI-digested A Ch21A with T4 ligase (21),

encapsulatedby in vitro packaging techniques (3), and plated on the recA- strain ED8767. Approximately 36,000plaqueswerescreenedfor viral DNA inserts by using the method of Benton and Davis (2) anda3p labeled RAV-2 viral RNA probe. Positiveplaques were isolated andpurified twice before amplification and extraction ofDNAfor restriction endonuclease anal-ysis.

Nuclease digestion. Restriction endonucleases

AluI, SalI,HindIII,XhoI,XbaI,KpnI,andPvuIwere purchased from New England Biolabs, Beverly, Mass. EcoRIwaspurchased from Miles Laboratories,

Elk-hart, Ind.Digestionswereperformedin 25 or 50

pl

of theappropriate buffer.

Gel electrophoresis, DNA transfer, and hy-bridization. Agarose gel electrophoresis and DNA transferwereperformedby the method ofSouthern

(28),aspreviously described (20). Hybridization pro-cedures havebeendescribed previously (20).For mo-lecular weight determinations, parallel lanes of the

gelscontainedHindIIIdigests of"C-labeledA DNA.

To determine the size of small DNA fragments

(<1,000 bp), digested DNA samples were analyzed by

electrophoresis invertical 6%polyacrylamide (acryl-amide/bisacrylamide ratio, 40:1) gels in TBE buffer (0.89 M Tris, pH 8.3,0.89 M boric acid, 0.01 M EDTA). Fragments of46X174 DNA digested withAluI were usedassizemarkers.

RESULTS

Molecular cloning of SR-RSV-B viral

DNA. Unintegrated covalently closed circular

proviralDNAwaspurifiedfrom chickenembryo fibroblasts infectedwithSR-RSV-Basdescribed above. The proviral DNA was digested with

restriction endonuclease Sall,whichwasfound tocleave the circular moleculesatasingle

loca-tion(Fig. 1). The cleaved DNAwasthenligated

totheAvectorCh21Aandpackagedintophage particles in vitro. Recombinant clones were

screened forvirus-specific inserts by using

32P-labeled RAV-2 viral RNA. SR-RSVand RAV-2

are approximately 80 to 90% homologous, as

determined by hybridization of SR-RSV 70S

RNA to complementary DNA prepared from

RAV-2 (14).

Cells infectedwith aviansarcomaviruses

con-tain nondefectiveand td DNAmolecules,which

have sizes of approximately 6.2 and 5 Md,

re-spectively (25). The td molecules are deletion mutantsderivedfromnondefective moleculesby loss of all or part of the viral src gene, which

codes for cellular transformation.Intheproviral DNApreparation used for cloning, td molecules represented approximately 50% of thecovalently closed circular molecules (data notshown). In

this report,recombinant clonescontaining only td moleculesarediscussed. Thecloning vehicle

Ch21A, in combination with the in vitro pack-aging procedure, selects for inserts witha

maxi-mum size of approximately 5.3 Md (8.2

kilo-bases) (lla). Because of its size limit, theuseof

Ch21Aallowedustoobtain clones withinserts

ofonly singletd molecules(Fig. 1and2). Restriction endonuclease analysis of XCh21A SR-RSV-B td (XSRBtd)

recombi-nant clones. A total of 12 independently de-rived clones containing viral DNA sequences

were isolated and plaque purified. DNA from

eachASRBtdclonewasdigested with Sall and

analyzed byagarose gel electrophoresis.

Diges-tion withSall generated thetwoarmsofCh21A

amI X Hindm XhoI SalI

I -I I

ll c

M

gag pol ."

t

EcoRI EcORI Kpn I

JL Xbol Xbal

ECKO

RI

| CO RL

PVUI PVUI

FIG. 1. Restrictionendonuclease map of ASRBtd

recombinant clones. Cleavage sites ofseven

restric-tionendonucleases inthelarger (5.2-Md) viral insert

of ASRBtdclonesareindicated. Theinsert isshown inorientationrwithadirectrepeat(shaded region). The relative orderof the viralgenesin the viral RNA

genome is: (5')gag, pol, env, C (3'). Ajagged line

indicatesthe siteofjoining of the repeatedsequences. VOL. 33,1980

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C. 3 4 5 2 3 4 -, 6 7 8

EsI~A

de ,_

9 10 11 6 7 8 9 (

' #i#

. . _

v_mi,,

*4

B

FIG. 2. SalI andHindIII digestionpatterns of ASRBtdclones. DNA from phage of XSRBtdcloneswas

extracted, digested, analyzed by electrophoresisin 0.7%agarose gels,and transferredtonitrocellulose filters

aspreviouslydescribed(20).Bandscontaining viralsequences weredetected after hybridization with

32P-labeled RAV-2 RNA (specific activity, -2x 107 to 4 x 107 Cerenkov cpm/lpg),prepared, and utilizedas

describedpreviously (20).Theleft panelsareethidiumbromide visualizations ofdigestedDNA from XSRBtd

clones,and therightpanelsareautoradiograms ofthegels after transfer andhybridization.(A)SalI digestion patterns. Lane1,marker"C-labeled DNAcleavedwithHindIII; lane2, Ch21A;lane3,XSRBtd-1; lane4, XSRBtd-2;lane5,ASRBtd-7.(B) HindIIIdigestionpatterns.Lane6,Ch21A; lane7,ASRBtd-1(orientation 1);

lane 8, XSRBtd-5 (orientation r); lane9, XSRBtd-2 (1); lane 10, XSRBtd-7(r); lane11,"C-labeled ADNA

cleaved withHindIII.

and the insertfragment(s) ineachrecombinant clone. Three types of clones could be distin-guished by differencesin theirdigestionpatterns

(Fig. 2A and Table 1). Digestion oftypeIclones (8of12) generatedaninsertfragment of 5.0 Md.

Type II clones (3 of 12) containedtwodifferent sizes of insert DNA(5.0 and5.2 Md). Onetype

IIIclone (XSRBtd-7) containedan insert of 5.2

Md.

Several XSRBtd cloneswere further charac-terizedby digestionwithanumber ofrestriction endonucleases. Figure 1 isa compositemap of

the larger viral insert in Ch21A, showing the

cleavage sites forsevenrestrictionenzymes. Al-though we observed differences in thenumber

and location ofsome ofthe HindIII and XbaI

sites, ourmapisingeneralagreement withthe

restriction maps reported for other avian sar-comavirus strains(16, 25).

With each of the clones, digestion with

HindIII,whichcleavesclosetothecenterofthe insertDNA(Fig.1)andoncewithin theAvector

leftarm,producedtwolarge fragments contain-ing virus-specificsequences.The sizes of the two

large fragments differed slightlyin the various

clones(Fig. 2B),asexpectediftheproviral DNA

TABLE 1. Recombinant DNAclonesof ASRBtd

Type XSRBtd Size of insert Orientation'

Tye clone (Md)a retain

I 1 5.0 1

3 5.0 l

4 5.0 l

5 5.0 r

6 5.0 r

8 5.0 r

11 5.0 r

12 5.0 r

II 2 5.0and5.2 1

9 5.0and5.2 r

10 5.0and5.2 r

III 7 5.2 r

aDeterminedby

SailI

digestion.

"DeterminedbyHindIIIdigestion.

was inserted in either one ofthe two possible

orientations. The orientationssuggested bythe

HindIIIdigestion patternswereverifiedby

anal-ysiswith other restriction endonucleases. Inserts

incorporatedsuch that the aviansarcomavirus

geneticmapis colinear withAlategenes

(tran-.1

Mr x 10-6

15.5-6

2-

43-

29-A

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scribed tothe right) are designated as being in

the r orientation (Table 1). Inserts in the r

orientation yielded virus-specific fragments of 8.65 and 8.0 Md when digested with HindIII,

and inserts in the left (1) orientation produced

fragments of 9.2 and 7.45 Md after digestion with

HindIII (Fig.2B).ResultsofHindIIIdigestions

ofindividual clones also indicatedthat none of

the clones containedmultipletandem inserts.

Smallerinserts intype II clones arise by

deletionoflarger inserts. Because of the size

limitations of the Ch21A vector and the data

from restrictionanalysis, it was clear that each

recombinantphagechromosome contained only

oneproviral genome. However, two sizes of

in-serts weredetectedinDNAfromtype II clones. It seemed possible, therefore, that the 5.0-Md

insertsin type II clones might arise by one of

the two following mechanisms: (i) the original

plaque containedtwo ormore different

recom-binantphage particles;or(ii) deletionof part of

thelarger insertgeneratedthesmaller one

dur-ing passage and amplification of the original

plaque-purifiedclone.

Todistinguish between thesetwopossibilities,

one type II clone (XSRBtd-2) was replated at

low density, and phage from 16 independent

plaqueswereisolatedandamplified. SalI

restric-tion endonuclease analysis of DNA from the

subclones revealed that eightwere identical to

the original parental clone and also contained

phage with the same twosizes ofinserts. The

relative proportions of the larger and smaller

inserts were variable among these clones, as

shown by the four representative examples in

Fig. 3, lanes3 through6. The other eight

sub-clonescontainedonly the smaller 5.0-Md insert,

as shown by the four clones in Fig. 3, lanes 7

through10. None of the 16subclonescontained

onlythe 5.2-Md insert.

If the typeII cloneshad containedamixture

ofparticles, then thesubsequent plaque

purifi-cation should have produced clones that

con-tained either the larger or the smaller insert.

Since no subclones contained only the larger

insert and 50% of the subclonescontained two

sizes ofinserts, it seemed most likely that the

smallerinsert arosefromthelargeroneby

dele-tion.Thus, weconclude thateach type IIclone

probably originated from a recombinant

mole-cule which contained a single unique insertof

5.2 Md. A subsequent specific deletion of

ap-proximately0.2Mdduringpassage ofeachclone

generated themixture ofmolecules. The

varia-tion inthe relative proportion ofthe twosizes of

inserts in subclones ofXSRBtd-2 (Fig. 3)

indi-catedthatthedeletioneventprobably occurred

atadifferent time for eachsubclone. Although

the deletionof a0.2-Mdfragmentoccurred

fre-2 3 4 5 6 7 8 9 10

Mr X106

15.5-

6.2-

4.3-

2.9-FIG. 3. SailI digestion patterns of subclones of

ASRBtd-2. The type II clone ASRBtd-2wasplaque purifiedonstrain ED8767.Phage fromindependent plaques were isolated and amplified. DNAs from

these subcloneswereanalyzed bySalIdigestionand electrophoresis in 0.7% agarose gels. This figure shows results with the parent cloneASRBtd-2 and eight representative subclones.Fragments were vis-ualized after ethidium bromide staining. Lane 1, marker XDNA cleaved withHindIII; lane 2, parent

cloneXSRBtd-2; lane 3,XSRBtd-2.3;lane 4,

XSRBtd-2.6; lane 5,XSRBtd-2.7;lane6,ASRBtd-2.11; lane 7,

XSRBtd-2.4; lane 8, XSRBtd-2.14; lane 9, XSRBtd-2.15;lane 10, XSRBtd-2.16.

quently, no additional deletions were detected

inany of theXSRBtd clonesorXSRBtd-2

sub-clones.

All recombinant phage particles were

origi-nallygrown andamplified in ED8767, a

recA-host. To determine whether the Rec function

couldacceleratedeletionformation,typeI,type

II, and type III clones were also grown in a

secondrecA- mutant (derivedfrom C600) and

arecA+ (DP50supF;')host. Nosignificant

differ-enceswere observedintherestrictionpatterns

ofthe DNAs isolatedin any ofthese

prepara-tions (datanotshown).

Size heterogeneity of the direct repeat. The size difference between the larger and

smaller inserts (0.2Md)in type IIclones

corre-spondstothe size ofthedirectrepeat present in

vivo in the larger circular DNA species and absent in the smaller one (16, 25). The direct repeat contains a centrallylocated EcoRI site

(Fig. 1) (16, 25). Therefore, digestion with this

enzymeshouldproduceafragmentequalin size

to the repeat unit. To determine whether the

putative deletion from thelargerinsert in type

II clones involved loss of one copy of the

re-peatedsequence, DNAsfromtypeI,typeII,and

type IIIclonesweredigestedwith EcoRI

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tion endonucleaseandanalyzed by

electropho-resis in 6%polyacrylamide gels (Fig.4).

DNAs from type II and type III clones did

produce anEcoRI fragment of approximately

300 bp (0.2Md). DNAs fromtype I clonesand

from subclones of ASRBtd-2 (type II) which

containedonly the 5.0-Md insert didnotcontain

this EcoRI fragment. Similar results were

ob-tained whenPvuIwasused, which also cleaves

withinthe directrepeat(datanotshown).

Analysis of three ARAV2clonesprepared from

RAV-2proviral DNA (Boone andSkalka,

man-uscript in preparation) yielded analogous data.

Two type II clones, XRAV2-2 and XRAV2-12

(Fig. 4, lanes 9 and 10), produced small

frag-ments when digested with EcoRI. No small

EcoRI fragment was detected when a type I

clone, XRAV2-1(Fig.4,lane4),wasdigested.

Unexpectedly, the size of the small EcoRI

fragment in XSRBtd and XRAV2 clones was

heterogeneous. Digestion of six differenttypeII

and type III clones with EcoRI yielded frag-ments that ranged in size from 200 to 340 bp

(Table 2). This sizeheterogeneitywasprobably

notthe result ofinstability duringpassageof the

recombinant phage particles, because each of

the eight subclones of XSRBtd-2 which

con-tained thelarger insert produceda340-bpEcoRI

fragment identical in size tothat oftheparent

clone. Three discrete sizes of thisfragmentwere

observed in the six clones examined: 200, 250,

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and 340 bp.Therefore,the sizeheterogeneityof the smallEcoRI fragmentappears to be limited.

DISCUSSION

Usingunintegrated proviral DNAof

SR-RSV-B and the A vector Ch21A, we generated,

iso-lated, and characterized 12recombinant clones

ofASRBtd.Analysisofthe12clonesby

restric-tion endonuclease digestion revealed three

classes of recombinantclones, all derived from

the tdcomponentof theviral DNA. DNA from

type Iclonescontainedaviralinsert of5.0Md, type II clones contained phage DNAwithtwo

sizesof inserts (5.0 and5.2Md), and thetype III

clone containedphage DNA with only the larger

insert (5.2Md).Arestrictionendonucleasemap

of the SR-RSV-B td insert showed extensive

homology with the maps previously described

forproviral DNA ofthePrague (subgroupsA,

B, andC) and B77 strains of RSV (16,25).

Itwasof interesttodeterminetherelationship

between the 5.0-and 5.2-Mdinserts, aswellas

the origin of the three classes of clones.For the

analysis of the XSRBtd clones,twoassumptions

were made. First, each recombinant molecule

was assumed tocontain onlya single insertat

thetime ofconception and throughoutpassage.

Thepossibility oftandemincorporation oftwo

ormoreviral insertswasunlikely,because of the

size limit imposed by the use of Ch21A and

because of the results of restriction analysis.

E TYPE

BASE PAIRS

--358

-337 -276

!258,

254

\247 \204 146

FIG. 4. Size ofrepeat unit in XSRBtd and XRAV2 clones. After digestion with EcoRI, DNAs from recombinantcloneswereanalyzed byelectrophoresisin6%polyacrylamide gels.Restrictionfragmentswere visualized by ethidium bromide staining. All XSRBtd clones andXRAV2-1 werederived by cleavage of proviral DNA withSalIbeforeinsertionintoCh21A. ARAV2-2andXRAV2-12werederived by cleavageof proviral DNA with HindIIIbeforeinsertion intoCh21A. Lanes1and12, marker cX174 DNA cleaved with

AluI;lane2,XSRBtd-1;lane3,ASRBtd-2.4; lane4,XRAV2-1;lane5,ASRBtd-2;lane6,XSRBtd-2.2;lane7, ASRBtd-9;lane8,ASRBtd-10;lane9,XRAV2-2;lane10,ARAV2-12;lane11, XSRBtd-7.

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TABLE 2. Sizeheterogeneity oftherepeated

sequence

Size ofsmall Clone Type

~~~(<1,000-bp)

Clone Type EcoRI fragment

(bp)

XSRBtd-1 I NPa

XSRBtd-5 I NP

ASRBtd-8 I NP

XSRBtd-2 II 340

ASRBtd-9 II 250

ASRBtd-10 II 340

XSRBtd-7 III 200

XSRBtd-2.4b I NP

XSRBtd-2.10 I NP

XSRBtd-2.2 II 340

ASRBtd-2.3 II 340

XSRBtd-2.6 II 340

XSRBtd-2.7 II 340

XSRBtd-2.8 II 340

XSRBtd-2.9 II 340

XSRBtd-2.11 II 340

XSRBtd-2.13 II 340

ARAV2-1 I NP

XRAV2-2 II 340

XRAV2-12 II 250

aNP, Not present.

bClones

XSRBtd-2.2

to

XSRBtd-2.13

aresubclones ofXSRBtd-2.

Second, weassumed thatSR-RSV-Bis

homol-ogous to the other RSV strains in its EcoRI

digestion pattern. Therefore, thesmall

(<1,000-bp) fragment thatappeared after EcoRI diges-tionpresumably containedsequencesfrom the

directrepeat,andthe size of thisfragment

cor-respondedtothe size of therepeatunitin each

clone. Analogous results from PvuI digestion

supported this assumption.

Fromthe datain Fig.2andthe assumptions

made,weconclude thateachXSRBtdclone

orig-inated from arecombinant molecule that

con-tained a single insert of either 5.0 or 5.2 Md.

During plaque purification and amplification,

the type II clones with 5.2-Md inserts lost a

portion of the viral DNA and generated some

molecules with a 5.0-Md insert. This deletion doesnot seem to occurmore rapidly in recA+ hosts. Itisnotknownwhether the

480

general recombinationfunction(RED), which is carried

ontheCh21Avector, plays a critical role in this

event.

The deletion apparently involved the loss of

all or part of one of the redundant sequences.

The DNA molecules with 5.2-Md inserts yielded

a small fragment upon digestion with EcoRI.

Thisfragment is the size of the terminal

redun-dancy of linear proviral DNA (-300 bp or0.2

Md).TypeIcloneswith 5.0-Md insertsdidnot

contain this EcoRIfragment. Although deletion

of this small repeat apparently occurred at a

highfrequency (in 50% of subclones of

XSRBtd-2), the 5.0-Md insertappeared tobe stable. No

otherdeletionsweredetected in either the Xor

the viral portion of the hybrid molecule. The

high frequency of deletion of the repeated

se-quence in the larger insert suggests that the

smaller circular DNA found in vivo may be

generated by the loss of this redundantsequence

from the larger circular species. Whether this

deletion is ofbiologicalsignificancein the viral

replicative cycle is unclear. Preliminary data

from DNA transfection experiments indicate

that the virus-specific inserts from both type I

andtypeIIclones havebiologicalactivity.

Stud-iesareunderwaytodetermine theefficiencyof

transfection andintegrationof viral DNA inserts

withand without therepeatedsequence.

The nature ofnucleotide sequences and the

mechanisms involved in the integration of

ret-rovirusesare notunderstood.Inprocaryotic

sys-tems,integrationoftranslocatable elements by

illegitimate recombination is associated with

di-rect orinvertedrepeatedDNAsequences.

Inser-tion elements and translocatable antibiotic

re-sistance elements contain inverted

complemen-tary repeats at their termini (6). In addition,

insertionandtranslocatable antibiotic resistance

elements and the bacteriophage Mu generate

smallduplicationsofhostsequencesatthe site

of insertion(1, 7, 12, 19).

Recently, translocatable elements in

eucar-yotic organismshave been described.These

ele-ments,which have been isolated fromyeast(8)

and fromDrosophila(23, 29),arestrikingly

sim-ilartothe retroviruses inseveral features. Like

the retroviruses, these elements contain direct

repeatsof0.25 to 0.5 kilobasesattheirtermini,

code foranabundantpolyadenylic

acid-contain-ing RNA, andcanintegrateatmanysitesin the

host genome. The presence of short repeated DNA sequences at the termini of procaryotic andeucaryotictranslocatable elementssuggests

that the direct repeats found in the proviral

DNAmay be essential forintegration and

pos-sibly translocation.

Deletionsarealsofrequently associated with

procaryotic translocatable elements.

Translocat-ableantibioticresistanceandinsertionelements

andbacteriophageMu generate

recA-independ-ent deletions ofnucleotide sequences (6). One

endpointof the observeddeletionsoccursat the

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repeated terminus of the translocatable element.

However, cloned insertsofrepeated

nontranslo-catable eucaryotic DNA sequences have also

been observed to undergo deletion and other

rearrangementswhen propagated inrecA-

pro-caryotic hosts (5, 11, 30). Thus, it is not clear

that the instability of the direct repeat in the

ASRBtdclones isrelatedtoitspresumed role in

recombination andintegration into the host

ge-nome.

Analysis oftherepeatfragmentgenerated by

EcoRI digestion of DNA from XSRBtd and

ARAV2 clones revealed someheterogeneity in

the size of therepeatunit. Of six clones

exam-ined, each hadaredundancy ofacharacteristic

size, ranging from200to 340bp (Table2). This

size difference didnot seem to be dueto

insta-bility duringpassage,since subclones ofa

partic-ular clone (XSRBtd-2)hadaredundancy of the

same size as the parentclone. Because we

ob-served the same sizes of the redundant

se-quencesinXRAV2 clones and inXSRBtdclones,

it isunlikely that the size heterogeneity ofthe

direct repeat ispeculiar only to SR-RSV-B td

molecules.

Atleast three discrete sizes of therepeatunit

seem to bepresent inproviral DNA. The type

IIIcloneXSRBtd-7 hadalargerinsert thatwas

apparently stable and did not generate

mole-cules with 5.0-Md inserts. This clone also had

thesmallest EcoRIfragment(200bp). It is

pos-sible that a repeat unit of minimal size may

result inamorestable redundantsequence.

The size heterogeneity of the small EcoRI

fragment is of interest. It implies that the

se-quences ofthe terminal repeat in the proviral

DNA are variable. Thus, the termination

event(s) whichgeneratestheend of the terminal

redundancy and/or the recombination event

whichresults in covalent closuretoforn circular

molecules maynot besite specific. Analysisof

the small EcoRI fragment from several clones

should allow us to determine whether any

se-quencesare common atthe recombination

junc-tion.

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