Physical mapping of a large-plaque mutation of adenovirus type 2.

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0022-538X/79/11-0623/06$02.00/0

Physical Mapping of

a

Large-Plaque Mutation of

Adenovirus

Type

2

G.CHINNADURAI,* SHANBAGAM CHINNADURAI, ANDJOHN BRUSCA

Institute forMolecularVirology,Saint LouisUniversity School of Medicine, St.Louis, Missouri63110

Received forpublication2April 1979

We have developed a simple method based on cotransfection of overlapping

DNArestriction fragments for construction of recombinants of adenovirustype

2(Ad2) and Ad5. When Ad2 DNA digested with restriction endonuclease EcoRI

was cotransfected with Ad5 DNA digested with SalI, recombination occurred

between Ad2 EcoRI-A(map position 0to59) and Ad5SalI-A (map position 45

to 100). Analysis of the recombinant DNAs by digestion with EcoRI orBamHI

restriction endonucleases indicatedthat,asexpected, recombination had occurred

in overlapping sequences (map position 45 to 59) between the Ad2 EcoRI-A

fragment and the Ad5 Sail-A fragment. By using this method, several

recombi-nantswereconstructed betweenalarge-plaque (Ip)mutantofAd2 and wild-type

Ad5. Cleavage of the recombinant genomes with restriction endonucleases BamHI, EcoRI,andHindIIIrevealed that theIpmutationis located withinthe left 41% of Ad2genome.

The 31 recognized human adenovirus sero-types form five groups (A to E) based upon genomicDNAhomologiesinmolecular hybrid-ization experiments (M. Green, J. K. Mackey, W. S. M.

Wold,

and P. Rigden, Virology, in press).TheDNAhomologies ofserotypeswithin eachgroup range from 48 to69% (groupA) to 99 to100% (groupC).Becauseofthisgroup-specific DNA homology, recombination between sero-types might be expectedto occur during infec-tions.Takemori (17) first demonstrated genetic recombination between cytocidal (cyt) mutants of adenovirustype 12 (Adl2) (group A). Subse-quently, Williams and co-workers (19) demon-strated recombinationamong temperature-sen-sitive (ts) mutants ofAd5 (group

C)

and con-structedageneticmapoftheAd5genomebased upon recombination

frequencies. Although

the genomes of Ad2 and Ad5 are

similar,

minor differencesin base sequences existbecausesome restriction endonucleases generate different fragments from Ad2 and Ad5DNA. These dif-ferences in

cleavage

patterns have been ex-ploitedtomapthecross-overpointsbetween ts mutants of Ad5andAd2+

ND,

(9, 14). Inthese studies, ts+ recombinants were isolated from cells coinfected with ts mutants of both Ad2+ ND1 and Ad5. In each case, the recombinant had Ad5 sequences at the site of theAd2+

ND,

tslesion and

Ad2+

ND,

sequencesatthe site of the Ad5 tslesion, thus explainingthe ts+ phe-notype.Similarmappingstudies have been done

withtsmutantsof Ad2 and Ad5(10).

Inthe present communication, wedescribe a simple method to construct recombinants be-tween Ad2andAd5 withwild-type DNA restric-tion fragments with overlapping sequence ho-mology. By using this method, we have con-structed a number of recombinants between a large-plaque (Ip) mutant ofAd2 and wild-type Ad5 and localized the Ip mutation on the Ad2 genome.Ourmethodfor construction of recom-binants between Ad2 and Ad5 will be valuable instudying aspectsofadenovirusrecombination aswell asin

experimnents

involving genetic ma-nipulations ofthe viral genome.

MATERIALS AND METHODS

Cells and viruses. Human cell lines KB and293

weregrownasdescribedpreviously (2).The293cells

are alineof humanembryokidneycells transformed

by transfection with sheared Ad5 DNA (5). Initial

stocks of Ad2 and Ad5wereobtained from M. Green.

The viruswaspropagatedin KBcells grown inSpinner

cultures. Viruswaspurifiedbythe methodof Green

and Pina (7), and the DNAwasextractedessentially

asdescribed elsewhere (8) with minormodifications.

Transfection.DNAtransfectionswerecarriedout on293cellsbythe"calcium-phosphate-precipitation"

method(6)asdescribedby Chinnaduraietal.(2).The 293cellswereused fortransfection because the

infec-tivity of Ad2DNA is about 50- to 100-foldhigheron

293cellsthan on KB cells (2, 5).Inmany cases, the

cellmonolayersweretreatedwith 20%Me2SO (2, 16).

Isolation ofplaquemorphologymutantof Ad2.

Ad2virus wasmutagenizedwith 1Mhydroxylamine

in phosphate-buffered saline for 16 h. Mutagenized

virus wasdialyzed againstphosphate-buffered saline

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andplaque assayedonKBcellsat33°C (2).Four

well-separated large plaqueswereisolated andreplaqued.

Theseisolates have beendesignated Ipl through Ip4.

Isolation andscreening of Ad2-Ad5

recombi-nants.Individualplaquesfrom 293 dishestransfected with Ad2 and Ad5 DNA fragments were aspirated with Pasteur pipettesinto 1.0 ml ofgrowthmedium

containing2% calfserum.Stocks of eachplaqueisolate

were prepared byinoculating 0.2 ml of each plaque suspension intoa60-mm dishcontainingabout 2x10' cells of 293 or KB cells. After complete cytopathic

effectwasobserved,cellsfrom each dishwerecollected

alongwiththegrowthmedium, sonicated,clarifiedby

low-speed centrifugation,and stored at-70°C.

To prepare32P-labeledviralDNA, about 106cells (KBor293) in 35-mm disheswereinfected with0.05 to0.1 ml of each virus stock. The infectedcellswere

labeled withcarrier-freeH332PO4 (250 ,Ci/ml)from20

to 36 h after infection. Viral DNA was selectively extractedbyamodificationof the method of Hirt(11).

Infected cells were lyzed with 0.8 ml ofa solution

containing0.6% sodium dodecyl sulfate,10 mM

Tris-hydrochloride (pH7.4),and 10 mM EDTA and treated

with500,ugofpronaseat37°Cfor1 h.

High-molecular-weightcellular DNAwasprecipitatedwith 0.2 ml of5

M NaClat4°C overnight.Virus DNAwasseparated

fromcellular DNAby centrifugationat15,000xgfor 20 min. The supernatant containing thevirus DNA wasextracted twice withphenolanddialyzed against

10mMTris-hydrochloride (pH 7.9) containing1mM

EDTA.Large quantities of virus DNAwasalso

pre-pared from virus purified by banding in two

subse-quentCsClequilibriumdensity gradients (7).

DigestionofviralDNAwithrestriction

endo-nucleases and analysis by agarose gel

electro-phoresis. VirusDNA was digested with restriction

endonuclease EcoRI(MilesLaboratories) in100 mM

Tris-hydrochloride (pH 7.9)-0.05 M KCl-10 mM

MgCl2-0.1 mM EDTA.Digestion ofvirus DNAwith

Sall (New England Biolabs) was carried out in 150

mMNaCl,7mM Tris-hydrochloride (pH 7.4),7mM

MgCl2, and 200,ugof bovine serum albumin perml.

Digestion with BamHI (Bethesda Research

Labora-tories) wascarriedoutin20 mM Tris-hydrochloride

(pH 7.4),7 mMMgCl2,and7mM,8-mercaptoethanol.

Digestion with HindIII (New England Biolabs) was

carriedoutin 60 mMNaCl,7mMTris-hydrochloride

(pH 7.9), and 7 mM MgCl2. The enzyme reactions

wereterminatedwith20mMEDTA.

Analyticalslabgelelectrophoresiswascarriedout

in 1or 1.4%agarose gelsorin2.2% acrylamide-0.7%

agarosecompositegels (1.5 by20by40mm).Thegels

werestained with 0.5 Mgof ethidiumbromide perml

(15) and photographed.When 32P-labeled DNAwas

used, gelswere air driedand autoradiographedwith

Kodak DF-85X-rayfilm.

RESULTS

Infectivity of restriction fragments of Ad2 and Ad5 with overlapping sequence

homology. Digestion of DNA digested with EcoRI or Sall virtually abolished transfection

infectivity in 293cells (Table 1). The low level of infectivity seen with DNA digested with

EcoRI may be due to the low levels of undi-gestedDNA. Ad2 DNA is cleaved by EcoRIfive timestoyieldsixfragments (13; Fig. 1). Ad2or

Ad5 DNA is cleaved by Sall three timestoyield four fragments (3, 4). We wished to determine whether cotransfection of cells with large

over-lapping DNA restriction fragments (EcoRI-A andSall-A)wouldresult in invivo recombina-tion between these fragments resulting in the production ofinfectious virus. As seen in Table 1,whenAd2DNAwasdigested with EcoRI and cotransfected with Ad2 or Ad5 DNA digested withSalI, considerableinfectivitycould be seen (3 to 9 PFU/,ug of genome equivalent). It is noteworthy that cotransfection of Ad2 DNA

di-gested with EcoRI and Sall gave consistently

more plaques (5 to 9

PFU/j,g)

compared with Ad5 DNA digested with SalI (3to 6 PFU/,ug). To check whether the infectivity is dueto true

genetic recombination between the restriction fragments, 15plaques were selected at random from dishes (inexperiments 3 and 4) transfected with Ad2 and Ad5 DNAfragments and multi-plied, and the DNA was analyzed by digestion with restriction endonucleases BamHI and EcoRI. Ad2 DNA is cleavedby BamHI at map positions 30, 42, and 59,making fourfragments, whereasAd5DNA is cleaved only once at map position 59.0 (12; Fig. 1). Therefore, this enzyme is suitable for discriminating the Ad2 andAd5 genome on the left half of the recombinant DNA. The righthalf of therecombinantDNA, on the otherhand, couldbe easily identified as

TABLE 1. Infectivity ofrestrictionfragments ofAd2

and Ad5DNAson 293cellsa

Expt

~~~~~~~~~~No.

of

Enxopt

DNA

(,tg/dish)

plaques per

dish

1 UndigestedAd2 (0.4) 137,117

Ad2, EcoRIdigested (4.0) 1,0 Ad2,SalIdigested (4.0) 0,0 Ad2, EcoRI digested+ (2.0+2.0) 12,13

Ad2, SalIdigested

2 Ad2, EcoRIdigested (4.0) 0,1 Ad2,SalI digested (4.0) 0, 0

Ad2,EcoRIdigested+ (4.0+4.0) 34,20

Ad2, SalIdigested

3 Ad2, EcoRIdigested (3.0) 0,0

Ad5, Sail digested (2.5) 0,0

Ad2, EcoRIdigested+ (3.0 +2.5) 12,8

Ad5,SalIdigested

4 Ad2, EcoRIdigested (2.5) 0,0 Ad5, Sail digested (3.0) 0,0

Ad2, EcoRIdigested+ (2.5+3.0) 15,12 Ad5, SalIdigested

Transfection of the DNA was carried outby theCa2` precipitation method (4) followed by an Me2SO boost as

describedelsewhere(2).

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625

0 10 20 30 40 50 60 70 80 90 100

A B F D E C

!~~~~~~~~~~~~~~~~

i E(

A C B

B D C A

B D C A

: B,

A B

F C B I J E A HL D G K

H

E C H D A B F I

G

*coRI

ialI A

3amHI

iindm B

C

FIG. 1. Mapsof restriction endonucleasecleavage

sites in Ad2 and Ad5 DNA. Thecleavagemapsfor

BamHI,EcoRI,andHindIII have beenpublishedby

Sambrooketal.(14).The mapsfor SailI cleavagesites

in Ad2and Ad5 DNA have beenpublishedbyChow

etal.(3) and Frost and Williams(4),

respectively.

Ad2or Ad5 bydigestion withEcoRI. This en-zymecleaves Ad2DNAfive timesand Ad5DNA twotimes (12-14). All the EcoRI cleavage sites arelocatedontheright half of thegenome.

Analysis of the DNA from 15 recombinants after cleavage withEcoRI andBamHI revealed that all the recombinants had identical struc-tures,

i.e.,

EcoRI

generated

three

fragments

sim-ilartoAd5 parentalDNAandBamHIgenerated four fragments similar to Ad2 parental DNA. The representative

pattern

ofone of these re-combinants is given in Fig. 2. These results indicate that the left half of the recombinant DNA isderived from Ad2 and the right halfis derivedfromAd5;recombinationapparently oc-curredsomewhere withinposition42.0and 58.5 between the Ad2 EcoRI-A fragment and Ad5 Sal-A

fragment.

Physical

mapping

ofanIpmutantof Ad2. Wehaveisolatedmutants ofAd2by mutageni-zation with

hydroxylamine

whichproduce large clear

plaques

onKB cells in7to8

days compared

with the wildtypewhichproduces small diffused plaques in 10 to 12 days. The

Ip

mutants de-scribedin the presentcommunicationaresimilar inphenotypetothecytmutantsofAd12 isolated by Takemorietal. (18).The

Ip

mutantsofAd2 exhibit the

large-plaque

phenotype at 33°C as wellasat

37°C.

However, the differencebetween the wild type and the mutant is greatly pro-nounced at33°C (Fig. 3).

Our DNA fragment transfection method for construction of recombinants isparticularly suit-able for construction of recombinants between

the Ad2

Ip

mutantandthe Ad5 wild type and

formappingthesite of the

Ip

lesion. This exper-iment is notpractical with mixedinfections of virions or intact DNAs because recombinants cannotbedistinguishedfrom thelargeexcessof

D E

F

C

2 3 2 3

FIG. 2. Gel electrophoresis of Ad2, Ad5, and a

recombinant DNAafter cleavage with restriction

en-donucleases EcoRI andBamHI.Electrophoresiswas

carriedoutin 1% agarosegelsasdescribed elsewhere

(2). Lane 1,Ad2; lane2, Ad5; and lane 3,

recombi-nant.

parentalplaquesthat would be produced.Since theinfectivityoftheparentalDNAis

abolished

bydigestionwith restrictionendonucleasesand only the recombinants can yield plaques, we haveconstructed severalrecombinants between an

Ip

mutant

(Ip3)

and the Ad5 wild type.These recombinantswere used to

physically

map the

Ip

mutation

by

the

approach

described

by

Grod-zickeretal. (9).

From twoexperimentsinwhich Ad2

Ip3

DNA

digested

with EcoRI and Ad5

wild-type

DNA digestedwith

SailI

werecotransfected, 10 well-separated

plaques

were isolated and multiplied and the DNA was analyzed by digestion with BamHI and EcoRI. All 10 isolates were found to be true recombinants and to exhibit the lp phenotype. Two types of recombinants were seen.

Eight

isolates(type

I)

hadBamHI

cleavage

patterns

identical to Ad2 and EcoRI cleavage

patterns

identical toAd5 (Fig. 4). Twoisolates (type II), after cleavage with BamHI, yielded onefragmentsimilar in sizetotheAd5BorAd2 Afragment and twootherfragmentssimilar in size toAd2 B

fragment (Fig.

4, right side, lane 4). Cleavageof DNA fromtype II recombinants with EcoRIyieldedthreefragmentsidentical to Ad5 EcoRI fragments (Fig. 4a, lane 4). These results indicate that all the recombinants might

a EcoR I

Ad2 Ad5 Ad2 Ad5 Ad2 Ad5 Ad2 Ad5

bBamH I

A A

B

B C ,.

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626 CHINNADURAI, CHINNADURAI, AND BRUSCA

FIG. 3. PlaquemorphologyofAd2 wildtype and Ad2lp3mutant. The wild type and themutantviruses

wereplaqueassayedonKB cellsat33°C (2)and stained withneutral red. The disheswerephotographed

with Polaroidfilmtype55 onday15.(A)Ad2wild type.(B)Ad2lp3.

-.., et ,.

.. wo0

rtf

'F1

i

-s *b B

;H( SO.A Cr -.

...-C

"I

45

FIG. 4. Autoradiogramof Ad2, Ad5,Ad2lp3,and recombinant DNAsafter cleavage with the restriction

endonucleases BamHI andEcoRL DNAfragmentswereresolvedin 1%agarosegelsandautoradiographed

asdescribedinthetext.Lane1, Ad2 wild type; lane 2, Ad2lp3; lane3, typeIrecombinantof Ad5 andAd2

lp3;lane 4, typeIIrecombinantofAd5 and Ad2

lp3;

lane5,Ad5 wild type.

have beengenerated byrecombination between crossover for type I recombinants may be lo-EcoRI-A of Ad2Ip3DNAandSalI-AorSalI-A cated within mappositions42to59

(Fig.

1)since

to -C (see below) ofAd5 DNA. The points of the DNAof these recombinants contain the Ad2

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LARGE-PLAQUE MUTANT OF Ad2 627

BamHI

cleavage

sites locatedatmap

positions

30 and 42.On theotherhand, sites ofcrossover fortype II recombinants may belocated within mapposition30and 42because the Ad2BamHI cleavage site at mapposition42(Fig. 1)isabsent inthe recombinant DNA, but thecleavagesite atmapposition30ispresent.Thesimplest mode bywhich these recombinantsmay havearisenis by asingle recombinationeventbetween EcoRI-Aof Ad2 Ip3 DNAandeithersmallportions of Ad5 DNAconsisting of Sall-Ato-C; these latter molecules may have been produced by incom-plete digestion with Sall or may represent a subpopulation that has lost the SailI cleavage siteatposition46.

Cleavage

oftype II recombi-nant DNAwithSallyielded the characteristic Ad2 orAd5pattern, i.e.,allfourfragmentswere produced (datanotshown), indicatingthat the formerinterpretationmay be correct.

All 10 recombinants selectedwere ofIp phe-notype indicating that

Ip

mutation of Ad2 is located anywhere between map positions 0 to 42. To further narrow down the locationof the Ipmutation, type II recombinant DNAwas di-gested with restriction endonuclease HindIII (Fig. 5) andcomparedwith Ad2 andAd5DNA fragmentsgeneratedbyHindIII. Withintheleft half ofthegenome,HindIII cleaves Ad2but not Ad5DNA atposition41 (Fig. 1). The recombi-nant genomehad identicalHindIII cleavage pat-tern asAd5 wildtype, i.e.,Ad2 Jfragment was notproduced

indicating

that the sequencesright ofmap position 41 are Ad5 and the sequences onthe leftareAd2.Therefore, the Ipmutation islocatedwithin theleft 41%of Ad2genome.

DISCUSSION

We have described a novel method for the constructionof recombinants betweenAd2 and Ad5. This method involves cotransfection of DNArestrictionfragmentswithoverlapping se-quencesfrom thetwoparentalDNAs to gener-ate in vivo recombinant DNAsresulting in in-fectious virus. The restriction fragments with overlapping sequence

homology

have been shown toundergo specificin vivorecombination to produce infectious DNA. In

principle,

this method could be appliedtootheradenoviruses that undergo genetic recombination. This ap-proach could also be used toconstruct recom-binantshaving the desiredsegmentsof thetwo parental DNAs by transfection ofappropriate restriction fragments. Such recombinants with definedsegmentsof thetwoparentalDNAs will bevaluable invariousgenetic studies.Wehave

successfully

exploited thismethod toisolate ad-enovirus mutants

lacking

specific restriction sites

(S.

Rajagopalan and G.

Chinnadurai,

man-uscript

inpreparation).

A

B-C

--D,E

Ft

G

-/-HI-

-/-- A

=-:

B

C

ID

E F,G

a-

H

- I

K-1

2

3

FIG. 5. Gelelectrophoresis of Ad2, Ad5, and type

IIrecombinantaftercleavagewith restriction

endo-nucleaseHindIII. Fragmentswereresolvedby

elec-trophoresison2.2%acrylamidegels containing0.7%

agarose (12) andphotographed. Lane 1, Ad2 wild

type; lane2, type IIrecombinant; lane 3, Ad5 wild

type.

Our results indicate that genetic recombina-tion may occur in the absence of viral DNA replication.It islikelythatthe recombination is carried outby the host enzymes orperhapsby virus proteins coded by the discontinuousvirus genome. Thismethod couldbe used to investi-gate aspects of adenovirus and cellular DNA recombination.

We have constructed several recombinants betweenanIpmutantofAd2 andAd5 wildtype. By dissection of the recombinantgenomes with restriction endonucleases that cleave Ad2 and Ad5 differently, wehavelocalized the Ip muta-tion to the left 41% of Ad2 genome. It is not possibleatpresentto more

precisely

maptheIp lesion within the left 41% of the viral genome, because norestriction endonucleaseshavebeen described that cleave Ad2 and Ad5 differently withinthisregion. The mapping approachthat wehave usedwasdeveloped by Sambrook,

Wil-liams,

and co-workers (9, 14) forphysical

map-ping

of thets mutantsof

Ad2+NDj

andAd5and has recently beenusedforphysical mappingof

tsmutantsofAd2

(10).

Recently,

ts mutants of Ad5 have been mapped by a marker rescue

technique by

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transfection of wild-typeDNA fragmentsandts

mutant DNA (1, 4). Application of modified

versions of this approach forphysical mapping

of plaque morphologymutantsofAd2 hasyet to

beexploited.

Inthe left 41% of Ad2 andAd5genomeseveral

tsand two hostrange (hr) mutants have been

mapped (1,4,10).One of thets mutants,H5ts36,

andtwohrmutantshave lesions inearlygenes,

whereas the other ts mutants, H5ts49, H5ts58,

and H2ts4,aredefective in lategenes. Itwill be

of interesttosee whether the Ipmutation isin anearlyorlategene.

ACKNOWLEDGMENTS

Thisinvestigationwassupported by research grants from

theNational Science Foundation (PCM77-12662), Missouri Cancer Programs, Inc., andaninstitutional grant from the American Cancer Society (77005). G.C. is an Established Investigator of the American Heart Association.

We thank Maurice Green for support andencouragement,

G. Gerard and W. S. M. Wold for critical reading of the manuscript, and Eric Frost for supplying the Ad5 SalI

cleav-agemapbeforepublication.

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Figure

FIG. 1.SambrookBamHI,sitesetin al. Ad2 Maps of restriction endonuclease cleavage in Ad2 and Ad5 DNA
FIG. 1.SambrookBamHI,sitesetin al. Ad2 Maps of restriction endonuclease cleavage in Ad2 and Ad5 DNA p.3
FIG. 2.recombinantdonucleasescarried(2). Gel electrophoresis of Ad2, Ad5, and a DNA after cleavage with restriction en- EcoRI and BamHI
FIG. 2.recombinantdonucleasescarried(2). Gel electrophoresis of Ad2, Ad5, and a DNA after cleavage with restriction en- EcoRI and BamHI p.3
FIG. 3.withwere Plaque morphology of Ad2 wild type and Ad2 lp3 mutant. The wild type and the mutant viruses plaque assayed on KB cells at 33°C (2) and stained with neutral red
FIG. 3.withwere Plaque morphology of Ad2 wild type and Ad2 lp3 mutant. The wild type and the mutant viruses plaque assayed on KB cells at 33°C (2) and stained with neutral red p.4
FIG. 4.endonucleasesaslp3; described Autoradiogram of Ad2, Ad5, Ad2 lp3, and recombinant DNAs after cleavage with the restriction BamHI and EcoRL DNA fragments were resolved in 1% agarose gels and autoradiographed in the text
FIG. 4.endonucleasesaslp3; described Autoradiogram of Ad2, Ad5, Ad2 lp3, and recombinant DNAs after cleavage with the restriction BamHI and EcoRL DNA fragments were resolved in 1% agarose gels and autoradiographed in the text p.4
FIG. 5.IInuclease recombinant Gel electrophoresis of Ad2, Ad5, and type after cleavage with restriction endo- HindIII
FIG. 5.IInuclease recombinant Gel electrophoresis of Ad2, Ad5, and type after cleavage with restriction endo- HindIII p.5