Physical Mapping of
Large-Plaque Mutation of
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. Althoughthe 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
cleavagepatterns 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,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
experimnentsinvolving genetic ma-nipulations ofthe viral genome.
MATERIALS AND METHODS
Cells and viruses. Human cell lines KB and293
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.
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
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
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).
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
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
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
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
2 Ad2, EcoRIdigested (4.0) 0,1 Ad2,SalI digested (4.0) 0, 0
Ad2,EcoRIdigested+ (4.0+4.0) 34,20
3 Ad2, EcoRIdigested (3.0) 0,0
Ad5, Sail digested (2.5) 0,0
Ad2, EcoRIdigested+ (3.0 +2.5) 12,8
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
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0 10 20 30 40 50 60 70 80 90 100
A B F D E C
A C B
B D C A
B D C A
F C B I J E A HL D G K
E C H D A B F I
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),
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,
fragmentssim-ilartoAd5 parentalDNAandBamHIgenerated four fragments similar to Ad2 parental DNA. The representative
patternofone 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
mappingofanIpmutantof Ad2. Wehaveisolatedmutants ofAd2by mutageni-zation with
hydroxylaminewhichproduce large clear
plaquesonKB cells in7to8
with the wildtypewhichproduces small diffused plaques in 10 to 12 days. The
Ipmutants de-scribedin the presentcommunicationaresimilar inphenotypetothecytmutantsofAd12 isolated by Takemorietal. (18).The
IpmutantsofAd2 exhibit the
large-plaquephenotype 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
Ipmutantandthe Ad5 wild type and
formappingthesite of the
Iplesion. This exper-iment is notpractical with mixedinfections of virions or intact DNAs because recombinants cannotbedistinguishedfrom thelargeexcessof
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,
parentalplaquesthat would be produced.Since theinfectivityoftheparentalDNAis
bydigestionwith restrictionendonucleasesand only the recombinants can yield plaques, we haveconstructed severalrecombinants between an
(Ip3)and the Ad5 wild type.These recombinantswere used to
From twoexperimentsinwhich Ad2
digestedwith EcoRI and Ad5
SailIwerecotransfected, 10 well-separated
plaqueswere 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.
patternsidentical to Ad2 and EcoRI cleavage
patternsidentical 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
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 ,.
-s *b B
;H( SO.A Cr -.
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
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
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.
Cleavageoftype 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
Ipmutation 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
indicatingthat the sequencesright ofmap position 41 are Ad5 and the sequences onthe leftareAd2.Therefore, the Ipmutation islocatedwithin theleft 41%of Ad2genome.
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
homologyhave 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
successfullyexploited thismethod toisolate ad-enovirus mutants
lackingspecific restriction sites
(S.Rajagopalan and G.
FIG. 5. Gelelectrophoresis of Ad2, Ad5, and type
agarose (12) andphotographed. Lane 1, Ad2 wild
type; lane2, type IIrecombinant; lane 3, Ad5 wild
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
preciselymaptheIp 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-pingof thets mutantsof
Ad2+NDjandAd5and has recently beenusedforphysical mappingof
Recently,ts mutants of Ad5 have been mapped by a marker rescue
on November 10, 2019 by guest
transfection of wild-typeDNA fragmentsandts
mutant DNA (1, 4). Application of modified
versions of this approach forphysical mapping
of plaque morphologymutantsofAd2 hasyet to
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.
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
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