Biochemical studies on bovine adenovirus type 3. III. Cleavage maps of viral DNA by restriction endoncleases EcoRI, BamHI, and HindIII.

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Biochemical Studies

on

Bovine Adenovirus

Type

3 III. Cleavage Maps

of

Viral DNA

by Restriction Endoncleases

EcoRI,

BamHI, and

HindIII

T.KUROKAWA,* K. IGARASHI,ANDY.SUGINO

BiologicalResearchLaboratories, CentralResearchDivision, Takeda ChemicalIndustries, Ltd.,

Osaka,Japan

Received forpublication 2 March1978

Cleavageof bovine adenovirustype3 (BAV3) DNAbyrestriction

endonucle-asesEcoRI, BamHI,and HindIIIyielded 7 (AtoG),5 (AtoE), and 12 (A to L) fragments, respectively.Theorderof thesefragmentshas been determined to be GDACBFEfor EcoRIfragments, AEBDCfor BamHIfragments, and JEBKAC-DHFGIL forHindIlI fragments, and cleavage sites of these enzymeshavebeen

mapped on the genome of BAV3. BAV3 preparation contains incomplete virus whosegenomehasadeletion of about13% ofcompletevirusgenome.Restriction

endonuclease digestionoftheincomplete virus DNA revealedthat EcoRI E and F, BamHI C and HindIll G, I, and Lfragments were deleted. Therefore, the deletedregion of incompletevirus DNAislocatedneartheright-handend ofthe BAV3DNAmolecule,aresult consistentwithourprevious electron-microscopic observationsonheteroduplexmoleculesformed betweencompleteandincomplete BAV3 DNA.

Genomecleavagemaps, constructedby

order-ingDNAfragmentsproduced by digestionwith

restriction endonucleases, are an essential tool

for thedetailedanalysisof the genomefunctions of various DNA tumor viruses and cellular trans-formation by their genomes. Cleavage maps have already been constructed in many DNA tumorviruses (2, 8, 11) andutilizedasthebasis for further studies.Cleavagemaps of adenovirus genomeshave also been producedin many hu-man(15, 17, 19, 19a,21) andmouse (14)

adeno-viruses.Accumulation of these data might give

some insight into the evolutional relationships

amongthese viruses.

Wehavepreviously reportedthebiochemical

properties of bovine adenovirus type3 (BAV3) (9, 16).The genome of BAV3 isalinearduplex

DNAmolecule withamolecularweightof 24 x

106. To provide specificDNAfragmentsforuse in further analysisof BAV3functions,we have

attemptedtoconstructcleavagemaps ofBAV3 DNA by the use of restriction endonucleases

EcoRI, BamHI, and HindIII. In this paper,

cleavage sites of these enzymes have been

mappedmainly by two methods: (i) analysis of

overlappingsetsofspecific fragments produced

by different endonucleases and (ii) cleavage of DNApreviouslyend-labeledbyT4 DNA

polym-erase.

Original stocks of BAV3 contain a unique population ofincomplete virus (16), whose

ge-nomehas a deletion whichoccupies about 13%

of the total genome of complete virus, as

re-vealed by electron-microscopic observation in our previous study (16). This observation was further confirmed by analysis of the deleted

fragmentsonrestriction endonucleasedigestion

of theincompletevirus DNA.

MATERIALS AND METHODS Virus andDNA. Two clonalviruses, BAV3-0 (16) and BAV3-1 (16), originating from BAV3 prototype

(WBR-1) (3) were growninconfluentmonolayers of

CKT-1 cells(12) (an establishedcelllinederived from

calf kidney) at an input multiplicity of about 10

PFU/cell.

The procedures for purification of complete and

incomplete BAV3 and for extraction of viral DNA

have been describedpreviously(16).

Enzymes. Restriction endonuclease EcoRI was

purifiedfromEscherichiacoliRY 13 by the method

of Greene et al. (7). Restriction endonuclease BamHI

from Bacillus amyloliquefaciens (22) and HindIII

fromHaemophilus influenzae(13) were gifts from H.

Shimojo and H. Ariga and from K. Fujinaga and K.

Sekikawa, respectively. T4 DNA polymerase was

kindly providedby M. Takanami.

Digestion ofDNA with restriction

endonucle-ase.Digestion ofBAV3 DNAwith restriction

endo-nucleasesEcoRI,BamHI,andHindIII wascarriedout

accordingtoPetterssonetal. (18), Wilson and Young (22), and Danna et al. (2), respectively. The reaction was stopped by the addition of 0.1 volume of 60%

glycerol containing0.2 M EDTA and 0.05% bromo-212

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phenol blue, and products ofthe reaction were sub-jectedtoanalysis byelectrophoresisongels.

Gelelectrophoresis.Twotypesofcylindrical gel (0.6 by 26 cm) were used: (i) 0.9 or 1.2% agarose (Seakem, BioProducts,Rockland, Maine) gel (20); (ii) 0.7% agarose-2.4% acrylamide composite gel (18). ElectrophoresiswasconductedwithEbuffer (40 mM Tris-hydrochloride, 5 mM sodium acetate, 1 mM

EDTA, pH 7.8)at 1.6V/cm for20h for both types of gel. TheDNAbandswerestained with ethidium bro-mide contained in thegel and E bufferatthe concen-tration of0.5 ,ug/ml and were visualized under UV light and photographed. Theradioactivity of gel slices was counted withtoluene-based scintillation cocktail aftersolubilization of gel slices with0.4 ml ofNCS (Amersham/Searle,Arlington Heights, Ill.).

For the extraction of DNA from agarose gel, gel slicesweremashed in buffer(10mM Tris-hydrochlo-ride, 1 mMEDTA, 0.1% sodium dodecyl sulfate, pH 8.0), and theresultingslurrywasincubatedovernight

at45°C. Thesupernatantwascollected and extracted threetimes withphenol, and then DNA was precipi-tatedby addition of2volumes of ethanol and dissolved inbuffer (10 mM Tris-hydrochloride, 1mM EDTA, pH7.8).

End-labeling of DNA by T4 DNA polymerase. Linear duplexDNAmoleculescanbe labeled witha

3H-labeleddeoxyribonucleosidetriphosphateattheir 3'terminiby T4 DNApolymerase(5). This method is basedonthe fact that T4 DNApolymerase contains

apolymerizingaswellasa3'-5'exonucleolytic activity. For theterminallabelingofBAV3DNA, the follow-ing reaction mixture (0.1ml)wasincubated for30min

at28°C:70mMTris-hydrochloride (pH 8.0),7mM

2-mercaptoethanol, 10,Lgof BAV3 DNA,and 0.2Uof

T4 DNApolymerase. Thena

10-pi

sample ofamixture of5 mMdeoxyribonucleotide triphosphates (dATP, dGTP,anddCTP)and100uCi of[3H]dTTP(2nmol, Daiichi Pure ChemicalCo.,Japan)wereaddedtothe

*mixture,and incubationwascarried out foranother1

h at28°C. DNA thus labeledwaspurified from the mixture by phenol extraction followed by passage throughaSephadex G-75 column (0.5by10cm). In thiscondition, 3H labelwasintroduced into about20

nucleotides from both 3' ends of the linear duplex moleculeof BAV3DNA.

RESULTS

Restriction endonuclease digestion of BAV3 DNA. BAV3 DNA was digested with restriction endonucleases EcoRI, BamHI, and

HindIII,

and each

digestion product

was

ana-lyzed by electrophoresis on0.9 or 1.2%agarose

gels (Fig. 1). Digestion of BAV3 DNA with

re-striction endonuclease EcoRIyieldedseven

frag-ments, which were

designated

in order of

de-creasing electrophoretic

mobility

asAtoG

ac-cording to Danna et al. (2). BamHI digestion yielded five (AtoE)

fragments.

HindIII

diges-tion product was separated into 11 bands by electrophoresison acolumn ofagarose

gel.

Fur-ther

analysis

showed that one of these bands

wascomposedof two DNAfragmentswith

sim-(a)

(b)

(C)

A

B C

D

E

F

G

A

B

C

D

B C

D

E E

F

G

H I

J

K

L

FIG. 1. Analysis of BAV3 DNA digested with re-striction endonucleaseEcoRI, BamHI, and HindIII. BAV3 DNA(about1

jig)

wasdigested with the three restrictionendonucleases, and the products were ap-plied togels. Electrophoresis wasconductedat 1.6

V/cm for 20h andphotographed under UV light. DNAfragment bands were designatedasA, B, C, andsoon, inorderof their size. (a) EcoRI digestion products on 0.9% agarosegel. (b) BamHIdigestion products on 1.2%agarosegel. (c)HindIII digestion products on 1.2% agarosegel.

ilar sizes (H and I

fragments). Therefore,

HindIII

digestion

gave 12

fragments (A

to

L).

HindIII

fragments

H andI could be

separated

onagarose-acrylamide

composite

gel (data

not

shown).

Estimation of molecular

weight

of each

fragment.

The size of each

fragment

as a per-centageof the whole DNAwasestimatedonthe basis of therelative

radioactivity

recovered in each fragment from

uniformly

labeled BAV3

[32P]DNA.

In Table 1, relative molecular weights of

EcoRI,

BamHI,

and HindIII

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mentsareshown. For somefragmentsthat were

difficult to separate from each other on gels (EcoRIAandBfragments andHindIII Hand

Ifragments), the sumof the values oftwo

frag-mentsis shown.

The molecular

weight

of each

fragment

was

estimated from its relative

mobility

on agarose

gel electrophoresis. The

relationship

between mobility and molecular

weight

was

plotted (Fig.

2) for some marker DNA

fragments,

and from

thisplot the molecularweightof eachfragment

wasdetermined(Table 1).

Reciprocal digestion of

specific

frag-ments

with

different restriction

[image:3.500.261.450.67.224.2]

endonucle-ases.

(i)

Combination ofEcoRI and Hindm. The orderof

fragments

on the DNA molecule couldbedetermined

by

overlapping

thespecific

TABLE 1. Molecularweights ofBAV3DNA fragments produced by restriction endonuclease

digestion

Molwt(% of BAV3

DNA) Mol wt(x

Fragment 105)

Radioac- Moilityb

tiVitya

Mobity

EcoRIfragment

A 46.9 24.9 57.2

B ) 46.9 2315 53.4

C 18.6 18.4 42.3

D 17.0 15.8 37.0

E 10.5 9.7 22.3

F 3.8 4.1 9.4

G 3.4 3.6 8.3

BamHIfragment

A 55.4 54.0 124.3

B 16.6 18.2 41.9

C 13.9 13.4 31.7

D 8.6 8.5 19.5

E 5.6 5.9 13.5

HindIIIfragment

A 22.2 22.4 51.5

B 15.1 15.0 34.5

C 11.4 11.3 26.0

D 10.3 9.4 21.6

E 6.5 6.9 15.9

F 6.4 6.1 14.0

G 5.6 5.7 13.1

H 10.5 5.2 12.0

I J5.2 12.0

J 5.0 5.0 11.5

K 4.7 4.5 10.4

L 2.4 3.4 7.8

aDetermined from the distributionof3Pradioactivityon

eachfragment.Gelsweresliced into1-mmslices, and

radio-activitywascounted. EcoRIAandBfragmentsandHindIII HandIfragmentswere notcompletely resolvedon electro-pherograms, and the numberof counts in bothfragmentswere addedtogether.

bDetermined from mobility on agarose gel. Molecular

weightwasestimated fromrelativemobilityofeachfragment on agarose gelelectrophoresis, andthe percentage ofviral genome was calculated fromthe molecular weightof each fragment, using2.4x107forthatof whole BAV3 DNA.

x10 6

20-10

6 a

4

LM

~~~~c

,012_ C D

E

:

11XI.*

o : 0.2 0.6 1.0 1.2

0.6 Mobility

FIG. 2. Relationship between mobility and molec-ularweight. In this experiment, 1.29o agarose gel was used. EcoRIfragments (A toF) and HindIII

frag-ments(a-d)ofhuman adenovirus type 2 DNA were

usedasmolecular weight markers.Molecular weights of these markers were adoptedfrom the values re-ported by Petterssonetal. (18) andDunn andHassell

(4).

fragments produced bytwodifferent restriction endonucleases. We applied thismethod to the combinationofEcoRIandHindIII. Successive digestion of BAV3 DNA with EcoRI and HindIII gave 17 fragment bands (A to Q) on

agarose gel (Fig. 3). Since the cleavage sitesof

both enzymesontheDNAmoleculewere differ-ent, 18fragments might be producedby double digestion with both enzymes. In our

experi-ments, one very small fragment, produced by closely located cleavage sites ofboth enzymes,

could be missed on thegel.

BAV3 DNA was cleaved with EcoRI or

HindIII, and each resulting fragment was

iso-lated from agarose gels. These fragments were

redigested with another endonuclease, i.e., EcoRIfragment with HindIII andHindIII frag-mentwith EcoRI, and the products were

ana-lyzed on agarose gels. From these results, we

examinedtherelation betweenthespecific frag-ments of EcoRI or HindIII and the

double-digestion fragments produced by additional digestion with reciprocalendonucleases (Table 2). The molecular weight of each specific frag-mentobtainedby thefirstenzyme digestionwas identical to or very close to the sum of the

molecular weightsof theproducts derivedfrom it by the second enzyme digestion (data not

shown). Accordingto thedata shown in Table 2, EcoRI and HindIII fragments could be

over-lapped throughdouble-digestion fragments, ex-cept at one site where one lost fragment of

double-digestion products mightbelinked. The orders of EcoRI andHindIIIfragments,deduced from their overlapping, were GDACB, EF and

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enzymes; one fragment could not bedetected, as in the case of the combination of EcoRI and HindIII (Fig. 3). The relation between specific fragments of EcoRI and BamHI and the double-digestion fragments produced by further diges-tion with reciprocal enzymes is presented in Table 3. The order of BamHI fragments was

A

deduced to be

AEBD,

C,

and that ofEcoRI

fragments

wasascertainedtobe

(GD)ACB, EF,

B thesame asinthe combination with HindIII.

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c Determination of

terminal

fragments.

TABLE 2. Relationbetween BAV3 DNA EcoRIor D HindIIIfragments and the fragmentsdouble

E digested with both restriction endonucleases

F Double-digested

Specific fragment

fjagment

EcoRIfragment

A.A,F,K

B.B,H,I,O

.C,D

D.G, E,Q

E.J, N,P F .L G

H

HindIIIfragment

A

B

C.

J D

E F

G ...

H ...

I...

J ...

K ...

L ...

A, C E,F

D,0

B G H L, P I

J

M, Q K

N

FIG. 3. Analysis of BA V3 DNA digested succes-sivelywithtworestrictionendonucleases. Conditions ofelectrophoresis were the same as in Fig. 1. (a)

EcoRIandHindIIIdigestion productson1.2%

aga-rosegel. (b) EcoRI and BamHI digestionproductson

1.2%agarosegel.

JEBKAC(DHF), GIL, respectively. Cleavage sites ofHindIIIthatyielded D,H, and F frag-mentsalllay in the EcoRIBfragment,andthe order of these three fragments could not be determined in thisexperiment.

(ii) Combination of EcoRI and BamHI. Thesameoverlapping procedurewasappliedto

the combination of EcoRI and BamHI. Ten fragment bands (A toJ) wereobtainedon

aga-rose gels by digestionof BAV3 DNAwith both

TABLE 3. Relation between BA V3 DNA EcoRI and BamHIfragments and thefragments double digested with both restriction endonucleases

Specificfrag-inent Double-digested

Specific fragment

fragnent EcoRIfragment

A ... A

B ... C,F C ... E, G,J D ... B E ... D F ... H G ... I BamHIfragment

A ... A,B, E,I B ... C,J C ... D,H

D ... F E ... G

(a)

(b)

A

B

C

D

E

F

G

H

IJ

K

L

M

N

0 p

Q

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BAV3 DNA waslabeled with

[3H]dTTP

in its 3'-terminalregionsbyT4DNA

polymerase.

The end-labeled BAV3 DNAwas

digested

with

re-striction endonucleases

EcoRI, BamHI,

and HindIII, and radioactivities of the

resulting

frag-ments were determined. The

radioactivity

of each fragment, expressed in terms of percent

distribution, is shown in Table 4. The radioac-tivitywasconcentratedat twoof thefragments produced by each restriction endonuclease in about equalproportions. From the data in Table

4,it isclear that the

fragments, EcoRI

E and

G,

BamHI A and C, and HindIII J and

L,

were

preferentially labeled and that these

fragments

[image:5.500.57.246.250.657.2]

werelocalizedatthetermini of theBAV3 DNA molecule.

TABLE 4. Distributionof

radioactivity

on the restriction endonucleasefragmentsof

terminal-labeled BAV3 and DNAa

Fragment

~~Distribution

of

ra-Fragment dioactivity(%)

EcoRIfragment

A .,

B .... ...

5.5

C ... 2.6

D ... 3.6

E ... 47.8

F.. .... 3.1

G.. .. 37.4

BamHIfragment

A ... 38.2

B .... 8.0

C .... 37.4

D . ... 8.5

E ... 7.8

HindIIIfragment

A .. ... 4.3

B ... 6.7

C ... 6.1

D.. .. 5.1

E ... 5.6

F ... 4.9

G ...) 5.8

H 8.0

J... 23.1

K ... 3.7

L ... .. 26.7

'Terminal-labeled

BAV3

DNA was

digested

with restriction endonucleases EcoRI, BamHI, and HindlIl,andthedigestion products of each were

sep-arated by agarose gel electrophoresis. Gels were

stained with ethidiumbromide, the gel slices contain-ingDNAbandswere cut out under UV light, and the radioactivities were counted. EcoRI A and B

frag-mentsandHindIII H and Ifragments were not

sepa-ratedongels, and for these fragments the sum of the values oftwo fragments is shown.

From these results, we concluded that the orders ofEcoRI, BamHI,andHindIII

fragments

were GDACBFE,

AEBDC,

and JEBKAC-(DHF)GIL, respectively. The cleavage mapsof

BAV3 DNA with restriction endonucleases

EcoRI and BamHI weretentativelyconstructed asshown inFig.4.

DeletedregionofincompleteBAV3 DNA. The BAV3preparation contains aunique

pop-ulation of incomplete virus (16). The DNA of this virus has a deletion of 13% of the genome of

completevirus. Incomplete virus DNA was

di-gestedwith restriction endonucleases and

ana-lyzed by agarose gel electrophoresis (Fig. 5). Compared withthe digestionproducts of

com-pletevirusDNA,those ofincomplete virusDNA

lacked EcoRI E and F fragments, BamHI C

fragment, and HindIII G, I, and L

fragments.

Therefore, the deleted region ofincompletevirus DNA was positioned at the right-hand end of

completevirusDNA, asdepicted inFig.4.

On gel electrophoresis, EcoRI B, BamHI D, and HindIII F fragments of incomplete virus

DNA were shownto beaccompanied by some

minor bands adjacent to them (Fig. 5). This

implied that thesefragments had minor hetero-geneities in their sizes, that is, the incomplete virus preparation containedseveral minor pop-ulations with differentdeletion

lengths,

and the deletedregions extended into the corresponding fragments.

The deletion of incomplete virus DNA was

thought tobe inone site; the presence of

par-tially deleted HindIIIFfragment in incomplete

virus DNA indicated that it was a neighborto thedeleted HindIII G fragment.

Moreover,from the analysis of partial diges-tion products of HindIII, it was resolved that HindIII C and D fragments were neighbors (data notshown).

Therefore,the order ofHindIIIfragmentswas concluded to be JEBKACDHFGIL, and from thisresult the cleavage map with HindIII was constructed as showninFig. 4.

DISCUSSION

In this paper, we showed cleavage maps of

BAV3DNAconstructedby use of three restric-tion endonucleases, EcoRI, BamHI, and

HindIII.

Ourexperiments showed that the gene nec-essary for cell transformation was located in EcoRI D orHindIII J-Efragments (10), and so we have tentatively placed these fragments at

theleft-handend ofthe maps shown in Fig. 4, as proposed in humanadenovirus(1).

Fragment orderswere determined mainlyby

overlapping ofspecific fragments produced by

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EcoRI [GI D BaffHI I

A C

A

B ,F, E

E B D C

Hindlil t1JI E I B IKI A C I D HIF G I IL 0 10 20 30 40 50 60 70 80 90 100

FIG. 4. Cleavage maps of BA V3 DNA. Maps are divided into 100 units. The deletion of incomplete BA V3

DNA is indicatedby (+-*).

differentrestrictionendonucleases. The order of

EcoRIfragments, asdeterminedbytwo differ-ent combinations of enzymes, EcoRI-HindlIl andEcoRI-BamHI,wasthesame.Three restric-tion endonucleases all cleaved BAV3 DNA at near 86 map units (Fig. 4). For this, one very

shortfragmentwasproduced,which wasmissing

on the analyses ofdouble-digestion fragments,

forboth endonucleasecombinations, making it

difficulttodeterminethefull order offragments by the overlapping method. The ordering of EcoRI andBamHIfragmentswasaccomplished bytheanalysisof thecleavage productsof end-labeled BAV3 DNA. Thefull order ofHindIII fragmentswasobtainedbytheaid of additional

observations: the analysis of partial digestion products and the examination of the deleted region ofincompletevirusDNA.

The partial digestion methodwas partly

ap-plied to the ordering of EcoRI and HindIII fragments, and the resultswereconsistent with the mapsdescribed

(data

notshown).

From the

analysis

ofcleavage

products

with

restrictionendonucleases,thedeletionof

incom-plete virus DNAcould be

mapped

tothe right-hand end oftheviralgenome. We had

previously

observed a

single-stranded

deletion

loop

occu-pying 13%ofthewhole genome in electron-mi-croscopic observation of the

heteroduplex

mol-ecule formed betweencomplete andincomplete BAV3 DNA (16).Attheendoftheloop, a short

double-strandedstem(about 1% ofthe genome) was observed, and therefore incomplete virus DNApossessed the samesequence is complete

virus DNA for both ends of themolecule. When complete and incompleteBAV3 DNA were denatured and then renatured, many

sin-gle-stranded circular DNAs were seen by the electronmicroscope (16).However, in those fig-ures, no double-stranded "panhandle" struc-tures weredetected; this is not the case in human adenovirus type 18 (6), which hasthe long

in-verted terminal repetitions (about 3% of the

genome)in its DNA. Thesefacts show that both

completeandincompleteBAV3 DNAcontained invertedterminal repetitions and that thelength ofrepetitionis rathershort.

Based on thecleavage maps shown inFig. 4,

(a)

(b)

(C)

A

A B

C

D

G

A B

D

B

C

D

E _E

F

H J K

FIG. 5. Analysis of incomplete BAV3 DNA di-gested with restriction endonucleases. Conditionsof electrophoresiswerethesame asinFig.1.(a) EcoRI digestionproducts on 0.9%oagarosegel. (b) BamHI

digestion products on

1.2%o

agarosegel. (c)HindIII

digestionproductson1.2%agarosegel.

theposition of thegene essential forcell trans-formation on BAV3 DNA was studiedby(i)

Cot

analysis of the viral genome harbored in trans-formedcells (Y. Niiyama,T.Kurokawa,K. Igar-ashi, and Y.Sugino,manuscript inpreparation)

28, 1978

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and (ii) celltransformationby infection of cells with fragmented DNA (10). These results are shown infollowingpapers.

ACKNOWLEDGMENTS

WearegratefultoK.Fujinaga and K. Sekikawa (Cancer Research Institute, Sapporo MedicalCollege, Sapporo,Japan) and H.Shimojo and H. Ariga (Institute of Medical Science, Tokyo University, Tokyo, Japan) for gifts of restriction

en-donucleasesHindIII andBamHI, respectively. Weare also

grateful toM. Takanami (Institute for ChemicalResearch, Kyoto University, Kyoto,Japan) foragift of T4DNA polym-erase.

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