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0022-538X/80/07-0176/08$02.00/0

Derivation of

a

Restriction Map of

Bacteriophage T3

DNA

and

Comparison with the Map of Bacteriophage T7 DNA

JEAN N.BAILEY, DAVID R. DEMBINSKI,AND WILLIAM T. McALLISTER*

Departmentof Microbiology,Rutgers Medical School, College of Medicineand Dentistry ofNew Jersey,

Piscataway, NewJersey 08854

The DNA of bacteriophage T3 was characterized by cleavage with seven

restriction endonucleases. AvaI, XbaI,Bgm,andHindIIIeachcutT3 DNAat1

site, KpnI cleaved itat2sites, MboI cleaved itat9sites, and HpaI cleaved itat

17 sites. The sizes of thefragments produced by digestion with these enzymes weredetermined by using restriction fragments of T7 DNAasmolecular weight

standards. Asaresult of this analysis, the size of T3 DNAwas estimatedtobe

38.74kilobases. The fragmentswereordered with respect to each other andto

the genetic map to produce a restriction map of T3 DNA. The location and

occurrence of the restriction sites in T3 DNAare compared with those inthe

DNA ofthe closely relatedbacteriophage T7.

T3 and T7 areclosely related bacteriophages

which sharemanyfeaturesbut differin interest-ing and potentially significant ways (7). For

ex-ample, both viruses elaborate phage-specified

RNA polymerases during infection which,

al-though theyare similar,do not transcribe the

heterologous DNA efficiently (3, 5). Although the two viruses have similar genetic organiza-tions(1, 7),adetaileddescriptionofthephysical

structureof thegenome(e.g., byrestriction anal-ysis) existed onlyforbacteriophageT7 (10, 12).

Restrictionmapsofboth viruses would be useful

forlocatingandcomparing regulatorysignals in

the DNAs and could serve as a basis for esti-mating the evolutionary divergence of thetwo

viruses. In thispaper, wedescribe thederivation ofarestriction mapforbacteriophage T3DNA and compareit with themapofT7DNA.

MATERIALS AND METHODS Bacterial andphage strains andpreparation of DNA.BacteriophageT3 Hausmann(15)and Esch-erichia coli B werefrom thelaboratory of E. K. F. Bautz. Deletion mutants of T3 (16) were obtained from F. W. Studier.

DNA wasisolated fromCsCl-purified virusparticles as previously described (9). For

[nP]DNA,

lysates

were prepared as previously described (9), and the phage particles wereharvested by centrifugation at

35,000 x g for 80min,suspendedin buffer (10mM Tris, pH 7.5,5mM

Mg9l2,

50mMNaCI),and clarified by centrifugation at 12,000 x g before isolation of DNA.

Cleavageof DNA andelectrophoresisof

frag-ments.AvaI, XbaI, KpnI,BglII,HindIII,MboI,and HpaI (11) were purchased from Bethesda Research Laboratories,Rockville,Md. MboIhas thesame spec-ificityasDpnII,whichwasusedpreviouslytomapT7 DNA(10, 11).Theconditionsofcleavagewerethose

recommendedby the supplier. For double digestions, the first enzymewasinactivated by the addition of 10 mM EDTA, followed by heating to650C for 5 min. The DNAwasprecipitated with ethanol and sodium acetate,washed with 70%ethanol, dried in vacuo, and taken up inbuffer suitable forcleavage by the second enzyme. DNAfragmentswereanalyzedby electropho-resisthrough agaroseorpolyacrylamide gels,as pre-viously described (10, 12).

Isolation ofindividualfragmentsfrom agarose gels.Individualfragmentswereresolved by electro-phoresis through 0.6% agarose gels and visualized by staining with ethidium bromide. The fragmentswere eluted from the gel by electrophoresis (17) or by adsorptionto glassbeadsasdescribedbyVogelstein andGillespie (19), except that Nal wasreplacedby NaCl04.

Endlabelingof DNA. T3 DNA and restriction fragments of T3 DNAwerelabeled attheir 5' ends with

'PO4

asdescribedby Maxam and Gilbert(8a).

L-imited exonuclease digestion. In a series of reactions (20

pl)

1,tgof T3 DNAwasincubated with increasing concentrations (0.06to 2 U) of bacterio-phage T7 gene 6 exonuclease (8) (the gift of J. J. Dunn) in50 mM Tris (pH 8.1)-20 mM KCI-5 mM MgCl2-1 mMdithiothreitol for30minat

370C.

The reactions were stopped by the addition of 10 mM EDTA, and the sampleswere heated to650Cfor 5 min. DNA wasprecipitated bythe additionof ethanol and sodium acetateandwascollected by

centrifuga-tion.Thepelletswerewashedtwice with70%ethanol, dried, andsuspendedin 20

pl

ofrestrictionbuffer.A 2-Uamountof restrictionendonucleasewasadded,and thesampleswereincubated for1hat370Cand

ana-lyzed bygel electrophoresis.

Hybridization of individual T3 earlymRNA's to restriction fragments of T3 DNA.

[nP]RNAs

isolated from T3-infectedcellswereseparatedby elec-trophoresis through 2.5% polyacrylamide-0.5% aga-rosecompositegels(14). IndividualRNAswereeluted from thegelsandhybridizedtoHpaIrestriction

frag-ments that had been immobilized on nitrocellulose 176

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35,1980

filterstrips aspreviously described (9). Gene0.7, 1, and 1.3 mRNA'swereisolatedfromcellsinfected with wild-type phage, gene0.3 mRNA wasisolated from cellsinfected with deletionmutantR14(16), andgene 1.1mRNAwasisolated fromcellsinfected with dele-tionmutantR16(16).

RESULTS

Therestrictionpatterns generated by

cleavage

of T3 DNA with thesevenenzymesconsidered inthis paperareshown inFig. 1. The enzymes AvaI, XbaI, BglII, and HindIll cut T3 DNA once,KpnIcutittwice, MboIcutit9times and HpaIcutit17times. The molecular

weights

of

the

resulting fragments

aresummarizedinTable

1. The sumof the sizes of thefragments (38.74 kilobases[kb]) is inagreementwith the range of sizes

previously

estimated for T3 DNA

(2,

4). As there issome

diversity

in

laboratory

strains of T3, itshould be noted that the

HpaI

restriction patternofourT3strain is

identical

tothat of T3 Hausmann (15).

Mapping

of AvaI,

XbaI,

BgM,,

HindM,

and

KpnI

sites. To

determine

the

relative

po-sitions of the

cleavage

sites for those enzymes thatcutT3DNAat

only

one or two

sites,

DNA

wasdigested with pairwise combinations ofthese

enzymes. The

cleavage site(s)

for eachenzyme wasdetermined by comparing theresulting dis-tributions of

fragments

with the

distributions

MAP OF T3 DNA 177

40

A- A- A-

A-~ ~ A

10

B-

A-B- B- B

w

G-tedaslgoH-e(nioae)adardniidb

H-

K-

N-

0-FIG. 1. Restriction fr-agments ofT3DNA.

Frag-mentsgeneratedbythe restriction enzymesare

plot-tedaslog ofsize (inkilobases) andareidentified by

letters. Thisprovidesanidealized representationof thepatternsobservedupongelelectrophoresis of the

fragments.

TABLE 1. Sizes of restrictionfragmentsof T3

DNA"

Restrictionendonuclease Fragment Size(basepairs) Restrictionendonuclease Fragment Size(basepairs)

HindIUI A 30,960 HpaI A 8,425

B 7,780 B 6,035

BglII A 21,675 C 5,900

B 17,065 D 4,570

KpnI A 30,925 E 3,045

B 6,715 F 2,515

C 1,100 G 2,100

XbaI A 32,840 H 1,200

B 5,900 I 1,010

AvaI A 33,825 J 905

B 4,915 K 815

MboI A 12,735 L 700

B 6,540 M 560

C 5,450 N 315

D 3,140 0 235

E 2,855 P 155

F 2,725 Q 135

G 2,350 R 100

H 1,390

I 840

J 715

aThe sizesofthe restriction fragments produced byeach enzyme were determined by comparing their electrophoretic mobilitieswiththoseofHpaI andMboIfragmentsof T7DNAthatwererun in the same gel. Sizes of many ofthe fragments were estimated more accurately by summing the sizes of the component fragments generatedbycleavagewith otherrestrictionendonucleases (see Fig. 3). As a result, the sum of the lengthsofthefragments foreachenzymeis38,740base pairs.

Hindul egi KPnI XbaI AaI MboI HpaI

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observed after digestion with eachenzymealone

(Table 2).

MboImap. Toassignthe MboI fragmentsto specific regions of the genome, T3 DNA was

digested with MboI and with one of the five enzymes whose cleavage siteswere mappedas

described above (Fig. 2 and Table 2). In addition, larger fragments defined by the HindIII, XbaI, andBglII cleavage siteswereseparated bygel

electrophoresis, eluted from the gels, and di-gested with MboI (Table 3).

From doubledigestions with MboI and KpnI, XbaI, orAvaI, the location and orientation of

MboI-A could be determined. The

HindIII/

MobI double digestion restriction pattern was

indistinguishable from thatproduced by MboI alone and thus indicates that HindIII cleaves

veryclosetooneof the MboI sites. The BglII/

MboI doubledigestion restriction pattern is

ex-pectedtobe identicaltothatproduced by MboI alone since theBglII recognitionsequence

con-tains the MboIsequence(11).

The locations of fragments G and E were

determined by MboI cleavage of

32PO4-end-la-beled DNA.Fragments B and Iwerelocatedby

MboI cleavage ofend-labeled

BgIII

fragments. From these data andthe results of MboI

diges-tions of isolated HindIII-B, XbaI-B, and HindIll/BglII fragments (Table 3), the loca-tions of all remaining fragmentsexceptJ and D

werededuced. Partial digestion with MboI

pro-ducedafragment (1,555 base pairs) which could

only beafusionproduct of MboI-I and MboI-J,

indicating that these two fragments must be adjacent in the T3genome.

Thelocation of the HindIII site relativetothe MboI siteatthe end ofMboI-Cwasdeducedby

end-labeling HinduI fragments and digesting with MboI. Since labelwasfound in association

with MboI-C but not with MboI-F (data not shown), theHindII sitemust beto the left of the MboI site.

HpaImap.Thepositions of HpaI fragments

G, B, D, and J were determined by double

digestionswithHpaIandHindIII, BglII, XbaI,

and AvaI, respectively (Fig. 2 and Table 2). From these data and the data in Table 3 the order ofHpaI fragments in the central areaof

the T3genomeis-G-A-B-C-D-(Q,M)-J-.

To orient theHpaIfragmentsnearthe termini

of the T3 DNA molecule, intact T3 DNAwas

5'-end-labeled andcleaved withHindIII, and the two HindIH fragments were separated by gel

[image:3.508.70.461.365.587.2]

electrophoresis toyield samples in which only

TABLE 2. Fragmentsgenerated bycleavageof T3 DNA with two restrictionendonuckasesa Firstenzyme Second en- Fragment(s) missing Sizes of newfragments (base pairs)

zyme

HindIII BglII HindIII-A 5,430,(20)d

KpnI HindIII-A 23,110, 6,715, 1,100

XbaI HindIII-A 25,060, 5,900

AvaI HindIII-A 26,045, 4,915

BglII KpnI BglII-A 13,860, 6,715, 1,100

XbaI BglII-A 15,775, 5,900

AvaI BglII-A 1,880,220

KpnI XbaI KpnI-B 5,900,815

AvaI KpnI-B 4,915, 2,800

XbaI AvaI XbaI-B 4,915,985

Mbojb HincdII MboI-C 2,720,(5)d

BglII None None

KpnI MboI-A 10,155, 2,470, 1,100

XbaI MboI-A 11,080, 1,655

AvaI MboI-A 12,065,670

Hpalb

HindIII HpaI-G 2,005,195

BglIII HpaI-B 5,395,640

KpnI HpaI-D 2,565, 1,100,905

XbaI HpaI-D 4,680,90

AvaI HpaI-J 705,200

MboI HpaI-F,-G, -A, -B, -J,-E 5,900, 2,700,2,525, 1,900,1,700, 870,815, 700, 640,345,200,(35)d

aT3 DNAwascleavedby the successive action of thetwoenzymesindicated,and theproductswereanalyzed

bygel electrophoresis.Fragments missingfromthepatternobserved aftercleavage bythe firstenzymealone andthesizesof anynewfragmentsareindicated. Sizes of thefragmentsweredeterminedasdescribedinTable 1,footnotea.

bSeeFig.2.

eHpaI

fragments B and Care not

well

resolved in thegelshowninFig.2; thepresenceof the

BglII

sitein HpaI-Bwasdeterminedby resolution of thesefragmentsina0.6%agarosegelthatwasrunat1V/cmfor12h.

dThisfragmentistoo

small

tobeobserved inthegelshowninFig.2.

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MAP OF T3 DNA 179

A

B

C

2 3 4 5 6 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

w

__

A--- B-

C-E D-G

H-

ii--A D

-E

-F

-G

-H

_=-K

_M

-H

_ K

-MNI

[image:4.508.59.453.68.236.2]

-N -0 --0

PO

FIG. 2. Gelelectrophoresis

offfragments

of T3DNA produced by double digestion. (A)T3DNA wasdigested with MboI (lane1)andthen withHindIII(lane2),BgllI(lane3),KpnI(lane4), Xbal(lae5),orAval(lane 6). (B andC)T3 DNAwasdigestedwith MboI(lane 1)orHpaI(lane2)orwithHpaIandoneotherenzyme, asfollows: MboI(lane3), HindIII(lane4),BglII(lane5),KpnI (lane 6), XbaI (lane7),orAvaI(lane8).DNA fragments were analyzed by electrophoresis in 0.8% agarose gels for 4 h at 50 V(A and B) or in 5% polyacrylamidegelsfor2hat10() V(C).

TABLE 3. Composition of individual restriction fragmentsa

Isolated fragment Recut Fragments observed

HindIII-B MboI C, G

HindIII/BglIIb MboI B, F

XbaI-A MboI B, C, D, F, G, I, J,

nll,080 XbaI-B MboI E, H,nl,665 HindIII-B HpaI F, H, K, L, N,0,R,

nl,880

HindIII/BgllIb

HpaI A, n640, n220

XbaI-A HpaI A, B, C, D, F, G, H,K,L, N,0,R

MboI-A HpaI C, D, M, Q,n700,n680 XbaI-B HpaI E, I, J, M, P, Q,n90

aThe restriction fragments indicated in the first

column were resolved by electrophoresis in agarose gels,eluted from thegels,anddigestedwith a second enzyme, asindicated. The resulting

fragments

were analyzed by gelelectrophoresistogether with a com-plete MboI or HpaI digest of T3 DNA. New fragments (not found in thecomplete digest) are identified by theletter "n"followedby the size (in base bairs).

b

HindIII/BgI

referstothefragmentof T3 DNA that isdefinedby the HindIII andBgllIsites (see Fig. 3).

one end contained

'P.

These fragments were then subjected to limited digestionwith HpaI

andanalyzed by gelelectrophoresis. The incre-ments in the sizes of theend-labeledfragments provide an indication of the arrangement of HpaIsites neartheends oftheT3 DNA

mole-cule (13). From the smallerHindm fragment,

the arrangement ofHpaI

fragments

at theleft

end of the map was determined to be R-O-N-F-. From the data in Table 3, the other HpaI fragmentsthat lie to the left of the HindIII site (inHpaI-G) arefragments H, K,andL. Diges-tionofDNA from previouslycharacterized

dele-tion mutants of T3 (see below) demonstrated

that HpaI fragments H and G are adjacent in the genome, and thus the order of the HpaI fragments from the left end must be R-O-N-F-(K,L)-H-G-. Partial digestion of the larger HindII end-labeledfragmentindicated thatthe orderoffragments at the right end of the genome is -E-P. The only other HpaI fragment that arises from DNA to the right of the XbaI site (in HpaI-J) is HpaI-I (Table 3), and thus the order of fragments at the right end must be -J-I-E-P.

To determine thepositionsofHpaIfragments

Q andM,T3 DNA was digested to completion with AvaI and thenpartially digestedwithHpaI. This resulted in the appearance of a 760-base pairfragment that was not present in either a completeHpaI digest or a partialHpaI digestof intact T3 DNA (data not shown). This new fragment couldonlyarisefrom thefusionof the 0.2-kbpart ofHpaI-Jthat lies to the left ofAvaI withHpaI-M (560 basepairs).Thus,HpaI-Mis adjacent to HpaI-J.

To orient HpaIfragments K andL,T3DNA was progressively shortened by

-exonuclease

treatment andsubsequently digestedto comple-tion with HpaI (data notshown). TheHpaI-K

fragment was more sensitive to exonuclease

digestion than HpaI-L and must therefore lie closer to the endof themolecule. This finding completestheHpaImap.

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180 BAILEY, DEMBINSKI, AND McALLISTER

Double digestion with

MboI

and HpaI confirms

the

physical

maps. With the excep-tion of the HpaIfragments that lie within MboI-A,theHpaI andMboI maps were derived inde-pendently of one another. The

arrangement

of fragments presented in Fig. 3 predicts the ap-pearance of 12 new bands in an MboI/HpaI double digestion of T3 DNA. With theexception of n12(which is too smalltobedetected in the gel analysis used

here),

all of these predicted bands were

observed

(Fig. 2 and Table 2).

There

areveryfew ways in which the

HpaI

andMboI mapscanbe redrawn whichare

consistent

with theresults of this

experiment,

andnoneofthese

alternates

isconsistent with the other data

pre-sented in this

report.

Orientation

of the genetic and

physical

maps.

Restriction

of DNA from

previously

characterized

mutantsofT3

(16)

indicated that

deletions which affect the T3

early

genes

(which

lieatthe left end of the T3

genetic map)

affect

the

migration

of

HpaI fragments F, G,

and H

B A

B A

A C B

I

A B

A B

G C F B I.J. D

iiI IIA I-H E

RON. F IIIKL H G A B C

I

5 7 4 11 3 1 9 8

L L &l I i lJ

D OMJ E P

U I I Hpal

612 10 2

LJ aswr Mbol/Hpa

[A07 1 1 E313 earygenes

10 15 20 25 30 kb

FIG. 3. RestrictionmapofT3DNA. Thecleavagesitesofsevenrestrictionenzymesareindicated. Thenew

fragmentsgeneratedby digestion ofT3 DNA with both MboI andHpaI (thosewhicharenotobservedupon

cleavage ofthe DNAbyeitherenzymealone)aredesignatedasnlthroughn12. Theorientationsofthegenetic andphysicalmapsandthelocationsofgenesintheearly regionarebaseduponargumentspresentedinthe text.Distancesfromtheleftendofthegenome(inkilobases)aregivenonthebottom line.

.., ~~~~~~~~~~~~~~~~~~~...

:-- ...

1,

-,--

---FIG. 4. HpaI digestion ofDNA from T3 deletion mutants. T3 DNA isolatedfrom mutantphages as indicated inthe textwasdigestedwithHpaIandanalyzed byelectrophoresisin 0.8%agarosegels(A)orin

5%polyacrylamide gels (B).Lane1,T3 ;lane2,R1;lane3,R16;lane4,LG123;lane5,R14;lane6,LG114. Thegenetic constitutionofthe mutants isgiveninTable4.

HindIII Bg111 Kpn

Xba Ava Mbo

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TABLE 4.Analysis of DNA fromdeletionmutantsofbacteriophage T3

taGeneticconstitu-

NAHpaI

Size ofnew Estimatedsize

Mutante

Mutamtioni

Geneticconatltu-

mRNAaffecteda fragment(s)

Pgps)b

fragment (base

(base

of deletion

pairs)'

Rl 0.3- 0.3, 0.7 F 2,100 415

R16 0.3-,0.7- 0.3,0.7,1.0 F 700 1,815

LG123 Ligase NSd H,G 1,400 1,900

R14 Lgase 1.0, 1.1,1.3 G 400 1,700

LG114 Ligase 1.3 G -e

aSeereference 16. bSee Fig. 4.

'Sizes of deletions were estimated by subtracting the

size

ofthe new fragment from the size(s) of the fragment(s)missing.

dNS, Not studied.

c-, It islikelythatthis deletionremovestheHpaIsite betweenHpaI fragmentsG and A and

results

inthe fusion of theremaining portionsof thetwofragments.Because of thelargesize ofHpaI-Atheextentofthis deletion hasnotbeen determined.

(Fig. 4 and Table 4). These fragments must

therefore arisefrom the genetic left end of T3 DNA(Fig. 3). Thephysicalextentofmostof the deletion mutations canbe estimated from the sizes of thenew

HpaI

fragments

observedafter cleavage ofmutantDNA (Table 4).

One of thedeletionmutants (LG123) affected

HpaI

fragments

H and G but no other HpaI

fragments; this result indicates that these two

fragmentsmustbeadjacent in the T3genome.

Physical boundaries of the T3 early

genes. Toaidinthe determinationof the phys-ical boundaries of the early genes, individual early mRNA's that had been isolated from in-fectedceliswerehybridizedto

HpaI

restriction fragments of T3 DNA (Fig. 5). Gene0.3and0.7

mRNA's hybridized to

HpaI-F;

gene 1 mRNA hybridized strongly to fragments K, L, and H

and

weakly

to

fragments

FandG; andgene 1.1

and 1.3mRNA'shybridized toHpaI-G butnot toHpaI-A.

Twodeletions (R14 and R16) which affect the size of thegene 1mRNA altered the

migration

of HpaI fragments F and G (Table 4). From these results and those described above, it is

apparent that gene 1 must extendinto regions of thegenomedefinedby HpaI

fragments

Fand G. The size of the gene 1 mRNA (ca. 3,200

nucleotides[15]) exceeds byca. 500nucleotides

the

coding

capacity ofHpaI fragments K, L, and

H

(which

lie between

HpaI-F

and HpaI-G).

Since the gene 1.1 and 1.3mRNA's can account for all but ca. 300 nucleotides of the coding

capacityofHpaI-G(15), gene 1 can extend into this fragment a maximum of 300 base pairs.

Thus, the position ofgene 1 onthe physicalmap canbeestimated withinca.300 base pairs.

If oneassumesthatthe coding regions for the T3earlygenes arecontiguous,asis thecase for T7 (6), thelocations of the other early genes

1

2

3

4

5

6

-F

z-G

~-H

-K

-L

FIG. 5. Hybridization ofT3earlymRNA'stoHpaI restrictionfragmentsof T3 DNA. Individual T3 early mRNA'sisolatedfrominfectedcellswerehybridized

toHpaIrestrictionfragments ofT3 DNA that had been denatured and immobilized on nitrocellulose filter strips.Lane 1, gene0.3mRNA;lane2,gene0. 7

mRNA;lane3, gene1mRNA;lane 4, gene1.1mRNA; lane 5, gene1.3mRNA; lane6, mixtureof samples fromlanes I through 5.

relative to gene 1 can be estimated from the sizes of theirrespectivemRNA's(Fig.3). These esti-mates are in agreement with the hybridization results presentedinFig.5.

DISCUSSION

The results of this workdefine the locations

of 32 recognition sites for various restriction MAP OF T3 DNA

181

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182

BAILEY, DEMBINSKI, AND McALLISTER

endonucleasesthatcleaveT3 DNA.Because T7 DNA iscut by manyofthese same enzymes (10, 12), thelocationandpresence of these physical reference pointsin the DNA molecules of these two bacteriophagesmay be used asmarkers in

considering the evolutionary divergence of the twoviruses.

The T3andT7 DNAmolecules shareasimilar geneticorganization (1,7)andexhibit extensive partialsequencehomology (4).Overcertain re-gionsofthe DNA,partialsequencehomologyin excessof85 to90% has been observed (4) (Fig.

6); one might expect that the restriction sites within these regions would be conserved. DNA molecules from both of these virusesarecleaved byHpaI, but inacomparison of the

electropho-retic patterns of thetwodigests very few of the

fragments appearedtocomigrate (15). The

gen-erationofidentically sized fragments from

cor-responding regions of tworelated DNA mole-cules requires that the restriction sites which define both ends of these fragments must be conserved and that no structural alterations (e.g., insertions or deletions) have taken place betweenthem. These conditionsareapparently

notsatisfied for mostof the

HpaI

sites in the

twomolecules.

The physical mapsof T3 and T7 DNAs are

compared in detail in Fig. 6. By summing the sizesof theHpaI fragments that arise from T3 DNA (Table 1),we estimate that the length of thismolecule is38.74kb; this is1.26kb less than thelength of theT7DNAmolecule (40 kb) (10). Electrophoresis of intact T3 and T7 DNAs in low-percentage agarose gels

confinns

that the

0 5

T3 genome issmallerthan that of T7 (datanot shown). Previousreportsindicatedthat the two DNAmolecules werenearly identicalin length

(4); thereasonsfor this discrepancyare notclear, but itmaybe duetodifferences in the strains of

T3from which the DNAwasisolated (15).

Re-gions of the T3 andT7DNAmoleculesin which

DNAcontentvaries have beendetected in

het-eroduplex analyses in the form of single-stranded loops of differing lengths (4); these occurjusttothe right ofgene 1 (near 6 kb on

the T7 map), near genes 3 and4(ca. 10.5 kb), nearthe left end ofgene 5 (13kb), and within gene 12 (near23kb). Despite the length

differ-encesbetween thetwoDNAs, somerestriction sites in the two molecules can reasonably be

proposed tocorrespond, as indicated in Fig. 6.

Given thehigh degree of partial homology de-tected byheteroduplex analysis, thereare

sur-prisingly few sites conserved. Of68sites mapped inthetwomolecules, only10 to14appear tobe related, andmostoftheseareinregions of high (>85%)partial homology.

If one assumes that restriction sites which

exhibit close positional correspondence are in

fact

related,

itispossibletoestimate theextent

of sequence

divergence

from the number of cleavage sites that have been lost. The fraction

(S)

of restriction sites

comprising

nnucleotides that would remain unchanged aftersome

pro-portion (p) of the nucleotides ina DNA mole-cule have

undergone

randomsubstitutionevents

is

given by

the

following

formulap=

-(1/n)lnS

(18). Considering the two DNA molecules

to-gether,

there are 54 sitesfor whichn = 6

(the

10 15 20 25 30 35 40 kb

3070 1 1.113

_ 00 0 @300o 00 aA wUJ @00s

vr In IIUnI a Ii v

v * * V *CDOVO v01 ** A 0 V @0 A @OM *@ (MD *

13 0.7 1 113

o

l

r

l

T3

T7

I

r:~

*Hpa oMbol AvaI &Kpn YBgl 11 Y Xba T HodIII

FIG. 6. Alignmentof the T3 andT7physicalmaps. The DNAsofT3and T7arerepresented bysolid lines whicharedrawntothesamescale; distancesfromtheleftendsofthe moleculesaregivenin kilobasepairs.

Positions of the recognitionsitesforthe enzymes indicated arerepresented bysymbolsgiven in thekey.

Cleavage sites which maycorrespond in thetwoDNAmoleculesareconnectedbysolid lines.Atthebottom of the figure theregionsofT7DNA which exhibitadegreeofpartialhomology with T3 DNAinexcessof85%,

asdetermined by heteroduplexanalysis (4),areindicatedbyopen boxes.Thelocationsofthe T3 and T7early

genes(genes0.3through1.3)arealsoindicated.

J. VIROL.

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[image:7.508.66.453.439.596.2]
(8)

VOL. 35,1980

MboI sites for whichn = 4arenotconsideredin

this

analysis).

Of

these,

12 are conserved, and

thusS=12/54.From therelationship described

above, the average basesubstitutionrateforthe twoDNAmolecules is 25%. Ifoneconsidersonly thehighly conserved regions from ca. 28 to 38 kb onthe T7 map (Fig. 6), there are 17 sites for which n=6,andof these 10 areconserved.The average substitutionrateforthisregion is there-fore9%.

Itshould be stressed that this analysismakes certainassumptions whichmay notbevalid. For example, as noted

above,

we do not know whether the cleavage sites which show close positionalcorrespondence in the two DNA

mol-eculesareinfactrelated.Similarly, the statisti-cal derivation of theaverage basesubstitution

rate assumesthat substitutionevents occur

ran-domly

(18). Onarandombasisonewould expect

that enzymes which recognize a six-base se-quence would cleave molecules the size of T3

andT7DNAsanaverageof9 to 10times (15); this is clearlynot the casefor mostof the en-zymesstudied here.

ACKNOWLEDGMENTS

This work wassupported by Public Health Service grant GM21783 to W.T.M. from the National Institute of General Medical Sciences and bygeneral research support funds from theCollegeof Medicine andDentistry of New Jersey. J.N.B. wassupported by a Rutgers University fellowship. D.R.D. was

supportedby the Summer Research Fellowship Program of Rutgers Medical School.

ADDENDUM

We havemapped two additional restriction enzyme sites. Bcll cleaves T3 DNA once, approximately 35 basepairstotheright of HindIII. BstEII cleaves once inHpaI-C, 27,160 base pairs from the left end of T3 DNA.

Thefollowingenzymes do not cut T3DNA: BamHI, EcoRI,PstI,SalI,SmaI, SstII,XhoI, BglI,andPvuII.

LITERATURE CITE

1.Beier, H., and R. Hausmann. 1973. Genetic map of bacteriophage T3. J. Virol. 12:417-419.

2. Bendet,I., E.Schachter,and M. A. Lauffer.1962.The sizeof T3 DNA. J. Mol. Biol.5:76-79.

MAP OF T3 DNA

183

3. Chamberlin, M., J. McGrath, andL Waskell.1970.

New RNApolymerase from Escherichiacoliinfected

withbacteriophageT7. Nature(London)228:227-231. 4.Davis, R. W., and R. W. Hyman. 1971. Astudy in

evolution: the DNA basesequencehomologybetween

coliphagesT7and T3. J.Mol. Biol. 62:287-301. 5. Dunn,J. J., F. A. Bautz, and E. K. F. Bautz.1971.

Different template specificities of phage T3 and T7 RNApolymerases. Nature(London)NewBiol. 230:94-96.

6. Dunn, J.J., and F. W. Studier. 1973.T7earlyRNAs and Escherichia coli ribosomal RNAs are cut from large precursor RNAs in vivo byribonucleaseIII. Proc. Natl.Acad. Sci. U.S.A. 70:3296-3300.

7. Hausmann,R. 1976. BacteriophageT7genetics. Curr. Top.Microbiol.Immunol.75:77-110.

8. Kerr,C.,and P. D.Sadowski.1972.Gene6ezonuclease

ofbacteriophage T7. I.Purificationandpropertiesof the enzyme.J. Biol. Chem. 247:305-310.

8a.Maxam, A.M.,and W.Gilbert1980.Sequenching

end-labelled DNA with base-specific chemical cleavages. Methods Enzymol.65:499-559.

9. McAllister,W.T., and C. L Barrett. 1977. Hybridiza-tionmapping of restriction fragments from the early region ofbacteriophageT7DNA. Virology 82:275-287. 10.McDonnell, M. W., M. N. Simon, and F. W. Studier. 1977.Analysis of restriction fragments of T7 DNA and

determinationofmolecular weights by electrophoresis inneutral and alkaline gels. J. Mol. Biol. 110:119-146. 11.Roberts, R. J. 1978. Restriction and modification

en-zymesand theirrecognition sequences. Gene 4:183-193. 12.Rosenberg,A.H.,M.N.Simon,F. W.Studier,and R. J.Roberts. 1980.Surveyandmappingof restriction endonucleasecleavagesites inbacteriophageT7DNA. J.Mol. Biol. 135:907-915.

13. Smith,H.O.,andM. LBirnstiel.1976.Asimplemethod for DNA restriction sitemapping. Nucleic Acids Res. 3:2387-2398.

14. Studier, F. W.1973.Analysisofbacteriophage T7early

RNAs andproteinsonslabgels.J.Mol.Biol. 79:237-248.

15. Studier, F. W. 1979. Relationships among different strains of T7 and among T7-related bacteriophages. Virology95:70-84.

16. Studier,F.W.,and N. R.Mowva.1976.SAMasegene of bacteriophage T3 is responsible for overcoming host restriction. J. Virol. 19:136-145.

17. Studier,F.W.,A.H.Rosenberg,M.N.Simon,and J. J. Dunn. 1980. Genetic andphysical mapping in the early region of bacteriophage T7 DNA. J. Mol. Biol. 135:917-937.

18. Upholt,W. B.1977.Estimationof DNA sequence diver-gence fromcomparison of restriction endonuclease di-gests. Nucleic Acids Res. 4:1257-1265.

19.Vogelstein,B., and D.Gillespie.1979.Preparative and analyticalpurification of DNA from agarose. Proc. Natl. Acad. Sci.U.S.A.76:615-619.

on November 10, 2019 by guest

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Figure

FIG.1.mentstheletters.ted Restriction fr-agmentsof T3 DNA. Frag- generated by the restriction enzymes are plot- as log This of provides size (in kilobases) and are identified by an idealized representation of patterns observed upon gel electrophoresis of thefragments.
TABLE 2. Fragments generated by cleavage of T3 DNA with two restriction endonuckasesa
FIG. 2.polyacrylamidefragmentsas6).with (B follows: Gel electrophoresis offfragments ofT3DNAproduced by double digestion
FIG. 3.fragmentsandtext.cleavage Restriction map of T3 DNA. The cleavage sites ofseven restriction enzymes are indicated
+3

References

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