Interruption-deficient mutants of bacteriophage T5. II. Properties of a mutant lacking a specific interruption.

0022-538X/79/02-0726/09$02.00/0

Interruption-Deficient

Mutants

of

Bacteriophage

T5

II.

Properties of

a

Mutant

Lacking

a

Specific

Interruptiont

STEPHEN G. ROGERS,: NANCY V. HAMLETT,§ANDMARC RHOADES*

BiologyDepartment, The Johns HopkinsUniversity, Baltimore,Maryland21218

Receivedfor publication15August1978

An examination wasmade of the properties ofT5HA4, a mutantof

bacterio-phageT5that lacks thesingle-chaininterruption thatoccurs at7.9% fromthe left

endof the genome. The DNAs of T5HA4 andthe wild type werecomparedby

electrophoresis in agarose gels of both single-stranded fragments produced by

denaturation and duplex fragments generated by sequential treatment with

exonuclease III and SI nuclease. These studies demonstrated that T5HA4 also

lacksan interruptionthat occurs at99.6% in wild-type DNA. The interruptions

at 7.9 and 99.6% thereforeoccur within the 8.3% of T5 DNAthat isterminally

repetitious. Evidence on the location of other interruptions within the terminal

repetition was also obtained. Analysis of T5HA4 DNA witha restriction

endo-nuclease indicated thatthe interruption deficiency is not due to a deletion or

addition mutation. Theinjectionof T5HA4DNAintoahost bacteriumwasfound

tooccur, as with the wild type,inatwo-step manner.Theinterruptionat7.9% is

thereforenotrequired for stoppingDNAtransferafter the initial 8%segmenthas

beeninjected.

The genome ofbacteriophage T5 contains a

number of site-specific single-chain

interrup-tions within one strand of its linear

double-stranded DNA. As described in the preceding

paper (18), mutants of T5 have been isolated

that eitherlackspecificinterruptionsorare

de-ficient forinterruptionsthroughout thegenome.

The latter class of mutations appears todefine

twogenes that participate in the formation of interruptions in T5 DNA. In contrast, the single-site mutants appearto haveundergone

altera-tions of therecognitionsequencesthat mark the

positions of the interruptions. Mutants have

been obtainedthat lack each of the four

princi-palinterruptions, whichoccur at7.9, 18.5, 32.6,

and64.8% from the left end ofT5DNA,aswell

as onesecondaryinterruption,locatedat45.3%.

The existence ofsingle-site

interruption-defi-cient T5 mutants permits direct testing of the

functionalsignificance ofspecific interruptions.

Thepresentreportisconcerned with the

prop-erties ofa mutantthatlacks theinterruptionat

7.9%. This site is of interest for several reasons. It occurs close to the boundary between the

repeated andnonrepeated segmentsatthe left

end of the genome. The terminal repetition in

t Contributionno.973 from the DepartmentofBiology, TheJohnsHopkinsUniversity,Baltimore,Md.

tPresentaddress:DepartmentofMicrobiology,TheJohns HopkinsUniversity School of Medicine,Baltimore,MD 21205. §Presentaddress:Department ofBiological Sciences, Tow-sonStateUniversity, Baltimore,MD 21204.

T5 DNA has been estimated tocomprise 8.3%

ofintact DNA (16, 17). The 7.9% interruption

also occurs close tothe boundary between the

first- and second-step transfer segments of T5

DNA. T5 is unique in that injection of its

ge-nomeintoahostbacteriumoccursbyatwo-step

process. Initially, an 8.0 to 8.5% segment, the

first-step transfer (FST) DNA, is injected (14).

Expression of at least two FST genes is then

required before the remaining 92% of thegenome

istransferred (11). The FSTsegment appears to

be at the left end of the T5 genome (10, 20).

Because of itslocation, the interruptionat 7.9%

is therefore a possible candidate for the signal

that terminatesthe firststageof DNA transfer.

The results obtained in this study have

con-firmed that the 7.9%interruptionlies within the

terminal repetition in T5 DNA. Mutants that

lack the 7.9%interruption are alsodeficientfor

an analogous site at 99.6%. In addition, these

studies haveprovided evidence onthe location

of various secondary interruptions that occur within the terminalrepetition.Theinterruption

at7.9%,however,doesnotappear toplayarole

in DNAtransfer.In theabsence of this

interrup-tion, injection occurs in the normal, two-step

manner.

MATERIALS AND METHODS

Bacteria and bacteriophage. The bacteria and

wild-type T5 phage used in this study have been

describedpreviously (18). T5st(102) isaviable,

heat-726

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stable deletion mutant that lacks 10.2% of T5st(+)

DNA (22). T5HA4,which lacks the 7.9%interruption,

was originallyisolated in T5st(102) as described in the

preceding paper (18). The original isolate wasable to

grow on Escherichia coli B23 and B40sul, but was

not able to grow on E.coli F. The inability to grow on

E. coli F was eliminated by backcrossing against

T5st(+). The recombinant obtained, however,

ad-sorbed poorly on E. coli F. A fast-adsorbing, HA4

recombinant was obtained by performing a second

backcrossagainst T5st(+). This stock was usedfor the

experimentsreported here.

Analysis of T5HA4 DNA. The procedures for electrophoretic analysis of T5 DNA have been

de-scribedpreviously. Single-strandedDNAwasanalyzed

in 5-mm-thick horizontal agarose slab gels (16, 19).

Double-stranded DNA underwent electrophoresis

similarlyorincylindrical vertical gels (16). Treatment

of T5 DNA with Aexonuclease, HpaIrestriction

en-donuclease, orexonuclease III and SI nuclease was

carried out as described (5, 16).

Measurement of two-step transfer of T5 DNA.

Theprocedure used to measure two-steptransfer of

T5 DNA was that of Lanni (11), except that shear

treatmentof infected cellswascarried outfor6minat

"HIGH"speed in a VirTis 45 homogenizer.32P-labeled

phage were purified onCsCl step gradients.

Isolation of intracellular DNA from FST

com-plexes.FST complexes (bacteria to which T5 phage

have been adsorbed and haveinjectedonly the FST

DNA), prepared and sheared as above, were

centri-fuged, washed, and resuspended in0.1MTris-0.1 M

NaCl-0.1 MEDTA, pH8.0. DNAwasextracted from

thecomplexes with sodium dodecyl sulfate (SDS) and

Pronase (2) and was dialyzed against 10 mM

Tris-hydrochloride-0.1 mM EDTA (pH 8.0). In one

exper-imentanattemptwasmadetoseparate theFST DNA

from the host DNA by using the

lysozyme-EDTA-Triton X-100 procedure used for plasmid DNA (9).

The FST DNA, however,wasfound associated with

the membrane-host DNA fraction. The FST DNA

wasreleased from thiscomplex by theSDS/Pronase

procedure used for intact cells (2) andwasseparated

from the bulk of the host DNA by sedimentation

through6 to20%neutralsucrosegradients.Thepooled

fractions were concentrated by pressure dialysis

against 10 mM Tris-hydrochloride-0.1 mM EDTA

(pH 8.0) beforeelectrophoretic analysis.

RESULTS

Thesearch forinterruption-deficientmutants

of T5describedintheprecedingpaperresulted

in theisolation ofseven mutantsthatlacked the

single-chaininterruptionat7.9% (18). These

mu-tants were identified because, after

denatura-tion, their DNAs lacked the single-chain

frag-mentsthat haveoneendat7.9%. These include

fragments 18, 21, and 22 (Fig. la and b). The

first mutant isolated in this category, T5HA4,

waschosen for furtherstudy.The HA4 mutation

hasalreadybeen shown to be neither dominant

norrecessivetothewild type(18). Phage

carry-ingthismutation are viableand have thesame

(a) 5

(b)

(C )

08 24 3.541 79 165 32 6

21 Is is3

39 22 i 4A 7 IA

29 26

27 29

A

391 .Ol

33 C

29 D

T

27 i E so

FIG. 1. Maps of single-chain interruptions and

fragments attheleft endof wild-typeT5 and T5HA4

DNAs.(a) Positionsof interruptionsinT5st(+) DNA,

expressedas apercentage of wild-type lengthfrom

theleft endofthe molecule. Thestrandpolaritiesand

interruptionsare shown asdetermined inreference

16. (b)Locationsofsingle-chain fragmentsattheleft

endof T5st(+) DNA. With theexceptionof fragment

30, whichoccurs between 0and3.3%o, allpreviously

mapped (16)fragments areshown. Thest(102)

dele-tion occurs between the18.5and 32.6ointerruptions.

(c) Locationsofsingle-chainfragmentsattheleftend

of T5HA4 DNA. The positions ofthe left ends of

fragments AthroughEwerecalculatedby

subtract-ing their molecularweights, giveninFig. 3, fromthe

molecularweightofthe intervalfrom0to 18.5%, as

determinedelectrophoretically (16).There isasmall

discrepancy between this value, 18.0%o of T5st(+)

DNA, and the valueof18.5%determined byelectron

microscopy(21).

averageburst sizeasthewildtype.

Analysis of single-stranded fragments of

T5HA4 DNA. As described below and in the

preceding paper (18), loss of fragment 18 and

manyof the small fragmentsatthe left end of

the genomeclearly indicatesthat T5HA4lacks

the interruption at 7.9%. However, since the

terminal repetition inT5 equals8.3% of intact

DNA, thesequencethatcontainsthe7.9%

inter-ruption should also occurclosetothe right end

of the molecule. Evidence has been obtained

that interruptions occur at analogous sites

within thetwocopies of the terminalrepetition

in T5 (16). Thus, if the previous analyses are correct, T5HA4 should lack twointerruptions,

one at 7.9% andone atapproximately99.6%.

The existence ofan interruption at 99.6% in

wild-type T5 DNA, and its absence in T5HA4

DNA, can be demonstrated by comparing the

single-chain fragments of the two DNAs. Only

the wild type should have a short fragment of

approximately450bases that occurs at the right

end of the interrupted strand of T5 DNA. To resolve the smaller single-chain fragments, we

subjecteddenaturedT5st(+) andT5st(+) HA4

DNAs to electrophoresis in 1.4% agarose slab 29,1979

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ROGERS, HAMLETT, AND RHOADES

gels. The results (Fig.2) showthat, forfragments

of less than 4% ofan intact strand, the single

chains of thetwoDNAs wereextremely similar.

However, thefastest migratingofthe detectable

1 2 3

TI'

FIG. 2. Electrophoresis ofthe small single-chain fragmentsof T5 DNA. Electrophoresis wascarried

outinahorizontal 1.4%agaroseslabgel.Thelength

ofthegelwas 15 cm. Each slot contained 15[Lg of

alkali-denatured DNA. Slot1,T5st(+)DNAafter1%

digestion with A exonuclease; slot2, T5st(+) DNA;

slot3, T5st(+)HA4DNA. Theupperarrowindicates

theposition, ingels1 and2, offragment28, which

equals 3.8% ofan intact strand. The lower arrow

indicates the 5'-terminalfragmentthat ismissingin

T5HA4DNA.

wild-type fragments appeared to be absent in

HA4 DNA. Since the external 5' end of the

interrupted strandoccurs attherightend ofthe

molecule (16),theterminallocation of this

frag-ment canbedemonstrated by limited digestion

ofwild-type DNA withAexonuclease,anenzyme

that isspecific for external5'termini(4,15). The

result ofdigestion of T5st(+) DNAto 1%

acid-soluble material withAexonuclease is shown in

slot 1 ofFig. 2. The only effect of exonuclease

treatment wasloss of the smallfragmentthat is

missingin T5HA4 DNA. Thisfragment

there-fore occurs atthe external 5' end of the

inter-ruptedstrand. Digestionto0.5% with A

exonu-cleasewas sufficient toyield the resultsshown

inFig.2.Identical electrophoreticpatternswere

also obtained with up to 5%digestion. The

re-sults with higher levels of digestion were not

surprising,since,withtheexception of thesmall

terminalfragment, allof thefragments that

oc-cur within the right terminal repetition also

occur atthe left end of theDNA. The size of the

5'-terminalfragmentidentified in Fig.2hasnot

been accurately measured. Its electrophoretic

mobility, however, is compatible with the

pre-vious estimates of450bases.

Analysis ofduplex fragments of T5HA4

DNA. Loss of theinterruptionsat 7.9and99.6%

should resultinalterations ofmanyofthe

single-chainfragments produced by denaturation of T5

DNA.Asnotedabove, fragments that haveone

terminusateitherinterruption should disappear

while newfragmentsthat spanthesesitesshould

become evident. Analysis of these new

frag-ments, manyofwhichwillhaveoneterminusat

a secondary interruption, can provide further

information on the structure of T5 DNA. In

principle, this informationcanbe obtainedfrom

analyses ofsingle-chain fragments as shownin

Fig. 2. However, the clearest results have been

obtained fromanalysis of the analogous duplex

fragments generated by sequential treatment

with exonucleaseIII ofE. coli and SI nuclease

of Aspergillus oryzae. Limited hydrolysis by

exonuclease III at the 3' side of each

interrup-tion, followed by digestionof thesingle-stranded

regions by SInuclease, resultsincleavageof the

duplexmolecule at the site of eachinterruption

(16). Since theelectrophoretic mobilitiesof

du-plexmolecules arealmostentirely dependenton

molecularweightinagarosegels (24), analysisof

these fragments yields an accurate mapofthe

interruptions.

The results of sequential exonuclease III-SI

nuclease digestion of T5st(+) and T5st(102)

DNA in thepresence and absence of the HA4 mutation are shown in Fig. 3. Electrophoresis

was carried out in 0.5% agarose gels toresolve

thelargerduplexfragments. Thefragmentsare

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2

8-9-.-..

10,A

-

B-

11- C-

D - 15-

16-3

-8

-9 -10 -11

-7

-15 -16

4

'-3 -4 -6

-7

-8

-9

-10

-11

-13 -15

-18

FIG. 3. Electrophoresis ofthelargeduplexfragments generated byexonucleaseIII-SI nucleasetreatment

ofT5 DNA.Electrophoresiswascarriedoutincylindrical0.5% agarosegels. Gel1, T5st(102)HA4DNA; gel

2,T5st(102) DNA; gel 3, T5st(+)HA4 DNA; gel 4, T5st(+) DNA. The fragments thatoccurin T5st(+) DNA

arenumbered in orderof decreasingsize(see 16). Electrophoresisin the presenceof referencemoleculeswas

employedtoidentifythe numberedfragmentsin the othersamples. The lettersAthroughEdenotefivenew

fragments that appear inT5st(102)HA4DNA. Theirmolecularweights,expressedas apercentageofT5st(+)

DNA,are17.4%for B, 15.8%for C, 14.6%for D,and14.0%forE.FragmentAcomigrateswithfragment10,

andtherefore equalsapproximately18.4%of T5st(+)DNA.

numbered in order of decreasing size as they

occurinT5st(+) DNA (gel 4). The deletionin

T5st(102) DNA does not remove an

interrup-tion.Therefore,theonlydetectable alterationin

thisDNAisloss oftwofragments,7and 13,that

span the deletion. Fragment 7', the shortened

version offragment7, canbeseeninFig.3 (gel

2). Fragment 13' istoosmalltobeseen inthis

experiment.

The HA4 mutation resultsinloss offragments

7, 18, 21, and 22 of those visible in Fig. 3. A

number ofnewfragmentsare evident.Analysis

ofT5st(102) DNAis most useful inthisregard

since severalof thenewfragmentsareobscured

by fragment 13in T5st(+) DNA. The five new

fragments detectableinT5st(102)HA4DNA are

labeledAthroughE inFig.3 (gel 1). The

exist-ence of fragment A is implied because of the

1

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730 ROGERS, HAMLETT, AND

increasedintensity ofthebandcontaining

frag-ment 10. Since no interruptions have been

lo-catedbetweentheprincipal interruptionsat 7.9

and 18.5% (16, 21),fragments Athrough E

pre-sumably extend leftward from 18.5% to various

sites near the left end of the molecule. The

positions of thesefragments, basedonthe above

assumption and their molecular weights, are

shown inFig. 1. FragmentA appears toextend

fromtheinterruption of 18.5%totheleft end of

the molecule. The other four fragments

termi-nate at 0.6, 2.2, 3.4, and 4.0%. These positions

agreewell with the locations of 0.8, 2.4,3.5,and

4.1%previously determined for secondary

inter-ruptions within the left terminalrepetition in T5

DNA(16).

Thesmaller fragments generated by

exonucle-ase III-SI nuclease treatment of T5st(+)HA4

DNA were analyzed in 1.0% agarose gels (Fig.

4). T5HA4 DNAwould be expected to lackall

shortfragments that extend leftward from 7.9%

inwild-type DNA. Fragments that extend

right-wardfrom the left end of theDNA tosecondary

interruptions in the terminal repetition should

not be altered by the HA4 mutation. As

indi-cated in Fig. 4, T5st(+)HA4 DNA lacks

frag-ments 21,22, 24, 26,and28andretainsfragments

27, 29, and30. This resultagrees with the

pre-viouslydetermined locations of thesefragments

(Fig. 1).

T5st(+)HA4DNAcontainsanumber ofnew

small duplexfragments (Fig. 4). Theseare

pre-sumablyderivedfrom therightend of the DNA

where loss of the 99.6% interruption will result

in fusion of the short terminal fragment

de-scribed above(Fig. 2)tothefragmentscontained

within the right terminal repetition. Thus,

slightlylonger versions of fragments 22,24, 26,

and 28should bepresentinT5HA4DNA. Bands

that could correspondtomostof the predicted

fragmentswereseen;however,itwasdifficultto

analyze theright end of T5 DNAbyuse ofthe

exonucleaseIII-SI nucleaseprocedure. Since the

3'end of the intact strand occurs atthe right

end of theDNA, fragments that extendto the

right end will be degraded from both sides by exonuclease III. Thiseffect, especiallyif

exonu-cleaseIII actspreferentiallyatduplex3'termini,

precludes accurate measurement of molecular

weights.

Analysis witharestriction endonuclease.

T5HA4 was isolated after hydroxylamine

mu-tagenesis,whichinducespointmutations.

How-ever, the procedure used for mutagenesis in-volved heating in the presence of a chelating

agent (23). Since T5 deletionmutations are

se-lected in asimilarmanner (8), theinterruption

deficiencyof T5HA4 could beduetoloss of the

region containing the 7.9 and 99.6%

interrup-J. VIROL.

18

21

22

24

26

27

28

29

30

FIG. 4. Electrophoresis of the small duplex

frag-mentsgeneratedby exonucleaseIII-SInuclease

treat-mentofT5 DNA.Electrophoresis was carried out in

ahorizontal1.0%oagaroseslab gel. The length of the

gel was 8.5 cm. Slot 1, wild-type T5 DNA; slot 2, T5HA4 DNA.

tions. Althougha deletion in HA4 DNAmight

have been detected by the studies described

above,thispossibilitycan bedirectlytestedby

restriction enzyme analysis. The mostdetailed

restriction map of T5currently availableisthat obtainedbycleavagewithanenzyme from

Hae-mophilusparainfluenzae, HpaI. The 26

frag-ments generated by HpaI cleavage ofT5st(+)

DNA have been ordered (5). The 11thand 17th

largestfragmentsare knownto contain the 7.9

and 99.6% interruptions, respectively. The

re-sults of HpaI digestion of T5st(+) and

-1

2

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SPECIFIC 731

T5st(+)HA4 DNA are shown in Fig. 5. Each

digestwasanalyzedin0.7 and 1.5%agarosegels

toresolvethe entire spectrum offragments.The

mobilitiesof all 26HpaI fragments, including11

and 17, didnotappeartobe altered in T5HA4

DNA. A small alteration infragment 11might

be difficultto detect since thisfragment

comi-1 2 3 4

2 3

20

21,22

1H112,

FIG. 5. Digestion of wild-type T5 and T5HA4

DNAs withHpaIendonuclease. Electrophoresiswas

carried out in 0.7%o(gels 1 and2) and 1.5% (gels3

and4) cylindricalagarosegels.Gels1and3,T5st(+)

DNA; gels2 and4,T5st(+)HA4 DNA.Fragment 11,

visibleingels1 and2, spansthe 7.9% interruption.

Fragment17,visible ingels3 and4,spansthe 99.6%

interruption.

grateswith fragments 12 and 13. Fragment 17,

however, is well separated from fragment 16,

which is only50basepairs larger. Consequently,

adeletion of20to30base pairs from fragment

17shouldhavebeeneasilydetected. Itis

there-fore likely that the HA4 mutation is a single,

hydroxylamine-induced base transition.

Two-step DNA transfer by T5HA4. The

interruptionat7.9%has been proposedtobea

signalormechanical barrierthatprevents

injec-tion beyond the FST (first-step transfer)

seg-ment of T5 DNA (1, 3). This hypothesis has

been questioned since mostT5DNA molecules

have additional interruptions between 0 and

7.9% (13, 16). It is also possible that, although

the 7.9%interruptionhasnoroleintermination

of further DNA transfer, it is the preferential

site ofbreakage when infectedcells aresubjected

toshearand thereforerepresentsoneend ofthe

FST segment as isolated. The HA4 mutation

provides a direct means to test both ofthese

hypotheses.

Two-step injection was demonstrated by

in-fecting E. coli F cells athigh concentration in

nutrient-free buffer. Under these conditionsonly

the FSTsegmentistransferredtothe host cell

(14). One portion of the FST complexes was

then subjected to shear forces to remove the

phage capsidand the remaining 92%ofthe

ge-nome. Only a small fraction of the infective

centers survived the shear treatment. Table 1

shows thatthefractionof infectivecenters

sur-viving shearing ofT5HA4 FST complexeswas

identical to that obtained for wild-type

com-plexes.Asecondportionof theFSTcomplexes

was not immediately sheared, but was diluted

into warmnutrientmedium and aerated for 15

min to allow transfer of the total DNA. With

thistreatmentboth theinfectivecentersand the

TABLE 1.

Transfer

into host cells

Conditions for transfer of: Expt Phagegenotype FST DNA Total DNA

no

IC%a 32p%b IC%,, 32p%b 1 Wildtype 7.5 - 100

HA4 5.3 - 111

-2 Wildtype 11.7 10 90 63

HA4 7.3 16 79 66

3 Wild type 0.2 14 106 71

HA4 2.4 12 100 67

aInfective centers after blending, expressed as a

percentage of infective centersinan unblended

con-trol.

b32P associated with thehostcells after blending, expressedas apercentageof32p initiallyadsorbed.

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majority of the 32P became resistant to shear in

bothwild-type andHA4infection.

Characterization of the FST DNA from

T5 wild-type and HA4 infections.

Intracel-lular DNA isolated from shear-treated FST

complexeswasanalyzed by electrophoresis (Fig.

6). T5st(+) HpaI fragmentswereused as

stan-dardsformolecular-weight determination. The

identity of the FST DNA was confirmed by

autoradiography. Thisanalysis showed that the

FST fragments from wild type- and

HA4-in-fectedcellsareidentical. Themajor 32P-labeled

fragment comigratedwith HpaIfragment4,

in-dicatingamolecular weight of6.0 x 106 (5) or

7.9% of T5st(+) DNA. This value agrees well

withpreviouslypublished values of6.3 x 106 for

duplex FST DNA (14) and 3.0 x 106 for FST

single strands (7). Therewas alsoaless

promi-nent 32P-labeled band that migrated ahead of

HpaI fragment 8 and thus has a molecular

weightof 3.8 x106(5). McCorquodaleandLanni

a l 2 3 4

(14) also observed a smallerfragment in DNA

isolated fromFSTcomplexes,but estimated its

sizetobe 1.5 x 106by sedimentation insucrose

gradients. McCorquodale (13) has attributed

thisfragmenttoshearing ofDNAextruded from

phage heads. We do notknow the significance

ororigin of thesmallerfragmentseenhereorits

relationship to the smaller fragment seen by

McCorquodaleand Lanni. Thelarge 32P-labeled

band appears to be intact T5 DNA which

es-capedshear.

The size of the major FST fragment is

ap-proximately equal to 7.9% of the T5 genome.

Thus,inwild-type infection, transferof the FST

DNA must terminate very near to the 7.9%

interruption, and sheartreatmentof the infected

complexes results in breakagenearthis

interrup-tion. The results obtained with T5HA4, which

lacks the 7.9% interruption, show thatthis

inter-ruption neither signals termination of FST

transfernorprovidesashear-sensitive site.

b

1

_-FIG. 6. Electrophoretic analysis ofintracellular DNAfrom FST complexes.(a)Appearance of gelstained

with ethidium bromide; slots 1 and 4, 32P-labeled HpaI-digested T5st(+) DNA; slot2, 32P-labeled DNA

isolatedfrom T5st(+)HA4 FST complexes;slot3,32P-labeled DNA isolatedfrom T5 wild-type FST complexes.

(b)Autoradiograph of thesamegel. Thearrowsindicate thepositions of the6.0x106- and3.8x106-dalton

phageDNAfragments. Thegelwasdriedundervacuum onfilterpaper and thenclamped againstKodak

RoyalBlueX-ray film for autoradiography.

.;.I-% ;%, n..t,v

I&',.

i. 7;.

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SPECIFIC INTERRUPTION-DEFICIENT 733

DISCUSSION

This report describes the properties of

T5HA4,amutantthat lacks oneof thespecific

sites where single-chain interruptions occur in

T5DNA.Analysis ofthesingle-chain fragments

produced by denaturation of T5HA4 DNA

clearly indicates that thismutantlacks the

prin-cipal single-chain interruption that occurs at

7.9%. Sincethis siteoccurswithin the 8.3% ofT5

DNA that is terminally repetitious, the HA4

mutation also results in loss of an analogous

interruptionat99.6%.

T5HA4wasisolated after hydroxylamine

mu-tagenesis andappears tobefree from detectable

deletionoraddition mutations. The interruption

deficiencyis therefore presumably dueto abase

substitution mutation thatoriginally occurredat

either 7.9 or 99.6%. As is the case for other

terminalredundancy heterozygotes, segregation

to homozygosity would be expected to occur

during subsequent growth. Alternatively, it is

possible that the HA4 mutation itself is not

located at 7.9and 99.6%. The observation that

HA4 is neither dominant nor recessive to the

wildtype only indicates thatthisparticular

in-terruption deficiency is not due to lack of a

diffusiblegeneproduct.Otherpossibilities,such

asinactivation ofadistalessential site, havenot

been eliminated. The only direct evidence on

this question has been obtained with mutants

thatlack the interruptionat32.6% (18). Several

mutations that resultinloss of the 32.6%

inter-ruption have been foundtobetightly linkedto

the nearby st(102) deletion (unpublisheddata).

Loss of the interruptionsat 7.9and99.6% in

T5HA4 DNAresults inanumber of changes in

the fragments produced by denaturation or

digestion with exonuclease III and SI nuclease.

Thesechangesreflect the loss of fragments that

normally terminate at 7.9 and 99.6% and the

appearance ofnew fragments that span these

sites. T5HA4 isparticularlyuseful for this kind

of analysis since the terminally repeated seg-ments of T5 DNA contain a high density of

secondary interruptions. These sites appear to

be clustered in the left half of the repeated

region between 0 to 4.1% and 91.7 to 95.8%.

Consequently, T5HA4 DNA contains virtually

all of thefragmentsof less than 4% ofanintact

strand(Fig. 2). These fragments extend from the

left end of the DNA to interruptions located

between 0.8 and 4.1%. Numerous fragments

whichequal4to8%ofT5st(+)DNA aremissing

in T5HA4 DNA. Thesefragmentsnormally

ex-tendleftwardfrom7.9%and 99.6%arereplaced

atthe left end of HA4 DNA, by a new set of

fragmentsthat extend left from 18.5% (Fig. 3).

The four secondary interruptions mapped by

thissetof fragments hadbeen previously located

byanalysis of restrictionfragments of T5 DNA

(16). The redundant regions contain additional

interruptions which couldbe mapped by more

extensiveanalysis ofT5HA4 DNA.

Itshould be noted, however, that at present

none of the new fragments that appear in

T5HA4 DNA have been unambiguously

mapped. It ispossible, for example, thatsome or

all of fragments A through E (Fig. 3) do not

extend as far rightward as the 18.5%

interrup-tion. The construction ofdouble mutants that

lack both the7.9and18.5% interruptions should

resolve thisquestion.

The observation that T5HA4 transfers its

DNA inanormal, two-step manner

unambigu-ously excludesarole for the 7.9%interruption in

this process. Because of its location, the 7.9% sitewas acandidate for thesignalthatinterrupts

transfer,provided that the left end is transferred

first. Although direct evidenceonthe direction

of T5 DNAinjection hasnotbeenobtained, the

available evidence ismostconsistent with initial

transfer of the left end (10, 20). If the rightend

istransferred first, a role forinterruptions can

also be excluded since the region immediately

around 92% islargelyfree fromevensecondary

interruptions (21). Normaltwo-steptransferhas

also been observed with T5HA23,amutantthat

iscompletelyfree frominterruptions(S. Rogers,

unpublisheddata).

The size of the FST segment isolated from

sheared T5HA4-infected cells is identicaltothat

obtained for wild-typeT5. Moreover, the band

containingthe HA4FSTsegmentisassharpas

thatseen forwildtype. Thus,eventhough the

FSTfragment equals7.9% of T5 DNA, the

in-terruption at7.9% doesnotprovideanessential

shear-sensitive site. This observation is

surpris-ing considering the known shear sensitivity of

single-chain interruptions (6) andsuggeststhat

the actual size of the initially transferred

seg-ment equals 7.9% of intact DNA. If the 7.9%

interruption had providedashear-sensitivesite,

the actual size of the initially transferred

seg-ment could have been substantially larger or

smallerthan 7.9%.

Avalue of 7.9%for T5 FST DNAimpliesthat

asmall portionof theterminalrepetitionisnot

included in the initially transferred segment.

Oneofthe functions oftheFST DNA is

exten-sivedegradationofhost DNA(12).Presumably,

the FSTDNA isprotected fromsimilar

degra-dationduring normal infectionalthough the

pro-tectivemechanismhasnotbeenidentified.Even

ifsuch amechanismexists,somedegradationof

FSTDNAcouldoccur.Therefore,byholdinga

portion oftherepeatedregioninreserve,T5has

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http://jvi.asm.org/

the potential for restoring, by recombination, a continuousgenome once DNA transfer has been completed.

ACKNOWLEDGMENTS

Thisresearch wassupported by grant PCM76-24036from theNational Science Foundation.S.G.R.wassupported bya training grant (HD-139) from the National Institutes of Health.

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