Physical map of Caulobacter crescentus bacteriophage phi Cd1 DNA.

JOURNAL OF VIROLOGY, May1980, p. 542-549

0022-538X/80/05-0542/08$02.00/0 Vol.34, No.2

Physical Map

of

Caulobacter

crescentus

Bacteriophage 4Cdl

DNA

BILHARABOY, LUCILLE SHAPIRO,* AND KEI AMEMIYA

Department ofMolecularBiology, Division ofBiological Sciences,Albert Einstein College of Medicine,

Bronx,

New York10461

A restrictionmapof the Caulobactercrescentusbacteriophage 4Cdlgenome wasconstructedby usingthe restriction endonucleasesHindIII and HpaI.Atotal of12fragments, ranging in molecular weight from7.7 x 106to0.25 x 106,were

produced byHindIII, and 7 fragments, ranging in molecular weight from 9.0 x

106 to0.24 x

106,

weregeneratedby HpaI. The molecular weight of thegenome was estimated to be approximately 28.8 x

10'

on the basis of the relative electrophoretic mobilities of the restriction fragments. The relative order of the cleavage fragments was determined by specific cleavage of isolated restriction fragments, terminal labeling of both the whole genomeand isolated fragments, and hybridization of isolated fragments to restriction fragments generated by other restriction enzymes. The genome of 4Cdl was found to be terminally repetitive, and analysis of previously determined in vivo and in vitro RNA transcripts showed that the restrictionmapcouldbe oriented such that transcrip-tionbeganonthe left andproceededtothe right end ofthegenome.

Caulobactercrescentusphage pCdlis asmall, icosohedron-shaped bacteriophage containinga linearduplexDNAmolecule of molecular weight 29 x 106 (23). Its host, C. crescentus, has a defined cellcycle (14, 19), which includes mor-phologically distinct swarmer cells and stalked cells. Unlike many otherCaulobacter bacterio-phages, which infect cells onlyat acertain de-velopmental stage (2, 19), both stalked and swarmer cells are sensitive to 4Cdl infection (23). We have studied the in vitrotranscription ofphage pCdlDNAbythe C. crescentusRNA polymerase (3) and the RNA processing ofthe phage RNA transcripts (6) by C. crescentus RNase III. It has been demonstrated that the host RNA

polymerase

is

responsible

for the in vivo transcription of a portion of

4Cdl

DNA

immediately

afterinfectionandthata rifampi-cin-resistant RNA

polymerase,

dependentonde novoproteinsynthesis,appearsearlyafter infec-tion (Amemiya, Raboy, and Shapiro, Virology, inpress). To map theregionofearly pCdlDNA transcriptionandtolocatethe

early

promoters,

wepreparedacleavagemap of thephageDNA byanalyzingthe sizes andpositionsof

fragments

produced by two restriction

endonucleases,

HpaI (7, 20) andHindIII (18,21),and

by

cleav-age of

4Cdl

DNApreviouslyendlabeledbyT4 DNA kinase. Wealso determined that the ends of

4Cdl

DNA contain terminally

repetitious

DNA sequences since partial degradation by exonuclease III and

annealing

results in the

con-version oflinear DNAtocircularmolecules(17).

MATERIALS AND METHODS

Bacteria andbacteriophage. C.crescentusCB13

was used in thisstudy. C. crescentus was grown in peptone yeast extractmedium (16) at30°C. Bacterio-phage4Cdl(23) was agift from N. Agabian.4Cdlwas

propagated in C. crescentusCB13, and concentrated

phage suspensions wereobtained by the method of

Yamamoto et al. (24). The phagewere precipitated with 6% (wt/vol)polyethylene glycolcontaining 0.5 M

NaCl andsubsequently purified in CsCl gradients(3).

32P-labeled<>Cdl DNAwas prepared by growing the host in the presenceof 10ytCi of32P, (ICN, Irvine, Calif.)perml foronegeneration before the additionof

thephage. Labeled phagewereharvested and concen-trated asdescribed above.

Bacteriophage DNA preparation. Bacterio-phage4Cdl obtained from CsCl gradientswasdialyzed overnightagainst bufferD(0.01MTris-hydrochloride, pH8.0, 0.02 MNaCl,0.1 mMEDTA, disodium salt) andthen extractedthree times with distilled phenol saturated with 2x SSC (lx SSC is0.15 M sodium chlorideplus0.015Msodiumcitrate). The DNAwas then dialyzed extensively against large volumes of

bufferDand storedat4°Coverchloroform. Restriction endonuclease digestion of DNA. 4CdlDNA(1to2

jig)

wasdigestedin 0.05-mlreaction volumes containing 0.01 M Tris-hydrochloride (pH

7.6),7mMMgCl2,1mMdithiothreitol,0.02MNaCl,

100,igofbovine serumalbuminperml,and1to3U of restriction endonuclease HpaI or HindIII (New England Biolabs, Inc., Beverly, Mass., or Bethesda ResearchLaboratories, Rockville,Md.).For prepara-tivegelsthe reaction volumeswereincreased upto

10-fold.Thereaction mixtureswereincubatedat37°Cfor

1to 3h,digestionwasstopped bytheaddition of0.2

volumes ofa solutioncontaining 25% (vol/vol)

glyc-542

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VOL. 34, 1980

erol, 5%(wt/vol) sodium dodecyl sulfate,and 0.025% (wt/vol) bromophenol blue, and incubationwas

con-tinuedat65°C for10min.

5'-Terminal labeling of fCdl DNA or 4Cdl

restrictionfragments. Intact <>CdlDNA orisolated HpaI fragmentsweretreated with bacterial alkaline

phosphatase to remove the5'-phosphorylgroup.The terminiwerethenlabeledbyusing [y-32P]ATP and T4 polynucleotide kinase (10).

Gel electrophoresis. 4Cdl restriction fragments

wereseparated bytwodifferent slabgel systems. In

one system the restrictionfragmentswereseparated

by electrophoresis on a 1% agarose slab gel as de-scribed elsewhere (Amemiya et al., in press). The restriction fragments were also analyzed on a 1.4% polyacrylamide-0.5% agarose composite slab gel (13 by 33 cm) (13). The electrophoresis buffer (1)

con-tained40mMTris-acetate (pH 7.8), 20mMsodium

acetate,and2mM EDTA(buffer E).Electrophoresis

wascarriedout at a constant voltage and period, as noted in thefigurelegends. The restrictionfragments

werelocatedeitherbyautoradiographyusing Kodak NS-5T X-ray filmorbysoaking the gel in ethidium bromide (1.0

[Lg/ml)

for 30min andvisualizing the fragments with a UV transilluminator (Ultraviolet Products,Inc., SanGabriel, Calif.).

Isolationof DNA restriction fragments. 32P-la-beledorunlabeled4CdlDNAwasdigested withHpaI andseparatedbyelectrophoresison apreparative 1%

agarose slab gel. The restriction fragments were lo-catedbystaining the right and left edges of the slab gel with ethidium bromide.Regionsofthegel

contain-ing each fragment were cut out, and the restriction

fragmentswerethen recovered byelectrophoretic elu-tion (11). Residualagarose inthe DNAsolutionwas removed by chromatography on 0.5-ml columns of acid-washed DEAE-cellulose(DE52) (Whatman, Inc., Clifton, N.J.),asdescribed byHirsh and Schleif(9).

ThepurifiedDNA was thenconcentratedby ethanol precipitation.

32P-labeled4CdlHpaIorHindIII restriction

frag-ments for hybridization experiments were obtained from 1.0%agarosehorizontal slabgels (16by28cm) by phenol extraction andethanol precipitation ofthe

electrophoreticallyeluted DNAsamples.

DNA-DNAhybridization. Stripsofnitrocellulose filterpapercontaining ¢Cdl HpaIorHindIII

restric-tionfragmentswerepreparedasdescribed (Amemiya et al., inpress).Before hybridization thefilter strips

wereplacedinplastic bags (Seal-N-Savebags;Sears, Roebuck &Co.),wetted with 3xSSC, andthen

incu-batedat65°C for3hinmodifiedDenhardt solution (5) containing 0.2% (wt/vol) bovine serum albumin,

0.2% (wt/vol) Ficoll400(Pharmacia FineChemicals, Inc., Piscataway, N.J.), and 0.2% (wt/vol)

polyvinyl-pyrrolidone (Calbiochem,LaJolla,Calif.) in 3x SSC.

32P-labeled

0Cd1

DNA in 0.1 M Tris-hydrochloride

(pH7.4)-i MNaCl-0.01 MEDTA-0.5% (wt/vol)

so-dium dodecyl sulfate-50% (vol/vol) formamide was then added to thefilters and incubated for 48 h at

37°Cwithgentlerocking.Thefilterswere then rinsed with 3xSSC,washed for 30 min at 65°C with 6x SSC

containing 0.02% (wt/vol) bovine serum albumin, 0.02% (wt/vol) Ficoll 400, and 0.02% (wt/vol)

polyvi-nylpyrrolidone,and finally washed with 3xSSC. After

CLEAVAGE MAP OF

oCdl

DNA 543

thefiltersweredried underaheatlamp, autoradiog-raphy wascarried out by using Kodak XR-5 X-ray film withaCronexQuanta IIIintensifyingscreen(Du

PontCo.,Wilmington, Del.).

Exonuclease III digestion and annealing of 4CdlDNA. Limiteddigestion of 4Cdl DNA from the

3' end of each strand (16) was accomplished with Escherichia coli exonuclease III (New England

Bio-labs), asdescribedby Ritchieetal. (17). The reaction mixtures(1.0ml)contained25mMTris-hydrochloride (pH8.0), 2.5 mMMgCl2,5mM2-mercaptoethanol, 7.5

,ugof32P-labeled

oCdl

DNA, and 20 U of exonuclease III.The mixture wasincubatedat37°C for 7.5 min, and the reactionwasstopped by the adding0.1mlof 20x SSC andcoolingto4°C. Under these conditions approximately5%of theDNAwasdigested (2.5% from

each end). Annealing of the partially digestedDNA was carried out at 65°C for 60 min. Samples were

placedonice before electronmicroscopy. For controls

weused(i) DNA which was incubated in the absence ofexonuclease IIIandcarriedthrough theannealing

procedure, and (ii) DNA which was digested with

exonucleaseIII but notannealed.

Electron microscopy. DNA samples were

pre-pared and spreadby the method of Davis etal. (4).

The DNA waspickedup oncarbon-coated (Parlodion-backed) grids, stained with uranyl acetate, and shadowed withplatinum-paladium. Sampleswere

ob-served withaSiemens 1A electron microscope.

RESULTS

Digestion of 4Cdl DNA with restriction endonuclease. 4Cdl DNA was digested with

restriction endonuclease HindIII or HpaI, and thecleavageproductswereseparated by electro-phoresis on composite 1.4% polyacrylamide-0.5% agarose or 1.0% agarose slab gels (Fig. 1). Theautoradiograms(Fig. 1,lanesaand c) were overexposed so that the smallest HindIII and HpaI fragmentscouldbe visualized.These small fragments are difficult to visualize by staining withethidium bromide.Digestion of

oCdl

DNA withHindlll produced12fragments,which were designated AtoLaccordingto increasing elec-trophoretic mobility. Electrophoresis of ¢Cdl HpaI cleavage products yieldedsixbands, des-ignatedAtoF.Increasingthe amount of HindIII

orHpaIendonucleaseduring digestion of4Cdl DNA didnot changethe number of restriction fragments. As discussedbelow, further analysis ofHpaIfragment Crevealed that it wasactually composed of two fragments of similar electro-phoretic mobility.

Determination of molecular weights of restriction fragments. To estimate the sizes of the

oCdl

HindIII andHpaI restriction frag-ments, theirmobilitiesonagarosegels were com-pared with the relative mobilities of A DNA HindIII restrictionfragments(12) and T7 DNA HpaIrestriction fragments (11).Table 1 shows the molecular weights obtained for the

oCdl

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544 RABOY, SHAPIRO, AND AMEMIYA

Hind

A

B: -A

E-G-

-;i_DC

H-1-_

E

-F

-G

-H

-J

E- F-

K-a

b

FIG. 1. Analysis of 4Cdl DNA

strictinn 1ndnnr -1'.;.QHin-dTTTtn

H/pa

I identical when the double band at HpaI

frag-mentCwas taken intoaccount.Themolecular A

weight

ofthe

4Cdl

genomedetermined

by

using

-B restrictionenzymeswas

similar

tothat derived

byWestetal.(23), who usedzonesedimentation.

c

Ordering

ofrestriction

fragments.

Several

D methods

(see

below)

were used to order the

4CdlHindIII and HpaI restrictionfragmentsin aphysicalmap.

(i) Digestion ofonerestriction fragment with the other restriction endonuclease. Thesimultaneous digestion of 4Cdl DNA with restriction endonucleases HindIII and HpaI yieldedeightnewfragments (designated by the prefix n) (Fig. 2, lane g) whichwerenot present

when

oCdl

DNAwasdigested with eachenzyme separately (Fig. 2, lanes a and b). These new fragments could be accounted for when each large HpaI fragment was redigested with HindIII(Fig. 2, lanescthrough f, and Table 2). IndividualHpaI fragmentswereanalyzed in or-dertodetermine whichHindIII fragmentswere located within the large HpaI fragments andto

c d helpidentify overlapping fragments.

Digestion of

¢pCdl

HpaI fragment A with

,digefsted

Awth

ren

HindlIl yieldedtwo fragments (Fig. 2, lane c).

mately1.0Pgof32P-labeled4CdlDNAwasdigested with HindIII orHpaI. Restriction fragments were

analyzedbyelectrophoresis, usinga1.4%

polyacryl-amide-0.5%agaroseslab gelrunataconstant50V

for16h, andfragments werevisualizedby

autora-diography (lanes aand c).Alternatively, restriction

fragmentswereanalyzedby electrophoresis, usinga

1%agarose slabgel, andfragments werevisualized

byethidium bromide staining (lanes band d). The

smallest HindIII fragments (K and L) and HpaI

fragments (E and F) are difficult to visualize by ethidiumbromidestaining. To visualizethe smallest

fragments,theautoradiogramswereoverexposed.

restrictionfragments. The molecular weights of

4CdlHpaI fragments ranged from9.0 x 106 to

0.24x106,and thesumof themolecularweights of theHpaI fragmentswas24.4x 106.The 4Cdl HindIII fragments ranged in molecular weight from 7.7x 106to0.25 x 106. However,thesum of the molecular weights of the HindIlI

frag-mentswas28.6x 106.Thediscrepancybetween thesumsof the molecular weightsof theHpaI and HindIII fragments was resolved when the

amountofradioactivityineachrestriction

frag-mentwasmeasured(Table 1).Itwasfound that HpaI fragment C contained twice as much

ra-dioactivelabel aswould be expectedfora

frag-mentof its size. These results suggest thatHpaI fragment C actuallyconsisted oftwo fragments of similar size (designated HpaI-C, and HpaI-C2). Thesums ofthe molecular weights of the HpaI and HindIII fragments were essentially

TABLE 1. Molecularweights of 4 CdI restriction fragments produced by digestion with HpaI and

HindIII

Fragment_Fragment % of

_~DNA"

oCdl

Mol

_(x1io)bwt

HpaI fragments

A 31.4 9.00

B 23.1 7.00

C(C +C2) 30.8 4.40(x2)

D 12.8 3.40

E 1.4 0.38

F 0.77 0.24

HindIII fragments

A 24.3 7.70

B 20.2 5.70

C 10.9 2.97

D 10.4 2.85

E 8.8 2.24

F 6.7 1.90

G 6.2 1.75

H 4.4 1.15

I 3.6 1.00

J 2.7 0.82

K 1.3 0.32

L 0.78 0.25

a Determined from the distribution of32P

radioac-tivity. Gelswerestained with ethidiumbromide, indi-vidual DNA bands were cutout, and radioactivities weremeasured.

bMolecularweightswere estimated from the rela-tive mobility of each fragment compared with the mobilities ofAHindIII(12)and T7HpaI (11)digestion

products. Gelcompositionandelectrophoresis condi-tions wereasdescribed in thelegendtoFig.1.

J. VIROL.

I A_8- - c-

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CLEAVAGE MAP OF pCdl DNA 545

A

-C

_

*

XS

n2

C

-

-

D

W-d 60MA

4_

D:W

E- *fl -n3

7

H-

_-- -PS..&*

a.m

-n4

nsl

-E

K-L- -F

[image:4.503.44.237.70.339.2]

ob

cd e f g

FIG. 2.Analysis of redigestion ofindividualHpaI fragments by HindIII. 32P-labeled¢Cdl DNA was digestedwithHindIII (lane a), HpaI (lane b),orboth HindIII andHpaI (lane g).ThefollowingHpaI frag-mentswereredigestedwithHindIII:HpaI-A (lane c), HpaI-B (lane d),HpaI-C,and-C2 (lane e),and HpaI-D(lanet).After redigestion byHindIII, thesamples (eachwithapproximately5x103 cpm)wereanalyzed by electrophoresis on a 1.4% polyacrylamide-0.5%

agarose slabgelwhichwas run ata constant 85 V for12 h. Thenewfragments produced bythedigestion of XCdlDNA bybothHpaIandHindIIIarelabeled (prefixed bythe lettern)ontheright.

One fragment was HindIII-C, and the other fragment was a new fragment, designated nl.

Thesumofthe molecularweights of thesetwo

fragments was 8.77 x 10', which was in close

agreementwiththemolecularweight of HpaI-A

(9.00X 106).

Digestion of pCdl HpaI fragment B with

HindIIIgaverisetoHindIII fragments Hand K andalsototwo newfragments, n2andn6 (Fig. 2,lane d). Thesumof themolecularweights of

these four fragments (6.83 x 106) agreed well with themolecularweight of HpaI fragment B

(7.0 x 106).

When HpaI fragments C1 and C2 were

di-gestedwithHindIII, sevenfragmentswere

pro-duced(Fig.2,lanee).Among thesewereHindIII

fragments E, F, G, and L. The new fragments

generated by HindIIIdigestionweren4, n5,and

n7. Unlike the HindIll digestion products of

HpaI fragments A and B, thesumof the molec-ularweights of the HpaI-C1 and -C2 digestion products (8.14x 106)wasslightlylowerthanthe

molecular weightof

HpaI-C,

plus-C2 (8.8x106).

Nevertheless, these results again indicate that

HpaI fragment C is composed of two fragments having similarmolecularweights.

Digestion ofHpaI fragment D with HindIII yielded HindIII fragment J and two new

frag-ments,n3andn8(Fig. 2,lanefl.Inthiscase,the

sumof themolecularweightsof theproductsof

redigestion withHindIII (3.30 x 106) was very close to the molecular weight of the original fragment (3.04 x 106).

(ii)5'-TerminallabelingofqCdl DNA and

isolated fragments.Todeterminewhich 4Cdl

HindIII and HpaI restriction fragments were

located at the termini of the genome, the 5'

termini were labeled with [_y-32P]ATP before digestion with HindIlI or HpaI. In addition,

pCdl HpaI fragments A, B, C, and D were labeled at their termini before being digested

withHindIII in order todetermine which

frag-mentswerelocatedatthe ends of theindividual HpaIfragments (datanotshown).

Digestion ofthe 5' terminally labeled pCdl

DNA with HindIIIrevealed thatHindIlI

frag-ments C and F were located at the ends, and

TABLE 2. RedigestionofthemajorcpCdlHpaI fragments with HindIIIa

Fragmentsgenerated IsolatedHpaI fragment by redigestionwith

HindIII

Molwt Molwt

Fragment

(x106)

Fragment

A 9.00 nl 5.80

C 2.97

B 7.00 n2 4.60

H 1.15

n6 0.76

K 0.32

C(C,+C2) 4.40(x2) E 2.24

F 1.90

G 1.75 n4 0.86

n5 0.80

n7 0.34

L 0.25

D 3.40 n3 2.15

J 0.82

n8 0.33

aThe

largest

HpaI

fragments were isolated and redigested withHindIII as described in the text. The molecular weights of the pCdl HpaI, HindIII, and

newfragments (designatedby theletter nfollowedby

anumber) produced by digestionswith bothHpaIand HindIII were determined as described in Table 1, footnote b.

VOL. 34,1980

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546 RABOY, SHAPIRO, AND AMEMIYA

cleavage of the labeled genome with HpaI showed that HpaI fragments A and

C,

were at theends. Theassignment ofHpaIfragment C1 to the end of the genome, rather than HpaI fragment C2, is discussed below. Both fragments fromtheHindIIIdigestion ofterminallylabeled HpaIfragmentAwerelabeledsince there were only twofragmentsproduced. Fragments n2 and n6 were found to be labeled when terminally labeled HpaI fragment B was digested with HindIlI. Digestion of terminally labeled HpaI fragments

C,

and C2 yielded fourlabeled frag-ments, namely fragments HindIII-F and n5 be-longing to

HpaI-C,

and fragments n4 and n7 belongingtoHpaI-C2. Finally,whenHpaI frag-ment Dwas terminallylabeledand digestedwith HindIII, fragments n3and n8 were found to be labeled.

(iii) Reciprocal DNA hybridization. To help identify overlapping fragments and their order in a physical map, individual

32P-labeled

fragments cut by one restriction enzyme were hybridizedto Southernblots (22) of restriction fragments generated by a second restriction en-zyme. The twosmallestHpaIand HindIII frag-mentswere not used as hybridization probes in thisstudy because of thedifficultiesinobtaining adequate quantities. In several instances they couldnotbeobserved after autoradiography of thehybridization filter, possiblybecause of the poor retention ofsuchsmallfragments to nitro-cellulosefilters (22).

Figure3shows thehybridization patternwhen individual

32P-labeled

oCdl

HpaI fragments were

hybridized

to the

oCdl

HindIII

cleavage

fragments. WhenHpaIfragmentA wasused as theprobe,it

hybridized

toHindIII

fragments A,

C,and F (Fig.3,lanea).Hybridizationwasalso observedtoHindIII

fragments

BandH,butwe believe thistobethe resultofsome contamina-tion ofHpaI fragmentAwithHpaI

fragment

B. This appears

likely

because when

HpaI-B

was

used as the

probe,

it

hybridized

strongly

to HindIIIfragmentsB and Hbesides

hybridizing

to HindIII fragments D andK

(Fig.

3, lane b). WhenHpaI

fragments

C,

and

C2

wereusedas a

probe,theyhybridizedtoHindIII fragments A, B, C, E,F,G,and I(Fig.3, lanec).When HpaI-D wastheprobe,it

hybridized

to HindIII

frag-mentsD,I,and J(Fig.3, lane d).

Figure 4 shows the

reciprocal

hybridizations

when individualHindIII

fragments

wereusedas probesagainstHpaI

cleavage

fragments.

Cross-contaminationcanbeseenbythe relative inten-sities of

hybridization

to

specific

HpaI

frag-ments. Nevertheless, the results from this

hy-bridization study and the

reciprocal study

de-scribed abovehelpeddefine the

overlapping

re-gions. In addition, homology between the two

J. VIROL.

e

A- _

B

C- D-E-P

F-

G-!

-Oso4

4-

K-(

FIG. 3. Hybridization of individual 32P-labeled HpaI fragmentstothe4Cdl HindIII restriction pat-tern. Approximately5 x 10 cpmof each HpaI

frag-ment was usedin thehybridization assay and

ana-lyzed as described in the text. The autoradiogram

showsthehybridization patterns withHpaI-A (lane a), HpaI-B (lane b),HpaI-C1 and-C2 (lane c), and HpaI-D (lane d). The HindIII restriction pattern of

4oCdl

DNAisshownschematicallyattheleft.

endsof the pCdl genome wasrevealed bythis study, since HindIII fragment F hybridized to HpaI fragments C1andC2 andtoHpaIfragment A (Fig. 4, lane fl and HindIII-C hybridized to

the samefragments (Fig.4, lane c). In the recip-rocal experiments, HpaI-A hybridized to both HindIIIfragmentsC and F (Fig.3, lanea),and HpaI fragments C1 and C2 (or whatwe desig-nated

HpaI-C,

as being at one end of the ge-nome) hybridized toboth HindIII fragments C andF (Fig.3,lanec).

With the information obtainedbythe proce-dures described above, the restriction map of

4Cdl DNA was constructed (Fig. 5). Based on

the analysis of in vivo and in vitro4Cdl tran-scripts (Amemiya et al.,in press), thephysical map of 4Cdl DNA has been oriented suchthat transcription of the genome begins at the left

end andproceedstoward theright. HindIII frag-ment F andHpaIfragmentC1,therefore,should contain the earlypromotersrecognized by the

host RNApolymerase (Amemiyaetal.,inpress). We know thatHpaIfragmentC

actually

consists

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[image:5.503.293.433.65.330.2]

B-B= * __{_-_-}- - _

- -_

E-

F-ci cc d e g h

FIG. 4. Hybridization of individual 32P-labeled HindIIIfragmentstothe 4CdlHpaIrestriction pat-tern. Approximately 2 x 104 cpm ofeach HindIII fragment was used in the hybridization assay and

analyzedasdescribedin thetext.Theautoradiogram shows thehybridizationpatternwith HindIII-A(lane a), HindIII-B(laneb), HindIII-C (lane c),HindIII-D

(lane d), HindIII-E (lane e), HindIII-F (lane

t),

HindIII-G (lane g), HindIII-H (lane h), HindIII-I (lane i), and HindIII-J (lanej). TheHpaIrestriction pattern of pCdlDNA isshownschematicallyatthe

left.

POSITION 0 20 40 60 80 100

T

Hndm f G 0 HAK 0 JI l E A C

T T T 7

[image:6.503.45.238.66.216.2]

Hpo I Cl E 0 0 C2 F A

FIG. 5. Restriction mapofpCdlDNA. The cleav-age mapofpCdlDNAconstructed withHindIIIand

HpaIrestriction endonucleases isshown.Restriction

fragments were labeled andorderedasdescribed in

thetext.Only the positionsof the smallestfragments (HindIII-L andHpaI-F) are uncertain. Early

tran-scriptionofthe genomebeginsattheleftandproceeds

totheright.

of two fragments (Cl and C2); one of these is

located at the leftterminus,and that fragment

wasdesignatedHpaI-Cl. HpaI-C2wasassigned

to the region further to the right. The exact

placement of the two smallest fragments,

HindIII-L andHpaI-F,remains inquestion. Terminally repetitious sequences. Two

different experiments indicated that the 4Cdl

genome might be terminally repetitious. The

results ofreciprocal hybridization studies (Fig.

3 and 4) showed that HindIll fragment F

hy-bridizedto bothHpaI-Aand-Cl (Fig. 4,lanef) and thatHindIII fragment C hybridizedto the

same fragments as HindIII-F (Fig. 4, lane c).

The second observation which indicated that thegenomemayhaveterminally repetitious se-quences camefrom invitro transcription

map-pingstudies (Amemiyaetal.,inpress).Asingle

RNAtranscript synthesizedinvitrobyC.

cres-centusRNApolymerase with 4Cdl DNAasthe template was found to hybridize to restriction fragments located at both termini. To determine whether terminally repetitious sequences were

present in 4Cdl DNA, the intact genome was

partially degraded (approximately 5%) with E. coli exonuclease III (16) and then allowed to

annealat65°C for 60min,asdescribedby Rit-chie et al. (17). Electron micrographs of the digested and annealed molecules are shown in Fig. 6. Only linear molecules were observed in controlsamples of

oCdl

DNA that hadnotbeen treated with exonuclease III but had otherwise beensubjected totheincubationandannealing conditions (Fig. 6A). Treatment of 4Cdl DNA with exonuclease III but without the annealing procedure also yielded only linear molecules (data not shown). However, when 4Cdl DNA was partially degraded by exonuclease III and allowedtoanneal,asignificant proportionof the molecules were circular (Fig. 6B). Approxi-mately 10% of the exonuclease III-treated and annealedmoleculeswerecircular,whereas in the

two controlsamplesno circularmoleculeswere

seen. These results, together with those dis-cussedabove,indicate that

oCdl

DNA contains terminallyrepetitiousnucleotidesequences.

DISCUSSION

A physical map of the Caulobacter phage pCdl genome wasconstructed by using restric-tionendonucleases HindIII and HpaI. A total of

12fragments were produced byHindIll, and 7 fragments were generated by HpaI. Although

somecross-contamination between the large re-strictionfragmentsoccurred during the isolation procedure, the relative order of the restriction fragmentscould be deduced. Only the positions of the smallestfragments(HpaI-F and HindIII-L) are still uncertain. The molecular weight of ¢Cdl DNA wasestimated to be approximately

28.8 x 106, asdetermined by the relative electro-phoreticmobilities of the restriction fragments. This is in close agreement with the results of Westetal. (23), who determinedthemolecular weighttobe29.0 x

10k

by zone sedimentation.

Hybridization studies with restriction frag-mentsand in vitro transcription mapping (Ame-miya et al., in press) indicated that common sequences were located at the termini of the DNA. The presence of terminally repetitious sequences was confirmed by the observation that circularmolecules were formed after limited exonuclease III digestion and annealing of the exposed single strands. Terminally repetitious sequenceshave been shown to be present in T3, T7 (17), and T5 (15) DNA. It has become ap-parentthat there are many similarities between

oCdl

andthe E. coli bacteriophage T7 (8, 25).

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t..-FIG. 6. Electronmicrograph ofexonucleaseIII-treatedo4CdlDNA.4Cdl DNAwaspartially digestedwith exonuclease III and then allowedtoannealasdescribed in thetext. (A) 4Cdl DNA withoutexonuclease III digestionbut incubated underannealingconditions. (B) oCdl DNA treated with exonuclease III (approxi-mately5%digestion)and allowedtoanneal. Bar= 1.0 Lm.

548

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oCd1

These phages have a small icosahedral head

(diameter, approximately 60nm) andpossess a short tail. Their genomes are double-stranded linear moleculeswith similar molecularweights (29.0x 106for

oCdl

and26.5x 106 forT7) (11). The ends of both

oCdl

DNA and T7 DNAare terminally redundant. Furthermore,atleasttwo aspectsof theinfection processare commonto

these lyticphages: (i) thehost RNApolymerase is involved in the early transcription program, and (ii) arifampicin-resistant RNA polymerase isrequired for lategeneexpression.

We have oriented the 4Cdl restriction map

such thatphage transcription beginsatthe left

end of the mapandproceedstotheright. Both in vivo and in vitro transcription studies have identified the restriction fragment where

tran-scription is initiated. Our investigation of the temporalprogramofphage transcription

(Ame-miya et al., in press) indicated that the host RNApolymerase is involvedinthetranscription of the phage genome early after infection and that a rifampicin-resistant RNA polymerase, whoseappearanceisdependentondenovo pro-teinsynthesis, is required for later transcription. With the construction of the physical map, we have identified whichregions of thegenome are involved in the early transcription program of thephage and which regions are dependent on denovoproteinsynthesis forgeneexpression. In vivo, theearly phage RNA transcripts produced by the host RNA polymerase hybridized pri-marily to HindIII fragments F, G, and B and HpaI fragmentsCi and B. RNAtranscripts

syn-thesized invitrohybridized tothe same region of thephysicalmap.Weare nowintheprocess oflocating thepromotersontherestriction

frag-mentsin theearly regionofthe 4>Cdl genome.

ACKNOWLEDGMENTS

Thisinvestigationwassupported by grantGB42545X from

the National ScienceFoundation(to L.S.), byPublic Health

Service grant GM 11301 from the National Institutes of

Health, and by PublicHealth ServicecorecancergrantNIH/ NCI P30CA113330-8from the National Institutes of Health.

L.S. isarecipient ofaHirschl TrustAward, and B.R. isa

recipient ofaMartinFellowship.

We thank N.Agabian for phage4Cdland JaneFant for

her excellent assistance with theelectronmicroscopy.

LITERATURE CITED

1. Aaij, C.,and P. Borst. 1972.Thegel electrophoresisof DNA.Biochim.Biophys.Acta269:192-200.

2. Agabian-Keshishian,N.,and L. Shapiro. 1971. Bac-terial differentiationandphageinfection.Virology44:

46-53.

3. Amemiya,K.,C. W.Wu,andL.Shapiro. 1977. Cau-lobactercrescentusRNApolymerase.Purification and characterizationofholoenzymeandcorepolymerase.J. Biol. Chem.252:4157-4165.

4. Davis, R. W., M. Simon, and N. Davidson. 1971. Elec-tron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids. MethodsEnzymol. 21:413-428.

5. Denhardt,D. T. 1966. A membranefiltertechniquefor thedetectionofcomplementaryDNA.Biochem. Bio-phys.Res.Commun. 23:641-646.

6. Dunn, J.J.,and F.W.Studier. 1973.T7earlyRNAs are generated by site-specific cleavages. Proc. Natl. Acad.Sci.U.S.A. 70:1559-1563.

7. Gromkova, R., and S. H. Goodgal. 1972. Action of Haemophilus endodeoxyribonuclease on biologically activedeoxyribonucleic.J.Bacteriol.109:987-992. 8. Hausmann, R. 1976.Bacteriophage T7genetics. Curr.

Top.Microbiol.Immunol.75:77-110.

9. Hirsh, J., and R. Schleif.1976.High resolution electron microscopic studies of geneticregulation.J.Mol. Biol. 108:471-480.

10. Maxam, A. M.,andW.Gilbert.1977. A newmethod for sequencing DNA. Proc.Natl. Acad. Sci. U.S.A. 74:560-564.

11. McDonell,M. W., M. N.Simon, and F. W.Studier. 1977. Analysis of restrictionfragmentsof T7 DNA and determination of molecular weights by electrophoresis inneutral and alkalinegels.J.Mol. Biol.110:119-146. 12. Murray, K., and N. E. Murray. 1975. Phage lambda

receptor chromosomes for DNAfragments madewith restrictionendonucleaseIIIofHaemophilus influenzae and restriction endonucleaseI ofEscherichia coli. J. Mol. Biol.98:551-564.

13. Peacock,A.C., and C.W.Dingham. 1968.Molecular weight estimationandseparationof ribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry7:668-674.

14. Poindexter,J.S. 1964.Biologicalproperties and classi-ficationof theCaulobacter group. Bacteriol. Rev. 28: 231-295.

15. Rhoades, M., andE. A.Rhoades.1972. Terminal repe-tition inthe DNA ofbacteriophage T5. J. Mol. Biol. 69:187-200.

16. Richardson, C. C., I. R. Lehman, and A. Kornberg. 1964. Adeoxyribonucleicacidphosphatase-exonuclease from Escherichia coli. J.Biol. Chem.239:251-258. 17. Ritchie,D.A., C.A.Thomas,Jr., L.A. MacHattie,

andP.C. Wensink.1967.Terminal repetitionin non-permuted T3 and T7bacteriophageDNA molecules. J. Mol. Biol.23:365-376.

18. Roy, P.H., and H.0. Smith. 1973. DNA methylases of Haemophilus influenzae Rd. I.Purification and prop-erties. J.Mol. Biol.81:427-444.

19. Shapiro, L.1976.Differentiationin the Caulobacter cell cycle. Annu. Rev. Microbiol.30:377-407.

20. Sharp, P. A., B. Sugden, and J. Sambrook. 1973. Detectionof two restriction endonuclease activities in Haemophilus parainfluenzaeusing analytical agarose-ethidium bromide electrophoresis. Biochemistry 12: 3055-3063.

21. Smith, H. O., and K. W. Wilcox. 1970. A restriction enzyme from Haemophilusinfluenzae. I. Purification andgeneral properties.J. Mol. Biol. 51:379-391. 22. Southern, E. M. 1975. Detection of specific sequences

amongDNAfragments separatedby gel electrophore-sis. J.Mol. Biol.98:503-517.

23. West,D.,C.Lagenaur,andN.Agabian. 1976. Isolation andcharacterizationofCaulobacter crescentus bacte-riophageoCd1.J.Virol. 17:568-575.

24. Yamamoto, K. R.,B. M. Alberts,R. Benzinger, L. Lawthorne, and G.Treiber. 1970. Rapid bacterio-phage sedimentation in the presence of polyethylene glycoland itsapplication to large-scale virus purifica-tion.Virology40:743-744.

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