Evolution of bacteriophage phi X174. IV. Restriction enzyme cleavage map of St-1.

0022-538X/78/0027-0738$02.00/0

Copyright©1978 AmericanSociety for Microbiology Printed in U.S.A.

Evolution of

Bacteriophage

45X174

IV. Restriction Enzyme Cleavage

Map

of

St-1

J. N. GRINDLEYt* ANDG. N. GODSON

Radiobiology Laboratories, Yale MedicalSchool,NewHaven,Connecticut06510

Received for publication7May1977

The St-1 genome is about 6,050 base pairs in size, approximately 10% larger than 4X174 (5,375 base pairs). The DNA fragmentsobtained by HincII, HaeIII, and EcoRIdigestionwereordered andaligned intoa

colinear

map,and thesingle BglI cleavage sitewaslocated.

St-1 is a

small,

icosahedral, single-stranded

DNA-containingbacteriophage whichwasfirst isolated by Bradley (4) and was further charac-terized by Bowes and Dowell (3). It infects K-12

strains of

Escherichia coli and is serologically

unrelated to 4X174

(4,

22).

Preliminary experiments

in

this

laboratory

have indicated that St-1 codes for proteins sim-ilar in number and size to those directed by

4X174, although

theSt-1

products presumed

to

be

equivalent

tothe

OX

gene

A, F,

andG

prod-ucts are

larger.

However, St-1 has much simpler

requirements for host proteins

involved in DNA

replication than 4X174. The in vitro conversion

ofSt-1 viral

single-stranded

DNA to

the RFII

(open circular,

double-stranded

form)

requires

only the dnaG and dnaE

gene

products

plus

DNA

unwinding protein and

elongation factors

I

and

11(25). In

this,

St-1

resembles G4

(18, 25).

In

vivo, however, both

St-1 Rf replication and

viral strand synthesis

can take

place

at the

non-permissive

temperature in

hosts carrying

tem-perature-sensitive lesions

in

the

dnaB or

dnaC/D

genes

(2;

G. N. Godson and J. N.

Grindley, unpublished

data).

Both

G4 and

OX174

are

unable

to

replicate under such

con-ditions (7, 12,

14,

23,

26).

As a

preliminary

to

investigating further the

differences

among

bacteriophages

4X174,

G4,

and

St-1,

we

have constructed

a

cleavage

map ofSt-1 DNA,

using

the restriction

endonucleases

from

Haemophilus influenzae

(HincII),

which

is an

isoschizomer

of

HindII,

from

Haemophilus

aegyptius

(HaeIII),

and that

specified

by the R

factor RI

(EcoRI).

We have also

located

the

single

cut

produced by

theenzyme from Bacillus

globiggi (BglI).

MATERIALS AND METHODS

Phageandbacterial stocks.Bacteriophage St-1

wasobtained from C. E.

Dowell.

The Escherichia coli

tPresent address:DepartmentofBiochemistry,School of Medicine, UniversityofPittsburgh,Pittsburgh,PA 15261.

K-12 host strainW3110wasobtained from K. Brooks Low.

Preparation of32P-labeled RFI DNA. W3110 was grownat40°C in TPG aminoacid, low phosphate-containing medium (19)toabout4 x

10'

cells per ml andinfected withSt-1at amultiplicity of infection of

3. After2to 3min, 30,ugofchloramphenicol per ml was added toinhibitsingle-strand DNA synthesis. The cells were labeled with10

,uCi

of[32P]phosphate (New EnglandNuclear)perml10minafterphageinfection andharvested65minlater.

Thecellsweresuspended in 10% sucrose (wt/vol) with 50 mMTris-hydrochloride (pH 8.0), treated with lysozyme in the presence of EDTA, and lysed with Sarkosyl in0.2MNaClasdescribed by Godson (10). Theclear viscouscell lysatewascentrifuged at 80,000

xgfor 45 min topelletmost of the host chromosomal DNA. The supernatant was treated with 25 yg of RNaseperml andphenol extracted and the DNAwas

precipitated with ethanol.St-lRFwasfurtherpurified on 5 to20% neutralsucrosegradients.

Preparation of restriction endonucleases.

HincllwasisolatedasdescribedbySmith and Wilcox (20) fromcells obtained from New England Biolabs. HaeIII and Bglwereprepared in thislaboratory by the methods ofSmith and Wilcox (20).EcoRIwas a

gift of W. Summers.

Enzymedigestion. Preparationofpartial and

ter-minal digestion products wasessentiallyasdescribed by Godson (9).Enzyme digests were analyzed on 3 to 5%acrylamide gelsby the buffer system of Maniatis (15) or on 0.7 to 2% agarose gels as described by Sugdenetal.(21).

DNA fragments were extracted from the gel by macerating the gel slice and soaking it in 0.2 M

NaCl-10mM Tris (pH 7.4). The acrylamidewas

re-movedby passage of the solutionthrough glass wool. SolutionscontainingDNA wereextracted withphenol, and the DNAwas precipitated with ethanol in the presence of tRNA as carrier where necessary. The DNAwasresuspended in enzyme digestion buffer for subsequent digestions.

Nomenclature. For

4X174,

the nomenclature of

Edgelletal. (8) for HincIIproducts and Middletonet

al.(16) for HaeIIIproductswasused. The limitdigest fragments of

St-i

were designated Hinclll to -9, HaeIII1to-12,andasindicatedintheResults section for theproducts ofotherenzymes.

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VOL. 27, 1978

RESULTS

Size

and order of

St-1

fragments

pro-duced by the H.

influenzae (HincH)

restric-tion

enzyme.

The

HinclI

enzyme cut

St-i

DNA

into nine

fragments, although only eight bands

were visible in the autoradiograph (Fig. 1) be-cause

the

smallest fragment, HincII9,

was not

retained

on

the

gel. Most of the

fragment sizes

shown in

Table

1 were

obtained

by comparing

their

mobility

with that of the DNA

fragments

generated by

HaeIII and HincII from

4X174,

whose sizes

are

well documented

(13, 16, 17).

However,

because the

largest

OX174

fragment

is

only 1,200 base pairs

(bp)

and because the

rela-tionship between

log molecular weight and

mo-bility

on

4%

polyacrylamide

gels

ceases to

be

a

strictly linear function for fragments

greater

than

1,000

bp

(9),

the

molecular

weights of such

fragments

are

based

where possible

onthe sum

of

the sizes of

fragments produced from the large

fragment by another

enzyme. We were

unable

to

determine the

exact

molecular

weight of

HincII3 because it

was not

subcut

by

the other

enzymes

used. With this

reservation, the total

nucleotide length of St-1 RF from addition of

the

HincII fragment sizes

was

about

6,120

nu-cleotides.

Incomplete digestion of

St-1 with HincII

gave

10

partial fragments whose terminal digestion

products

are

listed in Table

2.

From

these data

the order

1 2 -9-5 6 4 7 8 3

for

the

HincIl

ter-minal

fragments

was

established.

Size and order of

St-1

fragments

pro-duced

by the H.

aegyptius

(Haell

restric-tion

enzyme.

St-1 RF

was

cleaved

by

HaeIII

into

12

fragments whose nucleotide

lengths,

in-cluding that of

HaeIII12,

which is

not

visible in

Fig.

1, gave a

total of

about 6,010

nucleotides

(Table 1). The terminal digestion products

(Ta-ble 3) obtained from overlapping partial

frag-ments

indicated that the

order of the HaeIII

fragments

was 1 3 4 8 10, 5 9 2 7 6 11.

No

partial

which

overlapped

both

10

and

5 was

isolated, but these

were

deduced

to

be

contig-uous

from data described

below. The position of

HaeIII12

could

not

be

deduced from

partial

mapping,

nor

could

1 and 11 be shown to be

adjacent.

Correlation

of

the

HincH

and HaeM

re-striction

maps.

(i)

Location of

HaeT

sub-cuts

within

Hincd

fragments. Table

4shows

the

fragments

obtained when HincII terminal

products

are digested with

HaeIII.

Fractionation

of

St-1

DNA

digested

with both

HinclI

and

HaeIII

endonucleases

(data

not

shown)

con-firmed

that certain

of

the

HincII

or HaeIII

fragments

were cut

by

HaeIII

or

HincII,

respec-tively,

and were therefore

missing,

and that new

bands, those designated

as

deletions,

were

gen-erated.

Three intact HaeIII

fragments

were

produced

from

HincId1

by HaeIII digestion

(Table

4).

They were

identified

as

terminal

HaeIII

frag-ments on

the

basis of their relative

mobility

in

acrylamide

gels. The 850-bp fragment

was

des-St-1

Hae

mU

0X1 74

+

Hae

ruI

2-O

I-m4

-IS

6-*7

8-me

92__0tw

10- .-;

St-i

Hiiincc

_-1

2

*i*

v-V

2 3

4

-em-_-5

5 _ _* -6

6a _

6b

--7

7 ----

4

4bio_ 8 --- 1

11- '' ,. - _{9 -}

_A

-8

10--FIG. 1.Analysisofthe HincII and HaeIII cleav-ageproducts of

St-i

DNA on a4%

polyacrylamide

gel. ,X174DNAdigestedwith thesameendonuclease

was fractionated in parallel, although

only

the HaeIIIdigestis shown. The autoradiograph

of

the driedgelwasscanned withaJoyceLoebl microden-sitometer,and the molarratiosofthefragments from eachdigestwereestimatedfromtheprofiles.

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TABLE 1. Sizeof HincII, HaeIII restriction enzyme fragments of St-la

HinclI HaeIII

Product Size Product Size

1 1,710 1 1,840

2 1,460 2 920

3 1,050 3 715

4 720 4 600

5 460 5 410

6 328 6 355

7 263 7 323

8 78 8 258

9 50 9 230

10 175

11 115

12 70

aThe sizes in basepairsofmostof theseterminal

digestionproductswerededuced frommeasurements of theirmigration ina4%polyacrylamidegel,relative tothoseof4X174 digested bythesameenzyme (Fig.

1).The size ofHincdII and-2andHaeIII1,-2, and-3

wereobtained by addition of the sizes ofsubfragments

generated from thembyredigestionwith another re-striction enzyme(Tables4and5).

ignated as a deletion of HaeIII2 because the latter fragment mapped next to

HaeIII7

and becauseHaeIII2 has been shownto generatea fragment of this size when digested with HinclI (Table 5). The fourth fragment, 70 bp long, we designated HaeIII12. The map location of

HaeIIL12 wasnotresolvedbypartial mapping, but the sole position remaining was between HaeIII1 and -11. HaeIII12 probably does not

containanHinclI cleavage site becausea

frag-ment with the same mobility asHaeIII12 was

presentinthe

HincII

plus HaeIII digest of St-1. Under the conditions offractionation used (7% polyacrylamide gels),twofragments which differ by as little as 5 bp, which is the minimum possible distance between the cleavage sites of thetwoendonucleases, should have distinguish-able mobilities. TheHaeIII cleavage site demar-cating HaeIIIl from -12 probably is located within HincII3 andnot-1,asclosetothe bound-aryof thesetwo

Hincll

fragmentsaspossible. If this location is correct,afurthertinyfragment, possibly only 5 bp, which isadeletionof

HincII1,

[image:3.501.57.455.341.457.2]

mustbeproduced. Sucha fragment wouldnot

TABLE 2. Redigestion ofHincII partial digestion products withHincII

Partial size (bp) Observed redigestion fragments' Sum of fragment sizes (bp)

P1 1 2 1,710 + 1,460=3,170

P2 1 3 1,710 + 1,050=2,760

P3 2 9 5 1,460+50+460= 1,970

P4 6 4 7 328 + 720 + 263=1,311

P5 8 3 78+ 1,050=1,125

P6 970 4 7 720+263 =963

P7 850 9 5 6 50+460 + 328=838

P8 800 5 6 460+328=788

P9 535 9 5 50 + 460=510

Plo 345 7 8 263 + 78=341

1 2 9 5 6 4 7 8 3

a The composition of the HincII partial fragments was deduced from the molar ratios of the terminal fragments generated upon redigestion with HincII. The sizes, given in base pairs, of the partial fragments were measureddirectly from the gel.

bFragment deduced from a partial fragment present in the redigestion product.

TABLE 3. Redigestion of HaeIII partial digestion products with HaeIII

Partialsize (bp) Observed redigestionfragmentsa Sum of fragment sizes (bp)

P1 1 3 1,840+715= 2,555

P2 3 4 715+600= 1,315

P3 2 7 920+323= 1,243

P4 1,130 9 2 230+920= 1,150

P5 1,020 4 8 10 600 +258+175= 1,033

P6 860 4 8 600+ 258=858

P7 795 7 6 11 355+323+115=793

P8 670 7 6 355+323=678

P9 630 5 9 410+230=640

P10 480 6 11 355+115=470

Pll 440 8 10 258+ 175=433

1 3 4 8 10 5 9 2 7 6 11

aTheidentity of the terminal digestion products generated from eachpartial fragmentwasdeduced from their relative mobilitieson 4%polyacrylamide gels.

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TABLE 4. Digestion ofHincII fragments with HaeIII

HincII fragment (base Fragments obtained bydigestion

plair)a with HaeHI

HincIld

HincII2

HincII3(1,053) HincII4 (720)

HincII5(460)

HincII6 (328)

HincII7 (263)

850AHaeIII2

355 (HaeIII6)

323 (HaeIII7)

115 (HaeIII11)

70 (HaeIII12)b Total1,713

310AHaeIII4 410 (HaeIII5)

258 (HaeIII8) 230 (HaeIII9)

175 (HaeIII10) 75AHaeIII2 Total 1,458

Uncutb

573AHaeIII1

153AHaeIIf3

Total 726

228AHaeIII4

228AHaeIII3 Total 456

Uncut

Uncut

aThe size ofeachfragment produced bydigestion

bythe secondenzyme(HaeIII)wasobtained by

meas-uringitsmobilityonthegel,relativetothoseof St-I

HinclI orHaeIIIfragments. Some second digestion

products were presumed to be certain HaeIII frag-ments,asindicated in parentheses, ifthey coelectro-phoresed with that fragment. Other products were

inferred (see text) tobe deletions, indicated by A, of St-i HaeIIIfragments.

bSeetextfordetails.

be detected inourexperiments. However,a

pos-sibility remains that the 70-bp fragment is a deletion ofHaeIII12 thatretains themobilityof

the intact fragment. In this case the

HincII

cleavage site demarcating 1 from 3 would be within HaeIII12 very close to the HaeIII12/1 border. We could discover no further data to resolve thisquestion.

Table 4 showsthat four whole HaeIll

frag-mentswerecoveredby HincII2.The other two productswereprobably deletions oftheHaeIII fragments adjacentto8 and9, that is4and 2, respectively. Moreover, since 8, 10, 5, and9are the only complete HaeIII fragments produced by cleavage of HincII2, HaeIII5and -10 mustbe contiguous.

HincII6 and -7 were uncut by HaeIII and

must be from withinHaeIII fragments greater

than 328 and 263 nucleotides, respectively.

HincII8

and

-9 were not

subjected

to

HaeIII

digestion because of their small size, but

ap-peared

uncut

in

the

HincII plus

HaeIII

digest of

St-1.

(ii)

Location

of

Hincd

cleavages

within

HaeM fragments. Table

5

shows that

7

of the

12

HaeIII

fragments did

notpossess an

HinclI

cleavage

site.

Since the

uncut

fragments

HaeIII7,

-6,

and

-11were

shown

to

be

contiguous

(Table

3),

they

must

lie within

an

HinclI

frag-mentgreater

than

their

total

length (793

nucleo-tides),

i.e.,

HincIIl,

-2,

or-3.

Table

4

shows that

these

lie within

Hincdll.

Similarly HaeIII10,

-8,

-5,

and

-9,

which

are

together

on

the

map, must

be

generated by

HaeIll

cleavages within

a

frag-mentgreater

than

1,073

nucleotides which

must

be

HincII2.

HaeIII1

overlaps

HincII7 and

-8.

Because the

length of HincII3 could

not

be

accurately

as-sessed, for the

reasons

stated above,

we were not

TABLE 5. DigestionofHaeIIIfragmentswith HincII

Fragmentsobtainedby

diges-HaeIII

fragments tion with

HincIIa

HaeIIIl 930AHincII3b

573AHincII4 263 (HincII7)

78 (HincII8) Total 1,844

HaeIII2 850AHincII1

75 AHincII2 Total 925

HaeIII3(715) 330 (HincII6) 230AHincII5

153AHincII4 Total 713

HaeIII4 (620) 310AHincII2

228AHincII5

50 (HincII9) Total 588

HaeIII5 (410) Uncut HaeIII6 (355) Uncut HaeIII7 (323) Uncut HaeIII8(258) Uncut

HaeIII9(230) Uncut

HaeIII10 (175) Uncut HaeIII11 (115) Uncut

aThesizes, giveninnucleotides, of the products of

HincIIdigestion of HaeIIIfragmentswerecalculated from theirmobility relativetoSt-1 HincIIorHaeIII terminal digestion fragments. If they coelectropho-resed with knownHincIIfragments, theywere desig-natedassuch(indicatedinparentheses); productsthat

didnotwerethoughttobe deletions ofHincII frag-ments(A).

bSeetextfordetails.

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able

to

determine whether

the

HinclI site

divid-ing

HincIIl and

-3 was

located within

HaeIII1

or -12. If

it lies

within HaeIII1, the

fragment,

about

930

bp in size, will be intact HincII3,

and

an

undetected tiny

fragment which is

a

deletion

of the

adjacent

HincId

will also be

produced by

HincII digestion

of HaeIIIl. The second

deletion

fragment

produced from the other end of

HaeIIIl is

a

deletion of HincII4. HaeIII12

was

not

examined

on

its

own

for

an

HinclI

cleavage

site but,

as

discussed

above, appeared

intact

when St-1 RF

was

digested

with

both

enzymes.

On the basis of these

results,

we were

able

to

align the

two

restriction

maps as

shown in

Fig.

2.

EcoRI

and

Bgll

cleavage of

St-i.

The

po-sitions

on

the

map

of the three

EcoRI and

single

Bgl

cuts were

determined

by

digesting

St-1 RFI

with HincII

or

HaeIII

and

then with either

EcoRI

or

BglI.

Comparison of the banding

pat-tern obtained

by fractionation of these "double

digests"

on

acrylamide

gels with that of the

comparable single

enzyme

digest permitted

identification of the

particular fragment

cut

by

the

second enzyme. These

data

are

included

on

the

map

(Fig.

2).

Size of

St-1 RF. The

size of

St-1

RF DNA

was deduced to be about 6,050 bp in two ways:

first,

from the sum of the sizes of fragments

resulting from digestion of

St-i

RF by the

HinclI

or

HaeIII

enzyme; second, relative to that of

linear

4X174, G4,

or

S13

(Fig. 3). These were

also compared with

an EcoRI digest of

bacterio-phage

A DNA

whose fragment

sizes are well

documented

(24).

The

fast-moving

bands

pres-ent

in the

St-1

+

Bgl

and

OX174

+ Pst digests

were undigested

St-i

and

4X174

RFI,

respec-tively.

DISCUSSION

The

analysis of St-1

RF DNA

by restriction

endonucleases described

in

this

paper

has

con-firmed

the

observation, based

on

sedimentation

values, of Bowes and

Dowell

(3) that the St-1 genome

is

larger than that of

4X174.

Our

obser-vations

suggest

that,

at

about

6,050

nucleotide

pairs, the St-1

genome

is 10%

larger

than

OX174.

St-1

DNA is

also

larger than the DNA of the

related isometric

phages G4

and

S13 (Fig.

3)

[image:5.501.128.400.349.664.2]

and,

therefore,

possesses

the

largest

genome of

FIG. 2. Cleavage map of

St-I

DNA.

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VOL.27,1978

~~~~~~RESTRICTION

"F 'P

FIG. 3.

Comparison of

the

mobility

oflinearSt-i DNA

(produced

by

BglI cleavage)

with that

of

similarly

generated

linearG4,

4~X,

and S13 DNAs. An EcoRI

digest of

bacteriophage

A DNAonitsown,ormixed with

linearSt-i1or

4X174

DNA

before

loading

the

gel,

was

fractionated

in

parallel (three

slotsto

right of figure)

to

provide

the molecular

weight

markers noted in the

figure.

The

0.7%,o

agarose

gel

wasstained with ethidium

bromide

after electrophoresis

and

photographed

with UVillumination.

all the isometricphages reportedsofar. The sizes of thefragments producedfrom

St-1 DNA by cleavage with endonuclease HaeIII and Hincll (Fig. 1 and Table 1) arecompletely

different from thoseproducedfrom 4X174DNA. St-i DNA isalso cleavedbyendonucleasesBgl

(once) and EcoRI (three tizmes), which do not

cleave 4X174 DNA.Thus, the St-i genome not

onlyislargerthan that ofOX174,its DNAbase

sequence appears tobe different. However,the viral proteinscoded by St-i appear onsodium

dodecyl sulfate-acrylamidegelstobesimilar to

those codedfor

by 4X174, S13,

andG4

(Grindley

and

Godson,

unpublished

data).

This

imnplies

thatSt-1 has

kept

thesamebasicgenomestruck

tureasthe otherisometric

phages

sofar

exam-'

mned

(4X174, S13,

and

G4).

The extra DNA of

St-i1

may be due to an increase in size of its

intercistronic spacesor

p'erhaps

tothe

nonover-lap

in

St-i1

ofthose geneswhich have been shown

to

"overlap"

in

4X174, viz., D/E

and

A/B.

LikeG4

(18),

St-i

requires only dnaG, dnaE,

and DNA

binding protein

toinitiate and

synthe-size its

complementary

DNA strand in vitro

(14),

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744

but

unlike G4, it

does not require dnaB and

dnaC/D

proteins to replicate its

double-stranded DNA in vivo (6, 22, 23). At 42°C in

dnaB

and

dnaC/D

E. coli

cells,

St-1 synthesizes

normal amounts

of RF and

single-stranded

prog-eny DNA (Grindley and Godson,

unpublished

data) and

can

form

plaques normally

(2). These

differences

must

ultimately

reside in

differences

in the DNA

base sequence and structures of

protein

recognition

sites. The

generation

of the

St-1 restriction endonuclease

cleavage map

de-scribed in

this paper is a prelude to such a

sequencing study.

ACKNOWLEDGMENTS

This work wassupported by Public Health Service grants CA-06519 from the NationalCancer Institute and5RO1

AI-11633from theNational Institute of Allergy and Infectious Diseases.

LITERATURE CITED

1. Barrell, B. G., G. H. Air, and C. A. Hutchison III. 1976.Overlapping genes in bacteriophage4X174. Na-ture(London) 264:34-41.

2.Bowes, J. M. 1974. Replication ofbacteriophageSt-iin Escherichiacoli strains temperature sensitive in DNA synthesis. J. Virol.13:1400-1403.

3. Bowes, J. M.,andC. E. Dowell. 1974. Purification and some properties of bacteriophage St-1. J. Virol. 13:53-61.

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