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0022-538X/81/100133-09$02.00/0

Characterization

of

Bacteriophage N3 DNA

ELZBIETA S. FILIPSKA,'t DANIEL J. MOYNET,' AND FRANK M. DEFILIPPES2*

Laboratory of Streptococcal DiseaseslandLaboratory ofBiologyof Viruses,2 NationalInstitute ofAllergy

andInfectious Diseases, Bethesda,Maryland20205

Received5February 1981/Accepted9June1981

The DNA from

Haemophilus influenzae

temperate

phage N3

was

character-ized

by centrifugation

and

by

electrophoresis

after nuclease

digestion.

The

double-stranded

DNA,

witha massof 25.8x 10i daltons, had single-strand

cohesive

ends. Strand association through cohesion wasreduced

by

heatand removed by Si

nuclease

digestion.

N3 DNA

contained five EcoRI,

oneKpnI,two

SacI,

six XbaI,

and four XhoI cleavage sites. The cohesive end

segments were

identified

by

heating the digests before

electrophoresis. This

was

the first

stepin the

construc-tion of the

physical

mapsofthis DNA.

Haemophilus

influenzae bacteriophage

N3

grows in strain Rd9

of H.

influenzae. Although

the

phage does

not

adsorb

to

other

cells, DNA

extracted from the virus

transfects

different strains of H.

influenzae (7).

N3

prophage

in

strain Rd9 also

inhibits the transformation of

these cells. This inhibition is due to both a decrease in competence of

lysogenized

cells and

interference with the

expression

of the

reduced

quantity

of DNA which

is bound

to

the

cells

(11).

To

understand viral functions which may

effect this

inhibition,

as

well

as to

determine

the

physical

characteristics of N3 DNA which

may

be correlated with

genes,

we studied both ends of the

genome

and

established

restriction

en-zyme segment maps.

MATERIALS AND

METHODS

Purification of

bacteriophage

DNA. H.

influ-enzaeRd9 (N3)cells,lysogenic for temperate

bacte-riophage N3 (7),weregrownwith aerationat37°Cin

200ml of mediumcontaining 170mlofEugon broth

(Difco Laboratories), 30 ml of brain heart infusion

(BHI)broth(Difco), and1mMCaCl2,supplemented

with10ug of hemin(Calbiochem ) per ml and2ygof

NAD (Boehringer Mannheim Corp.) per ml. When

the cultures grewto5x108colony-formingunits/ml,

mitomycin C (Sigma Chemical Co.) was added to a

final concentration of0.1

jig/ml

toinduce the cells,

and the incubation was continued for 180 min.

Twomillicuries of

[methyl-3H]thymidine

(Amersham

Corp.,50Ci/mmol) or100uCi of

[methyl-"C]thymi-dine (Amersham Corp.,55mCi/mmol) was added to

somecultures with themitomycin C.Celldebris and

unlysed cellswere removed by three rounds of

cen-trifugation at 10,000 rpmfor 10 min. This step and

subsequent centrifugationswere at10°C. Phagewere

pelletedat35,000 rpm for90minin a 35 rotor

(Beck-man rotors wereusedthroughout), suspendedinBHI

tPresent address:InstituteofMicrobiology, Warsaw Uni-versity, Warsaw, Poland.

medium with1mMCaCl2(BHI-Ca),

afld

centrifuged

into a CsClgradientat25,000 rpmfor4h in anSW27.1

rotor.Thedensity gradient at pH 7.4, which contained

20mM Tris-hydrochloride and 8% sucrose (wt/vol),

wasformedbydiffusion from four 2-ml layers, which

werepreparedbydilutinga1.9-g/mlsolution of CsCl

withBHI-Catogivedensities of 1.7, 1.5, 1.35, and1.2

g/ml.The phage band, at a density of 1.47 g/ml, was

collected anddiluted with2volumes of BHI-Ca. Phage

werepelletedat40,000 rpmfor 6 h in a 50 Ti rotor and

suspendedin2mlofsolution H (1 M NaCl-100 mM

sodium phosphate-10 mM sodium-EDTA, pH 7.2).

The DNAwasextractedat24°Cby adding0.22mlof

10% sodiumdodecyl sulfatetothe suspension, which

wasthen heated at65°C for 5 min. After5 min at

0°C,0.33ml of2MKClwasadded,and thesolution

washeldat0°C for5min andcentrifugedat 10,000

rpmfor 20min (1).The supernatantwasmixed with

phenol,dialyzedagainstbuffer K (10 mMKCl-ImM

sodium-EDTA-10 mM Tris-hydrochloride, pH 8.3),

and storedat4°C. DNAfrombacteriophageA cI857

and adenovirus type2 waspurchasedfrom New

Eng-land BioLabs and Bethesda Research Laboratories,

respectively.

Centrifugation.

Lineargradientswereeither from

5to20% or 10to30%sucrose (wt/vol). Neutral

gra-dients usually contained 20mM Trs-hydrochloride,

10 mMsodium-EDTA, 0.5% Sarkosyl NL-97 (Geigy

ChemicalCorp.),anddifferentconcentrations of NaCl.

Alkalinegradients contained0.7MNaCl,0.3NNaOH,

10mMsodium-EDTA, and0.5% Sarkosyl. Centrifu-gationwasusuallyinanSW50.1orSW41 rotor at5°C.

Fractions were collected

through

the tube bottom.

Radioactive

samples

werecounted in

liquid

scintilla-tionfluid(Aquasol)inaBeckmanLS-350liquid

scin-tillationcounter.Alkalinesampleswereacidified with

acetic acid beforecounting.

CsCl equilibrium

gradient

solutions,

which

con-tained20mMsodium-EDTA,50mM

Tris-hydrochlo-ride,and5.63MCsCl(pH 8.3),hadaninitialdensity

of1.70g/mlat20°C.TheDNA,in 7

ml,

was

centri-fuged at40,000 rpm for 72 h in a 50 Ti rotor, and

fractionswerecollected from the bottom. The

refrac-133

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http://jvi.asm.org/

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134

tive index of every second fractionwasmeasured to

check that a lineargradient was established in the

middle of the tube where the DNAbandwaslocated.

Vaccinia virus DNAand Escherichia coli labeled with

[I4C]-

and [3H]thymidine, respectively, were used as standards of known buoyant density and guanine plus

cytosine content.The marker DNAs were included in

the tube with the[3H]thymidine-labeled N3 DNA and

alsobanded inaseparate tube without N3 DNA. The

N3 DNAwasalso banded inaseparatetubewithout

the other DNAs. Gradient fractionswerediluted with

1mlof water and counted as described above.

Thermal denaturation of N3 DNA. N3 DNA in

150 mM NaCl-15 mM sodium citrate (SSC) pH 7.0,

washeated in asealed quartz microcell in a Gilford

recording spectrophotometer. The temperature was

increased 1°C per min from 24 to 100°C, and the

absorbanceat 260 nm wasrecorded.T7oradenovirus

type2 DNAwasheated inaseparate cuvetteatthe

sametimetorecordabsorbancy changes for a DNA

with a knownmelting temperature and base

compo-sition.

Nuclease digestion. Restriction enzymes were

purchasedfrom NewEnglandBioLabs. N3 DNAwas

digested for4hat37°Cunderthe conditionsdescribed

by NewEnglandBioLabs for each enzyme. Thedigests

contained2U of enzyme per

jig

ofDNA.Thereaction

wasended by the addition of sodium dodecyl sulfate

andsodium-EDTAtofinal concentrations of 0.5% and

1mM.After extraction withphenol, the aqueous phase

wasdialyzed for4hagainst buffer K.

Nuclease Siwaspurchasedfrom Miles

Laborato-ries, Inc. The unit of enzymeactivityis thatdefined

by the manufacturer (1979). Digestion proceeded by

mixing 30

pi

of DNA inbuffer K with20,ulof bufferS

(600 mM NaCl-3.6 mMZnCl2-72mMsodium acetate,

pH4.6),containingeither8 or32U of enzyme per

jig

of DNA, and incubating at 47°C for 30 min. The

reactionwasstopped bycoolingat0°Cfor5minand

adding sodium-EDTA and Tris-hydrochloride buffer

(pH 8.3) to final concentrations of30 mM and 100

mM,respectively.

Gel electrophoresis. Agarose gels (0.6 or 1%;

SeaKem) containing buffer E (5 mM sodium

acetate-1mMsodium-EDTA40 mM Tris-hydrochloride, pH

7.95)wereformedashorizontal slabs(5 mm thick and

25by20cm).Electrophoresiswasat50Vfor16hat

24°C. DNA (0.4

jig)

inone-half-strengthbuffer E with

5%glycerolwasloaded in eachsamplewell. After the

first 2hofelectrophoresis, thegelslabwascovered

witha 2-mmlayerof buffer. Afterelectrophoresis,the gelwasimmersed in ethidium bromide (1,tg/ml) for

30 min and then photographedwith eithera

short-wave UV source (Mineralight R-52) or a 365-nm

source (C50 transilluminator, both from Ultraviolet

Products, Inc.) andaredfilter(23A, Tiffen).

In vitro labeling of DNA segments. Slices of

agarose gels which contained DNA segments were

dissolvedat24°Cin67% saturatedNaIsolution, and

the DNA wasrecoveredbythe procedure of

Vogel-steinandGillespie (21).DNAsegmentswerelabeled

with

[32P]dATP

for the nick translation procedure

(12). Labeled DNA wasseparated from precursor by

gelfiltration through agarose 1.5 m (Bio-Rad

Labo-ratories) andpurified byphenolextractionandethanol

precipitation.

VIROL.

Hybridization of labeled DNA. After

electropho-resis, the DNA in agarose gels wastransferred to a

nitrocellulose membrane filter (BA-85; Schleicher &

Schuell Co.) by the Southern procedure (18) and

im-mobilized on the membrane by heating at 60°C over-night. Membrane strips were preincubated in Den-hardt solution (4). A nick-translated DNA segment

was hybridized to theimmobilized DNA in 1 M NaCl,

10mM sodium-EDTA, 400 ,ug of bovine serum albumin

perml, 0.4% sodium dodecyl sulfate, 20 mM PIPES

[piperazine-N,N'-bis(2-ethanesulfonic acid)], pH 6.9,

at60°C for 20 h in a volume of 0.6 ml. The membrane

was washed first with a solution containing 0.5 M

NaCl, 10 mM sodium-EDTA, 0.4% sodium dodecyl sulfate, and 10 mM PIPES (pH 6.9) for 6 h at 60°C

and then with10mMTris-hydrochloride (pH 9.0) for

10 minat24°C. The membranes were dried at 24°C,

andthe hybridized[32P]DNA was located by

autora-diography. The location of the immobilized DNA on

the filter wasdetermined either from a ruled

photo-graph of the stained gel or by hybridizing a small

amountof sheared N3

[32P]DNA

to thefilter. In this latter case, hybridization of a unique DNA segment

producedaradioautograph with enhanced blackening

atspecificsites, which could be distinguished from the

"background" signal used to locate the immobilized

DNA.

RESULTS

Figure

1A

shows

an

alkaline

sucrose

gradient

centrifugation

pattern for N3 DNA. The

single-stranded chains sedimented at a

slightly

slower

rate

than did the

single strands from

A DNA,

which form

a

peak located by the

arrow.

In these

gradients,

N3 DNA

moved

at

the

same rate as

T7

DNA

(data

not

shown),

whichhas a

duplex

mass

of

26 x 106

daltons

(19), whereas

our A

DNA hasa

duplex

massof 32 x 106 daltons

(5,

13).

The

shoulder

onthe

low

mass

side increased

with

the

time of

storage of the

DNA,

indicating

radiation damage of

the

molecules.

When

N3

phage

DNA was

centrifuged

to

equi-librium in

a

cesium chloride

gradient,

a

single

symmetrical peak

was

observed

(Fig.

1B). The

buoyant

density of

N3

DNA,

as

estimated

by

a

linear

interpolation

between the

values

given

for

E.

coli and for vaccinia virus

DNA,

was 1.703

g/ml. This would correspond

to a

guanine

plus

cytosinecontentof43

mol%,

according

to

stand-ard

tables

(16,

20).

The

melting

temperature of

N3 DNA

(curve

not

shown)

was

87.3°C,

which

also

corresponds

to a

guanine

plus

cytosine

con-tentof43mol%

(10, 15).

The

single peaks

observed in

pattern

1were

notseen in

high-salt,

neutral-pH

velocity

sedi-mentation

gradients.

Centrifugation

of N3 DNA

at

neutral

pH

ina sucrose

gradient

containing

1

MNaCl

spread

the molecules into several frac-tions

(Fig. 2A).

Most of the N3 DNA moved more

rapidly

than T7

DNA,

whichsedimentsas

a

single

peak.

Since

analysis by

alkaline gra-dients

indicated

a

single

DNA

species,

we felt

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(3)

'(4

2

0

10 20 30 40 50 60 70 80

20

F

<

V B

I

n

1.71

15 1.70

-1.89

x10 1.68

5-10 2D 30 40 50 60

FRACTION NUMBER

FIG. 1. Centrifugation ofN3 DNA. (A)DNA was

sedimentedthroughanalkalinesucrosegradient. A

DNA, labeled with

['4C]thymidine,

was added to

buffer K, containingfreshly preparedN3DNA

la-beledwith[3H]thymidine. Thesolutionwaslayered

on the gradient, and the DNA was centrifuged at

25,000 rpm for 11 h at200C in a Beckman SW41

rotor. Thearrowindicates thepeakpositionofthe A

DNA. Symbol:

0,

N3

[3HJDNA.

The direction of

sedimentation in this and thefollowingpatternsfor

sedimentation velocityisfrom righttoleft. (B)

Equi-librium centrifugation in CsCl atpH8.3. N3 DNA

labeled with

[3H]thymidine

wasmixed with vaccinia

virus DNAlabeled with

['4C]thymidine

and E.coli

DNA labeled with [3H]thymidine. The number of

radioactivecountsofE. coliDNAaddedtothe

mix-ture was one quarter of the N3 DNA counts. The DNAwascentrifuged andcollectedasdescribed in

the text. Thecapital lettersimmediatelytotheleftof

eachpeak denote the DNA associatedwith thatpeak:

V,vaccinia virusDNA; N, N3 DNA; and C,E. coli

DNA. Symbols: A, N3DNA; 0, vaccinia or E. coli

DNA. Straight line represents density determined

fromrefractive index measurements.

'~6

o

2

5 10 15

20 -<

10

10 20 30 40

[image:3.496.270.427.52.378.2]

FRACTION NUMBER

FIG. 2. Centrifugation at pH 8.3. (A) N3 DNA

stored inbuffer Kat4°Cwascentrifuged through a

10 to30%sucrosegradientcontaining1MNaCl in a

BeckmanSW41 rotor at30,000rpmfor12hat5°C.

The arrowlocates T7 DNA, which was centrifuged

inanadjacent tube. (B) Centrifugation of N3 and T7

DNA in a sucrose gradient without added NaCl.

DNAwasheatedat68°C for5min,quickly cooled to

0°C,andimmediatelycentrifuged througha5to20%

sucrosegradientcontaining10mM

Tris-hydrochlo-ride andI mMsodium-EDTAat38,000 rpmfor3h

at5°C inaBeckmanSW50.1 rotor. Symbols:0, N3

[3H]DNA; 0, T7['4C]DNA.(C)Centrifugation of

Sl-treated DNA. N3 DNA inbuffer K was held at 70°C

for5min, cooledto47°C, and mixed, at 47°C, with

concentratedSlbuffer that contained32U of enzyme

per

pg

ofDNA. The solution wasincubatedat47°C

for 30 min. The reaction was terminated, and the

DNA wascentrifuged through a 10 to30% sucrose

gradient containing 0.1 MNaCI, at 30,000 rpm for 12

h at50C.

that the

results

of

Fig.

2A were due to the

aggregation

of

duplex

molecules. To reduce

ag-gregation,

a solution of N3 DNA was

kept

at

650C

for5 min and then cooled

rapidly

to

00C

and

examined

by

centrifugation

at50C

through

asucrose

gradient

containing

0.1M

NaCl.

That

profile

(data

not

shown)

showed

that, although

the short heattreatment and reduced

salt

on November 10, 2019 by guest

http://jvi.asm.org/

[image:3.496.43.237.55.455.2]
(4)

centration did not eliminate the spread of the molecules, they did redistribute the radioactive

countstowardthelowmasssideofthe gradient.

This result suggested that aggregation mightbe further reduced by othertreatments.Totestthe

effect of different conditions, weran aseries of

low-resolution, neutral-pH gradients. When the DNA was heated to 65°C for 5 min, quickly cooledto00C, and then sedimented througha5

to 20% sucrose gradient containing 1 mM so-dium-EDTA and 10 mM Tris-hydrochloride (pH 8.0), the profile of Fig. 2B was obtained.

With theseconditions,the N3 DNAsedimented

at the same rate as T7 DNA. Although the above conditions usually resulted in

centrifuga-tion patterns similarto those ofFig. 2B, there

were preparationsof N3 DNA whichproduced minor peaks thatwerelocatedonthehighmass

sideof the main peak. With these preparations, additional treatment ofthe DNA immediately

before centrifugation, such as heating to 700C

for 10 min ordigestion withproteinase Kfor4

h followed by heating at 700C for 10 min, did

notremovethefast-sedimenting material.

These experiments indicated that N3 DNA

has "sticky ends," which associate to combine intra- and intermolecular strands. Since heat

treatmentdid notalways produceonespecies of

linear duplex molecules, we assumed that the stickyends reacteddifferentlyfromthosefound

in A DNA (3). To verify the inferred

single-strand ends, we heated N3 DNA at70°C for 5

min; it was then cooled quickly to 00C and

treatedwith Si nucleaseat370C. Sedimentation

in 0.1 MNaCl didnot generateapatternwitha

single peak. However, when the DNA was

heated to700Cfor5min, cooled quicklyto470C,

and immediately digested with Si nuclease at 470C (22),the pattern ofFig.2C resulted. Here

the N3 DNApeakislocatedslightly behind (one

fraction) the position of T7 DNA, which was

sedimented inanadjacenttube.

DNAfromphage N3wasdigested by several restriction nucleases, including EcoRI, XbaI, XhoI, KpnI, and Sac. The massesof the

seg-ments produced by digestion with several

re-striction enzymes were determined froma

cali-bration curve, using adenovirus type 2 DNA

segmentsproducedby HindIII, BamI,orEcoRI

digestion as standards (9). The sizes listed in

Table 1 are average values, which were

deter-mined fromatleasttwodifferent gels. Thesum

of the masses oftheEcoRI segments is25.8 x

106 daltons, and that oftheXbaI segments is

25.75 x

106

daltons. Since these numbersagree

withourestimate fromcentrifugation, wechose

25.8x 106daltons forthemassofN3DNA.

Theelectrophoreticpatternsformed by

sepa-rating segments obtained by digestion with

TABLE 1. Molecularweightoftherestriction

enzyme segmentsof N3 DNA Mol wt(x106) of: Segment

EcoRI XbaI XhoI KpnI

A 6.8 13.2 8.45 24.78a

B 5.6 4.1 8.45 1.02

C 4.3 2.7 4.2

-D 4.1 2.0 3.5

-E 2.7 1.55 1.15

-F 2.3 1.25 -

-G - 0.95 -

-mol wt 25.8 25.75 25.75 25.8

aThe molecular weight ofthe A segment of the

KpnI digestwasobtained by deductingthe value of

the B segment from the intact DNA (25.8 - 1.02 = 24.78).

b

,None.

EcoRI,

XhoI, XbaI,

andKpnI are shown in Fig.

3. The

fainter

bands

denoted by

t are the

co-hered

end segments. The

major

segmentsfrom

a

single

enzyme

digest

are

designated

by capital

letters.

Aband ofdouble

brightness

isassigned

twoconsecutive

letters.

The convention for this and other

figures

is that lanes are numbered from rightto

left.

The cohesion of thetwoendshelpedto

iden-tify

the end segments, as

illustrated

in

Fig.

3

withan

XbaI

digest.

When DNA that had

been

stored fora

day

was

digested

for 120 min and

then

examined

by

electrophoresis, Xba-C

and

-D

did

not

show

(lane 5). Examination of

anXba

digest

that washeated at

68°C

for 10 min and

then

chilled

at

00C before

electrophoresis (lane

7) reveals that bands C and Daremuch

brighter

and that band t is diminished

compared

with

lane

5.

Since

themassof

the molecules

inband

tis thesumof the segments in bands

C

and

D,

we conclude that t contains molecules formed from the union of

C

and D.WhenanXba

digest

was

held

at

00C

and then

digested

with

S1

nuclease

at47°C for30

min,

mostof the

mole-cules

of bandtwere

eliminated

(lane

6).

Since

S1

nuclease

digests single-stranded

nucleic

acid (22),webelieve thatN3 DNAtermini cohere

by

association of

single

strands. The end segments presentinmostrestriction

digests

could be iden-tified

by

the effects of heat or

S1

treatment or both. The terminal segments in

Fig.

3areEco-A

and -B

(lane 1),

Xba-C and -D

(lane 2),

Xho-A

and -E

(lane 3),

and

Kpn-A

and -B

(lane 4).

The identification of end segments is the first step in

constructing

physical

maps. Mostof the data neededtoorder the segments

produced by

one enzyme were obtained

by

digesting

those segments withasecond restriction nuclease.

Fig-ure 4 shows

patterns

which were

generated

by

the

digestion

of EcoRI-cleaved DNA

by

asecond

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http://jvi.asm.org/

(5)

FIG. 3. Agarose gel ekctrophoresis of N3 DNA

restriction enzyme segments in a horizontal slab.

Lanesare ordered from rightto left. Theenzymes

usedfordigestionaregiven, with thegel lane showing

thedigestpattern.Lane 1,EcoRI; lane2, XbaI;lane

3,XhoI; lane4, KpnI. Lanes5to 7, electrophoretic

patterns afterSI nuclease digestion orheating or

bothofXbaIrestrictionenzymesegmentsofN3DNA.

Afterheattreatmentorenzymatic digestionorboth,

the samples were held at 0°C for 10 min before

electrophoresis. Lane 5, Xba digest ofDNA which

hadbeen storedat4°C in K buffer.Lane6,The Xba

digestwasheldat68°C for10min, cooledto47°C,

andimmediatelydigested with32 U ofSlnuclease

perugofDNAfor30minat47°C. Lane7, The Xba

digestwasheatedto68°Cfor 10min.

enzyme.Lanes 2 and 3showsegmentsproduced

by

cleavage with both XbaI

and

EcoRI,

flanked

by segments

produced

byEco(lane 1) and Xba

(lane 4). Double-digest bands are labeled by numbers; bands of double brightness are as-signedtwoconsecutive numbers.Double-digest bands which did not appear in single digests contained DNA definedby the overlap between

segments

produced

by individualenzymes.Since

EcosegmentsAand Bwere notpresentinthe

double digest and Xbasegments C and D had bandsatcorresponding levelsin the double di-gest,we had the expectedresult that C and D wereincludedinAandB.We concludethatEco segment

E,

atthe level of

Xba-C,

has an Xba

site

since the corresponding

double-digest band

3was not

twice

as

bright

as a

single band. This

reasoning could

not

be

applied

to

distinguish

between

the

survival of Eco-D

or

Xba-B

as an

intact

segment, one

of

which

might

be

contained

in

band

1

of the

double

digest.

Examination of

lanes

1

and

2

indicates that Eco

segments A, B,

C, and E contain Xba sites and that

segment D

might also contain

a

site.

By

comparing lanes

4

and

3,

we

conclude that Xba-A and Xba-F

con-tain Eco

sites and that Xba-B might contain

an

Eco

site.

The data

obtained by

digesting

N3 DNA

with

XhoI

(lane 8),

EcoRI

(lane

5),

and both enzymes

(lanes

6

and

7)

were

analyzed

in the

same

man-ner.Here

Eco-A

survived and

mustbe

included

in

Xho-A since the other

terminal

segment,

Xho-E,

was

smaller

than Eco-A. If

we

place

the

Eco-A

segment on

the

left side of the

map,

then

Eco-B,

which

was

cleaved since it includes

Xho-E,

is

at

the

right

side. Xho-E

was

the

only

Xho band

which

appeared

intact in the

double digest. Since

Eco-B,

-D,

and -F

were

cleaved

by

Xho and

since

Xho-A,

-B,

-C,

and -D

had Eco

sites,

seg-ments

contained in these bands

must

contain

overlaps.

Lanes

9 to 12

show digests by EcoRI

(lane

9),

EcoRI and

KpnI

(lanes

10

and

11),

and

KpnI

(lane 12).

This last

enzyme

had

one

site

in

N3

DNA, which

was

located in Eco-B.

If

intact

N3 DNA were

terminally

labeled,

KpnI

diges-tion

would

allow

the

construction of

maps

by

the

procedure

of Smith and

Birnstiel

(17).

Lanes 13 to 15

had

patterns produced

by digestion

with

EcoRI

(lane 13) and both

SacI and EcoRI (lanes

14

and 15).

Lane 16

contains

a

calibration

pat-tern

obtained from

a

partial

HindHI

digest of

adenovirus

type 2

DNA, for which the size of the

partial

products

was

known.

The

digest by SacI

alone, which is

not

shown,

produced

one

large

band of

double

brightness and

one very

small

band. The

presence

of

two

SacI sites

in N3 DNA

is

confirmed

in

Fig.

4

(lanes

13

and

14),

where

Eco

bands

B

and

D

(lane

13)

were not

present

in

the

double

digest

(lane 14).

SacI and

KpnI

cleaved Eco-B

at

the

right

terminus of

N3

DNA,

so

they

can

be used

to

distinguish end

segments

generated

by other

enzymes.

Physical

maps were

also

determined from

hy-bridization data.

Individual

segments

were

iso-lated from

gels,

labeled

by

nick

translation,

and

hybridized

to segments,

produced

by

a

second

enzyme, whichwere

immobilized

on

nitrocellu-lose

(18). Figure 5 shows a

radioautograph

in

which filter-bound

EcoRI

segments

were

hy-bridized

toXbaIsegments A

(lane 3),

B

(lane

2),

and C

(lane 1).

The

strips

indicate the

position

of

the

different

EcoRI

segments.

Xba

segment

A

hybridized

toEcosegments t,

A,

C,

D,

and F. Xba-B

hybridized

to

Eco-t,

-Band

-E,

and

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FIG. 4. Electrophoretic patterns ofrestrictionenzyme digests of EcoRI N3 DNAsegments. N3 DNA which

had been cleaved with EcoRI wasdigested with a secondrestriction nuclease. The DNA from the double

digest was, except for Saccleavage, placed in lanes between the DNA from each single-enzyme digestion. The

enzymesproducingthe DNApatterns were: lanes 1, 5, 9, and 13,EcoRI; lanes 2 and 3, XbaI-EcoRI; lane 4,

XbaI; lanes6and7,XhoI-EcoRI; lane 8, XhoI; lanes 10 and 11,

KpnI-EcoRI;

lane 12,

KpnI;

lanes 14 and 15,

SacI-KpnI; lane16, segmentsfrom adenovirus type2DNApartially digested by HindIII to provide DNA

segmentsofknownmass.

C

hybridized

toEco-tand -A.

Any

DNA which

hybridized

toEco-A or-B

also associated

with

Eco-t.

Since Xba-C

is a

terminal

segment,

it

must

be

on the

left, where it associates with

Eco-A. The other terminal

segment,

Xba-D,

is

atthe

right

end.Since Xba-A also

hybridized

to

Eco-A, it is to the

right

of Xba-C. Xba-A also

hybridized

toEco-C, -D, and -F, so they must

beto

the

right

of

Eco-A.

Xba

cleaved

Eco-A,-C,

and

possibly

-D

(Fig. 4),

sothe

right

end of

Xba-A must

overlap

the

left

side of

either

Eco-C or

-D.Band 1

(Fig.

4,

lanes

2and 3) atthelevel of

either Eco-D orXba-Bwas not

doubly bright,

soit iseither Xba-BnorEco-D. Itmustbe

Eco-D since the

right

terminus of Xba-A locates a site in Eco-C. We also conclude that Xba-B containsanEco site.Included between A and C

on the Eco map are segments D and

F,

whose

ordermustbedetermined.

Again,

Xho terminal

segmentAincluded Eco-A(Fig. 4),soit is atthe

leftend,whichplacesXho-E, the otherterminal

segment, at the right end. Since Eco-B was larger thanXho-E, it must contain an Xho site. The order ofXho-B, -C,and -Dmust be deter-mined. Since both Eco-D and -F were cleaved by Xho, Xho-B cannot be

adjacent

to Xho-A because in that position Xho-B would include either Eco-D or -F as an intact

piece.

Either Xho-C or -Dmustbe

adjacent

toXho-A. Xho-C and D were

sufficiently large

so that if either segmentwere

adjacent

totheright side of Xho-A, it would locate a second Xho site from the left terminus at a

position

which would allow

thecorrect

placement

of Eco segments D and F

next toEco-A.

Analysis

shows that thetwosmall

bands,

9 and 10, with massesof0.6 x

106

and

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(7)

was

confirmed by comparing

an

Xba digest

with

a

Sac-Xba double digest (data

not shown).

Xba-D, the

right terminal

segment

included

in

Eco-B, was cleaved by

Sac.

Furthermore, because

Sac-A and

-B

spanned

a

length

of the genome

equal

to at

least

24 x

106

daltons, the Sac site

on

the

right side

must

be

at

the right

end of

Eco-B.

One large Sac

segmentextended from the left

terminus for

at

least

a

length equal

to 11 x

106

daltons;

so

it includes

Eco-A and

either

Eco-F

orEco-D, depending on which segment is adja-cent to

Eco-A. Since Eco-D

was

cleaved

by SacI,

it

cannot

be

adjacent

to

Eco-A,

which

confimis

our

earlier conclusion.

This

Sac

site in Eco-D

must

also

be present in

Xba-A;

this was

dem-onstrated

in a

Sac-Xba

digest where

anew

seg-ment, 1,

with

a mass

of

about 10 x

106

daltons,

was

derived from

Xba-A.

In

Fig.

6,

labeled

segments from Xho-E (lane

1), Xho-D

(lane 2), Xho-C

(lane

3), Eco-F

(lane

4),

and

Eco-E

(lane

5) were

hybridized

to

im-mobilized Xba segments. Sheared, labeled

N3

DNA

was

used

to

locate each

Xba segment on

5

4

43

2

l

FIG. 5. Radioautograph showing the pattern of

hybridizationofN3 DNA XbaI segmentstoN3DNA

EcoRI segments immobilized on a nitrocellulose

strip. XbaI segments were labeled with32P bynick

translation andhybridizedtobound EcoRI segments

onindividualstrips.The letters locate thepositionof

the Ecosegments, with tdenotingthe cohered end

segments. The labeled segmentswere:lane 1,Xba-C;

lane 2,Xba-B;lane3, Xba-A.

0.55x

106

daltons

inthe

Xho-Eco double

digest

in

Fig.

4,

lanes

6and7, must

have been

generated

by Xho

cleavages of Eco-D and

-F when

they

were

arranged with

-F between -A

and

-D. If

Eco-D were

adjacent

to

Eco-A, then

one

small

band,

at

most, would

have beenproduced

by the

double

digest.

SacI cleaved

N3 DNA at two

sites and

pro-duced

an

electrophoretic pattern

which had

only

two bands

(data

not

shown).

The

lower

band,

with

segments

of 1.02 x

106

daltons, was the same as band 7 in the

Sac-Eco

double digest

(Fig.

4,

lanes

14 and 15). The upperband was

doubly bright

and contained two segments (A

andB) which moved

slightly

faster than Xba-A. Sac-A and -B each had a mass between 11.5 x

106

and 13.5 x

106

daltons. The Sac-Ecodigest did not contain a band at the

level

of

Eco-B,

whichlocates one of the Sac sites. This location

c

a

D A A *

... .

a r

4

_ a

S.

E so

F

0

a

I

0

la

I

C.

FIG. 6. Radioautograph showing thepattern of

hybridization ofN3DNA segmentsfrom XhoI and

EcoRIdigestsonXbaIsegmentsimmobilized on

ni-trocellulose strips. Xho and Eco segments, labeled

with32P,werehybridizedtobound Xbasegmentson

individualstrips.Theletters locatetheposition ofthe

Xba segments. Lane 1,Xho-E;lane2,Xho-D;lane3,

Xho-C;lane4,Eco-F; lane 5,Eco-E.

3

2

t

- I

A

.

C

-D

-E

-.

F

-|

t

_

-

B.

4

_. ~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...

i X:

r,

.A.&I

'WI,

-

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[image:7.496.62.206.69.366.2] [image:7.496.244.434.317.577.2]
(8)

A F D C E B

A

XbalM C E F B G D

Xhol tA C D B E,

Kpnl I A

B,

0 20 40 60 80 100

I

FIG. 7. Location

of

restriction endonuclease

cleavage

sites

of

N3 DNA.

the

nitrocellulose. The

hybridization of

a

partic-ular segment enhanced the

blackening

of

those

Xba

segments which contained

homologous

se-quences.

Xho-E associated

with

sequences

in

Xba-D and

-t,

producing

an

enhanced

signal.

This

result confirms

our

placement

of

Xho-E

at

the

right terminus of N3 DNA. Xho-D

hybrid-ized with Xba-A and -E,

whereas Xho-C covered

part

of Xba-A. These results

place

Xho-D

to

the

right of

Xho-C and

Xba-E to the

right

of

Xba-A.

Xho-C

was not

cleaved

byXba (data

not

shown),

which is consistent with its inclusion

in

Xba-A.

Xho-D, which

spanned Xba-A and

-E,

was cut

by Xba

at

the

junction of these

two

segments,

as

seen

in

an

Xba-Xho

digest (data

not

shown).

The

remaining Xho segment,

B,

must

be

placed

between Xho-D and the

right

terminal

segment

Xho-E.

In

this

position, Xho-B would include

an

intact Eco

segment

E and

Xba

segments

F, B,

and

G.

Lane

4

of

Fig.

6

shows that Eco-F

hybridized

only

to

Xba-A,

whereas Eco-E

covered Xba-B

and

Xba-F,

which links these

two

segments.

Xba-F, -B, and -G

are now

located between

Xba-E

and the terminal Xba-D.

Xba-G

must

then be

either

to

the

right

or

left of the linked

segments

F

and

B. We

know

that

Eco-E

and -Bare

linked

by their

hybridization

to

Xba-B

(Fig.

5),

which

locates

an

Eco site

at

the

left end of Eco-E. This

site,

the second

one

from the

right terminus,

would

cleave Xba-G if it

were

placed

to

the left

of

the linked Xbasegments B and F. Lane3 of

Fig.

4

indicates

that

Xba-G survived Eco

diges-tionso we

place

it between

Xba-B, -F,

and

Xba-D. Lane 3

also shows that Xba-F

was

cleaved

by

Eco,

which

places

ittothe

left

of Xba-B

since it

would be included in Eco-Bas anintact

piece

if it wereonthe

right

ofXba-B.

The

locations

of the

cleavage

sites for four

restriction

endonucleases, EcoRI, XbaI,

XhoI,

and

KpnI,

areshown in

Fig.

7. In

addition,

we

know that

SacI

has one

site

whichis

found

at a distance

corresponding

to 1.02 x

10'

daltons

from the

right

terminus

(96

units)

andanother

site in Eco-D

which

is

about

12 x

106

daltons

from

the

left

terminus.

DISCUSSION

N3

DNA aggregated when

stored at

40C. With

some

preparations, over 90%

of the

[3H]thymi-dine counts

could be

converted

into

linear

du-plexes which

sedimnented

at the rate of T7

DNA

by simply heating the sample

for 5

min

at

680C

andthen

rapidly cooling it to

00C.

This

fraction

of thenucleic acid

population seems

to

resemble

A DNA in that

unit-length linear molecules

are

obtained

by

the same

operation

(3, 6).

Other

preparations

of N3

DNA, stored

and tested

over

the same

range

of

concentrations

as

above, were

more

refractory

to

conversion

by the

above

treatment.

Frequently

25% or

more

ofthe

mol-ecules

sedimented

faster than unit-length

du-plexes, and this

fraction could

not be

further

converted by

incubation with proteinase

K

be-fore

heating

to

680C. This result indicates

that

cohesion of the terminal

region does

not

depend

on a

protein, but it

does

not

rule

out a

small

peptide that

may aid

cohesion

and be

protected

from

proteolytic digestion

by the

nucleic

acid.

Since the number of

cohered segments

was

greatly reduced by Si nuclease digestion,

we

conclude that

single-strand tails

cause

cohesion.

We do

not

know the

detailed

structure

of

the N3

DNA

termini,

but

we

assume that

they

resemble

those found in X DNA. With N3 DNA

prepara-tions that were

resistant

to

complete conversion,

we

could

not

increase

the number of

linear

mol-ecules

by

heating

at

750C for

10min.Thisresult

suggests that interactions

which occur

after

heating determine conformation.

We

plan

to

examine

aggregation

or

circle

formation

or

both

as a

function of

concentration and ionic

condi-tions. We know

from

equilibrium density

cen-trifugation

that N3 nucleic

acid

is not a

super-coil.

It should be noted that X DNA may be

readily

converted to a

linear

monomer after

heating

because the 12-base tail sequence can

fold

back on

itself

toform a

duplex hairpin

which

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[image:8.496.125.414.54.202.2]
(9)

retards

further association (3). The

possibility

remains that

not every

molecule has the

same

nucleotide

sequence

in

the

single-strand tail.

The electrophoretic patterns

obtained after

digestion

with several restriction

enzymes were

constant

for all

ourN3 DNA

preparations.

We

believe that the

single-strand tail is less than

100

nucleotides long since it

was not seen

in

the

electron

microscope. These observations

indi-cate

that

N3 DNA

is

a

unique duplex species

with

a

small tail

at

each

terminus, which

mayor

may not have a

unique nucleotide

sequence.

The

effect of

Si nuclease

digestion

onthe

efficiency

of

transfection of H.

influenzae Rd9 cells

can

be

tested

by both

plaque

assays

and radioactive

DNA

uptake. We know that dilute solutions of

N3

DNA

(0.1

ug/ml*+2

x

109

molecules/ml)

which

are

heated

to

800C

and fast-cooled

gen-erate 20

times

more

plaques

than DNA from

solutions which

are

heated and slow-cooled

(7).

Conditions which minimize circularization also

increase the

infectivity of

DNA

from

H.

influ-enzae

phage

HPlcl

(2).

The

tail

structuresmay

help

to

integrate

the

phage

DNA

into the

bac-terial

DNA,

as

with

Bacillus subtilis

phage

105

and Mu

phage

(14).

Genetic

analysis

of

N3 DNA

is limited

(7, 8).

Since the

presence

of N3

prophage

inhibits the

transformation of the Rd9 strain of

H.

influ-enzae

(11),

we may

be able

to

identify

gene

products in the

lysogen

which

affect

transfor-mation. To associate those

gene

products

with

a

given nucleotide sequence,

genetic

and

physi-cal

mapsmust

be

determined.

Asa

first step,

we

have

ordered the restriction

segments

of the

phage

DNA. Although

the

segments may not

have

the same order

in

the

prophage

as in the virus, we expect

only

minor

rearrangements

in

the

prophage

map.

ACKNOWLEDGMENTS

WethankRogerCole foracritical review of themanuscript

and JonRanhand for manyhelpful suggestions.We also thank TerryPopkinforhelpingwiththephotography.

LITERATURE CITED

1. Bautz,E. K.B.,and J. J. Dunn. 1971.DNA-cellulose

chromatography, p.743-747.InG. L. Cantoni and D. R. Davies (ed.),Proceduresinnucleic acids research, vol. 2.Harper and Row, New York.

2. Boling,M.E.,J. K.Setlow,andD. P.Allison.1972. Bacteriophage ofHaenophilusinfluenzae. I. Differ-ences between infection by whole phage, extracted phage DNA and prophage DNA extracted from lyso-genic cells. J. Mol. Biol.63:335-348.

3. Davidson, N.,and W.Szybalski. 1971. Physical and chemicalcharacteristics oflambda DNA, p. 45-82. In A.D.Hershey (ed.),Thebacteriophagelambda.Cold

Spring Harbor Laboratory, Cold Spring Harbor, New York.

4. Denhardt,D. T. 1966. A membrane filter technique for the detection of complementary DNA. Biochem. Bio-phys. Res. Commun. 23:641-646.

5. Fiandt, M., A. Honigman, E. C.Rosenvold, and W. Szybalski. 1977.Precise measurement of the b2 dele-tion incoliphage lambda. Gene 2:289-293.

6. Helling, R. B., H. A. Goodman,and H. W. Boyer. 1974.Analysis of endonuclease R. EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J. Virol. 14:1235-1244. 7. Jablonska, E., and A. Piekarowicz. 1976.

Bacterio-phage N3 ofHaemophilus influenzae. H. Infection of transformablecellsbybacteriophage DNA. Acta Mi-crobiol. Pol. 25:175-186.

8. Jablonska,E., G.Rzemek, and A. Piekarowicz. 1976. Bacteriophage N3 of Haemophilus influenzae. I. Inde-pendence of vegetative recombination among Haemo-philus influenzaebacteriophageon the host cell.Mol.

Gen. Genet. 147:111-114.

9.Larive,A., C. Pourcel, and P. Tiollais. 1979.

Localiza-tion of Streptomycesstanfordii endonuclease I (SstI) cleavagesitesongenomesofhumanadenovirus types twoand five. Gene 5:77-83.

10. Mandel,M., and J. Marmur. 1968. Use ofultraviolet absorbance-temperature profile for determining the guanine plus cytosine content of DNA. Methods En-zymol. 12:195-206.

11. Piekarowicz, A., and M. Siwinska. 1977. Inhibition of transformation and transfection inHaemophilus influ-enzaeRd9by lysogeny. J.Bacteriol. 129:22-29. 12. Rigby, P. W. J., M.Dieckmann, C. Rhodes, and P.

Berg. 1977. Labeling deoxyribonucleic acid to high

specific activity in vitrobynicktranslation with DNA

polymerase1.J. Mol.Biol. 113:237-251.

13. Rosenvold,E.C., and A.Honigman.1977. Mapping of AvaIand XmaIcleavage sites in bacteriophage DNA including a new technique of DNA digestion inagarose gels.Gene 2:273-288.

14. Scher, B.M., D. H. Dean, and A. J. Garro. 1977.

FragmentationofBaciUusbacteriophage0105DNA by

restrictionendonuclease EcoRI: evidence for comple-mentarysingle-strandedDNA in the cohesive ends of themolecule. J. Virol.23:377-383.

15. Schildkraut,C.,and S.Lifson.1966.Dependence of the

melting temperature of DNA on salt concentration.

Biopolymers3:195-208.

16. Schildkraut, C.,J.Marmur,andP.Doty.1962. Deter-mination ofthe base composition of deoxyribonucleic acid from itsbuoyantdensityinCsCl. J.Mol. Biol.4: 430-443.

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

18. Southern, E. M. 1975. Detection ofspecific sequences amongDNAfragmentsseparatedby gel electrophore-sis. J. Mol.Biol.98:503-517.

19. Studier, F. W.1965. Sedimentation studies of the size andshapeof DNA. J.Mol. Biol.11:373-390. 20. Szybalski, W.,and E.H. Szybalski.1971.Equilibrium

density gradient centrifugation, p. 311-354. In G. L. Cantoni and D.R.Davies(ed.),Procedures in nucleic acidresearch, vol. 2. Harper and Row, New York. 21. Vogelstein, G., and D. Gillespie. 1979. Preparative and

analytical purification of DNA from agarose. Proc. Natl. Acad.Sci. U.S.A.76:615-619.

22.Vogt, V. M. 1973.Purificationandfurtherproperties of single-strand specific nuclease fromAspergillus oryzae. Eur.J.Biochem. 33:192-200.

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Figure

FIG. 2.stored10 to Centrifugation at pH 8.3. (A) N3 DNA in buffer K at 4°C was centrifuged through a 30% sucrose gradient containing 1 M NaCl in a
FIG. 3.patternsperAfter3,restrictionLanesdigestthedigestandusedhadtheelectrophoresis.both XhoI; Agarose gel ekctrophoresis of N3 DNA enzyme segments in a horizontal slab
FIG. 4.XbaI;SacI-KpnI;segmentsdigesthadenzymes Electrophoretic patterns of restriction enzyme digests ofEcoRI N3 DNA segments
FIG. 5.EcoRIstrip.segments.hybridizationontranslationthelane individual Radioautograph showing the pattern of ofN3 DNA XbaI segments to N3 DNA segments immobilized on a nitrocellulose XbaI segments were labeled with 32P by nick and hybridized to bound EcoR
+2

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