Vol. 31, No. 1JOURNAL OF VIROLOGY, July 1979, p. 112-1230022-538X/79/07-0112/12$02.00/0

Cleavage Map of Bacteriophage T4 Cytosine-Containing DNAby Sequence-Specific Endonucleases SalI and KpnI

KARIN CARLSON* AND BERIT NICOLAISENInstitute ofMedical Biology, University of Troms0, 9001 Troms0, Norway

Received for publication 3 January 1979

Cytosine-containing T4 DNA from endoHIendo1V-dCTPase-alc2 phage grownin a sup' rB- mB host is cleaved by endo R .EcoRI and endo R-HindIII to >40fragments and by endo R Sall and endo R KpnI to 8 and 6 fragments, respec-tively. The latter two fragment sets have been correlated to each other to producea cleavage map of the genome. The sum of the molecular weights of the fragmentscalculated from electrophoretic mobility in agarose gels yields a genome molecularweight for cytosine-containing T4 DNA of 105 x 106.

Sequence-specific endonucleases ("restrictionenzymes") have been used in many systems todissect genomes into smaller, defined unitswhich allow much higher resolution in the anal-ysis of transcriptive and replicative patterns. Wewished to utilize this technique with bacterio-phage T4, the genetics of which is quite wellknown (review in reference 30) but for whichphysical mapping has proved very difficult dueto the large size and circular permutation (23) ofthe phage DNA molecules.The use of sequence-specific endonucleases

with DNA from bacteriophage T4 is hamperedby the glucosyl-hydroxymethylation of its cyto-sine residues, which prevents restriction enzymecleavage (11). T4 DNA which contains non-glu-cosylated hydroxymethyl-cytosine may becleaved by endoR . EcoRI (11), whereas cyto-sine-containing T4 DNA (T4 Cyt-DNA) may becleaved by several sequence-specific endonucle-ases (15, 27, 28; this communication).Although glucosylation does not appear to be

essential for phage growth in commonly usedlaboratory host strains (16), hydroxymethyla-tion is essential (13). Simultaneous mutations inphage genes coding for endonuclease II, endo-nuclease IV, and dCTPase, together with a mu-tation in the alc gene (20), however, allow thematuration of T4 Cyt-DNA (20). As a first stepin constructing a cleavage map of the genome ofthis phage to be used in further analysis, wereport in this paper the cleavage pattern result-ing from digestion of T4 Cyt-DNA with endo R.Sall and endo R.KpnI.

MATERIALS AND METHODSStrains. The phage strain T4DamE51saA9nd-

28alc2 (dCTPase- denB- denA-alc-) (20) was ob-tained from E. Kutter. DNA from this phage is re-ferred to as T4 Cyt-DNA in this paper. Strain

T4ogt57f)gtl4m- (8) was obtained from H. Revel, andstrain AcI857S7 was from W. Szybalski. Escherichiacoli strains K803, which is sup-2 rK-mK- rgr, andB834, which is sup' rB- mBs (29), were from E. Kutter.Strain W3101, sup-2 thy- trpA-, was from N. Franklin.The W3101G(cI857S7) lysogen was constructed by us-ing standard procedures. E. coli BRY13 and Serratiamarescens Sb were from B. Weisblum, and Strepto-myces albus Garcia was from J. M. Ghuysen; Xantho-monas amaranthicola (ATCC 11645) was obtainedfrom the American Type Culture Collection.T4 phage was grown under conditions of lysis in-

hibition, whereas lambda phage was obtained byinducing the W3101(XcI857S7) lysogen. TheamE51saA9nd28alc2 phage was propagated in thesup-2 host K803 to minimize accumulation of sponta-neous mutants (in this host, phage DNA becomesmodified due to suppression of the amber dCTPasemutation). Phage with cytosine-containing DNA wereprepared by one cycle of growth in the sup' rB mBhost B834, which is phenotypically Sup-, lacking am-ber suppressor.DNA was prepared by phenol extraction of CsCl-

purified phage, followed by exhaustive dialysis of theDNA against 10mM NaCl-10 mM Tris-hydrochloride,pH 7.4.Media. Bacteria and unlabeled phage stocks were

grown in LB medium (2). Isotope-labeled phage wasgrown in synthetic medium (GMC) containing 100mM Tris-hydrochloride (pH 7.4), 8.5 mM CaCl2, 0.54mM MgSO4, 0.1 mM CaSO4, 0.6,uM FeCI3, 5 mg of Pper liter as KH2PO4, 32p; (specific activity, 0.1 to 0.5mCi/mg of P) or [methyl-3H]dThd (5 mg/liter; specificactivity, 0.5 mCi/mg), 1 g of glucose per liter, and 0.5g of vitamin-free Casamino Acids (Nutritional Bio-chemicals Corp.) per liter.

Isotopes and chemicals. 32p; was obtained fromNew England Nuclear Corp. (NEX 054), and [methyl-3H]thymidine was from Amersham Corp. (TRK 300).Agarose was from Marine Colloids Inc. (SeaKem aga-rose, no. 2173) or Bio-Rad (no. 162 0100). Polymin Pwas a gift from BASF (WHOZ Hauptlaboratorium B9,Hochschullieferungen, Ludwigshafen/Rhein, Ger-many).

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RESTRICTION CLEAVAGE MAP OF PHAGE T4 DNA 113

Enzymes. (i) EcoRI was purified by the procedureof Tanaka and Weisblum (22).

(ii) For the preparation of SalI, 5 liters of Strepto-myces albus G was grown to late exponential phase.The cells were pelleted, washed with EXB (1 mM Na2-EDTA, 7 mM /i-mercaptoethanol, 10 mM potassiumphosphate buffer, pH 7.0), resuspended in about 100ml of EXB, and disrupted by sonication. After centri-fuging the sonic extract for 60 min at 70,000 x g(Spinco type 30 rotor), the supematant was made 0.7%with respect to polymin P (stock solution, 10% [vol/vol] in water, adjusted to pH 7.5 and clarified by low-speed centrifugation), and the precipitate was col-lected. This pellet was eluted with 55 ml of0.3M NaClin EXB. After centrifugation to remove undissolvedmaterial, 120 ml of saturated ammonium sulfate(Schwarz/Mann, enzyme grade, no. 901046) was addedto 60 ml of supernatant. The resulting precipitate wascollected by centrifugation, dissolved in EXB, anddiluted to 0.07 M NaCl-equivalents (as determined byconductometry). This material was chromatographedon a DEAE-cellulose column (Whatman DE52) equil-ibrated with 0.05 M NaCl in EXB. The column waseluted with a linear gradient from 0.05 to 1 M NaCl inEXB. The enzyme eluted between 0.13 and 0.4 MNaCl (as determined by assaying fractions for cleavageof phage lambda DNA). Fractions containing enzy-matic activity were pooled and dialyzed against stor-age buffer (0.3 M NaCl, 0.1 M Tris-hydrochloride, pH7.4, 1 mM fi-mercaptoethanol, 20% [vol/vol] glycerol)and stored at -70°C. Samples were thawed as neededand were stored at -20°C after thawing. The enzymepreparation was free from contaminating nucleases, asevidenced by the unchanged cleavage pattern obtainedby using three times more enzyme than needed forcomplete digestion of phage lambda DNA or by incu-bating the samples for three times longer than neededfor complete digestion of phage lambda DNA (cf. alsoFig. 3).

(iii) XamI and SmaI were purified by essentially thesame procedure, using 0.1 and 0.4% polymin P, respec-tively. These enzymes eluted off the DEAE-cellulosecolumn at 0.18 to 0.20 and 0.12 to 0.22 M NaCl,respectively.

(iv) BamHI, HpaII, HindIII, and KpnI were gen-erous gifts from U. Pettersson, and EcoRII was fromG. Wadell.

(v) XhoI was purchased from New England Biolabs.Enzyme assays. EcoRI digestions were carried out

in 50 mM NaCl-7.5 mM MgCl2-100 mM Tris-hydro-chloride, pH 7.5. Digestions with Sall, BamHI, andKpnI were carried out in 6 mM Tris-hydrochloride,pH 7.5-6 mM MgCl2-6 mM /8-mercaptoethanol.HindIII digestions were carried out in 7 mM Tris-hydrochloride, pH 7.4- 7mM MgCl2-0 mM NaCl. Allenzymes were assayed at 370C except KpnI, which wasassayed at 300C. Incubations were continued for 30min to 4 h and terminated by heating the samples to80°C for 2 min. The enzyme preparations were titratedperiodically to ensure limit digestion.

Electrophoresis. Electrophoresis was carried outin 0.5 to 0.7% agarose gels in a horizontal slab appa-ratus similar to the one described by Shinnick et al.(19), using 160 mM Tris acetate (pH 8.1)-SO mMsodium acetate-8 mM Na2 EDTA-72 mM NaCl as

electrophoresis buffer. The gel slabs were 10 by 15 by0.2 cm and were run at 40 mA/gel for 40 to 48 h. Thegels were then stained with ethidium bromide (EtBr)for photographing, dried for autoradiography, ortreated with alkali, neutralized, and blotted onto nitro-cellulose membrane filters for hybridization.

Isolation ofDNA from gels. Gel regions contain-ing EtBr-fluorescent material were cut out, maceratedthrough an 18-gauge needle, and soaked overnight inabout 2 ml of the electrophoresis buffer. The agarosewas removed by filtering the mush through a mem-brane filter (Mfilipore Corp., type HA, 0.45-am poresize). To the effluent was added 500 jig of yeast tRNA(Sigma) and subsequently 4 volumes of cold 96%ethanol. Samples were kept at -20°C until used. Forreanalysis the precipitate was spun out and dissolvedin a small volume (50 to 100 id) of 10 mM NaCl-10mM Tris-hydrochloride, pH 7.4.

Hybridization. Denatured fragment DNA wastransferred from agarose slabs to nitrocellulose mem-brane filters (Millipore HAWP or Schleicher & SchuellBA85), using the procedure of Southern (21). Themembrane was dried and cut into strips, each contain-ing DNA from one slot in the gel. These were soakedin 0.01% sodium lauryl sulfate-0.02% each polyvinyl-pyrrolidone, bovine serum albumen, and Ficoll, in 6xSSC (lx SSC - 0.15 M NaCl plus 0.015 M sodiumcitrate)(5), for 5 to 6 h and then transferred to dialysistubing pretreated with sonically treated and heat-de-natured calf thymus DNA (0.1 mg/ml in 10mM NaCl,10 mM Tris-hydrochloride, pH 7.4, 1 mM Na2EDTA).To each bag the sample to be hybridized (104 to 10lcpm of labeled DNA, sonically treated and heat de-natured and then added to 2 ml of the polymer solu-tion) was added, and the bag was tied and incubatedat 670C for 36 h with periodic agitation. The stripswere removed, washed exhaustively in 0.5% sodiumlauryl sulfate-2x SSC, and autoradiographed.

Detection of radioactivity. 32p label in dried gelsand on membrane filters was detected by autoradiog-raphy, using Kodak Autoprocess X-ray film (no. 3003688). The resulting films were scanned in an RFTTransidyne densitometer to determine the exact lo-cation of the bands and to quantitate the amount oflabel in each band.

Scintillation counting of radioactive samples in liq-uid was carried out in a Packard scintillation spectro-photometer, using a toluene-based scintillation fluid.

RESULTS

Cleavage pattern of T4 Cyt-DNA. Cyt-DNA from phage T4 was treated with severalsequence-specific endonucleases (Fig. 1). Theresulting digests were subjected to agarose gelelectrophoresis. As a comparison, DNA fromphage lambda was digested with several of theenzymes and electrophoresed in adjacent slots.After conclusion of the run, the gels were stainedwith EtBr and photographed. Figure 1A showsphotographs of the gels, and Fig. 1B shows den-sitometer tracings of a film negative.Enzymes EcoRI, HindM (not shown), SalI,

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FIG. 1. Cleavage pattern of T4 DNA (A and B) and of lambda DNA (A) with several sequence-specificendonucleases (EtBr-stained gels). (A) Photographs of representative gels. The BamHIpattern represents anincomplete digestion. (B) Densitometer tracings offilm negatives obtained from photographing gels like thosein (A). Letters and numbers identify individual fragments. From top to bottom: Cleavage of T4 Cyt-DNA withSall. with Knn and with Sail and KonI together.

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RESTRICTION CLEAVAGE MAP OF PHAGE T4 DNA 115

and KpnI all cleave T4 Cyt-DNA (Fig. 1),whereas enzymes SmaI, EcoRH, BamHI, XhoI,XamI, and HpaIl did not cleave this DNA (datanot shown). Sail yields 8 fragments, KpnI yields6 fragments, and the double digestion with bothof these enzymes simultaneously yields 13 frag-ments. Of the latter, seven represent new frag-ments (SK-1, SK-2, SK-4, SK-5, SK-7, SK-8,and SK-9), whereas six are found in the singleenzyme digests (but see below concerning frag-ment SK-11). (The following abbreviated nota-tions are used: SalI [etc.] = endo R-SaiI [etc.],S-G [etc.] = fragment SailI T4 G [etc.], K-F[etc.] = fragment KpnI T4 F [etc.], SK-11 [etc.]= fragment 11 [etc.] from a mixed digest of T4Cyt-DNA with SailI and KpnI.) The gels in Fig.1 were illuminated from the sides and the bot-tom to enhance the fluorescence from the small

fragments, a procedure which renders the peakheights in Fig. 1B somewhat uninformative. Itmay be noted, however, that fragment SK-11appears to be present in relatively largeramounts than fragment S-G.The rate of migration ofDNA in agarose gels

has been found to be inversely related to itsmolecular weight (24). Knowing the sizes of theBamHI (9), EcoRI (24), and Hindlll (17) frag-ments from phage lambda DNA, the approxi-mate sizes of the T4 fragments may be obtainedfrom a plot of molecular weight versus distancemigrated. The positions of the bands were de-termined from densitometer tracings of the filmnegative (EtBr-stained gels) or from tracings ofautoradiograms (3P-labeled DNA). FragmentsS-B and S-C, K-B and K-C, and SK-7 and SK-8 rarely form discernible peaks (see Fig. 1). Av-erage size and standard deviation were thereforecalculated from the composite bands S-B/C, K-

B/C, and SK-7/8 observed in most experiments.Fragment sizes shown in Table 1 were calculatedfrom several determinations involving, in thecase of SailI fragments, also an incompleteBamHI cleavage of lambda DNA, which pro-vided convenient markers in the high-molecular-weight range. These estimates have not beencorrected for the possibility that the rates ofmigration of lambda DNA and T4 DNA differdue to differences in the average base composi-tion (14).

Analysis of the SailI cleavage product by neu-

tral sucrose gradient centrifugation showed thatthe bulk of the material sedimented in a broadband with a sedimentation rate expected formolecules of approximately 20 x 106 daltons(Fig. 2). This analysis complements the electro-phoresis, in that sucrose gradients give a betterresolution in the high-molecular-weight range.The sucrose gradient also shows the effect ofmodification on the rate of migration of full-length genomes: The Cyt-DNA migrates some-what more slowly than the glucosyl-hydroxy-methylated DNA.Location of cleavage sites. Three ap-

proaches were used to locate the cleavage siteswith respect to each other: partial digestion,recleavage of isolated fragments, and hybridiza-tion of isolated fragments.

(i) Partial digestion. Complete digestion ofT4 Cyt-DNA is rarely observed. Most digestscontain some undigested or partially digestedDNA (cf. Fig. 1 and 3). SailI digests containvariable amounts of an extra band between S-Eand S-F [estimated molecular weight, (8.14 +0.14) x 106], whereas KpnI digests contain an

extra band between K-D and K-E [estimatedmolecular weight, (8.87 ± 0.35) x 106] (Fig. 1

TABLE 1. Approximate size of T4 Cyt-DNA SalI, KpnI, and (SailI + KpnI) fragmentsSall KpnI Sall + KpnI

Fragnent Mol wta (x 10-6+ SD) Fragment Mol wt (x 10-6 SD) Fragment Mol wt (x 10-i ± SD)A 23.18 ± 1.62 (8) A >23B/C 21.3 ± 1.3 (9) B/C -22 1 16.3 ± 1.5 (10)D 15.03 ± 0.65 (9) D 15.66 ± 1.08 (5) 2 14.5 ± 1.2 (10)E 9.71 ± 0.46 (9) 3 9.57 ± 0.56 (10)

4 8.61 ± 0.49 (10)5 8.33 ± 0.57 (10)

E 7.70 ± 0.23 (5) 6 7.90 ± 0.39 (10)7/8 6.64 ± 0.25 (10)9 6.3 ± 0.25 (10)

F 5.93 ± 0.18 (9) 10 5.70±0.17 (10)G 4.60 ± 0.17 (9) 11 4.48 ± 0.16 (10)H 2.53 ± 0.39 (7) 12 2.5± 0.4 (6)

F 1.67 ± 0.42 (3) 13 1.4± 0.2 (4)

Z 103.6 : 103.4a Molecular weights are calculated from electrophoretic mobility. SD, Standard deviation. Number of

determinations is given in parentheses.

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116 CARLSON AND NICOLAISEN

NSG

15

L-

0

010.

4.

c

u

L-

0

T4 wt

ABC D E F

T4Cy

.2 .4 .6

Fractional length of

FIG. 2. Sedimentation ofthe SalI ci

of T4 Cyt-DNA in a neutral sucrose

20% sucrose in 1 M NaCI, Spinco SWat 30,000 rpm). 32P-labeled T4 Cyt-DAdigest were analyzed separately, ingether with 3H-labeled DNA from T4(containing glucosylated hydroxynand the two patterns were superimpcthe upper part of the figure identifyBotton is to the left.

and 3). The product of the doubleboth enzymes contains two bandlower molar yields (labeled P1 a

1B) of approximate molecular we0.73) x 10W and (9.00 ± 0.51) x 10The amount of DNA in these foieach in the SailI and KpnI singleand P2 in the double digest) v

between different batches ofDNA3) but is not much affected by se

ations in the amount of enzyme aThese bands represent incomplDNA. Upon re-extraction, partDNA may be cleaved somewhat ficompletely (Fig. 5F).The estimated molecular weigh

tially digested fragments in thedigests correspond approximatelybe expected from doublets of S-F

E + K-F, respectively. The redigestion shown inFig. 5F confirns this with respect of S-F + S-H.We assume, therefore, that fragment S-F is lo-cated next to S-H and K-E is next to K-F in theuncleaved DNA.Other partially cleaved fragments, and un-

cleaved DNA, are found in variable amountsabove S-A, K-A, and SK-1, respectively (Fig. 1

F G H and 3). Redigestion of material from this regionof the gels generally produces all the specificcleavage products, and it has not been possibleto resolve the individual components (data not

t SalI shown).(ii) Recleavage of isolated fragments.

DNA was eluted from gel bands as described inMaterials and Methods. The eluted DNA wasreanalyzed without further treatment, aftertreatment with SalI, or after treatment withKpnI.

Figure 4 shows reanalysis of fragments froman original KpnI digest. A total digest ofT4 Cyt-DNA with Sall + KpnI was run in slots adjacent

6 / to the experimental ones to allow identificationof the bands. It can be seen that K-A, upon

wGI

digestion with Sall , gives rise to fragments| | SK-1 and SK-2 (panel A), and K-D gives rise to

.8 1.0 fragments SK-9 and SK-11 (panel D). Fragment

gradient K-E is not digested by SailI (panel E) and cor-~eavgepadi uct

responds to fragment SK-6. Fragment K-F waseavagenproduct not reanalyzed; it comigrates with SK-13.gradient (5 to Fragments K-B and K-C are difficult to sep-VA and its SailI arate from each other. Figure 4B and C showsboth cases to- redigestion of two preparations of K-B/C: panelwild-typephage B, a preparation from the upper part of thenethylcytosine), doublet band, presumably predominantly K-B;)sed. Letters in and panel C, a preparation from the lower partSaiI fragments. of the doublet band, presumably predominantly

K-C. Both panels show the presence of frag-ments SK-5, SK-8, and SK-10. Panel B shows

digestion with relatively more of fragments SK-3, SK-4, andIs obtained in SK-7 than does panel C, and in addition containsnd P2 in Fig. more SK-1 and SK-2, probably from contami-,ights (11.10 ± nating K-A. There is also considerably more6, respectively. undigested material in this analysis. From theseur bands (one results we attribute fragments SK-3, SK-4, anddigests and P1 SK-7 to K-B and fragments SK-5, SK-8, andaries strongly SK-10 to K-C. Since results described elsewherei (cf. Fig. 1 and indicate that fragment SK-12 (S-H) is located!veralfold vari- adjacent to fragment SK-10 (S-F) and is notidded (Fig. 3). cleaved by KpnI, we allocate SK-12 to K-C also.Letely cleaved Data shown in Fig. 5 are consistent with these;ially digested assignments. Perhaps the SailI sites in K-B areorther, but not less susceptible to cleavage than the sites in K-

C..ts for the par- In the SailI digest of K-D, fragment SK-1lsingle enzyme appears in much higher yields than expectedto what would from its size. This was noticed also in the total+ S-H and K- Sall + KpnI digest (Fig. 1B). It will be shown

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RESTRICTION CLEAVAGE MAP OF PHAGE T4 DNA 117

A B

FIG. 3. Enzyme titrations. 32P-labeled T4 Cyt-DNA was incubated with increasing amounts of either Sail(A) or Kpnl (B). From keft to right, DNA was digested with: (A) 0, 2,4, 6, 8,12,16, and 20 U of SaiI, or (B) 0,2,3,4, 5, 6, 7, and 8 U ofKpn. One unit of each enzyme is defined as that amount which digests compltelythe same quantity of lambda DNA under the same conditions. Increasing the amount of enzyme beyond thatshown here led to random degradation, probably due to nucleases contaminating the enzyme preparations.Results essentially similar to those shown here were obtained by using a constant amount of enzyme andvarying the time of incubation from 20 min to 4 h (data not shown).

below (Fig. 6) that "fragment SK-il" is in facta doublet of S-G and a new fragment of the samesize created by the double digestion. We denotethese fragments SK-lla and SK-llb. Both frag-ments were obtained by cleaving K-D with SalI.The results from recleavage of KpnI fragmentsby SailI are summarized in Table 2. This tablealso includes corrected molecular weights for thefragments, based on summation ofthe molecularweights of the respective double-digest frag-ments (from Table 1).A similar reanalysis of SailI fragments re-

cleaved by KpnI is shown in Fig. 5. All threelarge fragments are difficult to obtain pure sincethey migrate close to each other. They are alsolikely to be contaminated by various partiallydigested products, since SailI cleavage sites aregenerally less susceptible to cleavage than KpnIsites. A comparison of the relative amounts of

the different double-digest fragments resultingfrom digesting preparations of predominantly S-A (panel A), S-B (panel B), and S-C (panel C)with KpnI allows the assignment of fragmentsSK-2 and SK-4 to S-A, fragments SK-6, SK-8,and SK-9 (and SK-13) to S-B, and SK-1 andSK-lla to S-C. Fragment S-D gives rise to SK-5 and SK-7 upon KpnI digestion (panel D). Thereanalysis in panel F starts with the Sall frag-ment migrating between S-E and S-F, previouslyassumed to be a partially digested fragmentcomposed of S-F and S-H. The redigestion bySailI (dashed line in Fig. 5F) confirms this no-tion. Neither of these fragments is furthercleaved by KpnI (solid line in Fig. 5F). Fragmentassignments and recalculated molecular weightsare summarized in Table 2.Of the partially digested fragments from the

double digest with KpnI and Sall, SK-Ph most

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118 CARLSON AND NICOLAISEN

FIG. 4. Reanalysis of isolated KpnI fragments. 3P-labeled T4 Cyt-DNA was digested with KpnI, and thedigestion products were separated on agarose gels. Gel sections containing EtBr-fluorescent material werecut out, and DNA was eluted from the agarose. Eluted DNA was re-electrophoresed untreated and aftertreatment with SalI, the two samples being run in adjacent slots. After the run was concluded, the gels weredried and autoradiographed. The resulting filmswere scanned by densitometer. Tracings corresponding tountreated (dotted lines) and Sail-treated (solid lines) samples were superimposed in constructing the graphs.To avoid cluttering, the dotted lines have not been extended beyond regions where peaks are observed.Numbers and lines above each set oftracings indicatepositions offragments in a complete double digest withSalI and KpnI (run in another adjacent slot). Electrophoresis is from left to right. (A) Fragment K-A; (B)fragment K-B (+ K-C); (C) fragment K-C (+ K-B); (D) fragment K-D; (E) fragment K-E.

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RESTRICTION CLEAVAGE MAP OF PHAGE T4 DNA

3 4 5 6 78 9 10II

1 2 3 4 5 6 78 9 10I I

D 1 A2 3 4 5 6 789 10 11

II I X

E 1 2 4 5 6 789 10I i I I

11

1 1

12 13

119

FIG. 5. Reanalysis ofisolated SalIfragments. Fragments from SalI-cleaved T4 Cyt-DNA were obtained asdescribed in the legend to Fig. 4 and reanalyzed similarly. Dotted lines indicate samples retreated with SalI;solid lines indicate samples treated with KpnI. The graphs were constructed as described in the legend to Fig.4. (A) Fragment S-A (+ S-B/C); (B) fragment S-B (+ S-A and S-C); (C) fragment S-C (+ S-A and S-B); (D)fragment S-D; (E) fragment S-E; (F) partially digested fragment migrating between S-E and S-F; (G) fragmentS-G.

likely contains SK-9 and SK-llb (Fig. 4D),whereas SK-P2 probably contains SK-8 and SK-12 (Fig. 4C). It was not possible to obtain these

partially digested fragments pure enough forunambiguous assignment.

(iii) Hybridization. To resolve whether frag-

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120 CARLSON AND NICOLAISEN

TABLE 2. Recleavage of T4 Cyt-DNA fragmentsMolwt (xOriginal Corresponding double digest 10-6) (from

fragment fragment (Sail + KpnI) Table 1)

K-A SK-1, SK-2 30.8K-B SK-3, SK-4, SK-7 24.9K-C SK-5, SK-8, SK-10, SK-12 23.2K-D SK-9, SK-lla, SK-llb 15.3K-E SK-6 7.9K-F SK-13 1.4

S-A SK-2, SK-4 23.1S-B SK-6, SK-8, SK-9, SK-13 22.2S-C SK-1, SK-lla 20.8S-D SK-5, SK-7 15.0S-E SK-3 9.6S-F SK-10 5.7S-G SK-llb 4.5S-H SK-12 2.5

ment "SK-1l" in the double digest with SalIand KpnI indeed is a doublet, it was hybridizedto SailI and KpnI digestion products which hadbeen transferred to nitrocellulose membrane fil-ters by the Southern technique. In comparison,fragment S-G from a SailI single digest was alsoanalyzed. Figure 6 shows the results. As antici-pated from the recleavage data shown in Fig. 4and 5, fragments S-G and SK-11 both hybridizeto S-G and K-D. Fragment SK-11 also hybrid-izes to one of the large Sall fragments, confirm-ing the interpretation set forth above.Construction of the cleavage map. From

the recleavage and hybridization results sum-marized in Table 2 a composite map showingthe cleavage sites for the two enzymes relativeto each other may be constructed. This map isshown in Fig. 7. Letters and numbers within the

FIG. 6. Hybridization analysis of fragments S-G and SK-Il. 32P-labeled fragments were obtained asdescribed in the legend to Fig. 4. Denatured fragment DNA from digests of cold T4 Cyt-DNA was transferredfrom gels to nitrocellulose membrane filters. These were cut in strips the width of one slot in the original gel.Eluted 32P-labeled DNA was hybridized to these strips. The membrane filters were then autoradiographed,and the resulting films were scanned by densitometry. Randomly labeled T4 Cyt-DNA was hybridized toadjacent strips from the same sheet ofmembrane filters to determine the positions of the DNA bands. It wasnotpossible to distinguish the three large fragments (S-A, B, and C, and K-A, B, and C, respectively) in thesehybridizations. (A) Hybridization of fragment SK-1I to KpnI fragments (upper tracing) or SalI fragments(lower tracing). (B) Hybridization of S-G to KpnI fragments (upper tracing) or to SalI fragments (lowertracing).

A

ABC D E F

A BC D E F G-

BABC D E F

-y~~~~~~~~~~~~~~~~~~~I

I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

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RESTRICTION CLEAVAGE MAP OF PHAGE T4 DNA 121

three circles refer to fragments as identified inFig. 1. Cleavage sites for KpnI are shown insidethe circle, whereas cleavage sites for SailI areshown on the outside. The cleavage sites havenot been numbered, awaiting a correlation be-tween this physical map and the genetic map,which will allow clockwise numbering from thegenetic zero point between rIHA and rIIB (30).For each enzyme one cleavage site cannot belocated precisely, since the order of the internalfragments K-E and K-F within S-B, and S-F andS-H within K-C, has not been determined. How-ever, the assignments of SK-8 and SK-12 to SK-P2 (see above) suggests that the fragment orderin K-C is SK-5, SK-10, SK-12, and SK-8, asindicated in Fig. 7.

DISCUSSIONPreparation ofenzymes. This investigation

was initiated by a search for restriction-like en-donucleases cleaving the genome of phage T4into relatively few fragments, amenable to map-ping studies. It was shown previously (11) thatcompletely modified T4 DNA is not susceptibleto sequence-specific endonucleases, whereas theglucoseless hydroxymethylated DNA is cleavedonly by EcoRI. We therefore used the non-hy-droxymethylated (and non-glucosylated) DNAfrom T4 alc2saA9nd28amE51 (20) as the en-zyme substrate.To carry out the initial screening and subse-

Sail

FIG. 7. Cleavage map of T4 Cyt-DNA showing thelocations of cleavage sites for SailI and KpnI. Forfurther explanation, see text.

quent mapping, it was essential to obtain severalrestriction enzymes in high purity and largequantities by a rapid, simple procedure. Theprocedure described in Materials and Methodsfor purifying SailI was patterned on methodsdescribed for purification of RNA polymerase(3) and EcoRI (22). It has been utilized forseveral enzymes with good results; the entireprocedure takes less than 1 week, starting withgrowth of the bacteria. The key step is thepolymin P precipitation. This step was originallysuggested by Zillig and co-workers in a purifi-cation scheme for DNA-dependent RNA polym-erase from E. coli (31) and has been applied alsoby others in several purification procedures forDNA-metabolizing enzymes. For optimal resultsthe amount of polymin P to be used for precip-itation of a given enzyme has to be titrated (3).Screening of enzymes. The genome length

of T4 DNA has been estimated to be 166 ± 2kilobase pairs (12). With 65.6% adenine-thymine(A.T) (14) and a random nucleotide sequence,any specific hexanucleotide containing four gua-nosine-cytosine (G.C) pairs and two A-T pairsshould be expected to occur 16 times, whereas aspecific hexanucleotide containing four A. Tpairs and two G-C pairs should be expected 57times. The expected numbers for sequences withsix A *Ts or six G * Cs are 207 and 5, respectively.The initial survey, therefore, concentratedmainly on enzymes recognizing hexanucleotidesequences containing four or six G * C pairs.KpnI and SalI, both of which recognize se-

quences with four G- C pairs and two A *T pairs(1, 26), cleave T4 Cyt-DNA into six and eightfragments, respectively. It is possible that thedigestion pattern we observe with these twoenzymes is not complete (as discussed above,more cleavage sites might have been expected).The pattem is, however, unique and extremelyreproducible, allowing the arrangement of frag-ments into a physical map.One enzyme with a recognition sequence of

six GC pairs (five expected cleavage sites) wastested: SmaI (7). This enzyme did not cleave T4Cyt-DNA. The sites either are missing com-pletely or are protected by residual modification.Two enzymes with recognition sequences con-

taining four A-T pairs and two G.C pairs(EcoRI, 10, and HindIII, 18) both yielded >40fragments (57 sites expected). These have notbeen analyzed further, but will eventually allowthe construction of a more detailed physicalmap. EcoRI cleaves also hydroxymethylCyt-containing, non-glucosylated DNA (11, 15, 25;Fig. 1). It can be seen that smaller fragments areobtained from the non-hydroxymethylatedDNA (an alternative explanation that hydroxy-

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122 CARLSON AND NICOLAISEN

methylation drastically interferes with electro-phoretic migration is considered less likely).

Several other enzymes were tested and foundnot to cleave T4 Cyt-DNA. These includedEcoRII, BamHI, XhoI, XamI, and HpaII.Fragmentation pattern. Since T4 phage

DNA molecules are randomly circularly per-muted (23), digestion of each molecule will resultin two end fragments of variable size, dependingon where the physical ends of each molecule arelocated with respect to the cleavage sites. UsingSalI or KpnI, these end fragments will accountfor a measurable fraction of the DNA in therespective digests, but will not form discretebands upon agarose gel electrophoresis and,therefore, should not be noticeable. The ob-served bands should derive only from the "in-ternal" fragments in each molecule. Their sumshould therefore equal not the physical lengthof the mature DNA molecule (which contains a2% terminal redundancy), but the genomelength. With a genome length of 166 ± 2 kilobasepairs (12), and 65.6% A.T (14), this should equal102.5 megadaltons when the DNA contains cy-tosine instead of glucosyl-hydroxymethylcyto-sine. When approximate fragment molecularweights are calculated from electrophoretic mo-bility, their sum equals 103.5 x 10' (averagebetween determinations from SailI cleavage andfrom SailI + KpnI cleavage in Table 1). To thisshould be added the extent of the saA9 deletionin the DNA substrate, approximately 2,500 basepairs (6), or 1.5 x 106 daltons, resulting in a totalmass of 105 x 106 daltons for the genome of T4Cyt-DNA. We consider this to be in good agree-ment with the expected value, considering theerrors in the estimation of genome length byelectron microscopy (12) as well as in our deter-minations.The amount of enzyme needed to produce the

cleavage pattems illustrated in Fig. 1 through 3was higher than that needed to cleave com-pletely the same quantity of DNA from bacte-riophage lambda (data not shown), and, as evi-denced from Fig. 3, it was not possible to obtaincomplete cleavage of a given batch of DNAsimply by increasing the amount of enzymeadded. The SailI sites were noticeably less sus-ceptible to enzymatic cleavage than the KpnIsites. We interpret these results as indicative ofresidual modification of the DNA, either bysmall quantities of glucosyl-hydroxymethylcy-tosine or by methylcytosine and/or methylade-nine. Such modification might also account forthe fact that most enzymes tested did not cleavethe Cyt-DNA at all. If this interpretation iscorrect, the extent of residual modification var-ies from experiment to experiment, accountingfor intrinsic differences in susceptibility to cleav-

J. VIROL.

age of different batches of DNA. Since the par-tially digested DNA was not cleaved upon fur-ther direct addition of more enzyme (Fig. 3), butonly upon extraction from the reaction mixtureand renewed digestion with fresh enzyme (Fig.5F), we assume that the uncleaved sites areblocked by enzyme molecules which bind but donot cleave.Unusual resistance to endonucleolytic cleav-

age has been noted also in the case of EcoRIdigestion of Glc- hydroxymethylated Cyt-DNA(25). T4 DNA from a pseudorevertant ofANB5060(rll)amC87(42)amE51(56) was foundto be cleaved by XhoI to 15 fragments and byEcoRI and HindIII to about 60 fragments, butnot by BamHI (28). DNA from T4 nd28(denA)saA9(denB)amE51(56)amN55(42) was cleavedby EcoRI, HpaI, and HindIH to a larger numberof fragments, and by SaII to about six to sevenfragments (15). The different cleavage patternsmight be explained by different levels of modi-fication of the Cyt-DNA from these differentphage mutants.Physical map. The recleavage and hybridi-

zation results with isolated fragments have al-lowed the construction of a composite cleavagemap (Fig. 7). Two pairs of adjacent fragmentshave not been definitely ordered: S-F and S-H,and K-E and K-F. Since fragment SK-P2 mostlikely is composed of fragments SK-8 and SK-12(Fig. 40), the order ofthe former pair is probablyas indicated in Fig. 7, namely, S-D, S-F, S-H,and S-B.The present work does not allow a correlation

of the physical fragment map and the geneticmap. We are currently pursuing several ap-proaches to this problem. Preliminary experi-ments indicate that the deletion Atk2 (4), whichdeletes approximately 8,000 base pairs includingthe rI gene, removes the Sall cleavage site be-tween S-D and S-E. The map orientation shownin Fig. 7 is, of course, entirely arbitrary.

ACKNOWLEDGMENTSWe thank U. Pettersson, G. Wadell, E. Kutter, J. M.

Ghuysen, B. Weisblun, N. Franklin, and W. Szybalski for giftsof enzymes and strains, and E. Ljungqvist for drawing ourattention to the KpnI cleavage of T4 Cyt-DNA.

This investigation was partially supported by the Norwe-gian Research Council for General Science, grant C 17.14-16(awarded to B. H. Lindqvist).

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