Methylation Pattern of Lambda Deoxyribonucleic Acid

Copyright _©1972 AmericanSociety for Microbiology Printedin U.S.A.

Methylation

Pattern of Lambda

Deoxyribonucleic Acid

CECILIA HIDALGO' AND HOWARD A. NASH

LaboratoryofNeurochemistry, National Institute of Mental Health, Bethesda, Maryland 20014

Receivedforpublication 14July 1972

Deoxyribonucleic acid

(DNA)

extracted from phage X grown on Escherichia coli

K-12strain W4032 had 113 i 105-methylcytosine residues and215 i 206-methyl adenine residues per genome, asdetermined

by

threeindependent methods.These

methylated nucleotidesweredistributed equallyamong thetwo strandsofX DNA.

Shearing ofdouble-stranded DNA tohalf-length fragments revealed a

slight

defi-ciencyof 5-methyl cytosine in the55%guanineplus cytosine half. ShearingtheDNA

tofragments ofsmaller

length

showed that thedistribution ofmethylated nucleotides along the double helixwasuniform with the exception ofanundermethylated frag-ment arising from the center ofthe X DNA molecule. The implication of these

results for the function ofmethylated nucleotides in the X DNAmolecule is dis-cussed.

Methylated

bases havebeenfoundtobe present

in

deoxyribonucleic

acid

(DNA)

from

bacteria,

phages,and animal andplantcells. A small

frac-tion ofthe

methyl

groupspresentin bacterial and

phage DNA have been

implicated

in

host-con-trolled restriction and modification

(1).

No

bio-logical

role hasyet been found for therestof the

methylated

bases present in the DNA molecule. In anattempttoinfer therole of these other

meth-ylated bases, we determined the distribution of

methylated

nucleotides in the X DNAmolecule.

DNA from

phage

Xwaschosen because its

genet-ics have been

extensively

studied,

the

physical

localization

of most of its genes on the DNA

molecule is

known,

and

techniques

exist for the

physical

resolution of different

genetic regions

as well as the

separation

of

single

strands of the

phagegenome

(6, 10).

We found that X DNA extracted from

phage

grown in a K-12derivative of Escherichiacoli

con-tainsboth

5-methylcytosine

(5MG)

and

6-methyl-aminopurine

(6MAP),

in agreementwith

previous

studies(5,

9).

In

addition,

wehavedemonstrated that both strands have thesame

methyl

content,

and, along

the double

helix,

only

asmall

fragment

corresponding

to a

region

in the center of the linear genetic map shows a different degree of

methylationfrom therest.

MATERIALS AND METHODS

Bacterial and phage strains. E. coli K-12 strain W4032 (HfrC, lac A [complete], pro-, metA, T6R,

1Presentaddress: Facultad de Quimica y Farmacia, Uni-versidaddeChile, Vicuiia Mackenna20,Santiago,Chile.

described by Cook andLederberg [3]),wasobtained from R. Perlman. E. coli K-12 C600 was obtained from M. Gottesman. Thelambdaphage strains used were XcI857andXb2c1857.

Media and reagents. ModifiedTPA medium con-tained, per liter: NaCl, 0.5 g; KCI, 8.0 g; NH4Cl, 1.1 g; tris(hydroxymethyl)aminomethane (Tris), 12.1 g;KH2PO4,0.46 g;sodiumpyruvate,0.8 g; 19 L-amino acids, 150 mg each; L-methionine, 10 mg; 0.16 M Na2SO4,1.0ml;andadenine, 10 mg. Thefinal pH was 7.4, adjusted with HCl. Tris-Mg buffer was 0.015 M Tris-hydrochlorideand 0.01MMgSO4,pH 7.4.

Electrophoretically pure pancreatic deoxyribonu-cleasewasobtainedfromSigmaChemical Co. Snake venom phosphodiesterasewasobtained from Worth-ington Biochemical Corp.['H-methyl]-methionineand

[14C-methyl]-methionine were obtained from New EnglandNuclear Corp., Amersham-Searle, or Schwarz BioResearch, Inc. Specific activities of ['H-methyl]-methionine ranged from 2.6 to 7.6 Ci/mmole. [14C-methyl]-methionine specificactivities ranged from 12.8 to60mCi/mmole.Carrier-free32Pwasobtained from NewEnglandNuclear Corp.

DNApreparation. To obtain XDNA labeled either with [14C-methyl]-methionine or with "2P and [3H-methyl]-methionine,Xphagewasgrown onW4032 in modified TPA medium. Cells wereallowedtogrowin modified TPA mediumat37 C to a concentration of 3 X 108cells/ml, centrifuged,andresuspendedin 0.1 volume ofTris-Mgbuffer. The phage was added at a multiplicityof infection of1 to 3,andadsorption was allowedtoproceedfor 15minat 37 C. At thispoint, theoriginal volumewasrestoredby adding fresh modi-fied TPA medium with the radioactive isotopes, and the culturewas further incubated for 2.5 hr at 37 C withshaking. Chloroform was added to 1% volume, and,after shaking for 10moremin,the bacterialdebris wasremovedbycentrifugation and the clear lysate was

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HIDALGO AND NASH

treated with 10 g of polyethyleneglycol/100ml and 10 ml of 5 M NaCl/100 ml to precipitate the phage. After centrifugation for 15 min at 19,000 X g, the phage pellet was resuspended in 10 ml of Tris-Mg buffer, 7.76 gofCsCl was added, and the resulting solution was spunfor12 to 15 hr at 34,000rev/minin a Beck-manno. 40 rotor. The resulting band of phage was banded again in CsCland then dialyzed in Tris-Mg buffer. TheDNAwas extracted three times with redis-tilledphenol, and it was extensively dialyzed against 0.1 MNa2SO4orTris-Mg buffer.

Shearing ofXDNA andmercurycomplexes. DNA solutionswereusuallysheared at aconcentration of 25 ,ug/ml in0.1 MunbufferedNa2SO4.Atotal volumeof 22ml wasalwaysused.

The shearingtechnique described by Skalka,Burgi, and Hershey (10)was followed. To shear the DNA to one-half, one-tenth,andone-fifteenth lengthfragments, aGT-21 Hellermotor withcontroller unit wasused. One-twentieth length fragments were obtained by shearing the DNA for 30 min at 20,000 rev/min in a Virtis45 homogenizer.

The XDNA halveswereobtained byshearing for60 min at 1,500 rev/min. One-tenth and one-fifteenth lengthfragments were produced by shearing at 4,000 rev/minfor4hr or at8,000rev/minfor 1 hr, respec-tively. All DNA solutions were sheared in a Virtis stainless-steelcup at 4 C. Fragmentlengthswere char-acterized bysedimentation in 1 M NaClneutral 5 to 20%sucrosegradients withintact XDNAas marker. TheBurgi-Hershey formula (2) wasused for estima-tion ofmolecularweights.

After shearing oftheDNA, it was banded in Hg-Cs2SO4 density gradients according to Skalka et al. (10). Every timeanewpreparationof DNAwas ob-tained, atrial centrifugationwasmadetodeterminean appropriateRF.AllRFvaluesusedranged from0.20 to 0.30.Thesolutionscontained,perml,5to50,ug of DNA,0.05ml of0.1 Msodiumborate, pH 9.0,43to 45% byweightofCs2SO4 (Gallard-Schlesinger), and the amountof mercuryspecified above. The mercury was always added last. Volumes of 3.2 to5.3 ml of solution were used. The tubes were overlaid with mineraloiland spunfor36 hrat36,000rev/mmnina fixed-anglerotor,at4C. Atthe end of the run, frac-tions werecollecteddropwisefrom the bottomofthe tube.

Separation of strands. The technique described by

HradecnaandSzybalski(6) toseparate the strandsof X DNA was followed, with the use of

polyuridylic-polyguanylic acid[poly (UG)Iobtained from Schwarz BioResearch, Inc. (lot 6801). The purified labeled phage wasdialyzedovernightin 10-3M sodium

ethyl-enediaminetetraaceticacid,pH8.0,at4 C.Thephage

solution was then mixed withpoly (UG)andsarkosyl,

heat-denatured for3 minat95C,chilledonice, and banded in CsClatadensityof 1.72g/cm3inanSW39 rotor at30,000rev/minfor 60 hr. Fractionswere col-lecteddropwisefrom the bottom of the tube.

Hydrolysis of DNAandthin-layerchromatography: bases.ThepurifiedDNA(ca.50ug)washydrolyzedto

free bases with70%perchloricacidat100 Cfor 1 hr. The hydrolysate was neutralized with KOH and

centrifuged to remove the precipitated potassium

perchlorate and charred sugars. The resulting bases were separated by two-dimensional chromatography on commercial cellulose plates (Eastman Organic Chemicals) without fluorescent indicator. Unlabeled markers of 5MC and 6MAP were added. The first sol-vent system used was n-butanol-absolute methanol-concentrated ammonia-water (60:20:20:1); the sec-ond solvent was absolute methanol-concentrated hy-drochloric acid-water (65:17:18). The spots were located under ultraviolet light, scraped into liquid scintillationvials, and counteddirectly in a Beckman liquid scintillation counter.

Mononucleotides. DNAsolutionsin amounts of0.1 to 1ml containing a total of 50 ,ug of DNA in Tris-Mg buffer,pH 7.4, were hydrolyzed to mononucleotides by consecutive treatment with pancreatic deoxyribonu-clease and snake venom phosphodiesterase (8). A reactionmixturecontaining20M&gofpancreatic deoxy-ribonucleaseand DNA wasincubated at 37 C for 30 min. The enzyme was then inactivated by heating to 95 Cfor5min. The digest was mixed with 0.1volume of 1 M glycine buffer, pH 9.2, and 400 ,ug of snake venom phosphodiesterase, and was hydrolyzed to mononucleotides by incubating at 37 C for 30 rnin. Theresultingmixture was adsorbed to activated char-coal, washed three times with distilled water, and eluted bywashingthreetimeswith50%ethanol-0.5% concentrated ammonia. Theelutedsolution was con-centratedin vacuo to a final volumeof0.2ml,and the mononucleotides were separated by two-dimensional chromatography on commercial PEI-cellulose plates (Eastman).

The firstsolventused was 1 MLiCl,and the second was 0.5 Nacetic acid. In betweensolvents, theplates wereimmersedinabsolute methanolandshaken for 15 min to remove theLiCl.The spots were demonstrated bymeansofautoradiography withKodakNo-screen X-rayfilm.Nucleotidecompositionsweredetermined by scraping the radioactive spots and counting in a Beckman liquid scintillationcounter.

RESULTS

Methyl content of whole X DNA.The methyla-tion ofDNAobtained fromXc1857

phage

grown on W4032 was studied

by

several different

pro-cedures. Purified

[14C-methyl]-labeled

DNA was

hydrolyzed

eithertobasesor to

mononucleotides,

and theextentof

methylation

wasdetermined

by

assumingthe

specific activity

ofthe

methyl

groups intheDNA to beequaltothe

specific activity

of methionineinthe

growth

medium. Table1 shows

the results of severaldeterminations.

Autoradio-grams ofthin-layer chromatograms ofthese hy-drolysates demonstrated that 6MAP and 5MC

aretheonly

methyl-labeled compounds. Labeling

the DNA with

32P-orthophosphate

followed

by

hydrolysistomononucleotidesalsogave thesame total content of 5MC and 6MAP. The

determina-tion of the

methyl

content

by

direct

analysis

of the

32P-labeled mononucleotides did not

require

any

assumption

regarding

the

specific activity

ofthe

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TABLE 1. Methylation ofX cl857DNA grownon

W4032

5MC/X, 6MAP/X Ratio, D.NAprepn genomeA genomea 65MAC/

14C-methylbases... 114±t10(8) 213±18(8) 0.5440.05 4C-methyl

mononucle-otides.1107(4)I. 218+7(4) 0.50+0. 04 32P-mononucleotides.... 114+10(6) 215 i20(6)0.53±0.07 aContent of 5-methylcytosine (5MC) or 5-methylamino-purine (5MAP) per genome was calculated by using the molecular weight of X as 32 X 106 and the molar absorb-ancycoefficient(basedonphosphate)as6.4 X 103.

bNumbers in parentheses represent number of determina-tions.

phosphate,since thecountsobtained inthe

meth-ylated mononucleotide spots were directly

com-paredwith thecountsobtained in the four main mononucleotide spots. The fact that the same values for

methyl

content were obtained

by

32P

labeling

and

'4C-methyl labeling

validates the

assumption made

regarding

the

specific activity

of

themethylgroups.

The

methyl

contents for X DNA described above are

approximately

twofold

higher

than

thosereported

by Gough

and

Lederberg (5)

forX grown onK-12 strain C600. We have examined

themethylation ofXDNAgrownonC600onlow

phosphate medium

using orthophosphate labeling

and

hydrolysis

to mononucleotides. From the

averageoftwo

experiments,

wefound 5MC con-tentsof56moles/genomeand 6MAP contentsof

132 moles/genome, in

good

agreement with the

values reported

by Gough

and

Lederberg.

We

have no

explanation

forthe twofold

difference

in methylation forX grown onW4032 andC600. The

possibility

ofa different

restriction-modification

system betweenW4032 and C600 has been ruled

out directly

by

comparing plating

efficiencies of

phage X on each strain (data not shown). The

difference in

methylation might

reflect the pres-ence ofthe sexfactorin W4032 orits absencein C600, or both, but we have no evidence on this point.

Methyl content of X DNA single strands. The

proportion ofmethylatedto totalnucleotideswas

compared by

using

the separated strands of X DNA doubly labeled with

[3H-methyl]-methio-nine and 32p

phosphate.

A typical experiment is illustrated inFig. 1. Theratioof3Hto32pcounts was calculated for each DNA-containing

frac-tion; nosignificantdifferences werefoundbetween

therand 1strands, indicatingthat the total con-tent of methylated bases is the same for each strand (Table2).To examine thecontent ofeach

of the twomethylatedbases in eachstrand,DNA waslabeledbygrowingXc1857onW4032 inTPA

X030r 20+001 20±001 I020

010I

I

do

20-a.I

10 20 30 40 50

FRACTIONNUMBER

FIG. 1. Distribution of methylated base residues between the strands ofX DNA. Phage X was doubly labeled with ['H-methyl]-methionine and32p. The two

strands wereseparatedbyheatdenaturing the DNAin the presence ofpoly (UG) followed by equilibrium density centrifugation in CsCl. Theratiosbetween 3H and 32p counts were determined for each DNA-con-tainingfraction.

medium with

['4C-methyl]-methionine.

The

sepa-rated strands were

hydrolyzed

with perchloric acid and the bases were resolved

by thin-layer

chromatography.

The ratio between 5MC and 6MAP obtained for each of the two separated strands was the same within

experimental

error

(Table 3).Weconclude that thetwo strandsofX

DNAare

equally

methylated

for both 5MC and

6MAP.

Methylation along theDNA molecule.

Phage

X

c1857wasgrownonW4032 in TPAmedium

con-taining

['4C-methyl]-methionine.

The DNA was

sheared to halves and the

separated fragments

were

hydrolyzed

tobases. Table 4shows the aver-agevalues for

methylated

base content obtained inthree different

experiments.

Thefractional con-tentof 6MAP and 5MCpernucleotide (Table 4,

columns 2 and

3)

in each halfis similar to that

obtained

for theintact DNA molecule.

To

investigate

in further detail any possible

heterogeneity

in

methylation

along the DNA

molecule, smaller fragments were prepared by

shearing doubly labeled

[3H-methyl]-

and

32P-DNA to

one-tenth,

one-fifteenth,

and

one-twentieth molecular lengths and separating the

fragmentsby bandingthem in

Hg-Cs2SO4

density

gradients. Themethylation of each fragmentwas

inferred from theratios of3Hto

32p

countsin each DNA-containing fraction. Figure 2 illustrates a

typical

experiment

ofthis kind. In all threetypes of

gradients,

with any of the three fragment lengths,thesamegeneralpatternfor total nucleo-tides wasobtained:ashouldercorrespondingto a

regionof lowguaninepluscytosine (GC) content and twomajor peaks.

Skalkaetal. (10) haveshownthat, forXDNA

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HIDALGO AND NASH

sheared to one-tenth length fragments, the shoulder contains material of

37%

GC content

derived from the b2 region. We have confirmed

these results by shearingtogether X c1857 and X

b2c1857 DNA to one-tenthlengthfragments

fol-lowed by banding the fragments in Hg-Cs2SO4 gradients. The shoulder corresponding to a low

GCcompositionshownin Fig. 2 wasmissingin

thecaseofXb2c1857DNA (datanot shown). By

direct analysis of nucleotide composition of X

DNAsheared toone-tenthlengths,wefound that

the shoulder corresponds to

40%0

GC composi-tion, and the two peaks to 46 and 57%o

GC,

respectively.Figure3 shows agradient of X DNA sheared to one-fifteenth fragment length. From

Fig. 2 and 3, it can be seen that alongthe two

major

peaks

the 3Hto32Pratiosare

indistinguish-TABLE 2. Methylatiotn ofXDNA sinlgle strands

3H to32pratio Expta,

rstrand 1strand

1 0.34 i 0.02 (ll)b 0.34 + 0.01 (5) 2 0.44 + 0.01 (12) 0.44 i 0.02 (8) Since thephageusedtoobtain the strands had different specific activities of "P at the times at

which theexperimentsillustratedinthe tablewere

done,comparisonsofratiosfor1andrstrandare

valid only within each

experiment.

dThe numbers in parentheses indicate the number offractions

averaged

for each strand.

TABLE 3. Methylated S-meilhylcytosine (SMC) and 6-methylaminopurinie (6MAP) ineach stranzdof

XDNA

Counts/min

Strand Ratio

5MCa 6MAPa

r 283 +t 12 570 + 25 0.50 i 0.03

1 381 i 15 683 + 18 0.56 ± 0.03

aFive determinations were made for each strand.

able. However, the 3Hto32Pratio decreasedin the shoulder region. Thesameresultwasobtained for

one-twentieth fragment lengths. Table 5 shows a summaryofall resultsobtained by this method. The X DNA one-fifteenth fragments show more

dramatically the presenceofanundermethylated

region of DNA in thecentralpartof themolecule. However,owingtothe large variation indifferent gradients, it isnotpossibletocalculatetheexact

length or the absolute extent of methylation of thisregion.

DISCUSSION

The major portion of the methylated nucleo-tides ofX DNA havenoknownroleineither the

lytic or lysogenic phases of the bacteriophage X

life cycle. The results presented above rule out

several different models concerning the function ofmethylgroupsin X DNA.

After infection of E.coli by phage X, the

paren-talstrandsare foundto haveanasymmetric fate

during replication; the1strand is found in circular

monomersandtherstrandin multigenomelength molecules (7). Asymmetry in the fatesof comple-mentary strands is a predicted feature of the

rolling circle model of DNA replication andmay

be a widespread feature in the physiology of

viruses. Thefinding that both strandsareequally

methylated, both in terms of total content per

strand and composition in each strand, implies that the bulk ofmethylated basescannotbeused

as arecognition elementtosignal theror1strand

in directing asymmetric replication, or in other processes that distinguish between the two strands.

The genes of X are clustered in functional

groups onthephage DNA, and these clustersare

characterized by differing GC content (10). The head and tail genes makeup the left arm of the

phage chromosome, as it is usually written, and haveauniform GC content of57%. Mostof the rightarmofthephagemapistakenupwithgenes

concerned with replication, recombination, and immunity; this region has a distribution ofGC

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contentthat varies from43 to 48%.Theleftarm

TABLE 4. MethylatedbasesinzXDNA halvesa

DNAhalf 61IAP/1001\LNb 5MC/100MNb 5MC/6MIAP 6MAP/100AC 5-\fC 100CI1

456X70 GC half 0.23 ± 0.02 0.12 i 0.01 0.52 ± 0.06 0.83 + 0.06 0.52 4 0.02

55% GChalf 0.21 + 0.01 0.09 + 0.01 0.43 + 0.05 0.93 + 0.05 0.34 0.02

aTheresultsareaverages ofthreedifferent

experiments.

bValuesare100 X (moles ofmethylated nucleotide per mole ofmononucleotide ineach half). ¢Valuesare100 X (moles of6-methylaminopurine per mole of adenine in eachhalf).

dValuesare100 X (molesof

5-methylcytosine

permole ofcytosine in each half).

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METHYLATION PATIERN OF LAMBDA DNA

64±003 .72±0O02 .68±t004

0. 07

06

10 20 30 40

FRACTION NUMBER

FIG. 2. Distribution of methylated base residues

among one-tenth length fragments of differing GC

composition. Phage X DNA doubly labeledwith [3H-methyl]-methionine and 31P was sheared to one-tenth lengthfragments. Thefigure illustrates a typical

Hg-CS2SO4equilibrium centrifugation gradient. The ratios between 3H and 32P counts were calculated for all fractions corresponding to regions I, II, and III. The averageratio valuesaregivenforeach gradientzonte.

0-08

10000 8000 2

6000-2- 4000-2000

.54± 003 87±0 04 78±0 03

~~ ~

10 20 30 40 50 FRACTION NUMBER

FIG. 3. Distributioni of methylated base residues

among one-fifteenth kengthfragmenits of differing GC

composition. Phage X DNA doubly labeled with [3H-methyl]-methionineand 32Pwasshearedtoone-fifteenth length fragmentsandbanded inHg-CS2SO4asdescribed in the text. The ratios between 3H and 32pcounts were

calculatedforall fractions corresponding to regionsI, II,andIII.The averageratio valuesaregivenforeach gradientzone.

genesaretypicallytranscribed and translated late after induction or infection of X; the right arm

genes are expressed early. The data presented in Table 5 andFig. 2and 3show thattheseregions

cannotbedistinguished fromoneanother onthe basis ofmethylcontent,andthus ruleoutthe

pos-sibilitythatearlyversuslatetranscriptionis

regu-lated byrecognitionof sitesidentifiedby amajor

fraction of themethylgroupsin X.

Anextremehypothesis,i.e.,thatmethylationis featureless and thereforeplays norolein

recogni-TABLE 5. Methylation inXDNAfragments

No.of Normalized8Hto32Pratios XDNAshearedto 'deter- ingradientregiona

mina-tions

One-tenth 17' 911 6100 3 102 -4 5

One-fifteenth 7 76 -4- 9106 -4 10 103 ±~5 One-twentieth 6 90 4- 3104 4± 3 100 ±4 2 aRatiosbetween 3H and321 countsinall

DNA-containing fractions on any given gradient were

averaged,

thegradientwasdividedinthreeregions

from low to high GC content (the shoulder and the twomainpeaks illustrated onFig. 2 and Fig. 3), and the mean 3H to 32P ratio for each

region

was divided bythetotal gradient average.

tion ofspecific DNA elements in X, mayalso be ruledoutfromanexamination of the fine detail of

methylatedbasedistribution. Ifmethylationwere

not based on the interaction of methylating

en-zymeswith specific DNA sites,onewould expect

that themethylcontent ofaregion would reflect

thedensityofthepotential substratenucleotides,'

adenine and cytosine. The density ofmethylated nucleotides with respect to potential precursor nucleotides may be calculated from the data in Table 4by combining the density of methylated bases per nucleotide and thecompositionof each half. Such acalculation (Table 4, columns 5 and 6) revealsasimilardensityof6MAP/adenine

be-tween thetwohalves butavery different density of5MC/cytosine.Thismeansthatmethylationof

cytosine has proceeded by a mechanism that is

responsive to dinucleotide or longer sequences.

Thisfindingis inagreementwith earlier chemical

studies showing that the distribution of methyl

cytosines amongthe variouspyrimidine tractsof

coli DNA isnonrandom (4).

T'he results obtained by shearing the DNA to

small fragment lengths reveal a more striking asymmetrythanthat discussed for halves. A small

fragment, arisingfromthe centralportionof the

X genome, shows a decrease in methylation as

compared to the totalmethylation of the rest of

the DNAmolecule,which is itselffairlyconstant.

Ifweassumethat themethylationof adenine and

cytosineisonlyafunction of the absoluteamount

of each base present, as done above for half

molecules, we calculate that the ratio of

methyl-atedtototal nucleotides would beexpectedto be

%higherfor a40%0/ GCfragmentthan forthe

entire molecule.Instead,wefind that the40%,- GC

fragment is between 10 and25% lessmethylated

than the average molecule. In the other regions resolvedbythe CS2SO4-Hg gradient, the

nonuni-formityofmethylationis much less dramatic.

VOL. 10, 1972

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HIDALGO AND NASH

Thus, the distribution of methylated nucleo-tidesis not a simple reflection of either total nu-cleotides or the content of the potential acceptor

nucleotides, adenine and cytosine. It is possible

thatthisnonrandomness in methyl group distribu-tion reflects the sum of many sequence-specific methylated sites-a few each for modification, transcriptional starts and stops, and replication signals. The data ofearlier workerson modifica-tion andourdataondistribution of methylgroups simply rule out that one of these functions ac-counts for the majority of methyl groups in X DNA. On the other hand,a yet unknown single function for the bulk of the methylated

nucleo-tidesmay exist.

ACKNOWLEDGMENTS

We thank J. Little for his help in teaching us his shearing techniques. We also thankJ.Little,D.Neville, J.L.Rosner, and M.Shulman fortheir carefulreadingsof the manuscript.

LITERATURE CITED

1. Arber,W., and S. Linn.1969.DNAmodification and restric-tion. Annu. Rev. Biochem.38:467-500.

2. Burgi,E., and A. D.Hershey. 1963.Sedimentationrate as a measure ofmolecularweight of DNA. Biophys. J. 3:309-321.

3.Cook,A., and J. Lederberg. 1962.Recombination studies of lactose nonfermenting mutants of Escherichia coli K 12. Genetics47:1335-1353.

4. Dosko6il, J., and Z. Sormova. 1965. The sequences of 5-methylcytosineinthe DNA of Escherichia coli. Biochem. Biophys.Res.Commun. 20:334-339.

5. Gough,M., and S. Lederberg. 1966. Methylated basesinthe host-modified deoxyribonucleicacid ofEscherichia coli and bacteriophageX. J.Bacteriol.91:1460-1468.

6. Hradecna, Z., and W.Szybalski. 1967.Fractionation ofthe complementary strands of coliphageXDNAbasedon the asymmetric distribution ofthe Poly I, G-binding sites. Virology32:633-643.

7. Ihler,G., andY. Kawai. 1971.Alternate fates of the comple-mentarystrands of lambdaDNAafter infection of Esche-richiacoli. J. Mol.Biol. 61:311-328.

8. Kelly, T. J.,Jr., and H.0.Smith. 1970. A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. J.Mol.Biol. 51:379-391.

9. Ledinko, N. 1964. Occurrenceof5-methyldeoxycytidylate in the DNAof phage lambda.J.Mol. Biol. 9:834-835. 10. Skalka, A., E.Burgi, and A. D. Hershey. 1968.Segmental

distribution of nucleotidesin the DNA ofbacteriophage lambda.J.Mol.Biol. 34:1-16.

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