Proc. Natl. Acad. Sci. USAVol. 83, pp. 9070-9074, December 1986Genetics

Escherichia coli K-12 restricts DNA containing 5-methylcytosine(,rg1/rgB/molecular cloning)

ELISABETH A. RALEIGH AND GEOFFREY WILSONNew England Biolabs, Beverly, MA 01915

Communicated by Allan M. Campbell, July 21, 1986

ABSTRACT We have observed that plasmids containingcertain cloned modification methylase genes of type H restric-tion-modification systems cannot be transformed into manylaboratory strains of Escherichia coli K-12. The investigationof this phenomenon, reported here, has revealed (i) DNAcontaining 5-methylcytosine is biologically restricted by thesestrains, while DNA containing 6-methyladenine is not; (ii)restriction is due to two genetically distinct systems that differin their sequence specificities, which we have named mcrA andmcrB (for moiffied cytosine restriction). Since 5-methylcyto-sine containing DNA is widespread in nature, the Mcr systemsprobably have a broad biological role. Mcr restriction mayseriously interfere with molecular cloning of 5-methylcytosine-containing foreign DNAs. The Mcr phenotypes of some com-monly used strains of E. coli K-12 are reported.

When foreign DNA is introduced into bacteria, it is frequent-ly attacked by restriction systems encoded by the host cell(1-4). An important feature of the action of these systems isthat the survival of a DNA molecule in a restricting hostdepends not only on its sequence but also on its history: thesame sequence behaves differently depending on what its lasthost was. The particular host in which the molecule replicatesconfers on the DNA a modification, usually methylation ofanadenine or cytosine residue within the target sequence, thatprotects it against the cognate restriction functions; it is notprotected against heterologous restriction. It is generallyassumed that restriction provides a defense against invasionby foreign DNA, especially phage DNA, and that cognatemodification serves primarily to prevent suicidal attack onhost DNA.The first restriction systems to be described were the Rgl

(for restricts glucose-less phage) systems (1, 5, 6). Thesewere identified as functions that attack T-even phages, butonly when they contain 5-hydroxymethylcytosine in theirDNA. The T-even phages incorporate 5-hydroxymethylcy-tosine intoDNA at the mononucleotide level; glucosyl groupsare later transferred to 5-hydroxymethylcytosine in thepolynucleotide from UDP-glucose by phage-encoded en-zymes. The phage DNA is sensitive to the RglA and Rg1Brestriction functions only when the 5-hydroxymethylcytosineis not glucosylated, as when the glucosyltransferase enzymesare defective, or when the host lacks UDP-glucose. Mutantsof T4 that contain completely unmodified cytosine are notrestricted.We describe here two restriction systems present in Esch-

erichia coli K-12 that attack DNA containing methylatedcytosine in particular sequences. In the course of cloning themethylase genes associated with restriction-modificationsystems, we encountered difficulty in transforming certainclones into many common E. coli K-12 strains. We foundexisting variation among laboratory strains for this methyl-ase-rejection property, and we have begun a genetic study of

the phenomenon. This rejection has the formal properties ofhost-controlled restriction systems. It is probably identical toRgl restriction of nonglucosylated T-even phages (E.A.R., R.Trimarchi, and H. Revel, unpublished observations; also seebelow).

MATERIALS AND METHODSBacterial and Phage Strains. Strains were obtained from the

following sources: W3110, C600, and CR63 (7), N. Kleckneror N. Murray; HB101, RR1, and LE392 (8), our collection orN. Kleckner; K802 (9) and MM294 (10), our collection;JM107 (11) and JM107 MA2 (12), R. Blumenthal; JM101 (11),J. Messing; Y1084, Y1088, and Y1090 (13), R. Young via L.McReynolds; GM2163 (14), M. Marinus; MC1061 (15), R.Neve.HR110-HR112 were from H. Revel and are K-12/F+(X);

HR111 is also rglA-, and HR112 is rglA- rglB- (5, 6); HR111and HR112 are also PI' and sup' (H. Revel, personalcommunication). K802 is a conjugational descendant of C600and Hfr Cavalli (9) and probably carries the hsd region of HfrCavalli and the purB region of C600. It is phenotypicallyRglA- RglB- (and thus presumably rglA rglB) and McrA-McrB-. LCK8 (lac3 or lacYl galK2 galT22 metBI hsdR2zj202::TnlO supE44; from L. Comai via B. Bachmann) isK802 with a TnlO insertion between hsd and serB and has therestriction phenotype of K802. By definition established inthis work, LCK8 and K802 carry the mcrBl allele. ER1370(trp3l his] argG6 rpsLl04 tonA2 A(lacZ)rl supE44 xy17 mtl2metB1 serB28) is descended from JC1552 (7) via NK7254 (16)and several additional transductional steps. Utilizing theclose proximity of mcrB to hsd, zy202::TnJ0, and serB,mcrBl derivatives of ER1370, W3110, MM294, JM107, andY1084 were constructed, yielding some strains used in thiswork: ER1378 (mcrBl), ER1380 (mcrB1 zj202::TnlO) andER1381 (mcrB+) (all hsdR2 serB+, from ER1370); ER1398(from MM294, hsdR2 mcrBl); and ER1451 (from JM107,hsdR2 or R17, mcrBl). These constructions will be detailedelsewhere (E.A.R., R. Trimarchi, and H. Revel, unpublishedresults). Xr and Pl1i, were from our collection. Transduc-tions were by standard methods (17).

Methylase Titrations. Five units (as defined by the manu-facturer) of methylase (Alu I, Msp I, Hha I, Hph I, Taq I,dam, or Hpa II; obtained from New England Biolabs) wasadded to 0.9 ,ug of pBR322 DNA in a total vol of 30 ,ul; thissample was diluted serially by factors of 2 into additionalDNA samples, such that the total volume (15 ,ul) and amountof DNA (0.45 ,Ag) were maintained in all samples. Forconvenience, the concentration of methylase present in themost dilute sample was arbitrarily designated "1 unit" inFigs. 1 and 2; this is not the same as the manufacturer's unit.Two control samples were carried through the procedureunder the same conditions but lacked either methylase orS-adenosylmethionine. Buffer conditions were 66 mM

Abbreviation: M. Hae II, modification methylase associated with theHae II endonuclease; other methylases are designated accordingly.

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Tris HCl, pH 7.5/6.6 mM EDTA/0.33 mM 2-mercaptoetha-nol/0.15 mM S-adenosylmethionine. Incubation was for 1 hrat 370C, and the reaction was stopped by heating to 650C for10 min.Phage Growth and Plating Tests. Phage stocks were pre-

pared by confluent lysis. Hae II-modified X was obtained bygrowth on LCK8/pHaeII 4-11 (see below). Phage weretitered on bacteria grown in XYM on X plates (18). E.O.P. isthe ratio of the titer on the tested strain to the titer on LCK8.

Plasmids. Clones carrying the following modificationmethylases were isolated by G.W. and coworkers (unpub-lished data): Alu I, Ava I, Ava II, BamHI, Ban I, Ban II, BglI, Dde I, EcoRI, FnuDII, Hae II, Hae III, Hga I, HgiAI, HhaI, Hha II, HindII, HindIII, Hinfl, Hpa II, Msp I, Nla III, NlaIV, Pst I, Sal I, Taq I. All are carried on pBR322 (19) orpUC19 (11). pHaeII 4-11 carries both the Hae II endonucle-ase and methylase. pER82 was constructed from pHaeII 4-11by in vitro deletion of a portion of the endonuclease gene.pPvuMl.9 carrying the Pvu II methylase was a kind gift fromR. Blumenthal (12).

RESULTSRejection of a Clone Carrying the Hae H Methylase Gene.

The plasmid pHaeII 4-11 carries the complete Hae II restric-tion-modification system [recognition sequence, RGCGCY(R = puRine and Y = pYrimidine); see ref. 20] cloned intopBR322; it was isolated by using E. coli strain RR1 as host.The DNA from cells containing pHaeII 4-11 is completelyHae II-modified. pHaeII 4-11 could be efficiently trans-formed into LCK8, an unrelated strain derived from K802;however, neither pHaeII 4-11 nor pER82, a derivativeencoding only the methylase, M. Hae II, could be efficientlytransformed into several other laboratory strains ofE. coli K-12, including ER1380, ER1381, JM107, MM294, and W3110.Strains that rejected these plasmids could be transformed bythem at a frequency of -10-5 relative to transformation of apermissive strain. The rare transformants were of two types:those carrying deleted plasmids lacking methylase activity,and those carrying host mutations that eliminated the rejec-tion phenotype permanently. We designated such host mu-tants mcrB- (for methyl cytosine restriction; see below formcrA). In all subsequent work, the response to pER82,carrying M. Hae II, or to Hae JI-methylated Xvir (see below),was used to define the mcrB genotype. A detailed geneticanalysis of this locus, adjacent to hsd, will be publishedelsewhere (E.A.R., R. Trimarchi, and H. Revel, unpublisheddata).McrB Phenotypically Restricts Hae II-Modified X. The

plating efficiency of Hae II-methylated Xvir was comparedwith that of unmethylated Xvir on isogenic mcrBl and mcrB+strains (ER1378 and ER1381, respectively). Unmethylated Xplated with equal efficiency on both strains (Table 1, column1). Hae JI-methylated X phage plated at an efficiency of 0.03on the McrB+ strain when compared with plating on theMcrB- strain (column 2). The unmethylated progeny oftheseHae II-methylated phage (obtained by passage in the absenceofM. Hae II) once again plated normally on the McrB+ strain(column 3). This reacquisition of resistance to restriction wasvery efficient: four of four independent progeny stockspassaged in the absence of the Hae II methylase plated

Table 1. E. coli K-12 restricts Hae II-modified X

Plating efficiency relative to LCK8Of phage stock

On host strain XAC600 XALCK8/pHaeII 4-11 XALCK8

normally on the McrB+ strain. These progeny also regainedsensitivity to Hae II restriction (not shown), demonstratingloss ofHae II-specific methylation. Further passage througha strain carrying M. Hae II restores sensitivity to McrBrestriction at high efficiency (not shown). This reversibleepigenetic alteration in plating properties is formally identicalto classical host-controlled restriction (1-4).McrB Restriction Is Methylcytosine Dependent. Samples of

pBR322 DNA were methylated in vitro to increasing extentswith purified M. Alu I (which creates the sequence AGmCT),and were transformed into ER1381 (McrB+) and its McrB-sibling, ER1378 (Fig. 1). The efficiency oftransformation intoER1381 was found to be inversely related to the degree ofmethylation, while with ER1378 the efficiency of transfor-mation was independent of the degree of methylation. Max-imal restriction by the McrB+ strain occurred when the DNAwas fully protected from Alu I digestion (arrow). Similarexperiments using other purified methylases (Table 2) sug-gest that sensitivity to McrB restriction results from cytosinemethylation but not from adenine methylation.A Second Methylcytosine-Specific Restriction System Exists.

The McrA restriction system was recognized when wetransformed mcrB mutants with pBR322 methylated in vitroby M. Hpa II (CmCGG). We found restriction of thissubstrate to be independent of the mcrB genotype of thestrain (Fig. 2). Both ER1378 (mcrBl) and ER1381 (mcrB+)(triangles) restricted Hpa II-methylated pBR322; however,neither JM107 (mcrB+) nor its mcrBl derivative, ER1451,restricted Hpa II-methylated pBR322 (circles). Therefore,restriction of Hpa II-methylated DNA can be eliminatedgenetically without eliminating McrB restriction and viceversa. The locus encoding McrA is nearpurB (E.A.R. and E.Latimer, unpublished data).

McrB-1.0

0C

0

0EI

0, McrB~0.01 1E ~~--'

12 4 8 16 32 64

[A/u I Methylase] (Arbitrary Units)

FIG. 1. McrB restriction of pBR322 DNA methylated in vitro atAlu I sites. Samples (0.04 ,g) of pBR322 DNA, methylated toincreasing extents with Alu I methylase, were transformed (8) inquadruplicate into ER1381 (mcrB+ mcrA+) and ER1378 (mcrBlmcrA+). The average number of transformants per plate from thecontrol with no added methylase was 87 (ER1378) and 473 (ER1381).These values were taken as transformation efficiencies of 1. Averagevalues for all other samples were normalized to these values. Thetransformation efficiency is plotted versus the concentration (inarbitrary units) ofAlu I methylase. Error bars are standard deviationof the mean; for clarity of presentation, the standard deviation is notshown for ER1378. v, ER1381; v, ER1378. Portions of eachmethylated sample were digested with Alu I endonuclease andelectrophoresed in a 1% agarose gel to assess extent of methylation;complete protection from digestion was achieved at the concentra-tion of methylase indicated by the arrow.

ER1381 McrB+ 0.82 0.03 0.69ER1378 McrB- 0.88 1.2 0.91

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Table 2. Methylated sequences restricted or not restricted by mcrB

Methylatedsequence*Methylase

Hoststested Methylase

Methylatedsequence*

I. Restricted by McrB+ strainsA. Methylases of known specificityAlu Itt AG CTDde It OTNAGHae UiPt GG CCHha Itt G CGCMsp Itt CCGGPvu it CAG CTG

B. Methylases of unknown specificityAva It CYCGRGBan It GGYR CCBan iHt GRG CYCHae Hit RG CGCY or RGCG 6YBglIt G CCN(5)GGC or GCCN(5)GG CFnuDIIt CG 6GHgaIt G CGTC and/or GACG CHgi At G(A/T)G 6(A/T)CNla IIIt CATGNla IVt GGNN 6C

D, EDA, B, D, EA, BA, B, D, EE

DA, EA, DA, B, C, D, EA, DDA, DA, DDD

II. Not restricted by McrB+A. Methylases of known specificitydamt G ATCEcoRIt GA ATTCHha lIt G ANTCHindIlt GTYR ACHindIIIt A AGCTTPst It CTCG AGSal It GTCG ACTaq Itt TCG ABamHItt GGAT CCdcm C C(A/T)GGHph It T CACCHpa IIt# C CGG

B. Methylases of unknown specificityAva Ilt GG(A/T)CCHinfIt GANTC

pBR322 derivatives carrying cloned methylase genes (t) or pBR322 DNA methylated in vitro with purified methylase enzymes (t) weretransformed into various strains of E. coli K-12. Clones classified as not restricted transformed McrB+ and McrB- strains at approximately thesame frequency; clones classified as restricted transformed McrB+ hosts at a frequency of 10-3, or less, compared to McrB- hosts. In vitromethylated pBR322 was classified as restricted if the transformation efficiency dropped by at least a factor of 10 as the DNA became fullymethylated. Recognition sites for the methylase enzymes are assumed to be the same as the sites recognized by the cognate endonuclease andare from ref. 21; specific methylation sites are from ref. 22, except for dem (23), Pvu II-(12), Dde I (J. Brooks, personal communication). Therecognition sites are aligned at the position of known or proposed methylation. The dcm methylase has not been tested as a clone, but the hostis dcm' (as well as dam') and must, we assume, find this methylation acceptable. Letters refer to sets ofmcrB1 and mcrB- hosts. Set A: HR110(McrA+ McrB+), HR111 (A- B+), and HR112 (A- B-). Set B: C600 (A- B+) and K802 (A- B-). Set C: JM107 (A- B+) and ER1451 (A- B-).Set D: MM294 (A' B+) and ER1398 (A' B-). Set E: ER1381 (A' B+) and ER1378 (Al B-). Mcr phenotypes were defined on the basis ofacceptance of the Hae II methylase plasmid for McrB and of Hpa II-methylated pBR322 for McrA. HR110 is wild-type K-12; HR111 (rglA)and HR112 (rglA- B-) are nitrosoguanidine-induced derivatives of HR110. The McrB- strains in sets C-E are transductants carrying the mcrBIallele found in LCK8; all of these strains are also RglB-. The sets include at least two mcrA- alleles: that introduced by nitrosoguanidinemutagenesis into HR111, and that (those) found in C600 and JM107. C600 and JM107 are also phenotypically RglA-; RglB restriction is observed,although it is not as strong as that seen in HR111.*Inferred from the recognition sequence of the endonuclease and the known (^) or proposed (') position of methylation within that sequence.tTested for ability of cloned methylase gene to transform.*Tested for ability of in vitro methylated pBR322 to transform.

Specificity of McrB. The sequence specificity of the McrBrestriction function was examined further using transforma-tion by plasmids carrying cloned modification methylasegenes (Table 2). Many of the methylases that confer sensi-tivity to McrB restriction (part IA) are known to generate thesequence GmC. For the rest of the mnethylases that confersensitivity, the site of methylation is unknown (part IB); inthese cases, the sequence GmC potentially could be gener-ated. The methylases that do not confer sensitivity (part II)are either adenine methylases or are known not to generatethe sequence GmC. The simplest conclusion is that GmC orRmC is a necessary component of the McrB recognition site.Mcr and Rgl Are Probably Identical. At least two indepen-

dent rglA mutations and two independent rglB mutationswere present among the strains tested in Table 2. All rglBmutants displayed the McrB- phenotype and all rglA mutantsdisplayed the McrA- phenotype. This coincidence leads us tosuspect that Rgl and Mcr are identical.The specificity of Rg1B from E. coli B differs from that of

K-12, and its effect is substantially weaker (5, 6). We expectthat the McrB function will, similarly, have a differentspecificity and a weaker activity. With the exception of M.Hpa II, the methylase gene clones referred to above wereisolated by using RR1 as host; this hybrid strain carries therglB region from E. coli B and is phenotypically McrB-,consistent with our expectation. The M. Hpa II gene couldnot be cloned in RR1, but it could be cloned K802. This isconsistent with the RglA+/McrA+ phenotype of RR1, andthe RglA-/McrA- phenotype of K802.

Survey of Common Laboratory Strains of E. coli K-12.Table 3 summarizes the results oftests of the Mcr phenotypesof various strains commonly used in E. coli genetics and inmolecular cloning experiments. All tested strains commonlyused for cloning experiments carry at least one ofthe two Mcrsystems. The results described above suggest that restrictionproblems may be encountered when these strains are usedand the DNA to be cloned contains methylcytosine.

DISCUSSIONWe show here that E. coli strains biologically restrict DNAmethylated at some cytosine residues. Plasmids carryingcloned methylases (Table 2), phage methylated in vivo (Table1), or plasmids methylated in vitro (Figs. 1 and 2; Table 2) areall restricted. We regard the phenomenon as restrictionbecause it fits the classical definition: rejection, at least ofHae II-modified X, depends upon the host that the phage aregrown on and is reversible at very high frequency by growthof the phage on another host (Table 1; see text).The observed restriction requires methylcytosine in spe-

cific sequences. That pBR322 DNA methylated in vitro isrestricted demonstrates that restriction is not dependent onunknown products of, or sequences present in, cloned DNAfrom foreign species; nor are alterations ofDNA other thanmethylation required. We assume that the methylation re-quired is at the 5 position of cytosine. Of the methylases thatconfer sensitivity, only M. Msp I has been shown to meth-ylate the 5 position (24, 25). It has recently been shown that

Hoststested

CA, B, DA, B, DA, DDA, B, DDA, C, D, EA, B, DAll dcm'EA, C, D, E

DA, D

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c

.)4-

o 0.1L-E O

o0

enc

0.01

0

0

0 McrA0

v

V

0

V

124 8 16 32 7 64

[Hpo II Methylase] (Arbitrary Units)

FIG. 2. McrA restriction of Hpa I-methylated pBR322 DNA.Hpa JI-methylated pBR322 was transformed in duplicate intoER1381 (mcrB+ mcrA+), ER1378 (mcrB- mcrA+), JM107 (mcrB+mcrA-), and ER1451 (=JM107 mcrBI), and the transformationefficiencies at various methylation levels were determined as de-scribed in the legend to Fig. 1. v, ER1381; v, ER1378; e, JM107; o,ER1451. Arrow indicates complete protection from Hpa II endonu-clease digestion, as in Fig. 1.

some methylases modify the N4 position of cytosine (25, 26),and we cannot rule out the possibility that such methylationalso confers sensitivity.We have identified two different restriction specificities,

McrA and McrB. These specificities are genetically different,since mutations that eliminate one activity have no effect onthe other activity. Cytosine methylases exist that do notconfer sensitivity to either Mcr system (Ava II, BamHI, dcm,Hph I); the systems must therefore discriminate amongmethylated residues. We suggest that the McrB functionrecognizes the consensus sequence of GmC, or RmC. Therecognition sequence of the McrA function remains uncer-

Table 3. Mcr phenotypes of selected laboratory strains

Mcr phenotypeStrain A B

K-12 + +W3110* + +MM294* + +ER1370* + +JM101 + NTJM107* - +C600 - +Y1084* - +Y1088 -t +Y1090 -t +LE392 - +CR63 - +HB101 + -RR1 + -MC1061 NT -K802 - -GM2163 - -

NT, not tested.*mcrB derivatives are available.tInferred from phenotype of plasmidless ancestor.

tain, since only one methylated sequence, CmCGG, has beenfound to confer sensitivity. Since RR1, the host used forcloning most of the methylase genes, is McrA' McrB-, ourcollection probably contains only methylases that are insen-sitive to McrA activity.The identity of Mcr and Rgl functions is suggested by the

facts that both systems are active only on DNA that containsmodified cytosine, and that among the strains tested, Mcr andRgl phenotypes invariably coincided. Genetic analysis(E.A.R., R. Trimarchi, and H. Revel, unpublished data)shows that both rglB and mcrB mutants map close to hsd, at99 min; that mutants isolated for either phenotype exhibit theother; and that the two phenotypes are not separated bytransduction. In addition, both McrA and RglA are affectedby a mutation that maps near purB (E. Latimer and E.A.R.,unpublished data).The Mcr restriction systems present a potentially serious

problem for those engaged in molecular cloning cf foreignDNA into E. coli. Possession ofboth systems is the wild-typestate, since the original K-12 strain carries both, 4nd manycommon laboratory strains of E. coli K-12 carry at least oneof the systems (Table 3). The DNA of many organismscontains methylated cytosine (27) and consequently shouldbe sensitive to Mcr restriction, at least at some sites. WhenpBR322 is fully methylated with the Alu I methylase, -2% ofits cytosine residues are methylated-a level of methylationwell within the range found in eukaryotic organisms-and thenumber of transformants in McrB+ strains is reduced to 1%of the level found with unmethylated DNA. Our resultssuggest that the use of strains defective in both Mcr functionswould enhance recovery of transformants carrying somesequences from ligation mixtures containing genomic DNA.We do not know the molecular mechanism of M4r restric-

tion. The enzymology of Rgl restriction also remai s uncer-tain. A function present in RglB+ cells, but not in RgB- cells,has been shown to act on hemihydroxymethylated fd RFDNA: an Rg1B+ RecBC- extract altered the mobility of thesubstrate in a sucrose gradient and rendered it sensitive toRecBC exonuclease activity from an Rg1B- extract (28, 29).However, a partially purified fraction that rendered thesubstrate sensitive to RecBC did not alter its mobility ongradients; a heat-labile non-dialyzable factor from an RgIB-extract was required for this. It is possible that RglB functionrequires two or more different subunits, or that a secondindependent enzymatic activity is required for substrateconversion to the RecBC-sensitive form. In many other casesof phenotypic restriction, restriction is mediated by site-specific double-strand cleavage of the target DNA. Othermechanisms are possible, however. For exaniple, theuracil-DNA glycosylase (ung) ofE. coli, in combination withapyrimidinic endonucleases, can mediate restriction ofuracil-containing DNA (30).Two other methyl-specific restriction systems have been

reported. The best characterized system, Dpn I, restrictsphage in vivo, and consists of an otherwise ordinary restric-tion endonuclease that specifically recognizes and cleaves amethylated sequence (GmATC). In this case, the "modifi-cation" that protects DNA from cleavage is the absence ofmethylation at that sequence (31, 32). Several endonucleaseswith the same specificity have been found in bacteria otherthan the original Diplococcus pneumoniae species (21). Theother system, from Acholeplasma laidlawii, has so far notshown evidence of sequence specificity. It results in pheno-typic restriction of DNA that has been methylated in vivo orin vitro with any of several sequence-specific cytosinemethylases (33).The existence of methyl-requiring restriction systems en-

sures that methylation alone is not sufficient for defenseagainst restriction. Many phage use active antirestrictionmechanisms (34) or extensively modify their DNA, some

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with quite baroque substitutions (glucose, putrescine, gluta-mine, isopentene; see ref. 35), but very few have limitedthemselves to the relatively simple expedient of incorporat-ing a methylated base in place of the normal one. Thissuggests that systems specific for simple modifications maybe common. Demonstration of two previously unrecognizedmethyl-specific restriction systems in the well-studied orga-nism E. coli K-12 suggests that such restriction may indeedbe much more widespread than previously appreciated. Thepaucity of restriction systems that are known to requiremethylated DNA may reflect primarily the use of unmethyl-ated substrates in the search for restriction endonucleases,rather than the rarity of such systems.

The authors thank Helen Revel for strains; Barbara Bachmann,Jon Beckwith, Ashok Bhagwat, Bob Blumenthal, Joan Brooks, DonComb, Nancy Kleckner, Noreen Murray, Mike Nelson, HelenRevel, Denise Roberts, Rich Roberts, and Ira Schildkraut fordiscussion and for critical review ofthe early draft ofthis manuscript;Bob Blumenthal and Antal Kiss for communication of results priorto publication; and Russ Camp, Chuck Card, Rebecca Croft, TinaJager, Elizabeth Latimer, Keith Lunnen, Chris Taron, and RuthTrimarchi for help with transformation and strain construction.

1. Luria, S. E. & Human, M. L. (1952) J. Bacteriol. 64, 557-569.2. Arber, W. (1974) Prog. Nucleic Acid Res. Mol. Biol. 14, 1-37.3. Bickle, T. A. (1982) in Nucleases, eds. Linn, S. & Roberts,

R. J. (Cold Spring Harbor Laboratory, Cold Spring Harbor,NY), pp. 85-108.

4. Modrich, P. & Roberts, R. J. (1982) in Nucleases, eds. Linn,S. & Roberts, R. J. (Cold Spring Harbor Laboratory, ColdSpring Harbor, NY), pp. 109-154.

5. Revel, H. R. (1967) Virology 31, 688-701.6. Revel, H. R. (1983) in Bacteriophage T4, eds. Mathews,

C. K., Kutter, E. M., Mosig, G. & Berget, P. B. (Am. Soc.Microbiol., Washington, DC), pp. 156-165.

7. Bachmann, B. (1972) Bacteriol. Rev. 36, 525-557.8. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular

Cloning: A Laboratory Manual (Cold Spring Harbor Labora-tory, Cold Spring Harbor, NY).

9. Wood, W. B. (1966) J. Mol. Biol. 16, 118-133.10. Backman, K., Ptashne, M. & Gilbert, W. (1976) Proc. Natl.

Acad. Sci. USA 73, 4174-4178.

11. Yanisch-Perron, C., Vieira, J. & Messing, J. (1985) Gene 33,103-119.

12. Blumenthal, R. M., Gregory, S. A. & Cooperider, J. S. (1985)J. Bacteriol. 164, 501-509.

13. Young, R. A. & Davis, R. W. (1983) Science 222, 778-789.14. Marinus, M. G., Carraway, M., Frey, A. Z., Brown, L. &

Arraj, J. A. (1983) Mol. Gen. Genet. 192, 288-289.15. Casadaban, M. & Cohen, S. N. (1980) J. Mol. Biol. 138,

179-207.16. Roberts, D., Hoopes, B. C., McClure, W. R. & Kleckner, N.

(1985) Cell 43, 117-130.17. Miller, J. W. (1972) Experiments in Molecular Genetics (Cold

Spring Harbor Laboratory, Cold Spring Harbor, NY).18. Kleckner, N., Barker, D., Ross, D. & Botstein, D. (1978)

Genetics 90, 427-450.19. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C.,

Heyneker, H. L. & Boyer, H. W. (1977) Gene 2, 95-113.20. Tu, C.-P. D., Roychoudhury, R. & Wu, R. (1976) Biochem.

Biophys. Res. Commun. 72, 355-362.21. Roberts, R. J. (1984) Nucleic Acids Res. 12, r167-r204.22. Smith, H. 0. & Kelley, S. B. (1984) in DNA Methylation:

Biochemistry and Biological Significance, eds. Razin, A.,Cedar, H. & Riggs, A. D. (Springer, New York), pp. 39-71.

23. Hattman, S. (1977) J. Bacteriol. 129, 1330-1334.24. Walder, R. Y., Hartley, J. L., Donelson, J. E. & Walder,

J. A. (1983) J. Biol. Chem. 258, 1235-1241.25. Butkus, V., Klimasauskas, S., Kersulyte, D., Vaitkevicius,

D., Lebionka, A. & Janulaitis, A. (1985) Nucleic Acids Res.13, 5727-5746.

26. Janulaitis, A., Klimasauskas, S., Petrusyte, M. & Butkus, V.(1983) FEBS Lett. 161, 131-134.

27. Ehrlich, M. & Wang, R. Y. (1981) Science 212, 1350-1357.28. Fleischman, R. A. & Richardson, C. C. (1971) Proc. Natl.

Acad. Sci. USA 68, 2527-2531.29. Fleischman, R. A., Campbell, J. L. & Richardson, C. C.

(1976) J. Biol. Chem. 251, 1561-1570.30. Duncan, B. (1985) J. Bacteriol. 164, 689-695.31. Lacks, S. & Greenberg, B. (1975) J. Biol. Chem. 250,

4060-4066.32. Lacks, S. & Greenberg, B. (1977) J. Mol. Biol. 114, 153-168.33. Sladek, T. L., Nowak, J. A. & Maniloff, J. (1986) J. Bacteriol.

165, 219-225.34. Krfiger, D. H. & Bickle, T. A. (1983) Microbiol. Rev. 47,

345-360.35. Warren, R. A. J. (1980) Annu. Rev. Microbiol. 34, 137-158.

Proc. Natl. Acad Sci. USA 83 (1986)

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