Host-controlled restriction and modification of deoxyribonucleic acid in Escherichia coli

VIROLOGY

27, 378-387 (1965)

Host-Controlled

Restriction

and Modification

Acid in Escher&a SEYMOUR Department

of Riclogy,

Brown

of Deoxyribonucleic

co/i

LEDERBERG University,

Accepted July

Providence,

Rhode Island

29, 1966

In Escherichia coli strain C600 (which has the host specificity of KlZ), restrictions against infection by phage X of host specificities B and C have the same sensitivity to heat inactivation. Likewise, in strain B, restrictions against phage X of host specificities CGOO and C have an identical heat sensitivity. Strain CSW(P1) has a heat sensitivity for loss of its Pl-directed restriction different from that of its C600-con trolled restriction. Mutants of Kl2-type strains and of B which are impaired in their restriction and modification activities have been isolated. In K12 strains, restrictions toward phage X of host specificities C and B are impaired by the same mutation. In B, restrictions toward phage h of host specificities C and C600 also are lost simultaneously by mutation. The coordinate changes in restriction by heat treatment and by mutation indicate that a common mechanism for restriction of h of host specificities C and B operates in K12 strains, and that a single mechanism for restriction of phage X of host specificities C and K12 occurs in strain B. Restrictionless mutants of B, unlike their parent, act as fertile I? in crosses with K12 Hfr strains. Modificationless mutants of K12 Hfr, unlike their parent, are no longer fertile with restricting C600 strains. Thus, the same mutations affect the modification and/or restriction of the DNA of bacteria as well as phage. Models are proposed for host restriction and modification. The models visualize restriction as either a screening of new DNA by a DNA-site specific degrading activity-successful passage permitting the DNA to operate in that cell-or a scavenging of new DNA by a nonspecific degrading activity when such DNA fails to be complexed with a site-specific protecting agent. INTRODUCTION

Bacteriophage host-range variations which are determined by the bacterium in which the phage were produced have been called “host-induced modifications” (Luria and Human, 1952). Bacteriophage carry a given host specificity and may either be rejected or ac’cepted upon infection of a new host. For phages Tl, X, and e, rejection of phage by nonaccepting cells is accompanied by loss and degradation of the infecting phage DNA (Lederberg, 1957; Dussoix and Arber, 1962; Arber et al., 1963; Lederberg and Meselson, 1964; Uetake et al., 1964). In the case where a bacteriophage is accepted by a new host, a host specificity characteristic of that host can be acquired by the newly made phage. 378

Three types of host specificity are described in Table 1. First, a one-step loss or gain of phage host-range is produced between strains Escherichia coli C and K12. Second, the presence of Pl prophage in a strain confers an additional independent host specificity to phage host-range. Third, strains E. coli B and K12 produce mutually incompatible specificities. From such behavior we might infer two processes: one is a screening activity which identifies the origin of an infecting phage and serves to restrict phage multiplication depending on this origin; the other is a modifying activity by which a phage acquires specificity from its last host. Different phages exhibit these types of

host specificity in various ways. Thus, phagc X recognizes a host specificity between strains C and K12 (Bertani and Weigle, 1953), strains B and K12 (Dussoix and Arber, 1962), and Pl-lysogens and nonlysogens (Arber and Dussoix, 1962). Phage P2 recognizes a host specificity between strains Xhigella dysenteriae Sh and E. co&i (Bertani and Weigle, 1953) whereas phage Tl does not. Pl-lysogeny in strains B and Sh produces restriction and modification for phages Tl and P2, but only restriction without modification for phages T3 and T7 (Lederberg, 1957). Moreover, not all the Tl phage which succeed in multiplying in Pl-lysogens acquire Pl-host specificity. Hence, modification of progeny phage is not obligatorily linked to the screening process. The possibility was considered (Lederberg, 1957) that the mechanism of restriction lay in the specificity of activity or localization of nucleases in the host, and that the different phage responses, in turn, were governed by structural or compositional differences in their DNA. A new phase of work on host-induced modification was initiated by Arber and Dussoix (1962), who found that the host specificity conferred to phage X persisted with the original physical copy of the phage genome during the replication of phage in nonmodifying hosts. Since old phage protein coats were removed and new ones acquired in this infection, this experiment argued that the subject of modification did not lie in the phage coat, but, rather, was strongly bound to the phage DNA or was part of it. In another experiment, conserved phage DNA which itself did not replicate was modified by its host. Hence, new host specificity could be imparted to polymerized DNA. The development by Kaiser and Hogness (1960) of conditions permitting successful infection of cells by free DNA of phage made it possible to ask directly whether the screening and modifying devices act on the DNA of phage X. The answer was that the DNA obtained from phenol-treated preparations of phage X exhibited the same overall features toward host restriction and modification as did phage X (Dussoix and Arber, 1965; Meselson and Lederberg, unpublished experiments). This result, in turn,

K12 B c .[, k;FL K12(Pl) B(Pl) C(Pl)

1 10-1 IO-4 1 lo-” 10-d

IO-” 1 10-4 10-4 1 10-d

1 1 1 1 1 1

10-4 IO-’ 10-7 1 10-1 10-d

10-r 10-t 10-T 10-a 1 10-A

10-d 10-j 10-e 1 1 1

a The values are approximate relative titers of host-modified phage on different host strains (adapted from Lederberg, 1957, and Arber and Dussoix, 1962). The generalized scheme holds in toto for phage 1, and in part for phages Tl and P2 (see test). Kl2-type strains include K12, KlO, AB259, C600, W3110, W1895, and W4032. C-t)ype strains include C, mutants of Kl2-type strains, and mut)ants of B. Not all Kl2-type strains and phages have been tested in all combinations. The age of the cells, and the pH and salt content of the growth and plating media, may somewhat vary the efficiency of infection. Secondary host factors and presence of episomes may further influence these values (Watanabe et al., 1964; Arber and Morse, 1965). made possible an attempt to analyze at the molecular level the mechanisms involved in restriction and modification. As guide lines for this work, biochemical models of enzymology consistent with the observations on biological infectivity of phage were induced and are presented here. It would be useful for the formulation of such models to ascertain how many independent screening devices are employed in determining host specificity. Thus, one could imagine that strain B uses the same screening device toward phage grown on strain K12 as toward phage grown on strain C. Likewise, strain K12 might use the same screening device toward phage grown on strain B as toward phage grown on strain C. Alternatively, strain K12 might have separate anti-B and anti-C activities, and strain B might have separate anti-K12 and anti-C activities. The first situation, but not the second, predicts that physiological and genetic manipulation of the screening functions in strains B and K12 would affect responses

LEDERBERG

380

to other strain specificities en bloc. Experiments examining this prediction are reported.. MATERIALS

AND

METHODS

Bacterial #trains Several bacterial strains of Escherichia co& employed for phage infections and as a source of host restriction and modification mutants, were obtained from S. E. Luria and M. Meselson: B = Bc 251, a X-sensitive derivative (Arber and Lataste-Dorolle, 1961) of Bc (Cohen, 1959) ; C (Bertani and Weigle, 1953) ; C600 (Appleyard, 1954) ; C600 rm4, a mutant of C600 with the host specificity of strain C (Meselson, 1964) ; H = AB259, an Hfr Hayes (X)- derivative (collection of E. A. Adelberg); and W4032 (collection of ,J. Lederberg). Pl-lysogens of these strains were derived with the Plkc variant of Lennox (11955). Streptomycin-resistant or T6resistant mutants were isolated as survivors of exposure of these cultures to streptomycin or phage T6. Nutrition-deficient mutants were isolated after mutagenesis with lmethyl-3-nitro-I-nitrosoguanidine. Bacteriophages Bacteriophages used were X reference type (Kaiser, 1957) and a clear-plaque mutant, X cz6 (Ihler and Meselson, 1963). The host specificity of a X stock is designated with a dot followed by the name of the host strain (Lederberg, 1957). Mediu Liquid cultures were made in tryptone broth (tryptone 10 g/l, NaCl5 g/l). Routine assays were made on tryptone agar (tryptone broth with Difco Bacto-agar or Oxoid No. 3 agar 10 g/l). Tryptone broth with 0.6 g/l agar was used for a top agar layer for phage plating. Methods Pldings. Bacteria grown to about log/ml in tryptone broth, resuspended in 0.01 M Tris buffer, pH 7.3 with 0.01 M MgSO, and starved for about 30 minutes, were used as indicator cells for assays of phage X. Phage was preadsorbed to cells for 15 minutes at

37” prior to plating. Omission of the NaCl from the broth and top agar layer, and reduction of the AIg++ to 0.002 ilil, improved the quality of the plaques of phage X on derivatives of strain B. Heat treatment. Bacteria prepared as for indicator cells were gently agitated in a stainless steel vessel immersed in a water bath. The desired temperature was reached within 3 minutes by direct measurement. Samples were chilled to 37”, infected with phage X at multiplicities of about 0.1, and assayed for successful infective centers on appropriate indicator cells. Broth dilutions were incubated and assayed for free phage after lysis. These methods measure the host cell’s residual restricting and modifying activities, respectively. Correction for unadsorbed phage was made from assays of the supernatant liquid after centrifugation of mixtures of cells and phage. In some experiments, mixtures of genetically marked phage of different host specificities were used to infect the heat-treated cells. This procedure permitted a more direct estimate of any differential effect of heating on different restricting abilities of the host. Mutagenesis. Bacteria grown in tryptone broth to about 3 X 10s/ml were resuspended in $io volume 0.2 M K acetate, pH 5.0. 1-Methyl-3-nitro-l-nitrosoguanidine (Mandell and Greenberg, 1960) was added to 50-100 mg/l and the suspension was maintained at 37” for 30 minutes to 2 hours. The treated cells were washed and diluted 1: 40 into broth, incubated overnight to segregate out muteants, and plated on tryptone agar. Isolated colonies were transferred to broth, incubated, and spotted on tryptone agar plates seeded with about lo6 phage X ~6. Cells which restrict a given Xehost produce from 0 to 3 plaques per spot. Cells whose restriction is impaired produce proportionately more plaques or lysed areas. Cells whose modification ability is impaired, but whose restriction is intact, would be expected to be unable to perpetuate successive cycles of infection by ~.parent host, and hence, should appear to be resistant to lysis. Matings. Matings were carried out according to the method of deHaan and Gross (1962), except that tryptone broth was em-

381 ployed for cultures and dilutions. Rcconbination efficiencies were determined by platings of diluted mixes interrupted after 40 minutes of mating. Selection for Zeu+ or (thr-leu)+ or la& and counterselection with T6 was used in crosses with derivatives of C600 T6R. Selection for am+. thrfx, or lac+ and counterselection with T6 or streptomycin was used in crosses with mutants of strain B T6R strs or R lacking these characters. Abbreviations Used in the Text or Tables ara+, la&: ability to use L-arabinose or lactose, respectively, as carbon source; thr+, Ecu+: ability to synthesize L-threonine or L-leucine, respectively; T6R: resistant to phage T6 ; sW: resistant to streptomycin; 1-112: restriction and/or modification of DNA.

RESULTS

Heat Sensitivity

oj Restriction

The relative in viva thermal stability of the screening functions of these strains was tested by heating cells and infecting them with phage X. Figure IA shows that heated cells of strain B lose their restriction toward phage X. C600 as readily as toward X. C600 ma4 (CSOO~77~4 is a mutant of C600 with the host specificity of strain C). Heated cells of strain C600 behave in a similar fashion toward X .B and X. C600 ~1724with a slightly higher temperature for the midpoint of this transition (Fig. la). These temperatures are interpreted as the denaturation temperatures for specific screening devices against the DNA of phage X. Cells of strain C6OO(Pl) lose the Pl-de-

I. i t

STRAIN

I

45

B

1

I

I

47 49 51 53 PREINFECTION TEMPERATURE,"C

1

55

FIG. 1A frequency of cells that have been heated in buffer for 10 minutes at the temperatures indicated and then infected at 37°C with phage x of various host specificities. (A) Heat treatment of strain B. (B), heat treatment of strain C600. (C) Heat treatment of strain CXOO(P1).

FIG. 1. Yielder

382

LEDERBERG 1

0.1

zl ,z 5 0.01 E Ez cs d F

0.001

47

I 51

49

53

55

b 57

PREINFECTION TEMPERATURE,"c

FIG. 1B

penden.t restriction and the CBOO-controlled restriction somewhat independently (Fig. 1C). In these heat treatments, the host-specific modification activity as measured by the host range of the phage produced is not noticeably impaired. The restriction-deficient state persists for at least 1 hour in heated cells of strain C600 which have then been chilled. Incubation in broth at 37” of heated cells of strain C600 restores the restriction activity in this time. Heat sensitivity of the screening activity against host-specific phage has been reported. for Salmonella phage (Uetake et al.,

1964) and for Pseudomonas phage (Holloway, 1965). The kinetics of restoration of restriction appear similar for E. coli C600 and Salmonella, but differ for Pseudomonas. Host Mutations A$ecting Restriction and Modi$cation Activity Genetic variants completely lacking both the restricting and modifying activities of K12 strains were isolated by Meselson from mutagenized stocks of strain C600 (Meselson, personal communication, 1962). In order to obtain a more comprehensive picture of mutants for these characters, a system was devised to select mutants whose restriction

RESTRICTION

AND

MODIFICATION

383

OF DNA

1

STRAIN C6Ml (PI)

l-

I1 -

01’

I

I

I

I

I

I

45

47

49

51

53

55

PREINFECTIONTEMPERATURE,% FIG. 1C

activity was impaired so that 1 or more out of 500 phage infections would succeed (see Materials and Methods). The host-specific properties of these mutants are listed in Table 2. Some of these mutants also have been isolated from similar strains by a different selection procedure (Wood, 1965). We see in Table 2, first, that the extent of restriction can vary from a barely detectable impairment to complete loss. Pl-lysogenic derivatives of several such mutants were made and found to possess typical Pl-host specificity. Therefore, these mutants are not defective in generalized screening and modifying functions, but rather in host specific activities.

Mutants of C600, AB259, and W4032 were identified by concurrent examination of sensitivity to X.B and to X.CBOO rm4. In the cases thus far examined, restriction activities against phage X.B and against phage X. C are impaired to the same extent by the same mutations. Likewise, in strain B restriction activities toward phage of host specificity C and of host specificity C600 were lost by the same mutations. These observations indicate the the restrictions against host specificities C and B have a common genetic basis in a K12-type strain, and the restrictions against host specificities C and C600 have a common genetic basis in strain B. The fact that impairments occur

384

LEDERBERG TABLE

2

PROPERTIES OF RESTRICTIONMODIFICATION MUTANTS” Parent strain c600 T6R

AB259

W4032 B T6R B T6R leu-

B T6R

slrR

rm Mutant number 1 10 14 15 16 17 20 21 22 23 27 32 34 35 27 38 11 13 19 41 114 119 124 128 130 111 157

Yielder frequency 1 0.02 1 0.2 0.1 0.3 1 0.2 1 1 0.01 0.3 1 0.005 0.05 0.2 1 0.1 1 0.5 0.5 0.4 1 0.05 1 1 0.4

Modification frequency 1 1 0.02 1 0.8 1 0.1 1 0.01 lO.001 1 0.05 lO.001 1 1 1 1 1 lO.001 1 1 1 1 1 1
a Yielder frequencies refer to the fraction of infections by phage X.C-type which succeed in producing phage. Infections by X.B of mutants of K12-type strains and by ~.C600 of mutants of B-type strains gave values similar to infections by X.C-type. A yielder frequency of 1 indicates complete loss of restriction activity. Modification frequencies refer to the proportion of phage liberated from the infected mutant, which possess the host specificity of the bacterial strain which gave rise to that mutant. A modification frequency of lo.001 indicates complete loss of modifying ability. Mutants C600 T6R rm34 and rms3, AB259 rm19, and B T6R s&R rmlll are indistinguishable from strain C in their host specificity toward phage X.

quantitatively together argues against a mutant having multiple lesions at separate restriction loci. About half of the restriction-deficient mutants are impaired to varying degrees in their modification activity. In the same mutagenized cultures, auxotrophic mutants

collectively appear about 20 times more frequently than restriction-deficient mutants. Most of the latter are still prototrophs. Therefore, the observed joint changes in restriction and modification probably do not arise from multiple independent mutations, although some isolates might be of this type. Host Mutations Affecting Restriction of Bacterial DNA The mutants with altered host specificity for restriction and modification of phage handle bacterial DNA in a similar fashion. Thus, Hfr H strains whose phenotype may be termed R+ M+ (restriction, modification) mate with C600 F- strains which are R- M-, R- Mf, or R+ M+, to produce thr+ leuf recombinants with efficiencies of about 4-10 % per input male. Hfr H strains which are R- M- mate equally readily with the first two types of F- strains, but with an efficiency reduced to about 0.3 % with the third. Here, too, the outcome is determined by the match of modification carried by the transferred DNA and the restriction scheme of the recipient host isolated on the basis of phage restriction. Strain B behaves as a poor acceptor in crosses with K12 Hfr strains. Some of the hybrids acquire the ability to mate efficiently with K12 Hfr. The locus for this difference in mating efficiency maps near the locus for synthesis of threonine (Boyer, 1964). The R- M- mutants of B described here mate readily with HfrH strains to produce recombinants for the arabinose region with an efficiency of about 5 %, as compared to about 0.1% for the parent B strain. Measurements of the recombination frequency and the time of marker transfer in K12 Hfr X K12 F- and K12 Hfr X B Fcrosses indicate that the sites for restriction and for modification either compose one unit or are closely linked, and lie between the Hfr Hayes origin and the threonine-arabinose region. The results of matings between restriction mutants and the chromosomal linkage relations of the loci involved will be reported in detail elsewhere. DISCUSSION

Among the cardinal phenomena of host restriction and modification are (1) that one

RESTRICTION

AND MODIFICATION TABLE

385

OF DNA

3

MODELS PROPOSED FOR HOST RESTRICTION AND HOST MODIFICATION”

Model 1

Host

s

1 2 1,3 2,3 3

2, 3

-i K12 B C K12(Pl) BP11

1 2 1, 3 2, 3 3

CW)

Model 2 d

Model 3 d

1, 3 1,2,3 2 1 1, 2

1 2 1, 3 2, 3 3

P 1 2 1,3 2,3 3

Model 4 i

2, 3

1, 3 1, 2, 3 2 1 1, 2

s

i

2, 3 1, 3 1, 2, 3 2

2, 3 1, 3 1, 2, 3 2

1

1

1, 2

1, 2

a The two vertical columns under each model heading refer to specific restricting and modifying activities present in the hosts indicated: p, a protecting (modifying) activity; d, a degrading (restricting) activity; ,s, a degradation-sensitizing (unmodifying) activity; i, a degradation-inhibiting (derestricting) activity. The numbers under these columns refer to a DNA-site specificity possessed by these host acti-vities. The activities in models 1 and 2 and in models 3 and 4 are complemented by nonspecific DNA-complexing agents and by nonspecific nucleases respectively. According to these proposals, free DNA would carry the imprimatur of p or s activities.

strain, E. coIi C, is a “universal acceptor” for phage DNA from two other strains, E. coli B and E. coli K12; (2) that strains B and K12 produce phage incompatible with each other; and (3) that phage DNA made in the universal

acceptor

strain

is itself

in-

compatible with strains I3 and 1112. The present observations on coordinated thermal sensitivity of restrictions and on the joint loss of restrictions by mutation support models for host specificity in which a single screening device is used against two other strains. Four such models are outlined in Table 3. The first model, whose grid format was suggested by Meselson, depicts the host, cell as having degrading enzymes acting only at specific sites in phage DNA when these sites are un.protected. Different bacterial strains

may

have

different

arrays

of such

specific enzymes. As a counterpart to this activity, the cell is thought to have specific protecting enzymes which can modify otherwise sensitive sites to make these sites resistant to degradation. A DNA molecule entering such a cell would be screened for its host specificity by the particular battery

of degrading enzymes present. If these enzymes see their substrate protected, then the DNA molecule can go on to function as the situation warrants. If not, the fate of the DNA molecule is determined by whether it will be destroyed before any protecting enzymes can become effective. The frequency

of escape in such a situation would depend on the relative location and concentration in the cell of these opposing activities. One would imagine that a membrane location for the degrading activity would be most effective, and indeed this is consistent with the rapidity of the onset of degradation, about 0.5 minute from the time of addition of phage to a suspension of restricting cells (Lederberg and Meselson, 1964). Model 2 supposes the protection is created in an inverse manner-sites would be normally resistant, but are sensitized or exposed in a previous host. This is of relevance to an assay system one might employ to isolate the protecting enzyme activities. However, the screening program is essentially the same: a DNA molecule is first screened for specificity by particular degrading enzymes--only if the DNA molecule survives screening, does it, go on to function. Model 3 supposes a different behavior for a DNA molecule new to the cell. Here, the first stage of screening lies in different host structures which can detect and protect specific sites in DNA. This protection is temporary and does not accompany the DNA either through mating transfer or phage maturation or chemical extraction. Possible candidates for this activity are those cellular agents found associated or complexed with DNA: RNA, ribosomes, polycations, and enzymes acting on or with DNA. In addition, the cell protects other

386

LEDERBEN:

sites IINI~(: pxnmmltly in a way which can survive mating, maturation, :mti phenol cxtraction. If the combination of firmly bound and temporary devices protects all types of degradat,ion-sensitive sites present, then t’he DNA mill escape the degrading activities present. In this situation, the requisite mclease activity need not be host specific. The essent#ial difference of model 3 from models 1 and 2 is that in this model the DNA asks whether the host has the correct nuclense-blocking apparatus. Operationally, this difference means that reconstruction of this system in vitro calls for a three-way combination: host-specific DNA, a hostspecific DNA-complexing agent, and a deoxyribonuclease. Model 4 postulates sensit’ization rather than protection and otherwise is like model 3. Inasmuch as transduction, episomal transfer, and bacterial chromosome transfer exhibit similar host specificities, it is possible that the models apply to all DNA which enters or is formed within bacterial cells. If we turn to the frequent concomitant loss of restricting and modifying abilities by a single mutation, several explanations may be suggested. First, a common structure might be needed for these activities and the mutation would affect this entity. Here, the specificity of the DNA would choose the activity to be exercised. Second, another locus might suppress differentially these two activities. Finally, the genes responsible for restriction and modification might interact for their expression as part of an operon (Jacob et al., 1960). Although the heat sensitivity of the screening activity against host-specific phage may be ascribed to an unusually labile enzyme, another interpretation might be that a highly organized complex is involved. A complex which denatures readily upon heating might well also have its tertiary structure relatively sensitive to coding errors. This suggestion takes into account the earlier observation that streptomycin-resistant mutants are frequently impaired in their screening activities (Lederberg, 1957) since such mutants have an increased ambiguity of coding (Gorini and Kataja, 1964). Alternatively, these various observations on temperature sensitivity, rapidity of degradation,

and streptonlyc.iil suppression nl:~y bra :l(‘commodated by viewing ribosomes as part of the site of restriction. BCKNOWLEDGMENT The author is indebted to Dr. M. Meselson for proposing the grid format of model 1; this format was parent to the structure of the other models developed here. He also wishes to thank Drs. S. E. Luria and M. Meselson for the strains of bacteria and phages employed in this work, and Mrs. M. Stenberg and J. Singer for their t.echnical assist,ante. This work was supported by grant GB-1945 of the National Science Foundation. REFERENCES APPLEYARD, R. K. (1954). Segregation of new lysogenie types during growth of a doubly lysogenic strain derived from Escherichia co& Kl2. Genetics 39, 440-452. ARBER, W., and Dussorx, D. (1962). Host specificity of DNA produced by Escherichia co&i. I. Host controlled modification of bacteriophage X. J. Mol. Biol. 5, 18-36. ARBER, W., and LATASTE-DOROLLE, C. (1961). Erweiterung des Wirtsbereiches des Bakteriophagen X auf Escherichia cdi B. Pathol. Microbiol. 24, 1010-1018. ARBER, W., and MORSE, M. L. (1965). Host specificity of DNA produced by Escherichia coli. VI. Effects on bacterial conjugation. Genetics 51, 137-148. ARBER, W., HATTMAN, S., and Dussorx, D. (1963). On the host-controlled modification of bacteriophage X. Virology 21, 30-35. BERTANI, G., and WEIGLE, J. (1953). Host controlled variation in bacterial viruses. J. Bacteriol. 65, 113-121. BOYER, H. (1964). Genetic control of restriction and modification in Escherichia coli. J. Bacterio2. 88, 1652-1660. COHEN, D. (1959). A variant of phage P2 originating in Escherichia coli, strain B. Virology 7, 112-126. DEBAAN, P. G., and GROSS, J. D. (1962). Transfer delay and chromosome withdrawal during conjugation in Escherichia coli. Genet. Res. 3, 251272. Dusso~x, D., and ARBER, W. (1962). Host specificity of DNA produced by Escherichia coli. II. Control over acceptance of DNA from infecting phage X. J. Mol. Biol. 5, 3749. DUSSOIX, D., and ARBER, W. (1965). Host specificity of DNA produced by Escherichia coli. IV. Host specificity of infectious DNA from bacteriophage lambda. J. Mol. Biol. 11, 238-246.

RESTRICTION

AND

MODIFICATION

GORINI, L., and K~TAJ.~, E. (1964). Phenotypic repair by st’reptomycin of defective genotypes in E. coli. Proc. Natl. Acad. Sci. U.S. 51, 487493. HOLLOWAY, B. W. (1965). Variations in restriction and modificatia’n of bacteriophage following increase of growth temperature of Pseudomonas aerugincsa. Virology 25, 634-642. IHLER, G., and MESELSON, M. (1963). Genetic recombination in bacteriophage X by breakage and joining of DNA molecules. Virology 21, 7-10. JACOB, F., PERRIN, D., SANCHEZ, C., and MONOD, J. (1960). L’opB ron : groupe de genes % expression coordonbe par un operateur. Compte. Rend. Acad. Sci. 250, 1727-1729. KAISER, A. D. (1957). Mutations in a temperate bacteriophage affecting its ability to lysogenize Escherichia coli. Virology 3, 42-61. KAISER,A. D., ~~~HOGNESS, D. (1960). The transformation of E.scherichia co& with deoxyribonucleic acid isolated from bacteriophage Xdg. J. Mol. Ba’ol. 2, 392-415. LEDERBERG, S. (1957). Suppression of the multiplication of heterologous bacteriophages in lysogenie bacteria. Virology 3, 496-513. LEDERBERG, S., and MESELSON, M. (1964). Degradation of non-replicating bacteriophage DNA in non-accepting cells. J. Mol. Biol. 8, 623628.

OF DNA

387

LENNOX, E. S. (1955). Transduction of linked genetic characters of the host by bacteriophage Pl. Virology 1, 190-206. LURIA, S. E., and HUMAN, M. L. (1952). A nonhereditary, host-induced variation of bacterial viruses. J. Bacterial. 64,557-569. MANDELL, J. D., and GREENBERG, J. (1960). A new chemical mutagen for bacteria, l-methyl3-nitro-1-nitrosoguanidine. Biochem. Bkphys. Res. Commun. 3, 575577. MESELSON, M. (1964). On the mechanism of genetic recombination between DNA molecules. J. Mol. Biol. 9, 734-745. UETAKE, H., TOYAMA, S., and HAGIWARA, S. (1964). On the mechanism of host-induced modification. Multiplicity activation and thermolabile factor responsible for phage growth restriction. Virology 22, 202-213. WATANABE, T., NISHIDA, H., OGATA, C., ARAI, T., and SATO, S. (1964). Episome-mediated transfer of drug resistance in Enterobacteriaceae. VII. Two types of naturally occurring R factors. J. Bacterial. 88, 716-726. WOOD, W. B. (1965). Mutations in E. coli affecting the host-controlled modification of bacteriophage A. Pathol. Microbial. 28, 73-76.