J. Mol. Biol. (1968) 34, 261-271

Recombination in Bacteriophage h I. Mutants Deficient in General Recombination

E. R. SIGNER AND JON WEnt

Department of Biology, Massachusetts Institute of Technology Cambridge, Massachusetts, U.S.A.

and

Biological Laboratories, Harvard University Cambridge, Massachusetts, U.S.A.

(Received 13 December 1967)

Two types of recombination-deficient (red-) A mutants have been studied: point mutants isolated after hydroxylamine mutagenesis, and certain deletion mutants isolated as plaque-forming biotin-transducing strains (A&I). In a recombination- deficient (ret-) host, crosses of the hbio deletions, or the lea& leaky point mutant, give recombinants at less than l/100 the frequency of the wild-type. In a ret+ host, recombination is restored to l/10 to l/2 the wild-type frequency, depending on the particular mutant. The product of at least one red gene is diffusible. All the mutant sites lie on the X map between the int and ~111 loci. These results demonstrate that X produces at least one component of the system responsible for general vegetative phage recombination, and that the bacterial recombination system can replace this phage function.

1. Introduction The genetic recombination that occurs during vegetative growth of phage h could, in principle, be promoted either by the recombination system of the bacterial host or by a system synthesized under the direction of the infecting phage. Since h recombines normally in a recombination-deficient bacterial host (Van de Putte, Zwenk & RBrsch, 1966; Takano, 1966; Brooks BE Clark, 1967), the phage may direct the synthesis of at least one component of the recombination apparatus. This and other results have prompted the search for phage mutants analogous to the bacterial ret-$ mutants, and this paper describes the isolation and some of the properties of such mutants. These mutants are called red- (recombination-deficient) in order to avoid confusion with the bacterial ret- mutants.

Both point and deletion red- mutants have been studied. In a ret- host each mu- tant has a characteristic reduction in recombination frequency, ranging from fivefold

t Present address : Department of Molecular Biology, Vanderbilt University, Nashville, Term., U.S.A.

$ Abbreviations used: Phage markers : red, recombination-deficiency; int, integration-deficiency; imm, immunity; c, clear plaque; h, host range; ws, (amber) suppressor-sensitivity; bio, biotin- t,ransducing. Bacterial markers : TIC, recombination-deficiency; gal, Zac, mal, utilization of galactose, lactose, maltose; B,, Zeu, thr, thy, try, requirement for thiamine, leuoine, threonine, thymine, tryptophan; Sm, Spc, sensitivity (“) or reeistance (a) to streptomycin, spectinomycin; 8un, amber suppressor II. HA, hydroxylamine. Italics are used for genetic loci (e.g. red+ and red- alleles at the red locus) and Roman type for functional products (e.g. the Red system).

261

262 E. R. SIGNER AND J. WEIL

to greater than IOO-fold. In a ret + host the recombination frequency is significantly higher. Therefore, both the phage and its host can contribute to vegetative h recom- bination, and there are two pathways, at least partially independent, by which recombination can occur. The mutant sites are located in the int-cIII segment of the h map.

The accompanying paper by Echols & Gingery (1968) describes similar point mu- tants. Franklin (1967) has described a recombination-deficient deletion mutant of

the related hybrid phage P”hta.

2. Materials and Methods

(a) Bacterial strains

(i) Strains for vegetative recombimtion studies

A nearly isogenic pair of permissive ret- and ret + strains was constructed in the follow- ing way. Strain V2a, which is thy-su,, +I&-, was constructed by Dr S. Brenner by crossing out the thr- and Zeu- markers of strain CR34 in a cross with Hfr Hayes. V2a was mated with a mal- mutant of the P4X-type Hfr strain AR3 (Signer & Beckwith, 1966), which carries the Tee- mutation described by Fuerst & Siminovitch (1965); maZ+ thy+ recom- binants were selected and scored for ret by u.v.-sensitivity and for su by spotting with Asus. A ret-su+ recombinant (QR46) was chosen and, a Tee+ derivative (QR47) wa,s constructed by Pl-transduction from Hfr Hayes. Since QR46 grows more slowly than QR47, a faster-growing ret- derivative (QR48), selected by streaking QR46 on h agar, was used in the later experiments. h recombination frequencies were identical in the two ret- strains wherever tested.

(ii) Cryptic lysogens

From a cryptic lysogen (Fischer-Fantuzzi & Calef, 1964), gal-try-su+SmR (XcrysusBlh), obtained from Dr L. Fischer-Fantuzzi, ret+ and ret- derivatives were constructed in t,hc following way. The P4X-type Hfr AR5 (ret-SpcRSmS) was first constructed by mating Hfr AR3 (ret-SpcSSmR) with MR31 (Zac-SpcRSmS, Signer AZ Beckwith, 1966) and selecting Zac+Sp? recombinants. AR5 was then mated with the cryptic lysogen with selection for try+SmR to give the ret+ (QR14) and ret- (QR15) cryptic lysogens used here.

(iii) Indicator strains

Permissive indicators were strain C600 and a BI + derivative (Ql) isolated by Dr 8. Brenner, which were used interchangeably. Non-permissive indicator was strain 594 (Campbell, 1965). Indicator for Ah was strain CR63 (Appleyard, McGregor & Baird, 1956). Ql and 594 were also used as hosts for mapping crosses.

x+.

(b) Bacteriophage strains

xh without modifier, from Dr A. D. Kaiser. hs (Kaiser, 1955), obtained from Dr A. D. Kaiser as himm434s. hclll67 (Kaiser, 1957). hsus mutants E”96b, JF and Rri (Campbell, 1961). Ad857 (Sussman & Jacob, 1962). XcI857h susA32susBl from Dr W. F. Dove. XcI857int6 (Gottesman & Yarmolinsky, 1968) from Dr M. Parmolinsky. M857h int29c from Dr J. Zissler ; the int- mutation is amber. Xbio non-defective transducing phages isolated by Manly, Signer & Radding (manuscript

in preparation). Unless otherwise indicated, phage strains are from the collection of Dr S. Urenner. Ot’her appropriate strains were constructed from these by recombination. A map of A is shown in Fig. 1.

RECOMBIXATION-DEFICIENT A MUTANTS “6,3

susA SUSB

h sus F SUSJ int clu cl sus R

/ \

/ / \

/ \ /

/ int exo /j cm ',

bio I,11 i 1

bio 69,?2 r 1

bio 16,64 =x511

Fla. 1. Y&p of pertinent X markers, only roughly to scale. The extent of hbio deletions is from Manly et al., (manuscript in preparation); ezo and p refer to loci determining exonuclease and p-protein.

(c) Media

h broth contains tryptone (1.0%) and NaCl (0.250/,); top and bottom agar contain in addition 0.65 and 1.2% agar, respectively. In some experiments T broth, identical except in having twice as much NaCl, was used. For slow-growing strains yeast extract (0.005!/,) u as sometimes added.

SM is described by Weigle, Meselson 8; Paigen (1959).

(d) Methods

Spot test for marker rescue. The test determines the ability of a phage carrying the h+ allele to rescue the h allele from a cryptic lysogen (hcryh). The phage to be tested were spott)ed onto a plate containing the cryptic lysogen plus strain CR63, which is resistant t,o Ah, + but not to xh. If the phage can rescue, the developing spot contains some Ah phage, so t’hat bot,h bacterial strains are lysed and the spot is relatively strong. If the phage cannot rescue. the spot, is faint due to overgrowth of CR63.

The bacterial plating mixtures, made from overnight cult,uros grown in brot’h plus 0.2:; maltJose, were QR14 plus CR63 (1: 2) or QRl5 plus CR63 (l:l), of which 0.1 ml. \vas used per plate. Phage were eit)her stabbed from plaques with sterile toothpicks (this method was used in isolating t*he mutants), spotted from plaques picked with a capillary tube or Pastetlr pipet into 1 ml. of SM, or spotted from stocks diluted to lo6 to 107/ml. For the mutant isolation we rmxl the ret + cryptic lysogen QR14. However, red - mutants show a more complete rescue-deficiency (a fainter spot) when tested on the ret- cryptic lysogen QR15. The test syst,em of Echols & Gingery (1968), which involves rescue by a SNS- phage of a sz[s + marker from a non-permissive cryptic lpsogen, gave results oompar- able to our system.

Standurd crass. Ovc~rnight cult~lres grown in broth plus 0.2% maltose and 0.01 M-MgSO, were diluted 1 : 100 in t,he same medium, grown with aeration at 37°C to 2 x lO*/ml.. centrifuged anal resuspended in 0.02 nf-MgSO, at) bhe same concentrat,ion. Phage were tliluted in SM f o gi\-e a multiplicity of infection of 5 for each phage. The adsorption tnixture, consisting of equal volumes of phage and cells, was incubat,ed wit,h gentle agita- tion at 37-c’ for 20 lnin (at least 990!, adsorption). The mixt[tre was then diluted serially 1 : 40 and 1 : 50 in warlned broth plus 0.001 M-MgSO,; the I : 50 dilution was aerated at 37°C ‘ for 90 luin and t,hen chloroformed. Average burst) sizes by this method often reached several hunclred. For II.\.. crosses t,he 1 : 40 tlilution was made in 0.01 &I-MgSO,and irradiated for 30 set at 54 in. I’I*OII~ a (icneral Electric G15T8 germicidal lamp, and subsequent steps Lvere then carried orlt as above. In order to minimize the formation of recombinants on the select,ivr plat,es, no more t,han 5 Y 104 phaga were seeded per plate when SW+ recom- 1,inants were to br select,etl. Recombinatjion frequencies for the same cross on different qlavs varied -\vithin a factor of about, 2. However, when sets of crosses were repeated on tlifferent days, all crosses of the set tended to vary together.

Hydroxylamine mutagenesis. The procedure used was that of Hall & Tessman (1966) lnodified to include MgSO, (0.01 M) in all solutions. After 7.5 hr treatment at 37”C, 300/;,

lir

261 E. R. SIGNER AND J. WEIL

of the control phage (no HA) and 9% of the mutagenized phage survived. The quenched suspension was plated and individual plaques were picked and tested for marker rescue (see above). The mutagenized stock contained 0.4% clear-plaque mutants, and three red- mutants were isolated from approximately 2500 plaques tested.

Test for int phenotype was that described by Gottesman & Yarmolinsky (1968).

3. Results

(a) Origin of red - mutants

Recombination-deficient mutants are likely to be recessive. To detect t,hem one requires a test system in which only one of the genomes participating in recombina- tion can contribute functionally to the recombination process. The basis for such a test system is the cryptic bacterial lysogen (Fischer-Fantuzzi & Calef, 1964), in which a large portion of the prophage including the immunity region has been delet.ed. When h infects such a strain it can grow vegetatively because the cryptic lysogen is not immune. Recombination between the infecting phage and the prophage can bc detected by the appearance of prophage markers among the progeny phage (marker rescue). If the prophage deletion includes h genes involved in recombination, the appropriate test conditions are obtained.

Using a simple spot test based on this principle (see Materials and Methods), we isolated three recombination-deficient mutants (red.2, red3 and red5) from a phage stock mutagenized with hydroxylamine. We assume they represent point mutations. The mutant s isolated by Kaiser (1955) is also recombination-deficient and has been renamed real. Mutants real, red3 and red5 form smaller plaques than wild type and have reduced burst sizes.

We also tested six plaque-forming biotin-transducing h strains (X&o) for recombina- tion deficiency. In this type of phage, originally described by Wollman (1963), a segment of phage DNA containing no genes essential for vegetative growt’h is re- placed by bacterial DNA that includes the genes for biotin synthesis. The Xbio strains used here were isolated and characterized by Manly, Signer & Radding (manuscript in preparation), and the map extent of their deletions is shown in Figure 1.

(b) Deficiency in vegetative recombination

The effect of the red- mutants on vegetative recombination was determined in crosses between a susA- susB- and susB- derivative of each mutant. The results are presented in Table 1.

When the crosses were made in a ret- host, each point mutant had a characteristi- cally reduced recombination frequency, ranging from about five- to over 100-fold. Mutant red5 was unusual in that reductions ranging from zero to 20-fold were ob- served in different experiments. Among the deletion mutants, biol, bioll, bio69 and bio72 had a greater than loo-fold reduction in recombination frequency, whereas recombination was normal for biol6 and bio64.

In a ret+ host the mutants also had a reduced recombination frequency, but, with the apparent exception of red2, the reduct’ion was much less than that in the ret- host, Therefore, the host recombination system can participate in X recombina- t’ion, although not as efficiently a,s the phage system.

Similar results for t’he interval sus%susJ, in a ret- host, are shown in Table 2 for red3 and red5. Qualit’at,ively similar results for the interval h-cl were obbained with the Xbio deletions.

RECOMBINATION-DEFICIENT /\ MUTANTS

TABLE 1

susA susB x susF

265

Expt : Host :

Per cent recombination 1 2 3 4

TCC+ ret - ret+ rec- ret + ret - ret + ret -

red + red1 red2 red3 red5 biol bioll biold bio64 bio69 bio72

1.2 0.9 1.0 0.9 0.7 0.6 0.9 0.7 0.5 0.03

0.1 0.1 0.3 <O.Ol

0.2 0.4 _<O.Ol 0.4 10.005 1.3 1.0

0.9 0.7 0.2 10.007

0.2 (0.01

Typical results for the cross su.sA- susB- x SUSP-, expressed as frequency of sus+ recombinants x 2 x 100. Except for the red1 crosses, and the biol and bio69 crosses in the Tee+ host, each cross has been repeated at least once, and values for red + in this Table are typical of the variation encountered. All the bio’s, and some of the red’s, also carried the markers h, and cI857.

In this and subsequent Tables _< indicates that there appeared on the selection plates a few plaques from which the recombination frequency shown in the Table was calculated. These may be recombinants formed in the cross, or they may have arisen subsequently on the plates. < indicates that no recombinant plaques occurred.

TABLE 2

SUSF x SUSJ

Host :

Per cent recombination

reo -

red + 2.2 red3 <0.006 red5 0.3

Frequencies expressed as in Table 1; all SUSP- phages also carried hl, and cI857.

In our experiments the red + control crosses have consistently given slightly lower recombination values on the ret- host (whether QR46 or QR48, see Materials and Methods) than on the ret+ host.

(c) Reversion of red3 and red5

Two of the presumed point mutants, red3 and reds, were shown in the following way to revert to red + . The temperature-sensitive mutant AcI857 forms clear plaques at 36~5°C and above. However, M857red3 and hcI857red5 have a slightly shifted temperature threshold and at 36.5% form plaques that are distinctly turbid, although less so than that of hcI + . When either of these double mutants is plated at 36.572, clear plaques can arise in two ways: from phage that have sustained a second tem- perature-insensitive cl mutation and from phage that have reverted to red + . A stock of hcI857red5 was streaked at 36*5”C! to give confluent lysis, and phage from nine

266 E. R. SIGNER AND J. M’EIL

clear areas were isolated and further tested. Five of t,he isolates proved to 1~ IW~ f revertants. A revertant, of red3 was isolated in a similar WLV. These findings WIT consistent with the assumpCon that red3 and red5 are point, mutantIs.

Among the hbio strains only bioI6, bio64, bio69 and bio72 raise t8hc tcrnperat~urc~ threshold of ~1857. Thus the effect is not completely correlated with rrd- phenotype.

TABLE 3

Deletion mapping of red mutants

Per cant recomhinatioll

red bio: 11

Abio and hred, each at multiplicity of infection of approximately 6, were croasctl in ()I with u.v. bio69 crosses were plated non-selectively, and plaques were picked and scored for WC! : 1~1 cent recombination is twice the frequency of red + x 100. bioll crosses were plated on ~ec- to ctlimi- nate the bio parent (Manly et al., manuscript in preparation), and plaques were picked and scorctl for red; per cent recombination is frequency of red + x 100. Data for bio69 x red2 ant1 ~1.3 are based on 750 and 500 plaques, respectively, all others on approximately 1000 plaqurs.

TABLE 4

int6 ~11167 x red3

Genotype int red e Kumber Cross-over class

Parental : + -;’

525 539

Rerombinant: + - - -If

t + - 19 7 -. . . + 31 22

-t -i- + -1 0 0 o

1092

Order deduced: &t-red-cIII Interval Per cent recombination

M-red 22 - : 1092

2.0

red-c 6 - IT 1092

0.55

int-c 28 - zzx 1092

2.6

single

single

double

Map distance

0.075

0.015

0.1

Input multiplicities were 5.3 for red- and 3.3 for int-c- ; host was 594. Interferemae illtLex from these data 1<9. Map distances were obtained from the mapping function of Amati & Meselson (1965) and are expressed in Morgans. The same cross repeated on Ql gave recombinant frequencies which were approximately twice as high but in the same ratio as those on 594.

RECOMBINATION-DEFICIENT X MUTANTS

(d) Location of the mutant sites

267

We mapped the red- point mutants by crossing them with two red- hbio deletions in a ret+ host (Table 3). The red1 site lies within or very close to the region deleted in bio69. The remaining three point mutant sites lie outside the bio69 deletion but within or very close to the bioll deletion. These results, together with the positions and properties of the hbio deletions (Manly et al., manuscript in preparation; see Fig. 1) and the data of Tables 4 and 5, establish that the order of the genes in the center of the /\ map is h-b2--in&-red-cIII.

TABLE 5

~1857 susR5 x red5

Genotype red c Number Cross-over class sus

Parental : - + + 170 + - - 326

Recombinant: - + - 15 f-f 10 25 single

- - - 9 +++ 2 1 11 single

- + -

++- 1 double

535

Order deduced : red-cl-suaR

Interval Per cent recombination

red-c 14 535

= 2.6

c-sus 28 535 = 5.2

red-sus 36 535 = 6.7

Input multiplicity was approximately 5 for each phage; host W&S &I. Interference index from these data I = 4.

The approximate distances among these markers were measured in the cross Ah ~$857 x hint ~11167. Here one must take into account the site-specific recombina- tion promoted by the prophage integration system (Int), which normally distorts the map between h and int (see Weil & Signer, 1968; Echols, Gingery & Moore, 1968). To eliminate this the amber mutation int29c was put into the h-cl- parent. This cross was done in a non-permissive host (594) where both parents are phenotypically int-. The progeny phage were scored for int on a permissive host (CSOO), where only those phage with the int6 allele were scored as int-. From the results of this cross (Table 6), map distances were calculated with the mapping function of Amati &

268 E. R. SIGNER AND S. WEIL

Meselson (1965). Although there were relatively few recombinants, additivity is reasonably good.

Our results are summarized in the map presented in Figure 2.

TABLE 6

h, ~1857 x int6 ~I1167

Genotype h int CIII cz

Number Cross-over class

Parental: - + + - + - - +

Recombinant: + - - - - i- + +

+ - + - - + - +

+ + + - - - - +

- t - -I- - + +

- -

+ + + +

- - + - + + - +

+ + - - - - + +

Order deduced: h-int-cIII-cI

Interval Per cent recombination

h-int 43 - zz 1126

3.8

int-cIII 24 - = 1126

2.1

CIII-CI 14 1126 = 1.2

h-CIII 47 - = 1126

4.2

int-cI 30 - = 1126

2.7

h-cZ 53 -= 1126

4.7

597 462

single

single

single

double

double

double

triple

Map distances

0.17

0.08 0.29

0.04

0.2

0.11

0.23

I nput multiplicities were 4.3 for h-cI- and 5.0 for int-cIII- ; host was 594. Map distances as in Table 4. The h-cl- parent also carried the amber mutation int29c (see text). Interference indices from these data I = 11 (h-clll) and I = 14 (int-cl).

RECOMBINATION-DEFICIENT I MUTANTS 269

h int6 red3 ~11167 ~1857 -- I I I I I --

red1 red5 red2

bio I,17 CZ

bio 69,72 c:-

bio 16,64 r:z: 11

FIG. 2. Genetic map of the center of h. The relative positions of int and ~111 are derived from Table 6, and that of red3 from Table 4 by adjusting the &t-c111 distance of Table 4 to equal that of Table 6. The positions of the bio deletions are from Manly et al. (manuscript in preparation), and redl, red2 and red5 have been located in relation to the deletions according to Table 3.

(e) Diffusibility of the Red product In order to determine whether the red gene produces a diffusible product, we tested

the ability of a red + phage to promote recombination between two red- deletion phages, biollsusA-susB- and biolIsusF-, in a triparental cross (Table 7). In cross A, the helper phage (red+susA-susB-swF-) could not contribute sus+ markers to the selected sus’ recombinants, and thus could promote recombination only if it produced a diffusible substance. Cross B was a negative control in which the helper was omitted. Cross C was a positive control in which the helper (red+susA+susB+susF-) could contribute SW+ markers as well as Red function.

TABLE 7

T&parental crosses

Parental phage : bioll swA32 susB1 plus

bioll susF96b plus

helper

Cross Helper Per cent recombination A (trans helper) susA -SUB-susB- 0.2 B (no helper) none < 0.062 C (cis helper) SUSF - 0.4

Multiplicity was approximately 5 for each phege; host was QR48. Both bioll phages also carried the markers h, and cI857. Recombination in cross A was calculated as frequency of aas+ among bio parents (i.e. titer on strain 594 x 2 x 100 divided by titer of clear plaques on C600). bioll is int- (Manly et al., menuscript in preparation) and the red+ helpers were also made int- to elimi- nate the possibility of non-specific Int-promoted recombination (subsequently shown to be neg- ligible, Weil & Signer, 1968).

The recombination frequency with the red+susA-susB-susF- helper (cross A), was more than 100 times greater than that in the negative control (cross B), and 50% of that in the positive control (cross C). Since sequential recombination of the helper phage with each of the bioll parents is expected to occur at a much lower frequency (Kaiser, 1955), the results indicate that the red + phage produces a diffusible substance that is not produced in active form by the red - phage. The somewhat lower recombina- tion frequency observed in cross A compared to cross C might result from limited diffusion of the Red product within the infected cell.

270 E. R. SIGNER AND J. WEIL

4. Discussion

Our results demonstrate that X produces one or more diffusible products involved in vegetative h recombination. They also show that the bacterial recombination sys- tem can function in h recombination. Similar conclusions have been reached by Echols $ Gingery (1968) and Franklin (1967). It will be useful to deline two recombination systems: the Red system, which is at least partially phage-directed and promotes recombination of red + phage in our rec- host; and the Ret system, which promotes recombination of red- phage in the ret+ but not the ret- host.

A detailed comparison of the two systems will have to await further information. We should like especially to know whether Red is entirely phage-directed and there- fore completely distinct from Ret, or a complex of phage and bacterial enzymes. By itself Red promotes almost as much h recombination as that observed in the normal (red+rec+ ) situation, whereas Ret alone is less efficient. The relative contributions of the two systems in the normal situation remain to be determined.?

It would also be interesting to know how many red genes there are in h. We plan to investigate complementation of the red- mutants, both by one another and by different ret- hosts, and to compare our red- mutants with those isolated by Echols & Gingery (1968). We should point out that our selection of red- point mutants may have been biased toward a particular class. The mutants were isolated with a ret+ cryptic lysogen, so that what was scored was inefficient recombination by the Ret system rather than a deficiency in the Red system per se. A mutant like the one de- scribed by Franklin (1967), which is recombination-deficient in a ret- host but normal in ret+, would not have been detected. We are isolating additional mutants using a ret - cryptic lysogen.

The accompanying papers (Weil & Signer, 1968; Echols, Gingery & Moore, 1968) show that a third recombination system, the prophage integration system (Int),

can promote vegetative recombination, but only in the region of the phage attachment site. When the Red, Ret and Int systems are all inactivated by mutation, recombina- tion is reduced more than loo-fold over the entire map. Thus these are the only systems that promote vegetative h recombination.

The Red system is not essential for phage growth, since red-Abio deletion mutants are viable. Furthermore, since hbio69 and hbio’72, which are int - as well as red -, grow about as well in the ret- as in the ret+ host (Manly et al., manuscript in preparation), there seems to be no requirement for any form of recombination. There may, of course, be steps in the recombination process that are also essential for vegetative growth.

We have some indication of the biochemical basis of the red- phenotype. All the red mutants map within the small interval between the int and cIII loci. This region contains the genes for the X exonuclease and t,he ,&protein (Radding, Szpirer & Thomas, 1967; Manly et al., manuscript in preparation), whose functions are unknown.

t Two lines of evidence suggest that the Red system may also promote bacterial recombination. First, Signer & Beckwith (1966) observed that phage infection of a ret- host resulted in a non- specific increase (i.e. independent of prophage attachment sites) in the frequency of chromosome mobilization by an F/-factor. Second, in a ret- host that is partially diploid and heterogenetic for two non-complementing Zac- mutants, there is a slight but significant increase in the propor- tion of Zac+ cells among the survivors of X infection (Signer, unpublished experiments). In this case the only cells scored are those in which h has entered the lysogenic pathway, and the fact, that the stimulation of recombination is only slight would be explained if Red functions poorly in this situation, as suggested by Gottesman & Yarmolinsky (1968; see also Manly ef nl., manu- script in preparation).

RECOMBINATION-DEFICIENT h MUTANTS 27 1

Smong the six hbio strains discussed here there is a perfect correlation between red - phenotype and absence of exonuclease activity (Manly et al., manuscript in prepara- tion), and Franklin (1967) has observed a similar correlation for deletions in the related phage a . ‘p80h +’ Crude extracts from induced lysogens of red2, red3 and red5 have less exonuclease activity than ext’racts from red + lysogens. and that of red3 seems also t!o have no p-protein (C. Radding, personal communication). Furt’hermore, we have recently isolated three red- mutant’s that are t’emperature-sensitive bot’h for re- combination in aivo and for h exonuclease activity in crude extracts (M. Shulman, personal communication). These results strongly suggest that the X exonuclease is involved in general vegetative recombination. h may produce other prot,eins involverl in recombination, and the ,&protein, which is controlled co-ordinabely with X exe- nuclease (Radding & Schreffler, 1965), is an obvious possibility.

We thank K. Manly, R. D’Ari, S. Heinemann, M. Meselson, 8. Luria and D. Botst,ein for useful and st’imulating discussions; G. Mosig and D. Kaiser for critical advice during preparation of the manuscript; Miss Judith Sparer, Miss Mary Bradford and Mr Ralph Rosenberg for excellent technical assistance; and H. Echols, R. Gingery, N. Franklin, M. Gottesman, M. Yarmolinsky, K. Manly, C. Radding and M. Shulman for making their results available to us and allowing us to quote their findings before publication. ‘I’hi,s work was supported-by National Institutes of Health grant GM13426 to one of us (E. R. S.) who is a Cancer Development Awardee of the N.I.H. The ot’her author (J. W.) was a Sational Institutes of Health postdoctoral fellow (grant 5F2-GM-28-775) in the laborat,ory of M. Meselson, who supported this work in part from his grant from the National Science Foundat)ion (GB5305).

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