T1 mutants with increased transduction frequency are defective in host chromosome degradation

VIROLOGY

112,670-6’77 (1981)

Tl Mutants with Increased Transduction Frequency Host Chromosome Degradation

Are Defective

in

MARY DENTON ROBERTS’ AND HENRY DREXLER2 Department of Microbiology and Immunology, The Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, Nwth Carolina 27109 Accepted March 5, 1981 Mutants of Tl with enhanced transduction ability (tar mutants) were characterized by experiments designed to identify the cause for enhancement. Several bacterial markers and Mu PFU located in separate operons were transduced at different efficiencies. Infectious particles were produced in relatively normal amounts. Sensitivity to hydroxyurea (which inhibits de luluo synthesis of nucleotides by the host) by a majority of the mutants suggested that they were defective in degradation of the host chromosome. One of the mutants was unable to incorporate host cell nucleotides, confirming its ability to degrade the host chromosome. Complementation experiments suggested that the tar mutations define a new Tl gene. INTRODUCTION

In a previous report, we described a method to detect mutants of phage Tl that transduce at increased efficiencies (Roberts and Drexler, 1981). Using this method, we isolated seven mutants which transduce a X prophage 50 to 30,000 times more efficiently than wild type. The tar mutations (tar because they transduce at altered rates) were found to be closely linked, nonidentical mutations located on a region of the map believed to be involved in the replication and maturation of Tl DNA (Figurski and Christensen, 1974). An increase in the production of transducing particles by Tl might be achieved in three ways: (i) An alteration in the ability of the phage to recognize and initiate headfilling at specific sites on DNA would result in increased encapsulation of host DNA relative to phage DNA (Schmieger, 1972). (ii) A decrease in the amount of phage DNA available during maturation, caused either by a defect in phage DNA synthesis or by premature lysis of infected cells, would result in a higher ratio of

6642-6822/81/1666’70-06$62.66/O 8 1981 by Academic Press, Inc. of reproduction in any form reserved.

MATERIALS

AND METHODS

Bacterial and phage strains. All bacterial strains are listed in Table 1. Amber (am) mutants were obtained from W.

r Present address: Department of Biology, Radford University, Radford, Va. 24141. ‘To whom reprint requests should be addressed. Copytight All rights

transducing particles to PFU and therefore, a higher efficiency of transduction (EOT) (Kylberg et al., 1975; Borchert and Drexler, 1980). (iii) An increased availability of host DNA during maturation (e.g., if the phage fails to degrade the bacterial chromosome during infection) would result in the production of increased numbers of transducing particles. The results in this report suggest that the Tltur mutants are not significantly altered in the ability to preferentially initiate headfilling at specific sites. Determinations of Tltur latent periods and average burst sizes indicated that the tar mutants produce progeny in relatively normal amounts. However, we found that the majority of the mutants are unable to efficiently degrade the Esche-richia coli chromosome. The results in this report were submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree by Mary Denton Roberts at Wake Forest University.

670

Tl MUTANTS DEFECTIVE

671

IN HOST CHROMOSOME DEGRADATION TABLE 1 BACTERIAL STRAINS Reference

Pertinent phenotype

Source

Sup+; protrophic for all pertinent markers

W. Michalke

(Michalke, 1967)

w3350

Sup-, Gal-

E. Six

(Campbell, 1961)

W3350 variants

Bio- or Gall

(Drexler and Kylber, 1975)

W335Otorz.A

Tl resistant

(Drexler, 1970)

KB-Bbio::(Mucts&

Bio-

(Bendig and Drexler, 19’77)

KB-Bgal::(Mucts&

BaI-

(Bendig and Drexler, 19’77)

KB-3trp::(Mucts&

Trp-

(Bendig and Drexler, 19’77)

KB-3lac::(Mucts&

Lac-

(Bendig and Drexler, 19’77)

B-2

sup-

(Drexler and Christenson, 1961)

B-11

Sup+, Thy-

M. Center

(Studier, 1969)

K’704

Sup+, Bio+

R. Huskey

(Wood, 1966)

K724

Sup+, Bio+; deleted between galactose and att

Designation KB-3

Michalke (1967). Tl wild-type (Tl WT) was prepared in this laboratory (Roberts and Drexler, 1981). Media. L broth, L agar, SNB, TMG broth, TMG agar, EMB agar, and Tl resuspension media (Tl-RM) have all been described elsewhere (Drexler and Christensen, 1961; Melechan and Skaar, 1961; Drexler, 1970; Roberts and Drexler, 1980). TES buffer contained 1.21% Trizma base (Sigma Chemical Co.), 1.75% NaCl, and 0.25% EDTA (pH 7.5). Mackal’s buffer contained 1.2% Trizma base and 0.75% NaCl (pH 7.0). CsCl (Harshaw Chemical Co.) was 84% (w/v) in Mackal’s buffer. Lysate preporation. Lysates of Tl made on Mu lysogenic donors were prepared at 25” by the standard plate lysate method described previously (Bendig and Drexler, 1977). All other lysates were prepared at 35” by a modification of the “two-phase lysate” method (Figurski and Christensen, 1974). A 0.4-ml portion of an overnight culture of the host strain was added to a sterile 125-ml flask containing 30 ml of

(Drexler, 1977)

solidified L agar and 15 ml SNB. The flask was shaken at 100 rpm for 140 min. The culture was infected with about 1 X lo6 phage by stabbing a well-isolated plague with an inoculating needle and swirling the needle in the culture. Incubation was continued for 2 to 3 hr or until lysis was evident. The Tl lysate was concentrated by centrifugation and then filtered. Transduction. The procedures for bacterial marker and prophage transduction experiments are described in the footnotes for Table 2 and Table 3, respectively, and have been described in detail elsewhere (Drexler and Kylberg, 1975; Bendig and Drexler, 1977). Averagp burst size. The method for the determination of average burst sizes has been reported before (Borchert and Drexler, 1980) and is briefly described in the footnote for Table 5. Hydroxgurea experiwwnts. B-l cells were grown overnight in TMG broth, concentrated to about 1 X l@ cells/ml, and divided into two tubes. Phage was added

672

ROBERTS

AND DREXLER

to each tube at a m.o.i. of about 0.1 (greater than 95% of the phage adsorbed to host cells) and fresh hydroxyurea (HU) was added to one of the tubes at a concentration of 2.3 mg/ml. The tubes were aerated at 35’ for 1 hr and sterilized by the addition of chloroform. The lysates were titered for progeny and burst sizes were calculated by dividing the concentration of progeny phage by the concentration of input phage. Radioactive labeling of phage. B-11 cells were grown in TMG broth supplemented with 1 pg/ml deoxythymidine (TdR) (Sigma) and 10 &i/ml [methyG3H]deoxythymidine ([%I)TdR) (New England Nuclear, specific activity of 20 Ci/mmol) to a titer of about 1 X 10’ cells/ml. The cells were washed three times in TES buffer and phage was added at a m.o.i. of about 5. After 15 min incubation at 35”, the cells were washed two times to remove unadsorbed phage and resuspended to the original volume in TMG broth supplemented with 10 @g/ml TdR. The culture was shaken at 35” for 1 hr and centrifuged at 5000 Q for 15 min in a Sorvall RC 2-B centrifuge to remove debris. After filtration through 0.8- and 0.65-grn Millipore filters, the lysate was concentrated to a lml volume by centrifugation at high speed TABLE

(27,000 9) for 2 hr and then treated with 1 ml of 1 mg/ml bovine pancreatic DNAase (Sigma) for 1 hr at 35”. The phage were centrifuged at high speed and then banded in CsCl by ultracentrifugation in a Beckman Ti 50 rotor at 100,OOOgin a Spinco Model L ultracentrifuge. The phage band was withdrawn in a wide bore syringe and dialyzed against three changes of TES buffer. Phage DNA was extracted two times by mixing with TES-saturated phenol and collecting the aqueous layer. The DNA was dialyzed against TES buffer and DNA determinations made using the diphenylamine assay (Habel and Salzman, 1968). Samples (0.1 ml) of phage or phage DNA were mixed with 10 ml Aquasol scintillation cocktail (New England Nuclear) and counted in a Beckman liquid scintillation counter, Model LS-233 (counting efficiency of about 50%). BUdR labeling. B-11 cells were grown to a titer of 1 X lO’/ml in TMG broth supplemented with 10 Fg/ml bromodeoxyuridine (BUdR) or TdR. The cells were washed, infected, and washed again as described above. The infected cells were resuspended in TMG broth supplemented with 10 pg/ml BUdR or TdR. The cultures were shaken at 35” for 1 hr and sterilized with chloroform. 2

TRANSDUCNONOFBACTERIALMARKERS" EOT Strain Tlamb Tlamtwl

Tlamtar2 Tlamtar3 Tlamtar4 Tlamtar5 Tlamtaffi Tlamtal?

Bio+ 1 1 1 1 1 8 8 3

x x x x x x X x

10-5 10-3 1o-4 10-3 lo+ 10-4 1O-4 lo-’

Gal+

Trp+ 1 2 2 2 1 3 1 3

x x x x x x x x

10-6 1o-4 1o-5 lo-’ 10-4 lo-’ 10-4 10-4

3 2 1 2 2 1 1 5

x x x x x x x x

10-7 1o-4 lo+ 1o-4 lo+ 1o-4 lo+ 1o-5

(1Auxotrophic variants of W3350 (Sup-) were infected with Tl . KB3 (Su+) at a m.o.i. of 1 or less. After a short incubation, the infected cells were spread on TMG or EMB agar plates. After 3 to 4 days incubation, the plates were scored for transductants. Each EOT value represents the average from experiments with at least two lysates. ’ Tlam is Tlam5amll.

Tl MUTANTS DEFECTIVE IN HOST CHROMOSOME DEGRADATION TABLE 3

Transduction of Mu PFU

TRANSDUCTIONOF Mu PFU BY Tlam tarl’ EOT MUC&,~ Location lac::(Mu) trp::(Mu) bio::(Mu) gal::(Mu)

Tlam 2x 5x 2x 3x

1o-7 1o-7 lo+ 1o-6

673

Tlamtarl 5x 7x 1x 2x

1o-4 10-d lo+ 10-3

a Tl lysates were prepared on KB-3 (Mu&s& donor strains at 25”. Samples of the lysates were plated with W3350 at a m.o.i. of 0.1 or less. Transduction of Mu was assayed by Mu plaque formation after incubation for 18 to 24 hr at 35”.

Ultraviolet irradiation of phage. Phage samples were diluted into Tl-RM and dispensed in 4-ml amounts into petri dishes (200-mm diameter). Irradiation was performed by swirling the dishes under a 15W General Electric germicidal lamp at a distance of 75 cm. Manipulations required for the titering and incubation of irradiated phage were performed only in the presence of yellow light or in the dark. RESULTS

Transduction of Bacterial Markers Tlam transduces a variety of bacterial markers at roughly three levels of efficiencies (Table 2), of which the EOT of Bio+ (high), Trp+ (intermediate), and Gal+ (low) are representative (Drexler and Kylberg, 1975). We examined the transduction of the three markers by the Tlam tar mutants to determine if individual tar mutants retained the specificity of host DNA encapsulation observed with Tlam. The data (Table 2) show that each tar mutant transduced the three markers at rates significantly higher than Tlam. Individual values for the EOT of any one marker may vary within a threefold range. However, it is evident each tar mutant transduced certain markers more efficiently than others, although they did not exhibit precisely the same transduction specificities as Tlam.

The transduction of Mu PFU from several different E. coli chromosomal locations provides a simple means of testing Tls ability to preferentially package specific regions of host DNA. When Tlam transduces Mu from several different operons (Table 3), the EOT of Mu varies 15fold (Bendig and Drexler, 19’7’7).Tlam tar1 transduced Mu PFU from the same operons with efficiencies that varied only fourfold. Nevertheless, there was a consistent variation in EOT of Mu by Tlam tar1 when Mu is integrated in four separate operons and the variation paralleled that observed with Tlam.

Transduction of Bio’ Tlam transduces the Bio+ marker more efficiently than other bacterial markers because Tlam initiates headfilling of Bio+ at a specific site (esp) located between the galactose operon and the A attachment site (Drexler, 1977). It is likely that Tlam tar1 also initiates headfilling at esp because the transduction of Bio+ by Tlam tar1 from an esp-deleted donor was less efficient than from an esp intact donor (Table 4). However, the data indicated that the presence of esp is not as important to the transduction of Bio+ by Tlam tar1 as by Tlam.

Average Burst Sixes A reduction in the amount of phage DNA synthesized would be expected to enable host DNA to compete more favorably for the phage encapsulation machinery. The failure of Tl to synthesize DNA would most likely be reflected in a significant decrease in the number of infective phage produced. The results (Table 5) from determinations of Tltar bursts sizes indicate that all of Tltar mutants produced less infectious phage per cell than Tl WT. Tltur2 produced bursts more nearly equal to the average burst of 56 determined for Tl WT, whereas the remaining mutants produced bursts as small as one-third that of Tl WT.

ROBERTS AND DREXLER

674

However, if a decrease in the production of PFU is the sole cause of increases in EOT by the Tltar mutants, we would expect to observe much smaller increases in EOT than those demonstrated. We also tested the average latent periods of the individual tar mutants. Using one-step growth experiments, we found that none of the mutants exhibited latent periods of significantly different lengths from Tl WT (data not shown).

Sensitititg of Tl to Hydroxgurea Hydroxyurea (HU) inhibits the enzyme, ribonucleoside diphosphate reductase (Sinha and Snustad, 1972), which converts ribonucleoside diphosphates to the corresponding deoxyribonucleoside diphosphates. Sixty to seventy percent of Tl nucleotides are derived from the degradation of the host chromosome (Labaw, 1952; J. R. Christensen, personal communication). If cells are infected in minimum medium containing HU, the only supply of deoxyribonucleotides for Tl DNA is provided by the degradation of the host chromosome. We tested the ability of the tar mutants to grow in the presence of HU. Unlike Tl WT, which is able to degrade the E. co& chromosome, the majority of the mutants were found to be quite sensitive to HU (Table 6). Two mutants, Tltur2 and Tltar4, were found to be only slightly sensitive to HU. The observation that the growth of the Tltur mutants, normally nonlethal muTABLE 4 TRANSDUCTION OF BIO+ FROM K704 AND K724”

EOT Donor x-704 K724b

Tlam 1.9 x 10-S 2.0 x 10-6

Tlamtarl 1.3 x 1o-9 4.1 x 1o-4

n See the footnote to Table 2 for experimental details. * K724 is deleted for about two-thirds of the DNA between galactose and ata. esp, the essential site for efficient packaging by Tl is deleted in this strain.

TABLE 5 AVERAGE BURSTSIZESFORTltar STRAINS”

Strain Tl WT Tl tar1 Tltar2 Tltar3 Tltar4 Tltar5 Tl tar6 Tltar7

Average burst size 56 (3)b

25 (5) 43 (5) 34 (3) 20 (3) 22 (3) 31 (3) 20 (3)

’ KB-3 was infected with Tl at a m.o.i. of 5. After a lo-min adsorption at 35”, the infected cells were diluted to about 1 infected cell per tube and incubated for 1 hr at 35’. The contents of each tube were plated with KB-3 and counted the next day. Averge burst sizes were calculated as described under Materials and Methods. “Numbers in parentheses indicate the number of individual experiments used in calculating average burst sizes.

tants, was inhibited in the presence of HU, provided the basis of complementation tests between the tar mutants and neighboring a~ mutants (dat not shown). Positive complementation between the tar mutants and Tlati (or Tla;M?) in Supcells in the presence of HU led us to conclude that the mutations define a new Tl gene or genes.

Trasqfer of Host L&e1 to Progeny Phage In order to confirm that the Tltur mutants fail to degrade the chromosome to nucleotides during infection, we used two different methods to examine the degree to which labeled nucleotides were transferred from E. co& DNA to progeny Tl. First, host cells were prelabeled with raH]TdR and infected with Tl in medium containing only TdR. The amount of radioactivity contained in lysate particles and lysate DNA was determined (Table 7). It is evident that the particles and DNA in a lysate of Tltarl contained less than one-third the radioactivity present in a Tl WT lysate. A Tl lysate contains both infectious and

Tl MUTANTS DEFECTIVE TABLE 6 SENSITIVITY OF Tltar

Strain

STRAINS TO HYDROXYUREA” Burst size + HU Burst size - HU

Tl WT

1.00

Tl tar1 Tltar2 Tltar3 Tllar4

0.02 0.43 0.03 0.31

Tlta6 Tltar6

Tl tar7

675

IN HOST CHROMOSOME DEGRADATION

0.04

0.02 0.02

‘B-1 cells were infected in TMG broth with or without hydroxyurea (HU), aerated for 1 hr, and sterilized with chloroform. Progeny phage were titered on KB-3. Tl bursts were calculated by dividing the concentration of progeny phage by the concentration of input phage.

transducing particles, Therefore, a measure of lysate radioactivity does not provide a precise account of the radioactivity incorporated only into infectious particles. We performed an experiment which measured the incorporation of host-labeled nucleotides only into PFU. E. coli was prelabeled with either TdR or BUdR and infected with Tl in medium containing either nucleoside. The sensitivity of Tl to inactivation by ultraviolet light, a measure of the BUdR content of phage DNA, was then tested. Because Tl acquires only part of its nucleotides from the host (Labaw, 1952; J. R. Christensen, personal communication), it was not surprising that cells prelabeled with one nucleoside and infected in the presence of the other produced phage intermediate in uv sensitivity between fully BUdR-substituted and unsubstituted controls (Fig. 1). On the other hand, Tltarl appeared to acquire only the nucleoside added exogenously at the time of infection.

We have done several other experiments to further characterize the tar mutants. Although the data from these studies are not presented in this report, the results are briefly summarized:

The tar mutants share physical characteristics in common with Tl WT. Their densities in CsCl gradients were similar to those of Tl WT. They were inactivated at equal rates by exposure to uv light. Tl WT and Tltar particles appear to contain similar amounts of DNA since the efficiencies with which Tltar+ and Tltar package X prophages were equally influenced by the length of the prophages in donor cells. It has been shown that Tl transduces markers with slightly higher efficiencies from X lysogenic donors than from nonlysogens (Kylberg et al., 1975). This is due to the fact that Tl-transducing particles are made early in the maturation period and Tl-infected X lysogens lyse prematurely before the majority of infectious particles are made. We did not observe any time dependence with transduction efficiencies by Tltar. The EOTs of markers by Tltar grown on h lysogens and on nonlysogenic donors were identical. DISCUSSION

Using a simple mixed indicator plating technique, we isolated mutants of Tl that exhibit enhanced frequencies of transduction (Roberts and Drexler, 1980). The tar mutations were shown to be closely linked and located in a region of the Tl genome identified as being important in Tl DNA synthesis. Because there is a clustering of related functions on the Tl chromosome, TABLE ‘7 INCORPORATION OF E. coli [3HJTdR INTO PHAGE DNA

[3HJI?dR incorporated (cpm) Strains Tl WT Tltarl

Phage 8.6 X lo-‘/PFU 2.9 x lo-‘/PFU

DNA 42,94O/mg 14,61O/mg

’ B-11 cells were grown in [‘HJrdR supplemented TMG broth and infected in TdR-supplemented medium. Progeny phage were filtered, concentrated, and banded in CsCl. DNA was harvested by phenol extraction and the concentration determined by a colorimetric assay.

676

ROBERTS AND DREXLER

we suspected that the tar mutations caused some alteration in the early development of Tl. The experiments discussed in this report examined the possibilities that the tar mutations affect either the specificity of DNA packaging or that they result in altered amounts of host or phage DNA available. The tar mutants transduced various bacterial markers at higher efficiencies than Tlam and not always with the same degree of specificity as Tlum. For example, Tlam transduced three markers (Bio+, Trp+, and Gal’) at three different efficiencies (Drexler and Kylberg, 1975), whereas Tlam tar’7 transduced Bio+ and Trp+ at the same rate but Gal+ was transduced at a significantly lower rate. Nevertheless, we believe that the tar mutants, without exception, have retained the ability to distinguish between certain regions of the host chromosome and to ‘preferentially transduce certain markers. The Tlturl mutant, similar to Tltar+, appeared to package Bio+ specifically from the esp site. Studies using heteroduplex analysis of Tlturl DNA have shown that Tlturl encapsulates phage DNA in the same sequential fashion reported for Tl WT DNA (G. Gill, personal communication). In order words, Tlturl does not seem to alter the specificity with which Tl packages DNA. A determination of Tltur average burst sizes revealed no gross alteration in the ability of the mutants to synthesize phage DNA, but four of the seven mutants did produce fewer than half of the number of infectious particles synthesized in Tl WTinfected cells. It is possible that small bursts alone could result in as much as threefold increases in the ratio of transducing particles to infectious particles (i.e., the EOT) but the large increases in EOT by Tltur must also be caused by additional factors. The sensitivity of the majority of the mutants to HU suggested that they require nucleotides exogenously supplied or synthesized de wvo. Therefore, the mutants are impaired in the degradation of the host chromosome during infection. The ability of HU-treated Tltuti to pro-

FIG. 1. Inactivation of Tl by ultraviolet light. B-11 cells were grown in the presence of TdR or BUdR and infected in the presence of either nucleoside. Progeny phage were irradiated and titered. The proportion of phage inactivated by less than 10 set exposure was similar in each sample and not indicated above. (A) Tl WT; (B) Tltarl. (0) Cells grown in TdR, infected in TdR; (0) cells grown in BUdR, infected in BUdR; (A) cells grown in TdR, infected in BUdR; (A) Cells grown in BUdR, infected in TdR.

duce progeny in numbers only slightly less than Tl WT could possibly be attributed to partial impairment or a delay in host chromosome breakdown. This may explain why Tltur2 transduced with higher efficiencies than Tltar+ but significantly less than the remaining mutants. However, we are at a loss to explain the relative insensitivity of Tltur4 to HU because this mutant transduced with efficiencies equal to those of, for example, Tlturl. The observation that Tltarl may have acquired as much as one-third of the host radioactive labeled nucleotides contained in Tl WT would suggest that Tltarl might be capable of inefficient degradation. A reasonable alternative explanation is that the radioactivity in a Tlturl lysate is contained almost exclusively in transducing particles which are present in much higher numbers than in Tl WT lysates. In fact, the BUdR labeling studies suggested that host-labeled nucleotides probably comprised an insignificant portion of Tl tar1 DNA. Therefore, we believe that the majority

Tl MUTANTS DEFECTIVE

IN HOST CHROMOSOME DEGRADATION

of the tar mutations affect a protein (or proteins) which enzymatically degrades the E. coli chromosome and provides a plentiful supply of nucleotides for incorporation into Tl DNA. Although this proposed protein may not participate directly in the assembly of transducing particles, it indirectly influences the process by controlling the availability of host DNA. The elimination of the phage’s degradative ability results in the increased production of transducing particles and may be the reason for several other observations: Transduction efficiencies by Tltar+, unlike Tlam tarl, are time dependent and are influenced by Tl’s ability to rapidly package certain markers before host chromosome degradation (Kylberg et al., 1975). If host DNA is available through the latent period, the time dependence of host DNA packaging is removed and a partial equalization of transduction rates would be observed. We have demonstrated that the Tl tar mutants transduce various markers with less variability than Tltur+. It has also been shown that the Tltur mutants produce smaller numbers of progeny phage. We think this is very likely a result of the reduction of the plentiful supply of nucleotides derived from the host chromosome. ACKNOWLEDGMENTS This work was supported by Grant PCM 77-26639 from the National Science Foundation. We would like to thank J. R. Christensen for several stimulating discussions of this work and for his valuable suggestions. REFERENCES BENDIG,M. M., and DREXLER,H. (1977). Transduction of bacteriophage Mu by bacteriophage Tl. J. Viral 22,640~645. BORCHERT,L., and DREXLER, H. (1989). Tl genes which affect transduction. J. Viral 33,1X2-1128. CAMPBELL,A. (1961). Sensitive mutants of bacteriophage X. Virom 14, 22-32,

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DREXLER,H. (1970). Transduction by bacteriophage Tl. Proc. Nat. Acad. Sci. %‘A 66.1083-1088.

DREXLER,H. (1977). Specialized transduction of the biotin region of Escherichia coli by phage Tl. MoL Gen. Gmt.

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SCHMIEGER,H. (1972). Phage P22 mutants with increased or decreased transduction abilities. Mol. Gen. Genet. 119.7588. SINHA, N., and SNUSTAD,D. (1972). Mechanism of inhibition of deoxyribonucleic acid synthesis in Escherichia coli by hydrozyurea. J. BwterioL 112, 1321-1334. STUDIER,F., W. (1969). The genetics and physiology of bacteriophage T7. Virology 39,562-574. TYE, B.-K. (1976). A mutant of phage P22 with randomly permuted DNA. J. MoL BioL 169,421~426. WOOD,W. B. (1966). Host specificity of DNA produced by Escherichia colti Bacterial mutants affecting the restriction and modification of DNA. J. Mol. BioL 16,118-133.