Phage lambda receptor chromosomes for DNA fragments made with restriction endonuclease III of Haemophilus influenzae and restriction endonuclease I of Escherichia coli

J. Mol. Biol. (1975) 98, 551-564

Phage Lambda Receptor Chromosomes for D N A Fragments made with Restriction Endonuclease HI of Haemophilus influenzae and Restriction Endonuclease I of Escherichia coli KENNETH MURRAY AND NOREEN E. ~IURRAY

Department of MoZe~ular Biology UniversiSy of Edinburgh Kings Buildings Edinburgh 9 Sc~t~ (Received ~7 March 1975) Phage A DNA yields seven fragments on digestion with endo R.HindIII. These seven fragments and the 12 fragments resulting from digestion of A DNA with both endo R.Hi~uiIII and endo R.mcoRI have been ordered within the map of the A genome. The positions of the six R.Hi~]_III targets have been defined and all of the targets, other than shindTTTA6, can be removed by the combination of a known deletion mutation with the substitution of imm 21 for imm ~. shindIIIA6, which is close to or within gene Q, can be removed by the substitution of a small region of the h chromosome with phi 80 DNA. This permits the construction of receptor chromosomes for DNA fragments generated by endo R.HindIII. One receptor phage, which has about 20% of its DNA deleted, can accommodate DNA fragments within the central region of its chromosome. Other receptor chromosomes are described for DNA fragments from endo R.HindIII or endo R.EcoRI digests, in w~ich nearly 40% of the ADNA has been deleted.

1. Introduction Four of the restriction endonucleases known at present, R . E c o R I , \ R . E c o R I I , R . R i n d I I I and R . HapII (which has the same specificity as R . H p a I I ) make staggered breaks in doub le-stranded DNA molecules to form fragments with short single-stranded projections at their 5' termlu~ (Hedgpeth et al., 1972~; Bigger e~ al., I973; Boyer e~ al., 1973; Old et al., 1975; Sugisaki & Takanami, 1973; Garfin & Goodman, 1974). Such fragments can associate one with another b y annealing of these cohesive ends and the joints closed covalently with polynueleotide ligase. This sequence of reactions has been exploited in the formation of recombinant DNA molecules in v~ro. Of special interest are those recombinants where additional fragments of DNA have been inserted into a plasmid or phage chromosome, which then serves as a vector for transmission of the fragment into Escherichia coliin a form such t h a t the fragment m a y be replicated along with its vector. The E. coli restriction enzyme, endo R . E c o R I (nomenclature of Smith & Nathans, 1973) has been used in these experiments. Fragments of DNA containing the ribosomal genes from X e n ~ s ~ev~ (Morrow d aL, 1974) or the penicillln-resistanee determinants of/~taphy~c~c~u,~ war~s (Chang & Cohen., 1974) 551

552

K. MURRAY AND N. E. MURRAY

have been cloned v/a the plasmid pSC101, while the plasmid co/E1 has been used to amplify thb~produets of the trp operon of E. coli through the cloning of DNA fragments from a phi 80trp phage (Hershfleld e~ ~., 1974). Phage A has been manipulated so t h a t it m a y function as a receptor and transfer agent for DNA fragments generated with endonuclease R . E c o R I and part of the trlv operon of E. coli and fragments of cukaryotic nuclear DNA (Murray & Murray, 1974; Thomas et al., 1974) have been amplified in E. coli vi~ transfectants formed from the recombinant DNA preparations. The usefulness of phage ~t as a vehicle for the transfer of DNA fragments to E. coli relies on the deletion of DNA from the inessential central region of the phage chromosome. I n this paper we describe further examples of receptors for the endo R.EcoRT system which permit the accommodation of DNA fragments with molecular weights up to about 15 • 10 e, but our main concern is to describe the development of the phage chromosome to serve as a receptor for DNA fragments formed b y digestion with restriction cndonuelcase I I I of Haemo~hi~u~ i~fluenzae, or endo R . H ~ n d I I I in the nomenclature of Smith & Nathans (1973).

2. Materials and M e t h o d s (a) Phage~ The following were used. Wild-type ~t and derivatives with supII-suppressible amber mutations: Pare80, Qam73 and l~am5 (Campbell, 1961). A heat-inducible (imm~cI857) derivative made defective in lysis by the supIII-suppressible mutation am7 in gone S (Goldberg & Howe, 1969). Derivatives of ~cIam509, with deletions in the central region of the ~t genome: ~b189, ~b538, ~tb519, ~b506, ~tb527 and ~b508 (Parl~in~on, 1971) and the deletion ;tb2 (Kellenberger et aJ., 1960). ;~inR5, which has a small deletion permitting N-independent growth (Court & Sate, 1969) and two derivatives of ~Pam80, T~H54 and KH70, having deletions within the immunity region (Blattner e~ aJ., 1974). Phage Awith the immunity of phage 434, A~m~n4s4 (Kaiser & Jacob, 1957) or with the immunity of phage 21, ~t~mm21 (Liedke-Kulke & Kaiser, 1967). Wild-type ~80, Cambridge strain (Fr,.ntrlln & Dove, 1969) and an he~ ~ recombinant, he~ (Murray et aJ., 1973). Derivatives of ~t with reduced numbers of targete for R.EcoRI (Murray & Murray, 1974). All other strains were recombinants isolated from conventional crosses. (b) B a . U r ~ The bacterial strains used are listed in Table 1. Where necessary, ~-resistant, phi 80resistant or lysogenic derivatives were isolated. (e) Med/a The rich medium was L broth (Lennox, 1955) eontR;n;-g (in g/l): Difeo Bacto-Tryptone, 10; Difeo Bacto yeast extract, 5; NaC1, 5; glucose, 1; adjusted to p H 7.2. Phage stocks for genetic analysis were prepared on L broth agar solidified by Difco agar (10 gfl). Phage assays were made on Baltimore Biological Laboratories Tryptiease agar, contalnlng (in g/l): Trypticase, 10; NaC1, 5; Difco agar, 10 for plates and 6.5 for top layers (Par~n~en, 1968). Phages were diluted and resuspended in phage buffer eon~ining (in g/l) : Na2HPO4, 7; g~r2PO4, 3; NaC1, 5; MgSO4,7H20, 0.25; CaCI~, 0.015; gelatin, 0-01. The low phosphate medium used for preparing 8~P-labelled phage was made as follows (John Abelson, personal communication). Difco Bacto-Peptone (20%) was titrated to pH 9 with NH4OH, the precipitate was removed and the pH of the supernatant adjusted to 7.5 with HC1. This Bacto-Peptone was diluted (10 ml to 1 1) with a medium containing (in g/l): KC1, 1"5; NaC1, 5.0; NH4C1, 1-0; Tris base, 12.1; pH adjusted to 7.5 with HC1. After sterilization, glucose to give 0.4% (w/v) and MgSO4 to give 10 -8 M were added.

A

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A ~K L~

§ I0

554

K. MURRAY

AND

N. E. MURRAY

(d) 6/sner02 techn/ques The methods for preparing and plating cells and phage stocks, assaying phage and making phage crosses have been described by Murray ee 02. (1973).

(e) Phage Zyaate~for D N A prepuru~o~ Preparations of phage were made by infection of exponentially growing cultures orE. co/i C600 in L broth. Growth was followed spectrophotometrieally and when the A680.m reached a mln~lTluril (usually about 9. h a~ter infection) lysis was completed by addition of CHC18 (0.5 inl/l) and 15 m~n later the lysate was clarified by eentrifugation (10 min at 10,000 g). Phage were recovered by eentrifugation, resuspended and treated with DNAase and RNAase (10 ~g/ml each, 9. h at room temperature), pelleted, resuspended and recovered by equilibrium centrifugation in 41-5% (w/w) caesium chloride solution (Kaiser & Hogness, 1960); the caesium chloride step was repeated, szP-labelled phage were prepared from the lysogen W1485 (AcI857SamT) which was grown (50 ml culture) to a cell density of 5 • 108/ml in L both, induced at 42~ for 10 m~n, then tranaferred to low phosphate medium and 32Pl (1 mCi) added. After 9. to 3 h growth, the cells were centrifuged, resuspended in phage buffer (3 ml) lysed with CHC18 (2 drops) and the phage recovered by 2 cycles of equilibrium centrifugation in 41.5% (w/w) CsC1.

(f) D N A preparat/ion Phage preparations were diluted to about 5 • 1011 to 5 • 1012 plaque-formlng units/ml and dialysed against 10 mM-Tris. HC1 (pH 8.0), 1 mM-EDTA. DNA was extracted by gentle rolling with freshly distilled phenol (Kaiser & Hogness, 1960) and phenol was removed from the aqueous phase by dialysis against 10 mM-Tris.HC1 (pH 8.0), 1 mM-EDTA (4 changes in about 9.4 h). 5' terminally labelled ADNA was prepared by dephosphorylation with bacterial alkaline phosphatase followed by treatment with polynucleotide kinase as described by Murray (1973). (g) Enzymes and chem~02s Pancreatic DNAase and RNAase were purchased from Worthington Biochemical Corporation, Freehold, N.J., U.S.A.; bacterial alkaline phosphatase was from Whatman Biochemicals Ltd, Maidstone, Kent, U.K. Polynucleotide kinase was prepared as described previously (Richardson; 1965; Murray, 1973); restriction endonuclease R.EcoRI was prepared essentially as described by Yoshimori (1971) and endo R.HindIII was prepared either as described by H. O. Smith (Old e~ 02., 1975) or Philippsen st 02. (1974). Some of the endo R.HindHT preparations used were generously provided by Dr P. Philippsen and by Dr H. Cook. Wherever possible the chemicals used were of AR grade; caesium chloride, caesium sulphate and agarose were from BDH Ltd, Peele, Dorset, U.K. (h) Res~rictio~ e~on~lease digestion and g6Z electropho're~s The quantity of the restriction endonucleases required for complete digestion (37~ 30 m~n) of 1 ~g of A+ DNA was determined in trial experiments with a series of digests, which were analysed by electrophoresis in 1% (w/v) agarose gels (in 0.04 M-Tris-acetate (pH 8.0), containing 0.4 ~g ethidium bromide/l; Sharp e~ 02., 1973). For analysis of the products of digestion of the various DNA preparations with the restriction endonueleases, samples of 1 to 2 ~g in about 20 ~1 0.01 M-Tris.HC1 (pH 7.5), 0.01 ~-MgC12, 0.01 ~-2mercaptoethanol (and, for R.EcoRI digests, 0.1 M-NaC1) were incubated with the appropriate quantity of enzyme at 37~ for 30 to 60 m~n, heated at 70~ for 10 rain, cooled in ice, mixed with 5 ~1 50% (v/v) glycerol containing bromophenol blue (about 0.1%), concentrated to about 10 ~1 in a vacuum desiccator and applied to wells in an agarose slab gel (40 cm • 20 cm • 0.3 cm; Sharp e~ 02., 1973) for electrophoresis, usually for about 18 h with a constant current of 40 mA. Gels were photographed under ultraviolet light on Ilford FP4 Alto (4 • red filter), which was developed (9 m;n at 18~ in Microphen (Ilford Ltd, Ilford, Essex, U.K.).

PHAGE k RECEPTOR

CHROMOSOMES

555

(i) Transfec~ion of E. cell w/th restr/cted ~hag~ DNA Cells (usually 803 e u p L I I ) competen~ for transfeetion with phage DBIA were grown in P m e d i u m (Kaiser, 1962) supplemented with glucose, 1 m g / m l (and me~hionine, 100 vg/ ml) a n d s t a r v e d in 0.1 ~-CaCI= a t 0~ as described b y Mandel & H i g a (1970). Cold D N A solutions (1 ~g/ml in saline/sodium citrate) were m ~ e d with t h e suspension of cells in CaCI= in the ratio 1 : 2 (v/v), placed in a w a t e r b a t h a t 37~ for a b o u t 30 s a n d r e t u r n e d to a n ice-bath for a t least 1 h before mixing with top layer agar for plating (A. J a c o b a n d S. Hobbs, personal communication).

Results Location of the targets for R. H i n d l I I in the Thage k genome 3.

(a)

T h e g e n o t y p e s o f t h e p h a g e s discussed i n t h i s s e c t i o n a n d t h e e l e e t r o p h o r e t i e s e p a r a t i o n o f f r a g m e n t s d e r i v e d on d i g e s t i o n o f t h e i r I ) N A w i t h e n d o n u c l e a s e R . HindIII a r e i l l u s t r a t e d in F i g u r e 1. D i g e s t i o n o f w i l d - t y p e k D N A w i t h endo R . H i n d I I I gives s e v e n f r a g m e n t s , A t o G i n o r d e r o f i n c r e a s i n g e l e c t r o p h o r e t i e m o b i l i t y in a g a r o s e gels. T h e size o f t h e fragments was estimated from their electrophoretic mobility and from the relative a b u n d a n c e o f r a d i o a c t i v i t y in f r a g m e n t s s e p a r a t e d f r o m digests o f u n i f o r m l y l a b e l l e d D N A (Table 2). W i t h 5' t e r m l n a l l y l a b e l l e d k D N A , o n l y b a n d s A a n d D were r a d i o a c t i v e , a n d i n a d o u b l e d i g e s t w i t h endo R . H ~ n d I I I a n d e n d o R . E c o R I t h e b a n d s A ' a n d F ' o f t h e c h a r a c t e r i s t i c endo R . E c o R I p a t t e r n (Fig. 2(b)) were r a d i o a c t i v e , s h o w i n g t h a t H ~ n d I I I b a n d A is l o c a t e d a t t h e l e f t - h a n d t e r m i n u s o f k D N A a n d ,4

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FIG. 1. Electrophoretio analysis of endo R . H i n d I I I digests of DNA from various strains to give the linear order of the fragments from k § DNA. The left-hand part of the Figure describes the genotypes of the phages; the gaps indicate the positions of deletions and the double lines represent substitutions. The scale 0 to 100 represents the length of k § DNA. The right-hand par~ of the Figure shows the electrophoretio separations on 1% (w/v) agarose gels of the fragments formed on digestion of the DNA with endo R. HindIII. The positions on the gels of new fragments, with respect to the standard pattern of fragments from ~+ DNA (A to G in order of inoreasing mobility), are shown by broken lines. The DNA samples were from the following phages, (a) ~+ ; (b) k+, but 5' a2P-labelled (only the radioactive fragments are shown); (e) ~5538; (d) kbS08; (e) ~tb2ninR5 (phage no. 457); (f) ~1~54, ~mmunity deletion; (g) fragment a from phage no. VI (Murray & Murray, 1974) which carries only targets 1 and 2 for endo R.EcoRI; (h) ~5506~mm=1 ~inR6shi~dIII~6 ~ (phage no. 545) in which ahn-6t was removed by a small substitution from phi80. The order of the fragments (and ~he positions of targets for R.HindllT) in ~+ DNA is shown at the lower left of the Figure. t See footnote on p. 569.

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FIG. 2. Elsotrophoretio analysis of double digests of D N A from various ;t strains with endonuoleasos R . H ~ d T f T and R . E c o R L As in Fig. 1, the left-hand part of the Figure describes the genotypos of the various phages, and the right-hand part the elcetrophoretio separation of D N A fragments in restriction enzyme digests of the DNA. For convenience, the positions of the targets in ~+ DlgA for endo R . E c o R I (Allot e~ ~., 1973; Thomas & Davis, 1975) and endo R . H i n d ~ I are included at the top of the list of phage genotypes, but are shown in more detail at the bottom of the Figure together with the order of the 12 fragments formed on digestion of ~+ DlgA with endo R . E c o R I and endo R.Hi~dl-II. ;t + DNA was used for digests (a) and (b), which were made with ondo R . H ~ n d I I I and endo R . E o o R I , respectively. The other digests were made with both restriction enzymes together and the DlgA used was from the phages listed below, the R o m a n numbers being the description used previously for some of those phages (Murray & Murray, 1974): (c) ~+; (d) X I ; (e) X ; if) ~ r f ; (g) phage no. 500, which is ~5519~vg~RS~rI;t4~176 (h) V-I; (i) fragment a from phage ~'I;

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(k) r~.

The D N A used in digest (d) contained only one target, 8rI~4, for endo R . E C e R I and carries the b538 deletion. I n the double digest of this DlgA, fragment C of the ondo R . H i n d l - i I produote is replaced by the fragments ~ and j ; the sizes of these products (Table 2) and of fragment D and the position of #rIA4 require that ~ precedes j in sequence left to right. Similarly, with the D N A of digest (o), whose only target for endo R . E c o R I is #rI~5, fragment D is replaced b y fragments r and l and, since the latter is the fragment right of ~rI~5, fragment ~ must separate fragments I and C. The DtgA of digest (f) has the region between ~rI~l and ~rI~2 deleted end has no other targets for endo R . E c o R L The only new fragment recognized in this digest was fragment I~, which means that fragment p is in the right-hand region of fragment E because digestion of the D N A segment between a r i e l and erI~t2 (i.e. fragment a of phage ~-I) with endo R .H~dTTT alone gives fragment 1r but not fragment E (Fig. 1). The DbTA used for digest (g) was from phage no. 500, (which contained targets erI~2 and ariA3 and had the deletions b519 and r and gave a smaller fragment C (C*, see Fig. 1 and Table 2) with endo R . H g ~ d T ~ by virtue of the ~vgnR5 deletion and in the double digest, fragments B and E are replaced by fragments ~, k, p and q. The sizes of those fragments and of fragment E (Table 2) can only be reconciled if fragment E comprises fragments 1o and q, which means that q precedes p and t h a t fragments A and/~ are derived b y cleavage of fragment B at #r~3. D N A containing only targets ~rI~l and ~rI~2 for endo R . ~ c o R I was used for digest 0a), which retained fragment F, but not fragment E, and also had fragments rn, p and q: since q and p constitute fragment E, fragment m must precede fragment F. This assignment was confirmed by the presence of fragments ~n, F and q in digest (i) of the fragment botwcen ~rl~l and ~rI~2 (fragment Via) with ondo R . H g n d I I I , and b y the occurrence of fragments 3', ~- and p, b u t not of fragment m, in digest (j), since this D N A (phage V I I I ) contained only

PIIAGE

~ RECEPTOR

CHROMOSOMES

659

H i ~ / I I I band D at the right-hand terminus. I n digests of DNA from the ~b538 deletion strain, only fragments A, C, D and G were found, which means t h a t three targets for endo R . H i n d I I I and the fragments E, F and part of B must lie within the confines of this deletion. DNA from the deletion strains b2 and b189 gave the same pattern, but with DNA from strain 5508, only one band, B, was missing and was replaced b y a slightly faster moving band, B". Similarly, with I)NA from a ninR5 deletion strain, band C alone was lost and was replaced b y a smaller fragment (C~'). The targets for endo R . H i n d I I I have been mapped in the immunity region of phage (Allot & Solem, 1974; Blattuer etal., 1974) at positions corresponding to 76.2 and 77.3 physical map units from the left-hand end of the wild-type ~ chromosome; fragment G is the only one t h a t could be placed between these two targets and this assignment is confirmed b y the pattern of fragments obtained with DNA from the immunity deletion strains K H 5 4 and K1~70 (see also Blattner e/aZ., 1974), in which bands B, G and C are replaced b y a single band with a slower mobility t h a n B. Taken together, these results establish the linear order of fragments of ~ I)NA produced b y the action of endo R . H i n d I I I as A(E,F)BGCI). Since fragments E and F lie within the region of the ~ chromosome covered b y the b2 and b538 deletions, an endonuclease R . E c o R I digest of DNA from a phage containing only targets ~rlA1 and srI?~2 (see Fig. 1) (phage no. VI, Murray & Murray, 1974) was used to determine the order of fragments E and F. A phage lacking the DNA between ~rlA1 and srlA2 (phage no. XII, Murray & l~Iurray, 1974) did not yield fragments E and F when its DNA was treated with endo R . H i n d I I I , and no new fragment was detected, although fragment A must be altered. In an endo R . H i n d I I I digest of the purified fragment contained between targets 8rIA1 and 8rlA2, fragment F persisted, but fragment E was missing. Fragment F, therefore, precedes fragment E in the linear order given above, and the approximate positions of the six targets for endo R . H i n d I I I which punctuate the seven fragments were inferred from the results listed in Table 2. The positions of the targets, which are shown in Figure 2, were confirmed b y the following observations. The fragments observed on agarose gel electrophoresis of endo R . H i n d I I I digests of DNA from the deletion strain b519 place shindIIIA1 to the right of this deletion (40.7 to 47-0%) and those from the deletion strains b189 or b2 place shindIIIA3 to the left of the crossover point within the attachment site (57.4~) (Davis & Parl~inson, 1971) ; confirmation of this is provided from digests of DNA from the b508 deletion which retain fragment E~. Similar analysis with the deletion b506 shows t h a t shn-2 The nomenclature for targets or sites within a given genome for restriction enzymes employs the abbreviation for the enzyme (Smith & l~Tathans, 1973) followed by the genome concerned and the number (or location) of the site within the genome. Thus, sites for endo R.HindIII in the phage Agenome are described by shindIIIA1, shindIIIA2, etc. Even this description is cumbersome and so in this paper, since we are concerned only with targets in ADNA for endonuelease III ofH. influenzae serotype d, we often use the further abbreviated form ahn-1, sh~-2, etc.

erI~2 of the endo R.EcoRI targets and also had the b519 deletion. Finally, digest (k), in which the DNA retained only srI~3 and ~rI~4 of the endo R.EcoRI targets and had the b538 deletion, contained fragments h and j, but not k. (Fragment n was replaced by a slightly slower moving fragment, n', because this phage differs from phage IX in being cIam rather than ci867; Blattner eCaL, 1974.) These results give the order of fragments from the double digest of )t+ DNA shown at the bottom of the Figure, which together with the results in Table 2 form the basis of the location of the targets for endo R .iiindIII, which are the averages of results from l(d) and 2(d) of Table 2. 37

560

K. MURRAY

AND

N. E. MURRAY

and shn-3 are to the right of this deletion (44.5 to 52.6~). The results of these gel electrophoreses axe illustrated in Figure 1. The size of fragments E and 1~ deduced from their relative yields in digests of n~i~ormly ~P-labelled )~+ DNA (and also from mobilities on gel eleotrophoresis) cannot be reconciled precisely with the allocation of positions of sites relative to the locations of deletions which have been obtained from electron microscopy of heteroduplex DNA (Fiandt et a~., 1971). To reduce these discrepancies, DNA from appropriate phages was digested with both endo R.HindlTI and endo R.EcoRI. Analyses of the resulting DNA fragments and the positions of the targets deduced from these experiments are given in Figure 2 and Table 2. (b) Manilau~ztion of ~ site 6for endo R. HindlII (i.e. shindlII~6) All the endo R.Hinct~7 targets in phage h DNA, other than shn.6, can be removed b y the combination of a known deletion mutation together with the substitution of imm ~1for imma (Fig. 1). Since hb2imm 2~DNA has only one target for endo R .HindIII (i.e. shn-6), mutations leading to loss of this target should be selected among the DNA moleenles that survive treatment with endo R.HindIII. This approach, however, was not successful and since shn-6 is very close to gone Q, we turned to the alternative of mA.l~ng DNA substitntious. Hybrid phages with the host range of phiS0 and the immunity of h, i.e. (hphls0 imm~) are unable to plaque efficiently on a phi80 lysogen, but derivatives that do so are selected readily and, generally, have acquired the Q, S and R genes from the prophage (Murray e~ aL, 1973). We selected Q+ derivatives of an hph18~imm~-Qam73 phage as plaques formed on a 8ulv~ strain. Disappointingly, those we examined either retained 8hn-6 or acquired a new target withln the phi80 DNA. l~ortuitously, we did recover a shn-6 ~ strain as a Q+ recombinant from a cross of ~tQam73 by an h phla~ immaQphis~ phage; concomitant with the formation of a hybrid Q gone the closely llnl~ed shn-6 was apparently lost. This interpretation was substantiated by screeu~ng derivatives of ~Qam73 in which the lesion in gone Q was repaired t~y marker rescue from a phi80 prophage. We found two classes of Q+ recombinants; the first had retained P ' R of phage ~, since they could be transaetivated by the ~Q gone; the second had acquired a more extensive region of the phi80 chromosome, presumably including P ' R from phage phi80, since they were not transactivated by the Q gone of phage ~)~. DNA was prepared from one representative of each class and that from the first no longer had an endo R.Hind.III target between immunity and the right-hand termlnus of the DNA, but it had retained srI)~5 (the endo R.EcoRI target close to P'RX). DNA from the second class of Q+ reeombinants had an endo R.HindIII target in the Q region of the chromosome, but it had lost stir5, shn-6 ~ can readily be transferred to any Q- derivative of phage h.

(o) Gonst~J~c~ionof r ~ t o r l~hag~ for D N A fragments made with endo R. ~i~8,III The essential features of a phage chromosome that is to serve as a receptor for fragments of DNA are that it rdains only one target for the restriction endonuclease used to make the fragments (this may be effected by elimlnation of inessential DNA between two targets); that an appreciable portion of inessential genetie material has been deleted to provide sufficient space for the accommodation of a large fragment of DNA, and that interruption of the chromosome by insertion of the additional DNA

PHAGE A RECEPTOR CHROMOSOMES

661

a t t h e r e s t r i c t i o n t a r g e t does n o t d e s t r o y an essential f u n c t i o n ( M u r r a y & ] ~ u r r s y ,

1974; R a m b a c h & TioUais, 1974; Thomas e~ ~ . , 1974). The locations of the various targets in ~ D N A for endo R . H i n d I I I and the availability of appropriate deletion strains (Fig. 1), together with the ability to manipulate ~hn-6 permit the construction of suitable receptor chromosomes. One of these (Fig. 3(a)) relies on the use of shn-3 in a phage t h a t has the region between ~rI~l and ~I;L2 (i.e. targets 1 and 2 in ~ D N A for endo R.EcoRI) deleted (phage X I I , ]Kurray & Murray, 1974), has the i m m u n i t y of phage 21, and has lost ~hn-6. E x t r a space is generated b y the n~nR5 deletion, which m a y be crossed into a n y ~ m m ~ Q - phage together with i m ~ ~z and Q+s/~i~lIII;~6 ~ using an l ~ ~ 1 7 6 phage to generate / ~ m m m O + recombinants. This receptor has lost a b o u t 20~o of the ~ chromosome and, since ~hn-3 is to the left of ~ (K. Borck, personal communication), the insertion of D N A at this site should leave a n integration proficient phage. An alternative receptor system is provided b y using only the left a r m of the phage described above and the right a r m from a ~b2~mmxninR5shindIIL~6 ~ phage. The A ; ' io

J

~/ fed ~ cI ' 6b ' ~o

io I

s~/x

2

I

shind ITIk

3

I I I I

4

I

II

123

5

I

I

45

I

6

V srl ~.I-2

(o)

O ' ,&

nin R5

I

imm-"-~l

(b)

b2

(c)

Vsr[), I-2

--II

-

ninR5

mnR5 I1"--

Vsr[kl-2

(d)

J

Iv I ninR5

/•4•4

Vsr/~l-2

(f)

I

I

0

I

I

I

20

40

ninR5 /mm4S4

Iv

I

I

60

I

1

80

I

I

100

FIG. 3. The genetic maps of additional receptor chromosomes for DNA fragments generated by endonucleases R.HindTTT and R.•coRI. The first map shows the positions of some genetic markers on the physical map of the ~ chromosome. OL and PL are the operator and promoter region at which transcription of the leftward message is initiated. The second map shows the positions of the restriction targets; those for endo R.EcoRI are above the map, those for cndo R.Hind_III are below it. Phage (a) to (c) provide receptors for the R.Hi~.BTTT system and (d) to (f) for the R.EcoRI system. Gaps indicate DNA deletions, double lines DNA substitutions, and 9 the position of the phage attachment region. Phage (a) AVsrI~tl-2sl~flTl~;t3+a~+imm=zni~RSsh~ndIII;t6 ~ (phage no. 540). Phage (b) ~b2immXVshi~dl~4-Sni~RSshi~dT[IA6 ~ (phage no. 573, which has a single target for endo R.Hi~dTTT within ~mmx). The fragment left of 8h~-3 (from phage (a)) and the fragment right of ahn-5 (from phage (b)) together form a receptor chromosome with a 36% deletion. Phage (c) AV~I~tl-2sh~dTTT~3+a~+imm~Vsh~ndTTT~4-6n~nRSsh~d~6 ~ (phage no. 554), provides the same receptor by loss of the DNA between shn-3 and shn-5. Phage (d) AVsrIA1-2a~+srIA3~176 ~ (phage no. 426). Phage (e) ~rrI~lOb527arIA3O~mm484(arI434+)#rIA4~ ~ (phage no. 518). The fragmen6 left of ariel (from phage (d)) and that right of srI434 (from phage (e)) together give a receptor lacking approximately 40% of the A chromosome. Phage (f) AV~'IA1-2o~ff+arI~3~ +)arIA4~ ~ (phage no. 567) provides the same receptor by loss of the DNA fragment be~wcen ariA1 and the R.EcoRI target in ~mm"s~.

569.

K. MURRAY

AND

N. E. MURRAY

chromosome shown in Figure 3(b) is also deleted for the DNA comprising fragment G, so that only two fragments result from the action of endo R.HindIII. This system relies on the use of separated fragments (Skulks, 1971; Murray & Murray, 1974) but permits the deletion of nearly 4 0 ~ of the ~ chromosome and demands the incorporation of an appreciable fragment of DNA to produce a chromosome that can be packaged into a phage head (BeUett et aL, 1971; Thomas e~ al., 1974). The two fragments for this receptor can be provided from a single phage (see Fig. 3(c)) by replacement of its central fragment of DNA. (d) Other receTtor phages for D N A fragments made with endo R.EcoRI A system analogous to that described above is available for endo R.EcoRI and uses the left-hand fragment from an 8rlhl + phage (Fig. 3(d)) and the right-hand fragment from a ~imm~34srI~4~176 phage (Fig. 3(e)) ; the latter fragment results from breakage at the endo R.EcoRI target within imm ~8~. Again, nearly 4 0 ~ of the )~ genome is deleted. Alternatively, the left and right-hand fragments of a single phage (Fig. 3(f)) will provide the same receptor. It is, of course, possible to insert DNA fragments generated by digestion with both endo R.EcoRI and endo R.HindIII between an appropriate combination of receptor fragments. 4. Discussion Phage h has been shown to provide receptor chromosomes for fragments of DNA generated by endo R.EooRI (Murray & Murray, 1974; Rambaeh & Tiollais, 1974; Thomas e~el., 1974). With this system the insertion of DNA at 8rI~3 (Fig. 3) destroys a phage recombination gene, redA, and the resulting Red- phage may be detected by their inability to form plaques on a polA strain of E. ooli (Murray & Murray, 1974). Alternatively, transducing phages may be selected by demanding the incorporation of extra DNA in order that the phage chromosome can be packaged (Thomas et eL, 1974). The transducing phages so isolated have the donor DNA inserted in the N operon of phage ~, which is transcribed from the efficient h promoter, P~. (Frs.nlr]~n, 1971; Davison es eL, 1974) but they have lost the phage attachment region and are therefore unable to integrate into the E. coli chromosome in the normal way. 1%r the following reasons we have developed an additional receptor system using DNA fragments generated by endo R.HindIII. First, to provide a receptor for a different spectrum of DNA fragments. Second, the cohesive ends generated by endo R.HindIII contain two G. C pairs and therefore form more stable hydrogen-bonded joints. As predicted, we have found that fragments generated by endo R.HindIII join more readily than those generated by endo R.EcoRI (J. D. Beggs, W. J. Brs.mmar, K. Murray & N. E. Murray, unpublished observations), and this has provided a more efficient recovery of transducing phages. Third, the transducing phages in which DNA has been inserted at shn-3 should retain a normal attachment region (K. Borck, personal communication) and hence should be able to integrate into and excise from the host chromosome. In a )t lysogcn the bacterial genes beyond the attachment region can be transcribed from the )~ promoter, P~., under the control of the N gene protein (Adhya et el., 1974). We would expect that bacterial genes inserted into phage to the left of the attachment region would be similarily expressed. The other receptors we describe for both the EcoRI and HindIII systems are of interest because they permit the incorporation of large fragments of DNA (Mr values

P H A G E h R E C E P T O R CHROMOSOMES

563

up to 15 • 106). Replacement of the central fragment of DNA from either phage 3c or phage 3f will delete not only part of the cI gene, cIII and gene/V, but all the recombination genes (see Fig. 3). The resulting phages lose the ability to plate on a recA strain (i.e. they are l~ec-), but lmlil~e )~+ they form plaques on a phage P2 lysogen (i.e. they are Spi-) (Zissler e~ al., 1971). This provides a ready means of screening for those phages t h a t have replaced the )~ DNA fragment with donor DNA (W. J. Brammar, personal communication), but the inability to grow on a recA host is disadvantageous for studies of repetitive I)NA sequences. I t should, however, be noted t h a t most such Spi- phages make very small plaques until they acquire a chi mutation (Henderson & Weft, 1975; Lam e~ al., 1974). An alternative Fec § receptor for endo R.EcoRI fragments in which the DNA between R.EcoRI targets 1 and 3 is deleted in the presence of imm 21 and the ninR5 deletion provides space for DNA fragments with molecular weights up to about 12 • l0 s. We are grateful to those colleagues who generously provided phage and bacterial strains, to Sandra Bruce and Graham Brown for excellent technical assistance and to other members of the Department, particularly W. J. Brammar, for constructive criticism and discussion. The work was supported in part by the Science Research Council and the Medical Research Council. Note added in proof: Since this manuscript was submitted for publication, AUet & Bukhari (1975) have published values for the positions of targets in h § DNA for endo R . H i n d I I I which agree well with our results.

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Franklin, N. C. (1971). I n The Bact~dolvhage Lambda (Hershey, A. D., ed.), pp. 621-638, Cold Spring Harbor Laboratories, New York. Fr~.nk|in, N. C. & Dove, W. (1969). Genet. Res. 14, 151-157. Garfin, D. E. & Goodman, H. M. (1974). Biochem. Biophya. 1~es. Gommun. 50, 108-116. Goldberg, A. R. & Howe, M. (1959). Virology, 38, 200-202. Georgopoulos, C. P. & Herskowitz, I. (1971). In The Bacte~ophage Lambda (Hershey, A. D., ed.), pp. 553-564, Cold Spring Harbor Laboratories, New York. Hedgpeth, J., Goodman, H. M. & Boyer, H. W. (1972). Prec. N ~ . Acad. Sc~., U.S.A. 69, 3448-3542. Henderson, D. A. & Weft, J. (1975). Gene~/c~, 70, 143-174. Hershfield, V., Boyer, H. W., Yanofsky, C., Lovett, M. A. & Helinski, D. R. (1974). Prec. Nc~. Acad. Sei., U.S.A. 71, 3455-3459. Kaiser, A. D. (1962). J . MoL Biol. 4, 275-287. Kaiser, A. D. & Hogness, D. S. (1960). J . Mol. BIOL 2, 392-415. Kaiser, A. D. & Jacob, F. (1957). Virology, 4, 509-521. Kellenberger, G., Ziehichi, M. L. & Weigle, J. (1960). Nature (London), 187, 161-164. Lam, S. T., Stahl, M. M., MeMi]in, K. D. & Stahl, F. W. (1974). (~enetica, 77, 425-433. Lennox, E. S. (1955). Virology, 1, 190-206. Liedke-Kulke, M. & Kaiser, A. D. (1967). Virology, 32, 475-481. Mandel, M. & Higa, A. (1970). J . Mol. Biol. 53, 159-162. Morrow, J., Cohen, S. N., Chang, A. C. Y., Boyer, H. W., Goodman, H. M. & Hefting, R. (1974). Prec. Nc~. Acad. Sci., U.S.A. 71, 1743--1747. Murray, K. (1973). Bioehem. J . 131, 569-583. Murray, N. E. & Murray, K. (1974). Nature (London), 251, 476-481. Murray, N. E., Manduca de Ritis, P. & Foster, L. A. (1973). Mol. Gen. Genet. 120, 261281. Old, R., Murray, K. & Roizes, R. (1975). J . Mol. Biol. 92, 331-339. Par~naon, J. S. (1968). Genet/cs, 59, 311-325. Park~n~on, J. S. (1971). J . Mol. Biol. 56, 385-401. Philippsen, P., Streeck, R. E. & Zachau, H. G. (1974). E~r. J. Biochem. 45, 479-488. Rambach, A. & Tiollais, P. (1974). Prec. Na~. Acad. Sd., U.S.A. 71, 3927-3930. Richardson, C. C. (1965). Prec. Na~. Acad. Sci., U.S.A. 54, 158-165. Sharp, P. A., Sugden, B. & Sambrook, J. (1973). Biochemi,st~'y, 12, 3055-3063. Skalka, A. (1971). I n Me~heds in Enzymology (C