Physical studies of lysogeny

J. Mol. Biol. (1968) 35, 413--438

Physical Studies o f Lysogeny HI. A Biophysical Study of k Lysogeny using P1 Transduction R o s s B. HODGETTS AND IRWIN RUBENSTEIN

Department of Molecular Biophyslas, Yale Univeraity New Haven, Connecticut, U.S.A. (Received 20 December 1967, and in revved form 29 April 1968) The temperate phage P1 is capable of excising fragments from the bacterial genome and including these fragments in P1 phage protein coats. By conAnlng our attention to those particles containing the galactose gene of the bacterium, we exR.mlned events at the A prophage attachment site. A phage, with DNA labeled with 5-bromodeoxyuridine and therefore heavier than unsubstituted DI~A, were used to infect a sensitive strain of Esch~z/ch/a col~ K I 2 under conditions yielding a high lysogenic response. A lysate of P1 was then prepared b y superinfecting these cells with P1. The density of those P1 phage containing the galactose region of the chromosome and the adjacent A prophage was investigated in a CsC1 gradient. From the density profile the following conclusions were drawn. Parental ADNA can be found in the prophage state. The size of this parental fragment was 0.8 -4- 0.2 ADNA equivalents if one assumed the DNA was a parental molecule which had undergone a single round of replication either before or after being reduced to the prophage state. The other alternative, that the fragment consisted of a piece of unreplicated parental DNA, 0.4 Agenome long, was not excluded. The percentage of the total number of prophages excised by P1 that contained a hybrid parental fragment was in the range of 2 to 31%, depending on the multiplicity of A infection. 1. I n t r o d u c t i o n The physical experiments of Hoffman & Rubenstein (1968a,b) have demonstrated a n association between parental A D N A and large pieces of intracellular D N A present in cells destined to become stable lysogens. The term, parental DNA, designates A D N A from either one or both of the strands of D N A t h a t were present in the A phage used to infect the sensitive host bacterium. These authors did not show t h a t the interaction t h e y detected involved a specific association of phage and bacterial DNA. I t is conceivable that, in part, the phenomenon studied b y Hoffman & Rubenstein (1968a,b) involved an interaction of parental virus D N A and a concatenation of newly synthesized phage D N A (Smith & Skalka, 1966), since such a structure would have been physically indistinguishable from the bacterial DNA. I n addition, their approach could not h a v e indicated whether the association occurred at the A~prophage a t t a c h m e n t site. Therefore, using P1 transduction, this work was initiated in an a t t e m p t to detect in the system of Hoffman & Rubenstein (1968a,b) a specific interaction of parental virus D N A with the galactose region of the chromosome. Finding such a specific interaction would i m p l y t h a t the physical association between viral ~nd intracellular DI~A t h a t 413

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R. B. HODGETTS AND I. R U B E N S T E I ~

they studied involved, in part, s non,random interaction of virus DNA with the bacterial chromosome. Furthermore, it was felt that the technique of P1 transduction could be extended to ascerta/n the frequency of occurrence of prophages contalnlng parental DNA and the size of the parental fragment.

2. Rationale Due to their use of the phenol method of extracting intracellular DNA from ~infected bacteria and to the pattern of isotopic labeling used, Hofrman & Rubenstein (1968a,b) were unable to differentiate between bacterial DNA and any newly synthesized viral DNA structure that physically resembled it. Our approach to the problem involved the use of the generalized transducing phage PI. The ability of this phage to excise fragments of lmlform length from the bacterial DNA and include these in normal phage coats (Ikeda & Tomlzawa, 1965a) enables one to exarolne events at a specific site on the bacterial DNA. These P1 phage, containing bacterial DNA, are called transducing particles and have been shown b y Ikeda & Tomizawa (1965a) to contain little or no P1 DNA. In our work, attention was focused at the ~ prophage attachment site which occurs near the galactose (gal) fermentation gone (Lederberg & Lederberg, 1953). Therefore transducing particles carrying this gone, Pl-gal, formed during the growth of P1 on a bacterium newly lysogenized b y ~, were examlued genetically and physically. It was reasoned that if parental ~ DNA, labeled with BUdRt, could be reduced to the prophage state, Pl-ga/particles resulting from the superinfection b y P1 of newly established ~ lysogeus would reflect by their density the pro~imlty of BUdR DNA to the galactose gone. Hence the presence of parental ~ DNA adjacent to the galactose gone could be detected by comparing the buoyant density of the Pl-gal particles formed in cells recently lysogenized b y B U d R ~ phage with the density of those Pl-gai particles formed in cells infected with unlabeled ~ phage. More detailed information as to the nature of the ~ prophage can be obtained by studying P1 transducing particles carrying both the ~ prophage and the galactose gone. The DNA fragment in P1 transducing particles has a uniform length which is approYimately equal to twice the length of the )t genome, and it is possible (Jacob, 1955; Rothman, 1965) to find joint P1 transducing~particles (Pl-gal-~) carrying both the gal gone and s functional )t genome. Let us suppose the ~ prophage could consist of any one of the following: ~ DNA in which both strands were labeled with B U d R (heavy); ~ DNA in which just one strand was BUdR-labeled (hybrid) and )~ DNA in which neither strand contained BUdR (light). The Pl-gal-~ joint transducing particles formed in lysogens containing such prophages have unique densities, which differ from each other by equal increments. Thus the presence of a given density class of prophage would in theory be discernible from a CsC1 density-gradient profile of the P'l-ga/-~ particles. The shape of this profile should enable one to draw conclusions regarding the ability of certain replicated forms of parental )~ DNA to associate with the chromosome and allow one to estimate the relative frequency of different prophage density classes; assumlug of course that P1 can excise, incorporate and transduce BUdR fragments of DNA with the same ef~ciency as unlabeled I)NA. t Abbreviations used: BUd_R, 5-bromodeoxyuridine; m.o.i., multiplicity of iBfection; p.f.u., plaque-forming unit; et.m.u,t atomic mats u~ite.

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3. Materials and Methods (a) B a c ~ r ~ l a ~ r ~ Most of t h e b~cterial strains used in this w o r k were d e r i v e d in our l a b o r a t o r y from. single parent. This w a s an Esch6r/ch/a co~i 1~12 derivative, s t r a ~ W3110, which was~ obtained from Miss K a y Fields a t M.I.T. This prototrophic strain was sensitive ~t o b o t h A a n d P1 phage. The gaZ- a n d / v / s - strA.in~ were spontaneously revertable a t m u t a t i o n rates of 4 to 6 × 10-11/bacterium/generation. Strain CR34(A)/A t h y - was also o b t a i n e d from Miss K a y Fields a t M.I.T. Since t h e strain was a thymlne-less m u t a n t , i t was convenient to use it for t h e p r e p a r a t i o n of B U d R as well as light A phage stocks. C600 was used to assay for t h e A lysogenlC response (Hoffman & Rubenstein, 1968@). (b) Bact~iol~h~7~ s~ra~ns The ~ phage used was ~ " w i l d - t y p e " of K a i s e r (1957), which F o x & Meselson (1963) r e p o r t is t h e phage carried b y CR34 (A)/A. The P1 phage used was P1 kc o b t a i n e d from D r F . R o t h m a n a t Brown University.

(c) M e d ~ phage stocks were p r e p a r e d in t h e " A " m e d i u m of F o x & Meselson (1963). This m e d i u m was also used as t h e pre-infection growth m e d i u m in t h e early superlnfection experiments. B o t h A a n d P I stocks were stored in t h e phage resuspension m e d i u m o f Weigle, Meselson & Paigen (1959). T h e complete m e d i a used were T r y p t o n e b r o t h (Weigle e~ a/;, 1959) a n d L u r i a b r o t h as modified b y F r a s e r (1957). The rn;n~mal m e d i u m used was t h a t of Burgi (1963). I n order to use t h e m~n~rnal meditun for bacterial growth prior to A lysogeniza~ion, it was desirable to a d d to it an a.m~uo acid e x t r a c t p r e p a r e d from ~ . c~Zi. The procedure inv o l v e d growing 100 to 200 ml. of W3110 to s a t u r a t i o n in m;n;mal medium. The bacteria were t h e n centrifuged a n d resuspended in 10 to 20 ml. of 0.85% saline a n d p o u r e d into 200 ml. of chilled acetone. A f t e r 10 rn~n in an ice b a t h , t h e precipitate was collected on W h a t m a n no. 1 filter paper, washed w i t h 25 ml. of ether a n d dried. The residue was scraped off t h e filter p a p e r a n d dissolved in 10 ml. o f 6 ~-HC1 a n d h e a t e d a t l l 0 ° C for 18 h r in an e v a c u a t e d vial. The m a t e r i a l was t h e n lyophillzed a n d t h e residue t a k e n up in 5 to 10 ml. of distilled water. To this, 0.04 g of N o r i t A charcoal (Fisher) was a d d e d per 10 ml. liquid a n d stirred into t h e e x t r a c t for 30 rn;n. The e x t r a c t was t h e n freed of t h e charcoal b y centrifugation a n d sterilized b y MAll:ipore filtration. The concentration of t h e e x t r a c t in t h e rn~n~rnal m e d i u m was 23~/o(W/W), b a s e d on a n optical deusit~ of 1.3 a t t h e 273 m ~ p e a k in t h e ultraviolet spectrum of t h e extract. The rn;u;mR.l a g a r was p r e p a r e d according to R o t h m a n (1965) w i t h o u t t h e indicator d y e a n d t h e A agar according to HoffInan & R u b e n s t e i n (1968a). P1 b o t t o m l a y e r agar was t h e b o t t o m layer a g a r described b y R u b e n s t e i n (manuscript in preparation), a n d t h e t o p layer was ~denticalexcept for a 0.65~o agar concentration. f

(d) P~'elgara$ion of bacteriophage stocks (i) Ligh$ ~ bacSsriophage Stocks of light ~ were p r e p a r e d b y induction of CR34(~)/~ using t h e procedure of Hoffman & Rubeustein (1968a) a n d characterized b y their optical cross-section a t 260 m~, uncorrected for light scattering. Values between 0.6 a n d 0 . 8 × 10 -xx cma/p,fiu, were routinely observed.

(ii) Heavy (BUdR) ~ bact~riopha4s CR34(~)/~ was grown to 2 × 10 ~ bacteria/ml, in A m e d i u m supplemented w i t h 3 ~g thymidine/ml. The culture was centrifuged, resuspended in A m e d i u m with 0 . 7 ~ g t h y m i dine/ml, a n d shaken for 60 min a t 37°C. (It was found a t ~a late stage in this w o r k t h a t highly s u b s t i t u t e d phage w i t h absorbances of 0.8 × 10-~cm~/p.f.u. could be o b t a i n e d b y omitting t h e 60-min growth period in a low-thymidlne medium. Most of t h e experim e n t s r e p o r t e d here, however, were conducted w i t h less v i a b l e B U d R stocks, t h e optical cross-sections of which r a n g e d between 1.2 a n d 1.9 × 10-~em~/p.f.u.) The culture was t h e n centrifuged, resuspended in A m e d i u m a n d given a 130-sec inducing dose o f

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ultraviolet light, 82 om from a Sylvania G15T8 lamp. To t h e culture were t h e n a d d e d 10 gg 5-bromodeoxyuridine]ml., 7 gg deoxyuridine/mh a n d 0.35 gg thymidine/ml, a n d a p h a g e lysate was allowed to develop with vigorous shalcing in a 37°C incubator. Purification of the lysate w a s the same as for the light X stocks. The D N A of such phage stocks b a n d e d in a relatively homogeneous p e a k (Ho~rnA.n & Rubenstein, 1968a), a n d t h e density of this DI~A was determined b y analyfiioal equilibrium sett;mentation to be 0.081 g c m - s heavier t h a n unsubstituted ~ D N A .

(iii) 17o~-rad~oactivs P1 bac~r~hags Stocks of P I were p r e p a r e d b y t h e agar scraping technique (Adams, 1959) 7 h r after the plates h a d been poured. T h e y were stored in modified L u r i a broth, after two centrlfugations a t 6000 rev./min h a d eliminated most of t h e agar. The titers of 1 to 2 × 10 x° p.f.u./ml, remained constant for a t least 3 weeks when t h e stocks were stored a t 4°C. Before using such stocks, t h e y were heated a t 45°C for 3 min to eliminate aggregation of t h e phage. (iv) Radioactive a~P.lab~lexZP1 bacX~ophag6 Such phage were prepared according to the m e t h o d of I k e d a & Tomlzawa (1965a) using minimal m e d i u m supplemented with 0.05% Difco CasA.mlno acids. L y s a t e s were harvested 2 h r after P1 adsorption b y t h e addition of lysozyme to a final concentration of 10 ttg/ml. The lysate was freed of bacterial debris b y a low-speed centrifugation a n d t h e phage were concentrated b y a 45-rain 20,000 rev./min centrifugation, a n d then extensively dialyzed against t h e phage resuspension medium. These stocks h a d a b o u t 10 -4 ct/mln/p.f.u. Since such stocks were used solely as density markers, t h e y were i r r a d i a t e d with ultraviolet light down to a surviving fraction of 10 -'q.

(e) Hra.n~dv~io~ techn61v~ The procedure used was essentially t h a t of Lennox (1955). However, for the analysis of Pl-ga~ particles in t h e later experiments, which involved t h e determination of transducing a c t i v i t y in the m a n y fractious collected from a gradient, it became desirable to eliminate t h e eentrifugation steps, which consumed a great deal of time a n d introduced large q u a n t i t a t i v e errors. Therefore, routine analysis of a gradient involved assaying 12 fractious a t a t i m e b y adding to each an equal volume o f a s a t u r a t e d W S l 1 0 9al-A-]~ recipient culture in modified L u r i a b r o t h + 5 × 10 - s M-CaCI~ a n d incubating 20 min a t 37°C without agitation. The samples were t h e n p u t directly into ice a n d portions p l a t e d on minimal agar -t- galactose. The n u m b e r of t r a n s d u c t a n t s d i d n o t v a r y from platings m a d e immediately after icing to platings m a d e after 60 min in ice. Control experiments showed t h a t even as much as 0.4 mh of t h e infected recipients could be spread w i t h o u t appreciable background growth occurring on t h e plates. To assay for Pl-ga~-~ particles two methods were employed. One simply involved picking t h e ga/+ t r a n s d u c t a n t s with sterile toothpicks a n d spotting t h e m on a ~ agar p l a t e on which a t o p layer agar containing W3110/P1 indicator bacteria h a d j u s t been poured. After 150 colonies h a d been spotted, t h e plates were given a 80-see dose of ultraviolet light a t a distance of 82 cm from a Sylvania G15T8 lamp. U p o n incubation a t 87°C, large halos a p p e a r e d around the areas on which joint ga~-;t t r a n s d u c t a n t s h a d been spotted. A far less laborious technique involved irradiating (as above) t h e minimal plates on which t h e gaZ+ colonies were growing a n d t h e n overlaying t h e m w i t h 2.5 ml. of top-layer agar containing W3110/P1. A small experimentally determined a m o u n t of anti-~ serum was a d d e d to t h e t o p layer j u s t before pouring to confine t h e halos which subsequently developed to t h e vicinity of the transduced 9al+ clone. Regardless of t h e assay used, an a t t e m p t was m a d e to keep t h e n u m b e r of gal + transd u c t a n t s p e r plate between 100 and 150. W h e n higher numbers occurred, spreading o f from lysogenie clones caused non-lysogenic clones to a p p e a r as lysogeus a n d a b n o r m a l l y high joint transducing frequencies were noted. Reconstruction experiments w i t h W3110 a n d W3110(~) indicated t h a t when the n u m b e r of colonies per plate was less t h a n 150, t h e colonies were generally sufficiently well separated so t h a t no problems of cross-cont a m i n a t i o n arose.

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(f) CaOl de~y.grad~n~ cent~fuga~ion I n order to b a n d P1 phage n e a r t h e center of t h e gradient in either t h e Spineo 50 swinging b u c k e t rotor or t h e 50 angle-head rotor, t h e following formula was used: 3.582 g of a s a t u r a t e d (at 25°C) CsC1 solution in phage resuspension medium, a n d 1.868 g P1 lysate in phage resuspension medium. All gradients were r u n a t 20°C in t h e Spineo model L2-65 centrifuge, t h e rotor being allowed to come to rest w i t h o u t braking. W h e n t h e SWS0 rotor was used, 16 to 18 h r a t 23,000 rev./mln was sufficient to bring t h e phage to equilibrlum. The gradient so o b t a i n e d was 0.0014 g cm-3/fraction, each fraction representing 5 drops collected through a cannula which was a size 21 needle w i t h squared ends. W h e n the 50 angie-head rotor was used, t h e eentrifugations were carried out a t 32,000 rev./min a n d equilibrium was a t t a i n e d in 12 to 15 hr. The gradient, unlike t h a t in t h e swinging b u c k e t rotor, was n o t linear over the whole t u b e ; this agrees with a similar observation of F]Amm, B o n d & B u r r (1966). The average value of t h e gradient in t h e region between 70 a n d 200 drops was 0.00088 g c m - 8 / 5 - d r o p fraction. (g) E:~periment~d procedurefor the P1 super~nfecZion ~xpor~n~nts Following t h e m e t h o d of Hoffman & R u b e n s t e i n (1968a) for a t t ~ n l n g a high ~tlysogenic response, a s a t u r a t e d overnight culture of W3110 in A m e d i u m was diluted 1/20 into the same m e d i u m a n d allowed to grow to a concentration of 5 × 108 bacteria/ml. The culture was centrifuged, washed a n d resuspended in 0.02 M-MgSO4 a t a concentration of 109 bacteria/ml. Following a s t a r v a t i o n period of 60 rnln in a 37°C incubator-shaker, ;I phage were adsorbed w i t h o u t agitation to t h e e x t e n t of 99o//o of t h e i n p u t in a 37°C w a t e r b a t h for 16 rain a t t h e desired multiplicity. Growth conditions were established for t h e infected cells b y t h e a d d i t i o n of an equal volume of 2 × T r y p t o n e b r o t h ; th~s was d e ~ s ~ n ~ d a~ time zero. The ~ response was assayed b y t h e two-layer technique of Hoffman & R u b e n stein (1968a) a t 28 rain. A t i m e of 30 mln was chosen for P1 superinfection (see Results) a n d t h e phage were adsorbed for 15 mln in t h e T r y p t o n e broth, which was m a d e 2"5 X 10-'~M in CaC12. Following centrifugation a t room t e m p e r a t u r e a n d resuspension in w a r m e d medium, which t o o k 10 min, t h e doubly infected b a c t e r i a were shaken in T r y p t o n e b r o t h a t a concentration of 1 to 3 X 107 bacteria/ml, in order to maYirnlze t h e yield of P1 phage. After a growth period of 80 min (see Results), t h e unlysed bacteria were t h e n centrifuged a w a y from t h e P1 phage (a t o t a l of 105 rn;n after t h e a d d i t i o n of P1). W h e n density-gradient analyses were to be carried o u t on t h e P1 lysate, the supern a t a n t fraction was concentrated b y a 45-rnln centrifugation a t 20,000 rev./m~n in a Spinco 30 angie-head rotor. Resuspension of t h e phage pellet occurred in phage reSuspension m e d i u m a t 4°C for 1 h r in t h e presence of a 1/100 dilution of anti-~ serum (Kvalue, 24 r a i n - l ) . A final low-speed centrifugation e]imlnated m o s t of t h e remaining bacterial debris. Since we discovered t h a t Pl-ga~ transducing particles were u n s t a b l e in storage, CsC1 ~anding of P1 lysates a n d analysis of biological a c t i v i t y in t h e gradient fractions were performed w i t h o u t delay. The above experimental system was used t h r o u g h o u t t h e early par6 of this work, in which an a t t e m p t was m a d e to detect p a r e n t a l ~ D N A associated with the prophage a t t a c h m e n t site in P l - g a l transducing particles. The conditions for t h e establishment of lysogeny were similar to those used b y Hoffman & Rubenstein (1968a). However, in t h e l a t e r experiments w i t h Pl-gaZ-~ particles t h e physiological conditions were altered in order to enable us to investigate t h e n a t u r e of bacterial D N A synthesis in newly es. tablished ~ lysogeus a n d to l a y t h e groundwork for future physical experiments. A defined growth m e d i u m was used prior to lysogenization to p e r m i t t h e use of 15N as a bacterial D N A m a r k e r ; this allows one to follow t h e replication of t h e bacterial chromosome after infection. However, u p o n substitution of minimal for A m e d i u m as t h e pro-infection growth medium, a high lysogenic response was n o t obtained until t h e s t a r v a t i o n period in MgSO4 was increased from 60 to 80 rnln a n d t h e post;infection growth m e d i u m was changed from T r y p t o n e b r o t h to t h e much richer modified L u r i a broth. Although a d e q u a t e lysogenie responses were obtained using these conditions, a further i m p r o v e m e n t was obtained b y supplementing t h e minimal m e d i u m with a bacterial Amino acid e x t r a c t (see Materials a n d Methods). These alterations in t h e physiological conditions m a d e i t necessary to reduce t h e P1 superlnfection t i m e from 30 to 20 m l , a n d t h e P1 adsorption t i m e from 15 to 10 min as discussed in Results.

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4. R e s u l t s (a) Determination of the biologicalparameters in the superinfection system Before density-gradient analyses could be carried out on P1 lysates formed b y the superinfection of newly established h lysogens, several important biological parameters had to be determined. I t was essential to minimize the time interval during which P1 could excise the )t prophage region of the chromosome since bacterial DNA replication, either before the addition of P1 or during the growth of the P1 lysate, would tend to dilute the amount of B U d R DNA in the h prophage region. I t was therefore desirable to harvest P1 lysates after a single growth cycle, which necessitated the determination of the one-step growth curve for P1 on newly established h lysogens. The results indicated t h a t when a superinfection time of 30 minutes was used in the system of Hoffman & Rnbenstein (1968a): (a) an average burst size of 43 P1 phage]bacterium was obta4ued; (b) the latent period was completed 60 to 65 minutes after the addition of P1; (c) the rise period was essentially complete 120 minutes after the addition of P1. On the basis of this experiment, it was decided to harvest P1 lysates 105 minutes after the addition of P1, at which time 80~/o of the P1 progeny had been liberated. Next, the optimum time for superinfection was determined. Since exclusion phenomena are known to exist amongst different phages infecting the same host (Luria & Delbriick, 1942; French, Lesley, Graham & van Rooyen, 195I), it was conceivable t h a t P1 could interfere with the normal course of events leading to the establishment of ~t lysogeny; or alternatively, ~ might interfere with the growth of the P1 phage. I n Hoffman & Rubenstein (1968b), at all but v e r y high A multiplicities ( ~ 10), the association detected was complete b y 30 minutes; therefore it was felt t h a t the superinfection time should be chosen as close as possible to 30 minutes consistent with the production of sufficient P1 phage to enable quantitative experiments to be done. The first experiment showed t h a t as the time between the ~ and P1 infections was increased from 0 to 60 minutes, the titers of the P1 lysates harvested 105 minutes after the addition of P1 increased nearly tenfold to a value of 10 l° phage/ml. The titers of lysates initiated at superinfection times greater t h a n 60 minutes remained constant at the elevated value. However, since the ~t multiplicity had no apparent effect upon the kinetics of P1 phage production, it was felt t h a t the increase in the P1 titers which accompanied the increase in the superinfection times was not due to exclusion of P1 b y ~, but rather was a result of the starvation period. That this was indeed the case was shown b y an experiment in which no ~ phage were added; the P1 titers in lysates initiated at increasing times after the starvation period increased in much the same manner as previously. In view of the marked variation in the P1 titers it was important to determine the kinetics of formation of the transducing particles, since it was this population in which we were ultimately interested. I t was found that, as a function of the superinfection time, the production of Pl-gal particles did not parallel the production of infective phage. This was true regardless of whether or not the starved ceils were infected with ~t. The frequency of transducing particles in the P1 lysate decreased from 10-6 to 10 -7 at time zero to a constant value in the range of 10 -7 to 10 -6 at a superinfeetion time of 75 mlnutes. The final value corresponded to t h a t obtained in lysates formed in a sample of the bacteria removed and infected prior to the starvation period. We concluded that, regardless of whether or not the ceils were infected with ~, a

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60-minute period of starvation in i~IgS04 significantlyenhanced the capacity of P1 to excise chromosomal markers. Finally, we examined the gal + transductants for the presence of a functional A genome; it was found t h a t the percentage of Pl-gal particles carrying the A prophage varied with the superinfection time as shown in Figure l(a). The final steady value of

t

o

.c_

o

~

• 0 I

I

I

I

25

50

75

IOO

(b) -G

E

~

100

g 0

,2

Time of P1 superinfection (min)

FIG. 1. (a) Percentage of Pl-gal-A particles in the Pl-gal population as a function of the P1 superinfeetion time. - - O - - O--, Am.o.i. = 17; - - @-- • --, Am.o.i. = 5; - - A - - A--, Am.o.i. ----3. (b) Variation in the number of Pl-gal-A particles present in P1 lysates as a function of the superinfection time. A m.o.i. = 17. 5 to 12% agrees well with the value of 5~/o obtained b y R o t h m a n (1965) using P1 transduction of A lysogens which had not been starved in MgS04. B y multiplying the percentage of Pl-gal particles carrying the A prophage b y the n u m b e r of Pl-gal particles in the lysates, the n u m b e r of Pl-gal-A particles in the lysates was obtained (Fig. l(b)). The density shift we expected to see in the Pl-gal population as a result of B U d R A-infection is directly proportional to the n u m b e r of PI-gaLA particles in the P1 lysate. Thus, choosing a P1 superinfection time of 30 minutes maximized the n u m b e r of Pl-gal-A particles and allowed completion of the association of parental A D N A detected b y Hoffman & Rubenstein (1968b). An interesting conclusion m a y be drawn from the fact t h a t there was a finite n u m b e r of Pl-gal-A particles in P1 lysates the growth of which was initiated at time zero (Fig. l(b)). As was mentioned earlier in this section, the P1 latent period was complete 65 minutes after the addition of the superinfecting phage. Therefore, in

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AND

I. RUBENSTEIN

those lysates initiated at time zero, the first P1 progeny had made their appearance b y 65 minutes. Since it is very lmHkely t h a t m a n y transducing phage were formed after the end of the latent period, it m a y be concluded that most of the joint transductants were formed in the first 65 minutes following A infection. Thus we m a y state t h a t A m a y associatewith the bacterial chromosomewithinthe first 65 minutes after infection. (b) Results at the galactose site on the chromosome The investigations outlined so far resulted in the experimental protocol described at the beginning of part (g) in the Materials and Methods section. This experiment was carried out three times using B U d R A infection and twice using light A infection. The resulting P1 lysates were banded in a CsC1 gradient in the Spinco SWS0 rotor, and when equilibrium had been attained, the material was unloaded b y puncturing the tube and dribbling out five drops per fraction into tubes containing 1 ml. of modified Luria broth. Although 60 fractions were collected from the gradient, determinations of P1 infective a n d Pl-gal transducing activity were only made on fractions 20 to 45. I t was unnecessary to sterilize the fractions before assaying for the transducing partitles, since no bacteria were ever found in the region of the gradient occupied b y the P1 band. The results obtained using B U d R A m a y be compared to the results obtained using light A (see Fig. 2). Since such small density shifts were encountered, the most ,

i

l

l

~

i

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i

i

,

i

i

b 5C

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30

/ ,- P1 infective phage 7 /P1 infedcive phage

IO

,,d ,,~ 25

.......

L / I ./"~-...

27

29

31

o 33 27 29 Fraction no.

31

33

35

37

FIG. 2. R e s u l t s a t t h e ga]actose site on t h e c h r o m o s o m e . , L i g h t A infection, m.o.i. = 10; , h e a v y A infection, m.o.i. = 10.

illustrative method of plotting the data turned out to be the cumulative distribution. To obtain this, one starts at t h e h e a v y side of the peak with the fractionwhere biological activity first occurs and, proceeding across the peak, plots the cumulative sum of the material obtained as a function of the fraction number. The pair of curves on the left corresponds to infection with light A, the pair on the right with B U d R A. I t is to be noted that the P1 infective phage curves act as reference density markers in both situations. Although one m a y calculate displacements from these curves in terms of the distances between the centers of mass of the peaks, very nearly the same results are obtained from the horizontal displacements in the

A BIOPHYSICAL STUDY OF ~ LYSOGENY

421

cumulative distributions at the 5 0 ~ point on the ordinate. Since this is so simple and is accurate enough for the present purposes, all results will be given in terms of this parameter. The Pl-gal particle density was displaced 0.5 fraction to the high-density side of the P1 infective phage when light A were used. This non-coincidence in the densities of the Pl-gal and P1 infective particles was routinely observed and has been reported by Ikcda & Tomizawa (1965a) for other markers. With BUdR A infection the displacement between the densities of the Pl-gal and P1 infective populations increased to 1.1 fractions. The presence of BUdR A resulted in an increased mean density of the Pl-gal population compared to the control experiment with light A. (c) Results at the hiztidine site on the chromosome It may be argued that the increased density of the Pl-gal population resulted from the breakdown of some of the BUdR Aphage DNA and the non-specific re-incorporation of BUdR into regions scattered around the bacterial chromosome. This should increase the average density of all P1 transducing particles. That this is not so was demonstrated by examining events at the histidine site, a region far removed from the A prophage attachment site. As a preliminary control experiment, an attempt to co-transduce the his marker and the Aprophage was made. Co-transduction was not demonstrable, in agreement with the finding of Jacob (1955), Lennox (1955) and others that P1 can only co-transduce closely linked markers. The results obtained by analyzing the CsC1 gradient fractions for Pl-his particles instead of the Pl-gal particles are shown in Figure 3. Again the pair of curves on the .

.

.

.

~, .

7a

.

.

.

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~, Pl-hls particles

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.

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22

24

.

,

,

26

28

30 31

33

o//P1 infective phage

35

,

,

,

37

39

41

Fraction no. FIG. 3. Results at, the histidine site on the chromosome. . . . . . . . , L i g h t A infection, m.o.i. =

5;

, heavy

A infection, m.o.i. =

6.

left represents infection with light A; the pair on the right, infection with BUdR A. Unlike the Pl-gal particles, the Pl-his particles in the case of the light Ainfection do not band at a significantly higher density than the P1 infective phage. This was routinely observed, although there is no ready explanation as to why the Pl-h/s population should be less dense than the Pl-gal population grown under identical conditions. The important thing to note is that the presence of BUdR Aphage makes

422

R. B, H O D G E T T S A N D I. R U B E N S T E I N

v e r y little difference in the m e a n density of the Pl-hls particles. Hence breakdown of parental phage label and its r a n d o m re-incorporation into the bacterial chromosome was not detected. Furthermore, later experiments designed to determine the approxim a t e size of the piece of parental D N A in the prophage indicated the presence on the chromosome near the gal site of far too m u c h phage label to be explained b y a breakdown and repair phenomenon. Table 1 indicates the results of all the experiments performed and Table 2 presents the biological parameters involved in these experiments. To summarize the data of Table 1, there was a signfficant density increase in the Pl-gal population when P1 TABLE 1

Banding l~ositions of P l - g a l and P l - h i s particles relative to that of the P1 infective phage in cells lysogenized by light and heavy h bacteriophage

Exp.

A phage

1

Heavy

2

Heavy

3

Heavy

4

Light

5a

Light

6 5b

Heavy Light

Mean banding position of P1 transducing particles relative to P1 infective phage P l-gal Pl-h@ particles particles 1.2 fractions heavier 1-0 fractions heavier 1.1 fractions heavier 0.5 fractions heavier 0.7 fractions heavier -

-

--

-----0-0 fractions 0"1 fractions lighter

The average increased density in the banding positions of the Pl-gal and Pl-h/s transducing particles in bacteria lysogenized with BUdR Awas 0.5 and 0.1 fraction, respectively.

phage were grown on cells recently lysogenized b y h e a v y A phage as compared to light ~. I n addition, Pl-his particles had essentially the same density whether h e a v y or light h phage were used to lysogenize the sensitive bacteria. These results are consistent with an association of parental A D N A with the galactose region of the bacterial chromosome. (d) Bacterial D N A metabolism in newly established ~ lysogens. Modifications of the

physiological conditions Since it was determined t h a t parental )t D N A could be found in the prophage state, we wished to s t u d y the frequency of the event and the approximate size of the piece involved. I t was felt t h a t the biological technique of P1 transduction was still appropriate. However, several changes in the physiological and experimental conditions were made in order to investigate additional properties of newly established lysogens and to increase the resolution in CsC1 gradient analysis.

A BIOPHYSICAL

STUDY

423

O F ~l L Y S O G E N Y

TABLE 2

Biological parameters for the experiments ~ressnted in Table I Response assayed at ~ = 28 rain Exp.

1 2 3 4 5a 6 5b

~ m:o.i.

6 10 I0 5 5 6 5

P1 m.o.i.

2 4 6 2 4 2 4

% lyric bacteria 9 8 19 5 4 8 4

% surviving % lysogeny % lysogenic bacteria in survivors response t 58 56 67 76 63 65 63

95 100 97 97 99 94 99

82 88 76 91 93 86 93

The recovery of bacteria (lyrically bound % surviving) was on the average 73%. Therefore 27% of the infected bacteria were killed by A infection; it is not known whether such cells are capable of yielding P1 progeny. Table 2 demonstrates that generally more than 95% of the survivors contained the ~ prophage. I n ~he later experiments, this value decreased to a value of about 75% (Table 3). ~f % lysogenic response

no. lysogenic survivors × 100 no. lyric bacteria % no. lysogenic survivors ~ no. refractory survivors

The biological system was changed from that described earlier because, for reasons t h a t will b e a p p a r e n t l a t e r , w e w a n t e d t o e s t a b l i s h g r o w t h c o n d i t i o n s t h a t w o u l d e n a b l e experiments concerning the nature of the bacterial DNA replication following k phage i n f e c t i o n t o b e p e r f o r m e d . ]If t h e u n i n f e c t e d b a c t e r i a a r e g r o w n i n a m i n i m a l m e d i u m u s i n g I~N as t h e sole s o u r c e o f n i t r o g e n , t h e n p r o v i d e d t h e p o s t - i n f e c t i o n g r o w t h m e d i u m c o n t a i n s o n l y 14N, t h e d e g r e e o f b a c t e r i a l ] ) N A r e p l i c a t i o n t h a t h a s o c c u r r e d at any given time can be measured by the amount of initially heavy DNA that has s h i f t e d t o t h e h y b r i d - d e n s i t y p o s i t i o n i n a CsCI g r a d i e n t . TXBLE 3

Biological responses in experiments performed with the modified physiological conditions Response at ~ = 30 rnln ~l phage

~ m.o.L

Heavy Heavy Heavy Heavy Heavy Heavy Heavy Heavy Heavy Light Light

3 4 7 8 8 14 15 23 29 7 9

P1 m.o.i.

3 4 5 4 6 6 4 4 6 5 4

% lyric bacteria --29t -23 ----33t --

% surviving % lysogeny % lysogenic bacteria in survivors response 55 59 79 90 64 63 65 49 81 93 88

77 68 82 80 80 78 84 86 58 80 50

--60 -59 -w -59 --

t Since the recovery in these two experiments was ~ 100%, in all probability the lyric response was too high due to a failure to inactivate complete]y some unadsorbed ~ phage. 28

30 ~

30

20

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35 40 Fraction no,

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0

45

FIG. 4. Cesium chloride density-gradient profiles of bacterial DNA extracted from cells infected with ~ bacteriophage. W3110 t h y - bacteria were grown overnight in minimal medium w i t h 3 Fg thymidine/ml, and zsN as the sole source of nitrogen. (At this point the technique of adding a h e a v y amino acid extract h a d n o t been developed. However, the biological response was adequate without the ext r a c t supplement.) The bacteria were diluted 1/20 in the same medium and grown to 3.5 X 108/ml., a t which time [SH]thymidine was added to a specific activity of 13.9 mc/mg. The bacteria were allowed to grow for 40 rnlu, during which time the titer increased to 6 X 10S/ml. They were t h e n centrifuged, washed and starved 80 rain in 0-02 r~-tV/gSO4 a t a concentration of 7 × 10S/m]. Before A infection, a portion was removed for DNA extraction. To the remainder was added unlabeled ~ phage at a multiplicity of 11 phage/bacterium. After 15 min adsorption, the concentration of infected bacteria was reduced to 7 x 107/ml. b y the addition of appropriate amounts of sterile water and 2 X modified Luria broth. This was, as usual, designated as time zero. The culture was t h e n shaken a t 37°C and portions removed at 5, 20, 35, 50, 70 and 120 min for DNA extraction (according to Hoffman & Rubenstein, 1968¢). The bacterial colony formers had doubled b y 120 mir, and, when tested a t 25 rain, 85% of the surviving bacteria were lysogenie for A and 81% of the infected bacteria h a d survived infection and were able to form colonies. W i t h the exception of the uninfected control sample, the DNA samples were t h e n sheared a t 3000 rev./min for 30 mln to reduce the D N A size to about 30 × 103 a.m.u, as determined b y sucrose sedimentation analyses (Hoffman & Rubenstein, 1968a). There were two reasons for this step. First, it was found t h a t unsheared DNA tended to aggregate in the CsC1 gradient---especially in samples t a k e n after 70 rain. Such aggregation resulted in the 3H profile being skewed towards t h e light end of the gradient. Reducing the a m o u n t of loaded material to as little as 0-01 Fg failed to ellminate this interaction, whereas shearing to 30 X l0 s a.m.u, was successful. The second reason for decreasing the size of the D N A to be b a n d e d in CsC1 was to increase the sensitivity of detecting small amounts of replication. The gradients were formed in a Spinco 50 angle-head rotor, using a speed of 42,000 rev./min and a temperature of about 7°C. Equilibrium was attained in 21 hr. Five-drop fractions were collected through a size 21 needle onto filter paper discs, the DNA precipitated with cold trichloroacetic acid and the samples counted in a sclntillatlon counter (Hoffman & Rubenstein, 1968a). Density increases towards the left. . . . . . . . ,33P-labeled light ;I D N A marker; - - , all-labeled bacterial DNA. (a) Pre-infection bacterial DNA (not sheared) versua A DNA; (b) 35 rnln bacterial DNA sample (sheared) veraua A DNA; (c) 70 rain bacterial DNA sample (sheared) v e r ~ s A DNA.

A BIOPHYSICAL

STUDY

O1~ ~ L Y S O G E N Y

425

However, growing the bacteria in a mlu~mal medium prior to starvation resulted in a drastic increase from 10 to 80% in the lyric response. B y increasing the starvation period in 0.02 M-MgSO~ from 60 to 80 minutes, changing the post-infection growth medium from T r y p t o n e b r o t h to modified Luria broth and supplementing the minlmel medium with a bacterial amlno acid extract (see Materials a n d Methods), the lysogenic response was greatly improved. I t was never possible to duplicate the v e r y h/gh lysogenic responses resulting when A medium was the pre-infection growth medium, but lysogen/e responses averaging 60~/o were obtained (compare Tables 2 and 3). Although studies on bacterial D N A synthesis in newly established A lysogens h a v e been done (S~chaud, 1960; F r y & Gros, 1959), the physiological conditions seem to aJ~ect the results significantly. Since our conditions were different from those described b y these workers, we felt it was necessary to determine the course of bacterial D N A metabolism in our system. The experimental rationale outlined above is presented in detail in the legend to Figure 4. This Figure shows the CsC1 profiles obtained b y anglehead centrifugation of the lminfected bacterial DNA, the 35-mluute and the 70-mlnute samples. A light 32P-labeled A D N A marker was admixed with the extracted bacterial D N A to facilitate a simple identification of the h e a v y and hybrid peaks. I n the uninfected sample there is no bacterial D N A in the hybrid position. All the bacterial SH label is banding in a sharp p e a k in the h e a v y position of the gradient. B y 35 minutes bacterial D N A has begun to appear in the hybrid position, indicating t h a t replication has started. B y 70 minutes the original h e a v y position of the bacterial D N A is represented b y a small shoulder on a large hybrid p e a k of DNA.

70

B 50

3C

IC 0

40

8'0

'

~20

Time after ~ infection (rain) FzG. 5. Bacterial DNA replication following Ainfection. The curve indicates the percentage of bacterial DNA that has undergone a single round of replication as a function of the time after A infection. About 85% of the surviving bacteria were ]ysogenic for ~t. To esthnate the percentage of the material that at any given time has shifted to the hybrid position, the following procedure was adopted. Using th e heavy side of the heavy peak as a guide, the light side of this peak was drawn so that a symmetrical peak emerged. (This was justified by the symmetrical shape of the uninfected bacterial DNA peak.) A vertical line representing the exact center of this peak was then drawn. By multiplying the amount of the material to the left of this line by 2 and subtracting the number from the total amount of sH label in the heavy and hybrid positions, we obtained the amount of replicated DNA. This was expressed as a percentage of the SH label recovered from the heavy and the hybrid positions.

~

•

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0

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A B I O P H Y S I C A L STUDY OF A L Y S O G E N Y

427

Figure 5 shows a plot of the percentage of the bacterial D N A t h a t has undergone replication v e r s ~ the time after )~infection. I t is apparent t h a t in a bacterial population destined for lysogeny, bacterial D N A replication resumes almost immediately after growth conditions are re-established. Nearly 95~/o of the D N A has replicated b y 75 minutes, although the colony formers surviving infection do not double until 120 ~nutes. Because of the rapid resumption of bacterial D N A synthesis after A infection, the following modifications in the earlier procedure were made. The P1 superinfection time was reduced from 30 to 20 minutes, and the P1 adsorption time from 15 to 10 minutes. I n this way the amount of bacterial D N A replication was minimized consistent with adequate P1 adsorption and a sufficient interval to allow )~lysogen/zation to proceed essentially to completion before P1 infection. I n addition, the following steps were taken to improve the resolution of the earlier experiments. First, we decided to s t u d y the CsC1 profile of the PI-g~-A particles formed during the growth of P1 on cells recently lysogen/zed with BUdR-labeled phage. The advantage of studying this profile as opposed to the Pl-gal profile is t h a t Pl-gal particles carrying the ~ prophage comprise less t h a n 40% of the Pl-gal population (Fig. 1). Therefore, if prophages containing parental A D N A comprise only a small part of the total number of prophages, t h e y will be more easily detected if the Pl-gal-~, as opposed to the Pl-gal population, is examined. Second, b y carrying out the centrifugation in the Spinco 50 angle-head rotor, we hoped to improve the separation between PI-gal-A particles of different density (Flamm et al., 1966). Third, b y decreasing the fraction size from 5 to 2 drops, we could gain a large improvement in the resolution. A final alteration involved the use of the ultraviolet-inactivated ~P-labeled P1 phage as the reference density marker (see Materials and Methods). A preliminary experiment was carried out t o ascertain whether these alterations in the experimental conditions altered quantitatively the previous results shown in Table 1. Using this modified system, the banding positions of the Pl-gal and P l - h ~ FIG. 6. Percentage of Pl-gal particles carrying the ~t prophage as a function of the fraction number. A si~ificant number of the gal + transductants (and indeed all of them in the tails of the distribution) in every fraction were tested for co-transduetion of ~t. The percentage of joint transductants amongst those gal + transductants tested was plotted against the fraction number. Each point on this curve represented the probability at that density that a phage particle carrying the gul + gene would also carry the ~tprophage. In addition, the normalized total of gaZ+ transduetants in each fraction--including both those tested and those not tested--was obtained by expressing the number in each fraction as a percentage of the total number of gaZ+ transductants recovered from the gradient. This percentage was also plotted as a function of fraction number. In this and all subsequent experiments (i) density increases to the left; (ii)"the vertical arrow indicates the position of the s2P-labeled P1 marker phage; (fii) the biological parameters are given in Table 3; (iv) the horizontal arrow indicates the joint Pl-gal-~ transducing frequency in the lysates before density-gradient centrifugation; (v) the vertical lines indicate one standard devia. tion of the statistical counting errors for a given experimental point; (vi) a Spinco 50 angle-head rotor was used in a Spineo L2 centrifuge and the run performed at 32,000 rev./m~n and 20°C. Two-drop fractions were collected into 1 ml. of modified Luria broth. (a),(b) BUdR ~ m.o.i.'s were 8 and 15, respectively. - - C)-- C)--, Banding profile of the Pl-gaZ particles (normalized to a percentage of the total recovery); - - O ~ O - , probability (expressed as a percentage of the Pl-gaZ particles) of finding a Pl-gal-~t particle at a density corresponding to the fraction numbers on the abscissae. (c) Results of the control experiment with light A (m.o.i. = 9). - - O - - Q - - , Pl-gal-A particles assayed by the picking technique; --C)--C)--, Pl-gaZ-~ particles assayed by the overlaying technique, two separate banding runs being made on the same P1 lysate.

428

R . B. H O D G E T T S

A N D I. R U B E N S T E I N

transductants relative to the 8~p marker were determined. We found once again that "the presence of heavy ~ affected the density of the Pl-gal particles but did not alter the density of the Pl-h/s particles. The increased density in the Pl-gal case corresponded to 0-7 fraction. This is in close agreement with the shift of 0-8 fraction which one would have expected, since the gradient in the angle head was only six-tenths that in the swinging bucket and five-drop fractions were collected in each case.

(e) Resolution of a peak of Pl.gal-~ particles carrying a ~ prophage containing parental bacteriophage DNA Having ascertained that parental )~ DNA was still associating with the bacterial chromosome, several experiments with newly established lysogens were performed and analyzed according to the procedures outlined in the previous section. The P1 lysates were banded in CsC1 and the profile of the percentage of Pl-gal particles carrying the ~ prophage is shown in Figure 6(a) and (b). These Figures present the results obtained using the same stock of BUdR )t phage at two different multiplicities of infection. In Figure 6(a) and (b) the probability (the number of Pl-gal3t expressed as a percentage of the number of galactose transductants) of finding a joint gal-~ transductant, rises to a definite peak on the dense side of the Pl-gal peak which is also plotted in these Figures. In the peak region of the Pl.gal profile, the probability decreases to the frequency found in the loaded material; this input frequency of Pl-ga/-~ particles in the Pl-gal population is designated by the horizontal arrow in both cases. The Pl-gal profile is a symmetrical curve. However, when one plots the product of the two curves seen in Figure 6(a) or (b) to obtain the Pl-gal-;t profile, one finds a distinctive shoulder on the heavy side of the Pl-gal-~ peak. The main peak was caused by Pl-ga/-)~ particles in which a light )~prophage was present. This statement is substantiated b y the banding position of Pl-ga/-~ particles in the control experiments using light )L infection (see the following section). The heavy shoulder was apparently caused by the inclusion of parental ~ DNA in the prophage of the Pl-gal.~ particles banding in this position. The results of the control experiments with light 2 phage are shown in Figure 6(c). This Figure shows the probability curves indicating how the frequency of Pl-gal-~ particles in the Pl-gal population varies with density. The top curve represents the analysis of a gradient in which the gal + transductants were picked in order to be tested for the ~ prophage. The bottom curve represents a second banding of the same P1 lysate, but this time the overlaying technique was used to assay for the Pl-gal-h particles_. (In the results described so far, using BUdR phage, only the first method was used.) The probability curves in Figure 6(c) show very little deviation from the input value. The high values which occurred in the denser fractions when BUdR )~ was used are conspicuously absent. If BUdR )t DNA has associated with the bacterial chromosome near the galactose site, as implied by the above results, then replication of the bacterial chromosome should reduce the fraction of dense-banding Pl.gal-h particles in the Pl-gal-h population. This was tested by infecting an unlabeled bacterial culture with BUdR )~ (m.o.i. ---- 8). The culture was divided into two parts; one was infected with P1 at 20 minutes; the other, 130 minutes after h infection. By this later time the number of cells had doubled. The fraction of dense-banding Pl-gal-~ particles in the early lysate was determined, as will be described later, and was approximately twice that in the

A BIOPHYSICAL

STUDY

OF)t

LYSOGENY

429

late lysate. This result is consistent with the above prediction and implies that the relative amount of chromosomal BUdR label directly influences the fraction of dense-banding Pl-gal-~ particles. It may be concluded that it was the presence in the Pl-gal-~ particles of BUdRlabeled parental A DNA which caused the peaking in the heavy fractions of the Pl-gal-~ probability profiles (see ~ig. 6(a) and (b)). (f) Quantitative analysis of the nature of the ~arental DNA in the ~ propl~]e We had expected the Pl-gal-~ profile to indicate the nature of the ~ prophage; that is, whether it represented unreplicated or once replicated parental DNA, or two or more times replicated ~ DNA. Two classes of Pl-gal-~ particles were detected, namely, those corresponding to the presence of a parental prophage and a fight prophagc. We were uncertain as to which class of parental DNA the material in the Pl-gal-~ shoulder corresponded. Did it represent a prophage containing an unreplicared parental DNA molecule, or a once replicated genome ? Another way of phrasing the question and ultimately the form to which our answers were fitted was this: Given that the fragment of parental ~ DNA on the prophage is a heavy or hybrid molecule, how long must the fragment be in order to yield a Pl-gal-~ particle of the density detected ? Let us now turn to the problem of determining the size of this fragment. The size and density of the parental fragment determlne the resultant density of the DNA in the Pl-gal.~ transducing particle. Therefore, with the knowledge that P1 transducing particles contain DNA twice the length of the ~ genome and from the Pl-gal-2 DNA density, one can determine the size of the parental fragment if an assumption is made about the density of the fragment. However, it was impossible to obtain a sufficient number of Pl-gal-~ particles containing parental DNA to allow us to extract the DNA and make a density measurement on it. As a result we could not obtain the size of the parental fragment directly. The indirect approach we employed involved determining the experimental relationship between the DNA density and the resultant P1 transducing particle density. This relationship allowed us to estimate the DNA density in a Pl-gal-~ particle from the experimentally determinable particle density. To arrive at the relationship, we needed to generate a P1 transducing particle the DNA density of which could be accurately measured and differed from that of normal light bacterial DNA. These prerequisites were ful~Hed by growing P1 on bacteria with chromosomes composed of BUdR-labeled DNA molecules. The density of this DNA could be determined by analytical CsC1 equilibrium centrifugation in a sample extracted before P1 infection. Growth of P1 on such bacteria would result in P1 transducing particles carrying this DNA. The density of these particles could then be measured by preparative CsC1 equilibrium centrifugation. One would then know the change in the P1 particle density that had resulted from a known change in the DNA density. An experiment was therefore designed to produce a bacterial population conta]nlng BUdR-labeled DNA. The experimental details are given in the legend to Figure 7. The BUdR-labeled DNA extracted from a sample of the bacteria just prior to the addition of the P1 phage was banded in a CsC1gradient in the model E ultracentrifuge with fight ~ reference DNA (density 1-707 g cm-3t}. The DNA banded in two peaks This value was taken from the data of Schildkraut, Marmur & Dory (1962) after correcting for an inaccurate estimate of the isoconcentrationradius (Ifft, Voet & Vinograd, 1961).

430

R. B. HODGETTS

AND I. RUBENSTEIN

(see F i g . 7(a) ); t h e d e n s i t y o f t h e l i g h t e r o f t h e t w o p e a k s w a s f o u n d t o b e 1.724 g c m - 3 a n d t h a t o f t h e h e a v i e r 1-739 g c m - 3. N o d e t e c t a b l e a m o u n t o f l i g h t - d e n s i t y b a c t e r i a l D N A w a s f o u n d w h e n t h e m a t e r i a l w a s r u n w i t h o u t t h e l i g h t )~ r e f e r e n c e D N A . T h e peaks were identified by performing another experiment in which DNA extractions

(a)

I Position of heavyA Lighttl reference

I oNA |

1"739 gcm-3

A,,24 II

\

i

(b)

160

9"-Peak 111 /~

"~ 80 d z 417 0

i

i

/~eakII °/

o\ .

oo#O

o ~'

~.

30

h

II

Peaklo

"°° ° 50

70 90 Fraction no.

IlO

1~o. 7. Determ;n~tion of the relationship between P1 DlqA and P1 transducing particle densities. An overnight culture of W3110 thy- in A medium plus 3/~g thymidine/ml, was diluted 1/20 into 40 ml. of the same medium. The culture was grown to 2 × 10e/rn]. The bacteria were centrifuged and the pellet resuspended in 40 ml. A medium plus 10/~g BUdR/ml., 7/~g deoxyuridine/ znl. and 0.35/~g thymidine/ml. The concentrations of these nueleotides were the same as those used for growth of the B U d R ~ phage. The bacteria were grown for 140 rain in this medium, during which time the colony-formers doubled. After one complete generation, 20 ml. of bacteria were removed, killed and the D N A extracted for the purpose of determining its density. Light D N A was added as a density marker and the centrifugation was carried out in a Spinco model E ultracentrifuge at 44,700 rev./min and a temperature of 23-5°C. Pictures with the ultraviolet optics were taken 12]~ hr after the run started and (a) shows the densitometer tracing of the photographs. To the remainder of the bacteria, P1 phage were added at a multiplicity of 5 P 1 phage/bacterium. Adsorption of the phage was carried out for 20 rain in the presence of 3 × 10 -s I~-NaCN2 and 2.5 × I0 -3 x-CaC12. Following adsorption, the culture was centrifuged and resuspended in 60 ml. of A medium plus 3/~g thymidine/ml. After 80 rnln of growth at 37°C, lysozyme was added to a final concentration of 15/~g/ml. and the culture incubated a further 20 mlu, The ]ysate was then freed of bacterial debris by centrifugation and concentrated by a 20,000 rev./rnln~ 50 min centrifugation in the Spineo 30 rotor. Resuspension was carried out in the phage resuspension medium for 25 rnln~ and a final low-speed eentrifugagion eliminated the remaining bacterial debris. The P1 titer was 2.4× 101° phage/ml. About 9 × 102 of these phage were mixed with "q2P-labeled marker P1 phage and banded in CsC1 in a Spineo 50 rotor at 32,000 rev./rnln and 20°C for 17 hr. The Pl-gal particles and the 82P-labeled phage were then assayed in the 2-drop fractions collected. The banding profile of the Pl-gat particles is shown in (b). --C)--C)--, Pl-ga~ particles assayed ~mmediately a f a r CsC1 centrifugation (curve A); ~$--Q~, Pl-ga/ particles assayed sever~ days (at 4°(3) after CsC1 oentrifugation (curve B),

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were made on samples of a bacterial culture taken at various times after the bacteria had been switched from the BUdR-free medium to the one conta~nlng BUdR. The density profiles of the DNA samples, again obtained by equilibrium ultracentrifugation, allowed us to identify the DNA in the denser of the two peaks of Figure 7(a) as DNA in which both strands were BUdR-labeled; the peak containing DNA of density 1-724 g cm -3 corresponded to DNA which was BUdR-labeled in one strand only. Although the density shift resulting from BUdR substitution into both strands of the bacterial DNA was only about one-half the density increment resulting from twostrand substitution of BUdR into A DNA, this is not unusual. Ikeda & Tomizawa (1965a) also found a difference in bacterial and phage (P1) DNA densities, although both had been grown in the same BUdR medium. The density profile of Pl-gal particles grown on such bacteria is given in Figure 7(b). It is apparent from this Figure, curve A, that three distinct peaks were resolved in the CsC1gradient profile of the Pl-gal particles. We concluded from the position of the 32P-labeled P1 marker phage in Figure 7(b) that peak I corresponded to Pl-gal particles with totally light DNA. We explain the presence of light DNA in some of the Pl-gal particles by bacterial DNA replication during P1 growth (Ikeda & Tomizawa, 1965a). Such replication in the BUdR-free post-infection growth medium generated some light DNA which was then excised by P1. The other two peaks apparently corresponded to Pl-gal particles containing the two density classes of DNA detected in the uninfected BUdR-labeled bacteria. Peak II represented Pl-gal particles which had incorporated the bacterial DNA of density 1-724 gcm - a. Peak I I I represented P 1-gal particles which had incorporated the bacterial DNA of density 1.739 g cm-8. One might wonder why the small peak in the Pl-gaZ profile (III) resulted from Pl-gal particles which contained the majority class of bacterial DNA (p ~ 1.739 g era- 3). Two factors could explainthe apparent discrepancy. First, bacterial DNA replication before P1 excision would tend to reduce the amount of totally substituted DNA and increase the amount of hybrid DNA if both species replicated at the same rate. Second, the inclusion in a Pl-gal particle of the more highly substituted BUdR DNA (p ---- 1.739 g cm -3) resulted in an increased lability of the Pl-gal population containing this DNA. Curve B in Figure 7(b) shows the results of re-assaying for the Pl-gal particles after the collection tubes of curve A had been in the refrigerator for several days. It may be seen that whereas the viability of the Pl-gal particles containing light bacterial DNA (peak I) decreased to 7 0 ~ of its value in curve A, the viability of peaks I I and I I I decreased to 25~/o and 15~ of their values in curve A, respectively. The three Pl-gal density classes, I, II and III, banded in fractions 104, 72 and 48 respectively. From a knowledge of the index of refraction gradient the density differences between these three classes of Pl-gal particles were determined (Ifft et al., 1961) and the results were:A p (I,II) ---- 0.0092 g c m - 3 ; ~ p (II,IH) -----0.0082 g cm-8; and /kp (I,III) ---- 0.017 g cm -s. The DNA density was plotted against the density of the transducing particle contaln~ng this DNA and the results are shown in Figure 8. The density of the Pl-gal particle containing light DNA was determined to be 1-470 g c m -3 relative to Aphage the density of which was taken to be 1.50 g c m -3 (Kaiser & Hoguess, 1960). It is to be noticed that the points in Figure 8 fall on a straight line. This con~rms our assignment of the three density classes of Pl-gal particles to the three classes of DNA. For comparisons the dashed lines in Figure 8 show the theoretical variation in

R. B. H O D G E T T S AND I. R U B E N S T E I N

432

i u

1

l

1.750

I

j

s ~

1-74C Z £3

1'73C

u

ao. 1.72c Q.

1-71G I

1"47(

1-475

I

I

1"480 1.48,5 Density of PI particle (cl cm-~)

I

1-490

Fro. 8. Relationship between the DNA density and the P1 transducing particle density. The solid line indicates the experimentally determined relationship. The dashed lines indicate the theoretical relationships when one assumes phage protein densities of (curve A) 1.30 and (curve B) 1.20 g c m -a, respectively.

transducing particle density as a function of the DNA density, assuming t h a t the volume fractions of DNA in the P1 phage and the P1 transducing particles are the same and the volume fractions of B U d R and unlabeled DNA are the same. Two curves are presented to show the effect of varying the value of the phage protein density. B y assuming that the phage protein had a density of 1.20 to 1-30 g c m - 3 a relatively good fit was obtained to the experimental data. I t is now possible to determine where a Pl-gaL~ particle contaln]ng a heavy prophage would band in the CsC1 gradient. The DNA in such a particle is composed of two density classes: B U d R A phage DNA of density 1.788 g cm-3; and normal bacterial DNA of density 1.707 g cm -8. The density of a Pl-gal-~ genome (the total length of which is 2 ADNA molecules) containing a heavy ~ prophage would therefore be 1.748 g c m - 3. Figure 8 indicates the density of a P1 particle with such DNA would be 0.0225 g cm -3 heavier than the Pl-gal-A (light) particlet; in the density gradients generated in these experiments, such a particle would band about 60 fractions heavier than a Pl-ga/-~ (light) particle. Since this was considerably further down the gradient than analyses up to this point had been carried out, an experiment with B U d R ~ (m.o.i. -~ 14) was done similar to those recorded in Figure 6(a) and (b), and analyses were carried out to ensure the detection of Pl-ga/-A (heavy) particles. The results indicated t h a t there were particles containing parental DNA in much the same position as had been detected before, namely, about 21 to 22 fractions removed from the light position. However, there were absolutely no Pl-gal (and therefore no Pl-ga/-~) particles banding heavier than fraction 47. Since the Pl-gal-~ (light) peak occurred in fraction 95, one would have expected Pl-gal-~ (heavy) phage to band in fraction 34.

t Pl-gal-~ particles containing a light A prophage are designated Pl-gal-~ (light), those contalnlng a hybrid prophage are designated Pl-gal-~ (hybrid) and those with a fully BUdR-substituted prophage are designated as Pl-gal-~ (heavy).

A BIOPHYSICAL

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Thus it may be concluded that, under the experimental conditions used, only one class of Pl-gal-2 transducing particle containing parental ~ DNA was found. (i) Size of the ~arental DNA fragment The experimental curve shown in Figure 8 allows us to estimate the size of the parental fragment in the ~ prophage from the experimentally determinable density of the Pl-ga/-h particle containing this prophage. Let us confine our attention to the experiment presented in Figure 6(a). The most accurate estimate of the position of the peak containing parental DNA is obtained from the Pl-gal-~ probability curve. The separation between the Pl-gal-~ (light) peak and the Pl-gal-~ peak corresponding to the inclusion of BUdR parental ~ DNA was 20.5 fractions, which represents a density increase of 0.0068 g c m - 3 in the Pl-ga/-~ particle. From Figure 8 we see that such an increase in the transducing particle density corresponds to an increase in the DNA density of 0-013 g cm -8 (1-707 to 1-720 g cm-8). Let us assume that the fragment of parental BUdR ~ DNA responsible for this increase in the density of the Pl-gal-~ particle is composed of hybrid BUdR-substituted DNA. The density of this hybrid DNA is 1.748 g cm -3. Hence we may write: 1.720 =1/2 z (1.748) -}-1/2 (2 -- z) (1.707) where x is the length of the parental DNA fragment (expressed as a fraction of the length of the Agenome) in the Pl-gal-A particle. Solving this equation yields x = 0.63. When similar calculations were carried out on five other experiments at various multiplicities, the data presented in Table 4 were obtained. T~BL~. 4

Size of the parental fragment in the ~ Trophage m.o.i.

Size of t h e p a r e n t a l fragmen~ (expressed as a fraction of the length of the ~l genome)

4 8 8

0.7 0.6 1.0

14 15 23

0.6 Average 0-8 ± 0"2 0.9 1.1

Under the experimental conditions used, we were able to detect only a single density class of parental DNA in the ~ prophage. The average size of this fragment was 0.8 h genome, if one assumed it was composed of a hybrid (one-strand BUdRlabeled) DNA molecule. There was no apparent correlation between the ~ multiplicity of infection and the size of the parental fragment. (ii) Frequency of occurrence of prophages containing ~arental DNA In order to determine the frequency of prophages containing parental DNA, amongst all the prophages excised by P1, one needs to be able to estimate the size of the peak of Pl-gal-h particles containing parental 2 DNA, relative to the size of the peak of Pl-ga/-~ (light) particles. The method which was developed provided an approximate estimate and is described in the legend of Table 5. In seven experiments

434

R.B.

HODGETTS

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I. RUBENSTEIN

at various values of m.o.i., the relative amounts of parental prophages were computed a n d t h e r e s u l t s a r e s h o w n i n T a b l e 5.

T.~Lv. 5

Relative.frequency of ~ l~rol~hages containing 1~arental DNA ~l m.o.i.

% o£ prophages containing parental ~ D N A

Stock n o . t

4 8 8 14 15 23

5 11 8 31 23

3 1 3 2 1

3 2

3 2

29

t Stock 1 h a d a n optical cross-section: 1-0× 10-11cm2/p.f.u. Stock 2 h a d a n optical cross-section: 1.4 × 10-11em~/p.f.u. Stock 3 h a d a n optical cross-section: 0.8 × 10-11cm2/p.f.u. The following m e t h o d was developed to provide a n approximate estimate of the percentage of ~ prophages containing p a r e n t a l DNA. The Pl-gal-~ profile was generated b y multiplying together the Pl-gal-A probability curve a n d the Pl-gal curve (e.g. Fig. 6(a) a n d (b)). A base line •was d r a w n across the b o t t o m of t h e Pl-gal-~ profile in a n a t t e m p t to correct for t h e trailing tendencies seen in most gradients. Lines parallel to this were drawn across the peak. l~ear the top of the light peak, the midpoints of these lines were m a r k e d a n d a vertical line drawn averaging these points. This line intersected t h e base line a t nearly 90 °. A symmetrical light peak was constructed, a n d the p a r e n t a l peak was drawn b y t a k i n g the difference between the complete profile a n d the constructed light peak. Since the p a r e n t a l peak so generated was nearly symmetrical a n d occurred within several fractions of t h e peak on t h e probability curve, the m e t h o d was considered valid. The complete profile a n d t h e n t h e p a r e n t a l peak were cut out a n d weighed. I n this m a n n e r t h e percentage of t h e parental prophages amongst the t o t a l n u m b e r excised b y P1 was obtained.

As a general conclusion, under the conditions used in these experiments, there was a variation in the frequency of prophages containing parental DNA as the multiplicity of ~ infection varied. Low (~4) and very high (~20) multiplicities resulted in few parental prophages (~-~2 to 5%). The maximum frequency was obtained with m.o.i, values in the range 8 to 15, when 8 to 31% of the prophages excised by P1 contained parental ~ DNA. The:percentages listed in Table 5 may not represent the state of affairs immediately after prophage insertion. It was possible under our conditions for a single round of bacterial DNA replication to have occurred before P1 excision of the ~ prophage region (see l~ig. 5). Therefore at the time of prophage insertion, the percentage of prophages containing parental DNA could have been as high as twice those given in Table 5.

5. Discussion and Conclusions In this work we set out to answer two main questions: (1) Is it possible in the system described by Hoffman & Rubenstein (1968a,5) to detect a specific association of parental A DNA with the galactose region of the bacterial chromosome ? (2) If parental DNA can be found in the prophage state, what can be said about the size of the fragment incorporated and the frequency of this event ?

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We chose to employ the method of P1 superinfection of newly established Alysogens to answer these questions. The presence of abnormally dense-banding Pl-gal particles in lysates grown on cells infected with BUdR A phage was demonstrated. We interpreted this as implying an association of parental ~ DNA with the galactose region of the bacterial chromosome. It may be argued that the finding of the heavy Pl-gal particles does not in itself indicate the presence within these particles of a certain amount, or indeed, of any BUdR A DNA; their altered density could be wholly or partially attributable to an altered proteln/DNA ratio. That the heavy Pl-gal particles do contain some BUdR can be inferred from the following arguments. Ikeda & Tomi~.awa (1965a) found heavy P1 transducing particles only if the bacterial DNA was labeled with BUdR prior to infection. This suggests that the formation of heavy Pl-gal particles in our experiments was a direct result of the inclusion in these particles of BUdR DNA excised from the chromosome. In the experiment in which BUdR A-infected cells were superinfected with P1 before and after chromosomal replication, the fraction of dense-banding Pl-gal-A particles in the lysate initiated prior to replication was twice that in the lysate initiated after bacterial replication. One explanation of this correlation between bacterial replication and the reduction of the fraction of dense-banding Pl-gal-A is that chromosomal replication decreased the relative density of the galactose region, thereby decreasing the fraction of dense Pl-gal-A particles. Although we have presented our reasons for assuming the dense-banding Pl-gal particles contained BUdR DNA, we have not yet examined the possibility that the increased density of these particles was in part due to an altered protein/DNA ratio. Morphological variants of P1 have been described by Walker & Anderson (personal communication) and Ikeda & Tomizawa (19655). However, the density of the major variant class, comprising about 2 5 ~ of the P1 population, is 0.04 to 0-05 g c m -a less than that of the normal P1 infective particle. It seems unlikely that we observed an abnormally dense P1 variant induced by BUdR-labeled A for the following reasons. We found that Pl-gal particles had the same density regardless of whether or not the cells were infected with light A. This indicated that Ainfection Terse was not inducing the formation of Pl-gal particles with an abnormal protein/DNA ratio. That the presence of BUdR ADNA in the cytoplasm was not indirectly affecting the density of the particles is substantiated by the fact that Pl-his particles had the same density regardless of whether or not they were formed in cells infected with BUdR or light A. Further evidence that the BUdR-labeled DNA did not cause an altered protein/DNA ratio in the abnormally dense Pl-gal-A particles was given by the experiment in which the dependence of the Pl-gal particle density upon the bacterial DNA density--altered by partial or total BUdR substitution--was examined. A linear relationship was found (Fig. 8) which implies that particles containing unsubstituted and BUdR DNA have the same protein/DNA ratio, but does not rule out more complex explanations. From the arguments above we conclude that the dense-bandiug Pl-gal particles contain a BUdR-labeled fragment of the A prophage. Since the biological conditions for the establishment of A lysogeny were identical to those employed by Hoffman & Rubenstein (1968a,b), we further conclude that a specific association of parental A DNA with the galactose region of the bacterial chromosome can be detected in their system. Having answered the first question in the affirmative, we improved the resolution of our techniques in an attempt to answer the second question. First, we investigated

436

R. B. H O D G E T T S

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the nature of the bacterial DNA replication following the establishment of ~ ]ysogeny. Under the physiological conditions in which the pre-infection growth medium was a defined medium, only 15~/o of the bacterial DNA had replicated by 30 minutes and at least one round of bacterial I)NA replication was completed 75 to 80 minutes after ~ infection. Second, the parental DNA found in the prophage appeared to be 0.8 -4-0-2 ~t genome long when one assumed that the parental ~ genome had undergone a single round of replication. No prophages containing unreplicated parental DNA of this size class were found. At this point, it is necessary to amplify the above statement. The results of Hoffman & Rubenstein (1968b) indicated that the size of the parental fragment was in the range 0.3 to 2.0 ~ DNA equivalents long. If their lower estimate is correct, then the results of our work could be interpreted in a different manner. We could have assumed the parental fragment consisted of DNA labeled with B U d R in both strands, in which case its length would have been 0.4 instead of 0.8 ~ genome long. If this was actually the case, one would have expected the first replication product of such a fragment to be present. However, our failure to find two classes of prophages containing parental DNA does not rule out this alternative, since we could not have distinguished between Pl-ga/-~ particles carrying a hybrid parental fragment 0.4 genome long and Pl.gal-~ (light) particles. The inclusion in the prophage of a heavy fragment of parental DNA 0.4 ~ genome long would indicate that extensive breakdown and repair phenomena had occurred in the integration of the prophage. Alternatively, one could imagine that a recombination event involving the parental genome and a newly synthesized ~ genome had occurred either before or after prophage integration, and had generated a molecule containing 40% of the parental and 60% of the light DNA. Due to uncertain evidence concerning the physical steps involved in prophage integration, it is perhaps best to state the simplest explanation for our results and the one we used as a working hypothesis : a fragment of parental 2 DNA, nearly equal in length to a mature phage genome and which had undergone a single round of replication either before or after insertion, was found in 2 to 31°/o of the prophages excised by P1 from newly established ~ lysogens. Let us now compare the findings in this work with previous observations. The finding that, except at intermediate values of m.o.i. (8 to 15), the majority of the prophages excised by P1 contain little parental D N A is supported by a report of Stent & Fuerst (1956). On the other hand, the results of Hoffman & Rubenstein (1968b) indicated that 1 to 3 parental phage equivalents of BUdR-labeled DNA per bacterial nucleus were banding in the light position of the CsC1 density-gradient. Their results imply the presence on the bacterial genome of far more parental ~ DNA than was detected in the present work. However, their value is based on the premise that the parental ~ DNA is only associating with light bacterial I)NA. Due to the possibility that the parental ~ I)NA might be associating with high molecular weight forms of intracellular light ~ DNA (Smith & Skalka, 1966), their estimate of the number of parental phage equivalents per bacterial nucleus may be high. S~chaud (1960) found that in newly established lysogens, the amount of ~ DNA synthesized prior to the resumption of bacterial DNA synthesis was approximately equal to the amount of input parental DNA. How then does one account for our finding that a considerable proportion of the prophages excised by P1 contained no parental DNA ? In our experiments, at the multiplicity of 7 phage/bacterium used by S~chaud, about 10% (see Table 5) of the ~ prophages excised by P1 contained parental

A BIOPHYSICAL

S T U D Y OF ~ L Y S O G E N Y

437

DNA, which we have assumed was a hybrid (once replicated) structure. The value of 10% m a y not represent the proportion of parental prophages at the time of insertion, since P1 excision did not necessarily occur immediately after prophage insertion, and any bacterial replication in the prophage region during this interval would reduce the average amount of parental label on the chromosome. Since a t most a single round of replication could have occurred before excision, no more t h a n 20% of the prophages could have contained parental DNA at the time of insertion. H a d any heavy (unreplicated) prophages been present at the time of insertion, or if every prophage region had not undergone one round of replication b y the time of P1 excision, the percentage of prophages containing parental DNA at the time of insertion would have been between the values of 10 and 20. Table 5 therefore presents a minimum estimate of the proportion of prophages containing parental material; the actual proportions at the time of insertion could assume values up to twice those given in the Table. We m a y conclude from the foregoing t h a t at the same m.o.i, t h a t S~chaud used, 80 to 90% of the h prophages contained no parental DNA. This would seem to be at odds with her work, since she demonstrated very little DNA replication prior to lysogenization. However, the disparity is not so large as it might appear. Hoffman & Rubenstein (19685) showed t h a t at an m.o.i. ---- 7, 50% of the parental ADNA neither replicated nor associated with the chromosome. Another 20% replicated once but did not associate. Hence the remaining 30% of the input genomes must account for 8 0 o of the replication seen b y S~chaud. Under these conditions, one would estimate t h a t on the basis of equal probabilities for all the genomes in the pool, 50~/o of the prophages should contain no parental DNA. Considering t h a t data from two experimental systems quite different from the one used in this work were used to obtain this figure, the result is not greatly different from the 80 to 9 0 0 value found in the present work. The disparity m a y just be a reflection of the increased lability of P1 particles containing B U d R DNA. Alternatively, it m a y indicate t h a t P1 is unable to excise, incorporate or transduce B U d R DNA with the same efficiency as light DNA. The results of these experiments, together with those of the other workers, lead us to the following statement. Although relatively few of the parental DNA molecules replicate prior to lysogenization, those which do m a y play an important part in determining which are destined for lysogenization. We are indebted to Dr F. Forro, Jr. for his helpful suggestions and constructive criticism of the manuscript. This work is part of a dissertation presented by one of us (R. B. H.) to Yale University in partial fulfillment of the requirements for the Ph.D. degree. The work was supported by National Science Foundation grants GB-3244 and GB-5972. REFERENCES Adams, M. H. (1959). B a c ~ e p ~ j ~ , New York: Interscience Publishers, Inc. Burgi, E. (1963). Prec. Nat. A c ~ . Sci., W~h. 49, 151. Flamm, V~. G., Bond, H. E. & Burr, H. E. (1966). Biochim. bi~hys, tlc~, 129, Sl0. Fox, E. & Meselson, M. (1963). J. _Mol. Biol. 7, 583. Fraser, D. (1957). Firology, 3, 527. French, C. R., Lesley, S. M., Graham, A. F. & van Rooyen, C. E. (1951). Gan. J. _Sled. Sci. 29, 144. Fry, B. A. & Gros, F. (1959). J. G~n. _MicrobioL 21, 685. Hoffman, D. B., Jr. & Rubenstein, I. (1968a). J. _~lrol.Bio~. S~, 375. Hoff~nan, D. B., Jr. & Rubenstein, I. (19685). J. Mo~. Bio~. S~, 401.

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Ifft, J. B., Voet, D. H. & Vinograd, J. (1961). J. Phys. Ohem. 65, 1138. Ikeda, H. & Tomizawa, J. (1965a). J. Mol. Biol. 14, 85. Ikeda, H. & Tomizawa, J. (1965b). J. Mol. Biol. 14, 120. Jacob, F. (1955). Virology, 1, 207. Kaiser, A. D. (1957). Virology, 8, 42. Kaiser, A. D. & 1Wogness, D. S. (1960). J. Mol. Biol. 2, 392. Lederberg, E. M. & Lederberg, J. (1953). Genera, 38, 51. Lennox, E. S. (1955). Virology, 1, 190. Luria, S. E. & Delbriick, M. (1942). Arch. Bioch~m. 1, 207. Rothman, J. L. (1965). J. Mol. Biol. 12, 892. S~chaud, J. (1960). Arch. ~ci. GBne.va, 13, 427. Schildkraut, C. L., Marmur, J. & Dory, P. (1962). J. Mol. Biol. 4, 430. Smith, M. G. & Skalka, A. (1966). J. Gsn. Physiol. 49, part 2, 127. Stent, G. S. & l~uerst, C. R. (1956). Virology, 2, 737. Weigle, J, J., Meselson, M. & Paigen, K. (1959). J. Mot. Biol. 1, 379.

Note added in proof: A recent paper by Merminger, Wright, Menninger & Meselson (1968) describes the finding of at least one parental strand of A DNA in the A prophage, which is in accord with the results of the present work. REFERENCE Menn]nger, J. R., Wright, M., Menn~nger, L. & Meselson, M. (1968). J. Mol. Biol. 32, 631.