BIOCHIMIE, 1974, 56, 1491-1496.

Some unsolved general problems of phage x development.

Harr ison EeHOLS. Department of Molecular Biology , University of Cali[ornia,

Berkeley, Cali[ornia 9~720, USA.

INTRODUCTION.

Dur ing the last ten years, there has been enormous progress in our knowledge of bacte- r iophage growth. Bacteriophage k has been stu- died wi th par t icuIar i n t e n s i t y ; the efforts of many people using many approaches have led to a remarkable general unde r s t and ing of the com- plex life-style of this virus.

Given this level of unders tand ing , one may be tempted to assume that the r ema in ing research problems in regulat ion of bacter iophage ~ deve- lopment involve solely the b iochemica l elucida- t ion of the mechanism by wh ich known genes work, and that there are no u n k n o w n genes. The purpose of this art icle is to point out that Ibere are !arge gaps in our unde r s t and ing of X develop- ment that may indicate the need for unident i f ied phage or host genes or for a re in te rpre ta t ion of the role of known genes. This i nqu i ry seems to me par t icu la r ly appropr ia te for an issue dedi- cated to Denise Imzzati, whose work necessitated a drastic re in te rpre ta t ion of our ideas some seven years ago. The << unsolved problems >> that I point out are cer ta inly not the only ones in ?. develop- ment, but are presented as examples of our igno- rance of even this ¢ wel l -unders tood ,> proka- ryotic biological system. I believe that one general message of such a discussion is that much more nvork with pro karyot ic regulat ion is needed, and that a complete switch to eukaryot ic development is both u n w a r r a n t e d and un!ikely to be very successful in the absence of a more complete knowledge of developmental aspecls of proka- ryotic regulation.

A BRIEF OUTLINE OF WHAT WE THINK WE KNOW.

Before cons ider ing unresolved problems, I shall summarize briefly those aspects of phage ). deve- lopment that seem to be unders tood in general terms. However, it should be noted that our unde r s t and ing has progressed to a b iochemical level for only one aspect of k development, the repression activity essential for the ma in tenance of lysogeny. For a more detailed discussion, the

reader should see the recent review articles on the lytic (Echols, 1971 a, Thomas, 1971) or lyso- genie pathways (Echols, 1971b, 1972; Ptashme, 1971). Other more general review articles are also avai!abte (Echols, 1973 ; Herskowitz, 1973).

A. The Lyric Pathwag.

l ,ytic development by phage ?~ involves a repli- cat ion-or iented early phase and an encapsula t ion- oriented late phase. This temporal pat tern involves three known regulatory genes - - N, Q and cro - - whose products , p resumably proteins, act to control t r anscr ip t ion from ~ DNA. The N and Q proteins provide sequential positive regu- lation to activate first the repl icat ion and recom- b ina t ion genes and then the genes for head, tail, and lysis proteins. The cro pro te in is a negative regulatory element wh ich acts to tu rn off the recombina t ion and repl ica t ion genes du r ing the late stage of viral development. Because ~. genes for reIated funct ions are clustered, the regulatory pat tern is quite simple ; it is shown diagramma- t ically in figure 1.

B. The Lysogenic Pathway.

The lysogenic response clearly involves a tu rn- off of the lytic pathway. However, the regulatory requi rements differ for the es tabl ishment and ma in tenance of l y soge ny ; not surpr is ingly , the molecular mechanisms also differ.

The simplest stage is the ma in tenance of lyso- geny. The regulatory problem is the ma in tenance of an established prophage through a repression of the genes for lytie development and for the reversible in tegrat ion-excis ion system. The main- tenanee of lysogeny is accomplished by a single ~, protein, the eI protein, w h i c h turns off the lyric pa thway through its capaci ty to repress the earliest stage of lytie development - - BNA syn- thesis ini t ia ted at the early promoter sites PL and Pa (fig. 1 and 2). The eI prote in also acts to provide for t r ansc r ip t ion of the eI gene ini t ia- ted at the ma in tenance promoter P.~t. Thus the eI prote in carries out negative regulat ion of genes essential for lyt ie growth and posit ive regulat ion of its own synthesis.

1 4 9 2 H. E c h o l s .

A r e m a r k a b l e f e a t u r e of ~ d e v e l o p m e n t is t h e e x i s t e n c e of t w o << r e p r e s s o r >> p r o t e i n s , c I a n d Cro, w h i c h s e e m to ac t i n a l m o s t t h e s a m e w a y (fig. 2) b u t h a v e t o t a l l y d i f f e r e n t p h y s i o l o g i c a l ro les . T h e cI p r o t e i n m a i n t a i n s l y s o g e n y , w h e r e a s C ro p o t e n t i a t e s t h e l y r i c r e s p o n s e t h r o u g h a s h u t o f f of t h e r e p l i c a t i o n , r e c o m b i n a t i o n , a n d r e g u l a t i o n genes , a c t i v e d u r i n g t h e e a r l y s tage of d e v e l o p m e n t . T h e Cro p r o t e i n d o e s d i f f e r f r o m t h e c I p r o t e i n i n i t s a b s e n c e of a p o s i t i v e r egu - l a t o r y a c t i v i t y fo r t h e cI gene .

F o r t h e e s t a b l i s h m e n t of l y s o g e n y , t h e r egu - l a t o r y p r o b l e m is m o r e c o m p l e x t h a n f o r m a i n t e - n a n c e b e c a u s e of t w o c o n s i d e r a t i o n s : e s t a b l i s h - m e n t r e q u i r e s no t o n l y r e p r e s s i o n of v i r a l genes , b u t a n e f f i c i en t i n t e g r a t i o n e v e n t ; a n d t h e p o p u - l a t i o n of i n f e c t e d ce l l s e x e r c i s e s a c h o i c e b e t w e e n l y t i c a n d l y s o g e n i c r e s p o n s e s . T h e r e q u i r e m e n t f o r i n t e g r a t i v e r e c o m b i n a t i o n m e a n s t h a t t h e i n t g e n e of t h e r e c o m b i n a t i o n r e g i o n m u s t b e effi- c i e n t l y t r a n s c r i b e d . I n a d d i t i o n , i n t e g r a t i o n is p r o b a b l y e n h a n c e d b y t h e m u l t i p l e g e n o m c

¢I repression

i n t c,rlJ " N c I c ro y e l i 0 P O

Heod Tail Recomb Reg DNA Lysis t t~ t,l

N oclivotion C/ octivation

FIG. 1. - - Transcript ion events during lyt ic develop- men t by phage ~. Approximate DNA regions t r ansc r i - bed dur ing the different stages of lyric g rowth are shown : ( ~ ' ~ - ~ ) represents the immedia te -ea r ly stage of RNA synthesis , pe r fo rmed solely by the hos t t r ans - c r ip t ion mach ine ry ; ( ~ ) repYesents the delayed- ear ly stage of RNA synthesis , in wh ich N pro te in acti- vates t r ansc r ip t i on of the cIII to in t and cII to Q re- gions ( ......... ~-) represents the la te stage of RNA syn- thesis, in which Q pro te in act ivates t r ansc r ip t ion of the lysis, head, and ta i l regions. Since X DNA exists in a c i rcular or eoncatemeric form dur ing much of i ts in t ra - ce l lu lar life, i t is l ike ly t h a t the ac tual un i t of t r ans - c r ip t ion is DNA w i t h the lysis region jo ined to the head region, r a t h e r t h a n the l inea r molecule extracted f rom phage and indicated here. The probable sites at wh ich N- and Q-act ivat ion occur are indica ted by the npavard ver t ical ar rows (A) . The sites a t wh ich the cI p ro te in acts to m a i n t a i n the repress ion essent ia l for lysogeny are indicated by the downward ver t ical a r rows (~ ' ) . The genetic o rgan iza t ion of the ~ DNA molecule is ind ica ted by the generic des ignat ion heloav the X DNA. Specific genes of the << regu la t ion region >> - - cIII, N, cI, cro, cII - - are indica ted above the << h DNA >>, as are the in tegra t ive r ecombina t ion gene int, the DNA repl i - ca t ion genes OP, and the la te regu la tory gene Q. The m a p is not d r awn to s c a l e ; int to the r igh t end is 43 p. cent of the k genome.

c o p i e s p r o v i d e d b y D N A r e p l i c a t i o n . T h u s , t h e i n t e g r a t i o n e v e n t d e p e n d s on t r a n s c r i p t i o n of t h e r e p l i c a t i o n a n d r e c o m b i n a t i o n genes . As a c o n s e - q u e n c e , v i r a l d e v e l o p m e n t m u s t p r o c e e d t h r o u g h

t he e a r l y s tage b u t s t op b e f o r e an i r r e v e r s i b l e c o m m i t m e n t to l y t i c g r o w t h .

T h e e s t a b l i s h m e n t of r e p r e s s i o n is a c c o m p l i s h e d m a i n l y t h r o u g h t h e a c t i o n of t w o p r o t e i n s , t h e p r o d u c t s of t h e c I I a n d c I I [ genes . T h e s e p r o t e i n s c a r r y ou t t w o r e g u l a t o r y a c t i v i t i e s : a t u r n on of l e f t w a r d RNA s y n t h e s i s for t h e cI gene a n d t h e r e - fo re a n a c t i v a t i o n of s y n t h e s i s of t h e m a i n t e n a n c e r e p r e s s o r ; a n i n h i b i t i o n of r i g h t w a r d t r a n s c r i p - t i o n f r o m ly r i c g e n e s a n d t h e r e f o r e a d e l a y in t h e o n s e t of t he la te s tage of l y r i c d e v e l o p m e n t (see fig. 1). T h i s b i f u n c t i o n a l ro le of t h e c I I / c I I I p r o t e i n s m a y i n v o l v e a c t i o n at a s i n g l e s i t e in t h e << y - r e g i o n >> of ~ DNA (fig. 1) to a c t i v a t e a n e w p r o m o t e r f o r l e f t w a r d t r a n s c r i p t i o n (Ps of fig. 2)

cIII N . / c l ~,~ c r o cII 0 P d)

P[ 0 L ~ O R 0

Pu

Fro. 2. - - S a m m a r g of genes and sites (proven or postulated) concerned w i th the es tab l i shment and maintenance of repression. To m a i n t a i n repression, the c[ pro te in acts at oL and on to repress le f tward and r igh tward t r ansc r ip t ion in i t i a t ed at the ear ly p romoter sites PL and PR respect ively ; the cI p ro te in p robab ly also act ivates le f tward t r ansc r ip t ion of the cI g e n e in i t ia ted at a ma in tenance p romote r pu. To es tab l i sh repression, the eII and eIII pro te ins are p resumed to act a t an opera tor site os to provide for cI gene t r ans - c r ip t ion in i t ia ted at an e s t ab l i shmen t p r emo te r pn. The two puta t ive promoters for cI gene t r ansc r ip t ion are separated by the cro gene, which is p resumed to be t r ansc r ibed as pa r t of r i gh tward RNA synthes is in i t i a ted at p~ (see fig. 1), The Cro product is l ike ly to act a t or near or, and o, to repress RNA synthes i s in i t ia ted at PL and PR and possibly at PM.

a n d to r e p r e s s r i g h t w a r d e a r l y t r a n s c r i p t i o n . T h i s r e p r e s s i o n a c t i v i t y i n t u r n m i g h t i n h i b i t l a t e gene t r a n s c r i p t i o n in a t l e a s t t w o w a y s ; t h e s y n t h e s i s of Q p r o t e i n m i g h t b e i n s u f f i c i e n t to a c t i v a t e e f f i c i e n t l y t h e l a t e s t age of R N A s y n t h e s i s ; t h e p r i o r s tage of RNA s y n t h e s i s i t s e l f m i g h t b e r e q u i - r e d to a c t i v a t e fu l ly t h e p r o m o t e r s i t e s f o r t h e n e x t s t age .

T h e l ea s t u n d e r s t o o d a s p e c t of t h e l y s o g e n i c p a t h w a y is t h e i n d u c t i o n r e s p o n s e . A v a r i e t y of t r e a t m e n t s w h i c h i n h i b i t or d e r a n g e h o s t D N A r e p l i c a t i o n r e l e a s e r e p r e s s i o n of t h e v i r a l DNA a n d p e r m i t l y t i c d e v e l o p m e n t . T h e m e c h a n i s m fo r t h e r e l e a s e of r e p r e s s i o n is a n i n a c t i v a t i o n of t h e cI p r o t e i n . T h e i n a c t i v a t i o n is n o t c a u s e d d i r e c t l y b y t h e e x t e r n a l i n d u c i n g agen t , b u t r e q u i r e s a n i n t r a c e l l u l a r s e q u e n c e of e v e n t s t h a t o c c u p i e s a b o u t 30 m i n u t e s a n d l e a d s to t h e p r o d u c t i o n o r

BIOCHIMIE, 1974, 56, n ° 11~12.

Some unsolved general problems of phage )~ deoelopmeut . 1493

ac t iva t ion of an i nduce r substance. This ¢ induc- t ion p a t h w a y ~ involves severa l bac t e r i a l genes also conce rned w i t h DNA repa i r . Thus the phage p r o b a b l y steals a ce l lu la r s ignal of DNA damage and uses it to ac t ivate an escape route. Once repress ion is re leased, v i ra l deve lopmen t is iden- t ical to the ly t ic p a t h w a y a l r eady desc r ibed except that the phage DNA is exc ised f rom the host DNA by a s i te-specif ic r ecombina t i on event that r equ i res the Int p ro t e in needed for in tegra- t ion and an add i t i ona l excis ion-speci f ic p ro te in t e rmed Xis.

An add i t i ona l aspect of regu la t ion du r ing the lysogenic p a t h w a y is cont ro l of the d i rec t ion of the in teg ra t ion-exc i s ion reac t ion . In tegra t ive re- combina t ion de te rmines the s table state unde r cond i t ions in w h i c h r ep res s ion is es tab l i shed in an in fec ted cell, and a low level of in tegra t ive (but not excis ive) r ecombina t i on pers i s t s even in a r epressed lysogen (p resumab ly due to a const i - tut ive p r o m o t e r p~ not subject to cI p ro te in) . The basis for this regula t ion of r e combina t i ona l events is not c lear ; it may involve regula t ion at the level of synthes i s of Int and Xis p ro t e ins or the i r act i- vi ty or both.

SOME GAPS IN OUR KNOWLEDGE.

A. Regulation of DNA Replication.

Expe r imen t s ca r r i ed out over the pas t severa l years have shown that phage ~ can r ep l i ca t e in two m o d e s : an ea r ly mode cha rac l e r i z ed by a c i r cu l a r r ep l i ca t ing molecule , and a late mode cha rac t e r i zed by the p r o d u c t i o n of a l inear , mult i - mer i c DNA molecule , p r e s u m a b l y genera ted by a ro l l ing c i rc le mechan i sm (Tomizawa and Ogawa, 1968 ; Smi th and Skalka, 19.66 ; Skalka, 1971 ; ScbnSs and Inman, 1970). The mu l t ime r i c DNA molecule is the p r e c u r s o r for ma tu ra t i on of phage pa r t i c l es (Stahl et at., 1972 ; Enquis t and Skalka, 1973). The ea r ly mode of r ep l i ca t ion is charac - ter ized by two features, a unique or ig in used for each round of r ep l i ca t ion and s y m m e t r i c b id i r ec - t ional r ep l i ca t ion (Dove et al., 1971 ; Schn6s and Inman, 1970). The la te mode of r ep l i ca t ion pro- bab ly differs f rom the ear ly in both these p ro - per t ies . The capac i ty of the c i r cu l a r molecule to genera te a long l inea r s t ruc ture by a r ep l i ca t ive route impl ies a swi t ch to a new rep l i ca t ion scheme (most l ike ly a ro i l ing c i rc le) . In add i t ion , I p r e sume tha t du r ing the late mode of r ep l i ca t ion the m e c h a n i s m for ea r ly in i t i a t ion is at least pa r - t ia l ly inh ib i t ed because e lec t ron m i c r o g r a p h s of the long l inea r mu l t imer s show no ind i ca t i on of r e in i t i a t ion at the or ig in cha rac t e r i s t i c of ea r ly r ep l i ca t ion (Skalka et at., 1971 ; Wake el al., 1971).

The ear ly to late swi t ch in DNA rep l i ca t ion poses two general p rob l ems of in teres t . F i r s t , wha t is the na ture of the r egu la to ry m e c h a n i s m that po ten t ia tes the swi tch ? Second, wha t se rv ice does an ear ly- to- la te swi tch p e r f o r m for the phage ? The la t te r cons ide ra t ion may in p r i n c i p l e lead us to some fu r the r ins ights conce rn ing regu- la t ion of v i ra l matura t ion .

If we assume that the t r ans i t ion f rom the ea r ly mode of r ep l i ca t ion to the late involves phage- med ia t ed regula t ion , two regu la to ry func t ions are i nd i ca t ed : a negat ive r egu la to ry ac t iv i ty that inh ib i t s ea r ly in i t i a t ion and a pos i t ive r egu la to ry ac t iv i ty that turns on the a symme t r i c ro l l ing c i rc le type of rep l ica t ion . Al though there is no compel - l ing evidence at p resen t for the exis tence of such r egu la to ry act ivi t ies , I w o u l d be s u r p r i s e d if they d id not exist in view of the extens ive regula t ion of every o ther aspect of phage Ws life cycle.

F o r pu rposes of d iscuss ion, I w i l l cal l the genes for these hypo the t i c a l fnnct ions codA and codB, where cod s tands for cont ro l of DNA. Wha t wou ld be the p h e n o t y p e of muta t ions in these hypothe- t ica l genes ? A codB- mutant defect ive in pos i t ive regula t ion of !ate r ep l i ca t ion wou ld be charac te - r ized by an absolute dependence upon r ecombi - na t ion for the fo rmat ion of ma ture phage par - t icles, because r ecombina t i on p rov ides an al ter- nat ive route to the uml t ip le - length DNA molecules r equ i r ed for packaging . One class of mutan ts w h i c h have this p h e n o t y p e for ano the r reason has a l r e a dy been found and charac te r i zed . These mutan t s are defect ive in the X gain gene, w h i c h specif ies a p ro te in essent ia l for the late phase of r ep l i ca t ion th rough its c apac i ty to antagonize the RecBC DNase, a degrada t ive i n h i b i t o r of late r ep l i ca t ion (Unger and Clark, 1972; Sakaki et at., 1973 ; Enquis t and Skalka, 1973). Another class of r e c o m b i n a t i o n - d e p e n d e n t mutan ts not conce rned wi th RecBC DNase has been found but not ye t cha rac t e r i zed at the level of DNA rep l i ca t ion (Echols, unpubl i shed) .

The expec ted p h e n o t y p e for a codA- mutan t defect ive in negat ive regula t ion of ea r ly rep l ica - t ion is much more compl ica ted . A codA- mutan t should p r o b a b l y begin the la te phase of r ep l i ca - t ion but cont inue to in i t i a te on both the c i r cu l a r and l inea r forms of the ro i l ing c i rc le . Thus a com- p l i ca t ed p h y s i c a l s t ruc ture wou ld be p roduced , and r ep l i ca t ion might be inh ib i t ed or the DNA r e n d e r e d incapab le of encapsu la t ion as a conse- quence of excess ive a b e r r a n t in i t ia t ion . W h e t h e r such a b e r r a n t DNA could be eff ic ient ly rescued by r e c ombina t i on is unclear .

There is a k n o w n ~ gene tha t might c a r r y out the codA funct ion . As noted in the prev ious

BIOCHIM1E, 1974, 56, n ° 11-12.

1494 H. E c h o l s .

section, the cro gene product funct ions to poten- tiate the late stage of lyric growth through a turnoff of the early genes for repl icat ion, regu- lat ion and recombinat ion . The Cro prote in pro- bably carries out this activity through a mecha- nism very much like that of the cI prote in : a negative regulat ion of the early proinoter sites (see Echols, 1972). The act ion of the cI pro te in is known to provide for an inh ib i t ion of the in i t ia t ion of DNA repl ica t ion (Thomas and Ber- rant, 1964 ; Ptashne, 1965 ; Dove et aL, 1971) ; the mechan i sm is unclear . I assume that the Cro prote in might effect a s imi lar i nh ib i t ion if it acts in a s imilar way to the cI protein.

There is some exper imenta l evidence that is in terpre table in terms of this hypothesis (although it is also in terpre table in terms of a n u m b e r of other possibil i t ies). Mutations in the cro gene are lethal f o r lyric growth if combined wi th muta- t ions in the cI gene (Folkmanis et aL, 1975). Thus at least a low level of repression (el or cro-medi - ated) is required for successful lyric development. The p r imary cause of this << cro- lethal i ty >> appears to be a severe inh ib i t ion of the normal process of DNA repl ica t ion (Folkmanis et aL, 1975). Thus the Cro protein has an unexpected par t ic ipa t ion in DNA repl icat ion. Such part ic i- pat ion is consis tent wi th (but of course does not prove) the idea that the Cro prote in may part i- cipate direct ly in the regulat ion of DNA repli- cation.

Why should phage k go through what appears to be a very complex process of beg inn ing to repl icate in a c i rcular mode and s 'wiiching to the p roduc t ion of mul t imer ic l inear molecules late in viral development ? I wil! state a few possibil i t ies and leave out fur ther considera t ion of them for lack of available evidence. First , (and I believe least likely) the long molecule might be an evolu- t ionary vestige of a t ime in which phage ?~ matu- ra t ion involved a headfull packaging mechan i sm l'ike that for phage DNA with c i rcular ly permuted and te rmina l ly r e d u n d a n t sequences. Second, the two modes of repl ica t ion might provide for diffe- rent DNA substrates that help dis t inguish between the lyric and the lysogenic pathways. Successful inser t ion of prophage DNA in the lysogenic pa thway must utilize a c i rcular DNA molecule and the in tegrat ion enzymes might require this from of DNA (perhaps in supercoi led form). If the packaging substrate for the lyric pa thway is a long l inear molecule, the virus wi l l have a regu- latory mechan i sm by which integrat io n wil l not often occur dur ing the lyric pa thway nor wil l packaging of prophage DNA occur dur ing the lysogenic pathway. Final ly , p roduct ion of multi-

BIOCHIMIE, 1974, 56, n ° 11-12.

merle DNA from a single infect ing virus as a prelude to packaging might be the means by which the virus insures that some unpackaged DNA remains for fur ther repl ica t ion and that at least one addi t ional copy of itself will he pro- duced dur ing the infect ion (a way of de te rmin ing that the infect ing virus will surely have at least one heir).

B. Regula t ion of R e p r e s s i o n : Ma in t enance and Induc t i on .

The aspect of k development that ~'c know the most about is not surpr i s ing ly the simplest, the ma in tenance of repression. As noted in the pre- vious section, we believe that lysogeny is main- ta ined through the action of a single k prote in , the cI protein, 'which acts to prevent the in i t ia t ion of RNA synthesis from the early promoter sites ; the cI prote in probably also acts to ma in t a in its own synthesis (see Ptashne, 1971 ; Echols, 1971 b, 1972). The repress ion activity of the c1 protein has been demonstra ted in vi tro with purif ied components and is thus clearly defined and rela- t ively well unders tood (Steinberg and Ptashne, 1971 ; Wu et at., 1972 a, b) ; the capaci ty of the cI prote in to activate the cI gene has not been demonstra ted in vitro. The failure of the cI pro- tein to carry out its p resmned positive regulatory role in vi tro may be si inply a problem of impro- per condi t ions in the in vi tro system. Alterna- tively, there might be an addi t ional prote in , phage- or host-specified, that par t ic ipates in the positive regulatory funct ion of the cI pro- tein but not in the repression funct ion. Mutants defective in such a ma in tenance funct ion should have the capaci ty to synthesize cI prote in nor- really du r ing the establ ishment phase of lyso- genic development because of normal act ivi ty of the clI and clII proteins. H~wever, such mutan ts should be unable to ma in ta in repression and thus would be character ized by a loss of capaci ty for stable lysogeny. Thus the phenotype of mainte- nance-defect ive (rant-) mutants should be very much like that of the previous ly found integra- t ion-defective (int-) mutants , except that rant- mutants should be able to exhibi t t rans ien t inser- t ion into the host genome.

Some muta t ions w i th a maintenance-defec t ive phenotype have been found (Strack et at., 1970 ; Yen and Gussin, 1973). One is most easily inter- preted as a muta t ion in the site at wh ich the ma in tenance t ransc r ip t ion of the cI gene is initiat~ed (Yen a nd Gussin, 1973~). The other muta- t ions have not been extensively characterized. Thus there is no evidence at present for the existence of a m n t gene. However, addi t iona l s tudy of maintenance-defec t ive muta t ions may be

S o m e u n s o l v e d genera l p r o b l e m s of phage ), d e o e l o p m e n t . 1495

appropr ia te . T h i n k i n g in evolut ionary terms, the capaci ty to repress and to ma in t a in repress ion through a low const i tut ive synthesis of repressor may have been the first to evolve. Subsequent ly the capaci ty for i m m u n i t y to super infec t ing phage, wh ich necessitates a higher level of repres- sion, may have developed through an act ivat ion funct ion for the cI gene. Phage P22 has a gcne essent ial for the ma in tenance of lysogeny. Although general ly in te rpre ted as a repressor of an << ant i - repressor >> gene (Levine el at., 1974; Botstein et aI., 1974), the P22 mnl gene can be in terpre ted along the l ines just described.

A s imi lar l ine of reasoning leads one to specu- late about the existence of possible addi t ional func t ions involved in induct ion , since the induc- t ion process may be a later evolut ionary stage. As noted in the Summary Section on ~ develop- ment, induc t ion involves the inac t iva t ion of the cI prote in through an << induc t ion pa thway >> trig- gered by the loss of the capaci ty for host DNA repl ica t ion. Conceivably the coupl ing between tile host repa i r efforts and the prophage eI pro- tein might involve an addi t ional phage-specified protein. Since lambdologists general ly employ thermal induc t ion of phage w i th a temperature- sensit ive muta t ion in the cI gene, the physiology and genetics of << real induc t ion >> has not been extensively studied, and thus such a regulatory feature (or more subtle ones) may have been missed.

C. Host Interactions.

Phage ~ interacts wi th the host in at least three vaguely d e f n e d ways : i nh ib i t ion of host pro te in synthesis , host~killing, and choice between lysis and lysogeny. I wil l discuss the first two and omit the latter because the mysteries of lysis vs. lyso- geny have been discussed at some length recent ly (Echols, 1972 ; Court et at., 1975).

As expected f o r a temperate phage, bacterio- phage k does not effect the complete aboli t ion of host pro te in synthesis and the degradat ion of host DNA carr ied out by typical v i ru len t viruses. On the other hand, phage ~ infect ion is associated wi th at least some inh ib i t ion of host pro te in syn- thesis, and phage ), does have a funct ion potien- t ial ly lethal for the host (in addi t ion to the obvious case of lysis enzymes). The na ture of these funct ions represents a large gap in our knowledge of ~, development, par t ly because we do not know how they work and par t ly because we do not know wha t role, if any, they play in viral deve- lopment . One effect on host pro te in synthesis is clearly revealed if host damage is l imited by thermal induc t ion of a DNA-defective prophage.

For this si tuation, the inh ib i t ion of ;6-galactosidase synthesis is reversible by the addi t ion of exoge- nous cycl ic AMP and thus represents a specific effect on catabolic operons (Wu et at., 1971). I wil l call this func t ion Car, for catabolic gene repres- sion. There may also be a general i nh ib i t ion of host pro lc in synthesis which is revealed by infec- t ion or more extensive viral development (Terzi and l ,evinthal , 1967; Cohen and Chang, 1970) (Car funct ion is p resumably a component of the Hin funct ion defined by Cohen and Chang, 1970).

Why should ), car ry out an inh ib i t i on of host catabolic operons ? Is the effect an impor tan t one for viral development, or is it only a byproduc t of some other aspect of ~ growth ? If the lat ter is so, might the effect really represent an effort by the cell (or a popula t ion of cells) to get r id of a viral infect ion ? The Car funct ion seems to involve a deplet ion of in t race l lu lar cyclic AMP (Green, March, and Echols, unpubl i shed) . How impor tan t is this for the phage ? Tile cyclic AMP level du r ing typical viral development does not appear to exert a crucial role in regulat ion of lyric development or in the decision of lysis vs. lysogeny (Jordan et at., 1973). On the other hand, complete abol i t ion of the cataboli te gene activa- t ion system through muta t ion does produce a small deplet ion in f requency of lysogeny (Grod- zicker et al., 1972) ; this effect can be markedly enhanced for a cIII- mutan t of )~ (Belfort and Wulff, 1974). Since these effects of extreme condi- t ions seem to me to be most l ikely ind i rec t ones on prote in synthesis before infect ion, I presume that the cyclic AMP level during infect ion is not of cr i t ical impor tance to the virus.

In the absence of an obvious ut i l i ty of the Car funct ion to the phage, it seems useful to ask whether this act ivi ty is a manifes ta t ion of a << d e l i b e r a t e , cellular response to viral infect ion. Lambdologists (myself included) have tended to th ink of the bac ter ium as a ra ther passive creature upon w h i c h the phage imposes its deve- lopmenta l pat tern. Perhaps there are host defense mechanisms in w h i c h viral infect ion is sensed and countered in an analogous way to the sensing and repa i r of DNA damage. The fol lowing discus- sion is derived from an analysis by Wil lard (1971).

There are two classes of potent ia l bacter ia l defense mechanisms : cellular and populat ion. In a cel lular mechanism, the cell senses some aspect of viral development and responds wi th an anta- gonist. For example, the extensive genetic recom- b ina t ion character is t ic of viral infect ion might l iberate an excess of deoxynucleot ides, which nfight affect the nucleot ide pools (of cAMP or other molecules) in such a way as 1o induce or

BIOCHIMIE, 1974, 56, n ° 11-12.

1 4 9 6 H, E c h o l s .

a c t i v a t e a n i n h i b i t o r of v i r a l DNA r e p l i c a t i o n ( the i n h i b i t o r y a c t i o n of R e c B C D N a s e ( S e c t i o n A) is a m o d e l fo r s u c h a s y s t e m ) . F o r a p o p u l a t i o n m e c h a n i s m , o n e h a s to t h i n k of t h e e v o l u t i o n a r y p r e s s u r e f o r t h e s u r v i v a l of a << c o m m u n i t y >> of ce l l s r a t h e r t h a n t h e i n f e c t e d ce l l i t se l f . I n t h i s t y p e of m e c h a n i s m , t h e i n f e c t e d ce l l m i g h t e x c r e t e a n e g a t i v e l y c h e n m t a c t i c s u b s t a n c e t h a t d r i v e s o t h e r p o t e n t i o n a l l y u n i n f e c t e d ce l l s i n t h e o p p o - s i t e d i r e c t i o n : a n u n l i k e l y m e c h a n i s m i n t h e f l ask o f t h e m o l e c u l a r b i o l o g i s t b u t n o t so u n l i k e l y p e r h a p s fo r a s e w e r o r p o n d .

T h e h o s t - k i l l i n g f u n c t i o n (Kil) r e p r e s e n t s a n o t h e r m y s t e r i o u s p h a g e - s p e c i f i e d a c t i v i t y of n o o b v i o u s v a l u e to t h e v i r u s . L ike Car , t h e Ki l f u n c t i o n is m o s t e a s i l y s t u d i e d b y t h e r m a l i n d u c - t i o n of a D N A - d e f e c t i v e p r o p h a g e ( p r o p h a g e DNA- s y n t h e s i s is i t s e l f l e t h a l to t h e h o s t - - P e r e i r a d a S i lva et al. (1968). O t h e r t h a n h o s t k i l l i n g t h e o n l y w e l l - d e f i n e d p h e n o t y p e a s s o c i a t e d w i t h t h e Ki l f u n c t i o n is f i l a m e n t - f o r m a t i o n b y t h e h o s t (Gree r , 1974). No m a r k e d ,effect of k i l - m u t a t i o n o n v i r a l d e v e l o p m e n t h a s b e e n f o u n d (Gveer , 1974). B o t h thee C a r a n d K i l f u n c t i o n s a re a s s o c i a t e d w i t h t h e r e c o m b i n a t i o n r e g i o n of ). D N A ( W i l l a r d , 1 9 7 1 ; Gree r , 1974). H o w e v e r , t h e t w o a r e p r o - b a b l y n o t t h e s a m e b e c a u s e k i l - m u t a n t s c a r r y o u t Ca r f u n c t i o n ( G r ee t , M a r c h , u n p u b l i s h e d ) . P e r h a p s t he K i l f u n c t i o n d e r i v e s f r o m a n ,effort b y t h e h o s t to i n h i b i t v i r a l d e v e l o p m e n t t h a t b e c o m e s l e t h a l f o r t h e h o s t a f t e r a v e r y l o n g t i m e (or is a n a b e r r a n t m a n i f e s t a t i o n of a v i r a l e f fo r t to i n f l u e n c e t h e h o s t e n v i r o n m e n t i n t h e c a u s e of m o r e f r e q u e n t lys i s o r l y s o g e n y ) .

C o n c l u s i o n a n d a c k n o w l e d g e m e n t .

As n o t e d i n t h e I n t r o d u c t i o n , t h e f o r e g o i n g d i s c u s s i o n r e p r e s e n t s a n e f fo r t to p o i n t ou t s o m e a r e a s of p h a g e ~ d e v e l o p m e n t in w h i c h m o r e u n d e r s t a n d i n g is g r i e v o u s l y n e e d e d . I b e l i e v e t h a t t h e s e e x a m p l e s a l so r e p r e s e n t a r e a s i n ~ , h i c h a d d i t i o n a l u n d e r s t a n d i n g ~onay i l l u m i n a t e i m p o r - t a n t g e n e r a l r e g u l a t o r y m e c h a n i s m s . R e p l i c a t i o n s w i t c h e s a n d s w i t c h - t o - o f f a n d h o l d - o f f t r a n s c r i p - t i o n c o n t r o l a l l h a v e o b v i o u s g e n e r a l a p p l i c a b i l i t y a s do v i r a l i n d u c t i o n a n d v i r a l i n t e r f e r e n c e .

T h e i d e a s r e p r e s e n t e d in t h i s d i s c u s s i o n h a v e e v o l v e d f r o m d i scuss , ions w i t h m a n y peop l e . I a m g r a t e f u l to a l l o f t h e s e i n d i v i d n a l s . I o w e a n e s p e c i a l d e b t to D e n i s e L u z z a t i fo r s h o w i n g m e l o n g ago in a w a r m a n d f r i e n d l y w a y t h a t I d i d no t :know n e a r l y as m u c h as I t h o u g h t .

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