Bacteriophages as Genetic and Biochemical Systems

Bacteriophages as Genetic and Biochemical Systems A. D. HERSHEY

Department of Ginetics, Carnegie Institution of Washington, Cold Spring Harbor, New York

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Summary of Facts and Ideas.. . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . , . , . ............................. 111. Initial Steps of Infection. . . A. Summary.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Primary Attachment. . .................

..................

IV. Lysogeny ..... . . . . . . . . . . . . . . . . .. . . . . . . .. .. .. . . . . .. . ......... .. ......... ................................... A. Lysogenization. . . . . . B. Virulence.. . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . .... , . .. ....... . . . ....... .

......................................

D. Induction.. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . .. , E. Lysogeny, as Bacterial Heredity. . . . . . . . . . . F. Imperfect Prophage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Relation between Bacteriophage and Bacterial Nucleus. . . . . . . . . . . . . H. Transduction and Phage Structure.. . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . , V. Phage Genetics.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . A. Genetic Recombination. ................ B. Mutation.. . . . . ...... . . . . . . . . . . . . . . . . . . . . .. . . . . ......... ....... . ... C. Genetic Fine Structure. ....................... D. Radiogenetics . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , , . . . .. . .................... VI. Chemistry of Vegetative Growth.. A. The Priming Material. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

C. Phage Precursor Nucleic Acid. . . . . . . . . . . . . . . . . . . . . . , . . , . . . . . . , . . . , , D. The DNA-Synthesizing Mechanism. . VII. Chemistry of Maturation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , , , . . , , A. Protein Synthesis.. . . . . . . . . . . . . . . . . . . , . , , . . .

.............................

VIII. Conclusi References. . . .

.............................

25 28 28 28 29 32 33 34 35 36 36 37 38 38 39

40 42 42 47

48

49

MI 51 51 52 53 55 56 56 59 59

I. INTRODUCTION Numerous reviews of special phases of research on bacteriophages have appeared recently. It may be useful to have in addition a concise but comprehensive statement of current trends in this field. The following essay aims to supply it. 25

26

A. D. HERSHEY

Several sections, notably I11 and VII B, depart from this aim in offering arbitrary interpretations of some experiments about which corrective controversy seems desirable. The casual reader may be contented with the introductory summaries to these sections. Only one general remark seems necessary. Work with bacteriophages is no longer of interest exclusively or primarily to virologists. It has become a branch of genetics, or rather two branches, formal and biochemical. This realignment was probably determined by the nature of the material, not by the bias of investigators. If so it may be presumed to reflect one of the directions in which virology as a whole will have to advance. This situation makes the task of the reviewer interesting but difficult. The task of his readers is also likely to prove difficult. Many virologists will meet with unfamiliar or arbitrary ideas; many other biologists will find familiar ideas obscured by strange materials; still others may encounter both difficulties. These difficulties are unavoidable. They dictate the manner of this review, which is written for all interested biologists. Its failure as a review should be measured in terms of its failure to bring a somewhat precious school of thought before this audience; not in terms of the doubtless numerous errors of fact and interpretation it contains.

11. SUMMARY OF FACTS AND IDEAS A few ideas generally accepted among phage workers will assist the reader and are presented here in brief, Many of the underlying facts are known to be true for only a few bacteriophages. As yet one need not question the generality of available information except to keep in mind that most bacteriophages, particularly the smallest ones, are unknown quantities. The known phage particles are tadpole-shaped structures consisting principally of a core of deoxyribonucleicacid (DNA) enclosed by a protein sheath to which the tail is attached. The head portion of T2 is hexagonal in cross section and its sheath tends to retain this shape when emptied. The largest species form particles about 0.1 p in diameter, the smallest perhaps 0.02 p. The particles are metabolically inert until brought into contact with a specifically receptive host bacterium. They are best called resting particles, to distinguish them from the quite different structures that are responsible for intracellular virus activity. 1 Specific attachment to the bacterium occurs through the agency of specialized reccptor sites on the surface of the bacterial cell, and a specialized attachment organ at the tail-tip of the phage particle. This is the best known but riot the only point at which virus-host specificity expresses itself. If the interaction is to lead to infection, primary attachment must

BACTERIOPHAGES A S GENETIC A N D BIOCHEMICAL SYSTEMS

27

be followed by the injection of the nucleic acid core into the bacterium. The empty protein sheath remains a t t8hecell surface, apparently without further function. Beginning with the injection, if the bacterium is of appropriate kind and suitably nourished, viral growth begins along either of two divergent channels. Following one alternative, the virus may Zysogenize the bacterium, in which case the infected cell survives to form a lysogenic clone of descendents. The essential feature of the lysogenic state is the continued reproduction of infected cells. Following its second alternative, the infecting virus particle may cause the cell to dissolve, the virus reproducing itself many-fold in the process. Hence the name bacteriophage. Both temperate and virulent phages may cause lysis; only temperate phages (by definition) can lysogenize bacteria. The difference between temperate and virulent phages may be only one of degree, depending on whether the frequency of lysogenization is detectable or not. In any case, a minor genetic change may transform a temperate into a hereditarily virulent phage. The reverse change has not been detected. It should be noted that, for practical reasons, no attempt is made to recognize the fundamental categories: phages none of whose close relatives is temperate, and phages some of whose close relatives are temperate. The first category is hypothetical, but it has been suggested that T2 may be an example (Lwoff, 1953), and indeed T2 appears to belong to a unique class in several respects. New phage particles are produced only during the lytic phase of development, which is therefore essential in somewhat different senses to the persistence of either temperate or virulent phages. It is often assumed, on the other hand, that the lysogenic phase is essential to the long-term survival of the virus. This assumption is unjustified: the relatives of T2, none of which is known to be temperate, form the most abundant (or most easily detected) family of coliphages in natural environments. The multiplying form of bacteriophage in lysogenic bacteria is called prophage, the study of which is essentially a branch of bacterial genetics. Prophage is to be distinguished sharply from the multiplying form of virus, called vegetative phage, responsible for the lytic process, the study of which belongs more properly to virology. These ideas serve to define several areas of investigation: the mechanics and chemistry of the initial steps of infection, the nature of prophage and vegetative phage, the transition from prophage to vegetative phage (called induction), the conversion of vegetative into resting phage (called maturation), and the relations between virus and host, both biochemical and genetic. These areas were first outlined in their present form in connec-

28

A. D. HERSHEY

tion with sympoc;ia at Royaumont in 1952 and at Cold Spring Harbor i n 1953.

Bacteriophages are so called because they infect bacteria exclusively. Any distinguishing features they possess must reflect this restricted host specificity. Only two points of difference between bacteriophages and other viruses can be noticed. First, bacteriophages contain a large proportion, nearly 50%, of nucleic acid that is exclusively of the deoxypentose type. Other viruses contain smaller, sometimes very small, proportions of nucleic acid, frequently of the ribose type (Cohen, 1955). Second, bacteriophages are tailed viruses. Other viruses do not possess comparable

c-7

multiplication

prophag!

/

/

,'lyrogmiration

IL

\

\\@duction \

FIQ.1. Terminology of life cycle. All stages are intrabacterial except the one represented in a beaker. The broken lines indicate processes restricted to temperate phages. organs of attachment. The significance of the large content of DNA is obscure, but should be thought of in connection with special relationships between bacteriophage and bacterial nucleus, and special roles in bacterial heredity. The unique attachment organ can be ascribed to the need for bacteriophages to penetrate cells with sturdy walls. Fig. 1 summarizes the life cycle alluded to above.

III.

INITIAL STEP;

OF INFECTION

A . Summary The initial steps of infection include a sharply specific primary attachment, mechanochemical puncture of the cell wall, and passage of DNA from phage particle into bacterium. The primary attachment may or may not pass through an obligatory reversible stage, but soon becomes irreversible in the course of infection. The DNA presumably enters through a

BACTERIOPHAGES AS GENETIC ANI) BIOCHEMICAL SYSTEMS

29

tubular tail, but neither stimulus nor driving force to the injection is understood.

B. Primary Attachment The extraordinary specificity of attachment of viruses to bacteria is generally taken to imply that receptor substances on the bacterial surface and the attachment organ a t the tail-tip of the virus particle present complementary surfaces of appreciable area. The specificity becomes somewhat ambiguous if one attempts to homologize the infective process and the attachment of phages to negatively charged surfaces like glass and cationic exchange resins (Puck and Sagik, 1953). This second kind of attachment must reflect properties of proteins in general, rather than specific properties of the tail protein of phage particles. The fact, long known by users of bacteriological and membrane filters, that peptone and other proteins compete with the attachment to nonspecific surfaces, but not to bacteria, suggests that the two kinds of attachment have only misleading features in common. Puck, Garen, and Cline (1951) present evidence for the contrary view. Puck (1953) and his collaborators studied the effect of amino and carboxyl blocking reagents on attachment and suggest that the specific features of the interacting surfaces reside in complementary patterns of ionizable groups. Other topographical features, likewise affected by the reagents used, must be important as well. The attachment requires electrolytes, and in part these must serve to reduce electrostatic repulsion between virus and bacterium. Since the requirements are cation-specific for different phages, cations must also act in other ways (Puck, 1953). The rapid rate of attachment of phage to bacteria is usually interpreted as evidence that receptors for a given phage are present everywhere on the cell surface, and that an appreciable fraction of collisions results in attachment (Stent and Wollman, 1952). This interpretation raises the difficulty that there would have to be quite general overlapping of receptors for different phages. Even if this were so, specificity of attachment implies requirements for orientation that could be satisfied only rarely by random collisions. It is not clear how this difficulty is minimized by supposing that the attachment involves principally ionized groups (Puck, 1953). Although the attraction between individual unlike charges is of relatively long range, the attraction between two surfaces presenting complementary patterns of mixed charges of like net sign would diminish very rapidly with distance of separation (reviewers’ opinion). To avoid these difficulties, I propose that the efficiency of attachment per collision is in fact very low. The contrary conclusion was reached by

30

A. D. HERSHEY

comparing the observed rate of attachment, usually 3 X ml. per bacterium per minute, with a theoretical collision frequency of about 5 X per minute computed for a perfect absorber with diffusion as the ratelimiting process (Stent and Wollman, 1952). A high efficiency of attachment per collision is not indicated by this comparison for two reasons. First, the theoretical rate computed for a stationary absorber is probably too low, because bacteria are subject to brownian and convective motions that invalidate the theory. More important, even if the observed and theoretical rates disagree only by a small factor, the theory is no longer competent to estimate collision frequency. According to the theoretical model, the steady-state concentration of phage at a distance equal t o 0.1 the bacterial radius from the cell surface is only 9% of the initial concentration; at closer distances much less (Delbruck, 1940). With an observed rate half the theoretical rate, the same model calls for a concentration of phage nowhere less than 50 % of the initial concentration, and the collision frequency is incalculably higher than that for a perfect absorber. It follows that the attachment of phage to bacteria is not diffusion-limited and the rate measurements do not permit any conclusions about sites or mechanisms of attachment. A phage particle probably has to collide many times with a bacterium before finding and fastening to a specific receptor. The number of receptors is nevertheless rather large. Some 200 particles of T2 can attach to a single cell, and bacteria saturated with T2 particles cannot adsorb TG (Watson, 1950). This relationship is not reciprocal: bacteria saturated with T4 or T6 can adsorb T2, and bacteria nearly saturated with T2 adsorb T4 better than T2 (Hershey and Chase, 1952; Weidel, 1953a). This method of analysis has not been exploited sufficiently to permit definite conclusions about distribution and overlapping of receptors, particularly because it is not certain that the observed effects are due solely t o mechanical blocking of receptors (Weidel, 1953b). The important work of Puck, Garen, and Cline (1951) seems to show that for T1, and perhaps other phages, primary attachment is reversible, and may or may not be followed by irreversible reactions depending on temperature, ionic environment, and other factors. Actually it has not been proved that the observed reversible attachment has anything to do with the process of infection. Since this experimental defect has been universally ignored, the pertinent facts and interpretations will be presented here in some detail. Phage T1 attaches irreversibly a t optimal rate to sensitive strain B of Escherichia coli at 37" C. in 0.0005 molar solutions of magnesium or calcium salts. At 0" C. under the same conditions the attachment is partly reversible. A phage-resistant bacterial mutant (B/l), or cells of B that

BACTERIOPHAGES A 8 GENETIC AND BIOCHEMICAL SYSTEMS

31

have been altered by irradiation or in other ways, adsorb T1 only reversibly. Reversible attachment of T1 does not occur, however, to bacteria in general. Garen and Puck (1951) conclude that infection involves a temperature-independent primary attachment that is reversible, followed by a temperature-dependent (enzymatic?) irreversible step. The alternative interpretation is that reversible and irreversible attachments involve different bacterial receptors, or different parts of the phage particle, in which caae the two kinds of attachment are not steps in a single process, but competing processes, neither one of which is enzymatic. The two alternatives were clearly pointed out by Stent and Wollman (1952). Garen (1954) studied the reaction between T1 and B/l. He found that it behaves in important respects like a reversible reaction between homogeneous reactants. The apparent equilibrium constant (about 3 X lo-* ml. per bacterium) is such that half the phage is attached at about 3 X lo7 bacteria per ml., independently of temperature. The duration of reversible attachment, according to an estimate involving several assumptions, appears to be several minutes. If this estimate is applicable to the attachment of T1 to B at 37" C., a simple test of the stepwise nature of the infective process is possible. Phage reversibly attached to B at low temperature should invariably make an irreversible attachment when the suspension is suddenly diluted by a large factor into warm attachment medium. Satisfactory experiments of this type are lacking. It may be added that the reversible attachment to either B or B/1 is greatly inhibited by peptone, thus resembling the attachment to glass rather than the attachment to B that results in infection. I n view of this fact it may be a mistake to study receptor activity in simple salt solutions. According to Garen's (1954) interpretation, the function of the reversible attachment is to allow time for slower, possibly enzymatic, ensuing reactions essential for the infective process. B/1 differs from B only with respect to the hypothetical ensuing reactions. It is not suggested here that this interpretation is incorrect, but that the alternative interpretation, namely, that B/1 retains one of two different kinds of receptor present in B, has not been excluded. Puck (1953) and Adams (1955) have shown that when T2 attaches to B at 0' C. an appreciable number of phage particles is lost without succesfully infecting bacteria. The reviewer has observed the same for TI. This result, together with the others mentioned above, suggests that phage particles attach to bacteria at alternative sites, or in alternative ways, reversibly and irreversibly, by several competing reactions. One or another kind of attachment predominates, depending on temperature and on the presence of electrolytes and substances like peptone. For the interesting kind, namely infection, there is little evidence for a characterist,ic re-

32

A.

D. HERSHEY

versible step. The irreversible attachment that competes with infection may explain in part the fact that not all phage particles are infective even under tJhe best conditions (Luria, Williams, arid Backus, 1951). It also helps to explain why the titer of a phage stock depends on both heritable and nonheritable variations in the host bacterium (Hershey and Davidson, 1951). For a different interpretation of the same facts, Puck’s (1953) review should be consulted.

C . Irreversible Attachment and Receptor Activity The nature of the primary attachment of phage to bacteria becomes important in connection with attempts to learn something about receptor sites by chemical fractionation of bacterial cells. If the primary reaction is reversible, receptor activity must be redefined in terms of tests capable of recognizing reversible interactions. Such tests have never been employed (Weidel, 1953b). Thus B/1 and irradiated B, were supposed to lack receptors for T1, whereas the work of Garen and Puck (1951) shows that some kind of receptor is present. Whether this kind of receptor is interesting depends on its relation to the infective process, which has not been elucidated. The existing information about isolated receptor substances comes from experiments using irreversible inactivation of phage as a test for receptor activity. If different phages attach to different receptor sites on the bacterial surface, it ought to be possible to separate them physically. This may have been accomplished in one instance. Weidel et al. (1954) extracted T5 receptor from E. coli, leaving receptors for T1 (reversible adsorption) and T2 behind. A clean separation was not attempted, however. Extraction of protein from the isolated T5 receptor substance with 90% phenol destroyed T5 receptor activity but unmasked a previously undetectable activity against T3, T4, and T7 in the insoluble residue. Material extracted from B/1,5 (which lacks T5 receptor) was similar in other respects but failed to inactivate T5. This complicated pattern of results is typical of all the work on receptor substances. Goebel and Jesaitis (1953) extracted from Shigella sonnei a fraction exhibiting all the receptor activity of the original cell, inactivating T2, T3, T4, T6, and T7. Extraction of protein from the complex left behind a lipocarbohydrate that retained receptor activity only against T3, T4, and T7. This material is presumably similar to the comparable material subsequently obtained from E. coli by Weidel et al. (1954). The T5 receptor and the T2 - - T7 receptor are similar, fairly homogeneous, fractions of complex composition and large particle size. Blocking experiments with individual phages applied to such preparations might be a useful means of testing whether the several antiviral activities are asso-

BACTERIOPHAGES AS GENETIC AND BIOCHEMICAL SYSTEMS

33

ciated with the same or different particles in the preparation. As will be seen below, the activities of these receptor substances do not end with simple combination with the phage. Irreversible attachment of phage particles to receptor sites, as distinct from ensuing reactions, is readily demonstrable only with isotopically labeled phage, or by electron microscopy. Irreversible attachment of T1, T2,and T4 has been clearly demonstrated (Anderson, 1953; Hershey and Chase, 1952; Christensen and Tolmach, 1955). Details of the attachment of T2 and T4 have been recently clarified by Kellenberger and Arber (1955). These workers show by beautiful electron micrographs that the tail of T2 consists of a tubular sheath through which runs a central pin. Following attachment of the phage to bacteria, the terminal portion of the tail-sheath frays out as slender filaments. These filaments probably represent the cementing substance for primary attachment (Williams and Fraser, personal communication). The exposed portion of the central pin punctures the cell wall up to a shoulder formed by the proximal half of the tail-sheath, which remains intact. Fraser (personal communication) suggests a mechanical model in which the driving force for the process is derived from the adhesion of tail-filaments to cell wall, activated by brownian motion. According to this model reversible attachment does not enter as an obligatory step, and it is understandable that a particle might attach irreversibly, at the extreme edge of a receptor site for instance, without being able to puncture the cell wall. The same description does not apply to T5. This phage is inactivated by attachment to receptor substance, particle for particle, at the tip of the tail. No visible alteration of tail structure ensues (Weidel and Kellenberger, 1955).

D. Injection Basic information about the structure of phage particles comes from osmotic shock experiments with phages like T2 (Anderson, 1953) and from heat-inactivation of T5 (Lark and Adams, 1953). These treatments release part or all of the DNA from the particles, and by further treatment with deoxyribonuclease one obtains nucleic acid-free ghosts containing most of the phage protein (Herriott, 1951). Ghosts of T2 adsorb to and kill bacteria. The tail structure of the ghosts is intact, and adsorption to bacteria causes the same changes in tail structure that are seen for whole phages (Kellenberger and Arber, 1955). It is unlikely, therefore, that DNA is released through the tail during osmotic shock. Nevertheless, the the release of DNA by osmotic shock provides a convenient model of the similar process that occurs at the start of infection of bacteria by phage. Infection calls for the passage of DNA from the phage particle into the

34

A.

D. HERSHEY

bacterium, presumably through the tail (Hershey and Chase, 1952; Anderson, 1953; Kellenberger and Arber, 1955). A second model of this process is observed when phage particles react with isolated receptor materials (Hershey and Chase, 1952; Anderson, 1953; Jesaitis and Goebel, 1953; Weidel and Kellenberger, 1955). In this instance the DNA is released into the medium as a consequence of a more-or-less normal interaction between the receptor substance and the tail-tip of the phage particle (Weidel and Kellenberger, 1955). The attachment to receptor substance is not invariably followed by release of DNA. T2 retains its DNA partially or completely after interaction with certain preparations of bacterial membranes (Anderson, 1953; Kellenberger and Arber, 1956). T5 attaches to bacteria perfectly well in the absence of calcium ions, but injection requires this cofactor (Luria and Steiner, 1954). Jesaitis and Goebel (1953) report cofactor activity toward T4 for several fatty acids, though whether for primary attachment or release of DNA is not clear. A clue to the mechanism of release of DNA may come from study of simpler models. Lark and Adams (1953) found that deprivation of calcium ions, and perhaps direct interaction with citrate, caused simultaneous loss of ability of T5 to attach to bacteria, and release of DNA from the particles. Pyrophosphate and certain complex ions have similar effects on T2 (Herriott, personal communication; Kozloff and Henderson, 1955). Like the infective process itself, these results suggest that the stimulus to release of DNA occurs at the tip of the tail. How it is released is not clear.

IV. LYSOGENY Several definitive reviews of lysogeny are available (Lwoff, 1953; Bertani, 1953; Jacob, 1954a). The main concepts were arrived at somewhat as follows. A bacterial culture is said to be lysogenic if large clones grown from single cells regularly contain bacteriophage. The single cells do not contain bacteriophage, hence the definition of lysogeny as the hereditary potentiality to produce bacteriophage without infection. This potentiality is biospecific, that is, a lysogenic culture perpetuates one or a few characteristic types of virus, hence the notion of prophage as the invisible, virus-specific, precursor. These ideas are not entirely new, but their adequate experimental justification and clarification dates from the independent work of Lwoff and his associates and Bertani. Lysogenic cultures are found in the first instance among natural populations. Lysogenic cultures identical to the natural ones can be produced by infecting sensitive bacteria with the appropriate phage. This shows that prophage specificity is separable from the phage-producing equipment of

BACTERIOPHAGES AS GENETIC AND BIOCHEMICAL SYSTEMS

35

the lysogenic bacterium. Much of the early interest in lysogeny centered about controversy concerning this distinction. The point at issue, equivalent to asking about the origin of the first prophage, now seems meaningless.

A . Lysogenization Given the facts already stated, naturally lysogenic cultures become relatively uninteresting, and one turns to the experimental study of lysogenization, that is, to the transition from resting phage to prophage (Fig. 1). The reagents needed are a lysogenic culture as a source of temperate phage, and a sensitive bacterial culture, necessarily closely related to the lysogenic one. The sensitive culture must play a dual role, first as the subject of experiments on lysogenization, second as a tester strain for the presence and quantitative assay of phage. When the sensitive culture is infected with the temperate phage, some of the bacteria lyse, and some of the others become lysogenic (Fig. 1). What determines the division into these two classes? Evidently the condition of Y the process the bacterium is one factor, and simple tests show not O K ~ that of lysogenization depends on the condition of the bacterium at the time of infection, but that it is subject to experimental interference for a considerable time thereafter (Lieb, 1953; Bertani, 1953). Changes of temperature or chemical interference applied during the first hour or less can alter the decision between lysogenization and lysis. Experiments along these lines define a prelysogenic state during which the material from the infecting phage is still plastic, in contrast to the relatively stable prophage state reached later on in that fraction of the bacteria becoming lysogenic.

B . Virulence Those factors influencing the decision between lysogenization and lysis that seem to reside in the phage are called virulent or temperate character. Like the predisposition of the bacterium, virulence can be analyzed only by very simple tests. For example, if a bacterium is infected at the same time with a virulent and closely related temperate Salmonella phage, the latter may protect against the former, and a lysogenic clone results. This seems to show that some temperate phages are temperate because they produce a prelysogenic immunity to their own lytic development. This notion is strengthened because the same temperate phages produce a higher frequency of lysogenization and a lower frequency of lytic response the greater the number of infecting phages per bacterium (Lieb, 1953; Boyd, 1951; Garen, personal communication). Not all temperate phages show these characteristics. however.

36

A.

D. HERSHEY

ltccent experiments by Leviiie (1955) analyze the phenomenon of prelysogenic immunity in a novel way. He finds that virulent mutants of a temperate Salmonella phage fall into two groups. Infection with phages belonging to either group produces only lytic responses. Mixed infection with any phage of group I and any phage of group I1 produces many lysogenic responses. This suggests that mutants of each class are unable to establish prelysogenic immunity because they are deficient in different, mutually complementary, ways. Both types of deficiency can be traced to a single locus by genetic crosses (Section V, A). Moreover, bacteria lysogenized by mixed infection prove t o carry prophages of group I only; they are lysogenic for a virulent phage. This clearly distinguishes prelysogenic immunity from the immunity that characterizes the lysogenic state. It also causes the definition of virulent phages to break down. Another type of virulent phage (strong) can induce the development of the lytic cycle in bacteria lysogenic for a related temperate phage (Fig. 1). A strong virulent mutant of lambda gives rise to mixed yields of virus under these conditions. The virulence of such phages can be attributed to a self-inducing property; they overcome, and hence cannot establish, the immunity to lytic development that is a necessary part of the lysogenic condition. This t,ype of virulence in lambda depends on multiple genetic factors (Wollman and Jacob, 1954). Evidently both the prelysogenic and subsequent immunity are essential to lysogenization, and the inability of a phage to establish either one is sufficient to explain virulence. A third type of virulence, based on lack of homology between genetic material of bacterium and virus, may be postulated (Section IV, G). In contrast to the examples cited above, this type of virulence may be a species character as in T2, a phage none of whose close relatives seems to be temperate. These attempts to systematize the origin of virulence may be carried too far. Perhaps the only safe conclusion is that many factors determine the habit of a given phage, which is only another way of saying that habit is genetically determined.

C, Prophage The central problem presented by the phenomenon of lysogeny has to do with the condition of prophage in the lysogenic bacterium. The facts call for an efficient mechanism of transmission of prophage to both daughter cells at each cell division. If the bacterium contains many identical prophages, the segregation could be random. If each cell contains one or a few, regular segregation is called for, and multiplication of prophage would have to be coupled to that of other bacterial organelles. All t,ypes of

BACTERIOPHAGES AS GENETIC AND BIOCHEMICAL SYSTEMS

37

evidence point to a limited number of prophages and, indeed, to chromosomal localization (Bertani, 1953). First, when a lysogenic bacterium is superinfected with a second phage (closely related to the carried prophage and genetically marked), the superinfecting phage is generally excluded. Occasionally it substitutes for the carried prophage. Only rarely does a mixed infection result. The latter exception proves the rule because in a doubly lysogenic bacterium one can distinguish between the sites occupied by the first and second prophages (Bertani, 1956). Thus the sites available to a given prophage are limited in number and can be seriated with respect to accessibility. Second, in crosses between lysogenic and nonlysogenic bacteria, lysogeny for lambda segregates with markers for galactose fermentation, suggesting that the prophage itself can profitably be thought of as occupying an obligatory site on the bacterial chromosome (Lederberg and Lederberg, 1953 ; Wollman, 1953). This idea may not prove quite adequate, however; the Lederbergs note that some other prophages failed to segregate in crosses, and Bertani’s results do not indicate a singular site. Third, if the conversion prophage to vegetative phage is induced in a lysogenic bacterium (Fig. l ) , which is at the same time superinfected with three or four particles of a second genetically marked phage, the viral yield contains equal numbers of the two phage types, suggesting that the carried prophage itself contributes the genetic equivalent of only a few phage particles per bacterium to the competitive growth (Jacob and Wollman, 1953).

D. Induction The term induction refers to the fact that most of the cells in cultures of certain lysogenic bacteria can be caused, by experimental interference, to enter the lytic cycle of phage development at the same time. The phenomenon has been reviewed by Jacob and Wollman (1953). Ultraviolet light, X-rays, and certain chemicals are effective inducers. Inducibility is in general characteristic of the prophage, not of the type of bacterium in which it propagates. Inducing agents probably act indirectly through the bacterial metabolism, not directly on the prophage. Following induction by ultraviolet light, there is an interval during which the induction ran be reversed by visible light. These facts suggest a few simplifying hypotheses. 13y defiliition, induction is the massive change-over of the culture from the lysogenic condition to the lytic cycle of development : prophage becomes vegetative phage, the latter multiplies and matures, and the cells lyse, liberating phage partivles. By analogy, one can suppose that the same sequence of events that occurs spontaneously in an occasional bacterium, and by which a lysogenic culture

38

A. D. HERSHEY

is recognized, is caused by some random fluctuation of bacterial metabolism similar to that caused by inducing agents. Since certain phages are themselves inducing agents, one class of virulent mutants (strong virulents) may be thought of as temperate phages that carry their own inducing agents. In fact some, if not all, virulent and temperate phages may follow a common pathway for some time after infection (Bertani, 1953;Lieb, 1953).

E. Lysogeny as Bacterial Heredity If prophage attaches to, or is incorporated into, the bacterial chromosome, the addition or substitution of new genetic material should have direct and side effects on bacterial function. To be sure, this scarcely amounts to a prediction, since the same could be said for all forms of symbiosis (Lederberg, 1955). Such effects have been known for a long time; only recently have they been viewed in a systematic way. Among direct effects one can recognize only the lysogeny itself, the prophage-specific immunity to spontaneous lytic development on which lysogeny depends, and the immunity to superinfection by phages related to the carried prophage. Perhaps one should add the exceptional sensitivity to ultraviolet light exhibited by bacteria that carry an inducible prophage. Such bacteria may be killed by radiation doses too small to affect comparable nonlysogenic strains, but indirectly, in consequence of the lytic development of the virus (Jacob, 1954a). The indirect effects are more numerous. For example, E. coli K12, only provided it carries the prophage lambda, does not support the growth of a certain class of mutants of the unrelated phage T4 (Benzer, 1955). Certain host-induced modifications of superinfecting phage are probably dependent on the pre-existing lysogenic condition of the host (reviewed by Luria, 1953). Toxin production by the diphtheria bacillus, and the production of certain antigens by Salmonella, are dependent in some way on carried prophages (Freeman, 1951 ; Lederberg, 1955; Bertani, 1956). These examples must be sharply distinguished from the unrelated phenomenon called transduction (see below). Hcre we are dealing with bacterial functions determined by genetic material perpetuated in the form of prophage. One suspects that examples of this kind may be profitably investigated in terms of problems that are at the same time as fundamental and as practical as any known to biology: first, as potential clues to the relation between genetic structure and function; second, as models for alterations in cellular heredity not explicable in terms of mutation or segregation alone. F. Imperfect Prophaye Lysogeny c w i be defined with precision; a lysogenic bacterium harbors one or more prophages. A nonlysogenic bacterium cannot be defined, ex-

BACTERIOPHAGES A S GENETIC A N D BIOCHEMICAL SYSTEMS

39

cept for the absence of a particular prophage. There is no test for an unknown prophage: only random tests. The ambiguity has multiplied through the discovery of imperfect prophages (Jacob, 1954a). These may be obtained by subjecting typical lysogenic bacteria to large doses of ultraviolet light and selecting among the survivors clones that resemble the lysogenic parent with respect to immunity to phage and sensitivity t o lysis by ultraviolet light, but which seldom or never produce phage. The step called maturation is blocked, directly or indirectly, in such bacteria (Fig. 1). The production of lightinducible, specific antibiotics (bacteriocins) bears a certain analogy to the imperfect prophage state (Jacob, 1954a). Another analogy may be the unproductive lysis of T2-infected bacteria in the presence of proflavin (DeMars, 1955). By extension of the idea of imperfect prophage, one can imagine a bacterium that carries phage-specific material, but no longer exhibits lysogeny, inducibility, nor immunity to homologous superinfecting phage. By further extension, one can imagine that all bacteria, if not actually lysogenic, harbor the equivalent of imperfect prophages. The most general notion is that of genetic homology between bacterium and virus. At this point the notion is idle: it will reappear in another context in the following paragraphs.

G . Relation between Bacteriophage and Bacterial Nucleus It is evident from the ideas already outlined that the invasion of a bacterium by a virus might be thought of as an addition of new genetic material, as a substitution of new for old, or-in the case of a strictly virulent phage-as more or less complete replacement. Ideas of this kind have a long history (Lwoff, 1953), culminating, perhaps, in the rational program of research outlined by Luria (1950), who should be read in the original. One of Luria’s points of departure, the cytology of virus-infected cells, suggested valuable ideas but does not at the moment offer much scope for discussion. One commonly sees a literal disruption of the visible nuclei followed, in the case of T2, by the chemical substitution of viral for bacterial DNA (Luria, 1950; Hershey et al., 1953). This seems to be an adequate preparation for the destruction of a bacterium by a virulent phage. Temperate phages may also produce characteristic cytological changes which, in the event of lysogenizstion, are only transient (Whitfield and Murray, 1954). The phenomenon of lysogeny evidently denotes genetic compatibility between virus and bacterium and some kind of integration of genetic materials from the two sources. One theoretical basis for such integration depends on partial (or complete) genetic homology (Bertani, 1953).

40

A . D. HERSHEY

Fraser first looked for evidence of such homology in terms of possible genetic transfer from bacterium to virus, with partial success (Hershey et al., 1954). She worked with the virulent phage T3. Further studies along this line continue to show promise (Zinder, personal communication). Radiobiological confirmation of the idea of genetic homology has been obtained by Garen and Zinder (1955). It is based on the following facts. First, most bacteriophages, compared with T2, are highly resistant to ultraviolet light, both absolutely and relative t o DNA content per particle. Second, all of them, including T2, are equally sensitive, per unit DNA content, to inactivation by decay of incorporated radiophosphorus. Third, the sensitivity of all of them to ultraviolet light approaches that of T2 when infectivity is measured not on healthy bacteria, but on bacteria that have themselves been irradiated. Caren and Zinder interpret these facts in the following way. The relative resistance to ultraviolet light of the DNA in most phages is not intrinsic but can be ascribed to an efficient merhsnism by which damaged DNA in the phage is replaced by homologous undamaged DNA in the host. The replacement fails for obvious reasons when the host DNA is also damaged, and for unknown reasons when the damage to the phage results from decay of radiophosphorus. In T2 one measures intrinsic sensitivity of DNA to ultraviolet light because homology betwecn T2 and its host is ruled out by chemical differences between the bact,erial and viral DNA. This is one of several reasons for placing phage T2 in a class by itself.

H . Transduction and Phage Structure Transduction is a process by which the hereditary potential of an acceptor bacterium is modified toward that of a donor bacterium as the result of the indirect transfer of presumably nuclear genetic subunits from bacterium to bacterium. The vector of transfer is a bacteriophage (Zinder, 1953, 1955; Lederberg, 1955). Transduction is instructive chiefly in relation to other genetic mechanisms in bacteria. As such it lies outside the scope of this review. However, it also says something about bacteriophage. Transduction is mediated, in Salmonella and Escherichia, by either certain temperate or virulent phages. The system employing temperate phage is simpler. For transduction experiments one needs it vector phage propagated on a genetically marked donor bacterium, and acceptor bacteria which are subsequently exposed to the phage. One observes the substitution of genetic markers from the donor bacterium for genetic markers in the acceptor bacteria, usually one at a time and in very few of the surviving cells. The phage particle is a passive vector in two senses. First, the trans-

BACTERIOPHAGES AS GENETIC A N D BIOCHEMICAL SYSTEMS

41

ducing agents it carries arc derived exclusively from the bacterium in which the phage underwent its most recent cycle of growth: the agents are bacteria-specific, not phage-specific. Transduction, unlike lysogeny, does not fuse bacterial inheritance aud phage inheritance. Second, implantation of transducing agent into acceptor bacterium by phage is independent of infection by phage. It can be brought about by phages rendered noninfective by ultraviolet light and in bacteria immune to infection. Specific attachment of phage to bacterium and injection of DNA, however, are inferred to be essential. One asks what is the relation between the prophage sites on the bacterial chromosome and the susceptibility of bacterial markers to transduction. The answers are clear but contradictory. In Salmonella, and in E. coli K12 transductions by phages other than lambda, there appears to be none: all bacterial markers are transducible (Zinder, 1955; Lennox, 1955; Jacob, 1955). Transductions in E. coli K12 mediated by phage lambda, however, are restricted to markers for galactose fermentation adjacent to the prophage site (Lederberg, 1955). One asks next how the transducing agent is organized within the phage particle. As already mentioned, it seems to be carried as material superfluous to the infective property of the phage particle. The majority of phage particles must contain neither a given bacterial marker, nor any homologue t o it. For example, phage propagated on bacteria incapable of synthesizing histidine include very few particles transducing this property and none competent for the reverse transduction (Zinder, 1955; for technical reasons the actual demonstration is more complicated). The transducing agent is extremely resistant to ultraviolet light and to inactivation by decay of assimilated P32 (Garen and Zinder, 1955). If it is DNA it is a small piece of DNA, consistent with the idea of a casual DNA contaminant. This idea is complicated by the discovery of transductions of lysogeny (Lennox, 1955; Jacob, 1955). A vector phage can carry an (unrelated) prophage from a lysogenic donor bacterium to a nonlysogenic acceptor bacterium. Like other examples of transduction for which the test can be made, this works both ways: the vector can also carry nonlysogeny. Both properties, lysogeny or nonlysogeny, tend to be transduced Fimultaneously with the expected galactose fermentation marker, showing that the transferred material is not prophage as such, but a bacterial chromosome fragment including prophage and adjacent material. On the one hand these results confirm expectation, since the prophage itself can be regarded as a bacterial marker which, in the light of Lennox’ work (1955), ought t o show correlated transductions with markers linked to it. On the other hand, the finding that one phage skin can contain

42

A. D. HERSHEY

determinants of two or even three different prophages, one proper to thc vector phage, others as transducing agents, raises new questions about the relation between germinal substance and casual genetic contaminants in the phage particle. At the least it suggests that the germinal substance may represent a small part of the total material in the particle. The present results do not force this conclusion. Following the lead of Garen and Zinder (1955) one visualizes phage structure in the following manner. The particle contains its proper germinal substance, that is, a large obligatory piece of genetic material that infects bacteria with high probability and forms the material basis of the host-independent germ line of the virus. In addition the phage particle contains small pieces of accessory material that play only nonspecific roles in viral perpetuation, but occasionally function specifically in infective heredity of bacteria. In order to admit the possibility that the accessory material can also occasionally perpetuate prophage, one is forced back on Garen and Zinder’s doctrine of phage-bacterium homology; some of the germinal substance of the phage particle, although genetically functional, is potentially dispensable during infection because the bacterium already contains material equivalent to it in general function. Transduced prophages may be partial structures forced to rely on such material. One alternative view can be excluded. If intact, physiologically equivalent chromosomes intended for two phage particles could readily get into one, the discovery would have been made long ago in phage crosses (Section v, A). These ideas about phage structure are more than a way out of a dilemma; they are subject to many tests mostly still untried.

V. PHAGE GENETICS A . Genetic Recombination Genetic recombination between phages was first studied in the related species T2 and T4. Recently T1 and lambda have been investigated in a very satisfactory manner (Wollman and Jacob, 1954; Kaiser, 1955; Bresch, 1955). Each system studied has called for technical innovations and produced new information, most of which can be fitted into a single description. Genetic recombination has been observed in lysogenic bacteria (Bertani, 1953), but is usually studied by mixed infections producing prompt 1ysis. Genetic recombination is interesting for at least two reasons. In the first place, elucidation of mechanism might be expected to have a bearing on questions about phylogenetic status of viruses. Secondly, it was long suspected, apparently with some justification, that phage multiplied in the form of naked genetic material and that replication and recombination

BACTERIOPHAGES AS GENETIC AND BIOCHEMICAL SYSTEMS

43

were somehow linked together. Thus one could hope t o bridge the gap between chromosome mechanics a t the visible level and genetic function a t the molecular level, the prime subject of interest being growth rather than recombination. Progress along these lines has been solid rather than spectacular. No major question has been answered unambiguously, but ways and means have grown steadily more powerful. A review of basic facts will not be attempted here; other reviews are available (Hershey, 1953a; Doermann, 1953). These facts led to the conclusion that viral inheritance is based on a linear linkage system comparable to that of other organisms. One new fact seems to be emerging: it is likely that phages contain a single linkage structure, joining the three linkage groups first recognized in T2 (Wollman and Jacob, 1954; Streisinger, personal communication). The first major accomplishment of recombination analysis showed that growth and recombination preceded the maturation of phage particles (Fig. 1). This led to the concept of vegetative phage as a pool of multiplying and interacting noninfective particles (Doermann, 1953). The second major accomplishment of recombination analysis led to the formulation of a quantitative theory of recombinant production (Visconti and Delbruck, 1953). According to this theory, vegetative phage particles interact pairwise (mate) a t random in a mating pool from which individual particles are withdrawn, also a t random, to enter the maturation cycle. Depending on the time of withdrawal and on chance, individual particles will stem from lines in which none or several mating opportunities have occurred. This time-dependent number of rounds of mating, the composition of the mating population, and the linkage relations between the markers under observation, determine the measured frequency of recombination. There is no doubt that the theory adequately represents the facts it deals with. On the other hand, it does not specify the nature of the mating event, and in this sense says nothing about the mechanism of recombination. One conclusion of the authors, that “our theory tends to place the genetics of phage very much in line with orthodox genetJictheory,” is true only to the extent that pairwise interaction between multiply-marked structures is common to both (Hershey and Chase, 1951). The third major accomplishment of recombination analysis suggested a relation between multiplication and recombination and proposed a mechanism for the latter (Levinthal and Visconti, 1953; Levinthal, 1954). These basic contributions can be understood in the following way. The fact that rccornbinant frequency is higher among phage particles maturing late than among phage particles maturing early could be explained in two ways: first by supposing that mating probability per particle is dependent on time (progressive mixing of the two parental types in the

44

A. D. HERSHEY

mating pool) or by supposing that mating events accumulate in the pool (successivc rounds of mating). On the first supposition, the drift toward genetic equilibrium would be independent of linkage, that is, recombination frequency involving linked and unlinked markers would show the same proportionate rise with time of sampling, and neither would approach the 50 % equilibrium point. In fact, recombination frequency for unlinked markers in T2 and T4 approaches equilibrium very quickly, and the kinetics of drift toward equilibrium for linked markers (Levinthal and Visconti, 1953;Bresch and Trautner, 1955) suggests a constant mating probability per interacting particle per unit time. These facts reveal a pool in which mating events accumulate but do not distinguish between a mating pool of replicating particles and a mating pool of particles that have stopped replicating and entered the maturation cycle. Do the products of recombination replicate? Unmistakable clones of recombinants are not found in normal crosses (Kaiser, 1955;Stahl, 1956). They are found, however, in crosses involving phage damaged by radiation (Jacob and Wollman, 1955;Stahl, 1956). Rather than postulate two independent mechanisms of recombination, it seems preferable to conclude that recombinants do multiply. Whether the multiplication produces detectable clones of recombinants then depends on how early recombinants are formed; damaged phages recombine early (Jacob and Wollman, 1955). Since yields from single bacteria infected with two marked phage particles never consist predominantly of recombinants, matings do not characteristically precede multiplication. Since the first particles to mature already contain a large proportion of recombinants, both replicating and mating particles must be noninfective. If, as suggested above, matings are also followed by replication, the concept of vegetative phage as a pool of replicating and interacting particles is confirmed. I n this pool replication and genetic recombination might be parts of the same process or alternating processes (Doermann, 1953;Levinthal, 1954). This brings us to the main question that recombination experiments with phage promise to answer. Before considering it, one should recall in a superficial way the correRponding problem presented by recombination in higher organisms. The essential features are: 1. Recombination is pairwise but involves four strands, two parental and two daughter, of the interacting chromosomes. Which pairs arc involved, and whether recombination occurs during or after replication of parental strands, is not clear. However, all models call for breakage of parental strands and for restrictions as to partners. 2. Recombination is equational, that is, a recombinant differs from its parent only as the mutant differsfrom wild-type, indicating that recombination points are exactly matched in the two interacting strands.

BACTERIOPHAGES AS GENETlC AND BIOCHEMICAL SYSTEMS

45

3. The complementary (reciprocal) recombinants can be produced by a single event, as demonstrated for example in Ascomycetes, in which all the products of a single meiotic cycle can be recovered. These facts call for a complicated model (Schwartz, 1954). What are the corresponding facts about recombination in phage? 1. No information is available about number of strands involved in a single mating, and no decisive information relating recombination to replication of strands. Whether parental strands are broken is also unknown, but information on this point is likely to emerge soon from other sources (Section VI, B). 2. Recombination is equational (Hershey and Rotman, 1949) except for transient local doublings reflected in the structure of heterozygotes (Hershey and Chase, 1951). 3. Complementary recombinants probably are not produced by a single event, first, because no correlated production of complements in single bacteria can be detected (Wollman and Jacob, 1954; Bresch, 1955; Kaiser, 1955), and second, because recombination products (heterozygotes) are known that characteristically produce single recombinants. The force of the first evidence is debatable because two factors are known that might obscure correlated production of recombinants : statistical heterogeneity in the system producing a weak false correlation (Bresch, 1955; cf. Kaiser, 1955) and randomization introduced by sampling from the mating pool at maturation (Visconti and Delbruck, 1953). However, disturbances of the second kind are minimized in systems like T1 and lambda, where the number of rounds of mating, and by inference the size of the mating pool, are small compared with the corresponding measures for T2 and T4. All four phages fail to show evidence of correlated production of recombinants. The elegant experiments of Bresch (1955) with T1 seem to prove, as he concluded, that complementary recombinants are produced by independent acts. It should be added that differences between the genetic behavior of lambda and T2 or T4 have not been entirely accounted for within the framework of current ideas (Wollman and Jacob, 1954). The striking difference in recombination frequency can be explained in terms of neither linkage nor growth habit, but only in terms of number of rounds of mating, A difference in structure of the mating pools seems to be implied. This difference, again, seems to set T2 and T4 apart from all other phages for which comparable genetic information is available. The evidence from heterozygotes bearing on mechanism of recombination also calls for reservations. The properties of heterozygotes indicate that particles of phage T2 are haploid structures that regularly contain bits of superfluous genetic material. When the superfluous bits are ge-

46

A. D. HERSHEY

netically marked, the particle is heterozygous, and when additional markers are present, the structure is revealed as a strand of biparental origin with short overlapping regions (Levinthal, 1954). Both parts of the overlap necessarily function during the growth cycle of the heterozygote, and Levinthal shows by brilliant and plausible analysis that the same process by which heterozygotes are formed can account for production of recombinants in T2. This process yields one parent and one recombinant, or occasionally two noncomplementary recombinants from triply marked parents, per mating act. In other systems the number of heterozygotes is much smaller (Jacob and Wollman, 1954; Bresch, 1955). To explain this, one may ask why the overlap region in heterozygous particles is short. The only explanation to suggest itself is that longer overlaps interfere mechanically with maturation. This thought weakens the particulars of Levinthal’s analysis because it means that the length and frequency of overlaps in mature heterozygous particles do not reflect directly the corresponding properties of their vegetative precursors. On the other hand it explains within the general framework of Levinthal’s theory why there is no direct correlation among different phages between number of heterozygotes and recombination frequency. As a matter of fact, all subsequent studies of phage recombination have suggested general ideas of mechanism very similar to those favored by Levinthal. What are the alternatives? There are basically two both deriving from classical genetic theory, and both requiring slightly modified statements to emphasize the advantages and limitations of biochemical and genetic experimentation with phage (Jacob and Wollman, 1955). Model 1 specifies exchange of parts between completed strands, as in the chiasmatype theory of crossing over. It predicts fragmentation of parental strands and (in simple form) simultaneous production of complementary recombinants. Model 2 specifies biparental production of a single strand during replication, without loss of integrity of parental strands. The model requires that matings occur during the vegetative cycle, not during maturation, It derives from ideas first proposed by Belling and has been called the partial replica hypothesis (Hershey, 1952) or, perhaps better, the copying choice mechanism (Lederberg, 1955). More detailed models have been considered, especially by Bresch (1955). Besides nonfragmentation of template strands, the model (in simple form) predicts production of rccombinants one at a time by independent acts. As already indicated, this model is favored by all phage geneticists beginning with Levinthal (1954). It should be mentioned that a model embodying features of both mechanisms (or rather two independent mechanisms) has been proposed by

BACTERIOPHAGES AS GENETIC A N D BIOCHEMICAL SYSTEMS

47

Schwartz (1954) t o account for experiments with Drosophila. It is possible that phage genetics will be reconciled with the classic materials without serious difficulty. In summary, the information favoring model 2 is of the following nature: 1. Heterozygotes of the structure described above are formed. 2. Production of complementary recombinants in single bacteria is uncorrelated (Wollman and Jacob, 1954; Bresch, 1955). 3. Two recombinations in a single mating act are frequent (Kaiser, 1955). 4. Radiation damage to one of the parental phages entering a cross increases probability of recombination per mating opportunity. Under these conditions the irradiated parent serves as a donor of short segments of genetic material (Jacob and Wollman, 1955; Doermann, Chase, and Stahl, 1955). 5. Under conditions (infection with one particle of unirradiated parent and several particles of a second irradiated parent) in which early, multiple recombinations involving the minority parent are forced, the latter nevertheless survives to form a clone (Jacob and Wollman, 1955). 6. Large numbers of lesions caused by decay of incorporated P32 in the genetic material of T4 do not greatly set back the replication of a single marker that has been rescued by recombination with undamaged phage (Stent, 1953; Stahl, 1956). Like some of the results mentioned above, this suggests a high frequency of double recombinations in a single precocious mating act involving the damaged phage. A mechanism based on model 2 can explain the facts of genetic recombination in a simple manner, whereas an explanation based on model 1 would have to be complicated. This might be considered adequate support for model 2 except for one circumstance: a biochemical test of the material integrity of the parental genome remains t o be achieved or proved impossible.

B. Mutation Mutants are essential for the study of genetic recombination, and publications cited in that connection should be consulted for descriptions of mutants available. Studies of the mutational process are few. Luria (1951) showed that mutations occur during the vegetative phase of viral growth, giving rise to mixed yields containing branching mutant clones. This is interpreted to mean that each vegetative phage particle is the potential parent of a clone. DeMars (1953) observed a mutagenic effect of proflavin on multiplying T2. Weigle (1953), in an important paper, described mutagenic action of

48

A. D. HERSHEY

ultraviolet light on phage lambda, for which separate irradiation of bacterium and phage was necessary. T3 behaved in the same way (Weigle and Dulbecco, 1953). I n contrast to Weigle’s results, Jacob (195413) obtained mutations in phage by irradiating only the bacteria before infection. The interpretation of these results is complicated by the idea of genetic homology between phage and bacterium (Section IV, G): is this mutation or genetic recombination between phage and bacterium? Recombination between phages is also encouraged by irradiation (Jacob and Wollman, 1955). It continues t o appear doubtful or unlikely that viable mutations can occur in resting phage particles.

C . Genetic Fine Structure Benzer (1955) has described a new selective method for distinguishing T4 from certain of its mutants that is widely applicable to problems of both genetic recombination and mutation. It is based on the fact that E. coli K12 carrying lambda prophage is resistant to one class of mutants of T4, but sensitive t o other mutants or to the wild-type phage. The mutants thus set apart identify a region of many closely linked loci a t which mutations produce similar effects (plaque type r). The mutants are not all functionally equivalent, however; by testing pairs of them two classes can be identified. Any member of either class can grow in lysogenic K12 if assisted by a second mutant of the other class, but is not helped by a member of its own class. The individual mutants are readily distinguished and mapped by recombination tests, and the two phenotypic classes can be assigned on this basis to adjacent segments of the map. Owing to the selective nature of the test for the wild-type recombinant, recombination frequencies down to about 10-* can be measured. This is 106 times below the limit imposed by the use of nonselective methods and brings a powerful microscope to the examination of genetic structure. By rough estimates that are acceptable in principle, Benzer points out corresponds to t>hatthe minimal recombination frequency observed ( the 480 thousandth part of the total map distance in T 4 or, in terms of DNA, a maximum of 13 nucleotide pairs separating the two sites of mutation. By similar considerations, certain mutational effects appear to span a considerable distance, and the length of the functional segments referred to above is about 4000 nucleotide pairs each. The most dubious assumption involved is that recombination frequency is an uncomplicated measure of physical distance. However this may be, Benzer’s deliberations clarify considerably general notions about genetic structure. With respect to the tools of genetic analysis, he shows that the mutational site must itself have R certain map length

BACTERIOPHAQES AS GENETIC AND BIOCIiEMIC.4L SYSTEMS

49

that cannot fall below the elementary unit of measurement of length, for mample, a single pair of nucleotides. What the elementary unit measurable by recombination tests really is cannot be independently determined. With respect to functional units, considered, for example, in terms of genetic specification of protein (enzyme) structure, he observes a t the top a class of effects (all r mutations) indistinguishable when T4 is tested on E. coli B and produced by mutations widely distributed throughout the chromosome. When these are subdivided by additional tests on K12, a more homogeneous class of linked mutational sites is discerned, which can be further subdivided by tests of mixed infection on K12 into two regions covering about 4000 nucleotide pairs each-still rather large (2% of the total map distance) to devote to one subclass of one kind of mutational effect. The next anticipatable kind of functional unit, spanning presumably a few nucleotide pairs, might be considered the ultimate functional unit : namely, a region specifying sequence at a single peptide bond, itself subject to multiple mutations possibly separable by recombination. The ultimate unit of recombination, by hypothesis a single nucleotide pair, will probably remain hypothetical for a long time. In the light of Benzer’s ideas many controversies about genetic structure evaporate (Hershey, 1953a; Beadle, 1955). This important gain from Benzer’s work will probably remain intact even if some of his specific assumptions prove incorrect.

D . Radiogenetics It has been known since the early work of H. J. Muller and L. J. Stadler

that radiations can produce local lesions (mutations and chromosome breaks) in genetic material. Chiefly from work with microorganisms, the idea subsequently developed that the main effects of radiation on cells could be explained on this basis. As long as observations were limited to kinetic studies of cell killing, this idea was bound to remain elusive and has, in fact, been periodically abandoned. Recent genetic experiments with phage have proved that it is essentially correct, that is, radiations produce mainly local lesions in the genetic material of phage particles, probably as a consequence of single quantum acts, and the undamaged portions of such dead particles can be reclaimed by genetic recombination with healthy vegetative phage particles. This idea was first proposed by Luria and is to be contrasted with the idea of some unspecified type of damage subject to repair, which also contains elements of truth (Dulbecco; see Bowen, 1953). The experimental discrimination between these two ideas is suvh a remarkable accomplishment that other aspects of the pertinent work will not be touched on here. Some of them have already been mentioned in Section V, A. It should be stated, however, that radiogcnetirs also pro-

50

A. D. HERSHEY

vides new tools for attacking several of the central problems of growth and inheritance. The main facts, revealed for example by experiments of Doermann et al. (1955) with T4 killed by ultraviolet light, can be summarized as follows. First, yields of phage from individual bacteria infected with one or more live plus one irradiated particle regularly show some but not others of genetic markers coming from the irradiated particle. Individual markers are considerably more resistant to radiation than is the infective property of the whole phage particle. Second, for moderate radiation doses, markers derived from irradiated particles are either absent entirely or present in numerous copies, in yields from individual bacteria. Third, markers known from genetic tests to be unlinked are inactivated independently of each other. Fourth, genetically linked markers can be inactivated together. These and other facts show that radiations can kill phage particles by producing local lesions in genetic material, and that undamaged parts can be rescued away from distant lesions by genetic recombination with live phage. It might be argued that the local lesion idea for the killing of phage particles cannot be extended to cells. This argument can be countered by a second remarkable fact: in favorable instances the radiosensitivity of the recently infected bacterium with respect to its ability to grow phage is equal to that of the free phage particle (Bowen, 1953; Stent, 1955). The cellular metabolic apparatus on which phage growth depends is relatively insensitive. So are the attachment and injection mechanisms of the phage particle, a t least to ultraviolet light. This must be due in part to a specific radiosensitivity of DNA. The excellent review of Bowen (1953) should be consulted for background information. The principal radiogenetic experiments are reported by Stent (1953), Doermann et al. (1955), Jacob and Wollman (1955), and Stahl (1 950).

VI. CHEMISTRY OF

THE

VEGETATIVECYCLE

By necessity, the chemistry of viral growth has been studied exclusively during the lytic cycle of phage growth (Fig. 1). During this cycle vegetative replication und maturation proceed more or less simultaneously. By hypothesis one imagines them nevertheless to be independent processes. They will be so treated here, arid the biochemical facts will be considered chiefly in terms of compatibility with this hypothesis. The background information has been reviewed recently from the same point of view (Hershey, 1956a) and for different purposes by Cohen (1955) and Putnam (1953), both pioneers in the biochemical attack on problems of viral growth.

BACTERIOPHAGES AS GENETIC AND BIOCHEMICAL SYSTEMS

51

A . The Priming Material The direct initiator of viral growth must be the material injected into the bacterium a t the start (Hershey, 1956b). As a general hypothesis, one should consider this priming material as composed of two parts, a germinal substance and some accessory substances. Neither part should be called a phage nucleus or a precursor of vegetative phage particles, because it is conceivable that neither functions as a template for the production of new phage material (Stent, 1955; Hershey, 195613; Tomizawa and Sunakawa, 1956). Even the term germinal substance must be stripped of connotations except one: the germinal substance carries genetic markers from the phage particle into the bacterium. Independently of template function, the germinal substance or the accessory substances, or both, might or might not be incorporated into offspring particles, and might or might not undergo fragmentation in the process. If fragmented, the fragments might retain specific functions or not. The transfer from parental to offspring phage furnishes a major clue to the function of germinal substance, but a clue that remains to be deciphered. Until it is understood, all attempts to analyze the germinal substance of the phage particle must be regarded as preliminary. For this reason the reviewer mildly deplores some of the conclusions that have been drawn from the blendor experiment. What it shows is that the sheath of the phage particle is dispensable after infection, and that the crude priming material is chiefly DNA but also contains small amounts of protein, one component of which has been detected (Hershey, 1955). About the composition of the germinal substance little can be said, except that for many reasons one expects it to contain an appreciable fraction of the total viral DNA (Stent and Fuerst, 1955; Benzer, 1955; Doermann et al., 1955; Stahl, 1956). What are the reasons for differentiating the primer into germinal and accessory substances? First, to make room for the agents of transduction in phages exhibiting this phenomenon (Section IV, H). Second, to account for some metabolic effects of infection that vary from phage to phage (Jacob, 1954a). It is possible, though, that these can be explained by action localized a t the site of attachment (Puck and Lee, 1955; French and Siminovitch, 1955). Third, as a working hypothesis that is essential if the facts are to be learned.

B. Material Transfer from Parental to Ofspring Phage The transfer of labeled atoms from parental to offspring phage, first observed by Putnam and Kozloff, presents a key problem bearing equally on questions about mechanism of genetic recombination, composition of the germinal substance, and mode of replication. Since the priming ma-

$2

A. D. HERSHEY

terial is chiefly DNA, the transferred atoms are chiefly those of DNA. The mechanism of transfer is largely unknown. Kozloff (1953) suggests that it involves breakdown and resynthesis. Hershey (1956a) summarizes old and new facts and suggests that this pessimism is unjustified. Stelit and Jeriie (1955) recently showed that most of the transferred atonis enter into very few of the offspring particles. This and other current developments promise to settle the main issues. At this time further discussion is unnecessary.

C . Phage-Precursor Nucleic Acid Factual references to the following summary will be found in previous reviews (Hershey, 1956a,b). However, what follows is a little more than a condensation of them. One of the ultimate objectives of precursor analysis is to learn something about the chemical composition and mode of reproduction of vegetative phage. At the start a new definition of vegetative phage must be introduced, namely, the physical structure with which new germinal substance is associated during vegetative reproduction. It is evident that “germinal substance” and “reproduction” refer back to genetic experiments, hence chemical and genetic methods are inseparable. This is a t once the novel and the difficult feature of the program, and even partial successes are worth recording. The considerations outlined at once invalidate any attempt to isolate vegetative phage in the literal sense: it could not be recognized among the isolates. Nevertheless, it is interesting that such attempts lead to the same suggestion as other methods: phage precursor nucleic acid seems to be free nucleic acid. The alternative methods employ kinetic tracer analysis of phage precursor materials to bring the process of reproduction under a microscope of a unique kind that is not supposed to interfere with the specimen under observation (cf. Delbruck, 1949). Whatever pessimism might be felt in advance about the potentials of this method, it should be recalled that the first tracer experiments with phage were published by Cohen in 1918 and it is unthinkable that the possibilities have been exhausted in the iiitervening years. It is instructive to recall that work with transforming DNA (Ilotchkiss, 1954) and work with phage employ exact methodological complements to similar ends. Transforming DNA is biologically active in the isolated condition but is, so far, invisible in the replicating condition. It may be expected that the results will also complement each other. Tracer experiments establish that the DNA that is to enter phage T2 particles is synthesized in advance of the particles. The direct information

BACTERIOPHAGES AS GENETIC AND BIOCHEMICAL SYSTEMS

53

leaves one question untouched: is this germinal substance, accessory DNA, or both? In a general way it appears to be both, because kinetic tracer experiments suggest a unitary pool containing about 25 phage equivalents of DNA per bacterium (in glucose-ammonia cultures), in which DNA from different sources is mixed, and from which DNA is drawn at randoni to form mature phage particles. The different sources tested include DNA of parental origin, DNA arising by reorganization of bacterial DNA, and DNA newly synthesized after infection. Moreover, genetic experiments show that the formation of germinal substance precedes the formation of phage particles (Section V, A), and likewise reveal a unitary pool (Visconti and Garen, 1953). One can safely conclude that the chemical precursors include germinal substance, and that germinal substance contains some of the precursor DNA. In order to define vegetative phage chemically it is necessary to know what else is inseparably associated with germinal substance during its replication. This question cannot be answered unambiguously at this time. However, one can determine whether phage precursor DNA in general is associated with phage precursor protein in general, which gives a partial answer. Chloraniphenicol inhibits protein synthesis but does not inhibit DNA synthesis. Phage precursor DNA formed in the presence of chloramphenicol (and incorporated into phage particles after removal of chloramphenicol) behaves exactly like normal phage precursor DNA. This means that phage precursor DNA is not synthesized inside a phage precursor protein membrane. I t is still uncertain whether the DNA so synthesized includes the germinal substance, because corroborating genetic experiments are lacking. Moreover, it is not certain whether chloramphenicol stops all types of protein synthesis equally, though this question can be postponed until the genetic questions are answered. Subject to these uncertainties the following conclusions can be drawn (Hershey, 1956b). First, vegetative phage does not multiply in the form of particles enclosed in a phage precursor protein membrane, which would require that most of the precursors be formed simultaneously. Second, the bulk of the phage precursor DNA, and by inference the germinal substance, is synthesized in the absence of concurrent protein synthesis, suggesting that genetic specificity resides in protein-free DNA. I do not wish to stress the reliability of these conclusions. They are important precisely because of the possibility of disproving them.

D. The DNA-Synthesizing Mechanism Three models of DNA synthesis should be considered pertinent to other

questions dealt with in this review. Model 1. Replicated molecules of DNA serve as independent centers

54

A. D. HERSHEY

for their further replication, along lines proposed by Watson and Crick (1953). The mechanism is geometric. Model 2. The injected DNA from the parental phage particle sets up one or a few DNA-synthesizing centers (containing DNA or not) that subsequently turn out new DNA by a linear mechanism. Model 3. In the first two models it is implied that the newly synthesized DNA is genetically specific. Suppose this is not so, but raw DNA is produced by either a geometric or linear mechanism, serving subsequently as a precursor for the replication of vegetative phage. If the latter process is geometric, Model 3 is equivalent to Model 1 for the purposes of genetic experiments, but not for chemical experiments. One fact probably excludes Model 2. Luria (1951) showed that the distribution of numbers of spontaneous phage mutants in single cell yields of virus is clonal, indicating a geometric mechanism of genetic replication. However, the interpretation of his result is complicated by the pool sampling problem (Section V, A; Levinthal, personal communication). Luria’s experiments might well be extended to include a phage like lambda, for which the vegetative pool may be smaller. Several facts tend to exclude Model 1. Stent (1955) found a totally unexpected stabilization of the phage-producing ability of infected bacteria toward the destructive effects of decay of incorporated radiophosphorus. To demonstrate this, he employed an ingenious and widely applicable experimental principle. The infected bacteria, containing radiophosphorus in the parental phage DNA, in newly synthesized DNA, or in both, were allowed to develop to the desired point in the cycle of phage growth and were then frozen. After a suitable interval for decay of radiophosphorus, the culture was thawed, and the cells were tested for ability to resume phage production. The evolution of response to radioactive decay paralleled the evolution of response to ultraviolet irradiation put in evidence by LuriaLatarjet experiments (Bowen, 1953). Both indicate that the phage-producing mechanism is remarkably resistant to radiochemical damage to DNA. Tomizawa and Sunakawa (1956) have provided complementary evidence along the same lines. They showed that phage DNA accumulated in the presence of chloramphenicol has little or no effect on the response of infected bacteria to ultraviolet light. Taken together, these two results seem to show that the radiation-sensitive targets characterizing the infected bacterium are not DNA at all, or contain only a small part of the total intrabacterial DNA somehow stabilized against radiochemical damage. As such they point to DNA synthesis along the lines of Model 2. Perhaps unfortunately, phage T2 was used

BACTERIOPHAGES AS GENETIC AND BIOCHEWCAL SYSTEMS

55

in these experiments. Luria-Latarjet experiments with this phage seem to be complicated by side-effects that do not reflect basic features of phage growth in general (Bowen, 1953). Another clue tending to exclude Model 1 for the synthesis of DNA was first described by Burton (1955) and studied independently by Melechen (1955) and Tomizawa and Sunakawa (1956). If protein synthesis is inhibited (in one of several ways) at the time of infection, synthesis of phage DNA fails to start. If the inhibition is imposed some minutes later, DNA synthesis is not prevented. The rate finally achieved is linear, and apparently depends on the amount of protein allowed to accumulate during the first few minutes after infection. Such protein could be template material or enzyme or something else, if the distinctions are permissible at this time. The facts summarized above, taken together, tend to exclude both models 1 and 2 for DNA synthesis. Probably no one would suggest that they do so decisively. Moreover, to fit them into Model 3 would call for further elaboration to account for the resistance to radiochemical action in the middle of the latent period. Nevertheless, consideration of Model 3 suggests extreme caution in accepting any conclusions about mechanisms of DNA synthesis or vegetative reproduction at this time. It also suggests three critical questions. Does genetically potent DNA accumulate in the presence of chloramphenicol? If so, is its point by point specificity determined by prior protein synthesis? Finally, does chloramphenicol block the synthesis of all types of protein equally? Experiments that could answer these questions are perfectly feasible. VII. CHEMISTRYOF MATURATION In Section VI we pursued the hypothesis that the vegetative cycle of phage growth is chiefly characterized by the synthesis of phage precursor DNA. By the same hypothesis, synthesis of phage precursor protein must be assigned to the maturation cycle. Neglecting minor components about which we know little (Hershey, 1955), phage precursor protein is the coat material of future phage particles. Even when forced into this possibly oversimplified scheme, the maturation process presents a more complicated problem, conceptually at least, than vegetative reproduction. In this respect it resembles, of course, morphogenetic problems in general. So far this aspect of phage growth has been attacked only as a side issue to what have seemed to be more promising opportunities. Considered simply as protein synthesis, on the other hand, the maturation cycle probably offers its own unique advantages.

56

A. D. HERSHEY

A . Prolein Sylthesis What proteins must be accounted for? The coat of phage T2 conlains two antigens, one probably corresponding to a specific structure in the tail (Lanni and Lanni, 1953). The tail structure is complex, consisting of a tubular sheath and a central pin (Kellenberger and Arber, 1955). The tail-sheath itself consists of two materials, a distal portion probably representing the substance responsible for primary attachment, and a proximal portion that can be detached from both head structure and tail-tip (Fraser and Williams, personal communication). As yet no single component can be isolated in bulk; hence only the general conditions of protein synthesis, rather than genetic problems of structure determination, are accessible for study. The specific function of the attachment organ nevertheless offers opportunities along this line (Streisinger, 1956). The rather opaque nature of the general problem of phage maturation is best illustrated by the effects of proflavin on phage growth (DeMars, 1955). All the known constituents of phage particles are manufactured, but no phage particles. I n the absence of proflavin, similar parts are found along with intact phage particles when infected bacteria are broken open by artificial means. All or most of these parts are precursors of phage particles, or are derived from more complex precursor particles too unstable to permit isolation. Phage precursor protein is synthesized only a few minutes before its incorporation into phage particles and is probably already serologically specific at the moment of synthesis. This information derives from morphological, serological, and biochemical studies initiated by Levinthal, Luria, Maalge, and collaborators. The numerous references will be found in a recent review (Hershey, 1956~). Infected bacteria also make large amounts of protein that is not phage precursor (Hershey et al., 1954). Its function is unknown.

B. Role of Ribonucleic Acid The phage-infected bacterium presents an unusual opportunity to explore the role of ribonucleic acid (RNA) synthesis in biological systems. The problem is virtually untouched. Volkin and Astrachan (1956) have shown that some labeled phosphorus from the medium enters RNA after infection of bacteria by T2. The nucleotides so labeled differ in purine-pyrimidine composition from the whole intrabacterial RNA, which does not show progressive change in composition. The remainder of this discussion may or may not seem informative, depending on the temper of the reader and his familiarity with the problem. During growth, uninfected cells of E. coli make RNA about five times faster than DNA. After infection with T2, accumulation of RNA stops more or less completely, and externally supplied phosphorus and carbon

BACTERIOPHAGES AS GENETIC AND BIOCHEMICAL SYSTEMS

57

are not incorporated into RNA at anything like the rate observed before infection. Do these facts mean that RNA plays no specific role in phage growth? That they should not be interpreted in this way can be argued on general principles bolstered, very recently, by the demonstration that some RNA synthesis occurs. Hershey (1953b) proposed that phage growth might be accompanied by a relatively rapid rate of RNA synthesis. Cohen (1955) pointed out, quite correctly, that Hershey’s evidence for RNA synthesis was inadequate. I with to counter here by showing that the contrary evidence was and is even less adequate. Further discussion may serve a useful purpose, first because the model of RNA synthesis originally proposed seems not to have been understood, second because someone should be induced to study what promises to be a fascinating and important relationship. To begin with, one might well adopt the widespread view that RNA does play a role in protein synthesis, possibly a specific one, and ask how such a role might be proved or disproved. This approach changes the nature of the problem considerably. We know that the bacterium on infection with T2 stops making certain bacterial (enzyme) proteins (Benzer, 1953) but continues making other proteins a t a rapid rate and soon starts to make phage precursor proteins. Even more abruptly, it stops making bacterial DNA and starts making phage DNA. It seems to stop making bacterial RNA. Does it start to make a new kind of RNA specifically needed for phage growth? If so, the amount needed would probably be small compared with the amount already present in the bacterium, because the new RNA would have only a limited, temporary task; it is not a phage precursor and only a few new types of protein are needed. The process might thus resemble the course of DNA synthesis when a small-particle phage like T7 infects a bacterium: there is no net change in DNA content but, by analogy with T2, one supposes that bacterial DNA is being converted into a different, specific viral DNA (Kozloff, 1953). In T2,where the conversion has been studied, it involves very little if any exchange of phosphorus or carbon with non-DNA precursors, and very little exchange with externally supplied DNA precursors except a moderate exchange with externally supplied thymidine (Hershey et al., 1954). Thus intermediates that are common precursors of RNA and DNA (purines, pyrimidines, their ribosides and deoxyribosides other than thymine and thymidine) are not involved or do not enter pathways accessible to competition. If some of or all the bacterial RNA were converted into a new kind of RNA by an analogous process, it could not be detected as a net change in amount of intrabacterial RNA, nor could it be recognized, except perhaps at a very low level, as exchange of phosphorus or carbon between RNA

58

A. D. HERSHEY

and medium. One might anticipate an appreciable transfer of carbon from RNA to DNA (since most of the known precursors of both are common or interconvertible) and this is in fact observed (Hershey et al., 1954). The model so far considered is therefore plausible but useless. A practicable basis for investigating this model was demonstrated by Hershey (1953b), subject only to uncertainty about his methods. RNA was assayed as the difference between labeled Schmidt-Thannhauser RNA phosphorus in boiled or acid-precipitated cells before and after treatment with ribonuclease (or as phosphorus made acid-soluble by the enzyme). The results suggested that a small pool of RNA, metabolically segregated from the bulk of the bacterial RNA, is rapidly built up after infection or, less likely, exists before infection and continues to function after. The identifying characteristics of this pool are that phosphorus enters it from the medium and leaves it again (possibly to enter DNA) at equal rates roughly equivalent to half the linear rate of DNA synthesis after infection. Its minimum phosphorus content corresponds to about 25 phage-particleequivalents per bacterium, or less than 10% of the total intrabacterial RNA. Its actual phosphorus content may be larger, since it probably receives phosphorus from bacterial RNA and DNA. Additional features of the pool of metabolically active RNA have been investigated by further (inadequate) experiments employing chloramphenicol to stop protein synthesis (Hershey, Siminovitch, and Graham, unpublished). This substance, when added to cultures infected with T2 some minutes earlier, does not block the incorporation of labeled phosphorus into RNA but seems to block the flow out of it. RNA labeled under these conditions continues to pile up at about half the rate of DNA synthesis. Like the postinfection RNA detected by Volkin and Astrachan, the labeled RNA is distinctive in composition. When chloramphenicol and radiophosphate are subsequently removed from the medium so that protein synthesis resumes, much of the labeled phosphorus previously incorporated into RNA leaves it, and there is a simultaneous further rise in labeled DNA, suggesting breakdown of RNA to the level of common RNA and DNA precursors. These results suggest a correlation between breakdown of RNA and synthesis of protein. A comparable pool of metabolically active RNA in uninfected bacteria could not be detected, because of the rapid synthesis of RNA that does not turn over (Hershey, 1954; Siminovitch and Graham, unpublished). The generality of the model for RNA synthesis in infected bacteria, as well as its specific relationship to phage growth, therefore remains undefined. The model proposed here is offered not for its factual value, but to show that there is ample scope for a thorough study of RNA synthesis in infected

BACTERIOPHAGES AS GENETIC AND BIOCHEMICAL SYSTEMS

59

bacteria. The ultimate aim is to establish structure and process relationships for DNA, RNA, and protein. The infected bacterium affords a unique opportunity to do this, because a starting point in the germinal substance of the phage particle and a terminus in its protein coat can be explicitly assumed. Analysis of the role, if any, of RNA in the growth of a virus that does not contain RNA should be instructive in many respects. VIII. CONCLUSION The history of lysogeny parallels the history of research with virulent phages. Both originated in the desire to learn something about viruses. Both have led to very similar (provisional) conclusions in one respect. Prophage and vegetative phage probably represent two modes of replication of the naked genetic material of the virus, chiefly DNA. The two histories do not, however, duplicate each other, first, because lysogeny is a different phenomenon from the acute phase of viral infection, and second, because the two present different opportunities that can be exploited only in different, almost mutually exclusive ways. Both are rapidly becoming branches of genetics. To anticipate what is not yet proved, work with the lytic cycle of viral growth is becoming a branch of chemical genetics: it emphasizes the molecular level of genetic determination. Lysogeny deals more exclusively at the cellular level of genetic determination, both through necessity and competence. I n transduction one observes an unclassified phenomenon somewhere between. No phage worker suggests that all virology is likely to find room among the tight little categories by which the life cycle of bacteriophages is presently described. On the other hand, many virologists are prepared to find the phage model helpful in dealing with certain problems of cellular differentiation that have, as yet, no visible kinship to virology. Somewhere in this still unclaimed territory surrounding molecular genetics, cellular genetics, and virology someone may, just possibly, bridge a gap between theoretical and practical problems. REFERENCES Adam, M. H. (1955). Virology 1, 336. Anderson, T. F. (1953). Cold Spring Harbor Symposia Quant. Biol. 18,197. Beadle, G . W. (1955). Bulletin Amer. Institute of Biological Sciences 6 , 15. Benzer, 9. (1953). Biochim. et. Biophya. Acta 11, 383. Benzer, S. (1955). Proc. Natl. Acad. Sci. ( V . S.) 41, 344. Bertani, G . (1953). Cold Spring Harbor Symposia Quant. Biol. 18, 65. Bertani, G . (1956). Brookhaven Symposia Biol. No. 8, 50. Bowen, G. H. (1953). Cold Spring Harbor Symposia Quant. Biol. 18, 245. Boyd, J. 5.K . (1951). J . Pathol. Bacteriol. 63, 601. Bresch, C. (1955). 2. Naturforach. lob, 545. Bresch, C., and Trautner, T. (1955). 2. Naturforsch. lob, 436.

60

A. D. HERBHEY

Burton, K. (1955). Biochem. J. 61, 473. Christensen, J. R., and Tolmach, L. J. (1955). Arch. Biochem. and Biophys. 67, 195. Cohen, 6. S. (1955). Advances in virus Research 3, 1. Delbriick, M. (1940). J. Uen. PbysiOl. 23, 631. Delbriick, M. (1949). Trans. Connecticut Acad. Sci. 88, 173. DeMars, R. I. (1953). Nature 172, 964. DeMars, R . I. (1955). Virology 1, 83. Doermann, A. H. (1953). Cold Spring Harbor Symposia Quant. Biol. 18, 3. Doermann, A. H., Chase, M., and Stahl, F. W. (1955). 1.Cellular Comp. Physiol. 46, Suppl. 2, 51. Freeman, V. J. (1951). J . Bacteriol. 61, 675. French, R. C., and Siminovitch, L. (1955). Canadian J. Microbiol. 1, 754. Garen, A. (1954). Biochirn. et Biophys. Acta 14, 163. Garen, A., and Puck, T. T. (1951). J. Ezptl. Med. 84, 177. Garen, A,, and Zinder, N. D. (1956). Virology 1,347. Goebel, W.F., and Jesaitis, M. A. (1953). Ann. inst. Pasteur 84,66. Herriott, R. M. (1951). J. Bacteriol. 61, 752. Hershey, A. D. (1952). Intern. Rev. Cytol. 1,119. Hershey, A. D. (1953a). Advances i n Uenet. 6,89. Hershey, A. D. (1953b). J . Uen. Physiol. 37,l. Hershey, A. D. (1954). J. Uen. Physiol. 38,145. Hershey, A. D. (1955). Virology 1,108. Hershey, A. D. (1956a). Zn “Currents in Biochemical Research” (D. Green, ed.), Interscience, New York. Hershey, A. D. (1956b). Brookhaven Symposia Biol. No. 8,6. Hershey, A. D. (19560). Zn “Enzymes: Units of Biological Structure and FuncH. Gaebler, ed.), p. 109. Academic Press, New York. tion” (0. Hershey, A. D., and Chase, M. (1951). Cold Spring Harbor Symposia Quant. Biol. 18, 471. Hershey, A. D., and Chase, M. (1952). J. Uen. PhysiOl. 36,39. Hershey, A. D., and Davidson, H. (1951). Uenetics 86,667. Hershey, A. D., and Rotman, R. (1949). Uenetics 34,44. Hershey, A. D., Dixon, J., and Chase, M. (1953). J. Uen Physiol. 36,777. Hershey, A. D., Garen, A., Fraser, D., and Hudis, J. D. (1954). Carnegie Znst. Wash. Yearbook 63, 210. Hotchkiss, R. D. (1954). Harvey Lectures Ser. 48, 124. Jacob, F. (1954a). “Les Batteries Lysogbes et la Notion de Provirus.” Masson, Paris. Jacob, F. (1954b). Compt. rend. soc. biol. 233, 732. Jacob, F. (1955). Virology1,207. Jacob, F., and Wollman, E. L. (1953). Cold Spring Harbor Symposia Quant. Biol. 18, 101. Jacob, F., and Wollman, E. L. (1964). Ann. inst. Pasteur 87, 653. Jacob, F., and Wollman, E. L. (1965). Ann. inat. Pasteur 88, 724. Jesaitis, M. A., and Goebel, W. F. (1953). Cold Spring Harbor Symposia Quant. Biol. 18, 205. Kaiser, A. D. (1955). Virology 1, 424. Kellenberger, E., and Arber, W. (1955). 2.Naturforech. lob, 698. Kozloff, L. M. (1953). Cold Spring Harbor Symposia Quant. Biol. 18,207 Ko5loff, L. M., and Henderson, K. (1965). Nature (London) 176, 1189.

BACTERIOPHAGES AS GENETIC AND BIOCHEMICAL SYSTEMS

61

Lanni, F.,and Lanni, Y.T. (1953). Cold Spring Harbor Symposia Qwmt. Biol. 18, 159.

Lark, K. G., and Adams, M. H. (1963). Cold Spring Harbor Symposia Quant. Biol. 18, 171.

Lederberg, E. M., and Lederberg, J. (1953). Genetics 38,51. Lederberg, J. (1955). J. Cellular Comp. Physiol. 46, Suppl. 2,75. Lennox, E. S. (1955). Virology 1, 190. Levine, M. (1955). Genetics 66, 582. Levinthal, C. (1954). Genetics 39, 169. Levinthal, C., and Visconti, N. (1953). Genetics 38, 500. Lieb, M. (1953). Cold Spring Harbor Symposia Quant. Biol. 18,71. Luria, S . E. (1950). Science 111,507. Luria, S . E. (1951). Cold Spring Harbor Symposia Quant. Biol. 16,463. Luria, 5.E. (1953). Cold Spring Harbor Symposia Quant. Biol. 18,237. Luria, S . E., and Steiner, D. L. (1954). J. Bacteriol. 67,635. Luria, S . E. , Williams, R. C., and Backue, R. C. (1951). J . Bacteriol. 61,179. Lwoff, A. (1953). Bacteriol. Revs. 17, no. Melechen, N. (1955). Genetics 40, 684. Puck, T. T. (1953). Cold Spring Harbor Symposia Quant. Biol. 18,149. Puck, T. T., and Lee, H. H. (1955). J . Ezptl. Med. 101,151. Puck, T. T., and Sagik, B. (1953). J . Ezptl. Med. 97,807. Puck, T. T., Garen, A., and Cline, J. (1951). J . Ezptl. Med. 93,65. Putnam, F.W. (1953). Advances in Protein Chem. 8, 175. Schwartr, D. (1954). Genetics 39. 692. Stahl, F. W. (1956). Virology 2, 206. Stent, G. S. (1953). Proc. Natl. Acad. Sci. (U. 5.) 39, 1234. Stent, G. S. (1955). J. Gen. Physiol. 38,853. Stent, G. S., and Fuerst, C. R. (1955). J . Gen. Physiol. 38,441. Stent, G. S., and Jerne, N. K. (1965). Proc. Natl. Acad. Sci. (U.8.)41,704. Stent, G. S., and Wollman, E. L. (1952). Biochim. et Biophys. Acta 8,260. Streisinger, G. (1956). Virology in press. Tomirawa, J., and Sunakawa, 9. (1956).. J. Gen. Physiol. 39, 553. Visconti, N., and Delbruck, M. (1953). Genetics 38, 5. Visconti, N., and Garen, A. (1953). Proc. Natl. Acad. Sci. (U.S.)39,620. Volkin, E., and Astrachan, L. (1956). Virology 2, 149. Watson, J. D. (1950). J. Bacteriol. 60, 697. Watson, J. D., and Crick, F. H. C. (1953). Cold Spring Harbor Symposia Qwmt. Biol. 18, 123. Weidel, W. (1953a). Ann. inst. Pasteur 84, 60. Weidel, W. (1953b). Cold Spring Harbor Symposia Quant. Biol. 18,155. Weidel, W., and Kellenberger, E. (1955). Biochim. et Biophys. Acta 17.1. Weidel, W., Koch, G., and Bobosch, K. (1954). Z. Naturjorsch. 9b, 573. Weigle, J. J. (1953). Proc. Natl. Acad. Sci. (U.5.)39,628. Weigle, J. J., and Dulbecco, R. (1953). Ezperientia 9,372. Whitfield, J. F., and Murray, R. G. E. (1954). Can. J . Microbiol. 1,216. Wollman, E. L. (1953). Ann. inat. Pasteur 84,281. Wollman, E . L., and Jacob, F. (1954). Ann. inst. Pasteur 87,674. Zinder, N . D. (1953). Cold Spring Harbor Symposia Quant. Biol. 18,261. Zinder, N . D. (1955). J. Cellular Comp. Physiol. 46. Suppl. 2,23.