STUDIES OF HEAT-INDUCIBLE h PHAGE MUTATIONS IN CISTRON N AFJ3ECTING HEAT INDUCTION1

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1 STUDIES OF HEAT-INDUCIBLE h PHAGE MUTATIONS IN CISTRON N AFJ3ECTING HEAT INDUCTION1 MARGARET LIEB Department of Mircobiology, Uniuersity of Southern California School of Medicine and Los Angeles County General Hospital, Los Angeles, California Received April 29, 1966 INDUCTION of a lysogenic bacterium can be defined as an event that results in a change in the host phenotype attributable to the presence of a prophage. The consequences of induction of K-12 (A) include loss of immunity to A, cell death, and synthesis of phage-specified enzymes. Increased synthesis of h mrna occurs shortly after exposure of a lysogen to an inducing agent (ATTARDI et at. 1963; SLY, ECHOLS and ADLER 1964) and has been observed in lysogens in which cell killing and phage replication are blocked by chloramphenicol (GREEN 1966). Studies of h mutants suggested that the protein product of gene CI prevents X induction (JACOB and CAMPBELL 1959; JACOB, SUSSMAN and MONOD 1962). It has been proposed that the CI product represses the synthesis of mrna for one or more enzymes required for h replication (JACOB and MONOD 1961). Mutations that result in induction when X lysogens are heated above 35 C map in the CI gene of X and are called CIt. A correlation between the locations of the t mutant sites on the Cr map and the properties of the corresponding lysogens led me to propose a model for the repression of h ( LIEB 1965,1966a). It was suggested that the C, product acts as a repressor when it is combined with a nascent protein, which is the product of an operator gene. The nascent protein was assumed to be an enzyme whose activity was inhibited by the Cr product. Mutations in the gene for this protein might be expected to influence its interaction with the C, product, and thus affect the heat-inducibility of prophages with mutations in CI. In an attempt to locate the gene for this hypothetical protein, lysogens containing mutations in CI and also in genes N, 0, P and R were prepared. The effects on heat induction of mutations in genes 0, P, and R are clearly distinguishable from those of N mutations, and will be reported elsewhere. This study concerns heat induction of X prophages with mutations in both Cr and N. Mutations in gene N result in a deficiency in the amount of a specific exonuclease which appears in X lysogens after the application of inducing agents (KORN and WEISSBACH 1963; RADDING 1964a, b). As shown below, these mutations alter the ability of prophages containing CIt to be induced at high temperatures. 1 This research was supported by grants from the National Science Foundation (G-24165) and the Public Health Service (AI and &K3-GM-1546). Genetics 5.1: September 1 366

2 836 M. LIEB MATERIALS AND METHODS Media Lysogenic cultures were grown with aeration in 1% Bacto tryptone plus 0.5% NaCl (TB) to a concentration of 2 to 4 x 108 bacteria/ml. Dilutions were made in 0.9% saline containing 10% TB. Bacteria were assayed by spreading on TA plates (TB plus 1.2% Bacto agar). Bacterial and phage strains: The sources of the bacteria and phages used in this study are listed in Table 1. The relative positions of the phage mutations on the 1 prophage map are shown in Figure 1. sus (suppressor sensitive) mutants are able to grow and yield mature progeny in M3, a permissive (pmf) host, but not in 594, a nonpermissive (pm-) host. Although the sus mutants cannot grow in pm-, the latter can be lysogenized by h sus, forming defective lysogens. h Crt mutants lysogenize at 36", but the prophages are induced when the respective lysogens are grown at 42 to 45". The prefix "Ci' will be omitted in referring to the heat-inducible mutants. Isolation of double mutants and preparation of lysogens: Double mutants containing a mutation in both C, and N were obtained by infecting pm+ with a multiplicity of about five of each of two phages. The phages had been exposed to approximately 300 ergs/"* ultraviolet light (W) from a Westinghouse Sterilamp. The lysate from the cross was plated on strain M3 at 42". Phage from clear plaques (indicating heat-inducibility) was spotted to pm- to test for the presence of the N sus gene. For each cross, three independent double mutant recombinants were picked. Pm+ lysogens containing each of the recombinants were used to prepare a high titer stock of the respective phage. Pm- lysogens were then prepared by infecting starved stationaryphase pm- with a multiplicity of 5h at 25". Two independently-isolated pm- lysogens were obtained from each double mutant phage stock. Thus, six different pm- lysogens were tested for each double mutant combination. The results presented are those from representative lysogens. Supezinfectiom Lysogenic cultures containing 2 to 4 x 108 bacteria/ml were superinfected by diluting 0.5 ml ml phage. After 10 min at 36", 5 ml TB was added and the culture aerated for 20 min at 36". The superinfected cultures were stored on ice for not more than 30 min and then diluted 1:lOO into warm medium. Mechanical stirring was used to maintain the temperature of the water bath within 0.5"C of the desired temperature. Measurement of induetiom Bacteria were grown in TB to about 2 x 108 bacteria/ml. To start induction, they were diluted 1:lOO into prewarmed TB. Samples were assayed at intervals. When heated at 43", almost 100% of the heat-inducible lysogenic bacteria in a culture become plaque-formers. However, it is more convenient, for several reasons, to study heat induction by measuring the loss of the colony-forming ability of lysogenic cells. Prolonged incubation at temperatures over 43" results in a loss of plaque-formers. Moreover, sus mutants only rarely produce mature progeny in pm- bacteria. sus mutations in gene N prevent or reduce replication of h DNA in pm- bacteria (BROOKS 1965; DOVE 1966; JOYNER, ISAACS and ECHOIS 1966). If normal phage replication were a necessary step in the killing of the host, it would not be possible TABLE 1 Origins of strains used Described by Obtained from E. coli strains M3 = pm+ LIEB = pm- Derivative of 3350; CAMPBELL 1961 J. WEIGLE A strains A+ (or Cr+ - Nf) J. WEIGLE C,tl;C,t2 LIEB 1966a C,c47; C,c71 KAISER 1957 J. WEIGLE N sus 7 CAMPBELL 1961 J. WEIGLE N sus 53 CAMPBELL 1961 A. CAMPBELL

3 NEAT INDUCTION OF PHAGE A 837 sus7 sus 53 t2 ti t L-JLLL-4 E. coli cm N - FIGURE 1.-Relative approximate. PART A PART B O,P,etC. ci order of genes in the N - C, region of 1. The map distances are very to study heat induction of N sus mutants in pm- by measuring cell death. As reported below, prophages with mutations in gene N that block phage production can, nevertheless, kill pmbacteria. permitting death of the lysogen to be used as an indicator of induction. For UV induction, bacteria were diluted 1 : 100 in saline, Small samples were irradiated under a Westinghouse Sterilamp with constant shaking. RESULTS UV induction of lysogens with double mutant A prophages: CAMPBELL (1961) called A N sus mutants uninducible in pm- because the UV-irradiated lysogens did not produce lysozyme, and thus the cultures did not lyse after UV. However, lysogens containing A N sus mutants were more sensitive to UV killing than nonlysogenic bacteria, suggesting that N sus did not prevent induction of some X function (K. BROOKS, personal communication). The high sensitivity to UV induction of some XCrt mutants (LIEB 1964) makes it easier to determine whether mutations in gene N (or any other gene) affect inducibility. The UV killing curves of pm-(at2 - Nf) and pm-(htz - N sus 7) are compared with nonlysogenic pm- in Figure 2. It is clear that lysogens containing N sus 7 prophages were Of- 5 E 3 2,001- a: U) 3 FIGURE 2.-Ultraviolet induction of pm- (kt1-a +) and pm-(at1 - N sus 7). Two independently-isolated lysogens of each type, and also a nonlysogenic culture of pm- were irradiated simultaneously M pm-(atl-n+) c - 4 pm-(ltl- Nsus 7 ).OOOO~ I20 UV DOSE (seconds)

4 838 M. LIEB sensitive to UV induction. Pm-(htl - N sus 7) lysogens did not form plaques when plated on pm+ after UV, so that killing cannot be attributed to a recovery of the normal function of Gene N. Mutation t2 results in a A prophage that is extremely sensitive to UV induction (LIEB 1966a). The UV sensitivity of pm-(at2 - N sus 7) lysogens was similar to that of pm-(ht2 - N+) (LIEB, unpublished observations). It is interesting that neither htl - N sus 7 or At2 - N sus 7 killed pm- when added to noniysogenic bacteria at multiplicities of 5 to IO, but their prophages killed pm- after UV irradiation. Heat induction of' At1 - N sus 7 prophage in pm+ and pm-: Prophages At1 - N+ and At2 - N+ are heat-inducible in both pm- and pm+. Pm+ lysogens containing prophages with mutations in both CI and N were induced when heated to 43" or 45" (Figures 3, 4). On the other hand, pm-(htl - N sus 7) lysogens multiplied at 43", and were killed very slowly at 45" (Figure 3). The bacteria in colonies formed by pm-(at1 - N sus 7) after heating were not killed by superinfection with hc47, indicating that the bacteria were still lysogenic. One cannot attribute the lack of induction of prophage At1 - N sus 7 in the pm- host solely to the absence of the normal product of gene N. It has already been shown that pm-(htl - N sus 7) can be induced by UV. In addition, pm- (ht2- N sus 7) was induced at both 43" and 45" (Figure 4). h-( At2 - N SUS 7) lysogens did not form plaques when plated on pm+ after heating, indicating that gene N had not regained its normal function. Zmmunity of pm-(htl - N sus 7) after heating: To test whether heating de- \ \.: 45*.oo I 0 I 43. I I I MINUTES AT 43' OR 45' FIGURE 3.-Heat induction of it1 - N sus 7 prophage in pm+ and pm- hosts. Procedure given in MATERIALS AND METHOD MINUTES AT 43' OR 45' FIGURE 4.-Heat induction of it2 - N sus 7 prophage in pm+ and pm-.

5 HEAT INDUCTION OF PHAGE h 839 stroyed the ability of the prophage to prevent the replication of other genomes, aliquots of a pm-(htl - N sus 7) culture were infected with At1 - N+ and ht2 - N+. The bacteria were first superinfected and then heated; a loss of immunity upon heating would be expected to allow the added genomes to kill the lysogenic host. Pm-(htl - N sus 7) cultures superinfected with Xtl - N+ or At2 - N+ did not die at 43" (Figure 5). About 90% of the bacteria in superinfected cultures were killed at 45", but the majority of nonsuperinfected lysogens survived heating for 60 minutes. The addition of N+ phages to nonsuperinfected lysogens after heating did not kill the 43" or 45" survivors. The results indicate that pm-(ad - Nsus7) lysogens retained their immunity at 43", and that even at 45", immunity was maintained in most of the lysogens that contained no N+ superinfecting phages. Effect of superinfection with htl - N sus 7 on heat induction of At2 - N sus 7 prophages in pm-: The observation that pm-(htl - N sus 7) bacteria retained their immunity at 43' suggested that the C, product made by At1 - N sus 7 in pm- was more heat-stable than the CI product of htl - N+. It has been shown that after the addition of h+, which produces a heat-stable CI product, a lysogen containing a heat-inducible prophage rapidly becomes resistant to heat induction (LIEB 1966b). Pm-(ht2 - N sus 7) bacteria were superinfected at equal multiplicities with either A+, htl - N sus 7 or heat-inducible N+ mutants (Figure 6). The superinfected lysogens were then placed at 43". Although not as effective as A+, htl - N sus 7 inhibited heat induction of pm-(at2 - N sus 7 ). In several similar experiments, At1 - N sus 7 afforded more protection against heat induction at 43" than did htl - N+, suggesting that complementation between CI mutants tl and t2 does not account fully for the protective effect. Heat inducibility of other CI-N double mutant prophages: Double mutants containing tl or t2 and N sus 53 have been prepared. "-(At1 - N sus 53) lysogens were not killed at 43" or 45". However, the UV killing curves indicated that pm- (At1- N sus 53) was sensitive to UV induction, After heating, pm-(htp - I pm-iati-nsus 7) :+:- ji 0.1 i '% 0.05 :'. TB 45' =-. -- ' 0 A l l 45- 'dr kl I FIGURE 5.-Heat induction of pm-(atl - N sus 7) superinfected with At1 - N+ or At2 - N+. The method of superinfection is given in MATERIALS AND METHODS. At1 = At1 - N+;AtZ = At2 - N+. TB = not superinfected. It should be noted that a small fraction of bacteria always escapes infection when exposed to A for only a short time, and that this may account for the leveling off of the 45" killing curves of superinfected bacteria. MINUTES AT 43'OR 45'

6 840 M. LIEB phage added 4 FIGURE 6.-Heat induction of pm-(xt2 - N sus 7) after superinfection with various phages. TB = not superinfected.,000 I I MINUTES AT 43' N sus 53) bacteria were no longer immune to superinfection, but there was little killing. Studies of these lysogens will be reported elsewhere. DISCUSSION The presence of a mutation in gene N does not abolish the ability of a prophage to kill its host. This is shown by the UV induction of pm-(at1 - N sus 7) and the heat induction of pm-(at2 - N sus 7). RADDING and SCHREFFLER (1966) have developed a sensitive assay for A-specific exonuclease in lysogens treated with the Immunity is lost TABLE 2 Induction of pm- lysogens Prophage Added phage Killing cr N c, N 43" tl tl sus7-3. tl sus53 4. t t2 sus7 6. t2 sus53 7. tl sus 7 tl + 8. tl -+- tl sus7 9. t2 sus7 tl sus7 10. t2 sus7 tl sus53 -c * e - 2 * + + +

7 HEAT INDUCTION OF PHAGE h 84 1 inducing agent mitomycin. They report that pm-(hc,+ - N SUS 53) has IO%, and pm-(hci+ - N sus 7) has 3% of the exonuclease activity found in induced lysogens containing wild-type h. It has been shown here that pm- lysogens containing it2 - N sus 7 or htl - N sus 53 are induced by UV. Therefore, normal production of exonuclease is not required for the killing of a h lysogen by a prophage. Although the exonuclease whose synthesis is controlled by gene N is apparently not required for induction, the presence of a mutation in N can prevent A induction, as measured by killing. Table 2 summarizes the results of several types of experiments. In pm- bacteria,,it2 prophages containing N sus 7 are not induced at 43. However, pm-(ht2 - N sus 7) lysogens are killed when heated to 43. Thus, the site of mutation in C, may determine whether N sus 7 inhibits heat induction in the pm- host. The site of mutation in gene N also affects the response of the lysogen to heat. Pm-(ht2 - N sus 7) lysogens are killed at 43, but pm- (At2- N sus 53) lysogens survive. It should be noted that at 37, mutation N sus 53 is more leaky than N sus 7 (RADDING and SCHREFFLER 1966). Since mutations in gene N affect a phage phenotype (e.g. heat-inducibility) that has been associated with gene Cry it is important to establish that CI and N are, indeed, separate cistrons, producing different products. XC, mutants do not appear to lack any gene product required for replication, since phage production is normal after infection or induction. In pm- bacteria, CI sus mutants that are unable to lysogenize can complement x s with sus mutations in N (LIEB, unpublished). CI+ - N sus mutants, when added to pm-(xit - Nf), prevent subsequent heat induction, and the rate of synthesis of CI+ product by CI+ - N sus 7 and by Cf + - N sus 53 is identical in pm- and pm+ (LIEB, unpublished). Thus, gene CI and gene N appear to function independently. Although N sus 7 is epistatic to CItl when pm-(htl - N sus 7) is incubated at 43, N sus7 does not prevent UV induction of the same lysogen. This can be understood if one assumes that N sus 7 does not affect the interaction of the CI product and the inducer produced in UV-irradiated bacteria ( GOLDTHWAITE and JACOB 1964). Since C, mutations which profoundly affect UV-inducibility map in region A of CI (LIEB 1966a), one may infer that the corresponding part of the Cf product contains the active site for interaction with the inducer. In all double mutants that have been tested, the presence of N sus 7 did not affect the heat-inducibility of h prophages with t mutations in C, region A, but prevented heat-induction of X s with mutations in Cr region B (CROSS, personal communication). Formally, N sus 7 can be regarded as a suppressor of heat-inducible mutations mapping in CI region B, but not those in C, region A. HOW can mutations in gene N prevent the heat induction of prophages with mutations in a particular part of gene CI? Suppressor-sensitive mutations of the amber type, which includes N sus 7 and N sus 53, are known to have two effects on gene action in a nonpermissive host. A nonsense codon at the site of the sus mutation terminates synthesis of the protein specified by the mutated gene (STRETTON and BRENNER 1965). In addition, the nonsense codon can prevent the synthesis of protein products by cistrons distal to the sus mutation, which are

8 842 M. LIEB presumed to be translated from the same (polycistronic) messenger (BENZER and CHAMPE 1962; AMES and HARTMAN 1963). This polar effect of sus mutations is the basis of the first of two hypotheses offered as possible explanations of the interaction of mutations in genes C, and N. 1. Polarity hypothesis: Protein synthesis during heating is required for the killing of At mutants mapping in Cr region B, but not those in Cr region A (LIEB 1966a; GREEN 1966). In CrB mutants, one may assume that a protein required for killing must be made while the lysogens are at the high temperature. The composition of this protein may be specified by a cistron distal to N in the same operon. The polarity hypothesis states that pm-(hcrb - N sus) bacteria are not killed at 43 because a nonsense codon in the message for the N product blocks translation of the RNA message for a killing protein. N sus mutations do not affect the synthesis of the product of C,, the gene adjacent to N in the right-hand direction on the prophage map (Figure 1). Thus, any polar effect of N sus would be expected to extend to genes to the left of N. Since the structural gene for the killing protein has not been identified, one is free to imagine that it maps to the left of N. However, there are data that are difficult to reconcile with the polarity hypothesis. One must invent special hypotheses to account for the synthesis of enough inducing protein in pm-(~2-- N sus 7) bacteria to account for their sensitivity to heat-killing. Furthermore, a polar mutant would not be expected to interfere with the expression of genes located in a trans position. Pm-(htl - N sus 7) bacteria containing htl - N+ superinfecting phage are not induced at 43, indicating that N sus 7 has an effect on the expression of genes in a different phage genome. A polar effect also fails to explain the protective effect of htl - N sus 7 on heat induction of pm-(ht2 - N sus 7). The fact that pm-(atl - N sus 7) lysogens multiply at 43, but die slowly at 45, might be attributed to an increase in the probability of translation of the N sus genome at the higher temperature. However, no increase in the fraction of bacteria that yield phage has been detected in cultures heated at Product interaction hypothesis: Let us suppose that gene N produces a product that combines with the C, product in lysogenic bacteria. Certain alterations in the structure of the N product would be expected to affect the configuration, and thus the heat-lability, of the Cf product. To account for the trans effect of Nsus mutations, some N product is assumed to be present in the bacterial cytoplasm. CI is the only h gene whose product has been shown to be present in lysogenic bacteria. There is no detectable h exonuclease in uninduced lysogens (RADDING and SHREFFLER 1966). However, additional studies by these authors suggest that N is not the structural gene for h exonuclease. Thus, there are no data that preclude the assumption that the prophage produces a small amount of N product in h lysogens. It may be noted that in superinfection heterozygotes containing htl- N sus 7 and Atl-N+, N sus 7 prevents heat induction more effectively when it is in the prophage than when it is in superinfecting phage (Table 2, lines 7 and 8). The C, gene, on the other hand, seems to function very effectively both in prophage and in superinfecting phage, and a single superinfecting Cr+ phage can prevent heat induction of a lysogen containing h CIt prophage (LIEB 1966b). The

9 HEAT INDUCTION OF PHAGE h 843 dominance of CI+ was attributed to the stabilizing effect of a single heat-stable subunit in an oligomer containing several molecules of C,t product. If the N product combines with the CI product in lysogenic bacteria, the substance that prevents h induction may be composed of subunits containing products of both gene N and gene CI. It is conceivable that while the presence of a single molecule of heat-stable C,+ product makes the oligomer heat-stable, more than one molecule of N sus product is required to achieve the same effect. In the superinfection experiments reported here, the multiplicity of superinfecting genomes may not have been sufficiently high to produce the concentration of N sus product required to stabilize the oligomer. The finding that N sus 7 interferes with the heat induction of XCIt mutants mapping in region B, but not those mapping in region A, can be understood if one assumes that the product of gene N interacts primarily with region B of the C, product. A major change in the size or structure of the N product might inhibit configurational changes in the CI product that are required to destroy its repressor activity. It is conceivable, for example, that the abbreviated products made by N sus 7 and N sus 53 in pm- hosts retard dissociation of the N product and region B of the C, product. The heat-inactivation of CI product with a mutation in region A may not be influenced by the N product associated with region B. Although both the polarity and product interaction hypotheses rest on some unsupported assumptions, the latter notion appears to account more satisfactorily for the effect of N sus mutations on heat induction. No enzymatic function for the N product is known, but it is not the killing enzyme. Thus, there is no information to support the notion that the C, product combines with a nascent enzyme (LIEB 1966a). However, the properties of CIt-N sus mutants indicate that the protein product of gene N participates in the regulation of h functions. SUMMARY Induction of h prophages has been studied by measuring killing of lysogenic Escherichia coli K-l2(h). Double mutants of h contained a t mutation in gene CI, resulting in heat-inducibility, and also a sus (suppressor-sensitive) mutation in gene N. N sus mutations permit normal synthesis of h exonuclease in permissive (pm+), but enzymatic activity is greatly reduced in nonpermissive (pm-) bacteria. The double mutant prophages were heat-inducible in pm+, but heatinducibility in pm- was dependent on the site of the mutation in C,. F m- (htl-n sus 7) was not induced at 43. Heat induction of pm- (W-N sus 7) was inhibited by infection with htl-n sus 7 before heating. The finding that sus mutations in gene N can prevent induction suggests that the products of both C, and N are involved in the repression of h prophage. AMES, B. N., and P. E. HARTMAN, 1963 Quant. Biol. 28: LITERATURE CITED The histidine operon. Cold Spring Harbor Symp. AITARDI, G., S. NAONO, J. ROUVIERE, F, JACOB, and F. GROS, 1963 Production of messenger - RNA and regulation of protein synthesis. Cold Spring Harbor Symp. Quant. Biol. 28:

10 844 M. LIEB BENZEFC, S., and S. P. CHAMPE, 1962 A change from nonsense to sense in the genetic code. Proc. Natl. Acad. Sci. U.S. 48: BROOKS, K., 1965 Studies in the physiological genetics of some suppressor-sensitive mutants of bacteriophage 1. Virology 26: CAMPBELL, A., 1961 Sensitive mutants of bacteriophage 1. Virology 14: DOVE, W., 1966 The action of the lambda chromosome. I. The control of functions late in phage development. J. Mol. Biol. (In Press). GOLDTHWAITE, D., and F. JACOB, Sur le mkcanisme de l'induction du dkveloppment du prophage chez les bactkries lysoghes. Compt. Rend. 259 : GREEN, M. H., 1966 Inactivation of the prophage X repressor without induction. J. Mol. Biol. 16: JACOB, F., and A. CAMPBELL, 1959 Sur le systkme de rkpression assurant l'immunitk chez les bactkries lysogknes. Compt. Rend. 248 : JACOB, F., and J. MONOD, 1961 Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3: JACOB, F., R. SUSSMAN, and J. MONOD, 1962 Sur la nature du rkpresseur assurant l'immunit6 des bactkries lysoghes. Compt. Rend. 254: JOYNER, A., L. N. ISAACS, and R. ECHOLS, 1966 DNA replication and messenger RNA production after induction of wild-type X phage and A mutants. J. Mol. Biol. (In press). KAISER, A. D., 1957 Mutations in a temperate bacteriophage affecting its ability to lysogenize Escherichia coli. Virology 3: KORN, D., and A. WEISSBACH, 1963 The effect of lysogenic induction on the deoxyribonucleases of Escherichia coli K12h. I. Appearance of a new exonuclease activity. J. Biol. Chem. 238: 33w3394. LIEB, M., 1964 Ultraviolet sensitivity of Escherichia coli containing heat-inducible h prophages. Science 145: A model of the X repressor based on studies of heat- inducible X mutants. (Abstr.) Genetics 52: a Studies of heat-inducible phage. I. Order of genetic sites and properties of mutant prophages. J. Mol. Biol. 16: b Studies of heat-inducible A phage. 11. Production of C, product by superinfecting 1 f in heat-inducible lysogens. Virology. 29 : RADDING, C. M., 1964.a Nuclease activity in defective lysogens of phage A. Biochem. Biophys. Res. CO". 15: b Nuclease activity in defective lysogens of phage A. 11. A hyperactive mutant. Proc. Natl. Acad. Sci. U.S. 52: RADDING, C. M., and D. C. SHREFFLER, 1966 Regulation of h exonuclease. 11. Increased synthesis of two proteins by X Til. J. Mol. Biol. (In press) SLY, W.S., H. ECHOLS, and J. ~ LEFC, 1964 Control of viral messenger RNA after lambda phage infection and induction. Proc. Natl. Acad Sci. U.S. 53: STRETTON, A. 0. W., and S. BRENNER, 1965 Molecular consequences of the amber mutation and its suppression. J. Mol. Biol. 12: