Action of the lambda chromosome

J. Mol. Biol. (1966) 19, 187-201

Action of the Lambda Chromosome I. Control of Functions Late in Bacteriophage Development F. DOVE McArdle Laboratory for Cancer Research Madison, Wisconsin, U.s.A. WILLIAM

(Received 8 March 1966, and in revised form 15 April 1966) The genetic control of the synthesis of DNA, bacteriophage antigen and lysozyme, and of the production of infectious DNA (DNA maturation) has been studied, using a set of suppressible (sus) mutants isolated by Campbell. These results clarify the previous studies of the control of functions late in the lytic growth of lambda. They demonstrate that late gene action is dependent both upon chromosomal replication and a general inducer of late functions, a product of gene Q. General models are presented for such a dual control system. A cluster of six DNA maturation genes in the A to F interval is reported. The distribution of genes on the lambda chromosome is discussed briefly.

1. Introduction The course of lytic phage development after infection by the coliphage lambda, or after induction of the lambda prophage, consists of a stage of synthesis of proteins necessary for the replication of the phage chromosome, a stage of DNA replication, and a stage of phage maturation culminated by cell lysis (Thomas, 1959; Sechaud, 1960). Lambda also exhibits intracellular stages of repression, both as a prophage and as a phage superinfecting an immune lysogen. The available evidence shows that high levels of phage proteins are not made under repression; further, the levels of phagespecific messenger RNA are not higher than 2% of these found during active phage development (Attardi, Naono, Gros, Buttin & Jacob, 1963; Sly, Echols & Adler, 1965; Green, 1966). Jacob, Fuerst & Wollman (1957) initiated the study of the genetic control of this development by studying a set of phage mutants which were propagated as prophages and were defective at one or another stage in phage development. Subsequently the body of work on the genetic control of development in phage T4 has clarified some of the control principles used in the lytic development of T4 (see, for example, Epstein et al., 1963; Wiberg, Dirksen, Epstein, Luria & Buchanan, 1962; Edlin, 1965). These principles are: (1) There exists a set of genes the function of which is necessary for phage chromosomal replication ("early proteins"). A defect in any of these genes not only prevents replication, but also prevents the synthesis of any of the maturation products and the phage lysozyme. 187

]88

W. F. DOVE

(2) There exist at least two genes, 33 and 55, which control the expression of all the known maturation genes without controlling the level of replication of the phage chromosome. (3) Inhibition of replication, either by a defect in any of the pre-replication genes, or by ultraviolet irradiation of an infecting phage, results in a prolonged expression of all intact pre-replication genes beyond the time at whieh they normally cease to act. An additional principle of chromosome structure has emerged from this work on phage T4. Genes for related functions tend to cluster on the genetic map. This clustering is not complete, however, and it is therefore unlikely to provide a molecular basis for the control principles stated above. The results of Jacob et al. (1957), of Karamata, Kellenberger, Kellenberger & Terzi (1962), and of Weigle (unpublished experiments) have indicated that structural phage proteins and lysozyme can be made in the absence of phage DNA synthesis. This implies that control principle (1) does not act in the lytic development oflambda. Further, these studies have rovealed no case demonstrating principle (2). Finally, Radding (1964) has evidence which implies that principle (3) does not act in lambda growth. I have reinvestigated this situation, using primarily a set of suppressorsensitive mutants isolated by Campbell. This paper is written to collate these new data with the classical work of Jacob et al., the recently published work of Brooks (1965) and the accompanying work of Joyner, Isaacs, Echols & Sly (1966). The data to be presented demonstrate that in lambda the expression of late phage functions is controlled both by phage chromosomal replication and by an inducer of late functions, a product of gene Q. In addition, these data demonstrate extensive clustering of related functions on the chromosome of lambda.

2. Materials and Methods (a) Strains

Bacteria. Derivatives of Escherichia coii K12 for growth and plating of lambda: C600 thr-leu- B l - lac - T I .5r SUII+;\SF - via S. Brenner; W3101 galtl- prototroph BU- via A. D. Kaiser; W3350 gall - gal2 - prototroph BU - ;\SF - via S. Brenner; Hfr CA24 galt - prototroph su - ;\S ex S. Brenner; W5274 thy-(;\)8U-F+ ex E. Lederberg; W5274-0 thy-;\Ssu-F+ cured of;\ by superinfection with ;\i4 3 4 ; CR34 thy-thr-leu- B l - Tlr SUII+ ;\SF- via S. Brenner; CR63 Tl" BUr + ;\rF + indicator for M, via S. Brenner; E. coli B prototroph for lysozyme assay via J. Wiberg. Phages. Derivatives of;\+ (lambda wild type, Kaiser 1957): ;\C72' cr mutant ex A. D. Kaiser; ;\ind-c857 (Sussman & Jacob. 1962) via S. Brenner; M, host-range mutant ex A. D. Kaiser; ;\i2 l ex M. Kulke; SU8 mutants of lambda wild type, numbers listed in Tables, all ex A. Campbell except BU8 N 7, 8U8 F 3 and 8U8 R 5 via W. Arber and BU8 029' Q2l' Q 1l7 and R 5 4 via A. D. Kaiser. Mutant 96B was obtained as a clear-plaque mutant from Campbell and crossed with ;\ to obtain BUS F 0 6 S e ", Synthesis of recombinant phages ;\C8578U8j and ;\C857 BU8 R 5 4 8U81 (where i = A through Q): Recombinants were made by standard crosses between an infecting phage, either ind-c857 or C857 SU8 R 5 4 • and an induced prophage 8U81 in C600. Clear plaques. isolated by plating the burst on C600, were tested for their 8U8 characters by plating on

+

FUNCTIONS LATE IN LAMBDA DEVELOPMENT

189

W3101 and W3350. The mutation sus R 54 is suppressed by this strain of W3350 (B. Egan, pers. commun.). The assignment of sus characters was confirmed by complementation on W3101 with ASUBI and with ASUSt+l> and by the lack of a lysozyme halo around a plaque arising from a BUS R 54 phage on C600. The ind- character was not scored in these recombinants. Synthesis of defective lysogenic strains of BUB mutants: Defective lysogenic strains for sus rnutants carrying the c " character were made by gal transduction. A low-frequency transducing lysate was made in the suppressing strain C600. It was adsorbed at high multiplicity to HfrCA24 gal- and the culture plated on TTC Gal plates (Hogness & Simmons, 1964). Gal. + colonies were picked and purified. They were shown to be heterogenotes by their capacity to segregate Gal- colonies on eosin-methylene blue-galactose plates. They were shown to be lysogenic for SUSt by their capacity to form plaques on a lawn of C600 but not on a lawn of a su - strain. The plaques on C600 presumably form by zygotic induction on the plate. Defective lysogenic strains for BUS mutants carrying the CB57 character were made by adsorbing a lysate made by induction of a C600 lysogen. The adsorption mixture was plated at 30°C and the resulting colonies were screened for their inability to grow at 40°C. Colonies unable to grow at 40°C, presumably carrying a temperature-inducible prophage, were purified arid tested for immunity to AC72' (b) Initiation of lytic growth Infection. Sensitive bacteria were grown at 37°C in M9 (Adams, 1959) supplemented with 0'5% Casamino acids (henceforth called M9aa). At a titer of 4 X lOB they were centrifuged, resuspended at 1 X 10 9 in 10- 2 M-MgSOcl0- 2 M-Tris (pH 8) and starved at 37°C for 1 hr. Phages were adsorbed at a multiplicity of about 5 at 37°C for 15 min, leaving :( 1% nonadsorbed phages. The infective centers were diluted fourfold into 37°C M9aa and grown at 37°0 with aeration. The latent period for A under these conditions is 50 min. Temperature induction. Lysogenies for phages carrying the C857 character were grown in M9aa at 30°C to 4 X 10 8 • They were induced by exposure to 45°0 for 10 min and were then aerated at 37°C. The fraction non-induced was usually about 10- 3 • The latent period for AC857 is 50 min under these conditions. Ultraviolet induction. Lysogenies for phages carrying the c+ character were grown in M9aa at 37°C to 4 X 10 8 and induced by exposure to ultraviolet irradiation. They were then protected from visible light and aerated at 37°C. The latent period for A + under these conditions is 100 min. (c) Measurement of DNA synthesis Lysogens of temperature-inducible SUB defective phages in the thymineless strain W5274-0 were grown in M9aa supplemented with 10/kg/mI. thymidine. They were temperature-induced and then aerated at 37°0 in the presence of methyl-tritiated thymidine (New England Nuclear, NET 027). At different times during phage development, 0·2-mI. samples were withdrawn, precipitated by cold 5% trichloroacetic acid, filtered, washed and counted by scintillation. Of such material synthesized by W5274-0 in 45 min, 67% is stable to treatment with 0·2 M-KOH for 18 hr at 37°C, and 77% is sensitive to hydrolysis with 6 tLg/mI. pancreatic DNase (Worthington once crystallized) at 37°C in 0·1 M-MgS0 4 at pH 8 for 30 min. For strain W5274-0 (AC857 BUS R 54) the alkali-stable fraction is 92% and the DNase-sensitive fraction is 85%. The net DNA synthesis was calculated from the specific activity, the known counting efficiency and the bacterial titer. Synthesis is expressed in phage units by using a molecular weight of 3 X 10 7 for lambda DNA.

+

(d) Measurement of infectioUB DNA Bacteria lysogenic for a SUB defective prophage were induced and allowed to grow in M9aa until times which would be late in normal phage development. Complexes were lysed by adding EDTA to 0·1 Mand sodium dodecylsulfate to 1 % and heating at 45°C for 1 min. The lysate was deproteinized by one treatment with 'I'ris-saturated phenol at 4°C and was

190

W.F. DOVE

dialyzed against 3 changes of 300 vol. of O'1111-Tris--0'001 M-EDTA (pH 8). Sterility was maintained throughout to prevent the introduction of nucleaaes. DNA recovery by this procedure is about 80%. Smith & Burton (1966) have shown that this lysis procedure will not liberate pulse-labeled DNA into the aqueous phase after phenol extraction; we have confirmed this for lambda infective centers. It is therefore likely that the DNA obtained by this procedure does not include the actively replicating material. The physical nature of this "progeny DNA" is under study. Infectious DNA was as sayed by the method of Kaiser (1957), using the immunity character as a selective marker. The efficiencies of the assay range' from 10 - 4 to 10 - 3 plaques/phage equivalent. (e) Measurement of lysozyme synthesis

The method of J'acob et al , (1957) was used to compare synthesis of lysozyme in nonlysing cultures with that in a wild-type culture. Infected cultures in M9aa at 37°0 were lysed by sonication for 1 min (M.S.E. Ultrasonic Disintegrator, 20 kc /s, 210 w, ! in. probe). Test bacteria, E. coli B, were grown in Tryptone broth at 37°0 to 4 X 108 , sensitized by resuspending in 0·1 M-EDTA-l'O M-Tris (pH 8'0) for 5 min at room temperature, and kept at 0°0 in water. Equal volumes of bacteria and a dilution of the lysate to be assayed were mixed and the optical density at 540 mf-L was followed at room temperature. Lysozyme activity is calculated from the rate of fall of optical density. The background rate of lysis for these sensitized bacteria in the absence of added lysozyme is 1·3%/min. A resistant fraction of 10% of the bacteria do not lyse. Extracts containing lysozyme activity produce a rate of lysis above the background rate which is proportional to the concentration of the extract, in the range from 10 to 40% lysis per min. All assays were performed in this range. (f) Measurement of tail antigen synthuis

Lysates were prepared for the measurement of lysozyme synthesis, using sonication to complete lysis even when the cultures had lysed. Rabbit antiserum of titer 380 min was donated by Dr V. Bode. Antiserum dilutions of titer 4 X 10 - 2 min -1 were used. All assays were carried out in suspension medium (Weigle, Meselson & Paigen, 1959) supplemented with 0'5I1l-NaCI, using a procedure derived from DeMars (1955). Antiserum (0,5 ml.) was incubated overnight at 47°C with a dilution of the lysate (0'1 ml.), A test phage MCB57 was then added (0,1 ml.) and inactivated for 100 min at 37°C. The test phage was selectively scored on OR63. A standard curve was run in parallel with each assay, using purified ,\i21 as a standard. If R is the inactivation ratio Initial titer of MCB57fFinaititer of MCB57' then standard curves of R versus log titer phage antigen were obtained as in Fig. 1. The plating error was always assumed to be not less than 20%, and the error envelope was drawn by eye by compounding the plating error and the scatter between duplicate standard values. r

'

3. Results (a) DNA synthesis

In order to interpret the results of thymidine incorporation after phage induction, control studies were carried out by measuring incorporation in (a) a non-lysogenic strain W5274-0 subjected to a mock induction, (b) two strains lysogenic for the early defective 8US N 7 , and (c) a strain lysogenic for the lysozyme defective sus R S4 which allows synthesis but not release of mature phage particles. These controls are shown in Fig. 2(a) . It can be seen that in an early defective no incorporation occurs after the initial synthesis, during the first 15 minutes after induction, of about 100 phage units per cell. In contrast, mutants which allow normal synthesis of phage DNA show continued synthesis for at least 40 minutes after induction, reaching a level of about 350 phage units per cell. This level is higher than the level of synthesis exhibited by a non-lysogenic strain under these conditions.

FUN CTIONS L AT E IN LAMBDA D EVEL OPME N T

191

65 .--- - - - - - - - - - - - - - - - - - - - - - - ..., 60 5S

N

-: 50 '" 4S ~ Q.

~

.,'"

40

(; 35

~ 30 o

.~

25

:5

20

';J

~

';J

15

:5 10

.E

------

S

o

9

Ixl09 2xl09 4xl0 Log concentration of standard

FIG. 1. Phage antigen assey-s-stendard curve. Standar d blocking phage : purified M21, titered by plaque-forming unit s. 0·1 rnl, added to 0·5 ml. anti serum and in cubated 18 hr at 46°C. Test phage : M eSS7' I nactivation ratio = Titer of t est phage at t = OjTiter of test phage at t = 100 min at 37°C. - - ', Mean value ; ----, probable error envelope .

Defectives in cistrons G through }I allow synt hesis of infecti ous lambda DNA; consequently they were not st udied fur ther . Defecti ves in cistrons A, B, C, E , F and Q were st udied; results with t hree representative st rains fr om t his set are record ed in Fi g. 2(b). All are abl e to make large amounts of DNA after temperature inducti on. The results of Brooks (1965) and most conclusively t hose of J oyn er , I saa cs, E chols & Sly (1966) show tha t of t he sus cistrons, only N , 0 and P control phage DNA synthesis. Further, t hese results show t hat a Adg in which t he entire A t o .M region is delet ed shows phage DNA replicati on. Thus t here can be no unk nown replicati on gene in this region of the lambda chromosome. Th e net DNA synthesis results pre sented here ar e in accord with those assignments. They demonstrate that DNA·positive mutants show enha nced net DNA synthesis compared to a DNA-negative mutant after a time about 15 minutes after temperature induction. These data also supply presumptive evidence that th ere is host DNA synthesis after temperature induction of lambda. Thi s synthesis is of course not repair syn t hesis of a conventional sort , since ultraviolet inducti on was not used. (b) S ynthesis of infectious DN A

D efective lysogenic strai ns were induced and grown until t imes near t he end of t he phage eclipse period. In all te mperature- induction experiments the mu tati on sus R 5 4 was present in the phage to prevent lysis of the infecti ve cent ers, since lysat es in which nu cleases ar e not inactivated show losses in infectivity of phage DNA. These cultures were lysed by EDTA-sodium dodecyl sulfate and deproteinized with phenol as described. Infectio us DNA was assayed by t he method of Kaiser (1957), scoring for the i A character. The results are shown in Table 1.

W.F.DOVE

192

400r--------------, (a)

300 200 E ::l

V)

-Vi Q)

L.

.oJ

-1: 4.l .oJ

U 0

CoD

i;;-<, 400 ~:~ a §

~=====:::==~==:;O

.., '" Z ]'300 0..

200 100

10

20

30

40

Time after temperature induction

FIG. 2. Total DNA synthesis in defective lysogenies. Strain W5274-Q thy-/J'U- and lysogenic derivatives of it were grown at 30 0 0 in M9aa with 10 p.g!mI. thymidine. They were induced at 45°0 for 10 min and then aerated at 37°0 in the presence of tritiated thymidine. DNA synthesis was measured by the net incorporation of thymidine into material insoluble in cold 5% trichloroacetic acid. (a): (0), 5274;; (0). 5274-(R5 4 b ; (.6). 5274-(N)a; (@), 5274-(N)b; (b): (0). B; (0), Q; (.6). A.

These results demonstrate that genes A through F and gene Q control the conversion from newly synthesized lambda DNA to DNA infectious in the Kaiser assay. Since, as will be shown, gene Q controls expression of all late functions in lambda growth, its control over the infectivity of newly synthesized DNA is probably indirect. Thus it is likely that genes A through F, which have no general control function, are structural genes for the proteins involved in DNA maturation. The molecular changes hypothesized for DNA maturation have been discussed by Dove & Weigle (1965) and are under study. (See note (1) added in proof.) (c) Synthesis of lysozyme

°

Campbell (1961) and Brooks (1965) have reported the lysis phenotypes of defective lysogens carrying sus mutants. The replication-defective mutants in cistrons N, and P fail to lyse. In addition, Q and R mutants fail to lyse. Campben & del CampilloCampbell (1963) have reported a temperature-sensitive mutant hS1 2 9 which maps within the R cistron and which makes a heat-labile lysozyme. It is thus quite likely that R is the structural gene for the phage lysozyme. I have confirmed the observations of Campbell and of Brooks and have in addition shown that the failure of N, 0, P and Q defectives to lyse is reflected by reduced levels of synthesis of phage lysozyme. Sonicates of infective centers late in the development of these defective phages were assayed for lysozyme by the method of Jacob et al. (1957). In Table 2 are shown the lysozyme activities of sonicates of cultures infected with lambda mutants.

FUNCTIONS LATE IN LAMBDA DEVELOPMENT TABLE

193

1

Synthesis of infectious lambda DNA by defective mutants of lambda

Mutant

8U8

Au B,

Host

Mode of Growth

W5274-0

Tt induction

W5274-0

T induction

sus 0 2 0

W5274-0

T induction

sus D ' 5

W3101

Infection

sus E ' 3

W5274-0

T induction

sus F 9 6B

W5274-0

T induction

sus G 9

W5274-0

T induction

HfrCA24

u.v. induction

sus H ' 2

HfrOA24

u.v, induction

sus 1 2

HfrCA24

u.v. induction

susJ a sus K 2 4 8U8 L a 3

HfrCA24 HfrCA24 HfrCA24

u.v, induction u.v. induction u.v, induction

sus M a 7

HfrCA24

u.v, induction

QU7

W5274-0

T induction

SUII

IIUS

Time (min)

40 55 40 55 40 55 40 55 40 55 40 55 40 55 50 70 50 70 50 70 60 60 50 70 50 70 40 55

Burst (phages/cell)

10- 5

< 10- 3 < 10- 3 < 10- 3 < 10- 3 < 10- 3 10- 5

< 10- 3 < 10- 3

10- 2 0·05

< 10- 3 < 10- 3 0·5

Infectious DNA Phage equivalents/cell

0·014 0·018 0·032 0·045 0·008 0·017 < 0·2 < 0·2 0·023 0·047 0·31 0·35 1 10 5 10 1 10 1 10 2 10 1 2 1 1 0·16 1·5

Lysogens in HfrCA24 are lysogenic heterogenotes. W5274-0 derivatives are lysogenic for a phage of the type Ca57 8U8 R 5 4 SUS l, where i = A through G, and Q. The latent period for wild-type lambda after infection or temperature induction is 50 min; after ultraviolet induction it is 100 min. t T, Temperature.

It is clear that infections by 0 and P defectives at low multiplicity give rise to a lower burst size than infections at high multiplicity. Furthermore, in those cases in which a defective mutant gives a burst size greater than one, high levels of lysozyme synthesis are observed. No such multiplicity effect is observed for Q defectives. Sus R defectives were not tested (see Campbell & del Campillo-Campbell, 1963). (See note (2) added in proof.) (d) Synthesis of phage antigen Cultures of induced defective lysogens were lysed by sonication and assayed for phage antigen by their capacity to block phage-neutralizing antiserum. Work with other phages (Lanni & Lanni, 1953; Franklin, 1961) indicates that the phage antigen relevant to this assay is a tail antigen. 13

194

W.F. DOVE TABLE

2

Synthesis of lysozyme by defective mutants of lambda Infection at multiplicity 5: Mutant

.\+ .\8'U8 N 7 .\B1U

0 29

.\8U8 P a .\8U8 Q21 .\8'U8 QU7

Burst (phagesjcell) 1

2

68

38

< 0·01 < 0·01 3·4 4·4

1-1 2·6

Lysozyme level

Infection at multiplicity 0·5: Burst (phagesjcell )

Lysozyme level

2

1·00

1·00

69

75

< 0·05

< 0·01

< 0·01

< 0·05

0·24 0·28 0·22 0·19

0·17 1·6 3·3

0·23 1·5 2·1 3·8

0·06 0·11 0·20 0·12

1·4 3·3 0·6 2·5

4·4

All phages carry the C857 character. Infection was carried out in W3101 and growth in M9aa at 37°C. Cultures were sonicated at 80 min. The latent period for .\ under these conditions is 50 min. The burst was determined (1) by lysis with chloroform alone; and (2) by lysis with lysozyme-EDTA followed by chloroform. Lysozyme levels were calculated relative to the synthesis exhibited by A-} at the same multiplicity of infection.

+

The results presented in Table 3 demonstrate the existence of a class of mutants which make high levels of phage antigen, of the order of 100 phage equivalents per bacterium. This class includes all mutants in cistron R and in cistrons A to M, with the clear exception of cistron J and the possible exceptions of cistrons F and M. There also exists a class of mutants which, under some conditions, make very low levels of phage antigen, ofthe order often phage equivalents per bacterium. This class includes eistrons J, N, 0, P and Q, as well as A+ induced in CR34 and growing at 10- 3 JLgJml. thymidine. It should also be noted that conditions allowing sus 0 2 9 and sus P 3 to give bursts near one per cell result in rather large amounts of phage antigen. These data indicate that cistron J is one of the structural genes for the phage antigen. In addition, they demonstrate that phage antigen cannot be synthesized by either the replication-defective mutants N, and P, or by Q-defective mutants. However, when conditions permit such mutants to grow to the extent of giving a burst in nearly every cell, then semi-normal levels of phage antigen are synthesized. The earlier work of Jacob et al. (1957), of Karamata et al. (1962) and of Weigle indicated that normal amounts of late phage proteins could be made in the absence of phage DNA synthesis. In the former study, the leakiness of the mutants was not determined because there is no suppressing host on which to titer them. The experiments of Karamata et al. (1962) and of Weigle were done by multiple infection of a thymineless strain, yielding burst sizes near one. The data presented here show that when conditions permit replication defective mutants to grow to the extent of giving a burst size near one, 10 to 30% levels of phage antigen and lysozyme are synthesized. This artifact may shed some light on the way in which replication controls late gene action in lambda development. The synthesis of phage antigen by the defective mutants employed by Jacob et al. (1957) has been checked; the results are listed in Table 4. In general, these revised

°

FUNCTIONS LATE IN LAMBDA DEVELOPMENT

TABLE

195

3

Synthesis of phage antigen by defective mutants of lambda Mutant

1 1

"C72

"+ sus Au sus B I sus C2 0 sus D I 5 sus E I 3

sus F 9 Bs sus G 9 sus H I 2 sus 12 sus J B sus K 2 • sus L B3 sus Ma 7 sus N 7

sus 0

92

sus P 3

sus

Expt

Q2I

BUS QU7

sus R 5 • susN70 2 9

1 2 1 2 1 2 1 1 2 3 1 1 2 1 1 1 2 1 1 2 1 2 1 2 3 1 2 1 2 3 1 1 1 2 1

Host

Mode of growth

Time (min)

Burst

Phage antigen

80

84

92 ± 40

130

80

200 ± 20

130 130 70 130 70 130 70 130 130 150 70 130 130 70 130 130 130 150 130 130 70 130 70 130 150 80 130 80 130 150 80 80 80 150 70 70

10- 2 10- 5

10 ± 1 110 ± 70 70 ± 40 150 ± 10 150 ± 80 190 ± 10 140 ± 70 140 ± 15 85 ± 15 96 ± 33 50 ± 30 65 ± 45 180 ± 45 160 ± 70 130 ± 20 80 ± 20 23 ± 3 9 ± 2 140 ± 10 110 ± 20 220 ± 80 55 ± 10 65 ± 35 5 ± 1 5 ± 5 2 ± 1 10 ± 1 36 ± 7 40 ± 1 20 ± 3 5±2 10 ± 5 5±3 37 ± 13 111 ± 26 10 ± 2

infection

W3101 CR34

u.v, induction,

HfrCA24 W5274-0 HfrCA24 W5274-0 HfrCA24 W5274-0 HfrCA24 HfrCA24

10 fLg/ml.thymidine 10 - 3 fLg/ml. thymidine u.v, induction Tt induction u.v. induction T induction u.v, induction T induction u.v, induction u.v, induction

W5274-0 HfrCA24 HfrCA24 W5274-0 HfrCA24 HfrCA24 HfrCA24

T induction u.v. induction UoV. induction T induction u.v, induction UoV. induction u.v. induction

HfrCA24 HfrCA24 W3101 HfrCA24 W3101 HfrCA24

UoV.

induction

u.v, induction

T induction UoV. induction T induction UoV. induction

W3101 HfrCA24 W3101 HfrCA24

infection u.v. induction infection UoV. induction

W3101 W3101 W3101 HfrCA24 W3101 W3101

infection infection infection u.v, induction T induction T induction

Phage antigen is expressed in phage units per infective center. t T, Temperature

13*

0

< 10- 3 < 10- 3 < 10- 3 < 10- 3 < 10- 5 < 10- 3 10- 5

< 10- 3 < 10- 3

<

10- 2 10- 5 10- 1 10- 3

< 10- 3 10- 3 10- 3 10- 3 0·02 0 0 02

03

<

10- 2 10- 3 10- 2 3 2 10- 5

196

W.F.DOVE TABLE

4

Properties of defective prophage mutants of Jacob et al. (1957) Reported Strain replication

lysozyme

Observed phage antigen

replication

lysis

phage antigen

DOmin KB P22 P30 P32 P34

0 0 0 0

0 0

+ +

+0

0·5 1 1

0

+0 +

0

+ +

73 <1 19 26 68

± ± ± ±

150 min

11

114

3 6 21

1 32 60 68

± 40 ± 0·5 ±8 ± 13 ± 21

Lytic growth of lambda was initiated by ultraviolet irradiation. The latent period of lambda wild type is 100 min under these conditions. Replication data are from Joyner et al. (1966), and are expressed as the differential rate of synthesis of phage-specific DNA at 50 min after mitomycin induction, relative to the rate observed for lambda wild type. Phage antigen levels are expressed in phage equivalents per infective center.

data do not contradict the results presented here. Some question remains about strain P30, which produces somewhat reduced levels of phage antigen, fails to lyse, and yet replicates well. This anomaly has been clarified by finding that the defect in strain P30 is actually in gene R (R. Thomas, unpublished work, communicated by F. Jacob and E. Signer).

4. Discussion These results and the work of Brooks (1965) and of Joyner et al. (1957), give the following assignment of function to 8US cistrons. h ABC

D E

F GH(KL)IJ

'm 'I'n N

0

P

o

~I\JI/ I DNA maturation

Inducer of late functions FIG. 3. Distribution of lambda functions. Ph9ge antigen

DNA replication

Lysozyme

In addition, this work establishes that at least two principles control the expression of the genes for functions late in phage development. (1) Replication-defective mutants N, 0 and P make low levels of lysozyme and phage antigen. (2) Cistron Q controls a product necessary for all known post-replicative functions: DNA maturation, and synthesis of phage antigen and phage lysozyme. The results of Joyner et al. (1966) accompanying this report, demonstrate that both ofthese control phenomena result in reduced levels oflate lambda messenger synthesis.

FUNCTIONS LATE IN LAMBDA DEVELOPMENT

197

These principles apply to lytic growth both after infection and after induction. The advantage conferred by principle (1) seems quite clear. Were a developing phage to begin synthesizing the proteins which terminate the development before replication of the phage chromosome, the organism would not achieve net replication in that lytic growth phase. We can only speculate at present about the advantage conferred by the existence of a general direct inducer of late functions, gene Q. There may be signals other than successful phage chromosome replication which influence cessation of phage development. Were this the case, an enzyme directed by gene Q could be controlled by such a signal. A general feature of such a dual control system is to permit highly non-linear kinetics of synthesis of late proteins. Suppose that the rate of expression of late genes, L, is a linear function of R, the level of replication (the gene dosage hypothesis), and that it is also a linear function of the level of I, the Q gene product. Suppose also that the level of replication is a linear function of time (cf. Fig. 2) and that the level of the Q gene product is also a linear function of time (cf. the kinetics oflambda exonuclease synthesis, Korn & Weissbach, 1963). Under these conditions the level oflate proteins will be a cubic function of time: if R =a.t and 1= fJt and L

= rRI

then t: = a.fJrt2 or L = 1/3 a.fJrt3 The functional form of the kinetics of synthesis of late proteins in phage development cannot be decided at present because of heterogeneity among infective centers and because of possible saturation phenomena. It can be said, however, that there is an apparent delay in the appearance of detectable levels of phage maturation proteins and lysozyme (a maturation "clock"). In principle, this feature of phage development can reside in a multiplicity of controls over late functions, and/or in a multiplicity of gene products which must interact to form the mature phage particle. It is useful at this time to present testable models for the mechanisms by which replication and a general inducer interact in late gene expression. Joyner et al. (1966) have clearly demonstrated that Q in some way controls the level of late phage messenger synthesis. This suggests, but does not prove, that Q controls a new RNA polymerase. Alternatively, Q might act at anyone of several points in protein synthesis, if messenger RNA synthesis is coupled strongly with protein synthesis (Ames & Hartman, 1963; Brenner & Beckwith, 1965; but see Gros, Naono, Bouviere & Shedlovsky, 1964, Proc. 6th Int. Oongr. Biochem.; Green, 1966). For purposes of discussion, however, let us assume that Q controls a new phage RNA polymerase. First, let us consider a sequential control model for the interaction of replication and the Q gene product. In this model, gene Q is activated by the very process of replication and is the first post-replicative function. The mechanisms by which replication might activate a gene previously inaccessible to the host RNA polymerase are considered below. In the sequential control model this Q gene product then acts to promote the transcription of the genes for the late phage functions.

W. F. DOVE j

198

Sequential control model Injected chromosome - - - -

!

~

Replication enzymes

Replication Host RNA~~ polymerase

1

o

product

FIG. 4. Sequential control model. The injected chromosome is proposed to be the template for the synthesis of mRNA for the replication enzymes (-..). A replication product of this chromosome is then the template for the synthesis of mRNA for the Q gene product, by the action of the host RNA polymerase.

Second, we may consider a set of parallel control models for the interaction of replication and the Q gene product. Suppose that gene Q is expressed independently of replication and is thereby an "early function". In this case, the mechanism by which replication activates late gene expression can be one of at least three sorts. (a) Gene dosage (Edlin, 1965). The rate of synthesis oflate messenger is proportional to the number of copies of the genes for these late functions. (b) The process of replication. The Q gene product is physically coupled with the replication machinery, and transcribes genes otherwise inaccessible to the host transcription apparatus (Edgar, personal communication). Or, the conformation of the replicative form in some way allows transcription by the Q gene product (e.g. polymerase) but not by the host polymerase. The conformations to be considered under this hypothesis are the circular lambda forms (Hershey, Burgi & Ingraham, 1963; Young & Sinsheimer, 1964; Bode & Kaiser, 1965) and the crystallographic A form (Franklin & Gosling, 1953). (c) The product ofreplication. Suppose that mature lambda DNA (Dove & Weigle, 1965) is chemically modified with respect to newly synthesized DNA. This chemical modification prevents transcription both by the host and by the Q apparatus. Replication produces immature chromosomes, as yet unmodified, and these are accessible to transcription by the Q apparatus. This model is strictly hypothetical at the present time, since it is not yet known what the molecular events in lambda DNA maturation are. Three pieces of evidence from this work contribute to the preliminary evaluation of these models. First, the fact that there is little if any multiplicity effect on the leakiness of sus Q mutants is presumptive evidence that Q is a post-replicative function as supposed in the sequential control model. Second, the gene dosage mechanism by which replication might control late gene expression seems unlikely, for amounts of replication allowing a burst size of only one phage per cell allow near-normal synthesis of phage antigen and lysozyme. Finally, the immature DNA hypothesis for the unmasking of late genes requires a special postulate to account for the course of development after induction of a prophage. Such a postulate is required because, naively, one would expect that a prophage is immature with respect to a chemical modification. Finally, it is pertinent to consider the evidence on the distribution of functions on the lambda chromosome. We have seen that there is not only a clustering of the

FUNCTIONS LATE IN LAMBDA DEVELOPMENT

199

Parallel control models assume that the 0 gene is expressed independently of replication (A) Gene dosage Injected chromosome 0 product

l /.......-.

Progeny chromosomes

t

Maturation proteins (B) The process of replication Injected chromosomes----~ 0 product ~

!~~

Replicating chromosomes

!

Maturation proteins

(e) The product of replication

~

Injected Chromosome-i)(f-:!""",,)(lf-7 product Newly synthetized chromosomes

4*=

Maturat ion proteins FIG. 5. Parallel control models. The injected chromosome is proposed to be the template for the synthesis of mRNA for the replication enzymes and the Q product. In case (A), the rate of synthesis of mRNA for late functions is considered to be limited by the number of copies of the relevant genes. The Q product is able to promote transcription of these genes. In case (B), the Q product (m!) has affinity for the DNA replication machinery, and thereby is able to promote transcription of genes otherwise inaccessible to it. In case (C), the injected chromosome is considered to carry chemical modifications (X) produced by DNA maturation in the previous cycle of growth. The Q product cannot promote transcription of genes modified in such a way, but can operate on non-modified DNA ( - - ) .

replication functions around the Cr locus, but also a clustering of six DNA maturation genes in the A to F interval. Preliminary evidence (Dove, unpublished experiments) shows that defectives in genes M, I and J are able to make phage heads. This suggests that at least the segment M, (KL), I, J controls phage tail synthesis. This clustering is considered significant at the present stage of knowledge oflambda because it is certain now that there are no replication genes whatsoever in the A to M interval and because the density of identified cistrons in the A to M interval is about 0,8, if the average cistron is 103 nucleotide pairs long. It seems likely that such complete clustering is not a reflection of the course of evolution of the phage chromosome, but that it is caused by

200

W. F. DOVE

some functional requirement of the phage chromosome (see, for example, Hershey, 1964; Stahl & Murray, 1966). In addition, the distribution of functions observed and the control principles established facilitate the mechanism of lambda repression. All the known replication genes are closely linked to the Cr locus. Furthermore, there can be no unknown replication genes in the A to M interval. The entire phage Chromosome can be repressed simply by repressing the expression of the replication genes and gene Q. Thus, complete repression of phage-specific messenger RNA synthesis and protein synthesis can be gained by controlling the transcription of a small segment of the lambda chromosome closely linked to the Cr locus. It should be borne in mind, however, that the repression of lambda cannot be explained simply by a repression of phage-specific RNA and protein synthesis (Thomas & Bertani, 1964). I thank A. Campbell and F. Jacob for the gift of strains. The hospitality of Professors Arthur Kornberg and A. D. Kaiser at Stanford is particularly appreciated. This work has benefited greatly from the general technical assistance of Mrs Mary Walker and from a. generous exchange with Harrison Echols and his colleagues. A portion of this work was done at the Medical Research Council Unit for Molecular Biology, Cambridge, England, with the support of a postdoctoral fellowship from the National Science Foundation. A large part was done at the Department of Biochemistry, Stanford University Medical Center, with the support of a fellowship from the U.S. Public Health Service. The work at Wisconsin is supported by a program-project grant (CA-07l75) of the U.S. Public Health Service and by the Alexander and Margaret Stewart Trust Fund.

Notes added in proof: (1) J. Weigle (to be published) has shown that genes A through E control the synthesis of phage heads, while genes G through M control the synthesis of phage tails. (2) J. Protass & D. Korn «1966). Proc. Nat. Acad. Sei., Wash. 55,1089) have reported similar results for lysozyme synthesis. REFERENCES Adams, M. H. (1959). Bacteriophages. New York: Interscience. Ames, B. N. & Hartman, P. E. (1963). Cold Spr, Harb. Symp. Quant. Biol 28, 349. Attardi, G., Naono, S., Gros, F., Buttin, G. & Jacob, F. (1963). C. R. Acad. Sci. Paris, 256,805. Bode, V. C. & Kaiser, A. D. (1965). J. Mol. Biol. 14, 399. Brenner, S. & Beckwith, J. R (1965). J. Mol. Biol. 13, 629. Brooks, K. (1965). Virology, 26, 489. Campbell, A. (1961). Virology, 14, 22. Campbell, A. & del Campillo-Campbell, A. (1963). J. Bact. 85, 1202. DeMars, R 1. (1955). Virology, 1, 83. Dove, W. F. & Weigle, J. J. (1965). J. Mol. Biol. 12, 620. Edlin, G. (1965). J. Mol. Biol. 12, 363. Epstein, R. H., Bolle, A., Steinberg, C. M., Kellenberger, E., Boy de la Tour, E., Chevalley, R, Edgar, R S., Susman, M., Denhardt, G. H. & Lielausis, A. (1963). Cold Spr. Harb. Symp. Quant. Biol. 28,375. Franklin, N. C. (1961). Virology, 14, 417. Franklin, R E. & Gosling, R G. (1953). Nature, 172, 156. Green, M. (1966). J. Mol. Biol. 16, 134. Hershey, A. D. (1964). Yearb. Carnegie Instn Wash. 63, 580. Hershey, A. D., Burgi, E. & Ingraham, L. (1963). Proc. Nat. Acad. Sci., Wash. 49, 748. Hogness, D. S. & Simmons, J. R (1964). J. Mol. Biol. 9, 411. Jacob, F., Fuerst, C. & Wollman, E. (1957). Ann. Lnet, Pasteur, 93, 724. Joyner, A., Isaacs, L. N., Echols, H. & Sly, W. S. (1966). J. Mol. Biol. 19, 174.

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Kaiser, A. D. (1957). Virology, 3, 42. Karamata., D., Kellenberger, E., Kellenberger, G. & Terzi, M. (1962). Path. Microbiol. 25, 575. Kom, D. & Weissbach, A. (1963). J. Biol. Ohern, 238,3390. Lanni, F. & Lanni, Y. T. (1953). Cold. Spr, Harb. Symp. Quant. Biol. 18, 159. Radding, C. M. (1964). Proc. Nat. Acad. Sci., Wa8h. 52, 965. Sechaud, .T. (1960). Arch. Sei., Geneve, 13, 428. Sly, W. S., Echols, G. H. & Adler, J. (1965). Proc. Nat. Acad. Sci., Wa8h. 53,378. Smith, M. G. & Burton, K. (1966). Biochem, J. 98, 229. Stahl, F. W. & Murray, N. (1966). Genetics, 53, 569. Sussman, R. & Jacob, F. (1962). C. R. Acad. Sci. Pari8, 254,1517. Thomas, R. (1959). Virology, 9, 275. Thomas, R. & Bertani, L. E. (1964). Virology, 24, 241. Weigle, J. J., Meselson, M. & Paigen, K. (1959). J. Mol. Biol. 1, 379. Wiberg, J. S., Dirksen, M-L., Epstein, R. R., Luria, S. E. & Buchanan, J. M. (1962). Proc. Nat. Acad. Sci., Wash. 48, 293. Young, E. T. & Sinsheimer, R. L. (1964). J. Mol. Biol. 10, 562.