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On the function of the N cistron in phage lambda
VIROLOGY
40,
23-33 (1970)
On
the
Function
of the MAXIME
Department
of Biochemistry
N Cistron
in
Phage
Lambda
SCHWARTZ’
and Molecular Biology, The Biological Cambridge, Massachusetts
Laboratories,
Harvard
University,
Accepted August II, 1969 An in vivo assay for the product of the N gene of bacteriophage lambda is described. Using this assay we show that (1) expression of the N gene starts during the first minute of the vegetative cycle; (2) the N product is functionally unstable between 30” and 35” but stable at 4”; (3) the phage repressor prevents expression of the early genes located to the left of N, even in the presence of N product; (4) the amount of N product synthesized by wild-type phage lambda is in large excess of what is required to obtain maximum expression of the early genes located to the left of N. INTRODUCTION
genes located between N and J (Kourilsky All known bacteriophages, with the possible et al., 196S), nor the protein products of exception of the very small ones, show an genesexo and fi (Radding and Echols, 1968) elaborate pattern of gene expression during can be detected. To the right of CI, genes their vegetative development. Some cistrons CII and 0, although expressed (Pereira da Silva and Jacob, 1968) are apparently tranplay a critical role in controlling this pattern in that the activity of their product is re- scribed only in a very limited fashion quired for the expression of other genes. Cis- (Szybalski, personal communication), and tron N in coliphage lambda is an example of gene Q is not transcribed (Kourilsky et al., 1968 and personal communication). Finally such a gene. none of the late genes are expressed in the The early genes of phage lambda are distributed on both sides of gene CI, the st,ruc- absence of N product, and this is probably due to the lack of expression of the Q gene tural gene of the phage repressor. Their location, as well as their direction of trans- (Dove, 1966). Thus the N product appears cription during lytic infection, is indicated to be required to turn on almost all the other genes of the phage. This paper deals with in Fig. 1. In a lysogenic cell, the CI product prevents transcription of these early genes someproperties of this product in vtio. by interacting with two operators located on MATERIAL AND METHODS each side of CI and defined by mutations Back&a. Strain 159 is the nonpermissive Vz to the left, VI and Va to the right (Ptashne (m-), gal,- &r-r, and UV-sensitive strain of and Hopkins, 1968). Expression of the early genesupon inactivation of the CI repressor, Escherichia coli K12 used by Ptashne (1967). as well as during the lytic infection of a sensi- CA169, obtained from J. Beckwith, is a detive cell, requires the activity of the N rivative of Hfr H carrying the ochre supproduct. When the phage carries a mutation pressor sue+ (Gallucci and Garen, 1966; in its N gene, the following pattern of tran- Stretton et al., 1966). Strain 159 .suc+ was scription is observed: to the left of CI only a obtained from a cross between 159 and small region, perhaps restricted to the N gene CAl69, as a gal+ str-r recombinant able to alone, is transcribed; no mRNA from the support growth of X.SUSN~~. Derivatives of the above strains lysogenic for different x 1 Present address : Depar tement de Biologie Moleculaire, Institut Pasteur, Paris, France. mutants have been prepared. The usual sym23
24
SCHWARTZ
b2-
*
bio,,bio@i434FIG. 1. Partial genetic map of bacteriophage X. The “left” half of the map, from cistron A to cistron J, is omitted. The gene nomenclature is that of Campbell (1961) except for the following: int, exo, and p are the structural genes for integrase (Zissler, 1967), X-exonuclease and p-protein (Radding et al., 1967); att is the attachment site @Veil and Signer, 1968); VI, V2, and V3 are mutations in the operators controlled by the phage repressor; x is a genet.ic region defined (Eisen et al., 1966) as being inside the region of nonhomology with Xi &, but outside and to the right of gene CI. The extent of various deletions or transpositions discussed in the text is indicated under the map (Manly et al., 1969; Kellenberger et al., 1961; Manly and Signer, personal communication). The arrows indicate the direction of transcription of the different parts of the genome (Taylor et al., 1967).
bol 159(X) will be used to represent strain 159 lysogenized by phage X. Bacteriophages. Strains X+, XmNh3, XsusN,, XsusN,susNa, XC&, and XC&, (Campbell, 1961; Ptashne, 1967; Kaiser, 1957; Sussman and Jacob, 1962) were given to us by M. Ptashne and N. Hopkins. The original CIsu, mutation was isolated in the non W-inducible mutant X ind- (Jacob and Campbell, 1959) ; all the X strains used in this work carrying the CIss7 mutation also carry the in& mutation. Xb&ioll and XC&,bzbiolo (Manly et al., 1969, and in preparation) were given to us by E. Signer and K. Manly; Xf.sNs, XCIS~TSUSN,SUSN~~ and XC&,susN,susNs$ls (Brown and Arber, 1964; Pereira da Silva et al., 1968) by H. Eisen and L. Pereira da Silva (the latter phages were obtained as prophages in W3350) ; Xi4”sUsNI (Kaiser and Jacob, 1957) by V. Pirrotta. The other phages were prepared as follows: xC&,SUSN~~ was selected as a clear recombinant at 40” on a a+ host, from a cross between XCI~~~SUSN~SUSN~~ and XsusNst (XsusNss but not XsusN, or XsusN~susNba, gives plaques on an sue+ host) ; XC185m3, the product of a cross between XC160 and xCI~~~SUSN,SUSN~SX~~,was selected as a prophage which cures at 42’; Xi!.sN#.&I~~, the product of a cross between XtsNg and XsusCI~~SUSN,SUSN~~ (Ptashne, 1957), was selected as clear and temperature sensitive on a SK host.
Media, bufers, preparation of phage stocks.
As in Ptashne (1967). Uniformly 14C-labeled L-leucine at a specific activity of about 200 mCi/mmole was from Schwarz BioResearch Inc. Irradiation. Cells were irradiated for 5 min at a distance of 25 cm from two G.E. 15 W germicidal lamps. The incident dose at this distance is about 140 erg mm-2 set-‘; 20-25 ml of cells (depending on the experiments), at a concentration of 3 X lo8 cells/ ml, were irradiated at 4’ in petri dishes of diameter 9 cm with swirling. A typical experiment: kinetics of N product formation upon thermoincluction. A culture
of strain 159 (XC&) growing exponentially at 34’ at a cell density of about lo9 cells/ml is transferred into a prewarmed flask incubating in a 42” shaking bath. When the temperature of the culture reaches 37” (i.e., after 10-15 set), the temperature at which the XC& repressor is inactivated, a t = 0 sample is withdrawn. The culture reaches 42’ at about t = 30 sec. The samples, taken after various times of incubation, are transferred into cold flasks containing enough cold (4”) medium to give a final volume of 20 ml of cells at a density of 3 X lo* cells/ml. The different diluted samples are then transferred into cold petri dishes and W irradiated. Onemilliliter samples of irradiated cells are transferred into incubation tubes containing 0.1 ml of 2 X 10-l M MgSOd, 0.1 ml of phage suspen-
FUNCTION
OF N CISTRON
sion in phage buffer, 0.5 ml of A medium made 2 X W2 M MgSOd and containing 0.2 to 0.5 PCi of leucine-14C. The tubes are incubated for 60 min at 42” with gentle shaking. Then 1 ml of 50% trichloroacetic acid is added, the samples are filtered on Millipore filters, and the filters are dried and counted in an end-window, low-background, gasflow Nuclear Chicago counter. RESULTS
Protein Synthesis in UV-Irradiated
Cells
As shown by M. Ptashne (Ptashne, 1967) infection under proper conditions of heavily UV irradiated E. coli with phage X results in a lo- to 20-fold increase of labeled amino acid incorporation into acid precipitable material (Fig. 2). However, infection of the irradiated cells with the mutant X phages XC&,rb2biolo or Xbzbioll in which most of the genes between N and J are deleted, stimulates protein synthesis very poorly (Fig. 3). This suggeststhat most of the protein synthesis observed in irradiated cells infected with X+ corresponds to the expression of the early genes located between N and 5. Hendrix and Schwartz (in preparation) have
4C cpm x lo-3
IN PHAGE
25
LAMBDA
in fact shown by disc electrophoresis analysis that most of the proteins synthesized upon infection of UV-irradiated cells by h+ are the products of X genes which are deleted in hb2 (Kellenberger et al., 1961) or in Abio, (Manly et al., 1969). Figure 3 also shows that, as reported by Ptashne (1967) no stimulation is observed upon infection with phages carrying suppressor sensitive (sus) mutations or temperature-sensitive (ts) mutations in gene N, under nonpermissive conditions. This result confirms that activity of the N gene product is required for expression of the early genes located between N and J. In Vivo “Assay” of the N GeneProduct The experiment in Fig. 4 shows that delayed complementation of the N function can be measured in this system. A lysogenic host is induced for a short time prior to W irradiation. N product accumulates inside the cells during this period as shown by the fact that, after UV irradiation, these cells can synthesize proteins upon infection with an N mutant. In order to facilitate timing of the experiments, the host was chosen to be
14C cpm x 1G3 n
multipliciti
of infectior
” ‘” time of%ubation
($c)
FIG. 2. Protein synthesis in UV-irradiated 159 upon infection with A+. Protein synthesis is measured as the amount of leucine-14C incorporated by the cells into TCA-precipitable material. Incubation is with 0.5 pCi. of leucine-i4C permilliliter of suspension containing 3 X 108 UV-irradiated cells. (A) TCAprecipitable 14C counts after 60 min of incubation at 37” as a function of multiplicity of infection with X+. (B) TCA precipitable W counts after various times of incubation at 37”. O-0, without added 0, after addition of X+ at an moi of 5. phage, O-
26
SCHWARTZ
Kinetics of Induction of N Product Synthesis
14C cpm x lo-3
I
0
25
multi(;licity
5
75
of infedtion
IO
FIG. 3. Protein synthesis in UV-irradiated 159 upon infection with various X mutants. TCAprecipitable 14C radioactivity after 60 min of incubation at 42” of 3 X lo* UV-irradiated cells with 0.4 &!i of leucine-W and various quantities of phages. O---e, L~u.sN~susN~~; O-0, XtsN9susCIa; A-A, XC&~bzbioto; A -A, Xbzbioll; m--o, x+.
lysogenic for the thermoinducible mutant xa35,. The repressor of this phage is immediately, but reversibly, inactivated at temperatures between 37” and 45” (Sussman and Jacob, 1962; Naono and Gros, 1967; Konrad, 1968). If the lysogenic cells are incubated for 10 min at 42” prior to UV irradiation (Fig. 4 B and D), then superinfection at 42” by an N mutant after UV irradiation stimulates protein synthesis 40 % as much as does Xf. (The same protein pattern is observed upon disc electrophoresis of the proteins synthesized by Xf and the N mutant in these conditions.) In the control experiment, where the cellswere not thermoinduced prior to UV irradiation, no such stimulation is observed (Fig. 4, A and C). In other experiments, not illustrated here, no complementation was observed when the prophage carried a mutation in gene N (also seeexperiment in Fig. 9).
The experiment in Fig. 5A shows that it takes only about 1 min of incubation at a temperature above 37” for the lysogenic host to display some N complementing ability, and that this ability increases only for 4-5 min. The ability of a superinfecting N mutant to stimulate protein synthesis is determined after the host has been induced for different lengths of time. In order to minimize the variation resulting from small differences in the UV dose received by the cells, the amount of protein synthesis observed upon infection by the N mutant is normalized to the amount of synthesis observed when identically treated cells are infected with X+. In this type of experiment, the N complementing ability always stops increasing after 5 min, in spite of the fact that complementation is then only about 40% (i.e., XN- stimulates protein synthesis only 40% as much as does A+). It is known that phagesbearing certain mutations in the II: region (see Fig. l), synthesize exonuclease and /3proteins in much larger quantities than does X+ (Eisen et al., 1966; Radding, 1966; Radding and Shreffler, 1966). Even with a host lysogenic for such a phage, complementation never reaches 100 % (Fig. 5B). However, complementation is reproducibly a little higher in this casethan with a wild-type prophage. Functional Instability of the N Product In the experiments, described above, where delayed N complementation could be demonstrated, the cells were immediately chilled and UV-irradiated after the period of thermoinduction. The experiments described in Fig. 6, however, show that if, just after the period of induction, the cells are incubated for a few minutes at a temperature where further synthesis of N product is repressed (30-35’)) no complementation is observed after UV irradiation. This result indicates that the N product activity produced during the heat induction period then decays during the further incubation at 3035”. A half-life of N complementing ability can be estimated as about 5 min at 35”. It can be seenthat the same result, is obtained when the prophage carries an x mutation.
FUNCTION
OF N CISTRON
I 14C ct3m x lom3
0 IO
0
15
IO
5
30
IN PHAGE
60
I 1 I
0 IO
time of incubation
:I 1
5
27
A
14C cpm x i -3
0
LAMBDA
30
hid
60
2. 14C cpm x 10~~
I
I
mulVplicity of infe%tion
5
IO
FIN. 4. Delayed complementation of XsusNsa by M&r. TCA-precipitable *C radioactivity incorporated by 159 (XC!I8~,) at 42°C in the presence of 0.5 PCi of leucineJ4C for 3 X 108 cells. m-0, uninfected; C-O, infected with XsusN~s;~---A, infected with X+. (A and B) Time course of incorporation for an moi = 10. (C and D) Amount of incorporation after 60 min of incubation for various moi. Strain 159 (XC&,) was cultivated at 32°C and UV-irradiated immediately (A and C) or incubated at 42” for 10 min prior to UV irradiation (B and D).
In contrast, no loss of complementing ability is observed after a 30 min incubation at 4’. This last circumstance actually made the whole study possible since it provides a means of stopping N product synthesis at any moment without losing the activity of the N product synthesized until that moment. The results presented so far concern the stability of the N product in the cells before W irradiation. Attempts have also been made to determine its stability after irradiation. These studies are difficult because the ability of the W-irradiated cells to synthesize proteins even after X+ infection decays rather rapidly. However, at 30”, the N com-
plementing ability seemedto be as unstable after W irradiation as it is before. This is also what the kinetics of incorporation of Fig. 4B suggest. In this experiment, the incorporation stimulated by XN- in preinduced cells appears to stop much earlier than does the incorporation stimulated by XN+ as if the supply of N product provided at the beginning of the incubation had been exhausted after 30 min. E$ect of the CI Repressorin the Presenceof N Product The effect of the CI product on protein synthesis was avoided in the experiments described so far by incubating the W-ir-
SCHWARTZ
28
incorporation 40
5o % of wild type incorporation
A
0
B
not stimulate protein synthesis. Thus, in the presence of repressor, the N product cannot turn on protein synthesis from the superinfecting phage. This, however, is true only if the super-infecting phage is homoimmune with the prophage. As shown on Fig. 7 C, the repressor synthesized by M&,~ is unable to repress the protein synthesis directed by Xi434.Xi434is a recombinant phage in which a small region of the X genomecomprising gene CI and the operators on which it acts has been replaced by the corresponding, but nonhomologous, region of the related phage 434 (see map in Fig. 1) (Kaiser and Jacob, 1957; Westmoreland et al., 1969). Overproduction of N Product by X+
In Fig. 8A it can be seenagain that, under nonpermissive conditions, N mutants are unable to stimulate protein synthesis in 30UV-irradiated cells. The experiment in Fig. 8B shows that, in the presence of the ochre suppressor sue+ the ability of three phages 20 :i;.rcarrying .susNmutations to stimulate protein . synthesis is restored. It is restored to essen1 I 0 IOftially 100% for XauaNhs,a phage known to give plaques on sue+hosts and only partially 012345 IO for XsusN7 and XSUSN~SUSN~~, neither of which gives plaques on s%+ hosts. Disc duration of thermoinductionbnir electrophoresis of the proteins synthesized Fra. 5. Kinetics of induction of N-complementby the susN phages and Xf have been shown ing ability. Suspensions of 159 @CI& (A) or to give identical patterns. Since sue+ is 159 (ML~zr13) (B) are incubated for various lengths of time at 42”, chilled, W-irradiated, and known (Gallucci and Garen, 1966; Stretton incubated for 60 min at 42” with leucine-Xl and et al., 1966) to restore translation with a 16 % either no phage or X+ or XsusNsa at an moi of 5. efficiency at most, this result seemsto imply The amount of TCA-precipitable radioactivity that a quantity of N product only 16 % (and incorporated by uninfected cells (@---a) or perhaps much less) of that synthesized by cells infected with XSUSN~S (O-O) is plotted as X+ is sufficient to fully turn on the genes loa percentage of the amount incorporat,ed by X+ cated between N and J. An alternative possiinfected cells. bility would be, however, that the amount of N product made by X-l- is regulated by a radiated lysogens at a temperature (42”) negative feedback mechanismto a level simiwhere the prophage’s repressor was inactive. lar to that obtained upon ochre suppression The prophage’s N product, if provided by of XSUSN~~. Thus, in order to conclude that preinduction, was then shown to be able to X+ overproduces N product, it is necessary turn on protein synthesis in the superinfect- to demonstrate directly that, in an suof host, ing phage (seethis also on Fig. 7, B and D). less N product is synthesized by XsusN~S If one now considers what happens in the than by X+. In an attempt to do so, the in presence of active repressor (i.e., at 35’) it vivo “assay” system described above was appears that, whether N product had been used to compare the amounts of N product provided by preinduction (Fig. 7C) or not synthesized by X+ and XsusN53in 159su,+. (Fig. 7A), infection by X+ and XSUSN~~ does The two phages were independently induced 40-
FUNCTION
IN PHAGE
1,
% of wild type incorporation
50 4
OF N CISTRON
29
LAMBDA
A
30 20
SJQOd0.
I
, duraficn of incubation 5
IO
before uyhir
35O--
% of wild type incorporation 6 -----a.* ----------z---+ 4O
I
lOba----,, 0
L &4 04
--s-w
_
durajion of incubation ‘O 3z00r40
before u\c(min 30
)
FIG. 6. Instability of the N-complementing ability. (A) A suspension of 159 (XCIa5,) is incubated for 5 min at 42”, and then for various lengths of time at 35”. At different times during this incubation samples are chilled, UV-irradiated, and incubated for 60 min at 42” with leucine-1% and either no phage or X+ or XSUSN~~ at an moi of 5. The amount of radioactivity incorporated by uninfected cells (@---0) or cells infected with XSUSN~~ (O--O) is plotted as a percentage of the amount incorporated by X+ infected cells. (B) Same experiment with a suspension of 159 (XC&,,x13) except that the incubation at 42’was for 15 min, and t,he subsequent incubations at either 32’ (solid lines) or 4” (dashed lines).
in 159 SW+, and the degree of complementation of a temperature sensitive mutant was determined. (The presence of sue+ in the host precluded the use of a susN mutant for superinfection.) It can be seenin Fig. 9 that the amount of complementation provided by preinducing the XsusNE3phage is about 5 times lessthan the amount provided by preinducing the wild-type phage. In this experiment the killing upon heating at 42°C was the same in the two cultures, and both suspensions were verified to contain more than 99 % lysogenic cells just before thermoinduction. The result is in agreement with
the hypothesis that, in a SW+ host XsusNs~ provides lessN product than does A+. DISCUSSION
This paper describes an in vivo assay for the N gene product. This assay is based on the following observations : (i) in adequately UV irradiated cells AN+ phage, but not ANphage, expressesits early geneslocated to the left of N (on the genetic map, Fig. 1) ; (ii) induction in the host of a AN+ prophage, but not of a AN- prophage, before UV irradiation, restores the ability of a superinfecting AN- phage to express its early genes lo-
30
SCHWARTZ
15
IO
5
15
IO
5
FIO. 7. Effect of the phage repressor on protein synthesis in the presence and the absence of N product. A culture of 159 (XC&) growing exponentially at 32’ is UV-irradiated (A and B) or preincubated for 5 min at 42” and then UV-irradiated (C and D). In each case it is then incubated for 60 min at 35” (A and C) or at 42” (B and D) with leucine-1% and no phage or Xf or ~susN 53 or G%usN, at an moi of 3. The ordinate indicates the amount of radioactivity incorporated into TCA-precipitable material by 3 X lo8 cells. F
159 su-
I
0 uninfected F=l X susN7N53 <
X wild type incorporation
100
lEl XsusN7 h susN,, PZZJx+ LUII h tsNgsusCI~~
-
Fro. 8. Effect of the suppression of N mutations by sus+. Cultures of 159 or 159 sue+ are UV-irradiated and then incubated for 60 min at 42’ with leucine-r4C and either no phage or the phages indicated on the figure at an moi of 5. The amount of radioactivity incorporated into TCA-precipitable material is plotted as a percentage of the amount incorporated by h+-infected cells.
FUNCTION
OF N CISTRON
IN PHAGE
4 % of A susNS3 incorporation
31
LAMBDA
e 6.
% of A susNS3 incorporation
60-
50 40-
30-
0
I 5
1 IO
0
duration of thermoinduction
I 5
I IO
(min.)
9. N-complementing ability provided by h+ and hsusN63 in an su$ host. Suspensions of 159 sue+ (XC&) (panel A) or of 159 sucf (XC18B~ susN& (panel B) exponentially growing at 32’ are incubated for 0, 5, or 10 min at 42”, chilled, UV-irradiated, and incubated at 42” for 60 min with leucine-W and either no phage or XsusNa3 or XtsNssusCIsl at an moi of 5. The amount of radioactivity incorporated into TCA-preoipitable material by uninfected cells (U-0) or cells infected with ht.sNgsusCIt~ (e---e) is plotted as a percentage of the amount incorporated by XsusNsz-infected cells. FIG.
cated to the left of N. The ability of a ANphage to stimulate protein synthesis in UVirradiated cells can thus be taken as an index of the presence of N product inside these cells. Making use of this assay, some aspects of N product function are investigated. We present evidence for the following facts: 1. Synthesis of the N product occurs very early in the vegetative cycle of the phage. 2. The N product is functionally unstable at physiological temperatures, but stable at low temperatures. 3. The CI repressor prevents expression of the early genes located to the left of the N gene, even in the presence of N product. 4. The minimum amount of N product required to obtain maximum expression of the early genes located to the left of N is much smaller than the amount synthesized by wild-type phage. After considering the problems involved in quantitating the N product assay, we shall discuss each of these conclusions in turn. Quantitation of the relationship between
the amount of N product in the cells and the degree of complementation of an N- mutant is difficult. In any attempt to quantitate this relationship one would have to understand, among other things, what limits the amount of complementation observed. It is a fact that no matter how long a wild-type prophage is induced in the host before UV irradiation, superinfection by an N- mutant can stimulate protein synthesis only 40 % as much as doessuperinfection by an N+ phage. It is not well understood why an amount of N product which is sufficient to ensure maximum protein synthesis when provided by the infecting phage itself, becomeslimiting when provided by a preinduced prophage. This may be due to the functional instability of the N product (seebelow) since the kinetics of protein synthesis (Fig. 4B) suggest that the N product activity decays during the incubation following UV irradiation. However, alternative or additional reasons, involving some compartmentalization for instance, could easily be found. In view of these uncertainties, no attempt will be made,
32
SCHWARTZ
in the discussion presented below, to quanti(in preparation), who found that exonuclease tate the amount of N product present in the synthesis cannot be turned on in an N- exe+ cells. We shall only assume that any in- prophage by a heteroimmune Nf exe- supercrease in complementation corresponds to an infecting phage unless the prophage reincrease in N product concentration. pressor is inactivated. One must then con1. The data presented here show that at clude that the early genes located to the left 42”, N starts being expressed within the first of N are controlled in cis by an operator subminute after repressor inactivation. The con- ject to repression by the CI product. A centration of N product then increases at priori one might have postulated the existleast until the fifth minute. No further in- ence of such an operator to the left of N. crease in complementation is observed after However, since it is shown here that protein 5 min, but this does not necessarily imply synthesis directed by the heteroimmune that N product synthesis actually stops at phage Xi434is insensitive to the presence of X that time. Since the N product is functionrepressor, one must conclude that the operaally unstable (see below), the apparent tor is in the region of nonhomology between stoppage in net N product synthesis may re- X and Xi434and is thus presumably identical flect the establishment of a steady state be- with the operator for the N gene, defined by tween synthesis and decay. the Vz mutation. By this criterion aloneThe presence of an x mutation, known to common control in cis by a single operatorlead to the overproduction of exonuclease the N gene and the other early genes located and fi protein (Eisen et al., 1966; Radding, to its left would appear to be part of a single 1966; Radding and Shreffler, 1966) leads to operon (Jacob and Monod, 1961). However, a slightly increased N complementing ability this whole group of genes does not behave of the prophage. This may indicate that z as a ‘(single unit of coordinated expression” mutants overproduce N product as well as since in the absence of active N product, exonuclease and /3protein. only the proximal part of this set of genes is 2. Upon incubation of the cells between transcribed (Kourilsky et al., 1968). Ap32” and 35” in the absence of any inhibitor, parently derepression is required for tranthe N complementing ability produced by scription to start at the Vz operator, and acinduction of the prophage has a half-life of tivation by the N product is required for about 5 min. In a previous study of the rate transcription to proceed further than a point of production of X-specific mRNA, Konrad located to the left of the N gene. (1968) also came to the conclusion that an 4. Finally we find that upon infection of a “inducing protein”, presumably the N host carrying the ochre suppressor sue+ product, had a functional half-life of this or- by a phage carrying an amber mutation in der of magnitude. In his experiments, how- the N gene, protein synthesis is the same as ever, chloramphenicol was added to stop upon infection by X+. This is merely an exfurther synthesis of the “inducing protein”, tension of the result of Radding and Echols and, consequently, the results could have (1968), who showed that upon ochre supbeen interpreted to mean that the drug in- pression of an amber N mutant, wild-type terfered with N product activity. amounts of exonuclease and 0 protein are It is of interest that the N product of an x produced. Under the simplest hypothesis, mutant is as unstable as that of wild type. the amount of N product synthesized upon One may conclude that the functional inochre suppression of an amber mutant should stability of the N product does not result from a specific inactivation mediated by the not be more than 16% the amount syntheproduct of a late gene or an early gene lo- sized by wild type (Gallucci and Garen, 1966; Stretton et al., 1966). In fact, we precated to the right of CI, since an x mutant evidence in this sent “semiquantitative” does not express any of these genes. 3. In the presence of active repressor, the paper that this hypothesis is probably correct. In agreement with Radding and Echols, N product cannot turn on the genes located to the left of N. This is in agreement with the we would then conclude that X-t seems to proresults of Luzzati (in preparation) and Pero duce at least 6 times more N product than is
FUNCTION
OF
N CISTRON
required to turn on the early genes located to the left of N. ACKNOWLEDGMENTS The support, direction, and encouragement of Professor J. D. Watson throughout the course of this work, the helpful advice of Drs. E. Signer, W. Gilbert, and M. Ptashne, and the skillful technical assistance of Mrs. Henrietta W. Tranum are gratefully acknowledged. These experiments were performed while the author was a junior fellow of the Society of Fellows of Harvard University. REFERENCES BROWN, A., and ARBER, W. (1964). Temperature-sensitive mutants of coliphage lambda. Virology 24, 237-239. CAMPBELL, A. (1961). Sensitive mutants of bacteriophage h. Virology 14, 22-32. DOVE, W. F. (1966). Action of the lambda chromosome. I. Control of functions late in bacteriophage development. J. Mol. Biol. 19, 187-201. EISEN, H. A., FUERST, C. R., SIMINOVITCH, L., THOMAS, R., LAMBERT, L., PEREIRA DA SILVA, Genetics and physiL., and JACOB, F. (1966). ology of defective lysogeny in K12 (h) : Studies of early mutants. Virology 30, 224241. GALLUCCI, E., and GAREN, A. (1966). Suppressor genes for nonsense mutations. II. The su-4 and su-5 suppressor genes of Escherichia coli. J. Mol. Biol. 15, 193-200. JACOB, F., and CAMPBELL, A. (1959). Sur le syst&me de repression assurant l’immunitk chez les batteries lysogenes. Compt. Rend. Acad. Sci. 248, 3219-3221. JACOB, F., and MONOD, J. (1961). Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3, 318. KAISER, A. D. (1957). Mutations in a temperate bacteriophage affecting its ability to lysogenize E. coli. Virology 3.42-61. KAISER, A. D., and JACOB, F. (1957). Recombination between related temperate bacteriophages and the genetic control of immunity and prophage localization. Virology 4, 509621. KELLENBERQER, G., ZICHICHI, M. L., and WEIOLE, J. (1961). A mutation affecting the DNA content of bacteriophage lambda and its lysogenic properties J. Mol. Biol. 3, 399408. KONRAD, M. W. (1968). Dependence of “early” bacteriophage RNA synthesis on bacteriophagedirected protein synthesis. Proc. Natl. Acad. Sci. U.S. 59, 171-178. KOURILSKY, P., MARCAUD, L., SHELDRICK, P., LUZZATI, D., and GROS, F. (1968). Studies on the messenger RNA of bacteriophage X. I. Various species synthesized early after induc-
IN
PHAGE
LAMBDA
33
tion of the prophage. Proc. Null. Acad. Sci. U.S. 61, 1013-1020. MANLY, K., SIGNER, E. R., and RADDING, C. M. (1969). Nonessential functions of bacteriophage X. Virology 37, 177-188. NAONO, S., and GROS, F. (1967). On the mechanism of transcription of the x genome during induction of lysogenic bacteria. J. Mol. Biol. 25, 517-536. PEREIRA DA SILVA, L. H., and JACOB, F. (1968). Induction of CII and 0 functions in early defective lambda prophages. Virology 33, Bl& 624. PEREIRA DA SILVA, L. EISEN, H., and JACOB, F. (1968). Sur la replication du bacteriophage X. Compt. Rend. Acad. Sci. 266, 926-928. PTASHNE, M. (1967). Isolation of the X phage repressor. Proc. Natl. Acad. Sci. U.S. 57, 306-313. PTASHNE, M., and HOPKINS, N. (1968). The operators controlled by the x phage repressor. Proc. Natl. Acad. Sdi. U.S. 60,1282-1287. RADDING, C. M. (1966). Regulation of X exonuclease. I. Properties of x exonuclease purified from lysogens of At,,, and wild type. J. Mol. Biol. 18, 235-250. RADDING, C. M., and ECHOLS, H. (1968). The role of the N gene of phage X in the synthesis of two phage specified proteins. Proc. hlatl. Acad. Sci. U.S. 60, 707-712. RADDING, C. M., and SHREFFLER, D. C. (1966). Regulation of X exonuclease. II. Joint regulation of exonuclease and a new X antigen. J. Mol. Biol. 18,251-261. RADDING, C. M., SZPIRER, J., and THOMAS, R. (1967). The structural gene for X exonuclease. Proc. Natl. Acad. Sci. U.S. 57, 277-283. STRETTON, A. 0. W., KAPLAN, S., and BRENNER, S. (1966). Nonsense codons. Cold Spring Harbor Symp. Quant. Biol. 31, 173-179. SUSSMAN, R., and JACOB, F. (1962). Sur un systeme de repression thermosensible chez le bacteriophage X d’Escherichia coli. Compt. Rend. Acod. Sci. 254, 1517-1520. TAYLOR, K., HRADECNA, Z., and SZYBALSKI, W. (1967). Asymmetric distribution of the transcribing regions on the complementary strands of coliphage X DNA. Proc. Natl. Acod. Sci. U.S. 57, 1618-1625. WEIL, J., and SIGNER, E. R. (1968). Recombination in bacteriophage X. II. Site specific recombination promoted by the integration system. J. Mol. Biol. 34, 273-279. WESTMORELAND, B. C., SZYBALSKI, W., and RIS, H. (1969). Mapping of deletions and substitutions in heteroduplex DNA molecules of bacteriophage lambda by electron microscopy. Science 163, 1343-1348. ZISSLER, J. (1967). Integration negative (int) mutants of phage X. Virology 31, 189.
40,
23-33 (1970)
On
the
Function
of the MAXIME
Department
of Biochemistry
N Cistron
in
Phage
Lambda
SCHWARTZ’
and Molecular Biology, The Biological Cambridge, Massachusetts
Laboratories,
Harvard
University,
Accepted August II, 1969 An in vivo assay for the product of the N gene of bacteriophage lambda is described. Using this assay we show that (1) expression of the N gene starts during the first minute of the vegetative cycle; (2) the N product is functionally unstable between 30” and 35” but stable at 4”; (3) the phage repressor prevents expression of the early genes located to the left of N, even in the presence of N product; (4) the amount of N product synthesized by wild-type phage lambda is in large excess of what is required to obtain maximum expression of the early genes located to the left of N. INTRODUCTION
genes located between N and J (Kourilsky All known bacteriophages, with the possible et al., 196S), nor the protein products of exception of the very small ones, show an genesexo and fi (Radding and Echols, 1968) elaborate pattern of gene expression during can be detected. To the right of CI, genes their vegetative development. Some cistrons CII and 0, although expressed (Pereira da Silva and Jacob, 1968) are apparently tranplay a critical role in controlling this pattern in that the activity of their product is re- scribed only in a very limited fashion quired for the expression of other genes. Cis- (Szybalski, personal communication), and tron N in coliphage lambda is an example of gene Q is not transcribed (Kourilsky et al., 1968 and personal communication). Finally such a gene. none of the late genes are expressed in the The early genes of phage lambda are distributed on both sides of gene CI, the st,ruc- absence of N product, and this is probably due to the lack of expression of the Q gene tural gene of the phage repressor. Their location, as well as their direction of trans- (Dove, 1966). Thus the N product appears cription during lytic infection, is indicated to be required to turn on almost all the other genes of the phage. This paper deals with in Fig. 1. In a lysogenic cell, the CI product prevents transcription of these early genes someproperties of this product in vtio. by interacting with two operators located on MATERIAL AND METHODS each side of CI and defined by mutations Back&a. Strain 159 is the nonpermissive Vz to the left, VI and Va to the right (Ptashne (m-), gal,- &r-r, and UV-sensitive strain of and Hopkins, 1968). Expression of the early genesupon inactivation of the CI repressor, Escherichia coli K12 used by Ptashne (1967). as well as during the lytic infection of a sensi- CA169, obtained from J. Beckwith, is a detive cell, requires the activity of the N rivative of Hfr H carrying the ochre supproduct. When the phage carries a mutation pressor sue+ (Gallucci and Garen, 1966; in its N gene, the following pattern of tran- Stretton et al., 1966). Strain 159 .suc+ was scription is observed: to the left of CI only a obtained from a cross between 159 and small region, perhaps restricted to the N gene CAl69, as a gal+ str-r recombinant able to alone, is transcribed; no mRNA from the support growth of X.SUSN~~. Derivatives of the above strains lysogenic for different x 1 Present address : Depar tement de Biologie Moleculaire, Institut Pasteur, Paris, France. mutants have been prepared. The usual sym23
24
SCHWARTZ
b2-
*
bio,,bio@i434FIG. 1. Partial genetic map of bacteriophage X. The “left” half of the map, from cistron A to cistron J, is omitted. The gene nomenclature is that of Campbell (1961) except for the following: int, exo, and p are the structural genes for integrase (Zissler, 1967), X-exonuclease and p-protein (Radding et al., 1967); att is the attachment site @Veil and Signer, 1968); VI, V2, and V3 are mutations in the operators controlled by the phage repressor; x is a genet.ic region defined (Eisen et al., 1966) as being inside the region of nonhomology with Xi &, but outside and to the right of gene CI. The extent of various deletions or transpositions discussed in the text is indicated under the map (Manly et al., 1969; Kellenberger et al., 1961; Manly and Signer, personal communication). The arrows indicate the direction of transcription of the different parts of the genome (Taylor et al., 1967).
bol 159(X) will be used to represent strain 159 lysogenized by phage X. Bacteriophages. Strains X+, XmNh3, XsusN,, XsusN,susNa, XC&, and XC&, (Campbell, 1961; Ptashne, 1967; Kaiser, 1957; Sussman and Jacob, 1962) were given to us by M. Ptashne and N. Hopkins. The original CIsu, mutation was isolated in the non W-inducible mutant X ind- (Jacob and Campbell, 1959) ; all the X strains used in this work carrying the CIss7 mutation also carry the in& mutation. Xb&ioll and XC&,bzbiolo (Manly et al., 1969, and in preparation) were given to us by E. Signer and K. Manly; Xf.sNs, XCIS~TSUSN,SUSN~~ and XC&,susN,susNs$ls (Brown and Arber, 1964; Pereira da Silva et al., 1968) by H. Eisen and L. Pereira da Silva (the latter phages were obtained as prophages in W3350) ; Xi4”sUsNI (Kaiser and Jacob, 1957) by V. Pirrotta. The other phages were prepared as follows: xC&,SUSN~~ was selected as a clear recombinant at 40” on a a+ host, from a cross between XCI~~~SUSN~SUSN~~ and XsusNst (XsusNss but not XsusN, or XsusN~susNba, gives plaques on an sue+ host) ; XC185m3, the product of a cross between XC160 and xCI~~~SUSN,SUSN~SX~~,was selected as a prophage which cures at 42’; Xi!.sN#.&I~~, the product of a cross between XtsNg and XsusCI~~SUSN,SUSN~~ (Ptashne, 1957), was selected as clear and temperature sensitive on a SK host.
Media, bufers, preparation of phage stocks.
As in Ptashne (1967). Uniformly 14C-labeled L-leucine at a specific activity of about 200 mCi/mmole was from Schwarz BioResearch Inc. Irradiation. Cells were irradiated for 5 min at a distance of 25 cm from two G.E. 15 W germicidal lamps. The incident dose at this distance is about 140 erg mm-2 set-‘; 20-25 ml of cells (depending on the experiments), at a concentration of 3 X lo8 cells/ ml, were irradiated at 4’ in petri dishes of diameter 9 cm with swirling. A typical experiment: kinetics of N product formation upon thermoincluction. A culture
of strain 159 (XC&) growing exponentially at 34’ at a cell density of about lo9 cells/ml is transferred into a prewarmed flask incubating in a 42” shaking bath. When the temperature of the culture reaches 37” (i.e., after 10-15 set), the temperature at which the XC& repressor is inactivated, a t = 0 sample is withdrawn. The culture reaches 42’ at about t = 30 sec. The samples, taken after various times of incubation, are transferred into cold flasks containing enough cold (4”) medium to give a final volume of 20 ml of cells at a density of 3 X lo* cells/ml. The different diluted samples are then transferred into cold petri dishes and W irradiated. Onemilliliter samples of irradiated cells are transferred into incubation tubes containing 0.1 ml of 2 X 10-l M MgSOd, 0.1 ml of phage suspen-
FUNCTION
OF N CISTRON
sion in phage buffer, 0.5 ml of A medium made 2 X W2 M MgSOd and containing 0.2 to 0.5 PCi of leucine-14C. The tubes are incubated for 60 min at 42” with gentle shaking. Then 1 ml of 50% trichloroacetic acid is added, the samples are filtered on Millipore filters, and the filters are dried and counted in an end-window, low-background, gasflow Nuclear Chicago counter. RESULTS
Protein Synthesis in UV-Irradiated
Cells
As shown by M. Ptashne (Ptashne, 1967) infection under proper conditions of heavily UV irradiated E. coli with phage X results in a lo- to 20-fold increase of labeled amino acid incorporation into acid precipitable material (Fig. 2). However, infection of the irradiated cells with the mutant X phages XC&,rb2biolo or Xbzbioll in which most of the genes between N and J are deleted, stimulates protein synthesis very poorly (Fig. 3). This suggeststhat most of the protein synthesis observed in irradiated cells infected with X+ corresponds to the expression of the early genes located between N and 5. Hendrix and Schwartz (in preparation) have
4C cpm x lo-3
IN PHAGE
25
LAMBDA
in fact shown by disc electrophoresis analysis that most of the proteins synthesized upon infection of UV-irradiated cells by h+ are the products of X genes which are deleted in hb2 (Kellenberger et al., 1961) or in Abio, (Manly et al., 1969). Figure 3 also shows that, as reported by Ptashne (1967) no stimulation is observed upon infection with phages carrying suppressor sensitive (sus) mutations or temperature-sensitive (ts) mutations in gene N, under nonpermissive conditions. This result confirms that activity of the N gene product is required for expression of the early genes located between N and J. In Vivo “Assay” of the N GeneProduct The experiment in Fig. 4 shows that delayed complementation of the N function can be measured in this system. A lysogenic host is induced for a short time prior to W irradiation. N product accumulates inside the cells during this period as shown by the fact that, after UV irradiation, these cells can synthesize proteins upon infection with an N mutant. In order to facilitate timing of the experiments, the host was chosen to be
14C cpm x 1G3 n
multipliciti
of infectior
” ‘” time of%ubation
($c)
FIG. 2. Protein synthesis in UV-irradiated 159 upon infection with A+. Protein synthesis is measured as the amount of leucine-14C incorporated by the cells into TCA-precipitable material. Incubation is with 0.5 pCi. of leucine-i4C permilliliter of suspension containing 3 X 108 UV-irradiated cells. (A) TCAprecipitable 14C counts after 60 min of incubation at 37” as a function of multiplicity of infection with X+. (B) TCA precipitable W counts after various times of incubation at 37”. O-0, without added 0, after addition of X+ at an moi of 5. phage, O-
26
SCHWARTZ
Kinetics of Induction of N Product Synthesis
14C cpm x lo-3
I
0
25
multi(;licity
5
75
of infedtion
IO
FIG. 3. Protein synthesis in UV-irradiated 159 upon infection with various X mutants. TCAprecipitable 14C radioactivity after 60 min of incubation at 42” of 3 X lo* UV-irradiated cells with 0.4 &!i of leucine-W and various quantities of phages. O---e, L~u.sN~susN~~; O-0, XtsN9susCIa; A-A, XC&~bzbioto; A -A, Xbzbioll; m--o, x+.
lysogenic for the thermoinducible mutant xa35,. The repressor of this phage is immediately, but reversibly, inactivated at temperatures between 37” and 45” (Sussman and Jacob, 1962; Naono and Gros, 1967; Konrad, 1968). If the lysogenic cells are incubated for 10 min at 42” prior to UV irradiation (Fig. 4 B and D), then superinfection at 42” by an N mutant after UV irradiation stimulates protein synthesis 40 % as much as does Xf. (The same protein pattern is observed upon disc electrophoresis of the proteins synthesized by Xf and the N mutant in these conditions.) In the control experiment, where the cellswere not thermoinduced prior to UV irradiation, no such stimulation is observed (Fig. 4, A and C). In other experiments, not illustrated here, no complementation was observed when the prophage carried a mutation in gene N (also seeexperiment in Fig. 9).
The experiment in Fig. 5A shows that it takes only about 1 min of incubation at a temperature above 37” for the lysogenic host to display some N complementing ability, and that this ability increases only for 4-5 min. The ability of a superinfecting N mutant to stimulate protein synthesis is determined after the host has been induced for different lengths of time. In order to minimize the variation resulting from small differences in the UV dose received by the cells, the amount of protein synthesis observed upon infection by the N mutant is normalized to the amount of synthesis observed when identically treated cells are infected with X+. In this type of experiment, the N complementing ability always stops increasing after 5 min, in spite of the fact that complementation is then only about 40% (i.e., XN- stimulates protein synthesis only 40% as much as does A+). It is known that phagesbearing certain mutations in the II: region (see Fig. l), synthesize exonuclease and /3proteins in much larger quantities than does X+ (Eisen et al., 1966; Radding, 1966; Radding and Shreffler, 1966). Even with a host lysogenic for such a phage, complementation never reaches 100 % (Fig. 5B). However, complementation is reproducibly a little higher in this casethan with a wild-type prophage. Functional Instability of the N Product In the experiments, described above, where delayed N complementation could be demonstrated, the cells were immediately chilled and UV-irradiated after the period of thermoinduction. The experiments described in Fig. 6, however, show that if, just after the period of induction, the cells are incubated for a few minutes at a temperature where further synthesis of N product is repressed (30-35’)) no complementation is observed after UV irradiation. This result indicates that the N product activity produced during the heat induction period then decays during the further incubation at 3035”. A half-life of N complementing ability can be estimated as about 5 min at 35”. It can be seenthat the same result, is obtained when the prophage carries an x mutation.
FUNCTION
OF N CISTRON
I 14C ct3m x lom3
0 IO
0
15
IO
5
30
IN PHAGE
60
I 1 I
0 IO
time of incubation
:I 1
5
27
A
14C cpm x i -3
0
LAMBDA
30
hid
60
2. 14C cpm x 10~~
I
I
mulVplicity of infe%tion
5
IO
FIN. 4. Delayed complementation of XsusNsa by M&r. TCA-precipitable *C radioactivity incorporated by 159 (XC!I8~,) at 42°C in the presence of 0.5 PCi of leucineJ4C for 3 X 108 cells. m-0, uninfected; C-O, infected with XsusN~s;~---A, infected with X+. (A and B) Time course of incorporation for an moi = 10. (C and D) Amount of incorporation after 60 min of incubation for various moi. Strain 159 (XC&,) was cultivated at 32°C and UV-irradiated immediately (A and C) or incubated at 42” for 10 min prior to UV irradiation (B and D).
In contrast, no loss of complementing ability is observed after a 30 min incubation at 4’. This last circumstance actually made the whole study possible since it provides a means of stopping N product synthesis at any moment without losing the activity of the N product synthesized until that moment. The results presented so far concern the stability of the N product in the cells before W irradiation. Attempts have also been made to determine its stability after irradiation. These studies are difficult because the ability of the W-irradiated cells to synthesize proteins even after X+ infection decays rather rapidly. However, at 30”, the N com-
plementing ability seemedto be as unstable after W irradiation as it is before. This is also what the kinetics of incorporation of Fig. 4B suggest. In this experiment, the incorporation stimulated by XN- in preinduced cells appears to stop much earlier than does the incorporation stimulated by XN+ as if the supply of N product provided at the beginning of the incubation had been exhausted after 30 min. E$ect of the CI Repressorin the Presenceof N Product The effect of the CI product on protein synthesis was avoided in the experiments described so far by incubating the W-ir-
SCHWARTZ
28
incorporation 40
5o % of wild type incorporation
A
0
B
not stimulate protein synthesis. Thus, in the presence of repressor, the N product cannot turn on protein synthesis from the superinfecting phage. This, however, is true only if the super-infecting phage is homoimmune with the prophage. As shown on Fig. 7 C, the repressor synthesized by M&,~ is unable to repress the protein synthesis directed by Xi434.Xi434is a recombinant phage in which a small region of the X genomecomprising gene CI and the operators on which it acts has been replaced by the corresponding, but nonhomologous, region of the related phage 434 (see map in Fig. 1) (Kaiser and Jacob, 1957; Westmoreland et al., 1969). Overproduction of N Product by X+
In Fig. 8A it can be seenagain that, under nonpermissive conditions, N mutants are unable to stimulate protein synthesis in 30UV-irradiated cells. The experiment in Fig. 8B shows that, in the presence of the ochre suppressor sue+ the ability of three phages 20 :i;.rcarrying .susNmutations to stimulate protein . synthesis is restored. It is restored to essen1 I 0 IOftially 100% for XauaNhs,a phage known to give plaques on sue+hosts and only partially 012345 IO for XsusN7 and XSUSN~SUSN~~, neither of which gives plaques on s%+ hosts. Disc duration of thermoinductionbnir electrophoresis of the proteins synthesized Fra. 5. Kinetics of induction of N-complementby the susN phages and Xf have been shown ing ability. Suspensions of 159 @CI& (A) or to give identical patterns. Since sue+ is 159 (ML~zr13) (B) are incubated for various lengths of time at 42”, chilled, W-irradiated, and known (Gallucci and Garen, 1966; Stretton incubated for 60 min at 42” with leucine-Xl and et al., 1966) to restore translation with a 16 % either no phage or X+ or XsusNsa at an moi of 5. efficiency at most, this result seemsto imply The amount of TCA-precipitable radioactivity that a quantity of N product only 16 % (and incorporated by uninfected cells (@---a) or perhaps much less) of that synthesized by cells infected with XSUSN~S (O-O) is plotted as X+ is sufficient to fully turn on the genes loa percentage of the amount incorporat,ed by X+ cated between N and J. An alternative possiinfected cells. bility would be, however, that the amount of N product made by X-l- is regulated by a radiated lysogens at a temperature (42”) negative feedback mechanismto a level simiwhere the prophage’s repressor was inactive. lar to that obtained upon ochre suppression The prophage’s N product, if provided by of XSUSN~~. Thus, in order to conclude that preinduction, was then shown to be able to X+ overproduces N product, it is necessary turn on protein synthesis in the superinfect- to demonstrate directly that, in an suof host, ing phage (seethis also on Fig. 7, B and D). less N product is synthesized by XsusN~S If one now considers what happens in the than by X+. In an attempt to do so, the in presence of active repressor (i.e., at 35’) it vivo “assay” system described above was appears that, whether N product had been used to compare the amounts of N product provided by preinduction (Fig. 7C) or not synthesized by X+ and XsusN53in 159su,+. (Fig. 7A), infection by X+ and XSUSN~~ does The two phages were independently induced 40-
FUNCTION
IN PHAGE
1,
% of wild type incorporation
50 4
OF N CISTRON
29
LAMBDA
A
30 20
SJQOd0.
I
, duraficn of incubation 5
IO
before uyhir
35O--
% of wild type incorporation 6 -----a.* ----------z---+ 4O
I
lOba----,, 0
L &4 04
--s-w
_
durajion of incubation ‘O 3z00r40
before u\c(min 30
)
FIG. 6. Instability of the N-complementing ability. (A) A suspension of 159 (XCIa5,) is incubated for 5 min at 42”, and then for various lengths of time at 35”. At different times during this incubation samples are chilled, UV-irradiated, and incubated for 60 min at 42” with leucine-1% and either no phage or X+ or XSUSN~~ at an moi of 5. The amount of radioactivity incorporated by uninfected cells (@---0) or cells infected with XSUSN~~ (O--O) is plotted as a percentage of the amount incorporated by X+ infected cells. (B) Same experiment with a suspension of 159 (XC&,,x13) except that the incubation at 42’was for 15 min, and t,he subsequent incubations at either 32’ (solid lines) or 4” (dashed lines).
in 159 SW+, and the degree of complementation of a temperature sensitive mutant was determined. (The presence of sue+ in the host precluded the use of a susN mutant for superinfection.) It can be seenin Fig. 9 that the amount of complementation provided by preinducing the XsusNE3phage is about 5 times lessthan the amount provided by preinducing the wild-type phage. In this experiment the killing upon heating at 42°C was the same in the two cultures, and both suspensions were verified to contain more than 99 % lysogenic cells just before thermoinduction. The result is in agreement with
the hypothesis that, in a SW+ host XsusNs~ provides lessN product than does A+. DISCUSSION
This paper describes an in vivo assay for the N gene product. This assay is based on the following observations : (i) in adequately UV irradiated cells AN+ phage, but not ANphage, expressesits early geneslocated to the left of N (on the genetic map, Fig. 1) ; (ii) induction in the host of a AN+ prophage, but not of a AN- prophage, before UV irradiation, restores the ability of a superinfecting AN- phage to express its early genes lo-
30
SCHWARTZ
15
IO
5
15
IO
5
FIO. 7. Effect of the phage repressor on protein synthesis in the presence and the absence of N product. A culture of 159 (XC&) growing exponentially at 32’ is UV-irradiated (A and B) or preincubated for 5 min at 42” and then UV-irradiated (C and D). In each case it is then incubated for 60 min at 35” (A and C) or at 42” (B and D) with leucine-1% and no phage or Xf or ~susN 53 or G%usN, at an moi of 3. The ordinate indicates the amount of radioactivity incorporated into TCA-precipitable material by 3 X lo8 cells. F
159 su-
I
0 uninfected F=l X susN7N53 <
X wild type incorporation
100
lEl XsusN7 h susN,, PZZJx+ LUII h tsNgsusCI~~
-
Fro. 8. Effect of the suppression of N mutations by sus+. Cultures of 159 or 159 sue+ are UV-irradiated and then incubated for 60 min at 42’ with leucine-r4C and either no phage or the phages indicated on the figure at an moi of 5. The amount of radioactivity incorporated into TCA-precipitable material is plotted as a percentage of the amount incorporated by h+-infected cells.
FUNCTION
OF N CISTRON
IN PHAGE
4 % of A susNS3 incorporation
31
LAMBDA
e 6.
% of A susNS3 incorporation
60-
50 40-
30-
0
I 5
1 IO
0
duration of thermoinduction
I 5
I IO
(min.)
9. N-complementing ability provided by h+ and hsusN63 in an su$ host. Suspensions of 159 sue+ (XC&) (panel A) or of 159 sucf (XC18B~ susN& (panel B) exponentially growing at 32’ are incubated for 0, 5, or 10 min at 42”, chilled, UV-irradiated, and incubated at 42” for 60 min with leucine-W and either no phage or XsusNa3 or XtsNssusCIsl at an moi of 5. The amount of radioactivity incorporated into TCA-preoipitable material by uninfected cells (U-0) or cells infected with ht.sNgsusCIt~ (e---e) is plotted as a percentage of the amount incorporated by XsusNsz-infected cells. FIG.
cated to the left of N. The ability of a ANphage to stimulate protein synthesis in UVirradiated cells can thus be taken as an index of the presence of N product inside these cells. Making use of this assay, some aspects of N product function are investigated. We present evidence for the following facts: 1. Synthesis of the N product occurs very early in the vegetative cycle of the phage. 2. The N product is functionally unstable at physiological temperatures, but stable at low temperatures. 3. The CI repressor prevents expression of the early genes located to the left of the N gene, even in the presence of N product. 4. The minimum amount of N product required to obtain maximum expression of the early genes located to the left of N is much smaller than the amount synthesized by wild-type phage. After considering the problems involved in quantitating the N product assay, we shall discuss each of these conclusions in turn. Quantitation of the relationship between
the amount of N product in the cells and the degree of complementation of an N- mutant is difficult. In any attempt to quantitate this relationship one would have to understand, among other things, what limits the amount of complementation observed. It is a fact that no matter how long a wild-type prophage is induced in the host before UV irradiation, superinfection by an N- mutant can stimulate protein synthesis only 40 % as much as doessuperinfection by an N+ phage. It is not well understood why an amount of N product which is sufficient to ensure maximum protein synthesis when provided by the infecting phage itself, becomeslimiting when provided by a preinduced prophage. This may be due to the functional instability of the N product (seebelow) since the kinetics of protein synthesis (Fig. 4B) suggest that the N product activity decays during the incubation following UV irradiation. However, alternative or additional reasons, involving some compartmentalization for instance, could easily be found. In view of these uncertainties, no attempt will be made,
32
SCHWARTZ
in the discussion presented below, to quanti(in preparation), who found that exonuclease tate the amount of N product present in the synthesis cannot be turned on in an N- exe+ cells. We shall only assume that any in- prophage by a heteroimmune Nf exe- supercrease in complementation corresponds to an infecting phage unless the prophage reincrease in N product concentration. pressor is inactivated. One must then con1. The data presented here show that at clude that the early genes located to the left 42”, N starts being expressed within the first of N are controlled in cis by an operator subminute after repressor inactivation. The con- ject to repression by the CI product. A centration of N product then increases at priori one might have postulated the existleast until the fifth minute. No further in- ence of such an operator to the left of N. crease in complementation is observed after However, since it is shown here that protein 5 min, but this does not necessarily imply synthesis directed by the heteroimmune that N product synthesis actually stops at phage Xi434is insensitive to the presence of X that time. Since the N product is functionrepressor, one must conclude that the operaally unstable (see below), the apparent tor is in the region of nonhomology between stoppage in net N product synthesis may re- X and Xi434and is thus presumably identical flect the establishment of a steady state be- with the operator for the N gene, defined by tween synthesis and decay. the Vz mutation. By this criterion aloneThe presence of an x mutation, known to common control in cis by a single operatorlead to the overproduction of exonuclease the N gene and the other early genes located and fi protein (Eisen et al., 1966; Radding, to its left would appear to be part of a single 1966; Radding and Shreffler, 1966) leads to operon (Jacob and Monod, 1961). However, a slightly increased N complementing ability this whole group of genes does not behave of the prophage. This may indicate that z as a ‘(single unit of coordinated expression” mutants overproduce N product as well as since in the absence of active N product, exonuclease and /3protein. only the proximal part of this set of genes is 2. Upon incubation of the cells between transcribed (Kourilsky et al., 1968). Ap32” and 35” in the absence of any inhibitor, parently derepression is required for tranthe N complementing ability produced by scription to start at the Vz operator, and acinduction of the prophage has a half-life of tivation by the N product is required for about 5 min. In a previous study of the rate transcription to proceed further than a point of production of X-specific mRNA, Konrad located to the left of the N gene. (1968) also came to the conclusion that an 4. Finally we find that upon infection of a “inducing protein”, presumably the N host carrying the ochre suppressor sue+ product, had a functional half-life of this or- by a phage carrying an amber mutation in der of magnitude. In his experiments, how- the N gene, protein synthesis is the same as ever, chloramphenicol was added to stop upon infection by X+. This is merely an exfurther synthesis of the “inducing protein”, tension of the result of Radding and Echols and, consequently, the results could have (1968), who showed that upon ochre supbeen interpreted to mean that the drug in- pression of an amber N mutant, wild-type terfered with N product activity. amounts of exonuclease and 0 protein are It is of interest that the N product of an x produced. Under the simplest hypothesis, mutant is as unstable as that of wild type. the amount of N product synthesized upon One may conclude that the functional inochre suppression of an amber mutant should stability of the N product does not result from a specific inactivation mediated by the not be more than 16% the amount syntheproduct of a late gene or an early gene lo- sized by wild type (Gallucci and Garen, 1966; Stretton et al., 1966). In fact, we precated to the right of CI, since an x mutant evidence in this sent “semiquantitative” does not express any of these genes. 3. In the presence of active repressor, the paper that this hypothesis is probably correct. In agreement with Radding and Echols, N product cannot turn on the genes located to the left of N. This is in agreement with the we would then conclude that X-t seems to proresults of Luzzati (in preparation) and Pero duce at least 6 times more N product than is
FUNCTION
OF
N CISTRON
required to turn on the early genes located to the left of N. ACKNOWLEDGMENTS The support, direction, and encouragement of Professor J. D. Watson throughout the course of this work, the helpful advice of Drs. E. Signer, W. Gilbert, and M. Ptashne, and the skillful technical assistance of Mrs. Henrietta W. Tranum are gratefully acknowledged. These experiments were performed while the author was a junior fellow of the Society of Fellows of Harvard University. REFERENCES BROWN, A., and ARBER, W. (1964). Temperature-sensitive mutants of coliphage lambda. Virology 24, 237-239. CAMPBELL, A. (1961). Sensitive mutants of bacteriophage h. Virology 14, 22-32. DOVE, W. F. (1966). Action of the lambda chromosome. I. Control of functions late in bacteriophage development. J. Mol. Biol. 19, 187-201. EISEN, H. A., FUERST, C. R., SIMINOVITCH, L., THOMAS, R., LAMBERT, L., PEREIRA DA SILVA, Genetics and physiL., and JACOB, F. (1966). ology of defective lysogeny in K12 (h) : Studies of early mutants. Virology 30, 224241. GALLUCCI, E., and GAREN, A. (1966). Suppressor genes for nonsense mutations. II. The su-4 and su-5 suppressor genes of Escherichia coli. J. Mol. Biol. 15, 193-200. JACOB, F., and CAMPBELL, A. (1959). Sur le syst&me de repression assurant l’immunitk chez les batteries lysogenes. Compt. Rend. Acad. Sci. 248, 3219-3221. JACOB, F., and MONOD, J. (1961). Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3, 318. KAISER, A. D. (1957). Mutations in a temperate bacteriophage affecting its ability to lysogenize E. coli. Virology 3.42-61. KAISER, A. D., and JACOB, F. (1957). Recombination between related temperate bacteriophages and the genetic control of immunity and prophage localization. Virology 4, 509621. KELLENBERQER, G., ZICHICHI, M. L., and WEIOLE, J. (1961). A mutation affecting the DNA content of bacteriophage lambda and its lysogenic properties J. Mol. Biol. 3, 399408. KONRAD, M. W. (1968). Dependence of “early” bacteriophage RNA synthesis on bacteriophagedirected protein synthesis. Proc. Natl. Acad. Sci. U.S. 59, 171-178. KOURILSKY, P., MARCAUD, L., SHELDRICK, P., LUZZATI, D., and GROS, F. (1968). Studies on the messenger RNA of bacteriophage X. I. Various species synthesized early after induc-
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