On the mechanism of transcription of the lambda genome during induction of lysogenic bacteria

J. ilId.

Rid. (1967) 25, 517-536

the Mechanism of Transcription of the Lambda Geno during Induction of Lysogenic Bacteria SHIRO NAONO AND FRANQOIS GROS de Physiologic Microbienne Institut de Biologie Physicochimique Paris, France

Laboratoire

(Received11 July 1966, and in revisedform 12 January 1966) When a thymine-requiring lysogen carrying a thermoinducible prophage (hEWi) is heated at 42°C in a medium lacking thymine and containing chloramphenicol, the capacity to synthesize phage-specific RNA is immediately stimulated. The maximum de-repression thus achieved corresponds to a lo- to 20-fold increasein the initial rate of h RNA synthesis. If the mutation causing heat sensitivity of the prophage repressor is of the C, B type (Lieb, 1966) this de-repression in RNA synthesis is partially (Xtl) or fully (h857) reversible after cooling to 34”C, even in the presence of chloramphenicol. In contrast, if lysogens belong to the C, A type (X tU32, XtIJ37) de-repression remains maximum after cooling. The A-specific RNA fraction induced in the absence of DNA and protein synthesis (“restricted RNA”) and the one synthesized during the replicative period of phage growth have been compared for t#heir annealing characteristics. “Restricted” A RNA has the properties of a GC-poor polynucleotide fraction, whereas “vegetative” r\,RNA behaves as a mixture of GC-poor and GC-rich polynucleotides species. Finally “restricted” A RNA displays a close sequence homology to h specific RNA’s from mitomycininduced lysogenscarrying early defective prophages. It can thns be concluded that lysogen induction, in conditions precluding prophage replication and protein synthesis, involves, as an immediate consequenoe, the enhanced transcription of certain X genesnecessaryfor early viral functions.

1. Introduction In bacteria carrying a Xprophage the maintenance of the lysogenic state requires the functioning of a particular region of the prophage genome called “Cr” (Jacob $ ollman, 1961). It has been proposed by Jacob & Monod (1961) in the framework of their general model on regulation that C, manufactures a repressor preventing transcription of the Xgeneswhoserole is to initiate the processof vegetative multiplication. That the Cr product actually repressesX mRNA synthesis was fist suggestedby experiments of Attardi, Naono, Jacob, Rouviere & Gros (1963) who found that in heat-inducible lysogens grown at non-inducing temperatures, levels of phagespecific RNA are not higher than 2% of those reached after induction. However, since no attempt was made to prevent phage replication, part of the enhancement following thermo-induction was presumably related to a gene dosage effect. That, a marked stimulation in the transcription rate of the X genome could result from a pure de-repression phenomenon was later established by Sly, Echols $ Adler (1965). They 517

518

S. NAONO

AND

F.

GROS

observed that infection of a sensitive host with a virulent mutant permitted the viral genome to be immediately transcribed at a much faster rate than infection of a lysogenio host using a similar multiplicity. However, since prophage induction is a complex phenomenon (Weisberg & Gallant, 1967) possibly involving a succession of steps mediated by different gene products (Thomas, personal communication), it rema.ined of interest to specify the region of the prophage to which transcription is confined as the immediate consequence of inactivation of the C, repressor. An approach to this study was offered by Green’s (1966) observations. Using a lysogen carrying htl, a prophage with a heat-sensitive repressor, he showed that induction even in the presence of cbloramphenicol enhances the synthesis of X-specific RNA without causing death of the lysogen. Whether a small fraction of the prophage genome was transcribed at a full rate or the entire genome de-repressed at a reduced rate was not however examined. These considerations prompted us to investigate the kinetics of X RNA synthesis during thermo-induction of a lysogen, while preventing both viral replication and protein synthesis, and to compare the characteristics of viral RNA thus formed to those of RNA transcribed during the normal vegetative cycle. Our results indicate that an appreciable synthesis of X-specific RNA follows thermoinduction in the absence of thymine and presence of chloramphenicol. The category of RNA formed under these conditions corresponds to a much smaller number of genetic sites than phage messenger RNA synthesized during late viral growth. According to its melting behaviour after hybridization at a given ionic strength, this RNA has the properties of a G-C-poor polynucleotide fraction, suggesting its transcription from the region of X DNA containing genes for early functions. De-repression of this GC-poor X-specific RNA seems to represent one of the first events caused by heat inact.ivation of the C, product. This is shown by the kinetics of this phenomenon throughout the heating period and by its complete reversibility in strains where the altered repressor function is restorable by cooling (lysogens .of the CI B group).

2. Materials (a) Media

and

and Methods bacterial

strains

We have used throughout the synthetic medium 63 (Monod, Cohen-Bazire, Papenheimer & Cohen, 1951) containing 0.4% glycerol and supplemented with 0.4% vitaminfree Casamino acids (Difco), plus 2 pg/ml. thiamine. When necessary, thymine (lo-& M) was added. Bacterial growth was followed by measuring optical density at 420 rnp, an O.D. of 1.0 being equivalent to 5 X lo8 cells/ml. Escherichia coli strains C600 (h857) and Hfr U1858 (X857) thy-, as well as the nonlysogenic counterparts, C600 and Hfr U, were obtained from Dr F. Jacob. In what follows, the thymine auxotroph strain Hfr U1858 (X357) will be abbreviatedU1858 (X357). h857 is a phage carrying a double mutation in the C, region leading to inducibility of the corresponding lysogen by heat treatment (Sussman & Jacob, 1962) but not by ultraviolet irradiation (Jacob & Campbell, 1959) nor by thymine starvation (Sicard & Devoret, 1962). Strains U1858 lysogenized for prophages h tl, X t U32 and h t U37 have also been prepared, the corresponding phage mutants being gifts from Dr M. Lieb (Lieb, 1966.) We have also used C600 lysogens carrying the defective h prophages tll (Radding, 1964a,b), t27, t66, as well as P22 (Jacob et al., 1957) and the strain W602 (X t75) for which we are indebted to Dr Fuerst. Figure 1 represents a simplified genetic map of the early region of X phage, with the specific locations of the various “defective” mutations (Eisen et al., 1966) and the order of genetic sites of C, mutant prophages (Lieb, 1966) utilized in this work.

TRANSCRIPTION

OF

X GENOXE

519

Frc. 1. Diagrammatic representation of the C region of lambda bacteriophage (after Eisen et al., 1966). tll, t66 and P22 are point mutations. The arrow at the locus of t75 indicates one end of the deleted region of defective prophage t75. This prophage does not contain the biotin region of E’. co% but includes h genes on the left-hand half of the vegetative map (A to M). Region X is an area in which many mutations exert a polarity effect on adjacent genes C,, and 0 (Eisen et al., 1966). Mutations within region Y are not polar with respect to CII and 0; the mutants in this region are not defective and do not complement Cn or CI mutants. The enlarged representation of the CI region shows the locations of the prophage mutants with a thermolabile repressor which we ha,ve used throughout this work (Lieb, 1966). (b) Preparation

of phage

h DNA

3Olitres of C600 (A867) in medium 63 were cultivated at 35°C in fermenters with vigorous stirring. When the cell density was 1.5 x 10Q, enough of the same medium at 70% was carefully added to raise the temperature to 42°C. After 30 min, a new addition of 0.40/, Gasamino acids was made and the temperature lowered to 37°C with cold medium. Incubation was continued until lysis appeared to be complete. The phages were then purified and banded in CsCl for 40 hr according to Kaiser & Hogness (lQ60). After dialysis against standard saline citrate buffer (1 x SSC, Marmur, 1961), the purified phage suspension was deproteinized by means of cold phenol following the method of Kaiser & Nogness (1960). The DNA after ethanol precipitation was finally dissolved at a concentration of approximately 500 pg/ m 1. in 0.1 x SSC buffer, a drop of chloroform added and the solut’ion kept at 2°C. (e) Thermo-induction

of lysogenic

cultures

and pulse-&belling

oj induced

cells

Unless otherwise specified, an exponentially growing culture of thermoinducible bacteria (5 x l.08 cells/ml.) at 34°C was heated to 42°C by vigorous shaking in a 75°C water bath (the time required to heat up 100 ml. of culture was of the order of 50 see). The heated culture was immediately placed in a 42% water bath and incubation continued with shaking. After various intervals, portions of the heat-treated suspension were pulselabelled, either at the same temperature, or after rapid cooling to 38°C. To label the cells, 2 ccc/ml. of C3H]uridine (5 to 8.5 c/m-mole) and in a few cases [“Hluracil (8 to 12.5 c/m-mole) were added with constant shaking for 30 set (42’C), or 45 set (38”C), and the cells then rapidly poured on to crushed, frozen growth medium containing low2 M-NaN3. After centrifugation the bacterial pellet was washed once with 10e2 M-Tris--HCl buffer, pW: 7.4, containing 10m3 x-MgCl,, (TM3), and kept frozen. (d) RNA

extraction

The labelled cells were resuspended in 2.5 times their wet weight of cold TM3. To this suspension 100 pg/ml. lysozyme and 25 pg/ml. “RNase-free” DNase were added (RNase, 2 “g/ml., had been heated at pH 5.0 for 10 min at 85°C). This was followed by sever,%1 successive cycles of freezing in an acetone-dry-ice bath and thawing at 30°C. A further incubation was carried out for 5 min at 25°C and an equal volume of water-saturated phenol was added. After mechanical shaking for 30 min the aqueous phase was carefully pipetted off and set aside. The phenolic phase was washed once with half a volume of T&B and the wash combined with the aqueous phase. A second phenol treatment was carried out, following which the aqueous phase was precipitated by 2 vol. of cold ethanol in the presence of 0.1 M-NaCI and treated once more with cold phenol. Finally, the deproteinized

520

S. NAONO

AND

F. GROS

RNA solution was precipitated by ethanol, redissolved in 2 x SSC buffer and dialysed for 12 hr at about 4°C against the same buffer. The specific activities of RNA’s prepared from cells labelled with [3H]uridine and from cells labelled with [3H]uracil were usually 1. to 1~5 x lo6 and 2 to 2.5 x lo6 cts/min/mg, respectively. (e) Measurements of phage DNA synthesis during the thermo-induction. of C600 A857 An exponentially growing culture of this lysogenio strain (cell density, 5 x lOs/ml.) at 35°C was transferred to 42°C and received[14C]thymine (5 x 10e5x,25*5 po/pmole) plus 50 pg/ml. of deoxyadenosine. At intervals, portions of the culture were sampled, supplemented immediately with 5 X 10m3 M-[r2c]thymine plus 50 pg of deoxyadenosine, and the incubation continued at 42°C for 30 min. This was followed by a further incubation at 38°C until complete lysis occurred. The total lysates were treated with chloroform and centrifuged for 15 min at 10,000 g. To the supernatant, 25 yg/ml. of DNase plus 25 pg/ml. of RNase were added and the suspension was incubated for 30 min at 37°C. At the end of this treatment, both the phage titre and the 5% trichloroacetio acid-precipitable radioactive material were measured. (f) Hybridization of X ape&k messenger RNA The Millipore filtration technique of Nygaard & Hall (1964) modified by Green (1963) was used. Generally, to a de6nite amount of heat-denatured DNA, ranging from 20 to 30 p-8, various amounts of labelled RNA’s were added and the ionic strength of the medium was adjusted to 2 x SSC (final volume 0.3 ml.). The reaction mixtures were incubated at 63°C for 4 hr. After cooling to 37”C, 0.7 ml. of “DNase-free” RNase solution (10 pg/ml.) was added. RNase incubation proceeded for 20 min at 35°C. The digested samples were transferred to an ice water bath and diluted with 2 ml. of cold 5 x SSC buffer. Each sample was then filtered on nitrocellulose membranes (Schleicher and Schuell B6) and washed on the filters with 60 ml. of cold 2 x SSC buffer. After drying, the radioactivity remaining on the membranes was determined using a TriCarb liquid scintillation counter. Backgrounds, due to passive retention of radioactive RNA samples, were subtracted from each value. In one case (experiment described in Fig. 11) the method of Gillespie & Spiegehnan (1965) was used. (g) Measurements of X-speci$c exonuclease activity Extracts for measuring Xspeciflc exonuclease have been prepared according to Radding (19643) by sonicating suspensions of heat-induced lysogenic bacteria in a 5 x 10e2 Mglycylglycine (pH 10-O) buffer containing 10m3 M-glutathione. Usually the cells recovered from 20 ml. of culture (at a density of 7 x 10s bacteria/ml.) were dispersed in 1 ml. of the buffer and kept frozen until the sonication step. Sonic&ion was performed with a Mullard (MSE) sonic disintegrator in the cold for 1 min. The cell debris was discarded by centrifugation and the exonuclease determined using the following reaction mixture : bacterial extract (about 650 pg protein/ml.), from 20 to 100 ~1.; MgCI,, 1 pmole; glyoine buffer, pH E. co% DNA (specific radioactivity, 1.5 x lo6 cts/min/mg; 10.0, 10 pmoles; 3H-labelled 400 pg/ml.), 40 ~1.; total volume, 0.3 ml. The incubation was carried out for 30 min at 37”C, at which time the reaction was stopped by immersing tubes in ice water and by adding 0.5 ml. of 3~5% HCIOs plus O-2 ml. unlabelled thymus DNA solution (0.25%) as carrier. After standing 15 min in the cold the tubes were centrifuged and 0.3 ml. of supernatant fraction were mixed in counting vials with 5 ml. of the following scintillation mixture : 60 g naphthalene ; 4 g POP ; 0.2 g POPOP ; 20 ml. ethylene glycol; 100 ml. methanol, enough dioxane to adjust to a litre. Corrections were made for the acid-soluble radioactive material found at time zero (before incubation).

3. Results (a) Kinetics When inducing

a lysogenic temperature,

of RNA

synthesis

upon thermoinduction

strain carrying a thermoinducible prophage its capacity to synthesize h-specific RNA

is placed at an increases almost

TRANSCRIPTION

OF

X GENOME

521

immediately upon heating, i.e. before one can detect any important rise in the number of viral genomes. This is demonstrated in the following experiment. A culture of C600 (h857) grown in medium 63 plus Casamino acids (34°C) was transferred directly to 41°C and incubated at this temperature for various periods folloting which portions were given pulse labels of [3H]uridine. The percentages of unfraetionated pulse-labelled RNA hybridizable with an excess of X DNA versus time of incubation at 42°C is plotted in Fig. 2. On the same Figure is also plotted the rate of A DNA synthesis, measured as described in Materials and Methods. Comparison of the two curves reveals that de-repression of RNA synthesis begins at the maximum rate promptly after heating, while DNA synthesis does not achieve its maximum rate until after five minutes. To take into account the possible influence of viral replication on the rate of de-repression in phage RNA synthesis, a new series of thermo-induction studies was performed with the auxotrophic strain Hfr U1858 (X857) in the presence or absence of

5000

a %? x .; . v(

4000 2

e .o‘;’ E 3000 Fh 2a 2000 ;

5 2 ,” 1000 :; .-mx 2

Time ofaddition (DNA) or time

of

of [“Clthymine induction (RNA)

PIG. 2. Synthesis of viral DNA and RNA upon thermo-induction of a A lysogen. On the same graph are plotted results of two independent experiments in which rates of /\ DNA or X RNA synthesis were determined in the course of thermo-induction. Deter&%&o?% of newly formed h DNA: a culture of C600 (X857) grown at 34% (medium 63 plus Casamino acids) was heated at 42% at time t = zero while receiving a mixture of [14C$hymine and deoxyadenosine (see Materials and Methods). At the indicated periods, the tracer was diluted by addition of excess cold thymine and incubation continued at the same temperature so as to ensure for each portion a 30-min incubation at 42’C. After cooling to 37’C, incubation was cont,inued until complete lysis. Phage particles were purified as outlined in the section on Materials and Methods and the radioactivity present in a DNA equivalent of lo10 phages was determined, D&r&nation of newly fomed X RNA: cells of the strain C600 (X857) grown at 34% in medium 63 plus Casamino acids (to an O.D. of 1.2 at 420 mp) were quickly heated to 42%. Incubation at this temperature was continued for different lengths of time. At the end of each incubation period., ,% 10O,.ml. sample received a 40-see pulse of [aH]uridine and after chilling and washing, RNA was extracted and the percentage of labelled material hybridizable to an excess of h DNA was determine --O--, Percentage of pulse-labelled RNA hybridized t,o X DNA; -O----O--, radioactivity found in phage fraction.

522

S. NAONO

AND

E‘.

GROS ifr

5 IO 15 20 Incubation

time at 42’C

25

1858

A857

1

30 (mid

FIG. 3. Synthesis of X RNA upon heat induction in the presence or absenoe of thymine. Cells of the strain U1858 (h857), grown in mineral medium plus Casamino acids, glycerol and thymine were starved for thymine for 20 min at 34°C and the suspension divided into equal halves. One received thymine (10T4 M); it was heated to 42% and incubated at this temperature for different lengths of time. At the end of each incubation period, a 45-see pulse of [3H]uridine was given, the cells rapidly chilled and processed for RNA extraction. The other half was heated at 42” C in the absence of thymine, pulse-labelled with [3H]uridme after various time intervals and the total RNA extracted as before. Both series of RNA preparations were hybridized to en excess of denatured X DNA. The percentage of labelled RNA hybridized is plotted versus the duration of incubation period. (0) Culture heated in the presence of thymine; (0) culture heated in the absence of thymine; ( x ) heating in the absence of thymine and in the presence of 50 pg/ml. chloramphenicol (CAP) ; (A) sitme experiment with non-lysogenic strains CEO0 and Hfr U.

thymine. Induction ofthe control sample (heating in complete medium plus thymine) gave results very similar to those obtained in Fig. 2 with the prototrophic lysogen. The percentage of newly formed RNA which is h specific increased almost linearly with time from the beginning of the heat treatment. In contrast, when the thyminerequiring lysogen was first starved for this pyrimidine, and induced in its absence, the cell capacity to form X-specific RNA became constant after five minutes and reached a value such that, in this experiment, about 5% of the pulse-labelled RNA was homologous to h DNA.? As also noted on Fig. 3, addition of chloramphenicol (25 fcg/ml.) at the same time as the temperature shift did not affect the rate or the extent of de-repression observed in the absence of thymine without chloramphenicol. The value of 5% represents quite a significant enhancement in h RNA synthesis considering that in the unheated lysogen and in the non-lysogenic strains (CSOO or Hfr U) the fractions of the pulse RNA’s showing homology for h DNA respectively amounted to O-3 and O*2o/o. However, the factor of enhancement varied somewhat in t In an accompanying paper, using the same Weisberg & &llant (1967) have observed that about 93 y0 9

strain and nearly same conditions thymine deprivation reduces DNA

of starvation, synthesis by

TRANSCRIPTION

Time

OF

after

523

h GENOME

transfer

at 34 “C (nin)

(a)

,Non-induced

(0.16

“/o)

I

IO Time

after

transfer

20 at

34°C

bin)

(b)

FIG. 4. Decay of A RNA-forming capacity upon transfer of a heat-induced lysogen to 34°C. (a) A culture of U1858 (X857), previously starved for thymine at 34%, was transferred to 41’c. Mter 16 min, a IOO-ml. portion was taken, placed at 37°C for 30 sea and pulse-labelled for 45 SW;, using 2 me/ml. [3H]uridine (0.8 mc/pmole). The rest of the heat-induced culture was cooled to 34°C and after the indicated intervals, loo-ml. samples were pulse-labelled as described above. RNA was extracted from each sample and hybridized to an excess of previously denaxured h DKA. (b) Same experiment as in (a), save that the pretreated culture was divided into two parts, one of which was cooled to 34% in the presence of 50 pg/ml. ehloramphenicol (crosses) and the other in the absence of inhibitor (filled circles),

524

S. NAONO

AND

F.

GROS

different experiments, the chief variable being, presumably, the condition involved in pulse-labelling. Values corresponding to ten minutes of thermo-induction (labelling period 30 seconds) were between 1.2 and 5.8% with an average of 25% in ten independent experiments. (b) Loss of total X RNA-forming capacity upon transfer of a heat-induced X857 lysogen to non-inducing temperatures In IX858 (h857) the capacity to synthesize X-specific RNAafter thermo-inductionin the absence of thymine almost completely decayed in a few minutes when cells of this lysogen were returned from 41°C to 34°C still in the absence of thymine (Fig. 4(a)). Furthermore, the same rate of decay was observed when chloramphenicol was present during the heat treatment and subsequent incubation at low temperature, a result suggesting that the heat-altered repressor can recover activity upon cooling, even in the absence of protein synthesis (Fig. 4(b)). Table 1 relates the extent of this recovery to the temperature at which the cooling step was carried out in a range between 34 and 39°C. The thermal transition required for restoring the repressor function appeared very sharp (i.e. after cooling from 40 to 39°C in the presence of chlorambhenicol, a 78% reduction in the rate of X RNA synthesis was observed). (c) Decay of exonuclease-specijc messenger RNA upon transfer to sub-inducing temperatures Another observation supporting the rapid renaturation of the C, repressor function of h857 upon transfer to a non-inducing temperature can be derived from experiments on the fate of the messenger RNA speciik for exonuclease production. TABLE 1 Reappearance of C, repressor fun&ion upon cooling a heat-pulsed tysogen to variozcs temperatures RNA

Percentage RNA

samples

X DNA

Cooled

Zero timei culture samples 10 min 39T 10 37 10 36 10 34 10 34

( + thymine)

of newly hybridized

formed to

E. co% DNA

Relative

X /E.

14

5.4

0*280

0.48 0.012 o-009 0 0

‘7.68 6.0 6.6 6.6 5.2

0.064 0.022 0.014 0 0

hybridization coefficients Percentage coli of control 100 22 7.8 5.0 0 0

t 40°C 20 min in thymineless medium with chloramphenicol. A culture of U1858 (X857) was starved of thymine for 20 min at 34’C, and transferred to 40% for 20 min in the absence of thymine and presence of chloramphenicol (50 pg/ml.). At the end of the thermo-induction period, a portion was submitted to a pulse of [3H]uridine for 40 see, and the RNA extracted as usual (zero time). Other portions of the preheated suspension were transferred to various temperatures ranging from 34 to 39% and, after 10 min equilibration, were also pulselabelled with r3H]uridine (the sample at 39°C for 40 see; samples at 37 and 36’C for 50 set, and samples at 34°C for 60 see). RNA’s were prepared from each labelled sample and hybridized either to h or to E. coli DNA (using in each case the equivalent of 20,000 ots/min of newly formed 3H-labelled RNA). In the sample designated 34°C ( + thymine), thymine (10m4 al) was added at the onset of the cooling period.

TRANSCRIPTION

Tryptophan

OF

X GENOME

525

Tryptophan I 20

IO Time after

returning

to 34°C

(mid

FIG. 5. Fate of the exonuclease-speciEc messenger RNA during transfer of a heat-induced iysogen to a subcritical temperature. A culture of U1858 (X857) was grown on 63 medium plus thymine and glycerol, supplemented with 0.2% of charcoal-treated Casamino acid. When the cell concentration reached 3.5 x 10s cells/ml., the bacteria were washed and starved for thymine for 20 min at 34°C. 5 x lo-* M-5-methyltryptophan (5MT) was added and cells were transferred to 41%. 15 mm later, a 20-ml. sample was taken and chilled in the presence of ehloramphenicoi. The remainder was divided in half and placed at 34°C. One-half received 10-s &r-n-tryptophan plus lo-* M-thymine immediately after cooling, whereas the other half received these additions 10 min after cooling. 20-ml. samples were taken at intervals following the addition of tryptophan in both cases. The exonuclease activity in each sample was determined as described in Materials a.nd Methods. Control determinations were made on cells heat-induced in the absence of Smethyltryptophan, as well as on uninduced cells.

A culture of thymine-starved lysogen was thermally induced at 41-Y for 15 minutes in the presence of 5methyltryptophan and the expression of the messenger .RNA specific for exonuclease was studied at 34°C in the presence of tryptophan (Pig. 5). When tryptophan was added just after cooling, there was an immediate increase in exonuclease activity, indicating that a supply of specific messenger had accumulated during thermo-induction. Conversely, if tryptophan was added ten minutes after cooling, praoticaLly no increase in exonuclease activity was found. Since it is likely that aearly all of the messenger RNA made during the heat induction will have decayed during the ten minutes at 34”C, this result would suggest that the repressor function of h857 can rapidly re-form at this temperature. Thus, the interpretation of this result is in agreement with the conclusion drawn from the hybridization experiments. (d) Xffect of cooling on total h RNA-forming capacity in U1858 containing other Cl mutant prophages The results obtained with U1858 containing h857prompted us to study how thermoinduction affects the rate of phage RNA synthesis in t.he same strain lysogenic for other prophage mutants of types C, A (X t U32, h t U37) or Cr B (X tl) (Lieb, 1966) and to examine the effects of subsequent cooling on this phenomenon. Table 2 summarizes results obtained by measuring the initial rates of h RNA synthesis either immedkte~y, at the end of a ten-minute heating period in complete medium plus ch.loramphenicoB, or five and ten minutes after cooling to 34°C. Phage RNA synthesis was determined,

526

S. NAONO

AND TABLE

Renaturability

Expt no.

heated

of Cr mutants

43°C 5min

cooled

34%

heated

43OC

cooled

34%

heated

43°C

cooled

34’C<

10 mill 5 min

c

i

GROS

2

of thermolabile repressor functions in Hfr U1858 lysogenic for various C, mutayzts of lambda prophage

Types

r

F.

cooled

10 min C5 min 1o min

34’C

1o min 1

Amount of h hybridizable RNA normalized for lo4 cts/min material hybridized to E. coli DNA

Renaturation index

3.900 43

98.0

zero 7.837 3.780

lOO*O 51.8

3.720 4.902 6.052

62.5 zero

5.640 12.076 5.400

zero 55.2

4.800 11.200 9-771

60.1 12.7

10~100

9.8

Cultures of strains U1858, lysogenic for X857, h tl, X U32 and X U37 were grown in Casamino acid 63 medium plus glycerol, thymine and B1. Each culture (6 x 108 oells/ml.)received 25 pg/ml. of chloramphenicol and was heated to 43%. After lo-min incubation at this temperature, a lOO-ml. portion in each heated sample was labelled with 3 ~c/ml. of [aHJuracil (10 mc/pmole) for 40 set and quickly chilled in a flask containing 0.1 M sodium azide. The remainder of each preheated culture was rapidly cooled to 34’C using an ice water bath, and incubation was continued at this temperature for 5 and 10 min, respectively, after which times loo-ml. portions were similarly pulse-labelled for 70 sec. RNA’s were extracted from each of the pulse-labelled samples. The specific radioactivities of the final RNA preparations were between 5 and 7.0 x 1Oe cts/min/mg. An amount of sH-labelled RNA equivalent to 5 x lo5 cts/min was annealed to excess denatured h or E. co& DNA. Assuming z and y correspond, respectively, to the amount of radioactivity found in hybrid (z - y) x 100 form, before and after cooling, we calculate the renaturation index by: z

as before, by hybridization. However, to take into account possible variations in cell capacity to synthesize RNA arising from thermal transitions, the percentages of material annealable to XDNA were normalized to a fixed amount of material hybridizable with E. coli DNA. Experiments were also repeated with U1858 (X857) for the sake of comparison. As expected, all lysogens synthesized phage-specific RNA upon thermo-induction in chloramphenicol, but depending upon the site of the C, mutation the shutoff of X RNA synthesis caused by cooling appeared to be complete (X357), partial (Xtl), or negligible (Xt U32, Xt U37). Lambda mRNA synthesized by thermoinduced and cooled X tl lysogensapparently correspondsto the expression of a smaller number of genetic sites than those expressed before cooling. This is suggested by comparing the saturation levels reached of hybridizable RNA from the induced h tl lysogen that was labelled immediately before or ten minutes after cooling to 34°C (Fig. 6).

TRANSCRIPTION

OF

X GENOME

RNA pg added (cts/min)

r3Hh

Fm. 6. Comparative levels of h DNA saturation by [3H]-labelled RNA’s from thermo-induced IJ1858 (X tl) before and after cooling.RNA’s were prepared from a thermo-induced culture of IJlE58 (h tl), labelled with [3H]uracil, either at the end of the heating period in the presence of chloramphenicol (sample A), or after heat induction in this medium followed by a cooling period of 10 min at 34°C (sample B). These preparations were the same as those used in the first experiment of Table 2. Increasing amounts were hybridized to a fixed amount (2 pg) of heat-denatured X DXA. The specific radio-activities of the RNA preparations from heated and. cooled samples were respectively 6.76 x 10” ots/min and 7.65 x lOe/mg total RNA.

j 2J 10,000

1

I

l ye

-

Vegetative

1

/’

2

VI E 25

/

2.G -z

$

.

/

5000-

g G 3

;

Restrictel

z I 500 i3HIRNA

I 1000 kg

added

FIG. 7. Kinetics of X DNA saturation by 3H-labelled RNA’s from a thermo-inducible lysogen induced in the presence or absence of thymine. Sample A (restricted) was starved for thy-mine, and heated to 42% for 15 mm in the presence of 50 pg/ml. chloramphenicol. Its temperature was quickly lowered to 38°C and it was labelled by 4 &ml. [3H]uridine (1 pc/pmole) for 30 set, Sample B (vegetative) was heated for 20 mm to 42W, in the presence of thymine and absence of cbloramphenicol and labelled under identical conditions. Increasing amounts of RNA from A and .R were hybridized to 10 pg of denatured h DNA.

525

S. NAONO

AND

F.

GROS

(e) Comnpawdive rcmwaling properties of X-specijic RNA’s formed in the preeence of thymine or in the a,bsence of thymine plus ehloramphenicol E. co& U1858 (X857) previously starved for thymine was heated to 42°C in the presenceof ohloramphenicol and absenceof thymine following which the temperature was lowered to 38°C and the culture immediately pulse-labelled with [3H]uridine. A parallel sample of the original culture, unstarved, was directly heated in the presence of thymine and absenceof chloramphenicol, then pulse-labelled. Increasing amounts of RNA’s derived from starved or unstarved cultures were hybridized to a fixed amount of denatured h DNA. Figure 7 showsthat the saturation level was about six times greater with the unstarved RNA preparation than with the starved ones.Thus the population of mRNA made during active phage growth includes about six times more molecular speciesthan the population of mRNA induced in absenceof replication and protein synthesis. For convenience and in view of these results, we shall refer to these two categories of RNA’s as “vegetative” and “restricted” RNA, respectively. It was interesting to examine by competitive annealing studies to what extent speciesconstituting restricted RNA are present among the vegetative RNA fraction. A fixed amount of pulse-labelled RNA of vegetative type was hybridized to homologous DNA in the presence of various amounts of unlabelled restricted RNA: the level of competition was of the order of 25% (Fig. 8(a)). Conversely, an excess of

I

I

I

100

250

500

/

73 Competitor

(0)

3 added

/

I

1

I

100

250

500

750

(pg) (b)

8. Competition between restricted and vegetative X RNA’s in:hybridization experiments. (a) Pulse-labelled (vegetative) RNA was isolated from U1858 (X857) thermo-induced in 63 medium plus thymine (same conditions of labelling as for sample B of Fig. 7). 25 pg of this RNA were hybridized to 2.5 pg of X DNA in the presence of restricted uulabelled RNA. This latter RNA was prepared from the same lysogen heated for 15 min at 42% in the absence of thymine plus chloramphenicol (50 &ml.). A symmetrical competition experiment was performed using 25 pg labelled restricted RNA (prepared as for sample A in experiment of Fig. 7) in the presence of unlabelled vegetative RNA prepared as above for labelled vegetative RNA. (b) This figure shows t,he results of two experiments in which labelled restricted or vegetative RNA’s were hybridized in the presence of homologous RNA’s as cold competitors. FIG.

TRANSCRIPTION

OF

X GIENOME

520

~~&be~ed vegetative RNA competed as much as 85% for the annealing of restricted labelled RNA. Competitions between homologous RNA’s were found to amount to at least 80% (Fig. 8(b)). These results suggest ‘that nearly all the restricted RNA species are present in the vegetative RNA fraction. (f) Cbmparative e&ion beliaviour of restricted and vegetative RNA’s after hybridization on a DNA-agar column Restricted RNA was obtained from U1858 (X857) that was first thymine-starved, then heat-induced in “thymine-free” medium plus chloramphenicol and puiselabelled 25 minutes later with [3H]uridine. Vegetative RNA was made from the same strain that had been similarly starved but induced in the presence of thymine and absence of chloramphenicol, and finally pulse-labelled 25 minutes later with [‘“C]uracil. A mixture of these two RNA’s was annealed at 63°C to an excess of denatured h DNA previously immobilized in an agar column. At the end of the annealing

Vegetative

AmRNA

l4C

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2 -$ - Y) -2

AmRNA 3H

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4000-

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2000A

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1000 -

Temperature (a)

of elution

(“C) (bi

Fm. 9. Differential elution pattern of restricted and vegetative RNA’s after hybridization on a DNA-agar column. (a) A mixture of 31-I-labelled restricted and 14C-labelled vegetative RNA’s in 2 X SSC buffer was inoubated with 250 yg denatured /\ DNA previously immobilized in 60 mg of an agar ge1 maintained at 63% in a jacketed, heated column. 1.2mg(6.5 x 105cts/min/mg) of restricted RNA (15 min incubation at 42°C in the absence of thymine and in presence of chloramphonicol) and 6.8 mg (6.7 x lo* cts/min/mg) vegetative RNA (heat induction for 25 min in complete medium) were used. After a 16-hr annealing period, enough 2 x SSC preheated at 63°C was passed through the column to remove the non-hybridized RNA fractions. The temperature was then lowered to 50% and RNA eluted by adding 10 ml. of 0.1 x SSC buffer heated at temperatures between 52.5 and 80% (raising the temperature in steps of 25%). A, B and C stand :for the 3 subfractions with well-defined peaks of elution at 57.5, 70 and 75”C, respectively (see text for details). (b) This figure describes the thermal elution behaviour of sH-labelled RNA from U1858 (a%?) induced in the absence of thymiue and chloramphenicol (CAP). After 20 min of starvation for thymine (35%) the culture was quickly heated to 42°C in the same medium, and 15 min later a 1 -minpulseof[3H]uridinewas given. 1.46 mg of unfractionated RNA (1.20 x lo5 cts/miq’mg) were hybridized with 350 pg X DNA immobilized in 60 mg agar. Same conditions as those described in (a) for washings and elution.

530

S. NAONO

AND

F.

GROS

period, the non-reacting RNA was washed out by 2 x SSC buffer at 63°C. The temperature was then lowered to 51°C and elution of the annealed material carried out by applying a gradient of increasing temperatures in a range between 50 to 80°C while maintaining a fixed ionic strength (O-1 x SSC) (Bolton & McCarthy, 1964). Figure 9 allows one to compare the elution profile of restricted and vegetative RNA’s. Restricted RNA is characterized by the presence of a large proportion of molecules eluted between 55 and 57°C (fraction A) but also includes a certain amount of material that can be eluted at higher temperatures.? The behaviour of restricted RNA contrasts with that of vegetative (14C-labelled) RNA (Fig. 9(a)) whose content in molecules of type A does not exceed 15% but which comprises two main subfractions: one eluted in the range of 65 to 70°C (fraction B) and another in the range of 75 to 80% (fraction C). Finally, Fig. 9(b) illustrates an elution pattern obtained after hybridizing the pulse-labelled RNA from U1858 (X857) induced in the absence of both thymine and chloramphenicol (contrary to the conditions involved in the experiment just described where ohloramphenicol was present). Here again, as for the RNA preparation described on Fig. 9(a), material of type A appears predominant, but B and C fractions are detectable although in much smaller proportion than in vegetative RNA. The synthesis of this surplus of hybridizable material is presumably due to partial leakiness of the thymine requirement (whose effect could be overcome by chloramphenicol). The possibility exists, however, that certain genes of h prophage to be induced require protein synthesis even when DNA replication is blocked. Although we have performed no direct chemical analysis on the various subfractions A, B and C, it is known that the melting temperature of a given DNA, at a definite ionic strength, is a direct function of its GC content (Marmur & Doty, 1959) and that a similar relationship exists between the temperature at which DNA-RNA hybrids dissociate and the GC content of the RNA chain hybridized. This latter point has been largely documented by Bolton & McCarthy (1964) in studies on E. coli DNAE. coli mRNA hybrids previously formed on a DNA-agar gel column. It thus appears likely that the GC content of the various eluted fractions described above (Fig. 9(a)), increasesfrom A to C. Using the chart published by Bolton & McCarthy (1964), the following GC contents for fractions A, B and C (regarded at their peak regions) have been estimated: fraction A (57@C), closeto 40%; fraction B (70°C) 48%; fraction C (75”C), 53%. Although these calculations can only be considered as indicative, it is interesting to note that the GC content of fraction C comesout closeto that exhibited by the left-hand half of X DNA (54%), as prepared after controlled stirring of the molecule (Hogness& Simmons, 1964; Hershey, Burgi & Davern, 1965). Fraction A is poorer in GC than the right half (45*2%), and fraction B displays a composition intermediate between those of each specific half. (g) Hybridization

studies with RNA’s from early functions

phage

strains

with

defective

To investigate the nature of h-specific RNA’s synthesized in conditions where prophage replication is either completely, or at least greatly inhibited because of T In some other experiments, however, the content of this latter material appeared negligible and the hybridized RNA was recovered from the column as almost pure fraction A. These variations can possibly be explained by differences in the time required to warm up the culture to 42°C before adding chloramphenicol.

TRANSCRIPTION

2% .N L .I? *$

[3H]RNA=restricted

OF

[3HlRNA=vegetativeo 00

'\

1

IO [3~l~~~+~~ld (a)

631

X GENOME

/

/- -&----

IO RNA/[~H~RNA (b)

FIG. 10. Competition between restricted or vegetative (labelled) RNA’s and unlabelled RNA’s from lysogens carrying defective prophages. (a) 50 pg (4 x lo3 cts/min) of restricted 3H-labelled RNA from U1858 (h857) were hybridized with 2 pg of dena.tured DNA in the presence of unlabelled RNA’s from defective lysogens treated for 60 min with 3 pg/ml. mitomycin (37’C). Control experiments were also carried out, using as induced with mitomycin for 20 cold competitors early RNA from a normal X lysogen (CSOO(h)) min, and restricted unlabelled RNA from U1858 (h857). (X ) tll; (0) t27; (a) t66; (0) t75; (0) P22; (A) CSOO(h); (0) U1858 (h&57). (b) 6 pg (7 x lo3 cts/min) of vegetative sH-labelled RNA from U1858 (h857) were hybridized with P pg of X DNA in the presence of the same competitors as those described in (a).

Fm. Il. Competition between vegetative labelled RNA and unlabdled RNA’s from CSOO(X) induced with mitomycin. Vegetative RNA was prepared from U1858 01857) heated to 42% fog 25 min in the presence ofthymine, and pulse-labelledwith [3H]uracilfor 60 seo. 150 pg of vegetative RNA were hybridized with 2 pg /\ denatured DNA previously immobilized on membrane titers (Gillespie & Spiegelman, 1965). RNA from CSOO(X) induced 60 min with mitomycin, was used ae competitor (-a----@-) and its effect compared with that of vegetative unlabelled RKA (--o--o-). 36

532

S. NAONO

AND

F.

GROS

mutations in genes for early functions, we have used RNA’s from defective lysogens induced by mitomycin. The defective strains involved in our experiments had mutations in region x or in cistron N. Another strain was characterized by a large deletion including (at least) genes N and C,,. In a first series of experiments, RNA’s from each of these mitomycin-treated strains were tested for ability to compete in hybridization experiments with 3Hlabelled restricted or vegetative mRNA’s prepared from U1858 (X357). It is clear (Fig. 10) that in none of the mutant strains studied did the RNA made during a 60-minute induction period contain more than 20 to 30% of the molecular species present in vegetative [3H]RNA. On the other hand, the same RNA’s could prevent at least 70% of restricted [3H]RNA from being annealed. Control experiments were carried out using, as competitor, RNA from a strain lysogenic for a normal prophage (CSOO(X)) after 60 minutes of treatment with mitomycin. As shown in Fig. 11 the corresponding RNA competed to a 70% level with 3H-labelled vegetative RNA (albeit with different kinetics than when homologous unlabelled RNA was involved). In a second series of experiments using the Bolton & McCarthy (1964) technique, we analysed the thermal elution profiles of 3H-labelled RNA’s from two mitomycininduced defective strains which are respectively lysogenic for X tll and X t75. It is clear that hybridizable RNA from these two lysogens displays a very similar elution behaviour to that of restricted RNA from U1858 (h857) (cf. Fig, 9(a)), since fraction A appears prominent in the recovered material. (h) Temporal gene expression during

synthesisof vegetative mRNA

The question arosewhether subfractions B and C that comprise the bulk of vegetative RNA (Fig. 9) are transcribed from regions of the Xgenomethat would be expressed at different times following prophage induction. Figure 12 shows that the thermal elution patterns of h-specific RNA’s made respectively after 25 and 25 minutes heat induction in the presence of thymine are markedly different. The 2*5-minute RNA includes, in addition to molecules of the A type, many that belong to group B but practically none from group C. This last category appearsto be transcribed relatively late after the beginning of the heat treatment (25 minutes) in the presenceof thymine. 4. Discussion Thermoinduction of a lysogen carrying a h prophage, under conditions where the viral genomecan neither replicate nor synthesize proteins stimulates the rate of ARNA synthesis about lo- to 20-fold. Although induction of lysogenic bacteria involves several successivesteps (Thomas, personal communication)-and there seemto exist two sequential functions under direct Cr control, one of these is entangled with replication (Weisberg & Gallant, 1967)-de-repressionof phage RNAsynthesis is likely to represent the immediate event that follows inactivation of the C, repressor. Accordingly, using Cr B type lysogens in which heat treatment in the presence of chloramphenicol does not cause killing (Lieb, 1966) we observe that de-repressionof h RNA synthesis is a rapidly reversibleprocessat non-inducing temperature. Contrarywise, using C, A type lysogens in which thermoinduction in the presenceof chloramphenicol is lethal, the capacity to synthesize h specific RNA is maintained at maximal rate even after cooling in the presence of chloramphenicol. The extent to which the heat-altered Cr product can recover its inhibitory activity on h DNA transcription

TRANSCRIPTION

I

I

1 (til)

OF J

’

’

’

X GENOME

I

I

I

533

11-I

l----l

2000.

iooo-

/ I__l-i----__L_i / 1 / 52.5 55 575 60 62.5 65 675 70 725 75 77.5 %?= 2 E 1; .s 2

200(

iOO(

Temperature

of elution (b)

(“C)

FIQ. 12. Thermal elation proties of I\ specific defective prophages. E. COG strains CSOO(Xtll) and plus glycerol and Casamino acids were induced by this period, each culture was pulse-labelled with RNA’s were extracted and hybridized to 500 pg of

After 16hr of annealingat

RNA’s derived from lysogens carrying early W602(X t76), grown at 37°C on mineral medium 3 pg/ml. mitomycin for 60 min. At the end of 3 pa/ml. [3H]Uridine (10 mc/pmole) for 60 sec. denatured X DNA previously embedded in agar. 63”C,washingsand stepwiseelutionswere caked out as describad jin

the legend for Fig. 9* (a) 4.25 mg of h tll E3H]RNA, (b) 3-O mg of h t76 [3H]RNA

(2.18 x lo5 cts/min) (1.29 x 106 cts/min)

were hybridized were hybridized

to h DNA. to X D’NA.

thus mainly influences the “commitment” of a lysogen that has been heated in the presence of chlora,mphenicol. The RNA made in responseto prophage induction in the absenceof both DXA and protein synthesis appears by several criteria to be transcribed from a restricted portion of the prophage genome. Annealing experiments indicate that the number of genetic sites homologousto this type of RNA is about five to six times smaller than the number sf sites transcribed during late phage growth (cf. saturation and competition st Figs 7 and 8). For these reasons(and in view of the conditions of starvation involved

S. NAONO

534

AND

F. GROS

during its synthesis), we have used the term restricted RNA to distinguish mRNA formed after inducing thymine-starved lysogens in the presence of chloramphenicol, from the mRNA synthesized during vegetative multiplication. From examining the melting patterns of complementary hybrids between X DNA and restricted RNA, at gradually increasing temperatures and fixed ionic strength (Bolton & McCarthy, 1964), we can conclude that this category of RNA contains predominantly GC-poor molecules. In contrast, vegetative RNA has been resolved into three relatively well-defined fractions : (1) fraction A, which doesnot amount to more than 15% of the population, resembles closely GC-poor restricted RNA; (2) fraction B has a higher apparent GC content than fraction A and is transcribed early in the course of phage development (after 150 secondsinduction in complete medium (seeFig. 12)) ; (3) fraction C, which has a higher GC content than fraction B, is made relatively late during the vegetative cycle (Fig. 12). It is well known that the h genome contains determinants whose expression takes place, respectively, early or late in the chronology of phage development. If one considers a physiological map, all known genes necessary for phage chromosomal replication (the early genes) appear grouped in the right half of the DNA, whereas most of the sofar recognized late genesinvolved in the maturation processare located in the other half (seeFig. 1) (Dove, 1966) (with the exception of gene R coding for the phage endolysin, which is at the extreme right). Furthermore, comparative analyses of both halves of A DNA, produced by controlled stirring of the molecule, show that the right segment has a lower GC content (45%) than the left one (54%) (Hershey et al., 1965; Wang, Nandi, Hogness& Davidson, 1965). From all these considerations, it appears likely that restricted RNA is transcribed from a region containing early genes, whereas vegetative RNA is transcribed from regions containing late aswell as early genes. II

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25 min Induction

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I

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of elution

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FIQ. 13. Thermal elution profiles of X specific RNA’s from U1858 (X357) heat-induced for various periods in the presence of thymine. Two samples of the thymine-requiring lysogen (grown at 34°C) were transferred to 42’C and pulse-labelledfor 1 min with 3 &ml. [3H]uridine (4 o/m-mole), 2.5 and 25 min after heating. RNA’s were extracted from the pulse-labelled cells, annealed on a h DNA-agar column and eluted according to the usual procedure. See Fig. 9 for explanation of A, B and C.

TRANSCRIPTION

OF

X GENOME

535

The view that RNA made after prophage induction in the absence of DNA and protein synthesis (i.e. mostly what we defined as fraction A) represents early gene products is strengthened by the following observations: (a) among the RNA species thus formed is included the messenger for the production of a h-specitlc exonuclease, known to be an early enzyme; (b) the DNA-agar elution pattern of restricted RNA bears a close resemblance to that of RNA made by certain defective lysogens (X tlZ> X t75) in which induction is not accompanied by prophage replication (Radding, 1964b; Fuerst, personal communication) nor leads to the expression of late functions (Joyner, Isaacs, Echols & Sly, 1966; Fuerst, personal communication); (c) competition studies in annealing experiments indicate that RNA’s from various early defective lysogens display general sequence homology to restricted RNA from U1858 (h857). Finally (d), we have recently observed (Naono & Gros, 1966) that restricted RNA from U1858 (X857) is hybridizable with the right half of sheared X DNA but not wit the left half, whereas h RNA transcribed during vegetative deve’iopment hybridizes equally well with both halves. The present data do not allow us to specify what portion of the early region is transcribed when induction occurs in the absence of DNA and protein synthesis. That this portion is smaller than the right half of phage DNA, as a whole, is suggestedby the fact that fraction A has a lower GC content than the average content of this specific half. Work is in progress to clarify the genetic origin of restricted RNA. In any caseour observations are in good agreement with results :reported by SkaUsa (1966). She found that viral RNA synthesized a short period after infection of a sensitive host (five to seven minutes) has a low GC content (45.9%) and can only ameal with the AT rich half of h DNA; in contrast, h-specific RNA ma,de late during th.e viral growth (32 minutes) has a high GC content (54.6%) and hybridizes well with both h.alves. That our subfraction C (cf. Pig. 9) is identical to the category of RNA she defined by “‘late” is likely : this is suggestedby its characteristic GC content (53%) and late chronology of synthesis after induction. Fraction B, since it is formed early in. the course of induction (25 minutes), at a period when little replication has occurred, is likely to be transcribed from genesinvolved in early functions. Its estimated GC content (47.9%) could suggest a possible origin from the extreme right quarter of X NA whose content in this particular base pair is about 47.0% as calculated from nsity measurementsof Hershey (1963). Fraction B would then represent a classof early mRNA whose induction requires protein synthesis since it is.practically absent from speciescomposing restricted RNA.? This work was supported by the Fonds de DeveloppementdB la RechercheScientifique et Technique, the Centre National de la Recherche Scientifique, the CommissariatB !‘Energie Atomique, the Ligue Nationale Francaise contre le Cancer, the Fondation pour la Recherehe Wdicale Fraqaise, and grants from the Minis&e des Affaires Soeiales.

i 111 previous work, Geiduschek, Snyder, Colvill & Sarnat (1966), extending earlier observations by K&sin, Gorlinko, Sheyakin, Bass & Prozorov (1963) have shown that E. co& RNA polymecase selectively transcribes the early region of native T4 Dl\rA in vitro. The same observation has been recently extended by Cohen, Maitra & Hurwitz (1966) as well as by ourselves (Naono & Gross 1966) to the extracellular transcription of native A DNA. It appears therefore very likely that the host RNA polymerese displays a much higher affinity for the initiation sites commanding tba transcription of early genes than for the sites corresponding to late genes. Restrictive transcription of X DNA during the pre-replicative period (absence of thymine, chlorampheniool) conld thus lie on specific punctuation features within this DNA.

536

S. NAONO

AND

F. GRQS

REFERENCES Attardi, G., Naono, S., Jacob, F., Rouviere, J. & Gras, F. (1963). Colld Spr. Ha&. Sym/p. Quant. Biol. 28, 363. Bolton, E. T. & McCarthy, B. J. (1964). J. Mol. Biol. 8, 201. Cohen, S., Maitra, M. & Hurwitz, J. (1966). CoZd Spr. Harb. Syrq~. &ant. Biol. in the press. Dove, W. F. (1966). J. Mol. Biol. 19, 187. Eisen, H. A., Fuerst, C. R., Siminovitch, L., Pereira da Silva, L., Thomas, R. & Jacob, F. (1966). Erology, 30, 224. Geiduschek, E. P., Snyder, L., Colvill, A. J. E. & Sarnat, M. (1966). J. Mol. BioE. 19, 541. Gillespie, D. & Spiegelman, S. (1965). J. Mol. Biol. 12, $29. Green, M. (1963). Proc. Nat. Acad. Sci., Wash. 50, 1177. Green, M. (1966). J. Mol. Biol. 16, 134. Hershey, A. D. (1963). Yearb. Carnegie In&n. 63, 577. Hershey, A. D., Burgi, E. & Davern, C. I. (1965). Biochem. Biophys. Res. Comm. 18, 675. Hogness, D. S. and Simmons, J. R. (1964). J. Mol. Biol. 9, 411. Jacob, F. & Campbell, A. (1959). C.R. Acad. Sci., Park, 248,3219. Jacob, F., Fuerst, C. & Wollman, E. (1957). Ann. Inst. Pasteup, 93, 724. Jacob, F. & Monod, J. (1961). J. Mol. Biol. 3, 318. Jacob, F. & Wollman, E. L. (1961). Sexuality and the Genetics of Bacteria. New York: Academic Press. Joyner, A., Isa&w, A. L. N., Echols, H. & Sly, N. S. (1966). J. Mol. Biol. 19, 174. Kaiser, A. D. & Hogness, D. S. (1960). J. MOE. BioZ. 2, 392. Khesin, R. B., Gorlinko, Zh. M., Sheyakin, M. F., Bass, I. A. & Prozorov, A. A. (1963). Biokhimiya, 28, 1070. Lieb, M. (1966). J. Mol. BioZ. 16, 149. Marmur, J. (1961). J. Mol. Biol. 3, 208. Marmur, J. 8-z Doty, P. (1959). Nature, 183, 1427. Monod, J., Cohen-Bazire, G., Papenheimer, A. M. & Cohen, M. (1951). Biochim. biophys. A&, 7, 585. Naono, S. & Gros, F. (1966). Cold Spr. Harb. Symp. Quant. Biol., in the press. Nygaard, A. P. & Hall, B. D. (1964). J. Mol. Biol. 9, 125. Radding, C. M. (1964a). Biochem. Biophys. Res. Comm. 15, 8. Radding, C. M. (1964b). Proc. Nat. Acad Sci., Wash. 52, 965. Sicard, N. & Devoret, R. (1962). C.R. Acczd. Sci. Paris, 255, 1417. Skalka, A., (1966). Proc. Nat. Accd. Sci., Wash. 55, 1190. Sly, W. S., Echols, H. & Adler, J. (1965). Proc. Nat. Acad. Soi., Wash. 53, 378. Sussman, R. & Jacob. F. (1962). C.R. dead. SC& Paris, 254, 1517. Thomas, R. (1966). J. Mol. Biol. 22, 79. Wang, J. C., Nandi, U. S., Hogness, D. S. & Davidson, N. (1965). Biochemistry, 4, 1687. Weisberg, R. & Gallant, J. (1967). J. Mol. Biol. 25, 537.