Interference of bacteriophage T4 in the reproduction of RNA-phage M12

J. Mol. Biol. (1967) 29, 173-190

Interference of Bacteriophage T4 in the Reproduction of RNA-phage Ml2 STANLEY HATTMAN AND PETER HANS HOFSCHNEIDER

Max-Plan&-Iwtitut

fiir Biochemie, Munich, Germany

(Received 19 April 1967, and in revised form 12 June 1967) The RNA-phage Ml2 is excluded from producing progeny phage following mixed infection with phage T4 (or with T4am mutants). A small fraction of the infected cells produce some Ml2 (“escape yielders”), but the burst size is drastically reduced. The failure of most cells to liberate Ml2 is not due to an inhibition of lysis. A single T4 particle is capable of excluding M1.2; exclusion occurs in RCstr, RCrel and RNase I-less strains. In simultaneous-mixed infection, little or no synthesis of infectious single- or double-stranded Ml2 RNA occurs; furthermore, the parental Ml2 RNA does not enter the RNase-resistant structure normally observed in control infections. Exclusion does not appear to involve degradation of the Ml2 RNA: recovery of radioactivity from cells infected with 32P-labelled Ml2 alone or with 32Plabelled Ml2 and T4 is the same and the RNA remains intact. When T4 is added about twenty minutes after Ml2 infection, little or no synthesis of cellular RNA is observed, whereas Ml2 RNA synthesis occurs in the absence of infectious phage production. The Ml2 RNA does not become incorporated into biologically inactive phage particles. Synthesis of Ml2 coat protein antigen is also markedly inhibited in mixedly infected cells. It is proposed that T4 exerts its control over phage Ml2 production by interfering with the functioning of Ml2 RNA in directing protein synthesis.

1. Introduction The infection of Escherichia co.%bacteria with T-even phages is attended by spectacular changes in the synthesis of macromolecules; all host nucleic acid production comes rapidly to a halt (Cohen, 1947,1948; Volkin & Astraohan, 1956; Nomura, Hall & Spiegelman, 1960; Brenner, Jacob & Meselson, 1961; Hall & Spiegelman, 1961). The inhibition of cellular DNA and mRNA production requires protein synthesis (Hayward & Green, 1966; Nomura, Witten, Mantei & Echols, 1966). Due to the rapidity with which control is expressed, neither breakdown of host DNA (Nomura, Matsubara, Okamoto & Fujimura, 1962), nor synthesis of an inhibitor of the DNA-dependent RNA-polymerase (Skiild & Buchanan, 1964) appears to be implicated. Although the over-all rate of protein synthesis is not significantly reduced (Cohen, 1947; Hershey, Garen, Fraser & Hudis, 1954), host-cell enzymes cannot be induced following phage infection (Monod & Wollman, 1947; Benzer, 1953), and none of the proteins made before infection continues to be made after infection (Levin & Burton, 1961). Termination of cell mRNA transcription must lead to a shut-off of cell protein synthesis when the mRNA molecules present at the time of phage infection have functionally decayed. It has in fact been observed that /3-galactosidase synthesis continues for some time after T6 infection (Kaempfer, 1965). 173

174

S. HATTMAN

AND

P. H.

HOPSCHNEIDER

The present investigation was undertaken in an attempt to elucidate the mechanism by which T-even phage controls RNA and protein synthesis determined by genetic elements other than its own. Cells mixedly infected with T4 (or mutants of T4) and the RNA-phage Ml2 were chosen as a model system.

2. Materials and Methods (a) Phage and bacterial strains strain 112-12 (hh) was obtained from Dr D. Pratt; E. coli K12 strain AB301 (X) meth--RNase I-less was kindly supplied by Dr R. Gesteland; E. coli WG.Cpl19X’ meth- his- th Y - RCps* was obtained from Dr Maalee. Phage T40s was used instead of wild-type T4 (and will be referred to as T4 hereafter); T4am55 is defective in its ability to synthesize the enzyme deoxycytidylate hydroxymethylase (gene 42) in those strains which do not carry a specific suppressor mutation (Epstein et al., 1963); T4am50 (a mutant in gene 20) synthesizes DNA and lyses non-permissive hosts (Epstein et al., 1963); Ml2 was from our own stock (Hofschneider, 1963).

E. coli K12

(b) Media and chemicaZs F-medium is the medium of Fraser & Jerrel (1953). D-medium has been described previously by Francke & Hofschneider (1966). Synthetic medium for 35S incorporation contained: 3 g KH,PO,; 7 g NaaHPO,; 0.5 g NaCl; 1 g NH&l; 4 g glucose; 11 mg &Cl,; 120 mg MgCls and 1 g Casamino acids in 11. of water. Tris buffer: 1.21 g Tris, 0.47 g sucoinic acid, 2.46 g MgS04,7Hz0/1. Phosphate buffer: 0.05 M-phosphate buffer (pH 7.0) + 0.15 M-NaCl + 0.01 M-MgSG,. Acetate buffer: 7.7 g ammonium acetate/l. and adjusted to pH 5 with HClO,. as sulphate were purchased from [5-3H]Uridine (3.54 c/m-mole) and 36S (carrier-free) The Radiochemical Centre, Amersham, England. [32P]Orthophosphate (carrier-free) was obtained from Hoechst, Frankfurt/M. in Germany.

(0) Preparation of crude Zysates Samples of infected cells were transferred to thick-walled Servall tubes in the cold and lysozyme added to O-4 mg/ml. The samples were frozen and thawed 3 times in a methanoldry ice bath; lysis was completed by adding sodium dodecyl sulphate to O-1 to 0.2% and shaking. Further clarification was obtained by low-speed centrifugation in the cold. The supernatant fractions were stored at -20°C. Ml2 phage was not inactivated by freezing and thawing. In all experiments to be reported, exponentially growing cells were infected at a titre of 1 to 3 x lO*/ml. with 5 to 10 phage particles per cell. (d) Extraction

of nucleic acids

3- to 6-ml. samples of infected cells were diluted three- to sixfold in cold Tris buffer in plastic Servall centrifuge tubes and sedimented 2 or 3 times at 10,000 rev./min for 10 min at 0°C. After the last oentrifugation, the pellets were resuspended in 5 ml. cold Tris buffer and transferred to thick-walled glass Servall tubes (all subsequent operations were performed in the cold). Lysis was carried out as described in (0); immediately after lysis was observed, 4 to 5 ml. of phenol (saturated with Tris buffer) and 20 mg Frankonit were added. The tubes were shaken for 10 min and then centrifuged 5 min at 10,000 rev./mm; the aqueous phase was removed and extracted again with phenol. After the second extraction, the aqueous phase was mixed with one-tenth vol. of 10 x acetate buffer and 2 vol. of ethanol and placed at -20°C for at least 2 hr. The samples were then centrifuged at 0°C for 30 min at 13,000 rev./mm The pellets were resuspended in 2 to 3 ml. acetate buffer and stored at -2O’C. (e) Fractionation

of Ml2

single- and double-stranded RNA by sezective adsorption to methyZuted albumin silicic acid

This procedure has already been described (Francke & Hofsohneider, tion was accomplished by adsorption in 1.1 M-salt (1.08 M-sodium sodium acetate, pH 5.0).

1966). Fractionachloride in 0.02 M-

.TC INTERFERENCE (f) Lysosyme

WITH

Ml2

175

spheroplasts; biological assay for infectious RNA

Preparation of spheroplasts and Francke & Hofschneider (1966).

assys

for

infectivity

were

performed

according

to

(g) Preparation of labelled Ml2 3H-labelled Ml2 was the generous gift of Mr Bertold Francke, and prepared as follows: E. coli K12 112-12 was grown to 2 x 108/ml. in D medium and infected with M12. [6-aH]Uracil was added to 100 +/ml. and incubation continued for 4 hr at 37°C; sodium dodecyl sulphate was added to complete lysis. Debris was removed by low-speed sedimentation and the supernatant fraction dialysed overnight at 4°C against 0.1 M-Tris+ 0.1 MNaCl (pH 7.5). MgSO, and DNase were added to the dialysate to final concentrations of 1 mg/ml. and 20 pg/ml., respectively. After 30 min incubation at 37”C, the mixture was dialysed again as described above, CsCl was added to p = 1.45 to 1.46 g/ml. and the mixture centrifuged for 24 hr in a Spinco model L SW39 rotor, 37,000 rev./min at 15°C. The tubes were punctured, fractions collected and diluted in the same buffer. Portions were taken to measure 3H and active phage; the Ml2 peak fractions were pooled and dialysed as before. The lysate contained 6 X lOlo active M12/ml. and 1.2 x lo5 cts/min/ml. E. coli AB301 was grown overnight in 32P-labelled Ml2 was prepared as follows. medium D containing additional inorganic phosphate to 0.01 M. A fresh culture was grown after a 1: 80 dilution into 30 ml. fresh medium D. The cells were infected at a concentration 32P-labelled orthophosphate was added to give of 2 x 108/ml. with about 10 to 20 MlB/cell. a concentration of about 10 PC/ml. and the cells were incubated for 4+ hr at 37%. The unlysed cells were sedimented for 10 min at 15,000 rev./mm; the supernatant fraction was collected and the pellet resuspended in 3.0 ml. TM buffer (0.01 M-Tris + 0.005 n/r-MgSO,, pH 7.5) and sodium dodecyl sulphate added to 0~5% to complete lysis. After centrifugation, the supernatant fraction was combined with the one from above and 2.6 g ammonium 1 + hr standing at 4”C, the precipitate was sulphate was added/l0 ml. of lysate. After harvested by centrifugation in the cold and resuspended in 2 ml. TM buffer. The sample was then dialysed overnight against TM buffer in the cold. After DNase was added, the solution was incubated for 30 min at 37°C. Samples (about 1.8 ml.) were layered on a 26-ml. 5 to 20% sucrose gradient ( 10-4~-Mg2+) and centrifuged in a Spinco model L SW25 rotor for 4 hr at 24,000 rev./min in the cold. The tubes were punctured and 30 fractions collected; 05 ml. of 0.01 M-phosphate buffer (pH 6.8) was added to each fraction. Portions were taken for determination of 32P- and Ml2-activity (the latter by spot tests of serial dilutions). The Ml2 peak fractions were pooled and dialysed against the same phosphate buffer; the final lysate was at 2.4 x lOlo M12/ml. and 7 x IO4 cts/min/ml. Both the 3H-labelled and 32P-labelled Ml2 lysates formed single homogeneous bands in sucrose gradients. (h) Preparation

of 32P-labelled

ribosomal RNA

E. coli AB301

was grown overnight in F medium containing 3.3 x low4 M-inorganic phosphate and 1 put/ml. 32P. The cells were collected by filtration, washed, resuspended in cold Tris buffer, and disrupted by sonication for 2 min in an MSE ultrasonic disintegrator. Debris was removed by 60 min centrifugation at 12,000 rev./min. The supernatant fraction was collected and centrifuged again for 120 min at 50,000 rev./min in a Spinco model L R50 rotor. The pellet of ribosomes was resuspended in cold Tris buffer and extracted with phenol according to the procedure detailed in (d) above. The preparation was stored at -20°C. (i) Sazrose gradient

centrifugation

26 ml. 5 to 2O”/0 (w/v) linear sucrose gradients were used in all cases. For centrifugation of nucleic acids, the sucrose was buffered in tenfold diluted acetate buffer containing 0.1 M-N&cl f 10-e M-MgSO,; for centrifugation of crude lysates, the sucrose was not buffered and contained LO-4 M-MgSo,. Crude lysates were centrifuged for 54 hr and nucleic acids for 134 to 14 hr at 25,000 rev./min in a Spinco model L SW25 rotor at 0 to 5°C. Tubes were punctured and drops collected. Cold trichloroacetic acid was added to 5%, and aft,er standing in the cold for at least 30 min, the contents were filtered on to membrane filters (Group 50, Gtittingen), washed, dried and counted.

176

S. HATTMAN (j) %S-label&g

AND

and immune

P. H.

HOFSCHNEIDER of Ml2

precipitation

coat protein

Samples of 4 to 5 ml. of uninfected or infected cells labelled with 35S were chilled, and 0.1 ml. of 0.5 M-MgSo+ + 0.1 ml. 20 mg lysozyme/ml. were added. The cells were frozen and thawed 3 times in methanol-dry ice and lysed by adding sodium dodecyl sulphate to 0.05%. After centrifugation at 15,000 rev./min for 30 min in the cold, the supernatant fractions were collected and stored at -20°C. Samples of 0.2 ml. and 0.4 ml. were pipetted into conical serum tubes containing 1-O ml. cold phosphate buffer; 0.05 ml. of carrier Ml2 (1 x lOI2 active phage/ml.) + 4.0 ml. cold phosphate buffer were then added. 0.15-ml. portions of anti-Ml2 serum exhausted against E. coli (K N 400) were added and the tubes incubated overnight at 0 to 4°C. After 30 min centrifugation at 4500 rsv./min in the cold, the supernatant fractions were discarded and the pellets resuspended in cold phosphate buffer. The samples were centrifuged again, and the pellets were resuspended in 0.5 ml. phosphate buffer; then 5.0 ml. cold 5% trichloroacetic acid + 0.01 M-MgSO, were added. After another cycle of centrifugation, the pellets were dissolved with 3 drops of 2 N-NaOH and precipitated with 5 ml. cold 5% trichloroacetic acid + 0.01 M-MgSO,. The samples were filtered on to membrane filters, washed, dried and counted. The amount of label precipitated was directly proportional to the amount of lysate added. Addition

of excess unlabelled cipitation.

Non-immune

lysate

from uninfected

cells did not reduce the non-specific

serum gave even lower (k) Cozcnting

levels

of non-specific

pre-

precipitation.

of radioactivity

3H, z2P and a55 were counted in a Packard Tri-Carb scintillation toluene scintillator was used in all experiments (3.6 g of 2,5-diphenyloxazol 1,4-bis-2-(4-methyl-5-phenyloxyzoyl)-benzol per litre).

spectrometer. A and 45 mg of

3. Results (a) General features of the exclusion system Preliminary experiments showed that addition of phage T2 or T4 to MlB-infected cells led to an almost complete inhibition of Ml2 progeny phage formation. This by T-even phages confirmed previous reports that the RNA phage f2 is excluded (Zinder, 1963; Neubauer & ZBvada, 1965). There is no loss of T phage-producing capacity as a consequence of mixed infection. Amber mutants of T4 that are unable to synthesize DNA in non-permissive host cells are also capable of excluding M12. Thus, the inhibition of Ml2 phage production is not necessarily due to T4-induced lysis prior to Ml2 maturation. Examination of the exclusion frequency as a function of T4am multiplicity (Fig. 1) showed that adsorption of a single particle is sufficient to establish exclusion. A small fraction of the infected cells, variable from experiment to experiment, is still capable of producing M12. That these cells are probably mixedly infected is indicated by the marked reduction in the average burst size of Ml2 (Table 1). Thus, T4am may exclude Ml2 either completely or depress its yield markedly. Whether these two effects are due to the same or different mechanisms was not investigated. Other experiments showed that exclusion of Ml2 by T4am occurs in RW strains (Borek, Ryan & Rockenbach, 1955; Stent & Brenner, 1961) as well as RCstr strains. This suggests that exclusion is not exerted through an inhibition of RNA synthesis by a process mimicking amino acid starvation in RCSt’ strains, even though RNAphage growth is subject to the same controls that regulate RNA synthesis in uninfected cells (Friesen, 1965). Effective exclusion and yield depression are also observed in cells lacking the enzyme RNase I. This rules out the possibility that T4 infection causes this enzyme

Multiplicity

Fro. 1. Excluding

and killing

ability

of infection

of T4am55 as a function

of its multiplicity

of infection.

E. coli K12 112-12 was grown to about 2 x108/ml. in F medium. Samples of 0.9 ml. were distributed into 8 Servall tubes at room temperature; at intervals of 30 see, O.l-ml. samples of various phage mixtures were added and the tubes transferred to 37°C. After 8 min adsorption, 4 ml. of cold medium was added and the samples sedimented in the cold. The supernatant fractions were collected, shaken with a few drops of chloroform and the unadsorbed phage determined; the pellets were resuspended in 1.0 ml. of anti-Ml2 serum (K - 2) and incubated 7 min at 37”C, after which’time 4 ml. of cold medium was added. Serial dilutions were made in the cold and samples taken for measurement of colony formers or Ml2-infective centres. All values are normalized to 100% for the Ml2 infection without added T4am. It should be noted that a large proportion of cells survive Ml2 infection as colony formers, even though they ultimately yield phage. -O-O-, Colony formers; - X-X -, infective oentres.

1

TABLE

Ml%yielders, Infection

colony formers and progeny Ml2

Adsorbed phad cell

Ml2

4.5

Ml2 + T4am55

5.6

in control and mixedly

M12yielding cells/ml.

Relative per cent yielders

Colonyforming cells/ml.

6.3 x lo7

100 (39)

1.2 x 108

8.3 x 106

1.3

9.5 x 105

infected cells

Relative gr;Igt;s

Average izj2:

22”

100 (75)

5.4 x 101’

8570

0.8

3.8 x 107

46

IO.0

E. co&i K12 strain 112-12 was grown to 1.7 X lO*/ml. in F medium. Portions of 0.9 ml. were added to centrifuge tubes at 37°C containing 0.1 ml. of the indicated phages. After 7 mm adsorption, 4.0 ml. of cold medium was added and the cultures sedimented in 0°C. The supernatant fractions were collected, shaken with a few drops of chloroform and eesayed for unadsorbed free phage. The pellets were resuspended in 1.0 ml. anti-Ml2 serum (K N 1 to 2) and incubated for 3 mm at 37°C; the tubes were chilled and 4.0 ml. cold medium wss added. Samples were either diluted serially in cold medium or into warmed medium (1: 1000) at 37°C. Portions of the cold dilutions were either plated with indicator bacteria to measure MlB-yielders, or spread on agar plates to measure colony formers. The samples diluted into warm medium were incubated 2 hr with aeration. After adding several drops of chloroform, the cultures were assayed for Ml2 progeny phage. The values shown above were determined from these assays. The figures in parentheses are the percentage yielders and survivors in the control infection based on the colony-former assay of cells just prior to infection; for convenience, the values for the control infection are normalized to 100%.

12

178

S. HATTMAN

AND

P. H.

HOFSCHNEIDER

to degrade Ml2 RNA; it will be shown later that exclusion does not involve destruction of the Ml2 RNA (see section (e) of Results). (b) Does exclusion involve interference with Ml2 RNA penetration? It is known that following adsorption of phage f2 and prior to cell penetration, the RNA is sensitive to exogenous RNase (Loeb, 1961; Valentine & Wedel, 1965). Whereas T4 does not interfere with Ml2 adsorption, it is conceivable that exclusion may be established during the RNase-sensitive phase. To test this possibility, Ml2 was adsorbed to cells in the cold, a condition which prevents RNA injection (Valentine & Wedel, 1965). The culture was then warmed to 37°C and portions transferred at intervals into anti-Ml2 serum containing either RNase, T4am or medium. After a suitable interval, the samples were chilled and diluted in the cold for plating before lysis. As can be seen from Pig. 2, there was a rapid increase in resistance to the presence of exogenous RNase. By 10 minutes after warming to 37”C, all the potential 100 A-A I -k /

Minutes

after

dilution

to 37’C

FIG 2. Kinetics of Ml2 penetration as measured by RNase resistance, E. coli AB301 was grown to 4x lOs/ml. in F medium. Cells and Ml2 were equilibrated at icebath temperature and mixed; after 10 min adsorption, an equal volume of warmed, fresh medium was added and aeration begun (t = 0) at 37°C. At 1, 5 and 10 mm, triplicate l.O-ml. samples were removed and mixed with 1.0.ml. volumes of medium containing anti-Ml2 serum (.K N 5 to 10) supplemented either with RNase (100 pg/ml.), T4am50 (4 x lOe/ml.), or F medium. At 20 min, 8.0 ml. cold medium was added and serial dilutions made at ice-bath temperature. Appropriate samples were plated for MlB-infective centres. Note that in the control the number of infeotive centres remained constant, indicating that adsorption was completed in the cold and that -A--A-, Control; - x-x -, RNase-treated; adsorbed Ml2 are not affected by anti-serum. - @-- l --, T4am50 superinfected.

Ml2 yielders were recoverable, suggesting that at least one functional Ml2 chromosome had penetrated each of these cells. Nevertheless, there was no appreciable increase in the proportion of escape-yielders; addition of T4am at 10 minutes still produced exclusion in well over 95% of the cells. These results show that exclusion probably occurs at some intracellular stage. Superinfection at later times was carried out to determine whether Ml2 becomes refractory to the effects of T4 at some later phase of its replication cycle. As shown in

T4 INTERFERENCE

WITH

Ml2

179

Table 2, superinfection with T4am55 as late as 25 minutes after Ml2 still produced a big reduction in the number of phage-producing cells and average burst size. Thus, there is no early MlS-specific intermediate structure(s) which, when formed, allows escape of exclusion. TABLE 2

Kinetics of Ml2 e.scupeof exclusion in cells superinfected with T4am55 Superinfection time (mm)

5 10 15 20 25 -

Infective centres

1.4 x 5.5 x 9.0 x 1.4 x 3.0 x 6.0 x

106 105 10s 106 10s lo7

Relative per cent yielders

2.2 0.9 1.4 2.2

Progeny @age

1.5 x 9.5 x 7.0 x 1.1 x 2.3 x 8.0 x

107 106 107 108 log 1O1’

Average burst size

Relative per cent burst

11 17 78 75 786 1.33 x 104

0.09 0.1 0.6 0.6

E. coli AR301 was grown to log phase in F medium, infected with Ml2 and divided into 6 cultures at 37°C. At the times indicated, T4am55 was added to each culture; anti-Ml2 serum was added to each tube at 12 min (K final N 1 to 2). At 29 mm the tubes were chilled in an ice bath and 4 vol. cold medium added. Samples were taken and diluted 1: 100 into fresh medium at 37°C; after 2Q hr incubation, lysis was performed by adding sodium dodecyl sulphate to 0.4%. Infective centres were assayed before lysis by plating appropriate dilutions (kept in the cold); progeny phage were assayed from the sodium dodeoyl sulphate lysates. All assays were performed on Al3301 as indicator.

It should also be noted in the experiment above (Table 2), that lysis of the cells was completed by adding sodium dodecyl sulphate to the cultures at 2=Jhours. Since this treatment did not liberate large amounts of progeny M12, it can also be concluded that exclusion is not an inhibition of cell lysis. (c) Infectious

Ml2 RNA synthesis in simultaneous mixed infections

The results described in the preceding sections concern only the effect of T4 on the capacity of Ml2 to synthesize biologically active phage. Experiments were then carried out to determine whether exclusion affected Ml2 RNA production. For this purpose, use was made of the ability of Ml2 RNA to infect protoplasts. The results shown in Table 3 demonstrate that T4 and T4am strongly inhibit the formation of both infectious single-stranded and double-stranded Ml2 RNA. (d) Fate of parental Ml2 RNA Nucleic acids were extracted from cells 15 minutes after infection with 32P-labelled Ml2 and analysed for the amount of acid-insoluble label and by sucrose gradient sedimentation (Fig. 3). The recoveries of parental label were the same for both control and mixed infections, indicating that no extensive degradation to acidsoluble material occurred. The gradient profiles depicted in Fig. 3(a) and (b) show that the parental RNA from the mixed infection sedimented homogeneously, even though exclusion was observed in 97% of the cells. That the 32P-label recovered from both cultures consists of uniform intact phage RNA molecules was shown in a separate experiment. A sample of the 32P-label analysed in Fig. 3(a) was sedimented with

S. HATTMAN

180

AND

P. H.

HOFSCHNEIDER

TABLE 3

Measurement of infectious RNA formation in Ml2 and mixed infectionsT

Phage(s)

Exp.

Infectious singlestrandedt RNA titre

Per cent infectivity relative to control infection

Infectious doublestranded$ 3 RNA titre

Per cent infectivity relative to control infection

1 Ml2 Ml2 + T4

Exp.

Time after infection (min)

1.0

N2X106 6~10~ 2.2 x 10s

- 0.9 (10’07

< 106 -8X106 -1x107

- 3.6 - 4.5

-3X106 5x107

-

10 20 30

3.5 x 109 2.4 x lOlo 3.5 x 1011

10 20 30

6.4 x lOa 4.9 x 109 1.6 x lOlo

0.2 1.3 4.5

15 25

1.6 x lo9 4.8 x 1010

(l”o”o)

15

6.4 x lo-’

0.1

< 106

-

25

4.4 x 108

0.3

-3X106

~6.0

(1:;

2 Ml2 Ml2 + T4am50

6.0 (100)

t Exp. 1 Ml2-yielding cells/ml.: control = 3.5 x 107; mixed infection = 3.6 x 10s; relative yielders = 10.2%. Exp. 2 M12-yielding cells/ml.: control = 1.4 x 107; mixed infection = 4.7 x 106; relative yielders = 3.3% $ Calculated from the infectivity efficiency of a standard Ml2 RNA preparation (plaques/active Ml2 phage before RNA extraction) in the protoplast assay (= 1.3 x 10-s in the above experiments). The figures listed are corrected for dilutions and given as titres in the original infected culture. § Double-stranded RNA was separated from single strands by selective adsorption to methylated albumin silicic acid (see Materials and Methods). Prior to heat den&u&ion, the doublestranded form is non-infectious: the values here correspond to the infectious RNA titres measured after heat treatment.

3H-labelled RNA isolated from M12-infected cells. The profiles obtained (Fig. 3(c)) show that the [32P]RNA sediments at about 27 to 28 S; this is the sedimentation coefficient expected for intact Ml2 RNA (Delius, 1966). In other experiments, deproteinization was omitted and lysates were analysed directly in sucrose gradients. No evidence for Ml2 RNA degradation was obtained (manuscript in preparation). These observations still do not rule out the possibility that a very small amount of degradation of parental Ml2 RNA may occur (e.g. at the end(s) of the molecule) which does not significantly affect the sedimentation properties of a resistant core. (e) Conversion of parental RNA to an RNase-resistant form The results described above indicate that whereas the parental Ml2 RNA appears to remain intact during exclusion, there is no extensive synthesis of infectious progeny RNA. To determine whether any replicative intermediates are synthesized, the conversion of parental-labelled RNA to an RNase-resistant form was studied.

T4 INTERFERENCE I

800

I

1

I

1

WITH

Ml2

I

I

181

(a)

ai 160 F J ” 120 ”

fraction

80

.?? .;

40

g

no.

FIG. 3. Sucrose gradient analysis of nucleic acids isolated from cells 15 min after infection with 32P-labelled Ml2 alone (control) or with 3ZP-labelled Ml2 and T4am.55. E. coli AB301 was grown to 2 x lO*/ml. in F medium and infected with 32P-labelled Ml2 in the presence or absence of T4am55. Samples were taken at 7 min for biological assays; in the mixed infection, only 3% of the cells produced M12, relative to the control. At 15 mm, the cultures were diluted 1: 3 into cold Tris buffer; washing, extraction and centrifugation are described in Materials and Methods. Included in Fig. 3(c) is LLseparate centrifugation made with an artificial mixture of the 32P-labelled Ml2 control nucleic acid fraction and one from MIB-infected cells incubated in the presence of E3H]uridine. (a) “cP-labelled Ml2 control; (b) 32P-labelled Ml2 + T4czm55~; (c) --O--O--, 32P-labelled cells incubated with [3H]uridiie. Ml2 control; -X - x -, MlB-infected

Cells were infected with 3H-labe.Ued Ml2 in the presence and absence of T4am50, and samples were taken at various intervals. The nucleic acids were prepared and assayed for total acid-insoluble and RNase-resistant acid-insoluble 3H-label. The results depicted in Fig. 4 show that T&m markedly depresses (or completely eliminates) the synthesis of any RNase-resistant structure(s) containing parental Ml2 RNA. The kinetics observed in the control Ml2 infection are similar to those reported for the RNA phage R17 (Erikson, Fenwick & E%anklin, 1964). As in the experiment described above (section (d)), recoveries of parental label in the nucleic acid fractions from control and mixedly infected cells were not significantly

182

S. HATTMAN

AND

Time after

P. H.

HOFSCHNEIDER

M 12 Infection

(min)

FIG. 4. Kinetics of conversion of 3H-labelled Ml2 RNA to an RNase-resistant form innormal and mixed infections. E. wli 112-12 was grown to 4 x lOs/ml. in D medium and infected with 3H-labelled Ml2 alone or in combination with T4u~50. At the times indicated, samples were removed and diluted 1: 5 into cold 0.3&r-NaCl containing 0.003~-EDTA and kept in ice-water until all samples were collected. The cuhures were washed 3 times in the cold by centrifugation in N&Cl-EDTA solution. The cells were lysed with 1% sodium dodecyl sulphate and extracted 3 times with phenol at room temperature; after the third extraction, the aqueous phase was removed and precipitated overnight in ethanol at -20°C. After sedimenting 30 min at 10,000 rev./mm in a refrigerated Servall centrifuge, the pellet was resuspended in 0.003 M-EDTA and stored at -20°C. Measurement of RNase-resistant 3H label was as follows: 1.0 ml. of sample was mixed at 37% with 1.0 ml. of solution containing 4 pg RNase/ml. in 0.02 u-MgCl, + 0.2 ivr-NaCl + 0.02 x-sodium citrate and incubated for 25 mm. The reaction was stopped by adding cold trichloroacetic acid to 5%. The acid-insoluble 3H label was then filtered on to membranes, washed, dried and counted. 100% in the Figures would correspond to 700 &s/mm. The results shown are from two independent expsriments: - x-x -, 3H-labelled Ml2 control; -O--O-, 3H-labelled Ml2 + T4am50 at t = 0; ----A.--, “H-labelled Ml2 + T4am50 at 2 min.

different. Furthermore, since the washing procedure used prior to phenol extraction would have been sufficient to remove even extracellularly adsorbed Ml2 (Ippen & Valentine, 1965; Valentine, Wedel & Ippen, 1965), the label must have been recovered from intracellular Ml2 RNA. Therefore, these observations suggest that one of the earliest observable sites of T4 interference with Ml2 replication in simultaneous mixed infection is an inhibition of the synthesis of Ml2 double-stranded RNA. (f) Synthesis of Ml2 RNA irt superinfected cells The interference with synthesis of Ml2 double-stranded RNA cannot be the sole process by which exclusion of Ml2 by T4 is mediated. This is evident from the experiments described in Table 2. Exclusion is still effectively established when T4am superinfection occurs at times when Ml2 double-stranded RNA synthesis is already in progress. It was of interest, therefore, to determine whether MlS-specific RNA synthesis could occur in cells superinfected with T4am at times when double-stranded RNA (as well as the specific RNA-synthesizing enzyme(s)) is already present. The patterns of RNA synthesis in cells infected with Ml2 alone or superinfected at various times were examined in sucrose gradients (Fig. 5). For the Ml2 control infection, one sees

T4 INTERFERENCE

WITH

Ml.2

183

the typical 4, 16 and 23 s cell RNA peaks, plus the Ml2 RNA peak at 27 to 28 s (Fig. 5(a)). This pattern is similar to that shown with R17 infection (Paranchych & Ellis, 1964) under conditions where there was no interference with cellular RNA production. With T4am superinfection at 1 minute, an almost complete suppression of cellular and Ml2 RNA synthesis occurred (Fig. 5(c)). On the other hand, addition of T4am at 21 minutes did not eliminate MlS-specific RNA formation (Fig. 5(b)), although exclusion occurred in 99% of the cells. It should be noted that [3H]uridine was added at 30 minutes; i.e. 9 minutes after infection with T4am. This eliminates the possibility that what is observed is merely residual Ml2 RNA synthesis occurring during a lag in the expression of exclusion. 2000

1 (a)

ISOO-

/

1

I

I

I

I

I

805

xx - 600 II

- 200 - 100 ",Q 0 30

20 Fraction

40

no.

FIQ. 5. Sucrose gradient analysis of RNA synthesized in Ml2 and T4orn superinfected cells. E. co& AR301 was grown to 2 x lOs/ml. in F medium, infected with Ml2 and divided into 3 equal portions. At 1 mm, one of the cultures was superinfected with T4am55, and at 21 min a second culture was superinfected. Relative to the control infection, only 0.3% of the 1 min- and 0.7% of the 21 mm-superinfected cells produced M12. [5-sH]lJridine was added to 0.4 @/ml. at 30 min and cold uracil (100 pg/ml.) was added at 45 min. At 50 min, the cells were harvested in the cold by centrifugation, washed, and nuc]eic acids prepared as described in the Materials and Methods. Samples were taken for determination of incorporation of 3H into acid-insoluble material or for infectious RNA. Normalized to the control infection, the relative incorporation of “H was about 1.7 and 15%, and the infectivity for protoplasts was ~0.4 and 25% for the early and late superinfections, respectively. Sucrose gradients were run as described in Materials and Methods; aaP-labelled ribosomal RNA was included in each tube as a marker. (a) 0.1 ml. of the Ml2 control; (b) 0.4 ml. of the 21-mm superinfection; (c) 0.5 ml. of the I-min superinfection. Radioactivity: 3H counts, - x-x-; 32P oounts, -o--o--.

184

S. HATTMAN

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Assays for infectious RNA at 50 minutes showed infectivities of 0.4 and 25% for the superinfections at 1 and 21 minutes, respectively, relative to the control. Since the amount of infectious RNA present at 20 minutes is only a small fraction of that synthesized from 20 to 50 minutes in controls, then the infectious RNA measured at 50 minutes represents net synthesis of Ml2 RNA. From the sucrose gradient profiles shown in Fig. 5, one can determine the amount of 3H-label incorporated into Ml2 RNA. The ratio of infectious RNA to MlS-specific tritium label was essentially the same for both the control and Sl-minute superinfected cultures. It is also concluded that there is no large-scale production of non-infectious Ml2 single-stranded RNA in the superinfected cells. That normal Ml2 RNA is produced in the cells super-infected with T4am at later times lends support to the notion that parental Ml2 RNA remains intact during exclusion in simultaneous mixed infection. It should be pointed out that, in the culture superinfected at 21 minutes, about 10% of the RNA produced was RNaseresistant; thus, double-stranded Ml2 RNA is also synthesized under these conditions. Yarosh & Levinthal (personal communication), studying mixed infections with MS2 and T4, observed no net synthesis of infectious MS2 RNA following T4 superinfection at 20 minutes; in fact, they measured a consistent loss of infectious RNA activity after addition of T4. This phenomenon is apparently not a consequence of the T4 adsorption process, since the presence of chloramphenicol at the time of T4 superinfection inhibits the loss of MS2 RNA infectivity. We have confirmed the initial reduction of infectious RNA infectivity in the M12-T4 system (the rate and extent of decrease was extremely variable, and sometimes not observed); nevertheless, there is a rapid rise in the production of infectious Ml2 RNA starting five to ten minutes after addition of superinfecting T4am. Thus, the long-term experiments described above, using [3H]uridine labelling and infectivity measurements, would not have detected the transient effect of T4am superinfection on Ml2 RNA synthesis. Perhaps the most striking features of these results is that cellular RNA synthesis was strongly inhibited in both superinfected cultures. The fact that cellular and Ml2 RNA syntheses become uncoupled under these experimental conditions is an indication that multiple control mechanisms are operative under the direction of the T4 genes. (g) Does the infectious Ml%progeny RNA made in late-superinfection become incorporated into non-viable particles?

In an experiment analogous to that above, T4am55 was added to MlS-infected cells at 19 minutes and i3H]uridine was added at 35 minutes. At 60 minutes samples were taken for either extraction with phenol or for crude lysates; the crude lysates were further clarified by two low-speed centrifugations. The nucleic acids exhibited a sedimentation pattern similar to that shown in Fig. 5. Biological assay of the lysates showed that the yield of active Ml2 phage in the mixed infection was only O*2o/o of the control at 60 minutes; however, the yield of infectious RNA after phenol extraction was 17% of the control. These results confirm that infectious Ml2 RNA synthesis can occur under appropriate superinfection conditions, although no viable phage particles are made. To characterize further the sedimentation properties of the RNA produced in the presence of T4am, the 3H-labelled crude lysates were examined by sucrose gradient centrifugation. In the superinfected cells, no detectable 3H-label is found under the

T4 INTERFERENCE

WITH

Ml2

185

phage peak (I) or under the 50 s ribosome peak (II) of the control (Fig. 6). All label is localized under the peak (III) corresponding to a mixture of 28 s and 23 s RNA, as well as 30 s ribosomes, which are not separated in the short time allowed for centrifugation.

5000 -

4000.c F II) 5 s x Y .z ” .-x

3000-

2

2000-

iooo-

Fraction

FIG. 6. Sucrose gradient

analysis

no.

of crude lysates from M12- and T4amsuperinfected

cells.

E. COGAl3301 was grown and infected as described in the legend to Fig. 5. At 19 min, one-half the culture was superinfected with T4anz5.5; at 35 min, [&sH]uridine was added to each culture to 0.6 to 0.7 PC/ml. In the superinfected culture, the MILLyielder frequency was 6% that of the control infection. At 60 min, samples were taken for either production of crude lysates or preparation of nucleic acids (see Materials and Methods). The nucleic acids were assayed for infectious RNA, and the crude lysates assayed for acid-insoluble 3H and Ml2 phage. Taking the control values as loo’+& the following values were obtained for the superinfected culture: infectious RNA = 17%; Ml2 progeny phage = 0.2%; sH incorporated = 18%. Shown in the Figure above are the superposed distributions of 3H label after sedimentation of the crude lysates in sucrose gradients (see Materials and Methods). Ml2 phage (peak I) was located in separate experiments using purified sH-labelled Ml2 as a marker. - x - x -, Control Ml2 infeotion; --m--m--, superinfection.

An additional experiment was done to determine whether transfer of Ml2 RNA into inactive particles might occur at times later than 60 minutes. M12-infected cells were superinfected with T4am at 19 minutes, and [3H]usidine was added at 35 minutes. At 60 and 120 minutes, lysates were made from. the control- and superinfected cultures. The results of sucrose gradient analysis of these samples are shown in Fig. 7(a). In the superinfected culture, the final yields of active Ml2 phage and of

186

S. HATTMAN

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HOPSCHNEIDER

total 3H incorporation at 120 minutes were 0.5 and 20%, respectively, relative to the control. As in Fig. 6, no 3H label is observed under the phage peak. From the 120-minute lysates, nucleic acid fractions were prepared and analysed by sucrose gradient centrifugation (Fig. 7(b)). The majority of 3H label from the superinfected cells sediments as normal phage RNA. It is concluded that the Ml2 RNA synthesized in late superinfections does not become incorporated into inactive particles of size similar to Ml2 phage. It is not excluded that highly labile defective particles are formed which are dissociated by the lysis procedure employed here. An alternative explanation for the absence of Ml2 particle formation is that maturation is prevented by an inhibition of coat protein synthesis.

2500 b)

fraction

I q % I: (II: 8,

/

,

no.

FIG. 7. Sucrose gradient analysis of crude lysates and nucleic acids from Ml2 control and T4am superinfected cells. The procedure is essentially the same 8s that described iu Fig. 6. The relative Ml2 yielder frequency = 3%; relative 3H incorporation = 26%; relative Ml2 progeny phage = 0.5%. Shown in (a) are the superposed distributions of 3H label of the crude lysates in sucrose gradients; in (b) are the superposed distributions after centrifugation of the 3H-labelled nucleic acids (see Materials and Methods). Control Ml2 infection: -x-x-, at 60 min; and --O--O---, at 120 mm. Superinfection: --m--m-, at 120 min. Centrifugation was as described in section (i) of Materials and Methods.

(h) Are Ml2-speci$c proteins synthesized? To study whether T4 infection also affects MlZ-specific protein synthesis, the formation of Ml2 coat protein antigen was measured by 35S-labelling and immuneserum precipitation. The results summarized in Table 4 show that addition of T4am50 inhibits the formation of Ml2 coat protein antigen at least fourfold, even when added as late as 45 minutes after M12. The extent of specific inhibition is probably greater

T4 INTERFERENCE

WITH

Ml2

187

TABLB~

Measurement of formation of Ml2 coat-protein antigen in control and mixed infectionst L&belling period (mi4

Infection

Exp. 1 Ml2 control Ml2 + T4am at 15 min Ml2 control Ml2 + T4am at 25 min Ml2 control Ml2 + Tlam at 35 min Uninfected cells

16 to 30 16 to 30 26 to 40

26to 36 to 36 to 25 to

40

20to 20to 30to 30to 40to 40to 50 to 50 to 50 to 50 to

30 30 40 40 50 50

50 50 60

Total =S incorporated (cts/min/ml.)

33,900 10,760

37.400 12;900 41,900 12,940 59,580

Total per cent precipitated

Per cent$ Ml2 specifk label

9.2 4.3 26.0 7.1 32.1 8.4 3.3

5.9 1.0 22.7 3.8 28.8 5.1 -

11.3

7.3 0.0 10.0 1.6 21.3 5.4 29.9 6.6 -

Exp. 2 Ml2 control Ml2 f T4am at 15 Ml2 control Ml2 + TCam at 25 Ml2 control Ml2 + T4am at 35 Ml2 control Ml2 + T4am at 45 Uninfected cells T4am infected cells

min min min min

60 60 60 60

29,800 15,600

16,400 8,400 30,600 11,500 37,500 15,600 29,000 18,400

3.8 14.0 5.6 25.3 9.4 33.9 10.6 4.0 4.0

$ E. COGAB301 was grown to log phase in synthetic medium and infected with M12. At the times indicated, duplicate samples were removed into tubes with T4am50 (input ratio 5 to 10 phage/cell) or with medium. 35S was then added to a kal activity of 1 to 2 PC/ml. Preparation of lysates and serum precipitation procedure is described in section (j) of Materials and Methods. 2 The percentage Ml2 specific label was calculated by subtracting the fraction of label precipitated in uninfected cell lysates from that for the test lysates.

than seen from Table 4, because no corrections were made for the contribution from the escape yielders to coat synthesis. In addition to this specific inhibition, the absolute level of Ml2 coat protein per oell is reduced by an additional factor of 2 or 3, i.e. the factor corresponding to the reduction in over-all rate of protein synthesis produced by infecting with T4am. Similar results were obtained using [Wlphenylalanine as the radioactive marker, or with T4am55 as the superinfecting phage. Additional superinfection experiments also showed that the inhibition of coat antigen synthesis is considerably greater than the reduction in synthesis of infectious Ml2 RNA.

4. Discussion The salient features of the exclusion observed in mixed infection with T4 and Ml2 may be summarized as follows: (a) in simultaneous mixed infection, Ml2 RNA synthesis is inhibited to the extent that the input strands do not become converted to the double-stranded form; however, the parental RNA remains intact; (b) if Ml2 is added 20 minutes prior to T4, then MlZ-infectious RNA is made in the absence of phage production or cell RNA synthesis. Under these conditions, the RNA does not

188

S. HATTMAN

AND

P. H.

HOFSCHNEIDER

become incorporated into defective particles and production of Ml2 coat-protein antigen is strongly inhibited. Although alternative models are by no means excluded, for the present, we favour the notion that T4 establishes control over Ml2 replication by interfering with the capacity of Ml2 RNA to function as mRNA. In RNA phage infection, the first protein(s) to be synthesized is related to the replication of the RNA. Any interference with the capacity to produce such protein(s) would be manifested by the absence of synthesis of replicat’ive intermediates and progeny strands. Nevertheless, parental single strands could be efficiently converted to an RNase-resistant form in the absence of measurable polymerase synthesis when SU- cells were infected with SU-3 mutants of phage f2 (Lodish & Zinder, 1966). The fact that simultaneous mixed infection with T4 effectively blocks formation of double-stranded Ml2 RNA suggests that the inhibition of MlS-protein synthesis is essentially complete. If, on the other hand, T4 superinfection occurs at a time when Ml2 RNA polymerase(s) synthesis is already in progress, then Ml2 progeny RNA formation should continue. This is observed, albeit after a short delay. Furthermore, the results described in section (g) showed also that synthesis of coat-protein antigen is markedly limited after addition of TCam to M12-infected cells. That the inhibition is not complete may be due to a type of “gene-dosage” effect; i.e. interference with protein synthesis is more efficient in superinfections at times when fewer Ml2 molecules are active in translation. This is suggested by the results shown in Table 4, as well as by the apparent absence of polymerase production in simultaneous mixed infection. The serum precipitation experiments have to be regarded with some reservations, since it is not known whether nascent coat protein on ribosomes or free coat protein have f&t to undergo folding (e.g. only during the maturation process) before they can be recognized by the antibody. However, the fact some antigen production can be measured under conditions where no Ml2 particles are formed suggests that the procedure can also detect coat protein not yet incorporated into mature phage. It should also be pointed out that an in vitro inhibition of synthesis of RNA-phage R17 coat protein can be demonstrated in extracts of T4-infected cells (Salser & Gesteland, personal communication). The absence of progeny Ml2 formation in superinfections at 20 minutes seems surprising in view of the incomplete block in coat-protein formation. However, if the assembly of complete phage particles were dependent on a higher than linear order of subunit concentration, then the extent of inhibition observed would be consistent with the absence of progeny or inactive phage production. A reduction in Ml2 RNA synthesis in cells superinfected at 20 minutes is not to be expected if T4 were to act only by inhibiting protein synthesis; interference with protein synthesis by addition of antibiotics at this time does not diminish subsequent RNA formation with phage R17 (Brownstein, 1964; Paranchych & Ellis, 1964) or f2 (Cooper & Zinder, 1963). A pause in RNA synthesis following T4 superinfection, due to a temporary sharp reduction in infectious RNA titre (Yarosh & Levinthal, personal communication), would account for the lower infectivities that we measured. It is possible that the manner in which T4 exerts inhibition of protein synthesis (being different from the mechanism of action of antibiotics) leads to a trapping of a portion of the phage RNA population in a non-infectious form. This is supported by the results of Yarosh & Levinthal (personal communication), who have observed in 20-minute superinfected cultures an unreduced synthesis of double-stranded RNA;

T4 INTERFERENCE

WITH

Ml2

189

however, this material had a sedimentation value of about 8 S, indicating a breakdown of the normal replicative forms. We can explain these results also in terms of our model of T4 interference with M12-messenger function. An inhbition at the level of the formation of MIS-specific polysomes appears to be ruled out by our most recent experiments (manuscript in preparation). Rather, we think the inhibition occurs at the level of translation of the Ml2 genome, and that the polysomes formed are non-functional in the presence of T4, but are not necessarily broken down. Under these conditions, if the RNAsynthesizing enzyme(s) is present, as in the 20-minute superinfection experiments, then an abortive synthesis of the polysome-bound RNA may ensue; e.g. the RNA is replicated in the regions between ribosomes, until the attachment site is reached, at which point breakage occurs producing small double-stranded molecules. The resumption in infectious RNA synthesis would be a consequence of the replication of the non-polysome-bound phage RNA. It is important to note that in simultaneous mixed infection, there is no breakdown of parental Ml2 RNA. This is attributed to the absence of RNA-polymerase synthesis, which precludes abortive synthesis of polysome-bound RNA. The superinfection experiments indicate that T4 does not interfere with the function of RNA polymerase. Assuming that T4 interferes with the messenger function of Ml2 RNA, the question of whether such a fate applies to host-cell mRNA must also be considered. As pointed out earlier, the functional decay of mRNA specific for /?-galactosidase appears not to be potentiated by T6 infection (Kaempfer, 1965). It is still not ruled out that other cell mRNA molecules are sensitive to the same process(es) that appears to affect the functioning of Ml2 RNA. In so far as RNA synthesis is concerned, that host-RNA synthesis is effectively eliminated where Ml2 RNA (as well as T4 mRNA) synthesis can proceed in superinfected cells suggests that a multiplicity of control mechanisms are operative in T4-infected cells. Consideration of animal virus-infected cells reveals several rather interesting features which may also apply to phage-infected bacteria, but which have not been examined to date. For example, host-specific polyribosomes are rapidly and specifically destroyed following infection of HeLa cells with poliovirus (Penman, Scherrer, Becker & Darnell, 1963; Scharff, Shatkin C Levintow, 1963; Willems & Penman, 1966) or of L-cells with vaccinia- or mengo-virus (Joklik & Merigan, 1966). In the latter system, however, the destruction of cell pol,yribosomes may not be a function determined by the invading viral genomes. Of further interest are the recent studies concerned with the mode of action of interferon. It has been suggested that the molecular mechanism can be attributed to the induction of a protein which either blocks the formation of viral-specific polyribosomes (Joklik 8t Merigan, 1966), or inhibits viral mRNA translation without loss of polysome stability (Marcus $ Salb, 1966). For the present, we like to think that T4 produces a protein (analogous to the interferon-induced protein) that exerts its action at the level of functioning of Ml2 polyribosomes (and possibly also bacterial polysomes). Such a model is amenable to experimental study, and the various possibilities are being investigated. It would also be of interest to determine the fate of host-specific polyribosomes after T4 infection. If there were to be a disruption in functioning of cellular protein producing polysomes, then this might actually serve as a feedback-control mechanism

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in shutting off further cell mRNA transcription. ceivably be involved in the regulation of enzyme

Lastly, such a process could consynthesis by T-even phage.

The able technical assistance of Miss Christa Seufert is gratefully acknowledged. We are indebted to Professor 8. E. Luria for his valuable comments and criticisms of the manuscript. We thank Professor A. Butenandt for his continued interest and Dr P. Knolle for suggesting the method for extraction of RNA. One of us (S. H.) is a post-doctoral fellow of The Helen Hay Whitney Foundation. This work was further supported by a grant from the Deutsche Forschungsgemeinschaft. REFERENCES Benzer, S. (1953). Biochim. biophys. Acta, 11, 383. Borek, E. A., Ryan, A. & Rookenbach, J. (1955). J. Bact. 69, 460. Brenner, S., Jacob, F. & Meselson, M. (1961). NatzLre, 190, 5’76. Brownstein, B. L. (1964). Proc. Nat. Acad. Sci., Wash. 52, 1045. Cohen, S. S. (1947). Cold Spr. Harb. Symp. Quant. Biol. 12, 35. Cohen, S. S. (1948). J. Biol. Chem. 174, 281. Cooper, S. & Zinder, N. D. (1963). Virology, 20, 605. Delius, H. (1966). Ph.D. Thesis, Ludwig-Maximilians-Universitiit, Mtichen. Epstein, R. H., Belle, A., Steinberg, C. M., Kellenberger, E., Boy de la Tour, C., Chevalley, R., Edgar, R. S., Susman, M., Denhardt, G. H. & Leilausis, I. (1963). Cold Spr. Ha&. Symp. Quant. Biol. 28, 375. Erikson, R. L., Fenwick, M. L. & Franklin, R. M. (1964). J. Mol. BioE. 10, 519. Francke, B. & Hofsohneider, P. H. (1966). J. Mol. Biol. 16, 544. Fraser, D. & Jerrel, E. A. (1953). J. BioZ. Chem. 205, 291. Friesen, J. D. (1965). J. Mol. BioZ. 13, 220. Hall, B. D. & Spiegelman, S. (1961). Proc. Nat. Acd Sci., Wash. 47, 137. Hayward, W. S. & Green, M. (1965). Proc. Nat. Acad. Sci., Wash. 54, 1675. Hershey, A. D., Garen, A., Fraser, D. K. & Hudis, J. D. (1954). Yearb. Carnegie Instn 53, 210. Hofschneider, P. H. (1963). 2. Naturf. lSb, 203. Ippen, K. A. & Valentine, R. C. (1965). Biochem. Biophys. Res. Comm. 21, 21. Joklik, W. K. & Merigan, T. C. (1966). Proc. Nat. Acad. Sci., Wash. 56, 558. Kaempfer, R. (1965). Ph.D. Thesis, Massachusetts Institute of Technology. Levin, A. P. & Burton, K. (1961). J. Gen. Microbial. 25, 307. Lodish, H. F. & Zinder, N. D. (1966). J. MOE. BioZ. 19, 333. Loeb, T. (1961). Ph.D. Thesis, The Rockefeller Institute. Marcus, P. & Salb, 5. (1966). I”iroZogy, 30, 502.1 Monod, J. & Wollman, E. L. (1947). Ann. Inst. Pasteur, 73, 937. Neubauer, Z. & Zavada, V. (1965). Biochem. Biophys. Res. Comm. 20, 1. Nomura, M., Hall, B. D. & Spiegehnan, S. (1960). J. Mol. BioZ. 2, 306. Nomura, M., Matsubara, K., Okamoto, K. & Fijimura, R. (1962). J. Mol. BioE. 5, 535. Nomura, M., Witten, C., Mantei, N. & Echols, H. (1966). J. Mol. BioZ. 17, 273. Paranchych, W. & Ellis, D. B. (1964). Virology, 14, 635. Penman, S., Scherrer, K., Becker, Y. 62;Darnell, J. E. (1963). Proc. Nat. Acad. Sci., Wash. 49, 654. Scharff, M. D., Shatkin, A. J. & Levintow, L. (1963). Proc. Nat. Acud. Sci., Wash. 50, 686. Skold, 0. & Buchanan, J. M. (1964). Proc. Nat. Acad. Sci., Wash. 51, 533. Stent, G. S. & Brenner, S. (1961). Proc. Nat. Acad. Sci., Wash. 47, 2005. Valentine, R. C. & Wedel, H. (1965). Biochem. Biophya. Res. Comm. 21, 106. Valentine, R. C., Wedel, H. & Ippen, K. A. (1965). Biochem. Biophys. Res. Comm. 21, 277. Volkin, E. & Astrachan, L. (1956). Virology, 2, 149. Willems, M. & Penman, S. (1966). V&rology, 30, 348. Zinder, N. D. (1963). In Perspectives in Virology, vol. 3, p. 58.