DNA Replication of Bacteriophage T5. 1. Fractionation of ... - CiteSeerX

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J. gen. Virot. (198o), 47, I23-T32 Printed in Great Britain

D N A Replication of Bacteriophage T5. 1. Fractionation of Intracellular T5 D N A by Agarose Gel Electrophoresis By R O G E R

D. E V E R E T T *

AND M A R Y

R. L U N T

Microbiology Unit, Department of Biochemistry, South Parks Road, Oxford OXI 3QU, U.K. (Accepted 9 October I979) SUMMARY

Two forms of DNA may be isolated from gently lysed T5-infected bacteria. In neutral sucrose gradients a fast sedimenting form (fsf) is distinguishable from a slow sedimenting form (ssf) which moves at a rate similar to that of DNA extracted from mature T 5 virus particles. A method of agarose gel electrophoresis is described which gives complete separation of the slow sedimenting from the fast sedimenting form. The DNA was extracted from the agarose using potassium iodide and isopycnic centrifugation; samples of the slow sedimenting form prepared by these methods were suitable for detailed structural studies.

INTRODUCTION

The replication of bacteriophage D N A has been shown to proceed generally via multigenome length or concatemeric intermediates. One of the fundamental questions posed by this finding is how mature length DNA molecules are excised from the concatemer and packaged into phage heads. We have studied this problem using bacteriophage T5. The mature virion DNA of T5 is a linear duplex of tool. wt. approx. 74 × to6. The molecules are of unique sequence (Thomas & Rubenstein, I964) and contain the largest terminal redundancy of any known phage, amounting to about 9 % of the total length (Rhoades & Rhoades, I972). A further peculiarity of T5 DNA is that one strand contains a number of interruptions, or 'nicks', at genetically defined locations (Abelson & Thomas I966; Chen & Bremer, I976). These nicks can be sealed by treatment with DNA ligase alone and therefore consist simply of a missing phosphodiester bond (Jacquemin-Sablon & Richardson, 197o). Packaging of T 5 DNA into the phage head probably occurs in a defined manner since one specific end of the molecule was found to be associated with the phage tail after disruption of mature virus particles (Saigo, I975). The method of excision f r o m concatemers and packaging of DNA, recently reviewed by Murialdo & Becket (I978), must vary between different groups of phages. The processes may be relatively simple in those phages with circularly permuted genomes, such as T2 and T4. According to the 'headful' hypothesis (Streisinger et al. t967) DNA enters the phage capsid until it is full, at which point endonuclease activity releases the filled head from the concatemer. In T2 the sites of initiation of packaging and endonuclease activity are probably random, so that populations of T2 phage contain a complete set of random circular permutations of the genome; there appears to be no preferred starting point for packaging (MacHattie et al. 1967). * Present address: Department of Molecular Biology, King's Building, Mayfield Road, Edinburgh EH9 3JR, Scotland. OO22-I317/80/0OOO-3812 $02.00 (~) 1980 SGM

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A somewhat more sophisticated version of this model assumes that packaging initiates at a specific site, but endonuclease cleavage is again random. Subsequent packaging initiates at the site of the previous termination and therefore a number of families of discreet but circularly permuted genomes will arise in the phage population. It is likely that the number of these families will depend on the average length of the intracellular concatemer. Examples of phages with these limited circular permutations are P22, which can package up to ten genomes progressively in a unique direction from a single concatemer ('rye et al. I974; Jackson et al. I978) and TI which contains three permutations (Gill & MacHattie 1976). Both these versions of the headful model lead to two predictions. First, if the genome size is reduced by deletion, the length of the terminal redundancy must be correspondingly increased, and second, since the site of termination is not specific any population of DNA molecules from these phages will have a slight heterodispersity of length. These predictions have been experimentally verified (Kim & Davidson, 1974; Tye et al. I974; Gill & MacHattie, 1976; MacHattie & Gill, 1977). A basic feature of the headful model is that packaging of DNA uses concatemeric DNA directly; excision and head-filling must be intimately linked. In support of this hypothesis, intracellular pools of mature length DNA have not been found in bacteria infected with phages with circularly permuted genomes (Botstein, I968; Frenkel, 1968; Carrington & Lunt, 1973). Those phages with unique sequence genomes must employ a site-specific packaging mechanism. In contrast to the circularly permuted phages, deletion mutants of T5, T7 and ,~ contain reduced amounts of DNA ; the length of the terminal redundancy remains constant (Ritchie & Malcolm, 197o; Parkinson & Huskey, I97I). Despite this difference from the circularly permuted phages, it appears that packaging of both T7 and A DNA uses the concatemer directly. A pool of phage-sized intracellular DNA was not detected in T7infected bacteria (Serwer, I974a, b) while concatemeric DNA was found to be the preferred substrate for the in vitro packaging of both A and T7 DNA (Kaiser et al. I975; Masker et al. 1978). Furthermore, it has been shown that the activity of the phage ,~ concatemer cutting enzyme is dependent on the amount of DNA packaged into the head (Henderson & Weil, I977), thus emphasizing the relationship between packaging and concatemer excisionl There is evidence that packaging of ,~ DNA resembles that of P22 in that initiation of packaging occurs at a specific site and then proceeds in a unique direction along the concatemer. Packaging termination is of course site-specific during /~ maturation (Emmons, 1974; Feiss & Bublitz, 1975). Therefore, the observation that intracellular DNA from T5-infected bacteria contains a pool of molecules, the slow sedimenting form (ssf) of similar sedimentation rate to that of mature T5 virion DNA (Carrington & Lunt, 1973) implies that the mechanism of packaging of T 5 DNA may be different from that of other similar phage systems. Two lines of investigation indicated a productive role for the ssf: during pulse-chase experiments it appeared to be an intermediate between concatameric DNA and phage heads, while a T5 strain defective in late DNA replication was unable to produce the ssf or phage heads (Carrington & Lunt, 1973). However, the structure of the ssf remained uncertain. It was shown to contain a similar but not identical pattern of single-strand interruptions to that of mature DNA, and it bound to nitrocellulose whereas mature T 5 DNA did not (Carrington & Lunt, I973). In view of the potential importance of the ssf, we considered that a detailed study of its structure was required. This paper describes a method for the preparation of the ssf in a relatively pure form. The following paper describes in more detail the structure and properties of the ssf and discusses its role in T5 replication.

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METHODS

Bacteria and bacteriophage. Bacteriophage T 5 was the same strain as that used by Cartington & Lunt (1973). It was grown and assayed on Escherichia coli strain B/2, and purity checked with the resistant strains B/I, 5 and B/3 , 4, 5, 7. E. coli C3ooo thi- was used for all experiments involving the isolation of intracellular T 5 phage DNA. All bacteria and phage were grown in aerated cultures at 37 °C. Media and buffers. Tryptone broth and phage dilution fluid were those used by Fielding & Lunt (197o). Low phosphate minimal medium was that of Hershey (1955). For infections with phage T5, media were made I mM with respect to CaCI~. TES buffer contained o.oi M-tris-HC1, pH 8.1, o.OOlM-EDTA, O.Oli-NaC1. Buffer A (high salt) contained O'O5Mtris-HC1, pH 7"8, o-ooi M-EDTA, I.oM-NaC1. Buffer B (low salt) contained o.o5i-tris-HCl, pH 7"8, o.ooi M-EDTA, o-o2 M-NaCI. Preparation of 3zP-labelled DNA from T5 virions. E. coli B/2 was grown to a cell density of 3 × IoS/ml when carrier free a2P-phosphate was added to give 2/~Ci/ml. When the culture reached 5 × lo8/ml the bacteria were infected with T 5 at a m.o.i, of IO. Aeration was continued until lysis, which was usually complete at 6o to 7o rain p.i. The phage were purified by differential centrifugation, followed by banding on a CsCI step-gradient prepared in phage dilution buffer (Thomas & Abelson, 1966). The phage were dialysed and D N A isolated by phenol deproteinization followed by extensive dialysis against TES buffer. Preparation ofintracellular T5 DNA. E. coli C3ooo was grown in low phosphate medium supplemented with thiamine (2/~g/ml) to a cell density of 5 × Io8/ml and infected with T5 at a m.o.i, of IO. After addition of 3H-thymidine (see later), samples (5 ml) of the culture were pipetted on to frozen o-I M-NaCI to stop further DNA synthesis and the bacteria collected by centrifuging at 350o g for 5 rain. The bacteria were resuspended in 0"4 ml of 15 ~ (w/v) sucrose in o.oi M-tris-HCl, pH 8-1 and 0-2 ml of a I : I mixture of lysozyme (I mg/ml in 0"25 M-tris-HC1 pH 8.1) and the disodium salt of EDTA (2"7 mg/ml) was added. The suspension was kept on ice for 5 min. The bacteria were then lysed by the addition of o'4 ml of a I : I mixture of Brij 58 (5 %, w/v) and sodium deoxycholate (2 %, w/v), each detergent being dissolved in o.I M-tris-HC1, pH 8.1, lO% sucrose. Lysis was complete within 3 to 5 min at 4 °C. The procedure is based on that of Godson & Sinsheimer (1967) but with solutions containing sucrose at densities adjusted to facilitate mixing. Agarose gel electrophoresis. Agarose was dissolved in electrophoresis buffer (O'O4Mtris-HC1, o.o2M-sodium acetate, o.OO2M-EDTA, pH 8"3) by heating in a boiling water bath for 15 rain. The molten gel was poured into glass tubes (15 × I cm internal diam.) and allowed to set at 4 °C. Before electrophoresis, the bottom of each tube was covered with muslin secured by a rubber collar and the top surface levelled with a razor blade. All gels were adjusted to a constant length of 14 cm. Electrophoresis was carried out at 5o V for approx. I6 h at 4 °C without buffer circulation. Recovery ofDNAfrom agarose gels. The method of Blin et al. (1975) was adopted. Agarose slices which contained DNA were dissolved in a saturated solution of KI in o'05 M-tris-HC1, pH 7"6, o.oi M-EDTA and the solution adjusted to a density of I-5 g/ml by weighing. After centrifuging at 42ooo rev/min for 24 to 4o h in the Beckman SW5o. r rotor, fractions of 0"25 ml were collected by upward displacement (see later) with saturated KI solution. The samples which contained DNA were quickly identified by spotting lO #1 amounts on to Whatman G F / A papers which were dried, placed in scintillation fluid and radioactivity measured in a scintillation counter. The KI was removed from the DNA of the appropriate pooled samples by dialysis against TES buffer. Sucrose density gradient centrifugation. Five ml linear gradients of io to 25 ~ (w/v) sucrose in either buffer A or buffer B were prepared. Centrifuging was done in the SW5o. I

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rotor of a Beckman L2-65B centrifuge as described in the text and figure legends. Gradient fractionations were usually drop-collected on to glass fibre paper strips, but when samples were required for further analysis, to avoid shear the gradient fractions were collected through a wide-bore adapter from the top of the tube. Details of these procedures, including the preparation of samples for estimating radioactivity are described by Carrington & Lunt (I973). Radioactive materials and their measurement. Carrier-free a2P-phosphate and 6-8Hthymidine (24 Ci/mmol) and U-14C-L-leucine (348 mCi/mmol) were obtained from The Radiochemical Centre, Amersham, Bucks. Gradient samples on glass fibre paper were placed in scintillation vials with 2. 5 ml of a solution which contained 0"5 ~ butyl PBD in toluene and radioactivity measured in a Beckman or LKB scintillation counter. Agarose gels were cut into I mm slices with a Mickle gel slicer; two-slice samples were dissolved by heating in o.I ml of I M-HC104 at 7o °C for 3o min and mixed with 3 ml of a scintiUant which contained 5"5 g PPO, o'I g POPOP, 333 ml Triton X-~oo and 667 ml toluene. Radioactivity was measured in a scintillation counter; counts were corrected for background, and where necessary for overlap between channels. Reagents. Brij 58 (polyoxyethylenecetyl ether) and Triton X-Ioo were from Sigma (London) Chemical Co. Butyl PBD [5-(4-biphenyl)-z-(4-tert-butylphenyl)-~-oxa-3:4 diazole] was supplied by Ciba (Duxford, Cambs). PPO (2,5-diphenyloxazole), POPOP [I,4-bis-z(5-phenyloxazolyl)benzene], sodium deoxycholate and agarose (electrophoresis grade) were from BDH (Poole, Dorset). Enzymes. DNase I (EC 3 . 1 . 4 . 5 ) was from Seravac Laboratories (Maidenhead, Berks). Ribonuclease A (EC 3. r . 4 . 2 2 ) and egg white lysozyme (EC 3.2.~. I7) grade I were from Sigma. RESULTS

Agarose gel electrophoresis of intracellular T5 DNA A culture of E. coli C3ooo in low phosphate medium was infected with T 5 phage as already described. At 3o min p.i., ~H-thymidine was added to give 2/zCi/ml and at 35 min a 5 ml portion of the culture was removed and the bacteria harvested and lysed by the lysozyme-Brij-deoxycholate procedure. A o.2 ml sample of the lysate was carefully layered on top of a o'5 ~ (w/v) agarose gel together with 2/zg of 32P-labelled T5 virion DNA. After electrophoresis, the gel was cut into I mm slices, and adjacent two-slice samples were combined, dissolved and their radioactivity measured as described under Methods. As shown in Fig. t a large proportion (about 8o ~ ) of the labelled material was trapped at the top of the gel (peak I), but two peaks of material were found to have migrated into the gel: a major peak, peak II, moving more slowly than mature T5 virion DNA, and a minor shoulder, peak III, moving at the same rate as, or slightly faster than, the marker of virion DNA. The proportion of radioactivity which migrated into the gel was similar to that found in the ssf region after a high salt sucrose density gradient analysis of an identically prepared lysate. To show that peaks II and III retain their mobilities on re-running, duplicate gels were prepared as above, and after electrophoresis one gel was sliced and radioactivity estimated to locate the positions of the peaks. The corresponding slices from the other gel were then separately layered directly on top of further o'5 ~ gels and again subjected to electrophoresis. Peak II material was reproducibly slow-moving; no breakdown to peak III material occurred. Peak III and mature virion DNA were coincident on re-running (results not shown). To confirm that peaks II and III contained DNA, the lysate was digested with DNase I (37 °C for 2 h) before electrophoresis. After this treatment no radioactivity was detected

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Fig. I. Agarose gel electrophoresis of intracellular T5 DNA. Electrophoresis was at 5o V for 16"25h at 4 °C. O-G, SH intracellular DNA; O-Q, 3zp mature T5 virion DNA. in the gel, although some DNase-resistant material was observed in the top two samples. Using a preparation of intact T5 which had been purified on a CsC1 gradient it was found that mature phage could penetrate the gel to a depth of up to I cm under our conditions; no material was found in the region where peak II would have been expected, and it is therefore unlikely that peak II represents a breakdown product of pre-formed phage or phage heads. Similarly it was shown that peak II was probably not a complex of DNA and RNA since after treatment of a lysate with RNase (IO #g/ml; 37 °C for I h) electrophoresis gave the same gel peaks as were obtained without RNase treatment.

Electrophoresis of the fast-sedimenting and slow-sedimenting forms of intracellular T5 DNA To discover a possible relationship between the components resolved by electrophoresis and the ssf and fsf of intracellular T 5 DNA, a Brij-deoxycholate lysate of infected bacteria was prepared and o-2 ml samples were analysed by centrifugation through to to 25 ~ linear gradients of sucrose dissolved in the high salt buffer A. After centrifuging, alternate I and 3 drop fractions were collected by upward displacement, and radioactivity in the single drop fractions was estimated (Fig. 2). Fractions which contained the fsf and ssfwere then layered directly on to separate o'5 ~ agarose gels for electrophoresis. The results (Fig. 3) showed that, as anticipated, the fsf was retained almost quantitatively at the top of the gel; only a small proportion of the radioactivity (about 3 ~ ) co-migrates with mature virion DNA. In contrast, the ssf fraction moved coincidently with T5 virion DNA, although about zo ~o of the counts were retained at the top of the gel; this latter material probably consisted of 5

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Fig. 2. Sucrose gradient centrifugation of intracellular T5 DNA at high ionic strength. Centrifugation was at 38o00 rev/min for I'75 h in the Beckman SW5o. I rotor of a model L centrifuge. O-C), 3H intracellular DNA; O - O , 32p mature T5 virion DNA. Arrows show fsf and ssf (fast and slow sedimenting forms) respectively. Fig. 3. Agarose gel electrophoresis of isolated ssf and fsf components of intracellular T5 DNA. Electrophoresis was at 5o V for I6 h at 4 °C. ( 3 - 0 , 8H fsf; 0 - 0 , 3H ssf; A - i , , 32p mature T5 virion DNA. Arrow shows position of peak I! on a parallel gel. c o n t a m i n a t i n g fsf a n d also some capsid-associated D N A (R. D. Everett, u n p u b l i s h e d data). M a t e r i a l o f electrophoretic m o b i l i t y characteristic o f p e a k II was n o t detected in a n y fraction f r o m a sucrose g r a d i e n t p r e p a r e d in the high salt buffer A.

Analysis of intracellular T 4 DNA on sucrose density gradients at low ionic strength Thus p e a k I I which migrates m o r e slowly t h a n T5 virion D N A can be recovered after gel electrophoresis o f cell lysates, b u t n o t if the lysates are first fractioned on sucrose density gradients which contain I M-NaCI. The latter gradients yield ssf D N A which is c h a r a c t e r ized by a s e d i m e n t a t i o n rate and e l e c t r o p h o r e t i c m o b i l i t y close to t h a t o f virion D N A . The m a j o r difference between the conditions o f gel electrophoresis a n d the sucrose gradients so far described is t h a t o f the ionic strengths o f the buffers used. A c c o r d i n g l y , we decided to see whether p e a k I I material could be recovered from sucrose gradients m a d e up in a solution o f low ionic strength, n a m e l y buffer B. Intracellular T5 D N A was labelled with aH-thymidine as a l r e a d y described a n d a o.2 ml sample o f the subsequent lysate was layered on a lo to 25 ~ sucrose gradient p r e p a r e d in buffer B. A f t e r centrifuging, fractions o f 0.2 ml were collected by u p w a r d displacement and 2o #1 p o r t i o n s o f each fraction were taken for estimation o f radioactivity. As shown in Fig. 4, all the intracellular D N A sedimented faster than T 5 virion D N A , and resolution into separate peaks was poor. Selected fractions from the g r a d i e n t were next analysed by agarose gel electrophoresis.

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Fig. 4. Sucrose gradient centrifugation of intracellular T5 D N A at low ionic strength. Centrifugation was at 38000 rev/min for 1.75 h in the Beckman S W 5 o . i rotor of a model L centrifuge. Arrow

shows position of 3~P-phosphate-labeUed mature T5 virion D N A loaded on the same gradient. O - C ) , ~H i n t r a c e l l u l a r D N A . Fig. 5. Agarose gel electrophoresis of intracellular T5 D N A isolated by sucrose gradient centrifugation at low ionic strength (for details see text). C ) - © , 3H i n t r a c e l l u l a r D N A ; 0 - 0 , 32p mature virion D N A .

It was found that material corresponding to peak II could be recovered from the fast sedimenting gradient fractions; a typical result (using fraction 9 from the gradient of Fig. 4) is shown in Fig. 5. When samples from these fast sedimenting positions on a low ionic strength sucrose gradient were re-centrifuged through high ionic strength (buffer A) sucrose gradients, the 3H-labelled D N A co-sedimented with T5 virion DNA. By contrast, the fast sedimenting properties were retained if the gradients had been prepared in the low salt buffer B. It was concluded that peak II and the ssf isolated from high salt sucrose gradients contain the same D N A component. The differences in electrophoretic mobilities disappear after cell lysates or the isolated fractions have been subjected to conditions of high ionic strength either on sucrose gradients in buffer A or by exposure to the high concentrations of KI used in elution of D N A from agarose gels, as described later (see also Everett & Lunt, 198o). In gradients made in solutions of high ionic strength, previous experiments had shown that no protein could be detected in association with the intracellular T5 D N A (Carrington & Lunt, I973).

Recovery of DNA from agarose gels The elution of DNA from agarose gels was attempted by several methods of which the most successful was the KI method of Blin et al. (1975). The method was useful in avoiding shear and recoveries were high (over 9o %). A typical result of the KI banding of the D N A from peak lI is shown in Fig. 6. The material from two gels could be isolated on a single 5 ml gradient, giving a yield of I to 2 #g of peak II DNA. 5-2

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Fig. 6. Potassium iodide gradient centrifugation of peak II DNA. Centrifugation was at 36ooo rev/min for 4o h at 2o °C in the SW5o. I rotor of a Beckman L2-65B ultracentrifuge. © - Q , 3H peak II D N A ; O - O , density (g/ml). Fig. 7. Sucrose gradient centrifugation of peak II DNA. Centri(ugation was at 4oooo rev/min for 2 h in the SW5o. I rotor. © - O , 3H peak II DNA; O - O , 3~p mature T5 virion DNA.

Sedimentation properties of peak H DNA Peak II DNA labelled with 3H-thymidine was isolated by gel electrophoresis and KI density gradient centrifugation as described above. A o.I ml sample of this preparation was centrifuged through a 5 to 2o % sucrose gradient in buffer A, together with o'5 #g of a2p_ labelled T5 virion DNA. The result (Fig. 7) shows that peak II DNA sedimented as a homogeneous band with a slight trailing edge of lower tool. wt. material. This was probably due to breakage during handling since the amount of this trailing edge varied between preparations. In this and other experiments the sedimentation of peak II DNA was very similar but not identical to that of virion DNA which moved slightly faster. Therefore, by comparison with virion DNA, peak II DNA and the ssf appear to have essentially similar sedimentation rates, although any small differences could be masked by the heterogeneity of the ssf. In a similar experiment, peak III DNA was shown to contain material of heterogeneous mol. wt., mostly slower sedimenting than T5 virion DNA. Since this peak was present in small and variable amounts, it is likely that it represents shear degradation products of other forms of intracellular T 5 DNA produced during isolation. Association of protein with peak II DNA When it is maintained in solutions of low ionic strength, peak II DNA has been shown to have an unusually low electrophoretic mobility and high sedimentation rate compared with that expected from the mol. wt. of the DNA. Preliminary experiments were therefore done to test the possibility that these characteristics were due to the presence of protein associated with the DNA.

T5 D N A replication 1

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Accordingly, it was found that material labelled with 14C-leucine during T5 infection cosedimented with peak II DNA, provided the sucrose gradients contained low, not high, concentrations of NaCI. Similarly, 14C-leucine-labelled material co-migrated with peak II DNA on agarose gels. However, if the gel slices which contained the peak II material were incubated with the high ionic strength buffer A and then re-electrophoresed, the peak II DNA showed the mobility of T 5 virion DNA and the ~C-labelled material did not appear in the gel. Characterization of the proteins associated with peak II is described in the accompanying paper (Everett & Lunt, I98O).

DISCUSSION

This paper describes a new method of isolating and detecting the ssf of intracellular T5 DNA. The difference in electrophoretic mobility between peak II DNA and deproteinized T5 virion DNA (Fig. I) and the absence of material with the mobility characteristic of peak II in fractions from high ionic strength sucrose density gradients of intracellular T5 DNA (Fig. 3) is explained by the association of protein with peak II DNA at low ionic strength (Everett & Lunt, I98O). Following isolation in conditions of low ionic strength, either on sucrose gradients or on gels, peak II DNA reverts to the sedimentation behaviour of ssf DNA on treatment with high salt buffers, as shown in Fig. 7. We therefore believe that peak II and the ssf contain the same DNA component. Although agarose gel electrophoresis has been used extensively in recent years to analyse mixtures containing relatively small DNA fragments (see McDonell et al. I977) the method described here has been applied to high mol. wt. DNA. However, the basis of the separation in these experiments cannot be primarily due to a difference in the tool. wt. of the ssf and fsf since double-stranded DNAs larger than 3o × Io~ mol. wt. are poorly resolved by this method (Hayward & Smith, I972). Instead the branched and complex nature of the fsf (Everett, I978) precludes its entry into the gel (Fig. 3). When combined with the KI elution technique, the method allows the isolation of intact, homogeneous preparations of ssf which are suitable for detailed structural studies, as described in the following paper. R. D. E. thanks the Medical Research Council for a studentship held during the course of this work. REFERENCES ABELSON, J. & THOMAS, C. A. JUN. (I966). The anatomy of the T5 bacteriophage D N A molecule. Journal of Molecular Biology i8, 262-29L BLIN, N., OABAIN,A. V. ~ BUJARD,H. (I975)- Isolation of large molecular weight D N A from agarose gels for further digestion by restriction enzymes. FEBS Letters 53, 84-86. BOTSTEIN, D. (I968). Synthesis and maturation of phage P22 DNA. I. Identification of intermediates. Journal of Molecular Biology 34, 621-64I. CARRINGTON, J. M. & LUNT, M. R. (I973). Studies on the replication of bacteriophage T5. Journal of General Virology 18, 9I-lO9. CHEN, C. & BREMER,M. (I976). Identification of single-stranded D N A fragments of bacteriophage T5. Virology 74, Io4-I I5. EMt~ONS, S. W. 0974). Bacteriophage lambda derivatives carrying two copies of the cohesive end site. Journal of Molecular Biology 83, 5I 1-525. EVERETT,g. D. ([978). Studies on the DNA replication of bacteriophage "1"5.D.Phil. thesis, University of Oxford. EVERETT, R. O. & LUNT, ~,~. g. (I980). DNA replication of bacteriophage T5. z. Structure and properties of the slow sedimenting form of intracellular T5 DNA. Journal of General Virology 47, 133-I 50. FEISS,M. & BUaLITZ, g. (I975)- Polarised packaging of the bacteriophage ,~-chromosome. Journal ef Molecular Biology 94, 583-594. FIELDING, P. E. & LUNT, M. R. 0970). The relation between breakdown of superinfecting virus deoxyribonucleic acid and temporal exclusion induced by T4 and T5 bacteriophages. Journal of General Virology 6, 333-34z.

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(Received I2 June

I979)