In vitro packaging of bacteriophage T4 DNA

VIROLOGY

113, 336-344 (1981)

In Vitro Packaging

of Bacteriophage

T4 DNA

LINDSAY W. BLACK Department of Biological Chemistry, Univasity

of Maryland

Medical School, Baltimore, Maryland 21201

Received January 23, 1981; accepted March 25, 1981 Bacteriophage T4 can be formed in an in vitro DNA packaging system which requires addition of exogenous T4 DNA. Extracts prepared from bacteria infected with mutants 2Ocs, 17es, 16am, 1’7am, or 16am-17am contain processed proheads. These appear to be efficient DNA acceptors in the in vitro system. Packaging of mature T4 DNA is more efficient than of concatemeric T4 DNA. The phage yield is proportional to the quantity of DNA and prohead-containing extract added. Activity requires addition of nucleoside triphosphate, Mp, and is inhibited by g-amino acridine. gp16 and gp17, the primary T4 DNA packaging gene products, are essential, but 16am and 17am mutants appear not to complement each other in vitro. Many T4 recombinants, including multiple recombinants, appear to be generated by in vitro recombination in this system. In vitro T4 DNA packaging conforms to expectations based upon analysis of T4 mutants and head assembly in vivo and displays fundamental similarity to that of other complex double-stranded DNA phages.

INTRODUCTION

DNA packaging in bacteriophage T4 is thought to be fundamentally the same as in the other complex double-stranded DNA containing bacteriophages. The same three factors interact and are essential for packaging: an active, processed prohead; replicated, concatemeric DNA substrate; and certain nonstructural gene products specific to packaging. Phage T4 differs from phages such as X in lacking unique end sequences. Phage T4 appears to have variable DNA ends as a consequence of filling according to a strict headful mechanism, normally encapsidating 102% of a genome equivalent of DNA from a concatemer, and filling to constant DNA density. As a consequence the T4 chromosome is circularly permuted and terminally redundant. It. is therefore probable that phage T4 can package DNA nonspecifically from many initiating sequences, and it can become a generalized transducing phage. (Frankel, 1968, Streisinger et aZ.,1967; Wilson et al., 1979; see Murialdo and Becker, 1978). The underlying mechanism of DNA translocation and condensation into the 0042-6822/81/110336-09$02,00/O Copyright All rights

0 1981 by Academic Press. Inc. of reproduction in any form reserved.

336

capsid remains a fundamental problem in studies of all bacteriophages. Although in vitro encapsidation of T4 phage DNA has not been previously reported, this bacteriophage represents an especially favorable system for studying this problem. The phage T4 head structure is particularly well established (Aebi et aZ.,1974; Ishii and Yanagida, 1975; Laemmli et al., 1976; Muller-Salamin et aZ., 1977; see Eiserling, 1980). The role of some of the capsid structural proteins in the packaging process is understood, as is the sequence of proteolytic processing reactions which leads to the prohead structure competent for packaging (Hsiao and Black, 1977,1978). Other complex T4 features (unusual DNA and variable

chromosome

end

structure,

au-

tonomous DNA development, and prohead protein processing) may actually be of advantage in revealing general mechanisms of DNA encapsidation. The bacteriophage T4 head begins as a complex core-containing prohead (Tauparticle) which is assembled upon the cytoplasmic membrane and then proteolytically processed (Fig. 1) (Simon, 1972; Showe and Black, 1973; Kellenberger et aZ.,

BACTERIOPHAGE

337

T4 DNA

1 341s toil fiber I

a

231s major 43tsDNA polymemse 321s HD protein - - ----30ts DNA ligase

49ts

recombination

copsid

22tr major core 211s proteinore 241s minor 31 ts caprid

+

capsid assembly

T4

I l

b

p21 protein

cleavage

FIG. 1. (a) Order of function of phage T4 gene products in bacteriophage T4 assembly using csts temperature shifts (Jarvik and Botstein, 19’73; Hsiao and Black 1977,19’78; and unpublished). ts mutants to the left of 29~s act before, those to the right, after WCS. Gene 16 and 17ts mutants interact with 26cs, or suppress it, suggesting these gene products act in the same step. (b) Schematic diagram of the pathway of bacteriophage T4 head assembly and of DNA packaging, genes involved in this process, gene product locations in the head structure, and functions of genes used in the present study. This pathway is consistent with the genetic results summarized in (a) and with other work. Unprocessed prohead (Tau-particle) is assembled on the cytoplasmic membrane. Following proteolytic processing of the prohead and detachment from the membrane, DNA encapsidation is initiated. and the filled head is matured (see Murialdo and Becker, 1978).

1968; Laemmli and Favre, 1973). Order of function studies establish, however, that DNA packaging is initiated after protein processing and prohead detachment from the cytoplasmic membrane, and that DNA replication is not coupled to DNA encapsidation in wivo (Hsiao and Black, 19’7’7). A capsid structural protein (gp20) situated at the DNA entrance vertex of the head appears to act directly in DNA filling, by linking prohead, DNA, and specific T4 DNA packaging gene products (gp16 and gp17) (Fig. 1). Cold-sensitive mutations in gene 20 produce processed proheads containing gp20 which mature to viable phage when the temperature is raised (Hsiao and Black, 1977, 1978). A physical interaction between the DNA entrance vertex protein (gp20) and the specialized T4 packaging gene products (gp16 and gp17) during head filling is suggested from the interdependence of mutations in these three genes (Fig. 1). Mutants which overproduce gp16 and gp17 have been isolated and these

gene products partially purified. Gene 16 and gene 17 products appear not to be head structural proteins but may bind to DNA (in preparation). Therefore the order of events in T4 DNA packaging, the components involved, and the gene products which are directly implicated in packaging are well characterized. In this paper an in vitro packaging system for phage T4 is described which utilizes some of the mutants described above to prepare extracts which will package exogenous T4 DNA. This system is used to analyze the mechanism of T4 DNA packaging in vitro. MATERIALS

AND METHODS

Bacterial strains. E. coli strains l34OSu+’ and the isogenic P30l(pm-) were permissive and nonpermissive strains, respectively, for growth of T4 amber mutants (Strigini and Gorini, 1970). E. coli strains CA180 (HfrH (X) lacy14B;Su+2), CA265

(HfrH (X) lacr,1zsB;Su+3)(S. Brenner), CR63 (A) su+‘, and W3102 (X) pm- (this laboratory) were nonpermissive for T4 rll mutants, and permissive or nonpermissive for various T4 amber mutants. SG5010 (endol- RNase- recB pm-) was a gift of Dr. Susan Gottesman. Bucteriophuge. The following amber mutants of phage T4 1’7(um NG178), 17(am A465) 20(am N50)-23(am Hll), 16(am N66-am N87), 16(am N66)-17(am A465), 24(am NG433), 55(um BL292), and coldsensitive mutants 17(csF3) and 2O(csN33) were combined with rll deletion dZrllA(H88) through standard complementation and recombination tests with bacterial strains listed above. Preparation of DNA. CsCl step gradient purified wild type T4 phage or amber mutants of phage T4 were extracted with an equal volume of redistilled phenol (saturated with buffer and adjusted to pH 7.4) and one-half volume of chloroform-isoamylalcohol (24:l) (volume:volume) until the aqueous layer cleared following lowspeed centrifugation. The aqueous layer was then dialyzed against 10 mMTris, 0.5 mM EDTA, pH 7.4. Concatemeric T4 DNA was prepared by infecting E. coli SG5010 or E. coli P301 with T4 17(um NG178), T4 17(um A465), or 24(um NG433). After infection and growth for 30 min at 37”, 60 ml of infected bacteria were centrifuged from H-broth and resuspended in 0.05 M Tris-Cl, pH 7.5, 25% sucrose (1 ml). Following addition of 200 ~1 egg white lysozyme (1 mg/ml) and 50 ~1of 0.25 M EDTA, pH 8 for 30 min at 4”, 1 ml of detergent solution (7.5 ml 0.25 M EDTA, pH 8,1.5 ml 1 M Tris-Cl, pH 8, 0.7 ml water, 0.3 ml 10% Triton X-100) was added and gently mixed. The solution was then treated with phenol-chloroform-isoamylalcohol and dialyzed as described above. DNA solutions were stored at 4” over chloroform, and DNA concentrations were determined by their absorptions at 260 nm. Preparation of irlfected extracts fm in vitro packaging. E. coli P301 was grown exponentially to 2 X lO*/ml in a mixture of 1 vol H-broth1 vol M9S. Following addition of L-tryptophan to 40 pg/ml, bacteria were infected at a multiplicity of 4,

and superinfected with the same multiplicity following 14 min of growth at 22”. At 90 min after the initial infection, 750ccl portions of the infected culture were centrifuged for 12 min at 7000 rpm at 4” in sterile 1500-~1 Eppendorf capped centrifuge tubes. The supernatant was completely removed with a fine aspirator tip while maintaining the tube at the temperature of ice. Twenty microliters of reaction buffer were added, and the infected pellet gently dispersed by tapping during 30 min on ice. Several microliters of DNA solution were then added, and the tubes immediately placed at the temperature of incubation, generally either 30 or 37“. The reaction was ended by adding one drop of chloroform and 500 ~1 of dilution buffer containing 20 @g/ml pancreatic DNase. The mixed solution was then incubated for 15 min at 3’7” before dilution for plating. In vitro complementation was carried out by mixing together at 4”, 750-~1 portions of infected cultures, and preparing mixed extracts by the procedure outlined above. Quick freezing of bacteria, in the presence or absence of sucrose or glycerol, in dry ice-ethanol or liquid Nz, appeared to destroy or greatly reduce activity. For a given batch of DNA, the reproducibility of the phage yield was generally high ( rt 50% ) and duplicate tubes were run to determine values given. For each series of experiments, cs20rll extract was prepared and used to establish a standard yield. Media and t&.Yfers.The standard reaction buffer (RB) was prepared as follows: sterile, freshly prepared buffer of the following composition: 50 mM Tris-HCl, pH 7.4,20 mM MgClz, 3 mM mercaptoethanol, 6 mM spermidine trihydrochloride, 10 mM ATP, 1000 ~1, was saturated with 20 ~1 of chloroform at 4” with repeated mixing over about 1 hr, and the aqueous layer used for the reaction. 1OmMATP appeared to be optimum for the reaction. Dilution buffer (containing 2 mM Me), H-broth, and M9S have been previously described (Black and Ahmad-Zadeh, 1971). Nucleoside triphosphates were dissolved to 250 mM in 1 M Tris base. Other procedures have been described previously. Materials. Pancreatic DNase I, chlor-

BACTERIOPHAGE

amphenicol, 9-aminoacridine, and ethidium bromide were from Sigma Biochemicals. Nucleoside and deoxynucleoside triphosphates were from PL Biochemicals. Coumermycin Ai was a gift of Dr. W. F. Minor of Bristol Laboratories. Rifampicin and nalidixic acid were purchased from Sigma Biochemicals. Antiserum against bacteriophage T4 was prepared by injecting rabbits with purified phage. RESULTS

339

T4 DNA TABLE

1

PHAGE T4 IN VITRO PACKAGINGREQUIREMENTS Components cs20-rll extract, complete -DNA -Extract

Phage yield 1.4 x 10’ 0 0

-ATP (10 mM) +EDTA (50 ml@

0 0

+DNase (20 pg) (added at 0 min) +T4 antiserum (added following incubation)

0 140

Bacteriophage T4 DNA packaging in

vitro has not been previously reported. A number of factors may create difficulties: instability of the phage precursor components, inefficiency of the reaction, high levels of unabsorbed phage or of phage progeny due to leakiness of the mutant infections used to form extracts, and inability to uncouple synthesis of late phage components required for packaging from DNA replication in viva. In relation to the problem of instability, the T4 packaging system appears to have different requirements from other phage systems due to sensitivity to freezing and thawing; chloroform can however be used to prepare the extracts. Phages which can be propagated as defective lysogens (e.g. X or P22) (Kaiser and Masuda, 1973; Hohn, 1975; Poteete et al., 1979), or in which late structures can be synthesized without concurrent synthesis of endogenous competing DNA (e.g., T7 and T3) (Kerr and Sadowski, 1974; Fujisawa et aZ.,1978), achieve very low background levels of phages against which to measure’increases from in vitro packaging. This can be achieved for phage T4 by combining the T4 head assembly mutants of interest with rll deletion mutants. Then by plating upon X lysogens, exogenous rll+ DNA packaging into active phage structures can be selectively scored in an in vitro system similar to that originally introduced for X by Kaiser and Masuda (1973). The requirements of T4+ DNA packaging are shown in Table 1. A processed prohead-containing extract, rll+ mature or concatemeric DNA, and ATP and Mgz are all necessary for phage formation.

Addition of T4 antiserum following the incubation, or of pancreatic DNase at the beginning of incubation, reduces or eliminates phage formation, suggesting that the assay on the X lysogen does indeed measure in vitro formation of normal phage structures from exogenous DNA. The cs20-rll mutant-infected bacterial extract, which contains all essential components for phage formation, was the reference extract for further characterization of the in vitro system. The phage yield is a linear function of the amount of rll+ DNA added until a plateau is reached (Fig. 2). The yield depends also upon the amount of infected extract added, until a maximum value is reached, with some decline in yield at higher concentration of extract (Fig. 3). One microgram of mature T4 DNA yields about lo4 to lo5 phage in this assay. In addition, it can be calculated that about 10e5of those proheads present in the extract at 90 min can be filled with exogenous DNA to give

FIG. 2. The formation of rll+ T4 phage (titered on E. coli CAlgO) is measured as a function of the quantity of mature phage T4+ DNA added in the standard in vitro packaging system.

LINDSAY

340

(II+

W. BLACK

12.3

Phage -10’ 6,4

I

400

!

800

1200

pi Bacterial

Extroci

FIG. 3. The formation

1 1600

of rll+ T4 phage (titered

on

E. coli CAl80) is measured as a function of the quantity of infected bacteria (2 X 108 ml) used to prepare the standard in vitro packaging system. A constant amount of exogenous rI1’ DNA was added.

a plaque. This can be estimated from the 20-40 phage/infected cell which are formed in an 20cs-2lts-tam infection at 90 min following shift to high temperature (Black and Silverman, 1978). This calculation may underestimate the efficiency of de novo packaging of all DNA, since there is considerable endogenous concatemeric DNA in the extract which may compete. Exogenous concatemeric DNA isolated from a gl7am or g24am infection is also packaged in the in vitro system, but with at least 20- to loo-fold lower efficiency than mature DNA (Table 2). Concatemeric DNA was isolated from several mutant infections which accumulate T4 concatemers at late times. In addition, a nucleasedeficient E. coli host (SG5010), as well as the host (E. coli P301) used for in vitro extract formation, gave similar results for concatemeric T4 DNA preparation (see Table 2). Therefore it would be expected TABLE

IN VITROPACKAGING CONCATE-MERIC DNA

2 OF MATURE T4 DNA

Source

Amount (4

AND

Phage yield

‘r4+

Phage particle

1.6

1.4 x

24hzNG433)

Phage particle

0.5

5.7 x lo’

24(amNG433)

Phage particle

1.3

1.2 x 106

24amNG433)

Concatemeric (E. coliP301)

0.9

4.8

x 16

ZWamNG433)

Concatemeric (E. m.!i SG5010) Coneatemeric (IiT. coli SG.5010)

1.5

3.3

x lo”

1.8

9.7 x lop

1’7(amNG1’78)

106

3

I5

30 45

60

75

90 105 120

hllfl

FIG. 4. The formation of rll+ T4 phage in the standard in vitro packaging system is measured as a function of time of incubation at 3’7”. The reaction was terminated by the addition of pancreatic DNase containing dilution buffer and chloroform at the indicated times.

that this DNA should be isolated in a competent, intact state for packaging. The rate of phage formation in the in vitro packaging system upon incubation at 37” (Fig. 4), is comparable to the rate of maturation of es20 proheads in vivo at 3’7” (Black and Silverman, 1978). After a 2-min lag period, the first phage is produced at 3 min following incubation at 37”. A plateau value is reached by 30 min at 37’. Phage formation in the in vitro system is somewhat temperature sensitive even though the cs20 mutant grows well at higher temperatures. Relative yields at 25, 30, 37, 39.5, 42, and 46” were 47, 100, 38, 16, 1.2, and 0% respectively. Addition of spermidine or polyamines is not apparently required for in vitro encapsidation, and in fact, at higher concentrations inhibited formation of phage (Table 3 and data not shown). However, ATP or other nueleoside triphosphate is absolutely required for phage formation; dATP is equivalent to or superior to ATP, and CTP and UTP were much less effective, but also give appreciable yields. The other four nucleoside and deoxynucleoside triphosphates were not active. Protein syn-

BACTERIOPHAGE

T4 DNA

341

fected cell can donate gp16 and gp17, but not the prohead. Thus it can be concluded PHAGE T4 IN VITRO PACKAGINGREQUIREMENTS that gpl6 and gpl? are required for in viANDINHIBITOR~ tro packaging, and are apparently supplied by the g20am-g23am-infected extract. It Relative phage yield also appears that the processed T4 proComponents m,) head found to accumulate in gene 16 and 17 and 20~smutant-infected bacteria is the 100 -Spermidine substrate for DNA packaging (Table 4). 0 -ATP (IO mM) This conclusion is also supported by glyc-ATP + dATP (10 mM) 100 erol gradient centrifugation of radioactive 5 -ATP + UTP (10 mM) infected extracts, which suggested that 3 -ATP + CTP (10 mM) packaging activity coincides with the pro100 +Chloramphenicol (67 Ng/ml) cessed prohead peak (data not shown). 65 +Coumermycin (30 &ml) However the yield has so far been ex+Rifampicin (67 pg/ml) 100 10 +Ethidium bromide (3.3 pg/ml) tremely low (about 10 phage) upon frac12 +9-Aminoacridine (12 kg/ml) tionation of the extracts. Requirement for 0 +9-Aminoacridine (40 ag/ml) the gene 16 product is apparently slightly leaky in vitro as in vivo (Granboulan et al., 1971), as is seen by a low but significant thesis (chloramphenicol), RNA synthesis phage yield with the gene 16 double amber (rifampicin), and E. coli DNA gyrase in- mutant (Table 4, line 4). Gene 16am and hibitors (coumermycin) do not signifigene 17ctm mutant-infected bacteria apcantly inhibit phage yield at concentra- pear not to complement each other in vitro tions known to be effective. However, (Table 4, line lo), although of course these ethidium bromide and 9-aminoacridine in- two genes complement in viva, suggesting hibit phage formation in vitro quite both gene products must be synthesized strongly (Table 3). 9-Aminoacridine is a in the same cell to obtain efficient in vitro known inhibitor of the phage T4 DNA packaging step in viva (Piechowski and TABLE 4 Susman, 1967). PHAGE T4 IN VITRO PACKAGINGBY IN VITRO By mixing infected bacteria, in vitro COMPLEMENTATIONOF DEFECTIVE EXTRACTS complementation of extracts can be used to investigate the T4 late components rePhage yield quired for in vitro packaging. An extract lacking late T4 components (g55am infecExtract(s) -DNA +DNA tion) is inactive (Table 4, line 13). A gene 20~s or gene 1’7~smutant-infected extract 1. g20csrll 0 2.0 x lo4 2. gl7csrll 4.0 x 103 0 contains all essential components for phage 3. gl7umrll 0 0 formation, however gl?am, gl6am, g24am, 4. gl6amamrll 0 25 and g20am-g23am mutant-infected ex5. gl6am-gl7amrll 0 0 tracts cannot give rise to significant phage 6. g20am-g23amrll 0 0 yields. However, gl6am, gl’lam, and 7. g20am-g23amrll gl6am-gl’7um mutant-infected extracts -t gl7amrll 0 5.5 x lo3 can be complemented by g20am-g23am 8. g20am-g23amrll mutant infected bacteria (Table 4, lines 7+ gl6amamrll 0 6.1 x ld 9). gl6am, gl’lam, gl7cs, and g2Ocsmutant 9. g20am-g23amrll infections result in accumulation of pro+ gl6am-gl7amrll 0 2.1 x lo4 cessed proheads and concatemeric DNA 10. gl6amamrll -t gl7amrll 0 25 in vivo (Wunderli et al., 1977; Hsiao and 0 22 Black, 1978; unpublished) and a g2Oum- 11. g24amrll g23am mutant infection accumulates no 12. g24amrll + g20amg23amrll 0 130 known head structure (Laemmli et aZ., 13. g55amrll 0 0 1970). Therefore the g20am-g23am-inTABLE

3

LINDSAYW.BLACK TABLE5

vitro recombination seen are comparable to those occurring in vivo. The requirement for phage T4 early gene products for this in vitro genetic recombination is presCAlsOSuf ently being investigated.

PHAGE ~4 IN VITRO RECOMBINATIONOCCURSDURING IN VITRO PACKAGING OF EXOGENOUS DNA’

wslo2(x)sU1.PhageT4 2.c&?&l1 extract+ T4+ DNA 3.&?orIIextract+ 24(amNG43S)DNA

213

333

881

1111

160

1155

Genotypes of 100plaques onCA1130

82m.24(NG433) r11+ca+ 14am+ r11+c* 4ant+ rllf cs+ 100

‘In 2,T4+matureDNA,andin 3,24ambermatureDNA,wereused 8sexogenous DNA8in the standardin vitro packagingsystem.In 3, 100plaquespickedst randomfrom B CA180plate weretestedfor genotype (rll-es20-24anNG43.3 markers)bystandardgrowthandcx~mplementation spottestingwith theresultsshown.It is likely that the c&9-24am NG433 genotype isselected againstundertheCA180 growth conditions.

packaging. This could be explained if g16 or g17 product formed a defective complex in the absence of the other protein. Order of function studies appear to show that both of these gene products act in the same step in DNA phage morphogenesis and interact with the gene 20~s mutation (Fig. 1). Finally, gene 24 mutant infections accumulate the early, unprocessed proheads (Tau-particles) (see Fig. 1) which in the case of gene 24ts mutant infections can be matured in viva (Bijlenga et cd., 1973). A gene 24am mutant which is known to give rise to the unprocessed T4 prohead (Tauparticle), appears to form a very much lower but possibly significant level of phage in this assay upon complementation with g20am-g23am, suggesting that the unprocessed prohead can also be matured in vitro at much lower efficiency (Table 4, lines 11-12). A high level of genetic recombination occurs during T4 DNA packaging in vitro between genetic markers in the exogenous, mature DNA and in the endogenous concatemeric DNA. In addition, recombination occurs between closely linked markers and multiple recombinants are formed (Table 5). Recombination also occurs if concatemeric DNA is the packaging substrate (data not shown). The levels of in

DISCUSSION

In vitro encapsidation of phage T4 DNA displays the same basic requirements as are seen for most other phage DNA packaging systems. This is consistent with other studies of phage T4 head assembly, which suggest T4 head formation and DNA packaging are fundamentally the same as in the other complex, doublestranded DNA phages (see Murialdo and Becker, 1978). The essential components are the processed, active prohead (~OCS, 17cs, 16am, or 17um mutant-infected bacterial extract), exogenous DNA, the T4 packaging/linkage proteins (gp16 and gp17), and an energy source, supplied by ATP or other nucleoside or deoxynucleoside triphosphates (dATP, and to a lesser extent UTP or CTP). DNA encapsidation is inhibited by DNA intercalcating compounds (9-aminoacridine, ethidium bromide) known to prevent packaging in vivo. The efficiency of DNA packaging into the T4 prohead measured in this system is poor (lob5 with respect to prohead and 10M5 to 10e6with respect to DNA), but not qualitatively different from that displayed by most other in vitro packaging systems (e.g., P22, 10e3, Poteete et cd., (1979). Fractionation of a radioactive extract on sucrose or glycerol gradients has so far led to very substantial losses in capacity to form phages. Therefore, the conclusion that the processed prohead is the precursor for DNA packaging is presently only weakly supported by direct identification of a radioactive prohead active in vitro. However, this conclusion is strongly supported by the in vitro complementation experiments which show extracts accumulating such proheads are active in packaging DNA in vitro. In addition, the unprocessed T4 prohead (Tau-particle) can possibly be matured with much lower efficiency (Table 4). It is likely that the exogenous mature

BACTERIOPHAGE

T4 DNA supplied is largely, if not entirely, packaged followingrecombination into the endogenous concatemeric DNA. Analysis of T4 mutant infections strongly suggests that concatemeric DNA is the natural substrate for packaging. For example, g16, g17, and 20~s mutant infections, which are apparently blocked immediately before packaging, result in accumulation of concatemeric DNA as well as cleaved proheads (Fujisawa and Minagawa, 1971; Hsiao and Black, 1978). Therefore it is probable that the exogenous DNA recombines into a concatemer which is competent for packaging. Preferential packaging efficiency of exogenous mature T4 DNA over exogenous concatemeric DNA could reflect difficulties in isolating the latter DNA without damage, although this was carefully isolated from an endonucleasedeficient host. We have found that following normal replication, a requirement for DNA ligase is maintained at late times to allow completion of T4 DNA packaging in viva, and infer that nicks in the DNA apparently reversibly interrupt completion of DNA encapsidation (Hsiao and Black, 1977; Zachary and Black, 1981; Black et ah, 1981). Therefore, since T4 late concatemerit DNA is probably nicked in viva (Cascino et& 1970), there is likely to be a requirement for DNA repair processes to successfully package such DNA in vitro. Consequently, the mature, exogenous DNA might be more efficiently packaged because it has fewer nicks. However, it is also possible that the mature T4 DNA is more efficiently or preferentially packaged into the proheads in these extracts because the higher concentration of DNA ends more efficiently initiates packaging. The high level of genetic recombination observed in the in vitro system is consistent with the very active T4 in vivo recombination system and suggests the system may be of use as an assay for T4 in vitro recombination. Such recombination is consistent with the hypothesis that the added mature DNA probably recombines with the endogenous concatemeric DNA to enter the prohead. It is possible therefore, that the assay depends upon and measures in vitro recombination as well

T4 DNA

343

as DNA packaging. However, the evidence is strong that all of the plaques counted result from packaging of exogenous DNA, so that the system appears to be an accurate assay for DNA encapsidation. Whether and to what extent genetic recombination is required for T4 DNA encapsidation, and how this relates to T4 functions required for recombination in Go, are presently under investigation. ACKNOWLEDGMENT

This work was supported by NIH grant AI-11676. REFERENCES

AEBI, U., BIJLENCA, R., BROEK,J. v.d., BROEK,R. v.d., EISERLING,F., KELLENBERGER,C., KELLENBERGER, E., MESYANZHINOV,V., MULLER, L., SHOWE, M., SMITH, R., and STEVEN, A. (1974). The Transformation of Tau particles into T4 Heads. J. SupromoL strllct. 2.253-275. BIJLENGA, R. K. L., SCRABA,D., and KELLENBERGER, E. (19’73).Studies on the morphogenesis of the head of T-even phage IX. Tau-particles: Their morphology, kinetics of appearance and possible precursor function. VirdoQy 56, 250-267. BLACK, L. W., and AHMAD-ZADEH, C. (1971). Internal proteins of bacteriophage T4D: Their characterization and relation to head structure and assembly. J. Mol. Biol. 51. 71-92. BLACK, L. W., and SILVERMAN,D. J. (1978). Model for DNA packaging into bacteriophage T4 heads. J. ViroL 28,643-655. BLACK, L. W., ZACHARY,A. L., and MANNE, V. (1981). Studies of the Mechanism of Bacteriophage T4 DNA Encapsidation in Phage Morphogenesis, M. DuBow ed., Alan Liss, N. Y., in press. CASCINO,A., RIVA, S., and GEIDUSCHEK,E. P. (1970). DNA ligation and the coupling of T4 late transcription to replication. Cold Spring Harbor Sgm Quunt. Biol. 35, 213-226. EISERLING,F. A. (1980). Bacteriophage structure. In “Comprehensive Virology” (H. Frankel-Conrad and R. R. Wagner, eds.), Vol. 13, pp. 543-575. Plenum, New York. FRANKEL, F. R. (1968). Studies on the nature of replicating DNA in T4-infected Escherichia coli. J. MoL Bid 18.127-143. FUJISAWA, H., and MINAGAWA, T. (1971). Genetic control of the DNA maturation in the process of phage morphogenesis. Virw 45.289-291. FUJISAWA, H., MIYAZAKI, J., and MINAGAWA, T. (1978). In vitro packaging of phage T3 DNA. Viro~87.394-466. GRANBOULAN, P., SECHAUD, J., and KELLENBER-

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