Effects of Genome Size on Bacteriophage 6x174 DNA Packaging in Vitro *

Effects of the size of template DNA on the DNA packaging reaction of bacteriophage 4x1 74 were studied using plasmids of various sizes which contain the #X 174 origin of DNA replication and the in vitro phage synthesizing system (Aoyama, A., Hamatake, R. K., Hayashi, M. Proc. Natl. Acad. Sci. U. S. A. 80, 4195-4199). DNA between 78.5% and 101% of the length of 4x174 DNA produced infectious particles efficiently. Packaging of DNA smaller or larger than this range produced uninfectious defective particles. Although these particles contained circular single-stranded DNA, they suffered structural changes which altered the sedimentation properties or the ability to adsorb to the cells. Mutant phage were found from the packaging reaction of DNA larger than 101% of 4x1 74 DNA. These mutants deleted the termination region of DNA, suggesting that they were produced by early termination of the phage synthesizing reaction.

viral strand between nucleotides 4305 and 4306 (14,15). To understand the mechanism of the DNA packaging process during stage 111, the size of the genome packagable to the phage capsid is one of the important morphogenetic parameters. This problem was studied in vivo in several laboratories using recombinant DNA technique (16-18). van der Ende et ul. (16) cloned various parts of 4x174 RF DNA with 4x174 HueIII-Z6B fragment which contains the origin of 4x174 viral DNA synthesis into plasmid pACYC 177 to make recombinant plasmids with various sizes (16). When Escherichia coli cells that carry these recombinant plasmids were infected with 6x174, phage particles that contain recombinant plasmid ssDNA are co-produced with 4x174 phage. Using this assay method, they showed that the lower limit of the genome size is between 3.96 kb and 4.41 kb and the upper limit is between 5.58 kb and 5.70 kb. DNA smaller than this limit could be packaged but the resultant phage particles were uninfectious. The upper limit of the genome size was studied by Muller and Wells (17) and Russell and Muller (18) in more detail. The mutants of 4x174 that have the insertion of DNA fragments in the intercistronic region between genes J and F of the genome were used for this purpose. They found that the upper limit could be at least 6.09 kb. However, the phage particles with genomes larger than 5.55 kb were highly unstable. Thus, the size of genome packagable within the capsid has been established.
In this report, the packaging of DNA smaller or larger than the limit was studied using the in vitro phage synthesizing system developed in our laboratory (19). The system is composed of the purified E. coli and the viral enzymes and is capable of synthesizing infectious phage with the addition of 4x174 RF I DNA. Recombinant plasmids of various sizes were constructed by cloning 4x174 HincII-3 DNA fragment, which contains the origin of 4x174 viral DNA synthesis, into plasmid pBR322, pBR325, or their derivatives. These were used as template in the in vitro phage synthesizing system. During these experiments, we found mutants which had deleted DNA. Characterization of these deletion mutants as well as other phage particles synthesized in the in vitro system are described.

MATERIALS AND METHODS
Bacteria and Plusmids-Escherichia coli strains used were HF4704 (6x174') and C600 SF8 (recBC-Jopll). Plasmid pKJB51, a derivative of plasmid pBR322 with a deletion in the DNA sequence between the BamHI site and the PuuII site (nucleotides 375-2069 of pBR322), was constructed by Dr. K. J. Buckley in our laboratory. Plasmid pAS976, also a derivative of plasmid pBR322 with a deletion in DNA sequence between the EcoRI site and the BamHI site, was a gift from Dr. A. Shafferman in Dr. D. R. Helinski's laboratory. Plasmid pAS976 was derived by digesting pBR322 with EcoRI, filling the 3"recessed ends, ligation with BamHI linkers, and cleaving with BamHI and religation. Therefore, this plasmid has both an EcoRI site (restored by the end C nucleotide of the BamHI linker) and a BamHI site (20).
Isolation of DNA and Enzymes-The RF I DNA of 4x174 and plasmids were purified as described previously (21). The 6x174 gene A protein, gene C protein, gene J protein, prohead, E. coli DNA polymerase 111 holoenzyme, rep protein, and dUTPase were purified as described previously (19).

Construction of Various Sizes of Recombinant Plasmids Containing
Origin of 4x174 Viral DNA Synthesis-The 4x174 HincII-3 DNA fragment (nucleotides 4200-4812) (1) has been previously cloned into the EcoRI site of the plasmid pACYC184 using EcoRI linkers (21). The resulting recombinant plasmid, pH24, was used as a source of rbX174 HincII-3 DNA fragments flanked by EcoRI linkers. The 6x174 HincII-3 DNA fragment was reisolated by digesting the recombinant plasmid pH24 with EcoRI, followed by gel electrophoresis. The DNA fragment was introduced into the EcoRI site of plasmid pKJB51, a pBR322 derivative with a deletion between the BamHI and the PuuII sites (nucleotides 375-2069 of pBR322), pAS976, a pBR322 derivative with a deletion bet,ween the EcoRI and the BamHI sites, pBR322, or pBR325 DNA. E. coli C600 SF8 cells were transformed by the ligated products. Transformants with an ampicillinresistant phenotype were selected and their DNA was isolated by an alkaline SDS method (22). The recombinant DNA that contained the 4x174 gene A protein cleavage site contiguous with the L strand of the plasmids were selected based on the digestion pattern of the restriction enzymes Hinfl and EcoRI. The recombinant plasmids derived from pAS976 (~4x4500) and pBR325 (~4 x 6 6 0 0 ) were further cut by AuaI and digested by exonuclease Bal31 for various time periods, and the digested DNA molecules were ligated. E. coli C600 SF8 cells were transformed by the final ligated products and the transformants with an Amp-resistant phenotype were selected. The DNA was purified by an alkaline SDS method and their sizes were identified by gel electrophoresis. Eleven recombinant plasmids were selected from our collection for studies. Each recombinant plasmid contains the +X174 HincII-3 DNA fragment in the same orientation and carries Amp resistance, but varies in size from 60% to 123% of the size of 6x174 RFI DNA.
In Vitro Stage 1 1 1 Reaction-The complete reaction mixture (25 pl) cont.ained 50 mM Tris-HCI (pH 7.5), 10 mM 2-mercaptoethanol, 20 mM MgCI2, 0.09 mM concentration each of dATP, dGTP, dCTP, and [3H]dTTP (400-1600 cpm/pmol of deoxyribonucleotides), 0.8 mM rATP, 0.1 mg/ml bovine serum albumin, 0.1 pmol of template DNA, 280 ng of the 6x174 gene A protein, 75 ng of the 4x174 gene C protein, 48 ng of the 4x174 gene J protein, 20 pg of the prohead, 120 ng of E. coli DNA polymerase 111 holoenzyme, 7 ng of E. coli rep protein, and 4 units of E. coli dUTPase. The reaction mixture was incubated a t 30 "C for 30 min and cooled on ice. To examine the DNA synthesis or the packaging reaction, acid-insoluble radioactivities or DNase-resistant, acid-insoluble radioactivities of the 7-pl reaction mixture were determined as described previously (19). The infectivity of the product was determined by the titration of the diluted samples from the reaction mixture as previously described (21).
I n Vitro Stage [I(+) Reaction-The complete reaction mixture (25 p l ) contained 50 mM Tris-HCI (pH 7.5), 10 mM 2-mercaptoethanol, 5  dTTP as a radioactive precursor and the reaction mixture was four times larger than the standard reaction mixture. After incubating a t 30 "C for 30 min, the reaction mixture was cooled on ice and loaded onto a 15-30'36 sucrose density gradient made with a buffer containing 50 mM Tris-HCI (pH 7.5), 10 mM MgClz, and 0.1 M NaCl with the cushion of 50% sucrose made in the same buffer. The sample was centrifuged at 49,000 rpm for 70 min in a Beckman SW 50.1 rotor at 4 "C. The sample was fractionated into glass tubes (0.13 ml/tube). The trichloroacetic acid-insoluble radioactivities and the infectivities of the fractions were determined as described previously (19).
Adsorption Experiment-The procedure was the modified method of Newbold and Sinsheimer (23). E. coli HF4704 cells were grown in a tryptone/KCl (1% and 0.576, respectively) medium containing 10 mM sodium phosphate (pH 7.2) and 20 pg/ml thymidine to Asonrn = 0.8 (4-5 X 10' cells/ml) at 37 "C. The 32P-labeled stage 111 products were added in less than a 100-pl volume to 3 ml of the cell culture in a centrifuge tube a t 37 "C. The mixture was brought to 10 mM MgC12 and 5 mM CaCI2 and incubated a t 37 "C for 10 min. A 1.5-ml portion was removed to assay total radioactivity and the remaining 1.5 ml was centrifuged to pellet the cells. The supernatant fluid was removed and the pellet was resuspended in 1.5 ml of the tryptone/KCl broth. The supernatant fraction was added to a previously prepared pellet containing the original number of uninfected cells. The three 1.5-ml samples were mixed with 1/10 volume of cold 80% trichloroacetic acid and allowed to stand at 0 "C for 1 h. The precipitate was collected by filtration through a Whatman glass filter disc, rinsed with cold 6% trichloroacetic acid, dried, and counted.

Stage 111 Reactions Using Recombinant Plasmid DNA as
Template-The in vitro stage I11 system contains purified 4x174 gene A protein, gene C protein, gene J protein, prohead, E. coli rep protein, DNA polymerase I11 holoenzyme, and dUTPase (19). The system synthesizes and packages 4x174 viral strand DNA when 4x174 RF I DNA is added as template (4x174 system). The final product of this reaction is infectious 6x174 phage particles which contain 4x174 circular viral strand DNA. Previously we showed that the 4x174 HincII-3 DNA fragment, which contains the origin region of 4x174 DNA replication, cloned into the plasmid pACYC184 supports the stage 111 reaction when added to the stage I11 system as template (21). The final product of this reaction was the 6x174 phage particles that contained either strand of the recombinant plasmid DNA depending on the orientation of the 4x174 HincII-3 DNA fragment in the template DNA. The chimeric phage particles were infectious. The recombinant plasmid ssDNA was transferred to the host cell upon infection and converted to dsDNA in the cell. This process yielded the Amp-resistant transformants. To study the effects of the size of the template DNA in the stage I11 reaction, recombinant DNA with various sizes were constructed by cloning the 4x174 HincII-3 DNA fragment into EcoRI site of plasmid pBR322, pBR325, or their derivatives. Eleven recombinant plasmids that have 4x174 HincII-3 DNA fragment in the same orientation were selected for studies (Fig. 1). The size of DNA appears in base pairs in the name of each plasmid. The ability to produce the infectious phage particles in the in vitro system was dependent on the size of the template DNA (Fig. 2). The upper and the lower limits of the size of the template DNA which can produce the infectious phage particles as efficiently as the 4x174 RF I DNA were between 101% (~4x5400) and 78.5% (~4 x 4 2 0 0 ) of 6x174 RF I DNA. The efficiency of producing infectious phage particles dropped rapidly when the template DNA was larger than 101% or smaller than 78.5% of 4x174 RF I DNA. However, all recombinant plasmid DNA tested were as active as 4x174 RF I DNA in supporting DNA synthesis or packaging the DNA into prohead (Table  I). These results indicate that uninfectious defective phage particles are made during the packaging reaction of DNA larger than 101% or smaller than 78.5% of 4x174 RF DNA.
Characterization of Products of Stage ZZI Reaction Using Recombinant Plasmid DNA as Template-The recombinant plasmid DNA were classified into three groups according to the size (Fig. 1) and the ability to support the production of infectious phage (Fig. 2): group A includes ~4 x 3 2 5 0 , 3350, and 3650; group B includes ~4 x 4 2 0 0 , 4500, 4850, and 5400; group C includes p4X5650,5950,6450, and 6600. The recombinant plasmids from group A (p$X3250), group B (~4 x 4 2 0 0 , 4850, 5400), and group C (~4 x 6 6 0 0 ) were used to examine the structure of the products. As previously shown (19), the product of the 4x174 system contained materials sedimenting with the s value of 114 S and 90 S (Fig. 3a). The 114 S materials (fractions 6 to 9) were associated with infectivity as in uiuo phage. The 90 S materials (fractions 15 to 19) were defective particles with no infectivity. The plasmids from group B produced a similar sedimentation profile (Fig. 3, c, d,  and e). However, the plasmids from group A or C produced only one major peak that showed no infectivity (Fig. 3, b or f). The infectious phage was produced with low efficiency in these systems and sedimented faster than the major peak of defective phage particles. The DNA extracted from the defective particles of group A or C system as well as those from infectious or defective particles in group B system migrated with the marker ss circular DNA of the respective template plasmid during gel electrophoresis (data not shown). The DNA in the defective particles of group B or C was susceptible to DNase I, whereas the DNA in the infectious particles of group B was not (Fig. 4). The degradation products from the DNase I treatment contained a mixture of relatively long DNA fragments and short DNA fragments. The DNA in the defective particles in group A was resistant to DNase I.
The phage made in the group B system (Fig. 5c, fractions 7 to 10) adsorbed to the cells as efficiently as 4x174 114 S phage (Fig. 5a, fractions 6 to 9). The products of the group A system showed little ability to adsorb to the cells (Fig. 5b,  fractions 11 to 16). The slow sedimenting defective particles observed in the 6x174 system (Fig. 5a, fractions 15 to 18), group B system (Fig. 5c, fractions 13 to 16), or group C system (Fig. 5d, fractions 13 to 16) showed lower adsorption to the cells when compared to the infectious phage.
Deletion Mutants Made in Group C System-To study the nature of infectious phage made in low efficiency in group A or C system, the DNA were isolated from cells infected with these products and analyzed by gel electrophoresis (Fig. 6).  The DNA isolated from the cells infected with group A products co-migrated with the respective template DNA as did those of group B products (Fig. 6a, I to 7). However, the reaction mixtures containing various template DNA were incubated and analyzed by sucrose density gradient centrifugation as described under "Materials and Methods." The trichloroacetic acid-insoluble radioactivities (0) and the infectivities (0) of aliquots were determined as described previously. The infectivity were represented as described in the legend to Fig. 2. The template DNA used were: a, 4x174; b, ~6x3250; c, ~6x4200; d, ~6x4850; e, ~6x5400; and f, ~6x6600.
DNA from the cells infected with the group C products migrated faster than the respective template DNA (Fig. 6u, 8  to 11). In order to examine the difference between in vivo DNA and the template DNA used for the reaction mixture, the DNA was digested with EcoRI ( Fig. 6b), with Hinfl, or double-digested with EcoRI and Hinfl (Fig. 6, c and d). The DNA from the cells infected with the group Aproducts carried the 6x174 HincII-3 DNA fragments (Fig. 6b, 1 to 7) and showed identical restriction enzyme digestion patterns to the respective template DNA as did those of group B system (Fig.  6 , l t o 7 in c and d). However, the DNA from the cells infected with the group C products lost one of the EcoRI sites, which resulted in the loss of the 4x174 HincII-3 DNA fragments (Fig. 66, 8 to 11). The restriction enzyme digestion analyses by Hinfl and EcoRI showed that these DNA lost one or more of the restriction fragments adjacent to the termination point of the viral DNA synthesis (Fig. 6 , s t o 11 in c and d).
The DNA sequences of the initiation/termination region of four DNA isolated from the cells infected with the products of group C system were analyzed (Fig. 7). All DNA contained the 5'-end portion of the 4x174 HincII-3 DNA fragment from the nucleotide 4306 (the initiation site of the viral DNA synthesis) but lacked the 3'-end portion from the nucleotide 4305. The nucleotide 4306 of the 4x174 HincII-3 DNA fragment fused to various sites of the plasmid pBR325 DNA portion. This introduced various changes in the DNA sequence of the initiation/termination region of stage I11 reaction. The template activities of these DNA were examined in the stage I11 system (Table 11). The DNA D-14, D-34, and D-36 showed considerable template activities to support the synthesis and the packaging of the DNA and the production of infectious phage particles. The DNA D-4 showed little template activity.

DISCUSSION
Effects of the size of template DNA on the DNA packaging of 4x174 has been studied. The summary of the results are  Fig. 3 were digested with 10 pg/ml DNase I at 30 "C for 20 min. The DNA was extracted by incubating the sample with 0.5 mg/ml of pronase in 0.1% sodium dodecyl sulfate solution at 25 "C for 1 h. The samples were run through 1% agarose gel as described previously (21). The gel was dried under vacuum and the autoradiography was carried out. The markers were prepared by extracting DNA from the in vitro stage II(+) reaction mixtures (see "Materials and Methods") containing various template DNA. The in vitro stage II(+) system is capable of synthesizing circular ssDNA when 6x174 RF I DNA (25) or the recombinant plasmid DNA which carries 4x174 HincII-3 DNA fragment was added as template? Columns 1, 2, 3, marker, fractions 6 to 9, 15 to 19 in Fig. 313, respectively, for 6x174. Columns 4, 5, 6, marker, fractions 11 to 13, 14 to 17 in b, respectively, for ~6x3250. Columns 7, 8, 9, marker, fractions 8 to 11, 13 to 17 in d, respectively, for ~~~5x4850. Columns IO, 11, marker, fractions 13 to 17 in f, respectively, for ~6x6600.
shown in Table 111. The size of DNA packagable in the 6x174 capsid was previously determined by in vivo studies by van der Ende et ul. and Muller and co-workers (16-18). The lower limit is between 3.96 kb and 4.41 kb (74% and 82% of 4x174 DNA) and the upper limit between 5.50 kb and 5.70 kb (102% and 106%). A similar result has been obtained in the in vitro phage synthesizing system. Only group B plasmid, whose genome sizes are between 4.20 kb and 5.40 kb (78.5% and 101%), could produce infectious phage as efficiently as 4x174 RF I DNA. The packagable size of the genome has been thus established both in in vivo and in vitro DNA packaging systems.
Although only group B DNA could produce infectious phage efficiently, group A, whose genome sizes are between 3.25 kb and 3.65 kb, or group C , whose genome sizes are between 5.65 kb and 6.60 kb, could also produce phage particles. However these particles were not infectious. This may be due to the A. Aoyama  infected with stage 111 products. A single colony with Amp-resistant phenotype was randomly selected and the cells were grown in LB broth (26) containing 80 pg/ml ampicillin. DNA was extracted by an alkaline-SDS method and analyzed by agarose gel electrophoresis as described previously (21). The gel was stained with 1 pg/ml ethidium bromide solution and a photograph was taken under the ultraviolet lamp. a, DNA with no treatment. b, DNA digested with EcoRI. c, DNA digested with Hinfl. d, DNA digested with Hinfl and EcoRI. I, ~6x3250; 2, ~6x3350; 3, ~6x3650; 4, ~6x4200; 5, ~6x4500; 6, ~6x4850; 7, ~6x5400; 8, ~6x5650; 9, ~4x5950; IO, ~6x6450; 11, ~6x6600. Left and right lunes of each number show the template DNA for the reaction mixture and the in vivo DNA isolated from the cells, respectively. inability to adsorb to the cells of these particles (Fig. 5), but not due to the problem on the packaged DNA because these particles contained ss circular DNA? The particles suffered A. Aoyama and M. Hayashi   structural changes that affected the sedimentation properties (Fig. 3). Understanding the structure of these defective particles may be helpful to elucidate both the structure of phage and the mechanism of the DNA packaging. Since the prohead contains 4x174 gene F, G , H, B, and D proteins (3), while the mature phage contains gene F, G, H, and J proteins, gene B and D proteins must be removed from the prohead structure, and gene J protein must be added to the phage particle during or after DNA packaging. Some of these processes may be affected by packaging DNA of group A or C, producing defective particles. In these particles, gene B or D proteins could remain, or the amount of gene J protein could be altered.
Further studies should clarify this problem. Defective particles similar to those in group C system were observed in group B system (Fig. 3). DNA in these particles were susceptible to the action of DNase I (Fig. 4), indicating that DNA is partially exposed to the outside of the particles. Similar defective particles were observed in gene H mutant infected cells (24).
Therefore, these deficient particles could have a similar structure to the particles made in gene H mutant infected cells.
During the studies of the DNA isolated from the chimeric phage infected cells, we found that the group C system produced deletion mutants. All infectious particles tested in group C contained DNA smaller than the template plasmid  DNA (Fig. 6). DNA sequence analyses showed that the deletion started at various points on the plasmid portion of the recombinant plasmid and ended at the initiation/termination point of 4 x 1 7 4 viral DNA synthesis (nucleotide 4306) (Fig.  7 ) . This indicates that the early termination of stage 111 DNA replication occurred a t various points on the template DNA.
This early termination produces a phage particle that contains ss circular DNA smaller than the unit length of the template DNA. The sizes of all such deleted DNA were similar to those of group B DNA' (see Fig. 6). Only the phage particles carrying DNA with sizes similar to those of group B retained infectivity because of the size requirement for infectious phage production (Fig. 2). Such early termination of stage I11 reaction may occur either by ligating the pre-existing nick on the template DNA or by nicking/closing activity of 4 x 1 7 4 gene A protein. In either case, this produces the recombinant plasmids that have mutations in the origin region of 4 x 1 7 4 DNA replication. These mutant DNA produced infectious phage with several different efficiencies (Table 11). Therefore, these mutants may be useful to study the interaction between origin region of DNA and gene A protein or other proteins required for stage 111 reaction.