Fate of cloned bacteriophage T4 DNA after phage T4 infection of clone-bearing cells

J. Mol. Biol. (1983) 170, 343-355

Fate of Cloned Bacteriophage T4 D N A after Phage T4 Infection of Clone-bearing Cells T. MATTSON, G. VAN HOUWE, A. BOLLE AND R. EPSTEIN

Department of Molecular Biology, University of Geneva 30, Quai Ernest-Ansermet 1211 Gen~ve 4, Switzerland (Received 26 April 1983) Plasmid pBR322 replication is inhibited after bacteriophage T4 infection. If no T4 DNA had been cloned into this plasmid vector, the kinetics of inhibition are similar to those observed for the inhibition of Escherichia coli chromosomal DNA. However, if T4 DNA has been cloned into pBR322, plasmid DNA synthesis is initially inhibited but then resumes approximately at the time that phage DNA replication begins. The T4 insert-dependent synthesis of pBR322 DNA is not observed if the infecting phage are deleted for the T4 DNA cloned in the plasmid. Thus, this T4 homology-dependent synthesis of pIasmid DNA probably reflects recombination between plasmids and infecting phage genomes. However, this recombination-dependent synthesis of pBR322 D N A does not require the T4 gene 46 product, which is essential for T4 generalized recombination. The effect of T4 infection on the degradation of plasmid DNA is also examined. Plasmid DNA degradation, like E. coli chromosomal DNA degradation, occurs in wild-type and denB mutant infections. However, neither plasmid or chromosomal degradation can be detected in denA mutant infections by the method of DNADNA hybridization on nitrocellulose filters.

1. Introduction In studying the expression of cloned bacteriophage T4 genes it is i m p o r t a n t to carry out experiments in T4-infected cells. Normal expression of T4 genes occurs in an environment in which the characteristics of the host's DNA transcription and replication systems have been profoundly changed. Phage infection-induced alterations include T4-encoded positive regulatory functions necessary for normal T4 gene expression and alterations t h a t inhibit transcription and replication of Escherichia coli chromosomal DNA. Thus, at present, only phage infection can provide a normal environment for the expression of cloned T4 genes. Several effects of phage infection on host DNA t h a t are not normally i m p o r t a n t for the expression of T4 genes m a y be i m p o r t a n t in studies on the expression of cloned T4 genes. F o r example, expression of normal, h y d r o x y m e t h y l e y t o s i n e containing T4 genes is a p p a r e n t l y unaffected by the state of the ale gene. In contrast, transcription of cytosine-containing genes on either E. coli or T4 phage 343 0022-2836/83/300343-13 $03.00/0

© 1983 Academic Press Inc. (London) Ltd.

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genomes is strongly inhibited by the wild-type alc gene product (Snyder et al., 1976; Sirotkin et al., 1977; Kutter et al., 1981). Consequently, if phage infection inhibits the replication system of a plasmid (as it does, see below) efficient expression of cloned T4 genes on autonomous plasmid molecules may not be possibl e in wild-type alc gene infections. A different kind of complication arises from the possibility that infecting phage genomes can have a positive role in the expression of cloned T4 genes other than merely providing trans-acting factors necessary for the expression of the plasmidborne cloned genes. That is, the cloned genes can be expressed after recombination with infecting phage genomes as well as being expressed from autonomous plasmid molecules. It is important to distinguish between these two pathways for the expression of cloned T4 genes, termed recombinational and plasmid-borne gene expression pathways. The study of the expression of cloned T4 genes might not offer significant advantages over the study of the expression of the same genes on normal phage chromosomes unless most of the cloned gene expression is coming from the plasmid-borne genes. Although several studies concerned with the expression of cloned T4 genes in phage-infected cells have been published (Mattson et al., 1977; Huang, 1978; Velton & Abelson, 1980; Vorozheikina et al., 1980; Lloyd & Hanawalt, 1981; Krisch & Seizer, 1981; Revel, 1981; Jacobs et al., 1981; Jacobs & Geiduschek, 1981; Oliver et al., 1981; VSlker et al., 1982a,b), none has been focused on the fate of either the cloning vector or the cloned T4 DNA. In this paper we examine the effects of phage infection on the stability and replication of the plasmid DNA used as the cloning vehicle. The results of these experiments suggest that significant replication of plasmid DNA after phage infection requires recombination between the infecting genomes and plasmid molecules that contain cloned T4 DNA. This plasmid-phage recombination apparently results in the integration of entire plasmid molecules into phage genomes (Mattson et al., 1983, accompanying paper).

2. Materials and Methods (a) Biological strains

Plasmid pVH691, which contains a single T4 EcoRI restriction fragment coding for genes 21 to 23, is a T4-pBR322 chimeric plasmid derived from plasmid pVH652 after EcoRI digestion and differs from it only in the orientation of the T4 insert. In pVH691 gene 23 is closer to the tetracycline-resistant determinant than gene 21. Plasmid pVH652 was derived from plasmid pVH503 as described (Young et al., 1980). Plasmid pTBI0-2, which contains a HindIII restriction fragment coding for parts of the rIIA and rIIB cistrons, is a pBR313 derivative (Seizer et al., 1978). The host for pBR322 and these chimeric plasmids was E. coli BE, a non-permissive strain for amber mutants. Permissive and non-permissive plating bacteria for amber mutant phage were CR63 and S/6, respectively. The host for making phage stocks and genetic crosses was CR63. Bacteriophage T4D mutations used in the combinations indicated in the figure legends were 23amA489, 44amN82, 46amN130, denA Sl12, alc TB1 and the denB deletion SaA9. The T4B extended rII deletion 2226 was used to eliminate the denB gene in some of the experiments presented in Fig. 3 because it also covers the cloned rII DNA present in

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plasmid TB10-2. The alc gene mutation, TB1, was isolated in this laboratory by a modification of the method first used to isolate ale gene mutations (Snyder et al., 1976). A quadruple m u t a n t phage (23amA489-56amE51-denA Sll2-denB SaA9) was plated on a lawn of B834 (sup r~m~) bacteria bearing the plasmid pVH652. Although unlikely, the alc gene mutant isolated in this selection could have incorporated pBR322 DNA into the phage genome. A D N A - D N A hybridization test showed t h a t this particular isolate did not (see Results and accompanying paper). (b) Identification of phage mutations The deletion SaA9 (Depew et al., 1975) was detected in individual phage plaques by its acridine-resistance phenotype in spot tests using CR63 plating bacteria and l pg acriflavin/mI in the bottom agar. denA m u t a n t phage were identified first by their sensitivity to hydroxyurea (Hercules et al., 1971) and then confirmed by a D N A - D N A hybridization test. The sensitivity to hydroxyurea of presumed denA mutants was judged qualitatively from the results of spot tests and quantitatively as efficiencies of plating of 10°/o or less on plates with 40 to 60 mg hydroxyurea in the top agar. The D N A - D N A hybridization test involved labelling E. coli DNA before phage infection (see section (d), below), and then 15min after phage infection measuring the extent of host DNA degradation, determined by hybridization to E. coli filters. In addition, the extent of reutilization of degraded host DNA was determined by hybridization to T4 DNA filters (see section (e), below). Although both hybridization criteria produce consistent results, the extent of hybridization to T4 filters is a more sensitive single criterion. Thus, a wild-type denA gene in comparison to a m u t a n t allele causes at least a 5-fold increase in the extent of hybridization to T4 filters but decreases the extent of hybridization to E. coli DNA filters by only 2- to 3-fold. These hybridization tests were often done with CR63-tabelled cells for phage stocks containing amber mutations in T4 replication functions. Occasionally, phage stocks considered to be denA mutants by the hydroxyurea sensitivity criteria were found to contain a wild-type denA gene, by the D N A - D N A hybridization test (see also, Hercules et al., 1971 ). The ale TB1 mutation was detected in phage stocks by its ability to donate a marker allowing a gene 56 (amE51)-denA-denB triple m u t a n t phage to plate on B834 (Snyder et al., 1976). Lysates of crosses made to combine this alc mutation with other mutations were plated on CR63 and " t e s t " stocks were then made from a number of individual plaques. These test stocks were crossed to the 56amE51-denA-denB triple m u t a n t and the progeny were plated on B834. Individual progeny plaques from these crosses were tested for the presence of the gene 56am mutation on a lawn of S/6 containing gene 56am m u t a n t seed phage. If the gene 56 amber mutation was found in plaques from the B834 plates, the alc gene mutation must have been present in the test stock. B834 was frequently reisolated from single colonies as this strain accumulates amber suppressor mutations in our hands. The presence of amber mutations in phage stocks was determined by standard spot tests. (c) Bacterial growth and phage infection Growth and radioactive labelling of uninfected and infected cell cultures was done at 37°C. A primary culture, started from a single bacterial colony, was grown by shaking in M9S medium (Bolle el al., 1968) containing 5 # g ampicillin/ml, or in the absence of drug if no plasmid was present, to 1 x l0 s to 2 × l08 cells/ml. A low concentration of ampicillin was used because the T4 gene 21 to 23 EcoRI restriction fragment, like many T4 EcoRI restriction fragments cloned in pBR322, significantly reduces the ampicillin-resistance level of the cells (unpublished results). (Saturated overnight cultures were avoided because cells producing abnormal colony morphology on LA plates (Maniatis et al., 1982) often accumulate in saturated cultures.) The presence of specific cloned restriction fragments in the primary cultures was verified genetically by marker rescue spot tests (Mattson et al., 1977). Exponentially growing cells were prepared for all experiments by diluting the

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T. MATTSON E T A L .

primary culture 50-fold into M9S supplemented, if appropriate, with 5/~g of freshly prepared ampicillin/ml. This culture was grown by shaking to a cell density of approximately 2 x 108/ml as measured with a Petroff Hausser bacteria counter. On the basis of colony-forming assays made just before and between 3 and 5 min after infection at multiplicities of approximately 10, more than 90°/o of the bacteria were infected in all experiments. (d) D N A labelling. Cellular DNAs that were to be analysed by DNA-DNA hybridization were labelled as described by Warner & Hobbs (1967) with [2-14C]uracil (1/~Ci/ml, 0-018/~mol/ml; New England Nuclear) added at the time of dilution of the primary culture and pulse-labelled for 2 min with [3H]thymidine (5/~Ci/ml, 0"25 #mol/ml; New England Nuclear). One minor exception to this protocol is noted in the legend to Fig. 1. Pulse-labeltings were terminated at the times indicated in the Figures by adding 5 vol. of ice-cold Tris/salt/EDTA (500 mMNaC1; 50 mM-EDTA (pH 8-0); l0 mM-Tris-HCI (pH 8-0)). Cell samples were collected by centrifugation, resuspended in 1.5 vol. of ice-cold Tris/salt/EDTA, again pelleted and finally resuspended in 0-25 vol. of Tris/salt/EDTA before being processed further (see section (e), below) or stored at -20°C. Duplicate l-ml samples of uninfected cells were taken shortly before infection and single 1-ml samples were taken at various times after infection. (e) Nucleic acid hybridization

The methods used for collecting cell samples and for hybridizing labelled DNA to nitrocellulose filters charged with denatured DNA were adapted from the method of Frey et al. (1979). Cells or DNA in 0.25 ml of Tris/salt/EDTA (see section (d), above) were mixed with 0-25 ml of 1 M-NaOH. After 5 min in a boiling water bath, 0.55 ml of a neutralization mix was added (10 ml of I M~HCI; 8 ml of 20 x SSC (SSC is 0-15 M-NaCI, 0.015 M-sodium citrate); 2 ml of l M-Tris. HC1 (pH 8"0) and 2 ml of water) and the pH was adjusted to 7.0+0.1. A 50-ttl portion was set aside for acid precipitation and then 1.0 ml of formamide (A270 less than 0.15) was added. This procedure produces short fragments of singlestranded DNA (Frey et al., 1979) and completely hydrolyses RNA, thus eliminating the possibility of labelled RNA hybridizing to the filters. After transferring these mixtures to glass scintillation vials, 4 pencil-marked nitrocellulose filters, prepared (Young et al., 1980) with different purified species of DNA, were wetted in 6 × SSC and added to the vials. Hybridizations were done in a shaking water bath for 65 to 70 h at 42°C. In each vial there were separate nitrocellulose filters for E. coli chromosomal DNA (8 pg of phenol-extracted type VIII E. coli DNA purchased from Sigma), T4D wild-type phage particle DNA (8 pg), pBR322 DNA (0-8/lg of caesium chloride/ethidium bromide density gradient purified), and either calf thymus (4#g of phenol-extracted type A calf thymus DNA purchased from Calbiochem) or salmon sperm DNA (a gift from L. Moran). After hybridization the filters were batch-washed by gentle shaking at room temperature for 30 rain in 50~/o (w/v) formamide/2 × SSC and then again for 15 min in this solution, then l0 rain in 2 × SSC and finally 5 min in 0.2 × SSC. Dried filters were counted in a toluene-based scintillator. All hybridization data presented in the Figures have been corrected for non-specific binding of labelled DNA to calf thymus filters and in double-label experiments, for crossover from the 14C into the 3H channel. The rationale for labelling the DNA with both 14C and 3H and for presenting the hybridization data as a ratio of 3H to 14C has been discussed previously {Bird et al., 1976). The efficiencies of hybridization for labelled E. coli chromosomal DNA, T4 DNA and pBR322 DNA are usually between 5 and 10%, 15 to 25% and probably greater than 25%, respectively. The value for E. coli chromosomal DNA is the efficiency of hybridization of the total DNA extracted from uninfected cells. The values given for T4 DNA, which are the percentages of total DNA labelled after infection that hybridizes to T4 filters, may

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somewhat underestimate the true values, because a significant fraction of the DNA labelled after infection is E. coli chromosomal DNA. The efficiency of hybridization of labelled pBR322 DNA is probably greater than for T4 DNA. This can be seen from the hybridization results obtained with the labelled DNA from uninfected ceils containing the plasmid pVH691. In this situation 2 to 3 times as much label hybridizes to the pBR322 filters as hybridizes to the T4 filters. Thus, assuming similar sizes for the T4 restriction fragment (3-6 x 103 bases) (O'Farrell et al., 1980) and the plasmid vector (4-3 × 103 bases) but neglecting any possible preferential incorporation of labelled thymidine into the T4 DNA (T4 DNA is A+T-rich), it is possible that as much as 50% of the pBR322 DNA labelled in these experiments is detected by the hybridization method used. Under our conditions, it is expected that pBR322 DNA would hybridize more efficiently than T4 DNA because the molar ratio of the DNAs fixed onto the 2 nitrocellulose filters differs by almost a factor of 4.

3. Results (a) Degradation of plasmid D N A Wild-type T-even bacteriophage infections of E. coli lead to the degradation of the host's chromosomal DNA. Mutations in T4 genes involved in this degradation have been isolated and an outline of the degradation p a t h w a y has been established (for a recent review, see Koerner & Snustad, 1979). The d a t a presented in Figure 1 demonstrate t h a t T4 infection also induces the breakdown of plasmid pBR322 DNA and show t h a t the T4 denA gene product, which is involved in degrading E. coli chromosomal DNA, is also involved in degrading pBR322 DNA. For these experiments, plasmid and E. coli chromosomal DNAs were labelled before phage infection. The fate of these DNA species after phage infection was followed by the method of D N A - D N A hybridization on nitrocellulose filters. This was done by hybridizing samples of total cellular DNA in vials containing four filters, each charged with a different purified, denatured DNA: E. coli chromosomal DNA, pBR322 vector DNA, wild-type T4 DNA and calf t h y m u s DNA. We consider t h a t degradation has been detected only if the a m o u n t s of label hybridizing in samples taken a t several different times after phage infection are significantly less than the amounts hybridizing in the duplicate samples taken before infection. (It should be noted t h a t partially degraded, labelled DNA can still hybridize with the unlabelled DNA bound to the filters. Thus, in some instances (e.g. gene 46 m u t a n t infections) this assay m a y significantly underestimate both the e x t e n t of degradation and the time at which it begins.) The d a t a presented in Figure 1 show t h a t the hybridization assay can be used to detect differences in the stability of E. coli chromosomal DNA t h a t are usually detected by other methods ( K u t t e r & Wiberg, 1968; W a r n e r et al., 1970). Thus, this assay detects the phage-induced chromosomal DNA breakdown occurring in wild-type and denB m u t a n t infections and shows t h a t this degradation can be prevented if the infecting phage are m u t a n t in the denA gene. Since D N A - D N A hybridization can be used to characterize the stability of chromosomal DNA, it can presumably also be used to follow the fate of plasmid DNA in wild type, denA and denB m u t a n t infections. As shown in Figure l(a) and (b), similar patterns are observed for plasmid and

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Fro. l. Degradation of DNA after phage infection. Plasmid and chromosomal DNA in E. coil strains carrying only the plasmid vector pBR322 (B E. pBR322) or carrying the T4 late genes 21 to 23 cloned into pBR322 (B E. pVH691) was labelled before phage infection with 14C (given as {2-14Cluracil) as described in Materials and Methods, except that in one of the experiments involving B E. pVH691, three times as much labelled uracil was added. Separate portions of each culture were infected with the phage mutants desc,ibed below. Samples of total cellular DNA collected before and at the times indicated in the Figure after phage infection were hybridized to filter-bound DNA as described in Materials and Methods. (a) E. coil filters with B E.pBR322 cellular DNA; (b) pBR322 filters with B E-pBR322 cellular DNA; (c)pBR322 filters with B E.pVH691 cellular DNA; ( d ) T 4 filters with B E. pVH691 cellular DNA. ( 0 ) Wild-type alleles of both the denA and denB genes; (O) denA mutant; (Z~) denB mutant; ([~) denA-denB double mutant. Each of these phage stocks contained a gene 44 amber mutation in order to block synthesis of phage DNA and thus cell lysis and a gene 23 amber mutation, which is unimportant for this experiment. The gene 44 amber mutation (amN82) has no apparent effect on the degradation of host chromosomal DNA (Warner et al., 1970). The values presented in the Figure are the average of the values obtained from either 2 ((c) and (d)) or 3 ((a) and (b)) experiments. The radioactivity attached to the pBR322 filters in the uninfected cell samples varied from 352 to 774 cts/min after subtraction of the background. These specific hybridization values were 170 to 316 cts/min for the T4 filters hybridized to B E. pVH691 cellular DNA, and from 4973 to 28334 cts/min for E. coil filters. Background subtractions were usually l0 to 20% of the total counts bound to the pBR322 filters and exceeded 25% for only 3 filters, which were ibr wild-type and denB mutant-infected cell samples collected at 26 and 36 min after infection. Background subtraction for the E. coil filters was always less than 10% of the total counts bound. This value for the T4 filters hybridized with B E. pVH691 DNA was usually between 20 and 30% . All of the data presented have been corrected for these background hybridization values and have then been normalized to the values obtained in the uninfected cell samples.

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FATE OF CLONED PHAGE T4 DNA

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double mutation in preventing plasmid DNA degradation, we are not certain of this conclusion. This is because the hybridization assay is not sensitive enough to detect a small, previously described effect of a denB mutation on the extent of chromosomal DNA degradation (Souther et al., 1972). When T4 DNA is cloned into the plasmid, the fate of both the vector portion and the T4 portion of the chimeric plasmid molecules can be followed. The cloned T4 DNA does not appear to alter either the kinetics or the extent of breakdown of the vector portion of the recombinant plasmid molecules (Fig. l(c)). In addition, the genetic requirements for preventing the degradation of the vector DNA remain unchanged. That is, a denA mutation is still necessary to ensure the stability of pBR322 DNA and it may also be sufficient for this purpose. The fate of the cloned T4 restriction fragment is shown in Figure l(d). The cloned T4 DNA is degraded when the vector DNA is degraded and remains stable when the vector DNA remains stable. That is, a denA but not a denB mutation is needed to ensure stability of the cloned T4 DNA. It is surprising that the cloned T4 DNA is stable after infection with phage that are able to produce the denB gene product, endonuclease IV, but are mutant in the denA gene (i.e. denA--denB+). In vitro, purified endonuclease IV prefers a single-stranded, cytosine-containing DNA substrate (Sadowski & Hurwitz, 1969b) and in vivo it has an essential role in degrading T4 cytosine-containing DNA but only a minor rote in degrading E. coli chromosomal DNA (Bruner et al., 1972; Souther et al., 1972). Thus, one might expect that endonuclease IV could attack at least the T4 DNA portion of the chimeric plasmid molecules and that this might lead to degradation of both the vector portion and the T4 portion of these plasmids. However, the results obtained do not confirm this prediction. Possible explanations for these results are given in the Discussion. In contrast, results presented in Figure 1 show that both the T4 DNA portion and the vector portion of the chimeric plasmid molecules are degraded after infection with phage that are able to produce the denA gene product, Endo II, but are mutant for the denB gene (i.e. denA+-denB-). I n vitro, purified Endo II prefers double-stranded, cytosine-containing DNA (Sadowski & Hurwitz, 1969a) and in vivo it plays an important role in degrading E. coli chromosomal DNA but has only a minor effect on cytosine containing T4 DNA (Warner et al., 1970; Hercules et al., 1971; Kutter et al., 1975). Thus, even if Endo II attacks only the vector portion of the chimeric plasmids, degradation of both the T4 and the vector portions of the plasmids could be expected. In the experiments described in Figure l, where plasmid DNA was labelled only before infection with phage that were unable to replicate, the fate of the cloned T4 DNA could be followed independently of the fate of the vector portion of the chimeric plasmids. However, for experiments involving DNA labetlings done after infection with phage that can replicate, the fate of most cloned T4 restriction fragments cannot be followed with the DNA-DNA hybridization assay because label entering the cloned T4 DNA cannot easily be distinguished from label entering homologous DNA present in the infecting phage genomes. Consequently, in experiments involving DNA labellings done after infection we will assume that the fate of the T4 portion of the chimeric plasmids is the same as the fate of the

T. M A T T S O N E T A L .

350

v e c t o r p o r t i o n o f t h e c h i m e r i c p l a s m i d . T h e r e s u l t s r e p o r t e d in F i g u r e 1 s u g g e s t t h a t t h i s is a r e a s o n a b l e a s s u m p t i o n . (b) I n h i b i t i o n o f p l a s m i d replication (i) P l a s m i d vector alone R e p l i c a t i o n o f E. coil c h r o m o s o m a l D N A is i n h i b i t e d a f t e r T 4 i n f e c t i o n , e v e n if d e g r a d a t i o n o f t h e c h r o m o s o m a l D N A is b l o c k e d ( S o u t h e r et al., 1972). D a t a

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FIG. 2. Synthesis of pBR322 DNA after phage infection. Relative rates of DNA synthesis in E. coli strains BE. pBR322 and BE. pVH691, labelled before infection with [2-14C]uracil and pulse-labelled for 2 min with [3H]thymidine before and at several different times after phage infection, were determined by DNA-DNA hybridization as described in Materials and Methods. The specific hybridization data, which are plotted at the end of each pulse period given after infection, are expressed as a ratio of 3H to t4C for the counts bound to the pBR322 and E. coli filters. These ratios have been normalized to the ratios obtained with the uninfected cell samples. (a) E. coil filters with B E. pBR322 cellular DNA; (b) pBR322 filters with B E. pBR322 cellular DNA; (c) pBR322 filters with B E. pVH691 cellular DNA; (d) T4 filters with BE-pVH691 cellular DNA. (O)Mutant in gene 44; ( ~ ) m u t a n t in gene 46; (0) wild-type alleles for both genes 44 and 46. Each of these phage strains also contained mutations in genes 23, denA, denB (SaA9) and alc. Specific hybridization values tbr pBR322 filters with DNA from uninfected pBR322 cells were 480cts/min of 14C and 2110cts/min of all. These values were 473 cts/min of 14(, and 3203 cts/min of 3H for the pVH691 plasmid cell samples. Specific hybridization values for E. coli filters with uninfected cell samples were 5591 and 4690 cts/min of 14C and 40,829 and 22,000cts/min of 3H for pBR322 and pVH691 plasmid-bearing cells, respectively. Background corrections for the pBR322 filters were tess than 20% except for some of the 3H hybridization results obtained with samples from infected B E. pBR322 cells, where background corrections were up to 65% of the total counts bound.

FATE OF CLONED PHAGE T4 DNA

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presented in Figure 2 show that T4 infection also inhibits plasmid pBR322 replication. The infecting phage used in these experiments were mutant in both the denA and denB genes. It can be seen that the kinetics and extent of inhibition are similar for plasmid and chromosomal DNA. (ii) Chimeric plasmids The presence of cloned T4 DNA in pBR322 significantly alters the effect of phage infection on the synthesis of plasmid DNA. In this case, synthesis of plasmid DNA is also initially inhibited but then resumes a few minutes later (Fig. 2(c)). This is a specific, cis effect of the cloned T4 DNA because replication of the host chromosomal DNA is not affected by the cloned T4 DNA (data not shown). A similar effect of T4 infection on the synthesis of pBR322 DNA has been observed for more than 30 different T4 chimeric plasmids, with a plasmid containing T4 genes 21 to 23 cloned in the opposite orientation, and with infecting phage that are not mutant in the alc gene (data not shown). A comparison shows that pBR322 DNA synthesis resumes at the time phage synthesis begins (Fig. 2(c) and (d)). One possible explanation for these observations is that reciprocal recombination between cloned T4 DNA and homologous sequences on infecting phage genomes leads to the integration of a complete plasmid molecule into a phage genome and that this recombination can be followed by replication of the plasmid vector DNA as part of a phage genome. Thus, we are proposing that the T4 insert-dependent synthesis of pBR322 DNA is recombination-dependent. Although other explanations for this pBR322 DNA synthesis are possible, we will present evidence showing that it can be adequately explained by this recombinational pathway model. (c) Requirements for insert-dependent synthesis of pBR322 DNA Homologous recombination involving cloned T4 DNA could occur between plasmid and phage genomes or between two plasmids. If the T4 insert-dependent synthesis of pBR322 DNA depends exclusively on homologous plasmid-phage recombination, it should be eliminated if the homologous sequences are deleted from the infecting phage genomes. However, if this synthesis of pBR322 DNA depends on plasmid-plasmid recombination, deletion of homologous sequences from the infecting phage genomes should have little or no effect. Experiments designed to test these possibilities are presented in Figure 3. In bacteria containing a cloned rII restriction fragment and infected with phage carrying a deletion covering the cloned fragment, T4 insert-dependent synthesis of pBR322 DNA was not observed (Fig. 3(a)). In contrast, plasmid DNA synthesis did resume when the rII region was present in the infecting phage {Fig. 3(c)). Control experiments showed that the rII deletion did not significantly affect either the phage infection-induced inhibition of plasmid replication or the T4 insert-dependent resumption of pBR322 DNA synthesis (Fig. 3(a) and (b)). Thus, these experiments show that the T4 insert-dependent synthesis of pBR322 DNA 12

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Time after mfecti0nat 3T°C (rain)

Fro. 3. Homology-dependent synthesis of pBR322 DNA. The DNA of E. coil strains B E. pBR322, B E. pVH69] and B E. pTBl0-2 was labelled with [2-14C]uracil and [3H]thymidine as described in the legend to Fig, 2, and analysed by DNA-DNA hybridization as described in Materials and Methods. All of the data presented are from hybridizations to pBR322 filters. (a) DNA from B E. pBR322; (b) DNA from BE.pVH691; (c)DNA from BE-pTBI0-2. All of the phage strains used in this experiment contained 4 mutations: a gene 23 amber mutation, an ale gene mutation, a denA mutation, and a denB deletion. They differ from one another only in the deletion used to eliminate the denB gene. ~ , denB deletion is SaA9; - - O - - O - - , de~B deletion is the rIl deletion 2226. Specific hybridization values for the uninfected cell samples from BE-pBR322, BE.pVH691 and BE.pTB10-2 were 397, 307 and 420cts/min of 14C and 1472, 3717 and 2185cts/min of 3H, respectively. Background corrections were less than 15°/o for all of the laC hybridization results and for all of the aH hybridization results obtained with B E. pVH691. Background subtractions for 3H hybridization results obtained with infected cell samples of B E. pBR322 and B E. pTBl0-2 were as much as 60%.

detected in these experiments is a T4 homology-dependent process. In addition, these experiments imply that there are some types of phage-plasmid interactions and that equivalent plasmid-plasmid interactions lead to little, if any, synthesis. The results of these rII deletion experiments do not allow us to distinguish between the possibility that significant amounts of T4 homology-dependent, plasmid-plasmid recombination does not occur and the possibility that plasmidplasmid recombination occurs but does not lead directly to the synthesis of detectable amounts of plasmid DNA. However, these results may be of interest in regard to certain models proposed for T4 origins of replication (see Discussion). If the T4 insert-dependent synthesis of pBR322 DNA occurs only after integration of plasmid molecules into phage genomes, it might depend on T4 replication functions. The data presented in Figure 2(c) show that this pBR322 DNA synthesis is, in fact, dependent on a phage function affecting replication. The effect of a T4 gene 46 amber mutation on T4 homology-dependent synthesis of pBR322 DNA is shown in Figure 2(c). Plasmid vector DNA synthesis resumes after gene 46 mutant infection, but is turned off again a few minutes later. Comparison of this DNA synthesis profile with the phage DNA synthesis profile (Fig. 2(d)) suggests that the gene 46 product is not essential for the formation of the proposed plasmid-phage recombinant genomes. However, its absence may limit replication of the plasmid-phage recombinant genomes because gene 46 mutations lead to a general arrest of phage-specific DNA synthesis. We do not know the significance, if any, of the earlier arrest of pBR322 DNA in this experiment. Additional evidence supporting the recombination pathway proposal is presented in the accompanying paper.

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4. Discussion

(a) Degradation of plasmid D N A The results reported here show that previously described effects of the T4 denA and denB gene products on the degradation of E. coli chromosomal DNA can be extended to the DNA of the plasmid pBR322. Specifically, the denA gene product is involved in the degradation of pBR322 DNA and the denB product appears to ]lave no major role in the degradation of this plasmid. Extensive degradation of plasmid DNA after phage infection could be an important factor in studies on the expression of cloned DNA. Thus, when plasmid DNA is degraded, it may be difficult to establish clearly that cloned genes are being expressed from intact plasmid molecules. Degradation of chimeric plasmid DNA can be reduced by using phage mutant in the denA gene. In contrast, marker rescue frequencies among viable progeny phage do not appear to be strongly influenced by either mutant or wild-type alleles in the denA and denB genes (unpublished results). Although the T4 denB gene product, Endo IV, is known to play a major role in degrading cytosine-containing DNA of phage genomes, we do not detect a similar effect on cloned, cytosine-containing T4 DNA (Fig. l(b)). Our observation could be explained simply if Endo IV attacks cloned T4 DNA but does not cause it to be extensively degraded, or if this particular restriction fragment does not provide a suitable substrate for Endo IV (Sadowski & Hurwitz, 1969b). Alternatively, Endo IV might preferentially degrade replicating cytosine-containing DNA in vivo. This proposal, which has been made previously (Elliott et al., 1973; Kutter et al., 1975), could explain why Endo IV plays only a minor role in degrading host DNA. That is, host chromosomal and plasmid DNAs may not be suitable substrates for Endo IV, only because their replication is blocked after phage infection. Additional in vivo evidence consistent with this possibility comes from experiments showing that Endo IV can play an important role in degrading E. coli DNA after infection with mutants causing a delay in the inhibition of host replication (Parson & Snustad, 1975; Snustad et al., 1976). (b) Inhibition of plasmid replication The data presented in Figure 2 show that replication of pBR322 and the E. coli chromosome are inhibited, with similar kinetics, after phage infection. Analysis of the effect of T4 infection on other E. coli plasmids might provide additional information on the identity of the target(s) of this inhibition. In any case, the rapid inhibition of E. coli chromosomal replication strongly suggests that the inhibition is probably not limited to an effect on initiation of replication. Inhibition of plasmid replication has important implications for studies on the expression of' T4 genes cloned in these plasmids. Since T4 genes in autonomous l)lasmid molecules would continue to contain cytosine after phage infection instead of the normal hydroxymethylcytosine, efficient expression of plasmidborne cloned genes should require an alc gene mutation in the infecting phage genomes (Snyder etal., 1976; Kutter etal., 1981; and unpublished results).

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Although evidence apparently contradicting this expectation has been presented (Jacobs et al., 1981; Jacobs & Geiduscheck, 1981), we have obtained evidence supporting this prediction (Mattson et al., unpublished results). (c) P lasmid-phage recombination The results reported here suggest that plasmid vector DNA as well as cloned T4 DNA can become integrated into vegetative phage genomes. Previously, it has generally been assumed that only the T4 portion of the chimeric plasmid molecules become integrated into phage genomes. This notion was derived from the genetic analysis of viable progeny phage produced in clone-bearing cells. Among viable progeny phage, wild-type alleles present in the cloned DNA appear to be substituted for a small fraction of the mutant alleles present in the infecting phage genomes. The observations described here can be adequately explained by integration of entire plasmid molecules into phage genomes. The experiments presented in Figure 3 show that T4 insert-dependent synthesis of plasmid DNA cannot result from an interaction between two plasmid molecules or from autonomous replication of individual chimeric plasmid molecules. The most likely remaining possibility is plasmid-phage recombination leading to integration of plasmid vector DNA into phage genomes, presumably in the region of T4 homology. Evidence supporting integrative recombination (Campbell, 1962) is presented in the accompanying paper. The plasmid-phage recombination proposed as a prerequisite for the T4 insertdependent synthesis of pBR322 DNA is an unusual kind of T4 recombination. Although the T4 gene 46 product appears to play a major role in generalized recombination between infecting phage genomes, it does not appear to affect the type of plasmid-phage recombination described here (Fig. 2(c)). In addition, this type of plasmid-phage recombination, unlike marker rescue recombination, is not detected unless the infecting phage carry a deletion covering the denB gene (unpublished results). Recombinational intermediates have been proposed to act as T4 origins of replication (Luder & Mosig, 1982). In regard to this suggestion, it will be interesting to study plasmid-plasmid recombination in T4-infected cells. There is at present no reason to believe that such recombination could not occur in the infected cells, at least between the ~loned T4 restriction fragments. However, in the rII deletion experiments presented here, recombination between nonreplicating plasmid molecules, if it occurred, did not lead to detectable synthesis of plasmid DNA. This work was supported by grant no. 3.078.81 from the Swiss National Science Foundation. REFERENCES Bird, R., Chandler, M. & Caro, L. (1976). J. Bacteriol. 125, 1215-1223. Bolle, A, Epstein, R. H., Salzer, W. & Geiduschek, E. P. (1968). J. Mol. Biol. 31,325-348.

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Edited by J. M i l l e r