Proc. Nat. Acad. Sci USA Vol. 73, No. 3, pp. 692-696, March 1976

Biochemistry

Genetic recombination of bacteriophage T7 DNA in vitro [DNA packaging/17 exonuclease/17 DNA nucleotidyltransferase (polymerase)/density-labeled

DNA/heterozygotes]

PAUL D. SADOWSKI AND DAN VETTER Departments of Pathology and Medical Genetics, University of Toronto, Toronto, Canada M5S 1A8

Communicated by Jerard Hurwitz, November 11, 1975

ABSTRACT

We have demonstrated recombination of

MATERIALS AND METHODS Bacterial and Phage Strains. Escherichla coli B was used as the nonpermissive host and E. coli 011' (2) and E. coli BBW/1 (11) were the permissive hosts for amber mutants. E. coli BL2 [iig-, no suppressor activity (Su0)] was from F. W. Studier (12). Shigella sonnei D2 571-48 (Su0) and a Su+ derivative (Shigella sonnei 3-18) were obtained from R. Hausmann (13). These strains restrict the growth of wildtype T7 (T7+) phage bearing the ss+ allele (ss = suicide on Shigella, see ref. 13). Phage mutants arise spontaneously (ss- mutants) which are able to form plaques on these Shigella strains. An ss- T7 phage was obtained from R. Hausmann. The following T7 amber and temperature-sensitive mutants were used, (the gene numbers are indicated in brackets): am HA1S, (gene 1.3); am64, (gene 2); am29, (gene 3); am2O, (gene 4); am28, (gene 5); am147, (gene 6); aml40, (gene 14); amSl, (gene 15); am290, (gene 17); tsll, (gene 5). Amber and temperature-sensitive T7 mutants were obtained from F. W. Studier (2) and multiple mutants were constructed by standard phage crosses (2). Media and Chemicals. LB broth contained 10 g of Bacto tryptone, 5 g of yeast extract, and 5 g of NaCl per liter. The pH was adjusted to 7.4 with NaOH. The concentrations of tryptone and yeast extract were doubled to make 2 X LB. T7 diluent consisted of 0.02 M Tris-HCl buffer, pH 7.4, 10 mM MgSO4, 0.5% NaCl, and 10 ttg/ml of gelatin (1, 14). CA medium contained per liter 2.0 g of vitamin-free casamino acids, 5.0 g of NaCl, 1.0 g of NH4Cl, 12.0 g of Tris base. The pH was adjusted to 7.4 with HC1 and after autoclaving 2 ml of 20% glucose was added per liter of medium. Fluorodeoxyuridine (FdUrd) was obtained from Sigma and 5-bromodeoxyuridine (BrdUrd) was purchased from Calbiochem. Carrier-free [32P]orthophosphoric acid was from New England Nuclear. Growth of Phage Stocks. High titer stocks were prepared in 2 X LB medium as described previously (14). To obtain 32P-, density-labeled phage, overnight cultures of E. coli B or BBW/1 grown in CA medium were diluted 50-fold into 250 ml of CA medium and grown at 300 to a cell density of about 1 X 108 per ml (OD65o = 0.08). FdUrd and BrdUrd were added to final concentrations of 10 ,ug/ml and 200 jig/ml, respectively, and H332P04 was added at a concentration of 5 ,uCi/ml. Shaking was continued for 3.5 hr, by which time cell growth ceased (OD65o = 0.35). The appropriate phage was added at a multiplicity of 10 phages per cell. The culture was shaken vigorously until lysis occurred about 75 min later; phage titers of 1 to 2 X 1010 per ml were obtained. Density-labeled phage had a buoyant density in CsCl 0.025-0.030 g/ml greater than light phage. Phage were purified by differential centrifugation and banding in CsCl gradients and DNA was isolated by phenol extraction and dialyzed against 0.01 M Tris-HCl pH 7.4, 1 mM EDTA, 0.05 M NaCl (15).

bacteriophage T7 DNA in vitro. An extract of Escherichia coli B cells infected with wild-type T7 (17+) is incubated with mature DNA extracted from T7 phage. Packaging of the exogenous DNA within the phage head a ppears to be preceded by recombination of exogenously added DNA with DNA present in the extracts. In order to detect the recombination, we used an exogenous DNA bearing a marker (ss-) such that progeny phage which have packaged this marker are able to plate on Shigella sonnei D2 57148, whereas T7+ phage present in the extracts do not. The recombinational process bears many of the character-

istics of in vivo recombination. The exogenous DNA is not packaged intact but undergoes fragmentation to a length of about 3000 base pairs before being incorporated into a mature DNA molecule. If ss- DNA bearing an amber mutation is used in the assay, the frequency of amber+ progeny produced varies with the distance of the amber marker from the ss- marker. When DNA bearing three mutations is used in the reaction, phage heterozygous for the unselected marker are readily detected. Finally the products of phage genes 4 (DNA replication protein), 5(DNA polymerase), and 6(exonuclease), genes previously implicated in recombination in vivo, are required for the in vitro reaction.

We recently described an efficient in vitro complementation system which was capable of packaging and maturing bacteriophage T7 DNA (1). In this assay, the immature DNA present in a phage-infected cell extract lacking head precursors was packaged within head precursors of a complementing T7-infected extract, leading to the formation of mature infectious phage. In the course of extending this work, we also found that it was possible to detect the encapsulation of mature DNA extracted from phage particles. To our surprise, the mature DNA appeared not to be packaged intact, but was found to recombine with DNA present in a cell-free extract prior to encapsulation within the phage head. Phage T7 may be a useful system in which to study the mechanism of recombination in vitro because of the high frequencies of recombination exhibited in vivo, the relatively small size of its DNA, and the availability of a wide range of mutants (2, 3). Many of the gene products which have been implicated in T7 recombination in vivo are available in purified form (4-9). A study of in vitro recombination would be greatly aided by a sensitive biological assay to detect genetic recombinants. However, infectivity assays of T7 DNA are very inefficient (10) and appear to lack the sensitivity which might be needed. The system described here appears to have the required sensitivity in that recombinant DNA is detected as infectious DNA which is encapsulated within viable phage

particles. Abbreviations: Suo and Su+, wild-type and suppressor-positive phenotypes, respectively; am, amber mutation; ts, temperature-sensitive mutation.

692

Biochemistry:

Sadowski and Vetter

Proc. Nat. Acad. Sci. USA 73 (1976)

693

10

10

0

0.

1.54

10'

CPl go 0

/IS

I

*1.50

*1

a

1.46 ,

0

FE

109

0l

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co

phage

DNA equivalents per ml FIG. 1. Dependence of ss- phage titer upon the amount of ssDNA added. Twenty microliters of a sonicated T7+-infected cell extract were incubated with 2 ul of various concentrations of T7 ss- DNA as described in the Materials and Methods. The titer of ss- phage produced per ml of reaction mixture was determined and plotted against the number of ss- DNA molecules per ml added. The background in the absence of added ss- DNA is less than 105 ss- phage per ml.

Preparation of Extracts. The procedure for preparation of extracts was the same as that described previously (1). Logarithmically growing E. colh B cells were infected at a multiplicity of 10-20 phage per cell at 300 and the cells were harvested by centrifugation 18.5 min after infection. The cell pellet was resuspended in 1/200 the original culture volume of T7 diluent, frozen in a dry ice/acetone bath, and thawed at 300. The extract was then sonicated (three bursts of 10 see each with a Branson sonicator using the microtip). Sonication increased the efficiency of the assay about 10fold. The activity of these extracts is relatively stable if stored at 0° (50-90% loss in 24 hr) but is unstable to freezing and thawing (greater than 99.9% loss). All experiments reported were done with freshly made extracts. Fractionation of Extracts. One milliliter sonicated extract was fractionated by differential centrifugation as follows. The supernatant obtained after centrifugation at 12,000 X g for 10 min (Sorvall SS34 rotor) was centrifuged at 100,000 X g for 1 hr in the IEC A270 rotor. The pellet was resuspended in 0.1 ml of T7 diluent and dialyzed Table

Genetic requirements of T7 recombination reaction

1.

ss- phage Phage

Phenotype Exp.

Host

DNAendonuclease IDNADNA polymerase& exonucleaseExp. 2

T7+ T7+ 1.3 amber 1.3 amber

ligaseligase-

X

106

1

T7+ 2 amber 3 amber 4 amber 5 amber 6 amber

per ml

B B B B B B

7,100

B BL2 (lig-) B BL2 (lig-)

16,000 9,000 300 30

0

1.78

*1.70

20

10 fraction

FIG. 2. (a) Buoyant density of phage which packaged 32p_, BrdUrd-labeled T7 DNA in vitro. The reaction mixture contained 3.5 nmol of 32p_, BrdUrd T7+ DNA (14,000 cpm/nmol), 50 gl of a dialyzed high-speed pellet fraction, 100 Ml of a dialyzed, ammonium-sulfate-concentrated high-speed supernatant fraction (both prepared as in Materials and Methods), 5 mM ATP, and T7 diluent to a final volume of 0.45 ml. After 60 min at 300, 100 ,ug of pancreatic DNase (Worthington) were added and incubation was continued at 370 for 15 min. Light [3H]thymidine-labeled marker phage was added. The solution was diluted to 7 ml with T7 diluent, the density was adjusted to about 1.50 g/cm3 by addition of 5 g of solid CsCl, and the tube was centrifuged at 38,000 rpm in the IEC A270 rotor for 16 hr at 5°. The gradient was fractionated, the fractions were acid precipitated with trichloroacetic acid, and the precipitates were collected on glass-fliber filters. Radioactivity was determined in a liquid scintillation counter. The arrow indicates the expected banding position of fully dense phage. (b) An identical reaction to that used in (a) was carried out and the resulting phage were banded in CsCl. Aliquots of the fractions were acid precipitated and the peak fractions of 32p were pooled and extracted with phenol. After dialysis against 0.01 M Tris.HCl, pH 7.4, 0.05 M NaCl, 1 mM EDTA, the DNA was diluted in the same buffer, and the density was adjusted to about 1.70 g/cm3 by addition of solid CsCl. Light 3H-marker T7 DNA was added and the tube was centrifuged 48 hr at 150 and 38,000 rpm in the IEC A270 rotor. The gradient was fractionated and counted as above. The arrow indicates the position of fully heavy DNA as determined in a separate gradient. 32P radioactivity 0-0; 3H radioactivity -; buoyant density in g/cm3 ...... ----

1,900

6,500 75