Lambda and T4 bacteriophage hybrids

J. Mol.

Biol.

(1977) 113, 567-572 LETTERS

TO THE EDITOR

Lambda and T4 Bacteriophage Hybrids Four hybrid h bacteriophages containing T4 DNA fragments have been obtained after digestion of hgt-hC DNA and non-glucosylated T4 DNA with endo R. EcoRl endonucleasesfollowed by covalent joining with T4 polynucleotide ligase and transfection of Escherichia coli cells. The sizesof the inserted T4 DNA fragments have been estimated from their electrophoretic mobilities in agarosegels. The T4 geneslocated in the inserted fragments of two hybrid phageshave been identified. It has been shown that the endo R. EcoRI endonucleasecleaves t,he non-glucosylated T4 DNA (Kaplan &zNierlich, 1975; Li et al., 1975). This observation raised the possibility of cloning of T4 DNA fragments generated by endo R. EcoRI cleavage in vector DNA molecules. However, it is not clear whether the fragments would be replicated in vector DNA molecules because DNA of T-even phages contains 5-hydroxymethylcytosine instead of cytosine (Wyatt & Cohen. 1953) and is replicated with phage DNA polymerase (de Waard et al., 1965). In this communication we show that hybrid h phagescontaining T4 DNA fragments can be obtained. We also find that recombination of h hybrid and T4 phages takes place. These experiments do not create novel biohazards and were performed using normal sterile microbiological procedures. hgt-hC! DNA (Thomas et al., 1974) was prepared by phenol treatment (McHattie et al., 1967) of phagespurified by differential centrifugation followed by equilibrium centrifugation (Bovre et al., 1971). Cleavage of DNA with endo R. EcoRI was performed under the conditions described by Polisky et al. (1975). Endo R. EcoRI was prepared as described by Yoshimori (1971) from E. coli strain llOO/RI (Murray & Murray, 1975). The DNA fragments of hg&hC were separated in a glycerol gradient (Ihler & Kawai, 1971). The preparation of DNA was not heated before the centrifugation, therefore we could observe four peaks in the gradient (Fig. 1). Peak 1 was formed by the end fragments joined at the natural “cohesive” ends and peak 4 by the central fragment of hgt-X. The fractions marked by a heavy line were collected and used in ligase reaction. There was no noticeable contamination of the end fragments by the central fragment, as was shown by electrophoresis through an agarose gel. The T4 phage ccgtam8,PgtamlO (Georgeopoulos & Revel, 1971) was purified by three cycles of differential centrifugation, and non-glucosylated DNA w-asprepared by phenol treatment. Non-glucosylated T4 DNA is more resistant t,o endo R. EcoRI treatment than ADNA. In our conditions we have not achieved the complete cleavage of T4 DNA despite the extensive digestion. The mixture of the end fragments of Xgt-hC DNA and T4 DNA fragments was covalently joined by T4 DNA ligase and used to infect E. coli 802 (rK-, mK+) (Wood, 1966) treated with CaCl, according to Mandel & Higa (1970). While we obtained 27 plaques after ligation and transfection with a mixture of h and T4 DNA, only one plaque was obtained if the ligation step was omitted. The phages from the plaques were plated on E. coli ligts7 at 37°C to check the Red+ phenotype (Gottesman et al., 1973) and were characterized by the 3x 567

51%

L.

I’.

‘I’lliHOJl~liO\‘:\

fS’17’

.IL.

I

-Lf0Lki+kA* Fraction

.

.

I.0

of gradient

FIG. 1. Separation of the Xgt-hC DNA fragments. The fragments were separated in a glycerol gradient at 32,000 revs/min, rotor SW41, 14 h at 15”(‘, ultracentrifuge LS-50. After the run was completed the gradient solution was passed from t,he bot,tom of the tube through an Analabs u.v.-monitor. The fractions marked by a heavy line were collected and used in the ligase reaction.

EMRO test to check the ability of h phages to form stable lysogens (Gottesman & Yarmolinsky, 1968). Seven of the 27 phages were able to grow on E. coli ligts7 cells and produced stable lysogens. This suggests that they contain the EcoRI-C fragment in the same orientation as in h + . They were not analysed further. The other phages were not able to grow on E. coli ligts7 and therefore had a Red- phenotype. Sixteen of them produced lysogens as was shown by the EMBO test and had restriction ratios of 30 to 50, while the restriction ratios for hb2 and hgt,-hC under our conditions were equal to 2086 and 78, respectively. It was shown by the electrophoretic separation of the fragments generated by endo R. EcoRI that they contained the fragments of the same molecular weight as the EcoRI-C fragment. As has been shown by Thomas et al. (1974) the phages containing the EcoRl-C fragment inverted, phages hgt-hC’: form stable lysogens and reconstituted EcoRI restriction sites are still cleaved by endo R. EcoRI. This suggests that all 16 phages contained the EcoRI-C fragment in inverse orientation, genotype Xgt-AC’, and they were not further analysed. We propose they result from the contamination of the end fragment preparation with the EcoRI-C fragment even though its concentration was too small to observe in gel electrophoresis. The last four phages were not able to lysogenize E. c&i cells and had various restriction ratios. Their DNAs were able to hybridize with T4 [3H]DNA and they contained from one to three foreign fragments (Table 1). The electrophoretic separation pattern of the DNA fragments produced by endo R. EcoRI is shown in Figure 2. Tt was concluded that the phages contained T4 DNA fragments. Molecular weights of the inserted fragments were estimated from the electrophoretic mobilities of the fragments in comparison with mobilities of h DNA fragments of known molecular weights (Thomas & Davis, 1975). The standard curve for h DNA fragments was drawn on the basis of nine electrophoretic separations (Pig. 3) in l”/; agarose gel (voltage 2 V/cm). The fragment inserted in the genome of Xgt-T4, hybrid phage was not separated from the EcoRI-E fragment (Fig. 2,I). Therefore it has a

LETTERS

TO

THE

TABLE Prop&es

Restriction ratio

(‘lrmc

1

of Agt-T4

569

1 hybrid phages

Number of fmgments

Molecular weights f IO-”

1 3 2 2

3.0 3.0;1.3;0.8 1.8 :(I.3 2.4 :I)-9

12 479 152 154 78

2 3 4 hgt,-h(’ (cwltrol)

EDITOR

I)SADNA? hybridization

T4 genes inserted

0.064 0.13” CbO52 lW72 IJ.OO?

26.27.51 1,6,60

t Ratios of H3 label retained on the filters immobilized with hybrid phage DNA to 3H label on the filter immobilized with T4 DNA. DNA-DNA hybridization was performed as described by Green et al. (1969). A total of 3-6 rg of DNA was immobilized on 24-mm filter circles and incubated for 24 hours at 60°C with T4 t3H]DNA of specific activity 51,000 cts/min prl pg DNA.

molecular weight of about 3.0 x 106. One of the fragments inserted in the genome of Xgt-T4, had a high mobility in agarose gel, so its molecular weight was calculated by electrophoresis of incomplete cleavage fragments (Fig. 2,V). There is a good correlation between the total molecular weight of the inserted fragments of each phage and hybridization of the DNA of these phages to T4 f3H] DNA. Three hybrid phages contain more than one fragment. Most probably the inserted I to)

Ibl

II (cl

(01

2

A

Ib)

Ip

RI (cl

(0)

(b)(c)

(01

Ib)

Y (cl

(01

(b)

Cc)

and

XrT85i

4

A B

.B C D E F

C

FIG. 2. Electrophoretic separations of the DNA fregment,s of the hybrid phagss Ram7, through 1% agarose gels. The conditions of electrophoresiv were as described by Tikhomirova el ctl. (1976). I. dh. voltage 10 V/cm; (a) X gt-T4,, (b) X gt-T4, + hcI867Sam7, (c) hcI857Sam7. V. 9 h. voltage 2 V/cm. II (a) Agt-T4,, (b) Agt-T4, + XcI857Sam7. III (a) hgt-Tii,, (b) hcT857Ram7. IV (a) hg&T4,, (b) /\gt-T4, + hcI857Sam7. V, Incomplrte rnleavage (a) hgt T4,, (b) gt,-T4, + hcI857Sam7, (c) hcI857Sam7.

II, III, IV, Agt--T4, + rlf hgt-T4:,;

670

Relotwe

distance

FIG. 3. Determination of the molecular weights of the inserted curve. The inserted fragments are designated by number of clone decreasing size.

fragments and capital

from ! t he standard letters in order of

fragments of each hybrid phage are neighbouring since the products of incomplete endo R. EcoRI cleavage of T4 DNA were used. However, we cannot exclude the possibility that they originate from different parts of the T4 DNA molecule. In order to identify the T4 genes located in the inserted fragments a spot-test experiment was carried out. Loopfuls of 53 different T4 amber mutant phage preparations were plated on a lawn of W3350 (hsusA19) cells infected with hybrid phages (about 1 x lo0 per plate). Amber mutants with an index of reversion of less than 10m4 were used. There was a great increase in the number of plaques in the spots where recombination between T4 amber mutants and hybrid phage took place. With the hybrid phage Xgt-T4, confluent lysis was observed with the T4 amber mutants for genes 26, 27 and 51. The samepicture was observed for hgt-T4, and T4 TABLE

2

Recombinationbetween hybrid phages and T4 amber mutants T4 mutant

Gene

t About

Added

26 (N131)

Gent

27 (N120)

Gene

51 (869)

2.5 2.5x lx 2x 2 x

Gene

4 (N112)

3x 5x

Gene

5 (N135)

Gene

50 (11458)

1 x lo9 hybrid

phages

Number am+

to plate?

x 10’ IO’ + gt,-T4, 107 lo5 + gt-T4, 108 1Oj + gtkT4, 107

5x lo7 + gt-T4, 3x 106 3 x 10” + gt-T4, lx 106 2 x lo4 -+ gt-T4, were added

to plates

where

19 382

13 120 11 159 0 06 14 232 4

13 indicated.

of

LETTERS

TO THE

FIG. 4. The location of the genes inserted into Linkage map of T4 bacteriophage was compiled

EDITOR

571

the A phage genome on the genetic from Champe (1974).

map

of T4B.

amber mutants for genes 4, 5 and 50. We failed to identify the T4 genes located in the inserted fragments of hybrid /\gt-T4, and hgt-T4,. The spot-test data were confirmed in the experiments when W3350 (hsusA19) cells were coinfected with the hybrid phages and T4 amber mutants and plated on separate plates. The results of the experiments are presented in Table 2. The frequencies of T4 am+ phages when the cells were coinfected with the hybrid phages and T4 amber mutants exceeded the frequencies of spontaneous revertants by two to four orders of magnitude. Phages from the plaques were able to grow on E. c&i B cells. These results evidently show that recombination between T4 phages and hybrid phages takes place and that the hybrid phageshgt-T41 and hgt-T4, contain the T4 genes 26, 27, 51 and 4, 5, 50 involved in the synthesis of the base-plate (Champe, 1974) (Fig. 4). We thank for

technical

Institute Physiology

A. S. Solonin assistance. of Biochemistry of Microorganisms

for the T4 polynucleotide

ligase

preparation

and of the

U.S.S.R.

Academy of Sciences,Pushchino-on-Oka, Moscow Received

region,

142292,

4 October

1976,

U.S.S.R. and

in

revised form 14 January 1977

and

0. M. Selivanova

LAIMA P. TIKHOMIROVA DARA P. VOROZHEIKINA NINA I. PUTINTCEVA NICOLAI I. MATVIENKO

572

1,.

1'. '~lliHOhlIROV~\

ET

/I I;.

REFERENCES K., Loxeron. H. A. & Szyhalski, \V. (1971). In ~~lethods i)t I’iro/ogy!/, \-()I. 5. 1~1~. 271-m292. Champe, H. P. (1974). In Handbook oj Jficro6iology (Lasking. A. I. & Le~~h~~vali~~~~. H. A.. eds), vol. 4, pp. 605.-608, CRC press, (‘leveland, Ohio. de Waard, A., Paul, A. 1’. & Lehman, 1. Ii. (1965). J’roc. Rat. Acad. Sci., I:.S.A. 54, 1241-1248. Goorgepoulos, C. P. & Revel, H. K. (1971). l’irology, 44, 271- 285. Uottesman, M. E. & Yarmolinsky, M. B. (1968). .I. 1l1ol. Hiol. 31, 478-505. Gottesman, M. M., Hicks, M. & Gellert, M. (1973). J. 12ilol. Biol. 77, 531 ~547. Green, M., Fujinaga, K. & Pina, M. (1969). In Fundamental Techniques in l’irology, Academic Press, New X-ork. Ihler, G. & Kawai, Y. (197 I). .J. Nol. Hid. 61. 311X328. Kaplan, D. A. & Nierlich, D. P. (1975). ,J. Bid. Ch.em. 250, 2395-2397. Li, L., Tanyashin, V. l., Matvienko, N. I. di BayelI, A. A. (1975). Dokl. Acad. 1Vauk SSXK, 223, 1262-1265 (in Russian). McHattie, L. A., Ritchie, D. A. HL Thomas. (1. A. (1967). ./. Mol. Hiol. 23, 355-363. Mandel, M. & Higa, A. ( 1970). J. Mol. Biol. 53, 159 162. Murray, K. & Murray. N. E. (I 975). J. &lo/. Sol. 98, 551- 564. Polisky, B., Greene. T’., Garfitl, 1). E., McCarthy, H. J ., Goodman, H. M. bz Bayer, H. M’. (1975). hoc. h’at. ilcarl. Sci., 1 T.S.A. 72, 3310- 3314. Thomas, M. & Davis, K. W. (1975). J. Mol. Rio/. 31, 487. 505. Thomas, M., Cameron, .I. It. CI- Davis, H. \V. (1974). I’roc. Nat. Acad. Sci., ci.S.il. 71, 4579~-4583. Tikhomirova, L. P., Solonin, A. S., Kscnzcnko. V. Ni. dt Matvienko, N. 1. (1976). Nucleic Acid Res. 3, 24% 2490. Wood, W. (1966). J. Mol. Bid. 16, 118-133. Wyatt, G. R. dz Cohere, S. S. (1953). Biochem. J. 55, 774-782. Yoshimori, K. N. (1971). Ph. D. dissertation. University of California.

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