Phasmids: hybrids between Co1E1 plasmids and E. coli bacteriophage lambda

27

Gene, ll(l982) 21-44 Elsevier Biomedical Press

Phasmids: hybrids between ColEl plasmids and E. coli bacteriophage (“Lifting”

phages; att sites; recombinant

lambda

DNA; pACL vectors)

Sydney Brenner,Gianni Cesareni+ and Jonathan Karn Medical Research Council Laboratory of Molecular Biology, HillsRoad, Cambridge, CB2 2QH (U.K.) (Received July 18th, 1981) (Accepted November 3rd, 1981)

SUMMARY

Plasmids carrying cloned X att sites may be integrated into the bacteriophage genome by the site-specific recombination mechanism of h. The cross, referred to as “lifting” the plasmid, requires mixed infection of an Escherichia coli strain carrying the plasmid with two appropriately constructed “lifting” X phages. One phage donates a short left arm and the other donates a short right arm. These two short arms are of insufficient length to produce a viable phage genome and yield no recombinants when crossed on standard bacteria. However, viable recombinants are obtained when the genome length is extended by integration of one or more plasmids. We call these recombmants phasmids. They contain multiple att sites introduced at the ends of the integrated plasmids, and in the presence of integrase, recombination between these att sites can be exploited to effect release of the plasmid components. These novel genetic elements can be used in a variety of ways as vectors in genetic manipulation experiments. Sequences cloned in phasmids may be studied as a component of either a plasmid and or of a phage, and easily interconverted between the two states.

INTRODUCTION

Small plasmids related to ColEl and derivatives of phage h have been the vectors mainly used for the construction of in vitro DNA recombinants propagated in E. coli. To exploit the virtues of each vector

* Present address: European Molecular Biology laboratory, 69 Heidelberg, Postfach 10.2209 (F.R.G.). Abbreviations: am, amber mutations; att, phage A attachment site; bp, base pairs; A, deletion; Fee-, “feckless”, inability of h phage to grow on RecA strains; imm, bacteriophage immunity; inv, inversion; kb, kilobase pairs; s, restriction site; 0378-1119/82/0000-0000/$02.75

0 Elsevier Biomedical Press

and to study any sequence as a component both of a plasmid and of a phage, it is desirable to have quick and easy methods for interconversion between the two states. We have been able to accomplish this by exploiting the site-specific recombination mechanisms of phage h and have constructed plasmids with cloned h att sites which may be reversibly integrated and X, insertion; Spi-, P2-interference-less, h mutants that are gam-, red- and are able to grow on strains lysogenic for P2; su’, suppressor minus; su+, suppressor mutations; retR, tetracycIine resistance; tonA and tonB, the ton abbreviation refers to Tl resistance which is equivalent to resistance to 080 (tow4 and tonB are two resistance genes of E. coli); vir, vku-, lence; %h, 485 bp; < >, att sites generated by a lifting cross; (), indicates prophage in lysogen.

28

excised from specially constructed

phage genomes in

vivo by this process.

ing tonA mutants after selection with 48Ovir. This phage was chosen to prevent recombination with the prophage. Releasing strains are lysogens of EQ82 carrying the appropriate immunity and the constitutive int-c226 mutation (these strains are also xis-). Strain WY1 is the standard lysogen used for h immunity. The releasing strain WY22 is a derivative of WY21 which also carries the int-c226 prophage. This

MATERIALS AND METHODS

(a) Bacterial strains

is used for release from lysates of lifting crosses. Bacterial strains used to derive selective indicators in the construction of phages are listed in Table I. Derivatives include lysogens and a set resistant to different host range phages. All strains are derivatives of K-12 with the exception of CQ3, derived from E. cali C, which is naturally resistant to ti434. Strains resistant to @SO,isolated as the survivors after plating with $80 uir, were invariably tonA, and resistant to Tl. h-resistant hosts were picked as maltoseE. cdi

negative ’ colonies on McConkey maltose agar after plating with excess of hvir or k1. A recombinant hh434Xvir was used to isolate strains resistant to this host range. A fraction of these mutants was also resistant to Xhh. To select resistant variants of P2 lysogens, bio250K phage with the appropriate host range were used. Lysogens were tested either by induction (UV or high temperature) or by immunity to CI mutants of the phage, repurified and retested. Strains used for selection of phasmids in standard lifting crosses carry 81 immunity and are resistant to phage $80. WY21 was constructed first by lysogenizing EQ82 with ~80att80imm8lc’Sam7, and then isolat-

(b) Nomenclature

for bacteriophages

In general, our nomenclature

is similar to that used

by others. Restriction sites are named according to the form suggested by Borck et al. (1976) where SRI, sBam, sHindII1 (or sH3) refer to sites for EcoRI, BumHI and Hind111 cleavage. The sites are numbered sequentially from the right end of the lambda map (e.g., sR1 * 1, sH3 .6”, where superscript ’ indicates inactive

restriction

site). All sites appear in the wild-

type X sequence unless otherwise designated. Deletions either have the trivial name of origin (such as b189) or are named A [gr - gZ] where gl and g2 are either the genes bounding the ends or, in those cases where the deletions were made in vitro, the restriction sites from which DNA was excised. Inversions are denoted inv [s2 - sl] where sr and s2 are the sites flanking the fragment in the original phage. In phasmid structures open brackets < > are used to denote att sites generated by a lifting cross. In some cases, more than one plasmid may be included in the actual

TABLE I Parental bacterial strains Strain

Relevant features

M327 Q219 wxo EQ82 WR3 Q276 Q342

su

CQ3 4360 wx7 1

o

G sut11 su$u~IIhsr~hsmj: recA1 sue recA 1 su iI recA1 mfI mirI E. coli C(P2) miI(P2) aiII(P2)

WNO

groNSl2

Q203 Q241

WoP SUt POL41

WX24

RI plasmid

Source or ref. Cambridge collection Cambridge collection Ymel, C. Yanofsky; Taylor and Yanofsky (1964) ED8654, N. Murray; Borck et al. (1976) N100,M. Gottesman; Gottesman and Yarmolinsky (1968) Cambridge collection Cambridge collection G. Bertani Cambridge collection I. Herskowitz Georgopoulos (197 1) Georgopoulos and Herskowitz

(1971)

Zissler et al. (1971) Brammar et al. (1974); Yoshimori et al. (1972)

29

phasmid

but only a single plasmid is denoted.

positions

of restriction

coordinates Szybalski,

(i.e.,

The

sites are given in the text in h

in %X units;

see Szybalski

and

1979).

constructed

as follows.

Unlike

X, 81 phage (which

differ in their P genes) plate with full efficiency

tonA strains from crosses of $81 and hhti538 imm~. From these, $80[att-cIII]%mm81 phage were obtained by crossing with G80att80immh and selecting

(c) Construction of lifting phages Table II lists the bacteriophages used as donors of components of the lifting phages and phasmids speci-

on a groP strain resistant to tihx. The restriction

fied in Table III. Wherever possible, crosses were designed to generate unique recombinants and donors of various markers were standardized. Markers with

and this was used to confirm the structures

not easily selectable

phenotypes,

such as mutations

removing restriction sites, were introduced by close linkage to other mutations. We outline below the significant steps in the construction of more important and unusual combinations

on

groP strains (Georgopoulos and Herskowitz, 197 1). Xhti538imm81 recombinants were selected on groP

of phage markers.

(i) #80att80 tonB trpD’atth This long left arm is used to balance the very short right arms in our experiments. Hopkins et al. (1976) cloned HindIII fra~ents of the E. coli tryptophan operon with a functional trpD gene into an acceptor phage (hhA [SRI . 1-SRI * 2]imm2lc”ninS sHindII1 6”). We crossed one of these phages, 478 (Table HE) with the tr~sducing phage 483, ~8~tt8Ot~n~ trpDEutth Nam7am53cI”ninS [see (iv) below], and selected for Fee+ recombinants on a YecA strain resistant to A. Recombination occurs through the trp homology to generate an expanded phage, 545, #8O~tt8OtunB trpD’ atth 21~’ nin5 sHindIII.6”. To confirm the structure of this phage, a derivative with host range of h was made and lysogens of a tonB strain isolated. Such lysogens acquired a tonB* phenotype, regaining sensitivity to #SO phages and wild-type growth on media containing chromium ions. (ii) [fftt-cIII]81j~~81 and [att-cIII] ?‘&7rm81 Heteroduplex experiments by Niwa et al. (1978) showed that h and 81 are homologous in the att-xis region and at a few other sites, notably in the regions of the cIII and & genes. The ~~tt-cIII]81i~~81 arm was obtained from our original 81 strain, which differs in the QSR region from the Niwa et al. (1978) strain. This arm can be uniquely recombined with varous left arms by site-specific recombination with an appropriate bio250 phage, using a recA selecting strain. The donor of [att-cIII]$rm81

ton_4 was

of the att-cII1

maps

regions of h and 81 differ markedly of these

arms. (iii) h434b189

and h434Z&A[sRI

* l-sRIX

* 21

These strains are important ~te~e~ates for the introduction of amber and other mutations into the left arm. The published coordinates suggested that the h434 substitution (Simon et al., 1971; Rambach and Tiollais, 1974; Davis and Parkinson, 1971; Szybalski and Szybalski, 1979) might overlap the b189 deletion and the lac5 substitution, but crosses showed that recombination was possible with both. First, the h434 substitution was introduced into a h background, by crossing with X253, hXA [&IX - IsRIh - 2]inv[sRIX * 3-attWP’]spi6c1857 SRI * 4” nin5 SRI * 5” where spi6 is a substitution of the attP’ to ~111 region of phage 113 (Table HE) by the attB to gal region of E. coli in a naturally occurring gal transducing derivative of phage 113. The required recombinant selected on h-resistant P2 lysogen is 269, spi6 h434A [sRIh . 1-sRIX .2]inv [&IX * 3+ttP’] ~1857 sRIh. 4” nin5 SRI& - 5’. When 269 is crossed with b189 ~I857 or hh luc5 A [SRI . I--&IX -21 ~1857, rare recombinants with Fee+ phenotype (Zissler et al., 1971) were found

on X-resistant TeeA-

bacteria. (iv)sBam~l”b189andsBam~ l”hXlac5 A[sRI. lSRIA * 21 The sBam - 1’ marker (Klein and Murray, 1979) was introduced from a donor, 884, h&Barn -1” art80 tonB trpDE N7am53 ~1’ nin5, made by crossing 436 with 869 (Table IID). This was crossed into recipient b 189 or h&c5 arms carrying Wam403EamlOO mutations (from phage 526, Table II) and selecting for recombinants on su- hosts. (v) Removal of restriction

sites from the right arm

Modified restriction sites were introduced by linkage to known markers as follows: sRIX - 4’, Oam’1005 (Furth et al., 1977) sRIA - 5’, Sam7 (Murray

30 TABLE II Bacteriophage Number

Genotype

Source

hb 189cIam tiu~5A[~RI~l-sRIh~2]sRI3~cI857sRI~~4~PamsRIh~5~ hb522cIam hABAM cIam )ilac5A[sRIh~2aft]cIKH54sRIh~4°nin5 Qam73sRIh.S0

J. Parkinson P. Tiollais J. Parkinson M. Gottesman N. Murray

(A) Deletions 129 397 256 249 187

(B) Substitutions @80att~am7Nam53~1857Pam3ninS 258 h434art434imm2lcItsPam3 130 hatr80imm21cItsPam3 194 hpgllcI857Sam7 302 8limmSlcI; 178: 8limm8lcIts 92 8limm8lcIts am16 336

118 80

J. Salstrom Cambridge Cambridge 1. Herskowitz Cambridge

himm2lcIts

R. Thomas Cambridge

himm434c+

(C) Amber mutations

526

P. Leder

497

hWam403Eam1100 a[sRIh.l-21 inv[sRIh.3-21 ~I857 sRIh.4°nin5 sRIh.5’ Sam100 Uam439 Kam424 b522 red3 ~I857

725 119 231 111

AcI857 Oam1005 see E hA[sRI~~l-2]imm21c+Pam1001nin5 see D

M. Furth F. Stahl Cambridge

(D) Restriction 869 101 111 106

site alterations UBarn.1’ redam imm2lc+ hb519 ~1857 sRIh.4’ nin5sRIh.S” hA[sRIA*l-21 ~1857 ~RIh.4~ninS sRIh.S’Sam7 AA[sRIh.l-21 imm2lc+nin5 sH3h.6’

J. Parkinson

K. N. N. N.

(E) Other phage hint-c226 c+ 413 hcIIchil53 Pam902 119 Ab1319 ~I857 chi3 116 hA[sRIh.I-21 inv [sRIh.3-21 ~I857 sRIh.4’ nin5 sRIh.5’ 113 tiott80 tonB trpDl? Nam7 Nam53 AcI+ninS 415 hA[sRIh.I-21 trpCl0 imm2lcI*ninS sH3k.6’ 478 303

Aredam gam am210 hc+

474

kgal49 bio250 cI857

and Murray, 1974) sHindIIIh * 6’, Sam7 or Qam73 (Murray and Murray, 1975). Often the absence of EcoRI sites could be verified by measuring the efficiency of plating of phage on WX24, a strain containing the EcoRI restriction and modification enzymes (Yoshimori et al., 1972; Brammar et al., 1974). The

Murray Murray Murray Murray

L. Enquist F. Stahl J. Weil R. Davis N. Murray N. Murray F. Stahl H. Nash

structures of arms were confirmed enzyme mapping of phage DNAs.

by restriction

(vi) imm434cIIchil53 and immkI857cIIchil53 The mutation, chi153, is located in the ~11 gene and confers a cII- phenotype (Stahl and Stahl, 1975).

TABLE II (cont~ued) Number

Mutations

(A) Deletions 129 397

b189 A[sRI*l-sRIa.21

b522 A [ int and pACL33(B * B’) by the phage 260 (h80 attX A[intcIII]cI857) and 264 (hX bl89 [P’-cIII]sl imm81). The phasmids generated in these crosses contain only single plasmid inserts. In these experiments, 1.7 X low2 of infected cells containing pACL29 yield phas-

31 TABLE III Lifting phage, substitution arms, and phasmids Stock numbers are specified for the various phage. (A) Left arm donors, host range, deletion, [att-cIII], and immunity Host range

Left arm

Deletion

[att-CIII]

Immunity

81 81 81 A A A A A A

8lcIts 8lcIts 8lcIts 8lcIts 8lcIts 8lcIts 8lcIts 8lcIts 81cI+

hh lac5 A [sRIA . 1 - sRIA 21

b189

Stock numbers hA hh hh hh h434 hh hh hh hh

Lam439 Kam424 Warn403 Earn 100 sBam .

lo

sBam * lo

264 393 653 664 834 862 876 1094 911

648 656 827 861 878 972 970

(B) Right arm donors, $80 aft80 fonB TrpD’ aTfA A[int-CIII], immunity,

[ O-cosR ]

Immunity ~~1857 cIIchilS3

[O-cosR]

AKH54

AKH54 cIIchil53

434 cIIchil5 3

Stock numbers 767 584 782 598

754 586 783 558

817 801

559 574

593 1113 1104

802

738

1277

1274

(C) Substitution arms, 080 atr80 tonB trpD’,

atth

ninS chi3

Pam1005 nin5 chi3 nin5 Sam7 sRIA ‘4’ nin5 sRIA ’ 5’ sRIA .4O nin5 sRIA . 5’ sRIA . do nin5 sHindIIIA

deletion, immunitysosR ImmunitycosR

Deletion b522

wild type Pam902 Oam 1005

A [art-red]

b1319

Stock numbers 922 1335 1001

932 1336 1002

(D) Substitution

arms, h80 att80 2lcIts [O-cosR]

1118

~I857 sRIA .a0 nin5 sRIA ‘5’ Sam7 2lcIts sRIh .4O Pam1001 nin5 sHindIIIA . 6’ 2lcIts sRIh .4’ nin5 chi3

Stock numbers

[O-cosR]

673 1050 961 1114 1052 1036 1276 1167

sRIA .4O Pam1001 nin5 sHindIIIA .6’ SRIA .4’ nin5 chi3 SRIA .4O nin5 sHindIIIA .6’ sRIA . Jo nin5 sRIh 5’ sRIA .4o nin5 sRIA . SoSam7 sRIA .4’ nin5 Qam73 sRIA .5” sRIA . 4O nin5 sHindIIIA . 6” sRIA 5O

OamlOOS

Sam7 ‘6’

Sam7 am16 am16 am16

Sam7 Sam7

38 TABLE III (continued). (E) Phasmids with defined restriction endonuclease immunity_cosR h

pACL29

pACL46

maps. Stock numbers are shown for phasmids with the structure hh (pACL)

pACL5 3

pACL6 1

ImmunitysosR

Stock numbers sBamh sBamh

lo b189 lo b189

sBamA lo tiac5 A SRI. l+RIh . 2

1139 1129 1415 1039 1321

1323 1084 1324

1130 1122 1103

1417

1006 1127 1095 1008 1089 914

A[&cIII]cI8S7cIIchi nin5 chi3 A[int_cIII]cI857cIIchi sRIh 4’ninS sRIh ‘5’ A[inf-cIII]cI857cIIchi sRIh donin sRIh.5” Sam7 A lint-c1111cIK54 sRIh .4’ nin5 chi3 A[int-cIII] KH54 cIIchi sRIh 4Onin5 SRIA 5’ sHindIIIh 6’ b522 2lcIts sRIA ‘4’ nin5 chi3 b522 2lcIts SRIA .4O nin5 sHindIIIh 6’ 6522 2lcIts sRIh .4O nin5 SRI A .5O Sam7 A[int-red] 2lcIts sRIh .4O nin5 chi3 A[int-red] 2lcIts sRIh . b” nin5 sRIh . 5OSam7 A[inf-cIII] ~I857 cIIchinin5 chi3 A[ini-red]

951

when assayed on the indicator strain WX56. With pACL30 the efficiency was 1.2 X lo-’ but with pACL33 the yield was reduced tenfold to 1.2 X 10m3. The inefficient lifting of the B * B’ plasmid was eventually traced to the [P’cIII] region of phage 8 1. Substitution of [P'-~1111” region with the corresponding mids

region from h yielded hh lac5 A[sRI * 1-sRIh * 21 phage which lifted B . B’ plasmids efficiently. HOWever, these phage show somewhat

reduced efficiency

with P - P’ plasmids. Lifting is int-dependent;

recombinants

no

are

found with int- phage. In certain configurations, it is strongly x&dependent. In general, lifting of pACL29(P . P’) can dispense with xis but pACL33(B * B’) requires xis for efficient lifting. We have also carried out experiments

with lyso-

genie recipients for lifting. When a double lysogen of 393 and 593 (Table III) is transfected with pACL29, and plated at 39”C, more than 80% of the cells plated yield plaques on WY21 which are phasmids. Plating of the lysogen strain alone at 39’C gives no plaques on WY21 (< lo8 of cells plated). In this case, the cells have a chance to divide before they reach the temperature at which the phage are induced. Many of the phasmids have 81 immunity because at this temperature they can grow as virulent phage on the indicator. These represent single lifting events and are more abundant. In summary, conditions can be found for a given plasmid which give lifting efficiencies of 0.1 to 1%. It

2lcItssRIh

.4’nin5 sRIh

5” Sam7

is difficult to analyse the process in any detail since it involves multiple pathways for phasmid assembly and decomposition. (d) Growth properties of phasmids Phasmids may be grown 1yticalIy as phage or they can be’ propagated as plasmids in bacteria. In the latter case, the X replication functions must be repressed and the active repressor gene must be carried on the phasmid to avoid copy number escape virulence (Cesareni et al., 1981; Kahn and Helinski, 1978). After infection of cells with phasmids with a temperature sensitive repressor, such as Xc1857 or 2lcIts, colonies can be isolated at low temperature which are both resistant to ampicillin and immune to phage infection. These carry complete phasmids and lyse with a burst of phasmids after heat induction. No transductants are found on the polA mutant indicating that the maintenance of the phasmid with repressed X replication functions is by ColEl replication which is known to be dependent onpoZA (Sakakibara and Tomizawa, 1974).

(e) “Release” of the plasmid component

of phasmids

Efficient release of plasmids from phasmids requires both integrase and repressor to prevent replication of the phage genome. We have constructed a series of releasing strains which harbour a prophage carrying an appropriate immunity region and the int~226 mutation (Shimada and Campbell, 1974). These

39

10

0

20

40

30

80

RBaH

H R

Ba

100 % A

90

I 40

I _ 30 I

Ba

70

60

210

10

0

50

I I R Sa Sa

50 Kb I 6a

BaHH

H R

H

R

b189 17 b522

A

11

[ant -cllI] I-1 2, cIts

B nln 5 0

X 1127 : hX&amI’

b 189CpACL 29 > b522 21 cIts svRI4’nin 5 s_HllI 6” P.P’

21 cIts

PQA’

BaI

HO h 1129 : hWE3am I%189 < pACL 29 > A[ int - c X]CI 857 cll[chi sRI4”nin 5 SRI 5’ A-P’

PP’

P-P r

Ba

h.P’

HH

H

HH

H

P.P’

Ba I Fig. 4. Structure of some phasmids designed for use in genetic manipulation experiments. The restriction maps of h1127, All29 and Al 130 are compared with that of wild-type h DNA (top line). Bars underneath the restriction map of h indicate the map positions of the deletions (open bars) and substitutions (stippled bars) used in the construction of these phasmids. Bars underneath the restriction maps of the phasmids indicate the regions of DNA that may be substituted in genetic manipulation experiments after cleavage of the phasmid with the indicated enzyme (see legend to Fig. 2 for enzyme abbreviations).

phage

are all xis- but produce

stitutive

promoter

The efficiency frequency

with

integrase

from

a con-

in the xis gene. of release which

to a releasing

the infected

has been measured

phasmids

resistance

transduce

as the

ampicillin

cells yield

The releasing

efficiency

21 immunity

with

strain.

Approximately

ampicillin with strains

the appropriate

resistant

10% of colonies.

carrying

434 and

phasmid

is com-

40

parable to that obtained

with h. Release may also be

effected

with a helper phage of the

by coinfection

same immunity

which provides int and xis functions

but which itself does not lysogenize

or also in some cases by homologous

bination

between tandemly

recom-

plasmids.

Phasmids phages

listed

are constructed in Table

by crossing the lifting

III (sections

A and B) on

jklmnopqrst

abcdefghi ‘v-

ments

such as a b2

mutant,

integrated

(f) Construction of phasmids with defined structures for use as vectors in genetic manipulation experi-

*

%’

Fig. 5. Analysis of phasmid DNA structures by restriction mapping. DNA from the phasmids A914 (lanes a, g, m), Al127 @, h, n), h1129 (c, i, o) and ~1130 (d, j, p) and the plasmids pACL29 (e, k, q) and pACL53 (f, 1, r) were digested with BumHI (lanes a-f), EcoRI (g-l) or Hind111 (m-r). Lanes s and 1 show ~1130 DNA, respectively, digested with WI, and pACL53. The genotypes of phasmids and A are given in Table III, and Fig. 4. Plasmid pACL5 3 is a derivative of pBR322 carrying a h att from the Hind111 site at 56.8 to the &mHI site at 57.8 %h cloned in the Hind111 and BumHI sites in the tetracyclineresistance gene of the plasmid. The restriction fragments were end-labeled by incubation with AMV polymerase in the presence of 1.0 &i [&*P]dATP, and 100 &l dCTP, dGTP and dTTP, electrophoresed on 1% agarose gel and autoradiographed.

41

A.

Cloning

Restriction

(h1127-

BarnHI)

Fragments

&P

BamHI Cleave

with

Barn HI,

ligate

to 5-15

kb

BamHI

a

P-P’

Phasmid

P-n’

BamHI Select

Vector

21cI ts

Phasmld

inserted

fragments A-P’

Infect

with

with

DNA

i

P*A’ 21 cIts .:.:.:.~.:.:.:.:.:.:.:.:: .x::::::::.:.:.: ........ . .. . IBamHI

h integrase stra in

const itutive

P- P’

Select

ampicillin

resistant

B.

Plasmid

bacteria

inserted

Cloning

Restriction

Phasmid

Vector

(h

Fragments

BamHI

with

ligate

to10-20

with

Nonreleasing

PP’

P.P’

BamHI

BamHI

BamHI, kb

a

DNA

1129 - Barn HI>

A*P’

Cleave

contains

Select fragments A=P’ BamHI

phage with

inserted

DNA

7

BamHI

Fig. 6. Schematic diagram illustrating the use of phasmids as cloning vectors. (A) Cloning of BarnHI fragments in h1127. The cloned fragments are inserted into the BarnHI site of the lifted plasmid pACL29. This produces a recombinant phasmid with two tandemly placed h att sites from which a recombinant plasmid may be released. (B) Cloning of BumHI fragments in ~1129. After cleavage of this vector with BarnHI, the lifted pACL plasmids are removed and substitution of this DNA with new fragments yields a phage with a single atth site.

42

bacteria carrying pACL plasmids. The right arms of such phasmids can then be substituted by other arms employing standard phage crosses using the donor phage listed in Table III (sections C and D). A series of phasmids with specific restriction nuclease sites have been constructed manipulation structures

experiments

for use in genetic

(Table III, section E). The

of some of these phasmids

Fig. 4 and restriction

endo-

endonuclease

are shown in

digests of these

phage are shown in Fig. 5. Depending on the arrangement of restriction endonuclease sites, the product of a cloning experiment a recombinant

is either a phasmid from which

pACL plasmid may be released, or a

phage which retains a single attachment

site.

Xl 127 (Figs. 4, 5, 6) is an example of a phasmid which may be used to clone 5 kb to 15 kb BarnHI or Hind111 fragments. Phasmids carrying inserts and a single plasmid replicon are reconstituted when the phage ‘arms are ligated to appropriate restriction fragments (Fig. 5). Recombinant plasmids may then be released from these phasmids by infecting an appropriate releasing strain as described above. Phasmid vectors suitable for insertion of 5 kb to 15 kb EcoRI (X1129, 1130, 1323, 1324), BarnHI (1006, 1127), Hind111 (h1127, 1324) or Sal1 (h1130) fragments have also been constructed. Vectors capable of maintaining fragments up to 24 kb have also been produced; however, in these vectors the cleavage sites for restriction enzymes have been arranged such that only one attachment site remains in the recombinant phage and each of the plasmids is removed. An example of a phage vector of this type is Xl 139 used in conjunction with BarnHI.

DISCUSSION

We have exploited the site-specific recombination mechanism of bacteriophage h to affect natural recombination between plasmids and bacteriophage. We have called the resulting combinations “phasmids”, a term which has also been applied to combinations of bacteriophage and plasmids made by in vitro methods (Kahn and Helinski, 1978). Phasmids have properties of both plasmids and bacteriophage. They contain functional origins of replication of ColEl and h and may be propagated lytically as a

bacteriophage presence

of

or non-lytically integrase

as a plasmid.

recombination

takes

In the place

between the multiple att sites and this can be used to effect release of integrated plasmids. The genetic elements

described in this paper have

been used in a variety of ways in genetic manipulation experiments. pACL plasmids

Nematode

DNA has been cloned in

which have been transferred

into h

vectors by lifting crosses to facilitate screening and storage of the clones. Plasmids of interest were then recovered by release from selected phasmids for amplification and analysis of the inserted DNA (Karn, J. and Brenner, S., unpublished). Alternatively, phasmids have been used as phage substitution vectors. Depending on the placement of restriction sites with respect to the attachment sites the product of a cloning experiment is either a phage or a phasmid from which a recombinant plasmid may be released. Because of the modular nature of the phasmids we have been able to construct an extensive range of phage which accommodate different sizes, and types of restriction fragments. Recently this collection has been improved and expanded by the development of derivative vectors which permit genetic selection for inserted DNA (Karn et al., 1980). Phasmids have also been extensively used in the genetic analysis of plasmid-coded genes. A line structure map of the fl-lactamase gene has been constructed, and mutants affecting plasmid replication have been selected and mapped in phasmids. The mutant plasmids were recovered by “release”, (Castagnoli et al., 1981; Cesareni et al., 1981). The methods described here for “lifting” and “release” of miniColE plasmids by site-specific recombination should also be applicable to systems involving other replicons and DNA sequences. Recent experiments have demonstrated h integrase activity in vitro (Kikuchi and Nash, 1978, 1979; Hsu et al., 1980; Mizuchi and Mizuchi, 1979). It seems likely that these techniques could be used to effect in vitro release of an insert from a phasmid and this could be used to recover fragments that cannot replicate in E. coli or lack a suitable selective marker.

ACKNOWLEDGEMENTS

We thank our many colleagues who send us bacterial and phage strains (see Tables I and II). George

43

Pieczenick

constructed

pACL46

constructed

pACL61.

preparation

of this manuscript.

Leslie

and Rita Fishpool

Barnett Gianni

aided in the Cesarini was

supported by a fellowship from the EMBO and Jonathan Karn was supported by a fellowship from the Helen Hay Whitney Foundation.

REFERENCES Blattner, F.R., Fiandt, M., Hass, K.K., Twose, P.A. and Szybalski, W.: Deletions and insertions in the Immunity region of coliphage h: revised measurements of the promoter-startpoint distance. Virology 62 (1974) 458-471. Borck, K., Beggs, J.D., Brammar, W.J., Hopkins, AS. and Murray, N.E.: The construction in vitro of transducing derivatives of phage h. Mol. Gen. Genet. 146 (1976) 199207. Brammar, W.J., Murray, N.E. and Winston, S.: Restriction of hrrp bacteriophages by E. coli K. J. Mol. Biol. 90 (1974) 633-647. Castagnoli, L., Cesarini, G. and Brenner, S.: The phasmid as a tool for plasmid genetics, I. Fine structure of the p-lactamase gene. Genet. Res. (1982) submitted. Cesarini, G., Castagnoli, L. and Brenner, S.: The phasmid as a tool for plasmid genetics, II. Isolation of point mutations that affect replication of a ColEl related plasmid. Genet. Res. (1982) submitted. Davis, R.W. and Parkinson, J.S.: Deletion mutants of bacteriophage h. III. Physical structure of attP. J. Mol. Biol. 56 (1971) 403-423. Enquist, L.W. and Weisberg, R.A.: A genetic analysis of the att-int-xis region of coliphage h. J. Mol. Biol. 111 (1977) 97-120. Fiandt, M., Hradecna, Z., Lozeron, H.A. and Szybalski, W.: Electron micrographic mapping of deletions, insertions, inversions and homologies in the DNAs of coliphages h and phi80, in Hershey, A.D. (Ed.), The Bacteriophage Lambda. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1971, pp. 329-354. Fiandt, M., Gottesman, M.E., Shulman, M.J., Szybalski, E.H., Szybalski, W. and Weisberg, R.A.: Physical mapping of coliphage hatt*. Virology 72 (1976) 6-12. Furth, M.E., Blattner, F.R., McLeester, C. and Dove, W.F.: Genetic structure of the replication origin of bacteriophagelambda. Science 198 (1977) 1046-1051. Georgopoulos, C.P.: Bacterial mutants in which the gene N function of bacteriophage lambda is blocked have an altered RNA polymerase. Proc. Natl. Acad. Sci. USA 68 (1971) 2977-2981. Georgopoulos, C.P. and Herskowitz, I.: Escherichia coli mutants blocked in lambda DNA synthesis, in Hershey, A.D. (Ed.), The Bacteriophage Lambda, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1971, pp. 553-564.

Gottesman, M. and Yarmolinsky, M.: The integration and excision of the bacteriophage lambda genome. Cold Spring Harbor Symp. Quant. Biol. 33 (1968) 735-748. Heffron, F., Bedinger, P., Champoux, J.J. and Falkow, S.: Deletions affecting the transposition of an antibiotic resistance gene. Proc. Natl. Acad. Sci. USA 74 (1977) 702706. Henderson, D. and Weil, J.: Recombinationdeficient deletions in bacteriophage h and their interaction with chi mutations. Genetics 79 (1975) 143-174. Hopkins, A.S., Murray, N.E. and Brammar, W.J.: Characterization of htrp-transducing bacteriophages made in vitro. J. Mol. Biol. 107 (1976) 549-569. Hsu, P.-L., Ross, W. and Landy, A.: The lambda phage att site: functional limits and interaction with Int protein. Nature 285 (1980) 85-91. Kahn, M. and Helinski, D.R.: Construction of a novel plasmid-phage hybrid: Use of the hybrid to demonstrate ColEl DNA replication in vivo in the absence of a ColElspecified protein. Proc. Natl. Acad. Sci. USA 75 (1978) 2200-2204. Karn, J., Brenner, S., Barnett, L. and Cesareni, G.: Novel bacteriophage lambda cloning vector. Proc. Natl. Acad. Sci. USA 77 (1980) 5172-5176. Kikuchi, Y. and Nash, H.A.: The bacteriophage lambda int gene product. J. Biol. Chem. 253 (1978) 7149-7157. Kikuchi, Y. and Nash, H.A.: Nicking-closing activity associated with bacteriophage lambda int gene product. Proc. Natl. Acad. Sci. USA 76 (1979) 3760-3764. Klein, B. and Murray, K.: Phage lambda receptor chromosomes for DNA fragments made with restriction endonuclease I of Bacillus amyloliquefaciens H. J. Mol. BioL 133 (1979) 289-294. Mizuchi, K. and Mizuchi, M.: Integrative recombination of bacteriophage lambda: in vitro study of the intermolecular reaction. Cold Spring Harbor Symp. Quant. Biol. 43 (1979) 1111-1114. Murray, K. and Murray, N.E.: Phage lambda receptor chromosomes for DNA fragments made with restriction endonuclease III of Haemophilus influenzae and restriction endonuclease I of Escherichia coli. J. Mol. Biol. 98 (1975) 551-564. Murray, N.E. and Murray, K.: Manipulation of restriction targets in phage h to form receptor chromosomes for DNA fragments. Nature (Lond.) 251 (1974) 476-481. Nash, H.A.: AatfBattP, a h derivative containing both sites involved in integrated recombination. Virology 56 (1974) 207-216. Niwa, O., Yamagishi, H. and Ozeki, H.: Sequence homology in DNA molecules of temperate phages 081, $80 and h. Mol. Gen. Genet. 159 (1978) 259-268. O’CalIaghan, C.H., Morris, A., Kirby, S.M. and Shingler, A.H.: Novel method for detection of p-lactamases by using a chromogenic cephalosporin substrate. Antimicrob. Agents Chemother. 1 (1972) 283-288. Parkinson, J.S.: Deletion mutants of bacteriophage lambda, II. Genetic properties of attdefective mutants. J. Mol.’ Biol. 56 (1971) 385-401.

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Rambach, A. and Tiollais, P.: Bacteriophage X having EcoRI endonuclease sites only in the non essential region of the genome. Proc. Natl. Acad. Sci. USA 71 (1974) 39273920. Sakakibara, Y. and Tomizawa, J.-I.: Replication of colicin El plasmid DNA in cell extracts: IL Selective synthesis of early replicative intermediates. Proc. Natl. Acad. Sci. USA 71 (1974) 1403-1407. Shimada, K. and Campbell, A.: Knt~onstitutive mu~nts of bacteriophage lambda. Proc. Natl. Acad. Sci. USA 71 (1974) 237-241. Simon, M.N., Davis, R.W. and Davidson, N.: Heteroduplexes of DNA molecules of lambdoid phages: physical mapping of their base sequence relationship by electron microscopy, in Hershey, A.D. (Ed.), The Bacteriophage Lambda. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1971,~~. 313-328. Stahl, F.W., Craseman, J.M. and Stahl, M.M.: Reomediated recombinational hot spot activity in bacteriophage lambda, III. Chi mutations are site-mutations stimulating ret-mediated recombination. J. Mol. BioL 94 (1975) 203~212. Stahl, F.W. and Stahl, M.M.: Ret-mediated recombinational hot spot activity in bacteriophage h, IV. Effect of heterology on chi-stimulated crossing over. Mol. Gen. Genet. 140 (1975) 29-37. Sternberg, N. and Weisberg, R.: Packaging of prophage and host DNA by colipbage lambda. Nature 256 (19759 97103. Szybalski, E.H. and Szybalski, W.: Physical mapping of the

at&N region of coliphage lambda: apparent oversaturation of coding capacity in the gum-ral segment. Biochimie 56 (1974) 1497-1503. Szybalski, E.H. and Szybalski, W.: A comprehensive molecular map of bacteriophage lambda. Gene 7 (1979) 217270. Taylor, M. and Yanofsky, C.: Transformation of bacterial markers and transfer of phage markers with DNA isolated from a h-&O hybrid phage carrying the tryptophan genes of E. co& Biochem. Biophys. Res. Commun. 17 (1964) 798-804. Thomas, D., Cameron, J. and Davis, R.W.: Viable molecular hybrids of bacteriophage lambda and eukaryotic DNA. Proc. Natl. Acad. Sci. USA 71 (1974) 4579-4583. Tiemeier, D.L., Enquist, L. and Leder, P.: Improved derivative of a pbage hEK2 vector for cloning recombinant DNA. Nature (Land.) 263 (1976) 526-527. We& J., Cunningham, R., Martin III, R., Mitchell, E. and Bolling, B.: Characteristics of Xp4, a h derivative containing 9% excess DNA. Virology 50 (1972) 373-380. Yoshimori, R., Roulland-Dussoix, D. and Boyer, H.W.: R. Factor-~ontroB~ restriction and mod~i~tion of DNA: restriction mutants. J. Bacterial. 112 (1972) 1275-1279. Zissler, J., Signer, E. and Schaefer, F.: The role of recombination in growth of bacteriophage lambda: I. The gamma gene, in Hershey, A.D. (Ed.), The Bacteriophage Lambda. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1971, pp. 455-475. Communicated by W. Szybalski.