Proc. Nati. Acad. Sci. USAVol. 84, pp. 4379-4383, July 1987Biochemistry

Syrinx 2A: An improved X phage vector designed for screeningDNA libraries by recombination in vivo

(gene cloning/phage-plasmid cointegrates/homologous recombination)

CHARLES T. LUTZ*t§, WILLIAM C. HOLLIFIELD*, BRIAN SEEDS, JOSEPH M. DAVIE*¶,AND HENRY V. HUANG**Department of Microbiology and Immunology and tDivision of Laboratory Medicine, Washington University School of Medicine, St. Louis, MO 63110;and tDepartment of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114

Communicated by David M. Kipnis, March 9, 1987

ABSTRACT The Syrinx 2A phage and irAN13 plasmidwere designed for screening of DNA libraries by homologousrecombination in vivo. Syrinx 2A carries multiple cloning sitesand a recently identified X gene, rap (recombination adept withplasmid), required for efficient phage-plasmid recombination.We describe a rapid, reliable, and technically easy method toscreen Syrinx 2A libraries, expand the resulting phage-plasmidcointegrates, and subclone plasmid in as little as 2 days.Recombination screening allows one specific member of aclosely related multigene family to be isolated selectively.

alent phenotype is encoded by the QSR region of phage 480,which renders Charon 4A and related phage Rap+. Wedescribe here the construction of Syrinx 2A, a A cloningvector that is Rap' and that contains multiple cloning sites(BamHI, EcoRI, Sal I, Sst I, andXba I). We also describe theminiplasmid, TAN13, which is of high copy number forimproved recombination screening of phage libraries. Wepresent efficient strategies to screen phage libraries, subclonetarget DNA, and isolate specifically one member of a closelyrelated multigene family.

Specific clones from a bacteriophage A library can be selectedwith nucleic acid probes by one of two methods. TheBenton-Davis method relies on hybridization of a DNA orRNA probe to phage DNA that has been immobilized on anitrocellulose or other filter (1). The in vivo recombinationmethod of Seed relies on homologous recombination betweenphage DNA and a probe plasmid. Phage-plasmid cointe-grates are isolated by selection for a marker carried by theplasmid (2). Library screening by in vivo recombinationpotentially offers five important advantages over traditionalhybridization methods: (i) recombination screening can iden-tify selectively one gene from a family of closely relatedgenes, without the need for special hybridization washingconditions; (ii) in vivo recombination is fast, requiring only 10hr to obtain specific clones; (iii) an enormous number ofphage (109) from one or several libraries can be screenedeasily in parallel in a single experiment; (iv) the point ofplasmid integration allows one to map the phage clones easilyand to locate the sequence of interest within the phage; (v)plasmid subclones of target sequences can be obtained easilyby appropriate restriction enzyme digestion of cointegrateDNA, followed by ligation and transformation.

Despite these potential advantages, recombination screen-ing has not been widely adopted. First, the plasmid irVX,originally used as the vector for recombination screening, isof low copy number, due to partial deletion of the replicationprimer promoter (2, 3). This results in a low frequency ofrecombination. Second, and more importantly, phage-plas-mid recombination is inefficient with most commonly used Acloning vectors. Exceptions include Charon 4A and otherphage vectors that either carry the 480 QSR region orphage-encoded recombination functions, but these phagevectors suffer from a paucity of cloning sites (3-5).A bacteriophage A gene, rap (recombination adept with

plasmid), is required for efficient phage-plasmid recombina-tion (W.C.H., Evan N. Kaplan, H.V.H., unpublished re-sults). rap maps to orf-204 (ninG) and is deleted by the nin5deletion from most A cloning vectors. A functionally equiv-

MATERIALS AND METHODSReagents. L broth, NZMg agar, NZMg top agar, and SM

buffer have been described (7). TE is 10 mM Tris, pH 7.6/0.1mM EDTA, pH 7. Isopropyl B-D-thiogalactoside (IPTG,Sigma) and 5-bromo-4-chloro-3-indolyl P-D-galactoside (X-Gal, Bachem Fine Chemicals, Torrance, CA) were used asdescribed (7). NZYMg broth is NZMg supplemented with 5g of Bacto yeast extract per liter. Restriction enzymes(Bethesda Research Laboratories, New England Biolabs,and Boehringer Mannheim) were used as described (ref. 8,pp. 98-106) with minor modifications. Pancreatic DNase I(Sigma) and RNase A (Boehringer Mannheim) were dis-solved in 10 mM Tris (pH 7.6) at 5 mg/ml and stored frozen.Proteinase K (Boehringer Mannheim) was dissolved in TE at2 mg/ml and used immediately.

Bacterial Strains. All strains are Escherichia coli K-12 deriv-atives; LE392 (ref. 8, p. 504) is supEsupF; JF242 is a recB21-recC22 derivative of LE392. The recBC- phenotype of JF242cultures was routinely checked using sensitivity to UV irradi-ation, as described (7). MC1061 (9) is sup°; MC1061/p3 (2) iskanamycinR, amber mutation in ampicillinR, amber mutation intetracyclineR (where R is resistant or resistance). MC1061/p3transformed with irAN7, irANl3, or their derivatives weregrown in the presence of ampicillin and tetracycline. LG75 (7)is supo, lacZam.

Plasmids and Phage. 7AN7 is an 885-base-pair (bp) plasmidcontaining the pBR322 replicon (FnuDII-Dde I, 2521-3159,ref. 10). A HindIlI linker (CAAGCTTG) was inserted into theFnuDII site and was cut with HindIII and ligated to theHindIlI site of a polylinker sequence (GAA TTC CCG GGGATC CGT CGA CCT GCA GAT CTCTAG AAG CTT). TheEcoRI site of the polylinker was ligated to the EcoRI site onthe promoter side of the synthetic tRNA supF gene of 7VX(2); the other EcoRI site of supF was filled in and joined withthe pBR322 replicon at the Dde I site, leaving vAN7 with aunique EcoRI site in the polylinker. (See the 1986 New

Abbreviations: IPTG, isopropyl P-D-thiogalactoside; X-Gal, 5-bromo-4-chloro-3-indolyl B-D-galactoside.§Present address: Department of Pathology, University of Iowa,Iowa City, IA 52242.TPresent address: G. D. Searle and Co., Skokie, IL 60077.

4379

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Proc. Natl. Acad. Sci. USA 84 (1987)

England Biolabs catalog for a complete restriction map ofirAN7.) iTAN13 is an 895-bp plasmid that carries theEcoRI-HindIII polylinker of plasmid pUC13/phageM13mpll (GAA TTC GAG CTC GCC CGG GGA TCC TCTAGA GTC GAC CTG CAG CCC AAG CTT) in place of theIrAN7 polylinker. The plasmid rMTSXSX contains a 268-bpXba I-Sst I fragment of X DNA (24,508-24,776, ref. 11) andwas used to test recombination frequency with phage vec-tors. Syrinx 2A (Fig. 1) was constructed by standard methodsand consists ofthe left arm, from cosL to EcoRI, ofEMBL3A(12), and the right arm, from EcoRI to cosR, of XR76d2. TheXR76d2 right arm was derived from the EcoRI-Sst II (linkerat ca. 34,499-40,389, ref. 12) fragment of EMBL3A, the SstII-HindIII fragment of XcI857Sam7 (40,389-44,141, with aninternal deletion of HindII-Nru I, 40,942-41,810, ref. 11),which confers the Rap' phenotype, and the HindIII (44,141)-cosR fragment of XgtWES-XB (16). The internal or "stuffer"region of Syrinx 2A consists of (from left to right) the6.6-kilobase (kb) EcoRI lacS fragment (which is invertedrelative to Charon 4A) and the 7.7-kb EcoRI bio256 fragment,both from Charon 4A (4). Syrinx 2A is Aam32, Baml (fromEMBL3A), Sam100 (from XgtWES-XB).Phage Library Construction and Amplification. Genomic

DNA libraries were constructed and packaged by standardmethods (ref. 8, pp. 269-294). HGAC 47 hybridoma 1.9- to2.4-kb Sst I DNA fragments were isolated on low-melting-temperature agarose gels, ligated to dephosphorylated Syrinx2A Sst I cloning arms, and packaged in vitro to produce 8.2x 105 phage. This primary library was amplified by plate lysison 150-mm NZMg agar plates using about 109 (1-2 ml offreshovernight culture) JF242 cells and 1 x 105 phage per plate toyield 4.9 x 1010 phage. The combination ofa recBC- host anda red- gam- phage minimizes deletion of DNA sequencesdue to recombination during library amplification and elim-inates the selective advantage of those clones that, bychance, carry chi sequences in their inserts (ref. 8, pp.24-25).Recombination Screening and Rapid Subcloning of Target

DNA. The strategy is outlined in Table 1, with which a Syrinx2A library can be screened, phage-plasmid cointegrates canbe expanded, and target DNA sequences can be subcloned,all in as little as 2 days.To screen a library, one or multiple aliquots of Syrinx 2A

library phage (AamBamSam) are used to infect MC1061/p3/irAN13-probe plasmid cells at a multiplicity ofinfection ofabout 0.1. All phage will productively infect this supF host,but only Syrinx 2A library phage with significant homologyto the probe will recombine in vivo to form phage-plasmidcointegrates. (Cointegrates may contain plasmid dimers-e.g., due to phage recombining with dimeric plasmids.) Theinfected cells are then plated with a 10- to 20-fold excessnumber of sup' MC1061 cells. Under these conditions, onlyphage-plasmid cointegrates or, rarely, phage that have re-verted all three amber mutations will form plaques. Thecointegrate plaques may be small or irregular. Plaques areinoculated into 2 ml of NZYMg and shaken overnight. After

cos L_

SBR H

Aam Bam

lysing bacteria and clearing debris these "minilysates" typ-ically have titers of 1-10 x 108 plaque-forming units/ml.Phage-plasmid cointegrates are further expanded by in-

fecting about 9 x 108 JF242 cells with 0.5 ml of minilysate.Debris typically appear in 1.5-3 hr. After lysing remainingbacteria and pelleting debris, aliquots can be stored at 4°C asphage stocks and the remainder of these midilysates can beused to prepare phage DNA.During the midilysate procedure, putative cointegrates are

tested for supF by spotting a sample of minilysate onto afreshly poured lawn of LG75 (sup°, lacZar) plus IPTG andX-Gal. Only cointegrates will form blue plaques.Phage-plasmid cointegrate DNA is isolated by clearing

debris from 1 ml of phage midilysate in a microcentrifuge,digesting free nucleic acids with DNase I and RNase A,precipitating the phage with PEG/NaCl, and disrupting thephage with EDTA/NaDodSO4/proteinase K. The DNA isextracted, precipitated, and resuspended in TE.

Target DNA can be rapidly subcloned by digesting 0.1 ,ugor 0.1 vol of the DNA preparation with an appropriaterestriction endonuclease, such that 1rAN13 and flankingsequences are included on one fragment. In the cointegrateshown in Fig. 2, ITAN13 and the rearranged V,,-2547 genesegment to the left of irAN13 were contained on a singleBamHI or HindIII fragment. Fragments are circularized byligation in dilute conditions and are used to transformMC1061/p3 cells.

Southern Blotting. Southern blotting was performed essen-tially as described (13). Hybridization probe sequences wereseparated from plasmid vector sequences by restrictionendonuclease digestion and electrophoresis through low-melting-temperature agarose gels and were then labeled byrandom hexamer primer extension (14). The 3' JK recombi-nation probe is a Rsa I-Sst I 446-bp fragment derived fromA/J liver DNA, which lies immediately downstream of theJ.Ks gene segment (bases 2105-2551, ref. 15), inserted into theSma I-Sst I sites of 7TAN13. The JKS hybridization probe isa 333-bp Hae III-Sau3AI fragment derived from BALB/cliver DNA (bases 1995-2328, ref. 15). The 5' VK_25_39 and 5'VK-2547 probes were 547-bp and 535-bp EcoRI-Nsi I frag-ments derived from the rearranged VK gene segments ofHGAC 39 and HGAC 47, respectively (13). These 5' VKprobes were inserted into the EcoRI-Pst I sites of mAN7,replacing linker sequences homologous to polylinker se-quences in Syrinx 2A (see Discussion and ref. 3).

RESULTSSyrinx 2A Phage. Syrinx 2A was constructed as described

in Materials and Methods; its derivation is outlined in Fig. 1.Syrinx 2A is AamBamSam and requires supF for lyticgrowth; supE does not efficiently suppress SamlOO (16).Syrinx 2A is Rap' for efficient recombination with plasmid.Foreign DNA can be cloned between Sal I, BamHI, EcoRI,Sst I, and Xba I sites. Syrinx 2A is red-, int-, gam- and istherefore deficient in phage-encoded recombination func-tions. Syrinx 2A recombined with test plasmid, irMT5XSX, at

RBI rI I iii'

Si sI xloc 5

Origin:I I EMBL3Ai-----I Charon 4A9MIM XcI857S7

XgtWES *X B

SI bio256

1kb

FIG. 1. Syrinx 2A map. Shown are the phage from which Syrinx 2A was derived. Also shown are relevant genes, the region encoding theRap' phenotype, and restriction endonuclease sites, Sal I (S), BamHI (B), EcoRI (R), HindIII (H), Xba I (X), Sst I (SI), and Sst II (SII). TheEcoRI lacS fragment is inverted relative to Charon 4A.

4380 Biochemistry: Lutz et al.

.s SE H cos R.le a

IRap+ Sam

Proc. Natl. Acad. Sci. USA 84 (1987) 4381

Table 1. Outline of recombination screening andrapid subcloning

Day Step

1 Library screening1. Incubate aliquots of 5 x 106 Syrinx 2A library

phage with 50 1.l of fresh overnight culture ofMC1061/p3/rAN13-probe plasmid at 370C, 20 min

2. Add 1 ml of fresh overnight culture of MC1061and 10 ml of melted NZMg top agar, pour onto150-mm NZMg agar plate, incubate at 370C, 10-12hr

Minilysate1. Pick plaques with Pasteur pipette, inoculate into 2

ml of NZYMg, shake overnight at 370C2 2. Lyse remaining bacteria with three drops of

CHCl3, pellet debris at 1000-2000 x g, 10 minMidilysate

1. Add 1 ml of fresh overnight culture of JF242 to 1.5ml of NZYMg, shake at 370C, 15-30 min

2. Add 0.5 ml of minilysate, incubate at 370C, 15 min3. Shake at 370C until debris appears, for a maximum

of 3 hr4. Lyse remaining bacteria with five drops of CHCl3,

pellet debris at 1000-2000 x g, 10 minTest for phage-plasmid cointegrates

1. Mix 0.1 ml of fresh overnight culture of LG75 with10 ,ul of 0.1 M IPTG, add 3 ml of melted NZMgtop agar and 50 Al of X-Gal (20 mg/ml), pour onto100-mm NZMg agar plate

2. Spot minilysates on LG75 lawn with steriletoothpicks

3. Incubate at 37°C, 10-24 hr, score for blue plaquesIsolation of DNA

1. Clear 1 ml of midilysate in microcentrifuge at15,000 X g, 5 min

2. Recover supernatant, add 25 ,g of DNase I and 5,g of RNase A, mix and incubate at 37°C, 30 min

3. Mix by inversion with 0.5 ml of 30% PEG/3 MNaCl/10 mM MgCl2

4. Incubate at 0°C, 1 hr5. Precipitate phage in microcentrifuge at 4°C, 5 min,

and aspirate supernatant6. To the phage pellet add 75 ,l of 0.33 M EDTA/

0.33% NaDodSO4, add 0.2 ml of TE andresuspend vigorously

7. Add 25 Al of freshly dissolved proteinase K (2mg/ml in TE) and incubate at 37°C, 30 min

8. Extract twice with equilibrated phenol and twicewith diethyl ether

9. Precipitate DNA with 0.5 vol of 7.5 M NH4OAc, 3vol of ethanol

10. Resuspend pellet in TERapid subcloning

1. Digest 0.1 vol of DNA with appropriate restrictionendonuclease, extract, precipitate, resuspend DNAin TE

2. Ligate with T4 DNA ligase in 30 ,ul at 16-25°C, 2hr

3. Transform frozen, transformation-competentMC1061/p3 cells, select for ampicillin plustetracycline resistance

a frequency of 0.05 per infected cell, whereas Charon 4Arecombined with irMT5XSX at a frequency of 0.17. Incontrast, XgtWES-XBAgam recombines with irMTSXSX at amuch lower frequency (by a factor of 5000).Recombination Screening Using Syrinx 2A: Immunoglobulin

VK Gene Segments. Using mouse immunoglobulin K genes asa model system, we tested how efficiently and specifically

.42.1kb

SI SIl

Si

2.1 kb

H B X Si

lO1 bp

SI H B SI

_ _ 1.3 kb _

I ZI Target DNA97///z Probe DNA

ITAN 13 plasmid vector DNA

FIG. 2. Recombination of probe plasmid with target DNA pro-duces a phage-plasmid cointegrate. Shown is the predicted config-uration of cointegrate DNA after the 3' JK probe recombined with therearranged HGAC 47 V,,/JK gene segments. The indefinite borders ofthe 3' JK probe sequences and homologous target sequences in thecointegrate indicate that the recombination event may occur any-where within the probe region but would still be expected to produceSst I fragments of 2.1 and 1.3 kb. Restriction endonuclease sites areSst I (SI), HindIlI (H), and BamHI (B).

Syrinx 2A libraries could be screened by recombination. Theproductively rearranged VK_-2547/JK gene segment of hybrid-oma HGAC 47 resides on a 2.1-kb Sst I fragment (13).Homologous recombination between a plasmid, which con-tains the 3' JK Rsa I-Sst I probe, and the rearranged VK/JKgene segment should generate a 2.1-kb Sst I fragment,containing VK-2547 and 3' J,, sequences, and a 1.3-kb Sst Ifragment, containing 3' J,, sequences (Fig. 2). We used Syrinx2A to clone 1.9- to 2.4-kb Sst I fragments ofHGAC 47 DNA,size selected to include the productively rearranged VK-2547gene segment but not related VK gene segments or other JKgene segments (ref. 13; C.T.L., unpublished data). Wescreened 6.25 x 106 phage from the amplified HGAC 47library by in vivo recombination with the 3' JK probe. Sixputative phage-plasmid cointegrates were recovered, each ofwhich produced a blue plaque on LG75 (Table 2) andtherefore carried a suppressor gene. DNA from five of thesecointegrates contained the expected 2.1- and 1.3-kb Sst Ifragments (Figs. 2 and 3). (In addition to the phage cloningarms, four of the clones also contained extra Sst I fragments,due to the tandem insertion of more than one Sst I fragmentduring library construction.) The 2.1-kb Sst I fragment inthese five cointegrates hybridized to a 5' VK-25-39 probe(Figs. 2 and 3) that cross-hybridizes with VK-2547 (13). Aspredicted, all five cointegrates also carried 3' JK sequences ontwo Sst I fragments as demonstrated by hybridization with aJ,5 probe, which overlaps with the 3' J,, recombination probe(Figs. 2 and 3). Thus, these five clones contained theappropriate target DNA. The plasmid plus flanking se-quences from one of the cointegrates was subcloned (seeMaterials and Methods, Table 1): The cointegrate DNA (40

Table 2. Recombination screening of the HGAC 47-Syrinx 2Aphage library

Recombination Frequency of trueExperiment probe positives x 10-1

1 3' JK 802 5' VK-25-39 4

5' VK-_2547 152

Biochemistry: Lutz et al.

Proc. Natl. Acad. Sci. USA 84 (1987)

In

a

C 0

o _E0

u

0

200'

0 iu c

01 1 ~U

kb- 23.1

- 9.42-

*:-6.56-

-4.37-

_E0 ,00 -5,±a

IzC _Oc

CL CL

9

2.32-2.03 -

- 1.35- _

- 108-

5'VK_25 -39PROBE

JK5 PROBE

FIG. 3. Phage-plasmid cointegrates contain expected targetDNA. (Left) One hundred nanograms ofDNA from the cointegratesand from a control phage was digested with Sst I, Southern blotted,hybridized to the 5' VK-25-39 probe, and exposed to Kodak XAR-5film. (Right) The filter was then washed free of all radioactivity asdescribed (13), hybridized to the J,,5 probe, and exposed to film.HindIII-digested X DNA and Hae III-digested 4OX174 DNA wereused as molecular size standards, listed in kb.

ng) was digested with BamHI, ligated, and used to transformMC1061/p3 and gave 1700 transformants. All 10 plasmidsubclones examined had restriction maps consistent with themap of the cointegrate.The remaining cointegrate contained insert Sst I fragments

of 6.1 and 1.9 kb, rather than the expected fragments of 2.1and 1.3 kb, and did not hybridize to the 5' VK-25-39 probe. TheJS probe detected a single band that comigrated with the SstI cloning arms (about 17.5 kb). It is likely that the plasmid inthis case integrated into phage DNA outside the borders ofthe hybridoma insert DNA.Recombination Screening Selects One Member of a

Multigene Family. Shen and Huang (7) demonstrated thatphage and plasmid that share a region of 100% sequencehomology recombine some 40 times more frequently thanphage-plasmid pairs only 90% homologous, consistent withresults reported by Seed (2). To test whether one member ofa multigene family in a Syrinx 2A library could be isolatedselectively, we screened 5 x 107 phage from the HGAC 47library with the 100% homologous 5' VK-2547 probe or withthe ca. 95% homologous (C.T.L., unpublished data) 5'VK-25-39 probe (Table 2). (The 5' VK-25-39 and 5' VK-2547probes strongly cross-hybridize under stringent conditions:60 mM NaCl/6 mM sodium citrate at 68°C, Fig. 3, ref. 13.)Eighty-three putative phage-plasmid cointegrates were re-covered with the 100% homologous 5' VK-2547 probe; 11 of12 randomly selected cointegrates tested grew in minilysate,for a calculated recovery of76 cointegrates per 5 x 107 phage,or 152 x 10-8, a frequency similar to that of the 3' JK probe(Table 2). In contrast, the heterologous VK-25-39 probe gaveonly 2 phage-plasmid cointegrates for a frequency of4 x 10-8(Table 1). All 10 of the homologous cointegrates that weretested and both of the heterologous cointegrates containedthe desired VK-2547/JK rearranged gene segment, since they

JK5 PROBE

FIG. 4. Heterologous and homologous phage-plasmid cointe-grates contain expected target DNA. Cointegrates were isolated fromthe HGAC 47 library with the heterologous 5' VK-25-39 probe or thehomologous 5' VK-2547 probe and 50 ng of cointegrate DNA wasdigested with Pst I, Southern blotted, and hybridized to the J,,5probe. Hae III-digested 4X174 DNA was used as molecular sizestandard, listed in bp.

contained a 780-bp JK+5 Pst I fragment (Fig. 4) identical to theJKS-hybridizing Pst I fragment of the productively rearrangedgene segment in HGAC 47 (C.T.L., unpublished data). Thus,the 100% homologous 5' VK-2547 probe recombined 38 timesmore frequently with the rearranged HGAC 47 Vj/J genesegment than did the :'90% homologous 5' VK-25-39 probe(Table 2).

DISCUSSIONRecombination screening is a rapid, reliable, and technicallyeasy method to select specific clones from bacteriophage X

libraries. Using our protocol (see Materials and Methods,Table 1), phage-plasmid cointegrates can be isolated andexpanded and plasmid can be subcloned in 2 days. We reporthere Syrinx 2A, a X phage vector, and rAN13, a plasmidvector, designed for recombination screening. Syrinx 2A is aversatile cloning vector for inserts of 8-21 kb if clonedbetween the BamHI, EcoRI, or Sal I sites. (The BamHI sitecan be used to clone partially Mbo I- or Sau3AI-digestedDNA to approximate uniform distribution of genomic DNAin the library.) Syrinx 2A can also accept fragments of 0-11.9kb at the Sst I sites and fragments of 0-6.6 kb at the Xba Isite.The Syrinx 2A and 7AN13 vectors were designed to fulfill

several requirements for efficient recombination screening.(i) Plasmid and phage vectors must not be homologous toeach other. This extends to polylinker sequences, since Shenand Huang (7) found a recombination frequency of 2 x 10',when phage and plasmid share only a 19-bp homologousregion. Indeed, we recovered 50 phage-plasmid cointegrateswhen 107 phage with the Syrinx 2A polylinker were screenedwith irAN7 (C.T.L., unpublished data). The predicted se-

quence of the Syrinx 2A polylinkers is identical to the irAN7polylinker for a stretch of 13 bp flanked by homologous

bp

-1353-1078- 872- 603

4382 Biochemistry: Lutz et al.

Proc. Natl. Acad. Sci. USA 84 (1987) 4383

stretches of 6 and 8 bp (12). IrAN13 was designed to share nosignificant homology with Syrinx 2A.A second requirement (ii) for efficient recombination

screening is that the X phage vector should carry rap, presentin Syrinx 2A and Charon 4A. X phage without rap recombinewith plasmid less efficiently (by a factor of 100-5000). (iii)The plasmid should have a high copy number. Plasmids withcopy number of ca. 3 (e.g., 7rVX) recombine less frequently(by a factor of at least 10) than plasmids with a copy numberof ca. 30 (e.g., 7TAN7 and rrAN13; H.V.H., unpublisheddata). (iv) The phage-plasmid cointegrate DNA must notexceed the size limit for efficient packaging of X phage DNA,about 51 kb. Because the rAN13 plasmid is very small, 895bp, and plasmids that carry 250- to 400-bp probe regionsrecombine at an adequate frequency, this does not signifi-cantly constrain the size of insert DNA used for libraryconstruction. For 200- to 400-bp probes, inserts ofup to about19.8 kb can be cloned between the BamHI, EcoRI, or Sal Isites of Syrinx 2A. (v) The plasmid must carry a selectablemarker. The supF tRNA gene is small and suppresses allthree amber mutations in the Syrinx 2A. The multiple ambermutations of Syrinx 2A minimize the frequency of amberrevertants that are supF-independent; we routinely screenphage lysates for amber revertants (<10-8) before preparingstocks of phage vector DNA. More importantly, packagingextracts must not contribute supF-independent phage. Wehave encountered commercial packaging extracts that, whenused to package libraries, gave rise to supF-independentphage. A simple solution to this problem would be toUV-irradiate packaging strains (17).The frequency of cointegrates can be predicted as follows.

Based on the size of the mammalian genome (about 3 x 106kb; ref. 6), a library of 1.7 x 105 phage with an average insertof 17.5 kb corresponds to one haploid equivalent of DNA.Screening such a library with a 270-bp probe 100% homolo-gous to target sequences (e.g., a cDNA fragment) shouldyield phage-plasmid cointegrates at a rate of 5.8 x 10-6 x 5.2x 10-2 or 3 x 10-7 (frequency of a single copy sequence inthe library multiplied by the frequency of recombination fora 100% homologous 270-bp sequence, ref. 7). Thus, about 30phage-plasmid cointegrates should appear when 108 phageare screened. Because the frequency of recombination cor-relates with length of probe (7), screening libraries with shortprobes requires that more phage be screened. A mammalianDNA library screened with a 44-bp probe should yieldphage-plasmid cointegrates at a rate of 5.8 x 10-6 x 3.6 xi0-3 or 2 x 10-8 (frequency of a single copy sequence in thelibrary multiplied by the frequency of recombination for a100% homologous 44-bp sequence, ref. 7). Thus, about twocointegrates should appear when 108 phage are screened.When 100% homologous probes are not available (e.g.,

probes derived from a different species), two strategies cancompensate for the lower frequency of recombination. (i)Longer probe sequences increase the likelihood of longstretches of perfect homology. For example, a 405-bp probe90% homologous to phage sequences recombined at a fre-quency of 2.9 x 1O-3, comparable to the recombinationfrequency for a 100% homologous 44-bp probe (7). (ii) Analiquot of the phage library can be expanded by plate lysis onbacteria that carry the probe plasmid to provide multipleopportunities for recombination, followed by selection ofcointegrates of sup° bacteria (2).Complete characterization of phage-plasmid cointegrates

includes DNA sequence determination, but preliminary in-formation might be obtained as follows. First, since aberrantrecombination events are uncommon (this paper and C.T.L.,unpublished data), clones with identical or overlapping re-striction maps are likely to contain the desired target se-quences. Second, the restriction map of the clones should be

consistent with the restriction map obtained by Southernblotting of genomic DNA. More simply, if the probe plasmidcontains a unique restriction endonuclease site in the proberegion, then the desired cointegrates can be quickly distin-guished from aberrant cointegrates by possession of a re-striction fragment identical in size to the probe plasmid. Forexample, homologous Sst I sites in the 3' JK probe and thetarget sequence (Fig. 2) allowed us to predict a 1.3-kb Sst Ifragment in the resulting cointegrates.Gene conversion occurs frequently during phage-plasmid

recombination (W.C.H., unpublished data) and could alterthe target DNA sequences in the region of recombination ifprobe and target are not 100% homologous. A simple strategyis to screen the library in parallel with two nonoverlappingprobes. Target sequence can then be deduced from the twosets of cointegrates.Recombination screening may be most useful to isolate one

specific member of a closely related multigene family. It isquite difficult, using hybridization techniques, to differenti-ate sequences that are >90% homologous to each other.Thus, many members of a multigene family will usually bedetected by hybridization screening, each of which needs tobe characterized to find the specific member. With recom-bination screening, in contrast, the sequence with the bestmatch will be isolated at the highest frequency (Table 2). Ifall members of a multigene family must be isolated, hybrid-ization screening will identify the most clones. If one specificmember of a multigene family must be isolated, recombina-tion screening will produce the clone most homologous to theprobe. Thus, hybridization screening and recombinationscreening complement each other. Recombination screeningmay be most useful when the gene that encodes a cDNA issought or when a specific locus or even a specific allele mustbe isolated from multiple genomic libraries. Recombinationscreening of Syrinx 2A libraries with ITAN13 probe plasmidallows specific members of multigene families to be isolatedrapidly and selectively.We thank Mrs. Janice Cole for cheerful preparation of this

manuscript and Dr. N. Murray for EMBL3A phage. This work wassupported by National Institutes of Health Grants GM31838 andAI-15926 and by a grant from Hoechst AG. C.T.L. is supported byNational Institute of General Medical Sciences Training GrantGM-07157.

1. Benton, W. D. & Davis, R. W. (1977) Science 196, 180-182.2. Seed, B. (1980) Nucleic Acids Res. 11, 2427-2445.3. Huang, H. V., Little, P. F. R. & Seed, B. (1987) in Vectors: A Survey of

Molecular Cloning Vectors and Their Uses, ed. Rodriguiz, R. (Butter-worth, Stoneham, MA), in press.

4. Williams, B. G. & Blattner, F. R. (1979) J. Virol. 29, 555-575.5. Murray, N. (1983) in Lambda II, eds. Hendrix, R. W., Roberts, J. W.,

Stahl, F. W. & Weisberg, R. A. (Cold Spring Harbor Laboratory, ColdSpring Harbor, NY), pp. 677-683.

6. Lewin, B. (1980) Gene Expression: Eukaryotic Chromosomes (Wiley,New York), Vol. 2, 2nd Ed., pp. 488-492.

7. Shen, P. & Huang, H. V. (1986) Genetics 112, 441-457.8. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1983) Molecular Cloning: A

Laboratory Manual (Cold Spring Harbor Laboratory, Cold SpringHarbor, NY).

9. Casadaban, M. J. & Cohen, S. N. (1980) J. Mol. Biol. 138, 179-207.10. Sutcliffe, J. G. (1979) Cold Spring Harbor Symp. Quant. Biol. 43, 77-90.11. Daniels, D. L., Schroeder, J. L., Szyblaski, W., Sanger, F., Coulson,

A. R., Hong, G. F., Hill, D. F., Petersen, G. B. & Blattner, F. R. (1983)in Lambda II, eds. Hendrix, R. W., Roberts, J. W., Stahl, F. W. &Weisberg, R. A. (Cold Spring Harbor Laboratory, Cold Spring Harbor,NY), pp. 519-676.

12. Frischauf, A.-M., Lehrach, H., Poustka, A. & Murray, N. (1983) J. Mol.Biol. 170, 827-842.

13. Lutz, C. T., Bartholow, T. L., Greenspan, N. S., Fulton, R. J.,Monafo, W. J., Perlmutter, R. M., Huang, H. V. & Davie, J. M. (1987)J. Exp. Med. 165, 531-545.

14. Feinberg, A. P. & Vogelstein, B. (1984) Anal. Biochem. 137, 266-267.15. Max, E. E., Maizel, J. V., Jr., & Leder, P. (1981) J. Biol. Chem. 256,

5116-5120.16. Leder, P., Tiemeier, D. & Enquist, L. (1977) Science 196, 175-177.17. Maniatis, T., Hardison, R. C., Lacy, E., Lauer, J., O'Connell, C.,

Quan, D., Sim, G. K. & Efstratiadis, A. (1978) Cell 15, 687-701.

Biochemistry: Lutz et al.