Site-Specific RecombinationDirected by Single-Stranded Crossover Linkers:Specific Deletion of the Amino-Terminal

Region of the \g=b\-Galactosidase Gene in pUC Plasmids

WING L. SUNG and DIANA M. ZAHAB

ABSTRACT

The "duplex crossover linker" technique was simplified and used to delete the \g=b\-galactosidase (\g=b\-Gal)-codingsequence upstream from the multiple restriction sites in pUC plasmids. A single-stranded crossover linker,with a homology-searching sequence as short as 5 bases, was initially ligated to a linearized plasmid. InsideEscherichia coli, the plasmid was circularized by intramolecular, homologous recombination between the(5\m='\-or 3\m='\-) protruding homology-searching sequence and a targeted region in the opposite terminus. As a con-sequence, sequences beyond the point of integration were deleted. Specific deletion of sequences up to 1472bp was demonstrated. The single-stranded linkers apparently avoided generation of undesirable mutants as-sociated with the usage of duplex linkers. A mechanism has been proposed for the intramolecular recombina-tion directed by the crossover linkers. It principally involves either 3\m='\- or 5\m='\-exonucleolytic breakdown of thehomologous terminus of the plasmid, circularization by spontaneous pairing of the exposed complementarystrands, and subsequent degradation of any redundant sequence.

INTRODUCTION

PLASMiD pUC8 (Vieira and Messing, 1982), a.commoncloning vector, is efficient for gene expression (Sung et

al., 1986a). However, with all of its unique restriction sitesclustered within a /3-galactosidase (/3-Gal) gene, the ex-

pressed product of any cloned DNA would be a polypep-tide fused to an amino-terminal /3-Gal sequence (Sung etal., 1986a). For direct expression of any foreign DNAcloned in pUC8, it would therefore require precise deletionof this short /3-Gal-coding sequence upstream from the re-gion of multiple restriction sites.Recently a crossover linker technique for specific gene

modification has been developed (Sung et al., 1986b).Essentially it involves an in vivo intramolecular recombina-tion between the homologous redundant termini of a plas-mid intermediate. The construction of this key intermedi-ate requires a double-stranded linker that has (i) a cohesive

Division of Biological Sciences, National Research Council of

end for an initial ligation with a terminus of the linearizedplasmid, (ii) a middle region with a sequence promotingspecific modification, and (iii) a homology-searching se-

quence that is homologous to a specific domain in the op-posite plasmid terminus targeted for subsequent recombi-nation.The crossover linker technique has been modified and

used to delete the short ^-Gal-coding sequence between themultiple restriction sites and the ribosome-binding site inplasmid pUC8. We establish that the crossover linkers canbe used as single-stranded instead of as duplex linkers pre-viously reported (Sung et al., 1986b). As a demonstrationof their efficiency, these single-stranded linkers directedspecific deletion of a human parathyroid hormone (PTH)cDNA (299 bp) and a tetracycline resistance (TcR) gene(1472 bp) located within pUC plasmids in separate experi-ments.

, Ottawa, Canada K1A OR6.

MATERIALS AND METHODSMaterialsAU enzymes were purchased from Bethesda Research

Laboratories (BRL) or Boehringer Mannheim. Bacterialstrain Escherichia coli JM103 was used in all transforma-tions. Plasmid pUC8 was purchased from BRL (Fig. 1A).Short oligonucleotides COL-1, COL-2, COL-3, COL-2B,COL-Ps/ I, COL-12 (Fig. IB), COL-2C (11-mer, 3'-CTTT-GTCCTAG-5', Bam HI end underlined) and COL-2E (9-mer, 3'-TTGTCCTAG-5') homologous to the lac operator

(lacZpo), were prepared by a DNA synthesizer (model380A, Applied Biosystem). Dideoxynucleotide chain-termi-nation kit for DNA sequence was from New England Bio-labs. Plasmid pPTH-84 was prepared previously by inser-tion of human parathyroid hormone (PTH) cDNA betweenthe Eco RI and Hind III sites (Fig. 1C) (Sung et al.. 1986c).Plasmid pUC9-TcR (Pharmacia) was derived from inser-tion of the pBR322 tetracycline resistance gene between theEco RI and Sma I sites of pUC9 (Fig. ID) (Vieira andMessing, 1982). All plasmids possessed an ampicillin-resis-tance (ApR) gene.

A. Plasmid pUC8

lacZpo e-Gal2200 2210 2220 start 2230 2240

1 i met thr met i le thr asn ser arg.TGAGCGGATA'ACAATTTCAC ACAGGAAACA GCT ATG ACC ATG ATT ACG AAT TCC CGG.ACTCGCCTAT TGTTAAAGTG TGTCCTTTGT CGA TAC TGG TAC TAA TGC TTA AGG GCC

RBS2250 2260gly ser val asp leu ginGGA TCC GTC GAC CTG CAG.CCT AGG CAG CTG GAC GTC.BamHI PstI

EcoRI

B. Crossover linkers

C0L-1COL-2

COL-3

C0L-2B

COL-PstIC0L-12

22001 2210 2220 |TACAATTTCAC ACAGGAAACA GT TGTTAAAGTG TGTCCTTTGT CCTAG-51 BamHI or BglllT TGTTAAAGTG TGTCCTTTGT CTTAA-5' EcoRI

CCTTTGT CCTAG-5' BamHI

ACAATTTCAC ACAGGAAACA GTGCA-3' PstIAGTG TGTCCTTTGT C

C. Plasmid pPTH-84

lacZpo { \

D. Plasmid pUC9-Tc

lacZpo \

B-Gal PTH

i-299 bp-

?-Gal Tc

1472 bp-

BglllHindlll

EcoRII

FIG. 1. A. Plasmid pUC8. The amino-terminal region of the /3-Gal gene that was targeted for modification ispresented. B. Synthetic crossover linkers COL-1 :COL-2, COL-3, COL-2B and COL-Pst LCOL-12. Numbers above thelinkers indicate the region of homology in the /3-Gal gene of pUC8. The linkers are also aligned according to their se-quence homology. C. Plasmid pPTH-84. D. Plasmid pUC9-TcR. Regions between vertical arrows indicate the lac pro-moter/operator (lacZpo) sequence (position 2201-2221) homologous to the synthetic crossover linkers.

Construction of plasmid p WS-1 RESULTS

Synthetic oligonucleotides COL-1 and COL-2 (0.3pmole, 1 pi, sixfold molar excess) were phosphorylated in-dividually in a solution (22.5 pi) containing 0.13 mM ATP,70 mM Tris-HCl pH 7.6, 10 mM MgCl2, 100 mM KC1, 5mM DTT and 15 U of T4 DNA kinase at 37°C for 1 hr.The two phosphorylation solutions were combined andheated at 70°C for 10 min. After cooling slowly to roomtemperature, the solution was added to a solution (8 pi)containing 75 mM Tris-HCl, 7.5 mM MgCl2, 12.5 mMDTT, 0.5 mM ATP, 1 U of T4 DNA ligase, and 100 ng (2pi) of Eco Rl-Bam HI-treated plasmid pUC8. After incu-bation at 12°C for 16 hr, the ligation mixture was used totransform E. coli JM103 on YT plates (8 g Bacto tryptone,5 g Bacto yeast extract, 5 g NaCl, 15 g Bacto-agar in 1 liter)containing 100 pg Ap/ml. Transformants were colorimetri-cally selected for the loss of /3-Gal activity on XGal andIPTG, and analyzed by hybridization to 32P-labeled probeCOL-2. Filters were washed at 58°C, then 63°C. Coloniesthat hybridized with COL-2 were selected for plasmid prep-aration. The operator and amino-terminal regions of thelacZ gene of the plasmids were sequenced by the dideoxy-nucleotide chain-termination method.The modification of Eco Rl-Bam HI-treated plasmid

pUC8 described above was repeated. Substituting duplexCOL-1 :COL-2 (Fig. IB), single-stranded linker COL-2,COL-2B (Fig. IB), COL-2C or COL-2E at various molarratios (6-, 84-, 500-, and 2500-fold excess) was used in theconstruction of plasmid pWS-1.

Construction of other pWS plasmidsThe general approach described, involving phosphoryla-

tion, ligation of a crossover linker with an appropriate plas-mid, hybridization, and DNA sequencing, had beenadopted for the construction of the other pWS-type plas-mids.To construct plasmid pWS-3 , pUC8 was linearized with

Eco RI and dephosphorylated with calf intestinal alkalinephosphatase (CIAP). In separate experiments, the linear-ized plasmid was subsequently modified by linker COL-3(0.3 pmole or sixfold molar excess), with or without thecomplementary fragment COL-1.To construct plasmid pWS-4, pPTH-84 was linearized

with Bgl II and dephosphorylated with CIAP. LinkerCOL-2 (4 pmole or 66-fold molar excess) was used tomodify the linearized plasmid pPTH-84.To construct plasmid pWS-5, pUC9-TcR was linearized

with Eco RI and dephosphorylated with CIAP. LinkerCOL-3 (4 pmole or 66-fold molar excess) was used tomodify the linearized plasmid pUC9-TcR.Construction of plasmid pWS-6 was accomplished by

modifying 100 ng of Bam Hl-Pst I-treated pUC8 with un-phosphorylated single-stranded linker COh-Pst I (2.4pmoles, 40-fold molar excess), with or without the phos-phorylated complementary fragment COL-12.

Construction of plasmid pWS-1 with duplexlinker COL-1 :COL-2

Crossover linker duplex COL-1 :COL-2, which possessesa Bam HI end and a 22-base homology with bases 2201-2221 of pUC8 (Fig. IB), was phosphorylated and ligated tothe Bam HI terminus of the Eco Rl-Bam HI-linearizedpUC8 (Fig. 2). Among 293 transformants generated, 22white colonies (on a background of blue colonies) resultingfrom the loss of the /3-Gal activity, were selected for hy-bridization with probe COL-2. At 58°C, 18 transformantcandidates demonstrated positive affinity to probe COL-2.However at 63°C, only 11 of the 18 candidates maintainedtheir affinity for COL-2. DNA sequencing of plasmids in-dicated that the group of 11 candidates (positive at 63°C)did possess plasmid pWS-1 (11 mutants per 293 transfor-mants, or 4% mutant population). Nucleotide sequencingconfirmed that bases 2222-2248 was deleted (Fig. 3). Theother seven candidates (positive at 58°C but negative at

63°C) harbored plasmid pWS-2 (mutant population 2.5%),which had lost an A:T base pair in the region of triple A:Tbase pairs around position 2216, in addition to the designeddeletion of the sequence encoding the amino-terminal of/3-Gal (Fig. 3). In both plasmids pWS-1 and pWS-2, themultiple restriction sites are immediately downstream fromthe two ribosome-binding sites (at positions 2214 and2221).

Construction ofplasmid pWS-3Further test of the specificity of crossover linker was

conducted in a linearized plasmid possessing identical re-striction ends. A new crossover linker COL-1:COL-3 (Fig.IB), with a homology-searching sequence identical toCOL-1 :COL-2, had an Eco RI end for ligation with theEco RI/CIAP-treated pUC8 (Fig. 4). In the biosyntheticpathway envisaged, two molecular equivalents of theCOL-1 :COL-3 duplex were probably ligated to both EcoRI ends of the plasmid to yield intermediate Z for the sub-sequent intramolecular recombination (Fig. 4, path A).The first duplex linker, ligated to the end containing the lacoperator, could not initiate homologous recombination be-cause of its undesirable orientation. However, the secondduplex linker, ligated to the other end of the plasmid,would be able to recombine intramolecularly with the op-posite lac operator to yield plasmid pWS-3. Mutant trans-formants were identified by both hybridization and nucleo-tide sequencing (23 mutants per 218 transformants, or11%). All restriction sites of the precursor plasmid pUC8had been preserved in the new plasmid pWS-3 (Fig. 3).After further analysis, we suspected that the initial de-

phosphorylation of the 5'-terminus of the linearized plas-mid designed primarily to suppress regeneration of theplasmid pUC8 might also have prevented the ligation ofCOL-1 to the plasmid intermediate. This would thereforeimply that only COL-3 of the COL-1 :COL-3 duplex had

EcoRI \ BamHI EcoRI BamHI

nucleases BamHI,EcoRI

BamHI

recombinationin E. coli

FIG. 2. Specific deletion of the sequence encoding /3-Gal (hatched) by crossover linkers COL-1 (stippled), and COL-2(black). A duplex of COL-1 :COL-2, which was homologous to the lac operator (stippled and black), was ligated to theBam HI end of Eco Rl-Bam HI-treated pUC8 to give a terminally redundant intermediate. Intramolecular recombinationbetween homologous regions yielded plasmid pWS-1.

been joined covalently to the plasmid. Subsequent deletionmight be achieved through a different intermediate Z' (Fig.4, path B), instead of Z (Fig. 4, path A).To test the feasibility of achieving the same modification

by a single-stranded crossover linker, the previous conver-sion of plasmid pUC8 to pWS-3 was repeated with onlylinker COL-3 (Fig. 4, path B), instead of duplex COL-1:COL-3. Hybridization and nucleotide sequencing identifiedmutant transformants bearing plasmid pWS-3 (42 mutantsper 1160 transformants, or 3.6%).

Construction of plasmid pWS-4: Specificdeletion of cloned PFH cDNALinker COL-2 with its Bam HI end was ligated to the

complementary Bgl II ends of Bgl II/CIAP-treated pPTH-84 (Fig. 1C) for the precise deletion of a 299 bp sequencecomprising both PTH cDNA and the /3-Gal gene residue.Of 1200 transformants, 22 hybridized to probe COL-2(mutant population 1.9%). DNA sequencing of plasmidpWS-4 prepared from these transformants indicated thatthe 299 bp sequence had been deleted accurately (Fig. 3).

Construction of plasmid p WS-1 withsingle-stranded linker COL-2Conversion of Eco Rl-Bam HI-treated plasmid pUC8 to

pWS-1 was repeated with the single-stranded linker COL-2.Mutant transformants harboring plasmid pWS-1 were

identified (Fig. 3). Increasing the molar excess of linkerCOL-2 improved the yield of mutants (2.8% with seven-

fold molar excess, 17% with 500-fold, and 16% with 2500-fold). The unexpected mutant pWS-2, which was isolatedin the study with duplex linker COL-1 :COL-2, was not de-tected. In a related experiment (not shown), linker COL-2directed deletion of the same sequence in pUC8 bearing dif-ferent versions of the PTH cDNA between the Bam HI andHind III sites, resulting in a mutant population as high as

91%.

Construction of plasmid pWS-5: Specific deletionof a cloned tetracycline resistance geneLinker COL-3 was ligated to the Eco RI terminus of the

linearized plasmid pUC9-TcR (Fig. ID). Of 550 transfor-mants chosen without prescreening of tetracycline-resis-tance, only one hybridized to probe COL-3. DNA sequenc-ing of the new plasmid pSW-5 isolated from this transfor-mant indicated that the 1472-bp sequence comprising bothtetracycline resistance gene and the /3-Gal gene residue hadbeen deleted specifically (mutant population 0.2%) (Fig.3).

Construction of plasmid pWS-6The intramolecular recombination process described so

far involved plasmid intermediates possessing either a

Plasmid pWS-1lacZpo2200J 2210 2220 I 2260

.ACAGGAAACA GGA TCCGTCGACC TGCAG.

.TGTCCTTTGT CCT AGGCAGCTGG ACGTC.RBS BamHI PstI

Plasmid pWS-2 same as pWS-1, but with an A:T bp at position-2216deleted.I l 2250

Plasmid pwS-3 _!.ACAGGAAACA GAA TTCCCGGGGA TCCGTC_.TGTCCTTTGT CTT AAGGGCCCCT AGGCAG....

EcoRI BamHI

Plasmid pwS-4

Plasmid pWS-5

Plasmid pWS-6

.ACAGGAAACA GGA TCT TGA AGC TT.

.TGTCCTTTGT CCT AGA ACT TCG AA.original HindiIIBgl11 site

I.ACAGGAAACA GAA TTCACTGGCC..TGTCCTTTGT CTT AAGTGACCGG.

EcoRI

| 2265.ACAGGAAACA GTGCAG..TGTCCTTTGT CACGTC.

originalPstI site

FIG. 3. New plasmids pWS-1, pWS-2, pWS-3, pWS-4, pWS-5, and pWS-6 derived from pUC-type plasmids. PlasmidpWS-1 has bases 2222-2248 deleted. Plasmid pWS-2 is the same as pWS-1, but with an additional deletion of an A:T basepair at position 2216. Plasmid pWS-3 has bases 2222-2238 deleted. Plasmid pWS-4 has a 299-base deletion comprisingboth PTH cDNA and the short upstream portion of the /3-Gal gene. Plasmid pWS-5 has the deletion of a 1472-bp se-quence comprising both TcR gene and the short upstream portion of the /3-Gal gene. Plasmid pWS-6 has bases 2222-2260deleted. Vertical arrows indicate the targeted homologous sequences in the lac operator (lacZpo) targeted by the linkers.

COL-1:C0L-3

ligasePath A

in vivorecombination

in vivorecombination

FIG. 4. Specific deletion of sequences encoding /3-Gal (hatched) by crossover linkers COL-1 (stippled):COL-3 (black),which were homologous to the lac operator (stippled and black). Path A. Two molecular equivalents of duplex COL-1 :COL-3 were ligated to both Eco RI ends of Eco RI/CIAP-treated pUC8 to give intermediate Z. Intramolecular recombi-nation between homologous regions of the plasmid ends in Z yielded plasmid pWS-3. Path B. Linker COL-3 (black)converted linearized pUC8 to pWS-B through a different intermediate, Z'.

double-stranded or 5'-protruding homology-searching se-

quence. For a better understanding of the mechanism ofthis process in vivo, the possibility of recombination via anintermediate with a 3'-protruding homology-searching se-

quence was also investigated. Linker COL-Pst I (Fig. IB)was used to delete the DNA sequence between positions2222 and 2260 of Bam Hl-Pst I-linearized pUC8. Hybrid-ization and nucleotide sequencing showed that 2 of 227transformants possessed the new plasmid pWS-6 (Fig. 3)(mutant population 1%). For comparison between duplexand single-stranded linkers, the whole process was repeatedwith COL-Pst I that had been annealed to a phosphorylatedcomplementary fragment COL-12 (Fig. IB). Of 119 trans-formants analyzed, three colonies possessed plasmidpWS-6 (Fig. 3) (mutant population 2.8%).

Construction of plasmid p WS-1 with linkersCOL-2B, COL-2C, and COL-2EFor establishing the homology requirement for recombi-

nation, a short linker, COL-2B, was used to repeat the con-struction of plasmid pWS-1. Its homology-searching se-

quence of only 8 bases (Fig. IB) was considerably shorterthan that (22 bases) of COL-2 or other minimal homologyrequirements (20 bp duplex) previously reported (Lin et al.,1984; Sung et al., 1986b; Watt et al., 1985). Hybridizationand nucleotide sequencing successfully identified mutanttransformants possessing plasmid pWS-1 (Fig. 3). As in thecase of the longer linker, COL-2, the percentage of the mu-tant (pWS-1) population was generally improved with anincreased amount of linker COL-2B (5% with 84-foldmolar excess, 18% with 500-fold molar excess). Shorterlinkers, COL-2C and COL-2E, with homology-searchingsequences of 7 bases and 5 bases, were also used in the con-version of pUC8 to pWS-1. At 90-fold molar excess, COL-2C and COL-2E yielded mutant (pWS-1) populations of1% and 2.7%, respectively.

DISCUSSION

Using different crossover linkers, we have convertedpUC-type plasmids into new plasmid vectors pWS-1,pWS-2, pWS-3, pWS-4, pWS-5, and pWS-6 with multiplerestriction sites immediately downstream from the lac pro-moter-operator (Fig. 3). This arrangement would permitdirect expression of any cloned DNA. We have demon-strated that an effective homology-searching sequence forthese linkers could be a single-stranded sequence as short as5 bases, as compared with a duplex of 20 bp or longer re-ported previously (Weber and Weissmann, 1983; Lin et al.,1984; Watte/ al., 1985; Sung et al., 1986b). For reasons un-known, the single-stranded linkers (COL-2, COL-2B, andCOL-3) had the additional advantage of avoiding the gen-eration of mutant plasmids associated with the usage of du-plex linker. Specific deletion of sequences up to 1472 bpwere achieved with these single-stranded linkers with a ho-mology-searching sequence of 5-22 bases.

In our previous study, crossover linkers were used solelyin plasmids that had been linearized at two unique restric-tion sites upstream or downstream from the region to bealtered (Sung et al., 1986b). Our present results indicatethat linearization at a single unique restriction site was ade-quate for the modification. In spite of identical cohesiveends created within such linearized plasmids, the linker,which was ligated to the plasmid at both ends, had oper-ated only in the specific orientation predetermined by itshomology-searching sequence (Fig. 4, path B). This generalapproach of integrating DNA sequences by way of shorthomology-searching sequence would overcome some com-mon problems of "gene cassette" insertion in plasmids thatpossess identical cohesive ends. Under normal circum-stances, cloning of a DNA duplex designed with the identi-cal cohesive ends would certainly yield plasmids bearing theinsert in both possible orientations and, often undesirably,in multiple copies (Sung et al., 1986a).The mechanism for recombination directed by the cross-

over linker may be a variation of pathways originally pro-posed for intramolecular recombination in bacteriophage Xand mouse L cells (Cassuto et al., 1971; Radding, 1971; Linet al., 1984). After infection, both 5'-ends of the linear XDNA are degraded by the specific X exonuclease (Little,1967). The intact 3'-ended strands were subsequently ex-posed and annealed intramolecularly, resulting in the circu-larization of the nicked phage DNA duplex. A similar path-way of recombination via 5'-exonucleolytic breakdown ofplasmid termini is probably used by the single-strandedlinkers COL-2, COL-2B, and COL-3. The intermediate,initially generated by ligation of such linkers to the linear-ized plasmid, would obviously require a 5'-exonucleolyticdegradation of the terminus containing lacZpo to expose its3'-ended complementary strand (i.e., of lacZpo) for ho-mologous, intramolecular pairing with the linker sequencein the other terminus. Our data also indicate a competingpathway involving a 3'-exonucleolytic action for a differentset of single-stranded linkers. In the modification of pUC8with the single-stranded linker COL-Pst I, the plasmid in-termediate, which was derived from the ligation of thislinker to the linearized plasmid, would possess a 5'-pro-truding homology-searching sequence constituted by thelinker. A proposed 3'-exonucleolytic degradation of the ter-minus containing lacZpo would then expose its 5'-endedcomplementary sequence (i.e., of lacZpo) for spontaneouspairing with the homology-searching sequence in the otherterminus. Subsequently, an exonuclease or endonucleasemay release any redundant sequences at the junction be-tween the paired and unpaired regions, resulting in a circu-lar plasmid with nicks (Cassuto et al., 1971; Radding,1971). Conversely, a 5'-exonucleolytic degradation of theterminus containing lacZpo would destroy the 5'-endedcomplementary sequence of the lac operator, which is in-dispensible for the proper recircularization of the plasmid.However, in the case of plasmid intermediates constructedwith a duplex crossover linker (e.g., COL-2:COL-l, andCOL-Psí LCOL-12), both 3'- and 5'-exonucleolytic path-ways can be envisioned to generate identical mutant plas-mid. A 3'-exonucleolytic breakdown of both homologoustermini would expose two 5'-ended complementary strands

in the plasmid intermediate. Similarly, 5'-exonucleolyticbreakdown of both homologous termini would expose two3'-ended complementary strands. Intramolecular pairing ofthe complementary strands in these degraded plasmidswould simultaneously yield the same plasmid with differentnicks. This apparent capability of duplex linkers using twoseparate pathways may be responsible for more efficientmodification of pUC8, as compared to a single-strandedlinker (2.8% with COL-Pst LCOL-12 vs. 1% with COL-Pst I; a combined 6.5% with COL-2:COL-l vs. 2.8% withCOL-2).

ACKNOWLEDGMENTS

We thank Dr. Saran Narang for his generosity in allow-ing us access to the DNA synthesizer and Cathy Mac-Donald for technical assistance. The article is NRCC publi-cation No. 27924.

REFERENCES

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LIN, F.-L., SPERLE, K., and STERNBERG, N. (1984). Modelfor homologous recombination during transfer of DNA intomouse L cells: Role for DNA ends in the recombination pro-cess. Mol. Cell. Biol. 4, 1020-1034.

LITTLE, J.W. (1967). An exonuclease induced by bacteriophage

X. II. Nature of the enzymatic reaction. J. Biol. Chem. 242,679-686.

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SUNG, W.L., ZAHAB, D.M., MACDONALD, CA., and TAM,C.S. (1986b). Synthesis of mutant parathyroid hormone genesvia site-specific recombination directed by crossover linkers.Gene 47, 261-267.

SUNG, W.L., ZAHAB, D.M., YAO, F.-L., and TAM, C.S.(1986c). Hybrid gene synthesis: Its application to the assemblyof DNA sequences encoding the human parathyroid hormonesand analogues. Biochem. Cell Biol. 64, 133-138.

VIEIRA, J., and MESSING, J. (1982). The pUC plasmids, anM13 mp 7-derived system for insertion mutagenesis and se-

quencing with synthetic universal primers. Gene 19, 259-268.WATT, V.M., INGLES, C.J., URDEA, M.S., and RUTTER,W.J. (1985). Homology requirements for recombination inEscherichia coli. Proc. Nati. Acad. Sei. USA 82, 4768-4772.

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Address reprint requests to:Dr. Wing L. Sung

Division of Biological SciencesNational Research Council of Canada

Ottawa, Canada K1A OR6

Received for publication January 30, 1987, and in revised formApril 23, 1987.