System in the Yeast Saccharomyces cerevisiae

MOLECULAR AND CELLULAR BIOLOGY, June 1987, p. 2087-2096 Vol. 7, No. 60270-7306/87/062087-10$02.00/0Copyright C 1987, American Society for Microbiology

Functional Expression of the cre-lox Site-Specific RecombinationSystem in the Yeast Saccharomyces cerevisiae

BRIAN SAUERE. I. du Pont de Nemours & Co., Inc., Central Research and Development Department, Experimental Station,

Wilmington, Delaware 19898

Received 25 November 1986/Accepted 4 March 1987

The procaryotic cre-lox site-specific recombination system of coliphage P1 was shown to function in anefficient manner in a eucaryote, the yeast Saccharomyces cerevisiae. The cre gene, which codes for a site-specificrecombinase, was placed under control of the yeast GAL] promoter. lox sites flanking the LEU2 gene wereintegrated into two different chromosomes in both orientations. Excisive recombination at the lox sites (asmeasured by loss of the LEU2 gene) was promoted efficiently and accurately by the Cre protein and wasdependent upon induction by galactose. These results demonstrate that a procaryotic recombinase can enter aeucaryotic nucleus and, moreover, that the ability of the Cre recombinase to perform precise recombinationevents on the chromosomes of S. cerevisiae is unimpaired by chromatin strujcture.

In general, the genome of eucaryotic cells is larger andmore structurally complex than that of procaryotic cells.Moreover, the eucaryotic genome is composed of multiplelinear chromosomes, whereas bacteria tend to have a singlecircular chromosome. A distinctive eucaryotic feature is thatthe genomic DNA is organized into nucleosomes by intimateassociation with histones and other proteins to form chro-matin. Treatment of chromatin with micrococcal nucleaseresults in a characteristic nucleosome repeat structure (16).Moreover, chromatin can exist in both transcriptionallyactive and inactive forms. This difference is most likely dueto differences in accessibility of the DNA, as measured bysusceptibility to nuclease digestion (34).The ability of an enzyme to access DNA in chromatin may

not be a property intrinsic to its eucaryotic source. Certainlyeucaryotic DNA polymerases, RNA polymerases, andtopoisomerases can act on naked DNA in vitro. Conversely,procaryotic proteins such as the lexA repressor and theEcoRI endonuclease can act in vivo at specific DNA se-quences on the chromosomes of the yeast Saccharomycescerevisiae (3, 5). These observations suggest that procary-otic proteins may also be able to conduct more sophisticatedtransactions on the DNA of a eucaryotic chromosome. Forinstance, can DNA recombination events in eucaryotes bepromoted by a bacterial protein? Such events demand notonly recognition ofDNA sequences but also synapsis, DNAcleavage, strand exchange, and religation. In particular, cana site-specific recombination system of Escherichia colifunction in a eucaryotic cell? The cre-lox site-specific recom-bination system of coliphage P1 is well suited to answer suchquestions.Phage P1 encodes an efficient site-specific recombination

system consisting of a short asymmetric DNA sequencecalled loxP and a 38-kilodalton protein called Cre (1, 12, 29).The loxP site is a 34-base-pair sequence composed of two13-base-pair inverted repeats separated by an asymmetric8-base-pair core sequence. Recombination between loxPsites (i) can occur either inter- or intramolecularly, (ii) canoccur when the sites are present on either supercoiled orlinear DNA, and (iii) is independent of the relative orienta-tion of the loxP sites on the DNA molecular. Thus, recom-bination between two directly repeated sites on the sameDNA molecule results in deletion of the DNA segment lying

between the sites. Similarly, on a molecule on which theloxP sites are in an inverted orientation, recombinationresults in inversion of the intervening DNA segment. Re-combination between loxP sites requires only the Cre pro-tein. There is no requirement for any host factors. Thesefeatures of the cre-lox system may thus be sufficient to allowthis site-specific recombination system to function in aeucaryotic cell.To perform recombination, the Cre protein must locate

and bind to the loxP site, perform synapsis of DNA at twosuch sites, and then break and rejoin the DNA to generate arecombinant molecule(s). Experiments presented here showthat Cre can enter the nucleus of the yeast S. cerevisiae toefficiently recognize and cause recombination at loxP sitesplaced in native chromosomes.

MATERIALS AND METHODSBacterial strains, media, and methods. The E. coli recA

strains DH1 (10) and DH5 AlacU169 (a gift from M. Berman,Bionetics Research, Inc.) were used to propagate recombi-nant plasmids. Bacteria were transformed with plasmidDNA by the method of either Hanahan (10) or Mandel andHiga (18) and propagated on L broth (19) with appropriatedrug selection. Small amounts of plasmid DNA were pre-pared by the boiling method of Holmes and Quigley (13).Larger amounts of plasmid DNA were prepared by a cesiumchloride centrifugation method (19).

Yeast strains, media, and methods. The S. cerevisiaestrains used are described in Table 1. Media, methods ofmating, and other yeast procedures are described by Sher-man et al. (27). Briefly, cells were grown on either the richmedium YEPD (2% Bacto-Peptone, 2% glu.cose, 1% yeastextract; Difco Laboratories, Detroit, Mich4.) or the definedgrowth medium S supplemented with the appropriate growthrequirements and containing either 2% glucose (SD), 2%galactose (SG), or 2% raffinose as the carbon source. YeastDNA was isolated as described by Davis et al. (7). Yeastspheroplasts were transformed with DNA as described byHinnen et al. (11).Enzymes and DNA procedures. Enzymes were obtained

from New England BioLabs, Inc., Boehringer MannheimBiochemicals, or Bethesda Research Laboratories, Inc., andused in accordance with supplier instructions. Southern blot

2087

2088 SAUER

TABLE 1. S. cerevisiae strains

Strain Genotype

DBY745a. ... a adel leu2 ura3DBY931a. ... a his4 leu2 ura3 met8 can)BSY3.... DBY745(pBS49)BSY4.... DBY931 VII::pBS42 LEU2BSY16.... DBY931 VII::pBS43 LEU2BSY23.... DBY931 XIII: ILV2::pBS44 LEU2BSY27.... DBY931 XIII: ILV2::pBS47 LEU2BSY31..... BSY3 x BSY23BSY35.... BSY3 x BSY27BSY38.... BSY3 x BSY4BSY45.... BSY3 x BSY16BSY56.... DBY745 x BSY23BSY59.... DBY745 x BSY27BSY63.... DBY745 x BSY 4BSY70.... DBY745 x BSY16BSY90.... DBY745(pBS39)BSY91.... BSY90 x 13SY4BSY92.... DBY745(pBS77)BSY93.... BSY92 x BSY4

a Obtained from S. C. Falco (Du Pont).

analysis (28) was performed with dextran sulfate (33) byusing DNA probes nick translated with [at-32P]dCTP (23).Dideoxynucleotide DNA sequencing (25) was performeddirectly from double-stranded plasmid DNA (35), by usingthe (clockwise) sequencing primer at the pBR322 EcoRI site(GTATCACGAGGCCCT), obtained from New EnglapdBioLabs. Linkers were obtained from Collaborative Re-search, Inc.

Construction of recombinant plasmids. The P1 cre genewas cloned into pBM150 (14) (Fig. 1). The starting plasmidpBS7, which contains the cre gene, is identical to theconstruct pRH103-A6 described previously (30) except thatthe EcoRI site is replaced by a Sall site. Plasmid pBS7 was

digested with XhoI and ligated to the self-complementaryBglII-XhoI adapter sequence TCGAGTAGATCTAC (syn-thesized by Ellen Doran, Du Pont Co.). Subsequent cleavageby Bgfl and Sall produced a 1.3-kilobase (kb) fragmentcontaining the cre gene with a 5' BglII site and a 3' SalI site.Since the 5' overhangs generated by BgllI and BamHI are

identical, this fragment was cloned into pBM150 previouslydigested with BamHI and SalI to yield pBS39. In the relatedplasmid pBS49, a portion of the mouse metallothionein geneMT-I (21) lies 3' to the cre gene. The XhoI-Sall cre genefragment from pBS7 was cloned into the unique XhoI site ofpBMTx (obtained from George Pavlakis, Frederick CancerResearch Facility) such that the 3' polyadenylation signal ofthe MT-I gene would lie downstream from the cre gene. TheEcoRI site 3' to the MT-I gene was converted to a Sall site,and the SaII-BamHI fragment was cloned into the BamHIand Sall sites of pBS39 to generate pBS49.The Cre- plasmid pBS77 was derived from pBS49 by

digesting pBS49 with BamHI, which cuts in the middle of thecre structural gene (30), filling in the ends with the largeKlenow fragment of DNA polymerase I, and religating theends to generate a plasmid which has no Cre activity in E.coli.

Integrative yeast plasmids containing the LEU2 gene

flanked by similarly oriented lox sites were constructed (Fig.2). Plasmid pBS30 was generated by destroying the HindlIlsite of pRH499, a derivative of pRH43 (1) obtained from RonHoess (Du Pont). The XhoI-linearized pBS30 was theninserted in both orientations into the unique XhoI site of a

circularized (by T4 ligase) 5.5-kb Hindlll fragment of pJM53

which is homologous to a region lying between TRP5 andLEU1 on yeast chromosome VII (J. E. Golin, S. C. Falco,and J. P. Margolskee, Genetics, in press) to generate plas-mids pBS42 and pBS43. An additional pair of integrativeplasmids containing two directly repeated lox sites was alsoconstructed. The ILV2 gene from yeast chromosome XIII

-is inserted into the XhoI site of pBS30 in both orientations- first attaching XhoI linkers to the ClaI-HindIII fragmentvm pCP2-4-10 (9) and ligating the resulting fragment toA)I-digested pBS30 to yield plasmids pBS44 and pBS47Fig. 4).

-lasmid DNA on chromosome VII can be retrieved from;;Y4, BSY16, and their derivatives because the insert with

, iacent chromosome VII sequences is flanked by Hindlll's (see Fig. 5). Therefore, 2.5 ,ug of genomic DNA from

.ch independently obtained Leu- derivative of BSY38 wasdigested to completion with HindIII, and the resulting DNAwas religated at a concentration of 3 ,ug/ml to circularize theHindIII fragments. One-fourth of the ligation mixture wasthen used to transform E. coli DH5 Alac to obtain 1 to 10Ampr colonies. Because pBS49 could also be recovered bythis procedure, plasmids so obtained were screened byrestriction analysis to confirm that they were derived fromchromosome VII.

Antibody and Western blots. Purified Cre protein, a gift ofKen Abremski (Du Pont), was used to immunize a rabbit.Affinity-purified rabbit antibody to Cre was prepared byMarian Kelley (Du Pont) and further purified by preadsorb-tion to E. coli. Yeast spheroplasts were generated by incu-bation with Zymolyase 60,000 (Miles Laboratories, Inc.).Protein extracts were prepared by suspending thespheroplasts in 0.5% sodium dodecyl sulfate-50 mMEDTA-50 mM Tris hydrochloride (pH 6.8)-i mMphenylmethylsulfonyl fluoride (Sigma)-10% ,-mercaptoeth-anol (J. T. Baker Chemical Co.)-20 ,ug of leupeptin (Sigma)per ml. After being boiled for 5 min, the supernatant fractionwas either used immediately or stored at -70°C. Proteinconcentrations were determined either by the method ofBradford (4) or by using the BCA protein assay reagent(Pierce Chemical Co.).

Polyacrylamide gel electrophoresis in the presence ofsodium dodecyl sulfate was performed as described byLaemmli (17). Western blot analyses were performed aspreviously described (26), after electrophoretic transfer tonitrocellulose in 15 mM sodium phosphate, pH 6.5.[125I]protein A was obtained from Amersham Corp.

RESULTS

Expression of the Cre protein in S. cerevisiae. In theconstruction of a yeast vector which would express the Creprotein, the following two factors were deemed necessaryboth to facilitate subsequent analysis and to ensure that theresulting construct be generally useful: (i) that cre be ex-pressed at high levels when desired, and (ii) that cre beswitched off when desired. The yeast GAL] gene displaysboth of these attributes. Cells grown in the presence ofgalactose express the GAL] gene at high levels, whereascells grown in glucose do not (2, 8). This regulation occursprimarily at the transcriptional level (31).The cre gene of coliphage P1 was placed under control of

the yeast GAL] promoter region (Fig. 1). The parentalsingle-copy yeast vector pBM150 contains a centromere (theCEN4 sequence), the ARSJ sequence to permit autonomousreplication in S. cerevisiae, the URA3 gene for selection in

MOL. CELL. BIOL.

cre-lox IN S. CEREVISIAE 2089

Bam HI Sal IMet Ser Asn + 4+

TGAGTGTTAAATG-10 I CRE -

I Xho IXho I, Sal I 2. Ligate Bgl fl-Xho I adapter

3. Bgl I, Sal IBam HI Xho I Bam HI

Xho I Sal I Bgl a Sal I1.3 kb 1.3 kb

Eco

Ligate

AmpR

4Bam HI iSal I

Bam HICRE

Sal I

ARSIURA3

CRE

CENN4:~~ARSI URA3

FIG. 1. Construction of yeast plasmids expressing the P1 cre gene. The relevant portion of pBS7, which contains the P1 cre gene on a1.3-kb XhoI-Sall fragment, is shown. The XhoI site is located 56 nucleotides upstream from the first ATG initiating methionine of the cre gene;the Sall site lies just 3' to the end of the cre gene (30). This fragment was cloned into both pBM150 at the BamHl site (which was destroyed)and the mouse metallothionein gene MT-I at the XhoI site, as described in Materials and Methods, to generate pBS39 and pBS31, respectively.Plasmid pBS49 was derived from pBS39 by replacing the small BamHI-SalI of pBS39, which contains the carboxy-terminal portion of the cregene, with the BamHI-EcoRI fragment of pBS31, which contains not only the carboxy-terminal portion of the cre gene but also the MT-I geneand the flanking 3' region.

S. cerevisiae, and the inducible GAL] promoter of S. cere-visiae (14). The cre gene fragment placed downstream fromthe GAL] promoter was chosen such that no other ATGsequence would like between the transcription start site andthe initiating ATG methionine codon of the cre gene (15, 30).Two yeast vectors capable of expressing cre were produced:plasmid pBS39, which simply contains the properly orientedcre gene fragment downstream from the GAL] promoter,and pBS49, a derivative of pBS39 which contains thepolyadenylation signals of the mouse metallothionein geneMT-I inserted 3' to the cre gene.

Plasmid pBS49 was transformed into the leu2 ura3 yeaststrain DBY745 to yield the Ura+ transformant BSY3. To

determine whether or not the Cre protein could be producedin S. cerevisiae, BSY3 was grown in medium containingglucose and shifted to medium containing galactose. Whole-cell protein extracts were prepared from samples taken atvarious times during the induction period and subjected toWestern blot analysis as described in Materials and Meth-ods. Figure 3 shows that Cre protein was produced 4 h aftergalactose induction and attained reasonably high levels by 24h after induction. In particular, the antibody was Cre specificand recognized no other protein in these yeast extracts.Before induction, no Cre protein was detectable nor was anyCre protein detectable in cells grown in galactose but lackingplasmid pBS49. Thus, the cre gene was placed under control

Xho I

-56

GallpromoterBBom HISal I

- URA3

1. Eco RI2. Fill-in3. Ligate4. Sal I,

VOL. 7, 1987

MOL. CELL. BIOL.

lox Hind

pJM53 InsertR

Amp

Hind m Xho I Hind m

j 1. Ligate4t2. Xho I

h HindAiXho I Hind m Xho I

Ligate

AmpRorl /

Xho I

ori

RAmp

ori

Xho I

LEU2

|Bam HIXhoxXho I

AmpRorl

Xho I C

Hind mHind m

FIG. 2. Construction of plasmids pBS42 and pBS43. The HindlIl site of pRH499 was destroyed by digestion with HindlIl, filling in of thesticky ends with the Klenow fragment of DNA polymerase I, and religation to generate pBS30. The 5.5-kb HindlIl fragment of pJM53depicted by the large open arrow was cloned by Jeanne Margolskee and is homologous to a segment of chromsome VII lying between LEUIand TRPS, where the head of the arrow points away from the centromere (Golin et al., in press). It was released from pJM53, circularizedwith ligase, and then digested with XhoI, which cleaves once within this sequence. The resulting fragment was ligated into the XhoI site ofpBS30 to generate pB42 and pBS43.

of the GAL] promoter such that its expression in S. cerevi-siae was regulated by the carbon source.Chromosomes used to measure Cre activity. Cre causes

site-specific DNA recombination between loxP sites in bac-teria and in vitro (1). To adress the question of whether ornot Cre could act on a chromosome in a eucaryotic cell, asimple genetic test was devised. The yeast selectable markergene LEU2 was placed between two lox sites in directorientation (a lox2 LEU2 construction). Recombination atthe lox sites results in excision of the LEU2 gene. BecauseLEU2 does not contain an ARS sequence it is not maintainedin S. cerevisiae unless it is integrated into a yeast chromo-some (11). Such a lox2 LEU2 construction can be integratedinto a chromosome of a leu2 yeast strain. Hence, cellscontaining an integrated LEU2 gene flanked by lox sites inthe same orientation (lox2 LEU2) should become Leu- if Cre

is functionally active in S. cerevisiae, because of LEU2excision.

Yeast chromosome XIII was modified to contain the lox2LEU2 construct. Plasmids pBS44 and pBS47 contain boththe lOx2 LEU2 construct and the ILV2 gene from chromo-some XIII (Fig. 4). The two plasmids differ only in theorientation of the ILV2 gene. Yeast transformants wereobtained by homologous recombination of these plasmidsinto chromosome XIII by using the homology provided bythe ILV2 gene to obtain chromosomes in which the lox2LEU2 construct is nested in a duplication of a 5.0-kb regionof DNA containing the ILV2 gene. The two chromosomesdiffer from each other only in the orientation of the insertedlox2 LEU2 construct and the accompanying pBR322 se-quences.

In addition, a lox2 LEU2 cassette was placed on chromo-

2090 SAUER


some VII. Because it was not known whether Cre would actefficiently in a eucaryotic cell and, since homologous recom-bination events at the duplication could also lead to loss ofthe LEU2 gene (albeit at a low frequency), the lox' LEU2construct was integrated into chromosome VII such that noduplication of chromosomal sequences occurred (Fig. 5).This was accomplished in a single step by integration ofeither plasmid pBS42 (Fig. 5a) or pBS43 (Fig. 5b) into aregion of chromosome VII between LEUI and TRP5. BothpBS42 and pBS43 contain the lox' LEU2 construct and a5.5-kb HindIII fragment of DNA which lies centromeredistal to LEUI. The HindIII fragment was rearranged headto tail about an internal XhoI site such that linearization ofeither pBS42 or pBS43 at the unique HindIII site (which layat the head-to-tail junction) resulted in a DNA moleculewhich could stably integrate into chromosome VII by one-step gene transplacement (24) and which resulted in themodified chromosomes shown in Fig. 5. Because the abso-lute orientation of the DNA fragment from chromosome VIIwith respect to the centromere is known and because pBS42and pBS43 differ only in the orientation of this fragment in

1 2 3 4 5 6

200-

97.4

68-

43

25.7 -

+ + + - pBS49

o 4 24 24 Hrs + galactoseFIG. 3. Induction of the Cre protein in S. cerevisiae by galac-

tose. Whole yeast extracts were prepared as described in Materialsand Methods, and 50 ,ug of protein of each extract was run on an 8%polyacrylamide gel containing sodium dodecyl sulfate. Westernanalysis was performed by using affinity-purified Cre-specific rabbitantibody and '25I-labeled protein A. Molecular weights of markerproteins (Bethesda Research Laboratories) are shown in kilo-daltons. Lanes: 1, 2 ng of purified Cre protein; 2, 1 ng of purified Creprotein; 3, BSY3 uninduced; 4, BSY3 4 h after galactose induction;5, BSY3 24 h after galactose induction; 6, DBY745 24 h aftergalactose induction.

CRE

the plasmid, integration of pBS42 (Fig. 5a) resulted in amodified chromosome VII containing a lOx2 LEU2 constructin which the lox sites point away from the centromere.Conversely, integration of pBS43 resulted in a modifiedchromosome VII containing a lox2 LEU2 construct in whichthe lox sites point toward the centromere.

Induction ofLEU2 loss by Cre expression. If the Cre proteinis indeed functional in S. cerevisiae, induction of Cre ex-pression by galactose should lead to deletion of the LEU2gene from its position on either chromosome XIII or VII inthe constructions described above because of Cre-catalyzedrecombination at the flanking loxP sites. Because the excisedcircular fragment of DNA containing the LEU2 gene carriesno ARS sequence it is not maintained in S. cerevisiae. Thus,excision of the LEU2 gene by recombination at the flankingloxP sites should result in a Leu- phenotype.The cre expression plasmid pBS49 can be conveniently

introduced into the lOx2 LEU2 strains by mating. Diploidstrains were therefore constructed by mating BSY3, whichcontains plasmid pBS49, to leu2 strains containing the 1ox2LEU2 construct integrated in each orientation on chromo-some VII. The stability of the LEU2 gene in the resultingdiploids was determined by plating cells on plates containingeither glucose or galactose and replicating the resultingcolonies to leucine omission plates to determine the numberof leucine auxotrophs. The results are shown in Table 2.Yeast strains which contain the lox2 LEU2 construct on

chromosome VII, in either orientation with respect to thecentromere (Table 2), but which do not contain the creexpression plasmid pBS49 maintain a stable Leu+ phenotypewhen grown in either glucose or galactose. Similarly, suchstrains containing pBS49 display a Leu+ phenotype whengrown on glucose with pBS49. Thus, under conditions inwhich no Cre protein is expressed, there was no recombina-tion between the lox sites flanking the LEU2 gene to causedeletion of the LEU2 gene. However, when such strainscontaining pBS49 were grown on galactose to induce pro-duction of the Cre protein, all of the resulting colonies wereleucine auxotrophs (Table 2). This is very likely the result ofCre-mediated recombination at the lox sites flanking theLEU2 gene. Similar results occurred with haploid strainscontaining pBS49 and the lox2 LEU2 construct (data notshown). Hence, the Cre protein is functional in S. cerevisiaeand can act on an authentic yeast chromosome.To ensure that generation of leucine auxotrophs is due to

production of Cre protein and not some other property ofpBS49, plasmids related to pBS49 were tested for theirability to generate leucine auxotrophs in a lox2 LEU2 yeaststrain grown on galactose (Table 3). Three independentlyobtained galactose-grown colonies of each strain tested weredispersed in water, individual cells were plated to nonselec-tive media, and the leucine auxotrophy of the resultingcolonies was determined. Plasmid pBS39 is identical topBS49 except that it lacks mouse metallothionein sequenceswhich are 3' to the cre gene on pBS49. That pBS39 is alsoable to produce functional Cre in S. cerevisiae is indicatedby the efficient generation of leucine auxotrophs. PlasmidpBS77 is a Cre- derivative of pBS49. It contains a frameshiftmutation in the cre gene generated by cleavage with BamHIat the unique BamHI site in the cre gene, filling in theresulting single-stranded ends with the Klenow fragment ofDNA polymerase I and religation. pBS77 did not fostergeneration of leucine auxotrophs in a yeast strain containingthe lox2 LEU2 construct (Table 3). The ability of pBS49 togenerate leucine auxotrophs in appropriate yeast strainsgrown on galactose is therefore due to functional expression

VOL. 7, 1987

MOL. CELL. BIOL.

R jIOXAmp .

ori I

XhoI LEU2pBS44

.-V ~~~~loxILV2 Xho I

ILV2.r}

t t

HomologousRecombinotion

ori Amp LEU2lox lox

_ _ _ _

Xho I--I

t + XhoI

4 lox

ILV2

tb

I HomologousRecombinotion

LEU2 AmpR orilox lox

+ t Xho I + +

JLV2

Xho I

FIG. 4. Integration of lox sites into chromosome XIII at the ILV2 locus. Plasmids pBS44 and PBS47 differ only in the orientation of the5.0-kb ILV2 fragment, as indicated by the asymmetric positions of the EcoRI sites (light arrows). The lox sites and their orientation are shownby heavy arrows and are separated by 2.5 kb of DNA which includes the LEU2 gene. Leu+ transformants of DBY931 were confirmed bySouthern analysis to have the structure shown in A after transformation with pBS44 or the structure shown in B after transformation withpBS47. Note that the integrants in A and B differ only in the orientation of the LEU2 gene and the associated lox sites on the chromosome.

of the cre gene. Moreover, the experiment also indicates thatmost cells in a galactose-grown colony of a Cre+ strain areindeed Leu-.The ability of the Cre protein to act at lox sites in a yeast

chromosome is not limited to the locus described above onchromosome VII. Diploids were constructed by matingstrains containing the lox2 LEU2 plasmid pBS44 or pBS47,integrated at ILV2 on chromosome XIII (Fig. 4), with thepBS49-containing strain or its isogenic parent which lackspBS49. The resulting strains were tested for stability of theLEU2 gene when grown on galactose (Table 4). Only thediploid strains containing pBS49 allowed efficient productionof leucine auxotrophs. Thus, it is likely that Cre is able to actat lox sites present in any yeast chromosome.

Kinetics of marker loss. The ability of Cre to catalyzerecombination at a chromosomal lox site was further exam-ined by determining the efficiency with which the LEU2marker is lost after cre induction by galactose. StrainBSY38, which contains both the cre expression plasmidpBS49 and also a lox2 LEU2 construct integrated on chro-mosome VII, was grown to mid-log phase in syntheticmedium containing leucine with glucose as the carbonsource and then shifted to the same medium containinggalactose as the carbon source. The culture was sampled atvarious times after induction to assess loss of the LEU2gene. The results are shown in Fig. 6. Leucine auxotrophswere first detected at 8 h after cre induction. By 24 h afterinduction, 98% of the cells in the induced culture were

A.

ILV2 ILV2

B.

i

2092 SAUER


A. or! A4n4 LiEUtb7lx in

HndI Xho I Xho I Hind Z+

Hind Xho I Hind x

IH-RRecombination

ori Amp LEU2

Hind X Xho I XhoI Hind

B. LEU2 Ar4 ori

Hind x Xho I Xho I Hind m

Hind X Xho I Hind m

ne otnion

dl

LEU2 Amp orllox lox

4-- n m i9Xho I Xho I Hind

FIG. 5. Integration of lox sites into chromosome VII withoutduplication of chromosome VII DNA. Plasmids pBS42 (panel A)and pBS43 (panel B) were linearized with HindIll before transfor-mation of DY931. Leu+ transformants were shown by Southernanalysis to have the structure indicated and were generated by genereplacement (24). Because the orientation with respect to thecentromere (represented by the black circle) of the segment ofchromosome VII DNA present on pBS42 and pBS43 (derived frompJM53 and shown as a large open arrow) is known (Golin et al., inpress), the orientation of the lox sites on chromosome VII of theresulting Leu+ transformants is known and is as indicated.

leucine auxotrophs by this assay. The kinetics of commit-ment to loss of the LEU2 gene, shown here, closely paral-leled the induction of galactokinase, the product of the GAL]gene (2), and correlated with production of Cre protein. Inparticular, there was a characteristic lag period after galac-

TABLE 2. Generation of LEU2 loss on chromosome VIIby expression of Cre

Strain and lox2 No. of leucine

LEU2 integrated Plasmid with Carbon auxotrophs/

plasmid cre gene source total no. ofcolonies

BSY70(pBS43) None Glucose 0/80Galactose 0/100

BSY45(pBS43) pBS49 (Cre+) Glucose 0/77Galactose 100/100

BSY63(pBS42) None Glucose 0/86Galactose 0/80

BSY38(pBS42) pBS49 (Cre+) Glucose 0/610Galactose 610/610

a Cells were grown to mid-log phase in selective medium containing glucoseand plated on minimal medium supplemented with leucine and the indicatedcarbon source. Auxotrophy was determined by replication to the appropriateselective medium.

TABLE 3. Dependence of LEU2 loss on a functional cre gene

Strain, plasmid with creGalactose- No. of leucine

Strain, plasmid withe rea grown auxotrophs/total no.gene, and phenotypea cooyo ooiSbcolony of colonies

BSY63 Cre- A 0/177B 0/108C 0/211

BSY38(pBS49) Cre+ A 114/11B 129/12'C 86/86

BSY91(pBS39) Cre' A 165/165B 96/%C 123/123

BSY93(pBS77) Cre- A 0/199B 0/348C 0/200

a All strains contain plasmid pBS42 (which contains the lox2 LEU2 cassette)integrated on chromosome VII.

b Each strain was streaked on SG minimal medium supplemented withleucine (plus uracil for uracil auxotrophs) and incubated at 30°C for 4 days.Three individual galactose-grown colonies (labeled A, B, and C) from eachstrain were picked and separately dispersed in water. Individual cells fromeach colony were then plated on YEPD plates. The resulting colonies werereplicated to appropriate media to determine auxotrophy for leucine.

tose induction to allow abolition of glucose repression. Thislag period could be eliminated by pregrowing cells on anonglucose carbon source. In fact, galactose induction ofcells containing the lox2 LEU2 construct and pBS49pregrown on raffinose resulted in over 90% of the cellscommitted to leucine auxotrophy within 1 h after induction(data not shown). Thus, Cre appears to be efficient in itsability to recognize and recombine chromosomal lox sites inS. cerevisiae.Marker loss occurs by site-specific recombination. Induc-

tion of Cre in strain BSY38 resulted in production of leucineauxotrophs, presumably by recQmbination of the lox sitesflanking the LEU2 gene. To verify that this was indeed thecase, DNA was isolated from seven independently obtainedleucine auxotrophs derived from BSY38 after induction ofCre. The DNA was digested with EcoRI, subjected toagarose gel electrophoresis, transferred to nitrocellulose,and hybridized to radiolabeled pBS78 DNA. Plasmid pBS78was derived from pBS42 by Cre-mediated recombination inE. coli and hence contains sequences homologous to pBR322and to the locus on chromosome VII at which pBS42 isintegrated but lacks homology to the LEU2 gene. Thus, aunique 3.4-kb fragment (Fig. 7) is recognized by this probe inchromosomes containing an integrated copy of pBS42 and isabsent in chromosomes which have deleted the LEU2 geneon chromosome VII. Moreover, site-specific recombinationat the lox sites flanking LEU2 predicts the appearance of anew 1.8-kb fragment in such chromosomes.

Figure 7 indicates that all of the leucine auxotrophsgenerated after induction of Cre which were analyzed oc-curred by the anticipated site-specific recombination event.Lane 1 shows the parental strain, which lacks both pBS49and a modified chromosome VII locus. Integration of pBS42into chromosome VII resulted in production of two newfragments (of 4.5 and 3.4 kb) and loss of the endogenous4.0-kb fragment (lane 2). The diploid strains BSY63, whichlacks plasmid pBS49, and BSY38 are shown in lanes 3 and 4,respectively. Lanes 5 to 11 show the seven leucineauxotrophs. All lack the 3.4-kb fragment and exhibit a new

VOL. 7, 1987

Hins

MOL. CELL. BIOL.

TABLE 4. Generation of LEU2 loss on chromosome XIII byexpression of Cre

Strain and Plasmid with Galactose- No. of leucineintegrated lox2 cre gene grown auxotrophs/LEU2 plasmid (phenotype) colony total no. of

BSY56(pBS44) None A 0/125B 1/137C 0/142

BSY31(pBS44) pBS49 (Cre+) A 101/101B 122/122C 125/125

BSY59(pBS47) None A 0/173B 1/125C 0/125

BSY35(pBS47) pBS49 (Cre+) A 173/173B 129/129C 89/89

a Leucine auxotrophy of cells from three individual galactose-grown colo-nies (A, B, and C) of each strain was determined as described in Table 3,footnote b.

1.8-kb fragment, implying that recombination has occurredat or very near the lox sites flanking the LEU2 gene inBSY38. A similar analysis with a LEU2 probe led to thesame conclusion (data not shown). Because diploid strainswere used, it is clear that no DNA rearrangement eventsoccurred on the chromosome VII homolog which did notcontaip the lox2 LEU2 cassette.

Similarly, Southern analysis indicated that Cre-mediatedloss of LEU2 at the ILV2 locus on chromosome XIII alsooccurred by recombination at the lox sites (data notshown).To demonstrate further that recombination had taken

place at the lox sites flanking the LEU2 gene on chromosomeVII, DNAs containing this region from two independentlyobtained leucine auxotrophs were cloned into E. coli, and a110-base-pair region spanning the single remaining lox sitewas sequenced. In both cases, the resulting sequence wasidentical to that obtained from pBS78, which had beengenerated from pBS42 by Cre-mediated recombination in E.coli. Therefore, cre catalyzes precise recombination of loxsites on a chromosome in S. cerevisiae.

DISCUSSION

The cre gene of coliphage P1, which encodes a site-specific recombinase, can be expressed in S. cerevisiae byfusing the cre structural gene to the inducible GAL] pro-moter. Induction by galactose leads to production of activeCre protein which is able to perform efficient, preciserecombination of DNA at lox sites placed in an authenticyeast chromosome. Thus, the Cre protein must enter theyeast nucleus, recognize chromosomal lox sites (which arepresumably present in some type of chromatin structure),and then perform synapsis and recombination events. Inparticular, there is no barrier posed by the structure of aeucaryotic chromosome to the proper functioning of a pro-caryotic recombinase.The ability of Cre to enter the nucleus may be a conse-

quence of its size since it seems unlikely that Cre contains aspecific signal to direct nuclear localization in a eucaryote.The size of the nuclear pore is sufficient to allow passage of

a spherical protein of 50 to 60 kilodaltons (20, 22). Cre has amolecular weight of 38 kilodaltons and thus may use thisroute to enter the nucleus. Both the EcoRI endonuclease (3)and lexA (5) are similarly surmised to enter the yeast nucleusin this manner.Because the yeast strains used all contain the 2,um plas-

mid, these experiments also indicate that the site-specificrecombination system of the endogenous 2,um plasmid of S.cerevisiae, which is very similar to the cre-lox system (6),exerts no significant influence on the ability of the cre-loxsystem to function in S. cerevisiae. Since the lox2 LEU2constructs are stable in these strains in the absence of Creprotein, the 2,um FLP recombinase does not cause theirrecombination. Conversely, the presence of 2p.m plasmidcontaining FLP-binding sites does not significantly (if at all)impede the ability of Cre to recombine lox sites.The alacrity and precision with which Cre can recombine

chromosomal lox sites may offer a novel and useful methodof effecting rearrangement of the eucaryotic genome. In theconstructions described here, a simple chemical agent caninduce efficient and precise excision of a predeterminedchromosomal segment. Because the Cre protein has alsobeen shown both in E. coli and in vitro to cause inversion ofa DNA segment lying between inverted lox sites, it should bepossible to effect both chromosomal inversions and deletionsof large segments of a yeast chromosome by judiciousplacement and orientation of lox sites. Moreover, the abilityof Cre to perform intermolecular recombination suggests

100

80

60

-a0

40

20

0 8 16 24Hours After Induction

FIG. 6. Kinetics of commitment to Cre-mediated recombination.The diploid strain BSY38 was grown in 2% glucose plus leucine to adensity of 107 cells per ml. Cells were washed twice in water,suspended at 107 cells per ml in SG plus leucine, and incubated for24 h (which allows three to four mass doublings after a long lagperiod). At the indicated time after galactose induction, 150 to 250cells were plated out on YEPD plates (which contain glucose). Theresulting colonies were then replicated to selective media to deter-mine whether or not they were auxotrophic for leucine. In anidentically performed control experiment (data not shown), thisprocedure generated no Leu- auxotrophs from BSY63, which lacksthe Cre expression plasmid pBS49.

2094 SAUER


3 4 5 6 7 8 9 1011

LouSegregants

ori Amp LEU2lox lox

t2.3 + 4.5 + 1.0+ 3.4 tl.0+

iCreori AmpR

lox

4.5 1.8 1.0

Probe

FIG. 7. Southern analysis of Cre-generated leucine auxotrophs.The structure of the lox2 LEU2 region of chromosome VII in strainsBSY4 and BSY38 is depicted (see also Fig. 5), as well as theanticipated structure after Cre-mediated recombination at the loxsites. EcoRI sites are indicated by small vertical arrows. The sizes(in kilobases) of the DNA fragments generated by EcoRI digestionare shown between the flanking EcoRI sites. For the Southern blotin the upper half of the figure, lanes 1 to 11 contained 1 ,ug of yeastgenomic DNA digested with EcoRI; 500 pg of EcoRI-digestedplasmid DNA was loaded in the lanes labeled pBS42 and pBS78.Sizes (in kilobases) of the relevant bands are shown alongside theautoradiogram. The 11.5-kb band represents plasmid pBS49, whichhas a single EcoRI site. The band immediately below the 1.8-kbband in plasmid lanes pBS78 and pBS42 represents a junctionfragment of the plasmids containing sequences in the genomic DNApresent on the 2.3- and 1.0-kb bands. This band was not resolvedwell from the 1.8-kb band in the pBS78 lane. Lanes 1, DBY931; 2,BSY4 containing the pBS42 gene disruption; 3, BSY63, a diploidcontaining both the normal and the disrupted chromosome VII; 4,BSY38, a diploid which contains both forms of chromosome VII andalso pBS49; 5 to 11, seven independently isolated leucineauxotrophs generated after Cre induction.

that the cre-lox system may be useful in the generation ofchromosomal translocations and also other unusual struc-tures such as circular, dicentric, and acentric chromosomes.

Retrieval of DNA from a yeast chromosome could befacilitated by flanking the desired DNA fragment with cor-rectly oriented lox sites and placing an E. coli replicationorigin and selectable marker within that fragment. Cre-mediated recombination would excise a circular moleculecapable of transforming E. coli.The cre-lox system may also be useful in yeast strain

construction involving DNA transformation. For example, aselectable marker gene could be recycled so that the samemarker could be used in multiple independent DNA trans-formations. This could be accomplished by flanking themarker gene with lox sites and then excising the marker withCre before the next DNA transformation.The ability of Cre to function in S. cerevisiae bodes well

for the possibility that the cre-lox system will be able tofunction in other eucaryotes. In particular, such a site-specific recombination system would be of considerablescientific interest in cultured mammalian cells. Site-specificrecombination may be able to integrate exogenous DNAefficiently at a predetermined DNA location after placementof lox sites on both an endogenous chromosome and theDNA molecular being transfected. Integration of the trans-forming (circular) DNA would occur by transient cre expres-sion and the resulting lox x lox recombination. Preliminaryexperiments indicate that such events can be made to occurin S. cerevisiae.The cre-lox system may also be of benefit in understanding

aspects of chromatin structure and the study of homologousrecombination. For instance, are there chromosomal loca-tions or contexts at which lox sites are not efficientlyrecognized? Does recombination at lox sites stimulate theoccurrence of homologous recombination events in adjacentsequences? The ability of double-strand breaks to stimulaterecombination events in S. cerevisiae (32) suggests thatbreakage of chromosomal DNA by Cre in the course ofsite-specific recombination may lead to stimulation of ho-mologous recombination events. Work to address a varietyof these issues is currently in progress.

ACKNOWLEDGMENTSI thank my colleagues at Du Pont for useful discussions and

advice, Bob Zagursky for assistance with DNA sequencing, andRolf Menzel and Deborah Chaleff for critical comments on themanuscript.

LITERATURE CITED

1. Abremski, K., R. Hoess, and N. Sternberg. 1983. Studies on theproperties of P1 site-specific recombination: evidence for topo-logically unlinked products following recombination. Cell32:1301-1311.

2. Adams, B. G. 1972. Induction of galactokinase in Saccharomy-ces cerevisiae: kinetics of induction and glucose effects. J.Bacteriol. 111:308-315.

3. Barnes, G., and J. Rine. 1985. Regulated expression of endonu-clease EcoRI in Saccharomyces cerevisiae: nuclear entry andbiological consequences. Proc. Natl. Acad. Sci. USA 82:1354-1358.

4. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.

5. Brent, R., and M. Ptashne. 1984. A bacterial repressor proteinon a yeast transcriptional terminator can block upstream acti-vation of a yeast gene. Nature (London) 312:612-615.

6. Broach, J. R. 1982. The yeast plasmid 2 ,. circle. Cell28:203-204.

pBS pBS78 42 1 2

VOL. 7, 1987

2.3

MOL. CELL. BIOL.

7. Davis, R. W., M. Thomas, J. Cameron, T. P. St. John, S. Sherer,and R. A. Padget. 1980. Rapid DNA isolation for enzymatic andhybridization analysis. Methods Enzymol. 65:404 411.

8. Douglas, H, C., and D. C. Hawthorne. 1964. Enzymatic expres-sion and genetic linkage of genes controlling galactose utiliza-tion in Saccharomyces. Genetics 49:837-844.

9. Falco, S. C., and K. S. Dumas. 1985. Genetic analysis of mutantsof Saccharomyces cerevisiae resistant to the herbicide sulfo-meturon methyl. Genetics 109:21-35.

10. Hanahan, D. 1983. Studies on transformation of E. coli withplasmids. J. Mol. Biol. 166:557-580.

11. Hinnen, A., J. B. Hicks, and G. R. Fink. 1978. Transformation ofyeast. Proc. Natl. Acad. Sci. USA 75:1929-1933.

12. Hoess, R., and K. Abremski. 1985. Mechanism of strand cleav-age and exchange in the cre-lox site-specific recombinationsystem. J. Mol. Biol. 181:351-362.

13. Holmes, D. S., and M. Quigley. 1981. A rapid boiling method forthe preparation of bacterial plasmids. Anal. Biochem. 114:193-197.

14. Johnston, M., and R. W. Davis. 1984. Sequences that regulatethe divergent GALJ-GALIO promoter in Saccharomyces cere-visiae. Mol. Cell. Biol. 4:1440-1448.

15. Kozak, M. 1983. Comparison of initiation of protein synthesis inprocaryotes, eucaryotes, and organelles. Microbiol. Rev.47:1-45.

16. Lacy, E., and R. Axel. 1975. Analysis of DNA of isolatedchromatin subunits. Proc. Natl. Acad. Sci. USA 72:3978-3982:

17. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

18. Mandel, M., and A. Higa. 1970. Calcium dependent bacterio-phage DNA infection. J. Mol. Biol. 53:154-162.

19. Maniatis, T., E. F. Fritisch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

20. Paine, P. L., L. C. Moore, and S. B. Horowitz. 1975. Nuclear-envelope permeability. Nature (London) 254:109-114.

21. Pavlakis, G. N., and D. H. Hamer. 1983. Regulation of ametallothionein-growth hormone hybrid gene in bovine papil-loma virus. Proc. Natl. Acad. Sci. USA 80:397-401.

22. Peters, R. 1983. Nuclear-envelope permeability measured byfluorescence microphotolysis of single liver-cell nuclei. J. Biol.

Chem. 258:11427-11429.23. Rigby, P. W. J., M. Dieckmann, C. Rhoades, and P. Berg. 1977.

Labeling deoxyribonucleic acid to high specific activity in vitroby nick translation with DNA polymerase I. J. Mol. Biol.113:237-251.

24. Rothstein, R. J. 1983. One-step gene disruption in yeast. Meth-ods Enzymol. 101:202-211.

25. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci.USA 74:5463-5467.

26. Sauer, B., and I. R. Lehman. 1982. Immunological comparisonof purified DNA polymerase a from embryos of Drosophilamelanogaster with forms of the enzyme present in vivo. J. Biol.Chem. 257:12394-12398.

27. Sherman, F., G. R. Fink, and C. Lawrence. 1974. Methods inyeast genetics. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

28. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by agarose gel electrophoresis. J.Mol. Biol. 98:503-507.

29. Sternberg, N,, and D. Hamilton. 1981. Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. J.Mol. Biol. 150:467-486.

30. Sternberg, N., B. Sauer, R. Hoess, and K. Abremski. 1986.Bacteriophage P1 cre gene and its regulatory region: evidencefor multiple promoters and for regulation by DNA methylation.J. Mol. Biol. 187:197-212.

31. St. John, T. P., and R. W. Davis. 1979. Isolation of galactose-inducible DNA sequence from Saccharomyces cerevisiae bydifferential plaque filter hybridization. Cell 16:443-452.

32. Szostak, J. W., T. L. Orr-Weaver, R. J. Rothstein, and F. W.Stahl. 1983. The double-strand-break model for recombination.Cell 33:25-35.

33. Wahl, G. M., M. Stern, and G. R. Stark. 1979. Efficient transferof large DNA fragments from agarose gels to diazobenzyloxy-methyl-paper and rapid hybridization by using dextran sulfate.Proc. Natl. Acad. Sci. USA 76:3683-3687.

34. Weintraub, H,, and M. Groudine. 1976. Chromosomal subunitsin active genes have an altered conformation. Science 193:848-856.

35. Zagursky, R., K. Baumeister, N. Lomax, and M. Berman. 1985.Rapid and easy sequencing of large linear double-stranded DNAand supercoiled plasmid DNA. Gene Anal. Tech. 2:89-94.

2096 SAUER

Documents

System in the Yeast Saccharomyces cerevisiae