Vol. 172, No. 2JOURNAL OF BACTERIOLOGY, Feb. 1990, p. 610-6180021-9193/90/020610-09$02.00/0Copyright X) 1990, American Society for Microbiology

Chromosome Engineering in Saccharomyces cerevisiae by Using aSite-Specific Recombination System of a Yeast PlasmidHIROAKI MATSUZAKI, RYOICHI NAKAJIMA, JUNKO NISHIYAMA,t HIROYUKI ARAKI,

AND YASUJI OSHIMA*

Department of Fermentation Technology, Faculty of Engineering, Osaka University, 2-1 Yamadaoka,Suita-shi, Osaka 565, Japan

Received 28 June 1989/Accepted 30 October 1989

We have developed an effective method to delete or invert a chromosomal segment and to create reciprocalrecombination between two nonhomologous chromosomes in Saccharomyces cerevisiae, using the site-specificrecombination system of pSR1, a circular cryptic DNA plasmid resembling 2,um DNA of S. cerevisiae butoriginating from another yeast, Zygosaccharomyces rouxii. A 2.1-kilobase-pair DNA fragment bearing thespecific recombination site on the inverted repeats of pSR1 was inserted at target sites on a single or twodifferent chromosomes of S. cerevisiae by using integrative vectors. The cells were then transformed with aplasmid bearing the R gene of pSR1, which encodes the site-specific recombination enzyme and is placeddownstream of the GAL] promoter. When the transformants were cultivated in galactose medium, therecombination enzyme produced by expression of the R gene created the modified chromosome(s) byrecombination between two specific recombination sites inserted on the chromosome(s).

Although recombinant DNA technology is effective forcreating novel strains in various organisms, the DNA thatcan be manipulated in a vector is limited to a few genes or aDNA fragment less than 100 kilobase pairs (kb) long, and thevector is, in general, unstable. To overcome these diffi-culties, the artificial chromosome vector YAC, which is ableto clone an exogenous DNA fragment of several hundredkilobase pairs and is maintained stably, has been developedin Saccharomyces cerevisiae (6). Another possible proce-dure for integrating large exogenous DNA efficiently, andalso to delete, invert, and translocate a chromosomal seg-ment at a predetermined location on a chromosome by usingthe cre-lox site-specific recombination system of bacterio-phage P1, has been suggested by Sauer (27). We havesucceeded in effecting such chromosomal modifications in S.cerevisiae by using a site-specific recombination system ofplasmid pSR1, which was isolated from a strain of Zygosac-charomyces rouxii.Plasmid pSR1 (1, 35) is a 6,251-base-pair (bp) circular

DNA molecule having an architecture similar to that of 2,umDNA (3), the plasmid widely distributed in S. cerevisiaestrains. The DNA sequence of the pSRl molecule has nosimilarities with that of 2,um DNA (1) or genomic DNA of S.cerevisiae (our unpublished results). The pSR1 molecule hasa pair of inverted repeats, each composed of a 959-bpsequence, which divide the plasmid molecule into twounique regions of 2,654 and 1,679 bp (Fig. la). pSRl canreplicate in S. cerevisiae as well as in its native host andexists in two isomeric forms generated by intramolecularrecombination at the inverted repeats. The intramolecularrecombination is initiated at a restricted region, at most a58-bp sequence, in the inverted repeats and is catalyzed bythe R protein encoded by the plasmid molecule (19).The site-specific recombination system of pSRl has dis-

tinct advantages in modification of S. cerevisiae chromo-

* Corresponding author.t Present address: Department of Natural Science, Faculty of

Arts and Sciences, Osaka Women's University, Sakai-shi, Osaka590, Japan.

somes because it operates efficiently in S. cerevisiae and isindependent of the host recombination system. Recombina-tion at the 58-bp sequence strictly depends on the function ofthe R protein and is different in specificity from the similarrecombination system of 2,um DNA. Thus, the resident 2p.mDNA in a S. cerevisiae cell does not have to be removed inapplication of the pSR1 system.

This communication deals with the procedure for deletionor inversion of a chromosomal segment or reciprocal recom-bination between two nonhomologous chromosomes. Theprocedure consists of insertion of the recombination sites(RSs) of pSR1 at target sites on a single chromosome or twodifferent chromosomes with the aid of yeast integrativevectors, transformation with a plasmid carrying the R geneof pSR1 inserted downstream of the GAL] promoter, andcultivation in galactose medium to express the R gene. TheR protein thus produced effectively brings about recombina-tion between the two RSs.

MATERIALS AND METHODSMicroorganisms and plasmids. Three haploid S. cerevisiae

strains, NBW5 (MATo ade2ochre leu2-3,112 ura3-1,2 his3-532 trpl -289 pho3-1), NA87-11A (MATct leu2-3,112 his3 trplpho5-J [1, 19]), and SH986 (MATa ade2-101Phre hisA3leu2-3,112 trpAl ura3-52), were selected from our stockcultures and used as the hosts for chromosomal modifica-tion. Since the plasmids used were connected with thereplication origin of ColEl, they were propagated in Esche-richia coli JA221 (9). Various DNA fragments bearing the S.cerevisiae genes were prepared from plasmids YIpl (32),YIp5 (32), YIp32 (2), YEp51 (5), and YRp7 (32). Plasmidp286 (details not shown), bearing a 685-bp EcoRI-BamHIfragment of the GAL] promoter originated from pBM150(18), was obtained from A. G. Hinnebusch of the NationalInstitutes of Health, Bethesda, Md. The other two plasmids,pRAS1 and p237 (Fig. lb), which carry the RAS] gene (10)or the URA3-SUPJJ construct, were gifts from M. Yama-moto of the Institute of Medical Science, University ofTokyo, and P. Hieter of The Johns Hopkins UniversitySchool of Medicine, Baltimore, Md., respectively.

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FIG. 1. Structures of pSR1 and the principal plasmids used. (a)Structure of pSR1 and of the 2.1-kb RS fragment. The linearportions of pSR1 represent inverted repeats, the thick lines markedP, R, and S indicate the open reading frames, and the tapered endsindicate the 3' ends of the frame (1). A cross connecting the invertedrepeats (IR1 and IR2) shows the approximate position of the specificRS that is located within a 58-bp sequence from nucleotide positions497 to 554 on IR1 and from 3624 to 3681 on IR2 (19). Numbers at therestriction sites represent positions with respect to G of the EcoRIsite as position 1 (1). The 2.1-kb Sall fragment extending fromnucleotide positions 3469 to ca. 5520 (exact position not known) was

used as the RS fragment, and the arrowhead in the fragmentindicates the specific RS. Both Sall ends of the RS fragment were

created by linker insertion on pSRT322 and pSRT310 (16). (b)Structures of the principal plasmids used. Plasmid pRAS1 was usedas the source of the RASI DNA. Plasmid pUS1 was constructed byligation of the shorter PstI fragments of p237 and YIp5. pHM153was constructed with the DNAs from YIp32, YEp51, p286, andpSR1 and used for generation of R protein. Although details of theother DNA fragments composed of pHM153 are not described here,the pSR1 fragment (1,920 bp) connected with the 6-bp Sall linkersequence was derived from the region between nucleotide positions5607 (the EcoT14I site at nucleotide position +11 from the ATGcodon of the R open reading frame) and 3687 at 458 bp down-stream from the stop codon of R (the Sall site created by the linkerinsertion in IR1 of pSRT327 and converted to IR2 by recom-bination-associated gene conversion [19]). The 1,920-bp pSR1fragment was connected with a short synthetic oligonucleotide,G-T,TTATACGCGAC at the EcoT14I site and then ligatedto the BamHI end of the 685-bp GAL] promoter DNA. The Sall endof the 1,920-bp R fragment was ligated with the Sall end of acomposite LEU2 DNA constructed from YIp32 and YEp51 DNAs.The arrowhead in parentheses on the R region of pHM153 repre-sents the site of the R-gene disruption made by restriction of a BglIIsite, filling in with Klenow fragment, and ligation. pHM149 was

Media and genetic and biochemical methods. Nutrientmedium (YPAD) and synthetic medium SGlu (formerly SD[1]) for yeast cells and media for E. coli were as describedpreviously (1, 35) except for SGal medium, in which galac-tose (20 g/liter) was substituted for glucose in SGlu medium.To analyze the red-colored phenotype of yeast colonies dueto the ade2 mutation, YPD medium was prepared by omit-ting adenine from YPAD medium. General methods for cellcultivation, DNA preparation, gel electrophoresis, diges-tion, ligation and filling in of DNA fragments, and transfor-mation of yeast cells by the lithium acetate method (15) andfor E. coli cells were as described previously (1, 19, 35).Insertion of a DNA fragment on a chromosome (23, 26) andhybridization of yeast cells (29) were performed by standardprocedures. To separate and analyze the yeast chromo-somes, an apparatus (model 2015 Pulsaphor system; LKBProdukter AB, Bromma, Sweden) for pulsed-field gel elec-trophoresis (PFGE; 28) was employed, using a point elec-trode or hexagonal electrode array (8) as specified by themanufacturer. The chromosome samples for PFGE wereprepared by the embedded-agarose method (7). Slab gels (10by 10 by 0.5 cm for the point electrode array and 15 by 15 by0.5 cm for the hexagonal electrode array) were prepared bypouring melted agarose (1%) in 0.5x TBE buffer (lx TBEbuffer is 90 mM Tris, 90 mM boric acid, and 2.5 mM EDTA,pH 8.3). PFGE was carried out at 450 V for 16 h with a 55-spulse time for the point electrode array or 170 V for 41 h witha 100-s pulse time for the hexagonal electrode array. Afterstaining in ethidium bromide solution (0.5 jg/ml), the gel wasplaced in 0.25 M HCl for 30 min and then incubated for 30min in denaturating solution (0.5 M NaOH and 1.5 M NaCl)with gentle shaking and for 30 min more in 0.5 M Trishydrochloride-1.5 M NaCl solution (pH 7.5). The DNA wasblotted onto a Biodyne nylon membrane (Pall Biosupport,East Hills, N.Y.) with 20x SSC (lx SSC is 0.15 M NaClplus 0.015 M sodium citrate) for 16 h by the method ofSouthern (30) and hybridized with probe DNA at 65°C for 16h in a hybridization solution (5x Denhardt buffer [11], 5xSSPE [20x SSPE is 3.6 M NaCl, 0.2 M sodium phosphate,and 0.02 M EDTA, pH 8.3], 0.2% [wt/vol] sodium dodecylsulfate, 100 jig of denatured calf thymus DNA per ml) asrecommended by Pall Biosupport. The 32P-labeled probeDNAs were prepared by the random hexanucleotide primingmethod (13).

RESULTS

Function of the site-specific recombination system on achromosome. To examine whether the site-specific recombi-nation system of pSRI functions on a chromosome in S.cerevisiae, strain YHM201 (MA Tot ade2ochre ura3 leu2-3,112his3::pHM149 [RS-URA3-SUPJJ-RS]) was constructed byinsertion of a chimeric DNA fragment, RS-URA3-SUPI I-RS

constructed from pUS1 and the other DNA fragments as describedin the legend to Fig. 2 and used for integration of the RS-URA3-SUPJI-RS fragment at the HIS3 locus. The regions marked Apr andori are the DNA fragments of pBR322 except for the fragment ofpBR327 in pRASI. Regions marked HIS3, LEU2, RAS], SUPII,URA3, and GAL] promoter are the DNAs from S. cerevisiae; R isfrom pSR1, and REP3 and ARS are from 2,um DNA. The boxmarked RS is the 2.1-kb RS fragment. Those marked Y'a and Y'band CEN4 are chromosomal fragments of S. cerevisiae bearingtelomere (Y'a and Y'b) and centromere (CEN4). Abbreviations forrestriction endonuclease sites: B, BamHI; E, EcoRI; H, HindlIl; M,MluI; P, PstI; S, Sall; St, StuIl; and X, XhoI.

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FIG. 2. Excision of a short DNA fragment from a chromosome.(a) The procedure for constructing strain YHM201 used for theexcision experiment. Plasmid pHM149 bearing two copies of the2.1-kb RS fragment (Fig. la) in the same direction and two HIS3DNA fragments, derived from a 1.7-kb S. cerevisiae DNA bearing asingle complete HIS3 gene prepared from YIpl but ligated at theBamHI ends to form the head-to-tail arrangement after cutting at theXhoI site in the gene, was constructed by the following procedure.The 1.7-kb BamHI HIS3 fragment prepared from YIpl was split atthe XhoI site in the HIS3 locus, and each fragment was connectedwith the 2.1-kb RS fragment by XhoI-SalI ligation. Thus, the5'-HIS3-RS and RS-HIS3-3' chimeric DNA fragments were ob-tained. The two chimeric fragments have the same relative nucleo-tide directions of the RS fragment as that of HIS3 and have theBamHI end at the HIS3 side and the Sall end at the RS side. Thesetwo fragments were inserted into the modified pUS1 (Fig. lb; theHindlll site was converted to a XhoI site by restriction withHindIII, filling in with Klenow fragment, and ligation with the XhoIlinker); the 5'-HIS3-RS fragment was inserted at the SalI-BamHIgap of the modified pUS1, and the RS-HIS3-3' fragment wasinserted at the XhoI-BamHI gap. The resultant plasmid, pHM149,has a structure in which the URA3 and SUPHI genes of S. cerevisiaederived from p237 are located between the direct repeats of two RSfragments and the head-to-tail configuration of the HIS3 gene is

(Fig. 2a), at the his3 locus on chromosome XV (21) of strainNBW5. The ade2ochre mutation is suppressed by SUPJJ(14), and two RS fragments (Fig. la) were arranged in thesame direction. By recombination at the two RSs, a 7.6-kbfragment bearing the URA3 and SUPJJ genes should beexcised from the integrated pHM149 molecule; such clonesshould be detectable by examination of the adenine (Ade)and uracil (Ura) phenotypes on SGlu plates, since theexcised fragment would be lost from the cell (although theSUPII DNA has autonomously replicating sequence [ARS]function, it is extremely inefficient [14]).

Strain YHM201 was transformed to the leucine pro-totrophic phenotype (Leu+) with pHM153, which carries theR gene downstream of the GAL] promoter and is replicatedwith the 2,urm DNA replicon (Fig. lb). Transformants wererecovered on an SGlu plate. After appropriate dilution, someof the transformant colonies were spread on SGal platessupplemented with the necessary nutrients but not leucine.After 4 days of incubation at 30°C, colonies were examinedfor growth by replicating on SGlu plates lacking eitherleucine and adenine or leucine and uracil. We found threeclasses of colonies. Class I (81 of 121 total colonies exam-ined) consisted of Ade- Ura- cells; class II (38 of 121)formed papilla at the spotted area, which might have beendue to a mixed population of Ade- Ura- and Ade+ Ura+cells; and class III, the remaining two colonies, showed theAde+ Ura+ phenotype. No Ade+ Ura- or Ade- Ura+ cellswere found. All clones examined of classes I (four clones)and II (seven clones) contained a new 3.8-kb BamHI frag-ment hybridizable with a probe bearing the HIS3 gene,whereas the original YHM201 showed a hybridization bandat a molecular size of 11.4 kb (Fig. 2b). Since the 11.4-kbband was hardly detected in the cell population of the classII clones, deletion of the 7.6-kb fragment might proceedcontinuously in the cells.

In addition to the 3.8-kb band, a faint band showingslightly faster migration than the 11.4-kb band was observedin all samples except those of the original strains, NBW5 andYHM201. The most plausible explanation for this faint bandis that it is due to a 9.3-kb BamHI fragment derived from aminor fraction of the cells having chromosome XV withdeletion of the 7.6-kb RS-URA3-SUPJI-RS region but inser-tion of the pHM153 molecule by homologous recombinationbetween the R region of the RS fragments remaining on thechromosome and the R region of pHM153. This structureshould create 9.3- and 3.5-kb fragments upon digestion withBamHI, both of which carry a portion of the HIS3 DNA.The 3.5-kb fragment might overlap on the gel with the 3.8-kbBamHI fragment derived from the major fraction of the cellpopulation.

located on the other side of the RS direct repeats. Thus, BamHl (B)restriction of pHM149 results in a linear DNA molecule having the5'-terminal region of the HIS3 DNA at one end and the 3'-terminalend of HIS3 at the other end. This configuration is favorable forsingle-copy insertion of pHM149 at the HIS3 locus in chromosomeXV. The linearized pHM149 molecule was integrated at the HIS3locus of strain NBW5 as described in the text. The resultant strain,YHM201, having the Ade+ Ura+ phenotype, was then transformedwith pHM153 (Fig. lb) and cultivated in SGal medium to excise the7.6-kb fragment between the two RSs. (b) Genomic DNAs preparedfrom four class I clones showing the Ade- Ura- phenotype andseven class II clones having a mixed population of Ade+ Ura+ andAde- Ura- cells were restricted with BamHI, electrophoresed,blotted onto a nylon membrane, and hybridized with the 32P-labeled1.7-kb HIS3 fragment.

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When the original YHM201 cells transformed withpHM153 and cultivated on SGlu were spread on an SGluplate, 112 of 118 colonies showed the Ade+ Ura+ phenotype,whereas the remaining 6 were Ade- Ura-, probably becauseof leaky expression of the R gene. No such Ade- Ura-colonies appeared when the pHM153 plasmid carrying adisrupted R gene (Fig. lb) was used for transformation eventhough the transformants were cultivated on SGal medium(122 colonies examined). These findings indicate that dele-tion of the 7.6-kb RS-URA3-SUPII-RS segment is due torecombination of the two RSs catalyzed by the R protein andthat pHM153 is effective for production of R protein in SGalmedium. Thus, the site-specific recombination system ofpSR1 is effective for the S. cerevisiae chromosome.

Deletion and inversion of a chromosomal segment. Next weexamined deletion or inversion of a chromosomal segment.One copy of the RS fragment was inserted at each of theRAS] and HIS3 loci on chromosome XV of the ADE2+haploid strain NA87-11A (MATt), having the leu2-3,112,his3, and trpl mutations, using YIp vectors carrying, respec-tively, RAS] and HIS3 DNA (Fig. 3a). The distance betweenthe RAS] and HIS3 loci is about 60 centimorgans (21), whichis equivalent to 180 kb (20), and the region contains theADE2 locus. Four different strains having different relativedirections of the inserted RS fragments at the RAS] andHIS3 loci were made by constructing six YIp plasmids withdifferent relative orientations of the RAS] and HIS3 DNAsto the RS fragment. These four strains were mated with theade2 haploid strain SH986 (MATa ade2 leu2-3,112 hisA3;isogenic with NA87-11A) harboring pHM153. The four re-sultant diploids, YRN-A, YRN-B, YRN-C, and YRN-D,have the ADE2Iade2 genotype and form white (Ade+) colo-nies on a YPD plate.Upon cultivation of the diploids in SGal medium, recom-

bination at RSs should excise the 180-kb RASJ-HIS3 regionif the RS fragments are inserted in the same direction, as inthe case of strains YRN-A and YRN-B (Fig. 3a), and theADE2 gene should be lost from the cells. When the two RSsare inserted in opposite directions relative to each other, asin strains YRN-C and YRN-D, then an inversion is ex-pected. These possibilities were supported by the observa-tions that YRN-A and YRN-B strains produced red (Ade-)colonies at high frequencies when cultivated in SGal mediumbut only rarely when cultivated in SGlu medium (Table 1).Strains YRN-C and YRN-D also produced red colonies atlow frequencies. Some of the red and white colonies ofYRN-A and YRN-C were examined for chromosomal migra-tion by PFGE, using the point electrode array and Southernhybridization with a 32P-labeled HIS3 fragment as a probe(Fig. 3b). All of the red clones from YRN-A produced twohybridization bands (30 clones tested, of which an exampleis illustrated in Fig. 3b), one corresponding to chromosomeXV and the other, a more rapidly migrating one, correspond-ing to chromosome XV with a 180-kb deletion. The hybrid-ization bands with the deletion chromosomes always showedsomewhat higher densities than those with an intact chro-mosome XV, possibly because the intact ade2 chromosomeXV originating from SH986 has a Ahis3 deletion allele,whereas the chromosome having the 180-kb deletion mighthave originated from strain NA87-11A and should have afull-length HIS3 DNA. The white clones of YRN-A andYRN-C and a red clone from YRN-C showed a hybridizationsignal only at the band corresponding to chromosome XV.The results indicate that the red clone of YRN-A has a180-kb deletion on chromosome XV, whereas the red clonefrom the YRN-C does not. This finding suggests that the red

clones from YRN-C might have lost the whole of chromo-some XV bearing the wild-type ADE2 gene or that geneconversion to the ade2lade2 configuration might have oc-curred.

Strains YRN-C and YRN-D are expected to have aninversion at the RASJ-HIS3 segment. Since this modificationmight not be detectable on PFGE, restriction analysis wasperformed. The RS fragment has one MluI site (Fig. la), andthe XbaI and XhoI sites are located outside of the RSfragmenlt inserted at the RAS] an HIS3 loci (Fig. 4a). Whena deletion occurs between these two RSs (YRN-A andYRN-B), the MluI-XhoI and MluI-XbaI fragments locatedinside of the RS-RS region should disappear on the gel,whereas those located outside of the region should bedetected. In the case of inversion (YRN-C and YRN-D), nodifferences in these MluI-XbaI and MluI-XhoI fragmentswould be expected for either the glucose- or galactose-growncells. However, single restriction with XbaI or XhoI shouldresult in differences between the original and inverted chro-mosomes. These possibilities were examined by using DNAsprepared from cells of strains YRN-A and YRN-C grown onSGlu and SGal media. The DNAs were digested doubly withMluI and XhoI or singly with XbaI or XhoI, electrophoresed,blotted, and hybridized with the 32P-labeled RS fragment asa probe (Fig. 4b). The results supported the expectation, asYRN-A had a deletion and YRN-C had an inversion at theRS-RS region by recombination at the RSs, at least in the2.1-kb RS fragment. The hybridization bands of the DNAsamples from the recombinant clones were faint in compar-ison with those of the original clones. This finding suggeststhat the RS fragment is lost from the recombinant chromo-some in some fraction of the cell population, although thereason is not known.Recombination between nonhomologous chromosomes. To

examine reciprocal recombination between nonhomologouschromosomes, the RS fragment was inserted at the URA3locus of chromosome V and at the HIS3 locus of chromo-some XV. We constructed two haploid strains, YRN-1 andYRN-2, from stiain NBW5 (ade2ochre ura3 his3 leu2-3,112;Fig. 2a). Since the transcriptional directions of the URA3and HIS3 loci relative to their respective centromeres are

known (17), we could insert the RS fragments at those loci inthe same direction relative to the centromere in YRN-1 andin the opposite direction in YRN-2 (Fig. 5a). Recombinationbetween the two RSs should create two monocentric chro-mosomes in YRN-1 and one dicentric and one acentricrecombinant chromosome in YRN-2. Thus, the recombina-tion will be harmless to the cell in YRN-1 but lethal inYRN-2. The molecular sizes of chromosomes V and XVwere calculated to be 710 and 1,130 kb, respectively, accord-ing to the map position of these genes and the chromosomelength (20, 21), whereas those of the two recombinantchromosomes produced in the YRN-1 cells should be 910and 930 kb, respectively.

Plasmid pHM153 was introduced into these strains. Theresultant Leu+ transformants were cultivated in SGal or

SGlu medium overnight, and the cells were spread on SGluplates. Several colonies that developed on the plates were

isolated at random and examined for chromosomal patternon PFGE, using the point electrode array. When the trans-formant of YRN-1 was cultivated in SGal medium, 11 of the55 clones examined showed a new band with a migrationdistance corresponding to a chromosome having about 970kb of DNA (Fig. Sb; only 1 of the 11 samples showing thenew band is shown). Since several chromosomes comigratedon PFGE with the point electrode array (for example,

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FIG. 3. Deletion and inversion of a chromosomal segment. (a) Strategies to delete or invert the RASI-HIS3 region of chromosome XV.The RS fragments were inserted at the RAS] and HIS3 loci of chromosome XV by using pBR322-based YIp plasmids. For insertion of theRS fragment at the RAS] locus, a YIp plasmid was constructed by insertion of a 2.9-kb EcoRI-BamHI fragment bearing the RAS] gene (10)derived from pRAS1 (Fig. lb) at the short EcoRI-BamHI gap of pBR322. Then the chimeric plasmid was ligated with a 1.6-kb PstI fragmentof YRp7' (i.e., the 1.4-kb EcoRI fragment bearing ARSI and TRPI from S. cerevisiae of YRp7 was inverted) bearing the TRPI gene at thePstI site as a selection marker and the RS fragment at the SalI site of the pBR322 moiety. For insertion of the RS fragment at the HIS3 locus,another YIp plasmid was constructed by ligation of a 1.7-kb BamHI fragment bearing the HIS3 gene prepared from YIpl and the RS fragment,respectively, at BamHI and Sall sites of pBR322. Two types of plasmids were constructed for the RASI plasmid and four types wereconstructed for the HIS3 plasmid with different relative orientations of RS to the RAS] or HIS3 DNA. DNAs of these chimeric plasmid wereprepared in E. coli and linearized by restriction at the StuI site of the RAS] DNA and at the XhoI site of the HIS3 DNA of the respectiveplasmids. These StuI and XhoI sites are unique in the respective chimeric plasmids. (b) PFGE patterns of chromosomes. One clone eachshowing the red (Ade-) or white (Ade+) phenotype was isolated at random from YRN-A (having two RSs in the same direction) and YRN-C(having two RSs inverted). Slots were added with DNA prepared from equivalent amounts of cells. The chromosomal bands (left) werehybridized with the 32P-labeled 1.7-kb HIS3 fragment (31) after blotting onto a nylon membrane (right). The arrowhead on the left indicatesthe site of a new chromosomal band.

chromosomes V and VIII and chromosomes VII and XVcomigrated in strain NBW5), the chromosomal bands werehybridized with 32P-labeled DNA fragments bearing theHIS3, URA3, or RAS] gene or with the RS fragment as aprobe. When the chromosomal pattern of the galactose-grown cells showing the new chromosome band was exam-ined (Fig. 5b; lanes R), all probes except the RS fragmenthybridized strongly with the new band and weakly with theband corresponding to chromosome V or XV. The chromo-somal pattern of the same transformant cultivated in SGlu

medium (lanes 0) showed hybridization with these probes ofURA3, RAS], and HIS3 only at the band corresponding tochromosome V or XV or, with the RS probe, to both. Theseresults indicate that the new band consisted of two recom-binant chromosomes created by recombination betweenchromosomes V and XV at the RSs inserted at the URA3and HIS3 loci and that the two recombinant chromosomesmight have comigrated, probably because they were similarin molecular size, as was predicted. When YRN-1 cellstransformed with pHM153 were cultivated in SGlu medium,

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TABLE 1. Appearance of red and white colonies in cells havingthe RS insertion at the RAS] and HIS3 loci of chromosome XVa

Relativedirection of No. of colonies

Strain RS Medium Red (%)

RAS] HIS3 Red White Total

YRN-A SGlu 2 280 282 0.7SGal 318 53 371 85.7

YRN-B SGlu 1 312 313 0.3SGal 259 34 293 88.4

YRN-C SGlu 0 296 296 0SGal 12 279 291 4.1

YRN-D SGlu 0 267 267 0SGal 16 294 310 5.2

a Cells of each strain were inoculated from SGlu medium supplementedwith necessary nutrients but not leucine to SGlu or SGal broth supplementedwith the same nutrients and cultivated at 30°C for 2 days. The cells were thenspread on YPD plates after appropriate dilution, and all colonies that appearedon three plates were scored for red (Ade-) and white (Ade+) phenotypes after4 days of incubation at 30°C.

the frequency of clones having the recombinant chromo-somes was severely reduced, with only 2 of 54 clonesexamined showing a band of the recombinant chromosomes.However, weak hybridization signals were always de-

tected at the migration site of chromosome V or XV in therecombinant clones with the URA3, RAS], or HIS3 DNA asthe probe. This result suggests persistence of the originalconfiguration of chromosomes V and XV in a minor fractionof the galactose-grown cells. These signals gradually in-creased upon cultivation of the recombinant cells in YPADmedium after curing of the R plasmid (data not shown).Since the clones examined were isolated by repeated single-colony isolation, these observations suggest the in vivoreconstruction of the original configuration of chromosomesV and XV, probably from the two recombinant chromo-somes.Although we do not know the mechanisms underlying the

observations presented above, it is possible to speculatefollowing. The URA3 locus of chromosome V with the RSfragment inserted has a --URA3-SUPII-RS-URA3-- struc-ture, since the RS fragment was inserted at this locus byconnecting it to the modified pUS1 plasmid (Fig. lb), andone of the recombinant chromosomes should have a--URA3-SUPI I-RS-HIS3-- arrangement at the RS. Thus, theSUPII DNA having a weak ARS function (14) is locatedadjacent to a chromosome breaking point. This type ofDNAstructure may result in another event besides recombination,similar to the high frequency of gene conversion in the pSR1molecule (19) or switching of the mating-type locus (4).These arguments suggest that these unexpected events mightbe the result of the SUP]J DNA.To examine this possibility, the RS fragment was inserted

at the URA3 locus of chromosome V in strain NBW5 (leu2his3 ura3) with a YIp plasmid constructed by ligation of the3.2-kb PstI fragment of YIp5 bearing a major region of theURA3 gene (32) and the 4.5-kb PstI fragment prepared fromthe plasmid constructed by insertion of the 2.1-kb RSfragment at the XhoI site (originally HindIll) of the modifiedpUS1 (Fig. lb) prepared in the experiments described above.The Ura+ transformant was then transformed with the sameHIS3 DNA construct bearing the RS fragment used in theexperiments illustrated in Fig. 3 and 5. The resultant Ura+His' transformant was designated YRN-3. The RS frag-ments at the URA3 and HIS3 loci in YRN-3 should have thesame direction relative to the respective centromere, but the

RS fragment inserted at the URA3 locus should lack theSUPJJ DNA. YRN-3 was transformed with pHM153 to theLeu+ phenotype. The Ura+ His' Leu+ transformant wascultivated in SGal medium, and the cells were spread on anSGlu plate. Thirty colonies were isolated at random andexamined for chromosomal migration on the PFGE with ahexagonal electrode array. We found that 2 of the 30 clonesshowed a band at the position of the recombinant chromo-some(s), as observed with YRN-1. Chromosomal migrationon the PFGE gel with the hexagonal electrode array gavebetter resolution than did that with the point electrode arrayand could separate chromosomes V and VIII and chromo-somes VII and XV. The PFGE gels loaded with the DNAsample prepared from one of the two recombinant clones,along with the clone having the original chromosomal con-figuration, were electrophoresed, blotted onto a nylon mem-brane, and hybridized with the same URA3, RAS], HIS3, orRS probe as was used for the analysis of YRN-1 (Fig. 5). Theresults clearly indicated that the URA3, RAS], and HIS3probes could hybridize only to the recombinant chromo-some(s); no hybridization signals were detected at the posi-tion of putative chromosome V or XV on the DNA sample of

aYRN-A

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* ~~~~~~2.0-rFIG. 4. Restriction analysis of modified chromosome XV. (a)

Restriction maps of MIuI, XbaI, and XhoI sites flanking the RSfragment inserted at the RAS] and HIS3 loci of chromosome XVand of the modified chromosomes resulting from the recombinationbetween two RSs. !:3 represents the 2.1-kb RS fragment. Abbre-viations for restriction sites: M, MluI; X, XhoI; Xb, XbaI. (b)Results of restriction analysis of the region flanking the RS insertionsites of chromosome XV. The genomic DNAs were prepared fromcells showing the original chromosomal pattern (0) and the cloneshowing the recombinant chromosome (R) determined by prelimi-nary experiments with strains YRN-A and YRN-C, respectively.The DNA samples were digested doubly with MluI and XhoI (M/X)or singly with XhoI (X) or XbaI (Xb), electrophoresed on an agarosegel, blotted, and hybridized with the 32P-labeled 2.1-kb RS fragmentas a probe. The molecular sizes of the DNA fragments wereestimated with X phage DNA digested with HindIIl.

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616 MATSUZAKI ET AL.

'hr- XV

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FIG. 5. Recombination between nonhomologous chromosomes. (a) Strategy of recombination between chromosomes V and XV. Forinsertion of the RS fragment at the URA3 locus on chromosome V, a YIp-type chimeric plasmid was constructed with a modified pUS1, inwhich the HindIII site was converted to XhoI during the preparation of pHM149 (Fig. 2a). The RS fragment was connected at the XhoI siteof the modified pUS1 bearing the URA3-SUPHJ DNA. Two HIS3 plasmids constructed in the deletion and inversion experiments (Fig. 3) wereused for integration of the RS on chromosome XV. Since these two HIS3 plasmids have the RS fragment in opposite directions, one of thetransformants, YRN-1, has two RS fragments at URA3 and HIS3 in the same direction relative to the respective centromeres and the other,YRN-2, has them in the opposite directions. The plasmid DNAs were linearized by restriction before transformation at the unique NcoI sitein the URA3 DNA for the URA3 plasmid and at the XhoI site in the HIS3 DNA as described in the legend to Fig. 3a. (b) Detection ofrecombinant chromosomes by PFGE and Southern hybridization with a 32P-labeled 0.85-kb PstI-NsiI fragment (25) of YIp5 (32) bearingURA3, a 1.8-kb HindlIl fragment ofpRAS1 bearing RASI, the 1.7-kb BamHI fragment of YIpl bearing HIS3, and the RS fragment as probes.Four sets of the same PFGE gels for chromosome separation were prepared by using the point electrode array. Each slot on the gels wascharged with a DNA sample prepared from cells of YRN-1 having the original configuration of chromosomes V and XV (0) or fromgalactose-grown cells having the recombinant chromosomes and cured for pHM153 (R). These gels were blotted and hybridized with the fourprobes as indicated. The arrowhead on the left marks the site of a new chromosomal band; the migration sites for chromosomes VII and XVand for chromosomes V and VIII are indicated.

the recombinant clone (Fig. 6). The cells having the originalchromosomal configuration showed hybridization signals atchromosome V or XV with the URA3, RAS], and HIS3probes and at both with the RS probe, as expected. Thus, theweak hybridization signals observed in the recombinantclone of YRN-1 on the putative reconstructed chromosomeV or XV (Fig. Sb) might have been caused by the SUPJJDNA, although the exact mechanism is not known.No recombinant bands were detected in the 52 indepen-

dent Leu+ transformants of YRN-2 so far examined,whether cultivated in SGal or in SGlu medium. In addition,the YRN-2 transformants showed slow growth on SGalmedium but normal growth in SGlu medium. These results

agree well with the prediction that the YRN-2 cells createacentric and dicentric chromosomes by the recombination(Fig. Sa).To examine whether the recombination indeed occurred at

the two RSs, genomic DNAs prepared from the originalYRN-3 and that having the recombinant chromosomes weresubjected to restriction analysis. The RS fragment has oneMluI site; chromosomes V and XV have two PstI sitesflanking and sandwiching the inserted RS fragment. Bothsides of the RS fragment on chromosome V have two Sallrestriction sites, and the RS fragment inserted on chromo-some XV was linked to the carrier plasmid YIp at the Sallcohesive ends (Fig. 7a). Therefore, Mlul-PstI fragments

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CHROMOSOME ENGINEERING IN S. CEREVISIAE 617

URA3 RASI H153 RS0 R O R O R O R

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FIG. 6. Detection of recombinant chromosomes by PFGE andSouthern hybridization. A chromosomal sample prepared from cellsof YRN-3 having the original configuration of chromosomes V andXV was loaded in lanes marked 0, and lanes marked R were loadedwith another chromosomal sample prepared from a clone showingthe recombinant chromosomes after pHM153 had been cured. Thegel was blotted onto a nylon filter, cut into four strips, each of whichhad a pair of the 0 and R lanes, and hybridized with the 32P-labeledURA3, RAS], or H153 DNA or with the RS fragment used in theexperiments shown in Fig. 5 as a probe. The arrowhead on the leftmarks the site of a new chromosomal band; bands for chromosomesV and XV are also indicated.

should be the same regardless of whether recombinationoccurred at the RS. When the same DNA samples aredigested with PstI or Sail, the restriction fragments shoulddiffer between the original and recombinant chromosomes.Results of Southern hybridization to detect these fragmentswith the RS fragment as a probe agreed well with thesepredictions (Fig. 7b). Similar results were obtained forrestriction analysis with the YRN-1 cells having the originalor recombinant chromosomes (data not shown). Thus, re-

ciprocal recombination occurred at the RS, at least in the2.1-kb RS fragment.

DISCUSSION

By using the site-specific recombination system of pSRl,we were able efficiently to modify yeast chromosomes, i.e.,delete or invert a large chromosomal segment or achievereciprocal recombination between two nonhomologouschromosomes. Construction of various modified chromo-somes with deletions (22, 34), recombination between non-

homologous chromosomes (17, 24, 33), and fragmentation ofa chromosome by YCF vectors (36) in S. cerevisiae havebeen described. Since the methods used for modification ofchromosomes are based on the homologous (general) recom-bination mechanism, isolation of yeast strains having such a

modified chromosome requires an appropriate selectiontechnique. Although insertion of the RS at a target site on a

chromosome with the aid of the YIp vector also depends on

homologous recombination, the recombination frequenciesbetween two inserted RS sites catalyzed by the R enzymeare high enough for isolation of the recombinants by randomselection. Because various chromosomal genes have beencloned in S. cerevisiae, it is possible to insert the RSfragment at various target sites and to select effectively byconnecting the DNA fragments of the cloned genes on a YIpvector bearing the RS fragment. Thus, a wide variety of

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FIG. 7. Restriction analysis of the chromosomes created byrecombination between chromosomes V and XV. (a) Restrictionmaps of the MluI, PstI, and Sall sites in the flanking regions to theRS fragments inserted at the URA3 and HIS3 loci on chromosomesV and XV. Abbreviations for restriction sites are as for Fig. 1 and 4.(b) Analysis of DNA samples prepared from cells of YRN-3 havingthe original configuration of chromosomes V and XV withoutpHM153 (0) and from those having the recombinant chromosomesafter pHM153 had been cured (R). The DNA samples were digestedwith Sall or PstI or doubly with MluI and PstI, electrophoresed on

an agarose gel, blotted, and hybridized with the 32P-labeled 2.1-kbRS fragment as probe. The molecular sizes of the DNA fragmentswere estimated with phage DNA digested with HindIIl.

modifications and recombinations should be possible byusing hybrid cells constructed by mating two cells havingdifferent insertions of the RS fragment.Although the exact mechanism is still obscure, the SUP] I

DNA flanking the RS seems to disturb chromosome modifi-cation by this method (Fig. 5). The faint hybridization bandmigrating in front of the 11.4-kb band observed in the earlierexperiment (Fig. 2b) has been explained by integration of thepHM153 molecule at the RS fragment remaining on the HIS3locus. The SUPJJ DNA was also connected to the insertedDNA construct and might be involved in creation of thesupposed 9.3-kb BamHI fragment.The modified chromosome(s) may have one or two copies

of the RS, and these sites will cause further recombination.This might be prevented by curing the R plasmid, since theyeast chimeric plasmids were easily lost by cultivation of thetransformants under nonselective conditions, but the possi-bility of homologous recombination would remain. Althoughnot known exactly, the frequencies of homologous recombi-nation between two inserted RS regions should be of thesame level as that caused by the Ty elements.

This method can be used to insert a large DNA fragmentinto a chromosome or to eliminate a certain chromosome bycoupling it with the technique of 2,um DNA mapping (12). Itcan also be used to recover a large chromosomal segment in

O R O R 0 R 0 R

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618 MATSUZAKI ET AL. J. BACTERIOL.

an S. cerevisiae or E. coli host as a plasmid by inserting areplication origin of a yeast or E. coli plasmid in a regionbetween two RS sites inserted on a chromosome. Thus, itwill open new avenues in recombinant DNA technology,such as transplantation of a metabolic pathway by introduc-ing a large number of genes, construction of a sterile cloneby creating a chromosomal aberration, and creating poten-tially useful organisms that, after construction, do not con-tain a vector molecule. If insertion of the RS on a chromo-some is possible, the procedure should be applicable toplants and other organisms.

ACKNOWLEDGMENTS

We thank Allan G. Hinnebusch of the National Institutes ofHealth, Bethesda, Md., Masayuki Yamamoto of the Institute ofMedical Science, University of Tokyo, and Philip Hieter of TheJohns Hopkins University School of Medicine, Baltimore, Md., fortheir generous supply of plasmids. Our thanks are also extended toReed B. Wickner of the National Institutes of Health for criticalreading of the manuscript and Peter Hawkes for assistance withrevising the manuscript.

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