Copyright 0 1990 by the Genetics Society of America

Chromosomal Assignment of Mutations by Specific Loss in the Yeast Saccharomyces cerevisiae

L. Paul Wakem* and Fred Sherman**+

Departments of *Biochemistry and tBiophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Manuscript received December 20, 1989 Accepted for publication March 3, 1990

ABSTRACT Yeast 2-pm plasmids were integrated near the centromereof a different chromosomein each of 16 cir' mapping strains of Saccharomyces cerevisiae. The specific containing the integrated 2-pm plasmid DNA were lost at a high frequency after crossing the cir' strains to cir+ strains. A recessive mutation in a cir+ strain can then be easily assigned to its chromosome using this set of mapping strains, since the phenotype of the recessive mutation will be manifested only in diploids having the integrated2-pm plasmid and the unmapped mutationon homologous chromosomes.

HE large number of chromosomes and the high pm DNA repeat sequence of the integrant and cata- T frequency of meiotic recombination in yeast lyze a site specific recombination event. This results make it difficult tomap a simplyby tetrad in the loss of the integrant as well as chromosomal analysis. Numerous procedures have been developed DNA distal to the site of integration.If the 2-pm to circumventthese difficulties by first assigning a plasmid DNA is integrated at or near the centromere, mutation to its chromosome (LIRASet al. 1978; KA- the entire chromosome is lost at a high frequency. WASAKI 1979; WICKNER1979; MORTIMER,CONTO- These properties of chromosomes with integrated 2- POULOU and SCHILD198 1; SCHILDand MORTIMER pm plasmid DNA have been described (FALCOet al. 1985; KLAPHOLZand ESPOSITO1982; WOOD 1982). 1982; FALCO,ROSE and BOTSTEIN1983; FALCOand Some of these methods rely on constructinga diploid BOTSTEIN1983). heterozygous for the unmapped mutation andvarious A recessive mutation in a cir+ straincan be assigned chromosome markers and subsequently the random to its chromosome by crossing to the set of cir' map- loss of chromosomes caused by chemical agents or ping strains. Subclones of the cir'/cir+ diploids will certain mutations. However,most of these procedures lose the integrated 2-pm plasmid DNA plus the chro- require that the unmapped mutation be crossed into mosome into which integration occurred at a high a particular yeast background. In addition, chromo- frequency. The recessive mutation will be expressed some lossis nonspecific and multiplechromosome only in the diploid containing thecir' mapping strain losses can occur, which further complicates the chro- with an integrant at the centromere of the chromo- mosomal assignment. Thispaper describesa new some that is homologous to the chromosome that the method for thechromosomal assignment of recessive mutation is on. The mutation will remain heterozy- mutations that does not require strain constructions gous in all of the other diploid strains. Once a muta- and that involves the specific loss of individual chro- tion has been assigned to its chromosome, thelocation mosomes. The method relies on a set of cir' tester on the chromosome can be determined by conven- strains, each containing2-pm plasmid DNA integrated tional meiotic analysis. at ornear the centromereof a different chromosome. Plasmids containingthe 2-pm invertedrepeat se- MATERIALS AND METHODS quence and either random or definedsegments of Genetic methods: Key yeast strains and plasmids used in yeast DNA were integrated into the yeast genome of this study are listed, respectively, in Tables 1 and 2. The cir' strains atthe regionof homology. The 2-pm mediaand genetic procedures including genetic crosses, plasmid DNA is stably maintained as an integrant sporulation, dissection, tetrad analysis and the scoring of nutritional markers have been described (SHERMAN,FINK because the plasmid DNA lacks the 2-pm FLP (flip) and HICKS 1987). The medium containing 5-fluoroorotic gene required for 2-pm site specific recombination acid (1 mg/ml) is described by BOEKE,LACROUTE and FINK and the cir' cells contain no resident 2-pm circles to (1984). provide the FLP function. Specific mitotic chromo- Molecular biology methods: Methods used in the con- struction of plasmids, including restriction enzyme digests, some loss can be induced aftera cir' X cir+ cross. The separation of plasmid DNA and restriction fragments on FLP recombinationfunction provided by the 2-pm agarose gels, ligation of DNA fragments, and the isolation circles of the cir+ parent can recognize a site in the 2- of plasmid DNA are described in MANIATIS, FRITSCHand

Genetics 125: 333-340 (June, 1990) 334 L. P. Wakem and F. Sherman

TABLE 1 Yeast strains

Strain Genotype Source

3482-16-1 car' MATa ura3-52 leu2-3,112 trpl-289 his3-AImet2 CyhR D. LIVINGSTON B-7926 cir+ MATa cdc28-1cycl-72 glcl lys2-I87 tyrl This study B-80 13 cir" MATa ura3-52leu2-3,112 trpl-289 met2 This study K382-19D cir+ MATa ura3 can1 cyh2 ade2 his7 hom3 tyrl spoll KLAPHOLZand ESPOSITO(1 982) K381-9D cir+ MATa ura3adeb arg4 aro7 asp5 met14 lys2 pet17 trpl spol I KLAPHOLZand ESPOSITO (1982) K393-35C cir+ MATa ura3 his2 leul lyslmet4 pet8 spoll KLAPHOLZand BPOSITO(1 982) K396-22B cir+ MATa ura3adel his1 leu2 lys7 met3trp5 KLAPHOLZand ESPOSITO(1 982) GT153-6A cir+ MATa ura3adel ade2 arg4 aro7 asp5 gal7 his2 his6 ilv3 leul leu2 lys7 CAMBELLet al. (1981) lys9 met2 met14 trpl A298-6 1 D cir' MATa ade8arg4 arolC leu2-3 pet2 ma3trp4 Thr- Ura- GABERet al. (1 983) MC-30 1 cir+ MATa ade5cryl-13 his4-15 inol-13 ino4-8 leu2 lys2 MAL2 thr4 M. CULBERTSON

SAMBROOK(1982). Yeast transformations were performed These 46 transformants were further characterized by the spheroplasting method(BEGGS 1982) andEscherichia by identifying the chromosome into which the 2-pm coli transformations were performed with the CaCl method (COHEN,CHANG and Hsu 1972). plasmid DNA was integrated in each strain. Each transformant was crossed to the following cir+ multi- RESULTS ply marked mapping strains containingrecessive aux- otrophic mutations linked to the centromeres of all A set of cir' 2p mapping strains was constructed by 16 chromosomes: K382-19D, K381-9D, K393-35C, integrating derivatives of the YEp24 plasmid (BOT- K396-22B, GT153-6A, MC-301 and A298-61D. Dip- STEIN et 1979) at or near the centromere of each al. loids fromthese crosses were isolated on selective yeast chromosome in a cir' strain. The derivatives of medium,the diploids were plated on nonselective the YEp24 plasmid contained the gene, the 2- URA3+ medium at a concentration resulting in 50-100 colo- pm circle DNA repeat sequence, pBR322 sequences nies per plate, and then assayed on various types of anddifferent yeast segments which correspondto omission media forthe expression of auxotrophic regions at or near the centromere of each of the 16 markers. The chromosome intowhich integration had chromosomes. The yeast DNA segments wereob- occurred was identified for a particular transformant tained from either a random yeast DNA bank (CARL- when an auxotrophic marker was expressed. Since SON and BOTSTEIN1982) or various cloned yeast DNA cir'/cir+ diploids will lose the integrated 2-pm plasmid fragments encompassing centromeric regionsor DNA plus all or part of the chromosome containing which map close to certain centromeres, as summa- theintegrant at a high frequency,an auxotrophic rized in Table 2. marker will only be expressed in a diploid containing Integration of 2-pm plasmid DNA containingran- a transformant with 2-pm plasmid DNA integrated in dom yeast DNA sequences: Mapping strains for chro- mosomes ZZ, ZZZ, VZI,VZZZ, ZX, XI and XV were con- the homologous chromosome. Of the 46 transform- structed by transforming the cir' strain,3482-16-1 ants characterized by this method, 7 were found to with the YEp24 plasmid library containing inserts of have integrations in the following chromosomes: ZZ, random yeast DNA segments (CARLSONand BOTSTEIN VZZ, VZZZ, ZX (2), XI and XV (Table 2). The remaining 1982). URA3+ transformants were selected on me- transformants were not identified because no auxo- dium lacking uracil, and screened by first plating on trophic markers were expressed in diploids containing nonselective medium, andthen spotting on 5-fluo- these strains. This suggests that integration in these roorotic acid (FOA) medium. FOA medium inhibits strainsdid not occur near thecentromere. In this URA3+ cells, but allows growth of ura3- cells (BOEKE, case, the loss of the 2-pm plasmid DNA would not LACROUTEand FINK 1984). Those transformants destabilize the entire chromosome at high frequency, which contained 2-pm plasmid DNA integrated into but only chromosomal sequences distal to thepoint of the genome, about 1%,were unable to grow on FOA integration. If integration occurred near a telomere medium, since the 2-pm plasmid DNA containing the or on a chromosomal arm which is not well marked URA3+ generemained stably integrated in the ge- by auxotrophic mutations, the chromosome carrying nome incir' cells. However,confluent growth on the integrant would not be identified. FOA medium is observed for transformants contain- The transformation of strain 3482-1 6-1 using the ing autonomously replicating 2-pm plasmids, which random DNA bank also allowed for the direct selec- are unstable and lost at a high frequency in cir' cells. tion of a strain with an integrant near the centromere Out of 6183 URA3+ transformants, 46 contained 2- on chromosome ZZZ. Transformants complementing pm plasmid DNA stably integrated into the genomes. the leu2-3, I12 mutation were selected on medium C hromosomal AssignmentChromosomal in Yeast 335 lacking both leucine and uracil. These transformants each overnight culture, subcloned on a nonselective were tested on medium containing FOA to detect a medium, then plated on medium containing FOA to strain with 2-pm plasmid DNA stably integrated at the select for transformants which contained integrated LEU2 gene near the centromereof chromosome ZZZ. 2-pm plasmid DNA. The stable integrants isolated Integration of 2-pm plasmidDNA containing from transformations with each of the plasmids de- cloned yeastcentromeric-linked DNA sequences: scribedabove were crossed tostrains K382-19D, Mapping strains for chromosomes I, ZV, V, VI, X, XZZ, K381-9D, K393-35C, K396-22B and A298-61D.A XZZZ, XZV and XVZ were constructed using the defined diploid from each cross was isolated, subcloned on a yeast DNA sequences shown in Table 2. The 2-pm nonselective medium and then diluted and plated on DNA containing plasmids ZA50 (TEEMand ROSBASH a nonselective medium at a concentration to produce 1983) and pYE5 1B (ABOVICHand ROSBASH1984), 50-100 colonies per plate. These plates were replica- which contain clones of the RP5lA and RP5lBgenes, plated on various omission media to test that only respectively, were used to transform strain 3482-16- auxotrophic markers on the same chromosome con- 1. Integrants of the plasmid ZA50 containing the taining the integrated 2-pm plasmid DNA were ex- RP51A gene integrated into chromosome XZZZ, while pressed. integrants of the plasmid pYE51B containing the Genetic analysis of 2~ mappingstrains: The RP51B gene integrated into chromosomeV. All other strains containing integrated2-pm plasmid DNA were defined sequences were transferred into the plasmid crossed to the cir' strain, B-8013; the diploids were pAB659 which was constructed by replacing the sporulated and dissected, and the tetrads from each EcoRl-Hind111 fragment of YIp5 with the 2.1-kb cross analyzed. The URA3+ marker segregated 2:2 in EcoRI-Hind111 2-~mB fragment containing the 2-pm all crosses, consistent with the view thatthe 2-pm DNA repeat sequence.The HindIII-Hind111 centrom- plasmid DNA (URA3+) is stably integrated into the eric regions for chromosomes I, ZV, X,XZV and XVZ yeast genome. A set of MATa and MATa 2p mapping were transferred from plasmids p181, pll3, p180, strains werechosen from among the tetradsof crosses p203 and p179, respectively (HIETERet al. 1985; P. with B-8013, as shown in Table 3. The strains B-7 179 HIETER,personal communication), to the HindIIIsite and B-7590 are separate strains which contain 2-pm of pAB659,resulting in, respectively, the plasmids plasmid DNA integrated at different sites in chromo- pAB660, pAB661, pAB662, pAB663 and pAB664. some V. Similarly, the strains B-7175 and B-7176 Also fragments encompassing the genes GCN4, contain 2-pm plasmid DNA integrated at different SUPll-1 and PPRl were insertedinto pAB649. A sites in chromosome ZX. The strains B-7603 and B- 4.5-5.0-kb BamHI-Hind111 fragment containing the 7607 are the MATa equivalents of B-7590 and B- GCN4 gene was transferredfrom pAH2O (HINNE- 7 175, respectively. BUSCH and FINK 1983) andligated in the BamHI and The frequency at which the integrated 2-pm plas- HindIII sites of pAB649, resulting in pAB665. Part mid DNA is lost from the genome in each of the 2p of the PPRl gene contained in a 2.5-kb EcoRI-EcoRI mappingstrains was determined by crossing each fragment (LILJELUNDet al. 1984; F. LACROUTE,per- strain to the ci~+ura3- strain K381-9D and assaying sonal communication) was transferred into the EcoRI for the percentage of Ura- diploid subclones. The site of pAB645, resulting in pAB666. The plasmid loss of the integrated 2-pm plasmid DNA also results pAB667 was constructed by replacing the 4.0-kb SalI- in the loss of the URA3+ gene containedin the plasmid NdeI fragment of pAB645 with the 5.4-kb MI-NdeI DNA. The 2p mapping strains lose the integrated 2- fragment of p179(HIETER et al. 1985). The two pm plasmid DNA at frequencies ranging from 10 to fragments are the same except the fragment from 50% (Table 2), and lose the entire chromosome con- p179 also contains a 1.35-kb fragment encompassing taining the integrated 2-pm plasmid DNA at frequen- the SUP11-1 gene. cies ranging from 1 to 50% (Figure 1). The variation Plasmids ZA50, pYE5 lB, pAB660,pAB661, in the frequency of chromosomal loss in the different pAB662, pAB663, pAB664, pAB665, pAB666 and mapping strains may be due to the difference in the pAB667 were used to transform strain 3482-16-1 to sites of integration on different chromosomes. In ad- uracil prototrophy. Transformants, in which plasmids dition, the frequency of chromosomal lossof each had stably integrated into the genome, were selected mapping strain can also vary somewhat when they are by first enriching for integrants. Transformants from crossed to different cir+ strains (data notshown). each transformation were pooled in lots containing Each mapping strain was examined for its efficiency about100 transformant colonies and each lot was in the chromosomal assignment of mutations along used to inoculate an overnight culture in YPD non- the entire length of the chromosome containing the selective medium. Samples were diluted and plated integrated 2-pm plasmid. The strains were crossed to on medium lacking uracil to select for URA3+ trans- 28 multiply marked mapping strains, some of which formants. Ten URA3+ colonies were isolated from are listed in Table 1 and which included the56 336 P. L. Wakem and F. Sherman

TABLE 2 Plasmids integrated into the2p mapping strains and the frequenciesof loss of the plasmidDNA

~~ Integrated plasmid

~ ~~~ Plasmid Source of yeast segment Frequency of plasmid loss strain ChromosomeYeast strain No, SegmentReference Plasmid Reference (% ura3-) - B-7588 I pAB660 This study CENl p181 HIETERet al. (1 985) 44 B-7 170 11 Undefined DNA bank CARLSONand BOTSTEIN Not recovered 18 (1 982) B-7171 111 LEU2 DNA bank CARLSONand BOTSTEIN Not recovered 20 (1982) B-7589 IV pAB66 1 This study CEN4 pl13 HIETERet al. (1985) 40 B-7590 V pAB665 This study GCN4 YpAH20 HINNEBUSCHand FINK 21 (1 983) B-7 179 V pYE5 1 B ABOVICHand ROSBACH RP5IB pYE5 1 B ABOVICHand ROSBASH 12 (1984) (1 984) B-7591 VI pAB667 This study SUP11-1 p179 HIETERet al. (1985) 34 B-7 173 VII Undefined DNA bank CARLSONand BOTSTEIN 33 Not recovered (1982) B-7 174 VIII Undefined DNA bank CARLSONand BOTSTEIN 17 Not recovered (1 982) B-7 175 IX Undefined DNA bank CARLSONand BOTSTEIN 18 Not recovered (1 982) B-7 176 IX Undefined DNA bank CARLSONand BOTSTEIN 11 Not recovered (1 982) B-7593 X pAB662 This study CENI 0 p180 HIETERet al. (1985) 25 B-7 178 XI Undefined DNA bank CARLSONand BOTSTEIN 12 Not recovered (1 982) B-7595 XII pAB666 This study PPR I puc8-ppr 1 LILJELUNDet al. (1984) 48 B-7255 XIII ZA50 TEEMand ROSBASH RP51A ZA50 TEEMand ROSBASH 50 (1 983) (1983) B-7596 XIV pAB663 This study CENl4 p203 HIETERet al. (1 985) 45 B-7 180 xv Undefined DNA bank CARLSONand BOTSTEIN 12 Not recovered (1 982) B-7598 XVI pAB664 This study CENI 6 pl79 HIETERet al. (1 985) 43 markers shown in Figure 1. Diploids from each cross taining the integrated DNA at ahigher frequency were assayed for the frequency at which various re- than the opposite arm. For example, ura3- diploids cessive auxotrophicmutations which map onboth from crosses of B-7171 and cir+ ura3- leu2- strains, arms of a chromosome were expressed. A summary were always Zeu2-, since the integrated 2-pm plasmid of thefrequencies at which mutationsalong each in B-7171, which integrated at the LEU2 gene (left chromosome arm are expressed in the appropriate arm of chromosome ZZZ), contained both the URA3+ mapping strain are shown in Figure 1. In most cases, and LEU2+ genes. All leu2- diploids from a cross of the strains express mutations onboth arms of the B-’7 1’71 and MC-30 1 were his#- (left arm of chromo- chromosome containing the integrated 2-pm plasmid some ZZZ), while only 15% were both hid- and thr4- DNA. The frequency of loss of the left arms of chro- (right armof chromosome ZZZ). This also suggests that mosomes VZZZ and XZZ, in strains B-7 174 andB-7595, mutations on the same chromosome arm and closely respectively, were notdetermined because easily linked to thesite containing the 2-pm plasmid are lost scored recessive-mutations on these two chromosome at the same frequency as the integrated DNA. Muta- arms were not available. In general, strains with an tions which are on the same chromosome arm, but integration very near the centromere lose both arms further from the site of integration, may be lost at of the chromosome containing the integrant at asim- lower frequencies. In crosses of B-7170 and B-7926, ilar frequency (e.g.,B-7589, B-7590, B-7591, B-7593, 32 out of 294 diploids were lys2-cdc28- tyrl-; 4 B-7596 and B-7598). We believe both arms of chro- diploids were lys2- cdc28- TYRl+ and 1 diploid was mosome Z are lost at a frequency of loss of 10 to 50% lys2- CDC28+ TYRI+. Although the exact site of in- in B-7588, even though only the adel marker was tegration of the 2-pm plasmid is unknown in this tested;the adel marker maps on the right arm of strain, it probably lies between the centromere and chromosome I, closely linked tothe centromere. the LYS2 gene since the integration must be close Strains with an integration,which is not as near to the enough to the centromere to result in both chromo- centromere, tend to lose the chromosomal arm con- some arms being lost. Chromosomal Assignment in Yeast 337 TABLE 3

SIP mapping strains

Strain" Insert Strain" Genotype B-7588 CHRl::URA3+cir' MATa ura3-52 leu2-3,112 trpl-289 met2 E7170 CHR2::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7171 CHR3::URA3+ LEU2+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7589 CHR4::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7590 CHR5::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7 179 CHR5::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7591 CHR6::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 B-7 173 CHR7::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7 174 CHR8::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7 175 CHR9::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3dlmet2 CyhR B-7 176 CHR9::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7593 CHRlO::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 B-7CHRl 178 l::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7595 CHR12::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 met2 CyhR B-7255 CHRI3::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7596 CHR14::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7 180 CHR15::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7598 CHRl6::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 met2 CyhR B-7599 CHRl::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 met2 B-7600 CHR2::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7601 CHR3::URA3+ LEU2+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7602 CHR4::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7603 CHR5::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7604 CHR6::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7605 CHR7::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7606 CHR8::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7607 CHR9::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 Cyh' B-7608 CHRlO::URA3+ cir' MATa ura3-52 1eu2-3,112 trpl-289 his3-A1 met2 CyhR B-7609 CHRl l::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-76CHR 10 12::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 met2 CyhR B-7611 CHRl3::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-7612 CHR14::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 met2 B-7613 CHRl5::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 his3-A1 met2 CyhR B-76 14 CHRlG::URA3+ cir' MATa ura3-52 leu2-3,112 trpl-289 met2 CyhR * These strains have been deposited with the Yeast Genetic Stock Center (Donner Laboratory,University of California, Berkeley, California 94720).

In the studies described above with cir+ ura3- mut- colonies per plate. Several hundred colonies of each strains, the following classes of diploids were found: isolateddiploid are replica-plated ontoa medium URA3+ MUT+; ura3- MUT+; and ura3- mut- (where which detects the phenotype of the unmapped muta- mut- denotes various auxotrophic mutations). Only tion, mut-. The recessive mut- mutation will only be auxotrophic mutations on the chromosome homolo- manifested in the diploid having the integrated 2 pm gous tothe 2-pmplasmid containing chromosome plasmid and the mut- mutation on homologous chro- were expressed. In addition, no URA3+ mut- diploids mosomes. A recessive mutation should be expressed were found and no auxotrophic mutations on chro- at a frequency of between 1 to 50%, depending on mosomes nonhomologous to the 2-pm plasmid con- both the particular chromosomal mappingstrain used taining chromosome were expressed (

&I - some VII because about 2 1 % of the diploid colonies -Y)CM fromcross aof pet54 with B-7605 were-Pet-. Meiotic mappingconfirmed that the PET54 gene maps on

cdc4 - awl I& odr8 ma3 chromosome VII, 3.2 cM distal to SUFI5 (M. COS- TANZO, personal communication; COSTANZO, SEAVER 8-755U and FOX 1989). The PMRI gene was also assigned to

8-7179 chromosome VII using the 2p mapping method. This chromosomal assignment was confirmed by hybridi- 8-7591 zation of the cloned PMRI gene to a chromosomal 8-7113 blot prepared by OFAGE and by meiotic mapping of 8.7174 the PMRI gene, which located it 8.2 cM distal to LYS5

8-7175 (RUDOLPHet al. 1989). In addition,the CYSZ and CYS4 genes, which are closely linked to each other B-7176 (ONOet al. 1988), were assigned to chromosome VI1 8.7593 since a cys2- cys4- double mutation was manifested 8-7178 only in diploids constructed with B-7 173. Subsequent

B-7W mapping localized the CYSZ and CYS4 genes on the right arm of chromosome VII, centromere proximal B-NS to ADE3 (B. ONO,personal communication). 8.7596

B.7lsO DISCUSSION

8.759% The 2p mapping method described in this paper is FIGC~RE1 .-The frequency of expression ofauxotrophic markers a rapid and efficient procedure for the chromosomal located along the arms of each chrornosonle in each of the 2p assignment of unmapped recessive mutations. Com- Inapping strains listed on the lelt of the figure. The 2p mapping strains were crossed to 28 multiply marked strains and the diploids pared to other chromosomal assignment procedures, from each cross were assaved for expression of the various auxo- the 2p mapping method has several advantages. First, ~ropllicnlarkrrs shown. The numbered ellipses represent chromo- each 2p mapping strain can be inducedto specifically somal centromeres. The frequencies at which the various mutations lose a particular chromosome. This eliminates ambi- were expressed are denoted as follows: solid areas denote a fre- guities due to multiple chromosomeloss events which quency of IO-.iO%: stippled areas denote a frequency of 1-10%: and open areas denote untested regions of the chromosome. occur in other chromosomal assignment methods re- lying on mitotic chromosome loss (LIRASet al. 1978; 7175, mutations on the left arm of chromosome IX KAWASAKI1979; MORTIMER, CONTOPOULOUand are expressed at frequencies greater than lo%, while SCHILD 198 1; SCHILDand MORTIMER1985; WOOD mutations on the right arm are expressed at frequen- 1982). Secondly, the 2p methoddoes not require cies of less than 10%. In contrast, in diploids contain- preliminarystrain constructions to couple required ing B-7176, mutations on the left and right arm of alleles with the unmapped mutation (LIRASet al. 1978; chromosome IX are expressed at frequencies of less KAWASAKI1979; MORTIMER, CONTOPOULOUand than and greater than lo%, respectively. SCHILD 198 1 ; SCHILDand MORTIMER1985; KLA- After assigning the mut- mutation to its chromo- PHOLZ and ESPOSITO1982). The 2p mapping method some, its exact location on the chromosome can be is most useful for genes which have not been cloned. determined by conventionalmeiotic analysiswith A clonedgene can be conveniently assigned to its genes whose mapping position on the chromosome is chromosome by hybridization to electrophoretically known. separatedchromosomes or by mappingmethods Chromosomal assignment of unmappedmuta- whichinvolve eitherchromosome fragmentation tions by the 2~ mapping method: Several different (VOLLRATHet al. 1988) or the integration of a 2-pm laboratorieshave successfully used the 2p mapping plasmid containing a cloned yeast fragment into the strainsdescribed in this paper to assignpreviously yeast genome (FALCOand BOTSTEIN,1983). unmapped mutations to their chromosome. The mu- Each 2p mapping strain loses the arms of the chro- tation ptll was assigned to chromosomeXV using the mosome containing the integrated2-pm plasmid DNA 2p mapping strains, since onlya cross of B-7180 with at frequencies ranging from1 to 50% (Figure 1). The a strain containing ptll yielded temperature sensitive frequency of loss depends both on the mapping strain ptlI- diploid subclones. Its position on chromosome andthe chromosomal arm. Mapping strains, which XV was confirmed by meiotic mapping which showed were constructed by targeting the2-pm plasmid DNA that the PTLI gene maps about 25 cM distal to the to integratevery near the centromere, inas the strains HIS3 gene (J. TOYN, personal communication;TOYN B-7588, B-7589, B-7590, B-7591, B-7593, B-7596 et al. 1988). A pet54 mutant was assigned to chromo- and B-7598, resulted in strains in which both arms of Ch romo som al AssignmentChromosomal in Yeast 339 the particular chromosome were lost at a similar fre- CAMBELL,D., J. S. DOCTOR,J. H. FEURERSANCERand M. M. quencyranging from 10 to 50%. The strains con- DOOLI~LE,1981 Differentialmitotic stability of yeast di- somes derived from triploid meiosis. Genetics 98: 239-255. structed using arandom yeast DNA bank (e.g., B- CARLSON,M., and D. BOTSTEIN,1982 Two differentially regu- 7170, B-7171, B-7173, B-7174, B-7175, B-7176, B- lated mRNAs with different 5’ endsencode secreted and 71 78 and B-7180) lose one chromosome arm at a intracellular forms ofyeast invertase. Cell 28: 145-154. higher frequency than the other. This suggests that COHEN, S. A.N., C. Y. CHANCand L. HSU, 1972 integration in these strains occurred further from the Nonchromosomalantibiotic resistance in bacteria: genetic transformation ofEscherichia coli by R-factor DNA.Proc. centromere than in the strains which lose both arms Natl. Acad. Sci. USA 69 21 10-21 14. at a similar frequency. Even though the exact sites of COSTANZO,M., E. C. SEAVERand T. D. FOX, 1989 The PET54 integration in thesestrains are unknown, the sites gene of Saccharomyces cerevisiae: characterization of a nuclear were sufficiently close to the centromereso that both gene encoding a mitochondrial translational activator and sub- chromosomearms were still lost. Since integration cellular localization of its product. Genetics 122: 297-305. FALCO,S. C., and D. BOTSTEIN,1983 A rapidchromosome- occurred at the LEU2 gene, which is located on the mapping method for cloned fragmentsof yeast DNA. Genetics left arm of chromosome ZZZ, and because strain B- 105: 857-872. 7 17 1loses the left chromosome arm at a higher fre- FALCO,S. C., M.ROSE and D. BOTSTEIN,1983 Homologous quencythan theright arm, the chromosomearm recombination between episomal plasmids and chromosomes which is lost at a higher frequency apparently contains in yeast. Genetics 105: 843-856. FALCO,S. C., Y. LI, J. R. BROACHand D. BOTSTEIN,1982 Genetic the integrated 2 pm plasmid DNA. Presumably, the properties of chromosomally integrated 2p plasmid DNA in 2-pm plasmid DNA was integrated into the left and yeast. Cell 29: 573-584. right arm of chromosome ZX in, respectively B-7175 GABER,R. F., L. MATHISON,I. EDELMANand M. R. CULBERTSON, and B-7 176. The results with strains B-7 179 and B- 1983 Frameshiftsuppression in Saccharomyces cerevisiae. VI. 7255, which were constructed by integrating 2-pm Complete genetic map of twenty-five suppressor genes. Ge- netics 103: 389-407. plasmids containing the RPSlB and RP5lA genes, HIETER, P., D. PRIDMORE,J, H. HECEMANN,M. THOMAS,R. W. respectively, suggests thatthe RP5lB and RP5lA DAVIS andP. PHILIPPSEN,1985 Functionalselection and genes are located on the right arms of, respectively, analysis of yeast centromeric DNA. Cell 42: 9 13-92 1. chromosome V and chromosome XZZI. HINNEBUSCH,A. G., and G. R. FINK, 1983 Positive regulation in The 2p mapping method has been successfully used the general aminoacid control ofSaccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 80: 5374-5378. to show that the gene PTLl (TOYNet al. 1988); and KAWASAKI,G., 1979 Karyotypic instability andcarbon source the genes PET54 (COSTANZO,SEAVER and Fox 1989), effects in cell cycle mutants of Saccharomyces cereuisiae. Ph.D. PMRI (RUDOLPHet al. 1989), CYS2 and CYS4 (ONOet thesis, University of Washington, Seattle. al. 1988) map on chromosomes XV and VII, respec- KLAPHOLZ,S., and R. E. ESPOSITO,1982 A new mapping method tively. Subsequent meiotic mapping confirmed these employing a meiotic rec- mutant of yeast. Genetics 100: 387- 412. assignments. Since PET54 (COSTANZO,SEAVER and LILJELUND,P., R. LOSSON,B. KAMMERERand F. LACROUTE, FOX 1989), CYS2 and CYS4 (ONOet al. 1988) are 1984 Yeast regulatory gene PPRI. 11. Chromosomal localiza- located on the right arm and PMRI (RUDOLPHet al. tion, meiotic map, suppressibility, /recessivity and 1989) is located on the left arm of chromosome VZZ, dosage effect. J. Mol. Biol. 180: 251-265. this method has been used to assign genes which are LIRAS, P.,J. MCCUSKER, S. MASCIOLI andJ. E. HABER, located on either armof a chromosome. 1978 Characterization of a mutation in yeast causing nonran- dom chromosome loss during mitosis. Genetics 88: 651-671. MANIATIS,T., E. F. FRITSCHand J. SAMBROOK, 1982Molecular This investigation was supported by the U.S. Public Health Cloning: A Laboratory Manual.Cold Spring Harbor Laboratory, Research grantR01 GM12702 from the National Institutesof Cold Spring Harbor, N.Y. Health. We thank GITA SHARMAandJEFFREY COHENfor assistance MORTIMER,R. K., R. CoNToPoULouand D. SCHILD,1981 Mitotic in testing some of the random inserts. chromosome loss in aradiation-sensitive strain of the yeast Saccharomyces cerevisiae. Proc. Natl. Acad. 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