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Chromosomal Assignment of Mutations by Specific Chromosome Loss in the Yeast Saccharomyces Cerevisiae

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Chromosomal Assignment of Mutations by Specific Chromosome Loss in the Yeast Saccharomyces Cerevisiae Copyright 0 1990 by the Genetics Society of America Chromosomal Assignment of Mutations by Specific Chromosome 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 chromosomes 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 gene 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 defined segments 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 genes 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+ gene remained 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.
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