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YEAST VOL. 11: 53-55 (1995) Construction of a Set of Convenient Saccharomyces cerevisiae Strains that are Isogenic to S288C FRED WINSTON*, CATHERINE DOLLARD AND STEPHANIE L. RICUPERO-HOVASSE Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, M A 02115, USA Received 2 May 1994; accepted 12 September 1994 A set of GAL2' yeast strains that are isogenic to strain S288C have been constructed. They contain non-reverting mutations in genes commonly used for selection for recombinant plasmids. Strains from this collection are being used for the European Union Yeast Genome Sequencing Programme. Representative strains from this collection have been deposited with the ATCC. K ~ WORDS Y - S288C; isogenic; Saccharomyces cerevisiae. Beginning with strain S288C (Mortimer, 1993; Mortimer and Johnston, 1986), we have constructed an isogenic set of strains by gene replacement. The strains are GAL2' and contain non-reverting mutations in URA3, HIS3, LEU2, L YS2 and TRPl. They have already been distributed to many laboratories and have been used in our laboratory for recent studies (for examples, see Arndt et al., 1992 and Hirschhorn ei al., 1992). A diploid derived from two of these strains is the source of DNA for the European Union Yeast Genome Sequencing Programme (Dujon, 1994; Dujon rt al., 1994; Thierry and Dujon, 1992; A. Thierry. C. Fairhead and B. Dujon, personal communication). Several plasmids were used to recombine mutations into S288C. The plasmid used for recombining ura3-52 into the genome was pMRFW2 (Rose and Winston, 1984). The plasmids used for recombining his3A200, leu2A1 and trplA63 into the genome were YRp141his3A200, YRp15Ileu2Al and YRp 14ltrplA63, respectively (Sikorski and Hieter, 1989). The plasmid used for recombining lvs2A202 into the genome was pJF210 (J. Fassler and F. Winston, unpublished results). This plasmid is a derivative of plasmid pSL42-2 (Simchen et a/., 1984) that contains a deletion of the XhoIHpaI fragment of L YS2. Plasmid YCp5O-HO was used to convert mating type and was generously provided by Rob Jensen and Ira Herskowitz. The *Corresponding authoi CCC 0749- 501)</95/010053-03 ( 1995 bq John Wiley ti Sons Ltd plasmid to transform strains to GAL,?' was pAAl (generously provided by A. Antebe and G. R. Fink). This plasmid, derived from a GALL?' clone described by Tschopp et al. (1986), contains an EcoRI-Hind111 fragment that contains GAL2' in the plasmid YIp5. All media and genetic methods are described in Rose et al. (1990). Transformations were done essentially as described by Schiestl and Gietz (1 989). The first step in the strain constructions was to recombine ura3-52 into S288C. This was done as follows: (1) 5 pg of plasmid pMRFW2 was digested with BamHI and SmaI; (2) the BamHISmaI fragment that contains ura3-52 was used to transform strain S288C; (3) after the final step in the transformation procedure (the incubation in PEG), the cells were pelleted, resuspended in 5 ml of YPD, and grown overnight; (4) the next day, the cells were washed twice in water and resuspend in 5 ml of water; and (5) dilutions of the cultures were plated on 5-fluoro-orotic acid (5-FOA) plates. Several 5-FOAR colonies were purified and Southern analysis was performed to verify that they contained the ura3-52 mutation. One of these strains was saved and designated F Y 1. The second step was to construct a GAL2' derivative of FY1. This step was accomplished using plasmid pAAl and two-step gene replacement (Scherer and Davis, 1979). One GAL2' strain was saved and designated FY2 (Table 1). All subsequent strains were derived from FY2. 54 Table 1. Yeast strains. F. WINSTON I'T \L. comparing the strains FY2, FY3 and S288C. All chromosomes comigrated (data not shown). Strain Genotype We tested the frequency at which strains FY1 and FY2 can be transformed by the plasmid YCp50. Using the standard LiAc transformation MA Ta ura3-52 FY2 method using denatured calf thymus DNA as the FY3 M A Ta ura3-52 carrier (Schiestl and Gietz, 1989), transformation FY4 MATa MA Ta ura3-52 trpl A63 frequencies ranged from 1.4 x lo4 to 2.4 x lo4 FY7 FY8 M A Ta ura3-52 l.ys2A202 transformants/pg of YCp50 DNA. Using fresh FYlO MATa ura3-52 Ieu2AI overnight cultures, the transformation frequencies MA Ta ura3-52 his3A200 FY22 were substantially less, ranging from 350 to 1200 FY23 MATa ura3-52 trplA63 leu2A1 transformants/pg of YCp50 DNA. Experiments FY67 MATa trplA63 with other isogenic strains have shown that they FY69 MATa leu2AI can be transformed at the same approximate FYI3 MA Ta his3A200 ura3-52 FY833 MATa his3A200 ura3-52 leu2A1 Ij*s2A202 frequencies (data not shown). This set of strains is similar to a set previously trp 1A63 FY834 M.4 Ta his3A200 ura3-52 leu2A1 lj)s2A202 described (Sikorski and Hieter, 1989). There are trpl A63 two differences between the two sets of strains. First, we began with S288C and made all constructions by gene replacement methods; in the previous constructions, the beginning strain, YNN216, is To obtain a MATa version of FY2, we trans- congenic to S288C (see Sikorski and Hieter. 1989). formed FY2 with YCp5O-HO (generously pro- Second, the Galf phenotype of YNN216 is the vided by Rob Jensen and Ira Herskowitz). The result of reversion of the ga/2 mutation found in Uraf transformants were then streaked on 5-FOA S288C background, rather than a gene replaceplates and the single colonies were tested for their ment by the GAL2' gene (M. Carlson. personal mating behavior. One MATa derivative was saved communication). and designated FY3. One significant advantage to using these strains Auxotrophic markers for HIS3, LEU2, LYS2 is that a diploid made by mating strains FY23 and and TRPl were recombined into strain FY2, re- FY73 (and designated stain FY 1679) was used as placing the wild-type alleles, by two-step gene the source of DNA for a library that is being used replacement. For each marker, we determined that for the European Union Yeast Genome Sequencthe auxotrophy segregated 2 : 2 in tetrads (data not ing Programme. This DNA has been or is curshown) and confirmed the gene replacement by rently being used for sequencing chromosomes Southern analysis (data not shown). All strains VII, X, XI, XIV, XV and parts of IV and XI1 (B. that contain combinations of these markers Dujon, personal communication; Dujon rt a/.. (strains with the FY designation; Table 1) were 1994). Strains derived from strain FY I679 have constructed by genetic crosses. also been used for several other genome-related studies (for example, see Thierry and Dujon. 1993). Struin cliuracterizations To make these strains easily available. strains To ensure that these strains were not disomic for FY23, FY67, FY69, FY73, FY833 and FY834 any chromosome, we crossed FY2 and FY3 by a have been deposited with the ATCC. The ATCC set of strains to follow segregation of at least one numbers are as follows: FY23, 90840; FY67. marker on each of the 16 yeast chromosomes. FY2 90841; FY69, 90842; FY73, 90843: FY833, 90844: and FY3 were crossed by strains previously de- and FY834, 90845. scribed by Gaber et al. (1983) and by Klapholz and Esposito (1982). The results demonstrated that every marker segregated 2 : 2 (data not shown). ACKNOWLEDGEMENTS Therefore, each chromosome is monosomic in We thank Phil Hieter and Mark Rose tor helpful these two strains. To address if there were any discussions during the Cold Spring Harbor Yeast major chromosomal rearrangements that might Course that provided the motivation to make not have been detected by these crosses, we exam- these strains. We thank Bernard Dujon for ined the chromosomes by C H E F gel analysis, communicating unpublished results. This work YEAST STRAINS ISOGENIC TO S288C was supported by NIH grants GM32967 and GM45720 to F.W. REFERENCES Arndt, K. M., Ricupero, S. L., Eisemann, D. M. and Winston, F. (1992). Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivn and DNA binding in vitro. Mol. Cell. Biol. 12, 2372-2382. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K. (1988). 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