Abstract
Saccharomyces cerevisiae is an ideal model eukaryotic system for the systematic analysis of gene function due to the ease and precision with which its genome can be manipulated. The ability of budding yeast to undergo efficient homologous recombination with short stretches of sequence homology has led to an explosion of PCR-based methods to delete and mutate yeast genes and to create fusions to epitope tags and fluorescent proteins. Here, we describe commonly used methods to generate gene deletions, to integrate mutated versions of a gene into the yeast genome, and to construct N- and C-terminal gene fusions. Using a high-efficiency yeast transformation protocol, DNA fragments with as little as 40 bp of homology can accurately target integration into a particular region of the yeast genome.
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References
Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101:202–211
Elledge SJ, Davis RW (1988) A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae. Gene 70:303–312
Cormack B, Castano I (2002) Introduction of point mutations into cloned genes. Methods Enzymol 350:199–218
Rothstein R (1991) Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol 194:281–301
Wach A, Brachat A, Pohlmann R et al (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10:1793–1808
Goldstein AL, McCusker JH (1999) Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15:1541–1553
Gueldener U, Heinisch J, Koehler GJ et al (2002) A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res 30:e23
Wach A, Brachat A, Alberti-Segui C et al (1997) Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae. Yeast 13:1065–1075
Goldstein AL, Pan X, McCusker JH (1999) Heterologous URA3MX cassettes for gene replacement in Saccharomyces cerevisiae. Yeast 15:507–511
Ito-Harashima S, McCusker JH (2004) Positive and negative selection LYS5MX gene replacement cassettes for use in Saccharomyces cerevisiae. Yeast 21:53–61
Janke C, Magiera MM, Rathfelder N et al (2004) A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21:947–962
Longtine MS, McKenzie A 3rd, Demarini DJ et al (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14:953–961
Gueldener U, Heck S, Fielder T et al (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24:2519–2524
Delneri D, Tomlin GC, Wixon JL et al (2000) Exploring redundancy in the yeast genome: an improved strategy for use of the cre-loxP system. Gene 252:127–135
Chee MK, Haase SB (2012) New and Redesigned pRS Plasmid Shuttle Vectors for Genetic Manipulation of Saccharomyces cerevisiae. G3 (Bethesda) 2:515–526
Sung MK, Ha CW, Huh WK (2008) A vector system for efficient and economical switching of C-terminal epitope tags in Saccharomyces cerevisiae. Yeast 25:301–311
Hegemann JH, Heick SB (2011) Delete and repeat: a comprehensive toolkit for sequential gene knockout in the budding yeast Saccharomyces cerevisiae. Methods Mol Biol 765:189–206
Shoemaker DD, Lashkari DA, Morris D et al (1996) Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar-coding strategy. Nat Genet 14:450–456
Winzeler EA, Shoemaker DD, Astromoff A et al (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285:901–906
Smith AM, Heisler LE, Mellor J et al (2009) Quantitative phenotyping via deep barcode sequencing. Genome Res 19:1836–1842
Smith AM, Durbic T, Kittanakom S et al (2012) Barcode sequencing for understanding drug-gene interactions. Methods Mol Biol 910:55–69
Manivasakam P, Weber SC, McElver J et al (1995) Micro-homology mediated PCR targeting in Saccharomyces cerevisiae. Nucleic Acids Res 23:2799–2800
Hughes TR, Roberts CJ, Dai H et al (2000) Widespread aneuploidy revealed by DNA microarray expression profiling. Nat Genet 25:333–337
Lehner KR, Stone MM, Farber RA et al (2007) Ninety-six haploid yeast strains with individual disruptions of open reading frames between YOR097C and YOR192C, constructed for the Saccharomyces genome deletion project, have an additional mutation in the mismatch repair gene MSH3. Genetics 177:1951–1953
Copic A, Latham CF, Horlbeck MA et al (2012) ER cargo properties specify a requirement for COPII coat rigidity mediated by Sec13p. Science 335:1359–1362
Hartwell LH, Culotti J, Reid B (1970) Genetic control of the cell-division cycle in yeast. I. Detection of mutants. Proc Natl Acad Sci U S A 66:352–359
Reid RJ, Lisby M, Rothstein R (2002) Cloning-free genome alterations in Saccharomyces cerevisiae using adaptamer-mediated PCR. Methods Enzymol 350:258–277
Langle-Rouault F, Jacobs E (1995) A method for performing precise alterations in the yeast genome using a recycable selectable marker. Nucleic Acids Res 23:3079–3081
Maeder CI, Maier P, Knop M (2007) A guided tour to PCR-based genomic manipulations of S. cerevisiae (PCR-targeting). Methods Microbiol 36:55–78
Prein B, Natter K, Kohlwein SD (2000) A novel strategy for constructing N-terminal chromosomal fusions to green fluorescent protein in the yeast Saccharomyces cerevisiae. FEBS Lett 485:29–34
Gauss R, Trautwein M, Sommer T et al (2005) New modules for the repeated internal and N-terminal epitope tagging of genes in Saccharomyces cerevisiae. Yeast 22:1–12
Knop M, Siegers K, Pereira G et al (1999) Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines. Yeast 15:963–972
Funakoshi M, Hochstrasser M (2009) Small epitope-linker modules for PCR-based C-terminal tagging in Saccharomyces cerevisiae. Yeast 26:185–192
Van Driessche B, Tafforeau L, Hentges P et al (2005) Additional vectors for PCR-based gene tagging in Saccharomyces cerevisiae and Schizosaccharomyces pombe using nourseothricin resistance. Yeast 22:1061–1068
Sheff MA, Thorn KS (2004) Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. Yeast 21:661–670
Tamm T (2009) Plasmids with E2 epitope tags: tagging modules for N- and C-terminal PCR-based gene targeting in both budding and fission yeast, and inducible expression vectors for fission yeast. Yeast 26:55–66
Gadal O, Strauss D, Braspenning J et al (2001) A nuclear AAA-type ATPase (Rix7p) is required for biogenesis and nuclear export of 60S ribosomal subunits. EMBO J 20:3695–3704
Gadal O, Strauss D, Petfalski E et al (2002) Rlp7p is associated with 60S preribosomes, restricted to the granular component of the nucleolus, and required for pre-rRNA processing. J Cell Biol 157:941–951
Lee S, Lim WA, Thorn KA (2013) Improved blue, green, and red fluorescent protein tagging vectors for S. cerevisiae. PLoS One 8:e67902
Schneider BL, Seufert W, Steiner B et al (1995) Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae. Yeast 11:1265–1274
Moqtaderi Z, Struhl K (2008) Expanding the repertoire of plasmids for PCR-mediated epitope tagging in yeast. Yeast 25:287–292
Webster TD, Dickson RC (1983) Direct selection of Saccharomyces cerevisiae resistant to the antibiotic G418 following transformation with a DNA vector carrying the kanamycin-resistance gene of Tn903. Gene 26:243–252
Brachmann CB, Davies A, Cost GJ et al (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14:115–132
Wieczorke R, Krampe S, Weierstall T et al (1999) Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Lett 464:123–128
Baudin A, Ozier-Kalogeropoulos O, Denouel A et al (1993) A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res 21:3329–3330
Johnston M, Riles L, Hegemann JH (2002) Gene disruption. Methods Enzymol 350:290–315
Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96
Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272
Slaughter BD, Schwartz JW, Li R (2007) Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging. Proc Natl Acad Sci U S A 104:20320–20325
Onischenko E, Stanton LH, Madrid AS et al (2009) Role of the Ndc1 interaction network in yeast nuclear pore complex assembly and maintenance. J Cell Biol 185:475–491
Hailey DW, Davis TN, Muller EG (2002) Fluorescence resonance energy transfer using color variants of green fluorescent protein. Methods Enzymol 351:34–49
Acknowledgements
We are indebted to participants of the Cold Spring Harbor Yeast Genetics and Genomics Course for their insights into yeast cell manipulation. We thank members of the Jaspersen lab for comments on the manuscript. S.L.J. is supported by the Stowers Institute for Medical Research and the American Cancer Society (RSG-11-030-01-CSM).
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Gardner, J.M., Jaspersen, S.L. (2014). Manipulating the Yeast Genome: Deletion, Mutation, and Tagging by PCR. In: Smith, J., Burke, D. (eds) Yeast Genetics. Methods in Molecular Biology, vol 1205. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1363-3_5
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DOI: https://doi.org/10.1007/978-1-4939-1363-3_5
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