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Cited by 3,953Fungal and yeast genetics researchBioinformatics and Genomic NetworksMicrobial Metabolic Engineering and Bioproduction

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Functional Characterization of the S. cerevisiae Genome by Gene Deletion and Parallel Analysis

Winzeler, Elizabeth A., Shoemaker, Daniel D., Astromoff, Anna, Liang, Hong, Anderson, Keith, Andre, Bruno, Bangham, Rhonda, Benito, Rocio, Boeke, Jef D., Bussey, Howard, Chu, Angela M., Connelly, Carla, Davis, Karen, Dietrich, Fred, Dow, Sally Whelen, El Bakkoury, Mohamed, Foury, Françoise, Friend, Stephen H., Gentalen, Erik, Giaever, Guri, Hegemann, Johannes H., Jones, Ted, Laub, Michael, Liao, Hong, Liebundguth, Nicole, Lockhart, David J., Lucau-Danila, Anca, Lussier, Marc, M'Rabet, Nasiha, Menard, Patrice, Mittmann, Michael, Pai, Chai, Rebischung, Corinne, Revuelta, Jose L., Riles, Linda, Roberts, Christopher J., Ross-MacDonald, Petra, Scherens, Bart, Snyder, Michael, Sookhai-Mahadeo, Sharon, Storms, Reginald K., Véronneau, Steeve, Voet, Marleen, Volckaert, Guido, Ward, Teresa R., Wysocki, Robert, Yen, Grace S., Yu, Kexin, Zimmermann, Katja, Philippsen, Peter, Johnston, Mark, Davis, Ronald W.·1999·Science
DOI 10.1126/science.285.5429.901
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Abstract

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

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  1. To construct deletion strains two long oligonucleotide primers are synthesized each containing (3′ to 5′) 18 or 19 bases of homology to the antibiotic resistance cassette KanMX4 (U1 D1) a unique 20-bp tag sequence an 18-bp tag priming site (U2 or D2) and 18 bases of sequence complementary to the region upstream or downstream of the yeast ORF being targeted (including the start codon or stop codon; see ). These 74-mers are used to amplify the heterologous KanMX4 module which contains a constitutive efficient promoter from a related yeast strain Ashbya gosspii fused to the kanamycin resistance gene nptI (5). Because oligonucleotide synthesis is 3′ to 5′ and the fraction of full-size molecules decreases with increasing length improved targeting is achieved by performing a second round of PCR using primers bearing 45 bases of homology to the region upstream and downstream of a particular ORF. Transformation with the PCR product results in replacement of the targeted gene upon selection for G418 resistance. The unique 20-mer tag sequences are covalently linked to the sequence that targets them to the yeast genome creating a permanent association and genetic linkage between a particular deletion strain and the tag sequence. The use of two tags increases confidence in the analyses and the redundancy is useful in case one of the tags carries a mutation or performs poorly in hybridization assays. To verify correct integration of the deletion cassette genomic DNA was prepared from the resistant strains and used as template in PCR reactions using two primers common to the KanMX4 module (KanB (5′-CTGCAGCGAGGAGCCGTAAT-3′) and KanC (5′-TGATTTTGATGACGAGCGTAAT-3′) and four ORF-specific primers (A B C and D). A and D are from regions 200 to 400 bases upstream or downstream of the start codon whereas B and C are from within the ORF (see ). For verification both the A-KanB and the D-KanC PCR reactions were required to give the correct size product when analyzed by gel electrophoresis. If one of either the A-KanB or D-KanC reactions failed to yield a product the identification of the correctly-sized AD product could suffice. In addition haploid deletion strains were tested for the disappearance of the wild-type AB and CD products. All ORFs encoding proteins greater than 100 amino acids in size were initially selected for deletion. The deletion cassettes were designed to remove the entire coding sequence for a given ORF but to leave the start and stop codon intact. Although ∼10% of ORFs in S. cerevisiae overlap one another the positions of the deletions were not adjusted nor was any attempt made to avoid essential genes genes in which a previous deletion had been constructed or genes with a well-defined function. Genes represented multiple times in the genome (telomeric ORF Ty-elements) were usually not deleted as their targeted disruption would pose a challenge due to the conservation of upstream and downstream regions. Some smaller nonannotated ORFs (NORFs) will be deleted in the future. Transcripts from many of these NORFs have been detected in SAGE analysis warranting their inclusion in the study [
  2. The UPTAG and DOWNTAG sequences were separately amplified from genomic DNA by means of PCR using primers B-U2-comp (5′ biotin-GTCGACCTGCAGCGTACG-3′) and U1 (5′-GATGTCCACGAGGTCTCT-3′) or primers B-D2-comp (5′-biotin-CGAGCTCGAATTCATCG-3′) and D1 (5′-CGGTGTCGGTCTCGTAG-3′). In both cases a twofold molar excess of biotinylated primers was used in the reactions. The amplified UPTAG and DOWNTAG sequences were combined and hybridized to high-density oligonucleotide arrays in 200 μl of 6×SSPE [1 M NaCl 66 mM NaH 2 PO –4 6.6 mM EDTA (pH 7.4)] containing 0.005% Triton X-100 (SSPE-T) 200 pmol U1 200 pmol U2 (5′-CGTACGCTGCAGGTCGAC-3′) 200 pmol D1 and 200 pmol primer D2 (5′-CGATGAATTCGAGCTCG-3′). The addition of complementary primers to the hybridization mix was shown to improve the signal-to-noise ratio [D. D. Shoemaker thesis Stanford University Stanford CA (1998)]. Samples were heated to 100°C for two min and then cooled on ice before being applied to the array. Samples were hybridized for 1 hour at 42°C. The arrays were washed two times with six changes of 6×SSPE-T. The arrays were then stained at 42°C for 10 min with 6×SSPE-T containing 2 μg/ml phycoerythrin-streptavidin (Molecular Probes) and 1 μg/ml acetylated bovine serum albumin washed two times with five changes 6×SSPE-T and scanned at an emission wavelength of 560 nm using an Affymetrix GeneChip Scanner. Of the strains analyzed 157 contained only a single tag sequence (UPTAG). Six of these strains were not detected in the hybridization mix. Of the strains represented with two-tag sequences 98.5% exhibited either an UPTAG or DOWNTAG bar code hybridization signal that was greater than threefold over background. Sequencing of 186 deletion regions tags showed that 25% of mutations in the tags or tag priming sites resulted in a nonfunctional tag that could not be amplified or detected by hybridization or both. In only 1.1% of cases was a complete lack of hybridization signal not associated with a mutation in the tag or tag priming site. Mutations were most often found in the tags or tag priming sites (0.85% per base) and were less frequent in the regions of yeast homology (0.25% per base) most likely due to selection against the mutated PCR products during the recombination event or to the two-step PCR strategy.
  3. EUROFAN projects B0 and B9 at www.mips.biochem.mpg.de/proj/eurofan/index.html; R. Niedenthal et al. Yeast in press.

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Authors
Winzeler, E. A., Shoemaker, D. D., Astromoff, A., Liang, H., Anderson, K., Andre, B., Bangham, R., Benito, R., Boeke, J. D., Bussey, H., Chu, A. M., Connelly, C., Davis, K., Dietrich, F., Dow, S. W., El Bakkoury, M., Foury, F., Friend, S. H., Gentalen, E., Giaever, G., Hegemann, J. H., Jones, T., Laub, M., Liao, H., Liebundguth, N., Lockhart, D. J., Lucau-Danila, A., Lussier, M., M'Rabet, N., Menard, P., Mittmann, M., Pai, C., Rebischung, C., Revuelta, J. L., Riles, L., Roberts, C. J., Ross-MacDonald, P., Scherens, B., Snyder, M., Sookhai-Mahadeo, S., Storms, R. K., Véronneau, S., Voet, M., Volckaert, G., Ward, T. R., Wysocki, R., Yen, G. S., Yu, K., Zimmermann, K., Philippsen, P., Johnston, M., & Davis, R. W.
Affiliations
  • Stanford Medicine (US)
  • Merck & Co., Inc., Rahway, NJ, USA (United States) (US)
  • Universite Libre de Bruxelles, Laboratoire de Physiologie Cellulaire et de Genetique des Levures, Campus Plaine, Brussels CP244, Belgium.
  • Yale University (US)
  • Dipartimento de Microbiologia y Genetica, Universidad de Salamanca, Edificio Depatamental 323/CSIC, Campus Miguel de Unamuno, E-37007 Salamanca, Spain.
  • Johns Hopkins University (US)
  • McGill University (CA)
  • University of Basel (CH)
  • IRMW-ULB, Avenue E. Gryson, 1, B-1070 Brussels, Belgium.
  • FYSA-UCL, Place Croix du Sud, 2/20, 1348-Louvain-la-Neuve, Belgium.
  • Affymetrix (United States) (US)
  • Heinrich Heine University Düsseldorf (DE)
  • Department of Biology, McGill University, Montreal, PQ, Canada H3A 1B1.
  • Washington University in St. Louis (US)
  • Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, CT 06520–8103, USA.
  • Université Libre de Bruxelles (BE)
  • KU Leuven (BE)
  • UCLouvain (BE)
ORCID
Venue
Science
Publisher
American Association for the Advancement of Science (AAAS)
Published
1999-08-06
Volume
285
Issue
5429
Pages
901-906
ISSN
  • 0036-8075 (ISSN-L)
  • 1095-9203
DOI
10.1126/science.285.5429.901
OpenAlex
W2140948740
Cited by
3,953
References
35
Citations by year
2026: 41 · 2025: 60 · 2024: 90 · 2023: 103 · 2022: 97 · 2021: 122 · 2020: 133 · 2019: 114 · 2018: 147 · 2017: 146 · 2016: 143 · 2015: 146 · 2014: 157 · 2013: 199 · 2012: 199
FWCI
46.82
Type
journal article
Language
en
Open access
closed
Locations
  • Science (publishedVersion)
  • PubMed (publishedVersion)
  • DIAL (Catholic University of Leuven) (submittedVersion)
  • Digital Access to Libraries (submittedVersion)
  • Dépôt institutionnel de l'Université libre de Bruxelles (Université Libre de Bruxelles) (submittedVersion)
Funders
  • National Institutes of Health (1r01hg001633-01, 5r01hg001627-03)
  • National Human Genome Research Institute (HG-01627, HG00185-02, HG01633)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bioinformatics and Genomic Networks
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
Keywords
Saccharomyces cerevisiae, gene deletion, open reading frames, gene function, essential genes, growth defects
MeSH terms
Culture Media, Genes, Fungal, Phenotype, Recombination, Genetic, Saccharomyces cerevisiae, Gene Expression Regulation, Fungal, Polymerase Chain Reaction, Open Reading Frames, Genome, Fungal, Gene Deletion, Gene Targeting, Genes, Essential

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