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New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae.

Achim Wach(1), Arndt Brachat, Rainer Pöhlmann and Peter Philipspsen.

(1) Institut fur Angewandte Mikrobiologie, Universitat Basel, Switzerland.

We have constructed and tested a dominant resistance module, for selection of S. cerevisiae transformants, which entirely consists of heterologous DNA. This kanMX module contains the known kanr open reading-frame of the E. coli transposon Tn903 fused to transcriptional and translational control sequences of the TEF gene of the filamentous fungus Ashbya gossypii. This hybrid module permits efficient selection of transformants resistant against geneticin (G418). We also constructed a lacZMT reporter module in which the open reading-frame of the E. coli lacZ gene (lacking the first 9 codons) is fused at its 3' end to the S. cerevisiae ADH1 terminator. KanMX and the lacZMT module, or both modules together, were cloned in the center of a new multiple cloning sequence comprising 18 unique restriction sites flanked by Not I sites. Using the double module for constructions of in-frame substitutions of genes, only one transformation experiment is necessary to test the activity of the promotor and to search for phenotypes due to inactivation of this gene. To allow for repeated use of the G418 selection some kanMX modules are flanked by 470 bp direct repeats, promoting in vivo excision with frequencies of 10(-3)-10(-4). The 1.4 kb kanMX module was also shown to be very useful for PCR based gene disruptions. In an experiment in which a gene disruption was done with DNA molecules carrying PCR-added terminal sequences of only 35 bases homology to each target site, all twelve tested geneticin-resistant colonies carried the correctly integrated kanMX module.

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Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar-coding strategy

Daniel D. Shoemaker (1), Deval A. Lashkari, Don Morris, Mike Mittmann & Ronald W. Davis (1)

(1) Department of Biochemistry, Beckman Center, Stanford University Medical Center, Stanford, CA 94305, USA

A quantitative and highly parallel method for analysing deletion mutants has been developed to aid in determining the biological function of thousands of newly identified open reading frames (ORFs) in Saccharomyces cerevisiae. This approach uses a PCR targeting strategy to generate large numbers of deletion strains. Each deletion strain is labelled with a unique20-base tag sequence that can be detected by hybridization to a high-density oligonucleotide array. The tags serve as unique identifiers (molecular bar codes) that allow analysis of large numbers of deletion strains simultaneously through selective growth conditions. Hybridization experiments show that the arrays are specific, sensitive and quantitative. A pilot study with 11 known yeast genes suggests that the method can be extended to include all of the ORFs in the yeast genome, allowing whole genome analysis with a single selectivegrowth condition and a single hybridization.

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Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications

Carrie Baker Brachmann, Adrian Davies, Gregory J. Cost, Emerita Caputo, Joachim Li, Philip Hieter, Jef D. Boeke(1)

(1) Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A

A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.

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Genomic profiling of drug sensitivities via induced haploinsufficiency

Guri Giaever(1), Daniel D. Shoemaker, Ted W. Jones, Hong Liang, Elizabeth A. Winzeler, A. Astromoff & Ronald W. Davis(1)

(1) Department of Biochemistry, Beckman Center, Stanford University Medical Center, Stanford, CA 94305, USA

Lowering the dosage of a single gene from two copies to one copy in diploid yeast results in a heterozygote that is sensitized to any drug that acts on the product of this gene. This haploinsufficient phenotype thereby identifies the gene product of the heterozygous locus as the likely drug target. We exploited this finding in a genomic approach to drug target identification. Genome sequence information was used to generate molecularly tagged heterozygous yeast strains that were pooled, grown competitively in drug, and analyzed for drug sensitivity using high-density oligonucleotide arrays. Individual heterozygous strain analysis verified six known drug targets. Parallel analysis identified the known target and two hypersensitive loci in a mixed culture of 233 strains in the presence of the drug tunicamycin. Our discovery that both drug target and hypersensitive loci exhibit drug-induced haploinsufficiency may have important consequences in pharmacogenomics and variable drug toxicity observed in human populations.

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

Elizabeth A. Winzeler* Daniel D. Shoemaker*, Anna Astromoff*, Hong Liang*, Keith Anderson, Bruno Andre, Rhonda Bangham, Rocio Benito, Jef D. Boeke, Howard Bussey, Angela M. Chu, Carla Connelly, Karen Davis, Fred Dietrich, Sally Whelen Dow, Mohamed El Bakkoury, Françoise Foury,Stephen H. Friend, Erik Gentalen, Guri Giaever, Johannes H. Hegemann, Ted Jones, Michael Laub, Hong Liao, Nicole Liebundguth, David J. Lockhart, Anca Lucau-Danila, Marc Lussier, Nasiha M'Rabet, Patrice Menard, Michael Mittmann, Li Ni, Chai Pai, Corinne Rebischung, Jose L. Revuelta, Linda Riles, Christopher J. Roberts, Petra Ross-MacDonald, Bart Scherens, Michael Snyder, Sharon Sookhai-Mahadeo, Reginald K. Storms, Steeve Véronneau, Marleen Voet, Guido Volckaert, Teresa R. Ward, Robert Wysocki, Grace S. Yen, Kexin Yu, Katja Zimmermann, Peter Philippsen, Mark Johnston, and Ronald W. Davis (1)

(1) Department of Biochemistry, Beckman Center, Stanford University Medical Center, Stanford, CA 94305, USA
* These authors contributed equally to this work.

The functions of most open reading frames (ORFs) identified in genome-sequencing projects is unknown. New, whole genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-thoughput 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.
[supplementary data click here]

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Widespread aneuploidy revealed by DNA microarray expression profiling

Timothy R. Hughes (1), Christopher J. Roberts, Hongyue Dai, Allan R. Jones, Michael R. Meyer, David Slade, Julja Burchard, Sally Dow, Teresa R. Ward, Matthew J. Kidd, Stephen H. Friend and Matthew J. Marton

(1) Rosetta Inpharmatics, Inc., Kirkland, Washington, USA.

Expression profiling using DNA microarrays holds great promise for a variety of research applications, including the systematic characterization of genes discovered by sequencing projects1, 2. To demonstrate the general usefulness of this approach, we recently obtained expression profiles for nearly 300 Saccharomyces cerevisiae deletion mutants3. Approximately 8% of the mutants profiled exhibited chromosome-wide expression biases, leading to spurious correlations among profiles. Competitive hybridization of genomic DNA from the mutant strains and their isogenic parental wild-type strains showed they were aneuploid for whole chromosomes or chromosomal segments. Expression profile data published by several other laboratories also suggest the use of aneuploid strains. In five separate cases, the extra chromosome harboured a close homologue of the deleted gene; in two cases, a clear growth advantage for cells acquiring the extra chromosome was demonstrated. Our results have implications for interpreting whole-genome expression data, particularly from cells known to suffer genomic instability, such as malignant or immortalized cells.

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Functional Profiling of the Saccharomyces cerevisiae Genome

Guri Giaever (¹⁾, Angela M. Chu, Li Ni, Carla Connelly, Linda Riles, Steeve Véronneau, Sally Dow, Ankuta Lucau-Danila, Keith Anderson, Bruno André, Adam P. Arkin, Anna Astromoff, Mohamed el Bakkoury, Rhonda Bangham, Rocio Benito, Sophie Brachat, Stefano Campanaro, Matt Curtiss, Karen Davis, Adam Deutschbauer, Karl-Dieter Entian, Patrick Flaherty, Francoise Foury, David J. Garfinkel, Mark Gerstein, Deanna Gotte, Ulrich Güldener, Johannes H. Hegemann, Svenja Hempel, Zelek Herman, Daniel F. Jaramillo, Diane E. Kelly, Steven L. Kelly, Peter Kötter, Darlene LaBonte, David D. Lamb, Ning Lan, Hong Liang, Hong Liao, Lucy Liu, Chuanyun Luo, Marc Lussier, Rong Mao, Patrice Menard, Siew Loon Ooi, Jose L. Revuelta, Christopher J. Roberts, Matthias Rose, Petra Ross-Macdonald, Bart Scherens, Greg Schimmack, Brenda Shafer, Daniel D. Shoemaker, Sharon Sookhai-Mahadeo, Reginald K. Storms, Jeffrey N. Strathern, Giorgio Valle, Marleen Voet, Guido Volckaert, Ching-Yun Wang, Teresa R. Ward, Julie Wilhelmy, Elizabeth A. Winzeler, Yonghong Yang, Grace Yen, Elaine Youngman, Kexin Yu, Howard Bussey, Jef D. Boeke, Michael Snyder, Peter Philippsen ⁽¹³⁾, Ronald W. Davis (^1,2) & Mark Johnston (⁵⁾

⁽¹⁾ Stanford Genome Technology Center, Palo Alto, California 94303, USA
⁽²⁾ Department of Biochemistry, Stanford University School of Medicine, Stanford, California  94305-5307, USA
⁽⁵⁾ Department of Genetics, Washington University Medical School, St. Louis, MO 63110, USA
⁽¹³⁾ Biozentrum, Department of Molecular Microbiology, Biozentrum, University of Basel, Switzerland

Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.
[supplementary data click here]

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Systematic screen for human disease genes in yeast

Lars M. Steinmetz (1, 3*) Curt Scharfe (2,3*), Adam M. Deutschbauer, Dejana Mokranjac, Zelek S. Herman, Ted Jones, Angela M. Chu, Guri Giaever, Holger Prokisch, Peter J. Oefner & Ronald W. Davis (1, 2, 3)

(1) Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA.
(2) Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305, USA.
(3)Stanford Genome Technology Center, Palo Alto, California 94304, USA.
* These authors contributed equally to this work.

High similarity between yeast and human mitochondria allows functional genomic study of Saccharomyces cerevisiae to be used to identify human genes involved in disease. So far, 102 heritable disorders have been attributed to defects in a quarter of the known nuclear-encoded mitochondrial proteins in humans. Many mitochondrial diseases remain unexplained, however, in part because only 40-60% of the presumed 700-1,000 proteins involved in mitochondrial function and biogenesis have been identified. Here we apply a systematic functional screen using the pre-existing whole-genome pool of yeast deletion mutants to identify mitochondrial proteins. Three million measurements of strain fitness identified 466 genes whose deletions impaired mitochondrial respiration, of which 265 were new. Our approach gave higher selection than other systematic approaches, including fivefold greater selection than gene expression analysis. To apply these advantages to human disorders involving mitochondria, human orthologs were identified and linked to heritable diseases using genomic map positions.
[supplementary data click here]

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Parallel phenotypic analysis of sporulation and postgermination growth in Saccharomyces cerevisiae

Adam M. Deutschbauer (1), Roy M. Williams, Angela M. Chu, and Ronald W. Davis (1*)

(1) Departments of Genetics and *Biochemistry, Stanford University School of Medicine, Stanford, CA 94305

We have quantitatively monitored the sporulation and germination efficiencies of 4,200 yeast deletion strains in parallel by using a molecular bar coding strategy. In a single study, we doubled the number of genes functionally implicated in sporulation to 400, identifying both positive and negative regulators. Our set of 261 sporulation-deficient genes illustrates the importance of autophagy, carbon utilization, and transcriptional machinery during sporulation. These general cellular factors are more likely to exhibit fitness defects when deleted and less likely to be transcriptionally regulated than sporulation-specific genes. Our postgermination screening assay identified recombination/chromosome segregation genes, aneuploid strains, and possible germination-specific factors. Finally, our results facilitate a genome-wide comparison of expression pattern and mutant phenotype for a developmental process and suggest that 16% of genes differentially expressed during sporulation confer altered efficiency of spore production or defective postgermination growth when disrupted.
[supplementary data click here]

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Evolutionary Rate in the Protein Interaction Network

Hunter B. Fraser (1*), Aaron E. Hirsh (2*), Lars M. Steinmetz, Curt Scharfe, Marcus W. Feldman

(1) Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
(2) Center for Computational Genetics and Biological Modeling, Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.
* These authors contributed equally to this work.

High-throughput screens have begun to reveal the protein interaction network that underpins most cellular functions in the yeast Saccharomyces cerevisiae. How the organization of this network affects the evolution of the proteins that compose it is a fundamental question in molecular evolution. We show that the connectivity of well-conserved proteins in the network is negatively correlated with their rate of evolution. Proteins with more interactors evolve more slowly not because they are more important to the organism, but because a greater proportion of the protein is directly involved in its function. At sites important for interaction between proteins, evolutionary changes may occur largely by coevolution, in which substitutions in one protein result in selection pressure for reciprocal changes in interacting partners. We confirm one predicted outcome of this process--namely, that interacting proteins evolve at similar rates.
[supplementary data click here]

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Role of duplicate genes in genetic robustness against null mutations

Zhenglong Gu (1*), Lars M. Steinmetz(2*), Xun Gu, Curt Scharfe, Ronald W. Davis and Wen-Hsiung Li(1)

(1)Department of Ecology & Evolution, University of Chicago, 1101 East 57th Street, Chicago, Illinois 60637, USA
(2) Department of Biochemistry, and Stanford Genome Technology Center, Stanford University School of Medicine, Stanford, California 94305, USA
* These authors contributed equally to this work

Deleting a gene in an organism often has little phenotypic effect, owing to two mechanisms of compensation. The first is the existence of duplicate genes: that is, the loss of function in one copy can be compensated by the other copy or copies. The second mechanism of compensation stems from alternative metabolic pathways, regulatory networks, and so on. The relative importance of the two mechanisms has not been investigated except for a limited study, which suggested that the role of duplicate genes in compensation is negligible. The availability of fitness data for a nearly complete set of single-gene-deletion mutants of the Saccharomyces cerevisiae genome has enabled us to carry out a genome-wide evaluation of the role of duplicate genes in genetic robustness against null mutations. Here we show that there is a significantly higher probability of functional compensation for a duplicate gene than for a singleton, a high correlation between the frequency of compensation and the sequence similarity of two duplicates, and a higher probability of a severe fitness effect when the duplicate copy that is more highly expressed is deleted. We estimate that in S. cerevisiae at least a quarter of those gene deletions that have no phenotype are compensated by duplicate genes.
[supplementary data click here]

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