Petra Ross-Macdonald

Functional profiling of the Saccharomyces cerevisiae genome

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.

Large-scale analysis of the yeast genome by transposon tagging and gene disruption

Economical methods by which gene function may be analysed on a genomic scale are relatively scarce. To fill this need, we have developed a transposon-tagging strategy for the genome-wide analysis of disruption phenotypes, gene expression and protein localization, and have applied this method to the large-scale analysis of gene function in the budding yeast Saccharomyces cerevisiae. Here we present the largest collection of defined yeast mutants ever generated within a single genetic background—a collection of over 11,000 strains, each carrying a transposon inserted within a region of the genome expressed during vegetative growth and/or sporulation. These insertions affect nearly 2,000 annotated genes, representing about one-third of the 6,200 predicted genes in the yeast genome. We have used this collection to determine disruption phenotypes for nearly 8,000 strains using 20 different growth conditions; the resulting data sets were clustered to identify groups of functionally related genes. We have also identified over 300 previously non-annotated open reading frames and analysed by indirect immunofluorescence over 1,300 transposon-tagged proteins. In total, our study encompasses over 260,000 data points, constituting the largest functional analysis of the yeast genome ever undertaken.

TRIPLES: a database of gene function in Saccharomyces cerevisiae

Using a novel multipurpose mini-transposon, we have generated a collection of defined mutant alleles for the analysis of disruption phenotypes, protein localization, and gene expression in Saccharomyces cerevisiae. To catalog this unique data set, we have developed TRIPLES, a Web-accessible database of TRansposon-Insertion Phenotypes, Localization and Expression in Saccharomyces. Encompassing over 250 000 data points, TRIPLES provides convenient access to information from nearly 7800 transposon-mutagenized yeast strains; within TRIPLES, complete data reports of each strain may be viewed in table format, or if desired, downloaded as tab-delimited text files. Each report contains external links to corresponding entries within the Saccharomyces Genome Database and International Nucleic Acid Sequence Data Library (GenBank). Unlike other yeast databases, TRIPLES also provides on-line order forms linked to each clone report; users may immediately request any desired strain free-of-charge by submitting a completed form. In addition to presenting a wealth of information for over 2300 open reading frames, TRIPLES constitutes an important medium for the distribution of useful reagents throughout the yeast scientific community. Maintained by the Yale Genome Analysis Center, TRIPLES may be accessed at http://ycmi.med.yale.edu/ygac/triples.htm

Transposon mutagenesis for the analysis of protein production, function, and localization.

Publisher Summary This chapter describes a transposon mutagenesis system that produces multipurpose constructs for the monitoring of protein production, localization, and function. A single mutagenesis generates a large spectrum of alleles, including null, hypomorphic, and conditional alleles, reporter fusions, and epitope-insertion alleles. The system, therefore, provides the basis for a wide variety of studies of gene and protein function. The chapter provides comprehensive instructions for use of the new transposons to mutagenize a gene of interest, and for use of the transposon insertion libraries to mutagenize the yeast genome. While the application of these specific transposons is limited to organisms in which the Saccharomyces cerevisiae selectable marker URA3 can be used, the approach is generally applicable to mutagenesis of DNA from any organism for which a transformation and selection system exists.

10 Transposon Tagging I: A Novel System for Monitoring Protein Production, Function and Localization

Publisher Summary The use of transposons allows the rapid construction of a large number of the alleles of a gene of interest. Transposon insertion libraries can be used for both the mutagenesis and identification of genes regulated by particular growth conditions and strain backgrounds. This chapter presents an overview of a newly developed multipurpose transposon mutagenesis system that allows the monitoring of protein production, function, and localization in yeast. The system uses two basic types of transposon, designated as mTn-3×HA/lacZ and mTn 3×HA/GFP. The transposon system was tested by the mutagenesis of several individual yeast genes. The HAT tag was successfully used to analyze the localization of the Spa2, Arp100, and Sao1 proteins. The new transposons expand the repertoire of insertions that may be generated to include GFT fusions and epitope tags. It is expected that shuttle mutagenesis will continue to be an important tool for the characterization of individual genes and their products and for systematic analysis of the entire yeast genome.