With its tractable genetics and fully-sequenced genome, the budding yeast Saccharomyces cerevisiae
is an ideal model organism for functional genomics; several large-scale studies are already in place to identify cellular functions for each of the 6200 predicted genes within the Saccharomyces
). These genomic studies, however, possess finite limitations: it is unlikely that any single large-scale project will be completely successful in exhaustively characterizing gene function, as many genes do not generate easily observable phenotypes upon disruption (4
). Functional genomic approaches, therefore, will need to be augmented with traditional research from individual laboratories studying a single gene or pathway (4
). Following this paradigm, we have developed a transposon-based mutagenesis system facilitating both genomic and traditional research approaches: applied to S.cerevisiae
, our method has yielded an unprecedented quantity of functional data while generating thousands of reagents for further use by researchers throughout the yeast community.
Our transposon-tagging strategy utilizes a multifunctional minitransposon (mTn) to mutagenize a plasmid-based library of yeast genomic DNA in Escherichia coli
). Transposon-mutagenized genomic DNA is subsequently transformed into yeast, generating a collection of mutant strains, each carrying a single mTn insertion at a defined site within the Saccharomyces
genome. This strain collection (available from our web site) can facilitate a wide variety of functional studies. Using mTn-encoded lacZ
as a reporter, we can determine when each transposon-tagged gene is expressed during the yeast life cycle (e.g., during vegetative growth or sporulation). Additionally, mTn insertion will create a truncation of the mutagenized gene, thereby generating disruption alleles for phenotypic analysis. Finally, the inserted transposon can be modified in situ
, reducing the mTn to a 93-codon in-frame tag encoding three tandem copies of an epitope from the influenza virus hemagglutinin protein. Epitope-tagged proteins may be localized within the cell by indirect immunofluorescence. A single transposon insertion, therefore, is sufficient to generate three types of data regarding gene function: expression profiles, disruption phenotypes and protein localization.
To catalog these three data sets, we have developed TRIPLES, an on-line database of TRansposon-Insertion Phenotypes, Localization and Expression in Saccharomyces.
The TRIPLES database has been designed to offer easy access to all data generated from the functional analysis of our strain collection. At present, this collection encompasses nearly 7800 mTn-insertion alleles available in each of three forms: as diploid yeast strains containing lacZ
-fusions generated from mTn insertion, as diploid yeast strains containing mTn-encoded epitope tags, and as bacterial clones carrying plasmid-borne mTn-mutagenized yeast DNA. Users may request these strains free of charge from order forms linked to TRIPLES. The TRIPLES database also contains external links to the Saccharomyces
Genome Database (SGD) (8
) and GenBank (9
), thereby providing full background literature concerning all transposon-tagged genes within our collection. Finally, TRIPLES allows access to a set of strains carrying mTn insertions identifying non-annotated open reading frames (NORFs) within the yeast genome; these putative genes represent a potentially rich source of novel proteins in Saccharomyces
). Collectively, the data and reagents made available through TRIPLES constitute a unique information resource designed to promote ongoing research within the yeast community.