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

Abstract

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.

Lethality and centrality in protein networks

“…This simulated tolerance against random mutation is in agreement with systematic mutagenesis studies, which identified a striking capacity of yeast to tolerate the deletion of a substantial number of individual proteins from its proteome 9,10 . Yet, if indeed this is due to a topological component to error tolerance, on average less connected proteins should prove less essential than highly connected ones.…”

SSP2 and OSW1, Two Sporulation-Specific Genes Involved in Spore Morphogenesis in Saccharomyces cerevisiae

“…Identification of the SSP2 and OSW1 genes: We previously identified transposon insertions that generate in-frame lacZ fusion genes expressed specifically during sporulation (Burns et al 1994;Ross-Macdonald et al 1999). Diploids homozygous for these insertions were constructed and screened for sporulation competence.…”

A Bayesian system integrating expression data with sequence patterns for localizing proteins: comprehensive application to the yeast genome 1 1Edited by F. Cohen

“…We extensively examined a variety of other possible priors, based on our other possible training sets (i.e. the Localized-465, Localized-704, and Localized-2013), the overall composition of the MIPS database, and the experimental data from the Snyder lab (Ross-Macdonald et al, 1999). These are shown in the various subpanels of figure 4.…”

“…Moreover, we feel it is quite reasonable for some proteins not to have a definite localization. For instance, several proteins have been found experimentally in more than one compartment (Hodges et al, 1999;Ross-Macdonald et al, 1999). In particular, the transcription factor complex NFλB is known to shuttle between an inactive form in the cytoplasm and an active form in the nucleus (Kopp & Ghosh, 1994), and various structural proteins also appear both in the nucleus and the cytoplasm (see TUB4 example below).…”