Abstract
Yeast is a powerful model for systems genetics. We present a versatile, time- and labor-efficient method to functionally explore the Saccharomyces cerevisiae genome using saturated transposon mutagenesis coupled to high-throughput sequencing. SAturated Transposon Analysis in Yeast (SATAY) allows one-step mapping of all genetic loci in which transposons can insert without disrupting essential functions. SATAY is particularly suited to discover loci important for growth under various conditions. SATAY (1) reveals positive and negative genetic interactions in single and multiple mutant strains, (2) can identify drug targets, (3) detects not only essential genes, but also essential protein domains, (4) generates both null and other informative alleles. In a SATAY screen for rapamycin-resistant mutants, we identify Pib2 (PhosphoInositide-Binding 2) as a master regulator of TORC1. We describe two antagonistic TORC1-activating and -inhibiting activities located on opposite ends of Pib2. Thus, SATAY allows to easily explore the yeast genome at unprecedented resolution and throughput.
Highlights
Saccharomyces cerevisiae is an invaluable model for cell biology (Weissman, 2010)
We present a novel method based on random transposon insertion and next-generation sequencing, to functionally screen the genome of Saccharomyces cerevisiae
SAturated Transposon Analysis in Yeast (SATAY) can in principle be implemented in any strain background, since it does not rely on the existence of available deletion libraries and does not necessitate markers to follow genetic traits
Summary
Saccharomyces cerevisiae is an invaluable model for cell biology (Weissman, 2010). Despite the simplicity of its genome, its inner working mechanisms are similar to that of higher eukaryotes. Its ease of handling allows large-scale screenings. Yeast genetic screens have classically been performed by random mutagenesis, followed by a selection process that identifies interesting mutants. Elegant the ‘tricks’ implemented to expose the sought-after mutants, this selection phase remains a tedious process of finding a needle-in-a-haystack (Weissman, 2010). The selection phase can limit the throughput and the saturation of classical yeast genetic screens
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