Functional mapping of yeast genomes by saturated transposition

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.

DOI: http://dx.doi.org/10.7554/eLife.23570.001

eLife digest

Genes are stretches of DNA that carry the instructions to build and maintain cells. Many studies in genetics involve inactivating one or more genes and observing the consequences. If the loss of a gene kills the cell, that gene is likely to be vital for life. If it does not, the gene may not be essential, or a similar gene may be able to take over its role.

Baker’s yeast is a simple organism that shares many characteristics with human cells. Many yeast genes have a counterpart among human genes, and so studying baker’s yeast can reveal clues about our own genetics. Michel et al. report an adaptation for baker’s yeast of a technique called “Transposon sequencing”, which had been used in other single-celled organisms to study the effects of interrupting genes. Briefly, a virus-like piece of DNA, called a transposon, inserts randomly into the genetic material and switches off individual genes. The DNA is then sequenced to reveal every gene that can be disrupted without killing the cell, and remaining genes are inferred to be essential for life.

The approach, named SATAY (which is short for “saturated transposon analysis in yeast”), uses this strategy to create millions of baker’s yeast cells, each with a different gene switched off. Because the number of cells generated this way vastly exceeds the number of genes, every gene will be switched off by several independent transposons. Therefore the technique allows all yeast genes to be inactivated several times in one single experiment. The cells can be grown in varying conditions during the experiment, revealing the genes needed for survival in different situations. Non-essential genes can also be inactivated beforehand to uncover if any genes might be compensating for their absence.

In the future, this technique may be used to better understand human diseases, such as cancer, since many disease-causing genes in humans have counterparts in yeast.

DOI: http://dx.doi.org/10.7554/eLife.23570.002