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

Indirect monitoring of TORC1 signalling pathway reveals molecular diversity among different yeast strains

“…Furthermore, constitutive activity of EGOC fails to suppress the TORC1 signalling defect under ammonium deprivation (Binda et al, 2009). Thus, there is an alternative mechanism of TORC1 activation independent of EGOC in which the participating proteins have not been fully determined (Chantranupong, Wolfson, & Sabatini, 2015;Gonzalez & Hall, 2017), with only suggested actors such as Pib2 protein (Kim & Cunningham, 2015;Michel et al, 2017;Tanigawa & Maeda, 2017;Ukai et al, 2018;Varlakhanova, Mihalevic, Bernstein, & Ford, 2017).…”

Identification of essential genes and fluconazole resistance genes inCandida glabrataby profiling ofHermestransposon insertions

“…2C, red). These findings suggest Hermes-NATr insertions in C. glabrata have little or no enhancer and promoter activities, which is similar to Hermes-NATr in S. cerevisiae (21) and distinct from mini-Ac/Ds that contains such activities (16).…”

“…Next-generation DNA sequencing technology facilitates en masse analyses of very large pools of insertion mutants (14)(15)(16). Typically, genomic DNA adjacent to each insertion site is directly amplified by PCR and sequenced using Illumina technology, and then the reads are mapped to precise sites in the genome and tabulated.…”

“…In contrast to CRISPR/Cas9 screening approaches that are user-guided and indirect, transposon insertion profiling enables direct observation and quantitation of each mutation in the population and full coverage of the genome. In S. cerevisiae, millions of independent insertions have been obtained using derivatives of the maize Activator/Dissociation (mini-Ac/Ds) transposon (16) and the housefly Hermes transposon (17).…”

Insertion Pool Sequencing for Insertional Mutant Analysis in Complex Host‐Microbe Interactions

“…The analysis of insertional mutant libraries is well established for bacteria (Gawronski et al, 2009;Goodman et al, 2009;Langridge et al, 2009;van Opijnen et al, 2009) but has not been applied extensively to eukaryotic microorganisms. Genome-wide insertional mutant libraries were generated successfully for baker's yeast (Saccharomyces cerevisiae) by homologous recombination (Winzeler et al, 1999) and, more recently, by transposition (Michel et al, 2017) and for the rice pathogenic fungus Magnaporthe oryzae by the kinase ATM (Jeon et al, 2007). However, tools that allow for efficient highthroughput analysis of a negative depletion screen in the context of a host, for instance in the case of M. oryzae and rice (Jeon et al, 2007), were not available until recently.…”