Unknown,Transcriptomics,Genomics,Proteomics

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Rapid High-Resolution Functional Mapping of Yeast Genomes by Saturated Transposition

ABSTRACT: The yeast Saccharomyces cerevisiae is a highly powerful model for systems genetics. While the advent of ordered deletion libraries has considerably facilitated yeast screening, it has also narrowed the diversity of screenable variants and precluded the exploration of the non-coding genome. Here, we present a versatile, time- and work-efficient method to functionally explore the yeast genome at an unprecedented throughput and resolution, using saturated transposon mutagenesis coupled to high-throughput sequencing. SAturated Transposon Analysis in Yeast (SATAY) allows the one-step mapping of all genetic loci in which a transposon can be inserted without disrupting a function necessary for cell growth. SATAY is especially suited to discover loci important for growth in various conditions. Here, we demonstrate that SATAY can be used to (2) reveal genetic interactions in single and multiple mutant strains, (1) reveal drug-resistant and -sensitive mutants, (3) detect not only essential genes, but also essential functional protein domains, and (4) generate not only null alleles, but also other informative genetic variants. Thus SATAY allows to easily explore the yeast genome at an unprecedented resolution and throughput.

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Benoit Kornmann

PROVIDER: E-MTAB-4885 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Lipid intermediates derived from sphingolipid metabolism are crucial regulators of mitochondrial function from yeast to humans. Among these intermediates, trans-2-hexadecenal (t-2hex) within the sphingolipid degradation pathway exhibits remarkable efficiency in inducing mitochondria-mediated cell death. In yeast cell cultures, the addition of t-2-hex triggers complete disintegration of the mitochondrial network, leading to subsequent cell death. This effect is particularly pronounced in yeast cells lacking the activity of the t-2-hex degrading enzyme, Hfd1. However, the molecular mechanisms of t-2-hex induction of mitochondrial dysfunction are completely unknown. In this project, we want to exploit the unprecedented power of yeast genetics to unveil novel genetic determinants involved in t-2-hex's pro-apoptotic function. To accomplish this, we employed the SATAY method, which combines saturated transposon mutagenesis with high-throughput sequencing to functionally explore the yeast genome. In our screening approach, hfd1 mutant cells harboring a plasmid-encoded inducible MiniDs transposon were induced by galactose, resulting in extensive integration of the transposon throughout the yeast genome. Cells with the plasmid excised and the transposon genomically integrated were pooled together, creating a high-density transposon library comprising approximately 2.3E+06 independent insertion mutants. Subsequently, the pooled mutant library was subjected to treatment with the mitochondria-mediated death inducer, t-2-hexadecenal. As a control, cells were also incubated with the solvent dimethyl sulfoxide (DMSO), in which hexadecenal is dissolved. Following the treatments, cells were collected for genomic DNA extraction and digestion, using restriction enzymes with frequent four-base pair recognition sites. The resulting library fragments were circularized using T4 DNA ligase, and the transposon-genome junctions were selectively amplified through PCR with outward-facing primers specific to the transposon. Finally, the pooled and purified amplicons were subjected to massive sequencing on an Illumina MiSeq platform. The obtained sequences were then aligned to the reference genome of Saccharomyces cerevisiae, allowing for the mapping of transposon insertions and the calculation of transposon counts per gene. This project enabled the identification of genes required for the resistance and toxicity to t-2hex.

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Rapid High-Resolution Functional Mapping of Yeast Genomes by Saturated Transposition

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