The Saccharomyces cereivisiae gene deletion project revealed that approximately 20% of yeast genes are required for viability. The analysis of essential genes traditionally relies on conditional mutants, typically temperature-sensitive (ts) alleles. We developed a systematic approach (termed "diploid shuffle") useful for generating a ts allele for each essential gene in S. cerevisiae and for improved genetic manipulation of mutant alleles and gene constructs in general. Importantly, each ts allele resides at its normal genomic locus, flanked by specific cognate UPTAG and DNTAG bar codes. A subset of 250 ts mutants, including ts alleles for all uncharacterized essential genes and prioritized for genes with human counterparts, is now ready for distribution. The importance of this collection is demonstrated by biochemical and genetic screens that reveal essential genes involved in RNA processing and maintenance of chromosomal stability.
Bibliographical noteFunding Information:
We thank A. Carter, M. Tao, M. Kofoed, B. Le, and L. Tang for technical assistance, M. Kupiec, K. McManus, G. Simchen, and X. Pan for helpful discussions; and J. Woolford for helpful comments on an earlier version of the manuscript. Supported in part by National Institutes of Health (NIH) grant P01 CA16519 to P.H. and J.D.B., Canadian Institute for Health Research (CIHR) grant MOP-38096 to P.H., and NIH roadmap grant U54 RR020839 (to J.D.B.). S.B.-A. was supported by an Human Frontier Science Program (HFSP) long-term fellowship and by a research grant from the Killam trust.