RRC ID 34917
Author Tanaka S, Miyazawa-Onami M, Iida T, Araki H.
Title iAID: an improved auxin-inducible degron system for the construction of a 'tight' conditional mutant in the budding yeast Saccharomyces cerevisiae.
Journal Yeast
Abstract Isolation of a 'tight' conditional mutant of a gene of interest is an effective way of studying the functions of essential genes. Strategies that use ubiquitin-mediated protein degradation to eliminate the product of a gene of interest, such as heat-inducible degron (td) and auxin-inducible degron (AID), are powerful methods for constructing conditional mutants. However, these methods do not work with some genes. Here, we describe an improved AID system (iAID) for isolating tight conditional mutants in the budding yeast Saccharomyces cerevisiae. In this method, transcriptional repression by the 'Tet-OFF' promoter is combined with proteolytic elimination of the target protein by the AID system. To provide examples, we describe the construction of tight mutants of the replication factors Dpb11 and Mcm10, dpb11-iAID, and mcm10-iAID. Because Dpb11 and Mcm10 are required for the initiation of DNA replication, their tight mutants are unable to enter S phase. This is the case for dpb11-iAID and mcm10-iAID cells after the addition of tetracycline and auxin. Both the 'Tet-OFF' promoter and the AID system have been shown to work in model eukaryotes other than budding yeast. Therefore, the iAID system is not only useful in budding yeast, but also can be applied to other model systems to isolate tight conditional mutants.
Volume 32(8)
Pages 567-81
Published 2015-8-1
DOI 10.1002/yea.3080
PMID 26081484
MeSH Gene Targeting / methods* Indoleacetic Acids / pharmacology* Mutagenesis Mutation* Promoter Regions, Genetic Saccharomyces cerevisiae / drug effects Saccharomyces cerevisiae / genetics* Saccharomyces cerevisiae Proteins / genetics*
IF 3.143
Times Cited 14
Yeast BY28260 BY28261 BY28262 BY28263 BY28264 BY28265 BY28266 BY28267 BY28268 BY28269 BY28270 BY28271 BY28272 BY28273 BY28274 BY28275 BY28276 BY28277 BY28278 BY28279 BY28280 BY28281 BY28282 BY28283 BY28284 BY28285 BY28286 BY28287 BY28288 BY28289 BY28290 BY28291 BY28292 BY28293 BY28294 BY28295