title: Conditional Budding Yeast Mutants with Temperature-Sensitive and Auxin-Inducible Degrons for Screening of Suppressor Genes.
abstract: The conditional control of protein expression is useful to characterize the function of proteins, especially of those that are essential for cell viability. Two degron-based systems, temperature-sensitive and auxin-inducible degrons, can be used to generate conditional mutants of budding yeast, simply by transforming appropriate cells with PCR-amplified DNA. We describe a protocol for the generation of temperature-sensitive and auxin-inducible degron mutants. We also show that a conditional mutant with few spontaneous revertants was generated by combining two degron systems for the Inn1 protein. Finally, we describe a suppressor screening method that uses the dual degron-Inn1 mutant to identify mutant proteins that suppress Inn1-K31A, which has a defect in cytokinesis.
authors.affiliation: Department of Molecular Biology and Genetics, Canik Basari University, Gürgenyatak Köyü, Samsun, 55080, Turkey. asli.devrekanli@gmail.com.
authors.forename: Asli
authors.initials: A
authors.lastname: Devrekanli
authors.affiliation: Center of Frontier Research, National Institute of Genetics, Research Organization of Information and Systems, SOKENDAI, Yata 1111, Mishima, Shizuoka, 411-8540, Japan. mkanemak@nig.ac.jp.|Department of Genetics, SOKENDAI, Mishima, Shizuoka, Japan. mkanemak@nig.ac.jp.|JST, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan. mkanemak@nig.ac.jp.
authors.forename: Masato T
authors.initials: MT
authors.lastname: Kanemaki
mesh_terms: D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects*; D015723:Gene Library; D005800:Genes, Fungal; D016153:Genes, Suppressor; D007210:Indoleacetic Acids / Q000494:pharmacology*; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D004718:Saccharomycetales / Q000235:genetics*; D013696:Temperature; D014170:Transformation, Genetic
chemical_list: D007210:Indoleacetic Acids
keywords: Auxin-inducible degron; Conditional mutant; Inn1; Protein degradation; Suppressor screen ing; Temperature-sensitive degron