Chapter title |
Conditional Budding Yeast Mutants with Temperature-Sensitive and Auxin-Inducible Degrons for Screening of Suppressor Genes.
|
---|---|
Chapter number | 18 |
Book title |
Yeast Cytokinesis
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3145-3_18 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3144-6, 978-1-4939-3145-3
|
Authors |
Devrekanli, Asli, Kanemaki, Masato T, Asli Devrekanli, Masato T. Kanemaki |
Editors |
Alberto Sanchez-Diaz, Pilar Perez |
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. |
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