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      An expanded tool kit for the auxin-inducible degron system in budding yeast

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          Abstract

          Fusion of inducible degradation signals, so-called degrons, to cellular proteins is an elegant method of controlling protein levels in vivo. Recently, a degron system relying on the plant hormone auxin has been described for use in yeast and vertebrate cells. We now report the construction of a series of vectors that significantly enhance the versatility of this auxin-inducible degron (AID) system in Saccharomyces cerevisiae. We have minimized the size of the degron and appended a series of additional epitope tags, allowing detection by commercial antibodies or fluorescence microscopy. The vectors are compatible with PCR-based genomic tagging strategies, allow for C- or N-terminal fusion of the degron, and provide a range of selection markers. Application to a series of yeast proteins, including essential replication factors, provides evidence for a general usefulness of the system. © 2013 The Authors. Yeast published by John Wiley & Sons, Ltd.

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          Most cited references17

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          Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines.

          Epitope tagging of proteins as a strategy for the analysis of function, interactions and the subcellular distribution of proteins has become widely used. In the yeast Saccharomyces cerevisiae, molecular biological techniques have been developed that use a simple PCR-based strategy to introduce epitope tags to chromosomal loci (Wach et al., 1994). To further employ the power of this strategy, a variety of novel tags was constructed. These tags were combined with different selectable marker genes, resulting in PCR amplificable modules. Only one set of primers is required for the amplification of any module. Furthermore, convenient laboratory techniques are described that facilitate the genetic manipulations of yeast strains, as well as the analysis of the epitope-tagged proteins. Copyright 1999 John Wiley & Sons, Ltd.
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            Getting started with yeast.

            F Sherman (1991)
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              The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes.

              The anchor-away (AA) technique depletes the nucleus of Saccharomyces cerevisiae of a protein of interest (the target) by conditional tethering to an abundant cytoplasmic protein (the anchor) by appropriate gene tagging and rapamycin-dependent heterodimerization. Taking advantage of the massive flow of ribosomal proteins through the nucleus during maturation, a protein of the large subunit was chosen as the anchor. Addition of rapamycin, due to formation of the ternary complex, composed of the anchor, rapamycin, and the target, then results in the rapid depletion of the target from the nucleus. All 43 tested genes displayed on rapamycin plates the expected defective growth phenotype. In addition, when examined functionally, specific mutant phenotypes were obtained within minutes. These are genes involved in protein import, RNA export, transcription, sister chromatid cohesion, and gene silencing. The AA technique is a powerful tool for nuclear biology to dissect the function of individual or gene pairs in synthetic, lethal situations.
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                Author and article information

                Journal
                Yeast
                Yeast
                yea
                Yeast (Chichester, England)
                Blackwell Publishing Ltd (Oxford, UK )
                0749-503X
                1097-0061
                September 2013
                23 July 2013
                : 30
                : 9
                : 341-351
                Affiliations
                [1 ]Cancer Research UK London Research Institute, Clare Hall Laboratories South Mimms, UK
                [2 ]Institute of Molecular Biology Ackermannweg 4, 55128, Mainz, Germany
                Author notes
                *Correspondence to:H. D. Ulrich, Institute of Molecular Biology, Ackermannweg 4, 55128, Mainz, Germany., E-mail: h.ulrich@ 123456imb-mainz.de
                Article
                10.1002/yea.2967
                4171812
                23836714
                2c6446df-d814-4945-889a-c75c26b155a4
                Copyright © 2013 John Wiley & Sons, Ltd.

                This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

                History
                : 08 March 2013
                : 28 June 2013
                Categories
                Research Articles

                Molecular biology
                degron,auxin,budding yeast,protein degradation,protein stability
                Molecular biology
                degron, auxin, budding yeast, protein degradation, protein stability

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