An expanded tool kit for the auxin‐inducible degron system in budding yeast
Reads0
Chats0
TLDR
The construction of a series of vectors is reported that significantly enhance the versatility of this auxin‐inducible degron (AID) system in Saccharomyces cerevisiae and provides evidence for a general usefulness of the system.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.read more
Citations
More filters
Posted ContentDOI
Rim aperture of autophagic membranes balances cargo inclusion with vesicle maturation
Oren Shatz,Milana Fraiberg,Alexandra Polyansky,Eyal Shimoni,Tali Dadosh,Sharon G. Wolf,Zvulun Elazar +6 more
TL;DR: In this article , an obligate complex of Atg24/Snx4 with Atg20 or Snx41 assembles locally at the rim in a spatially-extended manner that specifically depends on autophagic PI(3)P. This assembly stabilizes the open rim to promote autoophagic sequestration of large cargo in correlation with vesicle inflation.
Posted ContentDOI
Mps1 expression is critical for chromosome orientation and segregation in yeast with high ploidy
TL;DR: It is reported that cells with elevated chromosome number are particularly sensitive to the reduction of Mps1 level, and this phenomenon may be relevant in understanding why hyperdiploid cancer cells become excessively reliant on high MPS1 expression for successful chromosome segregation.
Journal ArticleDOI
Development of AlissAID system targeting GFP or mCherry fusion protein
TL;DR: In this paper , an Affinity-linker based super-sensitive auxin-inducible degron (AlissAID) system in budding yeast by using a single domain antibody (a nanobody).
Posted ContentDOI
Rapid, efficient auxin-inducible protein degradation in Candida pathogens
Kedric L. Milholland,Justin B. Gregor,Smriti Hoda,Encarnación Dueñas-Santero,Bao G. Vu,Krishna Patel,W. Scott Moye-Rowley,Carlos R. Vázquez de Aldana,Jaime Correa-Bordes,Scott D. Briggs,Mark C. Hall +10 more
TL;DR: In this article , the authors developed a collection of plasmids that support AID system use in laboratory strains of these pathogens and demonstrated that the original AID and the second generation AID2 systems work efficiently and rapidly in the human pathogenic yeasts Candida albicans and Candida glabrata.
Posted ContentDOI
CTCF and transcription influence chromatin structure re-configuration after mitosis
Haoyue Zhang,Jessica Lam,Di Zhang,Marit W. Vermunt,Yemin Lan,Cheryl A. Keller,Belinda Giardine,Ross C. Hardison,Gerd A. Blobel +8 more
TL;DR: In this article, the authors exploited the M-to-G1-phase progression to interrogate the contributions of the architectural factor CTCF and the process of transcription to re-sculpting the genome in newborn nuclei.
References
More filters
Journal ArticleDOI
Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae
Mark S. Longtine,Amos Mckenzie,Douglas J. Demarini,Nirav Shah,Achim Wach,Arndt Brachat,Peter Philippsen,John R. Pringle +7 more
TL;DR: A new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications that should further facilitate the rapid analysis of gene function in S. cerevisiae.
Book ChapterDOI
Getting started with yeast.
TL;DR: The yeast Saccharomyces cerevisiae is now recognized as a model system representing a simple eukaryote whose genome can be easily manipulated and made particularly accessible to gene cloning and genetic engineering techniques.
Journal ArticleDOI
An auxin-based degron system for the rapid depletion of proteins in nonplant cells
TL;DR: The auxin-inducible degron (AID) system allowed rapid and reversible degradation of target proteins in response to auxin and enabled us to generate efficient conditional mutants of essential proteins in yeast as well as cell lines derived from chicken, mouse, hamster, monkey and human cells, thus offering a powerful tool to control protein expression and study protein function.
Journal ArticleDOI
Epitope tagging of yeast genes using a PCR‐based strategy: more tags and improved practical routines
Michael Knop,Katja Siegers,Gislene Pereira,Wolfgang Zachariae,Barbara Winsor,Kim Nasmyth,Elmar Schiebel +6 more
TL;DR: 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 to produce PCR amplificable modules.
Journal ArticleDOI
Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation
Philipp Stelter,Helle D. Ulrich +1 more
TL;DR: These findings assign a function to SUMO during S phase and demonstrate how ubiquitin and SUMO, by regulating the accuracy of replication and repair, contribute to overall genomic stability.