Recombination-Induced tag exchange (RITE) cassette series to monitor protein dynamics in Saccharomyces cerevisiae
| Autor: | Fred van Leeuwen, Marit Terweij, Sjoerd J. van Deventer, Victoria Menendez-Benito, Tibor van Welsem, Pedro A. San-Segundo, Kitty F. Verzijlbergen, Jacques Neefjes, David Ontoso |
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| Přispěvatelé: | Netherlands Genomics Initiative, Netherlands Organization for Scientific Research |
| Rok vydání: | 2013 |
| Předmět: |
Chromatin Immunoprecipitation
Rite Protein turnover Saccharomyces cerevisiae Proteins Genetic Vectors Saccharomyces cerevisiae Protein inheritance ComputingMilieux_LEGALASPECTSOFCOMPUTING Computational biology Investigations Histones 03 medical and health sciences Genetics Epitope tag Gene Knock-In Techniques Molecular Biology Genetics (clinical) 030304 developmental biology Recombination Genetic 0303 health sciences Integrases biology Protein dynamics 030302 biochemistry & molecular biology Inheritance (genetic algorithm) biology.organism_classification 3. Good health Histone biology.protein Pulse-chase Chromatin immunoprecipitation Function (biology) |
| Zdroj: | Digital.CSIC. Repositorio Institucional del CSIC instname G3: Genes|Genomes|Genetics |
| Popis: | This is an open-access article distributed under the terms of the Creative Commons Attribution Unported License. Proteins are not static entities. They are highly mobile and their steady state levels are achieved by a balance between ongoing synthesis and degradation. The dynamic properties of a protein can have important consequences for its function. For example, when a protein is degraded and replaced by a newly synthesized one, post-translational modifications are lost and need to be reincorporated in the new molecules. Protein stability and mobility are also relevant for duplication of macromolecular structures or organelles, which involves coordination of protein inheritance with the synthesis and assembly of newly synthesized proteins. To measure protein dynamics we recently developed a genetic pulse-chase assay called Recombination-Induced Tag Exchange (RITE). RITE has been successfully used in Saccharomyces cerevisiae to measure turnover and inheritance of histone proteins, to study changes in post-translational modifications on aging proteins, and to visualize the spatiotemporal inheritance of protein complexes and organelles in dividing cells. Here we describe a series of successful RITE cassettes that are designed for biochemical analyses, genomics studies, as well as single cell fluorescence applications. Importantly, the genetic nature and the stability of the tag-switch offer the unique possibility to combine RITE with high-throughput screening for protein dynamics mutants and mechanisms. The RITE cassettes are widely applicable, modular by design, and can therefore be easily adapted for use in other cell types or organisms. This project was sponsored by the Netherlands Genomics Initiative and by The Netherlands Organization for Scientific Research. |
| Databáze: | OpenAIRE |
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