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dc.contributor.authorSanchez-Diaz, Alberto
dc.contributor.authorKanemaki, Masato
dc.contributor.authorMarchesi, Vanessa
dc.contributor.authorLabib, Karim
dc.date.accessioned2009-10-19T15:36:38Z
dc.date.available2009-10-19T15:36:38Z
dc.date.issued2004-03-09
dc.identifier.citationRapid depletion of budding yeast proteins by fusion to a heat-inducible degron. 2004, 2004 (223):PL8 Sci. STKEen
dc.identifier.issn1525-8882
dc.identifier.pmid15010550
dc.identifier.doi10.1126/stke.2232004pl8
dc.identifier.urihttp://hdl.handle.net/10541/84408
dc.description.abstractOne effective way to study the biological function of a protein in vivo is to inactivate it and see what happens to the cell. For proteins that are dispensable for cell viability, the corresponding gene can simply be deleted from its chromosomal locus. The study of essential proteins is more challenging, however, because the function of the protein must be inactivated conditionally. Here, we describe a method that allows the target protein to be depleted rapidly and conditionally, so that the immediate effects on the cell can be examined. The chromosomal locus of a budding yeast gene is modified so that a "heat-inducible degron cassette" is added to the N terminus of the encoded protein, causing it to be degraded by a specific ubiquitin-mediated pathway when cells are shifted from 24 degrees to 37 degrees C. Degradation requires recognition of the degron cassette by the evolutionarily conserved Ubr1 protein, which is associated with a ubiquitin-conjugating enzyme. To promote rapid and conditional depletion of the target protein, we use a yeast strain in which expression of the UBR1 gene can be either repressed or strongly induced. Degron strains are constructed by a simple "one-step" approach using the polymerase chain reaction.
dc.language.isoenen
dc.subject.meshBase Sequence
dc.subject.meshCell Survival
dc.subject.meshChromosome Mapping
dc.subject.meshChromosomes, Fungal
dc.subject.meshDNA, Fungal
dc.subject.meshGene Expression Regulation, Fungal
dc.subject.meshGenes, Essential
dc.subject.meshGenes, Fungal
dc.subject.meshHaploidy
dc.subject.meshHot Temperature
dc.subject.meshMolecular Sequence Data
dc.subject.meshMutagenesis, Insertional
dc.subject.meshNucleic Acid Amplification Techniques
dc.subject.meshOligonucleotides
dc.subject.meshPolymerase Chain Reaction
dc.subject.meshProtein Processing, Post-Translational
dc.subject.meshRecombinant Fusion Proteins
dc.subject.meshSaccharomyces cerevisiae
dc.subject.meshSaccharomyces cerevisiae Proteins
dc.subject.meshTemperature
dc.subject.meshTransformation, Genetic
dc.titleRapid depletion of budding yeast proteins by fusion to a heat-inducible degron.en
dc.contributor.departmentCancer Research U.K., Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK.en
dc.identifier.journalScience's STKEen
html.description.abstractOne effective way to study the biological function of a protein in vivo is to inactivate it and see what happens to the cell. For proteins that are dispensable for cell viability, the corresponding gene can simply be deleted from its chromosomal locus. The study of essential proteins is more challenging, however, because the function of the protein must be inactivated conditionally. Here, we describe a method that allows the target protein to be depleted rapidly and conditionally, so that the immediate effects on the cell can be examined. The chromosomal locus of a budding yeast gene is modified so that a "heat-inducible degron cassette" is added to the N terminus of the encoded protein, causing it to be degraded by a specific ubiquitin-mediated pathway when cells are shifted from 24 degrees to 37 degrees C. Degradation requires recognition of the degron cassette by the evolutionarily conserved Ubr1 protein, which is associated with a ubiquitin-conjugating enzyme. To promote rapid and conditional depletion of the target protein, we use a yeast strain in which expression of the UBR1 gene can be either repressed or strongly induced. Degron strains are constructed by a simple "one-step" approach using the polymerase chain reaction.


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