title: Optimisation of the Schizosaccharomyces pombe urg1 expression system.
abstract: The ability to study protein function in vivo often relies on systems that regulate the presence and absence of the protein of interest. Two limitations for previously described transcriptional control systems that are used to regulate protein expression in fission yeast are: the time taken for inducing conditions to initiate transcription and the ability to achieve very low basal transcription in the "OFF-state". In previous work, we described a Cre recombination-mediated system that allows the rapid and efficient regulation of any gene of interest by the urg1 promoter, which has a dynamic range of approximately 75-fold and which is induced within 30-60 minutes of uracil addition. In this report we describe easy-to-use and versatile modules that can be exploited to significantly tune down Purg1 "OFF-levels" while maintaining an equivalent dynamic range. We also provide plasmids and tools for combining Purg1 transcriptional control with the auxin degron tag to help maintain a null-like phenotype. We demonstrate the utility of this system by improved regulation of HO-dependent site-specific DSB formation, by the regulation Rtf1-dependent replication fork arrest and by controlling Rhp18(Rad18)-dependent post replication repair.
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, East Sussex, United Kingdom.
authors.forename: Adam T
authors.initials: AT
authors.lastname: Watson
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, East Sussex, United Kingdom.
authors.forename: Yasukazu
authors.initials: Y
authors.lastname: Daigaku
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, East Sussex, United Kingdom.
authors.forename: Saed
authors.initials: S
authors.lastname: Mohebi
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, East Sussex, United Kingdom.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Etheridge
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, East Sussex, United Kingdom.
authors.forename: Charly
authors.initials: C
authors.lastname: Chahwan
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, East Sussex, United Kingdom.
authors.forename: Johanne M
authors.initials: JM
authors.lastname: Murray
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, East Sussex, United Kingdom.
authors.forename: Antony M
authors.initials: AM
authors.lastname: Carr
mesh_terms: D001120:Arginine / Q000494:pharmacology; D004720:Endonucleases / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D005818:Genetic Engineering / Q000379:methods*; D056426:Genetic Loci / Q000235:genetics; D006918:Hydroxyurea / Q000494:pharmacology; D007210:Indoleacetic Acids / Q000494:pharmacology; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000187:drug effects / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D039221:mRNA Cleavage and Polyadenylation Factors / Q000235:genetics
chemical_list: D007210:Indoleacetic Acids; C512176:Mmi1 protein, S pombe; D012333:RNA, Messenger; D029702:Schizosaccharomyces pombe Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; D001120:Arginine; D004720:Endonucleases; D006918:Hydroxyurea