Optimisation of the Schizosaccharomyces pombe urg1 Expression System

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 indu...

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Vydáno v:PloS one Ročník 8; číslo 12; s. e83800
Hlavní autoři: Watson, Adam T., Daigaku, Yasukazu, Mohebi, Saed, Etheridge, Thomas J., Chahwan, Charly, Murray, Johanne M., Carr, Antony M.
Médium: Journal Article
Jazyk:angličtina
Vydáno: United States Public Library of Science 20.12.2013
Public Library of Science (PLoS)
Témata:
Antibiotics
Arginine - pharmacology
Cell cycle
Control systems
Dynamic range
Endonucleases - metabolism
Gene expression
Gene Expression - drug effects
Genetic engineering
Genetic Engineering - methods
Genetic Loci - genetics
Genomes
Genomics
Hydroxyurea - pharmacology
In vivo methods and tests
Indoleacetic Acids - pharmacology
Life sciences
Molecular biology
mRNA Cleavage and Polyadenylation Factors - genetics
Mutation
Phenotype
Plasmids
Plasmids - genetics
Promoter Regions, Genetic - genetics
Proteins
Pyrimidines
Recombination
Replication
RNA Stability
RNA, Messenger - chemistry
RNA, Messenger - genetics
RNA, Messenger - metabolism
Schizosaccharomyces - drug effects
Schizosaccharomyces - genetics
Schizosaccharomyces pombe Proteins - genetics
Transcription
Transcription, Genetic - drug effects
Uracil
Yeast
ISSN:1932-6203, 1932-6203
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Shrnutí: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.
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Conceived and designed the experiments: AMC ATW JMM YD CC. Performed the experiments: ATW YD TJE SM. Analyzed the data: AMC ATW JMM. Wrote the paper: ATW AMC.
Competing Interests: The authors have declared that no competing interests exist.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0083800