Regulation of gene expression at the fission yeast Schizosaccharomyces pombe urg1 locus

The lack of a rapid and efficient system to regulate transcriptional induction in the fission yeast Schizosaccharomyces pombe is currently a limitation of this model eukaryote. The commonly used nmt1 promoter has excellent dynamic range and a low "off-state" transcription, but takes 14-16 hours to induce upon thiamine withdrawal. Conversely, other induction systems have rapid response times, but suffer from a limited dynamic range and/or relatively high levels of off-state transcription. Recently, the urg1 gene was identified as a rapidly induced transcript, responding to uracil addition in similar to 30 min and exhibiting low off-state transcription and high dynamic range. However, attempts to reproduce this ectopically result in a significant increase in off-state transcription, severely limiting utility. To overcome this, we have adapted the Cre/lox recombination-mediated cassette exchange (RCME) system to facilitate easy insertion of sequences at the urg1 locus. We show that the P-urg1 induction kinetics are maintained when ectopic open reading frames (ORFs) replace the native urg1 ORF. As proof of principle, we characterise HO-endonuclease expression in cells harbouring a novel S. pombe single-strand annealing (SSA) assay. After 60 min induction we observe clear double-strand breaks, demonstrate >90% of cells are committed to SSA and show that the Rad22(Rad52) repair protein associates robustly with sequences adjacent to the DSB. This inducible system will be a valuable tool for future studies in S. pombe. (C) 2011 Elsevier B.V. All rights reserved.