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[摘要]：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. |
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