Regulation of gene expression at the fission yeast Schizosaccharomyces pombe urg1 locus. 2011

Adam T Watson, and Petra Werler, and Antony M Carr
Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Sussex, BN1 9RQ , UK.

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 ~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.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D004720 Endonucleases Enzymes that catalyze the hydrolysis of the internal bonds and thereby the formation of polynucleotides or oligonucleotides from ribo- or deoxyribonucleotide chains. EC 3.1.-. Endonuclease
D005786 Gene Expression Regulation Any of the processes by which nuclear, cytoplasmic, or intercellular factors influence the differential control (induction or repression) of gene action at the level of transcription or translation. Gene Action Regulation,Regulation of Gene Expression,Expression Regulation, Gene,Regulation, Gene Action,Regulation, Gene Expression
D005800 Genes, Fungal The functional hereditary units of FUNGI. Fungal Genes,Fungal Gene,Gene, Fungal
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D012568 Schizosaccharomyces A genus of ascomycetous fungi of the family Schizosaccharomycetaceae, order Schizosaccharomycetales. Fission Yeast,Schizosaccharomyces malidevorans,Schizosaccharomyces pombe,Yeast, Fission,S pombe,Fission Yeasts

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