Watt, S; Mata, J; Lopez-Maury, L; Marguerat, S; Burns, G; Bahler, J; (2008) urg1: A Uracil-Regulatable Promoter System for Fission Yeast with Short Induction and Repression Times. PLOS ONE , 3 (1) , Article e1428. 10.1371/journal.pone.0001428.

Preview

PDF 76078.pdf Download (442kB)

Abstract

Background. The fission yeast Schizosaccharomyces pombe is a popular genetic model organism with powerful experimental tools. The thiamine-regulatable nmt1 promoter and derivatives, which take > 15 hours for full induction, are most commonly used for controlled expression of ectopic genes. Given the short cell cycle of fission yeast, however, a promoter system that can be rapidly regulated, similar to the GAL system for budding yeast, would provide a key advantage for many experiments. Methodology/Principal Findings. We used S. pombe microarrays to identify three neighbouring genes (urg1, urg2, and urg3) whose transcript levels rapidly and strongly increased in response to uracil, a condition which otherwise had little effect on global gene expression. We cloned the promoter of urg1(uracil-regulatable gene) to create several PCR-based gene targeting modules for replacing native promoters with the urg1 promoter (P urg1) in the normal chromosomal locations of genes of interest. The kanMX6 and natMX6 markers allow selection under urg1 induced and repressed conditions, respectively. Some modules also allow N-terminal tagging of gene products placed under urg1 control. Using pom1 as a proof-of-principle, we observed a maximal increase of P urg1-pom1 transcripts after uracil addition within less than 30 minutes, and a similarly rapid decrease after uracil removal. The induced and repressed transcriptional states remained stable over 24-hour periods. RT-PCR comparisons showed that both induced and repressed P urg1- pom1 transcript levels were lower than corresponding P3 nmt1-pom1 levels (wild-type nmt1 promoter) but higher than P81 nmt1-pom1 levels (weak nmt1 derivative). Conclusions/Significance. We exploited the urg1 promoter system to rapidly induce pom1 expression at defined cell-cycle stages, showing that ectopic pom1 expression leads to cell branching in G2-phase but much less so in G1-phase. The high temporal resolution provided by the urg1 promoter should facilitate experimental design and improve the genetic toolbox for the fission yeast community.

Download activity - last month

Export as XMLCSVJSON

Download activity - last 12 months

Export as CSVJSONXML

Downloads by country - last 12 months

Export as CSVJSONXML

Archive Staff Only

View Item