Methods paperRegulation of gene expression at the fission yeast Schizosaccharomyces pombe urg1 locus
Introduction
The fission yeast Schizosaccharomyces pombe is a single celled eukaryote commonly used as a model organism for the study of a wide variety of basic cellular processes. S. pombe has a number of features which distinguish it from the alternative, and more widely used, single-celled eukaryotic model, Saccharomyces cerevisiae. These include the conservation of several pathways present in vertebrates that have been lost (or are significantly diverged) in S. cerevisiae and a more representative regulation of progress through the cell cycle (Nurse, 2000). However, the tools that have been developed in S. pombe are generally less advanced than those developed for S. cerevisiae. One example of this, which has lead to significant limitations in the exploitation of fission yeast for basic research, is the lack of method to rapidly induce transcription of a chosen gene from a low to a high level. Such a tool is highly desirable for the analysis of gene function within a single cell cycle and also permits the rapid induction of a chosen exogenous protein to alter or manipulate cell state.
The most widely used transcriptional induction system in S. pombe is based on the promoter of the nmt1 gene (Maundrell, 1993). Pnmt1 responds to the lack of exogenous thiamine and is induced approximately 75-fold when thiamine is removed from the growth media. Pnmt1 can be integrated ectopically to drive transcription of any gene of interest or can be used on an episome. In both scenarios, Pnmt1 maintains a similar dynamic range and low off-state transcription to that seen for endogenous Pnmt1. Both a low off-state level of transcription and a broad dynamic range are essential features for the control of biological activity. Modifications of the TATA box of Pnmt1 have been made and characterised (Basi et al., 1993) which simultaneously reduce both off-state and on-state transcription, thus maintaining the dynamic range. These variants, known as Pnmt4 and Pnmt8, have proved extremely useful since they allow researchers to choose the desired level of expression. However, the main disadvantage of the Pnmt series is that the induction of transcription requires 14–16 hours and occurs relatively asynchronously following the removal of thiamine from the growth medium.
To overcome the slow induction kinetics and asynchrony of Pnmt1, several alternative promoter systems have been developed. The glucose-repressed inv1 promoter is rapidly induced by a shift from glucose to sucrose-based culture medium (Iacovoni et al., 1999), but suffers from a relatively high off-state level of transcription and is also induced significantly as cells approach saturation. The regulation of the CaMV promoter by tetracycline (Forsburg, 1993) provides an alternative approach with relatively low off-state transcription, but lacks significant dynamic range.
In a search for genes regulated by simple media manipulations, transcriptome analysis identified urg1. The urg1 transcript is rapidly induced from levels undetectable by microarray analysis to high levels upon the addition of uracil to the medium (Watt et al., 2008). Uracil addition regulated only a few transcripts in S. pombe and the urg1 gene, while of unknown function, is inessential for growth and its loss does not unduly perturb known cellular functions. However, despite these advantages, use of Purg1 has proved disappointing because when the promoter fragment is transferred to a plasmid or placed ectopically in the genome to regulate a gene of interest, its off-state transcription is significantly elevated and the dynamic range consequently reduced from several hundred fold to approximately 10 (unpublished data). In this study we attempt to overcome this limitation by replacing the endogenous urg1 open reading frame (ORF) with alternative ORFs, thus maintaining Purg1 at its native locus. We demonstrate that several different genes can be rapidly induced with an excellent dynamic range and, as proof of principle, characterise the production of a single double-strand break in the S. pombe genome via the induction of the HO-endonuclease. To facilitate rapid and simple manipulation of urg1 locus, we have developed a Cre recombinase and lox recombination site-based recombination-mediated cassette exchange (RCME) system.
Section snippets
Strains and growth conditions
The media composition was as described by Moreno et al. (1991). For selection of G418, hygromycin (HPH) and nourseothricin (NAT) resistant cells, G418 disulsuphate (Melford), hygromycin B (Melford) and nourseothricin-dihydrogen sulphate (Melford) were added to YEA plates at a final concentration of 200ug/ml, 200ug/ml and 100ug/ml respectively. EMM media was supplemented with amino acids leucine (L), uracil (U) and histidine (H) at 100ug/ml as required. Cell pre-cultures for Purg1 induction
Adaptation of an S. pombe recombinase-mediated cassette exchange (RMCE) system for the insertion of gene sequences at the urg1 locus
The Cre-lox RMCE system described by Watson et al.(2008) is an effective tool to easily and quickly insert gene sequences at a defined S. pombe genomic locus. We chose to adapt this system to insert sequences at the urg1 locus. This involved the construction of an urg1 ‘base strain’ where a genetic marker, flanked by the two incompatible lox sites, loxP and loxM3, replaces sequences chosen within the urg1 locus. This genetic marker can then be exchanged for any plasmid-borne sequence flanked by
Discussion
In this report we describe a set of tools that allow the facile replacement of the urg1 ORF of fission yeast with any gene of interest. Our characterisation of the induction kinetics of the endogenous urg1 and the heterologous HO-endonuclease genes inserted into the urg1 locus using this system demonstrates that the induction kinetics are largely conserved. This offers, for the first time in S. pombe, a rapid method to regulate transcription that will allow biological experiments which have, to
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