Combined use of two transcriptional reporters improves signalling assays receptors in fission yeast
Das, Anamika, Forfar, Rachel, Ladds, Graham and Davey, John. (2006) Combined use of two transcriptional reporters improves signalling assays receptors in fission yeast. YEAST, 23 (12). pp. 889-897. ISSN 0749-503XFull text not available from this repository.
Official URL: http://dx.doi.org/10.1002/yea.1402
The biochemical and genetic tractability of yeasts make them ideal hosts for the analysis of signalling from G protein-coupled receptors (GPCRs). Selected modifications to the strains allow the introduction of non-yeast components, while signal-dependent expression of reporter genes provides growth selection or enzyme read-out as assays for signalling. One issue with such systems is reporter expression in the absence of stimulation, usually because of spontaneous activation of intracellular signalling components and/or incomplete repression of the signal-dependent promoter. This limits the difference between reporter activity in the presence and absence of stimulation, often referred to as the signal: background ratio. In an effort to extend the applicability of the yeast system, we generated a Schizosaccharomyces pombe strain containing pheromone-dependent reporters for both growth selection and beta-galactosidase production. Simultaneous use of the two reporters provided several advantages over strains expressing only one reporter, particularly when coupled to the use of a competitive inhibitor of the nutritional reporter. For example, the beta-galactosidase signal: background ratio following stimulation with 10(-6) m P-factor increased from 35 for a strain containing a single lacZ reporter to almost 2500 for the double reporter. The sensitivity of the system was also improved, with higher signal: background ratios allowing detection of lower concentrations of beta-factor. Although we have used Sz. pombe and focused on GPCR-based induction of P-galactosidase, the principles described can be applied to other yeasts, different signalling pathways and alternative reporters. Copyright (c) 2006 John Wiley & Sons, Ltd.
|Item Type:||Journal Article|
|Subjects:||Q Science > QD Chemistry
T Technology > TP Chemical technology
Q Science > QR Microbiology
Q Science > QK Botany
|Journal or Publication Title:||YEAST|
|Publisher:||JOHN WILEY & SONS LTD|
|Number of Pages:||9|
|Page Range:||pp. 889-897|
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