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 0749-503X

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Abstract

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
ISSN:	0749-503X
Date:	September 2006
Volume:	23
Number:	12
Number of Pages:	9
Page Range:	pp. 889-897
Identification Number:	10.1002/yea.1402
Publication Status:	Published
URI:	http://wrap.warwick.ac.uk/id/eprint/32794

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