| Title: | Combined use of two transcriptional reporters improves signalling assays for G protein-coupled receptors in fission yeast |
| Author(s): | Das A; Forfar R; Ladds G; Davey J; |
| Address: | "Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK" |
| ISSN/ISBN: | 0749-503X (Print) 0749-503X (Linking) |
| 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 P-factor. Although we have used Sz. pombe and focused on GPCR-based induction of beta-galactosidase, the principles described can be applied to other yeasts, different signalling pathways and alternative reporters" |
| Keywords: | "DNA, Fungal/chemistry/genetics Gene Expression Regulation, Fungal Genes, Reporter/genetics/*physiology Lac Operon/genetics/physiology Mutagenesis, Insertional Receptors, G-Protein-Coupled/*physiology Schizosaccharomyces/genetics/*physiology Schizosaccharo;" |
| Notes: | "MedlineDas, Anamika Forfar, Rachel Ladds, Graham Davey, John eng Research Support, Non-U.S. Gov't England 2006/09/27 Yeast. 2006 Sep; 23(12):889-97. doi: 10.1002/yea.1402" |
