Combined use of two transcriptional reporters improves signalling assays for G protein-coupled receptors in fission yeast.
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.
Das
Anamika
A
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
Forfar
Rachel
R
Ladds
Graham
G
Davey
John
J
D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter / Q000235:genetics / Q000502:physiology*; D007763:Lac Operon / Q000235:genetics / Q000502:physiology; D016254:Mutagenesis, Insertional; D043562:Receptors, G-Protein-Coupled / Q000502:physiology*; D012568:Schizosaccharomyces / Q000235:genetics / Q000502:physiology*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology; D014498:Uracil / Q000031:analogs & derivatives / Q000502:physiology
D004271:DNA, Fungal; C085846:P-factor, S pombe; D043562:Receptors, G-Protein-Coupled; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; C400810:ura4 protein, S pombe; D014498:Uracil; C100248:azauracil