| Title: |
Combined use of two transcriptional reporters improves signalling assays receptors in fission yeast |
| Authors: |
Das, Anamika; Forfar, Rachel; Ladds, Graham; Davey, John |
| Publisher Information: |
John Wiley & Sons Ltd. |
| Publication Year: |
2006 |
| Collection: |
The University of Warwick: WRAP - Warwick Research Archive Portal |
| Subject Terms: |
QD Chemistry; TP Chemical technology; QR Microbiology; QK Botany |
| Description: |
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. |
| Document Type: |
article in journal/newspaper |
| Language: |
unknown |
| ISSN: |
0749-503X |
| Relation: |
Das, Anamika, Forfar, Rachel, Ladds, Graham and Davey, John (2006) Combined use of two transcriptional reporters improves signalling assays receptors in fission yeast. Yeast, Volume 23 (Number 12). pp. 889-897. doi:10.1002/yea.1402 ISSN 0749-503X. |
| DOI: |
10.1002/yea.1402 |
| Availability: |
https://wrap.warwick.ac.uk/id/eprint/32794/; https://doi.org/10.1002/yea.1402 |
| Accession Number: |
edsbas.4182DA73 |
| Database: |
BASE |