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Combined use of two transcriptional reporters improves signalling assays receptors in fission yeast
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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.
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Official URL: http://dx.doi.org/10.1002/yea.1402
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 | ||||
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| Subjects: | Q Science > QD Chemistry T Technology > TP Chemical technology Q Science > QR Microbiology Q Science > QK Botany |
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| Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) > Biological Sciences ( -2010) Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School > Health Sciences Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School |
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| Journal or Publication Title: | Yeast | ||||
| Publisher: | John Wiley & Sons Ltd. | ||||
| ISSN: | 0749-503X | ||||
| Official Date: | September 2006 | ||||
| Dates: |
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| Volume: | Volume 23 | ||||
| Number: | Number 12 | ||||
| Number of Pages: | 9 | ||||
| Page Range: | pp. 889-897 | ||||
| DOI: | 10.1002/yea.1402 | ||||
| Status: | Peer Reviewed | ||||
| Publication Status: | Published |
Data sourced from Thomson Reuters' Web of Knowledge
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