Development of a semi-quantitative plate-based α-galactosidase gene reporter for Schizosaccharomyces pombe and its use to isolate a constitutively active Mam2

Goddard, Alan and Ladds, Graham and Davey, John (2005) Development of a semi-quantitative plate-based α-galactosidase gene reporter for Schizosaccharomyces pombe and its use to isolate a constitutively active Mam2. Yeast, 22 (1). pp. 31-41. ISSN 0749-503X

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Official URL: http://dx.doi.org/10.1002/yea.1190

Abstract

To extend the tools available for biochemical and genetical analysis in the fission yeast Schizosaccharomyces pombe we have investigated the development of gene reporter systems using the secreted α-galactosidase encoded by the Sz. pombe ORF SPAC869.07c (CAB60017, which we propose naming Mel1p to reflect its structural and functional similarity to MEL1p in Saccharomyces cerevisiae. The α-galactosidase activity can be monitored in liquid assays and converted the colourless substrate 5-bromo-4-chloro-3-indolyl-α -D-galactopyranoside (X-α-gal) into an insoluble blue product that was suitable for semi quantitative plate-based assays; colonies expressing the highest levels of α-galactosidase developed the most intense blue colour. Unlike assays based on β-galactosidase, the Sz. pombe colonies develop the blue colouration under normal growth conditions, avoiding the need to replicate colonies to fresh plates for analysis. It is therefore suitable for screening large numbers of colonies. To illustrate the use of mel1 as a reporter we linked expression to the sxa2 gene promoter to provide a convenient readout for signalling through the pheromone response pathway. The sxa2>mel1 strain identified constitutively active Mam2 pheromone receptors from a randomly mutagenised library. There was an approximate correlation between the intensity of the blue colour developed by each mutant colony and its level of constitutive activity and we identified a subset of mutants with low constitutive activity that could not have been isolated by a previous screen using nutritional selection. The mel1 α -galactosidase activity identified and characterised in this study can be easily adapted to provide a gene reporter for many biological processes and is a new addition to the research tools available in Sz. pombe.

Item Type:Article
Additional Information:To extend the tools available for biochemical and genetical analysis in the fission yeast Schizosaccharomyces pombe we have investigated the development of gene reporter systems using the secreted α-galactosidase encoded by the Sz. pombe ORF SPAC869.07c (CAB60017, which we propose naming Mel1p to reflect its structural and functional similarity to MEL1p in Saccharomyces cerevisiae. The α-galactosidase activity can be monitored in liquid assays and converted the colourless substrate 5-bromo-4-chloro-3-indolyl-α -D-galactopyranoside (X-α-gal) into an insoluble blue product that was suitable for semi quantitative plate-based assays; colonies expressing the highest levels of α-galactosidase developed the most intense blue colour. Unlike assays based on β-galactosidase, the Sz. pombe colonies develop the blue colouration under normal growth conditions, avoiding the need to replicate colonies to fresh plates for analysis. It is therefore suitable for screening large numbers of colonies. To illustrate the use of mel1 as a reporter we linked expression to the sxa2 gene promoter to provide a convenient readout for signalling through the pheromone response pathway. The sxa2>mel1 strain identified constitutively active Mam2 pheromone receptors from a randomly mutagenised library. There was an approximate correlation between the intensity of the blue colour developed by each mutant colony and its level of constitutive activity and we identified a subset of mutants with low constitutive activity that could not have been isolated by a previous screen using nutritional selection. The mel1 α -galactosidase activity identified and characterised in this study can be easily adapted to provide a gene reporter for many biological processes and is a new addition to the research tools available in Sz. pombe.
Keywords:α-galactosidase, Gene reporter, mel1, Plate assay, Sz. pombe
Subjects:C Biological Sciences > C710 Applied Molecular Biology, Biophysics and Biochemistry
Divisions:College of Science > School of Life Sciences
ID Code:6825
Deposited By: Alan Goddard
Deposited On:15 Nov 2012 16:18
Last Modified:15 Nov 2012 16:18

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