Development of a semi-quantitative plate-based alpha-galactosidase gene reporter for Schizosaccharomyces pombe and its use to isolate a constitutively active Mam2.
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 alpha-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 alpha-galactosidase activity can be monitored in liquid assays and converted the colourless substrate 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside (X-alpha-gal) into an insoluble blue product that was suitable for semi quantitative plate-based assays; colonies expressing the highest levels of alpha-galactosidase developed the most intense blue colour. Unlike assays based on beta-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 alpha-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.
Goddard
Alan
A
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. adgoddard@bio.warwick.ac.uk
Ladds
Graham
G
Davey
John
J
D000595:Amino Acid Sequence; D005656:Fungal Proteins; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D010675:Pheromones / Q000494:pharmacology*; D043563:Receptors, Pheromone / Q000302:isolation & purification*; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000302:isolation & purification*; D015398:Signal Transduction; D014157:Transcription Factors / Q000494:pharmacology*; D000519:alpha-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D005656:Fungal Proteins; D010675:Pheromones; D043563:Receptors, Pheromone; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; C085847:map2 protein, S pombe; D000519:alpha-Galactosidase