A rapid method to determine the stress status of Saccharomyces cerevisiae by monitoring the expression of a Hsp12:green fluorescent protein (GFP) construct under the control of the Hsp12 promoter.
The gene for the green fluorescent protein (GFP) was fused in-frame to the 3' end of HSP12. This construct was regulated by the HSP12 promoter in a pYES2 yeast expression vector. No fluorescence was observed in yeast growing exponentially in glucose-containing medium, but fluorescence was observed when the yeast entered the stationary phase. Fluorescence microscopy indicated that the fusion protein was localized to the peripheral regions of the cell as well as to the cytoplasm and the tonoplast. Subjecting the yeast to a variety of stresses known to induce HSP12 transcription, including salt, osmotic, ethanol, and heat stress, resulted in a time-dependent increase in GFP fluorescence. The use of this system as a method to assess the general stress status of yeast growing in an industrial application is proposed.
Karreman
Robert J
RJ
Department of Molecular and Cellular Biology, University of Cape Town, Rondebosch 7701, South Africa.
Lindsey
George G
GG
D001483:Base Sequence; D015966:Gene Expression Regulation, Fungal; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D006360:Heat-Shock Proteins / Q000032:analysis / Q000235:genetics*; D007218:Industrial Microbiology / Q000379:methods*; D008969:Molecular Sequence Data; D009997:Osmotic Pressure; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000235:genetics*; D012965:Sodium Chloride / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
C112591:HSP12 protein, S cerevisiae; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012965:Sodium Chloride