APPLICATION OF MESSENGER-RNA DIFFERENTIAL DISPLAY TO INVESTIGATE GENE-EXPRESSION IN THERMOTOLERANT CELLS OF SACCHAROMYCES-CEREVISIAE

We have described the use of differential display of PCR-amplified rev erse transcribed mRNA (DDRT-PCR) to survey changes in gene expression profiles induced by heat shock and carbon catabolite derepression in S accharomyces cerevisiae. It is well established that either of these s tates elicits thermotolerant phenotypes. An initial analysis conducted on cells of an inherently thermosensitive strain (Ysen) indicated tha t approximately 10% of the total number of cDNAs detected were either up or down regulated following heat shock at 37 degrees C (30 min) in comparison to control cells (25 degrees C). In addition, whereas 7% of all PCR products were preferentially expressed during derepressive gr owth, approximately 2% were found to be common to both heat-shocked an d derepressed cells. A repeat analysis, performed on all three cell ty pes of Ysen as well as cells of a relatively thermoresistant strain (Y res) yielded 30 differentially displayed cDNA fragments common to heat -shocked and derepressed cells of both strains. Eighteen of these gene rated signals on Northern blots, of which three were confirmed as regu lated. Five amplicons, including one not detected by Northern analysis and another from the derepressed state, were cloned and sequenced. Th ree of these exhibited homology to S. cerevisiae genes with well-chara cterized protein products: HSP 90, HXK1 and STA1. The remaining two ap plicons showed nucleotide identity to YTIS11, a homolog of the mammali an TIS11 and putative transcriptional activator, and an orphan gene en coding a hypothetical transmembrane protein belonging to the multi-dru g resistance translocase family. Our novel application of DDRT-PCR has identified new and known genes that may be further evaluated as facto rs involved in stress regulation and has demonstrated the potential of the technique to systematically analyse gene expression in yeast. (C) 1998 John Wiley & Sons, Ltd.