Functional analysis of some yeast genes

Abstract

A series of mutant strains of the yeast Saccharomyces cerevisiae that are sensitive to osmotic stress and also have a defect in vacuolar biogenesis have been isolated (M. Latterich, PhD Thesis 1992). The mutations that cause this pleiotropic phenotype are termed ssv, for salt sensitive vacuolar mutants. Complementation analysis has revealed that ssv mutations fall into one of 18 complementation groups. A MAP kinase related signal transduction pathway, termed the HOG pathway for High Osmolarity Glycerol, has been identified in yeast. This pathway senses osmotic stress and invokes the cellular response, one aspect of which is the accumulation of intracellular glycerol (Brewster et. al, 1993). Mutations in the HOG pathway often cause an osmosensitive phenotype similar to that shown by ssv mutations. This work sets out to characterise several ssv strains for defects in the HOG pathway. These strains were subjected to osmotic stress and the intracellular and extracellular glycerol determined and compared to control strains and conditions. Many of the strains showed reduced, or even elevated in one case, glycerol levels compared to wild-type strains. No correlation could be made between these glycerol levels and the activity of the rate-limiting enzyme, glycerol-3-phosphate dehydrogenase (GPDH) determined in an independent study. Transcription of the GPDH gene is under the control of the HOG pathway. In a separate study, the nucleotide sequence of a short region of yeast chromosome VII was determined. Approximately 11,000 bases of DNA from the right sub-telomeric region was sequenced. Analysis of the DNA sequence showed four potential open reading frames. One of these encoded the YORl gene and another a protein related to PAU1 The remaining two ORFs, termed ORFl and ORF2, encoded potential proteins of unknown function. Disruption cassettes containing the LEU2 selectable marker were constructed for both ORFl and ORF2. Successful disruption of ORFl was achieved, but no viable transformants were ever recovered after attempted disruption of 0RF2..ORFl gene knockouts are viable and show no observable phenotype under a range of growth conditions. Subsequent analysis of ORFl and 0RF2 after the completion of the Yeast Genome Project, shows that both ORFl and 0RF2 are members of different sub- telomeric associated gene families. 0RF2 encodes a putative Y' protein.