Analysis of a set of stationary phase genes in Saccharomyces cerevisiae.

This thesis describes the characterisation of a set of six genes of the baker's yeast Saccharomyces cerevisiae, that are induced when a molasses-grown culture reaches the end of rapid fermentative growth. The rationale for the isolation of such genes was that their promoters may be useful in a biotechnological application, where the regulated expression of heterologous proteins is required. Additionally, their analysis should provide information about how yeast cells adapt to changing environmental situations. Therefore the aims of this project were to identify the unknown members of this set of genes and to determine the transcriptional regulatory mechanisms acting on all six. Six cDNA clones, which showed differential hybridisation to mRNAs of exponential and stationary phase cells, were used as probes to recover the cognate genes from a library of yeast genomic DNA fragments carried in the bacteriophage vector, EMBL3. Two had been identified as the previously described genes, HSP26 and HXKl encoding the 26kDa heat shock protein and the glucose-repressible hexokinase, respectively. A further two, previously undescribed genes, encode a second small, 12kDa, stress-induced protein (HSP12) and a thiamine biosynthetic enzyme (THI4). DNA sequence analysis carried out in this study has characterised the two remaining genes as HXT4=LGT1, a low affinity hexose transporter and a new thiamine regulated gene, THI5. Transcription of these six genes has been examined under a variety of growth conditions and in various mutant backgrounds. The two heat-shock genes, HSP12 and HSP26, are subject to glucose control via a cAMP-dependent protein kinase and become induced when the cells experience stress or starvation; presumably they have a role in maintaining the integrity of the cell under these conditions. The genes involved in hexose metabolism, HXKl and HXT4, are regulated by glucose repression through the TUP1/CYC8/SNF1 pathway and are derepressed by exhaustion of glucose from the growth medium; their products enable the cell to utilise alternative hexoses as growth substrates. Both of the thiamine genes, THI4 and THI5, are subject to repression by exogenous thiamine and become derepressed on depletion of the vitamin from the growth medium. Thus, although these genes show apparent coordinate induction they are regulated via different transcriptional control mechanisms. DNA sequence analysis revealed the THI5 gene to encode a second thiamine biosynthetic enzyme homologous to the product of the Schizosaccharomyces pombe gene nmtl. THI5 is present within the yeast genome in multiple copies located on chromosomes VI (THI5), X (THI11) and XIV (THI12). Evidence is presented to show that all three copies potentially express a functional product. Likely mechanisms by which the multiple copies of this gene have arisen and subsequent conclusions about the evolution of the yeast genome are discussed.