The MET3 promoter: a new tool for Candida albicans molecular genetics.

A central technique used to investigate the role of a Candida albicans gene is to study the phenotype of a cell in which both copies of the gene have been deleted. To date, such investigations can only be undertaken if the gene is not essential. We describe the use of the Candida albicans MET3 promoter to express conditionally an essential gene, so that the consequences of depletion of the gene product may be investigated. The effects of environmental conditions on its expression were investigated, using GFP as a reporter gene. The promoter showed an approximately 85-fold range of expression, according to the presence or absence of either methionine or cysteine in concentrations in excess of 1 mM. In the presence of either amino acid, expression was reduced to levels that were close to background. We used URA3 as a model to demonstrate that the MET3 promoter could control the expression of an essential gene, provided that a mixture of both methionine and cysteine was used to repress the promoter. We describe an expression vector that may be used to express any gene under the control of the MET3 promoter and a vector that may be used to disrupt a gene and simultaneously place an intact copy under the control of the MET3 promoter. During the course of these experiments, we discovered that directed integration into the RP10 locus gives a high frequency of transformation, providing a means to solve a long-standing problem in this field.

The yeast PRS3 gene is required for cell integrity, cell cycle arrest upon nutrient deprivation, ion homeostasis and the proper organization of the actin cytoskeleton.

PRS3 is one of a family of five genes encoding phosphoribosylpyrophosphate synthetase, an enzyme which catalyses the first step in a variety of biosynthetic pathways, including purine and pyrimidine biosynthesis. We report here that prs3Delta mutants have a number of phenotypes that suggest an unexpected role for PRS3 in linking nutrient availability to cell cycle progression, cell integrity and the actin cytoskeleton. Upon nutrient limitation, prs3Delta mutants fail to arrest in G(1)-cells remain budded and a significant fraction have a G(2) DNA content. Furthermore, in such conditions, prs3Delta mutants have a disorganized actin cytoskeleton: actin accumulates in one or two intensely staining clumps per cell. Prs3Delta mutants also show defects in ion homeostasis and cell integrity. They fail to grow on medium containing 1.0 M NaCl, 5 mM caffeine or when incubated at 37 degrees C. The caffeine and temperature sensitivity are rescued by supplementing the growth medium with 1.0 M sorbitol. These phenotypes resemble those of whi2Delta mutations and indeed, a prs3 allele was recovered in a colony-sectoring screen for mutations that are co-lethal with whi2Delta. However, further investigation showed that the prs3Delta whi2Delta double mutant was viable, with no obvious growth defect compared to either single mutant. In the same colony-sectoring assay, an mpk1 allele was also recovered. Multicopy PRS3 rescued the caffeine sensitivity of this mpk1 allele.

Filamentous growth of the budding yeast Saccharomyces cerevisiae induced by overexpression of the WHi2 gene.

The WHI2 gene of the budding yeast Saccharomyces cerevisiae is required for the arrest of cell proliferation upon nutrient exhaustion: whi2 mutants carry on dividing and in the absence of growth become abnormally small. It is reported here that overexpression of Whi2 from the GAL1 promoter results in filamentous growth - cells fail to complete cytokinesis, the budding pattern changes from axial to polar, cells become elongated and cell size increases threefold. In many ways, these filaments resemble the pseudohyphae which result from nitrogen-limited growth and the filaments seen during the invasive growth of haploids. However, Whi2-induced filament formation is reduced, but not blocked, by mutations in STE7, STE12 or STE20 which do block pseudohypha formation. Furthermore, pseudohypha formation can still occur in a diploid in which both copies of the WHI2 gene have been deleted. Thus Whi2-induced filament formation and pseudohypha formation must come about through the action of different pathways. Despite this, a mutation in the STE11 gene, which is required for pseudohypha formation, does block Whi2-induced filament formation. Concanavalin A pulse-chase experiments show that new cell wall material is incorporated only into the tips of the apical cells. An extragenic suppressor of the Whi2 allele also results in filamentous growth. These results suggest that Whi2 negatively regulates a function required for the budding mode of cell proliferation including cytokinesis. This function is defined wholly or in part by the fsw1 allele.

Deregulation of CLN1 and CLN2 in the Saccharomyces cerevisiae whi2 mutant.

Wild-type cells of the budding yeast Saccharbmyces cerevisiae arrest in G1 upon nutrient exhaustion. Cell cycle arrest requires the WHI2 gene since whi2 mutants continue to divide and become abnormally small as nutrients are depleted. Here we show that CLN1 and CLN2 transcript levels in a whi2 strain are higher during exponential growth, and persist longer upon starvation, than in an isogenic wild-type strain. In contrast to CLN1 and CLN2, CLN3 levels declined only at very high cell density and were unaffected by the whi2 mutation. Elevated CLN expression is sufficient to explain the whi2 phenotype since ectopic expression of CLN1 in a nutrient-depleted culture caused cells to continue dividing and interfered with the acquisition of heat resistance. These observations show that, either directly or indirectly, Whi2 negatively regulates G1 cyclin expression. Interestingly extremely high levels of Cln1 induced filamentous growth upon nutrient deprivation, suggesting a direct connection between G1 cyclin activity and morphological responses to poor nutrient conditions.

Transcript characterisation, gene disruption and nucleotide sequence of the Saccharomyces cerevisiae WH12 gene.

WH12 is a gene which plays a prominent role in regulating growth and proliferation in Saccharomyces cerevisiae. It is expressed as a 2.0-kb mRNA transcript. Disruption of this transcript in a WH12+ cell results in the mutant phenotype being displayed. The nucleotide sequence of the cloned gene has been determined and found to include a 487-codon long open reading frame coding for a 55.3-kDa protein. The protein showed no extensive homologies to any previously identified protein. The 5' and 3' noncoding regions contained many of the features found in other yeast genes.

Defective metabolism of metoprolol in poor hydroxylators of debrisoquine.

Eight healthy volunteers received oral metoprolol 200 mg once daily for a week. The AUC, half-life and duration of beta-adrenoceptor blockade on day 7 was much greater in two subjects than in the remaining six. This suggested that the metabolism of metoprolol was impaired in two and the effect was therefore prolonged. Subsequent testing of oxidation phenotype with oral debrisoquine showed that the subjects with high metoprolol availability were also poor hydroxylators of debrisoquine. The urinary debrisoquine/4-hydroxydebrisoquine ratio was highly correlated with metoprolol AUC, half-life and beta-adrenoceptor blockade at 24 h. Thus patients with a genetic defect in drug oxidation, when treated with metoprolol, are likely to have high plasma concentrations and a prolonged effect.