QUANTITATIVE-ANALYSIS OF YEAST GENE-FUNCTION USING COMPETITION EXPERIMENTS IN CONTINUOUS-CULTURE

One possible route to the evaluation of gene function is a quantitativ e approach based on the concepts of metabolic control analysis (MCA). An important first step in such an analysis is to determine the effect of deleting individual genes on the growth rate (or fitness) of S. ce revisiae. Since the specific growth-rate effects of most genes are lik ely to be small, we employed competition experiments in chemostat cult ure to measure the proportion of deletion mutants relative to that of a standard strain by using a quantitative PCR method. In this paper, w e show that both densitometry and GeneScan(TM) analysis can be used wi th similar accuracy and reproducibility to determine the proportions o f (at least) two strains simultaneously, in the range 10-90% of the to tal cell population. Furthermore, we report on a model competition exp eriment between two diploid nuclear petite mutants, homozygous for del etions in the cox5a or pet191 genes, and the standard strain (ho::kanM X4/ho::kanMX4) in chemostat cultures under six different physiological conditions. The results indicate that competition experiments in cont inuous culture are a suitable method to distinguish quantitatively bet ween deletion mutants that qualitatively exhibit the same phenotype. ( C) 1998 John Wiley & Sons, Ltd.