Quantitative analysis of yeast gene function using competition experiments in continuous culture.
One possible route to the evaluation of gene function is a quantitative 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. cerevisiae. Since the specific growth-rate effects of most genes are likely to be small, we employed competition experiments in chemostat culture to measure the proportion of deletion mutants relative to that of a standard strain by using a quantitative PCR method. In this paper, we show that both densitometry and GeneScan analysis can be used with similar accuracy and reproducibility to determine the proportions of (at least) two strains simultaneously, in the range 10-90% of the total cell population. Furthermore, we report on a model competition experiment between two diploid nuclear petite mutants, homozygous for deletions in the cox5a or pet191 genes, and the standard strain (ho::kanMX4/ho::kanMX4) in chemostat cultures under six different physiological conditions. The results indicate that competition experiments is continuous culture are a suitable method to distinguish quantitatively between deletion mutants that qualitatively exhibit the same phenotype.
Baganz
F
F
Department of Biomolecular Sciences, UMIST, Manchester, U.K.
Hayes
A
A
Farquhar
R
R
Butler
P R
PR
Gardner
D C
DC
Oliver
S G
SG
D003470:Culture Media; D004271:DNA, Fungal / Q000032:analysis; D003720:Densitometry; D017353:Gene Deletion; D005800:Genes, Fungal / Q000502:physiology*; D009154:Mutation; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods; D012984:Software
D003470:Culture Media; D004271:DNA, Fungal