SUITABILITY OF REPLACEMENT MARKERS FOR FUNCTIONAL-ANALYSIS STUDIES INSACCHAROMYCES-CEREVISIAE

The complete yeast sequence contains a large proportion of genes whose biological function is completely unknown. One approach to elucidatin g the function of these novel genes is by quantitative methods that ex ploit the concepts of metabolic control analysis. An important first s tep in such an analysis is to determine the effects of deleting indivi dual genes on the growth rate (or fitness) of Saccharomyces cerevisiae . Since the specific growth-rate effects of most genes are likely to b e small, they are most readily determined by competition against a sta ndard strain in chemostat cultures where the true steady state demande d by metabolic control analysis may be achieved. We have constructed t wo different standard strains in which the HO gene is replaced by eith er HIS3 or kanMX. We demonstrate that HO is a selectively neutral site for gene replacement. However, there is a significant marker effect a ssociated with HIS3 which, moreover, is dependent on the physiological conditions used for the competition experiments. In contrast, the kan MX marker exhibited only a small effect on specific growth rate (less than or equal to +/- 4%). These data suggest that nutritional markers should not be used to generate deletion mutants for the quantitative a nalysis of gene function in yeast but that kanMX replacements may be u sed, with confidence, for such studies. (C) 1997 John Wiley & Sons, Lt d.