LARGE-SCALE PHENOTYPIC ANALYSIS - THE PILOT PROJECT ON YEAST CHROMOSOME-III

In 1993, a pilot project for the functional analysis of newly discover ed open reading frames, presumably coding for proteins, from yeast chr omosome III was launched by the European Community. In the frame of th is programme, we have developed a large-scale screening for the identi fication of gene/protein functions via systematic phenotypic analysis. To this end, some 80 haploid mutant yeast strains were constructed, e ach carrying a targeted deletion of a single gene obtained by HIS3 or TRP1 transplacement in the W303 background and a panel of some 100 gro wth conditions was established, ranging from growth substrates, stress to, predominantly, specific inhibitors and drugs acting on various ce llular processes. Furthermore, co-segregation of the targeted deletion and the observed phenotype(s) in meiotic products has been verified. The experimental procedure, using microtiter plates for phenotypic ana lysis of yeast mutants, can be applied on a large scale, either on sol id or in liquid media. Since the minimal working unit of one 96-well m icrotiter plate allows the simultaneous analysis of at least 60 mutant strains, hundreds of strains can be handled in parallel. The high num ber of monotropic and pleiotropic phenotypes (62%) obtained, together with the acquired practical experience, have shown this approach to be simple, inexpensive and reproducible. It provides a useful tool for t he yeast community for the systematic search of biochemical and physio logical functions of unknown genes accounting for about a half of the 6000 genes of the complete yeast genome. (C) 1997 John Wiley & Sons, L td.