IDENTIFICATION OF GENES WITH NUTRIENT-CONTROLLED EXPRESSION BY PCR-MAPPING IN THE YEAST SACCHAROMYCES-CEREVISIAE

We have used RNA fingerprinting by the mRNA Differential Display techn ique to identify new genes in the yeast Saccharomyces cerevisiae, expr ession of which is controlled by specific nutrient conditions. mRNA wa s isolated from cells grown on glucose medium into exponential and sta tionary phase, and from cells starved for nitrogen on glucose-containi ng medium. To avoid interference with the large number of glucose-repr essible genes, a glucose-repression-deficient strain was used. Twenty different sets of arbitrary primers chosen at random were used for PCR -amplification of reverse transcriptase generated cDNAs, which resulte d in six highly reproducible gene expression patterns. The validity of the approach was confirmed by sequencing PCR products of genes with k nown expression patterns, SUP44/RPS4, CTT1, SSA3, HSP30 and HSP104, an d genes with related functions, TEF1 and TEF3, encoding translation el ongation factors. In all cases the specificity of the responses was co nfirmed by Northern blot analysis. The results show that the PCR-mappi ng method is highly useful for the identification of new genes express ed under specific conditions in the yeast S. cerevisiae. (C) 1997 by J ohn Wiley & Sons, Ltd.