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 technique to identify new genes in the yeast Saccharomyces cerevisiae, expression of which is controlled by specific nutrient conditions. mRNA was isolated from cells grown on glucose medium into exponential and stationary phase, and from cells starved for nitrogen on glucose-containing medium. To avoid interference with the large number of glucose-repressible 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 resulted in six highly reproducible gene expression patterns. The validity of the approach was confirmed by sequencing PCR products of genes with known expression patterns, SUP44/RPS4, CTT1, SSA3, HSP30 and HSP104, and genes with related functions, TEF1 and TEF3, encoding translation elongation factors. In all cases the specificity of the responses was confirmed by Northern blot analysis. The results show that the PCR-mapping method is highly useful for the identification of new genes expressed under specific conditions in the yeast S. cerevisiae.
Crauwels
M
M
Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven, Leuven-Heverlee, Flanders, Belgium.
Winderickx
J
J
de Winde
J H
JH
Thevelein
J M
JM
D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D012333:RNA, Messenger