We experimentally validated these three predictions. The GEV system can achieve at most a 10-fold increase in protein expression from a single inducible allele [11]. We created haploid GEV yeast strains carrying high-copy 2 μ plasmids with a GEV-inducible allele (P GAL1-GENE) of a gene of interest, which allowed us to test the model prediction that massive overexpression of Ssk1, but not of Ypd1, should lead to inappropriate HOG pathway activation. These high-copy yeast plasmids are estimated to be present at 15-50 copies per cell [25-28].