Isolation of the yeast regulatory gene GAL4 and analysis of its dosage effects on the galactose/melibiose regulon.

Abstract

GAL4 is a classically defined positive regulatory gene controlling the 5 inducible structural genes of galactose/melibiose utilization in yeast. The positive regulatory function of the GAL4 gene product in turn is controlled by the product of another gene, the negative regulator GAL80. A 3.1-kilobase fragment containing GAL4 was cloned by homologus complementation using the multicopy chimeric vector YEp24 and it was demonstrated that multiple copies of GAL4 in yeast have pronounced dosage effects on the expression of the structural genes. Yeast transformed with GAL4-bearing plasmid become constitutive for expression of the galactose/melibiose genes, even in normally repressing (glucose) medium. Multiple copies of the GAL4 plasmid also increase expression of the structural genes in inducing (galactose) medium and can partially overcome the effects of a dominant super-repressor mutant, GAL80s. Using an internal deletion in GAL4, it was demonstrated that these dosage effects are due to overproduction of GAL4 positive regulatory product rather than an effect of the flanking sequences titrating out a negative regulator. These results point to the importance of competitive interplay between the positive and negative regulatory proteins in the control of this system. The dosage effect of GAL4 plasmid was also used in combination with different GAL4 and GAL80 alleles to create new phenotypes. These phenotypes are interpreted as indicating that the repressing effects of glucose, at least in part, are mediated by the product of the negative regulatory gene, GAL80, and the GAL80 protein may have specific interactions with the control regions of the structural genes.