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

GAL4 is a classically defined positive regulatory gene controlling the five 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. We have cloned a 3.1-kilobase fragment containing GAL4 by homologous complementation using the multicopy chimeric vector YEp24 and 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, we have 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. We have also used the dosage effect of GAL4 plasmid in combination with different GAL4 and GAL80 alleles to create new phenotypes. We interpret these phenotypes as indicating that (i) the repressing effects of glucose, at least in part, are mediated by the product of the negative regulatory gene, GAL80, and (ii) the GAL80 protein may have specific interactions with the control regions of the structural genes.     

Amino terminus of the yeast GAL4 gene product is sufficient for nuclear localization.

We have studied the intracellular compartmentalization in yeast of Escherichia coli beta-galactosidase bearing heterologous amino acid sequences at its amino terminus. Chimeras containing as few as 74 NH2-terminal amino acids of GAL4, a yeast positive regulatory protein, at the amino terminus accumulate in the cell nucleus. This and other results are consistent with the proposal that the GAL4 gene product mediates positive control by binding to DNA and that the information for nuclear localization resides in its amino terminus. The amino acid sequence of the GAL4 amino terminus does not agree with the previously proposed consensus sequences responsible for nuclear localization. The beta-galactosidase activity in cells bearing the non-nuclear chimeric proteins is 10-fold greater than in cells bearing chimeric proteins that specifically concentrate in the nucleus.