Lactose or galactose induces the expression of the lactose-galactose regulon in Kluyveromyces lactis. We show here that the regulon is not induced in strains defective in L4C9. We demonstrate that this gene codes for a regulatory protein that acts in a positive manner to induce transcription. The LAC9 gene was isolated by complementation of a lac9 defective strain. DNA sequence analysis of the gene gave a deduced protein of 865 amino acids. Comparison of this sequence with that of the GAL4 protein of Saccharomyces cerevisiae revealed three regions of homology. One region of about 90 amino acid occurs at the amino terminus, which is known to mediate binding of GAL4 protein to upstream activator sequences. We speculate that a portion of this region, adjacent to the "metal-binding finger," specffies DNA binding. We discuss possible functions of the two other regions of homology. The functional implicatiodis of these structural similarities were examined. When LAC9 was introduced into a gal4 defective strain of S. cerevisiae it complemented the mutation and activated the galactose-melibiose regulon. However, LAC9 did not simply mimic GAL4. Unlike normal S. cerevisiae carrying GAL4, the strain carrying LAC9 gave constitutive expression of GAL) and MEL], two genes in the regulon. The strain did show glucose repression of the regulon, but repression was less severe with LAC9 than with GAL4. We discuss the implications of these results and how they may facilitate our understanding of the LAC9 and GAL4 regulatory proteins.Elucidation of genetic regulatory systems has relied heavily on the isolation of mutations in regulatory and structural genes. We are utilizing this approach to detetmine how lactose or galactose induces the lactose-galactose regulon in the yeast Kluyveromyces lactis. The regulon contains five known structural genes whose products are: LAC4, P-galactosidase (EC 3.2.1.23) (44); GAL], galactokinase (EC 2.7.1.6) (37); GAL7, galactose-1-phosphate uridyltransferase (EC 2.7.7.10) (37); GALIO, uridine diphosphoglucose-4-epimerase (EC 5.1.3.2) (37); and LAC12, a lactose permease (45). 3-Galactosidase activity can be induced over 100-fold above a moderate basal level (12). Increased enzyme activity results from increased transcription of the ,B-galactosidase structural gene, indicating that induction is regulated at the level of transcription (26). Mutations in LACIO cause constitutive expression of the regulon, suggesting that the gene functions in a negative fashion to regulate transcription (14).To further understand how lactose induces gene expression we characterized in more detail our previously isolated Lac-mutants (43). We found that mutants defective in lac9 are uninducible for all the enzymes of the lactose-galactose regulon. These and other data indicate that LAC9 is a positive regulatory gene that acts in trans to control transcription of target genes. This gene is the first positive-acting regulatory function that has been identified for the lactosegalactose regulon of K. lactis.We previously noted ...