We have suggested previously from Northern blot analysis that transcription of the negative regulatory gene GAL80 was controlled positively by another regulatory gene GALA, and negatively by GAL80 itself, in similar way to GAL1, GAL7 and GAL10 genes encoding galactosemetabolizing enzymes in Saccharomyces cerevisiae. To study further the controlled expression of GAL80, we have exploited the gene fusion technique. We constructed gene fusions consisting of 5′ fragments of GAL80 and a 5′ truncated lacZ of Escherichia coli, and introduced the GAL80'-'lacZ fusions into wild-type yeast or various GALA or GAL80 mutants using multiple-copy or single-copy plasmid vectors. We then studied β-galactosidase activity in the resultant transformants under uninduced, induced or glucose-repressed conditions. Expression of the GAL80'-'lacZ fusions was clearly under the control of Gal4/Gal80. Next we constructed GAL7'-'lacZ fusions, whose upstream activating sequence (UAS) from GAL7 was replaced with a GAL80 fragment containing a UAS-like sequence located in the 5′ flanking region of GAL80. Synthesis of β-galactosidase directed by the hybrid genes was inducible by galactose exactly like the original GAL7'-'lacZ fusion with a UAS from GAL7. Finally we constructed a GAL7-GAL80 hybrid gene, in which the entire 5′ flanking region was derived from GAL7. When the chromosomal GAL80 gene in wild-type yeast was replaced with the hybrid gene, the uninduced level, but not the induced level, of the GAL10-encoded enzyme (uridine diphosphoglucose-4-epimerase) was significantly increased.
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