In Saccharomyces cerevisiae, utilization of galactose requires four inducible enzyme activities. Three of these activities (galactose-1-phosphate uridyl transferase, EC 2.7.7.10; uridine diphosphogalactose 4-epimerase, EC 5.1.3.2; and galactokinase, EC 2.7.1.6) are specified by three tightly linked genes (GAL7, GAL10, and GAL1, respectively) on chromosome II, whereas the fourth, galactose transport, is specified by a gene (GAL2) located on chromosome XII. Although classic genetic analysis has revealed both positive and negative regulatory genes that coordinately affect the appearance of all four enzyme activities, neither the basic events leading to the appearance of enzyme activities nor the roles of the regulatory genes have yet been determined. Regulation of inducible enzyme activity could be mediated by events related to transcription, translation, or enzyme activation. For the purpose of studying galactose pathway induction and its regulation, we have developed an immunoprecipitation assay that enables us to detect the GAL7 specified uridyl transferase polypeptide in yeast extracts and among the polypeptides synthesized in an RNA-dependent in vitro translation system. Use of this immunoprecipitation assay in conjunction with in vivo labeling experiments demonstrates the presence of [(3)H]leucine-labeled transferase in extracts prepared from cells grown in galactose but not from cells grown in glucose. This galactose-specific induction of transferase polypeptide is mediated by the de novo appearance of a functional mRNA species whose synthetic capacity is detectable by the combination of in vitro translation and immunoprecipitation. The appearance of functional transferase mRNA depends on wild-type expression of the positive regulatory gene, GAL4. Cells carrying a nonsense (amber) mutation in the GAL4 gene fail to produce the transferase mRNA, whereas a nonsense suppressor of the GAL4 amber mutant regains the galactose-specific mRNA response. Our results establish that the induction of the GAL7 specified uridyl transferase activity is mediated by de novo appearance of a functional mRNA and that this galactose-specific response is dependent on a wild-type GAL4 gene product.

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Journal Article | Research Support, U.S. Gov't, P.H.S.

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Hopper JE, Broach JR, Rowe LB

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