Isolation of the structural gene for alcohol dehydrogenase by genetic complementation in yeast

Abstract

Alcohol dehydrogenase (ADH) catalyses the interconversion of acetaldehyde and ethanol. This reaction is the final step during alcoholic fermentation in yeast and the initial step in the metabolism of ethanol in a wide variety of organisms. The regulation of ADH is now being studied in several organisms, notably yeast1–4, Drosophila5, maize6 and man7,8. These organisms contain multiple ADH isozymes which are differentially expressed. The budding yeast Saccharomyces cerevisiae is particularly suitable for study because of the potential for genetic analysis of enzyme regulation and structure. Yeast possesses three distinct ADH isozymes2. In the presence of glucose ADHI (the ‘classical’ yeast ADH) is expressed whereas ADHII is repressed. A third enzyme, mADH, is associated with mitochondria. Mutants lacking each of these enzymes have been isolated by virtue of their greater tolerance to allyl alcohol1, which is converted by ADH to the toxin, acrolein. There is no evidence as to whether the regulation of any one of these enzymes is under transcriptional control. Availability of recombinant DNA clones containing the respective genes would be of obvious value in resolving this problem. We describe here the use of the newly developed technique of yeast transformation9–11 to isolate recombinant plasmids that contain the structural gene for ADHI (ADC1). These plasmids were selected from a pool of plasmids containing yeast DNA by virtue of their ability to complement the growth defect of yeast strains completely lacking ADH activity. Using DNA sequencing, they have been shown to contain the structural gene for ADHI.

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