Cloning and transcriptional control of a eucaryotic permease gene

The uracil permease gene of the yeast Saccharomyces cerevisiae was cloned on a hybrid plasmid which replicates autonomously in both yeast and Escherichia coli. Cloning was carried out by complementation in yeast. The smallest DNA fragment found to complement the uracil permease deficiency in recipient yeast cells measured approximately 2.3 kilobases. In strains transformed by the plasmid with the uracil permease gene inserted, initial rates of uracil uptake increased up to 25 times more than the rates found in the wild type. Using DNA probes carrying several regions of the cloned gene, I showed that a strain carrying the dhul-I mutation, which is not linked to the permease structural gene and is responsible for enhanced uptake velocity of uracil, had enhanced transcription of the permease gene. By using DNA probes recloned in phage M13 mp7, the direction of transcription of the permease gene relative to the restriction map was deduced. A half-life of 2 min was found for the permease mRNA in labeling kinetics experiments.