Cloning and restriction mapping of the yeast URA2 gene coding for the carbamyl phosphate synthetase aspartate-transcarbamylase complex

Abstract

Two yeast DNA pools inserted in a hybrid Escherichia coli-yeast vector pFL1 were used to transform E. coli and yeast aspartate-transcarbamylase-less strains to prototrophy. From the first pool--a BamHI yeast DNA digest--a 6.4 kb BamHI fragment was recovered that gave good complementation of the E. coli auxotrophy but poor complementation of the yeast auxotrophy. From the second pool--a partial Sau3A yeast DNA digest--five independent plasmids complementing either E. coli, yeast, or both were recovered. Each of the five plasmids possessed sequences in common with the 6.4 kb BamHI fragment. One of these plasmids, which complemented the two URA2 activities in yeast and which produced a carbamyl-phosphate synthetase, aspartate-transcarbamylase complex sensitive to UTP feedback inhibition contained the full URA2 gene. A restriction map of the URA2 gene has been constructed and seven different consecutive segments have been recloned in pBR322 to measure their hybridization with URA2 messenger RNA, allowing us to estimate the limits of the gene.