Cloning of a eukaryotic regulatory gene

Abstract

From a pool of hybrid plasmids carrying Sau3A fragments representing the entire yeast (S. cerevisiae) genome, a DNA fragment containing the regulatory gene PPR1 was cloned by complementation of a non-inducible ppr1 mutation which confers to the cells an increased sensitivity to 6-azauracil. Cells containing the cloned DNA regained the ability to induce the synthesis of URA1 and URA3 gene products controlled by PPR1. A physical map has been constructed and the study of subcloned restriction endonuclease fragments from the original yeast DNA fragment allowed us to localize the wild-type PPR1 regulatory gene within a 3 kilobase-pair region. The ppr1 RNA level was measured and the hybridization data indicate in a wild-type strain a low efficiency of transcription of PPR1 as compared to the structural URA3 gene, without effect of inducing conditions.