New shuttle vectors for direct cloning in Saccharomyces cerevisiae

Abstract

We have constructed new shuttle vectors to facilitate the screening of recombinant plasmids after direct transformation of yeast cells. The vectors are pBluescript-based shuttle vectors in which the lacZ marker has been replaced by an analogous system based on the Saccharomyces cerevisiae URA3 gene. DNA fragments are inserted in a Polylinker located after the beginning of the URA3 coding sequence. Transformants are selected either by Trp or Leu prototrophy. Plasmids bearing an insert are selected by growth on 5-fluoro-orotic acid (5-FOA), a uracil analog toxic to cells containing a functional URA3 + gene (thus, this method requires the recipient strain to be ura3 -); only cells containing a plasmid with an insert that disrupts the functional continuity of the URA3 gene can grow on medium containing 5-FOA. Using these plasmids, we were able to directly redone the ACE 1 gene from genomic DNA by directly transforming a strain deleted for ACE 1. These vectors can be used for a variety of purposes including rapid cloning of genes by complementation or expression of fusion genes driven from the URA3 promoter.