New shuttle vectors for direct cloning in Saccharomyces cerevisiae.
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 The vectors are pBluescript-based shuttle vectors in which the lacZ marker has been replaced by an analogous system based on the Saccharomyces cerevisiae URA3 gene.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 reclone the ACE1 gene from genomic DNA by directly transforming a strain deleted for ACE1. 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.
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109.
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Thiele
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D005800:Genes, Fungal; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D010957:Plasmids; D015183:Restriction Mapping; D012190:Retroviridae / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*
D009963:Orotic Acid; C001242:5-fluoroorotic acid