USE OF POLYMERASE CHAIN-REACTION EPITOPE TAGGING FOR PROTEIN TAGGING IN SACCHAROMYCES-CEREVISIAE

Epitope tagging is the insertion of a short stretch of amino acids con stituting an epitope into another protein. Tagged proteins can be iden tified by Western, immunoprecipitation and immunofluorescence assays u sing pre-existing antibodies. We have designed vectors containing the URA3 gene flanked by direct repeats of epitope tags. We use the polyme rase chain reaction (PCR) to amplify the tag-URA3-tag cassette such th at the ends of the PCR fragments possess homology to the gene of inter est. In vivo recombination is then used to direct integration of the f ragment to the location of interest, and transformants are selected by their Ura(+) phenotype. Finally, selection for Ura(-) cells on 5-fluo ro-orotic acid plates yields cells where recombination between the rep eated epitopes has 'popped out' the URA3 gene, leaving a single copy o f the epitope at the desired location. PCR epitope tagging (PET) provi des a rapid and direct technique for tagging that does not require any cloning steps. We have used PET to tag three Saccharomyces cerevisiae proteins, Cln1, Sic1 and Est1.