Exploring redundancy in the yeast genome: an improved strategy for use of the cre-loxP system

Gene families having more than three members are a common phenomenon in the Saccharomyces cerevisiae genome. As yeast research enters the post-genome era, the development of existing deletion strategies is crucial for tacklin g this apparent redundancy, hence a method for performing rapid multiple ge ne disruptions in this organism has been developed. We constructed three re placement cassettes in which different selectable markers were placed betwe en two loxP loci. Multiple deletions (of members of a gene family) were gen erated, in one strain, using sequential integration of different replacemen t markers (kanMX, LYS2, KlURA3 and SpHIS5). Their excision from the genome was performed simultaneously, as the final step, using a new cre recombinas e vector, which carries the cycloheximide-resistance gene from Candida malt osa as a selectable marker. Our multiple gene deletion system significantly accelerates and facilitates the functional analysis process and is particu larly useful for studying gene families in either laboratory or industrial yeast strains. (C) 2000 Elsevier Science B.V. All rights reserved.