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

Abstract 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 tackling this apparent redundancy, hence a method for performing rapid multiple gene disruptions in this organism has been developed. We constructed three replacement cassettes in which different selectable markers were placed between two lox P loci. Multiple deletions (of members of a gene family) were generated, in one strain, using sequential integration of different replacement markers ( kan MX, LYS2 , KlURA3 and SpHIS5 ). Their excision from the genome was performed simultaneously, as the final step, using a new cre recombinase vector, which carries the cycloheximide-resistance gene from Candida maltosa as a selectable marker. Our multiple gene deletion system significantly accelerates and facilitates the functional analysis process and is particularly useful for studying gene families in either laboratory or industrial yeast strains.