Heterologous URA3MX cassettes for gene replacement in Saccharomyces cerevisiae

Heterologous gene replacement cassettes are powerful tools for dissecting g ene function in Saccharomyces cerevisiae. Their primary advantages over hom ologous gene replacement cassettes include reduced gene conversion (leading to efficient site-specific integration of the cassette) and greater indepe ndence of strain background. Perhaps the most widely used cassettes are the MX cassettes containing the dominant selectable kanamycin resistance gene (kan(r)), which confers resistance to G418 (Wach et al., 1994). One limitat ion of the kanMX cassettes is that they are not counterselectable and there fore not readily recyclable, which is important when constructing strains w ith more than one gene deletion. To address this limitation, and to expand the choices of heterologous markers, we have created two new MX cassettes b y replacing the kan(r) ORF from plasmids pFA6-kanMX3 and pFA6-kanMX4 with t he Candida albicans URA3 ORF. These plasmids, pAG60 (CaURA3MX4) and pAG61 ( CaURA3MX3) are identical to the kanMX cassettes in all other respects but h ave the added advantage of being counterselectable and therefore readily re cyclable in S. cerevisiae. Copyright (C) 1999 John Wiley & Sons, Ltd.