A high-efficiency method to replace essential genes with mutant alleles in yeast.

Temperature-sensitive (TS), internally deleted and truncated alleles are important tools to facilitate the characterization of essential genes. We have developed a straightforward method to replace a wild-type gene with a mutant allele at the endogenous locus. This method is an efficient alternative to the two-step method for integration of alleles that are compromised in function or contain multiple mutations. A strain is constructed that has the essential gene of interest disrupted by a selectable marker. Strain viability is maintained by a plasmid carrying a copy of the essential wild-type gene and the ADE3 gene. The mutant allele is cloned into an integratable vector carrying a selectable/counter-selectable marker, such as URA3. The plasmid is linearized and transformed, directing integration to the 5' or 3' region flanking the essential open reading frame (ORF). Transformants that have integrated the mutant gene at the endogenous locus can lose the autonomous plasmid carrying the wild-type copy of the essential gene and the ADE3 gene. These transformants are identifiable as white sectoring colonies, display the mutant phenotype and may be characterized. An optional second selection step on 5-fluoroorotic acid (5-FOA) selects for popouts of the integrating vector sequences, leaves the mutant allele at the endogenous locus, and recycles selectable markers. We have used this method to integrate a TS allele of SPC110 that could not be integrated by standard methods. (c) 2005 John Wiley & Sons, Ltd.