Direct selection of stabilised yeast URA3 transformants with 5- fluorouracil

Yeast expression vectors are generally based on the 2 micron plasmid and are not stably inherited, so that continued selection pressure must be applied to prevent the gradual accumulation of plasmid-free cells. A number of 'autoselection' systems have been proposed and perhaps the most versatile is the stabilisation of plasmids containing the URA3 gene in urc3 furl host strains (1). These strains are non-viable since they are blocked in uridine 5'-monophosphate synthesis by both the de novo and salvage pathways; maintenance of a WJ/13-containing plasmid is then obligatory for viability. Since the ura3 furl host strain is nonviable, the final transformant has been obtained by the lengthy procedure of mating a furl strain with a ura3 strain containing the URA3 plasmid, and selecting plasmid-containing ura3 furl progeny (1). It has been suggested (1) that spontaneous furl mutants of a transformant could be directly selected in one step by their ability to survive high concentrations of 5-fluorouracil (5-FU), and that these mutants would have 100% stable plasmids. We have tested this idea and provide a simple protocol for URA3 plasmid stabilisation.