Direct selection of stabilised yeast URA3 transformants with 5-fluorouracil

• Published in: Nucleic acids research 1991-01, Vol.19 (1), p.197-197
• Main Authors: Romanos, MA, Beesley, K M, Clare, J J
• Format: Article
• Language: English
• Subjects: Saccharomyces cerevisiae
• ISSN: 0305-1048

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 ura3 fur1 host strains. 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 URA3-containing plasmid is then obligatory for viability. Since the ura3 fur1 host strain is nonviable, the final transformant has been obtained by the lengthy procedure of mating a fur1 strain with a ura3 strain containing the URA3 plasmid, and selecting plasmid-containing ura3 fur1 progeny. It has been suggested that spontaneous fur1 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.