A positive selection for plasmid loss in Saccharomyces cerevisiae using galactose-inducible growth inhibitory sequences

Counter-selections for the loss of introduced plasmid sequences are useful for gene manipulations in yeast. We have used GAL10 promoter-mediated overe xpression of GIN sequences, which inhibit the growth of cells, to develop a novel counter-selection system. Yeast cells carrying a GIN sequence grow n ormally on glucose medium but are unable to grow On galactose medium, where as derivatives that have lost the GIN sequence are able to grow in the pres ence of galactose. We constructed autonomously replicating, integrating, an d disruption plasmids carrying GIN sequences and tested their use to select for loss of the plasmid. Thr results showed that the GIN sequences provide a selection for efficient loss of plasmids or integrated constructs from y east during growth on galactose medium, indicating that this system can be used for plasmid shuffling, gene replacements and marker gene recycling. Th is counter-selection system has wide application, because any Gal(+) strain and a wide variety of marker genes can be used. In addition, counter-selec tion systems using growth-inhibitory sequences should be applicable to othe r yeasts and possibly to other organisms. Copyright (C) 1999 John Wiley & S ons, Ltd.