ISOLATION OF GLYCOSYLATION MUTANTS IN DICTYOSTELIUM-DISCOIDEUM USING FLOW-CYTOMETRY

Fluorescence-activated cell sorting was used to isolate five spores of the soil amoeba Dictyostelium discoideum that carried new glycosylati on mutations, which were produced by restriction enzyme-mediated integ ration (REMI)-induced gene disruption and which occurred at frequencie s of around 10(-5). These mutations were identified by the loss of an O-glycosylation epitope found on surface proteins of wild type D. disc oideum spores that is recognised by the monoclonal antibody MUD62. A s econdary antibody conjugated to the fluorochrome fluorescein isothiocy anate identified MUD62 bound to spores. Spores lacking this epitope di d not fluoresce, allowing this population to be separated. Samples wer e found to contain around 0.1% of viable spores that were wild type bu t lacked the MUD62 epitope at the time of sorting, To remove these spo res from the unlabelled population, samples were labelled with monoclo nal antibody MUD50, which recognises surface proteins on immature spor es and proteins exposed from an inner coat layer, Double labelling wit h MUD50 and MUD62 reduced the unlabelled viable population to less tha n 0.002%, allowing the glycosylation-defective spores to be isolated, This is the first use of a selective approach to isolate nonmorphologi cal REMI-induced mutants in D, discoideum. This study also characteris es the surface properties of spore types found in mature fruiting bodi es of D. discoideum. (C) 1996 Wiley-Liss, Inc.