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 glycosylation mutations, which were produced by restriction enzyme-mediated integration (REMI)-induced gene disruption and which occurred at frequencies of around 10(-5). These mutations were identified by the loss of an O-glycosylation epitope found on surface proteins of wild type D. discoideum spores that is recognised by the monoclonal antibody MUD62. A secondary antibody conjugated to the fluorochrome fluorescein isothiocyanate identified MUD62 bound to spores. Spores lacking this epitope did not fluoresce, allowing this population to be separated. Samples were found to contain around 0.1% of viable spores that were wild type but lacked the MUD62 epitope at the time of sorting. To remove these spores from the unlabelled population, samples were labelled with monoclonal antibody MUD50, which recognises surface proteins on immature spores and proteins exposed from an inner coat layer. Double labelling with MUD50 and MUD62 reduced the unlabelled viable population to less than 0.002%, allowing the glycosylation-defective spores to be isolated. This is the first use of a selective approach to isolate nonmorphological REMI-induced mutants in D. discoideum. This study also characterises the surface properties of spore types found in mature fruiting bodies of D. discoideum.
Macquarie University Centre for Analytical Biotechnology, School of Biological Sciences, Macquarie University, Sydney, New South Wales, Australia.
K R
KR
Griffiths
A C
AC
Champion
G
G
Vesey
K L
KL
Williams
D000818:Animals; D000911:Antibodies, Monoclonal; D004023:Dictyostelium / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D000939:Epitopes / Q000096:biosynthesis / Q000276:immunology; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000096:biosynthesis / Q000276:immunology; D006031:Glycosylation; D008565:Membrane Proteins / Q000096:biosynthesis / Q000276:immunology; D016296:Mutagenesis; D015800:Protozoan Proteins; D013170:Spores
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005656:Fungal Proteins; D008565:Membrane Proteins; D015800:Protozoan Proteins; C061925:spore coat proteins, Dictyostelium