Isolation of Dictyostelium discoideum cytokinesis mutants by restriction enzyme-mediated integration of the blasticidin S resistance marker

Abstract

We have developed an improved REMI (restriction enzyme-mediated integration) system for generating mutant Dictyostelium cells quickly and efficiently for systematic screening of cytokinesis mutants. By means of this system, three cytokinesis mutants that grow as giant and multinucleate cells were isolated from 2,000 Dictyostelium transformants. Southern blot analysis of these mutants revealed that a single copy of the tag DNA was integrated into each genome. The tag with flanking genomic DNA at both ends was rescued from one of the mutants and reintroduced into the parental Ax2 strain. Homologous recombination of the rescued gene and the Dictyostelium genome led to the phenotypical changes expected for cytokinesis mutants.