Parasexual genetics of Dictyostelium gene disruptions: identification of a ras pathway using diploids

We describe new techniques for parasexual fusion of strains in liquid medium, selection and maintenance of the resulting stable diploid strains, and segregation to make recombined haploids. We have used these techniques to isolate rasS/gefB double nulls. The phenotypes of these mutants are no more severe than either parent, with movement, phagocytosis and fluid-phase endocytosis affected to the same degree as in rasS or gefB single nulls. In addition, we have produced diploids from one AX2- and one AX3-derived parent, providing an axenic strain with fewer secondary phenotypes than has been previously available.The phenotype of the rasS/gefB double mutant suggests that the RasS and GefB proteins lie on the same linear pathway. In addition, axenic diploids and the techniques to generate, maintain and segregate them will be productive tools for future work on Dictyostelium. They will particularly facilitate generation of multiple mutants and manuipulation of essential genes.A large body of work in the 1970s and 1980s showed that parasexual recombination of haploid Dictyostelium discoideum strains was a potent tool for generating multiple mutants and constructing relatively complex genetic experiments [1-3]. During normal starvation, pairs of haploid cells can occasionally fuse, apparently at random, to give diploid progeny. These are stable enough to grow, develop and form spores while remaining diploid. If cells of two different strains, each carrying a different selectable marker, are starved together, diploids will be formed from one cell of each parental strain. These can be separated from the haploid background by applying both selections simultaneously, so each haploid parent is killed but diploids survive. As long as selection is maintained, the diploids may remain reasonably stable, but there is a continual process of haploidization in which individual lines lose one chromosome of each diploid pair. This segregation is apparently random, which means that diploi