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

Abstract

Background: The relative ease of targeted gene disruption in the social amoeba Dictyostelium has stimulated its widespread use as an experimental organism for cell and developmental biology. However, the field has been hamstrung by the lack of techniques to recombine disrupted genes.

Results: 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.

Conclusions: 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 manipulation of essential genes.

Figure 1

Selection of diploid strains. The figure shows a schematic representation of the genotype on chromosome 3.

Figure 2

The ploidy of DH1, JH10 and DIR1 cells was verified by (A) FACS analysis of DNA content, and (B) cytological staining of mitotic nuclei to visualise the chromomsomes. Strains are: (i) DH1, (ii) JH10, (iii) & (iv) DIR1. Panel (v) shows a possible mitotic non-disjunction event after exposure to thiabendazole. Bars indicate 2 μm.

Figure 3

Growth of DIR1 diploids in FM medium in (A) dishes and (B) shaken flasks. Circles – DH1 in FM + uracil; diamonds – JH10 in FM + thymidine; triangles – DIR1 in FM.

Figure 4

Segregation of diploids. Diploid cells were grown in axenic medium in the presence of (a) 10 μg/ml benomyl and (b & c) 5 μg/ml thiabendazole. DIR1 cells were grown for the times indicated in axenic medium supplemented with thymidine and uracil. Ploidy was measured by cytological staining and scoring 100 cells for chromosome number. Diamonds indicate diploid cells, squares indicate haploid cells, and triangles indicate aneuploids. Note that the true level of aneuploidy is overstated because of overlaps between chromosomes in the diploid images.

Figure 5

Diploid hybrids between AX2 and AX3. (A) Growth of DIR2 (AX2/AX3) diploids in shaken flasks (squares) and dishes (diamonds) and AX2 cells in dishes (triangles) in HL-5 medium. (B) Fruiting body morphology of cells after development on clearing plates.

Figure 6

Production of a rasS/gefB double mutant by parasexual recombination. (A) Schematic diagram showing the recombination process (B) PCR screen for rasS disruption. Wild-type alleles give a 450 bp product, disrupted alleles give a product of 3000 bp.(C) PCR screen for gefB disruption. Wild-type alleles give a 480 bp product, disrupted alleles give a product of 1550 bp.

Figure 7

Relative rates of (a) motility and (b) phagocytosis of rasS^-, gefB^- and double mutant strains. Rates of phagocytosis were determined by the decrease in optical density of a bacterial suspension as bacteria are removed. The rate of phagocytosis relative to wild-type cells is plotted in the inset. Motility values are the means ± SD of 10 cells. OD values are the means ± SD of triplicate experiments.