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

King J, Insall RH - BMC Genet. (2003)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK. jasonking50@yahoo.co.uk

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 s. 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 s. 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.

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Related in: MedlinePlus

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.
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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.

Mentions: Since the aim of this work was to avoid the need for bacterial growth, we also tested the effects of benomyl and thiabendazole during axenic growth. DIR1 cells were grown in the presence of 10 μg/ml benomyl or 5 μg/ml thiabendazole, and samples were taken every day and cytologically stained to count chromosomes. Fig. 4 shows the process of segregation of diploid cells. After two days growing in benomyl or thiabendazole, as many as 69% of the cells were aneuploid, with the rest split between diploid and haploid. Four days of treatment resulted in a population in which nearly all cells were haploid. It is therefore clear that segregation under axenic conditions is an efficient way of generating haploid progeny from diploid parents.


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

King J, Insall RH - BMC Genet. (2003)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC183827&req=5

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.
Mentions: Since the aim of this work was to avoid the need for bacterial growth, we also tested the effects of benomyl and thiabendazole during axenic growth. DIR1 cells were grown in the presence of 10 μg/ml benomyl or 5 μg/ml thiabendazole, and samples were taken every day and cytologically stained to count chromosomes. Fig. 4 shows the process of segregation of diploid cells. After two days growing in benomyl or thiabendazole, as many as 69% of the cells were aneuploid, with the rest split between diploid and haploid. Four days of treatment resulted in a population in which nearly all cells were haploid. It is therefore clear that segregation under axenic conditions is an efficient way of generating haploid progeny from diploid parents.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK. jasonking50@yahoo.co.uk

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 s. 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 s. 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.

Show MeSH
Related in: MedlinePlus