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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

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.
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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.

Mentions: Examination of the double mutants revealed phenotypes very similar to either parent. In particular, there was no sign of a synthetic phenotype – in each case, the double mutants were no worse affected than their parents. Colonies resembled both parents in the morphology of their feeding fronts, producing fruiting bodies which resembled those produced by the gefB- parent (data not shown). In two assays which have been used to define the phenotypes of rasS- and gefB- mutants – unstimulated cell speed and phagocytosis – the double mutants were as severely affected as each parent, but no worse. Figure 7 shows two such assays. In fig. 7a, the cell speed of unstimulated, bacterially-grown double mutants is around three times the speed of wild type cells, comparable with both rasS- and gefB- parents. Fig. 7b shows the rate at which cells clear a suspension of bacteria. Wild type cells diminished the OD600 from 0.45 to below 0.2 in 5 hours. In the same interval, rasS- and gefB- mutants only cleared the OD600 to around 0.4. Both double mutants behaved in the same way, revealing a serious defect in phagocytosis in all mutant strains, but one that is not worsened by the double disruption.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: Examination of the double mutants revealed phenotypes very similar to either parent. In particular, there was no sign of a synthetic phenotype – in each case, the double mutants were no worse affected than their parents. Colonies resembled both parents in the morphology of their feeding fronts, producing fruiting bodies which resembled those produced by the gefB- parent (data not shown). In two assays which have been used to define the phenotypes of rasS- and gefB- mutants – unstimulated cell speed and phagocytosis – the double mutants were as severely affected as each parent, but no worse. Figure 7 shows two such assays. In fig. 7a, the cell speed of unstimulated, bacterially-grown double mutants is around three times the speed of wild type cells, comparable with both rasS- and gefB- parents. Fig. 7b shows the rate at which cells clear a suspension of bacteria. Wild type cells diminished the OD600 from 0.45 to below 0.2 in 5 hours. In the same interval, rasS- and gefB- mutants only cleared the OD600 to around 0.4. Both double mutants behaved in the same way, revealing a serious defect in phagocytosis in all mutant strains, but one that is not worsened by the double disruption.

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