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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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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.
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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.

Mentions: We generated diploids with one AX2 and one AX3 parent in order to confirm that the mechanisms of axenic growth were compatible between the two strains (figure 5). Several researchers have observed major differences in behaviour between AX2 and AX3, including cell size, macropinosome formation, and the phenotypes of mutations which affect proteins such as IQGAPs [30,31] and RasS (R.H.I., unpublished data). These differences led us to wonder whether additional mutations were needed for optimal growth in addition to the three known axenic loci, and these were different in AX2 and AX3. In this case, diploids with one AX2 and one AX3-derived parent would grow more slowly than either parent, or even not at all. To our initial surprise, AX2/AX3 diploids (here named DIR2) are as healthy or healthier than either parent, giving robust growth in axenic medium, and producing large colonies which sometimes produce larger fruiting bodies than either parent (fig. 5). In retrospect this observation is less surprising. AX2 and AX3 are known to carry the same three axenic mutations. Each was generated independently by heavy mutagenesis of NC4. They therefore presumably both carry a large number of secondary mutations, but in different loci. In AX2/AX3 diploids like DIR2, most of the secondary mutations from each parent are likely to be complemented by intact genes in the other.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: We generated diploids with one AX2 and one AX3 parent in order to confirm that the mechanisms of axenic growth were compatible between the two strains (figure 5). Several researchers have observed major differences in behaviour between AX2 and AX3, including cell size, macropinosome formation, and the phenotypes of mutations which affect proteins such as IQGAPs [30,31] and RasS (R.H.I., unpublished data). These differences led us to wonder whether additional mutations were needed for optimal growth in addition to the three known axenic loci, and these were different in AX2 and AX3. In this case, diploids with one AX2 and one AX3-derived parent would grow more slowly than either parent, or even not at all. To our initial surprise, AX2/AX3 diploids (here named DIR2) are as healthy or healthier than either parent, giving robust growth in axenic medium, and producing large colonies which sometimes produce larger fruiting bodies than either parent (fig. 5). In retrospect this observation is less surprising. AX2 and AX3 are known to carry the same three axenic mutations. Each was generated independently by heavy mutagenesis of NC4. They therefore presumably both carry a large number of secondary mutations, but in different loci. In AX2/AX3 diploids like DIR2, most of the secondary mutations from each parent are likely to be complemented by intact genes in the other.

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