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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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Selection of diploid strains. The figure shows a schematic representation of the genotype on chromosome 3.
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Figure 1: Selection of diploid strains. The figure shows a schematic representation of the genotype on chromosome 3.

Mentions: Previous attempts at growing diploid Dictyostelium in axenic culture have not been productive. This is mainly caused by incompatibility between axenic growth and the selectable markers used to maintain the strains. Diploids are formed at low efficiency during normal Dictyostelium starvation [17]. Experimental isolation of diploids therefore requires genetic selections which allow diploids, but not haploids, to grow. Typical selections involve two parents, each of which carries a different recessive mutation, so neither parent can grow under appropriate selective conditions [7]. In diploids both mutants are complemented, so growth is possible. Some selections, for example growth on Bacillus subtilis, cannot be used without bacteria, while temperature sensitivity selections are apparently too harsh for efficient axenic growth of diploids [9]. We therefore designed schemes for selecting diploids which work optimally under axenic conditions. Fig. 1 shows diagrams of two such schemes. In fig. 1a one parent, DH1, carries a deletion in the pyr56 gene and thus requires exogenous uracil for growth [18]. The other parent is JH10, which requires added thymidine because of an insertion of pyr56 in the thyA gene [19]. Thus neither parent is able to grow in FM minimal medium. Diploids formed by the fusion of JH10 and DH1 cells carry a single copy of both pyr56 and thyA, however, and are thus able to grow in FM.


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

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

Selection of diploid strains. The figure shows a schematic representation of the genotype on chromosome 3.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Selection of diploid strains. The figure shows a schematic representation of the genotype on chromosome 3.
Mentions: Previous attempts at growing diploid Dictyostelium in axenic culture have not been productive. This is mainly caused by incompatibility between axenic growth and the selectable markers used to maintain the strains. Diploids are formed at low efficiency during normal Dictyostelium starvation [17]. Experimental isolation of diploids therefore requires genetic selections which allow diploids, but not haploids, to grow. Typical selections involve two parents, each of which carries a different recessive mutation, so neither parent can grow under appropriate selective conditions [7]. In diploids both mutants are complemented, so growth is possible. Some selections, for example growth on Bacillus subtilis, cannot be used without bacteria, while temperature sensitivity selections are apparently too harsh for efficient axenic growth of diploids [9]. We therefore designed schemes for selecting diploids which work optimally under axenic conditions. Fig. 1 shows diagrams of two such schemes. In fig. 1a one parent, DH1, carries a deletion in the pyr56 gene and thus requires exogenous uracil for growth [18]. The other parent is JH10, which requires added thymidine because of an insertion of pyr56 in the thyA gene [19]. Thus neither parent is able to grow in FM minimal medium. Diploids formed by the fusion of JH10 and DH1 cells carry a single copy of both pyr56 and thyA, however, and are thus able to grow in FM.

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