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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

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.
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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.

Mentions: We used the Thy/G418 system to generate rasS/gefB double s (the scheme is shown in fig. 6a), because as described earlier it allowed us to cross pre-existing strains. First, a gefB strain was crossed with the Thy/G418 strain IR110 to give a gefB-/gefBwt thyAG418/thyAwt diploid. This was segregated to give haploids, and a gefB- thyAG418 clone was selected. This in turn was crossed with a rasS to give a rasS-/rasSwt gefB-/gefBwt thyAG418/thyAwt diploid. When this diploid was segregated, all possible combinations of mutant and wild type in rasS and gefB were found, as shown by PCR screens (fig. 6b &6c). We selected two rasS- gefB- double mutant clones and named them JK41 and JK42. The whole procedure took a little over one month, with relatively few hours of hands-on work during that period.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: We used the Thy/G418 system to generate rasS/gefB double s (the scheme is shown in fig. 6a), because as described earlier it allowed us to cross pre-existing strains. First, a gefB strain was crossed with the Thy/G418 strain IR110 to give a gefB-/gefBwt thyAG418/thyAwt diploid. This was segregated to give haploids, and a gefB- thyAG418 clone was selected. This in turn was crossed with a rasS to give a rasS-/rasSwt gefB-/gefBwt thyAG418/thyAwt diploid. When this diploid was segregated, all possible combinations of mutant and wild type in rasS and gefB were found, as shown by PCR screens (fig. 6b &6c). We selected two rasS- gefB- double mutant clones and named them JK41 and JK42. The whole procedure took a little over one month, with relatively few hours of hands-on work during that period.

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