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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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The ploidy of DH1, JH10 and DIR1 cells was verified by (A) FACS analysis of DNA content, and (B) cytological staining of mitotic nuclei to visualise the chromomsomes. Strains are: (i) DH1, (ii) JH10, (iii) & (iv) DIR1. Panel (v) shows a possible mitotic non-disjunction event after exposure to thiabendazole. Bars indicate 2 μm.
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Figure 2: The ploidy of DH1, JH10 and DIR1 cells was verified by (A) FACS analysis of DNA content, and (B) cytological staining of mitotic nuclei to visualise the chromomsomes. Strains are: (i) DH1, (ii) JH10, (iii) & (iv) DIR1. Panel (v) shows a possible mitotic non-disjunction event after exposure to thiabendazole. Bars indicate 2 μm.

Mentions: We verified that DIR1 cells were true diploids using fluorescence activated cell sorting (FACS) and chromosome staining. Fig. 2a shows a typical FACS result. After propidium iodide staining, both haploid parents exhibit a peak intensity at a DNA content of about 181 ± 13, with a smaller additional peak at 380 ± 29. DIR1 diploids, on the other hand, contained a large peak at 307 ± 14 with a smaller peak at 650 ± 42, an increase of ~70% for both peaks. This is as expected as the mitochondria contribute almost 30% of the total cellular DNA [20].


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

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

The ploidy of DH1, JH10 and DIR1 cells was verified by (A) FACS analysis of DNA content, and (B) cytological staining of mitotic nuclei to visualise the chromomsomes. Strains are: (i) DH1, (ii) JH10, (iii) & (iv) DIR1. Panel (v) shows a possible mitotic non-disjunction event after exposure to thiabendazole. Bars indicate 2 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: The ploidy of DH1, JH10 and DIR1 cells was verified by (A) FACS analysis of DNA content, and (B) cytological staining of mitotic nuclei to visualise the chromomsomes. Strains are: (i) DH1, (ii) JH10, (iii) & (iv) DIR1. Panel (v) shows a possible mitotic non-disjunction event after exposure to thiabendazole. Bars indicate 2 μm.
Mentions: We verified that DIR1 cells were true diploids using fluorescence activated cell sorting (FACS) and chromosome staining. Fig. 2a shows a typical FACS result. After propidium iodide staining, both haploid parents exhibit a peak intensity at a DNA content of about 181 ± 13, with a smaller additional peak at 380 ± 29. DIR1 diploids, on the other hand, contained a large peak at 307 ± 14 with a smaller peak at 650 ± 42, an increase of ~70% for both peaks. This is as expected as the mitochondria contribute almost 30% of the total cellular DNA [20].

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