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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

Growth of DIR1 diploids in FM medium in (A) dishes and (B) shaken flasks. Circles – DH1 in FM + uracil; diamonds – JH10 in FM + thymidine; triangles – DIR1 in FM.
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Figure 3: Growth of DIR1 diploids in FM medium in (A) dishes and (B) shaken flasks. Circles – DH1 in FM + uracil; diamonds – JH10 in FM + thymidine; triangles – DIR1 in FM.

Mentions: Diploid cells grow well in axenic medium under a variety of conditions (Fig. 3). In petri dishes, DIR1 cells grow as rapidly under selective conditions (without additions) as either parent (with thymidine or uracil added as appropriate). The doubling time under these conditions is just over 20 hrs for DIR1 diploids and both parents. When cells are grown in shaken culture, the growth rate of diploids is also similar to that of haploids. Shaken DIR1 double every 21 hours, compared with approximately 19 hours for JH10 and DH1. The long-term stability and ploidy of DIR1 diploids is ensured by the way in which the JH10 parental strain was generated. JH8, the strain from which JH10 was made, contains an deletion in the pyr56 gene and JH10 was then generated from this by re-insertion of the intact pyr56 gene into thyA. As indicated in fig. 1a, this means that the intact pyr56 gene in JH10 is situated at the same genetic locus as the thyA gene in DH1. This means that no normal segregation can generate a haploid that is able to grow in FM minimal medium. Only an unequal crossover or similar, extremely rare, event can generate haploid cells containing both thyA and pyr56. DIR1 diploids are thus exceedingly stable when grown under selective conditions. The growth of diploids is strongly favoured even in milder conditions – thyA- haploids die and pyr56- cells grow extremely poorly in normal, unsupplemented axenic medium (data not shown). Even after 30 generations of growth under these conditions, >60% cells are diploid. It is important to note that, due to the difficulty in getting perfectly separate chromosomes, the proportion of diploid cells estimated by cytological staining is likely to be underestimated – many of the cells scored as having 12 or 13 chromosomes are actually diploid.


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

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

Growth of DIR1 diploids in FM medium in (A) dishes and (B) shaken flasks. Circles – DH1 in FM + uracil; diamonds – JH10 in FM + thymidine; triangles – DIR1 in FM.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Growth of DIR1 diploids in FM medium in (A) dishes and (B) shaken flasks. Circles – DH1 in FM + uracil; diamonds – JH10 in FM + thymidine; triangles – DIR1 in FM.
Mentions: Diploid cells grow well in axenic medium under a variety of conditions (Fig. 3). In petri dishes, DIR1 cells grow as rapidly under selective conditions (without additions) as either parent (with thymidine or uracil added as appropriate). The doubling time under these conditions is just over 20 hrs for DIR1 diploids and both parents. When cells are grown in shaken culture, the growth rate of diploids is also similar to that of haploids. Shaken DIR1 double every 21 hours, compared with approximately 19 hours for JH10 and DH1. The long-term stability and ploidy of DIR1 diploids is ensured by the way in which the JH10 parental strain was generated. JH8, the strain from which JH10 was made, contains an deletion in the pyr56 gene and JH10 was then generated from this by re-insertion of the intact pyr56 gene into thyA. As indicated in fig. 1a, this means that the intact pyr56 gene in JH10 is situated at the same genetic locus as the thyA gene in DH1. This means that no normal segregation can generate a haploid that is able to grow in FM minimal medium. Only an unequal crossover or similar, extremely rare, event can generate haploid cells containing both thyA and pyr56. DIR1 diploids are thus exceedingly stable when grown under selective conditions. The growth of diploids is strongly favoured even in milder conditions – thyA- haploids die and pyr56- cells grow extremely poorly in normal, unsupplemented axenic medium (data not shown). Even after 30 generations of growth under these conditions, >60% cells are diploid. It is important to note that, due to the difficulty in getting perfectly separate chromosomes, the proportion of diploid cells estimated by cytological staining is likely to be underestimated – many of the cells scored as having 12 or 13 chromosomes are actually diploid.

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