Abstract
Background Dictyostelium discoideum amoebae are haploid and, as they share many features with animal cells, should be an ideal creature for studying basic processes such as cell locomotion. Isolation of mutants in this amoeba has largely been limited to non-essential genes: nsfA—the gene for NEM-sensitive factor—remains the only essential gene for which conditional (ts) mutants exist. These ts mutants were generated by gene replacement using a library of mutagenised nsfA containing a selectable marker: transformants were then screened for temperature sensitivity. The success of this approach depended on the high level of homologous recombination prevailing at this locus: ∼95% of selected clones were homologous recombinants. This is unusually high for Dictyostelium: homologous recombination at other loci is usually much less, usually between 0–30%, making the isolation of ts mutants much more tedious.Methodology/Principal FindingsIn trying to make ts mutants in sec1A, homologous recombination was found to be only ∼25%. A new approach, involving single loxP sites, was investigated. LoxP sites are 34 bp sequences recognised by Cre recombinase and between which this enzyme catalyses recombination. A Dictyostelium line containing a single loxP site adjacent to the 3′ end of the sec1A gene was engineered. A sec1A replacement DNA also containing a single loxP site in a homologous position was then introduced into this cell line. In the presence of CRE recombinase, homologous recombination increased to ∼80% at this locus, presumably largely driven by intermolecular recombination between the two single loxP sites.Conclusions/SignificanceA route to increase the rate of homologous recombination at a specific locus, sec1A, is described which enabled the isolation of 30 ts mutants in sec1A. One of these, sec1Ats1,has been studied and found to cease moving at the restrictive temperature. The approach described here may be valuable for enhancing homologous recombination at specified loci and thus for introducing mutations into specific genes in Dictyostelium and other creatures.
Highlights
With a haploid genome and known genomic sequence, the D. discoideum amoeba is perhaps the most attractive organism for studying the motile machinery and how this is integrated with signalling processes in chemotaxis
Methods have been available for some time for removing non-essential genes in Dictyostelium and many of these, those associated with the cytoskeleton, have been inactivated [1]
The isolation of these ts mutants depended both on the earlier demonstration that homologous recombination can occur between an introduced replacement vector and the genome [3,4] and on the unusually high level of homologous recombination which was found at this site:,95% of isolated transformants were found to be homologous replacements [2]
Summary
With a haploid genome and known genomic sequence, the D. discoideum amoeba is perhaps the most attractive organism for studying the motile machinery and how this is integrated with signalling processes in chemotaxis. NSF is required for cell locomotion [2], supporting the view for a dynamic role for membranes in movement The isolation of these ts mutants depended both on the earlier demonstration that homologous recombination can occur between an introduced replacement vector and the genome [3,4] and on the unusually high level of homologous recombination which was found at this site: ,95% of isolated transformants were found to be homologous replacements [2]. These ts mutants were generated by gene replacement using a library of mutagenised nsfA containing a selectable marker: transformants were screened for temperature sensitivity The success of this approach depended on the high level of homologous recombination prevailing at this locus: ,95% of selected clones were homologous recombinants. The approach described here may be valuable for enhancing homologous recombination at specified loci and for introducing mutations into specific genes in Dictyostelium and other creatures
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