%0 Journal Article
%T Optimization of a large-scale gene disruption protocol in Dictyostelium and analysis of conserved genes of unknown function
%A Patricia Torija
%A Alicia Robles
%A Ricardo Escalante
%J BMC Microbiology
%D 2006
%I BioMed Central
%R 10.1186/1471-2180-6-75
%X Here we present an optimized protocol based on the previously described construction of gene disruption vectors by in vitro transposition. Our method allows a rapid selection of the construct by a simple PCR approach and subsequent sequencing. Disruption constructs were amplified by PCR and the products were directly transformed in Dictyostelium cells. The selection of homologous recombination events was also performed by PCR. We have constructed 41 disruption vectors to target genes of unknown function, highly conserved between Dictyostelium and human, but absent from the genomes of S. cerevisiae and S. pombe. 28 genes were successfully disrupted.This is the first step towards the understanding of the function of these conserved genes and exemplifies the easiness to undertake large-scale disruption analysis in Dictyostelium.Comparative genomics is based on the conservation of the molecular function of genes in different organisms throughout evolution. Orthologous genes can be studied in simple, genetically tractable model systems, as a first step to address their function in higher organisms, including humans and evaluate their possible roles in diseases. The completion of Dictyostelium genome now offers the opportunity to study the function of conserved genes present in the social amoeba and other organisms in a systematic way [1].Dictyostelium is a primitive eukaryote, living as a single cell organism while bacteria, its source of nutrients, are present in the soil. When bacteria are consumed, starvation triggers a complex response allowing the cells to aggregate by chemotaxis and form a multicellular structure. Many different aspects of its biology including motility, chemotaxis, cytokinesis, cell-differentiation and morphogenesis among others, are more closely related to those in higher organisms than to unicellular models, such as yeasts [2-4]. We have performed a systematic knock-out approach to begin to address the function of genes of unknown function prese
%U http://www.biomedcentral.com/1471-2180/6/75