RESUMO
Here, we describe the identification and characterization of the cytokinesis-deficient mutant cell line 17HG5, which was generated in a restriction enzyme-mediated integration mutagenesis screen designed to isolate genes required for cytokinesis in Dictyostelium discoideum. Phenotypic characterization of the 17HG5 cell line revealed no apparent defects in the global functionality of the actomyosin cytoskeleton except for the observed cytokinesis defect when grown in suspension culture. Plasmid rescue was used to identify the disrupted gene locus (pats1; protein associated with the transduction of signal 1). that caused the cytokinesis defect. Disruption of the pats1 locus was recreated through homologous recombination in several independent cell lines, each recapitulating the cytokinesis-defective phenotype and thereby confirming that this gene locus is important for proper cytokinesis. Sequence data obtained by analysis of the genomic region flanking the inserted restriction enzyme-mediated integration plasmid revealed an 8892-bp genomic open reading frame encoding a 2964-amino-acid protein. The putative pats1 protein contains 3 regulatory domains (RI-phosphatase, RII-GTP-binding, R-III protein kinase), 13 leucine-rich repeats, and 8 WD-40 repeats. These regulatory domains coupled with the protein-protein interacting domains suggest that pats1 is involved in signal transduction during cytokinesis in Dictyostelium.
