Role of Mdr-Related Genes in Dictyostelium Development

Kuspa, Adam

Abstract

In multicellular organisms the proportioning of differentiated cell-types must be carefully controlled such that the ultimate tissue is of the appropriate size for optimal function within the mature organism. While the development of Dictyostelium is simpler than that of metazoans, resulting in a ball of spores held on a cellular stalk, proportioning of the major cell types is tightly controlled in this organism. Dictyostelium will be studied as a model organism to gain an understanding of cell differentiation and the proportioning of cell types during multicellular development. The tagB gene is required for the differentiation of one of the three main prestalk cell subtypes, Pst A cells, from an established pool of prestalk cells. The predicted tagB protein product is very similar in its primary structure to the product of the human multidrug resistance gene, MDRI, which encodes P-glycoprotein (P-gp). P-gp was originally described in drug resistant tumor cells as an ATP-dependent efflux pump whose overexpression leads to reduced drug accumulation in cells. A developmentally regulated a rhodamine (Rh) transporter is also expressed in prespore cells with properties similar to human P-gp. Physical genomic mapping techniques identified six additional loci in Dictyostelium which containing tagB-related genes. The role of tagB in cell differentiation will be determined by a variety of experiments. First w will determine whether tagB encodes an ATP-dependent transporter. We will also attempt to functionally substitute the tagB gene with the human MDRI gene which would indicate the tagB is an MDR homolog. The TagB protein will be altered, by site-directed mutagenesis of its gene, in amino acid residues known to be important for mammalian P-gp function, followed by a functional analysis of the altered tagB gene in tagB mutant cells. Genetic tests of tagB function will be carried out in an attempt to determine the cell autonomous function of tagB in specifying the pstA cell fate. The precise localization of tabB expression within the developing organism will be determined using reporter genes fused to the tagB promoter. Ectopic expression of tagB will also provide useful information regarding tagB function. Using reporter gene constructs and selected double mutants, we will determine the epistatic relationships of tagB to other genes involved in PstA cell formation. The tagB-related genes will be cloned by PCR or by positional cloning in hopes of characterizing additional MDR-related genes. The expression patterns will be determined for these genes with the goal of finding a candidate gene for the prespore Rh transporter. If found, it will be deleted from the genome by homologous recombination in order to determine its role in development and in Rh transport in prespore cells. The prespore Rh transporter will be characterized biochemically to clarify its role in development ad to aid in the identification of its gene. Additional genes which regulate cell-type proportioning in Dictyostelium will be isolated and characterized with respect to tagB and the other MDR-related genes. Multidrug resistance is a major obstacle in the chemotherapy of human cancer where cross resistance of tumor cells to a large variety of functionally unrelated drugs is mediated by P-gp. The normal function of p-gp is poorly understood. The study of this gene family in a simple eukaryote should help to further our understanding of the roles of human MDR genes in normal and malignant cell physiology.