The ability to target specific mutations into the yeast genome has been an extremely powerful tool for the study of many cellular processes. The application of this methodology has been limited exclusively to yeast. Our initial description of gene targeting in Dictyostelium has made possible the use of this approach in an organism that displays many cellular processes that are not found in yeast. Dictyostelium is an excellent candidate for biochemical and molecular genetic studies of many cell biological processes found in higher eukaryotes. The two major goals of this proposal are to explore in detail the phenomenon of plasmid integration by homologous recombination in Dictyostelium and to use this approach to study the function of conventional myosin and single-headed myosin in vivo. 1. The requirements for efficient targeted integration will be determined: we will study the efficiency of targeted integration by linear and circular plasmids, the relationship of integration efficiency and homologous insert length, the integration efficiency of different portions of the mhcA gene, the use of carrier DNA to improve transformation efficiency, and the development of other selectable markers. 2. Gene-targeting will be used for the study of myosin function in vivo: myosin heavy chain (mhcA) null mutants will be constructed either by gene replacement or by plasmid integration, mutant mhcA genes will be introduced into the null mutant strains and the effect of specific site-directed mutations and also random mutations will be studied, the myosin I (single-headed myosin) heavy chain gene will be isolated and used to construct a null mutant, all the myosin mutants will be characterized by studying their cytoskeletal structure by electron microscopy and their behavior during growth, chemotaxis, and development by time- lapse video microscopy and by Nomarski DIC video microscopy.
Showing the most recent 10 out of 24 publications
