This proposal originates from our discovery in 1987 that the eukaryote Dictyostelium can be used for high-efficiency gene targeting. At that time disruption of the myosin heavy chain gene provided the first elucidation of the phenotype of a cell lacking this form of myosin. Since then Dictyostelium molecular genetic tools have been developed that have allowed the exploration in Dictyostelium of the in vivo roles of a variety of proteins in ways that have been readily accessible only in yeast. Whereas yeast lacks many phenotypes common to higher eukaryotic cells, including a multicellular developmental pathway, Dictyostelium manifests cellular and developmental behavioral aspects which are very similar to those of mammalian cells. The long term goal of this proposal is to exploit Dictyostelium as a model system to study the molecular basis of fundamental cell and developmental processes. The primary aims for the next five years are: (1) to define structure/function relationships of myosin II in regard to its in vivo roles in cytokinesis and other myosin II-dependent events, (2) to identify and isolate genes that code for proteins that are intimately involved in myosin II-dependent events, and (3) to study the in metro and in vivo properties of a protein tyrosine kinase that is essential for the final stages of the Dictyostelium developmental cycle. In the context of these three research foci, we will continue to expand further the repertoire of molecular genetic methods available for Dictyostelium.
Aim 1 will involve both site-directed and random mutagenesis of Dictyostelium myosin II, including the isolation of cold-sensitive missense mutations obtained by a novel azide selection scheme.
Aim 2 will involve three approaches for identifying and cloning new genes involved in myosin-related functions: use of the azide selection scheme to generate mutants that behave phenotypically as a myosin-minus cell but are mutations in other loci; application of the REMI (restriction enzyme mediated integration) technique for knocking out and tagging genes having myosin-associated function; and development of an antisense-driven technique for identifying and cloning genes involved in cytokinesis and other myosin II-dependent functions.
Aim 3 will involve application of the repertoire of molecular genetic methods available for Dictyostelium to study an important member of the protein tyrosine kinase family.
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