Genetic Techniques for Enhancing Biochemical and Structural Characterization of Dictyostelium Myosin II

Myosin mutants and their suppressors can provide information about conformational states of the myosin motor and their biochemical properties. Appropriate mutations can give rise to motors that arrest or overoccupy otherwise inaccessible states in the motor cycle. Intragenic (in the same gene) suppressor mutations that counteract mutations of known properties represent "fixes" or counters to the defect of the starting mutation and thus contain information about driving transitions or stabilizing states of the motor. Due to its variety of myosin-dependent phenotypes, Dictyostelium is a powerful tool for the identification of conditional mutants as well as selection of large numbers of intragenic revertants of a mutant of interest. Techniques are presented that allow isolation and identification of cold-sensitive myosin mutants in Dictyostelium as well as facile selection of revertants and identification of their suppressing mutation.