We propose to use molecular, genetic, physiological and biochemical approaches to determine the function of alternative isoforms of the protein myosin heavy chain (MHC) which is a major component of thick filaments of muscle and is directly involved in generating muscle contraction. Drosophila melanogaster has a single gene coding for muscle MHC and produces up to 480 forms of the protein by alternative RNA splicing. We have identified null mutations that prevent MHC accumulation in all muscle types or that selectively prevent MHC accumulation in adult muscles inessential to viability. Using these mutants in conjunction with germline transformation of in vitro mutagenized genes, we propose to study how regions of the myosin head affect the properties of myosin protein both in vivo and in vitro. We will express embryonic myosin isoforms in adults and examine their nucleotide and actin binding ability, ATPase activity and competence to induce in vitro motility and force generation. The myosin biochemistry studies will be conducted in collaboration with Dr. Michael Geeves (Max Planck, Dortmund). We will also express adult thoracic MHC containing substitutions of single regions of the embryonic myosin head in order to determine how in vivo muscle function, ultrastructural features and mechanical properties are affected by each of these regions. Muscle mechanical studies will be conducted in collaboration with Drs. David White and John Sparrow (University of York). The in vitro assays will be performed on MHC isolated from these transformants as well. We have shown that the wild-type Mhc gene introduced by germline transformation rescues all defects associated with a MHC-null allele and that we can express high levels of an embryonic cDNA in a null background, demonstrating the feasibility of our proposed studies. We will also determine the molecular, biochemical and ultrastructural defects associated with four Mhc mutations that likely affect key amino acid residues. These mutations, along with the specific differences among the myosin isoforms, will be studied in relation to the three-dimensional structure of the myosin molecule and a proposed model for the mechanochemical cycle (in collaboration with Dr. Ron Milligan, Scripps Research Institute). Overall, our studies should lead to insights into how the myosin protein functions in muscle and permit testing of models for the mechanochemical cycle. Since mutations in the myosin head are involved in defects in human cardiac and skeletal muscle, these studies are relevant to understanding human myopathies. Finally, we will continue our analysis of the mechanism of alternative RNA splicing by developing genetic approaches to isolating trans-acting factors involved in this process. Several human disorders result from defects in splicing and determining the factors involved in alternative RNA splicing may therefore have implications for understanding human disease.