We are utilizing molecular and genetic approaches to define the functional significance of regions of the muscle protein myosin heavy chain (MHC). We have previously shown that Drosophila melanogaster has a single gene coding for muscle MHC. Alternative splicing of the RNA coding regions (exons) from this gene leads to the production of up to 480 alternative forms (isoforms) of the MHC protein. During the forthcoming period we propose to directly examine the functional differences among MHC isoforms and define regions of the protein that give rise to these differences. We will utilize cDNA clones along with the MHC gene promoter to develop vectors that express high levels of individual isoforms. These vectors will be inserted into the germline of mutants that are unable to synthesize MHC in the bulk of their thoracic muscles. MHC isolated from the thoraces of these transgenic flies will be biochemically and functionally analyzed in order to determine the distinct properties encoded by alternative exons. In another series of experiments we will examine the in vivo function of specific alternative regions of the MHC protein by constructing MHC genes that lack a specific alternative exon and inserting these genes into mutants which fail to synthesize either thoracic MHC or all MHC isoforms. We will examine muscle function and ultrastructure in these transgenic organisms. We will map the location of amino acid residues that differ among the MHC isoforms on to the recently determined three-dimensional structure of the myosin head. We will mutate specific residues in the molecule and test how this affects muscle structure, function and biochemical properties of the molecule. This will allow mapping of functional domains of the protein and help explain how the three- dimensional structure of the molecule contributes to these functions. We will also determine the molecular defects in several flightless mutants which fail to synthesize MHC in their thoracic muscles or that accumulate aberrant MHC and display defects in myosin function. This will serve to define regions of the gene that are important for muscle- specific protein accumulation or myosin function in vivo. Finally, we will examine the regulatory regions of the MHC gene in an effort to understand the basis for its expression in all muscle types. The presence of a single MHC gene in Drosophila, the availability of MHC mutants, and the use of vectors capable of expressing the MHC gene via germline transformation makes this a powerful approach to examining the function of MHC protein isoforms and should lead to insights into how the contractile apparatus functions in normal and mutant muscles. Funds are sought for one undergraduate and two graduate students. The undergraduate will have a two year training period in which he/she will first be closely supervised by a postdoctoral fellow or advanced Ph.D. student. This will be followed by an independent research project designed in conjunction with the P.I. The graduate students will receive training directly from the P.I. and other laboratory personnel and learn to be independent scientists. All students will be required to attend weekly laboratory meetings, journal clubs and seminars.
