This is a proposal to continue systematic investigation of the formation and functioning of Drosophila melanogaster indirect flight muscles (IFM). During the two previous grant periods we used recombinant DNA methodology to isolate and characterize genes which encode actins and tropomyosins of flight muscles. In addition, we isolated null alleles of IFM-specific actin and tropomyosin genes, and demonstrated that muscle defects associated with these alleles can be rescued by germ line transformation using wild type genes. Finally, as part of our germ line transformation work, we have demonstrated that the regulated expression of transposed actin and tropomyosin genes is identical to that of their """"""""resident"""""""" counterparts. We now intend to extend this work into more biochemical, physiological, and developmental areas. In the first part of the project we will employ a combination of in vitro mutagenesis and germ line transformation methodologies to further elucidate structure/function relationships for both actin and tropomyosin. In the case of actin, we have already constructed and partially characterized a suitable series of dysfunctional alleles. We intend to generate an analogous array of mutant tropomyosins, and to characterize functional defects in detail. In a second project we will investigate regulation of flight muscle contractility by isolation and characterization of genes encoding Drosophila troponins. To achieve this end we will screen suitable genomic or cDNA libraries using both antibody and nucleic acid probes. The ultimate goal of this portion of the proposal is to elucidate the mechanistic basis of stretch-activated contractility. In a third part of our project we will employ classical genetic screening to isolate mutations which prevent normal determination and differentiation of indirect flight muscles. We will recognize such mutations by their ability to suppress accumulation of alcohol dehydrogenase (ADH) encoded by a fusion gene wherein the ADH structural gene is governed by the regulatory portion of the flight muscle-specific actin gene. The long-term goals of this project are to enhance our understanding of mechanisms which regulate eucaryotic genes, and to help establish the hierarchy of molecular interactions which mediates the assembly and contractility of myofibrils. Such work may lead to better therapy for congenital muscular disorders, particularly muscular dystrophies.
