In this proposal we will continue to systematically investigate genetic regulation of myofibril assembly in differentiating indirect flight muscle (IFM) of Drosophila melanogaster. During the previous grant period we used recombinant DNA methodology to isolate several genes encoding myofibrillar proteins of the IFM and to determine their chromosomal arrangement and temporal pattern of expresssion. For two such genes we have evidence that defective alleles result in abnormal IFM myofibril formation. We now intend to use both classical and molecular genetic techniques to further elucidate regulation and function of particular contractile protein genes and to 'correct' effects of mutant alleles, thus restoring normal myofibril morphology. This research will be divided among three lines. In the first, we will mutagenize or delete particular regions of cloned contractile protein genes and subsequently assess their ability to function in a regulated manner when re-introduced into the germline. This strategy should allow both regulatory and protein coding functions to be described in more detail than has been possible previously. In a second project the germline transformation technique will be used to investigate synthesis of contractile protein isoforms. We will establish whether genes encoding particular actin isoforms can be substituted for one another and thus more critically evaluate the selective value of the actin multigene family. In a third project we will rescue particular defective alleles by integrating cloned wild type genes into chromosomes of the mutant strain. For each such correction observed we will document the ratio of normal to defective alleles required and thus establish formal genetic bases for correction of mutations in structural protein genes. The long term goals of this project are to enhance our understanding of mechanisms which regulate activities of eucaryotic genes, to help establish the hierarchy of molecular interactions which mediates the assembly of myofibrils, and to document how particular defects in a developmental program can be genetically corrected. Such work will likely lead to better therapy for congenital muscular disorders, particularly muscular dystrophies.
