We are utilizing molecular and genetic approach to examine the generation of diversity of the muscle protein myosin heavy chain (MHC) and to define the functional significance of his diversity. 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 may lead to the production of up to 384 alternative forms (isoforms) of the MHC protein. During the forthcoming period we propose to examine the diversity of MHC isoforms by isolating and analyzing cDNA clones made from larval and adult RNA and by observing the hybridization pattern of alternative exon specific oligonucleotides to tissue sections from organisms at these stages of development. We will also determine the molecular defects in several flightless mutants which fail to synthesize HC in their thoracic muscles. this will serve to define regions of the gene that are important for muscle-specific protein accumulation. 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 n 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. We will also use this approach to begin to elucidate the functional differences between muscle MHC and cytoplasmic MHC. 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 MHVC isoforms. We will examine muscle function and ultrastructure in these transgenic organisms. The presence of a single MHC gene in Drosophila, the availability of MHC mutants, and the use of vectors capable of expressing he MHC gene via germline transformation makes this a powerful approach to examining the functional diversity of the MHC protein and should lead to insights into how the contractile apparatus functions in normal an mutant muscles. Finally, we will continue to analyze alternative RNA splicing, the process which leads to MHC isoform diversity. Drosophila is also a useful organism in which to study alternative RNA splicing since germline transformation is available for determining which cis-acting sequences are important for this process to occur in a tissue-and stage- specific fashion. Furthermore, the use of genetic manipulation (in conjunction with germline transformation) should eventually permit mutation of trans-acting factor genes in order to study their structural domains. Using minigene vectors that can be inserted to the Drosophila germline or transfected into tissue culture cells, we will determine the cis-acting sequences responsible for muscle-specific alternative splicing of MHC transcripts. Using a Drosophila cell-free splicing system, we will begin to examine the trans-acting factors required for this process. Several human disorders result from defects in splicing and understanding the factors involved in alternative RNA splicing may therefore have implications for understanding human disease.
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