The objectives of the experiments outlined in this proposal are to determine the mechanisms and controls regulating developmental gene expression. We have determined the structure and expression of the muscle tropomyosin I (mTm I) gene from Drosophila melanogaster. The gene consists of 5 exons and 4 introns. During myogenesis, the gene is developmentally regulated and undergoes alternative splicing in different muscle of the fly. A set of experiments are proposed to study promoter function of the mTm I gene by P element mediated transformation. The expression of the normal and in vitro mutated gene will be determined in transformed flies in order to define the physical limits of the gene and DNA sequences involved in expression. Promoter function will also be studied in an in vitro cell-free transcription system to identify and characterize trans-acting regulatory factors. A second series of experiments will focus on the regulation and mechanisms that control alternative splicing. These experiments will involve an analysis of intron, exon and DNA sequence requirements necessary for proper splicing in different muscle. Normal and in vitro mutated gene transcription and RNA processing will be studied in transformed flies and cell-free extracts. The rationale for these experiments parallels the proposed studies on promoter function. A third objective of this proposal is to finish our analysis of the cytoplasmic-muscle tropomyosin II gene complex. These experiments will involve cDNA isolation, S1 nuclease mapping and DNA sequencing. Finally, the identity of a putative troponin C clone will be confirmed and the gene structure determined.
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