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Description
Drosophila melanogaster is a well characterized genetic system that can be easily manipulated to generate mutants affecting muscle assembly, structure and/or function. Coupling mutants with the ability to reintroduce genes into the Drosophila genome provides a powerful approach in which to study sequence specific function as it relates to muscle structural proteins. In chapter I we show that Drosophila melanogaster muscles contain the standard form of the thick filament protein paramyosin, as well as a novel paramyosin isoform, which we call miniparamyosin. Standard paramyosin is ≈105 kD and cross-reacts with polyclonal antibodies made against Caenorhabditis elegans or Heliocopris dilloni paramyosin. The Heliocopris antibody also crossreacts with a ≈55 kD protein likely to be miniparamyosin. We have cloned and sequenced cDNA's encoding both Drosophila isoforms. The C-terminal 363 amino acids are identical in standard and miniparamyosin. However, the smaller isoform has 114 residues at the N terminus that are unique as compared to the current protein sequence database. In-situ hybridization to Drosophila tissue sections shows that standard paramyosin is expressed in all larval and adult muscle tissues whereas miniparamyosin is restricted to a subset of the adult musculature. In chapter two we characterize the partial structure of the Drosophila paramyosin gene. This gene is single copy per haploid genome and utilizes two promoters to generate mRNA's that have either of two different 5' coding sequences joined to common 3' exons. We also show that a chromosomal deficiency eliminates the function of the Drosophila paramyosin gene. This deficiency is homozygous lethal at the first larval instar stage and has a morphological phenotype which suggests that muscle function is impaired. Finally, in chapter 3 we describe the cloning of two different genomic DNA fragments that each contain the heavy chain gene (MHC) of Drosophila melanogaster. Both of these clones have been used to create transgenic lines. The expression level from these MHC transgenes has been determined to be from 0 to 60% of the wild type haploid amount. However, even multiple copies of the transgenes are unable to rescue the dominant flightless and recessive lethal phenotypes associated with Mhc hypomorphic mutants.
