By comparing the structure of wild-type and mutant muscle myosin heavy chain (MHC) genes of Drosophila melanogaster, we have identified the defect in the homozygous-viable, flightless mutant Mhc 10. The mutation is within the 3' splice acceptor of an alternative exon (exon 15a) that encodes the central region of the MHC hinge. The splice acceptor defect prevents the accumulation of mRNAs containing exon 15a, whereas transcripts with a divergent copy of this exon (exon 15b) are unaffected by the mutation. In situ hybridization and Northern blot analysis of wild-type organisms reveals that exon 15b is used in larval MHCs, whereas exons 15a and/or 15b are used in adult tissues. Because Mhc ~~ mutants fail to accumulate transcripts encoding MHC protein with hinge region a, analysis of their muscle-specific reduction in thick filament number serves as a sensitive assay system for determining the pattern of accumulation of MI-ICs with alternative hinge regions. Electron microscopic comparisons of various muscles from wild-type and Mhc ~~ adults reveals that those that contract rapidly or develop high levels of tension utilize only hinge region a, those that contract at moderate rates accumulate MHCs of both types, and those that are slowly contracting have MI-ICs with hinge region b. The presence of alternative hinge-coding exons and their highly tissue-specific usage suggests that this portion of the MHC molecule is important to the isoform-specific properties of MHC that lead to the different physiological and ultrastructural characteristics of various Drosophila muscle types. The absence of other alternative exons in the rod-coding region, aside from those shown previously to encode alternative carboxyl termini, demonstrates that the bulk of the myosin rod is not involved in the generation of isoform-specific properties of the MHC molecule.
We describe the ultrastructural and initial molecular characterization of four homozygous-viable, dominantflightless mutants of Drosophila melanogaster. Genetic mapping indicates that these mutations are inseparable from the known muscle myosin heavy-chain (MHC) allele Mhc^, and each mutation results in a muscle-specific reduction in MHC protein accumulation. The indirect flight muscles (IFMs) of each of these homozygous mutants fail to accumulate MHC, lack thick filaments, and do not display normal cylindrical myofibrils. As opposed to the null phenotype observed in the IFM, normal amounts of MHC accumulate in the leg muscles of three of these mutants, whereas the fourth mutant shows a 45% reduction in leg muscle MHC. The ultrastructure of the tergal depressor of the trochanter muscle (TDT, or jump muscle] is normal in one mutant, completely lacks thick filaments in a second mutant, and displays a reduction of thick filaments in two mutants. The thick filament reduction in this latter class of mutants is limited to the four smaller anterior cells of the TDT, indicating that the TDT is a mixed fiber-type muscle. Because all isoforms of muscle MHC are encoded by alternative splicing of transcripts from a single gene, our results suggest that there is a complex pattern of MHC isoform accumulation in Drosophila, The phenotypes of the homozygous-viable mutants provide evidence for the differential localization of MHC isoforms in different muscles, within the same muscle, and even within a single muscle cell. The mutant characteristics also suggest that the use of some alternative exons is shared among the IFM, TDT, and additional muscles whereas the use of others is unique to the IFM.[Key Words: Drosophila-, muscle mutants; myosin heavy chain; alternative RNA splicing]
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