Specific myosin heavy chain mutations suppress troponin I defects in Drosophila muscles.

We show that specific mutations in the head of the thick filament molecule myosin heavy chain prevent a degenerative muscle syndrome resulting from the hdp2 mutation in the thin filament protein troponin I. One mutation deletes eight residues from the actin binding loop of myosin, while a second affects a residue at the base of this loop. Two other mutations affect amino acids near the site of nucleotide entry and exit in the motor domain. We document the degree of phenotypic rescue each suppressor permits and show that other point mutations in myosin, as well as null mutations, fail to suppress the hdp2 phenotype. We discuss mechanisms by which the hdp2 phenotypes are suppressed and conclude that the specific residues we identified in myosin are important in regulating thick and thin filament interactions. This in vivo approach to dissecting the contractile cycle defines novel molecular processes that may be difficult to uncover by biochemical and structural analysis. Our study illustrates how expression of genetic defects are dependent upon genetic background, and therefore could have implications for understanding gene interactions in human disease.

Transformation of Drosophila melanogaster with the wild-type myosin heavy-chain gene: rescue of mutant phenotypes and analysis of defects caused by overexpression.

We have transformed Drosophila melanogaster with a genomic construct containing the entire wild-type myosin heavy-chain gene, Mhc, together with approximately 9 kb of flanking DNA on each side. Three independent lines stably express myosin heavy-chain protein (MHC) at approximately wild-type levels. The MHC produced is functional since it rescues the mutant phenotypes of a number of different Mhc alleles: the amorphic allele Mhc1, the indirect flight muscle and jump muscle-specific amorphic allele Mhc10, and the hypomorphic allele Mhc2. We show that the Mhc2 mutation is due to the insertion of a transposable element in an intron of Mhc. Since a reduction in MHC in the indirect flight muscles alters the myosin/actin protein ratio and results in myofibrillar defects, we determined the effects of an increase in the effective copy number of Mhc. The presence of four copies of Mhc results in overabundance of the protein and a flightless phenotype. Electron microscopy reveals concomitant defects in the indirect flight muscles, with excess thick filaments at the periphery of the myofibrils. Further increases in copy number are lethal. These results demonstrate the usefulness and potential of the transgenic system to study myosin function in Drosophila. They also show that overexpression of wild-type protein in muscle may disrupt the function of not only the indirect flight but also other muscles of the organism.

A charge change in an evolutionarily-conserved region of the myosin globular head prevents myosin and thick filament accumulation in Drosophila.

We have determined the molecular lesion in Mhc9, a homozygous-viable mutant of the Drosophila muscle myosin heavy chain gene. This mutation is in an adult-specific alternative exon (exon 9a) which encodes a portion of the myosin head that is highly conserved among both cytoplasmic and muscle myosins of all organisms. The mutation results in a charge change in the evolutionarily invariant amino acid residue 482. The phenotype of the homozygous mutant is identical to that of an organism having a stop codon within alternative exon 9a, i.e. lack of thick filaments in the indirect flight muscles and a greatly reduced number of thick filaments in the small cells of the jump muscles. This phenotype correlates with the known expression pattern of exon 9a. Genetic, biochemical and ultrastructural analyses show that the failure to accumulate thick filaments in the mutant is not a result of aberrant interactions with thin filaments and that the mutant myosin heavy chain does not poison assembly of wild-type thick filaments. Our results, in conjunction with recent structural and mutant studies by others, indicate that residue 482 is important for generating ATPase activity and for myosin stability in muscle.

Defects in the Drosophila myosin rod permit sarcomere assembly but cause flight muscle degeneration.

We have determined the molecular and ultrastructural defects associated with three homozygous-viable myosin heavy chain mutations of Drosophila melanogaster. These mutations cause a dominant flightless phenotype but allow relatively normal assembly of indirect flight muscle myofibrils. As adults age, the contents of the indirect flight muscle myofibers are pulled to one end of the thorax. This apparently results from myofibril "hyper-contraction", and leads to sarcomere rupture and random myofilament orientation. All three mutations cause single amino acid changes in the light meromyosin region of the myosin rod. Two change the same glutamic acid to a lysine residue and the third affects an amino acid five residues away, substituting histidine for arginine. Both affected residues are conserved in muscle myosins, cytoplasmic myosins and paramyosins. The mutations are associated with age-dependent, site-specific degradation of myosin heavy chain and failure to accumulate phosphorylated forms of flightin, an indirect flight muscle-specific protein previously localized to the thick filament. Given the repeating nature of the hydrophobic and charged amino acid residues of the myosin rod and the near-normal assembly of myofibrils in the indirect flight muscle of these mutants, it is remarkable that single amino acid changes in the rod cause such severe defects. It is also interesting that these severe defects are not apparent in other muscles. These phenomena likely arise from the highly organized nature and rigorous performance requirements of indirect flight muscle, and perhaps from the interaction of myosin with flightin, a protein specific to this muscle type.

Determining structure/function relationships for sarcomeric myosin heavy chain by genetic and transgenic manipulation of Drosophila.

Drosophila melanogaster is an excellent system for examining the structure/function relationships of myosin. It yields insights into the roles of myosin in assembly and stability of myofibrils, in defining the mechanical properties of muscle fibers, and in dictating locomotory abilities. Drosophila has a single gene encoding muscle myosin heavy chain (MHC), with alternative RNA splicing resulting in stage- and tissue-specific isoform production. Localization of the alternative domains of Drosophila MHC on a three-dimensional molecular model suggests how they may determine functional differences between isoforms. We are testing these predictions directly by using biophysical and biochemical techniques to characterize myosin isolated from transgenic organisms. Null and missense mutations help define specific amino acid residues important in actin binding and ATP hydrolysis and the function of MHC in thick filament and myofibril assembly. Insights into the interaction of thick and thin filaments result from studying mutations in MHC that suppress ultrastructural defects induced by a troponin I mutation. Analysis of transgenic organisms expressing engineered versions of MHC shows that the native isoform of myosin is not critical for myofibril assembly but is essential for muscle function and maintenance of muscle integrity. We show that the C-terminus of MHC plays a pivotal role in the maintenance of muscle integrity. Transgenic studies using headless myosin reveal that the head is important for some, but not all, aspects of myofibril assembly. The integrative approach described here provides a multi-level understanding of the function of the myosin molecular motor.

Analysis of the 5' end of the Drosophila muscle myosin heavy chain gene. Alternatively spliced transcripts initiate at a single site and intron locations are conserved compared to myosin genes of other organisms.

We have localized the transcription start site of the Drosophila melanogaster muscle myosin heavy chain (MHC) gene and find that all forms of the alternatively spliced MHC mRNA initiate at the same location. Therefore the alternative inclusion/exclusion of the 3' penultimate exon in transcripts from this gene (Bernstein, S.I., Hansen, C.J., Becker, K.D., Wassenberg, D.R., II, Roche, E.S., Donady, J.J., and Emerson, C. P., Jr. (1986) Mol. Cell. Biol. 6, 2511-2519; Rozek, C.E., and Davidson, N. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2128-2134) does not result from the use of different 5' transcription initiation sites. This gene is the first invertebrate MHC gene shown to have TATA and CAAT box consensus sequences and a noncoding 5' exon, properties that are shared with some vertebrate and invertebrate contractile protein genes. The intron that splits the 5' noncoding region of the Drosophila MHC gene contains no major conserved elements relative to other Drosophila contractile protein genes. The introns within the coding region near the 5' end of the Drosophila MHC gene are located at the same sites as nematode and vertebrate MHC gene introns, indicating that these MHC genes are derived from a common ancestral sequence. The putative ATP binding domain encoded in the fourth exon of the Drosophila MHC gene is highly conserved relative to vertebrate, invertebrate, and non-muscle MHC genes suggesting that each of these myosins bind ATP by the same mechanism. Two divergent copies of the third exon are present within the 5' region of the Drosophila MHC gene, suggesting that alternative splicing produces MHC isoforms with different globular head regions.

Muscle-specific accumulation of Drosophila myosin heavy chains: a splicing mutation in an alternative exon results in an isoform substitution.

We show that the molecular lesions in two homozygousviable mutants of the Drosophila muscle myosin heavy chain gene affect an alternative exon (exon 9a) which encodes a portion of the myosin head that is highly conserved among both cytoplasmic and muscle myosins of all organisms. In situ hybridization and Northern blotting analysis in wild-type organisms indicates that exon 9a is used in indirect flight muscles whereas both exons 9a and 9b are utilized in jump muscles. Alternative exons 9b and 9c are used in other larval and adult muscles. One of the mutations in exon 9a is a nonsense allele that greatly reduces myosin RNA stability. It prevents thick filament accumulation in indirect flight muscles and severely reduces the number of thick filaments in a subset of cells of the jump muscles. The second mutation affects the 5' splice site of exon 9a. This results in production of an aberrantly spliced transcript in indirect flight muscles, which prevents thick filament accumulation. Jump muscles of this mutant substitute exon 9b for exon 9a and consequently have normal levels of thick filaments in this muscle type. This isoform substitution does not obviously affect the ultrastructure or function of the jump muscle. Analysis of this mutant illustrates that indirect flight muscles and jump muscles utilize different mechanisms for alternative RNA splicing.

Altered turnover of allelic variants of hypoxanthine phosphoribosyltransferase is associated with N-terminal amino acid sequence variation.

The results of our previous studies suggested that differences in the primary structures of the hypoxanthine phosphoribosyltransferase (HPRT) A and B proteins (EC 2.4.2.8) of mice are associated with altered turnover of these proteins in reticulocytes. On the basis of nucleotide sequence comparisons of their corresponding cDNAs, we show here that the HPRT A and B proteins differ at two positions; there is an alanine/proline substitution at amino acid position 2 and a valine/alanine substitution at amino acid position 29 (HPRT A/B proteins, respectively; total protein length, 218 amino acids). On the basis of results obtained from sequencing of the N termini of the purified HPRT A and B proteins, we also show that these amino acid substitutions are associated with differences in processing of the proteins; HPRT B, which is encoded as N-terminal Met-Pro, has a free N-terminal proline residue; HPRT A, which is encoded as N-terminal Met-Ala, lacks a free N-terminal alpha-amino group and is presumed to be acetylated following removal of the N-terminal methionine (i.e. AcO-Ala). These observations are discussed in reference to the idea that the N terminus of a protein plays a role in determining the rate at which the protein is degraded in erythroid cells.

Alternative myosin hinge regions are utilized in a tissue-specific fashion that correlates with muscle contraction speed.

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 Mhc10. 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 Mhc10 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 MHCs with alternative hinge regions. Electron microscopic comparisons of various muscles from wild-type and Mhc10 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 MHCs 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.