Abstract
Using a combination of molecular and genetic techniques we demonstrate that Ifm(2)2 is an allele of the single-copy sarcomeric myosin heavy chain gene. Flies homozygous for this allele accumulate wild-type levels of mRNA and protein in tubular muscle of adults, but fail to accumulate detectable amounts of myosin heavy chain mRNA or protein in the indirect flight muscle. We propose that the mutation interferes with either transcription of the gene or splicing of the primary transcript in the indirect flight muscle and not in other muscle tissues. Biochemical and electron microscopic analysis of flies homozygous for this mutation has revealed that thick filament assembly is abolished in the indirect flight muscle resulting in the instability of wild-type thick filament proteins. In contrast, thin filament and Z disc assembly are marginally affected. We discuss a working hypothesis for sarcomere assembly and define and experimental approach to test the predictions of this proposed pathway for sarcomere assembly.
Full Text
The Full Text of this article is available as a PDF (4.0 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Arrigo A. P., Tanaka K., Goldberg A. L., Welch W. J. Identity of the 19S 'prosome' particle with the large multifunctional protease complex of mammalian cells (the proteasome). Nature. 1988 Jan 14;331(6152):192–194. doi: 10.1038/331192a0. [DOI] [PubMed] [Google Scholar]
- Bainbridge S. P., Bownes M. Staging the metamorphosis of Drosophila melanogaster. J Embryol Exp Morphol. 1981 Dec;66:57–80. [PubMed] [Google Scholar]
- Bernstein S. I., Hansen C. J., Becker K. D., Wassenberg D. R., 2nd, Roche E. S., Donady J. J., Emerson C. P., Jr Alternative RNA splicing generates transcripts encoding a thorax-specific isoform of Drosophila melanogaster myosin heavy chain. Mol Cell Biol. 1986 Jul;6(7):2511–2519. doi: 10.1128/mcb.6.7.2511. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bernstein S. I., Mogami K., Donady J. J., Emerson C. P., Jr Drosophila muscle myosin heavy chain encoded by a single gene in a cluster of muscle mutations. 1983 Mar 31-Apr 6Nature. 302(5907):393–397. doi: 10.1038/302393a0. [DOI] [PubMed] [Google Scholar]
- Boggs B., Cabral F. Mutations affecting assembly and stability of tubulin: evidence for a nonessential beta-tubulin in CHO cells. Mol Cell Biol. 1987 Aug;7(8):2700–2707. doi: 10.1128/mcb.7.8.2700. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bonner J. J., Parks C., Parker-Thornburg J., Mortin M. A., Pelham H. R. The use of promoter fusions in Drosophila genetics: isolation of mutations affecting the heat shock response. Cell. 1984 Jul;37(3):979–991. doi: 10.1016/0092-8674(84)90432-x. [DOI] [PubMed] [Google Scholar]
- Dibb N. J., Brown D. M., Karn J., Moerman D. G., Bolten S. L., Waterston R. H. Sequence analysis of mutations that affect the synthesis, assembly and enzymatic activity of the unc-54 myosin heavy chain of Caenorhabditis elegans. J Mol Biol. 1985 Jun 25;183(4):543–551. doi: 10.1016/0022-2836(85)90170-6. [DOI] [PubMed] [Google Scholar]
- Falkenburg P. E., Haass C., Kloetzel P. M., Niedel B., Kopp F., Kuehn L., Dahlmann B. Drosophila small cytoplasmic 19S ribonucleoprotein is homologous to the rat multicatalytic proteinase. Nature. 1988 Jan 14;331(6152):190–192. doi: 10.1038/331190a0. [DOI] [PubMed] [Google Scholar]
- Falkenthal S., Graham M., Wilkinson J. The indirect flight muscle of Drosophila accumulates a unique myosin alkali light chain isoform. Dev Biol. 1987 May;121(1):263–272. doi: 10.1016/0012-1606(87)90158-8. [DOI] [PubMed] [Google Scholar]
- Falkenthal S., Parker V. P., Davidson N. Developmental variations in the splicing pattern of transcripts from the Drosophila gene encoding myosin alkali light chain result in different carboxyl-terminal amino acid sequences. Proc Natl Acad Sci U S A. 1985 Jan;82(2):449–453. doi: 10.1073/pnas.82.2.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Falkenthal S., Parker V. P., Mattox W. W., Davidson N. Drosophila melanogaster has only one myosin alkali light-chain gene which encodes a protein with considerable amino acid sequence homology to chicken myosin alkali light chains. Mol Cell Biol. 1984 May;4(5):956–965. doi: 10.1128/mcb.4.5.956. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Homyk T., Jr, Emerson C. P., Jr Functional interactions between unlinked muscle genes within haploinsufficient regions of the Drosophila genome. Genetics. 1988 May;119(1):105–121. doi: 10.1093/genetics/119.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kemphues K. J., Kaufman T. C., Raff R. A., Raff E. C. The testis-specific beta-tubulin subunit in Drosophila melanogaster has multiple functions in spermatogenesis. Cell. 1982 Dec;31(3 Pt 2):655–670. doi: 10.1016/0092-8674(82)90321-x. [DOI] [PubMed] [Google Scholar]
- Kidd S., Lockett T. J., Young M. W. The Notch locus of Drosophila melanogaster. Cell. 1983 Sep;34(2):421–433. doi: 10.1016/0092-8674(83)90376-8. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Mahaffey J. W., Coutu M. D., Fyrberg E. A., Inwood W. The flightless Drosophila mutant raised has two distinct genetic lesions affecting accumulation of myofibrillar proteins in flight muscles. Cell. 1985 Jan;40(1):101–110. doi: 10.1016/0092-8674(85)90313-7. [DOI] [PubMed] [Google Scholar]
- Mogami K., Fujita S. C., Hotta Y. Identification of Drosophila indirect flight muscle myofibrillar proteins by means of two-dimensional electrophoresis. J Biochem. 1982 Feb;91(2):643–650. doi: 10.1093/oxfordjournals.jbchem.a133736. [DOI] [PubMed] [Google Scholar]
- Mogami K., Hotta Y. Isolation of Drosophila flightless mutants which affect myofibrillar proteins of indirect flight muscle. Mol Gen Genet. 1981;183(3):409–417. doi: 10.1007/BF00268758. [DOI] [PubMed] [Google Scholar]
- Mogami K., O'Donnell P. T., Bernstein S. I., Wright T. R., Emerson C. P., Jr Mutations of the Drosophila myosin heavy-chain gene: effects on transcription, myosin accumulation, and muscle function. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1393–1397. doi: 10.1073/pnas.83.5.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mullins J. I., Casey J. W., Nicolson M. O., Davidson N. Sequence organization of feline leukemia virus DNA in infected cells. Nucleic Acids Res. 1980 Aug 11;8(15):3287–3305. doi: 10.1093/nar/8.15.3287. [DOI] [PMC free article] [PubMed] [Google Scholar]
- O'Donnell P. T., Bernstein S. I. Molecular and ultrastructural defects in a Drosophila myosin heavy chain mutant: differential effects on muscle function produced by similar thick filament abnormalities. J Cell Biol. 1988 Dec;107(6 Pt 2):2601–2612. doi: 10.1083/jcb.107.6.2601. [DOI] [PMC free article] [PubMed] [Google Scholar]
- O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
- Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
- Rozek C. E., Davidson N. Differential processing of RNA transcribed from the single-copy Drosophila myosin heavy chain gene produces four mRNAs that encode two polypeptides. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2128–2132. doi: 10.1073/pnas.83.7.2128. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rozek C. E., Davidson N. Drosophila has one myosin heavy-chain gene with three developmentally regulated transcripts. Cell. 1983 Jan;32(1):23–34. doi: 10.1016/0092-8674(83)90493-2. [DOI] [PubMed] [Google Scholar]
- SHAFIQ S. A. Electron microscopic studies on the indirect flight muscles of Drosophila melanogaster. II. Differentiation of myofibrils. J Cell Biol. 1963 May;17:363–373. doi: 10.1083/jcb.17.2.363. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Takano-Ohmuro H., Hirose G., Mikawa T. Separation and identification of Drosophila myosin light chains. J Biochem. 1983 Sep;94(3):967–974. doi: 10.1093/oxfordjournals.jbchem.a134440. [DOI] [PubMed] [Google Scholar]
- Taylor K. A., Reedy M. C., Córdova L., Reedy M. K. Three-dimensional reconstruction of rigor insect flight muscle from tilted thin sections. 1984 Jul 26-Aug 1Nature. 310(5975):285–291. doi: 10.1038/310285a0. [DOI] [PubMed] [Google Scholar]
- Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wassenberg D. R., 2nd, Kronert W. A., O'Donnell P. T., Bernstein S. I. 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. J Biol Chem. 1987 Aug 5;262(22):10741–10747. [PubMed] [Google Scholar]
- Waterston R. H., Thomson J. N., Brenner S. Mutants with altered muscle structure of Caenorhabditis elegans. Dev Biol. 1980 Jun 15;77(2):271–302. doi: 10.1016/0012-1606(80)90475-3. [DOI] [PubMed] [Google Scholar]
- Zengel J. M., Epstein H. F. Identification of genetic elements associated with muscle structure in the nematode Caenorhabditis elegans. Cell Motil. 1980;1(1):73–97. doi: 10.1002/cm.970010107. [DOI] [PubMed] [Google Scholar]