The dystrophin / utrophin homologues in Drosophila and in sea urchin☆
Introduction
The gene which is defective in duchenne muscular dystrophy (DMD) is the largest gene known to date, spanning over 2500 kb of the X chromosome. The product of the gene in the muscle, dystrophin, is a 427 kDa rod-shaped protein consisting of four domains: N-terminal actin binding domain, 24 triple helix spectrin-like repeats with four hinge regions, a cysteine-rich domain with two potential calcium binding motifs, and a unique C-terminal domain (Koenig et al., 1988). Dystrophin forms a linkage between the cytoskeletal actin and a group of membrane proteins, as well as with a number of non-membranal proteins (collectively called dystrophin associated proteins; DAPs) (Yoshida and Ozawa, 1990, Ervasti and Campbell, 1991). The association with the DAPs is mediated mainly by the cysteine-rich and C terminal domains of dystrophin (Suzuki et al., 1994, Jung et al., 1995). One of the DAPs, α dystroglycan, binds laminin. Thus, in the muscle this complex links the cytoskeleton, the sarcolemma and the extracellular matrix (Ahn and Kunkel, 1993, Campbell, 1995, Ozawa et al., 1995).
The DMD gene also codes for two non muscle isoforms of dystrophin, each controlled by a different promoter located in the 5′ end region of the gene; the brain type (Nudel et al., 1989, Barnea et al., 1990, Boyce et al., 1991) and Purkinje cell type dystrophins (Gorecki et al., 1992). In addition, internal promoters located within introns further downstream in the huge DMD gene regulate the expression of smaller products. Dp71, a 70.8 kDa protein, consists of only the cysteine-rich and C-terminal domains of dystrophin (Bar et al., 1990, Lederfein et al., 1992). It is the most abundant non-muscle product of the DMD gene. The highest levels of Dp71 are found in the brain (Rapaport et al., 1992, Greenberg et al., 1996). The other known small products of the DMD gene consist of the cysteine-rich and C-terminal domains with various extensions into the spectrin like repeats domain (reviewed in Yaffe et al., 1996). These products are Dp116 (Byers et al., 1993), Dp140 (Lidov et al., 1995) and Dp260 (D'Souza et al., 1995), which are expressed mainly in Schwann cells, brain, and retina, respectively, and have molecular weights of 116, 140 and 260 kDa. The functions of the non-muscle dystrophins and of the smaller products of the DMD gene are not known.
Several genes encoding proteins with various levels of sequence identity with dystrophin were characterized. Utrophin (DRP1) is encoded by an autosomal gene and consists of all four domains of dystrophin. At the amino acid level the two proteins show ∼51% identity. The exon/intron structures of the two genes are also very similar (Love et al., 1989, Pearce et al., 1993). The utrophin gene also encodes smaller products transcribed from downstream promoters (Fabbrizio et al., 1995, Blake et al., 1995, Wilson et al., 1999). Sequence comparisons suggested that the dystrophin and utrophin genes were separated by duplication during early evolution of the vertebrates (Wang et al., 1998, Roberts and Bobrow, 1998).
Bessou et al. (1998) described a gene in C. elegans encoding a dystrophin related protein. Only a single product was described. Mutations in the gene which is expressed mainly in muscle resulted in hypersensitivity to acetylcholine and hyperactivity of the worms.
The other known genes that seem to be evolutionary related to the DMD gene are much smaller, each encodes a single protein with low but significant similarity to the cysteine-rich and C-terminal domains of dystrophin. These include the torpedo 87 kDa phosphoprotein and its mammalian homologues dystrobrevin (Carr et al., 1989, Blake et al., 1996, Sadoulet-Puccio et al., 1996) and the Drosophila 75 kDa protein, Dah (Zhang et al., 1996). A small and simple structured gene encoding a 110 kDa protein (DRP2) which consist of two spectrin-like repeats, the cysteine-rich and the C-terminal domains (like in Dp116), has also been characterized in vertebrates (Roberts et al., 1996).
Roberts and Bobrow (1998), using PCR with redundant primers, cloned fragments from the 3′ end region of the mRNAs of dystrophin related proteins from several organisms, including Drosphila, suggesting the widespread existence of such proteins in invertebrates. In a previous publication we reported on the identification of a sea urchin gene encoding a protein which is most likely an evolutionary homologue of Dp116, one of the small products of human dystrophin gene. The same gene also encoded a partially sequenced larger product, which we assumed to be a homologue of the full-length human dystrophin (Wang et al., 1998). Sea urchins are deuterostomes which diverged early from the evolutionary branch leading to primitive chordates and to vertebrates. To get a further insight into the evolution of the DMD gene and the function of its products, we attempted to characterize the dystrophin gene homologue of the protostome Drosophila. The molecular genetic information on Drosophila and the available genetic methodologies provide an excellent model system for a structural and functional analysis of the gene and its products.
In the present communication we report on the characterization of a Drosophila homologue of dystrophin gene and on the completion of the sequencing of the mRNA encoding the sea urchin dystrophin-like protein. Both genes encode large products homologous to the full-length dystrophin and utrophin, as well as smaller products transcribed from internal promoters. Like the smaller products of dystrophin and utrophin genes, the small products of the sea urchin and Drosophila genes lack the N-terminal actin-binding domain and a substantial part of the spectrin-like repeats. The complex structure of the gene, encoding both large full-length dystrophin-like proteins and smaller truncated products, with different patterns of expression, is very ancient and existed before the divergence between the protostomes and deuterostomes.
Section snippets
Isolation and sequencing of cDNA clones
A Drosophila 8–12 h embryos cDNA library in γgt11 was obtained from K. Zinn (Zinn et al., 1988). The library was screened using a 32P cDNA probe based on the partial sequence of Drosophila mRNA encoding a polypeptide with great similarity to the C-terminal region of dystrophin (accession no. X99757). Positive phages were purified, their cDNA inserts were amplified by PCR (expended high fidelity kit, Boeringer) using vector based primers, and cloned in the plasmid pGEMT (Promega). DNA was
The Drosophila homologue of the dystrophin gene
Using sequence data on the 3′ end coding region of a potential Drosophila homologue of dystrophin (Roberts and Bobrow, 1998), we isolated from a cDNA library of 8–12 h Drosophila embryos, partially overlapping cDNA clones of the corresponding mRNA. Among the isolated cDNA clones, which consisted of the previously described sequence, two contained additional 5′ sequences with a large open reading frame (ORF). The 3′ part of these two clones encoded an amino acid sequence with significant
The Drosophila and sea urchin dystrophin-like proteins
The present communication describes a Drosophila gene which seems to be a genuine homologue of the human dystrophin and utrophin genes. Three other Drosophila genes encoding proteins that share sequence similarity with regions in human dystrophin have been described earlier. One of them is a small and simple structured gene encoding a 75 kDa protein (Dah) with low sequence similarity to the C-terminal part of human dystrophin (Zhang et al., 1996). The other two genes encode large proteins
Note added in proof
Greener and Roberts (2000) reported recently on the squencing of a single product of the gene. It is a shorter, alternatively spliced isoform of dmDLP2 which is described in the present communication.
Acknowledgements
We wish to thank Dr R.G. Roberts for the unpublished sequence data (34), Dr Eyal Schejter for drosophila embryos and flies, Dr Adi Zalzberg for helping with the chromosomal mapping, Ora Fuchs for technical assistance and Vivienne Laufer for editorial assistance. This work was supported by the Israeli Academy of Science and Humanities, the Muscular Dystrophy Association, USA and the AFM, France. U.N. is the incumbent of the Elias Sourasky Professorial Chair at the Weizmann Institute of Science.
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Retinal dystrophins and the retinopathy of Duchenne muscular dystrophy
2023, Progress in Retinal and Eye ResearchCitation Excerpt :The current knowledge and working hypotheses regarding the roles of the distinct dystrophins in retinal physiology and visual functions is detailed in the next sections. Animal models of dystrophinopathies include non-mammals, such as the dystrophic fruit fly (Drosophila melanogaster; see Kreipke et al., 2017 for review) or zebrafish with a nonsense mutation at the N-terminal domain of the dystrophin (Berger and Currie, 2012), which have helped to understand some of the molecular interactions and biochemical functions of dystrophins (de León et al., 2005; Neuman et al., 2001; Plantié et al., 2015). They have also allowed testing small molecules to modulate phenotypes and exon-skipping strategies to rescue dystrophins expression (Berger et al., 2011; Kawahara and Kunkel, 2013; Kunkel et al., 2006; Pantoja and Ruohola-Baker, 2013; Zaynitdinova et al., 2021).
Animal models of Duchenne muscular dystrophy, with special reference to the mdx mouse
2012, Biocybernetics and Biomedical EngineeringCitation Excerpt :Smaller breeds of dog such as the beagle and the King Charles Cavalier spaniel with mutations in dystrophin have been identified or generated [45, 46] but this only goes a small way to address the problems of using dogs in MD research. Although mammalian models of DMD play an important role in preclinical studies of the disease, orthologues of dystrophin have been identified in lower vertebrates such as zebrafish and invertebrates such as Drosophila and the nematode Caenorhab-ditis elegans (C. elegans) [47, 48] (Table 2). These organisms are cheap and easy to maintain, while being easy to manipulate genetically.
Altered presynaptic ultrastructure in excitatory hippocampal synapses of mice lacking dystrophins Dp427 or Dp71
2011, Neurobiology of DiseaseCitation Excerpt :While dystrophins appear to be selectively expressed postsynaptically in brain, studies in mutant Drosophila recently stressed a possible involvement of this family of proteins in the retrograde regulation of synaptic transmitter release (Pilgram et al., 2010). The complex dystrophin-gene structure and production of multiple products of different sizes is conserved in evolutionary remote organisms, but sequence homology is limited across species (Neuman et al., 2001, 2005). Here we show for the first time that loss of the mammalian brain dystrophins, Dp427 and Dp71, is associated with ultrastructural presynaptic modifications of excitatory, most likely glutamatergic, synapses.
From action potential to contraction: Neural control and excitation-contraction coupling in larval muscles of Drosophila
2009, Comparative Biochemistry and Physiology - A Molecular and Integrative PhysiologyBiology of the Striated Muscle Dystrophin-Glycoprotein Complex
2008, International Review of CytologyCitation Excerpt :Thus, studies in C. elegans may help explain why defects in the dystrophin–glycoprotein complex cause perturbations in the neuromuscular junction without apparent functional consequence in mammals (Section 3.2). As in C. elegans, the Drosophila genome encodes homologues for dystrophin, dystroglycan, dystrobrevin, sarcoglycans, and syntrophin (Roberts and Bobrow, 1998; Neuman et al., 2001; Greener and Roberts, 2000). Consistent with studies in mammals (Section 3) and worms (Section 4.1), genetic and RNAi-mediated knockdown of dystrophin or dystroglycan in Drosophila causes decreased mobility and age-dependent muscle degeneration (Shcherbata et al., 2007).
Drosophila Dystrophin is required for integrity of the musculature
2007, Mechanisms of Development
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Accession numbers of sequences described here are: dmDp186 – AF300294; dmDLP2 – AF297644; the 5′ end regions of dmDLP1 and dmDLP3 – AF300456, AF302236, respectively; suDLP – AF304204.
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Present address: Kaplan Hospital, Rehovot, Israel.