The dystrophin / utrophin homologues in Drosophila and in sea urchin.

The gene which is defective in Duchenne muscular dystrophy (DMD) is the largest known gene containing at least 79 introns, some of which are extremely large. The product of the gene in muscle, dystrophin, is a 427 kDa protein. The same gene encodes at least two additional non-muscle full length dystrophin isoforms transcribed from different promoters located in the 5'-end region of the gene, and four smaller proteins transcribed from internal promoters located further downstream, and lack important domains of dystrophin. Several other genes, encoding evolutionarily related proteins, have been identified. To study the evolution of the DMD gene and the significance of its various products, we have searched for genes encoding dystrophin-like proteins in sea urchin and in Drosophila. We previously reported on the characterization of a sea urchin gene encoding a protein which is an evolutionary homologue of Dp116, one of the small products of the mammalian DMD gene, and on the partial sequencing of a large product of the same gene. Here we describe the full-length product which shows strong structural similarity and sequence identity to human dystrophin and utrophin. We also describe a Drosophila gene closely related to the human dystrophin gene. Like the human gene, the Drosophila gene encodes at least three isoforms of full length dystrophin-like proteins (dmDLP1, dmDLP2 and dmDLP3,), regulated by different promoters located at the 5' end of the gene, and a smaller product regulated by an internal promoter (dmDp186). As in mammals, dmDp186 and the dmDLPs share the same C-terminal and cysteine-rich domains which are very similar to the corresponding domains in human dystrophin and utrophin. In addition, dmDp186 contains four of the spectrin-like repeats of the dmDLPs and a unique N-terminal region of 512 amino acids encoded by a single exon. The full length products and the small product have distinct patterns of expression. Thus, the complex structure of the dystrophin gene, encoding several large dystrophin-like isoforms and smaller truncated products with different patterns of expression, existed before the divergence between the protostomes and deuterostomes. The conservation of this gene structure in such distantly related organisms, points to important distinct functions of the multiple products.

The cellular prion protein colocalizes with the dystroglycan complex in the brain.

The function of PrP(C), the cellular prion protein (PrP), is still unknown. Like other glycophosphatidylinositol-anchored proteins, PrP resides on Triton-insoluble, cholesterol-rich membranous microdomains, termed rafts. We have recently shown that the activity and subcellular localization of the neuronal isoform of nitric oxide synthase (nNOS) are impaired in adult PrP(0/0) mice as well as in scrapie-infected mice. In this study, we sought to determine whether PrP and nNOS are part of the same functional complex and, if so, to identify additional components of such a complex. To this aim, we looked for proteins that coimmunoprecipitated with PrP in the presence of detergents either that completely dissociate rafts, to identify stronger interactions, or that preserve the raft structure, to identify weaker interactions. Using this detergent-dependent immunoprecipitation protocol we found that PrP interacts strongly with dystroglycan, a transmembrane protein that is the core of the dystrophin-glycoprotein complex (DGC). Additional results suggest that PrP also interacts with additional members of the DGC, including nNOS. PrP coprecipitated only with established presynaptic proteins, consistent with recent findings suggesting that PrP is a presynaptic protein.

Targeted inactivation of Dp71, the major non-muscle product of the DMD gene: differential activity of the Dp71 promoter during development.

The dystrophin gene, which is defective in Duchenne muscular dystrophy (DMD), also encodes a number of smaller products controlled by internal promoters. Dp71, which consists of the two C-terminal domains of dystrophin, is the most abundant product of the gene in non-muscle tissues and is the major product in adult brain. To study the possible function of Dp71 and its expression during development, we specifically inactivated the expression of Dp71 by replacing its first and unique exon and a part of the concomitant intron with a beta-galactosidase reporter gene. X-Gal staining of Dp71-null mouse embryos and tissues revealed a very stage- and cell type-specific activity of the Dp71 promoter during development and during differentiation of various tissues, including the nervous system, eyes, limb buds, lungs, blood vessels, vibrissae and hair follicles. High activity of the Dp71 promoter often seemed to be associated with morphogenic events and terminal differentiation. In some tissues the activity greatly increased towards birth.

A sea urchin gene encoding dystrophin-related proteins.

The gene which is defective in Duchenne muscular dystrophy (DMD) is the largest known gene. The product of the gene in muscle, dystrophin, is a 427 kDa protein. The same gene encodes at least six additional products: two non-muscle dystrophin isoforms transcribed from promoters located in the 5'-end region of the gene and four smaller proteins transcribed from internal promoters located further downstream. Several other genes, encoding evolutionarily related proteins, have been identified. These include a structurally very similar gene in vertebrates encoding utrophin (DRP1), which is closely related to dystrophin, and a number of small and simple genes in vertebrates or invertebrates encoding proteins similar to some of the small products of the DMD gene. We have isolated a sea urchin gene showing very strong sequence and structural homology with the DMD and utrophin genes. Sequence and intron/exon structure similarities suggest that this gene is related to a precursor of both the DMD gene and the gene encoding utrophin. The sea urchin gene has the unique complex structure of the DMD gene. There is at least one, and possibly more, product(s) transcribed from internal promoters, as well as a large product of >300 kDa containing at least three of the four major domains of dystrophin. The small product seems to be evolutionarily related to Dp116, one of the small products of the human DMD gene. Partial characterization of this gene helped us to construct an evolutionary tree connecting the vertebrate dystrophin gene family with related genes in invertebrates. The constructed evolutionary tree also implies that the vertebrate small and simple structured gene encoding a Dp71-like protein, called DRP2 , evolved from the dystrophin/utrophin ancestral large and complex gene by a duplication of only a small part of the gene.

A Leuconostoc lactis protein with homology to ribosomal protein S1 shares common epitopes and common DNA binding properties with a mammalian DNA binding nuclear factor.

A mouse testis cDNA expression library (Clontech) was screened with a synthetic oligonucleotide ligand containing CT-rich motifs derived from the rat skeletal muscle actin gene promoter. These motifs bind nuclear proteins, and seem to be involved in the regulation of the gene. Analysis of isolated clones, which expressed proteins that specifically bind the oligonucleotide, indicated that they were derived from a single gene. This gene was identified as a contaminant of bacterial origin (Leuconostoc lactis). The cloned gene from L. lactis encodes a protein with significant homology to bacterial ribosomal protein S1, which we designated LrpS1-L. Band shift analysis and competition experiments indicated that both the bacterial protein and a mouse nuclear protein specifically bind to the same CT-rich motif of the skeletal muscle actin promoter. Furthermore, antibodies against the recombinant bacterial protein interfered with the formation of complex between the CT-rich element and the mouse nuclear protein. These results indicate that the bacterial LrpS1-L protein and the mammalian protein bind the same CT-rich motif and share common antigenic epitopes.

Reduced levels of dystrophin associated proteins in the brains of mice deficient for Dp71.

Duchenne muscular dystrophy (DMD) is a progressive degenerative lethal muscle disease. A significant proportion of DMD affected children suffer also from mental retardation. The rod shaped protein, dystrophin, which is absent from or defective in the muscle of DMD patients, binds to a number of membrane associated proteins (known collectively as dystrophin associated proteins [DAPs]). The levels of DAPs is greatly reduced in the muscle of DMD patients and mdx mice, which lack dystrophin. In addition to dystrophin isoforms, the DMD gene codes also for several smaller proteins. One of the small proteins, Dp71, is expressed in most or all non-muscle tissues and is the major DMD gene product in the brain. The function of the small DMD gene products is unknown. Here we show that mutant mice which do not express the smaller non-muscle products of the DMD gene have a reduced level of DAPs in their brain. This suggests that Dp71 is important for the formation and/or stabilization of a DAPs complex in brain.

Exogenous Dp71 restores the levels of dystrophin associated proteins but does not alleviate muscle damage in mdx mice.

Dp71 is a non-muscle product of the Duchenne muscular dystrophy gene. It consists of the cysteine-rich and C-terminal domains of dystrophin. We have generated transgenic mdx mice which do not have dystrophin but express Dp71 in their muscle. In these mice, Dp71 was localized to the plasma membrane and restored normal levels of dystrophin associated proteins (DAPs), indicating that Dp71 is capable of interacting with the DAPs in a similar manner to dystrophin. However, the presence of Dp71 and DAPs in the muscle fibres of mdx mice was not sufficient to alleviate symptoms of muscle degeneration.

Characterization and localization of a 77 kDa protein related to the dystrophin gene family.

The Duchenne muscular dystrophy gene gives rise to transcripts of several lengths. These mRNAs differ in their coding content and tissue distribution. The 14 kb mRNA encodes dystrophin, a 427 kDa protein found in muscle and brain, and the short transcripts described encode DP71, a 77 kDa protein found in various organs. These short transcripts have many features common to the deduced primary structure of dystrophin, especially in the cysteine-rich specific C-terminal domains. The dystrophin C-terminal domain could be involved in membrane anchorage via the glycoprotein complex, but such a functional role for these short transcript products has yet to be demonstrated. Here we report the first isolation of a short transcript product from saponin-solubilized cardiac muscle membranes using alkaline buffer and affinity chromatography procedures. This molecule was found to be glycosylated and could be easily dissociated from cardiac muscle and other non-muscle tissues such as brain and liver. DP71-specific monoclonal antibody helped to identify this molecule as being related to the dystrophin gene family. Immunofluorescence analysis of bovine or chicken cardiac muscle showed a periodic distribution of DP71 in transverse T tubules and this protein was co-localized with the dystrophin glycoprotein complex in the Z-disk area.

Detection of Duchenne muscular dystrophy gene products in amniotic fluid and chorionic villus sampling cells.

We have examined the expression of several Duchenne muscular dystrophy (DMD) gene products in amniotic fluid (AF) and chorionic villus sampling (CVS) cells. Variable amounts of dystrophin could be detected in most CVS and AF samples by immunoprecipitation followed by Western blot analysis. PCR analysis demonstrated the presence of the muscle type dystrophin mRNA in all AF cell cultures. The brain type dystrophin mRNA was also detected in some of these cultures. These DMD gene transcripts are of fetal origin and are produced by most or all clonable AF cells. The results may facilitate the development of a method for prenatal diagnosis of DMD, based on the expression of the gene in AF and CVS cells.

A housekeeping type promoter, located in the 3' region of the Duchenne muscular dystrophy gene, controls the expression of Dp71, a major product of the gene.

The 70.8 kDa protein product of the distal part of the giant Duchenne muscular dystrophy (DMD) gene, Dp71, is expressed in many cell types and tissues. Anchored PCR, primer extension and functional analysis of transfected constructs were used to determine the 5' end of the mRNA and characterize the promoter of this major DMD gene product. The 5' untranslated region (5'UTR) of Dp71 is transcribed from a single exon; the promoter does not contain a TATA box, and has a very high GC content and several potential Sp1 binding sites. It is located more than 2000 kb 3' to the muscle and brain type dystrophin promoters and only 150 kb from the 3' end of the gene, suggesting that in most DMD patients the expression of Dp71 is unaffected.

Dp71, the nonmuscle product of the Duchenne muscular dystrophy gene is associated with the cell membrane.

The 70.8 kDa protein, Dp71, is the major Duchenne muscular dystrophy (DMD) gene product in many nonmuscle tissues including the brain. Dp71 shares most of the C-terminal and cysteine-rich domains with the dystrophins but lacks the entire large rod shaped domain of spectrin-like repeats, and the N-terminal actin-binding domain. The function of Dp71 is unknown. Using subcellular fractionation and immunostaining we show that Dp71 is associated with the plasma membrane. Dp71 is also associated with the plasma membrane in mdx myogenic cells transfected with a vector expressing Dp71.

Expression of the Duchenne muscular dystrophy gene products in embryonic stem cells and their differentiated derivatives.

Three protein products of the Duchenne muscular dystrophy (DMD) gene were identified so far. These include the two very similar muscle and brain type dystrophins, which are encoded by 14-kilobase (kb) mRNAs, and Dp71, which is much smaller. Dp71 is encoded by a 6.5-kb mRNA, which is transcribed from approximately 6% of the giant dystrophin gene. The present investigation shows that Dp71 is the first product of the DMD gene detectable during development. It is already expressed in the pluripotent embryonic stem cells. The two 14-kb mRNAs encoding the dystrophins are detectable only after differentiation of specialized cell types. The possible implication of these findings with regard to the ontogenetic activation and the evolution of the DMD gene are discussed.

A 71-kilodalton protein is a major product of the Duchenne muscular dystrophy gene in brain and other nonmuscle tissues.

The known Duchenne muscular dystrophy (DMD) gene products, the muscle- and brain-type dystrophin isoforms, are 427-kDa proteins translated from 14-kilobase (kb) mRNAs. Recently we described a 6.5-kb mRNA that also is transcribed from the DMD gene. Cloning and in vitro transcription and translation of the entire coding region show that the 6.5-kb mRNA encodes a 70.8-kDa protein that is a major product of the DMD gene. It contains the C-terminal and the cysteine-rich domains of dystrophin, seven additional amino acids at the N terminus, and some modifications formed by alternative splicing in the C-terminal domain. It lacks the entire large domain of spectrin-like repeats and the actin-binding N-terminal domain of dystrophin. This protein is the major DMD gene product in brain and other nonmuscle tissues but is undetectable in skeletal muscle extracts.

Characterization and cell type distribution of a novel, major transcript of the Duchenne muscular dystrophy gene.

Previously we identified a novel 6.5 kb mRNA transcribed from the Duchenne muscular dystrophy (DMD) gene. This mRNA differs in coding content and tissue distribution from the known muscle type and brain type 14 kb DMD mRNAs which code for dystrophin. The novel transcript shares with dystrophin most of the sequence coding for the cysteine-rich and C-terminal domains. Here we used cDNA cloning to identify the divergence point between the common region and the sequence unique to the novel mRNA at the 5' end of the sequence encoding the cysteine-rich domain of dystrophin. This unique sequence containing the translation initiation site is located in a new exon in the intron between exons 62 and 63 of the dystrophin gene. Using probes containing RNA sequences specific to the novel mRNA, we investigated the expression of this mRNA in various tissues and cell types. The study reveals that this mRNA is the main DMD gene product detectable in a variety of nonmuscle tissues including brain cells. The amount of this mRNA in some tissues is comparable to the amount of dystrophin mRNA in the muscle. The expression of the 6.5 kb mRNA is down-regulated during differentiation of myogenic cells; it is present in small amounts in proliferating myoblasts but is undetected in differentiated muscle cultures depleted of mononucleated cells.

Multiple products of the Duchenne muscular dystrophy gene.

The gene which is defective in Duchenne Muscular Dystrophy (DMD) extends over 2300 kb of the X chromosome. Its product in the muscle is a 14 kb mRNA encoding a 427 kd rod-shaped protein called dystrophin. A 14 kb transcript encoding a very similar isoform of dystrophin is produced in the brain. The brain 14 kb mRNA is transcribed from the same gene but controlled by a different promoter, located at least 75 kb upstream from the muscle dystrophin promoter. The regulation of these promoters is very stringently controlled. The muscle-type but not the brain-type dystrophin mRNA is found in cloned skeletal muscle cells and its presence is correlated with the appearance of multinucleated fibers. The brain type is expressed in neurons, while in glia cells the muscle-type promoter is active. A third DMD gene transcript which is only 6.5 kb long has been identified. It contains the sequence coding for the C-terminal domain and the cysteine-rich domain of dystrophin but not the large region encoding the spectrin-like repeats and the N-terminal domain. The cell type distribution of this transcript is also very different from that of the two 14 kb mRNA isoforms. It is the major product of the DMD gene in many nonmuscle tissues including brain. Using monoclonal antibodies we have identified a 77 Kd protein which seems to be the translation product of this mRNA. As expected from the distribution of the 6.5 Kb mRNA, this protein is the major DMD gene product detectable in brain and many other nonmuscle tissues; it is undetectable in skeletal muscle but is present in the heart and stomach (as is the 6.5 Kb mRNA).

A deletion including the brain promoter of the Duchenne muscular dystrophy gene is not associated with mental retardation.

A total of 161 unrelated Duchenne (DMD) and Becker muscular dystrophy (BMD) patients were screened for deletions in the brain promoter region of the dystrophin gene. Southern blot analysis using a probe for the brain promoter detected a deletion in this region in only one of the DMD families, in a patient with normal intelligence. This deletion also included the promoter of the muscle-type dystrophin and the exons encoding the actin-binding and part of the spectrin-like domains. Our data suggest that deletions in the brain promoter region are rare in DMD and are compatible with normal intelligence.

Mapping of dystrophin brain promoter: a deletion of this region is compatible with normal intellect.

Using a mouse genomic fragment containing the brain-specific promoter region of the dystrophin gene, we have located the brain promoter 75-300 kb proximal of the muscle promoter. Within our DMD-families we detected a patient who lacks both the brain-specific and muscle-specific promoter sequences. The normal intellectual capabilities of the patient argue against an indispensable role of the brain-specific first exon in mental functioning. The possibility exists that a NH2-terminally truncated dystrophin has taken over the function of the normal dystrophins in brain and/or muscle.

Brain-type and muscle-type promoters of the dystrophin gene differ greatly in structure.

The promoter of the 14 kb mRNA encoding the brain isoform of dystrophin in the mouse has been isolated and partially characterized. Unlike the promoter of the muscle dystrophin isoform, it does not contain a TATA box or other consensus sequences characteristic of the proximal region upstream of the cap sites of eukaryotic genes. Yet, it has a major initiation of transcription start site located 266 bp upstream from the first ATG which is in frame with the dystrophin coding sequence. The 5' untranslated region contains nine additional ATG triplets which are not in-frame with the coding sequence or are followed by stop codons. A DNA fragment extending from bp -1149 to +11 is sufficient to activate a reporter gene lacking a promoter in transfected neuroblastoma cells.

A novel product of the Duchenne muscular dystrophy gene which greatly differs from the known isoforms in its structure and tissue distribution.

A novel transcript of the Duchenne muscular dystrophy gene has been identified. This 6.5 kb mRNA contains sequences from the 3' untranslated region of dystrophin mRNA and from the regions coding for the C-terminal and the cysteine-rich domains. However, probes for the regions encoding the spectrin-like repeats and the actin-binding domain, as well as probes for the first exons of the muscle- and brain-type dystrophin mRNA, did not hybridize with this new mRNA. Significant amounts of the 6.5 kb mRNA were found in a variety of non-muscle tissues, such as liver, testis, lung and kidney, but not in skeletal muscle. The abundance of this mRNA in the brain is at least as high as that of the previously described 14 kb brain-type dystrophin mRNA.