Desmin-related myopathies (DRM) are inherited neuromuscular disorders characterized by adult onse... more Desmin-related myopathies (DRM) are inherited neuromuscular disorders characterized by adult onset and delayed accumulation of aggregates of desmin, a protein belonging to the type III intermediate filament family, in the sarcoplasma of skeletal and cardiac muscles. In this paper, we have mapped the locus for DRM in a large French pedigree to a 26-cM interval in chromosome 11q21–23. This region contains the αB-crystallin gene (CRYAB), a candidate gene encoding a 20-kD protein that is abundant in lens and is also present in a number of non-ocular tissues, including cardiac and skeletal muscle. αB-crystallin is a member of the small heat shock protein (shsp) family and possesses molecular chaperone activity. We identified an R120G missense mutation in CRYAB that co-segregates with the disease phenotype in this family. Muscle cell lines transfected with the mutant CRYAB cDNA showed intracellular aggregates that contain both desmin and αB-crystallin as observed in muscle fibers from DRM patients. These results are the first to identify a defect in a molecular chaperone as a cause for an inherited human muscle disorder.
In mouse cells transformed with the ts-a mutant of polyoma virus (ts-a-3T3), only low amounts of ... more In mouse cells transformed with the ts-a mutant of polyoma virus (ts-a-3T3), only low amounts of the virus-specific T antigen were synthesized at high temperature (39 C). After a shift-down to the permissive temperature (31 C), these cells exhibited the same level of T-antigen production as wild-type polyoma transformants. The T antigen produced by ts-a-transformed cells was inactivated at 39 C in vitro at a faster rate than that produced by wild-type-transformed cells. These observations indicate that T antigen is, or includes, a virus-coded peptide.
We have previously reported that high level human desmin expression depends on a 280-base pair mu... more We have previously reported that high level human desmin expression depends on a 280-base pair muscle-specific enhancer which can function not only in myotubes, but can also activate gene expression in myoblasts. We report here that this enhancer contains two different regions, one active in myotubes and the other in myoblasts. In the myotube-specific region, one MyoD1 site and one MEF2 site are necessary for full enhancer activity. Site-directed mutation of the MyoD1 binding site revealed that the intact site is essential for gene expression in myotubes and for transactivation by MyoD1 or myogenin in co-transfected fibroblasts. In the myoblast-specific region, four regions are protected by nuclear factors from the myogenic cell line C2, 7; three regions contain a GC-rich sequence sharing homology with the Krox binding site. Deletion and site-directed mutation experiments demonstrated that at least two Krox-like sequences are required for enhancer activity in myoblasts. In addition, another GC-rich sequence, designated Mb, is also required for full enhancer activity in myoblasts.
Transgenic C57 mice bearing a transgene of the desmin gene linked to the lacZ reporter gene which... more Transgenic C57 mice bearing a transgene of the desmin gene linked to the lacZ reporter gene which encoded for the enzyme beta-galactosidase were used. In the muscle cell, a blue nuclear product appearing in the presence of the X-gal substrate for the enzyme provided evidence of the expression of the desmin gene. However, no transgene expression was observed 2 weeks postnatal in skeletal muscles, even though endogenous desmin was present. In order to investigate the regulatory mechanisms of the desmin gene during regeneration, adult pectoralis fragments (without expression of the desmin transgene) from transgenic mice were implanted into the tibialis anterior of 4 day or 6 week old Swiss mice. Adult pectoralis transplants reexpressed the transgene from day 4 to 10 after implantation. In addition, lesions were performed in adult transgenic pectoralis and transgenic expression in injured muscles was observed 2 days later. This new transgenic mouse is a powerful tool for the study of the various steps of skeletal muscle regeneration.
Desmin-related myopathies (DRM) are inherited neuromuscular disorders characterized by adult onse... more Desmin-related myopathies (DRM) are inherited neuromuscular disorders characterized by adult onset and delayed accumulation of aggregates of desmin, a protein belonging to the type III intermediate filament family, in the sarcoplasma of skeletal and cardiac muscles. In this paper, we have mapped the locus for DRM in a large French pedigree to a 26-cM interval in chromosome 11q21–23. This region contains the αB-crystallin gene (CRYAB), a candidate gene encoding a 20-kD protein that is abundant in lens and is also present in a number of non-ocular tissues, including cardiac and skeletal muscle. αB-crystallin is a member of the small heat shock protein (shsp) family and possesses molecular chaperone activity. We identified an R120G missense mutation in CRYAB that co-segregates with the disease phenotype in this family. Muscle cell lines transfected with the mutant CRYAB cDNA showed intracellular aggregates that contain both desmin and αB-crystallin as observed in muscle fibers from DRM patients. These results are the first to identify a defect in a molecular chaperone as a cause for an inherited human muscle disorder.
In mouse cells transformed with the ts-a mutant of polyoma virus (ts-a-3T3), only low amounts of ... more In mouse cells transformed with the ts-a mutant of polyoma virus (ts-a-3T3), only low amounts of the virus-specific T antigen were synthesized at high temperature (39 C). After a shift-down to the permissive temperature (31 C), these cells exhibited the same level of T-antigen production as wild-type polyoma transformants. The T antigen produced by ts-a-transformed cells was inactivated at 39 C in vitro at a faster rate than that produced by wild-type-transformed cells. These observations indicate that T antigen is, or includes, a virus-coded peptide.
We have previously reported that high level human desmin expression depends on a 280-base pair mu... more We have previously reported that high level human desmin expression depends on a 280-base pair muscle-specific enhancer which can function not only in myotubes, but can also activate gene expression in myoblasts. We report here that this enhancer contains two different regions, one active in myotubes and the other in myoblasts. In the myotube-specific region, one MyoD1 site and one MEF2 site are necessary for full enhancer activity. Site-directed mutation of the MyoD1 binding site revealed that the intact site is essential for gene expression in myotubes and for transactivation by MyoD1 or myogenin in co-transfected fibroblasts. In the myoblast-specific region, four regions are protected by nuclear factors from the myogenic cell line C2, 7; three regions contain a GC-rich sequence sharing homology with the Krox binding site. Deletion and site-directed mutation experiments demonstrated that at least two Krox-like sequences are required for enhancer activity in myoblasts. In addition, another GC-rich sequence, designated Mb, is also required for full enhancer activity in myoblasts.
Transgenic C57 mice bearing a transgene of the desmin gene linked to the lacZ reporter gene which... more Transgenic C57 mice bearing a transgene of the desmin gene linked to the lacZ reporter gene which encoded for the enzyme beta-galactosidase were used. In the muscle cell, a blue nuclear product appearing in the presence of the X-gal substrate for the enzyme provided evidence of the expression of the desmin gene. However, no transgene expression was observed 2 weeks postnatal in skeletal muscles, even though endogenous desmin was present. In order to investigate the regulatory mechanisms of the desmin gene during regeneration, adult pectoralis fragments (without expression of the desmin transgene) from transgenic mice were implanted into the tibialis anterior of 4 day or 6 week old Swiss mice. Adult pectoralis transplants reexpressed the transgene from day 4 to 10 after implantation. In addition, lesions were performed in adult transgenic pectoralis and transgenic expression in injured muscles was observed 2 days later. This new transgenic mouse is a powerful tool for the study of the various steps of skeletal muscle regeneration.
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