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Yuji Mizuno - One of the best experts on this subject based on the ideXlab platform.
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genomic organization and single nucleotide polymorphism map of desmuslin a novel Intermediate Filament Protein on chromosome 15q26 3
BMC Genetics, 2001Co-Authors: Annibale A Puca, Yuji Mizuno, Alan H Beggs, Kristine F Obrien, Louis M KunkelAbstract:Desmuslin is an α-dystrobrevin-interacting Protein expressed primarily in heart and skeletal muscle. The desmuslin Protein interacts with and is closely related to desmin, a Protein encoded by a locus mutated in some forms of hereditary distal myopathy. As a muscle-specific Intermediate Filament Protein, desmuslin is also a candidate for myopathies of unknown etiology. The desmuslin gene was localized to chromosome 15q26.3 by electronic screening of the human DNA sequence database. Primer pairs were designed to amplify the 5 exons of the desmuslin gene in 11 overlapping DNA segments. The desmuslin gene was screened for mutations in 71 patients with various forms of myopathy for which there was no known cause. In this analysis, 10 common and 2 rare amino acid altering single-nucleotide polymorphisms were identified, all of which were seen in a control population of individuals thus making these unlikely causes of the phenotype. Interestingly, one of the single-nucleotide polymorphisms found in a patient resulted in a premature stop codon in the first exon. The nonsense mutation was also detected in the patient's unaffected father and one unaffected control; it was detected in 0.44% (2/454) of unrelated chromosomes and is therefore predicted to have a homozygous frequency of 0.002%. No causative mutations were found in the desmuslin gene. However, the single-nucleotide polymorphisms mapped in this study represent a well-mapped group that can be used for disequilibrium studies of this region of chromosome 15q26.3.
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Genomic organization and single-nucleotide polymorphism map of desmuslin, a novel Intermediate Filament Protein on chromosome 15q26.3
BMC Genetics, 2001Co-Authors: Yuji Mizuno, Annibale A Puca, Kristine F O'brien, Alan H Beggs, Louis M KunkelAbstract:Background Desmuslin is an α-dystrobrevin-interacting Protein expressed primarily in heart and skeletal muscle. The desmuslin Protein interacts with and is closely related to desmin, a Protein encoded by a locus mutated in some forms of hereditary distal myopathy. As a muscle-specific Intermediate Filament Protein, desmuslin is also a candidate for myopathies of unknown etiology. Results The desmuslin gene was localized to chromosome 15q26.3 by electronic screening of the human DNA sequence database. Primer pairs were designed to amplify the 5 exons of the desmuslin gene in 11 overlapping DNA segments. The desmuslin gene was screened for mutations in 71 patients with various forms of myopathy for which there was no known cause. In this analysis, 10 common and 2 rare amino acid altering single-nucleotide polymorphisms were identified, all of which were seen in a control population of individuals thus making these unlikely causes of the phenotype. Interestingly, one of the single-nucleotide polymorphisms found in a patient resulted in a premature stop codon in the first exon. The nonsense mutation was also detected in the patient's unaffected father and one unaffected control; it was detected in 0.44% (2/454) of unrelated chromosomes and is therefore predicted to have a homozygous frequency of 0.002%. Conclusion No causative mutations were found in the desmuslin gene. However, the single-nucleotide polymorphisms mapped in this study represent a well-mapped group that can be used for disequilibrium studies of this region of chromosome 15q26.3.
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Desmuslin, an Intermediate Filament Protein that interacts with α-dystrobrevin and desmin
Proceedings of the National Academy of Sciences, 2001Co-Authors: Yuji Mizuno, Michihiro Imamura, Eijiro Ozawa, Terri G. Thompson, Jeffrey Robert Guyon, Hart G.w. Lidov, Melissa Brosius, Simon C. Watkins, Louis M KunkelAbstract:Abstract Dystrobrevin is a component of the dystrophin-associated Protein complex and has been shown to interact directly with dystrophin, α1-syntrophin, and the sarcoglycan complex. The precise role of α-dystrobrevin in skeletal muscle has not yet been determined. To study α-dystrobrevin's function in skeletal muscle, we used the yeast two-hybrid approach to look for interacting Proteins. Three overlapping clones were identified that encoded an Intermediate Filament Protein we subsequently named desmuslin (DMN). Sequence analysis revealed that DMN has a short N-terminal domain, a conserved rod domain, and a long C-terminal domain, all common features of type 6 Intermediate Filament Proteins. A positive interaction between DMN and α-dystrobrevin was confirmed with an in vitro coimmunoprecipitation assay. By Northern blot analysis, we find that DMN is expressed mainly in heart and skeletal muscle, although there is some expression in brain. Western blotting detected a 160-kDa Protein in heart and skeletal muscle. Immunofluorescent microscopy localizes DMN in a stripe-like pattern in longitudinal sections and in a mosaic pattern in cross sections of skeletal muscle. Electron microscopic analysis shows DMN colocalized with desmin at the Z-lines. Subsequent coimmunoprecipitation experiments confirmed an interaction with desmin. Our findings suggest that DMN may serve as a direct linkage between the extracellular matrix and the Z-discs (through plectin) and may play an important role in maintaining muscle cell integrity.
Louis M Kunkel - One of the best experts on this subject based on the ideXlab platform.
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genomic organization and single nucleotide polymorphism map of desmuslin a novel Intermediate Filament Protein on chromosome 15q26 3
BMC Genetics, 2001Co-Authors: Annibale A Puca, Yuji Mizuno, Alan H Beggs, Kristine F Obrien, Louis M KunkelAbstract:Desmuslin is an α-dystrobrevin-interacting Protein expressed primarily in heart and skeletal muscle. The desmuslin Protein interacts with and is closely related to desmin, a Protein encoded by a locus mutated in some forms of hereditary distal myopathy. As a muscle-specific Intermediate Filament Protein, desmuslin is also a candidate for myopathies of unknown etiology. The desmuslin gene was localized to chromosome 15q26.3 by electronic screening of the human DNA sequence database. Primer pairs were designed to amplify the 5 exons of the desmuslin gene in 11 overlapping DNA segments. The desmuslin gene was screened for mutations in 71 patients with various forms of myopathy for which there was no known cause. In this analysis, 10 common and 2 rare amino acid altering single-nucleotide polymorphisms were identified, all of which were seen in a control population of individuals thus making these unlikely causes of the phenotype. Interestingly, one of the single-nucleotide polymorphisms found in a patient resulted in a premature stop codon in the first exon. The nonsense mutation was also detected in the patient's unaffected father and one unaffected control; it was detected in 0.44% (2/454) of unrelated chromosomes and is therefore predicted to have a homozygous frequency of 0.002%. No causative mutations were found in the desmuslin gene. However, the single-nucleotide polymorphisms mapped in this study represent a well-mapped group that can be used for disequilibrium studies of this region of chromosome 15q26.3.
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Genomic organization and single-nucleotide polymorphism map of desmuslin, a novel Intermediate Filament Protein on chromosome 15q26.3
BMC Genetics, 2001Co-Authors: Yuji Mizuno, Annibale A Puca, Kristine F O'brien, Alan H Beggs, Louis M KunkelAbstract:Background Desmuslin is an α-dystrobrevin-interacting Protein expressed primarily in heart and skeletal muscle. The desmuslin Protein interacts with and is closely related to desmin, a Protein encoded by a locus mutated in some forms of hereditary distal myopathy. As a muscle-specific Intermediate Filament Protein, desmuslin is also a candidate for myopathies of unknown etiology. Results The desmuslin gene was localized to chromosome 15q26.3 by electronic screening of the human DNA sequence database. Primer pairs were designed to amplify the 5 exons of the desmuslin gene in 11 overlapping DNA segments. The desmuslin gene was screened for mutations in 71 patients with various forms of myopathy for which there was no known cause. In this analysis, 10 common and 2 rare amino acid altering single-nucleotide polymorphisms were identified, all of which were seen in a control population of individuals thus making these unlikely causes of the phenotype. Interestingly, one of the single-nucleotide polymorphisms found in a patient resulted in a premature stop codon in the first exon. The nonsense mutation was also detected in the patient's unaffected father and one unaffected control; it was detected in 0.44% (2/454) of unrelated chromosomes and is therefore predicted to have a homozygous frequency of 0.002%. Conclusion No causative mutations were found in the desmuslin gene. However, the single-nucleotide polymorphisms mapped in this study represent a well-mapped group that can be used for disequilibrium studies of this region of chromosome 15q26.3.
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Desmuslin, an Intermediate Filament Protein that interacts with α-dystrobrevin and desmin
Proceedings of the National Academy of Sciences, 2001Co-Authors: Yuji Mizuno, Michihiro Imamura, Eijiro Ozawa, Terri G. Thompson, Jeffrey Robert Guyon, Hart G.w. Lidov, Melissa Brosius, Simon C. Watkins, Louis M KunkelAbstract:Abstract Dystrobrevin is a component of the dystrophin-associated Protein complex and has been shown to interact directly with dystrophin, α1-syntrophin, and the sarcoglycan complex. The precise role of α-dystrobrevin in skeletal muscle has not yet been determined. To study α-dystrobrevin's function in skeletal muscle, we used the yeast two-hybrid approach to look for interacting Proteins. Three overlapping clones were identified that encoded an Intermediate Filament Protein we subsequently named desmuslin (DMN). Sequence analysis revealed that DMN has a short N-terminal domain, a conserved rod domain, and a long C-terminal domain, all common features of type 6 Intermediate Filament Proteins. A positive interaction between DMN and α-dystrobrevin was confirmed with an in vitro coimmunoprecipitation assay. By Northern blot analysis, we find that DMN is expressed mainly in heart and skeletal muscle, although there is some expression in brain. Western blotting detected a 160-kDa Protein in heart and skeletal muscle. Immunofluorescent microscopy localizes DMN in a stripe-like pattern in longitudinal sections and in a mosaic pattern in cross sections of skeletal muscle. Electron microscopic analysis shows DMN colocalized with desmin at the Z-lines. Subsequent coimmunoprecipitation experiments confirmed an interaction with desmin. Our findings suggest that DMN may serve as a direct linkage between the extracellular matrix and the Z-discs (through plectin) and may play an important role in maintaining muscle cell integrity.
Annibale A Puca - One of the best experts on this subject based on the ideXlab platform.
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Genomic organization and single-nucleotide polymorphism map of desmuslin, a novel Intermediate Filament Protein on chromosome 15q26.3
BMC Genetics, 2001Co-Authors: Yuji Mizuno, Annibale A Puca, Kristine F O'brien, Alan H Beggs, Louis M KunkelAbstract:Background Desmuslin is an α-dystrobrevin-interacting Protein expressed primarily in heart and skeletal muscle. The desmuslin Protein interacts with and is closely related to desmin, a Protein encoded by a locus mutated in some forms of hereditary distal myopathy. As a muscle-specific Intermediate Filament Protein, desmuslin is also a candidate for myopathies of unknown etiology. Results The desmuslin gene was localized to chromosome 15q26.3 by electronic screening of the human DNA sequence database. Primer pairs were designed to amplify the 5 exons of the desmuslin gene in 11 overlapping DNA segments. The desmuslin gene was screened for mutations in 71 patients with various forms of myopathy for which there was no known cause. In this analysis, 10 common and 2 rare amino acid altering single-nucleotide polymorphisms were identified, all of which were seen in a control population of individuals thus making these unlikely causes of the phenotype. Interestingly, one of the single-nucleotide polymorphisms found in a patient resulted in a premature stop codon in the first exon. The nonsense mutation was also detected in the patient's unaffected father and one unaffected control; it was detected in 0.44% (2/454) of unrelated chromosomes and is therefore predicted to have a homozygous frequency of 0.002%. Conclusion No causative mutations were found in the desmuslin gene. However, the single-nucleotide polymorphisms mapped in this study represent a well-mapped group that can be used for disequilibrium studies of this region of chromosome 15q26.3.
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genomic organization and single nucleotide polymorphism map of desmuslin a novel Intermediate Filament Protein on chromosome 15q26 3
BMC Genetics, 2001Co-Authors: Annibale A Puca, Yuji Mizuno, Alan H Beggs, Kristine F Obrien, Louis M KunkelAbstract:Desmuslin is an α-dystrobrevin-interacting Protein expressed primarily in heart and skeletal muscle. The desmuslin Protein interacts with and is closely related to desmin, a Protein encoded by a locus mutated in some forms of hereditary distal myopathy. As a muscle-specific Intermediate Filament Protein, desmuslin is also a candidate for myopathies of unknown etiology. The desmuslin gene was localized to chromosome 15q26.3 by electronic screening of the human DNA sequence database. Primer pairs were designed to amplify the 5 exons of the desmuslin gene in 11 overlapping DNA segments. The desmuslin gene was screened for mutations in 71 patients with various forms of myopathy for which there was no known cause. In this analysis, 10 common and 2 rare amino acid altering single-nucleotide polymorphisms were identified, all of which were seen in a control population of individuals thus making these unlikely causes of the phenotype. Interestingly, one of the single-nucleotide polymorphisms found in a patient resulted in a premature stop codon in the first exon. The nonsense mutation was also detected in the patient's unaffected father and one unaffected control; it was detected in 0.44% (2/454) of unrelated chromosomes and is therefore predicted to have a homozygous frequency of 0.002%. No causative mutations were found in the desmuslin gene. However, the single-nucleotide polymorphisms mapped in this study represent a well-mapped group that can be used for disequilibrium studies of this region of chromosome 15q26.3.
Alan H Beggs - One of the best experts on this subject based on the ideXlab platform.
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Genomic organization and single-nucleotide polymorphism map of desmuslin, a novel Intermediate Filament Protein on chromosome 15q26.3
BMC Genetics, 2001Co-Authors: Yuji Mizuno, Annibale A Puca, Kristine F O'brien, Alan H Beggs, Louis M KunkelAbstract:Background Desmuslin is an α-dystrobrevin-interacting Protein expressed primarily in heart and skeletal muscle. The desmuslin Protein interacts with and is closely related to desmin, a Protein encoded by a locus mutated in some forms of hereditary distal myopathy. As a muscle-specific Intermediate Filament Protein, desmuslin is also a candidate for myopathies of unknown etiology. Results The desmuslin gene was localized to chromosome 15q26.3 by electronic screening of the human DNA sequence database. Primer pairs were designed to amplify the 5 exons of the desmuslin gene in 11 overlapping DNA segments. The desmuslin gene was screened for mutations in 71 patients with various forms of myopathy for which there was no known cause. In this analysis, 10 common and 2 rare amino acid altering single-nucleotide polymorphisms were identified, all of which were seen in a control population of individuals thus making these unlikely causes of the phenotype. Interestingly, one of the single-nucleotide polymorphisms found in a patient resulted in a premature stop codon in the first exon. The nonsense mutation was also detected in the patient's unaffected father and one unaffected control; it was detected in 0.44% (2/454) of unrelated chromosomes and is therefore predicted to have a homozygous frequency of 0.002%. Conclusion No causative mutations were found in the desmuslin gene. However, the single-nucleotide polymorphisms mapped in this study represent a well-mapped group that can be used for disequilibrium studies of this region of chromosome 15q26.3.
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genomic organization and single nucleotide polymorphism map of desmuslin a novel Intermediate Filament Protein on chromosome 15q26 3
BMC Genetics, 2001Co-Authors: Annibale A Puca, Yuji Mizuno, Alan H Beggs, Kristine F Obrien, Louis M KunkelAbstract:Desmuslin is an α-dystrobrevin-interacting Protein expressed primarily in heart and skeletal muscle. The desmuslin Protein interacts with and is closely related to desmin, a Protein encoded by a locus mutated in some forms of hereditary distal myopathy. As a muscle-specific Intermediate Filament Protein, desmuslin is also a candidate for myopathies of unknown etiology. The desmuslin gene was localized to chromosome 15q26.3 by electronic screening of the human DNA sequence database. Primer pairs were designed to amplify the 5 exons of the desmuslin gene in 11 overlapping DNA segments. The desmuslin gene was screened for mutations in 71 patients with various forms of myopathy for which there was no known cause. In this analysis, 10 common and 2 rare amino acid altering single-nucleotide polymorphisms were identified, all of which were seen in a control population of individuals thus making these unlikely causes of the phenotype. Interestingly, one of the single-nucleotide polymorphisms found in a patient resulted in a premature stop codon in the first exon. The nonsense mutation was also detected in the patient's unaffected father and one unaffected control; it was detected in 0.44% (2/454) of unrelated chromosomes and is therefore predicted to have a homozygous frequency of 0.002%. No causative mutations were found in the desmuslin gene. However, the single-nucleotide polymorphisms mapped in this study represent a well-mapped group that can be used for disequilibrium studies of this region of chromosome 15q26.3.
Paul G Fitzgerald - One of the best experts on this subject based on the ideXlab platform.
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targeted deletion of the lens fiber cell specific Intermediate Filament Protein filensin
Investigative Ophthalmology & Visual Science, 2003Co-Authors: Azita Alizadeh, John F Hess, John I Clark, Teri Seeberger, Thomas N Blankenship, Paul G FitzgeraldAbstract:PURPOSE. To determine the function of the lens fiber cell‐ specific cytoskeletal Protein, filensin, in lens biology. METHODS. Targeted genomic deletion was used to delete exon 1 and the transcriptional start site of the filensin gene. Resultant chimeric animals were bred to homozygosity for the mutant allele. These animals were outbred to mice bearing the wild-type CP49 alleles to eliminate the mutant CP49 gene carried by the 129 strain of mice. Animals homozygous for the mutated filensin gene and wild-type CP49 gene were compared with wild-type and heterozygous animals by Northern and Western blot analyses, light and electron microscopy, and slit lamp microscopy. RESULTS. Disruption of the filensin gene successfully blocked production of filensin mRNA, reduced levels of filensin’s assembly partner CP49, and prevented the assembly of beaded Filaments. Despite the absence of beaded Filaments, lenses did not show obvious changes in fetal development, nor in the differentiation of epithelial cells into mature fiber cells, as judged by light microscopic analysis. Filensin knockouts began to show evidence of light-scattering by 2 months and worsened with age. Heterozygous animals exhibited an Intermediate phenotype, showing a reduction in filensin transcript and moderate light-scattering at 5 months. CONCLUSIONS. The lens fiber cell‐specific Intermediate Filament Protein filensin is essential for beaded Filament assembly. However, although beaded Filaments are not needed for normal lens fetal development or fiber cell differentiation, they appear to be necessary for the long-term maintenance of optical clarity. The mechanism by which the absence of filensin and the beaded Filament affects optical clarity has yet to be defined. (Invest Ophthalmol Vis Sci. 2003;44:5252‐5258) DOI: 10.1167/iovs.03-0224
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targeted genomic deletion of the lens specific Intermediate Filament Protein cp49
Investigative Ophthalmology & Visual Science, 2002Co-Authors: Azita Alizadeh, John F Hess, John I Clark, Teri Seeberger, Thomas N Blankenship, Andrew P Spicer, Paul G FitzgeraldAbstract:PURPOSE. To deduce the function of the lens-specific cytoskeletal structure, the beaded Filament, by blocking expression of the fiber cell-specific beaded Filament Protein CP49. METHODS. The first exon of the mouse CP49 gene was deleted by using targeted genomic deletion techniques. Gene deletion was assessed through Southern blot analysis and PCR. Translation and Protein expression were characterized by Northern and Western blot analysis of both CP49 and its assembly partner filensin. The architecture of knockout lenses was compared with that of wild-type lenses at the histologic level by light microscopy. Lens clarity was assessed in situ by direct ophthalmic examination and slit lamp microscopy. RESULTS. Transcription and translation of CP49 were successfully negated in knockout animals. Lenses homozygous for the CP49 deletion showed no obvious changes in lens architecture at the light microscope level. Filensin levels were sharply reduced, although filensin mRNA levels appeared unchanged. Direct examination of lenses showed no obvious loss of lens clarity, but slit lamp examination revealed the emergence of opacification in even the youngest animals. The opacification worsened with age. CONCLUSIONS. The absence of CP49 causes a subtle loss of optical clarity in the ocular lens, a loss that worsens with age. However, CP49 is not essential for the assumption or maintenance of overall fiber cell shape or long-range order of fiber cells. CP49 appears to regulate the Protein levels of its assembly partner filensin, suggesting a mechanism for the regulation of beaded Filament Protein stoichiometry.
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Primary sequence, secondary structure, gene structure, and assembly properties suggests that the lens-specific cytoskeletal Protein filensin represents a novel class of Intermediate Filament Protein.
Experimental eye research, 1998Co-Authors: John F Hess, Jodi T. Casselman, Allen Kong, Paul G FitzgeraldAbstract:The ocular lens fiber cell assembles a novel cytoskeletal element, the Beaded Filament, from CP49 and filensin, two Proteins expressed only in the differentiated lens fiber cell. We report the primary sequence, secondary structural analysis, gene structure and Yeast Two Hybrid interaction data for human filensin, and develop a consensus model of filensin from the human and previously reported bovine and chicken filensin sequences. This consensus model, combined with gene structure and Yeast Two Hybrid studies establish that filensin is a member of the Intermediate Filament family of Proteins. Specifically, filensin exhibits (1) divergence at amino acid sequence motifs otherwise highly conserved among Intermediate Filament Proteins, (2) a loss of 29 amino acids from the central rod domain which is unique among cytoplasmic Intermediate Filament Proteins, (3) an absence of sequence identity with any existing class of Intermediate Filament Protein, (4) a gene structure unique among Intermediate Filament family, (5) an inability to dimerize with representatives of Type I, II, and III Intermediate Filament Proteins. Thus, at each level of analysis, we find that filensin is similar to the consensus model of Intermediate Filament Proteins, supporting our conclusion that filensin's relatedness to the IF family is not the consequence of convergent evolution. However, filensin also shows unique or extreme distinctions from the consensus Intermediate Filament Protein at each level of analysis, indicating that filensin constitutes a novel class of IF Protein. Some of filensin's unique features are incompatible with current models of IF assembly. Analysis of filensin gene structure suggests that the 29 amino acid reduction in the central rod domain was not the result of a single splice site mutation, the mechanism suggested for the transition between nuclear lamins and cytoplasmic Intermediate Filament Proteins.
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Gene structure and cDNA sequence identify the beaded Filament Protein CP49 as a highly divergent type I Intermediate Filament Protein.
The Journal of biological chemistry, 1996Co-Authors: John F Hess, Jodi T. Casselman, Paul G FitzgeraldAbstract:Abstract The fiber cell of the vertebrate ocular lens assembles a cytoskeletal structure, the beaded Filament, which contains two Proteins unique to the fiber cell: CP49 (phakinin) and CP115/CP95 (filensin). We report here the complete primary sequence and gene structure for human CP49. These data show that CP49 is a member of the Intermediate Filament family, but highly unusual in several regards. 1) CP49 primary sequence does not permit unambiguous assignment to any existing class of Intermediate Filament Protein, but exhibits a gene structure that is identical to the Type I cytokeratins. 2) CP49 essentially lacks one of the three major domains that characterize all Intermediate Filament Proteins, the carboxyl-terminal tail domain. 3) CP49 shows substitutions at 3 of 4 residues in the otherwise highly conserved Intermediate Filament Protein motif LNDR. Notably, this divergence includes an Arg to Cys substitution that has only been observed in the mutant human cytokeratin K14, a mutation shown to cause the skin blistering seen in the genetic disorder Dowling-Meara epidermolysis bullosa simplex.