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At least 541 records · Page 30Linked to original sources
PubMed2002

Gene expression profiling in dysferlinopathies using a dedicated muscle microarray.

We have performed expression profiling to define the molecular changes in dysferlinopathy using a novel dedicated microarray platform made with 3'-end skeletal muscle cDNAs. Eight dysferlinopathy patients, defined by western blot, immunohistochemistry and mutation analysis, were investigated with this technology. In a first experiment RNAs from different limb-girdle muscular dystrophy type 2B patients were pooled and compared with normal muscle RNA to characterize the general transcription pattern of this muscular disorder. Then the expression profiles of patients with different clinical traits were independently obtained and hierarchical clustering was applied to discover patient-specific gene variations. MHC class I genes and genes involved in protein biosynthesis were up-regulated in relation to muscle histopathological features. Conversely, the expression of genes codifying the sarcomeric proteins titin, nebulin and telethonin was down-regulated. Neither calpain-3 nor caveolin, a sarcolemmal protein interacting with dysferlin, was consistently reduced. There was a major up-regulation of proteins interacting with calcium, namely S100 calcium-binding proteins and sarcolipin, a sarcoplasmic calcium regulator.

Adolescent↗
PubMed1989

Rescue of excitation-contraction coupling in dysgenic muscle by addition of fibroblasts in vitro.

Muscular dysgenesis (mdg) in mice causes the failure of excitation-contraction (E-C) coupling in skeletal muscle. Cultured dysgenic muscle fails to contract upon depolarization, lacks typical muscle ultrastructure, including normal triads, and lacks functional voltage-dependent slow calcium channels. We show that normal rodent fibroblasts and 3T3 fibroblasts "rescue" dysgenic myotubes, reestablishing contractions (i.e., E-C coupling), normal ultrastructure, and functional slow calcium channels. These results support the finding that the expression of the slow calcium channel is affected in the mdg mutation and that this protein is essential for E-C coupling. Additionally, fibroblast rescue provides a system for examining the mechanisms of heterotypic cellular influence on cell function.

Action Potentials↗
PubMed1987

Induction of myofibrillogenesis in cardiac lethal mutant axolotl hearts rescued by RNA derived from normal endoderm.

A strain of axolotl, Ambystoma mexicanum, that carries the cardiac lethal or c gene presents an excellent model system in which to study inductive interactions during heart development. Embryos homozygous for gene c contain hearts that fail to beat and do not form sarcomeric myofibrils even though muscle proteins are present. Although they can survive for approximately three weeks, mutant embryos inevitably die due to lack of circulation. Embryonic axolotl hearts can be maintained easily in organ culture using only Holtfreter's solution as a culture medium. Mutant hearts can be induced to differentiate in vitro into functional cardiac muscle containing sarcomeric myofibrils by coculturing the mutant heart tube with anterior endoderm from a normal embryo. The induction of muscle differentiation can also be mediated through organ culture of mutant heart tubes in medium 'conditioned' by normal anterior endoderm. Ribonuclease was shown to abolish the ability of endoderm-conditioned medium to induce cardiac muscle differentiation. The addition of RNA extracted from normal early embryonic anterior endoderm to organ cultures of mutant hearts stimulated the differentiation of these tissues into contractile cardiac muscle containing well-organized sarcomeric myofibrils, while RNA extracted from early embryonic liver or neural tube did not induce either muscular contraction or myofibrillogenesis. Thus, RNA from anterior endoderm of normal embryos induces myofibrillogenesis and the development of contractile activity in mutant hearts, thereby correcting the genetic defect.

Ambystoma↗
PubMedDate not supplied

[Myogenic differentiation in experimental malignant schwannomas (an immunohistochemical and molecular genetic study of the "triton" tumor)].

Spontaneous myogenic differentiation was observed in 2 out of 15 monolayer cultures from schwannomas induced in BD1X rats by transplacental exposure to N-enthyl-N-nitrosourea (ENU). Cells were sorted following fluorescence-activating method with monoclonal antibody (Mab) 217c. Myotubes and numerous mononucleated cells no longer expressed the Schwann cell antigens 217c and S-100 protein, but rather revealed the presence of desmin, the alpha-sarcomeric form (alpha-sr) of actin, and the cell surface antigen specified by Mab RB21-7, a glycoprotein sharing an epitope with the neural cell adhesion molecule (N-CAM). Subcutaneous reimplantation of such cells into syngeneic animals resulted in the appearance of tumours composed of both S-100 positive Schwann cells and desmin and alpha-sr-actin positive rhabdomyoblasts, thus closely resembling the human "triton" tumour. With the use of the polymerase chain reaction and allele-specific oligonucleotide hybridization, DNA isolated from individual myotubes was analysed for the presence of a T-->A transversion mutation at nucleotide 2007 of the neu gene, which is diagnostic of ENU-induced rat schwannomas. All of the amplified DNA isolated contained the mutant neu allele, thus providing direct genetic proof for the capacity of mammalian neuroectodermal cells for myogenic differentiation.

Animals↗
PubMed1996

Formation of triads without the dihydropyridine receptor alpha subunits in cell lines from dysgenic skeletal muscle.

Muscular dysgenesis (mdg/mdg), a mutation of the skeletal muscle dihydropyridine receptor (DHPR) alpha 1 subunit, has served as a model to study the functions of the DHPR in excitation-contraction coupling and its role in triad formation. We have investigated the question of whether the lack of the DHPR in dysgenic skeletal muscle results in a failure of triad formation, using cell lines (GLT and NLT) derived from dysgenic (mdg/mdg) and normal (+/+) muscle, respectively. The lines were generated by transfection of myoblasts with a plasmid encoding a Large T antigen. Both cell lines express muscle-specific proteins and begin organization of sarcomeres as demonstrated by immunocytochemistry. Similar to primary cultures, dysgenic (GLT) myoblasts show a higher incidence of cell fusion than their normal counterparts (NLT). NLT myotubes develop spontaneous contractile activity, and fluorescent Ca2+ recordings show Ca2+ release in response to depolarization. In contrast, GLTs show neither spontaneous nor depolarization-induced Ca2+ transients, but do release Ca2+ from the sarcoplasmic reticulum (SR) in response to caffeine. Despite normal transverse tubule (T-tubule) formation, GLT myotubes lack the alpha 1 subunit of the skeletal muscle DHPR, and the alpha 2 subunit is mistargeted. Nevertheless, the ryanodine receptor (RyR) frequently develops its normal, clustered organization in the absence of both DHPR alpha subunits in the T-tubules. In EM, these RyR clusters correspond to T-tubule/SR junctions with regularly spaced feet. These findings provide conclusive evidence that interactions between the DHPR and RyR are not involved in the formation of triad junctions or in the normal organization of the RyR in the junctional SR.

Animals↗
PubMed1991

Rat model of the human "Triton" tumor: direct genetic evidence for the myogenic differentiation capacity of schwannoma cells using the mutant neu gene as a cell lineage marker.

Spontaneous myogenic differentiation was observed in 2 out of 15 cases when cells from schwannomas induced in the offspring of BDIX rats by transplacental exposure to N-ethyl-N-nitrosourea (EtNU) were grown in monolayer culture following fluorescence-activated cell sorting with monoclonal antibody (Mab) 217c. Myotubes and numerous mononucleated cells no longer expressed the Schwann cell antigens 217c and S-100 protein, but rather revealed the presence of desmin, the alpha-sarcomeric form (alpha-sr) of actin, and the cell surface antigen specified by Mab RB21-7, a 250 kD glycoprotein sharing an epitope with the neural cell adhesion molecule (N-CAM). Subcutaneous reimplantation of such cells into syngeneic animals led to the appearance of tumors composed of both S-100 positive Schwann cells and desmin and alpha-sr-actin positive rhabdomyoblasts, thus closely resembling the human "Triton" tumor. With the use of the polymerase chain reaction and allele-specific oligonucleotide hybridization, DNA isolated from individual myotubes was analyzed for the presence of a T----A transversion mutation at nucleotide 2012 of the neu gene, which is diagnostic of EtNU-induced rat schwannomas. All of the amplified DNA isolates contained the mutant neu allele, thus providing direct genetic proof for the capacity of mammalian neuroectodermal cells for myogenic differentiation.

Animals↗
PubMed1999

Association of mitochondria with plectin and desmin intermediate filaments in striated muscle.

Plectin (M(r) > 500,000) is a versatile and widely expressed cytolinker protein. In striated muscle it is predominantly found at the Z-disc level where it colocalizes with the intermediate filament protein desmin. Both proteins show altered labeling patterns in tissues of muscular dystrophy patients. Moreover, mutations in the plectin gene lead to the autosomal recessive human disorder epidermolysis bullosa simplex with muscular dystrophy, and defects in the desmin gene have been shown to cause familiar cardiac and skeletal myopathy. Since intermediate filaments (IFs) in striated muscle tissue have been found to be intimately associated with mitochondria, we investigated whether plectin is involved in this association. Using postembedding immunogold labeling of Lowicryl sections and immunogold labeling of ultrathin cryosections, we show that plectin is associated with desmin IFs linking myofibrils to mitochondria at the level of the Z-disc and along the entire length of the sarcomere. The localization of plectin label at the mitochondrial membrane itself was consistent with a putative linker function of plectin between desmin IFs and the mitochondrial surface. In mitochondrion-rich muscle fibers, both plectin and desmin were part of an ordered arrangement of mitochondrial side branches, which wound around myofibrils adjacent to the Z-discs and were anchored into a filamentous network transversing from one fibril to the other. The association of mitochondria with plectin and IFs was seen also in tissues without regular distribution patterns of mitochondria, such as heart muscle and neonatal skeletal muscle tissues. These data were supplemented with in vitro binding assays showing direct interaction of plectin with desmin via its carboxy-terminal IF-binding domain. As a cytolinker protein associated with mitochondria and desmin IFs, plectin could play an important role in the positioning and shape formation, in particular branching, of mitochondrial organelles in striated muscle tissues.

Animals↗
PubMed1997

Mitogen-activated protein kinase phosphatase 1 inhibits the stimulation of gene expression by hypertrophic agonists in cardiac myocytes.

The effect of constitutive expression of mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) on gene expression in response to hypertrophic agonists was examined in cultured neonatal rat ventricular myocytes. Luciferase (LUX) reporter genes linked to promoters for atrial natriuretic factor, ventricular myosin light chain 2, beta-myosin heavy chain, skeletal muscle alpha-actin (SkM alpha-actin) and serum response element-regulated c-fos (c-fos-SRE) were transfected into cardiomyocytes. Phenylephrine (PE; 10 microM), phorbol 12-myristate 13-acetate (1 microM) and endothelin 1 (10 nM) stimulated the expression of these various reporter genes by 2. 5-20-fold. MKP-1 inhibited these effects by 60-85%. In contrast, MKP-1 had no effect on the expression of a constitutively active Rous sarcoma virus-LUX reporter gene. A catalytically inactive mutant MKP-1CS (cysteine-->serine mutation) and the broad-specificity protein tyrosine phosphatase 1B (PTP-1B) had no significant effect on any reporter gene tested. MKP-1 had much less effect on the morphological features accompanying agonist-induced cardiac hypertrophy. PE (10 microM) increased myocyte area by 59% but this effect was only decreased by one-third by MKP-1 and was also partly decreased (by 25%) by expression of PTP-1B. PE also altered cell shape but this was unaffected by MKP-1. There was also no clear effect of MKP-1 on the organization of the contractile apparatus into sarcomeric structures in the presence of 10 microM PE. We conclude that the transcriptional responses accompanying cardiac myocyte hypertrophy are dependent on an MKP-1-sensitive step, presumably the activation of one or members of the MAPK family, but that cell size, shape and myofibrillar organization are much less sensitive to inhibition by MKP-1.

Animals↗
PubMed1995

Mechanical function of dystrophin in muscle cells.

We have directly measured the contribution of dystrophin to the cortical stiffness of living muscle cells and have demonstrated that lack of dystrophin causes a substantial reduction in stiffness. The inferred molecular structure of dystrophin, its preferential localization underlying the cell surface, and the apparent fragility of muscle cells which lack this protein suggest that dystrophin stabilizes the sarcolemma and protects the myofiber from disruption during contraction. Lacking dystrophin, the muscle cells of persons with Duchenne muscular dystrophy (DMD) are abnormally vulnerable. These facts suggest that muscle cells with dystrophin should be stiffer than similar cells which lack this protein. We have tested this hypothesis by measuring the local stiffness of the membrane skeleton of myotubes cultured from mdx mice and normal controls. Like humans with DMD mdx mice lack dystrophin due to an x-linked mutation and provide a good model for the human disease. Deformability was measured as the resistance to indentation of a small area of the cell surface (to a depth of 1 micron) by a glass probe 1 micron in radius. The stiffness of the membrane skeleton was evaluated as the increment of force (mdyne) per micron of indentation. Normal myotubes with an average stiffness value of 1.23 +/- 0.04 (SE) mdyne/micron were about fourfold stiffer than myotubes cultured from mdx mice (0.34 +/- 0.014 mdyne/micron). We verified by immunofluorescence that both normal and mdx myotubes, which were at a similar developmental stage, expressed sarcomeric myosin, and that dystrophin was detected, diffusely distributed, only in normal, not in mdx myotubes. These results confirm that dystrophin and its associated proteins can reinforce the myotube membrane skeleton by increasing its stiffness and that dystrophin function and, therefore, the efficiency of therapeutic restoration of dystrophin can be assayed through its mechanical effects on muscle cells.

Animals↗
PubMed2003

Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles.

Assembly of specialized membrane domains, both of the plasma membrane and of the ER, is necessary for the physiological activity of striated muscle cells. The mechanisms that mediate the structural organization of the sarcoplasmic reticulum with respect to the myofibrils are, however, not known. We report here that ank1.5, a small splice variant of the ank1 gene localized on the sarcoplasmic reticulum membrane, is capable of interacting with a sequence of 25 aa located at the COOH terminus of obscurin. Obscurin is a giant sarcomeric protein of approximately 800 kD that binds to titin and has been proposed to mediate interactions between myofibrils and other cellular structures. The binding sites and the critical aa required in the interaction between ank1.5 and obscurin were characterized using the yeast two-hybrid system, in in vitro pull-down assays and in experiments in heterologous cells. In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin. The M line localization of ank1.5 required a functional obscurin-binding site, because mutations of this domain resulted in a diffused distribution of the mutant ank1.5 protein in skeletal muscle cells. The interaction between ank1.5 and obscurin represents the first direct evidence of two proteins that may provide a direct link between the sarcoplasmic reticulum and myofibrils. In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum. Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin. Based on reported evidence showing that the COOH terminus of ank2.2 is necessary for the localization of ryanodine receptors and InsP3 receptors in the sarcoplasmic reticulum, we propose that obscurin, through multiple interactions with ank1.5 and ank2.2 isoforms, may assemble a large protein complex that, in addition to a structural function, may play a role in the organization of specific subdomains in the sarcoplasmic reticulum.

3T3 Cells↗
PubMed2000

Molecular genetics of hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM), a serious and often tragic disorder, is characterized by hypertrophy of the interventricular septum and left ventricular wall, hypercontractile systolic function with diastolic dysfunction, and in some cases, left ventricular outflow tract obstruction. On histopathologic examination, myofiber disarray is common. The genes for familial cases of hypertrophic cardiomyopathy are known to encode members of the sarcomere and to date nine genes have been identified (beta-myosin heavy chain, alpha-tropomyosin, cardiac troponin T, troponin I, myosin binding protein-C, regulatory myosin light chain, essential myosin light chain, cardiac actin, and titin) for this genetically and clinically heterogeneous disease. In this review the genetic basis of HCM is discussed.

Adult↗
PubMed2005

Sudden death and hypertrophic cardiomyopathy: a review.

Hypertrophic cardiomyopathy is a genetic disease that affects the cardiac sarcomere, resulting in myocardial hypertrophy and disarray. Affected patients have a predisposition for malignant ventricular tachyarrhythmias and, consequently, sudden cardiac death. With the availability of therapeutic measures that prevent sudden death, the identification of high-risk patients is now of greater importance. Clinical risk factors for sudden death (ie, age, syncope, family history of sudden cardiac death, cardiac arrest survivor, nonsustained ventricular tachycardia and abnormal blood pressure response to exercise) have been identified. The clinical electrophysiological study is of limited use for stratifying these patients. More recently, increased attention has been given to the degree of echocardiographically documented left ventricular hypertrophy and prognostically significant genetic mutations. Once a high-risk patient is identified, prophylactic treatment is warranted. For this purpose, amiodarone has been supplanted by the implantable cardioverter-defibrillator. Implantable cardioverter-defibrillator treatment appears to reduce the risk of sudden cardiac death in both primary and secondary prevention settings. Thus, tools are now available to identify and treat high-risk patients with hypertrophic cardiomyopathy.

Amiodarone↗
PubMed1994

Immunofluorescent studies on titin and myosin in developing hearts of normal and cardiac mutant axolotls.

Homozygous recessive cardiac mutant gene c in the axolotl, Ambystoma mexicanum, results in a failure of the embryonic heart to initiate beating. Previous studies show that mutant axolotl hearts fail to form sarcomeric myofibrils even though hearts from their normal siblings exhibit organized myofibrils beginning at stage 34-35. In the present study, the proteins titin and myosin are studied using normal (+/+) axolotl embryonic hearts at stages 26-35. Additionally, titin is examined in normal (+/c) and cardiac mutant (c/c) embryonic axolotl hearts using immunofluorescent microscopy at stages 35-42. At tailbud stage 26, the ventromedially migrating sheets of precardiac mesoderm appear as two-cell-layers. Myosin shows periodic staining at the cell peripheries of the presumptive heart cells at this stage, whereas titin is not yet detectable by immunofluorescent microscopy. At preheartbeat stages 32-33, a myocardial tube begins to form around the endocardial tube. In some areas, periodic myosin staining is found to be separated from the titin staining; other areas in the heart at this stage show a co-localization of the two proteins. Both titin and myosin begin to incorporate into myofibrils at stage 35, when normal hearts initiate beating. Additionally, areas with amorphous staining for both proteins are observed at this stage. These observations indicate that titin and myosin accumulate independently at very early premyofibril stages; the two proteins then appear to associate closely just before assembly into myofibrils. Staining for titin in freshly frozen and paraffin-embedded tissues of normal embryonic hearts at stages 35, 39, and 41 reveals an increased organization of the protein into sarcomeres as development progresses. The mutant siblings, however, first show titin staining only limited to the peripheries of yolk platelets. Although substantial quantities of titin accumulate in mutant hearts at later stages of development (39 and 41), it does not become organized into myofibrils as in normal cells at these stages.

Aging↗
PubMed2006

Lysosomal dysfunction, cellular pathology and clinical symptoms: basic principles.

UNLABELLED: Between 40 and 50 lysosomal storage disorders are known at present. Fine details of the pathogenic process involved are in general not known. This overview highlights the basic principles of lysosomal pathogenesis and the clinical consequences of defective genes involved in lysosomal functions. The subject is discussed in the context of the possibility of prevention and reversal of cellular and organ damage by enzyme replacement therapy. Also presented is a mechanical model for the muscle pathology observed in Pompe disease. Direct mechanical effects of the non-contractile inclusions - glycogen-loaded lysosomes - seem to be a key factor in the loss of force during both early and late stages of the disease. CONCLUSION: Each lysosomal storage disorder and each patient with a given lysosomal disorder has unique molecular, pathological and clinical features. But, the order of pathological events is largely the same. Mutations in a gene cause lysosomal dysfunction which, in turn, results in cellular pathology affecting organ structure and function. Clinical symptoms are the ultimate manifestation. The reversibility of symptoms with enzyme replacement therapy will vary according to the disease, as well as the nature and stage of organ pathology.

Animals↗
PubMed2005

Paramyosin phosphorylation site disruption affects indirect flight muscle stiffness and power generation in Drosophila melanogaster.

The phosphoprotein paramyosin is a major structural component of invertebrate muscle thick filaments. To investigate the importance of paramyosin phosphorylation, we produced transgenic Drosophila melanogaster in which one, three, or four phosphorylatable serine residues in the N-terminal nonhelical domain were replaced by alanines. Depending on the residues mutated, transgenic lines were either unaffected or severely flight impaired. Flight-impaired strains had decreases in the most acidic paramyosin isoforms, with a corresponding increase in more basic isoforms. Surprisingly, ultrastructure of indirect flight muscle myofibrils was normal, indicating N-terminal phosphorylation is not important for myofibril assembly. However, mechanical studies of active indirect flight muscle fibers revealed that phosphorylation site mutations reduced elastic and viscous moduli by 21-59% and maximum power output by up to 42%. Significant reductions also occurred under relaxed and rigor conditions, indicating that the phosphorylation-dependent changes are independent of strong crossbridge attachment and likely arise from alterations in thick filament backbone properties. Further, normal crossbridge kinetics were observed, demonstrating that myosin motor function is unaffected in the mutants. We conclude that N-terminal phosphorylation of Drosophila paramyosin is essential for optimal force and oscillatory power transduction within the muscle fiber and is key to the high passive stiffness of asynchronous insect flight muscles. Phosphorylation may reinforce interactions between myosin rod domains, enhance thick filament connections to the central M-line of the sarcomere and/or stabilize thick filament interactions with proteins that contribute to fiber stiffness.

Analysis of Variance↗
PubMed2005

Contribution of sequence variation in Drosophila actins to their incorporation into actin-based structures in vivo.

Actin is a highly conserved protein important for many cellular functions including motility, contraction in muscles and intracellular transport. Many eukaryotic genomes encode multiple actin protein isoforms that differ from each other by only a few residues. We addressed whether the sequence differences between actin paralogues in one species affect their ability to integrate into the large variety of structures generated by filamentous actin. We thus ectopically expressed all six Drosophila actins as fusion proteins with green fluorescent protein (GFP) in a variety of embryonic, larval and adult fly tissues. We found that each actin was able to integrate into most actin structures analysed. For example, in contrast to studies in mammalian cells, the two Drosophila cytoplasmic actins were incorporated into muscle sarcomeres. However, there were differences in the efficiency with which each actin was incorporated into specific actin structures. The most striking difference was observed within the Z-lines of the sarcomeres: one actin was specifically excluded and we mapped this feature to one or both of two residues within the C-terminal half of the protein. Thus, in Drosophila, the primary sequence of different actins does affect their ability to incorporate into actin structures, and so specific GFPactins may be used to label certain actin structures particularly well.

Actins↗
PubMed2006

Identification of putative in vivo substrates of calpain 3 by comparative proteomics of overexpressing transgenic and nontransgenic mice.

Calpain 3 (CAPN3) is a calcium-dependent protease, mutations in which cause limb girdle muscular dystrophy type 2A. To explore the physiological function of CAPN3, we compared the proteomes of transgenic mice that overexpress CAPN3 (CAPN3 Tg) and their nontransgenic (non-Tg) counterparts. We first examined known muscular dystrophy-related proteins to determine if overexpression of CAPN3 results in a change in their distribution or concentration. This analysis did not identify any known muscular dystrophy proteins as substrates of CAPN3. Next, we used a proteomic approach to compare and identify differentially represented proteins in 2-DE of CAPN3 Tg and non-Tg mice. LC-MS/MS analysis led to the identification of ten possible substrates for CAPN3, classified into two major functional categories: metabolic and myofibrillar. Myosin light chain 1 (MLC1) was focused upon because our previous studies suggested a role for CAPN3 in sarcomere remodeling. In this study, CAPN3 was shown to proteolyze MLC1 in vitro. These studies are the first to identify possible substrates for CAPN3 in an in vivo system and support a role for CAPN3 in sarcomere remodeling by cleavage of myofibrillar proteins such as MLC1. In addition, these data also suggest a role for CAPN3 in mitochondrial protein turnover.

Animals↗
PubMed1999

Fine mapping and genomic structure of ACTN2, the human gene coding for the sarcomeric isoform of alpha-actinin-2, expressed in skeletal and cardiac muscle.

The present paper reports on the fine mapping of the ACTN2 gene and on the reconstruction of its genomic structure. By radiation hybrid mapping, the gene was located about 912 cR from the 1p-telomere. ACTN2 was placed between the marker WI-9317 (alias D1S2421) and the marker AFMA045ZC5, within the chromosomal band 1q43. The gene was detected in YAC 955 c 12. This YAC was used as template DNA for long-distance and Alu-PCR, using a set of putative exonic primers, designed on the cDNA sequence of alpha-actinin-2, in order to characterize the ACTN2 intron-exon boundaries. The entire genomic structure of the gene was reconstructed. The ACTN2 gene contained 21 exons, in a segment spanning about 40 kb of genomic DNA. Only the proximal part of the gene shows a high conservation through evolution, whereas in the remaining part a divergence from the genomic organization of C. elegans and D. melanogaster was noticed. A series of intronic primers was specifically designed and produced, to amplify all the exons of ACTN2, directly from genomic DNA. This will enable mutation screening in patients affected with hereditary diseases linked to the marker CA4F/R, a polymorphism in the last intron of the alpha-actinin-2 gene.

Actinin↗