|Nonneman, Danny - Dan|
Submitted to: Animal Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/26/1999
Publication Date: N/A
Citation: N/A Interpretive Summary: The calpain system is a ubiquitous and well-characterized cytosolic proteolytic system. The involvement of the calcium-dependent proteases, the calpains, and their specific inhibitor, calpastatin, in muscle growth and postmortem meat tenderization is well documented. The expression of calpains and calpastatin during prenatal muscle growth and development are well described, as well as the requirement of m-calpain for myoblast fusion. The skeletal muscle specific calpain (skm-calpain), however, is poorly characterized, and its involvement in muscle growth and meat tenderization is unknown. An important role in muscle growth and development is anticipated since a mutation in the skm-calpain gene in humans leads to limb-girdle muscular dystrophy (type 2A), and treatment of pigs with a beta- adrenergic agonist, which increases muscle protein accretion, decreases skm-calpain mRNA expression. Skm-calpain has been found to associate with titin, degradation of which contributes to postmortem tenderization of meat. The interaction of the skm-calpain with calcium, substrates, calpastatin, cellular compartments and regulatory proteins is unknown because of a lack of molecular tools and the difficulty to develop biochemical assays for this short-lived protease. In this study, we sequenced and mapped the bovine and sheep skm-calpain. This information will allow the design and production of specific molecular probes that should help us to elucidate the role of skm-calpain in muscle growth and meat tenderness. Single nucleotide polymorphisms were used to map bovine and sheep skm-calpain, and will allow linkage analysis for quantitative trait loci in beef and lamb.
Technical Abstract: The coding regions of the bovine and sheep skeletal muscle-specific calpains (CANP3 or p94) were cloned and sequenced by RT-PCR. Direct sequencing confirmed 2466 bp of open reading frame for both species, and bovine and sheep CANP3 shared 98.5% identity in their amino acid code. These sequences were greater than 88% identical to human, pig, rat, and mouse CANP3 nucleotide sequences, and greater than 93% identical for the amino acid code. Single nucleotide polymorphisms were used to map the bovine and sheep CANP3 genes in two steps. The genes were placed into linkage groups based on two-point LOD scores (>3.0) and the best order was determined with multi-point linkage analysis (CRI-MAP v. 2.4). Bovine CANP3 mapped to bovine chromosome 10, relative position 33.9cM with linkage to nine markers; LOD scores ranged from 4.89 - 8.61 (order, BMS2349-BL1035-RME25-SKMCALP-BM6305- BMS861-ILSTS053-BMS2742-CA090-BMS529). Ovine CANP3 mapped to chromosome 7, relative position 58cM, with linkage to only one marker, BMS861 (a bovine microsatellite that has been used in sheep), with no recombination and a LOD score of 5.72. The observed heterozygosity was 50% for both CANP3 markers in our pedigrees. The addition of the cDNA sequences of the bovine and ovine CANP3, and the mapping of this anchored type I marker will assist physiological and genetic studies on the role of CANP3 in muscle development in these species.