Characterization of two porcine genes involved in skeletal muscle growth

The differentiation and subsequent growth of muscle cells are two important processes in skeletal muscle development. Myogenin is a muscle regulatory factor responsible for the differentiation of proliferating myoblasts into myotubes. During embryogenesis, the absence of myogenin results in severe musclar deficiency. Skeletal muscle calpain is a nonlysosomal cysteine protease expressed only in skeletal muscle. Skeletal muscle calpain is involved in growth and protein accretion and is present in both the nucleus and cytosol. The goal of the present research was to obtain porcine genomic DNA clones for each of these skeletal muscle-specific nuclear factors and to make a chromosomal assignment of these genes to the pig gene map. Knowledge of the regulation of these genes may help in the production of lean meat and the mapping of these genes will make them available for marker-assisted selection of economically important traits. A pig genomic library was constructed in a cosmid vector to allow large DNA insert screening of myogenin and skeletal muscle calpain. For myogenin, a 5,300 bp fragment was sequenced and found to contain the entire myogenin coding region with 2,400 bp of upstream sequence and 902 bp of downstream sequence. The coding region of pig myogenin is 90% homologous to that of human and mouse. Transfection of CH310T1/2 fibroblasts with the porcine myogenin resulted in 27.4% myogenic conversion indicating that the cloned gene was functional. A 3.8 kb Eco RI fragment was sequenced from cosmids obtained from the skeletal muscle calpain screening. This fragment contained 409 bp of coding region interrupted by four introns. In this part of the coding region, the pig sequence was 98.5% similar to rat skeletal muscle calpain. Based on the intron/exon structure, a set of primers were designed that will amplify the skeletal muscle calpain sequence from both mRNA and genomic DNA to provide a sequence tagged site for gene mapping assays. Pig skeletal muscle calpain was mapped to chromosome 1q15-17 by in situ hybridization. This is the first structural gene mapped to the 1q region in the pig. The assignment to chromosome 1 was verified via PCR amplification of flow sorted chromosomes