Molecular characterization of a developmentally regulated porcine skeletal myosin heavy chain gene and its 5′ regulatory region

Abstract

Members of the myosin heavy chain (MyHC) gene family show developmental stage- and spatial-specificity of expression. We report on the characterization and identification of a porcine skeletal fast MyHC gene, including its corresponding 5′ end cDNA and 5′ regulatory region. This MyHC isoform was found exclusively in skeletal muscles from about the last quarter of gestation through to adulthood. Expression of this isoform was higher postnatally and its spatial distribution resembled a rosette cluster; each with a ring of fast fibres surrounding a central slow fibre. This rosette pattern was absent in the adult diaphragm but about 20% of the fibres continued to express this MyHC isoform. Further in vivo expression studies, in a variety of morphologically and functionally diverse muscles, showed that this particular skeletal MyHC isoform was expressed in fast oxidative-glycolytic fibres, suggesting that it was the equivalent of the fast IIA isoform. Two domains in the upstream regulatory region were found to confer differentiation-specific expression on C2 myotubes (−1007 to -828 and -455 to -101), based on in vitro transient expression assays using the chloramphenicol acetyltransferase (CAT) reporter gene. Interestingly, for high levels of CAT expression to occur, a 3′ region, extending from the transcriptional start site to part. of intron 2, must be present in all the DNA constructs used.