Structure and regulation of the porcine skeletal alpha-actin-encoding gene

The postnatal increase in skeletal alpha-actin (Sk-alpha-Act) synthesis in pigs is due, in part, to increased transcription. To characterize the factors responsible for its transcriptional regulation, we have cloned and determined the nucleotide sequence of a 5.2-kb HindIII genomic DNA fragment which contains the complete coding region of Sk-alpha-Act distributed over seven exons, plus 1.9 kb of 5' flanking region and 0.5 kb of 3' flanking sequence. The major transcription start point (tsp) of Sk-alpha-Act was determined to be 840 bp 5' to the ATG start codon by primer extension and RNase protection analysis. To demonstrate that the Sk-alpha-Act promoter was functional, L6 myoblasts, C2C12 myoblasts and HeLa cells were transfected with a construct (pPSKAFL-CAT) linking the 5' Sk-alpha-Act promoter to the chloramphenicol acetyltransferase reporter gene (cat). Cell lysates from L6 myoblasts, L6 myotubes, C2C12 myoblasts, C2C12 myotubes, and HeLa cells were analyzed for CAT activity. CAT activity was detected only in C2C12 myotubes. Thus, the porcine Sk-alpha-Act promoter is regulated in a developmental and cell-type specific manner.