Serial deletion constructs of human cardiac myosin heavy chain genes generated by PCR amplification

Serial deletion constructs derived from the 5'-flanking regions of the human cardiac alpha- and beta-myosin heavy chain genes were generated by polymerase chain reaction (PCR) amplifications. Generation of different length chimeric constructs were based on the complete sequence of the human cardiac myosin heavy chain genes. The primers were synthesized with HindIII and BamH1 sites and were linked to any designed nucleotide of the 5' flanking sequence of the myosin heavy chain gene(s). Following the PCR amplification and the site-directed mutagenesis, the PCR products were verified by DNA sequencing and subsequently ligated to the chloramphenicol acetyltransferase (pBLCAT3) reporter gene which was restricted with Hind III and BamH1. Neonatal rat cardiocytes were used to assay the promotor activity (i.e. CAT activity) of different lengths of the chimeric constructs of the gene.