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 diff erent length chimeric constructs were based on the complete sequence o f the human cardiac myosin heavy chain genes [1, 2]. The primers were synthesized with HindIII and BamH1 sites and were linked to any design ed nucleotide of the 5' flanking sequence of the myosin heavy chain ge ne(s). Following the PCR amplification and the site-directed mutagenes is, the PCR products were verified by DNA sequencing and subsequently ligated to the chloramphenical acetyltransferase (pBLCAT3) reporter ge ne which was restricted with Hind III and BamH1. Neonatal rat cardiocy tes were used to assay the promotor activity (i.e. CAT activity) of di fferent lengths of the chimeric constructs of the gene.