Characterization of rabbit cardiac sarco(endo)plasmic reticulum Ca2(+)-ATPase gene

We have isolated three overlapping genomic clones extending over 39 kilobases (kb), which encodes the rabbit cardiac sarco(endo)plasmic reticulum Ca2(+)-ATPase gene (SERCA2). S1 nuclease mapping and primer extension analysis of the 5' end of the cardiac/slow-twitch (SERCA2a) and smooth/non-muscle (SERCA2b) mRNAs showed that both transcripts are initiated from the same transcription initiation site, located 528 base pairs (bp) upstream of the translation initiation codon AUG. The putative promoter revealed a "TATA box" like element at -24 bp and a "CAAT box" at -78 bp relative to the cap site. A number of DNA sequence elements that could bind trans-acting factors were also found within the 1.8 kb of DNA sequence upstream from the transcription initiation site. To determine the DNA sequences governing transcriptional regulation, we have stably transfected the myogenic cell line C2C12 with a plasmid containing the putative promoter and 946 bp upstream sequence of the SERCA2 gene, coupled to the chloramphenicol acetyltransferase gene. Our results show that this chimeric plasmid construct exhibits appropriate activation and coordinate expression with the endogenous SERCA2 gene during the terminal differentiation of myoblasts into myotubes, suggesting that it contains the promoter and upstream sequence elements required for the regulated expression of the SERCA2 gene.