Characterization of the regulatory regions of murine alpha 2C2 adrenoceptor subtype gene

In order to delineate the regulatory mechanisms underlying the control of alpha 2 adrenoceptor expression, the sequence of 5' (1145 bp) and 3' (2682 bp) flanking regions of murine alpha 2C2 subtype gene were determined and characterized from a genomic phage clone MA2C2. The 5' flanking region has no TATA box yet with high GC content. The 3' flanking region is marked by the presence of a polyadenylation signal 2.3 kb down stream from the stop codon. The transcription start site was mapped by 5' rapid amplification of cDNA ends (RACE) and primer extension assays at nucleotide A, 415 bp upstream from the first initiation codon and resides in a motif resembling the consensus sequence of initiator found in many TATA-less promoters. An NcoI fragment (4.7 kb) immediately upstream from the translation initiation site was linked to a reporter gene lacZ. Using an in vitro transfection assay, cell lines of renal or neural origin were identified as permissive hosts for alpha 2C2 subtype expression. With its core promoter clearly defined and sequence of the regulatory regions at hand, this in vitro gene transfer system will facilitate the identification of putative cis-elements and transcription factors key to alpha 2C2 adrenoceptor expression.