MOLECULAR-CLONING, SEQUENCING AND FUNCTIONAL-STUDY OF THE PROMOTER REGION OF THE HUMAN ALPHA(2)C4-ADRENERGIC RECEPTOR GENE

Screening of a human foetal brain genomic DNA library allowed us to is olate an EcoRI-EcoRI fragment containing 6 kb of the 5'-flanking regio n, the open reading frame and 4 kb of the 3'-flanking region of the al pha(2)C4 gene. Analysis of the sequenced region (4850 bp) revealed tha t the first 900 bp 5' to the start codon are very rich in GC (84%), co ntain several Sp1-binding sites and lack a consensus TATA box. The 5'- and 3'-ends of the alpha(2)C4 transcript were determined by RNase-pro tection assays carried out with a series of antisense probes. The data obtained with cellular RNA from HepG2 cells demonstrated that transcr iption is initiated 891 bases upstream of the translation-start site a nd that the polyadenylation site is located 550 bases downstream of th e stop codon. These results are consistent with the existence of a non -conventional TATA box (TTAGAAA) and the presence of a unique polyaden ylation signal (AATAAA). They also fit with the size of alpha(2)C4-RNA found by Northern-blot analysis (2.9 kb). The transcriptional activit y of the alpha(2)C4 promoter region was investigated by transfecting s everal cell types with chimaeric constructs containing various fragmen ts of the 5'-non-coding region and luciferase as a reporter gene. The activity of the construct containing the entire 5'-non-coding region a ppeared to depend on the host cell. Removal of the 5'-untranslated reg ion resulted in loss of cell specificity and a concomitant increase in luciferase activity. Transfection of HepG2 and SK-N-MC cells with con structs deleted of additional 5'-flanking fragments permitted the defi nition of a minimal 200 bp promoter fragment containing the pseudo-TAT A box and two putative SP1-binding sites.