title: Cloning, characterisation and identification of several polymorphisms in the promoter region of the human alpha2B-adrenergic receptor gene.
abstract: Screening of a foetal brain genomic DNA library allowed to isolate a 10-kb fragment of the gene encoding the human alpha2B-adrenergic receptor, that contained 5.5 kb of the 5'-flanking region, the open reading frame and 2.9 kb of the 3'-flanking region. The 1-kb fragment upstream from the start codon was rich in GC, lacked consensus TATA or CAAT box, but contained several Sp1-binding sites. Other potential cis-regulatory elements found in the 5'-flanking region included AP2, USF, Stat-6, NFkappaB and Olf-1. A single canonical polyadenylation signal (AATAAA) was found at position +3252/+3257 and the polyadenylation site was 3274 nucleotides downstream from ATG. Transfection experiments with chimeric luciferase constructs containing various truncated fragments of the 5'-region showed that the fragment -3160/+3 exhibited promoter activity in all tested cell lines and permitted the definition of a minimal 200-bp promoter (-603/-411) containing three putative Sp1-binding sites and two initiator elements. Transcriptional activity of this region was inhibited by the addition of mithramycin, a specific inhibitor of Sp1 binding to GC-rich sequences. The search for sequence variants within a fragment covering 1.7 kb of 5'-flanking region and the coding region allowed us to identify five novel single nucleotide polymorphisms. Interestingly, the G/C substitution at position -98 relative to the start codon was common and in complete linkage with a previously identified insertion/deletion polymorphism in the coding region which was showed to affect alpha2B-adrenergic receptor function. Based on transfection data and computer-assisted sequence analysis, the -98 G/C single nucleotide polymorphism was located within a portion of the 5'-UTR (-127/+3) affecting luciferase activity and it created additional putative binding site for Sp1. However, G/C substitution had no significant incidence on promoter activity in BHK-21 or HeLa cells.
authors.affiliation: INSERM U388, Institut Louis Bugnard, CHU Rangueil, 31403 Toulouse, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Cayla
authors.forename: Paula
authors.initials: P
authors.lastname: Heinonen
authors.forename: Liisa
authors.initials: L
authors.lastname: Viikari
authors.forename: Stéphane
authors.initials: S
authors.lastname: Schaak
authors.forename: Amir
authors.initials: A
authors.lastname: Snapir
authors.forename: Anne
authors.initials: A
authors.lastname: Bouloumié
authors.forename: Matti K
authors.initials: MK
authors.lastname: Karvonen
authors.forename: Ullamari
authors.initials: U
authors.lastname: Pesonen
authors.forename: Mika
authors.initials: M
authors.lastname: Scheinin
authors.forename: Hervé
authors.initials: H
authors.lastname: Paris
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D006801:Humans; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic / Q000235:genetics*; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics*
chemical_list: C547771:ADRA2B protein, human; D018341:Receptors, Adrenergic, alpha-2; D004247:DNA