Molecular cloning, sequencing and functional study of the promoter region of the human alpha2C4-adrenergic receptor gene.
Screening of a human foetal brain genomic DNA library allowed us to isolate an EcoRI-EcoRI fragment containing 6 kb of the 5'-flanking region, the open reading frame and 4 kb of the 3'-flanking region of the alpha2C4 gene. Analysis of the sequenced region (4850 bp) revealed that the first 900 bp 5' to the start codon are very rich in GC (84%), contain several Sp1-binding sites and lack a consensus TATA box. The 5'- and 3'-ends of the alpha2C4 transcript were determined by RNase-protection assays carried out with a series of antisense probes. The data obtained with cellular RNA from HepG2 cells demonstrated that transcription is initiated 891 bases upstream of the translation-start site and that the polyadenylation site is located 550 bases downstream of the stop codon. These results are consistent with the existence of a non-conventional TATA box (TTAGAAA) and the presence of a unique polyadenylation signal (AATAAA). They also fit with the size of alpha2C4-RNA found by Northern-blot analysis (2.9 kb). The transcriptional activity of the alpha2C4 promoter region was investigated by transfecting several cell types with chimaeric constructs containing various fragments of the 5'-non-coding region and luciferase as a reporter gene. The activity of the construct containing the entire 5'-non-coding region appeared to depend on the host cell. Removal of the 5'-untranslated region resulted in loss of cell specificity and a concomitant increase in luciferase activity. Transfection of HepG2 and SK-N-MC cells with constructs deleted of additional 5'-flanking fragments permitted the definition of a minimal 200 bp promoter fragment containing the pseudo-TATA box and two putative SP1-binding sites.
Schaak
S
S
Institut National de la Santé et de la Recherche Médicale U.317, Institut Louis Bugnard, CHU Rangueil, 31403 Toulouse Cedex 4, France.
Devedjian
J C
JC
Cayla
C
C
Sender
Y
Y
Paris
H
H
D001482:Base Composition; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D015698:Genomic Library; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D018341:Receptors, Adrenergic, alpha-2 / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
C547774:ADRA2C protein, human; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D018341:Receptors, Adrenergic, alpha-2; D011993:Recombinant Fusion Proteins