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Molecular cloning, sequencing and functional study of the promoter region of the human alpha2C4-adrenergic receptor gene.
- Source :
-
The Biochemical journal [Biochem J] 1997 Dec 01; Vol. 328 ( Pt 2), pp. 431-8. - Publication Year :
- 1997
-
Abstract
- 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.
- Subjects :
- Base Composition
Base Sequence
Carcinoma, Hepatocellular metabolism
Gene Expression
Genes, Reporter
Genomic Library
Humans
Molecular Sequence Data
RNA, Messenger genetics
RNA, Neoplasm genetics
Receptors, Adrenergic, alpha-2 biosynthesis
Recombinant Fusion Proteins biosynthesis
Sequence Analysis, DNA
Transcription, Genetic
Tumor Cells, Cultured
Promoter Regions, Genetic
Receptors, Adrenergic, alpha-2 genetics
Subjects
Details
- Language :
- English
- ISSN :
- 0264-6021
- Volume :
- 328 ( Pt 2)
- Database :
- MEDLINE
- Journal :
- The Biochemical journal
- Publication Type :
- Academic Journal
- Accession number :
- 9371698
- Full Text :
- https://doi.org/10.1042/bj3280431