Identification of a neuron-specific promoter of human aromatic L-amino acid decarboxylase gene.
We have cloned the 5' region of human aromatic L-amino acid decarboxylase (AADC) gene in a cosmid and an overlapping lambda clone, and sequenced the first five exons. A 61 base pair (bp) non-coding, first exon containing for the 5' end of a human pheochromocytoma AADC cDNA was localized 16 kb upstream of exon 2, in which translation is initiated. The transcription start site was localized by RNAse mapping, primer extension and reverse transcription-PCR. The non-conventional cap site was preceded by a modified TATA box at position -29. A strong promoter was characterized in the 560 bp region upstream of the cap site by linkage to the reporter gene LacZ, and transfection in human neuroblastoma SK-N-BE and SK-N-BE-K2 cells. Using a series of constructs bearing a varying length of 5' flanking region, three positive regulatory elements have been localized in the -560 to -394, -244 to -200 and -147 to -1 regions. Negative regulatory elements were localized in the -9000 to -560 and -394 to -316 regions. Surprisingly, constructs comprising all or the major part of intron 1 were inactive, suggesting the presence of a silencer in the first intron, or incorrect splicing events. The construct containing 560 bp of 5' flanking sequence did not express in human cholinergic neuroepithelioma cells MC-I-XC, and in three non-neuronal cell lines which displayed high AADC activities: human pancreatic carcinoma cells AsPC-1, rat insulinoma cells RINm5F and mouse anterior pituitary cells AtT20. These data suggest that we have identified a neuron-specific AADC promoter. An extensive search for a second promoter responsible for AADC gene expression in non-neuronal cells only gave negative results.
Le Van Thai
A
A
Laboratoire de Biologie Moléculaire des Eucaryotes, Centre National de la Recherche Scientifique, Toulouse, France.
Coste
E
E
Allen
J M
JM
Palmiter
R D
RD
Weber
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009474:Neurons / Q000502:physiology*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D001142:Aromatic-L-Amino-Acid Decarboxylases