The TATA-less rat GAD65 promoter can be activated by Sp1 through non-consensus elements.
Glutamic acid decarboxylase (GAD) 65 is one of two homologous proteins responsible for the synthesis of gamma-aminobutyric acid, the most ubiquitous inhibitory neurotransmitter. In order to characterize the DNA elements responsible for controlling GAD65 expression, we cloned the 5' flanking region of the rat GAD65 gene. A major, proximal and a minor, distal region of transcription initiation were located by RACE experiments. Sequence analysis revealed that the initiation sites are located within a region devoid of TATA boxes. We investigated the functional organization of the promoter by measuring the ability of 5' deletion mutants to drive the expression of a luciferase reporter gene. The major promoter was found to be located in the region encompassing the 100bp immediately upstream of the proximal transcription initiation site. A number of near consensus GC boxes and initiator elements are found in this region, but gel-shift assays suggest that they play only a minor role in transcription initiation. However, gel-shift assays and reporter gene assays suggest that Sp1 can bind to a region devoid of consensus Sp1 binding sites.
Skak
K
K
Hagedorn Research Institute, Niels Steensensvej 6, 2820, Gentofte, Denmark.
Michelsen
B K
BK
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D004586:Electrophoresis; D017930:Genes, Reporter; D005968:Glutamate Decarboxylase / Q000235:genetics*; D007515:Islets of Langerhans / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016914:Ribonuclease H / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000502:physiology*; D016385:TATA Box / Q000502:physiology*; D014018:Tissue Distribution; D014158:Transcription, Genetic; D014162:Transfection
D016329:Sp1 Transcription Factor; D016914:Ribonuclease H; D005968:Glutamate Decarboxylase