title: Transcriptional regulation of GABAA receptor gamma2 subunit gene.
abstract: We have cloned the promoter regions of the genes for the mouse and human gamma2 subunits of the type A receptors for gamma-aminobutyric acid (GABA). For the mouse, the two major transcription start sites were at +1 (by definition) and +43, as established by rapid amplification of cDNA ends (RACE) and primer extension. This numbering places the start methionine at +297. There was no TATA or CCAAT box. Both mouse and human sequences have a candidate neuron-restrictive silencer element (NRSE) site in the first intron (+956 in mouse). We made assorted mouse-based promoter/reporter (luciferase) constructs starting from a core extending from -331 to +136, varying sizes at both ends, and including and excluding the putative NRSE and more proximal sequences. These were tested by transient transfection in several neuron-like and non-neuronal cell lines. Both proximal and distal downstream elements appeared to help direct expression to neuron-like cells, the NRSE in the intron, by repression in non-neurons, and a 24-bp portion of the 5' untranslated region starting at +113 (named GPE1) by preferentially promoting expression in neuron-like cells. Cotransfected human NRSF (transcription factor for NRSE) reduced reporter expression in neuron-like cells for constructs containing the NRSE in two locations. In gel mobility shift assays, the mouse gamma2 NRSE and a consensus NRSE both bound in vitro translated NRSF very similarly, and the NRSF gave the same major shifted band with the mouse gamma2 NRSE as was observed with nuclear extracts.
authors: Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD 21201-1559, USA.
authors: W
authors: W
authors: Mu
authors: D R
authors: DR
authors: Burt
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001569:Benzodiazepines / Q000494:pharmacology; D001923:Brain Chemistry / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary; D005786:Gene Expression Regulation / Q000502:physiology; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000502:physiology; D017434:Protein Structure, Tertiary; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology*; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000502:physiology
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D011963:Receptors, GABA-A; D001569:Benzodiazepines; D005680:gamma-Aminobutyric Acid; D008156:Luciferases