Mu W, Burt D R
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD 21201-1559, USA.
Brain Res Mol Brain Res. 1999 Apr 6;67(1):137-47. doi: 10.1016/s0169-328x(99)00049-2.
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.
我们已经克隆了小鼠和人类γ-氨基丁酸（GABA）A型受体γ2亚基基因的启动子区域。对于小鼠，通过cDNA末端快速扩增（RACE）和引物延伸确定，两个主要转录起始位点分别位于+1（根据定义）和+43。这种编号方式将起始甲硫氨酸定位在+297。没有TATA或CCAAT框。小鼠和人类序列在第一个内含子中（小鼠为+956）都有一个候选的神经元限制性沉默元件（NRSE）位点。我们构建了各种基于小鼠的启动子/报告基因（荧光素酶）构建体，起始于从-331到+136的核心区域，两端大小不同，包括和排除推定的NRSE及更靠近近端的序列。这些构建体在几种神经元样和非神经元细胞系中通过瞬时转染进行了测试。近端和远端下游元件似乎都有助于将表达导向神经元样细胞，内含子中的NRSE通过在非神经元中发挥抑制作用，以及从+113开始的5'非翻译区的24个碱基部分（命名为GPE1）通过优先促进在神经元样细胞中的表达来实现。共转染的人类NRSF（NRSE的转录因子）降低了含有两个位置NRSE的构建体在神经元样细胞中的报告基因表达。在凝胶迁移率变动分析中，小鼠γ2 NRSE和一个共有NRSE在体外翻译的NRSF结合方面非常相似，并且NRSF与小鼠γ2 NRSE产生的主要迁移带与用核提取物观察到的相同。
