Sakimura K, Kushiya E, Ogura A, Kudo Y, Katagiri T, Takahashi Y
Department of Neuropharmacology, Niigata University, Japan.
Brain Res Mol Brain Res. 1995 Jan;28(1):19-28. doi: 10.1016/0169-328x(94)00177-g.
Neuron-specific enolase (NSE) occurs in mature neurons and paraneurons. We have isolated the genomic clone coding for rat NSE and clarified its gene structure. In order to analyze the regulatory sequence in the 5'-upstream region and introns, we carried out transient expression experiments of NSE genomic DNA fragments fused to chloramphenicol acetyltransferase (CAT) gene which were transfected into several cultured cells. The used cells were primary cultured rat neurons, PC12, neuroblastoma 35, neuroblastoma 103, C6, primary cultured rat glial cells and HeLa cells. The promoter sequence (190 bp) upstream to the transcription initiation site was important in the expression of CAT gene in these cells. From the experiments with external and internal deletion mutants of the fusion gene, the cis-acting regulatory region responsible for the enhanced expression of the CAT activity in the primary cultured neuron and PC12 cells was found to be localized at upstream 500 bp sequence of the intron 1 and 1.5 kbp upstream sequence of the transcription initiation site. In the upstream important sequences, there were the nearest sequences for AP-1 binding motif, AP-2 binding element, SP-1 binding sequence, cAMP response element, half site of glucocorticoid receptor (GRE) binding sequence, half site of thyroid hormor receptor (TR) or retinoic acid receptor (RAR) binding sequence and MTF-1 binding sequence. Furthermore, Octamer-6 binding motifs also were found. In the intron 1, 5' end upstream 50 bp and downstream 100 bp were the most important sequences. We found the nearest sequences for cAMP response element, E2F binding sequence, early growth response (EGR)-1 binding motif, half site of TCF-1 binding sequence and a neuron-specific element-like sequence in the intron 1.
神经元特异性烯醇化酶（NSE）存在于成熟神经元和神经旁细胞中。我们分离出了编码大鼠NSE的基因组克隆，并阐明了其基因结构。为了分析5'上游区域和内含子中的调控序列，我们进行了与氯霉素乙酰转移酶（CAT）基因融合的NSE基因组DNA片段的瞬时表达实验，这些片段被转染到几种培养细胞中。所用细胞包括原代培养的大鼠神经元、PC12、神经母细胞瘤35、神经母细胞瘤103、C6、原代培养的大鼠神经胶质细胞和HeLa细胞。转录起始位点上游的启动子序列（190 bp）对这些细胞中CAT基因的表达很重要。通过对融合基因的外部和内部缺失突变体进行实验，发现负责原代培养神经元和PC12细胞中CAT活性增强表达的顺式作用调控区域位于内含子1上游500 bp序列和转录起始位点上游1.5 kbp序列处。在上游重要序列中，存在与AP-1结合基序、AP-2结合元件、SP-1结合序列、cAMP反应元件、糖皮质激素受体（GRE）结合序列半位点、甲状腺激素受体（TR）或视黄酸受体（RAR）结合序列半位点以及MTF-1结合序列最接近的序列。此外，还发现了八聚体-6结合基序。在内含子1中，5'端上游50 bp和下游100 bp是最重要的序列。我们在内含子1中发现了与cAMP反应元件、E2F结合序列、早期生长反应（EGR）-1结合基序、TCF-1结合序列半位点以及一个神经元特异性元件样序列最接近的序列。
