Hitoshi S, Kusunoki S, Kanazawa I, Tsuji S
Molecular Glycobiology, Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-0198, Japan.
J Biol Chem. 1999 Jan 1;274(1):389-96. doi: 10.1074/jbc.274.1.389.
The rabbit H-blood type alpha1,2-fucosyltransferase (RFT-I), gene and its biosynthetic products, H antigens (Fucalpha1,2Galbeta), are abundantly expressed in a subset of dorsal root ganglia (DRG) neurons. To investigate the regulatory mechanisms for the RFT-I gene expression, we determined the genomic structure and promoter activity of this gene. PCR amplification of the 5' cDNA end analysis revealed two transcriptional start sites, 498 and 82 nucleotides upstream of the translational initiation codon, the latter site yielding a major 3.1-kb transcript specifically expressed in DRG, as revealed by Northern blotting. Promoter analysis of the 5'-flanking region of the RFT-I gene using a luciferase gene reporter system demonstrated strong promoter activity in PC12 cells, which express the rat H-type alpha1,2-fucosyltransferase gene, and Neuro2a mouse neuroblastoma cells. Deletion analysis revealed the 704-base pair minimal promoter region flanking the translational initiation codon, for which two distinct promoter activities were detected and differentially used in PC12 and Neuro2a cells. The minimal promoter region contained a GC-rich domain (GC content 80%), in which a Sp1 binding sequence and a GSG-like nerve growth factor-responsive element were found, but lacked TATA- and CAAT-boxes. Promoter analysis with a primary culture of DRG neurons demonstrated that the minimal promoter region of the RFT-I gene was sufficient for the expression of a reporter gene in DRG neurons. We conclude that the TATA-less GC-rich minimal promoter region of the RFT-I gene controls DRG small neuron-specific expression of the RFT-I gene.
兔H血型α1,2-岩藻糖基转移酶（RFT-I）、其基因及其生物合成产物H抗原（Fucα1,2Galβ）在背根神经节（DRG）神经元的一个亚群中大量表达。为了研究RFT-I基因表达的调控机制，我们确定了该基因的基因组结构和启动子活性。5' cDNA末端分析的PCR扩增显示了两个转录起始位点，分别位于翻译起始密码子上游498和82个核苷酸处，如Northern印迹所示，后一个位点产生了一个主要的3.1-kb转录本，在DRG中特异性表达。使用荧光素酶基因报告系统对RFT-I基因5'侧翼区域进行启动子分析，结果表明在表达大鼠H型α1,2-岩藻糖基转移酶基因的PC12细胞和Neuro2a小鼠神经母细胞瘤细胞中具有强大的启动子活性。缺失分析揭示了翻译起始密码子侧翼的704碱基对最小启动子区域，在PC12和Neuro2a细胞中检测到两种不同的启动子活性并被差异使用。最小启动子区域包含一个富含GC的结构域（GC含量80%），其中发现了一个Sp1结合序列和一个GSG样神经生长因子反应元件，但缺乏TATA盒和CAAT盒。对DRG神经元原代培养物进行的启动子分析表明，RFT-I基因的最小启动子区域足以在DRG神经元中表达报告基因。我们得出结论，RFT-I基因的无TATA富含GC的最小启动子区域控制着RFT-I基因在DRG小神经元中的特异性表达。
