Grant A L, Jones A, Thomas K L, Wisden W
MRC Laboratory of Molecular Biology, Cambridge, U.K.
Neuroscience. 1996 Dec;75(4):1099-115. doi: 10.1016/0306-4522(96)00344-2.
Hippocalcin is an EF-hand [Persechini A. et al. (1989) Trends Neurosci. 12, 462-467] Ca2+ binding protein encoded by a neuron-specific gene. A detailed atlas of hippocalcin messenger RNA expression in the adult rat brain was complied using in situ hybridization. Highest levels of messenger RNA are found in the hippocampus, where messenger RNA is localized in proximal dendrites of CA pyramidal cells. Expression is also seen in other brain regions, including the neocortex, caudate-putamen, taenia tecti, claustrum, olfactory tubercle, anterior olfactory nucleus, and granule cell and glomerular layers of the olfactory bulb. The rat hippocalcin gene spans approximately 9 kb and consists of three exons, separated by introns of 6.7 and 0.25 kb. Sequence analysis of the putative proximal promoter region identified two clusters of multiple E-box sites which may regulate the cell-specific expression. Two lacZ fusion constructs carrying 0.9 and 3.4 kb of rat hippocalcin gene upstream region were used to create transgenic mice. With the 3.4 kb construct, transgene expression varied between founder mice, but was always found in the dentate gyrus and CA1-CA4 regions of the hippocampus, thus partly mimicking the expression of the endogenous gene. For the 0.9 kb construct, the levels of lacZ expression were weaker and more variable. Neither construct showed expression in any peripheral tissues examined. To establish an in vitro model of transcriptional regulation, the 3.4 and 0.9 kb 5' upstream regions were fused to a promoterless reporter gene encoding chloramphenicol acetyltransferase and transiently transfected into the hippocalcin-positive NG-108 cells. The 3.4 kb construct was strongly expressed, whilst the 0.9 kb construct was not expressed. In this paper, we describe the detailed expression pattern of the rat hippocalcin gene, the gene structure and its neuron-specific promoter.
海马钙结合蛋白是一种由神经元特异性基因编码的EF手型[佩尔塞基尼A.等人（1989年）《神经科学趋势》12卷，462 - 467页]钙结合蛋白。利用原位杂交技术绘制了成年大鼠脑海马钙结合蛋白信使核糖核酸表达的详细图谱。在海马体中发现信使核糖核酸水平最高，其中信使核糖核酸定位于CA锥体细胞的近端树突。在其他脑区也可见表达，包括新皮层、尾状核 - 壳核、带状终板、屏状核、嗅结节、前嗅核以及嗅球的颗粒细胞层和小球层。大鼠海马钙结合蛋白基因跨度约9千碱基对，由三个外显子组成，被6.7千碱基对和0.25千碱基对的内含子隔开。对假定的近端启动子区域的序列分析确定了两个多个E - 盒位点的簇，它们可能调节细胞特异性表达。使用携带大鼠海马钙结合蛋白基因上游区域0.9千碱基对和3.4千碱基对的两个乳糖操纵子融合构建体来创建转基因小鼠。对于3.4千碱基对的构建体，转基因表达在创始小鼠之间有所不同，但总是在海马体的齿状回和CA1 - CA4区域中发现，因此部分模拟了内源性基因的表达。对于0.9千碱基对的构建体，乳糖操纵子表达水平较弱且变化较大。在检测的任何外周组织中，这两种构建体均未显示表达。为了建立转录调控的体外模型，将3.4千碱基对和0.9千碱基对的5'上游区域与编码氯霉素乙酰转移酶的无启动子报告基因融合，并瞬时转染到海马钙结合蛋白阳性的NG - 108细胞中。3.4千碱基对的构建体强烈表达，而0.9千碱基对的构建体未表达。在本文中，我们描述了大鼠海马钙结合蛋白基因的详细表达模式、基因结构及其神经元特异性启动子。
