Shimoda K, Ikeshima H, Matsuo K, Hata J, Maejima K, Takano T
Laboratory Animal Center, Keio University School of Medicine, Tokyo, Japan.
Brain Res Mol Brain Res. 1995 Jul;31(1-2):61-70. doi: 10.1016/0169-328x(95)00032-n.
Three non-allelic rat calmodulin (CaM) genes CaMI, CaMII and CaMIII, which share no homology in their 5'-upstream regions, are coordinately expressed in neurons of the central nervous system (CNS). Deletion analysis of the CaMIII promoter showed that the upstream segments longer than 700 bases functioned as efficient promoters, and that the sequence from -133 to -65 was required for the activity of house-keeping type promoter in transient expression assays on a mouse glioma cell line C6. However, the transient expression seemed not to be cell type specific. To determine the temporal and spatial specificity of the promoter function, we produced transgenic mice carrying a fusion gene of the CaMIII segment from -877 to +103 and the lacZ reporter gene. In CNS of the adult transgenic mice, the localization of transgene expression was similar to that of endogenous CaMIII transcripts analyzed by in situ hybridization. The transgene was expressed prominently in pyramidal cells of the cerebral neocortex and the hippocampal regions CA1 to CA3, in Purkinje cells of the cerebellar cortex, and in neurons of the spinal cord, and moderately in granule cells of the dentate gyrus and the cerebellar cortex. In the developing CNS, the overall profiles of neuron-specific expression were also similar for both transgene and endogenous CaMIII that were expressed in the mantle layer and the dorsal root ganglia of the embryonal spinal cord. These results indicated that the neuron-specific expression of rat CaMIII was directed by this 877-base promoter sequence. The CaMIII segment used for the promoter of transgene contained a 29-bp sequence at -410, namely H3, which was conserved in the upstream regions of vertebrate CaMII and CaMIII. H3 seemed to play a pivotal role in the temporal and spatial expression of transgene in CNS, although the deletion of H3 did not decrease CAT activity in the transient expression. The transgene expression was not observed in the external granular cells of the developing cerebellum and in some neurons of the embryonic sensory ganglia in which the endogenous CaMIII was obviously expressed. Therefore, the other cis-acting element(s) located outside of this 877-bp segment seemed to be required for the temporal regulation of CaMIII in certain rudimentary neurons.
三个非等位基因的大鼠钙调蛋白（CaM）基因CaMI、CaMII和CaMIII，它们在5'上游区域没有同源性，在中枢神经系统（CNS）的神经元中协同表达。对CaMIII启动子的缺失分析表明，长度超过700个碱基的上游片段可作为有效的启动子，并且在对小鼠胶质瘤细胞系C6进行的瞬时表达试验中，从-133到-65的序列是维持型启动子活性所必需的。然而，瞬时表达似乎不是细胞类型特异性的。为了确定启动子功能的时空特异性，我们制备了携带从-877到+103的CaMIII片段与lacZ报告基因融合基因的转基因小鼠。在成年转基因小鼠的中枢神经系统中，转基因表达的定位与通过原位杂交分析的内源性CaMIII转录本的定位相似。转基因在大脑新皮质的锥体细胞和海马区域CA1至CA3、小脑皮质的浦肯野细胞以及脊髓神经元中显著表达，在齿状回颗粒细胞和小脑皮质中适度表达。在发育中的中枢神经系统中，转基因和内源性CaMIII在胚胎脊髓的套层和背根神经节中表达的神经元特异性表达总体情况也相似。这些结果表明，大鼠CaMIII的神经元特异性表达是由这个877个碱基的启动子序列指导的。用于转基因启动子的CaMIII片段在-410处包含一个29个碱基的序列，即H3，它在脊椎动物CaMII和CaMIII的上游区域是保守的。H3似乎在中枢神经系统中转基因的时空表达中起关键作用，尽管在瞬时表达中删除H3并没有降低CAT活性。在发育中的小脑的外颗粒细胞和胚胎感觉神经节的一些神经元中未观察到转基因表达，而在这些细胞中内源性CaMIII明显表达。因此，在某些原始神经元中，CaMIII的时间调控似乎需要位于这个877个碱基片段之外的其他顺式作用元件。
