Analysis of the ascidian inward rectifier K+ channel gene promoter activity in cleavage-arrested embryonic cell pairs using GFP reporter gene

1.内向整流钾通道（Inward rectifier K+ channel，IRK）是海鞘发育早期神经分化过程的定量标志之一。利用噬斑杂交技术从罗氏盐爪菌基因组文库中筛选IRK基因，并克隆了该基因。(1)我们测定了基因组IRK基因（TuIRKA）的全序列，包括其编码区，4.8kb的5 '端启动子区和2.5kb的3'端非翻译区。(2)我们使用GFP报告基因检测了TuIRKA对来自8细胞胚胎的切割停滞细胞对aa、bb和aA的启动子活性。结果发现，活性更显着检测在表皮分化的aa和bb对比在神经分化的aA对。(3)BLAST 2参考文献表明TuIRKA氨基酸序列与人IRK的同源性高于果蝇和C.优雅而TuIRKA基因组中的多个剪接位点与C.优雅。在海鞘发育早期，来自8细胞胚胎的前神经外胚层细胞（a4.2）注定要成为幼虫的前神经结构，而推定的脊索神经细胞（A4.1）成为幼虫的后神经结构。来自4细胞胚胎的前象限细胞（A3）是它们的母细胞。我们制备了新的相互作用的裂解阻滞细胞三联体系统，它由A3和一对a4.2或A4.1细胞组成。在A3-aa三联体中，三联体中的所有三个细胞根据细胞接触时间分化成表皮细胞或神经元细胞。当分离的A3细胞神经化与枯草杆菌蛋白酶，蛋白酶，他们主要成为前神经型。同时，保持分离的A3在培养基中含有bFGF，后神经化的促进。

1. Inward rectifier K+ channel (IRK) is known as one of the quantitative markers in the early neural differentiation process of the ascidian development. We carried out the screening of the genomic IRK gene from Halocynthia roretzi genomic library by the plaque hybridization, and cloned the gene. (1) We determined the total sequence of the genomic IRK gene (TuIRKA) including its coding region, 4.8 kb 5'-proximal promoter region and 2.5 kb 3'-UTR. (2) We examined the promoter activity of TuIRKA on the cleavage-arrested cell pairs, aa, bb and aA, from the 8-cell embryo, using GFP reporter. It was found that the activity was more remarkably detected in the epidermally differentiated aa and bb pairs than in the neurally differentiated aA pair. (3) BLAST2 reference represented that the TuIRKA amino acid sequence was more homologous to that of human IRK than those of Drosophila and C. elegans. While, the multiple splice sites in TuIRKA genomic gene, which existed in its membrane-spanning and pore segments as well as cytoplasmic regions, were considerably homologous to those of C. elegans.2. During early ascidian development, anterior neuroectoderm cells (a4.2) from the 8-cell embryo are destined to become larval anterior neural structures, while presumptive notochordal neural cells (A4.1) become larval posterior neural structures. An anterior quadrant cell (A3) from the 4-cell embryo is their parent cell. We prepared the new mutually interactive system of cleavage-arrested cell triplets, which consisted of A3 and a pair of a4.2 or A4.1 cells. In the A3-aa triplet, all three cells in the triplet differentiated into either epidermal or neuronal cells according to the cell contact timing. When isolated A3 cells were neuralized with subtilisin, a protease, they predominantly became the anterior neural type. While, keeping isolated A3 in medium containing a bFGF, posterior neuralization was facilitated.