In situ visualization of the signal proteins in the nervous system of the ascidian embryo

为了研究海鞘胚胎神经系统的G蛋白偶联信号系统，我们分离了G蛋白α和β亚基的cDNA克隆。从海鞘幼虫cDNA文库中分离到5个G蛋白α亚基的cDNA克隆。HrGi、HrGq和HrGs的氨基酸序列分别与人Gi、Gq和Gs的氨基酸序列有较高的同源性。另外两个（HrGn和HrGx）分别被认为是Gi和Gq家族中的一个新的亚家族，原位杂交和北方印迹分析表明，这两个G蛋白α亚基在胚胎发育和成体器官中都有表达，并具有不同的时空表达模式。将构建体注射入海鞘受精卵中，结果表明，在HrGn翻译起始位点上游约1.4Kb的基因组片段中含有HrGn的启动子区，并构建了1.4-HrGn/HrGn-coding/GFP融合基因，该融合基因含有HrGn α亚基和GFP的全长编码区。本课题的最终目的是对该构建体进行显微注射实验，目前正在进行中。

In order to investigate G protein-coupling signaling system of neural system of ascidian embryo, we have isolated CDNA clones of G protein alpha and beta subunit. Five different CDNA clones of G protein alpha subunit were isolated from a CDNA library of ascidian larvae. The deduced amino acid sequence of three of them (HrGi, HrGq and HrGs) showed high homology to those of human Gi, Gq and Gs, respectively. The other two (HrGn and HrGx) were supposed to define a novel sub family within the Gi abd Gq families, respectively.Whole-mount in situ hybridization and northern blot analysis revealed that the messages of all these G protein alpha subunits were found during embryogenesis and in adult organs with distinct spatial and temporal expression patterns.We cloned 5'-franking sequence of HrGn and constructed HrGn/GFP fusion gene. The microinjection of the construct into fertilized eggs of ascidian revealed that the genomic fragment containing 1.4 Kb upstream of the putative translation start site contains the promoter region responsible for the expression pattern characteristic for HrGn.Then, we constructed 1.4-HrGn/HrGn-coding/GFP fusion gene, which has the full length coding region of HrGn alpha subunit and GFP. Microinjection experiment of this construct is the final purpose of this project and now on going.