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个不同的G蛋白α亚基CDNA克隆。其中HrGi、HrGq和HrGs氨基酸序列分别与人类Gi、Gq和Gs氨基酸序列具有较高的同源性。另外两个（HrGn和HrGx）被认为分别在Gi和Gq家族中定义了一个新的亚家族。全安装原位杂交和northern blot分析表明，所有这些G蛋白α亚基的信息都存在于胚胎发生和成体器官中，并具有不同的时空表达模式。我们克隆了HrGn的5′-franking序列，构建了HrGn/GFP融合基因。将该构建体显微注射到海鞘受精卵中发现，在假定的翻译起始位点上游含有1.4 Kb的基因组片段包含负责HrGn表达模式特征的启动子区域。然后，我们构建了1.4-HrGn/HrGn-编码/GFP融合基因，该基因具有HrGn α亚基和GFP的全长编码区。该结构的显微注射实验是本项目的最终目的，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.