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

海鞘胚胎神经系统信号蛋白的原位可视化

• 批准号: 11680663
• 负责人: IWASA Tatsuo
• 金额: $ 2.24万
• 依托单位: MURORAN INSTITUTE OF TECHNOLOGY (2000-2001)Himeji Institute of Technology (1999)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680663/
• 关键词: G protein; Signal transduction; Molecular cloning; Microinjection; Protochordata; GFP; Embryogenesis; Regulation of gene expression; ホヤ; 卵発生; DNA配列

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.

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。从海鞘幼虫的CDNA文库中分离到5个不同的G蛋白α亚基的CDNA克隆。其中HrGi、HrGq和HrGs的推导氨基酸序列分别与人Gi、Gq和Gs的氨基酸序列具有较高的同源性。另外两个亚基(HrGn和HrGx)分别被认为是Gi和Gq家族中一个新的亚家族。原位杂交和Northern印迹分析表明，所有这些G蛋白α亚基的信息都存在于胚胎发育过程中和成体器官中，并具有不同的时空表达模式。经显微注射入海鞘受精卵，发现在推测的翻译起始点上游1.4kb的基因组片段中含有决定HrGn表达模式特征的启动子区域，构建了HrGnα亚基和GFP全长编码区的1.4-HrGn/HrGn编码/GFP融合基因。该结构的显微注射实验是该项目的最终目的，目前正在进行中。

项目成果

I. Shimizu, Y. Yamakawa, Y. Shimazaki, T. Iwasa: "Molecular Cloning of Bombyx Cerebral Opsin (Boceropsin) and Cellular Localization of Its Expression in the Silkworm Brain"Biochem. Biophys. Res. Commun.. 287. 27-34 (2001)

I. Shimizu、Y. Yamakawa、Y. Shimazaki、T. Iwasa：“家蚕脑视蛋白（Boceropsin）的分子克隆及其在蚕脑中表达的细胞定位”Biochem。

吉田麗子 他4名: "ホヤ幼生の脳神経系で特異的に発現しているGタンパク質遺伝子"生物物理. 40Sup.. S213 (200)

Reiko Yoshida 等 4 人：“G 蛋白基因在海鞘幼虫的大脑和神经系统中特异性表达”，生物物理学 40Sup.. S213 (200)

I. Shimizu, T. Iwasa: "Bombyx cerebral opsin (boceropsin) and its expression in the silkworm brain"Zool. Sci.. 18s. 109-109 (2001)

I. Shimizu，T. Iwasa：“家蚕脑视蛋白（boceropsin）及其在蚕脑中的表达”Zool。

Tatsuo Iwasa et al.: "Heterotrimeric G Protein α and β Subunit Genes of the Ascidian,Halocynthia roretzi.in"Biology of Ascidians""Springer-Verlag Tokyo. 6 (2001)

Tatsuo Iwasa 等人：“海鞘异三聚体 G 蛋白 α 和 β 亚基基因，Halocynthia roretzi.in“海鞘生物学”，Springer-Verlag Tokyo 6 (2001)。

M. Takao, T. Iwasa, H. Yamamoto, T. Takeuchi, F. Tokunaga: "Anti-Bovine Rhodopsin Monoclonal Antibody Recognizing Light-Dependent Structural Change"Zool. Sci.. (in press). (2002)

M. Takao、T. Iwasa、H. Yamamoto、T. Takeuchi、F. Tokunaga：“识别光依赖性结构变化的抗牛视紫红质单克隆抗体”Zool。