CLONING OF T-TYPE CALCIUM CHANNEL GENE

T型钙通道基因的克隆

基本信息

批准号：
04670108
负责人：
GONOI Tohru
金额：
$ 1.28万
依托单位：
CHIBA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04670108/
关键词：
T-type Ca channel L-type Ca channel Xenopus Oocyte CHO cell Somatostatin Receptor PACAP receptor Patch-clamp method T型カルシウムチャネル L型カルシウムチャネル Caチャンネル遺伝子クローニングミエローマ細胞 Oocyte PCR法

项目摘要

A mouse myeloma cell line, PAI, which functionally expresses T-type Ca channel, but no L-type Ca channels, was used as a source material for cloning a T-type Ca channel gene. cDNA was made from the cells using several pairs of degenerate oligo DNA primers, which were designed based on L-type Ca channel gene sequences, were used for the polymerase chain reactions to amplify a possibly T-type Ca channel gene. We obtained a unique sequence which has homologous parts to already known L-type Ca channel genes but is different from any DNA sequences reported to date. Work of sequensing full length of the gene is now undertaken.We have also made a Xenopus o oocyte-expression system to identify functional properties of the genes we cloned. We have isolated genes of 2 alpha-subunits (4A and 4B) and 3 beta-subunits of L-type Ca channel from rat brain and pancreas. The subunit genes were expressed in different combinations in frog g oocytes and Chinese hamster ovary cells, and properties of the channels were investigated electrophysiologically. Ca channels currents induced by 4A and beta2 genes showed extremely longer inactivation time than those of the other combinations of the genes. The injection of an alpha-subunit gene alone did not induce any detectable Ca channel current.A gene of GTP-binding protein coupling membrane receptor protein has been closed by low-stringency screening of cDNA library of a mouse clonal cell line of a pancreatic beta-cell MIN6. The frog oocyte expression system was used to identify that the gene encodes the 3rd type of PACAP (pituitary adenylate cyclase-activating polypeptide) receptor.

小鼠骨髓瘤细胞系PAI在功能上表达T型Ca通道，但没有L型Ca通道，用作克隆T型Ca通道基因的来源材料。使用几对简并基于L型Ca通道基因序列设计的简并寡核DNA引物制成cDNA，用于聚合酶链反应，以扩增可能是T型CA通道基因。我们获得了一个独特的序列，该序列与已知的L型CA通道基因具有同源部分，但与迄今报道的任何DNA序列不同。现在正在进行序列全长的序列工作。我们还制造了一个爪蟾卵母细胞表达系统，以识别我们克隆的基因的功能特性。我们有2个α-亚基（4a和4b）的孤立基因，以及来自大鼠脑和胰腺的L型CA通道的3个β-亚基。亚基基因在青蛙卵母细胞和中国仓鼠卵巢细胞中以不同的组合表示，并在电生理学上研究了通道的特性。由4A和β2基因诱导的Ca通道电流显示出极长的灭活时间，而其基因的其他组合的流动时间非常长。仅注射α-亚基基因并不能诱导任何可检测的CA通道电流。GTP结合蛋白偶联膜受体蛋白的基因已通过对胰腺β-细胞MIN6的小鼠克隆细胞系列的cDNA库的低静脉筛选而封闭。青蛙卵母细胞表达系统用于识别该基因编码第三种类型的PACAP（垂体腺苷酸环化酶激活多肽）受体。