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.

功能性表达T型Ca通道但不表达L型Ca通道的小鼠骨髓瘤细胞系PAI被用作克隆T型Ca通道基因的源材料。使用几对简并寡 DNA 引物从细胞中制备 cDNA，这些引物是根据 L 型 Ca 通道基因序列设计的，用于聚合酶链反应以扩增可能的 T 型 Ca 通道基因。我们获得了一个独特的序列，它与已知的 L 型 Ca 通道基因具有同源部分，但与迄今为止报道的任何 DNA 序列不同。目前正在对该基因进行全长测序。我们还制作了非洲爪蟾卵母细胞表达系统来鉴定我们克隆的基因的功能特性。我们从大鼠脑和胰腺中分离出了 L 型 Ca 通道的 2 个 α 亚基（4A 和 4B）和 3 个 β 亚基基因。亚基基因在青蛙g卵母细胞和中国仓鼠卵巢细胞中以不同组合表达，并通过电生理学研究了通道的特性。 4A 和 beta2 基因诱导的 Ca 通道电流显示出比其他基因组合更长的失活时间。单独注射α亚基基因不会诱导任何可检测到的Ca通道电流。通过低严格筛选小鼠胰腺β克隆细胞系的cDNA文库，已封闭了GTP结合蛋白偶联膜受体蛋白的基因- 单元格 MIN6。利用青蛙卵母细胞表达系统鉴定该基因编码第三类PACAP（垂体腺苷酸环化酶激活多肽）受体。