Isolation of a clone coding for the alpha-subunit of a mouse acetylcholine receptor
Isolation of a clone coding for the alpha-subunit of a mouse acetylcholine receptor
复制标题
编码小鼠乙酰胆碱受体α亚基的克隆的分离
作者：
J. Boulter;W. Luyten;K. Evans;P. Mason;M. Ballivet;D. Goldman;S. Stengelin;G. Martin;S. Heinemann;J. Patrick
小鼠细胞系BC3H-I合成了一种乙酰胆碱受体(AChR)，具有肌肉尼古丁胆碱能受体的药理学特性。我们已经从该细胞系中提纯了mRNA，并使用大小分级的Poly(A)+RNA产生了一个约有50,000个克隆的cDNA文库。用含有编码鸡乙酰胆碱受体α亚基推测的乙酰胆碱结合位点的基因组序列的亚克隆筛选文库。我们获得了一个含有1,717个碱基对插入片段的质粒pMARα15。插入的cDNA在5‘端有26个核苷酸，编码部分信号肽，紧随其后的是1,311个核苷酸的单一开放阅读框架，编码一个49,896道尔顿的蛋白质。该插入物有377个碱基的3‘-非翻译序列，带有3个多聚腺苷酸化位点。用放射性标记的质粒DNA对BC3H-I细胞中Poly(A)+选择的RNA进行Northern印迹分析，鉴定了约2kb的同源RNA物种。在神经支配的小鼠横隔膜和腿部肌肉以及小鼠和大鼠的脑中也发现了类似大小的mRNA。将小鼠AChRα亚基的氨基酸序列与鱼雷、卡氏梭子虫、鸡、人和小牛的氨基酸序列进行比较，其同源性分别为80%、80%、86%、96%和95%。更详细的分析揭示了氨基酸替换在几个结构域中的非随机分布。基于半胱氨酸残基的绝对保守性，提出了α-亚基胞外部分二硫键排列的新模型。
The mouse cell line BC3H-I synthesizes an acetylcholine receptor (AChR) with the pharmacological properties of a muscle nicotinic cholinergic receptor. We have purified mRNA from this cell line and used the size- fractionated poly(A)+RNA to produce a cDNA library of approximately 50,000 clones. The library was screened with a subclone containing genomic sequences coding for the putative acetylcholine-binding site of the alpha-subunit of chicken AChR. We obtained a plasmid, pMAR alpha 15, with a 1,717-base pair insert. The insert cDNA has 26 nucleotides at the 5′-end which code for a portion of the signal peptide followed by a single open reading frame of 1,311 nucleotides which code for a protein of 49,896 daltons. The insert has 377 bases of 3′-untranslated sequence with 3 polyadenylation sites. Radiolabeled plasmid DNA has been used to identify homologous RNA species of about 2 kilobases in Northern blot analyses of poly(A)+ selected RNA from BC3H-I cells. A similar size mRNA is seen in innervated mouse diaphragm and leg muscle, and both mouse and rat brain. Comparisons of the deduced amino acid sequence of the mouse AChR alpha-subunit with Torpedo marmorata, T. californica, chicken, human, and calf sequences show overall homologies of 80%, 80%, 86%, 96%, and 95%, respectively. More detailed analyses reveal a non-random distribution of amino acid substitutions in several structural domains. Based on the absolute conservation of cysteine residues, a new model for the arrangement of the disulfide bonds in the extracellular portion of the alpha-subunit is proposed.