Merlie J P, Sebbane R, Gardner S, Lindstrom J
Proc Natl Acad Sci U S A. 1983 Jun;80(12):3845-9. doi: 10.1073/pnas.80.12.3845.
Sequences from a gene coding for mouse acetylcholine receptor alpha subunit have been inserted into a recombinant plasmid and cloned in Escherichia coli. mRNAs for acetylcholine receptors occur in low abundance in vertebrate muscle. To clone the mouse alpha-subunit cDNA, we made use of (i) a cell line, BC3H-1, that overproduces the alpha-subunit mRNA and (ii) a polysome fractionation procedure that results in enrichment of alpha-subunit mRNA. Polyadenylylated RNA was used to construct a cDNA library of 750 recombinant clones. Acetylcholine receptor-specific sequences were identified by hybrid-selected translation, followed by monoclonal antibody precipitation and peptide mapping of the translation product. One clone (pA59) that fit these criteria was found in the first 80 isolates. It had a 700-base-pair insert that was excised with Pst I. Blot-hybridization experiments with nick-translated pA59 DNA showed that BC3H-1 cells contain 100-1,000 times more alpha-subunit mRNA than does newborn or adult mouse muscle. Blot hybridization of restriction digests of mouse liver DNA revealed that pA59 is homologous to a very small number (probably one) of genomic sequences.
编码小鼠乙酰胆碱受体α亚基的基因序列已被插入到重组质粒中，并在大肠杆菌中进行克隆。乙酰胆碱受体的mRNA在脊椎动物肌肉中的丰度较低。为了克隆小鼠α亚基cDNA，我们利用了（i）一种过量产生α亚基mRNA的细胞系BC3H-1，以及（ii）一种导致α亚基mRNA富集的多核糖体分级分离程序。使用聚腺苷酸化RNA构建了一个包含750个重组克隆的cDNA文库。通过杂交选择翻译，随后进行单克隆抗体沉淀和翻译产物的肽图谱分析，鉴定出乙酰胆碱受体特异性序列。在前80个分离株中发现了一个符合这些标准的克隆（pA59）。它有一个700个碱基对的插入片段，用Pst I酶切下来。用缺口平移的pA59 DNA进行印迹杂交实验表明，BC3H-1细胞中α亚基mRNA的含量比新生或成年小鼠肌肉中的多100 - 1000倍。对小鼠肝脏DNA限制性消化产物进行印迹杂交显示，pA59与极少数（可能一个）基因组序列同源。
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