Itoh N, Slemmon J R, Hawke D H, Williamson R, Morita E, Itakura K, Roberts E, Shively J E, Crawford G D, Salvaterra P M
Proc Natl Acad Sci U S A. 1986 Jun;83(11):4081-5. doi: 10.1073/pnas.83.11.4081.
Choline acetyltransferase (EC 2.3.1.6) is the biosynthetic enzyme for the neurotransmitter acetylcholine. To isolate choline acetyltransferase cDNA clones, a cDNA library was constructed from poly(A)+ RNA of Drosophila melanogaster heads, these being one of the richest known sources of the enzyme. By screening the cDNA library with a mixture of three different monoclonal antibodies to Drosophila choline acetyltransferase, we isolated 14 positive clones. Only 1 of these clones was identified to be a Drosophila choline acetyltransferase cDNA clone based on the following evidence. (i) The amino acid sequence deduced from the nucleotide sequence of the cDNA insert completely corresponded to that of several tryptic peptides from choline acetyltransferase. (ii) The cDNA insert hybridized specifically to only the region on Drosophila polytene chromosomes that had been identified as the site of the choline acetyltransferase (Cha) gene by cytogenetic analysis. The cDNA insert consisted of a coding region 2190 nucleotides long, a 3'-noncoding region 284 nucleotides long, and EcoRI linkers. RNA analysis of Drosophila head poly(A)+ RNA with the cDNA insert as a probe showed the choline acetyltransferase mRNA to be approximately equal to 4700 nucleotides long.
胆碱乙酰转移酶（EC 2.3.1.6）是神经递质乙酰胆碱的生物合成酶。为了分离胆碱乙酰转移酶的cDNA克隆，用黑腹果蝇头部的聚腺苷酸加尾RNA构建了一个cDNA文库，果蝇头部是已知该酶最丰富的来源之一。通过用三种不同的抗果蝇胆碱乙酰转移酶单克隆抗体混合物筛选该cDNA文库，我们分离出了14个阳性克隆。基于以下证据，这些克隆中只有1个被鉴定为果蝇胆碱乙酰转移酶cDNA克隆。（i）从cDNA插入片段的核苷酸序列推导的氨基酸序列与胆碱乙酰转移酶的几个胰蛋白酶肽段的序列完全一致。（ii）cDNA插入片段仅与果蝇多线染色体上通过细胞遗传学分析已被确定为胆碱乙酰转移酶（Cha）基因位点的区域特异性杂交。cDNA插入片段由一个2190个核苷酸长的编码区、一个284个核苷酸长的3'非编码区和EcoRI接头组成。以cDNA插入片段为探针，对果蝇头部聚腺苷酸加尾RNA进行RNA分析，结果显示胆碱乙酰转移酶mRNA长度约为4700个核苷酸。
