Molecular cloning of a full-length cDNA for human alcohol dehydrogenase.
Molecular cloning of a full-length cDNA for human alcohol dehydrogenase.
复制标题
人乙醇脱氢酶全长 cDNA 的分子克隆。
DOI：
10.1073/pnas.82.9.2703
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发表时间：
1985
影响因子：
11.1
通讯作者：
Yoshida,A
中科院分区：
文献类型：
--
作者：
Ikuta,T;Fujiyoshi,T;Kurachi,K;Yoshida,A
我们从λ gt 11噬菌体构建的人肝cDNA文库中克隆了编码人乙醇脱氢酶（ADH;乙醇：NAD+氧化还原酶，EC 1.1.1.1）的全长cDNA。通过使用抗人ADH的兔抗体作为第一探针，通过Young和Davis的改良方法[Young，R. A.和戴维斯河W.等人（1983）Proc. Acad. Sci. USA 80，1194-1198]。使用编码Asp-Asp-His-瓦尔-瓦尔和Gln-Cys-Gly-Lys-Cys的混合14-mer合成寡核苷酸作为第二探针。这些氨基酸序列被认为在由ADH 1、ADH 2和ADH 3基因座控制的所有三个亚基（α、β和γ）中是常见的。通过抗体筛选获得的35个阳性噬菌斑的10个λ gt 11重组体含有1.5-2.4个λ gt 11酶对的插入cDNA，并通过与合成探针杂交发现其显示阳性信号。其中一个插入了1631个碱基对的cDNA，含有编码人β 1亚基的374个氨基酸残基的序列，一个链起始密码子，一个链终止密码子，以及另外的3'和5'非翻译区。从cDNA推导出人β 1亚基的完整氨基酸序列。
We have cloned a full-length cDNA coding for human alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) from a human liver cDNA library constructed in phage lambda gt11. The library was screened by using a rabbit antibody against human ADH as a first probe, by the modified method of Young and Davis [Young, R. A. & Davis, R. W. (1983) Proc. Natl. Acad. Sci. USA 80, 1194-1198]. Mixed 14-mer synthetic oligonucleotides encoding Asp-Asp-His-Val-Val and Gln-Cys-Gly-Lys-Cys were used as a second probe. These amino acid sequences are considered to be common in all three subunits (alpha, beta, and gamma) controlled by the ADH1, ADH2, and ADH3 loci. Ten lambda gt11 recombinants of 35 positive plaques obtained by antibody screening contained inserted cDNAs of 1.5-2.4 kilobase pairs and were found to exhibit positive signals by hybridization with synthetic probes. One of them, with an inserted cDNA of 1631 base pairs, contained a sequence that encodes 374 amino acid residues of the human beta 1 subunit, a chain initiation codon, a chain termination codon, and additional 3' and 5' untranslated regions. A complete amino acid sequence of the human beta 1 subunit was deduced from the cDNA.