CLONING OF CDNAS FOR HUMAN ALDEHYDE DEHYDROGENASE-1 AND DEHYDROGENASE-2
CLONING OF CDNAS FOR HUMAN ALDEHYDE DEHYDROGENASE-1 AND DEHYDROGENASE-2
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DOI：
10.1073/pnas.82.11.3771
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发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
YOSHIDA, A
中科院分区：
文献类型：
--
作者：
HSU, LC;TANI, K;YOSHIDA, A
从在噬菌体λgt11中构建的人肝cDNA文库分离编码人胞质醛脱氢酶(ALDH1)和线粒体醛脱氢酶(ALDH2)的部分c互补DNA克隆。使用针对 ALDH1 和 ALDH2 的兔抗体筛选表达文库。随后用与ALDH1和ALDH2的肽序列相容的混合合成寡核苷酸筛选如此获得的阳性克隆。 ALDH1 的阳性克隆之一包含 1.6 千碱基对 (kbp) 的插入。该插入片段编码 340 个氨基酸残基，并具有 538 bp 的 3'' 非编码区和 Poly(A) 片段。从cDNA序列推导的氨基酸序列与报道的人ALDH1的氨基酸序列一致，只是在推导的序列中发现之前的氨基酸序列研究中161位的缬氨酸是异亮氨酸。由于 ALDH2 的氨基酸序列未知，因此分离并测序了人 ALDH2 的 33 个胰蛋白酶肽。基于由此获得的氨基酸序列数据，制备混合寡核苷酸探针。两个阳性克隆，λALDH2-21和λALDH2-36，含有相同的1.2kbp插入片段。另一个克隆，λALDH2-22，含有1.3kbp的插入片段。这 2 个插入片段包含 0.9 kbp 的重叠区域。组合的 cDNA 包含编码 399 个氨基酸残基的序列、一个链终止密码子、一个 403 bp 的 3'' 非翻译区和一个 Poly(A) 片段。推导的氨基酸序列与胰蛋白酶肽的氨基酸序列相一致。人类 ALDH1 和 ALDH2 cDNA 编码区的同源性程度为 66%，蛋白质水平的同源性为 69%。在它们的 3''非翻译区中没有发现显着的同源性。
Partial c[complementary]DNA clones encoding human cytosolic aldehye dehydrogenase (ALDH1) and mitochondrial aldehyde dehydrogenase (ALDH2) were isolated from a human liver cDNA library constructed in phage .lambda.gt11. The expression library was screened by using rabbit antibodies against ALDH1 and ALDH2. Positive clones thus obtained were subsequently screened with mixed synthetic oligonucleotides compatible with peptide sequences of ALDH1 and ALDH2. One of the positive clones for ALDH1 contained an insertion of 1.6 kilobase pairs (kbp). The insert encoded 340 amino acid residues and had a 3'' noncoding region of 538 bp and a poly(A) segment. The amino acid sequence deduced from the cDNA sequence coincided with the reported amino acid sequence of human ALDH1, except that valine at position 161 in the previous amino acid sequence study was found to be isoleucine in the deduced sequence. Since the amino acid sequence of ALDH2 was unknown, 33 tryptic peptides of human ALDH2 were isolated and sequenced. Based on the amino acid sequence data thus obtained, a mixed oligonucleotide probe was prepared. Two positive clones, .lambda.ALDH2-21 and .lambda.ALDH2-36, contained the same insert of 1.2 kbp. Another clone, .lambda.ALDH2-22, contained an insert of 1.3 kbp. These 2 inserts contained an overlap region of 0.9 kbp. The combined cDNA contained a sequence that encodes 399 amino acid residues, a chain-termination codon, a 3'' untranslated region of 403 bp, and a poly(A) segment. The deduced amino acid sequence was compatible with the amino acid sequences of the tryptic peptides. The degree of homology between human ALDH1 and ALDH2 is 66% for the coding regions of their cDNA and 69% at the protein level. No significant homology was found in their 3'' untranslated regions.