Cloning and complete nucleotide sequence of a full-length cDNA encoding a catalytically functional tumor-associated aldehyde dehydrogenase.
Cloning and complete nucleotide sequence of a full-length cDNA encoding a catalytically functional tumor-associated aldehyde dehydrogenase.
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编码具有催化功能的肿瘤相关醛脱氢酶的全长 cDNA 的克隆和完整核苷酸序列。
DOI：
10.1073/pnas.85.6.1782
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发表时间：
1988
影响因子：
11.1
通讯作者：
Lindahl,R
中科院分区：
文献类型：
--
作者：
JonesJr,DE;Brennan,MD;Hempel,J;Lindahl,R
为探讨S调控大鼠肝癌发生过程中肿瘤相关乙醛脱氢酶表达的机制，克隆了编码该同工酶的cDNA，并用抗体探针进行了鉴定。从HTC大鼠肝癌细胞中提取的含有Poly(A)的RNA被证明具有高水平的肿瘤ALDH，并以此为模板合成了双链cDNA。该基因被甲基化以保护其内部位点。为了确保pUC8的lac启动子正确的表达方向，将两个不同的合成DNA连接物依次添加到cDNA3中。获得了一个包含100,000个独立成员的文库，其插入片段超过1千个碱基。从这个文库中，用间接免疫探针鉴定了两个明显相同的肿瘤ALDH克隆，只是大小不同。选择其中较大的克隆pTALDH进行进一步的研究。有趣的是，由于肿瘤ALDH是一种二聚体酶，pTALDH在细菌细胞中指导合成功能性肿瘤ALDH。通过与从HTC细胞中提纯的肿瘤ALDH的氨基酸序列比较，证实了该序列的正确性。
To study the mechanism(s) controlling expression of the tumor-associated aldehyde dehydrogenase (tumor ALDH), which appears during rat hepatocarcinogenesis, cDNAs encoding this isozyme were cloned and identified with an antibody probe. Poly(A)-containing RNA from HTC rat hepatoma cells, which have been shown to possess high levels of tumor ALDH, was used as template to synthesize double-stranded cDNA. The cDNA was methylated to protect internal sites. Two different synthetic DNA linkers were added sequentially to the cDNA to insure correct orientation for expression from the lac promoter of pUC8. A library of 100,000 independent members carrying inserts greater than 1 kilobase was obtained. From this library, two apparently identical tumor ALDH clones, differing only in size, were identified with an indirect immunological probe. The larger of the cDNA clones identified, pTALDH, was chosen for further study. Interestingly, since tumor ALDH is a dimeric enzyme, pTALDH directs synthesis of a functional tumor ALDH in the bacterial cell. The cDNA sequence has been confirmed by comparison to the amino acid sequence of tumor ALDH purified from HTC cells.