Molecular cloning and construction of the coding region for human acetylcholinesterase reveals a G + C-rich attenuating structure.
Molecular cloning and construction of the coding region for human acetylcholinesterase reveals a G + C-rich attenuating structure.
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人乙酰胆碱酯酶编码区的分子克隆和构建揭示了富含 G C 的减毒结构。
DOI：
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发表时间：
1990
影响因子：
11.1
通讯作者：
Y. Lipidot
中科院分区：
文献类型：
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作者：
H. Soreq;R. Ben;C. Prody;Shlomo Seidman;A. Gnatt;L. Neville;J. Lieman;E. Lev;D. Ginzberg;Y. Lipidot
为了研究人乙酰胆碱酯酶（AcChoEase; EC 3.1.1.7）的初级结构及其基因的表达和扩增，我们从表达卵母细胞可翻译AcChoEase mRNA的人组织中构建cDNA文库，并用标记的寡脱氧核苷酸探针进行筛选。多个cDNA克隆所编码的多肽与Torpedo AcChoEase和human butyylcholinesterase （BtChoEase; EC 3.1.1.8）的氨基酸排列相同度大于或等于50%。然而，这些cDNA克隆都被截断在一个300核苷酸长的富含G + c的区域内，其二级结构模式在编码区预期的5'端下游具有高吉布斯自由能（-117 kcal/mol）。基因组DNA文库筛选发现缺失的5'结构域。当与cDNA连接并构建成转录载体时，该序列编码的合成mRNA在微注射卵母细胞中翻译成具有催化活性的AcChoEase，其作为底物对乙酰硫胆碱的偏好优于丁基硫胆碱，对AcChoEase抑制剂BW284C51的抑制敏感，并且对BtChoEase抑制剂四异丙基焦磷酰胺具有抗性。对携带扩增AcChoEase基因的不同个体的基因组DNA进行Blot杂交，发现在预测的富含G + c结构的上游和下游区域的探针显示出不同的强度和限制性模式。因此，人类AcChoEase基因包括一个假定的富含G + c的衰减域，并且在AcChoEase基因扩增的情况下会发生结构改变。
To study the primary structure of human acetylcholinesterase (AcChoEase; EC 3.1.1.7) and its gene expression and amplification, cDNA libraries from human tissues expressing oocyte-translatable AcChoEase mRNA were constructed and screened with labeled oligodeoxynucleotide probes. Several cDNA clones were isolated that encoded a polypeptide with greater than or equal to 50% identically aligned amino acids to Torpedo AcChoEase and human butyrylcholinesterase (BtChoEase; EC 3.1.1.8). However, these cDNA clones were all truncated within a 300-nucleotide-long G + C-rich region with a predicted pattern of secondary structure having a high Gibbs free energy (-117 kcal/mol) downstream from the expected 5' end of the coding region. Screening of a genomic DNA library revealed the missing 5' domain. When ligated to the cDNA and constructed into a transcription vector, this sequence encoded a synthetic mRNA translated in microinjected oocytes into catalytically active AcChoEase with marked preference for acetylthiocholine over butyrylthiocholine as a substrate, susceptibility to inhibition by the AcChoEase inhibitor BW284C51, and resistance to the BtChoEase inhibitor tetraisopropylpyrophosphoramide. Blot hybridization of genomic DNA from different individuals carrying amplified AcChoEase genes revealed variable intensities and restriction patterns with probes from the regions upstream and downstream from the predicted G + C-rich structure. Thus, the human AcChoEase gene includes a putative G + C-rich attenuator domain and is subject to structural alterations in cases of AcChoEase gene amplification.