Molecular cloning and construction of the coding region for human acetylcholinesterase reveals a G+C-rich attenuating structure

Hermona Soreq*, Revital Ben-Aziz, Catherine A. Prody, Shlomo Seidman, Averell Gnatt, Lewis Neville, Judith Lieman-Hurwitz, Efrat Lev-Lehman, Dalia Ginzberg, Yaron Lapidot-Lifson, Haim Zakut

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

205 Scopus citations

Abstract

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 ≥50% identically aligned amino acids to Torpedo AcChoEase and human butyrylcholinesterase (BtCho-Ease; 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. (.

Original languageEnglish
Pages (from-to)9688-9692
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume87
Issue number24
DOIs
StatePublished - 1990

Keywords

  • Attenuator sequence
  • Butyrylcholinesterase
  • DNA secondary structure
  • Gene amplification

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