Molecular Cloning and Construction of the Coding Region for Human Acetylcholinesterase Reveals a G + C-Rich Attenuating Structure

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 87; no. 24; pp. 9688 - 9692
• Main Authors: Soreq, Hermona, Ben-Aziz, Revital, Prody, Catherine A., Seidman, Shlomo, Gnatt, Averell, Neville, Lewis, Lieman-Hurwitz, Judith, lev-Lehman, Efrat, Ginzberg, Dalia, Lapidot-Lifson, Yaron, Zakut, Haim
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.12.1990; National Acad Sciences
• Subjects: 550201 - Biochemistry- Tracer Techniques; ACETYLCHOLINE; acetylcholinesterase; Acetylcholinesterase - genetics; Acetylcholinesterase - metabolism; AMINES; AMINO ACID SEQUENCE; Amino acids; AMMONIUM COMPOUNDS; ANIMALS; AUTONOMIC NERVOUS SYSTEM AGENTS; Base Sequence; BASIC BIOLOGICAL SCIENCES; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; Biological and medical sciences; CARBOXYLESTERASES; cDNA; cholinesterase; CLONING; Cloning, Molecular; Complementary DNA; Cytosine; DAYS LIVING RADIOISOTOPES; DNA; DNA HYBRIDIZATION; DNA probes; DNA SEQUENCING; DNA-CLONING; DRUGS; ENZYMES; ESTERASES; ESTERS; Female; Fundamental and applied biological sciences. Psychology; GENE AMPLIFICATION; Gene expression; Gene Library; Genomics; Guanine; Humans; HYBRIDIZATION; HYDROLASES; ISOTOPES; LIGHT NUCLEI; MAMMALS; MAN; MESSENGER-RNA; Models, Molecular; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; MOLECULAR STRUCTURE; NEUROREGULATORS; NUCLEI; Nucleic Acid Conformation; NUCLEIC ACIDS; Nucleotides; ODD-ODD NUCLEI; Oligonucleotide Probes; OLIGONUCLEOTIDES; Oocytes; Oocytes - enzymology; ORGANIC COMPOUNDS; PARASYMPATHOMIMETICS; PHOSPHORUS 32; PHOSPHORUS ISOTOPES; PRIMATES; Protein Biosynthesis; QUATERNARY COMPOUNDS; RADIOISOTOPES; RECOMBINANT DNA; restriction fragment length polymorphism; Restriction Mapping; RNA; RNA, Messenger - genetics; Sequence Homology, Nucleic Acid; Sequencing; STRUCTURAL CHEMICAL ANALYSIS; Torpedo; Transcriptional regulatory elements; VERTEBRATES; Xenopus; Human; Gene amplification; Molecular processing; Messenger RNA; Complementary DNA; Nucleotide sequence; Enzyme; Chromosome DNA; Acetylcholinesterase; Molecular cloning; Gene expression; Secondary structure
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.87.24.9688

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 (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.