TY - JOUR
T1 - Molecular cloning and construction of the coding region for human acetylcholinesterase reveals a G+C-rich attenuating structure
AU - Soreq, Hermona
AU - Ben-Aziz, Revital
AU - Prody, Catherine A.
AU - Seidman, Shlomo
AU - Gnatt, Averell
AU - Neville, Lewis
AU - Lieman-Hurwitz, Judith
AU - Lev-Lehman, Efrat
AU - Ginzberg, Dalia
AU - Lapidot-Lifson, Yaron
AU - Zakut, Haim
PY - 1990
Y1 - 1990
N2 - 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. (.
AB - 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. (.
KW - Attenuator sequence
KW - Butyrylcholinesterase
KW - DNA secondary structure
KW - Gene amplification
UR - http://www.scopus.com/inward/record.url?scp=0025596995&partnerID=8YFLogxK
U2 - 10.1073/pnas.87.24.9688
DO - 10.1073/pnas.87.24.9688
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C2 - 2263619
AN - SCOPUS:0025596995
SN - 0027-8424
VL - 87
SP - 9688
EP - 9692
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 24
ER -