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. 1990 Dec;87(24):9688–9692. doi: 10.1073/pnas.87.24.9688

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

H Soreq 1, R Ben-Aziz 1, C A Prody 1, S Seidman 1, A Gnatt 1, L Neville 1, J Lieman-Hurwitz 1, E Lev-Lehman 1, D Ginzberg 1, Y Lipidot-Lifson 1, et al.
PMCID: PMC55238  PMID: 2263619

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 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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arpagaus M., Kott M., Vatsis K. P., Bartels C. F., La Du B. N., Lockridge O. Structure of the gene for human butyrylcholinesterase. Evidence for a single copy. Biochemistry. 1990 Jan 9;29(1):124–131. doi: 10.1021/bi00453a015. [DOI] [PubMed] [Google Scholar]
  2. Burstein S. A., Boyd C. N., Dale G. L. Quantitation of megakaryocytopoiesis in liquid culture by enzymatic determination of acetylcholinesterase. J Cell Physiol. 1985 Jan;122(1):159–165. doi: 10.1002/jcp.1041220124. [DOI] [PubMed] [Google Scholar]
  3. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dreyfus P., Zevin-Sonkin D., Seidman S., Prody C., Zisling R., Zakut H., Soreq H. Cross-homologies and structural differences between human cholinesterases revealed by antibodies against cDNA-produced human butyrylcholinesterase peptides. J Neurochem. 1988 Dec;51(6):1858–1867. doi: 10.1111/j.1471-4159.1988.tb01169.x. [DOI] [PubMed] [Google Scholar]
  5. Hall L. M., Spierer P. The Ace locus of Drosophila melanogaster: structural gene for acetylcholinesterase with an unusual 5' leader. EMBO J. 1986 Nov;5(11):2949–2954. doi: 10.1002/j.1460-2075.1986.tb04591.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kessler M., Ben-Asher E., Aloni Y. Elements modulating the block of transcription elongation at the adenovirus 2 attenuation site. J Biol Chem. 1989 Jun 15;264(17):9785–9790. [PubMed] [Google Scholar]
  7. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  8. Lapidot-Lifson Y., Prody C. A., Ginzberg D., Meytes D., Zakut H., Soreq H. Coamplification of human acetylcholinesterase and butyrylcholinesterase genes in blood cells: correlation with various leukemias and abnormal megakaryocytopoiesis. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4715–4719. doi: 10.1073/pnas.86.12.4715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Layer P. G., Rommel S., Bülthoff H., Hengstenberg R. Independent spatial waves of biochemical differentiation along the surface of chicken brain as revealed by the sequential expression of acetylcholinesterase. Cell Tissue Res. 1988 Mar;251(3):587–595. doi: 10.1007/BF00214007. [DOI] [PubMed] [Google Scholar]
  10. Lockridge O., Bartels C. F., Vaughan T. A., Wong C. K., Norton S. E., Johnson L. L. Complete amino acid sequence of human serum cholinesterase. J Biol Chem. 1987 Jan 15;262(2):549–557. [PubMed] [Google Scholar]
  11. Maulet Y., Camp S., Gibney G., Rachinsky T. L., Ekström T. J., Taylor P. Single gene encodes glycophospholipid-anchored and asymmetric acetylcholinesterase forms: alternative coding exons contain inverted repeat sequences. Neuron. 1990 Feb;4(2):289–301. doi: 10.1016/0896-6273(90)90103-m. [DOI] [PubMed] [Google Scholar]
  12. McTiernan C., Adkins S., Chatonnet A., Vaughan T. A., Bartels C. F., Kott M., Rosenberry T. L., La Du B. N., Lockridge O. Brain cDNA clone for human cholinesterase. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6682–6686. doi: 10.1073/pnas.84.19.6682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mercken L., Simons M. J., Swillens S., Massaer M., Vassart G. Primary structure of bovine thyroglobulin deduced from the sequence of its 8,431-base complementary DNA. Nature. 1985 Aug 15;316(6029):647–651. doi: 10.1038/316647a0. [DOI] [PubMed] [Google Scholar]
  14. Prody C. A., Zevin-Sonkin D., Gnatt A., Goldberg O., Soreq H. Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3555–3559. doi: 10.1073/pnas.84.11.3555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rakonczay Z., Brimijoin S. Biochemistry and pathophysiology of the molecular forms of cholinesterases. Subcell Biochem. 1988;12:335–378. doi: 10.1007/978-1-4899-1681-5_10. [DOI] [PubMed] [Google Scholar]
  16. Schumacher M., Camp S., Maulet Y., Newton M., MacPhee-Quigley K., Taylor S. S., Friedmann T., Taylor P. Primary structure of Torpedo californica acetylcholinesterase deduced from its cDNA sequence. 1986 Jan 30-Feb 5Nature. 319(6052):407–409. doi: 10.1038/319407a0. [DOI] [PubMed] [Google Scholar]
  17. Sikorav J. L., Duval N., Anselmet A., Bon S., Krejci E., Legay C., Osterlund M., Reimund B., Massoulié J. Complex alternative splicing of acetylcholinesterase transcripts in Torpedo electric organ; primary structure of the precursor of the glycolipid-anchored dimeric form. EMBO J. 1988 Oct;7(10):2983–2993. doi: 10.1002/j.1460-2075.1988.tb03161.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Soreq H., Seidman S., Dreyfus P. A., Zevin-Sonkin D., Zakut H. Expression and tissue-specific assembly of human butyrylcholine esterase in microinjected Xenopus laevis oocytes. J Biol Chem. 1989 Jun 25;264(18):10608–10613. [PubMed] [Google Scholar]
  19. Soreq H., Zevin-Sonkin D., Razon N. Expression of cholinesterase gene(s) in human brain tissues: translational evidence for multiple mRNA species. EMBO J. 1984 Jun;3(6):1371–1375. doi: 10.1002/j.1460-2075.1984.tb01979.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Zakut H., Ehrlich G., Ayalon A., Prody C. A., Malinger G., Seidman S., Ginzberg D., Kehlenbach R., Soreq H. Acetylcholinesterase and butyrylcholinesterase genes coamplify in primary ovarian carcinomas. J Clin Invest. 1990 Sep;86(3):900–908. doi: 10.1172/JCI114791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Zakut H., Even L., Birkenfeld S., Malinger G., Zisling R., Soreq H. Modified properties of serum cholinesterases in primary carcinomas. Cancer. 1988 Feb 15;61(4):727–737. doi: 10.1002/1097-0142(19880215)61:4<727::aid-cncr2820610416>3.0.co;2-l. [DOI] [PubMed] [Google Scholar]

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