Cloning of a human α(1,3)-fucosyltransferase gene that encodes ELFT but does not confer ELAM-1 recognition on Chinese hamster ovary cell transfectants

R. Kumar, B. Potvin, W. A. Muller, P. Stanley

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Abstract

In previous studies, Chinese hamster ovary (CHO) cell genomic DNA transfectants that expressed a human α(1,3)-fucosyltransferase (α(1,3)Fuc-T) gene were isolated and shown to possess a common ~7.5-kilobase (kb) EcoRI fragment that hybridized to an Alu probe (Potvin, B., Kumar, R., Howard, D. R., and Stanley, P. (1990) J. Biol. Chem. 265, 1615-1622). One of these transfectants was used to make a genomic DNA library in λZAP-II from EcoRI-digested, size-selected (6-8 kb) DNA, and plaques that hybridized to an Alu probe were purified. After in vivo excision, two plasmids with DNA inserts ≥6 kb were obtained and one of these (D2.1) conferred human α(1,3)-Fuc-T activity on CHO transfectants. A partial restriction map of this clone revealed an ~3.6-kb PstI fragment that contained an Alu sequence. This fragment was subcloned into pGEM-3Zf(+) and compared by restriction analyses with a previously described ~3.6-kb PstI DNA fragment isolated from a human peripheral blood lymphocyte library and shown to encode an α(1,3)-Fuc-T gene (Lowe, J. B., Stoolman, L. M., Nair, R. P., Larsen, R. D., Berhend, T. L., and Marks, R. M. (1990) Cell 63, 475-484). Both ~3.6-kb fragments gave identical restriction patterns. In addition, they both caused CHO transfectants to synthesize the Le(x) determinant Galβ(1,4)[Fucα(1,3)]GlcNAcβ1 but not the α(2,3)-sialyl-Le(x) determinant. As expected, these transfectants did not bind to ELAM-1 on activated endothelial cells, since sialyl-Le(x) is a carbohydrate ligand recognized by ELAM-1. Surprisingly, however, an open reading frame encoded within the ~3.6-kb PstI fragment had a sequence identical to that of ELFT, an α(1,3)-Fuc-T previously reported to confer ELAM-1 binding on a CHO transfectant (Goelz, S. E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B., Chi-Rosso, G., and Lobb, R., (1990) Cell 63, 1349-1356). Possible explanations for these apparently disparate results are discussed.

Original languageEnglish (US)
Pages (from-to)21777-21783
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number32
StatePublished - Jan 1 1991

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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