TY - JOUR
T1 - 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
AU - Kumar, Ravindra
AU - Potvin, Barry
AU - Muller, William A.
AU - Stanleyl, Pamela
PY - 1991
Y1 - 1991
N2 - 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 Lex determinant Galβ(1,4)[Fucα(1,3)]GlcNAcβ1 but not the α(2,3)-sialyl-Lex determinant. As expected, these transfectants did not bind to ELAM-1 on activated endothelial cells, since sialyl-Lex 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.
AB - 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 Lex determinant Galβ(1,4)[Fucα(1,3)]GlcNAcβ1 but not the α(2,3)-sialyl-Lex determinant. As expected, these transfectants did not bind to ELAM-1 on activated endothelial cells, since sialyl-Lex 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.
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M3 - Article
C2 - 1718983
AN - SCOPUS:0025720695
SN - 0021-9258
VL - 266
SP - 21777
EP - 21783
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 32
ER -