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

Ravindra Kumar, Barry Potvin, William A. Muller, Pamela 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 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.

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

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galactoside 3-fucosyltransferase
E-Selectin
Cloning
Cricetulus
Organism Cloning
Ovary
Genes
Cells
DNA
Lymphocytes
Genomic Library
Endothelial cells
Gene Library
Open Reading Frames
Plasmids
Blood
Endothelial Cells
Clone Cells
Carbohydrates
Ligands

ASJC Scopus subject areas

  • Biochemistry

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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. / Kumar, Ravindra; Potvin, Barry; Muller, William A.; Stanley, Pamela.

In: Journal of Biological Chemistry, Vol. 266, No. 32, 1991, p. 21777-21783.

Research output: Contribution to journalArticle

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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 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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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

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AU - Stanley, Pamela

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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.

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