Cloning of a human alpha(1,3)-fucosyltransferase gene that encodes ELFT but does not confer ELAM-1 recognition on Chinese hamster ovary cell transfectants.
In previous studies, Chinese hamster ovary (CHO) cell genomic DNA transfectants that expressed a human alpha(1,3)-fucosyltransferase (alpha(1,3)Fuc-T) gene were isolated and shown to possess a common approximately 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 lambda ZAP-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 greater than or equal to 6 kb were obtained and one of these (D2.1) conferred human alpha(1,3)-Fuc-T activity on CHO transfectants. A partial restriction map of this clone revealed an approximately 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 approximately 3.6-kb PstI DNA fragment isolated from a human peripheral blood lymphocyte library and shown to encode an alpha(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 approximately 3.6-kb fragments gave identical restriction patterns. In addition, they both caused CHO transfectants to synthesize the Lex determinant Gal beta(1,4)[Fuc alpha(1,3)]GlcNAc beta 1 but not the alpha(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 approximately 3.6-kb PstI fragment had a sequence identical to that of ELFT, an alpha(1,3)-Fuc-T previously reported to confer ELAM-1 binding on a 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.
Kumar
R
R
Department of Cell Biology, Albert Einstein College of Medicine, New York 10461.
Potvin
B
B
Muller
W A
WA
Stanley
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001483:Base Sequence; D016466:CHO Cells; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D006224:Cricetinae; D019040:E-Selectin; D004730:Endothelium, Vascular / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005647:Fucosyltransferases / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D015698:Genomic Library; D006801:Humans; D016256:Lewis X Antigen / Q000032:analysis; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping; D014162:Transfection
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D015815:Cell Adhesion Molecules; D019040:E-Selectin; D016256:Lewis X Antigen; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase