A cloned human DNA restriction fragment determines expression of a GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase in transfected cells. Evidence for isolation and transfer of the human H blood group locus.
We have described previously a gene transfer system for the isolation of human DNA sequences that determine expression of a mammalian GDP-fucose: beta-D-galactoside-2-alpha-L-fucosyltransferase (alpha-(1,2)-fucosyltransferase) (Ernst, L. K., Rajan, V. P., Larsen, R. D., Ruff, M. M., and Lowe, J. B. (1989) J. Biol. Chem. 264, 3436-3447). With this system, we found that de novo expression of the fucosyltransferase in mouse recipient cells was associated with the transfer and stable genomic integration of characteristic human DNA restriction fragments. We report here the results of experiments designed to determine the genetic origin of the fucosyltransferase determined by these sequences. First, we characterize the fucosyltransferases found in these mouse transfectants and in the human cell line used as a DNA donor. We compare their properties to those displayed by the human H and Secretor blood group fucosyltransferases. We find that the enzymes in the transfected cells have properties similar or identical to those of the human H alpha-(1,2)-fucosyltransferase. However, their properties differ significantly from the properties of the human Secretor alpha-(1,2)-fucosyltransferase and are also distinct from the properties of a murine alpha-(1,2)-fucosyltransferase. To confirm further that these transfected human sequences determine the H phenotype of the transfectants, we cloned the two human EcoRI restriction fragments common to each H-expressing secondary transfectant. The larger of these two fragments directs de novo expression of an alpha-(1,2)-fucosyltransferase when transfected into COS-1 cells. The pH activity profile of this alpha-(1,2)-fucosyltransferase and its apparent Michaelis constants for substrate and acceptor mirror those we determined for the human H alpha-(1,2)-fucosyltransferase. We conclude that genetic information sufficient to determine expression of this alpha-(1,2)-fucosyltransferase resides within the 3.4-kilobase pair human EcoRI restriction fragment and that this most likely represents the human H blood group locus.
Rajan
V P
VP
Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor 48109-0650.
Larsen
R D
RD
Ajmera
S
S
Ernst
L K
LK
Lowe
J B
JB
D000017:ABO Blood-Group System / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D015246:Deoxyribonuclease EcoRI; D005647:Fucosyltransferases / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006602:Hexosyltransferases / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000201:enzymology; D007422:Intestines / Q000201:enzymology; D007700:Kinetics; D007739:L Cells / Q000201:enzymology; D051379:Mice; D008809:Mice, Inbred C3H; D013154:Spleen / Q000201:enzymology; D014162:Transfection; D000097631:Galactoside 2-alpha-L-fucosyltransferase
D000017:ABO Blood-Group System; D004247:DNA; D015246:Deoxyribonuclease EcoRI; D005647:Fucosyltransferases; D006602:Hexosyltransferases; D000097631:Galactoside 2-alpha-L-fucosyltransferase