Stable expression of blood group H determinants and GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase in mouse cells after transfection with human DNA.
We report here the application of a genetic approach to identify and isolate human DNA sequences controlling the expression of a GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase [alpha-1,2)fucosyltransferase). Mouse L cells were chosen as host cells for this scheme since they express the necessary substrate and acceptor molecules for surface display of blood group H Fuc alpha 1----2 G al linkages constructed by (alpha-1,2) fucosyltransferases. However, they do not express cell surface blood group H structures nor detectable (alpha-1,2)fucosyltransferase activity. We therefore asked if (alpha-1,2)fucosyltransferase activity could be expressed and detected in these cells after transfection with human DNA sequences. These cells were transfected with genomic DNA isolated from a human cell line (A431) that expresses (alpha-1,2)fucosyltransferase. A panning procedure and fluorescence-activated cell sorting were used to isolate a mouse transfectant cell line that expresses cell surface H Fuc alpha 1----2 Gal linkages and a cognate (alpha-1,2)fucosyltransferase. Southern blot analysis showed that the genome of this cell line contains several hundred kilobase pairs of human DNA. Genomic DNA from this primary transfectant was used to transfect mouse L cells, and several independent, H-expressing secondary transfectants were isolated by immunological selection. Each expresses an (alpha-1,2)fucosyltransferase. Southern blot analysis demonstrated that the genome of each secondary transfectant contains common, characteristic human DNA restriction fragments. These results show that transfected human DNA sequences determine expression of the (alpha-1,2)fucosyltransferases in the mouse transfectants, that these sequences represent a single locus, and that they are within or linked to specific human restriction fragments identifiable in each secondary transfectant. These sequences may represent a human (alpha-1,2)fucosyltransferase gene.
Ernst
L K
LK
Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor 48109-0650.
Rajan
V P
VP
Larsen
R D
RD
Ruff
M M
MM
Lowe
J B
JB
D000017:ABO Blood-Group System / Q000235:genetics*; D000818:Animals; D000911:Antibodies, Monoclonal; D015139:Blotting, Southern; D002236:Carbohydrate Conformation; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes; D000939:Epitopes / Q000235:genetics*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005647:Fucosyltransferases / Q000235:genetics*; D005786:Gene Expression Regulation; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism; D006602:Hexosyltransferases / Q000235:genetics*; D006801:Humans; D007739:L Cells; D051379:Mice; D009693:Nucleic Acid Hybridization; D014162:Transfection; D000097631:Galactoside 2-alpha-L-fucosyltransferase
D000017:ABO Blood-Group System; D000911:Antibodies, Monoclonal; D004247:DNA; D004262:DNA Restriction Enzymes; D000939:Epitopes; D005647:Fucosyltransferases; D006154:Guanosine Diphosphate Fucose; D006602:Hexosyltransferases; D000097631:Galactoside 2-alpha-L-fucosyltransferase