In order to isolate a human gene encoding an α-(1,3)fucosyltransferase (α-(1,3)Fuc-T), genomic DNA from HL-60 cells was transfected by several methods into Chinese hamster ovary (CHO) cells. Colonies expressing α-(1,3)Fuc-T activity were identified by their ability to bind a monoclonal antibody (anti-SSEA-1) that recognizes the carbohydrate product of α-(1,3)Fuc-T action. CHO cells do not express α-(1,3)Fuc-T activity but contain at least two, silent α-(1,3)Fuc-T genes previously identified by their activation in the rare, dominant mutants LEC11 and LEC12. These CHO enzymes were shown to be distinguishable from the α-(1,3)Fuc-T activity of HL-60 cells by the latter's comparative inability to transfer fucose to paragloboside and fetuin. Based on these criteria, only 11 isolates from more than 70 putative transfectants examined were found to stably express an α-(1,3)Fuc-T activity typical of HL-60 cells. Genomic DNA from two of these isolates was used to generate five independent secondary transfectants with HL-60-like α-(1,3)Fuc-T activity. Southern analysis revealed a common DNA fragment that hybridized to an Alu probe in each secondary, providing evidence that a human α-(1,3)Fuc-T gene had been transfected. However, in all transfection experiments, isolates that expressed α-(1,3)Fuc-T activities similar to CHO-encoded enzymes were also obtained. Several lines of evidence indicated that these cells arose from activation of endogenous CHO α-(1,3)Fuc-T genes as a consequence of DNA transfection. These false positives complicated the identification of transfectants expressing a human α-(1,3)Fuc-T gene and represent an important consideration in experiments to transfect other glycosyltransferase genes.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1990|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology