Abstract
In animal cells, the enzyme α(1,3)-mannoside-β(1,2)-N-acetylglucosaminyltransferase I (GlcNAc-TI, EC.2.4.1.101) catalyzes the addition of N-acetylglucosamine to the ASN-linked Man GlcNAc oligosaccharide. The Chinese hamster ovary (CHO) mutant cell line Lec1 is deficient in this enzyme activity and, therefore, accumulates mannose-terminating cell surface ASN-linked oligosaccharides. Consequently, Lec1 cells are sensitive to the cytotoxic effects of the mannose-binding lectin Concanavalin A (Con A). Lec1 cells were co-transformed with human DNA form A431 cells and eukaryotic expression plasmids containing the bacterial neo gene by calcium phosphate/DNA-mediated transformation. Co-transformants were selected for resistance to Con A and G-418. DNA from a primary co-transformant was purified and used to transform Lec1 cells, resulting in secondary co-transformants. Both primary and secondary co-transformants exhibited in vitro GlcNAc-TI-specific enzyme activity. DNA gel blot analysis indicated that secondary co-transformants contained both human and neo sequences.
Original language | English (US) |
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Pages (from-to) | 117-122 |
Number of pages | 6 |
Journal | Journal of cellular biochemistry |
Volume | 42 |
Issue number | 3 |
DOIs | |
State | Published - Mar 1990 |
Keywords
- DNA-mediated transformation
- Gene transfer
- GlcNAc-TI
- Glycosyltransferase
- Man GlcNAc
ASJC Scopus subject areas
- Biochemistry
- Cell Biology