Analysis of Human Genomic DNA Transformants That Have Recovered from the Deffect of Lipid-Linked Oligosaccharide Synthesis in Mouse G258 Mutant

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We have isolated a temperature-sensitive (<ts>___-) Asn-linked glycosylation mutant, designated G258,from a mouse mammary carcinoma cell line FM3A by [2-^3H] mannose suicide selection. G258 mutant is not only <ts>___- for Asn-linked glycosylation, but also <ts>___- for cell growth. The biochemical defect in G258 mutant resides in the formation of lipid-linked oligosaccharide. At 33℃, G258 mutant synthesized full-sized Glc_3Man_9GlcNAc_2-P-P-Dol, but at 39℃, G258 mutant is able to synthesize merely medium-sized lipid-linked oligosaccharides (up to Man_3GlcNAc_2-P-P-Dol), but unable to elongate the larger lipid-linked oligosaccharides. Analysis of spontaneous revertants revealed that temperature-sensitivities for both cell growth and lipid-linked oligosaccharide synthesis of G258 mutant were derived from a single gene mutation. Therefore the DNA that complements the temperature-sensitivity for cell growth of G258 mutant may contain the gene that complements its temperature-sensitivity for lipid-linked oligosaccharide synthesis. We have transfected human genomic DNA fragments into G258 mutant by the radiation hybrid method, and isolated primary transformants (XR-2,-6 and -8) which showed the recovery from temperature-sensitivities for both cell growth and Asn-linked glycosylation, and the presence of human DNA fragments. Subsequently, we have transfected gonomic DNA fragments from one of the primary transformants (XR-6) into G258 mutant by the radiation hybrid method, and isolated secondary transformants (WXR6-2,-7 and -9). In all WXR clones, there was a common 1.3 kb human DNA fragment. This fragment should serve as a marker DNA for isolating the human gene that complements the deffect of lipid-linked oligosaccharide synthesis in G258 mutant.
東海大学の論文

Analysis of Human Genomic DNA Transformants That Have Recovered from the Deffect of Lipid-Linked Oligosaccharide Synthesis in Mouse G258 Mutant

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