Complementation of a defect in the asparagine-linked glycosylation of a mouse FM3A mutant G258 cell line by spheroplast fusion of a human mega YAC clone 923f5.
Mouse G258 mutant stopped both cell growth and the synthesis of lipid-linked oligosaccharide at the Man(3)GlcNAc(2)-P-P-Dolichol at a restricted temperature with a single gene mutation. To clarify the lesion in the G258 mutant, we isolated human genomic DNA transformants of the G258 mutant, which recovered from both defects by way of cell hybridization with X-ray irradiated HeLa cells. We detected a common 1.3-kb product by inter-human specific sequence in the L1 (L1Hs) PCR in the transformants (Kataoka et al., Somat. Cell Mol. Genet., 24, 235-243 (1998)). In the present study, we screened a human mega yeast artificial chromosome (YAC) library by PCR with primers designed according to the 1.3-kb DNA, and selected YAC clone 923f5. Moreover, we found by spheroplast fusion that YAC clone 923f5 complemented both defects of the G258 mutant. Since the human counterpart of the yeast ALG11 gene is localized in the region, the G258 mutant might have a defect in the mouse ALG11 gene.
Department of Applied Chemistry, School of Engineering, Tokai University, Hiratsuka, Kanagawa, Japan.
Takahisa
T
Masuda
Masayuki
M
Moriya
Kensuke
K
Kataoka
Yoshihisa
Y
Nishikawa
D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D001483:Base Sequence; D002240:Carbohydrate Sequence; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D013104:Spheroplasts / Q000235:genetics*; D013696:Temperature; D014170:Transformation, Genetic
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D001216:Asparagine; D008364:Mannosyltransferases