cDNA Cloning of the Putative Human β-1,4 Mannosyltransferase Gene which is Involved in Lipid-linked Oligosaccharide Synthesis

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The assembly of the lipid-linked oligosaccharide, Glc_3Man_9GlcNAc_2-P-P-Dol, occurs on the rough ER membrane in the ordered stepwise manner by fourteen glycosyltransferases. The process is highly conserved among eukaryotes. In order to isolate the human β-1,4 mannosyltransferase (β-1,4 MT) gene which is involved in lipid-linked oligosaccharide synthesis, we used the yeast ALGl gene, which has been already cloned and characterized as the β-1,4 MT gene. First, we performed homology search in the EST database on the basis of the amino acid sequence of the ALGl gene product. As a result, seven related human cDNA clones were detected and their nucleotide sequences were used to design gene-specific primers (A1-1F and A1-2R). Using these primers, we amplified by PCR the part of cDNA (about 420 bp) from human fetal brain cDNA library, and cloned it. Using this amplified DNA as a probe, we rescreened the cDNA library and isolated two cDNA clones (HR1-3 and HR1-4) which were likely to be the parts of human β-1,4 MT gene. Sequence analysis of them revealed that the putative human β-1,4 MT possessed several highly conserved regions shared with yeast β-1,4 MT.
東海大学の論文