The Improvement of the Homology Cloning Method for the Isolation of the Human Gene Involved in Lipid-linked Oligosaccharide Synthesis

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The lipid-linked oligosaccharide, Glc_3Man_9GlcNAc_2-P-P-Dol, is the precursor for the Asn-linked oligosaccharde of glycoprotein. Its biosynthesis is carried out by fourteen glycosyltransferases on the rough ER membrane. Although this process is highly conserved among eukaryotes, most of the genes for these glycosyltransferases have not yet been identified in higher eukaryotes including human. In order to isolate a human mannosyltransferase gene which is involved in lipid-linked oligosaccharide synthesis, we applied two independent approaches using the yeast ALG2 gene, which encoded the GDP-Man : Man_2GlcNAc_2-P-P-Dol α-1,3 mannosyltransferase. First, we cloned the entire yeast ALG2 gene by PCR from yeast genome. Using this cloned ALG2 gene as a probe, we screened the human liver cDNA library by plaque hybridization under the very low strigency condition, and picked up two cDNA clones. Although these cloned cDNAs showed some homology to the yeast ALG2 gene, they were turned out to be unrelated to the yeast ALG2 gene. Secondly, we performed homology search in the EST database on the basis of the amino acid sequence deduced from the yeast ALG2 gene. As a result, several related human cDNA clones were detected in the database, and they were used to design the gene-specific primers. Using these primers, we screened the human liver cDNA library by PCR, and cloned the part of cDNA. It was revealed that the amino acid sequence deduced from the part of cDNA shared several regions with yeast α-1,3 mannosyltransferase, suggesting that it was the part of the human α-1,3 mannosyltransferase gene. These results suggested that the latter approach should be very useful for isolating the unidentified human genes homologous to the already sequenced genes of other species.
東海大学の論文

The Improvement of the Homology Cloning Method for the Isolation of the Human Gene Involved in Lipid-linked Oligosaccharide Synthesis

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