Cloning of the human cDNA which can complement the defect of the yeast mannosyltransferase I-deficient mutant alg 1
The assembly of the lipid-linked oligosaccharide, Glc 3 Man 9 GlcNAc 2 -P-P-Dol, occurs on the rough ER membrane in an ordered stepwise manner. The process is highly conserved among eukaryotes. In order to isolate the human mannosyltransferase I (MT-I) gene involved in the process, we used the Saccharomyces cerevisiae MT-I gene (ALG1), which has already been cloned. On searching the EST database with the amino acid sequence of the ALG1 gene product, we detected seven related human EST clones. A
... human EST clones. A human fetal brain cDNA library was screened by PCR using gene-specific primers based on the EST nucleotide sequences and a 430 bp cDNA fragment was amplified. The cDNA library was rescreened with this 430 bp cDNA, and two cDNA clones (HR1-3 and HR1-4) were isolated and sequenced. On a homology search of the EST database with the nucleotide sequence of HR1-3, we detected a novel human EST clone, AA675921 (GenBank accession number). Based on the nucleotide sequences of AA675921 and HR1-4, we designed gene-specific PCR primers, which allowed to amplify a 1.8 kb cDNA from human fetal brain cDNA. This cDNA was cloned and shown to contain an ORF encoding a protein of 464 amino acids. We designated this ORF as Hmat-1. The amino acid sequence deduced from the Hmat-1 gene showed several highly conserved regions shared with the yeast and nematode MT-I sequences. Furthermore, this 1.8 kb cDNA successfully complemented the S.cerevisiae alg1-1 mutation, indicating that the Hmat-1 gene encodes the human MT-I and that the function of this enzyme was conserved between yeast and human.