Amplification and molecular cloning of the hamster tunicamycin-sensitive N-acetylglucosamine-1-phosphate transferase gene. The hamster and yeast enzymes share a common peptide sequence

M. A. Lehrman, X. Zhu, S. Khounlo

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Abstract

The first step in the assembly of the dolichol-linked oligosaccharides required for asparagine-linked glycosylation in eukaryotes is catalyzed by a tunicamycin-sensitive, dolichol phosphate-dependent N-acetylglucosamine-1-phosphate transferase (GPT). A fragment of the gene encoding the enzyme from Chinese hamster ovary (CHO) cells was partially cloned and characterized by a novel strategy. By stepwise selection, CHO cells were made 80-fold resistant to tunicamycin and found to have 10-fold elevated levels of GPT activity. Using a cloned segment of the yeast ALG-7 gene, which encodes the putative GPT from yeast, an amplified gene was identified by Southern blotting of the CHO DNA and a 6.6-kilobase segment of the gene was molecularly cloned. A family of RNA molecules in the 2.0-2.2-kilobase range identified with a probe from this gene was overexpressed in the resistant cells. The cloned DNA revealed a 24-amino acid residue sequence that was 92% conserved with the corresponding yeast sequence.

Original languageEnglish (US)
Pages (from-to)19796-19803
Number of pages8
JournalJournal of Biological Chemistry
Volume263
Issue number36
Publication statusPublished - 1988

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ASJC Scopus subject areas

  • Biochemistry

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