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.
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.
Lehrman
M A
MA
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Zhu
X Y
XY
Khounlo
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D003001:Cloning, Molecular; D004351:Drug Resistance; D005784:Gene Amplification; D005796:Genes / Q000187:drug effects*; D007700:Kinetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010770:Phosphotransferases / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013045:Species Specificity; D017855:Transferases (Other Substituted Phosphate Groups); D014415:Tunicamycin / Q000494:pharmacology*
D014415:Tunicamycin; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C023222:UDPacetylglucosamine-dolichyl-phosphate acetylglucosamine-1-phosphate transferase