The immediate goals of this work are to obtain Chinese Hamster Ovary (CHO) cells that overexpress five different enzymes of N- linked glycosylation and to obtain molecular clones of these enzymes. This work will require a cytotoxic selection for resistance to (1) tunicamycin, an inhibitor of N- acetylglucosamine-1-phosphate transferase, (2) castanospermine, an inhibitor of glucosidases I and II, (3) deoxymannojirimycin, an inhibitor of mannosidase I, and (4) swainsonine, an inhibitor of mannosidase II. Selection for tunicamycin resistance is direct since this inhibitor is toxic to CHO cells. The other three inhibitors are not cytotoxic alone, but have been found to be cytotoxic in the presence of concentrations of concanavalin A that are normally harmless to CHO cells. Selection protocols will be designed to obtain resistance by gene amplification and to eliminate other undesirable forms of resistance. Cells that overexpress these enzymes will make it possible to examine the role of enzyme level in regulation of N-linked glycosylation. Furthermore, the cells' nucleic acids will be used to prepare highly enriched recombinant-DNA libraries in lambda and plasmid vectors which will yield molecular clones after either in-gel renaturation or screening by differential hybridization. the identities of clones will be verified by both immunological and gene-transfer approaches that are independent of the initial screening procedures. These clones will allow future study of the regulation of the enzymes of N-linked glycosylation, their mechanisms of sorting, and the functions of the oligosaccharide chains.
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