MOLECULAR BIOLOGY OF ASPARAGINE-LINKED GLYCOSYLATION

天冬酰胺连接糖基化的分子生物学

• 批准号: 3295049
• 负责人: Mark Lehrman
• 金额: $ 11.41万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1990-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3295049
• 关键词: O glycosidase; antibiotics; antibody; asparagine; cytotoxicity; gel electrophoresis; gene expression; genetic library; genetic manipulation; genome; glycosyltransferase; mannosidase; messenger RNA; molecular cloning; molecular genetics; natural gene amplification; plasmids; tissue /cell culture

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.

这项工作的直接目标是获得中国仓鼠 卵巢（CHO）细胞过表达五种不同的n-酶 连接的糖基化并获得这些分子克隆 酶。 这项工作将需要细胞毒性选择 抗（1）衣霉素的耐药性，N-的抑制剂 乙酰葡萄糖1-磷酸转移酶（（2）Castanospermine， 葡萄糖苷酶I和II的抑制剂 甘露糖苷酶I的抑制剂和（4）Swainsonine，一种抑制剂 甘露糖苷酶II。 绝对性的穿媒霉素耐药性是直接的 由于该抑制剂对CHO细胞有毒。 其他三个 抑制剂不是单独的细胞毒性，而是发现 在存在浓度的浓蛋白A的情况下，细胞毒性 通常对CHO细胞无害。 选择协议将 设计以通过基因扩增获得抗性和 消除其他不良形式的阻力。 细胞 过表达这些酶将有可能检查 酶水平在N连接糖基化调节中的作用。 此外，细胞的核酸将用于制备 Lambda和质粒中高度富集的重组DNA库 将在任何凝胶后产生分子克隆的载体 通过差异杂交进行恢复或筛选。 这 克隆的身份将通过免疫学和 基因转移方法独立于初始 筛选程序。 这些克隆将允许将来研究 调节N连接糖基化的酶，它们 排序机制和寡糖的功能 链。