COMPLEMENT C5 DEFICIENCY--MOLECULAR ANALYSIS

补充C5缺乏症--分子分析

• 批准号: 3070957
• 负责人: RICK A. WETSEL
• 金额: $ 6.16万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3070957
• 关键词: cell free system; complement deficiency; genetic library; genetic manipulation; genetic markers; human subject; laboratory mouse; messenger RNA; molecular cloning; molecular pathology; nucleic acid sequence; protein structure; restriction fragment length polymorphism; restriction mapping; secretion; tissue /cell culture; transfection

The fifth component of complement (C5) is a serum glycoprotein that mediates important inflammatory and cytolytic processes. Sera from C5-deficient individuals lack bactericidal activity and have severely impaired ability to induce chemotaxis. The recent isola- tion and characterization of a full-length cDNA for mouse C5, have thus far allowed us to demonstrate: (a) C5D mice synthesize small amounts (10-20% of normal) but do not secrete single chain intracellular C5 protein; (b) C5D mRNA was quantitatively (lO-fold less) and qualitatively (6.0 and 6.5 kb species in cytoplasm) different from sufficient (C5S) mRNA; (c) restriction fragment length polymorphisms (Hind III and Pvu II) between the C5S and C5D genes correlated with the protein deficiency. The experiments outlined in this proposal will extend these preliminary findings in the mouse and will initiate a parallel study regarding C5 deficiency in humans. We will define the structural abnormalities in the protein synthesized by the C5D cells that interfere with the secretion of the protein. cDNA libraries will be prepared from C5S and C5D mRNA and the C5 specific cDNA will be isolated and characterized to analyze the mRNA sequence abnormality(ies) responsible for production of the abnormal C5 protein. In addition, the full-length C5D cDNAs will be employed in in vitro and in vivo translational systems to determine which C5D mRNA is translated into the non-secreted C5D protein. Genomic libraries (cosmid or YAC) will be prepared from C5S and C5D DNA and C5 specific clones will be isolated and characterized to analyze the structural abnormalities in the C5D gene responsible for the abnormalities in the C5D mRNA. The C5S and C5D genomic clones will be transfected into mouse L-cells and the expression of the genes in these cells will be studied to examine the potential role of the C5D cells in producing the abnormalities in protein secretion. Finally, the defects in the human C5 gene which cause the C5 protein deficiency will be determined from restriction fragment length polymorphisms which correlate with the disease. Also, C5S and C5D genes will be isolated and characterized for structural defects which are ultimately responsible for the protein deficiency.

补体的第五个成分（C5）是一种血清糖蛋白， 介导重要的炎症和细胞溶解过程。 血清来自 缺乏C5的个体缺乏杀菌活性，并且具有 严重受损的诱导趋化性的能力。 最近的隔离 用于小鼠C5的全长cDNA的表征和表征 到目前为止，我们可以证明：（a）C5D小鼠合成小 金额（正常的10-20％），但不分泌单链 细胞内C5蛋白； （b）定量C5D mRNA（lo倍） 较少）和定性（细胞质中的6.0和6.5 kb物种） 不同于足够的（C5）mRNA； （c）限制片段 C5S和C5D之间的长度多态性（Hind III和PVU II） 基因与蛋白质缺乏相关。 实验 该提案中概述将延长这些初步发现 在小鼠中，将启动有关C5的平行研究 人类的不足。 我们将定义结构异常 在由C5D细胞合成的蛋白质中 蛋白质的分泌。 将从C5s准备cDNA库 将分离C5D mRNA和C5特异性cDNA，并 特征是分析mRNA序列异常（IES） 负责产生异常C5蛋白。 在 此外，全长C5D cDNA将用于体外 和体内翻译系统以确定哪种C5D mRNA为 翻译成未分泌的C5D蛋白。 基因组库 （Cosmid或YAC）将从C5S和C5D DNA和C5制备 特定的克隆将被隔离并表征以分析 C5D基因的结构异常负责 C5D mRNA异常。 C5S和C5D基因组克隆将 被转染到小鼠L细胞和基因的表达 在这些细胞中将研究以检查 C5D细胞产生蛋白质分泌异常。 最后，引起C5的人C5基因的缺陷 蛋白质缺乏症将从限制片段确定 与疾病相关的长度多态性。 另外，C5S 和C5D基因将被隔离并以结构为特征 最终导致蛋白质的缺陷 不足。