COMPLEMENT C5 DEFICIENCY--MOLECULAR ANALYSIS

补体的第五组分（C5）是血清糖蛋白， 介导重要的炎症和细胞溶解过程。 血清 C5缺乏的个体缺乏杀菌活性， 诱导趋化性的能力严重受损。 最近的Isola- 小鼠C5全长cDNA的分离和表征 到目前为止，我们证明：（a）C5 D小鼠合成小 量（正常的10-20%），但不分泌单链 细胞内C5蛋白;（B）C5 D mRNA定量（10倍 更少）和定性（细胞质中6.0和6.5 kb的物质） 不同于足够的（C5 S）mRNA;（c）限制性片段 C5 S和C5 D之间的长度多态性（Hind III和Pvu II） 与蛋白质缺乏相关的基因。 实验 本提案中概述的内容将扩展这些初步调查结果， 并将启动关于C5的平行研究 人类的缺陷。 我们将定义结构异常 在C5 D细胞合成的蛋白质中， 蛋白质的分泌。 将从C5 S制备cDNA文库 分离C5 D mRNA和C5特异性cDNA， 表征以分析mRNA序列异常 负责产生异常的C5蛋白。 在 此外，全长C5 D cDNA将在体外使用， 和体内翻译系统，以确定哪种C5 D mRNA 翻译成非分泌型C5 D蛋白。 基因组文库 将从C5 S和C5 D DNA以及C5 将分离特异性克隆并进行表征， C5 D基因的结构异常， C5 D mRNA的异常。 C5 S和C5 D基因组克隆将 转染到小鼠L-细胞中， 在这些细胞中，将进行研究，以检查潜在的作用， C5 D细胞在产生蛋白分泌异常。 最后，人类C5基因的缺陷导致C5 蛋白质缺乏将由限制性片段确定 与疾病相关的长度多态性。 此外，C5 S 和C5 D基因将被分离并表征其结构 最终导致蛋白质 缺陷

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.