COMPLEMENT C5 DEFICIENCY--MOLECULAR ANALYSIS

补体的第五组分(C5)是一种血清糖蛋白，它 调节重要的炎症和细胞溶解过程。来自中国的血清 缺乏C5的个体缺乏杀菌活性，并具有 严重损害了诱导趋化性的能力。最近的Isola- 小鼠C5、Have全长基因的克隆与鉴定 到目前为止，我们已经证明：(A)C5D小鼠合成小分子 数量(正常的10-20%)，但不分泌单链 细胞内C5蛋白；(B)C5D mRNA定量(10倍) 较少)和定性(细胞质中有6.0kb和6.5kb的物种) 不同于足够的(C5s)mRNA；(C)限制片段 C5S和C5D之间的长度多态(Hind III和Pvu II) 与蛋白质缺乏相关的基因。这些实验 这份提案中概述的内容将扩展这些初步发现 并将启动一项关于C5的平行研究 人类的缺乏症。我们将定义结构异常 在C5D细胞合成的蛋白质中干扰 蛋白质的分泌。将从C5s中制备cDNA文库 并将分离C5D mRNA和C5特异性cDNA，并 以分析m RNA序列异常为特征 负责异常C5蛋白的产生。在……里面 此外，全长C5D cDNA将在体外使用 和体内翻译系统来确定哪一个C5D mRNA是 翻译成不分泌的C5D蛋白。基因组文库 (COSMID或YAC)将由C5S和C5D DNA和C5制备 将分离特定克隆并对其进行鉴定以分析 C5D基因的结构异常导致了 C5D基因表达异常。C5S和C5D基因组克隆将 转染小鼠L细胞及其基因的表达 在这些细胞中的作用将被研究，以检验 C5D细胞产生蛋白质分泌异常。 最后，人类C5基因中导致C5的缺陷 蛋白质缺乏症将从限制性片段中确定 与疾病相关的长度多态。还有，C5 C5D基因将被分离并鉴定为结构 对蛋白质负有最终责任的缺陷 缺乏症。

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.