STRUCTURE AND EXPRESSION OF COMPLEMENT GENES C3 AND C4

该项目建议研究(A)编码基因的结构 补体蛋白C3，(B)调控该基因转录成 RNA，(C)导致遗传性C3缺陷的分子缺陷 人和(D)小鼠c DNA的分离。这项研究将利用 小鼠C3基因克隆和C3基因组DNA克隆已在 申请人的实验室，并将延长目前正在进行的工作。第一, C3的组织特异性表达将被研究。补体C3基因 浓度将在肝细胞、巨噬细胞和 C3产生和不产生的组织培养细胞系的多样性和 与基因甲基化状态相关。肝脏的5‘端 和巨噬细胞C3的mRNAs进行比较，以确定是否 基因在两个组织中从相同的启动子转录。它的目的是 建立C3mRNA的全核苷酸序列，为下一步的研究奠定基础 对C3多肽特定区域的研究，如结合 细胞C3d受体的位置。第二部分建议分离出一株 从基因文库中获得人类C3基因的完整拷贝 余弦向量。将对人类基因进行表征和分析 通过将其导入组织培养细胞而发挥作用。有缺陷的等位基因 由纯合子C3缺陷的荷兰家庭成员携带 分析过了。在第三部分中，描述了一种生产方法 克隆小鼠补体C4的基因克隆。最后一节概述了C4cDNA克隆是如何 将与C4变异体的cDNA克隆进行鉴定和区分 在小鼠中，SLP蛋白。这些克隆将用于筛选小鼠的基因 C4基因组克隆文库。人类C4基因的研究工作将紧随其后 根据用小鼠C4基因获得的初步结果。这个 对C3和C4基因的拟议研究将提供关于 这些基因的结构和调控以及对遗传性人类的影响 与这些基因相关的疾病。

The project proposes to study (a) the structure of the gene encoding complement protein C3, (b) the controls of transcription of this gene into RNA, (c) the molecular defects that lead to inherited C3 deficiencies in man and (d) the isolation of mouse cDNA. The study will take advantage of mouse C3 cDNA clones and C3 genomic DNA clones that have been prepared in the applicant's laboratory and will extend currently ongoing work. First, the tissue specific expression of C3 will be investigated. C3 mRNA concentrations will be titrated in hepatocytes, macrophages and in a variety of C3 producing and non-producing tissue culture cell lines and correlated with the state of methylation of the gene. The 5'-ends of liver and macrophage C3 mRNAs will be compared in order to determine whether the gene is transcribed in both tissues from the same promoter. It is intended to establish the total nucleotide sequence of C3 mRNA as a basis for future studies of particular regions of the C3 polypeptide, such as the binding sites for cellular C3d receptors. The second part proposes to isolate one intact copy of the human C3 gene from a gene library to be constructed in cosmid vectors. The human gene will be characterized and assayed for function by transfection into tissue culture cells. A defective allele carried by homozygous C3 deficient members of a Dutch family will be analyzed. In the third part an approach is described for the production of cloned mouse C4 cDNA clones. The last section outlines how C4 cDNA clones will be characterized and distinguished from cDNA clones for the C4 variant of mice, the Slp-protein. These clones will be used to screen mouse gene libraries for C4 genomic clones. Work on the human C4 gene will follow according to the initial results obtained with the mouse C4 gene. The proposed studies on the C3 and C4 genes will provide basic information on the structure and the regulation of these genes and also on inherited human disorders associated with these genes.