STRUCTURE AND EXPRESSION OF COMPLEMENT GENES

补体基因的结构和表达

• 批准号: 3277514
• 负责人: Ronald T Ogata
• 金额: $ 20.24万
• 依托单位: MEDICAL BIOLOGY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-04-01 至 1992-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3277514
• 关键词: autoradiography; biochemical evolution; complement; gel electrophoresis; gene expression; genetic library; genetic mapping; genetic transcription; hormone regulation /control mechanism; laboratory mouse; major histocompatibility complex; molecular cloning; molecular genetics; natural gene amplification; nucleic acid hybridization; nucleic acid sequence; point mutation; radiotracer; recombinant DNA; testosterone; tissue /cell culture; transfection

The S region of the murine histocompatibility complex (H-2) was defined almost 25 years ago as a genetic locus controlling the quantity of Ss (serum substance) in mouse serum. Subsequent work from a number of laboratories established that Ss is composed of two distinct proteins: C4, the murine fourth component of complement, and sex-limited protein (Slp), a protein that shares extensive structural and biochemical identity with C4 but which lacks C4 activity. Slp is notable in that its expression is testosterone-dependent and hence limited to males in some strains while in other strains expression is either independent of testosterone or conversely completely undetectable. Recent nucleic acid cloning and sequencing studies in our laboratory and others have demonstrated that C4 and Slp are encoded by distinct structural genes that lie in the S region and are doubtless the products of gene duplication. The goals of the proposed program are (a) to determine and compare the structural organization of the C4 and Slp genes; (b) to understand the structural and evolutionary relationships between these genes and other genes, in particular the class I and class II genes of the H-2 complex and the genes for the related complement proteins C3 and C5; and (c) to characterize the molecular mechanisms controlling sex-, tissue-, and strain- specific expression of the C4 and Slp genes. We propose to use recombinant DNA cloning and sequencing methods (a) to sequence the entire transcribed regions of the C4 and Slp genes; (b) to extend our study of upstream regions of the C4 and Slp genes by mapping restriction enzyme cleavage sites and by DNA sequencing; (c) to sequence regions upstream of the transcription initiation sites of C4 and Slp genes from mouse strains that exhibit varied C4 and Slp expression phenotypes; and (d) to use site-specific mutation and DNA reconstruction methods, together with methods for expression in cell culture to identify the gene segments responsible for regulating C4 and Slp expression.

小鼠组织相容性复合物（H-2）的S区是 大约25年前被定义为控制 小鼠血清中Ss（血清物质）的量。 后续 许多实验室的工作证明， 由两种不同的蛋白质组成：C4，小鼠的第四种蛋白质， 补体成分和性别限制蛋白（Slp）， 与C4有着广泛的结构和生化特性 但缺乏C4活性 Slp的显著之处在于它的表达式 是睾丸激素依赖性的，因此在某些情况下仅限于男性。 菌株，而在其他菌株中表达是独立的， 睾丸激素或者完全检测不到 最近 我们实验室的核酸克隆和测序研究， 其他人已经证明，C4和Slp由不同的基因编码， 位于S区的结构基因，毫无疑问， 基因复制的产物。 该计划的目标是（a）确定和 比较C4和Slp基因的结构组织;（B） 去理解它们的结构和进化关系 在这些基因和其他基因之间，特别是I类和 H-2复合体的II类基因和相关的 补体蛋白C3和C5;和（c）表征所述补体蛋白C3和C5的特征， 控制性别、组织和应变的分子机制 C4和Slp基因的特异性表达。 我们建议使用重组DNA克隆和测序 方法（a）对C4的整个转录区进行测序， 和Slp基因;（B）为了扩展我们的研究， C4和Slp基因限制性酶切位点定位 和通过DNA测序;（c）对所述DNA序列的上游区域进行测序。 小鼠C4和Slp基因的转录起始位点 表现出不同的C4和Slp表达表型的菌株;和 (d)使用位点特异性突变和DNA重建方法， 以及在细胞培养物中表达以鉴定 负责调节C4和Slp的基因片段 表情