CURESPONSIVE TRANSCRIPTIONAL REGULATION

本申请中描述的研究计划涉及 铜反应基因表达的机制。实验性的 模型是绿藻衣藻，这是理想的 适用于铜和铜蛋白代谢的研究。 以前的工作已经表明，几种蛋白质的积累 在生物体内的含量取决于体内铜的浓度 灵媒。其中之一是塑料蓝蛋白，一种丰富的铜- 含有光合作用电子转移催化剂，其铜- 依赖蓄积受金属特异性全蛋白的影响 铜蛋白的形成和相对稳定性的提高 载脂蛋白。另一种是细胞色素C6(一种含有 替代电子转移蛋白)，其合成是诱导的 其基因转录激活在铜缺乏细胞中的表达 介导的铜反应元件(CURE)和固化结合 蛋白质(BP)。第三种是辅酶原(Coprogen)。 氧化物酶--血红素生物合成后期的催化剂 途径，与细胞色素C6在铜中协同诱导。 缺乏细胞。 计划在下一个项目期进行的研究目标 是通过生物化学和反向的方法来鉴定和表征 铜依赖转录的遗传学成分 在这个系统中的反应，并阐明分子基础 它的一些主要特征，特别是灵敏度和 金属调节剂相对于其金属的选择性-和 DNA结合位点(S)。重点将放在治愈BP上，该BP 将根据其序列特异性结合到 先前发现的与细胞色素c6基因相关的治疗方法。 生化和生物物理分析将被用来理解 其铜响应功能的结构基础。CDNAs和 编码CURE-BP的基因组序列将通过 传统的方法。在平行实验中，“标记的”调控 将通过筛选插入-的菌落来鉴定突变体 致突变细胞对铜的反应缺失A基因表达 记者基恩。标记基因的野生型等位基因将是 克隆和表达这样一种反向遗传方法 用于金属调节剂的功能分析。一个 第二个目标是分离和分子鉴定 辅酶原氧化酶基因上的铜反应元件与 确定共同调控途径是否起作用的目标 几个铜反应基因，如果是的话，是否治愈了BP 在不同的基因上表现出对治疗的优先利用。 这项研究计划的长期目标是 了解真核细胞中铜的动态平衡 尤其是铜对细胞内基因表达的调控 适应铜缺乏的背景。拟议中的项目 将扩大我们对响应机制的了解，通过 有机体分配营养有限的资源，以便 维持新陈代谢的基本功能，并通过这样做，将 有助于理解人类铜的失衡，这可以 由摄入量减少、排泄过多或遗传引起 疾病。

The research program described in this application deals with the mechanism of copper-responsive gene expression. The experimental model is the green alga Chlamydomonas reinhardtii which is ideally suited for studies of copper and copper-protein metabolism. Previous work has shown that the accumulation of several proteins in this organism is dependent upon the concentration of copper in the medium. One of these is plastocyanin, an abundant copper- containing photosynthetic electron transfer catalyst, whose copper- dependent accumulation is effected by metal-specific holoprotein formation and increased stability of the copper-protein relative to the apoprotein. Another is cytochrome C6 (a heme containing substitute electron transfer protein), whose synthesis is induced in copper-deficient cells by transcriptional activation of its gene mediated -a copper-responsive elements (CuREs) and a CuRE-binding protein (BP). A third is the enzyme coproporphyrinogen (coprogen) oxidase, a catalyst of a late step in the heme biosynthetic pathway, which is induced coordinately with cyt c6 in copper- deficient cells. The objective of the research planned for the next project period is to identify and characterize biochemically and by reverse genetics the components of the copper-dependent transcriptional response in this system, and to elucidate the molecular basis for some of its key features, particularly the sensitivity and selectivity of the metalloregulator with respect to its metal- and DNA-binding site(s). Emphasis will be placed on the CuRE-BP which will be purified on the basis of its sequence specific binding to previously identified CuREs associated with the cyt c6 gene. Biochemical and biophysical analyses will be employed to understand the structural basis of its copper-responsive function. cDNA and genomic sequences encoding the CuRE-BP will be cloned by conventional methods. In parallel experiments, "tagged' regulatory mutants will be identified by screening colonies of insertionally- mutagenized cells for loss of copper-responsive expression of a reporter gene. The wild-type alleles of the tagged genes will be cloned and expressed so that a reverse genetic approach can be exploited for the functional analysis of the metalloregulator. A second aim is the isolation and molecular characterization of copper responsive elements on the coprogen oxidase gene with the objective of determining whether a common regulatory pathway serves several copper-responsive genes and, if so, whether the CuRE-BP displays preferential utilization of CuREs on different genes. The long term objective of this research program is the understanding of copper homeostasis in eukaryotic cells, specifically the regulation of gene expression by copper in the context of adaptation to copper deficiency. The proposed project will expand our knowledge of the response mechanisms by which organisms allocate nutritionally-limited resources so as to maintain metabolically-essential functions, and in so doing, will contribute to the understanding of human copper imbalance which can result from reduced intake, excessive excretion or genetic diseases.