Genomic Approaches to Divining Mechanisms for Acclimation Cu Defiency

描述(由申请人提供)： 人类缺铜与出生缺陷和心血管疾病有关。金属营养研究最容易在微生物中进行研究，因为有定义明确的介质，允许控制铜含量以进行生长。莱茵衣藻是研究铜缺乏的理想模式生物，因为衣藻提供了在简单、明确的盐介质中生长的优势和遗传亲和性。带有注释的基因组的可用性与经典和分子遗传学相结合，使衣藻成为更容易获得的模型。以往对衣藻对铜缺乏反应的研究表明：1)衣藻对铜的利用有一个层次，其中更重要的蛋白质如细胞色素氧化酶优先利用；2)在铜细胞中表达铜独立的“备份”代谢途径，以补偿铜酶功能的丧失；3)从活跃降解的非必需铜酶中回收铜。这些反应由一种新的转录因子CRR1控制，该转录因子与先前定义的固化核心结合。在过去的十年里，传统的遗传和差异表达方法已经揭示了这一信号转导途径中的近12个基因。由于基因组分析预测衣藻中铜酶的数量将比以前记录的多一个数量级，很可能还有更多的铜缺乏反应基因有待发现。缺铜的靶标将通过使用Illumina的Solexa测序平台的数字信使核糖核酸测序来确定，而不是传统的微阵列，用于对信使核糖核酸种群进行更深层次和更定量的采样。提出了三种类型的实验：A)比较野生型-铜和+铜驯化细胞的mRNA谱；B)比较野生型细胞从富铜到缺铜和从缺铜到缺铜的mRNAs；以及C)比较缺铜CRRL突变细胞和缺铜野生型细胞。这些数据将在已知CRR1和铜缺乏靶标的表达模式的背景下进行分析，以确定产生反应组的主要反应基因。在对候选铜应答蛋白的功能和位置进行验证和预测的基础上，将利用RNAi敲除技术对其亚集进行功能分析，以推断它们参与铜的动态平衡。铜是人体生理所必需的，但在缺乏和过量的情况下，它会引起代谢紊乱。该项目的目标是确定对铜缺乏的反应和适应机制。

DESCRIPTION (provided by applicant): Copper-deficiency in humans has been linked to birth defects and cardiovascular disease. Metal nutrition studies have most easily been studied in microorganisms due to the availability of well-defined media that allow for the manipulation of copper content for growth. Chlamydomonas reinhardtii is an ideal model organism for the study of copper-deficiency because Chlamydomonas offers the advantage of growth in a simple, well-defined salts medium, and genetic amenability. The availability of an annotated genome coupled with classical and molecular genetics make Chlamydomonas an even more accessible model. Previous studies of copper-deficiency responses in Chlamydomonas have demonstrated 1) a hierarchy of copper utlization in Chlamydomonas with more essential proteins like cytochrome oxidase prioritized, 2) the occurrence of copper-independent "back-up" metabolic routes that are expressed in -Cu cells to compensate for the loss of function of cuproenzymes, and 3) re-cycling of Cu salvaged from actively degraded non-essential cuproenzmes. These responses are controlled by a novel transcription factor CRR1 that binds to the previously-defined core of a CuRE. Conventional genetic and differential expression approaches over the last decade have revealed nearly a dozen genes in this signal transduction pathway. Since genome analysis predicts the occurrence of an order of magnitude more cuproenzymes in Chlamydomonas than documented previously, it is likely that there are many more copper-deficiency response genes remaining to be discovered. Targets of copper-deficiency will be identified via digital mRNA profiling using Illumina's Solexa sequencing platform in lieu of conventional microarrays for deeper and more quantitative sampling of the mRNA population. Three types of experiments are proposed: A) comparison of mRNA profiles from wild-type -Cu vs. +Cu acclimated cells, B) comparison of mRNAs isolated from wild-type cells as they transition from copper-replete to copper-deficient and vice-versa, and C) comparison of copper-deficient crrl mutant cells to copper-deficient wild-type cells. The data will be analyzed in the context of the pattern of expression of known CRR1 and Cu-deficiency targets, to identify the primary response genes to generate groups of responses. Based on the validation and prediction of function and location of candidate copper-responsive proteins, a subset will be analyzed functionally by RNAi knock-down techniques to deduce their participation in copper homeostasis. Copper is essential for human physiology, but in deficient and excess concentrations it causes metabolic disorders. The project's goal is to identify the responsive and adaptive mechanisms to copper-deficiency.