Genomic Approaches to Divining Mechanisms for Acclimation Cu Defiency

描述（由申请人提供）： 人类的铜缺乏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，这是因为有明确的培养基，可以控制生长所需的铜含量。莱茵衣藻是研究铜缺乏的理想模式生物，因为衣藻提供了一个简单的，明确的盐培养基中生长的优势，和遗传的顺从性。加上经典和分子遗传学的注释基因组的可用性使衣原体成为更容易获得的模型。以前的研究表明，铜缺乏反应的衣原体1）层次的铜利用更重要的蛋白质，如细胞色素氧化酶优先，2）铜的非依赖性的“备份”代谢途径的发生，表达在-铜细胞，以弥补铜酶的功能丧失，和3）再循环的铜抢救积极降解非必需的铜酶。这些反应由一种新的转录因子CRR 1控制，该因子与先前定义的CuRE核心结合。在过去的十年中，传统的遗传和差异表达方法已经揭示了近十几个基因在这个信号转导通路。由于基因组分析预测衣原体中铜酶的数量级比以前记录的多，因此可能还有更多的铜缺乏反应基因有待发现。铜缺乏的靶点将通过使用Illumina的Solexa测序平台的数字mRNA分析来识别，以代替传统的微阵列，从而对mRNA群体进行更深入和更定量的采样。提出了三种类型的实验：A）来自野生型-Cu与+Cu适应细胞的mRNA谱的比较，B）当野生型细胞从铜充足转变为铜缺乏时从野生型细胞分离的mRNA的比较，反之亦然，和C）铜缺乏crrl突变细胞与铜缺乏野生型细胞的比较。将在已知CRR 1和Cu缺乏靶点表达模式的背景下分析数据，以鉴定产生应答组的主要应答基因。基于对候选铜响应蛋白的功能和位置的验证和预测，将通过RNAi敲低技术对一个子集进行功能分析，以推断其参与铜稳态。铜是人体生理所必需的，但在缺乏和过量的浓度，它会导致代谢紊乱。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.