Genomic Approaches to Divining Mechanisms for Acclimation Cu Defiency

预测铜缺乏症适应机制的基因组方法

• 批准号: 7544376
• 负责人: Madeli Castruita
• 金额: $ 4.68万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-11-20 至 2010-11-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7544376
• 关键词: Acclimatization; Algae; Anabolism; Animal Model; B-Lymphocytes; Back; Binding; Biology; Cardiovascular Diseases; Cells; Chlamydomonas; Chlamydomonas reinhardtii; Chloroplasts; Congenital Abnormality; Copper; Coupled; Cytochromes c6; Data; Depth; Endopeptidases; Environment; Enzymes; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Techniques; Genome; Genomics; Goals; Growth; Hemeproteins; Homeostasis; Human; Hydroxyl Radical; Indium; Iron; Life; Link; Location; Malnutrition; Manganese; Messenger RNA; Metabolic; Metabolic Diseases; Metals; Methods; Micronutrients; Mitochondria; Modeling; Molecular Genetics; Nutritional; Organism; Oxidation-Reduction; Pattern; Peptide Hydrolases; Physiology; Plastocyanin; Population; Production; Proteins; RNA Interference; Range; Reaction; Reactive Oxygen Species; Recycling; Response Elements; Route; Salts; Sampling; Signal Transduction; Signal Transduction Pathway; System; Techniques; Tetrapyrroles; Time; Trace metal; Transition Elements; Validation; Work; Zinc; base; cell type; chemical property; cofactor; cytochrome c oxidase; digital; genome sequencing; hypocupremia; interest; knock-down; loss of function; microorganism; mutant; novel; nutrition; positional cloning; protein degradation; research study; response; transcription factor

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.

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，因为可以使用明确的培养基来控制铜含量以促进生长。莱茵衣藻是研究铜缺乏症的理想模型生物，因为衣藻具有在简单、明确的盐培养基中生长的优势以及遗传适应性。带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。先前对衣藻中铜缺乏反应的研究表明：1）衣藻中铜的利用具有层次结构，优先考虑细胞色素氧化酶等更重要的蛋白质，2）铜独立的“备用”代谢途径的出现，以-Cu表达细胞补偿铜酶功能的丧失，以及3）回收从主动降解的非必需铜酶中回收的铜。这些反应由一种新型转录因子 CRR1 控制，该转录因子与先前定义的 CuRE 核心结合。过去十年的传统遗传和差异表达方法已经揭示了该信号转导途径中的近十几个基因。由于基因组分析预测衣藻中铜酶的出现量比之前记录的要多一个数量级，因此可能还有更多的缺铜反应基因有待发现。将使用 Illumina 的 Solexa 测序平台代替传统的微阵列，通过数字 mRNA 分析来识别缺铜的目标，从而对 mRNA 群体进行更深入、更定量的采样。提出了三种类型的实验：A) 比较野生型 -Cu 与 +Cu 适应细胞的 mRNA 图谱，B) 比较从野生型细胞中分离的 mRNA，因为它们从铜富集转变为铜缺乏，反之亦然。 -反之亦然，以及C)缺铜crrl突变细胞与缺铜野生型细胞的比较。将在已知 CRR1 和 Cu 缺乏靶标的表达模式的背景下分析数据，以确定产生反应组的主要反应基因。基于对候选铜反应蛋白的功能和位置的验证和预测，将通过 RNAi 敲除技术对一个子集进行功能分析，以推断它们对铜稳态的参与。铜对于人体生理至关重要，但浓度不足和过量都会导致代谢紊乱。该项目的目标是确定对铜缺乏的响应和适应机制。