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: 7741723
• 负责人: Madeli Castruita
• 金额: $ 5.05万
• 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/7741723
• 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; 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; 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; genome-wide; hypocupremia; interest; knock-down; loss of function; meetings; 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）： 人类的铜缺陷与先天缺陷和心血管疾病有关。金属营养研究最容易在微生物中研究，这是由于定义明确的培养基可以操纵铜含量以进行生长。 Chlamydomonas Reinhardtii是研究铜缺陷的理想模型生物，因为衣原体在简单，定义明确的盐培养基中提供了生长的优势和遗传性不足。带注释的基因组的可用性以及经典和分子遗传学的可用性使衣原体成为更容易获得的模型。先前关于衣原体中铜缺陷反应的研究表明1）在衣原体中的铜尿布化层次结构，具有更重要的蛋白质，具有更重要的蛋白质，例如已被化为细胞色素氧化酶，2）在-cu细胞中表达的铜和cu-cu细胞中表达的铜和3个损失cubliency-cuby cume cuncy conforcy conforcy cumpoy cumi cumigy cumi cumigy cumigy cumpy noce cumi cuncory cumigy conformige cumpy nocie cumigy conforcy conformiN cucky nocie，从积极退化的非必需的库氏菌中挽救。这些响应由新型转录因子CRR1控制，该转录因子CRR1与治疗的先前定义的核心结合。在过去十年中，常规的遗传和差异表达方法在该信号转导途径中揭示了近十二个基因。由于基因组分析可以预测，在衣原体中的库酶的发生比以前的文献多，因此可能还有更多的铜缺陷响应基因要发现。将使用Illumina的Solexa测序平台来代替传统的微阵列，通过数字mRNA分析来确定铜缺陷的目标，以代替传统的微阵列，以更深入，更定量的mRNA种群。 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.将在已知CRR1和CU缺陷靶标表达模式的背景下进行分析，以识别主要响应基因以生成响应组。根据候选铜响应蛋白的功能和位置的验证和预测，将通过RNAi敲低技术来分析子集，以推断其参与铜稳态。铜对于人类生理学是必不可少的，但由于不足和过度浓度会引起代谢障碍。该项目的目标是确定对铜缺陷的响应和适应性机制。