CHARACTERIZATION OF SMALL MOLECULE METABOLITES

小分子代谢物的表征

基本信息

批准号：
8365852
负责人：
STANLEY FIELDS
金额：
$ 2.18万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2012-06-30
项目状态：
已结题

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Metabolism encompasses all the processes by which a cell generates energy and other essential molecules from nutrients. These pathways rely on hundreds of genes and involve thousands of small molecule intermediates, vitamins and cofactors. To characterize small molecule metabolites in the yeast Saccharomyces cerevisiae, we used chemical derivatization in combination with capillary electrophoresis. We quantified primary amine-containing metabolites in ~4500 yeast strains, each lacking a single non-essential gene, and clustered the strains based on the similarities among profiles to identify genes involved in related processes. Using this approach we found that strains lacking genes involved in arginine biosynthesis all show a similar accumulation of lysine, demonstrating that the clustering approach allows related genes to be assigned to pathways. In addition to the clustering approach, we identified a number of strains exhibiting unique and interesting profiles. Many of the genes identified in these analyses have not been previously characterized; others encode components of well-known complexes. This approach has already yielded a few interesting discoveries relevant to human disease. We observed a dramatic accumulation of an unknown metabolite in strains lacking the gene RPS19, and found that deletion of the genes for many ribosomal proteins results in accumulation of the same metabolite. Haploinsufficiency of RPS19 in humans leads to Blackfan?s anemia, while loss of other ribosomal proteins can lead to other varieties of anemia. The identification of the metabolite accumulation in the rps19 mutant may allow better understanding of this disease in humans. Continuing work will focus on profiling yeast strains in each of which, one of 1000 of the essential genes can be turned off after exposure to the drug doxycycline. As a complementary approach, these strains will also be profiled using a new technology, GCxGC-TOFMS. This method has been shown to allow identification and relative quantification of several hundred metabolites in each sample. Mass spectrometry will also be used to further characterize some of the interesting mutant strains identified using the capillary electrophoresis.

该子项目是利用资源的众多研究子项目之一由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持并且子项目的主要研究者可能是由其他来源提供的，包括其他 NIH 来源。子项目可能列出的总成本代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。新陈代谢涵盖了细胞从营养物质中产生能量和其他必需分子的所有过程。这些途径依赖于数百个基因，涉及数千种小分子中间体、维生素和辅因子。为了表征酿酒酵母中的小分子代谢物，我们使用化学衍生化与毛细管电泳相结合。我们对约 4500 个酵母菌株中的含伯胺代谢物进行了定量，每个酵母菌株都缺乏一个非必需基因，并根据谱图之间的相似性对菌株进行聚类，以识别参与相关过程的基因。使用这种方法，我们发现缺乏参与精氨酸生物合成的基因的菌株都表现出相似的赖氨酸积累，证明聚类方法允许将相关基因分配给途径。除了聚类方法之外，我们还鉴定了许多表现出独特且有趣的特征的菌株。这些分析中鉴定出的许多基因以前从未被表征过；其他编码众所周知的复合物的成分。这种方法已经产生了一些与人类疾病相关的有趣发现。我们观察到缺乏 RPS19 基因的菌株中未知代谢物的急剧积累，并发现许多核糖体蛋白基因的缺失会导致相同代谢物的积累。人类 RPS19 的单倍体不足会导致 Blackfan 贫血，而其他核糖体蛋白的缺失则可能导致其他类型的贫血。 rps19突变体中代谢物积累的鉴定可能有助于更好地了解人类的这种疾病。后续工作将集中于对酵母菌株进行分析，其中 1000 个必需基因中的一个在接触强力霉素药物后会被关闭。作为补充方法，这些菌株还将使用新技术 GCxGC-TOFMS 进行分析。该方法已被证明可以对每个样品中的数百种代谢物进行鉴定和相对定量。质谱分析还将用于进一步表征使用毛细管电泳鉴定的一些有趣的突变菌株。