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.

该副本是利用资源的众多研究子项目之一由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持而且，副投影的主要研究员可能是其他来源提供的包括其他NIH来源。列出的总费用可能代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。代谢涵盖了细胞从营养中产生能量和其他必要分子的所有过程。这些途径依靠数百个基因，涉及数千个小分子中间体，维生素和辅因子。为了表征酿酒酵母中酵母糖粉中的小分子代谢产物，我们将化学衍生化与毛细管电泳结合使用。我们在〜4500个酵母菌菌株中量化了原代胺的代谢产物，每个代谢物缺乏一个非必需的基因，并基于概况之间的相似性聚集了菌株，以识别相关过程中涉及的基因。使用这种方法，我们发现缺乏精氨酸生物合成基因的菌株均显示出类似的赖氨酸积累，这表明聚类方法允许将相关的基因分配到途径。除了聚类方法外，我们还确定了许多菌株，这些菌株表现出独特而有趣的概况。这些分析中鉴定的许多基因以前尚未表征。其他编码众所周知的复合物的组成部分。这种方法已经产生了一些与人类疾病有关的有趣发现。我们观察到缺乏基因RPS19的菌株中未知代谢产物的急剧积累，发现许多核糖体蛋白的基因缺失导致同一代谢产物的积累。 RPS19在人类中的单倍不足会导致黑人贫血，而其他核糖体蛋白的损失会导致其他贫血的其他品种。 RPS19突变体中代谢物积累的鉴定可能可以更好地了解人类的这种疾病。持续的工作将集中于分析酵母菌菌株，其中每种菌株都可以在暴露于药物强力霉素后关闭1000个基本基因。作为一种互补的方法，这些菌株还将使用新技术GCXGC-TOFM进行介绍。已显示该方法允许在每个样品中对数百个代谢产物的识别和相对定量。质谱法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。