CHARACTERIZATION OF SMALL MOLECULE METABOLITES

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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.