| 研究実績の概要 |
We developed a highly parallel and unbiased yeast chemical-genetic screening system. We constructed an optimized, diagnostic mutant collection representing all major yeast biological processes in a drug-sensitive genetic background. And then, we implemented a multiplexed (768-plex) barcode sequencing protocol, enabling assembly of thousands of chemical-genetic profiles. By FY2016, we applied this chemical-genetic pipeline to annotate 10,000 compounds from the RIKEN NPDepo library, which is composed largely of purified natural products or natural product derivatives, in an unbiased and systematic manner. We observed compounds targeting 17 distinct biological processes represented in the global genetic interaction similarity map. Among them, we examined the activity of 25 compounds annotated to cell wall-related biological processes utilizing several different cell biological readouts including the image-profiling program CalMorph. Microscopic examination of fluorescent staining of two different cell wall polymers, beta-1,3-glucan and chitin, revealed that 8/25 (32%) cell wall predicted compounds induced abnormal cell wall composition, and 10/25 (40%) caused increased bud neck width, a common phenotype of cell-wall-targeting agents.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
We already established the high-throughput chemical-genomic pipeline and screened ~10,000 NPDepo compounds for measuring chemical genetic interactions. And we validated the compounds annotated to cell wall-related biological processes using the image-processing program, CalMorph. Thus, we so far proceeded as we planned.
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| 今後の研究の推進方策 |
We already dealt with our genetic and chemical-genetic data analysis using our database and computational system for ~10,000 compounds in the NPDepo library. We already analyzed morphological profiles using CalMorph for part of the compounds annotated to cell wall-related biological processes. We will further validate target-predicted compounds including not only cell wall but also glycosylation and others using CalMorph. We also plan to characterize protein binding through immunoprecipitation of unique compounds that have been biotinylated or have been UV-crosslinked to affinity beads, the bound proteins may then be identified via mass spectrometry. Finally, we will generate an active database, housing all our chemical-genetic screens and validation of target predictions.
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