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Enzymatic and chemical mapping of nucleosome distribution in purified micro- and macronuclei of the ciliated model organism, Tetrahymenathermophila

查看全文 作　　者：Xiao [1]Chen;Shan [1,2]Gao;Yifan [3]Liu;Yuanyuan [1]Wang;Yurui [1]Wang;Weibo [1]Song 高影响力作者 机构地区：[1]Institute of Evolution & Marine Biodiversity, Ocean University of China;[2]Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology;[3]Department of Pathology, University of Michigan高影响力机构 出　　处：《Science China(Life Sciences)》索引2016年第59卷第9期,共11页高影响力期刊 基　　金：supported by the Natural Science Foundation of China(31522051,31470064);the funding awarded to Weibo Song(15-12-1-1-jch);the Qingdao National Laboratory for Marine Science and Technology,China;Yifan Liu was supported by National Sanitation Foundation(MCB 1411565);National Institute of Health(R01 GM087343);the Department of Pathology at the University of Michigan 摘　　要：Genomic distribution of the nucleosome, the basic unit of chromatin, contains important epigenetic information. To map nucleosome distribution in structurally and functionally differentiated micronucleus(MIC) and macronucleus(MAC) of the ciliate Tetrahymena thermophila, we have purified MIC and MAC and performed micrococcal nuclease(MNase) digestion as well as hydroxyl radical cleavage. Different factors that may affect MNase digestion were examined, to optimize mono-nucleosome production. Mono-nucleosome purity was further improved by ultracentrifugation in a sucrose gradient. As MNase concentration increased, nucleosomal DNA sizes in MIC and MAC converged on 147 bp, as expected for the nucleosome core particle. Both MNase digestion and hydroxyl radical cleavage consistently showed a nucleosome repeat length of^200 bp in MAC of Tetrahymena, supporting ~50 bp of linker DNA. Our work has systematically tested methods currently available for mapping nucleosome distribution in Tetrahymena, and provided a solid foundation for future epigenetic studies in this ciliated model organism. 关 键 词：核小体 模式生物 核酸酶 纤毛虫 纯化 羟基自由基 DNA连接 化学
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