Rapid Isolation of Centromeres from Scheffersomyces stipitis
Rapid Isolation of Centromeres from Scheffersomyces stipitis
复制标题
从树干树干酵母中快速分离着丝粒
DOI：
10.1021/acssynbio.7b00166
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发表时间：
2017
影响因子：
4.7
通讯作者：
Shao, Zengyi
中科院分区：
文献类型：
--
作者：
Cao, Mingfeng;Seetharam, Arun Somwarpet;Severin, Andrew Josef;Shao, Zengyi
着丝粒（CENs）是染色体区域促进动粒形成忠实的染色体分离。在酵母中，CEN已被认为是染色体外DNA稳定化的基本要素。然而，CEN的表观遗传性使得它们在单个染色体上的定位非常具有挑战性，特别是在许多未充分研究的非常规酵母物种中。在此之前，我们应用逐步的方法从Schillosomiastipitischromosome5中鉴定了一个500 bp的CEN5，并通过实验证实了它在提高质粒稳定性方面的关键作用。在这里，我们报告了一个基于文库的策略，集成了silicoGC 3染色体扫描和高通量功能筛选，这使得分离所有8个S. stipitisentromeres与16 000倍的序列减少非常有效。进一步鉴定的125 bp的CEN核心序列，出现在每个染色体上多次，但都在独特的签名GC3谷表明，CEN的位置可能是准确地辨别其本地GC3百分比在一个亚组的酵母。
Centromeres (CENs) are the chromosomal regions promoting kinetochore formation for faithful chromosome segregation. In yeasts, CENs have been recognized as the essential elements for extra-chromosomal DNA stabilization. However, the epigeneticity of CENs makes their localization on individual chromosomes very challenging, especially in many not well-studied nonconventional yeast species. Previously, we applied a stepwise method to identify a 500-bp CEN5 fromScheffersomyces stipitischromosome 5 and experimentally confirmed its critical role on improving plasmid stability. Here we report a library-based strategy that integratesin silicoGC3chromosome scanning and high-throughput functional screening, which enabled the isolation of all eightS.stipitiscentromeres with a 16 000-fold reduction in sequence very efficiently. Further identification of a 125-bp CEN core sequence that appears multiple times on each chromosome but all in the unique signature GC3-valley indicates that CEN location might be accurately discerned by their local GC3percentages in a subgroup of yeasts.