Department of Molecular Biology and Genetics, Division of Biostatistics and Bioinformatics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Proc Natl Acad Sci U S A. 2010 Dec 21;107(51):21966-72. doi: 10.1073/pnas.1016382107. Epub 2010 Dec 3.
Transposons are mobile genetic elements that are an important source of genetic variation and are useful tools for genome engineering, mutagenesis screens, and vectors for transgenesis including gene therapy. We have used second-generation sequencing to analyze ≈2 × 10(5) unique de novo transposon insertion sites of the transposon Hermes in the Saccharomyces cerevisiae genome from both in vitro transposition reactions by using purified yeast genomic DNA, to better characterize intrinsic sequence specificity, and sites recovered from in vivo transposition events, to characterize the effect of intracellular factors such as chromatin on target site selection. We find that Hermes transposon targeting in vivo is profoundly affected by chromatin structure: The subset of genome-wide target sites used in vivo is strongly associated (P < 2e-16 by Fisher's exact test) with nucleosome-free chromatin. Our characterization of the insertion site preferences of Hermes not only assists in the future use of this transposon as a molecular biology tool but also establishes methods to more fully determine targeting mechanisms of other transposons. We have also discovered a long-range sequence motif that defines S. cerevisiae nucleosome-free regions.
转座子是移动的遗传元件，是遗传变异的重要来源，也是基因组工程、诱变筛选以及包括基因治疗在内的转基因载体的有用工具。我们使用第二代测序技术，对来自酵母基因组的纯化 DNA 的体外转座反应，以及体内转座事件中回收的转座子 Hermes 在酿酒酵母基因组中的约 2×10(5)个独特的从头插入位点进行了分析，以更好地描述内在序列特异性，以及描述染色质等细胞内因素对靶位选择的影响。我们发现 Hermes 转座子在体内的靶向作用受到染色质结构的深刻影响：体内使用的全基因组靶位子集与无核小体染色质强烈相关（Fisher 确切检验的 P<2e-16）。我们对 Hermes 插入位点偏好性的描述不仅有助于将来将该转座子用作分子生物学工具，而且还建立了更全面地确定其他转座子靶向机制的方法。我们还发现了一个长序列基序，它定义了酿酒酵母无核小体区域。
