The Howard Hughes Medical Institute and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Proc Natl Acad Sci U S A. 2013 Feb 5;110(6):E478-87. doi: 10.1073/pnas.1121543109. Epub 2012 Oct 22.
Chromosome structure and function are influenced by transposable elements, which are mobile DNA segments that can move from place to place. hAT elements are a superfamily of DNA cut and paste elements that move by excision and integration. We have characterized two hAT elements, TcBuster and Space Invaders (SPIN), that are members of a recently described subfamily of hAT elements called Buster elements. We show that TcBuster, from the red flour beetle Tribolium castaneum, is highly active in human cells. SPIN elements are currently inactive elements that were recently highly active in multiple vertebrate genomes, and the high level of sequence similarity across widely diverged species and patchy phylogenetic distribution suggest that they may have moved between genomes by horizontal transfer. We have generated an intact version of this element, SPIN(ON), which is highly active in human cells. In vitro analysis of TcBuster and SPIN(ON) shows that no proteins other than transposase are essential for recombination, a property that may contribute to the ability of SPIN to successfully invade multiple organisms. We also analyze the target site preferences of de novo insertions in the human genome of TcBuster and SPIN(ON) and compare them with the preferences of Sleeping Beauty and piggyBac, showing that each superfamily has a distinctive pattern of insertion. The high-frequency transposition of both TcBuster and SPIN(ON) suggests that these transposon systems offer powerful tools for genome engineering. Finally, we describe a Saccharomyces cerevisiae assay for TcBuster that will provide a means for isolation of hyperactive and other interesting classes of transposase mutants.
染色体的结构和功能受转座元件的影响，转座元件是可以在不同位置移动的可移动 DNA 片段。hAT 元件是一种 DNA 切割和粘贴元件的超家族，通过切除和整合进行移动。我们已经鉴定了两个 hAT 元件，TcBuster 和 Space Invaders (SPIN)，它们是最近描述的 hAT 元件亚家族 Buster 元件的成员。我们表明，来自红粉甲虫 Tribolium castaneum 的 TcBuster 在人类细胞中非常活跃。SPIN 元件目前是不活跃的元件，最近在多个脊椎动物基因组中高度活跃，并且广泛分化的物种之间以及斑驳的系统发育分布中存在高度的序列相似性表明它们可能通过水平转移在基因组之间移动。我们生成了这个元件的完整版本 SPIN(ON)，它在人类细胞中非常活跃。对 TcBuster 和 SPIN(ON)的体外分析表明，除转座酶之外，没有其他蛋白质是重组所必需的，这种特性可能有助于 SPIN 成功入侵多个生物体。我们还分析了 TcBuster 和 SPIN(ON)在人类基因组中新插入的靶位点偏好，并将它们与 Sleeping Beauty 和 piggyBac 的偏好进行比较，表明每个超家族都具有独特的插入模式。TcBuster 和 SPIN(ON)的高频转位表明这些转座子系统为基因组工程提供了强大的工具。最后，我们描述了一种用于 TcBuster 的酿酒酵母测定法，该方法将为分离超活性和其他有趣类别的转座酶突变体提供一种手段。
