Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition
Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition
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DOI：
10.1534/genetics.106.060244
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发表时间：
2006-10-01
期刊：
影响因子：
3.3
通讯作者：
Kawakami, Koichi
中科院分区：
文献类型：
--
作者：
Urasaki, Akihiro;Morvan, Ghislaine;Kawakami, Koichi
Tol2元件是在脊椎动物基因组中发现的自然存在的活性转座元件。Tol2转座子系统在鱼类和哺乳动物中都很活跃，被认为是脊椎动物中有用的基因转移载体。然而，对转位至关重要的顺式序列尚未被表征。在这里，我们报道了Tol2元素的最小顺式序列的表征。我们构建了含有不同长度DNA的Tol2载体，分别来自左端（5‘）和右端（3’），并通过斑马鱼胚胎的瞬时切除实验和斑马鱼生殖谱系中的染色体转位分析来测试它们的转位活性。我们证明了距离左端200 bp和右端150 bp的Tol2载体能够在不降低转座效率的情况下进行转座，并发现这些序列，包括末端倒置重复序列（TIRs）和亚末端区域，是转座所必需的。左右两端不能互换。携带> 11kb插入物的Tol2载体可以转置，但在左右端之间需要一定长度的间隔物（< 276但> 18bp）才能切除。此外，我们发现一个5-bp序列，5'-(a /G)AGTA-3'，在基本亚末端区域重复了33次。亚末端区13个不同位点重复序列的突变以及tir的突变严重降低了切除活性，表明它们在转位中起重要作用。Tol2元件最小顺式序列的确定和mini-Tol2载体的构建将有助于开发有用的转座子工具。同时，本研究为进一步了解亚末端重复序列在转位中的作用奠定了生物化学和分子生物学的基础。
The Tol2 element is a naturally occurring active transposable element found in vertebrate genomes. The Tol2 transposon system has been to be active from fish to mammals and considered to be a useful gene transfer vector in vertebrates. However, cis-sequences essential for transposition have not been characterized. Here we report the characterization of the minimal cis-sequence of the Tol2 element. We constructed Tol2 vectors containing various lengths of DNA from both the left (5') and the right (3') ends and tested their transpositional activities both by the transient excision assay using zebrafish embryos and by analyzing chromosomal transposition in the zebrafish germ lineage. We demonstrated that Tol2 vectors with 200 bp from the left end and 150 bp from the right end were capable of transposition without reducing the transpositional efficiency and found that these sequences, including the terminal inverted repeats (TIRs) and the subterminal regions, are sufficient and required for transposition. The left and right ends were not interchangeable. The Tol2 vector carrying an insert of > 11 kb could transpose, but a certain length of spacer, < 276 but > 18 bp, between the left and right ends was necessary for excision. Furthermore, we found that a 5-bp sequence, 5'-(A/G)AGTA-3', is repeated 33 times in the essential subterminal region. Mutations in the repeat sequence at 13 different sites in the subterminal region, as well as mutations in TIRs, severely reduced the excision activity, indicating that they play important roles in transposition. The identification of the minimal cis-sequence of the Tol2 element and the construction of mini-Tol2 vectors will facilitate development of useful transposon tools in vertebrates. Also, our study established a basis for further biochemical and molecular biological studies for understanding roles of the repetitive sequence in the subterminal region in transposition.