Transposition mechanism of DNA-type transposable elements of the medaka fish

为了研究青冈鱼转座元件Tol2的转座机制，我们首先建立了一套实验系统，以质粒载体上的细菌LacZ基因作为切割指标，获得该元件的切除率。在这个系统中，由于Tol2元件的切除而丢失的结果是蓝色细菌菌落。接下来，使用这个系统，我们鉴定了一个自主的Tol2拷贝，Tol2存在于青花鱼(O.lasipe)的基因组中，但不存在于与青花鱼密切相关的吕宋青花鱼(O.luzonensis)中。因此，切除引入吕宋青花鱼的单一元素表明该元素是自治的。成功地检测到了切除，因此，所使用的拷贝被发现是一个自主元件。在这些准备工作之后，我们开始检查自主Tol2拷贝的部分，以确定它们对转座功能的重要性。我们制作了各种携带序列改变或缺失的克隆，并估计了这些克隆的转座率。结果表明：(1)转座需要Tol2元件的末端反向重复序列。(2)在该元件的中心区存在一个转座所必需的因子。(3)元件长度与转座率呈正相关。最后两件事是Tol2所独有的，也就是说，到目前为止所研究的其他转座元件没有这些特征。我们还研究了青冈鱼自然种群中Tol2元件结构的变化。发现Tol2在长度和结构上高度均一。这与上述转座机制的结果是一致的。

For the purpose of studying the transposition mechanisms of the medaka fish transposable element Tol2, we first established an experimental system to obtain the excision rate of the element, in which the bacterial lacZ gene on a plasmid vector was used as an indicator of excision. In this system, loss of the Tol2 element due to its excision results in blue bacterial colonies. Next, using this system, we identified an autonomous Tol2 copy, Tol2 is present in the genome of the medaka fish (O.latipes) but is not in the Luzon medaka fish (O.luzonensis) which is closely related to the medaka fish. Therefore, excision of a single element introduced into the Luzon medaka fish indicates that the element is autonomous. It was successful to detect excision, and, thus, the copy used was found to be an autonomous element.After these preparations, we started examining portions of the autonomous Tol2 copy for their importance on the transposition function. We made various clones that carry sequence alterations or deletions, and estimated the transposition rate of the clones. The following results were obtained : (1) The terminal inverted repeat of the Tol2 element are required for transposition. (2) A factor necessary for transposition is present in the central region of the element. (3) The length of the element is positively correlated with the transposition rate. The last two things are unique to Tol2, that is, other transposable elements so far examined do not have these characters.We also examined variation in the structure of Tol2 elements in a medaka fish natural population. Tol2 was found to be highly homogeneous in length and the structure. This is consistent with the results about the transposition mechanisms described above.