Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio
Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio
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DOI：
10.1016/s0378-1119(99)00444-8
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发表时间：
1999-11-15
期刊：
影响因子：
3.5
通讯作者：
Shima, A
中科院分区：
文献类型：
--
作者：
Kawakami, K;Shima, A
Tol 2元件存在于青鳉鱼（Oryzias latipes）的基因组中，含有与hAT家族转座子相似的DNA序列。先前，我们已经开发了斑马鱼中的瞬时胚胎切除测定，其中用含有Tol 2元件的质粒DNA注射斑马鱼胚胎，并且已经表明Tol 2元件可从注射的质粒DNA中切除（Kawakami，K.，古贺，A.，Hori，H.，Shima，A.，1998.青鳉Tol 2转座因子在斑马鱼体内的切除Gene 225，17-22）。虽然Tol 2元件被认为是自主的，但尚未鉴定出由Tol 2元件编码的活性转座酶。在这里，我们报告的鉴定和分析的mRNA转录的Tol 2元件在斑马鱼胚胎。Tol 2转录物具有编码649个氨基酸的蛋白质的能力，其氨基酸序列与hAT家族的转座酶的氨基酸序列相似。为了确定转录是否编码活性酶，我们开发了一种新的瞬时胚胎切除试验，在该试验中，斑马鱼受精卵共注射RNA转录体外使用的Tol 2 cDNA作为模板和质粒DNA携带的非自主Tol 2元件，其中有一个缺失的转座酶编码区。只有当与Tol 2 RNA共注射时，才能在斑马鱼中有效地切除非自主Tol 2元件。这一结果表明，Tol 2转录编码的活性酶，一个可能的转座酶，可以催化切除反应的反式。此外，通过共注射分析，我们发现，Tol 2序列缺乏转座酶基因的第一内含子序列不能被切除，这表明它可能含有必要的顺式元件。(C)1999 Elsevier Science B. V.保留所有权利。
The Tol2 element is found in the genome of the medaka fish, Oryzias latipes, and contains DNA sequences similar to those of transposons of the hAT family. Previously, we have developed a transient embryonic excision assay in zebrafish, in which zebrafish embryos were injected with a plasmid DNA harboring the Tol2 element, and have shown that the Tol2 element is excisable from the injected plasmid DNA (Kawakami, K., Koga, A., Hori, H., Shima, A., 1998. Excision of the Tol2 transposable element of the medaka fish, Oryzias latipes, in zebrafish, Danio rerio. Gene 225, 17-22). Although the Tol2 element is thought to be autonomous, an active transposase encoded by the Tol2 element has not been identified. Here we report the identification and analysis of mRNA transcribed from the Tol2 element in zebrafish embryos. The Tol2 transcript has the capacity to encode a protein of 649 amino acids, whose amino acid sequence is similar to those of transposases of the hAT family. To determine whether the transcript encodes an active enzyme, we developed a novel transient embryonic excision assay in which zebrafish fertilized eggs were co-injected with RNA transcribed in vitro using the Tol2 cDNA as a template and a plasmid DNA harboring a nonautonomous Tol2 element, which has a deletion in the transposase coding region. The nonautonomous Tol2 element could be efficiently excised in the zebrafsh only when co-injected with the Tol2 RNA. This result indicates that the Tol2 transcript encodes an active enzyme, a probable transposase, that can catalyze the excision reaction in trans. Further, by the co-injection analysis, we found that the Tol2 sequence lacking the first intron sequence of the transposase gene could not be excised, suggesting that it may contain essential cis-elements. (C) 1999 Elsevier Science B.V. All rights reserved.