A Tetrahymena thermophila ribozyme-based indicator gene to detect transposition of marked retroelements in mammalian cells -: art. no. e49
A Tetrahymena thermophila ribozyme-based indicator gene to detect transposition of marked retroelements in mammalian cells -: art. no. e49
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DOI：
10.1093/nar/30.11.e49
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发表时间：
2002-06-01
影响因子：
14.9
通讯作者：
Heidmann, T
中科院分区：
文献类型：
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作者：
Esnault, C;Casella, JF;Heidmann, T
我们基于一种自催化核酶元件——嗜热四膜虫23S rRNA I类内含子，设计了一种用于反转录转座的指示基因。该内含子可在体外自我剪接，并且与核mRNA内含子不同，它不需要任何特定的途径和细胞组分来完成剪接过程。我们构建了几种载体，对嗜热四膜虫内含子进行了适当修饰，以便在内含子剪接后能恢复新霉素编码序列的条件下，将其插入含新霉素的盒式结构内的不同位置，并在培养的哺乳动物细胞中检测其剪接效率。通过Northern杂交分析以及在逆转录病毒转导盒式结构后新霉素活性的恢复情况，我们表明剪接效率既取决于局部碱基配对，也取决于内含子在新霉素转录本中的整体位置，并且一些构建体是有功能的。我们进一步表明，当作为对照用人类LINE反转录转座子进行检测时，它们能够有效地筛选出反转录转座事件。这些指示基因对于阐明一系列与不易进行核mRNA内含子剪接的转录本相关的反转录元件的转座机制将有很大帮助，并且这些反转录元件以前无法进行任何反转录转座检测。
We devised an indicator gene for retrotransposition based on an autocatalytic ribozyme element-the Tetrahymena thermophila 23S rRNA group I intron-which can self-splice in vitro and does not require-at variance with nuclear mRNA introns-any specific pathway and cellular component for the completion of the splicing process. Several constructs, with the Tetrahymena intron adequately modified so as to be inserted at various positions within a neomycin-containing cassette under conditions that restore the neomycin-coding sequence after splicing out of the intron, were assayed for splicing efficiency in mammalian cells in culture. We show, both by northern blot analysis and by the recovery of neomycin activity upon retroviral transduction of the cassettes, that splicing efficiency depends on both the local base pairing and the global position of the intron within the neomycin transcript, and that some constructs are functional. We further show that they allow the efficient sorting out of retrotransposition events when assayed, as a control, with a human LINE retrotransposon. These indicator genes should be of great help in elucidating the mechanisms of transposition of a series of retro-elements associated with transcripts not prone to nuclear mRNA intron splicing and previously not opened to any retrotransposition assay.