Han Jeffrey S, Boeke Jef D
Department of Molecular Biology and Genetics and High Throughput Biology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Nature. 2004 May 20;429(6989):314-8. doi: 10.1038/nature02535.
LINE-1 (L1) elements are retrotransposons that comprise large fractions of mammalian genomes. Transcription through L1 open reading frames is inefficient owing to an elongation defect, inhibiting the robust expression of L1 RNA and proteins, the substrate and enzyme(s) for retrotransposition. This elongation defect probably controls L1 transposition frequency in mammalian cells. Here we report bypassing this transcriptional defect by synthesizing the open reading frames of L1 from synthetic oligonucleotides, altering 24% of the nucleic acid sequence without changing the amino acid sequence. Such resynthesis led to greatly enhanced steady-state L1 RNA and protein levels. Remarkably, when the synthetic open reading frames were substituted for the wild-type open reading frames in an established retrotransposition assay, transposition levels increased more than 200-fold. This indicates that there are probably no large, rigidly conserved cis-acting nucleic acid sequences required for retrotransposition within L1 coding regions. These synthetic retrotransposons are also the most highly active L1 elements known so far and have potential as practical tools for manipulating mammalian genomes.
LINE-1（L1）元件是逆转录转座子，在哺乳动物基因组中占很大比例。由于延伸缺陷，通过L1开放阅读框的转录效率低下，这抑制了L1 RNA和蛋白质（逆转录转座的底物和酶）的强劲表达。这种延伸缺陷可能控制着哺乳动物细胞中L1的转座频率。在此，我们报告通过从合成寡核苷酸合成L1的开放阅读框来绕过这种转录缺陷，在不改变氨基酸序列的情况下改变了24%的核酸序列。这种重新合成导致稳态L1 RNA和蛋白质水平大大提高。值得注意的是，当在既定的逆转录转座试验中用合成开放阅读框替代野生型开放阅读框时，转座水平增加了200多倍。这表明在L1编码区内进行逆转录转座可能不需要大的、严格保守的顺式作用核酸序列。这些合成逆转录转座子也是迄今为止已知的活性最高的L1元件，具有作为操纵哺乳动物基因组的实用工具的潜力。
