Kirilyuk Alexander, Tolstonog Genrich V, Damert Annette, Held Ulrike, Hahn Silvia, Löwer Roswitha, Buschmann Christian, Horn Axel V, Traub Peter, Schumann Gerald G
Max-Planck-Institut für Zellbiologie, Rosenhof, D-68526 Ladenburg, Germany.
Nucleic Acids Res. 2008 Feb;36(2):648-65. doi: 10.1093/nar/gkm1045. Epub 2007 Dec 10.
LINE-1 (L1) is a highly successful autonomous non-LTR retrotransposon and a major force shaping mammalian genomes. Although there are about 600 000 L1 copies covering 23% of the rat genome, full-length rat L1s (L1Rn) with intact open reading frames (ORFs) representing functional master copies for retrotransposition have not been identified yet. In conjunction with studies to elucidate the role of L1 retrotransposons in tumorigenesis, we isolated and characterized 10 different cDNAs from transcribed full-length L1Rn elements in rat chloroleukemia (RCL) cells, each encoding intact ORF1 proteins (ORF1p). We identified the first functional L1Rn retrotransposon from this pool of cDNAs, determined its activity in HeLa cells and in the RCL cell line the cDNAs originated from and demonstrate that it is mobilized in the tumor cell line in which it is expressed. Furthermore, we generated monoclonal antibodies directed against L1Rn ORF1 and ORF2-encoded recombinant proteins, analyzed the expression of L1-encoded proteins and found ORF1p predominantly in the nucleus. Our results support the hypothesis that the reported explosive amplification of genomic L1Rn sequences after their transcriptional activation in RCL cells is based on L1 retrotransposition. Therefore, L1 activity might be one cause for genomic instability observed during the progression of leukemia.
LINE-1（L1）是一种非常成功的自主非长末端重复逆转录转座子，也是塑造哺乳动物基因组的主要力量。尽管大鼠基因组中约有60万个L1拷贝，覆盖了23%的基因组，但尚未鉴定出具有完整开放阅读框（ORF）的全长大鼠L1（L1Rn），而这些完整开放阅读框代表着逆转录转座的功能性主拷贝。结合阐明L1逆转录转座子在肿瘤发生中作用的研究，我们从大鼠氯白血病（RCL）细胞中转录的全长L1Rn元件中分离并鉴定了10种不同的cDNA，每个cDNA都编码完整的ORF1蛋白（ORF1p）。我们从这个cDNA库中鉴定出了第一个功能性L1Rn逆转录转座子，确定了它在HeLa细胞和其来源的RCL细胞系中的活性，并证明它在表达它的肿瘤细胞系中被激活。此外，我们制备了针对L1Rn ORF1和ORF2编码的重组蛋白的单克隆抗体，分析了L1编码蛋白的表达情况，发现ORF1p主要存在于细胞核中。我们的结果支持这样一种假说，即报道的RCL细胞中基因组L1Rn序列转录激活后的爆发性扩增是基于L1逆转录转座。因此，L1活性可能是白血病进展过程中观察到的基因组不稳定的一个原因。
