Transplantation of target site specificity by swapping the endonuclease domains of two LINEs

被引:54
作者
Takahashi, H
Fujiwara, H [1 ]
机构
[1] Johns Hopkins Sch Med, Dept Mol Biol & Genet, Baltimore, MD 21205 USA
[2] Univ Tokyo, Grad Sch Frontier Sci, Dept Integrat Biol, Kashiwa, Chiba 2778562, Japan
关键词
endonuclease; LINE; non-LTR retrotransposon; SART1; tolomeric repeat;
D O I
10.1093/emboj/21.3.408
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Long interspersed elements (LINEs) are ubiquitous genomic elements in higher eukaryotes. Here we develop a novel assay to analyze in viro LINE retrotransposition using the telomeric repeat-specific elements SART1 and TRAS1. We demonstrate by, PCR that silkworm SART1, which is expressed from a recombinant baculovirus, transposes in Sf9 cells into the chromosomal (TTAGG)(n) sequences, at the same specific nucleotide position as in the silkworm genome. Thus authentic retrotransposition by complete reverse transcription of the entire RNA transcription unit and occasional 5' truncation is observed. The retrotransposition requires conserved domains in both open reading frames (ORFs), including the ORF1 cysteine-histidine motifs. In contrast to human L1, recognition of the 3' untranslated region sequence is crucial for SART1 retrotransposition, which results in efficient trans-complementation. Swapping the endonuclease domain from TRAS1 into SART1 converts insertion specificity to that of TRAS1. Thus the primary determinant of in vivo target selection is the endonuclease domain, suggesting that modified LINEs could be used as gene therapy vectors, which deliver only genes of interest but not retrotransposons themselves in trans to specific genomic locations.
引用
收藏
页码:408 / 417
页数:10
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