Transplantation of target site specificity by swapping the endonuclease domains of two LINEs.
Long interspersed elements (LINEs) are ubiquitous genomic elements in higher eukaryotes. Here we develop a novel assay to analyze in vivo 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.
Takahashi
Hidekazu
H
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bioscience Building 501, Kashiwa, Chiba 277-8562, Japan.
Fujiwara
Haruhiko
H
D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D002460:Cell Line; D004720:Endonucleases / Q000235:genetics; D005786:Gene Expression Regulation; D017344:Genes, Insect; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics*; D008969:Molecular Sequence Data; D016615:Telomere / Q000235:genetics
D004720:Endonucleases