Published online 3 March 2004
Nucleic Acids Research, 2004, Vol. 32, No. 4 1555-1565
© 2004 Oxford University Press
Integration of the 5' end of the retrotransposon, R2Bm, can be complemented by homologous recombination
1 Division of Radiological Protection and Biology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan, 2 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan and 3 Research Center for Emerging Infectious Diseases, Research Institute for Microbial Diseases Osaka University, Yamadaoka 3-1, Suita, Osaka 565-0871, Japan
*To whom correspondence should be addressed. Tel: +81 3 5285 1111; Fax: +81 3 5285 1194; Email: hidemae{at}nih.go.jp
Present address:
Kazuo Hashido, Division of Radiation Protection, National Institute of Neuroscience, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan
R2Bm is a non-long-terminal-repeat (non-LTR) retrotransposon that was identified at a specific target site in the 28S rRNA genes of the silkworm, Bombyx mori. Although in vitro analysis has revealed that the 3' end of R2Bm is integrated into the target site by means of target-primed reverse transcription (TPRT), the mechanism of the 5' end integration is not well understood. We established a novel in vivo system to assay the insertion mechanism of R2Bm using a cultured cell line, C65, and a baculovirus, AcNPV, as host and vector, respectively. The 3' end of R2Bm integrated at the target site in the rRNA genes of C65 cells when an AcNPV containing both the full-length 3' UTR and the entire open reading frame (ORF) of R2Bm was introduced while the 5' end integration was incorrect. The 5' end of R2Bm was integrated, however, when the 28S gene sequence upstream of the R2Bm target site was added to the R2Bm sequence. Thus, in our assay, homologous sequences were likely essential for the successful integration of the entire R2Bm into the host cell genome. We also demonstrated that the failure to integrate caused by a frame-shifted ORF was rescued by co-infection with a helper virus that contained only the R2Bm ORF. This indicates that R2 retrotransposition can be complemented in trans. These findings suggest that the host’s mechanism for DNA repair may be necessary for the integration of the 5' end of R2Bm and that R2Bm protein may only have the ability to integrate the 3' end of the element by TPRT.
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