2000 Nucleic Acids Research, 2005, Vol. 33, No. 6 Figure 5. Effects of downstream sequences on the R1 retrotransposition. (A) Diagram of the R1 constructs with various 30 end structures; 14 and 50 nt of the downstream 28S gene are added to the R1WT construct. The 14 bp sequence of TSD is indicated by an open box. Note that the 30 termini of each construct is followed by the AcNPV-derived polyhedrin 30UTR. Transcription start, +1. (B) PCR amplification of the 30 boundaries between the transposed R1 copies and the 28S gene. DNA extraction and PCR reaction are basically same as in Figure 3. The primer set used for PCR was +5121 and 28S(+109). 2D680A, RT-deficient mutant. (C) 72 h.p.i. 30 junctions obtained with various R1 construct in (A). Shown at the top of each figure is a diagram of the 30 end structure of the constructs. Sequences derived from R1Bm and additional 28S downstream sequences are indicated by shaded and hatched boxes, respectively. The vertical dotted lines represent the boundary between the cDNA region and the target DNA region. The initiation site for reverse transcription (left of the dotted vertical lines) is indicated by the numbers. Boxes to the right of the vertical lines represent the 14 bp of TSD. The number of clones containing each insertion type is indicated in the right-most column. Two clones amplified from the + (A)5 + 14nt construct contained mutations at the 30 end of 30UTR (G of GAA corresponds to the position +5446). construct, indicating that addition of poly (A) tail resulted in a severe defect in retrotransposition even if the poly (A) tract was followed by downstream 28S sequence. Among 5 sequenced clones, three showed the reverse transcription from the poly (A) tract. Two clones showed the reverse transcription from +5459 and some aberrations inside the R1 sequence. The above results indicate that R1Bm transcription pass through into the 28S rDNA region in vivo, that transcripts are base-paired strongly with the target DNA sequence, and that the minimal interval between the base-paired region and the predestinate RT start site is important for efﬁcient and accurate retrotransposition of R1 (Figure 6). DISCUSSION The newly cloned R1 from the silkworm genome was found to be active for retrotransposition, and it turned out that the baculovirus-based in vivo retrotransposition assay could be applied to the study of various site-speciﬁc LINEs (17,26). The most distinctive feature of R1 retrotransposition is its