in the initial process of TPRT (see Figure 6). According to this hypothesis, the reverse transcription should start from the position next to the TGT or TG sequences. Among 40 clones, only two clones (Figure 3B; denoted as +) inserted into the sequence other than R1-speciﬁc integration site, which is 180 bp upstream of TSD. We also generated the recombinant R1Bm constructs with single-amino-acid substitutions at the conserved motif in EN domain and RT domain (Figure 2A). While 2D680A mutation abolished in vivo R1 retrotransposition as shown for several LINEs (Figure 3A, lane 10–13) (16,17), very faint bands were observed for the 2H209A mutant with a non-sense mutation in the catalytic motif of EN domain at 72 h.p.i. (Figure 3A, lane 9). The two recovered fragments from lane 9 indicated the R1 integration into the 180 bp upstream of TSD (Figure 3C), which is identical for two clones of wild-type integration (Figure 3B). These results indicate that a DNA nick has been already pre-formed at 180 of the R1 target site in 28S rDNA, and R1Bmks use this nick for the endonuclease-independent retrotransposition. 50-junction of integrated R1Bmks elements PCR ampliﬁcation of the 50-junction using the primers com- plementary to the 28S rDNA and polyhedrin promoter gave rise to faint bands, ranging from 100 to 500 bp (Figure 4A, lane 4). The sizes of these bands were much shorter than expected from the putative full-length product, 590 bp plus the 28S rDNA (Figure 2A). To clarify the ﬁgure, we trans- ferred the PCR products to a nylone membrane and performed Southern hybridization with a GST probe (Figure 4C). There were signals in good accordance to full-length products (590 + 137 bp) to 50 truncated products (>137 bp). To invest- igate the 50 junction sequences, we cloned and sequenced the PCR products from Figure 4A, lane 4. All the sequenced clones turned out to be variable 50-truncated R1 elements connected with the 28S gene sequence (Figure 4B). The vari- able 50 truncation was shown to be resulting from a frequent dissociation of RT from the RNA template before completion of the reverse transcription; a diagnostic feature of LINE retrotransposition (24). Some clones contained precise 14 bp TSD. The others, however, showed some aberrations such as deletions in the 28S rDNA gene, non-templated nucleotides addition, and 13 bp insertions of downstream sequence. To detect the full-length R1 insertion, we used another primer complementary to the sequence upstream of +231. One of the resulting fragments (Figure 4B, top) contained the precise junction between TSD and the polyhedrin RNA 50-terminal sequence (AAG, Figure 2A). One clone included additional CAC nucleotides which was identical to the 28S gene Nucleic Acids Research, 2005, Vol. 33, No. 6 1999 sequence just upstream of TSD (Figure 4B, underlined). Sev- eral clones showed aberrations in the 28S gene and/or non- templated nucleotide additions. While these variations shown above were somewhat different from those found in endogen- ous R1 elements from the silkworm or Drosophila genomic DNA (9,25), retrotransposition of full-length R1 sequence into the speciﬁc site indicates the authentic de novo retrotransposi- tion of R1Bmks via its RNA transcript. Sequence in the 30-terminus affects efficiency and accuracy of retrotransposition R2 element, 28S rDNA-speciﬁc LINE with a single ORF, requires several regions in 30UTR for initiating the target- primed reverse transcription in vitro (11). To clarify the func- tion of 30UTR in the R1 retrotransposition, we made an R1 D30UTR-AcNPV construct which lacks the entire R1 30UTR (Figures 5A and C). Note that this construct retains a down- stream polyhedrin 30UTR. As shown in Figure 5B (D30UTR; lane 4), the band was very faint, but two clones representing the authentic reverse transcription from ORF2 sequence of R1 were recovered from the PCR product (Figure 5C, D30UTR). This result suggests that 30UTR is not necessary absolutely for the R1 retrotransposition although loss of the sequence lowered severely the retrotransposition efﬁciency. As shown above, the short UG or UGU sequences in RNA of WT R1 are potentially base-paired with the target DNA during initial process of TPRT (refer to Figure 6). Then, we next tested whether the long base-pairing between template RNA and target DNA raises the efﬁciency or accuracy of R1 retrotransposition. We made recombinant baculoviruses with various length of 28S rDNA sequence located downstream of R1 elements (Figure 5A, +14 nt, +50 nt, and + (A)5 + 14 nt). In striking contrast to R1WT, addition of downstream 14 and 50 nt from 28S rDNA showed a strong single band represent- ing the retrotransposition (Figure 5B, lanes 5 and 6). All the sequenced clones (38 clones in total) from lanes 5 and 6 showed that the transposed copies have the precise junction between the 30 end of 30UTR of R1 and the 28S target sequence as in the endogenous R1Bm elements (Figure 5C, +14 nt, +50 nt). These ﬁndings suggest that the downstream 28S sequence greatly enhanced the accurate initiation of reverse transcription from the EN-digested site. Site-speciﬁc LINEs usually have poly (A) tract at their 30 tail, but R1Bmks contains only two A nucleotides at its 30-terminus (data not shown). To clarify the functional role of the 30-terminal sequence of R1, ﬁve A nucleotides (A5) were added to the +14 nt construct between the R1-28S junction (Figure 5A). As shown in Figure 5B, the retrotransposition efﬁciency was drastically reduced, compared with the +14 nt Figure 4. 50-junction analysis for retrotransposed R1 elements. (A) PCR amplification of the boundaries of the transposed R1 50 ends with the 28S gene. The DNA extraction and PCR analyses were basically same as in Figure 3. A pair of primers, +590 and 28S( 62) was used for amplifying the 50 junctions. 2D680A, RT-deficient mutant (Figure 2A). (B) 72 h.p.i. 50 junction obtained with wild-type R1. Shown at the top of the figure is a diagram of the 50 end structure of R1WT- pAcGHLT. Sequences derived from the pAcGHLTB vector are indicated by shaded boxes. The numbers on the left and the right of the shaded boxes correspond to the nucleotide position numbered with the transcription initiation site. The gray boxes on the right are cDNA and the open boxes on the left are the 28S rDNA regions. Extra nucleotides at the junction that are not derived from either the 28S gene or the R1 construct are given between two vertical lines. Boxes to the left of the vertical lines represent the 14 bp of TSD. Duplicated nucleotides are underlined. Two of the insertions contained 13 bp downstream sequences at the insertion site. The number of clones containing each insertion type is indicated in the right-most column. The top five sequences are from PCR with primer +231, and the bottom eight sequences are from PCR with primer +590. (C) Southern hybridization of the retrotransposed R1 50 junction PCR. The PCR reaction was conducted by primers +590 and 28S ( 137). The PCR products were transferred to a nylon membrane and hybridized with the GST probe (Figure 2A). The DNA size marker was electrophoresed simultaneously and shown on the left.