1996 Nucleic Acids Research, 2005, Vol. 33, No. 6 Figure 2. In vivo retrotransposition assay for R1 elements using recombinant baculoviruses. (A) Diagram of the PCR assay for retrotransposition. Shown at the top of the figure is a diagram of the R1 element expressed from AcNPV. The 50UTR sequence derived from the polyhedrin promoter and ORF1/ORF2 of R1 are shaded in black and gray, respectively. Nucleotide position is numbered with the transcription initiation site (A of TAAG) defined as +1. EN and RT denote the endonuclease and reverse transcriptase domains, respectively. The amino acid position of each mis-sense mutant is also shown; the 2H209A mutant represents the substitution of the 209th histidine (H) in the ORF2 for alanine (A), and the 2D680A mutant represents the substitution of the 680th aspartic acid (D) in the ORF2 for alanine (A). Shown at the bottom is a diagram of an rDNA unit showing the location of R1 insertion; 14 bp of the target site duplication (TSD) created upon R1 insertion is boxed. The putative cleavage sites by R1-EN are indicated by black arrowheads. Arrows represent the primer set to amplify the 50 and 30 junctions between the R1 sequence and the 28S sequence. The thick bar above the GST region which is fused to the R1 ORF1 indicates the probe used for Southern hybridization in Figure 4. (B) Expression of R1 proteins with a baculovirus-based expression system. The total proteins were extracted from Sf9 cells infected with recombinant viruses shown above and run on SDS–PAGE; the predicted molecular weight for R1 GST/(His)6/ORF1 was 78 170. Lane M; size markers. of ORF2 protein shorter by 12 amino acids (1047 ! 1035 amino acids). As shown in Figure 1C, the amino acid sequences and the length of the C-terminal regions of ORF2 are also not conserved among three relatively close insect species. The alterations of amino acid sequence shown above were limited to the non-conserved regions between R1 elements. R1Bmks retrotransposes into the specific site of 28S rDNA To ﬁgure out whether R1Bmks is active for retrotransposition, we have introduced in vivo R1Bm retrotransposition assay using baculovirus-based expression system (17). We have expressed R1Bmks with its 50UTR replaced by glutathione S-transferase (GST) and histidine tag, (His)6 under the control of AcNPV polyhedrin promoter in the Sf9 cells. De novo retrotranspositions into genomic DNA of Sf9 cells were detec- ted by 40 cycles of PCR using primers designed to amplify the junction regions between R1 sequence and 28S rDNA sequence (Figure 2A). GST-His-R1Bmks ORF1 fusion protein expressions comprising the molecular size of 78 kDa were conﬁrmed by SDS–PAGE for every R1Bmks recombinant viruses (Figure 2B). The band derived from ORF2 expression in the calculated size, 116 kDa was not observed in wild-type and mutated R1 elements. ORF2 expression is also not observed in other non-LTR retrotransposons such as SART1 in Coomassie staining (unpublished data), and in L1, it is only observed by immunohistochemistry with anti-ORF2 protein antibody (23). However, retrotransposition activity is observed for both SART1 and L1, therefore, it is suggested that even though the quantity is small, enough amount of ORF2 is expressed for retrotransposition. First, the 30 junctions between the retrotransposed R1 ele- ment and the 28S gene were analyzed (Figure 3). A single round of PCR gave rise to the bands indicating the retrotrans- position events at 48 and 72 h.p.i. of recombinant AcNPV constructs (Figure 3A). As seen in lane 4, there were only two bands of 400 and 600 bp at 48 h.p.i. At 72 h.p.i., the total intensity and the number of bands increased to the range of 200–1000 bp as in lane 5. In order to know whether these bands represent retrotransposed copies, we have cloned total PCR products into a cloning vector, and sequenced 16 clones for lane 4 (data no shown) and 40 clones for lane 5 (Figure 3B). For 48 h.p.i., 15 clones were integrated into the speciﬁc site of 28S rDNA with 14 bp of target-site duplications. Endogen- ous R1 elements from the silkworm are shown to be ﬂanked by a speciﬁc 14 bp TSD of 28S rDNA (9), suggesting that R1Bmks cleaved the speciﬁc target site and integrated into the identical position to the endogenous R1 elements. It is of interest that the 50 end of TSD was adjoined to various sequences of R1; there were 5 clones which adjoined ATACA+5249, 7 clones for AGGAC+5286, 1 clone for GACGG+5361 and 2 clones which adjoined the R1 30 end. For 72 h.p.i., 38 out of 40 clones were integrated into the speciﬁc site of 28S rDNA with the TSD. There was a wide variety in the R1 sites adjoining the 50 end of TSD, suggesting that reverse transcription initiated from various positions of template RNA. Some clones contained the downstream AcNPV vector region, implying reverse transcription from read-through transcripts with the non-R1 sequence. It is