Table 1. List of primers Name R1 S519 R1 S4861 R1 A4992 R1 A5136 R1 2H209A R1 2D680A R1-(14 nt) R1-(50 nt) R1-(A)7-(14 nt) +231 +590 +5121 +4941 28S ( 9) 28S ( 137) 28S (+57) 28S (+109) pAcGHLTB-S3304 pAcGHLTB-S2183 Nucleic Acids Research, 2005, Vol. 33, No. 6 1995 Sequence (50!30) AAAAAGAATTCAGATGTCGGAGGAGGAGAGGGAGCTATTTTCCCCT GTTTACCACGCGGACCTGGT TCACCCACATTCTTCGTCGCCAGGCAGTCC AAAAACTGCAGTTCCCACCACCTCCCATGGTCCCA CATCGAGCGAT GC CCGGCTCATTGTGTTTGGGGTG GAGATGGTCGCCTATGCCG TC GACGTGACGGTACTG AAAAACTGCAGAGATAGTAGATAGGGACATTCCCACCACC AAAAACTGCAGTCCCAAGCCCGTTCCCTTGGCT AAAAACTGCAGAGATAGTAGATAGGGACATTTTTTTCCCACCACC GGAAGATTGGGAAACTCCAAACCCAATTCAAAC ATTTTGGGAACGCATCCAGGCACATTGGGT CCTCTCTATGACGAAATCCGGGGCAGGATGCTCGATGGAA CCCGATGTTGCGGACAGGAA ACGCGCATGAATGGATTAACGA GTTGACGCAATGTGATTTCTGCC AGGGTCTTCTTTCCCCGCT CTCCTTACAATGCCAGACTAGAGTCAAGCTCAACAGGGTC CTGCAGGGTACCCCCGGGAGATCTGTACCG CCTATAAATACGGATCTGTATTCATGTCCC Underlined letters indicate restriction sites used for subcloning. Mutagenized nucleotides are boxed. Recombinant AcNPV generation and protein extraction Southern hybridization Sf9 cell was propagated as monolayer cultures at 27 C in TC-100 supplemented with 10% fetal bovine serum (Katakura Co., Nagano, Japan) in the presence of penicillin/streptomycin (Gibco-BRL). The recombinant baculovirus was produced as described previously (17). Brieﬂy, R1WT/mutant-pAcGHLTB plasmid was co-transfected with BaculoGoldTM DNA (Phar- Mingen) into the Sf9 cells using the Tfx-20 reagent (Promega). The subsequent virus was plaque-puriﬁed and ampliﬁed, according to the manufacturer’s instructions (PharMingen). Protein extraction and in vivo retrotransposition assay Approximately 5 · 105 of Sf9 cells were infected in a 12-well plate with a R1-containing AcNPV at a multiplicity of 10 plaque forming units (p.f.u.) per cell. At the 12, 24, 48, 72 h post-infection (h.p.i.), the total genomic DNAs from Sf9 cells were extracted with the Puregene DNA isolation kit (Gentra Systems). PCR ampliﬁcations were carried out with LA-taq DNA polymerase (TaKaRa), anti-taq antibody (Clontech), 2.0 ng of Sf9 genomic DNA, and the primer sets shown below. 30 junctions of R1 integrations were amp- liﬁed by the primer sets, +4941/+5121 and 28S (+109). 50 junctions were ampliﬁed using the primer sets, 28S ( 9)/ ( 137) and +231/+590. The reaction mixture was denatured at 96 C for 2 min, followed by 40 cycles of 98 C for 30 s, 62 C for 30 s and 72 C for 30 s. The PCR products were loaded on 2.5% agarose gel, electrophoresed and stained with ethidium bromide. The ampliﬁed fragments were directly cloned into the pGEM-T Easy vectors (Promega) and sequenced with an ABI 3100 genetic analyzer (Applied Biosystems). Sequence ana- lyses were conducted with the software, Vector NTI (World Fusion). For the protein analysis, the collected Sf9 cells were washed twice with phosphate-buffered saline (PBS) and sub- jected to the suspension by the sample buffer containing 50 mM Tris–HCl (pH 6.8), 10% glycerol, 10 mM b-mercaptoethanol and 2% sodium dodecyl sulfate (SDS). The samples were loaded onto the 8% polyacrylamide gel electrophoresis, and the resulting gel was stained with Quick CBB (Wako). 50 junction PCR products were electrophoresed on 2.5% agarose gels and blotted onto nylone membrane (Biodyne B Membrane; PALL BioSupport) in 0.4 N NaOH. After pre- hybridization, the membrane was hybridized with a 550 bp probe corresponding to Glutathione S-transferase (GST) region, which is fused to N-terminus of ORF1 (Figure 2A), at 42 C overnight in 50% formamide, 10· Denhardt’s solution (0.2% each of BSA, Ficoll and polyvinylpyrrolidone), 5· SSC, 250 mg/ml salmon sperm DNA and 50 mM NaPO4 (pH 7.0). The probe was labeled with [a-32P]dCTP using Ex Taq poly- merase (TaKaRa) by PCR. The primers used for generating the probe are +590 and pAcGHLT-B-S2183. RESULTS Structural features of newly cloned R1Bm We have newly cloned a complete sequence of R1 (4618 bp) from the genomic DNA of the silkworm, Bombyx mori to develop retrotransposition assay speciﬁc for 28S rDNA. As shown in Figure 1A, this clone [named ‘R1Bmks’ (R1Bm clone of the silkworm strain, Kinshu · Showa), accession number; AB182560] contained a synonymous mutation in the position 4857 (GAGGTG!GAGGTA; reading frame is underlined) and two non-synonymous mutations in the position 1318 (CCCGGG!CCCGCG; Gly!Ala) and 1345 (GCTGCC!GCTGTC; Ala!Val) (shown as open arrow- heads in Figure 1A), compared with the R1Bm sequence pub- lished previously [M19755, (9)] Additional 3 nt were also inserted in the ORF2 (Figure 1A, solid arrowhead in the middle), resulting in the alteration of the amino acid sequence inside the RT domain (Figure 1B, 754-VSSTIASLSRH-764 ! 754-VFDDCLSFAPH-765). These regions, which are located just downstream the seventh highly conserved block between RT domains (6), are not conserved among three insect species (Figure 1B). In R1Bmks clone, 1 nt insertion at the end of ORF2 altered the reading frame (Figure 1A, the right solid arrowhead), which makes the length