1994 Nucleic Acids Research, 2005, Vol. 33, No. 6 Figure 1. Identification of a new subtype of the R1 element from the silkworm, Bombyx mori. (A) Schematic representation of 28S gene-specific LINEs. Shown in the middle is a diagram of the rDNA unit of Bombyx mori. The newly identified R1 clone (named R1Bmks) is integrated into the sequence 74 bp downstream of the R2 insertion site. Open reading frames (ORFs) are depicted by open boxes. The positions of non-synonymous mutations in the ORF1 and nucleotide insertions in the ORF2 (see B and C) are shown by white and black arrowheads, respectively. (B and C) Altered amino acid sequence of R1Bmks in the ORF2. Insertion of 3 nt within the RT domain results in alteration of 10 amino acids (indicated by bold line), compared with R1Bm (B). Addition of a single nucleotide in R1Bmks alters the amino acid sequences and made the length of ORF2 shorter by 12 amino acids, compared with R1Bm (C). The amino acids conserved between more than two elements are indicated by bold letters. Putative translational stop was indicated by asterisks. R1Bm clones are aligned with R1Dm (X51968; accession number) from D.melanogaster; R1Sc (L00945) from Sciara coprophila. but poly (A) tract seems essential (16). In addition, active L1 elements possess the ability to transduce non-L1 DNA ﬂank- ing their 30 ends to new genomic locations (3,18,19). In con- trast, the recognition of 30UTR by SART1 proteins is crucial for the SART1 retrotransposition (17), and the loss of poly (A) tract resulted in a severe loss of the efﬁciency for the initiation of reverse transcription (20). R1Bm is a member of recently-branched subtype, and inser- ted speciﬁcally into the 28S rDNA of the silkworm (9,21). The R1 insertion site is located 74 bp downstream of the R2 site (Figure 1A) (22). R1Bm is ﬂanked by speciﬁc 14 bp target site duplication (TSD), and lacks the poly (A) tract at its 30 end. To understand the basal mechanisms of TPRT, it is of great interest to know how this poly (A)-less R1 initiates its reverse transcription. In this study, we have newly cloned the R1 element from the silkworm genome, and ﬁrstly succeeded in retrotransposing the R1 sequence into the 28S rDNA target of the host cells with recombinant baculovirus, AcNPV. Using the in vivo retrotransposition system of R1, we studied the functional roles of 30UTR of R1 RNA and co-expressed 28S rRNA downstream of R1 sequence. MATERIALS AND METHODS Plasmid construction The R1 ORF1/ORF2/30UTR portion was directly ampliﬁed by PCR with Pfu Turbo DNA polymerase (Stratagene) using primers R1 S519, R1 A5136 (Table 1) and 5 ng of the genomic DNA extracted from Bombyx mori strain, Kinshu · Showa. The reaction mixture was denatured at 94 C for 1 min, fol- lowed by 30 cycles of 98 C for 30 s, 60 C for 30 s, and 72 C for 12 min. The PCR products were loaded on 0.6% agarose gel, and electrophoresed. The DNA band in the calculated size was excised from the gel and puriﬁed by the QIAquick Gel Extraction Kit (Qiagen). The recovered product was digested with EcoRI and PstI, subcloned between the EcoRI and PstI sites of the pAcGHLTB plasmid (PharMingen). The resulting plasmid, named R1WT-pAcGHLTB, contained the 64 bp polyhedrin 50UTR and the GST-X5-(His)6-X29 coding gene fused in-frame with MSEEERE of the R1 ORF1, followed by R1 ORF2/30UTR and the polyhedrin 30UTR. Point muta- tions were introduced into R1WT-pAcGHLTB with primer sets listed in Table 1 using QuickChangeTM Mutagenesis Kit (Stratagene). The mutation of each plasmid was conﬁrmed by DNA sequencing. The D30UTR mutant was constructed by self-ligation of the DNA fragment ampliﬁed from R1WT- pAcGHLTB with the inverse PCR using 50-phosphorylated primers, R1 A4992 and pAcGHLTB-S3304. R1WT- pAcGHLTB plasmids with various 30 tails were constructed as follows. First, 333 bp fragment of 30 junction between R1 and 28S rDNA was ampliﬁed by PCR with a primer set, R1 S4861 and 28S rDNA(+57) using the silkworm genomic DNA as a template and was directly cloned into pGEM-T vector (Promega). Second, PCR reaction was conducted between R1 S4861 and the various primers with PstI sites (Table 1), and the resulting fragments were cloned into the NcoI and PstI sites of R1WT-pAcGHLTB.