Nucleic Acids Research, 2005, Vol. 33, No. 6 1997 Figure 3. 30-junction analysis for retrotransposed R1 elements. (A) PCR amplification of the 30 boundaries between the transposed R1 and the 28S rDNA gene. Sf9 genomic DNAs were extracted 12, 24, 48, and 72 h post-infection (h.p.i.) with AcNPV expressing wild-type R1, 2H209A (EN-deficient mutant) and 2D680A (RT-deficient mutant). The purified DNA was used as template for PCR amplification with a pair of primers, +4941 and 28S(+109) (Figure 2A). The PCR products were subjected to 2.5% agarose electrophoresis and stained with ethidium bromide. The molecular size marker is loaded alongside and each size in base pair (bp) was shown on the left of the picture. (B and C) 72 h.p.i. 30 junction clones obtained with wild-type R1 (B) and endonuclease-deficient R1 (2H209A) (C). Shown at the top of each figure is a diagram of the 30 end structure of the construct. Sequences derived from the R1Bm and the pAcGHLTB vectors are indicated by shaded and open boxes, respectively. The initiation sites for reverse transcription (left of the dotted vertical lines) are indicated by nucleotide numbers. The target DNA regions are shown on the right of the dotted vertical lines. Extra nucleotides at the junction that are not derived from either the 28S gene or the R1 construct are given between the two vertical lines (non-templated). 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 and the most major type is indicated by an asterisk. The TGT or TG sequences on the 30 end of the R1 template that can base-pair with the target DNA are also indicated (Figure 6). +, insertions into the site 180 bp upstream of TSD observed for wild-type and endonuclease-deficient R1.