FIG. 1.
Simple sequence alterations at the 5′ genomic DNA/L1 junction. A. Rationale of the assay. The 3′ UTR of a human RC-L1 was tagged with a reporter cassette designed to detect retrotransposition events. Open rectangles indicate L1 ORF1 and L1 ORF2, respectively. The relative positions of the endonuclease (EN), RT, and cysteine-rich domains (C) are indicated. The position of the L1 promoter (P) and the simian virus 40 late polyadenylation signal (pA) needed for L1 expression also are indicated. The mneoI gene and ColE1 bacterial origin of replication are indicated by light and dark gray rectangles, respectively. The relative positions of the prokaryotic/eukaryotic promoter (P′) and the thymidine kinase polyadenylation signal (A′) required for reporter gene expression also are shown. The mneoI gene is interrupted by an intron (γ-globin intron 2) in the same transcriptional orientation as the L1. SD and SA indicate the splice donor and splice acceptor sites, respectively. This arrangement ensures that a functional NEO transcript will be translated only following L1 retrotransposition. The putative structure of a resultant retrotransposition event that confers G418 resistance (G418r) to HeLa cells is shown at the bottom. Horizontal arrows flanking the resultant insertion indicate the TSD. Black lines flanking the insertion indicate HeLa genomic DNA, and RE represents the cleavage site for the restriction enzyme present in flanking genomic DNA. B. Extra sequences at the 5′ genomic DNA/L1 junction. The top center panel indicates a Y-branched L1 retrotransposition intermediate following the initial stages of TPRT. A variety of sequence alterations were found at the 5′ genomic DNA/L1 junction including the addition of single “untemplated” nucleotides (indicated by the “N” in the bottom left panel), the formation of a palindromic repeat (indicated by the arrows in the bottom middle panel), and the addition of “filler” DNA (indicated by the open rectangle in the bottom right panel).