Linear Group II Intron Retrohoming in Drosophila reverse transcription of the intron RNA template from the annealed DNA primer and extends it to the 59 end of the Ll.LtrB RNA template, where it can then switch to a second 40-nt DNA or RNA template with the nucleotide sequence of exon 1 (E1 RNA or DNA, red and black, lanes 5 and 6, respectively). The 39 end of the Ll.LtrB RNA has an aminoblock to impede the RT from switching to a second molecule of the initial template. Figure 6, lanes 5 and 6 show that the Ll.LtrB RT efficiently extends the annealed primer c (Pri c) to the end of the intron RNA template, yielding major labeled products of ,60-nt, which were resolved as a doublet, along with smaller amounts of larger products of the size expected for template switching to the exon 1 (E1) DNA or RNA (,100 nt) or to a second molecule of Ll.LtrB RNA despite the presence of the aminoblock (,120 nt). Controls show that no labeled products were detected after incubating the RT with primer c in the presence or absence of the exon 1 RNA or DNA (lanes 2–4). Cloning and sequencing of the gel bands confirmed that the major ,60-nt products (bands a and b in lane 5 and h and i in lanes 6) correspond to cDNAs extending to or near the 59 end of the intron RNA, with the doublet reflecting the addition of extra nucleotide residues, mostly A-residues, to the 39 end of the cDNA upon reaching the end of the Ll.LtrB RNA template (Figure 7A and 7B). Such non-templated nucleotide addition is a common property of DNA polymerases and RTs [35,36,45–48]. The first set of larger gel bands (90–110 nts; band c–e in lane 5 and j–l in lane 6) corresponds to products resulting from template switching from the 59 end of the intron to the 39 end of exon 1 DNA or RNA (Figure 7A and 7B), as well as products resulting from template switching to the 39 end or internal regions of the Ll.LtrB intron (Figure S1). Many of the template switches to exon 1 DNA occurred seamlessly, but small numbers of extra nucleotide residues, mostly A residues, were found at some junctions, as well as at the 39 end of the cDNAs (Figure 7A; bands c–e). The Figure 7. DNA sequences resulting from template switching of LtrA from Ll.LtrB RNA to exon 1 DNA or RNA. (A) and (B) Sequences of DNA products resulting from template switching of the Ll.LtrB RT (LtrA protein) from the 59 end of the Ll.LtrB intron RNA template/Pri c DNA substrate to exon 1 DNA (lane 5) or RNA (lane 6), respectively. (C) Sequences of DNA products resulting from template switching of the Ll.LtrB RT from the 59 end of Ll.LtrB RNA/primer c substrate to double-stranded exon 1 DNA with a 59 bottom-strand overhang (AS+9; lane 9). Bands were excised from the gel, cloned, and sequenced, as described in Materials and Methods. The substrate and expected cDNA or DNA product sequences are shown boxed above each set of experimentally determined DNA product sequences. Extra or mutant nucleotide residues are shown in lower case letters. Freq., frequency of occurrence; *, 32P-label at 59 end of primer c. doi:10.1371/journal.pgen.1002534.g007 PLoS Genetics | www.plosgenetics.org 9 February 2012 | Volume 8 | Issue 2 | e1002534