R) by PCR using primers flanking the junctions, followed by cloning and sequencing of the PCR products (Figure 3 and Figure 4). The 59- and 39-integration junctions from lariat intron retro- homing and the 39-integration junctions from linear intron retrohoming result from reverse-splicing reactions (see Figure 1), and as expected, the PCRs for these junctions gave single prominent products, with no differences between the wild-type and mutant strains (Figure 3, bottom gels; in each case, the expected precise junction sequence was confirmed by sequencing; see legend for details). By contrast, the 59-integration junctions resulting from linear intron RNA retrohoming were heterogeneous in all strains, with a major band of the size expected for full-length intron insertion and smaller bands, which appeared most prominent in the lig42; P{lig4+} and polQ2 embryos (Figure 3, top gels). DNA sequences of the 59-integration junctions resulting from linear intron retrohoming in wild-type w1118 and lig42, ku702, and lig42; P{lig4+} embryos are summarized in Figure 4A–4D, and their characteristics are compared by the bar graphs in Figure 5. As found previously [25], the 59-integration junctions for linear intron RNA retrohoming in the wild-type embryos were heterogeneous with different combinations of 59-exon deletions, 59-intron truncations, and extra nucleotide additions (Figure 4A). Some of the junctions show evidence of DNA repair at regions of microhomology (parentheses), and in some cases, the extra nucleotides inserted at the junctions match and were presumably copied from neighboring sequences in the 59 exon or intron (underlined). Linear Group II Intron Retrohoming in Drosophila The 59 junctions resulting from linear intron retrohoming in the lig42, ku702, and lig42; P{lig4 + } mutants were generally similar to those in the wild type w1118, the more closely related wild-type strain, with no large differences in the percentage of junctions with 59-exon deletions, 59-intron truncations, extra nucleotide addi- tions, or microhomologies (Figure 4 and Figure 5). Compared to the other strains assayed in parallel, the proportion of long 59- intron truncations appears to be somewhat lower in the ku702 embryos and higher in the lig42 and lig42; P{lig4+} embryos, but the significance of these findings is unclear, as the differences were not large and the proportions of full-length and shorter 59-junction products in each stock were somewhat variable in different experiments. The similarity of the junction sequences resulting from linear intron RNA retrohoming in the lig4 and ku70 mutants to those resulting from double-strand break repair in these mutants [31,39] supports the hypothesis that Lig4-independent linear intron retrohoming occurs predominantly by using components of the alt-EJ pathway. The polQ mutation decreases extra nucleotide addition and increases long microhomologies at 59-integration junctions The DNA repair polymerase h (PolQ) has been shown to function in DNA end-joining repair in Drosophila, including a role for extra nucleotide addition at the repaired junctions [38]. To Figure 3. PCR analysis of integration junctions from lariat and linear intron RNA retrohoming in wild-type and mutant strains. Retrohoming assays with lariat and linear RNPs were done as described in Figure 2A and Materials and Methods, and DNA was extracted from 80 pooled embryos for each strain. 59- and 39-integration junctions were amplified by PCR, using primers that flank the junction (59junction, forward primer P1 and reverse primer LtrB933a; 39 junction, forward primer P3 and reverse primer P4; see Materials and Methods). The PCR products were analyzed in a 1% agarose gel, which was stained with ethidium bromide. Precise 59 and 39 junctions for lariat intron RNA retrohoming and precise 39 junctions for linear intron RNA retrohoming were confirmed by sequencing junctions from at least 10 randomly selected TetR+AmpR colonies or PCR products from pooled embryos for all strains (not shown). doi:10.1371/journal.pgen.1002534.g003 PLoS Genetics | www.plosgenetics.org 5 February 2012 | Volume 8 | Issue 2 | e1002534