FIGURE 5.

Schematic alignments of GUW1 and AF18 with their 5′ flanking sequence obtained by genome walking. Genome DNA samples from the same three pairs of male and female pupae as in Figure 4A were completely digested by MspI and ligated to MspI adaptor. The ligation mixtures were used as the templates to PCR-amplify the 5′ unknown flanking sequences of GUW1 and AF18 using the general primer MspI-a and the gene-specific primers GUW1-118-R and AF18-GSP (Supplementary Table S2), respectively (A). The obtained genome walking PCR products of GUW1 (left panel in B) and AF18 (right panel in B) from the three pairs of pupae were separated on 2% agarose gels (B). The two genome walking bands of AF18 from each of the six pupae and the single walking band of GUW1 from the three female pupae were sequenced and schematically aligned with the reported and cloned AF18 (C) and GUW1 sequences (D), respectively. Filled black boxes represent the known (reported) sequences of AF18 or GUW1, whereas clear (GUW1), cross-striped (AF18 band 1) or dashed (AF18 band 2) boxes represent the 5′ flanking sequences of the two markers identified by our genome walking experiment. The lengths of the 5′ flanking and reported (known) parts of the three walking sequence are placed above each box. The total length of each sequence is shown on the right side of each box. The positions of start and stop codons of the GUW1 open reading frame are shown below the walking sequence box. Std, molecular weight marker.