Intron length and alternative splicing of Bru-3 in distantly related Drosophila species. (A) Splice choices identified in D. melanogaster, D. pseudoobscura, D. persimilis and D. virilis. Only lengths of exons (boxes), but not introns (lines connecting exons), are drawn to scale. The ORF is shaded. The untranslated regions (UTRs) of Bru-3 were not analyzed in D. virilis. For simplicity, exon skipping and alternative acceptor sites, but not intron splicing per se, are depicted. While many splice choices in Bru-3 were found conserved between tested species, the gene underwent evolution at the exon level. Exon 8 were created de nove from intronic sequence in D. pseudoobscura and D. persimilis sibling species pair, and exon 6 lost coding potential in D. melanogaster. (B) The gene structure of Bru-3 in D. pseudoobscura. All exons and the introns that are shorter than 2 kb are drawn to scale. The lengths of the other introns are presented. (C) The intron length and splicing of adjacent exons in Bru-3. The column heights are proportional to the natural logarithm of intron lengths in D. melanogaster, D. pseudoobscura and D. virilis. The columns are aligned between corresponding exons. The circles enclose the number of exons that included into the majority of ASTs. The horizontal line depicts the suggested cut-off, 250 nucleotides, between short- and long- introns [12, 54]. The shorter introns are likely spliced out through the intron definition mechanism whereas the longer introns relay on the exon definition mechanism for their splicing . The intron definition mechanism of splicing was shown to be more precise and efficient than the exon definition mechanism . We found that the exons adjacent to long-introns were frequently skipped in ASTs of Bru-3, whereas the exons connected by short-introns - exons 9, 10 and 11, and exons 13 and 14 (black bars) - were always spliced together.