To delineate the exact boundaries of the newly identified human exons, pairwise comparisons and evolutionary rate estimation were performed for different functional domains of the gene using OWEN alignments (19). Evolutionary divergence was estimated in separate pairwise alignments for different functional domains of the OPRM1 gene (Ks, Ka, Ku for exons and Ki for introns) for closely related rodent (mouse and rat) and primate (human and macaca) species, and for distantly related species (human and mouse). Both flanking (50 nucleotides) and core regions of functional domains were considered (Table 1). The analysis revealed significant selective pressure associated with exonic regions and their vicinities further supporting the existence of the expanded human OPRM1 gene locus, which is ∼90 kb longer than was thought previously. Analysis of evolutionary divergence and conservation of regulatory splicing sites suggest that some of the new exons in primates are extended or shortened relative to rodent exons. These data are consistent with the generally low conservation of exonic boundaries in alternative splicing patterns in the human and mouse genomes (45).
