Analysis of the L fluviatilis nprl3 gene. (A) Structure of the L fluviatilis nprl3 gene. The predicted exons (green, untranslated regions; purple, coding regions) are numbered. Primers used for RT-PCR are shown, with expected fragment sizes in base pairs (genomic/cDNA). (B) Expression of L fluviatilis NPRL3 mRNA assessed by RT-PCR, using primers shown in panel A, amplifying exons 2 to 5 or 13 to 14. Purple asterisks indicate fragments of expected size for cDNA amplicons; blue asterisk indicates fragment of expected size for genomic amplicon. Bacteriophage λ DNA digested with Pst1 was used as marker (λxPstI). (C) Phylogenetic relationship of human (Homo sapiens [Hs]), mouse (Mus musculus [Mm]), chicken (Gallus gallus [Gg]), zebra finch (Taeniopygia guttata [Tg]), pufferfish (Tetraodon nigroviridis [Tn]), river lamprey (L fluviatilis [Lf]), sea lamprey (Pmarinus [Pm]), fruit fly (Drosophila melanogaster [Dm]), and zebrafish (Danio rerio [Dr]), and NPRL3 proteins inferred by using the maximum likelihood method. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Sizes of proteins are indicated as number of amino acids (aa). (D) VISTA plot displaying alignments of the L fluviatilis, T nigroviridis, and H sapiens NPRL3 genes. Note that the first (noncoding) exon of L fluviatilis nprl3 is not included in the drawing. UTR, untranslated region.