Tes. Furthermore, the phylogenetic origin of the vertebrate visual cycle is still unclear. Recently, it was proposed that a prototype of the vertebrate visual cycle is operational in the tunicate Ciona intestinalis [12] when Tsuda and coworkers identified CRALBP, BCMO1 and opsin orthologs in Ciona intestinalis larva and a presumed RPE65 ortholog in adult animals [13]. Though these authors did not test for enzymatic activity of this presumed RPE65 ortholog, they later reported in a review article [14] that they could not detect such activity, though no data was presented. BCMO1 orthologs are also found in arthropods [15] and are essential for chromophore production [16], but this alone does not indicate a vertebrate visual cycle. While a CRALBP-like homolog is found in the Drosophila genome [17], its precise function and whether it can actually bind 11-cis retinal has not been determined. Mammalian RPE65 activity was demonstrated only after 12 years of thorough biochemical work and so the Rubusoside biological activity absence of activity for FCCP presumptive Ciona RPE65 in itself may not serve as evidence of different function. However, in neither case did they address whether LRAT was present or not. RPE65 is the only known member of the carotenoid oxygenase family to use retinyl ester instead of a carotenoid as substrate. Therefore, it is reasonable to hypothesize that an enzyme that could reliably provide this novel substrate for RPE65 would appear contemporaneously in evolution with an ancestral RPE65 to facilitate this new enzymatic function for a carotenoid oxygenase. To clarify these questions we performed phylogenetic analysis for both the RPE65 and the LRAT families. We found that a gene for an LRAT ortholog is not present in the curated genomes of either Ciona intestinalis or the cephalochordate Branchiostoma floridae. These results for nonvertebrate chordates are consistent with the in silico studies of Albalat [18]. However, we have extended these studies of Albalat [18] 1676428 to provide experimental data for functions of these proteins. The first chordate LRAT orthologs we found were in the sea lamprey Petromyzon marinus (which has two copies of LRATLRATa and LRATb- as does the teleost 24272870 Danio). We confirmed our findings with determination of the enzymatic activity of the recombinant proteins and immunofluorescence studies of RPE65 in RPE, showing that functional lamprey LRATb and RPE65 are present in lamprey RPE. We also demonstrated that Ciona BCMOa (annotated as RPE65 in the Ciona draft genome) has carotenoid oxygenase cleavage activity, but no discernable RPE65 activity, rendering unlikely the premise that a vertebrate visual cycle arose before the last common ancestor of the jawless and jawed vertebrates.Results Phylogenetic Analysis of the RPE65/BCMO SuperfamilyA maximum likelihood (ML) phylogenetic tree of the RPE65/ BCMO superfamily is shown in Figure 1. The topologies of ML, NJ (neighbor-joining), MP (maximum parsimony) and ME (minimum evolution) trees are slightly different- however these differences do not affect the results and conclusions of the phylogenetic analysis (Figure S1). The ML tree is rooted using seaanemone (Nematostella vicentis) BCMO sequences (Figure 1). The Ciona BCMOb sequence forms a well-supported clade with the vertebrate BCMO1 sequences (the bootstrap value is 79; Figure 1). The Branchiostoma floridae (Cephalochordata) BCMOa and the Ciona intenstinalis/Ciona savignyi BCMOa (Ci-RPE65) form a clade with the RPE65 family (Figure 1). Howe.Tes. Furthermore, the phylogenetic origin of the vertebrate visual cycle is still unclear. Recently, it was proposed that a prototype of the vertebrate visual cycle is operational in the tunicate Ciona intestinalis [12] when Tsuda and coworkers identified CRALBP, BCMO1 and opsin orthologs in Ciona intestinalis larva and a presumed RPE65 ortholog in adult animals [13]. Though these authors did not test for enzymatic activity of this presumed RPE65 ortholog, they later reported in a review article [14] that they could not detect such activity, though no data was presented. BCMO1 orthologs are also found in arthropods [15] and are essential for chromophore production [16], but this alone does not indicate a vertebrate visual cycle. While a CRALBP-like homolog is found in the Drosophila genome [17], its precise function and whether it can actually bind 11-cis retinal has not been determined. Mammalian RPE65 activity was demonstrated only after 12 years of thorough biochemical work and so the absence of activity for presumptive Ciona RPE65 in itself may not serve as evidence of different function. However, in neither case did they address whether LRAT was present or not. RPE65 is the only known member of the carotenoid oxygenase family to use retinyl ester instead of a carotenoid as substrate. Therefore, it is reasonable to hypothesize that an enzyme that could reliably provide this novel substrate for RPE65 would appear contemporaneously in evolution with an ancestral RPE65 to facilitate this new enzymatic function for a carotenoid oxygenase. To clarify these questions we performed phylogenetic analysis for both the RPE65 and the LRAT families. We found that a gene for an LRAT ortholog is not present in the curated genomes of either Ciona intestinalis or the cephalochordate Branchiostoma floridae. These results for nonvertebrate chordates are consistent with the in silico studies of Albalat [18]. However, we have extended these studies of Albalat [18] 1676428 to provide experimental data for functions of these proteins. The first chordate LRAT orthologs we found were in the sea lamprey Petromyzon marinus (which has two copies of LRATLRATa and LRATb- as does the teleost 24272870 Danio). We confirmed our findings with determination of the enzymatic activity of the recombinant proteins and immunofluorescence studies of RPE65 in RPE, showing that functional lamprey LRATb and RPE65 are present in lamprey RPE. We also demonstrated that Ciona BCMOa (annotated as RPE65 in the Ciona draft genome) has carotenoid oxygenase cleavage activity, but no discernable RPE65 activity, rendering unlikely the premise that a vertebrate visual cycle arose before the last common ancestor of the jawless and jawed vertebrates.Results Phylogenetic Analysis of the RPE65/BCMO SuperfamilyA maximum likelihood (ML) phylogenetic tree of the RPE65/ BCMO superfamily is shown in Figure 1. The topologies of ML, NJ (neighbor-joining), MP (maximum parsimony) and ME (minimum evolution) trees are slightly different- however these differences do not affect the results and conclusions of the phylogenetic analysis (Figure S1). The ML tree is rooted using seaanemone (Nematostella vicentis) BCMO sequences (Figure 1). The Ciona BCMOb sequence forms a well-supported clade with the vertebrate BCMO1 sequences (the bootstrap value is 79; Figure 1). The Branchiostoma floridae (Cephalochordata) BCMOa and the Ciona intenstinalis/Ciona savignyi BCMOa (Ci-RPE65) form a clade with the RPE65 family (Figure 1). Howe.
