N.Materials and Methods Ethics StatementSea Lamprey tissues for this study were collected under an Animal Study Protocol approved by the National Eye Institute (NIH) Animal Care and Use Committee.Datasets and Phylogenetic Analysis and ModelingProtein sequences were downloaded from the NCBI and ENSEMBL web sites. Similarity searches were 842-07-9 performed using the non-redundant protein sequence database at the NCBI and the gapped BLAST program. Multiple protein sequence alignments were constructed using the Muscle program and then adjusted by hand (details available upon request from Eugenia Poliakov, [email protected]). Phylogenetic trees based on multiple alignments of protein sequences were constructed using the maximum-likelihood, neighbor-joining, minimum-economy and maximum-parsimony methods as implemented in MEGACloning of Lamprey LRAT and RPE65 for Expression in HEK293F CellsRPE was carved out from frozen lamprey heads. Total RNA from lamprey RPE was purified using TRIzolH reagent (Invitrogen) according to the manufacturer’s instructions. Briefly, 2 RPEs were homogenized in 1 ml of TRIzol and incubated at room temperature for 5 minutes. 0.2 ml of chloroform was added and the tube was shaken for 15 seconds following by 3 minutes incubation at room temperature. The sample was centrifuged at 12, 0006g for 15 minutes at 4uC. The upper aqueous phase wasOrigin and Evolution of Vertebrate Visual CycleFigure 7. Localization of RPE65 in the Lamprey RPE/retina. Cy3 (green) staining of frozen sections of fixed lamprey retina/RPE with rabbit polyclonal antibodies to A: RPE65; B: arrestin; C: blue cone opsin SWS2 [38,39]. Nuclear DAPI staining in blue. Lamprey 4EGI-1 site Retina histology D: 20X magnification; E: 40X magnification of semithin sections stained with toluidine blue. F: Immunoblot of Lamprey retina and RPE extracts. Retina and RPE were prepared as described in Methods. Lanes from left to right: lane 1, marker Spectra Multicolored Broad Range Protein ladder (Fermentas), lane 2, retina extract, lane 3, RPE extract. doi:10.1371/journal.pone.0049975.gremoved and placed into a new tube. 0.5 ml of 100 isopropanol was added to the aqueous phase and mixed. After 10 minutes incubation at room temperature the sample was centrifuged at 12, 0006g for 10 minutes. The supernatant was removed from the tube and the RNA pellet was washed with 1 ml of 75 ethanol. The sample was vortexed briefly and centrifuged at 75006g for 5 minutes at 4uC. The wash was discarded and the RNA was air dried for 5 minutes. The RNA pellet was resuspended in 20 ml RNase-free water. SMARTerTM RACE cDNA Amplification kit (Clontech) was used to clone lamprey RPE65 and LRAT following manufactur-er’s instructions. Phusion Flash II DNA Polymerase (Finnzymes) was used for PCR amplification. For RPE65 cloning, 1 mg of total RNA from RPE was reverse transcribed in 10 ml reaction by SMARTScribeTM Reverse Transcriptase with 59-RACE CDS Primer A 59?T)25VN?9 and SMARTer II A Oligonucleotide 59 AGCAGTGGTATCAACGCAGAGTACXXXXX?9 at 42uC for 90 min. This firststrand reaction product was diluted with Tricine-EDTA buffer to 100 ml. The recommended program for touchdown PCR was used with PhusionTM Flash High-Fidelity PCR Master Mix (Finnzymes) with Universal Primer A Mix (UPM) Long (0.4 mM), Short (2 mM)Origin and Evolution of Vertebrate Visual Cycleand lamprey_RPE65 59-RACE primer (59-GACAAGGATGAGGGAGGCCCAACTCGTAG-39) that was designed 1662274 based on partial genomic DNA sequence from contig39407, Petromyzo.N.Materials and Methods Ethics StatementSea Lamprey tissues for this study were collected under an Animal Study Protocol approved by the National Eye Institute (NIH) Animal Care and Use Committee.Datasets and Phylogenetic Analysis and ModelingProtein sequences were downloaded from the NCBI and ENSEMBL web sites. Similarity searches were performed using the non-redundant protein sequence database at the NCBI and the gapped BLAST program. Multiple protein sequence alignments were constructed using the Muscle program and then adjusted by hand (details available upon request from Eugenia Poliakov, [email protected]). Phylogenetic trees based on multiple alignments of protein sequences were constructed using the maximum-likelihood, neighbor-joining, minimum-economy and maximum-parsimony methods as implemented in MEGACloning of Lamprey LRAT and RPE65 for Expression in HEK293F CellsRPE was carved out from frozen lamprey heads. Total RNA from lamprey RPE was purified using TRIzolH reagent (Invitrogen) according to the manufacturer’s instructions. Briefly, 2 RPEs were homogenized in 1 ml of TRIzol and incubated at room temperature for 5 minutes. 0.2 ml of chloroform was added and the tube was shaken for 15 seconds following by 3 minutes incubation at room temperature. The sample was centrifuged at 12, 0006g for 15 minutes at 4uC. The upper aqueous phase wasOrigin and Evolution of Vertebrate Visual CycleFigure 7. Localization of RPE65 in the Lamprey RPE/retina. Cy3 (green) staining of frozen sections of fixed lamprey retina/RPE with rabbit polyclonal antibodies to A: RPE65; B: arrestin; C: blue cone opsin SWS2 [38,39]. Nuclear DAPI staining in blue. Lamprey retina histology D: 20X magnification; E: 40X magnification of semithin sections stained with toluidine blue. F: Immunoblot of Lamprey retina and RPE extracts. Retina and RPE were prepared as described in Methods. Lanes from left to right: lane 1, marker Spectra Multicolored Broad Range Protein ladder (Fermentas), lane 2, retina extract, lane 3, RPE extract. doi:10.1371/journal.pone.0049975.gremoved and placed into a new tube. 0.5 ml of 100 isopropanol was added to the aqueous phase and mixed. After 10 minutes incubation at room temperature the sample was centrifuged at 12, 0006g for 10 minutes. The supernatant was removed from the tube and the RNA pellet was washed with 1 ml of 75 ethanol. The sample was vortexed briefly and centrifuged at 75006g for 5 minutes at 4uC. The wash was discarded and the RNA was air dried for 5 minutes. The RNA pellet was resuspended in 20 ml RNase-free water. SMARTerTM RACE cDNA Amplification kit (Clontech) was used to clone lamprey RPE65 and LRAT following manufactur-er’s instructions. Phusion Flash II DNA Polymerase (Finnzymes) was used for PCR amplification. For RPE65 cloning, 1 mg of total RNA from RPE was reverse transcribed in 10 ml reaction by SMARTScribeTM Reverse Transcriptase with 59-RACE CDS Primer A 59?T)25VN?9 and SMARTer II A Oligonucleotide 59 AGCAGTGGTATCAACGCAGAGTACXXXXX?9 at 42uC for 90 min. This firststrand reaction product was diluted with Tricine-EDTA buffer to 100 ml. The recommended program for touchdown PCR was used with PhusionTM Flash High-Fidelity PCR Master Mix (Finnzymes) with Universal Primer A Mix (UPM) Long (0.4 mM), Short (2 mM)Origin and Evolution of Vertebrate Visual Cycleand lamprey_RPE65 59-RACE primer (59-GACAAGGATGAGGGAGGCCCAACTCGTAG-39) that was designed 1662274 based on partial genomic DNA sequence from contig39407, Petromyzo.