cladeD_fav_1 cladeD_fav_n cladeD_rest_1 cladeD_rest_n cladeD_agar_1 cladeD_agar_n cladeB_1 cladeB_n cladeC_rest_1 cladeC_rest_n cladeC_zoan_1A cladeC_zoan_nA cladeC_zoan_1B cladeC_zoan_nB cladeC_styl_1 cladeC_styl_n cladeB2_1 cladeB2_n cladeD2_1 cladeD2_n cladeC2_fung_n cladeC2_fung_1 ATHWTYTCDGTGGATGSYTCCCAYTT TYMGGRAACRWCTGCTTGAAATA ATCTCAATGCRRTGGTCCACAAA TASTTTTRAAGTCACAYMGGTAAT AATGCGGTGGTCTATGAAGTGA TGCTTGAAAWAGTCTGGTATGT TAACMGGTCCATYRGRAGGAAAGTT AAATGGCARAGGTCCNCCCTTGGT TTACRGGTCCATCAGCRGGAAAGTT WWGABAGWATGTCMAAGGAGAAT TTTCATCACAGGTCCATCAGCAGGAAA ACAYGARTTCTTGAAAWAGTCAAC TTGCTATCACTGGTCCATTGTCAGGAAA AAAAGATGGCTTGAAAWAGTCAAC CATYAYGGGTCCRTYAGCRGGAAAGTT AGWATGTCAAAGGARAATGSAAGG GTCTTCYTYTGCAYAACRGGTCCAT ACCRTCTTCRAAGKKCATGRHCCKYTCC- CAT WTCTCAGTGGATGGYTCCCA RYRAAHRCYCTGTTKCCRTA CRGGRAAGTTVACSCCRACAAATT CCRCCTCCYDCVAGCTTKAGGWACAT cladeC_zoanred_1 TTCTTAGCATCGCTGCGGTCTT cladeC_zoanred_n ACCATCCTTCAGAAGGAGGTACAT The ‘‘1’’ primers were used in the first 5’ RACE PCR reaction together with 5prox (5’ proximal adaptor primer [56]) for 12 cDNA samples from different organisms. The PCR reaction was performed in 96-well plate. The cycling parameters were: 94uC 400- 55uC 300-72uC 19, 30 cycles. The product of the first PCR was used in the nested PCR reaction with ‘‘n’’ primers and Udist (universal adaptor primer, [56]). The cycling parameters: 94uC 400- 55uC 300-72uC 19, 18 cycles. The RACE products were cloned into pGEM-T vector (Promega) and sequenced (6–8 clones per each product). The sequences were assembled in SeqMan II software (Lasergene) and the contigs were checked for homology using BLASTX [57]. A pair of 3’-RACE primers were designed for each FP-related contig to amplify a complete open reading frame (ORF). The nested 3’- RACE primer corresponded to the very beginning of the ORF and contained a ‘‘translation initiation heel’’ [58]. After identifi- cation and sequencing of fluorescent clones, the inserts from them were re-amplified using the same 3’-RACE nested primer and a primer corresponding to the C-terminus of the ORF with six histidine codons inserted in front of the termination codon, as we described earlier [9]. The product of this amplification was cloned into pGEM-T vector and used to produce a heterologously References 1. Lukyanov KA, Chudakov DA, Fradkov AF, Labas YA, Matz MV, et al. (2006) Discovery and properties of GFP-like proteins from nonbioluminescent Anthozoa. Methods Biochem Anal 47: 121–138. 2. Chudakov DM, Lukyanov S, Lukyanov KA (2005) Fluorescent proteins as a toolkit for in vivo imaging. Trends in Biotechnology 23: 605–613. 3. Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nature Methods 2: 905–909. 4. Wachter RM (2006) The family of GFP-like proteins: Structure, function, photophysics and biosensor applications. Photochem Photobiol 82: 339– 344. Coral Fluorescent Proteins expressed protein that could be purified by metal-affinity chromatography using Ni-NTA agarose (Qiagen). Spectroscopy The fluorescent properties of isolated proteins were determined using LS-50B spectrofluorometer (Perkin Elmer Instruments); emission spectra were corrected for the dependence of photo- multiplier sensitivity on the wavelength. Molar extinction coefficients of native proteins were determined from the absorption of the chromophore in denaturing conditions (in 1M NaOH) assuming a molar extinction coefficient of 44,000 for cyan, green, chromoproteins and DsRed-like red proteins[25] and 28,000 for Kaede-like proteins [10]. Quantum yields were determined by using either fluorescein (QY = 0.97) or sulforhoda- mine 101 (QY = 0.90) as a reference standard. Phylogenetic analysis The coding DNA sequence alignment of the fluorescent proteins was assembled following the protein sequence alignment, that was in turn constructed by appending the previously reported alignment [25]. The appropriate model of evolution was identified as GTR+G+I [59] with the help of Modeltest software [60]. The phylogenetic analysis was performed using MrBayes 3.1 [61]. The MCMCMC chain was run for 1,500,000 iterations with a sample frequency of 200 resulting in 7,500 trees, of which the first 6,000 were discarded while summarizing the data. The analysis was run three times to ensure convergence. Probabilistic ancestral reconstruction To reconstruct ancestral proteins sequences for the common ancestor of all coral proteins (‘‘all-coral ancestor’’) and the ancestor of all proteins from representatives of the Faviina suborder (‘‘all- Faviina ancestor’’) we used MrBayes 3.1 with the fixed amino acid-based model JTT [62]. Five sequences per ancestral gene were probabilistically assembled by drawing the site states from the underlying posterior distribution of state probabilities and synthesized individually as described earlier [6]. Supporting Information Table S1 List of GenBank accession numbers for all the sequences used in the phylogenetic analysis. The newly cloned sequences are shown in blue. Found at: doi:10.1371/journal.pone.0002680.s001 (0.04 MB PDF) File S1 FASTA-formatted alignment of the coding sequences used for the phylogenetic analysis. Found at: doi:10.1371/journal.pone.0002680.s002 (0.10 MB DOC) Author Contributions Conceived and designed the experiments: DJM JW AS MVM. Performed the experiments: NOA KAK SFF EAM MEH VBR. Analyzed the data: NOA MEH JW AS MVM. Wrote the paper: JW MVM. 5. Shagin DA, Barsova EV, Yanushevich YG, Fradkov AF, Lukyanov KA, et al. (2004) GFP-like proteins as ubiquitous metazoan superfamily: Evolution of functional features and structural complexity. Mol Biol Evol 21: 841–850. 6. Ugalde JA, Chang BSW, Matz MV (2004) Evolution of coral pigments recreated. Science 305: 1433. 7. Dove SG, Hoegh-Guldberg O, Ranganathan S (2001) Major colour patterns of reef-building corals are due to a family of GFP-like proteins. Coral Reefs 19: 197–204. 8. Matz MV, Marshall NJ, Vorobyev M (2006) Are corals colorful? Photochem Photobiol 82: 345–350. PLoS ONE | www.plosone.org 11 July 2008 | Volume 3 | Issue 7 | e2680