David F. Gruber; Jean P. Gaffney; Shaadi Mehr; Rob DeSalle; John S. Sparks; Jelena Platisa; Vincent A. Pieribone

“ Marine organisms biofluoresce by absorbing the dominant blue light, and reemitting it at a longer, lower energy wavelength, visually resulting in green, orange, and red fluorescence. Following the seminal discovery of green fluorescent protein (GFP) from a hydrozoan jellyfish in 1962 [3] , fluorescent proteins (FPs) have been found in numerous anthozoans, primarily scleractinian corals [4] , [5] , copepods [6] , amphioxus [7] , [8] , ctenophores [9] and most recently, fishes [10] , [11] . ”

“ The UnaG, Chlopsid FP I and Chlopsid FP II were used as bait for other vertebrate FABPs. FABP DNA and protein sequences were used for analysis (S1 Table) . Phylogenetic trees were generated and we found that eel FPs from the families Anguillidae and Chlopsidae are either sister to the FABP7 brain clade, which diverged from primitive fishes, or nested within it. Although we found non-fluorescent FABPs in Kaupichthys and in the transcriptome of Anguilla, these FABPs do not contain the Gly-Pro-Pro tri-peptide motif. ”

“ This difference in amino acid sequence can potentially explain the fluorescence shift, due to an increase in the π conjugation of the system. This change in fluorescence emission spectra demonstrates that it is possible to make changes to the amino acids around the conjugated bilirubin, which can alter the fluorescence spectrum of the protein. ”

“ Alignment of human brain FABP-7, UnaG and Chlopsid FP I. Red rectangles show residues that are inserted in the eel FP. Green rectangles show residues under dN/dS skew. Grey rectangles indicate polarity index. Blue highlights secondary structure factors. Pink highlights volume. Light green highlights refractivity/heat capacity and purple highlights charge/iso-electric point. When multiple colors appear at a site, this means that more than a single method detected changes or skew at those positions. ”

“ Muscle tissue was homogenized in TriZol reagent (Life Technologies, Carlsbad, CA) , and the total RNA was precipitated with isopropanol and dissolved in ddH2O. The quality of RNA was assessed on a 2100 Bioanalyzer and with agarose gel electrophoresis. ”

“ Major colour patterns of reef building corals are due to a family of GFP-like proteins. Coral Reefs 19: 197–204 .↑ Lukyanov KA, Fradkov AF, Gurskaya NG, Matz MV, Labas YA, Savitsky AP (2000) Natural animal coloration can Be determined by a nonfluorescent green fluorescent protein homolog. The Journal of Biological Chemistry 275: 25879–25882 . pmid:10852900↑ Mazel CH, Fuchs E (2003) Contribution of fluorescence to the spectral signature and perceived color of corals. ”

“ Two photon image of eel fluorescence.UnaG distribution in HEK293 cells (top panel) , two photon localization of Chlopsid FP I in HEK293 cells (bottom panel) and confocal images of CiVSP-UnaG fusions in HEK293 cells. Discussion
The marine environment has proven to be the richest reservoir of novel FPs on the planet [40] . The upper photic ocean is stably monochromatic with downwelling daylight becoming primarily blue (470-490nm) in color with increasing depth [1] . ”

“ The human fatty acid-binding protein family: evolutionary divergences and functions. Hum Genomics 5: 170–191 . pmid:21504868↑ Tsukamoto K (2006) Oceanic biology: spawning of eels near a seamount. Nature 439: 929. pmid:16495988↑ Lee TW, Miller MJ Hwang HB, Wouthuyzen S, Tsukamoto K (2008) Distribution and early life history of Kaupichthys leptocephali (family of Chlopsidae) in the central Indonesian Seas. Marine Biology 153: 285–295 .↑ Bowmaker JK, Semo M, Hunt DM, Jeffery G (2008) Eel visual pigments revisited: the fate of retinal cones during metamorphosis. ”