CHANG, BS; JONSSON, K; KAZMI, MA; DONOGHUE, MJ; SAKMAR, TS; Rockefeller University, New York; Rockefeller University, New York; Rockefeller University, New York; Yale University, Connecticut; Rockefeller University, New York: Recreating a Functional Ancestral Archosaur Visual Pigment

Phylogenetic ancestral reconstruction methods hold much promise for probing the biochemistry and physiology of animals long vanished from the earth. We explore these methods for investigating visual proteins in the ancestors of the archosaurs, a major branch of the diapsid reptiles, would have originated more than 240 million years ago near the dawn of the Triassic Period. We used maximum likelihood phylogenetic ancestral reconstruction methods, and explored different models of evolution in inferring the amino acid sequence of a putative ancestral archosaur visual pigment. Ancestral reconstructions of the ancestral archosaur node using the best-fitting models of each type were found to be in agreement except for three amino acid residues, at which one reconstruction differed from the other two. In order to determine if these ancestral pigments would be functionally active, the corresponding genes were chemically synthesized, and then expressed in a mammalian cell line in tissue culture. The expressed artificial genes were all found to bind 11-cis-retinal to yield stable photoactive pigments with lmax values of about 508 nm, which is slightly red-shifted relative to that of many extant vertebrate pigments. The ancestral archosaur pigments also activated the retinal G protein transducin, as measured in a fluorescence assay. Our results show that ancestral genes from ancient organisms can be reconstructed de novo and tested for function using a combination of phylogenetic and biochemical methods.