*Laboratory of Molecular Biology & Biochemistry, The Rockefeller University, New York;
Department of Ecology & Evolutionary Biology, Yale University
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Abstract |
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Introduction |
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One experimental approach used in studies of molecular evolution is the use of phylogenetic methods to infer ancestral sequences of biological molecules with the aim of recreating extinct genes or proteins in the laboratory (Chang and Donoghue 2000
). This approach shows much promise for investigating the function and evolution of ancient proteins (Malcolm et al. 1990
; Adey et al. 1994
; Chandrasekharan et al. 1996
; Dean and Golding 1997
; Bishop, Dean, and Mitchell-Olds 2000
), and perhaps even the organisms in which they existed (Jermann et al. 1995
; Messier and Stewart 1997
; Nei, Zhang, and Yokoyama 1997
; Boissinot et al. 1998
; Galtier, Tourasse, and Gouy 1999
). But in taking these studies of ancestral proteins into the laboratory, few experimental studies have explored the use of maximum likelihood methods of ancestral reconstruction, particularly in the light of the plethora of likelihood models now available. Using maximum likelihood methods (Felsenstein 1981
; Yang, Kumar, and Nei 1995
) we explored different models for reconstructing an ancestral archosaur rhodopsin. Once inferred, the phylogenetically reconstructed archosaur rhodopsin gene sequences were then synthesized, expressed, and assayed for function in the laboratory. The ancestral archosaurs were chosen as a test case for this type of molecular paleontological approach for two reasons. First, although the archosaur lineage gave rise to some of the largest reptiles to walk the earth, including the late Cretaceous carnivorous dinosaurs, little is known yet of the physiology and behavior of their ancestors. Because visual pigments constitute the critical first step in the visual phototransduction cascade in the eye and rhodopsin in particular is essential for vision at low light levels, recreating the inferred visual pigments of the archosaur ancestors in the laboratory should be an important initial step toward a better understanding of their visual capabilities that is difficult to obtain using other means. Second, divergences among extant archosaur rhodopsin protein sequences are no more than 16%, levels within the range at which likelihood methods of ancestral reconstruction should work reasonably well.
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Materials and Methods |
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Synthetic Gene Design, Construction, and Expression
The artificial archosaur gene was synthesized in large fragments (168 to 230-mers) on a solid-phase oligonucleotide synthesizer (Applied Biosystems, model 392). The synthesized fragments were amplified using the Pfu polymerase (Stratagene), cloned into the pCR-Blunt vector (Invitrogen), pieced together using unique restriction sites, and then cloned into a mammalian expression vector (pMT). The artificial archosaur gene was expressed and purified by previously described methods (Han et al. 1996
), essentially by transient transfection into COS cells using Lipofectamine Plus (Life Technologies), harvested after 48 h, regenerated in 5 µM 11-cis retinal, solubilized in 1% n-dodecyl-ß-d-maltoside detergent, and immunoaffinity purified using the 1D4 monoclonal antibody. Absorbance spectroscopy was performed at 25°C using a Perkin-Elmer Lambda 800 spectrophotometer, using quartz cuvettes with a 1-cm pathlength. Transducin fluorescence was monitored at 10°C using an SPEX spectrofluorometer equipped with a Xenon arc lamp by methods described previously (Marin et al. 2000
).
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Results and Discussion |
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The purified ancestral archosaur rhodopsin bound to 11-cis retinal to produce a stable pigment with a visible absorption maximum at 508 nm (fig. 3A
), which is redshifted from that of most mammalian and fish rhodopsins but within the higher end of the range of values reported for reptiles and particularly birds, which tend to have longer wavelengthabsorbing rhodopsins (see fig. 1
). Upon bleaching with light, the visible absorption peak shifted to 383 nm, which is characteristic of the active conformation of metarhodopsin II (inset, fig. 3A
). To determine if the light-activated conformation of the ancestral archosaur rhodopsin was functionally active, a fluorescence assay was used to measure guanine-nucleotide uptake by the heterotrimeric G-protein transducin. The photolyzed archosaur pigment activated transducin at a rate similar to that of bovine rhodopsin (86% normalized relative to bovine rhodopsin; fig. 3B
). Similar experiments were carried out on the ancestral archosaur rhodopsin variants (T213I, T217A, and V218I), which represent all possible alternate reconstructions. These variants showed similar results both in terms of spectral properties (max = 508 for all three) and transducin activation rates (83%, 74%, and 79%, respectively). A triple-replacement variant was also found to have spectral properties similar to the archosaur rhodopsin (
max = 509).
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Fossils preserved well enough to shed light on physiology and behavior are extremely rare (Ruben et al. 1999
; Fisher et al. 2000
). Attempts to amplify ancient DNA from exceptional samples preserved in amber or from dinosaur bone extracted from Cretaceous period coal beds have met with questionable success; in fact, material older than several hundred thousand years may not prove to be a reliable source of DNA, except under highly unusual circumstances (Hoss et al. 1996
). An entirely different approach is to use phylogenetic methods to infer ancestral sequences (Yang, Kumar, and Nei 1995
). One elegant study (Jermann et al. 1995
) recreated in the laboratory the molecular evolution of ribonuclease, specifically in the artiodactyl lineage, whose ancestor was estimated to have lived approximately 40 MYA. These types of approaches combine phylogenetic inference of ancestral gene structure with gene synthesis methods to obtain biological molecules that can be characterized in detail to provide a better understanding of the biology of ancient animals.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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Keywords: phylogenetic ancestral reconstruction
rhodopsin
biochemical evolution
vertebrate vision G proteincoupled receptor
Address for correspondence and reprints: Thomas P. Sakmar, Laboratory of Molecular Biology & Biochemistry, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, Box 284, New York, New York 10021. E-mail: sakmar{at}mail.rockefeller.edu
or changb{at}mail.rockefeller.edu
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