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MBE Advance Access published online on January 12, 2005
Molecular Biology and Evolution, doi:10.1093/molbev/msi086
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Molecular Biology and Evolution © Society for Molecular Biology and Evolution 2005; all rights reserved.
Accepted January 3, 2005

Research Article

Reconstitution of Ancestral Green Visual Pigments of Zebrafish and Molecular Mechanism of their Spectral Differentiation

Akito Chinen 1, Yoshifumi Matsumoto 1, and Shoji Kawamura 1*

1 Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo

* To whom correspondence should be addressed.
Shoji Kawamura, E-mail: kawamura{at}k.u-tokyo.ac.jp


  Abstract

We previously reported that zebrafish has four tandemly duplicated green (RH2) opsin genes (RH2-1, RH2-2, RH2-3 and RH2-4). Absorption spectra vary widely among the four photopigments reconstituted with 11-cis retinal, their peak absorption spectra ({lambda}max) being 467, 476, 488 and 505 nm, respectively. In this study, we inferred the ancestral amino acid sequences of the zebrafish RH2 opsins by likelihood-based Bayesian statistics and reconstituted the ancestral opsins by site-directed mutagenesis. The ancestral pigment to all the four zebrafish RH2 (A1) and that to RH2-3 and RH2-4 (A3) showed {lambda}max at 506 nm, while that to RH2-1 and RH2-2 (A2) at 474 nm, indicating that spectral shift occurred toward shorter wavelength on the evolutionary lineages A1 to A2 by 32 nm, A2 to RH2-1 by 7 nm and A3 to RH2-3 by 18 nm. Pigment chimeras and site-directed mutagenesis revealed large contribution (~15 nm) of glutamic acid to glutamine substitution at residue 122 (E122Q) to the A1 to A2 and A3 to RH2-3 spectral shifts. However, the rest of the spectral differences appear to result from complex interactive effects of numbers of amino acid replacements each of which has only a minor spectral contribution (1-3 nm). The four zebrafish RH2 pigments cover nearly entire range of {lambda}max distribution among vertebrate RH2 pigments and must provide an excellent model to study spectral tuning mechanism of RH2 in vertebrates.

Keywords: zebrafish; RH2 opsins; visual pigments; gene duplication; spectral differentiation; ancestral sequence.
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