Shozo Yokoyama

Molecular evolution of vertebrate visual pigments.

Dramatic improvement of our understanding of the genetic basis of vision was brought by the molecular characterization of the bovine rhodopsin gene and the human rhodopsin and color opsin genes (Nathans and Hogness, 1983; Nathans et al., 1984, 1986a,b). The availability of cDNA clones from these studies has facilitated the isolation of retinal and nonretinal opsin genes and cDNA clones from a large variety of species. Today, the number of genomic and cDNA clones of opsin genes isolated from different vertebrate species exceeds 100 and is increasing rapidly. The opsin gene sequences reveal the importance of the origin and differentiation of various opsins and visual pigments. To understand the molecular genetic basis of spectral tuning of visual pigments, it is essential to establish correlations between a series of the sequences of visual pigments and their lambda(max) values. The potentially important amino acid changes identified in this way have to be tested whether they are in fact responsible for the lambda(max)-shifts using site-directed mutagenesis and cultured cells. A major goal of molecular evolutionary genetics is to understand the molecular mechanisms involved in functional adaptations of organisms to different environments, including the mechanisms of the regulation of the spectral absorption. Therefore, both molecular evolutionary analyses of visual pigments and vision science have an important common goal.

Elucidation of phenotypic adaptations: Molecular analyses of dim-light vision proteins in vertebrates

Vertebrate ancestors appeared in a uniform, shallow water environment, but modern species flourish in highly variable niches. A striking array of phenotypes exhibited by contemporary animals is assumed to have evolved by accumulating a series of selectively advantageous mutations. However, the experimental test of such adaptive events at the molecular level is remarkably difficult. One testable phenotype, dim-light vision, is mediated by rhodopsins. Here, we engineered 11 ancestral rhodopsins and show that those in early ancestors absorbed light maximally (λmax) at 500 nm, from which contemporary rhodopsins with variable λmaxs of 480–525 nm evolved on at least 18 separate occasions. These highly environment-specific adaptations seem to have occurred largely by amino acid replacements at 12 sites, and most of those at the remaining 191 (≈94%) sites have undergone neutral evolution. The comparison between these results and those inferred by commonly-used parsimony and Bayesian methods demonstrates that statistical tests of positive selection can be misleading without experimental support and that the molecular basis of spectral tuning in rhodopsins should be elucidated by mutagenesis analyses using ancestral pigments.

Molecular analysis of the evolutionary significance of ultraviolet vision in vertebrates

Many fish, amphibians, reptiles, birds, and some mammals use UV vision for such basic activities as foraging, mate selection, and communication. UV vision is mediated by UV pigments in the short wavelength-sensitive type 1 (SWS1) group that absorb light maximally (λmax) at ≈360 nm. Reconstructed SWS1 pigments of most vertebrate ancestors have λmax values of ≈360 nm, whereas the ancestral avian pigment has a λmax value of 393 nm. In the nonavian lineage, UV vision in many modern species is inherited directly from the vertebrate ancestor, whereas violet vision in others has evolved by different amino acid replacements at ≈10 specific sites. In the avian lineage, the origin of the violet pigment and the subsequent restoration of UV pigments in some species are caused by amino acid replacements F49V/F86S/L116V/S118A and S90C, respectively. The use of UV vision is associated strongly with UV-dependent behaviors of organisms. When UV light is not available or is unimportant to organisms, the SWS1 gene can become nonfunctional, as exemplified by coelacanth and dolphin.

Evolution of Dim-Light and Color Vision Pigments

A striking level of diversity of visual systems in different species reflects their adaptive responses to various light environments. To study the adaptive evolution of visual systems, we need to understand how visual pigments, the light-sensitive molecules, have tuned their wavelengths of light absorption. The molecular basis of spectral tuning in visual pigments, a central unsolved problem in phototransduction, can be understood only by studying how different species have adapted to various light environments. Certain amino acid replacements at 30 residues explain some dim-light and color vision in vertebrates. To better understand the molecular and functional adaptations of visual pigments, we must identify all critical amino acid replacements that are involved in the spectral tuning and elucidate the effects of their interactions on the spectral shifts.

DNA elimination in tetrahymena: A developmental process involving extensive breakage and rejoining of DNA at defined sites

Elimination of specific DNA sequences occurs during macronuclear development in the ciliate Tetrahymena thermophila. Recombinant DNA clones containing a segment of micronuclear (germinal) DNA involved in elimination and the corresponding segment of macronuclear (somatic) DNA produced after elimination were isolated. Detailed comparisons of the cloned DNAs, as well as the genomic DNAs, by hybridization indicated that DNA elimination is accompanied by specific DNA rearrangements. In this 9.5 kb region three defined DNA segments are deleted and the remaining sequences are linked together as one contiguous piece in the macronucleus. Specific DNA rearrangement of this kind occurs widely in the genome. Analysis of 20 randomly selected DNA clones suggests that there are more than 5000 such rearrangement sites in the genome. Thus specific breakage and rejoining of DNA occurs extensively during development, and might play an essential role in nuclear differentiation.

Molecular genetics and the evolution of ultraviolet vision in vertebrates

Despite the biological importance of UV vision, its molecular bases are not well understood. Here, we present evidence that UV vision in vertebrates is determined by eight specific amino acids in the UV pigments. Amino acid sequence analyses show that contemporary UV pigments inherited their UV sensitivities from the vertebrate ancestor by retaining most of these eight amino acids. In the avian lineage, the ancestral pigment lost UV sensitivity, but some descendants regained it by one amino acid change. Our results also strongly support the hypothesis that UV pigments have an unprotonated Schiff base-linked chromophore.

Molecular evolution of color vision in vertebrates

Visual systems of vertebrates exhibit a striking level of diversity, reflecting their adaptive responses to various color environments. The photosensitive molecules, visual pigments, can be synthesized in vitro and their absorption spectra can be determined. Comparing the amino acid sequences and absorption spectra of various visual pigments, we can identify amino acid changes that have modified the absorption spectra of visual pigments. These hypotheses can then be tested using the in vitro assay. This approach has been a powerful tool in elucidating not only the molecular bases of color vision, but the processes of adaptive evolution at the molecular level.

Functional characterization of visual and nonvisual pigments of American chameleon (Anolis carolinensis)

Using only 11-cis 3, 4-dehydroretinal as a chromophore in the pure-cone retina, American chameleon (Anolis carolinensis) detects a wide range of color from ultraviolet (UV) to infrared. We previously characterized its visual opsin genes sws1Ac, sws2Ac, rh1Ac, rh2Ac, and LwsAc that encode SWS1Ac, SWS2Ac, RH1Ac, RH2Ac, and LWSAc opsins, respectively, and the pineal gland-specific opsin (PAc) gene. Here we present the light absorption profiles of the visual pigments obtained by expressing these opsins and reconstituting them with 11-cis retinal using the COS1 cell cDNA expression system. The purified SWS1Ac, SWS2Ac, RH1Ac, RH2Ac, LWSAc, and PAc pigments have the wavelengths of maximal absorption at 358, 437, 491, 495, 560, and 482 nm, respectively. SWS1Ac is the first vertebrate UV opsin whose spectral sensitivity has been directly evaluated. RH1 pigments, orthologous to the rod pigments of other vertebrates, are sensitive to hydroxylamine in the dark, exhibiting a cone pigment-like characteristic, probably reflecting their adaptation to the pure cone retina. Interestingly, the blue-sensitive SWS2Ac pigment shows an exceptionally low level of sensitivity to hydroxylamine, possessing a rod pigment-like characteristic.

Genetics and evolution of ultraviolet vision in vertebrates

Various vertebrates use ultraviolet (UV) vision for such basic behaviors as mating, foraging, and predation. We have successfully interchanged the color‐sensitivities of the mouse UV pigment and the human blue pigment by introducing forward and reverse mutations at five sites. This unveils for the first time the general mechanism of UV vision. Most contemporary UV pigments in vertebrates have maintained their ancestral functions by accumulating no more than one of the five specific amino acid changes. The avian lineage is an exception, where the ancestral pigment lost UV‐sensitivity but some descendants regained it by one amino acid replacement at an entirely different site.

REGENERATION OF ULTRAVIOLET PIGMENTS OF VERTEBRATES

We report here the regeneration of the visual pigments of mouse, rat, goldfish and pigeon, which have wavelengths of maximal absorption at 359 nm, 358 nm, 359 nm, and 393 nm, respectively. The construction and functional assays of the ultraviolet or near‐ultraviolet pigments from a wide range of vertebrate species will allow us to study the molecular bases of ultraviolet vision for the first time.