* Department of Medicine, University of Washington, Seattle
Research School of Biological Sciences
Centre for Bioinformation Science, The Australian National University, Canberra, Australia
Department of Biology, Syracuse University
Correspondence: E-mail: sdeeb{at}u.washington.edu.
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Abstract |
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Key Words: wallaby visual pigments cones sequence
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Introduction |
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Very few studies have been performed on the color vision of marsupials, and none of the marsupial visual pigment sequence is known. Studies on these metatherian mammals are likely to yield additional important information on mechanisms of spectral tuning and adaptive evolution and of their visual pigments since they diverged from the eutherian mammals around 130 MYA.
The SWS pigments are evolutionarily much closer to the rhodopsins than to the MWS and LWS pigments. The mammalian SWS pigments belong to class 1 cone pigments, which range in max from UV to blue (360 nm to 460 nm) (reviewed in Yokoyama and Shi [2000]). Recent phylogenetic and mutagenesis experiments indicated that the F86Y substitution is largely responsible for shifting the
max of the UVS pigments of teleost fish, amphibia, reptiles, and rodents from about 360 nm to the 430 nm of the blue sensitive pigments of ungulates (Cowing et al. 2002; Fasick, Applebury, and Oprian 2002).
Variation in max among the mammalian MWS/LWS pigments ranges from 507nm to 560 nm and appears to be determined by differences at only five amino acid sites. The S180A, H197Y, Y277F, T285A, and A308S substitutions shift, in a largely additive manner, the
max values towards blue by approximately 7, 28, 7, 15, and 16 nm, respectively (Neitz, Neitz, and Jacobs 1991; Merbs and Nathans 1992; Asenjo, Rim, and Oprian 1994; Yokoyama and Radlwimmer 2001; Yokoyama 2002).
Behavioral studies on the Virginia opossum (Didelphis virginiana) indicated evidence for color vision (Friedman, Becker, and Bachman 1967). However, ERG measurements identified only a single LWS cone type (Jacobs 1993). Immunocytochemical studies on the North American opossum (Monodelphis domestica) (Wikler and Rakic 1990) and the South American opossum (Didelphis marsupialis aurita) (Ahnelt, Hokoc, and Rohlich 1995) indicated the presence of SWS cones in their retinae but at low density. Behavioral studies on an Australian marsupial, the tammar wallaby (Macropus eugenii), indicated capacity for dichromatic color vision based on SWS and MWS pigments with peak sensitivities (max) at 420 nm and 539 nm, respectively (Hemmi 1999), as did immunocytochemical studies (Hemmi and Grunert 1999). However, again, MSP measurements detected only an MWS cone pigment (Hemmi, Maddess, and Mark 2000). ERG and MSP methodologies were perhaps inadequate to detect SWS cones because of their scarcity (<10% of all cones) in the retinae of these animals. Of great interest is the MSP detection of SWS, MWS, and LWS photopigments in the retinae of two other Australian marsupials, the honey possum (Tarsipes rostratus) and the fat-tailed dunnart (Sminthopsis crassicaudata) (Arrese et al. 2002), suggesting potentially trichromatic color vision. Interestingly, the honey possum belongs to the same marsupial taxonomic division (diprotodontata) as the dichromatic tammar wallaby.
Here, we have determined the coding sequences of the SWS and MWS cone pigment genes and inferred the sequence of the corresponding opsins. Further, we expressed and reconstituted both pigments in vitro and determined their spectra. We propose that the tammar wallaby most likely has dichromatic color vision. We also compared the sequences of the cone pigments to those of other species and inferred amino acid residues and mechanisms of spectral tuning.
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Materials and Methods |
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Reverse Transcription and PCR Amplification
Reverse transcription of total retinal RNA was performed with Superscript II reverse transcriptase and a random hexamer as primer (Stratagene, La Jolla, Calif.). The choice of primers used in amplification of the wallaby SWS and MWS/LWS pigment cDNA sequences was based on comparisons among known coding sequences of these pigment cDNAs of a number of species. For the SWS-specific primers, comparisons were made among the human, mouse, gorilla, bottle-nosed dolphin, chicken, domestic pigeon, Humbolt's penguin, common canary, and salamander. The sequence of primers we designed to amplify the wallaby SWS coding sequence are given in table 1 and their positions indicated in figure 1. The PCR amplification reaction (in a total volume of 50 ml) included an initial denaturation step of 2 min at 94°C, followed by 35 cycles of denaturation at 94°C for 15 s, annealing at 64°C for 30 s, and polymerization at 72°C for 30 s. Primers MS1F and MS1R amplified a single DNA fragment of approximately 540 bp and MS2F and MS2R amplified a single fragment of approximately 620 bp. The PCR products were purified on QiaQuick spin columns as described by the manufacturer (Qiagen, Valencia, Calif.) and sequenced on an ABI Model 3100.
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Rapid Amplification of cDNA Ends (RACE)
Based on the wallaby partial cDNA sequences obtained above, primers were designed to determine the rest of the cDNA sequences employing 5' and 3' RACE analysis with the Smart II Race kit according to the manufacturer's protocol (Clontech, Palo Alto, Calif.). WS1F and WS1R were used as internal primers for SWS first-strand cDNA synthesis, and primers WG4F and WG4R were used for MWS/LWS first-strand synthesis. The 5' and 3' external primers were supplied by the manufacturer. The RACE cDNA products were used as templates for PCR amplification of the 3' and 5' ends of wallaby SWS cDNA using primers WS2F and WS1R, respectively (table 1 and figure 1) and sequenced with primers WS2F, WS5F, and WS1R. The 3' and 5' ends of wallaby MWS/LWS sequences were amplified and sequenced with primers WG5F and WG3R, respectively. The external primer for all these amplification reactions was supplied by the manufacturer and is complementary to the external primer used for first-strand cDNA synthesis. Amplification was performed using the Advantage II PCR kit as described by the manufacturer (Clontech). The PCR products were purified and sequenced as described above.
Expression and Spectral Analysis of Pigments
The SWS and MWS cDNA clones were amplified by PCR by using the following primers: 5'-ACAATCGAATTCCACCATGTCAGGGGACGAGGAG-3' (F) and 5'-GGGATCGTCGACCTAGGGCCAACTTGGCTGG-3' (R) (for SWS) and 5'-AGAAAGGAATTCCACCATGACACAGGCATGGGAC-3' (F) and 5'-TGGGGAGTCGACGCAGGCGCCACAGAGGACAC-3' (R) (MWS).
These cDNAs in an expression vector, pMT5, were expressed in COS1 cells by transient transfection (Yokoyama 2000b). The visual pigments were regenerated by incubating these opsins with 11-cis-retinal (Storm Eye Institute, Medical University of South Carolina, Charleston) in the dark. The resulting visual pigments were then purified by immunoaffinity chromatography by using monoclonal antibody 1D4 Sepharose 4B (The Cell Culture Center, Minneapolis, Minn.) in buffer consisting of 50 mM N-(2-hydroxyethyl) piperazine- N'-2-ethanesulfonic acid (pH 6.6), 140 mM NaCl, 3 mM MgCl2, 20% (wt/vol) glycerol, and 0.1% dodecylmaltoside. The absorption spectra of visual pigments were recorded at 20°C using a Hitachi (Tokyo) U-3000 dual-beam spectrophotometer. Recorded spectra were analyzed by using SigmaPlot software (Jandel, San Rafael, Calif.).
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Results |
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Sequence and Spectral Tuning of the Wallaby SWS Pigment
The nucleotide sequence of the cDNA and the deduced amino acid sequence are shown in figure 1. The open reading frame encodes an opsin of 346 amino acids. The wallaby sequence shares 86.5%, 85.5%, and 83% identity with SWS pigments of the mouse, bovine, and human, respectively. Alignment of SWS sequences of various species is shown in figure 2. Notably, low sequence identity is found in the 30 amino-terminal residues. Comparisons at informative amino acid positions, including the eight sites proposed by Yokoyama and colleagues (Yokoyama and Shi 2000; Shi, Radlwimmer, and Yokoyama 2001) to be involved in tuning in the primate pigments are shown in table 2. The max of the wallaby SWS pigment was previously determined by behavioral methods to be about 420 nm (Hemmi 1999), which is similar to that of the human pigment. However, the wallaby and human pigments differ at seven of the eight sites involved in spectral tuning. Remarkably, the wallaby and bovine pigments differ at only three of these sites, given the vast evolutionary distance between them. The wallaby has Y at position 86, indicating that it belongs to the blue class of pigments exemplified by those of the ungulates (Cowing et al. 2002; Fasick, Applebury, and Oprian 2002).
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Structure of the Wallaby MWS Pigment Gene
We determined the intron/exon structure of the wallaby MWS pigment gene by amplification with wallaby coding sequence primers chosen to lie within the corresponding exons of the human and mouse pigment genes. The primer pairs used to amplify across introns 1 to 5 are WG1F and WG2R, WG2F and WG3R, WG3F and WG4R, WG4F and WG5R, and WG5F and WG6R, respectively (table 1 and fig. 4). The amplification conditions were an initial 2 min of denaturation at 94°C followed by 35 cycles of denaturation at 94°C for 15 s, annealing at 64°C for 30 s, and polymerization. This was followed by a final step of polymerization at 72°C for 4 min. The amplification products were directly sequenced to identify the intron/exon boundaries. The size of introns was estimated by gel electrophoresis. The position of introns is indicated on the cDNA sequence shown in figure 4A. The wallaby MWS pigment gene spans approximately 19 kb and, like all other MWS and LWS pigment genes, is composed of six exons. Introns interrupt the coding sequences of the wallaby, mouse, and human MWS pigment sequences at the same corresponding nucleotide positions. However, the length of introns varies widely among these species (fig. 4B).
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Discussion |
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The cone pigments of four marsupials, the wallaby, the honey possum, the fat-tailed dunnart, and the South American opossum, have so far been studied by behavioral, MSP, or ERG methods. There is evidence to suggest that the wallaby and the South American opossum have dichromatic color vision. However, more studies are needed to establish the absence of a third class of cone. Interestingly, MSP measurements detected three classes of cone in both the honey possum and the fat-tailed dunnart, suggesting a potential for trichromatic color vision (Arrese et al. 2002). The tammar wallaby and the honey possum belong to the same taxonomic group, the Diprotodonta.
Our study is the first to determine the sequence of marsupial cone pigments and address the question of the potential for trichromatic color vision in the tammar wallaby and to compare mechanisms of spectral tuning with other mammalian species. Exhaustive molecular analysis that involved PCR amplification using a variety of primers complementary to coding sequences and both cDNA and genomic DNA templates, indicated the presence in the retina and in genomic DNA of only two cone pigment sequences, SWS and MWS. The annealing temperatures in amplification reactions were selected to be of low stringency to allow amplification of homologous but not identical sequences. We could have easily detected the presence of a LWS pigment sequence since, in other species, it is known to be highly homologous to MWS. This result is consistent with the conclusion that the tammar wallaby has dichromatic color vision as determined by behavioral methodology (Hemmi 1999).
Based on sequence comparisons, the wallaby SWS pigment belongs to the SWS1 evolutionary cluster. This is expected since all other known mammalian SWS pigments belong to this class (Yokoyama 2000a). In order to infer the max of the wallaby SWS pigment, we compared the amino acids at positions previously determined to be important in spectral tuning of this class of pigments. Yokoyama and coworkers (Yokoyama and Shi 2000; Shi, Radlwimmer, and Yokoyama 2001) proposed that the spectral difference (66 nm) between mouse ultraviolet (UV) and human blue SWS1 pigments is due to substitutions at five sites. Following the numbering of bovine rhodopsin, the T52F, F86L, T93P, A114G, and S118T substitutions synergistically account for the shift of
max from 358 nm (mouse UV) to 424 nm (human blue). Interestingly, substitution at these five sites separately did not shift the
max of the mouse pigment (Yokoyama and Shi 2000; Shi, Radlwimmer, and Yokoyama 2001). Subsequently, Fasick, Applebury, and Oprian (2002) and Cowing et al. (2002) showed by site-directed mutagenesis of SWS1 pigments that the amino acid substitution F86Y by itself accounts for the majority (66 nm, or 80%) of the difference between the bovine blue (436 nm) and mouse UV (358 nm) pigments. The mechanism by which this amino acid substitution shifts the spectrum towards red involves protonation of the chromophore Schiff base (Cowing et al. 2002; Fasick, Applebury, and Oprian 2002). Substitutions at three sites (F86Y/I93T/A97T) shifted the
max of the mouse SWS1 pigment by 72 nm (90%) to 430 nm. In contrast, the F86L substitution by itself had little effect in blue-shifting the spectrum of the human SWS1 pigment. Thus, a different mechanism of spectral tuning of SWS1 pigments seems to have evolved in primates.
The wallaby SWS1 pigment differs at positions 86 (Y), 97 (S), and 114 (G) from that of the mouse and at positions 93, 114, and 118 from that of the cow (table 2). Since position 86 shifts the spectrum by 66 nm and that the Gly114Ala substitution by itself results in no change in the max of the mouse SWS1 pigment (Yokoyama and Shi 2000; Shi, Radlwimmer, and Yokoyama 2001), the wallaby SWS1 pigment is estimated to have a
max of near 424 nm (358 + 66). To confirm this value, we expressed and reconstituted the wallaby SWS pigment in vitro and determined its
max to be 420 ± 2 nm. This is in agreement with the
max of near 420 previously determined by behavioral methods (Hemmi 1999). This small difference could be related to the different methods used. We conclude that the mechanism of spectral tuning in the wallaby involves the F86Y substitution observed in SWS pigments of ungulates and differs significantly from the five amino acid mechanism of primates. It is possible that spectral tuning in the SWS1 pigments using the F86Y mechanism existed before divergence of the metatherian and eutherian mammals some 130 MYA (Graves and Westerman 2002). However, it is more likely that the F86Y substitution could have occurred independently in the ungulate and wallaby lineages as a result of convergent evolution. Convergent evolution of blue/UV shifts in the spectral of pigments of other species has been previously proposed and discussed (Hunt et al. 2001; Shi, Radlwimmer, and Yokoyama 2001). Interestingly, the SWS pigments found in the fat-tailed dunnart and the honey possum were of the UV type, with
max values of around 350 nm (Arrese et al. 2002). The UV pigments in birds seem to have evolved from blue pigments by yet a third mechanism. A single amino acid substitution, S90C (according to the numbering in bovine rhodopsin), shifts the
max of avian SWS1 pigments from the UV to the blue regions of the spectrum (Wilkie et al. 2000; Yokoyama and Radlwimmer 2001).
The max of vertebrate MWS and LWS cone pigments ranges from 508 nm to 575 nm (reviewed in Yokoyama 2000a; Ebrey and Koutalos 2001; Yokoyama and Radlwimmer 2001). The MWS and LWS pigments in Old World primates differ in
max by about 30 nm. It has been shown that amino acid differences at three sites largely account for this difference (Yokoyama and Yokoyama 1990; Neitz, Neitz, and Jacobs 1991; Merbs and Nathans 1992; Asenjo, Rim, and Oprian 1994). The A180S, F277Y, and A285T substitutions shift the spectrum of the MWS pigment towards the red by approximately 7, 8, and 15 nm, respectively (see Yokoyama and Radlwimmer [2001]). Extensive evolutionary analysis of the pigments of a number of vertebrate species revealed the involvement of two additional sites (H197Y and A308S) in spectral tuning of the MWS and LWS pigments (Sun, Macke, and Nathans 1997; Radlwimmer and Yokoyama 1998). The H197Y substitution shifts the
max by about 28 nm towards the blue, as seen in rodents. Residue 197 is part of a chloride-binding site when occupied by H. The A308S change shifts the
max by about 18 nm towards the blue. The changes in
max due to substitutions at the above five sites seem to be additive.
Based on the "five-site rule," we inferred that the wallaby has a MWS pigment with a max of 530 nm. We confirmed this by measuring the in vitroexpressed pigment, which has a
max of 528 ± 1 nm). However, ERG measurements on the wallaby indicated a MWS pigment with a
max of 539 nm. ERG measurements have been observed to vary in
max from those on in vitroexpressed pigments. For example, whereas the ERG-determined
max values of MWS pigments of the white-tailed deer, guinea pig, and grey squirrel violated the five-site rule, those determined by in vitro measurements were different and did conform to the rule (Yokoyama and Radlwimmer 2001).
The MWS pigments of the honey possum and the fat-tailed dunnart were shown by MSP to have max values of 509 nm (suggested to belong to the RH2 group) and 535 nm, respectively (Arrese et al. 2002). However, the wallaby MWS pigment belongs to the MWS/LWS group. It would be interesting to correlate the spectral sensitivity of the cone pigments of these marsupials with ecological parameters. Although the tammar wallaby is often described as crepuscular or nocturnal, it is also active in the daylight hours in the late afternoon, grazing on grass and low tree branches (Hemmi, Maddess, and Mark 2000; personal observations). The retinal distribution of SWS and MWS cones differs, with ratios in dorsal retina (ventral visual field) optimal for distinguishing features with different shades of green such as grasses.
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Acknowledgements |
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Footnotes |
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