Wild-type Pigment Research Articles

An in vivo plant platform to assess genes encoding native and synthetic enzymes for carotenoid biosynthesis.

This chapter provides an overview of how to test enzyme function in plants using candidate genes encoding carotenoid biosynthetic enzymes and/or engineered enzyme versions for synthetic biology applications. A step-by-step guide explains transformation and screening of Agrobacterium tumefaciens containing a binary vector, preparation of inoculum, and transient expression of candidate or retooled enzymes in Nicotiana benthamiana leaf tissue. In planta expression is especially useful for screening candidate carotenoid biosynthetic enzymes because many of the substrates that these enzymes would use are already present in the leaf. In this assay pigments are extracted from leaf discs expressing transgenes and changes in pigment profiles resulting from the activity of these introduced enzymes are scored using High Performance Liquid Chromatography (HPLC). If a transgene product modifies a carotenoid substrate, differences are easily revealed by comparison against wildtype pigment profiles. Additionally, this in planta method presents the researcher with an opportunity to evaluate candidate carotenoid biosynthetic enzyme function in the existing context of N. benthamiana photosynthetic chloroplasts. Photosynthetic phenotypes that arise from changes in pigment composition can be determined by Pulse Amplitude Modulated (PAM) fluorescence measurements, if desired.

New Insights into the Melanophilin (MLPH) Gene Affecting Coat Color Dilution in Rabbits.

Coat color dilution corresponds to a specific pigmentation phenotype that leads to a dilution of wild type pigments. It affects both eumelanin and pheomelanin containing melanosomes. The mode of inheritance of the dilution phenotype is autosomal recessive. Candidate gene approaches focused on the melanophilin (MLPH) gene highlighted two variants associated with the dilution phenotype in rabbits: The c.111-5C>A variant that is located in an acceptor splice site or the c.585delG variant, a frameshift mutation. On the transcript level, the skipping of two exons has been reported as the molecular mechanism responsible for the coat color dilution. To clarify, which of the two variants represents the causal variant, (i) we analyzed their allelic segregation by genotyping Castor and Chinchilla populations, and (ii) we evaluated their functional effects on the stability of MLPH transcripts in skin samples of animals with diluted or wild type coat color. Firstly, we showed that the c.585delG variant showed perfect association with the dilution phenotype in contrast to the intronic c.111-5C>A variant. Secondly, we identified three different MLPH isoforms including the wild type isoform, the exon-skipping isoform and a retained intron isoform. Thirdly, we observed a drastic and significant decrease of MLPH transcript levels in rabbits with a coat color dilution (p-values ranging from 10−03 to 10−06). Together, our results bring new insights into the coat color dilution trait.

A Grapevine TTG2-Like WRKY Transcription Factor Is Involved in Regulating Vacuolar Transport and Flavonoid Biosynthesis.

A small set of TTG2-like homolog proteins from different species belonging to the WRKY family of transcription factors were shown to share a similar mechanism of action and to control partially conserved biochemical/developmental processes in their native species. In particular, by activating P-ATPases residing on the tonoplast, PH3 from Petunia hybrida promotes vacuolar acidification in petal epidermal cells whereas TTG2 from Arabidopsis thaliana enables the accumulation of proanthocyanidins in the seed coat. In this work we functionally characterized VvWRKY26 identified as the closest grapevine homolog of PhPH3 and AtTTG2. When constitutively expressed in petunia ph3 mutant, VvWRKY26 can fulfill the PH3 function in the regulation of vacuolar pH and restores the wild type pigmentation phenotype. By a global correlation analysis of gene expression and by transient over-expression in Vitis vinifera, we showed transcriptomic relationships of VvWRKY26 with many genes related to vacuolar acidification and transport in grapevine. Moreover, our results indicate an involvement in flavonoid pathway possibly restricted to the control of proanthocyanidin biosynthesis that is consistent with its expression pattern in grape berry tissues. Overall, the results show that, in addition to regulative mechanisms and biological roles shared with TTG2-like orthologs, VvWRKY26 can play roles in fleshy fruit development that have not been previously reported in studies from dry fruit species. This study paves the way toward the comprehension of the regulatory network controlling vacuolar acidification and flavonoid accumulation mechanisms that contribute to the final berry quality traits in grapevine.

Hydrophobin genes of the entomopathogenic fungus, Metarhizium brunneum, are differentially expressed and corresponding mutants are decreased in virulence

Hydrophobins are small, cysteine-rich, secreted proteins, ubiquitously produced by filamentous fungi that are speculated to function in fungal growth, cell surface properties, and development, although this has been rigorously tested for only a few species. Herein, we report identification of three hydrophobin genes from the entomopathogenic fungus, Metarhizium brunneum, and functional characterization of strains lacking these genes. One gene (HYD1/ssgA) encodes a class I hydrophobin identified previously. Two new genes, HYD3 and HYD2, encode a class I and class II hydrophobin, respectively. To examine function, we deleted all three separately, from the M. brunneum strain KTU-60 genome, using Agrobacterium tumefaciens-mediated transformation. Deletion strains were screened for alterations in developmental phenotypes including growth, sporulation, pigmentation, colony surface properties, and virulence to insects. All deletion strains were reduced in their ability to sporulate and showed alterations in wild-type pigmentation, but all retained wild-type hydrophobicity, except for one individual hyd3 mutant. Complementation with the wild-type HYD3 gene restored hydrophobicity. Each gene, present as a single copy in the genome, showed differential expression patterns dependent on the developmental stage of the fungus. When Spodoptera exigua (beet armyworm) larvae were treated with either conidia or blastospores of each hyd mutant, reductions in virulence and delayed mortality were observed as compared to WT. Together, these results suggest that hydrophobins are differentially expressed and may have distinct, but compensating roles, in conidiation, pigmentation, hydrophobicity, and virulence.

Temporal responses of wild-type pigmentation and RcaE-deficient strains of Fremyella diplosiphon during light transitions.

A temporal study was conducted to evaluate the dynamic complementary chromatic adaptation (CCA) response of two Fremyella diplosiphon strains-wild-type pigmentation strain SF33 and an RcaE-deficient (ΔrcaE) strain, which lacks the photosensor that regulates CCA. SF33 and ΔrcaE cultures were monitored for 15 days after transition of green-light (GL) acclimated cultures to red light (RL) and vice versa. SF33 showed similar growth irrespective of the external light quality; however, a ΔrcaE strain grew slower than SF33 under both RL and GL. Chlorophyll a (chla) content increased in both strains over time and was not much different under RL and GL indicating that chla biosynthesis is not affected significantly by light quality or RcaE function. Phycoerythrin is the sole pigment to absorb GL, whereas several pigments, i.e., allophycocyanin, phycocyanin and chla, function under RL to drive photosynthesis. SF33 compensates for this by synthesizing a higher percentage of PE under GL. The final pigment distribution in the ΔrcaE mutant was found to be more different from SF33 under GL than under RL indicating that RcaE is needed for a transitional response to RL and RL-dependent repression of PE accumulation, yet RcaE is virtually critical for both transitioning to and a full adaptation to GL.

Interactions between zebrafish pigment cells responsible for the generation of Turing patterns.

The reaction-diffusion system is one of the most studied nonlinear mechanisms that generate spatially periodic structures autonomous. On the basis of many mathematical studies using computer simulations, it is assumed that animal skin patterns are the most typical examples of the Turing pattern (stationary periodic pattern produced by the reaction-diffusion system). However, the mechanism underlying pattern formation remains unknown because the molecular or cellular basis of the phenomenon has yet to be identified. In this study, we identified the interaction network between the pigment cells of zebrafish, and showed that this interaction network possesses the properties necessary to form the Turing pattern. When the pigment cells in a restricted region were killed with laser treatment, new pigment cells developed to regenerate the striped pattern. We also found that the development and survival of the cells were influenced by the positioning of the surrounding cells. When melanophores and xanthophores were located at adjacent positions, these cells excluded one another. However, melanophores required a mass of xanthophores distributed in a more distant region for both differentiation and survival. Interestingly, the local effect of these cells is opposite to that of their effects long range. This relationship satisfies the necessary conditions required for stable pattern formation in the reaction-diffusion model. Simulation calculations for the deduced network generated wild-type pigment patterns as well as other mutant patterns. Our findings here allow further investigation of Turing pattern formation within the context of cell biology.

Association of dsRNA to Down-Regulation of Perithecial Synthesis in Monosporascus cannonballus

Monosporascus cannonballus is a soilborne fungal pathogen that causes vine-decline of muskmelon which re- sults in reduced yield in many melon growing areas around the world. M. cannonballus isolates were collected from dif- ferent provinces in Spain and experiments conducted to determine pigmentation, perithecial formation, and the presence of cellular dsRNA. Thirty-one isolates were grouped based on dsRNA fragment sizes using cluster analysis and Euclidean distances. Three distinct dsRNA groupings were observed. Group 2 isolates containing 2, 3, and 3.5 kb dsRNA appeared to exhibit a decrease in perithecia production compared to the other groups. Group 1 isolates exhibited yellow pigmenta- tion only, while Group 3 isolates expressed grey (wild-type) and yellow (degenerate) pigmentation. Isolates that did not contain dsRNA (Group 4) exhibited wild-type pigmentation. Down-regulation and variation in phenotype are hypothe- sized to be due to dsRNA/iRNA interactions with the fungal transcription mechanisms. Future research will focus on elu- cidating the mechanisms of regulation by the dsRNA of M. cannonballus.

The Green-absorbing Drosophila Rh6 Visual Pigment Contains a Blue-shifting Amino Acid Substitution That Is Conserved in Vertebrates

The molecular mechanisms that regulate invertebrate visual pigment absorption are poorly understood. Through sequence analysis and functional investigation of vertebrate visual pigments, numerous amino acid substitutions important for this adaptive process have been identified. Here we describe a serine/alanine (S/A) substitution in long wavelength-absorbing Drosophila visual pigments that occurs at a site corresponding to Ala-292 in bovine rhodopsin. This S/A substitution accounts for a 10-17-nm absorption shift in visual pigments of this class. Additionally, we demonstrate that substitution of a cysteine at the same site, as occurs in the blue-absorbing Rh5 pigment, accounts for a 4-nm shift. Substitutions at this site are the first spectrally significant amino acid changes to be identified for invertebrate pigments sensitive to visible light and are the first evidence of a conserved tuning mechanism in vertebrate and invertebrate pigments of this class.

Characterization and zeste reaction of spontaneously reduced transposable elements in Drosophila melanogaster

Seven transposable elements (TE), selected from parental stock w 239 Cy, have been characterized. They all carry w+ but have lost both rst+ and the two polytene chromosome bands of a complete TE and are thus cytologically invisible. In situ hybridization with probes from the white-roughest interval shows that the reduced TE's are truncated in the same region, 40-90 kb upstream of the white locus, well proximal to the zeste interaction site. They express the white gene in wild type fashion in all tissues studied (eyes, testis sheaths, and Malpighian tubules) but give zeste reactions varying from yellow to bright red. Thus, by the criterion of the zeste reaction, 40 kb of TE sequences 5' to the transcription start site cannot insulate the white gene from position effects. One single spontaneously reduced TE (TE203) originated from parental stock w TE100 ct. This TE is truncated closer to the white locus (+ 18- +26 kb), but still well separated from the zeste interaction site. In a z1w11E4 background, homozygous TE203 flies have an uneven eye pigmentation, while heterozygotes have eyes that are yellowish red. The Malpighian tubules and testes show wild type pigmentation in both cases, like normal TE's.

Epigenetic Inactivation of Chalcone Synthase-A Transgene Transcription in Petunia Leads to a Reversion of the Post-Transcriptional Gene Silencing Phenotype

Petunia plants that exhibit a white-flowering phenotype as a consequence of chalcone synthase transgene-induced silencing occasionally give rise to revertant branches that produce flowers with wild-type pigmentation. Transcription run-on assays confirmed that the production of white flowers is caused by post-transcriptional gene silencing (PTGS), and indicated that transgene transcription is repressed in the revertant plants, providing evidence that induction of PTGS depends on the transcription rate. Transcriptional repression of the transgene was associated with cytosine methylation at CpG, CpNpG and CpNpN sites, and the expression was restored by treatment with either 5-azacytidine or trichostatin A. These results demonstrate that epigenetic changes occurred in the PTGS line, and these changes interfere with the initiation of transgene transcription, leading to a reversion of the PTGS phenotype.

Inheritance of a Dominant Spotted Melanic Mutation in the Livebearing Fish Phalloceros caudimaculatus var. reticulata from "Banados del Este" Reserve of Biosphere Site in Uruguay

An alternative chromatic variation to the wild-type phenotype of the livebearing fish Phalloceros caudimaculatus (Phalloceros caudimaculatus var. reticulata) was detected in a single female from “Banados del Este” Reserve of Biosphere Site, in Uruguay. The founder mutant female, which presented a melanic spotted pattern overlapping the wild-type pigmentation, produced laboratory breed strains. Four phenotypes showing different degrees of spotted patterns were obtained. In order to determine the inheritance of this chromatic mutation, experimental crosses and cytogenetic analyses of these laboratory strains were carried out. The breeding experiments among the spotted phenotypic classes and backcrosses suggest the dominant and non-sex linked inheritance of this mutation. Statistical tests demonstrated that the inheritance mechanism of this mutation does not correspond to a 2-gene independent segregation hypothesis as it was proposed in Poecilia sphenops, or to a single dominant gene hypothesis. Additionally, cytogenetic analyses detected a partially heterochromatic biarmed chromosome associated to the inheritance of the pigmentation pattern in laboratory breed strains.

Reversion mutation of ib oculocutaneous albinism to wild‐type pigmentation in medaka fish

We have previously identified three naturally occurring mutations in the medaka fish tyrosinase gene caused by transposable element insertions. Tyr-i(b) is one of these, containing the Tol2 element in the promoter region. Its homozygous carriers exhibit a weak oculocutaneous albino phenotype. We report here spontaneous reversion of the albino phenotype to the wild-type pigmentation, associated with excision of the Tol2 element. The newly arising mutant gene is inherited in the Mendelian fashion. Thus, oculocutaneous albinism is not strictly irreversible, at least in this organism and the results also indicate that the insertion of the Tol2 element is the main, and possibly the only, cause of the i(b) albinism. Importantly our data also suggest that medaka fish possess an active transposase.

Polymorphisms within the canine MLPH gene are associated with dilute coat color in dogs

BackgroundPinschers and other dogs with coat color dilution show a characteristic pigmentation phenotype. The fur colors are a lighter shade, e.g. silvery grey (blue) instead of black and a sandy color (Isabella fawn) instead of red or brown. In some dogs the coat color dilution is sometimes accompanied by hair loss and recurrent skin inflammation, the so called color dilution alopecia (CDA) or black hair follicular dysplasia (BHFD). In humans and mice a comparable pigmentation phenotype without any documented hair loss is caused by mutations within the melanophilin gene (MLPH).ResultsWe sequenced the canine MLPH gene and performed a mutation analysis of the MLPH exons in 6 Doberman Pinschers and 5 German Pinschers. A total of 48 sequence variations was identified within and between the breeds. Three families of dogs showed co-segregation for at least one polymorphism in an MLPH exon and the dilute phenotype. No single polymorphism was identified in the coding sequences or at splice sites that is likely to be causative for the dilute phenotype of all dogs examined. In 18 German Pinschers a mutation in exon 7 (R199H) was consistently associated with the dilute phenotype. However, as this mutation was present in homozygous state in four dogs of other breeds with wildtype pigmentation, it seems unlikely that this mutation is truly causative for coat color dilution. In Doberman Pinschers as well as in Large Munsterlanders with BHFD, a set of single nucleotide polymorphisms (SNPs) around exon 2 was identified that show a highly significant association to the dilute phenotype.ConclusionThis study provides evidence that coat color dilution is caused by one or more mutations within or near the MLPH gene in several dog breeds. The data on polymorphisms that are strongly associated with the dilute phenotype will allow the genetic testing of Pinschers to facilitate the breeding of dogs with defined coat colors and to select against Large Munsterlanders carrying BHFD.

A Dark and Constitutively Active Mutant of the Tiger Salamander UV Pigment

A triple mutant (F86L/T93P/S118T; bovine rhodopsin numbering) of the tiger salamander UV cone pigment appears to be trapped in an open conformation that is metarhodopsin-II-like. The pigment is able to activate transducin in the dark, and the ligand-free apoprotein is also able to activate transducin constitutively. The pigment permits protons and chloride ions from solution access to the active site as it displays a pH- and NaCl-dependent absorption spectrum not observed with the wild-type pigment. However, the wild-type properties of light-dependent activity and a pH-independent absorption spectrum are recovered upon reconstitution of the triple mutant with 11-cis-9-demethyl retinal. These results suggest that binding the native chromophore cannot deactivate the protein because of steric interactions between the protein, possibly residue 118, and the 9-methyl group of the chromophore. Furthermore, the absorption spectrum of the 9-demethyl retinal regenerated pigment exhibits a band broader and with lower extinction at the absorption maximum than either the human blue or salamander UV wild-type pigments generated with the same retinal analogue. The broad spectrum appears to be comprised of two or more species and can be well-fit by a sum of scaled spectra of the two wild-type pigments. Binding the chromophore appears to trap the pigment in two or more conformations. The triple mutant reported here represents the first example of a dark-active cone pigment and constitutively active cone opsin.

Structural changes in the photoactive site of proteorhodopsin during the primary photoreaction.

Proteorhodopsin (PR), found in marine gamma-proteobacteria, is a newly discovered light-driven proton pump similar to bacteriorhodopsin (BR). Because of the widespread distribution of proteobacteria in the worldwide oceanic waters, this pigment may contribute significantly to the global solar energy input in the biosphere. We examined structural changes that occur during the primary photoreaction (PR --> K) of wild-type pigment and two mutants using low-temperature FTIR difference spectroscopy. Several vibrations detected in the 3500-3700 cm(-1) region are assigned on the basis of H(2)O --> H(2)(18)O exchange to the perturbation of one or more internal water molecules. Substitution of the negatively charged Schiff base counterion, Asp97, with the neutral asparagine caused a downshift of the ethylenic (C=C) and Schiff base (C=N) stretching modes, in agreement with the 27 nm red shift of the visible lambda(max). However, this replacement did not alter the normal all-trans to 13-cis isomerization of the chromophore or the environment of the detected water molecule(s). In contrast, substitution of Asn230, which is in a position to interact with the Schiff base, with Ala induces a 5 nm red shift of the visible lambda(max) and alters the PR chromophore structure, its isomerization to K, and the environment of the detected internal water molecules. The combination of FTIR and site-directed mutagenesis establishes that both Asp97 and Asn230 are perturbed during the primary phototransition. The environment of Asn230 is further altered during the thermal decay of K. These results suggest that significant differences exist in the conformational changes which occur in the photoactive sites of proteorhodopsin and bacteriorhodopsin during the primary photoreaction.

Cloning and Characterization of a New Polyketide Synthase Gene Cluster inStreptomyces aureofaciensCCM 3239

We cloned a new polyketide gene cluster, aur2, in Streptomyces aureofaciens CCM3239. Sequence analysis of the 9531-bp DNA fragment revealed 10 open reading frames, majority of which showed high similarity to the previously characterized type II polyketide synthase (PKS) genes. An unusual feature of the aur2 cluster is a disconnected organization of minimal PKS genes; ACP is located apart from the genes for ketosynthases KSalpha and KSbeta. The aur2 gene cluster was disrupted in S. aureofaciens CCM3239 by a homologous recombination, replacing the four genes (aur2A, E, F, G) including ketosynthase KSalpha, with antibiotic resistance marker gene. The disruption did not affect growth and differentiation, and disrupted strain produced spores with wild-type grey-pink pigmentation. The biochromatographic analysis of the culture extracts from S. aureofaciens wild type and aur2-disrupted strains did not reveal any difference in the pattern of antibacterial compounds.

Zebrafish puma mutant decouples pigment pattern and somatic metamorphosis

The genetic and developmental bases for trait expression and variation in adults are largely unknown. One system in which genes and cell behaviors underlying adult traits can be elucidated is the larval-to-adult transformation of zebrafish, Danio rerio. Metamorphosis in this and many other teleost fishes resembles amphibian metamorphosis, as a variety of larval traits (e.g., fins, skin, digestive tract, sensory systems) are remodeled in a coordinated manner to generate the adult form. Among these traits is the pigment pattern, which comprises several neural crest-derived pigment cell classes, including black melanophores, yellow xanthophores, and iridescent iridophores. D. rerio embryos and early larvae exhibit a relatively simple pattern of melanophore stripes, but this pattern is transformed during metamorphosis into the more complex pattern of the adult, consisting of alternating dark (melanophore, iridophore) and light (xanthophore, iridophore) horizontal stripes. While it is clear that some pigment cells differentiate de novo during pigment pattern metamorphosis, the extent to which larval and adult pigment patterns are developmentally independent has not been known. In this study, we show that a subset of embryonic/early larval melanophores persists into adult stages in wild-type fish; thus, larval and adult pigment patterns are not completely independent in this species. We also analyze puma mutant zebrafish, derived from a forward genetic screen to isolate mutations affecting postembryonic development. In puma mutants, a wild-type embryonic/early larval pigment pattern forms, but supernumerary early larval melanophores persist in ectopic locations through juvenile and adult stages. We then show that, although puma mutants undergo a somatic metamorphosis at the same time as wild-type fish, metamorphic melanophores that normally appear during these stages are absent. The puma mutation thus decouples metamorphosis of the pigment pattern from the metamorphosis of many other traits. Nevertheless, puma mutants ultimately recover large numbers of melanophores and exhibit extensive pattern regulation during juvenile development, when the wild-type pigment pattern already would be completed. Finally, we demonstrate that the puma mutant is both temperature-sensitive and growth-sensitive: extremely severe pigment pattern defects result at a high temperature, a high growth rate, or both; whereas a wild-type pigment pattern can be rescued at a low temperature and a low growth rate. Taken together, these results provide new insights into zebrafish pigment pattern metamorphosis and the capacity for pattern regulation when normal patterning mechanisms go awry.

Regulatory mechanism for the stability of the meta II intermediate of pinopsin.

Pinopsin is a chicken pineal photoreceptive molecule with a possible role in photoentrainment of the circadian clock. Sequence comparison among members of the rhodopsin family has suggested that pinopsin might have properties more similar to cone visual pigments than to rhodopsin, but the lifetime of the physiologically active intermediate (meta II) of pinopsin is rather similar to that of metarhodopsin II, which is far more stable than meta II intermediates of cone visual pigments [Nakamura, A. et al., (1999) Biochemistry 38, 14738-14745]. In the present study, we investigated the amino acid residue(s) contributing to this unique property of pinopsin by using site-directed mutagenesis to pinopsin-specific structural features, (i) Ser171, (ii) Asn184, and (iii) the second extracellular loop two-amino acids shorter than that of cone visual pigments. The meta II stability of the 171/184 double mutant of pinopsin (S171R/N184D) is almost the same as that of wild-type pinopsin. In contrast, the meta II lifetime is markedly shortened (one third) by introduction of the third mutation (replacement of a six-amino acid stretch, 188-193, by the corresponding eight residues of chicken green-sensitive cone pigment) to the 171/184 double mutant of pinopsin. Consistently, meta II of the green-sensitive pigment mutant, in which the eight-amino acid stretch is inversely replaced by the corresponding six residues of pinopsin, is more stable than meta II of the wild-type pigment. These results strongly suggest that the specific sequence and/or the number of residues at amino acids 188-193 in pinopsin play an important role in the stabilization of the meta II intermediate.

Mosaic: a position-effect variegation eye-color mutant in the mosquito Anopheles gambiae.

The Mosaic (Mos) mutation, isolated in the F1 of 60Co-irradiated mosquitoes, confers variegated eye color to third and fourth instar larvae, pupae, and adults of the mosquito Anopheles gambiae. Mos is recessive in wild pink eye (p+) individuals, but is dominant and confers areas of wild-type pigment in mutant pink eye backgrounds. Mos is located 14.4 cM from pink eye on the X chromosome and is associated with a duplication of division 2B euchromatin that has been inserted into division 6 heterochromatin. Various combinations of Mos, pink eye alleles, and the autosomal mutation red eye were produced. In all cases, the darker pigmented regions of the eye in Mos individuals show the phenotypic interactions expected if the phenotype of those regions is due to expression of a p+ allele. Expression of Mos is suppressed by rearing larvae at 32 degrees C relative to 22 degrees C. All of these characteristics are consistent with Mos being a duplicated wild copy of the pink eye gene undergoing position-effect variegation.

21] Phylogenetic analysis and experimental approaches to study color vision in vertebrates

To elucidate the molecular mechanisms of vertebrate color vision, it is essential to establish associations between amino acid substitutions and the directions of lambda max shifts of visual pigments. In this way, we can identify critical amino acid changes that may be responsible for lambda max shifts of visual pigments. In this process, we may consider only highly conserved residues, simply because the evolutionary conservation often implies functional importance. Using such an "evolutionary model" as a convenient tool in designing mutagenesis experiments, we can test specific hypotheses on the molecular mechanisms that are responsible for color vision in vertebrates. Virtually any vertebrate opsin cDNA can be expressed in COS cells, reconstituted with 11-cis-retinal, and the lambda max values of the regenerated pigments can be measured rather easily. By constructing mutant pigments with desired amino acid changes and conducting the in vitro assay and comparing their lambda max values with those of corresponding wild-type pigments, we can elucidate the molecular mechanisms of lambda max shifts--and color vision--of vertebrates rigorously.