Red Pigment Genes Research Articles

Unexpected conservation of the X-linked color vision gene in nocturnal prosimians: evidence from two bush babies.

Bush babies have had a long history of nocturnal life and it would be interesting to know whether their color vision genes have become degenerate. Therefore, we used PCR techniques to sequence the X-linked pigment gene of two of these nocturnal prosimians: Galago senegalensis and Otolemur garnettii. Southern hybridization of genomic DNA of G. senegalensis showed a single X-linked pigment gene. Interestingly, the deduced pigment sequences of the two bush babies are identical. By comparing the X-linked pigments of bush baby, human, squirrel monkey, and marmoset, 38 variable positions were identified. At those positions that may cause a spectral shift, the bush baby pigment has identical or biochemically similar residues to those of the marmoset cone pigment with a spectral peak of 543 nm. This result is consistent with the estimate of 544-545 nm for the spectral peak of the X-linked pigment of Otolemur crassicaudatus, which is closely related to Otolemur garnettii. The neighbor-joining tree of mammalian X-linked pigments showed a significantly shorter branch in the bush baby lineage than in other primate lineages. A relative rate test showed that the nonsynonymous substitution rate of the bush baby X-linked pigment gene is about three times slower than that of the human red pigment gene, though the synonymous substitution rates of the two genes are similar. The slower nonsynonymous rate in the bush baby lineage suggests that the bush baby X-linked pigment gene is under functional constraints, in spite of its nocturnal life. Two radical changes at positions in the intradiskal surface next to the sixth transmembrane domain were observed in the X-linked cone pigment of bush babies but not in other primates. They are changes from Ala to Ser and from Asn to His, which are similar in function to the corresponding residues in rhodopsins. These two changes may be of importance for dim light sensitivity, which is consistent with our proposal that the evolution of the bush baby X-linked pigment gene is under selective pressure. In addition, the 2.5% divergence in introns 2 and 5 of the X-linked pigment gene between the two bush babies supports their classification into two separate genera.

Cloning and expression of the red visual pigment gene of goat ( Capra hircus)

We have cloned and sequenced the red opsin gene, r Ch , of goat ( Capra hircus). When r Ch is expressed in cultured cells and reconstituted with 11- cis retinal, the resulting visual pigment has a wavelength of maximal absorption ( λmax) of 553 nm. This result and the deduced aa sequence of the goat red opsin suggest that the λmax values of the red pigments of goat and human are based mainly on Y277 and T285. The slightly lower λmax value of the red pigment in goat than in human (~560 nm) can be explained by a single aa difference between the goat (A180) and human (S180) red pigments.

Identification of a Gene within the Tandem Array of Red and Green Color Pigment Genes

The tandem array of color pigment genes on chromosome Xq28 contains nested exons of a distinct gene. This gene (termed TEX28) is composed of five exons that span almost the entire distance between the protein-coding regions of the color pigment genes and a transketolase-related gene. Although most of the TEX28 gene is repeated within the color pigment gene array, the exclusion of exon 1 from the array is predicted to restrict transcription to a single copy of the gene. The TEX28 gene encodes a polypeptide of 410 amino acid residues. This polypeptide does not display significant homology with any known proteins in public databases. Transcripts of the gene (1.8 kb) were detected in testes, but not in any other tissue examined. Color vision disorders that result from the deletion of color pigment genes should be reappraised for associated phenotypes that may derive from disruption of the TEX28 gene.

Visual pigment gene structure and expression in human retinae.

We determined the genotypes of the X-chromosome-linked red/green color vision genes by a novel PCR/SSCP-based method and assessed expression by mRNA analysis in retinae of 51 unselected post mortem eye specimens from Caucasian males of unknown color vision status. All individuals had a single red (long-wave) pigment gene and one or more (an average of two) green (middle-wave) pigment genes. Four males had 5'green-red3' hybrid genes in addition to normal red and green pigment genes. These findings are consistent with earlier studies on human visual pigment gene structure using Southern blotting and with a recent study using pulsed-field electrophoresis. We interpret claims of much larger numbers of red, green and green-red hybrid genes to be technical artifacts. The ratio of expressed red to green pigment retinal mRNA varied widely (1-10 with a mode of 4) and was not correlated with that of red to green pigment genes. In one individual with a green-red hybrid gene in addition to normal red and green pigment genes, the normal red pigment gene and the hybrid gene were both expressed, but the normal green gene was not. This person presumably had deuteranomalous color vision. Two with green-red hybrid genes expressed the normal red and green pigment genes, but not the hybrid genes. These two individuals presumably had normal color vision. We interpret the failure to express their green-red hybrid genes to be caused by their location at a more distal position in the visual pigment gene array.

Sequence and evolutionary history of the length polymorphism in intron 1 of the human red photopigment gene.

The length of intron 1 of the red pigment gene is dimorphic among Africans but not among Caucasians or Asians. This dimorphism was found to result from the presence or absence of a block of 1,284 bp comprised of three Alu elements and 328 bp of intervening unique-sequence DNA. This additional sequence in the "long" intron 1 of the red pigment gene was shown to have inserted into a fourth and older Alu element present in the "short" from of intron 1. Furthermore, the size and sequence of the "short" intron 1 of the red pigment gene is equivalent to that of the adjacent green pigment gene. The block containing the three Alu elements was not found in intron 1 of the red or green pigment genes of Old World monkeys and orangutans but was present in intron 1 of both the green and red pigment genes of gorillas and chimpanzees. The nucleotide sequence of this block in Old World primates and the estimated ages of the three elements suggest that their insertion occurred sequentially in the Old World monkey lineage prior to duplication of the ancestral X-chromosome-linked pigment gene. After gene duplication, deletion of the entire block containing the three Alu elements from one of the genes created the "short" intron variant. Unequal recombination between the adjacent and highly homologous red and green pigment genes may have resulted in the formation of the "short" intron variant of the other gene.

Gene Conversion between Red and Defective Green Opsin Gene in Blue Cone Monochromacy

Blue cone monochromacy is an X-linked condition in which the function of both the red pigment gene (RCP) and the green pigment gene (GCP) is impaired. Blue cone monochromacy can be due to a red/green gene array rearrangement existing of a single red/green hybrid gene and an inactivating C203R point mutation in GCP. We describe here a family with blue cone monochromacy due to the presence of the C203R mutation in both RCP and GCP. The flanking sequences of the C203R mutation in exon 4 of RCP were characteristic for GCP, indicating that this mutation was transferred from GCP into RCP by gene conversion.

Molecular patterns and sequence polymorphisms in the red and green visual pigment genes of Japanese men.

The red-green pigment gene arrays of 203 (101 from a previous study and 102 from this study) randomly selected men of Japanese ancestry from the Seattle area were screened for the abnormal molecular patterns (deletions and red/green or green/red hybrid genes) that are usually associated with defective color vision. Such molecular patterns were found in approximately 5% of these individuals, which is equivalent to the frequency of phenotypic color vision defects in Japanese males in Japan. Thus, the majority of hybrid genes carried by Japanese males appear to be associated with defective color vision. In contrast, the frequency of hybrid genes among Caucasians and African-Americans is approximately two and five times the frequency of color vision defects in these two ethnic groups, respectively. The coding sequences of 50 males of Japanese ancestry were determined. All the polymorphisms in the red and green pigment genes that were detected in the Japanese sample had been observed in Caucasians and African-Americans. The same polymorphisms of the red pigment gene were present in the green pigment gene, suggesting that gene conversion contributes to sequence homogenization between these pigment genes. As is the case for Caucasians, exon 3 of the red and green pigment genes was observed to be a hot spot for recombination and gene conversion. Fewer polymorphic sites (4 vs 11) and haplotypes (5 vs 14) of the red pigment gene were observed in Japanese than in Caucasians. The Japanese population was more uniform with respect to the red pigment gene, with 70% of individuals having the same haplotype, as compared with the 43% for the Caucasian population. This difference was largely due to the lower degree of polymorphism at position 180 of the red pigment gene in Japanese (84% Ser and 16% Ala vs 62% Ser and 38% Ala.) The number of polymorphic sites and haplotypes in the green pigment gene was similar in the two populations. Nevertheless, the Japanese population was more uniform with 65% having the same haplotype. The difference in the frequency of alleles at position 283 accounted for this difference in haplotype distribution.

In the eye of the beholder: Visual pigments and inherited variation in human vision

Jeremy Nathans Howard Hughes Medical Institute Departments of Molecular Biology and Genetics, Neuroscience, and Ophthalmology Johns Hopkins University School of Medicine Baltimore, Maryland 21205 Each of us inhabits an internally consistent visual world. In everyday life we take the constancy of this world for granted and assume that others share it. Although this assumption is roughly valid when averaged over the popu- lation, psychophysical experiments over the past two cen- turies have revealed significant differences among individ- ual humans in many aspects of visual function. One source of these differences has recently been identified at a mo- lecular level: sequence variation in the genes encoding the visual pigments, the light-absorbing proteins in the rods and cones that initiate phototransduction. In humans there are four visual pigments. Each visual pigment consists of an integral membrane apoprotein bound to a chromophore, 1 l-cis retinal. Rhodopsin, the rod pigment, mediates vision in dim light. The three cone pigments, referred to as blue, green, and red, mediate color vision and function only in bright light. The absorption spectra of the four human visual pigments are shown in Figure 1. Inherited Variation in Co/or Vision The most common variation in human color vision arises from a serinelalanine polymorphism at position 180 in the red pigment gene. The absorption spectrum of the red(Ser- 180) allele is red-shifted 4-5 nm relative to the red(Ala-180) allele (Merbs and Nathans, 1992a; Asenjo et al., 1994). Males who carry the red(Ser-180) allele have a corre- spondingly greater sensitivity to long wavelength lights than do those who carry the red(Ala-180) allele (Wind- erickx et al., 1992b). In the Caucasian gene pool, the red(Ser-180) and red(Ala-180) alleles are found at frequen- cies of 82% and 38%, respectively. The common variations in red-green color vision that are colloquially referred to as color blindness arise from loss of either the red or the green cone pigment (dichro- macy) or from the production of a visual pigment with a shifted absorption spectrum (anomalous trichromacy). The high frequencies of these variations in males-approxi- mately 2% for dichromacy and, depending on the popula- tion, between 2% and 8% for anomalous trichromacy- reflect the X chromosome location of the red and green pigment genes, a relaxation of selective pressure for nor- mal trichromacy, and an unusually facile mutational mech- anism. The red and green pigment genes are organized in a head-to-tail tandem array in which the transcription units and intergenic spacer DNA are 98% identical at the nucleotide level, an arrangement that predisposes them to homologous but unequal recombination (reviewed by Nathans et al., 1992). Unequal intergenic recombination generates variant arrays in which entire repeat units are

Sequence divergence of the red and green visual pigments in great apes and humans.

We have determined the coding sequences of red and green visual pigment genes of the chimpanzee, gorilla, and orangutan. The deduced amino acid sequences of these pigments are highly homologous to the equivalent human pigments. None of the amino acid differences occurred at sites that were previously shown to influence pigment absorption characteristics. Therefore, we predict the spectra of red and green pigments of the apes to have wavelengths of maximum absorption that differ by < 2 nm from the equivalent human pigments and that color vision in these nonhuman primates will be very similar, if not identical, to that in humans. A total of 14 within-species polymorphisms (6 involving silent substitutions) were observed in the coding sequences of the red and green pigment genes of the great apes. Remarkably, the polymorphisms at 6 of these sites had been observed in human populations, suggesting that they predated the evolution of higher primates. Alleles at polymorphic sites were often shared between the red and green pigment genes. The average synonymous rate of divergence of red from green sequences was approximately 1/10th that estimated for other proteins of higher primates, indicating the involvement of gene conversion in generating these polymorphisms. The high degree of homology and juxtaposition of these two genes on the X chromosome has promoted unequal recombination and/or gene conversion that led to sequence homogenization. However, natural selection operated to maintain the degree of separation in peak absorbance between the red and green pigments that resulted in optimal chromatic discrimination. This represents a unique case of molecular coevolution between two homologous genes that functionally interact at the behavioral level.

Stability of YACs containing ribosomal or RCP/GCP locus DNA in wild-type S. cerevisiae and RAD mutant strains.

About 2% of human YAC clones, including tandemly repeated segments color vision pigment DNA, ribosomal DNA and alphoid DNA have been reported to be inherently unstable in yeast hosts, producing more stable deletion products. YACs containing color vision red pigment gene DNA or 1.5 rDNA tandem repeat units were transformed into hosts bearing lesions at the RAD1, RAD6, RAD51, or RAD52 loci. YACs susceptible to deletion during outgrowth of wild-type cells (or in preliminary experiments, in RAD6 transformants) were stable for up to 100 generations or more in the other strains. Thus both the RAD1 and RAD51/RAD52 epistatic pathways are apparently involved in the instability of YACs containing tandem repeat loci, presumably during recombination-based deletion formation; and a yeast host disarmed in these pathways will likely maintain YACs intact that are otherwise unstable.

Molecular characterization of a blue visual pigment gene in the fish Astyanax fasciatus

We report here the isolation and sequence determination of a gene closely linked to the Astyanax red visual pigment gene. Reverse transcription polymerase chain reaction assays show that this new gene ( B23 Aƒ ) and the previously characterized red and green visual pigment genes of Astyanax are all expressed in the eye. Phylogenetic analysis shows that B23 Aƒ belongs to the group consisting of short wavelength-sensitive pigment genes from different species and is most closely related to the goldfish blue visual pigment gene.

Molecular characterization of the red visual pigment gene of the American chameleon ( Anolis carolinensis)

The red sensitive visual pigment of the American chameleon, Anolis carolinensis, is unique in having absorption maximum some 50 nm further into the red than any other terrestrial vertebrate examined. We report here the isolation and sequence determination of the genomic DNA clone for the Anolis red visual pigment gene. Phylogenetic analysis shows that this gene is most closely related to the gecko green and chicken red visual pigment genes. We identified nine Anolis-specific amino acid replacements, seven of which reside in transmembrane domains and might contribute to the red-shift of the Anolis visual pigments.

BglII RFLP in DXS 498 between the pigment gene repeat unit, RCP and GCP.

The red (RCP) and green (GCP) color pigment genes are located in Xq28, a chromosomal region implicated in many genetic disorders. The restriction fragment length polymorphism (RFLP) we describe here will be useful for linkage analysis in these disorders.

Absorption spectra of the hybrid pigments responsible for anomalous color vision.

Unequal homologous recombination events between green and red cone pigment genes produce the red-green or green-red hybrid pigment genes found in many individuals with variant color vision. Photobleaching difference absorption spectroscopy of hybrid pigments produced in cultured cells shows that the spectral sensitivity of each hybrid pigment is intermediate between the parental green and red pigment sensitivities. Amino acids encoded by exons 2, 3, 4, and 5 produce spectral shifts at the wavelength of maximal absorbance of 0 to 4, 0 to 4, 3 to 4, and 15 to 21 nanometers, respectively, the exact value depending on the identities of amino acids elsewhere in the hybrid.

Selective expression of human X chromosome-linked green opsin genes.

The human red and green photopigments are specified by genes on the long arm of the X chromosome (Xq28). In individuals with normal color vision, the locus was proposed to consist of a single red pigment gene upstream of one or more copies of green pigment genes. The presence of a single red pigment gene in the array was confirmed by demonstration of only one retinal mRNA transcript coding for the red opsin. In individuals with multiple green pigment genes, it is unknown whether all genes are expressed. We analyzed the sequence of red- and green-specific mRNA from retinas of individuals with multiple green pigment genes in comparison with the corresponding genomic DNA sequences. The data showed that only a single green pigment gene is expressed. We therefore suggest that a locus control-like element, already known to be located 3.8 kilobases upstream of the transcription initiation site of the red pigment gene, allows transcription of only a single copy of the green pigment genes, probably the most proximal copy. This finding provides an explanation for the not-infrequent presence of 5' green-red hybrid genes in individuals with normal color vision. Such hybrid genes are usually associated with defective color vision. We suggest that 5' green-red hybrid genes produce defective color vision only when their position in the gene array allows expression in the retinal cone cells.

A locus control region adjacent to the human red and green visual pigment genes

Deletion of sequences 5′ of the human red and green pigment gene array results in blue cone monochromacy, a disorder in which both red and green cone function are absent. To test whether these sequences are required for transcription of the adjacent visual pigment genes in cone photoreceptors, we produced transgenic mice carrying sequences upstream of the red and green pigment genes fused to a β-galactosidase reporter. The patterns of transgene expression indicate that the human sequences direct expression to both long and short wave-sensitive cones in the mouse retina and that a region between 3.1 kb and 3.7 kb 5′ of the red pigment gene transcription initiation site is essential for expression. Sequences within this region are highly conserved among humans, mice, and cattle, even though the latter two species have only a single visual pigment gene at this locus. These experiments suggest a model in which an interaction between the conserved 5′ region and either the red or the green pigment gene promoter determines which of the two genes a given cone expresses.

Linkage of emery-dreifuss muscular dystrophy to the red/green cone pigment (RGCP) genes, proximal to factor VIII

Further DNA linkage studies on two previously described X-linked recessive Emery-Dreifuss muscular dystrophy (EMD) families are reported, which refine the localization of the gene responsible for EMD. Two recombination events indicate that the most likely localization for the EMD gene lies in the interval between DXS15/DXS52 and F8C. A maximum LOD score of 3.44 at θ = 0 is obtained for EMD vs the red and green cone pigment genes (RCP and GCP). Our data provide additional support for one of the two proposed orientations of genes and markers distal to DXS15/DXS52, with respect to the telomere. Given this favoured orientation, our data best fit a localization of EMD to within a 2 megabase (Mb) interval between DXS15/DXS52 and F8C.

Convergent evolution of the red- and green-like visual pigment genes in fish, Astyanax fasciatus, and human.

We have isolated and sequenced genes from the blind cave fish, Astyanax fasciatus, that are homologous to the human red and green visual pigment genes. The data strongly suggest that, like human, these fish have one red-like visual pigment gene and multiple green-like visual pigment genes. By comparing the DNA sequences of the human and fish visual pigment genes and knowing their phylogenetic relationship, one can infer the direction of amino acid substitutions in the red and green visual pigments. The results indicate that the red pigments in human and fish evolved from the green pigment independently by identical amino acid substitutions in only a few key positions.

Molecular genetics of X chromosome-linked color vision among populations of African and Japanese ancestry: high frequency of a shortened red pigment gene among Afro-Americans.

Red-green color vision in humans is mediated by the X chromosome-linked highly homologous red and green pigment genes. Color vision defects are caused by deletions and fusions involving these genes. However, we found the frequency of molecular abnormalities among Caucasians to be twice as high as that of phenotypic color vision defects. Among Japanese the frequency of phenotypic and molecular color vision defects was similar (approximately 5%). Among Afro-Americans, molecular defects (largely green-red fusion genes) were at least five times more frequent (21%) than phenotypic color vision defects (approximately 4%). In addition, 35% of Afro-Americans, 2% of Japanese, and less than 1% of Caucasians had a shortened red pigment gene not associated with phenotypic color vision defects. This gene lacked 1.9 kilobases in its first intron and had the identical size as the green pigment gene from which it presumably originated by gene conversion in an ancestral African population. This gene and the closely linked glucose-6-phosphate dehydrogenase A+ variant were in linkage equilibrium. A model for the evolutionary origin of the color vision pigment genes in higher primates is portrayed.

A 195-kb cosmid walk encompassing the human Xq28 color vision pigment genes

By using cosmid walking, we have cloned a 195-kb region from chromosome band Xq28 that encompasses the red and green color pigment genes and 85 kb of flanking sequences. This has allowed us to confirm that the color pigment genes are within very homologous units arranged in tandem array. Each unit contains two BssHII sites and one NruI site that are frequently methylated in male leukocyte DNA. A NotI and an EagI site are present 6 kb upstream from the red pigment gene promoter; the NotI site was shown to be unmethylated in the active X chromosome in leukocytes and may represent a CpG island for the whole cluster. We have identified another CpG island, 61 kb 3′ from the last green pigment gene, that is unmethylated in leukocytes on the active X chromosome, but methylated on the inactive X. This island is flanked by sequences conserved in evolution and may thus correspond to an expressed gene. We also describe an informative three-allele restriction fragment length polymorphism within the pigment gene cluster.