Visual Pigment Genes Research Articles

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.

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.

Molecular evolution of the rhodopsin gene of marine lamprey, Petromyzon marinus

Visual pigment genes have been isolated from a marine lamprey, Petromyzon marinus. We report here the rhodopsin gene, spanning 21.2 kb from start to stop codons, making it the longest opsin gene known in vertebrates. Southern analysis suggests that the lamprey genome contains a single rhodopsin gene. The amino acid (aa) sequence deduced from this gene has 92% sequence similarity with that of the river lamprey rhodopsin. The data reveal that aa substitutions occurred more often in the transmembrane region than in the non-transmembrane region, possibly reflecting functional adaptation of the rhodopsin during the last 500 million years of the jawless fish evolution.

Visual pigment gene structure and the severity of color vision defects.

Rearrangements of the visual pigment genes are associated with defective color vision and with differences between types of red-green color blindness. Among individuals within the most common category of defective color vision, deuteranomaly, there is a large variation in the severity of color vision loss. An examination of specific photopigment gene sites responsible for tuning photopigment absorption spectra revealed differences that predict these variations in the color defect. The results indicate that the severity of the defect in deuteranomalous color vision depends on the degree of similarity among the residual photopigments that serve vision in the color-anomalous eye.

Temporal and spatial patterns of opsin gene expression in zebrafish (Danio rerio).

In zebrafish, the first class of cone photoreceptor to become morphologically distinct is the ultraviolet-sensitive short single cone, at 4 days postfertilization, whereas the last class, the red- and green-sensitive double cone, becomes distinct at 10 days postfertilization. We have examined the time course of visual pigment gene expression in zebrafish using whole-mount in situ hybridization. Within the retina, opsins may be detected as early as 40 h postfertilization with the ultraviolet and rod visual pigments being expressed before the blue- (48 h) and red- (60 h) sensitive pigments. In the pineal, red-sensitive opsin is expressed at 48 h postfertilization. Visual pigment expression provides a useful tool for investigations of early cell fate in zebrafish.

Polymorphism in the number of genes encoding long-wavelength-sensitive cone pigments among males with normal color vision

Examination by direct DNA sequence analysis of the X-linked visual pigment genes in 27 males with normal color vision reveals that almost half have two or more different genes encoding a long-wavelength-sensitive cone pigment. This is counter to the conventional theory proposed from results of Southern hybridization studies that there is a single long-wave pigment gene per X-chromosome. Further, the sequences and consideration of the structure of the X-linked pigment gene array suggest that the majority of the observers (as many as 2/3) have hybrid (or fusion) genes like those that have been proposed to underlie color anomaly. In some observers the long-wave hybrid genes contain a substantial amount of middle-wave sequence, e.g. five observers have hybrid long-wave genes that contain middle-wave sequences that include exon 4. Three of those five have the hybrid as their only long-wave gene, and thus have no other gene that could potentially encode a long-wave pigment. In these subjects, it is the hybrid gene that produces their normal long-wavelength-sensitive cone pigment. The high frequency of hybrid genes indicates that they are normal variant forms of the long-wave gene. Contrary to what is commonly believed, the introduction and the expression of hybrid genes is not sufficient to cause color vision defects.

Polymorphism in the number of genes encoding long-wavelength-sensitive cone pigments among males with normal color vision

Examination by direct DNA sequence analysis of the X-linked visual pigment genes in 27 males with normal color vision reveals that almost half have two or more different genes encoding a long-wavelength-sensitive cone pigment. This is counter to the conventional theory proposed from results of Southern hybridization studies that there is a single long-wave pigment gene per X-chromosome. Further, the sequences and consideration of the structure of the X-linked pigment gene array suggest that the majority of the observers (as many as 2/3 ) have hybrid (or fusion) genes like those that have been proposed to underlie color anomaly. In some observers the long-wave hybrid genes contain a substantial amount of middle-wave sequence, e.g. five observers have hybrid long-wave genes that contain middle-wave sequences that include exon 4. Three of those five have the hybrid as their only long-wave gene, and thus have no other gene that could potentially encode a long-wave pigment. In these subjects, it is the hybrid gene that produces their normal long-wavelength-sensitive cone pigment. The high frequency of hybrid genes indicates that they are normal variant forms of the long-wave gene. Contrary to what is commonly believed, the introduction and the expression of hybrid genes is not sufficient to cause color vision defects.

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.

Comparison of DNA Sequences of Exon-4 or Exon-5 in Visual Pigment Genes between Amphidromous and Landlocked Ayu-fish, Plecoglossus Altivelis.

Nucleotide sequences of exon-4 or -5 encoding one region of visual pigments in Plecoglossus altivelis were determined with the amplified fragments. One nuclotide was substituted in the exon-4 of rhodopsin pigment gene between the amphidromous and landlocked form, and one colon (ATC) in exon-4 of amphidromous form was changed to GTC in that of landlocked form. There was no difference in the exon-5 of the sequence in the red-sensitive pigment gene between the two forms.

A human opsin-related gene that encodes a retinaldehyde-binding protein.

The ligand-binding property of a cytoplasmic membrane-bound protein from bovine retinal pigment epithelium (RPE) has been demonstrated. The putative RPE-retinal G protein coupled receptor (RGR) covalently binds both all-trans- and 11-cis-retinal after reduction by sodium borohydride. The 32-kDa receptor binds all-trans-retinal preferentially, rather than the 11-cis isomer. The amino acid sequence of the opsin-related protein in humans is 86% identical to that of bovine RGR, and a lysine residue, analogous to the retinaldehyde attachment site of rhodopsin, is conserved in the seventh transmembrane domain of RGR in both species. The human gene that encodes the novel retinaldehyde receptor spans 14.8 kb and is split into seven exons. The structure of the gene is distinct from that of the visual pigment genes. These findings support the notion that the rgr gene represents the earliest independent branch of the vertebrate opsin gene family. A second form of human RGR in retina is predicted by alternative splicing of its precursor mRNA. This RGR variant results from the alternative use of an internal acceptor splice site in the second intron of the human gene, and it contains an insertion of four amino acids in the connecting loop between the second and thrid transmembrane domains. Since RGR binds all-trans-retinal preferentially, one of its functions may be to catalyze isomerization of the chromophore by a retinochrome-like mechanism.

Sequence divergence, polymorphism and evolution of the middle-wave and long-wave visual pigment genes of great apes and old world monkeys

In man, the spectral shift between the middle-wave (MW) and long-wave (LW) visual pigments is largely achieved by amino acid substitution at two codons, both located in exon 5. A third amino acid site coded by exon 3 is polymorphic between pigments. We have studied the equivalent regions of the cone opsin genes in two members of the Hominidea (the gorilla, Gorilla gorilla and the chimpanzee, Pan troglodytes) and in three members of the Cercopithecoidea family of Old World primates (the diana monkey, Cercopithecus diana, the talapoin monkey, Miopithecus talapoin, and the crab-eating macaque, Macaca fascicularis). No variation in the codons that specify the amino acids involved in spectral tuning were found. We predict therefore that the MW and LW pigments of gorilla and chimpanzee have similar spectral characteristics to those of man. Multiple copies of the same opsin gene sequence were identified in the chimpanzee, talapoin and macaque and we also show that non-human Old World primates are similar to man in showing a bunching of polymorphic sites in exon 3. We discuss the ancestry of the separate MW and LW genes of Old World primates and the equivalent polymorphic gene of the marmoset, a New World primate.

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.

A sequence upstream of the mouse blue visual pigment gene directs blue cone-specific transgene expression in mouse retinas.

A 6.4-kb sequence upstream of the mouse blue visual pigment gene has been assayed in transgenic mice for the ability to direct cell-type-specific expression of a linked beta-galactosidase (lacZ) reporter. The construct is expressed specifically in cone photoreceptors in three independent lines. Transgene expression is found in the developing retina on the first postnatal day, increases rapidly in subsequent days, and persists through adulthood. A gradient of transgene expression is observed across the retina, with the transgene-expressing cones found almost exclusively in the lower retina and rarely in the upper retina, a pattern that parallels the distribution of blue cones in the mouse retina. Double-labeling with anti-cone pigment antibodies shows that transgene expression is confined to blue cones. These results imply that all of the sequence elements necessary for the control of blue cone-specific expression are encoded within the 6.4-kb DNA fragment tested.

Blue cones and cone bipolar cells share transcriptional specificity as determined by expression of human blue visual pigment-derived transgenes

Sequences 5' of the human blue visual pigment gene have been assayed in transgenic mice for their ability to direct cell-type-specific expression of linked beta-galactosidase (lacZ) and placental alkaline phosphatase (P ALP1) reporters. Constructs containing either 5.4 kilobases (kb) or 0.47 kb of 5' flanking DNA direct expression exclusively to the retina. Within the retina, transgene expression is confined to blue cones and cone bipolar cells, as determined, respectively, by double labeling with anti-cone pigment antibodies and by morphologic analyses. These results imply that blue cones and cone bipolar cells have partially overlapping transcriptional specificities.

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.

A Revised Map of the Human Chromosomes

HIS paper presents an updated of the human T chromosomes, building on the plain English map previously published (Offner 1992). I believe that regular revisions are important because the field of human genetics is expanding so rapidly. Teachers wishing to use this in the classroom should use it in conjunction with the previously published in The American Biology Teacher (Offner 1992). As of early 1992, more than 2500 genes had been assigned to specific loci (McKusick 1992), several hundred more than when the last was constructed. For example, the gene for Marfan Syndrome has been added to the map. In addition, new advances in genetics have made it possible to teach about previously mapped genes in a meaningful way to high school and college students. This is true of p53, a gene that prevents cancer, and Prader-Willi/Angelman Syndromes, both of which have been added to the map. Finally, there has been some exciting new research on some genes already on the (cystic fibrosis and LH, beta chain). A brief summary of this research has been included in the gene explanations accompanying the map. Some genes such as those for ribosomal RNA, transfer RNA and retinoblastoma have been added in response to requests from users of the first map. And the genes for myoglobin and blue cone pigment were added because they are part of gene families (the globin genes and the visual pigment genes) which are of great interest in studying evolution. The study of this yields several important insights. It is tempting to present genes as things in the cell that cause disease. And while it is important to teach about genetic diseases, a study of the shows that genes are actually a set of instructions for assembling an organism. The genes for amylase, trypsin and chymotrypsin tell your digestive system how to make enzymes to digest your food; collagen genes are instructions for making bone; antibody genes are instructions for building an immune system; hemoglobin genes are instructions for making oxygen-carrying proteins in red blood cells. Even the cystic fibrosis gene, in its normal form, is an instruction for building a channel to let chloride ions out of the cell. It is only when there is a mistake in these instructions that a genetic disease results. So while studying genetic diseases is an excellent way of capturing student interest and getting students to think about what happens when there is an error in the normal processes, we should always emphasize that genes exist even where there is no disease. Another interesting insight that comes from the study of the is the answer to the question of why some genes are dominant and some are recessive. It turns out that whether a gene is dominant or recessive depends on the particular details of how the gene functions. Hence, a mutation in the p53 gene that causes an abnormal protein to be made is dominant because the protein happens to function as a tetramer, so one abnormal monomer can ruin an entire molecule. (Assuming the monomers are assembled randomly into finished protein, only one finished protein in 16 would have four normal monomers and no abnormal ones.) At the same time, the protein made by the retinoblastoma gene functions as a monomer, so a mutation in this gene is recessive in a single cell, since enough functional protein is made by the healthy gene if the cell is heterozygous. It is only when a cell loses its one functional gene that a cancer results, as described in the gene explanations. This, however, means that the disorder is dominant since, in the millions of cells in a human retina, it is likely that one or two cells will lose their one functional gene (Oppenheimer 1991). I hope that this updated will be useful in the classroom. I am planning to continue to revise the and would appreciate feedback from its users.

Characterization of the Gene Encoding Human Cone Transducin α-Subunit (GNAT2)

The human cone transducin α-subunit (GNAT2) gene has been completely characterized. The human GNAT2 transcription unit is 9967-bp in length and consists of eight exons with seven introns. The eight exons are identical to the reported cDNA sequence (Lerea et al., 1989). Northern blot analysis of RNA from human retinas and a retinoblastoma cell line, WERI-RB1, reveals a 1.7-kb transcript for GNAT2. Multiple transcription initiation sites were mapped for human retina and WERI-RB1 RNA by primer extension and S1 nuclease protection assays. This gene has seven initiation sites spanning 31 bp. The sequence upstream of the GNAT2 gene shows a TATA box consensus sequence at -29, a CCAAT box consensus sequence at -58 (reverse orientation), and a sequence (CCATAT) similar to the CCAAT box consensus at -76. The GNAT2 upstream sequence shows no significant identity with the upstream region of the human rod transducin α-subunit gene (GNAT1) or with the upstream regions of the color visual pigment genes, indicating that the expression of GNAT2 may be regulated differently than these other rod- and cone-specific proteins.

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.

Detecting gene conversion: primate visual pigment genes.

The effects of gene conversion can be detected in the DNA sequences of multigene families. We develop a permutation test of the significance of patterns of sequence mismatches, and apply it to the sequences of the red- and green-sensitive visual pigment genes of human and the diana monkey. Whereas conventional tests of the rate of sequence divergence are equivocal, the permutation test convincingly excludes divergence in the absence of gene conversion (p = 10(-6)).

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.