Short-wavelength Sensitive Opsin Research Articles

A Phe-rich region in short-wavelength sensitive opsins is responsible for their aggregation in the absence of 11-cis-retinal

Human blue and mouse S-opsin are prone to aggregation in the absence of 11-cis-retinal, which underlie the rapid cone degeneration in human patients and animal models of Leber congenital amaurosis (LCA). By in silico analysis and domain swapping experiments, we show that a Phe-rich region in short-wavelength sensitive (SWS) opsins, but not in medium/long-wavelength sensitive opsins, is responsible for SWS opsin aggregation. Mutagenesis studies suggest that Phe residues in this region are critical in mediating protein aggregation. Fusing the Phe-rich region of SWS opsins to GFP causes the latter to aggregate. Our findings suggest that new therapeutics can be designed to disrupt the Phe-rich region in preventing cone degeneration due to S-opsin aggregation in LCA.

Rod monochromacy and the coevolution of cetacean retinal opsins.

Cetaceans have a long history of commitment to a fully aquatic lifestyle that extends back to the Eocene. Extant species have evolved a spectacular array of adaptations in conjunction with their deployment into a diverse array of aquatic habitats. Sensory systems are among those that have experienced radical transformations in the evolutionary history of this clade. In the case of vision, previous studies have demonstrated important changes in the genes encoding rod opsin (RH1), short-wavelength sensitive opsin 1 (SWS1), and long-wavelength sensitive opsin (LWS) in selected cetaceans, but have not examined the full complement of opsin genes across the complete range of cetacean families. Here, we report protein-coding sequences for RH1 and both color opsin genes (SWS1, LWS) from representatives of all extant cetacean families. We examine competing hypotheses pertaining to the timing of blue shifts in RH1 relative to SWS1 inactivation in the early history of Cetacea, and we test the hypothesis that some cetaceans are rod monochomats. Molecular evolutionary analyses contradict the “coastal” hypothesis, wherein SWS1 was pseudogenized in the common ancestor of Cetacea, and instead suggest that RH1 was blue-shifted in the common ancestor of Cetacea before SWS1 was independently knocked out in baleen whales (Mysticeti) and in toothed whales (Odontoceti). Further, molecular evidence implies that LWS was inactivated convergently on at least five occasions in Cetacea: (1) Balaenidae (bowhead and right whales), (2) Balaenopteroidea (rorquals plus gray whale), (3) Mesoplodon bidens (Sowerby's beaked whale), (4) Physeter macrocephalus (giant sperm whale), and (5) Kogia breviceps (pygmy sperm whale). All of these cetaceans are known to dive to depths of at least 100 m where the underwater light field is dim and dominated by blue light. The knockout of both SWS1 and LWS in multiple cetacean lineages renders these taxa rod monochromats, a condition previously unknown among mammalian species.

Expression and Evolution of Short Wavelength Sensitive Opsins in Colugos: A Nocturnal Lineage That Informs Debate on Primate Origins.

A nocturnal activity pattern is central to almost all hypotheses on the adaptive origins of primates. This enduring view has been challenged in recent years on the basis of variation in the opsin genes of nocturnal primates. A correspondence between the opsin genes and activity patterns of species in Euarchonta—the superordinal group that includes the orders Primates, Dermoptera (colugos), and Scandentia (treeshrews)—could prove instructive, yet the basic biology of the dermopteran visual system is practically unknown. Here we show that the eye of the Sunda colugo (Galeopterus variegatus) lacks a tapetum lucidum and has an avascular retina, and we report on the expression and spectral sensitivity of cone photopigments. We found that Sunda colugos have intact short wavelength sensitive (S-) and long wavelength sensitive (L-) opsin genes, and that both opsins are expressed in cone photoreceptors of the retina. The inferred peak spectral sensitivities are 451 and 562 nm, respectively. In line with adaptation to nocturnal vision, cone densities are low. Surprisingly, a majority of S-cones coexpress some L-opsin. We also show that the ratio of rates of nonsynonymous to synonymous substitutions of exon 1 of the S-opsin gene is indicative of purifying selection. Taken together, our results suggest that natural selection has favored a functional S-opsin in a nocturnal lineage for at least 45 million years. Accordingly, a nocturnal activity pattern remains the most likely ancestral character state of euprimates.Electronic supplementary materialThe online version of this article (doi:10.1007/s11692-013-9230-y) contains supplementary material, which is available to authorized users.

The basis of polychromatic ultraviolet vision in mantis shrimp

Event Abstract Back to Event The basis of polychromatic ultraviolet vision in mantis shrimp Michael J. Bok1*, Megan L. Porter1 and Thomas W. Cronin1 1 University of Maryland, Baltimore County, United States Stomatopod crustaceans, or mantis shrimp, possess some of the most notable visual systems known to biology. Their visual ecology has been characterized in great detail, showing that many species of this order are capable of advanced color and polarization discrimination. The photoreceptors of stomatopod compound eyes are maximally sensitive to at least sixteen separate wavelengths of light in the human visible range, from 400 to 700 nm. Stomatopod photoreceptors have also been shown to be maximally sensitive to at least five discrete wavelengths of ultraviolet (UV) light, between 310 and 380 nm, as well as orthogonal polarization angles of UV light. Little is known about the visual pigments and spectral tuning mechanisms at work in these UV-sensitive photoreceptors, or their implication to the visual ecology of mantis shrimp. Here I report on molecular investigations of short wavelength-sensitive opsin transcripts in the retina of Neogonodactylus oerstedii, and spectroscopic measurements from the optical components of the eye. These findings suggest that multiple UV-absorbing visual pigments and novel UV optical filters are responsible for the surprising diversity of spectral sensitivities observed in stomatopod UV photoreceptors. Furthermore, comparative analysis of these spectral tuning components alongside preliminary behavioral experiments may indicate a diversity of UV photoreceptor spectral arrays, and UV visual capabilities, across different species of stomatopods. Keywords: Eye, Filters, Mantis Shrimp, Opsin, optics, Stomatopod, Ultraviolet, Vision Conference: Tenth International Congress of Neuroethology, College Park. Maryland USA, United States, 5 Aug - 10 Aug, 2012. Presentation Type: Poster (but consider for participant symposium and student poster award) Topic: Sensory: Vision Citation: Bok MJ, Porter ML and Cronin TW (2012). The basis of polychromatic ultraviolet vision in mantis shrimp. Conference Abstract: Tenth International Congress of Neuroethology. doi: 10.3389/conf.fnbeh.2012.27.00373 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 01 May 2012; Published Online: 07 Jul 2012. * Correspondence: Mr. Michael J Bok, University of Maryland, Baltimore County, Baltimore, United States, mikebok@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Michael J Bok Megan L Porter Thomas W Cronin Google Michael J Bok Megan L Porter Thomas W Cronin Google Scholar Michael J Bok Megan L Porter Thomas W Cronin PubMed Michael J Bok Megan L Porter Thomas W Cronin Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Intra-retinal variation of opsin gene expression in the guppy (Poecilia reticulata)

Although behavioural experiments demonstrate that colouration influences mate choice in many species, a complete understanding of this form of signalling requires information about colour vision in the species under investigation. The guppy (Poecilia reticulata) has become a model species for the study of colour-based sexual selection. To investigate the role of opsin gene duplication and divergence in the evolution of colour-based mate choice, we used in situ hybridization to determine where the guppy's nine cone opsins are expressed in the retina. Long wavelength-sensitive (LWS) opsins were more abundant in the dorsal retina than in the ventral retina. One of the middle wavelength-sensitive opsins (RH2-1) exhibited the opposite pattern, while the other middle wavelength-sensitive opsin (RH2-2) and the short wavelength-sensitive opsins (SWS1, SWS2A and SWS2B) were expressed throughout the retina. We also found variation in LWS opsin expression among individuals. These observations suggest that regions of the guppy retina are specialized with respect to wavelength discrimination and/or sensitivity. Intra-retinal variability in opsin expression, which has been observed in several fish species, might be an adaptation to variation in the strength and spectral composition of light entering the eye from above and below. The discovery that opsin expression varies in the guppy retina may motivate new behavioural experiments designed to study its role in mate choice.

UVS is rare in seabirds

Ultraviolet-sensitive vision (UVS), believed to have evolved from an ancestral state of violet-sensitive vision (VS), is widespread among terrestrial birds, where it is thought to play a role in orientation, foraging, and sexual selection. Less is known, however, about the distribution and significance of UVS in seabirds. To date UVS has been definitively demonstrated only in two families (Laridae and Sternidae), although indirect evidence has been used to argue for a more widespread occurrence. In this study we analyzed short-wavelength sensitive (SWS1) opsin DNA sequences to determine the distribution of ancestral (VS) and derived (UVS) amino acid spectral tuning sites in 16 seabird species representing 8 families with diverse ecological niches. Our results revealed sequences associated with UVS pigments (UVSs) in the Black-backed gull ( Larus dominicanus), providing further evidence of its widespread occurrence within the Laridae. The Caspian tern ( Hydroprogne caspia) and White-fronted tern ( Sterna striata), however, were found to have VSs, suggesting an evolutionary reversion to the ancestral state within Sternidae. VSs were also detected in an additional six families. Our results raise interesting questions about the functions of UV vision in marine environments.

Phylogenetic analyses in cetacean species of the family Delphinidae using a short wavelength sensitive opsin gene sequence

The short wavelength sensitive (SWS) opsin gene is expected to contain informative sites for understanding the speciation of the family Delphinidae, because it is not functional in cetaceans. We determined partial SWS gene sequences from 15 delphinid species of 12 genera and from harbor porpoise for comparison. We found a 39-bp insertion that was shared by six species (the insertion group: Delphinus delphis, Delphinus capensis, Stenella longirostris, Stenella coeruleoalba, Lagenodelphis hosei, and Sousa chinensis) and common base substitutions shared by eight species (Stenella frontalis, Tursiops truncatus, and six species of the insertion group). As these insertions and substitutions are not found in the other seven delphinids or in the cloven-hoofed mammals (which are close to cetaceans), it is suggested that these eight species are more closely related to each other than to the other species. This hypothesis is supported by phylogenetic analyses. The eight species with the substitutions formed a clade containing two sister clades, one consisting of the insertion group and the other consisting of the two other species, in both neighbor-joining and Bayes analyses. Phylogenetic analyses also showed that Lissodelphis borealis and Lagenorhynchus obliquidens are closely related and that their common ancestor diverged from the others at an early stage of delphinid evolution.

Evolution of opsin genes reveals a functional role of vision in the echolocating little brown bat ( Myotis lucifugus)

Echolocating bats are able to orientate, navigate and forage without visual cues. To probe the role of vision in bats, we studied the visual opsin genes from the echolocating little brown bat ( Myotis lucifugus). Short-wavelength sensitive ( SWS1) opsin, middle/long-wavelength sensitive ( M/ LWS) opsin and rhodopsin cDNA sequences were identified from the Ensembl database and validated by the sequencing of genomic DNA. We retrieved the published orthologous genes from eleven additional representative species of mammals from GenBank and conducted an evolutionary analysis. We found that the M/ LWS opsin and rhodopsin genes were both under strong purifying selection, whereas the SWS1 opsin gene has undergone positive selection at two amino acid sites and one lineage, though the main evolutionary force is still purifying selection. Two-ratio model of the SWS1 opsin gene revealed that the ω ratio for the little brown bat lineage was nearly three times lower than the background ratio, suggesting a much stronger functional constraint. Our relative rate tests show the little brown bat has a lower nonsynonymous substitution rate than those in other mammals (on average 32% lower) for the SWS1 opsin gene. However, no such significant differences were detected for the M/ LWS opsin and rhodopsin genes. The results of the relative ratio tests are consistent with that of tests for selection, showing a history of purifying selection on the little brown bat opsin genes. These findings suggest a functional role of vision in the little brown bat despite being nocturnal and using echolocation. We speculate that this echolocating bat may be able to use visual cues to orientate, navigate and forage at night, to discriminate color under moonlight and starlight conditions, or to avoid predation by diurnal raptors.

Signatures of Functional Constraint at Aye-aye Opsin Genes: The Potential of Adaptive Color Vision in a Nocturnal Primate

While color vision perception is thought to be adaptively correlated with foraging efficiency for diurnal mammals, those that forage exclusively at night may not need color vision nor have the capacity for it. Indeed, although the basic condition for mammals is dichromacy, diverse nocturnal mammals have only monochromatic vision, resulting from functional loss of the short-wavelength sensitive opsin gene. However, many nocturnal primates maintain intact two opsin genes and thus have dichromatic capacity. The evolutionary significance of this surprising observation has not yet been elucidated. We used a molecular population genetics approach to test evolutionary hypotheses for the two intact opsin genes of the fully nocturnal aye-aye (Daubentonia madagascariensis), a highly unusual and endangered Madagascar primate. No evidence of gene degradation in either opsin gene was observed for any of 8 aye-aye individuals examined. Furthermore, levels of nucleotide diversity for opsin gene functional sites were lower than those for 15 neutrally evolving intergenic regions (>25 kb in total), which is consistent with a history of purifying selection on aye-aye opsin genes. The most likely explanation for these findings is that dichromacy is advantageous for aye-ayes despite their nocturnal activity pattern. We speculate that dichromatic nocturnal primates may be able to perceive color while foraging under moonlight conditions, and suggest that behavioral and ecological comparisons among dichromatic and monochromatic nocturnal primates will help to elucidate the specific activities for which color vision perception is advantageous.

Demonstrating the Theory of Ecological Speciation in Cichlids

Demonstrating the Theory of Ecological Speciation in Cichlids

Short-wavelength sensitive opsin (SWS1) as a new marker for vertebrate phylogenetics

BackgroundVertebrate SWS1 visual pigments mediate visual transduction in response to light at short wavelengths. Due to their importance in vision, SWS1 genes have been isolated from a surprisingly wide range of vertebrates, including lampreys, teleosts, amphibians, reptiles, birds, and mammals. The SWS1 genes exhibit many of the characteristics of genes typically targeted for phylogenetic analyses. This study investigates both the utility of SWS1 as a marker for inferring vertebrate phylogenetic relationships, and the characteristics of the gene that contribute to its phylogenetic utility.ResultsPhylogenetic analyses of vertebrate SWS1 genes produced topologies that were remarkably congruent with generally accepted hypotheses of vertebrate evolution at both higher and lower taxonomic levels. The few exceptions were generally associated with areas of poor taxonomic sampling, or relationships that have been difficult to resolve using other molecular markers. The SWS1 data set was characterized by a substantial amount of among-site rate variation, and a relatively unskewed substitution rate matrix, even when the data were partitioned into different codon sites and individual taxonomic groups. Although there were nucleotide biases in some groups at third positions, these biases were not convergent across different taxonomic groups.ConclusionOur results suggest that SWS1 may be a good marker for vertebrate phylogenetics due to the variable yet consistent patterns of sequence evolution exhibited across fairly wide taxonomic groups. This may result from constraints imposed by the functional role of SWS1 pigments in visual transduction.

Regulation of gene expression in the mammalian eye and its relevance to eye disease

We used expression quantitative trait locus mapping in the laboratory rat (Rattus norvegicus) to gain a broad perspective of gene regulation in the mammalian eye and to identify genetic variation relevant to human eye disease. Of >31,000 gene probes represented on an Affymetrix expression microarray, 18,976 exhibited sufficient signal for reliable analysis and at least 2-fold variation in expression among 120 F(2) rats generated from an SR/JrHsd x SHRSP intercross. Genome-wide linkage analysis with 399 genetic markers revealed significant linkage with at least one marker for 1,300 probes (alpha = 0.001; estimated empirical false discovery rate = 2%). Both contiguous and noncontiguous loci were found to be important in regulating mammalian eye gene expression. We investigated one locus of each type in greater detail and identified putative transcription-altering variations in both cases. We found an inserted cREL binding sequence in the 5' flanking sequence of the Abca4 gene associated with an increased expression level of that gene, and we found a mutation of the gene encoding thyroid hormone receptor beta2 associated with a decreased expression level of the gene encoding short-wavelength sensitive opsin (Opn1sw). In addition to these positional studies, we performed a pairwise analysis of gene expression to identify genes that are regulated in a coordinated manner and used this approach to validate two previously undescribed genes involved in the human disease Bardet-Biedl syndrome. These data and analytical approaches can be used to facilitate the discovery of additional genes and regulatory elements involved in human eye disease.

Visual pigments of marine carnivores: pinnipeds, polar bear, and sea otter

Rod and cone visual pigments of 11 marine carnivores were evaluated. Rod, middle/long-wavelength sensitive (M/L) cone, and short-wavelength sensitive (S) cone opsin (if present) sequences were obtained from retinal mRNA. Spectral sensitivity was inferred through evaluation of known spectral tuning residues. The rod pigments of all but one of the pinnipeds were similar to those of the sea otter, polar bear, and most other terrestrial carnivores with spectral peak sensitivities (lambda(max)) of 499 or 501 nm. Similarly, the M/L cone pigments of the pinnipeds, polar bear, and otter had inferred lambda(max) of 545 to 560 nm. Only the rod opsin sequence of the elephant seal had sensitivity characteristic of adaptation for vision in the marine environment, with an inferred lambda(max) of 487 nm. No evidence of S cones was found for any of the pinnipeds. The polar bear and otter had S cones with inferred lambda(max) of approximately 440 nm. Flicker-photometric ERG was additionally used to examine the in situ sensitivities of three species of pinniped. Despite the use of conditions previously shown to evoke cone responses in other mammals, no cone responses could be elicited from any of these pinnipeds. Rod photoreceptor responses for all three species were as predicted by the genetic data.

Regulation of phototransduction in short-wavelength cone visual pigments via the retinylidene Schiff base counterion.

Short-wavelength visual pigments (SWS1) have lambda(max) values that range from the ultraviolet to the blue. Like all visual pigments, this class has an 11-cis-retinal chromophore attached through a Schiff base linkage to a lysine residue of opsin apoprotein. We have characterized a series of site-specific mutants at a conserved acidic residue in transmembrane helix 3 in the Xenopus short-wavelength sensitive cone opsin (VCOP, lambda(max) approximately 427 nm). We report the identification of D108 as the counterion to the protonated retinylidene Schiff base. This residue regulates the pK(a) of the Schiff base and, neutralizing this charge, converts the violet sensitive pigment into one that absorbs maximally in the ultraviolet region. Changes to this position cause the pigment to exhibit two chromophore absorbance bands, a major band with a lambda(max) of approximately 352-372 nm and a minor, broad shoulder centered around 480 nm. The behavior of these two absorbance bands suggests that these represent unprotonated and protonated Schiff base forms of the pigment. The D108A mutant does not activate bovine rod transducin in the dark but has a significantly prolonged lifetime of the active MetaII state. The data suggest that in short-wavelength sensitive cone visual pigments, the counterion is necessary for the characteristic rapid production and decay of the active MetaII state.

Antibody labeling of the blue-sensitive cones in the retinae of teleost fishes

Characterization of visual opsins can be achieved with the use of specific polyclonal or monoclonal antibodies. In this study, antiserum was raised against a conjugate protein including an N-terminal sequence of the short wavelength sensitive (SWS, commonly referred to as blue-sensitive) visual pigment opsin in goldfish (Carrasius auratus). The antiserum showed immunoreactivity to retinae of phylogenetically distant teleosts, including the goldfish, rainbow trout (Oncorhynchus mykiss), and bluehead wrasse (Thalassoma bisasciatum). Complementary DNA (cDNA) was synthesized from goldfish retinal messenger RNA. An N-terminal sequence of the SWS opsin was PCR-amplified from the cDNA and subsequently cloned into an expression vector. Expressed protein was purified by metal chelate affinity chromatography and used to immunize New Zealand white rabbits. Immune serum was collected and used in immunocytochemical assays, which revealed that the antiserum contains antibodies specific for an opsin that resides in single-cone photoreceptors of the goldfish, rainbow trout, and bluehead wrasse. The results confirm those from earlier studies of goldfish in which short-wavelength sensitivity was localized to single cones, and suggest that the association between short-wavelength sensitivity and single cones may be a feature common to many teleosts. The production of antiserum specific for SWS opsins provides a useful tool in the characterization of opsin expression within teleost retinae.

Localization of protein kinase C to UV-sensitive photoreceptors in the mouse retina.

The present study has identified a population of cone photoreceptors in the murine retina that are uniquely immunoreactive for protein kinase C (PKC). Wavelength-sensitive cone subtypes are segregated along the dorso-ventral axis in the mouse retina with ventral retina occupied exclusively by ultraviolet wavelength-sensitive (UVWS) cones, and dorsal retina dominated by middle wavelength-sensitive cones. PKC-positive cones are found primarily in the ventral retina, and double-label immunocytochemistry using a short wavelength-sensitive opsin antibody confirms that they specifically correspond to the UVWS cone subtype. The PKC antibody, as documented in other mammals, also identifies rod bipolar cells in the mouse retina. UVWS cones and bipolar cells have previously been shown to share transcriptional regulatory elements, as observed in transgenic mice encoding a portion of the human SWS-opsin promoter controlling the lacZ reporter gene. In such mice, the transgene product, beta-galactosidase, is expressed in populations of both cones and bipolar cells. The present study confirms that lacZ-expressing photoreceptors are indeed PKC-positive photoreceptors, but that the lacZ-expressing bipolar cells are not the PKC-positive rod bipolar cells. These cells must correspond to a type of cone bipolar cell.

Short wavelength-sensitive opsins from the Saharan silver and carpenter ants.

We have previously cloned the opsins coding for the long-wavelength visual pigments from the Saharan silver ant and carpenter ant. Here we report two new cDNA clones isolated from cDNA libraries which also code for opsin proteins. These cDNAs code for deduced proteins with 369 amino acids which are 91% identical to each other, but only 38% identical to the previously cloned opsins. Phyletic comparisons suggest that these opsins are likely the ultraviolet sensitive visual pigments, a conclusion that is supported by the presence of a phenylalanine at the counterion position in the third transmembrane segment.

Positional information and opsin identity in retinal cones.

To test the hypothesis that local environmental cues regulate the expression of middle wavelength-sensitive (MWS) and short wavelength-sensitive (SWS) opsins in cone photoreceptors, we examined the development of the neonatal mouse retina in an organotypic culture system. The segregation of MWS and SWS cones into dorsal and ventral fields in the mouse retina offers an opportunity to isolate a phenotypically homogeneous population of immature cones prior to opsin expression. Retinae were harvested from mice ranging in age from birth (P0) to P18 and maintained in vitro for up to 4 weeks. Cones from newborn mice were first immunoreactive to SWS opsin-specific antibodies (OS-2 and JH455) after 5 days in vitro, which corresponds to a time course similar to that in vivo. The topographic separation of SWS cones into distinct dorsal and ventral fields was also obvious in retinal explants from newborn mice. However, the MWS opsin, identified by polyclonal antibody JH492, was expressed only in vitro when dorsal explants were harvested from P3 or older pups. Despite the absence of MWS opsin expression in newborn retinal cultures, there was no evidence of an increase in the numbers of SWS cones. To test if local diffusable cues could induce immature cones to express an aberrant opsin, dorsal and ventral retinal explants at different stages of maturation were cocultured during the incubation period. Neither the emergence of the cone fields nor the difference in the regional and temporal development of the MWS and SWS opsins was affected in these experiments. These results suggest that positional information in the retina and the opsin identity of cones is determined prior to birth and argue against the hypothesis that postnatal cones can be induced to express an aberrant opsin.

Phylogenetic relationships among short wavelength-sensitive opsins of American chameleon (Anolis carolinensis) and other vertebrates.

The vertebrate opsins have been classified into four major phylogenetic groups. One of them, a short wavelength-sensitive (SWS) opsin group, is further divided into two subgroups, SWS-I and SWS-II, having the wavelengths of maximal absorption of about 420 and 450 nm, respectively. Here we report the DNA sequences of the SWS-I and SWS-II genes from the lizard Anolis carolinensis. The shorter wavelengths of absorption by the two SWS subgroup opsins seem to be achieved by different sets of amino acid replacements in the transmembrane regions.