Synchronized Expansion and Contraction of Olfactory, Vomeronasal, and Taste Receptor Gene Families in Hystricomorph Rodents.

Chemical senses, including olfaction, pheromones, and taste, are crucial for the survival of most animals. There has long been a debate about whether different types of senses might influence each other. For instance, primates with a strong sense of vision are thought to have weakened olfactory abilities, although the oversimplified trade-off theory is now being questioned. It is uncertain whether such interactions between different chemical senses occur during evolution. To address this question, we examined four receptor gene families related to olfaction, pheromones, and taste: olfactory receptor (OR), vomeronasal receptor type 1 and type 2 (V1R and V2R), and bitter taste receptor (T2R) genes in Hystricomorpha, which is morphologically and ecologically the most diverse group of rodents. We also sequenced and assembled the genome of the grasscutter, Thryonomys swinderianus. By examining 16 available genome assemblies alongside the grasscutter genome, we identified orthologous gene groups among hystricomorph rodents for these gene families to separate the gene gain and loss events in each phylogenetic branch of the Hystricomorpha evolutionary tree. Our analysis revealed that the expansion or contraction of the four gene families occurred synchronously, indicating that when one chemical sense develops or deteriorates, the others follow suit. The results also showed that V1R/V2R genes underwent the fastest evolution, followed by OR genes, and T2R genes were the most evolutionarily stable. This variation likely reflects the difference in ligands of V1R/V2Rs, ORs, and T2Rs: species-specific pheromones, environment-based scents, and toxic substances common to many animals, respectively.

The genomics of ecological flexibility, large brains, and long lives in capuchin monkeys revealed with fecalFACS.

Ecological flexibility, extended lifespans, and large brains have long intrigued evolutionary biologists, and comparative genomics offers an efficient and effective tool for generating new insights into the evolution of such traits. Studies of capuchin monkeys are particularly well situated to shed light on the selective pressures and genetic underpinnings of local adaptation to diverse habitats, longevity, and brain development. Distributed widely across Central and South America, they are inventive and extractive foragers, known for their sensorimotor intelligence. Capuchins have among the largest relative brain size of any monkey and a lifespan that exceeds 50 y, despite their small (3 to 5 kg) body size. We assemble and annotate a de novo reference genome for Cebus imitator Through high-depth sequencing of DNA derived from blood, various tissues, and feces via fluorescence-activated cell sorting (fecalFACS) to isolate monkey epithelial cells, we compared genomes of capuchin populations from tropical dry forests and lowland rainforests and identified population divergence in genes involved in water balance, kidney function, and metabolism. Through a comparative genomics approach spanning a wide diversity of mammals, we identified genes under positive selection associated with longevity and brain development. Additionally, we provide a technological advancement in the use of noninvasive genomics for studies of free-ranging mammals. Our intra- and interspecific comparative study of capuchin genomics provides insights into processes underlying local adaptation to diverse and physiologically challenging environments, as well as the molecular basis of brain evolution and longevity.

Structural model for ligand binding and channel opening of an insect gustatory receptor.

Insect gustatory receptors play roles in sensing tastants, such as sugars and bitter substances. We previously demonstrated that the BmGr9 silkworm gustatory receptor is a d-fructose-gated ion channel receptor. However, the molecular mechanism of how d-fructose could initiate channel opening were unclear. Herein, we present a structural model for a channel pore and a d-fructose-binding site in BmGr9. Since the membrane topology and oligomeric state of BmGr9 appeared to be similar to those of an insect odorant receptor coreceptor, Orco, we constructed a structural model of BmGr9 based on the cryo-EM Orco structure. Our site-directed mutagenesis data suggested that the transmembrane region 7 forms channel pore and controls channel gating. This model also suggested that a pocket formed by transmembrane helices 2 to 4 and 6 binds d-fructose. Using mutagenesis experiments in combination with docking simulations, we were able to determine the potent binding mode of d-fructose. Finally, based on these data, we propose a conformational change that leads to channel opening upon d-fructose binding. Taken together, these findings detail the molecular mechanism by which an insect gustatory receptor can be activated by its ligand molecule.

Genetic variation in the human olfactory receptor OR5AN1 associates with the perception of musks.

Humans have significant individual variations in odor perception, derived from their experience or sometimes from differences in the olfactory receptor (OR) gene repertoire. In several cases, the genetic variation of a single OR affects the perception of its cognate odor ligand. Musks are widely used for fragrance and are known to demonstrate specific anosmia. It, however, remains to be elucidated whether the OR polymorphism contributes to individual variations in musk odor perception. Previous studies reported that responses of the human musk receptor OR5AN1 to a variety of musks in vitro correlated well with perceptual sensitivity to those odors in humans and that the mouse ortholog, Olfr1440 (MOR215-1), plays a critical role in muscone perception. Here, we took advantage of genetic variation in OR5AN1 to examine how changes in receptor sensitivity are associated with human musk perception. We investigated the functional differences between OR5AN1 variants in an in vitro assay and measured both perceived intensity and detection threshold in human subjects with different OR5AN1 genotypes. Human subjects homozygous for the more sensitive L289F allele had a lower detection threshold for muscone and found macrocyclic musks to be more intense than subjects homozygous for the reference allele. These results demonstrate that the genetic variation in OR5AN1 contributes to perceptual differences for some musks. In addition, we found that the more functional variant of OR5A1, a receptor involved in ß-ionone perception, is associated with the less functional variant of OR5AN1, suggesting that the perceived intensities of macrocyclic musks and ß-ionone are inversely correlated.

Origin and Evolution of the Gene Family of Proteinaceous Pheromones, the Exocrine Gland-Secreting Peptides, in Rodents.

The exocrine-gland secreting peptide (ESP)gene family encodes proteinaceous pheromones that are recognized by the vomeronasal organ in mice. For example, ESP1 is a male pheromone secreted in tear fluid that regulates socio-sexual behavior, and ESP22 is a juvenile pheromone that suppresses adult sexual behavior. The family consists of multiple genes and has been identified only in mouse and rat genomes. The coding region of a mouse ESP gene is separated into two exons, each encoding signal and mature sequences. Here, we report the origin and evolution of the ESP gene family. ESP genes were found only in the Muridea and Cricetidae families of rodents, suggesting a recent origin of ESP genes in the common ancestor of murids and cricetids. ESP genes show a great diversity in number, length, and sequence among different species as well as mouse strains. Some ESPs in rats and golden hamsters are expressed in the lacrimal gland and the salivary gland. We also found that a mature sequence of an ESP gene showed overall sequence similarity to the α-globin gene. The ancestral ESP gene seems to be generated by recombination of a retrotransposed α-globin gene with the signal-encoding exon of the CRISP2 gene located adjacent to the ESP gene cluster. This study provides an intriguing example of molecular tinkering in rapidly evolving species-specific proteinaceous pheromone genes.

Acceleration of Olfactory Receptor Gene Loss in Primate Evolution: Possible Link to Anatomical Change in Sensory Systems and Dietary Transition.

Primates have traditionally been regarded as vision-oriented animals with low olfactory ability, though this "microsmatic primates" view has been challenged recently. To clarify when and how degeneration of the olfactory system occurred and to specify the relevant factors during primate evolution, we here examined the olfactory receptor (OR) genes from 24 phylogenetically and ecologically diverse primate species. The results revealed that strepsirrhines with curved noses had functional OR gene repertoires that were nearly twice as large as those for haplorhines with simple noses. Neither activity pattern (nocturnal/diurnal) nor color vision system showed significant correlation with the number of functional OR genes while phylogeny and nose structure (haplorhine/strepsirrhine) are statistically controlled, but extent of folivory did. We traced the evolutionary fates of individual OR genes by identifying orthologous gene groups, demonstrating that the rates of OR gene losses were accelerated at the ancestral branch of haplorhines, which coincided with the acquisition of acute vision. The highest rate of OR gene loss was observed at the ancestral branch of leaf-eating colobines; this reduction is possibly linked with the dietary transition from frugivory to folivory because odor information is essential for fruit foraging but less so for leaf foraging. Intriguingly, we found accelerations of OR gene losses in an external branch to every hominoid species examined. These findings suggest that the current OR gene repertoire in each species has been shaped by a complex interplay of phylogeny, anatomy, and habitat; therefore, multiple factors may contribute to the olfactory degeneration in primates.

Metabolism of Odorant Molecules in Human Nasal/Oral Cavity Affects the Odorant Perception.

In this study, we examined the mode of metabolism of food odorant molecules in the human nasal/oral cavity in vitro and in vivo. We selected 4 odorants, 2-furfurylthiol (2-FT), hexanal, benzyl acetate, and methyl raspberry ketone, which are potentially important for designing food flavors. In vitro metabolic assays of odorants with saliva/nasal mucus analyzed by gas chromatography mass spectrometry revealed that human saliva and nasal mucus exhibit the following 3 enzymatic activities: (i) methylation of 2-FT into furfuryl methylsulfide (FMS); (ii) reduction of hexanal into hexanol; and (iii) hydrolysis of benzyl acetate into benzyl alcohol. However, (iv) demethylation of methyl raspberry ketone was not observed. Real-time in vivo analysis using proton transfer reaction-mass spectrometry demonstrated that the application of 2-FT and hexanal through 3 different pathways via the nostril or through the mouth generated the metabolites FMS and hexanol within a few seconds. The concentration of FMS and hexanol in the exhaled air was above the perception threshold. A cross-adaptation study based on the activation pattern of human odorant receptors suggested that this metabolism affects odor perception. These results suggest that some odorants in food are metabolized in the human nasal mucus/saliva, and the resulting metabolites are perceived as part of the odor quality of the substrates. Our results help improve the understanding of the mechanism of food odor perception and may enable improved design and development of foods in relation to odor.

Comparative genomic analysis of translation initiation mechanisms for genes lacking the Shine-Dalgarno sequence in prokaryotes.

In prokaryotes, translation initiation is believed to occur through an interaction between the 3΄ tail of a 16S rRNA and a corresponding Shine-Dalgarno (SD) sequence in the 5΄ untranslated region (UTR) of an mRNA. However, some genes lack SD sequences (non-SD genes), and the fraction of non-SD genes in a genome varies depending on the prokaryotic species. To elucidate non-SD translation initiation mechanisms in prokaryotes from an evolutionary perspective, we statistically examined the nucleotide frequencies around the initiation codons in non-SD genes from 260 prokaryotes (235 bacteria and 25 archaea). We identified distinct nucleotide frequency biases upstream of the initiation codon in bacteria and archaea, likely because of the presence of leaderless mRNAs lacking a 5΄ UTR. Moreover, we observed overall similarities in the nucleotide patterns between upstream and downstream regions of the initiation codon in all examined phyla. Symmetric nucleotide frequency biases might facilitate translation initiation by preventing the formation of secondary structures around the initiation codon. These features are more prominent in species' genomes that harbor large fractions of non-SD sequences, suggesting that a reduced stability around the initiation codon is important for efficient translation initiation in prokaryotes.

Ligand Specificity and Evolution of Mammalian Musk Odor Receptors: Effect of Single Receptor Deletion on Odor Detection.

Musk odors have been used widely for fragrance and medicine for >2000 years because of their fascinating scent and physiological effects. Therefore, fragrance manufacturers have been eager to develop high-quality musk compounds that are safe and easily synthesized. We recently identified muscone-responsive olfactory receptors (ORs) MOR215-1 and OR5AN1 in mice and humans, respectively (Shirasu et al., 2014). In this study, we identified musk ORs that are evolutionarily closely related to MOR215-1 or OR5AN1 in various primates and investigated structure-activity relationships for various musk odorants and related compounds. We found that each species has one or two functional musk ORs that exhibit specific ligand spectra to musk compounds. Some of them, including the human OR5AN1, responded to nitro musks with chemical properties distinct from muscone. The ligand specificity of OR5AN1 reflects the perception of musk odors in humans. Genetic deletion of MOR215-1 in mice resulted in drastic reduction of sensitivity to muscone, suggesting that MOR215-1 plays a critical role in muscone perception. Therefore, the current study reveals a clear link between the identified OR and muscone perception. Moreover, the strategy established for screening ligands for the muscone OR may facilitate the development of novel and commercially useful musk odors. SIGNIFICANCE STATEMENT: The long-sought musk odor receptor family in mammals was discovered and found to be well conserved and narrowly tuned to musk odors. In mice, deletion of the most sensitive musk receptor resulted in drastic reduction in sensitivity to muscone, demonstrating a strong link between receptor and odor perception. In humans, we found one musk receptor that recognized both macrocyclic and nitro musks that had distinct chemical structures. The structure-activity relationships were in a good agreement with human sensory perception and therefore may be used to develop novel musk aroma in fragrance fields. Finally, identification of a natural ligand(s) for musk receptors in mammals other than musk deer would reveal an evolutionarily pivotal role in each species in the future.

Extreme expansion of the olfactory receptor gene repertoire in African elephants and evolutionary dynamics of orthologous gene groups in 13 placental mammals.

Olfactory receptors (ORs) detect odors in the environment, and OR genes constitute the largest multigene family in mammals. Numbers of OR genes vary greatly among species--reflecting the respective species' lifestyles--and this variation is caused by frequent gene gains and losses during evolution. However, whether the extent of gene gains/losses varies among individual gene lineages and what might generate such variation is unknown. To answer these questions, we used a newly developed phylogeny-based method to classify >10,000 intact OR genes from 13 placental mammal species into 781 orthologous gene groups (OGGs); we then compared the OGGs. Interestingly, African elephants had a surprisingly large repertoire (∼ 2000) of functional OR genes encoded in enlarged gene clusters. Additionally, OR gene lineages that experienced more gene duplication had weaker purifying selection, and Class II OR genes have evolved more dynamically than those in Class I. Some OGGs were highly expanded in a lineage-specific manner, while only three OGGs showed complete one-to-one orthology among the 13 species without any gene gains/losses. These three OGGs also exhibited highly conserved amino acid sequences; therefore, ORs in these OGGs may have physiologically important functions common to every placental mammal. This study provides a basis for inferring OR functions from evolutionary trajectory.

The evolution of animal chemosensory receptor gene repertoires: roles of chance and necessity.

Chemosensory receptors are essential for the survival of organisms that range from bacteria to mammals. Recent studies have shown that the numbers of functional chemosensory receptor genes and pseudogenes vary enormously among the genomes of different animal species. Although much of the variation can be explained by the adaptation of organisms to different environments, it has become clear that a substantial portion is generated by genomic drift, a random process of gene duplication and deletion. Genomic drift also generates a substantial amount of copy-number variation in chemosensory receptor genes within species. It seems that mutation by gene duplication and inactivation has important roles in both the adaptive and non-adaptive evolution of chemosensation.

Dynamic evolution of translation initiation mechanisms in prokaryotes.

It is generally believed that prokaryotic translation is initiated by the interaction between the Shine-Dalgarno (SD) sequence in the 5' UTR of an mRNA and the anti-SD sequence in the 3' end of a 16S ribosomal RNA. However, there are two exceptional mechanisms, which do not require the SD sequence for translation initiation: one is mediated by a ribosomal protein S1 (RPS1) and the other used leaderless mRNA that lacks its 5' UTR. To understand the evolutionary changes of the mechanisms of translation initiation, we examined how universal the SD sequence is as an effective initiator for translation among prokaryotes. We identified the SD sequence from 277 species (249 eubacteria and 28 archaebacteria). We also devised an SD index that is a proportion of SD-containing genes in which the differences of GC contents are taken into account. We found that the SD indices varied among prokaryotic species, but were similar within each phylum. Although the anti-SD sequence is conserved among species, loss of the SD sequence seems to have occurred multiple times, independently, in different phyla. For those phyla, RPS1-mediated or leaderless mRNA-used mechanisms of translation initiation are considered to be working to a greater extent. Moreover, we also found that some species, such as Cyanobacteria, may acquire new mechanisms of translation initiation. Our findings indicate that, although translation initiation is indispensable for all protein-coding genes in the genome of every species, its mechanisms have dynamically changed during evolution.

Degeneration of olfactory receptor gene repertories in primates: no direct link to full trichromatic vision.

Odor molecules in the environment are detected by olfactory receptors (ORs), being encoded by a large multigene family in mammalian genomes. It is generally thought that primates are vision oriented and dependent weakly on olfaction. Previous studies suggested that Old World monkeys (OWMs) and hominoids lost many functional OR genes after the divergence from New World monkeys (NWMs) due to the acquisition of well-developed trichromatic vision. To examine this hypothesis, here we analyzed OR gene repertoires of five primate species including NWMs, OWMs, and hominoids for which high-coverage genome sequences are available, together with two prosimians and tree shrews with low-coverage genomes. The results showed no significant differences in the number of functional OR genes between NWMs (marmosets) and OWMs/hominoids. Two independent analyses, identification of orthologous genes among the five primates and estimation of the numbers of ancestral genes by the reconciled tree method, did not support a sudden loss of OR genes at the branch of the OWMs/hominoids ancestor but suggested a gradual loss in every lineage. Moreover, we found that humans retain larger numbers of ancestral OR genes that were in the common ancestor of NWMs/OWMs/hominoids than orangutans and macaques and that the OR gene repertoire in humans is more similar to that of marmosets than those of orangutans and macaques. These results suggest that the degeneration of OR genes in primates cannot simply be explained by the acquisition of trichromatic vision, and our sense of smell may not be inferior to other primate species.

Difference in gene duplicability may explain the difference in overall structure of protein-protein interaction networks among eukaryotes.

BACKGROUND: A protein-protein interaction network (PIN) was suggested to be a disassortative network, in which interactions between high- and low-degree nodes are favored while hub-hub interactions are suppressed. It was postulated that a disassortative structure minimizes unfavorable cross-talks between different hub-centric functional modules and was positively selected in evolution. However, by re-examining yeast PIN data, several researchers reported that the disassortative structure observed in a PIN might be an experimental artifact. Therefore, the existence of a disassortative structure and its possible evolutionary mechanism remains unclear. RESULTS: In this study, we investigated PINs from the yeast, worm, fly, human, and malaria parasite including four different yeast PIN datasets. The analyses showed that the yeast, worm, fly, and human PINs are disassortative while the malaria parasite PIN is not. By conducting simulation studies on the basis of a duplication-divergence model, we demonstrated that a preferential duplication of low- and high-degree nodes can generate disassortative and non-disassortative networks, respectively. From this observation, we hypothesized that the difference in degree dependence on gene duplications accounts for the difference in assortativity of PINs among species. Comparison of 55 proteomes in eukaryotes revealed that genes with lower degrees showed higher gene duplicabilities in the yeast, worm, and fly, while high-degree genes tend to have high duplicabilities in the malaria parasite, supporting the above hypothesis. CONCLUSIONS: These results suggest that disassortative structures observed in PINs are merely a byproduct of preferential duplications of low-degree genes, which might be caused by an organism's living environment.

Evolutionary dynamics of olfactory receptor genes in chordates: interaction between environments and genomic contents.

Olfaction is essential for the survival of animals. Versatile odour molecules in the environment are received by olfactory receptors (ORs), which form the largest multigene family in vertebrates. Identification of the entire repertories of OR genes using bioinformatics methods from the whole-genome sequences of diverse organisms revealed that the numbers of OR genes vary enormously, ranging from approximately 1,200 in rats and approximately 400 in humans to approximately 150 in zebrafish and approximately 15 in pufferfish. Most species have a considerable fraction of pseudogenes. Extensive phylogenetic analyses have suggested that the numbers of gene gains and losses are extremely large in the OR gene family, which is a striking example of the birth-and-death evolution. It appears that OR gene repertoires change dynamically, depending on each organism's living environment. For example, higher primates equipped with a well-developed vision system have lost a large number of OR genes. Moreover, two groups of OR genes for detecting airborne odorants greatly expanded after the time of terrestrial adaption in the tetrapod lineage, whereas fishes retain diverse repertoires of genes that were present in aquatic ancestral species. The origin of vertebrate OR genes can be traced back to the common ancestor of all chordate species, but insects, nematodes and echinoderms utilise distinctive families of chemoreceptors, suggesting that chemoreceptor genes have evolved many times independently in animal evolution.

Diversity of preferred nucleotide sequences around the translation initiation codon in eukaryote genomes.

Understanding regulatory mechanisms of protein synthesis in eukaryotes is essential for the accurate annotation of genome sequences. Kozak reported that the nucleotide sequence GCCGCC(A/G)CCAUGG (AUG is the initiation codon) was frequently observed in vertebrate genes and that this 'consensus' sequence enhanced translation initiation. However, later studies using invertebrate, fungal and plant genes reported different 'consensus' sequences. In this study, we conducted extensive comparative analyses of nucleotide sequences around the initiation codon by using genomic data from 47 eukaryote species including animals, fungi, plants and protists. The analyses revealed that preferred nucleotide sequences are quite diverse among different species, but differences between patterns of nucleotide bias roughly reflect the evolutionary relationships of the species. We also found strong biases of A/G at position -3, A/C at position -2 and C at position +5 that were commonly observed in all species examined. Genes with higher expression levels showed stronger signals, suggesting that these nucleotides are responsible for the regulation of translation initiation. The diversity of preferred nucleotide sequences around the initiation codon might be explained by differences in relative contributions from two distinct patterns, GCCGCCAUG and AAAAAAAUG, which implies the presence of multiple molecular mechanisms for controlling translation initiation.

Comparative genomic analyses illuminate the distinct evolution of megabats within Chiroptera.

The revision of the sub-order Microchiroptera is one of the most intriguing outcomes in recent mammalian molecular phylogeny. The unexpected sister-taxon relationship between rhinolophoid microbats and megabats, with the exclusion of other microbats, suggests that megabats arose in a relatively short period of time from a microbat-like ancestor. In order to understand the genetic mechanism underlying adaptive evolution in megabats, we determined the whole-genome sequences of two rousette megabats, Leschenault's rousette (Rousettus leschenaultia) and the Egyptian fruit bat (R. aegyptiacus). The sequences were compared with those of 22 other mammals, including nine bats, available in the database. We identified that megabat genomes are distinct in that they have extremely low activity of SINE retrotranspositions, expansion of two chemosensory gene families, including the trace amine receptor (TAAR) and olfactory receptor (OR), and elevation of the dN/dS ratio in genes for immunity and protein catabolism. The adaptive signatures discovered in the genomes of megabats may provide crucial insight into their distinct evolution, including key processes such as virus resistance, loss of echolocation, and frugivorous feeding.

Similar numbers but different repertoires of olfactory receptor genes in humans and chimpanzees.

Animals recognize their external world through the detection of tens of thousands of chemical odorants. Olfactory receptor (OR) genes encode proteins for detecting odorant molecules and form the largest multigene family in mammals. It is known that humans have fewer OR genes and a higher fraction of OR pseudogenes than mice or dogs. To investigate whether these features are human specific or common to all higher primates, we identified nearly complete sets of OR genes from the chimpanzee and macaque genomes and compared them with the human OR genes. In contrast to previous studies, here we show that the number of OR genes ( approximately 810) and the fraction of pseudogenes (51%) in chimpanzees are very similar to those in humans, though macaques have considerably fewer OR genes. The pseudogenization rates and the numbers of genes affected by positive selection are also similar between humans and chimpanzees. Moreover, the most recent common ancestor between humans and chimpanzees had a larger number of functional OR genes (>500) and a lower fraction of pseudogenes (41%) than its descendents, suggesting that the OR gene repertoires are in a phase of deterioration in both lineages. Interestingly, despite the close evolutionary relationship between the 2 species, approximately 25% of their functional gene repertoires are species specific due to massive gene losses. These findings suggest that the tempo of evolution of OR genes is similar between humans and chimpanzees, but the OR gene repertoires are quite different between them. This difference might be responsible for the species-specific ability of odor perception.

A long-range cis-regulatory element for class I odorant receptor genes.

Individual olfactory sensory neurons express a single odorant receptor gene from either class I genes residing in a single cluster on a single chromosome or class II genes spread over multiple clusters on multiple chromosomes. Here, we identify an enhancer element for mouse class I genes, the J element, that is conserved through mammalian species from the platypus to humans. The J element regulates most class I genes expression by exerting an effect over ~ 3 megabases within the whole cluster. Deletion of the trans J element increases the expression frequencies of class I genes from the intact J allele, indicating that the allelic exclusion of class I genes depends on the activity of the J element. Our data reveal a long-range cis-regulatory element that governs the singular class I gene expression and has been phylogenetically preserved to retain a single cluster organization of class I genes in mammals."Each olfactory sensory neuron expresses a single odorant receptor gene from either class I or class II genes. Here, the authors identify an enhancer for mouse class I genes, that is highly conserved, and regulates most class I genes expression by acting over ~ 3 megabases within the whole cluster."