Identification and characterization of cichlid TAAR genes and comparison with other teleost TAAR repertoires

Background

TAARs (trace amine-associated receptors) are among the principal receptors expressed by the olfactory epithelium. We used the recent BROAD Institute release of the genome sequences of five representative fishes of the cichlid family to establish the complete TAAR repertoires of these species and to compare them with five other fish TAAR repertoires.

Results

The genome sequences of O. niloticus, P. nyererei, H. burtoni, N. brichardi and M. zebra were analyzed by exhaustive TBLASTN searches with a set of published TAAR gene sequences used as positive bait. A second TBLASTN analysis was then performed on the candidate genes, with a set of non-TAAR class A GPCR (G protein-coupled receptors) used as negative bait. The resulting cichlid repertoire contained 44 complete TAAR genes from O. niloticus, 18 from P. nyererei, 23 from H. burtoni, 12 from N. brichardi and 20 from M. zebra, plus a number of pseudogenes, edge genes and fragments. A large proportion of these sequences (80%) consisted of two coding exons, separated in all but two cases by an intron in the interloop 1 coding sequence. We constructed phylogenetic trees. These trees indicated that TAARs constitute a distinct clade, well separated from ORs (olfactory receptors) and other class A GPCRs. Also these repertoires consist of several families and subfamilies, a number of which are common to fugu, tetraodon, stickleback and medaka. Like all other TAARs identified to date, cichlid TAARs have a characteristic two-dimensional structure and contain a number of amino-acid motifs or amino acids, such cysteine, in particular conserved positions.

Conclusions

Little is known about the functions of TAARs: in most cases their ligands have yet to be identified, partly because appropriate methods for such investigations have not been developed. Sequences analyses and comparisons of TAARs in several animal species, here fishes living in the same environment, should help reveal their roles and whether they are complementary to that of ORs.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1478-4) contains supplementary material, which is available to authorized users.     

The dog and rat olfactory receptor repertoires

Background

Dogs and rats have a highly developed capability to detect and identify odorant molecules, even at minute concentrations. Previous analyses have shown that the olfactory receptors (ORs) that specifically bind odorant molecules are encoded by the largest gene family sequenced in mammals so far.

Results

We identified five amino acid patterns characteristic of ORs in the recently sequenced boxer dog and brown Norway rat genomes. Using these patterns, we retrieved 1,094 dog genes and 1,493 rat genes from these shotgun sequences. The retrieved sequences constitute the olfactory receptor repertoires of these two animals. Subsets of 20.3% (for the dog) and 19.5% (for the rat) of these genes were annotated as pseudogenes as they had one or several mutations interrupting their open reading frames. We performed phylogenetic studies and organized these two repertoires into classes, families and subfamilies.

Conclusion

We have established a complete or almost complete list of OR genes in the dog and the rat and have compared the sequences of these genes within and between the two species. Our results provide insight into the evolutionary development of these genes and the local amplifications that have led to the specific amplification of many subfamilies. We have also compared the human and rat ORs with the human and mouse OR repertoires.     

Mesencephalic dopaminergic neurons express a repertoire of olfactory receptors and respond to odorant-like molecules

Background

The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the Substantia Nigra (SN) (A9 neurons) and the Ventral Tegmental Area (VTA) (A10 cells). Selective degeneration of A9 neurons occurs in Parkinson’s disease (PD) while abnormal function of A10 cells has been linked to schizophrenia, attention deficit and addiction. The molecular basis that underlies selective vulnerability of A9 and A10 neurons is presently unknown.

Results

By taking advantage of transgenic labeling, laser capture microdissection coupled to nano Cap-Analysis of Gene Expression (nanoCAGE) technology on isolated A9 and A10 cells, we found that a subset of Olfactory Receptors (OR)s is expressed in mDA neurons. Gene expression analysis was integrated with the FANTOM5 Helicos CAGE sequencing datasets, showing the presence of these ORs in selected tissues and brain areas outside of the olfactory epithelium. OR expression in the mesencephalon was validated by RT-PCR and in situ hybridization. By screening 16 potential ligands on 5 mDA ORs recombinantly expressed in an heterologous in vitro system, we identified carvone enantiomers as agonists at Olfr287 and able to evoke an intracellular Ca²⁺ increase in solitary mDA neurons. ORs were found expressed in human SN and down-regulated in PD post mortem brains.

Conclusions

Our study indicates that mDA neurons express ORs and respond to odor-like molecules providing new opportunities for pharmacological intervention in disease.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-729) contains supplementary material, which is available to authorized users.     

Copy number variation in the speciation of pigs: a possible prominent role for olfactory receptors

Background

Unraveling the genetic mechanisms associated with reduced gene flow between genetically differentiated populations is key to understand speciation. Different types of structural variations (SVs) have been found as a source of genetic diversity in a wide range of species. Previous studies provided detailed knowledge on the potential evolutionary role of SVs, especially copy number variations (CNVs), between well diverged species of e.g. primates. However, our understanding of their significance during ongoing speciation processes is limited due to the lack of CNV data from closely related species. The genus Sus (pig and its close relatives) which started to diverge ~4 Mya presents an excellent model for studying the role of CNVs during ongoing speciation.

Results

In this study, we identified 1408 CNV regions (CNVRs) across the genus Sus. These CNVRs encompass 624 genes and were found to evolve ~2.5 times faster than single nucleotide polymorphisms (SNPs). The majority of these copy number variable genes are olfactory receptors (ORs) known to play a prominent role in food foraging and mate recognition in Sus. Phylogenetic analyses, including novel Bayesian analysis, based on CNVRs that overlap ORs retain the well-accepted topology of the genus Sus whereas CNVRs overlapping genes other than ORs show evidence for random drift and/or admixture.

Conclusion

We hypothesize that inter-specific variation in copy number of ORs provided the means for rapid adaptation to different environments during the diversification of the genus Sus in the Pliocene. Furthermore, these regions might have acted as barriers preventing massive gene flow between these species during the multiple hybridization events that took place later in the Pleistocene suggesting a possible prominent role of ORs in the ongoing Sus speciation.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1449-9) contains supplementary material, which is available to authorized users.     

Heteromeric Anopheline odorant receptors exhibit distinct channel properties

Background

Insect odorant receptors (ORs) function as odorant-gated ion channels consisting of a conventional, odorant-binding OR and the Orco coreceptor. While Orco can function as a homomeric ion channel, the role(s) of the conventional OR in heteromeric OR complexes has largely focused only on odorant recognition.

Results

To investigate other roles of odorant-binding ORs, we have employed patch clamp electrophysiology to investigate the properties of the channel pore of several OR complexes formed by a range of different odorant-specific Anopheles gambiae ORs (AgOrs) each paired with AgOrco. These studies reveal significant differences in cation permeability and ruthenium red susceptibility among different AgOr complexes.

Conclusions

With observable differences in channel function, the data support a model in which the odorant-binding OR also affects the channel pore. The variable effect contributed by the conventional OR on the conductive properties of odorant-gated sensory channels adds additional complexity to insect olfactory signaling, with differences in odor coding beginning with ORs on the periphery of the olfactory system.     

Comparison of the canine and human olfactory receptor gene repertoires

Background

Olfactory receptors (ORs), the first dedicated molecules with which odorants physically interact to arouse an olfactory sensation, constitute the largest gene family in vertebrates, including around 900 genes in human and 1,500 in the mouse. Whereas dogs, like many other mammals, have a much keener olfactory potential than humans, only 21 canine OR genes have been described to date.

Results

In this study, 817 novel canine OR sequences were identified, and 640 have been characterized. Of the 661 characterized OR sequences, representing half of the canine repertoire, 18% are predicted to be pseudogenes, compared with 63% in human and 20% in mouse. Phylogenetic analysis of 403 canine OR sequences identified 51 families, and radiation-hybrid mapping of 562 showed that they are distributed on 24 dog chromosomes, in 37 distinct regions. Most of these regions constitute clusters of 2 to 124 closely linked genes. The two largest clusters (124 and 109 OR genes) are located on canine chromosomes 18 and 21. They are orthologous to human clusters located on human chromosomes 11q11-q13 and HSA11p15, containing 174 and 115 ORs respectively.

Conclusions

This study shows a strongly conserved genomic distribution of OR genes between dog and human, suggesting that OR genes evolved from a common mammalian ancestral repertoire by successive duplications. In addition, the dog repertoire appears to have expanded relative to that of humans, leading to the emergence of specific canine OR genes.

     

An improved genome reference for the African cichlid,Metriaclima zebra

Background

Problems associated with using draft genome assemblies are well documented and have become more pronounced with the use of short read data for de novo genome assembly. We set out to improve the draft genome assembly of the African cichlid fish, Metriaclima zebra, using a set of Pacific Biosciences SMRT sequencing reads corresponding to 16.5× coverage of the genome. Here we characterize the improvements that these long reads allowed us to make to the state-of-the-art draft genome previously assembled from short read data.

Results

Our new assembly closed 68 % of the existing gaps and added 90.6Mbp of new non-gap sequence to the existing draft assembly of M. zebra. Comparison of the new assembly to the sequence of several bacterial artificial chromosome clones confirmed the accuracy of the new assembly. The closure of sequence gaps revealed thousands of new exons, allowing significant improvement in gene models. We corrected one known misassembly, and identified and fixed other likely misassemblies. 63.5 Mbp (70 %) of the new sequence was classified as repetitive and the new sequence allowed for the assembly of many more transposable elements.

Conclusions

Our improvements to the M. zebra draft genome suggest that a reasonable investment in long reads could greatly improve many comparable vertebrate draft genome assemblies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1930-5) contains supplementary material, which is available to authorized users.     

The Arabidopsis Kinome: phylogeny and evolutionary insights into functional diversification

Background

Protein kinases constitute a particularly large protein family in Arabidopsis with important functions in cellular signal transduction networks. At the same time Arabidopsis is a model plant with high frequencies of gene duplications. Here, we have conducted a systematic analysis of the Arabidopsis kinase complement, the kinome, with particular focus on gene duplication events. We matched Arabidopsis proteins to a Hidden-Markov Model of eukaryotic kinases and computed a phylogeny of 942 Arabidopsis protein kinase domains and mapped their origin by gene duplication.

Results

The phylogeny showed two major clades of receptor kinases and soluble kinases, each of which was divided into functional subclades. Based on this phylogeny, association of yet uncharacterized kinases to families was possible which extended functional annotation of unknowns. Classification of gene duplications within these protein kinases revealed that representatives of cytosolic subfamilies showed a tendency to maintain segmentally duplicated genes, while some subfamilies of the receptor kinases were enriched for tandem duplicates. Although functional diversification is observed throughout most subfamilies, some instances of functional conservation among genes transposed from the same ancestor were observed. In general, a significant enrichment of essential genes was found among genes encoding for protein kinases.

Conclusions

The inferred phylogeny allowed classification and annotation of yet uncharacterized kinases. The prediction and analysis of syntenic blocks and duplication events within gene families of interest can be used to link functional biology to insights from an evolutionary viewpoint. The approach undertaken here can be applied to any gene family in any organism with an annotated genome.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-548) contains supplementary material, which is available to authorized users.     

Identification of Cichlid Fishes from Lake Malawi Using Computer Vision

Background

The explosively radiating evolution of cichlid fishes of Lake Malawi has yielded an amazing number of haplochromine species estimated as many as 500 to 800 with a surprising degree of diversity not only in color and stripe pattern but also in the shape of jaw and body among them. As these morphological diversities have been a central subject of adaptive speciation and taxonomic classification, such high diversity could serve as a foundation for automation of species identification of cichlids.

Methodology/Principal Finding

Here we demonstrate a method for automatic classification of the Lake Malawi cichlids based on computer vision and geometric morphometrics. For this end we developed a pipeline that integrates multiple image processing tools to automatically extract informative features of color and stripe patterns from a large set of photographic images of wild cichlids. The extracted information was evaluated by statistical classifiers Support Vector Machine and Random Forests. Both classifiers performed better when body shape information was added to the feature of color and stripe. Besides the coloration and stripe pattern, body shape variables boosted the accuracy of classification by about 10%. The programs were able to classify 594 live cichlid individuals belonging to 12 different classes (species and sexes) with an average accuracy of 78%, contrasting to a mere 42% success rate by human eyes. The variables that contributed most to the accuracy were body height and the hue of the most frequent color.

Conclusions

Computer vision showed a notable performance in extracting information from the color and stripe patterns of Lake Malawi cichlids although the information was not enough for errorless species identification. Our results indicate that there appears an unavoidable difficulty in automatic species identification of cichlid fishes, which may arise from short divergence times and gene flow between closely related species.     

Sex-linked pheromone receptor genes of the European corn borer, Ostrinia nubilalis, are in tandem arrays

Background

Tuning of the olfactory system of male moths to conspecific female sex pheromones is crucial for correct species recognition; however, little is known about the genetic changes that drive speciation in this system. Moths of the genus Ostrinia are good models to elucidate this question, since significant differences in pheromone blends are observed within and among species. Odorant receptors (ORs) play a critical role in recognition of female sex pheromones; eight types of OR genes expressed in male antennae were previously reported in Ostrinia moths.

Methodology/Principal Findings

We screened an O. nubilalis bacterial artificial chromosome (BAC) library by PCR, and constructed three contigs from isolated clones containing the reported OR genes. Fluorescence in situ hybridization (FISH) analysis using these clones as probes demonstrated that the largest contig, which contained eight OR genes, was located on the Z chromosome; two others harboring two and one OR genes were found on two autosomes. Sequence determination of BAC clones revealed the Z-linked OR genes were closely related and tandemly arrayed; moreover, four of them shared 181-bp direct repeats spanning exon 7 and intron 7.

Conclusions/Significance

This is the first report of tandemly arrayed sex pheromone receptor genes in Lepidoptera. The localization of an OR gene cluster on the Z chromosome agrees with previous findings for a Z-linked locus responsible for O. nubilalis male behavioral response to sex pheromone. The 181-bp direct repeats might enhance gene duplications by unequal crossovers. An autosomal locus responsible for male response to sex pheromone in Heliothis virescens and H. subflexa was recently reported to contain at least four OR genes. Taken together, these findings support the hypothesis that generation of additional copies of OR genes can increase the potential for male moths to acquire altered specificity for pheromone components, and accordingly, facilitate differentiation of sex pheromones.     

Innate Immunity and Non-Hodgkin’s Lymphoma (NHL) Related Genes in a Nested Case-Control Study for Gastric Cancer Risk

Objective

Genetic variants regulating the host immune system may contribute to the susceptibility for the development of gastric cancer. Little is known about the role of the innate immunity- and non-Hodgkin’s lymphoma (NHL)-related genes for gastric cancer risk. This nested case-control study was conducted to identify candidate genes for gastric cancer risk for future studies.

Methods

In the Discovery phase, 3,072 SNPs in 203 innate immunity- and 264 NHL-related genes using the Illumine GoldenGateTM OPA Panel were analyzed in 42 matched case-control sets selected from the Korean Multi-center Cancer Cohort (KMCC). Six significant SNPs in four innate immunity (DEFA6, DEFB1, JAK3, and ACAA1) and 11 SNPs in nine NHL-related genes (INSL3, CHMP7, BCL2L11, TNFRSF8, RAD50, CASP7, CHUK, CD79B, and CLDN9) with a permutated p-value <0.01 were re-genotyped in the Replication phase among 386 cases and 348 controls. Odds ratios (ORs) for gastric cancer risk were estimated adjusting for age, smoking status, and H. pylori and CagA sero-positivity. Summarized ORs in the total study population (428 cases and 390 controls) are presented using pooled- and meta-analyses.

Results

Four SNPs had no heterogeneity across the phases: in the meta-analysis, DEFA6 rs13275170 and DEFB1 rs2738169 had both a 1.3-fold increased odds ratio (OR) for gastric cancer (95% CIs = 1.1–1.6; and 1.1–1.5, respectively). INSL3 rs10421916 and rs11088680 had both a 0.8-fold decreased OR for gastric cancer (95% CIs = 0.7–0.97; and 0.7–0.9, respectively).

Conclusions

Our findings suggest that certain variants in the innate immunity and NHL-related genes affect the gastric cancer risk, perhaps by modulating infection-inflammation-immunity mechanisms that remain to be defined.     

Parallel evolution in Ugandan crater lakes: repeated evolution of limnetic body shapes in haplochromine cichlid fish

Background

The enormous diversity found in East African cichlid fishes in terms of morphology, coloration, and behavior have made them a model for the study of speciation and adaptive evolution. In particular, haplochromine cichlids, by far the most species-rich lineage of cichlids, are a well-known textbook example for parallel evolution. Southwestern Uganda is an area of high tectonic activity, and is home to numerous crater lakes. Many Ugandan crater lakes were colonized, apparently independently, by a single lineage of haplochromine cichlids. Thereby, this system could be considered a natural experiment in which one can study the interaction between geographical isolation and natural selection promoting phenotypic diversification.

Results

We sampled 13 crater lakes and six potentially-ancestral older lakes and, using both mitochondrial and microsatellite markers, discovered strong genetic and morphological differentiation whereby (a) geographically close lakes tend to be genetically more similar and (b) three different geographic areas seem to have been colonized by three independent waves of colonization from the same source population. Using a geometric morphometric approach, we found that body shape elongation (i.e. a limnetic morphology) evolved repeatedly from the ancestral deeper-bodied benthic morphology in the clear and deep crater lake habitats.

Conclusions

A pattern of strong genetic and morphological differentiation was observed in the Ugandan crater lakes. Our data suggest that body shape changes have repeatedly evolved into a more limnetic-like form in several Ugandan crater lakes after independent waves of colonization from the same source population. The observed morphological changes in crater lake cichlids are likely to result from a common selective regime.

Electronic supplementary material

The online version of this article (doi:10.1186/s12862-015-0287-3) contains supplementary material, which is available to authorized users.     

An Odorant Receptor from the Southern House Mosquito Culex pipiens quinquefasciatus Sensitive to Oviposition Attractants

Background

Insect odorant receptors (ORs) are heteromers comprised of highly variable odorant-binding subunits associated with one conserved co-receptor. They are potential molecular targets for the development of novel mosquito attractants and repellents. ORs have been identified in the malaria mosquito, Anopheles gambiae, and in the yellow fever mosquito, Aedes aegypti. However, they are still unknown in the Southern house mosquito, Culex quinquefasciatus, which transmits pathogens that cause human diseases throughout the world, including West Nile Virus in the United States.

Methodology

We have employed a combination of bioinformatics, molecular cloning and electrophysiology approaches to identify and characterize the response profile of an OR in Cx. quinquefasciatus. First, we have unveiled a large multigenic family of one-hundred-fifty-eight putative ORs in this species, including a subgroup of conserved ORs in three mosquito species. Using the Xenopus oocytes expression system, we have determined the response profile of CquiOR2, an antennae-specific OR, which shares high identity with putative orthologs in Anopheles gambiae (AgamOR2) and Aedes aegypti (AaegOR2).

Conclusion

We show that CquiOR2 is highly sensitive to indole, an oviposition attractant for Cx. quinquefasciatus. The response profile of CquiOR2 expressed in Xenopus oocytes resembles that of an olfactory receptor neuron housed in the antennal short blunt-tipped sensilla (A2) of Cx. quinquefasciatus, which are natural detectors for oviposition attractants. This first Culex OR de-orphanized is, therefore, a potential molecular target for screening oviposition attractants.     

Nicotinic acetylcholine receptor variants associated with susceptibility to chronic obstructive pulmonary disease: a meta-analysis

Background

Only 10-15% of smokers develop chronic obstructive pulmonary disease (COPD) which indicates genetic susceptibility to the disease. Recent studies suggested an association between COPD and polymorphisms in CHRNA coding subunits of nicotinic acetylcholine receptor. Herein, we performed a meta-analysis to clarify the impact of CHRNA variants on COPD.

Methods

We searched Web of Knowledge and Medline from 1990 through June 2011 for COPD gene studies reporting variants on CHRNA. Pooled odds ratios (ORs) were calculated using the major allele or genotype as reference group.

Results

Among seven reported variants in CHRNA, rs1051730 was finally analyzed with sufficient studies. Totally 3460 COPD and 11437 controls from 7 individual studies were pooled-analyzed. A-allele of rs1051730 was associated with an increased risk of COPD regardless of smoking exposure (pooled OR = 1.26, 95% CI 1.18-1.34, p < 10^-5). At the genotypic level, the ORs gradually increased per A-allele (OR = 1.27 and 1.50 for GA and AA respectively, p < 10^-5). Besides, AA genotype exhibited an association with reduced FEV1% predicted (mean difference 3.51%, 95%CI 0.87-6.16%, p = 0.009) and increased risk of emphysema (OR 1.93, 95%CI 1.29-2.90, p = 0.001).

Conclusions

Our findings suggest that rs1051730 in CHRNA is a susceptibility variant for COPD, in terms of both airway obstruction and parenchyma destruction.     

Trichomonas Transmembrane Cyclases Result from Massive Gene Duplication and Concomitant Development of Pseudogenes

Background

Trichomonas vaginalis has an unusually large genome (∼160 Mb) encoding ∼60,000 proteins. With the goal of beginning to understand why some Trichomonas genes are present in so many copies, we characterized here a family of ∼123 Trichomonas genes that encode transmembrane adenylyl cyclases (TMACs).

Methodology/Principal Findings

The large family of TMACs genes is the result of recent duplications of a small set of ancestral genes that appear to be unique to trichomonads. Duplicated TMAC genes are not closely associated with repetitive elements, and duplications of flanking sequences are rare. However, there is evidence for TMAC gene replacements by homologous recombination. A high percentage of TMAC genes (∼46%) are pseudogenes, as they contain stop codons and/or frame shifts, or the genes are truncated. Numerous stop codons present in the genome project G3 strain are not present in orthologous genes of two other Trichomonas strains (S1 and B7RC2). Each TMAC is composed of a series of N-terminal transmembrane helices and a single C-terminal cyclase domain that has adenylyl cyclase activity. Multiple TMAC genes are transcribed by Trichomonas cloned by limiting dilution.

Conclusions/Significance

We conclude that one reason for the unusually large genome of Trichomonas is the presence of unstable families of genes such as those encoding TMACs that are undergoing massive gene duplication and concomitant development of pseudogenes.     

Insect Repellents: Modulators of Mosquito Odorant Receptor Activity

Background

DEET, 2-undecanone (2-U), IR3535 and Picaridin are widely used as insect repellents to prevent interactions between humans and many arthropods including mosquitoes. Their molecular action has only recently been studied, yielding seemingly contradictory theories including odorant-dependent inhibitory and odorant-independent excitatory activities on insect olfactory sensory neurons (OSNs) and odorant receptor proteins (ORs).

Methodology/Principal Findings

Here we characterize the action of these repellents on two Aedes aegypti ORs, AaOR2 and AaOR8, individually co-expressed with the common co-receptor AaOR7 in Xenopus oocytes; these ORs are respectively activated by the odors indole (AaOR2) and (R)-(−)-1-octen3-ol (AaOR8), odorants used to locate oviposition sites and host animals. In the absence of odorants, DEET activates AaOR2 but not AaOR8, while 2-U activates AaOR8 but not AaOR2; IR3535 and Picaridin do not activate these ORs. In the presence of odors, DEET strongly inhibits AaOR8 but not AaOR2, while 2-U strongly inhibits AaOR2 but not AaOR8; IR3535 and Picaridin strongly inhibit both ORs.

Conclusions/Significance

These data demonstrate that repellents can act as olfactory agonists or antagonists, thus modulating OR activity, bringing concordance to conflicting models.