Functional dissection of Odorant binding protein genes in Drosophila melanogaster

Most organisms rely on olfaction for survival and reproduction. The olfactory system of Drosophila melanogaster is one of the best characterized chemosensory systems and serves as a prototype for understanding insect olfaction. Olfaction in Drosophila is mediated by multigene families of odorant receptors and odorant binding proteins (OBPs). Although molecular response profiles of odorant receptors have been well documented, the contributions of OBPs to olfactory behavior remain largely unknown. Here, we used RNAi-mediated suppression of Obp gene expression and measurements of behavioral responses to 16 ecologically relevant odorants to systematically dissect the functions of 17 OBPs. We quantified the effectiveness of RNAi-mediated suppression by quantitative real-time polymerase chain reaction and used a proteomic liquid chromatography and tandem mass spectrometry procedure to show target-specific suppression of OBPs expressed in the antennae. Flies in which expression of a specific OBP is suppressed often show altered behavioral responses to more than one, but not all, odorants, in a sex-dependent manner. Similarly, responses to a specific odorant are frequently affected by suppression of expression of multiple, but not all, OBPs. These results show that OBPs are essential for mediating olfactory behavioral responses and suggest that OBP-dependent odorant recognition is combinatorial.     

Contribution of olfactory and gustatory sensations of octanoic acid in the oviposition behavior of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> (Diptera: Drosophilidae)

Selection of oviposition sites in insects represents an important part of their ecological adaptation. In Drosophila fruit flies, adult preference for a particular oviposition site determines larval food, affecting fitness throughout the entire life cycle. Two odorant-binding proteins (OBPs) OBP57d and OBP57e were identified to be involved in the evolution of specific preference for the toxic plant Morinda citrifolia L. in D. sechellia Tsacas &; Bächli. D. melanogaster Meigen mutants for Obp57d and Obp57e showed enhanced preference for octanoic acid, but still not as much as D. sechellia does, indicating that other genes are also involved in the behavioral evolution of D. sechellia. Here, by using an improved method for behavioral assay, we found that the ablation of antenna enhanced the preference for octanoic acid in the Obp57d and Obp57e mutants to a level comparable with D. sechellia, suggesting that both olfactory and gustatory sensations are involved in oviposition site selection in response to octanoic acid. Behavioral analysis of gene-knockout strains revealed that Odorant receptor co-receptor (Orco) has little contribution compared with the effect of antennal ablation. These data suggest that in addition to Obp57d and Obp57e, the evolution of D. sechellia involves genetic changes in olfactory genes that function independently from Orco.     

The PI/GLO-like locus in orchids: duplication and purifying selection at synonymous sites within Orchidinae (Orchidaceae)

Positive selection and relaxation of purifying constraints after duplication events have driven the functional diversification of gene families involved in development. One example of this occurred within the plant MADS-box genes. The evolution of the orchid flower was driven by duplication events followed by sub- and neo-functionalization of class B DEF-like MADS-box genes, which are present at three to four copies in the orchid genome. In contrast, the orchid PI/GLO-like class B MADS-box genes have been reported thus far as single-copy loci, with the only exception of Habenaria radiata.We isolated a novel PI/GLO-like gene (OrcPI2) in Orchis italica, which is different than the previously characterized OrcPI locus. The presence of two functional paralogs of PI/GLO-like genes in orchids is detectable only within the tribe Orchidinae. Evolutionary analyses revealed an apparent relaxation of purifying selection acting on the two PI/GLO-like paralogs of the Orchidinae when compared to the single-copy PI/GLO-like genes found in other orchid species. Furthermore, by measuring dN/dS (ω) ratios, we show that a high percentage of sites between the two PI/GLO-like paralogs have different evolutionary pressures. Interestingly, the apparent relaxation of selective constraints on the two PI/GLO-like paralogs is due to strong purifying selection at synonymous sites rather than to a high value of nonsynonymous substitution rate. This peculiar evolutionary pattern might be related to molecular processes such as mRNA folding and/or translational efficiency control. These processes could potentially be involved in or predate the functional diversification of the two PI/GLO-like paralogs within Orchidinae.     

Modeling human epilepsy by TALEN targeting of mouse sodium channel Scn8a

To evaluate the efficiency of TALEN technology for introducing mutations into the mouse genome we targeted Scn8a, a member of a multigene family with nine closely related paralogs. Our goal was to generate a model of early onset epileptic encephalopathy by introduction of the Scn8a missense mutation p.Asn1768Asp. We used a pair of TALENs that were highly active in transfected cells. The targeting template for homologous recombination contained a 4 kb genomic fragment. Microinjection of TALENs with the targeting construct into the pronucleus of 350 fertilized mouse eggs generated 67 live‐born potential founders, of which 5 were heterozygous for the pathogenic mutation, a yield of 7% correctly targeted mice. Twenty‐four mice carried one or two Scn8a indels, including 12 frameshift mutations and the novel amino acid deletion p.Asn1759del. Nine off‐site mutations in the paralogs sodium channel genes Scn5a and Scn4a were identified. The data demonstrate the feasibility and efficiency of targeting members of multigene families using TALENs. The Scn8a^tm1768DMm mouse model will be useful for investigation of the pathogenesis and therapy of early onset seizure disorders. genesis 52:141–148. © 2013 The Authors genesis Published by Wiley Periodicals, Inc.     

Long-range looping of a locus control region drives tissue-specific chromatin packing within a multigene cluster

The relationships of higher order chromatin organization to mammalian gene expression remain incompletely defined. The human Growth Hormone (hGH) multigene cluster contains five gene paralogs. These genes are selectively activated in either the pituitary or the placenta by distinct components of a remote locus control region (LCR). Prior studies have revealed that appropriate activation of the placental genes is dependent not only on the actions of the LCR, but also on the multigene composition of the cluster itself. Here, we demonstrate that the hGH LCR ‘loops’ over a distance of 28 kb in primary placental nuclei to make specific contacts with the promoters of the two GH genes in the cluster. This long-range interaction sequesters the GH genes from the three hCS genes which co-assemble into a tightly packed ‘hCS chromatin hub’. Elimination of the long-range looping, via specific deletion of the placental LCR components, triggers a dramatic disruption of the hCS chromatin hub. These data reveal a higher-order structural pathway by which long-range looping from an LCR impacts on local chromatin architecture that is linked to tissue-specific gene regulation within a multigene cluster.     

Natural Variation in Odorant Recognition Among Odorant-Binding Proteins in Drosophila melanogaster

Chemical recognition is essential for survival and reproduction. Adaptive evolution has resulted in diverse chemoreceptor families, in which polymorphisms contribute to individual variation in chemosensation. To gain insights into the genetic determinants of individual variation in odorant recognition, we measured olfactory responses to two structurally similar odorants in a population of wild-derived inbred lines of Drosophila melanogaster. Odorant-binding proteins (OBPs) are the first components of the insect olfactory system to encounter odorants. Previously four single-nucleotide polymorphisms (SNPs) in the Obp99 group were associated with variation in olfactory responses to benzaldehyde. Here, we identify six different SNPs that are associated with variation in responses to a structurally similar odorant, acetophenone, in the same Obp genes. Five SNPs are in coding regions of Obp99b and Obp99d and one SNP is in the 3′-untranslated region of Obp99a (A610G). We found that the 610G allele is associated with higher response scores to acetophenone than the 610A allele, but with lower expression of Obp99a, suggesting that binding of acetophenone to Opb99a might limit rather than facilitate access to odorant receptors. Our results show that overlapping sets of OBPs contribute to odorant recognition for structurally similar odorants, but that different SNPs are associated with odorant-specific individual variation. Thus, dual olfactory recognition where OBPs regulate odorant access to receptors may enhance olfactory discrimination.ADAPTIVE evolution in diverse chemical environments has resulted in large multigene chemoreceptor families, including odorant-binding protein (Obp) genes, odorant receptor (Or) genes, and gustatory receptor (Gr) genes (Hekmat-Scafe et al. 2002; Robertson et al. 2003; Nozawa and Nei 2007; Nei et al. 2008; Su et al. 2009). Polymorphisms in these chemoreceptor genes contribute to individual variation in chemosensory behavior (Keller et al. 2007; Wang et al. 2007). At the same time, combinatorial recognition of odorants may contribute functional redundancy, which allows individual variation without compromising overall olfactory ability. This may be the reason why segregating null alleles of chemoreceptor genes can be maintained within a population (Takahashi and Takano-Shimizu 2005; Wang et al. 2007). Drosophila melanogaster presents a favorable model for investigating the genetic basis of individual variation in olfactory discrimination, because the genome can be manipulated readily. Furthermore, flies can be inbred, which enables repeated behavioral measurements on identical genotypes under controlled environmental conditions. In addition, both the olfactory and the gustatory systems of Drosophila have been well characterized (Su et al. 2009; Yarmolinsky et al. 2009). Convergent projections of olfactory neurons expressing distinct odorant receptors have been mapped to specific glomeruli in the antennal lobe (Gao et al. 2000; Vosshall et al. 2000), and detailed electrophysiological studies on transgenic flies have identified molecular response profiles of a large fraction of the odorant receptor repertoire (de Bruyne et al. 2001). Surprisingly, however, behavioral responses to odorants do not necessarily conform to predictions based on electrophysiological response profiles (Keller and Vosshall 2007).Whereas Drosophila odorant receptors have been studied extensively, less is known about the function of odorant-binding proteins (OBPs) in mediating odor recognition and olfactory discrimination. OBPs are secreted by support cells in olfactory sensilla into the aqueous perilymph that surrounds olfactory dendrites and are thought to facilitate solubilization and transport of hydrophobic odorants, thereby either promoting or limiting access of odorants to odorant receptors (Steinbrecht 1998). For example, the pheromone-binding protein of the silk moth, Bombyx mori, binds and releases bombykol in a pH-dependent manner at the membrane interface (Wojtasek and Leal 1999; Sakurai et al. 2004). In D. melanogaster, an OBP, Lush, is essential for activation of the Or67d receptor by the pheromone cis-vaccenyl acetate in trichoid sensilla of the Drosophila third antennal segment (Ha and Smith 2006; Kurtovic et al. 2007). Binding of the pheromone causes a conformational transition in Lush, which enables this OBP to activate the Or67d receptor (Laughlin et al. 2008). Lush also interacts with short chain alcohols (Kim et al. 1998), but recognition of alcohols by Lush does not involve a conformation change and, thus, proceeds via a different mechanism (Stower and Logan 2008).Polymorphisms in Obp genes can serve as a substrate for natural selection and contribute to speciation. A polymorphism in Obp57e is responsible for differences in host plant preference between D. sechellia and D. melanogaster. D. melanogaster flies lacking the Obp57e and Obp57d genes were no longer repelled by hexanoic and octanoic acid, toxins produced by Morinda citrifolia, the host plant for D. sechellia. Here, inactivation of an Obp gene has enabled D. sechellia to occupy a specialist evolutionary niche (Matsuo et al. 2007). Differences in expression levels between Ors and Obps between D. sechellia and D. simulans have also been reported and postulated to contribute to the evolution of host plant preferences (Kopp et al. 2008).Despite the demonstrated importance of OBPs in pheromone and host plant recognition, little is known about how naturally occurring allelic variation in Obp genes affects individual variation in olfactory behavior. Previously, we identified polymorphisms associated with natural variation in olfactory behavior in response to benzaldehyde in Obp99a, Obp99c, and Obp99d in a population of wild-derived inbred lines of D. melanogaster (Wang et al. 2007). These studies indicated that these OBPs are likely to recognize benzaldehyde in a combinatorial manner, similar to odorant recognition by mammalian odorant receptors (Malnic et al. 1999). This observation enables us to begin to explore OBP odorant response profiles using a population genetics approach that capitalizes on naturally occurring mutations that affect behavior. As a first step, we asked whether variation in responses to odorants that are chemically similar would be associated with the same or overlapping sets of OBPs and, if so, whether the same or different polymorphisms in these OBPs would contribute to individual variation for olfactory behavior in response to these odorants. We focused on genes of the Obp99 group, previously associated with phenotypic variation in response to benzaldehyde. We obtained complete sequences of these genes from 297 inbred lines from the same wild-derived inbred population of D. melanogaster and measured variation in olfactory behavior in response to acetophenone, which is structurally similar to benzaldehyde. These odorants occur in fruits from host plants on which flies from the Raleigh population feed (e.g., apples and peaches). We find that overlapping sets of OBPs contribute to recognition of these two odorants, but that different SNPs are associated with odorant-specific individual variation.     

Molecular population genetics of the OBP83 genomic region in Drosophila subobscura and D. guanche: contrasting the effects of natural selection and gene arrangement expansion in the patterns of nucleotide variation

Chromosomal inversion polymorphism play a major role in the evolutionary dynamics of populations and species because of their effects on the patterns of genetic variability in the genomic regions within inversions. Though there is compelling evidence for the adaptive character of chromosomal polymorphisms, the mechanisms responsible for their maintenance in natural populations is not fully understood. For this type of analysis, Drosophila subobscura is a good model species as it has a rich and extensively studied chromosomal inversion polymorphism system. Here, we examine the patterns of DNA variation in two natural populations segregating for chromosomal arrangements that differentially affect the surveyed genomic region; in particular, we analyse both nucleotide substitutions and insertion/deletion variations in the genomic region encompassing the odorant-binding protein genes Obp83a and Obp83b (Obp83 region). We show that the two main gene arrangements are genetically differentiated, but are consistent with a monophyletic origin of inversions. Nevertheless, these arrangements interchange some genetic information, likely by gene conversion. We also find that the frequency spectrum-based tests indicate that the pattern of nucleotide variation is not at equilibrium; this feature probably reflects the rapid increase in the frequency of the new gene arrangement promoted by positive selection (that is an adaptive change). Furthermore, a comparative analysis of polymorphism and divergence patterns reveals a relaxation of the functional constraints at the Obp83b gene, which might be associated with particular ecological or demographic features of the Canary island endemic species D. guanche     

Odorant-binding proteins OBP57d and OBP57e affect taste perception and host-plant preference in Drosophila sechellia

Despite its morphological similarity to the other species in the Drosophila melanogaster species complex, D. sechellia has evolved distinct physiological and behavioral adaptations to its host plant Morinda citrifolia, commonly known as Tahitian Noni. The odor of the ripe fruit of M. citrifolia originates from hexanoic and octanoic acid. D. sechellia is attracted to these two fatty acids, whereas the other species in the complex are repelled. Here, using interspecies hybrids between D. melanogaster deficiency mutants and D. sechellia, we showed that the Odorant-binding protein 57e (Obp57e) gene is involved in the behavioral difference between the species. D. melanogaster knock-out flies for Obp57e and Obp57d showed altered behavioral responses to hexanoic acid and octanoic acid. Furthermore, the introduction of Obp57d and Obp57e from D. simulans and D. sechellia shifted the oviposition site preference of D. melanogaster Obp57d/e^KO flies to that of the original species, confirming the contribution of these genes to D. sechellia's specialization to M. citrifolia. Our finding of the genes involved in host-plant determination may lead to further understanding of mechanisms underlying taste perception, evolution of plant–herbivore interactions, and speciation.     

Comparative genomics and transcriptomics in ants provide new insights into the evolution and function of odorant binding and chemosensory proteins

Background

The complex societies of ants and other social insects rely on sophisticated chemical communication. Two families of small soluble proteins, the odorant binding and chemosensory proteins (OBPs and CSPs), are believed to be important in insect chemosensation. To better understand the role of these proteins in ant olfaction, we examined their evolution and expression across the ants using phylogenetics and sex- and tissue-specific RNA-seq.

Results

We find that subsets of both OBPs and CSPs are expressed in the antennae, contradicting the previous hypothesis that CSPs have replaced OBPs in ant olfaction. Both protein families have several highly conserved clades with a single ortholog in all eusocial hymenopterans, as well as clades with more dynamic evolution and many taxon-specific radiations. The dynamically evolving OBPs and CSPs have been hypothesized to function in chemical communication. Intriguingly, we find that seven members of the conserved clades are expressed specifically in the antennae of the clonal raider ant Cerapachys biroi, whereas only one dynamically evolving CSP is antenna specific. The orthologs of the conserved, antenna-specific C. biroi genes are also expressed in antennae of the ants Camponotus floridanus and Harpegnathos saltator, indicating that antenna-specific expression of these OBPs and CSPs is conserved across ants. Most members of the dynamically evolving clades in both protein families are expressed primarily in non-chemosensory tissues and thus likely do not fulfill chemosensory functions.

Conclusions

Our results identify candidate OBPs and CSPs that are likely involved in conserved aspects of ant olfaction, and suggest that OBPs and CSPs may not rapidly evolve to recognize species-specific signals.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-718) contains supplementary material, which is available to authorized users.     

The Roles of Gene Duplication,Gene Conversion and Positive Selection in Rodent Esp and Mup Pheromone Gene Families with Comparison to the Abp Family

Three proteinaceous pheromone families, the androgen-binding proteins (ABPs), the exocrine-gland secreting peptides (ESPs) and the major urinary proteins (MUPs) are encoded by large gene families in the genomes of Mus musculus and Rattus norvegicus. We studied the evolutionary histories of the Mup and Esp genes and compared them with what is known about the Abp genes. Apparently gene conversion has played little if any role in the expansion of the mouse Class A and Class B Mup genes and pseudogenes, and the rat Mups. By contrast, we found evidence of extensive gene conversion in many Esp genes although not in all of them. Our studies of selection identified at least two amino acid sites in β-sheets as having evolved under positive selection in the mouse Class A and Class B MUPs and in rat MUPs. We show that selection may have acted on the ESPs by determining K_a/K_s for Exon 3 sequences with and without the converted sequence segment. While it appears that purifying selection acted on the ESP signal peptides, the secreted portions of the ESPs probably have undergone much more rapid evolution. When the inner gene converted fragment sequences were removed, eleven Esp paralogs were present in two or more pairs with K_a/K_s >1.0 and thus we propose that positive selection is detectable by this means in at least some mouse Esp paralogs. We compare and contrast the evolutionary histories of all three mouse pheromone gene families in light of their proposed functions in mouse communication.     

Transgenic Arabidopsis expressing osmolyte glycine betaine synthesizing enzymes from halophilic methanogen promote tolerance to drought and salt stress

Gene duplication events exert key functions on gene innovations during the evolution of the eukaryotic genomes. A large portion of the total gene content in plants arose from tandem duplications events, which often result in paralog genes with high sequence identity. Ubiquitin ligases or E3 enzymes are components of the ubiquitin proteasome system that function during the transfer of the ubiquitin molecule to the substrate. In plants, several E3s have expanded in their genomes as multigene families. To gain insight into the consequences of gene duplications on the expansion and diversification of E3s, we examined the evolutionary basis of a cluster of six genes, duplC-ATLs, which arose from segmental and tandem duplication events in Brassicaceae. The assessment of the expression suggested two patterns that are supported by lineage. While retention of expression domains was observed, an apparent absence or reduction of expression was also inferred. We found that two duplC-ATL genes underwent pseudogenization and that, in one case, gene expression is probably regained. Our findings provide insights into the evolution of gene families in plants, defining key events on the expansion of the Arabidopsis Tóxicos en Levadura family of E3 ligases.     

Dynamic Evolution of Oryza Genomes Is Revealed by Comparative Genomic Analysis of a Genus-Wide Vertical Data Set

Oryza (23 species; 10 genome types) contains the world's most important food crop — rice. Although the rice genome serves as an essential tool for biological research, little is known about the evolution of the other Oryza genome types. They contain a historical record of genomic changes that led to diversification of this genus around the world as well as an untapped reservoir of agriculturally important traits. To investigate the evolution of the collective Oryza genome, we sequenced and compared nine orthologous genomic regions encompassing the Adh1-Adh2 genes (from six diploid genome types) with the rice reference sequence. Our analysis revealed the architectural complexities and dynamic evolution of this region that have occurred over the past ~15 million years. Of the 46 intact genes and four pseudogenes in the japonica genome, 38 (76%) fell into eight multigene families. Analysis of the evolutionary history of each family revealed independent and lineage-specific gain and loss of gene family members as frequent causes of synteny disruption. Transposable elements were shown to mediate massive replacement of intergenic space (>95%), gene disruption, and gene/gene fragment movement. Three cases of long-range structural variation (inversions/deletions) spanning several hundred kilobases were identified that contributed significantly to genome diversification.     

The Cohesive Population Genetics of Molecular Drive

The long-term population genetics of multigene families is influenced by several biased and unbiased mechanisms of nonreciprocal exchanges (gene conversion, unequal exchanges, transposition) between member genes, often distributed on several chromosomes. These mechanisms cause fluctuations in the copy number of variant genes in an individual and lead to a gradual replacement of an original family of n genes (A) in N number of individuals by a variant gene (a). The process for spreading a variant gene through a family and through a population is called molecular drive. Consideration of the known slow rates of nonreciprocal exchanges predicts that the population variance in the copy number of gene a per individual is small at any given generation during molecular drive. Genotypes at a given generation are expected only to range over a small section of all possible genotypes from one extreme (n number of A) to the other (n number of a). A theory is developed for estimating the size of the population variance by using the concept of identity coefficients. In particular, the variance in the course of spreading of a single mutant gene of a multigene family was investigated in detail, and the theory of identity coefficients at the state of steady decay of genetic variability proved to be useful. Monte Carlo simulations and numerical analysis based on realistic rates of exchange in families of known size reveal the correctness of the theoretical prediction and also assess the effect of bias in turnover. The population dynamics of molecular drive in gradually increasing the mean copy number of a variant gene without the generation of a large variance (population cohesion) is of significance regarding potential interactions between natural selection and molecular drive.     

Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions

Background  

The PE and PPE multigene families of Mycobacterium tuberculosis comprise about 10% of the coding potential of the genome. The function of the proteins encoded by these large gene families remains unknown, although they have been proposed to be involved in antigenic variation and disease pathogenesis. Interestingly, some members of the PE and PPE families are associated with the ESAT-6 (esx) gene cluster regions, which are regions of immunopathogenic importance, and encode a system dedicated to the secretion of members of the potent T-cell antigen ESAT-6 family. This study investigates the duplication characteristics of the PE and PPE gene families and their association with the ESAT-6 gene clusters, using a combination of phylogenetic analyses, DNA hybridization, and comparative genomics, in order to gain insight into their evolutionary history and distribution in the genus Mycobacterium.     

Exploring the evolutionary differences of SBP-box genes targeted by miR156 and miR529 in plants

The combinatorial control of one target by multiple miRNAs brings big challenges to elucidate its precise evolutionary mechanism. Squamosa promoter binding protein-like (SBP) gene family exhibits the different regulatory patterns, in which some members are only regulated by miR156 and others by miR156 and miR529. Here, we explored the different evolutionary patterns and rates between miR156 targets and miR529 ones in three species (moss, rice, and maize). Our work found that the miR529 targets were members of miR156 target dataset, indicative of cooperative control. Further phylogenetic analyses as well as gene structure features demonstrated that miR529 targets derived from a monophyletic branch of miR156 targets which evolved into two independent branches duo to the ancient gene duplication. Moreover, inspection of evolutionary rate parameters (d_N/d_S, d_N and d_S) for miR156 targets and miR529 ones revealed they were under different selection strength. MiR529 targets were more constraint by strong purifying selection and evolved conservatively with a slow rate. By contrast, miR156 targets evolved more rapidly and experienced more relaxed purifying selection, which may contribute to their functional diversification. Our results will enhance the understanding of different evolutionary fates of SBP-box genes regulated by the different numbers of miRNA families before functional studies.