LUSH odorant-binding protein mediates chemosensory responses to alcohols in Drosophila melanogaster.

The molecular mechanisms mediating chemosensory discrimination in insects are unknown. Using the enhancer trapping approach, we identified a new Drosophila mutant, lush, with odorant-specific defects in olfactory behavior. lush mutant flies are abnormally attracted to high concentrations of ethanol, propanol, and butanol but have normal chemosensory responses to other odorants. We show that wild-type flies have an active olfactory avoidance mechanism to prevent attraction to concentrated alcohol, and this response is defective in lush mutants. This suggests that the defective olfactory behavior associated with the lush mutation may result from a specific defect in chemoavoidance. lush mutants have a 3-kb deletion that produces a null allele of a new member of the invertebrate odorant-binding protein family, LUSH. LUSH is normally expressed exclusively in a subset of trichoid chemosensory sensilla located on the ventral-lateral surface of the third antennal segment. LUSH is secreted from nonneuronal support cells into the sensillum lymph that bathes the olfactory neurons within these sensilla. Reintroduction of a cloned wild-type copy of lush into the mutant background completely restores wild-type olfactory behavior, demonstrating that this odorant-binding protein is required in a subset of sensilla for normal chemosensory behavior to a subset of odorants. These findings provide direct evidence that odorant-binding proteins are required for normal chemosensory behavior in Drosophila and may partially determine the chemical specificity of olfactory neurons in vivo.     

The invertebrate odorant-binding protein LUSH is required for normal olfactory behavior in Drosophila

The invertebrate odorant-binding proteins consist of a large family of low-molecular-weight, highly divergent proteins expressed exclusively in the chemosensory sensilla of insects. Each member of this family studied to date is secreted into the sensillum lymph of a small subset of sensilla by non-neuronal support cells. These expression patterns suggests an odor-specific function for these proteins as opposed to a general role in sensillum biology. Consistent with this notion, mutants defective for LUSH, a Drosophila member of this family, have odor-specific defects in olfactory behavior. The Drosophila genome contains at least 32 members of this gene family, rivaling the number of odorant receptors in this species. The relationship between these two protein families and how they act to determine odor specificity of olfactory neurons will be the topic of future studies.     

Structure of a specific alcohol-binding site defined by the odorant binding protein LUSH from Drosophila melanogaster

We have solved the high-resolution crystal structures of the Drosophila melanogaster alcohol-binding protein LUSH in complex with a series of short-chain n-alcohols. LUSH is the first known nonenzyme protein with a defined in vivo alcohol-binding function. The structure of LUSH reveals a set of molecular interactions that define a specific alcohol-binding site. A group of amino acids, Thr57, Ser52 and Thr48, form a network of concerted hydrogen bonds between the protein and the alcohol that provides a structural motif to increase alcohol-binding affinity at this site. This motif seems to be conserved in a number of mammalian ligand-gated ion channels that are directly implicated in the pharmacological effects of alcohol. Further, these sequences are found in regions of ion channels that are known to confer alcohol sensitivity. We suggest that the alcohol-binding site in LUSH represents a general model for alcohol-binding sites in proteins.     

Gustatory organs of Drosophila melanogaster: fine structure and expression of the putative odorant-binding protein PBPRP2

In Drosophila, as in most insects, gustation is mediated by sensory hairs located on the external and internal parts of the proboscis and on the legs and wings. We describe in detail the organization and ultrastructure of the gustatory sensilla on the labellum and legs and the distribution of PBPRP2, a putative odorant-binding protein, in the gustatory organs of Drosophila. The labellum carries two kinds of sensilla: taste bristles and taste pegs. The former have the typical morphology of gustatory sensilla and can be further subdivided into three morphological subtypes, each with a stereotyped distribution and innervation. Taste pegs have a unique morphology and are innervated by two receptor cells: one mechanoreceptor and the other a putative chemoreceptor cell. PBPRP2 is abundantly expressed in all adult gustatory organs on labellum, legs, and wings and in the internal taste organs on the proboscis. In contrast to olfactory organs, where PBPRP2 is expressed in the epidermis, this protein is absent from the epidermis of labial palps and legs. In the taste bristles of the labellum and legs, PBPRP2 is localized in the crescent-shaped lumen of the sensilla, and not in the lumen where the dendrites of the gustatory neurons are found, making a function in stimulus transport unlikely in these sensilla. In contrast, PBPRP2 in peg sensilla is expressed in the inner sensillum-lymph cavity and is in contact with the dendrites. Thus, PBPRP2 could be involved as a carrier for hydrophobic ligands, e.g., bitter tastants, in these sensilla.     

Single particle EM studies of the Drosophila melanogaster origin recognition complex and evidence for DNA wrapping

Hyperphosphorylation of the Drosophila melanogaster origin recognition complex (DmORC) by cyclin dependent kinases (CDKs) allows nucleotide binding but inhibits the ATPase activity of Orc1, and ablates the ATP-dependent interaction of ORC with DNA. Here we present single particle electron microscopy (EM) studies of ORC bound to nucleotide in both the dephosphorylated and hyper-phosphorylated states. 3D image reconstructions show that nucleotide binding gives rise to an analogous conformation independent of phosphorylation state. At the intermediate resolution achieved in our studies, ATP promotes changes along the toroidal core of the complex with negligible differences contributed by phosphorylation. Thus, hyperphosphorylation of DmORC does not induce meso-scale rearrangement of the ORC structure. To better understand ORC's role in origin remodeling, we performed atomic force microscopy (AFM) studies that show the contour length of a 688bp linear DNA fragment shortens by the equivalent of approximately 130bp upon ORC binding. This data, coupled with previous studies that showed a linking number change in circular DNA upon ORC binding, suggests that ORC may wrap the DNA in a manner akin to DnaA. Based on existing data and our structures, we propose a subunit arrangement for the AAA+ and winged helix domains, and in addition, speculate on a path of the 133bp of DNA around the ORC complex.     

Three odorant-binding proteins are co-expressed in sensilla trichodea of Drosophila melanogaster

Odorant-binding proteins (OBPs) are small soluble proteins present in the aqueous medium surrounding olfactory receptor neurones. In this study we examine the expression patterns of three Drosophila OBPs (LUSH=OBP76a, OS-E=OBP83b and OS-F=OBP83a), using post-embedding immunocytochemistry. All three OBPs are co-expressed in sensilla trichodea whereas sensilla intermedia show co-expression of OS-E and OS-F only, but not of LUSH. Thus, it is confirmed that an individual sensillum can contain more than one OBP, even if it comprises only a single receptor neurone, such as the subtype T-1. In s. trichodea of lush⁻ mutants, expression of OS-E and OS-F is not impaired. No other sensillum type on antenna or maxillary palp (e.g. sensilla basiconica, sensilla coeloconica) expresses LUSH, OS-E or OS-F. Within the s. trichodea the three OBPs show the same labelling pattern: the extracellular sensillum lymph in the hair lumen and the sensillum-lymph cavities are heavily labelled. Intracellularly, the three OBPs are co-localised in a variety of dense granules in all auxiliary cells, and also in the receptor neurones. Immunocytochemical data from antennal sections of flies where lush gene expression has been tagged with the reporter gene lacZ suggest that LUSH is synthesised only in the trichogen and the thecogen cells. Thus, LUSH OBP is produced and secreted by two auxiliary cells, whereas its turnover and decomposition does not appear to be restricted to these auxiliary cells but may also occur in the tormogen and receptor cells. The immunocytochemical results are discussed with respect to current concepts of the function of odorant-binding proteins.     

A high-frequency null mutant of an odorant-binding protein gene, Obp57e, in Drosophila melanogaster

We have found a null mutant of an odorant-binding protein, Obp57e, in Drosophila melanogaster. This frameshift mutation, which is a 10-bp deletion in the coding region, is at a high frequency in the Kyoto population and is also present in Taiwan and Africa. We have sequenced a 1.5-kb region including the tandemly duplicated gene, Obp57d, from 16 inbred lines sampled in Kyoto, Japan. The analyses showed a peak of nucleotide diversity and strong linkage disequilibrium around this mutation. This pattern suggests an elevated mutation rate or an influence of balancing selection in this region. The level of nucleotide divergence between D. melanogaster and D. simulans does not support the former possibility. Thus, this presence/absence polymorphism may be due to balancing selection, which takes advantage of the relatively weak functional constraint in members of a large gene family. In addition, the Obp57d gene region showed an excess of high-frequency-derived mutants that is consistent with a pattern predicted under positive natural selection.     

Patterns of nucleotide polymorphism and divergence in the odorant-binding protein genes OS-E and OS-F: analysis in the melanogaster species subgroup of Drosophila

The Olfactory Specific-E and -F genes (OS-E and OS-F) belong to the odorant-binding protein gene family, which includes the general odorant-binding proteins and the pheromone-binding proteins. In Drosophila melanogaster, these genes are arranged in tandem in a genomic region near the centromere of chromosome arm 3R. We examined the pattern of DNA sequence variation in an approximately 7-kb genomic region encompassing the two OS genes in four species of the melanogaster subgroup of Drosophila and in a population sample of D. melanogaster. We found that both the OS-E and the OS-F gene are present in all surveyed species. Nucleotide divergence estimates would support that the two genes are functional, although they diverge in their functional constraint. The pattern of nucleotide variation in D. melanogaster also differed between genes. Variation in the OS-E gene region exhibited an unusual and distinctive pattern: (i) a relatively high number of fixed amino acid replacements in the encoded protein and (ii) a peak of nucleotide polymorphism around the OS-E gene. These results are unlikely under the neutral model and suggest the action of natural selection in the evolution of the two odorant-binding protein genes.     

Behavioral analyses of mutants for two odorant-binding protein genes, Obp57d and Obp57e, in Drosophila melanogaster

The odorant-binding protein (OBP) functions in chemosensation in insects. Two OBP genes, Obp57d and Obp57e, are involved in the evolution of the unique host-plant preference in Drosophila sechellia. Comparative analysis of the Obp57d/e genomic sequences in the Drosophila melanogaster species group has revealed that the rapid evolution of Obp57d and Obp57e has resulted in functional divergence between the two genes. Here, using D. melanogaster knockout strains generated by gene targeting, we examined the roles of Obp57d and Obp57e in behavioral response to a series of fatty acids. In the taste-based oviposition-site preference assay, the knockout flies showed stronger preference for acids than wild-type flies, indicating that the normal functions of Obp57d and Obp57e are associated with the suppression of positive preference for C6-C9 acids. Heterozygotes for each knockout haplotype also exhibited a significantly different behavioral response compared with wild-type flies, suggesting that Obp57d and Obp57e have a gene dosage effect on behavior. In contrast, the wild-type and knockout flies exhibited similar responses in the feeding assay and the odor-based free-walking assay, suggesting that the two OBPs' contribution to feeding and olfactory behaviors is small. Taken together, our results demonstrated that each of Obp57d and Obp57e in D. melanogaster contributes to the determination of reproductive sites, suggesting that the two OBP genes play an important role in ecological adaptation of Drosophila.     

Effect of n-alcohols on the structure and stability of the Drosophila odorant binding protein LUSH

LUSH is an odorant binding protein expressed in the olfactory organs of Drosophila melanogaster that is required for the detection of alcohol in adult flies. Here we demonstrate that, in the absence of ligand, in vitro LUSH exists in a partial molten globule state. The presence of short-chain n-alcohols at pharmacologically relevant concentrations less than 50 mM shifts the conformational equilibrium to a more compact state that exhibits reduced binding of the fluorescent dye 1-anilino-8-naphthalenesulfonic acid. Equilibrium unfolding studies of LUSH-alcohol complexes reveal that, for a series of short-chain n-alcohols, each methylene group can contribute approximately 1 K cal mol(-1) to the overall stability of the protein-alcohol complex. Using NMR spectroscopy, we have identified the regions of LUSH that show increased conformational stability on binding alcohols. These residues primarily line the alcohol-binding pocket. The results presented here provide a direct measure of the degree of stability that alcohol imparts on LUSH. These observations may represent a model for how ethanol can stabilize alternative protein conformations in alcohol-sensitive human proteins and ultimately lead to the observed changes in higher order function throughout the central nervous system.     

Molecular evolution of odorant-binding protein genes OS-E and OS-F in Drosophila

The Drosophila olfactory genes OS-E and OS-F are members of a family of genes that encode insect odorant-binding proteins (OBPs). OBPs are believed to transport hydrophobic odorants through the aqueous fluid within olfactory sensilla to the underlying receptor proteins. The recent discovery of a large family of olfactory receptor genes in Drosophila raises new questions about the function, diversity, regulation, and evolution of the OBP family. We have investigated the OS-E and OS-F genes in a variety of Drosophila species. These studies highlight potential regions of functional significance in the OS-E and OS-F proteins, which may include a region required for interaction with receptor proteins. Our results suggest that the two genes arose by an ancient gene duplication, and that in some lineages, one or the other gene has been lost. In D. virilis, the OS-F gene shows a different spatial pattern of expression than in D. melanogaster. One of the OS-F introns shows a striking degree of conservation between the two species, and we identify a putative regulatory sequence within this intron. Finally, a phylogenetic analysis places both OS-E and OS-F within a large family of insect OBPs and OBP-like proteins.     

Rapid evolution of two odorant-binding protein genes, Obp57d and Obp57e, in the Drosophila melanogaster species group

Genes encoding odorant-binding protein (OBP) form a large family in an insect genome. Two OBP genes, Obp57d and Obp57e, were previously identified to be involved in host-plant recognition in Drosophila sechellia. Here, by comparing the genomic sequences at the Obp57d/e locus from 27 Drosophila species, we found large differences in gene number between species. Phylogenetic analysis revealed that Obp57d and Obp57e in the D. melanogaster species group arose by gene duplication of an ancestral OBP gene that remains single in the obscura species group. Further gain and loss of OBP genes were observed in several lineages in the melanogaster group. Site-specific analysis of evolutionary rate suggests that Obp57d and Obp57e have functionally diverged from each other. Thus, there are two classes of gene number differences in the Obp57d/e region: the difference of the genes that have functionally diverged from each other and the difference of the genes that appear to be functionally identical. Our analyses demonstrate that these two classes of differences can be distinguished by comparisons of many genomic sequences from closely related species.     

Drosophila OBP LUSH is required for activity of pheromone-sensitive neurons

Odorant binding proteins (OBPs) are extracellular proteins localized to the chemosensory systems of most terrestrial species. OBPs are expressed by nonneuronal cells and secreted into the fluid bathing olfactory neuron dendrites. Several members have been shown to interact directly with odorants, but the significance of this is not clear. We show that the Drosophila OBP lush is completely devoid of evoked activity to the pheromone 11-cis vaccenyl acetate (VA), revealing that this binding protein is absolutely required for activation of pheromone-sensitive chemosensory neurons. lush mutants are also defective for pheromone-evoked behavior. Importantly, we identify a genetic interaction between lush and spontaneous activity in VA-sensitive neurons in the absence of pheromone. The defects in spontaneous activity and VA sensitivity are reversed by germline transformation with a lush transgene or by introducing recombinant LUSH protein into mutant sensilla. These studies directly link pheromone-induced behavior with OBP-dependent activation of a subset of olfactory neurons.     

Site-specific instability in Drosophila melanogaster: evidence for transposition of destabilizing element

In this study, we show that at least one lethal mutation at the 3F-4A region of the X chromosome can generate an array of chromosome rearrangements, all with one chromosome break in the 3F-4A region. The mutation at 3F-4A (secondary mutation) was detected in an X chromosome carrying a reverse mutation of an unstable lethal mutation, which was mapped in the 6F1-2 doublet (primary mutation). The primary lethal mutation at 6F1-2 had occurred in an unstable chromosome (Uc) described previously (LIM 1979). Prior to reversion, the fF1-2 doublet was normal and stable, as was the 3F-4A region in the X chromosome carrying the primary lethal mutation. The disappearance of the instability having a set of genetic properties at one region (6F1-2) accompanied by its appearance elsewhere in the chromosome (3F-4A) implies that a transposition of the destabilizing element took place. The mutant at 3F-4A and other secondary mutants exhibited all but one (reinversion of an inversion to the normal sequence) of the eight properties of the primary lethal mutations. These observations support the view that a transposable destabilizing element is responsible for the hypermutability observed in the unstable chromosome and its derivatives.     

No evidence for postcopulatory inbreeding avoidance in Drosophila melanogaster

Selection to avoid inbreeding is predicted to vary across species due to differences in population structure and reproductive biology. Over the past decade, there have been numerous investigations of postcopulatory inbreeding avoidance, a phenomenon that first requires discrimination of mate (or sperm) relatedness and then requires mechanisms of male ejaculate tailoring and/or cryptic female choice to avoid kin. The number of studies that have found a negative association between male-female genetic relatedness and competitive fertilization success is roughly equal to the number of studies that have not found such a relationship. In the former case, the underlying mechanisms are largely unknown. The present study was undertaken to verify and expand upon a previous report of postcopulatory inbreeding avoidance in D. melanogaster, as well as to resolve underlying mechanisms of inbreeding avoidance using transgenic flies that express a sperm head-specific fluorescent tag. However, siblings did not have a lower fertilization success as compared to unrelated males in either the first (P(1) ) or second (P(2) ) mate role in sperm competition with a standard unrelated competitor male in our study population of D. melanogaster. Analyses of mating latency, copulation duration, egg production rate, and remating interval further revealed no evidence for inbreeding avoidance.