WO2003020913A2

WO2003020913A2 - Anopheles gambiae odorant receptors and genes

Info

Publication number: WO2003020913A2
Application number: PCT/US2002/028315
Authority: WO
Inventors: Kevin J. Lee; Jane Ong; Thuy-Ai T. Nguyen; Brian Kloss
Original assignee: Sentigen Corp.
Priority date: 2001-09-04
Filing date: 2002-09-04
Publication date: 2003-03-13
Also published as: WO2003020913A3; AU2002326834A8; AU2002326834A1

Abstract

The present invention relates to Anopheles gambiae odorant receptor genes and methods for identifying odorant receptor genes. The invention provides nucleotide sequences of Anopheles gambiae odorant receptor genes, amino acid sequences of their encoded proteins (including peptides or polypeptides), and related products and methods. The nucleic acids of the invention may be operatively linked to promoter sequences and transformed into host cells. Methods of production of an odorant receptor protein (e.g., by recombinant means), and derivatives and analogs thereof, are provided. Antibodies to an odorant receptor protein, and derivatives and analogs thereof, are provided. Methods for identifying molecules that bind or modulate the activity of these odorant receptor genes are provided. Molecules found to bind or modulate the activity of odorant receptor genes may be formulated into pest control agents by providing a carrier. In a preferred embodiment, molecules that bind or modulate the activity of an odorant receptor gene from Anopheles gambiae but not others is desired. Methods to modify the insect behavior by modifying an odorant receptor are also provided.

Description

NUCLEIC ACIDS AND PROTEINS OF ANOPHELES GAMBIAE ODORANT RECEPTOR GENES AND USES THEREOF

This application claims priority to U.S. provisional application no.

60/317,401, filed September 4, 2001, which is incorporated by reference herein in its entirety.

1. INTRODUCTION The present invention relates to Anopheles gambiae odorant receptor genes and methods for identifying odorant receptor genes that are related to the Anopheles gambiae odorant receptor genes. The invention provides nucleotide sequences of Anopheles gambiae odorant receptor genes, amino acid sequences of their encoded proteins (including peptide or polypeptide), and derivatives (e.g., fragments) and analogs thereof. The invention further relates to fragments (and derivatives and analogs thereof) of

Anopheles gambiae odorant receptor proteins. The present invention yet further relates to odorant receptor genes and proteins from insects such as Culex spp. or Aedes aegypti that relate to the Anopheles gambiae odorant receptor genes and proteins disclosed herein. Methods of production of an Anopheles gαmbiαe odorant receptor protein (e.g., by recombinant means), and derivatives and analogs thereof, are provided. Antibodies to an Anopheles gαmbiαe odorant receptor protein, and derivatives and analogs thereof, are provided. Methods for identifying molecules that bind or modulate the activity of these odorant receptor genes are provided. Molecules found to bind or modulate the activity of these odorant receptor proteins may be formulated into pest control agents by providing a canier. In a prefened embodiment, molecules that bind or modulate the activity of an odorant receptor protein from Anopheles gαmbiαe but not from other insect species is desired. Methods of modifying insect behavior by modifying the activity of an Anopheles gαmbiαe odorant receptor are also provided.

2. BACKGROUND OF THE INVENTION

Insects have a profound impact upon human health throughout the world. Mosquitoes and other biting insects transmit a number of devastating infectious diseases to man and have a profound influence on economic growth and human welfare worldwide. In addition, quarantines imposed to control the spread of insect pests severely impinge on world trade and the import and export of agricultmal products. The insect-bome disease of greatest concern, malaria, kills more than 1 million people each year. This situation may worsen as strains of the malaria parasite that are resistant to anti-malarial drugs have arisen and have the potential to spread rapidly. Other significant insect-bome diseases include Dengue fever/Dengue Hemonhagic Fever, lymphatic filariasis, West Nile and St. Louis encephalitis.

Presently, pests posing a danger to human health are targeted with the widespread spraying of insecticides in or near residential areas. The use of conventional pesticides, however, is associated with significant hazards to the environment, human health, and non-renewable natmal resources. Thus while chemical insecticides are designed to kill insects, their non-selective effects on human health, the environment and other animal species make them damaging and controversial. As a result, governments throughout the world are placing increasingly severe restrictions and bans on the use of chemical pesticides. Moreover, insects develop resistance to pesticides after prolonged use, necessitating the spraying of increased levels of pesticide, or the development of new, more potent, pesticide formulations.

Clearly, traditional insecticide-based methods to control biting insects, especially those that transmit disease, have not been sufficient to manage pest populations and prevent the spread of malaria and other diseases. The most common active ingredient in commercial insect repellents is N, N-diethyl-m-toluamide, or DEET. Although this compound is relatively safe for human use, to be effective, it must be used at high concentrations. It is also reactive with some plastics and paints. Moreover, these methods may become even less effective, as insecticide-resistant disease vectors are becoming more widespread, and many insecticides are being restricted due to concerns for human health and environmental safety. Increased research efforts on malaria and Dengue fever promise new opportunities for vaccine and drug development, but it is likely that new treatments may take many years to develop. Thus, there is a critical need to develop potent, cost-effective tools to reduce and ultimately prevent the spread of insect-bome disease as soon as possible. Alternative repellents that would remain effective for long periods of time and which are comparable in cost and potency to insecticide-treated bed netting would provide valuable tools for vector insect control. One potential approach is to exploit knowledge of insect behavior and recent exciting advances in the molecular neurobiology of insect olfaction to develop novel strategies for insect control.

The identification of odorant receptor genes in pest insect species such as Anopheles gambiae can be used to develop new methods of intervention for insect control. Disrupting the ability of insects to recognize environmental cues will effectively block harmful insect behavior and will provide a safe and selective means to prevent insect damage. In addition, innate olfactory-driven behaviors, such as host attraction and egg laying in response to odor cues, can be utilized to control pest insect species.

2.1. INSECT OLFACTORY BEHAVIOR

The behavior of all animals, including humans, involves the perception of events in the environment by visual, auditory and other sensory systems and the translation of these sensory stimuli into appropriate muscle responses. In simpler organisms such as insects, the recognition of sensory stimuli results in very stereotyped or "hard- wired" behaviors. Thus, by modifying or blocking the perception of environmental cues, it is possible to alter the behavior of such animals in a predictable way. Such alterations afford a powerful means to interfere with or divert innate behaviors that have a destmctive effect on human health and welfare, such as the host-finding behavior of biting insects and agricultmal pests. Many insect behaviors, such as the location and selection of mating partners, food sources and suitable places for egg laying, are driven by the recognition of specific odors in the environment. For example, the male hawkworm moth, Manduca sexta, can detect extremely low concentrations of an attractive odor, called a pheromone, produced by females of the same species, and uses this sense to pmsue females over large distances (Hildebrand, 1995, Proc. Natl Acad. Sci. U.S.A. 92:67-74). Female navel orangeworm moths, Amyelois transitella, a pest of almonds in California, are attracted to and lay eggs on their prefened host plant in response to volatile odors emitted by almond fruits and by larvae feeding on the almonds (Curtis and Clark, 1979, Environ. Entomol. 8:330-333; Phelan et al, 1991, J. Chem. Ecol. 17:599-614). Social insects, such as ants, make extensive use of chemical cues in communication, for example in the recognition and attack of intruder ants from other colonies (Holldobler and Wilson, 1990, The Ants, Belknap Press of Harvard University Press, Cambridge, MA). Finally, female mosquitoes of many species, including Anopheles gambiae, the principal malaria canier, orient toward and locate human hosts by detecting human-specific scents (Takken and Knols, 1999, Annu. Rev. Entomol. 44 : 131 - 157; Bock and Cardew, eds. , 1996, Olfaction in Mosquito-Host Interactions, (Ciba Foundation Symposium 200), Wiley, Chichester). Recent progress in the understanding of the molecular basis of the sense of smell provides important new insight into the mechanisms by which these odor cues elicit specific behaviors. These advances provide an exciting opportunity to develop new tools for the behavior-based control of destmctive insect species. 2.2. THE MOLECULAR BIOLOGY OF INSECT OLFACTION

Insects recognize odors in the environment using specialized olfactory organs: i) the antenna, a highly evolved stmcture that extends from the head and can r attain a size equivalent to the length of the organism; and ii) the maxillary palps, a pair of club-shaped structures adjacent to the proboscis. The antenna and maxillary palps are covered with tiny sensory hairs that contain nerve cells with specialized machinery that can detect odorants often at vanishingly low concentrations. The initial step in the detection of odors requires the binding of odorants to specific receptor molecules that reside on the ι _β smface of these nerve cells.

Recently, a family of roughly 60 genes encoding odorant receptors has been identified in the genome of the model insect, the fruit fly Drosophila melanogaster (Vosshall et al, 1999, Cell, 96:725-736; Clyne et al, 1999, Neuron 22:327-338; Gao and Chess, 1999, Genomics 60:31-39; Vosshall et al, 2000, Cell 102:147-159). These odorant ι r receptors have seven predicted transmembrane domains and belong to the large superfamily of proteins termed G-protein coupled receptors (GPCRs). The expression of 42 of these receptor genes has been detected in small, non-overlapping subsets of olfactory neurons in the antenna or maxillary palp (Vosshall et al, 2000, Cell 102:147-159). The large size of this gene family, their predicted identity as seven transmembrane domain-containing n GPCRs, and their selective expression in olfactory nemons strongly implicate them in the process of olfactory recognition in the fly. More recently, functional studies have identified a candidate ligand for one of these Drosophila odorant receptor gene products, confirming their identity as receptors for behaviorally relevant odorants (Wetzel et al, 2001, Proc. Natl. Acad. Sci. USA 98:9377-9380; Stδrtkuhl and Kettler, 2001, Proc. Natl. Acad. Sci. USA

₂₅ 98:9381-9385).

One exception to the mle that an individual olfactory nemon expresses a single odorant receptor gene is the odorant receptor Or83b (previously known as A45; Vosshall et al, 1999, Cell 96:725-736; see also U.S. provisional application no. 60/312,319, filed August 14, 2001, which is incorporated by reference herein in its entirety),

2Q which is expressed by most, if not all, olfactory nemons in the antenna and maxillary palp. Thus, it appears that olfactory nemons actually express two odorant receptor genes: the "ubiquitous" odorant receptor gene Or83b, and one of the other "classical" odorant receptor genes.

Molecular genetic studies in Drosophila have also provided insight into the

2 logic of olfactory processing in insects and have revealed how information about the molecular identity of odors detected by the antenna is transmitted to the insect brain. Expression studies have revealed that individual olfactory nemons are functionally distinct, in that each nerve cell expresses only one of the odorant receptor genes (Vosshall et al, 1999, Cell 96:725-736). Olfactory nemons expressing the same receptor and therefore responsive to the same odor extend axons that converge on a fixed point in the brain (Vosshall et al, 2000, Cell 102:147-159). Different neurons converge on different points. It immediately follows that a given odor will activate a small group of nemons in the antenna that in turn will activate distinct spatial patterns in the insect brain. The quality of a perceived odor is therefore determined by spatial patterns of activation in the brain. These patterns are then interpreted to elicit appropriate behavioral responses such as attraction, repulsion, flight, mating and feeding. Odorants that modulate such behaviors in harmful insect species, such as Anopheles gambiae, will be of great value in managing populations of these harmful insects.

Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present inventors have identified a number of genes in Anopheles gambiae that are related to the "classical" Drosophila odorant receptor genes. Compounds that bind to Anopheles gambiae odorant receptors and/or modulate the activity of Anopheles gambiae odorant receptors will have utility in the control of this insect species that is injmious to human health and welfare. Natural or synthetic compounds that stimulate or block Anopheles gambiae odorant receptor activity will dis pt olfactory-driven behaviors and will be useful as novel tools for the control and management of pest insect species.

The identification of odorant receptors in Anopheles gambiae by the present inventors permits the following strategy for the development of safe and effective insect control products: First, functional Anopheles gambiae odorant receptor molecules are produced in cultmed cells or in Xenopus laevis oocytes, or overexpressed in transgenic insects. Cells expressing Anopheles gambiae odorant receptors can be used as a screening tool for the rapid, efficient discovery of novel compounds that interact with Anopheles gambiae odorant receptors. This screening methodology can be used to identify compounds act as "super-agonists," that is, compounds that bind to receptors with higher affinity than the natural agonists. In addition, similar screening techniques can be used to isolate compounds that inactivate or antagonize receptor function, providing potent and selective chemicals to interfere with olfactory-driven behaviors. The compounds identified in such screens may be used for attracting insects to traps or to localized toxins, for monitoring pests, for repelling insects from individuals or from residential areas, or for interfering with the function of the olfactory system such that insects are unable to locate food and hosts. Since different species of insects have highly specialized food and host preferences and the odorant receptors that mediate these behaviors are extremely variable between species, control strategies that target olfaction offer powerful and selective approaches to combat pest insects. In contrast to non-selective pesticides, such products have broad applicability as pest control agents. Whether used for pesticides, repellants or attractants, these agents selectively target disease vectors and can be expected to be harmless to beneficial species of insects, insect predators and other animals. Moreover, as behaviorally-based strategies present less selective pressme than chemical pesticides and genetically engineered crops, these strategies are expected to help reduce the appearance of pesticide-resistant insect vectors. Thus, the compounds identified using this methodology will offer novel approaches to control insect damage and the spread of disease, and will significantly reduce dependence on toxic pesticides, having a direct and immediate impact on coordinated insect management programs.

Further, the Anopheles gambiae olfactory receptor genes identified herein have elucidated a sequence motif present at the C-terminus of Drosophila and Anopheles olfactory receptor gene products that will be useful in identifying odorant receptors from other insects, including but not limited to Culex spp. or Aedes aegypti.

The present invention relates to purified polypeptides that are insect olfactory receptors comprising an amino acid sequence having at least 80%, 90% or 95% identity to the amino acid sequence of SEQ ID NO:25. In prefened embodiments, the insect olfactory receptor is a Culex spp. or Aedes aegypti olfactory receptor. In a most prefened embodiment, the insect olfactory receptor is an Anopheles gambiae olfactory receptor.

The present invention further relates to purified polypeptides that relate to the K09 subfamily of olfactory receptors comprising an amino acid sequence having at least 70%, 80%, 90% or 95% identity to a 20, 30, 50, 70 or 100 amino acid fragment of SEQ ID NO:26. In prefened embodiments, the insect olfactory receptor is a Culex spp. or Aedes aegypti olfactory receptor. In a most prefened embodiment, the insect olfactory receptor is an Anopheles gambiae olfactory receptor.

The present invention provides purified polypeptides comprising an amino acid sequence having at least 80%, 90% or 95% identity to the amino acid sequence of SEQ ID NO:25. In one embodiment, the amino acid sequence is not found in SEQ ID NOS:35- 95. In another embodiment, the amino acid sequence does not comprise any of SEQ ID NOS:35-95, or a fragment thereof of 20, 30, 50 or 100 amino acids. In certain specific embodiments, such polypeptides comprise at least 20, 30, or 50 contiguous amino acids of the sequence as set forth in SEQ ID NO:2, or all of the amino acid sequence as set forth in SEQ ID NO:2. In other specific embodiments, such polypeptides comprise at least 20, 30, or 50 contiguous amino acids of the sequence as set forth in SEQ ID NO:4, or all of the amino acid sequence as set forth in SEQ ID NO:4. In other specific embodiments, such polypeptides comprise at least 20, 30, or 50 contiguous amino acids of the sequence as set forth in SEQ ID NO:6, or all of the amino acid sequence as set forth in SEQ ID NO:6. In yet other specific embodiments, such polypeptides comprise at least 20, 30, or 50 contiguous amino acids of the sequence as set forth in SEQ ID NO: 8, or all of the amino acid sequence as set forth in SEQ ID NO: 8. In yet other specific embodiments, such polypeptides comprise at least 20, 30, or 50 contiguous amino acids of the sequence as set forth in SEQ ID NO:10, or all of the amino acid sequence as set forth in SEQ ID NO:10. In yet other specific embodiments, such polypeptides comprise at least 20, 30, or 50 contiguous amino acids of the sequence as set forth in SEQ ID NO: 12, or all of the amino acid sequence as set forth in SEQ ID NO: 12.

The present invention further provides pmified polypeptides comprising an amino acid sequence having at least 80%, 90% or 95% identity to the amino acid sequence of SEQ ID NO:25, wherein the amino acid sequence is not found in SEQ ID NO:35-95, wherein the polypeptides are capable of being bound by an antibody that also binds to a polypeptide defined by an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, and SEQ ID NO:12. The present invention further provides purified polypeptides comprising an amino acid sequence having at least 70%, 80%, 90% or 95% identity to a 20, 30, 50, 70 or 100 amino acid fragment of SEQ ID NO:26, wherein the polypeptides are capable of being bound by an antibody that also binds to a polypeptide defined by an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO: 12. Further details of the polypeptides of the invention are provided in Section

5.3 below. A table indicating to which nucleotide or amino acid sequence each SEQ ID NO conesponds is presented below:

Table 1

The present invention further provides nucleic acids encoding any of the foregoing proteins, as well as nucleic acids that are complementary to nucleic acids encoding any of the foregoing proteins. In certain specific embodiments, the nucleic acid comprises a nucleotide sequence as set forth in any of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:l 1. In other specific embodiments, the nucleic acid encodes the polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, or SEQ ID NO: 12.

The nucleic acids of the invention may further comprise an origin of replication. The nucleic acids of the invention may also be operatively linked to a promoter. Further details of the nucleic acids of the invention are provided in Section 5.1 below. A table indicating to which odorant receptor-related sequence each SEQ ID NO. conesponds is presented at page 7 above.

In prefened embodiments of the invention, a polypeptide of the invention does not comprise a Drosophila odorant receptor, i.e., does not comprise any of SEQ ID NO:35-95. In other prefened embodiments of the invention, a polypeptide of the invention does not comprise an amino acid sequence that is found in a Drosophila odorant receptor, i.e., is not fom d SEQ ID NO:35-95. In other prefened embodiments of the invention, a polypeptide of the invention does not comprise an amino acid sequence that is found in a 20, 30, 50, 70 or 100 amino acid fragment of a Drosophila odorant receptor, i.e., is not found in a 20-, 30-, 50- or 100- amino acid fragment of any of SEQ ID NO:35-95. With respect to the nucleic acids of the invention, in a prefened embodiments of the invention, a nucleic acid of the invention does not comprise a nucleic acid sequence encoding the open reading frame of Drosophila odorant receptor gene, i.e., does not encode any of SEQ ID NO:35-95. In yet other prefened embodiments of the invention, a nucleic acid of the invention does not comprise a nucleotide sequence that is present in the open reading frame of a Drosophila odorant receptor, /^'. e. , does not comprise a sequence that encodes any of SEQ ID NO:35-95. In yet other prefened embodiments, a nucleic acid of the invention does not encode an amino acid sequence that is found in a 20, 30, 50 or 100 amino acid fragment of a Drosophila odorant receptor, i. e. , does not encode an amino acid sequence that is found in a 20-, 30-, 50- or 100- amino acid fragment of any of SEQ ID NO:35-95. Table 2 below summarizes known Drosophila odorant receptor genes, and the conesponding SEQ ID NOS.

The present invention yet further provides host cells comprising any of the foregoing nucleic acids. Host cells comprising a nucleic acid encoding a polypeptide of the invention may further comprise a nucleic acid encoding an Or83b receptor, for example the Anopheles gambiae Or83b receptor whose amino acid sequence is set forth in SEQ ID NO:14.

The present invention further provides methods of identifying molecules that bind to and/or modulate the activity of the olfactory receptors of the invention, most preferably the activity of an Anopheles gambiae olfactory receptor. Briefly, molecules that modulate that activity of the olfactory receptors of the invention can be agonists or antagonists. Modulation of G protein activity can be assayed by measming G protein activity or calcium concentration in a cell. The screening methods of the invention are further described in Sections 5.7 and 5.8, infra. In one method of identifying a molecule that binds to an insect olfactory receptor, the olfactory receptor comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:25, wherein the amino acid sequence is not fomid in any of SEQ ID NO:35-95, or alternatively, does not comprise any of SEQ ID NO: 35-95, the method comprising (a) contacting a first cell and a second cell with a test molecule under conditions conducive to binding between the olfactory receptor and the test molecule, wherein the first cell expresses the insect olfactory receptor and the second cell does not express the insect olfactory receptor, and wherein the first cell and the second cell are of the same cell type; and (b) determining whether the test molecule binds to the first cell or the second cell; wherein a molecule that binds to the first cell but not the second cell is a molecule that binds to the olfactory receptor. The first cell and optionally the second cell can further comprise an Or83b receptor. In one embodiment, the insect is Anopheles gambiae. In other embodiments, the insect is Culex spp. ox Aedes aegypti.

The present invention further provides methods of identifying a modulator of an insect olfactory receptor, the olfactory receptor comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:25, wherein the amino acid sequence is not found in any of SEQ ID NO:35-95, or alternatively, does not comprise the amino acid sequence of any of SEQ ID NO:35-95, the method comprising (a) contacting a first cell and a second cell with a test molecule under conditions conducive to binding between the olfactory receptor and the test molecule, wherein the first cell expresses the olfactory receptor and the second cell does not express the olfactory receptor, and wherein the first cell and the second cell are of the same cell type; and (b) determining whether the test molecule modulates G-protein activity in the first cell or second cell, wherein a molecule that modulates G-protein activity in the first cell but not in the second cell is a modulator of the insect olfactory receptor. The first the second cell can further comprise an Or83b receptor. In one embodiment, the insect is Anopheles gambiae. In other embodiments, the insect is Culex spp. or Aedes aegypti.

The present invention yet further provides methods of identifying a molecule that binds to an olfactory receptor from Anopheles gambiae but not to a second olfactory receptor from another species, the method comprising (a) contacting a first cell that expresses an Anopheles gambiae receptor with a test molecule under conditions conducive to binding between the Anopheles gambiae receptor and the test molecule; (b) determining whether the test molecule binds to the first cell; (c) contacting a second cell that expresses the second olfactory receptor with the test molecule under conditions conducive to binding between the second receptor and the test molecule, wherein the second cell is of the same cell type as the first cell; and (d) determining whether the test molecule binds to the second cell, wherein a test molecule that binds to the first cell but not to the second cell binds to the Anopheles gambiae olfactory receptor but not to the olfactory receptor from the other species. The present invention yet further provides methods of identifying a modulator of an olfactory receptor from Anopheles gambiae but not a second olfactory receptor from a second species, the method comprising (a) contacting a first cell that expresses an Anopheles gambiae receptor with a test molecule under conditions conducive to binding between the Anopheles gambiae receptor and the test molecule; (b) determining whether the test molecule modulates G-protein activity in the first cell; (c) contacting a second cell that expresses the second olfactory receptor with the test molecule under conditions conducive to binding between the second receptor and the test molecule, wherein the second cell is of the same cell type as the first cell and; and (d) determining whether the test molecule modulates G-protein activity in the second cell, wherein a test molecule that modulates G-protein activity in the first cell but not in the second cell modulates the

Anopheles gambiae olfactory receptor but not the olfactory receptor from the other species. The present invention yet further provides methods of identifying an odorant that binds to a first Anopheles gambiae olfactory receptor but not to a second Anopheles gambiae olfactory receptor, the method comprising (a) contacting a first cell that expresses the first Anopheles gambiae olfactory receptor with a test molecule, the first olfactory receptor comprising an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8_s SEQ ID NO:10, or SEQ ID NO:12 under conditions conducive to binding between the first Anopheles gambiae olfactory receptor and the test molecule; (b) determining whether the test molecule binds to the first cell; (c) contacting a second cell that expresses the second Anopheles gambiae olfactory receptor with the test molecule, the second olfactory receptor comprising an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO: 12, under conditions conducive to binding between the second receptor and the test molecule, wherein the second cell is of the same cell type as the first cell and the first olfactory receptor is different from the second olfactory receptor; and (d) determining whether the test molecule binds to the second cell, wherein a test molecule that binds to the first cell but not to the second cell is an odorant that binds to the first Anopheles gambiae olfactory receptor but not to the second Anopheles gambiae olfactory receptor.

The present invention yet further provides methods of identifying an olfactory that modulates the activity of a first Anopheles gambiae olfactory receptor but not the activity of a second Anopheles gambiae olfactory receptor, the method comprising (a) contacting a first cell that expresses the first Anopheles gambiae olfactory receptor with a test molecule, the first olfactory receptor comprising an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10 or SEQ ID NO: 12, under conditions conducive to binding between the first Anopheles gambiae olfactory receptor and the test molecule; (b) determining whether the test molecule modulates G-protein activity in the first cell; (c) contacting a second cell that expresses the second Anopheles gambiae olfactory receptor with the test molecule, the second olfactory receptor comprising an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12, under conditions conducive to binding between the second receptor and the test molecule, wherein the second cell is of the same cell type as the first cell and the first olfactory receptor is different from the second olfactory receptor; and (d) determining whether the test molecule modulates G-protein activity in the second cell, wherein a test molecule that modulates G-protein activity in the first cell but not in the second cell modulates the activity of the first Anopheles gambiae olfactory receptor but not the second Anopheles gambiae olfactory receptor.

In yet other aspects of the present invention, insect control agent formulations, comprising one or more of the foregoing Or83b binding molecules or modulators and a suitable canier are provided. In one embodiment, the insect control agent is an insect repellent. In another embodiment, the insect control agent is an insect attractant. The canier can be a solid canier or a liquid canier. Examples of suitable caniers are described in Section 5.9, infra.

The present invention further provides methods for protecting a mammal against malaria comprising contacting the mammal with a repellent identified by any of the foregoing methods. The present invention further provides methods for reducing populations of Anopheles gambiae mosquitos comprising placing a trap comprising an attractant identified by any of the foregoing methods in an area where such population reduction is desired. Further details on the uses of the modulators of the receptors of the invention are provides in Section 5.9, infra.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Amino acid sequence alignment of the predicted Anopheles gambiae odorant receptor genes. Protein sequences, as predicted by GENSCAN and RT-PCR from antennal mRNA, were aligned using ClustalW (Vector NTI Suite, AlignX program,

Informax, Inc.) Amino acids that are identical in three or more predicted genes are shown in black; similar amino acids are shaded in light gray. The shaded boxes beneath the amino acid sequence indicate the approximate positions of the seven transmembrane domains. The line with anowheads indicates the position of the conserved C-terminal consensus motif region.

FIG. 2. The sequence of the C-terminal consensus motif conserved among Anopheles gambiae odorant receptor genes is shown here. The character "_" indicates ambiguous amino acid positions.

FIG. 3. An alignment of the Anopheles gambiae odorant receptors (SEQ ID NOS: 4, 6 and 12) belonging to the K09 subfamily of Anopheles gambiae odorant receptors and consensus K09 amino acid sequence (SEQ ID NO:26) are shown.

FIG. 4. Amino acid sequence comparison of the predicted Anopheles gambiae odorant receptor genes and the ten most highly related Drosophila melanogaster odorant receptor genes. Amino acids that are identical in 8 or more of the listed genes are shown in black; amino acids that are similar in 8 or more are shown in light gray.

FIG. 5. Phylogenetic tree showing sequence relationships among the predicted Anopheles odorant receptor genes and the 10 most highly related Drosophila odorant receptor genes. The phylogenetic tree was constructed using the Neighbor Joining method (as implemented by AlignX program, Vector NTI Suite, Informax, Inc.) FIG. 6. The sequence of the Anopheles gambiae Or83b cDNA (SEQ ID NO: 13) and its predicted encoded protein (SEQ ID NO: 14) are shown.

FIG. 7A-7L. FIGS. 7A, 7C, 7E, 7G, 71 and 7K are hydrophilicity plots of the Anopheles gambiae C19, K0920.3, K0927.1, M09, N03 and K09 7.1 receptors, respectively (as described in Kyte and Doolittle, 1962, J. Mol. Biol, 157:105-132). FIGS. 7B, 7D, 7F, 7H, 7J and 7L are transmembrane domain predictions of the Anopheles gambiae C19, K0920.3, K0927.1, M09, N03 and K09 7.1 receptors, respectively (as described in von Heijne, 1992, J. Mol. Biol. 225: 487-494).

5. DETAILED DESCRIPTION OF THE INVENTION

Described herein are novel Anopheles gambiae odorant receptor genes and their encoded proteins and protein derivatives. The Anopheles gambiae receptor genes disclosed herein encode Anopheles gambiae odorant receptor proteins related to the odorant receptors of Drosophila melanogaster. These new Anopheles gambiae odorant receptor genes constitute very useful targets for pest control agents. Sequence analysis of the Anopheles gambiae odorant receptor proteins described herein has elucidated an amino acid motif present in the C terminus of the Anopheles gambiae odorant receptors disclosed herein and in the C terminus of the most closely related Drosophila melanogaster odorant receptors. This motif is expected to be useful for identifying related odorant receptor genes in other insect species, such as the mosquitos Culex spp. and Aedes aegypti. Like Anopheles gambiae, Culex spp. and Aedes aegypti transmit diseases that are harmful to human health and thus the identification of odorant receptors from Culex spp. and Aedes aegypti species will be useful in screening for odorants that can be used to control the behaviors of these species.

The present invention thus provides proteins encoded by and nucleotide sequences of Anopheles gambiae odorant receptor genes. The invention further relates to fragments and other derivatives and analogs of such Anopheles gambiae odorant receptor proteins. Nucleic acids encoding such fragments or derivatives are also within the scope of the invention. Production of the foregoing proteins, e.g., by recombinant methods, is provided.

Antibodies to an Anopheles gambiae odorant receptor protein, its derivatives and analogs, are additionally provided. The invention is illustrated by way of examples set forth in Section 6 below which disclose, inter alia, the cloning and characterization of the Anopheles gambiae odorant receptor genes.

The nucleic acids and polypeptides of the invention may be isolated or pmified.

"Isolated" or "pmified" when used herein to describe a nucleic acid molecule or nucleotide sequence, refers to a nucleic acid molecule or nucleotide sequence which is separated from other nucleic acid molecules which are present in the natural somce of the nucleic acid molecule. Preferably, an "isolated" nucleic acid molecule is free of sequences (preferably protein encoding sequences) which naturally flank the nucleic acid (i. e. , sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. As used herein, an isolated nucleic acid does not encompass a nucleic acid present in a library, such as a cDNA, genomic, or expression library. In a particular embodiment, the isolated nucleic acid of the invention does not contain introns. In another embodiment, the isolated nucleic acid of the invention does not have the sequence set forth in Genbank Accession No. AL152274. In another embodiment, the isolated nucleic acid of the invention is not the cloning vector and insert identified as clone 24C19 of NotreDamel library from strain PEST of Anopheles gambiae in Genbank Accession No. AL152274. In another embodiment, the isolated nucleic acid of the invention does not have the sequence set forth in Genbank Accession No. AL153125. In another embodiment, the isolated nucleic acid of the invention is not the cloning vector and insert identified as clone 25K12 of NotreDamel library from strain PEST of Anopheles gambiae in Genbank Accession No. AL153125. In another embodiment, the isolated nucleic acid of the invention does not have the sequence set forth in Genbank Accession No. AL 144370. In another embodiment, the isolated nucleic acid of the invention is not the cloning vector and insert identified as clone 08K09 of NotreDamel library from strain PEST of Anopheles gambiae in Genbank Accession No. AL144370. In another embodiment, the isolated nucleic acid of the invention does not have the sequence set forth in Genbank Accession No. AL156632. In another embodiment, the isolated nucleic acid of the invention is not the cloning vector and insert identified as clone 31M09 of NotreDamel library from strain PEST of Anopheles gambiae in Genbank Accession No. AL156632. In another embodiment, the isolated nucleic acid of the invention does not have the sequence set forth in Genbank Accession No. AL155543. In another embodiment, the isolated nucleic acid of the invention is not the cloning vector and insert identified as clone 29N03 of NotreDamel library from strain PEST of Anopheles gambiae in Genbank Accession No. AL155543. "Isolated" or "pmified" when used herein to describe a protein or biologically active portion thereof (i. e. , a polypeptide or peptide fragment) refers to a protein or biologically active portion thereof substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. A protein or biologically active portion thereof (i. e. , a polypeptide or peptide fragment) that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous, or contaminating, protein. The term "olfactory receptor'Or "odorant receptor" refers to a polypeptide involved in olfactory sensation. An "olfactory receptor nucleic acid" is a nucleic acid encoding a polypeptide involved in olfactory sensation. Insects have two classes of odorant receptor genes: the Or83b class of receptors expressed by most, if not all, olfactory nemons in the antenna and maxillary palp, and the "classical" odorant receptor genes which are expressed in small, non-overlapping subsets of olfactory nemons. The term "Anopheles gambiae olfactory receptor" or "Anopheles gambiae odorant receptor" as used herein refers to an Anopheles gambiae-encoded classical olfactory receptor. Such receptors are characterized by the following features:

(a) has a hydrophilicity profile substantially as shown in FIG. 7A, FIG. 7C, FIG. 7E, FIG. 7G, FIG. 71, or FIG. 7K e.g., has the same or a similar distribution of hydrophobic peaks as shown in FIG. 7A, FIG. 7C, FIG. 7E, FIG. 7G, FIG. 71, or FIG. 7K, or a transmembrane domain distribution substantially as shown in FIG. 7B, FIG. 7D, FIG. 7F, FIG. 7H, FIG. 7J, or FIG. 7L i.e., has the same or a similar distribution of transmembrane domains as shown in FIG. 7B, FIG. 7D, FIG. 7F, FIG. 7H, FIG. 7J or FIG. 7L;

(b) is expressed in a small subset of olfactory nemons in the antenna and maxillary palp of Anopheles gambiae in a non-overlapping manner with other Anopheles gambiae olfactory receptors; and

(c) comprises a protein sequence that is at least 65% identical to the Anopheles gambiae classical receptor consensus sequence (SEQ ID NO:25).

The term "K09 olfactory receptor'Or "K09 odorant receptor" refers to a member of the K09 subfamily of insect odorant receptors exemplified by K0920.3 (SEQ ID NO:4), K0927.1 (SEQ ID NO:6), and K097.1 (SEQ ID NO:12). A "K09 olfactory receptor nucleic acid" is a nucleic acid encoding a K09 odorant receptors, as exemplified by the coding regions of the Anopheles gambiae K0920.3 (SEQ ID NO:3), K0927.1 (SEQ ID NO:5), and K097.1 (SEQ ID NO:l 1) genes. Such receptors are characterized by the following features: (a) has a hydrophilicity profile substantially as shown in FIG. 7C, FIG. 7E, or FIG. 7K e.g., has the same or a similar distribution of hydrophobic peaks as shown in FIG. 7C, FIG. 7E, or FIG. 7K, or a transmembrane domain distribution substantially as shown in FIG. 7D, FIG. 7F, or FIG. 7L i.e., has the same or a similar distribution of transmembrane domains as shown in FIG. 7D, FIG. 7F, or FIG. 7L; (b) is expressed in a small subset of olfactory nemons in the antenna and maxillary palp of Anopheles gambiae in a non-overlapping manner with other Anopheles gambiae olfactory receptors; and

(c) comprises a protein sequence that is at least 50% identical to the K09 consensus sequence (SEQ ID NO:26). The Anopheles gambiae odorant receptor genes and encoded proteins of the invention may be used for the development of safe and effective insect control products using the following strategy: First, conventional gene expression techniques will utilize these cloned genes to produce functional Anopheles gambiae odorant receptor molecules in cultured cells. Cell lines expressing these receptors will be used as a screening tool for the rapid, efficient discovery of novel compounds that interact with pest insect Anopheles gambiae odorant receptors. This screening methodology can be used to identify compounds that act as "super-agonists", that is, compounds that bind to receptors with higher affinity than the natural agonists. In addition, similar screening techniques can be used to isolate compounds that inactivate or antagonize receptor function, providing potent and selective chemicals to interfere with olfactory-driven behaviors.

To identify ligands that interact with these Anopheles gambiae odorant receptors, cell cultme based systems can be used for the functional expression of the Anopheles odorant receptors identified above. These methodologies have been successfully employed to identify chemical ligands of several mammalian odorant receptors and numerous other GPCRs (Krautwmst et al. , 1998, Cell 95:917-926; Howard et al. , 2001, Trends Pharmacol. Sci. 22:132-140).

Conventional cell cultme and gene transfection techniques can be used to express Anopheles odorant receptors in defined mammalian and insect cell lines and monitor trafficking of the receptors to the cell surface. Receptor activation can be visualized by changes in the concentration of intracellular calcium, as monitored with calcium sensitive dyes such as FURA-2.

Candidate odorants that have been shown to produce behavioral or physiological responses in the mosquito can be assayed in this system. Examples of such 5 compounds include acetone, l-octen-3-ol, L-lactic acid, phenols, indoles, and carboxylic acids (Takken and Knols, 1999, Annu. Rev. Entomol. 44:131-157). These methods can be used to identify a specific chemical or small subset of chemicals that activate a given receptor in cultured cells.

For clarity of disclosure, and not by way of limitation, the detailed 10 description of the invention is divided into the subsections which follow.

5.1. ANOPHELES GAMBIAE ODORANT RECEPTOR NUCLEIC ACIDS

Anopheles gambiae odorant receptor nucleic acids are described herein. As ι ^ used herein, an Anopheles gambiae odorant receptor gene or gene sequence refers to: (a) at least one of the nucleotide sequences and/or fragments thereof that are depicted herein in FIG. 1 (SEQ ID NOS:l, 3, 5, 7, 9 and 11); (b) any nucleotide sequence or fragment thereof that encodes the amino acid sequences that are depicted in FIG. 1 (SEQ ID NOS:2, 4, 6, 8, 10 and 12); (c) any nucleotide sequence that hybridizes to the complement of one of the

20 coding nucleotide sequences depicted herein in FIG. 1 (SEQ ID NOS:l, 3, 5, 7, 9 and 11) under stringent conditions, e.g., hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45° C followed by one or more washes in 0.2xSSC/0.1% SDS at about 50-65° C, or hybridization to filter-bound DNA in 0.5 M sodium pyrophosphate/7% SDS at about 65 ° C followed by one or more washes in

25 0.2xSSC/l% SDS at about 42-55 ° C, or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F.M. et al, eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3); (d) any nucleotide sequence that hybridizes to the complement of one of the coding nucleotide sequences

₃₀ depicted herein in FIG. 1 (SEQ ID NOS: 1, 3, 5, 7, 9, and 11) under highly stringent conditions, e.g., hybridization to filter-bound nucleic acid in 6xSSC at about 45° C followed by one or more washes in O.lxSSC/0.2% SDS at about 68° C, or hybridization to filter- bound DNA in 0.5 M sodium pyrophosphate/7% SDS at about 65 ° C followed by one or more washes in 0.2xSSC/l% SDS at about 68 ° C, or under other stringent hybridization $ conditions which are known to those of skill in the art (see, for example, Ausubel, F.M. et al, eds. , 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3), including such other hybridization conditions as those described herein; and (e) the complement of any of the Anopheles gambiae odorant receptor genes or gene sequences recited in (a)-(d) above. Preferably, the nucleic acid molecules that hybridize to the complements of the Anopheles gambiae odorant receptor gene sequence disclosed herein are the same length or about the same length as the Anopheles gambiae odorant receptor gene sequence disclosed herein (i.e., about 1100 nucleotides in length) and/or also encode gene products, e.g. , gene products that are the same length or about the same length as an Anopheles gambiae odorant receptor gene product encoded by a nucleotide sequence of (a) above (i. e. , approximately 350 to 386 amino acid residues in length) and/or are functionally equivalent to an Anopheles gambiae odorant receptor gene product encoded by a nucleotide sequence of (a), above. "Functionally equivalent," as the term is used herein, can refer to, in certain embodiments, a gene product (e.g., a polypeptide) capable of exhibiting a substantially similar in vivo activity as an endogenous Anopheles gambiae odorant receptor gene product encoded by one or more of the above-recited Anopheles gambiae odorant receptor gene sequences. Alternatively, and in certain other embodiments, as when utilized as part of assays such as those described hereinbelow, "functionally equivalent" can refer to peptides or other molecules capable of interacting with other cellular or extracellular molecules in a manner substantially similar to the way in which the conesponding portion of the endogenous Anopheles gambiae odorant receptor gene product would. Functionally equivalent gene products can therefore include naturally occuning Anopheles gambiae odorant receptor gene products. Functionally equivalent Anopheles gambiae odorant receptor gene products also include gene products that retain at least one of the biological activities of an Anopheles gambiae odorant receptor gene product described above (e.g. , which is capable of being bound by an antibody that also binds to a polypeptide defined by an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12; which is capable of modulating G protein activity; or which is capable of binding ligand). Thus, the functionally equivalent Anopheles gambiae odorant receptor gene products of the invention also include gene products which are recognized by and bind to antibodies (polyclonal or monoclonal) directed against one or more of Anopheles gambiae odorant receptor gene products described above (e.g., which are encoded by the coding sequences depicted herein in FIGS. 2, 4, 6, 8, and 10). Further, and as those skilled in the art readily appreciate, an amino acid sequence encoded by a given nucleic acid sequence may also be encoded by a number of "degenerate" nucleic acid sequence which are apparent to those skilled in the art. Thus, the Anopheles gambiae odorant receptor gene sequences of the present invention also include degenerate variants of the sequences described in (a) through (e), above.

The Anopheles gambiae odorant receptor gene nucleotide sequences of the invention also encompass: (a) nucleotides that encode an Anopheles gambiae odorant receptor gene product; (b) nucleotides that encode portions of an Anopheles gαmbiαe odorant receptor gene product that conesponds to one or more of its functional domains including, but not limited to, a signal sequence domain, an extracellular domain (ECD), a transmembrane domain (TM), a cytoplasmic domain (CD) or an intracellular domain (ID), and one or more odorant-binding domains; (c) nucleotide sequences that encode one or more splice variants of an Anopheles gαmbiαe odorant receptor gene product including, for example, sequences that encode a splice variant of an Anopheles gαmbiαe odorant receptor gene product; and (d) nucleotide sequences that encode mutants of an Anopheles gαmbiαe odorant receptor gene product in which all or part of one of its domains is deleted or altered including, but not limited to, mutants which encode soluble forms of u e Anopheles gαmbiαe odorant receptor gene product in which all or a portion of the TM domain is deleted, and nonfunctional receptors in which all or a portion of a CD is deleted. The Anopheles gαmbiαe odorant receptor gene nucleotide sequences of the invention still further include nucleotide sequences that encode fusion proteins, such as fusion proteins containing any one or more of the Anopheles gαmbiαe odorant receptor gene products described in (a)-(e) supra fused to another polypeptide. A fusion protein comprises all or part (preferably biologically active) of a polypeptide encoded by an Anopheles gambiae odorant receptor nucleotide sequence operably linked to a heterologous polypeptide (i.e., a polypeptide other than the same polypeptide of the invention). An exemplary Anopheles gambiae odorant receptor fusion protein comprises the amino- terminus of a chaperone protein, such as rhodopsin, and Anopheles gambiae odorant receptor TM domains II- VII . The Anopheles gambiae odorant receptor gene nucleotide sequences of the invention still further include nucleotide sequences conesponding to the above described Anopheles gambiae odorant receptor gene nucleotide sequences (i.e., the sequences described in (a)-(e) above and fusion proteins thereof) wherein one or more of the exons or fragments thereof, have been deleted. Still further, the Anopheles gambiae odorant receptor gene nucleotide sequences of the invention also include nucleotide sequence that have at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more nucleotide sequence identity to one or more of the Anopheles gambiae odorant receptor gene nucleotide sequences of (a)-(e) above. The Anopheles gambiae odorant receptor gene nucleotide sequences of the invention also include nucleotide sequences encoding polypeptides that have at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more amino acid sequence identity to one or more of the polypeptides encoded by any of the Anopheles gambiae odorant receptor gene nucleotide sequences of (a)-(e) above. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at conesponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the conesponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical overlapping positions/total # of positions x 100%). In one embodiment, the two sequences are the same length.

The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A prefened, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al, 1990, J. Mol. Biol. 215:403-0. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program parameters set, e.g. , to score- 50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI- Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., http://www.ncbi.nlm.nih.gov). Another prefened, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

The methods and compositions of the invention also encompass nucleic acid molecules, preferably DNA molecules, that hybridize to and are therefore the complements of the Anopheles gambiae odorant receptor gene nucleotide sequences (a) through (e) in the preceding paragraph. Such hybridization conditions can be highly stringent or less higlily stringent, as described above. The nucleic acid molecules of the invention that hybridize to the above described DNA sequences include oligodeoxynucleotides ("oligos") which hybridize under highly stringent or stringent conditions to the DNA sequences (a) through (e) in the preceding paragraph. In general, for oligos between 14 and 70 nucleotides in length the melting temperature (Tm) is calculated using the formula:

Tm(°C) = 81.5 + 16.6(log[monovalent cations (molar)] + 0.41 (% G+C) - (500/N), where N is the length of the probe. If the hybridization is canied out in a solution containing formamide, the melting temperature may be calculated using the equation: Tm(°C) = 81.5 + 16.6(log[monovalent cations (molar)]) + 0.41(% G+C) - (0.61% formamide) - (500/N) where N is the length of the probe. In general, hybridization is canied out at about 20-25 degrees below Tm (for DNA-DNA hybrids) or about 10-15 degrees below Tm (for RNA-DNA hybrids). Other exemplary highly stringent conditions may refer, e.g., to washing in 6xSSC/0.05% sodium pyrophosphate at 37°C (for 14-base oligos), 48°C (for 17-base oligos), 55°C (for 20-base oligos), and 60°C (for 23-base oligos). These nucleic acid molecules can be used in the methods or compositions of the invention, e.g., as Anopheles gambiae odorant receptor gene antisense molecules which are useful, for example, in Anopheles gambiae odorant receptor gene regulation. The sequences can also be used as antisense primers, e.g., in amplification reactions of an Anopheles gambiae odorant receptor gene nucleic acid sequence. Further, such complementary sequences can be used as part of ribozyme and/or triple helix sequence, also useful for Anopheles gambiae odorant receptor gene regulation.

Fragments of the Anopheles gambiae odorant receptor gene and Anopheles gambiae odorant receptor gene nucleotide sequences of the invention can be at least 10 nucleotides in length. In alternative embodiments, the fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or more contiguous nucleotides in length. Alternatively, the fragments can comprise sequences that encode at least 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more contiguous amino acid residues of the Anopheles gambiae odorant receptor gene products. Fragments of the Anopheles gambiae odorant receptor gene nucleic acid molecules of the invention can also refer to exons or introns of the above described nucleic acid molecules, as well as portions of the coding regions of such nucleic acid molecules that encode domains such as extracellular domains (ECD), transmembrane domains (TM) and cytoplasmic domains (CD). In certain specific embodiment, the invention provides pmified or isolated nucleic acids consisting of at least 8 nucleotides (e.g., a hybridizable portion) of an Anopheles gambiae odorant receptor gene sequence; in other embodiments, the nucleic acids consist of at least 12 (continuous) nucleotides, 15 nucleotides, 18 nucleotides, 25 nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, or 200 nucleotides of an Anopheles gambiae odorant receptor sequence, or a full-length Anopheles gambiae odorant receptor coding sequence. In other embodiments, the nucleic acids consist of at least 120 (continuous) nucleotides, 160 nucleotides, 250 nucleotides, 420 nucleotides, 550 nucleotides, 625 nucleotides, 750 nucleotides, or 1000 nucleotides of an Anopheles gαmbiαe odorant receptor sequence. In yet other embodiments, the nucleic acids are smaller than 35, 75, 200, 450, 525, or 610 nucleotides in length. In other embodiments, the nucleic acids are 100-200, 200-400, 400-600, 600-800, or larger than 800 nucleotides in length. Nucleic acids can be single or double stranded. As stated above, the invention also relates to nucleic acids hybridizable to or complementary to the foregoing sequences or their reverse complements. In specific aspects, nucleic acids are provided which comprise a sequence complementary to at least 10, 25, 50, 100, or 200 nucleotides or the entire coding region of an Anopheles gαmbiαe odorant receptor gene. In specific aspects, nucleic acids are provided which comprise a sequence complementary to at least 75, 125, 250, 500, or 650 nucleotides or the entire coding region of an Anopheles gαmbiαe odorant receptor gene.

In the above or alternative embodiments, the nucleic acids of the invention consist of a nucleotide sequence of not more than 2, 5, 7, 10, 15, or 20 kilobases. The methods and compositions of the invention also use, and therefore encompass, (a) DNA vectors that contain any of the foregoing coding sequences and/or their complements (i.e., antisense); (b) DNA expression vectors that contain any of the foregoing coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences; and (c) genetically engineered host cells that contain any of the foregoing coding sequences operatively associated with a regulatory element, such as a heterologous regulatory element, that directs the expression of the coding sequences in the host cell. As used herein, regulatory elements include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression. Such regulatory elements include but are not limited to the cytomegalovirus hCMV immediate early gene, the early or late promoters of S V40 adenovims, the lac system, the trrj system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast α-mating factors. The invention includes fragments of any of the DNA sequences disclosed herein.

In addition to the gene sequences described above, homologs of these gene sequences and/or full length coding sequences of these genes, as can be present in the same or other species, can be identified and isolated, without undue experimentation, by molecular biological techniques well known in the art.

For example, Anopheles gambiae odorant receptor gene sequences can be labeled and used to screen a cDNA or genomic library from a different insect, such as Culex spp. or Aedes aegypti.

According to this invention, a "genomic DNA library" is a clone library which contains representative nucleotide sequences from the DNA of a given genome. It is constmcted using various techniques that are well known in the art, for instance, by enzymatically or mechanically fragmenting the DNA from an organism, organ, or tissue of interest, linking the fragments to a suitable vector, and introducing the vector into appropriate cells so as to establish the genomic library. A genomic library contains both transcribed DNA fragments as well as nontranscribed DNA fragments.

In comparison, a "cDNA library" is a clone library that differs from a genomic library in that it contains only transcribed DNA sequences and no nontranscribed DNA sequences. It is established using techniques that are well known in the art, i.e., selection of mRNA (e.g., by polyA) making single stranded DNA from a population of cytoplasmic mRNA molecules using the enzyme RNA-dependent DNA polymerase (i. e. , reverse transcriptase), converting the single-stranded DNA into double-stranded DNA, cloning the resultant molecules into a vector, and introducing the vector into appropriate cells so as to establish the cDNA library. Alternately, a cDNA library need not be cloned into a vector and/or established in cells, but can be screened using PCR with gene-specific primers, as is well known in the art. Particularly useful types of cDNA libraries for identifying odorant receptors from Anopheles gambiae, Culex spp., and Aedes aegypti are antennal or maxillary palp cDNA libraries from Anopheles gambiae, Culex spp. and Aedes aegypti, respectively. cDNA screening can also identify clones derived from alternatively spliced Anopheles gambiae odorant receptor transcripts or related gene sequences from Anopheles gambiae or other insect species, such as but not limited to Culex spp. and Aedes aegypti. Low and moderate stringency conditions will be well known to those of skill in the art, and will vary predictably depending on the specific insects from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.; and Ausubel et al, 1989, Cunent Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, N.Y.). For example, low stringency conditions include the following: Filters containing DNA are pretreated for 6 h at 40 °C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCI (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatmed salmon sperm DNA. Hybridizations are canied out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 10⁶ cpm ³²P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40 °C, and then washed for 1.5 h at 55° C in a solution containing 2X SSC, 25 mM Tris-HCI (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60 °C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68 °C and re-exposed to film. Increasing the stringency can be accomplished by use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) more stringent that those described above. Exemplary moderate stringency conditions include overnight incubation at 37 °C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatmed sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C. Further, a previously unknown Anopheles gambiae odorant receptor gene sequence or a related gene sequence from another insect species, such as but not limited to Culex spp. and Aedes aegypti, can be isolated by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of nucleotide sequences within one or more of the above described known Anopheles gambiae odorant receptor gene sequences. A "polymerase chain reaction" ("PCR") is a reaction in which replicate copies are made of a target nucleic acid using one or more primers, and a catalyst of polymerization, such as a reverse transcriptase or a DNA polymerase, and particularly a thermally stable polymerase enzyme. Methods for PCR are taught in U.S. Patent Nos. 4,683,195 (Mullis) and 4,683,202 (Mullis et al). All processes of producing replicate copies of the same nucleic acid, such as PCR or gene cloning, are collectively refened to herein as "amplification."

The template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from insect antennal or maxillary palp tissue, or any cultmed tissues or cells that are suspected to express an insect odorant receptor gene product. The insect is preferably Anopheles gambiae, Culex spp. or Aedes aegypti. The PCR product can be subcloned and sequenced to ensme that the amplified sequences represent the sequences of the appropriate odorant receptor gene nucleic acid sequence. The design of PCR primer pairs is well known in the art. Primers suitable in the present invention are generally capable of encoding at least five, more preferably six contiguous amino acids of the sequences found in conserved motifs of an Anopheles gambiae odorant receptor. Thus, they are, at a minimum, 15 to 18 nucleotides in length. The primer pair is chosen such that the reverse primer is downstream of a forward primer. Prefened oligonucleotides for amplification of a portion of an insect odorant receptor gene or cDNA are pairs of degenerate oligonucleotide that serve as forward and reverse primers. Various commercially available programs for primer design are available, for example, MacVector (Oxford Molecular Ltd.) and Vector NTI Suite (Informax, Inc.). Forward and reverse primers are preferably selected such that amplification of an insect odorant receptor sequence results in a product of at least 100 nucleotides. Suitable conditions for amplification of an insect odorant receptor nucleic acid from Anopheles gambiae, Culex spp. or Aedes aegypti genomic or cDNA include, but are not limited to, using 1 μg of cDNA or genomic DNA template and 80 pmol each primer in a 50 μl reaction, cycled between 94°C for 1 min, 51°C for 1 min, 72°C for 1 min for a total of 40 cycles. The annealing temperature can be lowered, e.g., to 48°C, 45°C, 42°C, 40 °C or 37°C, to amplify sequences insect olfactory receptors that are distantly related to the sequences disclosed herein. The PCR fragment can then be used to isolate a full length cDNA clone by a variety of methods. For example, the amplified fragment can be used to screen a bacteriophage cDNA library. Alternatively, the labeled fragment can be used to screen a genomic library. PCR technology can also be utilized to isolate full length cDNA sequences.

For example, RNA can be isolated, following standard procedures, from an appropriate cellular or tissue source. A reverse transcription reaction can be performed on the RNA using an oligonucleotide primer specific for the most 3' end of the amplified fragment for the priming of first strand synthesis. The resulting RNA/DNA hybrid can then be "tailed" with guanines using a standard terminal fransferase reaction, the hybrid can be digested with RNAase H, and second strand synthesis can then be primed with a poly-C primer. Thus, cDNA sequences upstream of the amplified fragment can easily be isolated. For a review of cloning strategies which can be used, see e.g., Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.; and Ausubel et al, 1989, Cunent Protocols in Molecular Biology, (Green Publishing Associates and Wiley Interscience, N.Y.).

As will be appreciated by those skilled in the art, DNA sequence polymorphisms of an Anopheles gambiae odorant receptor gene identified by the methods of the present invention will typically exist within a population of individual insects (e.g., within a locust or medfly population). Such polymorphisms may exist, for example, among individual insects within a population due to natural allelic variation. Such polymorphisms include ones that lead to changes in amino acid sequence. An allele is one of a group of genes which occurs alternatively at a given genetic locus. Accordingly, as used herein, an "allelic variant" refers to a nucleotide sequence which occurs at a given locus or to a gene product encoded by the nucleotide sequence. Natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given gene. Certain allelic variations in the nucleotide sequence of a gene may be silent variations, i.e., do not encode a variant protein.

Alternative alleles or allelic variants can be identified by sequencing the gene of interest in a number of different insects of the same species. This can be readily canied out by using PCR amplification of Anopheles gambiae odorant receptor gene products from genomic DNA from individual insects.

As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide of the invention. The term can further include nucleic acid molecules comprising upstream and/or exon/intron sequences and structure.

With respect to allelic variants of the insect, e.g., Anopheles gambiae, odorant receptor genes and gene products of the present invention, any and all nucleotide variations and/or amino acid polymoφhisms or variations that are the result of natural allelic variation of insect odorant receptor genes and/or gene products are intended to be within the scope of the present invention. Such allelic variants include, but are not limited to, ones that do not alter the functional activity of a given odorant receptor gene products of the invention. Variants also include, but are not limited to "mutant alleles." As used herein, a "mutant allele" of an odorant receptor gene or gene product of the invention is an allelic variant which does alter the functional activity of the odorant receptor gene product. For example, a cDNA of a mutant Anopheles gambiae odorant receptor gene can be isolated by using PCR or by screening a genomic or cDNA library prepared from a population of insects that have the mutant allele. The normal Anopheles gambiae odorant receptor gene or any suitable fragment thereof can then be labeled and used as a probed to identify the conesponding mutant allele in the library. The clone containing this mutant Anopheles gambiae odorant receptor gene can then be purified through methods routinely practiced in the art, and subjected to sequence analysis.

Other allelic variants and/or mutant variants of the insect, e.g., Anopheles gambiae, odorant receptor genes of the invention include single nucleotide polymorphisms (SNPs), including biallelic SNPs or biallelic markers which have two alleles, both of which are present at a fairly high frequency in a population of organisms. Conventional techniques for detecting SNPs include, e.g., conventional dot blot analysis, single stranded conformational polymorphism (SSCP) analysis (see, e.g., Orita et al, 1989, Proc. Natl. Acad. Sci. USA 86:2766-2770), denat ing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and other routine techniques well known in the art (see, e.g., Sheffield et al, 1989, Proc. Natl. Acad. Sci. 86:5855-5892; Grompe, 1993, Nat e Genetics 5:111-117). Alternative, prefened methods of detecting and mapping SNPs involve microsequencing techniques wherein an SNP site in a target DNA is detected by a single nucleotide primer extension reaction (see, e.g. , Goelet et al. , PCT Publication No. WO 92/15712; Mundy, U.S. Patent No. 4,656,127; Vary and Diamond, U.S. Patent No. 4,851,331; Cohen etal, PCT Publication No. WO 91/02087; Chee et al, PCT Publication No. Wo 95/11995; Landegren et al, 1988, Science 241 :1077- 1080; Nicerson et al, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:9823-8927; Pastinen etal, 1997, Genome Res. 7:606-614; Pastinen et al, 1996, Clin. Chem. 42:1391-1397; Jalanko et al, 1992, Clin. Chem 38:39-43; Shumaker et al, 1996, Hum. Mutation 7:346-354; Caskey et al, PCT Publication No. 95/00669).

Fragments of the Anopheles gambiae, odorant receptor nucleic acids comprising regions conserved between (e.g., with homology to) other Anopheles gambiae odorant receptor nucleic acids are also provided. Sequence alignment of the amino acid sequences of Anopheles gambiae odorant receptor genes display conserved amino acid residues in the C terminus. In a prefened embodiment, fragments of Anopheles gambiae odorant receptor nucleic acids comprising regions conserved in other Anopheles gambiae odorant receptor nucleic acids contain at least 15, 20, 30 or 50 contiguous nucleotides encoding part or all of a conserved motif of an Anopheles gambiae odorant receptor.

In a specific embodiment, a novel Anopheles gambiae odorant receptor gene may be identified using a program such as the TBLASTN program (Altschul et al, 1997, Nuc.Acids Res. 25:3389-3402) to query the a database of interest (e.g., the Anopheles gambiae genome project, Genoscope/ Laboratory of Biochem. and Biol. Molec. of Insects, Institut Pasteur) with an Anopheles gambiae odorant receptor gene sequence in order to identify a contiguous sequence of interest. Individual EST sequences contributing to the contiguous sequence of interest may be identified and a RACE strategy (rapid amplification of cDNA ends) may be used to extend the available sequence to obtain the complete coding region encoded by the novel Anopheles gambiae odorant receptor gene. The above-described methods are not meant to be limiting with respect to the methods by which clones of Anopheles gambiae odorant receptor genes and related genes from other insects such as Culex spp. and Aedes aegypti may be obtained.

5.2. EXPRESSION OF ANOPHELES

GAMBIAE ODORANT RECEPTOR

GENES

The nucleotide sequence coding for an Anopheles gambiae odorant receptor protein or a functionally active analog or fragment or other derivative thereof, or for a related receptor from another species can be inserted into an appropriate expression vector, e.g., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. The necessary transcriptional and translational signals can also be supplied by the native odorant receptor gene and/or its flanking regions. Thus, the nucleotide sequence is operatively linked to a promoter. A variety of host- vector systems may be utilized to express the protein-coding sequence. These include but are not limited to mammalian cell systems infected with vims (e.g., vaccinia vims, adenovims, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used. In yet another embodiment, a fragment

5 of an Anopheles gambiae odorant receptor protein comprising one or more motifs of an Anopheles gambiae odorant receptor protein is expressed.

Any of the methods previously described for the insertion of DNA fragments into a vector may be used to constmct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and the protein coding

10 sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of a nucleic acid sequence encoding an Anopheles gambiae odorant receptor protein or peptide fragment may be regulated by a second nucleic acid sequence so that the Anopheles gambiae odorant receptor polypeptide is expressed in a host transformed with the recombinant DNA molecule. For

15 example, expression of an Anopheles gambiae odorant receptor protein may be controlled by any promoter/enhancer element known in the art. A promoter/enhancer may be homologous (e.g., native) or heterologous (e.g., not native). Promoters which may be used to control Anopheles gambiae odorant receptor gene expression include, but are not limited to, the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the

20 promoter contained in the 3' long terminal repeat of Rous sarcoma vims (Yamamoto et al. , 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al, 1982, Nature 296:39-42), the regulatory sequence of the human cytomegalovirus for expression in any tissues (Foecking and Hofstetter, 1986, Gene 45:101-

25 105; U.S. Patent No. 5,168,062), prokaryotic expression vectors such as the β-lactamase promoter (Villa-Kamaroff et al, 1978, Proc Natl. Acad. Sci. U.S.A. 75:3727-3731), or the lac promoter (DeBoer et al, 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25; Scientific American, 1980, 242:74-94), plant expression vectors comprising the nopaline synthetase promoter region (Henera-Estrella et al, Nature 303:209-213), the cauliflower mosaic vims

30 35S RNA promoter (Gardner et al, 1981, Nucl. Acids Res. 9:2871), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Henera-Estrella et al, 1984, Nature 310:115-120), promoter elements from yeast or other fungi such as the Gal4- responsive promoter, the ADH (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional

35 control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al, 1984, Cell 38:639-646; Ornitz et al, 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); a gene control region which is active in pancreatic beta cells (Hanahan, 1985, Natme 315:115-122), an immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al. , 1984, Cell 38:647- 658; Adames et al, 1985, Natme 318:533-538; Alexander et al, 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor vims control region which is active in testicular, breast, lymphoid and mast cells (Leder et al, 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al, 1987, Genes and Devel. 1 :268-276), alpha- fetoprotein gene control region which is active in liver (Krumlauf et al, 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al, 1987, Science 235:53-58), alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al. , 1987, Genes and Devel. 1:161- 171), beta-globin gene control region which is active in myeloid cells (Mogram et al, 1985, Nature 315:338-340; Kollias et al, 1986, Cell 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al, 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Natme 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al, 1986, Science 234: 1372-1378). In a specific embodiment, a vector is used that comprises a promoter operably linked to an Anopheles gambiae odorant receptor gene nucleic acid, one or more origins of replication, and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene).

In a specific embodiment, an expression construct is made by subcloning an Anopheles gambiae odorant receptor coding sequence into the EcoRI restriction site of each of the three pGEX vectors (Glutathione S-Transferase expression vectors; Smith and Johnson, 1988, Gene 7:31-40). This allows for the expression of the Anopheles gambiae odorant receptor protein product from the subclone in the conect reading frame.

In another specific embodiment, the promoter that is operably linked to the Anopheles gambiae odorant receptor gene is not the native Anopheles gambiae odorant receptor gene promoter (e.g., it is a heterologous promoter).

Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As previously explained, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal vimses such as vaccinia vims or adenovims; insect vimses such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda phage), and plasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engineered Anopheles gambiae odorant receptor protein may be controlled. Furthermore, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation of proteins). Appropriate cell lines or host systems can be chosen to ensme the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce a non- glycosylated core protein product. Expression in yeast will produce a glycosylated product. Expression in animal cells, including insect and mammalian cells and Xenopus oocytes, can be used to ensme "native" glycosylation of an Anopheles gαmbiαe odorant receptor protein. In other specific embodiments, the Anopheles gαmbiαe odorant receptor protein, fragment, analog, or derivative may be expressed as a fusion, or chimeric protein product (comprising the protein, fragment, analog, or derivative joined via a peptide bond to a heterologous protein sequence (of a different protein)). A chimeric protein may include fusion of the Anopheles gαmbiαe odorant receptor protein, fragment, analog, or derivative to a second protein or at least a portion thereof, wherein a portion is one (preferably 10, 15, 20, 30, or 50) or more amino acids of said second protein. The second protein, or one or more amino acid portion thereof, may be from a different Anopheles gαmbiαe odorant receptor protein, from an odorant receptor protein from another insect, or may be from a protein that is not an insect odorant receptor protein. Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art. Alternatively, such a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.

The foregoing expression methods can also be used for the expression of nucleic acids conesponding to odorant receptor genes from other species, such as Culex spp. or Aedes aegypti, that relate to the Anopheles gambiae receptor sequences disclosed herein in. 5.3. ANOPHELES GAMBIAE ODORANT RECEPTOR GENE PRODUCTS

In particular aspects, the invention provides amino acid sequences of

Anopheles gambiae odorant receptor proteins and fragments and derivatives thereof which r comprise an antigenic determinant (e.g. , can be recognized by an antibody) or which are otherwise functionally active, as well as nucleic acid sequences encoding the foregoing. "Functionally active" Anopheles gambiae odorant receptor material as used herein refers to that material displaying one or more functional activities associated with a full-length (wild- type) Anopheles gambiae odorant receptor protein, e.g., binding to an Anopheles gambiae

20 odorant receptor associated protein or binding to a specific nucleotide or DNA sequence antigenicity (binding to an anti-Anopheles gαmbiαe odorant receptor protein antibody), immunogenicity, modulating the activity of a G protein, and/or binding to an Anopheles gαmbiαe odorant receptor ligand.

In specific embodiments, the invention provides fragments of an Anopheles _j r gαmbiαe odorant receptor protein consisting of at least 10 amino acids, 20 amino acids, 50 amino acids, or of at least 75 amino acids. In other specific embodiments, the invention provides fragments of an Anopheles gαmbiαe odorant receptor protein consisting of at least 100 amino acids, 150 amino acids, 200 amino acids, 250 amino acids, or of at least 300 amino acids. In other specific embodiments, the invention provides fragments of an

20 Anopheles gαmbiαe odorant receptor protein consisting of at least 85 amino acids, 175 amino acids, 275 amino acids, 310 amino acids, or of at least 325 amino acids. Fragments, or proteins comprising fragments, lacking some or all of the foregoing regions of an Anopheles gαmbiαe odorant receptor protein are also provided. Nucleic acids encoding the foregoing are provided. In specific embodiments, the nucleic acids are less than 5 or 10

25 kilobases. In specific embodiments, the foregoing proteins or fragments are not more than 25, 50, 100, or 200 contiguous amino acids.

Once a recombinant which expresses the Anopheles gαmbiαe odorant receptor gene sequence is identified, the gene product can be analyzed. This is achieved by assays based on the physical or functional properties of the product, including radioactive

2o labeling of the product followed by analysis by gel electrophoresis, immunoassay, etc.

Once the Anopheles gαmbiαe odorant receptor protein is identified, it may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

35 Alternatively, once an Anopheles gαmbiαe odorant receptor protein produced by a recombinant is identified, the amino acid sequence of the protein can be deduced from the nucleotide sequence of the chimeric gene contained in the recombinant. As a result, the protein can be synthesized by standard chemical methods known in the art (e.g., see Hunkapiller et al, 1984, Nat e 310:105-111).

In another alternate embodiment, native Anopheles gambiae odorant receptor proteins can be pmified from natmal somces, by standard methods such as those described above (e.g., immunoaffinity purification).

In a specific embodiment of the present invention, such Anopheles gambiae odorant receptor proteins, whether produced by recombinant DNA techniques or by chemical synthetic methods or by purification of native proteins, include but are not limited to those containing, as a primary amino acid sequence, all or part of the amino acid sequence substantially as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12, as well as fragments and other derivatives, and analogs thereof, including proteins homologous thereto.

The invention further relates to Anopheles gambiae odorant receptor proteins, derivatives (including but not limited to fragments), analogs, and molecules of Anopheles gambiae odorant receptor proteins. As used herein, a molecule defined by a particular SEQ ID NO, shall be constmed to mean that the sequence of that molecule consists of that SEQ ID NO. Nucleic acids encoding Anopheles gambiae odorant receptor protein derivatives and protein analogs are also provided. In one embodiment, the Anopheles gambiae odorant receptor proteins are encoded by the Anopheles gambiae odorant receptor nucleic acids described in Section 5.1 above. In particular aspects, the proteins, derivatives, or analogs are of Anopheles gambiae odorant receptor proteins encoded by the sequence set forth in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:l 1. The production and use of derivatives and analogs related to an Anopheles gambiae odorant receptor protein are within the scope of the present invention. In a specific embodiment, the derivative or analog is functionally active, e.g., capable of exhibiting one or more functional activities associated with a full-length, wild-type Anopheles gambiae odorant receptor protein. As one example, such derivatives or analogs which have the desired immunogenicity or antigenicity can be used in immunoassays, for immunization, for inhibition of Anopheles gambiae odorant receptor activity, etc. As yet another example, such derivatives or analogs which have the desired binding activity can be used for binding to an odorant ligand (see e.g., Levine, A., et al, 1997, Cell 88:323-331). Derivatives or analogs that retain, or alternatively lack or inhibit, a desired Anopheles gambiae odorant receptor protein property-of-interest (e.g., binding to an Anopheles gambiae odorant receptor protein binding partner), can be used as inducers, or inhibitors, respectively, of such property and its physiological conelates. A specific embodiment relates to an Anopheles gambiae odorant receptor protein fragment that can be bound by an anti- Anopheles gambiae odorant receptor protein antibody. Derivatives or analogs of an Anopheles gambiae odorant receptor protein can be tested for the desired activity by procedures known in the art, including but not limited to the assays described below.

In particular, Anopheles gambiae odorant receptor derivatives can be made by altering Anopheles gambiae odorant receptor sequences by substitutions, additions (e.g., insertions) or deletions that provide for functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as an Anopheles gambiae odorant receptor gene may be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of an Anopheles gαmbiαe odorant receptor gene which is altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.

Likewise, the Anopheles gαmbiαe odorant receptor derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of an Anopheles gαmbiαe odorant receptor protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutions for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such substitutions are generally understood to be conservative substitutions. In a specific embodiment of the invention, proteins consisting of or comprising a fragment of an Anopheles gαmbiαe odorant receptor protein consisting of at least 10 (continuous) amino acids of an Anopheles gαmbiαe odorant receptor protein is provided. In other embodiments, the fragment consists of at least 20 or at least 30 or at least 50 amino acids of the Anopheles gαmbiαe odorant receptor protein. In specific embodiments, such fragments are not larger than 35, 100 or 200 amino acids. In specific embodiments, such fragments are 30-50, 50-100, 100-220, or 200-390 amino acids. Derivatives or analogs of Anopheles gambiae odorant receptor proteins include but are not limited to those molecules comprising regions that are substantially homologous to an protein or fragment thereof (e.g., in various embodiments, at least 60% or 70% or 80% or 90% or 95% identity over an amino acid sequence of identical size without any insertions or deletions or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art) or whose encoding nucleic acid is capable of hybridizing to a coding Anopheles gambiae odorant receptor gene sequence, under high stringency, moderate stringency, or low stringency conditions. Based on the sequence alignment, Anopheles gambiae odorant receptor genes are expected to encode proteins with stretches of conserved amino acid residues at the C-terminus, e.g., comprising SEQ ID NO:25.

The Anopheles gambiae odorant receptor derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, a cloned Anopheles gambiae odorant receptor gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al , 1989, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. In the production of a modified gene encoding a derivative or analog of an Anopheles gαmbiαe odorant receptor protein, care should be taken to ensure that the modified gene remains within the same translational reading frame as the native protein, uninterrupted by translational stop signals, in the gene region where the desired Anopheles gαmbiαe odorant receptor protein activity is encoded.

Additionally, an Anopheles gαmbiαe odorant receptor nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or to form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson et αl, 1978, J. Biol. Chem. 253:6551), use of TAB® linkers (Pharmacia), PCR with primers containing a mutation, etc.

Manipulations of an Anopheles gαmbiαe odorant receptor protein sequence may also be made at the protein level. Included within the scope of the invention are Anopheles gambiae odorant receptor protein fragments or other derivatives or analogs which are differentially modified d ing or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be canied out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.

In addition, analogs and derivatives of an Anopheles gαmbiαe odorant receptor protein can be chemically synthesized. For example, a peptide conesponding to a portion of an Anopheles gαmbiαe odorant receptor protein which comprises the desired domain, or which mediates the desired activity in vitro, can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the Anopheles gαmbiαe odorant receptor sequence. Non-classical amino acids include but are not limited to the D- isomers of the common amino acids, α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, omithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β- alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

In a specific embodiment, an Anopheles gαmbiαe odorant receptor protein derivative is a chimeric or fusion protein comprising an Anopheles gαmbiαe odorant receptor protein or fragment thereof (preferably consisting of at least a domain or motif of the Anopheles gαmbiαe odorant receptor protein, or at least 10 amino acids of the Anopheles gαmbiαe odorant receptor protein) joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein. In specific embodiments, the amino acid sequence of the different protein is at least 6, 10, 20 or 30 continuous amino acids of the different proteins or a portion of the different protein that is functionally active. In specific embodiments, the amino acid sequence of the different protein is at least 50, 75, 100, or 150 continuous amino acids of the different proteins or a portion of the different protein that is functionally active. In one embodiment, such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising an Anopheles gαmbiαe odorant receptor-coding sequence joined in-frame to a coding sequence for a different protein). Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art. Alternatively, such a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer. Chimeric genes comprising portions of an Anopheles gambiae odorant receptor gene fused to any heterologous protein- encoding sequences may be constructed. A specific embodiment relates to a chimeric protein comprising a fragment of an Anopheles gambiae odorant receptor protein of at least six amino acids, or a fragment that displays one or more functional activities of the Anopheles gambiae odorant receptor protein.

In another specific embodiment, the Anopheles gambiae odorant receptor derivative is a molecule comprising a region of homology with an Anopheles gambiae odorant receptor protein. By way of example, in various embodiments, a first protein region can be considered "homologous" to a second protein region when the amino acid sequence of the first region is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% identical, when compared to any sequence in the second region of an equal number of amino acids as the number contained in the first region without any insertions or deletions or when compared to an aligned sequence of the second region that has been aligned by a computer homology program known in the art. For example, a molecule can comprise one or more regions homologous to an Anopheles gambiae odorant receptor domain or a portion thereof. In a specific embodiment, the invention relates to Anopheles gambiae odorant receptor derivatives and analogs, in particular Anopheles gambiae odorant receptor fragments and derivatives of such fragments, that comprise, or alternatively consist of, one or more domains of an Anopheles gαmbiαe odorant receptor protein. In another specific embodiment, a molecule is provided that comprises one or more domains (or functional portion thereof) of an Anopheles gαmbiαe odorant receptor protein but that also lacks one or more domains (or functional portion thereof) of an Anopheles gαmbiαe odorant receptor protein. By way of example, such a protein may retain such domains separated by a spacer. In another embodiment, a molecule is provided that comprises one or more domains (or functional portion(s) thereof) of an Anopheles gαmbiαe odorant receptor protein, and that has one or more mutant (e.g., due to deletion or point mutation(s)) domains of an Anopheles gαmbiαe odorant receptor protein (e.g., such that the mutant domain has decreased or increased function compared to wild type).

The present invention yet further encompasses polypeptides that encode olfactory receptors that originate from insect species other than Anopheles gαmbiαe, for example Culex spp. and Aedes aegypti, and that relate in sequence, transmembrane distribution, hydrophilicity, and/or function to the Anopheles gambiae olfactory receptors disclosed herein.

5.4. STRUCTURE OF ANOPHELES

GAMBIAE ODORANT RECEPTOR GENES AND PROTEINS

The structure of Anopheles gambiae odorant receptor genes and proteins of the invention, and related odorant receptor genes and proteins of other insect species such as

Culex spp. and Aedes aegypti, can be analyzed by various methods known in the art. Some examples of such methods are described below.

5.4.1. GENETIC ANALYSIS

The cloned DNA or cDNA conesponding to an Anopheles gambiae odorant receptor gene can be analyzed by methods including but not limited to Southern hybridization (Southern, 1975, J. Mol. Biol. 98:503-517), Northern hybridization (see e.g., Freeman et al, 1983, Proc. Natl. Acad. Sci. U.S.A. 80:4094-4098), restriction endonuclease mapping (Maniatis, 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), and DNA sequence analysis. Accordingly, this invention provides nucleic acid probes recognizing an Anopheles gambiae odorant receptor gene. For example, polymerase chain reaction (PCR; U.S. Patent Nos. 4,683,202, 4,683,195 and 4,889,818; Gyllenstein et α/., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7652-7656; Ochman et α/., 1988, Genetics 120:621-623; oh etal, 1989, Science 243:217-220) followed by Southern hybridization with an Anopheles gambiae odorant receptor gene-specific probe can allow the detection of an Anopheles gambiae odorant receptor gene in DNA from various cell types, such as olfactory nemons in antennae and maxillary palps. Methods of amplification other than PCR are commonly known and can also be employed. In one embodiment, Southern hybridization can be used to determine the genetic linkage of an Anopheles gambiae odorant receptor gene. Northern hybridization analysis can be used to determine the expression of an Anopheles gambiae odorant receptor gene. The stringency of the hybridization conditions for both Southern and Northern hybridization can be manipulated to ensme detection of nucleic acids with the desired degree of relatedness to the specific Anopheles gambiae odorant receptor gene probe used. Modifications of these methods and other methods commonly known in the art can be used. In a prefened specific embodiment of the invention, Northern hybridization is performed using different poly(A)+ mRNA preparations (e.g., cells treated or untreated with DNA damaging agents) which were fractionated on an agarose gel along with size standards and blotted to a nylon membrane. A DNA fragment containing an Anopheles gambiae odorant receptor coding region is excised from a clone digested with an appropriate restriction endonuclease, separated by electrophoresis in an agarose gel, extracted from the gel, and ³²P-labeled by random-priming using the Rediprime labeling system (Amersham). Hybridization of the labeled probe to the mRNA blot is performed overnight. The blot is washed at high stringency (0.2x SSC/0.1% SDS at 65°C) and mRNA species that specifically hybridized to the probe are detected by autoradiography using X-ray film.

Restriction endonuclease mapping can be used to roughly determine the genetic structme of an Anopheles gαmbiαe odorant receptor gene. Restriction maps derived by restriction endonuclease cleavage can be confirmed by DNA sequence analysis. DNA sequence analysis can be performed by any techniques known in the art, including but not limited to the method of Maxam and Gilbert (1980, Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger et αl, 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463), the use of T7 DNA polymerase (Tabor and Richardson, U.S. Patent No. 4,795,699), or use of an automated DNA sequenator (e.g., Applied Biosystems, Foster City, California).

The genetic analysis methods described above for Anopheles gαmbiαe odorant receptor genes of the invention can also be used for analysis of related odorant receptor genes and proteins of other insect species such as Culex spp. and Aedes aegypti.

5.4.2. PROTEIN ANALYSIS

The amino acid sequence of an Anopheles gambiae odorant receptor protein, or a related protein from another insect species such as Culex spp. and Aedes aegypti can be derived by deduction from the DNA sequence, or alternatively, by direct sequencing of the protein, e.g., with an automated amino acid sequencer. An Anopheles gambiae odorant receptor protein sequence or a related sequence can be further characterized by a hydrophilicity analysis (Hopp and Woods, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824). A hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of the Anopheles gambiae odorant receptor protein or related protein and the conesponding regions of the gene sequence which encode such regions. Structural prediction analysis (Chou and Fasman, 1974, Biochemistry 13:222) can also be done, to identify regions of an Anopheles gambiae odorant receptor protein or related insect odorant receptor protein that assume specific secondary structures. Manipulation, translation, secondary structure prediction, open reading frame prediction and plotting, as well as determination of sequence homologies, can also be accomplished using computer software programs available in the art (see Section 5.2).

Other methods of stmctmal analysis can also be employed. These include but are not limited to X-ray crystallography (Engstom, 1974, Biochem. Exp. Biol. 11:7- 13), nuclear magnetic resonance spectroscopy (Clore and Gonenbom, 1989, CRC Crit. Rev. Biochem. 24:479-564) and computer modeling (Fletterick and Zoller, 1986, Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

5.5. ASSAYS OF ANOPHELES GAMBIAE ODORANT RECEPTOR PROTEIN

DERIVATIVES

The functional activity of Anopheles gambiae odorant receptor proteins, as well as the functional activity of related odorant receptor proteins of other insect species such as Culex spp. and Aedes aegypti, and derivatives and analogs of the foregoing, can be assayed by various methods known to one skilled in the art.

For example, in one embodiment, where one is assaying for the ability to bind to or compete with a wild-type Anopheles gambiae odorant receptor protein for binding to an anti-Anopheles gαmbiαe odorant receptor antibody, various immunoassays known in the art can be used, including but not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. In 03/020913

another embodiment, where an Anopheles gambiae odorant receptor-binding protein is identified, the binding can be assayed, e.g., by means well-known in the art.

In another embodiment, genetic studies can be done to study the phenotypic effect of an Anopheles gαmbiαe odorant receptor gene mutant that is a derivative or analog of a wild-type Anopheles gαmbiαe odorant receptor gene. Other such methods will be readily apparent to the skilled artisan and are within the scope of the invention.

In yet other embodiments, assays of Anopheles gαmbiαe odorant receptor derivatives and fragments can be assayed for their ability to modulate G protein activity, or to bind ligand, as described in the screening assays in Section 5.8, infra. The functional analysis methods described supra for Anopheles gambiae odorant receptors can be applied to related odorant receptor proteins of other insect species such as Culex spp. and Aedes aegypti.

5.6. ANTIBODIES According to the invention, an Anopheles gambiae odorant receptor protein, its fragments or other derivatives, or analogs thereof, may be used as an immunogen to generate antibodies which immunospecifically bind such an immunogen. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. In another embodiment, antibodies to a domain of an Anopheles gambiae odorant receptor protein are produced. In a specific embodiment, fragments of an Anopheles gαmbiαe odorant receptor protein identified as hydrophilic are used as immunogens for antibody production.

Various procedures known in the art may be used for the production of polyclonal antibodies to an Anopheles gαmbiαe odorant receptor protein or derivative or analog. In a particular embodiment, rabbit polyclonal antibodies to an epitope of an Anopheles gαmbiαe odorant receptor protein consisting of the sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12, or a subsequence thereof, can be obtained. For the production of antibody, various host animals can be immunized by injection with the native Anopheles gαmbiαe odorant receptor protein, or a synthetic version, or derivative (e.g., fragment) thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.

For preparation of monoclonal antibodies directed to an Anopheles gambiae odorant receptor protein sequence or analog thereof, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (Kohler and Milstein 1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al, 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole et al, 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ- free animals (see e.g., PCT/US90/022548). According to the invention, human antibodies may be used and can be obtained by using human hybridomas (Cole et al, 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV vims in vitro (Cole et al. , 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). In fact, according to the invention, techniques developed for the production of "chimeric antibodies" (Monison et α/., 1984, Proc. Nati. Acad. Sci. U.S.A. 81:6851- 6855; Neuberger et al, 1984, Nature 312:604-608; Takeda et al, 1985, Nature 314:452- 454) by splicing the genes from a mouse antibody molecule specific for an Anopheles gambiae odorant receptor protein together with genes from a human antibody molecule of appropriate biological activity can be used; such antibodies are within the scope of this invention. In another embodiment, "humanized" antibodies are also provided by the invention (U.S. Patent No. 5,225,539)

According to the invention, techniques described for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce Anopheles gambiae odorant receptor-specific single chain antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of Fab' expression libraries (Huse et al, 1989, Science 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for Anopheles gambiae odorant receptor proteins, derivatives, or analogs.

Antibody fragments which contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include but are not limited to, the F(ab')₂ fragment which can be produced by pepsin digestion of the antibody molecule, the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragment, the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent, and Fv fragments.

In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art (e.g., enzyme-linked immunosorbent assay or ELISA). For example, to select antibodies which recognize a specific domain of an

Anopheles gambiae odorant receptor protein, one may assay generated hybridomas for a product which binds to an Anopheles gambiae odorant receptor fragment containing such domain. For selection of an antibody that specifically binds a first Anopheles gambiae odorant receptor but which does not specifically bind a different Anopheles gambiae odorant receptor, one can select on the basis of positive binding to the first Anopheles gambiae odorant receptor and a lack of binding to the second Anopheles gambiae odorant receptor.

Antibodies specific to a domain of an Anopheles gαmbiαe odorant receptor protein are also provided. Antibodies specific to an epitope of an Anopheles gαmbiαe odorant receptor protein are also provided.

5.7. IDENTIFICATION OF COMPOUNDS

THAT BIND TO ANOPHELES GAMBIAE ODORANT RECEPTOR PROTEINS

This invention provides screening methodologies useful in the identification of proteins and other compounds which bind to, or otherwise directly interact with, Anopheles gαmbiαe odorant receptor genes and proteins. Such compounds will include molecules that agonize or antagonize Anopheles gαmbiαe odorant receptor function.

Screening methodologies are well known in the art (see e.g., PCT International Publication No. WO 96/34099, published October 31, 1996, which is incorporated by reference herein in its entirety). The proteins and compounds include endogenous cellular components which interact with the identified genes and proteins in vivo and which, therefore, may provide new targets for pharmaceutical and therapeutic interventions, as well as recombinant, synthetic, and otherwise exogenous compounds which may have binding capacity and, therefore, may be candidates for pharmaceutical agents. Thus, in one series of embodiments, cell lysates or tissue homogenates may be screened for proteins or other compounds which bind to one of the Anopheles gαmbiαe odorant receptor genes and proteins.

Alternatively, any of a variety of exogenous compounds, both naturally occuning and/or synthetic (e.g., libraries of small molecules or peptides), may be screened for binding capacity. Binding compounds can include, but are not limited to, other cellular proteins. Binding compounds can also include, but are not limited to, peptides such as, for example, soluble peptides, including, but not limited to, Ig-tailed fusion peptides (see, e.g., Lam et al, 1991, Nature 354:82-84; Houghten et al, 1991, Nature 354:84-86), antibodies (including, but not limited to polyclonal, monoclonal, human, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab')₂ and FAb expression library fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules. Such compounds can include organic molecules (e.g., peptidomimetics) that bind to the ECD and either mimic the activity triggered by the natural odorant ligand (i.e., agonists); as well as peptides, antibodies or fragments thereof, and other organic compounds that mimic the ECD (or a portion thereof) and bind to and "neutralize" natmal odorant ligand. Such compounds identified in a screen for binding to an Anopheles gambiae odorant receptor can be assayed for their effects on Anopheles gambiae odorant receptor signaling, as described in Section 5.8, infra.

Computer modeling and searching technologies permit identification of compounds that can modulate Anopheles gambiae odorant receptor activity. Having identified such a compound or composition, the active sites or regions are preferably identified. Such active sites might typically be odorant ligand binding sites, such as the interaction domains of odorant ligands with Anopheles gambiae odorant receptor polypeptides. The active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of Anopheles gambiae odorant receptor polypeptides with their natural ligands. In the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where on the Anopheles gambiae odorant receptor polypeptides the complexed odorant ligand is found. The three dimensional geometric structme of the active site can also be determined. This can be done by known methods, including X-ray crystallography, which can determine a complete molecular stmcture. Solid or liquid phase NMR can also be used to determine certain infra-molecular distances within the active site and/or in the odorant ligand/ Anopheles gambiae odorant receptor complex. Any other experimental method of structure determination can be used to obtain partial or complete geometric stmctures. The geometric stmctures may be measured with a complexed odorant ligand, natural or artificial, which may increase the accmacy of the active site stmcture determined.

Methods of computer based numerical modeling can be used to complete the stmcture (e.g., in embodiments wherein an incomplete or insufficiently accurate stmcture is determined) or to improve its accmacy. Any art recognized modeling method may be used, including, but not limited to, parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models. For most types of models, standard molecular force fields, representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry. Exemplary forcefields that are known in the art and can be used in such methods include, but are not limited to, the Constant Valence Force Field (CVFF), the AMBER force field and the CHARM force field. The incomplete or less accmate experimental structures can serve as constraints on the complete and more accmate stmctmes computed by these modeling methods.

Finally, having determined the stmcture of the active site, either experimentally, by modeling, or by a combination, candidate modulating compounds can be identified by searching databases containing compounds along with information on their molecular stmcture. Such a search seeks compounds having stmctures that match the determined active site stmcture and that interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. These compounds found from this search are potential target or pathway polypeptide modulating compounds.

Alternatively, these methods can be used to identify improved modulating compounds from an already known modulating compound or odorant ligand. The composition of the known compound can be modified and the structmal effects of modification can be determined using the experimental and computer modeling methods described above applied to the new composition. The altered stmcture is then compared to the active site structure of the compound to determine if an improved fit or interaction results. In this manner systematic variations in composition, such as by varying side groups, can be quickly evaluated to obtain modified binding compounds or odorant ligands of improved specificity or activity.

Examples of molecular modeling systems are the CHARMm and QUANTA programs (Polygen Corporation, Waltham, MA). CHARMm performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modeling and analysis of molecular stmcture. QUANTA allows interactive constmction, modification, visualization, and analysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive with specific proteins, such as Rotivinen et al, 1988, Acta Pharmaceutical Fennica 97:159-166; Ripka, June 16, 1988, New Scientist 54-57; McKinaly and Rossmann, 1989, Annu. Rev. Pharmacol. Toxiciol. 29:111-122; Perry and Davies, OSAR: Quantitative Stmcture- Activity Relationships in Drag Design pp. 189-193 (Alan R. Liss, Inc. 1989); and Lewis and Dean, 1989, Proc. R. Soc. Lond. 236:125-140 and 1-162. Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign, Inc. (Pasadena, CA.), Allelix, Inc. (Mississauga, Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario).

Although generally described above with reference to design and generation of compounds which could alter binding, one could also screen libraries of known compounds, including natural products or synthetic chemicals, and biologically active materials, including proteins, for compounds which bind to Anopheles gambiae odorant receptor polypeptides.

Assays for identifying additional compounds as well as for testing the effectiveness of compounds, identified by, for example, techniques, such as those described in Section 5.8, are discussed, below, in Section 5.8. As will be apparent to one of ordinary skill in the art, there are numerous other methods of screening individual proteins or other compounds, as well as large libraries of proteins or other compounds (e.g., phage display libraries) to identify molecules which bind to Anopheles gambiae odorant receptor proteins. All of these methods comprise the step of mixing an Anopheles gambiae odorant receptor protein, fragment or mutant, or a composition comprising said Anopheles gambiae odorant receptor protein, fragment or mutant, including but not limited to a cultured cell, with test compounds, allowing time for any binding to occur, and assaying for any bound complexes, as described in further detail below.

In prefened embodiments of the present invention, the cells used for screening for molecules that bind to and/or modulate activity of an Anopheles gambiae olfactory receptor also express an Or83b-class receptor, most preferably the Anopheles gambiae Or83b (SEQ ID NO: 14) protein.

5.7.1. SCREENING FOR SMALL MOLECULES THAT BIND TO ANOPHELES GAMBIAE ODORANT RECEPTORS IN VIVO

In particular, methods are provided for identifying a molecule that binds to an Anopheles gambiae odorant receptor protein. In one embodiment, the method comprises contacting a cell that expresses the Anopheles gambiae odorant receptor with a test molecule, or plurality of test molecules, under conditions conducive to binding between the receptor and the test molecule, and determining whether the test molecule binds to the cell. A molecule that binds to an Anopheles gambiae odorant receptor, but not to a counterpart cell that does not express an Anopheles gambiae odorant receptor, can be identified thereby.

In an alternative embodiment, a molecule that binds to an Anopheles gambiae odorant receptor from Anopheles gambiae but not from another species is identified. This method comprises contacting two different species of cells, one of which is Anopheles gambiae, that both express an Anopheles gambiae odorant receptor with a test molecule under conditions conducive to binding of the receptor and the test molecule. The binding of the test molecule to the cells is tested. Test molecules that bind to Anopheles gambiae odorant receptor on the first cell but not the second cell are identified. In an alternative embodiment, a method is provided for identifying a molecule that binds to a first insect olfactory receptor but not a second insect olfactory receptor. The method comprises contacting a first cell that expresses the Anopheles gambiae odorant receptor and a first olfactory receptor and a second cell that expresses the Anopheles gambiae odorant receptor and a second olfactory receptor, with a test molecule, or plurality of test molecules, under conditions conducive to binding between the receptor and the test molecule, and determining whether the test molecule binds to the cells. A molecule that binds to the first cell, and therefore first olfactory receptor, but not the second cell and second insect olfactory receptor can be identified thereby.

5.7.2. SCREENING FOR SMALL MOLECULES THAT

BIND TO ANOPHELES GAMBIAE ODORANT RECEPTORS IN VITRO

In vitro systems can be designed to identify compounds capable of binding the Anopheles gambiae odorant receptor polypeptides of the invention. Compounds identified can be useful, for example, in modulating the activity of wild type Anopheles gambiae odorant receptors, and thereby modulating Anopheles gambaie behavior.

The principle of the assays used to identify compounds that bind to Anopheles gambiae odorant receptor polypeptides involves preparing a reaction mixture of an Anopheles gambiae odorant receptor polypeptide and a test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways. For example, one method to conduct such an assay involves anchoring the Anopheles gambiae odorant receptor polypeptide or the test substance onto a solid phase and detecting Anopheles gambiae odorant receptor polypeptide/test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the Anopheles gambiae odorant receptor polypeptide can be anchored onto a solid smface, and the test compound, which is not anchored, can be labeled, either directly or indirectly. hi practice, microtiter plates can conveniently be utilized as the solid phase. The anchored component can be immobilized by non-covalent or covalent attachments. Non-covalent attachment can be accomplished by simply coating the solid smface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized can be used to anchor the protein to the solid smface. The surfaces can be prepared in advance and stored.

In order to conduct the assay, the nonimmobilized component is added to the coated smface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid smface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously nonimmobilized component is pre-labeled, the detection of label immobilized on the smface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the smface; e.g., using a labeled antibody specific for the previously nonimmobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for the Anopheles gambiae odorant receptor polypeptide or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes. As an example, and not by way of limitation, techniques such as those described in this section can be utilized to identify compounds which bind to an Anopheles gambiae odorant receptor polypeptide. For example, an Anopheles gambiae odorant receptor polypeptide can be contacted with a compound for a time sufficient to form an Anopheles gambiae odorant receptor polypeptide/compound complex and then such a complex can be detected.

Alternatively, the compound can be contacted with the Anopheles gambiae odorant receptor polypeptide in a reaction mixture for a time sufficient to form an Anopheles gambiae odorant receptor polypeptide/compound complex, and then such a complex can be separated from the reaction mixt e. 5.8. SCREENING FOR MOLECULES THAT MODULATE ANOPHELES GAMBIAE ODORANT RECEPTOR ACTIVITY

Particularly useful molecules that bind to or modulate Anopheles gambiae odorant receptor protein activity are small molecules, most preferably volatile small molecules, that function as odorant. The term "odorant" as employed herein refers to a molecule that has the potential to bind to an olfactory receptor. Equivalent terms employed herein include "odorant ligand", "odorant molecule" and "odorant compound". The term "binding" or "interaction" as used herein with respect to odorant ligands refers to the interaction of ligands with the receptor polypeptide where the ligands may serve as either agonists and/or antagonists of a given receptor or receptor function. This effect may not be direct, but merely by altering the binding of an Anopheles gαmbiαe odorant receptor to another ligand. An odorant ligand may thus directly cause a perception of odor (an agonist), or may block the perception of odor (an antagonist). An odorant ligand may include, but is not limited to, molecules which interact with polypeptides involved in olfactory sensation. Odorant ligands and molecules which interact with olfactory receptors are generally small, approximately 1 kD, more preferably approximately 0.75 kD, more preferably approximately 0.5 kD, or even more preferably approximately 0.3 kD, hydrophobic molecules with a variety of functional groups. Small changes in stmctme can induce profound changes in odorant ligand binding and hence in the odor perceived by an individual. In a specific embodiment, the odorant ligand is an Anopheles gαmbiαe odorant receptor ligand, i.e., a ligand that binds to an Anopheles gαmbiαe odorant receptor alone or an Anopheles gαmbiαe odorant receptor in conjunction with an Or83b related receptor.

Thus, the present invention provides methods for screening for molecules, more preferably small molecules, most preferably volatile molecules, that modulate Anopheles gαmbiαe odorant receptor activity. Methods for screening odorant compounds using Anopheles gαmbiαe odorant receptors in nemonal cells are known in the art (Firestein et αl., WO 98/50081; Duchamp-Viret et αl., 1999, Science 284:2171-2174; Sato et αl., 1994, J. Neurophys.72:2980-2989; Malnic et αl., 1999, Cell 96:713-723; Zhao et αl., 1998, Science 279:237-242). There are also methods which can be employed to screen odorant compounds which do not require nemonal cells and are known in the art (Kauvar et αl. , U. S. Pat. No. 5,798,275; Kiefer et αl., 1996, Biochemistry 35:16077-16084; Krautwmst et αl, 1998, Cell 95:917-926).

In a particular embodiment, a method is provided for identifying a modulator of an Anopheles gαmbiαe odorant receptor protein. The method comprises contacting a first cell that expresses the Anopheles gαmbiαe odorant receptor with a test molecule, or plmality of test molecules, under conditions conducive to binding between the receptor and the test molecule, and determining whether the test molecule modulates G-protein activity in the first cell but not in a second cell which does not express the Anopheles gambiae odorant receptor. A molecule that modulates an Anopheles gambiae odorant receptor can be identified thereby.

In an alternative embodiment, a molecule that modulates an Anopheles gambiae odorant receptor from Anopheles gambiae but not from another insect species is identified. This method comprises contacting two different species of cells, one of which is Anopheles gambiae, that both express an Anopheles gambiae odorant receptor with a test molecule under conditions conducive to binding of the receptor and the test molecule. The G-protein activity in the cells is measured. Test molecules that modulate G-protein activity of the Anopheles gambiae odorant receptor on the first cell but not the second cell are identified.

In an alternative embodiment, a method is provided for identifying an odorant that modulates the activity of first olfactory receptor but not a second insect olfactory receptor. The method comprises contacting a first cell that expresses the Anopheles gambiae odorant receptor and a first olfactory receptor and a second cell that expresses the Anopheles gambiae odorant receptor and a second olfactory receptor, with a test molecule, or plmality of test molecules, under conditions conducive to binding between the receptor and the test molecule, and determining whether the test molecule binds to the cells. A molecule that modulates the first cell, and therefore first olfactory receptor, but not the second cell and second insect olfactory receptor can be identified thereby.

Several methods of measming G-protein activity are known to those of skill in the art and can be used in conjunction with the methods of the present invention, including but not limited to measming calcium ion or cyclic AMP concentration in the cells. Such methods are described in Howard et al, 2001, Trends Pharmacol Sci. 22(3):132-40; Krautwmst et al, 1999, Cell 95:917-926; Chandrashekar et al, 2000, Cell.l00(6):703-11; and Oda et al, 2000, J Biol Chem. 275(47):36781-6, which are incorporated by reference herein in their entireties. In certain specific embodiments, intracellular calcium concentration is measmed in the screening assays of the instant application by using a Fluorometric Imaging Plate Reader ("FLIPR") system (Molecular Devices, Inc.), which provides the advantages automated, high-throughput screening, see also Sullivan et al, 1999, "Measurement of [Ca²⁺]i using the fluometric imaging plate reader (FLIPR)," In Calcium Signaling Protocols. ed Lambert, D.G., pp. 125 - 136 (New Jersey: Humana Press); or as described by Offermanns and Simon, 1995, J. Biol. Chem. 270(25):15175-80; Ungrin et al, 1999, Anal Biochem. 272(l):34-42; or in U.S. Patent No. 6,004,808, which employs Fura-PE3 (Molecular Probes, Inc., Eugene, OR) as a stain of calcium ions. Calcium ion activity is proportional to Anopheles gambiae odorant receptor G protein activity. In another specific embodiment, cAMP concentration is measmed in the screening assays of the instant application by the method of Fitzgerald et al, Anal. Biochem. 275(1):54-61. cAMP concentration is proportional to Anopheles gambiae odorant receptor activity.

The functional assay developed above may form the basis of a chemical screen to identify compounds that maximally activate a given receptor or that block receptor activation. Rapid, automated screening procedmes that allow the screening of greater than 100,000 candidate compounds are used widely in the pharmaceutical industry to search for new drags that target GPCRs and should be readily adaptable to Anopheles gambiae odorant receptor screening.

5.8.1. PROTEINS WHICH INTERACT WITH ANOPHELES GAMBIAE ODORANT RECEPTOR POLYPEPTIDES

The present invention further provides methods of identifying or screening for proteins which interact with Anopheles gambiae odorant receptor proteins, or derivatives, fragments, mutants or analogs thereof.

Any method suitable for detecting protein-protein interactions can be employed for identifying novel Anopheles gambiae odorant receptor protein-cellular protein interactions. Among the traditional methods which can be employed are co-immunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns. Utilizing procedmes such as these allows for the identification of proteins that interact with an Anopheles gambiae odorant receptor polypeptide. Once identified, such proteins can be used, for example, to dismpt Anopheles gambiae odorant receptor signaling through the endogenous cellular counterpart of the protein, thereby antagonizing Anopheles gambiae odorant receptor-induced behaviors. Once identified, such proteins that interact with an Anopheles gambiae odorant receptor polypeptide can also be used, in conjunction with standard techniques, to identify the conesponding gene that encodes the protein which interacts with the Anopheles gambiae odorant receptor polypeptide. For example, at least a portion of the amino acid sequence of the polypeptide can be ascertained using techniques well known to those of skill in the art, such as via the Edman degradation technique (see, e.g., Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N. Y., pp.34-49). The amino acid sequence obtained can be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for gene sequences. Screening can be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and for screening are well-known. (See, e.g. , Ausubel, supra., and PCR Protocols: A Guide to Methods and Applications, 1990, Innis, M. et al, eds. Academic Press, Inc., New York).

Additionally, methods can be employed which result in the simultaneous identification of genes which encode proteins interacting with an Anopheles gambiae odorant receptor polypeptide. These methods include, for example, probing expression libraries with labeled Anopheles gambiae odorant receptor polypeptide, using this protein in a manner similar to the well known technique of antibody probing of λgtl 1 libraries. One method which detects protein interactions in vivo, the two-hybrid system, is described in detail for illustration purposes only and not by way of limitation. One version of this system has been described (Chien et al, 1991, Proc. Natl. Aca. Sci. U.S.A. 88:9578-9582) and is commercially available from Clontech (Palo Alto, CA).

Briefly, utilizing such a system, plasmids are constmcted that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to a known protein, in this case, an Anopheles gambiae odorant receptor polypeptide, and the other consists of the activator protein's activation domain fused to an unknown protein that is encoded by a cDNA, preferably an insect antennal or maxillary palp cDNA, which has been recombined into this plasmid as part of a cDNA library. The plasmids are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., lacZ) whose regulatory region contains the transcription activator's binding sites. Either hybrid protein alone cannot activate transcription of the reporter gene, the DNA- binding domain hybrid cannot because it does not provide activation function, and the activation domain hybrid cannot because it cannot localize to the activator's binding sites. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter polypeptide.

The two-hybrid system or related methodology can be used to screen activation domain libraries for proteins that interact with a known "bait" polypeptide. By way of example, and not by way of limitation, Anopheles gambiae odorant receptor polypeptides can be used as the bait polypeptides. Total genomic or cDNA sequences are fused to the DNA encoding an activation domain. This library and a plasmid encoding a hybrid of the bait polypeptide fused to the DNA-binding domain are cotransformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene. For example, and not by way of limitation, the bait (e.g., Anopheles gambiae odorant receptor) gene can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein. These colonies are pmified and the library plasmids responsible for reporter gene expression are isolated. DNA sequencing is then used to identify the proteins encoded by the library plasmids.

A cDNA library of the cell line from which proteins that interact with bait (e.g., Anopheles gambiae odorant receptor) polypeptide are to be detected can be made using methods routinely practiced in the art. According to the particular system described herein, for example, the cDNA fragments can be inserted into a vector such that they are translationally fused to the activation domain of GAL4. This library can be co-transformed along with the bait gene-GAL4 fusion plasmid into a yeast strain which contains a lacZ gene driven by a promoter which contains GAL4 activation sequence. A cDNA encoded protein, fused to GAL4 activation domain, that interacts with bait polypeptide will reconstitute an active GAL4 protein and thereby drive expression of the lacZ gene. Colonies which express lacZ can be detected by their blue color in the presence of X-gal. The cDNA can then be purified from these strains, and used to produce and isolate the bait gene-interacting protein using techniques routinely practiced in the art.

5.8.2. ASSAYS FOR COMPOUNDS THAT

INTERFERE WITH ANOPHELES GAMBIAE ODORANT RECEPTOR SIGNALING

The Anopheles gambiae odorant receptor polypeptides of the invention can, in vivo, interact with one or more cellular macromolecules, such as proteins, including but not limited to G proteins. Such macromolecules can include, but are not limited to those proteins identified via methods such as those described, above, in Section 5.8. Compounds that dismpt such interactions can be useful in regulating the activity of an Anopheles gαmbiαe odorant receptor polypeptide, thereby modulating insect behavior. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and the like.

The basic principle of the assay systems used to identify compounds that interfere with the interaction between an Anopheles gαmbiαe odorant receptor polypeptide and its cellular binding partner or partners involves preparing a reaction mixture containing the Anopheles gαmbiαe odorant receptor polypeptide and the binding partner under conditions and for a time sufficient to allow the two to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of an Anopheles gαmbiαe odorant receptor polypeptide and its cellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the Anopheles gαmbiαe odorant receptor polypeptide and the cellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the Anopheles gαmbiαe odorant receptor polypeptide and the interactive binding partner.

The assay for compounds that interfere with the interaction of the Anopheles gαmbiαe odorant receptor polypeptides and binding partners can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the Anopheles gαmbiαe odorant receptor polypeptide or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is canied out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the Anopheles gαmbiαe odorant receptor polypeptides and the binding partners, e.g. , by competition, can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the Anopheles gαmbiαe odorant receptor polypeptide and interactive cellular binding partner. Alternatively, test compounds that dismpt preformed complexes, e.g. compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below.

In a heterogeneous assay system, either the Anopheles gαmbiαe odorant receptor polypeptide or the interactive cellular binding partner, is anchored onto a solid smface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtiter plates are conveniently utilized. The anchored species can be immobilized by non-covalent or covalent attachments. Non-covalent attachment can be accomplished simply by coating the solid smface with a solution of the Anopheles gαmbiαe odorant receptor polypeptide or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid smface. The surfaces can be prepared in advance and stored. In order to conduct the assay, the partner of the immobilized species is exposed to the coated smface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid smface. The detection of complexes anchored on the solid smface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the smface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds which inhibit complex or which dismpt preformed complexes can be identified. In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed complex of the Anopheles gambiae odorant receptor polypeptide and the interactive cellular binding partner is prepared in which either the Anopheles gambiae odorant receptor polypeptide or its binding partner is labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Patent No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances which dismpt Anopheles gambiae odorant receptor polypeptide/cellular binding partner interaction can be identified. In a particular embodiment, the target polypeptide can be prepared for immobilization using recombinant DNA techniques. For example, the Anopheles gambiae odorant receptor coding region can be fused to a glutathione-S-transferase (GST) gene using a fusion vector, such as pGEX-5X-l, in such a manner that its binding activity is maintained in the resulting fusion protein. The interactive cellular binding partner can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art and described above, in Section 5.6. This antibody can be labeled with the radioactive isotope ¹²⁵I, for example, by methods routinely practiced in the art. In a heterogeneous assay, e.g., the GST-Anopheles gambiae odorant receptor fusion protein can be anchored to glutathione-agarose beads. The interactive cellular binding partner can then be added, in the presence or absence of the test compound in a manner that allows interaction and binding to occm. At the end of the reaction period, unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components. The interaction between the Anopheles gambiae odorant receptor polypeptide and the interactive cellular binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compomid will result in a decrease in measmed radioactivity.

Alternatively, the GST-Anopheles gambiae odorant receptor fusion protein and the interactive cellular binding partner can be mixed together in liquid in the absence of the solid glutathione-agarose beads. The test compound can be added either d ing or after the species are allowed to interact. This mixture can then be added to the glutathione- agarose beads and unbound material is washed away. Again the extent of inhibition of the Anopheles gambiae odorant receptor polypeptide/binding partner interaction can be detected by adding the labeled antibody and measming the radioactivity associated with the beads. In another embodiment of the invention, these same techniques can be employed using peptide fragments that conespond to the binding domains of the Anopheles gambiae odorant receptor polypeptide and/or the interactive cellular binding partner (in cases where the binding partner is a protein), in place of one or both of the full length proteins. Any number of methods routinely practiced in the art can be used to identify and isolate the binding sites. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for disruption of binding in a co- immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that conespond to the region of the protein involved in interactive binding. Alternatively, one protein can be anchored to a solid smface, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain can remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding for the cellular binding partner is obtained, short gene segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized.

For example, and not by way of limitation, an Anopheles gambiae odorant receptor polypeptide can be anchored to a solid material as described, above, in this Section, by making a GST-Anopheles gambiae odorant receptor fusion protein and allowing it to bind to glutathione agarose beads. The interactive cellular binding partner can be labeled with a radioactive isotope, such as ³⁵S, and cleaved with a proteolytic enzyme such as trypsin. Cleavage products can then be added to the anchored GST-Anopheles gambiae odorant receptor fusion protein and allowed to bind. After washing away unbound peptides, labeled bound material, representing the cellular binding partner binding domain, can be eluted, pmified, and analyzed for amino acid sequence by well known methods. Peptides so identified can be produced synthetically or fused to appropriate facilitative proteins using well known recombinant DNA technology.

5.9. USES OF ANOPHELES GAMBIAE

ODORANT RECEPTOR MODULATORS

The compounds identified in such screens may be used for attracting Anopheles gambiae mosquitoes to traps or to localized toxins, for repelling Anopheles gambiae mosquitoes from individuals or from residential areas, or for interfering with the function of the olfactory system such that Anopheles gambiae mosquitoes are unable to locate food and hosts. Since different species of insects have highly specialized food and host preferences and the odorant receptors that mediate these behaviors are extremely variable between species, control strategies that selectively target olfaction in Anopheles gambiae mosquitoes offer powerful and selective approaches to combat Anopheles gambiae mosquitoes. In contrast to non-selective pesticides, these products can be expected to be harmless to beneficial species of insects, insect predators and other animals. Moreover, as behaviorally-based strategies present less selective pressme than chemical pesticides, these strategies are expected to help reduce the appearance of pesticide-resistant Anopheles gambiae mosquitoes. Thus, the compounds identified using this methodology will offer novel approaches to control the spread of malaria by Anopheles gambiae mosquitoes, and will significantly reduce dependence on toxic pesticides, having a direct and immediate impact on coordinated Anopheles gambiae management programs.

In another embodiment of the invention, Anopheles gambiae odorant receptor genes may be used in controlling this pest species. For example, odorant receptor modulators, including but not limited to small molecules, proteins and nucleic acids, can have activity in modifying the behavior growth, feeding and/or reproduction of Anopheles gambiae mosquitoes. Additionally, in general, effective control agents exert a disabling activity on the Anopheles gambiae mosquitoes such as attraction (for example into a bait or trap), repulsion, paralysis, blocked development, or cessation of feeding. Anopheles gambiae mosquito control agents may be classified as pesticides, repellants or attractants. Such pests include but are not limited to egg, larval, juvenile and adult forms of the Anopheles gambiae mosquito.

The compounds identified by the screens described in Sections 5.7 and 5.8, supra, are expected to fall into one of three possible categories: they may act as insect attractants, repellents, or blocking agents that interfere with the Anopheles mosquito's ability to sense odor stimuli. Behavioral assays can be used to determine whether the compounds have selective effects on Anopheles gambiae mosquitoes or whether they act more broadly on a larger number of insect species. Field testing can be used to monitor responses of Anopheles mosquitoes in a natmal setting to develop appropriate delivery systems for "real-world" Anopheles mosquito control applications. Agonistic or antagonistic recombinant or synthetic odorant receptor proteins, analogs, or derivatives, or nucleic acids encoding such agonistic or antagonistic recombinant or synthetic Anopheles gambiae odorant receptor proteins, analogs, or derivatives, can be assayed for attractant or repellent activity. Laboratory and field assays of insect attraction and repulsion are well known in the art, and those that may be used to test molecules that bind to or modulate odorant receptors include but are not limited to those described by Foster et al, 1997, Annu. Rev. Entomol. 42:123-46; "Insect Olfaction," B. S. Hansson, Ed., Springer Verlag, Berlin, Heidelberg, New York; Birkett et al, 2000, Proc. Nat'l Acad. Sci. U S A. 97(16):9329-34; De Moraes et al, 2001, Natme. 410(6828):577-80; Kline etal, 1990, Med Vet Entomol. 4(4):383-91; Phelan, 1987, J. Econ. Entomol. 80:779-783; and Leskey et al, 2001, J Chem Ecol. 27(1):1-17.

In one embodiment, Anopheles gambiae odorant receptor genes encoding dominant negative forms of Anopheles gambiae odorant receptor proteins can be tested as insect control agents in the form of recombinant vimses that direct the expression of a dominant negative Anopheles gambiae odorant receptor gene in the target pest. In a prefened embodiment, the insect species is Anopheles gambiae. Suitable recombinant vims systems for expression of proteins in infected insect cells include but are not limited to recombinant Semliki Forest virus (DiCiommo and Bremner, 1998, J. Biol. Chem. 273:18060-66), recombinant sindbis vims (Higgs et al, 1995, Insect Mol. Biol. 4:97-103; Seabaugh et al, 1998, Virology 243:99-112), recombinant pantropic retroviras (Matsubara et al, 1996, Proc. Natl. Acad. Sci. USA 93:6181-85; Jordan et al, 1998, Insect Mol. Biol. 7:215-22), and most preferably recombinant baculovims. Use of recombinant baculovimses as a means to engineer expression of proteins in insects, and as insect control agents, is well known in the art. This approach has a number of specific advantages including host specificity, environmental safety, the availability of easily manipulable vector systems, and the potential use of the recombinant vims directly as a pest control agent without the need for purification or formulation of the Anopheles gambiae odorant receptor protein (Cory and Bishop, 1997, Mol. Biotechnol. 7(3):303-13; U.S. Patent No. 5,470,735; U.S. Patent No. 5,352,451; U.S. Patent No. 5, 770, 192; U.S. Patent No. 5,759,809; U.S. PatentNo. 5,665,349; U.S. Patent No. 5,554,592). Thus, recombinant baculovimses that direct the expression of Anopheles gambiae odorant receptor genes can be used for both testing the pest control activity of the Anopheles gambiae odorant receptor proteins under controlled laboratory conditions, and as insect control agents in the field. Alternatively, for testing the dominant negative activity of Anopheles gambiae odorant receptor genes, transgenic insects can be made as taught by Handler, 2001 , Insect Biochem Mol Biol. 31 (2) : 111 -28, or by Atkinson et al, 2001, Annu. Rev. Entomol.46:317-46.

Anopheles gambiae odorant receptor proteins, nucleic acids, and most preferably ligands, such as those agonists and antagonists identified by the methods described in Section 5.7.1, supra, may be formulated with any acceptable excipients known in the art, including but not limited to vehicles, caniers, binders, UV blockers, adhesives, hemecants, thickeners, dispersing agents, preservatives and insect attractants. Thus the compositions of the invention may, for example, be formulated as a solid comprising the active agent and a finely divided solid canier. Alternatively, the active agent may be contained in liquid compositions including dispersions, emulsions and suspensions thereof. Any suitable final formulation may be used, including for example, granules, powder, bait pellets (a solid composition containing the active agent and an insect attractant or food substance), microcapsules, water dispersible granules, emulsions and emulsified concentrates.

Examples of solid caniers suitable for use with the present invention include but are not limited to starch, active carbon, soybean powder, wheat powder, wood powder, fish powder, powdered milk, talc, kaolin, bentonite, calcium carbonate, zeolite, diatomaceous earth, fine silica powder, clay, alumina, pyrophyllite, kieselguhr chalk, lime, fuller's earth, cottonseed hulls, pumice, tripoli, walnut shell flour, redwood flour, and lignin.

Examples of liquid caniers suitable for use with the present invention include but are not limited to water, isopropyl alcohol, ethylene glycol, cyclohexanone, methyl ethyl ketone, dioxane, tetrahydrofuran, kerosene, light oil, xylene, frimethylbenzene, tetramethylbenzene, methylnaphthalene, solvent naphtha, chlorobenzene, dimethylacetamide, a glycerin ester, an acetonitrile, or dimethylsulfoxide.

Insect repellent formulations for a non-human animal may be in the form of a pour-on formulation, a spot-on formulation, a spray, a shampoo, a dusting powder, an impregnated strip, a soap, an ear or tail tag or a gel. Insect repellent formulations for humans can be in the form of a powder, an ointment, a lotion, a wipe, a cream, a soap, an erodible stick or a clothes patch. The formulation may include antioxidants and UV absorbers. Creams and lotions are of particular interest, and may be adapted for application to the skin. For other uses, formulations containing the attractants of the invention may be formulated as lures, baits or traps. Odor-baited trapping systems may be used for the capt e and population reduction of mosquitoes and other pest insects. In effect, the traps consist of netting or some other type of enclosure that is appropriately colored to attract the target insect, and is laced with a compound or a mixture of compounds that are potent olfactory attractants. The traps may also contain a contact insecticide that kills the insects after trapping. Formulations containing repellants can be applied to the trees, plants or areas to be treated in the form of sprays, droplets, microfilms, microcapsules, or thin defined layers by using conventional devices known to those skilled in the art. Such formulations may be formulated for controlled release. The repellant formulation may be in the form of dispersion coating, film coating, spray coating, microencapsulated products, polymer slow release drops, globs, blocks, such as paraffin blocks, monoliths, puffers, and any such other similar form as known in the art. Various controlled-release systems are described in Controlled Delivery of Crop-Protection Agents, Taylor and Francis, New York, (1990), Editor R. M. Wilkins, especially chapters 3 and 9 and in Insect Suppression with Controlled Release Pheromone Systems, Vol. I and II, CRC Press, Boca Raton, Florida (1982).

Traps baited with olfactory attractants may be used in monitoring populations of mosquitoes. These monitoring systems can play a very important role in determining where and when disease and vector control treatments are required. Moreover, as different species are known to respond in different ways to olfactory cues, it may be possible to make such traps selective for disease-carrying species of mosquitoes, thus increasing the accmacy and value of the information provided by trap counts.

In addition to using olfactory cues for host location, mosquitoes use olfaction to locate sites for egg laying, or oviposition. The identification of these oviposition attractants and synthetic compounds that stimulate this behavior could be used to divert gravid female mosquitoes from their normal breeding sites to traps, thereby reducing mosquito populations while reducing pesticide use.

The following examples are provided merely as illustrative of various 5 aspects of the invention and shall not be construed to limit the invention in any way.

6. EXAMPLE: IDENTIFICATION AND SEQUENCE ANALYSIS OF cDNAs OF ANOPHELES GAMBIAE ODORANT RECEPTOR GENES

Using genomic database searching and gene cloning techniques, 5 ι o independent members of the Anopheles gambiae odorant receptor gene family in the malaria mosquito Anopheles gambiae have been identified. Aside from the conserved carboxy terminus and conserved protein topology, the proteins encoded by these genes are very divergent in sequence from each other and from the previously identified Drosophila melanogaster odorant receptors. Thus, compounds that target the activity of these receptors

15 will act as extremely selective and potent tools to control the behavior of Anopheles gambiae mosquitoes.

6.1. IDENTIFICATION OF ODORANT

RECEPTOR GENES IN ANOPHELES GAMBIAE

20 Recently, a large-scale effort has been initiated to determine the complete genome sequence of Anopheles gambiae, the principal mosquito vector responsible for the transmission of malaria. As part of the initial stages of this effort, researchers at Genoscope and the Institute Pastern, France have generated and released a database consisting of 17,000 random sequences, representing 12,000,000 base pairs, or roughly 5% of the total

25 mosquito genome.

(http://www.genoscope.cns.fr/exteme/English/Projets/Projet_AK7organisme_AK.html)

Protein sequences of each of the Drosophila melanogaster odorant receptors were compared to the six-frame conceptual translations of the Genoscope/Institute Pastern Anopheles gambiae genomic DNA sequences, using the Basic Local Alignment Sequencing

30 Tool (BLAST). A total of five genomic sequences, representing four different genomic loci, produced significant matches to multiple Drosophila odorant receptor gene sequences and were chosen for further analysis. (Sequences 24C19 and 25K12 are two independent, overlapping sequences conesponding the same genomic locus)

35 Table 3

For each of these fo genomic loci, the polymerase chain reaction (PCR) was used to amplify the conesponding DNA fragment from Anopheles gambiae genomic DNA. The PCR reactions were performed using 1 μg of genomic DNA template and 80 pmol each primer in a 50 μl reaction, cycled between 94°C for 1 min, 55°C for 1 min, 72°C for 1 min for a total of 25 cycles to produce a product of expected size. The primers used to amplify each genomic fragment and the resulting PCR product size are indicated in table 4, below:

Table 4

The sequences of the primers recited in Table 4 are provided in Table 5:

Table 5

These PCR products were subcloned and sequenced and then used to screen an Anopheles gambiae genomic DNA library. Positives identified from library screening were pmified and sequenced. Following the isolation and sequencing of several overlapping genomic DNA clones, potential open reading frames (ORFs) were identified by GENSCAN analysis. In the comse of analyzing genomic library isolates for one of these loci (08K09), an additional, higlily related odorant receptor gene was identified. The original gene, a fragment of which is present in the Genoscope/Pasteur database, is identified here as K0920.3, and the second gene is designated K0927.1.

To isolate additional odorant receptor genes related to K0920.3 and K0927.1, a 580 bp Nhel-Sacl DNA fragment from the K0927.1 gene (encoding the C-terminal 193 amino acids) was used to screen an Anopheles gambiae genomic DNA library at low stringency. Hybridization was canied out at 42° C in 5XSSCP, 25% formamide, followed by 2 washes of 30 minutes at 42° C in 0.5XSSC. One of the hybridizing clones identified in this manner conesponded to K09 7.1. Oligonucleotide primers flanking the predicted initiation and termination codons of each of these Anopheles gambiae genes were synthesized (presented in Table 6 below) and used to amplify the gene coding region by reverse transcription-PCR (RT-PCR) from poly A+ mRNA isolated from Anopheles gambiae heads and antennae. The resulting RT-PCR products were subcloned into pGEM^®-T Easy (Promega Corp., Madison, WI) and sequenced. The inserts could be excised from the vector using digestion with EcoRI (some of the genes also contained an internal EcoRI site).

Table 6

6.2. SEQUENCE ANALYSIS OF ANOPHELES GAMBIAE ODORANT RECEPTOR

GENES

A ClustalW alignment of the predicted amino acid sequence of these Anopheles gambiae odorant receptor genes is presented in FIG. 1. Overall sequence similarity between Anopheles odorant receptor genes is generally low (15-25%), with the exception of K0920.3 and K0927.1, which are 60.4% identical. However, there are several short stretches within the last 80 amino acids that show significantly higher sequence similarity. These regions of greater sequence similarity located in the C-terminal region of the predicted receptor sequences define a consensus motif that defines the odorant receptor gene family. This motif is presented in FIG. 2.

A sequence comparison of the identified Anopheles odorant receptor genes and a subset of the previously described Drosophila odorant receptor genes is shown in FIG. 3. The overall sequence similarity between Anopheles and Drosophila odorant receptor genes is similar to that between members of the odorant receptor gene family within a given species. In addition, a comparison of the gene families in the two species reveals significant conservation of the C-terminal signatme motifs between both insect species. For the most part, clear relationships between individual Anopheles odorant receptor genes and any of the Drosophila odorant receptor genes are not apparent. A phylogenetic tree showing sequence relationships between the Anopheles odorant receptor genes and the most highly related Drosophila odorant receptor genes is shown in FIG. 4. The highest degree of sequence similarity is between the Anopheles M09 sequence and two Drosophila odorant receptor sequences, Or49b and Or43a [M09 - Or49b: 55% similarity, 34.6% identity; M09 - Or43a: 52% similarity, 35.5% identity].

These studies demonstrate that odorant receptor gene sequences have diverged rapidly dming insect evolution and that each insect species utilizes a characteristic set of odorant receptors specialized for its behavioral repertoire. Due to the low degree of sequence conservation, identification of odorant receptor gene sequences in other insect species will depend on genomic technologies rather than conventional molecular biology techniques. As a result, these cross-species sequence comparisons provide an important tool to facilitate odorant receptor identification efforts.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Various references are cited herein above, including patent applications, patents, and publications, the disclosmes of which are hereby incorporated by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

1. A pmified polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:25, wherein the amino acid sequence is not found in SEQ ID NO:35-95.

2. The purified polypeptide of claim 1, wherein the polypeptide has at least 90% identity to the amino acid sequence of SEQ ID NO:25.

3. The purified polypeptide of claim 1 , wherein the polypeptide has at least 10 95% identity to the amino acid sequence of SEQ ID NO:25.

4. The purified polypeptide of claim 1, 2, or 3, comprising at least 20 contiguous amino acids of the sequence as set forth in SEQ ID NO:2.

15 5. The purified polypeptide of claim 4, comprising at least 30 contiguous amino acids of the sequence as set forth in SEQ ID NO:2.

6. The pmified polypeptide of claim 5, comprising at least 50 contiguous amino acids of the sequence as set forth in SEQ ID NO:2.

20

7. The pmified polypeptide of claim 1, 2, or 3 comprising an amino acid sequence as set forth in SEQ ID NO:2.

8. The purified polypeptide of claim 1, 2, or 3, comprising at least 20 25 contiguous amino acids of the sequence as set forth in SEQ ID NO:4.

9. The purified polypeptide of claim 8, comprising at least 30 contiguous amino acids of the sequence as set forth in SEQ ID NO:4.

30 10. The purified polypeptide of claim 9, comprising at least 50 contiguous amino acids of the sequence as set forth in SEQ ID NO:4.

11. The purified polypeptide of claim 1 , 2, or 3 comprising an amino acid sequence as set forth in SEQ ID NO:4.

35

12. The pmified polypeptide of claim 1, 2, or 3, comprising at least 20 contiguous amino acids of the sequence as set forth in SEQ ID NO: 6.

13. The purified polypeptide of claim 12, comprising at least 30 contiguous amino acids of the sequence as set forth in SEQ ID NO:6.

14. The pmified polypeptide of claim 14, comprising at least 50 contiguous amino acids of the sequence as set forth in SEQ ID NO:6.

15. The pmified polypeptide of claim 1, 2, or 3 comprising an amino acid sequence as set forth in SEQ ID NO: 6.

16. The purified polypeptide of claim 1, 2, or 3, comprising at least 20 contiguous amino acids of the sequence as set forth in SEQ ID NO: 8.

17. The pmified polypeptide of claim 16, comprising at least 30 contiguous amino acids of the sequence as set forth in SEQ ID NO:8.

18. The pmified polypeptide of claim 17, comprising at least 50 contiguous amino acids of the sequence as set forth in SEQ ID NO:8.

19. The pmified polypeptide of claim 1, 2, or 3 comprising an amino acid sequence as set forth in SEQ ID NO: 8.

20. The pmified polypeptide of claim 1, 2, or 3, comprising at least 20 contiguous amino acids of the sequence as set forth in SEQ ID NO: 10.

21. The pmified polypeptide of claim 20, comprising at least 30 contiguous amino acids of the sequence as set forth in SEQ ID NO: 10.

22. The pmified polypeptide of claim 21 , comprising at least 50 contiguous amino acids of the sequence as set forth in SEQ ID NO: 10.

23. The pmified polypeptide of claim 1, 2, or 3 comprising an amino acid sequence as set forth in SEQ ID NO : 10.

24. The pmified polypeptide of claim 1 , 2, or 3, comprising at least 20 contiguous amino acids of the sequence as set forth in SEQ ID NO: 12.

25. The pmified polypeptide of claim 24, comprising at least 30 contiguous amino acids of the sequence as set forth in SEQ ID NO: 12.

26. The pmified polypeptide of claim 25, comprising at least 50 contiguous amino acids of the sequence as set forth in SEQ ID NO:2.

27. The pmified polypeptide of claim 1, 2, or 3 comprising an amino acid sequence as set forth in SEQ ID NO: 12.

28. The pmified polypeptide of claim 1, 2, or 3 which is capable of being bound by an antibody that also binds to a polypeptide defined by an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 12.

29. The purified polypeptide of claim 1, 2, or 3 which is an insect olfactory receptor.

30. The pmified polypeptide of claim 1, 2, or 3 which is a Culex spp. or Aedes aegypti olfactory receptor.

31. The pmified polypeptide of claim 1 , 2, or 3 which is an Anopheles gambiae olfactory receptor.

32. A pmified polypeptide comprising an amino acid sequence having at least 70% identity to a fragment of SEQ ID NO:26 of at least 20 amino acids, wherein the amino acid sequence is not found in SEQ ID NO:35-95.

33. The pmified polypeptide of claim 32, wherein the polypeptide has at least 80% identity to the fragment of SEQ ID NO:26.

34. The purified polypeptide of claim 32, wherein the polypeptide has at least 90% identity to the fragment of SEQ ID NO:26.

35. The purified polypeptide of claim 32, wherein the polypeptide has at least 95% identity to the fragment of SEQ ID NO:26.

36. The purified polypeptide of claim 32, 33, 34, or 35, wherein the fragment of SEQ ID NO:26 is at least 30 amino acids.

37. The purified polypeptide of claim 32, 33, 34, or 35, wherein the fragment of

SEQ ID NO:26 is at least 50 amino acids.

38. The purified polypeptide of claim 32, 33, 34, or 35, wherein the fragment of SEQ ID NO:26 is at least 75 amino acids.

39. The pmified polypeptide of claim 32, 33, 34, or 35, wherein the fragment of SEQ ID NO:26 is at least 100 amino acids.

40. The pmified polypeptide of claim 32, 33, 34, or 35, comprising an amino acid sequence as set forth in SEQ ID NO:4.

41. The pmified polypeptide of claim 32, 33, 34, or 35, comprising at least 20 contiguous amino acids of the sequence as set forth in SEQ ID NO:4.

42. The purified polypeptide of claim 41 , comprising at least 30 contiguous amino acids of the sequence as set forth in SEQ ID NO:4.

43. The purified polypeptide of claim 42, comprising at least 50 contiguous amino acids of the sequence as set forth in SEQ ID NO:4.

44. The purified polypeptide of claim 32, 33, 34, or 35, comprising an amino acid sequence as set forth in SEQ ID NO: 6.

45. The purified polypeptide of claim 32, 33, 34, or 35, comprising at least 20 contiguous amino acids of the sequence as set forth in SEQ ID NO: 6.

46. The pmified polypeptide of claim 45, comprising at least 30 contiguous amino acids of the sequence as set forth in SEQ ID NO:6.

47. The pmified polypeptide of claim 46, comprising at least 50 contiguous amino acids of the sequence as set forth in SEQ ID NO:6.

48. The pmified polypeptide of claim 32, 33, 34, or 35, comprising an amino acid sequence as set forth in SEQ ID NO: 12.

49. The pmified polypeptide of claim 32, 33, 34, or 35, comprising at least 20 contiguous amino acids of the sequence as set forth in SEQ ID NO: 12.

50. The pmified polypeptide of claim 49, comprising at least 30 contiguous amino acids of the sequence as set forth in SEQ ID NO: 12.

51. The pmified polypeptide of claim 50, 52, comprising at least 50 contiguous amino acids of the sequence as set forth in SEQ ID NO: 12.

52. The purified polypeptide of claim 32, 33, 34, or 35 which is capable of being bound by an antibody that also binds to a polypeptide defined by an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:12.

53. The pmified polypeptide of claim 32, 33, 34, or 35 which is an insect olfactory receptor.

54. The pmified polypeptide of claim 32, 33, 34, or 35 which is a Culex spp. or Aedes aegypti olfactory receptor.

55. The purified polypeptide of claim 32, 33, 34, or 35 which is an Anopheles gambiae olfactory receptor.

56. An isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide of claim 1, 2, or 3.

57. The isolated nucleic acid of claim 56 which further comprises an origin of replication.

58. The isolated nucleic acid of claim 56 in which the nucleotide sequence is operatively linked to a promoter.

59. The isolated nucleic acid of claim 57 in which the nucleotide sequence is operatively linked to a promoter.

60. An isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12.

61. The isolated nucleic acid of claim 60, wherein the nucleic acid comprises a nucleotide sequence as set forth in SEQ ID NO:l.

62. The isolated nucleic acid of claim 60, wherein the nucleic acid comprises a nucleotide sequence as set forth in SEQ ID NO:3.

63. The isolated nucleic acid of claim 60, wherein the nucleic acid comprises a nucleotide sequence as set forth in SEQ ID NO:5.

64. The isolated nucleic acid of claim 60, wherein the nucleic acid comprises a nucleotide sequence as set forth in SEQ ID NO:7.

65. The isolated nucleic acid of claim 60, wherein the nucleic acid comprises a nucleotide sequence as set forth in SEQ ID NO: 9.

66. The isolated nucleic acid of claim 60, wherein the nucleic acid comprises a nucleotide sequence as set forth in SEQ ID NO:l 1.

67. The isolated nucleic acid of claim 60 which further comprises an origin of replication.

68. The isolated nucleic acid of claim 60 in which the nucleotide sequence is operatively linked to a promoter.

69. The isolated nucleic acid of claim 67 in which the nucleotide sequence is operatively linked to a promoter.

70. A host cell transformed with the nucleic acid of claim 28.

71. A host cell transformed with the nucleic acid of claim 60.

72. The host cell of claim 70 further comprising a nucleic acid encoding the polypeptide of SEQ ID NO: 14.

73. A host cell transformed with the nucleic acid of claim 60.

74. The host cell of claim 73 further comprising a nucleic acid encoding the polypeptide of SEQ ID NO: 14, wherein said isolated nucleic acid further comprises an origin of replication.

75. A method of identifying a molecule that binds to an insect olfactory receptor, said olfactory receptor comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:25, wherein the amino acid sequence is not found in SEQ ID 35-95, said method comprising:

(a) contacting a first cell and a second cell with a test molecule under conditions conducive to binding between the olfactory receptor and the test molecule, wherein the first cell expresses the insect olfactory receptor and the second cell does not express the insect olfactory receptor, and wherein the first cell and the second cell are of the same cell type; and (b) determining whether the test molecule binds to the first cell or the second cell; wherein a molecule that binds to the first cell but not the second cell is a molecule that binds to the olfactory receptor.

76. The method of claim 75 wherein said first cell and said second cell express a polypeptide comprising the amino acid sequence of SEQ ID NO: 14.

77. The method of claim 75 wherein the insect is Anopheles gambiae.

78. The method of claim 75 wherein the insect is Culex spp. or Aedes aegypti.

79. An insect control agent formulation comprising:

(a) a canier; and (b) a molecule identified by the method of claim 75, 76, 77, or 78.

80. The insect control agent of claim 79 which is a repellant.

81. The insect confrol agent of claim 79 which is an attractant.

82. A method of identifying a modulator of an insect olfactory receptor, said olfactory receptor comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:25, wherein the amino acid sequence is not found in SEQ ID 35-95, said method comprising: (a) contacting a first cell and a second cell with a test molecule under conditions conducive to binding between the olfactory receptor and the test molecule, wherein the first cell expresses the olfactory receptor and the second cell does not express the olfactory receptor, and wherein the first cell and the second cell are of the same cell type; and

(b) determining whether the test molecule modulates G-protein activity in said first cell or second cell, wherein a molecule that modulates G-protein activity in the first cell but not in the second cell is a modulator of the insect olfactory receptor.

83. The method of claim 82 wherein said first cell and said second cell express a polypeptide comprising the amino acid sequence of SEQ ID NO: 14.

84. The method of claim 82 wherein the insect is Anopheles gambiae.

85. The method of claim 82 wherein the insect is Culex spp. or Aedes aegypti.

86. The method of claim 82, wherein G-protein activity is determined by measming calcium ion or cyclic AMP concentration in the cell.

87. The method of claim 82, wherein the modulator is an agonist.

88. The method of claim 82, wherein the modulator is an antagonist.

89. An insect control agent formulation comprising:

(a) a canier; and

(b) a modulator identified by the method of claim 82, 83, 84, or 85.

90. The insect control agent of claim 89 which is a repellant.

91. The insect control agent of claim 89 which is an attractant.

92. A method of identifying a molecule that binds to an olfactory receptor from Anopheles gambiae but not to a second olfactory receptor from another species, said method comprising:

(a) contacting a first cell that expresses an Anopheles gambiae receptor with a test molecule under conditions conducive to binding between said Anopheles gambiae receptor and the test molecule;

(b) detennining whether the test molecule binds to said first cell; (c) contacting a second cell that expresses the second olfactory receptor with the test molecule under conditions conducive to binding between said second receptor and the test molecule, wherein said second cell is of the same cell type as the first cell; and (d) determining whether said test molecule binds to said second cell, wherein a test molecule that binds to the first cell but not to the second cell binds to the Anopheles gambiae olfactory receptor but not to the olfactory receptor from the other species.

93. An insect control agent formulation comprising: (a) a canier; and (b) a molecule identified by the method of claim 92.

94. The insect control agent of claim 93 which is a repellant.

95. The insect control agent of claim 93 which is an attractant.

96. A method of identifying a modulator of an olfactory receptor from Anopheles gambiae but not a second olfactory receptor from a second species, said method comprising: (a) contacting a first cell that expresses an Anopheles gambiae receptor with a test molecule under conditions conducive to binding between said Anopheles gambiae receptor and the test molecule; (b) determining whether the test molecule modulates G-protein activity in said first cell; (c) contacting a second cell that expresses the second olfactory receptor with the test molecule under conditions conducive to binding between said second receptor and the test molecule, wherein said second cell is of the same cell type as the first cell and; and (d) determining whether the test molecule modulates G-protein activity in said second cell, wherein a test molecule that modulates G-protein activity in the first cell but not in the second cell modulates the Anopheles gambiae olfactory receptor but not the olfactory receptor from the other species.

97. The method of claim 96, wherein G-protein activity is determined by measming calcium ion or cyclic AMP concentration in the cell.

98. The method of claim 96, wherein the modulator is an agonist.

99. The method of claim 96, wherein the modulator is an antagonist.

100. An insect control agent formulation comprising:

(a) a canier; and

(b) a modulator identified by the method of claim 96.

101. The insect control agent of claim 100 which is a repellant.

102. The insect control agent of claim 100 which is an attractant.

103. A method of identifying an olfactory that binds to a first Anopheles gambiae olfactory receptor but not to a second Anopheles gambiae olfactory receptor, said method comprising:

(a) contacting a first cell that expresses the first Anopheles gambiae olfactory receptor with a test molecule, said first olfactory receptor comprising an amino acid sequence as set forth in SEQ ID NO:2,

SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO: 12 under conditions conducive to binding between the first Anopheles gambiae olfactory receptor and the test molecule;

(b) determining whether the test molecule binds to said first cell; (c) contacting a second cell that expresses the second Anopheles gambiae olfactory receptor with the test molecule, said second olfactory receptor comprising an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO :10 or SEQ ID NO: 12, under conditions conducive to binding between the second receptor and the test molecule, wherein said second cell is of the same cell type as the first cell and said first olfactory receptor is different from said second olfactory receptor; and (d) determining whether the test molecule binds to said second cell, wherein a test molecule that binds to the first cell but not to the second cell is an olfactory that binds to the first Anopheles gambiae olfactory receptor but not to the second Anopheles gambiae olfactory receptor.

104. An insect control agent formulation comprising: (a) a canier; and

(b) a modulator identified by the method of claim 103.

105. The insect control agent of claim 104 which is a repellant.

106. The insect control agent of claim 104 which is an attractant.

107. A method of identifying an olfactory that modulates the activity of a first Anopheles gambiae olfactory receptor but not the activity of a second Anopheles gambiae olfactory receptor, said method comprising: (a) contacting a first cell that expresses the first Anopheles gambiae olfactory receptor with a test molecule, said first olfactory receptor comprising an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO: 12, under conditions conducive to binding between the first Anopheles gambiae olfactory receptor and the test molecule;

(b) determining whether the test molecule modulates G-protein activity in said first cell;

(c) contacting a second cell that expresses the second Anopheles gambiae olfactory receptor with the test molecule, said second olfactory receptor comprising an amino acid sequence as set forth in

SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10 or SEQ ID NO: 12, under conditions conducive to binding between the second receptor and the test molecule, wherein said second cell is of the same cell type as the first cell and said first olfactory receptor is different from said second olfactory receptor; and

(d) determining whether the test molecule modulates G-protein activity in said second cell, wherein a test molecule that modulates G-protein activity in the first cell but not in the second cell modulates the activity of the first Anopheles gambiae olfactory receptor but not the second Anopheles gambiae olfactory receptor.

108. The method of claim 107, wherein G-protein activity is determined by measming calcium ion or cyclic AMP concentration in the cell.

109. The method of claim 107, wherein the olfactory is an agonist.

110. The method of claim 107, wherein the olfactory is an antagonist.

111. An insect control agent formulation comprising: (a) a canier; and

(b) an olfactory identified by the method of claim 107.

112. The insect control agent of claim 111 which is a repellant.

113. The insect control agent of claim 111 which is an attractant.

114. The insect control agent formulation of claim 79, 89, 93, 100, 104, or 111, wherein the canier is selected from the group consisting of a solid canier and a liquid canier.

115. The insect control agent formulation of claim 114, wherein the solid canier is starch, active carbon, soybean powder, wheat powder, wood powder, fish powder, powdered milk, talc, kaolin, bentonite, calcium carbonate, zeolite, diatomaceous earth, fine silica powder, clay or alumina.

116. The insect control agent formulation of claim 114, wherein the liquid canier is water, isopropyl alcohol, ethylene glycol, cyclohexanone, methyl ethyl ketone, dioxane, tetrahydrof an, kerosene, light oil, xylene, frimethylbenzene, tetramethylbenzene, methylnaphthalene, solvent naphtha, chlorobenzene, dimethylacetamide, a glycerin ester, an acetonitrile, or dimethylsulfoxide.

117. A method for protecting a mammal against malaria comprising contacting said mammal with the repellent of claim 80, 90, 94, 101, 105, or 112.

118. A method for reducing a population of Anopheles gambiae mosquitos comprising placing a trap comprising attractant of claim 81, 91, 95, 102, 106, or 113 in an area where such population reduction is desired.