Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-01-25
2003-08-26
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C536S023500, C530S358000
Reexamination Certificate
active
06610511
ABSTRACT:
FIELD OF THE INVENTION
This invention pertains to novel olfactory receptors and to methods of using such receptors. More particularly, this invention pertains to the nucleic acids and amino acids of novel olfactory receptors in Drosophila and to methods of using such nucleic acids and amino acids.
BACKGROUND OF THE INVENTION
Animals can detect a vast array of odors with remarkable sensitivity and discrimination. Olfactory information is first received by olfactory receptor neurons (olfactory receptors), which transmit signals into the central nervous system (CNS) where they are processed, ultimately leading to behavioral responses. An enormous amount of investigation into olfactory function, organization, and development has been carried out in insect model systems for many years (Kaissling et al., (1987) Ann. NY Acad. Sci. 510, 104-112; Hildebrand (1995) Proc. Natl. Acad. Sci. USA 92, 67-74). However, a number of central questions have been refractory to incisive analysis because the receptor molecules to which odor molecules bind have not been identified, in any insect.
To investigate the molecular mechanisms of olfactory function and development, applicants studied the olfactory system of
Drosophila melanogaster
, which is highly sensitive and capable of odor discrimination (Siddiqi, (1991) Olfaction in Drosophila, in: Wysocki & Kare (ed.), Chemical Senses, Marcel Dekker; Carlson (1996) Trends Genet. 12, 175-180). There are two olfactory organs on the adult fly, the third segment of the antenna and the maxillary palp (FIG.
1
A). In both organs, olfactory receptors are housed in sensory hairs called sensilla. The organization of the approximately 1200 olfactory receptors of the antenna is complex but ordered. On the antenna there are different morphological categories of sensilla: s. trichodea, s. coeloconica, large s. basiconica, and small s. basiconica (FIG.
1
B). The different morphological categories of sensilla are distributed in overlapping patterns across the surface of the antenna (
FIGS. 1C-F
) (Venkatesh & Singh, (1984) Int. J. Insect Morphol. Embryol. 13, 51-63; Stocker, (1994) Roux's Arch. Dev. Biol. 205, 62-72).
Electrophysiological studies show that each morphological category of sensilla can be divided into different functional types (denoted by different colors in FIGS.
1
C-F), defined by the characteristic response profiles of their olfactory receptors (Rodrigues et al., (1991) Mol. Gen. Genet. 226, 265-276; Clyne et al., (1997) Invert. Neurosci. 3, 127-135; de Bruyne et al., unpublished results). For s. trichodea, the different functional types are segregated into zones on the surface of the antenna (FIG.
1
C); segregation is also observed for the different functional types of s. coeloconica (FIG.
1
D). This zonal organization is less conspicuous for the large and small s. basiconica, of which different functional types are intermingled (FIGS.
1
E-F). Electrophysiological data suggest that there are on the order of thirty different classes of olfactory receptors in the antenna, a rough estimate based upon the odor response profiles of individual olfactory receptors (and in a few cases, the assumption that the neurons of particular functional types of sensilla have unique response profiles).
In contrast to the antenna, the organization of the approximately 120 olfactory receptors of the maxillary palp is less complex. There are approximately 60 s. basiconica on the maxillary palp, each housing two olfactory receptors (Singh & Nayak, (1985) Int. J. Insect Morphol. Embryol. 14, 291-306). The 120 olfactory receptors fall into six different classes based upon their odorant response profiles (Clyne et al., (1999) Neuron 22, 339-347; de Bruyne et al., (1999) J. Neurosci. 19, 4520-4532). Neurons of the six ORN classes are always found in characteristic pairs in three functional types of s. basiconica, with the total number of neurons in each class being equal. Each class is distributed broadly over all, or almost all, of the olfactory surface of the maxillary palp.
Thus electrophysiological and anatomical studies suggest that there are on the order of thirty-five classes of olfactory receptors in the adult fly (approximately thirty on the antenna and six on the palp), each class with a distinct odor sensitivity. Classes of olfactory receptors found in the antenna are arrayed in zones, while the classes of olfactory receptors found in the maxillary palp are distributed in a less ordered fashion. olfactory receptors in both the maxillary palp and the antenna extend their axons to the antennal lobe of the brain, where first-order processing of olfactory information occurs. The lobe contains approximately forty olfactory glomeruli, spheroidal modules where ORN axons converge and where their terminal branches form synapses with the dendrites of their target interneurons (Stocker, (1994) Cell Tissue Res. 275, 3-26; Hildebrand & Shepherd, (1997) Annu. Rev. Neurosci. 20, 595-631).
One possibility underlying the molecular basis for distinct odor sensitivities for different classes of olfactory receptors is that each class of ORN expresses a unique odorant receptor, as has been proposed for vertebrate olfactory systems (Ngai et al., (1993) Cell 72, 667-680; Ressler et al., (1993) Cell 73, 597-609; Vassar et al., (1993) Cell 74, 309-318; Buck, (1996) Annu. Rev. Neurosci. 19, 517-544; Hildebrand & Shepherd, (1997) Annu. Rev. Neurosci. 20, 595-631). Alternatively, each class of ORN might express a unique combination of a large set of receptors, as found in chemosensory cells of the nematode,
C. elegans
(Troemel et al., (1995) Cell 83, 207-218). Both models call for a family of receptor genes, and several lines of evidence suggest that for insects such a family would belong to the superfamily of seven-transmembrane G protein-coupled receptors (GPCRs). First, there is evidence that insects generate responses to odorants via GPCR-activated second-messenger systems. For example, a rapid and transient increase in inositol 1,4,5-trisphosphate (IP3) has been observed in response to stimulation with pheromone and other odors using antennal preparations from various insect species (Breer et al., (1990) Nature 345, 65-68; Boekhoff et al., (1993) Insect Biochem. Mol. Biol. 23, 757-762; Wegener et al., (1993) J. Insect Physiol. 39, 153-163). This increase in IP3 can be blocked by pertussis toxin, implicating a G protein signaling cascade (Boekhoff et al., (1990) Cell. Signal. 2, 49-56). In Drosophila, norpA mutants, which lack the phospholipase C that is an essential component of phototransduction, also exhibit reduced olfactory responses of the maxillary palp (Riesgo-Escovar et al., (1995) J. Comp. Physiol. A180, 151-160). A second reason to suspect that odorant receptors in Drosophila are GPCRs is that GPCRs have been shown to be odorant receptors in both vertebrates and
C. elegans
; moreover, abundant evidence indicates that olfactory information in these other organisms is transduced by GPCR-activated second messenger systems (Buck, (1996) Annu. Rev. Neurosci. 19, 517-544; Bargmann & Kaplan, (1998) Annu. Rev. Neurosci. 21, 279-308). It would thus seem unlikely that a family of receptors that have a completely novel structure and that use a completely different transduction mechanism would have arisen in insects.
There have been extensive efforts to identify odorant and pheromone receptors in a variety of insects using a wide range of strategies. These efforts have been driven in part by interest in analyzing receptor genes in the context of highly tractable experimental systems in which there is a wealth of knowledge about olfactory function and organization. For example, Drosophila offers the advantages of a model genetic organism together with the ability to measure olfactory function conveniently in vivo, through either physiological or behavioral means. Interest in insect odorant receptors has also arisen because of the critical role of olfaction in the attraction of many insect pests to their plant hosts, of insect vectors of disease to their human hosts, and of in
Carlson John R.
Clyne Peter J.
Kim Junhyong
Warr Coral G.
Eyler Yvonne
Morgan & Lewis & Bockius, LLP
Murphy Joseph F.
Yale University
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