CA2106847A1 - Odorant receptors and uses thereof - Google Patents

Abstract

The invention provides an isolated nucleic acid, e.g. cDNA
encoding an odorant receptor. The invention further provides expression vectors containing such nucleic acid. Also provided is a purified protein encoding an odorant receptor, with the aforementioned expression vectors and the resulting transformed cell. The invention also provides methods of identifying odorant ligands and of identifying odorant receptors. The invention further provides methods of developing fragrances, of identifying appetite suppressant compounds, of controlling appetite, of controlling pest populations, of promoting and inhibiting fertility, and of detecting odors.

Description

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ODOR~NT RE:C~PTORE3 ANI~ 18E~ T}IBEl~OF
Backqrouna of the InvQntion This application is a continuation-in-part of IJ.S. Serial NoO681,880, filed April 5, 1~91, the contents of which are hereby incorporated by reference.
, Throughout this application, various publications are lo referenced by Arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
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In vertebrate sensory systems, periph~ral neurons respond to environmental stimuli and transmit these signals to higher sensory centers in the brain where they are processed to allow the discrimination of complex sensory information.
The delineation of the peripheral mechanisms by which environmental stimuli are transduced into neural information can provide insight into the logic underlying sensory processing. Our understanding of color vision, for example, emerged only after the observation that the discrimination of hue results from the blending of information from only three classes of photoreceptors ~1, 2, 3, 4). The basic logic underlying olfactory sensory perception, however, has remained elusive. Mammals possess an olfactory system of enormous discriminatory power (5, 6). Humans, for example, ! are thought to be capable of distinguishing among thousands of distinct odors. The specificity of odor recognition is emphasized by the observation that subtle alterations in the molecular structure of an odorant can lead to profound '' '.. '' ''' ~.'' ~ ': .

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changes in perceived odor.

The detection of chemically distinct odorant presumably results from the association of odorous ligands with specific receptors on olfactory neurons which reside in a specialized epithelium in the nose. Since these receptors have not been identified, it has been difficult to determine how odor discrimination might be achieved. It is possible that olfaction, by analogy with color vision, involves only a few odor receptors, each capable of interaction with multiple odorant molecules. Alternatively, the sense of smell may involve a large number of distinct receptors each i-capable of associating with one or a small number of odorant. In either case, the brain must distinguish which receptors or which neurons have been activated to allow the discrimination between different odorant stimuli. Xnsight into the mechanisms underlying olfactory perception is likeLY to depend upon the i~olation of the odorant receptors, and the characterization of their diversity, specificity, and patterns of expression. -~

The primary events in odor detection occur in a specialized ol~actory neuroepithelium located in the posterior recesses o~ the nasal cavity. Three cell types dominate this epithelium (Figure lA)- the olfactory ~ensory neuron, the sustentacular or supporting cell, and the basal cell which is a stem cell that generates olfactory neurons throughout life (7, 8). The olfactory sensory neuron is bipolar: a dendritic process extends to the mucosal surface where it gives rise to a number of specialized cilia which provide an ;
extensivè, receptive surface for the interaction of odors with olfactory sensory neurons. The olfactory neuron also ;~
gives rise to an axon which projects to the olfactory bulb of the brain, the first relay in the olfactory system. The axons of the olfactory bulb neurons, in turn, project to :

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WO92/1758' PCT/US~2/02741 ,~ ., subcortical and cortical ragions where higher l~vel processing of olfactory information allows the discrimination of odors by the brain.
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5 The initial events in odor discrimination are thought to involve the association of odors with specific receptors on the cilia of olfactory neurons. Selective removal of the cilia results in the loss of olfactory response (9).
Moreover, in fish, whose olfactory system senses amino acids as odors, the specific binding of amino acids to isolated cilia has been demonstrated (10, 11). The cilia are also the site o~ olfactory signal transduction. Exposure of isolated cilia from rat ol~actory epithelium to numerous odorant leads to the rapid sti~ulation of adenylyl cyclase and elevations in cyclic AMP (an elevation in IP3 in response to one odorant has also b~en observed) (12, 13, 14, 15). The activation of adenylyl cyclase is dependent on ~he presence of GTP and is therefore likely to be mediated by receptor-coupled GTP binding proteins ~G-proteins) tl6~.
Elevations in cyclic AMP, in turn,~are thought to elicit depolarization o~ olfactory neurons by direct activation of a cyclic nucleotide-gated, cation permeable channel (17, 18). This channel i5 opened upon binding of cyclic nucleotides to its cytoplasmic domain, and can therefore transduce changes in intracellular levels of cyclic AMP into alterations in the membrane potential. ~-.:
These observations suggest a pathway for olfactory signal transduction (Figure lB) in which the binding of odors to specific surface receptors activates specific G-proteins.
The G-proteins then initiate a cascade of intracellular signalling events leading to the generation of an action potential which is propagated along the olfactory sensory axon to the brain. A number of neurotransmitter and hormone receptors which transduce intracellular signals by ,..~ ~:

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activation of specific G-proteins have been identified.
Gene cloning has demonstrated that each of these receptors is a member of a large superfamily of surface recPptors which traverse the membrane seven times (19, 20). The S pathway of olfactory signal transduction (Figure lB) predicts that the odorant receptors might,also be members of this superfamily of receptor proteins. The detection of odors in the periphery is therefore likely to involve signalling mechanisms shared by other hormone or neurotransmitter systems, but the vast discriminat~ry power of the olfactory system will require higher order neural processing to permit the perception of individual odors.
This invention address the problem of olfactory perception at a molecular level. Eighteen different members of an extremely large multigene family have been cloned and characteri~ed which encodes seven transmembrane domain proteins whose expression is restricted to the olfactory epithelium. The members of this novel gene family encode ~ -the individual odorant receptors.

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, B~MM~RY OF T~E INVEiNTION

The invention provides an isolated nucleic acid, e.g. a DNA
and cDNA molecule, encoding an odorant receptor. The invention further provides expression vectors containing such nucleic acid. Also provided by the invention is a purified protein encoding an sdorant receptor. The invention Purther provides a method of transforming cells which comprises transfecting a suitable host cell with a suitable expression vector containing the nucleic acid encoding the odorant receptor.

The invention al50 provides methods of identifying odorant ligands and of identifying odorant receptors. 'rhe invention further provides methods of developing fragrances, oP
identi~ying appetite suppressant compounds, of controlling appetite. The invention also provides methods of controlling insect and other animal populations. The invention additionally provides a method of detecting odors such as the vapors emanating from Cocaine, Marijuana, Heroinl Hashish, Angel Dust, gasoIine, decayed human Plesh, alcohol, gun powder explosives, plastic explosives, firearms, poisonous or harmful smoke, or natural gas.

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Figure 1. The Olfactor~ NeuFoepithelium and a Pathwav for Olfactorv Sianal Transduction. A. T h e O l f a c t o r y Neuroepithe}ium. The initial event in odor perception occurs in the nasal cavity in a specialized neuroepithelium which is diagrammed here. Odors are belie~ed to interact with specific receptors on the cilia of olfactory sensory neurons. The signal generated by these initial binding events are propagated by olfactory neuron axons to the olfactory bulb. B. A Pathway of Olfactory Signal Transduction. In this scheme, the binding of an odorant molecule to an odor-specific transmembrane receptor leads to the interaction of the receptor with a GTP-binding protein (Gs[olf~)~ This interaction, in turn, leads to the release of the GTP-coupled ~-subunit of the G-protein, which then stimulates adenylyl cyclase to produce elevated levels of cAMP. The increase in cAMP opens nucleotide-gated cation channels, thus causing an alteration in membrane potential.
Figure 2. A PCR Am~lification Product_Containinq Mult Ple Species_of DNA. cDNA prepared from olfactory epithelium RNA
was subjected to PCR amplification with a series of different primer oligonucleotides and the DNA products of appropriate size were isolated, further amplified by PCR, and size ~ractionated on agarose gels (A) (For details, see text). Each of these semipurified PCR products was digested with the restriction enzyme, Hinf I, and analyzed by agarose gel electrophoresis. Lanes marked "M" contain size markers of 23.1, 9.4, 5.6, 4.4, 2.3, 2.0, 1.35, l.O~, 0.~37, 0.60, 0.31, 0.~8, 0.23, 0.19, 0.12 and 0.07kb. (B). Twenty-two of the 64 PCR products that were isolated and digested with Hinf I are shown here. Digestion of one of these, PCR
13, yielded a large number of fragments whose sizes su~med to a value much greater than that of the undigested PCR 13 .

WO9~/1758' 2 1 ~ ~ 8 ~ PCT/US92~02741 1, DNA, indicating that PCR 13 mig~t contain multiple species of DNA which are representatives of a multigene family.

- Figure 3. Northern Blot Anal~sis with a Mixture of Twenty Probes. One ~g of polyA+ RNA isolatPd from rat olfactory epithelium, brain, or spleen was size-fractionated in formaldehyde agarose, blotted onto a nylon membrane, and hybridized with a 32P-labeled mixture of segments of 20 cDNA
clones. The DNA segments were obtained by PCR using primers homologous to transmembrane domains 2 and 7.

Figure 4. The Protein Sequences Encoded bY Ten Diverqent cDNA Clones. Ten divergent cDNA clones were subjected to DNA sequence analyses and the protein sequence encoded by each was determined. Amino acid residues which are conserved in 60~ or more of the proteins are shaded. The presence of seven hydrophobic domains (I-VII), as well as short conserved motifs shared with other members of the superfamily, demonstrate that these proteins belong to the seven transmembrane domain protein superfamily. Motifs ~;~
conserved among members of the superfamily and the family of olfactory proteins include the GN in TM1 (transmembrane domain 1), the central W of TM4, the Y near the C-terminal end of TM5, and the NP in TM7. In addition, the DRY motif C-terminal to TM3 is common to many members of the G-protein-coupled superfamily. Nowever, all of the proteins shown here share sequence motifs not found in other members of this superfamily and are clearly members of a novel family of proteins.
Figure 5. Positlons of_ Greatest Variability in the Olfactorv_Protein FamiIv. In this diagram, the protein encoded by cDNA c~lone I15 is shown traversing the plasma membrane seven times with its N-terminus located extracellularly, and its C-terminus intracellularly. The , i :' .

WO92/1758~ PCT/U~92/02~41 2 1 0 6 8 ~ ~ ~

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vertical cylinders delineate the seven putative ~-helices spanning the membrane. Positions at which 60% or more of the 10 clones shown in Figure 4 share the same residue as I15 are shown as white balls. More variable residues are shown as black balls. The high degree of variability encountered in transmembrane domains III, IV, and V is evident in this schematic.

Figure 6. The Presence of Subfamilies in a Diver~ent Multiqene Family. Partial nucleotide sequences and deduced protein sequences were obtained for 18 different cDNA
clones. Transmembrane domain V along with the flanking :Loop sequences, including the entire cytoplasmic loop between transmembrane domains V and VI, are shown here for each protein. Amino acid residues ~ound in 60% or more of the clones in a given position are shaded (A). This region of the olfactory proteins (particularly transmembrane domain V) appears to be highly variable ~see Figure 4). These proteins, however, can be grouped into subfamilies (B,C,D~
in which the individual subfamily members share considerable homology in this divergent region of the protein.

Figure 7. Southern Blot Analyses w th Non-crossh~ridizinq Fraqments of Diverqent cDNAs. Five ~g of rat liver DNA was digested with Eco RI (A) or Hind III ~B), electrophoresed in 0.75% agarose, blotted onto a nylon membrane, and hybridized to the 32P-labeled probes indicated. The probes used were PCR-generated fragments of: 1, clone F9 (identical to F12 in Figure 4); 2, F5; 3, F6; 4, I3; 5, I7; 6, I14; or 7, I15.
The lane labeled "1-?" was hybridized to a mixture of the seven probes. The probes used showed either no crosshybridization or only trace crosshybridization with one another. The size markers on the left correspond to the four blots on the left (1-4) whereas the marker positions 3~5 noted on the right correspond to the four blots on the right . .

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Figure 8. Northern Blot ~naly_is with a Mix of_Seven Diverqent Clones. One ~g of polyA~ RNA from each of the tissues shown was size-fractionat~d, blotted onto a nylon membrane, and hybridized with a 32P-lclbeled mixture of segments of seven divergent cDNA clones (see Legend to Figure 7~. ~
, ~, ., 10 Figure 9. The amino acid and nucleic acid sequence of clone F3.

Figure 10. The amino acid and nucleic acid sequence of clone F5.
F.igure 11. The amino acid and nucleic acid sequence of clone F6.
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Figure 12. The amino acid and nucleic acid sequence of 20 clone F12.

Figure 13. The amino acid and nuc}eic acid sequence of clone I3.

25 Figure 14. The amino acid and nucleic acid sequence of clone I7.

Figure 15. The amino acid and nucleic acid sequence of clone I8.
Figure 16. ~he amino acid and nucleic acid sequence of clone I9.

Figur~ 17. The amino acid and nucleic acid sequence of ~ 35 clone I14. ~

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Figure 18. The amino acid and nucleic acid sequence of clone I15.

Figure 19. The amino acid and nucleic acid sequence of human clone H5.

Figure 20. The amino acid and nucleic: acid sequence of clone Jl, where the reading frame starts at nucleotide position 2.
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Figure 21. The amino acid and nucleic acid sequence of clone J2.

Figure 22. The amino acid and nucleic acid sequence of :
clone J4, where the reading frame starts at nucleotide position 2.

Figure 23. The amino acid and nucleic acid sequence of :~
clone J7, where the reading frame starts at nucleotide position 2.

Figure 24. The amino acid and nucleic acid se~uence of clone J~, where the reading frame starts at nucleotide : :
positon 2.
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Figure 25. The amino acid and nucleic acid sequence of clone J11. `:
:. ' ': ,, Figure 26. The amino acid and nucleic acid sequence of clone Jl~, where the reading frame starts at nucleotide -:
position 2.

Figure 27. The amino acid and nucleic acid sequence of clone J15, where the reading frame starts at nucleotide ::
psition 2. :.

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Figure 28. The amino acid and nucleic acid sequence ofclone J16, where the reading ~rame starts at nuoleotide positi.on 2.

Figure 29. The amino acid and nucleic acid sequence of clone J17, where the reading frame start~i at nucleotide position 2.

Figure 30. The amino acid and nucleic acid sequence of clone Jl9, where the reading frame starts at nucleotide position 2.

Fiyure 31. The amino acid and nucleic acid sequence of clone J20, where the reading frame starts at nucleotide position 2.

Figure 32. SOUTHERN BLOT: Five micrograms of DNA isolated from 1. Human placenta, 2. NCI H-1011 neuroblastoma cells, or 3. CHP 134 neuroblastoma cells were treated with the restriction enzyme A. Eco RI, B. Hind III, C. Bam HI, or D.
Pst I, and then electrophoresed on an agarose gel and blotted onto a nylon membrane. The blotted DNA was hybridized to the 32P-labeled H3/H5 sequence. An autoradiograph of the hybridized blot is shown with the sizes of co-electrophoresed size markers noted ir kilobases.

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W092/l75g~ PCT/~JS92/0274l 2 1 ~ 7 ~-12-Dat~ile~ D~cription of th~_I~vention The invention provides an isolated nucleic acid, e.g. a DNA
or cDNA molecule, encoding an odorant receptor. Such a receptor is a receptor which binds an odorant ligand and include but not limited to pheromone receptors. An odorant ligand may include, but is not limited to, molecules which interact with the olfactory sensory neuron, molecules which interact with the olfactory cilia, pheromones, and molecules which interact with structures within the vo~eronasal organ.

The invention specifically provides the isolated cDNAs encoding odorant receptors the sequences of which are shown in Figures 9-31. The nucleic acid is most typically a cDNA
and encodes an insect, a vertebrate, a fish or a mammalian odorant receptor. The mammalian odorant receptor is pr~ferably a human, rat, mouse or dog receptor. In an embodiment, human odorant receptor cDNA sequence and the correspondent protein is isolated (Figure 19).
In another embodiment, phermone recaptors are is~lated and shown as clones J1, J2, J4, J7, J8, J11, J14, J15, J16, J17, J19 and J20 (Figures 20-31).

The invention ~`urther provides e~pression vectors containing cDNA which encodes odorant receptors. Such expression vectors are well known in the art and include in addition to the nucleic acid the elements necessary for replication and ~-expression in a suitable hosts. Suitable hosts are well known in the art and include without limitation bacterial hosts such as E. coli, animal hosts such as CHO cells, insect cells, yeast cells and like.
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The invention also provides purified proteins encoding ~-odorant receptors. Such proteins may be prepared by ~
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~'09~/17~ 2 ~ ~ 6 ~ ~ 7 PCT/US9~/02741 expression of the forementioned exprassion vectors in sui.table host cells and recovery and purification of the receptors using methods well known in the art. Exa~ples of such proteins include those having the ami.no acid sequences shown in figures 9-3l.

The purified protein typically encc,des an insect, vertebrate, fish or mammalian odorant receptor. The mammalian odorant receptor may be a human, rat, mouse or dog.

In one embodiment the invention provides a novel purified protein which belong to a class of proteins which have 7 transmem~rane regions and a third cytoplasmic loop from the N-terminus which is approximately 17 amino acid long and to nucleic acid molecules encoding such proteins.

The invention provides methods of transforming cells which comprises transfecting a suitable host cell with a suitable 20. expression vector containing nucleic acid encoding of the odorant receptor. Techniques for carrying out such transformations on cells are well known to those skilled in the art. (41,42) Additionally, the resulting transformed cells are also provided by the invention. These transform~d cells may be either olfactory cell~ or non-olfactory cells.
one advantage of using transformed non-olfactory cells is that the desired odorant receptor will be the only odorant receptor expressed on the cell's surface.

In order to obtain cell lines that express a single receptor type, standard procedures may be used to clone individual cDNAs or genes into expression vectors and then transfect the cloned sequences into mammalian cell lines. This approach has been used with sequences encoding some othDr members of the seven transmembrane domain super~amily : ' ' ' ' WO92/1758~ P~T/US92/02741 2 1 1~ 7 ~

including the 5HTlc serotonin receptor. (43) The cited work illustrates how members of this superfamily transferred into cell lines may generate immortal cell lines that express high levels of the transfected receptor on the cell surface where it will bind ligand and that such abnormally expressed receptor molecules can transduce signals upon binding to ligand.

The invention also provides a method of identifying a desired odorant ligand which comprises contacting transformed non-olfactory cells expressing a known odorant receptor with a series of odorant ligands to determining which ligands bind to the receptors present on the non-olfactory cells.
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Additionally, the invention provides a method of identifying a desired odorant receptor comprising contacting a series of :
transformed non-ol~actory cells with a known odorant ligand and determining which odorant receptor binds with the odorant ligand.

The invention provides a method of detecting an odor which comprises: a) identifying a odorant receptor which binds the desired odorant ligand and; b) imbedding the receptor in a membrane such that when the odorant ligand binds to the receptor so identified a detectable signal is produced. In one embodiment of the invention the membrane used in this method is cellular, including a membrane of an olfactory cell or a synthetic mem~rane.
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The ligand tested for may be the vapors emanating from Cocaine, Marijuana, Heroin, Hashish, Angel Dust, gasoline, decayed human flesh, alcohol, gun powder explosives, plastic explosives or firearms. In another embodiment the ligand tested for may be natural gas, a pheromone, toxic fumes, ''' ..
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noxious fumes or dangerous fumes.

In one embocliment of the invention the det~ctable 6ignal is a lightbulb lighting up, a buzzer buzzing, a bell ringing, a color change, phosphorescence, or radioactivity.

The invention further provides a method of quantifying the amount of an odorant ligand present in a sample which comprises utilizing the above~mentionecl method for odor detection and then ~uantifying the amount of signal produced.

The invention further provides a method of developing fragrances which comprises identifying a desired odorant receptor by the above method, then contacting non-olfactory cells, which have been transfected with an expression vector containing nucleic acid encoding the desired odorant receptor such that the receptor is expressed upon the surface of the non-olfactory cell, with a series of compounds to determine which compound or co~pounds bind the receptor.

The invention provides to a method of identifying an "odorant fingerprint" which comprises contacting a series of' cells, which have been trans~ormed such that each express a known odorant rec~ptor, with a desired sample and determining the type and quantity of the odorant ligands present in the sample.

The invention provides a method of identifying odorant ligands which inhibit the activity of a desired odorant receptor which comprises contacting the desired odorant receptor with a series of compounds and determining which compounds inhibit the odorant ligand - odorant receptor ~ interaction.

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The invention also provides for a method of identifying appetite suppressant compounds which comprises identifying odorant ligands by the method mentioned in the preceding paragraph wherein the desired odorant receptor is that which is associated with the perception of food. Additionally, the invention provides a method of controlling appetite in a subject which comprises contacting the olfactory epithelium of the subject with these odorant ligands.
Further the invention provides a nasal spray, to control lo appetite comprising the compounds identified by the above method in a suitable carrier.
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The invention provides a method of trapping odors which comprises contacting a membrane which contains multiples of the desired odorant receptor, with a sample such that the desired odorant ligand is absorbed by the binding of the odorant ligand to the odorant receptor. The invention also provides an odor trap employing this method. ;~

The invention also provides a method of controlling pest populations which comprises identifying odorant ligands by the method mentioned above which are alarm odorant ligands and spraying the desired area with the identified odorant ligands. AdditionalIy, provided by the invention is a method of controlling a pest population which comprises identifying odorant ligands by the above mentioned method, which intQrfere with the interaction between the odorant ligands and the odorant receptors which are associated with fertility. In one embodiment the pest population is a population of insects or rodents, including mice and rats.

The invention also provides a method of promoting fertility which comprises identifying odorant ligands which interact with the odorant receptors associa~ed with fertility by the above mentioned method. Further, the invention provides a , , , . ... .

w o 9~/1758' PC~r/US92/02741~ 2:~6~'~7 method of inhibiting fertility which compris~s employing the above mentione~ method to identifying odorant ligands which inhibit the interaction between the odorant ligands and the odorant receptors associated with fertility.
This invention is illustrated in th~ Experimental Detail section which follow. These sections are set forth to aid in an understanding of the invention but are not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter.

~P~RI~ENTAL_DETAI~8 MATERIAL~ AND MET~2DS
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Polymerase Chain Reaction ~NA was prepared from the olfactory epithelia of Sprague Dawley rats according to Chirgwin et al. (40~ or using RNAzol B (Cinna/Biotecx) and then treated with DNase I (0.1 unit/~g RNA) (Promega). In order to obtain cDNA, this RNA
was incubated at 0.1 ~g/~l with 5 ~M random hexamers (Pharmacia) 1 mM each of dATP, dCTP, dGTP, TTP, and 2 units/~1 RNase inhibitor (Promega) in 10 mM TrisCl (pH 8.3), 50 mM KCl, 2.5 mM MgC12, and 0.001% gelatin for 10 min. at 22C, and then for a further 45 min. at 37C following the addition of 20 u./~1 of Moloney murine leukemia virus ; 30 reverse transcriptase (BRL). After heating at 95~C for 3 i~
min., cDNA prepared from 0.2 ~g of RNA was used in each of - ~`
a series of polymerase chain reactions (PCR) containing 10 mM TrisCl (pH 8.3), 50 mM KCl, 1.5 mM MgC12, 0.001% gelatin, 200 ~M each of dATP, dCTP, dGTP, and TTP, 2.5 u. Taq polymerase (Perkin Elmer Cetus), and 2 ~M of each PCR

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W092/1758~ ~CT/US92/0274l 2 1 ~ G 8 ~7 pri~er. PCR reactions were performed according to the following schedule: 96C for 45 sec., 55C for 4 min. (or 45C for 2 ~in.), 72C for 3 min. with 6 ~ecO extension per cycle for 48 cycles. The primers used for PCR were a series of degenerate oligonucleotides made according to the amino acid sequences found in transmembra~e domain 2 and 7 of a variety of different members of the 7 tranismembrane domain protein superfamily (19). The regions used corre~pond to amino acids number 60-70 and 286-295 of clone I15 (Figure 4). Each of five different 5' primers were used in PCR
reactions with each of six different 3' primers. The 5' primers had the sequences:

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C AC A C CT
Al, AATTGGATICTIGTIAATCTIGCIGTIGCIGCIGA;
C C CA A C C .. ~
A2, AATTATTTTCTIGTIAATCTIGCITTIGCIGA;
CCA CC A C ~:
A3, AATTTITTTATIATITCICTIGCITGIGCIGA;: :
A T C T ACT C
A4, CGITTICTIATGTGTAACCTITGCTTTGCIGA;
C CT TG
A5, ACIGTITATATIACICATCTIACIATIGCIGA.
The 3' primers were: .
TTA T CAG C C A
Bl, CTGICGGTTCATIAAIACATAIATIATIGGGTT;
TG GA G G A A :
B2, G~TCGTTIAGACAACAATAIATIATIGGGTT;
A G G A
B3, TCIATGTTAAAIGTIGTATAIATIATIGGGTT;
T G G A A . : . :
B4, GCCTTIGTAAAIATIGCATAIAGGAAIGGGTT;
G AGA G G G A : , B5, AAATCIGGGCTICGIC~ATAIATCAIIGGGTT; . .. .
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CT CT G G G G A .~ .
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~6, GAIGAICCIACAAAAAAATAIATAAAIGGGTT.

An aliquot of each PCR reaction was analyzed by agarose gel electrophoresis and bands of interest werP amplified ~urther by performing PCR reactions on pipet tip (approx. 1 ~l) plugs of the agarose gels containing those DNAs. Aliquots of the6e semi-purified PCR products were digested with the restriction enzymes Hae III or Hinf I and the digestion products were compared with the undigested DNAs on agarose gels.

Isolation and AnalYsis of cDNA Clones CDNA libraries were prepared according to standard procedures (41, 42) in the cloning vector, AZAP II
(Stratagene) using poly A+ RNA prepared from Sprague Dawley rat epithelia (see above) or from an enriched population of olfactory neurons which had been obtained by a 'panning' procedure, using an antibody against the H blood group antigen (Chembiomed) found on a large percentage of rat olfactory neurons. In initial library screens, 8.5 X 105 independent clones from the olfactory neuron library and 1.8 X 106 clones from the olfactory epithelium library were screened t41) with a 32P-labeled probe tPrime-it, Stratagene) consisting of a pool o~ gel-isolated PCR
products obtained using primers A4 and B6 ~see above) in PCR
reactions using as template, olfactory epithelium cDNA, rat liver DNA, or DNA prepared from the two cDNA libraries. In ~ later library screens, a mixture of PCR products obtained from 20 cDNA clones with the A4 and B6 primers was used as probe ('P1' probe). In initial screens, phage clones were analyzed by PCR using primers A4 and B6 and those which showed the appropriate size species were purified. In lat r screens, all position clones were purified, but only those that could be amplified with the B6 primer and a primer ~'092/1758~ 6 ~ ~ l PCT/US92/02741 ' :~

specific for vector sequence were analyzed further. To obtain plasmids from the isolated phage clones, phagemid rescue was performed according to ~he instructions of the '~
~anufacturer of AZAP II (Stratagene~. DNA sequence analysis was performed on plasmid DNAs using the Se~uenase system (USB), initially with the A4 and B6 primers and later with oligonucleotide primers made according to sequencas already obtained.
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Northern and Southern BIot Analvses , ~:
For Northern blots, poly A+ RNAs ~rom various tissues were prepared a~ described above or purchased from Clontech. One , ~g of each ~NA was size fractionated on formaldehyde agarose gels and blotted onto nylon membranes (41, 42). For, '' Southern blots, genomic DNA prepared from Sprague Dawley rat ' liver was digeqited with the restriction enzymes Eco RI or ' Hind III, sizie fractionated on agarose gels and blotted onto , nylon membranes ~41, 42). The memkranes were dried at 80C, ' and then prehybridized in 0.5 M sodium phosphate buffer (pH '~
7.3) containing 1~ bovine serum albumin and 4% sodium dodecyl sulfate. Hybridization was carried out in the same , ,' buffer at 65-70C for 14-20 hrs. with DNAs labeled with ,', .
32p. For the first Northern blot shown, the 'Pl' probe (see '' above under cDNA clone isolation) was u~ed. For the second Northern blot shown, a mix of PCR ~ragments from ~even ', , divergent cDNA clo~es was used. For Southern klots, the ;, ', region indicated in clone I15 by amino acids 118 through 251 ~' ' was amplified from a series of divergent cDNA clones using PCR. The primers used for these reactions had the~, . .
sequences~

Pl, ATGGCITATGATCGITATGTIGC, and ,~' .. . .
~ 35 P4, AAIAGIGAIACIATIGAIAGATGIGAICC ~
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W092/1758~ 6 8 ~ PCT/US92/02741 These DNAs ~or a DNA encompassing transmembrane domains 2 through 7 for clone F6) were labeled and tested for cro6shybridization at 700c. Those DNAs which did not show appreciable crosshybridizationwer~ hybridized individually, or as a pool to Southern blots at 70C.

Rat Sequences used to obtain similar sequences exeressed ln Humans There are genes similar to the rat genes discussed above present in humans, these genes may be readily isolated by screening human gene libraries with the cloned rate sequences or by performing PCR experiments on human genomic DNA with primers homologous to the rat se~uences~ First, PCR experiments were performed with genomic DNA from rat, human, mouse, and several other species. When primers homologous to transmembrane domains 2 and 6 (the A4/B6 primer set used to isolate the original rat sequences) were used, DNA of the appropriate sizs was amplified from rat, human and mouse DNAs. When these primary PCR reactions were subsequently diluted and subjected to PCR using primers to internal sequences tPl and P4 primers), smaller DNA species were amplified whose size was that seen when the ~ame primers were used in PCR reactions with the cloned rat cDNAs. Similarly, when the secondary PCR was performed with one outer primer together with one inner primer (ie. ~4/P4 or P1/B6), amplified DNAs were obtained whose sizes were also consistent with the amplification~of genes similar in sequence and organization to the cloned rat cDNAs. Second, a mix of segments from 20 of the rat cDNAs ('P1" probe) was used to screen libraries constructed from human genomic DNAs. Hybridization under high or low stringency conditions reveals the presence of a large number of cloned human DNA
segments that are homologous to the rat sequences.
Finally, RNA from a human olfactory tumor ~neuroesthesioma, .. :.

:
~ .. . .

.' -W~9~/l7~8~ PCT/~S92/02741 2 L ~

NCI-H-1011) cell line has been examined ~or ~equences homologous to those cloned in the rat. cDNA prepared from this RNA was sub~ected to PCR with the A4/B6 primer set and a DNA species of the appropriate size was seen. This DN~
was subcloned and partially sequenced and clearly encodes a member of the olfactory protein family identified in the rat.

The inserted sequence in human clones H3/H5 was amplified by PCR with the A4/B6 primers, gel purified, and then labeled with 32P. The labeled DNA was then hybridizecl to restriction enzyme human placenta. Multiple hybridizing species were observed with each DNA (See Figure 32). This observation is consistent with the presence of a family of odorant receptor genes in the human genome.

The sequence of clone H5 is hereby shown in Figure 19. In addition, the translated protein sequence is shown in Figure 19. ' ' :' `
In other to identify odorant receptors in other species, :'~
degenerated primer oligonucleotides homologous to conserved regions within the rat odorant receptor family may be used in PCR reactions with genomic DNA or with cDNA prepared from olfactory tissue RNA from those species.

RE8~L~8 Cloning the Gene Family A series of degenerate oligonucleotides were designated which could anneal to conserved regions of members of the superfamily of G-protein coupled seven transmembrane domain receptor genes. Five degenerate oliogonucleotides ~A1~5;
see Experimental Procedures) matching sequences within transmembrane domaln 2, and six degenerate oligonucleotides '~
' WO9~/1758~ 2 ~ PCT/US92/02741 (Bl-6) matching transmembrane domain 7 were used in all combinations in PCR reactions to amplify homologous sequences in cDNA prepared from rat olfactory epithelium RNA. The amplification products of each PCR reaction were then analyzed by agarose gel electrophoresis. Multiple bands were observed with each of the primer combinations.
The PCR products within the size range expected for this family of receptors ~600 to 1300 bp~ were subsequently picked and amplified further with the appropriate primer pair in order to isolate individual PCR bands. Sixty-four PCR bands isolated in this fashion revealed only one or a small number of bands upon agarose gel electrophoresis.
Representatives of these isolated PCR products are shown in Figure 2A.
The isolated PCR products were digested with the -~
endonuclease, Hae III or Hinf I, which recognize four base restriction sites and cut DNA at frequent intervals. In most instances, digestion of the PCR product with Hinf I
generated a set of fragments whose molecular weights sum to the size of the original DNA (Figure 2B). These PCR bands are therefore likely to each contain a single DNA species.
In some cases, however, restriction digestion yielded a series of frasments whose molecular weights sum to a value greater than that of the original PCR product. The most dramatic example is shown in Figure 2 where the 710 bp, PCR
13 DNA, is cleaved by Hinf I to yield a very large number of restriction fragments whose sizes sum to a value five- to ten-fold greater than that of the original PCR product.
These observations indicated that PCR product 13 consists of a number of different species of DNA, each of which could be amplified with the same pair of primer oligonucleotides. In addition, when PCR experiments similar to those described were performed using cDNA library DNAs as templates, a 710 bp PCR product was obtained with the PCRl3 primer pair ' ..~
.

W092/175~ PCT/US92/02741 ~6~7 -2~-(~4/B6) with DNA from olfactory cDNA libraries, but not a glioma cDNA library. Moreover, digestion of one of this 710 bp product also revealed the presence of multiple DNA
species. In other cases (see PCR product 20, ~or example), digestion yielded a series of restriction fragments whose molecular weights also sum to a size greater than the startin~ material. Further analysisr however, revealed that the original PCR product consisted of multiple bands of si~ilar but different sizes.
",. .. .
In order to determine whether the multiple DNA species preisent in PCR 13 encode members of a family of seven transmembrane domain proteins, PCR 13 DNA was cloned into the plasmid vector Bluescript and five individual clones were subjected to DNA sequence analysis. Each of the five clones exhibited a different DNA sequence, but each encoded a protein which displayed conserved features of the superfamily of seven transmembrane domain receptor proteins~
In addition, the proteins encoded by all five clones shared distinctive sequence motifs not found in other superfamily members indicating they were all members of a new family of receptors.
.: .
To obtain full-length cDNA clones, cDNA libraries prepared ;~-from olfactory epithelium RNA or from RNA of an enriched population of olfactory sensory neurons were screened. The probe used in these initial screens was a mixture of PCR 13 DNA as well as DNA obtained by amplication of rat genomic DNA or DNA from two olfactory cDNA libraries with the same pri~ers used to generate PCR 13 (A4 and B6 primers). :
Hybridizing plaques were subjected to PCR amplification with the A4/B6 primer set and only those giving a PCR product of the appropriate size ~approximately 710 bp) were purified.
The frequency of such positive clones in the enriched olfactory neuron cDNA library was approximately five times .
-.

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WO92/1758~ PC~/US92/~2741 greater than the ~requency in the olfactory epithelium cDNA
library. The increased frequency of positive clones observed in the olfactory neuron library is comparable to the enrichment in olfactory neurons ~enerally obtained in the purification procedure.

The original pair of primers used to amplify PCR 13 DNA were then used to amplify coding segments of 20 different cDNA
clones. A mix of these PCR products were labeled and used as prob~ for further cDNA library screens. This mixed probe was also used in a Northern blot ~Figure 3) to determine whether the expression of the gene family is restricted to the olfactory epithelium. The mixed probe detects two diffuse bands centered at 2 and 5 kb in RNA from olfactory }5 epithelium; no hybridization can be detected in brain or spleen. (Later experiments which examined a larger number of tissue RN~5 with a more restricted probe will be shown below.) Taken together, these data indicate the discovery of a novel multigene family encoding seven transmembrane domain proteins which are expressed in olfactory epithelium, and could be expressed predominantly or exclusively in olfactory neurons.

The Protein Se~uences of Numerous, OlfactorY-specific Members af the Seven Transmembrane Domain Superfamily Numerous clones were obtained upon screening cDNA libraries constructed from olfactory epithelium and olfactory neuron RNA at high stringency. Partial DNA sequences were obtained from 36 clones; 18 of these cDNA clones are different, but all of them encode proteins which exhibit shared sequence motifs indicating that they are members of the family identified in PCR 13 DNA. A complete nucleotide sequence was determined for coding regions of ten of the most 39 d~vergent clones (Figure 4). 1he deduced protein sequences ~

W O 92/175X' PC~r/US92/0274~
2 ~0~7 --2~-of these cDNAs defines a new multigene family which shares seguence and structural propertie~ with the superfamily of neurotransmitter and hormone receptors khat traverse the membrane seven times. This novel family, however, exhibit~
features different from any other member of the receptor superfamily thus far identified.
:: ' Each of the ten sequences contains seven hydrophobic stretches (19-26 amino acids) that represent potential transmembrane domains. These domains con~titute the regions of maximal sequence similarity to other members of the seven transmembrane domain superfamily (see legend to Figure 4).
On the basis of structural homologies with rhodopsin and the B-adranergic receptors, (19) it is likely that the amino termini of the olfactory proteins are located on the extracellular side of the plasma membrane and the carboxyl termini are located in the cytoplasm. In this scheme, three extracellular loops alternate with three intracellular loops to link the seven transmembrane domains (see Figure 5).
Analysis of the sequences in figure 4 demonstrates tha~ the ~-olfactory proteins, like other members of the receptor superfamily, display no evidence of an N-terminal signal sequence. As in several other super~amily members, a potential N-linked glycosylation site is present in all ten proteins within the~short N-terminal extracellular segment.
Other structural features conserved with previously identified members of the superfamily included cysteine residues at fixed positions within the ~irst and second extracellular loops that are thought to form a disulfide bond. Finally, many of the olfactory proteins reveal a conserved cysteine within the C-terminal domain which may serve as a palmitoylation site anchoring this domain to the membrane (21). These features, taken together with several short, conserved sequence motifs (see legend to Figure 4~, clearly define this new family as a member o~ tbP

WO92/1758~ PCT/US92/0~741 2 ~

superfamily of genes encoding the seven transmembrane domain recaptors.

There are, however, important differences between the olfactory protein family and the other seven transmembrane domain proteins described previously and these differences may be relevant to proposed function of these proteins in odor recognition. Structure-function experiments involving ln vitro mutayenesis suggest that adrenergic ligands interact with this class of receptor molecule by binding within the plane of the membrane (22, 20). Not surprisingly, small receptor families that bind the same class of ligands, such as the adrenergic and muscarinic acetylcholine receptor families exhibit ~aximum sequence conservation (often over 80%) within the transmembrane domains. In contrast, the family of receptors discussed in this application shows striking divergence within the third, fourth, and fifth transmembrane domains (Figure 4). The variability in the three central transmembrane domains is highlighted schematically in Figure 5. The divergence in potential ligand binding domains is consistent with the idea ~hat the family of molecules cloned is capable of associating with a large number of odorant of diverse molecular structure.
Receptors which belong to the superfamily of seven transmembrane domain proteins interact with G-proteins to generate intracellular signals. In _tro mutagenesis experiments indicate that one site of association between ~0 receptor and G-protein resides within the third cy~oplasmic loop (22, 23). The sequence of this cytoplasmic loop in 18 different clones we have characterized is shown in Figure 6A. This loop which is often quite long and of variable length in the receptor superfamily is relatively short (only 17 amino acids) and of fixed length in the 18 clones ~ ' ",, :.
~' :' :: , ' W09~/17~8~ PCT/US92/02741 ~ .
21~ 6 ~ ~ rS~

examined. Eleven of the 18 different clones exhibit the sequence motif K/R I V S S I (or a close relative) at the N-terminus of this loop. Two of the cDNA clones reveal a different H I T C/W A V motif at this site. If this short loop is a site of contact with G-proteins, it i~ii possible that the conserved motifs may reflect sites of interaction with different G-proteins that activate different intracellular signalling systems in response to odors. In addition, the receptors cloned reveal several serine or threonine residues within the third cytoplasmic loop. By analogy with other G-protein coupled receptors, these residues may represent sites of phosphorylation for specific receptor kinases involved in desensitization. (24) Subfamilies within the Multiqene Family ..
Figure 6A displays the sequences of the fifth transmembrane domain and the adjacent cytoplasmic loop encoded by L8 of the cDNA clones we have analyzed. As a group, the 18 sequences exhibit considerable divergence within this region. The multigene family, however, can be divided into subfamilies such that the members of a given subfamily share significant sequence conservation. In subfamily B, clones F12 and F13, for example, differ from one another at only four of 44 positions (91~i identify), and clearly define a subfamily. Clones F5 and Ill (subfamily D) differ from F12 and F13 at 34-36 positions within this region and clearly define a separate subfamily. Thus, this olfactory-specific multigene family consists of highly divergent subfamilies.
If these genes encode odor receptors, i~ is possible that members of the divergent subfamilies bind odorant of widely differing structural c~lasses. Members of the individual subfamiIies could therefore recognize more subtle dif~erences between molecules which belong to the same ~ -structural class of molecules structures. ~

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W092/l758~ PCT/US92/0~741 The Size of the Multiqene Family Genomic Southern blotting experiments were preformed and genomic libraries were screened to obtain an estimate o~ the sizes of the multigene ~amily a~d the member ~ubfamilie6 encoding the putative odor receptors. DNAs extending from the 3' end of transmembrane domain 3 to the middle of transmembrane domain 6 were synthesized by PCR from DNA of seven of the divergent cDNA clones (Figure 4). In initial experiments, these DNAs were labeled and hybridized to each other to define conditions under which minimal crosshybridization would be observed among the individual clones. At 70C, the seven DNAs showed no crosshybridization, or crosshybridized only very slightly.
The trace levels of crosshybridization observed are not likely to be apparent upon genomic Southern blot analysis where the amounts of DNA are far lower than in the test cross.

Probes derived from these seven DNAs were annealed under stringent conditions, either individually or as a group, to Southern blots of rat liver DNA digested with the restriction endonucleases Eco RI or Hind III (Figure 7).
Examination of the Southern blots reveals that all but one o~` the cDNAs detects a relatively large, distinctive array of bands in genomic DNA. Clone I15 (probe 7), for example, detects about 17 bands with each restriction endonuclease, whereas clone F9 (probe 1) detects only about 5-7 bands with each enzyme. A single band is obtained with clone I7 (probe 5~. PCR experiments using nested primers ~TM2/TM7 primers followed by primers to internal sequences) and genomic DNA
as template indicate that the coding regions of the members of this multigene family, liXe those of many members of the G-protein coupled superfamily, may not be interrupted by ~ -35 ~ introns. This observation, together wit~ the fact that most ~ :"--, WO92/1758' PC~/US92/02741 2 ~ 7 ` - `

of the probes only encompasse6 400 nucl~otides suggests that aach band observed in these e~periment~ is likely to represent a different ~ene. These data sugge~t that the individual probes chosen are representatives of subfamilies which range in size from a ~ingle member to as many as 17 members. A total of about 70 individual bands were detected in this analysis which could represent the presence of at least 70 different genes. Although the DNA probes u~ed in these blots did not crosshybridize appreciably with each other, it is possible that a given gene might hybridize to more than one probe, resulting in an overestimate of gene number. However, it is probable that the total number of bands on].y reflects a minimal estimate of gene number since it is unlikely that we have isolated representative cDNAs from all of the potential subfamilies and the hybridizations were performed under conditions of very high stringency.

A more accurate estimate of the size of the olfactory-specific gene family was obtained by screening rat genomic libraries. The mix of the seven divergent probes used in Southern blots, or the mix of 20 different probes used in our initial Northern blots (see Figure 3), were used as hybridization probes under high (65C) or lowered (55C) stringency conditions in these experiments. Nested PCR ~see above) was used to verify that the clones giving a positive siqnal under low stringency annealing conditions were indeed members of this gene family. It is estimated fr~m these studies that there are between 100 and 200 positive clones per haploid genome. The estimate of the size of the family obtain from screens of genQmic libraries again represents a lower limit. Given the si~e of the multigene family, one might anticipate that many of these genes are linked such that a given genomic clone may contain multiple genes. Thus the data from Southern blotting and screens of genomic libraries indicate that the multigene family identified . .

~092/17~8~ 2 ~ 7 PCT/US92/02741 consists of one to several hundred member genes which can be divided into multiple subfamilies.
.
It should be noted that the cDNA probes isolated may not be representative of the full complement of subfamilies within the larger family of olfactory proteins. The isolation of cDNAs, for example, relies heavily on PCR with primers from transmembrane domains 2 and 7 and biases our clones for homology within these regions. Thus, estimates of gene number as well as subsequent estimates of RNA abunclance should be considered as minimal.

Expression of the Members of this Multi~ene Familv Additional Northern blot analyses were preformed to demonstrate that expression of the members o~ this gene family is restricted to the olfactory epithelium. (Figure 8) Northern blot analysis with a mixed probe consisting of the seven di~ergent cDNAs u~ed above reveals two diffuse bands about 5 and 2 kb in length in olfactory epithelium RNA.
This pattern is the same as that seen previously with the mix of 20 DNAs. No annealing is observed to RNA from the brain or retina or other, nonneural tissues, including lung, liver, spleen, and kidney.
An ~stimate of the level of expression of this family can be obtained from screens of cDNA libraries. The frequency of positive clones in cDNA libraries made from olfactory epithelium RNA suggests that the abundance of the RNAs in the epithelium is about one in 20,000. The frequency of positive clones is approximately five-fold higher in a cDNA
library prepared from RNA from purified olfactory neurons (in which 75% of the cells are olfactory neurons). The increased frequency of positive clones obtained in the olfactory neuron cDNA library is compar~ble to the .
,'~'~. .
. . ' '' :

.: " . '' ' W092/l7~X~ PCT/US92/02741 enrichment we obtain upon purification of olfactory neurons.
These observation~ suggest that this multigene ~amily is expressed largely, if not solely, in olfactory neurons and may not be expressed in other cell types with.in the epithelium. If each ol~actory neuron contains 105 ~RN~
molecules, from the frequency of positive clones we predict that each neuron contains only 25-30 txanscripts derived from this gene family. Since the family of olfactory proteins consists of a minimum of a hundred genes, a given olfactory neuron could maximally express only a proportion of the many different family members. These values thus suggest that olfactory neurons will exhibit signif:icant diversity at the level of expression of these olfactory proteins.
Identification of ~heromone receptors in vomeronasal oraan The vomeronasal organ (vomeronasal gland) is an accessory olfactory structure that is located near the nasal cavity.
Like the olfactory epithelium of the nasal cavity, the olfactory epithelium of the vomeronasal organ contains olfactory sensory neurons. The vomeronasal organ is believed to play an important role in the sensing of pheromones in numerous species. Pheromones are believed to have profound effects on both physiol~gical and behavioral aspects of reproduction. the identification oP pheromone receptors would permit the identification of the pheromones themselves. It would also enable one to identify agonists or antagonists that would either mimic the pheromones or block the pheromone receptors from transducing pheromone signals.
Such information wouId be important to the development of species specific pesticides and, conversely, to animal husbandry. The identification of pheromone receptors in human could ultimately lead to the development of contraceptives or to treatments for infertility in humans.
It is likely that the identification of pheromone receptors WO92/1758' PCT/US92/02741 ~ 21~6~7 in low mammals such as rodents would lead to the identification of simil,r receptors in human.

In order to identify potential pheromc,ne receptors, we isolate RNA from the vomeronasal organs of fe~ale rats and prepared cDNA ~rom this RNA. The cDNA w;i~s subjected to PCR
with several different pairi of degenerate oligonucleotide primers that match ~equencPs pr~sent i.n the rat odorant receptor family. The PCR products were subcloned and the nucleotide sequences of the subcloned DNAs were determined.
Each of the subcloned DNAs encodes a protein that belongs to the odorant receptor family. The sequences of the following vomeronasal subclones are shown: Jl, J2, J4, J7, J8, Jll, Jl4, Jl5, Jl6, Jl7, Jl9, J20. In a few cases (~2, J4), the sa~e sequence was amplified with two different primer pairs and the sequence shown is a composite of the two sequences.
It is possible that one or more of these molecules, or closely related molecules, serv~ as pheromone receptors in the rat~
DISCUSSION
.' ,':~
The mammalian olfactory system can recognize and discriminate a large number of odorous molecules~ -Perception in this system, as in other s~nsory systems, ..
initially involves the recognition of external stimuli by primary sensory neurons. This sensory information is then transmitted to the brain where it is decoded to permit the discrimination of different odors. Elucidation of the logic -~
undierlying olfactory perception is likely ~o require the identification of the specific odorant receptors, the ~;
analysis of the extent ~of receptor diversity and receptor specificity, as well as an understanding of the pattern of receptor expression in the olfactory epithelium.
-" . :' :' ~'092/l7~ 2 i a ~ ~ ~ 7 PCT/US92/0~741 ~. . , The odorant receptors are thought to transduce intracellular signals by interacting with G-proteins which activate second messenger systems (12, 13, 14, 15). These proteins are clearly members of the family of G-protein coupled receptori which traverse the membrane seven times (19~. The odorant receptors should be expressed specifically in the tissue in which odorant are recognized. The family of olfactory proteins cloned is exprecised in the olfactory ~pithelium.
~ybridizing RNA is not detected in brai~ or retina, or in a lo host of nonneural tissues. Moreover, expression of this gene family the epithelium may be restricted to olfactory neurons. The ~amily of odorant receptors must be capable of interacting with extremely diverse molecular structures.
The genes cloned are members of any extremely large multigene family which exhibit variability in regions thought to be important in ligand binding. The possibility that each member of t~is large family of seven transmembrane proteins is capable of interacting with only one or a small number of odorant provides a plausible mechanism to accommodate the diversity of odor perception. The properties of the gene family identified suggests that this family is likely to encode a large number of distinot odorant receptors.

Size of the Multiqene Familv The size of the receptor repertoire is likely to reflect the range of detectable odors and the degree of structural specificity exhibited by the individual receptors. It has been estimated that humans can identify ov~r lO,OOO
structurally-distinct odorous ligands. However, this does not nececsarily imply that humans possess an equally large repertoire of odorant receptors. For example, binding studies in lower vertebrates suggest that structurally-related odorant may activate the same receptor molecules.

WO92/17~X~ 2 ~ a ~ 8 ~ 7 PCT/US92/02741 In fish which smell amino acids, the binding of alanine to isolated cilia can be competed by other small polar residues (threonine and serine), but not by the basic amino acids, lysine or arginine (ll). These data suggest that individual receptors are capahle of associatinlg with several structurally-related ligands, albeit with different affinities. Stereochemical models of olfactory recognition in mammals (25) (based largely on psychophysical, rather than biophysical data) have suggested existence of several primary odor groups including camphoraceous, musky, peppermint, ethereal, pungent, and putrid. In such a model, each yroup would contain odorant with common molecular configurations which bind to common receptors anA share similar odor qualities.
Screens of genomic libraries with mixed probes consisting of divergent family members detect approximately lO0 to 200 positive clones per genome. The present estimate of at least lO0 genes provides only a lower limit since it is likely that the probes used do not detect all of the possible subfamilies. Moreover, it is probable that many of these genes are linked such that a given genomic clone may contain multiple genes. It is therefore expected that the actual size of the gene fami]y may be considerably higher and this family of putative odorant receptors could constitute one of the largest gene families in the genome.

The characterization of a large multigene family encoding putative odorant receptors suggests that the olfactory system utilizes a far greater number of receptors than the visual system. Color vision, for example, allows the discrimination of several hundred hues, but is accomplished by only three different photoreceptors (l, 2, 3 and 4). The photoreceptors each have different, but overlapping 35~ absorption spectra which cover the entire spectrum of :
.~

W092/l7~8~ PCT/~S9~/02741 7 ~

visible wavelengths. Discrimination of color reiults from comparative processing of the information from these three classes of photoreceptors in the brain. Whereas three photoreceptors can absorb light across the entire visible spectrum, our data suggest that a ~mall number of odorant receptors cannot recognize and disicriminate the full spectrum of distinct molecular structures perceived by the mammalian olfactory system. R~ther, olfactory perception probably employs an extremely large number of receptors each capable of recognizing a small number of odorous ligands.

Diver ity within the Gene FamilY and the Specificity of_Odor Recoqnition The olfactory proteins identified in this application are clearly members of the superfamily of receptors which traverse the membrane seven time. Analysis of the proteins encoded by the 18 distinct cDNAs we have cloned reveals structural features which may render this family particularly well suited for the detection of a diverse array of structurally distinct odorant. Experiments with other members of this class of receptors suggest that the ligand binds to its receptor within the plane of the membrane such that the ligand contacts many, if not all of the transmembrane helices. The family of olfactory proteins can be divided into several different subfamilies which exhibit significant sequence divergence within the transmembrane domains. Nonconservative changes are commonly observed within blocks of residues in transmembrane regions 3, 4, and 5 (Figures 4, 5, 6); these blocks could reflect the sites of direct contact with odorous ligands. Some members, for example, have acidic residues in transmembrane domain 3, which in other families are thought to be essential for binding aminergic ligands (20) while other members maintain hydrophobic residues at these positions.
'' ' .
.

W092/17~ PCT/US92/02741 2~68~7 This divergence within transmembrane dDmain6 may reflect the fact that the members of the ~amily of odorant receptors must associate with odorant of widely diffarent molecular structures.
These observations suggest a model in which each of the individual subfamilies encode receptors which bind distinct structural classes of odorant~ Within a given subfamily, however, the sequence differenoes are far less dramatic and are often restricted to a small number of residues. Thus, the members of a subfamily may recognize more subtle variations among odor molecules of a given structural class.
At a practioal level, individual subfamilies may recogni~e grossly different structures isuch that one subfamily may associate, for example, with the aromatic compound, benzene and its derivatives, whereas a second subfamily may recognize odorous, short chain, aliphatic molecules. Subtle variations in the structure of the receptors within, for --example, the hypothetical benzene subfamily could facilitate the recognition and discrimination of various substituted derivatives such as toluene, xylene or phenol. It should be noted that such a model, unlike previous stereochemical models, does not necessarily predict that molecules with similar structures will have similar odors. The activation of distinct receptors with similar structures could elicit different odors, since perceived odor will depend upon higher order processing of primary sensory information.
.
Evolution of the Gene Familv and the Generation of Diversi~y Preliminary evidence from PCR analyses suggests that members of this family of olfactory proteins are conserved in lower vertebrates as well as invertebrates. This gene family ~;~
presumably expa~ded over evolutionary time providing mammals -with the abllity to recogniæe an increasing diversity of : :

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U'092/]75~ PCT/US92/02741 t. : ~

odorant. Examination of the sequences of the family members cloned from mammals provides some insight into the evolution of this multigene family. Although the chromosomal locî
encoding these genes has yet to be characterized, it is likely that at least some member genes will be tandemly arranged in a large cluster as is observed with other large multigene families. A tandem array of t~is sort provides a template for recombination events including unequal crossing over and gene conversion, that can lead to expansion and further diversification of the sort apparent among the family members we have cloned (26).

The multigene family encoding the olfactory proteins is large: all of the member genes clearly have a common ancestral origin, but have undergone considerable divergence such that individual genes encode proteins that share from 40-80% amino acid identity. Subfamilies are apparent with groups of genes sharing greater homology among themselves than with members of other subfamilies. Examination of th~
sequences of even the most divergent subfamilies, however, reveals a pattern in which several blocks of c~nserved residues are interspersed with variable regions. This segmental homology is conceptually similar to the organization of framework and hypervariable domains within the families of immunoglobulin and T cell receptor variable region sequences (27, 2~). This analogy goes beyond structural organization and may extend to the function of these two gene families: each family consists of a large number of genes which have diversified over evolutionary time to accommodate the binding of a highly diverse array of ligands. The evolutionary mechanisms responsible for the diversification and maintenance of ~hese large gene families ~may also oe similar. It has been sugges~ed that gene conversion has played a major role in the evolution of immunoglobulin and T cell receptor variable domains (29, 30 -: ; :'':, '.

- ' WO 92/17:~8` PC~`/US92/02741 .
' 2 1~ ~ 8 f ~ ~

and 31). Analysis of the sequence of the putative olfactory receptors reveals at least one instance where a motif from a variable region of one subfamily is found imbedded in the otherwi~e divergent ~equence o~ a second subfamily, suggesting that conversion has occurr~d. Such a mixing o~
motifs from one subfamily to another over evolutionary time would provide additional combinatorial pos~sibilities leading to the generation of diversity.

It should be noted, however, that the combinatorial joining of gene segments by DNA rearrangement during development, which is characteristic of immunoglobulin loci (27), is not a feature of the putative odor receptor gene family. No evidence for DNA rearrangement to generate the diversity of genes cloned has been observed. The entire coding region has been sequenced along with parts of the 5' and 3l untranslated regions of 10 different cDNA clones. The sequences of the coding regions are all different; no evidence has been obtained for constant regions that would suggest DNA rearrangement of the sort seen in the immune system. The observations indicate that the diversity olfactory proteins are coded by a large number of distinct .:. ~ :,.. .
gene seguences.
.
Although it is unlikely from the data that DNA rearrange~ent is responsible for the generation of diversity among the putative odorant receptors, it remains possible that DNA
rearrangements may be involved in the regulation of expression of this gene family. If each olfactory neuron expresses only one or a small number of genes, then a transcriptional control mechanism must be operative to choose which of the more than one hundred genes within the family will be expressed in a given ne~ron. ~ene conversion from one of multiple silent loci into a single active locus, as observed for the trypanosome-variable surf ace . .
: '' .:
. .
,, .

W092/17~8~ PCT/US92tO2741 2 ~ 7 glycoproteins (32), provides one attractive model. The yene conversion event could be stochastic, such that a given neuron could randomly express any one of several hundred receptor genes, or regulated (perhaps by positional information), such that a given neuron could only express one or a small number of predetermined receptor types.
Alternatively, it is possible that positional information in the olfactory epithelium controls the expression of the family of olfactory receptors by more classical mechanisms that do not involve DNA rearrangement. What ever mechanisms will regulate the expression of receptor genes within this large, multigene family, these mechanisms must accommodate the requirement that ol~actory neurons are regenerated every 30~60 days (8) and therefore the expression of the entire repertoire of receptors must be accomplished many times duri~g the life of an organism.

Receptor Diversity and_the Central Processina of Olfactory Information The results suggest the existence of a large family of distinct odorant receptors. Individual members of this receptor family are likely to be expressed by only a sm~ll set of the total number of olfactory neurons. The primary sensory ~eurons within th~ olfactory epithelium will therefore exhibit significant diversity at the level of receptor expression. The question then emerges as to whether neurons expressing the same receptoss are localized in the olfactory epithelium. Does the olfactory system employ a topographic map to discriminate among the numerous odorant? The spatial organization of distinct classes of olfactory sensory neurons, as defined by receptor expression, can now be determined by using the procedures of in situ hybridization and i~munohistochemistry with probes ~ 35 specific for the individual receptor subtypes. This ~;
': ~ , ~'' ::

WO 92/1758~ 2 1 ~ 6 ~ L~ 7 PCT/US92/02741 ~41--information should help to distinguish between different models that have b0en proposed to explain the coding of diverse odorant stimuli ~33).

In one model, sensory neurons that express a given receptor and respond to a given odorant may be localized within defined positions within the olfactory epithelium. This topographic arrangement would al50 be reflec~ed in the projection of olfactory sensory axons into discrete regions (glomeruli) within the olfactory bulb. In this scheme, the central coding to permit the discrimination of discrete odorant would depend, in part, on the spatial segregation of different receptor populations. Attempts to discern the topographic localization of specific receptors at the level of the olfactory epithelium has led to conflicting results.
In some studies, electrophysiological recordings have -revealed differences in olfactory re~ponses to distinct -~
odorant in different reyions of the olfactory epithelium (34, 35). However, these experiments have been difficult to interpret since the differences in response across the epithelium are often small and are not observed in all studies (36).

A second model argues that sensory neurons expressing distinct odorant receptors are randomly distributed in the epithelium but that neurons responsive to a given odorant project to restricted regions within the olfactory bulb. In this instance, the discrimination of odors would be a consequence of the position of second order ne.urons in the olfactory bulb, but would be independent of the site of origin of the afferent signals within the epithelium.
Mapping of the topographic projections of olfactory neurons has been performed by extracellular recordings from different regions of the bulb (37, 38) and by 2-deoxyglucose ~ -autoradiography to map regional activity after exposure to :
,:''.', :'., .. ..
',', ', ' : ~ , WO92/1758~ 2 ~ PCT/US9~/02741 ~,,1 ., diffe.rent odorant (39). These studies suggest that spatially localized groups of bulbar neur~ns preferentially respond to different odorant. The existence of specific odorant receptors, randomly distributed through the olfactory epithelium, which converge on a common target within the olfactory bulb, would raise additional questions about the recognition mechanisms usecl to guide these distinct axonal subsets to their central targets.

Other sensory systems also spatially segregate afferent input from primary sensory neurons. The spatial segregation of information employed, for example, ~y the visual and somatosensory systems, is used to define the location of the stimulus within the external environment as well as to indicate the quality of the stimulus. In contrast, olfactory processing does not extract spatial features of the odorant stimulus. Relieved of the necessity to encode information about the spatial localization of the sensory stimulus, it is possible that the olfactory system of mammals uses the spatial segregation of sensory input ~olely to enco~e the identity of the stimulus itself. The molecular identification of the genes likely to encode a large family of olfactory receptors should provide initial insights into the underlying logic of olfactory processing in the mammalian nervous system.

.
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W092/1758~ 2 ~ PCT/US92/~2741 REFBRENCE~

1. Rushton, W.A.H. (1965). A Foveal pigment i~ the -deuteranope. J. Physiol. 176, 24-37.
: . .
2. Rushton, W.A.H. (1955~. Foveal photopigments in normal and colour-blind. J. Physiol. 129, 41-42.
.. .. . .
3. Wald, G., Brown, P.K., and Smith, P.H. (1955). `
Iodopsin. J. Gen. Physiol. 38~ 623-681.
.
4. Nathans, J., Thomas, D., and Hogness, D.S. (1986).
Molecular genetics of human color vision: Th~ genes encoding blue, green and red pigments. Science 232, 193-20~.

5. Lancet, D. (1986). Vertebrate olfactory reception.
Ann. Rev. Neurosci. 9, 329-355.

6. Reed, R.R. (1990). How does the nose know?
Minireview. Cell 60, 1-2.

7. Moulton, D.G., and Beidler, L.M. (1967). Structure and function in the peripheral olfactory system. Physiol.
Rev. 47, 1-52.
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8. Graziadei, P.P.C., and Monti Graziadei, G.A. (1979).
Neurogenesis and neuron regeneration in the ol~actory system of mammals. I. ~orphological aspects of differentiation and structural organization of the olfactory sensory neurons. J. Neurocytol. 8i, 1-18.

9. Bronshtein, A.A., and Minor, A.V. (1977). Regeneration of olfactor~ flagella and restoration of the . . :,:

. ;. - :

~'092/1758' PCT/US92/02741 .
3 ~

electroolfactogram following application of triton X-~oo to the olfactory mucosa of frog~s. Tsitologiia 19, 33-39.

10. Rhein, L.D., and Cagan, R.H. (1980). Biochemical studies ~f olfaction: Isolation, characterization, and odorant binding activity of cilia *rcm rainbow trout olfactory ros~ttes. Proc. Natl. Acad. Sci. USA 77, 4412-4416.

11. Rhein, L.D., and Cagan, R H. ~1983). Biochemical studies of olfaction: Binding specificity of odorant to a cilia preparation f'rom rainbow trout olfactory rosettes. J. Neurochem. 41, 569-577.

12. Pace, ~., Hans~i, E., Salomon, Y., and Lancet, D.
(1985). Odorant-sensitive adenylate cyclase may mediate olfactory reception. Nature 316, 25S-258.

13. Sklar, P.B., Anholt, R.R.H., and Snyder, S.~. (19 6).
The odorant-sensitive adenylate cycla~e of olfactory receptor cells: Di~ferential stimulation by distinct `~`
classes of odorant. J. Biol. Chem. 261, 15538-15543.

14. Breer, H., Boekhoff, I., and Tar2ilus, E. (1990).
Rapid kinetics of second messenger formation in olfactory transduction. Nature 345, 65-68. -~

~; 15. Boekhoff, I., Tareilus, E., Strotmann, J., a~d Breer, H. (1990). Rapid activation of alternatlve second messenger pathways in olfactory cilia Prom rats by ~ different odorant. EMB0 J. g, 2453 -~

; ~ 16. Jones, D.T., and Raed, R.R. (1989). GD1~: an olfactory ;;
neuron specific-G-protein~lnvolved in odorant signal , .

W092/17;8~ ~ 1 0 6 ~ 4 7 PCT/US92/02741 -45~
transduction. Science 244, 790-795.

17. Nakamura, T. and Gold G. (1987). A cyclic nucleotide-gatad conductance in olfactory receptor cilia. Nature 325, 442-444.

18. Dhallan, R.S., Yau, K.-W., Schrader, R.A., and Reed, R.R. (l99o)- Primary structurle and functional expression of a cyclic nucleotide-activated channel from olfactory neurons. Nature 347, 184 187.

19. O'Dowd, B.F., Felkowitz, R.J., and Caron, M.G. (1989).
Structure of the adrenergic and related receptors.
Ann. Rev. Neurosci. 1~, 67-83.
20. Strader, C.D., Sigal, I.S., and Dixon, R.F. (1989).
Structural basis of ~ adrenergic receptor function FASEB J. 3, 1825-1832. ~
, '. : ' ' .
21. O'Down, B.F., Hnatowich, M., Caron, M.G., Lefkowitz, R.J., and Bouvier, M. (1989) Palmitoylation of the human ~2-adrenergic receptor. Mutation of C~S34l in the carboxyl tail leads to an uncoupled nonpalymitoylated form of the receptor. J. Biol. Chem.
264, 7564 7569.

22. ~obilka, B.KI., Kobilka, T.S., Daniel9 K., Regan, J.W., Caron, M.G., and Lefkowitz, R.J. (1~88) Chimeric ~2~B2-ldrenergic receptors: delineation of domains involved in ef~ector coupling and ligand binding speci~icity.
Science 240, 1310-1316.

23. Hamm, H E.,~Deretic, D., Arendt, A., Hargrove, P.A., Koenig, B., and Hofmann, X.P. (1988) Site of ~ protein binding to rhodopsin mapping with synthetic peptides to W O 92/1758' PC~r/US92/02741 S~ ~ ~3 ~ 7 ~

the ~ subunit. Science ?41, 832-835. (1988).

24. Bouvier, M.W., ~ausdorff, A., De Blasi, A., O'Dowd, B.F., Kobilka, B.K., Caron, M.G., and Lefkowitz, R.J.
(1988). Removal of phosphorylation sites frQm the ~-adrenergic receptor delays the onset of agonist-promoted desensitization. Nature (London) 333, 370-373.

25. Amoore, J.E. (1982). Odor Theory and Odor Classification, In Fragrance Chemistry (New York:
Academic Press, Inc.), pp. 27-73.

26. Maeda, N., and Smithies, 0. (1986). The evolution of multigene families: Human haptoglobin genes. Ann.
Rev. Genet. 20, 81-108.

27. Tonegawa, S. ~1983). Somatic generation of antibody diversity. Nature 302, 575-581.
28. Hood, L., Kro~enberg, M., and Hunkapiller, T. (19&5).
T cell antigen receptors and the i~munoglobulin supergene family. Cell 40, 225-229 29. Baltimore, D. (1981). Gene conversion: Some implications for immunoglobulin genes. Minireview.
Cell 24, 592-594.
., .
30. Egel, R. (1981). Intergenic conversion and reiterated genes~ Nature 290, 191-192.
. ~, .
31. Flanagan, J.G., Le~ranc, M.-P., and Rabbitts, T.H.
(1984). Mechanisms of divergence and converg~nce of ~-~
the human immunoglobulin ~1 and ~2 constant region gene .
35 ~ ~ sequences. Cell 36, 681-6~38.

.
::

~O 92/1758~ 2 ~ pc~r/us92/o274 -~7-32. Van der Ploeg, L.H.T. (1991). Antigenic variation in Afri~an trypanosomes: genetic recomb.ination and transcriptional control of VSG genes. In Gene Rearrangement. (New York: Oxford Universi~y Pre~s~, pp 51-98.

33. Shepherd, G.M. (1985). Are there labeled lines in the olfactory pathway? In Taste, Ol~action and the Central Nervous System (New York: The Rockefeller Vniversity Press), pp. 307-321.

34. Mackay-Sim, A., Shaman, P., and Moulton, D.G. (1982).
Topo~raphic coding of olfactory quality: odorant-specific patterns of epithelial responsivity in the Salamander. J. Neurophysiol. 48, 584~596.

35. Thommesen, G., and Doving, K.B. (1977). Spatial distribution of the EOG in the rat. A variation with odour stimulation. Acta physiol. scand. 99, 270~280.
36. Sicard, G. (1985). Olfactory discrimination of structurally related molecules: Receptor cell responses to camphoraceous odorant. Brain Res. 3~6, 203-212.

. .
37. Thommesen, G. (1978). The spatial distribution of odour induced potentials in the olfact~ry bulb or char and trout (Salmonidae). Acta Physiolv Scand. 102, 205-217.
38. Doving, K.B., Selset, R., and Thommesen, G. (1980).
Olfactory sensitivity to bile acids in salmonid fishes.
Acta Physiol. Scand. 108, 123-131.

3~ 39. Stewart, W.B., Kauer, JvS., and Shepherd, G.M. (1979).
, : ~
-: ':, W092/17~8~ PCr/US92/02741 Functional organization of the rat olfactory bulb analyzed by the 2-deoxyglucose method. J. Comp.
Neurol. 185, 715-734.

40. Chirgwin, J.M., Przbyla, A.E., ~acDonald, R.J., and Rutter, W.J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. :~
Biochemistry 18, 5294-529g.

41. Maniatis, T., Fritsch, E., and Sambrook, J. (1982).
Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).
~:
42. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press).

43. Julius et al., (1988). Molecular Charatization of a .- Functional cDNA Encoding a Seritonin lc Receptor.
. ~ Science 24l, 558-5~4.

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: : .: ' , :, , .
:,,~ . :: '.:' 21~6~7 W O 92/17~X~ PCT/US92/02741 .
. -49-. S~QUENC~ LISTING
( 1 ) GENERAL INFQRMAT~ON~
(1) APPLICANT: Columbla Unlv~r~ley ln tho cley o~ N Y
The Tru2~tee~ o~ , "
tll) TITLE OF INV~NTION~ ODORAN~ ~C2PTORS ~ND US~S TH~R~oF
(lll) NUM8~R OF SEQU~NC~Ss 36 (lv) CO~R~SPOND~NC~ ADDR~SS~ :
(A~ ADD~ESS~I COOP~R ~ DUNHAN
~8) STRE~T~ 30 Roc~f~llor Pl~z (C) CI~Ys N~w Yor~
(D) STATt s N - w Yo~k : : .
~B) COUNTRYt U .S.A.
(r) ZIP~ 10112 (v) COMPUTER ~EADABL~ ~ORM:
(A) MRDIU~ TYP~ Floppy dlak (a) COMPUTERI IS~ PC comp~tlbl~ .
(C) OP~RAT~NG SYST2M2 PC-DOS/~S-DOS
(D) SOrTWAR~: Pat~ntIn R~ln~a- ~1.0, Vor-lon ~1.25 (vll) PRIO~ APPLICATION DAT~
tA) APPLICAT~OW NUM8E~ US 6al,880 ~8) FILINC DAT~s 05-APR-1991 (vlll) ~TTO~NEYtAG~NT ~N~ORH~TION:
(A) NA~R~ W~lt~, John P. .:
(8) A~CISSR~TION NUM~Rs 28,678 : ~.
(C1 A~F~2NCE/DOCXBT NU~B2R: 3as86 (lx) TEL~C0HMUN~CATION INFO~MATION~ .
(A) T~L~PHONCI ~212) 977-9550 . :
a) T~LR~AX~ (212~ 664-05~5 .. :
~Xt ~212~ 422523 C00P UI : :
. .
(2) INFO~MATION ~OX S~Q ID NOsl~
~l) SEQU2NC~ CHAR~CT~RISTIC~:
(A) LBNG~NI 9S~ b~-o ~alr~ :
~1~ ) TYPle ~ nu~: lo !.C ~c id ~C) S~A~ND~DNES~ ln~l~
~D) TOPO~4~Y~ llno~r (11) MOLECUL~ TYP~s cDNA
(111) HYPO~ETICA~: Y~S
(iv) AN~I-SENS~: NO
(vi) ORIGINAL SOURCE~
(A) ORGAN$5H~ r~t olfactory eplthellum (B) STRAINJ Srpagu~-Dawl0y r~t (P~ TXSSU~ TYP~t olfactory epithellu~
~vll) IMMrDIATE SOURCZ-:

SUBSrlTUTE SHEEr :: :

wo 92/] 7~8~ PCI /US92/027~11 (B) CLON~: F12 (xl) SEQUENCE DESCRIPT~OH: SEQ ID NO:l:
ATGGAATCAG GGAACAGCAC AAGAACATTT TCAAGrrrTT TTCTSCTTGG ATTTACAGAA 60 AACCCACAAC T~CACTTCCT CATrT~TGC~ CTATTCCTGT CCATGTACCT CGTAACAGTG 120 CTTGGGAACC TGCTTATCAT TATGGCCATC ATC~CAC~GT CTCATTTGCA TAC~CCCATG lB0 TACTTTTTCC TTGCTAACCT ATCCTTTCTG GAC~TCTGTT TCACCTCCAC CACCATCCCA 240 AAGAT~TTGG TAAATATATA CACCCAGAGC AAGAGCATCA CCTATCAAGA CTCTATTAGC 300 CAGATGTGTG TCTSCTTGGT TTTCGCAGAA TTG5GC~ACT TTCTCCTGGC TGTGATGGCC 360 TATGAGCGAT ATGTGGCTAA CTGTCACCC~ CTGTGTTACA CAGTCATTGT GA~CCACCGG 420 CTCTGTATCC TGCTGCT~CT GCTGTCCTGG GTTATCAGCA TTTTCCATGC CTTCATAC~G 480 AATCTTC~AC CTGTTATC~T CGCACCCATT TCCTTCAGTG CCATCCTTTA C~CTTATTTC 660 AAGATAGTAT CC~CCATACA TTCTATCTCC ACACTTCAGC GCAACTACAA GGCATTTTCT 720 ~:
ACTTGTGCCT CTCACCTTTC CATTGTCTCC TTATTTTATA GTACAGGCCT C&CAGTGTAC 780 GTCAG~TCTG CTGTCCTCCA AACCTCACAT TCTGC~GCAA G~GCTTOGGT CATGTATACT 840 GCTCTGGAAA GACTGTTAGA AGGAAACTGT AAAcTacATc A~TGCACTGG ATGA 954 ` .
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENC~ CaARACTERISTICS:
(A) L~NGTH: I002 bAso p~ir~ :
t~ TYPE: nucloic ~cid (C) STRANDEDNgSS: ingl-tD) TOPOLOGY: lin~r ~ii) MOL~CULE TYP~: cDNA
(iii) HYPOTHETIC~L: YES
(iv) AN~I-SENSE: NO
~vi) ORIGINAL SOU~OE : .:
(A) ORGANISM: rat olf3~tory epith~lium ~. -(B) S~R~N: Srpagu~-Dawl~y rat (F) TISSUE TYP~: olfactory eplthelium (vii) IMMEDIA~E SOURCE: -(B) CLONE: F3 (xi~ SEQUENCE D~SCRIPTION: SEQ ID NO:2: ;~.
; ,.
.. .
., SVBSTITUTE SHEE~T
~ .

WO 92/17S8' ~ P~/US92/02741 i . ~ . . .
-5i- ;. ..
ATGGACTCAA GC~ACAGGAC ~AGAGTTTCA GAATTTCTTC TTCTTGGATT TGTAGAAAAC 60 . :.

GGAAACATAT CCATTA~TGT GGCTATCATT TCAGATCCCT GTCTGCACAC CCCCATGTAT 180 ATGTTACTGA AC~TCCAGAC CCAAA~CAAT GTCATCACCT ATCCAGGATG C~TTACCCAC 300 ATATACTTTT TCTTGCTCTT TGTAGAATTG GACA~CTTCT TGCTGACTAT CAT~GCCTAT 360 GACCGTTACG TAGCCATCTG TCACCCCATG CACTA QCAG TTATCA~GAA CTACAAGCTC 420 TGTGGATTTC TGGTTCTCGT ATCTTGGATT GTAACTGT~C TGCATGCCTT GTTTCAAAGC 480 TTCATGATGT TGGCGCTCCC CI~CTGCACA CATCTGGAAA TCCCACACTA CTTCTGTGAA 540 .;

ATAGTGTCCT CCATATGTGC TATATCGTCA GTTCATCGGA AGTACAAAGC ATTCTCCACC 720 .
TGTGC~TC~C ACC~TTCAGT CGTGTCTTTA TTTTACTGCA CAGGACTAGG AGTGTACCTC 780 GTTACCCC~A TGGTCAACCC TTTTATCTAT AGTCTTAGGA ATAAAGATGT TAAGAGTGTT 900 CTGAAAAAAA CTCTTTGTGA GGAAGTTATA AGGAG$CCAC CTTCCCTACT TCATTTCTTC 960 CTAGTGTTAT GTCA$CTCCC TTGTTTTATT TTTTGTTATT AA 1002 (2) INFORMATION FOR SEQ ID NO:3:
(1) SEQUENC~ CHARACTERISTICS:
~A) LBNCTH: 942 bnoe pair~
(~ TYP~: nuclolc acld ~C) STRAND~DNESS: lngle (D) ~OPOLCGY: linear (LL) MOLEC~LF TYPE: cDNA
(LLl) HYPCTHETICAL: YES
(i~) ANTI-SENSE: N~
(vi) ORIGINAL SOURCE:
~A) ORGANIS~: rat olfactory epith~llum ~) STRAIN: Srpague-Dawley rnt ~F) TISSUE TYPE: olfactory epithelLu~
~vi1) IMMEDIATE 50URCE:

~B) CLONE: F5 ~xL) SEQ~ENCE DESCRIPTION: SEQ ID N0:3: .
ATGAGCAGC~ CC~ACCAGTC CAGTGTCACC GAGTTCCTCC TCCTG&GACT CTCCAGCCAG 60 :
, .
CCC~CAGCAGC AGCAGCTCCT CTTCCTGCTC TTCCTCATCA TGTACCTGGC CACTGTCCTG 120 ;:~

: ~
: .:. . :
SUBSrITUTE SHFET
~ ' ':

WO 92/1758' PCI/US92/02741 2 ~ 52- ~-CGA~ACCTGC TCATCATCCT GGCTATTGGC ACAGACTCCC GCCTGCACAC CCCCATGTAC 180 TTCTTCCTCA GTAACCTGTC CTTTGTGGAT GTCTGCTTCT CCTCTACCAC TGTCCCTAAA 2~0 GTTCTGGCCA ACCATATACT TGGGAGTCAG GCCATTTCCT TCTCTG&GTG TCTCACCCAG 300 TGTCTCCTGC T~GTTGTGGG CTCATG~GTT GTAGCCAACA TGAATTGTCT GTTGCACATA 480 CTGCTCATGG CTC~ACTCTC CTTCTGTGCA GACAACATGA TCCCCC~CTT CTTCTGTSAT 540 W AACTCCCC TCCTGAAACT CTCCTCCTCA GACACAC~TC TCAATGAGCT GATGATTCTT 600 ATCACCTGTC CTGTCCTCAG AGTCTCATCC CCCAGGCGAG GATGGA~ATC CTTCTCCACC 720 (2) INFORMATION FOR SEQ ID NO:4:
(i~ SEQUENCE~ C~EARACT~RISTICS:
(A) LENGT~: g36 b~0e p~Lr~
(B) TYPE: nucl~Lc ~cld (C) ST~ANDEDNESS: ~lngl~
(D) TOPOLCGY: linear (ii) MOLECULE TYPE: cDNA
(i~i) ~YROTHETYC~L: Y~S
(i~) ANTI-SENSE: NO .:
(vl) OR~CINAL SOURC~:
(~) ORCANISM: rat olfactory 0plth~11um (B) STRAIN: Srp~gu~-Dawley rat (~) TISSU~ TYPE: olgactory opithel~um (vii) IMMEDIATE SOURCE: .
(~) CLON~: F6 (xi) SEQUEN OE D~SCRIPTION: SEQ ID NO:4: ~ ~-ATGGCTTGGA GTACTGGCCA GAACCTGTCC ACACCA&GAC CATTCATCTT GCTG&CCTTC 60 CCAGGGCC~A CGAGCATGCG CATTGGGCTC TTCCT~CTTT TCCTGGTCAT GTATCTGCTT 120 :~ ~
ACGGT~GrTG GAAACCTAGC CATCATC$CC CTGGTAGGTG CCCACAGATG CCTACAGACA 180 ;~ :
CCCATGTACT TCTTCCTCTG C~ACCTCTCC TTCCTGGAGA TCT W TTCAC CACAGCCTCC 240 GTACCCAAGA CCCTOGCCAC ATTTGCGCCT CGGGGTGGAG TCATTTCCTT GCCTGGCTGT 300 -~
:

.. -:.......
SUBSTITUTE SHF:ET
, WO9Z/1758`` ~ q 7 P~/US92/02741 , . .:

GCCACAC~GA TCTACTTTCT CTTTTCTTTG CGCTGTACCG AGTAC~CCT CCTGGCTCTG 360 ATGGCTTATG ~CCGCT~CCT GGCCATCTGC CTGCCACTGC GCTATGGT~; CATCATGACT 420 CCTGGGCTGG CGATGCGGTT GGCCCTGCGA TCCTGGCTGT GTGCGTTTTC TCCAATC~CA 4~0 GTTCCTGCTA CCCTCATTGC CCGCCTCTCT TTCTGTGGCr CACGT~TC~T CAACC~CTTC 540 TTCTGTGACA TTTCGCCCTC GATAGTGCTT TCCTGC~CCC ACACGCAGc'r CG~CGAACIG 600 GTGTCCTTTG CCATTGCCTT CTGTGTTATT C'rG&GCTCGT GTGGTATCAC ACTAGTCTCC 660 TATGCTTACA TCATCACTAC CATCATCAAG ATTCCCTCTG CCCGGGGCCG GcAccccGcc 720 TTC$CAACCT GCTCATCCCA TCTCACTGTG GTGcTGArTT GGTATCGCTC CACCATCTTC ~80 TTGCATGT~A GGACCTCG~T AGAGAGCTCC TTGGACCTCA CCAAAGCTAT CACAG~GCTC 840 ~2) INFORHATION FOR SEQ ID NO:S:
~i) SEQUENCE CHARACTERTSTICS:
~A) LENGTH: 939 b~se pair ft ( B ) TYPE s nuc 10 ~ c ~cld (C) STRANDEDN~SS t ~lngl~
~D) TOPOLCCYs llnear (ii) MOLLCUL~ TYP~: cDNA
) HYPOTHE~CAL: YES
(1~) ANTI-SENSE: NO
(vi3 ORIGINAL SOURCE:
(A) ORGANIS~: r~t olf2ctory ~pith~lium ( a ) STRAINs S~p~gu~-DawlQy rAt (F) TISSUE TYP~: olf~ctory eplthelium . .
(vil) IM~DIATX SOURCE:
(8) CLON~: I14 (xi) SEQUEN OE DESCRIPTION: SEQ ID NO:5:
ATGACTGGAA ATAACCAAAC TTTGATCT~G GAGTTCCTCC TCCTGGGTCT GCCCATCCCA 60 TCAGAGTATC ATCTCCTGTT CTATGCCCTG TTCCTGGCCA TGTACCTCAC CATCATCC~G 120 ~.
GGAAACC~GC TAATCATTGT CCTTGTTCGA CTGGACTCTC ATCTCCACAT GCCCATGSAC 180 CTGTACTTCT TTATGGTT$T TGJGAGATATG GAGAGCTTCC TTCT$GTGGT CATGGCCTAT 360 GACCGCTATG TGGCCATTTG CTTTCCT~TG CGTTACACCA CCATCATGAC CACCAACTTC 420 . ~
SUBSTITVTE SHEEI

W ~ 92/1758~ PCT/US92/027~1 CTACTCAT;rG CTAGATTGTC TTTTTGTGAG AAGAATGTGA TTCTTCACrT TTTCTCIGAC 540 ATCTTGGGTG GACTCATCAT TATTATCCCA TTCCTATTAA TTGTTAT~TC CTATGTTAGA 660 ATTTTCTTCT CCATTTTGAA GTTTCCATCT ~TTCAGGACA TCTACAAGGT ATTCTCAACC 720 TGTGGTTCCC ATCTCTCTGT GGTGACCTTG TrTTATGGGA CAATTTTTGG TATCTACTTA 780 TGTCCATCAG GTAATAATTC TACTCTGAAG GAGA~TGCC~ TGGCTATGAT GTACACAGTC 840 GTGACTCCCA TGCTCAATCC CTTC~TCTAC AGCCTGAGGA ACAGAGACAT G~AAAGGGCC 900 (2) INFORMATION POR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 945 b~se p~ir~
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: lin~r (ii) MOLECULE TYPE: cDNA
(i~i) HYPOTHETICAL: YES : :
(lv) ANTI-SENSE: NO
(~i) ORIGIN~L SOURCE:
(A) O~GANISM: rat olfactory ~piehalium :.
(8) STRA~N: Srpagu~-D~wley r~t (F) TISSUE TYPE: olf~ctory epithelium (vii) IMMEDIATE SOURCE: .
(B) CLONE: I15 .~
' , (xl) SEQUENCE DESCRIPTION: S~Q ID NOt6:
ATGACAGAAG AGAACCAAAC ~CTGATCTCC CAGTTCCTTC TCCT$~TCCT CCCCATCCCC 60 TCAGAGCACC AGCACGTGT~ CTACGCCCT~ rTCCTCTCCA TGTACCTCAC CACTGTCCTG 120 GGGAACCTCA TCATCATCAT CCTCATTCAC CTGCACTCCC ATCTCCACAC ACCCATGTAC lB0 TTGTTCCAGA ACATGCAGAG CCAAGT$CCA TCCATCCCCT TTGCAGGCTG CCTGACACAA 300 ::~

5V BSTITl 1TE SH EET
, . : .: .

W 0 92/1758' ~ PC~r/US92/~2741 : 55-CTCA~GGGAG GGCTTGTTAT TGTCATTCCA TTTGTCCTCA TCAIIGTATC TTATGCACGA 660 TGCGGCTCCC ATCTGTCTGT CG~GTCACTG TTCTATGGGA CAATCATTGG TCTCTACTTA 780 TOTCCGTCAG CTAAT~ACTC T~CTCTGAAG GAGACTGTCA TGGCCATGAT GTACACAGTG 840 (2) I~FORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTE~ISTICS:
(A) LENGTH: 933 b~ p~ir~
(8) TYP~: nucl~lc ~cld ~C) STRANDEDNESS: aingl0 (D) TOPOLOGY: lln~ar (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: YES
(iv) ~NT}-S~NSE: NO
(v1) ORIGINAL SOURCE:
(A) ORGANISM: r~t olfactory eplth~lLum (8) STRAIN: Srp~gue-D~wley r~t (F) TISSV~ TYPE: olfactory ~pithelium EDIATE SOURCE:
(B) CLONE: I3 (~i) SEQUENCE D~SCRIPTION: SEQ ID NO:7:
.ATGAACAATC AAACrISCAT CACCCAATTC CTTCTCCICC GACTCCCCAT CCCTGAAG~A 60 CATCAGCACC TCTTCTATGC CTTGTTCCTG CTCATCTACC TCACCAC QT CTTGGCA~AC 120 T$GCTAATCA TTGTACTTGT TCAAC~GGAC TCC Q CCTCC ACACACCTAT GTAT$TGTIT 180 CTCAGCAATT TGTC15TCTC TGATCTATG~ rTTTCC~CTG TCACAATGCC CAAGCTGC~G 240 TTCTTTATGG TTTTT&GAGA TATGGAGAGT TTCCTTCTTG TCGCCATG~C CTATGACCGC 360 TATGTGGCCA TC~GCT$CCC TCTGCATTAC ACCAGCATCA TGAGCCCCAA GCTCTGTACT 420 GCAGCAAGAT TGTCTTTTTG TGAGAACAAT GTGGTCCTCA ACTTCTTCTC TGACCTAT~T 540 SIJB5TITUTE S~EE:T
~ .

WO 92/175~:` 2 ~ ~3 ~ ~ ~ i PCT/US92/02741 ~.
TCCCATCTCT CTGTAGTATC ACTGTTCTAT GGGACAATTA TTGGTCTCTA CTTAT~TCCA 780 . .
GCAGGTAATA ATTCC~CTGT AAAAGAGATG GTCATCGCCA TGATGTACAC TGTGGTGACC 840 CCCATGCTGA ATCCCTTCAT CTACAGCCTA AGGAATAGAG ATA~GAAGAG GGCCCTAATA 900 ~2) INPORMATION FO~ SEQ ID NO:8:
(1) SEQU2NC2 C~ARACTeRISTICS:
(A) L~NGTH~ 984 ba~ palr~
IB) TYPE: nucl~Lc ~cld (C) STRANDEDNESS: ~ingl0 (D) TOPO~OGY: lin~ar ~ HOLECUL~ IYP~: cDNA
(ili) HYPOT~ETIC~L: YES
(iv) ANTI -SE~SE: NO ..
(vi) OR~GINAL SOURCE: -(A) ORCANISM: r~t olfactory ~pith~llum (B) STRAIN: Srpague-Dawl~y rat .;
(F) TISSUE TYPE: olfactory eplthellum (vll) IMMEDIAT~ SOURCE: ;
~) CLONE: I7 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
AT~GAGCGAA CGAACCACAG TGGGAGAGTG AGTGAATTTG TaTTGCTGGG TTTCCQ GCT 60 ACrGAAAACA ~GCTCATCAT TATAGCAATT AGGAACCACC CAACCCTCCA CAAACCCATC 180 ~ -TA m T~TCT ~GGCTAATAT GTCATTTCTG GAGATT~GGT ATGTCAC5GT T~CGATTCCT 240 ~, ~
AAGATGCTCG CTGGCTTCAT TGGTTCCAAC GAGAACCATG GACAGCTGAT CTCCrTTCAG 300 : :
GCATGCA~CA CACAACTCTA CTTTTSCCTC GGCrTGGGTT GCACAGAGTG TGTCCITCTT 360 GCTGTGATGG CCTATGACCC CTATGTGGCT ATCTGTCATC CACTCCACTA CCCCGTCAT~ 420 TCCATGGTTA ~GTTTTCCT TATTTCTCGC CTGTCTTACT GTGGCCCCAA CACCATCAAC 54Q ~: :
CACTTTTTC~ GTGATGTGTC TCCATTGCTC AACCTGTCAT GCACTGACAT GTCCACAGCA sbo AAAGCCTTTT CAACCTG~GC CTCCCACCTC ACTGTTGTGA TCATCTTCTA TGCAGCCAGT 780 ATTTTCATCT ATGCCAGGCC TAAGGCACTC TCAGCTTTTC ACACC~ACAA GCTGGTCTCT 840 ~. .-SIJBSTITLlTE SH~:~T ::
... .. .
.

WO 92/17~ PCT/US~2/02741 `' '.

GATGTCAAAA GAGCGCTACG TCGCAC~CTG CACCTGGCCC AGGACCAGGA GcccAATAcc 960 AACA~AGGC~ GCAAAATrCG ITAG 9~4 (2) INFORMATION POR SEQ ID NO:9:
(1) SEQU~NCE CHAPACTERISTICS:
(A) LENGrHI 939 baft~ pair~
~B) TYP~ n~cl~lc acid ~C) ST~AND~DNES3t ~lngl~
~D) TOPOLOGY: llnear ~ll) MOLECULE TYPE: cDNA
YPO~ETIC~L: YZS
~iv) ANTI-SENS~: NO
(vl) ORIGINAL SOURCR:
~A) ORGANIS~: rat ol~actory ~pith~lLum (B) STRAIN: Srp~gu~-Dawley rat (F) TISSUE TYPE: olf~ctory epith~lium ~i1) IMM~D~ATE SOURC2:
(B) CLON~: ~B
Ixl) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ATC~AC~ACA AAACTCTCA~ CACCCA m C CTCCTCCTGG GArrGCCCAT CCCCCCAGAG 60 CACCAGCAAC TGTTCISTGC CCTGTTCCTG ATCA~GTACC TCACCACC$T TC~ÇGGAA~C 120 CTGCTAArTC STCTCCrTGT ~C~ACTGGAG TCTCATCTCC ACACACCCAT GTACTTa m 180 C~CAGC~ACT TGTCCTTCTC TCATCTCTGC TTTTCCTCTC ~TACAATGCT GAAATTGCIG 2~0 CAAAATATAC AGAGCCAAGS ACC~TCSATA TCCTATGCAG GATGCCTGAC ACAGATAITC 300 TTCTT$TTGT TC m GGCTA CCT~GCGAA$ $TCCTSC55G SAGCCA$~GC CTA$GACCGC 360 rA$~TGGCCA TC~GC$SCCC TCTGC~TT~T ACCAACATCA TG~GCCATA~ GC$CTGTACT 420 $~TCTCCTGC $GGTATS$TC GATAATGACA TCATCTCATC CCATGATGCA CACCCTGCrT 4~30 CCAGCAAGAT T0TC5TS~TG TGAGAACAAT GTAC~CCTCA AC$TlTTCl~i $GACCTG m 540 GTT~TCC$AA AGT~GGCCTC CTCAGACACS TATC~$AATC AGT$G~TCA$ ACATATCASG 600 GGCG$GATCA TCATTGT$AT $CCAT$CCTG CTCAT~GSTA TATCCTATGC CAAGATCATC 660 $CCTCcArTC $TAAGGTTCC ATCTACTCAA AGCATTCACA AGGTCTTCTC CACTTGTGGT 720 TC$CATCTCT C$G$GGTGTC TC$GTTCTAC GGGACAATTA TTGGTC$CTA TTTA$GTCCA 780 TCAGGTGATA ~TTTTACTCT AAAGGGaiTcs GCCATGGCTA TGATGTACAC AG$CGTAACT 840 CCAATCCTGA ACCCGTTCAT CSACACCC$A AGAAACAGAG ACATG~AGCA CGCCCTAATA 900 AC~G$$ACCT CTAGCAAGAA AATC~CTCTGi CCATGGSAC 939 ~2) INFORM~TION FOR SEQ I9 NO:10:
':
SUBSTITUTE SHEFr hl .L U U (J
W O 92/1758~ PCT/~S92/0274 (l~ S~QUENCE CHARACTERISTICs:
(A) LENGT~: 945 b~8~3 palro (B) TYPE: nucl~lc ~ld (C) STR~NDEDNESS: ~lngl~ .
(D) TOPOLOGY: llnn~r (l~) MOLECULF TYP~: cDNA
(111) HY~OTXETIC~L~ YES
(iv) ANTI-SENSE: NO
~vl) ORIGINA~ SOURCE: :
(A) ORGANISM: rAt olfactory ~plth~llu~ .
~(B) STRAIN: Srp~gue-Dawl~y rat (F) TISSU~ TYPE: olf~ctory ~pith~llum (vii) IMMEDIAT~ SOU~C~:
(B) CLONE: I9 (xi) SEQUEN OE DESCRIPTION: SEQ ID NO:10:
ATGACTAGAA GAAACCAAAC TGCCATCTCT CAGTTCTTCC TTCTGGGCCT GCC~TTCCCC 60 ~:

G~GAACCTCA TCATCATCAT CCTCATTCTA CTGGACTCCC ATCTCCACAC ACCCATGTAC 180 , :.
TTC~TTCTCA GCAATT~ATC CTT~GCCGAC CTCTGTTTTT CCTCTGTCAC AAT~CCCAAG 240 TTGTTGCAGA ACATGCAGAG CC~ACTTCCA TCCATCCCCT ATGCAGGGTG CCTGGCAC~G 300 GACCGCTATG TG~CCATCTG CTTCCCCCTT CATTACATGA GCATCATGAG CCCCAAGCTC 420 TGTGTGAGTC TCGTGCTCCT GTCCTGGGTG CTGACT~CCT TCCATGCCAT GCTGCACACC 480 ~:
CTGCTCATGG CCAGATTGTC ATTCTGTGAG GACAGrGTG~ TCCCTCACTA TTTCTGTGAT 540 ~ :
ATGTCTACTC TGCTCAAAGT GGCTTGTTCT GACACCCATG ATAATGAATT AGCAATA m 6~0 ;~ :
ATC~TAG4GG GCCC~ATAGT TGTACTACCT TTCC~TCTQ TCATTCTTTC TTATGCAACA 660 .
ATTGTTTCCT CCATCTTCAA ~GTCCCTTCT TCTCAAAGCA TCCATAAAGC CTTCTCCACC 720 ..
TGTGGCTCCC ACCTGTCTGT GGTGTCACTG TTCTATGGGA CAGTCATTGG TCTCTACTTA 780 : .:
T~TCCTTCAG CTAATAACTC CACTGTGAAG GAGACTGTCA TGTCTTTGAT GTACACAATG 840 TTAGAAAAAA TAATGTGCAA AAAGCAAATT CCCTCCTTTC TATCA 945 .: :.
: (2) INFORHATION FOR SFQ ID NO~
~i) SEQUENCE CHARACTERISTICS: : :
(A) LENGT8: 645 baDe pairD
(B) TYPE: nucleic acid :
(C) STRANDEDNESS: aingle (D) TOPOLOGY: linear - ' ' '' '''' . ~

. ,~: .", .
SUBST!TUTE SHEET ~ ~
.
.
: .

WQ 92/l75~ 2 ~ 7 PCI/US92/0~741 _59_ ( 11 ) ~OL~CUI.E TYPB: cDNA
(111) HYPOT~EiTICAL: YES
~iv) ANTI-S~NSE: NO
( v 1 ) OR I G I NAL SOURCE s ~A) ORGANISH: homo~plen ~v11) I~ DIATF SOURCL:
~B) CLONE: H5 ~lx) FEATUREl ~ A ) NA~ EY: CDS
( B ) LOCATION t 1. . 645 (xl) ssQuEtacE DESCP~IPTIO}~: SEQ ID NO:ll:

Ile Cy~ Phe Val Ser Thr Thr Val Pro Ly~ Gln Leu Val A~n Il~ Gln ACA CAG AGC AGA CTC ATC ACC TAT GCA GAC TGC ATC ACC CAG ATGi TCC 96 Thr Gln Ser Arg Val Il~ Thr Tyr Ala A~p CYD Ilt~ Thr Cln Met Cy~

Phe Phe Ile Leu Phe Val V~l Lau A~p Ser Leu Leu Lau Thr Val Met GCC TAT GAC CGG T$T GTG GCC ~TC TGT CAC CCC C~rG CAC TAC ACA GTC 192 Al~ Tyr Asp Arg Phe V~l Ala Ile Cy~ Hil3 Pro Leu Hi~ Tyr Thr Val ATT ATC AGC TCC TCG C~C TGT GGA CrG Cl`G CTT C~G GTG TCC TGG ATC 240 Ile Met Ser Ser Trp Leu Cy~ Gly Leu Leu V~l Leu V~l Ser Trp Ilo Val Ser Ile Leu Tyr Ser Leu Leu Gln S~r Ile Met Ala Leu Gln Leu 85 90 95 . :
TCC ~TC TGT AC~ G~A C~Gi AAA ATC CCT C~A Tl~ TTC TGT GAA CTT AAT 336 Ssr Phe Cyfl Thr Glu LQu Lyo Ile Pro Gln Phe Ph~ Cyo Glu L~u Aen Gln Val Ile Hl~ Leu A1a CYB Ser A~p Thr Phe Ile A~n AfJp Het M/3t 11~ 120 125 ATG AAT TT~ ACA AGT GTG CTG CTG GGT GGG CGA TGC CTC GCT GCA ATA 432 Met Asn Phe Thr Ser V~l Leu Leu Gly Gly Gly Cy~ Leu A1~ Gly Ile Phe Tyr Xaa Tyr Phe Ly~ Ile Leu Cys Cy~ Ile Cy~ Ser Ile Ser Ser GCT CAG G~4 ATG AAT AAA GCA CTT TCC ACC IGT GCA TCT CAc CTC TC~ 525 Ala Gln Gly Met A~n Ly~ A1~ Leu Ser Thr Cy~ Ala Ser Hi~ Leu Ser ~65 170 175 SUBSTITUTE SHEET
:
:~ .

~'O 92/1758~ PC~r/~S92/07741 ~ 3 ~ 7 -60- ~
CTT GTC TCC TT~ TTT ~AT TGT ACA ~GC CT~ GCT G~G TAC CTT ACT TCT 576 Va1 VaI Ser L~U Ph~ TYr CY~ Thr Gly Val Gly V~l Tyr L~U S~r S~r A1a A1a Thr H1- A0n Ser Leu Ser Aan A1~ A1~ Sar V~1 H~t TYr ACT GTG GTC ~CC TCC ATG CTC 645 Thr Vn1 Va1 Thr Ser Met Lsu 210 ~15 (2) INFO~MATION ~OR SEQ ID NO:12:
(1) S~QUEN OE C~ARACTERISTICS:
(A) LENCTH~ 215 aminO aC1dr (S) TYPE: amlnO nC~d (D) TOPOLOGY: llnear .
(ii) MOLECULE TYP~: PrOteLn (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
I1e CY~ Ph~ Va1 Ser Thr Thr Va1 PrO LY~ G1n LeU Va1 A8n I1a G1n Thr Gln Ser Ar9 Va1 I1e Thr TYr A1a A~P CY~ I1e Thr G1n M~t Cya Phe Phe I1e LeU Phe Va1 Va1 LeU A~P Ser LeU LeU LeU Thr Va1 Met : . :.
35 40 45 . :~
A1a Tyr A~P Ar9 Ph~ Va1 A1a I1~ CY~ Hin PrO L~U HLS TYr Thr Va1 50 55 60 .
I1~ Met S~r S~r TrP LeU CY~ G1Y L8U LaU VA1 LeU Val S~r rrP I1e Va1 Ser Ile Leu Tyr Ser LeU Leu Gln Ser Ile M~t A1a L~U Gln Leu : `
85 90 95 .
Ser Phe Cy8 Thr Glu Leu LY~ Ile PrO Gln Phe Ph~ CY~ G1U LeU ADn 100 105 110 . .
Gln Val Ile His LeU A1a CYB Ser A~P Thr Phe Ile A~n A~p ~et Met Met A~ Phe Thr Ser Val Leu LeU Gly Gly G1Y CY~ Leu Ala G1Y I1e Phe TYr Xaa Tyr Ph~ LY8 Ile Leu Cy~ Cy~ Ile Cy~ Ser I1~ Ser Ser :: :
145 150 155 160 .
Ala Gln Gly Het ~n Lys Ala Leu Ser Thr Cy8 Ala Ser E~is Leu Ser I65 170 175 . ,-.
Val Val Ser LeU Phe TYr CY~ Thr G}y Val Gly Val TYr LeU Ser Ser 180 185 190 ~
Ala A1a Thr Hi~ A~n Ser LeU Ser Asn Ala Ala Ala Ser Val Met Tyr . ~;
195 200 205 .~
. ,.
,:
~ SUE~SrITUTE SffEET
....

'.,.
.. . :,, ,.,, .. , .. ,... " .. . :

W O 9~/1758~ 2 ~ 7 PCT/US92/02741 ~;- 61 Thr V~l Val Shr S~r Hat L~u (2) INFORMATION FOR SEQ ID NO:13:
(1) SEQU~NOEi CHARACTERISTICS:
(A) LENGTHI $40 ba~0 pslr~
(B) TYP~: nucl~lc hcld (C) ST~ANDEDN~SSI alngl~
(D) T0POLOGY~ l~near : :
(li) MOLECUL~ TYPBt cDNA
~li) HYPOTXRTICAL~ Y~S
(1~) ANTI-SENSB: NO
(vl) ORIGINAL SOU~CE:
(A) ORGANISM: rat olfactory epithelium B) STRAIN: Srpague-Dawlsy rat ~) TISSUE TYPF: olfactory epith~lium (vii) IMMRDIATE SOURCE:
(B) CLONE: Jl (lx) ~ATURE:
(A) NAMEI~EY: CDS
~B) LOCATION: 2.... 640 :

(xl) SBQUgNOE DESCRIPTION: S~Q ID NO:13:
C ATC TGC TlT ~CT TCT ~CT AGC ATC CCA AAG AT~ C~A GTG AAT ASA 46 Il~ Cyu Ph~ Thr Ser Als Ser I1~ Pro Lya Het Leu Val Aan CAG ACG AAG AAC AAG GTG ATC ACC TAT GAA GGC TGC ATC TCC CAA GT~ 94 Gln Thr Lya Aan LYB Val Ile Thr Tyr Glu Gly Cy3 Il~ Ser Gln Val TAC TTT TCA TAC TCT TTG GAC TrT TGG ACA ACT TTC T~C TCG ACT GT~ 142 Tyr Phe S~r Tyr Ser Leu Glu Pho Trp Thr Thr Ph~ Ph~ S~r Thr Val 3S 40 45 ~:
A~C GCC TAT GAC CGA TAT GTC CCC ATC ~CT CAC CCA TCT NAC TAC ~CA 190 M~t Ala Tyr h~p Arg Tyr Vnl Ala Ile Cy~ Hi~ Pro S~r Xaa Ty~ Thr Gly ~is Hi~ Glu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa~ Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xna Xaa Xaa Xaa Xai~ Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XAa xad Xa~ Xaa Xaa Xaa Xai~ X~a Xa~ Xaa Xaa Xaa 100 105 110 ..

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa SUBSTlTllTE SHEET

: ,, W O 9~/1758~ PCT/US92/02741 3 ~ 7 ~ ~

NNN N~N NNN NNN NNN N~IN NNN NNN NNN NNN N~ NNN NNN NNN NNN HTT 430 Xaa Xaa Xaa XaA Xaa Xaa Xaa Xaa Xaa Xaa X~a Xa~ Xna Xail Xaa Xaa . .
~30 135 140 : .
TAT TCT TAC TCT AAG ~TA GTT TCC TCC ATA CGA GAA ATC TC~ TCA TCA 478 Tyr Sor Iyr Ser Ly~ Val Sar S~r Il~ Arg Clu Ilo Sor Sor Sor 145 lSO 155 CAG GGA AAG TAC AAG NNA TTC TCC ACC TGT GCA TCC C~C CTC TCA GTT 526 Gln Gly Ly~ Tyr Lyu X~a Ph~ Ser Thr Cys Ala S~r Hl~ L~l S~r Val GTT TCA TTA TTC TAT ~CT ~CA CTT T~G GGT GTG TAC CTT AGT TC~ TCT 5 74 Val S~r Leu Phe Tyr S~r Thr Leu L~u Gly Yal Tyr Luu S~r S~r Ser TTT ACC CAA AAC TCA CAC TCA ACT GCA CGG GCA TC~ GTT ATG TAC AGT 622 Ph~ Thr Gln Asn S~r Hi~ S~r Thr Ala Arg Ala Ser Val M~t Tyr S~r ::
195 200 205 . ~ .

Val Val Thr Pro M~t Leu ~10 ' '' (2) 1NFO~MATION FOR SEQ ID NO:14: -(i~ SEQUENCE CHARACTERISTICS:
(A) L~NGT~: 213 amino acids ~B) TYPE: amino acid (D) TOPOLOGY: linear ..
(li) MOLECUL~ ~YP~: protein ~.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Il~ Cy~ Ph~ Thr S~r Ala Sar Ile Pro Lya ~Sat I.eu Val A~n Ile ~:;ln : .

Thr Ly~ Aan Ly8 Val Il~ Thr Tyr Glu G}y Cy~ S~r Gln Val Tyr ~.

Phe Ser Tyr Ser Leu Glu Phe Trp Thr Thr Ph~ Ph~ Ser Thr Val H0t Ala Tyr A3p Arg Tyr Val Ala Il~ Cy~ ~liB Pro Ser Xaa Tyr Thr Cly Hi~ Hi~ Glu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa ~5 70 75 80 ~ .
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xas Xaa Xa~ Xaa Xaa Xaa ~ ~
as go 95 , ,~, Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 ,;
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 12S .

,.

SUBSTITUTE SHEIET ~:
: ' , .
': ' U'O 92/1758~ 2 1~ 6 8 ~ PCT/US92/02741 Xa~ Xaa Xa-l X~a Xa~ X~a Xa~ Xa~ X~a Xaa Xaa X~ Xaa Xaa Xn~ Tyr S~r Tyr S~r LyY Il~ val SQr S0r Ile Arg Glu Ile S~r S~r S~r Gln 145 l50 l5~ 160 Gly LYJ Tyr Ly~ X~a Phs Ser Thr Cy8 Al~ S~r ffl~ L~u Si~r V~l Val 165 l~0 175 S~r Lau Phe Tyr S~r Thr Leu Leu Gly Val Tyr L~u S~r Sler S~r Ph-180 185 ~go Thr Gln A~n Ser Hl~ S~r Thr A1A Arg Al~ sar V~ t Tyr S~r V~l Val Thr Pro M~t Leu210 (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQU~NCE C~ARACT~RISTICS:
(A) LENGTH: 636 bas~ pair~
(B) TYPE: nucleic acid (C) ST~ANDEDNFSS: ~insle (D) TOPOLCGY: llnQar ~li) MOLECULL TYP~: prot~ln ~Lii) HYPOTs~TlCAL: YES
(iv) ANTI-SENSL: NO
(vi) ORIG~NAL SOURCE: , ~A) ORGANISM: rat olfactory epith~lium (B) STR~lN: arpagu~-D~wley rat (F) TISSU~ TYPE: olfa~tory epithelium (vii) I~WÆDIAT~ SOVRC~:
(B) CLONE: J2 ~lx) F~ATUR~:
: (A) NA~StXEY: CDS
~E~) LC)CASIONs 1..636 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l5:
ACC TCC ACC ACC ATC CCA A~C ATG CTG GT~ AAT ATA CAC ACC CAG AGC 48 Thr Ser Thr Thr Il~ Pr~ Ly~ H~t Leu V~l Aan Ilo ~i- Thr Gln S~r AA~ AC~ ATC ACC TAT GAA GAC TGT ATT TCC CAG ATG TrT G~A C~C TTG 96 A~n ~hr Il~ Thr Tyr Glu Af~p Cy~ I}0 S~r Gln Met ~he V~l Leu L~u 20 25 : 30 GTT STS GCA GAA CTG GAC AAC T~ CTC CTO CCT GTG AIC GCC TAT GAT 1~4 : Val Ph~ Gly Glu L~u A~p A~n Ph~ L~u L~u Al~ Val M~t Al~ Tyr Asp : 35 40 4~
CGA TAT GTG GCT ATC TGT CAC CCA CTG TAT TAC ACA GTC ATT GTG AAC lY2 Arg Tyr Val Ala Ile Cy~ H55 Pro Leu Ty y 60 .:
' ' ''. .
,: ~ : ..... . .

SUBSrlTWTE Sl lEEr , W O 9~/1758~ ~CT/~S92/02741 2 ~ 8 ~ 7 -64- ~, C~C CGA CTC TGT ATC CTG CIG CTT CTG CTG TCC TGG GTT GTC AGC ATT 240 HL~ Arg Leu Cy- Ilo L~u L~u L~u L~u L~u Ser Trp VA1 V~1 S~r l1D
65 70 75 ~0 TTA CAT CCC TTC TTA CAG ~GC TSA ATT GTA CT~ CAC TrG ACC TTC TGT 2B8 L~U H1D Ala P~o L~u Gln S-r L~u Ile Val L~u Cln L4u Thr Ph~ Cy~ :

GCA GAT GTG AAA ATC ccr c~c TTC TTC TGT GAG CTC AAT CAG CTG TCC 336 :
Gly A~p Val Lyo Il~ Pro Hl~ Phe Ph~ Cy~ Glu Leu Aan Gln L4u S~r 100 10~ 110 CAA CTC ACA TGT T Q GAC AAC m CCA AGT CAC CTC AC~ ATG CAT CTT 38d Gln L~u ~hr Cy- Ssr ABP A~n Phe Pro S~r Hla L~u Thr H~t Hi~ Leu GTA CCT GTT ATA TTT GCA GCT ATT ~CC CTC AGT CGT ATC Cl"r TAC TCT 432 Val Pro Val Ils Phe Ala Ala lle Ser Leu S0r Gly Il0 Lbu Tyr Ser Tyr Phe Ly~ Ile Val Ser Ser Ile Arg Ser Mea S~r Ser Val Gln Gly ::

Ly~ Tyr Ly~ Al~ Phe Ser Thr Cy~ Ala S~r Hi6 Leu Ser Ile Val S~r TTA TTT TAT AGT ACA GGC CTC GGG GTG TAC GTC ~GT TCT GCT CTG ATC 576 ~;
Leu Phe Syr Shr Thr Gly L~u Gly Vnl Tyr Val Ser S~r Ala V~
180 185 190 .:;~
CG~ AGC TCA CAC rcc TCT GCA AGT GCT TCG GTC ATG TAT A~S GTG GSC 624 Arg S~r Ser Hia Ser Ser Ala Ser Ala Ser Val Met Tyr Thr Val Val 195 200 205 :
ACC CCC ATC S$G 636 Thr Pro M~t Leu (2) INFORMATION FOR SEQ ID NO:16:
(i~ SEQUENCE CHARAC~ERISTICS:
(A) LENCTH: 212 ~mi~o ~eida B) TYPE: amino a~ld (D) TO~O~D5Y: linoar ~. :
(ll) MOL~CULL TYP~5 protoln (xi) 52QUENCE DLSCRIPSION: SEQ ID NO:16:
Thr Ser Thr Thr Il~ Pro Lya Het Leu Val A~n Ile Hi~ Thr Gln Ser -:

A~n Thr Ile Thr Tyr Glu A~p Cy~ Ile Ser Gln Met Phe V~l L~u Leu 2~ 30 Val Phe Gly Glu Leu Aap A~n Ph L~u Leu Ala V~l M t Ala Tyr ~p .

Arg Tyr V~l Al~ Ile Cy~ Hla Pro Leu Tyr Tyr Thr Val ILe Val A~n ~ ' - " , .

- .

W O 92/17~ 2 ~ 7 PCT/US92/02741 -6~-~ .

Hi~ Arg Leu Cy3 Ile Leu Leu Leu L~u L~u S~r Trp V~l VA1 S~r Il~

L~u Hi~ Ala Ph~ L~u Gln S~r Leu Il~ Val L~u Gln L~u Thr Ph~ Cy-Gly A~p V~l Ly~ Ile Pro Hl~ Ph~ Phe Cy~ Glu L~u A8n Gln Leu Ser Gln Leu Thr Cy~ 50r A~p Asn Phe Pro Sar Hla L~u Thr M~t Hl.~ L~u Val Pro Val Ile Ph~ Aln Ala Il~ S~r LQU S~ Gly Il~ ~u Tyr Se~

Tyr Phe Ly~ Ile Yal Ser ser Ile Arg Ser ~et Scr Ser Val Gln Gly Lys Tyr Ly~ Ala Ph~ S~r Thr Cys Ala Ser Hi~ Leu Scr Ile V~l S~r Leu Phe Tyr Ser Thr Gly Leu Gly Val Tyr VA1 Ser S~r Al~ Val Ile lao 1~5 190 Arg Ser S~r Hi~ Scr S~r Al~ Ser Ala Scr V~l M~t ~yr Thr Val Val Thr Pro Mut Leu (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTER~STICS:
~A) LENGTH: 646 ba~ palr~ .
~B) TYPE: nuclelc acid .
tC) STRANDEDNESS: 6ingl~
~D) TOPOLCGY: lin~ar ~ii) MOLYiCULF TYP~i: cDNA :
~lli) HYPO~HETICAL: YES
~iv~ ANTI-SENSE: NO
~vij ORIGINAB SOUROE :
~A) ORGANISM: rat olfactory epith~lium (B) STRAIN: ~rpague-Dawl~y rat (F) TISSUE TYPE: olfactory ~pithelium . .
, (vii) IMMEDIATE SOURCE: . .
(B~ CLONE: J4 (ix) F~ATU~
(A) NAME/REY: CDS
~; LOC~TION: 2..646 . ~.
(xl) SEQUENCE DESC~IPTION: SEQ ID NO:17: ::
. .
C ATA GGC TAT TCA T~T TCT GTC ACA CCC AA~ ATG CTT GTC AAC TTC 46 : :

: ' ' '' ' SUE~STITUTE SHEET - ~

: : ~ . . .
: ; ..

:

WO 92/17~X' PC~/US92/û2741 2 ~ ~ g~

Il~ Gly Iyr Sar S~r S~r V~l Thr Pro A~n H~t L~u V~l ADn Pho l 5 lO li CTT ATA AAG CAA AAT ACC ATC TCA TAC CM GCA TGT TC~ ATA CAC m 94 L~U I1H Ly~ Gln A~n Thr Il~ S~r Tyr Lou Gly Cy- Ser Il~ Gln Pho GGC TCA GCT GC'T T~G TTT CCA GG~ C~T GAA TGC TTC CrT CX GCT GCC 142 Gly Sar Al~ Al L~u Ph~ Gly Cly L u Glu Cy~ Ph~ LQU LOU Aln Al~ :

ATG GCG TAT GAT CGT T~T GTA GCA ATC TCC AAC CCA C~C CTT TAT ~CA l90 M~t A1A Tyr A~p Arg Ph~ Val Ala Il~ Cy- A~n Pro Leu L~u Tyr Ser 50 5 5 60 : .
ACC A~A ATG SCC ACA C~A GTC ~GT GTC CA~ S~G GTT CTG CGA TCT ~AT 23 8 Thr Ly~ Mat Sor Thr Gln Val Cy~ V~l Gln L~u V~l V~l Gly S~r Tyr .~.. .
65 70 75 ':;
ATA GGG GGA TTT CTT AAT GCC TCC TCT TTT ACC CTT TCC TTT TTT TCG 286 . ``Ila Gly Gly Pho Leu A~n Ala S~r Sor Ph~ Thr L~u Sar Ph~ Pho S~r 80 85 90 9S .:

Leu SQr Ph~ Cyx Gly Pro A~n Arg Il~ A-n ~i~ Ph~ Tyr Cyu Aop Pho 100 105 110 ', GCT CCC TTA GTA GAA C~T TCT TGC TC$ CAT GTC ACT GTT CC$ GAT GCT 382 Ala P~o L~u v~l Glu Leu Ser Cy~ Ser A~p V~l S~r V~l Pro A-p Al~
115 120 125 ' .:.
GTT ACC TCA TTT TCT GC~ GCC ~CA GT~T ACT AT~ CTC ~C~ GT~ TTT ATC 430 V~l Thr S~r Ph~ Ser Al~ Al~ Sar V~1 Thr ~ae L~u Thr V~1 Pho Xl~
130 135 140 ~ :
ATA CCC ATC TCC TA~ ACC TAT ATC CSC ATC ACC ASC CTG AAG ATC CGT 478 :.
Ila Al~ S~r Tyr Thr Tyr Il- ~u Il~ Th~ L~u ~y~ M~t Arg 145 ~50 155 TCC ACT GAG GCT CCA CAG AA~ GC~ rTC TCT ACC TGC ACT TCC CAC CTC 526 S-r ~hr Clu Gly Ar~ ~ln Ly- Al~ Pho Scr Thr Cy~ Thr S~r Hi~ L~u 160 165 17U 175 .
ACT GCA GTC ACT CTG ~C TAT GGA ACC ATC ACA TTC ATC TAT GTC ATC 574 Thr Ala Val Thr L-U Cy- Syr Cly T~r 11- Th~ Ph~ Tyr Val ~ot CCC A~G SCC AGC TAC TCC AC~ CAC CAG AAC AAC CTG GTC ~CT GT~ m 622 :
Pro Ly- SO~ S~r ~yr S~r T~r A~p Gln A-n Ly~ Val Val S2r Val Ph- .. .
l9S 20~ 205 ;: .
TAT A$G G~G ~TG ATC CCC ATC TSG 646 Tyr ~t Vll V~ Pro Hot L~u 210 215 ~ .~
' :'.' ' (2) INFO~MATION FOR SEQ ID NO: 18:

i ) SEQUENC~ C~ARACTER15TXCS~
(~ LEN~H: ? 15 amino acid~ ~ :
(3) TYPE: amino ~cid ~D) ~OPOLOGY: lin0ar ~ ~.

SUBSTlTl)TE SHEET

. .

W O 92/17~8~ 2 ~ 7 PCT/US92/02741 , :, (11) MOL~CJLE ~YPE: prQt~ln (xl) SEQUENCE D~SCRIPTION: SEQ ID NO:18:
Il~ Gly Tyr Ser Ser Ser V~l Thr Pro ~n ~et L~u V$1 Asn Ph~ ~u Il~ Lys Gln Asn Thr Ile S~r Tyr Leu Gly Cy~ S~r Il~ Gln Pha Gily S~r Als Aln L~u Ph0 Gly Gly Leu Glu Cy~ Pho L~u L~u Al~ Al~ ~t Ala Tyr A~p Arg Ph~ Val Aln Ile Cy~ Asn Pro Leu Leu Tyr S~r Thr Lyi3 ~et Ser Thr Gln Val Cys Val Gln Leu Val Val aly S~r Tyr Il~
i30 Gly Gly Phe Lsu Aen Ala Ser Ser Ph~ Thr L~u 5~r Ph~ Ph~ S~r Leu Ser Phe Cyia Cly Pro Aa~ Arq Ile Aan Hl~ Pho Tyr Cy~ Asp Phe A1 Pro Leu Val Glu Leu Ser Cy~ Ser Ailp Val Ses V~l Pro Aap Ala Val Thr S~r Phe Sar Ala Ala S~r Val Thr Mi~t Lau Thr Vsl Ph~ Ile I1 Ala Il~ Ser Tyr Thr Tyr Il~ L~u ~1~ T~r Il~ L~u Lyia Met Arg Sar ..
145 150 155 160 : ..
$hr Clu Gly Arg Gln Lyi~ Ala Ph~ ser Thr Cy3 Thr S~r Hi~ Lou Thr 165 170 175 ~ :
Ala Val T~r Lou cyi3 Tyr Gly Thr Ile Thr Ph~ Tyr Val Met Pro 180 185 190 : .
Lyi3 S~r Sor ~yr Sor Thr Aap Gln ~an Lyo Val Val sQr Val Ph~ Tyr l9S 200 205 : .
M~t Val Val Il~ Pro M~t Leu .
~2) INFORMAT~ON FO~ S~Q ID NO:l9:
(i) SEQU~NCE CHARACTERISTICS:
(A~ LENGTH: 481 ba~e pa~rs ~ .:
(B) TYP~: nu~leic acid tc) STRAND~DNESS: ain~l~
(D) ~OPOLOGY: llnear :
(li) MOLFCUL~ ~YP~: cDNA
(iii) HYR0THE~IC~L: YES
(iv) ANTI-SENS~: NO

~vi) ORIGINAL SOURCE:
(A) O~G~NISM: rat olfactory ~pithelLw~

:~:
Sl)E~STITUTE SHEET ~ .

WO 92/1758:~ PCl/US9~/0274 Q ~ r~

( E~ ) ST~AIN: srpAgu~-D~wley r~t IF) TISSU2 TYPE: o'factory splth~l1um (v11) IM~EDIATE SOU~CB: .
~8) CLONEI J7 , -(lx) FEA~UR~:
~A) NAH~/~EY: CDS
~B) LOCATION: 2..481 ~xl) SEQ~ENCE D~SC~IPTION: SEQ ID NO:19:
C ATC TGC AAG CCC CTG CAC TAC ACC ACC ATC ~TG AAT ~AC CGA GTG 46 Ile Cy~ Ly~ Pro Leu H1~ Syr Thr Thr Il~ Met A3n A~ Arg Val 1 5 l0 15 TGC ACA GTT CTA GTC CTC TCC TGT TGG m GCT GGC C~C T1~ ATC ATC 94 Cy~ Thr Val Leu Val Leu Ser Cys Trp Phe Ala Gly Leu Leu Il0 Ile CTC CCA CCT CTT GGT CAT GGC CTC CAG CTG GAG ITC TGT GAC TCC AAT l42 Leu Pro Pro Leu Gly His Gly Leu Gln Leu Glu Phe Cy~ A~p S~r Asn CTG ATT CAT C~T TTT GGC TGT GAT CCC TCT CCA ATT CTC CAG ATA ACC l90 Val Ile A8p ~1~ PhQ Cly Cy~ A8p Ala Ser Pro Ile Lau Gln Ile Thr TGC TCA GAC ACG GTA TTT ATA GAG A~A ATT GTC TTG GG~ m GCC ATA 238 Cy~ S~r A~p Thr Val Phe Ile Glu Lya Il~ Vfil Leu Ala Phe Ala Il~ .. :

5TG ACA CTC ATC ATT ACT CTG.GTA TGT GTT GTT CTC TCC TAC ACA TAC 286 ~::
Leu Thr Leu lle Ile Thr Leu Val Cy~ Val Val Leu Ser Tyr Thr Tyr :
8~ 85 90 95 .
ATC ATC AAG ACC ATT TTA AAG TTT CCT TC~ GCT C~A CAA ACA AAA AAG 334 ~, Ile Ile Ly~ ~hr Il~ L~u Ly~ Phe Pro Ser Al~ Gln Gln Arg Lys Ly~
l00 105 110 ccc m TCT ACA ~GT TCT TCC CAC ATG ATT CTG CTT TCC ATC ACC TAT 382 ,~
Ala Pho S~r Thr Cy~ Ser Sor HL~ M~t Ilo Val Val Ser Ile Thr Tyr 115 l~0 125 GCG AGC TGT ATT TTC ATC TAC ATC AAA CCT TCA GCG AAG G~A GGa GTA 430 Gly Ser Cy~ Ile Pha Ilo Tyr rIe Ly~ Pro Ser Ala Ly~ Glu Gly Val GCC ATC ~AT ~AG GTT GTA TCT GTG C~C ACA ACA TC~ GTC GCC CCT TTG 478 Ala I le ADn Ly~ Val Val Ser Val Leu Thr Thr Sor Val Ala Pro Leu C~C 481 Leu ~2) INFORMATION FOR SEQ ID NO:20 ~i) SEQUENCE CHARACTERISTICS:
~A) LENGT~: 160 amino acid0~ ;
.~
.
SUBSTITUTE SHEET
:: :
:

.

W O 92/]758~ 2 1 ~ ~ ~ 'i 7 PCT/~S92/027ql (B) TYP~: ~mlno acld (D) TOPOLOGY: lln~r (ll) MOLECULE TYP~: protaln ~xl) SEQUE~CE D~SCRIPTION: S~Q ID NO:20~
Ile Cy~ LYD Pro L~u Hl~ ~yr Thr Thr Il~ H~t A~n Asn Arg Val Cyo Thr V~l Eau Vll L~u S~r Cy~ Trp Phc Ala Gly L~u Leu Ilo ~lo L~

Pro Pro Leu Gly HL~ Gly L~u Cln L~u Glu Ph~ Cya A~p S~r A~n V~l Ile A~p Hl~ Ph~ Gly Cy~ A-p ~1~ S~r Pro ~1~ Leu Gln Ilo Thr Cy~

s~r A~p Thr VA1 Ph~ Ile Glu LYB Ile Val L~U Ala Phe Ala Il~ L~u Thr L~u Ila Ila Thr L~u V~l Cy~ V~l V~l Leu Ser Tyr Thr Tyr Il~

Ils Lys Thr Il~ L~u LY~ Ph~ Pro Ser Ala Gln Gln Arg Lyo LYO Ala ::
100 10S 110 .
Phe Ser Thr Cy~ S~r Ser Hls Met Ils Val Val Ser ~le Thr Tyr Gly 115 120 125 ' Ser Cy8 Ile Pho I1~ Tyr Ile Lyc Pro Ser Al~ Ly~ Glu Gly Val ~la 130 135 140 . ~.
Ila ~n Ly- V~1 Val S~r Val Lou Shr Thr S~r V~l Ala P~o L~u Lou 145 150 155 160 :: : .
: :
~2~ INFORMATION FOR S~ ID NO:21:
(l~ S~QUENCE CHARACTER~STICS~
(A) L~NGTH: 481 baoe pairD
(D) TYPL: nucl-Lc acid ~C) STRANDEDN~SS: ~lngl~
(D) TOPO~O~Y: lLne~r .
OLECUL2 TYPB: protein ~lli) XYPOTHE~IC~L: YES
( iv ) ANTI-SENSE: NO :
~vi) ORICINAL SOURCE~
~A) ORG~NIS~: rat olfactory epitbalium ::
(B) STRAIN: Srpa~ua-~awlay rat (F) TISSUE SYPE: olfactory epithslium vii) IMMEDIATE SOURCE:
(B) CLONE: J3 (ix) FEATURE: :.
(A) NAME/~EY: CD5 '~',, ' :
SUB~ITUTE SHE~ET

', " .' . '.;,~, ,'. " , ,.,~, `' ! ' .

V~O 92/17~8~ 2 .L O ~ X l~ 7 PCT/VS92/0~741 ~ B ) LOCATION: 2 . . 481 (~tl) S2QU~NC~ D~SCRIPTION: SP:Q ID HO~21~
C ATC TC2 C~C CCG CTC CAC TAC TCT CTT CTC A5G AaT CCT GAC ~AC 4 6 I 1O CY~ H19 PrO L~U ~ TYr S~r Le~l LOU M~t S~r PrO A~P A~n } 5 10 15 I~GT GCT GCT CTG GTA AC~ CTC TCC ~; G~Y; ACA ~; ~ rG CGC ACO CGC 9 4 CY~ A1a A~ U Va1 Thr V~1 S~r TSP Va1 ThX cly V~l G1Y $hr G1Y

$TC CTG CCT TCC C~C C~G ATT TCT AAC rrO CAC 5SC rGT Gt;C CCC AAC 142 Ph~ L~U PrO S r LOU L U I1- S~r LY L~ U A~P Ph~ CY~ G1Y P~O ~9n . ,:
CCC ATC AAC CAT $TC 'r$C TGT GAC Cl C CCT CCA TTA ATC CAG CSG TCC 1 9 0 Arg I10 A8n Hi~ PhO Phe CY8 A8P LeU PrO PrO LeU I1~9 G1n L~u S~r ~:C TCC ACC GrC T~ GTC ACA GAA ATC; GCC ATC m GTC CTG TCC A~C ~3 a CY~ Sar S~r Va1 Phn V~1 ~hr G1U tlnt A1a I1U Ph~ Vcl Lau S~r Ile 65 70 75 : ":
GCT CTC CTC TGC ATC TCT TTC CTC CTA ACC CNN NNN $CC TAC A1'T TTC 2F~6 A1a Va1 LeU CY~ I1el CY- PhO L~U LeU Thr Xaa Xa~ SeS 'ryr I1Q Ph~ .:
80 85 90 95 . :
A$A GTG TCC TCC Aq~r CTt; ACA ATC CCT TCC A~r ACC CGC AGG A5 G AAG 3 3 4 Il~ Val Ser S~r ~1Q L~u Arq I18 Pr~ S~r Thr Thr Gly Arg ~oe Ly~
100 105 llG
ACA TTT TCT ACA TG$ GGC TCC CAC CTG GCC GTC GTC ACC ATC TAC TAT 382 Thr Phe s~r Ths Cy~ Cly 54r His L~u Al- V~l V~l Thr Ilo Tyr Tyr ::
1~5 120 125 .
GCC ACC ATC ATC TCC A~i T?~T G'rC GGiC CCA AAT t;CC CA~ CrC ~CC CC&i 430 Gly Thr M~t Ilo S-r Mff~ ~yr V~1 Gly Pro A~n A1~ HLf~ L~u Ser. Pro 130 135 140 . : .
CAC C:SC AAC A~G CSC AT~ G'rC l~C ~AC AC~ CSGi ~SC: ACC CQ CSA 478 Glu ~u A~n Ly- Val I1- s-r V~l Phq Tyr ~hr Vlil Ilel Thr Pro L~u ;
145 150 ~ 155 Cl~i 481 ~.
L~u 160 :.
.:
(2) INFORMATION FOR SEQ ID 1~0:22:
i ) SEQUENCE CHARACTE~iIS~ICS:
(A~ LENGTH: 160 ~unino acids (~) TYPFi: iQmino illcid (D) TOPO~iY: lin~ar ( il) MOLECU~ TYPE: prot~in (xl) S~QU~iNCE DESCRIPTI4N: SEQ ID NO:22:
lle Cy~ Pro L-u Ni~ Tyr Setr Leu Leu Hat Ser Pro A~p ~sn Cy~

' : :

:8UBSTITUTE SHEET

~'0 92/1~8~ 2 ~ 7 PCT/US92/0~741 '~ .`

l 5 l0 l5 Al~ Ala Lou V 1 Thr Val S~r Trp Val Thr Gly Val Gly Thr Gly Ph~

L~u Pro Ser Leu Leu Il~ S~r Ly L~u A~p Phe Cy~ aly Pro A~n Arg Il~ A0n Hl~ Ph~ Pho Cy- ABP LOEU Pro Pro LHU Ile Gln Leu SHr Cy~ ~.

Ser Ssr V~l Ph0 V~l Thr Glu Met Ala Il~ Ph V~l Leu S~r Val Leu Cy~ Ile Cy Pha Leu Leu Thr Xaa X&~ Ser Tyr Il~ Ph Il~

Val Ser s~r Ile Leu Arg Ile Pro Ser Thr Thr Gly Arg Met Ly~ Thr Phe Ser Thr Cyr Gly Ser HiD l2au0 Ala Val Val Thr I25 Tyr Tyr Gly Thr Met Ile Ser ~et Tyr V13aS Gly Pro Aan ~l l40 Leu Ann Ly~ Val ll~ Sar Val Ph0 Tyr Thr V~ 0 Th~ Pro L~u L~u (2) INFO~MATION FO~ SEQ ID NO:23:
~i) SEQUENCg CaARACTERISTICS:
(A~ L2NGTM: 646 base pairB ~ ::
(~) TYPE: nuc}~ic acl~
(C) STRANDEDNFSS: 8lngl~
(D) ~OPOLOGY: lin~ar .
(ii) MOLE5ULL $YPE: pro~in (ili) ~YPOTH~TIC~L: YES
(iv) ANTI-SLNS~: NO
(vi) ORIGINA8 SOURCE~
(A) ORCANlSM: r8t ol~actory ~pithelium (B) STRAIN: Srpagua-Dawloy rat ~F) TISSU~ TYP~: olf~ctory epith~liu~
(vii) IMMEDIATE SOURCE: .
(8) CLONE: Jll .. :.
(ix) FEATU~E:
(A) NAMEt~Y: CDS
~8) LOCATION: 2..646 ~xi) SEQUENCL DESCRIPTION: SEQ ID NO:23:

Val Cy~ Phe Ser Ser Thr Thr Val Pro Ly~ Val Leu Ala A~n Hi~ : :
l 5 l0 15 ~ ,:

'' ~', W O 92/1758~ PCT/US92/027~1 ? ~ 68~2 ;~
ATA CTC AGT ~aT CAO GCC ~TT TCC rTC TCT GGG TGT CTA ACT C~C CTG 94 ~1~ Lsu S~r S~r G1n A1- I1a S-X Ph~ S-r C1Y CY~ L0U Thr C1n L0U

TAT ~rT CTC TGT C~C TCT CTG ~AT ATC GAC A~T T~C CTG C~ GCT G~C 142 Syr PhO LOU CY~ Va1 S~r Val A~n ~t A~p ~ ~ PhO L~U L~l~ A1a V~
35 40 4!5 AT~ GCC TAT G~C AGA ~rT GTG GCC ATA TCC C~C CC~ TT~ TAC T~C ACA 190 ~8t A1a Tyr A~p Arq Ph~ V~1 A1a I1~ CY~ Pr~ L~U ~Yr Tyr ~hr . ,.:
50 55 60 ...
A Q AAG ATG ACC C~C CAG CTC TGT GTC T~G Cr~ CTG TCT cGA TCA ~NN 238 Thr Lyo ~ae Thr HiC G1n L4U CY~ Va1 L~U L U Va1 S~r G~y s~r Xa~

NNN NNN NNN NNN NNN NNN NHN NNN NNN NNN NNN NNN ~NN NNN NNN NNN .286 .
Xaa Xa~ Xaa XAn Xa~ Xaa X~a X~a Xaa Xaa ~aa X~ X~a Xa~ X~ Xa~
ao 85 90 95 NNN NNN NNN NNN NNN NNN NNN NN~ NNN NNN NNN NNN NNN NNN NNN NNN 334 Xaa Xaa Xaa Xaa X~a Xaa Xaa Xaa Xaa XAa Xaa Xaa Xaa Xaa Xaa Xad X~h Xaa X~a X~ Xaa X~a X~a Xaa Xaa Xaa XaA Xaa xaa xe~ Xaa Xaa 115 120 ~ 125 :.
NNN NNN NNN NNN NNN ~NN NNT GrG .~LTC ~G GSC ACC CC~ ~ GTC TGC 430 X~a Xaa Xa~ X~A Xaa X~a Xa~ V~ Mot Val Thr Pro Phc Val Cy~ .
130 135 140 .. :
ATC crc ATC ~rCT TAC ATC S~C A~C ACC AAT GCA CTC crc AGA GTC TCA 47B
Ile Leu Il- Sor ~yr Ile ryr ~1~ Thr A~n Al~ Val ~eu Arg V~l S-r TCC TTT AGG GGA G~A TGG A~A GCC TTO TCC ~CC TCT CGC TCA CAC CTG 526 :~
Ser Phe Arg Gl y G1Y T~p Ly- Al~ Ph- Sær Thr Cy~ G1Y Snr Hi~ L~u ~:

GCT C~C GTC TGC CTC T~C SAT CGC A~C AT~ AT~ GCT GTG T~T TTC ~AT 574 Ala V~l Val Cy2 L~u Ph- Tyr Gly Thr Il~ Al~ Vhl Tyr Ph~ A~n 180 1~5 190 ;~ .
CCT GTA TCT SCC CA~ SCA TCT G~a AAG GAC ACT GCA GCA ACr GT5 CTA 622 Pro Vi~l S~r Sor Hl~ S~r Ser G1U Lys ~p Thr ~la Al~ Thr V~l L~u TAC AC~ GT~ GT~ ACT CCC ATG TTC 645 Tyr Thr Val Val Thr Pro Mot Lou (2) lN~ORMAT~ON FOR SEQ ID NO:24:
(i) SFQUENCF C~ACTFRISTICS:
~A) LFNGTH: 215 amino acids (ES) TYPR: amino acid (D) TopoLoay: linear tii) ~OLECULE TYPE: protein ' , .

; ' SUBS~ITUTE SHEET
. .
''~' .

W O 92/1758~ 2 ~ 7 PC~r/US92/02741 (x~) SEQU~NCE D~SCRIPTION: S~Q ID N0~24 Val Cy~ Pho S~r S~r ~hr Thr VA1 Pro Ly~ Val Lsu Ala A~n Hl~ Iln Lnu S~r S~r Cln Al~ S~r Ph0 Sur Cly Cy- L4u Thr Oln ~u Syr Pho L~u Cy~ V~l S~r Val A~n Mot ~op Aln Pho Lsu L~u Al~ Val M~t ~5 40 45 Al~ Tyr Anp Arg Pho V~l A55 IlQ CyD H1~ Pro 6uO Tyr ~yr Thr T r LY0 H~t Shr ~1- Gln L u Cy~ V~l Lou L~u Val S0r ~ly S-r ~a~ X~a ` :
~0 Xaa X5A Xaa Xaa XaA Xaa Xaa Xaa Xaa Xaa Xaa %aa Xan Xaa Xaa Xaa a5 ~0 gs Xaa Xaa Xaa Xaa X~a X~a Xa~ Xaa Xaa Xaa Xaa Xaa Xaa Xaa X~a Xaa Xaa Xaa Xaa Xaa Xaa X~a xaQ Xaa X~a XaA Xaa Xaa Xa~ X~a Xaa X~a Xaa X-~ XAa Xa4 Xa~ X~a Val Ilo Met Val Thr Pro Ph- Val Cy9 Il~
130 l~S 140 LBU Il~ Ser Tyr Ile Tyr Ilo Thr Arn Al~ Val ~nu Arg Val S~r S~r 145 lSO 155 160 Ph~ ~rg Gly Gly Trp Lyn Al ~ Ph~ Sar ~hr Cys Gly SQr Hi~ ~cu A1- :
165 1~0 175 :
Val Val Cys Lou Pho Tyr Gly Thr Il~ Al~ Val Tyr PhD A~n Pro 180 185 190 ` ` :.
Val S~r S~r ~i~ Sdr 50r Glu Ly- A-p ~Ar Al~ Al~ Thr V~l Lou ~yr i95 20~ 205 Thr Val Val Thr Pro M~t L~u (2) INFORMA~ION ~OR S8Q ID NO:25:
tl~ S8QULNCg C~UURAC$~RISTICS:
~A) LSSNG~H~ 646 b~o pair (~) TYPE: nuclolc ~cid ~C) STRANDEDN~SS~ ingls (~) TO~OLOCY~ lin~ar (ii) MOL~CUL~ TYP~: cDNA
HYPOIH~T~C~L: Y~S :
( 1V ) AN'r~-Sl~N5 : NO
(vi) ORIGINA~ SOURC~:
(A) ORGANISM: rat ol~actory ~plt~ u~
~8) ST~AaN: Srp~gu~-Dawl~y r~t ~F) TI55U~ TYP~: olfactory ~plth~liu~

. , . -.: .
: :: ::
SUB~3TITUl-E SH~ET
- ,~,., ~'0 92/17~ PCI'/US92/02741 ~ `,. :.
2 L g ~ 3 ~ rJ~ 74 vil ~ I~EDIAT~ SOURCB:
(B) CLONl~: J14 ( ix ) FEA'rURE:
~A) NA~/lU~Y: CDS
(B) LOCATION: 2..646 (xl) S~QUISNCE DESCRIPTION: SEQ ID No:2s~
T GTC ~GC ~C TCC TCC ACC AC~ CTC CCC ~C O~A C~; t3C:~ AAC CAC 46 Val Cy~ Ph~ S~r S~r Tl~r Tl~r V~l Pro I,y~ V~l L~u Al~ A-l~ Hi~

ATA CTC AGT AGT ~C GCC ATT TCC T~rc 'rC~ GGt; TGT ~A Ar ~G CTG 9 ~
Ils Luu S~r S~r Gln Al~ Ilo Sor Ph~ Sar Gly Cyo L ~u rhr C:ln l~u ~ .

TAT l'TT CTC ~GT GTC TCT GTG ~AT ATC GAC A1~'r rTC C~G CTO GCT GI~C I42 Tyr Ph~ L~u Cy~ Val Ser V~l A0n M~t A0p A~n Ph~ L~u l.~u Ala Val ArG GCC TAT GAC AGA TTT GTC GCC ATA TGC CAC CCT ~C TAC TAC ACA l90 Het Ala Tyr A~p Arg Ph~ V~l A~ Cy- Hi- Pro L4~u Tyr Tyr Thr ACA CCG ATG ACC CAC CAG CTC TGT GTC 'l~G CTG CTC ~CT GGA TCA NNN 2 38 Thr Pro Met Thr Hls Gln Leu Cyr V~l L~au L~u V~l 5~r Gly Ser Xa~
65 70 75 .:
NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NN~ NNN NNN NNN NNN NNN 286 Xaa Xaa Xa~ X~a Xaa X~ Xaa Xaa Xa~ Xa~ Xa~ Xa~ Xaa Xa~ Xa~ Xaa Xaa Xa~ Xaa X~ Xa~ Xa~ Xa~ X~ X~a X~ X~ X-a X~ Xa~ X~ XAA

N~N NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN N W NNN NNN NNN 382 Xa~ Xaa X~ Xa~ Xa~ Xa~ X~ Xa~ Xa~ Xaa Xa~ X~a X~ Xaa X

NNN NNN NNN NNN NNN NNN NNT GTG A~C ATG GTC ACC CCA STT C~C TGC 430 Xa~ X~ X~ X~a X~A X~a X~a V~l Il3 ff-t V~l ~hr Pro Ph- V~l Cy~
130 135 14C ..
A~C CTC ATC TCT TAC ATC TAC ATC ACC AAT OCA CTC CTC AGA GTC TCA 478 Il~ L-u Il~ 5~1r Tyr Il~ Tyr Ile Thr A~n ~la V~l Leu Arg Val Sor TCC TTT ACG GGA GGA ~CG AAA GCC ?TC TCC ACC TGT GGC TCA CAC CSC 526 Ser Phe Ars Gly G1Y Trp Ly~ Ala Phe S~r Thr Cy~ Cly S~r Hil~ ~u 160 165 170 17~ -GCT GTG CTC TGC C~C T~C TAT CGC ACC ASC A~T GCT GTG TAT TTC ~AT 574 Al~ Val Val Cy~ Leu Phe Tyr Gly Thr Ile Ila P~la Val ?yr Ph~ A~n 1~0 185 190 CCT GTA TC~ ~CC CAT ~C~ TC~ aAG AAG ~AC ACT CCA CCA ACT GTG CrA S22 Pro V~l Ser Ser Hi~ S~r Ser Glu Ly~l A~p Thr Ala ~ hr V~l Leu ::

:
: : :
.
; ~
SUElSrlTUTE SHEET

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','`'.' ' .' ',.. `',"`''''',''''' ~':,' ,;`, :,''.

WO 92/175~:~ 2 :1 0 6 8 L~ ~ P~/US92/02741 ~ . ~ ~75- :
~ .

Iyr Thr Val Val Thr Pro ~-t L~u ~2) IN~ORXA~ION FOR S~Q ~0 NO~26 L ) S~QUZNC~ CHARACT~RISTIC~
~A) LE~GT~s 215 ~mino acld~
(8) TYP~ lno a~ld (D) TOPOLOCYI llna~r (li~ ~OL~CU1~ ~YP~: prot~in (xl~ S~QU~NC~ D~SC~IPTIONI SEQ ID NO~26:
Val Cy8 Pho S~r Sar Thr Thr Val Pro Ly~ V~l Lou Ala A~n Hl~ Il0 Lou S~r S~r Gln Ala Il~ S~r Ph~ Sar Gly Cy~ Leu Thr Gln Leu Tyr Pho Leu Cy~ Val Sar Val A~n H~t A~p Asn Phc Leu L~u Al~ Val M~t . .
35 40 45 :.
Ala Tyr A~p ~rg Pho Val Ala Ilo Cy~ HL~ Pro Leu Tyr Tyr ~hr Thr 50 55 60 .
Pro M~t Thr Hi~ Gln Leu Cyr Val LQU Leu Val Ser Gly S~r Xaa Xaa ~5 ~0 75 80 Xaa X~a Xaa Xaa Xaa Xa~ X~ X~a X~a X~a Xa~ Xaa Xa~ X~a Xa~ X~

Xaa Xaa Xæa Xaa Xaa X8a Xaa Xaa Xaa Xaa Xa~ Xaa X~a Xa~ Xat~ X8a Xaa X3~ Xaa Xa~ Xa~ Xaa Xaa XA~ Xa~ Xaa X~a Xaa X~a Xaa X~ Xan Xaa X~a Xaa Xaa Xaa XAa Val Il- H~t Val Thr Pro Ph~ Val Cy~

Leu Ile S3r ~yr Il~ Tyr Ils Thr ~n A12 Val ~ u Arg VA1 S-r S-r Pho ~sg Gly Gly Trp LYD Ala Ph~ Ser Thr Cy~ Gly S~r H1~ Leu Al~ ~.

V~l V~l Cy~ Lou Pb~ ~yr Gly Thr Il~ Iln Ala V~l Tyr Ph~ A~n Pro ~
180 13~ 190 ~ ~ .
Vsl Sor S~r ~i~ SQr S~r Glu Ly~ A~p Thr Ala Ala Thr V~l L~u Tyr lgS 200 205 .
Thr V~l V~l Thr Pro M~t L~u :

~2) INFO~MATION FOR SEQ ID NO:27:

~1) SEQUENCE CHARACTE~ISTICS: :
-::
' ,'. "~
,~
;.',. : ~
SUE~STITUTE SHEE~T
' :

.

W O 92/1758' P~T/US92/~741 ~6~7 -76- ~.

(A) LENGTHI 4B1 b~O~t Pa1 ~) IYP~: nUC1~1C ~C1d (Cj STRAND3DNESSt R1~g1 ~D) TOPOLOOYt 1111~dr ~11) HOL~CUL~ TYPE~ CDNA
~1i1) HYPOTXETIC~Lt YES
(iV) ANTI_S~NS~ NO
(~1) O~IGINA~ 50URC~
(A) ORa~NI8~ r~t O1f~CtOrY OP1thG1iUnt ( B ) ST~AIN I SrP~t~tt~-DaW1eY r6t (F) TISSU~ ~YP~I O1~CtOrY aP1th~11U~t ~V11) I~XEDIATE SOURCEt (B) CLON~: J15 (iX) FEATUR~:
(A) NA~E/K~Y: CDS
(B) LOCAT~ON: 2..481 (X1) SEQUENCE DESCRIPTION: SEQ ID NO:27:
T ATC TGC AAC CCT CTC CCC TAC CQ GTC CTC ATa ACC GCC C~G CTa 46 I1e CYt3 At~tn PrO LeU Ar9 TYr PrO V~1 L~U M~t Ser G1Y Arg V~1 TGC CTC CTC ATC CTC GSC GCC TCC ~GG TTG CGA CGA TCC CTC AAC GCC 9 CY~ L~tU LaU MOt Vd1 V~1 A1~ S~r TrP LOU G1Y G1Y Sqr LOU A-n A1A
:
TCC AT~ C~C ACT TCT CTG ACC CTT CAG TTC CCC TAC TGT GGA TCA CGO 142 S~r I1e G1n Thr Sar L~U Thr Leu Cln Ph~ Pro Tyr CY- Cly S~r Arg AAG ATC TCC CAC TTC TTC ~GT GAO C~C CCC TCG CTC CTG ANN N~C CCC 1gO
LY~ Ser Hi0 Ph~ Ph- Cys Clu V~l PrO Ser I~u Lau Xa~ Xa~ Al r~

TGT CCA GAC ACS CAA CCC TAT GAC CAG GTA CTA ~ST CSC A Q GCC GTC 23~ .~
Cy~ Al Arp Thr Clu Al~ ~Yr G1U G1~ Va1 L~U Ph- Va1 ~hr ~}Y Va1 70 75 . .
CTG GTC CTC CrG CTG CCC AT~ ACA TTC ATT ACT GCC TCT T~T CCC CTC 286 V~1 VA1 LeU LOU V~1 PrO I1~ Thr PhO I18 Thr Al~ SOr TYr A1~ LeU
80 85 90 95 :
A~C C$G GCT GCT CSC CTC CG~ AIG CAC TCT GCG GAC CGG AOT CAC AAC 334 I1~ Leu Ala Al~ Val Lou ~rg l~e'c Hi~ Ser Al~ Olu C~y S~r Cln Ly~
100 105 110 , ' GCC C~ GCC ACA TGC TCC TCT CAC ~TG ACA G~C GTC AAT C~C T~ ~AT 382 A1~ L~U A1~ Thr CY~ Ser S~r His Leu Thr V~1 Ydl A~n ~u Phe Tyr 115 120 125 ~ :
GGG CCC CTT GTC TAC ACC TAC ATC TTA CCT GG~ TCC TA~ CAC ~CA CC~ 430 G1Y PrO L0U VII1 Tyr Thr Tyr M~t Leu Pro Ala Ser Tyr ~i- S~r Pro : ~ ' :
', SUBSTlTl)TE SHEET
', ,, ' , ' .' . . :

WC) 92/17~8' 210 6 ~ ~ ~ PCI/US92/0274]

G~C CAA GAC GAC ATA GrA TCC aTC rTT TAC ACC GTT crc AC~ CCC ATC 478 Gly Gln A~p Aap Il- Val S~r Val Ph~ Tyr ~hr V~l L4u Thr Pro ~t 481.:
L~u (2) INYORM~TIO~ ~OR 8~Q ID NOl28 (1) 8~QuæNc~ ~ARACT~RISTICS~
~A) LBNOTHs 160 amino acld~
(~) TYP~ amlno ~cld (D) TOPOLOGYs lln~ar (ll) ~OL~CUL~ TYP~: prot~n (xi) SEQU~NC2 DESCRIPTION: S~Q ID NO:28:
Ile Cy~ A~n Pro Lau Ar~ Tyr Pr~ Val Le~ H~t Ser Gly ~rg V~l Cyo Leu Leu Met VaL V~l Ala S0r Trp Lou Gly Cly S~r Lou ~n Al~ sQr Ile Gln Thr S~r Leu Thr L~u Gln Ph- Pro Tyr Cy~ Cly S~r Ary Ly-Il~ Ser Hl- Ph~ Pho Cy- Clu Vdl Pro S~r Lau L~u X~a XAa Al- Cy~ . .

Al~ A~p ~hr Glu Al~ Tyr Glu Cln Val L~u Pho V~l $hr Cly V~l V~l .
65 70 75 ~0 : ::
V~l L~u Lau Val Pro I1Y Thr Ph~ Thr Ala Sos ~yr Al~ L~u Il~
85 go ~5 ~ :
Leu Ala Al~ V~l Leu Asg ~t Hi~ S~r Ala Clu Gly Sor Cln Ly- Al~ -100 1~5 110 .:~
Leu Ala Thr Cy- S-r S~r Hl~ ~eu Thr Yal V~l A-n L~u Ph- ~yr Cly -.

.~ . .
Pro Leu V~l ryr Thr ~yr M~t Lou Pro Al~ S-r Tyr ~1- S~r Pro Cly 130 135 14~ :
Gln Asp AGP Ile Y~l S~r V~l Ph~ Tyr Thr V~l ~ u Thr Pro M~t Lnu :: . .
1~5 lS0 155 ~60 ' :.
.:
(2) INFORHATION YOR SFQ ID NO:29: -~1) SEQUFNC~ CHARAC~ERISTICS: .:::.
(A) ~ENC~H: ~81 ba~ pair~
~B) TYPE: nucl~i~ acit --: (C) STRANDEDN~SS: ~ingl~
(D) TOPOLOGY: line~r ~ OLECULE TYPg: eDNA .
(iil) ~YPDTH~iTICAL: Y~S

: .
:
.,.~
SUBSTITUTE Sl IEET ; .

~ ~ , ,'' ', ,"~,~,,,,,,,""-,".~",".`.

~'092/17~8' ~1~ 6(~3~7 PCl/US92/02741 --7~--(iV) ANTI_S~NS~I N0 ( V1 ) OAIGINAL ~OURCB I
~A) OP~GANI5M: r~t O1faCtOrY ~P1th~11Um 8 ) STRAIN ~ Srp~gu~-Dawley r~e ~F) TISSU~ TYP~I O1f~CtOrY ~pLthellu~
( V1L ) I~DIA1~ SOURCE:
(8) C~N2t J16 ( 1X ) F1S~TUR~ I
A ~ NAM~ / 2tll Y: CD S
~B) LOCATION~ 2..481 ~xl) S~9UENC~ D2SCRIPTI0NI SBQ ID NO-29:
C ASC SGT AGG CCS CTT CAC TAT CC~r ACC C~C A~G ACC CAC; A~ CTG 4 6 Ilu Cy~ Arg Pro L~u Hl~ Tyr Pro Thr Lou Moe Thr Cln Thr L~u T~r GCC AAG ATT GCC ACT GCT TGC TGG ~; G~;A GGC TTC G~T GGG CCA 94 Cyn Al~ Ly~ Ala Thr Gly Cy~ TrP LeU G1Y G1Y LqU A1~ G1Y PrO

GTG GTA CAA ATT TCC rSG GTG TCT CGT CTC Crr T5"T TGT GCC CCC AAT 142 V~1 V~1 G1U I1~ SOr L~U VA1 Ser Ar9 L U L9U Ph- CY~ Gly Pro A~n CAC ATT CAA CAC ATC S~T TCT GAT STC CCA CCT CTG CTG AGC T~G GCT l90 H1~ I1e C1n H~ Ph- CY~ A-p Pho Pro Pro V~l Leu sRr L~U A1~
50 55 60 , -TGT ACT GAT ACA TCA GTG AA~ GTC CTG G'rA GAIr ~ A1~ ATA AAC CSC 238 Cy~ Thr A~P ~hr S~r V~l A~n Vhl Leu V~l A-p Ph~ A-n L~u 65 70 75 ::
SGC AAC ATC CTG GCC ACC TSC CTG CTG ATC CTG AGC SCC TAC STC; CAG 2~6 Cy~ Lya Ilo Leu Ala Thr Pb- Lsu L~u Il~ Lou Ser Ser Syr LRU Gln 80 ~5 90 95 ATA ATC CGC AC~ CSG CTC AAG ArT CCT TQ CCT GCA CGC AAG A~C AAA 3 3 4 Ar9 Thr Va1 ~OU LY~ PrO S~r A1a A1;~ C;1Y LY~ LYU LY~

GC1~ l~C T~G ACT TGT GCC TCC CA'r C~C ACT CTG GTT CTC ~TC 'rTC TAT 3~2 A1a Phe S~r Thr CYr A1~ S~r ~lL~ L~U Thr V;~1 YII1 L~U I 1~ PhO TYr 115 120 125 .
GCX~; AGC ATC CTT TTC ATG TI~T Gl~G; CGG; CrG AAa AAG ACT TAC TCC CTT 430 G1Y Ser I1e L~U Phe ~9t TYr V;~1 Ar9 LeU LY~ LY~I Thr Tyr SOr LeU
130 135 140 - .
GAC TAC GAC AGA CCC TrC GCA GTA GTC TAC TCC GTG;; Gr~ ACC CCT STC 478 AaP TYr A~P Arg A1a L~U ~1a V;~1 Va1 TYr S~r V;91 Va1 Thr PrO Phe C~; ~81 LeU
160 . .

~2) INFO~ATION FO~ SEQ ICI NO:30: ~
. ~ .

SUBS~lTlJTE SHEET

W O 92/17~8~ 2 ~ 7 PCT/US92/02741 , (i) SEQUENC~ CHARACTE~ISTICS:
(A) LBNGr~s 160 a~no ~cldo (~) TYP~t ~lno ~cld 5D) TOpoLoGY~ lln~r ( 11 ) MOLBCULX TYP~: prot~ln txl) SEQUBNC~ D~SCRIPTION: S~Q ID No:30:
Ilo cy- Arg Pro ~su ~1~ ryr Pro Thr L~u ~t Thr Gln Thr L~u Cy~

Al~ Ly~ Ala Thr Gly Cy~ Trp Leu Gly Gly Lou Al~ Cly Pro V~l Val Glu Ila S~r Lau V~l S~r Arg L~u Lou Ph0 CY3 Gly Pro A~n H1 ~5 Il~ Gln Hi~ Phe Cy~ ~8p Phe Pro Pro V~l L~u Ser Leu Ala Cy-Thr A~p Shr Ser Val A~n V~l L~u Vnl ~p Pho ~ A~n L~u Cy~ :

Ly~ Il0 LQU Ala T~r Phe L0u Leu Il0 Lou S~r S~r Tyr Lou Gln Il~
85 90 95 .
Ile Arg Thr Val L~u Ly~ Ilo Pro S~r Ala Ala Gly Ly~ Ly~ Ly~ Al~
100 105 110 . :
Ph~ s~r Thr Cy~ Ala Ser Hl~ L~u Thr Val V~l L~U Il~ Ph~ ~yr Gly llS 120 125 Sor Il~ Lou Ph~ ~-t Tyr Val Arg L~u Ey~ Ly~ Thr Tyr S~r ~Qu A~p 130 135 1~0 Tyr A-p Arg Al~ L~u Al~ Val V~1 ~yr S~r Val Vsl Thr Pro Ph~ L0u 145 150 lSS 160 :-~ . .
52) INFOP~ATION FOR SLQ ID NO:31:
~1) SEQUZNC~ CNARACT~RISTICS:
(A) ~RNGTHs 481 b~30 pairD
~B) 5YPX- nucl~ic acid ~C) STRANDLDNLSSs ingl~
~D) SOPOLOGY: llnoar ~ii) MOLECU~E TYP~: cDNA
5iii) HYPOT~TICAL: YES
5iv) ANTS-SENSg: NO
~i) ORIG~NAL SOURC~
(A) ORGANISM: rat olfa~tory epithaliu~ ::
(B) STRAIN: Srp~gu~Dawlay rat ..
~F) TISSUE TYP~: olf~ctory ~pithellum (vii~ IMK~DIAT~ SOURC~
~) CLDNE: J17 .: , . .
- : " ' .
SU~STITUTE SHEET ;
: -: . -, ~
: . .

WO92/17~8~ 2~ 7 PCT/US92/û2741 (~X) FEATURE:
(A) NAHE/~Y: CDS
(B) LOCATIONI 2..481 (X1) S~QU2NC~ DESC~IPT1ON: S~Q ID NO:31 A ATC TGC AAC CQ CTC CTT TAT TCC ACC AA~ ATG TCC ACA CA~ GTC 46 I1~ CY~ A~ PrO LoU L~U TYr S-r Thr LY~ ~-t S~r Thr G1n V~1 ~GT ATC C~G S~C CTT ~CA CGA TCT TAT A~A GGG GGT m crT AAT ~CT 94 CY~ a1n LaU Va1 A1a G1Y S~r TYr I1~ G1Y G1Y Phe LOU ASn Thr 20 25 30 .
TGC CTC ATC ATG m TAC TTT ~TC TCS TTT CTC rTC ~CT CCC CC~ AAT 142 CY~ L~U I1Q Het Ph~ Tyr Pho Ph~ SOr Ph~ L~u Ph~ Cy- G1Y PrO Aan ATA GTT GAT CAT TTT TTC TGT GAT m GCT CCT TTN NTC GAA C~T TCG 190 I18 V~1 A~P Hi~ Ph~ Ph~ Cya A0p Phe A1a PrO X~a Xa~ Glu Leu S~r TGC TCT GAT GTG AGT CTC TCT GTA GTT GTT ATG TCA m TCT GCT G~C 238 CYD Ser Aap Val s~r Val S~r V~l Val Val M-lt Ser Ph~ SQr Ala Gly TCA CTT ACT ATG ATC ACA CTC m ATC ATA GCC ATC TCC TAT TCT TAC 286 S~r V~1 ~hr M~t I1~ Thr V~1 Ph~ IlQ Il~ Al~ S~r Tyr S0r Tyr 80 85 9~ 9~ -ATC CTC ATC RCC ATC CTC AA5 ATG TCC TCA ACS CA~ GGC CCT CAC AAG 33~ -.
I1e LeU I1~ Thr I1O L0U LY~ M~t 8~r Sar Thr G1U G1Y Arg Hir Ly~ : . :. .
100 105 110 : :
GCT TTC TCC ACA TGT ACC TCC CAC ~TC ACT GC~ GTC ACT CTC TAC TA~ 382 Ala Phe Ser ~hr Cy~ Thr Sar Hlr L0u Thr Ala V~l Thr L~u Tyr Tyr llS 120 125 GGC ACC ATT ACC rTc A~T TAT GTC AT~ CCC ~AC, TCC AC~ TAC SCS ~CA 430 Gly ~hr ~1~ T~r Phc Il~ Tyr V~l M~t Pso Lyn S-r ~hr Tyr S~r ~r G~C CAG AAC AAC GTG CTG TCT GTG ~ TAC A'rGi GTGi GTG ATC C~ ATC 47a A~p Gln Aun Ly~ V~l Val S~r V~l Ph0 Tyr ~t V~l Yal Iln Pro Mot TTG 431 ..
Leu .

(2) INFORMAT~ON FOR SEQ ID NO:32: :~
(i) SEQUENCE CNARACTE~IS~ICS:
~A) LENGTH: 160 amino ~cids (9) ~YPE: amino ~cid (D) TOPOLOGY: line~r (ii) ~OLECULE ~YPE: protein (xi) SEQUENCE DESCRIP~ION: SEQ ID NO:32:
- ~ ~

. :: ' .
SUBSTITUTE SHEET

W O 92/1758' 2 1 o ~ ~ 4 7 PC~r/US92/02741 Il~ Cy~ A~n Pro L~u L~u Tyr S~r Thr Ly~ M~t Sor Thr Gl~ V~l Cy~

Il~ Gln L~u Val A1A Gly 3ar Tyr Il~ Cly Gly Ph~ Lou Au~ Thr Cy~

L~u Il~ H~t Ph- Tyr Ph- Ph~ Sar P~ L~u Ph~ Cy~ Gly Pro A~n Il~

V~l A~p H1~ P~o Pho Cy~ A~p Ph- Al~ P~o Xaa X~ Clu L ~ S~r Cy~

S~r A~p VA1 S0r V~1 Sar Val V~l Val Hnt S~r Ph~ S~r Al~ Cly S~r ::
~0 ~5 80 V~l Thr H~t Il~ Thr Vnl Ph~ Ala Il~ SQr ~yr S~r Tyr Il~ :
~35 9~ 95 L0u Ile Thr Il~ L~u Lys Het S~r S~r Thr Glu Gly Arg Hi~ Ly~ Al~

Phe Ser Thr Cy~ Shr Ser Hia Leu Thr Ala V~l Thr L~u Tyr Tyr Gly 115 120 125 .:
Thr Ile ~hr Phe Ile Tyr V~l Met Pro Ly~ S~r Thr Tyr S~r Thr Asp Cln A~n Ly~ Va} Val Sqr Val Pho ~yr M~t V~l Val Ile Pro M~t Lo~

(2) INFORMASION FOR SEQ ID NO:33: ~-(i) SEQUENCE CHARACTERISTICS:
(A) LENG~H: 479 ~ p~ir~
~B) TYPL: nucl~ic acld (C) ST~ANDEDN2SS: ~ingl~
(D) ~OPOLOGY: llno~r ~Ll) MOL~CUL~ TYP~: cDNA
(lil) HYPoTE~TI QL: Y~
(iv) ANT~-SENS~: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: r~t olfactory epithollu~ .. :
(~) STR~IN: Srp~gu~-Dawl~y rat . :
(F) SISSUE SYP~: ol~actory ~pithelium lV~ MEDTA~E 50URC~: ~
(~ CLO~E: J19 :
(ix) F~ATU~E: ~
~A) NAME/REY: CDS . .
~) LOCATI0N: 2..479 '~ .: ' ' (xi) SEQUENC~ DESCRIPTION: SEQ ID No:33:

Ile Cy~ Hi~ Pro Lau Ly~ Tyr Thr Val lle M~t A~n Hi~ Tyr Phe ;
' ~

' ~:
Sl3BSTITUTE SHEET

U'O 92/17~ h ~ 5 ~ ~ 7 PCr/US92/02741 1 ~ 5 10 lS
TGT GTG ATG CTG CTG crc TTC TCT GTG ~TC GTT AGC ATT GCA CAT GCa 94 Cy~ V~l M~t Leu L~u Lau Ph~ Ser Val Ph~ Val S~r Il~ Al~ Hi~ Al~

TTG TTC CAC A'rT TTi~ i~TG GTG TTG ATA CTG ACT TTC AGC ACA i~A~ ACT 142 LQU Ph~ Ilo L~u M~t V~l Lsu I 1Q Lou Thr Ph- S~r Thr Ly~ Th:
35 40 4~
GAA ATC CC~ QC TTT rTC TGT Gi~G CSG GCT CAT i~TC i~TC A~A CTT ACC 190 Glu Ila Pro H1~ Ph~ Ph~ Cy~ Glu L~u Al~ B Ly~ L~u Thr TGT TCC GAT i~AT TTT i~TC iLAC TAT CTG CTG ATA TAC AC~ GAG TCT GTC 238 Cy~ Ser Asp Aan Phe Il~ A~n Tyr Leu L4u Ilo Tyr Thr Glu Ser Val TTA TTT TTT GGT GTT CAT ATT GTA GG~ ATC ATT TTG TCT TAT ATT TAC 286 L~u Phe Ph~ Gly V~l His Ile Val Gly Ile Ile Leu s~r Tyr Il~ Tyr ACT GTA TCC TCA GTT TTA AGA ATG TCA TTA TTG GGA GGA ATG TAT i~AA 334 Thr Val sor S~r Val Leu Arg Mct Ser Leu L~u Gly Gly M~t Tyr Ly~

Ala Ph~ S~r Thr Cy~ Gly S~r 8i~ Leu S~r Val Val S~r Val Leu Trp 115 ~20 125 ~i~ Arg Phe Trp Gly Thr Hi~ Lya L~u S~r Thr Tyr ~ Leu Ser Ly~

GAA GAC TGT AGT GGC TTC AGT GAT GTA CAC ~GT GGT TAC TC~ GAT GCT G 4 7 9 Glu Asp Cys ssr Gly Ph~ Scr Asp Val Hiu Cy~ Gly Tyr S~r A~p Al~

.' ''' (2) INFORMATION FOR SEQ ID NO:34:
(i) S~QUENCE C8ARACTERISTICS:
(A) LENGTH: 159 amino acids (~) TYPE: ~mino acid ~D) TOPOLOGY: linsar ) MOLECULE TYPE: PrOtCin ~Xi) SE~UF~CE DESCRIPTION: SEQ ID NO:34:
I1e CY~ Hi~ PrO L~U LY5 ~yr 'rhr Va1 I1e Met A~n H18 ~Yr Phe CY~

Va1 MetLeULeULeU Phe SerVa1PheVa1 Ser I1e A1a H1~ A1a LeU

Phe H18I1eLeU~et Va1 LeUI1eLeUThr Phe Ser Thr LY8 Thr G1U
:
I 1e PrO Hi~ Phe Phe CY~ G1U LeU A1A Hi~ Ile Ile LY8 L8U Thr CY~

SUBSTiTUTE SHEET

~'0 92/1758~ PC~r/US92/02741 ' Sor A~p A~n Ph~ Iln A3n Iyr Lou LQU Il~ Sy Ths Glu S~r v~l L~U

Pho Ph- Cly V~l ~1 Ila V~l Cly 51~ Ilo Lou s~r Tyr Ils ryr ~hr g0 g5 V~l S~r S-r Val LDU Ar~ ~2t g-r L u L~u Oly 01Y ~-t ~yr ~y~ Al~

Pbo S~r Thr Cy- aly A~r ~l~ Lsu 55r V-l Val ~r V~l L~u ~rp ~l~

~r~ Ph~ Trp Cly Shr 8l~ Ly~ Lou S~r ~hr ~y~ o L~u S~r ~y~ Glu 130 135 1~0 A~p Cy- s-r ~ly Ph~ 8-r ~Jp V~ Cy~ ~ly 5yr S~r ~ Al~ :
1~5 150 155 (2) ~NFOR~ATION rOR SBQ ID NO:35:
~1) SEQUEHC~ C~ARACTERIS~ICS~
(A) LENGSH: 481 b~au p~r~
~S) TYPE: nucl-lc acld (C) S~RAN~DN5SS: ~lnglo ( D ) TOPOLOCY: 1 lnoar ~ OL~CU~ ~YP~: cDNA
(ill) HYPOTH~TIC~L: YES ~ :
~1~) ANSI-S2NS~: NO
(v1) OR~CINAL SOURC$2 ~A) ORGAN~ rat ol~ctory uplth~lLu~ .:
(~) STR~N~ Srp~gu~-D~wl-y rat ~ ISSU~ TYP~ olfactory ~p~th~llu~
(v~ EDlASL SOURC~ .
~8) C~ON~ J20 A ) NAMR /IU Y 5 CDS
~111 ) S~CA~2011 t 2 . . 4B1 ~xL~ SLQU~NC~ D1~SC~1PSION~ S~:Q ID NO~35~
A ATC rCC rAC C~ A C~; AGG TAC Crr CTC A~C A~G ACC S~; G~G a'rG 46 I 10 CYU SYr PrO L U Ar9 TYr L~U L~U I 1~1 H-t SOr SrP V~l1 V~

CA GQ csa TCt: GTG CC~ A'rC ~CG CTC ATA GCC m TOT GCC SCC 94 GY~ Thr A1A L~U S~r Va1 A1a I1~ TrP Ya1 I10 G1Y Pb~ Cy~ Ala S~r :::.
20 25 30 ~:
GTS A~A CCT C~C TGI: q"sc AC:G A~C CTC: CQ C~C TG5' GG'r CC'r TAC GSC 1 ~ 2 ~
V~ Pro LQu Cy~ Ph~ T~r Il~ Lau Pro L-u Cy- Cly Pro Tyr VA~
35 40 45 ; ~.:
G1`T GAS TAT ~ ~C 'rGC GAG CSG CCC ATC ~ CSC CAC CTG l~C St;C 1 9 0 V~1 ArP Tyr L~U Pha CYO G1U L0U PrO I1al L4U I~U H1 L8U Ph8 Cy#
50 ~ 55 i:
ACA GAT ACA TCT C$0 CSC GAG N~N NN~I NNN 2mN NNN NNN NNN N~N NHN 23 Thr A~p Ttlr S~r L~u L~u alu X~a xda Xaa Xa~ X~l Xa~ X-a Xaa X~

SUBSTITUTE SHEET ~
, .
, .

W092/l75~ P~/US92/0274 NNN NNN NNN NNN N~N CCC l`TC CTC CIY: A~ t;TT CTC ~CC T~C ~T CG~ 286 X a Xa~ Xn~ XaA Xa~ Pro Pha Lau ~u I 1- V 1 L~BU Sor Tyr Lou Arsl ATC t~G GTG GCT GT~ AT~ ~CA ATA GAC TCA Gt~ t,AC t;CC At;A A~A ~AC 334 Ilo L~u V~l Al~ V 1 Ilo ~rg Ils A-p Sor Als Glu Cly Arg Ly Ly-GCC rrT TCA ACT Ta'r t~ TCA CAC rrG ct T t;~:; G~ ACC ATC T~C TAT 382 Al~ Pha sor Thr CYD ~111 S~r Hl~ ~0U Al~ Val V~l1 Thr I:Lo Tyr Tyr 115 120 1;25 t;GA ACA t}GC; CrC ATC AGG TAC TTC AtX; CCC AAt TCC t TT TAT TCC GCT 430 Gly Thr G Ly Leu I1IJ Arg Tyr L~u Arg Pro Ly~ S~r Leu Tyr S~r Al~ -GAG GGA t;AC A~ t T~; ATC TCT GTG l~C T~T GCl~ CTC ATS GC5: CCT G~ A 478 Glu Gly A~p Arg Lelu 11~ Ser V~1 Pho Tyr ~la V~ Gly Pro Al~ -Lau ~2) INFOR~AT~ON FOR SEQ ID NO:36:
(1) SEQUENClÇ CHARACT2RISTICS:
(A) L~NCTH: 160 amino acida ( 8 1 ~YP1S: amlno cc ld ~D) TOPOLOGY: lin~r (ii) MOLECUL~ TYP~: protoin (xL~ SEQUENCIS D~SC~IPTIOM: 52Q ID NO:36:
Tyr Pro ~u Arg Tyr Lou Lou I l~ Mae Snr ~rrp Val VA1 Cy~

Thr A1~ L~U S r Va1 A~ TrP V 1 l1O ClY Phal CYI~ A1D Sor Vo.l `~

- Pro Lalu Cy- Phr ~llr Il~ Lau PrO L~u Cyll aly Pro Tyr V~l Val .. .
~5 40 45 A~p Iyr L~u Ph0 Cy~ Glu L6~u Pro Il~ Leu L~u Hi- Leu Ph~ Thr Asp Thr Ser Leu Leu Glu Xu~l Xaa X~ Xa~ X~ X~a X~ Xad Xall Xa~l ~0 Xaa Xaa Xaa Xaa PrO Pho L~u Leu I 1~ Val Leu S~r Tyr Leu Arg I 1 gO 95 Lau Val Al~ Val Ilo Arg Il0 Aap Sor Ala Glu ~ly Arg Lyu Ly~ Alll 100 105 110 .
Ph~ Ser Tllr Cy~ Ala S~3r His Leu P.l~ Val V~l Thr I12 Tyr Tyr Gly 115 1~20 ~25 Thr Gly Leu Ilo Arg Tyr Leu Arg Pro Lya S~r L~u Tyr Ser Ala Glu Gly i~p Arg Leu Ilff Ssr Val Ph0 Tyr All Val Il~ Cly Pro Ala Lau -:
~UBSTITUrE S~EET
,

Claims (98)

What is claimed is:

1. An isolated nucleic acid molecule encoding an odorant receptor.

2. An isolated DNA of claim 1.

3. An isolated cDNA of claim 2.

4. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 9.

5. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 10.

6. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 11.

7. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 12.

8. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 13.

9. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 14.

10. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 15.

11. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 16.

12. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 17.

13. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 18.

14. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 19.

15. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 20.

16. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 21.

17. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 22.

18. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 23.

19. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 24.

20. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 25.

21. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 26.

22. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 27.

23. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 28.

24. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 29.

25. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 30.

26. An isolated cDNA of claim 3 wherein the sequence of the cDNA has the nucleotide sequence shown in Figure 31.

27. An isolated cDNA of claim 3 encoding an insect odorant receptor.

28. An isolated cDNA of claim 3 encoding a vertebrate odorant receptor.

29. An isolated cDNA of claim 3 encoding a fish odorant receptor.

30. An isolated cDNA of claim 3 encoding a mammalian odorant receptor.

31. An isolated cDNA of claim 30 wherein the mammalian odorant receptor is a human odorant receptor.

32. An isolated cDNA of claim 30 wherein the mammalian odorant receptor is a rat, dog or mouse odorant receptor.

33. An expression vector comprising the cDNA of claim 3 and the sequence elements necessary for replication and expression in a suitable host.

34. An expression vector comprising the cDNA of any of claims 4-19 and the sequence elements necessary for replication and expression in a suitable host.

35. A purified protein encoding an odorant receptor.

36. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 9.

37. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 10.

38. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 11.

39. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 12.

40. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 13.

41. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 14.

42. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 15.

43. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 16.

44. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 17.

45. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 18.

46. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 19.

47. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 20.

48. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 21.

49. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 22.

50. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 23.

51. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 24.

52. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 25.

53. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 26.

54. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 27.

55. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 28.

56. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 29.

57. A purified protein of claim 35 wherein the amino acid sequence is the sequence in Figure 31.

58. A purified protein of claim 35 encoding an insect odorant receptor.

59. A purified protein of claim 35 encoding a vertebrate odorant receptor.

60. A purified protein of claim 35 encoding a fish odorant receptor.

61. A purified protein of claim 35 encoding a mammalian odorant receptor.

62. A purified protein of claim 61 wherein the mammalian odorant receptor is a human odorant receptor.

63. A purified protein of claim 61 wherein the mammalian odorant receptor is a rat, dog or mouse odorant receptor.

64. A purified protein of claim 35 which has 7 transmembrane regions and whose third cytoplasmic loop from the N-terminus is approximately 17 amino acid long.

65. A method of transforming cells which comprises transfecting a host cell with a suitable expression vector of claim 33.

66. A method of transforming cells which comprises transfecting a host cell with a suitable expression vector of claim 34.

67. Cells transformed by the method of claim 65.

68. Transformed cells of claim 67 wherein the cells are olfactory cells.

69. Transformed cells of claim 67 wherein the cells are non-olfactory cells.

70. A method of identifying a desired odorant ligand comprising contacting transformed non-olfactory cells of claim 69, expressing a known odorant receptor with a series of odorant ligands and determining which ligands bind to the receptors present on the non-olfactory cells.

71. A method of identifying a desired odorant receptor comprising contacting a series of transformed non-olfactory cells of claim 69 with a known odorant ligand and determining which odorant receptor binds with the odorant ligand.

72. A method of detecting an odor which comprises:

a) identifying a odorant receptor which binds the desired odorant ligand by the method of claim 71 and;

b) imbedding the receptor in a membrane such that when the odorant ligand binds with the receptor identified in a) above, a detectable signal is produced.

73. A method of claim 72 wherein the desired odorant is a pheromone.

74. A method of claim 72 wherein the desired odorant ligand is the vapors emanating from ***e, marijuana, heroin, hashish, or angel dust.

75. A method of claim 72 wherein the desired odorant ligand is the vapors emanating from gasoline, natural gas or alcohol.

76. A method of claim 72 wherein the desired odorant ligand is the vapors emanating from decayed human flesh.

77. A method of claim 72 wherein the desired odorant ligand is the vapors emanating from explosives, plastic explosives, firearms, or gun powder,.

78. A method of claim 72 wherein the desired odorant ligand is toxic fumes, noxious fumes or dangerous fumes.

79. A method of claim 72 wherein the membrane is a cell membrane.

80. A method of claim 72 wherein the membrane is an olfactory cell membrane.

81. A method of claim 72 wherein the membrane is a synthetic membrane.

82. A method of claim 72 wherein the detectable signal is a color change, phosporescene, or radioactivity.

83. A method of quantifying the amount of an odorant ligand present in a sample which comprises the method of claim 72 wherein the detectable signal is quantified.

84. A method of developing fragrances which comprises identifying a desired odorant receptor by the method of claim 71 then contacting non-olfactory cells, which have been transfected with an expression vector containing the cDNA of the desired odorant receptor such that the receptor is expressed upon the surface of the non-olfactory cell, with a series of compounds to determine which ones bind with the receptor.

85. A method of identifying an odorant fingerprint which comprises contacting a series of cells, which have been transformed such that each express a known odorant receptor, with a desired sample and determining the type and quantity of the odorant ligands present in the sample.

86. A method of identifying odorant ligands which inhibit the activity of a desired odorant receptor which comprises contacting the desired odorant receptor with a series of compounds and determining which compounds inhibit the odorant ligand - odorant receptor interaction.

87. A method of identifying appetite suppressant compounds which comprises identifying odorant ligands by the method of claim 86 wherein the desired odorant receptor is that which is associated with the perception of food.

88. A method of controlling appetite in a subject which comprises contacting the olfactory epithelium of the subject with the odorant ligands identified by the method of claim 87.

89. A nasal spray, to control appetite comprising the compounds identified by the method of claim 87 in a suitable carrier.

90. A method of trapping odors which comprises contacting a membrane which contains multiples of the desired odorant receptor, with a sample such that the desired odorant ligand is absorbed by the binding of the odorant ligand to the odorant receptor.

91. An odor trap employing the method of claim 90.

92. A method of controlling pest populations which comprises identifying odorant ligands by the method of claim 70 which are alarm odorant ligands and spraying the desired area with the identified odorant ligands.

93. A method of controlling a pest population which comprises identifying odorant ligands by the method of claim 70 which interfere with the interaction between the odorant ligands and the odorant receptors which are associated with fertility.

94. A method of claim 92 or 93 wherein the pest population is a population of insects.

95. A method of claim 92 or 93 wherein the pest population is a population of rodents.

96. A method of claim 95 wherein the population of rodents is a population of mice or rats.

97. A method of promoting fertility which comprises employing the method of claim 70 to identify odorant ligands which interact with the odorant receptors associated with fertility and administering he identified odorant ligands to a subject.

98. A method of inhibiting fertility which comprises employing the method of claim 70 to identifying odorant ligands which inhibit the interaction between the odorant ligands and the odorant receptors associated with fertility and administering the identified odorant ligands to a subject.