HK1116217B - Modulators of odorant receptors - Google Patents

Description

Modulators of odorant receptors

Priority of this application to U.S. provisional application serial No. 60/581,087 filed on 18.6.2004 and to U.S. provisional application serial No. 60/582,011 filed on 22.6.2004, the contents of each of which are hereby incorporated by reference in their entirety.

The invention was made with government support under grant number DC05782 issued by the national institutes of health.

Technical Field

The present invention relates to polypeptides capable of promoting cell surface localization and functional expression of odorant receptors. The invention also provides assays for detecting ligands specific for a variety of odorant receptors. In addition, the invention provides methods of screening for odorant receptor accessory protein polymorphisms and mutations associated with disease states, as well as methods of screening for therapeutic agents, ligands, and modulators of such proteins.

Background

Olfactory dysfunction is caused by a variety of causes and profoundly affects the quality of life of patients. Approximately 200 million americans experience some type of olfactory dysfunction. Studies have shown that olfactory dysfunction affects at least 1% of the population under 65 years of age, and well over 50% of the population over 65 years of age. The sense of smell determines the aroma of foods and beverages and serves as a warning system to detect environmental hazards such as rotten food, leaking natural gas, smoke or airborne pollutants. Loss or distortion of smell can adversely affect food preference, food intake, and appetite.

Olfactory disorders are classified as: 1) loss of smell: inability to detect qualitative olfactory sensations (e.g., lack of olfactory function), 2) partial olfactory loss: some, but not all, odors can be perceived, 3) hyposmia or microsmia: reduced sensitivity to odor, 4) olfactory hypersensitivity: abnormally acute olfactory function, 5) olfactory disturbance (malodor or wrong sense of smell): distorted or distorted olfactory or odor stimuli, 6) phantom smell (phantosmia): olfactory obstructive sensations (also known as olfactory hallucinations) perceived without odor stimuli and 7) olfactory recognition cannot: the smell sensation cannot be recognized.

Olfactory dysfunction is further classified as: 1) conductive or trafficking injury caused by obstruction of the nasal passage (e.g., chronic rhinoinflammation, polyposis, etc.), damage to sensory nerves caused by destruction of neuroepithelium (e.g., viral infection, airborne toxins, etc.), 3) central olfactory nerve damage caused by central nervous system injury (e.g., tumors, bumps affecting the olfactory tract, neurodegenerative disorders, etc.). These classifications are not mutually exclusive. For example, viruses can cause damage to the olfactory nerve epithelium, and they can also be transported through the olfactory nerve into the central nervous system, causing damage to central elements of the olfactory system.

Olfactory capacity is initially determined by neurons in the olfactory epithelium, the olfactory sensory neurons (hereinafter "olfactory neurons"). In the olfactory neurons, odorant receptor (hereinafter "OR") proteins, which are members of the G-protein coupled receptor (hereinafter "GPCR") superfamily, are synthesized in the endoplasmic reticulum, transported and finally concentrated at the cell surface membrane of cilia at the tips of dendrites. Given the role of OR in the recognition of targets forming olfactory axons, OR proteins are also present at axon terminals (see, e.g., Mombaerts, P., (1996) Cell87, 675-. In rodents, odors are transduced by up to 1000 different ORs encoded by a multigene family (see, e.g., Axe1, R. (1995) Sci Am1273, 154-. Each olfactory neuron expresses only one type of OR, forming the cellular basis for olfactory neurons to discriminate between odors (see, e.g., Lewcock, J.W., and Reed, R.R. (2004) Proc Natl Acad Sci U S A; Malnic, B. et al, (1999) Cell96, 713-Asn 723; Serizawa, S. et al, (2003) Science302, 2088-Asn 2094; each of which is incorporated herein by reference in its entirety).

A better understanding of olfactory sensations is needed. There is also a need for a better understanding of the function of the odorant receptor.

Brief description of the invention

The present invention relates to polypeptides capable of promoting cell surface localization and functional expression of odorant receptors. The invention also provides assays for detecting ligands specific for a variety of odorant receptors. In addition, the invention provides methods of screening for odorant receptor accessory protein polymorphisms and mutations associated with disease states, as well as methods of screening for therapeutic agents, ligands and modulators of such proteins.

In a preferred embodiment, the present invention provides a method of identifying an odorant receptor ligand, comprising the steps of: a) providing i) a cell line or cell membrane thereof comprising an odorant receptor and a reporter reagent, and ii) a test compound; b) exposing the test compound to a cell line;and c) measuring the activity of the reporter agent. In some embodiments, the cell line expresses REEP1, RTP1, RTP2, RTP1-A, RTP1-B, RTP1-C, RTP1-D and RTP1-E, RTP1-A1, RTP1-D1, RTP-D2 and RTP 1-D3. In some embodiments, the cell line is a heterologous cell line or a native cell line. In some embodiments, the cell line is a 293T cell line. In a preferred embodiment, the odorant receptor is a human odorant receptor. In other preferred embodiments, the test compound is an odor molecule. In other embodiments, the reporter agent is modulated by a cAMP response element, and in preferred embodiments, the cell line further comprises G _αolf. In other embodiments, the odorant receptor is a murine odorant receptor. In other embodiments, the odorant receptor is a synthetic odorant receptor. In preferred embodiments, the odorant receptor comprises S6/79, S18, S46, S50, MOR23-1, MOR31-4, MOR31-6, MOR32-5 and/or MOR 32-11. In other embodiments, the reporter agent is a luminescent agent. In other embodiments, the luminescent agent is luciferase. In an alternative embodiment, the method further comprises the step of detecting the presence or absence of an odorant receptor ligand based on the activity of the reporter agent.

In a preferred embodiment, the invention provides a cell line expressing an odorant receptor, wherein the expression is localized on the surface of the cell. In a preferred embodiment, the cell line comprises a heterologous gene. In a preferred embodiment, the heterologous gene comprises one or more of REEP1, RTP1, and RTP 2. In other preferred embodiments, the cell line is a 293T cell line. In some embodiments, the odorant receptor is a human odorant receptor. In a preferred embodiment, the odorant receptor is labeled with a reporter reagent. In some embodiments, the reporter reagent is a luminescent reporter reagent. In some embodiments, the light-emitting reporter reagent comprises glutathione-S-transferase (GST), c-myc, 6-histidine (6X-His), Green Fluorescent Protein (GFP), Maltose Binding Protein (MBP), influenza A virus Hemagglutinin (HA), beta-galactosidase, or GAL 4. In a preferred embodiment, the cell line further comprises G _αolfAnd (4) expressing. In a preferred embodiment, the odorant receptor is a murine odorant receptor. In some embodimentsIn one embodiment, the odorant receptor is a synthetic odorant receptor. In a preferred embodiment, the odorant receptors comprise S6/79, S18, S46, S50, MOR23-1, MOR31-4, MOR31-6, MOR32-5 and MOR 32-11.

The invention also provides an isolated nucleic acid comprising a sequence encoding a polypeptide comprising SEQ id no: 21, 27, 33, 34, 37, 38 and 41-50, and proteins corresponding to SEQ ID NO: 21, 27, 33, 34, 37, 38 and 41-50 are at least 80% identical variants thereof. In a preferred embodiment, the sequence is operably linked to a heterologous promoter. In a preferred embodiment, the sequence is contained in a vector. In a preferred embodiment, the vector is in a host cell.

The invention also provides isolated and purified nucleic acid sequences that hybridize under high stringency conditions to a nucleic acid sequence comprising SEQ ID NO: 1, 7, 13, 14, 17 and/or 18. In a preferred embodiment, the sequence is operably linked to a heterologous promoter. In a preferred embodiment, the sequence is contained in a vector. In some embodiments, the host vector is in a host cell. In a further preferred embodiment, the host vector is expressed in a host cell. In a preferred embodiment, the host cell is located in an organism, wherein the organism is a non-human animal. In a preferred embodiment, the invention provides a polynucleotide sequence comprising at least 15 (e.g., 15, 18, 20, 21, 25, 50, 100, 1000..) nucleotides that hybridizes to an isolated nucleotide sequence under stringent conditions.

In a preferred embodiment, the present invention provides a polypeptide consisting of a sequence selected from SEQ ID NOs: 1, 7, 13, 14, 17 and 18 and nucleic acids corresponding to SEQ ID NO: 1, 7, 13, 14, 17 and 18, at least 80% identical to the polypeptide encoded by the variant thereof. In other embodiments, the protein is identical to SEQ ID NO: 1, 7, 13, 14, 17 and 18 are at least 90% identical. In other embodiments, the protein is identical to SEQ ID NO: 1, 7, 13, 14, 17 and 18 are at least 95% identical.

In a preferred embodiment, the present invention provides a composition comprising a nucleic acid that inhibits a polypeptide selected from the group consisting of SEQ ID NOs: 1, 7, 13, 14, 17 and 18 with their complementary sequence.

In a preferred embodiment, the present invention provides a method of detecting a variant REEP polypeptide in a subject, comprising providing a biological sample from the subject, wherein the biological sample comprises the REEP polypeptide; and detecting the presence or absence of the variant REEP polypeptide in the biological sample. In a preferred embodiment, the biological sample is selected from the group consisting of a blood sample, a tissue sample, a urine sample and an amniotic fluid sample. In other embodiments, the subject is selected from the group consisting of an embryo, a fetus, a newborn animal, and a young animal. In other embodiments, the animal is a human. In a preferred embodiment, the detection comprises differential antibody binding. In other embodiments, the detecting comprises western blotting. In some preferred embodiments, the variant REEP polypeptide is a variant REEP1 polypeptide. In other embodiments, detecting comprises detecting a REEP1 nucleic acid sequence.

In a preferred embodiment, the present invention provides a method of detecting a variant RTP polypeptide in a subject, comprising providing a biological sample from the subject, wherein the biological sample comprises the RTP polypeptide; and detecting the presence or absence of the variant RTP polypeptide in the biological sample. In a preferred embodiment, the biological sample is selected from the group consisting of a blood sample, a tissue sample, a urine sample and an amniotic fluid sample. In other embodiments, the subject is selected from the group consisting of an embryo, a fetus, a newborn animal, and a young animal. In other embodiments, the animal is a human. In a preferred embodiment, the detection comprises differential antibody binding. In other embodiments, the detecting comprises western blotting. In some preferred embodiments, the variant RTP polypeptide is a variant RTP1 and/or RTP2 polypeptide. In other embodiments, detecting comprises detecting an RTP1 and/or RTP2 nucleic acid sequence. In a preferred embodiment, the RTP1 variant is selected from the group consisting of RTP1-A1, RTP1-D1, and RTP 1-D3.

In a preferred embodiment, the present invention provides a kit comprising reagents for detecting the presence or absence of a variant REEP polypeptide in a biological sample. In some embodiments, the kit further comprises instructions for using the kit to detect the presence or absence of a variant REEP polypeptide in a biological sample. In a preferred embodiment, the REEP polypeptide is a REEP1 polypeptide. In other embodiments, the REEP polypeptide is selected from REEP 1-6. In a preferred embodiment, the instructions include in vitro diagnostic kit instructions as required by the U.S. food and drug administration. In a preferred embodiment, the agent is one or more antibodies. In a preferred embodiment, the biological sample is selected from the group consisting of a blood sample, a tissue sample, a urine sample and an amniotic fluid sample. In a preferred embodiment, the reagents are configured to detect a REEP1 nucleic acid sequence.

In a preferred embodiment, the present invention provides a kit comprising reagents for detecting the presence or absence of a variant RTP polypeptide in a biological sample. In some embodiments, the kit further comprises instructions for using the kit to detect the presence or absence of a variant RTP polypeptide in a biological sample. In a preferred embodiment, the RTP polypeptide is an RTP1 and/or RTP2 polypeptide. In other embodiments, the RTP polypeptide is selected from RTP 1-4. In a preferred embodiment, the instructions include in vitro diagnostic kit instructions as required by the U.S. food and drug administration. In a preferred embodiment, the agent is one or more antibodies. In a preferred embodiment, the biological sample is selected from the group consisting of a blood sample, a tissue sample, a urine sample and an amniotic fluid sample. In preferred embodiments, the reagents are configured to detect RTP1 and/or RTP2 nucleic acid sequences. In a preferred embodiment, the RTP1 polypeptide is a variant RTP1 polypeptide selected from the group consisting of RTP1-A1, RTP1-D1, and RTP 1-D3.

In a preferred embodiment, the present invention provides a method of screening for a compound comprising providing a sample expressing a heterologous REEP polypeptide and a test compound; and exposing the sample to a test compound and detecting the biological effect. In a preferred embodiment, the REEP polypeptide is selected from REEP 1-6. In a preferred embodiment, the sample comprises cells. In a preferred embodiment, the sample comprises a tissue. In preferred embodiments, the sample is found in a subject. In some embodiments, the biological effect comprises a change in REEP activity. In some embodiments, the biological effect comprises a change in REEP expression.

In a preferred embodiment, the present invention provides a method of screening for a compound comprising providing a sample expressing a heterologous RTP polypeptide and a test compound; and exposing the sample to a test compound and detecting the biological effect. In a preferred embodiment, the RTP polypeptide is selected from the group consisting of RTP1-4 and RTP1-A1, RTP1-D1 and RTP 1-D3. In a preferred embodiment, the sample comprises cells. In a preferred embodiment, the sample comprises a tissue. In preferred embodiments, the sample is found in a subject. In some embodiments, the biological effect comprises a change in RTP activity. In some embodiments, the biological effect comprises a change in RTP expression.

Drawings

Figure 1 shows a screening strategy for identifying molecules that promote cell surface expression of odorant receptors. REEP1 was obtained from the numerical differential display analysis. RTP1 was obtained from the SAGE library.

Figure 2 shows that REEP and/or RTP promotes cell surface expression of odorant receptors in 293T cells. (A) cDNAs encoding different ORs (MOR203-1, OREG, o1fr62, OR-S46 and rat I7) were transfected with OR without REEP1, RTP1 and/OR RTP 2. In cells co-expressing the helper protein, increased cell surface staining of the OR was observed. In contrast, in cells expressing β 2 adrenergic receptors, no difference in cell surface staining was observed. Using a live cell staining protocol, cell surface fluorescence signals were taken as characteristic spot staining. The scale bar is equal to 50 um. (B) The normalized number of labeled cells is shown for each transfection condition (N4918-15526). FACS analysis was performed after double immunofluorescent staining for Rho-labeled receptors and HA-labeled β 2 adrenergic receptors to quantify the immunopositive cells. The number of Rho-labeled receptor positive cells was normalized to the number of HA-labeled β 2 adrenergic receptor positive cells. In almost all cases, more immune positive cells were observed when different ORs were expressed with REEP1, RTP1, and/OR RTP 2. In contrast, no enhancement was observed when VR4 and mT2R5 receptors were used instead of OR. (C) Normalized mean fluorescence of labeled cells is shown. The mean fluorescence of β 2 adrenergic receptors was used as a control. Stronger fluorescence was observed when different ORs were expressed with REEP1, RTP1, and/OR RTP 2. In contrast, no enhancement was observed when VR4 and mT2R5 receptors were used instead of OR. (D) A summary of FACS analysis is shown.

Figure 3 shows that REEP and/or RTP do not promote cell surface expression of VR4 and mT2R5 in 293T cells. The cDNAs encoding VR4 and mT2R5 were transfected with or without REEP1, RTP1 and/or RTP 2. Unlike OR, in cells expressing these proteins, no increased cell surface staining was seen. BFP expression is shown to confirm the high (about 70%) transfection efficiency of VR4 transfected cells. Using a live cell staining protocol, cell surface fluorescence signals were taken as characteristic spot staining. The scale bar is equal to 50 um.

Fig. 4 shows fluorescence histogram data for REEP1, RTP1 and RTP2 expression with odorant receptors (a) o1fr62 and (B) mT2R 5.

Figure 5 shows the REEP and RTP families. (A) The putative amino acid sequence of REEP 1. Solid bars represent putative transmembrane regions (TM). The first TM region may function as a signal peptide. (B) A rootless system tree of REEP family members. On the mouse genome, at least 6 REEP family members (REEP1-6) were identified. Yeast YOP1P, barley HVA22 and human DP1 are homologous to the REEP protein. (C) Putative amino acid sequences of RTP1 and RTP 2. Solid bars represent putative transmembrane domains. The shaded amino acids are conserved between RTP1 and RTP 2. On the mouse genome, there were 2 more members (RTP3 and 4).

Fig. 6 shows the expression of REEP1, RTP1 and RTP 2. (A) Northern blot analysis. Total RNA was used for northern blot analysis. The olfactory epithelium, the vomeronasal organ and the brain showed an approximately 3.6kb band corresponding to the REEP1 mRNA. Only olfactory epithelium and vomeronasal RNA showed bands of about 3.5kb and about 2.6kb corresponding to RTP1 and RTP2mRNA, respectively. Ethidium bromide staining of 18S rRNA is shown as a control. (B) In situ hybridization analysis in olfactory epithelium. Among the REEP members, olfactory neurons specifically express REEP1 only. The support cells express REEP 6. Among RTP members, olfactory neurons strongly express RTP1 and RTP 2. Olfactory neurons also express RTP4, but at much lower levels. OMPs are markers of mature olfactory neurons. The high magnification of REEP1, RTP1 and RTP2 indicates that all olfactory neurons can express all 3 molecules. Scale bar: 200um (70 um in high magnification photograph). (C) In situ analysis of REEP1 in the brain. A small group of brain cells express REEP 1. Scale bar: 200 um.

Figure 7 shows the association of odorant receptors with REEP1 and RTP 1. (A) Control Western blot analysis indicating expression of HA-labeled MOR203-1, Flag-labeled REEP1, RTP1, and ICAP1 in 293T cells. (B) When Flag-RTP1 or Flag-REEP1 precipitated, the HA-MOR203-1 protein co-precipitated (lanes 1 and 2). However, when Flag-ICAP-1 (negative control protein) was precipitated, no HA-MOR203-1 protein was detected (lane 3). (C) When HA-MOR203-1 precipitated, Flag-REEP1 and Flag-RTP1 were co-purified upon co-expression (lanes 1 and 2). The negative control protein (Flag-ICAP-1) was not co-precipitated (lane 3). Asterisks indicate nonspecific Ig proteins. (D) When RTP1 was transfected into 293T cells, little cell surface expression was observed. However, when RTP1 and Odorant Receptor (OREG) were co-transfected, more RTP1 staining signal was observed. (E) When REEP1 was transfected into 293T cells, a small amount of cell surface signal was observed. Co-expression of OR (o1fr62) did not alter expression of REEP 1. The scale bar is equal to 50 um.

Figure 8 shows that expression of REEP1, RTP1 or RTP2 enhances odorant receptor activation. (A) A schematic diagram showing CAMP Response Element (CRE) and luciferase for monitoring OR activation is shown. Activation of OR increases cAMP, which enhances luciferase reporter gene expression by CRE. (B) Normalized luciferase activity ± SEM (N ═ 4). REEP1, RTP1, and RTP2, expressed in various combinations with OREG, enhanced luciferase activity compared to OR alone. (C) relative luciferase activity + SEM (N ═ 4). Use of OREG OR-S46 to test whether REEP1, RTP1 OR RTP2 would alter the ligand specificity of OR. To is coming toRelative activation to different odors was obtained, and luciferase activity to 300uM vanillin (OREG) OR capric acid (OR-S46) was considered as 1 under each expression condition. (D) Normalized luciferase activity + SEM (N ═ 8). Enhanced response in Hana3A cells (a stable cell line expressing REEP1, RTP1, RTP2 and Golf) when expressing 3 different ORs. (E) Measurement of cAMP. Enhanced cAMP production in Hana3A cells at different concentrations of eugenol when transfected with OREG. Conversely, when β 2 adrenergic receptors are transfected and isoproterenol is used, cAMP production is at the expression of G _αolfThere was no difference between Hana3A cells and 293T cells.

FIG. 9 shows RT-PCR analysis of Hana3A cells; + represents a PCR product using a cDNA sample from Hana3A cells as template DNA; -represents a negative control without reverse transcriptase; m represents a DNA marker.

Fig. 10 shows cell surface expression of odorant receptors in Hana3A and 293T cells. cDNAs encoding 3 OREGs (OREG, o1fr62 and OR-S46) were transfected into Hana3A cells OR 293T cells. In Hana3A cells, enhanced cell surface staining was observed. The scale bar is equal to 50 um.

Figure 11 shows the recognition spectrum of odorant receptors for odorants. (A) The test odor is shown on the left. Color indicates relative luciferase activity (N ═ 4). Each OR responds to a different subset of scents. (B) And (C) normalized luciferase activity (N ═ 4). 139 chemicals were used for primary ligand screening of MOR203-1 and o1fr 62. MOR203-1 responds to pelargonic acid. 01fr62 responds to five related aroma compounds.

Fig. 12 shows cell surface expression of 8 odorant receptors in Hana3A cells. The scale bar is equal to 50 um.

Figure 13 shows a model of the role of REEP and/or RTP in odorant receptor expression.

FIG. 14 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 21) of murine REEP 1.

FIG. 15 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 2) and amino acid sequence (SEQ ID NO: 22) of murine REEP 2.

FIG. 16 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 23) of murine REEP 3.

FIG. 17 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 4) and amino acid sequence (SEQ ID NO: 24) of murine REEP 4.

FIG. 18 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 5) and amino acid sequence (SEQ ID NO: 25) of murine REEP 5.

FIG. 19 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 6) and amino acid sequence (SEQ ID NO: 26) of murine REEP 6.

FIG. 20 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 7) and amino acid sequence (SEQ ID NO: 27) of human REEP 1.

FIG. 21 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 8) and amino acid sequence (SEQ ID NO: 28) of human REEP 2.

FIG. 22 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 9) and amino acid sequence (SEQ ID NO: 29) of human REEP 3.

FIG. 23 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 10) and amino acid sequence (SEQ ID NO: 30) of human REEP 4.

FIG. 24 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 11) and amino acid sequence (SEQ ID NO: 31) of human REEP 5.

FIG. 25 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 12) and amino acid sequence (SEQ ID NO: 32) of human REEP 6.

FIG. 26 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 13) and amino acid sequence (SEQ ID NO: 33) of murine RTP 1.

FIG. 27 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 14) and amino acid sequence (SEQ ID NO: 34) of murine RTP 2.

FIG. 28 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 15) and amino acid sequence (SEQ ID NO: 35) of murine RTP 3.

FIG. 29 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 16) and amino acid sequence (SEQ ID NO: 36) of murine RTP 4.

FIG. 30 shows the nucleic acid (mRNA) sequence of human RTP1-A1 (SEQ ID NO: 17) and the amino acid sequence of human RTP1 (SEQ ID NO: 37).

FIG. 31 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 18) and amino acid sequence (SEQ ID NO: 38) of human RTP 2.

FIG. 32 shows the nucleic acid (mRNA) sequence (SEQ ED NO: 19) and amino acid sequence (SEQ ID NO: 39) of human RTP 3.

FIG. 33 shows the nucleic acid (mRNA) sequence (SEQ ID NO: 20) and amino acid sequence (SEQ ID NO: 40) of human RTP 4.

Figure 34 shows the activation pattern of human odorant receptors in response to odorant exposure.

FIG. 35 illustrates amino acid fragments of RTP1-A, RTP1-B, RTP1-C, RTP1-D, and RTP1-E compared to RTP 1.

FIG. 36 shows the murine amino acid sequence of RTP1-A (SEQ ID NO: 41).

FIG. 37 shows the murine amino acid sequence of RTP1-B (SEQ ID NO: 42).

FIG. 38 shows the murine amino acid sequence of RTP1-C (SEQ ID NO: 43).

FIG. 39 shows the murine amino acid sequence of RTP1-D (SEQ ID NO: 44).

FIG. 40 shows the murine amino acid sequence of RTP1-E (SEQ ID NO: 45).

Fig. 41 shows cell surface expression of OLFR62 in Hana3A and 293T cells. cDNAs encoding RTP1, RTP1-A, RTP1-B, RTP1-C, RTP1-D and RTP1-E were transfected into Hana3A or 293T cells. Increased cell surface staining was observed in Hana3A cells and 293T cells expressing RTP 1-D.

Figure 42 illustrates a luciferase assay for monitoring OLFR62 activity. CAMP Response Element (CRE) and luciferase were used to monitor activation of OLFR 62. Activation of OLFR62 increased cAMP, which increased luciferase reporter gene expression by CRE.

FIG. 43 shows OLFR62 activity as indicated by luciferase expression in Hana3A and 293T cells expressing RTP1, RTP1-A, RTP1-B, RTP1-C, RTP1-D, RTP1-E and control pCI.

FIG. 44 illustrates amino acid fragments of RTP1-A1, RTP1-D1, RTP1-D2, and RTP1-D3 compared to RTP1-A and RTP1-D, respectively.

FIG. 45 shows the murine amino acid sequence of RTP1-A1 (SEQ ID NO: 46) and the human amino acid sequence of RTP1-A1 (SEQ ID NO: 47).

FIG. 46 shows the murine amino acid sequence of RTP1-D1 (SEQ ID NO: 48).

FIG. 47 shows the murine amino acid sequence of RTP-D2 (SEQ ID NO: 49).

FIG. 48 shows the murine amino acid sequence of RTP-D3 (SEQ ID NO: 50).

Fig. 49 shows cell surface expression of OLFR62 in 293T cells. cDNAs encoding RTP1, RTP1-A1, RTP1-D1, RTP1-D2 and RTP1-D3 and control pCI were transfected into 293T cells. Increased cell surface staining was observed in 293T cells expressing RTP1-A1, RTP1-D1, and RTP 1-D3.

FIG. 50 shows OLFR62, OREG, S6 and 23-1 activities as indicated by luciferase expression in 293T cells expressing RTP1, RTP1-A1, RTP1-D1, RTP1-D2 and RTP1-D3 and control pCI.

FIG. 51 shows OLFR62, OREG, S6 and 23-1 activities as indicated by luciferase expression in Hana3A cells expressing RTP1, RTP1-A1, RTP1-D1, RTP1-D2 and RTP1-D3 and control pCI.

FIG. 52 shows cell surface expression of OLFR62, OREG, MOR203-1, S6 and 23-1 in 293T cells co-transfected with RTP1, RTP1-A1 or control pCI. The cDNAs encoding RTP1, RTP1-A1, and control pCI were transfected into cells.

FIG. 53 shows schematically the amino acid fragments of RTP1-A1-A (chimera 1), RTP1-A1-D2 (chimera 2), RTP1-A1-D1 (chimera 3), RTP4-A1-A (chimera 4), RTP4-A1-D2 (chimera 5) and RTP4-A1-D1 (chimera 6).

Figure 54 shows cell surface expression of OR in cells expressing RTP1, RTP4, chimera 1, chimera 2, chimera 3, chimera 4, chimera 5, chimera 6 and control pCI. The cdnas encoding RTP1, RTP4, RTP1-a1, chimera 1, chimera 2, chimera 3, chimera 4, chimera 5, chimera 6 and control pCI were transfected into 293T cells.

FIG. 55 shows OLFR62, OREG, S6 and 23-1 activity as indicated by luciferase expression in 293T cells expressing RTP1, RTP4, RTP1-A1, RTP1-D1, RTP1-D2, chimera 1, chimera 2, chimera 3, chimera 4, chimera 5, chimera 6 and control pCI.

FIG. 56 shows the detection of RTP1, RTP1-A, RTP1-B, RTP1-C, RTP1-A1, RTP1-D, chimera 4, chimera 5, RTP1-D3, RTP1-D1, chimera 6 and RTP4 using anti-RTP 1.

Definition of

To facilitate an understanding of the present invention, a number of terms are defined below.

As used herein, the term "REEP" when used in reference to a protein or nucleic acid refers to a REEP protein or nucleic acid encoding a REEP protein of the present invention. The term REEP includes the same proteins as wild-type REEPs (e.g., REEP1, REEP2, REEP3, REEP4, REEP5, and REEP6) and those derived from wild-type REEPs (e.g., variants of the REEP polypeptides of the invention). In some embodiments, a "REEP" is a wild-type murine REEP nucleic acid (mRNA) (e.g., SEQ ID NOS: 1-6) or a polypeptide encoded by a wild-type murine REEP amino acid sequence (e.g., SEQ ID NOS: 21-26). In other embodiments, "REEP" is a wild-type human REEP nucleic acid (mRNA) (e.g., SEQ ID NOS: 7-12) or a polypeptide encoded by a wild-type human REEP amino acid sequence (e.g., SEQ ID NOS: 27-32). Examples of REEP proteins or nucleic acids include, but are not limited to, REEP1, REEP2, REEP3, REEP4, REEP5, and REEP 6.

As used herein, the term "RTP" when used in reference to a protein or nucleic acid refers to an RTP protein or nucleic acid encoding an RTP protein of the present invention. The term RTP includes proteins identical to wild-type RTP (e.g., RTP1, RTP2, RTP3, and RTP4) and those derived from wild-type RTP (e.g., variants of the RTP polypeptides of the invention, including but not limited to RTP1-a, RTP1-B, RTP1-C, RTP1-D, RTP1-E, RTP1-a1, RTP1-D1, RTP-D2, RTP-D3, or chimeric genes constructed with portions of the RTP1 coding region (e.g., RTP1-a1-a (chimera 1), RTP1-a1-D2 (chimera 2), RTP1-a1-D1 (chimera 3), RTP4-a1-a (chimera 4), RTP4-a1-D2 (chimera 5) and RTP4-a 39 1-D1 (chimera 6)). In some embodiments, an "RTP" is a wild-type murine RTP nucleic acid (mRNA) (e.g., SEQ ID NOS: 13-16) or a polypeptide encoded by a wild-type or variant murine RTP amino acid sequence (e.g., SEQ ID NOS: 33-36, 41-50). In other embodiments, "RTP" is a wild-type human RTP nucleic acid (mRNA) (e.g., SEQ ID NO: 17 for RTP 1-A1; and SEQ ID NOS: 18-20 for RTP2, RTP3, and RTP4) or a polypeptide encoded by a wild-type human RTP amino acid sequence (e.g., SEQ ID NOS: 37-40). Examples of RTP proteins or nucleic acids include, but are not limited to, RTP1, RTP2, RTP3, RTP4, RTP1-A, RTP1-B, RTP1-C, RTP1-D, RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A (chimera 1), RTP1-A1-D2 (chimera 2), RTP1-A1-D1 (chimera 3), RTP4-A1-A (chimera 4), RTP4-A1-D2 (chimera 5), and RTP4-A1-D1 (chimera 6).

As used herein, the term "odorant receptor" refers to an odorant receptor produced from olfactory sensory neurons. Examples of odorant receptors include, but are not limited to, S6/79, S18, S46, S50, MOR23-1, MOR31-4, MOR31-6, MOR32-5 and MOR 32-11.

As used herein, the term "odorant receptor cell surface localization" or equivalent terms refers to molecular transport of odorant receptors to cell surface membranes. Examples of cell surface localization include, but are not limited to, cilia, which are localized at the tips of dendrites, and axon terminals.

As used herein, the term "odorant receptor functional expression" or equivalent terms refers to the ability of an odorant receptor to interact with an odorant receptor ligand (e.g., an odorant molecule).

As used herein, the term "olfactory disorder", "olfactory dysfunction", "olfactory disease" or similar term refers to a disorder, malfunction or disease that results in diminished olfactory sensations (e.g., olfactory aberrations). Examples of olfactory disorders, dysfunctions and/or diseases include, but are not limited to, head trauma, upper respiratory tract infections, anterior cranial pit tumors, Kallmann syndrome, foster kennedy syndrome, parkinson's disease, alzheimer's disease, huntington's chorea and exposure to toxic chemicals or infections. The diminished olfactory sensations can be classified as: loss of smell-lack of sense of smell; hyposmia-diminished olfactory sensation; olfactory disorder-distortion of olfactory sensation; malodor-bad or foul odor sensation; and olfactory reversal-olfactory sensation without appropriate stimulation.

As used herein, the term "REEP 1" when used in reference to a protein or nucleic acid refers to a REEP1 protein or a nucleic acid encoding a REEP1 protein of the present invention. The term REEP1 includes the same proteins as wild-type REEP1 and those derived from wild-type REEP1 (e.g., variants of the REEP1 polypeptides of the invention) or chimeric genes constructed with portions of the REEP1 coding region). In some embodiments, "REEP 1" is a wild-type murine REEP1 nucleic acid (mRNA) (SEQ ID NO: 1) or a polypeptide encoded by a wild-type murine amino acid sequence (SEQ ID NO: 21). In other embodiments, "REEP 1" is a wild-type human REEP1 nucleic acid (mRNA) (SEQ ID NO: 7) or a polypeptide encoded by the wild-type human REEP1 amino acid sequence (SEQ ID NO: 27). In other embodiments, "REEP 1" is a variant or mutant nucleic acid or amino acid.

As used herein, the term "RTP 1" when used in reference to a protein or nucleic acid refers to the RTP1 protein or nucleic acid encoding the RTP1 protein of the invention. The term RTP1 includes the same proteins as wild-type RTP1 and those derived from wild-type RTP1 (e.g., variants of the RTP1 polypeptide of the invention, including but not limited to RTP1-a, RTP1-B, RTP1-C, RTP1-D, RTP1-E, RTP1-a1, RTP1-D1, RTP-D2, RTP-D3) or chimeric genes constructed with portions of the RTP1 coding region (e.g., RTP1-a1-a (chimera 1), RTP1-a1-D2 (chimera 2), RTP1-a1-D1 (chimera 3), RTP4-a1-a (chimera 4), RTP4-a1-D2 (chimera 5) and RTP4-a1-D1 (chimera 6)). In some embodiments, "RTP 1" is a wild-type murine RTP1 nucleic acid (mRNA) (SEQ ID NO: 13) or a polypeptide encoded by a wild-type murine amino acid sequence (SEQ ID NO: 33). In other embodiments, "RTP 1" is a wild-type human RTP1 nucleic acid (mRNA) (SEQ ID NO: 17, for RTP1-A1) or a polypeptide encoded by the wild-type human RTP1 amino acid sequence (SEQ ID NO: 37). In other embodiments, "RTP 1" is a variant or mutant nucleic acid or amino acid.

As used herein, the term "RTP 2" when used in reference to a protein or nucleic acid refers to the RTP2 protein or nucleic acid encoding the RTP2 protein of the invention. The term RTP2 includes the same proteins as wild-type RTP2 and those derived from wild-type RTP2 (e.g., variants of the RTP2 polypeptide of the invention) or chimeric genes constructed with portions of the RTP2 coding region). In some embodiments, "RTP 2" is a wild-type murine RTP2 nucleic acid (mRNA) (SEQ ID NO: 14) or a polypeptide encoded by a wild-type murine amino acid sequence (SEQ ID NO: 34). In other embodiments, "RTP 2" is a wild-type human RTP2 nucleic acid (mRNA) (SEQ ID NO: 18) or a polypeptide encoded by the wild-type human RTP2 amino acid sequence (SEQ ID NO: 38). In other embodiments, "RTP 2" is a variant or mutant nucleic acid or amino acid.

As used herein, the terms "subject" and "patient" refer to any animal, e.g., mammals such as dogs, cats, birds, livestock, and preferably humans. Specific examples of "subjects" and "patients" include, but are not limited to, individuals with olfactory disorders, and individuals with olfactory disorder-related characteristics or symptoms.

As used herein, the phrases "symptoms of an olfactory disorder" and "characteristics of an olfactory disorder" include, but are not limited to, diminished olfactory sensations (e.g., olfactory aberrations).

The phrase "under conditions that provide symptomatic relief" refers to any degree of qualitative or quantitative relief of detectable symptoms of the olfactory disorder, including, but not limited to, a detectable effect on the rate of disease recovery, or a relief of at least one symptom of the olfactory disorder.

The term "siRNA" refers to short interfering RNAs. Methods of using siRNA are described in U.S. patent application nos.: 20030148519/A1 (incorporated herein by reference). In some embodiments, the siRNA comprises a duplex or double-stranded region that is about 18-25 nucleotides long; siRNA often contain about 2 to 4 unpaired nucleotides at the 3' end of each strand. At least one strand of the duplex or double-stranded region of the siRNA is substantially homologous or substantially complementary to the target RNA molecule. The strand complementary to the target RNA molecule is the "antisense strand, the strand homologous to the target RNA molecule is the" sense strand, "and is also complementary to the siRNA antisense strand. The siRNA may also contain additional sequences; non-limiting examples of such sequences include linker sequences, or loops, as well as stems and other folded structures. sirnas appear to act as key mediators that trigger RNA interference in invertebrates and vertebrates, and sequence-specific RNA degradation during post-transcriptional gene silencing in plants.

The term "RNA interference" or "RNAi" refers to the silencing or reduction of gene expression by siRNA. It is a sequence-specific, post-transcriptional gene silencing process in animals and plants, initiated by sirnas homologous to the silenced gene sequence in its duplex region. The gene may be endogenous or exogenous to the organism, present integrated into the chromosome, or present in a transfection vector that is not integrated into the genome. The expression of the gene is completely or partially suppressed. RNAi can also be considered to inhibit the function of the target RNA; the function of the target RNA may be complete or partial.

As used herein, the terms "instructions for using the kit to detect the presence or absence of a variant REEP1 nucleic acid or polypeptide in the biological sample", "instructions for using the kit to detect the presence or absence of a variant RTP1 nucleic acid or polypeptide in the biological sample", "instructions for using the kit to detect the presence or absence of a variant RTP2 nucleic acid or polypeptide in the biological sample" include instructions for using the reagents contained in the kit to detect variant and wild-type REEP and/or RTP nucleic acids or polypeptides.

The term "gene" refers to a nucleic acid (e.g., DNA) sequence comprising coding sequences necessary for the production of a polypeptide, RNA (e.g., including, but not limited to, mRNA, tRNA, and rRNA), or precursor (e.g., REEP1, RTP1, or RTP 2). The polypeptide, RNA, or precursor can be encoded by a full-length coding sequence or any portion of a coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the full-length or fragment are maintained. The term also includes the coding region of the structural gene, and sequences located adjacent to the coding region at a distance of about 1kb from either end at the 5 'and 3' ends, such that the gene corresponds to the length of the full-length mRNA. Sequences located 5 'to the coding region and present on the mRNA are referred to as 5' untranslated sequences. Sequences located 3 'or downstream of the coding region and present on the mRNA are referred to as 3' untranslated sequences. The term "gene" includes both cDNA and genomic forms of a gene. Genomic forms or clones of a gene contain coding regions interrupted by non-coding sequences, referred to as "introns" or "intervening regions" or "intervening sequences". Introns are gene segments transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements, such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; messenger RNA (mRNA) transcripts therefore have no introns. mRNA plays a role in the translation process, defining the order or sequence of amino acids in the nascent polypeptide.

More specifically, the terms "REEP 1 gene", "RTP 1 gene", "RTP 1 gene(s)", "RTP 2 gene" or "RTP 2 gene(s)" refer to each full-length REEP and/or RTP nucleotide sequence (e.g., as contained in SEQ ID NOs: 1, 2 and 3). However, the term is also intended to include fragments of the REEP and/or RTP sequences (e.g., RTP1-a, RTP1-B, RTP1-C, RTP1-D and RTP1-E, RTP1-a1, RTP1-D1, RTP-D2, RTP-D3), chimeric genes constructed with portions of the RTP1 coding region (e.g., RTP1-a1-a (chimera 1), RTP1-a1-D2 (chimera 2), RTP1-a1-D1 (chimera 3), RTP4-a1-a (chimera 4), RTP4-a1-D2 (chimera 5) and RTP4-a1-D1 (chimera 6)), mutants of the REEP and/or RTP sequences, as well as other domains within the full-length REEP and/or RTP nucleotide sequences. Furthermore, the terms "REEP 1 nucleotide sequence", "REEP 1 polynucleotide sequence", "RTP 1 nucleotide sequence", "RTP 1 polynucleotide sequence", "RTP 2 nucleotide sequence" or "RTP 2 polynucleotide sequence" include DNA sequences, cDNA sequences, RNA (e.g., mRNA) sequences and related regulatory sequences.

When "amino acid sequence" is recited herein to refer to the amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and similar terms such as "polypeptide" or "protein" are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the protein molecule.

In addition to containing introns, genomic forms of a gene may also contain sequences located at the 5 'and 3' ends of the sequences present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (which flanking sequences are located 5 'or 3' to the untranslated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct transcription termination, post-transcriptional cleavage, and polyadenylation.

The term "wild-type" refers to a gene or gene product that has the characteristics of the gene or gene product when isolated from a naturally occurring source. Wild-type genes are the genes most frequently observed in a population, and thus the "normal" or "wild-type" form of a gene is arbitrarily designed. Conversely, the terms "modified", "mutant", "polymorphism" and "variant" refer to a gene or gene product that exhibits a modification (i.e., altered characteristic) in sequence and/or functional properties as compared to the wild-type gene or gene product. It should be noted that naturally occurring mutants may be isolated; they can be identified by the fact that they have altered properties compared to the wild-type gene or gene product.

As used herein, the terms "nucleic acid molecule encoding...," DNA sequence encoding.. and "DNA encoding.. refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus encodes an amino acid sequence.

DNA molecules are said to have "5 'ends" and "3' ends" because mononucleotides are reacted to prepare oligonucleotides or polynucleotides in such a way that the 5 'phosphate group of one mononucleotide pentose ring is bound to its adjacent 3' oxygen in one direction by a phosphodiester bond. Thus, if its 5 ' phosphate group is not linked to the 3 ' oxygen of the pentose ring of the mononucleotide, this end of the oligonucleotide or polynucleotide is called the "5 ' end"; a "3 ' terminus" is defined if its 3 ' oxygen does not have the 5 ' phosphate group attached to the pentose ring of the next mononucleotide. As used herein, a nucleic acid sequence may be referred to as having 5 'and 3' ends, even within a larger oligonucleotide or polynucleotide. In a linear or circular DNA molecule, discrete elements are referred to as being "upstream" or 5 'of "downstream" or 3' elements. This term reflects the fact that transcription proceeds in a 5 'to 3' manner along the DNA strand. Promoter and enhancer elements that direct the transcription of a linked gene are typically located 5' or upstream of the coding region. However, enhancer elements can exert their effects even when located 3' to the promoter element and coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.

As used herein, the terms "oligonucleotide having a nucleotide sequence encoding a gene" and "polynucleotide having a nucleotide sequence encoding a gene" refer to a nucleic acid sequence comprising the coding region of a gene, in other words, a nucleic acid sequence encoding a gene product. The coding region may be present in the form of cDNA, genomic DNA or RNA. When present in DNA form, the oligonucleotide or polynucleotide may be single-stranded (i.e., the sense strand) or double-stranded. If it is desired to allow proper initiation of transcription and/or proper processing of the primary RNA transcript, appropriate control elements (e.g., enhancers/promoters, splice junctions, polyadenylation signals, etc.) may be placed in close proximity to the coding region of the gene. Alternatively, the coding region used in the expression vectors of the invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc., or a combination of endogenous and exogenous control elements.

As used herein, the term "regulatory element" refers to a genetic element that controls some aspect of the expression of a nucleic acid sequence. For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements include splicing signals, polyadenylation signals, termination signals, and the like.

As used herein, the term "complementary" or "complementarity" is used to refer to polynucleotides (i.e., nucleotide sequences) related by the base-pairing rules. For example, for sequence 5 '- "A-G-T-3'", it is complementary to sequence 3 '- "T-C-A-5'". Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Alternatively, "complete" or "full" complementarity may exist between nucleic acids. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and detection methods that rely on binding between nucleic acids. Complementarity may include the formation of base pairs between any type of nucleotides, including non-natural bases, modified bases, synthetic bases, and the like.

The term "homology" refers to the degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence is a sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid, and is referred to using the functional term "substantially homologous". The term "inhibition of binding" when used in reference to binding of nucleic acids refers to inhibition of binding by competition of homologous sequences for binding to the target sequence. Inhibition of hybridization of a perfectly complementary sequence to a target sequence can be examined using hybridization assays under low stringency conditions (DNA or RNA blotting, solution hybridization, etc.). Under low stringency conditions, a substantially homologous sequence or probe will compete for and inhibit binding (i.e., hybridization) of the perfect homologue to the target. This is not to say that conditions of low stringency are those which allow non-specific binding; low stringency conditions require that the binding of 2 sequences to each other be a specific (i.e., selective) interaction. Using a second target that even lacks a partial degree of complementarity (e.g., less than about 30% identity), the absence of non-specific binding can be tested; in the absence of non-specific binding, the probe will not hybridize to a second non-complementary target.

It is recognized in the art that many equivalent conditions can be employed to constitute low stringency conditions; factors such as the length and nature of the probe (DNA, RNA, base composition) and the nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.), and the concentration of salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered, and the hybridization solution can be varied to produce different (but equivalent) low stringency hybridization conditions than those described above. In addition, conditions are known in the art that promote hybridization under high stringency conditions (e.g., increasing the temperature of the hybridization and/or washing steps, using formamide in the hybridization solution, etc.).

The term "substantially homologous" when used in reference to a double-stranded nucleic acid sequence (e.g., a cDNA or genomic clone) refers to any probe that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.

Genes can produce a variety of RNA species resulting from differential splicing of primary RNA transcripts. cDNAs that are splice variants of the same gene will contain regions of sequence identity or complete homology (representing the presence of the same exon or portions of the same exon on both cDNAs) and regions of complete non-identity (e.g., representing the presence of exon "A" on cDNA1, where cDNA2 instead contains exon "B"). Since 2 cDNAs contain regions of sequence identity, they all hybridize with probes derived from the entire gene or portions of the gene containing the sequences present on the 2 cDNAs; thus, the 2 splice variants are substantially homologous to such probes and to each other.

The term "substantially homologous" when used in reference to a single-stranded nucleic acid sequence refers to any probe that can hybridize to (i.e., is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.

As used herein, the term "competitive binding" is used to refer to the binding of a first polypeptide having activity to the same substrate as a second polypeptide having activity, wherein the second polypeptide is a variant of the first polypeptide or a related or dissimilar polypeptide. The binding efficiency (e.g., kinetics or thermodynamics) of a first polypeptide can be the same as, greater than, or less than the substrate binding efficiency of a second polypeptide. For example, the equilibrium binding constant (K) for 2 polypeptides to bind to a substrate_D) May be different. The term "Km" as used herein refers to the michaelis constant of an enzyme and is defined as the concentration of a particular substrate at which a given enzyme produces half of its maximum rate in an enzyme-catalyzed reaction.

As used herein, the term "hybridization" is used to refer to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the bond between nucleic acids) are affected by: for example, the degree of complementarity between nucleic acids, the stringency of the conditions involved, the T of the hybrids formed _mAnd the G: C ratio within the nucleic acid.

As used herein, the term "T_m"used to refer to" melting temperature ". Melting temperature is a population of double-stranded nucleic acid moleculesTemperature at which half of the molecules dissociate into single strands. It is well known in the art to calculate T for nucleic acids_mThe equation of (c). As indicated in the standard literature, when the nucleic acid is in 1M NaCl aqueous solution, can be through the following equation to calculate T_mSimple estimate of the value: t is_m81.5+0.41 (% G + C) (see, e.g., Anderson and Young, Quantitative Filter Hybridization, Nucleic Acid Hybridization [1985 ]]). Other documents include more complex calculations, which are at T_mThe structure and sequence characteristics are considered in the calculation of (2).

As used herein, the term "stringency" is used in reference to the temperature, ionic strength, conditions of presence of other compounds (e.g., organic solvents) at which nucleic acid hybridization is carried out. One skilled in the art will recognize that the "stringency" conditions can be varied by varying the above parameters individually or in concert. Using "high stringency" conditions, nucleic acid base pairing occurs only between nucleic acid fragments that have a high frequency of complementary base sequences (e.g., hybridization under "high stringency" conditions can occur between homologs that are about 85-100% identical, preferably about 70-100% identical). Using medium stringency conditions, nucleic acid base pairing can occur between nucleic acids having a medium frequency of complementary base sequences (e.g., hybridization under "medium stringency" conditions can occur between homologs having about 50-70% identity). Thus, nucleic acids derived from genetically different organisms often require conditions of "weak" or "low" stringency because the frequency of complementary sequences is generally lower.

"high stringency conditions" as used when referring to nucleic acid hybridization comprise conditions equivalent to: when a probe of about 500 nucleotides in length was used, the probe was purified by 5XSSPE (43.8g/1NaCl, 6.9g/1 NaH)₂PO₄ H₂O and 1.85g/1EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5 XDenhardt's reagent and 100. mu.g/ml denatured salmon sperm DNA at 42 ℃ binding or hybridization, followed by a 0.1 XSSPE, 1.0% SDS containing solution at 42 ℃.

When used in reference to nucleic acid hybridizationConditions of equal stringency "comprise conditions equivalent to: when a probe of about 500 nucleotides in length was used, the probe was purified by 5XSSPE (43.8g/1NaCl, 6.9g/1 NaH)₂PO₄ H₂O and 1.85g/1EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5 XDenhardt's reagent and 100g/ml denatured salmon sperm DNA at 42 ℃ binding or hybridization, followed by a solution containing 1.0 XSSPE, 1.0% SDS at 42 ℃ washing.

"Low stringency conditions" comprise conditions equivalent to: when a probe of about 500 nucleotides in length was used, the probe was purified by 5XSSPE (43.8g/l NaCl, 6.9g/l NaH)₂PO₄ H₂O and 1.85g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent [50X Denhardt's reagent per 500ml containing: 5g Ficoll (Type400, pharmacia), 5g BSA (Fraction V; Sigma) ]And 100. mu.g/ml denatured salmon sperm DNA at 42 ℃ followed by washing at 42 ℃ in a solution containing 5 XSSPE, 0.1% SDS.

The present invention is not limited to hybridization of probes that are about 500 nucleotides long. The invention encompasses the use of probes that are about 10 nucleotides in length up to thousands (e.g., at least 5000) of nucleotides in length. Those skilled in the relevant art will appreciate that stringency conditions can be varied for probes of other sizes (see, e.g., Anderson and Young, Quantitative Filter Hybridization, Nucleic Acid Hybridization [1985], Sambrook et al, molecular cloning: A Laboratory Manual, Cold Spring Harbor Press, NY [1989 ]).

The following terms are used to describe the sequence relationships between 2 or more polynucleotides: "reference sequence", "sequence identity", "percentage of sequence identity" and "substantial identity". A "reference sequence" is a particular sequence that serves as a basis for sequence alignment; the reference sequence may be a subset of a larger sequence, for example, as a fragment of the full-length cDNA sequence given in the sequence listing, or may comprise the entire gene sequence. Typically, the reference sequence is at least 20 nucleotides long, often at least 25 nucleotides long, often at least 50 nucleotides long. Since 2 polynucleotides may each (1) comprise a sequence that is similar between 2 polynucleotides (i.e., a portion of the complete polynucleotide sequence), and (2) may further comprise a sequence that differs between 2 polynucleotides, sequence alignments between 2 (or more) polynucleotides are typically performed by aligning the sequences of the 2 polynucleotides over an "alignment window" to identify and align local regions of sequence similarity. As used herein, a "comparison window" refers to a conceptual fragment of at least 20 contiguous nucleotide positions in which a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides, and in which the portion of the polynucleotide sequence in the comparison window may comprise 20% or fewer additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the 2 sequences. Local homology algorithms by Smith and Waterman [ Smith and Waterman, adv.appl.math.2: 482(1981), by the homology alignment algorithm of Needleman and Wunsch [ Needleman and Wunsch, J.mol.biol.48: 443(1970) ], by the search similarity method of Pearson and Lipman [ Pearson and Lipman, proc.natl.acad.sci. (u.s.a.) -85: 2444(1988) ], by computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA inter Wisconsin Genetics Software Package Release7.0, Genetics computer Group, 575Science Dr., Madison, Wis.) or by inspection, it is possible to make an optimal alignment of the sequences over the alignment window and to select the optimal alignment produced by various methods (i.e.producing the highest percentage of homology over the alignment window). The term "sequence identity" means that the 2 polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term "percent sequence identity" is calculated as follows: comparing 2 optimally aligned sequences over a comparison window, determining the number of positions at which the same nucleobase (e.g., a, T, C, G, U, or I) occurs in both sequences, generating the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100, generating the percentage of sequence identity. As used herein, the term "substantial identity" refers to a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence having at least 85% sequence identity, preferably at least 90-95% sequence identity, more often at least 99% sequence identity, as compared to a reference sequence in a window of comparison of at least 20 nucleotide positions (often a window of comparison of at least 25-50 nucleotides), wherein the percentage of sequence identity is calculated as follows: the reference sequence and the polynucleotide sequence are aligned over a window of alignment, which may comprise deletions or additions which together comprise 20% or less of the reference sequence. The reference sequence may be a subset of a larger sequence, for example, as a fragment of a full-length sequence (e.g., REEP1, RTP1, or RTP2) of the claimed compositions.

As applied to polypeptides, the term "substantial identity" means that 2 peptide sequences, when optimally aligned (e.g., by the programs GAP or BESTFIT, using default GAP weights), have at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity or greater (e.g., 99% sequence identity). Preferably, the residue positions that are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; one group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine and tryptophan; one group of amino acids having basic side chains is lysine, arginine and histidine; and a group of amino acids having sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine.

As used herein, the term "fragment" refers to a polypeptide having an amino-terminal and/or carboxy-terminal deletion as compared to the native protein, but wherein the remaining amino acid sequence is identical to the corresponding position in the amino acid sequence deduced from the full-length cDNA sequence. Fragments are typically at least 4 amino acids long, preferably at least 20 amino acids long, usually at least 50 amino acids long or longer, and span the portion of the polypeptide required for intermolecular binding of the composition (as claimed herein) with its different ligands and/or substrates.

The term "polymorphic advantage" is the presence of a locus in a population that exhibits variation between members of the population (i.e., the most common allele has a frequency of less than 0.95). In contrast, a "monomorphic locus" is a genetic locus where little or no variation is observed between members of a population (typically a locus where the most common allele in a population's gene pool has a frequency of more than 0.95).

As used herein, the term "genetic variation information" or "genetic variant information" refers to the presence or absence of one or more variant nucleic acid sequences (e.g., polymorphisms or mutations) in a given allele of a particular gene (e.g., the REEP and/or RTP genes of the invention).

As used herein, the term "detection assay" refers to an assay for detecting the presence or absence of a variant nucleic acid sequence (e.g., a polymorphism or mutation) in a given allele of a particular gene (e.g., a REEP and/or RTP gene).

As used herein, the term "naturally occurring" as applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and that has not been intentionally modified artificially in the laboratory is naturally occurring.

"amplification" is a special case involving template-specific nucleic acid replication. It is in contrast to non-specific template replication (i.e., template-dependent, but not specific template-dependent replication). Template specificity is distinguished herein from the fidelity of replication (i.e., synthesis of the appropriate polynucleotide sequence) and nucleotide (ribose-or deoxyribose-) specificity. Template specificity is often described in terms of "target" specificity. The target sequence is a "target" in the sense that it is sought to be sorted from other nucleic acids. Amplification techniques were originally designed for this sorting.

As used herein, the term "primer" refers to an oligonucleotide, whether naturally occurring (as in a purified restriction digest) or synthetically produced, that is capable of acting as a point of initiation of synthesis when placed under conditions (i.e., at a suitable temperature and pH in the presence of a nucleotide and an inducing agent, such as a DNA polymerase) that induce synthesis of a primer extension product complementary to a nucleic acid strand. For maximum amplification efficiency, the primer is preferably single-stranded, but may alternatively be double-stranded. If double stranded, the primer is first treated to separate its strands and then used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be long enough to prime the synthesis of extension products in the presence of an inducing agent. The exact length of the primer depends on many factors, including temperature, source of primer, and use of the method.

As used herein, the term "probe" refers to an oligonucleotide (i.e., a nucleotide sequence) that is capable of hybridizing to another oligonucleotide of interest, whether occurring naturally (e.g., in a purified restriction digest) or produced synthetically, recombinantly or by PCR amplification. The probe may be single-stranded or double-stranded. Probes can be used to detect, identify and isolate specific gene sequences. It is contemplated that any probe used in the present invention may be labeled with any "reporter" and therefore detectable in any detection system, including but not limited to enzymatic (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. The present invention is not intended to be limited to any particular detection system or label.

As used herein, the term "target" refers to a nucleic acid sequence or structure to be detected or characterized. Thus, attempts are made to sort out "targets" from other nucleic acid sequences. A "fragment" is defined as a region of nucleic acid within a target sequence.

As used herein, the term "amplification reagents" refers to those reagents (deoxyribonucleotide triphosphates, buffers, etc.) necessary for amplification in addition to primers, nucleic acid template, and amplification enzyme. Typically, amplification reagents, as well as other reaction components, are placed and contained in a reactor (test tube, microwell, etc.).

As used herein, the terms "restriction endonuclease" and "restriction enzyme" refer to bacterial enzymes, each of which cleaves double-stranded DNA at or near a specific nucleotide sequence.

As used herein, the term "recombinant DNA molecule" refers to a DNA molecule comprised of segments of DNA joined together by molecular biological techniques.

As used herein, the term "antisense" is used to refer to an RNA sequence that is complementary to a particular RNA sequence (e.g., mRNA). This definition includes antisense RNA ("asRNA") molecules that are involved in gene regulation of bacteria. Antisense RNA can be generated by any method, including synthesis by reverse splicing of the gene of interest to a viral promoter that allows synthesis of the coding strand. Once introduced into the embryo, the transcribed strand binds to the native mRNA produced by the embryo, forming a duplex. These duplexes then block further transcription of the mRNA or its translation, in this way mutant phenotypes can be generated. The term "antisense strand" is used to refer to a strand of nucleic acid that is complementary to the "sense" strand. Identification (-) (i.e., "negative") is sometimes used to refer to the antisense strand, and identification (+) is sometimes used to refer to the sense (i.e., "positive") strand.

The term "isolated" when used in conjunction with a nucleic acid, as in "isolated oligonucleotide" or "isolated polynucleotide," refers to a nucleic acid sequence that is identified and isolated from at least one contaminating nucleic acid with which it normally accompanies in its natural source. An isolated nucleic acid exists in a form or environment different from its native state. Conversely, non-isolated nucleic acids are nucleic acids such as DNA and RNA that are found in their naturally occurring state. For example, a given DNA sequence (e.g., a gene) is found in the chromosome of a host cell in the vicinity of a gene; RNA sequences, such as a particular mRNA sequence encoding a particular protein, are found in cells as a mixture with many other mrnas encoding a variety of proteins. However, as an example, an isolated nucleic acid encoding a REEP and/or RTP includes a nucleic acid in a cell that normally expresses a REEP and/or RTP, where the nucleic acid is at a chromosomal location different from that of the native cell, or is flanked by nucleic acid sequences different from those found in nature. An isolated nucleic acid, oligonucleotide or polynucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid, oligonucleotide or polynucleotide is used to express a protein, the oligonucleotide or polynucleotide contains at a minimum the sense or coding strand (i.e., the oligonucleotide or polynucleotide may be single-stranded), but may contain both the sense and antisense strands (i.e., the oligonucleotide or polynucleotide may be double-stranded).

As used herein, "part of a chromosome" refers to a discrete portion of a chromosome. Cytogeneticists divided the chromosomes into sites or parts as follows: the short (relative to the centromere) arm of the chromosome is called the "p" arm; the long arm is referred to as the "q" arm. Each arm was then divided into 2 regions, referred to as region 1 and region 2 (region 1 nearest to the centromere). Each region is further divided into bands. The tape may be further divided into sub-tapes. For example, part 11p15.5 of human chromosome 11 is the part located on sub-band 5(.5) of band 5 of region 1 (1) of the short arm (p) of chromosome 11 (11). A portion of a chromosome may be "altered"; for example, due to a deletion, the entire portion may be absent, or may be rearranged (e.g., inverted, translocated, expanded or contracted due to changes in repeat regions). In the case of a deletion, attempting to hybridize (i.e., specifically bind) to a probe homologous to a particular portion of a chromosome can produce a negative result (i.e., the probe cannot bind to a sample containing genetic material suspected of containing the deleted portion of the chromosome). Thus, hybridization of probes homologous to a particular portion of a chromosome can be used to detect a change in a portion of the chromosome.

The term "chromosome-associated sequence" refers to a preparation of a chromosome (e.g., the expansion of a metaphase chromosome), nucleic acids extracted from a sample containing chromosomal DNA (e.g., a preparation of genomic DNA), RNA generated by transcription of genes located on a chromosome (e.g., hnRNA and mRNA), and cDNA copies of RNA transcribed from DNA located on a chromosome. Chromosomal-related sequences can be detected by a number of techniques, including probing of DNA and RNA blots with probes containing sequences homologous to the nucleic acids in the preparations described above and in situ hybridization to RNA, DNA or metaphase chromosomes.

As used herein, the term "coding region" when used in reference to a structural gene refers to a nucleotide sequence that encodes an amino acid found in a nascent polypeptide as a result of translation of an mRNA molecule. In eukaryotes, the coding region is bounded on the 5 'side by the nucleotide triplet "ATG" that encodes the initiator methionine and on the 3' side by one of the three triplets (i.e., TAA, TAG, TGA) of the designated stop codon.

As used herein, the term "purified" or "purification" refers to the removal of contaminants from a sample. For example, REEP and/or RTP antibodies are purified by removing contaminating non-immunoglobulin albumin; they are also purified by removing immunoglobulins that do not bind REEP and/or RTP polypeptides. Removal of non-immunoglobulin proteins and/or removal of immunoglobulins that do not bind to REEP and/or RTP polypeptides results in an increase in the percentage of REEP1, RTP1, or RTP 2-reactive immunoglobulins in the sample. In another example, recombinant REEP and/or RTP polypeptides are expressed in bacterial host cells and the polypeptides are purified by removal of host cell proteins; thereby increasing the percentage of recombinant REEP and/or RTP polypeptides in the sample.

As used herein, the term "recombinant DNA molecule" refers to a DNA molecule comprised of segments of DNA joined together by molecular biological techniques.

The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule expressed from a recombinant DNA molecule.

As used herein, the term "native protein" is used to refer to a protein that does not contain amino acid residues encoded by a vector sequence; native proteins contain only those amino acids found in naturally occurring proteins. Native proteins may be produced recombinantly or may be isolated from naturally occurring sources.

As used herein, when referring to a protein (as in "a portion of a given protein"), the term "portion" refers to a fragment of that protein. The size of the fragment may be reduced by 1 amino acid from 4 consecutive amino acid residues to the entire amino acid sequence.

The term "southern blot" refers to the analysis of DNA on agarose or acrylamide gels to fractionate the DNA according to size, followed by transfer of the DNA from the gel to a solid support, such as nitrocellulose or nylon membranes. The immobilized DNA is then probed with a labeled probe to detect DNA species complementary to the probe used. The DNA may be cleaved with restriction enzymes prior to electrophoresis. After electrophoresis, the DNA may be partially depurinated and denatured prior to or during transfer to a solid support. Southern blotting is a standard tool for Molecular biologists (J.Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring harborPress, NY, pp9.31-9.58[1989 ]).

As used herein, the term "northern blot" refers to the analysis of RNA by running the RNA on an agarose gel to fractionate the RNA according to size, followed by transfer of the RNA from the gel to a solid support, such as a nitrocellulose or nylon membrane. The immobilized RNA is then probed with a labeled probe to detect RNA species complementary to the probe used. Northern blotting is a standard tool for molecular biologists (J.Sambrook, et al, supra, pp7.39-7.52[1989 ]).

The term "western blot" refers to the analysis of a protein (or polypeptide) immobilized to a support (e.g., nitrocellulose or membrane). Proteins are run on acrylamide gels to isolate the proteins, and then the proteins are transferred from the gel to a solid support, such as nitrocellulose or nylon membranes. The immobilized protein is then exposed to an antibody having reactivity to the antigen of interest. Binding of the antibody can be detected by a variety of methods, including the use of radiolabeled antibodies.

As used herein, the term "antigenic determinant" refers to the portion of an antigen (i.e., an epitope) that is contacted by a particular antibody. When a protein or protein fragment is used to immunize a host animal, many regions of the protein can elicit the production of antibodies that specifically bind to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants. An antigenic determinant may compete with the intact antigen (i.e., the "immunogen" used to elicit the immune response) for binding to an antibody.

As used herein, the term "transgene" refers to a foreign, heterologous, or autologous gene that is placed into an organism by introducing the gene into a newly fertilized egg or early embryo. The term "foreign gene" refers to any nucleic acid (e.g., gene sequence) that is introduced into the genome of an animal by experimental manipulation, and may include gene sequences found in the animal, so long as the introduced gene is not located at the same position as the naturally occurring gene. The term "autologous gene" is intended to include variants (e.g., polymorphisms or mutants) of naturally occurring genes. The term transgene thus includes a variant form in which a naturally occurring gene is replaced with the gene.

As used herein, the term "vector" is used to refer to a nucleic acid molecule that transfers a DNA segment from one cell to another. The term "medium" is sometimes used interchangeably with "carrier".

As used herein, the term "expression vector" refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotic cells generally include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

As used herein, the term "host cell" refers to any eukaryotic or prokaryotic cell (e.g., bacterial cells such as e.coli, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo. For example, the host cell can be located in a transgenic animal.

The term "overexpression" and grammatical equivalents are used to refer to the level of mRNA to indicate an expression level that is about 3-fold higher than that typically observed in a given tissue of a control or non-transgenic animal. The level of mRNA can be measured using any of a number of techniques known to those skilled in the art, including but not limited to northern blot analysis (see, example 10, protocol for performing northern blot analysis).

As used herein, the term "transfection" refers to the introduction of exogenous DNA into a eukaryotic cell. Transfection may be accomplished by a variety of means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, 1, 5-dimethyl-1, 5-diaza-undecamethylene polymethine bromide-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

The term "stable transfection" or "stably transfected" refers to the introduction and integration of foreign DNA into the genome of a transfected cell. The term "stable transfectant" refers to a cell that has stably integrated foreign DNA into genomic DNA.

The term "transient transfection" or "transient transfection" refers to the introduction of foreign DNA into a cell, wherein the foreign DNA is not integrated into the genome of the transfected cell. The foreign DNA remains in the nucleus of the transfected cells for several days. During this time, the foreign DNA is under regulatory control that governs the expression of the endogenous gene in the chromosome. The term "transient transfectant" refers to a cell that has taken up exogenous DNA, but does not integrate the DNA.

The term "calcium phosphate co-precipitation" refers to a technique for introducing nucleic acids into cells. When nucleic acids are presented as calcium phosphate-nucleic acid co-precipitates, uptake of nucleic acids by cells is enhanced. Several groups have improved the Graham and van der Eb original technology (Graham and van der Eb, Virol., 52: 456[1973]) to optimize the conditions for a particular cell type. Such various modifications are well known in the art.

As used herein, "a composition comprising a given polynucleotide sequence" broadly refers to any composition comprising a given polynucleotide sequence. The composition may comprise an aqueous solution. Compositions comprising polynucleotide sequences encoding REEP1, RTP1, or RTP2 (e.g., SEQ id nos 1, 2, and 3), or fragments thereof, can be employed as hybridization probes. In this case, the polynucleotide sequences encoding REEP and/or RTP are typically used in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS), and other components (e.g., Denhardt's solution, milk powder, salmon sperm DNA, etc.).

The term "test compound" refers to any chemical entity, drug, etc., that can be used to treat or prevent a disease or disorder of bodily function, or to alter the physiological or cellular state of a sample. Test compounds include known and potential therapeutic compounds. By screening using the screening methods of the invention, it can be determined that the test compound is therapeutic. "known therapeutic compound" refers to a therapeutic compound that has been demonstrated to be effective in such treatment or prevention (e.g., by animal testing or prior experience of administration to humans).

As used herein, the term "sample" is used in its broadest sense. A sample suspected of containing human chromosomes or sequences associated with human chromosomes can comprise cells, chromosomes isolated from cells (e.g., unfolding of metaphase chromosomes), genomic DNA (in solution or bound to a solid support, e.g., for southern blot analysis), RNA (in solution or bound to a solid support, e.g., for northern blot analysis), cDNA (in solution or bound to a solid support), and the like. A sample suspected of containing a protein may comprise a cell, a portion of a tissue, an extract containing one or more proteins, and the like.

As used herein, the term "reaction" when used in reference to an assay refers to the generation of a detectable signal (e.g., accumulation of a reporter protein, increase in ion concentration, accumulation of a detectable chemical product).

As used herein, the term "reporter gene" refers to a gene that encodes a protein that can be assayed. Examples of reporter genes include, but are not limited to, luciferase (see, e.g., deWet et al, mol. cell. biol.7: 725[1987] and U.S. Pat. Nos. 6,074,859, 5,976,796; 5,674,713; and 5,618,682; all of which are incorporated herein by reference), green fluorescent protein (e.g., GenBank accession No. U43284; many GFP variants are commercially available from CLONTECHLABORIES, Palo Alto, Calif.), chloramphenicol acetyltransferase, beta-galactosidase, alkaline phosphatase, and horseradish peroxidase.

General description

The inability to functionally express OR in heterologous cells to identify cognate ligands significantly hinders the continued progress in understanding olfactory encoding. To overcome this problem, experiments conducted in the course of the present invention searched for molecules contained in the cell surface expression of OR. 3 transmembrane proteins REEP1, RTP1 and RTP2, and variants thereof, that promote functional cell surface expression of OR in 293T cells were identified. Olfactory neurons in the olfactory epithelium specifically express REEP and/or RTP. REEP1 and RTP1 interact with OR proteins. Using cells expressing REEP1 and RTP1 and RTP2, novel ORs that react to aliphatic odorants were identified. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nevertheless, experiments carried out in the course of the present invention confirm the importance of the accessory proteins of OR in functional cell surface expression and decoding of OR-ligand specificity.

The identification and use of proteins involved in localization of OR provides for a number of research, diagnostic, drug screening and therapeutic applications. For example, the nucleic acids and proteins of the invention allow for the selective and controllable presentation of OR on test cells to specifically identify novel ORs, characterize ORs, identify OR ligands, correlate olfactory responses and potential molecular interactions of such responses, identify and characterize sets of ORs and ligands responsible for olfactory responses and health conditions, and identify, select and characterize modulators of OR responses to study and control olfactory responses. The present invention thus also provides a means of manipulating olfactory reactions and the molecular basis of such reactions in vitro and in vivo. Thereby enabling many commercial uses, including the production, characterization and use of in vitro OR in vivo cell arrays expressing the desired localized OR, for screening (e.g., high throughput screening) compounds OR as synthetic olfactory systems. The compositions and methods of the present invention may be particularly useful in any industry, including the food industry, health industry, cosmetics industry, military, health agencies, sniffing animals (e.g., for pharmaceuticals, explosives, accident victims, etc.).

Inhibitors of OR/ligand interactions (e.g., antibodies, small molecules, aptamers, etc.) identified by the methods of the invention have a variety of uses. For example, the present invention provides a systematic way to identify which receptors and ligands are responsible for a particular sense of smell (e.g., a perceived odor). Thus, for example, perceived odor may be controlled by blocking specific interactions (e.g., by a nasal spray containing an inhibitor) or enhancing specific interactions (e.g., by a nasal spray that provides certain ligands or provides a coating on the surface of a subject that emits certain ligands). Thus, undesirable odors can be blocked, covered, or modified (e.g., sniffing dogs can be treated to sniff only the target of interest, but not other confusing odors, sanitizers can be immunized against the odor of trash, etc.), and desirable odors can be enhanced.

The invention also provides novel gene and protein sequences and methods of their use. The following describes a detailed description of certain preferred embodiments and applications of the present invention. The present invention is not limited to these specific exemplary embodiments.

Detailed Description

The present invention relates to peptides capable of promoting cell surface localization and functional expression of odorant receptors. The invention also provides assays for detecting therapeutic agents and for detecting odorant receptor accessory protein polymorphisms and mutations associated with disease states. Exemplary embodiments of the invention are described below.

Exemplary compositions and methods of the invention are described in more detail in the following sections: I. olfactory sensation; reep and RTP polynucleotides; reep and RTP polypeptides; detection of reep and RTP alleles; production of reep and RTP antibodies; gene therapy using REEP and RTP; transgenic animals expressing exogenous REEP and RTP genes and homologues, mutants and variants thereof; drug screening using REEP and RTP; IX. pharmaceutical compositions containing REEP and RTP nucleic acids, peptides and analogs; rna interference (RNAi); RNAi to reep and RTP; identification of odorant receptor ligands.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are well explained in the literature, for example, "Molecular cloning: a laboratory manual "2 nd edition (Sambrook et al, 1989); "Oligonucleotide synthesis" (edited by m.j. gait, 1984); "Animal Cell culture" (ed. r.i. freshney, 1987); series "Methods in enzymology" (Academic Press, Inc.); "Handbook of experimental immunology" (eds. D.M.Weir & CC. Blackwell); "Gene transducers for mammalian cells" (eds. J.M.Miller & M.P.Calos, 1987); "Current protocols in molecular biology" (ed.F.M. Ausubel et al, eds., 1987 and periodic updates); "PCR: the polymerase chain reaction "(Mullis et al, 1994); and "Current protocols in immunology" (J.E.Coligan et al, 1991), each of which is incorporated herein by reference in its entirety.

I. Sense of smell

The olfactory system represents one of the oldest sensory modalities in the phylogenetic history of mammals. The human smell is less well developed than other mammals (e.g., rodents). As a chemical sensor, the olfactory system detects food and affects social and sexual behavior. Specialized olfactory epithelial cells characterize a unique group of neurons capable of regeneration. Activation occurs when the odorant molecule contacts a specialized protuberance called the olfactory bulb. In the nasal cavity, the turbinates or turbinates serve to direct inhaled air to the olfactory epithelium in the posterior superior region. This region (just a few centimeters wide) contains more than 1 billion olfactory receptor cells. These specialized epithelial cells produce an olfactory bulb containing the motile cilia, which serves as a site for stimulation of transduction.

There are 3 specialized nervous systems in the nasal cavity of humans: 1) the main olfactory system (cranial nerve I), 2) the trigeminal sensory system (cranial nerve V), 3) the terminal nerve (cranial nerve 0). Cranial nerve I mediates odorant perception. It is responsible for determining odor. Cranial nerve V mediates bodily sensations including burns, cold, irritation and itching. Cranial nerve 0 is the plexus with ganglia. It is distributed in most nasal mucosa and then passes through the lamina cribrosa into the forebrain inside the olfactory tract. The exact function of the human terminal nerve is not known.

The olfactory neuroepithelium is a pseudostratified columnar epithelium. Specialized olfactory epithelial cells are the only group of neurons capable of regeneration. The olfactory epithelium is located in the upper part of each nostril and comprises the parts of the lamina cribrosa, the superior turbinate, the superior septum and the middle turbinate. It contains sensory receptors of the main olfactory system and some cranial nerve V free nerve endings. The olfactory epithelium loses its general homogeneity after birth and islands of histotransformation of the respiratory-like epithelium occur as early as the first few weeks of life. The range of tissue transformation increases throughout life. It is presumed that this process is the result of stimuli from the environment (e.g., viruses, bacteria, and toxins).

There are 6 different cell types in the olfactory neuroepithelium: 1) bipolar sensory receptor neurons, 2) micro-villus hair cells, 3) supporting cells, 4) globular basal cells, 5) horizontal basal cells, 6) cells lining the olfactory glands. In adult olfactory neuroepithelium, there are approximately 6,000,000 bipolar neurons. They are thin dendritic cells with ciliated columns at one end and long central neurites forming olfactory filaments at the other end. Olfactory receptors are located at the ciliated dendritic ends. Unmyelinated axons were combined into 40 bundles, called olfactory filaments, which covered Schwann-like cells. The olfactory filaments cross the lamina cribrosa and enter the anterior cranial fossa and form the cranial nerve I. Microvilli cells are near the surface of the neuroepithelium, but the exact function of these cells is unclear. The supporting cells are also on the surface of the epithelium. They tightly connect neurons and micro-hair cells. They also extend microvilli into the mucus. Their functions include insulating the recipient cells from each other, regulating the composition of mucus, inactivating odorants, and protecting the epithelium from foreign agents. Basal cells are located near the basement membrane and are progenitor cells derived from other cell types. The olfactory gland is the main source of mucus in the olfactory epithelial region.

Odorant receptors are located on cilia of recipient cells. Each receptor cell expresses a single odorant receptor gene. There are now approximately 1,000 receptors. The olfactory receptor is linked to a stimulatory guanine nucleotide binding protein, Golf. When stimulated, it activates adenylate cyclase, generating the second messenger cAMP, with subsequent events leading to depolarization of the cell membrane and signal propagation. Although each recipient cell expresses only one type of receptor, each cell responds electrophysiologically to a wide but limited range of stimuli. This means that a single receptor accepts a range of molecular entities.

The olfactory bulb is located above the lamina cribosa at the base of the frontal lobe of the anterior cranial fossa. It accepts thousands of primary axons from olfactory receptor neurons. Within the olfactory bulb, these axes form synapses with a much smaller number of secondary neurons, which form the olfactory tract and extend to the olfactory cortex. The olfactory cortex includes the frontal and temporal lobes, the thalamus and hypothalamus.

Although mammalian ORs were identified more than 10 years ago, the selectivity of the different ORs to chemical stimuli was barely known, primarily because of the difficulty in expressing the ORs on the cell surface of heterologous cells and determining their ligand-binding specificity (see, e.g., Mombaerts, P. (2004) Nat Rev Neurosci 5, 263-278; herein incorporated by reference in its entirety). The reason is that the OR protein is retained in the ER and subsequently degraded in the proteosome (see, e.g., Lu, M. et al, (2003) Traffic4, 416-. Despite these difficulties, extensive efforts have been made to match, to varying degrees of certainty, about 20 ORs to homologous ligands (see, e.g., Bozza, T. et al, (2002) J Neurosci22, 3033-3043; Gaill, L et al, (2002) Eur J Neurosci15, 409-418; Hatt, H. et al, (1999) Cell Mol Biol45, 285-291; Kajiya, K. et al, (2001) JNeurosci21, 6018-6025; Krautwurst, D. et al, (1998) Cell95, 917-926; Malnic, B. et al, (1999) Cell96, 713-723; Raming, K. et al, (1993) Nature361, 353-356; Spehr, M. et al, (2003) Science 2058, 404, 2058; Touha. K. 723; Nature 20545, 1998; Nature 202, K. 279, K. 242; Nature361, K. 242; Nature K. 242, et al, cited in each, Nature, S.K. 242, K. 76, 1998, et al, incorporated by Nature K. No. 76, et al, incorporated herein, incorporated by reference, et al, et. The addition of the 20N-terminal amino acids of rhodopsin (e.g., Rho-tag) OR a foreign signal peptide to the N-terminus promotes surface expression of some OR in heterologous cells (see, e.g., Hatt, H., et al, (1999) Cell Mol Biol45, 285-. However, for most ORs, the modification does not reliably promote cell surface expression. For example, ODR-4, which is required for the proper localization of chemosensory receptors in Meloidogyne possesses little effect on promoting cell surface expression of one rat OR, but not the other (see, e.g., Gimelbrant, A.A., et al, (2001) J Biol Chem276, 7285-7290; incorporated herein by reference). These findings suggest that olfactory neurons have a selective molecular mechanism that facilitates the appropriate targeting of OR proteins to the cell surface, but the components of this mechanism have not been identified (see, e.g., Gimelbrant, A.A. et al, (2001) J Biol Chem276, 7285-.

For some GPCRs, accessory proteins are required for proper targeting to Cell surface membranes (see, e.g., Brady, A.E., and Limbird, L.E. (2002) Cell Signal14, 297-309; incorporated herein by reference in its entirety). These proteins include NinaA for the rhodopsin of the Drosophila (see, e.g., Baker, E.K. et al, (1994) Embo J13, 4886-4895; Shieh, B.H. et al, (1989) Nature338, 67-70; each of which is incorporated herein by reference in its entirety), RanBP2 for the mammalian cone opsin (see, e.g., Ferreira, P.A. et al, (1996) Nature383, 637-640; incorporated herein by reference in its entirety), RAMP for the mammalian calcitonin receptor-like receptor (CRLR) (see, e.g., McLatchee, L.M. et al, (1998) Nature, 333-339; incorporated herein by reference in its entirety) and finally the M10 family of MHC class I proteins and β 2 microglobulin for the V2R, V2R are putative mammalian pheromone receptors (see, e.g., Loto, J.393, 2003-cono et al, (2003) 112; incorporated herein by reference in its entirety). These helper proteins do not have any sequence homology to each other, except NinaA and RanBP 2; their only common feature is their association with the membrane.

The present invention provides novel proteins (e.g., REEP1, RTP1, RTP2, RTP1-A, RTP1-B, RTP1-C, RTP1-D, RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A (chimera 1), RTP1-A1-D2 (chimera 2), RTP1-A1-D1 (chimera 3), RTP4-A1-A (chimera 4), RTP4-A1-D2 (chimera 5) and RTP4-A1-D1 (chimera 6)) that promote OR cell surface localization and functional expression of OR, and a number of compositions and methods related to these findings.

Reep and RTP polynucleotides

As described above, the present invention provides novel proteins that promote odorant receptor cell surface localization and odorant receptor functional expression. More specifically, the present invention provides REEP genes and polypeptides (e.g., REEP1, REEP2, REEP3, REEP4, REEP5, and REEP6) and RTP genes and polypeptides (e.g., RTP1, RTP2, RTP3, RTP4, RTP1-a, RTP1-B, RTP1-C, RTP1-D, RTP1-E, RTP1-a1, RTP1-D1, RTP-D2, RTP-D3, RTP1-a1-a (chimera 1), RTP1-a1-D2 (chimera 2), RTP 2-a 2-D2 (chimera 3), RTP 2-a (chimera 4), RTP 2-a 2-D2 (chimera 5), and RTP 2-a 2-D2 (chimera 6)). In preferred embodiments, REEP1, RTP1, variants of RTP2 and RTP1 (e.g., RTP1-a, RTP1-B, RTP1-C, RTP1-D and RTP1-E, RTP1-a1, RTP1-D1, RTP-D2, RTP-D3, RTP1-a1-a (chimera 1), RTP1-a1-D2 (chimera 2), RTP1-a1-D1 (chimera 3), RTP4-a1-a (chimera 4), RTP4-a1-D2 (chimera 5) and RTP4-a1-D1 (chimera 6) promote odor receptor cell surface localization and odor receptor functional expression.

Accordingly, the present invention provides nucleic acids encoding REEP genes, homologs, variants (e.g., polymorphisms and mutants), including, but not limited to, SEQ ID NOs: 1-12. The present invention provides nucleic acids encoding RTP genes, homologs, variants (e.g., polymorphisms and mutants), including, but not limited to, SEQ ID NOs: 13-20. Table 1 describes exemplary REEP and RTP genes of the present invention. In some embodiments, the invention provides a polypeptide that hybridizes to SEQ ID NO: 1-20, provided that the polynucleotide sequence capable of hybridizing encodes a protein that retains the biological activity of the naturally occurring REEP and/or RTP protein. In some embodiments, the protein that retains the biological activity of a naturally occurring REEP and/or RTP is at% homology to wild-type REEP and/or RTP70, preferably at% homology to wild-type REEP and/or RTP80, more preferably at% homology to wild-type REEP and/or RTP90, and most preferably at% homology to wild-type REEP and/or RTP 95. In a preferred embodiment, the hybridization conditions are based on the melting temperature (T) of the nucleic acid binding complex _m) And with reference to "stringency" as defined above (see, e.g., Wahl et al, meth.enzymol., 152: 399-.

In other embodiments of the invention, additional alleles of REEP and/or RTP genes are provided. In preferred embodiments, the alleles are derived from polymorphisms or mutations (i.e., changes in nucleic acid sequence), and typically produce altered mrnas or polypeptides, which may or may not be altered in structure or function. Any given gene may have 0, 1 or more allelic forms. Common mutational changes that result in alleles are often attributed to deletions, additions or substitutions of nucleic acids. Each of these types of variations may occur one or more times in a given sequence, either alone or in combination with each other. Other examples include truncation mutations (e.g., such that the encoded mRNA does not produce the complete protein).

In other embodiments of the invention, the nucleotide sequences of the invention may be engineered to alter REEP and/or RTP coding sequences for a variety of reasons, including, but not limited to, alterations that modify the cloning, processing, and/or expression of the gene product. For example, mutations can be introduced using techniques well known in the art (e.g., site-directed mutagenesis to insert new restriction sites, change glycosylation patterns, change codon bias, etc.). Variants of RTP1 include, but are not limited to, RTP1-A, RTP1-B, RTP1-C, RTP1-D and RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A (chimera 1), RTP1-A1-D2 (chimera 2), RTP1-A1-D1 (chimera 3), RTP4-A1-A (chimera 4), RTP4-A1-D2 (chimera 5) and RTP4-A1-D1 (chimera 6).

In some embodiments of the invention, the polynucleotide sequence of REEP and/or RTP can be extended to detect upstream sequences such as promoters and regulatory elements using nucleotide sequences in a variety of methods known in the art. For example, it is contemplated that restriction site Polymerase Chain Reaction (PCR) may be used in the present invention. This is a direct method of using universal primers to recover unknown sequences adjacent to known loci (Gobinda et al, PCR Methods application, 2: 318-22 (1993); incorporated herein by reference in its entirety). First, genomic DNA is amplified in the presence of a primer having a linker sequence and a primer specific to a known region. Then, a second round of PCR is performed on the amplified sequences using the same ligation primers and primers of another specificity within the first. The products of each round of PCR are transcribed with the appropriate RNA polymerase and sequenced using reverse transcriptase.

In another embodiment, reverse PCR may be used to amplify or extend sequences using divergent primers based on known regions (Triglia et al, Nucleic Acids Res., 16: 8186[1988 ]). Primers can be designed to be 22-30 nucleotides long, have a GC content of 50% or higher, and anneal to the target sequence at a temperature of about 68-72 ℃, using oligo4.0(National Biosciences Inc, Plymouth Minn.) or another suitable procedure. This method uses several restriction enzymes to generate appropriate fragments in known gene regions. The fragment is then circularized by intramolecular ligation and used as a template for PCR. In other embodiments, step-wise PCR is used. Step-up PCR is a targeted gene-stepping method that allows for recovery of unknown sequences (Parker et al, Nucleic Acids Res., 19: 3055-60[1991 ]). The PROMOTTERFINDER kit (Clontech) uses PCR, nested primers, and a special library to "walk in" genomic DNA. This process does not require screening of libraries and can be used to find intron/exon junctions.

Preferred libraries for screening full-length cdnas include mammalian libraries that have been size-selected to contain larger cdnas. In addition, libraries of random primers are also preferred because they will contain more sequences containing both the 5' and upstream gene regions. In cases where the oligo d (T) library does not produce full-length cDNA, a library of random primers may be particularly useful. Genomic mammalian libraries can be used to obtain introns and to extend 5' sequences.

In other embodiments of the invention, variants of the disclosed REEP and/or RTP sequences are provided. In a preferred embodiment, the variant is derived from a polymorphism or mutation (i.e., a change in nucleic acid sequence), and typically produces an altered mRNA or polypeptide, which may or may not be altered in structure or function. Any given gene may have 0, 1 or multiple variant forms. Common mutational changes that produce variants are often attributed to deletions, additions or substitutions of nucleic acids. Each of these types of variations may occur one or more times in a given sequence, either alone or in combination with each other.

It is contemplated that the structure of a peptide having a function (e.g., REEP and/or RTP function) can be modified for purposes of, among others, altering biological activity (e.g., altered REEP and/or RTP function). Such modified peptides are considered as functional equivalents of peptides having the activity of REEP and/or RTP peptides as defined herein. Modified peptides may be generated in which the nucleotide sequence encoding the polypeptide has been altered, for example by substitution, deletion or addition, and in particularly preferred embodiments these modifications do not significantly reduce the biological activity of the modified REEP and/or RTP gene, in other words construct "X" may be assessed to determine whether it is a member of the functionally (rather than structurally) defined modified or variant REEP and/or RTP genera of the present invention. In preferred embodiments, the activity of a variant REEP and/or RTP polypeptide is assessed by the methods described herein (e.g., production of transgenic animals or use of signal transduction assays).

Moreover, as noted above, variant forms of the REEP and/or RTP genes are also considered to be equivalent to those peptide and DNA molecules detailed herein. For example, the following isolated substitutions are not expected to have a significant effect on the biological activity of the resulting molecule: a substitution of leucine for isoleucine or valine, an aspartic acid for glutamic acid, a threonine for serine, or a similar substitution of an amino acid for a structurally related amino acid (i.e., conservative mutation). Accordingly, some embodiments of the invention provide variants of REEP and/or RTP that contain conservative substitutions. Conservative substitutions are those that occur within families of amino acids that are related in their side chains. Genetically encoded amino acids can be divided into 4 families: (1) acidic (aspartic acid, glutamic acid); (2) basic (lysine, arginine, histidine); (3) non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In a similar manner, all amino acids can be divided into: (1) acidic (aspartic acid, glutamic acid); (2) basic (lysine, arginine, histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), optionally individually classified serine and threonine as aliphatic-hydroxy groups; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amides (asparagine, glutamine); and (6) sulfur-containing (cysteine and methionine) (e.g., eds. Stryer, Biochemistry, pp. 17-21, 2 nd edition, WH Freeman and Co., 1981). By assessing the ability of a variant peptide to function in a manner similar to the wild-type protein, it can be readily determined whether a change in the amino acid sequence of the peptide will result in a functional polypeptide. In the same way, peptides with more than one substitution can be easily tested.

More rarely, variants include "non-conservative" changes (e.g., replacement of glycine for tryptophan). Similar minor changes may also include amino acid deletions or insertions or both. Using computer programs (e.g., LASERGENE software, DNASTAR inc., Madison, Wis.), guidelines can be found for determining which amino acid residues can be substituted, inserted, or deleted without disrupting biological activity.

Variants can be generated by directed evolution or other techniques for generating combinatorial libraries of variants described in more detail below, as described in more detail below. In other embodiments of the invention, the nucleotide sequences of the invention may be engineered to alter REEP and/or RTP coding sequences, including, but not limited to, alterations that modify the cloning, processing, localization, secretion, and/or expression of the gene product. For example, mutations can be introduced using techniques well known in the art (e.g., site-directed mutagenesis to insert new restriction sites, change glycosylation patterns, or change codon preferences, etc.).

Variants of RTP1 include, but are not limited to, RTP1-A, RTP1-B, RTP1-C, RTP1-D and RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A (chimera 1), RTP1-A1-D2 (chimera 2), RTP1-A1-D1 (chimera 3), RTP4-A1-A (chimera 4), RTP4-A1-D2 (chimera 5) and RTP4-A1-D1 (chimera 6).

Reep and RTP polypeptides

In other embodiments, the invention provides REEP and/or RTP polynucleotide sequences encoding REEP and/or RTP polypeptide sequences (e.g., polypeptides of SEQ ID NOS: 21-40, 41-50, respectively). In a preferred embodiment, the present invention provides a polypeptide consisting of a sequence selected from SEQ ID NOs: 1, 7, 13, 14, 17 and 18 and nucleic acids corresponding to SEQ ID NO: 1, 7, 13, 14, 17 and 18, at least 80% identical to the polypeptide encoded by the variant thereof. In other embodiments, the protein is identical to SEQ ID NO: 1, 7, 13, 14, 17 and 18 are at least 90% identical. In other embodiments, the protein is identical to SEQ ID NO: 1, 7, 13, 14, 17 and 18 are at least 95% identical. Other embodiments of the invention provide fragments, fusion proteins or functional equivalents of a REEP and/or RTP protein (e.g., RTP1-A, RTP1-B, RTP1-C, RTP1-D and RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A (chimera 1), RTP1-A1-D2 (chimera 2), RTP1-A1-D1 (chimera 3), RTP4-A1-A (chimera 4), RTP4-A1-D2 (chimera 5) and RTP4-A1-D1 (chimera 6.) in some embodiments, mutants of a REEP and/or RTP polypeptide may be used in other embodiments of the invention, recombinant DNA molecules are produced that direct the expression of REEP and/or RTP variants, homologs, and mutants in appropriate host cells. In some embodiments of the invention, the polypeptide may be a naturally purified product. In other embodiments, it may be the product of a chemical synthetic process, and in other embodiments, it may be produced by recombinant techniques using prokaryotic or eukaryotic hosts (e.g., by cultured bacterial, yeast, higher plant, insect, and mammalian cells). In some embodiments, the polypeptides of the invention may be glycosylated or may be non-glycosylated depending on the host employed in the recombinant production method. In other embodiments, the polypeptides of the invention may also include an initial methionine amino acid residue.

In one embodiment of the invention, due to the inherent degeneracy of the genetic code, sequences other than SEQ ID NO: 21-50 to clone and express REEP and/or RTP proteins, typically such polynucleotide sequences will hybridize to the sequence of SEQ ID NO: 21-50. As will be appreciated by those skilled in the art, REEP and/or RTP-encoding nucleotide sequences having non-naturally occurring codons may be advantageously generated. Thus, in some preferred embodiments, codons preferred by a particular prokaryotic or eukaryotic host are selected (Murray et al, Nucl. acids Res., 17[1989]), for example, to increase the REEP and/or RTP expression rate, or to generate recombinant RNA transcripts with desirable properties (e.g., longer half-lives than transcripts produced by naturally occurring sequences).

In preferred embodiments, REEP1, RTP1 and RTP2 polypeptides promote odorant receptor cell surface localization and odorant receptor functional expression.

1. Carrier for the production of REEP and RTP

The polynucleotides of the invention may be used to produce polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be contained in any of a variety of expression vectors for expressing a polypeptide. In some embodiments of the invention, vectors include, but are not limited to, chromosomal, nonchromosomal and synthetic DNA sequences (e.g., SV40 derivatives, bacterial plasmids, phage DNA; baculovirus, yeast plasmids, vectors derived from a combination of plasmids and phage DNA, and viral DNA such as vaccinia, adenovirus, fowlpox virus and pseudorabies). It is contemplated that any vector may be used so long as it is replicable and viable in the host.

More specifically, some embodiments of the present invention provide recombinant constructs comprising one or more sequences as broadly described above (e.g., SEQ ID NOs: 1-20). In some embodiments of the invention, the construct comprises a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in either forward or reverse orientation. In other embodiments, at an appropriate stage, heterologous structural sequences (e.g., SEQ ID NOS: 1-20) are assembled with translation initiation and termination sequences. In a preferred embodiment of the invention, the appropriate DNA sequence is inserted into the vector using any of a variety of methods. Typically, the DNA sequence is inserted into an appropriate restriction endonuclease site by methods known in the art.

A large number of suitable vectors are known to those skilled in the art and are commercially available. Such vectors include, but are not limited to, the following: 1) of the bacterium: pQE70, pQE60, pQE-9(Qiagen), pBS, pD10, phagescript, psiX174, pbluescriptSK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); 2) eukaryotic-pWLNEO, pSV2CAT, pOG44, PXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia); and 3) baculovirus-pPbac and pMbac (Stratagene). Any other plasmids or vectors may be used as long as they can replicate and survive in the host. In some preferred embodiments of the invention, the mammalian expression vector comprises an origin of replication, a suitable promoter and enhancer, and any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences and 5' flanking nontranscribed sequences. In other embodiments, the DNA sequence is derived from SV40 splicing, and a polyadenylation site may be used to provide the required non-transcribed genetic elements.

In certain embodiments of the invention, the DNA sequence in the expression vector is operably linked to an appropriate expression control sequence (promoter) to direct mRNA synthesis. Promoters useful in the present invention include, but are not limited to, the LTR or SV40 promoter, the E.coli lac or trp, phage lambda P_LAnd P_RT3 and T7 promoters and Cytomegalovirus (CMV) immediate early, Herpes Simplex Virus (HSV) thymidine kinase and mouse metallothionein-I promoters and other promoters known to control gene expression in prokaryotic or eukaryotic cells or their viruses. In other embodiments of the invention, the recombinant expression vector includes an origin of replication and a selectable marker that allows for transformation of the host cell (e.g., dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in E.coli).

In some embodiments of the invention, transcription of DNA encoding a polypeptide of the invention by higher eukaryotes is enhanced by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300bp, that act on a promoter to enhance its transcription. Enhancers useful in the present invention include, but are not limited to, the SV40 enhancer at 100 to 270 bp posterior to the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer at the posterior side of the origin of replication, and adenovirus enhancers.

In other embodiments, the expression vector further comprises a ribosome binding site for translation initiation and a transcription terminator. In other embodiments of the invention, the vector may also comprise appropriate sequences for amplifying expression.

2. Host cells for production of REEP and RTP polypeptides

In another embodiment, the invention provides a host cell comprising the above construct. In some embodiments of the invention, the host cell is a higher eukaryote cell (e.g., a mammalian or insect cell). In other embodiments of the invention, the host cell is a lower eukaryote cell (e.g., a yeast cell). In other embodiments of the invention, the host cell may be a prokaryotic cell (e.g., a bacterial cell). Specific examples of host cells include, but are not limited to, Escherichia coli, Salmonella typhimurium, Bacillus subtilis and various species within the genera Pseudomonas, Streptomyces and Staphylococcus, as well as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila S2 cells, Spodoptera Sf9 cells, Chinese Hamster Ovary (CHO) cells, COS-7 lines of monkey kidney fibroblasts, (Gluzman, Cell 23: 175[1981]), C127, 3T3, 293, 293T, HeLa and BHK Cell lines.

The gene product encoded by the recombinant sequence may be produced in a conventional manner using a construct in a host cell. In some embodiments, the construct can be introduced into the host cell by calcium phosphate transfection, DEAE-dextran mediated transfection, or electroporation (see, e.g., Davis et al, Basic Methods in Molecular Biology, 1986). Alternatively, in some embodiments of the invention, the polypeptides of the invention may be produced synthetically by conventional peptide synthesizers.

The protein may be expressed in mammalian cells, yeast, bacteria or other cells under the control of a suitable promoter. Such proteins may also be produced using RNA derived from the DNA constructs of the invention using cell-free translation systems. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described in Sambrook, et al, molecular cloning: a Laboratory Manual, 2 nd edition, Cold Spring Harbor, N.Y., 1989.

In some embodiments of the invention, after transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by an appropriate means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time. In other embodiments of the invention, the cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. In other embodiments of the invention, the microbial cells employed in the expression of the protein may be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of a cell lysing agent.

In a preferred embodiment, the invention provides cell lines (e.g., heterologous 293T cell lines) comprising odorant receptors (e.g., human odorant receptors, murine odorant receptors, synthetic odorant receptors), REEP1, RTP1, RTP2 and G, which are localized on the cell surface_αolfExpression of (2). In some embodiments, the odorant receptor is labeled with a reporter reagent (e.g., glutathione-S-transferase (GST), c-myc, 6-histidine (6X-His), Green Fluorescent Protein (GFP), Maltose Binding Protein (MBP), influenza A virus Hemagglutinin (HA), β -galactosidase, and GAL 4). The cell lines described in this embodiment are not limited to a particular odorant receptor. In some embodiments, the odor expressed in the cell line is affected byBodies include, but are not limited to, S6/79, S18, S46, S50, MOR23-1, MOR31-4, MOR31-6, MOR32-5 and MOR 32-11. In a preferred embodiment, the cell line expressing the odorant receptor is used for classification of functional expression of the odorant receptor (e.g., ligand specificity). In other embodiments, the cell line expressing an odorant receptor is used for classification of olfactory sensations in an animal.

Purification of REEP and RTP polypeptides

The invention also provides methods for recovering and purifying REEP and/or RTP polypeptides from recombinant cell cultures, including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. In other embodiments of the invention, protein-refolding steps may be used as desired to complete the configuration of the mature protein. In other embodiments of the invention, High Performance Liquid Chromatography (HPLC) may be employed in the final purification step.

The invention also provides polynucleotides having the coding sequence of the REEP and/or RTP genes fused in frame to a marker sequence (e.g., SEQ ID NOs 1-20) that allows for purification of the polypeptides of the invention. A non-limiting example of a tag sequence is a hexa-histidine tag, which may be provided by a vector (preferably a pQE-9 vector), which in the case of a bacterial host provides for purification of the polypeptide fused to the tag; alternatively, for example, when a mammalian host (e.g., COS-7 cells) is used, the marker sequence may be a Hemagglutinin (HA) marker. The HA tag corresponds to an epitope derived from influenza hemagglutinin protein (Wilson et al, Cell, 37: 767[1984 ]).

Truncation mutants of REEP and RTP polypeptides

In addition, the invention provides fragments (i.e., truncation mutants) of REEP and/or RTP polypeptides. In some embodiments of the invention, when it is desired to express a portion of a REEP and/or RTP protein, it may be necessary to add an initiation codon (ATG) to the oligonucleotide fragment containing the target sequence to be expressed. It is well known in the art that methionine at the N-terminal position can be enzymatically cleaved using the enzyme Methionine Aminopeptidase (MAP). MAP has been cloned from E.coli (Ben-Bassat et al, J.Bacterid., 169: 751[1987]) and Salmonella typhimurium and its activity in vitro has been demonstrated on recombinant proteins (Miller et al, Proc. Natl.Acad.Sci.USA 84: 2718[1990 ]). Thus, if desired, the N-terminal methionine may be removed in vivo by expressing such a recombinant polypeptide in a MAP producing host (e.g.E.coli or CM89 or s.cerevisiae), or in vitro by using purified MAP.

5. Fusion proteins comprising REEP and RTP

The invention also provides fusion proteins incorporating all or part of a REEP and/or RTP polypeptide of the invention. Thus, in some embodiments of the invention, the coding sequence for a polypeptide may be integrated as part of a fusion gene comprising nucleotide sequences encoding different polypeptides. It is contemplated that such expression systems may be used under conditions in which it is desired to produce immunogenic fragments of REEP and/or RTP proteins. In some embodiments of the invention, the VP6 capsid protein of rotavirus is used as an immunological carrier protein for a portion of the REEP and/or RTP polypeptides, either in monomeric form or in the form of viral particles. In other embodiments of the invention, a nucleic acid sequence corresponding to a portion of a REEP and/or RTP polypeptide against which an antibody is to be raised may be incorporated into a fusion gene construct comprising a coding sequence for a late vaccinia virus structural protein to produce a population of recombinant viruses that express a fusion protein comprising a portion of a REEP and/or RTP as part of a virion. Immunogenic fusion proteins utilizing hepatitis B surface antigen fusion proteins have been used to demonstrate that recombinant hepatitis B virions can also be utilized in this role. Similarly, in other embodiments of the invention, chimeric constructs encoding fusion proteins comprising a portion of a REEP and/or RTP polypeptide and a poliovirus capsid protein are established to enhance the immunogenicity of the set of polypeptide antigens (see, e.g., European publication No. 025949; and Evans et al, Nature 339: 385[1989 ]; Huang et al, J.Virol., 62: 3855[1988 ]; and Schlienger et al, J.Virol., 66: 2[1992 ]).

In other embodiments of the invention, multiple antigen peptide systems for peptide-based immunization may be used. In this system, the desired REEP and/or a portion of RTP is obtained directly from the organic chemical synthesis of peptides on an oligomeric branched lysine core (see, e.g., Posnett et al, J.biol. chem., 263: 1719[1988 ]; and Nardelli et al, J.Immunol., 148: 914[1992 ]). In other embodiments of the invention, antigenic determinants of REEP and/or RTP proteins may also be expressed and presented by bacterial cells.

In addition to the use of fusion proteins to enhance immunogenicity, it is widely recognized that fusion proteins may also facilitate expression of proteins, such as the REEP and/or RTP proteins of the invention. Thus, in some embodiments of the invention, the REEP and/or RTP polypeptides may be produced as glutathione-S-transferase (i.e., a GST fusion protein). Such GST fusion proteins are expected to facilitate purification of REEP and/or RTP polypeptides, e.g., by using glutathione-derivatized matrices (see, e.g., Ausabel et al (ed.), Current protocols molecular biology, John Wiley& Sons，NY[1991]). In another embodiment of the invention, the fusion gene encoding the purified leader sequence may be allowed to pass through the use of Ni ²⁺Affinity chromatography on metal resins to purify the expressed REEP and/or RTP fusion proteins, e.g., a poly- (His)/enterokinase cleavage site sequence at the N-terminus of a portion of the desired REEP and/or RTP polypeptide. In another embodiment of the invention, the purification leader sequence may be subsequently removed by enterokinase treatment (see, e.g., Hochuli et al, J.Chromatogr., 411: 177[1987 ]](ii) a And Janknecht et al, proc.natl.acad.sci.usa88: 8972).

Techniques for preparing fusion genes are well known. Basically, ligation of multiple DNA fragments encoding different polypeptide sequences is performed according to conventional techniques using blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling-in of cohesive termini where appropriate, alkaline phosphatase treatment to avoid unwanted ligation, and enzymatic ligation. In another embodiment of the present invention, the fusion gene can be synthesized by conventional techniques, including an automated DNA synthesizer. Alternatively, in other embodiments of the invention, PCR amplification of gene fragments can be performed using anchor primers that create complementary overhangs between 2 adjacent gene fragments that can subsequently anneal to create a chimeric gene sequence (see, e.g., current protocols in Molecular Biology, supra).

Variants of REEP and RTP

Other embodiments of the invention provide mutant or variant forms (i.e., mutant proteins) of REEP and/or RTP polypeptides. The structure of peptides having the activity of REEP and/or RTP polypeptides of the invention may be modified for the purpose of enhancing therapeutic or prophylactic efficacy, inactivating proteins or stability (e.g., shelf life ex vivo and/or resistance to proteolytic degradation in vivo), etc. Such modified peptides are considered as functional equivalents of peptides having the activity of the subject REEP and/or RTP proteins as defined herein. Modified peptides may be produced in which the amino acid sequence has been altered, for example by amino acid substitution, deletion or addition.

Variant forms of RTP1 include, but are not limited to, RTP1-A, RTP1-B, RTP1-C, RTP1-D and RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A (chimera 1), RTP1-A1-D2 (chimera 2), RTP1-A1-D1 (chimera 3), RTP4-A1-A (chimera 4), RTP4-A1-D2 (chimera 5) and RTP4-A1-D1 (chimera 6).

Moreover, as noted above, variant forms (e.g., mutant or polymorphic sequences) of the subject REEP and/or RTP proteins are also contemplated as being equivalent to those peptide and DNA molecules described in detail. For example, as described above, the present invention includes mutant and variant proteins containing conservative or non-conservative amino acid substitutions.

The invention also includes methods of producing the combinatorial mutant panels and truncation mutants of the REEP and/or RTP proteins of the invention, and are particularly useful for identifying potential variant sequences (i.e., mutant or polymorphic sequences) that are involved in or are resistant to neurological disorders (e.g., olfactory disorders). The purpose of screening such combinatorial libraries is to generate new REEP and/or RTP variants that may, for example, act as agonists or antagonists or have new activities.

Thus, in some embodiments of the invention, REEP and/or RTP variants are engineered by the methods of the invention to provide altered (e.g., increased or decreased) biological activity. In other embodiments of the invention, combinatorially derived variants are produced that have selective potency compared to naturally occurring REEP and/or RTP. Such proteins, when expressed from recombinant DNA constructs, may be used in gene therapy protocols.

Other embodiments of the invention provide REEP and/or RTP variants that have a significantly different intracellular half-life than the corresponding wild-type protein. For example, the altered protein may be made more or less stable to proteolytic degradation or other cellular processes that result in destruction or otherwise inactivation of REEP and/or RTP polypeptides. Such variants and the genes encoding them can be used to alter the localization of REEP and/or RTP expression by modulating the half-life of the protein. For example, a short half-life may produce a more transient REEP and/or RTP bioeffect, which when part of an inducible expression system may allow tighter control of the REEP and/or RTP levels within the cell. As above, such proteins, particularly their recombinant nucleic acid constructs, may be used in gene therapy protocols.

In other embodiments of the invention, REEP and/or RTP variants are produced by recombinant methods, acting as antagonists, because they interfere with the ability of the corresponding wild-type protein to modulate cellular function.

In some embodiments of the combinatorial mutagenesis approach of the present invention, the amino acid sequences of a population of REEP and/or RTP homologs, variants, or other related proteins are aligned, preferably to promote the highest homology possible. Such a population of variants may include, for example, REEP and/or RTP homologs from one or more species, or REEP and/or RTP variants from the same species but differing due to mutations or polymorphisms. Amino acids appearing at each position of the aligned sequences are selected to create a degenerate set of combined sequences.

In a preferred embodiment of the invention, a combinatorial REEP and/or RTP library is produced by degenerate libraries of genes encoding a library of polypeptides, each of said polypeptides comprising at least a portion of a potential REEP and/or RTP protein sequence. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into a gene sequence such that a degenerate set of potential REEP and/or RTP sequences can be expressed as a single polypeptide, or, as a set of larger fusion proteins (e.g., for phage display) comprising a set of REEP and/or RTP sequences.

There are many methods of generating libraries of potential REEP and/or RTP homologues and variants from degenerate oligonucleotide sequences. In some embodiments, the chemical synthesis of a degenerate gene sequence is performed in an automated DNA synthesizer and the synthesized gene is ligated into an appropriate gene for expression. The purpose of the degenerate set of genes is to provide, in a mixture, all sequences encoding the desired set of potential REEP and/or RTP sequences. The synthesis of degenerate oligonucleotides is well known in the art (see, e.g., Narang, Tetrahedron Lett., 39: 39[1983 ]; Itakura et al, Recombinant DNA, in Walton (eds.), Proceedings of the3rd Cleveland Symposium on macromolecules, Elsevier, Amsterdam, pp273-289[1981 ]; Itakura et al, Annu. Rev. biochem., 53: 323[1984 ]; Itakura et al, Science 198: 1056[1984 ]; Ike et al, Nucl. acid Res., 11: 477[1983 ]). Such techniques have been used for directed evolution of other proteins (see, e.g., Scott et al, Science 249: 386[1980 ]; Roberts et al, Proc. Natl. Acad. Sci. USA 89: 2429[1992 ]; Devrin et al, Science 249: 404[1990 ]; Cwirla et al, Proc. Natl. Acad. Sci. USA 87: 6378[1990 ]; each of which is incorporated herein by reference; and U.S. Pat. Nos. 5,223,409, 5,198,346 and 5,096,815, each of which is incorporated herein by reference).

It is contemplated that REEP and/or RTP nucleic acids of the invention (e.g., SEQ ID NOS: 1-20 and fragments and variants thereof) can be used as starting nucleic acids for directed evolution. These techniques can be used to develop REEP and/or RTP variants with desirable properties (e.g., increased or decreased biological activity).

In some embodiments, artificial evolution is performed by random mutagenesis (e.g., by using error-prone PCR to introduce random mutations into a given coding sequence). This method requires fine tuning of the frequency of mutations. As a general rule, beneficial mutations are rare, while deleterious mutations are common. This is because the combination of deleterious mutations and beneficial mutations often results in inactive enzymes. The ideal number of base substitutions for a targeted gene is typically 1.5 to 5(Moore and Arnold, nat. Biotech., 14, 458[1996 ]; Leung et al, Technique, 1: 11[1989 ]; Eckert and Kunkel, PCR Methods appl., 1: 17-24[1991 ]; Caldwell and Joyce, PCR Methods appl., 2: 28[1992 ]; and Zhao and Arnold, Nuc.acids.Res., 25: 1307[1997 ]). Following mutagenesis, the resulting clones are selected for the desired activity (e.g., screening for REEP and/or RTP activity). Successive rounds of mutagenesis and selection are often required to develop enzymes with the desired properties. It should be noted that only useful mutations go into the next round of mutagenesis.

In other embodiments of the invention, the polynucleotides of the invention are used in gene shuffling or sexual PCR methods (e.g., Smith, Nature, 370: 324[1994 ]; U.S. Pat. No. 5,837,458; 5,830,721; 5,811,238; 5,733,731; all of which are incorporated herein by reference). Gene shuffling involves random fragmentation of several mutant DNAs, which are then reassembled into full-length molecules by PCR. Examples of various gene shuffling methods include, but are not limited to, assembly after DNase treatment, staggered extension process (STEP) and random primer in vitro recombination. In the DNase-mediated method, DNA fragments isolated from a pool of positive mutants are cut into random fragments with DNase I and subjected to multiple rounds of PCR without the addition of primers. As the PCR cycle progresses, the length of the random fragments approaches the length of the uncut fragments, resulting in mixing and accumulation of mutations present in different clones in some of the resulting sequences. Multiple rounds of selection and shuffling have resulted in enhanced function of several enzymes (Stemmer, Nature, 370: 398[1994 ]; Stemmer, proc. Natl.Acad.Sci.USA, 91: 10747[1994 ]; Crameri et al, Nat. Biotech., 14: 315[1996 ]; Zhang et al, proc. Natl.Acad.Sci.USA, 94: 4504[1997 ]; and Crameri et al, Nat. Biotech., 15: 436[1997 ]). Variants generated by directed evolution can be screened for REEP and/or RTP activity by the methods described herein.

Many techniques are known in the art for screening gene products of combinatorial libraries made by point mutations, and for screening cDNA libraries for gene products having certain properties. Such techniques can be generally adapted for rapid screening of gene libraries generated by combinatorial mutagenesis or recombination of REEP and/or RTP homologues or variants. The most widely used techniques for screening large gene libraries typically involve cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions that facilitate relatively easy isolation of the vector encoding the gene whose product is being tested for the desired activity.

Chemical Synthesis of REEP and/or RTP Polypeptides

In an alternative embodiment of the invention, the coding sequences for REEP and/or RTP are synthesized in whole or in part using chemical methods well known in the art (see, e.g., Caruthers et al, Nucl. acids Res. Symp. Ser., 7: 215[1980 ]; Crea and Horn, Nucl. acids Res., 9: 2331[1980 ]; Matteucci and Caruthers, Tetrahedron Lett., 21: 719[1980 ]; and Chow and Kempe, Nucl. acids Res., 9: 2807[1981 ]). In other embodiments of the invention, the protein itself is produced using chemical methods to synthesize the entire REEP and/or RTP amino acid sequence, or a portion thereof. For example, peptides can be synthesized by solid phase techniques, cleaved from resins, and purified by preparative high performance liquid chromatography (see, e.g., Creighton, Protein Structures and molecular principles, W H Freeman and Co, New York N.Y. [1983 ]). In other embodiments of the invention, the composition of the synthesized peptide is confirmed by amino acid analysis or sequencing (see, e.g., Creighton, supra).

Direct peptide synthesis can be performed using various solid phase techniques (Roberge et al, Science 269: 202[1995]), and automated synthesis can be achieved, for example, using an ABI431A peptide synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer. In addition, the amino acid sequence of the REEP and/or RTP polypeptides, or any portion thereof, can be altered during direct synthesis and/or combined with other sequences using chemical methods to produce variant polypeptides.

Detection of reep and RTP alleles

In some embodiments, the invention provides methods of detecting the presence of wild-type or variant (e.g., mutant or polymorphic) REEP and/or RTP nucleic acids or polypeptides. Detection of mutant REEP and/or RTP polypeptides can be used to diagnose a disease (e.g., an olfactory disorder).

A. Detection of variant REEP and/or RTP alleles

In some embodiments, the invention provides alleles of REEP and/or RTP that increase a patient's susceptibility to olfactory disorders (e.g., upper respiratory tract infections, anterior cranial fossa tumors, Kallmann syndrome, foster kennedy syndrome, parkinson's disease, alzheimer's disease, and huntington's chorea). Any mutation that results in an altered phenotype (e.g., diminished olfactory sensory ability) is within the scope of the present invention.

Accordingly, the present invention provides methods for determining whether a patient has an increased susceptibility to olfactory disorders (e.g., upper respiratory tract infections, anterior cranial fossa tumors and Kallmann syndrome, foster kennedy syndrome, parkinson's disease, alzheimer's disease, huntington's disease) by determining, directly or indirectly, whether the individual has a variant REEP and/or RTP allele. In other embodiments, the invention provides methods of providing an individual with a prognosis of an increased risk of an olfactory disorder based on the presence or absence of one or more variant REEP and/or RTP alleles.

A number of methods are available for analyzing variant (e.g., mutant or polymorphic) nucleic acid or polypeptide sequences. Assays for detecting variants (e.g., polymorphisms or mutations) by nucleic acid analysis can be divided into several categories, including, but not limited to, direct sequencing assays, fragment polymorphism assays, hybridization assays, and computer-based data analysis. A wide variety of protocols and commercially available kits or services are available for performing these assays. In some embodiments, assays are performed in combination or heterozygously (e.g., combining different reagents or techniques from several assays to produce one assay). The following are exemplary assays useful in the present invention: direct sequencing assays, PCR assays, mutation analysis by dHPLC (e.g., as available from Transgenomic, Omaha, NE or Varian, Palo Alto, Calif.), fragment length polymorphism assays (e.g., RFLP or CFLP (see, e.g., U.S. Pat. Nos. 5,843,654; 5,843,669; 5,719,208; and 5,888,780; each of which is incorporated herein by reference)), hybridization assays (e.g., direct detection of hybridization, detection of hybridization using DNA chips (see, e.g., U.S. Pat. Nos. 6,045,996; 5,925,525; 5,858,659; 6,017,696; 6,068,818; 6,051,380; 6,001,311; 5,985,551; 5,474,796; PCT publication WO99/67641 and WO 00/39587; each of which is incorporated herein by reference)), enzymatic detection of polymorphisms (see, e.g., U.S. Pat. Nos. 5,717,066,066,001,543,543; 6,919; PCT publication WO 387; WO 2/67641; and WO 3875/39587; each of which are incorporated herein by reference), indirect detection of polymorphisms (see, e.g., indirect detection of polymorphisms such as found in, e.g., indirect polymorphism sequences 3978,543,543, 3978; and 465,543,543,543 (see, incorporated herein, 2,543,090; and 465,543,543,543,543,97, other sequences in the SPG-6 locus may be used; this process is described in U.S. patent nos.: 5,612,179 (incorporated herein by reference)) and mass spectrometry.

In addition, assays that detect variant REEP and/or RTP proteins can be used in the present invention (e.g., cell-free translation methods, see, e.g., U.S. patent 6,303,337, incorporated herein by reference) and antibody binding assays. The generation of antibodies that specifically recognize mutant versus wild-type proteins is discussed below.

B. Kit for analyzing risk of olfactory disorder

The invention also provides kits for determining whether an individual contains a wild-type or variant (e.g., mutant or polymorphic) allele or polypeptide of REEP and/or RTP. In some embodiments, the kit can be used to determine whether a subject is at risk for developing an olfactory disorder (e.g., upper respiratory tract infection, anterior cranial fossa tumor and Kallmann syndrome, forster kennedy syndrome, parkinson's disease, alzheimer's disease, huntington's disease). Diagnostic kits are generated in a variety of ways. In some embodiments, the kit contains at least one reagent for specifically detecting mutant REEP and/or RTP alleles or proteins. In a preferred embodiment, the agent is a nucleic acid that will hybridize to a nucleic acid containing a mutation and will not bind to a nucleic acid that does not contain a mutation. In other embodiments, the reagent is a primer that is used to amplify a region of DNA containing a mutation. In other embodiments, the agent is an antibody that preferentially binds to wild-type or mutant REEP and/or RTP proteins.

In some embodiments, the kit contains instructions for determining whether a subject is at risk for an olfactory disorder (e.g., upper respiratory tract infection, anterior cranial fossa tumor and Kallmann syndrome, foster kennedy syndrome, parkinson's disease, alzheimer's disease, huntington's disease). In a preferred embodiment, the instructions state that the risk of developing an olfactory disorder is determined by detecting the presence or absence of a mutant REEP and/or RTP allele in a subject, wherein subjects having the mutant allele are at a higher risk of developing an olfactory disorder.

The presence or absence of disease-related mutations in the REEP and/or RTP genes can be used to make therapeutic or other medical decisions. For example, a couple with a family history of odorant receptor-related diseases may choose to become pregnant by in vitro fertilization and pre-implantation genetic screening. In this case, fertilized embryos are screened for mutant (e.g., disease-associated) alleles of the REEP and/or RTP genes, and only embryos with wild-type alleles are implanted into the uterus.

In other embodiments, an intrauterine screen (e.g., amniocentesis or chorionic villus screen) is performed on a developing fetus. In other embodiments, genetic screening for newborn infants or very young children is performed. Early detection of REEP and/or RTP alleles known to be associated with olfactory disorders allows early intervention (e.g., genetic or drug therapy).

In some embodiments, the kit includes auxiliary reagents such as buffers, nucleic acid stabilizers, protein stabilizers, and signal producing systems (e.g., fluorescence producing systems, such as the Fret system). The test kit may be packaged in any suitable manner, typically with the components in a single container or in multiple containers as desired, and an instruction for performing the test. In some embodiments, the kit also preferably comprises a positive control sample.

C. Bioinformatics

In some embodiments, the invention provides methods for determining an individual's risk of developing olfactory disorders (e.g., upper respiratory tract infections, anterior cranial fossa tumors and Kallmann syndrome, foster kennedy syndrome, parkinson's disease, alzheimer's disease, huntington's disease) based on the presence of one or more variant alleles of the REEP and/or RTP genes. In some embodiments, the analysis of variant data is performed by a computer using information stored in the computer (e.g., in a database). For example, in some embodiments, the present invention provides a bioinformatic research system comprising a plurality of computers running a multi-platform object-oriented programming language (see, e.g., U.S. Pat. No. 6,125,383; incorporated herein by reference). In some embodiments, one of the computers stores genetic data (e.g., the risk of exposure to REEP and/or RTP-associated olfactory disorders associated with a given polymorphism, and the sequence). In some embodiments, one of the computers stores an application (e.g., for analyzing the results of a detection assay). The results are then transmitted to the user (e.g., via one of the computers or via the internet).

For example, in some embodiments, raw data generated by a detection assay (e.g., the presence, absence, or amount of a given REEP and/or RTP allele or polypeptide) is translated into data that is predictive of the clinician using a computer-based analysis program. The clinician may access the predictive data using any suitable approach. Thus, in some preferred embodiments, the invention provides the additional benefit that clinicians who may not have been trained in genetics or molecular biology do not need to understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician can then immediately utilize the information in order to optimize the care of the subject.

The present invention includes any method capable of receiving, processing, and transmitting information to and from laboratories, information providers, medical personnel, and subjects where assays are performed. For example, in some embodiments of the invention, a sample (e.g., a biopsy or a serum or urine sample) is obtained from a subject and presented to a profiling service (e.g., a clinical laboratory in a medical facility, a genomic profiling business, etc.) located anywhere in the world (e.g., in a country other than the subject's country of residence, or a country other than the country where the information is ultimately used), resulting in raw data. When the sample comprises a tissue or other biological sample, the subject may visit a medical center to obtain the sample and send it to a profiling center, or the subject may collect the sample itself (e.g., a urine sample) and send it directly to the profiling center. When the sample contains previously determined biological information, the subject may send the information directly to the profiling service (e.g., an information card containing the information may be scanned by a computer and the data transferred to a computer at a profiling center using an electronic communication system). Once the profiling service is received, the sample is processed and a profile (i.e., the presence of wild-type or mutant REEP and/or RTP genes or polypeptides) specific to the diagnostic or prognostic information desired by the subject is generated.

The profile data is then formatted for interpretation by the attending clinician. For example, rather than providing raw data, the prepared format may represent a diagnosis or risk assessment for the subject (e.g., the likelihood of developing REEP and/or RTP related olfactory disorders), as well as a recommendation for a particular treatment option. The data may be displayed to the clinician by any suitable method. For example, in some embodiments, the profiling service generates a report that can be printed by the clinician (e.g., at a point of care) or displayed to the clinician on a computer display.

In some embodiments, the information is first analyzed at the point of care or at the regional facility. The raw data is then sent to a central processing facility for further analysis and/or converted into information useful to a clinician or patient. The central processing facility provides the following advantages: privacy (all data stored in a central facility with a uniform security scheme), consistency of speed and data analysis. The central processing facility can then control the fate of the data after treatment of the subject. For example, using an electronic communication system, a central facility may provide data to a clinician, subject, or researcher.

In some embodiments, the subject can acquire the data directly using an electronic communication system. Based on the results, the subject may select other interventions or counseling. In some embodiments, the data is used for research purposes. For example, the data may be used to further optimize the association of a given REEP and/or RTP allele with an olfactory disorder.

Production of reep and RTP antibodies

The invention provides isolated antibodies or antibody fragments (e.g., FAB fragments). Antibodies can be generated to allow detection of REEP and/or RTP proteins of the invention (e.g., wild-type or mutant). Antibodies can be prepared using a variety of immunogens. In one embodiment, the immunogen is a human REEP and/or RTP peptide to produce antibodies that recognize human REEP and/or RTP. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, Fab expression libraries, or recombinant (e.g., chimeric, humanized, etc.) antibodies, so long as it recognizes the protein. Antibodies can be produced according to conventional methods for producing antibodies or antisera using the protein of the present invention as an antigen.

Polyclonal antibodies to REEP and/or RTP polypeptides can be produced using a variety of methods known in the art. For antibody production, various host animals, including but not limited to rabbits, mice, rats, sheep, goats, etc., can be immunized by injection of peptides corresponding to REEP and/or RTP epitopes. In a preferred embodiment, the peptide is conjugated to an immunogenic carrier (e.g., diphtheria toxoid, Bovine Serum Albumin (BSA) or Keyhole Limpet Hemocyanin (KLH)). Depending on the host species, various adjuvants may be used to increase the immune response, including but not limited to Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum).

For the preparation of monoclonal Antibodies against REEP and/or RTP, it is contemplated that any technique for producing antibody molecules by continuous cell lines in culture may be used in the present invention (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). They include, but are not limited to, the hybridoma technology originally developed by Kohler and Milstein (Kohler and Milstein, Nature 256: 495-497[1975]), as well as the three-source hybridoma technology, the human B-cell hybridoma technology (see, e.g., Kozbor et al, Immunol.Tod., 4: 72[1983]), and the EBV-hybridoma technology to produce human monoclonal antibodies (Cole et al, in monoclonal antibodies and Cancer Therapy, Alan R.Liss, Inc., pp.77-96[1985 ]).

In another embodiment of the invention, monoclonal antibodies are produced in sterile animals using techniques such as those described in PCT/US 90/02545. Furthermore, human hybridomas are expected to produce human antibodies (Cote et al, Proc. Natl. Acad. Sci. USA 80: 2026-.

In addition, it is contemplated that the described techniques for producing single chain antibodies (U.S. Pat. No. 4,946,778; incorporated herein by reference) can be used to produce REEP and/or RTP specific single chain antibodies. Another embodiment of the present invention utilizes the described techniques for constructing Fab expression libraries (Huse et al, Science 246: 1275-1281[1989]), to allow for the rapid and easy identification of monoclonal Fab fragments with the desired specificity for REEP and/or RTP polypeptides.

In other embodiments, the invention includes recombinant antibodies or fragments thereof directed against the proteins of the invention. Recombinant antibodies include, but are not limited to, humanized and chimeric antibodies. Methods for producing recombinant Antibodies are known in the art (see, e.g., U.S. Pat. Nos. 6,180,370 and 6,277,969 and "Monoclonal Antibodies" H.Zola, BIOS Scientific Pub letters Limited2000.Springer-Verlay New York, Inc., New York; each of which is incorporated herein by reference).

It is contemplated that any technique suitable for producing antibody fragments may be used to produce antibody fragments containing the idiotype (antigen binding region) of the antibody molecule. For example, such fragments include, but are not limited to: f (ab') 2 fragments, which can be produced by pepsin digestion of antibody molecules; fab 'fragments, which can be produced by reducing the disulfide bond of F (ab') 2 fragments; and Fab fragments, which can be produced by treating the antibody molecule with papain and a reducing agent.

In the production of antibodies, it is contemplated that screening for the desired antibody may be accomplished by techniques known in the art (e.g., radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein a assays, and immunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a second antibody or reagent to the primary antibody. In another embodiment, the secondary antibody is labeled. Many methods for detecting binding in immunoassays are known in the art and are within the scope of the invention. As is well known in the art, the immunogenic peptides provided should be free of carrier molecules used in any immunization protocol. For example, if the peptide is conjugated to KLH, it may be conjugated to BSA, or used directly in a screening assay.

The foregoing antibodies may be used in methods known in the art relating to the localization and structure of REEP and/or RTP (e.g., western blotting), measuring their levels in an appropriate biological sample, and the like. The antibodies can be used to detect REEP and/or RTP in a biological sample from an individual. The biological sample may be a biological fluid containing cells, such as, but not limited to, blood, serum, plasma, interstitial fluid, urine, cerebrospinal fluid, and the like.

The biological sample can then be tested directly for the presence of human REEP and/or RTP using appropriate strategies (e.g., ELISA or radioimmunoassay) and formats (e.g., microwells, dipsticks (e.g., as described in international patent publication WO 93/03367), etc. alternatively, the proteins in the sample can be separated by size (e.g., by polyacrylamide gel electrophoresis (PAGE) in the presence or absence of Sodium Dodecyl Sulfate (SDS), and the presence of REEP and/or RTP detected by immunoblotting (western blotting).

Another approach uses antibodies as agents to alter signal transduction. Specific antibodies that bind to the binding domains of REEP and/or RTP or other proteins involved in intracellular signaling may be used to inhibit interactions between the various proteins and their interactions with other ligands. Antibodies that bind the complexes can also be used therapeutically to inhibit protein complex interactions in signal transduction pathways that result in various physiological and cellular effects of REEP and/or RTP. Such antibodies may also be used diagnostically to measure abnormal expression of REEP1 and/or RTP, or abnormal formation of protein complexes, which may be indicative of a disease state.

Gene therapy with REEP and RTP

The invention also provides methods and compositions suitable for gene therapy to alter REEP and/or RTP expression, production or function for research, generation of transgenic animals, and/or therapeutic uses. As described above, the present invention provides human REEP and/or RTP genes, and provides methods for obtaining REEP and/or RTP genes from other species. Thus, the methods described below are generally applicable to many species. In some embodiments, it is contemplated that gene therapy is performed by providing a subject with a wild-type allele of the REEP and/or RTP gene (i.e., an allele that does not contain a REEP and/or RTP disease allele (e.g., no disease causing polymorphism or mutation)). By the methods described above, a subject in need of such treatment is identified. In some embodiments, a transient or stable therapeutic nucleic acid (e.g., antisense oligonucleotide, siRNA) is used to reduce or prevent expression of the mutant protein. In other embodiments, the gene is deleted to reduce or block the desired olfactory sensation.

Viral vectors commonly used for targeting and therapeutic procedures in vivo or ex vivo are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (see, e.g., Miller and Rosman, BioTech., 7: 980-. Preferably, the viral vectors are replication-defective, that is, they are unable to replicate autonomously in the target cell. Typically, the genome of a replication-defective viral vector used within the scope of the present invention lacks at least 1 region of the virus necessary for replication in infected cells. These regions may be eliminated (in whole or in part), or may be rendered non-functional by any technique known to those skilled in the art. These techniques include total removal, substitution (with other sequences, especially inserted nucleic acids), partial deletion or addition of one or more bases to the region(s) necessary for replication. Such techniques can be performed in vitro (i.e., on isolated DNA) or in situ using genetic manipulation techniques, or by treatment with mutagens.

Preferably, the replication-defective virus retains its genomic sequence required for encapsidation of the viral particle. DNA viral vectors include attenuated or defective DNA viruses, including, but not limited to, Herpes Simplex Virus (HSV), papilloma virus, EB virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses that are completely or almost completely deleted in viral genes are preferred because defective viruses cannot be infected after introduction into cells. The use of defective viral vectors allows for administration to cells in specific localized areas without concern that the vector will infect other cells. Thus, specific tissues can be specifically targeted. Examples of specific vectors include, but are not limited to, defective herpes virus 1(HSV1) vectors (Kaplitt et al, mol. cell. Neurosci., 2: 320-330[1991]), defective herpes virus vectors lacking the glycoprotein L gene (see, for example, patent publication RD371005A) or other defective herpes virus vectors (see, for example, WO 94/21807; and WO 92/05263); attenuated adenoviral vectors such as those described by Stratford-Perricaudet et al (J.Clin. Invest., 90: 626-; and defective adeno-associated virus vectors (Samulski et al, J.Virol., 61: 3096-.

Preferably, for in vivo administration, an appropriate immunosuppressive treatment is employed in conjunction with the viral vector (e.g., adenoviral vector) to avoid immune inactivation of the viral vector and transfected cells. For example, immunosuppressive cytokines such as interleukin-12 (IL-12), interferon-gamma (IFN-gamma) or anti-CD 4 antibodies may be administered to block humoral or cellular immune responses to the viral vector. In addition, viral vectors engineered to express a minimal number of antigens may be advantageously employed.

In a preferred embodiment, the vector is an adenoviral vector. Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver nucleic acids of the invention to a variety of cell types. There are a variety of adenovirus serotypes. Of these serotypes, human adenoviruses of type 2 or 5 (Ad2 or Ad5) or adenoviruses of animal origin (see, for example, WO94/26914) are preferred within the scope of the invention. Those adenoviruses of animal origin that can be used within the scope of the invention include those of canine, bovine, murine (e.g., Mavl, Beard et al, virol., 75-81[1990]), ovine, porcine, avian, and simian (e.g., SAV) origin. Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus (e.g., Manhattan or strain A26/61 (ATCC VR-800)).

Preferably, the replication defective adenovirus vector of the invention comprises an ITR, a encapsidation sequence and a nucleic acid of interest. More preferably, at least the E1 region of the adenoviral vector is non-functional. The deletion in the E1 region preferably extends from nucleotide 455 to 3329(PvuII-Bg1II fragment) or from 382 to 3446(HinfII-Sau3A fragment) in the Ad5 adenoviral sequence. Other regions may also be modified, in particular the E3 region (e.g.WO 95/02697), the E2 region (e.g.WO 94/28938), the E4 region (e.g.WO 94/28152, WO94/12649 and WO95/02697) or any late gene L1-L5.

In a preferred embodiment, the adenoviral vector has a deletion in the E1 region (Ad1.0). Examples of E1-deleted adenoviruses are disclosed in EP185,573, the content of which is incorporated herein by reference. In another preferred embodiment, the adenoviral vector has deletions in the E1 and E4 regions (Ad3.0). Examples of E1/E4-deleted adenoviruses are disclosed in WO95/02697 and WO 96/22378. In another preferred embodiment, the adenoviral vector has a deletion in the E1 region, into which the E4 region and the nucleic acid sequence are inserted.

Replication-deficient recombinant adenoviruses according to the invention can be prepared by any technique known to those skilled in the art (see, e.g., Levrero et al, Gene 101: 195[1991 ]; EP 185573; and Graham, EMBO J., 3: 2917[1984 ]). More specifically, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the DNA sequence of interest. After co-transfection of adenovirus and plasmid into appropriate cell lines, homologous recombination is accomplished. The cell line employed should preferably be: (i) can be transformed by the element to be used, and (ii) contains sequences which complement this part of the genome of the replication-defective adenovirus, preferably in integrated form, in order to avoid the risk of recombination. Examples of cell lines which may be used are the human embryonic kidney cell line 293(Graham et al, J.Gen.Virol., 36: 59[1977]), which contains the left-hand part (12%) of the Ad5 adenovirus genome integrated into its genome, and cell lines which complement the function of E1 and E4, as described in applications WO94/26914 and WO 95/02697. The recombinant adenovirus is recovered and purified using standard molecular biology techniques well known to those of ordinary skill in the art.

Adeno-associated viruses (AAV) are DNA viruses of relatively small size that can integrate into the genome of the cells they infect in a stable and site-specific manner. They are capable of infecting a broad spectrum of cells without inducing any effect on cell growth, morphology or differentiation, and they do not appear to be involved in human pathologies. AAV genomes have been cloned and sequenced and characterized. It comprises about 4700 bases and contains an Inverted Terminal Repeat (ITR) region of about 145 bases at each end, which serves as the viral origin of replication. The remainder of the genome is divided into 2 essential regions carrying encapsidation functions: the left hand portion of the genome, which contains the rep gene involved in viral replication and viral gene expression; the right hand portion of the genome, which contains the cap gene encoding the capsid proteins of the virus.

The use of vectors derived from AAV for in vitro and in vivo gene transfer has been described (see, e.g., WO 91/18088; WO 93/09239; U.S. Pat. No. 4,797,368; U.S. Pat. No. 5,139,941; and EP 488528, both incorporated herein by reference). These publications describe a variety of AAV-derived constructs in which the rep and/or cap genes have been deleted and replaced by target genes, and the use of these constructs for transferring target genes in vitro (into cultured cells) or in vivo (directly into an organism). Replication-defective recombinant AAV according to the invention can be prepared by co-transfecting a plasmid containing a target nucleic acid sequence flanked by 2 AAV Inverted Terminal Repeat (ITR) regions, and a plasmid carrying AAV encapsidation genes (rep and cap genes) into a cell line infected with a human helper virus (e.g., adenovirus). The AAV recombinants produced are then purified by standard techniques.

In another embodiment, the gene may be introduced into a retroviral vector (e.g., as described in U.S. Pat. Nos. 5,399,346, 4,650,764, 4,980,289, and 5,124,263; all of which are incorporated herein by reference; Mann et al, Cell 33: 153[1983 ]; Markowitz et al, J.Virol., 62: 1120[1988 ]; PCT/US 95/14575; EP 453242; EP 178220; Bernstein et al, Genet. Eng., 7: 235[1985 ]; McCormick, BioTechnol., 3: 689[1985 ]; WO 95/07358; and Kuo et al, Blood 82: 845[1993 ]). Retroviruses are integrating viruses that infect dividing cells. The retroviral genome comprises 2 LTRs, encapsidation sequences and 3 coding regions (gag, pol and env). In recombinant retroviral vectors, the gag, pol and env genes are typically deleted in whole or in part and replaced with a heterologous nucleic acid sequence of interest. These vectors can be constructed from different types of retroviruses, for example, HIV, MoMuLV ("murine Moloney leukemia Virus" MSV ("murine Moloney sarcoma Virus"), HaSV ("Harvey sarcoma Virus"), SNV ("spleen necrosis Virus"), RSV ("Rous sarcoma Virus") and Friedel viruses, defective retroviral vectors are also disclosed in WO 95/02697.

Typically, to construct a recombinant retrovirus containing nucleic acid sequences, a plasmid containing the LTRs, encapsidation sequences, and coding sequences is constructed. This construct is used to transfect packaging cell lines that can reverse-provide plasmid-deficient retroviral functions. Typically, the packaging cell line is thus capable of expressing the gag, pol and env genes. Such packaging cell lines have been described in the prior art, in particular the cell line PA317 (U.S. Pat. No. 4,861,719, incorporated herein by reference), the PsiCRIP cell line (see, WO90/02806) and the GP + envAm-12 cell line (see, WO 89/07150). In addition, recombinant retroviral vectors may contain modifications within the LTR to inhibit transcriptional activity, as well as extensive encapsidation sequences, which may include a portion of the gag gene (Bender et al, J.Virol., 61: 1639[1987 ]). The recombinant retroviral vector is purified by standard techniques known to those of ordinary skill in the art.

Alternatively, the vector may be introduced in vivo by lipofection. The use of liposomes for the encapsulation and transfection of nucleic acids in vitro has increased over the past decade. Synthetic cationic lipids designed to limit the difficulties and risks encountered with liposome-mediated transfection may be used to prepare liposomes for in vivo transfection of marker-encoding genes (Feigner et al, Proc. Natl. Acad. Sci. USA 84: 7413-7417[1987 ]; see also, Mackey et al, Proc. Natl. Acad. Sci. USA 85: 8027-8031[1988 ]; Ulmer et al, Science 259: 1745-1748[1993 ]). The use of cationic lipids can facilitate encapsulation of negatively charged nucleic acids and also fusion with negatively charged cell membranes (Feigner and Ringold, Science 337: 387-388[1989 ]). Particularly useful lipid compounds and compositions for transferring nucleic acids are described in WO95/18863 and WO96/17823 and U.S. Pat. No. 5,459,127, which are incorporated herein by reference.

Other molecules may also be used to facilitate transfection of nucleic acids in vivo, such as cationic oligopeptides (e.g., WO95/21931), peptides derived from DNA binding proteins (e.g., WO96/25508) or cationic polymers (e.g., WO 95/21931).

The vector may be introduced in vivo as a naked DNA plasmid. Methods of formulating and administering naked DNA to mammalian muscle tissue are described in U.S. Pat. Nos. 5,580,859 and 5,589,466, both of which are incorporated herein by reference.

DNA vectors for gene therapy can be introduced into a desired host cell by methods known in the art, including, but not limited to, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector carrier (see, e.g., Wu et al, J.biol.chem., 267: 963[1992 ]; Wu and Wu, J.biol.chem., 263: 14621[1988 ]; and Williams et al, Proc.Natl.Acad.Sci.USA 88: 2726[1991 ]). Receptor-mediated DNA delivery methods may also be used (Curiel et al, hum. Gene ther., 3: 147[1992 ]; and Wu, J.biol. chem., 262: 4429[1987 ]).

Transgenic animals expressing exogenous REEP and RTP genes and homologs, mutants, and variants thereof

The invention includes the production of transgenic animals comprising an exogenous REEP and/or RTP gene or homolog, mutant or variant thereof. In preferred embodiments, the transgenic animal will exhibit an altered phenotype as compared to a wild-type animal. In some embodiments, the altered phenotype is overexpression of mRNA of a REEP and/or RTP gene as compared to wild-type levels of REEP and/or RTP expression. In other embodiments, the altered phenotype is decreased expression of mRNA of an endogenous REEP and/or RTP gene as compared to wild-type levels of endogenous REEP and/or RTP expression. In some preferred embodiments, the transgenic animal comprises a mutant allele of REEP and/or RTP. Methods for analyzing the presence or absence of such phenotypes include northern blotting, mRNA protection assays, and RT-PCR. In other embodiments, the transgenic mouse has a knockout mutation of the REEP and/or RTP gene. In preferred embodiments, the transgenic animal exhibits an altered susceptibility to olfactory disorders (e.g., upper respiratory tract infections, anterior cranial fossa tumors and Kallmann syndrome, foster kennedy syndrome, parkinson's disease, alzheimer's disease, huntington's disease).

Such animals may be used for research purposes (e.g., to identify signaling pathways in which REEP and/or RTP proteins are involved), as well as drug screening purposes (e.g., to screen for drugs that prevent or treat olfactory disorders). For example, in some embodiments, a test compound (e.g., a drug suspected of being useful for treating an olfactory disorder) is administered to a transgenic animal and a control animal having a wild-type REEP and/or RTP allele, and the effect is evaluated. The effect of the test and control compounds on disease symptoms was then assessed.

Transgenic animals can be produced by a variety of methods. In some embodiments, embryonic cells at different developmental stages are used to introduce transgenes for the production of transgenic animals. Depending on the developmental stage of the embryonic cells, different methods are used. Fertilized eggs are the best target for microinjection. In mice, the male pronuclei reach a size of about 20 microns in diameter, which allows for reproducible injections of 1-2 picoliters (p1) of DNA solution. The use of fertilized eggs as targets for gene transfer is of great advantage, since in most cases the injected DNA will integrate into the host genome before the first cleavage (Brinster et al, Proc. Natl. Acad. Sci. USA 82: 4438-. As a result, all cells of the transgenic non-human animal will carry the integrated transgene. This will also typically be reflected in efficient transfer of the transgene to the founder's offspring, since 50% of the germ cells will carry the transgene. U.S. Pat. No. 4,873,191 describes a method of microinjecting fertilized eggs; the contents of this patent are incorporated herein in their entirety.

In other embodiments, retroviral infection is used to introduce a transgene into a non-human animal. In some embodiments, the retroviral vector is used to transfect an oocyte by injecting the retroviral vector into the perivitelline space of the oocyte (U.S. Pat. No. 6,080,912, incorporated herein by reference). In other embodiments, the developing non-human embryo may be cultured in vitro to the blastocyst stage. At this stage, blastomeres may serve as targets for retroviral infection (Janenich, Proc. Natl. Acad. Sci. USA 73: 1260[1976 ]). Zona pellucida was removed by enzymatic treatment to obtain an effective infection of blastomeres (Hogan et al, InManipulating the Mouse Embryo, Cold Spring Harbor laboratory Press, Cold Spring Harbor, N.Y. [1986 ]). Viral vector systems for the introduction of transgenes are typically replication-defective retroviruses carrying the transgene (Jahner et al, Proc. Natl. Acad Sci. USA 82: 6927[1985 ]). Transfection can be easily and efficiently achieved by culturing blastomeres on monolayers of virus-producing cells (Van der Putten, supra; Stewart, et al, EMBO J., 6: 383[1987 ]). Alternatively, the infection may be performed at a later stage. The virus or virus-producing cell may be injected into the blastocoel (Jahner et al, Nature 298: 623[1982 ]). Most founders are chimeras of transgenes, as integration occurs only in a small group of cells forming transgenic animals. Furthermore, founders may contain multiple retroviral insertions of transgenes at different locations in the genome, usually isolated in progeny. Alternatively, transgenes can be introduced into the germline by intrauterine retroviral infection of the mid-gestation (midgetation) embryo, albeit with low efficiency (Jahner et al, supra [1982 ]). Other methods known in the art for creating transgenic animals using retroviral or retroviral vectors include microinjection of retroviral particles or mitomycin C-treated retrovirus-producing cells into the perivitelline space of fertilized eggs or early embryos (PCT International application WO90/08832[1990] and Haskell and Bowen, MoL reprod. Dev., 40: 386[1995 ]).

In other embodiments, the transgene is introduced into an embryonic stem cell, and the transfected stem cell is used to form an embryo. Embryonic stem cells are obtained by culturing the preimplantation embryos in vitro under appropriate conditions (Evans et al, Nature 292: 154[1981 ]; Bradley et al, Nature 309: 255[1984 ]; Gossler et al, Proc. Acad. Sci. USA 83: 9065[1986 ]; and Robertson et al, Nature 322: 445[1986 ]). Transgenes can be efficiently introduced into embryonic stem cells by DNA transfection by a variety of methods known in the art, including calcium phosphate co-precipitation, protoplast or spheroid fusion, lipofection, and DEAE-dextran-mediated transfection. Transgenes may also be introduced into embryonic stem cells by retrovirus-mediated transduction or by microinjection. Following their introduction into the blastocoel of blastocyst-stage embryos, such transfected embryonic stem cells can thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal (reviewed in Jaenisch, Science 240: 1468[1988 ]). Prior to introducing the transfected embryonic stem cells into the blastocoel, the transfected embryonic stem cells may be subjected to various selection experiments to enrich for embryonic stem cells that have incorporated the transgene, assuming the transgene would provide a means for such selection. Alternatively, the polymerase chain reaction can be used to screen embryonic stem cells that have integrated the transgene. This technique does not require the growth of transfected embryonic stem cells under appropriately selected conditions prior to transfer into the blastocoel.

In other embodiments, homologous recombination is used to knock out gene function or to create deletion mutants (e.g., mutants in which particular domains of REEP and/or RTP are deleted). Methods for homologous recombination are described in U.S. Pat. No. 5,614,396, incorporated herein by reference.

Screening of Compounds Using REEP and RTP

In some embodiments, isolated nucleic acids and polypeptides (e.g., SEQ ID NOs: 1-50) of the REEP and/or RTP genes of the present invention and related proteins and nucleic acids are used for drug screening of compounds that alter (e.g., enhance or inhibit) REEP and/or RTP activity and signaling. The invention also provides methods of identifying ligands and signaling pathways of the REEP and/or RTP proteins of the invention.

The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nevertheless, based on OR expression analysis experiments performed during the course of the present invention, REEP and/OR RTP family proteins are expected to play a role in promoting odorant receptor cell surface localization and functional expression.

In some embodiments, the invention provides methods of screening compounds for the ability to alter the activity of REEP and/or RTP mediated by a natural ligand (e.g., identified using the methods described above). Such compounds may be useful for treating diseases mediated by REEP and/or RTP (e.g., olfactory disorders), altering olfactory sensory responses, and the like.

In some embodiments, the present invention provides methods of screening compounds for the ability to interact with mutant REEP and/or RTP nucleic acids and/or mutant REEP and/or RTP polypeptides while not interacting with wild-type REEP and/or RTP nucleic acids (e.g., SEQ ID NOS: 1-20) and/or wild-type REEP and/or RTP polypeptides (e.g., SEQ ID NOS: 21-50). Such compounds may be used to treat olfactory disorders that are promoted by the presence of mutant forms of REEP and/or RTP nucleic acids and/or proteins.

In some embodiments, the activity of a cell surface-localized OR in a cell expressing an exogenous REEP OR RTP polypeptide caused by a compound (e.g., a candidate OR ligand OR inhibitor) is evaluated.

One technique uses REEP, RTP OR antibodies generated as described above. Such antibodies are capable of specifically binding to a REEP, RTP OR peptide and compete with the test compound for binding to the REEP, RTP OR peptide. Similar screens can be performed with small molecule libraries, aptamers, and the like.

The invention includes the use of cell lines transfected with REEP and/or RTP genes and variants thereof to screen compounds for activity, particularly from combinatorial libraries (e.g., containing more than 10 ⁴A library of seed compounds). The cell lines of the invention can be used in a variety of screening methods. In some embodiments, the cell can be used in a second signaling assay that monitors signal transduction following activation of a cell surface receptor. In other embodiments, the cells can be used in reporter assays that monitor cellular responses at the level of transcription/translation.

In the second messenger assay, the host cell is preferably transfected with a vector encoding REEP and/or RTP or a variant or mutant thereof, as described above. The host cells are then treated with one or more compounds (e.g., from a combinatorial library) and the presence or absence of a response is determined. It is contemplated that at least some of the compounds in the combinatorial library may be used as agonists, antagonists, activators, OR inhibitors of one OR more proteins encoded by the vector OR of an OR located on the cell membrane. It is also contemplated that at least some of the compounds in the combinatorial library may be used as agonists, antagonists, activators, or inhibitors of proteins that function upstream or downstream of the vector-encoded protein in a signal transduction pathway.

In some embodiments, the second messenger assay measures fluorescent signals from reporter molecules that pair ion channels gated by stimulatory membrane receptors (e.g., ligand-gated ion channels; see Denyer et al, Drug Discov. Today3: 323[1998 ] ](ii) a And Gonzales et al, drug. 431-39[1999]) Induced intracellular changes (e.g., Ca)²⁺Concentration, membrane potential, pH, IP₃cAMP, arachidonic acid release). Examples of reporter molecules include, but are not limited to, FRET (fluorescence resonance energy transfer) systems (e.g., Cuo-lipids and oxonium alcohol, EDAN/DABCYL), calcium-sensitive indicators (e.g., Fluo-3, FURA2, INDO1 and FLUO3/AM, BAPTAAM), chlorine-sensitive indicators (e.g., SPQ, SPA), potassium-sensitive indicators (e.g., PBFI), sodium-sensitive indicators (e.g., SBFI), and pH-sensitive indicators (e.g., BCECF).

Typically, the host cell is loaded with the indicator prior to exposure to the compound. Host cell responses to compound treatment can be detected by methods known in the art, including, but not limited to, fluorescence microscopy, confocal microscopy (e.g., FCS system), flow cytometry, microfluidic devices, FLIPR systems (see, e.g., Schroeder and needle, j.biomol. screening 1: 75[1996]) and plate reading systems. In some preferred embodiments, the response (e.g., increase in fluorescence intensity) caused by a compound of unknown activity is compared to the response produced by a known agonist and expressed as a percentage of the maximal response of the known agonist. The maximal response by a known agonist is defined as the 100% response. Similarly, the maximal response recorded after addition of agonist to a sample containing a known or test antagonist is detectably less than 100% response.

The cells may also be used in reporter gene assays. The reporter gene assay comprises a host cell transfected with a vector encoding a nucleic acid comprising the transcriptional control element of a target gene (i.e., a gene that controls the biological expression and function of a disease target) spliced to the coding sequence of the reporter gene. Thus, activation of the target gene results in activation of the reporter gene product.

Using any of a number of approaches in combinatorial library methods known in the art, including biological libraries; peptoid libraries (libraries of molecules with functional groups of peptides but with novel non-peptide backbones, which are resistant to enzymatic degradation but still retain biological activity; see, e.g., Zuckennan et al, J.Med.chem.37: 2678-85[1994 ]); a spatially addressable parallel solid or solution phase library; synthetic library methods requiring deconvolution; "one bead one compound" library method; and synthetic library methods using affinity chromatography selection, test compounds of the invention can be obtained. The biological library and peptoid library protocols are preferably used for peptide libraries, while the other 4 protocols are applicable to compounds of peptide, non-peptide oligomer or small molecule libraries (Lam (1997) Anticancer Drug Des.12: 145).

The ability of a test compound to modulate the binding of REEP and/or RTP to a compound (e.g., an odorant receptor) can also be evaluated. This can be achieved as follows: for example, by coupling a compound (e.g., substrate) to a radioisotope or enzyme label, binding of the compound (e.g., substrate) to REEP and/or RTP can be determined by detecting the labeled compound (e.g., substrate) in the complex.

Alternatively, REEP and/or RTP is coupled to a radioisotope or enzyme label to monitor the ability of the test compound to modulate the binding of REEP and/or RTP to a REEP and/or RTP substrate in the complex. For example, can use¹²⁵I，³⁵S¹⁴C or³H labels a compound (e.g., a substrate) directly or indirectly and detects the radioisotope by direct counting of radioactive emissions or by scintillation counting. Alternatively, horseradish peroxidase, for example, can be used,Alkaline phosphatase or luciferase enzymatically labels the compounds and the enzymatic label is detected by measuring the conversion of the appropriate substrate to the product.

The ability of a compound (e.g., an odorant receptor) to interact with REEP and/or RTP can be evaluated with or without labeling either of the interactors. For example, the interaction of a compound with REEP and/or RTP can be detected using a microphysiometer in the absence of labeled compound or REEP and/or RTP (McConnell et al Science 257: 1906-. As used herein, a "microphysiometer" (e.g., Cytosensor) is an analytical instrument that uses light-addressable potentiometric sensors (LAPSs) to measure the rate at which cells acidify their environment. This change in acidification rate can be used as an indicator of the interaction between the compound and the REEP and/or RTP polypeptide.

In another embodiment, a cell-free assay is provided in which a REEP and/or RTP protein, or biologically active portion thereof, is contacted with a test compound and the ability of the test compound to bind the REEP and/or RTP protein, or biologically active portion thereof, is assessed. Preferred biologically active portions of REEP and/or RTP proteins to be used in the assays of the invention include fragments that are involved in interactions with substrates or other proteins, e.g., fragments with a high surface probability score.

Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the 2 components to interact and bind, thereby forming a complex that can be removed and/or detected.

The interaction between the 2 molecules may also be detected, for example, using fluorescence energy transfer (FRET) (see, e.g., Lakowicz et al, U.S. Pat. No. 5,631,169; Stavrianopoulos et al, U.S. Pat. No. 4,968,103; each of which is incorporated herein by reference). The fluorophore labels are chosen such that the fluorescence emitted by the first donor molecule is absorbed by the fluorescent label on the second ` acceptor ` molecule, which in turn is capable of emitting fluorescence due to the absorbed energy.

Alternatively, the 'donor' protein molecule may simply utilise the natural fluorescence energy of tryptophan residues. Labels emitting light of different wavelengths are selected so that the ` acceptor ` molecular label can be distinguished from the ` donor `. Since the efficiency of energy transfer between labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In the case where binding occurs between molecules, the fluorescence emission of the 'receptor' molecular label in the assay should be highest. The FRET binding event may be conveniently measured by standard fluorescence detection methods well known in the art (e.g., using a fluorescence photometer).

Modulators of REEP and/or RTP expression may also be identified. For example, a cell or cell-free mixture is contacted with a candidate compound and expression of REEP and/or RTP mRNA or protein is assessed as compared to the expression level of REEP and/or RTP mRNA or protein in the absence of the candidate compound. Identifying the candidate compound as a stimulator of REEP and/or RTP mRNA or protein expression when the expression of REEP and/or RTP mRNA or protein is greater in the presence of the candidate compound than in its absence. Alternatively, a candidate compound is identified as an inhibitor of REEP and/or RTP mRNA or protein expression when its expression in the presence of the candidate compound is less (i.e., statistically significantly less) than its absence. The level of REEP and/or RTP mRNA or protein expression can be determined by the methods described herein for detecting REEP and/or RTP mRNA or protein.

Using cell-based or cell-free assays, modulators can be identified and the ability of the agent to modulate the activity of a REEP and/or RTP protein can be demonstrated in vivo, e.g., in an animal such as an animal model of a disease (e.g., an animal with a REEP and/or RTP associated olfactory disorder).

B. Therapeutic agents

The invention also relates to novel agents identified by the above screening assays. Thus, it is within the scope of the present invention to further use agents identified as described herein (e.g., REEP and/OR RTP modulators OR mimetics, REEP and/OR RTP specific antibodies, REEP and/OR RTP-binding partners OR agonists OR inhibitors) to determine the efficacy, toxicity, side effects, OR mechanism of action of treatment with such agents in an appropriate animal model (e.g., those described herein). Moreover, as described above, the novel agents identified by the above-described screening assays may be used, for example, to treat olfactory disorders (e.g., including, but not limited to, olfactory disorders).

IX. pharmaceutical compositions containing REEP and RTP nucleic acids, peptides and analogs

The present invention also provides pharmaceutical compositions that may comprise all or a portion of a REEP and/or RTP polynucleotide sequence, a REEP and/or RTP polypeptide, an inhibitor or antagonist of the biological activity of REEP and/or RTP, including an antibody, alone or in combination with at least one other agent (e.g., a stabilizing compound), and may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, glucose, and water.

The methods of the invention can be used to treat a disease or alter a physiological state characterized by mutant REEP and/or RTP alleles (e.g., upper respiratory tract infections, anterior cranial fossa tumors and Kallmann syndrome, foster kennedy syndrome, parkinson's disease, alzheimer's disease, huntington's disease). The peptide can be administered intravenously to the patient in a pharmaceutically acceptable carrier, such as physiological saline. Standard methods of intracellular delivery of the peptide (e.g., via liposome delivery) can be used. Such methods are well known to those of ordinary skill in the art. The formulations of the invention may be used for parenteral administration, for example, intravenous, subcutaneous, intramuscular and intraperitoneal. Intracellular therapeutic administration of the polypeptide may also be accomplished using gene therapy as described above.

As is well known in the medical arts, the dosage for any one patient depends on a number of factors, including the size of the patient, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and interaction with other drugs being administered concurrently.

Thus, in some embodiments of the invention, REEP and/or RTP nucleotides and REEP and/or RTP amino acid sequences may be administered to a patient, alone or in combination with other nucleotide sequences, drugs or hormones, or in a pharmaceutical composition in which it is mixed with an excipient or other pharmaceutically acceptable carrier. In one embodiment of the invention, the pharmaceutically acceptable carrier is pharmaceutically inert. In another embodiment of the invention, the REEP and/or RTP polynucleotide sequences or REEP and/or RTP amino acid sequences may be administered separately to an individual suffering from or suffering from a disease.

These pharmaceutical compositions may be formulated and administered systemically or locally depending on the condition being treated. Techniques for formulation and application can be found in the latest edition "Remington's Pharmaceutical Sciences" (MackPublishing Co, Easton Pa.). Suitable routes may, for example, include oral or transmucosal administration; and parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal or intranasal administration.

For injection, the pharmaceutical compositions of the present invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as hanks 'solution, ringer's solution or physiologically buffered saline. For tissue or cell administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

In other embodiments, the pharmaceutical compositions of the present invention may be formulated in dosages suitable for oral administration using pharmaceutically acceptable carriers well known in the art. Such carriers can allow the pharmaceutical compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral or nasal ingestion by a patient to be treated.

Pharmaceutical compositions suitable for use in the present invention include compositions comprising an effective amount of the active ingredient to achieve the intended purpose. For example, an effective amount of REEP and/or RTP can be an amount that inhibits symptoms associated with an olfactory disorder. Determination of an effective amount is within the ability of those skilled in the art, especially with reference to the disclosure provided herein.

In addition to the active ingredient, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, lozenges, capsules or solutions.

The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known (e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes).

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil or synthetic fatty acid esters such as ethyl oleate or triglycerides or liposomes. Aqueous injection suspensions may also contain substances which increase the viscosity of the suspension, for example sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained as follows: the active compound and the solid excipient are combined, the mixture obtained is optionally ground and, after adding suitable adjuvants if necessary, the mixture of granules is processed to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, for example sugars, including lactose, sucrose, mannitol or sorbitol; starches derived from corn, wheat, rice, potato, and the like; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose or sodium carboxymethylcellulose; and gums including gum arabic and gum tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.

The dragee cores are provided with suitable coatings, for example concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings as product identifiers, or to characterize the amount (i.e., dosage) of active compound.

Pharmaceutical products that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating (e.g., glycerol or sorbitol). Push-fit capsules can contain the active ingredients in admixture with fillers or binders (e.g., lactose or starches), lubricants (e.g., talc or magnesium stearate) and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, with or without stabilizers, for example fatty oils, liquid paraffin, or liquid polyethylene glycols.

Compositions comprising a compound of the invention formulated in a pharmaceutically acceptable carrier can be prepared, placed in an appropriate container, and labeled for a therapeutic indication. For polynucleotide or amino acid sequences of REEP and/or RTP, the condition indicated on the tag may comprise treatment of a condition associated with an olfactory disorder.

The pharmaceutical composition may be provided as a salt and may be formed with a number of acids, including but not limited to hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be more soluble in aqueous or other protic solvents than the corresponding free base forms. In other cases, a preferred preparation may be a lyophilized powder at a pH range of 4.5 to 5.5 in 1mM-50mM histidine, 0.1% -2% sucrose, 2% -7% mannitol, in combination with a buffer prior to use.

For any compound used in the methods of the invention, a therapeutically effective dose can be estimated initially from cell culture assays. Preferably, then, the dosage may be formulated in animal models (especially murine models) to achieve the desired circulating concentration range that modulates REEP and/or RTP levels.

A therapeutically effective dose refers to an amount of REEP and/or RTP that ameliorates the symptoms of a disease state. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD₅₀(dose lethal to 50% of the population) and ED₅₀(therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀. Compounds exhibiting a large therapeutic index are preferred. The data obtained from these cell culture assays and additional animal studies can be used to formulate a range of dosage for human use. The dosage of such compounds is preferably such that ED is included₅₀In a range of circulating concentrations of (a), with little or no toxicity. The dosage will vary within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.

The precise dosage is selected by the individual physician according to the patient to be treated. The dosage and administration are adjusted to provide a sufficient level of the active moiety, or to maintain the desired effect. Other factors that may be considered include the severity of the disease state; the age, weight and sex of the patient; diet, time and frequency of administration, drug combination, response sensitivity, and tolerance/response to treatment. Long acting pharmaceutical compositions may be administered 1 time every 3-4 days, weekly, or every 2 weeks, depending on the half-life and clearance of the particular formulation.

Depending on the route of administration, normal doses may range from 0.01 to 100,000 micrograms up to a total dose of about 1 g. Guidance regarding specific dosages and delivery methods is provided in the literature (see, e.g., U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212, all of which are incorporated herein by reference). Those skilled in the art will employ different formulations for REEP and/or RTP than for the inhibitors of REEP and/or RTP. Administration to the bone marrow may require delivery in a manner other than intravenous injection.

RNA interference (RNAi)

RNAi represents an evolutionarily conserved cellular defense that controls the expression of foreign genes in most eukaryotes, including humans. RNAi is triggered by double-stranded RNA (dsRNA) and responds to dsRNA causing sequence-specific mRNA degradation of homologous single-stranded target RNA. mediators of mRNA degradation are small interfering RNA duplexes (sirnas), which are typically generated from long dsrnas by enzymatic cleavage in the cell. sirnas are typically about 21 nucleotides long (e.g., 21-23 nucleotides long) and have a base-pairing structure characterized by a 2-nucleotide 3' -overhang. Following the introduction of small RNAs (or RNAi) into cells, the sequence is thought to be delivered to an enzyme complex called RISC (RNA-induced silencing complex). RISC recognizes the target and cleaves it with endonucleases. It should be noted that if larger RNA sequences are to be delivered to the cell, the RNase III enzyme (Dicer) converts the long dsRNA into a ds siRNA fragment of 21-23 nucleotides.

Chemically synthesized sirnas have become powerful reagents for genome-wide analysis of mammalian gene function in cultured somatic cells. In addition to their value in validating gene function, siRNAs also have great potential as gene-specific therapeutics (Tuschl and Borkhardt, Molecular Intervent.2002; 2 (3): 158-67, incorporated herein by reference).

Transfection of siRNA into animal cells results in efficient and durable post-transcriptional silencing of a particular gene (Caplen et al, Proc Natl Acad Sci U.S. A.2001; 98: 9742-7; Elbashir et al, Nature.2001; 411: 494-8; Elbashir et al, GeneDev.2001; 15: 188-. Methods and compositions for RNAi using siRNA are described, for example, in U.S. Pat. No. 6,506,559, which is incorporated herein by reference.

siRNA is very effective in reducing the amount of targeted RNA and often reaches undetectable levels by extending the protein. This silencing effect can last for months and is very specific, since a 1 nucleotide mismatch between the target RNA and the central region of the siRNA is often sufficient to prevent silencing. BruMme I kamp et al, Science 2002; 296: 550-3; and Holen et al, Nucleic Acids Res.2002; 30: 1757-66, both of which are incorporated herein by reference.

RNAi of RE and RTP

As discussed above, the present invention provides RNAi, OR a pathway component involved in the expression OR activity of a REEP and/OR RTP polypeptide for inhibiting expression of such a component in a cell.

A. RNAi design and testing for REEP and/or RTP

To design siRNA for REEP and/or RTP (e.g., targeting REEP and/or RTPmRNA), software design tools are available in the art (e.g., on the internet). For example, Oligoengine's Web page has a design tool that finds RNAi candidates based on the Elbashir's criteria (Elbashir et al, Methods 2002; 26: 199- "213, incorporated herein by reference). Other design tools, such as the CenixBioscience design tool provided by Ambion, may also be used. In addition, there are Si2 silencing duplexes provided by Oligoengine.

There are also available RNA folding software programs that allow for determining whether an mRNA has a tendency to fold on itself and form a "hairpin" (undesirable in the case of dsRNAi because one goal is to allow RNAi to bind to the mRNA, not to itself). One preferred configuration is an open configuration having 3 or fewer linkages. Generally, a positive Δ G is required to confirm that it does not tend to spontaneously fold upon itself.

Generated siRNA candidate molecules can be screened in animal models of, for example, olfactory disorders, using techniques similar to those described above to quantitatively assess REEP and/or RTP expression in vivo.

B. Expression cassette

The REEP and/or RTP specific siRNA of the present invention can be chemically synthesized. Chemical synthesis may be achieved by any method known or discovered in the art. Alternatively, the REEP and/or RTP specific siRNA of the present invention can be synthesized by a method comprising transcriptional synthesis. In some embodiments, transcription is in vitro, such as from a DNA template and a bacteriophage RNA polymerase promoter. In other embodiments, the synthesis is in vivo, such as from genes and promoters. The split-strand duplex siRNA may also be chemically synthesized by any method known or discovered in the art, wherein 2 strands are separately synthesized and annealed. Alternatively, ds siRNA is synthesized by a method comprising transcription synthesis. In some embodiments, 2 strands of the double-stranded region of the siRNA are expressed separately by 2 different expression cassettes, either in vitro (e.g., in a transcription system) or in vivo in a host cell, and then joined together to form a duplex.

Thus, in another aspect, the invention provides compositions comprising an expression cassette comprising a promoter and a gene encoding an siRNA specific for REEP and/or RTP. In some embodiments, the transcribed siRNA forms one strand of a split-strand duplex (or double-stranded or ds) siRNA that is about 18 to 25 base pairs long; thus, the formation of ds siRNA requires transcription of each of the 2 different strands of ds siRNA. The term "gene" in an expression cassette refers to a nucleic acid sequence comprising a coding sequence necessary for the production of an siRNA. Thus, a gene includes, but is not limited to, the coding sequence of one strand of ds siRNA.

Typically, the DNA expression cassette comprises a chemically synthesized or recombinant DNA molecule containing the coding sequence for at least one strand of the gene or desired ds siRNA, and appropriate nucleic acid sequences required for expression of the operably linked coding sequence in vitro or in vivo. In vitro expression may include expression in transcription systems and transcription/translation systems. In vivo expression may include expression in a particular host cell and/or organism. Nucleic acid sequences required for expression in prokaryotic cells or prokaryotic in vitro expression systems are well known and generally include promoters, operators and ribosome binding sites, often along with other sequences. Eukaryotic in vitro transcription systems and cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. Nucleic acid sequences (referred to in the art as transcription templates) required for expression by bacterial RNA polymerases (e.g., T3, T7, and SP6) include a template DNA strand having a polymerase promoter region followed by the complement of the desired RNA sequence (or coding sequence or gene for siRNA). To create a transcription template, the complementary strand is annealed to the promoter portion of the template strand.

In any of the above expression cassettes, the gene may encode a transcript that contains at least one cleavage site such that, when cleaved, at least 2 cleavage products are produced. Such a product may comprise 2 opposite strands of ds siRNA. In expression systems for expression in eukaryotic cells, the promoter may be constitutive or inducible; the promoter may also be tissue or organ specific (e.g., specific for the eye), or specific for a developmental stage. Preferably, the promoter is located 5' to the transcribed region. Other promoters are also contemplated; such promoters include other polymerase III promoters and microRNA promoters.

Preferably, the expression cassette of a eukaryote further comprises a transcription termination signal suitable for use with a promoter; for example, when a promoter is recognized by RNA polymerase III, the termination signal is an RNA polymerase III termination signal. The cassette may also include a site for stable integration into the host cell genome.

C. Carrier

In other aspects of the invention, the composition comprises a vector comprising a gene encoding an siRNA specific for REEP and/or RTP, or preferably at least one expression cassette comprising a promoter and a gene encoding a sequence required for the production of an siRNA specific for REEP and/or RTP (siRNA gene). The vector may also comprise a marker gene, a reporter gene, a selection gene or a gene of interest, e.g. an experimental gene. The vectors of the present invention include cloning vectors and expression vectors. The expression vector may be used in an in vitro transcription/translation system, as well as in vivo in a host cell. Expression vectors for use in vivo in host cells may be transiently or stably transfected into the host cell. Thus, the vector may also comprise a site for stable integration into the genome of the host cell.

In some embodiments, it is useful to clone the siRNA gene downstream of the bacteriophage RNA polymerase promoter into a multicopy plasmid. A variety of transcription vectors containing a bacteriophage RNA polymerase promoter (e.g., the T7 promoter) are available. Alternatively, DNA synthesis can be used to add a bacteriophage RNA polymerase promoter located upstream of the siRNA coding sequence. Cloned plasmid DNA linearized with restriction enzymes can then be used as transcription template (see, e.g., Milligan, JF and Uhlenbeck, OC (1989) Method in Enzymology 180: 51-64).

In other embodiments of the invention, vectors include, but are not limited to, chromosomal, non-chromosomal and synthetic DNA sequences (e.g., derivatives of viral DNA, such as vaccinia, adenovirus, fowlpox virus and pseudorabies). It is contemplated that any vector may be used so long as it is capable of being expressed in an appropriate system (whether in vitro or in vivo) and is capable of surviving in a host when used in vivo; these 2 criteria are sufficient for transient transfection. For stable transfection, the vector may also replicate in the host.

A large number of suitable vectors are known to those skilled in the art and are commercially available. In some embodiments of the invention, the mammalian expression vector comprises an origin of replication, a suitable promoter and enhancer, and any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences and 5' flanking nontranscribed sequences. In other embodiments, DNA sequences derived from SV40 splicing and polyadenylation sites may be used to provide the required non-transcribed genetic elements.

In certain embodiments of the invention, gene sequences in expression vectors that are not part of the expression cassette comprising the siRNA genes (specific for REEP1, RTP1, RTP2, RTP1-A, RTP1-B, RTP1-C, RTP1-D and RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A (chimera 1), RTP1-A1-D2 (chimera 2), RTP1-A1-D1 (chimera 3), RTP4-A1-A (chimera 4), RTP4-A1-D2 (chimera 5) and RTP4-A1-D1 (chimera 6)) can be operably linked to appropriate expression control sequences (promoters) to direct the synthesis of mRNA. In some embodiments, the gene sequence is a marker gene or a selection gene. Promoters useful in the present invention include, but are not limited to, the Cytomegalovirus (CMV) immediate early promoter, the Herpes Simplex Virus (HSV) thymidine kinase promoter and the mouse metallothionein promoter and other promoters known to control expression of genes in mammalian cells or their viruses. In other embodiments of the invention, the recombinant expression vector includes an origin of replication and a selectable marker that allows for transformation of the host cell (e.g., dihydrofolate reductase or neomycin resistance in eukaryotic cell culture).

In some embodiments of the invention, transcription of the DNA encoding the gene is enhanced by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to enhance its transcription. Enhancers useful in the present invention include, but are not limited to, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Preferably, the vector is designed to deliver RNAi for more permanent inhibition. For example, the pSilencer siRNA expression vector provided by Ambion, the pSuper RNAi system provided by Oligoengine, and the GnESilencer system provided by IMGENEX. They are both RNAi based plasmid vectors. BD Biosciences provides an RNAi-Ready pSIREN vector that allows for plasmid-based vectors and adenoviral or retroviral delivery formats. Adenoviral vectors that would release siRNA immediately were expected by Ambion. As regards the design of the vector, there is no restriction as to the folding pattern, since the formation of hairpins is not a concern, or at least no study has found any differences in properties related to the mRNA folding pattern. Thus, SEQ ID NO: 1-20, for example, can be used with vector (plasmid and viral) delivery systems.

It should be noted that Ambion provides a tool for vector design on their webpage and BDBiosciences provides a manual for vector design, both of which can be used to design vectors for siRNA.

D. Transfected cells

In other aspects, the invention provides compositions comprising a cell transfected with an expression cassette of the invention as described above or a vector of the invention, wherein the vector comprises an expression cassette of the invention (or just an siRNA gene) as described above. In some embodiments of the invention, the host cell is a mammalian cell. The transfected cells may be cultured cells or tissues, organs or biological cells. Specific examples of cultured host cells include, but are not limited to, Chinese Hamster Ovary (CHO) cells, COS-7 lines of monkey kidney fibroblasts, 293T, C127, 3T3, HeLa and BHK cell lines. Specific examples of in vivo host cells include tumor tissue and ocular tissue.

The cell may be transiently or stably transfected (e.g., DNA expressing the siRNA is stably integrated into and expressed by the genome of the host cell). Cells can also be transfected with the expression cassettes of the invention, or they can be transfected with the expression vectors of the invention. In some embodiments, the transfected cells are cultured mammalian cells, preferably human cells. In other embodiments, they are tissues, organs or biological cells.

In the present invention, cells to be transfected in vitro are typically cultured and then transfected according to methods well known in the art, for example, by the preferred method defined by the U.S. tissue culture center. In certain embodiments of the invention, cells are transfected with an siRNA synthesized exogenously (or in vitro, e.g., by chemical means or in vitro transcription methods), or they are transfected with an expression cassette or vector that expresses the siRNA in the transfected cells.

In some embodiments, the cells are transfected with siRNA by any method known or discovered in the art that allows the cells to take up exogenous RNA and remain viable. Non-limiting examples include electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, osmotic shock, temperature shock and electroporation and pressure treatment. In an alternative embodiment, the siRNA is introduced in vivo by lipofection, as has been reported (e.g., Elbashir et al (2001) Nature 411: 494-498, incorporated herein by reference).

In other embodiments, the expression cassette or vector comprising at least 1 expression cassette is introduced into the desired host cell by methods known in the art, including, but not limited to, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, using a gene gun or using a DNA vector carrier (see, e.g., Wu et al (1992) J.biol.chem., 267: 963; Wu and Wu (1988) J.biol.chem., 263: 14621; and Williams et al (1991) Proc.Natl.Acad.Sci.USA 88: 272). Receptor-mediated DNA delivery protocols (Curiel et al (1992) hum. Gene Ther., 3: 147; and Wu (1987) J.biol. chem., 262: 4429) can also be used. In some embodiments, various methods are used to enhance transfection of cells. These methods include, but are not limited to, osmotic shock, temperature shock and electroporation and pressure treatment.

Alternatively, the vector is introduced in vivo by lipofection. The use of liposomes for the encapsulation and transfection of nucleic acids in vitro has increased over the past decade. Synthetic cationic lipids designed to limit the difficulties and risks encountered with liposome-mediated transfection may be used to prepare liposomes for in vivo transfection of marker-encoding genes. The use of cationic lipids can facilitate encapsulation of negatively charged nucleic acids, and can also facilitate fusion with negatively charged cell membranes. Particularly useful lipid compounds and compositions for transferring nucleic acids are described in WO95/18863 and WO96/17823 and U.S. Pat. No. 5,459,127, which are incorporated herein by reference. Other molecules may also be used to facilitate transfection of nucleic acids in vivo, such as cationic oligopeptides (e.g., WO95/21931), peptides derived from DNA binding proteins (e.g., WO96/25508) or cationic polymers (e.g., WO 95/21931).

Sequences encoding siRNA may also be introduced in vivo as naked DNA, either as an expression cassette or as a vector. Methods of formulating and administering naked DNA to mammalian muscle tissue are described in U.S. Pat. Nos. 5,580,859 and 5,589,466, both of which are incorporated herein by reference.

Stable transfection typically requires the presence of a selectable marker in the vector used for transfection. The transfected cells are then subjected to a selection procedure. Typically, the selection involves growing the cells in a toxic substance (e.g., G418 or hygromycin B), such that only those cells that express the transfected marker gene that confers resistance to the toxic substance to the transfected cells survive and grow. Such selection techniques are well known in the art. Typical selectable markers are well known and include genes encoding resistance to G418 or hygromycin B.

In a preferred embodiment, the transfection agent is OLIGOFECTAMINE. OLIGOFECTAMINE is a lipid-based transfection agent. Other examples of lipid-based transfection agents designed to transfect dsRNAi are Transit-TKO agents supplied by Mirus (Madison, Wis.) and jetSI introduced by Polyplus-trasfection SAS. In addition, the Silencer siRNA transfection kit provided by Ambion includes siPORT Amine and siPORT Lipid transfection reagent. Roche provides a Fugene6 transfection reagent, which is also lipid-based. In cell culture, there are also options to use electroporation. Preferably, the plasmid vector delivery system is transfected into cells using OLIGOFECTAMINE supplied by Invitrogen or siPORTXP-I transfection reagent supplied by Ambion.

In certain embodiments, certain chemical modifications of dsRNAi can be employed, such as altering the lipophilicity of the molecule (e.g., binding of lipophilic residues to the 3' terminus of dsRNA). dsRNA can also be delivered into organisms using previously developed methods for antisense oligonucleotide applications, such as injection of liposome-encapsulated molecules.

E. Reagent kit

The invention also provides a kit comprising at least 1 expression cassette comprising an siRNA gene specific for REEP and/or RTP. In some aspects, transcripts from the expression cassette form double-stranded siRNAs about 18 to 25 base pairs long. In other embodiments, the expression cassette is contained in a vector as described above, wherein the vector can be used in an in vitro transcription or transcription/translation system, or in vivo for transiently or stably transfecting cells.

In other aspects, the kit comprises at least 2 expression cassettes, each comprising an siRNA gene, at least one gene encoding one strand of the siRNA that binds to a strand encoded by a second cassette to form a ds siRNA; the ds siRNA so generated is any of the embodiments described above. These cassettes may contain a promoter and a sequence encoding one strand of the ds siRNA. In some further embodiments, 2 expression cassettes are present in one vector; in other embodiments, 2 expression cassettes are present in 2 different vectors. A vector having at least one expression cassette, or 2 different vectors, each comprising one expression cassette, can be used in an in vitro transcription or transcription/translation system, or in vivo for transiently or stably transfecting cells.

In other aspects, the kit comprises at least one expression cassette comprising a gene encoding 2 separate strands of ds siRNA and a processing site located between the sequences encoding each strand, so that when the gene is transcribed, the transcript is processed, e.g., by cleavage, to produce 2 separate strands which can bind to form ds siRNA as described above.

In some embodiments, the present invention provides a kit comprising: a) a composition comprising a small interfering RNA duplex (siRNA) configured to inhibit expression of a REEP and/or RTP protein, and b) a printed material with instructions for using the composition to treat a target cell expressing a REEP and/or RTP protein by expression of a REEP and/or RTPmRNA under conditions that cleave or otherwise inactivate the REEP and/or RTP mRNA. In certain embodiments, the printed material comprises instructions for using the composition to treat an ocular disorder.

F. Production of REEP and/or RTP specific siRNA

The invention also provides methods of synthesizing sirnas specific for REEP and/or RTP (e.g., human REEP and/or RTP) or specific for mutant or wild-type forms of REEP and/or RTP. siRNA can be synthesized in vitro or in vivo. In vitro synthesis includes chemical synthesis and synthesis by in vitro transcription. In vitro transcription is effected in a transcription system, such as RNA polymerase from a bacteriophage or in a transcription/translation system, such as RNA polymerase from a eukaryote. In vivo synthesis occurs in transfected host cells.

In vitro synthesized siRNA (whether chemically or by transcription) is used to transfect cells. Accordingly, the invention also provides methods of transfecting a host cell with an in vitro synthesized siRNA; in particular embodiments, the siRNA is synthesized by in vitro transcription. The invention also provides methods for silencing REEP and/or RTP genes in vivo by transfecting cells with siRNA synthesized in vitro. In other methods, the siRNA is expressed in vitro in a transcription/translation system from an expression cassette or vector and an expression vector encoding and expressing a reporter gene.

The invention also provides methods for expressing siRNA in vivo by transfecting cells with an expression cassette or vector that directs the synthesis of siRNA in vivo. The invention also provides methods for silencing genes in vivo by transfecting cells with an expression cassette or vector that directs the synthesis of siRNA in vivo.

XII identification of odorant receptor ligands

The present invention provides methods for identifying ligands specific for odorant receptors. The present invention is not limited to a particular method of identifying ligands specific for odorant receptors. In a preferred embodiment, the invention provides cell lines (e.g., heterologous 293T cell lines) that express target odorant receptors (e.g., any human odorant receptor) localized on the cell surface, REEP1, RTP1 or variants thereof, RTP2 and G _αolf. Activation of odorant receptors results in an increase in cAMP. Thus, in some embodiments, the cell line further comprisesA cAMP response element associated with a reporter reagent (e.g., luciferase) for detecting odorant receptor activation. An odorant molecule (e.g., eugenol) is exposed to the cell line. If the odorant molecule is a ligand specific for an odorant receptor, luciferase expression or a change in luciferase expression is detectable (see, e.g., example 7).

Examples

To identify accessory proteins involved in targeting OR to the cell surface, genes were screened for the ability to induce functional cell surface expression of OR in HEK293T (293T) cells. It was found that REEP1, RTP1, RTP2, RTP1-A, RTP1-B, RTP1-C, RTP1-D and RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A (chimera 1), RTP1-A1-D2 (chimera 2), RTP1-A1-D1 (chimera 3), RTP4-A1-A (chimera 4), RTP4-A1-D2 (chimera 5) and RTP4-A1-D1 (chimera 6) promoted cell surface expression of OR. These proteins are expressed by olfactory neurons, interact with OR proteins, and enhance the response to odorants when co-expressed with OR in 293T cells. Moreover, this has allowed the construction of heterologous expression systems to identify novel ORs that respond to aliphatic odorants.

Example 1 identification of odorant receptor accessory proteins

Given that mammalian OR requires functional cell surface expression of accessory proteins, a study was conducted to detect such molecules. From individual olfactory neurons as well as neurons from the vomeronasal apparatus, Long-SAGE (serial analysis of gene expression) libraries were constructed (see, e.g., Saha, S. et al, (2002) Nat Biotechnol20, 508-. To identify candidate genes expressed by olfactory neurons, a numerical differential display was also used (see, e.g., http:// www.ncbi.nlm.nih.gov/UniGene/info _ ddd.shtml). The ORF of the candidate gene encoding the membrane-associated protein was investigated. Genes with similarities to known chaperones were selected and cDNA was cloned from olfactory epithelial cDNA. The mRNA expression of each gene was confirmed by in situ hybridization. After isolation and subcloning into mammalian expression vectors, each cDNA was transfected into 293T cells together with mouse OR (MOR203-1) labeled with the 20N-terminal amino acids of rhodopsin (Rho-tag). Measurements were made to assess whether these clones had an effect on cell surface expression of OR by staining live cells with antibodies against Rho-marker (see, e.g., Laird, D.W. and Molday, R.S. (1988) Invest Ophthalmol Vis Sci29, 419-428; herein incorporated by reference in its entirety). When MOR203-1 was transfected alone, antibody staining detected only weak cell surface expression in less than 1% of the cells. In fig. 1, a schematic diagram depicting a screening method used in the present invention is provided.

Example 2 REEP and/OR RTP enhances cell surface expression of OR

2 unrelated clones (out of 61 tested) enhanced the number of cell surface expression and staining intensity of MOR203-1 (see FIG. 2A). The proteins encoded by these clones were designated REEP1 (i.e., receptor expression enhancing protein 1) and RTP1 (i.e., receptor transporter 1). Subsequently, RTP2 was discovered, which is closely related to RTP 1. RTP2 also enhanced the cell surface expression of MOR 203-1. Next, REEP1, RTP1 and RTP2 were tested to detect similar effects in promoting cell surface expression of other ORs. 4 different ORs were expressed in 293T cells with OR without REEP1, RTP1 OR RTP2 (mouse OREG, mouse o1fr62, mouse OR-S46 and rat I7). Co-transfection of BFP or GFP demonstrated that the transfection efficiency was consistent (about 70%). In addition, the OR transfected with REEP and/OR RTP produced more immunofluorescent cells and stronger signals in positive cells than the OR without REEP and/OR RTP (see fig. 2A). When different ORs are used in each condition, the signal intensity and number of immune positive cells will vary. For example, in the case of rat I7, the surface expression was significantly lower than the other ORs tested. Nevertheless, only occasional immune-positive cells were observed when co-expressing the helper protein. The effect of RTP1 or RTP2 was consistently stronger than that of REEP 1. The enhancement of cell surface expression is specific for OR, but not for other GPCRs: neither REEP nor RTP enhances expression of the β 2 adrenergic receptor, mT2R5 (mouse bitter taste receptor) (see, e.g., Chandrashekar, J. et al (2000) Cell100, 703-711; incorporated herein by reference in its entirety) or the V2R pheromone receptor (VR4) (see, e.g., Matsunami, H. and Buck, L.B. (1997) Cell90, 775-784; incorporated herein by reference in its entirety) (see, e.g., FIGS. 2A and 3). Finally, when REEP and other members of the RTP family (REEP2 and RTP4) were co-expressed, no enhancement of cell surface expression of MOR203-1 was observed.

To quantify the number and intensity of the immunopositive cells, fluorescence-activated cell sorting (FACS) analysis was performed. To monitor transfection and staining efficiency, HA-labeled β 2 adrenergic receptors were used as controls. When OR is expressed with the helper protein, more cells are labeled and the fluorescence signal is higher (see fig. 2B and 2C and fig. 4).

Example 3 REEP and/or RTP genes encoding transmembrane proteins

The REEP1 gene encodes a 201 amino acid protein that contains 2 putative transmembrane domains (see fig. 5A). Immunostaining of the C-terminally labeled REEP1 protein indicated that the C-terminus was extracellular. BLAST searches identified homologous genes in different eukaryotic species. REEP1 showed limited similarity to yeast YOP1, barley HVA22 and human DP1/TB2 (see figure 5B). YOP1 are involved in vesicle trafficking (see, e.g., Brands, A. and Ho, T.H. (2002) Plant Physiol130, 1121, 1131; incorporated herein by reference in its entirety). Expression of HVA22 is induced by abscisic acid and is regulated by a variety of environmental stresses, such as extreme temperatures or dehydration (see, e.g., Chen, C.N. et al (2002) Plant Mol Biol49, 633-644; Shen, Q. et al (1993) J Biol Chem268, 23652-23660; each of which is incorporated herein by reference in its entirety). DP1/TB2 is encoded by genes deleted in colon cancer (see, e.g., Kinzler, K.W. et al (1991) Science253, 661-. In the mouse genome, REEP1 has at least 5 additional homologous genes (called REEP2-6) (see fig. 5B).

The RTP1 and RTP2 genes encoded proteins with 263 and 223 amino acids, respectively, and had 73% sequence identity at the amino acid level (see fig. 5C). Both proteins appear to have no signal sequence, but both have a putative transmembrane domain located near the C-terminus. Immunostaining of C-terminally labeled RTP1 indicated that the C-terminus was extracellular. BLAST searches of the mouse genome identified 2 additional members RTP3 and RTP 4. Outside of vertebrate species, there are no obvious RTP homologs. Nonetheless, the Meloidogyne Egypti ODR-4 (see, e.g., Dwyer, N.D. et al (1998) Cell93, 455-466; incorporated herein by reference in its entirety) appears to have the same membrane topology as RTP.

Example 4 specific expression of REEP and/or RTP in olfactory neurons

Northern blot analysis with RNA extracted from different mouse tissues showed that REEP1 and in particular RTP1 and RTP2 were most significantly expressed in the olfactory organs and nasal plow. In the brain, significant levels of REEP1RNA were also detected (see fig. 6A). Long-term exposure revealed weak signaling of RTP1 and RTP2 in the brain. No expression in testis was observed, where a subset of OR was expressed (see, e.g., Parmentier, M. et al (1992) Nature355, 453-.

In the olfactory epithelium, REEP and/or RTP are specifically expressed in olfactory neurons, as can be seen by comparing OMP expression (a marker for mature olfactory sensory neurons) (see fig. 6B). To avoid cross-hybridization between RTP1 and RTP2RNA (whose coding sequences are 87% identical at the nucleotide level), a non-homologous 3' UTR region was used as a probe, except for the probe corresponding to the open reading frame. The signals are identical. No expression of other REEP or RTP genes was detected in olfactory neurons, except that RTP4 was expressed at lower levels (see fig. 6B). Finally, a subset of brain cells expressed REEP1 (see fig. 6C).

Example 5 REEP1 and RTP1 can interact with OR

Given the ability of REEP and/OR RTP to promote cell surface expression of OR, it is postulated that they also interact with OR proteins. This can be assessed using a co-immunoprecipitation assay. HA-labeled MOR203-1 and Flag-labeled REEP1, RTP1 or ICAP-1 (negative controls) (see, e.g., Zawistowski, J.S. et al (2002) Hum MolGenet11, 389-396; incorporated herein by reference in its entirety) were transfected into 293T cells. After precipitation of the cell extract with anti-Flag antibody, the proteins were eluted in SDS-sample buffer at room temperature, followed by western blot analysis to detect OR protein. After precipitation of REEP1 OR RTP1, the OR protein was detected as a high molecular weight band (see fig. 7B, lanes 1 and 2). Using these elution conditions, most control GPCR- β 2 adrenergic receptors do not form high molecular weight oligomers. Similarly, when HA-MOR203-1 protein was precipitated, REEP1 or RTP1 proteins were co-precipitated, while ICAP-1 was not detected (see FIG. 7C, lanes 1, 2 and 3). The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nevertheless, these results show that REEP1 and RTP1 complex with OR.

Based on protein interactions, it is postulated that functional expression of helper proteins may be regulated by the OR protein. When only the C-terminal Flag-labeled RTP1 was transfected into 293T cells, little cell surface signal was detected, indicating that most of the RTP protein was intracellular. In contrast, co-transfection of RTP1 and OR greatly enhanced cell surface RTP1 (see fig. 7D). The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nevertheless, these results demonstrate the interdependence of OR and RTP1 for cell surface expression and indicate that efficient cell surface expression of OR and RTP1 requires the formation of a relatively stable receptor complex between the two. When expressing the C-terminal tagged REEP1, a small amount of cell surface REEP1 was observed. Unlike RTP1, co-expression of OR protein did not promote cell surface expression of REEP1 (see fig. 7E).

Example 6 REEP and/OR RTP enhanced OR functionality

The poor signalling activity induced by odorants in heterologous cell culture systems expressing OR has been attributed to the poor cell surface expression of OR. The identification of REEP and/or RTP allows direct assessment of the problem. Luciferase reporter gene assays were used in which CAMP Response Element (CRE) mediates luciferase gene expression (see fig. 8A). Because OR activation results in an increase in cAMP, activation of the mouse odorant receptor OREG by its ligand eugenol is measured in the presence and absence of REEP and/OR RTP (see, e.g., Kajiya, K. et al (2001) J Neurosci21, 6018-F6025; Touhara, K. et al (1999) Proc Natl Acad Sci U A96, 4040-F4045; each of which is incorporated herein by reference in its entirety). As previously reported, eugenol increases the level of OREG-dependent luciferase activity (see, e.g., Katada, S. et al (2003) Biochem Biophys Res Commun305, 964-969; incorporated herein by reference in its entirety). Co-expression of OREG with REEP and/or RTP significantly enhanced odorant-dependent luciferase activity (fig. 8B). Similar results were obtained when two other OREG ligands, vanillin or ethyl vanillin, were used. Since RTP4 is also expressed at low levels in the olfactory epithelium, this protein is co-expressed with OREG, but this does not produce a significant increase in luciferase reporter gene activity.

Depending on the helper protein, other GPCRs may exhibit changes in ligand specificity (see, e.g., McLatchie, L.M. et al (1998) Nature393, 333-. To investigate whether REEP1, RTP1 OR RTP2 would alter the ligand selectivity of OR, OREG and OR-S46 were tested, as well as their agonists and related chemicals. No substantial change in relative chemoselectivity was observed when the receptor was co-expressed with the helper protein (see figure 8C).

Example 7 construction of a functional assay to identify odorant-receptor interactions

To facilitate analysis of odorant-OR interactions, stable expression of REEP1, RTP1, RTP2 and G was established_αolf(coupled to the alpha subunit of the G protein of OR) 293T cell line (see, e.g., Belluscio, L. et al (1998) Neuron20, 69-81; Jones, D.T. and Reed, R.R (1989). Science244, 790-795; each of which is incorporated herein by reference in its entirety). To establish such cells, mice REEP1, RTP1, RTP2 and G were included_αolfThe linearized expression vector of the ORF is transfected into 293T cells with PGK-Pac (puromycin resistance gene) (see, e.g., Watanabe, S. et al (1995) biochem Biophys Res Commun213, 130-137; incorporated herein by reference in its entirety). Among puromycin resistant clones, clone 3A showed a strong response to eugenol when transfected with OREG, and was designated Hana 3A. RT-PCR analysis showed that Hana3A cells express exogenous REEP1, RTP1, RTP2 and G _αolf(see FIG. 9). When OREG OR other OR was transfected and immunostained in Hana3A cells, enhanced cell surface expression was observed (see fig. 10). To test whether Hana3A cells also increased ligand response in luciferase assays, CRE-luciferase reporter genes were co-transfected with OREG (HA-labeled), OR-S46, OR OR-S50 and the cells were stimulated with their ligands eugenol, pelargonic acid, and azelaic acid, respectively (see, e.g., Malnic, B. et al (1999) Cell96, 713-Asonic 723; Touhara, K. et al (1999) Proc Natl Acadsi U S A96, 4040-Asonic 4045; each of which is incorporated herein by reference in its entirety). Little luciferase induction was observed when HA-OREG was expressed in 293T cells. In contrast, when Hana3A cells were used, an increase in luciferase activity was observed following eugenol stimulation (see fig. 8D). Similar results were obtained using 2 other ORs (OR-S46 and OR-S50). The OR-S50 gene did not produce a luciferase reaction in 293T cells, whereas the same receptor transfected into Hana3A cells produced strong luciferase activity (see FIG. 8D). Using this assay, G alone was expressed in 293T cells_αolfWith little OR no effect on OR activationThe application is as follows.

To demonstrate the enhanced OR function in the presence of REEP1, RTP1 and RTP2, the expression G was used_αolf293T cells and Hana3A cells, the amount of cAMP after ligand stimulation was measured. When OREG was transfected and eugenol was added to stimulate OR, more cAMP was produced in Hana3A cells. In contrast, when β 2 adrenergic receptors were expressed and isoproterenol was used, no significant difference in cAMP production was observed (see fig. 8E). The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nevertheless, this further supports the specific role of the helper protein in functional OR expression.

Previous studies demonstrated that individual olfactory neurons activated by aliphatic alcohols and acids express specific OR, primarily class I (fish-like) OR (see, e.g., Malnic, B. et al (1999) Cell96, 713-133; Zhang, X. and Firestein, S. (2002) Nat Neurosci5, 124-133; each of which is incorporated herein by reference in its entirety). For the panel of assays for aliphatic alcohols, aldehydes and acids and some other odorants, 4 OR' S (S6/79, S18, S46 and S50) were tested previously determined using other techniques (see, e.g., Malnic, B. et al (1999) Cell96, 713-723; incorporated herein by reference in its entirety). In addition, 5 "orphan" class I ORs (MOR23-1, MOR31-4, MOR31-6, MOR32-5 and MOR32-11) were tested, and their cognate ligands were not known. At a suprathreshold concentration of 100uM, all of these ORs were odorant selective, responding only to a small subset of the odorants tested (see fig. 11A). This specificity is maintained at a lower, more physiologically relevant concentration. Many of these ORs react to odorants present in micromolar concentrations. Cell surface expression of these ORs was assessed by live cell immunofluorescence. Some ORs (S18, MOR31-4, MOR31-6 and MOR32-5) were strongly expressed, while others (S6, S50, MOR23-1, MOR32-11) were less strongly expressed (see FIG. 12). The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nevertheless, these results indicate that the expression is weak enough to produce a significant response to a physiologically relevant concentration of odorant. Finally, 2 additional orphan II class OR, MOR203-1 and o1fr62 were tested against a panel of 139 odorants. MOR203-1 reacted to high concentrations of pelargonic acid (see FIG. 11B). 01fr62 reacts to coumarin and piperonal (see FIG. 11C). Next, several related aromatic compounds were tested, identifying 2-coumaranone as the preferred ligand for o1fr62 (see fig. 11C). When the parental 293T cells of these ORs were used in the luciferase assay, little OR no response to odorants was observed. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nevertheless, these results further demonstrate the importance of REEP and/OR RTP in functional OR expression.

Example 8 REEP and/or RTP Functions during receptor folding, transport and/or odorant recognition

Expression of GPCRs is a complex process involving protein folding, post-translational modification, and transport through cellular compartments, including the ER and golgi apparatus. In addition, there is evidence that proper targeting of GPCRs to the plasma membrane may involve homo-or heterodimerization (see, e.g., Angers, S. et al (2002) Annu Rev Pharmacol cytosol Toxicol42, 409-435; incorporated herein by reference in its entirety). The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nevertheless, although REEP and/OR RTP can function in any of these OR expression steps, fig. 13 shows three possibilities with respect to their possible interactions.

First, REEP and/OR RTP promote proper folding of OR in the ER. NinaA, a cyclophilin homolog of Drosophila, was identified as a chaperone for rhodopsin and was thought to promote proper folding (see, e.g., Baker, E.K. et al (1994) Embo J13, 4886-. The plant homolog HVA22 of REEP1 is a stress-induced gene and can allow plants to tolerate adverse conditions (see, e.g., Chen, C.N. et al (2002) plantaMol Biol49, 633-23644; Shen, Q. et al (1993) J Biol Chem268, 23652-23660; each of which is incorporated herein by reference in its entirety). The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nevertheless, although the precise role of HVA22 is not known, since many stress-induced proteins (e.g., heat shock proteins) act as chaperones, it is conceivable that HVA22 and, by analogy, possibly REEP1, act as chaperones to promote folding.

Second, REEP1, RTP1 and RTP2 facilitate the transport of specific vesicles/cargo including OR. Consistent with this concept, REEP1 homolog YOP1P in yeast has been implicated in Rab-mediated vesicle trafficking (see, e.g., Brands, A. and Ho, T.H. (2002) plantaPhysiol 130, 1121-. In Meloidogyne americana, clathrin aptamer subunit UNC-101 mediates the transport of chemosensory receptors to olfactory cilia (see, e.g., Dwyer, N.D., et al, (2001) Neuron31, 277-287; incorporated herein by reference in its entirety).

Again, REEP1, RTP1 and RTP2 act as co-receptors for OR. As shown in fig. 7D, co-expression of OR enhanced RTP1 cell surface expression. OR may contain an ER retention signal that is masked by binding to RTP (OR REEP1), a mechanism similar to the regulation of cell surface expression of GABA (B) R1 receptors by binding to GABA (B) R2 (see, e.g., Jones, K.A. et al (1998) Nature396, 674-containing 679; Kaupmann, K.et al (1998) Nature396, 683-containing 687; White, J.H. et al (1998) Nature396, 679-containing 682; each of which is incorporated herein by reference in its entirety). REEP and RTP may have different or complementary effects, which is a hypothesis consistent with the lack of any amino acid sequence similarity or specific sequence motifs.

The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nevertheless, the three actions described above are reasonable for REEP1, RTP1 and RTP 2; however, other possible functions are not excluded. Although no changes in ligand specificity of OREG OR-S46 were observed when expressed with REEP1, RTP1 OR RTP2, it is likely that they do play a role in regulating the recognition profile of some ORs. For example, different members of RAMP will alter the ligand specificity of a calcitonin receptor-like receptor (CRLR), a member of GPCRs. CRLR expressed with RAMP1 functions as the CGRP receptor and CRLR expressed with RAMP2 functions as the adrenomedullin receptor (see, e.g., McLatchie, L.M. et al (1998) Nature393, 333-339; herein incorporated by reference in its entirety).

The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nevertheless, many GPCRs, including the V1R pheromone receptor (see, e.g., Dulac, C and Axel, R. (1995) Cell83, 195- "206; incorporated herein by reference in its entirety), the T2R taste receptor (see, e.g., Adler et al (2000) Cell100, 693-; Matsunami, H., Montmayeur, J.P., and Buck, L.B. (2000) Nature404, 601-; each incorporated herein by reference in its entirety), the α 2C adrenergic receptor (see, e.g., Hurt, C.M., et al (2000) J Biol Chem275, 35424-; incorporated herein by reference in its entirety) and the thyroid-stimulating hormone releasing hormone receptor (see, e.g., Yu, R., and Hinkle, P.M. (1997) Mol rmacol 51, 785 Pha 793; incorporated herein by reference in its entirety), appear to require expression of their Cell surface cofactors. Thus, REEP and RTP members can regulate the transport of such GPCRs. In situ hybridization analysis has demonstrated that REEP3, REEP5, RTP1 and RTP2 are all expressed by VNO neurons. In addition, REEP members are differentially expressed in brain cell subsets (m.m. and h.m., unpublished observations). In such cases strategies may be applied, using SAGE and/or numerical differential display, to build a list of genes expressed in a particular cell type and screen for genes that promote cell surface expression of the receptor.

Example 9 REEP and/or RTP enables the study of odorant receptor-odorant interactions

Expression systems have been established that allow rapid identification of ligands for OR. The system was tested with twelve ORs. 4 of the tested ORs (S6/S79, S18, S46 and S50) were expressed in a single olfactory neuron that responded to an aliphatic odorant (see, e.g., Malnic, B. et al (1999) Cell96, 713-723; herein incorporated by reference in its entirety). The response profile of OR-S50 (but not that of OR-S18) is consistent with previous reports (see, e.g., Malnic, B. et al (1999) Cell96, 713-723; incorporated herein by reference in its entirety). In previous studies, olfactory neurons S6 and S79 expressed the same OR (OR-S6/S79) and both responded to azelaic acid, although only olfactory neuron S79 responded to 2 odorants (heptanoic acid and octanoic acid) (see, e.g., Malnic, B. et al (1999) Cell96, 713-723; herein incorporated by reference in its entirety). In experiments performed during the course of the present invention, OR-S6/S79 responded to azelaic acid, but not to heptanoic acid OR octanoic acid. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nevertheless, these results support the olfactory neuron S6 response spectrum. The differences may be due to variations in response when recorded from a single olfactory neuron. When multiple single olfactory neurons expressing the same OR are recorded against the same set of odorants using calcium imaging, their response profiles are similar but different (see, e.g., Bozza, T. et al (2002) J Neurosci22, 3033-3043; incorporated herein by reference in their entirety).

7 new ORs were identified that responded to different odorants in the test group. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nevertheless, these results demonstrate the applicability of this system to decode ligand specificity of OR. The response profile of the OR to different odorants is consistent with the concept of a "combinatorial receptor code" in which one OR responds to multiple related odorants and one odorant activates multiple receptors (see, e.g., Kajiya, K. et al (2001) J Neurosci21, 6018-.

In experiments conducted during the course of the present invention, not only 3 types of OR class I (S46, MOR23-1, MOR31-4), but also MOR203-1 (a type II OR), responded to nonanoic acid. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nevertheless, these results indicate that very different ORs may respond to the same chemical species, since MOR203-1 is only 29-32% identical to other nonanoic acid ORs (MOR23-1, MOR31-4 and S46). 01fr62 is one of the closely related ORs involved in the perception of isovaleric acid located near OR at the IVA locus (see, e.g., Griff, I.C. and Reed, R.R. (1995) Cell83, 407 414; Zhang, X. and Firestein, S. (2002) Nat Neurosci5, 124-. In experiments performed during the course of the present invention, o1fr62 did not respond to isovaleric acid, but to coumarin and other related aromatic compounds (see fig. 11C). 8 other ORs located near the IVA locus were also tested, but none responded to isovalerate. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nevertheless, these results indicate that these ORs are not involved in isovaleric acid detection.

The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nevertheless, a functional OR expression system and annotation of nearly all ORs in the mouse and human genomes (see, e.g., Glusman, G. et al (2001) Genome Res11, 685-702; Young, J.M. et al (2002) Hum Mol Genet11, 535-546; Zhang, X. and Firestein, S. (2002) Nat Neurosci5, 124-133; Zozulya, S. et al (2001) Genome Biol2, 18; each of which is incorporated herein by reference in its entirety) provides a platform for studying mammalian OR-odorant interactions in an integrated manner.

Example 10 Single cell Long SAGE analysis

Single Cell RT-PCR was performed after modification as described (see, e.g., Brady, G. and Iscove, N.N. (1993) methods enzymol225, 611-. Briefly, adult mouse olfactory tissues were dissociated with dispase (Invitrogen) and collagenase (Invitrogen). Using a micromanipulator, in the reverse directionSingle cells were picked under the microscope and transferred into 4.75ul lysis mix (1xPCR buffer (Roche), 1.5mM MgCl) ₂50uM dNTPs, 200ng/mg anchor primer (biotin-TATAGAATTCGCGGCCGCTCGCGA(T) 24), 0.3U/ul Prime RNase inhibitor (Eppendorf) and 0.4U/ul rRNase (Promega). The PCR tube containing lysed cells was heated to 65 ℃ for 1min, cooled at 4 ℃, added with 0.25ul RT mix (170U/ul Superscript II (Invitrogen), 35U/ul Prime RNase inhibitor and 45U/ul rRNase.), and incubated at 37 ℃ for 10min, then at 65 ℃ for 10 min. To each tube 5ul of TdT mix (1 XPCR buffer (Roche), 1.5mM MgCl2, 3mM dATP, 1.25U/ul TdT (Roche), 0.05U RNase H (Roche)) was added and incubated at 37 ℃ for 20min and then at 65 ℃ for 10 min. Add 5ul of product to 50ul of PCR mix (1xEX Taq buffer (Takara), 0.25mM dNTP, 20ng/ul anchor primer, 2.5UEX Taq HS polymerase (Takara)), incubate at 95 ℃ for 2min, 37 ℃ for 5min, 72 ℃ for 20min, followed by 28 cycles: 30sec at 95 ℃, 1min at 67 ℃, 6min at 72 ℃ plus 6 sec extension (per cycle), then 10min at 72 ℃. The amplified PCR products were analyzed for content using the Long SAGE protocol (see, e.g., Saha, S. et al (2002) Nat Biotechnol20, 508-512; incorporated herein by reference in its entirety). Briefly, single cell PCR products were cut with nlaii (neb). After binding of biotinylated DNA to streptavidin magnetic beads (Dynal), the linker was ligated. The ligated DNA was cleaved with MmeI (NEB). The cleaved tags are ligated to form a ditag and amplified by PCR. The PCR product was cleaved with NlaIII and double-labeled to form concatemers. They were ligated into pZero-1 vector (Invitrogen) and transformed. Single colonies were picked and sequenced. Tag sequences were analyzed using SAGE2002 software and NCBI Blast search.

Example 11 vector construction

cDNA was amplified from olfactory epithelial cDNA using either HotstarTaq DNA polymerase (Qiagen) or KOD DNA polymerase (Toyobo/Novagen) and subcloned into pCI expression vector (Promega). The OR open reading frame was amplified from genomic DNA of C57BL6(MOR203-1 and S46), 129(S18) OR DBA2(o1fr62, S6/S79, S50, MOR23-1, MOR31-4, MOR31-6, MOR32-5 and MOR32-11) and subcloned into a pCI containing a Rho-marker.

Example 12 cell culture and immunocytochemistry

293T cells were maintained in minimal essential medium (M10) containing 10% fetal bovine serum. Transfection was performed using Lipofectamine2000 (Invitrogen). In live cell staining, cells were incubated for 1 hour at 4 ℃ in M10 containing the anti-rhodopsin antibody 4D2 (see, e.g., Laird, D.W., and Molday, R.S. (1988) Invest Ophthalmol Vis Sci29, 419-428; incorporated herein by reference in its entirety) and 15mM NaN3 16 hours post transfection. After washing, the cells were incubated with Cy 3-conjugated anti-mouse igg (jackson immunologicals), washed and mounted. For FACS analysis, 4D2 and PE-conjugated anti-mouse IgG (Jackson immunologicals) were used to monitor Rho-labeled receptor expression. HA-. beta.2 adrenergic receptors were stained using anti-HA rabbit antibody (Sigma) and Alexa488 conjugated anti-rabbit IgG (molecular probes). To establish Hana3a cells, selection was performed using 1ug/ml puromycin. 96 colonies were picked and assayed by luciferase assay using OREG.

Example 13 analysis of REEP and RTP

To predict the signal peptide and transmembrane regions of REEPs and RTP, signala 1P (see, e.g., Nielsen, h. et al (1997) Protein Eng10, 1-6; incorporated herein by reference in its entirety) and TMHMM, respectively, were used. To build the phylogenetic tree, Clusta1W was used.

Example 14 northern blotting and in situ hybridization

Total RNA was extracted from different tissues using Trizol reagent (Invitrogen) or Aurum Total RNA (Biorad). RNA was electrophoresed on a formaldehyde-agarose gel and transferred onto Hybond N membrane (Amersham). The Dig-labeled probe was hybridized to the membrane in Dig easy solution (Roche) at 65 ℃. After washing, anti-Dig AP (Roche) was applied and the membrane was washed. The signal was detected using CDP-Star (Roche). The same membrane was used for all 3 probes. In situ hybridization is performed as described (see, e.g., Matsunami, H. and Buck, L.B. (1997) Cell90, 775- & 784; Matsunami, H. et al (2000) Nature404, 601- & 604; Schaeren-Wiemers, N. and Gerfm-Moser, A. (1993) Histochemistry100, 431- & 440; each of which is incorporated herein by reference in its entirety). Briefly, Dig-labeled RNA probes were hybridized to fresh frozen sections of 3-week-old CD-1 mice. After washing, Dig probes were reacted with anti-Dig AP and signals were detected using NBT-BCIP.

Example 15 immunoprecipitation

293T cells in 100mm dishes were transfected with OR, REEP1 and/OR RTP1 cDNA. 16 hours after transfection, cells were lysed in lysis buffer (50mM Tris (7.4), 150mM NaCl, 1% NP-40, 0.5mM PMSF, 2mM Benzamidene, 0.5ug/ml leupeptin, 1.4ug/ml pepstatin A, 2.4ug/ml chymostatin, 15ug/ml aprotinin, 1mM sodium orthovanadate). Lysates were incubated with anti-Flag M2 affinity gel (Sigma) or anti-HA affinity matrix (Roche) for 2 hours at 4 ℃ and washed with lysis buffer. Subsequently, bound proteins were eluted by incubation with SDS sample buffer for 2 hours at room temperature. SDS-PAGE and Western blotting were performed according to Mini-Protean 3 Cell (Bio-Rad) instructions. Using ECL (Amersham), the protein on the membrane was detected.

Example 16 luciferase assay

Luciferase assays were performed using the Dua1-Glo system (Promega). CRE-luciferase (Stratagene) was used to measure receptor activity. A constitutively active SV40 promoter-driven Renilla luciferase (pRL-SV 40: Promega) was used as an internal control. Cells were plated onto poly-D-lysine coated 96-well plates (BIOCOAT, Beckton Dickinson). After 8 hours (for the experiments shown in FIGS. 8B and 8C) or 12 hours (for the experiments shown in FIGS. 8D and 11) post-transfection, the medium was replaced with CD293 chemically defined medium (Invitrogen) and the plates were incubated for 1 hour at 37 ℃. The medium was replaced with 50ul of odorant solution dissolved in CD293 and incubated at 37 ℃ for 10 hours (for the experiments shown in FIGS. 8B and 8C) or 4 hours (for the experiments shown in FIGS. 8D and 11). Following the manufacturer's instructions for measuring luciferase and Renilla luciferase activities. Luminescence was measured using a Wallac Victor 1420 (Perkin-Elmer). Normalized luciferase activity was calculated as: [ Luc (N) -Luc (0) ]/RL (N), where Luc (N) ═ luminescence counts in certain wells, Luc (0) ═ luminescence counts in the absence of odorants for each OR, and RL (N) ═ luminescence counts in each well for Renilla luciferase. For cAMP assay, cells are plated onto 24-well plates. OREG or OREG/Golf cDNA were transfected into Hana3a or HEK293-T cells, respectively. 14 hours post-transfection, cells were incubated in CD293 for 2 hours and exposed to eugenol or isoproterenol (in MEM containing 10mM Hepes and 500uM IBMX) for 5 min. cAMP levels were measured using the cAMP-Screen Direct System (Applied Biosystems). Data analysis was performed using Prism software (Graphpad).

EXAMPLE 17 chemical reagents

All odorants were purchased from Sigma, except octanoic acid, which was purchased from Calbiochem. In example 19, the chemical reagents used in the discovery of homologous ligands of MOR203-1 and olfr62 are provided.

Example 18 Genbank accession number

The genbank accession numbers for mouse REEP1-6 and RTP1-4, and for human REEP1-6 and RTP1-A1, RTP2, RTP3, and RTP4 are: AY562225-AY 562244.

EXAMPLE 19 supplementary Material

The chemical reagents used in the primary ligand screen for MOR203-1 and olfr62 were as follows: 1(+) -carvone, 2L-Canvone, 3(-) -cuminone, 4-citral, 5(1R) - (-) -cuminone, 6(+) -anisic ketone, 7 rosemary oil, 8(-) -Rose oxide, 9(+) -Rose oxide, 10(-) -camphor, 11(S) - (-) -limonene, 12(R) - (+) -1-phenylethanol, 13(S) - (+) -2-phenylbutyric acid, 14(R) - (-) -2-phenylbutyric acid, 152-hexanone, 161-pentanol, 171-heptanol, 18 (+ -) -2-butanol, 191-propanol, 201-hexanol, 21(-) -menthol, 22(R) - (-) -2-heptanol, 23(-) - α -terpineol, 24(+) -menthol, 252-methyl-2-heptanol, 26(S) - (+) -2-octanol, 27(S) - (+) -2-butanol, 28(S) - (+) -2-heptanol, 29(R) - (-) -2-octanol or P (+) -2-octanol, 301-decanol, 31(-) - β -citronellol, 32(S) - (-) -1-phenylethanol, 33 propanal, 34 undecanal, 35 octanal, 36 trans-cinnamaldehyde, 37 nonanal, 38 heptanol, 39 n-decanal, 40 hexanoic acid, 41 hexanoic acid, 42 heptanoic acid (enanthic acid), 43 valeric acid, 44 propionic acid, 45 butyric acid, 46 nonanoic acid, 47 methyl propionate, 48 ethyl butyrate, 49 butyl butyrate, 50 tert-butyl propionate, 51 methyl butyrate, 52 propyl butyrate, 53 amyl acetate, 54 dimethyl pyrazine, 55 isobutyl amine, 56 geraniol, 572-pentanone, 582-butanone, 59(1S) - (-) -alpha-pinene, 601, 4-cineole, 61 phenetole, 62 butyl methyl ether, 63(R) - (+) -pulegone, 64 benzene, 65 benzyl alcohol, 66 guaiacol, 67 isoamylamine, 68 g-caprolactone, 69 g-caprolactone, 70octen, 71 allyl heptanoate, 72 a-amyl cinnamaldehyde, 73 amyl hexanoate, 74 amyl butyrate, 75 anethole, 76 anisaldehyde, 77 benzophenone, 78 benzyl acetate, 79 benzyl salicylate, 80 butyl heptanoate, 81 Camphor ((+) -Camphor), 82Cedryl acetate, 83 cinnamyl alcohol, 84 cinnamyl aldehyde, 85(R) - (+) -citronellal, 86(S) - (-) -citronellal, 87 citronellol, 88 coumarin, 89 cyclohexanone, 90 p-thymol, 915, 5-dimethyl-1, 3-cyclohexanedione (Dimedone), 92 ethyl amyl ketone (3-octanone), 93 cineole, 94 heptylacetate isobutyrate, 95 hexyl acetate, 96 a-hexyl cinnamaldehyde, 97 isobornyl acetate, 98 linalool, 99Lyral (a-amyl cinnamic aldehyde dimethyl acetal), 100 hydroxycitronellal, 101 p-toluyl isobutyrate, 102 o-toluyl isobutyrate, 103 p-toluyl phenylacetate, 1042-methoxy-3-methyl-pyrazine, 1052-methoxypyrazine, 106 methyl salicylate, 107 myrcene, 108 w-pentadecanolide, 109 prenyl acetate, 1102-phenylethanol, 1112-phenethyl acetate, 112 piperonal, 113 pyrazine, 114 guaiac oil, 115 thymol, 116 triethylamine, 1172-heptanone, 118 methyl eugenol, 119 eugenol, 120 eugenol methyl ether, 121 butyraldehyde, 122 hexanal, 1231-pentanol, 124 valeraldehyde, 125 azelaic acid dichloride, 126 azelaic acid, 127 isovaleric acid, 128 decanoic acid, 129 vanillic acid, 1301-octanol, 1314-ethylphenol, 132 heptaldehyde, 1331-nonanol, 134 nonanal, 135 ethyl vanillin, 136 vanillin, 137 acetophenone, 1382-ethylphenol, 139 octanal.

The chemicals associated with coumarin and piperonal (used for o1fr 62) are: 140 benzaldehyde, 141 piperitol, 1424-hydroxycoumarin, 1434-dihydrochromone, 1442-coumaranone.

Example 20 activation Pattern of human odorant receptor

Hana3A cells (expressing mouse REEP1, RTP1, RTP2 and G) were used_αolf293T cell of (1). The odorant receptor activity was measured using CRE-luciferase (Stratagene). The following human odorant receptors were tested for expression patterns in response to different odorants: 36, 35, 11, 57, 58, 9, 3, 42, 81, 82, 66, 13, 87, 33, 44, 43, 77, 75, 64, 59, 12, 62, 60, 120, 90, 95, 160 and 106. The following odorants were used to test human odorant receptor expression patterns: pyridine, 2, 2' - (dithiodimethylene) furan, 1-decanol, 1-hexanol, (-) -cuminone, (+) -cuminone, geraniol, 2-pentanone, benzyl salicylate, (+) -menthol, (-) -menthol, benzene, undecanal, methyl butyrate, heptyl isobutyrate, p-tolyl isobutyrate, pentyl butyrate, ethyl butyrate, hexyl acetate, pentyl acetate, piperonyl acetate, (-) -b-citronellol, eugenol methyl ether, methyl eugenol, a-amyl cinnamaldehyde dimethyl acetal, cinnamaldehyde, a-hexyl cinnamaldehyde, hydroxycitronellal, citral, (R) - (+) -citronellal, (S) - (-) -citronellal, p-tolyl phenylacetate was added to the reaction mixture, Allyl phenylacetate, propionic acid, azelaic acid dichloride, isovaleric acid, (R) - (-) -2-phenylbutyric acid, (S) - (+) -2-phenylbutyric acid, heptanoic acid, octanoic acid, pentanoic acid, hexanoic acid and butyl butyrate. A constitutively active SV40 promoter-driven Renilla luciferase (pRL-SV 40: Promega) was used as an internal control. Luciferase assays were performed using the Dual-Glo System (Promega). Cells were plated onto poly-D-lysine coated 96-well plates (BIOCOAT, Beckton Dickinson). 12-16 hours after transfection, the medium was replaced with CD293 chemically defined medium (hrvitrogen) and the plates were incubated for 1 hour at 37 ℃. The medium was replaced with 50ul of an odorant solution dissolved in CD293 and incubated at 37 ℃ for 4 hours. Following the manufacturer's instructions for measuring luciferase and Renilla luciferase activities. Luminescence was measured using a Wallac Victor 1420 (Perkin-Elmer). Normalized luciferase activity was calculated as: [ Luc (N) -Luc (0)]Rl (n), where Luc (n) ═ luminescence counts in certain wells, Luc (0) ═ luminescence counts in the absence of odorants for each OR, and rl (n) ═ luminescence counts in each well for Renilla luciferase. Figure 34 shows the activation pattern of human odorant receptors resulting from odorant exposure.

Example 21 cell surface expression of V1RE11 in Hana3A and 293T cells

Transfection of cDNA encoding a putative pheromone receptor (V1RE11) into Hana3A cells (expressing REEP1, RTP1, RTP2 and G)_αolfHEK293T cells) or 293T cells. V1RE11 is a putative pheromone receptor for mice and its amino acid sequence is completely different from that of the odorant receptor. Hana3A cells supported cell surface expression of V1RE 11. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nevertheless, these results indicate that REEP1, RTP1 and RTP2 can support functional expression of receptors other than odorant receptors.

Example 22 ability of RTP1-A, RTP1-B, RTP1-C, RTP1-D, and RTP1-E to enhance OLFR62 cell surface expression and Activity

This example describes the generation of variants of RTP1, RTP1-A, RTP1-B, RTP1-C, RTP1-D and RTP1-E and their ability to enhance OLFR62 cell surface expression and activity. By deleting portions of RTP1, a variant of RTP1 was created. FIG. 35 schematically shows amino acid fragments of RTP1-A, RTP1-B, RTP1-C, RTP1-D and RTP1-E as compared to RTP 1. pCI is a control vector. FIG. 36 shows the murine amino acid sequence of RTP1-A (SEQ ID NO: 41), FIG. 37 shows the murine amino acid sequence of RTP1-B (SEQ ID NO: 42), FIG. 38 shows the murine amino acid sequence of RTP1-C (SEQ ID NO: 43), FIG. 39 shows the murine amino acid sequence of RTP1-D (SEQ ID NO: 44), and FIG. 40 shows the murine amino acid sequence of RTP1-E (SEQ ID NO: 45).

Fig. 41 demonstrates cell surface expression of OLFR62 in Hana3A and 293T cells. cDNAs encoding RTP1, RTP1-A, RTP1-B, RTP1-C, RTP1-D, RTP1-E and control pCI were transfected into Hana3A cells or 293T cells. Enhanced cell surface staining was observed in Hana3A cells and 293T cells expressing RTP 1-D.

Figure 42 schematically shows a luciferase assay for monitoring OLFR62 activity. CAMP Response Element (CRE) and luciferase were used to monitor activation of OLFR 62. Activation of OLFR62 increases cAMP, which enhances luciferase reporter gene expression by CRE.

FIG. 43 demonstrates OLFR62 activity indicated by luciferase expression in Hana3A and 293T cells expressing RTP1, RTP1-A, RTP1-B, RTP1-C, RTP1-D, RTP1-E and control pCI. An increased enhancement of OLFR62 activity was observed in 293T cells and Hana3A cells expressing RTP 1-D.

Example 23 the ability of RTP1-A1, RTP1-D1, RTP1-D2, and RTP1-D3 to enhance cell surface expression and activity of OLFR62

This example describes the generation of RTP1 variants RTP1-a1, RTP1-D1, RTP1-D2 and RTP1-D3 and their ability to enhance cell surface expression and activity of OLFR 62. By deleting portions of RTP1-A and RTP1-D, a variant of RTP1 was created. Specifically, the primer pair is placed at a specific position corresponding to the desired deleted fragment and amplified by PCR using KOD polymerase. FIG. 44 schematically shows amino acid fragments of RTP1-A1, RTP1-D1, RTP1-D2, and RTP1-D3, as compared to RTP1-A and RTP1-D, respectively. FIG. 45 shows the murine amino acid sequence of RTP1-A1 (SEQ ID NO: 46) and the human amino acid sequence of RTP1-A1 (SEQ ID NO: 47). FIG. 46 shows the murine amino acid sequence of RTP1-D1 (SEQ ID NO: 48). FIG. 47 shows the murine amino acid sequence of RTP-D2 (SEQ ID NO: 49). FIG. 48 shows the murine amino acid sequence of RTP-D3 (SEQ ID NO: 50).

Fig. 49 demonstrates cell surface expression of OLFR62 in 293T cells. cDNAs encoding RTP1, RTP1-A1, RTP1-D1, RTP1-D2 and RTP1-D3 and control pCI were transfected into 293T cells. Enhanced cell surface staining was observed in 293T cells expressing RTP1-A1, RTP1-D1, and RTP 1-D3.

FIG. 50 shows OLFR62, OREG, S6 and 23-1 activities indicated by luciferase expression in 293T cells expressing RTP1, RTP1-A1, RTP1-D1, RTP1-D2 and RTP1-D3 and control pCI. Increased enhancement of OLFR62, OREG, S6 and 23-1 activities was observed in 293T cells expressing RTP 1-A1.

FIG. 51 shows OLFR62, OREG, S6 and 23-1 activities indicated by luciferase expression in Hana3A cells expressing RTP1, RTP1-A1, RTP1-D1, RTP1-D2 and RTP1-D3 and control pCI.

FIG. 52 shows cell surface expression of OLFR62, OREG, MOR203-1, S6 and 23-1 in 293T cells co-transfected with RTP1, RTP1-A1 or control pCI. The cDNAs encoding RTP1, RTP1-A1, and control pCI were transfected into cells. Increased cell surface staining was observed in cells expressing RTP1-a 1.

Example 24 RTP1 and RTP4 chimeras

This example describes RTP1 and RTP4 chimeras produced using chimeric PCR. More specifically, at the junction of the RTP1 and RTP4 sequences, composite chimeric primers were designed. For each chimera, 2 pairs of primers (e.g., forward and composite, composite and reverse) are first amplified. Next, the 2 PCR products were used as templates in the subsequent megaprimer PCR, using the original forward and reverse primers, to give the desired chimera. FIG. 53 shows schematically the amino acid fragments of RTP1-A1-A (chimera 1), RTP1-A1-D2 (chimera 2), RTP1-A1-D1 (chimera 3), RTP4-A1-A (chimera 4), RTP14-A1-D2 (chimera 5) and RTP4-A1-D1 (chimera 6).

Figure 54 shows cell surface expression of OR in cells expressing RTP1, RTP4, chimera 1, chimera 2, chimera 3, chimera 4, chimera 5, chimera 6 and control pCI. The cdnas encoding RTP1, RTP4, RTP1-a1, chimera 1, chimera 2, chimera 3, chimera 4, chimera 5, chimera 6, and control pCI were transfected into 293T cells.

FIG. 55 shows OLFR62, OREG, S6 and 23-1 activity indicated by luciferase expression in 293T cells expressing RTP1, RTP4, RTP1-A1, RTP1-D1, RTP1-D2, chimera 1, chimera 2, chimera 3, chimera 4, chimera 5, chimera 6 and control pCI.

FIG. 56 shows detection of RTP1, RTP1-A, RTP1-B, RTP1-C, RTP1-A1, RTP1-D, chimera 4, chimera 5, RTP1-D3, RTP1-D1, chimera 6 and RTP4 using anti-RTP 1.

All publications and patents mentioned in the above specification are herein incorporated by reference. While the invention has been described with reference to certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Sequence listing

<110>Duke University

Matsunami，Hiroaki

Matsunami，Momoka

Saito，Harumi

Zhuang，Hanyi

<120> modulators of odorant receptors

<130>DUKE-14653

<140>US 11/811,050

<141>2007-06-08

<150>US 11/156,516

<151>2005-06-20

<150>US 60/582,011

<151>2004-06-22

<150>US 60/581,087

<151>2004-06-18

<160>51

<170>PatentIn version 3.5

<210>1

<211>606

<212>DNA

<213> mouse (Mus musculus)

<400>1

atggtgtcgt ggatcatctc caggctggtg gtgcttatat ttggcaccct ttatcctgca 60

tattattcat acaaggctgt gaagtccaag gacattaaag aatatgtcaa atggatgatg 120

tattggatta tatttgccct cttcaccacg gcagagacgt tcacagacat cttcctttgc 180

tggtttccat tctattatga actaaaaata gcgtttgtag cctggctgct gtctccctat 240

acaaaaggat ccagcctcct gtacaggaag tttgttcatc ccacattgtc ttcaaaagaa 300

aaggaaatcg atgactgcct ggtccaagca aaagatcgaa gctatgacgc ccttgtgcac 360

tttgggaagc ggggcttgaa tgtggcagcc actgcagctg tgatggctgc ctccaaggga 420

cagggtgcct tgtcagagag actccggagc ttcagcatgc aggacctcac caccatcagg 480

ggtgatggtg ctcctgctcc ctcgggccct cctccaccag ggactgggcg gtccagcggc 540

aaacacagcc agcccaagat gtccaggagt gcttctgaga gtgccggcag ctcgggcacc 600

gcctag 606

<210>2

<211>765

<212>DNA

<213> mice

<400>2

atggtgtcct ggatcatctc tcgcctggtg gtgctcatct ttggcaccct gtacccagcc 60

tattcttcct acaaggccgt gaagaccaaa aacgtgaagg aatacgtaaa atggatgatg 120

tattggatag tcttcgcctt cttcaccaca gctgagacac ttacagatat aatactgtcc 180

tggttcccct tctactttga gctcaagatt gcctttgtga tatggctgtt gtccccttac 240

accaagggct ccagtgtcct ctaccgcaag ttcgtgcacc caacactgtc caacaaggaa 300

aaggagatcg acgaatacat cacacaagct cgagacaaga gctatgagac gatgatgagg 360

gtgggcaaga ggggcctgaa cctggctgcc aatgctgcag tcacagctgc tgccaagggc 420

cagggggtgc tgtcggaaaa gctgcggagc ttcagcatgc aggacctgac tctcattcga 480

gatgaggatg cgttaccgct gcaggggcca gatggccgcc tccaacccgg ccccgtgggt 540

ctcctggaca ctattgagga cttaggagat gagcctgccc taagtctaag gtctagcaca 600

agccagccag atccccggac agagacctca gaagatgacc tgggagacaa ggcacccaag 660

aggaccaaac ctatcaaaaa agtacccaga gctgagccgc cggcttccaa gacactgaag 720

acccggccca agaagaagag ttctggaggg ggcgactcag catga 765

<210>3

<211>765

<212>DNA

<213> mice

<400>3

atggtgtcct ggatgatctc ccgagccgtg gtgctggtgt ttggaatgct ctatccagcg 60

tactattcct acaaagccgt gaagacgaaa aacgtcaagg aatacgttcg ctggatgatg 120

tattggatcg tctttgccct ctacactgtc attgaaacgg tggccgatca gacacttgca 180

tggtttcccc tgtactatga gctgaagatt gccttcgtca tttggctgct gtcgccctac 240

actagagggg cgagtttaat ctatagaaag ttccttcatc ccctgctgtc atcaaaggaa 300

agggaaattg atgattatat tgtccaagcc aaagaaagag gctatgagac aatggtgaat 360

tttggacggc aaggtttgaa tttagcagct gcagccgccg tcactgcagc agtgaagagc 420

caaggagcaa taacggagcg tctgcgaagt ttcagcatgc atgatctgac agctatccaa 480

ggggatgagc ccgtgggaca cagaccctac cagactttgc cagaagcaaa gaggaaaggc 540

aaacaagcca ccgagtcacc agcctatgga attccactga aagatggaag tgagcagaca 600

gacgaagaag cggaggggcc attctccgat gacgagatgg tgactcacaa ggcgctgagg 660

cgatcccaga gcatgaaatc tgtcaagacc atcaaaggcc gcaaagaggt gcggtatggc 720

tcactaaaat ataaagtgaa gaagagaccg caagtgtatt tttag 765

<210>4

<211>774

<212>DNA

<213> mice

<400>4

atggtgtcct ggatgatctg tcgcctggta gtgctcatat ttggcatgct gtatcctgcg 60

tatgcttcct acaaggccgt gaagagcaag aacattcgag aatatgtacg gtggatgatg 120

tattggattg tctttgcgat cttcatggca gcagaaacct tcacagacat cttcatttcc 180

tggttcccgt tttattacga gttcaagatg gcttttgtgc tgtggctgct ctcaccttac 240

accaaggggg ccagcctgct ttaccgaaag tttgtccacc catccctatc ccgccatgag 300

aaggagatcg acgcgtgtat cgtgcaggca aaggagcgca gctatgaaac catgctcagt 360

tttgggaagc ggagcctcaa catcgctgcc tcagctgctg tgcaggctgc taccaagagt 420

caaggcgctc tagctggaag gctgcggagt ttctctatgc aagacctgcg ctctatccct 480

gacacccctg tccccaccta ccaagatccc ctctacctgg aagaccaggt accccgacgt 540

agacccccta ttggataccg gccaggcggc ctgcagggca gtgacacaga ggatgagtgt 600

tggtcagaca atgagatcgt cccccagcca cctgttcggc cccgagagaa gcctctaggc 660

cgcagccaga gccttcgggt ggtcaagagg aagccattga ctcgagaggg cacctcacgc 720

tccctgaagg tccgaacccg gaaaaaggcc atgccctcag acatggacag ctag 774

<210>5

<211>570

<212>DNA

<213> mice

<400>5

atgtccgcag ccatgaggga gaggttcgac cggttcctgc acgagaagaa ctgcatgact 60

gatctcctcg ccaagctcga ggccaagacc ggagtgaacc ggagcttcat cgcgctcggt 120

gtcatcggac tggtggcttt gtatctggtg ttcggttatg gagcctctct cctctgcaac 180

ctgataggtt tcggataccc agcctacatc tcaatgaaag ccatcgagag tcccaacaaa 240

gatgatgaca cccagtggct gacgtactgg gtggtatatg gtgtgttcag cattgccgaa 300

ttcttctccg atctcttcct gtcctggttc cccttctact acatgctgaa gtgtggcttc 360

ctgctgtggt gcatggcccc cagcccggct aatggggctg agatgcgcta caggcgaatc 420

atccgtccta tcttcctcaa gcacgagtcc caggtagaca gtgtggtgaa ggacgtgaag 480

gacaaagcca aagagactgc agatgccatc agcaaagaag tcaagaaagc tacagtgaac 540

ttgctgggcg atgagaagaa gagcacctga 570

<210>6

<211>606

<212>DNA

<213> mice

<400>6

atggacggtc tgcgccagcg cttcgaacgt tttctggaac agaagaacgt ggccaccgaa 60

gcgctcgggg cgctcgaagc aaggaccggt gtagagaagc ggtatctcgc cgcgggagcc 120

ctcgcccttc taggcctgta tcttctgttc ggttacgggg cctctctact gtgcaatgtc 180

atcggatttg tataccccgc atatgcttca gtcaaagcta tcgagagccc aagcaaggaa 240

gacgacactg tgtggctaac ctactgggtg gtgtacgccc tgttcggtct ggtcgaattc 300

ttcagcgatc tactcctgtt ctggttccct ttctactacg cgggcaagtg cgccttcctg 360

ttattttgca tgacgcccgg accctggaac ggggcattac tactatacca tcgcgtcata 420

agaccactct ttctaaagca ccacatggct ctagacagcg ccgcgagcca gctaagcgga 480

agagcattgg acctagcagc tgggataacc cgggacgtac ttcaggcctt ggctcggggc 540

cgggctctcg tcaccccagc atcaacatcg gaacccccag ccgctctgga actggacccc 600

aagtaa 606

<210>7

<211>606

<212>DNA

<213> Intelligent (Homo sapiens)

<400>7

atggtgtcat ggatcatctc caggctggtg gtgcttatat ttggcaccct ttaccctgcg 60

tattattcct acaaggctgt gaaatcaaag gacattaagg aatatgtcaa atggatgatg 120

tactggatta tatttgcact tttcaccaca gcagagacat tcacagacat cttcctttgt 180

tggtttccat tctattatga actaaaaata gcatttgtag cctggctgct gtctccctac 240

acaaaaggct ccagcctcct gtacaggaag tttgtacatc ccacactatc ttcaaaagaa 300

aaggaaatcg atgattgtct ggtccaagca aaagaccgaa gttacgatgc ccttgtgcac 360

ttcgggaagc ggggcttgaa cgtggccgcc acagcggctg tgatggctgc ttccaaggga 420

cagggtgcct tatcggagag actgcggagc ttcagcatgc aggacctcac caccatcagg 480

ggagacggcg cccctgctcc ctcgggcccc ccaccaccgg ggtctgggcg ggccagcggc 540

aaacacggcc agcctaagat gtccaggagt gcttctgaga gcgctagcag ctcaggcacc 600

gcctag 606

<210>8

<211>759

<212>DNA

<213> Intelligent people

<400>8

atggtgtcct ggatcatctc tcgcctggtg gtgctcatct ttggcaccct gtacccagcc 60

tattcttcct acaaggccgt gaagacaaaa aacgtgaagg aatatgtgaa atggatgatg 120

tactggatcg tctttgcctt cttcaccacg gccgagacgc tcacggatat agtgctctcc 180

tggttcccct tctactttga actgaagatc gccttcgtga tatggctgct gtccccttac 240

accaagggct ccagcgtgct ctaccgcaag ttcgtgcacc caacgctgtc caacaaggag 300

aaggagatcg acgagtacat cacgcaggcc cgagacaaga gctatgagac catgatgagg 360

gtgggcaaga ggggcctgaa ccttgccgcc aatgctgcag tcacagctgc cgccaagggg 420

gtgctgtcag agaagctccg cagcttcagc atgcaggacc tgaccctgat ccgggacgag 480

gacgcactgc ccctgcagag gcctgacggc cgcctccgac ccagccctgg cagcctcctg 540

gacaccatcg aggacttagg agatgaccct gccctgagtc taaggtccag cacaaacccg 600

gcagattccc ggacagaggc ttctgaggat gacatgggag acaaagctcc caagagggcc 660

aaacccatca aaaaagcgcc caaagctgag ccactggctt ccaagacact gaagacccgg 720

cccaagaagaagacctctgg cgggggcgac tcagcttga 759

<210>9

<211>441

<212>DNA

<213> Intelligent people

<400>9

atggtgtcct ggatgatctc cagagccgtg gtgctggtgt ttggaatgct ttatcctgca 60

tattattcat acaaagctgt gaaaacaaaa aacgtgaagg aatatgttcg atggatgatg 120

tactggattg tttttgctct ctatactgtg attgaaacag tagccgatca aacagttgct 180

tggtttcccc tgtactatga gctgaagatt gcttttgtca tatggctgct ttctccctat 240

accaaaggag caagtttaat atatagaaaa ttccttcatccacttctttc ttcaaaggaa 300

agggagattg atgattatat tgtacaagca aaggaacgag gctatgaaac catggtaaac 360

tttggacggc aaggtttaaa ccttgcagct actgctgctg ttactgcagc agtaaaggta 420

attgttcatt taccttttta a 441

<210>10

<211>774

<212>DNA

<213> Intelligent people

<400>10

atggtgtcct ggatgatctg tcgcctggtg gtgctggtgt ttgggatgct gtgtccagct 60

tatgcttcct ataaggctgt gaagaccaag aacattcgtg aatatgtgcg gtggatgatg 120

tactggattg tttttgcact cttcatggca gcagagatcg ttacagacat ttttatctcc 180

tggttccctt tctactatga gatcaagatg gccttcgtgc tgtggctgct ctcaccctac 240

accaagggcg ccagcctgct ttaccgcaag tttgtccacc cgtccctgtc ccgccatgag 300

aaggagatcg acgcgtacat cgtgcaggcc aaggagcgca gctacgagac cgtgctcagc 360

ttcgggaagc ggggcctcaa cattgccgcc tccgctgctg tgcaggctgc caccaagagt 420

cagggggcgc tggccggcag gctgcggagc ttctccatgc aggacctgcg ctccatctct 480

gacgcacctg cccctgccta ccatgacccc ctctacctgg aggaccaggt gtcccaccgg 540

aggccaccca ttgggtaccg ggccgggggc ctgcaggaca gcgacaccga ggatgagtgt 600

tggtcagata ctgaggcagt cccccgggcg ccagcccggc cccgagagaa gcccctaatc 660

cgcagccaga gcctgcgtgt ggtcaagagg aagccaccgg tgcgggaggg cacctcgcgc 720

tccctgaagg ttcggacgag gaaaaagact gtgccctcag acgtggacag ctag 774

<210>11

<211>570

<212>DNA

<213> Intelligent people

<400>11

atgtctgcgg ccatgaggga gaggttcgac cggttcctgc acgagaagaa ctgcatgact 60

gaccttctgg ccaagctcga ggccaaaacc ggcgtgaaca ggagcttcat cgctcttggt 120

gtcatcggac tggtggcctt gtacctggtg ttcggttatg gagcctctct cctctgcaac 180

ctgataggat ttggctaccc agcctacatc tcaattaaag ctatagagag tcccaacaaa 240

gaagatgata cccagtggct gacctactgg gtagtgtatg gtgtgttcag cattgctgaa 300

ttcttctctg atatcttcct gtcatggttc cccttctact acatgctgaa gtgtggcttc 360

ctgttgtggt gcatggcccc gagcccttct aatggggctg aactgctcta caagcgcatc 420

atccgtcctt tcttcctgaa gcacgagtcc cagatggaca gtgtggtcaa ggaccttaaa 480

gacaaggcca aagagactgc agatgccatc actaaagaag cgaagaaagc taccgtgaat 540

ttactgggtg aagaaaagaa gagcacctaa 570

<210>12

<211>555

<212>DNA

<213> Intelligent people

<400>12

atggacggcc tgaggcagcg cgtggagcac ttcctggagc aaaggaacct ggtcaccgaa 60

gtgctggggg cgctggaggc caagaccggg gtggagaagc ggtatctggc tgcaggagcc 120

gtcactctgc taagcctgta tctgctgttc ggctacggag cgtctctgct gtgcaatctc 180

atcggatttg tgtaccccgc atatgcctca atcaaagcta tcgagagccc aagcaaggac 240

gacgacactg tgtggctcac ctactgggtg gtgtacgccc tgtttgggct ggccgagttc 300

ttcagcgatc tactcctgtc ctggttccct ttctactacg tgggcaagtg cgccttcctg 360

ttgttctgca tggctcccag gccctggaac ggggctctca tgctgtatca gcgcgtcgtg 420

cgtccgctgt tcctaaggca ccacggggcc gtagacagaa tcatgaacga cctcagcggg 480

cgagccctgg acgcggcggc cggaataacc aggaacgtca agccaagcca gaccccgcag 540

ccgaaggaca agtga 555

<210>13

<211>792

<212>DNA

<213> mice

<400>13

atgaggattt ttagaccgtg gagactgcgc tgccctgcct tacacttacc ctctttcccc 60

acgttctcta taaagtgtag tttgcctcct cttcccactg acgaagacat gtgtaagagt 120

gtgaccacag gtgagtggaa gaaggtcttc tacgagaaga tggaggaggt gaagccagcg 180

gacagctggg acttcatcat agaccccaac ctcaagcaca atgtgttggc ccctggctgg 240

aagcagtacc tggaacttca tgcctcaggc aggttccact gttcctggtg ctggcacacc 300

tggcagtcac cccatgtagt catcctcttc cacatgtacc tggacaaggc tcagcgcgct 360

ggttcggtgc gcatgcgtgt gttcaagcag ctctgctacg agtgcggtac agcacggctg 420

gatgagtcca gcatgctgga ggagaacatc gaaagcctgg tggacaacct catcaccagt 480

ttgcgagagc agtgctacgg ggagcgtggt ggccactacc gcatccatgt ggccagccgg 540

caggacaacc ggcgacaccg cggagagttc tgcgaggcct gccaggaagg catcgtgcac 600

tggaagccca gtgagaagct gctggaggag gaggcgacca cctacacctt ctcccgtgct 660

cccagcccca ccaaaccgca ggctgaaaca ggctcaggct gcaacttctg ctccattccc 720

tggtgcttat tttgggccac ggttttgatg ctcatcatct acctgcaatt ctccttccgt 780

acttctgtct aa 792

<210>14

<211>672

<212>DNA

<213> mice

<400>14

atgtccacca gcctgaccac ttgtgagtgg aagaaggtct tctacgagaa gatggaggtg 60

gccaagccag cggacagctg ggagctcatc atagacccca ccctcaagcc caatgagctg 120

ggccctggct ggaagcagta cctggagcaa catgcctcag gcaggttcca ctgttcctgg 180

tgttggcaca catggcaatc tgccaatgtc gtcattctct tccacatgca cctggaccgt 240

gcccagcgtg ttggctcagt gcgcatgcgc gtgttcaagc agctgtgcta tcagtgcggc 300

acgtcgcggc tggacgagtc cagcatgctg gaggagaata tcgagggcct ggtggacaac 360

ctcatcacca gtctgcgcga gcagtgttac gatgaggatg gtggccagta ccgcatccac 420

gtagccagcc ggccagacag cggattgcac cgcagtgagt tctgcgaggc ctgccaggaa 480

ggcatcgtgc actggaagcc cagcgaaaag ctgctggagg aggatgccgc ctataccgat 540

gcctccaaga agaagggcca ggctggtttt atctccagct tcttctcatt tcgttggtgc 600

ctgttctggg gcaccctctg cctggtcatt gtctacctgc agttcttccg aggccgctct 660

ggcttccttt ag 672

<210>15

<211>1425

<212>DNA

<213> mice

<400>15

atgatggaag aagacatagg agacacagag caatggcgac atgtgttcca ggagctaatg 60

caagaggtga aaccctggca caaatggacc ctcataccag acaagaacct tcttcccaac 120

gttttgaagc caggatggac gcaataccag caaaagacct ttgctaggtt ccactgtcct 180

tcctgctctc gaagttgggc atctggccga gttctgatag tcttccacat gcggtggtgt 240

gagaagaagg ccaaggggtg ggtgaagatg agggtgtttg ctcagagatg taatcagtgc 300

cccgagcctc catttgcaac tccagaagtc acttgggaca acatctcaag gatcttgaac 360

aacctgctct tccaaattct gaagaagtgc tataaagaag gatttaagca aatgggtgag 420

attcctttgc tagggaacac cagtctcgaa gggccacatg acagcagcaa ctgtgaggcc 480

tgtctcctgg gcttttgtgc tcagaatgac ttaggccaag cctcaaaacc accagcaccc 540

ccattatctc ctacctcctc aaagtcagcc agggagccca aggtcactgc cacctgtagc 600

aacatttcct cctcacagcc ctcctctaaa gtacagatgc cccaagcatc aaaagcgaac 660

ccccaagcca gtaaccctac caaaaatgac cccaaagtta gctgcacctc aaaaccacca 720

gcacccccat tatctcctac ctccttaaag tcagccaggg agcctaaggt cactgtcacc 780

tgtagcaaca tttcctcctc gcggtcctcc tctaaagtac agatgcccca agcatcaaaa 840

gtgaaccccc aaaccagtaa tcctaccaaa aatgacccca agattagctg tacctcaaaa 900

ccatcaacta ctccaagact gacaatacaa cagctgtcag tagtaagccc acctgcccct 960

gcccctacat gtgtcattca aatgccttct cccactccca tcgacggcag cagagcagca 1020

gatgtagcaa aggagaacac cagatccaag accccaaagg cattgctctc atccccttta 1080

tacgtcccac ccacttcctc ctatgtccca cccacttcct cctatgtccc acccacttcc 1140

tcctatgtcc cgcccacttc ctcttatgtc ccacccactt cctcctcagt tattgtgccc 1200

atttcctcct cgtggagact accagaaaac actatttgcc aagtagagag aaacagtcat 1260

atccacccgc aaagccagtc ttcctgctgt ggggcctgct gcgagtcctg gtgtgagatc 1320

ttcaggtact catgctgtga ggccgcctgt aattgcatgt cacagagtcc actgtgttgc 1380

ttggcctttc taatcttgtt cttattgctg tggtatttat tataa 1425

<210>16

<211>750

<212>DNA

<213> mice

<400>16

atgctgttcc ccgatgactt cagtacttgg gagcagacat ttcaagaact gatgcaggag 60

gagaagcccg gggccaagtg gagcctgcat ttggataaga acattgtacc agatggtgca 120

gccctgggat ggaggcagca ccagcagaca gtgcttggca ggttccagtg ttccagatgc 180

tgcagaagtt ggacctctgc tcaggtgatg atcttgtgcc acatgtaccc ggacactttg 240

aaatcgcagg gccaggcacg catgaggatc tttggtcaga agtgccagaa gtgttttgga 300

tgtcaatttg agactcccaa gttctccaca gagatcatca aaagaattct gaataaccta 360

gttaattata ttctgcagag atactatgga cacaggaaga tagcattgac ctcgaatgca 420

tctttgggtg agaaggtgac tttggatggg ccccacgaca cacgcaattg tgaggcatgc 480

agtctaaact ctcatggaag atgtgccctt gcacacaaag taaaaccacc cagatctcca 540

tctccattac caaatagttc ctccccatca aagagctgcc ctcctccgcc tcagacccgg 600

aatacggatt ttgggaataa aactcttcag gattttggga atagaacttt tcagggatgc 660

agagagcccc cccaacgtga aatagagcca ccactatttc tgtttttgtc tattgctgca 720

tttgcccttt ttagtctttt cactagataa 750

<210>17

<211>684

<212>DNA

<213> Intelligent people

<400>17

atgtgtaaaa gcgtgaccac agatgagtgg aagaaagtct tctatgagaa gatggaggag 60

gcaaagccgg ctgacagctg ggacctcatc atagacccca acctcaagca caatgtgctg 120

agccctggtt ggaagcagta cctggaattg catgcttcag gcaggttcca ctgctcctgg 180

tgctggcaca cctggcagtc gccctacgtg gtcatcctct tccacatgtt cctggaccgc 240

gcccagcggg cgggctcggt gcgcatgcgc gtcttcaagc agctgtgcta tgagtgcggc 300

acggcgcggc tggacgagtc cagcatgctg gaggagaaca tcgagggcct ggtggacaac 360

ctcatcacca gcctgcgcga gcagtgctac ggcgagcgtg gcggccagta ccgcatccac 420

gtggccagcc gccaggacaa ccggcggcac cgcggagagt tctgcgaggc ctgccaggag 480

ggcatcgtgc actggaagcc cagcgagaag ctgctggagg aggaggcgac cacctacacc 540

ttctcccggg cgcccagccc caccaagtcg caggaccaga cgggctcagg ctggaacttc 600

tgctctatcc cctggtgctt gttttgggcc acggtcctgc tgctgatcat ctacctgcag 660

ttctctttcc gtagctccgt ataa 684

<210>18

<211>678

<212>DNA

<213> Intelligent people

<400>18

atgtgtacca gcttgaccac ttgtgagtgg aagaaagtct tctatgagaa gatggaggtg 60

gcaaagccag cggacagctg ggagctcatc atagacccca acctcaagcc cagtgagctg 120

gcccctggct ggaagcagta cctggagcag cacgcctcag gcaggttcca ctgctcctgg 180

tgctggcaca cctggcagtc tgcccatgtg gtcatcctct tccacatgtt cctggaccgc 240

gcccagcggg cgggctcggt gcgcatgcgc gtcttcaagc agctgtgcta tgagtgcggc 300

acggcgcggc tggacgagtc cagcatgctg gaggagaaca tcgagggcct ggtggacaac 360

ctcatcacca gcctgcgcga gcagtgctac gaggaggatg gtggccagta ccgcatccac 420

gtggccagcc gcccggacag cgggccgcat cgtgcagagt tctgtgaggc ctgccaggag 480

ggcatcgttc actggaagcc cagcgagaag ctgctggagg aggaggtgac cacctacacc 540

tctgaagcct ccaagccgag ggcccaggcg ggatccggct acaacttctt gtctcttcgc 600

tggtgcctct tctgggcctc tctctgcctg ctcgttgttt acctgcagtt ctccttcctc 660

agtcctgcct tcttttag 678

<210>19

<211>699

<212>DNA

<213> Intelligent people

<400>19

atggctgggg acacagaagt gtggaagcaa atgtttcagg agttaatgcg ggaggtgaag 60

ccatggcaca ggtggaccct gagaccagac aagggccttc ttcccaacgt cctgaagcca 120

ggctggatgc aataccagca gtggaccttc gccaggttcc agtgctcctc ctgctctcgt 180

aactgggcct ctgcccaagt tctggtcctt ttccacatga actggagtga ggagaagtcc 240

aggggccagg tgaagatgag ggtgtttacc cagagatgta agaagtgccc ccaacctctg 300

tttgaggacc ctgagttcac acaagagaac atctcaagga tcctgaaaaa cctggtgttc 360

cgaattctga agaaatgcta tagaggaaga tttcagttga tagaggaggt tcctatgatc 420

aaggacatct ctcttgaagg gccacacaat agtgacaact gtgaggcatg tctgcagggc 480

ttctgtgctg ggcccataca ggttacaagc ctccccccat ctcagacccc aagagtacac 540

tccatttaca aggtggagga ggtagttaag ccctgggcct caggagagaa tgtctattcc 600

tacgcatgcc aaaaccacat ctgtaggaac ttaagcattt tctgctgttg tgtcattctc 660

attgttatcg tggtgattgt tgtaaaaact gctatatga 699

<210>20

<211>741

<212>DNA

<213> Intelligent people

<400>20

atggttgtag atttctggac ttgggagcag acatttcaag aactaatcca agaggcaaaa 60

ccccgggcca catggacgct gaagttggat ggcaaccttc agctagactg cctggctcaa 120

gggtggaagc aataccaaca gagagcattt ggctggttcc ggtgttcctc ctgccagcga 180

agttgggctt ccgccaagtt gcagattctg tgccacacgt actgggagca ctggacatcc 240

cagggtcagg tgcgtatgag gctctttggc caaaggtgcc agaagtgctc ctggtcccaa 300

tatgagatgc ctgagttctc ctcggatagc accatgagga ttctgagcaa cctggtgcag 360

catatactga agaaatacta tggaaatggc atgaggaagt ctccagaaat gccagtaatc 420

ctggaagtgt ccctggaagg atcccatgac acagccaatt gtgaggcatg cactttgggc 480

atatgtggac agggcttaaa aagctacatg acaaagccgt ccaaatccct actcccccac 540

ctaaagactg ggaattcctc acctggaatt ggtgctgtgt acctcgcaaa ccaagccaag 600

aaccagtcag atgaggcaaa agaggctaag gggagtgggt atgagaaatt agggcccagt 660

cgagacccag atccactgaa catctgtgtc tttattttgc tgcttgtatt tattgtagtc 720

aaatgcttta catcagaatg a 741

<210>21

<211>201

<212>PRT

<213> mice

<400>21

Met Val Ser Trp Ile Ile Ser Arg Leu Val Val Leu Ile Phe Gly Thr

1 5 10 15

Leu Tyr Pro Ala Tyr Tyr Ser Tyr Lys Ala Val Lys Ser Lys Asp Ile

20 25 30

Lys Glu Tyr Val Lys Trp Met Met Tyr Trp Ile Ile Phe Ala Leu Phe

35 40 45

Thr Thr Ala Glu Thr Phe Thr Asp Ile Phe Leu Cys Trp Phe Pro Phe

50 55 60

Tyr Tyr Glu Leu Lys Ile Ala Phe Val Ala Trp Leu Leu Ser Pro Tyr

65 70 75 80

Thr Lys Gly Ser Ser Leu Leu Tyr Arg Lys Phe Val His Pro Thr Leu

85 90 95

Ser Ser Lys Glu Lys Glu Ile Asp Asp Cys Leu Val Gln Ala Lys Asp

100 105 110

Arg Ser Tyr Asp Ala Leu Val His Phe Gly Lys Arg Gly Leu Asn Val

115 120 125

Ala Ala Thr Ala Ala Val Met Ala Ala Ser Lys Gly Gln Gly Ala Leu

130 135 140

Ser Glu Arg Leu Arg Ser Phe Ser Met Gln Asp Leu Thr Thr Ile Arg

145 150 155 160

Gly Asp Gly Ala Pro Ala Pro Ser Gly Pro Pro Pro Pro Gly Thr Gly

165 170 175

Arg Ser Ser Gly Lys His Ser Gln Pro Lys Met Ser Arg Ser Ala Ser

180 185 190

Glu Ser Ala G1y Ser Ser Gly Thr Ala

195 200

<210>22

<211>254

<212>PRT

<213> mice

<400>22

Met Val Ser Trp Ile Ile Ser Arg Leu Val Val Leu Ile Phe Gly Thr

1 5 10 15

Leu Tyr Pro Ala Tyr Ser Ser Tyr Lys Ala Val Lys Thr Lys Asn Val

20 25 30

Lys Glu Tyr Val Lys Trp Met Met Tyr Trp Ile Val Phe Ala Phe Phe

35 40 45

Thr Thr Ala Glu Thr Leu Thr Asp Ile Ile Leu Ser Trp Phe Pro Phe

50 55 60

Tyr Phe Glu Leu Lys Ile Ala Phe Val Ile Trp Leu Leu Ser Pro Tyr

65 70 75 80

Thr Lys Gly Ser Ser Val Leu Tyr Arg Lys Phe Val His Pro Thr Leu

85 90 95

Ser Asn Lys Glu Lys Glu Ile Asp Glu Tyr Ile Thr Gln Ala Arg Asp

100 105 110

Lys Ser Tyr Glu Thr Met Met Arg Val Gly Lys Arg Gly Leu Asn Leu

115 120 125

Ala Ala Asn Ala Ala Val Thr Ala Ala Ala Lys Gly Gln Gly Val Leu

130 135 140

Ser Glu Lys Leu Arg Ser Phe Ser Met Gln Asp Leu Thr Leu Ile Arg

145 150 155 160

Asp Glu Asp Ala Leu Pro Leu Gln Gly Pro Asp Gly Arg Leu Gln Pro

165 170 175

Gly Pro Val Gly Leu Leu Asp Thr Ile Glu Asp Leu Gly Asp Glu Pro

180 185 190

Ala Leu Ser Leu Arg Ser Ser Thr Ser Gln Pro Asp Pro Arg Thr Glu

195 200 205

Thr Ser Glu Asp Asp Leu Gly Asp Lys Ala Pro Lys Arg Thr Lys Pro

210 215 220

Ile Lys Lys Val Pro Arg Ala Glu Pro Pro Ala Ser Lys Thr Leu Lys

225 230 235 240

Thr Arg Pro Lys Lys Lys Ser Ser Gly Gly Gly Asp Ser Ala

245 250

<210>23

<211>254

<212>PRT

<213> mice

<400>23

Met Val Ser Trp Met Ile Ser Arg Ala Val Val Leu Val Phe Gly Met

1 5 10 15

Leu Tyr Pro Ala Tyr Tyr Ser Tyr Lys Ala Val Lys Thr Lys Asn Val

20 25 30

Lys Glu Tyr Val Arg Trp Met Met Tyr Trp Ile Val Phe Ala Leu Tyr

35 40 45

Thr Val Ile Glu Thr Val Ala Asp Gln Thr Leu Ala Trp Phe Pro Leu

50 55 60

Tyr Tyr Glu Leu Lys Ile Ala Phe Val Ile Trp Leu Leu Ser Pro Tyr

65 70 75 80

Thr Arg Gly Ala Ser Leu Ile Tyr Arg Lys Phe Leu His Pro Leu Leu

85 90 95

Ser Ser Lys Glu Arg Glu Ile Asp Asp Tyr Ile Val Gln Ala Lys Glu

100 105 110

Arg Gly Tyr Glu Thr Met Val Asn Phe Gly Arg Gln Gly Leu Asn Leu

115 120 125

Ala Ala Ala Ala Ala Val Thr Ala Ala Val Lys Ser Gln Gly Ala Ile

130 135 140

Thr Glu Arg Leu Arg Ser Phe Ser Met His Asp Leu Thr Ala Ile Gln

145 150 155 160

Gly Asp Glu Pro Val Gly His Arg Pro Tyr Gln Thr Leu Pro Glu Ala

165 170 175

Lys Arg Lys Gly Lys Gln Ala Thr Glu Ser Pro Ala Tyr Gly Ile Pro

180 185 190

Leu Lys Asp Gly Ser Glu Gln Thr Asp Glu Glu Ala Glu Gly Pro Phe

195 200 205

Ser Asp Asp Glu Met Val Thr His Lys Ala Leu Arg Arg Ser Gln Ser

210 215 220

Met Lys Ser Val Lys Thr Ile Lys Gly Arg Lys Glu Val Arg Tyr Gly

225 230 235 240

Ser Leu Lys Tyr Lys Val Lys Lys Arg Pro Gln Val Tyr Phe

245 250

<210>24

<211>257

<212>PRT

<213> mice

<400>24

Met Val Ser Trp Met Ile Cys Arg Leu Val Val Leu Ile Phe Gly Met

1 5 10 15

Leu Tyr Pro Ala Tyr Ala Ser Tyr Lys Ala Val Lys Ser Lys Asn Ile

20 25 30

Arg Glu Tyr Val Arg Trp Met Met Tyr Trp Ile Val Phe Ala Ile Phe

35 40 45

Met Ala Ala Glu Thr Phe Thr Asp Ile Phe Ile Ser Trp Phe Pro Phe

50 55 60

Tyr Tyr Glu Phe Lys Met Ala Phe Val Leu Trp Leu Leu Ser Pro Tyr

65 70 75 80

Thr Lys Gly Ala Ser Leu Leu Tyr Arg Lys Phe Val His Pro Ser Leu

85 90 95

Ser Arg His Glu Lys Glu Ile Asp Ala Cys Ile Val Gln Ala Lys Glu

100 105 110

Arg Ser Tyr Glu Thr Met Leu Ser Phe Gly Lys Arg Ser Leu Asn Ile

115 120 125

Ala Ala Ser Ala Ala Val Gln Ala Ala Thr Lys Ser Gln Gly Ala Leu

130 135 140

Ala Gly Arg Leu Arg Ser Phe Ser Met Gln Asp Leu Arg Ser Ile Pro

145 150 155 160

Asp Thr Pro Val Pro Thr Tyr Gln Asp Pro Leu Tyr Leu Glu Asp Gln

165 170 175

Val Pro Arg Arg Arg Pro Pro Ile Gly Tyr Arg Pro Gly Gly Leu Gln

180 185 190

Gly Ser Asp Thr Glu Asp Glu Cys Trp Ser Asp Asn Glu Ile Val Pro

195 200 205

Gln Pro Pro Val Arg Pro Arg Glu Lys Pro Leu Gly Arg Ser Gln Ser

210 215 220

Leu Arg Val Val Lys Arg Lys Pro Leu Thr Arg Glu Gly Thr Ser Arg

225 230 235 240

Ser Leu Lys Val Arg Thr Arg Lys Lys Ala Met Pro Ser Asp Met Asp

245 250 255

Ser

<210>25

<211>185

<212>PRT

<213> mice

<400>25

Met Arg Glu Arg Phe Asp Arg Phe Leu His Glu Lys Asn Cys Met Thr

1 5 10 15

Asp Leu Leu Ala Lys Leu Glu Ala Lys Thr Gly Val Asn Arg Ser Phe

20 25 30

Ile Ala Leu Gly Val Ile Gly Leu Val Ala Leu Tyr Leu Val Phe Gly

35 40 45

Tyr Gly Ala Ser Leu Leu Cys Asn Leu Ile Gly Phe Gly Tyr Pro Ala

50 55 60

Tyr Ile Ser Met Lys Ala Ile Glu Ser Pro Asn Lys Asp Asp Asp Thr

65 70 75 80

Gln Trp Leu Thr Tyr Trp Val Val Tyr Gly Val Phe Ser Ile Ala Glu

85 90 95

Phe Phe Ser Asp Leu Phe Leu Ser Trp Leu Pro Phe Tyr Tyr Met Leu

100 105 110

Lys Cys Gly Phe Leu Leu Trp Cys Met Ala Pro Ser Pro Ala Asn Gly

115 120 125

Ala Glu Met Leu Tyr Arg Arg Ile Ile Arg Pro Ile Phe Leu Arg His

130 135 140

Glu Ser Gln Val Asp Ser Val Val Lys Asp Val Lys Asp Lys Ala Lys

145 150 155 160

Glu Thr Ala Asp Ala Ile Ser Lys Glu Val Lys Lys Ala Thr Val Asn

165 170 175

Leu Leu Gly Asp Val Lys Lys Ser Thr

180 185

<210>26

<211>20l

<212>PRT

<213> mice

<400>26

Met Asp Gly Leu Arg Gln Arg Phe Glu Arg Phe Leu Glu Gln Lys Asn

1 5 10 15

Val Ala Thr Glu Ala Leu Gly Ala Leu Glu Ala Arg Thr Gly Val Glu

20 25 30

Lys Arg Tyr Leu Ala Ala Gly Ala Leu Ala Leu Leu Gly Leu Tyr Leu

35 40 45

Leu Phe Gly Tyr Gly Ala Ser Leu Leu Cys Asn Val Ile Gly Phe Val

50 55 60

Tyr Pro Ala Tyr Ala Ser Val Lys Ala Ile Glu Ser Pro Ser Lys Glu

65 70 75 80

Asp Asp Thr Val Trp Leu Thr Tyr Trp Val Val Tyr Ala Leu Phe Gly

85 90 95

Leu Val Glu Phe Phe Ser Asp Leu Leu Leu Phe Trp Phe Pro Phe Tyr

100 105 110

Tyr Ala Gly Lys Cys Ala Phe Leu Leu Phe Cys Met Thr Pro Gly Pro

115 120 125

Trp Asn Gly Ala Leu Leu Leu Tyr His Arg Val Ile Arg Pro Leu Phe

130 135 140

Leu Lys His His Met Ala Leu Asp Ser Ala Ala Ser Gln Leu Ser Gly

145 150 155 160

Arg Ala Leu Asp Leu Ala Ala Gly Ile Thr Arg Asp Val Leu Gln Ala

165 170 175

Leu Ala Arg Gly Arg Ala Leu Val Thr Pro Ala Ser Thr Ser Glu Pro

180 185 190

Pro Ala Ala Leu Glu Leu Asp Pro Lys

195 200

<210>27

<211>201

<212>PRT

<213> Intelligent people

<400>27

Met Val Ser Trp Ile Ile Ser Arg Leu Val Val Leu Ile Phe Gly Thr

1 5 10 15

Leu Tyr Pro Ala Tyr Tyr Ser Tyr Lys Ala Val Lys Ser Lys Asp Ile

20 25 30

Lys Glu Tyr Val Lys Trp Met Met Tyr Trp Ile Ile Phe Ala Leu Phe

35 40 45

Thr Thr Ala Glu Thr Phe Thr Asp Ile Phe Leu Cys Trp Phe Pro Phe

50 55 60

Tyr Tyr Glu Leu Lys Ile Ala Phe Val Ala Trp Leu Leu Ser Pro Tyr

65 70 75 80

Thr Lys Gly Ser Ser Leu Leu Tyr Arg Lys Phe Val His Pro Thr Leu

85 90 95

Ser Ser Lys Glu Lys Glu Ile Asp Asp Cys Leu Val Gln Ala Lys Asp

100 105 110

Arg Ser Tyr Asp AIa Leu Val His Phe Gly Lys Arg Gly Leu Asn Val

115 120 125

Ala Ala Thr Ala Ala Val Met Ala Ala Ser Lys Gly Gln Gly Ala Leu

130 135 140

Ser Glu Arg Leu Arg Ser Phe Ser Met Gln Asp Leu Thr Thr Ile Arg

145 150 155 160

Gly Asp Gly Ala Pro Ala Pro Ser Gly Pro Pro Pro Pro Gly Ser Gly

165 170 175

Arg Ala Ser Gly Lys His Gly Gln Pro Lys Met Ser Arg Ser Ala Ser

180 185 190

Glu Ser Ala Ser Ser Ser Gly Thr Ala

195 200

<210>28

<211>252

<212>PRT

<213> Intelligent people

<400>28

Met Val Ser Trp IIe Ile Ser Arg Leu Val Val Leu Ile Phe Gly Thr

1 5 10 15

Leu Tyr Pro Ala Tyr Ser Ser Tyr Lys Ala Val Lys Thr Lys Asn Val

20 25 30

Lys Glu Tyr Val Lys Trp Met Met Tyr Trp Ile Val Phe Ala Phe Phe

35 40 45

Thr Thr Ala Glu Thr Leu Thr Asp Ile Val Leu Ser Trp Phe Pro Phe

50 55 60

Tyr Phe Glu Leu Lys Ile Ala Phe Val Ile Trp Leu Leu Ser Pro Tyr

65 70 75 80

Thr Lys Gly Ser Ser Val Leu Tyr Arg Lys Phe Val His Pro Thr Leu

85 90 95

Ser Asn Lys Glu Lys Glu Ile Asp Glu Tyr Ile Thr Gln Ala Arg Asp

100 105 110

Lys Ser Tyr Glu Thr Met Met Arg Val Gly Lys Arg Gly Leu Asn Leu

115 120 125

Ala Ala Asn Ala Ala Val Thr Ala Ala Ala Lys Gly Val Leu Ser Glu

130 135 140

Lys Leu Arg Ser Phe Ser Met Gln Asp Leu Thr Leu Ile Arg Asp Glu

145 150 155 160

Asp Ala Leu Pro Leu Gln Arg Pro Asp Gly Arg Leu Arg Pro Ser Pro

165 170 175

Gly Ser Leu Leu Asp Thr Ile Glu Asp Leu Gly Asp Asp Pro Ala Leu

180 185 190

Ser Leu Arg Ser Ser Thr Asn Pro Ala Asp Ser Arg Thr Glu Ala Ser

195 200 205

Glu Asp Asp Met Gly Asp Lys Ala Pro Lys Arg Ala Lys Pro Ile Lys

210 215 220

Lys Ala Pro Lys Ala Glu Pro Leu Ala Ser Lys Thr Leu Lys Thr Arg

225 230 235 240

Pro Lys Lys Lys Thr Ser Gly Gly Gly Asp Ser Ala

245 250

<210>29

<211>146

<212>PRT

<213> Intelligent people

<400>29

Met Val Ser Trp Met Ile Ser Arg Ala Val Val Leu Val Phe Gly Met

1 5 10 15

Leu Tyr Pro Ala Tyr Tyr Ser Tyr Lys Ala Val Lys Thr Lys Asn Val

20 25 30

Lys Glu Tyr Val Arg Trp Met Met Tyr Trp Ile Val Phe Ala Leu Tyr

35 40 45

Thr Val Ile Glu Thr Val Ala Asp Gln Thr Val Ala Trp Phe Pro Leu

50 55 60

Tyr Tyr Glu Leu Lys Ile Ala Phe Val Ile Trp Leu Leu Ser Pro Tyr

65 70 75 80

Thr Lys Gly Ala Ser Leu Ile Tyr Arg Lys Phe Leu His Pro Leu Leu

85 90 95

Ser Ser Lys Glu Arg Glu Ile Asp Asp Tyr Ile Val Gln Ala Lys Glu

100 105 110

Arg Gly Tyr Glu Thr Met Val Asn Phe Gly Arg Gln Gly Leu Asn Leu

115 120 125

Ala Ala Thr Ala Ala Val Thr Ala Ala Val Lys Val Ile Val His Leu

130 135 140

Pro Phe

145

<210>30

<211>257

<212>PRT

<213> Intelligent people

<400>30

Met Val Ser Trp Met Ile Cys Arg Leu Val Val Leu Val Phe Gly Met

1 5 10 15

Leu Cys Pro Ala Tyr Ala Ser Tyr Lys Ala Val Lys Thr Lys Asn Ile

20 25 30

Arg Glu Tyr Val Arg Trp Met Met Tyr Trp Ile Val Phe Ala Leu Phe

35 40 45

Met Ala Ala Glu Ile Val Thr Asp Ile Phe Ile Ser Trp Phe Pro Phe

50 55 60

Tyr Tyr Glu Ile Lys Met Ala Phe Val Leu Trp Leu Leu Ser Pro Tyr

65 70 75 80

Thr Lys Gly Ala Ser Leu Leu Tyr Arg Lys Phe Val His Pro Ser Leu

85 90 95

Ser Arg His Glu Lys Glu Ile Asp Ala Tyr Ile Val Gln Ala Lys Glu

100 105 110

Arg Ser Tyr Glu Thr Val Leu Ser Phe Gly Lys Arg Gly Leu Asn Ile

115 120 125

Ala Ala Ser Ala Ala Val Gln Ala Ala Thr Lys Ser Gln Gly Ala Leu

130 135 140

Ala Gly Arg Leu Arg Ser Phe Ser Met Gln Asp Leu Arg Ser Ile Ser

145 150 155 160

Asp Ala Pro Ala Pro Ala Tyr His Asp Pro Leu Tyr Leu Glu Asp Gln

165 170 175

Val Ser His Arg Arg Pro Pro Ile Gly Tyr Arg Ala Gly Gly Leu Gln

180 185 190

Asp Ser Asp Thr Glu Asp Glu Cys Trp Ser Asp Thr Glu Ala Val Pro

195 200 205

Arg Ala Pro Ala Arg Pro Arg Glu Lys Pro Leu Ile Arg Ser Gln Ser

210 215 220

Leu Arg Val Val Lys Arg Lys Pro Pro Val Arg Glu Gly Thr Ser Arg

225 230 235 240

Ser Leu Lys Val Arg Thr Arg Lys Lys Thr Val Pro Ser Asp Val Asp

245 250 255

Ser

<210>31

<211>189

<212>PRT

<213> Intelligent people

<400>31

Met Ser Ala Ala Met Arg Glu Arg Phe Asp Arg Phe Leu His Glu Lys

1 5 10 15

Asn Cys Met Thr Asp Leu Leu Ala Lys Leu Glu Ala Lys Thr Gly Val

20 25 30

Asn Arg Ser Phe Ile Ala Leu Gly Val Ile Gly Leu Val Ala Leu Tyr

35 40 45

Leu Val Phe Gly Tyr Gly Ala Ser Leu Leu Cys Asn Leu Ile Gly Phe

50 55 60

Gly Tyr Pro Ala Tyr Ile Ser Ile Lys Ala Ile Glu Ser Pro Asn Lys

65 70 75 80

Glu Asp Asp Thr Gln Trp Leu Thr Tyr Trp Val Val Tyr Gly Val Phe

85 90 95

Ser Ile Ala Glu Phe Phe Ser Asp Ile Phe Leu Ser Trp Phe Pro Phe

100 105 110

Tyr Tyr Met Leu Lys Cys Gly Phe Leu Leu Trp Cys Met Ala Pro Ser

115 120 125

Pro Ser Asn Gly Ala Glu Leu Leu Tyr Lys Arg Ile Ile Arg Pro Phe

130 135 140

Phe Leu Lys His Glu Ser Gln Met Asp Ser Val Val Lys Asp Leu Lys

145 150 155 160

Asp Lys Ala Lys Glu Thr Ala Asp Ala Ile Thr Lys Glu Ala Lys Lys

165 170 175

Ala Thr Val Asn Leu Leu Gly Glu Glu Lys Lys Ser Thr

180 185

<210>32

<211>184

<212>PRT

<213> Intelligent people

<400>32

Met Asp Gly Leu Arg Gln Arg Val Glu His Phe Leu Glu Gln Arg Asn

1 5 10 15

Leu Val Thr Glu Val Leu Gly Ala Leu Glu Ala Lys Thr Gly Val Glu

20 25 30

Lys Arg Tyr Leu Ala Ala Gly Ala Val Thr Leu Leu Ser Leu Tyr Leu

35 40 45

Leu Phe Gly Tyr Gly Ala Ser Leu Leu Cys Asn Leu Ile Gly Phe Val

50 55 60

Tyr Pro Ala Tyr Ala Ser Ile Lys Ala Ile Glu Ser Pro Ser Lys Asp

65 70 75 80

Asp Asp Thr Val Trp Leu Thr Tyr Trp Val Val Tyr Ala Leu Phe Gly

85 90 95

Leu Ala Glu Phe Phe Ser Asp Leu Leu Leu Ser Trp Phe Pro Phe Tyr

100 105 110

Tyr Val Gly Lys Cys Ala Phe Leu Leu Phe Cys Met Ala Pro Arg Pro

115 120 125

Trp Asn Gly Ala Leu Met Leu Tyr Gln Arg Val Val Arg Pro Leu Phe

130 135 140

Leu Arg His His Gly Ala Val Asp Arg Ile Met Asn Asp Leu Ser Gly

145 150 155 160

Arg Ala Leu Asp Ala Ala Ala Gly Ile Thr Arg Asn Val Lys Pro Ser

165 170 175

Gln Thr Pro Gln Pro Lys Asp Lys

180

<210>33

<211>263

<212>PRT

<213> mice

<400>33

Met Arg Ile Phe Arg Pro Trp Arg Leu Arg Cys Pro Ala Leu His Leu

1 5 10 15

Pro Ser Phe Pro Thr Phe Ser Ile Lys Cys Ser Leu Pro Pro Leu Pro

20 25 30

Thr Asp Glu Asp Met Cys Lys Ser Val Thr Thr Gly Glu Trp Lys Lys

35 40 45

Val Phe Tyr Glu Lys Met Glu Glu Val Lys Pro Ala Asp Ser Trp Asp

50 55 60

Phe Ile Ile Asp Pro Asn Leu Lys His Asn Val Leu Ala Pro Gly Trp

65 70 75 80

Lys Gln Tyr Leu Glu Leu His Ala Ser Gly Arg Phe His Cys Ser Trp

85 90 95

Cys Trp His Thr Trp Gln Ser Pro His Val Val Ile Leu Phe His Met

100 105 110

Tyr Leu Asp Lys Ala Gln Arg Ala Gly Ser Val Arg Met Arg Val Phe

115 120 125

Lys Gln Leu Cys Tyr Glu Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser

130 135 140

Met Leu Glu Glu Asn Ile Glu Ser Leu Val Asp Asn Leu Ile Thr Ser

145 150 155 160

Leu Arg Glu Gln Cys Tyr Gly Glu Arg Gly Gly His Tyr Arg Ile His

165 170 175

Val Ala Ser Arg Gln Asp Asn Arg Arg His Arg Gly Glu Phe Cys Glu

180 185 190

Ala Cys Gln Glu Gly Ile Val His Trp Lys Pro Ser Glu Lys Leu Leu

195 200 205

Glu Glu Glu Ala Thr Thr Tyr Thr Phe Ser Arg Ala Pro Ser Pro Thr

210 215 220

Lys Pro Gln Ala Glu Thr Gly Ser Gly Cys Asn Phe Cys Ser Ile Pro

225 230 235 240

Trp Cys Leu Phe Trp Ala Thr Val Leu Met Leu Ile Ile Tyr Leu Gln

245 250 255

Phe Ser Phe Arg Thr Ser Val

260

<210>34

<211>223

<212>PRT

<213> mice

<400>34

Met Ser Thr Ser Leu Thr Thr Cys Glu Trp Lys Lys Val Phe Tyr Glu

1 5 10 15

Lys Met Glu Val Ala Lys Pro Ala Asp Ser Trp Glu Leu Ile Ile Asp

20 25 30

Pro Thr Leu Lys Pro Asn Glu Leu Gly Pro Gly Trp Lys Gln Tyr Leu

35 40 45

Glu Gln His Ala Ser Gly Arg Phe His Cys Ser Trp Cys Trp His Thr

50 55 60

Trp Gln Ser Ala Asn Val Val Ile Leu Phe His Met His Leu Asp Arg

65 70 75 80

Ala Gln Arg Val Gly Ser Val Arg Met Arg Val Phe Lys Gln Leu Cys

85 90 95

Tyr Gln Cys Gly Thr Ser Arg Leu Asp Glu Ser Ser Met Leu Glu Glu

100 105 110

Asn Ile Glu Gly Leu Val Asp Asn Leu Ile Thr Ser Leu Arg Glu Gln

115 120 125

Cys Tyr Asp Glu Asp Gly Gly Gln Tyr Arg Ile His Val Ala Ser Arg

130 135 140

Pro Asp Ser Gly Leu His Arg Ser Glu Phe Cys Glu Ala Cys Gln Glu

145 150 155 160

Gly Ile Val His Trp Lys Pro Ser Glu Lys Leu Leu Glu Glu Asp Ala

165 170 175

Ala Tyr Thr Asp Ala Ser Lys Lys Lys Gly Gln Ala Gly Phe Ile Ser

180 185 190

Ser Phe Phe Ser Phe Arg Trp Cys Leu Phe Trp Gly Thr Leu Cys Leu

195 200 205

Val Ile Val Tyr Leu Gln Phe Phe Arg Gly Arg Ser Gly Phe Leu

210 215 220

<210>35

<211>281

<212>PRT

<213> mice

<400>35

Met Met Glu Glu Asp Ile Gly Asp Thr Glu Gln Trp Arg His Val Phe

1 5 10 15

Gln Glu Leu Met Gln Glu Val Lys Pro Trp His Lys Trp Thr Leu Ile

20 25 30

Pro Asp Lys Asn Leu Leu Pro Asn Val Leu Lys Pro Gly Trp Thr Gln

35 40 45

Tyr Gln Gln Lys Thr Phe Ala Arg Phe His Cys Pro Ser Cys Ser Arg

50 55 60

Ser Trp Ala Ser Gly Arg Val Leu Ile Val Phe His Met Arg Trp Cys

65 70 75 80

Glu Lys Lys Ala Lys Gly Trp Val Lys Met Arg Val Phe Ala Gln Arg

85 90 95

Cys Asn Gln Cys Pro Glu Pro Pro Phe Ala Thr Pro Glu Val Thr Trp

100 105 110

Asp Asn Ile Ser Arg Ile Leu Asn Asn Leu Leu Phe Gln Ile Leu Lys

115 120 125

Lys Cys Tyr Lys Glu Gly Phe Lys Gln Met Gly Glu Ile Pro Leu Leu

130 135 140

Gly Asn Thr Ser Leu Glu Gly Pro His Asp Ser Ser Asn Cys Glu Ala

145 150 155 160

Cys Leu Leu Gly Phe Cys Ala Gln Asn Asp Leu Gly Gln Ala Ser Lys

165 170 175

Pro Pro Ala Pro Pro Leu Ser Pro Thr Ser Ser Lys Ser Ala Arg Glu

180 185 190

Pro Lys Val Thr Val Thr Cys Ser Asn Ile Ser Ser Ser Arg Pro Ser

195 200 205

Ser Lys Val Gln Met Pro Gln Ala Ser Lys Val Asn Pro Gln Ala Ser

210 215 220

Asn Pro Thr Lys Asn Asp Pro Lys Val Ser Cys Thr Ser Lys Pro Pro

225 230 235 240

Ala Pro Pro Leu Ser Pro Thr Ser Leu Lys Ser Ala Arg Glu Pro Lys

245 250 255

Val Thr Val Thr Cys Ser Asn Ile Ser Ser Ser Arg Pro Ser Ser Lys

260 265 270

Val Gln Met Pro Gln Ala Ser Lys Val

275 280

<210>36

<211>248

<212>PRT

<213> mice

<400>36

Met Leu Phe Pro Asp Asp Phe Ser Thr Trp Glu Gln Thr Phe Gln Glu

1 5 10 15

Leu Met Gln Glu Glu Lys Pro Gly Ala Lys Trp Ser Leu His Leu Asp

20 25 30

Lys Asn Ile Val Pro Asp Gly Ala Ala Leu Gly Trp Arg Gln His Gln

35 40 45

Gln Thr Val Gly Arg Phe Gln Cys Ser Arg Cys Cys Arg Ser Trp Thr

50 55 60

Ser Ala Gln Val Met Ile Leu Cys His Met Tyr Pro Asp Thr Leu Lys

65 70 75 80

Ser Gln Gly Gln Ala Arg Met Arg Ile Phe Gly Gln Lys Cys Gln Lys

85 90 95

Cys Phe Gly Cys Gln Phe Glu Thr Pro Lys Phe Ser Thr Glu Ile Ile

100 105 110

Lys Arg Ile Leu Asn Asn Leu Val Asn Tyr Ile Leu Gln Arg Tyr Tyr

115 120 125

Gly His Arg Lys Ile Ala Leu Thr Ser Asn Ala Ser Leu Gly Glu Lys

130 135 140

Val Thr Leu Asp Gly Pro His Asp Thr Arg Asn Cys Glu Ala Cys Ser

145 150 155 160

Leu Asn Ser His Gly Arg Cys Ala Leu Ala His Lys Val Lys Pro Pro

165 170 175

Arg Ser Pro Ser Pro Leu Pro Asn Ser Ser Ser Pro Ser Lys Ser Cys

180 185 190

Pro Pro Pro Pro Gln Thr Arg Asn Thr Asp Phe Gly Asn Lys Thr Leu

195 200 205

Gln Asp Phe Gly Asn Arg Thr Phe Gln Gly Cys Arg Glu Pro Pro Gln

210 215 220

Arg Glu Ile Glu Pro Pro Leu Phe Leu Phe Leu Ser Ile Ala Ala Phe

225 230 235 240

Ala Leu Phe Ser Leu Phe Thr Arg

245

<210>37

<211>263

<212>PRT

<213> Intelligent people

<400>37

Met Arg Ile Phe Arg Pro Trp Arg Leu Arg Cys Pro Ala Leu His Leu

1 5 10 15

Pro Ser Leu Ser Val Phe Ser Leu Arg Trp Lys Leu Pro Ser Leu Thr

20 25 30

Thr Asp Glu Thr Met Cys Lys Ser Val Thr Thr Asp Glu Trp Lys Lys

35 40 45

Val Phe Tyr Glu Lys Met Glu Glu Ala Lys Pro Ala Asp Ser Trp Asp

50 55 60

Leu Ile Ile Asp Pro Asn Leu Lys His Asn Val Leu Ser Pro Gly Trp

65 70 75 80

Lys Gln Tyr Leu Glu Leu His Ala Ser Gly Arg Phe His Cys Ser Trp

85 90 95

Cys Trp His Thr Trp Gln Ser Pro Tyr Val Val Ile Leu Phe His Met

100 105 110

Phe Leu Asp Arg Ala Gln Arg Ala Gly Ser Val Arg Met Arg Val Phe

115 120 125

Lys Gln Leu Cys Tyr Glu Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser

130 135 140

Met Leu Glu Glu Asn Ile Glu Gly Leu Val Asp Asn Leu Ile Thr Ser

145 150 155 160

Leu Arg Glu Gln Cys Tyr Gly Glu Arg Gly Gly Gln Tyr Arg Ile His

165 170 175

Val Ala Ser Arg Gln Asp Asn Arg Arg His Arg Gly Glu Phe Cys Glu

180 185 190

Ala Cys Gln Glu Gly Ile Val His Trp Lys Pro Ser Glu Lys Leu Leu

195 200 205

Glu Glu Glu Ala Thr Thr Tyr Thr Phe Ser Arg Ala Pro Ser Pro Thr

210 215 220

Lys Ser Gln Asp Gln Thr Gly Ser Gly Trp Asn Phe Cys Ser Ile Pro

225 230 235 240

Trp Cys Leu Phe Trp Ala Thr Val Leu Leu Leu Ile Ile Tyr Leu Gln

245 250 255

Phe Ser Phe Arg Ser Ser Val

260

<210>38

<211>225

<212>PRT

<213> Intelligent people

<400>38

Met Cys Thr Ser Leu Thr Thr Cys Glu Trp Lys Lys Val Phe Tyr Glu

1 5 10 15

Lys Met Glu Val Ala Lys Pro Ala Asp Ser Trp Glu Leu Ile Ile Asp

20 25 30

Pro Asn Leu Lys Pro Ser Glu Leu Ala Pro Gly Trp Lys Gln Tyr Leu

35 40 45

Glu Gln His Ala Ser Gly Arg Phe His Cys Ser Trp Cys Trp His Thr

50 55 60

Trp Gln Ser Ala His Val Val Ile Leu Phe His Met Phe Leu Asp Arg

65 70 75 80

Ala Gln Arg Ala Gly Ser Val Arg Met Arg Val Phe Lys Gln Leu Cys

85 90 95

Tyr Glu Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser Met Leu Glu Glu

100 105 110

Asn Ile Glu Gly Leu Val Asp Asn Leu Ile Thr Ser Leu Arg Glu Gln

115 120 125

Cys Tyr Glu Glu Asp Gly Gly Gln Tyr Arg Ile His Val Ala Ser Arg

130 135 140

Pro Asp Ser Gly Pro His Arg Ala Glu Phe Cys Glu Ala Cys Gln Glu

145 150 155 160

Gly Ile Val His Trp Lys Pro Ser Glu Lys Leu Leu Glu Glu Glu Val

165 170 175

Thr Thr Tyr Thr Ser Glu Ala Ser Lys Pro Arg Ala Gln Ala Gly Ser

180 185 190

Gly Tyr Asn Phe Leu Ser Leu Arg Trp Cys Leu Phe Trp Ala Ser Leu

195 200 205

Cys Leu Leu Val Val Tyr Leu Gln Phe Ser Phe Leu Ser Pro Ala Phe

210 215 220

Phe

225

<210>39

<211>232

<212>PRT

<213> Intelligent people

<400>39

Met Ala Gly Asp Thr Glu Val Trp Lys Gln Met Phe Gln Glu Leu Met

1 5 10 15

Arg Glu Val Lys Pro Trp His Arg Trp Thr Leu Arg Pro Asp Lys Gly

20 25 30

Leu Leu Pro Asn Val Leu Lys Pro Gly Trp Met Gln Tyr Gln Gln Trp

35 40 45

Thr Phe Ala Arg Phe Gln Cys Ser Ser Cys Ser Arg Asn Trp Ala Ser

50 55 60

Ala Gln Val Leu Val Leu Phe His Met Asn Trp Ser Glu Glu Lys Ser

65 70 75 80

Arg Gly Gln Val Lys Met Arg Val Phe Thr Gln Arg Cys Lys Lys Cys

85 90 95

Pro Gln Pro Leu Phe Glu Asp Pro Glu Phe Thr Gln Glu Asn Ile Ser

100 105 110

Arg Ile Leu Lys Asn Leu Val Phe Arg Ile Leu Lys Lys Cys Tyr Arg

115 120 125

Gly Arg Phe Gln Leu Ile Glu Glu Val Pro Met Ile Lys Asp Ile Ser

130 135 140

Leu Glu Gly Pro His Asn Ser Asp Asn Cys Glu Ala Cys Leu Gln Gly

145 150 155 160

Phe Cys Ala Gly Pro Ile Gln Val Thr Ser Leu Pro Pro Ser Gln Thr

165 170 175

Pro Arg Val His Ser Ile Tyr Lys Val Glu Glu Val Val Lys Pro Trp

180 185 190

Ala Ser Gly Glu Asn Val Tyr Ser Tyr Ala Cys Gln Asn His Ile Cys

195 200 205

Arg Asn Leu Ser Ile Phe Cys Cys Cys Val Ile Leu Ile Val Ile Val

210 215 220

Val Ile Val Val Lys Thr Ala Ile

225 230

<210>40

<211>246

<212>PRT

<213> Intelligent people

<400>40

Met Val Val Asp Phe Trp Thr Trp Glu Gln Thr Phe Gln Glu Leu Ile

1 5 10 15

Gln Glu Ala Lys Pro Arg Ala Thr Trp Thr Leu Lys Leu Asp Gly Asn

20 25 30

Leu Gln Leu Asp Cys Leu Ala Gln Gly Trp Lys Gln Tyr Gln Gln Arg

35 40 45

Ala Phe Gly Trp Phe Arg Cys Ser Ser Cys Gln Arg Ser Trp Ala Ser

50 55 60

Ala Lys Leu Gln Ile Leu Cys His Thr Tyr Trp Glu His Trp Thr Ser

65 70 75 80

Gln Gly Gln Val Arg Met Arg Leu Phe Gly Gln Arg Cys Gln Lys Cys

85 90 95

Ser Trp Ser Gln Tyr Glu Met Pro Glu Phe Ser Ser Asp Ser Thr Met

100 105 110

Arg Ile Leu Ser Asn Leu Val Gln His Ile Leu Lys Lys Tyr Tyr Gly

115 120 125

Asn Gly Met Arg Lys Ser Pro Glu Met Pro Val Ile Leu Glu Val Ser

130 135 140

Leu Glu Gly Ser His Asp Thr Ala Asn Cys Glu Ala Cys Thr Leu Gly

145 150 155 160

Ile Cys Gly Gln Gly Leu Lys Ser Tyr Met Thr Lys Pro Ser Lys Ser

165 170 175

Leu Leu Pro His Leu Lys Thr Gly Asn Ser Ser Pro Gly Ile Gly Ala

180 185 190

Val Tyr Leu Ala Asn Gln Ala Lys Asn Gln Ser Asp Glu Ala Lys Glu

195 200 205

Ala Lys Gly Ser Gly Tyr Glu Lys Leu Gly Pro Ser Arg Asp Pro Asp

210 215 220

Pro Leu Asn Ile Cys Val Phe Ile Leu Leu Leu Val Phe Ile Val Val

225 230 235 240

Lys Cys Phe Thr Ser Glu

245

<210>41

<211>210

<212>PRT

<213> mice

<400>41

Met Glu Glu Val Lys Pro Ala Asp Ser Trp Asp Phe Ile Ile Asp Pro

1 5 10 15

Asn Leu Lys His Asn Val Leu Ala Pro Gly Trp Lys Gln Tyr Leu Glu

20 25 30

Leu His Ala Ser Gly Arg Phe His Cys Ser Trp Cys Trp His Thr Trp

35 40 45

Gln Ser Pro His Val Val Ile Leu Phe His Met Tyr Leu Asp Lys Ala

50 55 60

Gln Arg Ala Gly Ser Val Arg Met Arg Val Phe Lys Gln Leu Cys Tyr

65 70 75 80

Glu Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser Met Leu Glu Glu Asn

85 90 95

Ile Glu Ser Leu Val Asp Asn Leu Ile Thr Ser Leu Arg Glu Gln Cys

100 105 110

Tyr Gly Glu Arg Gly Gly His Tyr Arg Ile His Val Ala Ser Arg Gln

115 120 125

Asp Asn Arg Arg His Arg Gly Glu Phe Cys Glu Ala Cys Gln Glu Gly

130 135 140

Ile Val His Trp Lys Pro Ser Glu Lys Leu Leu Glu Glu Glu Ala Thr

145 150 155 160

Thr Tyr Thr Phe Ser Arg Ala Pro Ser Pro Thr Lys Pro Gln Ala Glu

165 170 175

Thr Gly Ser Gly Cys Asn Phe Cys Ser Ile Pro Trp Cys Leu Phe Trp

180 185 190

Ala Thr Val Leu Met Leu Ile Ile Tyr Leu Gln Phe Ser Phe Arg Thr

195 200 205

Ser Val

210

<210>42

<211>152

<212>PRT

<213> mice

<400>42

Met Tyr Leu Asp Lys Ala Gln Arg Ala Gly Ser Val Arg Met Arg Val

1 5 10 15

Phe Lys Gln Leu Cys Tyr Glu Cys Gly Thr Ala Arg Leu Asp Glu Ser

20 25 30

Ser Met Leu Glu Glu Asn Ile Glu Ser Leu Val Asp Asn Leu Ile Thr

35 40 45

Ser Leu Arg Glu Gln Cys Tyr Gly Glu Arg Gly Gly His Tyr Arg Ile

50 55 60

His Val Ala Ser Arg Gln Asp Asn Arg Arg His Arg Gly Glu Phe Cys

65 70 75 80

Glu Ala Cys Gln Glu Gly Ile Val His Trp Lys Pro Ser Glu Lys Leu

85 90 95

Leu Glu Glu Glu Ala Thr Thr Tyr Thr Phe Ser Arg Ala Pro Ser Pro

100 105 110

Thr Lys Pro Gln Ala Glu Thr Gly Ser Gly Cys Asn Phe Cys Ser Ile

115 120 125

Pro Trp Cys Leu Phe Trp Ala Thr Val Leu Met Leu Ile Ile Tyr Leu

130 135 140

Gln Phe Ser Phe Arg Thr Ser Val

145 150

<210>43

<211>119

<212>PRT

<213> mice

<400>43

Met Leu Glu Glu Asn Ile Glu Ser Leu Val Asp Asn Leu Ile Thr Ser

1 5 10 15

Leu Arg Glu Gln Cys Tyr Gly Glu Arg Gly Gly His Tyr Arg Ile His

20 25 30

Val Ala Ser Arg Gln Asp Asn Arg Arg His Arg Gly Glu Phe Cys Glu

35 40 45

Ala Cys Gln Glu Gly Ile Val His Trp Lys Pro Ser Glu Lys Leu Leu

50 55 60

Glu Glu Glu Ala Thr Thr Tyr Thr Phe Ser Arg Ala Pro Ser Pro Thr

65 70 75 80

Lys Pro Gln Ala Glu Thr Gly Ser Gly Cys Asn Phe Cys Ser Ile Pro

85 90 95

Trp Cys Leu Phe Trp Ala Thr Val Leu Met Leu Ile Ile Tyr Leu Gln

100 105 110

Phe Ser Phe Arg Thr Ser Val

115

<210>44

<211>234

<212>PRT

<213> mice

<400>44

Met Arg Ile Phe Arg Pro Trp Arg Leu Arg Cys Pro Ala Leu His Leu

1 5 10 15

Pro Ser Phe Pro Thr Phe Ser Ile Lys Cys Ser Leu Pro Pro Leu Pro

20 25 30

Thr Asp Glu Asp Met Cys Lys Ser Val Thr Thr Gly Glu Trp Lys Lys

35 40 45

Val Phe Tyr Glu Lys Met Glu Glu Val Lys Pro Ala Asp Ser Trp Asp

50 55 60

Phe Ile Ile Asp Pro Asn Leu Lys His Asn Val Leu Ala Pro Gly Trp

65 70 75 80

Lys Gln Tyr Leu Glu Leu His Ala Ser Gly Arg Phe His Cys Ser Trp

85 90 95

Cys Trp His Thr Trp Gln Ser Pro His Val Val Ile Leu Phe His Met

100 105 110

Tyr Leu Asp Lys Ala Gln Arg Ala Gly Ser Val Arg Met Arg Val Phe

115 120 125

Lys Gln Leu Cys Tyr Glu Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser

130 135 140

Met Leu Glu Glu Asn Ile Glu Ser Leu Val Asp Asn Leu Ile Thr Ser

145 150 155 160

Leu Arg Glu Gln Cys Tyr Gly Glu Arg Gly Gly His Tyr Arg Ile His

165 170 175

Val Ala Ser Arg Gln Asp Asn Arg Arg His Arg Gly Glu Phe Cys Glu

180 185 190

Ala Cys Gln Glu Gly Ile Val His Trp Lys Pro Ser Glu Lys Leu Leu

195 200 205

Glu Glu Glu Ala Thr Thr Tyr Thr Phe Ser Arg Ala Pro Ser Pro Thr

210 215 220

Lys Pro Gln Ala Glu Thr Gly Ser Gly Cys

225 230

<210>45

<211>172

<212>PRT

<213> mice

<400>45

Met Arg Ile Phe Arg Pro Trp Arg Leu Arg Cys Pro Ala Leu His Leu

1 5 10 15

Pro Ser Phe Pro Thr Phe Ser Ile Lys Cys Ser Leu Pro Pro Leu Prc

20 25 30

Thr Asp Glu Asp Met Cys Lys Ser Val Thr Thr Gly Glu Trp Lys Lys

35 40 45

Val Phe Tyr Glu Lys Met Glu Glu Val Lys Pro Ala Asp Ser Trp Asp

50 55 60

Phe Ile Ile Asp Pro Asn Leu Lys His Asn Val Leu Ala Pro Gly Trp

65 70 75 80

Lys Gln Tyr Leu Glu Leu His Ala Ser Gly Arg Phe His Cys Ser Trp

85 90 95

Cys Trp His Thr Trp Gln Ser Pro His Val Val Ile Leu Phe His Met

100 105 110

Tyr Leu Asp Lys Ala Gln Arg Ala Gly Ser Val Arg Met Arg Val Phe

115 120 125

Lys Gln Leu Cys Tyr Glu Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser

130 135 140

Met Leu Glu Glu Asn Ile Glu Ser Leu Val Asp Asn Leu Ile Thr Ser

145 150 155 160

Leu Arg Glu Gln Cys Tyr Gly Glu Arg Gly Gly His

165 170

<210>46

<211>227

<212>PRT

<213> mice

<400>46

Met Cys Lys Ser Val Thr Thr Gly Glu Trp Lys Lys Val Phe Tyr Glu

1 5 10 15

Lys Met Glu Glu Val Lys Pro Ala Asp Ser Trp Asp Phe Ile Ile Asp

20 25 30

Pro Asn Leu Lys His Asn Val Leu Ala Pro Gly Trp Lys Gln Tyr Leu

35 40 45

Glu Leu His Ala Ser Gly Arg Phe His Cys Ser Trp Cys Trp His Thr

50 55 60

Trp Gln Ser Pro His Val Val Ile Leu Phe His Met Tyr Leu Asp Lys

65 70 75 80

Ala Gln Arg Ala Gly Ser Val Arg Met Arg Val Phe Lys Gln Leu Cys

85 90 95

Tyr Glu Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser Met Leu Glu Glu

100 105 110

Asn Ile Glu Ser Leu Val Asp Asn Leu Ile Thr Ser Leu Arg Glu Gln

115 120 125

Cys Tyr Gly Glu Arg Gly Gly His Tyr Arg Ile His Val Ala Ser Arg

130 135 140

Gln Asp Asn Arg Arg His Arg Gly Glu Phe Cys Glu Ala Cys Gln Glu

145 150 155 160

Gly Ile Val His Trp Lys Pro Ser Glu Lys Leu Leu Glu Glu Glu Ala

165 170 175

Thr Thr Tyr Thr Phe Ser Arg Ala Pro Ser Pro Thr Lys Pro Gln Ala

180 185 190

Glu Thr Gly Ser Gly Cys Asn Phe Cys Ser Ile Pro Trp Cys Leu Phe

195 200 205

Trp Ala Thr Val Leu Met Leu Ile Ile Tyr Leu Gln Phe Ser Phe Arg

210 215 220

Thr Ser Val

225

<210>47

<211>227

<212>PRT

<213> Intelligent people

<400>47

Met Cys Lys Ser Val Thr Thr Asp Glu Trp Lys Lys Val Phe Tyr Glu

1 5 10 15

Lys Met Glu Glu Ala Lys Pro Ala Asp Ser Trp Asp Leu Ile Ile Asp

20 25 30

Pro Asn Leu Lys His Asn Val Leu Ser Pro Gly Trp Lys Gln Tyr Leu

35 40 45

Glu Leu His Ala Ser Gly Arg Phe His Cys Ser Trp Cys Trp His Thr

50 55 60

Trp Gln Ser Pro Tyr Val Val Ile Leu Phe His Met Phe Leu Asp Arg

65 70 75 80

Ala Gln Arg Ala Gly Ser Val Arg Met Arg Val Phe Lys Gln Leu Cys

85 90 95

Tyr Glu Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser Met Leu Glu Glu

100 105 110

Asn Ile Glu Gly Leu Val Asp Asn Leu Ile Thr Ser Leu Arg Glu Gln

115 120 125

Cys Tyr Gly Glu Arg Gly Gly Gln Tyr Arg Ile His Val Ala Ser Arg

130 135 140

Gln Asp Asn Arg Arg His Arg Gly Glu Phe Cys Glu Ala Cys Gln Glu

145 150 155 160

Gly Ile Val His Trp Lys Pro Ser Glu Lys Leu Leu Glu Glu Glu Ala

165 170 175

Thr Thr Tyr Thr Phe Ser Arg Ala Pro Ser Pro Thr Lys Ser Gln Asp

180 185 190

Gln Thr Gly Ser Gly Trp Asn Phe Cys Ser Ile Pro Trp Cys Leu Phe

195 200 205

Trp Ala Thr Val Leu Leu Leu Ile Ile Tyr Leu Gln Phe Ser Phe Arg

210 215 220

Ser Ser Val

225

<210>48

<211>212

<212>PRT

<213> mice

<400>48

Met Arg Ile Phe Arg Pro Trp Arg Leu Arg Cys Pro Ala Leu His Leu

1 5 10 15

Pro Ser Phe Pro Thr Phe Ser Ile Lys Cys Ser Leu Pro Pro Leu Pro

20 25 30

Thr Asp Glu Asp Met Cys Lys Ser Val Thr Thr Gly Glu Trp Lys Lys

35 40 45

Val Phe Tyr Glu Lys Met Glu Glu Val Lys Pro Ala Asp Ser Trp Asp

50 55 60

Phe Ile Ile Asp Pro Asn Leu Lys His Asn Val Leu Ala Pro Gly Trp

65 70 75 80

Lys Gln Tyr Leu Glu Leu His Ala Ser Gly Arg Phe His Cys Ser Trp

85 90 95

Cys Trp His Thr Trp Gln Ser Pro His Val Val Ile Leu Phe His Met

100 105 110

Tyr Leu Asp Lys Ala Gln Arg Ala Gly Ser Val Arg Met Arg Val Phe

115 120 125

Lys Gln Leu Cys Tyr Glu Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser

130 135 140

Met Leu Glu Glu Asn Ile Glu Ser Leu Val Asp Asn Leu Ile Thr Ser

145 150 155 160

Leu Arg Glu Gln Cys Tyr Gly Glu Arg Gly Gly His Tyr Arg Ile His

165 170 175

Val Ala Ser Arg Gln Asp Asn Arg Arg His Arg Gly Glu Phe Cys Glu

180 185 190

Ala Cys Gln Glu Gly Ile Val His Trp Lys Pro Ser Glu Lys Leu Leu

195 200 205

Glu Glu Glu Ala

210

<210>49

<211>193

<212>PRT

<213> mice

<400>49

Met Arg Ile Phe Arg Pro Trp Arg Leu Arg Cys Pro Ala Leu His Leu

1 5 10 15

Pro Ser Phe Pro Thr Phe Ser Ile Lys Cys Ser Leu Pro Pro Leu Pro

20 25 30

Thr Asp Glu Asp Met Cys Lys Ser Val Thr Thr Gly Glu Trp Lys Lys

35 40 45

Val Phe Tyr Glu Lys Met Glu Glu Val Lys Pro Ala Asp Ser Trp Asp

50 55 60

Phe Ile Ile Asp Pro Asn Leu Lys His Asn Val Leu Ala Pro Gly Trp

65 70 75 80

Lys Gln Tyr Leu Glu Leu His Ala Ser Gly Arg Phe His Cys Ser Trp

85 90 95

Cys Trp His Thr Trp Gln Ser Pro His Val Val Ile Leu Phe His Met

100 105 110

Tyr Leu Asp Lys Ala Gln Arg Ala Gly Ser Val Arg Met Arg Val Phe

115 120 125

Lys Gln Leu Cys Tyr Glu Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser

130 135 140

Met Leu Glu Glu Asn Ile Glu Ser Leu Val Asp Asn Leu Ile Thr Ser

145 150 155 160

Leu Arg Glu Gln Cys Tyr Gly Glu Arg Gly Gly His Tyr Arg Ile His

165 170 175

Val Ala Ser Arg Gln Asp Asn Arg Arg His Arg Gly Glu Phe Cys Glu

180 185 190

Ala

<210>50

<211>253

<212>PRT

<213> mice

<400>50

Met Arg Ile Phe Arg Pro Trp Arg Leu Arg Cys Pro Ala Leu His Leu

1 5 10 15

Pro Ser Phe Pro Thr Phe Ser Ile Lys Cys Ser Leu Pro Pro Leu Pro

20 25 30

Thr Asp Glu Asp Met Cys Lys Ser Val Thr Thr Gly Glu Trp Lys Lys

35 40 45

Val Phe Tyr Glu Lys Met Glu Glu Val Lys Pro Ala Asp Ser Trp Asp

50 55 60

Phe Ile Ile Asp Pro Asn Leu Lys His Asn Val Leu Ala Pro Gly Trp

65 70 75 80

Lys Gln Tyr Leu Glu Leu His Ala Ser Gly Arg Phe His Cys Ser Trp

85 90 95

Cys Trp His Thr Trp Gln Ser Pro His Val Val Ile Leu Phe His Met

100 105 110

Tyr Leu Asp Lys Ala Gln Arg Ala Gly Ser Val Arg Met Arg Val Phe

115 120 125

Lys Gln Leu Cys Tyr Glu Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser

130 135 140

Met Leu Glu Glu Asn Ile Glu Ser Leu Val Asp Asn Leu Ile Thr Ser

145 150 155 160

Leu Arg Glu Gln Cys Tyr Gly Glu Arg Gly Gly His Tyr Arg Ile His

165 170 175

Val Ala Ser Arg Gln Asp Asn Arg Arg His Arg Gly Glu Phe Cys Glu

180 185 190

Ala Cys Gln Glu Gly Ile Val His Trp Lys Pro Ser Glu Lys Leu Leu

195 200 205

Glu Glu Glu Ala Thr Thr Tyr Thr Phe Ser Arg Ala Pro Ser Pro Thr

210 215 220

Lys Pro Gln Ala Glu Thr Gly Ser Gly Cys Asn Phe Cys Ser Ile Pro

225 230 235 240

Trp Cys Leu Phe Trp Ala Thr Val Leu Met Leu Ile Ile

245 250

<210>51

<211>48

<212>DNA

<213> Artificial

<220>

<223> synthetic

<400>51

tatagaattc gcggccgctc gcgatttttt tttttttttt tttttttt 48

Claims (25)

1. A method of identifying an odorant receptor ligand, comprising:

a) provide for

i) A cell line or cell membrane thereof comprising an odorant receptor, wherein said cell line expresses: encoding the amino acid sequence of SEQ ID NO: 37 or 47, and a nucleic acid sequence encoding SEQ ID NO: 38, and

ii) a test compound;

b) exposing the test compound to the cell line; and

c) detecting the activity of the odorant receptor.

2. The method of claim 1, wherein said detecting comprises detecting a reporter reagent.

3. The method of claim 1, wherein said cell line is a 293T cell line.

4. The method of claim 1 wherein said odorant receptor is a human odorant receptor.

5. The method of claim 1, wherein said test compound is an odorant molecule.

6. The method of claim 2, wherein said reporter agent is modulated by a cAMP response element.

7. The method of claim 1, wherein said cell line comprises G_αolf。

8. The method of claim 1 wherein said odorant receptor is a murine odorant receptor.

9. The method of claim 1 wherein said odorant receptor is a synthetic odorant receptor.

10. The method of claim 1 wherein said odorant receptor is S6/79, S18, S46, S50, MOR23-1, MOR31-4, MOR31-6, MOR32-5 or MOR 32-11.

11. The method of claim 2, wherein said reporter agent is a luminescent agent.

12. The method of claim 11, wherein said luminescent agent is luciferase.

13. The method of claim 1, further comprising the steps of:

d) detecting the presence or absence of an odorant receptor ligand based on said activity.

14. The method of claim 1, wherein said cell line is selected from the group consisting of a heterologous cell line and a native cell line.

15. A cell line expressing an odorant receptor, wherein said expression is localized on the surface of a cell, wherein said cell line expresses: encoding the amino acid sequence of SEQ ID NO: 37 or 47, and a nucleic acid sequence encoding SEQ ID NO: 38.

16. The cell line of claim 15, wherein said cell line is a 293T cell line.

17. The cell line of claim 15, wherein said odorant receptor is a human odorant receptor.

18. The cell line of claim 15, wherein said odorant receptor is labeled with a reporter agent.

19. The cell line of claim 18, wherein said reporter agent is a luminescent reporter agent.

20. The cell line of claim 19, wherein said light-emitting reporter agent is Green Fluorescent Protein (GFP).

21. The cell line of claim 18, wherein the reporter reagent is selected from the group consisting of glutathione-S-transferase (GST), c-myc, 6-histidine (6X-His), Maltose Binding Protein (MBP), influenza a virus Hemagglutinin (HA), β -galactosidase, and GAL 4.

22. In the application ofThe cell line of claim 15, wherein said cell line further comprises G_αolfAnd (4) expressing.

23. The cell line of claim 15, wherein said odorant receptor is a murine odorant receptor.

24. The cell line of claim 15, wherein said odorant receptor is a synthetic odorant receptor.

25. The cell line of claim 15, wherein said odorant receptor is S6/79, S18, S46, S50, MOR23-1, MOR31-4, MOR31-6, MOR32-5 or MOR 32-11.