US20020155444A1

US20020155444A1 - Human VNO cDNA libraries

Info

Publication number: US20020155444A1
Application number: US09/783,252
Authority: US
Inventors: Ronald Herman; David Berliner
Original assignee: Individual
Current assignee: Pherin Pharmaceuticals Inc
Priority date: 2000-02-17
Filing date: 2001-02-13
Publication date: 2002-10-24
Also published as: WO2001061046A2; WO2001061046A3; US7087430B2; AU2001238456A1; US20030224440A1

Abstract

This invention relates to DNA libraries, in particular a human VNO cDNA library is described. Pheromone receptor cDNA once isolated is transfected into competent cells. The transfected cell lines provide a scaleable source of homogeneous material to develop efficient, automated high throughput screening assays for new vomeropherins, and thereby reduce the ongoing need for human volunteers in the preclinical phases of drug discovery. Identification and characterization of the human VNO receptor(s) will facilitate the development and commercialization of vomeropherins with improved specificity, and enhanced therapeutic efficacy in the treatment of the target diseases.

Description

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/183,128, filed Feb. 17, 2000.[0001]

FIELD OF THE INVENTION

The present invention relates generally to the field of CDNA libraries, and more specifically to human vomeronasal organ libraries.

BACKGROUND

Small, volatile and non-volatile organic molecules, commonly referred to as pheromones, mediate species-specific chemical communication between terrestrial animals. Pheromones are present in the secretions and excretions from various organs and tissues, including the skin, and represent diverse families of chemical structures. Pheromones play essential roles in sexual activity, reproductive biology, and other innate animal behaviors (Luscher et al., (1959) Nature 18:55-56; Meredith (1983) in Pheromones and Reproduction in Mammals (Vandenbergh, ed.) pp.199-252, Academic Press; Stern et al., (1998) Nature 392:177-179; Wysocki, (1979) Neurosci. Biobehav. Rev. 3:301-341; Jacob et al., (2000) Hormones and Behavior 37:57-78; Grosser et al., (2000) Psychoneuroendocrinology 25:289-299). Some, but not all, terrestrial vertebrates detect pheromones in the vomeronasal organ (the VNO), also known as Jacobson's organ, a small dead-end tubular structure with an opening into the nasal cavity that is located bilaterally at the base of the nasal septum (Moran et al., (1991) J. Steroid Biochem. Molec. Biol. 39:545-552.).

The VNO was first identified in humans in 1703 but it was believed to be a vestigial organ without function in the adult. In the 1990s, the presence of a VNO was established, caudal to the nasal septal cartilage on both sides of the nasal septum, in more than 1700 normal male and female human subjects (Berliner, (1996) J. Steroid Biochem. Molec. Biol. 58:1-2; Gaafar et al., (1998) Acta Otolargyngol. 118:408-412; Smith et al., (1998) Micro. Res. Tech. 41:483-491) The VNO is physically separate and functionally distinct from the olfactory epithelium that detects the volatile odorants. Odorants do not bind to the VNO receptors.

The VNO is lined with neuroepithelial cells with a microvillar surface that is the presumptive site of pheromone receptors. Immunohistochemical staining of adult human VNO epithelium detects neuron-specific enolase and protein gene product (PGP) 9.5, both neuronal and neuroendocrine markers, in some bipolar cells with morphological similarities to olfactory receptor neurons (Takami et al., (1993) Neuroreport 4:375-378). More recent studies show that the majority of the cells lining the lumen of the human VNO stain with antibodies to synaptophysin or chromogranin which are also markers for neuronal and neuroendocrine cells. These data provide clear evidence for the existence of a neuroepithelium in the human VNO. However, Takami et al. (1993) do not detect olfactory marker protein (OMP) in the human VNO even though it is expressed in the VNO of other vertebrates including rodents. This may reflect an important and interesting species difference between humans and other vertebrates.

In animals, signals from the olfactory epithelium travel via the olfactory bulb to the olfactory cortex and then on to other regions of the brain. In contrast, signals from the VNO are transmitted through the accessory olfactory bulb to the amygdala and hypothalamus (Broadwell et al., (1975) J. Comp. Neurol. 163:329-346; Kevetter et al., (1981) J. Comp. Neurol. 197:81-98). Surgical ablation of the VNO in male rodents alters a variety of endocrine-mediated responses to female pheromones including androgen surges, vocalization, territorial marking, and inter-male aggression. Ablation of the VNO in female rodents delays or prevents activation of reproduction, abolishes the effects of over-crowding on sexual maturation, and reduces maternal responses to intruders (Wysocki et al., (1991) J. Steroid Biochem. Molec. Biol. 39:661-669). In humans, the defect(s) that causes the inherited hypogonadal disorder, Kallmann Syndrome, is also associated with defective development of the VNO-terminal is complex (Kallmann et al., (1943) Am. J. Ment. Defic. 48:203-236).

Application of only femtomole quantities of any of several proprietary, synthetic vomeropherins directly to the VNO of human volunteers rapidly induces reproducible negative voltage potentials that can be measured locally with a multifunctional miniprobe. The electrophysiological response in the VNO is characteristic of a mass receptor potential. The magnitude of the response is dose-dependent and is accompanied by changes in autonomic nervous system function, brain wave activity, gonadotropin secretion, and mood (Berliner et al., (1996) J Steroid Biochem, Molec. Biol. 58:259-265; Monti-Bloch et al. (1998a) J. Steroid Biochem. Molec. Biol. 65:237-242; Monti-Bloch et al., (1998b) Ann. N.Y. Acad. Sci. 855:373-389; Monti-Bloch et al., (1994) Pyschoneuroendocrinology 19:673-686; Monti-Bloch et al., (1991) J. Steroid Biochem. Molec. Biol. 39:573-582; Grosser et al., (2000) Psychoneuroendocrinology 25:289-299).

Recent fMRI studies detect dose-dependent activation of the anterior medial thalamus, the inferior frontal gyrus, and other regions of the human brain, in the absence of detectable odor, following administration of estra-1,3,5(10), 16-tetraen-3yl acetate (PH15) to human volunteers. Although Sobel et al. ((1999) Brain 122:209-217) deliver the compound non-specifically to the nasal cavity in these fMRI tests, Monti-Bloch et al. (1994) have demonstrated that this compound induces physiological responses in vivo only when applied specifically to the VNO but not when applied to either olfactory or respiratory epithelium of human subjects. Therefore, the fMRI data support the existence of a functional neurological connection between the VNO and the human brain which can be activated by a vomeropherin.

Administration of naturally occurring compounds of known structure such as estra-1,3,5(10), 16-tetraen-3-ol and androsta-4,16-dien-3-one to the human VNO induce bradycardia, bradypnea, increases in core body temperature, and other physiological responses. Stern et al. (1998) have demonstrated that odorless human pheromones, obtained from the axillae of women at different stages of the menstrual cycle, exert opposing effects on ovulation when applied above the lips where they can volatilize into the nasal cavity of recipient females. Some vomeropherins act exclusively in human females or in males, and others exert opposite effects on autonomic reflexes such as body temperature. Taken together, these data provide substantial support for the existence of a functional VNO in humans with the capacity to exert significant physiological effects in vivo.

The VNO system affords the unique opportunity to develop and market novel therapeutics to treat disease via previously unexploited targets and neurological pathways. This approach has substantial benefits for the patient over existing therapies including: (i) the ease of delivery to the VNO, (ii) the requirement for only picograms of drug, (iii) the rapid response to drug, and (iv) the apparent absence of the side-effects and toxicity frequently associated with systemic (e.g., oral) delivery of drug. Thus, targeting receptors in the human VNO for the treatment of disease is desirable.

The standard bioassay for screening candidate vomeropherins requires the participation of human volunteers because pheromones are species-specific. In this assay, the compounds are delivered directly to the VNO of volunteers under IRB-approved protocols, thus necessitating prior toxicological study of each candidate vomeropherin in rodents. This expensive and time-consuming process limits the number of compounds that can be tested and hampers the detailed structure-activity relationship (SAR) analyses that are essential to successful drug discovery.

Viable neuroepithelial cells may be harvested directly from the human VNO for testing in vitro. The harvested VNO cells retain their characteristic neuroepithelial morphology in culture and respond electrophysiologically to the application of vomeropherins in vitro, thereby demonstrating the existence of functional receptors in cells from the target tissue. Although this method still requires the participation of human volunteers, it increases the screening throughput and decreases the number of animals required for toxicological studies. However, only a limited number of non-dividing cells with a ˜2-week life-span are obtained from each volunteer, and thus we require an entirely new approach to meet the demands of modern high throughput drug screening and SAR.

Several groups have cloned receptor cDNAs that are expressed exclusively in the VNO of rats and mice, but, to date, no one has cloned human VNO receptor cDNAs. The sequence of the cloned rodent receptor cDNAs indicates that they belong to the superfamily of G protein-coupled receptors containing seven transmembrane domains, but they are unrelated to any of the G protein-coupled receptors expressed in the olfactory epithelium (Dulac et al., (1995) Cell 83:495-206; Herrada et al., (1997) Cell 90:763-773; Matsunami et al., (1997) Cell 90:775-784; Ryba et al., (1997) Neuron 19:371-379; Saito et al., (1998) Brain Res. Molec. Brain Res. 60:215-227). Database comparisons identify motifs common to Ca ²⁺-sensing and metabotropic glutamate receptors in some of the clones. The apparent lack of homology to olfactory receptors is consistent with the observation that many vomeropherins are inactive when applied specifically to human olfactory epithelium in vivo.

Each cloned rodent receptor messenger RNA (mRNA) is detected by in situ hybridization in only a small number of neuroepithelial cells that are dispersed throughout the rodent VNO, and it is likely that each cell expresses only a single receptor gene. (Dulac et al., 1995; Herrada et al., 1997; Matsunami et al., 1997; Ryba et al., 1997; Saito et al., 1998). Some of the cloned rodent receptors exhibit sexually dimorphic expression, i.e., they are expressed differently in males or females.

The rodent VNO receptors are assigned to separate multi-gene families by two criteria: (i) the length of the extracellular (N-terminal) protein domain, and (ii) the isoform of the signal-transducing G protein co-expressed in the same cell. Receptors in the “V1R” family have a relatively short extracellular N-terminal domain and are expressed primarily in cells that express a Gα _iisoform of G protein. Receptors in the “V2R” family have a long extracellular N-terminal domain and are expressed primarily in cells that express a Gα₀isoform of G protein. Differences at the N-terminus between the V1R and V2R families may reflect differences in the structure of the ligand and/or in the location of the ligand-binding domain. (Matsunami et al., 1997; Ryba et al., 1997; Krieger et al., (1999 J. Biol. Chem. 274:4656-4662). Neuroepithelial cells expressing these distinct G protein isoforms are spatially segregated in the VNO in separate apical and basal longitudinal zones, suggesting that there is true physiological significance to the differences between the V1R and V2R receptor families.

Krieger et al. (1999) have recently shown that G protein-coupled receptors expressed in the rodent VNO are functionally linked to signal transduction pathways. Their results demonstrate that volatile and non-volatile pheromonal components of male rat urine selectively activate the major Goc protein subtypes (G _iand G₀, respectively) expressed in the VNO of female rats. The data imply that V1R family receptors, which are co-expressed with G_i, respond to volatile compounds whereas V2R family receptors, which are co-expressed with G₀, respond to non-volatile protein components of urine.

Dulac and Axel (1995) estimate that, in total, the rat V1R family contains approximately 35 candidate pheromone receptors; Herrada and Dulac (1997) and Ryba and Tirindelli (1997) estimate that the rat V2R family contains an additional 100 receptors. Of the various rodent tissues tested, only mRNA from the VNO gives a positive signal on northern blots probed with the cloned ( ³²P-labeled) pheromone receptor cDNAs. At this limit of sensitivity, these results suggest that the pheromone receptors are expressed exclusively (primarily) in the VNO. At the present time, it is not known if each VNO receptor recognizes a distinct pheromone or if several receptors recognize the same compound.

At reduced stringency, the cloned rodent VNO receptor cDNAs cross-hybridize to human genomic DNA. Dulac and Axel (1995) detect approximately 15 human genes that cross-hybridize to rat V1R family probes, and Herrada and Dulac (1997) detect an additional ten human homologues that cross-hybridize to rat V2R family probes. The two sequenced human V1R genomic DNA clones have ˜40-50% identity with the closest rat homologue. However, both human genomic clones have a stop codon in the putative coding region and may thus be pseudogenes (Dulac and Axel, 1995). Nevertheless, cross-hybridization suggests the evolutionary conservation of G protein-coupled receptors in the VNO and thereby provides a means to isolate human receptor clones.

The presence of these pseudogenes does not preclude the existence of functional human VNO receptor genes, especially in view of our assays with cells harvested directly from the VNO (Monti-Bloch (1997) Chemical Senses 22:752). The past difficulties in isolating, characterizing and cloning a VNO receptor reinforce our assertion that an appropriate way to isolate functional clones of the human VNO receptors is via cDNA prepared directly from the target tissue. In fact, Cao et al. ((1998) Proc. Natl. Acad. Sci. USA 95:11987-11992) have successfully isolated homologues from a goldfish CDNA library using probes based on the rodent receptor sequences even though that species lacks a defined VNO. The presence of pseudogenes in the family has not prevented the successful cloning of olfactory or VNO receptors from a variety of species and they should present no greater obstacle to the cloning of human VNO receptors.

Thus, isolation and characterization of the human VNO receptors is desirable for the development of new drugs, high throughput assays and characterization of the receptors and their signal transduction pathways.

SUMMARY

In one aspect of the invention there is a cDNA library prepared from the normal human female VNO.

In a second aspect of the invention there is provided human VNO receptor cDNA sequences.

In a further aspect there is provided transformed cells expressing a functional human VNO receptor.

In another aspect of the invention there is provided a human VNO cell culture expressing a functional pheromone receptor.

In yet another aspect there is provided a high throughput drug screening assay.

DESCRIPTION OF THE FIGURES

FIG. 1 is an electrophysiological trace showing the effects of pertussis toxin on membrane currents induced by a vomeropherin. FIG. 1A is a tracing of the inward currents induced by 10[0026] ⁻⁷M androstadienone (ADO) in a female human VNO cell. FIG. 1B is a tracing from a cell that was incubated with 100 ng/ml pertusis toxin (PTX) blocking the inward currents. FIG. 1C indicates when the cells were exposed to ADO (i.e., ADO pulses).

DETAILED DESCRIPTION

The invention will now be described in detail by way of reference only using the following definitions and examples. All patents and publications referred to herein are expressly incorporated by reference. [0027]
The present invention provides a human female VNO-specific cDNA library, which is a unique resource for the identification and isolation of genes expressed in the VNO, specifically genes for pheromone receptors, ion channels and prospective reagents for high throughput assays. Although the human female VNO has been used and is described in detail herein, the male VNO may be subjected to the same methods and procedures to yield a similar cDNA library. Thus, identification and characterization of pheromone receptors, as well as the sexually dimorphic pheromone response, may be investigated. [0028]
Definitions [0029]
As used herein, the following terms or abbreviations, whether used in the singular or plural, will have the meanings indicated: [0030]
A “pheromone” is a biochemical produced by an animal or individual which elicits a specific physiological or behavioral response in another member of the same species. In addition to physiological responses, pheromones can be identified by their species specific binding to receptors in the vomeronasal organ (VNO). Thus, human pheromones bind to human receptors. This can be demonstrated by measuring the change in the summated potential of neuroepithelial tissue in the presence of the pheromone. Human pheromones induce a change of at least about −5 millivolts in human neuroepithelial tissue of the appropriate sex (The binding of pheromones is generally sexually dimorphic.). Naturally occurring human pheromones induce sexually dimorphic changes in receptor binding potential in vivo in the human VNO. Naturally occurring human pheromones can be extracted and purified from human skin and they can also be synthesized. “Human pheromones” are pheromones that are naturally occurring in humans and effective as a specifically binding ligand in human VNO tissue, regardless of how the pheromone was obtained. Thus, both a synthesized and purified molecule may be considered a human pheromone. Commonly, pheromones affect development, reproduction and related behaviors. [0031]
“Sexually dimorphic” refers to a difference in the effect of, or response to, a compound or composition between males and females of the same species. [0032]
“Vomeropherin” as used herein is a more general term which includes pheromones and describes a substance from any source which functions as a chemosensory messenger, binds to a specific vomeronasal neuroepithelial receptor, and induces a physiological or behavioral effect. The physiologic effect of a “vomeropherin” is mediated through the vomeronasal organ. Vomeropherins may be naturally occurring compounds, synthetic modifications of natural compounds or totally synthetic compounds. [0033]
The term “cDNA library” as used herein refers to a collection of cDNAs representing the messenger RNAs expressed in a cell or tissue type. [0034]
“cRNA” means synthetic RNA produced by transcription from a specific DNA template. [0035]
A “vector” or “plasmid” is a small circular DNA capable of replicating in a host cell and into which CDNA can be inserted. [0036]
Experiments with cultured human VNO neuroepithelial cells show that pertussis toxin (PTX) blocks the electrophysiological response to a vomeropherin in vitro (FIG. 1). PTX uncouples receptors from their heterotrimeric G proteins and thereby blocks signal transduction. Sensitivity to PTX is an accepted marker for pathways involving G protein-coupled receptors that decrease intracellular cAMP, regulate ion channels or activate phospholipases (i.e., couple to G[0037] _ior G₀). (For a review, see Simon et al., (1991) Science 252:802-808.) We have also implicated a specific type of ion channel in the response of human VNO cells to vomeropherins. These data are entirely consistent with those obtained by Krieger et al. (1999), and thus we provide the first link between a functional G protein-coupled receptor(s) and signal transduction in human VNO cells. However, as noted above, cultured VNO cells are of limited value as a screening tool due to the need to continually isolate new cells. Thus, construction of a cDNA library was desired in order to clone and express pheromone receptors in a cell line.
We constructed a cDNA library of the mRNAs expressed in human VNO tissue and screened it for clones of G protein-coupled receptors with homology to the rat V1R and V2R receptor families and to other G protein-coupled receptor families. Human VNO tissue specimens were collected for this purpose by a team of surgeons. Human VNO RNA is essential for cDNA library construction because: (i) the receptors are species-specific, (ii) the receptors are expressed exclusively in the VNO, and (iii) human genomic DNA contains receptor pseudogenes and introns. [0038]
A cDNA library was prepared from the normal human female VNO. In brief, RNA was extracted from pooled VNO specimens and reverse transcribed with SUPERSCRIPT II reverse transcriptase (Life Technologies) to make first-strand cDNA using a Not I-oligo(dT)[0039] _12-18primer. E. coli DNA polymerase and RNase H were used for second-strand synthesis. Sal I adapters were ligated to the ends and the double-stranded cDNA was digested with Sal I and Not I. The cDNA was directionally ligated into pCMV-Sport7.neo (Life Technologies) and transformed into E. coli.
Certain vomeropherins elicit sexually dimorphic responses and some of the receptors are expressed dimorphically. In consideration of these observations, we constructed our first VNO cDNA library with tissue obtained exclusively from human females. Although others have successfully prepared cDNA libraries from individual rodent VNO neuroepithelial cells, we used whole VNO tissue pooled from a number of donors in order to maximize the number, size, and diversity of receptor clones in our library. [0040]
The library provides an excellent source to search for novel genes, gene fragments, or other nucleotide sequences encoding proteins that are implicated in detection of pheromones or other vomeropherins in the human VNO. Plasmid vectors are currently available that can accommodate the directional cloning of cDNA such that T7 and SP6 RNA polymerase promoter sequences can be used to generate sense and antisense transcripts for subtractive hybridization and riboprobe synthesis. [0041]
Thus, the present invention provides a method of identifying a gene or gene fragment contained within a library of the invention. This method involves the synthesis of at least one unique polynucleotide or oligonucleotide probe sequence comprising a sequence at least partially homologous to a DNA sequence within a selected gene or gene fragment, and of a size to stably hybridize to that gene or fragment thereof. The polynucleotide or oligonucleotide probes may be cRNA, genomic DNA, synthetic DNA, cDNA and the like. [0042]
For example, cRNA molecules transcribed from appropriate sequences are useful as hybridization probes in a method for determining the presence or concentration of an oligo- or polynucleotide, e.g. DNA, of interest. Suitable cRNA molecules may be obtained by preparing an RNA molecule complementary to the oligo- or polynucleotide of interest by methods known in the art. According to one method of this invention a labeled cRNA molecule or derivative thereof is contacted with the inventive cDNA library under suitable conditions and for a sufficient period of time permitting complementary nucleotide segments to hybridize. The cRNA molecule or fragment thereof contains a nucleotide segment complementary to the oligo- or polynucleotide of interest. The presence or intensity of radioactivity in hybridized nucleotide segments is then determined and correlated with the presence or concentration of the oligo- or polynucleotide of interest. [0043]
Thus, the oligo- or polynucleotide probe is labeled and hybridized to the library of the invention. This label permits the identification of the gene or gene fragment. For example, a probe may be used to identify a nucleotide sequence that encodes a protein related to a VNO receptor. [0044]
Any polynucleotide sequence used as a probe and capable of hybridizing to the human VNO libraries of the invention under stringent hybridization conditions (see, Sambrook et al, Molecular Cloning (A Laboratory Manual), 2d edit., Cold Spring Harbor Laboratory (1989), pages 387 to 389) to the DNA sequences of the invention is also covered by this invention. An example of one such stringent hybridization condition is hybridization at 5×SSC at 65° C., followed by a washing in 0.1×SSC at 65° C. for an hour. Alternatively, another stringent hybridization is in 50% formamide, 5×SSC at 42° C. [0045]
DNA sequences that hybridize to the sequences of the invention under less stringent hybridization conditions are also encompassed within this invention. Examples of such low-stringency hybridization conditions are 5×SSC at 50° C. or hybridization with 30-40% formamide, 5×SSC at 42° C. [0046]
Degenerate primers for known VNO receptors or other family of receptors can be used for the identification and amplification of cDNA's to be analyzed. The technique is carried out through many cycles (usually 20-50) of melting the template at high temperature, allowing the primers to anneal to complementary sequences within the template and then replicating the template with DNA polymerase. PCR can be used to amplify both double and single stranded DNA. The template is mixed with specific or degenerate primers, dNTPs, polymerase buffer including MgCl[0047] ₂and thermostable DNA polymerase. The template is denatured at high temperature (e.g. 95° C.) and then cooled to a temperature that will allow optimal primer binding. The reaction temperature is then raised to that optimal for the DNA polymerase (e.g., 72° C.) whereby the primers are extended along the template. This series of steps leads to an exponential amplification of the target template.
Screening techniques other than PCR or hybridization are well known to those of skill in the art and the selection of the techniques does not limit the present invention. The procedures for isolating and identifying gene fragments are well known to those of skill in the art; see, e.g. T. Maniatis et al, Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982). [0048]
Once identified and sequenced, the nucleotide fragments of the genes of the invention may be readily synthesized by conventional means, e.g. Merrifield synthesis Merrifield, J.A.C.S., 85:2149-2154 (1963). Alternatively, the DNA may be produced by recombinant methods, then sequenced. Cloning procedures are conventional and are described by T. Maniatis et al, Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982). [0049]
Further, hybridization or PCR methods can be performed using known probes in order to determine whether or not a selected gene is expressed in a gender specific manner by one or more of the libraries of the invention. Genes for which the library is likely to be probed include, but not limited to, for example, pheromone receptors. [0050]
As described in the examples below, to date, the results obtained by probing these libraries with neuron and/or neuroepithelial specific probes indicates that the constructed human female library is derived from VNO-specific tissue without olfactory tissue contamination. [0051]
Cell lines that stably express a VNO gene may be engineered. The inventive VNO receptor gene sequence may be inserted into an expression plasmid comprising a selection marker and suitable regulatory elements, and transfected into a competent host cell. Following the introduction of the plasmid by methods known in the art (for example, calcium phosphate precipitation, electroporation and the like), engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the novel plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines that express the desired VNO gene product on the cell surface, and are particularly useful in screening candidate drugs. For example, these cell lines are used to develop automated high throughput screening assays for novel compounds with therapeutic utility in the treatment of psychiatric and endocrine disorders and diseases such as, but not limited to: premenstrual syndrome (PMS), anxiety and phobias, sleep disorders, appetite control, fertility, and hypothalamic-pituitary disorders. [0052]
The library of the present invention has been deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110, U.S.A. for patent purposes. The ATCC accession number of this library is as follows. ATCC #PTA-1213, and was deposited on Jan. 20, 2000. [0053]

EXAMPLES

The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof. [0054]

Example 1

Tissue Collection

Human VNO tissue specimens were collected for this purpose by a team of surgeons. The human VNO is located bilaterally in the nostrils, and has been associated, inter alia, with pheromone reception. The VNO is a small nasal organ with a central lumen and a pit opening to the nasal cavity. The VNO is a bilateral structure located supra palatial. The pit is approximately 1 to 1.5 mm in diameter and the lumen is approximately 1 to 1.5 cm deep. The lumen is lined with sensory neuroepithelia which constitute a distinct locus of pheromone receptors. [0055]
Collaborating otolaryngologists rinsed the human VNO specimens in sterile phosphate-buffered saline (PBS) immediately after resection to remove blood and other fluids. They rapidly excised extraneous tissue and snap-froze the VNO in liquid nitrogen. The frozen specimens were shipped on dry ice to the laboratory for RNA extraction. Thus, authentic VNO tissue specimens were collected under conditions that sought to minimize potential degradation of the RNA. [0056]

Example 2

Isolation of a mRNA

Total cellular RNA was extracted from the VNO specimens using Trizol (Life Technologies). This procedure is rapid, and minimizes RNA degradation. However, any method for RNA isolation may be used. [0057]
Tissue samples were homogenized in Gibco BRL Trizol Reagent using a glass-Teflon or power homogenizer. After incubation of the homogenized samples for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes, 0.2-ml chloroform was added per 1 ml Trizol Reagent. The samples were mixed vigorously and then centrifuged at 12,000×g for 15 minutes at 4° C. Centrifugation separated the biphasic mixtures into the lower red, phenol-chloroform phase and the upper colorless, aqueous phase. [0058]
The RNA was precipitated from the aqueous phase by mixing with 0.5 ml of isopropanol (for each initial milliliter of Trizol Reagent). The samples were incubated at room temperature for 10 minutes and centrifuged at 12,000×g for 10 minutes at 4° C. The supernatant was removed and the RNA pellet was washed once with 70% ethanol. The pellet was air dried and dissolved in diethyl pyrocarbonate (DEPC)-treated water. The RNA was quantitated by A[0059] ₂₆₀measurement.

Example 3

cDNA Synthesis

First-strand CDNA was prepared using SUPERSCRIPT II (RNase H[0060] ⁻) Reverse Transcriptase (Life Technologies) which had been optimized for maximum yield of long cDNA products. The reaction was primed with a Not I-oligo(dT)_12-18adapter-primer (Life Technologies) under conditions specified by the supplier. cDNA synthesis was primed by the oligo(dT)_12-18at the 3′-poly(A) end of the mRNA; the adapter adds a Not I restriction site to the 5′-end of the first-strand cDNA. The reaction was incubated at 45° C. to melt potential secondary structures in the template mRNA. The length of first-strand cDNA that was synthesized in small pilot reaction mixtures containing [α−³²P]dCTP was determined, relative to known DNA standards, by alkaline agarose gel electrophoresis and autoradiography to test the quality and performance of the materials and conditions.
Second-strand synthesis was catalyzed by [0061] E. coli DNA polymerase I in combination with RNase H and E. coli DNA ligase at 16° C. In this procedure, RNase H introduces nicks into the RNA of the mRNA:cDNA hybrids and DNA polymerase I synthesizes second-strands by nick-translation; the low temperature reduces spurious synthesis by DNA polymerase I which has a tendency to strand-displace (rather than nick-translate) at higher temperatures. DNA ligase repairs nicks in the second-strands and improves the yield of long cDNAs. In the final step, T4 DNA polymerase fills in and blunts the ends of the double-stranded cDNA. The double-stranded cDNA was then deproteinized by organic extraction and precipitated with ethanol.
An excess of the commercially available Sal I adapter was ligated to the blunt ends of the double-stranded cDNA from the Not-oligo(dT)-primed reaction. Subsequent digestion with Not I removed the Sal I adapter from one end yielding molecules with a Sal I and a Not I end suitable for directional cloning into a vector that has been double-cut with these two enzymes. The recognition sites for Not I and Sal I are extremely rare in human DNA and thus the double-stranded cDNAs should be cut internally by these enzymes only very infrequently, if at all. [0062]
Unligated adapters, low molecular-weight cDNA (<500 base pairs), deoxynucleoside triphosphates, etc. were subsequently removed by chromatography on Sephacryl® S-500 HR prior to ligation into the vector. The >500-bp cDNA was ligated into pCMV-Sport 7.neo (Life Technologies) although any of a number of suitable vectors could be used. This vector has been developed at Life Technologies for cloning SUPERSCRIPT cDNA libraries. Among its features are a selectable marker gene for bacteria (β-lactamase), T7 and SP6 promoters flanking the multiple cloning site for synthesis of single-stranded sense and anti-sense cRNAs, a cytomegalovirus (CMV) promoter and SV40 polyadenylation signal for eukaryotic expression of directionally cloned inserts, and a selectable marker gene for eukaryotic cells (neo[0063] ^r).
The double-stranded cDNA from the Not-oligo(dT)-primed reaction with Sal I and Not I ends was directionally cloned into pCMV-Sport 7 that had been cut with these two enzymes. After ligation to the vector, the DNA was transformed into a highly competent strain of [0064] E. coli such as DH10B (Life Technologies). Recombinants were selected on LB agar plates for resistance to ampicillin. The library was amplified as described in Example 4 and plates prepared for colony hybridization.

Example 4

Amplification of Primary Library

The primary library was amplified once under semi-solid conditions. Semi-solid amplification of primary cDNA transformants minimizes representational biases that can occur during the expansion of plasmid cDNA libraries. [0065]
Media Preparation [0066]
2×LB: 20 g Tryptone, 10 g Yeast Extract, 10 g NaCl in 1,000 mls H[0067] ₂O.
2×LB Glycerol (12.5%): 175 ml 2×LB, 25 ml Glycerol (100%). Filter sterilize and store for up to two months at room temperature. [0068]
Prepare 2 liters of 2×LB. Remove 200 mls of the 2×LB to make the 2×LB Glycerol. Place a large stir bar and 1.35 g SeaPrep (FMC) agarose into each of four 500-ml autoclavable bottles. Place bottles on stir plates. With the stir plate turned on, add 450 ml of 2×LB to each bottle, avoiding the formation of large clumps of agarose. Autoclave these bottles of 2×LB agarose for 30 min. Cool bottles in 37° C. water bath for approximately 2 hours until media reaches 37° C. After the media reaches 37° C., add Carbenicillin to 50 μg/ml (preferred antibiotic) or Ampicillin 200 μg/ml. Mix on stir plate. [0069]
Amplification [0070]
Briefly, 4×10[0071] ⁵to 6×10⁵primary cDNA transformants (colonies from original library) were added to each of the autoclaved bottles of 2×LB agarose and mixed thoroughly on a stir plate for 2 minutes. The caps were tightened and the bottles placed in an ice water bath (0° C.) such that the level of water in the bath is at the same level as the upper level of media in the bottle. The bottles were incubated for 1 hour in the ice bath. The bottles were gently removed from the ice bath and the excess water wiped off the outside of the bottles. The bottle caps were loosened and the bottles placed in a gravity flow incubator set at 30° C. The bottles were incubated for 40-60 hrs without disturbance.
Cell Harvest [0072]
The contents of the bottles were poured into GSA bottles and centrifuged at 8,000 rpm for 20 minutes at room temperature (Caution: Make sure that the rotor was set at room temperature for at least two hours before adding the GSA bottles. Rotors at 4° C. will cause solidification of agar.) The supernatant was decanted off and the cells resuspended in a total volume of 100 ml 2×LB Glycerol (12.5%). Two 100 μl aliquots were removed for plating, further analysis, and colony estimate. Cells were filtered through sterile cheesecloth to remove agarose clumps if present. [0073]
Cell Storage [0074]
The cells were subdivided into small aliquots (Note: It is useful to make a number of 1 ml and 100 μl aliquots.) and stored at −70° C. Frozen cells can then be used to prepare DNA for experiments or can be further amplified in liquid at 30° C. to obtain DNA. Use 2.5×10[0075] ⁹cells per 100-ml growth medium for further expansion of library.
Amplified Library [0076]
The amplified library contains ˜3.5×10[0077] ¹¹colony-forming units (CFU) representing ˜1×10⁷primary transformants. Inserts range from ≧300 to ≧3000 base pairs (bp) in length, with an average insert size of ˜1500 bp. For comparison, mRNAs in the rat V1R receptor family contain, on average, ˜915 bases in the open reading frame (ORF) and ˜230 bases in the 3′-untranslated region (UTR) (Dulac and Axel, 1995). Therefore, the inventive CDNA library will be a source of suitably sized clones for identification and characterization of numerous genes and gene fragments. We also point out that full-length cDNAs containing the precise 5′ end of the mRNA sequence, though scientifically interesting, are not essential provided that we obtain the entire full-length ORF (see below).

Example 5

Probes

(i) We designed PCR primer pairs based on the published sequences of the rodent VNO receptors using readily available software packages such as Oligo™ or Primers. Biosource (Foster City, Calif.) synthesized the primers on a standard “fee-for-service” basis. The primers flanked the region encoding transmembrane domains II through VI of the rat receptor sequence that does not appear to contain introns. A separate PCR reaction was set up for each primer pair and the target region amplified from commercially available rat genomic DNA (Clontech). The products were analyzed by agarose gel electrophoresis to assess size and purity. If necessary, products of the predicted size were gel-purified to remove any spurious species. The PCR amplicons were analyzed by restriction enzyme mapping and/or sequenced on a “fee-for-service” basis by ACGT, Inc. (Northbrook, Ill.); they were cloned into a suitable vector such as pGEM-T-Easy (Promega). This procedure was also used for human “VN6”, a sequence from GenBank, probably a testes cDNA, and for HG25X, a human VNO receptor pseudogene. [0078]
Each probe was labeled to high specific activity by including [α[0079] ³²P]dCTP in the RediPrime (Amersham) random-priming reaction. The specificity and identity of the labeled rodent PCR products was confirmed by Southern blotting, at low (55°) or high (68° C.) stringency, to restriction enzyme-digested rat genomic DNA and compared to the published hybridization pattern(s) for that clone. These PCR products were also separately hybridized to blots of human genomic DNA (Clontech) at low or high stringency to ensure that they successfully cross-hybridize to human sequences under the conditions used. The human PCR amplicons were tested by separately hybridizing each to blots of human genomic DNA at low or high stringency prior to use in screening the library.
(ii) We used short oligonucleotide probes based on regions generally conserved in G protein-coupled receptors to screen the library (Kel et al., 1998). We screened the library by colony hybridization using a mixture of 15 short oligonucleotides that should detect conserved sequences in most, if not all, G-protein coupled receptors. This varies from standard colony hybridization because the probes are very short, i.e., 8 nucleotides, and do not represent any specific mRNA sequence. The probes were labeled at the 5′ end with [[0080] ³²P]-ATP and hybridized at 4° C. followed by washing at 10° C. Clones PP40 and PP41 were isolated from this screen.
(iii) We designed degenerate PCR primers for the V2R family based on Cao et al. (1998) and for olfactory and taste receptors. The pairs of degenerate oligonucleotide primers based on conserved regions of the known receptors (e.g., within the first and third intracellular loops of the V2R family). These oligos were used to prime PCR on the amplified library to screen G[0081] ₀-coupled receptors. The resulting amplicons were sequenced to identify receptor fragments and then used to screen the VNO cDNA library.

Example 6

Characterization of Amplified Library

The library was screened for the presence of cloned cDNAs representing proteins whose expression in the human VNO has been determined by immunohistochemistry. Oligonucleotide primer pairs were designed based on the GenBank mRNA sequence for each of the proteins and were used to direct PCR with ˜10[0082] ⁷CFU from the amplified library as the template. When a unique band of the predicted size was detected by ethidium bromide staining of an agarose gel (Table 1) the results were scored positive. The PCR products can be restriction mapped and/or sequenced, if necessary, to confirm their identity. In each case, a parallel reaction containing the primer pair alone, in the absence of template, did not yield any significant PCR products.

TABLE 1

Protein Immuno PCR^a

Neuron-specific enolase + +

Protein gene product 9.5 + +

Olfactory marker protein − −

Synaptophysin + +
The data in Table 1 show that the library contains cDNA for proteins identified immunohistochemically in sections of intact human VNO. Thus, the inventive library displays characteristics consistent with those seen in the intact tissue. [0083]

Example 7

Protein Identification in Human VNO CDNA Library

As noted above, in vitro data (FIG. 1) indicate that cells isolated from the VNO of human volunteers respond electrophysiologically to a vomeropherin via a PTX-sensitive pathway, a hallmark of G protein-coupled receptor signaling. Thus, we anticipated that components of the pathway such as G proteins (e.g., G[0084] _iand G₀), adenylyl cyclase (e.g., type 3 and 7), and various ion channels are expressed in these cells. We assayed for expression of these proteins in the VNO by screening our library for cDNA clones of the corresponding mRNAs. Knowledge of the signaling components expressed in VNO neurons is essential to express the receptors functionally in tissue culture cells for high throughput drug screening assays.
The library was screened by PCR using primers for various known mRNAs to assess the signal transduction mechanism of the activated VNO receptor. The primers used for screening were generated from known sequences for either the human or rodent mRNA. The PCR primer pairs can be specific for individual mRNAs, such as G[0085] _ior G₀, or degenerate to allow simultaneous amplification of related sequences in the same family. Clones of amplicons obtained with a unique primer pair were sequenced directly. Clones of amplicons obtained with degenerate primers were distinguished by restriction mapping and representatives sequenced. BLAST analysis was used against GenBank to identify the sequences that were obtained and thereby learn about signal transduction mechanisms in the VNO.
The results of the screening are shown in Table 2. The cDNA library was positive for adenylyl cyclase type 2, 3 and 7, Gα1, 2 and 3-proteins, and Golf. These results show the presence of the Golf protein although this G-protein is thought to be uniquely associated with olfactory tissue and is not detected in rodent VNO. However, OMP is not detected in the inventive cDNA library. Thus, the Golf did not arise from contaminating olfactory tissue and may couple to novel receptors in the human VNO. Also of interest is the failure to detect Gα[0086] ₀. Based on work on the rodent receptors, the Gα₀was expected to be present if there is a V2R human homolog. The lack of detection of the Gα₀may indicate that the human V2R homolog utilize a G-protein other than Gα₀. Other explanations also exist.

Example 8

Screening for Receptor cDNA

We separately screened the cDNA library for clones that hybridize to the V1R probes. Pools of [0087] ³²P-labeled probes were hybridized at low stringency to nylon membranes containing ˜3×10⁴colonies. The filters were successively washed at low stringency, autoradiographed, washed at high stringency, and autoradiographed. Clones that were positive after each round of washing were identified, plated to yield single colonies, and retested to eliminate false-positives and to ensure purity.
The size of the insert in positive clones was determined after release from the vector by restriction enzyme digestion with Not I and Sal I. We initially selected the longest positive clones for further analyses. If the clones were deemed too short to contain a full-length open reading frame (ORF) (based on comparison to the rodent cDNAs), we can use one of several approaches to obtain the complete cDNA: As noted above, the coding regions of the rodent V1R VNO receptors do not contain introns. Therefore, it is possible to screen a commercially available human genomic library at high stringency using a probe derived from the 5′ end of a human receptor cDNA. We can identify overlapping genomic clones that extend the sequence upstream toward the 5′ end, and subsequently assemble plasmids containing the full-length ORF. [0088]
Alternatively, we can use one of various published methods of 5′-RACE to extend the cDNA clones toward the 5′ end. We do not need clones containing the precise 5′ end of the mRNA sequence to express the receptors, provided that we obtain the full-length ORF. [0089]
Alternatively, a randomly primed human VNO cDNA library is prepared. Mixed hexamers randomly primed first-strand cDNA synthesis along the poly(A)[0090] ⁺ human VNO mRNA; the reactions are incubated at 45° C. to melt potential secondary structures in the template mRNA. Second strands are synthesized using E. coli DNA polymerase I in combination with RNase H and DNA ligase as was done for the oligo(dT)-primed VNO cDNA library. In the final step, T4 DNA polymerase fills in and blunts the ends of the randomly primed double-stranded cDNA. The cDNA is ligated to an excess of commercially available Eco RI (Not, Sal) adapter. The adapter contains the recognition sites for Not I and Sal I to facilitate subsequent excision of the insert from the vector. (These enzymes will cut the cDNA inserts only infrequently, if at all.) The randomly primed double-stranded cDNA is non-directionally cloned into a suitable vector that has been linearized with Eco RI and treated with phosphatase. The ligated DNA is transformed into competent E. coli DH10B. The randomly primed library is screened at high stringency using a probe derived from the 5′ end of individual human receptor cDNAs to identify overlapping fragments that can be assembled into a full-length cDNA clone.

Example 9

cDNA Clones isolated from the Human VNO cDNA Library

The cDNA library was screened using probes based on published rat VNO receptors, human homolog of rat VN6 and human HG25X pseudogene sequences. See SEQ. ID Nos 7-15. Probes were hybridized with clones under low stringency conditions to maximize the number of possible candidates for the human VNO receptor. Table 3 summarizes a partial listing of the clones sequenced, their putative homolog based on known gene sequences from GenBank, and the homology between the isolated sequence and the homolog, i.e., known GenBank sequence. At least six novel sequences were identified. See SEQ ID No. 1-6, and 16-20.

TABLE 2


			Hu VNO
	Rodent	Rodent	cDNA	Hu VNO
PROTEIN	OE	VNO	library	Method

ADENYLYL		+	+	PCR/sequence
CYCLASE TYPE 2				(PP23)
ADENYLYL	+	+	+	PCR/sequence
CYCLASE TYPE 3		non-neural		(PP24)
		cells
ADENYLYL		+	+	PCR/sequence
CYCLASE TYPE 7				(PP39a)
Gα11		+	ND
Gα13		+	—	PCR
Gα14		+	—	PCR
Gαi1		—	+	PCR/sequence
				(PP18; PP20)
Gαi2	+	+	+	PCR/sequence
				(PP14a)
Gαi3		—	+	PCR/sequence
				(PP16a; PP17a)
Gαo	+	+	—	PCR
Gαq		+	ND
Gαs		+	ND
Golf	+	—	+	PCR/sequence
				(PP15a; PP15b)
Neuron-specific			+	PCR
enolase
OMP	+	+	—	PCR
PGP9.5			+	PCR
Synapotphysin			+	PCR
Trp2		+	—	PCR
Trp homologs			—	PCR

TABLE 3


Clone	Homolog	Function of homolog	Comments

PP21	human cDNA	human selenium	bp 54-897
	NM003944	binding protein	95% identical to
	(1428 bp)	mouse acetaminophen	587-1424
	mouse cDNA	binding protein	of human homolog
	AI573970
PP22	human fetal kidney	similar to RING Zn	bp 370-667
	HSM800147	finger proteins	98% identical to
	(1199 bp)		1-298 of homolog
PP26	human brain cDNA	related to	bp 13-643
	AB011108	serine/threonine	97% identical to
	(6680 bp)	protein kinases	3476-4102
			of homolog
PP27			SEQ ID No. 18
PP28			SEQ ID No. 19
PP29	human Ciz1 mRNA		bp 282-593
	AB030835		99% identical to
	(5936 bp)		249-560 of
			homolog. bp 1-281
			not in homolog.
			Unspliced?
			splice variant?
			SEQ ID No. 20
PP30	human erg2	transcription factor;	˜600 bp sequenced;
	M17254	protooncogene	identical
	(3166 bp)
PP31	human erg2	transcription factor;	˜550 bp sequenced;
	M17254	protooncogene	identical
	(3166 bp)
PP32			NOVEL;
			˜600 bp @ 5′;
			˜500 bp @ 3′;
			SEQ ID Nos.
			1 and 2
PP33			NOVEL;
			˜600 bp @ 5′;
			SEQ ID No. 3
PP34	human melanoma	cellular adhesion	˜600 bp sequenced;
	adhesion molecule		identical
	NM006500
	(MCAM) (3583 bp)
PP35	human umbilical		partial match:
	vein endothelial cell		145 bp match of
	EST AA296414		˜1100 sequenced;
	(270 bp)		SEQ ID No. 4 and 5
PP36			NOVEL;
			˜1000 bp @ 5′
			sequenced
PP38			NOVEL;
			˜500 bp @ 5′
			sequenced;
			SEQ ID No. 6
PP40	human PAC clonc	genomic DNA clone	NOVEL cDNA;
	RP5-1093o17		˜660 bp @ 5′
	(160687 bp)		end sequenced;
			bp 17-474
			98% identical
			SEQ ID No. 16
PP41	human ubiquitin-	ubiquitin-conjugating	NOVEL cDNA;
	conjugating enzyme;	enzyme	˜550 bp sequenced;
	AF085362.1		bp 296-431
	(1294 bp)		85% identical to
			be 276-410 of
			homolog;
			SEQ ID No. 17

Example 10

Sequencing

Single-stranded sequencing of selected (full-length) clones was done by standard methods. Oligonucleotides that are complementary to the T7 and SP6 promoters in the pCMV-Sport7.neo vector were used to prime sequencing reactions from each end of a cloned insert. Internal primers, based on newly acquired sequence data, were synthesized, as necessary, to sequence overlapping internal regions of the cloned cDNAs. [0093]
We examined the assembled sequences by computer for the presence of a potential full-length open reading frame. Clones containing an in-frame internal termination codon were excluded because they likely represent expressed pseudogenes. We used standard BLAST analysis to compare the human VNO clones to each other and to sequences in GenBank. Based on cross-hybridization to rodent VNO receptor cDNAs (used to screen the library) and our proprietary PTX data, the human VNO clones show homology to the superfamily of G protein-coupled receptors and have seven predicted transmembrane domains. By virtue of the selection method, they also fall into subfamilies with homology to either the rodent V1R or V2R family of receptors. Analysis of the lengths of the extracellular N-terminal domains determine if the differences between the rodent V1R and V2R families are conserved in humans. [0094]

Example 11

In situ Hybridization

Confirmation that the cloned receptors and components of the signal transduction cascade (identified by PCR) are expressed in the neuronal cells of the human VNO is by in situ hybridization as described in detail by Schaeren-Wiemers and Gerfin-Moser (1993). This approach also provides important information about the number and distribution of cells expressing these genes in the VNO. Human VNO tissue is fixed with Tissue-Tek embedding medium (Miles) immediately after surgical resection and frozen at −40° C. in 2-methyl-butane. Sections (15 μm) are cut on a cryostat, mounted on polylysine-coated slides, and processed as described (Schaeren-Wiemers and Gerfin-Moser, 1993). [0095]
Digoxigenin (DIG)-labeled sense and anti-sense cRNA probes are transcribed from the linearized pCMV-Sport7.neo cDNA clones in vitro using SP6 and T7 RNA polymerase, respectively, in the presence of DIG-11-UTP (Roche Molecular); cRNA probes transcribed from the 3′-untranslated region should offer the highest degree of specificity (Ryba and Tirindelli, 1997). We will determine the size of the DIG-labeled cRNA probes and confirm their detection prior to use for in situ hybridization as follows: The CRNA is electrophoresed in a 1% agarose gel containing formaldehyde and ethidium bromide. The 18S and 28S ribosomal RNAs present in the unbound fraction from the oligo(dT)-cellulose column (see above) can be run in a parallel lane as size standards and visualized by UV transillumination. [0096]
The gel is blotted overnight onto a nylon membrane (Zeta Probe membrane; BioRad) in 10×SSC, pH 7.0, rinsed in 2×SSC and fixed by UV cross-linking. After blocking non-specific sites on the membrane with Blocking Reagent (Roche Molecular), the transferred DIG-cRNA is bound to sheep anti-DIG Fab antibody fragments coupled to alkaline phosphatase (Roche Molecular), and detected by color reaction using 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indole-phosphate. The size of the cRNA transcripts will subsequently be reduced to −200 bp by limited alkaline hydrolysis prior to in situ hybridization as recommended by Schaeren-Wiemers and Gerfin-Moser (1993); the size reduction can be confirmed by gel electrophoresis and blotting as described above. [0097]
VNO tissue sections on slides are prehybridized in a buffer containing yeast RNA and herring sperm DNA (Roche Molecular) at room temperature for at least 6 hr. The buffer is replaced with a hybridization buffer containing DIG-labeled probe and hybridized overnight at 72° C. (Schaeren-Wiemers and Gerfin-Moser, 1993). Hybridized DIG-labeled probe is detected with anti-DIG antibodies coupled to alkaline phosphatase (Roche Molecular) and color reagent. The sections are counterstained with Hoechst 33258, which stains nuclei, and examined by light microscopy. [0098]
Each anti-sense cRNA receptor probe hybridizes specifically to a small number of neuroepithelial cells distributed through the human VNO section. In contrast, the corresponding sense cRNA probe yields no distinct signal when hybridized in parallel to an adjacent serial section, thus ruling out non-specific hybridization to RNA or hybridization to genomic DNA. Probes for components of the signal transduction cascade will vary in the number of cells to which they hybridize. For example, anti-sense probes for specific G proteins (e.g., G[0099] _i) that are detected in the cDNA library hybridize to a subset of neurons in the tissue section, whereas anti-sense probes for adenylyl cyclase(s) and ion channels hybridize to many or all neurons. These results confirm the expression of the cloned sequences in the VNO, identify the cell type(s) expressing these proteins, and provide insights into gene expression and signal transduction in this tissue.

Example 12

Tissue Specificity

The tissue-specificity of the cloned receptor cDNAs is assessed by northern blot hybridization. Commercially prepared multiple tissue northern blot membranes containing mRNA isolated from a spectrum of human tissues (Clontech) are hybridized at high stringency (42° C.; 50% formamide) to one or a mixture of [0100] ³²P-labeled VNO receptor probes. The probes are prepared by random-priming (RediPrime) the human cDNAs in the presence of [α−³²P]dCTP. It is essential to include a hybridization control in these experiments. The rodent VNO receptor probes do not hybridize at high stringency to mRNA isolated from other tissues (Matsunami and Buck, 1997), and the commercially available human multiple tissue northern blots do not contain VNO mRNA. We, therefore, include a ³²P-labeled probe for a common housekeeping mRNA such as human GAPDH in each hybridization. This control confirms that the conditions are adequate to detect hybridization and simultaneously verifies the quality and relative quantity of mRNA in each lane of the blot.
Within the limits of sensitivity, the multiple tissue northern blots define the profile of receptor expression in the tissues tested. Higher sensitivity can be obtained by RT-PCR, but this procedure requires sufficient sequence information on every clone to design specific primers, and template mRNA from many different human tissues. [0101]
Conclusion [0102]
We detect cloned cDNAs in our library by PCR for the 3 proteins that are detected by immunohistochemical staining of human VNO tissue sections. We do not detect OMP cDNA in the library and Takami et al. (1993) do not detect the protein in human VNO tissue sections by immunohistochemical staining, even though it is present in the rodent VNO. We obtain negative PCR results for OMP using several independent samplings of the library, whereas we always obtain a product of the predicted size when human genomic DNA (Clontech) is used as template with these primers in a parallel reaction. Because the OMP mRNA contains a very long 3′-UTR, we have also tested a second primer pair, designed to amplify a region adjacent to its 3′-poly(A) tail. This primer pair also does not amplify OMP cDNA sequences from the library but, nonetheless, amplifies a region of the predicted size using human genomic DNA as the template in a parallel reaction. The apparent absence of both detectable OMP protein and cDNA makes it unlikely that this is simply a failure to clone the mRNA. [0103]
We draw these conclusions: (i) our library contains cloned cDNAs for proteins expressed in neuronal/neuroendocrine cells; (ii) absence of OMP cDNA implies that the neuronal/neuroendocrine cDNAs are not derived from olfactory neurons which abundantly express this protein; (iii) the agreement between the PCR and immunohistochemistry suggests that the library reflects gene expression in the human VNO; (iv) the absence of detectable OMP cDNA and protein likely represents a real species difference between humans and rodents. [0104]
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. [0105]
1 20 1 651 DNA Homo sapiens 1 gtcgacccac gcgtccgaag tgagaccctg tcttgaaaaa aaaaaaaatt aaccaatatg 60 attaataata atggcagcat caagagcctg tacttcctat gtgtttccat gtgtgtaaat 120 gctctgtcac accgtctcat ttcacctcat ttccccataa agaacattct attaactggg 180 gttcagagag taacttgttc tgtcgctcac ccaagatcgc cgtgtggttc ccgagtgtaa 240 ggtgtgaagc caagtctctg tggccctggg gcccgagccc tcaactgccc tgctagggtc 300 caagctgacc actgcagggc cttagtctgg aggaacggct tgactccgga catctgcagg 360 agtgtttgct gtgttgagtt gagcccctct gccagacgtg tcaaaacaaa tgcttttgtg 420 tgtttactgc ctcacacgct cagccagaag ctcctgtttt atcatctagt ttagattgag 480 gggaagaggc ttcatcagta aggacctgtc tcactcttca tcccacggcc ctgggccatg 540 ccctgttagc ttcaagaagc agttatcctc agggtggtcc tgctcaggct gccccacccc 600 atcctgtgtc tgcgccagat atgtagattg atttcagtcg ctttatgcta a 651 2 469 DNA Homo sapiens 2 tttttttttt tttttaaatg gagtttcgct tttgttgccc aggctggagt gcaatggtgt 60 gatctcggct caccacaacc tccgccttac actgtttatc acgagggaga caagtggaga 120 accttggaaa tgtaaaggaa gatgagccca cgcctttcaa agagaaagag ccggagcagg 180 gaaaccctga tcgtggctaa ttggcccatc agggtcctgc cctggacaga cctaggtgag 240 ggcgtcttta aagaaaacgt cccacctccg cttgccacag agatttctaa ggtttgccca 300 ctgtcctttt gtaagtgcct gctgggtaag tgtggagata agatgagtat tacattatga 360 tgcttcctca tgcatgaaac tctgttttaa agagagtctg gagggggcca tcaggaaggg 420 agagcctgac cagtggaggt agaaggaagg ctgctttatt aagagaagt 469 3 604 DNA Homo sapiens 3 gtcgacccac gcgtccgagc ttttatggca gtgccccgtt ggcctactga taagaaaccg 60 tggctgctca ggcggctgct gcacctgctg cttttgccgt ttctttcctg cttgtgtaga 120 taaagccctg cggagctgag ctggtttcac cttcgtcatt acaactttga agccctctgg 180 aggctttaac aacatcttgc cagtcttatc ctagagagga cagctagttc tccttgctag 240 gtgggaaggc tgaagctgaa cttgggaatt ctcatcaggg ctgcccatag gaggtctcat 300 catttccagc agaggaaaga aacttgaaga aagaatggat ttaagtaatt gcctccaggc 360 agtcttctct ctctcctccc tctctttaaa aaaaatcatg ggatcatgta atttttcagc 420 ataaataatg gcaataatgg ttggaggaca aggtaagatt tctggaaatc tggcaactac 480 gcaggtgact caaaagaaaa aataatgacc aagctaatct ttaactccac acctactctt 540 gccttttccc aggcagcttt cctggtttta agagcaaggg ttccccaaac ctgcagtagg 600 tatc 604 4 666 DNA Homo sapiens 4 gtcgacccac gcgtccgggg actcttcaca cagctttcta gtccattccc aggaccactg 60 ggtacctgtc agttggcctg taaggaagtg aagggtgggg acacagaggc atgtgtcacg 120 ttcacttaga tcctcactga ccaaaaggca ggagggtttc cttaggaaac aatgtaaact 180 tgttttctat tggggtataa aatccacctc aggccagtgg ttattctcga tcaagtgggc 240 tccaggaggt ctgtctgtca gtattaagtg aaatgagagc ctcccctcca ggcctggccc 300 ccagtccggc ctcgcacccc tttccctgcc caccctcatg tttttggtct ttggctcatg 360 actgcaccgt ctcaccatgc tcttgtcccc ttccttgcag gatgatgcta tatttgggat 420 ccttaacaaa gtgaagcctt cctataaatc ctgtgccgac tgcatgtacc ctacagccag 480 cggggctctg aggcctccag ggagcgatgt gaggacccca atgctcccgc catctgcacc 540 cagccagcct ttctacccca catcacgtcc tcccctgtgg cccacttggc cagcaggtcc 600 gtgttccgga gaagccagcc tctggcccaa ccaaccttcc cccgttccta ccaccagcag 660 gctcca 666 5 755 DNA Homo sapiens 5 tttttttttt ttttttacaa ctccttaaaa aggtaaaagc catcgcttga gccagggagg 60 ttgatgctgc agtgagtcat gattgcacta ctgtactcca gcctaggtga cagagccagg 120 ccctgtctta aagaggaaaa accattccta gctcacgggg ccacatgcca tagtttgctg 180 acccctgaat tcaacctttc tgcttttctg caagctgcct ctctctcgaa atgttttgac 240 atctagcttg ctgacacctt tgattcaagc ctggcgcagc gggactggct gagctcacct 300 gagtcttaaa ggggccgccc acagagccag gcagcgacta cagatccctc ttatactccc 360 tcactgctgc tgggaagagc tggagggaaa caggaagcag taatctcact gcaggaaggg 420 gcaactgtag acatccggga agcatcccga cagtcccgtt cctttcgggg aagccgctga 480 aatctccttt cccttcccta gatgggccct agtggaccta agcatctggg ctctcagcag 540 gacgatgtgt ctcagaacca ccacctgagc cagacacttg agcaatttca aacctaaaca 600 caatcatgtg tttcagcagc agacactcaa caatgcaggg tgggcccttc cccttgagat 660 ttaaacttca gcattagcaa caactggaaa caacccatac atttttccca ccgggacccc 720 tgtgctggtc aaacccgtta caacacacca ggaca 755 6 445 DNA Homo sapiens 6 agcaggctgg taccggtccg gaattcccgg gatatcgtcg acccacgcgt ccgtattttt 60 atgagtgcag tttacagtcc acaggtatat tctttgtcac ttaactacag caaattcttg 120 atcattctct ttagaaaagt ctcagaaatc atggcacctt gaaaatggaa acatttcatt 180 agtaattttg gatgcaaact gctttcctgt gttcacagaa tgggcagagg tggaaccgtt 240 aacaccactt ccctctttag tgacttccat gccatcacca tcagtgtgac tcaagtaggt 300 tagtgcagca gaaatttcag tgacacttat aataataaaa aaaataaatg gagatcagcc 360 aaatgaaaac aagaaatgac tatgtatttt agctttgccc taggagggga attagccacc 420 atcacttatg tttggtggag actca 445 7 405 DNA Homo sapiens 7 gtccagttat ctacaggtac aggttgatga gaggcctctc catttccacc acctgcctgt 60 tgagtgtcct ccaggccatc aacctcaccc caaggagctc ccgtttggca atgttcagag 120 atcctcacat cagaaaccgc gttgctttct cttgctgtgg gtcttccaca tatccattag 180 tggaagcttc ttagtctcca ctcttccctc caaaaatgtt gcctcaaata gtgttacatt 240 tgtcactcaa tcctgctctg ctgggcccct gagttgcttc cttgggcaga caattttcac 300 actgatgaca tttcaggatg tctccttgca gctcatggcc cccttcagtg gatacatggt 360 gattctcttg tgcaggcata acaggcagtc tcagcatctt catag 405 8 414 DNA Homo sapiens 8 gaagtgagga gcaccagagg actgatcacg gcatagacat tgactacaag actctgaact 60 tgctggttga ttgggccata tgcccacaac attgctgagg agaaggagat gatgaaatcc 120 acccaacaga ggaccacaga gaaacttacc agcagcagta tggtctggat ggcccgtttc 180 tcaggggaag ctcctgggga aggaccattg ctgtgaaggt ggtgggatca cctctgaggc 240 ctgaataaga gagtcaccat gtatgcaatt aagaacagca gtattcctac caggaaagca 300 tccctaagtg ttgtcagaat aagaaacgtg gccctgagga tgaagctcat ggagaaaact 360 gagcagtact tacctatatt cagtacattt gtctggctca cctggaagca gcta 414 9 632 DNA Rat VNO receptor 9 tgcccattgg tctcttgtcc ctaatcaact tacttatgct actgatgacg gcattcatag 60 ccacagacac ttttatttct tggagagggt gggatgacat catatgtaaa tcccttctct 120 acctgtacag aacttttaga ggtctctctc tttgtaccag ctgcctgttg agtgtcctgc 180 aggccatcat cctcagtccc agaagctcct gtttagcaaa gttcaaacat aagccttccc 240 atcacatctc ctgtgccatt ctttctctga gtgtcctcta catgttcatt agcagtcacc 300 tcttagtatc catcattgcc accccaaatt tgaccacgaa tgactttatt catgttactc 360 agtggtgctc tattctaccc atgagttacc tcatgcaaag catgttttct acactgctgg 420 ccatcaggga tgtctttctt attagtctca tggtcctgtc aacatggtac atggtggctc 480 tcttgtgtag gcacaggaaa cagacccggc atcttcaggg taccagcctt tccccaaaag 540 catccccaga acaaagggcc acccgttcca tcctgatgct catgagctta tttgttctga 600 tgtctgtctt tgacagcatt gtctgcagct ca 632 10 628 DNA Rat VNO receptor 10 ctgcccattg gtctcctgtc cctaatcaac ttacttatgc tactgattat ggcatgtata 60 gccacagaca tttttatttc ttgtagacga tgggatgaca tcatatgtaa atcccttctc 120 tacctgtaca gaacttttag aggtctctct ctttctacta cctgcctgtt gagtgtcctt 180 caggccatca tcctcagtcc cagaagctcc tgtttagcaa agtacaaaca taagcctccc 240 catcacatct tctgtgccat gcttttcctg agtgtcctct acatgttcat tagcagtcac 300 ctcttactat ccatcattgc caccccaaat ttgaccacaa atgactttat tcatgttagt 360 cagtcctgct ctattctacc catgagttac ctcatgcaaa gcatgttttc tacactgctg 420 gccatcagga atgtctttct tattagcctc attgtcctct cgacatggta catggtggct 480 ctcttgtgta ggcacaggaa acagacccgg catcttcagg ataccagcct ttcccgaaaa 540 gcatctccag aacaaagggc cacccgttcc atcctgatgc tcaggagctt atttggtctg 600 atgtctatct tcgacagcat tgcctcct 628 11 632 DNA Rat VNO receptor 11 tgcccattgg tctcttgtcc ctaatccacc tactgatgct actgatgggg gcattcatag 60 ccatagacat ttttatttct tggaggggat gggatgacat catatgtaaa ttccttgtct 120 acttgtacag aagttttaga ggtctctctc tttgtaccac ctgcatgttg agtgtcctgc 180 aggccatcac cctcagcccc agaagctcct gtttagcaaa gttcaaacat aagtctcccc 240 atcacgtctc ctgtgccatt atttcgctga gcatcctcta catgttcatt agcagtcacc 300 tcttagtatc catcaatgcc acccccaatt tgaccacgaa caactttatg caagttactc 360 agtcctgcta cattataccc ttgagttacc tcatgcaaag catgttttct acacttctgg 420 ccatcagaga tatctctctt attagtctca tggtcctctc gacttgttac atggaggttc 480 tcttgtgtag gcacaggaat cagatccagc atcttcaagg gaccaacctt tccccaaaag 540 catctccaga acaaagggcc acacagacca tcctgatgct catgaccttc tttgtcctaa 600 tgtccatttt cgacagcatt gtctcctgtt ca 632 12 662 DNA Rat VNO receptor 12 ctacattgca tccttgtccc taacacaact aatgctgctt ataactatgg gactcatagc 60 tgctgacatg tttatttctc aggggatatg ggattctacc tcatgccagt cccttatcta 120 tttgcacagg ctttcgaggg gttttaccct tagtgctgcc tgtctgctga atgtcttttg 180 gatgatcact ctcagttcta aaaaatcctg tttaacaaag tttaaacata actctcccca 240 tcacatctca ggtgcctttc ttctcctctg tgttctctac atgtgtttta gcagtcacct 300 tattttatcg attattgcta cccctaactt gacctcagat aattttatgt atgttactaa 360 gtcctgttca tttctaccca tgtgttactc cagaacaagc atgttttcca caacaattgc 420 tgtcagggaa gcctttttta tcggtctcat ggccctgtcc agtgggtacc tggtggcttt 480 cctctggaga cacaggaagc aggcccagca tcttcacagc accggccttt cttcaaagtc 540 atctccagag caaagggcca ccgagaccat cctgctgctt atgagtttct ttgtggttct 600 ctacattttg gaaaatgttg tcttctactc aaggatgaag ttcaaggatg ggtcaacatt 660 ct 662 13 653 DNA Rat VNO receptor 13 ttgctttctt atccttaacc caactaatgc tgcttataac tattggactt atagctgcag 60 acatgtttat gtctcggggg agatgggatt ctaccacatg ccagtccctt atctatttgg 120 acaggctttt gaggggtttt accctttgtg ctacctgtct gctgaatgtc ctttggacca 180 tcactctcag tcctagaagc tcctgtttaa caacatttaa acataaatct ccccatcaca 240 tctcaggtgc ctttcttttc ttctgtgttc tctatatatc ttttggcagt cacctctttt 300 tatcaacaat tgctaccccc aatttgactt cagataattt tatgtatgtt actaaatcct 360 gttcatttct acccatgagt tactccagaa caagcatgtt ttccacacca atggccatca 420 gggaagccct tcttattggt ctcattggcc tgtccagtgg gtacatggtt gctttcctat 480 ggagacacaa gaatcaggcc cggcatcttc acagcaccag cctttcttca aaagtgtccc 540 cagagcaaag ggccaccagg accatcatga ttctcatgag cttctttgtg gttctctaca 600 ttttggaaaa tgttgtcttc tactctagga tgacattcaa ggatgggtca atg 653 14 628 DNA Rat VNO receptor 14 acctgatcat cagtctcttg gccctcatcc accttgggat gctaacagtc atgggattca 60 gagctgttga tatttttgca tctcagaatg tgtggaatga catcaaatgc aaatcccttg 120 cccacttaca cagacttttg aggggcctct ctctttgtgc tacctgtctg ctgagtatct 180 tccaggccat cacccttagc cccagaagct cctgtttagc aaagttcaaa tataaatcca 240 cacagcacag cctgtgttcc cttcttgtgc tctgggcctt ctacatgtcc tgtggtactc 300 actactcctt caccatcgtt gctgactaca acttctcttc acgcagtctc atatttgtca 360 ctgaatcctg cattatttta cccatggatt acatcaccag ggatttattt ttcatattgg 420 ggatatttcg ggatgtgtcc ttcataggtc tcatggccct ctccagcggg tacatggtgg 480 ccctcttgtg cagacacagg aaacaggccc agcatcttca caggaccagc ctttctccaa 540 aagcatcccc agagcaaagg gccaccagga ccatcctgtt gctcatgagc ttctttgtgt 600 tgatgtactg cttggactgc accatatc 628 15 636 DNA Rat VNO receptor 15 atctgtgcat tgctttctta tccttaaccc aactaatgct gcttgtaact atgggactca 60 tagctgcaga catgtttatg gctcagggga tatgggatat taccacatgc aggtccctta 120 tctattttca cagacttttg aggggtttca acctttgtgc tgcctgtcta ctgcatatcc 180 tttggacctt cactctcagt cctagaagct cctgtttaac aaagtttaaa cataaatctc 240 cccatcacat ctcaggtgcc tatcttttct tctgtgttct ctatatgtcc tttagcagtc 300 acctctttgt attggtcatt gctacctcca atttaacctc agatcatttt atgtatgtta 360 ctcagtcctg ctcacttcta cccatgagtt actccagaac aagcacgttt tccttactga 420 tggtcaccag ggaagtcttt cttatcagtc tcatggccct gtccagtggg tacatggtga 480 ctctcctatg gaggcacaag aagcaggccc agcatcttca cagcaccaga ctttcttcaa 540 aagcatcccc acagcaaagg gccaccagga ccatcctgct gcttatgacc ttctttgtgg 600 ttttctacat tttaggcact gttatcttcc actcaa 636 16 660 DNA Homo sapiens 16 ccacgcgtcc gtgatgattc tgtatattta ttcactatga caggtaaatg cctcaggaaa 60 gaaatactta tgtctacagt gagcaagaca gggctagcat cctaggctgt aagtagactg 120 gggttgactc aggagttgaa ccacgaatta aatttgtgat cctggcaaac tgctcaatct 180 ctcagtatct cggtatcctc atacataaga aaggggtgat aatactcatc tcacagagag 240 gtaatgagat aattcacact cagtccttat gccaatgttt tgctcaatat gaatcttcag 300 tgaatattat cagttattaa aatttatttg caagtgtgat gtttgcatta cccacgtttg 360 tcaatgcagt gtttctgtga tattcactgt attaaagaaa ccggagtttc cctttttatg 420 tcttcaattc ctttagttca aactttccat atcttttttt attccttgga ttttaatatt 480 tgttttctat tctttttctt tttaaggcag tattatatat agtcaaatgg acagacctta 540 catgtgcaat ttaatgagtt gtgacaaatc tgtacactta ggcattcaac atccctatca 600 ggatagaaaa cacttctata ctctcagaaa atttcctctc atgcccctct cagtcaatcc 660 17 546 DNA Homo sapiens 17 agcaggctgg taccggtccg gattccggga tatcgtcgac cacgcgtccg ttcggtgact 60 agacggtccg caggggacat cccgtccctg gggcctcccc agtctccctc cccctcgcgc 120 ctgggcagct ctctcccagg gcttcggctc gagcctgcga cctgcacgga cacccccccc 180 tcaggatcta aaatgtccac tgaggcacaa agagttgatg acagtccaag cactagtgga 240 ggaagttccg atggagatca acgtgaaagt gttcagcaag aaccagaaag agaacaagtt 300 cagcccaaga aaaaggaggg aaaaatatcc agcaaaaccg ctgctaaatt gtcaactagt 360 gctaaaagaa ttcagaagga acttgcagaa atcacattgg accctcctcc caactgtagt 420 gctggaccca aaggagacaa catttatgaa atggaggtca actatattgg gacccccagg 480 atctgtctat taaggagggg tgtttctttc ttgacattac cttttcccag actattcctt 540 ttaaac 546 18 2036 DNA Homo sapiens 18 tcgaccccgc gtccgcggac gcgtgggcaa ccatcaaatt gaattaaaaa aaaaaaaaag 60 aggagcagaa gtttcattgt aagcctttat agcatttgac taatggctat atgcagttct 120 ctcagtctct tctgcctttg ctggaaatgt atagagtgtt tcttcatcct taggttgaga 180 gagcataaat atattgaatg gatttgattt cctacaaaac aatattctgg cattttgatt 240 aattgacgag gacccttcct ttgcaatgta cccaactttt ccttccaaac attagaatgt 300 ggatcaccta catttgaaga ggtagagctg gataaatctt tgctgatgaa ctaaaaaggc 360 tttcacttca gtgtctggtg aagcaattaa tgctggaaga gtagcttggg gtattaccag 420 gatgcagcat atggcggtgg tttgctaaag tgatttccat ttggctaacc acttgatggc 480 aagccagagg cagtatctgg agagaaggta gagttgggaa atgggtcttg agtacatctt 540 gtcctcaagg cacagggtga tcacagtggt gccttctaag aatgtcagtt agcaaccctt 600 tctcctgcca ccagtgagac agggccattg ttcttcatct ggaagaagcc tctttccttg 660 ctgaaaggat taggctttga catcaaattc tggctttgac atcattttta agacatcctc 720 tgaatctaac cctagttttc tgaacaggca aagcctctcg cttaaattca aaattctcca 780 ggccaaagat gatgtcatgt agttttgaaa ggctccagtt cctggagtac taccaggaaa 840 agaaagtcat cttccttgaa ttcagtccac cctcaaggtg tcctgagaaa gaagctgttt 900 ctcagaacag cccaggcaac attgctttca ggcaaactct tctgttgact tcgtatttcc 960 tacacattct taagccactg aaagagttta agtctgaaag atttctgata cctatttcct 1020 caccaggctg caaaaatacc agaattattt cattcctgca gcctcaaaga tagagaaatc 1080 aaggctccaa gagcatgtct tgagctaaaa tagtgatttt ccactttttt taagtgacag 1140 gatattttca tccaataaaa ctgtggaagg gacagattat ttttccactc accagaccag 1200 tcttcttgac cggtgggcag tgtggagagt tactttcagg ctacctttaa aacgctacct 1260 gggttctaaa gacaatttat tttttttgtt ggttttttgt ttgtttttgt tttgttttgt 1320 ttgaggcgga gtccctctct gtgttaccca ggctggagtg cagtggcatg atcttggctc 1380 actgcatcct ccgcctccca tattccagct actcaggagg ctgaggcagg aagatcactt 1440 gaggccagga gttggagacc agcctgagta acatagcaag acctcattta ttaaaaaata 1500 aattaataca tagatgatat gattataatg ataaaatgat tataaacagg cacttaataa 1560 cagacaaaat atatgaacaa aaattgacag aattgagggg agaaatagac aattctacaa 1620 tagtagttgg agaattatac ccaatatata caggacactc tccccaacaa gataacacta 1680 cccaacaaca acaggattgc gtatgggaca ttttccagga tagaccatta gttatgccac 1740 aagttaattt caatagattt tttttaaaga taaatattaa agtatctttt ctgatcacag 1800 atgaagttag aaaacaataa ccaaaggaaa attggaaaat tcacaaattt gtggaaatca 1860 aacagcacac tcttaaataa ccagtggacc caaagaagaa aacacatagg taattattag 1920 aaaatactta gagacgaatg aaaacacaat gtaacaaaac ttatggcaca tgggggaaaa 1980 cagggcttaa ggaagaaatt tatggtataa atgcttatat taaaaaaaaa aaaaaa 2036 19 1965 DNA Homo sapiens 19 tcgacccacg cgtccgtgcc cagaaagcat ccacccacca tggaaatggc actgagtaac 60 cacgtagata aaacgacttg actggttgac atttgtcagc tttatcacca gccacccaga 120 gctgccatga taggcccatg agcatcatgg ccttggtggc aaagacagag gttaccaatg 180 agcccagcag catgtgcttc actcaccaag gtctgtttca ctactgctgt tttggaggga 240 atgcccagct tgtcagcaac agacaccagc actgagccct taccatggca cggttcccca 300 ggggaccaac agatgcttta gcagtgagtt gactctattg gctcttcccg tcttggaggg 360 gctggaggtt tgtcctcata gggatgggca cctcctctgg gtatgcgatc acctctccca 420 cttgcagagc ctcagctgtc caggcagctc tagaaagctc atgctggctg ttgaatggaa 480 actgggctgt attgagccgg ttgaagacca ggaggccatg gaaggagaca ctgcaacagc 540 ccagttaaga gttgataaga aatgatggct gggtgacagt agagatggtg aaataatagg 600 attcaagttt aaaataacga aaaggtaaac aatgtgttat gataaaagct tactttcttc 660 taaggcatct tagacccacc tttctacatt ttaatggaca atacgttctc tctgattttc 720 ctctgatcca atgaatcctt tgataaaatt gcaaacaatg ttttagggtc ccgcagacac 780 aaagaaagca gggtgagtat ctagtggcat tgtgcccaga aagggtgtta cttttggcaa 840 aatgaaccag agcattttcc aaagtagtat ttattctttt taaaattatg cacgcaacaa 900 atgtctgggt gagccgactt ctctaaccca tgtactaatg tgtgggtagg cttataattt 960 ggggcactca ctcaggaaat tctgaaatta agggtctttc agaaagtgtt gactcatccc 1020 ctccacactt tctgaatata tccatttaac acactaatta agtaattcta aattgcattc 1080 taaattctgc aggtgatttt ctgatgaaat ggtgcttcgc taattctggt gggtgttgtt 1140 tagaatttgc ttctgcattg aaaatagctt tcattttgct tttgataaaa atggaaacta 1200 ttagaaaagg tccatccaac tggatatgac actgtgactc catcacagtc tactagtcta 1260 tgaggtttgc attcaaatac ggcactcatg catctgtttt tcgcctttga agaaagcaag 1320 tccttggtac aggagagttt atgagaaaat cattgttttt aaatatctat gtgcaatgcc 1380 caagaaacat acatttaatg tactagacag tacacaggat atactctgta ccatgtatgt 1440 atttaatcca ccatttagta gtttcctgag actgatcaat tttctaccat caatgcctac 1500 tgcttgatgt caaactttaa ttctaattta aaactaatga tttcaaatct taaacaaaag 1560 taggtattcc tcactaggag gcatttacat agatctttaa gtgatgcaca aagaaagagt 1620 aggtttttgt tttttctttt tttttttttt tttcagattt ctatgttgga tgcatgtaga 1680 aagctttcat attgaagcag agttttcagt gaagttggaa aaagaagaac aaaggtgaag 1740 atatccactt agcaactctc atcatttgtg tgtcaccatg gcttcagaga cagggataca 1800 catttagtat gaaaaggagg cttggaggtt agcggagagt tggtggtggt atagagtaag 1860 aagacctttt caaagtttgc tttcttgaag agcactagtt tccctggcat ggccaatggg 1920 gtgtttgctg gtcagtagct ataacttaaa gtgcttaaaa ccaca 1965 20 593 DNA Homo sapiens 20 tcgacccacg cgtccgaggc cccggagtag cagcggggag gccgggagcc cgcgggccgg 60 agccgcccgg ccgaggcgtg ggggctgcgg ggccggccca tccgtggggg cgacttgagc 120 gttgagggcg cgcggggagg cgagccacca tgttcagcca gcagcagcag cagcagctcc 180 agcaacagca gcagcagctc cagcagttac agcagcagca gctccagcag cagcaattgc 240 agcagcagca gttactgcag ctccagcagc tgctccagca gtccccacca caggccccgt 300 tgcccatggc tgtcagccgg gggctccccc cgcagcagcc acagcagccg cttctgaatc 360 tccagggcac caactcagcc tccctcctca acggctccat gctgcagaga gctttgcttt 420 tacagcagtt gcaaggactg gaccagtttg caatgccacc agccacgtat gacactgccg 480 gtctcaccat gcccacagca acactgggta acctccgagg ctatggcatg gcatccccag 540 gcctcgcagc ccccagcctc acacccccac aactggccac tccaaatttg caa 593

Claims

1. A human VNO cDNA library constructed from female tissue ATCC # PTA-1213.

2. A human CDNA library of female VNO tissue characterized by the presence of cDNAs coding for neuron-specific enolase, protein gene product 9.5 and synaptophysin, and the absence of cDNA coding for olfactory marker protein, wherein cDNA inserts range from about 300 base pairs to about 3000 base pairs in length.

3. A human cDNA library of female VNO tissue characterized by the presence of cDNAs encoding Ga proteins G_i1, G_i2, G_i3and G_olf.

4. A human cDNA library of female VNO tissue characterized by the presence of cDNAs encoding adenylyl cyclase types 2, 3 and 7.

5. cDNA corresponding to PP32 comprising SEQ ID Nos. 1 and 2.

6. cDNA corresponding to PP33 comprising SEQ ID No. 3.

7. cDNA corresponding to PP35 comprising SEQ ID No. 4 and 5.

8. cDNA corresponding to PP38 comprising SEQ ID No.6.

9. cDNA corresponding to PP40 comprising SEQ ID No. 16.

10. cDNA corresponding to PP41 comprising SEQ ID No. 17.

11. A method of identifying cDNA inserts encoding pheromone receptors comprising:

(a) generating a cDNA library which contains clones carrying cDNA inserts from human female VNO;

(b) hybridzing nucleic acid molecules of the clones from the cDNA libraries generated in step (a) with probes prepared from the group consisting of G-protein-coupled receptors, rodent VNO receptors and human pseudogene sequences;

(c) selecting clones which hybridized with the probes; and

(d) isolating clones which carry the hybridized inserts, thereby identifying the inserts encoding pheromone receptors.

12. The method of claim 11, wherein the probes are cDNA probes.

13. The method of claim 11, wherein the probes are cRNA probes.

14. The method of claim 11, wherein the probes are genomic DNA probes.

15. The method of claim 11, wherein the probes are PCR amplicons.

16. The cDNA inserts identified by the method of claims 11, 12, 13, 14 or 15.

17. A method for identifying DNA inserts encoding pheromone receptors comprising:

(a) generating DNA libraries which contain clones carrying inserts from a sample containing human female vomeronasal organ tissue;

(b) contacting clones from the DNA libraries generated in step (a) with a nucleic acid molecule selected from the group consisting of G protein-coupled receptors, rodent VNO receptors and human pseudogene sequences, in appropriate conditions permitting hybridization of the cloned DNA and the nucleic acid molecule;

(c) selecting clones which hybridized with the nucleic acid molecule; and

(d) isolating the clones which carry the hybridized inserts, thereby identifying the inserts encoding the pheromone receptors.

18. A method to identify DNA inserts encoding pheromone receptors comprising:

(a) Generating DNA libraries which contain clones with inserts from a sample which contains at least one human female vomeronasal organ;

(b) Contacting the clones from the DNA libraries generated in step (a) with appropriate polymerase chain reaction primers capable of specifically binding to nucleic acid molecules encoding pheromone receptors in appropriate conditions permitting the amplification of the hybridized inserts by polymerase chain reaction;

(c) Selecting the amplified inserts; and

(d) Isolating the amplified inserts, thereby identifying the inserts encoding the pheromone receptors.

19. A method of claim 18, wherein the sample contains only human female vomeronasal organ cells.

20. A method of claim 17, wherein the libraries are cDNA libraries.

21. A method of claim 18, wherein the libraries are cDNA libraries.

22. A method of claim 17, wherein the libraries are genomic DNA libraries.

23. A method of claim 18, wherein the libraries are genomic DNA libraries.

24. DNA inserts identified by the method of claim 17, 18, 19, 20, 21, 22 or 23.

25. A method to isolate DNA molecules encoding pheromone receptors comprising:

(a) Contacting a biological sample known to contain nucleic acids with appropriate polymerase chain reaction primers capable of specifically binding to nucleic acid molecules encoding pheromone receptors in appropriate conditions permitting the amplification of the hybridized molecules by polymerase chain reaction; and

(b) Isolating the amplified molecules, thereby identifying the DNA molecules encoding the pheromone receptors.

26. A method of claim 25, wherein the nucleic acid contained in the sample is DNA.

27. A method of claim 26, wherein the nucleic acid contained in the sample is genomic DNA.

28. The nucleic acid molecules isolated by the method of claim 25, 26 or 27.

29. A method to isolate DNA molecules encoding pheromone receptors comprising:

(a) Contacting a biological sample known to contain nucleic acids with appropriate polymerase chain reaction primers capable of specifically binding to nucleic acid molecules encoding pheromone receptors in appropriate conditions permitting the amplification of the hybridized molecules by Reverse Transcriptase (RT)-polymerase chain reaction; and

30. A method of claim 29, wherein the nucleic acid contained in the sample is RNA.