Patent Grant
7527944
The present invention relates to the field of sensory mechanisms of the domestic cat, Felis catus. The invention relates, for example, to the discovery of several genes of Felis catus encoding taste receptors of the T1R family, Tas1r1, Tas1r2, and Tas1r3, the polypeptides encoded thereby (T1R1, T1R2, and T1R3), and methods and uses of the same.
The sense of taste is important for determining food choice, for regulating food intake, and for ensuring efficient use of ingested nutrients. Taste can act as a warning system for the presence of potentially harmful foods, by, for example, the aversive sensations of sourness or bitterness, and as an attractant to potentially nutrient-rich foods, by, for example, the appealing sensations of sweetness, saltiness, and umami.
Taste stimuli are received by taste receptor cells assembled into taste buds that are located in the epithelium of taste papillae of the tongue (Kitagawa et al., Bioch. Bioph. Res. Comm., 283:236-242 (2001)). The stimuli are believed to be transduced by taste receptors at the surface of the taste receptor cells (Id.). The taste receptors encoded by the genes of a given species are reflective of that species' food choices. For example, the “sweet receptors” of an herbivorous species are expected to be different from those of a carnivorous species, since the two consume completely different diets whose foods contain different primary stimuli. Since taste receptor specificity likely reflects food choice, it follows that receptor sequence homology among species may be as predictive or more predictive of food preferences of a given species as phylogenetic relatedness among species.
The behavior of the domestic cat (Felis catus), a carnivore, towards stimuli such as sweet carbohydrates, which it generally cannot taste, and towards L-amino acids, which it generally can taste, should be explicable by the specificity of taste receptors of other carnivores. Direct knowledge of taste receptor genes will allow insight into an animal's sensory world and may be useful for identifying modulators of the taste receptors encoded thereby to influence an animal's taste preferences.
Molecular receptors for the taste element of sweetness have been identified from human, mouse, and rat. Thus far, there are three known members of the T1R taste receptor family: T1R1, T1R2, and T1R3 (Montmayeur & Matsunami, Curr. Opin. Neurobiol., 12(4):366-371 (2002)). The T1R3 receptor gene is located within the Sac locus, the primary genetic locus controlling preference for sweet-tasting stimuli in mice (Li et al., Mamm. Genome, 12(1):13-16 (2001); Li et al., Mamm. Genome, 13(1):5-19 (2002)). The human syntenic region for mouse T1R3 gene is on 1p36.33 (1162-1186 kb). The gene for T1R1 is located on human 1p36.23 (6324-6349 kb), which is ˜5 Mb from T1R3, and that for T1R2 is located on human 1p36.13 (18483-18729 kb), which is ˜12 Mb from T1R1.
Most of the T1Rs are G-protein coupled receptors with long N-terminal extracellular domains believed to be involved in ligand binding (Montmayeur & Matsunami, Curr. Opin. Neurobiol., 12(4):366-371 (2002)). The T1R receptors have been shown to dimerize. For example, homodimerization of T1Rs has been detected (Zhao et al., Cell, 115:255-266 (2003)). The taste receptors also heterodimerize. For example, coupling of T1R3 with T1R1 or T1R2 has been detected. In mouse, the T1R1/T1R3 heterodimer functions as a receptor for selected amino acids. The T1R2/T1R3 heterodimer functions as a receptor for stimuli considered sweet by humans. Current data indicate that the T1R3 component of the T1R dimer couples the taste receptor to cellular signal transduction processes, thereby ensuring that the stimulus-binding event is transduced to a neural signal. Thus, knowledge of the T1R receptors will lead to better understanding of species-specific reactions to sapid stimuli.
Currently, mechanisms for identifying novel taste stimuli for the domestic cat are limited, for example, to exhaustive and difficult feeding studies in which a novel ingredient is paired with a control ingredient and intake of the two are compared. Considerable time, effort, and expense can be expended in the discovery of a single stimulus. Furthermore, feline illnesses often are exacerbated by a cat's refusal to eat. Additionally, the molecular features that define acceptable taste stimuli for domestic cat remain largely unknown, making rational computational design approaches for taste stimuli difficult. As a result, knowledge of the feline taste receptor and its ligands may lead to a better understanding of cat taste perception and modulation thereof.
The present invention provides novel genes encoding the feline taste receptors T1R1, T1R2, and T1R3, the polypeptides encoded thereby, and methods of use of the receptors to identify compounds that can stimulate, inhibit, or modify the ingestive responses or general behavior of a cat. The screening methods of the invention allow the rapid screening of binding partners, agonists, antagonists, and modulators of the T1R receptors of the domestic cat. The results of the feline T1R receptor studies reflect the unique taste profile of the domestic cat.
Certain embodiments of the present invention relate to polynucleotides encoding a T1R receptor, including, but not limited to polynucleotides having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:63, fragments of the polynucleotide of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63 encoding a polypeptide having substantially the same biological activity as a polypeptide encoded by the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63, respectively; variants of the polynucleotide of SEQ ID NO:1 or SEQ ID NO:99 having at least 80% homology to the polynucleotide of SEQ ID NO:1 or SEQ ID NO:99; variants of the polynucleotide of SEQ ID NO:59 or SEQ ID NO:60 having at least 85% homology to the polynucleotide of SEQ ID NO:59 or SEQ ID NO:60; variants of the polynucleotide of SEQ ID NO:62 or SEQ ID NO:63 having at least 75% homology to SEQ ID NO:62 or SEQ ID NO:63; polynucleotide variants of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63 encoding a polypeptide having substantially the same biological activity as a polypeptide encoded by the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63, respectively; variants of the polynucleotide of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63 encoding a polypeptide conferring modified taste perception to one or more taste stimuli relative to a polypeptide encoded by the polynucleotide of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63, respectively; nucleotide sequences encoding the amino acid sequence of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64; nucleotide sequences substantially complementary to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63; and nucleotide sequences that hybridize to the complement of the polynucleotide having SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63 under high stringency conditions. The biological activity of the polypeptides encoded by the polynucleotides of the invention may be determined, for example, by an in vitro binding assay, such as but not limited to assessing the level of binding of the polypeptide to a T1R dimerization partner. The polynucleotides of the invention may be DNA or RNA and may be single- or double-stranded. In some embodiments of the invention, the polynucleotide fragments have at least about 42 nucleotides. The polynucleotide fragments of the invention encode, for example, an extracellular domain of the polypeptide of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64; a transmembrane domain of the polypeptide of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64; or an intracellular domain of the polypeptide of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64. For example, the polynucleotides of the invention encoding extracellular domains of feline T1R receptors include nucleotides 1-1689, 1870-1905, 2101-2178, and 2341-2376 of SEQ ID NO:60; nucleotides 1-441 of SEQ ID NO:63; and nucleotides 1-1713, 1882-1923, 2113-2193, and 2359-2382 of SEQ ID NO:1 or SEQ ID NO:99. The polynucleotides of the invention encoding transmembrane domains of feline T1R receptors include nucleotides 1690-1767, 1810-1869, 1906-1980, 2041-2100, 2179-2244, 2281-2340, and 2379-2451 of SEQ ID NO:60; nucleotides 442-501 of SEQ ID NO:63; and nucleotides 1714-1782, 1828-1881, 1924-1992, 2041-2112, 2191-2262, 2299-2358, and 2383-2436 of SEQ ID NO:1 or SEQ ID NO:99. The polynucleotides of the invention encoding intracellular domains of feline T1R receptors include nucleotides 1768-1809, 1981-2040, 2245-2280, and 2452-2523 of SEQ ID NO:60; nucleotides 502-1173 of SEQ ID NO:63; nucleotides 1783-1827, 1993-2040, 2263-2298, and 2437-2566 of SEQ ID NO:1; and nucleotides 1783-1827, 1993-2040, 2263-2298, and 2437-2595 of SEQ ID NO:99. The polynucleotides of the invention also include any combination of the polynucleotides encoding the functional domains of the invention, such as combinations of the polynucleotides encoding the extracellular, transmembrane, and/or intracellular domains of the same or different feline T1R receptors. In other embodiments of the invention, the polynucleotide variants of the polynucleotide of SEQ ID NO:1 or SEQ ID NO:99 encoding an amino acid sequence of SEQ ID NO:2 have a nonconserved amino acid substitution, for example, at residue 59 and/or residue 64.
The invention also encompasses expression vectors containing the polynucleotides of the invention operably linked to a promoter. Another embodiment of the invention provides host cells containing the expression vector. The host cells may be prokaryotic, such as bacterial cells, or eukaryotic, such as yeast or mammalian cells, including human, murine, porcine, bovine, canine, or feline cells. The invention further encompasses cell cultures of the host cells. The invention also encompasses methods of producing a feline T1R receptor by culturing the host cells and recovering receptor therefrom.
Another embodiment of the invention includes T1R receptor polypeptides, including polypeptides encoded by the polynucleotides of the invention. The polypeptides of the invention include, for example, those having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64, fragments of at least 30 contiguous amino acids of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64, and variants thereof having substantially the same biological activity as the polypeptide of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64, respectively. The biological activity of the polypeptides of the invention may be determined, for example, by an in vitro binding assay, such as but not limited to assessing the level of binding of the polypeptide to a T1R dimerization partner. Biological activity of the polypeptides of the invention also may be determined by measuring ion conductance; ion flow; calcium imaging including with fura-2, green dextran activity, or aquorin activity; voltage measurement and/or voltage imaging with dyes or reporter genes such as β-luciferase, alkaline phosphatase, β-galactosidase, or β-lactamase; second messenger measurement, for example, IP3, cAMP, G-protein activation-based assays; or receptor phosphorylation. The variant polypeptides of the invention may have an amino acid sequence having at least one sequence variation of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64 that confers modified taste perception to one or more taste stimuli relative to a polypeptide of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64, respectively. The polypeptides of the invention further comprise functional domains of the T1R receptors of the invention, for example, extracellular, transmembrane, and intracellular domains of the receptors, and combinations thereof. Examples of functional domains of the T1R1 polypeptide of SEQ ID NO:61 include extracellular domains corresponding to residues 1-563, 624-635, 701-726, and 781-792; transmembrane domains corresponding to residues 564-589, 604-623, 636-660, 681-700, 727-748, 761-780, and 793-817; and intracellular domains corresponding to residues 590-603, 661-680, 749-760, and 818-841. Examples of functional domains of the T1R2 receptor of SEQ ID NO:64 include an extracellular domain corresponding to residues 1-147; a transmembrane domain corresponding to residues 148-167; and an intracellular domain corresponding to residues 168-391. Examples of functional domains of the T1R3 polypeptide of SEQ ID NO:2 include the extracellular domains (residues 1-571, 628-641, 705-730, and 787-794 of SEQ ID NO:2), the transmembrane domains (residues 572-594, 610-627, 642-664, 681-704, 731-754, 767-780, and 795-812 of SEQ ID NO:2), and the intracellular domains (residues 595-609, 665-680, 755-766, and 813-865 of SEQ ID NO:2). The polypeptides of the invention also include any combination of the functional domains of the polypeptides of the invention, such as combinations of the extracellular, transmembrane, and/or intracellular domains of the same or different feline T1R receptors.
The invention provides methods of identifying a feline T1R receptor variant that confers modified taste perception by expressing a variant of the polynucleotide of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63 homologous to the polynucleotide of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63, respectively, and detecting an increase or a decrease in the biological activity of the polypeptide encoded by the variant relative to the biological activity of the polypeptide encoded by SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63, respectively.
The invention further provides kits for the detection of polynucleotides encoding a feline T1R receptor including a polynucleotide that specifically hybridizes to a polynucleotide encoding a polypeptide having an amino acid sequence of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64, and instructions relating to detection thereof.
Also provided by the invention are antibodies that immunoreact specifically with at least one epitope of a polypeptide of the invention. The invention also includes kits for the detection of polypeptides encoding a feline T1R receptor including antibodies of the invention and instructions relating to detection.
Further provided by the invention are methods for identifying a compound that interacts with a feline T1R receptor by expressing a polynucleotide of the invention in the presence of a test compound, and detecting direct or indirect interaction between a polypeptide produced by the expression step with the compound. Also provided are methods for identifying compounds that interact with a feline T1R receptor by contacting a feline T1R receptor with a test compound, and detecting interaction between the receptor and the compound. The methods for detecting such interaction may be cell-based or cell-free assays. For example, a polynucleotide of the invention may be expressed in a heterologous expression system or in a cellular extract. The receptor may be bound to a solid support. In one aspect of the invention, the recognition sites of the receptor are coupled with a monitoring system, either electrical or optical. In another embodiment, the solid support is formulated into a feline-specific electronic tongue or biosensor.
The invention also provides methods for identifying agonists and antagonists of a feline T1R receptor. For example, the methods of the invention include identification of an agonist of a feline T1R receptor by expressing a polynucleotide of the invention in the presence of a test compound, and detecting increased transcription of said polynucleotide or increased biological activity of a polypeptide produced by the expression step in the presence of the compound relative to the rate of transcription or biological activity of the polypeptide in the absence of the compound. The biological activity detected may be an increase or decrease in the interaction between the T1R receptor and its T1R dimerization partner. For example, the T1R dimerization partner of a T1R1 or a T1R2 receptor may be T1R3 and vice versa. (In addition, T1R receptors may form homodimers which may have unique ligand binding properties. The T1R dimerization partner thus also may be a homodomerization partner. Also included are methods for identifying agonists of a feline T1R receptor by contacting a polypeptide of the invention with a test compound, and detecting an increase in biological activity of the polypeptide in the presence of the compound relative to biological activity of the polypeptide in the absence of the compound. The methods for identifying agonists of the cat T1R receptors may be cell-based or cell-free assays. For example, a polynucleotide of the invention may be expressed in a heterologous expression system or in a cellular extract. The receptor may be bound to a solid support. In one aspect of the invention, the recognition sites of the receptor are coupled with a monitoring system, either electrical or optical. In another embodiment, the solid support is formulated into a feline-specific electronic tongue or biosensor.
Methods for identifying antagonists of the polypeptides of the invention also are provided. For example, the invention provides methods for identifying antagonists of a feline T1R receptor by expressing a polynucleotide of the invention in the presence of a test compound, and detecting decreased transcription of said polynucleotide or decreased biological activity of a polypeptide produced by the expression step in the presence of the compound relative to the rate of transcription or biological activity of the polypeptide in the absence of the compound. Another example of methods for identifying an antagonist of a feline T1R receptor involves contacting a polypeptide of the invention with a test compound, and detecting a decrease in biological activity of the polypeptide in the presence of the compound relative to biological activity of the polypeptide in the absence of the compound. The methods for identifying the antagonists may be cell-based or cell-free assays. For example, a polynucleotide of the invention may be expressed in a heterologous expression system or in a cellular extract. The receptor may be bound to a solid support. In one aspect of the invention, the recognition sites of the receptor are coupled with a monitoring system, either electrical or optical. In another embodiment, the solid support is formulated into a feline-specific electronic tongue or biosensor.
Also encompassed by the invention are methods for predicting the taste perception of an organism such as a mammal, including but not limited to a cat. The methods may involve detection of a nucleotide sequence or amino acid sequence of the invention in a biological sample of the organism. For example, an organism having a polynucleotide or polypeptide of the invention may be attracted to one or more amino acids. An organism having a polynucleotide or polypeptide of the invention may show no preference for one or more carbohydrates or high-intensity sweeteners. An organism having a polynucleotide or polypeptide of the invention may be attracted to one or more amino acids and may exhibit no preference for one or more carbohydrates or high-intensity sweeteners.
Another embodiment of the invention includes compounds and compositions for modifying the taste perception of a mammal, such as a cat. The compounds and compositions may contain at least one of the polynucleotides of the invention, polypeptides of the invention, or compounds identified by the methods of the invention. Examples of the compositions of the invention include veterinary foods and drinks and pharmaceutical compositions. The compositions of the invention may include a pharmaceutically acceptable excipient. The compositions of the invention may be breed-specific. Methods for modifying the taste perception of a mammal (e.g., a cat) by administering to the mammal a polynucleotide of the invention, a polypeptide of the invention, and/or a compound identified according to the methods of the invention also are provided.
The invention further contemplates transgenic animals comprising a polynucleotide of the invention.
The materials, methods, and examples provided herein are illustrative only and are not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description and claims.
The reference works, patents, patent applications, and scientific literature that are referred to herein reflect in part the knowledge of those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter.
Standard reference works setting forth the general principles of recombinant DNA technology are known to those of skill in the art (Ausubel et al., C
As used herein, “T1R receptor” encompasses the taste receptors of the T1R1, T1R2, and T1R3 types, for example, the feline T1R1, T1R2, and T1R3 taste receptors of the invention.
As used herein, “taste perception” refers to a response (e.g., biochemical, behavioral) or sensitivity of a T1R receptor of the invention to a taste stimulus. “Taste stimulus” as used herein refers to any compound that elicits, for example at the biochemical level (e.g., activation or inhibition of a taste receptor) or behavioral level (e.g., preference, indifference, or distaste), a taste response which would be perceived by a mammal as at least one of the five taste elements, including sweet, salty, sour, bitter, and umami. “Taste perception” or “taste stimulus,” or variants thereof, does not require, though it does include, transmission of a neural signal resulting in in vivo sensation of taste by a mammal. Modification of taste perception includes an alteration of (enhancement of, reduction to, or change to) a biochemical response, an ingestive response, a taste preference, or general behavior of a mammal in response to a compound.
As used herein “polynucleotide” refers to a nucleic acid molecule and includes genomic DNA, cDNA, RNA, mRNA, mixed polymers, recombinant nucleic acids, fragments and variants thereof, and the like. Polynucleotide fragments of the invention comprise at least 10, and preferably at least 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 75, or 100 consecutive nucleotides of a reference polynucleotide. The polynucleotides of the invention include sense and antisense strands. The polynucleotides of the invention may be naturally occurring or non-naturally occurring polynucleotides. A “synthesized polynucleotide” as used herein refers to polynucleotides produced by purely chemical, as opposed to enzymatic, methods. “Wholly” synthesized DNA sequences are therefore produced entirely by chemical means, and “partially” synthesized DNAs embrace those wherein only portions of the resulting DNA were produced by chemical means. The polynucleotides of the invention may be single- or double-stranded. The polynucleotides of the invention may be chemically modified and may contain non-natural or derivatized nucleotide bases as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
“Recombinant nucleic acid” is a nucleic acid generated by combination of two segments of nucleotide sequence. The combination may be, for example, by chemical means or by genetic engineering.
As used herein, “polynucleotide amplification” refers to a broad range of techniques for increasing the number of copies of specific polynucleotide sequences. Typically, amplification of either or both strand(s) of the target nucleic acid comprises the use of one or more nucleic acid-modifying enzymes, such as a DNA polymerase, ligase, RNA polymerase, or RNA-dependent reverse transcriptase. Examples of polynucleotide amplification include, but are not limited to, polymerase chain reaction (PCR), nucleic acid sequence based amplification (NASB), self-sustained sequence replication (3SR), strand displacement activation (SDA), ligase chain reaction, Qβ replicase system, and the like. A wide variety of alternative cloning and in vitro amplification methodologies are well known to those skilled in the art. Examples of these techniques are found in, for example, Berger et al., Guide to Molecular Cloning Techniques, M
As used herein, the term “oligonucleotide” or “primer” refers to a series of linked nucleotide residues which has a sufficient number of bases to be used in a polymerase chain reaction (PCR). This short sequence is based on (or designed from) a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar, or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a nucleic acid sequence having at least about 10 nucleotides and as many as about 50 nucleotides, often about 12 or 15 to about 30 nucleotides. They are chemically synthesized and may be used as probes. “Primer pair” refers to a set of primers including a 5′ upstream primer that hybridizes with the 5′ end of a target sequence to be amplified and a 3′ downstream primer that hybridizes with the complement of the 3′ end of the target sequence to be amplified.
As used herein, the term “probe” refers to nucleic acid sequences of variable length, for example between at least about 10 and as many as about 6,000 nucleotides, depending on use. Probes are used in the detection of identical, similar, or complementary target nucleic acid sequences, which target sequences may be single- or double-stranded. Longer probes are usually obtained from a natural or recombinant source, are highly specific, and are much slower to hybridize than oligomers, or shorter probes. They may be single- or double-stranded and are carefully designed to have specificity in PCR, hybridization membrane-based, or ELISA-like technologies. An “overgo probe” is a DNA probe comprising two short, overlapping DNA sequences (e.g., 10-50 nucleotides each) with a complementary overlapping region (e.g., 5-15 nucleotides) that is used in an overgo hybridization strategy. For example, an overgo probe may be two 22mers with an 8 bp complementary overlap, resulting in a 36mer overgo probe. As another example, an overgo probe may be two 24mers with an 8 bp complementary overlap, resulting in a 40mer overgo probe.
As used herein, the phrase “stringent hybridization conditions” or “high stringency conditions” refers to conditions under which a probe, primer, or oligonucleotide will hybridize to its target sequence, but to a minimal number of or no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences will hybridize with specificity to their proper complements at higher temperatures. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present in excess, at Tm, 50% of the probes are hybridized to their complements at equilibrium. Stringent temperature conditions will generally include temperatures in excess of 30° C., typically in excess of 37° C., and may be in excess of 45° C. Stringent salt conditions will ordinarily be less than 1.0 M, typically less than 0.5 M, and may be less than 0.2 M. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes, primers, or oligonucleotides (e.g., 10 to 50 nucleotides) and at least about 60° C. for longer probes, primers, or oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
As used herein “antisense oligonucleotide” refers to a nucleic acid molecule that is complementary to at least a portion of a target nucleotide sequence of interest and specifically hybridizes to the target nucleotide sequence under physiological conditions. The term “double stranded RNA” or “dsRNA” as used herein refers to a double-stranded RNA molecule capable of RNA interference, including short interfering RNA (siRNA) (see for example, Bass, Nature, 411, 428-429 (2001); Elbashir et al., Nature, 411, 494-498 (2001)).
As used herein, the term “complementary” refers to Watson-Crick basepairing between nucleotide units of a nucleic acid molecule.
The term “marker gene” or “reporter gene” refers to a gene encoding a product that, when expressed, confers a phenotype at the physical, morphologic, or biochemical level on a transformed cell that is easily identifiable, either directly or indirectly, by standard techniques and includes, but is not limited to, genes encoding proteins that confer resistance to toxins or antibiotics such as ampicillin, neomycin, and methotroxate; genes encoding proteins that complement auxotrophic deficiencies; and genes encoding proteins that supply critical components not available from complex media. Examples of marker genes include green fluorescent protein (GFP), red fluorescent protein (DsRed), alkaline phosphatase (AP), β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neor, G418r) dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding β-galactosidase), β-lactamase, luciferase (luc), and xanthine guanine phosphoribosyltransferase (XGPRT). As with many of the standard procedures associated with the practice of the invention, skilled artisans will be aware of additional sequences that can serve the function of a marker or reporter. Thus, this list is merely meant to show examples of what can be used and is not meant to limit the invention.
As used herein, the term “promoter” refers to a regulatory element that regulates, controls, or drives expression of a nucleic acid molecule of interest and can be derived from sources such as from adenovirus, SV40, parvoviruses, vaccinia virus, cytomegalovirus, or mammalian genomic DNA. Examples of suitable promoters include, but are not limited to, CMV, MSH2, trp, lac, phage, and TRNA promoters. Suitable promoters that can be used in yeast include, but are not limited to, such constitutive promoters as 3-phosphoglycerate kinase and various other glycolytic enzyme gene promoters such as enolase or glyceraldehydes-3-phosphate dehydrogenase, or such inducible promoters as the alcohol dehydrogenase 2 promoter or metallothionine promoter. Again, as with many of the standard procedures associated with the practice of the invention, skilled artisans will be aware of additional promoters that can serve the function of directing the expression of a marker or reporter. Thus, the list is merely meant to show examples of what can be used and is not meant to limit the invention.
“Operably linked” refers to juxtaposition wherein the components are in a functional relationship. For example, a promoter is operably linked or connected to a coding sequence if it controls the transcription or expression of the sequence.
The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein. “Polypeptide” refers to a polymer of amino acids without referring to a specific length. Polypeptides of the invention include peptide fragments, derivatives, and fusion proteins. Peptide fragments preferably have at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acids. Some peptide fragments of the invention are biologically active. Biological activities include immunogenicity, ligand binding, dimerization. and activity associated with the reference peptide. Immunogenic peptides and fragments of the invention generate an epitope-specific immune response, wherein “epitope” refers to an immunogenic determinant of a peptide and preferably contains at least three, five, eight, nine, ten, fifteen, twenty, thirty, forty, forty-five, or fifty amino acids. Some immunogenic peptides of the invention generate an immune response specific to that peptide. Polypeptides of the invention include naturally occurring and non-naturally occurring peptides. The term includes modified polypeptides (wherein examples of such modifications include glycosylation, acetylation, phosphorylation, carboxylation, ubiquitination, labeling, etc.), analogs (such as non-naturally occurring amino acids, substituted linkages, etc.), and functional mimetics. A variety of methods for labeling polypeptides are well known in the art and include radioactive isotopes such as 32P or 35S, ligands that bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antligands.
As used herein, the term “amino acid” denotes a molecule containing both an amino group and a carboxyl group. In some embodiments, the amino acids are α-, β-, γ- or δ-amino acids, including their stereoisomers and racemates. As used herein the term “L-amino acid” denotes an α-amino acid having the L configuration around the α-carbon, that is, a carboxylic acid of general formula CH(COOH)(NH2)-(side chain), having the L-configuration. The term “D-amino acid” similarly denotes a carboxylic acid of general formula CH(COOH)(NH2)-(side chain), having the D-configuration around the α-carbon. Side chains of L-amino acids include naturally occurring and non-naturally occurring moieties. Non-naturally occurring (i.e., unnatural) amino acid side chains are moieties that are used in place of naturally occurring amino acid side chains in, for example, amino acid analogs. Amino acid substituents may be attached, for example, through their carbonyl groups through the oxygen or carbonyl carbon thereof, or through their amino groups, or through functionalities residing on their side chain portions.
The amino acid sequences are presented in the amino (N) to carboxy (C) direction, from left to right. The N-terminal α-amino group and the C-terminal β-carboxy groups are not depicted in the sequence. The nucleotide sequences are presented by single strands only, in the 5′ to 3′ direction, from left to right. Nucleotides and amino acids are represented in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or amino acids are represented by their three letters code designations.
As used herein, the term “antibody” is meant to refer to complete, intact antibodies, and Fab, Fab′, F(ab)2, Fv, and other fragments thereof. Complete, intact antibodies include antibodies such as polyclonal antibodies, monoclonal antibodies, chimeric antibodies, and humanized antibodies, felinized antibodies, and immunologic binding equivalents thereof. The antibodies of the invention may be labeled or unlabeled. Examples of labels of antibodies include, but are not limited to, radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles, and the like. Recombinant immunoglobulins are included in the invention.
As used herein, the term “binding” means the physical or chemical interaction between two proteins or compounds or associated proteins or compounds or combinations thereof. Binding includes ionic, non-ionic, Hydrogen bonds, Van der Waals, hydrophobic interactions, etc. The physical interaction, the binding, can be either direct or indirect, indirect being through or due to the effects of another protein or compound. Direct binding refers to interactions that do not take place through or due to the effect of another protein or compound but instead are without other substantial chemical intermediates. Binding may be detected in many different manners. As a non-limiting example, the physical binding interaction between two molecules can be detected using a labeled compound. Other methods of detecting binding are well-known to those of skill in the art.
As used herein, the term “contacting” means bringing together, either directly or indirectly, a compound into physical proximity to a molecule of interest. Contacting may occur, for example, in any number of buffers, salts, solutions, or in a cell or cell extract.
As used herein, the terms “modulates” or “modifies” means an increase or decrease in the amount, quality, or effect of a particular activity or protein. “Modulators” refer to any inhibitory or activating molecules identified using in vitro and in vivo assays for, e.g., agonists, antagonists, and their homologs, including fragments, variants, and mimetics, as defined herein, that exert substantially the same biological activity as the molecule. “Inhibitors” or “antagonists” are modulating compounds that reduce, decrease, block, prevent, delay activation, inactivate, desensitize, or downregulate the biological activity or expression of a molecule or pathway of interest. “Inducers,” “activators,” or “agonists” are modulating compounds that increase, induce, stimulate, open, activate, facilitate, enhance activation, sensitize, or upregulate a molecule or pathway of interest. In some preferred embodiments of the invention, the level of inhibition or upregulation of the expression or biological activity of a molecule or pathway of interest refers to a decrease (inhibition or downregulation) or increase (upregulation) of greater than about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The inhibition or upregulation may be direct, i.e., operate on the molecule or pathway of interest itself, or indirect, i.e., operate on a molecule or pathway that affects the molecule or pathway of interest.
A “purified” or “substantially purified” polynucleotide or polypeptide is substantially separated from other cellular components that naturally accompany a native (or wild-type) nucleic acid or polypeptide and/or from other impurities (e.g., agarose gel). A purified polypeptide or protein will comprise about 60% to more than 99% w/w of a sample, and may be about 90%, about 95%, or about 98% pure. As used herein, the term “isolated” refers to a molecule that has been removed from its native environment. Examples of isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules.
“About” as used herein refers to +/−10% of the reference value.
As used herein, “variant” nucleotide or amino acid sequences refer to homologs, including, for example, isoforms, species variants, allelic variants, and fragments of the sequence of interest. “Homologous nucleotide sequence” or “homologous amino acid sequence,” or variations thereof, refers to sequences characterized by a homology, at the nucleotide level or amino acid level, of at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, preferably at least about 90%, at least about 95%, at least about 98%, or at least about 99%, and more preferably 100%, to a reference sequence, or portion or fragment thereof encoding or having a functional domain. The reference sequence may include, for example, but is not limited to the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:63, or portions thereof which encode a functional domain of the encoded polypeptide, SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64, or the polypeptide having amino acid sequence SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64, or fragments thereof having functional domains of the full-length polypeptide. Functional domains of the T1R receptors of the invention include extracellular domains, transmembrane domains, and intracellular domains. Examples of functional domains of the T1R1 polypeptide of SEQ ID NO:61 include extracellular domains corresponding to residues 1-563, 624-635, 701-726, and 781-792; transmembrane domains corresponding to residues 564-589, 604-623, 636-660, 681-700, 727-748, 761-780, and 793-817; and intracellular domains corresponding to residues 590-603, 661-680, 749-760, and 818-841. Examples of functional domains of the T1R2 receptor of SEQ ID NO:64 include an extracellular domain corresponding to residues 1-147; a transmembrane domain corresponding to residues 148-167; and an intracellular domain corresponding to residues 168-391. Examples of functional domains of the T1R3 polypeptide of SEQ ID NO:2 include the extracellular domains (residues 1-571, 628-641, 705-730, and 787-794 of SEQ ID NO:2), the transmembrane domains (residues 572-594, 610-627, 642-664, 681-704, 731-754, 767-780, and 795-812 of SEQ ID NO:2), and the intracellular domains (residues 595-609, 665-680, 755-766, and 813-865 of SEQ ID NO:2). Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. Homologous nucleotide sequences include nucleotide sequences encoding for a species variant of a protein. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. Study of mutations and polymorphisms of the T1R receptor polynucleotide sequences may explain breed-specific and/or individual taste preferences of a mammal such as a cat. Additionally, sequence variants of the T1R receptors may be associated with specific disease states, such that knowledge of the genes allows diagnosis and treatment of T1R-associated disorders (e.g., obesity, diabetes). Homologous amino acid sequences include those amino acid sequences which encode conservative amino acid substitutions in polypeptides having an amino acid sequence of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64, as well as in polypeptides identified according to the methods of the invention. Percent homology may be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using the default settings, which uses the algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2: 482-489, 1981). Nucleic acid fragments of the invention preferably have at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 50, or at least about 100 nucleotides of the reference nucleotide sequence. The nucleic acid fragments of the invention may encode a polypeptide having at least one biological property, or function, that is substantially similar to a biological property of the polypeptide encoded by the full-length nucleic acid sequence.
As is well known in the art, because of the degeneracy of the genetic code, there are numerous DNA and RNA molecules that can code for the same polypeptide as that encoded by a nucleotide sequence of interest. The present invention, therefore, contemplates those other DNA and RNA molecules which, on expression, encode a polypeptide encoded by the nucleic acid molecule of interest. For example, nucleotide “insertions”, “substitutions” or “deletions” are changes to or within a nucleotide sequence. Such polynucleotide variants are within the scope of the invention. DNA and RNA molecules other than those specifically disclosed herein characterized simply by a change in a codon for a particular amino acid, are within the scope of this invention.
Amino acid “insertions”, “substitutions” or “deletions” are changes to or within an amino acid sequence. The variation allowed in a particular amino acid sequence may be experimentally determined by producing the peptide synthetically or by systematically making insertions, deletions, or substitutions of nucleotides in the nucleic acid sequence using recombinant DNA techniques. Alterations of the naturally occurring amino acid sequence can be accomplished by any of a number of known techniques. For example, mutations can be introduced into the polynucleotide encoding a polypeptide at particular locations by procedures well known to the skilled artisan, such as oligonucleotide-directed mutagenesis.
A polypeptide variant of the present invention may exhibit substantially the biological activity of a naturally occurring reference polypeptide. “Biological activity” as used herein refers to the level of a particular function (for example, enzymatic activity) of a molecule or pathway of interest in a biological system. “Wild-type biological activity” refers to the normal level of function of a molecule or pathway of interest. “Reduced biological activity” refers to a decreased level of function of a molecule or pathway of interest relative to a reference level of biological activity of that molecule or pathway. For example, reduced biological activity may refer to a decreased level of biological activity relative to the wild-type biological activity of a molecule or pathway of interest. “Increased biological activity” refers to an increased level of function of a molecule or pathway of interest relative to a reference level of biological activity of that molecule or pathway. For example, increased biological activity may refer to an increased level of biological activity relative to the wild-type biological activity of a molecule or pathway of interest. Reference to exhibiting “substantially the biological activity of a naturally occurring polypeptide” indicates that variants within the scope of the invention can comprise conservatively substituted sequences, meaning that one or more amino acid residues of a polypeptide are replaced by different residues that do not alter the secondary and/or tertiary structure of the polypeptide. Such substitutions may include the replacement of an amino acid by a residue having similar physicochemical properties, such as substituting one aliphatic residue (Ile, Val, Leu or Ala) for another, or substitution between basic residues Lys and Arg, acidic residues Glu and Asp, amide residues Gln and Asn, hydroxyl residues Ser and Tyr, or aromatic residues Phe and Tyr. Further information regarding making phenotypically silent amino acid exchanges are known in the art (Bowie et al., Science, 247: 1306-1310, 1990). Other polypeptide homologs which might retain substantially the biological activities of the reference polypeptide are those where amino acid substitutions have been made in areas outside functional regions of the protein. The biological activity may be assessed by, for example, measuring binding of a T1R receptor of the invention to a dimerization partner. Biological activity of the polypeptides of the invention also may be determined by measuring ion conductance; ion flow; calcium imaging including with fura-2, green dextran activity, or aquorin activity; voltage measurement and/or voltage imaging with dyes or reporter genes such as β-luciferase, alkaline phosphatase, β-galactosidase, or β-lactamase; second messenger measurement, for example, IP3, cAMP, G-protein activation-based assays; or receptor phosphorylation.
A nucleotide and/or amino acid sequence of a nucleic acid molecule or polypeptide employed in the invention or of a compound identified by the screening method of the invention may be used to search a nucleotide and amino acid sequence databank for regions of similarity using Gapped BLAST (Altschul et al., Nuc. Acids Res., 25: 3389, 1997). Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity (Altschul et al., J Mol. Biol., 215: 403-410, 1990). Software or performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., J Mol. Biol., 215: 403-410, 1990). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoff et al., Proc. Natl. Acad. Sci. USA, 89: 10915-10919, 1992) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5787, 1993) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a gene or cDNA if the smallest sum probability in comparison of the test nucleic acid to the reference nucleic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
The term “mimetic” as used herein refers to a compound that is sterically similar to a reference compound. Mimetics are structural and functional equivalents to the reference compounds.
The terms “patient” and “subject” are used interchangeably herein and include, but are not limited to, avians, felines, canines, bovines, ovines, porcines, equines, rodents, simians, and humans. “Host cell” includes, for example, a prokaryotic cell, such as a bacterial cell, or eukaryotic cell, such as a mammalian cell (e.g., human, rodent, canine, feline), a yeast cell, or a plant cell. “Rodents” include, for example, rats and mice.
The term “treatment” as used herein refers to any indicia of success of prevention, treatment, or amelioration of a disease or condition. Treatment includes any objective or subjective parameter, such as, but not limited to, abatement, remission, normalization of receptor activity, reduction in the number or severity of symptoms or side effects, or slowing of the rate of degeneration or decline of the patient. Treatment also includes a prevention of the onset of symptoms in a patient that may be at increased risk for or is suspected of having a disease or condition but does not yet experience or exhibit symptoms thereof.
As used herein, the term “compound” means any identifiable chemical or molecule, including, but not limited to a small molecule, peptide, protein, sugar, nucleotide, or nucleic acid. Such compound can be natural or synthetic.
Topologically, sensory GPCRs have various domains including one or more of an “N-terminal domain,” an “extracellular domain,” “transmembrane domain,” cytoplasmic and extracellular loops, “intracellular domain,” and a “C-terminal domain” (see, e.g., Hoon et al., Cell 96:541-551 (1999); Buck & Axel, Cell 65:175-187 (1991)). These domains can be structurally identified using methods known to those of skill in the art, such as sequence analysis programs that identify hydrophobic and hydrophilic domains (see, e.g., Stryer, Biochemistry (3rd ed. 1988). Such domains are useful for making chimeric proteins and for in vitro assays of the invention, e.g., ligand binding assays. In the polypeptides of the invention, the N-terminal domain is believed to be extracellular, while the C-terminal domain is believed to be cytoplasmic or intracellular.
Polynucleotides
The invention provides purified and isolated polynucleotides (e.g., cDNA, genomic DNA, synthetic DNA, RNA, or combinations thereof, whether single- or double-stranded) that comprise a nucleotide sequence encoding the amino acid sequence of the polypeptides of the invention. Such polynucleotides are useful for recombinantly expressing the receptor and also for detecting expression of the receptor in cells (e.g., using Northern hybridization and in situ hybridization assays). Such polynucleotides also are useful in the design of antisense and other molecules for the suppression of the expression of a T1R receptor in a cultured cell, a tissue, or an animal; for therapeutic purposes; or to provide a model for diseases or conditions characterized by aberrant T1R expression. Specifically excluded from the definition of polynucleotides of the invention are entire isolated, non-recombinant native chromosomes of host cells. Polynucleotides of the invention include the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, and SEQ ID NO:63. It will be appreciated that numerous other polynucleotide sequences exist that also encode the T1R receptors of the invention due to the well-known degeneracy of the universal genetic code. Such polynucleotides are included within the scope of the invention.
The invention also provides a purified and isolated polynucleotide comprising a nucleotide sequence that encodes a mammalian (e.g., feline) polypeptide, wherein the polynucleotide hybridizes to a polynucleotide having a sequence of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63 or the non-coding strand complementary thereto, under high stringency conditions.
Genomic DNA of the invention comprises the protein-coding region for a polypeptide of the invention and is also intended to include allelic variants thereof. It is widely understood that, for many genes, genomic DNA is transcribed into RNA transcripts that undergo one or more splicing events wherein introns (i.e., non-coding regions) of the transcripts are removed, or “spliced out.” RNA transcripts that can be spliced by alternative mechanisms, and therefore be subject to removal of different RNA sequences but still encode a T1R polypeptide, are referred to in the art as splice variants which are embraced by the invention. Splice variants comprehended by the invention therefore are encoded by the same original genomic DNA sequences but arise from distinct mRNA transcripts. Allelic variants are modified forms of a wild-type gene sequence, the modification resulting from recombination during chromosomal segregation or exposure to conditions which give rise to genetic mutation. Allelic variants, like wild type genes, are naturally occurring sequences (as opposed to non-naturally occurring variants that arise from in vitro manipulation).
The invention also comprehends cDNA that is obtained through reverse transcription of an RNA polynucleotide encoding a T1R receptor (conventionally followed by second strand synthesis of a complementary strand to provide a double-stranded DNA).
One embodiment of the DNA of the invention comprises a double-stranded molecule along with the complementary molecule (the “non-coding strand” or “complement”) having a sequence unambiguously deducible from the coding strand according to Watson-Crick base-pairing rules for DNA.
The present invention includes fragments of nucleotide sequences encoding a T1R receptor comprising at least 10, and preferably at least 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 75, or 100 consecutive nucleotides of a polynucleotide encoding a T1R receptor. Fragment polynucleotides of the invention may comprise sequences unique to the T1R-encoding polynucleotide sequence or sequences shared only by the feline T1R-encoding polynucleotide sequences of the invention, and therefore hybridize under highly stringent or moderately stringent conditions only (i.e., “specifically”) to polynucleotides encoding a feline T1R receptor (or fragments thereof). Polynucleotide fragments of genomic sequences of the invention comprise not only sequences unique to the coding region, but also include fragments of the full-length sequence derived from introns, regulatory regions, and/or other non-translated sequences. Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides, and can be identified through use of alignment programs routinely utilized in the art, e.g., those made available in public sequence databases. Such sequences also are recognizable from Southern hybridization analyses to determine the number of fragments of genomic DNA to which a polynucleotide will hybridize. Polynucleotides of the invention can be labeled in a manner that permits their detection, including radioactive, fluorescent, and enzymatic labeling. Polynucleotide fragments of the invention also include nucleotide sequences encoding functional domains of the polypeptides of the invention. For example, the polynucleotides of the invention encoding extracellular domains of feline T1R receptors include nucleotides 1-1689, 1870-1905, 2101-2178, and 2341-2376 of SEQ ID NO:60; nucleotides 1-441 of SEQ ID NO:63; and nucleotides 1-1713, 1882-1923, 2113-2193, and 2359-2382 of SEQ ID NO:1 or SEQ ID NO:99. The polynucleotides of the invention encoding transmembrane domains of feline T1R receptors include nucleotides 1690-1767, 1810-1869, 1906-1980, 2041-2100, 2179-2244, 2281-2340, and 2379-2451 of SEQ ID NO:60; nucleotides 442-501 of SEQ ID NO:63; and nucleotides 1714-1782, 1828-1881, 1924-1992, 2041-2112, 2191-2262, 2299-2358, and 2383-2436 of SEQ ID NO:1 or 99. The polynucleotides of the invention encoding intracellular domains of feline T1R receptors include nucleotides 1768-1809, 1981-2040, 2245-2280, and 2452-2523 of SEQ ID NO:60; nucleotides 502-1173 of SEQ ID NO:63; nucleotides 1783-1827, 1993-2040, 2263-2298, and 2437-2566 of SEQ ID NO:1; and nucleotides 1783-1827, 1993-2040, 2263-2298, and 2437-2595 of SEQ ID NO:99. The polynucleotides of the invention also include any combination of the polynucleotides encoding the functional domains of the invention, such as combinations of the polynucleotides encoding the extracellular, transmembrane, and/or intracellular domains of the same or different feline T1R receptors.
Fragment polynucleotides are particularly useful as probes for detection of full-length or fragments of T1R polynucleotides. One or more polynucleotides can be included in kits that are used to detect the presence of a polynucleotide encoding a T1R receptor, or used to detect variations in a polynucleotide sequence encoding a T1R receptor, for example a feline T1R receptor.
The invention also embraces DNAs encoding T1R polypeptides that hybridize under high stringency conditions to the non-coding strand, or complement, of the polynucleotides.
Exemplary highly stringent hybridization conditions are as follows: hybridization at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% Dextran sulfate, and washing twice for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described, for example, in Ausubel et al. (Eds.), P
With the knowledge of the nucleotide sequence information disclosed in the present invention, one skilled in the art can identify and obtain nucleotide sequences which encode T1R receptors from different sources (i.e., different tissues or different organisms) through a variety of means well known to the skilled artisan and as disclosed by, for example, Sambrook et al., M
For example, DNA that encodes a T1R receptor may be obtained by screening mRNA, cDNA, or genomic DNA with oligonucleotide probes generated from the T1R gene sequence information provided herein. Probes may be labeled with a detectable group, such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with procedures known to the skilled artisan and used in conventional hybridization assays, as described by, for example, Sambrook et al.
A nucleic acid molecule comprising a T1R nucleotide sequence can alternatively be synthesized by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers produced from the nucleotide sequences provided herein. The PCR reaction provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single- or double-stranded DNA. The essence of the method involves the use of two oligonucleotide probes to serve as primers for the template-dependent, polymerase mediated replication of a desired nucleic acid molecule.
A wide variety of alternative cloning and in vitro amplification methodologies are well known to those skilled in the art. Examples of these techniques are found in, for example, Berger et al., Guide to Molecular Cloning Techniques, M
The polynucleotides of the invention may be used in hybridization techniques known to those skilled in the art, including but not limited to, Northern and Southern blotting and overgo hybridization (see infra). For example, polynucleotide probes of the invention may be used in tissue distribution studies and diagnostic assays. The T1R receptors of the invention are likely to be present and active in tissues other than those involved in taste perception. It is therefore likely that the feline T1R receptors serve multiple functions in vivo, such as, for example, regulation of amino acid metabolism in addition to taste perception.
Automated sequencing methods can be used to obtain or verify the T1R receptor-encoding nucleotide sequence. The nucleotide sequences of the present invention are believed to be accurate. However, as is known in the art, nucleotide sequences obtained by automated methods may contain some errors. Nucleotide sequences determined by automation are typically at least about 90%, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of a given nucleic acid molecule. The actual sequence may be more precisely determined using manual sequencing methods, which are well known in the art. An error in a sequence which results in an insertion or deletion of one or more nucleotides may result in a frame shift in translation such that the predicted amino acid sequence will differ from that which would be predicted from the actual nucleotide sequence of the nucleic acid molecule, starting at the point of the mutation.
The nucleic acid molecules of the present invention, and fragments derived therefrom, are useful for screening for restriction fragment length polymorphisms (RFLP) associated with certain disorders, for genetic mapping, and for methods for predicting the taste perception of an organism such as a mammal involving detection of a nucleotide sequence of the invention in a biological sample of the organism. For example, a mammal having a polynucleotide of the invention may be attracted to the taste of one or more amino acids and/or be insensitive or indifferent to one or more carbohydrate or high-intensity sweeteners.
The polynucleotide sequence information provided by the invention makes possible large-scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art.
Vectors
Another aspect of the present invention is directed to vectors, or recombinant expression vectors, comprising any of the nucleic acid molecules described above. Vectors are used herein either to amplify DNA or RNA encoding a T1R receptor and/or to express DNA which encodes a T1R receptor. Examples of vectors include, but are not limited to, plasmids, phages, cosmids, episomes, viral particles or viruses, and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination). Examples of viral particles include, but are not limited to, adenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses, adeno-associated viruses, Semliki Forest viruses, vaccinia viruses, and retroviruses. Examples of expression vectors include, but are not limited to, pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech). Other expression vectors include, but are not limited to, pSPORT™ vectors, pGEM™ vectors (Promega), pPROEXvectors™ (LTI, Bethesda, Md.), Bluescript™ vectors (Stratagene), pQE™ vectors (Qiagen), pSE420™ (Invitrogen), and pYES2™(Invitrogen).
Expression constructs may comprise T1R-encoding polynucleotides operably linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator. Expression control DNA sequences include promoters, enhancers, operators, and regulatory element binding sites generally, and are typically selected based on the expression systems in which the expression construct is to be utilized. Promoter and enhancer sequences are generally selected for the ability to increase gene expression, while operator sequences are generally selected for the ability to regulate gene expression. Expression constructs of the invention may also include sequences encoding one or more selectable markers that permit identification of host cells bearing the construct. Expression constructs may also include sequences that facilitate, or promote, homologous recombination in a host cell. Constructs of the invention also may include sequences necessary for replication in a host cell.
Expression constructs may be utilized for production of an encoded protein, but may also be utilized simply to amplify a T1R-encoding polynucleotide sequence. In some embodiments, the vector is an expression vector wherein a polynucleotide of the invention is operably linked to a polynucleotide comprising an expression control sequence. Autonomously replicating recombinant expression constructs such as plasmid and viral DNA vectors incorporating polynucleotides of the invention are also provided. Some expression vectors are replicable DNA constructs in which a DNA sequence encoding a T1R receptor is operably linked or connected to suitable control sequence(s) capable of effecting the expression of the receptor in a suitable host. Amplification vectors do not require expression control domains, but rather need only the ability to replicate in a host, such as conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. The need for control sequences in the expression vector will vary depending upon the host selected and the transformation method chosen. Control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding, and sequences which control the termination of transcription and translation.
Vectors of the invention may contain a promoter that is recognized by the host organism. The promoter sequences of the present invention may be prokaryotic, eukaryotic, or viral. Examples of suitable prokaryotic sequences include the PR and PL promoters of bacteriophage lambda (T
Additional regulatory sequences can also be included in vectors of the invention. Examples of suitable regulatory sequences are represented by the Shine-Dalgarno of the replicase gene of the phage MS-2 and of the gene cII of bacteriophage lambda. The Shine-Dalgarno sequence may be directly followed by DNA encoding a T1R receptor, resulting in the expression of the mature protein.
Moreover, suitable expression vectors can include an appropriate marker that allows the screening of transformed host cells. The transformation of the selected host is carried out using any one of the various techniques well known to the expert in the art and described in Sambrook et al., supra.
An origin of replication or autonomously replicating sequence (ARS) can also be provided either by construction of the vector to include an exogenous origin or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient. Alternatively, rather than using vectors which contain viral origins of replication, one skilled in the art can transform mammalian cells by the method of co-transformation with a selectable marker and T1R DNA. An example of a suitable marker is dihydrofolate reductase (DHFR) or thymidine kinase (see U.S. Pat. No. 4,399,216).
Additional regulatory sequences that may be included in the polynucleotides of the invention include secretion signals which allow the encoded polypeptide to cross and/or lodge in cell membranes, or be secreted from the cell.
Nucleotide sequences encoding a T1R receptor may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulation are disclosed by Sambrook et al., supra and are well known in the art. Methods for construction of mammalian expression vectors are disclosed in, for example, Okayama et al., Mol. Cell. Biol., 1983, 3, 280, Cosman et al., Mol. Immunol., 1986, 23, 935, Cosman et al., Nature, 1984, 312, 768, EP-A-0367566, and WO 91/18982, each of which is incorporated herein by reference in its entirety.
Vectors of the invention are useful for expressing T1Rs in various cell systems. Overexpression of a T1R may, for example, be useful in screening for antagonists of the receptor as described herein. Stimulation of transcription of T1R polynucleotides may be used to analyze the effect of T1R expression on the expression of other taste receptors. Vectors may also be used to produce antisense polynucleotides that inhibit endogenous T1R expression to analyze the effect of a loss of the T1R gene.
Host Cells
According to another aspect of the invention, host cells are provided, including prokaryotic and eukaryotic cells, comprising a polynucleotide of the invention (or vector of the invention) in a manner that permits expression of the encoded T1R polypeptide. Polynucleotides of the invention may be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein-coding region or a viral vector. Methods for introducing DNA into the host cell that are well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. Expression systems of the invention include bacterial, yeast, fungal, plant, insect, invertebrate, vertebrate, and mammalian cell systems.
The invention provides host cells that are transformed or transfected (stably or transiently) with polynucleotides of the invention or vectors of the invention. As stated above, such host cells are useful for amplifying the polynucleotides and also for expressing a T1R polypeptide or fragment thereof encoded by the polynucleotide.
In still another related embodiment, the invention provides a method for producing a T1R polypeptide (or fragment thereof) comprising the steps of growing a host cell of the invention in a nutrient medium and isolating the polypeptide or variant thereof from the cell or the medium. Because the T1R receptor is a membrane-spanning polypeptide, it will be appreciated that, for some applications, such as certain activity assays, the preferable isolation may involve isolation of cell membranes containing the polypeptide embedded therein, whereas for other applications a more complete isolation may be preferable.
According to some aspects of the present invention, transformed host cells having an expression vector comprising any of the nucleic acid molecules described above are provided. Expression of the nucleotide sequence occurs when the expression vector is introduced into an appropriate host cell. Suitable host cells for expression of the polypeptides of the invention include, but are not limited to, prokaryotes, yeast, and eukaryotes. If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences. Suitable prokaryotic cells include, but are not limited to, bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces, and Staphylococcus.
If a eukaryotic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequence. Eukaryotic cells may be cells of higher eukaryotes. Suitable eukaryotic cells include, but are not limited to, non-human mammalian tissue culture cells and human tissue culture cells. Host cells include, but are not limited to, insect cells, HeLa cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), human HEK-293 cells, and murine 3T3 fibroblasts. Propagation of such cells in cell culture has become a routine procedure (see, T
In addition, a yeast host may be employed as a host cell. Yeast cells include, but are not limited to, the genera Saccharomyces, Pichia, and Kluveromyces. Yeast hosts may be S. cerevisiae and P. pastoris. Yeast vectors may contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replication sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Shuttle vectors for replication in both yeast and E. coli are also included herein.
Alternatively, insect cells may be used as host cells. In some embodiments, the polypeptides of the invention are expressed using a baculovirus expression system (see, Luckow et al., Bio/Technology, 1988, 6, 47; B
Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immunoreactive with the T1R receptor. Host cells of the invention also are useful in methods for the large-scale production of T1R polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells, or from the medium in which the cells are grown, by purification methods known in the art, e.g., conventional chromatographic methods including immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC, and the like. Still other methods of purification include those methods wherein the desired protein is expressed and purified as a fusion protein having a specific tag, label, or chelating moiety that is recognized by a specific binding partner or agent. The purified protein can be cleaved to yield the desired protein, or can be left as an intact fusion protein. Cleavage of the fusion component may produce a form of the desired protein having additional amino acid residues as a result of the cleavage process.
Knowledge of the feline T1R receptor-encoding nucleotide sequence allows for modification of cells to permit, or increase, expression of endogenous receptor. Cells can be modified (e.g., by homologous recombination) to provide increased expression by replacing, in whole or in part, the naturally occurring T1R promoter with all or part of a heterologous promoter so that the cells express the receptor at higher or lower levels. The heterologous promoter is inserted in such a manner that it is operably linked to endogenous T1R coding sequence. (See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No. WO 91/09955.) It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamoyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the T1R coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the T1R coding sequences in the cells.
Knock-Out and Transplacement Animals
The DNA sequence information provided by the present invention also makes possible the development (e.g., by homologous recombination strategies; see Capecchi, Science 244:1288-1292 (1989), which is incorporated herein by reference) of transgenic or gene-targeted animals, including, for example, animals that fail to express functional T1R (“knock-out”) or that express a variant thereof (“transplacement”). Such animals (especially small laboratory animals such as rats, rabbits, mice, and cats) are useful as models for studying the in vivo activities of T1R receptors and modulators of T1R receptors.
Antisense and siRNA
Also encompassed by the invention are antisense and short interfering polynucleotides that recognize and hybridize to polynucleotides encoding the T1R receptors of the invention. Full-length and fragment antisense polynucleotides are provided. Fragment antisense molecules of the invention include (i) those that specifically recognize and hybridize to T1R RNA (as determined by sequence comparison of DNA encoding T1R receptor to DNA encoding other known molecules). Identification of sequences unique to T1R-encoding polynucleotides can be deduced through use of any publicly available sequence database, and/or through use of commercially available sequence comparison programs. After identification of the desired sequences, isolation through restriction digestion or amplification using any of the various polymerase chain reaction techniques well known in the art can be performed. Antisense polynucleotides are particularly relevant to regulation of expression of T1R receptor by those cells expressing T1R mRNA.
Antisense nucleic acids (preferably 10 to 30 base-pair oligonucleotides) capable of specifically binding to T1R expression control sequences or T1R RNA are introduced into cells (e.g., by a viral vector or colloidal dispersion system such as a liposome). The antisense nucleic acid binds to the target nucleotide sequence in the cell and prevents transcription and/or translation of the target sequence. Phosphorothioate and methylphosphonate antisense oligonucleotides are specifically contemplated for therapeutic use by the invention. Locked nucleic acids are also specifically contemplated for therapeutic use by the present invention. (See, for example, Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 97(10), 5633-5638 (2000), which is incorporated by reference in its entirety) The antisense oligonucleotides may be further modified by adding poly-L-lysine, transferrin polylysine, or cholesterol moieties at their 5′ end. Suppression of T1R expression at either the transcriptional or translational level is useful to generate cellular or animal models for diseases/conditions characterized by aberrant T1R expression.
Antisense oligonucleotides to or fragments of nucleotide sequence of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63, or sequences complementary or homologous thereto, derived from the nucleotide sequences of the present invention encoding T1R receptors are useful as diagnostic tools for probing gene expression in various tissues. For example, tissue can be probed in situ with oligonucleotide probes carrying detectable groups by conventional autoradiography techniques to investigate native expression of this enzyme or pathological conditions relating thereto. Antisense oligonucleotides may be directed to regulatory regions of a T1R nucleotide sequence, or mRNA corresponding thereto, including, but not limited to, the initiation codon, TATA box, enhancer sequences, and the like.
Those of skill in the art recognize that the antisense oligonucleotides that inhibit the expression and/or biological activity of a T1R receptor of the invention may be predicted using any gene encoding a T1R receptor. Specifically, antisense nucleic acid molecules comprise a sequence preferably complementary to at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 250 or 500 nucleotides or an entire T1R receptor gene sequence. The antisense oligonucleotides may comprise a sequence complementary to about 15 consecutive nucleotides of the coding strand of the T1R receptor-encoding sequence.
In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding a T1R protein. The coding strand may also include regulatory regions of the T1R sequence. The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding a T1R protein. The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions (UTR)).
Antisense oligonucleotides may be directed to regulatory regions of a nucleotide sequence encoding a T1R protein, or mRNA corresponding thereto, including, but not limited to, the initiation codon, TATA box, enhancer sequences, and the like. Given the coding strand sequences provided herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of a T1R mRNA, but also may be an oligonucleotide that is antisense to only a portion of the coding or noncoding region of the mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of an mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
Another means to inhibit the activity of a T1R receptor according to the invention is via RNA interference (RNAi) (see e.g., Elbashir et al., Nature, 411:494-498 (2001); Elbashir et al., Genes Development, 15:188-200 (2001)). RNAi is the process of sequence-specific, post-transcriptional gene silencing, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene (e.g., is homologous in sequence to the sequence encoding a T1R receptor, for example but not limited to the sequence as set forth in SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63). siRNA-mediated silencing is thought to occur post-transcriptionally and/or transcriptionally. For example, siRNA duplexes may mediate post-transcriptional gene silencing by reconstitution of siRNA-protein complexes (siRNPs), which guide mRNA recognition and targeted cleavage.
Accordingly, another form of a T1R inhibitory compound of the invention is a short interfering RNA (siRNA) directed against a T1R-encoding sequence. Exemplary siRNAs are siRNA duplexes (for example, 10-25, preferably 20, 21, 22, 23, 24, or 25 residues in length) having a sequence homologous or identical to a fragment of the T1R sequence set forth as SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63 and having a symmetric 2-nucleotide 3′-overhang. The 2-nucleotide 3′ overhang may be composed of (2′-deoxy) thymidine because it reduces costs of RNA synthesis and may enhance nuclease resistance of siRNAs in the cell culture medium and within transfected cells. Substitution of uridine by thymidine in the 3′ overhang is also well tolerated in mammalian cells, and the sequence of the overhang appears not to contribute to target recognition.
Polypeptides
The invention also provides purified and isolated mammalian (e.g., feline) T1R receptor polypeptides and dimers. The T1R polypeptides of the invention may be encoded by a polynucleotide of the invention. Some embodiments include a feline T1R polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64, or fragments thereof comprising an epitope specific to the polypeptide. T1R receptors may form homodimers or heterodimers in taste buds and other tissues to sense molecules. The invention further comprises homodimeric and heterodimeric forms of T1R receptors wherein one T1R receptor molecule associates with a molecule of T1R1, T1R2, or T1R3. A reference to “epitope specific to” or “polypeptide-specific epitope,” or variations thereof, indicates that a portion of the T1R receptor, T1R receptor amino acid sequence, or T1R dimer is recognizable by an antibody that is specific for the T1R or amino acid sequence.
Included within the scope of the invention are polypeptides encoded by feline allelic variants of T1R. The allelic variants of the T1R receptor of the invention may modify the taste perception of a mammal, such as a cat, to a taste stimulus. Such functional amino acid sequence modifications may account for differences in intraspecies (e.g., breed-specific) taste perception.
Extracellular epitopes are useful for generating and screening for antibodies and other binding compounds that bind to a T1R receptor or dimer. Thus, in another embodiment, the invention provides a purified and isolated polypeptide comprising at least one extracellular domain of the T1R receptor or dimer. Also included is a polypeptide comprising a T1R receptor fragment selected from the group consisting of an extracellular domain of T1R3 (residues 1-571, 628-641, 705-730, and 787-794 of SEQ ID NO:2), a transmembrane domain of T1R3 (residues 572-594, 610-627, 642-664, 681-704, 731-754, 767-780, and 795-812 of SEQ ID NO:2), an intracellular domain of T1R3 (residues 595-609, 665-680, 755-766, and 813-865 of SEQ ID NO:2), an extracellular domain of the T1R1 receptor (residues 1-563, 624-635, 701-726, and 781-792 of SEQ ID NO:61), a transmembrane domain of the T1R1 receptor (residues 564-589, 604-623, 636-660, 681-700, 727-748, 761-780, and 793-817 of SEQ ID NO:61), an intracellular domain of the T1R1 receptor (residues 590-603, 661-680, 749-760, and 818-841 of SEQ ID NO:61), an extracellular domain of T1R2 (residues 1-147 of SEQ ID NO:64), a transmembrane domain of a T1R2 receptor (residues 148-167 of SEQ ID NO:64), and an intracellular domain of a T1R2 receptor (residues 168-391 of SEQ ID NO:64). Polypeptide fragments of the invention may be continuous portions of the native receptor. However, it will also be appreciated that knowledge of the T1R genes and protein sequences as provided herein permits recombination of various domains that are not contiguous in the native protein.
The invention embraces polypeptides that preferably have at least about 99%, at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 74%, at least about 73%, at least about 72%, at least about 71%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, or at least about 50% identity and/or homology to the polypeptides of the invention.
Polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention. Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention.
The invention also embraces variant T1R polypeptides. In one example, insertion variants are provided wherein one or more amino acid residues supplement a T1R amino acid sequence such as SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the amino acid sequence. Insertional variants with additional residues at either or both termini can include, for example, fusion proteins and proteins including amino acid tags or labels.
Insertion variants include T1R polypeptides wherein one or more amino acid residues are added to a biologically active fragment thereof. For example, the insertion variants of the invention include chimeric T1R receptors wherein at least one functional domain of a feline T1R receptor of the invention is present.
The invention also embraces T1R variants having additional amino acid residues that result from use of specific expression systems. For example, use of commercially available vectors that express a desired polypeptide as part of a glutathione-S-transferase (GST) fusion product provides the desired polypeptide having an additional glycine residue at position −1 after cleavage of the GST component from the desired polypeptide. Variants that result from expression in other vector systems are also contemplated.
In another aspect, the invention provides deletion variants wherein one or more amino acid residues in a T1R polypeptide are removed. Deletions can be effected at one or both termini of the T1R polypeptide, or with removal of one or more non-terminal amino acid residues of T1R. Deletion variants, therefore, include all fragments of a T1R polypeptide.
The invention also embraces polypeptide fragments that maintain biological (e.g., ligand binding, dimerization, receptor activity) and/or immunological properties of a T1R polypeptide.
As used in the present invention, polypeptide fragments preferably comprise at least 10, 15, 20, 25, 30, 35, 40, 45, or 50 consecutive amino acids of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64. Some polypeptide fragments display antigenic properties unique to, or specific for, a feline T1R receptor. Fragments of the invention having the desired biological and immunological properties can be prepared by any of the methods well known and routinely practiced in the art.
In still another aspect, the invention provides substitution variants of T1R polypeptides. Substitution variants include those polypeptides wherein one or more amino acid residues of a T1R polypeptide are removed and replaced with alternative residues. In one aspect, the substitutions are conservative in nature; however, the invention embraces substitutions that are also non-conservative. Conservative substitutions for this purpose may be defined as set out in Tables 1, 2, or 3 below.
Variant polypeptides include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table 1 (from WO 97/09433, page 10, published Mar. 13, 1997 (PCT/GB96/02197, filed Sep. 6, 1996), immediately below.
Alternatively, conservative amino acids can be grouped as described in Lehninger, [B
As still another alternative, exemplary conservative substitutions are set out in Table 3, below.
It should be understood that the definition of polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues. By way of example, the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and inorganic moieties. Such derivatives may be prepared to increase circulating half-life of a polypeptide, or may be designed to improve the targeting capacity of the polypeptide for desired cells, tissues, or organs. Similarly, the invention further embraces T1R polypeptides that have been covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. Variants that display ligand binding properties of native T1R and are expressed at higher levels, as well as variants that provide for constitutively active receptors, are particularly useful in assays of the invention; the variants are also useful in providing cellular, tissue and animal models of diseases/conditions characterized by aberrant T1R activity.
In a related embodiment, the present invention provides compositions comprising purified polypeptides of the invention. Some compositions comprise, in addition to the polypeptide of the invention, a pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluent that serves as a pharmaceutical vehicle, excipient, or medium. Any diluent known in the art may be used. Exemplary diluents include, but are not limited to, water, saline solutions, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, glycerol, calcium phosphate, mineral oil, and cocoa butter.
Variants that display ligand-binding properties of native T1R and are expressed at higher levels, as well as variants that provide for constitutively active receptors, are particularly useful in assays of the invention; the variants are also useful in assays of the invention and in providing cellular, tissue and animal models of diseases/conditions characterized by aberrant T1R activity.
Antibodies
Also included in the present invention are antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, felinized antibodies, feline antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for a T1R receptor of the invention, a fragment thereof, or dimers of the T1R receptors of the invention (e.g., T1R1-T1R3; T1R2-T1R3; and T1R3-T1R3 dimers). Antibody fragments, including Fab, Fab′, F(ab′)2, and Fv, are also provided by the invention. The term “specific for,” when used to describe antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind T1R polypeptides, preferably exclusively (i.e., are able to distinguish T1R polypeptides of the invention from other known polypeptides by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between T1R and such polypeptides). It will be understood that specific antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and, in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds.), A
The invention provides an antibody that is specific for the feline T1R receptors of the invention. Antibodies that can be generated from polypeptides that have previously been described in the literature and that are capable of fortuitously cross-reacting with feline T1R receptor (e.g., due to the fortuitous existence of a similar epitope in both polypeptides) are considered “cross-reactive” antibodies. Such cross-reactive antibodies are not antibodies that are “specific” for a feline T1R receptor. The determination of whether an antibody is specific for a feline T1R receptor or is cross-reactive with another known receptor is made using any of several assays, such as Western blotting assays, that are well known in the art. For identifying cells that express a T1R receptor and also for modulating T1R-ligand binding activity, antibodies that specifically bind to an extracellular epitope of the T1R receptor may be used.
In some embodiments of the invention, the antibodies specifically bind T1R polypeptides, or block ligands or binding partners from binding to the T1R polypeptides. These antibodies may also block the biological activity of the T1R polypeptides. In other embodiments, the antibodies preferentially bind T1R polypeptides of a certain species or family of T1R polypeptides.
In some variations, the invention provides monoclonal antibodies. Hybridomas that produce such antibodies also are intended as aspects of the invention. In yet another variation, the invention provides a felinized antibody. Felinized antibodies are useful for in vivo therapeutic indications.
In another variation, the invention provides a cell-free composition comprising polyclonal antibodies, wherein at least one of the antibodies is an antibody of the invention specific for T1R receptor. Antisera isolated from an animal is an exemplary composition, as is a composition comprising an antibody fraction of an antisera that has been resuspended in water or in another diluent, excipient, or carrier.
In still another related embodiment, the invention provides an anti-idiotypic antibody specific for an antibody that is specific for T1R receptor of the invention.
It is well known that antibodies contain relatively small antigen binding domains that can be isolated chemically or by recombinant techniques. Such domains are useful T1R receptor binding molecules themselves, and also may be reintroduced into other antibodies or fused to toxins or other polypeptides. Thus, in still another embodiment, the invention provides a polypeptide comprising a fragment of a T1R-specific antibody, wherein the fragment and the polypeptide bind to the T1R receptor. By way of non-limiting example, the invention provides polypeptides that are single chain antibodies and CDR-grafted antibodies.
Non-feline antibodies may be felinized by any of the methods known in the art. In one method, the non-feline CDRs are inserted into a feline antibody or consensus antibody framework sequence. Similarly, non-human antibodies may be humanized by methods known in the art. In one embodiment, non-human CDRs are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.
Antibodies of the invention are useful for, e.g., therapeutic purposes (such as by modulating activity of T1R receptor), diagnostic purposes (such as detecting or quantitating T1R receptor activity), and also for purification of T1R receptor. Kits comprising an antibody of the invention for any of the purposes described herein are also included within the scope of the invention. In general, a kit of the invention preferably includes a control antigen for which the antibody is immunospecific.
Compositions
Mutations in the feline T1R gene that result in loss of normal function of the T1R gene product underlie some T1R-related disease states. The invention comprehends gene and peptide therapy, for example, to restore T1R activity to treat those disease states. Delivery of a functional T1R gene to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). See, for example, Anderson, Nature, supplement to vol. 392, No. 6679, pp. 25-20 (1998). For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992). Alternatively, it is contemplated that in other disease states, preventing the expression of, or inhibiting the activity of, T1R receptor will be useful in treatment. It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of T1R receptor.
Another aspect of the present invention is directed to compositions, including pharmaceutical compositions, comprising any of the nucleic acid molecules or recombinant expression vectors described above and an acceptable carrier or diluent. The carrier or diluent may be pharmaceutically acceptable. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field, which is incorporated herein by reference in its entirety. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used. The formulations may be sterilized by commonly used techniques.
Also within the scope of the invention are compositions comprising polypeptides, polynucleotides, or antibodies of the invention that have been formulated with, e.g., a pharmaceutically acceptable carrier.
The invention also provides methods of using antibodies of the invention. For example, the invention provides a method for modulating ligand-binding of a T1R receptor comprising the step of contacting the receptor with an antibody specific for the T1R polypeptide, under conditions wherein the antibody binds the receptor.
Methods of Identifying Ligands and Modulators
The invention also provides assays to identify compounds that bind and/or modulate T1R receptor. A “T1R binding partner” is a compound that directly or indirectly binds a T1R polypeptide of the invention. One assay of the invention comprises the steps of: (a) contacting T1R receptor or T1R dimer of the invention with a compound suspected of binding T1R receptor or dimer (the test compound); and (b) measuring binding between the compound and the T1R receptor or dimer. In one variation, the composition comprises a cell expressing T1R receptor or dimer on its surface. In another variation, isolated T1R receptor or dimer or cell membranes comprising T1R receptor or dimer are employed. The binding may be measured directly, e.g., by using a labeled compound, or may be measured indirectly. Compounds identified as binding a T1R receptor or T1R dimer may be further tested in other assays including, but not limited to, T1R activity assays and/or in vivo models, in order to confirm or quantitate their activity.
Specific binding molecules, including natural ligands and synthetic compounds, can be identified or developed using isolated or recombinant T1R products, T1R variants, or preferably, cells expressing such products. Binding partners are useful for purifying T1R products and detection or quantification of T1R products in fluid and tissue samples using known immunological procedures. Binding molecules are also manifestly useful in modulating (i.e., blocking, inhibiting or stimulating) biological activities of T1R, especially those activities involved in signal transduction. Binding molecules also are useful in methods for predicting the taste perception of an organism such as a mammal by detecting a polypeptide of the invention in a biological sample of the organism. For example, an organism in which a polypeptide of the invention has been identified may be attracted to the taste of amino acids and/or indifferent to the taste of carbohydrate and high-intensity sweeteners.
The DNA and amino acid sequence information provided by the present invention also makes possible identification of binding partner compounds with which a T1R polypeptide or polynucleotide will interact. Methods to identify binding partner compounds include solution assays, in vitro assays wherein T1R polypeptides are immobilized, and cell-based assays. Identification of binding partner compounds of T1R polypeptides provides candidates for therapeutic or prophylactic intervention in pathologies associated with T1R normal and aberrant biological activity.
The invention includes several assay systems for identifying T1R-binding partners. In solution assays, methods of the invention comprise the steps of (a) contacting a T1R receptor or dimer with one or more candidate binding partner compounds and (b) identifying the compounds that bind to the T1R receptor or dimer. Identification of the compounds that bind the T1R receptor or dimer can be achieved by isolating the T1R polypeptide/binding partner complex, and separating the binding partner compound from the T1R polypeptide. An additional step of characterizing the physical, biological, and/or biochemical properties of the binding partner compound is also comprehended in another embodiment of the invention. In one aspect, the T1R polypeptide/binding partner complex is isolated using an antibody immunospecific for either the T1R receptor or dimer or the candidate binding partner compound.
In still other embodiments, either the T1R receptor or dimer or the candidate binding partner compound comprises a label or tag that facilitates its isolation, and methods of the invention to identify binding partner compounds include a step of isolating the T1R polypeptide/binding partner complex through interaction with the label or tag. An exemplary tag of this type is a poly-histidine sequence, generally around six histidine residues, that permits isolation of a compound so labeled using nickel chelation. Other labels and tags, such as the FLAG tag (Eastman Kodak, Rochester, N.Y.), well known and routinely used in the art, are embraced by the invention.
In one variation of an in vitro assay, the invention provides a method comprising the steps of (a) contacting an immobilized T1R receptor or dimer with a candidate binding partner compound and (b) detecting binding of the candidate compound to the T1R receptor or dimer. In an alternative embodiment, the candidate binding partner compound is immobilized and binding of T1R receptor or dimer is detected. Immobilization is accomplished using any of the methods well known in the art, including covalent bonding to a support, a bead, or a chromatographic resin, as well as non-covalent, high affinity interactions such as antibody binding, or use of streptavidin/biotin binding wherein the immobilized compound includes a biotin moiety. The support may, for example, be formulated into a feline-specific electronic tongue or biosensor. Detection of binding can be accomplished (i) using a radioactive label on the compound that is not immobilized, (ii) using a fluorescent label on the non-immobilized compound, (iii) using an antibody immunospecific for the non-immobilized compound, (iv) using a label on the non-immobilized compound that excites a fluorescent support to which the immobilized compound is attached, as well as other techniques well known and routinely practiced in the art.
The invention also provides cell-based assays to identify binding partner compounds of a T1R receptor or dimer. In one embodiment, the invention provides a method comprising the steps of contacting a T1R receptor or dimer expressed on the surface of a cell with a candidate binding partner compound and detecting binding of the candidate binding partner compound to the T1R receptor or dimer. In some embodiments, the detection comprises detecting physiological event in the cell caused by the binding of the molecule.
Another aspect of the present invention is directed to methods of identifying compounds that bind to either T1R receptor or dimer or nucleic acid molecules encoding T1R receptor, comprising contacting T1R receptor or dimer, or a nucleic acid molecule encoding the same, with a compound, and determining whether the compound binds T1R receptor or dimer or a nucleic acid molecule encoding the same. Binding can be determined by binding assays which are well known to the skilled artisan, including, but not limited to, gel-shift assays, Western blots, radiolabeled competition assay, phage-based expression cloning, co-fractionation by chromatography, co-precipitation, cross-linking, interaction trap/two-hybrid analysis, southwestern analysis, ELISA, and the like, which are described in, for example, C
In another embodiment of the invention, high throughput screening for compounds having suitable binding affinity to T1R receptor or dimer is employed. Briefly, large numbers of different small peptide test compounds are synthesized on a solid substrate. The peptide test compounds are contacted with T1R receptor or dimer and washed. Bound T1R receptor or dimer is then detected by methods well known in the art. Purified polypeptides of the invention can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the protein and immobilize it on the solid support.
Generally, an expressed T1R receptor can be used for HTS binding assays in conjunction with a ligand, such as an amino acid or carbohydrate. The identified peptide is labeled with a suitable radioisotope, including, but not limited to, 125I, 3H, 35S or 32P, by methods that are well known to those skilled in the art. Alternatively, the peptides may be labeled by well-known methods with a suitable fluorescent derivative (Baindur et al., Drug Dev. Res., 1994, 33, 373-398; Rogers, Drug Discovery Today, 1997, 2, 156-160). Radioactive ligand specifically bound to the receptor in membrane preparations made from the cell line expressing the recombinant protein can be detected in HTS assays in one of several standard ways, including filtration of the receptor-ligand complex to separate bound ligand from unbound ligand (Williams, Med. Res. Rev., 1991, 11, 147-184; Sweetnam et al., J. Natural Products, 1993, 56, 441-455). Alternative methods include a scintillation proximity assay (SPA) or a FlashPlate format in which such separation is unnecessary (Nakayama, Cur. Opinion Drug Disc. Dev., 1998, 1, 85-91; Bossé et al., J. Biomolecular Screening, 1998, 3, 285-292.). Binding of fluorescent ligands can be detected in various ways, including fluorescence energy transfer (FRET), direct spectrophotofluorometric analysis of bound ligand, or fluorescence polarization (Rogers, Drug Discovery Today, 1997, 2, 156-160; Hill, Cur. Opinion Drug Disc. Dev., 1998, 1, 92-97).
Other assays may be used to identify specific ligands of a T1R receptor or dimer, including assays that identify ligands of the target protein through measuring direct binding of test ligands to the target, as well as assays that identify ligands of target proteins through affinity ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods. Alternatively, such binding interactions are evaluated indirectly using the yeast two-hybrid system described in Fields et al., Nature, 340:245-246 (1989), and Fields et al., Trends in Genetics, 10:286-292 (1994), both of which are incorporated herein by reference. The two-hybrid system is a genetic assay for detecting interactions between two proteins or polypeptides. It can be used to identify proteins that bind to a known protein of interest, or to delineate domains or residues critical for an interaction. Variations on this methodology have been developed to clone genes that encode DNA binding proteins, to identify peptides that bind to a protein, and to screen for drugs. The two-hybrid system exploits the ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with a DNA binding domain that binds to an upstream activation sequence (UAS) of a reporter gene, and is generally performed in yeast. The assay requires the construction of two hybrid genes encoding (1) a DNA-binding domain that is fused to a first protein and (2) an activation domain fused to a second protein. The DNA-binding domain targets the first hybrid protein to the UAS of the reporter gene; however, because most proteins lack an activation domain, this DNA-binding hybrid protein does not activate transcription of the reporter gene. The second hybrid protein, which contains the activation domain, cannot by itself activate expression of the reporter gene because it does not bind the UAS. However, when both hybrid proteins are present, the noncovalent interaction of the first and second proteins tethers the activation domain to the UAS, activating transcription of the reporter gene. For example, when the first protein is a receptor, or fragment thereof, that is known to interact with another protein or nucleic acid, this assay can be used to detect agents that interfere with the binding interaction. Expression of the reporter gene is monitored as different test agents are added to the system. The presence of an inhibitory agent results in lack of a reporter signal.
The yeast two-hybrid assay can also be used to identify proteins that bind to the gene product. In an assay to identify proteins that bind to a T1R receptor, or fragment thereof, a fusion polynucleotide encoding both a T1R receptor (or fragment) and a UAS binding domain (i.e., a first protein) may be used. In addition, a large number of hybrid genes each encoding a different second protein fused to an activation domain are produced and screened in the assay. Typically, the second protein is encoded by one or more members of a total cDNA or genomic DNA fusion library, with each second protein-coding region being fused to the activation domain. This system is applicable to a wide variety of proteins, and it is not necessary to know the identity or function of the second binding protein. The system is highly sensitive and can detect interactions not revealed by other methods; even transient interactions may trigger transcription to produce a stable mRNA that can be repeatedly translated to yield the reporter protein.
Other assays may be used to search for agents that bind to the target protein. One such screening method to identify direct binding of test ligands to a target protein is described in U.S. Pat. No. 5,585,277, incorporated herein by reference. This method relies on the principle that proteins generally exist as a mixture of folded and unfolded states, and continually alternate between the two states. When a test ligand binds to the folded form of a target protein (i.e., when the test ligand is a ligand of the target protein), the target protein molecule bound by the ligand remains in its folded state. Thus, the folded target protein is present to a greater extent in the presence of a test ligand which binds the target protein, than in the absence of a ligand. Binding of the ligand to the target protein can be determined by any method that distinguishes between the folded and unfolded states of the target protein. The function of the target protein need not be known in order for this assay to be performed. Virtually any agent can be assessed by this method as a test ligand, including, but not limited to, metals, polypeptides, proteins, lipids, polysaccharides, polynucleotides and small organic molecules.
Another method for identifying ligands of a target protein is described in Wieboldt et al., Anal. Chem., 69:1683-1691 (1997), incorporated herein by reference. This technique screens combinatorial libraries of 20-30 agents at a time in solution phase for binding to the target protein. Agents that bind to the target protein are separated from other library components by simple membrane washing. The specifically selected molecules that are retained on the filter are subsequently liberated from the target protein and analyzed by HPLC and pneumatically assisted electrospray (ion spray) ionization mass spectroscopy. This procedure selects library components with the greatest affinity for the target protein, and is particularly useful for small molecule libraries.
Other embodiments of the invention comprise using competitive screening assays in which neutralizing antibodies capable of binding a polypeptide of the invention specifically compete with a test compound for binding to the polypeptide. In this manner, the antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with T1R receptor. Radiolabeled competitive binding studies are described in A. H. Lin et al., Antimicrobial Agents and Chemotherapy, 1997, 41(10): 2127-2131, the disclosure of which is incorporated herein by reference in its entirety.
Another aspect of the present invention is directed to methods of identifying compounds that modulate (i.e., increase or decrease) activity of T1R receptor or dimer comprising contacting T1R receptor or dimer with a compound, and determining whether the compound modifies activity of T1R receptor or dimer. The activity in the presence of the test compound is compared to the activity in the absence of the test compound. Where the activity of the sample containing the test compound is higher than the activity in the sample lacking the test compound, the compound is an agonist. Similarly, where the activity of the sample containing the test compound is lower than the activity in the sample lacking the test compound, the compound is an antagonist.
Agents that modulate (i.e., increase, decrease, or block) T1R receptor or dimer activity or expression also may be identified, for example, by incubating a putative modulator with a cell containing a T1R polypeptide, dimer, or polynucleotide and determining the effect of the putative modulator on T1R receptor activity or expression. The selectivity of a compound that modulates the activity of T1R receptor or dimer can be evaluated by comparing its effects on T1R receptor or dimer to its effect on other T1R receptors or dimers. Selective modulators may include, for example, antibodies and other proteins, peptides, or organic molecules that specifically bind to a T1R polypeptide or a T1R receptor-encoding nucleic acid. Modulators of T1R receptor activity will be therapeutically useful in treatment of diseases and physiological conditions in which normal or aberrant T1R receptor activity is involved. Compounds identified as modulating T1R receptor activity may be further tested in other assays including, but not limited to, in vivo models, in order to confirm or quantitate their activity.
The invention also provides methods for identifying a T1R receptor modulator by: (a) contacting a T1R receptor (or dimer) binding partner and a composition comprising a T1R receptor in the presence and in the absence of a putative modulator compound; (b) detecting binding between the binding partner and the T1R receptor; and (c) identifying a putative modulator compound or a modulator compound in view of decreased or increased binding between the binding partner and the T1R receptor in the presence of the putative modulator, as compared to binding in the absence of the putative modulator. Compounds identified as modulators of binding between T1R receptor and a T1R binding partner may be further tested in other assays including, but not limited to, in vivo models, in order to confirm or quantitate their activity.
The invention also includes within its scope high-throughput screening (HTS) assays to identify compounds that interact with, enhance, or inhibit biological activity (i.e., affect enzymatic activity, binding activity, etc.) of a T1R receptor or dimer. HTS assays permit screening of large numbers of compounds in an efficient manner. Cell-based HTS systems are contemplated to investigate T1R receptor-ligand interaction. HTS assays are designed to identify “hits” or “lead compounds” having the desired property, from which modifications can be designed to improve the desired property. Chemical modification of the “hit” or “lead compound” is often based on an identifiable structure/activity relationship between the “hit” and the T1R polypeptide.
For example, modulators of T1R receptor activity may be identified by expressing the T1R receptor in a heterologous cultured mammalian cell line, such as HEK cells, and detecting receptor activity in the presence and absence of a test compound by monitoring changes in intracellular calcium using a calcium-specific intracellular dye. In another embodiment, this process may be automated using a high-throughput screening device.
Candidate modulators contemplated by the invention include compounds selected from libraries of either potential activators or potential inhibitors. There are a number of different libraries used for the identification of small molecule modulators, including: (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides, or organic molecules. Chemical libraries consist of random chemical structures, some of which are analogs of known compounds or analogs of compounds that have been identified as “hits” or “leads” in other drug discovery screens, some of which are derived from natural products, and some of which arise from non-directed synthetic organic chemistry. Natural product libraries are collections of microorganisms, animals, plants, or marine organisms that are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant, or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof. For a review, see Science 282:63-68 (1998). Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture. These libraries are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or proprietary synthetic methods. Of particular interest are non-peptide combinatorial libraries. Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997). Identification of modulators through use of the various libraries described herein permits modification of the candidate “hit” (or “lead”) to optimize the capacity of the “hit” to modulate activity.
T1R receptor binding partners that stimulate T1R receptor activity are useful as agonists in disease states or conditions characterized by insufficient T1R receptor signaling (e.g., as a result of insufficient activity of a T1R receptor ligand). T1R receptor binding partners that block ligand-mediated T1R receptor signaling are useful as T1R receptor antagonists to treat disease states or conditions characterized by excessive T1R receptor signaling. Thus, in another aspect, the invention provides methods for treating a disease or abnormal condition by administering to a patient in need of such treatment a substance that modulates the activity or expression of a polypeptide having a sequence of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64, or exhibiting substantially the same biological activity as a polypeptide having a sequence of SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64, or dimers thereof.
In addition T1R receptor modulators in general, as well as T1R receptor encoding polynucleotides and polypeptides, are useful in diagnostic assays for such diseases or conditions.
The T1R polynucleotides of the invention may also be used to identify compounds for which an organism having the receptor exhibits a taste preference or indifference using cell signaling assays known in the art. In such assays, polynucleotide encoding a T1R is incorporated into an expression vector and transfected into a host cell. The expression of the T1R may be inducible or constitutive. The host cells expressing the T1R receptor are contacted with candidate compounds and the effect of each compound on the cells is assayed. Stimulation of a response is indicative of reactivity to the test compound and correlates with compounds associated with a taste preference. The assays that can be used to assess stimulation of T1R receptor include, but are not limited to assays measuring ion conductance, ion flow, calcium imaging (e.g., using fura-2, green dextran activity or aequorin activity), voltage measurement and or voltage imaging with dyes, expression of reporter genes (e.g., luciferase, alkaline phosphatase, beta-galactosidase, beta-lactamase, fluorescent binding protein), receptor binding assays, second messenger assays (e.g., IP3, cAMP), G-protein activation based assays (e.g., modulation of GTP-gamma-S binding), receptor phosphorylation measures, and the like. In some embodiments analysis of stimulation of T1R receptors and receptor dimers may be determined using a FLIPR® assay as described by the manufacturer (Molecular Devices, Corp.).
Screening of cells treated with dyes and fluorescent reagents is well known in the art. Genetic engineering of cells to produce fluorescent proteins, such as modified green fluorescent protein (GFP), as a reporter molecule is also well known in the art. Fluorescence-based reagents are useful for the assay of many cell functions including ion concentrations, membrane potential, specific translocations, enzyme activities, gene expression, as well as the presence, amounts and patterns of metabolites, proteins, lipids, carbohydrates, and nucleic acid sequences.
Cell signaling assays known in the art may also be used to identify T1R antagonists. Expression of G protein coupled receptors at very high concentration in a heterologous system has been shown to result in constitutive cell signaling. For example, but not by way of limitation, T1R receptor may be overexpressed in Spodoptera frugiperda (Sf9) cells. Alternatively, for example, T1R may be operably linked to a CMV promoter and expressed in COS or HEK293 cells. In the activated constitutive state, test compounds may be assayed for their ability to inhibit constitutive cell signaling activity. Suitable assays include, but are not limited to assays measuring ion conductance, ion flow, calcium imaging (e.g., using fura-2, green dextran activity or aequorin activity), voltage measurement and/or voltage imaging with dyes, expression of reporter genes (e.g., luciferase, alkaline phosphatase, beta-galactosidase, beta-lactamase, fluorescent binding protein), receptor binding assays, second messenger assays (e.g., IP3, cAMP), G-protein activation based assays (e.g., modulation of GTP-gamma-S binding), receptor phosphorylation measures, and the like.
Tissue Distribution
One skilled in the art may determine whether a T1R receptor is expressed in tissues other than taste bud tissues through routine experimentation following the guidance provided herein. Specifically, one may detect T1R RNA using Northern blots or reverse transcriptase polymerase chain reaction (rtPCR) and other methods known in the art. Protocols and procedures for such assays may be found, for example in Ausubel et al., C
One may also detect T1R protein using specific antibodies and probing tissue sections. For example, but not by way of limitation, one may obtain paraffin-embedded sections of tissues of interest (from human or other species) or use fresh tissue. Tissue sections may be treated with 0.3% H2O2 in methanol for 30 minutes to eliminate endogenous peroxidase activity. The tissues may then be incubated in anti-T1R antibody (or an antisera raised against T1R) at 4° C. for hours to days. The tissues may then be treated with a secondary antibody that specifically binds to the primary antibody (such as a heterologous species' serum raised against the primary species immunoglobulin). The secondary antibody may be conjugated to biotin, for example. The tissue sections are further incubated with peroxidase-conjugated streptavidin at room temperature. After each step, tissue sections are rinsed in 0.01 M phosphate buffered saline (pH 7.2) containing 0.05% Tween 20. Immunoreaction products may be visualized through reaction with 0.0125% diaminobenzidine (DAB) and 0.002% H2O2 in 0.05M Tris-HCl buffer (pH 7.6). The sections are counterstained with hematoxylin and viewed with a light microscope. Other antibody-based assays to examine tissues for T1R expression may be used. Various protocols are known in the art and may be found, for example, in Harlow et al. (Eds.), A
The presence of T1R receptor in tissues outside of taste buds suggests a role for T1R receptors other than taste perception. The invention provides methods for modulating these functions by increasing or decreasing the expression of T1R.
As such, the invention encompasses a method of modulating a T1R receptor family member by modulating expression of another T1R family receptor. For example, the Tas1r gene may be expressed such that an increased level of Tas1r RNA results in feedback inhibition of expression of at least one of T1R1, T1R2, and T1R3. In other embodiments of the invention, the Tas1r gene may be expressed such that an increased level of T1R RNA results in an increase in the expression of at least one of T1R1, T1R2, and T1R3. In other embodiments, the Tas1r gene may be repressed such that a decreased level of Tas1r RNA results in an increase in the expression of at least one of T1R1, T1R2, and T1R3. In other embodiments, the Tas1r gene may be repressed such that a decreased level of Tas1r RNA results in a concomitant decrease in the expression of at least one of T1R1, T1R2, and T1R3. The gene modulation may be tissue- or species-specific. Thus, expression of Tas1r may be regulated using the expression vectors of the invention using inducible promoters and tissue-specific promoters, for example. Alternatively, expression of Tas1r may be repressed in cells that produce T1R using antisense RNA and other inhibitory strategies described herein and as are known in the art. The modulation of the T1R receptor family in patients may be useful for altering metabolism, nutrient uptake, neural function, and to treat disorders associated with an abnormally low or high level of T1R expression in cells.
Mimetics
Mimetics or mimics of compounds identified herein (sterically similar compounds formulated to mimic the key portions of the structure) may be designed for pharmaceutical use. Mimetics may be used in the same manner as the compounds identified by the present invention that modulate the T1R receptor and hence are also functional equivalents. The generation of a structural-functional equivalent may be achieved by the techniques of modeling and chemical design known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
The design of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a “lead” compound. This is desirable where, for example, the active compound is difficult or expensive to synthesize, or where it is unsuitable for a particular method of administration, e.g., some peptides may be unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.
There are several steps commonly taken in the design of a mimetic. First, the particular parts of the compound that are critical and/or important in determining its T1R-modulating properties are determined. In the case of a polypeptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. Alanine scans of peptides are commonly used to refine such peptide motifs.
Once the active region of the compound has been identified, its structure is modeled according to its physical properties, e.g. stereochemistry, bonding, size, and/or charge, using data from a range of sources, such as, but not limited to, spectroscopic techniques, X-ray diffraction data, and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of the active region, rather than the bonding between atoms), and other techniques known to those of skill in the art can be used in this modeling process.
In a variant of this approach, the three-dimensional structure of the compound that modulates a T1R receptor and the active region of the T1R receptor are modeled. This can be especially useful where either or both of these compounds change conformation upon binding. Knowledge of the structure of the ligand-binding domain (for example, residues 1-571 of SEQ ID NO:2) of the receptor also allows the design of high potency ligands and/or modulators.
A template molecule is then selected onto which chemical groups that mimic the T1R modulator can be grafted. The template molecule and the chemical groups grafted onto it can conveniently be selected so that the mimetic is easy to synthesize, is pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. Alternatively, where the mimetic is peptide-based, further stability can be achieved by cyclizing the peptide, thereby increasing its rigidity. The mimetic or mimetics found by this approach can then be screened by the methods of the present invention to see whether they have the ability to modulate the T1R receptor. Further optimization or modification can then be performed to arrive at one or more final mimetics for in vivo or clinical testing.
Compositions of Binding and/or Modulating Compounds
Following identification of a compound that binds and/or or modulates a T1R receptor, the compound may be manufactured and/or used in preparation of compositions including, but not limited to, foods, drinks, and pharmaceutical compositions. The compositions are provided or administered to patients, including, but not limited to, avians, felines, canines, bovines, ovines, porcines, equines, rodents, simians, and humans.
Thus, the present invention extends, in various aspects, not only to compounds identified in accordance with the methods disclosed herein but also foods, drinks, pharmaceutical compositions, drugs, or other compositions comprising such a compound; methods comprising administration of such a composition to a patient, e.g. for treatment (which includes prophylactic treatment) of a T1R receptor-associated disorder (e.g., obesity, diabetes); uses of such a compound in the manufacture of a composition for administration to a patient; and methods of making a composition comprising admixing such a compound with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.
Some compositions of the invention comprise a taste-modifying amount of at least one or more binding or modulating compounds. A “taste-modifying amount” is a quantity sufficient to increase or decrease the perception of a taste stimulus by a given mammal. The food and drink compositions of the invention are formulated by the addition of a binding or modulating compound to a food or drink of the mammal. Such compositions may be individualized or breed-specific. For example, feline veterinary specialty diets may thus be made more palatable.
The pharmaceutical compositions of the invention comprise a therapeutically effective amount of a compound identified according to the methods disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
Pharmaceutically acceptable carriers include but are not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile. The formulation should suit the mode of administration.
The composition, if desired, can also contain minor amounts of wetting or emulsifying agents or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
The pharmaceutical compositions of the invention may further comprise a secondary compound for the treatment of a disorder unrelated to the T1R receptor, such as an antibiotic or other therapeutic agent, to improve the palatability of the pharmaceutical composition, thereby improving the ease of administration.
In one embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for oral (e.g., tablets, granules, syrups) or non-oral (e.g., ointments, injections) administration to the subject. Various delivery systems are known and can be used to administer a compound that modulates a T1R receptor, e.g., encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated endocytosis, construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, topical, and oral routes.
The compounds of the invention may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents, for example in HAART therapy. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery; topical application, e.g., in conjunction with a wound dressing after surgery; by injection; by means of a catheter; by means of a suppository; or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
The composition can be administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in R
Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry-lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
Methods for Predicting and Representing Taste Perception of an Organism
Methods for predicting the taste perception of an organism, such as a mammal, include detection of a nucleotide sequence (e.g., nucleotide sequence of SEQ ID NO:1, SEQ ID NO:99, SEQ ID NO:59; SEQ ID NO:60, SEQ ID NO:62, or SEQ ID NO:63, or fragments, variants, or homologues thereof encoding a polypeptide having an equivalent, substantially the same, or same biological activity as the polypeptide encoded by the reference sequence) or amino acid sequence (e.g., SEQ ID NO:2, SEQ ID NO:61, or SEQ ID NO:64, or fragments, variants, or homologues thereof having the same or substantially the same biological activity of the reference polypeptide) of the invention in a biological sample of the organism. Methods for detecting a polynucleotide or polypeptide of the invention in a biological sample may comprise any method known to the skilled artisan, including but not limited to the methods of detection mentioned herein. An organism having a polynucleotide or polypeptide of the invention in its biological sample is predicted to exhibit characteristics of taste perception of a domestic cat. For example, an organism having a polynucleotide or polypeptide of the invention, such as a polynucleotide encoding a feline T1R1 receptor or the polypeptide encoded thereby, will be attracted to one or more amino acids. An organism having a polynucleotide or polypeptide of the invention may show no preference for one or more carbohydrates or high-intensity sweeteners. As an example, an organism having a polynucleotide encoding a single transmembrane domain T1R2 receptor, for example the feline T1R2 receptor of the invention, or the polypeptide encoded thereby, would be predicted to show a reduced or no response to sweet taste stimuli relative to an organism having a 7-transmembrane domain T1R2 receptor An organism having a polynucleotide or polypeptide of the invention may be attracted to one or more amino acids and may exhibit no preference for one or more carbohydrates or high-intensity sweeteners. For example, an organism having a polynucleotide or polypeptide of the invention, such as a polynucleotide encoding a feline T1R1 receptor or the polypeptide encoded thereby, and further having a polynucleotide encoding a single transmembrane domain T1R2 receptor, for example the feline T1R2 receptor of the invention, or the polypeptide encoded thereby, will be attracted to one or more amino acids and will exhibit no preference for one or more carbohydrates or high-intensity sweeteners.
Also contemplated by the invention is a method for representing taste perception of a particular taste in a mammal, for example a cat, by providing values X1 to Xn representative of the quantitative stimulation of each of n T1R receptors or dimers of the invention, where n is greater than or equal to 2, and generating from the values a quantitative representation of taste perception of the mammal. The representation may constitute a point or a volume in n-dimensional space, may constitute a graph or a spectrum, and/or may constitute a matrix of quantitative representations. Also, the providing step may comprise contacting a plurality of T1R receptors or dimers of the invention with a test compound and quantitatively measuring the interaction of the compound with T1R receptors or dimers.
Also contemplated by the invention are methods for predicting the taste perception in a mammal generated by one or more molecules or combinations of molecules yielding unknown taste perception in a mammal, such as a cat, by providing values X1 to Xn representative of the quantitative stimulation of each of n T1R receptors of the invention, where n is greater than or equal to 2, for one or more molecules or combinations of molecules yielding known taste perception in a mammal; and generating from the values a quantitative representation of taste perception in a mammal for the one or more molecules or combinations of molecules yielding known taste perception in a mammal, providing values X1 to Xn representative of the quantitative stimulation of each of n T1R receptors of the invention, where n is greater than or equal to 2, for one or more molecules or combinations of molecules yielding unknown taste perception in a mammal; and generating from said values a quantitative representation of taste perception in a mammal for the one or more molecules or combinations of molecules yielding unknown taste perception in a mammal, and predicting the taste perception in a mammal generated by one or more molecules or combinations of molecules yielding unknown taste perception in a mammal by comparing the quantitative representation of taste perception in a mammal for the one or more molecules or combinations of molecules yielding unknown taste perception in a mammal to the quantitative representation of taste perception in a mammal for the one or more molecules or combinations of molecules yielding known taste perception in a mammal.
In another embodiment, novel molecules or combinations of molecules are generated which elicit a predetermined taste perception in a mammal by determining a value of taste perception in a mammal such as a cat for a known molecule or combinations of molecules as described above; determining a value of taste perception in a mammal for one or more unknown molecules or combinations of molecules as described above; comparing the value of taste perception in a mammal for one or more unknown compositions to the value of taste perception in a mammal for one or more known compositions; selecting a molecule or combination of molecules that elicits a predetermined taste perception in a mammal; and combining two or more unknown molecules or combinations of molecules to form a molecule or combination of molecules that elicits a predetermined taste perception in a mammal. The combining step yields a single molecule or a combination of molecules that elicits a predetermined taste perception in a mammal.
Treatment Methods
The invention provides methods of treatment of T1R receptor-associated disorders by administering to a subject or patient an effective amount of a compound that modulates the T1R receptor. In some aspects of the invention, the compounds or pharmaceutical compositions of the invention are administered to a patient having an increased risk of or having a disorder associated with the T1R receptor. The patient may be, for example, avian, feline, canine, bovine, ovine, porcine, equine, rodent, simian, or human.
Kits
A kit of the invention comprises a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising an element to be used in the methods of the invention. For example, one of the container means may comprise the a polynucleotide encoding a T1R receptor of the invention, a T1R receptor of the invention, or an antibody thereto. The kit may also have one or more conventional kit components, including, but not limited to, instructions, test tubes, Eppendorf™ tubes, labels, reagents helpful for quantification of marker gene expression, etc.
The following examples are meant to be illustrative of the present invention and are not intended to limit the scope thereof.
Cloning and Characterization of the Feline T1R3 Receptor
The discovery of feline taste receptor, T1R3, was achieved by using a molecular strategy termed “overgo” (Thomas, et al., Genome Res., 12:1277-1285 (2002); Vollrath, D., DNA markers for physical mapping In G
Overgo probes were designed from conserved regions of the chromosome 1 marker, “disheveled 1” (DVL1) and the G protein-coupled receptor, T1R3, by aligning DVL1 and T1R3 genomic sequences from several species. The overlapping sequences of the seven DVL1 overgo probes used in the present invention were as follows:
The overlapping sequences of the twelve T1R3 overgo probes used in the present invention were as follows:
The single-stranded overhangs were filled in with 32P labeled dATP and dCTP, and the overgo probes hybridized with BAC libraries.
The overgo strategy is considered to be more versatile than a PCR-based strategy by those skilled in the art of comparative physical mapping for the following reasons: (1) overgo probes are short (e.g., 36mers or 40mers), making the probability of good alignment from among many species more favorable; (2) overgo probes are more specific to the target genes compared with traditional cDNA and genomic DNA probes used by PCR; and (3) although overgo probes are short, they are not as restricted as traditional PCR probes, which cannot tolerate even a few mismatches, because they can be used in hybridization approaches with BACs or other libraries.
Screening a feline genomic BAC library. Seven DVL1 overgo probes (SEQ ID NOS:21-34) were used in screening a feline genomic BAC library. Probes were radioactively labeled by the random hexa-nucleotide method (Feinberg & Vogelstein, Analytical Biochemistry, 132:6-13 (1983)). Hybridization and washing of membranes followed standard protocols (Church & Gilbert, PNAS U.S.A., 81:1991-1995 (1984)). Thirty-nine positive BAC clones were identified. Several BAC ends were sequenced. One clone containing homologous sequence to human chromosome 1p36, BAC 552J19, was identified using bioinformatics tools.
Production of a shotgun library for BAC 552J19 and identification of a single clone containing feline T1R3. BAC DNA from 552J19 was prepared by using Qiagen Large Construct Kit. DNA was then digested by the restriction enzyme Sau3A1 and subcloned into pGEM+3Z (Promega) vector. After transformants were arrayed to a nylon membrane, two separate hybridizations were performed using seven DVL1 and twelve T1R3 overgo probes (SEQ ID NOS:35-58). Two clones positive for DVL1 and four clones positive for T1R3 were found. These clones were confirmed by sequencing. Because DVL1 is the neighboring gene of T1R3 in human and mouse, it is likely this also is the case in cat; therefore, the DVL1 positive clones verified that the BAC 552J19 is the correct BAC, that is, it is the one containing feline T1R3.
Cloning and Characterization of the Feline T1R1 and T1R2 Receptors
Eludication of the cat T1R1 and cat T1R2 receptors also was accomplished using an overgo strategy. Overgo probes from conserved coding regions were designed by aligning T1R1 and T1R2 sequences from many different species, including human, mouse, rat, cow, and pig. The single-stranded overhangs (14 bases) were filled in with 32P-labeled dATP and dCTP, and the overgo probes hybridized with BAC libraries. The overlapping sequences of the six cat T1R1 overgo probes were as follows:
The overlapping sequences of the seven cat T1R2 overgo probes were as follows:
Screening a feline genomic BAC library. The T1R1 and T1R2 overgo probes were used to screen a feline genomic BAC library. Probes were radioactively labeled by the random hexa-nucleotide method (Feinberg & Vogelstein, Analytical Biochemistry, 132(1):6-13 (1983)). Hybridization and washing of membranes followed standard protocols (Church & Gilbert, PNAS U.S.A., 81:1991-1995 (1984)). Six positive BAC clones for cat T1R1 and eight positive BAC clones for cat T1R2 were identified.
Production of shotgun libraries for BACs containing cat T1R1 and T1R2, and identification of small insert clones containing feline T1R1 and T1R2. Two BACs (150M6 and 233G22) containing cat T1R1 and three BACs (93C1, 240H9 and 400B1) containing cat T1R2 were used to prepare BAC DNAs using Qiagen Large Construct Kit. BAC DNAs were digested using the enzyme Sau3AI and the digested BAC DNA fragments were subcloned into pGEM+3Z (Promega) vector. After transformants were arrayed to a nylon membrane, two separate hybridizations were performed using pooled six T1R1 and seven T1R2 overgo probes. By sequencing positive clones from shotgun libraries and by using a chromosome walking strategy, the full coding region of the cat T1R1 and exon 3 to exon 6 of cat T1R2 were obtained.
Elucidation of exon 1 and exon 2 of the cat T1R2 by PCR strategy. Since exon 1 and exon 2 of cat T1R2 were not present in the three BACs selected above, PCR was performed using degenerate primers designed from T1R2 alignments from different species (human, rodents, and dog) and cat genomic DNA as template.
The PCR products were confirmed by sequencing. The feline BAC library was then re-screened using PCR products and four new BACs were retrieved (4O545, 2J533, 4F220 and 24D448). Using a chromosome walking strategy, the complete sequence of exon 1 and exon 2 from these four BAC clones were obtained.
Results
More than 3 kb of genomic sequences containing the open reading frame for domestic cat taste receptor, T1R3, were obtained. Approximately 10 kb of genomic sequence containing the open reading frame (ORF) for cat T1R1 and approximately 38 kb of genomic sequence containing the open reading frame for cat T1R2 were obtained.
Table 4 shows the percent homology among the members of the T1R family in relation to the cat T1R taste receptors. The portion of Table 1 to the left of the diagonal (in bold type) shows the percent homology based on the open reading frame of the nucleotide sequences obtained from
36
30
90
36
30
73
37
30
74
30
30
55
28
36
91
28
34
69
28
36
53
28
33
15
31
28
92
30
27
72
30
25
72
91
55
33
37
31
73
37
31
74
26
31
55
91
15
57
28
34
71
29
36
52
28
33
21
93
32
15
31
27
73
30
26
72
79
56
35
79
56
35
35
31
81
29
31
57
78
17
56
78
17
57
28
36
58
28
41
39
73
39
36
75
40
38
29
23
73
79
54
35
78
56
35
84
56
53
28
30
42
64
22
41
61
22
44
72
48
44
29
33
34
74
36
36
75
53
39
79
53
39
This application is the National Stage of International Application No. PCT/US2004/015136, filed May 13, 2004, which claims the benefit of the filing date of U.S. Provisional Application No. 60/482,992, filed Jun. 27, 2003, and U.S. Provisional Patent Application No. 60/554,751, filed Mar. 19, 2004. The entire contents of these patent applications are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2004/015136 | 5/13/2004 | WO | 00 | 8/31/2006 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2005/005480 | 1/20/2005 | WO | A |
| Number | Date | Country |
|---|---|---|
| 2 364 058 | Jan 2002 | GB |
| 9109955 | Jul 1991 | WO |
| 9220808 | Nov 1992 | WO |
| 9412650 | Jun 1994 | WO |
| 0006592 | Feb 2000 | WO |
| 0006593 | Feb 2000 | WO |
| 0230981 | Apr 2002 | WO |
| 02064631 | Aug 2002 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20080066195 A1 | Mar 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60554751 | Mar 2004 | US | |
| 60482992 | Jun 2003 | US |
