US20030232339A1

US20030232339A1 - Human TRPCC cation channel and uses

Info

Publication number: US20030232339A1
Application number: US10/167,631
Authority: US
Inventors: Youmin Shu; Xuan Li; Gilbert Jay
Original assignee: Individual
Current assignee: Individual
Priority date: 2002-04-01
Filing date: 2002-06-13
Publication date: 2003-12-18
Also published as: AU2003218483A1; AU2003218483A8; US20030186249A1; WO2003085095A2; US20030228658A1; WO2003085095A3; US20030235826A1; US7115393B2

Abstract

The present invention relates to all facets of a novel polynucleotide, TRPCC, the polypeptides it encodes, antibodies, and specific binding partners thereto, and their applications to research, diagnosis, drug discovery, therapy, clinical medicine, forensic science and medicine, etc. The polynucleotides are expressed in brain, kidney, pituitary, and other tissues, and are therefore useful in a variety of ways, including, but not limited to, as molecular markers, as drug targets, and for detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, determining predisposition to brain and/or kidney disease, including diseases and conditions such as hypomagnesemia, hypocalcemia, and amyotrophic lateral sclerosis with frontotemporal dementia, etc.

Description

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid alignments of a human transient receptor potential cation channel (TRPCC) with related channel family members. Human sequences are TRPM (SEQ ID NO 2), human AB046836 (SEQ ID NO 4), human XM _—036123 (SEQ ID NO 3), and mouse XM_—140575 (SEQ ID 5).

FIG. 2 shows the expression pattern of human TRPCC in human tissues. A twenty-four tissue panel was used (lanes from right to left): 1, adrenal gland; 2, bone marrow; 3, brain; 4, colon; 5, heart; 6, intestine; 7, kidney; 8, liver; 9, lung; 10, lymph node; 11, lymphocytes; 12, mammary gland; 13, muscle; 14, ovary; 15, pancreas; 16, pituitary; 17, prostate; 18, skin; 19, spleen; 20, stomach; 21, testis; 22, thymus; 23, thyroid; 24, uterus. The lane at the far left contains molecular weight standards. The results were obtained according to the following procedures:

Polyadenylated mRNA was isolated from tissue samples, and used as a template for first-strand cDNA synthesis. The resulting cDNA samples were normalized using beta-actin as a standard. For the normalization procedure, PCR was performed on aliquots of the first-strand cDNA using beta-actin specific primers. The PCR products were visualized on an ethidium bromide stained agarose gel to estimate the quantity of beta-actin cDNA present in each sample. Based on these estimates, each sample was diluted with buffer until each contained the same quantity of beta-actin cDNA per unit volume. [0003]
To detect gene expression, PCR was carried out on aliquots of the normalized tissue samples using a forward (SEQ ID NO 6) and reverse (SEQ ID NO 7) gene-specific primers. The reaction products were loaded on to an agarose (e.g., 1.5-2%) gel and separated electrophoretically. [0004]

DESCRIPTION OF THE INVENTION

The present invention relates to all facets of novel polynucleotides that code for a human transient receptor potential cation channel (“TRPCC”), the polypeptides they encode, antibodies and specific binding partners thereto, and their applications to research, diagnosis, drug discovery, therapy, clinical medicine, forensic science and medicine, etc. TRPCC is differentially expressed in brain, kidney, and pituitary, making it and the polypeptides it encodes useful in a variety of ways, including, but not limited to, as molecular markers, as linkage markers, as drug targets, and for detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, determining predisposition to, etc., diseases and conditions relating to disorders and conditions associated with these tissues, especially relating to cation regulation. The present invention also relates to methods of using the polynucleotides and related products (proteins, antibodies, etc.) in business and computer-related methods, e.g., advertising, displaying, offering, selling, etc., such products for sale, commercial use, licensing, etc. [0005]
Human Transient Receptor Potential Cation Channel Gene and Polypeptide [0006]
Human TRPCC codes for a polypeptide of 1707 amino acids. As shown in FIG. 2, it is selectively expressed in brain, kidney, and pituitary, with very low expression observed in testis and ovary. By the phrase “selectively expressed,” it is meant that a nucleic acid molecule, when produced as a transcript, is characteristic of the tissue or cell-type in which it is made. This can mean that the transcript is expressed only in that tissue and in no other tissue-type, or it can mean that the transcript is expressed preferentially, differentially, predominantly, and more abundantly (e.g., at least 5-fold, 10-fold, etc., or more) in that tissue when compared to other tissue-types. [0007]
The nucleotide and amino acid sequences of human TRPCC are shown in [0008] SEQ ID NOS 1 and 2. Analysis of its primary structure indicates the presence of six transmembrane domains at about amino acids 870-892, 901-1112, 904-921, 936-958, 971-990, 1005-1024, 1085-1107 of SEQ ID NO 2, however, by analogy to other ion channels, it is generally believed to have only six transmembrane spanning regions. See, e.g., Clapham et al., Nature Reviews, Neuroscience, 2:387, 2001. The ion transport domain comprises amino acids 901-1112. There is also a putative transmembrane domain at the N-terminus at about amino acids 5-24. According to the six-transmembrane model, both the N- and C-terminus of the protein are intracellular, and provide a scaffolding for interaction with other proteins.
The human TRPCC contains 25 exons. The present invention relates to any isolated introns and exons that are present in the gene. Intron and exon boundaries can be routinely determined, e.g., using the sequences disclosed herein. [0009]
Partial sequences for human TRPCC were previously identified (e.g., Accession Numbers AB046836 and XM[0010] _—036123). For example, human AB046836 (SEQ ID 4) is incomplete, coding for 1017 amino acids (See FIG. 1, AB046836), and lacks the first 690 amino acids of human TRPCC, but shares about 99% identity with TRPCC along the rest of its length. Another partial sequence, human XM_—036123 (SEQ ID NO 3) codes for 988 amino acids (See FIG. 1, XM_—036123), lacking the first 719 amino acids of human TRPCC, but shares 100% identity with TRPCC along the rest of its length (See FIG. 1). XM_—140575 (SEQ ID NO 5) appears to be a homolog of human TRPCC, and shares about 94% sequence identity from about amino acids 82-693, or about amino acids 345-956 of human TRPCC (SEQ ID NO 2). Amino acids 1-81 and 694-736 (see FIG. 1) of the mouse homolog have low sequence identity with human TRPCC. Alignment with mouse genomic DNA using Spidey (NCBI) indicates that amino acids 1-80 of XM_—140575 are derived from exons 1 and 2 of the genomic DNA, and amino acids 694-736 are derived from exon 7 of the mouse genomic DNA. XM_—140575 is located on mouse chromosome 19B.
TRPCC maps to chromosomal region 9q21.1. Strikingly, hypomagnesemia with hypocalcemia (OMIM 602014) are known to be determined by a mutation within 9q21 (Walder et al., [0011] Human Molecular Genetics, 6: 1491-1497, 1997), as would be expected with a channel responsible for cation conductance. Consistent with its expression in brain, a susceptibility to amyotrophic lateral sclerosis with frontotemporal dementia (OMIM 105550) was mapped to this same chromosomal locus (Pinsky et al., Clinical Genetics, 7:186-191, 1975; Hosler et al., JAMA, 284:1664-1669, 2000). In addition, schizophrenia (Hovatta et al., Am. J. Hum. Genet., 65:1114-24), and familial dyskinesia/facial myokymia (Fernandez et al., Ann. Neurol., 49:486-92, 2001) are also associated with this gene locus. Nucleic acids of the present invention can be used, e.g., as linkage markers, diagnostic targets, and therapeutic targets for any of the mentioned disorders, as well as any disorders or genes mapping in proximity of TRPCC.
TRCC polynucleotides, polypeptides, ligands, and binding partners thereto, can be used in a number of useful ways. For example, binding partners, such as antibodies and ligands, can be used to selectively target agents to brain, kidney, and other tissues in which it is expressed for purposes including, but not limited to, imaging, diagnostic, therapeutics, etc. Imaging of tissues can be facilitated using agents such as TRPCC antibodies that can be used to target contrast agents to a specific site in the body. Various imaging techniques have been used in this context, including, e.g., X-ray, CT, CAT, MRI, ultrasound, PET, SPECT, and scintographic. A reporter agent can be conjugated or associated routinely with a TRPCC antibody. Ultrasound contrast agents combined with ligands such as antibodies are described in, e.g., U.S. Pat. Nos. 6,264,917; 6,254,852; 6,245,318; and 6,139,819. MRI contrast agents, such as metal chelators, radionucleotides, paramagnetic ions, etc., combined with selective targeting agents are also described in the literature, e.g., in U.S. Pat. Nos. 6,280,706 and 6,221,334. The methods described therein can be used generally to associate TRPCC and ligands thereof with an agent for any desired purpose. [0012]
An active agent can be associated in any manner with an TRPCC ligand that is effective to achieve its delivery to the target. The association of the active agent and the ligand (“coupling”) can be direct, e.g., through chemical bonds between the binding ligand and the agent or via a linking agent, or the association can be less direct, e.g., where the active agent is in a liposome, or other carrier, and the ligand is associated with the liposome surface. In such case, the ligand can be oriented in such a way that it is able to bind to TRPCC on the surfaces of kidney or brain cells. [0013]
Useful human TRPCC polypeptides and corresponding nucleic acids include polypeptides comprising amino acids 1-88, 5-24, 1-690, 1-719, and fragments thereof (See [0014] SEQ ID NO 2 and FIG. 1). Nucleic acids and polypeptides can be used as probes (e.g., in PCR, in Northern blots, etc.), as diagnostic agents, to generate antibodies, as vaccines, to produce recombinant proteins, as antisense, etc.
TRPCC has a number of biological activities, including, e.g., cation transport, signal transduction, protein binding, etc. By “signal transduction” is meant the activation of a chain of events that alters the concentration of one or more small intracellular signaling molecules (second messengers), e.g., cyclic AMP, calcium ions, as described in Sabala et al., [0015] British Journal of Pharmacology, 132:393-402, 2001. By “cation transport” is meant the influx or efflux of a cation, e.g., calcium, magnesium, into or from a cell. Mizuno et al., Molecular Brain Research, 64:41-51, 1999. Protein binding indicates the ability of the protein to interact with other proteins, e.g., as the N-terminus interacts with intracellular proteins. These activities can be determined routinely. Signal transduction can be assessed by expression of TRPCC in cells, etc., and measurement of the concentrations of elicited second messengers or byproducts, e.g., Ca²⁺ or Mg or cAMP, inositol, etc., by, e.g., atomic absorption spectrometry (ThermoElemental SOLAAR AA spectrometers), radioimmunoassay, etc. Sano et al. Science, 293:1327-1330, 2001. Cation transport can be assessed by measurement of changes in ionic currents by whole-cell patch-clamp analysis. For instance, cells or oocytes can be transfected with a polynucleotide of the present invention and then analyzed for expression of calcium channel activity, e.g., using patch clamp, calcium activated dyes, etc. See, also, e.g., Strubing et al., Neuron, 29:645-655, 2001; Sano et al., Science, 293:1327, 2001; Ohki et al., J. Biol. Chem., 275:39055-39060, 2000; Boulay et al., J. Biol. Chem., 272:29672-29680, 1997
The present invention relates to an isolated polynucleotide comprising, e.g., a polynucleotide sequence coding without interruption for a human TRPCC polypeptide, or complement thereto, said TRPCC having 80%, 85%, 90%, 92%, 95%, 99%, or more amino acid sequence identity along its entire length to the sequence comprising amino acids 1-690 of [0016] SEQ ID NO 2, and 80%, 85%, 90%, 92%, 95%, 99%, or more amino acid sequence identity along its entire length to the sequence comprising from amino acids 691-1707 of SEQ ID NO 2, and which has, e.g., cation transport, signal transduction, or protein binding activity.
Nucleic Acids [0017]
A mammalian polynucleotide, or fragment thereof, of the present invention is a polynucleotide having a nucleotide sequence obtainable from a natural source. When the species name is used, e.g., human TRPCC, it indicates that the polynucleotide or polypeptide is obtainable from a natural source. It therefore includes naturally-occurring normal, naturally-occurring mutant, and naturally-occurring polymorphic alleles (e.g., SNPs), differentially-spliced transcripts, splice-variants, etc. By the term “naturally-occurring,” it is meant that the polynucleotide is obtainable from a natural source, e.g., animal tissue and cells, body fluids, tissue culture cells, forensic samples. Natural sources include, e.g., living cells obtained from tissues and whole organisms, tumors, cultured cell lines, including primary and immortalized cell lines. Naturally-occurring mutations can include deletions (e.g., a truncated amino- or carboxy-terminus), substitutions, inversions, or additions of nucleotide sequence. These genes can be detected and isolated by polynucleotide hybridization according to methods which one skilled in the art would know, e.g., as discussed below. [0018]
A polynucleotide according to the present invention can be obtained from a variety of different sources. It can be obtained from DNA or RNA, such as polyadenylated mRNA or total RNA, e.g., isolated from tissues, cells, or whole organism. The polynucleotide can be obtained directly from DNA or RNA, from a cDNA library, from a genomic library, etc. The polynucleotide can be obtained from a cell or tissue (e.g., from embryonic or adult tissues) at a particular stage of development, having a desired genotype, phenotype, disease status, etc. [0019]
A polynucleotide which “codes without interruption” refers to a polynucleotide having a continuous open reading frame (“ORF”) as compared to an ORF which is interrupted by introns or other noncoding sequences. [0020]
Polynucleotides and polypeptides (including any part of TRPCC) can be excluded as compositions from the present invention if, e.g., listed in a publicly available database on the day this application was filed and/or disclosed in a patent application having an earlier filing or priority date than this application and/or conceived and/or reduced to practice earlier than a polynucleotide in this application. [0021]
As described herein, the phrase “an isolated polynucleotide which is SEQ ID NO,” or “an isolated polynucleotide which is selected from SEQ ID NO,” refers to an isolated nucleic acid molecule from which the recited sequence was derived (e.g., a cDNA derived from MRNA; cDNA derived from genomic DNA). Because of sequencing errors, typographical errors, etc., the actual naturally-occurring sequence may differ from a SEQ ID listed herein. Thus, the phrase indicates the specific molecule from which the sequence was derived, rather than a molecule having that exact recited nucleotide sequence, analogously to how a culture depository number refers to a specific cloned fragment in a cryotube. [0022]
As explained in more detail below, a polynucleotide sequence of the invention can contain the complete sequence as shown in [0023] SEQ ID NO 1, degenerate sequences thereof, anti-sense, muteins thereof, genes comprising said sequences, full-length cDNAs comprising said sequences, complete genomic sequences, fragments thereof, homologs, primers, nucleic acid molecules which hybridize thereto, derivatives thereof, etc.
Genomic [0024]
The present invention also relates genomic DNA from which the polynucleotides of the present invention can be derived. A genomic DNA coding for a human, mouse, or other mammalian polynucleotide, can be obtained routinely, for example, by screening a genomic library (e.g., a YAC library) with a polynucleotide of the present invention, or by searching nucleotide databases, such as GenBank and EMBL, for matches. Promoter and other regulatory regions (including both 5′ and 3′ regions, as well introns) can be identified upstream or downstream of coding and expressed RNAs, and assayed routinely for activity, e.g., by joining to a reporter gene (e.g., CAT, GFP, alkaline phosphatase, luciferase, galatosidase). A promoter obtained from the TRPCC can be used, e.g., in gene therapy to obtain tissue-specific expression of a heterologous gene (e.g., coding for a therapeutic product or cytotoxin). 5′ and 3′ sequences (including, UTRs and introns) can be used to modulate or regulate stability, transcription, and translation of nucleic acids, including the sequence to which it is attached in nature, as well as heterologous nucleic acids. [0025]
Constructs [0026]
A polynucleotide of the present invention can comprise additional polynucleotide sequences, e.g., sequences to enhance expression, detection, uptake, cataloging, tagging, etc. A polynucleotide can include only coding sequence; a coding sequence and additional non-naturally occurring or heterologous coding sequence (e.g., sequences coding for leader, signal, secretory, targeting, enzymatic, fluorescent, antibiotic resistance, and other functional or diagnostic peptides); coding sequences and non-coding sequences, e.g., untranslated sequences at either a 5′ or 3′ end, or dispersed in the coding sequence, e.g., introns. [0027]
A polynucleotide according to the present invention also can comprise an expression control sequence operably linked to a polynucleotide as described above. The phrase “expression control sequence” means a polynucleotide sequence that regulates expression of a polypeptide coded for by a polynucleotide to which it is functionally (“operably”) linked. Expression can be regulated at the level of the mRNA or polypeptide. Thus, the expression control sequence includes mRNA-related elements and protein-related elements. Such elements include promoters, enhancers (viral or cellular), ribosome binding sequences, transcriptional terminators, etc. An expression control sequence is operably linked to a nucleotide coding sequence when the expression control sequence is positioned in such a manner to effect or achieve expression of the coding sequence. For example, when a promoter is operably linked 5′ to a coding sequence, expression of the coding sequence is driven by the promoter. Expression control sequences can include an initiation codon and additional nucleotides to place a partial nucleotide sequence of the present invention in-frame in order to produce a polypeptide (e.g., pET vectors from Promega have been designed to permit a molecule to be inserted into all three reading frames to identify the one that results in polypeptide expression). Expression control sequences can be heterologous or endogenous to the normal gene. [0028]
A polynucleotide of the present invention can also comprise nucleic acid vector sequences, e.g., for cloning, expression, amplification, selection, etc. Any effective vector can be used. A vector is, e.g., a polynucleotide molecule which can replicate autonomously in a host cell, e.g., containing an origin of replication. Vectors can be useful to perform manipulations, to propagate, and/or obtain large quantities of the recombinant molecule in a desired host. A skilled worker can select a vector depending on the purpose desired, e.g., to propagate the recombinant molecule in bacteria, yeast, insect, or mammalian cells. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, Phagescript, phix 174, pBK Phagemid, pNH8A, pNH16a, pNH18Z, pNH46A (Stratagene); Bluescript KS+II (Stratagene); ptrc99a, pKK223-3, pKK233-3, [0029] pDR54 0, pRIT5 (Pharmacia). Eukaryotic: PWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, PBPV, PMSG, pSVL (Pharmacia), pCR2.1/TOPO, pCRII/TOPO, pCR4/TOPO, pTrcHisB, pCMV6-XL4, etc. However, any other vector, e.g., plasmids, viruses, or parts thereof, may be used as long as they are replicable and viable in the desired host. The vector can also comprise sequences which enable it to replicate in the host whose genome is to be modified.
Hybridization [0030]
Polynucleotide hybridization, as discussed in more detail below, is useful in a variety of applications, including, in gene detection methods, for identifying mutations, for making mutations, to identify homologs in the same and different species, to identify related members of the same gene family, in diagnostic and prognostic assays, in therapeutic applications (e.g., where an antisense polynucleotide is used to inhibit expression), etc. [0031]
The ability of two single-stranded polynucleotide preparations to hybridize together is a measure of their nucleotide sequence complementarity, e.g., base-pairing between nucleotides, such as A-T, G-C, etc. The invention thus also relates to polynucleotides, and their complements, which hybridize to a polynucleotide comprising a nucleotide sequence as set forth in [0032] SEQ ID NO 1 and genomic sequences thereof. A nucleotide sequence hybridizing to the latter sequence will have a complementary polynucleotide strand, or act as a template for one in the presence of a polymerase (i.e., an appropriate polynucleotide synthesizing enzyme). The present invention includes both strands of polynucleotide, e.g., a sense strand and an anti-sense strand.
Hybridization conditions can be chosen to select polynucleotides which have a desired amount of nucleotide complementarity with the nucleotide sequences set forth in [0033] SEQ ID NO 1 and genomic sequences thereof. A polynucleotide capable of hybridizing to such sequence, preferably, possesses, e.g., about 70%, 75%, 80%, 85%, 87%, 90%, 92%, 95%, 97%, 99%, or 100% complementarity, between the sequences. The present invention particularly relates to polynucleotide sequences which hybridize to the nucleotide sequences set forth in SEQ ID NO 1 or genomic sequences thereof, under low or high stringency conditions. These conditions can be used, e.g., to select corresponding homologs in non-human species.
Polynucleotides which hybridize to polynucleotides of the present invention can be selected in various ways. Filter-type blots (i.e., matrices containing polynucleotide, such as nitrocellulose), glass chips, and other matrices and substrates comprising polynucleotides (short or long) of interest, can be incubated in a prehybridization solution (e.g., 6×SSC, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA, 5× Denhardt's solution, and 50% formamide), at 22-68° C., overnight, and then hybridized with a detectable polynucleotide probe under conditions appropriate to achieve the desired stringency. In general, when high homology or sequence identity is desired, a high temperature can be used (e.g., 65° C.). As the homology drops, lower washing temperatures are used. For salt concentrations, the lower the salt concentration, the higher the stringency. The length of the probe is another consideration. Very short probes (e.g., less than 100 base pairs) are washed at lower temperatures, even if the homology is high. With short probes, formamide can be omitted. See, e.g., [0034] Current Protocols in Molecular Biology, Chapter 6, Screening of Recombinant Libraries; Sambrook et al., Molecular Cloning, 1989, Chapter 9.
For instance, high stringency conditions can be achieved by incubating the blot overnight (e.g., at least 12 hours) with a long polynucleotide probe in a hybridization solution containing, e.g., about 5×SSC, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 50% formamide, at 42° C. Blots can be washed at high stringency conditions that allow, e.g., for less than 5% bp mismatch (e.g., wash twice in 0.1% SSC and 0.1% SDS for 30 min at 65° C.), i.e., selecting sequences having 95% or greater sequence identity. [0035]
Other non-limiting examples of high stringency conditions includes a final wash at 65° C. in aqueous buffer containing 30 mM NaCl and 0.5% SDS. Another example of high stringent conditions is hybridization in 7% SDS, 0.5 M NaPO[0036] ₄, pH 7, 1 mM EDTA at 50° C., e.g., overnight, followed by one or more washes with a 1% SDS solution at 42° C. Whereas high stringency washes can allow for less than 5% mismatch, reduced or low stringency conditions can permit up to 20% nucleotide mismatch. Hybridization at low stringency can be accomplished as above, but using lower formamide conditions, lower temperatures and/or lower salt concentrations, as well as longer periods of incubation time.
Hybridization can also be based on a calculation of melting temperature (Tm) of the hybrid formed between the probe and its target, as described in Sambrook et al. Generally, the temperature Tm at which a short oligonucleotide (containing 18 nucleotides or fewer) will melt from its target sequence is given by the following equation: Tm=(number of A's and T's)×2° C.+(number of C's and G's)×4° C. For longer molecules, Tm=81.5+16.6 logio[Na[0037] ⁺]+0.41(%GC)-600/N where [Na⁺] is the molar concentration of sodium ions, %GC is the percentage of GC base pairs in the probe, and N is the length. Hybridization can be carried out at several degrees below this temperature to ensure that the probe and target can hybridize. Mismatches can be allowed for by lowering the temperature even further.
Stringent conditions can be selected to isolate sequences, and their complements, which have, e.g., at least about 90%, 95%, or 97%, nucleotide complementarity between the probe (e.g., a short polynucleotide of [0038] SEQ ID NO 1 or genomic sequences thereof) and a target polynucleotide.
Other homologs of polynucleotides of the present invention can be obtained from mammalian and non-mammalian sources according to various methods. For example, hybridization with a polynucleotide can be employed to select homologs, e.g., as described in Sambrook et al., [0039] Molecular Cloning, Chapter 11, 1989. Such homologs can have varying amounts of nucleotide and amino acid sequence identity and similarity to such polynucleotides of the present invention. Mammalian organisms include, e.g., mice, rats, monkeys, pigs, cows, etc. Non-mammalian organisms include, e.g., vertebrates, invertebrates, zebra fish, chicken, Drosophila, C. elegans, Xenopus, yeast such as S. pombe, S. cerevisiae, roundworms, prokaryotes, plants, Arabidopsis, Artemia, viruses, etc.
Alignment [0040]
Alignments can be accomplished by using any effective algorithm. For pairwise alignments of DNA sequences, the methods described by Wilbur-Lipman (e.g., Wilbur and Lipman, [0041] Proc. Natl. Acad. Sci., 80:726-730, 1983) or Martinez/Needleman-Wunsch (e.g., Martinez, Nucleic Acid Res., 11:4629-4634, 1983) can be used. For instance, if the Martinez/Needleman-Wunsch DNA alignment is applied, the minimum match can be set at 9, gap penalty at 1.10, and gap length penalty at 0.33. The results can be calculated as a similarity index, equal to the sum of the matching residues divided by the sum of all residues and gap characters, and then multiplied by 100 to express as a percent. Similarity index for related genes at the nucleotide level in accordance with the present invention can be greater than 70%, 80%, 85%, 90%, 95%, 99%, or more. Pairs of protein sequences can be aligned by the Lipman-Pearson method (e.g., Lipman and Pearson, Science, 227:1435-1441, 1985) with k-tuple set at 2, gap penalty set at 4, and gap length penalty set at 12. Results can be expressed as percent similarity index, where related genes at the amino acid level in accordance with the present invention can be greater than 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. Various commercial and free sources of alignment programs are available, e.g., MegAlign by DNA Star, BLAST (National Center for Biotechnology Information), BCM (Baylor College of Medicine) Launcher, etc. BLAST can be used to calculate amino acid sequence identity, amino acid sequence homology, and nucleotide sequence identity. These calculations can be made along the entire length of each of the target sequences which are to be compared.
After two sequences have been aligned, a “percent sequence identity” can be determined. For these purposes, it is convenient to refer to a Reference Sequence and a Compared Sequence, where the Compared Sequence is compared to the Reference Sequence. Percent sequence identity can be determined according to the following formula: Percent Identity=100[1−(C/R)], wherein C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence where (i) each base or amino acid in the Reference Sequence that does not have a corresponding aligned base or amino acid in the Compared Sequence, (ii) each gap in the Reference Sequence, (iii) each aligned base or amino acid in the Reference Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference; and R is the number of bases or amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as a base or amino acid. [0042]
Percent sequence identity can also be determined by other conventional methods, e.g., as described in Altschul et al., [0043] Bull. Math. Bio. 48: 603-616, 1986 and Henikoff and Benikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992.
Specific Polynucleotide Probes [0044]
A polynucleotide of the present invention can comprise any continuous nucleotide sequence of [0045] SEQ ID NO 1, sequences that share sequence identity thereto, or complements thereof. The term “probe” refers to any substance that can be used to detect, identify, isolate, etc., another substance. A polynucleotide probe composed of nucleic acid can be used to detect, identify, etc., other nucleic acids, such as DNA and RNA.
These polynucleotides can be of any desired size that is effective to achieve the specificity desired. For example, a probe can be from about 7 or 8 nucleotides to several thousand nucleotides, depending upon its use and purpose. For instance, a probe used as a primer PCR can be shorter than a probe used in an ordered array of polynucleotide probes. Probe sizes vary, and the invention is not limited in any way by their size, e.g., probes can be from about 7-2000 nucleotides, 7-1000, 8-700, 8-600, 8-500, 8-400, 8-300, 8-150, 8-100, 8-75, 7-50, 10-25, 14-16, at least about 8, at least about 10, at least about 15, at least about 25, etc. The polynucleotides can have non-naturally-occurring nucleotides, e.g., inosine, AZT, 3TC, etc. The polynucleotides can have 100% sequence identity or complementarity to a sequence of [0046] SEQ ID NO 1, or it can have mismatches or nucleotide substitutions, e.g., 1, 2, 3, 4, or 5 substitutions. The probes can be single-stranded or double-stranded.
In accordance with the present invention, a polynucleotide can be present in a kit, where the kit includes, e.g., one or more polynucleotides, a desired buffer (e.g., phosphate, Tris, etc.), detection compositions, RNA or cDNA from different tissues to be used as controls, libraries, etc. The polynucleotide can be labeled or unlabeled, with radioactive or non-radioactive labels as known in the art. Kits can comprise one or more pairs of polynucleotides for amplifying nucleic acids specific for TRPCC, e.g., comprising a forward and reverse primer effective in PCR. These include both sense and anti-sense orientations. For instance, in PCR-based methods (such as RT-PCR), a pair of primers is typically used, one having a sense sequence and the other having an antisense sequence. [0047]
Another aspect of the present invention is a nucleotide sequence that is specific to, or for, a selective polynucleotide. The phrases “specific for” or “specific to” a polynucleotide have a functional meaning that the polynucleotide can be used to identify the presence of one or more target genes in a sample and distinguish them from non-target genes. It is specific in the sense that it can be used to detect polynucleotides above background noise (“non-specific binding”). A specific sequence is a defined order of nucleotides (or amino acid sequences, if it is a polypeptide sequence) which occurs in the polynucleotide, e.g., in the nucleotide sequences of [0048] SEQ ID NO 1, and which is characteristic of that target sequence and substantially no non-target sequences. A probe or mixture of probes can comprise a sequence or sequences that are specific to a plurality of target sequences, e.g., where the sequence is a consensus sequence, a functional domain, etc., e.g., capable of recognizing a family of related genes. Such sequences can be used as probes in any of the methods described herein or incorporated by reference. Both sense and antisense nucleotide sequences are included. A specific polynucleotide according to the present invention can be determined routinely. Examples are specific probes are SEQ ID NOS 6-9, e.g., where SEQ ID NOS 8 and 9 can be used as forward and reverse PCR primers, respectively, to amplify a portion of amino acid region 1-160 of SEQ ID NO 2.
A polynucleotide comprising a specific sequence can be used as a hybridization probe to identify the presence of, e.g., human or mouse polynucleotide, in a sample comprising a mixture of polynucleotides, e.g., on a Northern blot. Hybridization can be performed under high stringent conditions (see, above) to select polynucleotides (and their complements which can contain the coding sequence) having at least 90%, 95%, 99%, etc., identity (i.e., complementarity) to the probe, but less stringent conditions can also be used. A specific polynucleotide sequence can also be fused in-frame, at either its 5′ or 3′ end, to various nucleotide sequences as mentioned throughout the patent, including coding sequences for enzymes, detectable markers, GFP, etc, expression control sequences, etc. [0049]
Probes specific for polynucleotides of the present invention can also be prepared using transcription-based systems, e.g., incorporating an RNA polymerase promoter into a selective polynucleotide of the present invention, and then transcribing anti-sense RNA using the polynucleotide as a template. See, e.g., U.S. Pat. No. 5,545,522. [0050]
Polynucleotide Composition [0051]
A polynucleotide according to the present invention can comprise, e.g., DNA, RNA, synthetic polynucleotide, peptide polynucleotide, modified nucleotides, dsDNA, ssDNA, ssRNA, dsRNA, and mixtures thereof. A polynucleotide can be single- or double-stranded, triplex, DNA:RNA, duplexes, comprise hairpins, and other secondary structures, etc. Nucleotides comprising a polynucleotide can be joined via various known linkages, e.g., ester, sulfamate, sulfamide, phosphorothioate, phosphoramidate, methylphosphonate, carbamate, etc., depending on the desired purpose, e.g., resistance to nucleases, such as RNAse H, improved in vivo stability, etc. See, e.g., U.S. Pat. No. 5,378,825. Any desired nucleotide or nucleotide analog can be incorporated, e.g., 6-mercaptoguanine, 8-oxo-guanine, etc. [0052]
Various modifications can be made to the polynucleotides, such as attaching detectable markers (avidin, biotin, radioactive elements, fluorescent tags and dyes, energy transfer labels, energy-emitting labels, binding partners, etc.) or moieties which improve hybridization, detection, and/or stability. The polynucleotides can also be attached to solid supports, e.g., nitrocellulose, magnetic or paramagnetic microspheres (e.g., as described in U.S. Pat. No. 5,411,863; U.S. Pat. No. 5,543,289; for instance, comprising ferromagnetic, supermagnetic, paramagnetic, superparamagnetic, iron oxide and polysaccharide), nylon, agarose, diazotized cellulose, latex solid microspheres, polyacrylamides, etc., according to a desired method. See, e.g., U.S. Pat. Nos. 5,470,967, 5,476,925, and 5,478,893. [0053]
Polynucleotide according to the present invention can be labeled according to any desired method. The polynucleotide can be labeled using radioactive tracers such as [0054] ³²P, ³⁵S, ³H, or ¹⁴C, to mention some commonly used tracers. The radioactive labeling can be carried out according to any method, such as, for example, terminal labeling at the 3′ or 5′ end using a radiolabeled nucleotide, polynucleotide kinase (with or without dephosphorylation with a phosphatase) or a ligase (depending on the end to be labeled). A non-radioactive labeling can also be used, combining a polynucleotide of the present invention with residues having immunological properties (antigens, haptens), a specific affinity for certain reagents (ligands), properties enabling detectable enzyme reactions to be completed (enzymes or coenzymes, enzyme substrates, or other substances involved in an enzymatic reaction), or characteristic physical properties, such as fluorescence or the emission or absorption of light at a desired wavelength, etc.
Nucleic Acid Detection Methods [0055]
Another aspect of the present invention relates to methods and processes for detecting TRPCC. Detection methods have a variety of applications, including for diagnostic, prognostic, forensic, and research applications. To accomplish gene detection, a polynucleotide in accordance with the present invention can be used as a “probe.” The term “probe” or “polynucleotide probe” has its customary meaning in the art, e.g., a polynucleotide that is effective to identify (e.g., by hybridization), when used in an appropriate process, the presence of a target polynucleotide to which it is designed. Identification can involve simply determining presence or absence, or it can be quantitative, e.g., in assessing amounts of a gene or gene transcript present in a sample. Probes can be useful in a variety of ways, such as for diagnostic purposes, to identify homologs, and to detect, quantitate, or isolate a polynucleotide of the present invention in a test sample. [0056]
Assays can be utilized which permit quantification and/or presence/absence detection of a target nucleic acid in a sample. Assays can be performed at the single-cell level, or in a sample comprising many cells, where the assay is “averaging” expression over the entire collection of cells and tissue present in the sample. Any suitable assay format can be used, including, but not limited to, e.g., Southern blot analysis, Northern blot analysis, polymerase chain reaction (“PCR”) (e.g., Saiki et al., [0057] Science, 241:53, 1988; U.S. Pat. Nos. 4,683,195, 4,683,202, and 6,040,166; PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, New York, 1990), reverse transcriptase polymerase chain reaction (“RT-PCR”), anchored PCR, rapid amplification of cDNA ends (“RACE”) (e.g., Schaefer in Gene Cloning and Analysis: Current Innovations, Pages 99-115, 1997), ligase chain reaction (“LCR”) (EP 320 308), one-sided PCR (Ohara et al., Proc. Natl. Acad. Sci., 86:5673-5677, 1989), indexing methods (e.g., U.S. Pat. No. 5,508,169), in situ hybridization, differential display (e.g., Liang et al., Nucl. Acid. Res., 21:3269-3275, 1993; U.S. Pat. Nos. 5,262,311, 5,599,672 and 5,965,409; WO97/18454; Prashar and Weissman, Proc. Natl. Acad. Sci., 93:659-663, and U.S. Pat. Nos. 6,010,850 and 5,712,126; Welsh et al., Nucleic Acid Res., 20:4965-4970, 1992, and U.S. Pat. No. 5,487,985) and other RNA fingerprinting techniques, nucleic acid sequence based amplification (“NASBA”) and other transcription based amplification systems (e.g., U.S. Pat. Nos. 5,409,818 and 5,554,527; WO 88/10315), polynucleotide arrays (e.g., U.S. Pat. Nos. 5,143,854, 5,424,186; 5,700,637, 5,874,219, and 6,054,270; PCT WO 92/10092; PCT WO 90/15070), Qbeta Replicase (PCT/US87/00880), Strand Displacement Amplification (“SDA”), Repair Chain Reaction (“RCR”), nuclease protection assays, subtraction-based methods, Rapid-Scan™, etc. Additional useful methods include, but are not limited to, e.g., template-based amplification methods, competitive PCR (e.g., U.S. Pat. No. 5,747,251), redox-based assays (e.g., U.S. Pat. No. 5,871,918), Taqman-based assays (e.g., Holland et al., Proc. Natl. Acad, Sci., 88:7276-7280, 1991; U.S. Pat. Nos. 5,210,015 and 5,994,063), real-time fluorescence-based monitoring (e.g., U.S. Pat. No. 5,928,907), molecular energy transfer labels (e.g., U.S. Pat. Nos. 5,348,853, 5,532,129, 5,565,322, 6,030,787, and 6,117,635; Tyagi and Kramer, Nature Biotech., 14:303-309, 1996). Any method suitable for single cell analysis of gene or protein expression can be used, including in situ hybridization, immunocytochemistry, MACS, FACS, flow cytometry, etc. For single cell assays, expression products can be measured using antibodies, PCR, or other types of nucleic acid amplification (e.g., Brady et al., Methods Mol. & Cell. Biol. 2, 17-25, 1990; Eberwine et al., 1992, Proc. Natl. Acad. Sci., 89, 3010-3014, 1992; U.S. Pat. No. 5,723,290). These and other methods can be carried out conventionally, e.g., as described in the mentioned publications.
Many of such methods may require that the polynucleotide is labeled, or comprises a particular nucleotide type useful for detection. The present invention includes such modified polynucleotides that are necessary to carry out such methods. Thus, polynucleotides can be DNA, RNA, DNA:RNA hybrids, PNA, etc., and can comprise any modification or substituent which is effective to achieve detection. [0058]
Detection can be desirable for a variety of different purposes, including research, diagnostic, prognostic, and forensic. Diagnostic purposes included testing patients and their families for the presence of mutations associated with hypomagnesemia with hypocalcemia or amyotrophic lateral sclerosis with frontotemporal dementia. For diagnostic purposes, it may be desirable to identify the presence or quantity of a polynucleotide sequence in a sample, where the sample is obtained from tissue, cells, body fluids, etc. In a preferred method as described in more detail below, the present invention relates to a method of detecting a polynucleotide comprising, contacting a target polynucleotide in a test sample with a polynucleotide probe under conditions effective to achieve hybridization between the target and probe; and detecting hybridization. [0059]
Any test sample in which it is desired to identify a polynucleotide or polypeptide thereof can be used, including, e.g., blood, urine, saliva, stool (for extracting nucleic acid, see, e.g., U.S. Pat. No. 6,177,251), swabs comprising tissue, biopsied tissue, tissue sections, cultured cells, etc. [0060]
Detection can be accomplished in combination with polynucleotide probes for other genes, e.g., genes which are expressed in other disease states, tissues, cells, such as brain, heart, kidney, spleen, thymus, liver, stomach, small intestine, colon, muscle, lung, testis, placenta, pituitary, thyroid, skin, adrenal gland, pancreas, salivary gland, uterus, ovary, prostate gland, peripheral blood cells (T-cells, lymphocytes, etc.), embryo, normal breast fat, adult and embryonic stem cells, specific cell-types, such as endothelial, epithelial, myocytes, adipose, luminal epithelial, basoepithelial, myoepithelial, stromal cells, etc. [0061]
Polynucleotides can be used in wide range of methods and compositions, including for detecting, diagnosing, staging, grading, assessing, prognosticating, etc. diseases and disorders associated with TRPCC, for monitoring or assessing therapeutic and/or preventative measures, in ordered arrays, etc. Any method of detecting genes and polynucleotides of [0062] SEQ ID NO 1, and polymorphisms thereof, can be used; certainly, the present invention is not to be limited how such methods are implemented.
Along these lines, the present invention relates to methods of detecting TRPCC in a sample comprising nucleic acid. Such methods can comprise one or more the following steps in any effective order, e.g., contacting said sample with a polynucleotide probe under conditions effective for said probe to hybridize specifically to nucleic acid in said sample, and detecting the presence or absence of probe hybridized to nucleic acid in said sample, wherein said probe is a polynucleotide which is [0063] SEQ ID NO 1, a polynucleotide having, e.g., about 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity thereto, effective or specific fragments thereof, or complements thereto. The detection method can be applied to any sample, e.g., cultured primary, secondary, or established cell lines, tissue biopsy, blood, urine, stool, cerebral spinal fluid, and other bodily fluids, for any purpose.
Contacting the sample with probe can be carried out by any effective means in any effective environment. It can be accomplished in a solid, liquid, frozen, gaseous, amorphous, solidified, coagulated, colloid, etc., mixtures thereof, matrix. For instance, a probe in an aqueous medium can be contacted with a sample which is also in an aqueous medium, or which is affixed to a solid matrix, or vice-versa. [0064]
Generally, as used throughout the specification, the term “effective conditions” means, e.g., the particular milieu in which the desired effect is achieved. Such a milieu, includes, e.g., appropriate buffers, oxidizing agents, reducing agents, pH, co-factors, temperature, ion concentrations, suitable age and/or stage of cell (such as, in particular part of the cell cycle, or at a particular stage where particular genes are being expressed) where cells are being used, culture conditions (including substrate, oxygen, carbon dioxide, etc.). When hybridization is the chosen means of achieving detection, the probe and sample can be combined such that the resulting conditions are functional for said probe to hybridize specifically to nucleic acid in said sample. [0065]
The phrase “hybridize specifically” indicates that the hybridization between single-stranded polynucleotides is based on nucleotide sequence complementarity. The effective conditions are selected such that the probe hybridizes to a preselected and/or definite target nucleic acid in the sample. For instance, if detection of a polynucleotide set forth in [0066] SEQ ID NO 1 is desired, a probe can be selected which can hybridize to such target gene under high stringent conditions, without significant hybridization to other genes in the sample. To detect homologs of a polynucleotide set forth in SEQ ID NO 1, the effective hybridization conditions can be less stringent, and/or the probe can comprise codon degeneracy, such that a homolog is detected in the sample.
As already mentioned, the methods can be carried out by any effective process, e.g., by Northern blot analysis, polymerase chain reaction (PCR), reverse transcriptase PCR, RACE PCR, in situ hybridization, etc., as indicated above. When PCR based techniques are used, two or more probes are generally used. One probe can be specific for a defined sequence which is characteristic of a selective polynucleotide, but the other probe can be specific for the selective polynucleotide, or specific for a more general sequence, e.g., a sequence such as polyA which is characteristic of mRNA, a sequence which is specific for a promoter, ribosome binding site, or other transcriptional features, a consensus sequence (e.g., representing a functional domain). For the former aspects, 5′ and 3′ probes (e.g., polyA, Kozak, etc.) are preferred which are capable of specifically hybridizing to the ends of transcripts. When PCR is utilized, the probes can also be referred to as “primers” in that they can prime a DNA polymerase reaction. [0067]
In addition to testing for the presence or absence of polynucleotides, the present invention also relates to determining the amounts at which polynucleotides of the present invention are expressed in sample and determining the differential expression of such polynucleotides in samples. Such methods can involve substantially the same steps as described above for presence/absence detection, e.g., contacting with probe, hybridizing, and detecting hybridized probe, but using more quantitative methods and/or comparisons to standards. [0068]
The amount of hybridization between the probe and target can be determined by any suitable methods, e.g., PCR, RT-PCR, RACE PCR, Northern blot, polynucleotide microarrays, Rapid-Scan, etc., and includes both quantitative and qualitative measurements. For further details, see the hybridization methods described above and below. Determining by such hybridization whether the target is differentially expressed (e.g., up-regulated or down-regulated) in the sample can also be accomplished by any effective means. For instance, the target's expression pattern in the sample can be compared to its pattern in a known standard, such as in a normal tissue, or it can be compared to another gene in the same sample. When a second sample is utilized for the comparison, it can be a sample of normal tissue that is known not to contain diseased cells. The comparison can be performed on samples which contain the same amount of RNA (such as polyadenylated RNA or total RNA), or, on RNA extracted from the same amounts of starting tissue. Such a second sample can also be referred to as a control or standard. Hybridization can also be compared to a second target in the same tissue sample. Experiments can be performed that determine a ratio between the target nucleic acid and a second nucleic acid (a standard or control), e.g., in a normal tissue. When the ratio between the target and control are substantially the same in a normal and sample, the sample is determined or diagnosed not to contain cells. However, if the ratio is different between the normal and sample tissues, the sample is determined to contain diseased cells. The approaches can be combined, and one or more second samples, or second targets can be used. Any second target nucleic acid can be used as a comparison, including “housekeeping” genes, such as beta-actin, alcohol dehydrogenase, or any other gene whose expression does not vary depending upon the disease status of the cell. [0069]
Methods of Identifying Polymorphisms, Mutations, etc., of TRPCC [0070]
Polynucleotides of the present invention can also be utilized to identify mutant alleles, SNPS, gene rearrangements and modifications, and other polymorphisms of the wild-type gene. Mutant alleles, polymorphisms, SNPs, etc., can be identified and isolated from cancers that are known, or suspected to have, a genetic component. Identification of such genes can be carried out routinely (see, above for more guidance), e.g., using PCR, hybridization techniques, direct sequencing, mismatch reactions (see, e.g., above), RFLP analysis, SSCP (e.g., Orita et al., [0071] Proc. Natl. Acad. Sci., 86:2766, 1992), etc., where a polynucleotide having a sequence selected from SEQ ID NO 1 is used as a probe. The selected mutant alleles, SNPs, polymorphisms, etc., can be used diagnostically to determine whether a subject has, or is susceptible to a disorder associated with TRPCC, as well as to design therapies and predict the outcome of the disorder. Methods involve, e.g., diagnosing a disorder associated with TRPCC or determining susceptibility to a disorder, e.g., hypomagnesemia with hypocalcemia or amyotrophic lateral sclerosis with frontotemporal dementia, comprising, detecting the presence of a mutation in a TRPCC gene (such as a mutation in SEQ ID NO 1, or variants thereof.
The detecting can be carried out by any effective method, e.g., obtaining cells from a subject, determining the gene sequence or structure of a target gene (using, e.g., mRNA, cDNA, genomic DNA, etc), comparing the sequence or structure of the target gene to the structure of the normal gene, whereby a difference in sequence or structure indicates a mutation in the gene in the subject. Polynucleotides can also be used to test for mutations, SNPs, polymorphisms, etc., e.g., using mismatch DNA repair technology as described in U.S. Pat. No. 5,683,877; U.S. Pat. No. 5,656,430; Wu et al., [0072] Proc. Natl. Acad. Sci., 89:8779-8783, 1992.
The present invention also relates to methods of detecting polymorphisms or mutations in TRPCC, comprising, e.g., comparing the structure of: genomic DNA comprising all or part of TRPCC, mRNA comprising all or part of TRPCC, cDNA comprising all or part of TRPCC, or a polypeptide comprising all or part of TRPCC, with the structure of TRPCC set forth in [0073] SEQ ID NO 1, e.g., in a patient having amyotrophic lateral sclerosis with frontotemporal dementia, or a family member thereof, in a patient having hypomagnesemia with hypocalcemia, or a family member thereof, etc. For example, if a patient has been identified with amyotrophic sclerosis or hypomagnesemia, his DNA can be examined and compared to SEQ ID NO 1 to determine whether it is different, and whether any differences so identified is related to the condition. Further analysis can be done on the patient's family members (e.g., parents, grandparents, siblings, offspring, cousins, nieces, nephews, etc.) to determine whether the mutation is always associated with the phenotype.
The methods can be carried out on a sample from any source, e.g., cells, tissues, body fluids, blood, urine, stool, hair, egg, sperm, cerebral spinal fluid, etc. These methods can be implemented in many different ways. For example, “comparing the structure” steps include, but are not limited to, comparing restriction maps, nucleotide sequences, amino acid sequences, RFLPs, Dnase sites, DNA methylation fingerprints (e.g., U.S. Pat. No. 6,214,556), protein cleavage sites, molecular weights, electrophoretic mobilities, charges, ion mobility, etc., between a standard TRPCC and a test TRPCC. The term “structure” can refer to any physical characteristics or configurations which can be used to distinguish among nucleic acids or polypeptides. The methods and instruments used to accomplish the comparing step depends upon the physical characteristics which are to be compared. Thus, various techniques are contemplated, including, e.g., sequencing machines (both amino acid and polynucleotide), electrophoresis, mass spectrometer (U.S. Pat. Nos. 6,093,541, 6,002,127), liquid chromatography, HPLC, etc. [0074]
To carry out such methods, “all or part” of the gene or polypeptide can be compared. For example, if nucleotide sequencing is utilized, the entire gene can be sequenced, including promoter, introns, and exons, or only parts of it can be sequenced and compared, e.g., [0075] exon 1, exon 2, etc.
Mutagenesis [0076]
Mutated polynucleotide sequences of the present invention are useful for various purposes, e.g., to create mutations of the polypeptides they encode, to identify functional regions of genomic DNA, to produce probes for screening libraries, etc. Mutagenesis can be carried out routinely according to any effective method, e.g., oligonucleotide-directed (Smith, M., [0077] Ann. Rev. Genet. 19:423-463, 1985), degenerate oligonucleotide-directed (Hill et al., Method Enzymology, 155:558-568, 1987), region-specific (Myers et al., Science, 229:242-246, 1985; Derbyshire et al., Gene, 46:145, 1986; Ner et al., DNA, 7:127, 1988), linker-scanning (McKnight and Kingsbury, Science, 217:316-324, 1982), directed using PCR, recursive ensemble mutagenesis (Arkin and Yourvan, Proc. Natl. Acad. Sci., 89:7811-7815, 1992), random mutagenesis (e.g., U.S. Pat. Nos. 5,096,815; 5,198,346; and 5,223,409), site-directed mutagenesis (e.g., Walder et al., Gene, 42:133, 1986; Bauer et al., Gene, 37:73, 1985; Craik, Bio Techniques, January 1985, 12-19; Smith et al., Genetic Engineering: Principles and Methods, Plenum Press, 1981), phage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204), etc. Desired sequences can also be produced by the assembly of target sequences using mutually priming oligonucleotides (Uhlmann, Gene, 71:29-40, 1988). For directed mutagenesis methods, analysis of the three-dimensional structure of the TRPCC polypeptide can be used to guide and facilitate making mutants which effect polypeptide activity. Sites of substrate-enzyme interaction or other biological activities can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photoaffinity labeling. See, for example, de Vos et al., Science 255:306-312, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992.
In addition, libraries of TRPCC and fragments thereof can be used for screening and selection of TRPCC variants. For instance, a library of coding sequences can be generated by treating a double-stranded DNA with a nuclease under conditions where the nicking occurs, e.g., only once per molecule, denaturing the double-stranded DNA, renaturing it to double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single-stranded portions from reformed duplexes by treatment with [0078] S 1 nuclease, and ligating the resulting DNAs into an expression vector. By this method, expression libraries can be made comprising “mutagenized” TRPCC. The entire coding sequence or parts thereof can be used.
Polynucleotide Expression, Polypeptides Produced Thereby, and Specific-Binding Partners Thereto. [0079]
A polynucleotide according to the present invention can be expressed in a variety of different systems, in vitro and in vivo, according to the desired purpose. For example, a polynucleotide can be inserted into an expression vector, introduced into a desired host, and cultured under conditions effective to achieve expression of a polypeptide coded for by the polynucleotide, to search for specific binding partners. Effective conditions include any culture conditions suitable for achieving production of the polypeptide by the host cell, including effective temperatures, pH, medium, additives to the media in which the host cell is cultured (e.g., additives that amplify or induce expression such as butyrate, or methotrexate if the coding polynucleotide is adjacent to a dhfr gene), cyclohexamide, cell densities, culture dishes, etc. A polynucleotide can be introduced into the cell by any effective method including, e.g., naked DNA, calcium phosphate precipitation, electroporation, injection, DEAE-Dextran mediated transfection, fusion with liposomes, association with agents that enhance its uptake into cells, viral transfection. A cell into which a polynucleotide of the present invention has been introduced is a transformed host cell. The polynucleotide can be extrachromosomal or integrated into a chromosome(s) of the host cell. It can be stable or transient. An expression vector is selected for its compatibility with the host cell. Host cells include, mammalian cells, e.g., COS, CV1, BHK, CHO, HeLa, LTK, NIH 3T3, G-402 (ATCC CRL-1440), ACHN (ATCC CRL-1611), Vero (ATCC CCL-81), 786-0 (ATCC CRL-1932), 769-P (ATCC CRL-1933), CCD 1103 KIDTr (ATCC CRL-2304), CCD 1105 KIDTr (ATCC CRL-2305), Hs 835.T (ATCC CRL-7569), Hs 926.T (ATCC CRL-7678), Caki-1 (ATCC HTB-46), Caki-2 (ATCC HTB-47), SW 839 (ATCC HTB-49), LLC-MK2 (ATCC CCL-7), BHK-21 (ATCC CCL-10), MDBK, CV-1, (ATCC CRL-1573), KNRK (ATCC CRL-1569), NRK-49F (ATCC CRL-1570), A-704 (ATCC HTB-45), and other established and primary kidney lines, CNS neural stem cells (e.g., U.S. Pat. No. 6,103,530), IMR32, A172 (ATCC CRL-1620), T98G (ATCC CRL-1690), CCF-STTG1 (ATCC CRL-1718), DBTRG-05MG (ATCC CRL-2020), PFSK-1 (ATCC CRL-2060), SK-N-AS and other SK cell lines (ATCC CRL-2137), CHP-212 (ATCC CRL-2273), RG2 (ATCC CRL-2433), HCN-2 (ATCC CRL-10742), U-87 MG and other U MG cell lines (ATCC HTB-14), D283 Med (ATCC HTB-185), PC12, Neuro-2a (ATCC CCL-131), and other established and primary brain cell lines, insect cells, such as Sf9 ([0080] S. frugipeda) and Drosophila, bacteria, such as E. coli, Streptococcus, bacillus, yeast, such as Sacharomyces, S. cerevisiae, fungal cells, plant cells, embryonic or adult stem cells (e.g., mammalian, such as mouse or human).
Expression control sequences are similarly selected for host compatibility and a desired purpose, e.g., high copy number, high amounts, induction, amplification, controlled expression. Other sequences which can be employed include enhancers such as from SV40, CMV, RSV, inducible promoters, cell-type specific elements, or sequences which allow selective or specific cell expression. Promoters that can be used to drive its expression, include, e.g., the endogenous promoter, MMTV, SV40, trp, lac, tac, or T7 promoters for bacterial hosts; or alpha factor, alcohol oxidase, or PGH promoters for yeast. RNA promoters can be used to produced RNA transcripts, such as T7 or SP6. See, e.g., Melton et al., [0081] Polynucleotide Res., 12(18):7035-7056, 1984; Dunn and Studier. J. Mol. Bio., 166:477-435, 1984; U.S. Pat. No. 5,891,636; Studier et al., Gene Expression Technology, Methods in Enzymology, 85:60-89, 1987. In addition, as discussed above, translational signals (including in-frame insertions) can be included.
When a polynucleotide is expressed as a heterologous gene in a transfected cell line, the gene is introduced into a cell as described above, under effective conditions in which the gene is expressed. The term “heterologous” means that the gene has been introduced into the cell line by the “hand-of-man.” Introduction of a gene into a cell line is discussed above. The transfected (or transformed) cell expressing the gene can be lysed or the cell line can be used intact. [0082]
For expression and other purposes, a polynucleotide can contain codons found in a naturally-occurring gene, transcript, or cDNA, for example, e.g., as set forth in [0083] SEQ ID NO 1, or it can contain degenerate codons coding for the same amino acid sequences. For instance, it may be desirable to change the codons in the sequence to optimize the sequence for expression in a desired host. See, e.g., U.S. Pat. Nos. 5,567,600 and 5,567,862.
A polypeptide according to the present invention can be recovered from natural sources, transformed host cells (culture medium or cells) according to the usual methods, including detergent extraction (e.g., non-ionic detergent, Triton X-100, CHAPS, octylglucoside, Igepal CA-630), ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, lectin chromatography, gel electrophoresis. Protein refolding steps can be used, as necessary, in completing the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for purification steps. Another approach is to express the polypeptide recombinantly with an affinity tag (FLAG epitope, HA epitope, myc epitope, His tag, maltose binding protein, chitinase, etc) and then purify by anti-tag antibody-conjugated affinity chromatography. [0084]
The present invention also relates to polypeptides of TRPCC, e.g., an isolated human TRPCC polypeptide comprising or having the amino acid sequence set forth in [0085] SEQ ID NO 1, an isolated human TRPCC polypeptide comprising an amino acid sequence having more than 98%, 99%, or more amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO 2, and optionally having one or more of TRPCC activities, such as nucleotide binding, ligand binding, signal transduction, etc. Fragments specific to TRPCC can also be used, e.g., to produce antibodies or other immune responses, as competitors to nucleotide binding, ligand binding, etc. or as, e.g., inhibitors or stimuli in signal transduction pathways. These fragments can be referred to as being “specific for” TRPCC. The latter phrase, as already defined, indicates that the peptides are characteristic of TRPCC, and that the defined sequences are substantially absent from all other protein types. Such polypeptides can be of any size necessary to confer specificity, e.g., 5, 8, 10, 12, 15, 20, etc. Examples of polypeptides include but are not limited to polypeptides that comprise the following amino acid residues set forth in SEQ ID NO 2:2-60, 598-660 of SEQ ID NO 2. The present invention also relates to antibodies and other specific-binding partners are specific for polypeptides encoded by polynucleotides of the present invention, e.g., TRPCC. Antibodies, e.g., polyclonal, monoclonal, recombinant, chimeric, humanized, single-chain, Fab, and fragments thereof, can be prepared according to any desired method. See, also, screening recombinant immunoglobulin libraries (e.g., Orlandi et al., Proc. Natl. Acad. Sci., 86:3833-3837, 1989; Huse et al., Science, 256:1275-1281, 1989); in vitro stimulation of lymphocyte populations; Winter and Milstein, Nature, 349: 293-299, 1991. The antibodies can be IgM, IgG, subtypes, IgG2a, IgG1, etc. Antibodies, and immune responses, can also be generated by administering naked DNA. See, e.g., U.S. Pat. Nos. 5,703,055; 5,589,466; 5,580,859. Antibodies can be used from any source, including, goat, rabbit, mouse, chicken (e.g., IgY; see, Duan, WO/029444 for methods of making antibodies in avian hosts, and harvesting the antibodies from the eggs). An antibody specific for a polypeptide means that the antibody recognizes a defined sequence of amino acids within or including the polypeptide. Other specific binding partners include, e.g., aptamers and PNA. Antibodies can be prepared against specific epitopes or domains of TRPCC, e.g., amino acids 2-30, 773-789, 870-887, 905-913, 943-958, 969-986, 1005-1022, 1087-1114, 1125-1131, 789-870, 913-943, 986-1005, etc.
The preparation of polyclonal antibodies is well known to those skilled in the art. See, for example, Green et al., Production of Polyclonal Antisera, in IMMUNOCHEMICAL PROTOCOLS (Manson, ed.), pages 1-5 (Humana Press 1992); Coligan et al., Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in CURRENT PROTOCOLS IN IMMUNOLOGY, section 2.4.1 (1992). The preparation of monoclonal antibodies likewise is conventional. See, for example, Kohler & Milstein, Nature 256:495 (1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow et al., ANTIBODIES: A LABORATORY MANUAL, page 726 (Cold Spring Harbor Pub. 1988). [0086]
Antibodies can also be humanized, e.g., where they are to be used therapeutically. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al., Proc. Nat'l Acad. Sci. USA 86:3833 (1989), which is hereby incorporated in its entirety by reference. Techniques for producing humanized monoclonal antibodies are described, for example, in U.S. Pat. No. 6,054,297, Jones et al., Nature 321: 522 (1986); Riechmann et al., Nature 332: 323 (1988); Verhoeyen et al., Science 239: 1534 (1988); Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992); Sandhu, Crit. Rev. Biotech. 12: 437 (1992); and Singer et al., J. Immunol. 150: 2844 (1993). [0087]
Antibodies of the invention also may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 119 (1991); Winter et al., Ann. Rev. Immunol. 12: 433 (1994). Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained commercially, for example, from STRATAGENE Cloning Systems (La Jolla, Calif.). [0088]
In addition, antibodies of the present invention may be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been “engineered” to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens and can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described, e.g., in Green et al., Nature Genet. 7:13 (1994); Lonberg et al., Nature 368:856 (1994); and Taylor et al., Int. Immunol. 6:579 (1994). [0089]
Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in [0090] E. coli of nucleic acid encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab′).sub.2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab′ fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. No. 4,036,945 and No. 4,331,647, and references contained therein. These patents are hereby incorporated in their entireties by reference. See also Nisoiihoffet al., Arch. Biochem. Biophys. 89:230 (1960); Porter, Biochem. J. 73:119 (1959); Edelman et al, METHODS IN ENZYMOLOGY, VOL. 1, page 422 (Academic Press 1967); and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4.
Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques can also be used. For example, Fv fragments comprise an association of V.sub.H and V.sub.L chains. This association may be noncovalent, as described in Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu, supra. Preferably, the Fv fragments comprise V.sub.H and V.sub.L chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising nucleic acid sequences encoding the V.sub.H and V.sub.L domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 97 (1991); Bird et al., Science 242:423-426 (1988); Ladner et al., U.S. Pat. No. 4,946,778; Pack et al., Bio/Technology 11: 1271-77 (1993); and Sandhu, supra. [0091]
Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides (“minimal recognition units”) can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 106 (1991). [0092]
The term “antibody” as used herein includes intact molecules as well as fragments thereof, such as Fab, F(ab′)2, and Fv which are capable of binding to an epitopic determinant present in Bin1 polypeptide. Such antibody fragments retain some ability to selectively bind with its antigen or receptor. The term “epitope” refers to an antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Antibodies can be prepared against specific epitopes or polypeptide domains. [0093]
Antibodies that bind to TRPCC polypeptides of the present invention can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen. For example, it may be desirable to produce antibodies that specifically bind to the N- or C-terminal domains of TRPCC. The polypeptide or peptide used to immunize an animal, which is derived from translated cDNA or chemically synthesized, can be conjugated to a carrier protein, if desired. Commonly used carriers that are chemically coupled to the immunizing peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. [0094]
Polyclonal or monoclonal antibodies can be further purified, for example, by binding to and eluting from a matrix, to which is bound a polypeptide or peptide, against which the antibodies were raised. Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan, et al., [0095] Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994, incorporated by reference).
Anti-idiotype technology can also be used to produce invention monoclonal antibodies which mimic an epitope. For example, an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the “image” of the epitope bound by the first monoclonal antibody. [0096]
Methods of Detecting Polypeptides [0097]
Polypeptides coded for by TRPCC of the present invention can be detected, visualized, determined, quantitated, etc. according to any effective method. Useful methods include, e.g., but are not limited to, immunoassays, RIA (radioimmunoassay), ELISA, (enzyme-linked-immunosorbent assay), immunoflourescence, flow cytometry, histology, electron microscopy, light microscopy, in situ assays, immunoprecipitation, Western blot, far Western blot, Northwestern blot, etc. [0098]
Immunoassays may be carried out in liquid or on biological support. For instance, a sample (e.g., blood, stool, urine, cells, tissue, cerebral spinal fluid, body fluids, etc.) can be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support that is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled TRPCC-specific antibody. The solid phase support can then be washed with a buffer a second time to remove unbound antibody. The amount of bound label on solid support may then be detected by conventional means. [0099]
A “solid phase support or carrier” includes any support capable of binding an antigen, antibody, or other specific binding partner. Supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, and magnetite. A support material can have any structural or physical configuration. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads [0100]
One of the many ways in which gene peptide-specific antibody can be detectably labeled is by linking it to an enzyme and using it in an enzyme immunoassay (EIA). See, e.g., Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA),” 1978, [0101] Diagnostic Horizons 2, 1-7, Microbiological Associates Quarterly Publication, Walkersville, Md.); Voller, A. et al., 1978, J. Clin. Pathol. 31, 507-520; Butler, J. E., 1981, Meth. Enzymol. 73, 482-523; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla. The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes that can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, .alpha.-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta.-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by calorimetric methods that employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect TRPCC peptides through the use of a radioimmunoassay (RIA). See, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986. The radioactive isotope can be detected by a gamma counter or a scintillation counter or by phosphorimager or autoradiography. [0102]
It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. The antibody can also be detectably labeled using fluorescence-emitting metals such as those in the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). [0103]
The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. [0104]
Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin. [0105]
Diagnostic [0106]
The present invention also relates to methods and compositions for diagnosing diseases associated with TRPCC dysfunction (e.g., mutations in the TRPCC gene), or determining susceptibility to a disorder, using polynucleotides, polypeptides, and specific-binding partners of the present invention to detect, assess, determine, etc., TRPCC. As indicated above, disorders that can be diagnosed with TRPCC include, e.g., amyotrophic lateral sclerosis with frontotemporal dementia and hypomagnesemia with hypocalcemia. In such methods, the gene can serve as a marker for the disorder, e.g., where the gene, when mutant, is a direct cause of the disorder; where the gene is affected by another gene(s) that is directly responsible for the disorder, e.g., when the gene is part of the same signaling pathway as the directly responsible gene; and where the gene is chromosomally linked to the gene(s) directly responsible for the disorder and segregates with it. Many other situations are possible. To detect, assess, determine, etc., a probe specific for the gene can be employed as described above and below. Any method of detecting and/or assessing the gene can be used, including detecting expression of the gene using polynucleotides, antibodies, or other specific-binding partners. [0107]
The present invention relates to methods of diagnosing a disorder associated with TRPCC (e.g., in brain or kidney disease), or determining a subject's susceptibility to such disorder, including, e.g., assessing the expression of TRPCC in a tissue sample comprising tissue or cells suspected of having the disorder (e.g., where the sample comprises brain and kidney tissue). The phrase “diagnosing” indicates that it is determined whether the sample has the disorder. A “disorder” means, e.g., any abnormal condition as in a disease or malady. “Determining a subject's susceptibility to a disease or disorder” indicates that the subject is assessed for whether s/he is predisposed to get such a disease or disorder, where the predisposition is indicated by abnormal expression of the gene (e.g., gene mutation, gene expression pattern is not normal, etc.). Predisposition or susceptibility to a disease may result when such disease is influenced by epigenetic, environmental, etc., factors. This includes prenatal screening where samples from the fetus or embryo (e.g., via amniocentesis or CV sampling) are analyzed for the expression of the gene. Such diseases include hypomagnesemia and hypocalcemia and associated kidney disease and amyotrophic lateral sclerosis with frontotemporal dementia. [0108]
By the phrase “assessing expression of TRPCC,” it is meant that the functional status of the gene is evaluated. This includes, but is not limited to, measuring expression levels of said gene, determining the genomic structure of said gene, determining the mRNA structure of transcripts from said gene, or measuring the expression levels of polypeptide coded for by said gene. Thus, the term “assessing expression” includes evaluating all aspects of the transcriptional and translational machinery of the gene. For instance, if a promoter defect causes, or is suspected of causing, the disorder, then a sample can be evaluated (i.e., “assessed”) by looking (e.g., sequencing or restriction mapping) at the promoter sequence in the gene, by detecting transcription products (e.g., RNA), by detecting translation product (e.g., polypeptide). Any measure of whether the gene is functional can be used, including, polypeptide, polynucleotide, and functional assays for the gene's biological activity. [0109]
In making the assessment, it can be useful to compare the results to a normal gene, e.g., a gene that is not associated with the disorder. The nature of the comparison can be determined routinely, depending upon how the assessment is accomplished. If, for example, the mRNA levels of a sample are detected, then the mRNA levels of a normal or those of a gene known not to be affected by the disorder can serve as comparisons. Methods of detecting mRNA are well known, and discussed above, e.g., but not limited to, Northern blot analysis, polymerase chain reaction (PCR), reverse transcriptase PCR, RACE PCR, etc. Similarly, if polypeptide production is used to evaluate the gene, then the polypeptides in a normal tissue sample or polypeptides from a different gene whose expression is known not to be affected by the disorder can be used as comparisons. These are only examples of how such a method could be carried out. The sequences of TRPCC genes can also be compared, e.g., between a normal gene as shown in [0110] SEQ ID NO 1 and the sequence of a gene from a patient with the disorder, e.g., hypomagnesemia with hypocalcemia.
Assessing the effects of therapeutic and preventative interventions (e.g., administration of a drug, chemotherapy, radiation, etc.) on brain and kidney disease is a major effort in drug discovery, clinical medicine, and pharmacogenomics. The evaluation of therapeutic and preventative measures, whether experimental or already in clinical use, has broad applicability, e.g., in clinical trials, for monitoring the status of a patient, for analyzing and assessing animal models, and in any scenario involving treatment and prevention of hypomagnesemia and hypocalcemia and associated kidney diseases, amyotrophic lateral sclerosis with frontotemporal dementia, etc. Analyzing the expression profiles of polynucleotides of the present invention can be utilized as a parameter by which interventions are judged and measured. Treatment of a disorder can change the expression profile in a manner that is prognostic or indicative of the drug's effect on it. Changes in the profile can indicate, e.g., drug toxicity, return to a normal level, etc. Accordingly, the present invention also relates to methods of monitoring or assessing a therapeutic or preventative measure (e.g., chemotherapy, radiation, anti-neoplastic drugs, antibodies, etc.) in a subject with or susceptible to brain or kidney disease, comprising, e.g., detecting the expression levels of TRPCC. A subject can be a cell-based assay system, non-human animal model, human patient, etc. Detection can be accomplished as described for the methods above and below. By “therapeutic or preventative intervention,” it is meant, e.g., a drug administered to a patient, surgery, radiation, chemotherapy, and other measures taken to prevent, treat, or diagnose a disorder. [0111]
Expression can be assessed in any sample comprising any tissue or cell type, body fluid, etc., as discussed for other methods of the present invention, including cells from brain and kidney tissue or cells derived from brain and kidney tissue. By the phrase “cells derived from brain and kidney tissue,” it is meant that the derived cells originate from, e.g., when metastasis from a primary tumor site has occurred, when a progenitor-type or pluripotent cell gives rise to other cells, etc. [0112]
Identifying Agent Methods [0113]
The present invention also relates to methods of identifying agents, and the agents themselves, which modulate TRPCC. These agents can be used to modulate the biological activity of the polypeptide encoded for the gene, or the gene, itself. Agents that regulate the gene or its products are useful in a variety of different environments, including as medicinal agents to treat or prevent disorders associated with TRPCC and as research reagents to modify the functions of tissues and cells. [0114]
Methods of identifying agents generally comprise steps in which an agent is placed in contact with the gene, transcription product, translation product, or other target, and then a determination is performed to assess whether the agent “modulates” the target. The specific method utilized will depend upon a number of factors, including, e.g., the target (i.e., is it the gene or polypeptide encoded by it), the environment (e.g., in vitro or in vivo), the composition of the agent, etc. [0115]
For modulating the expression of TRPCC gene, a method can comprise, in any effective order, one or more of the following steps, e.g., contacting a TRPCC gene (e.g., in a cell population) with a test agent under conditions effective for said test agent to modulate the expression of TRPCC, and determining whether said test agent modulates said TRPCC. An agent can modulate expression of TRPCC at any level, including transcription, translation, and/or perdurance of the nucleic acid (e.g., degradation, stability, etc.) in the cell. For modulating the biological activity of TRPCC polypeptides, a method can comprise, in any effective order, one or more of the following steps, e.g., contacting a TRPCC polypeptide (e.g., in a cell, lysate, or isolated) with a test agent under conditions effective for said test agent to modulate the biological activity of said polypeptide, and determining whether said test agent modulates said biological activity. [0116]
Contacting TRPCC with the test agent can be accomplished by any suitable method and/or means that places the agent in a position to functionally control expression or biological activity of TRPCC present in the sample. Functional control indicates that the agent can exert its physiological effect on TRPCC through any effective mechanism. The choice of the method and/or means can depend upon the nature of the agent and the condition and type of environment in which the TRPCC is presented, e.g., lysate, isolated, or in a cell population (such as, in vivo, in vitro, organ explants, etc.). For instance, if the cell population is an in vitro cell culture, the agent can be contacted with the cells by adding it directly into the culture medium. If the agent cannot dissolve readily in an aqueous medium, it can be incorporated into liposomes, or another lipophilic carrier, and then administered to the cell culture. Contact can also be facilitated by incorporation of the agent with carriers and delivery molecules and complexes, by injection, by infusion, etc. [0117]
After the agent has been administered in such a way that it can gain access to TRPCC, it can be determined whether the test agent modulates TRPCC expression or biological activity. Modulation can be of any type, quality, or quantity, e.g., increase, facilitate, enhance, up-regulate, stimulate, activate, amplify, augment, induce, decrease, down-regulate, diminish, lessen, reduce, etc. The modulatory quantity can also encompass any value, e.g., 1%, 5%, 10%, 50%, 75%, 1-fold, 2-fold, 5-fold, 10-fold, 100-fold, etc. To modulate TRPCC expression means, e.g., that the test agent has an effect on its expression, e.g., to effect the amount of transcription, to effect RNA splicing, to effect translation of the RNA into polypeptide, to effect RNA or polypeptide stability, to effect polyadenylation or other processing of the RNA, to effect post-transcriptional or post-translational processing, etc. To modulate biological activity means, e.g., that a functional activity of the polypeptide is changed in comparison to its normal activity in the absence of the agent. This effect includes increase, decrease, block, inhibit, enhance, etc. [0118]
Biological activities of TRPCC include, e.g., cation channel activity, signal transduction activity, and protein binding activity. As discussed above, the biological activity of TRPCC can be measured routinely. For example, if agents are to be identified which modulate the channel activity of TRPCC either electrophysiology or calcium imaging can be used to assess their effects, e.g., using fluo-3, Fura-red, Ca-sensitive chemi-luminescent probes, etc. (e.g., kits are commercially available from Molecular Probes) and a laser scanning confocal microscope to visualize the changes in intracellular calcium as a result of modulation of TRPCC. [0119]
A test agent can be of any molecular composition, e.g., chemical compounds, biomolecules, such as polypeptides, lipids, nucleic acids (e.g., antisense to a polynucleotide sequence selected from SEQ ID NO 1), carbohydrates, antibodies, ribozymes, double-stranded RNA, aptamers, etc. For example, if a polypeptide to be modulated is a cell-surface molecule, a test agent can be an antibody that specifically recognizes it and, e.g., causes the polypeptide to be internalized, leading to its down regulation on the surface of the cell. Such an effect does not have to be permanent, but can require the presence of the antibody to continue the down-regulatory effect. Antibodies can also be used to modulate the biological activity of a polypeptide in a lysate or other cell-free form. Antisense TRPCC can also be used as test agents to modulate gene expression. [0120]
Therapeutics [0121]
Selective polynucleotides, polypeptides, and specific-binding partners thereto, can be utilized in therapeutic applications, especially to treat diseases and conditions of brain and kidney tissue. Useful methods include, but are not limited to, immunotherapy (e.g., using specific-binding partners to polypeptides), vaccination (e.g., using a selective polypeptide or a naked DNA encoding such polypeptide), protein or polypeptide replacement therapy, gene therapy (e.g., germ-line correction, antisense), etc. [0122]
Various immunotherapeutic approaches can be used. For instance, unlabeled antibody that specifically recognizes a tissue-specific antigen can be used to stimulate the body to destroy or attack the brain or kidney disease, to cause down-regulation, to produce complement-mediated lysis, to inhibit cell growth, etc., of target cells which display the antigen, e.g., analogously to how c-erbB-2 antibodies are used to treat brain or kidney disease. In addition, antibody can be labeled or conjugated to enhance its deleterious effect, e.g., with radionuclides and other energy emitting entities, toxins, such as ricin, exotoxin A (ETA), and diphtheria, cytotoxic or cytostatic agents, immunomodulators, chemotherapeutic agents, etc. See, e.g., U.S. Pat. No. 6,107,090. [0123]
An antibody or other specific-binding partner can be conjugated to a second molecule, such as a cytotoxic agent, and used for targeting the second molecule to a tissue-antigen positive cell (Vitetta, E. S. et al., 1993, Immunotoxin therapy, in DeVita, Jr., V. T. et al., eds, Cancer: Principles and Practice of Oncology, 4th ed., J. B. Lippincott Co., Philadelphia, 2624-2636). Examples of cytotoxic agents include, but are not limited to, antimetabolites, alkylating agents, anthracyclines, antibiotics, anti-mitotic agents, radioisotopes and chemotherapeutic agents. Further examples of cytotoxic agents include, but are not limited to ricin, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin D, 1-dehydrotestosterone, diptheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, elongation factor-2 and glucocorticoid. Techniques for conjugating therapeutic agents to antibodies are well. [0124]
In addition to immunotherapy, polynucleotides and polypeptides can be used as targets for non-immunotherapeutic applications, e.g., using compounds which interfere with function, expression (e.g., antisense as a therapeutic agent), assembly, etc. RNA interference can be used in vivtro and in vivo to silence TRPCC when its expression contributes to a disease (but also for other purposes, e.g., to identify the gene's function to change a developmental pathway of a cell, etc.). See, e.g., Sharp and Zamore, [0125] Science, 287:2431-2433, 2001; Grishok et al., Science, 287:2494, 2001.
Delivery of therapeutic agents can be achieved according to any effective method, including, liposomes, viruses, plasmid vectors, bacterial delivery systems, orally, systemically, etc. Therapeutic agents of the present invention can be administered in any form by any effective route, including, e.g., oral, parenteral, enteral, intraperitoneal, topical, transdermal (e.g., using any standard patch), ophthalmic, nasally, local, non-oral, such as aerosal, inhalation, subcutaneous, intramuscular, buccal, sublingual, rectal, vaginal, intra-arterial, and intrathecal, etc. They can be administered alone, or in combination with any ingredient(s), active or inactive. [0126]
In addition to therapeutics, per se, the present invention also relates to methods of treating a disease of brain, kidney, etc. tissues showing altered expression of TRPCC, comprising, e.g., administering to a subject in need thereof a therapeutic agent which is effective for regulating expression of said TRPCC and/or which is effective in treating said disease. The term “treating” is used conventionally, e.g., the management or care of a subject for the purpose of combating, alleviating, reducing, relieving, improving, etc., the condition of, e.g., a disease or disorder. By the phrase “altered expression,” it is meant that the disease is associated with a mutation in the gene, or any modification to the gene (or corresponding product) that affects its normal function. Thus, expression of TRPCC refers to, e.g., transcription, translation, splicing, stability of the mRNA or protein product, activity of the gene product, differential expression, etc. [0127]
Any agent that “treats” the disease can be used. Such an agent can be one that regulates the expression of the TRPCC. Expression refers to the same acts already mentioned, e.g. transcription, translation, splicing, stability of the mRNA or protein product, activity of the gene product, differential expression, etc. For instance, if the condition results from a complete deficiency of the gene product, administration of gene product to a patient would be said to treat the disease and regulate the gene's expression. Many other possible situations are possible, e.g., where the gene is aberrantly expressed, and the therapeutic agent regulates the aberrant expression by restoring its normal expression pattern. [0128]
The present invention also relates to methods of using binding partners for differentially-regulated genes, such as antibodies, to deliver active agents to the brain, kidney, and other tissues in which TRPCC is expressed, for a variety of different purposes, including, e.g., for diagnostic, therapeutic, and research purposes. Methods can involve delivering or administering an active agent to a target tissue, comprising, e.g., administering to a subject in need thereof, an effective amount of an active agent coupled to a binding partner specific for a differentially-regulated gene polypeptide, wherein said binding partner is effective to deliver said active agent specifically to said target tissue. [0129]
Any type of active agent can be used in combination with the binding partner, including, therapeutic, cytotoxic, cytostatic, chemotherapeutic, anti-neoplastic, anti-proliferative, anti-biotic, etc., agents. A chemotherapeutic agent can be, e.g., DNA-interactive agent, alkylating agent, antimetabolite, tubulin-interactive agent, hormonal agent, hydroxyurea, Cisplatin, Cyclophosphamide, Altretamine, Bleomycin, Dactinomycin, Doxorubicin, Etoposide, Teniposide, paclitaxel, cytoxan, 2-methoxycarbonylaminobenzimidazole, Plicamycin, Methotrexate, Fluorouracil, Fluorodeoxyuridin, CB3717, Azacitidine, Floxuridine, Mercapyopurine, 6-Thioguanine, Pentostatin, Cytarabine, Fludarabine, etc. Agents can also be contrast agents useful in imaging technology, e.g., X-ray, CT, CAT, MRI, ultrasound, PET, SPECT, and scintographic. [0130]
An active agent can be associated in any manner with a binding partner which is effective to achieve its delivery specifically to the target. Specific delivery or targeting indicates that the agent is provided to the target tissue, without being substantially provided to other tissues. This is useful especially where an agent is toxic, and specific targeting to the desired tissue enables the majority of the toxicity to be aimed at the target with as small as possible effect on other tissues in the body. The association of the active agent and the binding partner (“coupling) can be direct, e.g., through chemical bonds between the binding partner and the agent, or, via a linking agent, or the association can be less direct, e.g., where the active agent is in a liposome, or other carrier, and the binding partner is associated with the liposome surface. In such case, the binding partner can be oriented in such a way that it is able to bind to the gene product on brain and kidney tissue cell surface. Methods for delivery of DNA via a cell-surface receptor is described, e.g., in U.S. Pat. No. 6,339,139. [0131]
Antisense [0132]
Antisense polynucleotide (e.g., RNA) can also be prepared from a polynucleotide according to the present invention, such as anti-sense to a sequence of [0133] SEQ ID NO 1. Antisense polynucleotide can be used in various ways, such as to regulate or modulate expression of the polypeptides they encode, e.g., inhibit their expression, for in situ hybridization, for therapeutic purposes, for making targeted mutations (in vivo, triplex, etc.) etc. For guidance on administering and designing anti-sense, see, e.g., U.S. Pat. Nos. 6,200,960, 6,200,807, 6,197,584, 6,190,869, 6,190,661, 6,187,587, 6,168,950, 6,153,595, 6,150,162, 6,133,246, 6,117,847, 6,096,722, 6,087,343, 6,040,296, 6,005,095, 5,998,383, 5,994,230, 5,891,725, 5,885,970, and 5,840,708. An antisense polynucleotides can be operably linked to an expression control sequence. A total length of about 35 bp can be used in cell culture with cationic liposomes to facilitate cellular uptake, but for in vivo use, preferably shorter oligonucleotides are administered, e.g. 25 nucleotides.
Antisense polynucleotides can comprise modified, non-naturally-occurring nucleotides and linkages between the nucleotides (e.g., modification of the phosphate-sugar backbone; methyl phosphonate, phosphorothioate, or phosphorodithioate linkages; and 2′-O-methyl ribose sugar units), e.g., to enhance in vivo or in vitro stability, to confer nuclease resistance, to modulate uptake, to modulate cellular distribution and compartmentalization, etc. Any effective nucleotide or modification can be used, including those already mentioned, as known in the art, etc., e.g., disclosed in U.S. Pat. Nos. 6,133,438; 6,127,533; 6,124,445; 6,121,437; 5,218,103 (e.g., nucleoside thiophosphoramidites); 4,973,679; Sproat et al., “2′-O-Methyloligoribonucleotides: synthesis and applications,” Oligonucleotides and Analogs A Practical Approach, Eckstein (ed.), IRL Press, Oxford, 1991, 49-86; Iribarren et al., “2′O-Alkyl Oligoribonucleotides as Antisense Probes,” Proc. Natl. Acad. Sci. USA, 1990, 87, 7747-7751; Cofton et al., “2′-O-methyl, 2′-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonucleotides as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event,” Nucl. Acids Res., 1991, 19, 2629-2635. [0134]
Arrays [0135]
The present invention also relates to an ordered array of polynucleotide probes and specific-binding partners (e.g., antibodies) for detecting the expression of TRPCC in a sample, comprising, one or more polynucleotide probes or specific binding partners associated with a solid support, wherein each probe is specific for TRPCC, and the probes comprise, e.g., a nucleotide sequence of [0136] SEQ ID NO 1 which is specific for said gene, a nucleotide sequence having sequence identity to SEQ ID NO 1 which is specific for said gene or polynucleotide, or complements thereto, or a specific-binding partner which is specific for TRPCC.
The phrase “ordered array” indicates that the probes are arranged in an identifiable or position-addressable pattern, e.g., such as the arrays disclosed in U.S. Pat. Nos. 6,156,501, 6,077,673, 6,054,270, 5,723,320, 5,700,637, [0137] WO0991971 1, WO00023803. The probes are associated with the solid support in any effective way. For instance, the probes can be bound to the solid support, either by polymerizing the probes on the substrate, or by attaching a probe to the substrate. Association can be, covalent, electrostatic, noncovalent, hydrophobic, hydrophilic, noncovalent, coordination, adsorbed, absorbed, polar, etc. When fibers or hollow filaments are utilized for the array, the probes can fill the hollow orifice, be absorbed into the solid filament, be attached to the surface of the orifice, etc. Probes can be of any effective size, sequence identity, composition, etc., as already discussed. Ordered arrays can further comprise polynucleotide probes or specific-binding partners which are specific for other genes.
Transgenic Animals [0138]
The present invention also relates to transgenic animals comprising a TRPCC gene. Such genes, as discussed in more detail below, include, but are not limited to, functionally-disrupted genes, mutated genes, ectopically or selectively-expressed genes, inducible or regulatable genes, etc. These transgenic animals can be produced according to any suitable technique or method, including homologous recombination, mutagenesis (e.g., ENU, Rathkolb et al., [0139] Exp. Physiol., 85(6):635-644, 2000), and the tetracycline-regulated gene expression system (e.g., U.S. Pat. No. 6,242,667). The term “gene” as used herein includes any part of a gene, i.e., regulatory sequences, promoters, enhancers, exons, introns, coding sequences, etc. The TRPCC nucleic acid present in the construct or transgene can be naturally-occurring wild-type, polymorphic, or mutated, and includes any mammalian homolog of TRPCC, such as mouse (e.g., the gene represented by XM_—140575), pig, monkey, rat, etc.
Along these lines, polynucleotides of the present invention can be used to create transgenic animals, e.g. a non-human animal, comprising at least one cell whose genome comprises a functional disruption of TRPCC. By the phrases “functional disruption” or “functionally disrupted,” it is meant that the gene does not express a biologically-active product. It can be substantially deficient in at least one functional activity coded for by the gene. Expression of a polypeptide can be substantially absent, i.e., essentially undetectable amounts are made. However, polypeptide can also be made, but which is deficient in activity, e.g., where only an amino-terminal portion of the gene product is produced. Functional disruption can be achieved in any region of the gene, e.g., regulatory regions, promoter, amino acids 1-690, etc. [0140]
The transgenic animal can comprise one or more cells. When substantially all its cells contain the engineered gene, it can be referred to as a transgenic animal “whose genome comprises” the engineered gene. This indicates that the endogenous gene loci of the animal has been modified and substantially all cells contain such modification. [0141]
Functional disruption of the gene can be accomplished in any effective way, including, e.g., introduction of a stop codon into any part of the coding sequence such that the resulting polypeptide is biologically inactive (e.g., because it lacks a catalytic domain, a ligand binding domain, etc.), introduction of a mutation into a promoter or other regulatory sequence that is effective to turn it off, or reduce transcription of the gene, insertion of an exogenous sequence into the gene which inactivates it (e.g., which disrupts the production of a biologically-active polypeptide or which disrupts the promoter or other transcriptional machinery), deletion of sequences from the TRPCC gene, etc. Examples of transgenic animals having functionally disrupted genes are well known, e.g., as described in U.S. Pat. Nos. 6,239,326, 6,225,525, 6,207,878, 6,194,633, 6,187,992, 6,180,849, 6,177,610, 6,100,445, 6,087,555, 6,080,910, 6,069,297, 6,060,642, 6,028,244, 6,013,858, 5,981,830, 5,866,760, 5,859,314, 5,850,004, 5,817,912, 5,789,654, 5,777,195, and 5,569,824. A transgenic animal which comprises the functional disruption can also be referred to as a “knock-out” animal, since the biological activity of its TRPCC genes has been “knocked-out.” Knock-outs can be homozygous or heterozygous. [0142]
For creating functional disrupted genes, and other gene mutations, homologous recombination technology is of special interest since it allows specific regions of the genome to be targeted. Using homologous recombination methods, genes can be specifically-inactivated, specific mutations can be introduced, and exogenous sequences can be introduced at specific sites. These methods are well known in the art, e.g., as described in the patents above. See, also, Robertson, [0143] Biol. Reproduc., 44(2):238-245, 1991. Generally, the genetic engineering is performed in an embryonic stem (ES) cell, or other pluripotent cell line (e.g., adult stem cells, EG cells), and that genetically-modified cell (or nucleus) is used to create a whole organism. Nuclear transfer can be used in combination with homologous recombination technologies.
For example, the TRPCC locus can be disrupted in mouse ES cells using a positive-negative selection method (e.g., Mansour et al., [0144] Nature, 336:348-352, 1988). In this method, a targeting vector can be constructed which comprises a part of the gene to be targeted. A selectable marker, such as neomycin resistance genes, can be inserted into a TRPCC exon present in the targeting vector, disrupting it. When the vector recombines with the ES cell genome, it disrupts the function of the gene. The presence in the cell of the vector can be determined by expression of neomycin resistance. See, e.g., U.S. Pat. No. 6,239,326. Cells having at least one functionally disrupted gene can be used to make chimeric and germline animals, e.g., animals having somatic and/or germ cells comprising the engineered gene. Homozygous knock-out animals can be obtained from breeding heterozygous knock-out animals. See, e.g., U.S. Pat. No. 6,225,525.
A transgenic animal, or animal cell, lacking one or more functional TRPCC genes can be useful in a variety of applications, including, as an animal model for hypomagnesemia with secondary hypocalcemia, amyotrophic lateral sclerosis with frontotermporal dementia, etc., drug screening assays (e.g., for signal transduction mediated by agents other than TRPCC; by making a cell deficient in TRPCC, the contribution of other receptors to, e.g., Ca[0145] ²⁺ modulation can be specifically examined), as a source of tissues deficient in TRPCC activity, and any of the utilities mentioned in any issued U.S. Patent on transgenic animals, including, U.S. Pat. Nos. 6,239,326, 6,225,525, 6,207,878, 6,194,633, 6,187,992, 6,180,849, 6,177,610, 6,100,445, 6,087,555, 6,080,910, 6,069,297, 6,060,642, 6,028,244, 6,013,858, 5,981,830, 5,866,760, 5,859,314, 5,850,004, 5,817,912, 5,789,654, 5,777,195, and 5,569,824. Such an animal can show a defect in cation (e.g., calcium) conductance, e.g., an impairment in the permeation of an ion through the channel.
The present invention also relates to non-human, transgenic animal whose genome comprises recombinant TRPCC nucleic acid operatively linked to an expression control sequence effective to express said coding sequence, e.g., in brain and kidney tissue. Such a transgenic animal can also be referred to as a “knock-in” animal since an exogenous gene has been introduced, stably, into its genome. For example, the endogenous locus can be knocked out by inserting [0146] SEQ ID NO 1 into it.
A recombinant TRPCC nucleic acid refers to a gene that has been introduced into a target host cell and optionally modified, such as cells derived from animals, plants, bacteria, yeast, etc. A recombinant TRPCC includes completely synthetic nucleic acid sequences, semi-synthetic nucleic acid sequences, sequences derived from natural sources, and chimeras thereof. “Operable linkage” has the meaning used through the specification, i.e., placed in a functional relationship with another nucleic acid. When a gene is operably linked to an expression control sequence, as explained above, it indicates that the gene (e.g., coding sequence) is joined to the expression control sequence (e.g., promoter) in such a way that facilitates transcription and translation of the coding sequence. As described above, the phrase “genome” indicates that the genome of the cell has been modified. In this case, the recombinant TRPCC has been stably integrated into the genome of the animal. The TRPCC nucleic acid in operable linkage with the expression control sequence can also be referred to as a construct or transgene. [0147]
Any expression control sequence can be used depending on the purpose. For instance, if selective expression is desired, then expression control sequences that limit its expression can be selected. These include, e.g., tissue or cell-specific promoters, introns, enhancers, etc. For various methods of cell and tissue-specific expression, see, e.g., U.S. Pat. Nos. 6,215,040, 6,210,736, and 6,153,427. These also include the endogenous promoter, i.e., the coding sequence can be operably linked to its own promoter. Inducible and regulatable promoters can also be utilized. [0148]
The present invention also relates to a transgenic animal which contains a functionally disrupted and a transgene stably integrated into the animals genome. Such an animal can be constructed using combinations any of the above- and below-mentioned methods. Such animals have any of the aforementioned uses, including permitting the knock-out of the normal gene and its replacement with a mutated gene. Such a transgene can be integrated at the endogenous gene locus so that the functional disruption and “knock-in” are carried out in the same step. [0149]
In addition to the methods mentioned above, transgenic animals can be prepared according to known methods, including, e.g., by pronuclear injection of recombinant genes into pronuclei of 1-cell embryos, incorporating an artificial yeast chromosome into embryonic stem cells, gene targeting methods, embryonic stem cell methodology, cloning methods, nuclear transfer methods. See, also, e.g., U.S. Pat. Nos. 4,736,866; 4,873,191; 4,873,316; 5,082,779; 5,304,489; 5,174,986; 5,175,384; 5,175,385; 5,221,778; Gordon et al., Proc. Natl. Acad. Sci., 77:7380-7384, 1980; Palmiter et al., Cell, 41:343-345, 1985; Palmiter et al., Ann. Rev. Genet., 20:465-499, 1986; Askew et al., Mol. Cell. Bio., 13:4115-4124, 1993; Games et al. Nature, 373:523-527, 1995; Valancius and Smithies, Mol. Cell. Bio., 11:1402-1408, 1991; Stacey et al., Mol. Cell. Bio., 14:1009-1016, 1994; Hasty et al., Nature, 350:243-246, 1995; Rubinstein et al., Nucl. Acid Res., 21:2613-2617, 1993; Cibelli et al., Science, 280:1256-1258, 1998. For guidance on recombinase excision systems, see, e.g., U.S. Pat. Nos. 5,626,159, 5,527,695, and 5,434,066. See also, Orban, P. C., et al., “Tissue- and Site-Specific DNA Recombination in Transgenic Mice,” Proc. Natl. Acad. Sci. USA, 89:6861-6865 (1992); O'Gorman, S., et al., “Recombinase-Mediated Gene Activation and Site-Specific Integration in Mammalian Cells,” Science, 251:1351-1355 (1991); Sauer, B., et al., “Cre-stimulated recombination at loxP-Containing DNA sequences placed into the mammalian genome,” Polynucleotides Research, 17(1):147-161 (1989); Gagneten, S. et al. (1997) Nucl. Acids Res. 25:3326-3331; Xiao and Weaver (1997) Nucl. Acids Res. 25:2985-2991; Agah, R. et al. (1997) J. Clin. Invest. 100:169-179; Barlow, C. et al. (1997) Nucl. Acids Res. 25:2543-2545; Araki, K. et al. (1997) Nucl. Acids Res. 25:868-872; Mortensen, R. N. et al. (1992) Mol. Cell. Biol. 12:2391-2395 (G418 escalation method); Lakhlani, P. P. et al. (1997) Proc. Natl. Acad. Sci. USA 94:9950-9955 (“hit and run”); Westphal and Leder (1997) Curr. Biol. 7:530-533 (transposon-generated “knock-out” and “knock-in”); Templeton, N. S. et al. (1997) Gene Ther. 4:700-709 (methods for efficient gene targeting, allowing for a high frequency of homologous recombination events, e.g., without selectable markers); PCT International Publication WO 93/22443 (functionally-disrupted). [0150]
A polynucleotide according to the present invention can be introduced into any non-human animal, including a non-human mammal, mouse (Hogan et al., [0151] Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1986), pig (Hammer et al., Nature, 315:343-345, 1985), sheep (Hammer et al., Nature, 315:343-345, 1985), cattle, rat, or primate. See also, e.g., Church, 1987, Trends in Biotech. 5:13-19; Clark et al., Trends in Biotech. 5:20-24, 1987); and DePamphilis et al., BioTechniques, 6:662-680, 1988. Transgenic animals can be produced by the methods described in U.S. Pat. No. 5,994,618, and utilized for any of the utilities described therein.
Database [0152]
The present invention also relates to electronic forms of polynucleotides, polypeptides, etc., of the present invention, including computer-readable medium (e.g., magnetic, optical, etc., stored in any suitable format, such as flat files or hierarchical files) which comprise such sequences, or fragments thereof, e-commerce-related means, etc. Along these lines, the present invention relates to methods of retrieving gene sequences from a computer-readable medium, comprising, one or more of the following steps in any effective order, e.g., selecting a cell or gene expression profile, e.g., a profile that specifies that said gene is differentially expressed in brain, kidney, and pituitary tissues, and retrieving said differentially expressed gene sequences, where the gene sequences consist or comprise of the genes represented by [0153] SEQ ID NO 1.
A “gene expression profile” means the list of tissues, cells, etc., in which a defined gene is expressed (i.e, transcribed and/or translated). A “cell expression profile” means the genes which are expressed in the particular cell type. The profile can be a list of the tissues in which the gene is expressed, but can include additional information as well, including level of expression (e.g., a quantity as compared or normalized to a control gene), and information on temporal (e.g., at what point in the cell-cycle or developmental program) and spatial expression. By the phrase “selecting a gene or cell expression profile,” it is meant that a user decides what type of gene or cell expression pattern he is interested in retrieving, e.g., he may require that the gene is differentially expressed in a tissue, or he may require that the gene is not expressed in blood, but must be expressed in brain or kidney disease. Any pattern of expression preferences may be selected. The selecting can be performed by any effective method. In general, “selecting” refers to the process in which a user forms a query that is used to search a database of gene expression profiles. The step of retrieving involves searching for results in a database that correspond to the query set forth in the selecting step. Any suitable algorithm can be utilized to perform the search query, including algorithms that look for matches, or that perform optimization between query and data. The database is information that has been stored in an appropriate storage medium, having a suitable computer-readable format. Once results are retrieved, they can be displayed in any suitable format, such as HTML. [0154]
For instance, the user may be interested in identifying genes that are differentially expressed in brain or kidney disease tissue. He may not care whether small amounts of expression occur in other tissues, as long as such genes are not expressed in peripheral blood lymphocytes. A query is formed by the user to retrieve the set of genes from the database having the desired gene or cell expression profile. Once the query is input into the system, a search algorithm is used to interrogate the database, and retrieve results. [0155]
Advertising, Licensing, etc., Methods [0156]
The present invention also relates to methods of advertising, licensing, selling, purchasing, brokering, etc., genes, polynucleotides, specific-binding partners, antibodies, etc., of the present invention. Methods can comprise e.g., displaying a TRPCC gene, TRPCC polypeptide, or antibody specific for TRPCC in a printed or computer-readable medium (e.g., on the Web or Internet), accepting an offer to purchase said gene, polypeptide, or antibody. [0157]
Other [0158]
A polynucleotide, probe, polypeptide, antibody, specific-binding partner, etc., according to the present invention can be isolated. The term “isolated” means that the material is in a form in which it is not found in its original environment or in nature, e.g., more concentrated, more purified, separated from component, etc. An isolated polynucleotide includes, e.g., a polynucleotide having the sequenced separated from the chromosomal DNA found in a living animal, e.g., as the complete gene, a transcript, or a cDNA. This polynucleotide can be part of a vector or inserted into a chromosome (by specific gene-targeting or by random integration at a position other than its normal position) and still be isolated in that it is not in a form that is found in its natural environment. A polynucleotide, polypeptide, etc., of the present invention can also be substantially purified. By substantially purified, it is meant that polynucleotide or polypeptide is separated and is essentially free from other polynucleotides or polypeptides, i.e., the polynucleotide or polypeptide is the primary and active constituent. A polynucleotide can also be a recombinant molecule. By “recombinant,” it is meant that the polynucleotide is an arrangement or form which does not occur in nature. For instance, a recombinant molecule comprising a promoter sequence would not encompass the naturally-occurring gene, but would include the promoter operably linked to a coding sequence not associated with it in nature, e.g., a reporter gene, or a truncation of the normal coding sequence. [0159]
The term “marker” is used herein to indicate a means for detecting or labeling a target. A marker can be a polynucleotide (usually referred to as a “probe”), polypeptide (e.g., an antibody conjugated to a detectable label), PNA, or any effective material. [0160]
The topic headings set forth above are meant as guidance where certain information can be found in the application, but are not intended to be the only source in the application where information on such topic can be found. Reference materials [0161]
For other aspects of the polynucleotides, reference is made to standard textbooks of molecular biology. See, e.g., Hames et al., [0162] Polynucleotide Hybridization, IL Press, 1985; Davis et al., Basic Methods in Molecular Biology, Elsevir Sciences Publishing, Inc., New York, 1986; Sambrook et al., Molecular Cloning, CSH Press, 1989; Howe, Gene Cloning and Manipulation, Cambridge University Press, 1995; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1994-1998.
The preceding description, utilize the present invention to its fullest extent. The preceding preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limiting the remainder of the disclosure in any way whatsoever. The entire disclosure of all applications, patents and publications, cited above and in the figures are hereby incorporated by reference in their entirety. [0163]
1 9 1 6455 DNA Homo sapiens CDS (431)..(5554) 1 gctttgtgca agaaagtgca agtttcccgt tctggcttca tttttgttcc cttttgcaat 60 cctcctggct cccccccaaa ccaagctagc aaagcaatgg ccccggttcc cccccaacgc 120 ctgacctgcg tttactggga ggagagcggg agagggagcg cgcattctgg agcaggctgc 180 tttgactccg accacaggct gttttgtgca ggctgtccct cttcttcaaa atcgtgcatc 240 ccctccccga agcagcaggc agtgtgcctc cattcagcca catttggtat gcatgagcac 300 ggctgcagag agaggggagg tggctgtttt aagaaggttc aggggctcag gcaaggctac 360 ttgactagtc ttccaagttc caggaagcct ctgccctaat ggaatttgca ggtgtggaga 420 tgaccatggg atg cca gag ccg tgg ggg acc gtt tat ttt cta ggc att 469 Met Pro Glu Pro Trp Gly Thr Val Tyr Phe Leu Gly Ile 1 5 10 gct cag gtt ttc agt ttc ttg ttt tcc tgg tgg aat ttg gaa ggg gtc 517 Ala Gln Val Phe Ser Phe Leu Phe Ser Trp Trp Asn Leu Glu Gly Val 15 20 25 atg aat cag gct gat gct cct cga ccc cta aac tgg acc atc cgg aag 565 Met Asn Gln Ala Asp Ala Pro Arg Pro Leu Asn Trp Thr Ile Arg Lys 30 35 40 45 ctg tgc cac gca gcc ttt ctt cca tct gtc aga ctt ctg aag gct cag 613 Leu Cys His Ala Ala Phe Leu Pro Ser Val Arg Leu Leu Lys Ala Gln 50 55 60 aaa tcc tgg ata gaa aga gca ttt tat aaa aga gaa tgt gtc cac atc 661 Lys Ser Trp Ile Glu Arg Ala Phe Tyr Lys Arg Glu Cys Val His Ile 65 70 75 ata ccc agc acc aaa gac ccc cat agg tgt tgc tgt ggg cgt ctg ata 709 Ile Pro Ser Thr Lys Asp Pro His Arg Cys Cys Cys Gly Arg Leu Ile 80 85 90 ggc cag cat gtt ggc ctc acc ccc agt atc tcc gtg ctt cag aat gag 757 Gly Gln His Val Gly Leu Thr Pro Ser Ile Ser Val Leu Gln Asn Glu 95 100 105 aaa aat gaa agt cgc ctc tcc cga aat gac atc cag tct gaa aag tgg 805 Lys Asn Glu Ser Arg Leu Ser Arg Asn Asp Ile Gln Ser Glu Lys Trp 110 115 120 125 tcc atc agc aaa cac act caa ctc agc cct acg gat gct ttt ggg acc 853 Ser Ile Ser Lys His Thr Gln Leu Ser Pro Thr Asp Ala Phe Gly Thr 130 135 140 att gag ttc caa gga ggt ggc cat tcc aac aaa gcc atg tat gtg cga 901 Ile Glu Phe Gln Gly Gly Gly His Ser Asn Lys Ala Met Tyr Val Arg 145 150 155 gta tct ttt gat aca aaa cct gat ctc ctc tta cac ctg atg acc aag 949 Val Ser Phe Asp Thr Lys Pro Asp Leu Leu Leu His Leu Met Thr Lys 160 165 170 gaa tgg cag ttg gag ctt ccc aag ctt ctc atc tct gtc cat ggg ggc 997 Glu Trp Gln Leu Glu Leu Pro Lys Leu Leu Ile Ser Val His Gly Gly 175 180 185 ctg cag aac ttt gaa ctc cag cca aaa ctc aag caa gtc ttt ggg aaa 1045 Leu Gln Asn Phe Glu Leu Gln Pro Lys Leu Lys Gln Val Phe Gly Lys 190 195 200 205 ggg ctc atc aaa gca gca atg aca act gga gcg tgg ata ttc act gga 1093 Gly Leu Ile Lys Ala Ala Met Thr Thr Gly Ala Trp Ile Phe Thr Gly 210 215 220 ggg gtt aac aca ggt gtt att cgt cat gtt ggc gat gcc ttg aag gat 1141 Gly Val Asn Thr Gly Val Ile Arg His Val Gly Asp Ala Leu Lys Asp 225 230 235 cat gcc tct aag tct cga gga aag ata tgc acc ata ggt att gcc ccc 1189 His Ala Ser Lys Ser Arg Gly Lys Ile Cys Thr Ile Gly Ile Ala Pro 240 245 250 tgg gga att gtg gaa aac cag gag gac ctc att gga aga gat gtt gtc 1237 Trp Gly Ile Val Glu Asn Gln Glu Asp Leu Ile Gly Arg Asp Val Val 255 260 265 cgg cca tac cag acc atg tcc aat ccc atg agc aag ctc act gtt ctc 1285 Arg Pro Tyr Gln Thr Met Ser Asn Pro Met Ser Lys Leu Thr Val Leu 270 275 280 285 aac agc atg cat tcc cac ttc att ctg gct gac aac ggg acc act gga 1333 Asn Ser Met His Ser His Phe Ile Leu Ala Asp Asn Gly Thr Thr Gly 290 295 300 aaa tat gga gca gag gtg aaa ctt cga aga caa ctg gaa aag cat att 1381 Lys Tyr Gly Ala Glu Val Lys Leu Arg Arg Gln Leu Glu Lys His Ile 305 310 315 tca ctc cag aag ata aac aca aga atc ggt caa ggt gtt cct gtg gtg 1429 Ser Leu Gln Lys Ile Asn Thr Arg Ile Gly Gln Gly Val Pro Val Val 320 325 330 gca ctc ata gtg gaa gga gga ccc aat gtg atc tcg att gtt ttg gag 1477 Ala Leu Ile Val Glu Gly Gly Pro Asn Val Ile Ser Ile Val Leu Glu 335 340 345 tac ctt cga gac acc cct ccc gtg cca gtg gtt gtc tgt gat ggg agt 1525 Tyr Leu Arg Asp Thr Pro Pro Val Pro Val Val Val Cys Asp Gly Ser 350 355 360 365 gga cgg gca tcg gac atc ctg gcc ttt ggg cat aaa tac tca gaa gaa 1573 Gly Arg Ala Ser Asp Ile Leu Ala Phe Gly His Lys Tyr Ser Glu Glu 370 375 380 ggc gga ctg ata aat gaa tct ttg agg gac cag ctg ttg gtg act ata 1621 Gly Gly Leu Ile Asn Glu Ser Leu Arg Asp Gln Leu Leu Val Thr Ile 385 390 395 cag aag act ttc aca tac act cga acc caa gct cag cat ctg ttc atc 1669 Gln Lys Thr Phe Thr Tyr Thr Arg Thr Gln Ala Gln His Leu Phe Ile 400 405 410 atc ctc atg gag tgc atg aag aag aag gaa ttg att acg gta ttt cgg 1717 Ile Leu Met Glu Cys Met Lys Lys Lys Glu Leu Ile Thr Val Phe Arg 415 420 425 atg gga tca gaa gga cac cag gac att gat ttg gct atc ctg aca gct 1765 Met Gly Ser Glu Gly His Gln Asp Ile Asp Leu Ala Ile Leu Thr Ala 430 435 440 445 tta ctc aaa gga gcc aat gcc tcg gcc cca gac caa ctg agc tta gct 1813 Leu Leu Lys Gly Ala Asn Ala Ser Ala Pro Asp Gln Leu Ser Leu Ala 450 455 460 tta gcc tgg aac aga gtc gac atc gct cgc agc cag atc ttt att tac 1861 Leu Ala Trp Asn Arg Val Asp Ile Ala Arg Ser Gln Ile Phe Ile Tyr 465 470 475 ggg caa cag tgg ccg gtg gga tct ctg gag caa gcc atg ttg gat gcc 1909 Gly Gln Gln Trp Pro Val Gly Ser Leu Glu Gln Ala Met Leu Asp Ala 480 485 490 tta gtt ctg gac aga gtg gat ttt gtg aaa tta ctc ata gag aat gga 1957 Leu Val Leu Asp Arg Val Asp Phe Val Lys Leu Leu Ile Glu Asn Gly 495 500 505 gta agc atg cac cgt ttt ctc acc atc tcc aga cta gag gaa ttg tac 2005 Val Ser Met His Arg Phe Leu Thr Ile Ser Arg Leu Glu Glu Leu Tyr 510 515 520 525 aat acg aga cat ggg ccc tca aat aca ttg tac cac ttg gtc agg gat 2053 Asn Thr Arg His Gly Pro Ser Asn Thr Leu Tyr His Leu Val Arg Asp 530 535 540 gtc aaa aag ggg aac ctg ccc cca gac tac aga atc agc ctg att gac 2101 Val Lys Lys Gly Asn Leu Pro Pro Asp Tyr Arg Ile Ser Leu Ile Asp 545 550 555 atc ggc ctg gtg atc gag tac ctg atg ggc ggg gct tat cgc tgc aac 2149 Ile Gly Leu Val Ile Glu Tyr Leu Met Gly Gly Ala Tyr Arg Cys Asn 560 565 570 tac acg cgc aag cgc ttc cgg acc ctc tac cac aac ctc ttc ggc ccc 2197 Tyr Thr Arg Lys Arg Phe Arg Thr Leu Tyr His Asn Leu Phe Gly Pro 575 580 585 aag agg ccc aaa gcc ttg aaa ctg ctg gga atg gag gat gat att ccc 2245 Lys Arg Pro Lys Ala Leu Lys Leu Leu Gly Met Glu Asp Asp Ile Pro 590 595 600 605 ttg agg cga gga aga aag aca acc aag aaa cgt gaa gaa gag gtg gac 2293 Leu Arg Arg Gly Arg Lys Thr Thr Lys Lys Arg Glu Glu Glu Val Asp 610 615 620 att gac ttg gat gat cct gag atc aac cac ttc ccc ttc cct ttc cat 2341 Ile Asp Leu Asp Asp Pro Glu Ile Asn His Phe Pro Phe Pro Phe His 625 630 635 gag ctc atg gtg tgg gct gtt ctc atg aag cgg cag aag atg gcc ctg 2389 Glu Leu Met Val Trp Ala Val Leu Met Lys Arg Gln Lys Met Ala Leu 640 645 650 ttc ttc tgg cag cac ggt gag gag gcc atg gcc aag gcc ctg gtg gcc 2437 Phe Phe Trp Gln His Gly Glu Glu Ala Met Ala Lys Ala Leu Val Ala 655 660 665 tgc aag ctc tgc aaa gcc atg gct cat gag gcc tct gag aac gac atg 2485 Cys Lys Leu Cys Lys Ala Met Ala His Glu Ala Ser Glu Asn Asp Met 670 675 680 685 gtt gac gac att tcc cag gag ctg aat cac aat tcc aga gac ttt ggc 2533 Val Asp Asp Ile Ser Gln Glu Leu Asn His Asn Ser Arg Asp Phe Gly 690 695 700 cag ctg gct gtg gag ctc ctg gac cag tcc tac aag cag gac gaa cag 2581 Gln Leu Ala Val Glu Leu Leu Asp Gln Ser Tyr Lys Gln Asp Glu Gln 705 710 715 ctg gcc atg aaa ctg ctg acg tat gag ctg aag aac tgg agc aac gcc 2629 Leu Ala Met Lys Leu Leu Thr Tyr Glu Leu Lys Asn Trp Ser Asn Ala 720 725 730 acg tgc ctg cag ctt gcc gtg gct gcc aaa cac cgc gac ttc atc gcg 2677 Thr Cys Leu Gln Leu Ala Val Ala Ala Lys His Arg Asp Phe Ile Ala 735 740 745 cac acg tgc agc cag atg ctg ctc acc gac atg tgg atg ggc cgg ctc 2725 His Thr Cys Ser Gln Met Leu Leu Thr Asp Met Trp Met Gly Arg Leu 750 755 760 765 cgc atg cgc aag aac tca ggc ctc aag gta att ctg gga att cta ctt 2773 Arg Met Arg Lys Asn Ser Gly Leu Lys Val Ile Leu Gly Ile Leu Leu 770 775 780 cct cct tca att ctc agc ttg gag ttc aag aac aaa gac gac atg ccc 2821 Pro Pro Ser Ile Leu Ser Leu Glu Phe Lys Asn Lys Asp Asp Met Pro 785 790 795 tat atg tct cag gcc cag gaa atc cac ctc caa gag aag gag gca gaa 2869 Tyr Met Ser Gln Ala Gln Glu Ile His Leu Gln Glu Lys Glu Ala Glu 800 805 810 gaa cca gag aag ccc aca aag gaa aaa gag gaa gag gac atg gag ctc 2917 Glu Pro Glu Lys Pro Thr Lys Glu Lys Glu Glu Glu Asp Met Glu Leu 815 820 825 aca gca atg ttg gga cga aac aac ggg gag tcc tcc agg aag aag gat 2965 Thr Ala Met Leu Gly Arg Asn Asn Gly Glu Ser Ser Arg Lys Lys Asp 830 835 840 845 gaa gag gaa gtt cag agc aag cac cgg tta atc ccc ctc ggc aga aaa 3013 Glu Glu Glu Val Gln Ser Lys His Arg Leu Ile Pro Leu Gly Arg Lys 850 855 860 atc tat gaa ttc tac aat gca ccc atc gtg aag ttc tgg ttc tac aca 3061 Ile Tyr Glu Phe Tyr Asn Ala Pro Ile Val Lys Phe Trp Phe Tyr Thr 865 870 875 ctg gcg tat atc gga tac ctg atg ctc ttc aac tat atc gtg tta gtg 3109 Leu Ala Tyr Ile Gly Tyr Leu Met Leu Phe Asn Tyr Ile Val Leu Val 880 885 890 aag atg gaa cgc tgg ccg tcc acc cag gaa tgg atc gta atc tcc tat 3157 Lys Met Glu Arg Trp Pro Ser Thr Gln Glu Trp Ile Val Ile Ser Tyr 895 900 905 att ttc acc ctg gga ata gaa aag atg aga gag att ctg atg tca gag 3205 Ile Phe Thr Leu Gly Ile Glu Lys Met Arg Glu Ile Leu Met Ser Glu 910 915 920 925 cca ggg aag ttg cta cag aaa gtg aag gta tgg ctg cag gag tac tgg 3253 Pro Gly Lys Leu Leu Gln Lys Val Lys Val Trp Leu Gln Glu Tyr Trp 930 935 940 aat gtc acg gac ctc atc gcc atc ctt ctg ttt tct gtc gga atg atc 3301 Asn Val Thr Asp Leu Ile Ala Ile Leu Leu Phe Ser Val Gly Met Ile 945 950 955 ctt cgt ctc caa gac cag ccc ttc agg agt gac ggg agg gtc atc tac 3349 Leu Arg Leu Gln Asp Gln Pro Phe Arg Ser Asp Gly Arg Val Ile Tyr 960 965 970 tgc gtg aac atc att tac tgg tat atc cgt ctc cta gac atc ttc ggc 3397 Cys Val Asn Ile Ile Tyr Trp Tyr Ile Arg Leu Leu Asp Ile Phe Gly 975 980 985 gtg aac aag tat ttg ggc ccg tat gta atg atg att gga aaa atg atg 3445 Val Asn Lys Tyr Leu Gly Pro Tyr Val Met Met Ile Gly Lys Met Met 990 995 1000 1005 ata gac atg atg tac ttt gtc atc att atg ctg gtg gtt ctg atg 3490 Ile Asp Met Met Tyr Phe Val Ile Ile Met Leu Val Val Leu Met 1010 1015 1020 agc ttt ggg gtc gcc agg caa gcc atc ctt ttt ccc aat gag gag 3535 Ser Phe Gly Val Ala Arg Gln Ala Ile Leu Phe Pro Asn Glu Glu 1025 1030 1035 cca tca tgg aaa ctg gcc aag aac atc ttc tac atg ccc tat tgg 3580 Pro Ser Trp Lys Leu Ala Lys Asn Ile Phe Tyr Met Pro Tyr Trp 1040 1045 1050 atg att tat ggg gaa gtg ttt gcg gac cag ata gac cct ccc tgt 3625 Met Ile Tyr Gly Glu Val Phe Ala Asp Gln Ile Asp Pro Pro Cys 1055 1060 1065 gga cag aat gag acc cga gag gat ggt aaa ata atc cag ctg cct 3670 Gly Gln Asn Glu Thr Arg Glu Asp Gly Lys Ile Ile Gln Leu Pro 1070 1075 1080 ccc tgc aag aca gga gct tgg atc gtg ccg gcc atc atg gcc tgc 3715 Pro Cys Lys Thr Gly Ala Trp Ile Val Pro Ala Ile Met Ala Cys 1085 1090 1095 tac ctc tta gtg gca aac atc ttg ctg gtc aac ctc ctc att gct 3760 Tyr Leu Leu Val Ala Asn Ile Leu Leu Val Asn Leu Leu Ile Ala 1100 1105 1110 gtc ttt aac aat aca ttt ttt gaa gta aaa tcg ata tcc aac caa 3805 Val Phe Asn Asn Thr Phe Phe Glu Val Lys Ser Ile Ser Asn Gln 1115 1120 1125 gtc tgg aag ttt cag agg tat cag ctc atc atg act ttc cat gaa 3850 Val Trp Lys Phe Gln Arg Tyr Gln Leu Ile Met Thr Phe His Glu 1130 1135 1140 agg cca gtt ctg ccc cca cca ctg atc atc ttc agc cac atg acc 3895 Arg Pro Val Leu Pro Pro Pro Leu Ile Ile Phe Ser His Met Thr 1145 1150 1155 atg ata ttc cag cac ctg tgc tgc cga tgg agg aaa cac gag agc 3940 Met Ile Phe Gln His Leu Cys Cys Arg Trp Arg Lys His Glu Ser 1160 1165 1170 gac ccg gat gaa agg gac tac ggc ctg aaa ctc ttc ata acc gat 3985 Asp Pro Asp Glu Arg Asp Tyr Gly Leu Lys Leu Phe Ile Thr Asp 1175 1180 1185 gat gag ctc aag aaa gta cat gac ttt gaa gag caa tgc ata gaa 4030 Asp Glu Leu Lys Lys Val His Asp Phe Glu Glu Gln Cys Ile Glu 1190 1195 1200 gaa tac ttc aga gaa aag gat gat cgg ttc aac tca tct aat gat 4075 Glu Tyr Phe Arg Glu Lys Asp Asp Arg Phe Asn Ser Ser Asn Asp 1205 1210 1215 gag agg ata cgg gtg act tca gaa agg gtg gag aac atg tct atg 4120 Glu Arg Ile Arg Val Thr Ser Glu Arg Val Glu Asn Met Ser Met 1220 1225 1230 cgg ctg gag gaa gtc aac gag aga gag cac tcc atg aag gct tca 4165 Arg Leu Glu Glu Val Asn Glu Arg Glu His Ser Met Lys Ala Ser 1235 1240 1245 ctc cag acc gtg gac atc cgg ctg gcg cag ctg gaa gac ctt atc 4210 Leu Gln Thr Val Asp Ile Arg Leu Ala Gln Leu Glu Asp Leu Ile 1250 1255 1260 ggg cgc atg gcc acg gcc ctg gag cgc ctg aca ggt ctg gag cgg 4255 Gly Arg Met Ala Thr Ala Leu Glu Arg Leu Thr Gly Leu Glu Arg 1265 1270 1275 gcc gag tcc aac aaa atc cgc tcg agg acc tcg tca gac tgc acg 4300 Ala Glu Ser Asn Lys Ile Arg Ser Arg Thr Ser Ser Asp Cys Thr 1280 1285 1290 gac gcc gcc tac att gtc cgt cag agc agc ttc aac agc cag gaa 4345 Asp Ala Ala Tyr Ile Val Arg Gln Ser Ser Phe Asn Ser Gln Glu 1295 1300 1305 ggg aac acc ttc aag ctc caa gag agt ata gac cct gca ggt gag 4390 Gly Asn Thr Phe Lys Leu Gln Glu Ser Ile Asp Pro Ala Gly Glu 1310 1315 1320 gag acc atg tcc cca act tct cca acc tta atg ccc cgt atg cga 4435 Glu Thr Met Ser Pro Thr Ser Pro Thr Leu Met Pro Arg Met Arg 1325 1330 1335 agc cat tct ttc tat tcg gtc aat atg aaa gac aaa ggt ggt ata 4480 Ser His Ser Phe Tyr Ser Val Asn Met Lys Asp Lys Gly Gly Ile 1340 1345 1350 gaa aag ttg gaa agt att ttt aaa gaa agg tcc ctg agc cta cac 4525 Glu Lys Leu Glu Ser Ile Phe Lys Glu Arg Ser Leu Ser Leu His 1355 1360 1365 cgg gct act agt tcc cac tct gta gca aaa gaa ccc aaa gct cct 4570 Arg Ala Thr Ser Ser His Ser Val Ala Lys Glu Pro Lys Ala Pro 1370 1375 1380 gca gcc cct gcc aac acc ttg gcc att gtt cct gat tcc aga aga 4615 Ala Ala Pro Ala Asn Thr Leu Ala Ile Val Pro Asp Ser Arg Arg 1385 1390 1395 cca tca tcg tgt ata gac atc tat gtc tct gct atg gat gag ctc 4660 Pro Ser Ser Cys Ile Asp Ile Tyr Val Ser Ala Met Asp Glu Leu 1400 1405 1410 cac tgt gat ata gac cct ctg gac aat tcc gtg aac atc ctt ggg 4705 His Cys Asp Ile Asp Pro Leu Asp Asn Ser Val Asn Ile Leu Gly 1415 1420 1425 ctg ggc gag cca agc ttt tca act cca gta cct tcc aca gcc cct 4750 Leu Gly Glu Pro Ser Phe Ser Thr Pro Val Pro Ser Thr Ala Pro 1430 1435 1440 tca agt agt gcc tat gca aca ctt gca ccc aca gac aga cct cca 4795 Ser Ser Ser Ala Tyr Ala Thr Leu Ala Pro Thr Asp Arg Pro Pro 1445 1450 1455 agc cgg agc att gat ttt gag gac atc acc tcc atg gac act aga 4840 Ser Arg Ser Ile Asp Phe Glu Asp Ile Thr Ser Met Asp Thr Arg 1460 1465 1470 tct ttt tct tca gac tac acc cac ctc cca gaa tgc caa aac ccc 4885 Ser Phe Ser Ser Asp Tyr Thr His Leu Pro Glu Cys Gln Asn Pro 1475 1480 1485 tgg gac tca gag cct ccg atg tac cac acc att gag cgt tcc aaa 4930 Trp Asp Ser Glu Pro Pro Met Tyr His Thr Ile Glu Arg Ser Lys 1490 1495 1500 agt agc cgc tac cta gcc acc aca ccc ttt ctt cta gaa gag gct 4975 Ser Ser Arg Tyr Leu Ala Thr Thr Pro Phe Leu Leu Glu Glu Ala 1505 1510 1515 ccc att gtg aaa tct cat agc ttt atg ttt tcc ccc tca agg agc 5020 Pro Ile Val Lys Ser His Ser Phe Met Phe Ser Pro Ser Arg Ser 1520 1525 1530 tat tat gcc aac ttt ggg gtg cct gta aaa aca gca gaa tac aca 5065 Tyr Tyr Ala Asn Phe Gly Val Pro Val Lys Thr Ala Glu Tyr Thr 1535 1540 1545 agt att aca gac tgt att gac aca agg tgt gtc aat gcc cct caa 5110 Ser Ile Thr Asp Cys Ile Asp Thr Arg Cys Val Asn Ala Pro Gln 1550 1555 1560 gca att gcg gac aga gct gcc ttc cct gga ggt ctt gga gac aaa 5155 Ala Ile Ala Asp Arg Ala Ala Phe Pro Gly Gly Leu Gly Asp Lys 1565 1570 1575 gtg gag gac tta act tgc tgc cat cca gag cga gaa gca gaa ctg 5200 Val Glu Asp Leu Thr Cys Cys His Pro Glu Arg Glu Ala Glu Leu 1580 1585 1590 agt cac ccc agc tct gac agt gag gag aat gag gcc aaa ggc cgc 5245 Ser His Pro Ser Ser Asp Ser Glu Glu Asn Glu Ala Lys Gly Arg 1595 1600 1605 aga gcc acc att gca ata tcc tcc cag gag ggt gat aac tca gag 5290 Arg Ala Thr Ile Ala Ile Ser Ser Gln Glu Gly Asp Asn Ser Glu 1610 1615 1620 aga acc ctg tcc aac aac atc act gtt ccc aag ata gag cgc gcc 5335 Arg Thr Leu Ser Asn Asn Ile Thr Val Pro Lys Ile Glu Arg Ala 1625 1630 1635 aac agc tac tcg gca gag gag cca agt gcg cca tat gca cac acc 5380 Asn Ser Tyr Ser Ala Glu Glu Pro Ser Ala Pro Tyr Ala His Thr 1640 1645 1650 agg aag agc ttc tcc atc agt gac aaa ctc gac agg cag cgg aac 5425 Arg Lys Ser Phe Ser Ile Ser Asp Lys Leu Asp Arg Gln Arg Asn 1655 1660 1665 aca gca agc ctg cga aat ccc ttc cag aga agc aag tcc tcc aag 5470 Thr Ala Ser Leu Arg Asn Pro Phe Gln Arg Ser Lys Ser Ser Lys 1670 1675 1680 ccg gag ggc cga ggg gac agc ctg tcc atg agg aga ctg tcc aga 5515 Pro Glu Gly Arg Gly Asp Ser Leu Ser Met Arg Arg Leu Ser Arg 1685 1690 1695 aca tcg gct ttc caa agc ttt gaa agc aag cac aac taa accttcttaa 5564 Thr Ser Ala Phe Gln Ser Phe Glu Ser Lys His Asn 1700 1705 tatccgccac agaaggctca agaatccagc cctaaaattc tctccaactc cagtttttcc 5624 cctttccttg aatcatacct gctttattct tagctgagca aaacaagcaa tgctttggga 5684 ggtgttaact caaaggtgac ttctgggcca cagatcaaga aagcatttga tctgacccag 5744 tgccagacac aggggattta aggcatgttc acacttgctg ggcagggagg gggaagagag 5804 ggagaaggaa gggttagaga tgaatgtgta tccgcagtca cagcagaaag ccatgagagc 5864 aggggaaaca aggggcttcg agcacgctcc atgccaggag gcatctgttg atttctgacc 5924 attatcaaga gttgtaggat gcagggctaa attgcaaaat aaaataaaat agccagcgta 5984 cacaatgaga tattctaaac ttccattctg ttttcttttc acattggctc catcactggt 6044 gactgatgaa gagcatcctc tttattcagt ataagccggc agcaagcagt tctacctaac 6104 gtcccacatc cttctcatgc caacacttct gtaattgatc attataaaga aaaaacaagg 6164 taacagtcat agttcacctg tctcttatct attcacttct ggtgccacaa ctgtttatcc 6224 ttttttgaag aaaataaggg aacagaaatg cctttttgta ttgcaatcga aatgaaagaa 6284 gagttgatgt taaaaaaaca aaagtcaagt gatttattat atacagtggg cgttcaagtc 6344 tagtcgagca agctcaggag aatgtaatta aataatttta tattttttaa tttattttgt 6404 atctcacctg tcatggatga attcattcac tgaatatgta atattgaact t 6455 2 1707 PRT Homo sapiens 2 Met Pro Glu Pro Trp Gly Thr Val Tyr Phe Leu Gly Ile Ala Gln Val 1 5 10 15 Phe Ser Phe Leu Phe Ser Trp Trp Asn Leu Glu Gly Val Met Asn Gln 20 25 30 Ala Asp Ala Pro Arg Pro Leu Asn Trp Thr Ile Arg Lys Leu Cys His 35 40 45 Ala Ala Phe Leu Pro Ser Val Arg Leu Leu Lys Ala Gln Lys Ser Trp 50 55 60 Ile Glu Arg Ala Phe Tyr Lys Arg Glu Cys Val His Ile Ile Pro Ser 65 70 75 80 Thr Lys Asp Pro His Arg Cys Cys Cys Gly Arg Leu Ile Gly Gln His 85 90 95 Val Gly Leu Thr Pro Ser Ile Ser Val Leu Gln Asn Glu Lys Asn Glu 100 105 110 Ser Arg Leu Ser Arg Asn Asp Ile Gln Ser Glu Lys Trp Ser Ile Ser 115 120 125 Lys His Thr Gln Leu Ser Pro Thr Asp Ala Phe Gly Thr Ile Glu Phe 130 135 140 Gln Gly Gly Gly His Ser Asn Lys Ala Met Tyr Val Arg Val Ser Phe 145 150 155 160 Asp Thr Lys Pro Asp Leu Leu Leu His Leu Met Thr Lys Glu Trp Gln 165 170 175 Leu Glu Leu Pro Lys Leu Leu Ile Ser Val His Gly Gly Leu Gln Asn 180 185 190 Phe Glu Leu Gln Pro Lys Leu Lys Gln Val Phe Gly Lys Gly Leu Ile 195 200 205 Lys Ala Ala Met Thr Thr Gly Ala Trp Ile Phe Thr Gly Gly Val Asn 210 215 220 Thr Gly Val Ile Arg His Val Gly Asp Ala Leu Lys Asp His Ala Ser 225 230 235 240 Lys Ser Arg Gly Lys Ile Cys Thr Ile Gly Ile Ala Pro Trp Gly Ile 245 250 255 Val Glu Asn Gln Glu Asp Leu Ile Gly Arg Asp Val Val Arg Pro Tyr 260 265 270 Gln Thr Met Ser Asn Pro Met Ser Lys Leu Thr Val Leu Asn Ser Met 275 280 285 His Ser His Phe Ile Leu Ala Asp Asn Gly Thr Thr Gly Lys Tyr Gly 290 295 300 Ala Glu Val Lys Leu Arg Arg Gln Leu Glu Lys His Ile Ser Leu Gln 305 310 315 320 Lys Ile Asn Thr Arg Ile Gly Gln Gly Val Pro Val Val Ala Leu Ile 325 330 335 Val Glu Gly Gly Pro Asn Val Ile Ser Ile Val Leu Glu Tyr Leu Arg 340 345 350 Asp Thr Pro Pro Val Pro Val Val Val Cys Asp Gly Ser Gly Arg Ala 355 360 365 Ser Asp Ile Leu Ala Phe Gly His Lys Tyr Ser Glu Glu Gly Gly Leu 370 375 380 Ile Asn Glu Ser Leu Arg Asp Gln Leu Leu Val Thr Ile Gln Lys Thr 385 390 395 400 Phe Thr Tyr Thr Arg Thr Gln Ala Gln His Leu Phe Ile Ile Leu Met 405 410 415 Glu Cys Met Lys Lys Lys Glu Leu Ile Thr Val Phe Arg Met Gly Ser 420 425 430 Glu Gly His Gln Asp Ile Asp Leu Ala Ile Leu Thr Ala Leu Leu Lys 435 440 445 Gly Ala Asn Ala Ser Ala Pro Asp Gln Leu Ser Leu Ala Leu Ala Trp 450 455 460 Asn Arg Val Asp Ile Ala Arg Ser Gln Ile Phe Ile Tyr Gly Gln Gln 465 470 475 480 Trp Pro Val Gly Ser Leu Glu Gln Ala Met Leu Asp Ala Leu Val Leu 485 490 495 Asp Arg Val Asp Phe Val Lys Leu Leu Ile Glu Asn Gly Val Ser Met 500 505 510 His Arg Phe Leu Thr Ile Ser Arg Leu Glu Glu Leu Tyr Asn Thr Arg 515 520 525 His Gly Pro Ser Asn Thr Leu Tyr His Leu Val Arg Asp Val Lys Lys 530 535 540 Gly Asn Leu Pro Pro Asp Tyr Arg Ile Ser Leu Ile Asp Ile Gly Leu 545 550 555 560 Val Ile Glu Tyr Leu Met Gly Gly Ala Tyr Arg Cys Asn Tyr Thr Arg 565 570 575 Lys Arg Phe Arg Thr Leu Tyr His Asn Leu Phe Gly Pro Lys Arg Pro 580 585 590 Lys Ala Leu Lys Leu Leu Gly Met Glu Asp Asp Ile Pro Leu Arg Arg 595 600 605 Gly Arg Lys Thr Thr Lys Lys Arg Glu Glu Glu Val Asp Ile Asp Leu 610 615 620 Asp Asp Pro Glu Ile Asn His Phe Pro Phe Pro Phe His Glu Leu Met 625 630 635 640 Val Trp Ala Val Leu Met Lys Arg Gln Lys Met Ala Leu Phe Phe Trp 645 650 655 Gln His Gly Glu Glu Ala Met Ala Lys Ala Leu Val Ala Cys Lys Leu 660 665 670 Cys Lys Ala Met Ala His Glu Ala Ser Glu Asn Asp Met Val Asp Asp 675 680 685 Ile Ser Gln Glu Leu Asn His Asn Ser Arg Asp Phe Gly Gln Leu Ala 690 695 700 Val Glu Leu Leu Asp Gln Ser Tyr Lys Gln Asp Glu Gln Leu Ala Met 705 710 715 720 Lys Leu Leu Thr Tyr Glu Leu Lys Asn Trp Ser Asn Ala Thr Cys Leu 725 730 735 Gln Leu Ala Val Ala Ala Lys His Arg Asp Phe Ile Ala His Thr Cys 740 745 750 Ser Gln Met Leu Leu Thr Asp Met Trp Met Gly Arg Leu Arg Met Arg 755 760 765 Lys Asn Ser Gly Leu Lys Val Ile Leu Gly Ile Leu Leu Pro Pro Ser 770 775 780 Ile Leu Ser Leu Glu Phe Lys Asn Lys Asp Asp Met Pro Tyr Met Ser 785 790 795 800 Gln Ala Gln Glu Ile His Leu Gln Glu Lys Glu Ala Glu Glu Pro Glu 805 810 815 Lys Pro Thr Lys Glu Lys Glu Glu Glu Asp Met Glu Leu Thr Ala Met 820 825 830 Leu Gly Arg Asn Asn Gly Glu Ser Ser Arg Lys Lys Asp Glu Glu Glu 835 840 845 Val Gln Ser Lys His Arg Leu Ile Pro Leu Gly Arg Lys Ile Tyr Glu 850 855 860 Phe Tyr Asn Ala Pro Ile Val Lys Phe Trp Phe Tyr Thr Leu Ala Tyr 865 870 875 880 Ile Gly Tyr Leu Met Leu Phe Asn Tyr Ile Val Leu Val Lys Met Glu 885 890 895 Arg Trp Pro Ser Thr Gln Glu Trp Ile Val Ile Ser Tyr Ile Phe Thr 900 905 910 Leu Gly Ile Glu Lys Met Arg Glu Ile Leu Met Ser Glu Pro Gly Lys 915 920 925 Leu Leu Gln Lys Val Lys Val Trp Leu Gln Glu Tyr Trp Asn Val Thr 930 935 940 Asp Leu Ile Ala Ile Leu Leu Phe Ser Val Gly Met Ile Leu Arg Leu 945 950 955 960 Gln Asp Gln Pro Phe Arg Ser Asp Gly Arg Val Ile Tyr Cys Val Asn 965 970 975 Ile Ile Tyr Trp Tyr Ile Arg Leu Leu Asp Ile Phe Gly Val Asn Lys 980 985 990 Tyr Leu Gly Pro Tyr Val Met Met Ile Gly Lys Met Met Ile Asp Met 995 1000 1005 Met Tyr Phe Val Ile Ile Met Leu Val Val Leu Met Ser Phe Gly 1010 1015 1020 Val Ala Arg Gln Ala Ile Leu Phe Pro Asn Glu Glu Pro Ser Trp 1025 1030 1035 Lys Leu Ala Lys Asn Ile Phe Tyr Met Pro Tyr Trp Met Ile Tyr 1040 1045 1050 Gly Glu Val Phe Ala Asp Gln Ile Asp Pro Pro Cys Gly Gln Asn 1055 1060 1065 Glu Thr Arg Glu Asp Gly Lys Ile Ile Gln Leu Pro Pro Cys Lys 1070 1075 1080 Thr Gly Ala Trp Ile Val Pro Ala Ile Met Ala Cys Tyr Leu Leu 1085 1090 1095 Val Ala Asn Ile Leu Leu Val Asn Leu Leu Ile Ala Val Phe Asn 1100 1105 1110 Asn Thr Phe Phe Glu Val Lys Ser Ile Ser Asn Gln Val Trp Lys 1115 1120 1125 Phe Gln Arg Tyr Gln Leu Ile Met Thr Phe His Glu Arg Pro Val 1130 1135 1140 Leu Pro Pro Pro Leu Ile Ile Phe Ser His Met Thr Met Ile Phe 1145 1150 1155 Gln His Leu Cys Cys Arg Trp Arg Lys His Glu Ser Asp Pro Asp 1160 1165 1170 Glu Arg Asp Tyr Gly Leu Lys Leu Phe Ile Thr Asp Asp Glu Leu 1175 1180 1185 Lys Lys Val His Asp Phe Glu Glu Gln Cys Ile Glu Glu Tyr Phe 1190 1195 1200 Arg Glu Lys Asp Asp Arg Phe Asn Ser Ser Asn Asp Glu Arg Ile 1205 1210 1215 Arg Val Thr Ser Glu Arg Val Glu Asn Met Ser Met Arg Leu Glu 1220 1225 1230 Glu Val Asn Glu Arg Glu His Ser Met Lys Ala Ser Leu Gln Thr 1235 1240 1245 Val Asp Ile Arg Leu Ala Gln Leu Glu Asp Leu Ile Gly Arg Met 1250 1255 1260 Ala Thr Ala Leu Glu Arg Leu Thr Gly Leu Glu Arg Ala Glu Ser 1265 1270 1275 Asn Lys Ile Arg Ser Arg Thr Ser Ser Asp Cys Thr Asp Ala Ala 1280 1285 1290 Tyr Ile Val Arg Gln Ser Ser Phe Asn Ser Gln Glu Gly Asn Thr 1295 1300 1305 Phe Lys Leu Gln Glu Ser Ile Asp Pro Ala Gly Glu Glu Thr Met 1310 1315 1320 Ser Pro Thr Ser Pro Thr Leu Met Pro Arg Met Arg Ser His Ser 1325 1330 1335 Phe Tyr Ser Val Asn Met Lys Asp Lys Gly Gly Ile Glu Lys Leu 1340 1345 1350 Glu Ser Ile Phe Lys Glu Arg Ser Leu Ser Leu His Arg Ala Thr 1355 1360 1365 Ser Ser His Ser Val Ala Lys Glu Pro Lys Ala Pro Ala Ala Pro 1370 1375 1380 Ala Asn Thr Leu Ala Ile Val Pro Asp Ser Arg Arg Pro Ser Ser 1385 1390 1395 Cys Ile Asp Ile Tyr Val Ser Ala Met Asp Glu Leu His Cys Asp 1400 1405 1410 Ile Asp Pro Leu Asp Asn Ser Val Asn Ile Leu Gly Leu Gly Glu 1415 1420 1425 Pro Ser Phe Ser Thr Pro Val Pro Ser Thr Ala Pro Ser Ser Ser 1430 1435 1440 Ala Tyr Ala Thr Leu Ala Pro Thr Asp Arg Pro Pro Ser Arg Ser 1445 1450 1455 Ile Asp Phe Glu Asp Ile Thr Ser Met Asp Thr Arg Ser Phe Ser 1460 1465 1470 Ser Asp Tyr Thr His Leu Pro Glu Cys Gln Asn Pro Trp Asp Ser 1475 1480 1485 Glu Pro Pro Met Tyr His Thr Ile Glu Arg Ser Lys Ser Ser Arg 1490 1495 1500 Tyr Leu Ala Thr Thr Pro Phe Leu Leu Glu Glu Ala Pro Ile Val 1505 1510 1515 Lys Ser His Ser Phe Met Phe Ser Pro Ser Arg Ser Tyr Tyr Ala 1520 1525 1530 Asn Phe Gly Val Pro Val Lys Thr Ala Glu Tyr Thr Ser Ile Thr 1535 1540 1545 Asp Cys Ile Asp Thr Arg Cys Val Asn Ala Pro Gln Ala Ile Ala 1550 1555 1560 Asp Arg Ala Ala Phe Pro Gly Gly Leu Gly Asp Lys Val Glu Asp 1565 1570 1575 Leu Thr Cys Cys His Pro Glu Arg Glu Ala Glu Leu Ser His Pro 1580 1585 1590 Ser Ser Asp Ser Glu Glu Asn Glu Ala Lys Gly Arg Arg Ala Thr 1595 1600 1605 Ile Ala Ile Ser Ser Gln Glu Gly Asp Asn Ser Glu Arg Thr Leu 1610 1615 1620 Ser Asn Asn Ile Thr Val Pro Lys Ile Glu Arg Ala Asn Ser Tyr 1625 1630 1635 Ser Ala Glu Glu Pro Ser Ala Pro Tyr Ala His Thr Arg Lys Ser 1640 1645 1650 Phe Ser Ile Ser Asp Lys Leu Asp Arg Gln Arg Asn Thr Ala Ser 1655 1660 1665 Leu Arg Asn Pro Phe Gln Arg Ser Lys Ser Ser Lys Pro Glu Gly 1670 1675 1680 Arg Gly Asp Ser Leu Ser Met Arg Arg Leu Ser Arg Thr Ser Ala 1685 1690 1695 Phe Gln Ser Phe Glu Ser Lys His Asn 1700 1705 3 988 PRT Homo sapiens 3 Met Lys Leu Leu Thr Tyr Glu Leu Lys Asn Trp Ser Asn Ala Thr Cys 1 5 10 15 Leu Gln Leu Ala Val Ala Ala Lys His Arg Asp Phe Ile Ala His Thr 20 25 30 Cys Ser Gln Met Leu Leu Thr Asp Met Trp Met Gly Arg Leu Arg Met 35 40 45 Arg Lys Asn Ser Gly Leu Lys Val Ile Leu Gly Ile Leu Leu Pro Pro 50 55 60 Ser Ile Leu Ser Leu Glu Phe Lys Asn Lys Asp Asp Met Pro Tyr Met 65 70 75 80 Ser Gln Ala Gln Glu Ile His Leu Gln Glu Lys Glu Ala Glu Glu Pro 85 90 95 Glu Lys Pro Thr Lys Glu Lys Glu Glu Glu Asp Met Glu Leu Thr Ala 100 105 110 Met Leu Gly Arg Asn Asn Gly Glu Ser Ser Arg Lys Lys Asp Glu Glu 115 120 125 Glu Val Gln Ser Lys His Arg Leu Ile Pro Leu Gly Arg Lys Ile Tyr 130 135 140 Glu Phe Tyr Asn Ala Pro Ile Val Lys Phe Trp Phe Tyr Thr Leu Ala 145 150 155 160 Tyr Ile Gly Tyr Leu Met Leu Phe Asn Tyr Ile Val Leu Val Lys Met 165 170 175 Glu Arg Trp Pro Ser Thr Gln Glu Trp Ile Val Ile Ser Tyr Ile Phe 180 185 190 Thr Leu Gly Ile Glu Lys Met Arg Glu Ile Leu Met Ser Glu Pro Gly 195 200 205 Lys Leu Leu Gln Lys Val Lys Val Trp Leu Gln Glu Tyr Trp Asn Val 210 215 220 Thr Asp Leu Ile Ala Ile Leu Leu Phe Ser Val Gly Met Ile Leu Arg 225 230 235 240 Leu Gln Asp Gln Pro Phe Arg Ser Asp Gly Arg Val Ile Tyr Cys Val 245 250 255 Asn Ile Ile Tyr Trp Tyr Ile Arg Leu Leu Asp Ile Phe Gly Val Asn 260 265 270 Lys Tyr Leu Gly Pro Tyr Val Met Met Ile Gly Lys Met Met Ile Asp 275 280 285 Met Met Tyr Phe Val Ile Ile Met Leu Val Val Leu Met Ser Phe Gly 290 295 300 Val Ala Arg Gln Ala Ile Leu Phe Pro Asn Glu Glu Pro Ser Trp Lys 305 310 315 320 Leu Ala Lys Asn Ile Phe Tyr Met Pro Tyr Trp Met Ile Tyr Gly Glu 325 330 335 Val Phe Ala Asp Gln Ile Asp Pro Pro Cys Gly Gln Asn Glu Thr Arg 340 345 350 Glu Asp Gly Lys Ile Ile Gln Leu Pro Pro Cys Lys Thr Gly Ala Trp 355 360 365 Ile Val Pro Ala Ile Met Ala Cys Tyr Leu Leu Val Ala Asn Ile Leu 370 375 380 Leu Val Asn Leu Leu Ile Ala Val Phe Asn Asn Thr Phe Phe Glu Val 385 390 395 400 Lys Ser Ile Ser Asn Gln Val Trp Lys Phe Gln Arg Tyr Gln Leu Ile 405 410 415 Met Thr Phe His Glu Arg Pro Val Leu Pro Pro Pro Leu Ile Ile Phe 420 425 430 Ser His Met Thr Met Ile Phe Gln His Leu Cys Cys Arg Trp Arg Lys 435 440 445 His Glu Ser Asp Pro Asp Glu Arg Asp Tyr Gly Leu Lys Leu Phe Ile 450 455 460 Thr Asp Asp Glu Leu Lys Lys Val His Asp Phe Glu Glu Gln Cys Ile 465 470 475 480 Glu Glu Tyr Phe Arg Glu Lys Asp Asp Arg Phe Asn Ser Ser Asn Asp 485 490 495 Glu Arg Ile Arg Val Thr Ser Glu Arg Val Glu Asn Met Ser Met Arg 500 505 510 Leu Glu Glu Val Asn Glu Arg Glu His Ser Met Lys Ala Ser Leu Gln 515 520 525 Thr Val Asp Ile Arg Leu Ala Gln Leu Glu Asp Leu Ile Gly Arg Met 530 535 540 Ala Thr Ala Leu Glu Arg Leu Thr Gly Leu Glu Arg Ala Glu Ser Asn 545 550 555 560 Lys Ile Arg Ser Arg Thr Ser Ser Asp Cys Thr Asp Ala Ala Tyr Ile 565 570 575 Val Arg Gln Ser Ser Phe Asn Ser Gln Glu Gly Asn Thr Phe Lys Leu 580 585 590 Gln Glu Ser Ile Asp Pro Ala Gly Glu Glu Thr Met Ser Pro Thr Ser 595 600 605 Pro Thr Leu Met Pro Arg Met Arg Ser His Ser Phe Tyr Ser Val Asn 610 615 620 Met Lys Asp Lys Gly Gly Ile Glu Lys Leu Glu Ser Ile Phe Lys Glu 625 630 635 640 Arg Ser Leu Ser Leu His Arg Ala Thr Ser Ser His Ser Val Ala Lys 645 650 655 Glu Pro Lys Ala Pro Ala Ala Pro Ala Asn Thr Leu Ala Ile Val Pro 660 665 670 Asp Ser Arg Arg Pro Ser Ser Cys Ile Asp Ile Tyr Val Ser Ala Met 675 680 685 Asp Glu Leu His Cys Asp Ile Asp Pro Leu Asp Asn Ser Val Asn Ile 690 695 700 Leu Gly Leu Gly Glu Pro Ser Phe Ser Thr Pro Val Pro Ser Thr Ala 705 710 715 720 Pro Ser Ser Ser Ala Tyr Ala Thr Leu Ala Pro Thr Asp Arg Pro Pro 725 730 735 Ser Arg Ser Ile Asp Phe Glu Asp Ile Thr Ser Met Asp Thr Arg Ser 740 745 750 Phe Ser Ser Asp Tyr Thr His Leu Pro Glu Cys Gln Asn Pro Trp Asp 755 760 765 Ser Glu Pro Pro Met Tyr His Thr Ile Glu Arg Ser Lys Ser Ser Arg 770 775 780 Tyr Leu Ala Thr Thr Pro Phe Leu Leu Glu Glu Ala Pro Ile Val Lys 785 790 795 800 Ser His Ser Phe Met Phe Ser Pro Ser Arg Ser Tyr Tyr Ala Asn Phe 805 810 815 Gly Val Pro Val Lys Thr Ala Glu Tyr Thr Ser Ile Thr Asp Cys Ile 820 825 830 Asp Thr Arg Cys Val Asn Ala Pro Gln Ala Ile Ala Asp Arg Ala Ala 835 840 845 Phe Pro Gly Gly Leu Gly Asp Lys Val Glu Asp Leu Thr Cys Cys His 850 855 860 Pro Glu Arg Glu Ala Glu Leu Ser His Pro Ser Ser Asp Ser Glu Glu 865 870 875 880 Asn Glu Ala Lys Gly Arg Arg Ala Thr Ile Ala Ile Ser Ser Gln Glu 885 890 895 Gly Asp Asn Ser Glu Arg Thr Leu Ser Asn Asn Ile Thr Val Pro Lys 900 905 910 Ile Glu Arg Ala Asn Ser Tyr Ser Ala Glu Glu Pro Ser Ala Pro Tyr 915 920 925 Ala His Thr Arg Lys Ser Phe Ser Ile Ser Asp Lys Leu Asp Arg Gln 930 935 940 Arg Asn Thr Ala Ser Leu Arg Asn Pro Phe Gln Arg Ser Lys Ser Ser 945 950 955 960 Lys Pro Glu Gly Arg Gly Asp Ser Leu Ser Met Arg Arg Leu Ser Arg 965 970 975 Thr Ser Ala Phe Gln Ser Phe Glu Ser Lys His Asn 980 985 4 1017 PRT Homo sapiens 4 Gln Glu Leu Asn His Asn Ser Arg Asp Phe Gly Gln Leu Ala Val Glu 1 5 10 15 Leu Leu Asp Gln Ser Tyr Lys Gln Asp Glu Gln Leu Ala Met Lys Leu 20 25 30 Leu Thr Tyr Glu Leu Lys Asn Trp Ser Asn Ala Thr Cys Leu Gln Leu 35 40 45 Ala Val Ala Ala Lys His Arg Asp Phe Ile Ala His Thr Cys Ser Gln 50 55 60 Met Leu Leu Thr Asp Met Trp Met Gly Arg Leu Arg Met Arg Lys Asn 65 70 75 80 Ser Gly Leu Lys Val Ile Leu Gly Ile Leu Leu Pro Pro Ser Ile Leu 85 90 95 Ser Leu Glu Phe Lys Asn Lys Asp Asp Met Pro Tyr Met Ser Gln Ala 100 105 110 Gln Glu Ile His Leu Gln Glu Lys Glu Ala Glu Glu Pro Glu Lys Pro 115 120 125 Thr Lys Glu Lys Glu Glu Glu Asp Met Glu Leu Thr Ala Met Leu Gly 130 135 140 Arg Asn Asn Gly Glu Ser Ser Arg Lys Lys Asp Glu Glu Glu Val Gln 145 150 155 160 Ser Lys His Arg Leu Ile Pro Leu Gly Arg Lys Ile Tyr Glu Phe Tyr 165 170 175 Asn Ala Pro Ile Val Lys Phe Trp Phe Tyr Thr Leu Ala Tyr Ile Gly 180 185 190 Tyr Leu Met Leu Phe Asn Tyr Ile Val Leu Val Lys Met Glu Arg Trp 195 200 205 Pro Ser Thr Gln Glu Trp Ile Val Ile Ser Tyr Ile Phe Thr Leu Gly 210 215 220 Ile Glu Lys Met Arg Glu Ile Leu Met Ser Glu Pro Gly Lys Leu Leu 225 230 235 240 Gln Lys Val Lys Val Trp Leu Gln Glu Tyr Trp Asn Val Thr Asp Leu 245 250 255 Ile Ala Ile Leu Leu Phe Ser Val Gly Met Ile Leu Arg Leu Gln Asp 260 265 270 Gln Pro Phe Arg Ser Asp Gly Arg Val Ile Tyr Cys Val Asn Ile Ile 275 280 285 Tyr Trp Tyr Ile Arg Leu Leu Asp Ile Phe Gly Val Asn Lys Tyr Leu 290 295 300 Gly Pro Tyr Val Met Met Ile Gly Lys Met Met Ile Asp Met Met Tyr 305 310 315 320 Phe Val Ile Ile Met Leu Val Val Leu Met Ser Phe Gly Val Ala Arg 325 330 335 Gln Ala Ile Leu Phe Pro Asn Glu Glu Pro Ser Trp Lys Leu Ala Lys 340 345 350 Asn Ile Phe Tyr Met Pro Tyr Trp Met Ile Tyr Gly Glu Val Phe Ala 355 360 365 Asp Gln Ile Asp Pro Pro Cys Gly Gln Asn Glu Thr Arg Glu Asp Gly 370 375 380 Lys Ile Ile Gln Leu Pro Pro Cys Lys Thr Gly Ala Trp Ile Val Pro 385 390 395 400 Ala Ile Met Ala Cys Tyr Leu Leu Val Ala Asn Ile Leu Leu Val Asn 405 410 415 Leu Leu Ile Ala Val Phe Asn Asn Thr Phe Phe Glu Val Lys Ser Ile 420 425 430 Ser Asn Gln Val Trp Lys Phe Gln Arg Tyr Gln Leu Ile Met Thr Phe 435 440 445 His Glu Arg Pro Val Leu Pro Pro Pro Leu Ile Ile Phe Ser His Met 450 455 460 Thr Met Ile Phe Gln His Leu Cys Cys Arg Trp Arg Lys His Glu Ser 465 470 475 480 Asp Pro Asp Glu Arg Asp Tyr Gly Leu Lys Leu Phe Ile Thr Asp Asp 485 490 495 Glu Leu Lys Lys Val His Asp Phe Glu Glu Gln Cys Ile Glu Glu Tyr 500 505 510 Phe Arg Glu Lys Asp Asp Arg Phe Asn Ser Ser Asn Asp Glu Arg Ile 515 520 525 Arg Val Thr Ser Glu Arg Val Glu Asn Met Ser Met Arg Leu Glu Glu 530 535 540 Val Asn Glu Arg Glu His Ser Met Lys Ala Ser Leu Gln Thr Val Asp 545 550 555 560 Ile Arg Leu Ala Gln Leu Glu Asp Leu Ile Gly Arg Met Ala Thr Ala 565 570 575 Leu Glu Arg Leu Thr Gly Leu Glu Arg Ala Glu Ser Asn Lys Ile Arg 580 585 590 Ser Arg Thr Ser Ser Asp Cys Thr Asp Ala Ala Tyr Ile Val Arg Gln 595 600 605 Ser Ser Phe Asn Ser Gln Glu Gly Asn Thr Phe Lys Leu Gln Glu Ser 610 615 620 Ile Asp Pro Ala Gly Glu Glu Thr Met Ser Pro Thr Ser Pro Thr Leu 625 630 635 640 Met Pro Arg Met Arg Ser His Ser Phe Tyr Ser Val Asn Met Lys Asp 645 650 655 Lys Gly Gly Ile Glu Lys Leu Glu Ser Ile Phe Lys Glu Arg Ser Leu 660 665 670 Ser Leu His Arg Ala Thr Ser Ser His Ser Val Ala Lys Glu Pro Lys 675 680 685 Ala Pro Ala Ala Pro Ala Asn Thr Leu Ala Ile Val Pro Asp Ser Arg 690 695 700 Arg Pro Ser Ser Cys Ile Asp Ile Tyr Val Ser Ala Met Asp Glu Leu 705 710 715 720 His Cys Asp Ile Asp Pro Leu Asp Asn Ser Val Asn Ile Leu Gly Leu 725 730 735 Gly Glu Pro Ser Phe Ser Thr Pro Val Pro Ser Thr Ala Pro Ser Ser 740 745 750 Ser Ala Tyr Ala Thr Leu Ala Pro Thr Asp Arg Pro Pro Ser Arg Ser 755 760 765 Ile Asp Phe Glu Asp Ile Thr Ser Met Asp Thr Arg Ser Phe Ser Ser 770 775 780 Asp Tyr Thr His Leu Pro Glu Cys Gln Asn Pro Trp Asp Ser Glu Pro 785 790 795 800 Pro Met Tyr His Thr Ile Glu Arg Ser Lys Ser Ser Arg Tyr Leu Ala 805 810 815 Thr Thr Pro Phe Leu Leu Glu Glu Ala Pro Ile Val Lys Ser His Ser 820 825 830 Phe Met Phe Ser Pro Ser Arg Ser Tyr Tyr Ala Asn Phe Gly Val Pro 835 840 845 Val Lys Thr Ala Glu Tyr Thr Ser Ile Thr Asp Cys Ile Asp Thr Arg 850 855 860 Cys Val Asn Ala Pro Gln Ala Ile Ala Asp Arg Ala Ala Phe Pro Gly 865 870 875 880 Gly Leu Gly Asp Lys Val Glu Asp Leu Thr Cys Cys His Pro Glu Arg 885 890 895 Glu Ala Glu Leu Ser His Pro Ser Ser Asp Ser Glu Glu Asn Glu Ala 900 905 910 Lys Gly Arg Arg Ala Thr Ile Ala Ile Ser Ser Gln Glu Gly Asp Asn 915 920 925 Ser Glu Arg Thr Leu Ser Asn Asn Ile Thr Val Pro Lys Ile Glu Arg 930 935 940 Ala Asn Ser Tyr Ser Ala Glu Glu Pro Ser Ala Pro Tyr Ala His Thr 945 950 955 960 Arg Lys Ser Phe Ser Ile Ser Asp Lys Leu Asp Arg Gln Arg Asn Thr 965 970 975 Ala Ser Leu Arg Asn Pro Phe Gln Arg Ser Lys Ser Ser Lys Pro Glu 980 985 990 Gly Arg Gly Asp Ser Leu Ser Met Arg Lys Leu Ser Arg Thr Ser Ala 995 1000 1005 Phe Gln Ser Phe Glu Ser Lys His Thr 1010 1015 5 736 PRT Mus musculus 5 Met Val Leu Gly Thr Gly Thr Phe Leu Ser Ser Gln His Thr Ala Gly 1 5 10 15 Arg Leu Pro Pro Gly Ala Phe Ala Lys Gln Arg Leu Leu Cys Gly Ala 20 25 30 Ala Leu Leu Leu Tyr Val Ser Ala Asn Asn Pro Ile Gln Ala Gln Ser 35 40 45 Val Pro Ile Met Leu Ser Gln Arg Gly Leu Leu Ala Thr Cys Thr His 50 55 60 Ser Gly Val Phe Leu Leu Pro Tyr Arg Leu Pro Pro Tyr Thr Gln Leu 65 70 75 80 Ala Pro Cys Gly Gln Asn Glu Thr Arg Glu Asp Gly Lys Thr Ile Gln 85 90 95 Leu Pro Pro Cys Lys Thr Gly Ala Trp Ile Val Pro Ala Ile Met Ala 100 105 110 Cys Tyr Leu Leu Val Ala Asn Ile Leu Leu Val Asn Leu Leu Ile Ala 115 120 125 Val Phe Asn Asn Thr Phe Phe Glu Val Lys Ser Ile Ser Asn Gln Val 130 135 140 Trp Lys Phe Gln Arg Tyr Gln Leu Ile Met Thr Phe His Glu Arg Pro 145 150 155 160 Val Leu Pro Pro Pro Leu Ile Ile Phe Ser His Met Thr Met Ile Phe 165 170 175 Gln His Val Cys Cys Arg Trp Arg Lys His Glu Ser Asp Gln Asp Glu 180 185 190 Arg Asp Tyr Gly Leu Lys Phe Leu Ile Thr Gly Asp Glu Leu Arg Lys 195 200 205 Val His Asp Phe Glu Glu Gln Cys Ile Glu Glu Tyr Phe Arg Glu Lys 210 215 220 Asp Asp Arg Phe Asn Ser Ser Asn Asp Glu Arg Ile Arg Val Thr Ser 225 230 235 240 Glu Arg Val Glu Asn Met Ser Met Arg Leu Glu Glu Val Asn Glu Arg 245 250 255 Glu His Ser Met Lys Ala Ser Leu Gln Thr Val Asp Ile Arg Leu Ala 260 265 270 Gln Leu Glu Asp Leu Ile Gly Arg Met Ala Thr Ala Leu Glu Arg Leu 275 280 285 Thr Gly Leu Glu Arg Ala Glu Ser Asn Lys Ile Arg Ser Arg Thr Ser 290 295 300 Ser Asp Cys Thr Asp Ala Ala Tyr Ile Val Arg Gln Ser Ser Phe Asn 305 310 315 320 Ser Gln Glu Gly Asn Thr Phe Lys Leu Gln Glu Ser Ile Asp Pro Ala 325 330 335 Gly Glu Glu Thr Ile Ser Pro Thr Ser Pro Thr Leu Met Pro Arg Met 340 345 350 Arg Ser His Ser Phe Tyr Ser Val Asn Val Lys Asp Lys Gly Gly Ile 355 360 365 Glu Lys Leu Glu Ser Ile Phe Lys Glu Arg Ser Leu Ser Leu His Arg 370 375 380 Ala Thr Ser Ser His Ser Val Ala Lys Glu Pro Lys Ala Pro Ala Ala 385 390 395 400 Pro Ala Asn Thr Leu Ala Ile Val Pro Asp Ser Arg Arg Pro Ser Ser 405 410 415 Cys Ile Asp Ile Tyr Val Ser Ala Met Asp Glu Leu His Cys Asp Ile 420 425 430 Glu Pro Leu Asp Asn Ser Met Asn Ile Leu Gly Leu Gly Glu Pro Ser 435 440 445 Phe Ser Ala Leu Ala Pro Ser Thr Thr Pro Ser Ser Ser Ala Tyr Ala 450 455 460 Thr Leu Ala Pro Thr Asp Arg Pro Pro Ser Arg Ser Ile Asp Phe Glu 465 470 475 480 Asp Leu Thr Ser Met Asp Thr Arg Ser Phe Ser Ser Asp Tyr Thr His 485 490 495 Leu Pro Glu Cys Gln Asn Pro Trp Asp Thr Asp Pro Pro Thr Tyr His 500 505 510 Thr Ile Glu Arg Ser Lys Ser Ser Arg Tyr Leu Ala Thr Thr Pro Phe 515 520 525 Leu Leu Glu Glu Ala Pro Ile Val Lys Ser His Ser Phe Met Phe Ser 530 535 540 Pro Ser Arg Ser Tyr Tyr Ala Asn Phe Gly Val Pro Val Lys Thr Ala 545 550 555 560 Glu Tyr Thr Ser Ile Thr Asp Cys Ile Asp Thr Arg Cys Val Asn Ala 565 570 575 Pro Gln Ala Ile Ala Asp Arg Ala Thr Phe Pro Gly Gly Leu Gly Asp 580 585 590 Lys Val Glu Asp Leu Ser Cys Cys His Pro Glu Arg Glu Ala Glu Leu 595 600 605 Ser His Pro Ser Ser Asp Ser Glu Glu Asn Glu Ala Arg Gly Gln Arg 610 615 620 Ala Ala Asn Pro Ile Ser Ser Gln Glu Ala Glu Asn Ala Asp Arg Thr 625 630 635 640 Leu Ser Asn Asn Ile Thr Val Pro Lys Ile Glu Arg Ala Asn Ser Tyr 645 650 655 Ser Ala Glu Glu Pro Asn Val Pro Tyr Ala His Thr Arg Lys Ser Phe 660 665 670 Ser Ile Ser Asp Lys Leu Asp Arg Gln Arg Asn Thr Ala Ser Leu Arg 675 680 685 Asn Pro Phe Gln Arg Lys Thr Ile Leu Gln Tyr Thr Pro Asn Lys Leu 690 695 700 Tyr Pro Glu Cys Leu Leu Ser Ser Ser Thr Gly Ala Val Glu Leu Tyr 705 710 715 720 Asp Pro Ala Glu Ala Ile Leu Leu Ala Ala Phe Leu Asp Gly Gly Tyr 725 730 735 6 24 DNA Homo sapiens 6 gctttagcct ggaacagagt cgac 24 7 24 DNA Homo sapiens 7 gtctttcttc ctcgcctcaa ggga 24 8 23 DNA Homo sapiens 8 gaccaaggaa tggcagttgg agc 23 9 24 DNA Homo sapiens 9 gtggtcccgt tgtcagccag aatg 24

Claims

1. An isolated polynucleotide comprising, a polynucleotide sequence coding without interruption for a human TRPCC polypeptide, or complement thereto,

said TRPCC having 90% or more amino acid sequence identity along its entire length to the sequence comprising amino acids 1-690 of SEQ ID NO 2, and 90% or more amino acid sequence identity along its entire length to the sequence comprising from amino acids 691-1707 of SEQ ID NO 2,

and which has cation transport activity.

2. An isolated polynucleotide of claim 1,

said TRPCC having 90% or more amino acid sequence identity along its entire length to the sequence comprising amino acids 1-690 of SEQ ID NO 2, and 95% or more amino acid sequence identity along its entire length to the sequence comprising from amino acids 691-1707 of SEQ ID NO 2.

3. An isolated polynucleotide of claim 1, which codes for a human TRPCC of SEQ ID NO 2.

4. An isolated polynucleotide of claim 1, which is SEQ ID NO 1.

5. An isolated polynucleotide comprising a polynucleotide sequence coding for a human TRPCC polypeptide having 90% or more amino acid sequence identity along its entire length to the sequence coding for amino acids 1-690 of SEQ ID NO 2, or a fragment thereof, which polynucleotide is specific for said human TRPCC.

6. An isolated polynucleotide of claim 5, wherein said fragment is effective in a polymerase chain reaction.

7. An isolated polynucleotide of claim 5 consisting essentially of a polynucleotide sequence coding for amino acids 1-690 of SEQ ID NO 2, or a fragment thereof.

8. An isolated polynucleotide of claim 5, which is SEQ ID NO 6, 7, 8, or 9.

9. An isolated human TRPCC polypeptide having an amino acid sequence of claim 1.

10. An isolated human TRPCC polypeptide having the amino acid sequence of claim 3.

11. An isolated human TRPCC polypeptide having an amino acid sequence of claim 6.

12. An isolated human TRPCC polypeptide having an amino acid sequence of claim 8, or a fragment thereof which is specific for a human TRPCC.

13. A method of detecting expression of a gene coding for human TRPCC, comprising,

contacting a sample comprising nucleic acid with a polynucleotide probe specific for a human TRPCC of claim 1 under conditions effective for said probe to hybridize specifically with said human TRPCC, and

detecting hybridization between said probe and said human TRPCC.

14. A method of claim 14, wherein said detecting is performed by:

Northern blot analysis, polymerase chain reaction (PCR), reverse transcriptase PCR, RACE PCR, or in situ hybridization.

15. A method for identifying an agent that modulates the expression of a human TRPCC gene, or the biological activity of polypeptide encoded thereby, in cells expressing said gene, comprising,

contacting cells expressing human TRPCC of claim 1 with a test agent under conditions effective for said test agent to modulate the expression of a gene coding for said human TRPCC, or the biological activity of a polypeptide encoded thereby, and

determining whether said test agent modulates said human TRPCC.

16. A method of claim 15, wherein said agent is an antisense polynucleotide which is effective to inhibit translation of said human TRPCC.

17. A method of detecting polymorphisms in human TRPCC comprising:

comparing the structure of: genomic DNA comprising all or part of human TRPCC, mRNA comprising all or part of human TRPCC, cDNA comprising all or part of human TRPCC, or a polypeptide comprising all or part of human TRPCC, with the complete structure of human TRPCC as set forth in SEQ ID NO 1 of claim 1.

18. A method of claim 17, wherein said polymorphism is a nucleotide deletion, substitution, inversion, or transposition.

19. A method of claim 17, wherein said polymorphism is a mutation associated with hypomagnesemia with hypocalcemia.

20. A method of claim 17, wherein said polymorphism is a mutation associated with amyotrophic lateral sclerosis with frontotemporal dementia.

21. A method of identifying a mutation associated with hypomagnesemia with hypocalcemia, comprising:

comparing the structure of: genomic DNA comprising all or part of human TRPCC, MRNA comprising all or part of human TRPCC, cDNA comprising all or part of human TRPCC, or a polypeptide comprising all or part of human TRPCC, with the complete structure of human TRPCC as set forth in SEQ ID NO 1 of claim 1, in a patient having hypomagnesemia with hypocalcemia, or a family member thereof.

22. A method of identifying a mutation associated with amyotrophic lateral sclerosis with frontotemporal dementia, comprising:

comparing the structure of: genomic DNA comprising all or part of human TRPCC, mRNA comprising all or part of human TRPCC, cDNA comprising all or part of human TRPCC, or a polypeptide comprising all or part of human TRPCC, with the complete structure of human TRPCC as set forth in SEQ ID NO 1 of claim 1, in a patient having amyotrophic lateral sclerosis with frontotemporal dementia, or a family member thereof.

23. A mammalian cell whose genome comprises a functional disruption of the human TRPCC gene of claim 1 within a polynucleotide sequence coding for amino acid residues 1-690 of SEQ ID NO 2.

24. A non-human, transgenic mammal comprising a cell of claim 23, which has a defect in calcium conductance.

25. An antibody which is specific-for:

an epitope selected from a human TRPCC polypeptide of claim 12.

26. A method of selecting a human TRPCC polynucleotide or amino acid sequence from a database, comprising:

displaying, in a computer-readable medium, a polynucleotide sequence or polypeptide sequence for human TRPCC of claim 1, or complements to the polynucleotides sequence, wherein said displayed sequences have been retrieved from said database upon selection by a user.