EP1280896A2 - Isolated human transporter proteins, nucleic acid molecules encoding the same, and uses thereof - Google Patents

Abstract

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the transporter peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the transporter peptides, and methods of identifying modulators of the transporter peptides.

Description

ISOLATED HUMAN TRANSPORTER PROTEINS, NUCLEIC ACID MOLECULES ENCODING HUMAN TRANSPORTER PROTEINS, AND USES THEREOF

RELATED APPLICATIONS The present application claims priority to U.S. Serial No. 09/765,344, filed January 22, 2001

(Atty. Docket CL000746CIP), which is a continuation-in-part of U.S. Serial No. 09/630,719, filed August 2, 2000 (Atty. Docket CL000746), which claims priority to U.S. Serial No. 60/199,811, filed April 26, 2000 (Atty. Docket CL000486-PRON).

FIELD OF THE INVENTION

The present invention is in the field of transporter proteins that are related to the sulfate transporter subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins, representing two splice forms of a novel sulfate transporter, that effect ligand transport and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION

Transporters Transporter proteins regulate many different functions of a cell, including cell proliferation, differentiation, and signaling processes, by regulating the flow of molecules such as ions and macromolecules, into and out of cells. Transporters are found in the plasma membranes of virtually every cell in eukaryotic organisms. Transporters mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of molecules and ion across cell membranes. When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, transporters, such as chloride channels, also regulate organelle pH. For a review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.

Transporters are generally classified by structure and the type of mode of action. In addition, transporters are sometimes classified by the molecule type that is transported, for example, sugar transporters, chlorine channels, potassium channels, etc. There may be many classes of channels for transporting a single type of molecule (a detailed review of channel types can be found at Alexander, S.P.H. and J.A. Peters: Receptor and transporter nomenclature supplement. Trends Pharmacol. Sci., Elsevier, pp. 65-68 (1997) and http://www.biologv.ucsd.edu/~msaier/transport/titlepage2.html.

Ion channels

An important type of transporter is the ion channel. Ion channels regulate many different cell proliferation, differentiation, and signaling processes by regulating the flow of ions into and out of cells. Ion channels are found in the plasma membranes of virtually every cell in eukaryotic organisms. Ion channels mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of ion across epithelial membranes. When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, ion channels, such as chloride channels, also regulate organelle pH. For a review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.

Ion channels are generally classified by structure and the type of mode of action. For example, extracellular ligand gated channels (ELGs) are comprised of five polypeptide subunits, with each subunit having 4 membrane spanning domains, and are activated by the binding of an extracellular ligand to the channel. In addition, channels are sometimes classified by the ion type that is transported, for example, chlorine channels, potassium channels, etc. There may be many classes of channels for transporting a single type of ion (a detailed review of channel types can be found at Alexander, S.P.H. and J.A. Peters (1997). Receptor and ion channel nomenclature supplement. Trends Pharmacol. Sci., Elsevier, pp. 65-68 and http://www- biology.ucsd.edu/~msaier/transport/toc.html.

There are many types of ion channels based on structure. For example, many ion channels fall within one of the following groups: extracellular ligand-gated channels (ELG), intracellular ligand-gated channels (ILG), inward rectifying channels (INR), intercellular (gap junction) channels, and voltage gated channels (VIC). There are additionally recognized other channel families based on ion-type transported, cellular location and drug sensitivity. Detailed information on each of these, their activity, ligand type, ion type, disease association, drugability, and other information pertinent to the present invention, is well known in the art.

Anion transport proteins The present invention provides two splice forms of a novel human anion transport protein that shows a particularly high degree of similarity to sulfate transporters. The alternative splice forms are herein referred to as splice forms 1 and 2. Splice form 1 has been previously disclosed by applicant in U.S. application 09/630,719, filed August 2, 2000. Anion transport proteins in mammalian cells participate in a wide variety of cell and intracellular organelle functions, including regulation of electrical activity, pH, volume, and the transport of osmolites and metabolites. These proteins also have essential physiological roles in the control of immunological responses, cell migration, cell proliferation, and differentiation. Several classes of anion transporters have been characterized with varying molecular structures and mechanisms for mediating anion flux. One of the most prominent anion transporter super-families is the multiple membrane-spanning permeases, which include Na+- or H+-dependent anion coanion transporters (symporters), anion/anion exchangers (antiporters), and cation-independent anion uniporters. This super-family is also referred to as the Carrier-type anion transporters <http://www- biology.ucsd.edu/~msaier/transport/titlepage2.html>. Typically, proteins within this group contain 8-14 hydrophobic alpha-helical peptide segments that allow the protein to reside in the membrane bilayer. These helices also establish the pathway for ion translocation. Both broad-substrate and substrate-specific anion transporters are known: the former type enables multiple anion species (chloride, iodide, sulfate, bromide, etc.) to permeate the bounding membrane, while anion transporters in the latter class restrict ion movement to one chemical species.

Anion transporter genes and gene products are potential causative agents of disease and disease phenotypes may be actuated both by alterations in gene transcription and by mutations in the protein sequence. For example, the down-regulated in adenoma (DRA) gene was originally identified as a gene that was down-regulated in colon tumors. It encodes a protein with anion transporter function that is expressed in the intestinal tract (duodenum, ileum, cecum, distal colon), but not in the esophagus or stomach (Antalis T.M., Reeder J.A., Gotley D.C. et al, Clin Cancer Res (1998) Aug;4(8):1857-63; Byeon M.K., Westerman M.A., Maroulakou I.G. et al, Oncogene (1996) Jan 18;12(2):387-96). A second illustration of the biomedical significance of anion transporters is found with patients presenting severe hypothyroidism caused by a congenital lack of iodide transport. These individuals do not accumulate iodide in their thyroids. A single amino acid substitution in the thyroid Na+/I- symporter, where proline replaced threonine at position 354, has been identified as the cause of this condition in two independent patients (Levy O., Ginter C.S., De la Vieja A. et al, FEBS Lett (1998) Jun 5;429(l):36-40). Equally compelling are two well- documented autosomal recessive disorders Pendred syndrome and Diastrophic dysplasia (DTD). Pendred syndrome is the most common form of syndromic deafness and characterized by congenital sensorineural hearing loss and goitre. This disorder has been mapped to chromosome 7 and the gene product causing Pendred syndrome (PDS) has been identified as a anion transporter for iodide and chloride (Scott D.A., Wang R., Kreman T.M. et al, Nat Genet (1999)Apr;21(4):440- 3). DTD is a well-characterized osteochondrodysplasia with clinical features including dwarfism, spinal deformation, and specific joint abnormalities. The disease occurs in most populations. The gene has been mapped to distal chromosome 5q and it encodes a sulfate anion transporter (Hastbacka J., de la Chapelle A., Mahtani M.M. et al, Cell 1994 Sep 23;78(6):1073-87).

Issued US Patents that demonstrate the utility for this group of protein/DNA molecules include, but are not limited to, 6,054,558 "Compositions and methods for the treatment and diagnosis of cardiovascular disease using rchd534 as a target"; 6,048,709 "Compositions and methods for the treatment and diagnosis of cardiovascular disease"; 6,046,030 "Human LIG-1 homolog (HLIG-1)"; 6,025,160 "Polynucleotide and polypeptide sequences encoding rat mdrlb2 and screening methods thereof; 6 6,013,672 "Agonists of metabotropic glutamate receptors and uses thereof; 6,008,015 "Glycine transporter"; 5,989,825 "Excitatory amino acid transporter gene and uses"; and 5,928,926 "Isolation and cloning of the human ARSA-I gene and uses thereof.

Transporter proteins, particularly members of the sulfate transporter subfamily, are a major target for drug action and development, particularly members that are expressed in the tissue types noted in Figure 1 (e.g. neoplastic cells)). Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown transport proteins. The present invention advances the state of the art by providing a previously unidentified human transport protein.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of amino acid sequences of human transporter peptides and proteins that are related to the sulfate transporter subfamily, as well as allelic variants and other mammalian orthologs thereof. Specifically, the present invention provides two splice forms of a novel human sulfate transporter protein. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate transporter activity in cells and tissues that express the transporter. Independent lines of evidence show expression of splice form 1 in fetal tissues such as brain and kidney; differentiated tissues such as brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas; and neoplastic tissues such as germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendrogliomas, chronic lymphotic leukemic B- cells, glioblastomas, and well-differentiated endometrial adenocarcinomas. DESCRIPTION OF THE FIGURE SHEETS

FIGURE 1 provides the nucleotide sequences of cDNA molecules that encode splice forms 1 and 2 of the transporter protein of the present invention (splice form 1 = SEQ ID NO:l, splice form 2 = SEQ ID NO:4). In addition, structure and functional information is provided, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of the inventions based on these molecular sequences. Independent lines of evidence show expression of splice form 1 in fetal tissues such as brain and kidney; differentiated tissues such as brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas; and neoplastic tissues such as germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendrogliomas, chronic lymphotic leukemic B-cells, glioblastomas, and well-differentiated endometrial adenocarcinomas.

FIGURE 2 provides the predicted amino acid sequences of splice forms 1 and 2 of the transporter of the present invention (splice form 1 = SEQ ID NO:2, splice form 2 = SEQ ID NO:5). In addition, structure and functional information such as protein family, function, and modification sites is provided where available, allowing one to readily determine specific uses of the inventions based on these molecular sequences.

FIGURE 3 provides a genomic sequence SEQ ID NO:3) that spans the gene encoding splice forms 1 and 2 of the transporter protein of the present invention. In addition, structure and functional information, such as intron/exon structure, promoter location, etc., is provided where available, allowing one to readily determine specific uses of the inventions based on this molecular sequence. As illustrated in Figure 3, identified SNP variations include g30344a, a31170g, cl6256t, al3376t, tl2210c, gl2072c, gll922t, -11903a, cl0009g, c4519t, a4181g, a20952c, t20987c, g21620a, 121795c, a22753t, g22945a, g23032a, g23738a, t23952g, a24123g, c24527-, c24691t, g25015a, and g25191t. Figure 3 also provides structural information for splice form 2, derived from the Genewise computer program.

DETAILED DESCRIPTION OF THE INVENTION

General Description The present invention is based on the sequencing of the human genome. During the sequencing and assembly of the human genome, analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homology to protein peptide/domains identified and characterized within the art as being a transporter protein or part of a transporter protein and are related to the sulfate transporter subfamily. Specifically, the present invention provides two splice forms of a novel human sulfate transporter. Utilizing these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized. Based on this analysis, the present invention provides amino acid sequences of human transporter peptides and proteins that are related to the sulfate transporter subfamily, nucleic acid sequences in the form of transcript sequences, cDNA sequences and/or genomic sequences that encode these transporter peptides and proteins, nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known protein peptide/domain that has structural or sequence homology to the transporter of the present invention.

In addition to being previously unknown, the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology and/or structural relatedness to known transporter proteins of the sulfate transporter subfamily and the expression pattern observed. Independent lines of evidence show expression of splice form 1 in fetal tissues such as brain and kidney; differentiated tissues such as brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas; and neoplastic tissues such as germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendrogliomas, chronic lymphotic leukemic B-cells, glioblastomas, and well-differentiated endometrial adenocarcinomas.. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene. Some of the more specific features of the peptides of the present invention, and the uses thereof, are described herein, particularly in the Background of the Invention and in the annotation provided in the Figures, and/or are known within the art for each of the known sulfate transporter family or subfamily of transporter proteins.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the transporter family of proteins and are related to the sulfate transporter subfamily (protein sequences are provided in Figure 2, cDNA sequences are provided in Figures 1 and genomic sequences are provided in Figure 3). Specifically, the present invention provides two splice forms of a novel human sulfate transporter. The peptide sequences provided in Figure 2, as well as the obvious variants described herein, particularly allelic variants as identified herein and using the information in Figure 3, will be referred herein as the transporter peptides of the present invention, transporter peptides, or peptides/proteins of the present invention. The present invention provides isolated peptide and protein molecules that consist of, consist essentially of, or comprising the amino acid sequences of the transporter peptides disclosed in the Figure 2, (encoded by the nucleic acid molecule shown in Figure 1 , transcript/cDNA or Figure 3, genomic sequence), as well as all obvious variants of these peptides that are within the art to make and use. Some of these variants are described in detail below.

As used herein, a peptide is said to be "isolated" or "purified" when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below). In some uses, "substantially free of cellular material" includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation. The language "substantially free of chemical precursors or other chemicals" includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of the transporter peptide having less than about 30%) (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.

The isolated transporter peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. Independent lines of evidence show expression of splice form 1 in fetal tissues such as brain and Iddney; differentiated tissues such as brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas; and neoplastic tissues such as germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendrogliomas, chronic lymphotic leukemic B-cells, glioblastomas, and well- differentiated endometrial adenocarcinomas. For example, a nucleic acid molecule encoding the transporter peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Many of these techniques are described in detail below. Accordingly, the present invention provides proteins that consist of the amino acid sequences provided in Figure 2 (SEQ ID NOS:2 and 5), for example, proteins encoded by the cDNA nucleic acid sequences shown in Figure 1 (SEQ ID NOS:l and 4) and the genomic sequences provided in Figure 3 (SEQ ID NO:3). The amino acid sequence of such a protein is provided in Figure 2. A protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentially of the amino acid sequences provided in Figure 2 (SEQ ID NOS:2 and 5), for example, proteins encoded by the cDNA nucleic acid sequences shown in Figure 1 (SEQ ID NOS:l and 4) and the genomic sequences provided in Figure 3 (SEQ ID NO:3). A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein.

The present invention further provides proteins that comprise the amino acid sequences provided in Figure 2 (SEQ ID NOS:2 and 5), for example, proteins encoded by the cDNA nucleic acid sequences shown in Figure 1 (SEQ ID NOS:l and 4) and the genomic sequences provided in Figure 3 (SEQ ID NO:3). A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the transporter peptides of the present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be made/isolated is provided below.

The transporter peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins. Such chimeric and fusion proteins comprise a transporter peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the transporter peptide. "Operatively linked" indicates that the transporter peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the transporter peptide. hi some uses, the fusion protein does not affect the activity of the transporter peptide per se. For example, the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, Hi-tagged and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant transporter peptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re- amplified to generate a chimeric gene sequence (see Ausubel et al, Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A transporter peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the transporter peptide.

As mentioned above, the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.

Such variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the transporter peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%> or more of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al, Nucleic Acids Res. 12(1):3 1 (1984)) (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the transporter peptides of the present invention as well as being encoded by the same genetic locus as the transporter peptide provided herein. RH panel mapping shows the gene encoding the transporter proteins of the present invention is found on chromosome 17 near markers SHGC- 56719 and SHGC-58932 (LOD=15.9 and 15.68).

Allelic variants of a transporter peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the transporter peptide as well as being encoded by the same genetic locus as the transporter peptide provided herein. Genetic locus can readily be determined based on the genomic information provided in Figure 3, such as the genomic sequence mapped to the reference human. RH panel mapping shows the gene encoding the transporter proteins of the present invention is found on chromosome 17 near markers SHGC- 56719 and SHGC-58932 ^00=15.9 and 15.68). As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70- 80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a transporter peptide encoding nucleic acid molecule under stringent conditions as more fully described below.

Figure 3 provides information on SNPs that have been found in the gene encoding the transporter proteins of the present invention. The following variations were seen: g30344a, a31170g, cl6256t, al3376t, tl2210c, gl2072c, gll922t, -11903a, cl0009g, c4519t, a4181g, a20952c, t20987c, g21620a, t21795c, a22753t, g22945a, g23032a, g23738a, t23952g, a24123g, C24527-, c24691t, g25015a, and g25191t.

Paralogs of a transporter peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the transporter peptide, as being encoded by a gene from humans, and as having similar activity or function. Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a transporter peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.

Orthologs of a transporter peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the transporter peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a transporter peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins. Non-naturally occurring variants of the transporter peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the transporter peptide. For example, one class of substitutions are conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a transporter peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and He; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gin; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al, Science 247:1306-1310 (1990).

Variant transporter peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind ligand, ability to transport ligand, ability to mediate signaling, etc. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. Figure 2 provides the result of protein analysis and can be used to identify critical domains/regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al, Science 244:1081- 1085 (1989)), particularly using the results provided in Figure 2. The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as transporter activity or in assays such as an in vitro proliferative activity. Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol. Biol. 22 :899-904 (1992); de Nos et al. Science 255:306-312 (1992)). The present invention further provides fragments of the transporter peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in Figure 2. The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from a transporter peptide. Such fragments can be chosen based on the ability to retain one or more of the biological activities of the transporter peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen. Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length. Such fragments will typically comprise a domain or motif of the transporter peptide, e.g., active site, a transmembrane domain or a substrate-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis). The results of one such analysis are provided in Figure 2. Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in transporter peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art (some of these features are identified in Figure 2).

Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- ribosylation, for instance, are described in most basic texts, such as Proteins - Structure and Molecular Properties, 2nd Ed., T.E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol 182: 626-6A6 (1990)) and Rattan et al. (Ann. N Y. Acad. Sci. 663-.AS-62 (1992)).

Accordingly, the transporter peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature transporter peptide is fused with another compound, such as a compound to increase the half-life of the transporter peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature transporter peptide, such as a leader or secretory sequence or a sequence for purification of the mature transporter peptide or a pro- protein sequence.

Protein/Peptide Uses The proteins of the present invention can be used in substantial and specific assays related to the functional information provided in the Figures; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the protein binds or potentially binds to another protein or ligand (such as, for example, in a transporter-effector protein interaction or transporter-ligand interaction), the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any or all of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products.

Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include "Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are based primarily on the source of the protein as well as the class/action of the protein. For example, transporters isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the transporter. Screening of tissue specific cDNA libraries for cDNA retrieval showed expression of splice form 1 in fetal brain, brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, fetal kidney, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas. Additionally, BLAST hits to ESTs derived from tissue specific libraries shows expression of splice form 1 in germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendroglioma, chronic lymphotic leukemia B-cells, infant brain (73 days post natal), placenta, gliobloastoma, well-differentiated endometrial adenocarcinoma, and fetal liver and spleen. A large percentage of pharmaceutical agents are being developed that modulate the activity of transporter proteins, particularly members of the sulfate transporter subfamily (see Background of the Invention). The structural and functional information provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention, particularly in combination with the expression information provided in Figure 1. Independent lines of evidence show expression of splice form 1 in fetal tissues such as brain and kidney; differentiated tissues such as brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas; and neoplastic tissues such as germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendrogliomas, chronic lymphotic leukemic B-cells, glioblastomas, and well-differentiated endometrial adenocarcinomas. Such uses can readily be determined using the information provided herein, that known in the art and routine experimentation.

The proteins of the present invention (including variants and fragments that may have been disclosed prior to the present invention) are useful for biological assays related to transporters that are related to members of the sulfate transporter subfamily. Such assays involve any of the known transporter functions or activities or properties useful for diagnosis and treatment of transporter-related conditions that are specific for the subfamily of transporters that the one of the present invention belongs to, particularly in cells and tissues that express the transporter. Screening of tissue specific cDNA libraries for cDNA retrieval showed expression of splice form 1 in fetal brain, brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, fetal kidney, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas. Additionally, BLAST hits to ESTs derived from tissue specific libraries shows expression of splice form 1 in germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendroglioma, chronic lymphotic leukemia B-cells, infant brain (73 days post natal), placenta, gliobloastoma, well-differentiated endometrial adenocarcinoma, and fetal liver and spleen. The proteins of the present invention are also useful in drug screening assays, in cell-based or cell-free systems ((Hodgson, Bio/technology, 1992, Sept 10(9);973-80). Cell-based systems can be native, i.e., cells that normally express the transporter, as a biopsy or expanded in cell culture. Independent lines of evidence show expression of splice form 1 in fetal tissues such as brain and kidney; differentiated tissues such as brain, pituitary gland, heart, leulcocytes, kidney,, liver, thyroid, lung, placenta, skeletal muscle, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas; and neoplastic tissues such as germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendrogliomas, chronic lymphotic leukemic B-cells, glioblastomas, and well-differentiated endometrial adenocarcinomas. hi an alternate embodiment, cell- based assays involve recombinant host cells expressing the transporter protein. The polypeptides can be used to identify compounds that modulate transporter activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the transporter. Both the transporters of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the transporter. These compounds can be further screened against a functional transporter to determine the effect of the compound on the transporter activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the transporter to a desired degree.

Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the transporter protein and a molecule that normally interacts with the transporter protein, e.g. a substrate or a component of the signal pathway that the transporter protein normally interacts (for example, another transporter). Such assays typically include the steps of combining the transporter protein with a candidate compound under conditions that allow the transporter protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the transporter protein and the target, such as any of the associated effects of signal transduction such as changes in membrane potential, protein phosphorylation, cAMP turnover, and adenylate cyclase activation, etc.

Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig- tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al, Nature 554:82-84 (1991); Houghten et al, Nature 554:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L- configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al, Cell 72:161-11% (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab')₂, Fab expression library fragments, and epitope- binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).

One candidate compound is a soluble fragment of the receptor that competes for ligand binding. Other candidate compounds include mutant transporters or appropriate fragments containing mutations that affect transporter function and thus compete for ligand. Accordingly, a fragment that competes for ligand, for example with a higher affinity, or a fragment that binds ligand but does not allow release, is encompassed by the invention.

The invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) transporter activity. The assays typically involve an assay of events in the signal transduction pathway that indicate transporter activity. Thus, the transport of a ligand, change in cell membrane potential, activation of a protein, a change in the expression of genes that are up- or down- regulated in response to the transporter protein dependent signal cascade can be assayed.

Any of the biological or biochemical functions mediated by the transporter can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly Figure 2. Specifically, a biological function of a cell or tissues that expresses the transporter can be assayed. Screening of tissue specific cDNA libraries for cDNA retrieval showed expression of splice form 1 in fetal brain, brain, pituitary gland, heart, leukocytes, Iddney, liver, thyroid, lung, placenta, skeletal muscle, fetal kidney, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas. Additionally, BLAST hits to ESTs derived from tissue specific libraries shows expression of splice form 1 in germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendroglioma, chronic lymphotic leukemia B-cells, infant brain (73 days post natal), placenta, gliobloastoma, well-differentiated endometrial adenocarcinoma, and fetal liver and spleen.

Binding and/or activating compounds can also be screened by using chimeric transporter proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions. For example, a ligand-binding region can be used that interacts with a different ligand then that which is recognized by the native transporter. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in other than the specific host cell from which the transporter is derived. The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the transporter (e.g. binding partners and/or ligands). Thus, a compound is exposed to a transporter polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble transporter polypeptide is also added to the mixture. If the test compound interacts with the soluble transporter polypeptide, it decreases the amount of complex formed or activity from the transporter target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the transporter. Thus, the soluble polypeptide that competes with the target transporter region is designed to contain peptide sequences corresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable to immobilize either the transporter protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Techniques for immobilizing proteins on matrices can be used in the drug screening assays. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of transporter-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation.^' Preparations of a transporter-binding protein and a candidate compound are incubated in the transporter protein-presenting wells and the amount of complex trapped in the well can be .quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the transporter protein target molecule, or which are reactive with transporter protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

Agents that modulate one of the transporters of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.

Modulators of transporter protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the transporter pathway, by treating cells or tissues that express the transporter. Independent lines of evidence show expression of splice form 1 in fetal tissues such as brain and kidney; differentiated tissues such as brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas; and neoplastic tissues such as germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendrogliomas, chronic lymphotic leukemic B-cells, glioblastomas, and well-differentiated endometrial adenocarcinomas. These methods of treatment include the steps of administering a modulator of transporter activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein.

In yet another aspect of the invention, the transporter proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with the transporter and are involved in transporter activity. Such transporter-binding proteins are also likely to be involved in the propagation of signals by the transporter proteins or transporter targets as, for example, downstream elements of a transporter-mediated signaling pathway. Alternatively, such transporter- binding proteins are likely to be transporter inhibitors.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a transporter protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a transporter-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the transporter protein.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a transporter-modulating agent, an antisense transporter nucleic acid molecule, a transporter-specific antibody, or a transporter-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein. The transporter proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. Independent lines of evidence show expression of splice form 1 in fetal tissues such as brain and kidney; differentiated tissues such as brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas; and neoplastic tissues such as germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendrogliomas, chronic lymphotic leukemic B-cells, glioblastomas, and well- differentiated endometrial adenocarcinomas. The method involves contacting a biological sample with a compound capable of interacting with the transporter protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. The peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs. Thus, the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered transporter activity in cell-based or cell-free assay, alteration in ligand or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample. The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drag disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp. Pharmacol. Physiol. 23(10- l l):983-985 (1996)), and Linder, M.W. (Clin. Chem. 43(2):254-266 (1997)). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action. Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype. The discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the transporter protein in which one or more of the transporter functions in one population is different from those in another population. The peptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality. Thus, in a ligand-based treatment, polymorphism may give rise to amino terminal extracellular domains and/or other ligand-binding regions that are more or less active in ligand binding, and transporter activation. Accordingly, ligand dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism. As an alternative to genotyping, specific polymorphic peptides could be identified.

The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Independent lines of evidence show expression of splice form 1 in fetal tissues such as brain and Iddney; differentiated tissues such as brain, pituitaiy gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas; and neoplastic tissues such as germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendrogliomas, chronic lymphotic leukemic B-cells, glioblastomas, and well-differentiated endometrial adenocarcinomas. Accordingly, methods for treatment include the use of the transporter protein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof. As used herein, an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge. The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab') , and Fv fragments.

Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Hariow, Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. The full-length protein, an antigenic peptide fragment or a fusion protein can be used. Particularly important fragments are those covering functional domains, such as the domains identified in Figure 2, and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures. Antibodies are preferably prepared from regions or discrete fragments of the transporter proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or transporter/binding partner interaction. Figure 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness (see Figure 2).

Detection on an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵1, ¹³¹1, ³⁵S or ³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to detennine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Screening of tissue specific cDNA libraries for cDNA retrieval showed expression of splice form 1 in fetal brain, brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, fetal kidney, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas. Additionally, BLAST hits to ESTs derived from tissue specific libraries shows expression of splice form 1 in germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendroglioma, chronic lymphotic leukemia B-cells, infant brain (73 days post natal), placenta, gliobloastoma, well-differentiated endometrial adenocarcinoma, and fetal liver and spleen. Further, such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover.

Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. Independent lines of evidence show expression of splice form 1 in fetal tissues such as brain and Iddney; differentiated tissues such as brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas; and neoplastic tissues such as germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendrogliomas, chronic lymphotic leukemic B-cells, glioblastomas, and well-differentiated endometrial adenocarcinomas. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. Independent lines of evidence show expression of splice form 1 in fetal tissues such as brain and kidney; differentiated tissues such as brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas; and neoplastic tissues such as germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendrogliomas, chronic lymphotic leukemic B-cells, glioblastomas, and well-differentiated endometrial adenocarcinomas. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art. The antibodies are also useful for tissue typing. Independent lines of evidence show expression of splice form 1 in fetal tissues such as brain and Iddney; differentiated tissues such as brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas; and neoplastic tissues such as germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendrogliomas, chronic lymphotic leukemic B-cells, glioblastomas, and well-differentiated endometrial adenocarcinomas. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.

The antibodies are also useful for inhibiting protein function, for example, blocking the binding of the transporter peptide to a binding partner such as a ligand or protein binding partner. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See Figure 2 for structural information relating to the proteins of the present invention.

The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nucleic acid arrays and similar methods have been developed for antibody arrays.

Nucleic Acid Molecules The present invention further provides isolated nucleic acid molecules that encode a transporter peptide or protein of the present invention (cDNA, transcript and genomic sequence). Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the transporter peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof. As used herein, an "isolated" nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5KB, 4KB, 3KB, 2KB, or 1KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.

Moreover, an "isolated" nucleic acid molecule, such as a transcript/cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence shown in Figure 1 or 3 (SEQ ID NOS:l and 4, cDNA/transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in Figure 2, SEQ ID NOS:2 and 5. A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.

The present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence shown in Figure 1 or 3 (SEQ ID NOS:l and 4, cDNA/transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in Figure 2, SEQ ID NOS:2 and 5. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in Figure 1 or 3 (SEQ ID NOS:l and 4, cDN A/transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in Figure 2, SEQ ID NOS:2 and 5. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences. Such a nucleic acid molecule can have a few additional nucleotides or can comprise several hundred or more additional nucleotides. A brief description of how various types of these nucleic acid molecules can be readily made/isolated is provided below.

In Figures 1 and 3, both coding and non-coding sequences are provided. Because of the source of the present invention, humans genomic sequence (Figure 3) and cDNA/transcript sequences (Figure 1), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5' and 3' non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in Figures 1 and 3 or can readily be identified using computational tools known in the art. As discussed below, some of the non-coding regions, particularly gene regulatory elements such as promoters, are useful for a variety of purposes, e.g. control of heterologous gene expression, target for identifying gene activity modulating compounds, and are particularly claimed as fragments of the genomic sequence provided herein.

The isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half- life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the transporter peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5' and 3' sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification. Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form

DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand). The invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the transporter proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non- conservative amino acid substitutions.

The present invention further provides non-coding fragments of the nucleic acid molecules provided in Figures 1 and 3. Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents. A promoter can readily be identified as being 5' to the ATG start site in the genomic sequence provided in Figure 3.

A fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene. A probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence shown in the Figure sheets or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene. RH panel mapping shows the gene encoding the transporter proteins of the present invention is found oh chromosome 17 near markers SHGC-56719 and SHGC-58932 (LOD=15.9 and 15.68).

Figure 3 provides information on SNPs that have been found in the gene encoding the transporter proteins of the present invention. The following variations were seen: g30344a, a31170g, cl6256t, al3376t, tl2210c, gl2072c, gll922t, -11903a, cl0009g, c4519t, a4181g, a20952c, t20987c, g21620a, t21795c, a22753t, g22945a, g23032a, g23738a, t23952g, a24123g, C24527-, c24691t, g25015a, and g25191t.

As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65C. Examples of moderate to low stringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in Figure 2 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in Figure 2. As illustrated in Figure 3, identified SNP variations include g30344a, a31170g, cl6256t, al3376t, tl2210c, gl2072c, gl l922t, -11903a, cl0009g, c4519t, a4181g, a20952c, t20987c, g21620a, t21795c, a22753t, g22945a, g23032a, g23738a, t23952g, a24123g, c24527-, c24691t, g25015a, and g25191t.

The probe can correspond to any sequence along the entire length of the nucleic acid molecules provided in the Figures. Accordingly, it could be derived from 5' noncoding regions, the coding region, and 3' noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence. The nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence,, such as into the cellular genome, to alter in situ expression of a gene and/or gene product. For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.

The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods. RH panel mapping shows the gene encoding the transporter proteins of the present invention is found on chromosome 17 near markers SHGC-56719 and SHGC-58932 (LOD=15.9 and 15.68).

The nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.

The nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.

The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides. The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Screening of tissue specific cDNA libraries for cDNA retrieval showed expression of splice form 1 in fetal brain, brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, fetal kidney, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas. Additionally, BLAST hits to ESTs derived from tissue specific libraries shows expression of splice form 1 in germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendroglioma, chronic lymphotic leukemia B-cells, infant brain (73 days post natal), placenta, gliobloastoma, well-differentiated endometrial adenocarcinoma, and fetal liver and spleen.

Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in transporter protein expression relative to normal results.

In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA include Southern hybridizations and in situ hybridization.

Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a transporter protein, such as by measuring a level of a transporter-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a transporter gene has been mutated. Screening of tissue specific cDNA libraries for cDNA retrieval showed expression of splice form 1 in fetal brain, brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, fetal kidney, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas. Additionally, BLAST hits to ESTs derived from tissue specific libraries shows expression of splice form 1 in germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendroglioma, chronic lymphotic leukemia B-cells, infant brain (73 days post natal), placenta, gliobloastoma, well-differentiated endometrial adenocarcinoma, and fetal liver and spleen.

Nucleic acid expression assays are useful for drug screening to identify compounds that modulate transporter nucleic acid expression.

The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the transporter gene, particularly biological and pathological processes that are mediated by the transporter in cells and tissues that express it.

Independent lines of evidence show expression of splice form 1 in fetal tissues such as brain and kidney; differentiated tissues such as brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas; and neoplastic tissues such as germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendrogliomas, chronic lymphotic leukemic B-cells, glioblastomas, and well-differentiated endometrial adenocarcinomas. The method typically includes assaying the ability of the compound to modulate the expression of the transporter nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired transporter nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the transporter nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.

The assay for transporter nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the transporter protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.

Thus, modulators of transporter gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of transporter mRNA in the presence of the candidate compound is compared to the level of expression of transporter mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate transporter nucleic acid expression in cells and tissues that express the transporter. Screening of tissue specific cDNA libraries for cDNA retrieval showed expression of splice form 1 in fetal brain, brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, fetal kidney, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas. Additionally, BLAST hits to ESTs derived from tissue specific libraries shows expression of splice form 1 in germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendroglioma, chronic lymphotic leukemia B-cells, infant brain (73 days post natal), placenta, gliobloastoma, well-differentiated endometrial adenocarcinoma, and fetal liver and spleen. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

Alternatively, a modulator for transporter nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drag or small molecule inhibits the transporter nucleic acid expression in the cells and tissues that express the protein. Independent lines of evidence show expression of splice form 1 in fetal tissues such as brain and kidney; differentiated tissues such as brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas; and neoplastic tissues such as germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendrogliomas, chronic lymphotic leukemic B-cells, glioblastomas, and well-differentiated endometrial adenocarcinomas.

The nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the transporter gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays for qualitative changes in transporter nucleic acid expression, and particularly in qualitative changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in transporter genes and gene expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the transporter gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the transporter gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a transporter protein. Individuals carrying mutations in the transporter gene can be detected at the nucleic acid level by a variety of techniques. Figure 3 provides information on SNPs that have been found in the gene encoding the transporter proteins of the present invention. The following variations were seen: g30344a, a31170g, cl6256t, al3376t, tl2210c, gl2072c, gl l922t, -11903a, cl0009g, c4519t, a4181g, a20952c, t20987c, g21620a, t21795c, a22753t, g22945a, g23032a, g23738a, t23952g, a24123g, c24527-, c24691t, g25015a, and g25191t. RH panel mapping shows the gene encoding the transporter proteins of the present invention is found on chromosome 17 near markers SHGC-56719 and SHGC-58932 (LOD=15.9 and 15.68). Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al, Science 241:1011-1080 (1988); and Nakazawa et al, PNAS Pi:360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al. , Nucleic Acids Res. 25:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.

Alternatively, mutations in a transporter gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis. Further, sequence-specific ribozymes (U.S. Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S 1 protection or the chemical cleavage method. Furthermore, sequence differences between a mutant transporter gene and a wild-type gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C.W., (1995) Biotechniques 79:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al, Adv. Chromatogr. 36:121-162 (1996); and Griffin et al, Appl Biochem. Biotechnol 55:147-159 (1993)).

Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA DNA duplexes (Myers et al, Science 230: 1242 (1985)); Cotton et al, PNAS 55:4397 (1988); Saleeba et al, Meth. Enzymol. 277:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al, PNAS 86:2166 (1989); Cotto et al., Mutat. Res. 255:125-144 (1993); and Hayashi et al, Genet. Anal. Tech. Appl. 9:13-19 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et al, Nature 575:495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension. The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the transporter gene in an individual in order to select an appropriate compound or dosage regimen for treatment. Figure 3 provides information on SNPs that have been found in the gene encoding the transporter proteins of the present invention. The following variations were seen: g30344a, a31170g, cl6256t, al3376t, tl2210c, gl2072c, gl l922t, -11903a, cl0009g, c4519t, a4181g, a20952c, t20987c, g21620a, t21795c, a22753t, g22945a, g23032a, g23738a, t23952g, a24123g, c24527-, c24691t, g25015a, and g25191t.

Thus nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs to control transporter gene expression in cells, tissues, and organisms. A DNA antisense nucleic acid molecule is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of transporter protein. An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into transporter protein.

Alternatively, a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of transporter nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired transporter nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the transporter protein, such as ligand binding.

The nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in transporter gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired transporter protein to treat the individual.

The invention also encompasses kits for detecting the presence of a transporter nucleic acid in a biological sample. Screening of tissue specific cDNA libraries for cDNA retrieval showed expression of splice form 1 in fetal brain, brain, pituitary gland, heart, leukocytes, kidney, liver, thyroid, lung, placenta, skeletal muscle, fetal kidney, small intestine, prostate, testis, adrenal gland, bone marrow, and pancreas. Additionally, BLAST hits to ESTs derived from tissue specific libraries shows expression of splice form 1 in germ cell tumors, lung carcinoid tissue, brain anaplastic oligodendroglioma, chronic lymphotic leukemia B-cells, infant brain (73 days post natal), placenta, gliobloastoma, well-differentiated endometrial adenocarcinoma, and fetal liver and spleen. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting transporter nucleic acid in a biological sample; means for determining the amount of transporter nucleic acid in the sample; and means for comparing the amount of transporter nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect transporter protein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in Figures 1 and 3 (SEQ ID NOS:l, 3, and 4).

As used herein "Arrays" or "Microarrays" refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in US Patent 5,837,832, Chee et al, PCT application W095/11995 (Chee et al), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et al, US Patent No. 5,807,522.

The microarray or detection kit is preferably composed of a large number of unique, single- stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5', or 3', sequence, sequential oligonucleotides that cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5' or at the 3' end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The "pairs" will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation. In order to conduct sample analysis using a microarray or detection kit, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to deteπnine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit. The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.

Using such arrays, the present invention provides methods to identify the expression of the transporter proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the transporter gene of the present invention. Figure 3 provides information on SNPs that have been found in the gene encoding the transporter proteins of the present invention. The following variations were seen: g30344a, a31170g, cl6256t, al3376t, tl2210c, gl2072c, gl l922t, -11903a, cl0009g, c4519t, a4181g, a20952c, t20987c, g21620a, t21795c, a22753t, g22945a, g23032a, g23738a, t23952g, a24123g, c24527-, c24691t, g25015a, and g25191t.

Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al, Techniques in Immunocytochemistry, Academic Press, Orlando, FL Vol. 1 (1 982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.

Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.

In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified transporter gene of the present invention can be routinely identified using the sequence information disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.

Vectors/host cells

The invention also provides vectors containing the nucleic acid molecules described herein. The term "vector" refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules. The vectors can function in procaryotic or eukaryotic cells or in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.

The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retroviras long-terminal repeats.

In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the S V40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retroviras LTR enhancers.

In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989). A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovavirases such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al, Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989).

The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art. The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterotransporter. Typical fusion expression vectors include pGEX (Smith et al. , Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, Gene 69:301-315 (1988)) and pET l id (Studier et al, Gene Expression Technology: Methods in Enzymology 755:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli. (Wada et al. , Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include pYepSecl (Baldari, et al, EMBO J. 6-.229-23A (1987)), pMFa (Kurjan et al. , Cell 50:933-943(1982)), pJRY88 (Schultz et al. , Gene 54:113-123 (1987)), and pYES2 (Invifrogen Corporation, San Diego, CA).

The nucleic acid molecules can also be expressed in insect cells using, for example, baculoviras expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al, Mol. Cell Biol 5:2156-2165 (1983)) and the pVL series (Lucldow et al, Virology 170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 529:840(1987)) and pMT2PC (Kaufman et al, EMBO J. (5:187-195 (1987)).

The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non- coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).

The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells. The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.

Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell- free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.

Where secretion of the peptide is desired, which is difficult to achieve with multi- transmembrane domain containing proteins such as transporters, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides.

Where the peptide is not secreted into the medium, which is typically the case with transporters, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.

Uses of vectors and host cells The recombinant host cells expressing the peptides described herein have a variety of uses.

First, the cells are useful for producing a transporter protein or peptide that can be further purified to produce desired amounts of transporter protein or fragments. Thus, host cells containing expression vectors are useful for peptide production. Host cells are also useful for conducting cell-based assays involving the transporter protein or transporter protein fragments, such as those described above as well as other formats known in the art. Thus, a recombinant host cell expressing a native transporter protein is useful for assaying compounds that stimulate or inhibit transporter protein function. Host cells are also useful for identifying transporter protein mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant transporter protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native transporter protein. Genetically engineered host cells can be further used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a transporter protein and identifying and evaluating modulators of transporter protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the transporter protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the transporter protein to particular cells.

Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, both by Leder et al, U.S. Patent No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.

In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PI . For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS 59:6232-6236 (1992). Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 257:1351-1355 (1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. Nature 555:810-813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G₀ phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring bom of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect ligand binding, transporter protein activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays.

Accordingly, it is useful to provide non-human transgenic animals to assay in vivo transporter protein function, including ligand interaction, the effect of specific mutant transporter proteins on transporter protein function and ligand interaction, and the effect of chimeric transporter proteins. It is also possible to assess the effect of null mutations, that is mutations that substantially or completely eliminate one or more transporter protein functions.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

SEQUENCE LISTING

<110> Wei, Ming-Hui Ketchum, Karen DiFrancesco, Valentina Beasley, Ellen

<120> ISOLATED HUMAN TRANSPORTER PROTEINS,

NUCLEIC ACID MOLECULES ENCODING HUMAN TRANSPORTER PROTEINS, AND USES THEREOF

<130> CL000746PCT

<150> 09/765,344 <151> 2001-01-22

<150> 09/630,719 <151> 2000-08-22

<150> 60/199, 611 <151> 2000-04-26

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<400> 3 ggtccccgcg gccctcggcc ttgctcgggg ccaagggacc gcggacggtc aggtggcgca 60 gggtctcctc cggagacccc aggatccgga gccagcggcc ttgtgggcag gggccggggg 120 caggggagtg gattttgccc ggagcggagc aggccggggg cagtgggggg ctgggggtga 180 gggtggctgg ctctgcgcgc gggcgccggg gccctggaag atgctgcgca cctgaattaa 240 ccgggcgcct ctgatgtcct cccagaagca actagaactc cagggctgtg aaagccacag 300 gtgggggctg agcgaggctt ggcctcagga gcggaggacc cccccccccc ccccctcgag 360 cgccgcagtc caccgtagcg ggtggagccc gccttggtgc gcagttggaa aacctcggaa 420 gccccgctgg atctcctggc tgccacccgc accccccgcc agctacggtg cgcccgcggg 480 cccagcttct ctctgcgctg ctccccgtta aattccctgg ggagacggaa aaaaaggcaa 540 aggaagtcgg ttctccaggg gccagaagtg ttgagcctaa ttagtcttca gacttctcaa 600 tgaggaatcg cttatcagtt tcttatctgg gagagttgag gatggaggga cagaaggcac 660 ccaggatttg cacggggggg gattcaggga gagagggtga tgagggacgg ggtgggcctt 720 ccagtcttgg cccagtcccc atcttgcaca cattgttggc ttcctcttag agccgttcgc 780 ccccctgggg aggggagacc catagtgacc tctcctgaca cccgccgacc ctgaccagtg 840 ttgccgggtt cttcaaaggc cacgctctga ctgctggtct gtgtcacctg caccccccag 900 ccccaccgta gagatgcctt cttcggtgac ggcgctgggt caggccaggt cctctggccc 960 cgggatggcc ccgagcgcct gctgctgctc ccctgcggcc ctgcagagga ggctgcccat 1020 cctggcgtgg ctgcccagct actccctgca gtggctgaag atggatttcg tcgccggcct 1080 ctcagttggc ctcactgcca ttccccaggc gctggcctat gctgaagtgg ctggactccc 1140 gccccaggtg aggcgtctga ccctgctgcc agccatatct cagaaacagt gcagaataca 1200 cagtatcaat cccagacacc atcagcgatt ccaggtttcc agcccctggg ccccaaggaa 1260 cctttggttt acagtgtgtg acgcagattg tctctgggcc gacccaggct cctatgcctg 1320 tttggtacac acagacactg agctggttat ggaggggcca gcgagatgac tcatggaggc 1380 ctcaggagtt caagaccagc ccgaccaaaa tggtgaaacc ccgtctccac taaaaataca 1440 aaaattaggc tgggtgcggt ggctcaagcc tgtaatccca gcactttggg aggccgaggc 1500 aggcggatcg caaggtcagg agatggagac catcctggct aacagggtga aaccccgtct 1560 ctactaaaaa tacaaaaaat tagccaggtg tggtggcggg tgcctgtagt cccagctacc 1620 tgggaggctg aggcaggaga atggcgtgaa cccgggaggc ggagcttgca gtgagccgag 1680 actgtgccac tgccctccag cctgggcgac agagcgagac tccatctcaa aaaaaaaaaa 1740 aaaagaatgc ttcctcagac ttggacacag cacacgggcc tgtaccgacc cctctgcctg 1800 gctgtctgca ccctgaggcc ccagttgagt gctgctaaaa aagtggcctc ctgatcactg 1860 caggtccacc cacagggcag ggcggtgcac ctttaacctg ggcctggaca cagctgacac 1920 ccacacatcc cgagcttgga cacgcacact agggagctgg tggatgggcc tcggcctcct 1980 gagtgctcac caccctctct ccccacagta tggcctctac tctgccttca tgggctgctt 2040 cgtgtatttc ttcctgggca cctcccggga tgtgactctg ggccccaccg ccattatgtc 2100 cctcctggtc tccttctaca ccttccatga gcccgcctac gctgtgctgc tggccttcct 2160 gtccggctgc atccagctgg ccatgggggt cctgcgtttg ggtgaggctc taccttcttg 2220 ccaaggggat gccctcgacc tcagcatttg cttgtttgca tttcaagtct atccccgtgt 2280 gcgtgtgtgt gcgtgttgtg ggggtgtggg tatgtatgtg tgtgtgtgta ggtgggtggg 2340 tggtggaggg ggtggggcac ttggctcctt agtctactat tttactgatt agaggccagg 2400 acattggaga aagtgacctg tggctcagac cccatatgcc ccnnnnnnnn nnnnnnnnnn 2460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnttcatttc ctaccccgtc attaaagctt 2520 cacctctgct gctgccgtca ccatcggctt tggacagatc aaggtaggca cggcgcccac 2580 ccagggcact gctctttggc cactgctcgt tggcacaggg atggcgggag caggactgag 2640 gccagtcctg atccctgtgg ccagtggacg tcttgctgtt tcagattgtc ttccatgggt 2700 caagaagcac gcggtgctct catgggtccc ctgttaataa aatgaccctc ctgggaggga 2760 tgtcacgtga tggttggatt tcacagcggg taacttgggg gccggtaatt ccatccccct 2820 gctcttgccc gagtttccgt gccagtgtgc ttggctggct ctgtgacgtg gctctgttct 2880 ccctgcactg ggcacaccca gcaggcccca ccagtcatga gcatgctgct agaatttcta 2940 taggcaaatt atttccccat gccaatttag ttagatggtt ttgttttgtg cttttttttc 3000 tggtttctgt ttaatatttt ttaaatgcca tgccttttat attttctttt aaaacattta 3060 taataagtac taaaatcagt catcagctga gggtgtaatt tattctgttt ttgctgggtt 3120 gtaagttcct cgagggtagg aactgtcctt ctcccagcca tggccgccca gcattgggct 3180 ggtgcagtag gggggtgctc agtggggtgt gtgtggagtg aagtgagccc agttccacag 3240 atgggaccat gcggccctca tggcagacta gggtcacatg ctgcctcctg accctgtgtc 3300 actgcaggtc atcttcccca agccaggccc tgttccaggc tggcctgaga cagtcttccc 3360 tgatggaggt accatgagaa gaccaaggac aggagagtgt gtgtgagagt gtgtatgagt 3420 gtgtgtgtgt gtgaatgagt gagtgtgtga gagtgtgagt gggtttgagg gagtgagtgt 3480 gtgcgtgtgt gagtgagtgt gagtgtgggt gtgagtgagt gcgtgtgtgt gagagagtgt 3540 gtgagtgtgt atgagtgaga gtgtgagagt gggtgtgggt gttagggagt gtgtgagtgt 3600 gcgcgcgcgt gtgtgagtgt atgagtgtga gagtgagtgt gagtgagtga gagtgggtgt 3660 gggtgtgagt gtgcgtgtga gtgtgagagt gtgagtgtgt gagtgtgaga ataaagtaga 3720 cactttttgc actcttgcta cgtgcgaggc actgggcagg acactctcca tgtgtaattc 3780 tcaacacacc ccgggaggta gatttatcat tatttccatt tgagagagga gggaccaact 3840 taggtgtggg tgagcgtgat ttgtacgtta tctacacgca tctctcaggg ttagcttggc 3900 aaatgctgtt tcagggcatg gttggttctt taatctggaa acatcatttt tggtgtcaag 3960 aatgttcttt tgtaggatcc cagtgagagt ggagagcgga gagtggagag tggaaggcat 4020 cccttgttca tgccttcata cttggcaacc ctagccccgc ccagggactc tgcagccatc 4080 tggggggagg ggcgtcctcc tgacaggccc aggacagaag accctacccc aacagtccca 4140 gagtccgccc cccaggatgt cctaacccca cccaccctgt acgcagcagt tttagggcag 4200 ggtccttgag cctgtggcca tgggatccag ggcctcaact ttcccctctg actcctgttg 4260 ggtctcaggc gatgtaaaga actaaagtgc aagctgtgcc tgcactgtgg atcccagcta 4320 cgtgggaggc cgaggtggga ggatcactgg agcccaggag ttcgaatcca gcctgggcca 4380 cagagcaagc aagaccccca ccccccaacc ccccgccgct ctctaaagaa aaaaaaacta 4440 aactaaaaca caggatagag tgccttctcc tgcccaggac ctcagagctg gtatcgtggt 4500 gggaggctcc tactttgccg aggattcccc aagctggttt cttgaagccc ctcagagccc 4560 tccacatctg cacactacga agagattttt cctcccgcag cagcggagct ggggggtggc 4620 ggggcaccta gtgagggaga cattctcaag ccaatggcag caagggtcta cactgcaggg 4680 ggcctggctg ctgagtggcc tggagccaat gggggtgggg ccaggcagcc catggcccct 4740 ggccatcaag ctgtagaacc tgtctgcctg ctgtggggtc acctacattg ttttttatga 4800 gaaaagtaat ttagagaaaa acatatcact gacccagtaa ttgtgaagta ctgtctccca 4860 cgagggtagc tttgatctcc tgccctaggg ggcgttggga gtgggcaggt ggacccctga 4920 gcccctaaga tgggccccgg gaataagggt tgggagcagg gccgggggac actgtctcag 4980 ccctagggga ggtgggcggg gagctgggga cagatggcct tggtttggga gcatagcctc 5040 tgatcagcat ctctgtgttt ggacagaacc tgctgggact acagaacatc cccaggccgt 5100 tcttcctgca ggtgtaccac accttcctca ggattgcaga gaccaggtac cccgggcttt 5160 gttcctccct cctataagga agctccttct tccacacctc ctctcccggc ccccacctca 5220 gtttccccac ccctggtgac tgctcaaaca ggggtcccca gagcagcccc atcagcagca 5280 cctgcaagct ggcaagaaat gagactctca gtcctttgca gacctgccct atcggagcca 5340 gaggtttaac aggaggccgg tggtctgcgc gcaggggaga gttcaagaag ctctgctctg 5400 gggagctgga gcagggcaat gttcctctct ccacaaagct ttcttggggg acaggaatgg 5460 ggagagtctg tgtaaagaaa aagaaggcag tgtgtctccc ccaggctgtg atttgttaag 5520 gaggagaaca cagggcgtgg ggagctaacc cagacagaac gcaggtggtg cagggacggc 5580 aggtggagct gtgatgagag atgaggagac ccaagcactt ccctcaggtg gtttaaagag 5640 cctctgatgg ccggacactg tggctcacac ctgtaatccc agcactttgg gaagcccagg 5700 tgggcagatc atcttaggtc aggagttcaa gaccagcctg gtcaacatgg tgaaactccg 5760 tctctactaa aaatacaaaa attagtcggg tgtgatggtg cgtgcctgta gtcccagctg 5820 ttcaggaggc tgaggcagga ggattgcttg aacctgtgag gcggaggttg cagtgagctg 5880 agatcacgcc actgcactcc agcccgggtg atagagtgag actccatctc cgccctgccc 5940 tgcccccccg aaaagaaaag cctctgatga ggggtacctc cctgccagac catccagcgg 6000 gaaggcagga tgcctctcta cctctctggc tggaaggggc tggaggagga aatgatctag 6060 gggagctata gagatggctg cccagtgctg tggcctggag ggagtggggg cgatcctgga 6120 ccatcttccc ctcccctctg atctgctgcc gaagcttccc agcagctgaa cccagctgga 6180 agccagtggg ttctttgctc ttcagaggca ccagtgggca ggggtcggcc agatggggga 6240 gcagtgatta cggagcctga ccagtcagag agcgtcacgg gctgtcatgt aaaaggggcc 6300 ttactgaccg tttagtccta agccttctag aatgtctaga gcagggtgtc cagtcttttg 6360 tcttccctgg gccacacagg aagaattgtc ttaaaataca ctaatgatag ctgatgagct 6420 acaaaaaaat cattgaaaga actcataatg ttttaagaaa gtttacaaat ttgtgttggg 6480 ccacattcaa agccatccca ggctgcacat ggcccacagg ctgtgggttt ggcagacttg 6540 ttctagagcc acctgcagag agcatcagct gctcaggctg gtccccattc cctgtggtca 6600 ctcaccagat cctgttccat agacttgagt cccagagagc tacgggagtg aaaatgtgag 6660 cacgtcgctc tgccatcatc atgacccggg cctctccact gcctgtcctg cccaagcctg 6720 tgtacttact gaattttgaa ttgagttttg aagtagaaca gggatgcata ttgcttagga 6780 gaaaacatct cagcctagtc tgtgtttggt cttgcagagt agcttgctaa agttcctgag 6840 ctttagttaa gtgtctgtga aatggtgaaa atattgaaaa tgctttattt ccttgtgata 6900 ctcactgtgg tctgggacca gcagcctggg cattgggcct ccctgggagc ttcttagtgc 6960 ctctcagctt aagcagcagc aggacgcttg agtcagcagt aggcctgggc cgagctgggt 7020 ggtgaccagt cctctgcctg tccacagggt aggtgacgcc gtcctggggc tggtctgcat 7080 gctgctgctg ctggtgctga agctgatgcg ggaccacgtg cctcccgtcc accccgagat 7140 gccccctggt gtgcggctca gccgtgggct ggtctgggct gccacgacag gtgaggggcc 7200 tctggctgac atcgtatgca accttggctg caggttgggg tcacttgggg agtcctagtc 7260 ccaccctagg gattctcacg tcattggtct gggtgtcact tgagcattgg gacatttaaa 7320 acaccacacc aaactctggg acatgtactt tttatttaat taattaatta attatttttt 7380 gagacagagt ttcgctctgt cgcccaggct ggagtacagt ggtgtgatat tggctcactg 7440 caacctccgc ctcccaggtt catgtgattc tcctgcctca gcctcctgag tagctgggat 7500 tacaggcgca tgccaccacg ctcgactgat ttttgtattt atagtagaga cggggtttca 7560 ccatgttgac catactggtc tcaaactcct gacctcgagt gatctgcctg cctcggcctc 7620 ccgaagtgct gggattacag gcatgagcca ccgccccgac ccaaactcta gaacatttaa 7680 aactctgagc caaactcggt gggttctgat gcagggtcag agctgagaac tgatgcatgg 7740 ccagctgtgg ccgttctcaa cctgaagcag ttttgccccc ctggtgacat gtggcaatgt 7800 tgggaaacat ttttggtgga taccaccggg cgaggcagtt gctggcaact agcgaggagg 7860 ggccaggaat gccactaagc ccctgacggc gcacagcctt ccacagcaaa cagtgatccg 7920 gcccaaaatg tcggcggtgc cgaggtggag aaacacagtc tactggcagg tcctggggag 7980 acagaacaaa ttccggggag accaacaggc tctgtttgaa ttgtgcagct ttatgggggt 8040 acaacttcag cttcaggcgc ggaggctgca ggctgagcca caaccaacca ggggtccttt 8100 gagaaagggg tggtgagggc tgcgtcgtgg aggagcaggg ctgtcagcag gtgaaggacc 8160 accctggggg aggtggaagc ctcttctcag tgcatccggg atgggcatgg gtcccctggc 8220 tgagcaccgg gcagggatgc ccagagaaga aggcacaccc aggaaagtca ggaagcatag 8280 gtgactccaa accttctcag agccgagggg acagattaag ctcagaataa cccgagcttg 8340 gtcttgctgt ttgggggctg caaaatccaa cagccacaga agagagaggg tggctggatt 8400 tagctcttga gtcacttctg cctgcccagg ctggtctgtt ctctgtggtc tctccctgga 8460 tcctgttcag cagacttgag gccgtggtga acactgagag tctggtgggg tgcagaggat 8520 gctgggttta gagtggagat gtccagcttc cagtcccagc ctggcctggg tttagacttg 8580 caaagtagct tccaaagttc ctgagcttta gttcagttgt ccgtgaaata gtgaaaatgc 8640 tttgttatct ttgttatctt ttgttttttg tttttttttg gagacggtgt ctcactcctg 8700 ttgcccaggc tggagtgcac tggccgtgat ccttggctca ctgcaacctg cctcctggtt 8760 ttcaagcaat tattcccccc ttaaccctcc cggagtagcc tgggatttac aggccgtgca 8820 cccaccacac ccgggctaat tttttgtatt tttgagttca agatgggggt ttcaccattg 8880 ttggcccatg ctgggtctcg aaactcccnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8940 nnnnnnnnnn nnnnnnnncc ctaaccctga aaggcagtgt ctccgcagcc agaaaggtct 9000 tttaagatgt ttagacatcg gtaatgtcca gaaacttcag aaacatttta cagtgtaggg 9060 ctgctgactc ctttctcccc agtgtgtgta gcattgtctt aggaaactac ttaaaaagct 9120 tttgattctt ataacagctt tgagaatgag tttattattt tttccatttt gtcagtgagg 9180 aaagtgagcc tatgaaagat gaaaaataaa acagaacttc tcaaggccac acgactggga 9240 agaggcagaa caaggacttc acccgagtcg tctgtctttt tttctgaatg gtctgtctag 9300 aagtgtgtca atgttatgat cctctcaaag aaccagcttt tggtttcatt gatctccctt 9360 ttgctttggt tttctagttc attgatttct gcttgggcct ttattatttc ctttcttcta ^•9420 tttattttca gttaacttta tccttttttt tttttttttc aaaaaaaaat tttttttttt 9480 gagacggagt ttcgctcttg tcccccaggt tggagtgcaa tggtgtgatc tcagctcact 9540 gcaacctccg cctcccgggt tcaagtgatt ctcctgcctc agcctcccaa gtagctggga 9600 ttacaggcgt atgccaccat gcctggctaa ttttttgtat ttttaataga gacggggttt 9660 caccatgttg gccaggctgg tctcgaactc ctgaactcag gtgatccacc cgcctcagcc 9720 tcccaaagtg ctgggattac aggcgtgagc cactgtgccc ggcctttttt ttcagattct 9780 taaggtggaa gcctgggtct ttgatttgag acctttcttc ttttctaata tggttgttta 9840 aatgctgtaa atttcactcc tagcattact ttagctgcat cccacagttt ttatatatat 9900 gctgttcttt aatttttatt cggttctaaa tactttgtga tttccatttt gacttctcct 9960 ttgacctgtg gattatttag aagtggatta tatgatttct tctgtgaagc atgggagtta 10020 tttagtttcc aaatagttgg gggtttccca attagtactt tgccattgat ttctaattta 10080 gttccattgt gataagaaaa cgtacttagt gatttaaatc tgttttcctt tattgagagt 10140 tgtattatgg ccctgaatat ggtctatctt agtgaatatt ctgtgtgtgc ttgagaacat 10200 gttttctgcc attattgggt ggaatgttct gtaaatgtca gttaggtctg gttggttatc 10260 tcaggctact atattcttgc tgattttctt tctacttgtt ctgttgatta ttaaaagaag 10320 aatgttgaaa taacttcagc tataaccatg actttgtcta ttttcctttc ttacttacca 10380 gttattgttt catatatttt gaagctctgt tttttagtat aaaaacatat agaattataa 10440 ttttctcttg gaatgatact tttttttctg ggcaatattc tcttccctga aatctacttt 10500 gatactaaga gagctactcc agctttcatt agtgttagca tatcttttcc catcttttta 10560 cttttttttt tttgagacag aatctctctc tgttgcccag gctggagtgc aatggcgtga 10620 tcttggctca ctgcaacctc cacctcccag gttcaagcaa ttgttgtgcc tcagcctccc 10680 aagtagctgg aattacaggc ttgcaccacc aagcccagct aatttttgtg tttttagtag 10740 caatgaggtt tcaccatgtt ggccagcctg gtctcaaatt cttggcctca agtgatccac 10800 ctgccttggc ctcccaaagt gctgggacac cacacccagc ctcctatctt tttactttta 10860 acgtatttaa ttgatttgtg ttcttgtgtt taatggtatg ttttatgggc agcatataga 10920 tgggatttgt ttttaatctc ataatctctg ccttttaatt ggaatcttat gccatttata 10980 tttaatgtga ttattggtct ctttgacttt agatctacca tgttgctgtt tgtttcctgt 11040 ttgtctcatt tgttctttgt tcccttttcc ccctccttct acctctactg gattattttt 11100 tatgattcca ttttacatcc tttattggct gattagctgt aacggctata tttagttttt 11160 gtttgtttgt ttctaagtga ctgctttaag gtttgtggta aacatcttta tcttgctaca 11220 gtatacatta aacttcaact tcatgtgata taagaacctt ataacaggcc aggcgcagtg 11280 gctcacacct ataatcctag cactttggga ggctgaggga ggcagattgt ctgagctcag 11340 gagtctaaga ccagcctggg caacatggcg aaaccccatc tctaccaaaa atacaaaaaa 11400 ttagccaggc atggtggtgc atgcctgtaa tcccagctac tggggaggct gaggtgggag 11460 gattgcttga acctggaagg cagagattgc agtgagccaa gatccgtcac tgaagctgca 11520 aactcctggg ctcaagtaat tctcctgcct cagcctcctg agtagcttgg tctacaggca 11580 tgcaccacca catctggcta attttttaaa tttttttata gaaacgggct atgttgctca 11640 ggctgatcaa aggttcttgg cctcaagtga tgctcctgcc tcagccaacc aaagtgctga 11700 gattgcagat gtgagccacc attccctgca ggaacagtct tagatttatc cacgtagtca 11760 ctgtttctgg tgctcttaat tcctttgtac aaatccagat ttccatctgg tataattttc 11820 cttctacctg aaggatgtta ttttttcttc tgttgcaggc ctgttggtga ttaactcttt 11880 cagcattttt tttttttttt tatttgacag gattcactct gtcactcagg ctggaatgca 11940 gtggtacaat tatagttcac tgcagccttg aactcctaga cccaagcaat cctcagcctc 12000 ctgagtggct gggactatag gcatgcatca ccacgcctgg ataatttttt atttttactt 12060 tttgtagaga tggtgtcttc ctacgttgcc caggctggtc tcttactcct gggctcaagg 12120 gatcctccca cgagggatcc tcccactttg gcttcccaaa tgttgggatt acaggtgtga 12180 gccactactc ctggtcttct ttcaactttt gacttttgta tgtctgaaag tctattttgc 12240 cttcattttt caaagatagt tttgctggtt atagaattct agactttttt ttttctttca 12300 gtacttttta aaaaaaagtt atttattatt ttttttattt tgagacagtg tctcgctgtg 12360 tcacccaggc tggagtgcag tggcacgatc ttggctcact gcaatctctg cctcctgggt 12420 tcaagtgatt ctcctgtctc agcctcccag gtagatggaa ttacaggcgc atgctaccac 12480 gcccggctaa tttttgtact tttagcagag atggggtttt gccatgttgg ccaggctgct 12540 ttcgaactcc ctgacctcag ggtgatccgc cctccttggc ctcccaaagt gctaggatta 12600 caggcgtgag ccactttgcc tggcctcagg acttttaaag atgctattct gctgtcttct 12660 ggcccacttc gtttcaagaa gtgtgctggc attctttggt cctctgtatg taaccttttt 12720 atccaccgct tttaaaatat tctttatcac tggttttagg cagtttcata atgtggcttg 12780 atgtagtgtt tttttgtttg tttgtttttt gttttctgtg tttatgcttg tgattcattg 12840 aacttcttag acatgtgggt ttgcagtttc atcaaatttg gaaaataaac acccatcttt 12900 tcgtgaaatc tttgtactgt ccccctatgc gattccacag actttctttg cagaatcccc 12960 tgtctgtatt tcaggcacct cgaaggtgtt ctcacagctc acttgggatt ttcagccttc 13020 tcctttgtgt gtttcatttt ggatagtttc tgctgctaca ttttagtctt ttttttctgt 13080 aatgtctcag ctgttgtaat tgcaccaagt gcttgttccg tctcagacac tgtggttttc 13140 atttctagaa atccagtttg aatctttttt tttctttttt ttttttttta agatggagtt 13200 tcgctctgtt gtccaggctg gagtgcaatg gcgcggtctt ggctcactgt aacctctgcc 13260 tcccggcttc aagtggtttt cctgcctcag cctcccaggt agctgggatt ataggcatcc 13320 accaccatgc ccagctaatt tttgtatttt tagtaaagac agagcttcac tgtgtaggct 13380 gggccagtct cgaattcctg acttcaggtg attcagctgc ctcggcctcc caaagtgctg 13440 ggattacagg cgtgagctac cgtgcccagc ctcaatttga atcttttaaa atgtattttc 13500 tacatcttta cttaacagtt gcaatctttc ctctgttttt tttttaagta tatggaattt 13560 ggttacagtt attcttttaa tgtcctatta actaaattac atttggttac agttactctt 13620 ttaatgtcct gttaattctg tcatctttaa ctatgttact tttgggtctt ttttttattt 13680 tttattttta ttttttgaga cagggtctca ctgtgtcacc caggctagag tacagtggca 13740 tgatcatggt tcactatagc ctcaacctcc caggctcaag ttatcatctc acctcagccc 13800 cccaagtagc tgggactata ggcatatgcc accatgctca actaattttt tattttttgt 13860 ggagacagtg tctcactaat ttgctcaggc tggcctcaaa cttctgggct caagcagtcc 13920 tcctgtcttg gcttcccaaa gtgttgggat tacaggcatg ggccactgca cccagccata 13980 ttttcctgct tctttgcatg cctagtaatt tttgtttgga tggatgctag actttgtgag 14040 tttgaggtta ttgcgtgctg gatatttttg tattctataa atcttcttga gctttgttct 14100 gggatacagt taagttactt aggacagttc gatccattta ggtcttactt ttaagctttg 14160 tgagctggac cagagcagtg ttcattctag ggcaagccta atttggccca ccacttgatt 14220 ttgtaaataa agttttattg gaaaatcact cccattcatt tacatggtca atgtcaggtt 14280 ttatgcaata ataacagact tgagtagttg cgccagagac tttatggccc acaaagcaaa 14340 aagtttttag tatttagccc tttacagaaa acatttgcca accccaggcc ttgggctaat 14400 cttccccact gctgaggtaa aaccctctgg agtccctgat gctccatgaa ttatgagggt 14460 tttttactct ggtgtgaatc cttgggattg tcccttgtaa ccctctttgt gactcttccc 14520 cactgtgggt ttgggtcatt ttcgcccagc tttgcctcga ccagttctct gctgcacatc 14580 cgagggcacc tctgcagatc tctgcagctc tgtctccggc tgccctctct gctcctgtga 14640 ccttgcggtc agctccagct gcaggcctcc tcagacttcc tcccagcatt gccccctgaa 14700 ctcagggaaa cccctgggct ctgctagggg gtcccctcca tacacatggg gaccatcggg 14760 ggaaaccgta ggcagccggc ggggcagtga tagactcacc tcccttgttt ccctgtggcc 14820 gtcatggctg ctgtcccgcg tcttagtggc catcatgtcc actgtcccgc atcttggagg 14880 ccattgtttc tgttgtttgg gaaagggggt aaatctggtt tcttcctcct ttgtgctttg 14940 aggtggatgt gccctgggcc acctgatttc agagagtctt tgccgcggtg cacgatgtcc 15000 agtcacctgg agcttggcag caggtggcat gtgcacctgt ccgcagcctg cagctctgcc 15060 gtcccacctg cttatgctgc cacatagcat ttatgtctgt gttgtgtttc ccagagtgca 15120 gagtgaggac tgtggcccgg tgggggcgtc tcctgcctct gggatgtctg ccccagtaac 15180 ctggaggcag ccgcgctacc ccacactcgc cgggagcagg gtctcggacg cacctctcct 15240 tctctcctag ctcgcaacgc cctggtggtc tccttcgcag ccctggttgc gtactccttc 15300 gaggtgactg gataccagcc tttcatccta acaggggaga cagctgaggg gctccctcca 15360 gtccggatcc cgcccttctc agtgaccaca gccaacggga cgatctcctt caccgagatg 15420 gtgcaggtgg gcggagccgg gaggcaggat ggcgtggctg aggctgcagt ggcccctggc 15480 ctggctccta ccctgatgta tctgctgggt gccagggggt ctgaggtcag ttaggacagc 15540 tgagtcctca ggaacggaca tctcagttat taaagaatcc caggttggat gcaaactcag 15600 cgagctcagg gatgtcacgt ttgtgttcag gggcgcttct cctgttttgg actccagctg 15660 aggatgaatt taccgtgttc ctcccagcac ctggcgcctc ttcagacaag gaggcggatc 15720 ctgcagctga caagcacttg ctcctgttac ctgtggggcg gggtgggtcc ttgctgcttt 15780 catgggtcac tgctgggtcc taccccttag gaaggtcact caccatccct ctctcctctc 15840 tcaggacatg ggagccgggc tggccgtggt gcccctgatg ggcctcctgg agagcattgc 15900 ggtggccaaa gccttcggta agacgcctgt cacccacacc ccaggtctcc cagtgcgccg 15960 gctgggctag gcctgcctgc tttctagctt gcctttatcc gttactagtt ttagaaattt 16020 gaattcatat ccaagtaata catgctcatg atagatacat atgtattgtg tatatatgat 16080 aaaactggat ctataatgag gcatgccctc ccaccccatg gtgtgctggt gagtgttgta 16140 acagcctctg ctgtttgtgg aaataaaagg ttttgcttcg tggcccttgc cgatgtccac 16200 ggtgtaaacg ctgctgtctg attttaaggt aacgtcactg aaaggggagt ttgcacatgg 16260 agctgggttg agatctgcat gaacaatcat attctatggt gtctccacca tgtagataca 16320 gtgggtgcaa ataacctcat cagtagtagc caaatgccaa ataaattagg aagtgatgag 16380 ttttaagtat tatctttggg ccaggcatgg tggcccaggc ctgtaatccc aacactttgg 16440 gaggctgaga cgggaggatc gcttgagctc aggagtttga aacccaccta ggcaacgtaa 16500 cgagacctcg tttctattaa aaataaaaat tagctgggca tggagcacac ctgtggtacc 16560 agcttctcag aaggctgagg caggaggatc acttgagccc gggaggtcga ggtggcattg 16620 agctgtgata gtgccactgc actccagcct gggcagcaga acaagatcct gtattatctg 16680 tttaattgaa agttttaatt taattggtaa taatggctat gtttagtaat aggctcacaa 16740 aactcctaaa cattcagcaa catgctttat tcagctggct cagcccatcg gccagcccct 16800 cctcgccccc agggaggcag ggaacccttc gtctccttca gtgtctgttt tgagtcagca 16860 tctctagatt ccctccttgc agctccgtcc ttcactcgct ccactcctgc cagctttgta 16920 ttgtctgtgc ggatgacacc ttcgttctgt cttgtcaccg tcatttagtc cttcttgcgt 16980 ttgggtttat gttgattcta aaggttacag cccagttacc agtgtatgtt actgtgactg 17040 tgtaaatcgt gtttactgcc tagcccaggc tgtgccaggg taaagttcct tctccaaagt 17100 cccagtgctc taacccttac ccccttcaaa ggagtttttt ttgttttttt tttttttttt 17160 tttttggaga cagagtctca ctctgtggcc caggctggag tgcagtggca cgatctctgc 17220 tcactgcaac ctccgcttcc caggtttaag caattctcat gcctcagtct cccaggtagc 17280 tgggaataca ggcatgcgcc accacgccca actaatattc tatactttta gtagagacag 17340 ggtctcacta tgttggccag gctggtctca agtgatctgc ccacctgggc ctcccagtaa 17400 tttttttttt tttttttttt gagatagtgt ttcactcttg ttgcccatgc tgggtgcaat 17460 ggtgtgatct tggctcaccg caacctccgc cccccaggtt ccagcaatta tcctgcctca 17520 gcctcccaag tagctgggat tacgggtgtg caccaacatg cctggctaat tttgtatttt 17580 tagtagagat ggggtttctc catgttgatc agactggtct caaactcctg acctcaggta 17640 atccgcccac ctcagcctcc caaagtgctg ggattacagg tgtgagccac catgctcagc 17700 cctccagtaa tttctttttt tttttttttc gagacggagt ctcgctctgt cgcccaggct 17760 ggagtgcagt ggcgtgatct cggctcactg caagctccgc ctcctgggtt cacgccattc 17820 tcctgcctca acctccogag tagctgggac tacaggcgtc cgccaccacg cccagctaat 17880 tttttggatt tttagtaaaa acgggggttt caccgggggt ctcgatctcc tgacctcggg 17940 atctgccctg ccttaacctc tcaaagggct gggattacag gcgggagccc ctgggccccg 18000 gccgtaattt tttaatggaa aaacaagggc tcactttggg ggccaaggct gacctcaaac 18060 tcctgagttc aagggatcct cctgcctcgg gtttnnnnnn nnnnnnnnnn nnnnnnnnnn 18120 nnnnnnnnnn nnnnnnnnnn nnnntggtct gggctaattt ttgttttttt gtagagagag 18180 tatttgccat gttgcccagg ctggtctcga actcctgggc tcaagcgatc ctcctgcctt 18240 ggcctctaaa agtgctgcga ttatagctgt gagccaccat acctggcctg attacatggt 18300 actgtacctc tttccactct gtgatacatt caagcacatg tttacacgca catatatgtg 18360 aatattttag gtgtgtattc ttagatttac aaaacttatt gccgtgtgta gaaattcata 18420 atctgtgtta cattttgtga atactttttg agcgatgttg aatattactg accaggtctt 18480 taccgttggt tccctgtagc atctcagaat aattaccgca tcgatgccaa ccaggagctg 18540 ctggccatcg gtaagacccc agccgcggga aggaagacac cagctgtggg cctccagggt 18600 cccaggcctg cccctctgtg tctcctgcat tgtaggaaga ccatgatggt ggtgatgaac 18660 tgggagggca aaggtggccc cagatgggat cttctggaat atttagtttt gttttgggtt 18720 tttgagatgg agtctcactc tgttgcccag gcaggagtgc agtggcacga tctcggctca 18780 ctacaacctc tgcctcctgg gttcaagtga ttctcctgcc tcagcctccc aagtagctgg 18840 gattaaaggc atatgccacc atgcatggct aatttttgta ttttgagtag agatggggtt 18900 tcaccatgtt ggccaggctg gtctcgaact cctgacctca agtgatccgc ccgcctcagc 18960 ctcccaaagt gctgagatta taggcgtgag ccactgcgcc tggcctatgc cttgttatct 19020 taaaccttga gactcagagt gtggcctggg accagcagcc tgggcactgg gcatctcctg 19080 ggagcttact ggaaaggcag gatctcaggc cccaccccag atctccgaat caggatctgt 19140 attcttcagg gcacgcccag gggattcatg gggtcagctt agaagtcaaa gtttggaagc 19200 cgggcatggt ggctcacgcc tgtaatccca gcactttggg aggccgaggg aggcagatca 19260 cctgaggtca ggagtttgaa accagcttgg ccaaaccccg tctctactaa aaatacaaaa 19320 attagctggg tgtggtggtg tgtgcctgtg atcccagcta cttgggaggt tgaggcagga 19380 gaatcacttg aacccgggta ggcggaggtt atagtgagcc aagattgcac cactgcactc 19440 cagcctggca acagagcgag actccttctc aaaaaaaaaa aaaaaaaagt caaagtttgg 19500 gaaatgctta gagaccccat gtttttcaaa gacttgtttt gtatgacttt atgagatgat 19560 caagcaggtt tggccacaag gggagacagc caaaggctca ggaagataac cagttttttt 19620 ttactcaaag gtcccttagt taccagggag gccacagcat gcctacttag gcccatgtca 19680 gaggacacca ggggtgtccc cggaaggata aatcccaatc tgtgatttta ggcctccgcc 19740 ctttttcggg aattttcttt gcttaatgct aatttcaata aaggcccccg gcttaagact 19800 tttcatatgt tattaatttt aggcgggttc taactcctta atgaagaaat attctccctt 19860 ccctggattt ccgaatcaca ccatttcttc gagaaacgcc tcccacnnnn nnnnnnnnnn 19920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnngttt tttttttggg cacccggcca 19980 tcatctgggg aggcagcatt aaggcccatc actttgccct tcaatgtgac tgtcatcttt 20040 ttacacgaaa aagttttttg gtggaaaccc ggtggtcggc cccattacct cctgagtgcc 20100 tagggtttgt gttccgggcc atttatctca gctttcaccc cctcctggat ggtaagatgc 20160 tcccctctgt tctgcgtggg gaaggcgggt cctggtccca caggatagat tgggttcaaa 20220 gtgtttcaga gctaggatct cttccaggct ctcgtgggtc actcctgttt cccagcactt 20280 tgggagggta agccgaatgg gtcacctaag gtcaggcatt tgagaccaga ttggccagca 20340 tggcacactc cctgtgtgct actaaaaata gaaaaattac actgggtgtg gtggcacatg 20400 cccgtagtcc cagcactttg gcaggttcaa ccgggtggat cactgaggtc aggagtttga 20460 gaccagcctg gccaacatgg caaaactctg tctgtactaa aaatacagaa attagccagg 20520 cgtggtagtg tgtgcctgta atcccagcta ctcaggaggc tgaggcagga gaatcgcttg 20580 aactgggagg cggaggctgc agtgagctga gatcgcgcca ctgcattcca gcctgggcaa 20640 caaagccaga ctctgtctca aaaaaaaaaa aaaaaaaaaa aaaggctagg ctttacgtct 20700 gcaagaatgt ggcctgtttt cttccttccc tgaaggagtg cgtaggccca ggcccccagg 20760 atgattctcc cgagccctgc cttcctgcct cccttctggg tttttgtccc cctccccatc 20820 tcctttccct ccatcctgtg tgccttccct ccacgatcag cctgtcttgc ctcctcccca 20880 ggtctcacca acatgttggg ctccctcgtc tcctcctacc cggtcacagg cagctttgga 20940 cggtgagtga catgtccgcc tcttctgttt gcccacgttg gacgccttaa cgttgttacg 21000 ctgacaagga gtctgcctgc cctgaccccg gcgccccgtc ctccactgtg aacgctccgt 21060 ggagaggcag ggctgggggt cacccactgt cctccagggt gttctctgtt tctttattct 21120 catagatcgt cctgcagttt catactagaa agttccactg ggcattgtgg tacacccttg 21180 ttatcccatt tactctggag gcggaagcag gagaatcgct tgagcccagc aggatgaggc 21240 tgcagtgagt tgtgaccgca ccgctgcact ccagcctggg caacagagcg agaccctgtc 21300 tctaaaataa atactagaaa gttcccagca cgccaaagcc ctcctagctc ctggtgccag 21360 agtcaattcc tgaaaggacg tggagatagg aagggcctcg gctctgtcct gaagcagccg 21420 ggcatgaagc ttagcccaga tgccctacgg cccctcctca gtcaggacaa caggatggag 21480 gtgacctgtg gcttaaagga gagaaggagg cgtcgcctgg cactgcccag tcccccagct 21540 ggtgaccctt gccctgctgg gtatgggggc cccacctgga tgggggcagg agacagagtc 21600 ggcaggaacc tgaaaggaca cgtgcttcct gagcttcttc ctatagtcag ggtggcccaa 21660 gcgcggctgt ctgtgactgc accctaagtc tctttgcctc ggtccccttg cagtccccgc 21720 ctgcttccca agccgtgctg ggagctgacg tcccctcgga agatcagcca caggagtgtg 21780 gactgaggtc tcccttttcc cggcccctgg tgactgacgg tctctgtgtt gccttccagg 21840 acagccgtga acgctcagtc gggggtgtgc accccggcgg ggggcctggt gacgggtaag 21900 gccccccatc ttccccttgt gcccgcagcc ctgagagtgg gagaaaggga ggagggggcc 21960 cacagagacg tccctttggc tcatgggccg tgcgccccgg gactgcacag ggacttgggg 22020 ggccacacag gagtaggggg accacaggag actgagcagg ggctgggggc cttggcagtc 22080 gtcgccctac ccccacccct gtccccagtg ggctctgctg aacaagaggc tgctacgctg 22140 cgtgctgggg ggaccctgca ctcccgaggt cacctgtgtt cccgtgcccc gcaggagtgc 22200 tggtgctgct gtctctggac tacctgacct cactgttcta ctacatcccc aagtctgccc 22260 tggctgccgt catcatcatg gccgtggccc cgctgttcga caccaagatc ttcaggacgc 22320 tctggcgtgt taagagtacg tccttgtcct acaggggaga gcgctgtgat gcggtgtctg 22380 aacgcggagg gtgtcattta tgctacccca ttttcctgca gccccctctg tggggctggg 22440 actgggaagt tagggcagtc ccggaacaga gaagtggatg gccaggagat ggccccagag 22500 atggtcccga ggctcagtgg gaagagctgg agctccttgt cctgacacct ggggtcttga 22560 ggcgagcact gacccggggg agggtcccct cctgatcccc ctgcccccat ccctaccctc 22620 cttgccaccc gcctccagcc accactctgc ccggcccagc tggggggagg gacaggagac 22680 gtccctggtg accagcaggg ccagcggaac agccttgcac cctggctcag aatggcagtt 22740 cctttttttt ttattattat tatttttatt tttttttatt gatcattctt gggtgtttct 22800 tgcagagggg gatttggcag ggtcatagga caatatgggg ttgggggtaa ggtcacagat 22860 aacaggatcc caaggcagag gaatttttct tagtgcagaa caaaatgaaa agtctcccat 22920 gtctacttct ttctacacag acacagcaac catccgattt ctcaatcttt tccccacctt 22980 tccccccttt ctattccaca aaaccgccat tgtcatcatg gcgcgttctc agtgagctgt 23040 tgggtacacc tcccagacgg ggtggtggcc gggcagaggg gctcctcact tcccagtagg 23100 ggcggccggg cagaggcgcc cctcacctcc cggacgggag cagttccttt aacttaacac 23160 attttgtttt gtttgtggaa gaagcatgga gtgggttcac ctagcttact tggcattttc 23220 tggttagccc cagcaagttg ccaggtgaaa ggatggataa ttttcttgca tgcccgtcgc 23280 atgccagggc ctttcgcgtg ccagtgcgct ggggctttag aacagccctg aaacgtgttc 23340 atgtgccggc gtggaatggg atggccgctg ctgctagaaa ccaaggctca gccagggtgg 23400 ggcaggcctg acctatgcgt gcggtggaat cccccacagg gctaagcccg tgcactttgt 23460 ccccagggca ccttctcctt ggccaggtct caagggctca cgtggtccct gccccactcc 23520 tcaggccagc tctgtgccct gacaagcccc tgctgctgcc ctccctgagg ttggaggcca 23580 gggaccggcc ggcaggtctc acctgcgctc agctcagatg gggagggcat ttctttcttt 23640 cgacttgaag catggcctgg tcagcagctg ctgtccccaa gtcctcaggg gctgcttggg 23700 gtccatgagc acctttactc atatgtgggg ggcagaaggc tgtcccgctg gtcagcaggg 23760 ccatgttggg gcctcgggca gctgccgggc attcctcagc tgtgcccttc tcctagggct 23820 ggacctgctg cccctgtgcg tgaccttcct gctgtgcttc tgggaggtgc agtacggcat 23880 cctggccggg gccctggtgt ctctgctcat gctcctgcac tctgcagcca ggcctgagac 23940 caaggtaccc ctccgtggcc tctgagtggg gagtgtgctg ggggcaggat tcctgggcat 24000 ggtcttatgt tttgagggtc cggggtgatt gtggtcgtgg gtgctgctga aggggaccgc 24060 tcgctggcag gtgggcagtc accttgctat aaaccatggt gttctcccac tgtgtggggg 24120 ccgtgggggt ctccccttag cacccctctc ccggtcccct gcagcacgct aggttgggtg 24180 ggggcttccc gcttgggaca ggccaagcct ggtggaggcc acccggtcag acccgcctcc 24240 aggactcact cctccccaca ggtgtcagag gggccggttc tggtcctgca gccggccagc 24300 ggcctgtcct tccctgccat ggaggctctg cgggaggaga tcctaagccg ggccctggaa 24360 ggtgcatggg cgggggtcaa ggtggtctga ggtcactccc ctgtcctctg ccccccactc 24420 cctgctgttc aggaccccaa gaccctgtcc ccgacgctct ccagtccaca aggatgcagg 24480 catctctgag tgggctggac cgtcctctgt gggcctcagc cagtggctgc tgcagcaagg 24540 gtggtggctc cccacatatc actccttccc tgcccctaaa gtccggttcc tgtttctggg 24600 gggttgattt taggggagct aagggcctgt gagtcctagg agggaaacag ctgctgctgt 24660 caccaaacaa ttgtctctgg tcctgccacc cgaatccccc aactgggcga ctcagccgcc 24720 acgagatgga gcactctggc ctctctgtcc cctgcccctg gccagagcct cctttggcct 24780 ctgcagagca gctttgggct gctctgggtg gcgtgacctg gctcgggcct gtctccccag 24840 tgtccccgcc acgctgcctg gtcctggagt gcacccatgt ctgcagcatc gactacactg 24900 tggtgctggg actcggcgag ctcctccagg acttccagaa gcagggcgtc gccctggcct 24960 ttgtgggcct gcaggtgggt gtgcactggg atgccttagg ggttagcagc tgccggaagg 25020 ccttcctgtg cctgcctccc atggcgaatg tgacatctct gggctgtgat gctggacggc 25080 ccttcggccg gtgctggctc tgcttctgat ttaaacagtt cttgtcccca tctggccttc 25140 ctcgtccctc cctgtggaag ggggagcggt ggcccccagc cctccgaggg gtcacgttat 25200 ggcttctggt cactgccaca ctgtcctttg tgctggggac acacagtgaa cgagggtcag 25260 tccctgctct caggggaatt gtattttagg aaacaaaaag agacaggtga ggctaggcgt 25320 ggtggcacac tcctggaatc ccagcacttt gggaggccaa ggcgggcaga tcacctgagg 25380 tcaggagttc aagaccagcc tggccaacat ggtgaaaccc cgtctctact aaaaatacaa 25440 aaaaaactag tcaggtgtgg tggcgagcgc ctgtaattcc agctacatgg gaggctggga 25500 gaatcgcttg aacctgggag gtggaggttg cagtaagccg agatcccact actgtactcc 25560 agcctgggcg acagagtggg actccatctc aaaaacaaac aaacgaaaaa aaacaggtgc 25620 tcatagaatt tcatgaaaaa cgtattgtca gggcttccag aggctgaaga cgggtttcta 25680 tggaggccgt cctgttcaga gccgcaggta aagtgtaagg gctgggtccc aggccctgcg 25740 tcttaggcct cacctaggag ccttctgagc actgcagggt caacatccca ggggtgtggc 25800 cagtgtttgc agagaggcag gggtctctgt tgctgtggtt aaatgtgcgc tctctgccaa 25860 gtcctagatg gcagaacgtg ggggactagg gcgtgtcccc aggcgcccag aggagacatt 25920 catgaactag ccatggaaca ggaggccaag tgacccgtat accccaggtg tggaccacag 25980 ccgacccttg tcagagtttc cttcattccc aacctggccc acgaggctag tgttatcttc 26040 atccccatgt ccagaagagg tcactgaggc ccagagaagc caggtaatct gcccacggtc 26100 acacaggatg gtgggcccag cttcagattt gggcatgtgc cttcagaact tcgctcataa 26160 gtgttacgtc ctgtgtcata tttgcagagc acatgtcttc atggtctttg gagatgattc 26220 acttaaaaaa atactcctct gacccagaca tggtggctca cacctgtaat cccagcactt 26280 tgagaggccg aggcgggagg tcaagggttt gagaccagcc tggccaacat agtgaaactc 26340 cgtctctact aaaaatacaa aaattagacg ggcatggtgg cgcacgcctg taatcccagc 26400 tgctcagggg gctgaggcag gagaatcgct tgaacccggg aggcagaggt tgcagtgagc 26460 cgagatcgcg ccattgcact ccagcctggg taacaagagt gaaactccat ctcaaaaaca 26520 aacaaacaaa caaacaaaaa gtcctctggg ccaggcgctg tggctcacac ctgtaatccc 26580 agcactttgg gaggccgagg taggaggatc atttgaggcc aggagtttga gaccagcctg 26640 ggcaacatga cgaaacccca tcaaaaaaat aggaaaaaat agctggacgt ggtggtgtgc 26700 acctgtagct actcaggagg ctgaggtggg aggatcactt gagtctggga ggttgaggct 26760 gcagtgagcc atgatcgtgc cactgcactc cagcctgagc aatgagcaag accctgtctc 26820 aaaaaacaaa aaatttaaaa aaaaaaaatc ttcctctgac agcattcccc tggggctgcg 26880 tttcttctca ccattcactg gtatggaggt gaagccatac ctctccggga gactctgaga 26940 tggcatgtct accaggctgc cgacccgtgt gctacagagg aacatccctg ccctggctaa 27000 agtctgtctg tctctcaggt ccccgttctc cgtgtcctgc tgtccgctga cctgaagggg 27060 ttccagtact tctctaccct ggaagaagca ggtgggcaca gtcagacatc ctgtggcttt 27120 ggtgattttg taaaaatcat aaatgcttat tgtaaaaaat atgggaaacc agctgggcac 27180 agtggctcat gcctgtaatc ccagcacttt gggaggccga ggcaggtgga tcacctgagg 27240 tcaggagttc gagaccagcc tggccaacat ggtaaaacct catcagtact aaaaatacaa 27300 aaattagctg ggtgtggtgg catgcacctc tagtcccagc tattcgggag gctgaagcag 27360 cagaatcgct tgaacccagg aggcagaggt tgcactgagc cgagattgtg cctctgcact 27420 tccagagtag ggtgagagag ctagccatgt tgtccagaaa annnnnnnnn nnnnnnnnnn 27480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ncctggttga gcaggtataa tcgcttgcaa 27540 cccaggtagc agcagttgta ctgagtccga gaattgtgcc actgcactcc agcctgggtg 27600 agagcagcga gtactctgtc tcaaaaaaaa aaaaaacaaa aaaaacagga aaccacctcc 27660 accccagtcc actttggtga tcactccata cecctcccct aaacacgcat atgtacctgc 27720 ctgtcaggat gtggatatat gtttgcggtt ttacgtaaat gggaccattt catacctggt 27780 gctctggaac ccacattttt catgcagaag gttggaagga tgtccttcca gccgaaagtc 27840 cacatcccgg gggatcagga cagagcaggg ccgggtcagg agatccagaa gccccgggac 27900 agaaggtacg ggagggacag gagcagggtg ggcgctgacc cttgagacag caatcgcagg 27960 aggtctgagc cgcagcaggt gtcaacaaga ggatgggcca gagatgcaga gcatccaccc 28020 caggccacac agcagtggcc agagggtccc aggccccagt gctaggcctc ttcctcttcc 28080 actgaggtca cagctgaagc tgggtcagct ccgtgagagt gaggggtggc ggatgttgta 28140 ctgacttcct tggctcaatg tgacgtcagg gagattcacc catgttgttg tagaatcagc 28200 tcagatgcag tgcactggag gtggataagc agaatgtggc ctggccgtgg gatgggaggg 28260 taccctggag cagtaagaaa gggccgttag tcacctgaaa aatacgctta cagagactca 28320 ggtgagaccc tcatggagtt agtgacactg gcctgggtgg cccacagctc cttcctgcac 28380 accttccagg actctggaag gccctcctta atcccttcct gtgaactgac ccatcctcac 28440 ttctgagctt ttagtgcttg aaacatttat tgtattttct gcagagaagc acctgaggca 28500 ggagccaggg acccagccct acaacatcag agaagactcc attctggacc aaaaggttgc 28560 cctgctcaag gcataatggg gccacccgtg ggcatccaca gtttgcaggg tgttccggaa 28620 ggttcttgtc actgtgattg gatgctggat gccgcctgat agacatgctg gcctggctga 28680 gaaacccctg agcaggtaac ccagggaaga gaaggaagcc aggcctggag gtccacggca 28740 gtggggagtg gggctcactg gcttcctgtg ggatgactgg aaaatgacct cgctgctgtt 28800 ccctggcatg accctctttg gaagagtggt ttggagagag ccttctagaa tgacagactg 28860 tgcgaggaag caggggcagg ggtttccagc ccgggctgtg cgaggcatcc tggggctggc 28920 agcaccttcc cggctcacca gtgccacctg cgggggaggg acggggcagg caggagtctg 28980 ggaggcgggt ccgctcctct tgtctgcggc atctgtgctc tccgagagaa aaccaaggtg 29040 tgtcaaatga cgtcaagtct ctatttaaaa ataattttgt gttttctaaa tggaaaaagt 29100 gatagctttg gtgattttgt aaaagtcata aatgcttatt gtaaaaaata caggaaacca 29160 cccctcaccc tgtccacttg ggtgatcatt ccagacccct ccccaaacat gcatatgtac 29220 ctgtccgtca gtgtgtggat gtatgtttac agttctacat aaatgggatc attttataca 29280 tggtgctctg gaacccacat ttttcatgca gtcatttgca gtgaattatt tattgtgata 29340 ataaatagca ttagaataca agatttttaa tgtctgcgtg gtattttggt ctatatatgc 29400 atcataattg acttaccgag ccctctgttc aacgtgtgcg tgggttagag acgggatcgt 29460 gcctccttta gatgtgtcag ttgaagccct tgccctctat gtgactgcgt ttggaaacag 29520 ggcttttagg gggtaactag gtttaatgaa ggtcataaag gtgggccctg aaccgatagc 29580 tttagtgtcc tcgtgagaag agacgccaga gagctcgttc tctgcaccct cacaccccag 29640 ggaactccat gagaggacac ggcaaaacca ggccatgcgc ccaccaggaa gaaaggcctc 29700 atgaggaccc cagcctccaa aactgggaga agatgaatct ctgtgctagg ccccgcagcc 29760 tggggtgatc tgtgacggcg gcctgagcag gtgaggactg cctgcatgtt tgtattttta 29820 tgaatgcttt gattgagtct gggggtaaat ccctggaggc ctgtggcagc ctcagaggtg 29880 tgttctccct gcactttctt caagaagaat gtggcctgcc ctgctgagcc tcgttctgcc 29940 cgttctgccc gggcagtccc ggccaatgtc agcgcagcaa ggggagggcc tctgtaacca 30000 gggctgctgg ctgcggggct ccccactgga cacgggagcg gacattggag tgtccttcat 30060 ccgcgtcact cttccaggtc ccttgcctcc cattttcctt ccttccttcc tctttctttc 30120 cttccttcct ttcctccctt gctcccttcc ttttctttct tccttccgtt tttctttcct 30180 ttcttccttc ctttcttttt ttccttcccg ttctcccttt cttcctccct ccaccccttt 30240 cttctcactg tgttccccag gctggtcttc aactcctggg ctcaagcaat cctcttgcct 30300 cagcctctgg tgtggctggg accacagaca catgccaccg cgccaggcta atttgttaat 30360 tttttttata gagacagggt ctcactttgt ttcccaggct cgtctcaaac tcctgggctc 30420 aagtgatact ccagcttcag cctgaagtgc tggaattaag gtgtgagcca ccatgcctgg 30480 ccctctctca ttcacaagtg aaccgttcac ccctgccctc caatccatgt cgtttctgac 30540 ctcaggcagc ttctctttct acataaagtg aacctgtccc aaagctgtgc tcactgggcc 30600 cccctgccag ggctggaaga ggcagcagtt cacatttggc ttgcactcac ataccaaggg 30660 catccatgct tgaacctaga catggttcat tcacagggat gggtgaaggt aaacagactg 30720 tggcgggcac taggcactat cagtttcata aatctgcata ccacctgtga ctcagcagtt 30780 atgcttctcg gaatctactc aaacatgctt gagtcagcct tccaggaagc aggcacaagg 30840 acgtttgtga caacttggtc agtaatttta agaagtagga agaaacctga gtttctctga 30900 attcggtaac atcttgactg taggacacac gaataatgcc gtggaatatt gtgcagcagt 30960 taaaagaaat gaggtgaagg ccgggtgtgg cagctcacgc ctgttatctc agcactttgg 31020 gaggccgagg ctggtggatc acttaaggtc aggagtttga gaccagtctg gccaacatgg 31080 cgaaaccctg tctctactaa aaatacaaaa attagctgac ctctgtgaca gagtgagact 31140 ctgcctcaaa aaaaaaaaaa aaaaaaaaaa aaaaatgagg tgaagcttta agttgtaaca 31200 ctgattttgg ggtgcaataa agcaagttgc agaatgatac ccatgttaag atgctatttg 31260 agtgaacaca cggaccaaac aattctatgt tgggtacaaa tttaagaaag agttctgggc 31320 tgggcacggt ggctcacaac tgtaatccca gcactctggg agggtgagtc ggatggatca 31380 ggaggtcagg acttctagac tagcctggcc aatatggtga aaccccatct ctactaaaaa 31440 tacaaaaatt agccaggcgt gttggcgcgt gtctggagtc ccagctactc aggagactaa 31500 ggtaggagaa tcacttgaac ccgggaggca gaggttgcag tgagctgaga tcatgccact 31560 gcactctggc ctgggcaaca gagcgagact caaaaaataa ataaataaat aaataaaata 31620 aataagagtt ttctgcactt tgggaggcct gtagtcccag ctactctgga ggctgaggca 31680 agaggatcac ttgagcctgg gggggtcgag gctgcagtga gtcctgattg tgtcactgaa 31740 atccagcctg ggcaacagag tgagac 31766

<210> 4 <211> 2955 <212> DNA <213> HUMAN

<400> 4 gctgtggcga cgccaggaga ccccaagctg catcgccgag tggaagcaac tagaactcca 60 gggctgtgaa agccacaggt gggggctgag cgaggcgtgg cctcaggagc ggaggacccc 120 cccactctcc ctcgagcgcc gcagtccacc gtagcgggtg gagcccgcct tggtgcgcag 180 ttggaaaacc tcggagcccc gctggatctc ctggctgcca cccgcacccc ccgccagcct 240 acgccccacc gtagagatgc cttcttcggt gacggcgctg ggtcaggcca ggtcctctgg 300 ccccgggatg gccccgagcg cctgctgctg ctcccctgcg gccctgcaga ggaggctgcc 360 catcctggcg tggctgccca gctactccct gcagtggctg aagatggatt tcgtcgccgg 420 cctctcagtt ggcctcactg ccattcccca ggcgctggcc tatgctgaag tggctggact 480 cccgccccag tatggcctct actctgcctt catgggctgc ttcgtgtatt tcttcctggg 540 cacctcccgg gatgtgactc tgggccccac cgccattatg tccctcctgg tctccttcta 600 caccttccat gagcccgcct acgctgtgct gctggccttc ctgtccggct gcatccagct 660 ggccatgggg gtcctgcgtt tggctcacat ctcccctcat cctctgggac tgggtggagc 720 cgggaccagc tcgatgtccc ctcttggctg gccagggttc ctgctggact tcatttccta 780 ccccgtcatt aaaggcttca cctctgctgc tgccgtcacc atcggctttg gacagatcaa 840 gaacctgctg ggactacaga acatccccag gccgttcttc ctgcaggtgt accacacctt 900 cctcaggatt gcagagacca gggtaggtga cgccgtcctg gggctggtct gcatgctgct 960 gctgctggtg ctgaagctga tgcgggacca cgtgcctccc gtccaccccg agatgccccc 1020 tggtgtgcgg ctcagccgtg ggctggtctg ggctgccacg acagctcgca acgccctggt 1080 ggtctccttc gcagccctgg ttgcgtactc cttcgaggtg actggatacc agcctttcat 1140 cctaacaggg gagacagctg aggggctccc tccagtccgg atcccgccct tctcagtgac 1200 cacagccaac gggacgatct ccttcaccga gatggtgcag gacatgggag ccgggctggc 1260 cgtggtgccc ctgatgggcc tcctggagag cattgcggtg gccaaagcct tcgcatctca 1320 gaataattac cgcatcgatg ccaaccagga gctgctggcc atcggtctca ccaacatgtt 1380 gggctccctc gtctcctcct acccggtcac aggcagcttt ggacggacag ccgtgaacgc 1440 tcagtcgggg gtgtgcaccc cggcgggggg cctggtgacg ggagtgctgg tgctgctgtc 1500 tctggactac ctgacctcac tgttctacta catccccaag tctgccctgg ctgccgtcat 1560 catcatggcc gtggccccgc tgttcgacac caagatcttc aggacgctct ggcgtgttaa 1620 gaggctggac ctgctgcccc tgtgggtgac cttcctgctg tgcttctggg aggtgcagta 1680 cggcatcctg gccggggccc tggtgtctct gctcatgctc ctgcactctg cagccaggcc 1740 tgagaccaag gtgtcagagg ggccggttct ggtcctgcag ccggccagcg gcctgtcctt 1800 ccctgccatg gaggctctgc gggaggagat cctaagccgg gccctggaag tgtccccgcc 1860 acgctgcctg gtcctggagt gcacccatgt ctgcagcatc gactacactg tggtgctggg 1920 actcggcgag ctcctccagg acttccagaa gcagggcgtc gccctggcct ttgtgggcct 1980 gcaggtcccc gttctccgtg tcctgctgtc cgctgacctg aaggggttcc agtacttctc 2040 taccctggaa gaagcagaga agcacctgag gcaggagcca gggacccagc cctacaacat 2100 cagagaagac tccattctgg accaaaaggt tgccctgctc aaggcataat ggggccaccc 2160 gtgggcatcc acagtttgca gggtgttccg gaaggttctt gtcactgtga ttggatgctg 2220 gatgccgcct gatagacatg ctggcctggc tgagaaaccc ctgagcaggt aacccaggga 2280 agagaaggaa gccaggcctg gaggtccacg gcagtgggag tggggctcac tggcttcctg 2340 tgggatgact ggaaaatgac ctcgctgctg ttccctggca tgaccctctt tggaagagtg 2400 gtttggagag agccttctag aatgacagac tgtgcgagga agcaggggca ggggtttcca 2460 gcccgggctg tgcgaggcat cctggggctg gcagcacctt cccggctcac cagtgccacc 2520 tgcgggggag ggacggggca ggcaggagtc tgggaggcgg gtccgctcct cttgtctgcg 2580 gcatctgtgc tctccgagag aaaaccaagg tgtgtcaaat gacgtcaagt ctctatttaa 2640 aaataatttt gtgttttcta aatggaaaaa gtgatagctt tggtgatttt gtaaaagtca 2700 taaatgctta ttgtaaaaaa tacaggaaac cacccctcac cctgtccact tgggtgatca 2760 ttccagaccc ctccccaaac atgcatatgt acctgtccgt cagtgtgtgg atgtatgttt 2820 acagttctac ataaatggga tcattttata catggtgctc tggaacccac atttttcatg 2880 cagtcatttg cagtgaatta tttattgtga taataaatag cattagaata caaaaaaaaa 2940 aaaaaaaaaa aaaaa 2955

<210> 5 <211> 630 <212> PRT <213> HUMAN

<400> 5

Met Pro Ser Ser Val Thr Ala Leu Gly Gin Ala Arg Ser Ser Gly Pro

1 5 10 15

Gly Met Ala Pro Ser Ala Cys Cys Cys Ser Pro Ala Ala Leu Gin Arg

20 25 30

Arg Leu Pro He Leu Ala Trp Leu Pro Ser Tyr Ser Leu Gin Trp Leu

35 40 45

Lys Met Asp Phe Val Ala Gly Leu Ser Val Gly Leu Thr Ala He Pro

50 55 60

Gin Ala Leu Ala Tyr Ala Glu Val Ala Gly Leu Pro Pro Gin Tyr Gly 65 70 75 80

Leu Tyr Ser Ala Phe Met Gly Cys Phe Val Tyr Phe Phe Leu Gly Thr

85 90 95

Ser Arg Asp Val Thr Leu Gly Pro Thr Ala He Met Ser Leu Leu Val

100 105 110

Ser Phe Tyr Thr Phe His Glu Pro Ala Tyr Ala Val Leu Leu Ala Phe

115 120 125

Leu Ser Gly Cys He Gin Leu Ala Met Gly Val Leu Arg Leu Ala His

130 135 140

He Ser Pro His Pro Leu Gly Leu Gly Gly Ala Gly Thr Ser Ser Met 145 150 155 160

Ser Pro Leu Gly Trp Pro Gly Phe Leu Leu Asp Phe He Ser Tyr Pro

165 170 175

Val He Lys Gly Phe Thr Ser Ala Ala Ala Val Thr He Gly Phe Gly

180 185 190

Gin He Lys Asn Leu Leu Gly Leu Gin Asn He Pro Arg Pro Phe Phe

195 200 205

Leu Gin Val Tyr His Thr Phe Leu Arg He Ala Glu Thr Arg Val Gly

210 215 220

Asp Ala Val Leu Gly Leu Val Cys Met Leu Leu Leu Leu Val Leu Lys 225 .230 235 240

Leu Met Arg Asp His Val Pro Pro Val His Pro Glu Met Pro Pro Gly

245 250 255

Val Arg Leu Ser Arg Gly Leu Val Trp Ala Ala Thr Thr Ala Arg Asn

260 265 270

Ala Leu Val Val Ser Phe Ala Ala Leu Val Ala Tyr Ser Phe Glu Val

275 280 285

Thr Gly Tyr Gin Pro Phe He Leu Thr Gly Glu Thr Ala Glu Gly Leu

290 295 300

Pro Pro Val Arg He Pro Pro Phe Ser Val Thr Thr Ala Asn Gly Thr 305 310 315 320

He Ser Phe Thr Glu Met Val Gin Asp Met Gly Ala Gly Leu Ala Val

325 330 335

Val Pro Leu Met Gly Leu Leu Glu Ser He Ala Val Ala Lys Ala Phe

340 345 350

Ala Ser Gin Asn Asn Tyr Arg He Asp Ala Asn Gin Glu Leu Leu Ala

355 360 365

He Gly Leu Thr Asn Met Leu Gly Ser Leu Val Ser Ser Tyr Pro Val 370 375 380 Thr Gly Ser Phe Gly Arg Thr Ala Val Asn Ala Gin Ser Gly Val Cys

385 390 395 400

Thr Pro Ala Gly Gly Leu Val Thr Gly Val Leu Val Leu Leu Ser Leu

405 410 415

Asp Tyr Leu Thr Ser Leu Phe Tyr Tyr He Pro Lys Ser Ala Leu Ala 420 425 430

Ala Val He He Met Ala Val Ala Pro Leu Phe Asp Thr Lys He Phe 435 440 445

Arg Thr Leu Trp Arg Val Lys Arg Leu Asp Leu Leu Pro Leu Trp Val 450 455 460

Thr Phe Leu Leu Cys Phe Trp Glu Val Gin Tyr Gly He Leu Ala Gly

465 470 475 480

Ala Leu Val Ser Leu Leu Met Leu Leu His Ser Ala Ala Arg Pro Glu 485 490 495

Thr Lys Val Ser Glu Gly Pro Val Leu Val Leu Gin Pro Ala Ser Gly 500 505 510

Leu Ser Phe Pro Ala Met Glu Ala Leu Arg Glu Glu He Leu Ser Arg

515 520 525

Ala Leu Glu Val Ser Pro Pro Arg Cys Leu Val Leu Glu Cys Thr His 530 535 540

Val Cys Ser He Asp Tyr Thr Val Val Leu Gly Leu Gly Glu Leu Leu

545 550 555 560

Gin Asp Phe Gin Lys Gin Gly Val Ala Leu Ala Phe Val Gly Leu Gin 565 570 575

Val Pro Val Leu Arg Val Leu Leu Ser Ala Asp Leu Lys Gly Phe Gin 580 585 590

Tyr Phe Ser Thr Leu Glu Glu Ala Glu Lys His Leu Arg Gin Glu Pro 595 600 605

Gly Thr Gin Pro Tyr Asn He Arg Glu Asp Ser He Leu Asp Gin Lys 610 615 620

Val Ala Leu Leu Lys Ala

625 630

Claims

ClaimsThat which is claimed is:

1. An isolated peptide consisting of an amino acid sequence selected from the group consisting of:

(a) an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5;

(b) an amino acid sequence of an allelic variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of SEQ ID NOS : 1 , 3 , and 4;

(c) an amino acid sequence of an ortholog of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of SEQ ID NOS:l, 3, and 4; and

(d) a fragment of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said fragment comprises at least 10 contiguous amino acids.

2. An isolated peptide comprising an amino acid sequence selected from the group consisting of:

(a) an amino acid sequence selected from the group consisting of SEQ ID NOS :2 and 5;

(b) an amino acid sequence of an allelic variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of SEQ ID NOS:l, 3, and 4;

(c) an amino acid sequence of an ortholog of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of SEQ ID NOS: 1, 3, and A; and

(d) a fragment of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said fragment comprises at least 10 contiguous amino acids.

3. An isolated antibody that selectively binds to a peptide of claim 2.

4. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5;

(b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of SEQ ID NOS:l, 3, and 4;

(c) a nucleotide sequence that encodes an ortholog of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of SEQ ID NOS:l, 3, and 4;

(d) a nucleotide sequence that encodes a fragment of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said fragment comprises at least 10 contiguous amino acids; and

(e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d).

5. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5;

(b) a nucleotide sequence that encodes an allelic variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of SEQ ID NOS:l, 3, and 4;

(c) a nucleotide sequence that encodes an ortholog of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of SEQ ID NOS:l, 3, and 4;

(d) a nucleotide sequence that encodes a fragment of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of

(a)-(d).

6. A gene chip comprising a nucleic acid molecule of claim 5.

7. A transgenic non-human animal comprising a nucleic acid molecule of claim 5.

8. A nucleic acid vector comprising a nucleic acid molecule of claim 5.

9. A host cell containing the vector of claim 8.

10. A method for producing any of the peptides of claim 1 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.

11. A method for producing any of the peptides of claim 2 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.

12. A method for detecting the presence of any of the peptides of claim 2 in a sample, said method comprising contacting said sample with a detection agent that specifically allows detection of the presence of the peptide in the sample and then detecting the presence of the peptide.

13. A method for detecting the presence of a nucleic acid molecule of claim 5 in a sample, said method comprising contacting the sample with an oligonucleotide that hybridizes to said nucleic acid molecule under stringent conditions and determining whether the oligonucleotide binds to said nucleic acid molecule in the sample.

14. A method for identifying a modulator of a peptide of claim 2, said method comprising contacting said peptide with an agent and determining if said agent has modulated the function or activity of said peptide.

15. The method of claim 14, wherein said agent is administered to a host cell comprising an expression vector that expresses said peptide.

16. A method for identifying an agent that binds to any of the peptides of claim 2, said method comprising contacting the peptide with an agent and assaying the contacted mixture to determine whether a complex is formed with the agent bound to the peptide.

17. A pharmaceutical composition comprising an agent identified by the method of claim 16 and a pharmaceutically acceptable carrier therefor.

18. A method for treating a disease or condition mediated by a human transporter protein, said method comprising administering to a patient a pharmaceutically effective amount of an agent identified by the method of claim 16.

19. A method for identifying a modulator of the expression of a peptide of claim 2, said method comprising contacting a cell expressing said peptide with an agent, and determining if said agent has modulated the expression of said peptide.

20. An isolated human transporter peptide having an amino acid sequence that shares at least 70% homology with an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5.

21. A peptide according to claim 20 that shares at least 90 percent homology with an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5.

22. An isolated nucleic acid molecule encoding a human transporter peptide, said nucleic acid molecule sharing at least 80 percent homology with a nucleic acid molecule selected from the group consisting of SEQ ID NOS:l, 3, and 4.

23. A nucleic acid molecule according to claim 22 that shares at least 90 percent homology with a nucleic acid molecule selected from the group consisting of SEQ ID NOS.T, 3, and 4.