Abstract:
Polymorphisms are identified in a human gene which encodes a voltage-gated ion channel. The gene maps to the same portion of human chromosome 13q22 to which genes for schizophrenia susceptibility, Bipolar Disorder, and Panic Disorder Syndrome have been mapped. The polymorphisms are used to follow inheritance of the susceptibilities within families. They are also used to identify affected probands. A complete mRNA sequence and genomic structure provide insights into function and relationship to other ion channels.

Description:
[0001]    This application claims priority from Provisional Application Serial No. 60/300,101 filed Jun. 25, 2001, the contents of which are hereby incorporated by reference. 
     
    
     
       TECHNICAL FIELD OF THE INVENTION  
         [0002]    This invention is related to ion channels expressed in brain tissue. In particular it is related to ion channels which are associated with susceptibility to psychiatric disorders.  
         BACKGROUND OF THE INVENTION  
         [0003]    It has long been known that the flow of ions in and out of cells in the nervous system is crucial for their activity, and that specific ion channels are largely responsible for this movement. Ion channels are generally gated (with the exception of the K +  leak channels), either by ligand binding or by voltage. Ligand binding ion channels convert extracellular chemical signals into electrical signals, while voltage gated ion channels, particularly Na +  channels, play a key role in action potential propagation. The voltage gated Ca ++  channel provides the only known means of converting electrical signals into chemical signals. We describe here the isolation and characterization of BION1 (Brain Ion 1), a novel voltage gated channel protein of unknown ion specificity. Various lines of evidence are presented that link the gene encoding this protein with a schizophrenia susceptibility locus on chromosome 13q32.  
           [0004]    Schizophrenia is a serious disorder characterized by severe psychotic symptoms, and is fairly common, affecting ˜1% of the general population. The illness often develops in young adults who were previously normal, and is characterized by a constellation of symptoms including hallucinations and delusions (psychotic symptoms) and symptoms such as severely inappropriate emotional responses, disordered thinking and concentration, erratic behavior, as well as social and occupational deterioration (Andreasen, 1995). Family, twin and adoption studies indicate that schizophrenia is primarily (71%) genetic. The genetic component of schizophrenia is complex, polygenic, and involves epistatic interaction between loci. Mapping studies reveal that this genetic component can be mapped to several distinct genetic loci, including SCZD7 on 13q32 (Blouin et al. 1998, Pulver et al. 1998, Shaw et al. 1998, Brzustowicz et al. 1999, Brzustowicz et al. 2000).  
           [0005]    Recently, a genetic linkage between 13q32 and a second syndrome, Panic Disorder Syndrome (Weissman et al. 2000) has been established. Panic Disorder (anxiety neurosis) is also genetic, with a prevalence of 1-3%, and is symptomatically represented by recurrent episodes of sudden apprehension and associated autonomic symptoms involving the cardio-respiratory system, nervous system, and gastrointestinal system. Panic Disorder Syndrome occurs in a subset of Panic Disorder patients, and is additionally associated with an increased rate of mitral valve prolapse, bladder/kidney problems, serious headaches and/or thyroid problems. When these additional symptoms are scored, a high LOD (likelihood of linkage) score was obtained with one marker, D13S779, which is also at 13q32. Interestingly, when only Panic Disorder was considered, there was no significant LOD with this marker, suggesting that panic disorder is also polygenic (Knowles et al. 1998). While multiple case reports of familial association of schizophrenia and Panic Disorder exist in the literature, statistical association of the two disorders has not been performed. Bipolar Disorder, another serious psychiatric disorder, has also been found to be genetically linked to a locus at or near chromosome 13q32.  
           [0006]    There is a need in the art for additional tools for diagnosing and studying psychiatric disorders, including schizophrenia, Bipolar Disorder, and Panic Disorder Syndrome.  
         SUMMARY OF THE INVENTION  
         [0007]    It is an object of the present invention to provide an isolated and purified polynucleotide which encodes human BION1.  
           [0008]    It is another object of the present invention to provide a method of determining susceptibility to Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome.  
           [0009]    It is an object of the present invention to provide an isolated and purified polynucleotide comprising at least 18 contiguous nucleotides of a human BION1 coding sequence.  
           [0010]    Another object of the invention is to provide an isolated and purified human BION1 protein.  
           [0011]    Another object of the invention is to provide an isolated and purified human BION1 polypeptide comprising at least 6 amino acids of a human BION1 protein.  
           [0012]    Another object of the invention is to provide a method of producing human BION1.  
           [0013]    Another object of the invention is to provide a method of screening test substances for candidates useful in treating Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome.  
           [0014]    These and other objects of the invention are achieved by providing one or more of the embodiments shown below. In one embodiment of the invention an isolated and purified polynucleotide which encodes human BION1 is provided. The polynucleotide encodes an amino acid sequence as shown in SEQ ID NO:1 or a polymorphic variant found in a schizophrenic, Bipolar Disorder, or Panic Disorder Syndrome patient.  
           [0015]    Another aspect of the invention is an isolated and purified polynucleotide comprising at least 18 contiguous nucleotides of a human BION1 coding sequence. The polynucleotide comprises at least one codon identified in Table 1, which codon is for an amino acid found in human BION1 proteins at the corresponding position.  
           [0016]    Still another embodiment provided by the present invention is an isolated and purified human BION1 protein. The protein comprises an amino acid sequence as shown in SEQ ID NO:1 or a polymorphic variant found in Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome patients.  
           [0017]    According to yet another embodiment of the invention, an isolated and purified human BION1 polypeptide comprising at least 6 amino acids of a human BION1 protein is provided. The polypeptide comprises an amino acid sequence found in humans but not in rats as identified in Table 1.  
           [0018]    According to another embodiment of the invention an isolated and purified polynucleotide of a human BION1 gene is provided. The polynucleotide comprises a polymorphic nucleotide identified in Table 2 or Table 3.  
           [0019]    Even another embodiment provided by the present invention is a method of producing human BION1. A host cell comprising a vector which encodes a human BION1 protein is cultured under conditions for expression of BION1 from the vector. BION1 protein is then collected from the cultured host cells or culture medium.  
           [0020]    According to another aspect of the invention a method is provided for screening test substances for candidates useful in treating Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome. A human BION1 protein is contacted with a test substance. Test substances are determined which bind to the human BION1 protein. A test substance is identified as a candidate drug useful for treating Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome if it binds to human BION1.  
           [0021]    Thus the present invention provides diagnostic and drug development tools for addressing psychiatric disorders for which genetic and pharmacologic interventions are needed. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0022]    [0022]FIG. 1 a : cDNA sequence of human BION1. The complete coding sequence is underlined, running from nucleotide 230 to 5443..  
         [0023]    [0023]FIG. 1 b : Protein sequence of human BION1. Protein sequence of human BION1. The positions of the transmembrane domains and loops are shown underlined. The residues in the loop thought to be responsible for ion selectivity (EEKE) are in red bold face.  
         [0024]    [0024]FIG. 2 a : The predicted secondary structure of BION1, showing its predicted topology within the cell membrane. Additionally, the predicted pore structure of Bion1, Na v , and Ca v  channels is shown  
         [0025]    [0025]FIG. 2 b : Predicted Secondary structure of BION1, showing its predicted topology within the cell membrane. Additionally, the predicted pore structure of Bion1, Na v , and Ca v  channels is shown.  
         [0026]    [0026]FIG. 3 a : Amino acid residues thought to be crucial in determining ion selectivity demonstrate the relationship between BION1, Na +  channels (type II from brain), and Ca ++  channels (L-type from heart). Note the conserved DEKA in Na +  channels and EEEE in Ca ++  channels is replaced by the novel combination EEKE.  
         [0027]    [0027]FIG. 3 b : Phylogenetic tree relating different members of the voltage-gated ion channel superfamily. Alignment of the amino acid sequences of S4 regions of different members of the voltage-gated ion channels was generated using CLUSTALW.  
         [0028]    [0028]FIG. 4 a : Northern blot of rat poly(A) +  RNA isolated from a variety of tissues probed with Bion1. Highest expression is in brain and pancreas (not shown), although significant expression is also seen in heart, kidney, small intestine and placenta.  
         [0029]    [0029]FIG. 4 b : Northern blot of rat poly(A) +  RNA isolated from different parts of the brain probed with Bion1. Densitometry readings relative to thalamic expression are given.  
         [0030]    [0030]FIG. 5 a : In situ hybridization of BION1 to rat brain sections. Note the strong signal in the hippocampus (hip) in panel A.  
         [0031]    [0031]FIG. 5 b : Emulsion autoradiogram of in situ hybridization of BION1 to rat brain sections. Note strong signal over the neuronal layer of the hippocampus (panel A).  
         [0032]    [0032]FIG. 6 a : Fluorescent in situ hybridization with BION1 showing hybridization to human chromosome 13q22. DAPI DNA staining in blue and the FISH signal in red.  
         [0033]    [0033]FIG. 6 b : The same karyotype as in FIG. 6 a  has been alu-banded in green.  
         [0034]    [0034]FIG. 7: BION1 gene structure. Protein coding sequence is shown in shaded.  
         [0035]    [0035]FIG. 8: BION1 polymorphism analysis. The positions of all polymorphisms found are marked with an asterisk. Intronic polymorphisms are indicated by minus signs in the name (e.g., T(−221)C, indicating that it is 221 nt from the intron/exon boundary). 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0036]    We have determined the sequence of the human homolog of the rat BION1 gene, a voltage-gated ion channel with novel ion specificity sequence. We have localized this gene to human chromosome 13q32, where a schizophrenia susceptibility gene (SCZD7), Bipolar Disorder locus, and Panic Disorder Syndrome locus map genetically. Moreover, the closest marker to these genetic loci, D13S779, is contained within a YAC clone that also contains BION1. We have shown by Northern blot analysis that rat BION1 RNA is expressed in the brain, consistent with a neuroactive protein. Interestingly, it is also expressed moderately in the heart; the subset of Panic Disorder patients that comprise the Panic Disorder Syndrome exhibit a high incidence of mitral valve prolapse. In situ hybridization to rat brain sections confirmed that staining was chiefly confined to neuronal cell types. We have identified the genomic structure of BION1 for all 42 exons. Sequencing of 13 exons, which comprises 27% of the coding region has revealed 3 exonic, 4 intronic, and 4 5′UTR polymorphisms.  
         [0037]    An isolated and purified polynucleotide according to the present invention typically encodes human BION1, which has an amino acid sequence as shown in SEQ ID NO:1. Polymorphic variants found in a schizophrenic, Bipolar Disorder, or Panic Disorder Syndrome patient are also included within the polynucleotides contemplated. The polynucleotides can be cDNA, genomic DNA, RNA or other forms of polynucleotides. The polynucleotide can be in an expression vector or a non-expression vector, such as a YAC clone, a BAC clone, a P1 clone, and the like. Preferably a genomic clone also contains marker D135779, which has been found to be closely linked to Panic Disorder Syndrome and schizophrenia. Preferably the polynucleotide includes at least 12, 15, 18, 20, 22, 25, 30, 35, 50, 75, 100, 250, 500, or 1000 nucleotides. More preferably the entire amino acid-coding sequence shown in SEQ ID NO:2 is contained within the polynucleotide. In another embodiment the complete cDNA sequence shown in SEQ ID NO:2 is contained within the polynucleotide. If the polynucleotide is an RNA molecule one preferred embodiment is a 6.2 kb transcript.  
         [0038]    Polynucleotides according to the invention contain either the wild type sequence as shown in SEQ ID NO:2 or polymorphisms, such as those shown in Table 2 or Table 3. Other polymorphisms can be readily identified using the method shown below in the examples. Isolated and purified polynucleotides according to the present invention are separated from the sequences to which they are adjacent in the human genome. Thus such isolated polynucleotides comprise less than a full chromosome or other genomic element. Moreover, purified polynucleotides are not present in a mixture of total genomic DNA or a library comprising total genomic DNA or cDNA. Purified polynucleotides have been separated from other sequences, usually by a process such as cloning, hybridization, or amplification such that a population of polynucleotide molecules is predominantly (&gt;50%) the type of molecule which contains the polynucleotide containing BION1 sequences.  
         [0039]    Polynucleotides according to the present invention are conveniently maintained, propagated, and expressed in a vector. The vector can be any useful vector known in the art, whether bacterial or eukaryotic, viral or plasmid. Vectors can be maintained and grown in host cells which are suitable for replication of the particular vector chosen. Appropriate pairs of vectors and host cells are well known in the art. Such host cells carrying BION1 coding sequences can be used to prepare quantities of BION1 protein. Host cells can be grown under suitable growth and expression conditions. BION1 will typically be extracted from the cells, but under certain conditions and with certain genetic constructs, may be isolatable from the culture medium.  
         [0040]    Susceptibility to Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome can be determined according to one aspect of the invention by tracking a polymorphism identified in a proband through other family members. The proband is an affected individual, having either Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome. A nucleotide at a defined location in a BION1 gene of a human is determined to identify whether the test individual contains the polymorphism carried by the proband. The polymorphic nucleotide can be one of the polymorphisms indicated in Tables 2 and 3 or can be a different nucleotide in the same codon, or a different nucleotide in a different codon. The nucleotide at the defined location can be determined by any means known in the art, including but not limited to sequencing, restriction enzyme digestion, allele-specific ligation, allele-specific amplification. The nucleotide at the defined location of the human is compared to that of the affected family member (proband) having a polymorphism at the nucleotide. Such comparison can be by any means known in the art, such as by side-by-side testing, or by serial testing at two different times, with the same or a different technique. The testing and/or comparison can be mannually accomplished or can be done by a machine or computer implemented technique. The human being tested is identified as susceptible to schizophrenia or panic disorder if the determined nucleotide contains the polymorphism found in the affected family member. As is well known in the art, susceptibility does not indicate that there is a 100% probability that the individual will develop the disease symptoms. Other genetic factors and environmental factors are believed to be involved in determining disease elaboration.  
         [0041]    Another method of determining susceptibility to Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome requires knowledge of no other affected family member. Thus the tested patient may be the first in a family to be affected or may have no knowledge of prior affected members, or prior affected family members may not be available for sample donation. A nucleotide at a defined location in a BION1 gene of a human is determined. If the determined nucleotide is a polymorphism identified in Table 2 or Table 3, the human is identified as susceptible to Panic Disorder Syndrome, Bipolar Disorder, or Schizophrenia. As the collection of identified polymorphisms grows, this knowledge can be used to identify susceptibility in others. Thus the list of Table 2 or Table 3 will become more extensive as additional knowledge accumulates regarding other families and other polymorphisms. The expanded list of polymorphisms can be used, like Table 2 or Table 3 as provided herein. Thus Table 2 and Table 3 represent any set of polymorphisms in BION1 previously identified at the time of performing the method.  
         [0042]    Probes and primers for BION1 are also provided which are useful for performing diagnoses according to the invention. Polynucleotides for use as primers or probes typically comprise at least 18 contiguous nucleotides of a human BION1 coding sequence, however, primers and probes of at least 10, 12, 14, 16, 20, 22, or 25 nucleotides may be useful, as well. Polynucleotides encoding only a portion of BION1 can also be fused to other genes to form fusion genes encoding fusion proteins. Preferably the polynucleotide comprises at least one codon identified in Table 1, which codon is for an amino acid found in human BION1 proteins at the corresponding position. More preferably the polynucleotide contains no codons identified in Table 1 as being a rat BION1 codon.  
         [0043]    Human BION1 protein according to the invention is useful inter alia for screening for potential therapeutic agents. Typically such protein is isolated and purified away from whole human cells or recombinant cells, as well as away from other human proteins and possibly also other proteins from a recombinant BION1-producing cell. The BION1 proteins according to the invention typically comprise an amino acid sequence as shown in SEQ ID NO:1. Alternatively the sequence can be that of a polymorphic variant found in Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome patients. More preferably it can be the sequence of a polymorphic variant disclosed in Tables 2 or 3.  
         [0044]    BION1 polypeptides comprise at least 6 amino acids of a human BION1 protein, i.e., enought to form an epitope which can be used to generate antibodies or immune cells which are specific for BION1. The polypeptides can comprise at least 10, 15, 20, 25, 30, 50, or even 75 contiguous amino acids of the BION1 protein. Preferably the polypeptide comprises an amino acid sequence found in humans but not in rats as identified in Table 1. Among other uses, the antibodies to BION1 can be used in binding assays to test substances which may be useful therapeutically. The antibodeies, whether polyclonal or monoclonal, can be used to identify bound or unbound BION1 in an assay or as a competitor molecule for BION1 binding.  
         [0045]    Test substances can be screened for candidates useful in treating Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome by using the human BION1 protein. It is contacted with a test substance, preferably with a library of test substances serially or simultaneously in separate reaction mixtures. Binding of a test substance to the human BION1 protein is determined. The BION1 protein may be wild-type or polymorphic, including but not limited to one of the polymorphisms disclosed in Tables 2 and 3. Any binding assay known in the art can be used. In some binding assays one of the binding partners is immobilized on a solid support. In other binding assays one binding partner is labeled. In some binding assays competition with a known binder is used as an indicator of binding activity. Those of skill in the art will readily understand how to set up convenient binding assays. A test substance is identified as a candidate drug useful for treating Schizophrenia, Bipolar Disorder, or Panic Disorder Syndrome if it binds to human BION1.  
         [0046]    The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.  
       EXAMPLE 1  
       [0047]    Identification of  C. elegans  Homologs of Mammalian Voltage-Gated Ion Channels.  
         [0048]    Sequences were identified within the  C. elegans  cosmids c27f2.3 (U40419) and c11d2.6 (AF045640) in a search of the  C. elegans  genomic database for sequence fragments that resembled voltage gated sodium (NaV) and calcium (CaV) ion channels. These putative, novel genes were approximately 26% identical, 45% conserved (e-value 2e-48) to human voltage-dependent L-type Ca channel (al subunit).  
       EXAMPLE 2  
       [0049]    Cloning of Human BION1 cDNA.  
         [0050]    These  C. elegans  sequences displaying similarity to the ion channels were used to identify a homologous human GenBank EST (#590575, derived from a human pancreatic cell line cDNA library.) This was sequenced and found to contain an open reading frame (ORF) encoding part of the human homolog of the  C. elegans  genes. Further screening of a pancreatic cDNA library led to the isolation of a full-length human cDNA clone (BION1). The complete human cDNA sequence of the human BION1 gene (SEQ ID NO:2) and its conceptual translation product (SEQ ID NO:1) are shown in FIG. 1. The predicted human and rat protein sequences contain 1738 amino acids, and are highly conserved, showing only 24 cross-species amino acid substitutions (1.4%), 12 of which are conservative. The predicted BION1 protein is comprised of four domains (I-IV), each containing six putative membrane-spanning regions (S1-6) and a pore loop (P) between S5 and S6 (FIG. 2 a ). The predicted topology of the BION1 protein is shown diagrammatically in FIG. 2 b.   
                                 TABLE 1                           Amino acid substitutions between human and rat BION1 proteins.            Position of first   Amino acid   Amino acid           nucleotide in codon   in human   in rat   Conservative?               1058   Ala   Ser   Conservative       1241   Ser   Thr   Conservative       1949   Val   Leu   Conservative       2069   Val   Leu   Conservative       2261   Leu   Phe       2267   Ser   Ile       2276   Thr   Ser   Conservative       2300   His   Asn   Conservative       2303   Ser   Pro       2312   Ser   Pro       2315   Ala   Thr       2318   Ile   Val   Conservative       2909   Ile   Leu   Conservative       4687   Lys   Ser       4670   Arg   ?       5117   Asn   His   Conservative       5120   Ser   Asn   Conservative       5123   Met   Thr   Conservative       5135   Thr   Ser   Conservative       5207   Ala   Thr       5228   Phe   Ile       5297   Leu   Phe       5348   Thr   Pro       5354   Ala   Thr                  
 
       EXAMPLE 3  
       [0051]    Cloning of Rat BION1 cDNA.  
         [0052]    The human BION1 cDNA was used to screen a rat brain cDNA library in order to isolate a full-length rat BION1 channel cDNA. At the amino-acid level, this protein exhibited 98.6% identity to the existing human sequence. Recently, another group has reported cloning and sequencing the rat BION1 gene (AF078779) (Lee et al. 1999.)  
       EXAMPLE 4  
       [0053]    BION1 Protein Sequence is Divergent from Other Voltage-Gated Ion Channels.  
         [0054]    Sequence alignments of BION1 with other voltage gated ion channels indicate significant divergence. Amino-acid residues thought crucial for determining ion selectivity are in a novel arrangement. BION1 has EEKE selectivity amino acids in its pore loop region, in contrast to most mammalian Ca V  channels with EEEE at these positions, and Na V  with DEKA (FIG. 3 a ). Cladistic analysis of BION1 and its  C. elegans  homologs suggests that these comprise a distinct family of channels that diverged from Ca V  and Na V  before the duplication that gave rise to these two more closely related families (FIG. 3 b ). BION1 family members have a reduced number of positively charged amino-acid residues in the S4 regions, which are believed to act as part of the voltage-sensing apparatus in other families of voltage-dependent ion channels. This reduction in number of charges is particularly pronounced in domain IV, and is suggestive that the activation of this channel may be relatively slow compared to Ca V  and Na V . BION1 channels have a divergent domain III-IV linker, missing the amino-acid sequence IFM, which has been shown to be important in mammalian Na V  channel inactivation. This suggests that BION1 channel inactivation may be slow compared to Na V .  
       EXAMPLE 5  
       [0055]    Expression of BION1.  
         [0056]    Northern analyses were performed using the rat BION1 gene. BION1 was found to be expressed abundantly in rat brain, spinal cord, and pancreas as a ˜6.2 kb transcript (FIG. 4 a ). Significant expression is also seen in heart, kidney, small intestine and placenta. The expression in heart is of significance given the mitral valve prolapse phenotype of Panic Disorder Syndrome.  
         [0057]    In the brain, BION1 is expressed most highly in the thalamus, but is also expressed in other regions of the brain (FIG. 4 b ). This distribution has been confirmed by RT-PCR. In situ hybridization using BION1 cRNA shows primarily neuronal staining, with strong hybridization to the olfactory bulb, piriform cortex, hippocampal neuronal layers, suprachiasmatic nucleus, medial habenular nucleus, locus coeruleus, and subformical organ, and at low levels throughout the CNS (FIG. 5).  
       EXAMPLE 6  
       [0058]    Chromosomal Mapping of BION1.  
         [0059]    A BAC clone (2294N13) was shown to contain BION1 by PCR analysis and sequencing using BION1 primers. FISH analysis of the BION1 BAC clone in the Ward laboratory (Yale University) localized the human gene to human chromosomal band 13q32 (FIG. 6).  
         [0060]    A well-defined schizophrenia susceptibility locus (SCZD7) is located on 13q32 (Lin et al. 1995, Blouin et al. 1998). The genetic linkage marker with the highest LOD score was CHLC.ATA26D07 (D13S779)(Blouin et al. 1998). A second neuropsychiatric disorder, Panic Disorder, was also linked to the D13S779 marker (Weissman et al. 2000).  
         [0061]    A YAC contig covering the 13q32 region including and surrounding the D13S779 marker was developed in the Ward laboratory and 2 YACs from the contig were demonstrated to contain the BION1 gene by PCR analysis using BION1 cDNA primers. The 2 YACs (968g12 and 761a1) share a single STS marker, D13S919, which is immediately adjacent to D13S779 on the MIT genetic linkage map. D13S779 is found only in YAC 761al. Thus BION1 physically maps to the same YAC as D13S779, the genetic marker with the highest LOD score with the 13q32 schizophrenia susceptibility locus.  
       EXAMPLE 7  
       [0062]    Genomic Structure of BION1.  
         [0063]    In order to determine the genomic structure of BION1, the BION1 BAC clone was sequenced in its entirety and additional overlapping BACs were identified, both by screening various BAC libraries with the available ESTs, and by database analysis. Genomic sequence of a second BION1-containing BAC, RP11-45P5, was recently completed as part of the Human Genome Project (Birren et al., unpublished). Together, these BAC sequences have allowed us to completely define the genomic structure of BION1. The BION1 gene is composed of 42 exons (FIG. 7). The 3′ portion of this gene (corresponding to bp 1855-6942 of the BION1 cDNA) spans 175 kb of genomic DNA; sequencing of the 5′ introns is as yet incomplete. However, by analysis of sequence of the two BAC clones, and by genomic PCR and sequencing, intron-exon boundaries and intronic sequences flanking individual exons have been obtained for all 42 exons.  
       EXAMPLE 8  
       [0064]    Evidence Linking BION1 to SCZD7.  
         [0065]    Genetic co-localization of BION1 and SCZD7, together with congruent tissue distribution and predicted function of the BION1 protein, constitutes strong but indirect evidence that the BION1 gene is the 13q32 schizophrenia susceptibility gene SCZD7. In order to validate this hypothesis, mutational analysis of BION1 was undertaken in cohorts of schizophrenia and Panic Disorder Syndrome patients.  
         [0066]    Approach to Identify Mutations in BION1.  
         [0067]    Given the polygenic nature of schizophrenia susceptibility, sequencing of the BION1 exons from the genomic DNA of large numbers of unrelated patient probands (48 Panic Disorder and 48 Schizophrenic patient probands) was undertaken in an attempt to identify mutations.  
         [0068]    Panic Disorder Patient Selection.  
         [0069]    We were fortunate to have access to the same cohort that Weissman et al. (2000) used to map Panic Disorder Syndrome to 13q32. Families were initially accepted into this study if at least 3 relatives appeared affected by panic disorder. A detailed diagnosis and pedigree analysis were then performed, and the data blinded to name and family. Three senior clinical investigators independently diagnosed each individual, and any case that had a discrepant diagnosis among the physicians was flagged for additional data collection and review. Since these samples were closely linked to 13q32, it was expected that all of these patients would be defective at 13q32.  
         [0070]    Schizophrenia Patient Selection.  
         [0071]    Genomic DNA from DSM-III-R criteria (Diagnostic and Statistical Manual of Mental Disorders) schizophrenic patient probands was obtained from the NIMH Schizophrenia Genetics Initiative collection. Samples are included in this collection if several conditions are met. Firstly, at least two affected individuals must be biologically related as first-degree relatives diagnosed with DSM-III-R schizophrenia (SZ) or schizoaffective disorder depressive type (SADD). If this condition is met, a pedigree is established and extended. Two senior psychiatrists or clinical psychologists separately and independently assess data from clinical interviews and family history to arrive at a diagnosis. In the case of a disagreement, a third senior psychiatrist or clinical psychologist reviewed all available data and a written summary of the discrepant points of view. The third clinician acts as a tie-breaker. Since there are at least 9 schizophrenia loci, it was expected that about 10% of these patients would be defective at SCZD7.  
         [0072]    Method of Polymorphism ID.  
         [0073]    Polymorphisms were initially detected by sequencing the relevant exon. Sequencing in the second direction was performed routinely for all exonic regions. In addition, the redundancy of the study, sequencing 48 schizophrenia and 48 Panic Disorder Syndrome samples, assures that the same polymorphism is detected in multiple samples. We did not systematically include control DNA, reasoning that most exons would be normal at most positions, and that a mutation at on position would be evident on comparison to the BION1 cDNA sequence.  
         [0074]    Exons Scanned for Polymorphisms in Panic Disorder Syndrome Samples.  
         [0075]    A total of 13 exons have been scanned, as well as the 3′ UTR, for 48 Panic Disorder Syndrome samples. This represents 1405 bp out of the 5231 bp coding sequence (27%) and 3050 bp out of the 6942 bp cDNA (44%). We found 2 exonic, 3 intronic, and 4 3′UTR polymorphisms (Table 2) (see FIG. 8).  
                                                                             TABLE 2                           Polymorphisms found in Panic Disorder Syndrome gDNA samples                Allele Frequency                Exon   Polymorphism ID   A/A*   A/B*   B/B*                            C3   T(−251)A†   2   14   12               T(−221)C†   12   14   2           C13   G(−33)A†   10   21   9           C10   C1822T   24   13   11           C17   A2065G   39   4   0           3′UTR   G6355A   3   4   4               T6370C   11   11   21               C6442T   24   10   10               G6453A   43   0   1                                              
 
         [0076]    Exons Scanned for Polymorphisms in Schizophrenia Samples.  
         [0077]    The same 13 exons were scanned with the 48 Schizophrenia patient DNAs. This represents 1405 bp out of the 5231 bp coding sequence (27%). We found the same 2 exonic polymorphisms seen in the Panic Disorder samples, plus one novel polymorphism not seen in the previous scan (Table 3) (see FIG. 8).  
                                                                     TABLE 3                           Polymorphisms found in NIMH Schizophrenic gDNA samples                Allele Frequency            Exon   Polymorphism ID   A/A*   A/B*   B/B*                    C13   C1102T   32   1   0       C10   C1822T   28   18   1       C17   A2065G   34   3   0                  
 
         [0078]    Polymorphisms Identified.  
         [0079]    A2065G was found to be heterozygous in 3 schizophrenia samples (Repository IDs 90C00517, 90C03310, 90C03592) out of 37 (8%). This polymorphism was present in 4/43 (9.5%) of Panic Disorder Syndrome samples, so this polymorphism is consistently rare in both disorders. Interestingly, this consistency was not true when considering C1822T. In schizophrenia samples, this polymorphic nucleotide is T in only 1 sample (Repository ID 90C00518) out of 47 (2%). When the same polymorphism is examined in Panic Disorder Syndrome patients, {fraction (11/48)} (23%) show a T at this position. The C1822T and A2065G polymorphisms were confirmed by sequencing the relevant exon in both directions. The intronic and 5′UTR polymorphisms were sequenced in Panic Disorder Syndrome samples, and not schizophrenia samples. C1102T was only seen in 1 patient with schizophrenia (Repository ID 90C02418), and no Panic Disorder Syndrome samples. Although this difference was seen in a rerun of the same sample, sequencing in the second direction was not done, so the identification of this polymorphism is tentative.  
         [0080]    Confirmation of Polymorphisms.  
         [0081]    In cases where the polymorphism was found to have generated or destroyed a restriction endonuclease cleavage site, then the existence of the polymorphism could be confirmed by performing a restriction digest on the relevant PCR product. This was the case for C1822T, where the polymorphic nucleotide led to the destruction of a TaqI site. TaqI digestion and agarose gel analysis confirmed the prediction that sequence analysis allowed us to make.  
       REFERENCES  
       [0082]    Andreasen, N. C. (1995) Symptoms, signs, and diagnosis of schizophrenia. Lancet 346:477-481.  
         [0083]    Blouin, J-L et al. (1998) Schizophrenia susceptibility loci on chromosomes 13q32 and 8p21. Nature Genetics 20:70-73.  
         [0084]    Brzustowicz, L. M. et al. (2000) Location of a Major Susceptibility Locus for Familial Schizophrenia on Chromosome 1q21-q22. Science 288:678-682  
         [0085]    Knowles et al. (1998) Results of a genome-wide screen for panic disorder. Am J Med Genet (Neuropsychiatr Genet) 81:139-147.  
         [0086]    Lee, J-H et al. (1999) Cloning of a novel four repeat protein related to voltage-gated sodium and calcium channels. FEBS Letters 445:231-236.  
         [0087]    Millar, J. K. et al. (2000) Disruption of two novel genes by a translocation co-segregating with schizophrenia. Hum Mol Genet. 9:1415-1423.  
         [0088]    Weissman, M. S et al. (2000) Potential Panic Disorder Syndrome: Clinical and Genetic Linkage Evidence. American Journal of Medical Genetics (Neuropsychiatric Genetics) 96:24-35  
     
       
       
         1 
         
           
             3  
           
           
             1  
             1738  
             PRT  
             Homo sapiens  
           
            1 

Met Leu Lys Arg Lys Gln Ser Ser Arg Val Glu Ala Gln Pro Val Thr 
 1               5                  10                  15 

Asp Phe Gly Pro Asp Glu Ser Leu Ser Asp Asn Ala Asp Ile Leu Trp 
            20                  25                  30 

Ile Asn Lys Pro Trp Val His Ser Leu Leu Arg Ile Cys Ala Ile Ile 
        35                  40                  45 

Ser Val Ile Ser Val Cys Met Asn Thr Pro Met Thr Phe Glu His Tyr 
    50                  55                  60 

Pro Pro Leu Gln Tyr Val Thr Phe Thr Leu Asp Thr Leu Leu Met Phe 
65                  70                  75                  80 

Leu Tyr Thr Ala Glu Met Ile Ala Lys Met His Ile Arg Gly Ile Val 
                85                  90                  95 

Lys Gly Asp Ser Ser Tyr Val Lys Asp Arg Trp Cys Val Phe Asp Gly 
            100                 105                 110 

Phe Met Val Phe Cys Leu Trp Val Ser Leu Val Leu Gln Val Phe Glu 
        115                 120                 125 

Ile Ala Asp Ile Val Asp Gln Met Ser Pro Trp Gly Met Leu Arg Ile 
    130                 135                 140 

Pro Arg Pro Leu Ile Met Ile Arg Ala Phe Arg Ile Tyr Phe Arg Phe 
145                 150                 155                 160 

Glu Leu Pro Arg Thr Arg Ile Thr Asn Ile Leu Lys Arg Ser Gly Glu 
                165                 170                 175 

Gln Ile Trp Ser Val Ser Ile Phe Leu Leu Phe Phe Leu Leu Leu Tyr 
            180                 185                 190 

Gly Ile Leu Gly Val Gln Met Phe Gly Thr Phe Thr Tyr His Cys Val 
        195                 200                 205 

Val Asn Asp Thr Lys Pro Gly Asn Val Thr Trp Asn Ser Leu Ala Ile 
    210                 215                 220 

Pro Asp Thr His Cys Ser Pro Glu Leu Glu Glu Gly Tyr Gln Cys Pro 
225                 230                 235                 240 

Pro Gly Phe Lys Cys Met Asp Leu Glu Asp Leu Gly Leu Ser Arg Gln 
                245                 250                 255 

Glu Leu Gly Tyr Ser Gly Phe Asn Glu Ile Gly Thr Ser Ile Phe Thr 
            260                 265                 270 

Val Tyr Glu Ala Ala Ser Gln Glu Gly Trp Val Phe Leu Met Tyr Arg 
        275                 280                 285 

Ala Ile Asp Ser Phe Pro Arg Trp Arg Ser Tyr Phe Tyr Phe Ile Thr 
    290                 295                 300 

Leu Ile Phe Phe Leu Ala Trp Leu Val Lys Asn Val Phe Ile Ala Val 
305                 310                 315                 320 

Ile Ile Glu Thr Phe Ala Glu Ile Arg Val Gln Phe Gln Gln Met Trp 
                325                 330                 335 

Gly Ser Arg Ser Ser Thr Thr Ser Thr Ala Thr Thr Gln Met Phe His 
            340                 345                 350 

Glu Asp Ala Ala Gly Gly Trp Gln Leu Val Ala Val Asp Val Asn Lys 
        355                 360                 365 

Pro Gln Gly Arg Ala Pro Ala Cys Leu Gln Lys Met Met Arg Ser Ser 
    370                 375                 380 

Val Phe His Met Phe Ile Leu Ser Met Val Thr Val Asp Val Ile Val 
385                 390                 395                 400 

Ala Ala Ser Asn Tyr Tyr Lys Gly Glu Asn Phe Arg Arg Gln Tyr Asp 
                405                 410                 415 

Glu Phe Tyr Leu Ala Glu Val Ala Phe Thr Val Leu Phe Asp Leu Glu 
            420                 425                 430 

Ala Leu Leu Lys Ile Trp Cys Leu Gly Phe Thr Gly Tyr Ile Ser Ser 
        435                 440                 445 

Ser Leu His Lys Phe Glu Leu Leu Leu Val Ile Gly Thr Thr Leu His 
    450                 455                 460 

Val Tyr Pro Asp Leu Tyr His Ser Gln Phe Thr Tyr Phe Gln Val Leu 
465                 470                 475                 480 

Arg Val Val Arg Leu Ile Lys Ile Ser Pro Ala Leu Glu Asp Phe Val 
                485                 490                 495 

Tyr Lys Ile Phe Gly Pro Gly Lys Lys Leu Gly Ser Leu Val Val Phe 
            500                 505                 510 

Thr Ala Ser Leu Leu Ile Val Met Ser Ala Ile Ser Leu Gln Met Phe 
        515                 520                 525 

Cys Phe Val Glu Glu Leu Asp Arg Phe Thr Thr Phe Pro Arg Ala Phe 
    530                 535                 540 

Met Ser Met Phe Gln Ile Leu Thr Gln Glu Gly Trp Val Asp Val Met 
545                 550                 555                 560 

Asp Gln Thr Leu Asn Ala Val Gly His Met Trp Ala Pro Val Val Ala 
                565                 570                 575 

Ile Tyr Phe Ile Leu Tyr His Leu Phe Ala Thr Leu Ile Leu Leu Ser 
            580                 585                 590 

Leu Phe Val Ala Val Ile Leu Asp Asn Leu Glu Leu Asp Glu Asp Leu 
        595                 600                 605 

Lys Lys Leu Lys Gln Leu Lys Gln Ser Glu Ala Asn Ala Asp Thr Lys 
    610                 615                 620 

Glu Lys Leu Pro Leu Arg Leu Arg Ile Phe Glu Lys Phe Pro Asn Arg 
625                 630                 635                 640 

Pro Gln Met Val Lys Ile Ser Lys Leu Pro Ser Asp Phe Thr Val Pro 
                645                 650                 655 

Lys Ile Arg Glu Ser Phe Met Lys Gln Phe Ile Asp Arg Gln Gln Gln 
            660                 665                 670 

Asp Thr Cys Cys Leu Leu Arg Ser Leu Pro Thr Thr Ser Ser Ser Ser 
        675                 680                 685 

Cys Asp His Ser Lys Arg Ser Ala Ile Glu Asp Asn Lys Tyr Ile Asp 
    690                 695                 700 

Gln Lys Leu Arg Lys Ser Val Phe Ser Ile Arg Ala Arg Asn Leu Leu 
705                 710                 715                 720 

Glu Lys Glu Thr Ala Val Thr Lys Ile Leu Arg Ala Cys Thr Arg Gln 
                725                 730                 735 

Arg Met Leu Ser Gly Ser Phe Glu Gly Gln Pro Ala Lys Glu Arg Ser 
            740                 745                 750 

Ile Leu Ser Val Gln His His Ile Arg Gln Glu Arg Arg Ser Leu Arg 
        755                 760                 765 

His Gly Ser Asn Ser Gln Arg Ile Ser Arg Gly Lys Ser Leu Glu Thr 
    770                 775                 780 

Leu Thr Gln Asp His Ser Asn Thr Val Arg Tyr Arg Asn Ala Gln Arg 
785                 790                 795                 800 

Glu Asp Ser Glu Ile Lys Met Ile Gln Glu Lys Lys Glu Gln Ala Glu 
                805                 810                 815 

Met Lys Arg Lys Val Gln Glu Glu Glu Leu Arg Glu Asn His Pro Tyr 
            820                 825                 830 

Phe Asp Lys Pro Leu Phe Ile Val Gly Arg Glu His Arg Phe Arg Asn 
        835                 840                 845 

Phe Cys Arg Val Val Val Arg Ala Arg Phe Asn Ala Ser Lys Thr Asp 
    850                 855                 860 

Pro Val Thr Gly Ala Val Lys Asn Thr Lys Tyr His Gln Leu Tyr Asp 
865                 870                 875                 880 

Leu Leu Gly Leu Val Thr Tyr Leu Asp Trp Val Met Ile Ile Val Thr 
                885                 890                 895 

Ile Cys Ser Cys Ile Ser Met Met Phe Glu Ser Pro Phe Arg Arg Val 
            900                 905                 910 

Met His Ala Pro Thr Leu Gln Ile Ala Glu Tyr Val Phe Val Ile Phe 
        915                 920                 925 

Met Ser Ile Glu Leu Asn Leu Lys Ile Met Ala Asp Gly Leu Phe Phe 
    930                 935                 940 

Thr Pro Thr Ala Val Ile Arg Asp Phe Gly Gly Val Met Asp Ile Phe 
945                 950                 955                 960 

Ile Tyr Leu Val Ser Leu Ile Phe Leu Cys Trp Met Pro Gln Asn Val 
                965                 970                 975 

Pro Ala Glu Ser Gly Ala Gln Leu Leu Met Val Leu Arg Cys Leu Arg 
            980                 985                 990 

Pro Leu Arg Ile Phe Lys Leu Val Pro Gln Met Arg Lys Val Val Arg 
        995                 1000                1005 

Glu Leu Phe Ser Gly Phe Lys Glu Ile Phe Leu Val Ser Ile Leu Leu 
    1010                1015                1020 

Leu Thr Leu Met Leu Val Phe Ala Ser Phe Gly Val Gln Leu Phe Ala 
1025                1030                1035                1040 

Gly Lys Leu Ala Lys Cys Asn Asp Pro Asn Ile Ile Arg Arg Glu Asp 
                1045                1050                1055 

Cys Asn Gly Ile Phe Arg Ile Asn Val Ser Val Ser Lys Asn Leu Asn 
            1060                1065                1070 

Leu Lys Leu Arg Pro Gly Glu Lys Lys Pro Gly Phe Trp Val Pro Arg 
        1075                1080                1085 

Val Trp Ala Asn Pro Arg Asn Phe Asn Phe Asp Asn Val Gly Asn Ala 
    1090                1095                1100 

Met Leu Ala Leu Phe Glu Val Leu Ser Leu Lys Gly Trp Val Glu Val 
1105                1110                1115                1120 

Arg Asp Val Ile Ile His Arg Val Gly Pro Ile His Gly Ile Tyr Ile 
                1125                1130                1135 

His Val Phe Val Phe Leu Gly Cys Met Ile Gly Leu Thr Leu Phe Val 
            1140                1145                1150 

Gly Val Val Ile Ala Asn Phe Asn Glu Asn Lys Gly Thr Ala Leu Leu 
        1155                1160                1165 

Thr Val Asp Gln Arg Arg Trp Glu Asp Leu Lys Ser Arg Leu Lys Ile 
    1170                1175                1180 

Ala Gln Pro Leu His Leu Pro Pro Arg Pro Asp Asn Asp Gly Phe Arg 
1185                1190                1195                1200 

Ala Lys Met Tyr Asp Ile Thr Gln His Pro Phe Phe Lys Arg Thr Ile 
                1205                1210                1215 

Ala Leu Leu Val Leu Ala Gln Ser Val Leu Leu Ser Val Lys Trp Asp 
            1220                1225                1230 

Val Glu Asp Pro Val Thr Val Pro Leu Ala Thr Met Ser Val Val Phe 
        1235                1240                1245 

Thr Phe Ile Phe Val Leu Glu Val Thr Met Lys Ile Ile Ala Met Ser 
    1250                1255                1260 

Pro Ala Gly Phe Trp Gln Ser Arg Arg Asn Arg Tyr Asp Leu Leu Val 
1265                1270                1275                1280 

Thr Ser Leu Gly Val Val Trp Val Val Leu His Phe Ala Leu Leu Asn 
                1285                1290                1295 

Ala Tyr Thr Tyr Met Met Gly Ala Cys Val Ile Val Phe Arg Phe Phe 
            1300                1305                1310 

Ser Ile Cys Gly Lys His Val Thr Leu Lys Met Leu Leu Leu Thr Val 
        1315                1320                1325 

Val Val Ser Met Tyr Lys Ser Phe Phe Ile Ile Val Gly Met Phe Leu 
    1330                1335                1340 

Leu Leu Leu Cys Tyr Ala Phe Ala Gly Val Val Leu Phe Gly Thr Val 
1345                1350                1355                1360 

Lys Tyr Gly Glu Asn Ile Asn Arg His Ala Asn Phe Ser Ser Ala Gly 
                1365                1370                1375 

Lys Ala Ile Thr Val Leu Phe Arg Ile Val Thr Gly Glu Asp Trp Asn 
            1380                1385                1390 

Lys Ile Met His Asp Cys Met Val Gln Pro Pro Phe Cys Thr Pro Asp 
        1395                1400                1405 

Glu Phe Thr Tyr Trp Ala Thr Asp Cys Gly Asn Tyr Ala Gly Ala Leu 
    1410                1415                1420 

Met Tyr Phe Cys Ser Phe Tyr Val Ile Ile Ala Tyr Ile Met Leu Asn 
1425                1430                1435                1440 

Leu Leu Val Ala Ile Ile Val Glu Asn Phe Ser Leu Phe Tyr Ser Thr 
                1445                1450                1455 

Glu Glu Asp Gln Leu Leu Ser Tyr Asn Asp Leu Arg His Phe Gln Ile 
            1460                1465                1470 

Ile Trp Asn Met Val Asp Asp Lys Arg Glu Gly Val Ile Pro Thr Phe 
        1475                1480                1485 

Arg Val Lys Phe Leu Leu Arg Leu Leu Arg Gly Arg Leu Glu Val Asp 
    1490                1495                1500 

Leu Asp Lys Asp Lys Leu Leu Phe Lys His Met Cys Tyr Glu Met Glu 
1505                1510                1515                1520 

Arg Leu His Asn Gly Gly Asp Val Thr Phe His Asp Val Leu Ser Met 
                1525                1530                1535 

Leu Ser Tyr Arg Ser Val Asp Ile Arg Lys Ser Leu Gln Leu Glu Glu 
            1540                1545                1550 

Leu Leu Ala Arg Glu Gln Leu Glu Tyr Thr Ile Glu Glu Glu Val Ala 
        1555                1560                1565 

Lys Gln Thr Ile Arg Met Trp Leu Lys Lys Cys Leu Lys Arg Ile Arg 
    1570                1575                1580 

Ala Lys Gln Gln Gln Ser Cys Ser Ile Ile His Ser Leu Arg Glu Ser 
1585                1590                1595                1600 

Gln Gln Gln Glu Leu Ser Arg Phe Leu Asn Pro Pro Ser Ile Glu Thr 
                1605                1610                1615 

Thr Gln Pro Ser Glu Asp Thr Asn Ala Asn Ser Gln Asp Asn Ser Met 
            1620                1625                1630 

Gln Pro Glu Thr Ser Ser Gln Gln Gln Leu Leu Ser Pro Thr Leu Ser 
        1635                1640                1645 

Asp Arg Gly Gly Ser Arg Gln Asp Ala Ala Asp Ala Gly Lys Pro Gln 
    1650                1655                1660 

Arg Lys Phe Gly Gln Trp Arg Leu Pro Ser Ala Pro Lys Pro Ile Ser 
1665                1670                1675                1680 

His Ser Val Ser Ser Val Asn Leu Arg Leu Gly Gly Arg Thr Thr Met 
                1685                1690                1695 

Lys Ser Val Val Cys Lys Met Asn Pro Met Thr Asp Ala Ala Ser Cys 
            1700                1705                1710 

Gly Ser Glu Val Lys Lys Trp Trp Thr Arg Gln Leu Thr Val Glu Ser 
        1715                1720                1725 

Asp Glu Ser Gly Asp Asp Leu Leu Asp Ile 
    1730                1735 

 
           
             2  
             6952  
             DNA  
             Homo sapiens  
             
               CDS  
               (230)...(5443)  
             
           
            2 

ggcacgagcg agcgtgagcc gcggccccag ccgggccgag cgcgctgcct gagctgagcc     60 

gccgtaggtg aggggcccgc gtccccgccc gccctgggcg ccgcgcctgg cactgatcct    120 

gccggtcgcc cactgtcgcc gccgccgccg cccgcgggca ccatgacagc tctgagcgct    180 

ggggttacag actgtggttt tgtgcttgct caccaaagct aacctcagc atg ctc aaa    238 
                                                      Met Leu Lys 
                                                       1 

agg aag cag agt tcc agg gtg gaa gcc cag cca gtc act gac ttt ggt      286 
Arg Lys Gln Ser Ser Arg Val Glu Ala Gln Pro Val Thr Asp Phe Gly 
     5                   10                  15 

cct gat gag tct ctg tcg gat aat gct gac atc ctc tgg att aac aaa      334 
Pro Asp Glu Ser Leu Ser Asp Asn Ala Asp Ile Leu Trp Ile Asn Lys 
 20                  25                  30                  35 

cca tgg gtt cac tct ttg ctg cgc atc tgt gcc atc atc agc gtc att      382 
Pro Trp Val His Ser Leu Leu Arg Ile Cys Ala Ile Ile Ser Val Ile 
                 40                  45                  50 

tct gtt tgt atg aat acg cca atg acc ttc gag cac tat cct cca ctt      430 
Ser Val Cys Met Asn Thr Pro Met Thr Phe Glu His Tyr Pro Pro Leu 
             55                  60                  65 

cag tat gtg acc ttc act ttg gat aca tta ttg atg ttt ctc tac acg      478 
Gln Tyr Val Thr Phe Thr Leu Asp Thr Leu Leu Met Phe Leu Tyr Thr 
         70                  75                  80 

gca gag atg ata gca aaa atg cac atc cgg ggc att gtc aag ggg gat      526 
Ala Glu Met Ile Ala Lys Met His Ile Arg Gly Ile Val Lys Gly Asp 
     85                  90                  95 

agt tcc tat gtg aaa gat cgc tgg tgt gtt ttt gat gga ttt atg gtc      574 
Ser Ser Tyr Val Lys Asp Arg Trp Cys Val Phe Asp Gly Phe Met Val 
100                 105                 110                 115 

ttt tgc ctt tgg gtt tct ttg gtg cta cag gtg ttt gaa att gct gat      622 
Phe Cys Leu Trp Val Ser Leu Val Leu Gln Val Phe Glu Ile Ala Asp 
                120                 125                 130 

ata gtt gat cag atg tca cct tgg ggc atg ttg cgg att cca cgg cca      670 
Ile Val Asp Gln Met Ser Pro Trp Gly Met Leu Arg Ile Pro Arg Pro 
            135                 140                 145 

ctg att atg atc cga gca ttc cgg att tat ttc cga ttt gaa ctg cca      718 
Leu Ile Met Ile Arg Ala Phe Arg Ile Tyr Phe Arg Phe Glu Leu Pro 
        150                 155                 160 

agg acc aga att aca aat att tta aag cga tcg gga gaa caa ata tgg      766 
Arg Thr Arg Ile Thr Asn Ile Leu Lys Arg Ser Gly Glu Gln Ile Trp 
    165                 170                 175 

agt gtt tcc att ttt cta ctt ttc ttt cta ctt ctt tat gga att tta      814 
Ser Val Ser Ile Phe Leu Leu Phe Phe Leu Leu Leu Tyr Gly Ile Leu 
180                 185                 190                 195 

gga gtt cag atg ttt gga aca ttt act tat cac tgt gtt gta aat gac      862 
Gly Val Gln Met Phe Gly Thr Phe Thr Tyr His Cys Val Val Asn Asp 
                200                 205                 210 

aca aag cca ggg aat gta acc tgg aat agt tta gct att cca gac aca      910 
Thr Lys Pro Gly Asn Val Thr Trp Asn Ser Leu Ala Ile Pro Asp Thr 
            215                 220                 225 

cac tgc tca cca gag cta gaa gaa ggc tac cag tgc cca cct gga ttt      958 
His Cys Ser Pro Glu Leu Glu Glu Gly Tyr Gln Cys Pro Pro Gly Phe 
        230                 235                 240 

aaa tgc atg gac ctt gaa gat ctg gga ctt agc agg caa gag ctg ggc     1006 
Lys Cys Met Asp Leu Glu Asp Leu Gly Leu Ser Arg Gln Glu Leu Gly 
    245                 250                 255 

tac agt ggc ttt aat gag ata gga act agt ata ttc acc gtc tat gag     1054 
Tyr Ser Gly Phe Asn Glu Ile Gly Thr Ser Ile Phe Thr Val Tyr Glu 
260                 265                 270                 275 

gcc gcc tca cag gaa ggc tgg gtg ttc ctc atg tac aga gca att gac     1102 
Ala Ala Ser Gln Glu Gly Trp Val Phe Leu Met Tyr Arg Ala Ile Asp 
                280                 285                 290 

agc ttt ccc cgt tgg cgt tcc tac ttc tat ttc atc act ctc att ttc     1150 
Ser Phe Pro Arg Trp Arg Ser Tyr Phe Tyr Phe Ile Thr Leu Ile Phe 
            295                 300                 305 

ttc ctc gcc tgg ctt gtg aag aac gtg ttt att gct gtt atc att gaa     1198 
Phe Leu Ala Trp Leu Val Lys Asn Val Phe Ile Ala Val Ile Ile Glu 
        310                 315                 320 

aca ttt gca gaa atc aga gta cag ttt caa caa atg tgg gga tcg aga     1246 
Thr Phe Ala Glu Ile Arg Val Gln Phe Gln Gln Met Trp Gly Ser Arg 
    325                 330                 335 

agc agc act act tca aca gcc acc acc cag atg ttt cat gaa gat gct     1294 
Ser Ser Thr Thr Ser Thr Ala Thr Thr Gln Met Phe His Glu Asp Ala 
340                 345                 350                 355 

gct gga ggt tgg cag ctg gta gct gtg gat gtc aac aag ccc cag gga     1342 
Ala Gly Gly Trp Gln Leu Val Ala Val Asp Val Asn Lys Pro Gln Gly 
                360                 365                 370 

cgc gcc cca gcc tgc ctc cag aaa atg atg cgg tca tcc gtt ttc cac     1390 
Arg Ala Pro Ala Cys Leu Gln Lys Met Met Arg Ser Ser Val Phe His 
            375                 380                 385 

atg ttc atc ctg agc atg gtg acc gtg gac gtg atc gtg gcg gct agc     1438 
Met Phe Ile Leu Ser Met Val Thr Val Asp Val Ile Val Ala Ala Ser 
        390                 395                 400 

aac tac tac aaa gga gaa aac ttc agg agg cag tac gac gag ttc tac     1486 
Asn Tyr Tyr Lys Gly Glu Asn Phe Arg Arg Gln Tyr Asp Glu Phe Tyr 
    405                 410                 415 

ctg gcg gag gtg gct ttt aca gta ctt ttt gat ttg gaa gca ctt ctg     1534 
Leu Ala Glu Val Ala Phe Thr Val Leu Phe Asp Leu Glu Ala Leu Leu 
420                 425                 430                 435 

aag ata tgg tgt ttg gga ttt act gga tat att agc tca tct ctc cac     1582 
Lys Ile Trp Cys Leu Gly Phe Thr Gly Tyr Ile Ser Ser Ser Leu His 
                440                 445                 450 

aaa ttc gaa cta cta ctc gta att gga act act ctt cat gta tac cca     1630 
Lys Phe Glu Leu Leu Leu Val Ile Gly Thr Thr Leu His Val Tyr Pro 
            455                 460                 465 

gat ctt tat cat tca caa ttc acg tac ttt cag gtt ctc cga gta gtt     1678 
Asp Leu Tyr His Ser Gln Phe Thr Tyr Phe Gln Val Leu Arg Val Val 
        470                 475                 480 

cgg ctg att aag att tca cct gca tta gaa gac ttt gtg tac aag ata     1726 
Arg Leu Ile Lys Ile Ser Pro Ala Leu Glu Asp Phe Val Tyr Lys Ile 
    485                 490                 495 

ttt ggt cct gga aaa aag ctt ggg agt ttg gtt gta ttt act gcc agc     1774 
Phe Gly Pro Gly Lys Lys Leu Gly Ser Leu Val Val Phe Thr Ala Ser 
500                 505                 510                 515 

ctc ttg att gtt atg tca gca att agt ttg cag atg ttc tgc ttt gtc     1822 
Leu Leu Ile Val Met Ser Ala Ile Ser Leu Gln Met Phe Cys Phe Val 
                520                 525                 530 

gaa gaa ctg gac aga ttt act acg ttt ccg agg gca ttt atg tcc atg     1870 
Glu Glu Leu Asp Arg Phe Thr Thr Phe Pro Arg Ala Phe Met Ser Met 
            535                 540                 545 

ttc cag atc ctc acc cag gaa gga tgg gtg gac gta atg gac caa act     1918 
Phe Gln Ile Leu Thr Gln Glu Gly Trp Val Asp Val Met Asp Gln Thr 
        550                 555                 560 

cta aat gct gtg gga cat atg tgg gca ccc gtg gtt gcc atc tat ttc     1966 
Leu Asn Ala Val Gly His Met Trp Ala Pro Val Val Ala Ile Tyr Phe 
    565                 570                 575 

att ctc tat cat ctt ttt gcc act ctg atc ctc ctg agt ttg ttt gtt     2014 
Ile Leu Tyr His Leu Phe Ala Thr Leu Ile Leu Leu Ser Leu Phe Val 
580                 585                 590                 595 

gct gtt att ttg gac aac tta gaa ctt gat gaa gac cta aag aag ctt     2062 
Ala Val Ile Leu Asp Asn Leu Glu Leu Asp Glu Asp Leu Lys Lys Leu 
                600                 605                 610 

aaa caa tta aag caa agt gaa gca aat gcg gac acc aaa gaa aag ctc     2110 
Lys Gln Leu Lys Gln Ser Glu Ala Asn Ala Asp Thr Lys Glu Lys Leu 
            615                 620                 625 

cct tta cgc ctg cga atc ttt gaa aaa ttt cca aac aga cct caa atg     2158 
Pro Leu Arg Leu Arg Ile Phe Glu Lys Phe Pro Asn Arg Pro Gln Met 
        630                 635                 640 

gtg aaa atc tca aag ctt cct tca gat ttt aca gtt cct aaa atc agg     2206 
Val Lys Ile Ser Lys Leu Pro Ser Asp Phe Thr Val Pro Lys Ile Arg 
    645                 650                 655 

gag agt ttt atg aag cag ttt att gac cgc cag caa cag gac aca tgt     2254 
Glu Ser Phe Met Lys Gln Phe Ile Asp Arg Gln Gln Gln Asp Thr Cys 
660                 665                 670                 675 

tgc ctc ctg aga agc ctc ccg acc acc tct tcc tcc tcc tgc gac cac     2302 
Cys Leu Leu Arg Ser Leu Pro Thr Thr Ser Ser Ser Ser Cys Asp His 
                680                 685                 690 

tcc aaa cgc tca gca att gag gac aac aaa tac atc gac caa aaa ctt     2350 
Ser Lys Arg Ser Ala Ile Glu Asp Asn Lys Tyr Ile Asp Gln Lys Leu 
            695                 700                 705 

cgc aag tct gtt ttc agc atc agg gca agg aac ctt ctg gaa aag gag     2398 
Arg Lys Ser Val Phe Ser Ile Arg Ala Arg Asn Leu Leu Glu Lys Glu 
        710                 715                 720 

acc gca gtc act aaa atc tta aga gct tgc acc cga cag cgc atg ctg     2446 
Thr Ala Val Thr Lys Ile Leu Arg Ala Cys Thr Arg Gln Arg Met Leu 
    725                 730                 735 

agc gga tca ttt gag ggg cag ccc gca aag gag agg tca atc ctc agc     2494 
Ser Gly Ser Phe Glu Gly Gln Pro Ala Lys Glu Arg Ser Ile Leu Ser 
740                 745                 750                 755 

gtg cag cat cat atc cgc caa gag cgc agg tca cta aga cat gga tca     2542 
Val Gln His His Ile Arg Gln Glu Arg Arg Ser Leu Arg His Gly Ser 
                760                 765                 770 

aac agc cag agg atc agc agg gga aaa tct ctt gaa act ttg act caa     2590 
Asn Ser Gln Arg Ile Ser Arg Gly Lys Ser Leu Glu Thr Leu Thr Gln 
            775                 780                 785 

gat cat tcc aat aca gtg aga tat aga aat gca caa aga gaa gac agt     2638 
Asp His Ser Asn Thr Val Arg Tyr Arg Asn Ala Gln Arg Glu Asp Ser 
        790                 795                 800 

gaa ata aag atg att cag gaa aaa aag gag caa gca gag atg aaa agg     2686 
Glu Ile Lys Met Ile Gln Glu Lys Lys Glu Gln Ala Glu Met Lys Arg 
    805                 810                 815 

aaa gtg caa gaa gag gaa ctc aga gag aac cac cca tac ttc gat aag     2734 
Lys Val Gln Glu Glu Glu Leu Arg Glu Asn His Pro Tyr Phe Asp Lys 
820                 825                 830                 835 

cca ctg ttc att gtc ggg cga gaa cac agg ttc aga aac ttt tgc cgg     2782 
Pro Leu Phe Ile Val Gly Arg Glu His Arg Phe Arg Asn Phe Cys Arg 
                840                 845                 850 

gtg gtg gtc cga gca cgc ttc aac gca tct aaa aca gac cct gtc aca     2830 
Val Val Val Arg Ala Arg Phe Asn Ala Ser Lys Thr Asp Pro Val Thr 
            855                 860                 865 

gga gct gtg aaa aat aca aag tac cat caa ctt tat gat ttg ctg gga     2878 
Gly Ala Val Lys Asn Thr Lys Tyr His Gln Leu Tyr Asp Leu Leu Gly 
        870                 875                 880 

ttg gtc act tac ctg gac tgg gtc atg atc atc gta acc atc tgc tct     2926 
Leu Val Thr Tyr Leu Asp Trp Val Met Ile Ile Val Thr Ile Cys Ser 
    885                 890                 895 

tgc att tcc atg atg ttt gag tcc ccg ttt cga aga gtc atg cat gca     2974 
Cys Ile Ser Met Met Phe Glu Ser Pro Phe Arg Arg Val Met His Ala 
900                 905                 910                 915 

cct act ttg cag att gct gag tat gtg ttt gtg ata ttc atg agc att     3022 
Pro Thr Leu Gln Ile Ala Glu Tyr Val Phe Val Ile Phe Met Ser Ile 
                920                 925                 930 

gag ctt aat ctg aag att atg gca gat ggc tta ttt ttc act cca act     3070 
Glu Leu Asn Leu Lys Ile Met Ala Asp Gly Leu Phe Phe Thr Pro Thr 
            935                 940                 945 

gct gtc atc agg gac ttc ggt gga gta atg gac ata ttt ata tat ctt     3118 
Ala Val Ile Arg Asp Phe Gly Gly Val Met Asp Ile Phe Ile Tyr Leu 
        950                 955                 960 

gtg agc ttg ata ttt ctt tgt tgg atg cct caa aat gta cct gct gaa     3166 
Val Ser Leu Ile Phe Leu Cys Trp Met Pro Gln Asn Val Pro Ala Glu 
    965                 970                 975 

tcg gga gct cag ctt cta atg gtc ctt cgg tgc ctg aga cct ctg cgc     3214 
Ser Gly Ala Gln Leu Leu Met Val Leu Arg Cys Leu Arg Pro Leu Arg 
 980                 985                 990                 995 

ata ttc aaa ctg gtg ccc cag atg agg aaa gtt gtt cga gaa ctt ttc     3262 
Ile Phe Lys Leu Val Pro Gln Met Arg Lys Val Val Arg Glu Leu Phe 
                1000                1005                1010 

agc ggc ttc aag gaa att ttt ttg gtc tcc att ctt ttg ctg aca tta     3310 
Ser Gly Phe Lys Glu Ile Phe Leu Val Ser Ile Leu Leu Leu Thr Leu 
            1015                1020                1025 

atg ctc gtt ttt gca agc ttt gga gtt cag ctt ttt gct gga aaa ctg     3358 
Met Leu Val Phe Ala Ser Phe Gly Val Gln Leu Phe Ala Gly Lys Leu 
        1030                1035                1040 

gcc aag tgc aat gat ccc aac att att aga agg gaa gat tgc aat ggc     3406 
Ala Lys Cys Asn Asp Pro Asn Ile Ile Arg Arg Glu Asp Cys Asn Gly 
    1045                1050                1055 

ata ttc aga att aat gtc agt gtg tca aag aac tta aat tta aaa ttg     3454 
Ile Phe Arg Ile Asn Val Ser Val Ser Lys Asn Leu Asn Leu Lys Leu 
1060                1065                1070                1075 

agg cct gga gag aaa aaa cct gga ttt tgg gtg ccc cgt gtt tgg gcg     3502 
Arg Pro Gly Glu Lys Lys Pro Gly Phe Trp Val Pro Arg Val Trp Ala 
                1080                1085                1090 

aat cct cgg aac ttt aat ttc gac aat gtg gga aac gct atg ctg gcg     3550 
Asn Pro Arg Asn Phe Asn Phe Asp Asn Val Gly Asn Ala Met Leu Ala 
            1095                1100                1105 

ttg ttt gaa gtt ctc tcc ttg aaa ggc tgg gtg gaa gtg aga gat gtt     3598 
Leu Phe Glu Val Leu Ser Leu Lys Gly Trp Val Glu Val Arg Asp Val 
        1110                1115                1120 

att att cat cgt gtg ggg ccg atc cat gga atc tat att cat gtt ttt     3646 
Ile Ile His Arg Val Gly Pro Ile His Gly Ile Tyr Ile His Val Phe 
    1125                1130                1135 

gta ttc ctg ggt tgc atg att gga ctg acc ctt ttt gtt gga gta gtt     3694 
Val Phe Leu Gly Cys Met Ile Gly Leu Thr Leu Phe Val Gly Val Val 
1140                1145                1150                1155 

att gct aat ttc aat gaa aac aag ggg acg gct ttg ctg acc gtc gat     3742 
Ile Ala Asn Phe Asn Glu Asn Lys Gly Thr Ala Leu Leu Thr Val Asp 
                1160                1165                1170 

cag aga aga tgg gaa gac ctg aag agc cga ctg aag atc gca cag cct     3790 
Gln Arg Arg Trp Glu Asp Leu Lys Ser Arg Leu Lys Ile Ala Gln Pro 
            1175                1180                1185 

ctt cat ctc ccg cct cgc ccg gat aat gat ggt ttt aga gct aaa atg     3838 
Leu His Leu Pro Pro Arg Pro Asp Asn Asp Gly Phe Arg Ala Lys Met 
        1190                1195                1200 

tat gac ata acc cag cat cca ttt ttt aag agg aca atc gca tta ctc     3886 
Tyr Asp Ile Thr Gln His Pro Phe Phe Lys Arg Thr Ile Ala Leu Leu 
    1205                1210                1215 

gtc ctg gcc cag tcg gtg ttg ctc tct gtc aag tgg gac gtc gag gac     3934 
Val Leu Ala Gln Ser Val Leu Leu Ser Val Lys Trp Asp Val Glu Asp 
1220                1225                1230                1235 

ccg gtg acc gta cct ttg gca aca atg tca gtt gtt ttc acc ttc atc     3982 
Pro Val Thr Val Pro Leu Ala Thr Met Ser Val Val Phe Thr Phe Ile 
                1240                1245                1250 

ttt gtt ctg gag gtt acc atg aag atc ata gca atg tcg cct gct ggc     4030 
Phe Val Leu Glu Val Thr Met Lys Ile Ile Ala Met Ser Pro Ala Gly 
            1255                1260                1265 

ttc tgg caa agc aga aga aac cga tac gat ctc ctg gtg acg tcg ctt     4078 
Phe Trp Gln Ser Arg Arg Asn Arg Tyr Asp Leu Leu Val Thr Ser Leu 
        1270                1275                1280 

ggc gtt gta tgg gtg gtg ctt cac ttt gcc ctc ctg aat gca tat act     4126 
Gly Val Val Trp Val Val Leu His Phe Ala Leu Leu Asn Ala Tyr Thr 
    1285                1290                1295 

tac atg atg ggc gct tgt gtg att gta ttt agg ttt ttc tcc atc tgt     4174 
Tyr Met Met Gly Ala Cys Val Ile Val Phe Arg Phe Phe Ser Ile Cys 
1300                1305                1310                1315 

gga aaa cat gta acg cta aag atg ctc ctc ttg aca gtg gtc gtc agc     4222 
Gly Lys His Val Thr Leu Lys Met Leu Leu Leu Thr Val Val Val Ser 
                1320                1325                1330 

atg tac aag agc ttc ttt atc ata gta ggc atg ttt ctc ttg ctg ctg     4270 
Met Tyr Lys Ser Phe Phe Ile Ile Val Gly Met Phe Leu Leu Leu Leu 
            1335                1340                1345 

tgt tac gct ttt gct gga gtt gtt tta ttt ggt act gtg aaa tat ggg     4318 
Cys Tyr Ala Phe Ala Gly Val Val Leu Phe Gly Thr Val Lys Tyr Gly 
        1350                1355                1360 

gag aat att aac agg cat gca aat ttt tct tcg gct gga aaa gct att     4366 
Glu Asn Ile Asn Arg His Ala Asn Phe Ser Ser Ala Gly Lys Ala Ile 
    1365                1370                1375 

acc gta ctg ttc cga att gtc aca ggt gaa gac tgg aac aag att atg     4414 
Thr Val Leu Phe Arg Ile Val Thr Gly Glu Asp Trp Asn Lys Ile Met 
1380                1385                1390                1395 

cat gac tgt atg gtt cag cct ccg ttt tgt act cca gat gaa ttt aca     4462 
His Asp Cys Met Val Gln Pro Pro Phe Cys Thr Pro Asp Glu Phe Thr 
                1400                1405                1410 

tac tgg gca aca gac tgt gga aat tat gct ggg gca ctt atg tat ttc     4510 
Tyr Trp Ala Thr Asp Cys Gly Asn Tyr Ala Gly Ala Leu Met Tyr Phe 
            1415                1420                1425 

tgt tca ttt tat gtc atc att gcc tac atc atg cta aat ctg ctt gta     4558 
Cys Ser Phe Tyr Val Ile Ile Ala Tyr Ile Met Leu Asn Leu Leu Val 
        1430                1435                1440 

gcc ata att gtg gag aat ttc tcc ttg ttt tat tcc act gag gag gac     4606 
Ala Ile Ile Val Glu Asn Phe Ser Leu Phe Tyr Ser Thr Glu Glu Asp 
    1445                1450                1455 

cag ctt tta agt tac aat gat ctt cgc cac ttt caa atc ata tgg aac     4654 
Gln Leu Leu Ser Tyr Asn Asp Leu Arg His Phe Gln Ile Ile Trp Asn 
1460                1465                1470                1475 

atg gtg gat gat aaa aga gag ggg gtg atc ccc acg ttc cgc gtc aag     4702 
Met Val Asp Asp Lys Arg Glu Gly Val Ile Pro Thr Phe Arg Val Lys 
                1480                1485                1490 

ttc ctg ctg cgg cta ctg cgt ggg agg ctg gag gtg gac ctg gac aag     4750 
Phe Leu Leu Arg Leu Leu Arg Gly Arg Leu Glu Val Asp Leu Asp Lys 
            1495                1500                1505 

gac aag ctc ctg ttt aag cac atg tgc tac gaa atg gag agg ctc cac     4798 
Asp Lys Leu Leu Phe Lys His Met Cys Tyr Glu Met Glu Arg Leu His 
        1510                1515                1520 

aat ggc ggc gac gtc acc ttc cat gat gtc ctg agc atg ctt tca tac     4846 
Asn Gly Gly Asp Val Thr Phe His Asp Val Leu Ser Met Leu Ser Tyr 
    1525                1530                1535 

cgg tcc gtg gac atc cgg aag agc ttg cag ctg gag gaa ctc ctg gcg     4894 
Arg Ser Val Asp Ile Arg Lys Ser Leu Gln Leu Glu Glu Leu Leu Ala 
1540                1545                1550                1555 

agg gag cag ctg gag tac acc ata gag gag gag gtg gcc aag cag acc     4942 
Arg Glu Gln Leu Glu Tyr Thr Ile Glu Glu Glu Val Ala Lys Gln Thr 
                1560                1565                1570 

atc cgc atg tgg ctc aag aag tgc ctg aag cgc atc aga gct aaa cag     4990 
Ile Arg Met Trp Leu Lys Lys Cys Leu Lys Arg Ile Arg Ala Lys Gln 
            1575                1580                1585 

cag cag tcg tgc agt atc atc cac agc ctg aga gag agt cag cag caa     5038 
Gln Gln Ser Cys Ser Ile Ile His Ser Leu Arg Glu Ser Gln Gln Gln 
        1590                1595                1600 

gag ctg agc cgg ttt ctg aac ccg ccc agc atc gag acc acc cag ccc     5086 
Glu Leu Ser Arg Phe Leu Asn Pro Pro Ser Ile Glu Thr Thr Gln Pro 
    1605                1610                1615 

agt gag gac acg aat gcc aac agt cag gac aac agc atg caa cct gag     5134 
Ser Glu Asp Thr Asn Ala Asn Ser Gln Asp Asn Ser Met Gln Pro Glu 
1620                1625                1630                1635 

aca agc agc cag cag cag ctc ctg agc ccc acg ctg tcg gat aga gga     5182 
Thr Ser Ser Gln Gln Gln Leu Leu Ser Pro Thr Leu Ser Asp Arg Gly 
                1640                1645                1650 

gga agt cgg caa gat gca gcc gac gca ggg aaa ccc cag agg aaa ttt     5230 
Gly Ser Arg Gln Asp Ala Ala Asp Ala Gly Lys Pro Gln Arg Lys Phe 
            1655                1660                1665 

ggg cag tgg cgt ctg cca tca gcc cca aaa cca ata agc cat tca gtg     5278 
Gly Gln Trp Arg Leu Pro Ser Ala Pro Lys Pro Ile Ser His Ser Val 
        1670                1675                1680 

tcc tca gtc aac tta cgg tta gga gga agg aca acc atg aaa tct gtc     5326 
Ser Ser Val Asn Leu Arg Leu Gly Gly Arg Thr Thr Met Lys Ser Val 
    1685                1690                1695 

gtg tgc aaa atg aac ccc atg act gac gcg gct tcc tgc ggt tct gaa     5374 
Val Cys Lys Met Asn Pro Met Thr Asp Ala Ala Ser Cys Gly Ser Glu 
1700                1705                1710                1715 

gtt aag aag tgg tgg acc cgg cag ctg act gtg gag agc gac gaa agt     5422 
Val Lys Lys Trp Trp Thr Arg Gln Leu Thr Val Glu Ser Asp Glu Ser 
                1720                1725                1730 

ggg gat gac ctt ctg gat att taggtggatg tcaatgtaga tgaatttcta        5473 
Gly Asp Asp Leu Leu Asp Ile 
            1735 

gggtggaaac cgttttctaa taatgtcctt gattgtccag tgagcaatct gtaattgact   5533 

ataactgaat tccagcttgt cacaagatgt ttataaattg attttcatcc tgccacagaa   5593 

aggcataagc tgcagtatga tgggttacta tcaatcattg ctcaaaaaaa tttttgtata   5653 

atgacagtac tgataatatt agaaatgata ccgcaagcaa atgtatatca ttaaaaatgt   5713 

catatattct gtctgcgtaa actaagtata attcatattg ctctaatagt attatcaccg   5773 

ccccsmaaag agtgctaagc ccaaagtggc tgatatttag ggtacagggg ttatagcttt   5833 

agttcacatc tttcccattt ccactagaaa ttttctcgag agaatttatt atttatgatt   5893 

gatctgaaaa ggtcagcact gaacttatgc taaatgatag tagttttaca aactacagat   5953 

tctgaatttt aaaaagtatc ttctttttct cgtgttatat ttttaaatat acacaagaca   6013 

tttggtgacc agaacaagtt gatttctgtc ctcagttatg ttaatgaaac tgttgcctcc   6073 

ttctaagaaa attgtgtgtg caagcaccag gcaaagaaat ggactcagga tgcttagcgg   6133 

tttaaaacaa acctgtagat aaatcacttg agtgacatag ttgcgcaaag atgttaagtt   6193 

tcttaagaaa ccttttaata actgagttta gcaaaaagaa taaaactata tagctcaatt   6253 

tatttaaaaa aatctttttg catgtgtgat gttatcattg gcttcatttc ttacccaagg   6313 

tatgtctgtt ttgccataaa tcagcagagt catttcattc tgggtgatcc tgacacacca   6373 

ttgctatgtt agatttgaaa tgacatctct gttaaaagaa tcttctatgg aaataatggt   6433 

gccctgcaaa atctycctct gaactcacag gttagggatc acacaactta cttaatcgtt   6493 

ttttgttttt gtttttttyc cttatatgtc aatcggccca tgtcctccgg gaaaattaga   6553 

aaagcaaaat gattacaaag tgctgttaga tttcttgtgc tgggccagcc aagtagaagt   6613 

ggacttgact tggaccttta actattttat tacagattgg acatttgctg ttcagatgtt   6673 

ttttaacaga gggattatct cagaatcctg tgacctccag gttgttttat aatctatttt   6733 

tctctattta acattcctca gatagatagg caaataggac attccttctg tgtcacagaa   6793 

gtatcgtggt agtggcagtc tacagtttat atgattcatt gtaactatga gataaagaac   6853 

aaccagtcat gtggccaaaa ggattagatt tgattggatg ttcacttgga gtttactttt   6913 

tgtacataca agataaaata aatattggat ttgtaaaat                          6952 

 
           
             3  
             1738  
             PRT  
             Homo sapiens  
           
            3 

Met Leu Lys Arg Lys Gln Ser Ser Arg Val Glu Ala Gln Pro Val Thr 
 1               5                  10                  15 

Asp Phe Gly Pro Asp Glu Ser Leu Ser Asp Asn Ala Asp Ile Leu Trp 
            20                  25                  30 

Ile Asn Lys Pro Trp Val His Ser Leu Leu Arg Ile Cys Ala Ile Ile 
        35                  40                  45 

Ser Val Ile Ser Val Cys Met Asn Thr Pro Met Thr Phe Glu His Tyr 
    50                  55                  60 

Pro Pro Leu Gln Tyr Val Thr Phe Thr Leu Asp Thr Leu Leu Met Phe 
65                  70                  75                  80 

Leu Tyr Thr Ala Glu Met Ile Ala Lys Met His Ile Arg Gly Ile Val 
                85                  90                  95 

Lys Gly Asp Ser Ser Tyr Val Lys Asp Arg Trp Cys Val Phe Asp Gly 
            100                 105                 110 

Phe Met Val Phe Cys Leu Trp Val Ser Leu Val Leu Gln Val Phe Glu 
        115                 120                 125 

Ile Ala Asp Ile Val Asp Gln Met Ser Pro Trp Gly Met Leu Arg Ile 
    130                 135                 140 

Pro Arg Pro Leu Ile Met Ile Arg Ala Phe Arg Ile Tyr Phe Arg Phe 
145                 150                 155                 160 

Glu Leu Pro Arg Thr Arg Ile Thr Asn Ile Leu Lys Arg Ser Gly Glu 
                165                 170                 175 

Gln Ile Trp Ser Val Ser Ile Phe Leu Leu Phe Phe Leu Leu Leu Tyr 
            180                 185                 190 

Gly Ile Leu Gly Val Gln Met Phe Gly Thr Phe Thr Tyr His Cys Val 
        195                 200                 205 

Val Asn Asp Thr Lys Pro Gly Asn Val Thr Trp Asn Ser Leu Ala Ile 
    210                 215                 220 

Pro Asp Thr His Cys Ser Pro Glu Leu Glu Glu Gly Tyr Gln Cys Pro 
225                 230                 235                 240 

Pro Gly Phe Lys Cys Met Asp Leu Glu Asp Leu Gly Leu Ser Arg Gln 
                245                 250                 255 

Glu Leu Gly Tyr Ser Gly Phe Asn Glu Ile Gly Thr Ser Ile Phe Thr 
            260                 265                 270 

Val Tyr Glu Ala Ala Ser Gln Glu Gly Trp Val Phe Leu Met Tyr Arg 
        275                 280                 285 

Ala Ile Asp Ser Phe Pro Arg Trp Arg Ser Tyr Phe Tyr Phe Ile Thr 
    290                 295                 300 

Leu Ile Phe Phe Leu Ala Trp Leu Val Lys Asn Val Phe Ile Ala Val 
305                 310                 315                 320 

Ile Ile Glu Thr Phe Ala Glu Ile Arg Val Gln Phe Gln Gln Met Trp 
                325                 330                 335 

Gly Ser Arg Ser Ser Thr Thr Ser Thr Ala Thr Thr Gln Met Phe His 
            340                 345                 350 

Glu Asp Ala Ala Gly Gly Trp Gln Leu Val Ala Val Asp Val Asn Lys 
        355                 360                 365 

Pro Gln Gly Arg Ala Pro Ala Cys Leu Gln Lys Met Met Arg Ser Ser 
    370                 375                 380 

Val Phe His Met Phe Ile Leu Ser Met Val Thr Val Asp Val Ile Val 
385                 390                 395                 400 

Ala Ala Ser Asn Tyr Tyr Lys Gly Glu Asn Phe Arg Arg Gln Tyr Asp 
                405                 410                 415 

Glu Phe Tyr Leu Ala Glu Val Ala Phe Thr Val Leu Phe Asp Leu Glu 
            420                 425                 430 

Ala Leu Leu Lys Ile Trp Cys Leu Gly Phe Thr Gly Tyr Ile Ser Ser 
        435                 440                 445 

Ser Leu His Lys Phe Glu Leu Leu Leu Val Ile Gly Thr Thr Leu His 
    450                 455                 460 

Val Tyr Pro Asp Leu Tyr His Ser Gln Phe Thr Tyr Phe Gln Val Leu 
465                 470                 475                 480 

Arg Val Val Arg Leu Ile Lys Ile Ser Pro Ala Leu Glu Asp Phe Val 
                485                 490                 495 

Tyr Lys Ile Phe Gly Pro Gly Lys Lys Leu Gly Ser Leu Val Val Phe 
            500                 505                 510 

Thr Ala Ser Leu Leu Ile Val Met Ser Ala Ile Ser Leu Gln Met Phe 
        515                 520                 525 

Cys Phe Val Glu Glu Leu Asp Arg Phe Thr Thr Phe Pro Arg Ala Phe 
    530                 535                 540 

Met Ser Met Phe Gln Ile Leu Thr Gln Glu Gly Trp Val Asp Val Met 
545                 550                 555                 560 

Asp Gln Thr Leu Asn Ala Val Gly His Met Trp Ala Pro Val Val Ala 
                565                 570                 575 

Ile Tyr Phe Ile Leu Tyr His Leu Phe Ala Thr Leu Ile Leu Leu Ser 
            580                 585                 590 

Leu Phe Val Ala Val Ile Leu Asp Asn Leu Glu Leu Asp Glu Asp Leu 
        595                 600                 605 

Lys Lys Leu Lys Gln Leu Lys Gln Ser Glu Ala Asn Ala Asp Thr Lys 
    610                 615                 620 

Glu Lys Leu Pro Leu Arg Leu Arg Ile Phe Glu Lys Phe Pro Asn Arg 
625                 630                 635                 640 

Pro Gln Met Val Lys Ile Ser Lys Leu Pro Ser Asp Phe Thr Val Pro 
                645                 650                 655 

Lys Ile Arg Glu Ser Phe Met Lys Gln Phe Ile Asp Arg Gln Gln Gln 
            660                 665                 670 

Asp Thr Cys Cys Leu Leu Arg Ser Leu Pro Thr Thr Ser Ser Ser Ser 
        675                 680                 685 

Cys Asp His Ser Lys Arg Ser Ala Ile Glu Asp Asn Lys Tyr Ile Asp 
    690                 695                 700 

Gln Lys Leu Arg Lys Ser Val Phe Ser Ile Arg Ala Arg Asn Leu Leu 
705                 710                 715                 720 

Glu Lys Glu Thr Ala Val Thr Lys Ile Leu Arg Ala Cys Thr Arg Gln 
                725                 730                 735 

Arg Met Leu Ser Gly Ser Phe Glu Gly Gln Pro Ala Lys Glu Arg Ser 
            740                 745                 750 

Ile Leu Ser Val Gln His His Ile Arg Gln Glu Arg Arg Ser Leu Arg 
        755                 760                 765 

His Gly Ser Asn Ser Gln Arg Ile Ser Arg Gly Lys Ser Leu Glu Thr 
    770                 775                 780 

Leu Thr Gln Asp His Ser Asn Thr Val Arg Tyr Arg Asn Ala Gln Arg 
785                 790                 795                 800 

Glu Asp Ser Glu Ile Lys Met Ile Gln Glu Lys Lys Glu Gln Ala Glu 
                805                 810                 815 

Met Lys Arg Lys Val Gln Glu Glu Glu Leu Arg Glu Asn His Pro Tyr 
            820                 825                 830 

Phe Asp Lys Pro Leu Phe Ile Val Gly Arg Glu His Arg Phe Arg Asn 
        835                 840                 845 

Phe Cys Arg Val Val Val Arg Ala Arg Phe Asn Ala Ser Lys Thr Asp 
    850                 855                 860 

Pro Val Thr Gly Ala Val Lys Asn Thr Lys Tyr His Gln Leu Tyr Asp 
865                 870                 875                 880 

Leu Leu Gly Leu Val Thr Tyr Leu Asp Trp Val Met Ile Ile Val Thr 
                885                 890                 895 

Ile Cys Ser Cys Ile Ser Met Met Phe Glu Ser Pro Phe Arg Arg Val 
            900                 905                 910 

Met His Ala Pro Thr Leu Gln Ile Ala Glu Tyr Val Phe Val Ile Phe 
        915                 920                 925 

Met Ser Ile Glu Leu Asn Leu Lys Ile Met Ala Asp Gly Leu Phe Phe 
    930                 935                 940 

Thr Pro Thr Ala Val Ile Arg Asp Phe Gly Gly Val Met Asp Ile Phe 
945                 950                 955                 960 

Ile Tyr Leu Val Ser Leu Ile Phe Leu Cys Trp Met Pro Gln Asn Val 
                965                 970                 975 

Pro Ala Glu Ser Gly Ala Gln Leu Leu Met Val Leu Arg Cys Leu Arg 
            980                 985                 990 

Pro Leu Arg Ile Phe Lys Leu Val Pro Gln Met Arg Lys Val Val Arg 
        995                 1000                1005 

Glu Leu Phe Ser Gly Phe Lys Glu Ile Phe Leu Val Ser Ile Leu Leu 
    1010                1015                1020 

Leu Thr Leu Met Leu Val Phe Ala Ser Phe Gly Val Gln Leu Phe Ala 
1025                1030                1035                1040 

Gly Lys Leu Ala Lys Cys Asn Asp Pro Asn Ile Ile Arg Arg Glu Asp 
                1045                1050                1055 

Cys Asn Gly Ile Phe Arg Ile Asn Val Ser Val Ser Lys Asn Leu Asn 
            1060                1065                1070 

Leu Lys Leu Arg Pro Gly Glu Lys Lys Pro Gly Phe Trp Val Pro Arg 
        1075                1080                1085 

Val Trp Ala Asn Pro Arg Asn Phe Asn Phe Asp Asn Val Gly Asn Ala 
    1090                1095                1100 

Met Leu Ala Leu Phe Glu Val Leu Ser Leu Lys Gly Trp Val Glu Val 
1105                1110                1115                1120 

Arg Asp Val Ile Ile His Arg Val Gly Pro Ile His Gly Ile Tyr Ile 
                1125                1130                1135 

His Val Phe Val Phe Leu Gly Cys Met Ile Gly Leu Thr Leu Phe Val 
            1140                1145                1150 

Gly Val Val Ile Ala Asn Phe Asn Glu Asn Lys Gly Thr Ala Leu Leu 
        1155                1160                1165 

Thr Val Asp Gln Arg Arg Trp Glu Asp Leu Lys Ser Arg Leu Lys Ile 
    1170                1175                1180 

Ala Gln Pro Leu His Leu Pro Pro Arg Pro Asp Asn Asp Gly Phe Arg 
1185                1190                1195                1200 

Ala Lys Met Tyr Asp Ile Thr Gln His Pro Phe Phe Lys Arg Thr Ile 
                1205                1210                1215 

Ala Leu Leu Val Leu Ala Gln Ser Val Leu Leu Ser Val Lys Trp Asp 
            1220                1225                1230 

Val Glu Asp Pro Val Thr Val Pro Leu Ala Thr Met Ser Val Val Phe 
        1235                1240                1245 

Thr Phe Ile Phe Val Leu Glu Val Thr Met Lys Ile Ile Ala Met Ser 
    1250                1255                1260 

Pro Ala Gly Phe Trp Gln Ser Arg Arg Asn Arg Tyr Asp Leu Leu Val 
1265                1270                1275                1280 

Thr Ser Leu Gly Val Val Trp Val Val Leu His Phe Ala Leu Leu Asn 
                1285                1290                1295 

Ala Tyr Thr Tyr Met Met Gly Ala Cys Val Ile Val Phe Arg Phe Phe 
            1300                1305                1310 

Ser Ile Cys Gly Lys His Val Thr Leu Lys Met Leu Leu Leu Thr Val 
        1315                1320                1325 

Val Val Ser Met Tyr Lys Ser Phe Phe Ile Ile Val Gly Met Phe Leu 
    1330                1335                1340 

Leu Leu Leu Cys Tyr Ala Phe Ala Gly Val Val Leu Phe Gly Thr Val 
1345                1350                1355                1360 

Lys Tyr Gly Glu Asn Ile Asn Arg His Ala Asn Phe Ser Ser Ala Gly 
                1365                1370                1375 

Lys Ala Ile Thr Val Leu Phe Arg Ile Val Thr Gly Glu Asp Trp Asn 
            1380                1385                1390 

Lys Ile Met His Asp Cys Met Val Gln Pro Pro Phe Cys Thr Pro Asp 
        1395                1400                1405 

Glu Phe Thr Tyr Trp Ala Thr Asp Cys Gly Asn Tyr Ala Gly Ala Leu 
    1410                1415                1420 

Met Tyr Phe Cys Ser Phe Tyr Val Ile Ile Ala Tyr Ile Met Leu Asn 
1425                1430                1435                1440 

Leu Leu Val Ala Ile Ile Val Glu Asn Phe Ser Leu Phe Tyr Ser Thr 
                1445                1450                1455 

Glu Glu Asp Gln Leu Leu Ser Tyr Asn Asp Leu Arg His Phe Gln Ile 
            1460                1465                1470 

Ile Trp Asn Met Val Asp Asp Lys Arg Glu Gly Val Ile Pro Thr Phe 
        1475                1480                1485 

Arg Val Lys Phe Leu Leu Arg Leu Leu Arg Gly Arg Leu Glu Val Asp 
    1490                1495                1500 

Leu Asp Lys Asp Lys Leu Leu Phe Lys His Met Cys Tyr Glu Met Glu 
1505                1510                1515                1520 

Arg Leu His Asn Gly Gly Asp Val Thr Phe His Asp Val Leu Ser Met 
                1525                1530                1535 

Leu Ser Tyr Arg Ser Val Asp Ile Arg Lys Ser Leu Gln Leu Glu Glu 
            1540                1545                1550 

Leu Leu Ala Arg Glu Gln Leu Glu Tyr Thr Ile Glu Glu Glu Val Ala 
        1555                1560                1565 

Lys Gln Thr Ile Arg Met Trp Leu Lys Lys Cys Leu Lys Arg Ile Arg 
    1570                1575                1580 

Ala Lys Gln Gln Gln Ser Cys Ser Ile Ile His Ser Leu Arg Glu Ser 
1585                1590                1595                1600 

Gln Gln Gln Glu Leu Ser Arg Phe Leu Asn Pro Pro Ser Ile Glu Thr 
                1605                1610                1615 

Thr Gln Pro Ser Glu Asp Thr Asn Ala Asn Ser Gln Asp Asn Ser Met 
            1620                1625                1630 

Gln Pro Glu Thr Ser Ser Gln Gln Gln Leu Leu Ser Pro Thr Leu Ser 
        1635                1640                1645 

Asp Arg Gly Gly Ser Arg Gln Asp Ala Ala Asp Ala Gly Lys Pro Gln 
    1650                1655                1660 

Arg Lys Phe Gly Gln Trp Arg Leu Pro Ser Ala Pro Lys Pro Ile Ser 
1665                1670                1675                1680 

His Ser Val Ser Ser Val Asn Leu Arg Leu Gly Gly Arg Thr Thr Met 
                1685                1690                1695 

Lys Ser Val Val Cys Lys Met Asn Pro Met Thr Asp Ala Ala Ser Cys 
            1700                1705                1710 

Gly Ser Glu Val Lys Lys Trp Trp Thr Arg Gln Leu Thr Val Glu Ser 
        1715                1720                1725 

Asp Glu Ser Gly Asp Asp Leu Leu Asp Ile 
    1730                1735