Patent Publication Number: US-2002006639-A1

Title: Disease-associated gene

Description:
[0001] The present invention relates to a novel asthma-associated gene, designated AAG6, and to the protein molecule encoded by AAG6. The invention also relates to the use of AAG6 polynucleotide sequences for diagnostic and prognostic screening of patient populations and the use of the protein encoded by AAG6 as a therapeutic target.  
       [0002] Asthma is a very common lung disease with the following characteristics:  
       [0003] airways obstruction—this is usually reversible but often progressive  
       [0004] chronic bronchial inflammation—a condition characterised by inflammatory cell infiltration and activation, release of biochemical mediators and structural changes (airway remodelling)  
       [0005] bronchial hyperresponsiveness (BHR)—an exaggerated bronchoconstrictor response to a variety of immunologic, biochemical and physical stimuli.  
       [0006] Asthma is characterised clinically by chronic, intermittent airway obstruction with wheezing, coughing and breathlessness. Although asthma is typically associated with an obstructive impairment that is reversible, neither this finding nor any other single test or measure is adequate to diagnose asthma [Guidelines for the diagnosis and development of asthma, 1997, NIH Publication No. 97-4051]. Many diseases are associated with this pattern of abnormality. The patient&#39;s pattern of symptoms (along with other information from the patient&#39;s medical history) and exclusion of other possible diagnoses also are needed to establish a diagnosis of asthma. Clinical judgement is needed in conducting the assessment for asthma. Patients with asthma are heterogeneous and present signs and symptoms that vary widely from patient to patient as well as within each patient over time.  
       [0007] Many hypotheses have been advanced to explain the pathophysiology of asthma, including problems with airway smooth muscle, the role of inflammation, nervous innervation of the airways and mechanisms related to mediators. Although all of these factors may be important, it is unclear which are the primary (i.e. causative) defects and which are the secondary defects. It is generally agreed, however, that both the environment and genetics are important. Given the multifactorial nature of asthma, one approach to identifying the fundamental mechanisms is to discover asthma susceptibility genes that predispose individuals to develop asthma.  
       [0008] One method which can be used to identify asthma susceptibility genes is positional cloning. In this method, susceptibility genes are localised to a specific region of a human chromosome by using DNA markers to track the inheritance of the genes through families. DNA markers are fragments of DNA with a defined physical location on a chromosome, whose inheritance can be monitored. The closer a DNA marker is to a susceptibility gene, the greater the probability that the marker and the susceptibility gene will be passed together from parent to child. This phenomenon is called genetic linkage. Once linkage to a specific chromosomal region has been obtained, the size of the region is narrowed down using a combination of physical and genetic mapping until the region is small enough to be sequenced and the susceptibility gene can be identified. After identification of the susceptibility gene, any polymorphisms in this gene can be determined and an analysis performed to see whether these mutations occur with greater prevalence in asthmatics compared to non-asthmatics. The major advantages of positional cloning are that it is possible to identify novel genes even though the underlying factors causing the disease are unknown, and the genes identified are of direct pathological relevance (i.e. primary causative defects) because they make carriers directly susceptible to developing the disease.  
       [0009] In recent years a number of academic research groups have provided evidence for the presence of genes important in the regulation of asthmatic and allergic responses on human chromosome 5. In particular, evidence for the presence of susceptibility genes for BHR and elevated serum IgE levels on chromosome 5 in subregion 5q31-5q33 [Meyers et al., Genomics 23: 464-470; Postma et al., N. Eng. J. Med. 333:894-900; and Bleecker et al., Clin. Exp. Allergy 25:84-88] was obtained from genetic linkage analysis of 92 Dutch asthma families. Strong evidence for genetic linkage between marker D5S436, raised total serum IgE levels [Meyers et al., Genomics 23: 464-470; Postma et al., N. Eng. J. Med. 333:894-900; and Bleecker et al., Clin. Exp. Allergy 25:84-88] and BHR [Postma et al., N. Eng. J. Med. 333:894-900; and Bleecker et al., Clin. Exp. Allergy 25:84-88] was found in the Dutch families.  
       [0010] No asthma susceptibility gene has yet been identified, so there is a need in the art for the identification of such genes. Identification of asthma susceptibility genes would provide a fundamental understanding of the disease process from which a number of clinically important applications would arise. Susceptibility genes identified may lead to the development of therapeutics (small molecule drugs, antisense molecules, antibody molecules) directly targeted to the gene or protein product of the gene, or may target the biochemical pathway of which the protein product is a part at an upstream or downstream location if the development of such drugs is easier than directly targeting the gene or its protein product. Polynucleotide sequences comprising the gene, sequence variants thereof and protein products thereof may be used to develop a clinical diagnostic test for asthma and for the identification of individuals at high risk for the development of asthma. The results of such tests may also have prognostic value and may be used to predict patients who respond to and those who do not respond to drug therapy. Finally, information about the DNA sequences of asthma susceptibility genes and the amino acid sequences encoded by these genes facilitates large scale production of proteins by recombinant techniques and identification of the tissues/cells naturally producing the proteins. Such sequence information also permits the preparation of antibody substances or other novel binding molecules specifically reactive with the proteins encoded by the susceptibility genes that may be used in modulating the natural ligand/antiligand binding reactions in which the proteins may be involved and for diagnostic purposes.  
       [0011] Accordingly, the present invention provides, in one aspect, an isolated polynucleotide, hereinafter alternatively referred to as AAG6, comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 or a functionally equivalent variant of said amino acid sequence, i.e. a variant thereof which retains the biological or other functional activity thereof, e.g. a variant which is capable of raising an antibody which binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.  
       [0012] Terms used herein have the following meanings:  
       [0013] “Isolated” refers to material removed from its original environment.  
       [0014] “Hybridization” or “hybridizes” refers to any process by which a strand of a polynucleotide binds with a complementary strand through base pairing.  
       [0015] “Stringent conditions” refer to experimental conditions which allow up to 20% base pair mismatches, typically two 15 minute washes in 0.1×SSC (15mM NaCl, 1.5 mM sodium citrate, pH 7.0) at 65° C. “Homology” or “homologous” refers to a degree of similarity between nucleotide or amino acid sequences, which may be partial or, when sequences are identical, complete.  
       [0016] “Expression vector” refers to a linear or circular DNA molecule which comprises a segment encoding a polypeptide of interest operably linked to additional segments which provide for its transcription.  
       [0017] “Antisense” refers to selective inhibition of protein synthesis through hybridisation of an oligo- or polynucleotide to its complementary sequence in messenger RNA (mRNA) of the target protein. The antisense concept was first proposed by Zamecnik and Stephenson (Proc. Natl. Acad. Sci. USA 75:280-284; Proc. Natl. Acad. Sci. USA 75:285-288) and has subsequently found broad application both as an experimental tool and as a means of generating putative therapeutic molecules (Alama, A., Pharmacol. Res. 36:171-178; Dean, N. M., Biochem. Soc. Trans. 24:623-629; Bennet, C. F., J. Pharmacol. Exp. Ther. 280:988-1000; Crooke, S. T., Antisense Research and Applications, Springer).  
       [0018] The term “variant” as used herein means, in relation to amino acid sequences, an amino acid sequence that is altered by one or more amino acids. The changes may involve amino acid substitution, deletion or insertion. In relation to nucleotide sequences, the term “variant” as used herein means a nucleotide sequence that is altered by one or more nucleotides; the changes may involve nucleotide substitution, deletion or insertion. A preferred functionally equivalent variant of the amino acid sequence SEQ ID NO:2 or SEQ ID NO:4 is one having at least 80%, more preferably at least 90%, and especially more than 95% amino acid sequence identity to SEQ ID NO:2 or SEQ ID NO:4. In such preferred functionally equivalent variants, the regions of SEQ ID NO:2 (amino acids 1-334) or SEQ ID NO:4 (amino acids 1-710) corresponding to the extracellular domain are usually substantially conserved.  
       [0019] By an amino acid sequence having x% identity to a reference sequence such as SEQ ID NO:2 or SEQ ID NO:4, is meant a sequence which is identical to the reference sequence except that it may include up to 100-x amino acid alterations per each 100 amino acids of the reference sequence. For example, in a subject amino acid sequence having at least 80% identity to a reference sequence, up to 20% of the amino acid residues in the reference sequence may be substituted, deleted or inserted with another amino acid residue. Percentage identity between amino acid sequences can be determined conventionally using known computer programs, for example the FASTDB program based on the algorithm of Brutlag et al (Comp.App.Biosci. (1990) 6:237-245).  
       [0020] The isolated polynucleotide of the invention may be cDNA, genomic DNA or RNA. In particular embodiments, the isolated polynucleotide is cDNA comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, a genomic DNA comprising the nucleotide sequence of SEQ ID NO:5 or a DNA comprising a nucleotide sequence which hybridises to SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 under stringent conditions.  
       [0021] The invention also provides an isolated polynucleotide comprising a consecutive 20 base pair nucleotide portion identical in sequence to a consecutive 20 base pair portion of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5.  
       [0022] A polynucleotide of the invention may be isolated by bioinformatics analysis of DNA sequences from the subregion 5q31-5q33 on chromosome 5 determined by sequencing of yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) and/or P1 artificial chromosomes (PACs) to identify genes within that subregion, searching for a sequence having greater than 95% identity to the predicted exon for a selected gene and isolating cDNA from a human lung cDNA library by PCR using primers designed using that sequence.  
       [0023] A polynucleotide of the invention, for example having the sequence SEQ ID NO:1 or SEQ ID NO:3 may be prepared from the nucleotides which it comprises by chemical synthesis, e.g. automated solid phase synthesis using known procedures and apparatus.  
       [0024] In another aspect, the present invention provides an isolated polypeptide, particularly a recombinant polypeptide, comprising the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:4 or a functionally equivalent variant thereof. Such a polypeptide may be produced by cloning a polynucleotide sequence as hereinbefore described into an expression vector containing a promoter and other appropriate regulating elements for transcription, transferring into prokaryotic or eukaryotic host cells such as bacterial, plant, insect, yeast, animal or human cells, and culturing the host cells containing the recombinant expression vector under suitable conditions. Techniques for such recombinant expression of polypeptides are well known and are described, for example, in J. Sambrook et al, Molecular Cloning, second edition, Cold Spring Harbor Press, 1990. The polypeptide of the invention, i.e. the polypeptide encoded by the AAG6 polynucleotide of the invention, has high homology to cadherin proteins, which are important participants in cell-cell adhesion-see M. Takeichi, Annu. Rev. Biochem (1990), 58, 237-52.  
       [0025] Accordingly, the present invention also provides a method of producing a polypeptide of the invention which comprises culturing a host cell containing an expression vector containing a polynucleotide sequence of the invention as hereinbefore described under conditions suitable for expression of the polypeptide and recovering the polypeptide from the host cell culture.  
       [0026] In another aspect, the present invention provides an expression vector containing a polynucleotide sequence of the invention as hereinbefore described.  
       [0027] The invention also provides an isolated polypeptide comprising a consecutive 10 amino acid portion identical in sequence to a consecutive 10 amino acid portion of SEQ ID NO:2, or SEQ ID NO:4.  
       [0028] A polypeptide of the invention may be expressed as a recombinant fusion protein with one or more heterologous polypeptides, for example to facilitate purification. For example, it may be expressed as a recombinant fusion protein with a heterologous polypeptide such as a polyhistidine containing a cleavage site located between the polynucleotide sequence of the invention and the heterologous polypeptide sequence, so that the polypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 may be cleaved and purified away from the heterologous moiety using well known techniques.  
       [0029] A polypeptide of the invention may also be synthesised, in whole or in part, from the amino acids which it comprises using well known chemical methods, for example automated solid phase techniques.  
       [0030] Isolated polypeptides of the invention as hereinbefore described may be purified by well known standard procedures.  
       [0031] The present invention also provides an antibody which is immunoreactive with a polypeptide of the invention as hereinbefore described, or a variant of said polypeptide having a polymorphism correlated with a particular disease, e.g. asthma. The antibody may be a polyclonal or monoclonal antibody. Such antibodies may be prepared using conventional procedures. Methods for the production of polyclonal antibodies against purified antigen are well established (cf. Cooper and Paterson in Current Protocols in Molecular Biology, Ausubel et al. Eds., John Wiley and Sons Inc., Chapter 11). Typically, a host animal, such as a rabbit, or a mouse, is immunised with a purified polypeptide of the invention, or immunogenic portion thereof, as antigen and, following an appropriate time interval, the host serum is collected and tested for antibodies specific against the polypeptide. Methods for the production of monoclonal antibodies against purified antigen are well established (cf. Chapter 11, Current Protocols in Molecular Biology, Ausubel et al. Eds., John Wiley and Sons Inc.). For the production of a polyclonal antibody, the serum can be treated with saturated ammonium sulphate or DEAE Sephadex. For the production of a monoclonal antibody, the spleen or lymphocytes of the immunised animal are removed and immortalised or used to produce hybridomas by known methods. Antibodies secreted by the immortalised cells are screened to determine the clones which secrete antibodies of the desired specificity, for example using Western blot analysis. Humanised antibodies can be prepared by conventional procedures.  
       [0032] In another aspect, the present invention provides an antisense oligonucleotide comprising a nucleotide sequence complementary to that of a polynucleotide of the invention, in particular a nucleotide sequence complementary to SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5, or complementary to that of a polynucleotide encoding a variant of a polypeptide of the invention having a polymorphism correlated with a disease, e.g. asthma, in particular a nucleotide sequence complementary to such a polymorphic variant of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5. The antisense oligonucleotide may be DNA, an analogue of DNA such as a phosphorothioate or methylphosphonate analogue of DNA, RNA, an analogue of RNA, or a peptide nucleic acid (PNA). The antisense oligonucleotides may be synthesised by conventional methods, for example using automated solid phase techniques.  
       [0033] The present invention also provides a polynucleotide probe comprising at least 15 contiguous nucleotides of a polynucleotide of the invention or a complement thereof. The probe may be cDNA, genomic DNA or RNA. Usually it is a synthetic oligonucleotide comprising 15 to 50 nucleotides, which can be labelled, e.g. with a fluorophore, to provide a detectable signal.  
       [0034] The polynucleotide probe is capable of selectively hybridising under stringent conditions to a polynucleotide fragment having a sequence selected from the group consisting of SEQ IDS: 1, 3 and 5. The probe has a sequence such that under such hybridisation conditions it hybridizes only to its cognate sequence. DNA probes as described above are useful in a number of screening applications including Northern and Southern blot analyses, dot blot and slot blot analyses, and fluorescence in situ hybridisation (FISH).  
       [0035] The present invention also includes a pair of oligonucleotides having nucleotide sequences useful as primers for DNA amplification of a fragment of a polynucleotide of the invention, i.e. of the human AAG6 gene (hAAG6), wherein each primer of said pair is at least 15 nucleotides in length and said pair have sequences such that when used in a polymerase chain reaction (PCR) with either human genomic DNA or a suitable human cDNA target they result in synthesis of a DNA fragment containing all or preferably part of the sequence of hAAG6. The primer pair is preferably capable of amplifying at least one exon of hAAG6 (or portion thereof), such as an exon selected from those in SEQ ID NO:1, 3 or 5. Examples of such primer pairs are shown hereinafter in the Examples. Exemplary applications of such primer pairs include amplification of DNA fragments for use in the detection of changes to the polynucleotide sequence in asthmatic patients as shown hereinafter in the Examples.  
       [0036] The role of the polypeptide of the invention in asthma and other obstructive or inflammatory airways diseases characterised by bronchial hyperresponsiveness can be determined using conventional allergen driven animal models for bronchial hyperresponsiveness, e.g. the ovalbumin-induced BHR mouse model (Tsuyuki et al, J. Clin. Invest. 96:2924-2931) or the guinea pig model hereinafter described.  
       [0037] Polynucleotides, polypeptides, antibodies, antisense oligonucleotides or probes of the invention as hereinbefore described, hereinafter alternatively referred to collectively as agents of the invention, may be used in the treatment (prophylactic or symptomatic) or diagnosis of inflammatory or obstructive airways diseases. For example, a polypeptide of the invention may be used to treat a mammal, particularly a human, deficient in or otherwise in need of that polypeptide; a polynucleotide of the invention may be used in gene therapy where it is desired to increase AAG6 activity, for instance where a subject has a mutated or missing AAG6 gene; an antisense oligonucleotide of the invention may be used to inhibit AAG6 activity or activity of variants of the AAG6 gene having a polymorphism correlated with a disease, e.g. asthma, where this is desired; an antibody of the invention may be used to detect, or determine the level of expression of, AAG6 polypeptides or a disease-correlated polymorphic variant thereof, or to inhibit ligand/antiligand binding activities of AAG6 polypeptides; and a probe of the invention may be used to detect the presence or absence of the AAG6 gene, i.e. to detect genetic abnormality.  
       [0038] “Gene therapy” refers to an approach to the treatment of human disease based upon the transfer of genetic material into somatic cells of an individual. Gene transfer can be achieved directly in vivo by administration of gene-bearing viral or non-viral vectors into blood or tissues, or indirectly ex vivo through the introduction of genetic material into cells manipulated in the laboratory followed by delivery of the gene-containing cells back to the individual. By altering the genetic material within a cell, gene therapy may correct underlying disease pathophysiology. Suitable vectors, and procedures, for gene delivery to specific tissues and organ systems in animals are described in Dracopoli, N. C. et al., Current Protocols in Human Genetics. John Wiley and Sons Inc., Chapters 12 and 13 respectively. In relation to polynucleotides of the invention, gene therapy may involve delivery of a viral or non-viral gene therapy vector containing an expression cassette of the AAG6 gene under suitable control elements to the lungs of diseased individuals (eg. asthmatics) so that the underlying disease pathophysiology is corrected or ameliorated.  
       [0039] Accordingly, in further aspects, the present invention provides  
       [0040] a pharmaceutical composition comprising a polynucleotide, polypeptide, antibody or antisense oligonucleotide of the invention as hereinbefore described, optionally together with a pharmaceutically acceptable carrier;  
       [0041] a method of treating an inflammatory or obstructive airways disease which comprises administering to a subject in need thereof an effective amount of a polynucleotide, polypeptide, antibody or antisense oligonucleotide of the invention as hereinbefore described;  
       [0042] a method of detecting genetic abnormality in a subject which comprises incubating a genetic sample from the subject with a polynucleotide probe of the invention as hereinbefore defined, under conditions where the probe hybridises to complementary polynucleotide sequence, to produce a first reaction product, and comparing the first reaction product to a control reaction product obtained with a normal genetic sample, where a difference between the first reaction product and the control reaction product indicates a genetic abnormality in the subject or a predisposition to developing a disease such as asthma;  
       [0043] a method of detecting the presence of a polynucleotide of the invention, e.g. comprising SEQ ID NO:1, 3 or 5, in cells or tissues which comprises contacting DNA from the cell or tissue with a polynucleotide probe as hereinbefore defined under conditions where the probe is specifically hybridizable with a polynucleotide of the invention, and detecting whether hybridization occurs;  
       [0044] a method of detecting an abnormality in the nucleotide sequence of a polynucleotide of the invention in a patient which comprises amplifying a target nucleotide sequence in DNA isolated from the patient by a polymerase chain reaction using a pair of primers as hereinbefore described which target the sequence to be amplified and analysing the amplified sequence to determine any polymorphism present therein; and  
       [0045] a method of detecting polymorphism in a subject which comprises treating a tissue sample from the subject with an antibody to a polymorphic variant of a polypeptide of the invention and detecting binding of said antibody.  
       [0046] The term “polymorphism” means any sequence difference as compared with the sequence of a polynucleotide of the invention as hereinbefore described.  
       [0047] Information obtained using the diagnostic assays described herein (alone or in conjunction with information on another genetic defect, which contributes to the same disease) is useful for prognosing, diagnosing or confirming that a symptomatic subject has a genetic defect (e.g. in an AAG6 gene or in a gene that regulates the expression of an AAG6 gene), which causes or contributes to the particular disease or disorder. Alternatively, the information (alone or in conjunction with information on another genetic defect, which contributes to the same disease) can be used prognostically for predicting whether a non-symptomatic subject is likely to develop a disease or condition, which is caused by or contributed to by an abnormal AAG6 activity or protein level in a subject. In particular, the assays permit one to ascertain an individual&#39;s predilection to develop a condition associated with a mutation in or associated with AAG6, where the mutation is a polymorphism such as a single nucleotide polymorphism (SNP). Based on the prognostic information, a doctor can recommend a regimen e.g. a therapeutic protocol useful for preventing or delaying onset of asthma in the individual.  
       [0048] In addition, knowledge of the particular alteration or alterations, resulting in defective or deficient AAG6 genes or proteins in an individual, alone or in conjunction with information on other genetic defects contributing to the same disease (the genetic profile of the particular disease) allows customization of therapy to the individual&#39;s genetic profile, the goal of pharmacogenomics. For example, an individual&#39;s AAG6 genetic profile or the genetic profile of the asthma can enable a doctor to: 1) more effectively prescribe a drug that will address the molecular basis of asthma; and 2) better determine the appropriate dosage of a particular drug. For example, the expression level of AAG6 proteins, alone or in conjunction with the expression level of other genes known to be involved in asthma, can be measured in many patients at various stages of the disease to generate a transcriptional or expression profile of asthma. Expression patterns of individual patients can then be compared to the expression profile of asthma to determine the appropriate drug and dose to administer to the patient.  
       [0049] The ability to target populations expected to show the highest clinical benefit, based on the AAG6 or asthma genetic profile, can enable: 1) the repositioning of marketed drugs with disappointing market results; 2) the rescue of drug candidates whose clinical development has been discontinued as a result of safety or efficacy limitations, which are patient subgroup-specific; and 3) an accelerated and less costly development for drug candidates and more optimal drug labeling (e.g. since the use of AAG6 as a marker is useful for optimizing effective dose).  
       [0050] Hybridisation of a polynucleotide probe of the invention with complementary polynucleotide sequence may be detected using in situ (eg. FISH) hybridization, Northern or Southern blot analyses, dot blot or slot blot analyses. The abnormality may also be detected for example by conformation sensitive gel electrophoresis (CSGE) and DNA sequencing as described hereinafter in the Examples. The genetic abnormality may result in a change in the amino acid sequence of the individual&#39;s AAG6 protein relative to the the amino acid sequence of a normal hAAG6 protein, or loss of protein. Alternatively, the change may not alter the amino acid sequence but may instead alter expression of the AAG6 gene by altering the sequence of controlling elements either at the 5&#39;-, or 3&#39;-end of the gene, or altering the sequence of control elements within intronic regions of the gene. Changes may also affect the way the gene transcript is processed or translated. The invention also includes kits for the detection of an abnormality in the polynucleotide sequence of an individual&#39;s AAG6 gene. Hybridisation kits for such detection comprise a probe of the invention as hereinbefore described, which probe may be modified by incorporation of a detectable, e.g. chemiluminescent or fluorescent, label therein, and may include other reagents such as labelling reagents, i.e. reagents to incorporate a detectable label such as a radioactive isotope, chemiluminescent or fluorescent group into a hybridised product, and buffers. PCR amplification kits comprise primer pairs such as those described above together with a DNA polymerase such as Taq polymerase, and may include additional reagents, such as an amplification buffer and the like. Specific embodiments of the PCR amplification kits can include additional reagents specific for a number of techniques that detect polynucleotide changes, including CSGE and DNA sequencing.  
       [0051] The effectiveness of an agent of the invention in inhibiting or reversing airways hyperreactivity may be demonstrated in a guinea pig test model. The acute injection of preformed immune complex renders guinea pigs hyperreactive to histamine. Doses of histamine which cause only a small degree of bronchoconstriction prior to administration of immune complex cause a much stronger effect thereafter. Guinea-pigs (Dunkin-Hartley, male, 400-600 g) are anaesthetised with phenobarbital (100 mg/kg i.p.) and pentobarbital (30 mg/kg i.p.) and paralysed with gallamine (10 mg/kg i.m.) and ventilated with a mixture of air and oxygen (45:55), v/v). Animals are ventilated (8 ml/kg, 1 Hz) via a tracheal cannula. Ventilation is monitored by a flow transducer. When making measurements of flow, coincident pressure changes in the thorax are monitored directly via an intrathoracic trochar, permitting display of differential pressure relative to the trachea. From this information resistance and compliance are calculated at each inspiration. An allergic reaction is initiated by intravenous injection of preformed immune complexes (prepared by adding 30 μg of bovine gamma globulin in 0.05 ml of saline to 0.05 ml of guinea pig anti-bovine gamma globulin anti-serum) 3 times at 10 minute intervals. Intravenous injections of histamine (1.0-3.2 μg/kg at 10 minute intervals) are used to define the sensitivity of the airways prior to and following the last exposure to the immune complex. Airways hyperreactivity is expressed as the paired difference for the maximal value of lung resistance in response to histamine before and after repeated injection of immune-complex. The agents of the invention are administered intratracheally either as solutions or suspensions in tragacanth. The ED 50  values for reversal of airways hyperreactivity are determined graphically from the dose response curves and represent those doses which cause a 50% reduction of airways hyperreactivity.  
       [0052] Inflammatory or obstructive airways diseases to which the present invention is applicable include asthma of whatever type or genesis including both intrinsic (non-allergic) asthma and extrinsic (allergic) asthma. Treatment of asthma is also to be understood as embracing treatment of subjects, e.g. of less than 4 or 5 years of age, exhibiting wheezing symptoms and diagnosed or diagnosable as “wheezy infants”, an established patient category of major medical concern and now often identified as incipient or early-phase asthmatics. (For convenience this particular asthmatic condition is referred to as “wheezy-infant syndrome”.)  
       [0053] Prophylactic efficacy in the treatment of asthma will be evidenced by reduced frequency or severity of symptomatic attack, e.g. of acute asthmatic or bronchoconstrictor attack, improvement in lung function or reduced airways hyperreactivity. It may further be evidenced by reduced requirement for other, symptomatic therapy, i.e. therapy for or intended to restrict or abort symptomatic attack when it occurs, for example anti-inflammatory (e.g. corticosteroid) or bronchodilatory. Prophylactic benefit in asthma may in particular be apparent in subjects prone to “morning dipping”. “Morning dipping” is a recognised asthmatic syndrome, common to a substantial percentage of asthmatics and characterised by asthma attack, e.g. between the hours of about 4 to 6 am, i.e. at a time normally substantially distant form any previously administered symptomatic asthma therapy.  
       [0054] Other inflammatory or obstructive airways diseases and conditions to which the present invention is applicable include adult respiratory distress syndrome (ARDS), chronic obstructive pulmonary or airways disease (COPD or COAD), including chronic bronchitis, or dyspnea associated therewith, emphysema, as well as exacerbation of airways hyperreactivity consequent to other drug therapy, in particular other inhaled drug therapy. The invention is also applicable to the treatment of bronchitis of whatever type or genesis including, e.g., acute, arachidic, catarrhal, croupus, chronic or phthinoid bronchitis. Further inflammatory or obstructive airways diseases to which the present invention is applicable include pneumoconiosis (an inflammatory, commonly occupational, disease of the lungs, frequently accompanied by airways obstruction, whether chronic or acute, and occasioned by repeated inhalation of dusts) of whatever type or genesis, including, for example, aluminosis, anthracosis, asbestosis, chalicosis, ptilosis, siderosis, silicosis, tabacosis and byssinosis.  
       [0055] Having regard to their anti-inflammatory activity, in particular in relation to inhibition of eosinophil activation, agents of the invention are also useful in the treatment of eosinophil related disorders, e.g. eosinophilia, in particular eosinophil related disorders of the airways (e.g. involving morbid eosinophilic infiltration of pulmonary tissues) including hypereosinophilia as it effects the airways and/or lungs as well as, for example, eosinophil-related disorders of the airways consequential or concomitant to Löffler&#39;s syndrome, eosinophilic pneumonia, parasitic (in particular metazoan) infestation (including tropical eosinophilia), bronchopulmonary aspergillosis, polyarteritis nodosa (including Churg-Strauss syndrome), eosinophilic granuloma and eosinophil-related disorders affecting the airways occasioned by drug-reaction.  
       [0056] The agents of the invention may be administered by any appropriate route, e.g. orally, for example in the form of a tablet or capsule; parenterally, for example intravenously; topically, e.g. in an ointment or cream; transdermally, e.g. in a patch; by inhalation; or intranasally.  
       [0057] Pharmaceutical compositions containing agents of the invention may be prepared using conventional diluents or excipients and techniques known in the galenic art. Thus oral dosage forms may include tablets and capsules, and compositions for inhalation may comprise aerosol or other atomizable formulations or dry powder formulations.  
       [0058] The invention includes (A) an agent of the invention in inhalable form, e.g. in an aerosol or other atomizable composition or in inhalable particulate, e.g. micronised form, (B) an inhalable medicament comprising an agent of the invention in inhalable form; (C) a pharmaceutical product comprising such an agent of the invention in inhalable form in association with an inhalation device; and (D) an inhalation device containing an agent of the invention in inhalable form.  
       [0059] Dosages of agents of the invention employed in practising the present invention will of course vary depending, for example, on the particular condition to be treated, the effect desired and the mode of administration. In general, suitable daily dosages for administration by inhalation are of the order of 1μg to 10 mg/kg while for oral administration suitable daily doses are of the order of 0.1 mg to 1000 mg/kg.  
       [0060] The present invention also provides a variant of a polynucleotide of the invention as hereinbefore described, particularly a polynucleotide having a sequence SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5, which contains a sequence polymorphism correlated with asthma. The polymorphism may be an addition, deletion or replacement of one or more nucleotides. Single nucleotide polymorphisms (SNPs), as compared with SEQ ID NO:1 or SEQ ID NO:3, or SEQ ID NO:5, which have been identified in genetic samples from asthmatic patients are shown hereinafter in the Examples.  
       [0061] The present invention further provides a method of determining predisposition of a subject to asthma comprising determining the presence or absence in DNA from the subject of a sequence polymorphism in a polynucleotide of the invention which correlates with asthma.  
       [0062] In another aspect, the present invention provides a method of determining predisposition of a patient to asthma which comprises identifying in DNA from the patient a sequence polymorphism or haplotype in a polynucleotide of the invention, as compared with a normal control DNA from a non-asthmatic subject, which correlates with asthma. A haplotype is a set of polymorphisms which is inherited together as a group.  
       [0063] In a related aspect, the invention provides a method of determining predisposition of a patient to asthma which comprises identifying in DNA from the patient a sequence polymorphism or haplotype in a polynucleotide of the invention, as compared with SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 which correlates with asthma.  
       [0064] Identification of a sequence polymorphism may be effected by conventional sequencing and sequence analysis techniques, for example as described in Cotton, R. G. H., Mutation Detection, Oxford University Press, 1997; Landegren, U., Laboratory Protocols for Mutation Detection, Oxford University Press; and R. G. H. Cotton et al, Mutation Detection, Oxford University Press, 1998.  
       [0065] Sequence polymorphisms which correlate with asthma may alter the amino acid sequence in the encoded polypeptide or may affect expression levels of the polypeptide or the way in which a transcript is processed.  
       [0066] Certain sequence polymorphisms or haplotypes may correlate with the severity and/or nature of the asthmatic phenotype, e.g. with mild, moderate or severe asthma as defined by established clinical parameters. Identification of polymorphisms may therefore be useful for prognosis, determination of therapeutic strategy and prediction of patient responses to therapy.  
       [0067] In particular, the invention further features predictive medicines, which are based, at least in part, on the identity of the novel AAG6 gene and alterations in the genes and related pathway genes, which affect the expression level and/or function of the encoded AAG6 protein in a subject.  
       [0068] For example, as described herein, AAG6 mutations that are particularly likely to cause or contribute to the development of asthma or other inflammatory or obstructive airways diseases characterised by BHR are those mutations that negatively impact normal (wildtype) functioning of AAG6, in particular the extracellular domain which is involved in homotypic association and therefore cell-cell adhesion and the intracellular domain which interacts with structural proteins or signalling molecules. Examples of such mutations include: i) mutations that affect the level of transcripts produced; ii) missense mutations occurring within the intracellular, transmembrane or extracellular domain; and mutations which affect the way in which the transcript is processed.  
       [0069] The present methods provide means for determining if a subject has (diagnostic) or is at risk of developing (prognostic) a disease, condition or disorder that is associated with an aberrant AAG6 activity, e.g., an aberrant level of AAG6 protein or an aberrant bioactivity, such as results in the development of asthma.  
       [0070] Accordingly, the invention provides methods for determining whether a subject has or is likely to develop an obstructive or inflammatory airways disease such as asthma, comprising determining the level of an AAG6 gene or protein, an AAG6 bioactivity and/or the presence of a mutation or particular polymorphic variant in the AAG6 gene.  
       [0071] In one embodiment, the method comprises determining whether a subject has an abnormal mRNA and/or protein level of AAG6, such as by Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, immunoprecipitation, Western blot hybridization, or immunohistochemistry. According to the method, cells are obtained from a subject and the AAG6 protein or mRNA level is determined and compared to the level of AAG6 protein or mRNA level in a healthy subject. An abnormal level of AAG6 polypeptide or mRNA level is likely to be indicative of an aberrant AAG6 activity.  
       [0072] In another embodiment, the method comprises measuring at least one activity of AAG6. For example, calcium-dependent cell-cell adhesion can be measured, e.g. as described herein. Comparison of the results obtained with results from similar analysis performed on AAG6 proteins from healthy subjects is indicative of whether a subject has an abnormal AAG6 activity.  
       [0073] In preferred embodiments, the methods for determining whether a subject has or is at risk for developing a disease, which is caused by or contributed to by an aberrant AAG6 activity is characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of: (i) an alteration affecting the integrity of a gene encoding an AAG6 polypeptide, or (ii) the mis-expression of the AAG6 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of: (i) a deletion of one or more nucleotides from an AAG6 gene, (ii) an addition of one or more nucleotides to an AAG6 gene, (iii) a substitution of one or more nucleotides of an AAG6 gene, (iv) a gross chromosomal rearrangement of an AAG6 gene, (v) a gross alteration in the level of a messenger RNA transcript of an AAG6 gene, (vi) aberrant modification of an AAG6 gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild type splicing pattern of a messenger RNA transcript of an AAG6 gene, (viii) a non-wild type level of an AAG6 polypeptide, (ix) allelic loss of an AAG6 gene, and/or (x) inappropriate post-translational modification of an AAG6 polypeptide. The present invention provides a large number of assay techniques for detecting alterations in an AAG6 gene. These methods include, but are not limited to, methods involving sequence analysis, Southern blot hybridization, restriction enzyme site mapping, and methods involving detection of the absence of nucleotide pairing between the nucleic acid to be analyzed and a probe.  
       [0074] Specific diseases or disorders, e.g., genetic diseases or disorders, are associated with specific allelic variants of polymorphic regions of certain genes, which do not necessarily encode a mutated protein. Thus, the presence of a specific allelic variant of a polymorphic region of a gene, such as a single nucleotide polymorphism (“SNP”), in a subject can render the subject susceptible to developing a specific disease or disorder. Polymorphic regions in genes, e.g., AAG6 genes, can be identified, by determining the nucleotide sequence of genes in populations of individuals. If a polymorphic region, e.g., SNP is identified, then the link with a specific disease can be determined by studying specific populations of individuals, e.g., individuals which developed a specific disease, such as asthma. A polymorphic region can be located in any region of a gene, e.g., exons, in coding or non coding regions of exons, introns, and promoter region.  
       [0075] It is likely that AAG6 genes comprise polymorphic regions, specific alleles of which may be associated with specific diseases or conditions or with an increased likelihood of developing such diseases or conditions. Thus, the invention provides methods for determining the identity of the allele or allelic variant of a polymorphic region of an AAG6 gene in a subject, to thereby determine whether the subject has or is at risk of developing a disease or disorder that is associated with a specific allelic variant of a polymorphic region.  
       [0076] In an exemplary embodiment, there is provided a nucleic acid composition comprising a nucleic acid probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence of an AAG6 gene or naturally occurring mutants thereof, or 5′ or 3′ flanking sequences naturally associated with the subject AAG6 genes or naturally occurring mutants thereof. The nucleic acid of a cell is rendered accessible for hybridization, the probe is contacted with the nucleic acid of the sample, and the hybridization of the probe to the sample nucleic acid is detected. Such techniques can be used to detect alterations or allelic variants at either the genomic or mRNA level, including deletions, substitutions, etc., as well as to determine mRNA transcript levels.  
       [0077] A preferred detection method is allele specific hybridization using probes overlapping the mutation or polymorphic site and having about 5, 10, 20, 25, or 30 nucleotides around the mutation or polymorphic region. In a preferred embodiment of the invention, several probes capable of hybridizing specifically to allelic variants, such as single nucleotide polymorphisms, are attached to a solid phase support, e.g., a “chip”. Oligonucleotides can be bound to a solid support by a variety of processes, including lithography. For example a chip can hold up to about 250,000 oligonucleotides. Mutation detection analysis using these chips comprising oligonucleotides, also termed “DNA probe arrays” is described e.g., in Cronin et al. (1996) Human Mutation 7:244. In one embodiment, a chip comprises all the allelic variants of at least one polymorphic region of a gene. The solid phase support is then contacted with a test nucleic acid and hybridization to the specific probes is detected. Accordingly, the identity of numerous allelic variants of one or more genes can be identified in a simple hybridization experiment.  
       [0078] In certain embodiments, detection of the alteration comprises utilizing the probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligase chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS 91:360-364), the latter of which can be particularly useful for detecting point mutations in the AAG6 gene (see Abravaya et al. (1995) Nuc Acid Res 23:675-682). In a merely illustrative embodiment, the method includes the steps of (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, (iii) contacting the nucleic acid sample with one or more primers which specifically hybridize to an AAG6 gene under conditions such that hybridization and amplification of the AAG6 gene (if present) occurs, and (iv) 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. It is anticipated that PCR, LCR or any other amplification procedure (e.g. self sustained sequence replication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), or Q-Beta Replicase (Lizardi, P. M. et al., 1988, Bio/Technology 6:1197)), may be used as a preliminary step to increase the amount of sample on which can be performed any of the techniques for detecting mutations described herein.  
       [0079] Knowledge of the particular alteration or alterations, resulting in defective or deficient AAG6 genes or proteins in an individual (the AAG6 genetic profile), alone or in conjunction with information on other genetic defects contributing to the same disease (the genetic profile of the particular disease) allows a customization of the therapy for a particular disease to the individual&#39;s genetic profile, the goal of “pharmacogenomics”. For example, subjects having a specific allele of an AAG6 gene may or may not exhibit symptoms of a particular disease or be predisposed of developing symptoms of a particular disease. Further, if those subjects are symptomatic, they may or may not respond to a certain drug, e.g., a specific AAG6 therapeutic, but may respond to another. Thus, generation of an AAG6 genetic profile, (e.g., categorization of alterations in AAG6 genes which are associated with the development of asthma), from a population of subjects, who are symptomatic for a disease or condition that is caused by or contributed to by a defective and/or deficient AAG6 gene and/or protein (an AAG6 genetic population profile) and comparison of an individual&#39;s AAG6 profile to the population profile, permits the selection or design of drugs that are expected to be safe and efficacious for a particular patient or patient population (i.e., a group of patients having the same genetic alteration).  
       [0080] For example, an AAG6 population profile can be performed, by determining the AAG6 profile, e.g., the identity of AAG6 genes, in a patient population having a disease, which is caused by or contributed to by a defective or deficient AAG6 gene. Optionally, the AAG6 population profile can further include information relating to the response of the population to an AAG6 therapeutic, using any of a variety of methods, including, monitoring: 1) the severity of symptoms associated with the AAG6 related disease, 2) AAG6 gene expression level, 3) AAG6 mRNA level, and/or 4) AAG6 protein level. and (iii) dividing or categorizing the population based on the particular genetic alteration or alterations present in its AAG6 gene or an AAG6 pathway gene. The AAG6 genetic population profile can also, optionally, indicate those particular alterations in which the patient was either responsive or non-responsive to a particular therapeutic. This information or population profile, is then useful for predicting which individuals should respond to particular drugs, based on their individual AAG6 profile.  
       [0081] In a preferred embodiment, the AAG6 profile is a transcriptional or expression level profile and step (i) is comprised of determining the expression level of AAG6 proteins, alone or in conjunction with the expression level of other genes, known to contribute to the same disease. The AAG6 profile can be measured in many patients at various stages of the disease.  
       [0082] Pharmacogenomic studies can also be performed using transgenic animals. For example, one can produce transgenic mice, which contain a specific allelic variant of an AAG6 gene. These mice can be created, e.g., by replacing their wild-type AAG6 gene with an allele of the human AAG6 gene. The response of these mice to specific AAG6 therapeutics can then be determined.  
       [0083] The treatment of an individual with an AAG6 therapeutic can be monitored by determining AAG6 characteristics, such as AAG6 protein level or activity, AAG6 mRNA level, and/or AAG6 transcriptional level. These measurements will indicate whether the treatment is effective or whether it should be adjusted or optimized. Thus, AAG6 can be used as a marker for the efficacy of a drug during clinical trials.  
       [0084] In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a preadministration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of an AAG6 protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the AAG6 protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the AAG6 protein, mRNA, or genomic DNA in the preadministration sample with the AAG6 protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of AAG6 to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of AAG6 to lower levels than detected, i.e., to decrease the effectiveness of the agent.  
       [0085] Cells of a subject may also be obtained before and after administration of an AAG6 therapeutic to detect the level of expression of genes other than AAG6, to verify that the AAG6 therapeutic does not increase or decrease the expression of genes which could be deleterious. This can be done, e.g., by using the method of transcriptional profiling. Thus, mRNA from cells exposed in vivo to an AAG6 therapeutic and mRNA from the same type of cells that were not exposed to the AAG6 therapeutic could be reverse transcribed and hybridized to a chip containing DNA from numerous genes, to compare thereby the expression of genes in cells treated and not treated with an AAG6-therapeutic. If, for example an AAG6 therapeutic turns on the expression of a proto-oncogene in an individual, use of this particular AAG6 therapeutic may be undesirable.  
       [0086] A polypeptide of the invention, including a polypeptide encoded by a variant of a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising amino acid sequence SEQ ID NO:2 or SEQ ID NO:4, which variant contains a sequence polymorphism, can be used to identify enhancers (agonists) or inhibitors (antagonists) of its activity, i.e. to identify compounds useful in the treatment of inflammatory or obstructive airways diseases, particularly asthma. Accordingly, the invention also provides a method of identifying a substance which modulates the activity of a polypeptide of the invention comprising combining a candidate substance with a polypeptide of the invention and measuring the effect of the candidate substance on said activity. The activity of a polypeptide of the invention may be measured, for example, by promotion of homotypic Ca 2+  dependent aggregation and adhesion in Chinese hamster ovary transfectants e.g. as described by Telo et al, J. Biol. Chem. 273:17565-17572. The invention also includes a method of identifying a substance which binds to a polypeptide of the invention as hereinbefore described comprising mixing a candidate substance with a polypeptide of the invention and determining whether binding has occurred.  
       [0087] The invention is illustrated by the following Examples. Abbreviations used in the Examples have the following meanings:  
                                      AEBSF:   4-(2-aminoethyl)benzenesulfonyl fluoride       BAC:   bacterial artificial chromosome       BAP:   1,4-bis(acryloyl)piperazine       BLAST:   basic local alignment search tool       BSA:   bovine serum albumin       CSGE:   conformation sensitive gel electrophoresis       DNTP:   deoxynucleotide triphosphate       DTT:   dithiothreitol       EIA:   enzyme immunoassay       EST:   expressed sequence tag       FCS:   fetal calf serum       HUVEC:   human umbilical vein endothelial cell       MTN:   multiple tissue northern       MVEC2:   mouse vascular endothelial cadherin 2       NHBE:   normal human bronchial epithelial       ORF:   open reading frame       PAC:   P1 artificial chromosome       PBS:   phosphate buffered saline       PEG:   polyethylene glycol       PMSF:   phenylmethylsulfonyl fluoride       SDS-PAGE:   sodium dodecyl sulfate polyacrylamide gel           electrophoresis       SNP:   single nucleotide polymorphism       STS:   sequence tagged site       TIGR:   The Institute for Genome Research       TTE:   44 mM Tris, 14.5 mM taurine, 0.1 mM EDTA, pH 9.0                  
 
     
    
    
     EXAMPLE 1  
     [0088] Bacterial artificial chromosome (BAC) clones identified using physical map information for human chromosome 5q31-q33 publicly available on the Lawrence Berkley National Laboratory Genome Centre web site (LBNL; www-hgc.lbl.gov/biology/bacmap/2.gif) obtained as BAC clone numbers h164 (22f14), c5 (50g20), h187 (35k5), h167 (8e5) and h177 (32d16) from Research Genetics (Huntsville, Ala., USA), and a P1 artificial chromosome (PAC) isolated by PCR using primers with SEQ ID NOS:65 to 68 for the STS markers bac51007T (5′ end of BAC 50g20) and bac51330T (3′ end of BAC 22f14) available on the LBNL website (www hgc.lbl.gov/sts.html) by Genome Systems Inc. (St. Louis, Mo., USA), the BACs and PAC together covering a sub-region of human chromosonal region 5q31-5q33, are sequenced using conventional techniques for an ABI 377 sequence (http://www.pebio.com/ab/about/dna/377/). The resulting genomic DNA sequence is analysed using GENSCAN (Burge and Karlin, J. Mol. Biol. 268:78-94) and GENEMARK version 2.4 (Borodovsky and McIninch, Comp. Chem. 17:123-133) gene-finding programs and BLAST (Altschul et al., J. Mol. Biol. 215:403-410) homology searches against public protein, EST and DNA databases (SWISSPROT, SWISSPROTPLUS, GenBank, Genbank updates, EMBL, GENEMBLPLUS, GenBank EST, EMBL EST, GenBank STS, EMBL STS), the results of which are parsed into a human chromosome 5-specific version of ACeDb ( A   C .  e legans  D ata b ase; http://www.sanger.ac.uk/Software/Acedb/) for graphic display. From this graphic display significant regions (i.e. genes) are identified by predicted exons and aligned EST/protein hits. A gene AAG6 is initially identified on the graphic display as a GENSCAN-predicted gene covering 20 kb of genomic DNA and comprising 8 exons ranging in size from 69-2816 bp:  
                                           Nucleotide position in           GENSCAN-Predicted Exon   SEQ ID NO:5   Exon Size (bp)                                            1   1001-1084   84       2   4313-4534   222       3   8446-8551   106       4    8622-11437   2816       5   14819-14916   98       6   16039-16107   69       7   16826-16977   152       8   20604-21028   425                  
 
     [0089] The DNA sequences in GENSCAN-predicted exons 4, 5, 7 and 8 encode a protein having homologies to cadherin-type molecules in a range of organisms, including humans, which suggest that it is a member of the cadherin protein family. Whereas most of the protein homologies are around 40%, a homology of 80% is detected with mouse vascular endothelial cadherin 2 (MVEC2; Telo et al., J. Biol. Chem. 273:17565-17572) indicating that AAG6 may be the human equivalent of MVEC2. In addition, an 83% homology to the MVEC2 mRNA sequence is also detected. BLAST searches against the TIGR database (www.TIGR.ORG) using a 425 bp DNA sequence corresponding to GENSCAN-predicted exon 8 from AAG6 identified on the graphic display reveals a 478 bp EST (TIGR accession no: THC 204848) with 98% sequence identity to the predicted exon. A plasmid clone containing a ˜3.2 kilobase cDNA insert is isolated from a human lung cDNA library of Origene Technologies Inc. (Rockville, Md., USA) by PCR using primers designed using THC 20848 and having SEQ ID NOS: 64, 65 and 66.  
     [0090] The cDNA insert is sequenced using primer-directed walking. The resulting 3.09 kb of insert sequence (SEQ ID NO:1) is analysed using the EditSeq module of Lasergene software (DNASTAR, Inc., Madison, Wis., USA). The cDNA sequence maps to BAC DNA sequence corresponding to GENSCAN-predicted exons 4, 5, 7 and 8. Further EditSeq analysis reveals an open reading frame (ORF) of 2436 nucleotides coding for a protein of 811 amino acids in length (SEQ ID NO:2). Alignment of the AAG6 and MVEC2 protein sequences using the MegAlign module of Lasergene software reveals that both proteins are highly homologous but that 377 amino acids encoded by nucleotides 299-1427 of the MVEC2 cDNA (Telo et al., J. Biol. Chem. 273:17565-175) are absent from the translated AAG6 ORF, indicating that the AAG6 cDNA sequence isolated from the Origene library is incomplete. BLAST analysis using nucleotides 1-1427 of the MVEC2 cDNA sequence indicates that a 916 nucleotide region of genomic DNA (8550 to 9465 in SEQ ID NO:5) 5′ of the AAG6 cDNA sequence may code for the missing amino acids in the AAG6 protein sequence. Therefore, this DNA sequence is appended to the AAG6 cDNA sequence and the new sequence of 4.006 kb (SEQ ID NO:3) is analysed using EditSeq. An ORF of 3564 nucleotides is revealed (from nucleotide position 9-3572 in SEQ ID NO:3) which codes for a protein of 1187 amino acids in size (SEQ ID NO:4). Alignment of this protein sequence with the MVEC2 protein sequence using MegAlign reveals that the amino acids missing in the translation of the ORF of the initial 3.09 kb cDNA sequence are now present.  
     [0091] Analysis of the AAG6 protein sequence using the Protean module of the Lasergene software as well as comparison with the MVEC2 protein structure shows the presence of an extracellular region that can be divided into 6 domains. Five peptide motifs ([L,I,V]X[L,I,V]XDXND[N,H]XP) (where X is any amino acid) found in classical cadherin molecules are found in the extracellular domain. In addition, the protein contains a transmembrane domain and a large cytoplasmic region. The high homology with the MVEC2 protein indicates that AAG6 is the human equivalent of this protein and may display the same functional properties as MVEC2.  
     [0092] Using a 404 bp PCR fragment generated from human genomic DNA using primers having SEQ ID NOS:64 and 65, a northern blot of mRNA from a number of human tissues (human 12-lane MTN blot; Clontech Laboratories UK Ltd., Basingstoke, Hampshire, UK) is probed to examine the expression pattern of AAG6. A single hybridising band of ˜4.5 kb is detected in heart, spleen, placenta, kidney, skeletal muscle, liver and lung. Very faint or no hybridisation is detected for small intestine, brain, colon, thymus and peripheral blood lymphocytes. PCR analysis of first-strand cDNAs derived from 24 different tissues (Rapid-Scan™ Gene Expression Panel; OriGene Technologies Inc., Rockville, Md., USA) using primers having SEQ ID NOS:62 and 63 confirms the Northern blot results. Probing a northern blot of HUVEC and NHBE mRNA with the 404 bp probe shows that the AAG6 gene is expressed in endothelial cells but not in epithelial cells. The expression pattern observed for the AAG6 gene parallels that observed for MVEC2 (Telo et al., J. Biol. Chem. 273:17565-17572).  
     [0093] The size of the hybridising mRNA band detected on northern blots (˜4.5 kb) indicates that SEQ ID NO:3 (4.006 kb) may still not represent the entire messenger RNA from AAG6 and that further non-translated exonic sequence might exist, possibly in the chromosomal DNA corresponding to GENSCAN-predicted exons 1-3 which are not encompassed by SEQ ID NO:3 (SEQ ID NO:3 contains DNA sequences corresponding to GENSCAN-predicted exons 4,5,7 and 8).  
     EXAMPLE 2  
     [0094] In this example conformation sensitive gel electrophoresis (CSGE: Ganguly et al., Proc. Natl. Acad. Sci. USA 90:10325-10329; Ganguly and Williams, Hum. Mut. 9:339-343) is used to detect potential sequence changes in PCR-amplified DNA fragments from blood DNA isolated from asthmatic patients. Single base mismatches in DNA heteroduplexes are detected by polyacrylamide gel electrophoresis in the presence of mildly denaturing solvents which amplify the tendency of mismatches to produce conformational changes and result in differential migration of homo-duplexes and heteroduplexes. To generate heteroduplexes, amplified PCR products are thermally denatured, annealed, then analysed by polyacrylamide gel electrophoresis. DNA fragments are visualised by ethidium bromide staining. DNA fragments showing differential electrophoretic migration patterns are then sequenced to confirm the presence of a change to the polynucleotide sequence and the exact nature of this change.  
     [0095] SEQ ID NO:3 is aligned with SEQ ID NO: 5 using the Align module of the FASTA package (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448) and four exons are identified:  
                                               Nucleotide position   Nucleotide position           Exon   in SEQ ID NO:3   in SEQ ID NO:5   Exon Size (bp)                                                1     1-2888    8550-11437   2888       2   2889-2987   14819-14917   99       3   2988-3140   16827-16979   153       4   3141-3984   20606-21440   835                  
 
     [0096] PCR primer sets covering the exons (including the exon-intron boundaries), the first 1 kb of intron 1 and 1 kb of the 3′-end of the AAG6 gene are designed using SEQ ID NO:5 and Primer Express™ (version 1.0; Perkin Elmer, P/N 604313). These primer sets (SEQ ID NOS: 6-61) are:  
                                       Primer Set   Forward Primer   Reverse primer                  Exon 1.1   GCCGGCAGCTCTGGG   CTGAGCAAGCCTTCCTCAGAGT       Exon 1.2   CACTCTCACGGTGAAATACCAAGT   GGATCCGGGTTCGCAGA       Exon 1.3   CCTTTGATGTGCTTGCCACA   TTGGAGTCCAAGACGTTGACCT       Exon 1.4   TGTCATTGTGGGCCCTGAT   TCGACGCAGAATGACCTGG       Exon 1.5   CGCTTCTCATAAAACTGACCGC   TTGTGTCCTCAATCCAAGTCATCT       Exon 1.6   CAGCCCATTGCAAAGTTCTCA   GGTGCATTGTCGTTGATGTCAC       Exon 1.7   CAGGACACAATGGTTTGGTCC   CAATAGCTACTAAGTGAGCAACTGGG       Exon 1.8   TGACATCAACGACAATGCACC   ACGGAGAGGCTGGCTTTTC       Exon 1.9   GCTCACTTAGTAGCTATTGACTCCAACA   TGCCCCCGAGTCTGCA       Exon 1.10   CTCTCCGTGCTTGTGAATGC   CTGAGTCCCTCAGGTGGTCC       Exon 1.11   TTCATCCTCAACCCTCATACGG   GCACGAGGTGGATGTCTGC       Exon 1.12   TGTACTGTTGGGCATCTTCGG   GAGGTTGACCGTGTCTTGCAG       Exon 1.13   GGGCAGTCCCACAAAGATGT   TGAGATGTCGCTGCCGTCT       Exon 1.14   GAGAACCTGAACCTTCCCGAG   GAGGACTGTGCAGGTTATGGG       Intron 1.1   GTCCGGCTGTCTGTGGCT   GGCACCATAAATGCCTTATAGGA       Intron 1.2   CTTAGCTGTGTGATCCTAGGCAAAT   TGAGTACTGACTACATGTCAGATGGACA       Intron 1.3   ACTTCTTCCTAGCTCTGAACCACAG   AACTCCAATCTGCCTAACTGCTAAG       Intron 1.4   ATGAAGAAGGCCAGCATAGGC   GATATGGGTGTGAGATCAGGCA       Exon 2   ACCATCCCCCATCTGATCAC   GGATGCTGTCTGCTGCACC       Exon 3   CCAGGCTGTGTATCCAGTTCC   CCTCCACAGAAACAGAATGGC       Exon 4.1   CCCTTGCCCTGCTCATTG   CCTCCCGCAGACCGAGA       Exon 4.2   CTGAGCCCAACAGGCACG   AGTCACCCTAAAGTTCTCAGGCC       Exon 4.3   CCCAGGTGGAAAGACGGG   TCCTGCCTGTGATACTCCGTG       Exon 4.4   AAGAGCCCCAGGACTAACAGC   CTGTCCAGGCCCAGTGTGA       3′ 1.1   GGGTGCCAGGAAATGCTCT   GGGTTAATAATACCTCCCTCCCA       3′ 1.2   ATGTTACCAACTAATCTGCTTCTCAGC   TCCTTTCAACAGTGTTTCCCG       3′ 1.3   CTGGGCCAACACCAAAACC   GAAGGCTGGCTGCTGTCAGT       3′ 1.4   AAGTAGCTCTTCAAGTGCGGATG   TATGATGCACATTATCCCTAGTTGG                  
 
     [0097] Using the above primer sets, 28 polynucleotides are amplified from blood DNA samples from 16 asthmatic patients. PCR reactions are carried out in a reaction volume of 10 μl containing 1×GeneAmpe 10×PCR buffer (Perkin Elmer P/N N808-0240), 13 ng of template DNA, 400 μM of each dNTP (Amersham Life Science Nucleix Plus ™ 25 mM dNTP mix; Prod. No. US77119), 30 ng of each primer, 2 mM MgCl 2  and 0.5 u of AmpliTaq Gold™ polymerase (Perkin-Elmer P/N N808-0242).  
     [0098] Typical thermal cycling conditions using a Biometra UNO II cycler (Part No. 050-603; Anachem Ltd., Luton, UK) are as follows, the sequence Step 2-Step 3-Step 4 being repeated 36 times:  
                                          Step 1   95° C.   10 min       Step 2   92° C.    1 min       Step 3   60° C.    1 min       Step 4   72° C.    2 min       Step 5   72° C.   10 min                  
 
     [0099] To generate heteroduplexes, 2 μl of PCR product is denatured at 95° C. for 10 minutes and annealed at 68° C. for 30 minutes using a thermal cycler (eg. Biometra UNOII). 2 μl of 2×loading buffer (20% ethylene glycol, 30% formamide, 0.025% xylene cyanol, 0.025% bromphenol blue) is added to each sample before gel analysis.  
     [0100] A standard DNA sequencing apparatus (Owl Scientific S3S; Autogen Bioclear UK Ltd.) is used with a 60 sample comb (Owl Scientific S2S-60A; Autogen Bioclear UK Ltd.) and standard power supply (Biorad, Cat No. 165-5057) equipped with a temperature probe (Biorad, Cat No. 165-5058). A 0.4 mm thick 15% polyacrylamide gel is prepared using a 99:1 ratio of acrylamide to BAP cross linker, 10% ethylene glycol and 15% formamide in 0.5 ×TTE. Gels are pre-run for one hour at 30 watts, limiting the temperature to a maximum of 25ûC (using an electric fan to keep the temperature down if necessary eg. Jencons, Cat No. 292-004). After the pre-run, the wells are flushed with a pipette and the samples are loaded into the wells. The gel is then electrophoresed at 12 watts overnight (15 hours) at 25° C. Fragments greater than 350 bp remain on the gel.  
     [0101] After electrophoresis, the gel plates are separated. The gel is stained by placing the gel in 0.5×TTE containing 1 μg/ml ethidium bromide (Biorad, Cat No. 161-0433) for 10 minutes, followed by destaining in 0.5×TTE for 10 minutes. The gel is then photographed on a UV transilluminator (eg. UVP GDS 7500).  
     [0102] Potential polynucleotide changes are detected by CSGE in one or more of the 16 patients for  18   0 f the 28 PCR fragments. For each of these potential changes, the PCR fragment from all  16  patients is subjected to double stranded DNA sequencing on an ABI377 automated sequencer using standard methods (http://www.pebio.com/ab/about/dna/377/) and the resulting DNA sequence is analysed using CONSED software (Gordon et al., Genome Res. 8:195-202) to confirm the presence of a sequence change and to identify the exact base change. All of the 18 potential changes detected by CSGE are confirmed. These changes are shown in the table below, in which #patients indicates the number of patients exhibiting the polymorphism:  
                                           Exon   Alias   Polymorphism   # patients                                                385   Exon 1.1   C to T   8       386   Exon 1.2   A to G   14       390   Exon 1.6   A to C   12       393   Exon 1.9   G to C   1       394   Exon 1.10   G to A   6       395   Exon 1.11   T to C   12           Exon 1.11   C to T   10       399   Intron 1.1   A to G   15       400   Intron 1.2   C to T   5       401   Intron 1.3   C to T   1       402   Intron 1.4   C to T   2           Intron 1.4   C to T   3           Intron 1.4   1 bp deletion   11       403   Exon 2   G to A   3       406   Exon 4.2   9 bp insertion   16       408   Exon 4.4   G to A   15       411   3′.3   T to C   1       412   3′.4   G to C   1                  
 
     [0103] Sixteen of the detected polynucleotide changes are single nucleotide polymorphisms (SNPs), one is a deletion and the remaining change is a 9bp insertion EXAMPLE 3  
     [0104] This Example relates to the expression of full length AAG6 with a 6 histidine tag at the C-terminus using the Baculovirus system in  T.ni  Hi5 cells, and to the purification of the resulting polypeptide. A recombinant fragment spanning the extracellular domain of AAG6 is generated as described for MVEC2 (Telo et al., J. Biol. Chem. 273:17565-17572).  
     [0105] 1. Construction of a Recombinant AAG6 Baculovirus  
     [0106] A unique EcoRI site is incorporated 5′ to the AAG6 start codon by PCR amplification using the following primer:  
     [0107] 5ST  
     [0108] 5′-GAAGATCTTCG GAATTC CATC ATG ATGCAACTTCTGCAACTTCTG-3′ 
     [0109] Another primer is used to introduce 6 histidine residues immediately prior to the AAG6 stop codon. This primer also incorporates a unique KpnI site 3′ to the AAG6 stop codon.  
     [0110] 3ST  
     [0111] 5′-   
     [0112] AAGATCTTC GGTACCTCAATGGTGATGGTGATGGTG CAGGCACCTGCTGCTGCTG-3′ 
     [0113] The recombinant “His tagged” version of AAG6 is ligated as a 2467 bp EcoRI/KpnI fragment into EcoRI/Kpnl digested pFastbac1 baculovirus transfer vector (Life Sciences). The recombinant AAG6 sequence is transposed into Bacmid DNA carried by DH10Bac cells (Life Sciences; Bac to Bac Baculovirus expression system). AAG6 recombinant Bacmids are isolated from DH10Bac cells and transfected into Sf9 cells using published protocols (Bac to Bac baculovirus expression system manual; Life Sciences).  
     [0114] 2. Amplification of recombinant Baculovirus stocks  
     [0115] The recombinant baculovirus is amplified by infecting Sf9 cells (maintained in SF900 SFMII medium; Life Sciences) at a cell density of 0.5×10 6  cells/ml and a multiplicity of infection (moi) of 0.01 for 96 hours. Sf9 cells are then centrifuged at 1000×g for 5 minutes. The supernatants containing high titre virus are stored at 4° C.  
     [0116] 3. Expression of recombinant AAG6 in Hi5 Cells  
     [0117] Hi5 cells (Invitrogen), maintained at densities of between 3×10 5  and 3×10 6  cells/ml in Excell 401 medium (JRH Biosciences; distributed by AMS Biotechnology in either shaker flasks (rotated at 90 RPM) or spinner flasks (stirring at 75 RPM) are infected with the amplified recombinant Baculovirus at a cell density of 2.0×10 6  at an moi of 2.0 for 60 hours. Following infection Hi5 cells are centrifuged at 1000×g for 5 minutes, the supernatants poured off and the cell pellets frozen at −80° C.  
     [0118] 4. Crude lysate preparation  
     [0119] The cells (1×10 9 ) are resuspended in 100 ml lysis buffer (20 mM Hepes pH 7.5, 100 mM NaCl, 5% glycerol, 2 mM E-mercaptoethanol, 0.5 mM imidazole, 0.1% Nonidet P-40, 40 μg/ml AEBSF, 0.5 μg/ml leupeptin, 1 μg/ml aprotinin and 0.7 μg/ml pepstatin A). Cells are incubated on ice for 15 min then centrifuged at 39,000×g for 30 min at 4° C. The sample is filtered through a 0.22 μm filter immediately prior to use.  
     [0120] 5. Metal chelate affinity chromatography  
     [0121] Metal chelate affinity chromatography is carried out at room temperature with a column attached to a BioCAD chromatography workstation. A 20 ml Poros MC/M (16 mmD×100 mmL) column is charged with Ni 2+  prior to use and after each injection. To charge with Ni 2+ , the column is washed with 10 column volumes (CV) 50 mM EDTA pH 8, 1 M NaCl followed by 10CV water. The column is charged with 500 ml 0.1 M NiSO4 pH 4.5-5, washed with 10CV water, then any unbound Ni 2+ removed by washing with 5CV 0.3 M NaCl. All steps are performed with a flow rate of 20 ml/min. The charged MC/M column is equilibrated with 5CV Buffer B (20 mM Hepes pH 7.5, 500 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol, 1 mM PMSF, 5 mM imidazole) to saturate the sites followed by 10CV Buffer A (as Buffer B except 0.5 mM imidazole). 90-95 ml of the crude lysate is loaded onto the column per run at a flow rate of 20 ml/min. Subsequent steps are carried out with a flow rate of 30 ml/min. Any unbound material is removed by washing with 12 CV buffer A and AAG6 eluted by applying a 0-50% Buffer B gradient over 10 CV. Fractions (8 ml) are collected over the gradient. AAG6-containing fractions are combined and protease inhibitors added to the final concentrations described for the lysis buffer above. DTT is also added to a final concentration of 1 mM. The combined fractions are dialysed overnight against 4 liters 20 mM Tris-HCl pH 7.5, 1 mM DTT, 0.2 mM PMSF at 4° C.  
     [0122] 6. Ion Echange (Anion Exchange) Chromatography  
     [0123] Resource™ Q chromatography is carried out at 4° C. with a column attached to an FPLC workstation (Amersham Pharmacia Biotech). A 6 ml Resource™ Q column (16 mmD×30 mmL) is equilibrated with 10 CV Buffer C (20 mM Tris-HCl pH 7.5, 1 mM DTT) at a flow rate of 2 ml/min. The dialysed metal chelate eluate is applied to the column and washed with 10 CV Buffer C. The protein is eluted by applying a 0-100% Buffer D gradient (20 mM Tris-HCl pH 7.5, 1 mM DTT, 1 M NaCl) over 10 CV. Fractions (3 ml) are collected on eluting the column.  
     [0124] 7. Gel Filtration  
     [0125] Gel Filtration chromatography is carried out at 4° C. with a column attached to a BioCAD SPRINT chromatography workstation (PE Biosystems). A 24 ml (10 mmD×300 mmL) Superdex 200 HR (Amersham Pharmacia Biotech) column is equilibrated with 10 CV Buffer E (20 mM Tris-HCl pH7.5, 1 mM DTT, 150 mM NaCl) at a flow rate of 0.5 ml/min. The ion exchange eluate is applied to the column and eluted with Buffer E. Fractions ( 1 ml) throughout the purification run are collected and analysed.  
     [0126] 8. Sample Concentration  
     [0127] Samples are concentrated approximately 10-fold using a Millipore Ultrafree-15 centrifugation device (MW cut-off 50 kDa) at 4° C. The device is pre-rinsed with water prior to use. The final storage buffer used for long term storage at−80° C. is 20 mM Hepes pH 7.5, 1 mM DTT, 100 mM NaCl, 5% glycerol. Glycerol can be omitted from the sample for storage at 4° C.  
     EXAMPLE 4  
     [0128] This example relates to the production of polyclonal antibodies against AAG6 protein purified as described in Example 3. Polyclonal antibodies against a recombinant fragment spanning the extracellular domain are generated as described by Telo et al. (J. Biol. Chem. 273:17565-17572).  
     Immunisation of Rabbits  
     [0129] Dutch rabbits (Harlen-Olac) are immunised at 4 subcutaneous sites with 500 μg purified AAG6 protein in PBS according to the following schedule:  
                                   DAYS   IMMUNISATIONS                   0   1 st  immunisation 1:1 in complete Freund&#39;s adjuvant       15   1 st  boost 1:1 in incomplete Freund&#39;s adjuvant       45   2 nd  boost 1:1 in incomplete Freund&#39;s adjuvant       55   1 st  test bleed from the ear artery       Every month   Boost 1:1 in incomplete Freund&#39;s adjuvant until a good           antibody response is obtained                  
 
     [0130] Test bleeds (500 μl) are taken and the serum assessed for antibody titre. Serum is collected when a maximum titre is reached. This is done by collecting blood (10 ml) and allowing it to clot for 2 hours at 4° C. The blood is centrifuged at 1000×g for 5 minutes to separate the serum. The serum is removed and stored at −20° C. until assayed.  
     ELISA Screening  
     [0131] Nunc-Immuno Plate Maxisorp 96 well plates (Nunc, Fisher Scientific UK, Loughborough, UK) are used as a solid support and coated with the purified AAG6 protein (100 ng/well) overnight at 4° C. The plates are blocked for 3 hours at 37° C. with PBS containing 2% BSA (Sigma) and 0.02% NaN 3  (Sigma). After blocking, plates are incubated overnight at room temperature with serum in different dilutions of PBS. The presence of polyclonal antibodies is checked with both biotin labelled IgG-antibodies to rabbit (Goat anti-rabbit IgG antiserum, 1:25000 dilution), with an incubation time of 40 min. Alkaline phosphatase conjugated streptavidin (Immununo Research, Dianova, CH) is then added at a dilution of 1:10000. Development of the reaction is carried out by adding an alkaline phosphatase substrate (Sigma, f.c. 1 mg/ml) dissolved in diethanolamine. After 45 min. absorbance is read at 405 nm with a reference of 490 nm with an ELISA plate reader (Bio-rad laboratories Ltd., Hemel Hempstead, UK).  
     Purification  
     [0132] 5 ml protein A-agarose is poured into a chromatography column and washed with 6 column volumes of 0.1 M tris (hydroxymethyl) methylamine (Tris) buffer pH 7.5. The rabbit serum containing anti-AAG6 antibodies is diluted (½) with Tris buffer and added to the protein A-agarose. Unbound proteins are removed by washing the column with 6 volumes of Tris buffer. The IgG is eluted off the column with three column volumes of 0.1 M glycine buffer pH 3.0 and collected as 1 ml fractions into tubes containing 28 μl of 1 M Tris. The fractions which are positive for protein content are checked for purity by SDS-PAGE under reducing conditions. Two bands at 50 and 25 Kd are visualised corresponding to the heavy and light chains of an immunoglobulin molecule. Fractions containing only immunoglobulin are pooled, re-checked for protein concentration and stored at −20° C.  
     EXAMPLE 5  
     [0133] This example describes the preparation of monoclonal antibodies against AAG6 protein purified as described in Example 3.  
     Immunisation of Mice  
     [0134] Female Balb/c mice are immunised intraperitoneally with 100 μg of AAG6 protein in PBS according to the schedule given below:  
                                                   DAYS   IMMUNISATIONS                           1   1 st  immunisation 1:1 with complete               Freund&#39;s adjuvant           14   1 st  boost 1:1 with incomplete Freund&#39;s               adjuvant           21   2 nd  boost 1:1 with incomplete Freund&#39;s               adjuvant           28-30   Three final boosts in PBS           31   Fusion with mouse myeloma cells                      
 
     [0135] Serum is assessed for antibody titre by ELISA (Example 4) after the animal is sacrificed for the preparation of spleen cells for fusion. If antibody titre is sufficient, ({fraction (1/1000)} to {fraction (1/100,000)}), the hybridomas are screened, otherwise discarded.  
     Preparation of Myeloma Cells  
     [0136] Sp2/0 murine myeloma cells (ATCC #CRL 1581; maintained in culture medium containing 20 μg/ml 8-azaguanine) are cultivated for one week before fusion in RPMI 1640 (8-azaguanine is not included), 10% (v/v) FCS and 1% penicillin-streptomycin (50IU/ml and 50 μg/ml, respectively). The cells are harvested by centrifugation (200×g for 5 min) and washed three times in cold RPMI 1640. Approximately 2.5×10 6  cells are used per 96 well microtitre plate.  
     Preparation of Spleen Cell Suspension  
     [0137] The mouse is killed by an overdose of anesthetic (Forene), the spleen dissected and pressed through a cell strainer (70 μm mesh cell strainer; Becton &amp; Dickinson, Oxford, UK, Cat. No 2350). The cell suspension is washed three times in RPMI 1640 (as above) and counted: 5.10 6  cells/96 well plate are necessary.  
     Fusion of Myeloma Cells and Spleen Cells  
     [0138] The spleen and myeloma cells are mixed (2:1), centrifuged (200×g for 5 min) and the pellet warmed in a 37° C. water bath. Prewarmed polyethylene glycol 4000 ( 1 ml per 10 8  cells) is added slowly over one minute, then 20 ml of prewarmed wash medium over two minutes. After centrifugation the pellet is carefully resuspended in selection medium (RPMI 1640, 10% FCS, 1% penicillin-streptomycin, 10% BM condimed H1 (feeder cell replacement from Boehringer Mannheim, Lewes, UK; Cat. No. 1 088 947), 10% HAT-media supplement (hypoxanthine, aminopterin and thymidine to select against unfused myeloma cells; Boehringer Mannheim, Lewes, UK; Cat. No. 644 579) and plated, 200 μl/well of a 96 well microtitre plate.  
     [0139] After five days clusters of hybrid cells can be identified by examining the bottom of the microtitre wells with an inverted microscope. After 10-14 days the culture supernatant is tested for the presence of antibodies by ELISA (example 4). The positive clones are expanded in a 24 well assay plate and retested.  
     Cloning of Positive Hybridomas  
     [0140] The expanded clones which are still positive are cloned by limiting dilution. Cells are diluted serially in four dilutions steps in a 96 well microtitre plate; 5, 2, 1 and 0.5 cells/well. HAT-media supplement is replaced with HT-media supplement (Boehringer Mannheim, Lewes, UK; Cat. No. 623 091). After approximately one week the cells are screened by ELISA (Example 4). The cells of those wells containing a single positive clone are expanded.  
     Production of Monoclonal Antibody Supernatant  
     [0141] The cells are grown in culture flasks in standard medium (RPMI 1640, 10% (v/v) FCS and 1% penicillin-streptomycin) until the hybridomas overgrow and die. The debris is removed by centrifugation and the supernatant containing the antibodies is titred using ELISA (Example 4) before storing under sterile conditions at 4° C., −20° C. or −70° C. .  
    
     
       
         1 
         
           
             70  
           
           
             1  
             3090  
             DNA  
             Homo sapiens  
           
            1 

ggtcattctg cgtcgacctc tagactatga aaagaaccct gcctacgagg tggatgttca     60 

ggcaagggac ctgggtccca atcctatccc agcccattgc aaagttctca tcaaggttct    120 

ggatgtcaat gacaacatcc caagcatcca cgtcacatgg gcctcccagc catcactggt    180 

gtcagaagct cttcccaagg acagttttat tgctcttgtc atggcagatg acttggattc    240 

aggaaacaat ggtttggtcc actgctggct gagccaagag ctgggccact tcaggctgaa    300 

aagaactaat ggcaacacat acatgttgct aaccaatgcc acactggaca gagagcagtg    360 

gcccaaatat accctcactc tgttagccca agaccaagga ctccagccct tatcagccaa    420 

gaaacagctc agcattcaga tcagtgacat caacgacaat gcacctgtgt ttgagaaaag    480 

caggtatgaa gtctccacgc gggaaaacaa cttaccctct cttcacctca ttaccatcaa    540 

ggctcatgat gcagacttgg gcattaatgg aaaagtctca taccgcatcc aggactcccc    600 

agttgctcac ttagtagcta ttgactccaa cacaggagag gtcactgctc agaggtcact    660 

gaactatgaa gagatggccg gctttgagtt ccaggtgatc gcagaggaca gcgggcaacc    720 

catgcttgca tccagtgtct ctgtgtgggt cagcctcttg gatgccaatg ataatgcccc    780 

agaggtggtc cagcctgtgc tcagcgatgg aaaagccagc ctctccgtgc ttgtgaatgc    840 

ctccacaggc cacctgctgg tgcccatcga gactcccaat ggcttgggcc cagcgggcac    900 

tgacacacct ccactggcca ctcacagctc ccggccattc cttttgacaa ccattgtggc    960 

aagagatgca gactcggggg caaatggaga gcccctctac agcatccgca gtggaaatga   1020 

agcccacctc ttcatcctca accctcatac ggggcagctg ttcgtcaatg tcaccaatgc   1080 

cagcagcctc attgggagtg agtgggagct ggagatagta gtagaggacc agggaagccc   1140 

ccccttacag acccgagccc tgttgagggt catgtttgtc accagtgtgg accacctgag   1200 

ggactcagcc cgcaagcctg gggctttgag catgtcgatg ctgacggtga tctgcctggc   1260 

tgtactgctg ggcatcttcg ggttgatcct ggctttgttc atgtccatct gccggacaga   1320 

aaagaaggac aacagggcct acaactgtcg ggaggccgag tccacctacc gccagcagcc   1380 

caagaggccc cagaaacaca ttcagaaggc agacatccac ctcgtgcctg tgctcagggg   1440 

tcaggcaggt gagccttgtg aagtcgggca gtcccacaaa gatgtggaca aggaggcgat   1500 

gatggaagca ggctgggacc cctgcctgca ggcccccttc cacctcaccc cgaccctgta   1560 

caggacgctg cgtaatcaag gcaaccaagg agcaccggcg gagagccgag aggtgctgca   1620 

agacacggtc aacctccttt tcaaccatcc caggcagagg aatgcctccc gggagaacct   1680 

gaaccttccc gagccccagc ctgccacagg ccagccacgt tccaggcctc tgaaggttgc   1740 

aggcagcccc acagggaggc tggctggaga ccagggcagt gaggaagccc cacagaggcc   1800 

accagcctcc tctgcaaccc tgagacggca gcgacatctc aatggcaaag tgtcccctga   1860 

gaaagaatca gggccccgtc agatcctgcg gagcctggtc cggctgtctg tggctgcctt   1920 

cgccgagcgg aaccccgtgg aggagctcac tgtggattct cctcctgttc agcaaatctc   1980 

ccagctgctg tccttgctgc atcagggcca attccagccc aaaccaaacc accgaggaaa   2040 

taagtacttg gccaagccag gaggcagcag gagtgcaatc ccagacacag atggcccaag   2100 

tgcaagggct ggaggccaga cagacccaga acaggaggaa gggcctttgg atcctgaaga   2160 

ggacctctct gtgaagcaac tgctagaaga agagctgtca agtctgctgg accccagcac   2220 

aggtctggcc ctggaccggc tgagcgcccc tgacccggcc tggatggcga gactctcttt   2280 

gcccctcacc accaactacc gtgacaatgt gatctccccg gatgctgcag ccacggagga   2340 

gccgaggacc ttccagacgt tcggcaaggc agaggcacca gagctgagcc caacaggcac   2400 

gaggctggcc agcacctttg tctcggagat gagctcactg ctggagatgc tgctggaaca   2460 

gcgctccagc atgcccgtgg aggccgcctc cgaggcgctg cggcggctct cggtctgcgg   2520 

gaggaccctc agtttagact tggccaccag tgcagcctca ggcatgaaag tgcaagggga   2580 

cccaggtgga aagacgggga ctgagggcaa gagcagaggc agcagcagca gcagcagcag   2640 

cagcaggtgc ctgtgaacat acctcagacg cctctggatc caagaaccag gggcctgagg   2700 

atctgtggac aagagctggt ttctaaaatc ttgtaactca ctagctagcg gcggcctgag   2760 

aactttaggg tgactgatgc tacccccaca gaggaggcaa gagccccagg actaacagct   2820 

gactgaccaa agcagcccct tgtaagcagc tctgagtctt ttggaggaca gggacggttt   2880 

gtggctgaga taagtgtttc ctggcaaaac atatgtggag cacaaagggt cagtcctctg   2940 

gcagaacaga tgccacggag tatcacaggc aggaaaggat ggccttcttg ggtagcagga   3000 

gtcagggggc tgtaccctgg gggtgccagg aaatgctctc tgacctatca ataaaggaaa   3060 

agcagtgaaa aaaaaaaaaa aaaaaaaaaa                                    3090 

 
           
             2  
             811  
             PRT  
             Homo sapiens  
           
            2 

Met Ala Asp Asp Leu Asp Ser Gly Asn Asn Gly Leu Val His Cys Trp 
  1               5                  10                  15 

Leu Ser Gln Glu Leu Gly His Phe Arg Leu Lys Arg Thr Asn Gly Asn 
             20                  25                  30 

Thr Tyr Met Leu Leu Thr Asn Ala Thr Leu Asp Arg Glu Gln Trp Pro 
         35                  40                  45 

Lys Tyr Thr Leu Thr Leu Leu Ala Gln Asp Gln Gly Leu Gln Pro Leu 
     50                  55                  60 

Ser Ala Lys Lys Gln Leu Ser Ile Gln Ile Ser Asp Ile Asn Asp Asn 
 65                  70                  75                  80 

Ala Pro Val Phe Glu Lys Ser Arg Tyr Glu Val Ser Thr Arg Glu Asn 
                 85                  90                  95 

Asn Leu Pro Ser Leu His Leu Ile Thr Ile Lys Ala His Asp Ala Asp 
            100                 105                 110 

Leu Gly Ile Asn Gly Lys Val Ser Tyr Arg Ile Gln Asp Ser Pro Val 
        115                 120                 125 

Ala His Leu Val Ala Ile Asp Ser Asn Thr Gly Glu Val Thr Ala Gln 
    130                 135                 140 

Arg Ser Leu Asn Tyr Glu Glu Met Ala Gly Phe Glu Phe Gln Val Ile 
145                 150                 155                 160 

Ala Glu Asp Ser Gly Gln Pro Met Leu Ala Ser Ser Val Ser Val Trp 
                165                 170                 175 

Val Ser Leu Leu Asp Ala Asn Asp Asn Ala Pro Glu Val Val Gln Pro 
            180                 185                 190 

Val Leu Ser Asp Gly Lys Ala Ser Leu Ser Val Leu Val Asn Ala Ser 
        195                 200                 205 

Thr Gly His Leu Leu Val Pro Ile Glu Thr Pro Asn Gly Leu Gly Pro 
    210                 215                 220 

Ala Gly Thr Asp Thr Pro Pro Leu Ala Thr His Ser Ser Arg Pro Phe 
225                 230                 235                 240 

Leu Leu Thr Thr Ile Val Ala Arg Asp Ala Asp Ser Gly Ala Asn Gly 
                245                 250                 255 

Glu Pro Leu Tyr Ser Ile Arg Ser Gly Asn Glu Ala His Leu Phe Ile 
            260                 265                 270 

Leu Asn Pro His Thr Gly Gln Leu Phe Val Asn Val Thr Asn Ala Ser 
        275                 280                 285 

Ser Leu Ile Gly Ser Glu Trp Glu Leu Glu Ile Val Val Glu Asp Gln 
    290                 295                 300 

Gly Ser Pro Pro Leu Gln Thr Arg Ala Leu Leu Arg Val Met Phe Val 
305                 310                 315                 320 

Thr Ser Val Asp His Leu Arg Asp Ser Ala Arg Lys Pro Gly Ala Leu 
                325                 330                 335 

Ser Met Ser Met Leu Thr Val Ile Cys Leu Ala Val Leu Leu Gly Ile 
            340                 345                 350 

Phe Gly Leu Ile Leu Ala Leu Phe Met Ser Ile Cys Arg Thr Glu Lys 
        355                 360                 365 

Lys Asp Asn Arg Ala Tyr Asn Cys Arg Glu Ala Glu Ser Thr Tyr Arg 
    370                 375                 380 

Gln Gln Pro Lys Arg Pro Gln Lys His Ile Gln Lys Ala Asp Ile His 
385                 390                 395                 400 

Leu Val Pro Val Leu Arg Gly Gln Ala Gly Glu Pro Cys Glu Val Gly 
                405                 410                 415 

Gln Ser His Lys Asp Val Asp Lys Glu Ala Met Met Glu Ala Gly Trp 
            420                 425                 430 

Asp Pro Cys Leu Gln Ala Pro Phe His Leu Thr Pro Thr Leu Tyr Arg 
        435                 440                 445 

Thr Leu Arg Asn Gln Gly Asn Gln Gly Ala Pro Ala Glu Ser Arg Glu 
    450                 455                 460 

Val Leu Gln Asp Thr Val Asn Leu Leu Phe Asn His Pro Arg Gln Arg 
465                 470                 475                 480 

Asn Ala Ser Arg Glu Asn Leu Asn Leu Pro Glu Pro Gln Pro Ala Thr 
                485                 490                 495 

Gly Gln Pro Arg Ser Arg Pro Leu Lys Val Ala Gly Ser Pro Thr Gly 
            500                 505                 510 

Arg Leu Ala Gly Asp Gln Gly Ser Glu Glu Ala Pro Gln Arg Pro Pro 
        515                 520                 525 

Ala Ser Ser Ala Thr Leu Arg Arg Gln Arg His Leu Asn Gly Lys Val 
    530                 535                 540 

Ser Pro Glu Lys Glu Ser Gly Pro Arg Gln Ile Leu Arg Ser Leu Val 
545                 550                 555                 560 

Arg Leu Ser Val Ala Ala Phe Ala Glu Arg Asn Pro Val Glu Glu Leu 
                565                 570                 575 

Thr Val Asp Ser Pro Pro Val Gln Gln Ile Ser Gln Leu Leu Ser Leu 
            580                 585                 590 

Leu His Gln Gly Gln Phe Gln Pro Lys Pro Asn His Arg Gly Asn Lys 
        595                 600                 605 

Tyr Leu Ala Lys Pro Gly Gly Ser Arg Ser Ala Ile Pro Asp Thr Asp 
    610                 615                 620 

Gly Pro Ser Ala Arg Ala Gly Gly Gln Thr Asp Pro Glu Gln Glu Glu 
625                 630                 635                 640 

Gly Pro Leu Asp Pro Glu Glu Asp Leu Ser Val Lys Gln Leu Leu Glu 
                645                 650                 655 

Glu Glu Leu Ser Ser Leu Leu Asp Pro Ser Thr Gly Leu Ala Leu Asp 
            660                 665                 670 

Arg Leu Ser Ala Pro Asp Pro Ala Trp Met Ala Arg Leu Ser Leu Pro 
        675                 680                 685 

Leu Thr Thr Asn Tyr Arg Asp Asn Val Ile Ser Pro Asp Ala Ala Ala 
    690                 695                 700 

Thr Glu Glu Pro Arg Thr Phe Gln Thr Phe Gly Lys Ala Glu Ala Pro 
705                 710                 715                 720 

Glu Leu Ser Pro Thr Gly Thr Arg Leu Ala Ser Thr Phe Val Ser Glu 
                725                 730                 735 

Met Ser Ser Leu Leu Glu Met Leu Leu Glu Gln Arg Ser Ser Met Pro 
            740                 745                 750 

Val Glu Ala Ala Ser Glu Ala Leu Arg Arg Leu Ser Val Cys Gly Arg 
        755                 760                 765 

Thr Leu Ser Leu Asp Leu Ala Thr Ser Ala Ala Ser Gly Met Lys Val 
    770                 775                 780 

Gln Gly Asp Pro Gly Gly Lys Thr Gly Thr Glu Gly Lys Ser Arg Gly 
785                 790                 795                 800 

Ser Ser Ser Ser Ser Ser Ser Ser Arg Cys Leu 
                805                 810 

 
           
             3  
             4006  
             DNA  
             Homo sapiens  
           
            3 

cggtaagcat gatgcaactt ctgcaacttc tgctggggct tttggggcca ggtggctact     60 

tatttctttt aggggattgt caggaggtga ccactctcac ggtgaaatac caagtgtcag    120 

aggaagtgcc atctggtaca gtgatcggga agctgtccca ggaactgggc cgggaggaga    180 

ggcggaggca agctggggct gccttccagg tgttgcagct gcctcaggcg ctccccattc    240 

aggtggactc tgaggaaggc ttgctcagca caggcaggcg gctggatcga gagcagctgt    300 

gccgacagtg ggatccctgc ctggtttcct ttgatgtgct tgccacaggg gatttggctc    360 

tgatccatgt ggagatccaa gtgctggaca tcaatgacca ccagccacgg tttcccaaag    420 

gcgagcagga gctggaaatc tctgagagcg cctctctgcg aacccggatc cccctggaca    480 

gagctcttga cccagacaca ggccctaaca ccctgcacac ctacactctg tctcccagtg    540 

agcactttgc cttggatgtc attgtgggcc ctgatgagac caaacatgca gaactcatag    600 

tggtgaagga gctggacagg gaaatccatt cattttttga tctggtgtta actgcctatg    660 

acaatgggaa cccccccaag tcaggtacca gcttggtcaa ggtcaacgtc ttggactcca    720 

atgacaatag ccctgcgttt gctgagagtt cactggcact ggaaatccaa gaagatgctg    780 

cacctggtac gcttctcata aaactgaccg ccacagaccc tgaccaaggc cccaatgggg    840 

aggtggagtt cttcctcagt aagcacatgc ctccagaggt gctggacacc ttcagtattg    900 

atgccaagac aggccaggtc attctgcgtc gacctctaga ctatgaaaag aaccctgcct    960 

acgaggtgga tgttcaggca agggacctgg gtcccaatcc tatcccagcc cattgcaaag   1020 

ttctcatcaa ggttctggat gtcaatgaca acatcccaag catccacgtc acatgggcct   1080 

cccagccatc actggtgtca gaagctcttc ccaaggacag ttttattgct cttgtcatgg   1140 

cagatgactt ggattcagga aacaatggtt tggtccactg ctggctgagc caagagctgg   1200 

gccacttcag gctgaaaaga actaatggca acacatacat gttgctaacc aatgccacac   1260 

tggacagaga gcagtggccc aaatataccc tcactctgtt agcccaagac caaggactcc   1320 

agcccttatc agccaagaaa cagctcagca ttcagatcag tgacatcaac gacaatgcac   1380 

ctgtgtttga gaaaagcagg tatgaagtct ccacgcggga aaacaactta ccctctcttc   1440 

acctcattac catcaaggct catgatgcag acttgggcat taatggaaaa gtctcatacc   1500 

gcatccagga ctccccagtt gctcacttag tagctattga ctccaacaca ggagaggtca   1560 

ctgctcagag gtcactgaac tatgaagaga tggccggctt tgagttccag gtgatcgcag   1620 

aggacagcgg gcaacccatg cttgcatcca gtgtctctgt gtgggtcagc ctcttggatg   1680 

ccaatgataa tgccccagag gtggtccagc ctgtgctcag cgatggaaaa gccagcctct   1740 

ccgtgcttgt gaatgcctcc acaggccacc tgctggtgcc catcgagact cccaatggct   1800 

tgggcccagc gggcactgac acacctccac tggccactca cagctcccgg ccattccttt   1860 

tgacaaccat tgtggcaaga gatgcagact cgggggcaaa tggagagccc ctctacagca   1920 

tccgcagtgg aaatgaagcc cacctcttca tcctcaaccc tcatacgggg cagctgttcg   1980 

tcaatgtcac caatgccagc agcctcattg ggagtgagtg ggagctggag atagtagtag   2040 

aggaccaggg aagccccccc ttacagaccc gagccctgtt gagggtcatg tttgtcacca   2100 

gtgtggacca cctgagggac tcagcccgca agcctggggc tttgagcatg tcgatgctga   2160 

cggtgatctg cctggctgta ctgctgggca tcttcgggtt gatcctggct ttgttcatgt   2220 

ccatctgccg gacagaaaag aaggacaaca gggcctacaa ctgtcgggag gccgagtcca   2280 

cctaccgcca gcagcccaag aggccccaga aacacattca gaaggcagac atccacctcg   2340 

tgcctgtgct caggggtcag gcaggtgagc cttgtgaagt cgggcagtcc cacaaagatg   2400 

tggacaagga ggcgatgatg gaagcaggct gggacccctg cctgcaggcc cccttccacc   2460 

tcaccccgac cctgtacagg acgctgcgta atcaaggcaa ccaaggagca ccggcggaga   2520 

gccgagaggt gctgcaagac acggtcaacc tccttttcaa ccatcccagg cagaggaatg   2580 

cctcccggga gaacctgaac cttcccgagc cccagcctgc cacaggccag ccacgttcca   2640 

ggcctctgaa ggttgcaggc agccccacag ggaggctggc tggagaccag ggcagtgagg   2700 

aagccccaca gaggccacca gcctcctctg caaccctgag acggcagcga catctcaatg   2760 

gcaaagtgtc ccctgagaaa gaatcagggc cccgtcagat cctgcggagc ctggtccggc   2820 

tgtctgtggc tgccttcgcc gagcggaacc ccgtggagga gctcactgtg gattctcctc   2880 

ctgttcagca aatctcccag ctgctgtcct tgctgcatca gggccaattc cagcccaaac   2940 

caaaccaccg aggaaataag tacttggcca agccaggagg cagcaggagt gcaatcccag   3000 

acacagatgg cccaagtgca agggctggag gccagacaga cccagaacag gaggaagggc   3060 

ctttggatcc tgaagaggac ctctctgtga agcaactgct agaagaagag ctgtcaagtc   3120 

tgctggaccc cagcacaggt ctggccctgg accggctgag cgcccctgac ccggcctgga   3180 

tggcgagact ctctttgccc ctcaccacca actaccgtga caatgtgatc tccccggatg   3240 

ctgcagccac ggaggagccg aggaccttcc agacgttcgg caaggcagag gcaccagagc   3300 

tgagcccaac aggcacgagg ctggccagca cctttgtctc ggagatgagc tcactgctgg   3360 

agatgctgct ggaacagcgc tccagcatgc ccgtggaggc cgcctccgag gcgctgcggc   3420 

ggctctcggt ctgcgggagg accctcagtt tagacttggc caccagtgca gcctcaggca   3480 

tgaaagtgca aggggaccca ggtggaaaga cggggactga gggcaagagc agaggcagca   3540 

gcagcagcag cagcagcagc aggtgcctgt gaacatacct cagacgcctc tggatccaag   3600 

aaccaggggc ctgaggatct gtggacaaga gctggtttct aaaatcttgt aactcactag   3660 

ctagcggcgg cctgagaact ttagggtgac tgatgctacc cccacagagg aggcaagagc   3720 

cccaggacta acagctgact gaccaaagca gccccttgta agcagctctg agtcttttgg   3780 

aggacaggga cggtttgtgg ctgagataag tgtttcctgg caaaacatat gtggagcaca   3840 

aagggtcagt cctctggcag aacagatgcc acggagtatc acaggcagga aaggatggcc   3900 

ttcttgggta gcaggagtca gggggctgta ccctgggggt gccaggaaat gctctctgac   3960 

ctatcaataa aggaaaagca gtgaaaaaaa aaaaaaaaaa aaaaaa                  4006 

 
           
             4  
             1187  
             PRT  
             Homo sapiens  
           
            4 

Met Met Gln Leu Leu Gln Leu Leu Leu Gly Leu Leu Gly Pro Gly Gly 
  1               5                  10                  15 

Tyr Leu Phe Leu Leu Gly Asp Cys Gln Glu Val Thr Thr Leu Thr Val 
             20                  25                  30 

Lys Tyr Gln Val Ser Glu Glu Val Pro Ser Gly Thr Val Ile Gly Lys 
         35                  40                  45 

Leu Ser Gln Glu Leu Gly Arg Glu Glu Arg Arg Arg Gln Ala Gly Ala 
     50                  55                  60 

Ala Phe Gln Val Leu Gln Leu Pro Gln Ala Leu Pro Ile Gln Val Asp 
 65                  70                  75                  80 

Ser Glu Glu Gly Leu Leu Ser Thr Gly Arg Arg Leu Asp Arg Glu Gln 
                 85                  90                  95 

Leu Cys Arg Gln Trp Asp Pro Cys Leu Val Ser Phe Asp Val Leu Ala 
            100                 105                 110 

Thr Gly Asp Leu Ala Leu Ile His Val Glu Ile Gln Val Leu Asp Ile 
        115                 120                 125 

Asn Asp His Gln Pro Arg Phe Pro Lys Gly Glu Gln Glu Leu Glu Ile 
    130                 135                 140 

Ser Glu Ser Ala Ser Leu Arg Thr Arg Ile Pro Leu Asp Arg Ala Leu 
145                 150                 155                 160 

Asp Pro Asp Thr Gly Pro Asn Thr Leu His Thr Tyr Thr Leu Ser Pro 
                165                 170                 175 

Ser Glu His Phe Ala Leu Asp Val Ile Val Gly Pro Asp Glu Thr Lys 
            180                 185                 190 

His Ala Glu Leu Ile Val Val Lys Glu Leu Asp Arg Glu Ile His Ser 
        195                 200                 205 

Phe Phe Asp Leu Val Leu Thr Ala Tyr Asp Asn Gly Asn Pro Pro Lys 
    210                 215                 220 

Ser Gly Thr Ser Leu Val Lys Val Asn Val Leu Asp Ser Asn Asp Asn 
225                 230                 235                 240 

Ser Pro Ala Phe Ala Glu Ser Ser Leu Ala Leu Glu Ile Gln Glu Asp 
                245                 250                 255 

Ala Ala Pro Gly Thr Leu Leu Ile Lys Leu Thr Ala Thr Asp Pro Asp 
            260                 265                 270 

Gln Gly Pro Asn Gly Glu Val Glu Phe Phe Leu Ser Lys His Met Pro 
        275                 280                 285 

Pro Glu Val Leu Asp Thr Phe Ser Ile Asp Ala Lys Thr Gly Gln Val 
    290                 295                 300 

Ile Leu Arg Arg Pro Leu Asp Tyr Glu Lys Asn Pro Ala Tyr Glu Val 
305                 310                 315                 320 

Asp Val Gln Ala Arg Asp Leu Gly Pro Asn Pro Ile Pro Ala His Cys 
                325                 330                 335 

Lys Val Leu Ile Lys Val Leu Asp Val Asn Asp Asn Ile Pro Ser Ile 
            340                 345                 350 

His Val Thr Trp Ala Ser Gln Pro Ser Leu Val Ser Glu Ala Leu Pro 
        355                 360                 365 

Lys Asp Ser Phe Ile Ala Leu Val Met Ala Asp Asp Leu Asp Ser Gly 
    370                 375                 380 

Asn Asn Gly Leu Val His Cys Trp Leu Ser Gln Glu Leu Gly His Phe 
385                 390                 395                 400 

Arg Leu Lys Arg Thr Asn Gly Asn Thr Tyr Met Leu Leu Thr Asn Ala 
                405                 410                 415 

Thr Leu Asp Arg Glu Gln Trp Pro Lys Tyr Thr Leu Thr Leu Leu Ala 
            420                 425                 430 

Gln Asp Gln Gly Leu Gln Pro Leu Ser Ala Lys Lys Gln Leu Ser Ile 
        435                 440                 445 

Gln Ile Ser Asp Ile Asn Asp Asn Ala Pro Val Phe Glu Lys Ser Arg 
    450                 455                 460 

Tyr Glu Val Ser Thr Arg Glu Asn Asn Leu Pro Ser Leu His Leu Ile 
465                 470                 475                 480 

Thr Ile Lys Ala His Asp Ala Asp Leu Gly Ile Asn Gly Lys Val Ser 
                485                 490                 495 

Tyr Arg Ile Gln Asp Ser Pro Val Ala His Leu Val Ala Ile Asp Ser 
            500                 505                 510 

Asn Thr Gly Glu Val Thr Ala Gln Arg Ser Leu Asn Tyr Glu Glu Met 
        515                 520                 525 

Ala Gly Phe Glu Phe Gln Val Ile Ala Glu Asp Ser Gly Gln Pro Met 
    530                 535                 540 

Leu Ala Ser Ser Val Ser Val Trp Val Ser Leu Leu Asp Ala Asn Asp 
545                 550                 555                 560 

Asn Ala Pro Glu Val Val Gln Pro Val Leu Ser Asp Gly Lys Ala Ser 
                565                 570                 575 

Leu Ser Val Leu Val Asn Ala Ser Thr Gly His Leu Leu Val Pro Ile 
            580                 585                 590 

Glu Thr Pro Asn Gly Leu Gly Pro Ala Gly Thr Asp Thr Pro Pro Leu 
        595                 600                 605 

Ala Thr His Ser Ser Arg Pro Phe Leu Leu Thr Thr Ile Val Ala Arg 
    610                 615                 620 

Asp Ala Asp Ser Gly Ala Asn Gly Glu Pro Leu Tyr Ser Ile Arg Ser 
625                 630                 635                 640 

Gly Asn Glu Ala His Leu Phe Ile Leu Asn Pro His Thr Gly Gln Leu 
                645                 650                 655 

Phe Val Asn Val Thr Asn Ala Ser Ser Leu Ile Gly Ser Glu Trp Glu 
            660                 665                 670 

Leu Glu Ile Val Val Glu Asp Gln Gly Ser Pro Pro Leu Gln Thr Arg 
        675                 680                 685 

Ala Leu Leu Arg Val Met Phe Val Thr Ser Val Asp His Leu Arg Asp 
    690                 695                 700 

Ser Ala Arg Lys Pro Gly Ala Leu Ser Met Ser Met Leu Thr Val Ile 
705                 710                 715                 720 

Cys Leu Ala Val Leu Leu Gly Ile Phe Gly Leu Ile Leu Ala Leu Phe 
                725                 730                 735 

Met Ser Ile Cys Arg Thr Glu Lys Lys Asp Asn Arg Ala Tyr Asn Cys 
            740                 745                 750 

Arg Glu Ala Glu Ser Thr Tyr Arg Gln Gln Pro Lys Arg Pro Gln Lys 
        755                 760                 765 

His Ile Gln Lys Ala Asp Ile His Leu Val Pro Val Leu Arg Gly Gln 
    770                 775                 780 

Ala Gly Glu Pro Cys Glu Val Gly Gln Ser His Lys Asp Val Asp Lys 
785                 790                 795                 800 

Glu Ala Met Met Glu Ala Gly Trp Asp Pro Cys Leu Gln Ala Pro Phe 
                805                 810                 815 

His Leu Thr Pro Thr Leu Tyr Arg Thr Leu Arg Asn Gln Gly Asn Gln 
            820                 825                 830 

Gly Ala Pro Ala Glu Ser Arg Glu Val Leu Gln Asp Thr Val Asn Leu 
        835                 840                 845 

Leu Phe Asn His Pro Arg Gln Arg Asn Ala Ser Arg Glu Asn Leu Asn 
    850                 855                 860 

Leu Pro Glu Pro Gln Pro Ala Thr Gly Gln Pro Arg Ser Arg Pro Leu 
865                 870                 875                 880 

Lys Val Ala Gly Ser Pro Thr Gly Arg Leu Ala Gly Asp Gln Gly Ser 
                885                 890                 895 

Glu Glu Ala Pro Gln Arg Pro Pro Ala Ser Ser Ala Thr Leu Arg Arg 
            900                 905                 910 

Gln Arg His Leu Asn Gly Lys Val Ser Pro Glu Lys Glu Ser Gly Pro 
        915                 920                 925 

Arg Gln Ile Leu Arg Ser Leu Val Arg Leu Ser Val Ala Ala Phe Ala 
    930                 935                 940 

Glu Arg Asn Pro Val Glu Glu Leu Thr Val Asp Ser Pro Pro Val Gln 
945                 950                 955                 960 

Gln Ile Ser Gln Leu Leu Ser Leu Leu His Gln Gly Gln Phe Gln Pro 
                965                 970                 975 

Lys Pro Asn His Arg Gly Asn Lys Tyr Leu Ala Lys Pro Gly Gly Ser 
            980                 985                 990 

Arg Ser Ala Ile Pro Asp Thr Asp Gly Pro Ser Ala Arg Ala Gly Gly 
        995                1000                1005 

Gln Thr Asp Pro Glu Gln Glu Glu Gly Pro Leu Asp Pro Glu Glu Asp 
   1010                1015                1020 

Leu Ser Val Lys Gln Leu Leu Glu Glu Glu Leu Ser Ser Leu Leu Asp 
1025               1030                1035                1040 

Pro Ser Thr Gly Leu Ala Leu Asp Arg Leu Ser Ala Pro Asp Pro Ala 
               1045                1050                1055 

Trp Met Ala Arg Leu Ser Leu Pro Leu Thr Thr Asn Tyr Arg Asp Asn 
           1060                1065                1070 

Val Ile Ser Pro Asp Ala Ala Ala Thr Glu Glu Pro Arg Thr Phe Gln 
       1075                1080                1085 

Thr Phe Gly Lys Ala Glu Ala Pro Glu Leu Ser Pro Thr Gly Thr Arg 
   1090                1095                1100 

Leu Ala Ser Thr Phe Val Ser Glu Met Ser Ser Leu Leu Glu Met Leu 
1105               1110                1115                1120 

Leu Glu Gln Arg Ser Ser Met Pro Val Glu Ala Ala Ser Glu Ala Leu 
               1125                1130                1135 

Arg Arg Leu Ser Val Cys Gly Arg Thr Leu Ser Leu Asp Leu Ala Thr 
           1140                1145                1150 

Ser Ala Ala Ser Gly Met Lys Val Gln Gly Asp Pro Gly Gly Lys Thr 
       1155                1160                1165 

Gly Thr Glu Gly Lys Ser Arg Gly Ser Ser Ser Ser Ser Ser Ser Ser 
   1170                1175                1180 

Arg Cys Leu 
1185 

 
           
             5  
             22494  
             DNA  
             Homo sapiens  
           
            5 

tttacacaat tactatttat gacattttta tttattttta atataaaaag atgtaataaa     60 

agacagcaat gtcataaagt tgaagtcagg tgtattatac tttggggttt tgttttgttt    120 

ttgagaccga gtctcactct gtcacccagg ctagagtgca gtggcacaat ctcagctcac    180 

ggcaacctct gcctcccggt agagggttca agcgattctc gtgcctcagc ctcctgagta    240 

gctgggattc caggtgcaca ccaccacatc tggctaattt ttgtattttt agtagggatg    300 

gggtttcacc atgttgccca ggctggtctt gaactcctgg gctcaagtga tccacccgct    360 

ttggcttccc aaagtgccgc gactacagat gtgagccacg gcgcctgggc cacttttttt    420 

tttttttttt aacttttaag ttcatgggta catgtgcagg tttgttatgt aggtaaactc    480 

gtgtcacggg ggtttgttgt acagattatt tcgtcaccca gtgacgaaat attaagtcta    540 

gtacttttta ttcttcctga tcctctccct cctctcatcc tctcatcctc taccctgtag    600 

taggtaggcc ccagtgtgtg ttgttcccca ctttgtgacc atgtgttctc atcatttagc    660 

ttccacttac aagtgagaat atgtggtatt tggttttctg ttcctgtgct agtttgctaa    720 

agatgatgga ggcactaagg tttggccagt atcacacagt ggtggagcca gggttagagc    780 

cgattcctca tcagtctgac tctctccagg agcctgtcag agaataaggg ttttttgtaa    840 

caaattcaca gagagtaaaa tagttctggc cttaagaact aaacgggaag gccctgggga    900 

agttaggatt cgagtttatg acttaaggat aggtgagaat gagaacacag acagagaaag    960 

agacccaact cccaccccct gtccccaggg acaccggatg atgacggagc ctagggagag   1020 

gagaggttac agtgtaccac ctagaccaga ggtcgggacc caggccacgg agtggagagt   1080 

agaagtgagt cacaaactat ggcttgtcac tataggggtt agcagacctg acggtgcctg   1140 

catttgttcc catccatcac tggcatctgg tagctgtccc gaaactttgc tgaactcatc   1200 

ccaccatccc aggggggact gcctcctttt ccatggccac cactcgcaag cactcctgca   1260 

acaagccctt gaacacagag gggcagcagt gtggagagga aaaccctgcc caacttttgg   1320 

ataaaactga accttaatga gccctacccc agtacagaag tgtccctgac ttcttgctgg   1380 

tctcttgtca ttgtgtattt gctcatcatg tagatcttaa aagtctatta tcatgtgggc   1440 

ctctgaaaca ttgttttttt ttaaaaaaaa ttagaggttt caggccaggc gtggtggctc   1500 

atgcctgtta tcccagccct ttgggaggcc gaggcaggtg gaccacctga gatcaggagt   1560 

ttgagaccag cctggccaat gtggtgaaac cctgcttcta ctaaaaatac aaaaaaaaaa   1620 

aaaaaaaatt agccgggctt ggtggcgggc acctgtaatc ccagctactc gggaggccga   1680 

gacaggagaa tcacttgaac ccaagaggca gaggttgcgg tgagcccaga tcgcaccatt   1740 

gcactccagc ctgggcaaca gaatgagact ctgtctcaaa aaataaaaca aaatttaaaa   1800 

aaatagaggc ttcatcctaa aaaattggaa gtgcctacac tagaagcaca attataaata   1860 

acgcttaaag tgctacttat atcccaggcc ccactctaag tgctgtatgt taactaagct   1920 

aaacccaaca tcaaccctat aacatagcac catcatcatc atcatcattc ctattttaaa   1980 

gctaaggaaa ctgatcatag ataattgact tgccatcgtc acacagatgg taaattacag   2040 

agccaatccc tgaacccagg caattggtgc cctgggccca catcattaac cccattgcta   2100 

tgcttagacc aagtcaatac ttggctcaag gaaagtggtc agctgggtct gtaaggagaa   2160 

atagccagat tacctgcgtg accagcttcc ctggccagtg caacctaaac aataactcag   2220 

gtcaggtcag cccagggtta tataaagaca gactgctaca agggcttgag gtggctgaca   2280 

gtataaaatg ttttctgtgc ttttgatgca agctaaaatt tgatttacaa taataggcac   2340 

agaatgatca tgtttatact cgcaacctgg cattttatta ttattcccct gaataacagt   2400 

gaaaagagtt ttgtaacccc aaggtaacat ggctctgtaa ggaggaaaga ggggtgagat   2460 

ttagcagcag ctgccatcat aaatcagtgt gtggctgttt ctctcagagc tccaggctca   2520 

actcctggct gtcataaact tataaaggga agccccatga gaatttatgt aacgatggga   2580 

agactcattt tccttcctgc aagagccttt ctgaagcaag gagagaatta ggccgattag   2640 

attctatatg aagaaaaaaa ccaagccaat ggtttagagg aagggaaaat gtgggtcaac   2700 

ttgagctact tcccttgcca aaaaaatgac cttgtaaata gagaggaaca tgctaaattc   2760 

caccaaataa atgagtgact ggatgactga atgatttaat aaatgagtgt aatctaggct   2820 

tcctccaggg tggttctttc gtgtaaaaca gttcgatctt ggagtcaccc agaaatgctg   2880 

aacacataac aaggccaatc aacagagttc gcttctgaat attcatgcac agatgatgct   2940 

cccagtcaat atgtttgatt tttttttctg tttactaata gattttcaaa ttcactgtgc   3000 

aaaccaaaat tcttaaaaaa aaaaaaaaag gaacttgtgt tgaaagcata cattatttct   3060 

gggcagagtt gtcagattaa aatatttata ctaaaattat tcattgttta tctgaaattc   3120 

taatttaact gggcactctg tatttttatt tactaaatct ggcaacccct ttctggacca   3180 

ataaccctac gtagaggaaa ggacagaaga tagcttctag gggacatgcc tgcacctggg   3240 

agttagaagg aaggaaaacc aaagaaggag ccagagaggc agatagagga gtctgcagaa   3300 

atatggagag agaaccaggt gagtatggaa ctctataatg aatagtgaca cacatgtcag   3360 

ggagaggtgg ggaccatgag gatgctgggt taagaaaggc ctgatcgcta tagagtttgg   3420 

cctttgcaat gggggatggt gattctgaga atttctaata ggtggttctc gatgaagttg   3480 

gggtggagat aaggatgcat gtcggaatca cccagaatat atcaaaattg aaatctgtac   3540 

accagaagta gtgcccttcc tctcgaaggt tggggagaaa gacatgtgtg tgtaactctt   3600 

atcaacccag aactcaagtg gtccagtttt gggtccatgt tatggttact aacgcacaaa   3660 

ataaatttgg aaaagaacat acaaatcttt tcttgtattt ttcttgaaaa tcatgaagga   3720 

ggtctataat ttgcaataac gaattcaaaa gtccttttca tcttcattct acctccttca   3780 

cagcctctgc tgttgtctgc tgtattgctc tccagaattt tctcagattg cagaaatctc   3840 

cctcctcccg tctatttatt tttatcacgt aaatcaactt agcctctctt tccagcaaaa   3900 

aggtccaatc aaattcccca accctgtccc aaatgaaggg tgcggaactg agctggcaat   3960 

tacagactat tctttggcag tattctggga aagggaaaag tatgaatttt cttgccctgt   4020 

ccctggtatt ttgtaaagct ttccaagtga cttcgatatc tggacccttt tgagaactgc   4080 

cgctgttaac caagacataa agctttttca tttcttgaaa ggtcctattt cttggcaaat   4140 

ttctccctgc aaagtgcatt cttttttctg cactgacccc agaaacaggt gccattgtgt   4200 

gggagacagc acctgtcatc tgtctcaaca aagtccacaa agtgccctgc taggagcaga   4260 

taacccagaa catctacctg agtctcccag tcaagattcc tggtctgact aggaatctaa   4320 

cttcaacaag atcttcctga aaaaagacgc tgagcttgga cggtccaacc acctccctac   4380 

ctgggataag ccggaggatg cttcttggct tccccaaagc tgtcttggtg gtgatgctgt   4440 

ggcaaccaca ggggagattc acgaggagaa agcctggaag accagagccc tggaagtggg   4500 

gcagccagcc cagcgggaca ttcgtagggg cgaggtgtga tctcagcctt cattcagctg   4560 

gcctgggagt cctccaaggg tcagagtcac accttactag tctgacccct caaggttcct   4620 

taggactcca ggtgttttaa gaagattctc tgaaccccca cccttcccac ttcacccctc   4680 

agccctccct gtggttaaga acccaagtgt gaacaacccc aagtttggtg gttagtgagg   4740 

ccttcaagca caaaccctaa tcctgccctt ttaaaaaaaa tattttggac ttaagggagg   4800 

ctgtcctcaa ttgtttcaaa cctatttcag agtacctcca ctccttcaaa cctattttca   4860 

aaactcagga aaacgtcgcc tttccagacg aactctagtg acagctccat tcaccccttg   4920 

gcatttgact gctaaagatc agctgacccc tgcagccaga aggaggagag aaaattgcta   4980 

aatggggacc tcatttccag aggtgatcac cttaaaaaag tctggggcct tgtttgccag   5040 

ctttgaaatc tcaaagggta atcttagctt catgtcccca gtgtgaataa aacaaacaat   5100 

aaaaaccacc tatgaaatct aaattcaaac tttcttgcga cctatgcatc atttttttgc   5160 

attcacattc ccagtcaaaa cacagacaca cacacacaca cacccaacag atatataact   5220 

gcatgtaaaa tatatataga taaaacaatg tcctctgaag aatataaatg ttaacacaag   5280 

taaaattatc atgaattcca taactccttc tgtggccttg gtccacgtta ggttttccat   5340 

taagagatac tattgtcatc agtgagtagc ttctcctgtt gtattcttct gtgtagcacg   5400 

gggctggagc tgtggatatt tatgaacccc aggcacccac ttcctgtttt cgtggctgtc   5460 

cccggtccct gcctctgtgc cgttttctct gcctttggcc accagatggc gctctggaaa   5520 

cgagttttct gcatcaaagc tcccctgcat tactctcaac taggctccct ccctcttctc   5580 

tgttcccttc caagccattt tccagggtgc agccaaaagg cctttctgga gggcacatct   5640 

gtgtgatctt gggcaagtta cctaacgctg tgtgctcagc atcttcaatg tgatggtatg   5700 

gctgtgagtg cgtcatgaga ggatgtaggt aaagcactta ggacaaggcc tggcatgtgg   5760 

tcgctgttct cattatcctg cgtagagctt ttcaaaagca ctccacgacc ttcaggacaa   5820 

ggtctaaatt cttagccatg ggaacccagg ccactgcatc atctgaggac ccctgcttat   5880 

gtcccctgcc caaccttcct ccctacacta ctgcactgac cctcttgcag cacagccagg   5940 

agccagcctg cccacttcgg ggcctttgcc cattttgccc ctgccaccag ctttgcctgt   6000 

ctcctgccta ttcaacttcc tcgggctcac tcctccaggt ccgtcaggat tcagcgtgtg   6060 

ccctccatga ggagcctccc ggacccccag gcctgattcc agagccattc tactggcctc   6120 

tcgcctttca ttttccctgc ccccacctgc gactgtcagc cccttgcctt gttcactgtc   6180 

aggcacacag ggggacgatt gcggggcgga tagtgagact gtcacgtggg ctgccagcag   6240 

ggggcaatgg gcacccttgt gtccctgagc tcccaggggc tcctagtagc ttaggcagca   6300 

ccttgcccac accaagctga gggagacctg agaaatggga ttcctgccaa agaggcaaag   6360 

aaaaaagtga agggacaaat gcaaggccat ctttgccatt tgcgtatgag gcataaaact   6420 

tggaagataa taaacagcaa aatcatagtc cttatgtgct gagtgctttc tctgagccag   6480 

gccctgtgct gagtccttac cccaccttgt gagatcgatg acagccctca gagaaggaaa   6540 

cagaggctag gagaggttaa gtcccttgtg cagtgtctca gagcttgtga gtggcctgtc   6600 

tgtctccagg gccatgctgt aggaggtggc cctggattca ggccccttgc tatggtttaa   6660 

tgggtttcag tgaaatagtt gggaaacctc aggagtggga tatggagtta agggaaagaa   6720 

aagccagaga gaaagggaag gagtgcaggt gactgtccct tagccacccc agtcctgatg   6780 

accacaggcc tccatgccaa taagccctga ctagtgccac ttgggtccaa acatggcatc   6840 

tctggcccca agggcttagt agcaaacacc catctaggga agctggcgtt cattctcatc   6900 

acctcaaatg cttcatgagc ctcagggatc aaccttgaag tgggtataag gctgggagaa   6960 

tgttgggggc agcaaactga agggcacaat aagaagcaat aaggccacct caaagcccac   7020 

ccaagcaaac tgctcattca cctccttcct tcctgaattt caccttatga ggaggtgagt   7080 

ggaagatagg gtatccctta aaacatctaa aaggagagtt gggggcagca aaggagatgt   7140 

gcttcacggg actcttataa acaaaactgg ggagagaaga attggaggaa gggggaaaga   7200 

catagatgaa agggagggga aggtgtggga gagggaaacg tataaaagct tccaagagga   7260 

gtgggaggct gggggttccc cagacagaga ctcagtctgg accagatgca gagaacaatg   7320 

gacttcaagg ctggaggggg gcagaaggga agcgggagga gagccacacg gtcaagttgc   7380 

acaggttctt gcagcttctg gaatcaagac catgggcacc ctcataagtc agtgtgggca   7440 

gggactgccc cagggccaat ccaagatcca gaggtagcca tagggtgtga caagttgtgc   7500 

agattacaac actcacccct tgcaataacg tcactgcctg tgactcgggg ccaggcccag   7560 

gccaaagccc ttcctacatc atttcgttta atcctcacag tttcctgctg aaagggctac   7620 

tattcttact cccatcccca ctctacagat gaggtaatgg aggcccagga aagttaagtg   7680 

acttgtccca gatgacaccg ctggtaagtt gcaaagtcag aatttgaact caggcagttt   7740 

acctctgatg gctgctctgt taatcacagc tgctttccag tgagacaaaa acgggtgatc   7800 

agggcagagt caagacagag aggtaaacaa gattgggaaa aagacaggaa tgagagggga   7860 

acaatggggg aaaagatagg aacaaagaga gttggggaag gggagagaaa caggaaacat   7920 

gacttgcccg ggaggggcat cagtccacgt gcaagcaggt ggaggctcaa gttttctgct   7980 

cacttggtga tgcagaggct ccctttccct cagcagccgc cttgctgcgt ggacagcagc   8040 

ttcccatctg gcctgtcccc ggagccccgg cctcatcctc ctcagcggca ggccacttag   8100 

cttcacagga aatgctcttt ctctaattgg cattgaaact cacagccctc ccttttcctg   8160 

taggtggggt ttccatagga aaaagctgct tctctgtttc cccagcctag caactgtttg   8220 

gcagtcagag tcccacatcc tgctcaactg ggtcaggtcc ctcttagacc agctcttgtc   8280 

catcatttgc tgaagtggac caactagttc cccagtaggg ggtctcccct ggcaattctt   8340 

gatcggcgtt tggacatctc agatcgcttc caatgaagat ggccttgcct tggggtcctg   8400 

cttgtttcat aatcatctaa ctatgggaca aggttgtgcc ggcagctctg ggggaaggag   8460 

cacggggctg atcaagccat ccaggaaaca ctggaggact tgtccagcct tgaaagaact   8520 

ctagtggttt ctgaatctag cccacttggc ggtaagcatg atgcaacttc tgcaacttct   8580 

gctggggctt ttggggccag gtggctactt atttctttta ggggattgtc aggaggtgac   8640 

cactctcacg gtgaaatacc aagtgtcaga ggaagtgcca tctggtacag tgatcgggaa   8700 

gctgtcccag gaactgggcc gggaggagag gcggaggcaa gctggggctg ccttccaggt   8760 

gttgcagctg cctcaggcgc tccccattca ggtggactct gaggaaggct tgctcagcac   8820 

aggcaggcgg ctggatcgag agcagctgtg ccgacagtgg gatccctgcc tggtttcctt   8880 

tgatgtgctt gccacagggg atttggctct gatccatgtg gagatccaag tgctggacat   8940 

caatgaccac cagccacggt ttcccaaagg cgagcaggag ctggaaatct ctgagagcgc   9000 

ctctctgcga acccggatcc ccctggacag agctcttgac ccagacacag gccctaacac   9060 

cctgcacacc tacactctgt ctcccagtga gcactttgcc ttggatgtca ttgtgggccc   9120 

tgatgagacc aaacatgcag aactcatagt ggtgaaggag ctggacaggg aaatccattc   9180 

attttttgat ctggtgttaa ctgcctatga caatgggaac ccccccaagt caggtaccag   9240 

cttggtcaag gtcaacgtct tggactccaa tgacaatagc cctgcgtttg ctgagagttc   9300 

actggcactg gaaatccaag aagatgctgc acctggtacg cttctcataa aactgaccgc   9360 

cacagaccct gaccaaggcc ccaatgggga ggtggagttc ttcctcagta agcacatgcc   9420 

tccagaggtg ctggacacct tcagtattga tgccaagaca ggccaggtca ttctgcgtcg   9480 

acctctagac tatgaaaaga accctgccta cgaggtggat gttcaggcaa gggacctggg   9540 

tcccaatcct atcccagccc attgcaaagt tctcatcaag gttctggatg tcaatgacaa   9600 

catcccaagc atccacgtca catgggcctc ccagccatca ctggtgtcag aagctcttcc   9660 

caaggacagt tttattgctc ttgtcatggc agatgacttg gattcaggac acaatggttt   9720 

ggtccactgc tggctgagcc aagagctggg ccacttcagg ctgaaaagaa ctaatggcaa   9780 

cacatacatg ttgctaacca atgccacact ggacagagag cagtggccca aatataccct   9840 

cactctgtta gcccaagacc aaggactcca gcccttatca gccaagaaac agctcagcat   9900 

tcagatcagt gacatcaacg acaatgcacc tgtgtttgag aaaagcaggt atgaagtctc   9960 

cacgcgggaa aacaacttac cctctcttca cctcattacc atcaaggctc atgatgcaga  10020 

cttgggcatt aatggaaaag tctcataccg catccaggac tccccagttg ctcacttagt  10080 

agctattgac tccaacacag gagaggtcac tgctcagagg tcactgaact atgaagagat  10140 

ggccggcttt gagttccagg tgatcgcaga ggacagcggg caacccatgc ttgcatccag  10200 

tgtctctgtg tgggtcagcc tcttggatgc caatgataat gccccagagg tggtccagcc  10260 

tgtgctcagc gatggaaaag ccagcctctc cgtgcttgtg aatgcctcca caggccacct  10320 

gctggtgccc atcgagactc ccaatggctt gggcccagcg ggcactgaca cacctccact  10380 

ggccactcac agctcccggc cattcctttt gacaaccatt gtggcaagag atgcagactc  10440 

gggggcaaat ggagagcccc tctacagcat ccgcagtgga aatgaagccc acctcttcat  10500 

cctcaaccct catacggggc agctgttcgt caatgtcacc aatgccagca gcctcattgg  10560 

gagtgagtgg gagctggaga tagtagtaga ggaccaggga agccccccct tacagacccg  10620 

agccctgttg agggtcatgt ttgtcaccag tgtggaccac ctgagggact cagcccgcaa  10680 

gcctggggcc ttgagcatgt cgatgctgac ggtgatctgc ctggctgtac tgttgggcat  10740 

cttcgggttg atcctggctt tgttcatgtc catctgccgg acagaaaaga aggacaacag  10800 

ggcctacaac tgtcgggagg ccgagtccac ctaccgccag cagcccaaga ggccccagaa  10860 

acacattcag aaggcagaca tccacctcgt gcctgtgctc aggggtcagg caggtgagcc  10920 

ttgtgaagtc gggcagtccc acaaagatgt ggacaaggag gcgatgatgg aagcaggctg  10980 

ggacccctgc ctgcaggccc ccttccacct caccccgacc ctgtacagga cgctgcgtaa  11040 

tcaaggcaac cagggagcac cggcggagag ccgagaggtg ctgcaagaca cggtcaacct  11100 

ccttttcaac catcccaggc agaggaatgc ctcccgggag aacctgaacc ttcccgagcc  11160 

ccagcctgcc acaggccagc cacgttccag gcctctgaag gttgcaggca gccccacagg  11220 

gaggctggct ggagaccagg gcagtgagga agccccacag aggccaccag cctcctctgc  11280 

aaccctgaga cggcagcgac atctcaatgg caaagtgtcc cctgagaaag aatcagggcc  11340 

ccgtcagatc ctgcggagcc tggtccggct gtctgtggct gccttcgccg agcggaaccc  11400 

cgtggaggag ctcactgtgg attctcctcc tgttcaggta cctggggcat gggcagctct  11460 

ttctctggtc actcctggac caccagaaac agaatcagac acccccataa cctgcacagt  11520 

cctcatgttt cttccttagg ctcacctggc actttctgat gctttgggta gcaatggttc  11580 

agggcacagc tttgtgatcg gctgaactta gctgtgtgat cctaggcaaa ttccttactg  11640 

tctctgggcc ttagttgtct tatctgtaaa ttagagatga tagtaagagt atctaccttg  11700 

tagagttgtc cagaggattt gatcaattaa ttcctataag gcatttatgg tgcctcacaa  11760 

gtagacacac agcaaaggta gtgattaaga gtataagctt tgggcaaggt tacctgggtt  11820 

caaatcctgg ctccacttct tcctagctct gaaccacaga ggaagttaac atctctgttg  11880 

aagatgtcta tgttaacacc acccatcttc taagtttgct tgaaaataaa atgagtgaat  11940 

gctcttaatg cctgtagaac agtgtccatc tgacatgtag tcagtactca gcaaattatt  12000 

actattactt cctcaaagca cttctccatc tccactattt gcctgccttc atctcatctg  12060 

ggcaacttcc ctatgaagaa ggccagcata ggcatcattg cacccaaacc caggtgttgc  12120 

aaataatttg tctgagaccc cagagctttc cggctgggcc tgcggatggg gcagtggaaa  12180 

ttaccttaga acttagcagt taggcagatt ggagttcaga tttcaacaca gccacttagg  12240 

agctgggcaa ccttgtacac gtccttcaac atctgagcct cattttcctc atctataaaa  12300 

tggggattat gagaccaaac ttaccctgtg tctgcctcct tagagtgtga gggctaaatg  12360 

ggatgatgag tgtcaaaatg tctaaggacc tgggattttc tagacacaag tggggaaacc  12420 

tttccaggga tgatgcctga tctcacaccc atatctgctt ctgtgaacct cagatatttt  12480 

cactgtgcca ggctgcccgc ccaaggcaca cttctgtttg gccaggctat cagctcaggt  12540 

ttcctgtgaa gcaaaacaca gtttccttcc ctattccctc ctcaggaagc cacctggaga  12600 

tggcccagct gttctcttag gaatcttgcc ctgggatcgg tgcagaccca gaggctgcca  12660 

gagccaacca ttcatttatc cttccattca tccatccatt tatcaggtgc tttttggata  12720 

aactctgtgc tgaacactgt gctggctgct ggtagacagg acaacagaga ataggccctg  12780 

tgggtgaccc cgctgaggtg acagaaagaa aggtgcccat ttgggatcaa cttgagggta  12840 

acaggcaact tgcccaatgc tcatatcttt ccagcagcac atagagtcag atctgagtgt  12900 

aaattccagc cctcccacta atttctgtgt agcccctgtg agcctccaat tccacctctg  12960 

caaaatggcc ataatggtat ctcttccctt aggctgttgt aagaattaaa tgagattata  13020 

caccactgga caatcactgt ttatcatggg atttattaat aagctcattt aaacctcgta  13080 

acccaccata tgacctgggt ctcattaata tgcacttttt ttactgttga ggaaactgag  13140 

taattttctc agaatgctta agtaattcct caaggccaca ctgatgggaa ctggggcagg  13200 

gacatattta ggatttgaac ctgagtctga ctgattcgag tctgggctgt aactgtgaag  13260 

tgaccctgaa tgacagtgct cagcataggg cttggcatgt agggagtcat gtcatgaaaa  13320 

ggcagctgtc acttgagcaa agggtggcag gtatcttagg agggcttctg ggcctgggct  13380 

gggaggtggg atcttccctg aggcaggcaa taagaccccg agtctcccaa gtcacagtga  13440 

aaccccagtc ctggccatct cctcttccaa ctctcctctg aggttgacac ttcctacctc  13500 

aagctgcaca gaggggcatg gtggacctcc taagggagga atggggatgg acccaggagg  13560 

aggaagcagt gtttctagga ataacagtgc aaactccttg cttttgcaga ggatttttaa  13620 

ctttttcaaa ccatttaatc gtgacaacac tgagaagaag gcgtgtgggt atttctagcc  13680 

ccctattaga gatgaggaaa ctgaggtctg aggaggtcta aaaacctgtc taatgctata  13740 

caataagcta caaatggcct tgaactagaa ctccgcctcc caggacttga tctgttgcct  13800 

ctcccttcca atggcctata gggtctggcc gtgatatctc acccatcccc tactccattc  13860 

tccagtcttt ctgaaactct accttctctt ccagcctttc agtgtgcagt gcttctgcct  13920 

gaaaacctcc ccaccccttg cccagacctt cattctcttt ccacttgcca aactcctatg  13980 

gagcctttgg gtctcagcag aggtgcctgg catgtggtat gaactcagta acatctgttg  14040 

aaatgaatga attaacttaa tccagtacca cccccctgct tcctccctgc tccaagttgg  14100 

gttaggggcc ctctgctccc acagagcccc cattctttcc ctatgcccag aggcaacccc  14160 

tgtggtaaac actcttcgct gccctgatct ccagccagcc tccctgtggg gctgcctgca  14220 

accttcacat cctgacagat gtgaccacgc tgagggcagc agctggggct ccttctcctt  14280 

cttactccca tagcaaccac cctgcctggc acttattaag tacttgttga atgaatgaaa  14340 

agatgaataa atgaataatt caatgacacc attttgcctt ctctacaaca tagtcttaca  14400 

tctggtcaac aaaaattggc cgaatgtcca aatactgcta aaggggaaca taactcttta  14460 

agataatctt catcaaagta acaacaaatt gatggtcagg aaatgccttt ttcaccccat  14520 

ctctcagagt ccaccaggtg caggacactc ggaggaatga gtatgagggg acaggcccag  14580 

agggtcctct tgctctcctt cagacactca ggtcctgggg tgacatgaca gagcagggga  14640 

cctgcccgcc caggtccctg tctctgtcac tgggcaccat cccccatctg atcacacaaa  14700 

catctgaagc cagaggaaag actataaaaa gaaagcaccg gcattgaggg aaacatccgg  14760 

cgaggaagtg tgctgagtcg gaattgtgtg gagtcaacag atttttcctg tcttgtagca  14820 

aatctcccag ctgctgtcct tgctgcatca gggccaattc cagcccaaac caaaccaccg  14880 

aggaaataag tacttggcca agccaggagg cagcaggtaa gcagcacgta gcccccacag  14940 

gcatctcaag gcccctactg gccgcccctc atcactgcag cctctgtgag tgaggaaggg  15000 

tgagcacaag tcagacaccg gtcccctggg agggtgcagc agacagcatc catcctccca  15060 

ccctgggagt tttggtcgtg aggtgactgt attgatagta tttacaggac ctagaagtcc  15120 

tgtaggacta actagctgta ttgataaata ccatcaatac agtagtcacc aaggaaatga  15180 

cctcatccct gagactgtat ttgtcccaat tacagaggtg ctgtaaattg cctttatatc  15240 

tgtaactgtc acctctcttt ctcttgaccc ataaacataa tatgaaggct ccccactaaa  15300 

gacatggctg tgtgggcgtg tccctttggg caatcctaga attgaagaca ccttaagaga  15360 

acatcaaatc caacttcttg ttacggatgg agaaactgag gctctgagta gcttgattca  15420 

aaccagctat gtaaacttga gcaagtgaga taatctctca ggaccttggt ttccccatct  15480 

gtaaaacaga acccatccca tttcccttgt ggtaatgggg tgaggatttc ataatcaatt  15540 

gtattctaag cccatagccc agcaactgac acatagtagg tgcacacaca atgctaactc  15600 

ctttcccctt gaagcaattt tgctaaaggt catgtaacaa gtgagtggca aagctgggac  15660 

cagctctcag agttcctgac tcctgcatca gtgctcttgc cctgacaact tacagcttct  15720 

ttttacaagg atggtgagag gtagtgtact gcaggggtga gtccacacac tccagggtca  15780 

cactggcctg gatttgaagt cttgctttat ctcttaatct atggggacct tgggcaacct  15840 

gcaaaatctt cacaagtctt ggttttctca tttgtacaat gggttgatta aagagaaccc  15900 

cttacaggtg tggaaggttt gaataagacg gtgcatggac cttgcatcgc ataaggcctg  15960 

actcagactc actgctcaaa aacattagtt gtcattatca cccacacact tgccacctcc  16020 

ctgctcattt cttcacagca gcagaaatgg ctacagggga acaaaaccaa acaaacagaa  16080 

ggactcctac gccttaaaac ccctcaggta gccttcccac ctgctgggat gagggtccag  16140 

gcctgtcccc aagtgtctat gggagagagg gaccaggatg gagacagcag gagactgggg  16200 

tgcaggtagc agattctgag cagctttagg ctgcctcact ccggctgcac cctccatagg  16260 

tctgctcttc tggcctagga tttggcctaa agtagggagg aaggaatgtt cagtcactca  16320 

ttcagcaccc actgtgtgcc acgccctgtt ctgagcatca gtaagggagc aggcaatgag  16380 

acaggcaaaa atcccagcct tctcctacgg acggatggac ggctgcactg gctccactga  16440 

ggtttactaa actcctcctg tgtgccaggc aatacctaga catgggaggg gcaacgagaa  16500 

tgatatcaat gaactcatct tcacttctgt ctgctgagtg cttacaatgt gccaggctct  16560 

gtggtaggcc cctctaccta caacgtctca ttttaccctc acaacaccct gtgaggtggg  16620 

tgttatttat tactctcatt ttacaaatga ggaaacagag taagaggagg gcgggaatca  16680 

catgatccag gttggtctga ctctagaggc caggctgtgt atccagttcc ctatatgggt  16740 

aaatgaatgc aaacatgaat gggcatgaat gaatgaatct atatgggaat gtccctaccc  16800 

atggactggt tggtttcttt tgcaggagtg caatcccaga cacagatggc ccaagtgcaa  16860 

gggctggagg ccagacagac ccagaacagg aggaagggcc tttggatcct gaagaggacc  16920 

tctctgtgaa gcaactgcta gaagaagagc tgtcaagtct gctggacccc agcacaggta  16980 

gggaccccct ggagttacct tgaccctgct tctgccccat ggtgtccaag cttcagagtt  17040 

ctgctccaat ccactgcagg gaaaagccat tctgtttctg tggaggaaca tcagcaacat  17100 

ttaacttccc ttgcaccctt gcagcctcag aaagtatttt aaagaccagg ttctactttg  17160 

attttttttt tttttagaca gggtcttgct ctgtcaccca ggctggagta cagtggcatg  17220 

atcacagcat gatcacagct cactgcagca ttgacctcct gggctcaggt gatcctccca  17280 

ccttagcctc ccaagtagct gggactacag gtgagcacca ccaccacatc ccgctaattt  17340 

ttgatttttt tttttttttg tagggacagg gattcacctg ttgcccaggc tggtctccaa  17400 

ctcctgggct caagcaatcc acccacctca gcctcccaaa gtgctggcat tagaggcatg  17460 

agccacctgt aatcaaagcc aaagtaggca cctggcctac gttgattatt cattttaaaa  17520 

atcattgtaa aaatagtgaa catgccaggc actggggaag catcttatgt acatgatctc  17580 

atttaagcct ccaaccaatc ccctcaagta gataattatt acctcgaatt tacagatgag  17640 

caaaccgagg ctcaaaaaaa tcaagtagcc ttcccagagt cacccagctc accagtgatt  17700 

gaaggggagc caaaccctga gccagcctgt cctctatgga cttcttacca ctcattggcg  17760 

tttggtgtgt ttcaataccc tttcaccctg ggaaccaggt caactcttta atatgctaac  17820 

aattgcaatg aaaacaaaaa gatttttttg cagagaaata attagctttg acaataaata  17880 

cataatgtga acttgaattc aagaatctgc tagcgccccc cgcccctgct gtggacattg  17940 

gtcagcctcc tttggtaaca caccccacgg cagaccatcc ctgtgctgtg gttttctatc  18000 

tatgtagtta acactgtttc tcatgtgctc catctggctg cagacacact gaaggtatgg  18060 

cagggacttc tctttccttg gttaaaaaat attgctaatg tttattgggc actttctatg  18120 

tgctaggccc tgttctaagt acctcatgag aaaatcttat gagatagggg ctattcatta  18180 

tccccatttc ataaaggaaa aatgtgaggg tcaggggtgt ttcataattt gcccaaggtt  18240 

gtgcagctcg tggatggtta ttaacacagg cagtgtggct gtaaatttta gagctgtgtt  18300 

gttcaatgag gtagtcacta gcgacaagtg gctatttaaa tttaagtgaa ttaaaattaa  18360 

ataaaataaa atattcagtt ctgccatacc agccacattt caagtgctca ctggctatgt  18420 

atggctgggg gatatagctt tggattctat ctctattggt cagctctggc tggaacctgt  18480 

ggggtcagtt cccacacggt cctgcttgcc cacagcacaa agggtactga acacaggggc  18540 

actgtaagga gttgggggta aggaaagagg aggggcagaa aagcctttgc tgctcacagg  18600 

ctggactgcc atggggagac tgggatcttg ctgcttcaga tctgcttcag attgcttcag  18660 

atttgctgct tcggattcag attgccccac ttgagaatgc gaccatgata atgttttcct  18720 

ggttgcatga actcactgaa cctcacaaca actccacaag atacgtacta ctgtctcacc  18780 

acttcctaag aggaaacggg agcttagaga ggttaagcca ttttcccaag gtcacacaca  18840 

ctgcccctaa gctgcaaagc tgggaaatca aacccaggtt ggcccagtgc ccacctgcag  18900 

ctagaactgg tgggtgatgc tgggtgaatt cagcatcact tttcagctgt tctcatacgc  18960 

cccagtgatt ttctcattcc aagcttcact ccttctgaag ctctctgacc catgtaaaag  19020 

gaaaggcctg attctataac accaagagtt ccatccaacc ttatagcctc agagtctcaa  19080 

ggaactaagg tttgcatttc ccaaatgttg aaggttaata tttatgttca ttcaatcatt  19140 

caacaaacat tcactaagtc ccaggcatgg ggctaaaccc aaggatccag tagggcagga  19200 

gcccaacagt gtctcacaag ctgcacagtc cagaatgcag ggcagagatt catttatcag  19260 

tacaataaat gactacacat acttcggaat aacttgttca aattacataa gtaatatttg  19320 

gttaatatag aaaaacatag ataaacaaaa agagaatgga ttgcttctgc ccagaggtaa  19380 

tttcagttaa cattttgaca tatctccttc cagtctttca ttctgtgcgc agattttaaa  19440 

gtaactcacc ttaggcaaac attagataaa tgttaagtag atgttgtgct tttttttttt  19500 

tttttaattt ttttaagaca ctgtttctta actccaagac ttataatttc tttcctctgg  19560 

cttggtctct accatcaggc ctgggactgg tttcttgaaa caggattttc tgttaaaggg  19620 

atgggaaaag aagggtgact gtggcattgc aaatgtaggg gccagttttt tgaattccca  19680 

gccaccaggc tgacttgcca ccgccactac cccagtccct ctcctgggac agcccttgga  19740 

caggcagccc ctccccagcc tgactctagg ccagggttcc agcagcttga gagatgatgt  19800 

tgaggaggat ttgaggatct ggggccaggt ggtaacagtg cagaccttag gtagcctggg  19860 

aattccagag gccccagctg ggcacctgga gtatccaggg ggaggaaggg ccagctcttc  19920 

agagagaggg tagggtggtg ccatcatagg cttcaaagtc atcagacctg gcatcaaatt  19980 

gaggtttggt tgcctagcag atgtgtgact taacttcttt cagcttcagt ttcctcatcc  20040 

atcaaatggg aataatactc ttgacctcaa aggtttgtga agttaaatga aataattatg  20100 

taaattgctc atcacgtaag tacctaatat gtggtcccta ttattatgga tgggaaccaa  20160 

aaaaggcagc gctaagtcag gaactccagc cttggcaaag aaaagggatt ttccctccat  20220 

cctcagctag tggagccctg aggggagaag gcgccctgag gggtaaaaat cctggatgaa  20280 

atatagaaga cactgagact ggaaacacac gcccagattg ggaaattctt cagaaatcgg  20340 

caaatttcta cttctaggcc ttctcctcct cctgctgctt ccccacttgg ttcgcccggg  20400 

tgtgtcagtc gactgctttc ctacccaggg agcttccctg tcagggcctc ttccttcctg  20460 

tccccgtctg gccactctgt ctgtcccctg ccatttccct tccctccttt gtccactgcc  20520 

cttgccctgc tcattgcccc accctcaccc gcctggccct tcccggagcc tggccctcac  20580 

tgtgtcccct ccttccccca caggtctggc cctggaccgg ctgagcgccc ctgacccggc  20640 

ctggatggcg agactctctt tgcccctcac caccaactac cgtgacaatg tgatctcccc  20700 

ggatgctgca gccacggagg agccaaggac cttccagacg ttcggcaagg cagaggcacc  20760 

agagctgagc ccaacaggca cgaggctggc cagcaccttt gtctcggaga tgagctcact  20820 

gctggagatg ctgctggaac agcgctccag catgcccgtg gaggccgcct ccgaggcgct  20880 

gcggcggctc tcggtctgcg ggaggaccct cagtttagac ttggccacca gtgcagcctc  20940 

aggcatgaaa gtgcaagggg acccaggtgg aaagacgggg actgagggca agagcagagg  21000 

cagcagcagc agcagcaggt gcctgtgaac atacctcaga cgcctctgga tccaagaacc  21060 

aggggcctga ggatctgtgg acaagagctg gtttctaaaa tcttgtaact cactagctag  21120 

cggcggcctg agaactttag ggtgactgat gctaccccca cagaggaggc aagagcccca  21180 

ggactaacag ctgactgacc aaagcagccc cttgtaagca gctctgagtc ttttggagga  21240 

cagggacggt ttgtggctga gataagtgtt tcctggcaaa acatatgtgg agcacaaagg  21300 

gtcagtcctc tggcagaaca gatgccacgg agtatcacag gcaggaaagg gtggccttct  21360 

tgggtagcag gagtcagggg gctgtaccct gggggtgcca ggaaatgctc tctgacctat  21420 

caataaagga aaagcagtga ttcattctcc tgtttgcacc tgtcaagagg gagaagggaa  21480 

gagtaataga gtggggttta tttgtcatcc aatccctggc aagaccgtca cactgggcct  21540 

ggacaggagt ggggcacctt ggtacacagt aacaggtaca caaatagcag acatgtgtgc  21600 

tcttacccct gttcccctat cactatgtta ccaactaatc tgcttctcag ctgcaaaatt  21660 

agagctaaga aagcacaact atttataaag catttacatg agccaggcac caggctttac  21720 

aataggattt aatttgattt tccaacaatc ctgggaggga ggtattatta accccatttc  21780 

acagacacgg atatggggct tggagaggtc aagtggcttt cccaagatca agcagggagt  21840 

agagccagag ccaggatttg gacccaggct gggccaacac caaaacccat gccttcaacc  21900 

ttgacaccag cctgccaacc aaagacagga gaagggaagg ccctggaggt gagctgttgg  21960 

cagcagtgaa cttgctcgag ctccctcggg aaacactgtt gaaaggaaca tttccaaagc  22020 

catccaggcc cgcacctctc tacattccca agcacaaggc aaaaggaact tggatctgaa  22080 

ctgatgcaga cccgttcttt cccaacaagt agctcttcaa gtgcggatgt catctttccc  22140 

aaccccattt gcgggtaaac cttcctctgt cattgcaggt tcaggcttgt gaatcccaga  22200 

ggacgctgtg tgaactgggt ctatgaaatc ggcactgaca gcagccagcc ttccagggga  22260 

tgggggaggg aagaaacagc agatattttc ccaaggttca gctttataat tttcttggaa  22320 

atttccgagc agccaatcag ctagcttcta aatagtgtgg gtctctcctg gacactgcag  22380 

gcaaaagttc ctctatctgc tcctcttttt gtcctttctt ggagctcagt gctcatgttc  22440 

actgttcaag aaatgggcca atcatactcc caactaggga taatgtgcat cata        22494 

 
           
             6  
             15  
             DNA  
             Homo sapiens  
           
            6 

gccggcagct ctggg                                                      15 

 
           
             7  
             22  
             DNA  
             Homo sapiens  
           
            7 

ctgagcaagc cttcctcaga gt                                              22 

 
           
             8  
             24  
             DNA  
             Homo sapiens  
           
            8 

cactctcacg gtgaaatacc aagt                                            24 

 
           
             9  
             17  
             DNA  
             Homo sapiens  
           
            9 

ggatccgggt tcgcaga                                                    17 

 
           
             10  
             20  
             DNA  
             Homo sapiens  
           
            10 

cctttgatgt gcttgccaca                                                 20 

 
           
             11  
             22  
             DNA  
             Homo sapiens  
           
            11 

ttggagtcca agacgttgac ct                                              22 

 
           
             12  
             19  
             DNA  
             Homo sapiens  
           
            12 

tgtcattgtg ggccctgat                                                  19 

 
           
             13  
             19  
             DNA  
             Homo sapiens  
           
            13 

tcgacgcaga atgacctgg                                                  19 

 
           
             14  
             22  
             DNA  
             Homo sapiens  
           
            14 

cgcttctcat aaaactgacc gc                                              22 

 
           
             15  
             24  
             DNA  
             Homo sapiens  
           
            15 

ttgtgtcctc aatccaagtc atct                                            24 

 
           
             16  
             21  
             DNA  
             Homo sapiens  
           
            16 

cagcccattg caaagttctc a                                               21 

 
           
             17  
             22  
             DNA  
             Homo sapiens  
           
            17 

ggtgcattgt cgttgatgtc ac                                              22 

 
           
             18  
             21  
             DNA  
             Homo sapiens  
           
            18 

caggacacaa tggtttggtc c                                               21 

 
           
             19  
             26  
             DNA  
             Homo sapiens  
           
            19 

caatagctac taagtgagca actggg                                          26 

 
           
             20  
             21  
             DNA  
             Homo sapiens  
           
            20 

tgacatcaac gacaatgcac c                                               21 

 
           
             21  
             19  
             DNA  
             Homo sapiens  
           
            21 

acggagaggc tggcttttc                                                  19 

 
           
             22  
             28  
             DNA  
             Homo sapiens  
           
            22 

gctcacttag tagctattga ctccaaca                                        28 

 
           
             23  
             16  
             DNA  
             Homo sapiens  
           
            23 

tgcccccgag tctgca                                                     16 

 
           
             24  
             20  
             DNA  
             Homo sapiens  
           
            24 

ctctccgtgc ttgtgaatgc                                                 20 

 
           
             25  
             20  
             DNA  
             Homo sapiens  
           
            25 

ctgagtccct caggtggtcc                                                 20 

 
           
             26  
             22  
             DNA  
             Homo sapiens  
           
            26 

ttcatcctca accctcatac gg                                              22 

 
           
             27  
             19  
             DNA  
             Homo sapiens  
           
            27 

gcacgaggtg gatgtctgc                                                  19 

 
           
             28  
             21  
             DNA  
             Homo sapiens  
           
            28 

tgtactgttg ggcatcttcg g                                               21 

 
           
             29  
             21  
             DNA  
             Homo sapiens  
           
            29 

gaggttgacc gtgtcttgca g                                               21 

 
           
             30  
             20  
             DNA  
             Homo sapiens  
           
            30 

gggcagtccc acaaagatgt                                                 20 

 
           
             31  
             19  
             DNA  
             Homo sapiens  
           
            31 

tgagatgtcg ctgccgtct                                                  19 

 
           
             32  
             21  
             DNA  
             Homo sapiens  
           
            32 

gagaacctga accttcccga g                                               21 

 
           
             33  
             21  
             DNA  
             Homo sapiens  
           
            33 

gaggactgtg caggttatgg g                                               21 

 
           
             34  
             18  
             DNA  
             Homo sapiens  
           
            34 

gtccggctgt ctgtggct                                                   18 

 
           
             35  
             23  
             DNA  
             Homo sapiens  
           
            35 

ggcaccataa atgccttata gga                                             23 

 
           
             36  
             25  
             DNA  
             Homo sapiens  
           
            36 

cttagctgtg tgatcctagg caaat                                           25 

 
           
             37  
             28  
             DNA  
             Homo sapiens  
           
            37 

tgagtactga ctacatgtca gatggaca                                        28 

 
           
             38  
             25  
             DNA  
             Homo sapiens  
           
            38 

acttcttcct agctctgaac cacag                                           25 

 
           
             39  
             25  
             DNA  
             Homo sapiens  
           
            39 

aactccaatc tgcctaactg ctaag                                           25 

 
           
             40  
             21  
             DNA  
             Homo sapiens  
           
            40 

atgaagaagg ccagcatagg c                                               21 

 
           
             41  
             22  
             DNA  
             Homo sapiens  
           
            41 

gatatgggtg tgagatcagg ca                                              22 

 
           
             42  
             20  
             DNA  
             Homo sapiens  
           
            42 

accatccccc atctgatcac                                                 20 

 
           
             43  
             19  
             DNA  
             Homo sapiens  
           
            43 

ggatgctgtc tgctgcacc                                                  19 

 
           
             44  
             21  
             DNA  
             Homo sapiens  
           
            44 

ccaggctgtg tatccagttc c                                               21 

 
           
             45  
             21  
             DNA  
             Homo sapiens  
           
            45 

cctccacaga aacagaatgg c                                               21 

 
           
             46  
             18  
             DNA  
             Homo sapiens  
           
            46 

cccttgccct gctcattg                                                   18 

 
           
             47  
             17  
             DNA  
             Homo sapiens  
           
            47 

cctcccgcag accgaga                                                    17 

 
           
             48  
             18  
             DNA  
             Homo sapiens  
           
            48 

ctgagcccaa caggcacg                                                   18 

 
           
             49  
             23  
             DNA  
             Homo sapiens  
           
            49 

agtcacccta aagttctcag gcc                                             23 

 
           
             50  
             18  
             DNA  
             Homo sapiens  
           
            50 

cccaggtgga aagacggg                                                   18 

 
           
             51  
             21  
             DNA  
             Homo sapiens  
           
            51 

tcctgcctgt gatactccgt g                                               21 

 
           
             52  
             21  
             DNA  
             Homo sapiens  
           
            52 

aagagcccca ggactaacag c                                               21 

 
           
             53  
             19  
             DNA  
             Homo sapiens  
           
            53 

ctgtccaggc ccagtgtga                                                  19 

 
           
             54  
             19  
             DNA  
             Homo sapiens  
           
            54 

gggtgccagg aaatgctct                                                  19 

 
           
             55  
             23  
             DNA  
             Homo sapiens  
           
            55 

gggttaataa tacctccctc cca                                             23 

 
           
             56  
             27  
             DNA  
             Homo sapiens  
           
            56 

atgttaccaa ctaatctgct tctcagc                                         27 

 
           
             57  
             21  
             DNA  
             Homo sapiens  
           
            57 

tcctttcaac agtgtttccc g                                               21 

 
           
             58  
             19  
             DNA  
             Homo sapiens  
           
            58 

ctgggccaac accaaaacc                                                  19 

 
           
             59  
             20  
             DNA  
             Homo sapiens  
           
            59 

gaaggctggc tgctgtcagt                                                 20 

 
           
             60  
             23  
             DNA  
             Homo sapiens  
           
            60 

aagtagctct tcaagtgcgg atg                                             23 

 
           
             61  
             25  
             DNA  
             Homo sapiens  
           
            61 

tatgatgcac attatcccta gttgg                                           25 

 
           
             62  
             22  
             DNA  
             Homo sapiens  
           
            62 

ggtgcctgtg aacatacctc ag                                              22 

 
           
             63  
             24  
             DNA  
             Homo sapiens  
           
            63 

gataggtcag agagcatttc ctgg                                            24 

 
           
             64  
             22  
             DNA  
             Homo sapiens  
           
            64 

tgactcctgc tacccaagaa gg                                              22 

 
           
             65  
             23  
             DNA  
             Homo sapiens  
           
            65 

cttgccttag gcttatctcc ctt                                             23 

 
           
             66  
             22  
             DNA  
             Homo sapiens  
           
            66 

gcctcggaat gtcagctact tt                                              22 

 
           
             67  
             19  
             DNA  
             Homo sapiens  
           
            67 

ggtcatctgg tgcctttgg                                                  19 

 
           
             68  
             22  
             DNA  
             Homo sapiens  
           
            68 

ccagcctaac aatgctctcc tt                                              22 

 
           
             69  
             45  
             DNA  
             Homo sapiens  
           
            69 

gaagatcttc ggaattccat catgatgcaa cttctgcaac ttctg                     45 

 
           
             70  
             55  
             DNA  
             Homo sapiens  
           
            70 

aagatcttcg gtacctcaat ggtgatggtg atggtgcagg cacctgctgc tgctg          55