EP1049800A1 - Asthma related genes - Google Patents

Asthma related genes

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Publication number
EP1049800A1
EP1049800A1 EP98904649A EP98904649A EP1049800A1 EP 1049800 A1 EP1049800 A1 EP 1049800A1 EP 98904649 A EP98904649 A EP 98904649A EP 98904649 A EP98904649 A EP 98904649A EP 1049800 A1 EP1049800 A1 EP 1049800A1
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EP
European Patent Office
Prior art keywords
asth1
seq
sequence
gene
asthma
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EP98904649A
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German (de)
French (fr)
Other versions
EP1049800A4 (en
Inventor
Angela R. Brooks-Wilson
Alan Buckler
Lon Wellcome Trust Ctr for Human Genetics CARDON
Alisoun H. Carey
Margaret Galvin
Andrew Miller
Michael North
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Axys Pharmaceuticals Inc
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Axys Pharmaceuticals Inc
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Publication of EP1049800A1 publication Critical patent/EP1049800A1/en
Publication of EP1049800A4 publication Critical patent/EP1049800A4/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • INTRODUCTION Asthma is a disease of reversible bronchial obstruction, characterized by airway inflammation, epithelial damage, airway smooth muscle hypertrophy and bronchial hyperreactivity. Many asthma symptoms can be controlled by medical intervention, but incidence of asthma-related death and severe illness continue to rise in the United States. The approximately 4,800 deaths in 1989 marked a 46 percent increase since 1980. As many as 12 million people in the United States have asthma, up 66 percent since 1980, and annually, the disease's medical and indirect costs are estimated at over $6 billion.
  • Atopy is characterized by a predisposition to raise an IgE antibody response to common environmental antigens.
  • asthma symptoms and evidence of allergy such as a positive skin test to common allergens, are both present.
  • Non-atopic asthma may be defined as reversible airflow limitation in the absence of allergies.
  • bronchial hyperreactivity a characteristic of asthma thought to have a heritable component.
  • Studies have demonstrated a genetic predisposition to asthma by showing, for example, a greater concordance for this trait among monozygotic twins than among dizygotic twins.
  • the genetics of asthma is complex, however, and shows no simple pattern of inheritance. Environment also plays a role in asthma development, for example, children of smokers are more likely to develop asthma than are children of non-smokers.
  • -1- is little or no biochemical data concerning the encoded product.
  • genes that predispose to human diseases, such as cystic fibrosis, Huntington's disease, etc. are of interest because of their phenotypic effect.
  • Biochemical characterization of such genes may be secondary to genetic characterization.
  • a solution to this impasse has been found in combining classical genetic mapping with the ability to identify genes and, if necessary, to sequence large regions of chromosomes. Population and family studies enable genes associated with a trait of interest to be localized to a relatively small region of a chromosome. At this point, physical mapping can be used to identify candidate genes, and various molecular biology techniques used to pick out mutated genes in affected individuals.
  • Positional cloning This "top-down" approach to gene discovery has been termed positional cloning, because genes are identified based on position in the genome. Positional cloning is now being applied to complex genetic diseases, which affect a greater fraction of civilization than do the more simple and usually rarer single gene disorders. Such studies must take into account the contribution of both environmental and genetic factors to the development of disease, and must allow for contributions to the genetic component by more than one, and potentially many, genes. The clinical importance of asthma makes it of considerable interest to characterize genes that underlie a genetic predisposition to this disease. Positional cloning provides an approach to this goal.
  • the genes associated with a genetic predisposition to asthma are provided.
  • the genes, herein termed ASTH1I and ASTH1J, are located close to each other on human chromosome 11 p, have similar patterns of expression, and common sequence motifs.
  • the nucleic acid compositions are used to produce the encoded proteins, which may be employed for functional studies, as a therapeutic, and in studying associated physiological pathways.
  • the nucleic acid compositions and antibodies specific for the protein are useful as diagnostics to identify a hereditary predisposition to asthma.
  • ASTH1 genes and fragments thereof, encoded protein, ASTH1 genomic regulatory regions, and anti-/AS7H1 antibodies are useful in the identification of individuals predisposed to development of asthma, and for the modulation of gene activity in vivo for prophylactic and therapeutic purposes.
  • the encoded ASTl ⁇ l protein is useful as an immunogen to raise specific antibodies, in drug screening for compositions that mimic or modulate ASTH ⁇ activity or expression, including altered forms of ASTH protein, and as a therapeutic.
  • Asthma is reversible airflow limitation in a patient over a period of time.
  • the disease is characterized by increased airway responsiveness to a variety of stimuli, and airway inflammation.
  • a patient diagnosed as asthmatic will generally have multiple indications over time, including wheezing, asthmatic attacks, and a positive response to methacholine challenge, i.e. a PC 20 on methacholine challenge of less than about 4 mg/ml.
  • Guidelines for diagnosis may be found in the National Asthma Education Program Expert Panel. Guidelines for diagnosis and management of asthma. National Institutes of Health, 1991 ; Pub. #91-3042.
  • Atopy, respiratory infection and environmental predisposing factors may also be present, but are not necessary elements of an asthma diagnosis.
  • Asthma conditions strictly related to atopy are referred to as atopic asthma.
  • the human ASTH1I and ASTH1J gene sequences are provided, as are the genomic sequences 5' to ASTH1J.
  • the major sequences of interest provided in the sequence listing are as follows:
  • Microsatellite flanking sequences DNA SEQ ID NO:160-281
  • the ASTH1 locus has been mapped to human chromosome 11p.
  • the traits for a positive response to methacholine challenge and a clinical history of asthma were shown to be genetically linked in a genome scan of the population of Tristan da Cunha, a single large extended family with a high incidence of asthma (discussed in Zamel et al. (1996) Am. J. Respir. Crit. Care Med. 153:1902-1906).
  • the linkage finding was replicated in a set of Canadian asthmatic families.
  • the region of strongest linkage was the marker D11S907 on the short arm of chromosome 11. Additional markers were identified from the four megabase region surrounding D11S907 from public databases and by original cloning of new polymorphic microsatellite markers.
  • TDT transmission disequilibrium test
  • ASTH1 I produces a 2.8 kb mRNA expressed at high levels in trachea and prostate, and at lower levels in lung and kidney and possibly other tissues.
  • ASTH11 cDNA clones have also been identified in prostate, testis and lung libraries. Sequence polymorphisms are shown in Table 3.
  • ASTH11 has at least three alternate forms denoted as altl , alt2, and alt3. The alternative splicing and start codons give the three forms of ASTH1 I proteins different amino termini.
  • the ASTH1 I proteins, altl , alt2 and alt3 are 265, 255 and 164 amino acids in length, respectively.
  • a domain of the ASTH1 I and ASTH1 J proteins is similar in sequence to transcription factors of the ets family.
  • the ets family is a group of transcription factors that activate genes involved in a variety of immunological and other processes.
  • the family members most similar to ASTH11 and ASTH1 J are: ETS1 , ETS2, ESX, ELF, ELK1 , TEL, NET, SAP-1 , NERF and FLI.
  • the ASTH1 I and ASTH1 J proteins show similarity to each other. Over the ets domain they are 66% similar (ie. have amino acids with similar properties in the same positions) and 46% identical to each other. All forms of ASTH1 I and ASTH1J have a helix turn helix motif, characteristic of some transcription factors, located near the carboxy terminal end of the protein.
  • ASTH1J produces an approximately 6 kb mRNA expressed at high levels in the trachea, prostate and pancreas and at lower levels in colon, small intestine, lung and stomach.
  • ASTH1J has at least three forms, consisting of the altl , alt2 and alt3 forms.
  • the open reading frame is identical for the three forms, which differ only in the 5' UTR.
  • the protein encoded by ASTH1 J is 300 amino acids in length.
  • Mouse coding region sequence of asthlj is provided in SEQ ID NO:326, and the amino acid sequence is provided in SEQ ID NO:327.
  • the mouse and human proteins have 88.4% identity throughout their length. The match in the ets domain is 100%.
  • the mouse cDNA was identified by hybridization of a full-length human cDNA to a mouse lung cDNA library (Stratagene).
  • ASTH1 genes is herein used generically to designate ASTH1I and ASTH1J genes and their alternate forms. The two genes lie in opposite orientations on a native chromosome, with the 5' regulatory sequences between them. Part of the genomic sequence between the two coding regions is provided as SEQ ID NO:1.
  • the term "ASTH1 locus” is used herein to refer to the two genes in all alternate forms and the genomic sequence that lies between the two genes. Alternate forms include splicing variants, and polymorphisms in the sequence. Specific polymorphic sequences are provided in SEQ ID NOs:16-159. For some purposes the previously known EST sequences described herein may be excluded from the sequences defined as the ASTH1 locus.
  • the DNA sequence encoding ASTH1 may be cDNA or genomic DNA or a fragment thereof.
  • the term "ASTH1 gene” shall be intended to mean the open reading frame encoding specific ASTH1 polypeptides, introns, as well as adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression, up to about 1 kb beyond the coding region, but possibly further in either direction. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.
  • cDNA as used herein is intended to include all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3' and 5' non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns removed by nuclear RNA splicing, to create a continuous open reading frame encoding the ASTH1 protein.
  • genomic ASTH1 sequence has non-contiguous open reading frames, where introns interrupt the protein coding regions.
  • a genomic sequence of interest comprises the nucleic acid present between the initiation codon and the stop codon, as defined in the listed sequences, including all of the introns that are normally present in a native chromosome. It may further include the 3' and 5' untranslated regions found in the mature mRNA. It may further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc.,
  • Genomic regions of interest include the non-transcribed sequences 5' to ASTH1J, as provided in SEQ ID NO:1. This region of DNA contains the native promoter elements that direct expression of the linked ASTH1J gene. Usually a promoter region will have at least about 140 nt of sequence located 5' to the ASTH1 gene and further comprising a TATA box and CAAT box motif sequence (SEQ ID NO: 14, nt. 597-736).
  • the promoter region may further comprise a consensus ets binding motif, (C/A)GGA(A/T) (SEQ ID NO:14, nt 1-5).
  • a region of particular interest containing the ets binding motif, TATA box and CAAT box motifs 5' to the ASTH1J gene, is provided in SEQ ID NO:14.
  • the position of SEQ ID NO:14 within the larger sequence is SEQ ID NO:1 , nt 60359-61095.
  • the promoter sequence may comprise polymorphisms within the CAAT box region, for example those shown in SEQ ID NO: 12 and SEQ ID NO: 13, which have been shown to affect the function of the promoter.
  • the promoter region of interest may extend 5' to SEQ ID NO:14 within the larger sequence, e.g. SEQ ID NO:1 , nt 59000-61095; SEQ ID NO:1 , nt 5700-61095, etc.
  • sequence of this 5' region, and further 5' upstream sequences and 3' downstream sequences, may be utilized for promoter elements, including enhancer binding sites, that provide for expression in tissues where ASTH1J is expressed.
  • tissue specific expression is useful for determining the pattern of expression, and for providing promoters that mimic the native pattern of expression.
  • Naturally occurring polymorphisms in the promoter region are useful for determining natural variations in expression, particularly those that may be associated with disease. See, for example, SEQ ID NO: 12 and 13.
  • mutations may be introduced into the promoter region to determine the effect of altering expression in experimentally defined systems.
  • Methods for the identification of specific DNA motifs involved in the binding of transcriptional factors are known in the art, e.g. sequence similarity to known binding motifs, gel retardation studies, etc. For examples, see Blackwell et al. (1995) Mol Med 1: 194-205; Mortlock et al. (1996)
  • the regulatory sequences may be used to identify cis acting sequences required for transcriptional or translational regulation of ASTH1 expression, especially in different tissues or stages of development, and to identify cis acting sequences and trans acting factors that regulate or mediate ASTH1 expression.
  • Such transcription or translational control regions may be operably linked to a ASTH1 gene in order to promote expression of wild type or altered ASTH1 or other proteins of interest in cultured cells, or in embryonic, fetal or adult tissues, and for gene therapy.
  • the nucleic acid compositions of the subject invention may encode all or a part of the subject polypeptides. Fragments may be obtained of the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc. For the most part, DNA fragments will be of at least 15 nt, usually at least 18 nt, more usually at least about 50 nt. Such small DNA fragments are useful as primers for PCR, hybridization screening, etc. Larger DNA fragments, i.e. greater than 100 nt are useful for production of the encoded polypeptide. For use in amplification reactions, such as PCR, a pair of primers will be used.
  • primer sequences The exact composition of the primer sequences is not critical to the invention, but for most applications the primers will hybridize to the subject sequence under stringent conditions, as known in the art. It is preferable to choose a pair of primers that will generate an amplification product of at least about 50 nt, preferably at least about 100 nt. Algorithms for the selection of primer sequences are generally known, and are available in commercial software packages. Amplification primers hybridize to complementary strands of DNA, and will prime towards each other.
  • the ASTH1 genes are isolated and obtained in substantial purity, generally as other than an intact mammalian chromosome. Usually, the DNA will be obtained substantially free of other nucleic acid sequences that do not include an ASTH1 sequence or fragment thereof, generally being at least about 50%, usually at least about 90% pure and are typically "recombinant", i.e. flanked by one or more
  • the DNA sequences are used in a variety of ways. They may be used as probes for identifying ASTH1 related genes. Mammalian homologs have substantial sequence similarity to the subject sequences, i.e. at least 75%, usually at least 90%, more usually at least 95% sequence identity with the nucleotide sequence of the subject DNA sequence. Sequence similarity is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al. (1990) J Mol Biol 215:403-10.
  • Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50°C and 10XSSC (0.9 M saline/0.09 M sodium citrate) and remain bound when subjected to washing at 55°C in 1XSSC. Sequence identity may be determined by hybridization under stringent conditions, for example, at 50°C or higher and 0.1XSSC (9 mM saline/0.9 mM sodium citrate).
  • probes, particularly labeled probes of DNA sequences one can isolate homologous or related genes.
  • the source of homologous genes may be any species, e.g.
  • RNA is isolated from a cell sample. mRNA may be amplified by RT-PCR, using reverse transcriptase to form a complementary DNA strand, followed by polymerase chain reaction amplification using primers specific for the subject DNA sequences.
  • mRNA sample is separated by gel electrophoresis, transferred to a suitable support, e.g. nitrocellulose, nylon, etc., and then probed with a fragment of the subject DNA as a probe.
  • suitable support e.g. nitrocellulose, nylon, etc.
  • Other techniques such as oligonucleotide ligation assays, in situ
  • -10- hybridizations, and hybridization to DNA probes arrayed on a solid chip may also find use. Detection of mRNA hybridizing to the subject sequence is indicative of ASTH1 gene expression in the sample.
  • the subject nucleic acid sequences may be modified for a number of purposes, particularly where they will be used intracellularly, for example, by being joined to a nucleic acid cleaving agent, e.g. a chelated metal ion, such as iron or chromium for cleavage of the gene; or the like.
  • a nucleic acid cleaving agent e.g. a chelated metal ion, such as iron or chromium for cleavage of the gene; or the like.
  • sequence of the ASTH1 locus may be mutated in various ways known in the art to generate targeted changes in promoter strength, sequence of the encoded protein, etc.
  • the DNA sequence or product of such a mutation will be substantially similar to the sequences provided herein, i.e. will differ by at least one nucleotide or amino acid, respectively, and may differ by at least two but not more than about ten nucleotides or amino acids.
  • the sequence changes may be substitutions, insertions or deletions. Deletions may further include larger changes, such as deletions of a domain or exon.
  • Other modifications of interest include epitope tagging, e.g. with the FLAG system, HA, etc.
  • fusion proteins with green fluorescent proteins may be used.
  • GFP green fluorescent proteins
  • Such mutated genes may be used to study structure-function relationships of ASTH1 polypeptides, or to alter properties of the protein that affect its function or regulation.
  • constitutively active transcription factors, or a dominant negatively active protein that binds to the ASTH1 DNA target site without activating transcription may be created in this manner.
  • the subject gene may be employed for synthesis of a complete ASTH1 protein, or polypeptide fragments thereof, particularly fragments corresponding to functional domains; binding sites; etc.; and including fusions of the subject polypeptides to other proteins or parts thereof.
  • an expression cassette may be employed, providing for a transcriptional and translational initiation region, which may be inducible or constitutive, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region.
  • Various transcriptional initiation regions may be employed that are functional in the expression host.
  • the polypeptides may be expressed in prokaryotes or eukaryotes in accordance with conventional ways, depending upon the purpose for expression.
  • a unicellular organism such as E. coli, B. subtilis, S. cerevisiae, or cells of a higher organism such as vertebrates, particularly mammals, e.g. COS 7 cells, may be used as the expression host cells.
  • Small peptides can also be synthesized in the laboratory.
  • polypeptides may be isolated and purified in accordance with conventional ways.
  • a lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique.
  • the purified polypeptide will generally be at least about 80% pure, preferably at least about 90% pure, and may be up to and including 100% pure. Pure is intended to mean free of other proteins, as well as cellular debris.
  • the polypeptide is used for the production of antibodies, where short fragments provide for antibodies specific for the particular polypeptide, and larger fragments or the entire protein allow for the production of antibodies over the surface of the polypeptide.
  • Antibodies may be raised to the wild-type or variant
  • ASTH Antibodies may be raised to isolated peptides corresponding to these domains, or to the native protein, e.g. by immunization with cells expressing ASTH1 , immunization with liposomes having ASTH1 inserted in the membrane, etc.
  • Antibodies are prepared in accordance with conventional ways, where the expressed polypeptide or protein is used as an immunogen, by itself or conjugated to known immunogenic carriers, e.g. KLH, pre-S HBsAg, other viral or eukaryotic proteins, or the like.
  • immunogenic carriers e.g. KLH, pre-S HBsAg, other viral or eukaryotic proteins, or the like.
  • adjuvants may be employed, with a series of injections, as appropriate.
  • the spleen is isolated, the lymphocytes immortalized by cell fusion, and then screened for high affinity antibody binding.
  • the immortalized cells, i.e. hybridomas, producing the desired antibodies may then be expanded.
  • the mRNA encoding the heavy and light chains may be isolated and mutagenized by cloning in E. coli, and the heavy and light chains mixed to further enhance the affinity of the antibody.
  • Alternatives to in vivo immunization as a method of raising antibodies include binding to phage "display" libraries, usually in conjunction with in vitro affinity maturation.
  • RNA sequence and/or hybridization analysis of any convenient sample from a patient e.g. biopsy material, blood sample, scrapings from cheek, etc.
  • a nucleic acid sample from a patient having asthma that may be associated with ASTH1 is analyzed for the presence of a predisposing polymorphism in ASTHL
  • a typical patient genotype will have at least one predisposing mutation on at least one chromosome.
  • the presence of a polymorphic ASTH1 sequence that affects the activity or expression of the gene product, and confers an increased susceptibility to asthma is considered a predisposing polymorphism.
  • Individuals are screened by analyzing their DNA or mRNA for the presence of a predisposing polymorphism, as compared to an asthma neutral sequence.
  • Specific sequences of interest include any polymorphism that leads to clinical bronchial hyperreactivity or is otherwise associated with asthma, including, but not limited to, insertions, substitutions and
  • an ASTH1 predisposing polymorphism may be modulated by the patient genotype in other genes related to asthma and atopy, including, but not limited to, the Fc ⁇ receptor, Class I and Class II HLA antigens, T cell receptor and immunoglobulin genes, cytokines and cytokine receptors, and the like. Screening may also be based on the functional or antigenic characteristics of the protein. Immunoassays designed to detect predisposing polymorphisms in ASTH1 proteins may be used in screening. Where many diverse mutations lead to a particular disease phenotype, functional protein assays have proven to be effective screening tools.
  • Biochemical studies may be performed to determine whether a candidate sequence polymorphism in the ASTH1 coding region or control regions is associated with disease. For example, a change in the promoter or enhancer sequence that affects expression of ASTH1 may result in predisposition to asthma.
  • Expression levels of a candidate variant allele are compared to expression levels of the normal allele by various methods known in the art. Methods for determining promoter or enhancer strength include quantitation of the expressed natural protein; insertion of the variant control element into a vector with a reporter gene such as ⁇ -galactosidase, luciferase, chloramphenicol acetyltransferase, etc. that provides for convenient quantitation; and the like.
  • the activity of the encoded ASTH1 protein may be determined by comparison with the wild-type protein.
  • nucleic acids for the presence of a specific sequence. Where large amounts of DNA are available, genomic DNA is used directly. Alternatively, the region of interest is cloned into a suitable vector and grown in sufficient quantity for analysis. Cells that express ASTH1 genes, such as trachea cells, may be used as a source of mRNA, which may be assayed directly or reverse transcribed into cDNA for analysis. The nucleic acid may be amplified by conventional techniques, such as the polymerase chain reaction (PCR), to provide sufficient amounts for analysis. The use of the polymerase chain reaction is described in Saiki, et al.
  • Amplification may also be used to determine whether a polymorphism is present, by using a primer that is specific for the polymorphism.
  • various methods are known in the art that utilize oligonucleotide ligation as a means of detecting polymorphisms, for examples see Riley et al. (1990) N.A.R. 18:2887-2890; and Delahunty et al. (1996) Am. J. Hum. Genet. 58: 1239-1246.
  • a detectable label may be included in an amplification reaction.
  • Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive labels, e.g.
  • the label may be a two stage system, where the amplified DNA is conjugated to biotin, haptens, etc. having a high affinity binding partner, e.g. avidin, specific antibodies, etc., where the binding partner is conjugated to a detectable label.
  • the label may be conjugated to one or both of the primers.
  • the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product.
  • sample nucleic acid e.g. amplified or cloned fragment
  • the nucleic acid may be sequenced by dideoxy or other methods, and the sequence of bases compared to a neutral ASTH1 sequence. Hybridization with the variant sequence may also be used to
  • SSCP Single strand conformational polymorphism
  • DGGE denaturing gradient gel electrophoresis
  • mismatch cleavage detection and heteroduplex analysis in gel matrices are used to detect conformational changes created by DNA sequence variation as alterations in electrophoretic mobility.
  • a polymorphism creates or destroys a recognition site for a restriction endonuclease (restriction fragment length polymorphism, RFLP)
  • the sample is digested with that endonuclease, and the products size fractionated to determine whether the fragment was digested. Fractionation is performed by gel or capillary electrophoresis, particularly acrylamide or agarose gels.
  • an array of oligonucleotides are provided, where discrete positions on the array are complementary to at least a portion of mRNA or genomic DNA of the ASTH1 locus.
  • Such an array may comprise a series of oligonucleotides, each of which can specifically hybridize to a nucleic acid, e.g. mRNA, cDNA, genomic DNA, etc. from the ASTH1 locus.
  • An array may include all or a subset of the polymorphisms listed in Table 3 (SEQ ID NOs:16-126).
  • One or both polymorphic forms may be present in the array, for example the polymorphism of SEQ ID NO: 12 and 13 may be represented by either, or both, of the listed sequences.
  • Such an array will include at least 2 different polymorphic sequences, i.e. polymorphisms located at unique positions within the locus, usually at least about 5, more usually at least about 10, and may include as many as 50 to 100 different polymorphisms.
  • the oligonucleotide sequence on the array will usually be at least about 12 nt in length, may be the length of the provided polymorphic sequences, or may extend into the flanking regions to generate fragments of 100 to 200 nt in length.
  • arrays For examples of arrays,
  • Antibodies specific for ASTH1 polymorphisms may be used in screening immunoassays.
  • a reduction or increase in neutral ASTH1 and/or presence of asthma associated polymorphisms is indicative that asthma is ASTH1 -associated.
  • a sample is taken from a patient suspected of having ASTH1 -associated asthma.
  • Samples include biological fluids such as tracheal lavage, blood, cerebrospinal fluid, tears, saliva, lymph, dialysis fluid and the like; organ or tissue culture derived fluids; and fluids extracted from physiological tissues. Also included in the term are derivatives and fractions of such fluids. Biopsy samples are of particular interest, e.g. trachea scrapings, etc.
  • the number of cells in a sample will generally be at least about 10 3 , usually at least 10 more usually at least about 10 5 .
  • the cells may be dissociated, in the case of solid tissues, or tissue sections may be analyzed. Alternatively a lysate of the cells may be prepared.
  • Diagnosis may be performed by a number of methods. The different methods all determine the absence or presence or altered amounts of normal or abnormal ASTH1 in patient cells suspected of having a predisposing polymorphism in ASTHL For example, detection may utilize staining of cells or histological sections, performed in accordance with conventional methods.
  • the antibodies of interest are added to the cell sample, and incubated for a period of time sufficient to allow binding to the epitope, usually at least about 10 minutes.
  • the antibody may be labeled with radioisotopes, enzymes, fluorescers, chemiluminescers, or other labels for direct detection.
  • a second stage antibody or reagent is used to amplify the signal.
  • the primary antibody may be conjugated to biotin, with horseradish peroxidase- conjugated avidin added as a second stage reagent.
  • Final detection uses a substrate that undergoes a color change in the presence of the peroxidase.
  • the absence or presence of antibody binding may be determined by various methods, including flow cytometry of dissociated cells, microscopy, radiography, scintillation counting, etc.
  • An alternative method for diagnosis depends on the in vitro detection of binding between antibodies and ASTH1 in a lysate. Measuring the concentration of
  • -17- ASTH1 binding in a sample or fraction thereof may be accomplished by a variety of specific assays.
  • a conventional sandwich type assay may be used.
  • a sandwich assay may first attach ASTH1 -specific antibodies to an insoluble surface or support.
  • the particular manner of binding is not crucial so long as it is compatible with the reagents and overall methods of the invention. They may be bound to the plates covalently or non-covalently, preferably non-covalently.
  • the insoluble supports may be any compositions to which polypeptides can be bound, which is readily separated from soluble material, and which is otherwise compatible with the overall method.
  • the surface of such supports may be solid or porous and of any convenient shape.
  • suitable insoluble supports to which the receptor is bound include beads, e.g. magnetic beads, membranes and microtiter plates. These are typically made of glass, plastic (e.g. polystyrene), polysaccharides, nylon or nitrocellulose. Microtiter plates are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples.
  • Patient sample lysates are then added to separately assayable supports (for example, separate wells of a microtiter plate) containing antibodies.
  • a series of standards containing known concentrations of normal and/or abnormal ASTH1 is assayed in parallel with the samples or aliquots thereof to serve as controls.
  • each sample and standard will be added to multiple wells so that mean values can be obtained for each.
  • the incubation time should be sufficient for binding, generally, from about 0.1 to 3 hr is sufficient.
  • the insoluble support is generally washed of non-bound components.
  • a dilute non-ionic detergent medium at an appropriate pH, generally 7-8, is used as a wash medium. From one to six washes may be employed, with sufficient volume to thoroughly wash non-specifically bound proteins present in the sample.
  • a solution containing a second antibody is applied.
  • the antibody will bind ASTH1 with sufficient specificity such that it can be distinguished from other components present.
  • the second antibodies may be labeled to facilitate direct, or indirect quantification of binding. Examples of labels that permit direct measurement of second receptor binding include radiolabels, such as 3 H or 125 l, fluorescers, dyes, beads, chemilumninescers, colloidal particles, and the like.
  • labels which permit indirect measurement of binding include enzymes where the substrate may provide for a colored or fluorescent product.
  • the antibodies are labeled with a covalently bound enzyme capable of providing a detectable product signal after addition of suitable substrate.
  • suitable enzymes for use in conjugates include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase and the like. Where not commercially available, such antibody-enzyme conjugates are readily produced by techniques known to those skilled in the art.
  • the incubation time should be sufficient for the labeled ligand to bind available molecules. Generally, from about 0.1 to 3 hr is sufficient, usually 1 hr sufficing.
  • the insoluble support is again washed free of non-specifically bound material.
  • the signal produced by the bound conjugate is detected by conventional means. Where an enzyme conjugate is used, an appropriate enzyme substrate is provided so a detectable product is formed. Other immunoassays are known in the art and may find use as diagnostics.
  • Ouchterlony plates provide a simple determination of antibody binding.
  • Western blots may be performed on protein gels or protein spots on filters, using a detection system specific for ASTH1 as desired, conveniently using a labeling method as described for the sandwich assay.
  • Other diagnostic assays of interest are based on the functional properties of
  • ASTH1 proteins are particularly useful where a large number of different sequence changes lead to a common phenotype, i.e. altered protein function leading to bronchial hyperreactivity.
  • a functional assay may be based on the transcriptional changes mediated by ASTH1 gene products.
  • Other assays may, for example, detect conformational changes, size changes resulting from insertions, deletions or truncations, or changes in the subcellular localization of ASTH1 proteins.
  • PCR fragments amplified from the ASTH1 gene or its transcript are used as templates for in vivo transcription/translation reactions to generate protein products. Separation by gel electrophoresis is performed to determine whether the polymorphic gene encodes a truncated protein, where truncations may be associated with a loss of function.
  • -19- Diagnostic screening may also be performed for polymorphisms that are genetically linked to a predisposition for bronchial hyperreactivity, particularly through the use of microsatellite markers or single nucleotide polymorphisms. Frequently the microsatellite polymorphism itself is not phenotypically expressed, but is linked to sequences that result in a disease predisposition. However, in some cases the microsatellite sequence itself may affect gene expression. Microsatellite linkage analysis may be performed alone, or in combination with direct detection of polymorphisms, as described above. The use of microsatellite markers for genotyping is well documented. For examples, see Mansfield et al. (1994) Genomics 24:225-233; Ziegle et al. (1992) Genomics 14:1026-1031 ; Dib et al., supra.
  • Microsatellite loci that are useful in the subject methods have the general formula:
  • U (R) n U' where U and U' are non-repetitive flanking sequences that uniquely identify the particular locus, R is a repeat motif, and n is the number of repeats.
  • the repeat motif is at least 2 nucleotides in length, up to 7, usually 2-4 nucleotides in length. Repeats can be simple or complex.
  • the flanking sequences U and U' uniquely identify the microsatellite locus within the human genome.
  • U and U' are at least about 18 nucleotides in length, and may extend several hundred bases up to about 1 kb on either side of the repeat. Within U and U', sequences are selected for amplification primers.
  • primer sequences are not critical to the invention, but they must hybridize to the flanking sequences U and U', respectively, under stringent conditions. Criteria for selection of amplification primers are as previously discussed. To maximize the resolution of size differences at the locus, it is preferable to chose a primer sequence that is close to the repeat sequence, such that the total amplification product is between 100-500 nucleotides in length.
  • the number of repeats at a specific locus, n is polymorphic in a population, thereby generating individual differences in the length of DNA that lies between the amplification primers.
  • the number will vary from at least 1 repeat to as many as about 100 repeats or more.
  • the primers are used to amplify the region of genomic DNA that contains the repeats.
  • a detectable label will be included in the amplification reaction, as previously described.
  • Multiplex amplification may be performed in which several sets of primers are combined in the same reaction tube. This is particularly advantageous when limited amounts of sample DNA are available for analysis.
  • each of the sets of primers is labeled with a different fluorochrome.
  • the products are size fractionated. Fractionation may be performed by gel electrophoresis, particularly denaturing acrylamide or agarose gels.
  • gel electrophoresis particularly denaturing polyacrylamide gels in combination with an automated DNA sequencer, see Hunkapillar et al. (1991) Science 254:59-74. The automated sequencer is particularly useful with multiplex amplification or pooled products of separate PCR reactions.
  • Capillary electrophoresis may also be used for fractionation. A review of capillary electrophoresis may be found in Landers, et al. (1993) BioTechniques 14:98-111.
  • the size of the amplification product is proportional to the number of repeats (n) that are present at the locus specified by the primers. The size will be polymorphic in the population, and is therefore an allelic marker for that locus.
  • D11S2008 A number of markers in the region of the ASTH1 locus have been identified, and are listed in Table 1 in the Experimental section (SEQ ID NOs:160-273). Of particular interest for diagnostic purposes is the marker D11S2008, in which individuals having alleles C or F at this locus, particularly in combination with the CAAT box polymorphism and other polymorphisms, are predisposed to develop bronchial hyperreactivity or asthma.
  • the association of D11S2008 alleles is as follows: llele Association with asthma Number of TATC repeats relative to allele C (SEQ ID NO:15)
  • a DNA sequence of interest for diagnosis comprises the D11S2008 primer sequences shown in Table 1 (SEQ ID NO:242 and 243), flanking one or three repeats of SEQ ID NO: 15.
  • microsatellite markers of interest for diagnostic purposes are CA39_2; 774F; 774J; 7740; L19PENTA1 ; 65P14TE1 ; AFM205YG5; D11S907; D11S4200; 774N; CA11-11 ; 774L; AFM283WH9; ASMI14 and D11S1900 (primer sequences are provided in Table 1 , the repeats are provided in Table 1 B).
  • ASTH1 genes are useful for analysis of ASTH1 expression, e.g. in determining developmental and tissue specific patterns of expression, and for modulating expression in vitro and in vivo.
  • the regulatory region of SEQ ID NO:1 may also be used to investigate analysis of ASTH1 expression.
  • Vectors useful for introduction of the gene include plasmids and viral vectors. Of particular interest are retroviral-based vectors, e.g. Moloney murine leukemia virus and modified human immunodeficiency virus; adenovirus vectors, etc. that are maintained transiently or stably in mammalian cells.
  • retroviral-based vectors e.g. Moloney murine leukemia virus and modified human immunodeficiency virus
  • adenovirus vectors, etc. that are maintained transiently or stably in mammalian cells.
  • a wide variety of vectors can be employed for transfection and/or integration of the gene into the genome of the cells.
  • micro-injection may be employed, fusion, or the like for introduction of genes into a suitable host cell.
  • suitable host cell See, for example, Dhawan et al. (1991) Science 254:1509-1512 and Smith et al. (1990) Molecular and Cellular Biology 3268-3271.
  • Administration of vectors to the lungs is of particular interest. Frequently such methods utilize liposomal formulations, as described in Eastman et al. (1997) Hum Gene Ther 8:765-773: Oudrhiri et al. (1997) P.N.A.S. 94:1651-1656: McDonald et al. (1997) Hum Gene Ther 8:411-422.
  • the expression vector will have a transcriptional initiation region oriented to produce functional mRNA.
  • the native transcriptional initiation region e.g. SEQ ID NO: 14, or an exogenous transcriptional initiation region may be employed.
  • the promoter may be introduced by recombinant methods in vitro, or as the result of homologous integration of the sequence into a chromosome.
  • Many strong promoters are known in the art, including the ⁇ -actin promoter, SV40 early and late promoters, human cytomegalovirus promoter, retroviral LTRs, methallothionein responsive element (MRE), tetracycline-inducible promoter constructs, etc.
  • Transcription cassettes may be prepared comprising a transcription initiation region, the target gene or fragment thereof, and a transcriptional termination region.
  • the transcription cassettes may be introduced into a variety of vectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like, where the vectors are able to transiently or stably be maintained in the cells, usually for a period of at least about one day, more usually for a period of at least about several days to several weeks.
  • Antisense molecules are used to down-regulate expression of ASTH1 in cells.
  • the anti-sense reagent may be antisense oligonucleotides (ODN), particularly synthetic ODN having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA.
  • ODN antisense oligonucleotides
  • the antisense sequence is complementary to the mRNA of the targeted gene, and inhibits expression of the targeted gene products.
  • Antisense molecules inhibit gene expression through various mechanisms, e.g. by reducing the amount of mRNA available for translation, through activation of RNAse H, or steric hindrance.
  • One or a combination of antisense molecules may be administered, where a combination may comprise multiple different sequences.
  • Antisense molecules may be produced by expression of all or a part of the target gene sequence in an appropriate vector, where the transcriptional initiation is oriented such that an antisense strand is produced as an RNA molecule.
  • the antisense molecule is a synthetic oligonucleotide.
  • Antisense oligonucleotides will generally be at least about 7, usually at least about 12, more usually at least about 20 nucleotides in length, and not more than about 500, usually not more than about 50, more usually not more than about 35 nucleotides in length, where the length is governed by efficiency of inhibition, specificity, including absence of cross-reactivity, and the like. It has been found that short oligonucleotides, of from 7 to 8 bases in length, can be strong and selective inhibitors of gene expression (see Wagner et al. (1996) Nature Biotechnology 14:840-844).
  • a specific region or regions of the endogenous sense strand mRNA sequence is chosen to be complemented by the antisense sequence.
  • a specific sequence for the oligonucleotide may use an empirical method, where several candidate sequences are assayed for inhibition of expression of the target gene in an in vitro or animal model. A combination of sequences may also be used, where several regions of the mRNA sequence are selected for antisense complementation.
  • Antisense oligonucleotides may be chemically synthesized by methods known in the art (see Wagner et al. (1993) supra, and Milligan et al., supra.) Preferred oligonucleotides are chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases.
  • phosphorothioates Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates.
  • Achiral phosphate derivatives include 3'-O'-5'-S-phosphorothioate, 3'-S-5'-O- phosphorothioate, 3'-CH2-5'-O-phosphonate and 3'-NH-5'-O-phosphoroamidate.
  • Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage. Sugar modifications are also used to enhance stability and affinity.
  • the ⁇ -anomer of deoxyribose may be used, where the base is inverted with respect to the natural ⁇ -anomer.
  • the 2'-OH of the ribose sugar may be altered to form 2'- O-methyl or 2'-O-allyl sugars, which provides resistance to degradation without comprising affinity. Modification of the heterocyclic bases must maintain proper base pairing. Some useful substitutions include deoxyuridine for deoxythymidine; 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine. 5- propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and deoxycytidine, respectively.
  • catalytic nucleic acid compounds e.g. ribozymes, anti-sense conjugates, etc.
  • Ribozymes may be synthesized in vitro and administered to the patient, or may be encoded on an expression vector, from which the ribozyme is synthesized in the
  • -24- targeted cell for example, see International patent application WO 9523225, and Beigelman et al. (1995) Nucl. Acids Res 23:4434-42).
  • oligonucleotides with catalytic activity are described in WO 9506764.
  • Conjugates of anti-sense ODN with a metal complex, e.g. terpyridylCu(ll), capable of mediating mRNA hydrolysis are described in Bashkin et al. (1995) Appl Biochem Biotechnol 54:43-56.
  • ASTH1 Protein A host may be treated with intact ASTH1 protein, or an active fragment thereof to modulate or reduce bronchial hypereactivity. Desirably, the peptides will not induce an immune response, particularly an antibody response. Xenogeneic analogs may be screened for their ability to provide a therapeutic effect without raising an immune response. The protein or peptides may also be administered to in vitro cell cultures.
  • the polypeptide formulation may be given orally, or may be injected intravascularly, subcutaneously, peritoneally, etc. Methods of administration by inhalation are well-known in the art.
  • the dosage of the therapeutic formulation will vary widely, depending upon the nature of the disease, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like.
  • the initial dose may be larger, followed by smaller maintenance doses.
  • the dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, etc. to maintain an effective dosage level. In many cases, oral administration will require a higher dose than if administered intravenously.
  • the amide bonds, as well as the amino and carboxy termini, may be modified for greater stability on oral administration.
  • the subject peptides may be prepared as formulations at a pharmacologically effective dose in pharmaceutically acceptable media, for example normal saline, PBS, etc.
  • the additives may include bactericidal agents, stabilizers, buffers, or the like.
  • the peptides may be encapsulated, introduced into the lumen of liposomes, prepared as a colloid, or another conventional technique may be employed that provides for an extended lifetime of the peptides.
  • the peptides may be administered as a combination therapy with other pharmacologically active agents.
  • the additional drugs may be administered separately or in conjunction with the peptide compositions, and may be included in the same formulation.
  • the subject nucleic acids can be used to generate genetically modified non-human animals or site specific gene modifications in cell lines.
  • transgenic is intended to encompass genetically modified animals having a deletion or other knock-out of ASTH1 gene activity, having an exogenous ASTH1 gene that is stably transmitted in the host cells, or having an exogenous ASTH1 promoter operably linked to a reporter gene.
  • Transgenic animals may be made through homologous recombination, where the ASTH1 locus is altered.
  • a nucleic acid construct is randomly integrated into the genome.
  • Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like. Of interest are transgenic mammals, e.g. cows, pigs, goats, horses, etc., and particularly rodents, e.g. rats, mice, etc.
  • a "knock-out" animal is genetically manipulated to substantially reduce, or eliminate endogenous ASTH1 function. Different approaches may be used to achieve the "knock-out”.
  • a chromosomal deletion of all or part of the native ASTH1 homolog may be induced. Deletions of the non-coding regions, particularly the promoter region, 3' regulatory sequences, enhancers, or deletions of gene that activate expression of ASTH1 genes.
  • a functional knock-out may also be achieved by the introduction of an anti-sense construct that blocks expression of the native ASTH1 genes (for example, see Li and Cohen (1996) Cell 85:319-329).
  • Transgenic animals may be made having exogenous ASTH1 genes.
  • the exogenous gene is usually either from a different species than the animal host, or is otherwise altered in its coding or non-coding sequence.
  • the introduced gene may be a wild-type gene, naturally occurring polymorphism, or a genetically manipulated sequence, for example those previously described with deletions, substitutions or insertions in the coding or non-coding regions.
  • the introduced sequence may encode an ASTH1 polypeptide, or may utilize the ASTH1 promoter operably linked to a reporter gene. Where the introduced gene is a coding sequence, it usually
  • a promoter which may be constitutive or inducible, and other regulatory sequences required for expression in the host animal.
  • constructs of interest include anti-sense ASTH1, which will block ASTH1 expression, expression of dominant negative ASTH1 mutations, and over-expression of a ASTH1 gene.
  • a detectable marker such as lac Z may be introduced into the ASTH1 locus, where upregulation of ASTH1 expression will result in an easily detected change in phenotype.
  • Constructs utilizing the ASTH1 promoter region, e.g. SEQ ID NO:1 ; SEQ ID NO:14, in combination with a reporter gene or with the coding region of ASTH1J or ASTH1I are also of interest.
  • the modified cells or animals are useful in the study of ASTH1 function and regulation. Animals may be used in functional studies, drug screening, ef ⁇ , e.g. to determine the effect of a candidate drug on asthma. A series of small deletions and/or substitutions may be made in the ASTH1 gene to determine the role of different exons in DNA binding, transcriptional regulation, etc. By providing expression of ASTH1 protein in cells in which it is otherwise not normally produced, one can induce changes in cell behavior. These animals are also useful for exploring models of inheritance of asthma, e.g. dominant v. recessive; relative effects of different alleles and synergistic effects between ASTH1I and ASTH1J and other asthma genes elsewhere in the genome.
  • DNA constructs for homologous recombination will comprise at least a portion of the ASTH1 gene with the desired genetic modification, and will include regions of homology to the target locus.
  • DNA constructs for random integration need not include regions of homology to mediate recombination. Conveniently, markers for positive and negative selection are included. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For various techniques for transfecting mammalian cells, see Keown et al. (1990) Methods in Enzymology 185:527-537.
  • an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, e.g. mouse, rat, guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder layer or grown in the presence of appropriate growth factors, such as leukemia inhibiting factor (LIF).
  • LIF leukemia inhibiting factor
  • ES cells When ES cells have been transformed, they may be used to produce transgenic animals. After transformation, the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be detected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct. Those colonies that are positive may then be used for embryo manipulation and blastocyst injection. Blastocysts are obtained from 4 to 6 week old superovulated females. The ES cells are trypsinized, and the modified cells are injected into the blastocoel of the blastocyst.
  • blastocysts are returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting litters screened for mutant cells having the construct. By providing for a different phenotype of the blastocyst and the ES cells, chimeric progeny can be readily detected.
  • the chimeric animals are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allogeneic or congenic grafts or transplants, or in in vitro culture.
  • Transformation of genetic function may utilize non-mammalian models, particularly using those organisms that are biologically and genetically well-characterized, such as C. elegans, D. melanogaster and S. cerevisiae.
  • transposon (Tc1) insertions in the nematode homolog of an ASTH1 gene or promoter region may be made.
  • the subject gene sequences may be used to knock-out or to complement defined genetic lesions in order to determine the physiological and biochemical pathways involved in ASTH1 function.
  • a number of human genes have been shown to complement mutations in lower eukaryotes.
  • Drug screening may be performed in combination with the subject animal models.
  • Many mammalian genes have homologs in yeast and lower animals. The study of such homologs' physiological role and interactions with other proteins can facilitate understanding of biological function.
  • yeast has been shown to be a powerful tool for studying protein-protein interactions through the two hybrid system described in
  • Drug Screening Assays By providing for the production of large amounts of ASTH1 protein, one can identify ligands or substrates that bind to, modulate or mimic the action of ASTHL Areas of investigation are the development of asthma treatments. Drug screening identifies agents that provide a replacement or enhancement for ASTH1 function in affected cells. Conversely, agents that reverse or inhibit ASTH1 function may stimulate bronchial reactivity. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, protein-DNA binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, and the like. The purified protein may also be used for determination of three-dimensional crystal structure, which can be used for modeling intermolecular interactions, transcriptional regulation, etc.
  • agent as used herein describes any molecule, e.g. protein or pharmaceutical, with the capability of altering or mimicking the physiological function of ASTHL Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids,
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic Compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
  • the screening assay is a binding assay
  • the label can directly or indirectly provide a detectable signal.
  • Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e.g. magnetic particles, and the like.
  • Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc.
  • the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures.
  • reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used. The mixture of components are added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4 and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0.1 and 1 hours will be sufficient.
  • assays of interest detect agents that mimic ASTH1 function.
  • candidate agents are added to a cell that lacks functional ASTH1 , and screened for the ability to reproduce ASTH1 in a functional assay.
  • the compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host for treatment of asthma attributable to a defect in ASTH1 function.
  • the compounds may also be used to enhance ASTH1 function.
  • the therapeutic agents may be administered in a variety of ways, orally, topically, parenterally e.g. subcutaneously, intraperitoneally, by viral infection, intravascularly, etc. Inhaled treatments are of particular interest.
  • the compounds may be formulated in a variety of ways.
  • the concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt.%.
  • compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like.
  • Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds.
  • Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.
  • Pharmacogenetics is the linkage between an individual's genotype and that individual's ability to metabolize or react to a therapeutic agent. Differences in metabolism or target sensitivity can lead to severe toxicity or therapeutic failure by altering the relation between bioactive dose and blood concentration of the drug. In the past few years, numerous studies have established good relationships between polymorphisms in metabolic enzymes or drug targets, and both response and toxicity. These relationships can be used to individualize therapeutic dose administration.
  • Genotyping of polymorphic alleles is used to evaluate whether an individual will respond well to a particular therapeutic regimen.
  • the polymorphic sequences are used to evaluate whether an individual will respond well to a particular therapeutic regimen.
  • a candidate ASTH1 polymorphism is screened with a target therapy to determine whether there is an influence on the effectiveness in treating asthma.
  • Drug screening assays are performed as described above. Typically two or more different sequence polymorphisms are tested for response to a therapy.
  • Drugs currently used to treat asthma include beta 2-agonists, glucocorticoids, theophylline, cromones, and anticholinergic agents.
  • beta 2-agonists For acute, severe asthma, the inhaled beta 2-agonists are the most effective bronchodilators. Short-acting forms give rapid relief; long-acting agents provide sustained relief and help nocturnal asthma.
  • First-line therapy for chronic asthma is inhaled glucocorticoids, the only currently available agents that reduce airway inflammation.
  • Theophylline is a bronchodilator that is useful for severe and nocturnal asthma, but recent studies suggest that it may also have an immunomodulatory effect. Cromones work best for patients who have mild asthma: they have few adverse effects, but their activity is brief, so they must be given frequently.
  • Cysteinil leukotrienes are important mediators of asthma, and inhibition of their effects may represent a potential breakthrough in the therapy of allergic rhinitis and asthma.
  • diagnostic screening may be performed. Diagnostic methods have been described in detail in a preceding section. The presence of a particular polymorphism is detected, and used to develop an effective therapeutic strategy for the affected individual.
  • molecular weight is average molecular weight
  • temperature is in degrees centigrade
  • pressure is at or near atmospheric.
  • Tristan da Cunha Asthma phenotype measurements and blood samples were obtained from the inhabitants of Tristan da Cunha, an isolated island in the South Atlantic, and from asthma families in Toronto, Canada (see Zamel et al., (1996) supra.)
  • the 282 inhabitants of Tristan da Cunha form a single large extended family descended from 28 original founders. Settlement of Tristan da Cunha occurred beginning in 1817 with soldiers who remained behind when a British garrison was withdrawn from the island, followed by the survivors of several shipwrecks.
  • 1827 Five women from St. Helena, one with children, emigrated to Tristan da Cunha and married island men.
  • One of these women is said to have been asthmatic, and could be the origin of a genetic founder effect for asthma in this population. Inbreeding has resulted in kinship resemblances of at least first cousin levels for all individuals.
  • Tristan da Cunha family pedigrees were ascertained through review of baptismal, marriage and medical records, as well as reliably accurate historical records of the early inhabitants (Zamel (1995) Can. Respir. J. 2:18).
  • the prevalence of asthma on Tristan da Cunha is high; 23% had a definitive diagnosis of asthma.
  • the Toronto cohort included 59 small families having at least one affected individual. These were ascertained based on the following criteria: (i) an affected proband; (ii) availability of at least one sibling of the proband, either affected or unaffected; (iii) at least one living parent from whom DNA could be obtained. A set of 156 "triad" families consisting of an affected proband and his or her parents were also collected. Signed consent forms were obtained from each individual prior to commencement of phenotyping and blood sample collection. The Toronto patients were mainly of mixed European ancestry.
  • a standardized questionnaire based on that of the American Thoracic Society (American Lung Association recommended respiratory diseases questionnaire for use with adults and children in epidemiology research. 1978. American Review of Respiratory Disease 118(2):7-53) was used to record the presence of respiratory symptoms such as cough, sputum and wheezing; the presence of other chest disorders including recent upper respiratory tract infection, allergic history; asthmatic attacks including onset, offset, confirmation by a physician, prevalence, severity and precipitating factors; other illnesses and smoking history; and all medications used within the previous 3 months.
  • a physician-confirmed asthmatic attack was the principal criterion for a diagnosis of asthma.
  • Skin atopy was determined by skin prick tests to common allergens: A. fumigatus, Cladospohum, Alternaria, egg, milk, wheat, tree, dog, grass, horse, house dust, cat, feathers, house dust mite D. farinae, and house dust mite
  • D. pteronyssinus Atopy testing of Toronto subjects omitted D. pteronyssinus and added cockroach and ragweed allergens. Saline and histamine controls were also performed (Bencard Laboratories, Mississauga, Ontario). Antihistamines were withdrawn for at least 48 hours prior to testing. Wheal diameters were corrected by subtraction of the saline control wheal diameter, and a corrected wheal size of >3 mm recorded 10 min after application was considered a positive response.
  • Airway responsiveness was assessed by a methacholine challenge test in those subjects with a baseline FEV1 (forced exhalation volume in one second) > 70% of predicted (Crapo et al. (1981) Am. Rev. Respir. Pis. 123:659). Methacholine challenge response was determined using the tidal breathing method (Cockcroft et al. (1977) Clin. Allergy 7:235). Doubling doses of methacholine from 0.03 to 16 mg/ml were administered using a Wright nebulizer at 4-min intervals to measure the provocative concentration of methacholine producing a 20% fall in FEV1 (PC20).
  • PCR primer pairs were synthesized using Applied Biosystems 394 automated oligo synthesizer. The forward primer of each pair was labeled with either FAM, HEX, or TET phosphoramidites (Applied Biosystems). No oligo purification step was performed. Genomic DNA was extracted from whole blood. PCR was performed using
  • thermocyclers (MJ Research). Reactions contained 10 mM Tris-HCl, pH 8.3; 1.5-3.0 mM MgCI 2 ; 50 mM KCI; 0.01 % gelatin; 250 ⁇ M each dGTP, dATP, dTTP, dCTP; 20 ⁇ M each PCR primer; 20 ng genomic DNA; and 0.75 U Taq Polymerase (Perkin Elmer Cetus) in a final volume of 20 ⁇ l. Reactions were performed in 96 well polypropylene microtiter plates (Robbins Scientific) with an initial 94°C, 3 min. denaturation followed by 35 cycles of 30 sec. at 94°C, 30 sec. at the annealing temp., and 30 sec.
  • Dye label, annealing temperature, and final magnesium concentration were specific to the individual marker.
  • Dye label intensity and quantity of PCR product (as assessed on agarose gels) were used to determine the amount to be pooled for each marker locus. The pooled products were precipitated and the product pellets mixed with 0.4 ⁇ l Genescan 500 Tamra size standard, 2 ⁇ l formamide, and 1 ⁇ l ABI loading dye. Plates of PCR product pools were heated to 80°C for 5 minutes and immediately placed on ice prior to gel loading.
  • Amplitaq Gold (Perkin Elmer Cetus) and buffer D (2.5 mM MgCI 2 , 33.5 mM Tris-HCl pH 8.0, 8.3 mM (NH 4 ) 2 SO 4 , 25 mM KCI, 85 ⁇ g/ml BSA) were used in the PCR.
  • a 'touchdown' amplification profile was employed in which the annealing temperature began at 66°C and decreased one degree per cycle to a final 20 cycles at 56°C. Products were run on 4.25% polyacrylamide gels using ABI 377 instruments. The data was processed with Genescan 2.1 and Genotyper 1.1 software.
  • a genome scan was performed in the population of Tristan da Cunha using 274 polymorphic microsatellite markers chosen from among those developed at Oxford (Reed et al. (1994) Nature Genetics 7:390), Genethon (Dib et al. (1996) Nature 380:152) and the Cooperative Human Linkage Center (CHLC, Murray et al. (1994) Science 265:2049). Markers with heterozygosity values of 0.75 or greater were selected to cover all the human chromosomes, as well as for ease of genotyping and size of PCR product for multiplexing of markers on gels. Fifteen multiplexed sets were used to provide a ladder of PCR products in each of three dyes when separated by size. Published distances were used initially to estimate map resolution. More accurate genetic distances were calculated using the study population as the data was generated. The 274 markers gave an average 14 cM interval for the genome scan.
  • a 50 ml culture of each YAC was grown in 2 x AHC at 30°C.
  • the cells were pelleted by centrifugation and washed twice in sterile water. After resuspension of the cells in 4 ml of SCEM (1 M sorbitol, 0.1 M sodium citrate (pH 5.8), 10 mM
  • Plugs were rinsed 3 times in TE (10 mM Tris pH 8.0, 1 mM EDTA) and incubated twice for 12 hours each at 50°C in lysis solution (0.5 M EDTA, pH 8.0;
  • CHEF Mapper (BIO-RAD) and according to methods supplied by the manufacturer, then transferred to nitrocellulose. YACs which comigrated with yeast chromosomes were visualized by hybridization of the blot with radiolabelled YAC vector sequences (Scherer and Tsui (1991) supra.)
  • Size-purified YAC DNA was prepared by pulsed field gel electrophoresis on a low melting temperature Seaplaque GTG agarose (FMC) gel, purified by GeneClean (BIO101) and radiolabeled for 30 mins with 32 P-dCTP using the Prime-It II kit (Sfratagene). 50 ⁇ l of water was added and unincorporated nucleotide was removed by Quick Spin Column (Boehringer Mannheim). 23 ⁇ l of 11.2 mg/ml human placental DNA (Sigma) and 36 ⁇ l of 0.5 M Na 2 HPO 4 , pH 6.0 were added to the approximately 150 ⁇ l of eluant.
  • FMC Seaplaque GTG agarose
  • the probe was boiled for 5 mins and incubated at 65°C for exactly 3 hours, then added to the prehybridized gridded BAC (Shizuya et al. (1992) Proc. Natl. Acad. Sci. 89:8794; purchased from Research Genetics) or chromosome 11 cosmid [Resource Center/ Primary Database of the German Human Genome Project, Berlin; Lehrach et al. (1990), In Davies, K.E. and Tilghman, S.M. (eds.). Genome Analysis Volume 1 : Genetic and Physical Mapping. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp.
  • FISH Metaphase fluorescence in situ hybridization
  • DIRVISH direct visual in situ hybridisation
  • Metaphase FISH was carried out by standard methods (Heng and Tsui (1994) FISH detection on DAPI banded chromosomes. In Methods of Molecular Biolo ⁇ v: In Situ Hybridisation Protocols (K.H.A. Choo, ed.) pp. 35-49. Human Press, Clifton, N.J.). High resolution FISH, or DIRVISH, was used to map the relative positions of two or more clones on genomic DNA. The protocol used was as described by Parra and Windle (1993) Nature Genet. 5:17. Briefly, slides containing stretched DNA were prepared by adding 2 ⁇ l of a suspension of normal human lymphoblast cells at one end of a glass slide and allowing to dry. 8 ⁇ l lysis buffer (0.5% SDS, 50 mM EDTA, 200 mM Tris-HCL, pH 7.4) was added and the
  • Probes were labeled either with biotin or with digoxygenin by standard nick translation (Rigby et al. (1977) J. Mol. Biol. 113:237). Hybridization and detections were carried out using standard fluorescence in situ hybridization techniques (Heng and Tsui (1994) supra.). Results were visualised using a Mikrophot SA microscope (Nikon) equipped with a CCD camera (Photometries). Images were recorded using Smartcapture software (Vysis).
  • Clones flanking gaps in the map were end cloned by digestion with enzymes that do not cut the respective vector sequences (Nsil for BAC clones and Xbal for PAC clones), followed by religation and transformation into competent DH5 ⁇ . Clones which produced two end fragments and plasmid vector upon digestion with Notl and Nsil or Xbal were sequenced. Gaps in the tiling path were filled by screening a gridded BAC library with the end clone probes or by screening DNA pools of a human genomic PAC library (loannou et al. (1994) Nature Genetics 6:84; licensed from Health Research, Inc.) by PCR using primers designed from end clone sequences.
  • BioNick Labeling System Gibco BRL
  • Unincorporated biotin was removed by spin column chromatography.
  • Approximately 100 ng of biotinylated genomic DNA was denatured and allowed to hybridize to 1 ⁇ g of blocked cDNA in a total volume of 20 ⁇ l in 120 mM NaPO 4 for 60 hours at 60°C under mineral oil. After hybridization, the biotinylated DNA was captured on streptavidin-coated magnetic beads (Dynal) in 100 ⁇ l of binding buffer (1 M NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA) for 20 minutes at room temperature with constant rotation.
  • PCR was performed on 1 , 2, 5, and 10 ⁇ l of eluate with Mbolb primers. Amplified products were analyzed on a 1.4% agarose gel. The reaction with the cleanest bands and least background was scaled up to produce approximately 1 ⁇ g of primary selected cDNA. This amplified primary selected cDNA was blocked with 1 ⁇ g of COT1 at 60°C for 1 hour followed by a second round of hybridization to 100 ng of the appropriate genomic DNA under the same conditions as the first round of selection. Washing of the bound cDNA, elution, and PCR of the selected cDNA was identical to the first round. 1 ⁇ l of PCR amplified secondary selected cDNA was cloned using the TA cloning system according to the
  • ElectroMax HB101 cells (Gibco BRL) and plated on 20 cm diameter LB ampicillin plates.
  • DNA was prepared from plates with > 2000 colonies by collection of the bacteria in LB ampicillin liquid and plasmid DNA purification by a standard alkaline lysis protocol (Sambrook et al. (1989) supra.) 5 ⁇ g of DNA from each plasmid pool preparation were electroporated into Cos 7 cells (ATCC) and RNA harvested using TRIZOL (Gibco BRL) after 48 hours of growth.
  • RT-PCR products were digested with BstXI prior to a second PCR amplification. Products were cloned into pAMP10 (Gibco BRL) and transformed into DH5 cells (Gibco BRL). 96 colonies per BAC were picked and analyzed for insert size by PCR.
  • Phage cDNA libraries were plated and screened with radiolabeled probes (exon trapping or cDNA selection products amplified by PCR from plasmids containing these sequences) by standard methods (Sambrook et al. (1989) supra.)
  • RACE libraries were constructed using polyA+ RNA and the Marathon cDNA amplification kit (Clontech). Nested RACE primer sets were designed for each cDNA or potential gene fragment (trapped exon, predicted exon, conserved fragment, etc). The RACE libraries were tested by PCR using one primer pair for each potential gene fragment; the two strongly positive libraries were chosen for RACE experiments.
  • Genomic sequencing DNA from cosmid, PAC, and BAC clones was prepared using Qiagen DNA prep kits and further purified by CsCI gradient. DNA was sonicated and DNA fragments were repaired using nuclease BAL-31 and T4 DNA polymerase. DNA fragments of 0.8-2.2 kb were size-fractionated by agarose gel electrophoresis and ligated into pUC9 vector. Inserts of the plasmid clones were amplified by PCR and sequenced using standard ABI dye-primer chemistry.
  • ABI sample file data was reanalyzed using Phred (Phil Green, University of Washington) for base calling and quality analysis. Sequence assembly of reanalyzed sequence data was accomplished using Phrap (Phil Green, University of Washington). Physical gaps between assembled contigs and unjoined but overlapping contigs were identified by inspection of the assembled data using GFP (licensed from Baylor College of Medicine) and Consed (Phil Green, University of Washington). Material for sequence data generation across gaps was obtained by PCR amplification. Low coverage regions were resequenced using dye-primer and dye-terminator chemistries (ABI). Final base-perfect editing (to > 99% accuracy) was accomplished using Consed.
  • PCR primers flanking each exon of the ASTH1 I and ASTH1J genes, or more than one primer pair for large exons, were designed from genomic sequence generated using Primer (publicly available from the Whitehead Institute for Biomedical Research) or Oligo 4.0 (licensed from National Biosciences).
  • Radioactive SSCP was performed by the method of Orita et al. (1989, Proc. Natl. Acad. Sci. 86:2766). Briefly, radioactively labeled PCR products between 150 and 300 bp and spanning exons of the ASTH1 I and ASTH1 J genes were generated from a set of asthma patient and control genomic template DNAs, by incorporating ⁇ - 32 P dCTP in the PCR. PCR reactions (20 ⁇ l) included 1x reaction buffer, 100 ⁇ M dNTPs, 1 ⁇ M each forward and reverse primer, and 1 unit Taq DNA polymerase (Perkin-Elmer) and 1 ⁇ Ci ⁇ - 32 P dCTP.
  • Inserts of the plasmid clones were amplified by PCR and sequenced using standard ABI dye-primer chemistry to determine the nature of the sequence variant responsible for the conformational changes detected by SSCP.
  • Fluorescent SSCP was carried out according to the recommended ABI protocol (ABI User Bulletin entitled 'Multi Color Fluorescent SSCP').
  • Unlabeled PCR primers were used to amplify genomic DNA segments containing different exons of the ASTH11 or ASTH1 J genes, in patient or control DNA.
  • Nested fluorescently labeled (TET, FAM or HEX) primers were then used to amplify smaller products, 150 to 300 bp containing the exon or region of interest. Amplification was done using a " touchdown' PCR protocol, in which the annealing temperature
  • Unlabeled PCR primers were used to amplify genomic DNA segments containing different exons of the ASTH1 I or ASTH1J genes, from patient or control DNAs. A set of nested PCR primers was then used to reamplify the fragment. Unincorporated primers were removed from the PCR product by Centricon-100 column (Amicon), or by Centricon-30 column for products less than 130 bp. The nested primers and dye terminator sequencing chemistry (ABI PRISM dye terminator cycle sequencing ready reaction kit) were then used to cycle sequence the exon and flanking region. Volumes were scaled down to 5 ⁇ l and 10% DMSO added to increase peak height uniformity. Sequences were compared between samples and heterozygous positions detected by visual inspection of chromatograms and using Sequence Navigator (licensed from ABI).
  • PCR products were also compared by subcloning and sequencing, and comparison of sequences for ten or more clones.
  • a genome scan was performed using polymorphic microsatellite markers from throughout the human genome, and DNA isolated from blood samples drawn from the inhabitants of Tristan da Cunha.
  • Linkage analysis an established statistical method used to map the locations of genes and markers relative to other markers, was applied to verify the marker orders and relative distances between
  • Cunha population was confirmed by genotyping and analyzing data from several markers near D11S907, spaced at intervals no greater than 5 cM across a possible linked region of about 30 cM. Sib-pair and affected pedigree member linkage analyses of these markers yielded confirmatory evidence for linkage and refined the genetic interval.
  • Yeast artificial chromosome (YAC) clones were derived from the CEPH megaYAC library (Cohen et al. 1993 Nature 366:698). Individual YAC addresses were obtained from a public physical map of CEPH megaYAC STS (sequence tagged site; Olson et al. (1989) Science 245:1434) mapping data maintained by the Whitehead Institute and accessible through the world wide web (Cohen et al. 1993. supra.; http://www-genome.wi.mit.edu/cgi-bin/contig/phys_map).
  • YAC clones spanning or overlapping other YACs containing D11S907 were chosen for map construction; STSs mapping to these YACs were used for map and clone verification. Some YACs annotated in the public database as being chimeric were excluded from the analyses. Multiple colonies of each YAC, obtained from a freshly
  • the YAC map at ASTH1 provided continuous coverage of a 4 Mb region, the central 1 Mb of which was of greatest interest.
  • YAC clones comprising a minimal tiling path of this region were chosen, and the size purified artificial chromosomes were used as hybridization probes to identify BAC and cosmid clones.
  • Gridded filters of a 3x human genomic BAC library and of a human chromosome 11 -specific cosmid library were hybridized with radiolabeled purified YAC. Clones corresponding to the grid coordinates of the positives were streaked to colony
  • a minimal tiling path of BAC and cosmid clones was chosen for genomic sequencing. Over 1 Mb of genomic sequence was generated at ASTHL On average, sequencing was done to 12x coverage (12 times redundancy in sequences). Marker order was verified relative to the STS map.
  • BLAST searches Altschul et al. (1990) supra.) were performed to identify sequences in public databases that were related to those in the ASTH1 region. Sequence-based gene prediction was done with the GRAIL [Roberts (1991) Science 254:805] and Geneparser [Snyder and Stormo (1993) Nucleic Acids Res. 21 : 607] programs. Genomic sequence and feature data was stored in ACeBD.
  • -47- a pool of trinucleotide and tetranucleotide repeat oligonucleotides.
  • the plasmid inserts were sequenced, the set of sequences compared with those of the known microsatellite markers in the region, using Power assembler (ABI) or Sequencher (Alsbyte). Primer pairs flanking each novel microsatellite repeat were designed, and the heterozygosity of each new marker was tested by Batched Analysis of Genotypes (BAGs; LeDuc et al., 1995. PCR Methods and Applications 4:331). Additional microsatellites were found by analysis of the genomic sequence in AceDB. Table 1 lists all the microsatellite markers used for genotyping in the ASTH1 region and their repeat type, source and primers. Table 1 B lists some repeat sequences.
  • 253E6TR1 AGGTTTAGGGGACAGGGTTTGG 1 17711.. GTTTCTTTCCTGGCTAACACGGTGAAATC
  • AFM205YG5 (G) TATCAAGGTAATATAGTAGCCACGG
  • AFM206XB2 (G) ATTGCCAAAACTTGGAAGC
  • CA39_2 GAGACTCTGA CA
  • CA GAGACTCTGA
  • the microsatellite markers isolated from YACs from the ASTH1 region were genotyped in both the Tristan da Cunha and Toronto cohorts. Genetic refinement of the ASTH1 region was accomplished by applying the transmission/disequilibrium test (TDT; Spielman et al. (1993) Am. J. Hum. Genet. 52:506) to genetic data from the Tristan and Toronto populations, at markers throughout the ASTH1 region.
  • TDT transmission/disequilibrium test
  • FIG. 1 shows graphs of ⁇ 2 values for key ASTH1 region markers for both history of asthma with positive methacholine challenge, for the Toronto triad families, ⁇ 2 is plotted vs. genomic location of the marker on the physical map.
  • the relative risk of affection vs. normal for this allele is 5.25.
  • the markers defining the limits of linkage disequilibrium were D11 S907 and 65P14TE1.
  • the physical size of the refined region is approximately 100 kb.
  • a significant TDT test reflects the tendency of alleles of markers located near a disease locus (also said to be in "linkage disequilibrium" with the disease) to segregate with the disease locus, while alleles of markers located further from the disease locus segregate independently of affection status.
  • An expectation that derives from this is that a population for which a disease gene (ie a disease predisposing polymorphism) was recently introduced would show statistically significant TDT over a larger region surrounding the gene than would a population in which the mutant gene had been segregating for a greater length of time. In the latter case, time would have allowed more opportunity for markers in the vicinity of the disease gene to recombine with it. This expectation is fulfilled in our populations.
  • the Tristan da Cunha population founded only 10 generations ago, shows a broader TDT curve than does the set of Toronto families, which are mixed European in derivation and thus represent an older and more diverse, less recently established population.
  • the tiling path of BACs, cosmids and PAC clones was subjected to exon trapping and cDNA selection to isolate sequences derived from ASTH1 region genes.
  • Exon trap clones were isolated on the basis of size and ability to cross- hybridize. Approximately 300 putatively non-identical clones were sequenced.
  • cDNA selection was performed with adult and fetal lung RNA using pools of tiling path clones. The cDNA selection clones were sequenced and the sequences assembled with those of the exon trap clones. Representative exon trapping clones spanning each assembly were chosen, and arranged as "masterplates" (96- well microtitre dishes) of clones. Exon trap masterplate clones and cDNA selection clones were subjected to expression studies.
  • RNA species Human multi-tissue Northern blots were probed with PCR products of masterplate clones. In some cases, exon trapping clones did not detect RNA species, either because they did not represent expressed sequences, or represented genes with very restricted patterns of expression, or due to small size of the exon probe.
  • ASTH1 I and ASTH1J were detected by exon trapping.
  • ASTH1 I exons detected a 2.8 kb mRNA expressed at high levels in trachea and prostate, and at lower levels in lung and kidney.
  • ASTH1 I exons were used as probes to screen prostate, lung and testis cDNA libraries; positive clones were obtained from each of these libraries. Isolation of a ASTH11 cDNA clone from testis demonstrates that this gene is expressed in this tissue, and possibly others, at a level not detectable by Northern blot analysis.
  • ASTH1 J exons detected a 6.0 kb mRNA expressed at high levels in the trachea, prostate and pancreas and at lower levels in colon, small intestine, lung and stomach. Pancreas and prostate libraries were screened with exon clones
  • ASTH1 J has three splice forms consisting of the altl form, found in prostate and lung cDNA clones, and in which the exons (illustrated in Figure 1) are found in the order: 5' a, b, c, d, e, f, g, h, i 3'.
  • a second form, alt2, in which the exon order is: 5' a2, b, c, d, e, f, g, h, i 3' was seen in a pancreas cDNA clone.
  • a third form, alt3, contains an alternate exon, a3, between exons a2 and b.
  • the start codon is within exon b, so that the open reading frame is identical for the three forms, which differ only in the 5' UTR.
  • the ASTH1J cDNAs shown as SEQ ID NO:2 (form altl); SEQ ID NO:3 (form alt2); SEQ ID NO:4 (form alt3) are 5427, 5510 and 5667 bp in length, respectively.
  • the sequence of the entire protein coding region and alternate 5' UTRs are provided.
  • the 3' terminus, where the polyA tail is added, varies by 7 bp between clones.
  • the provided sequences are the longest of these variants.
  • the encoded protein product is provided as SEQ ID NO:5.
  • ASTH1 I was seen in three isoforms denoted as altl , alt2, and alt3.
  • the exons of ASTH1 I and ASTH1J were given letter designations before the directionality of the cDNA was known, the order is different for the two genes.
  • exons are in the following order: 5' i, f, e, d, c, b, a 3'.
  • an alternative 5' exon, j substitutes for exon i, with the following exon arrangement: 5' j, f, e, d, c, b, a 3'.
  • the alt3 form of the gene has the exon order: 5' f, k, h, g, e, d, c, b, a 3'.
  • the alternative splicing and start codons in each of exons i, f and e give the three forms of ASTH11 protein different amino termini.
  • the common stop codon is located in exon a, which also contains a long 3' UTR.
  • Two polyadenylation signals are present in the 3' UTR; some cDNA clones end with a polyA tract just after the first polyA signal and for others the polyA tract is at the end of the sequence shown. Since the sequences shown for the altl ,
  • nucleotide sequences of the altl , alt2 and alt3 forms of ASTH1J and the altl , alt2 and alt3 forms of ASTH1 I were used in BLAST searches against dbEST in order to identify EST sequences representing these genes. Perfect or near perfect matches were taken to represent sequence identity rather than relatedness. Accession numbers T65960, T64537, AA055924 and AA055327 represent the forward and reverse sequences of two clones which together span the last 546 bp (excluding the polyA tail) of the 3' UTR of ASTH1 I. No ESTs spanned any part of the coding region of this gene.
  • One colon cDNA clone (accession number AA149006) spanned 402 bp including the last 21 bp of the ASTH1 J coding region and part of the 3' UTR.
  • the genomic organization of genes in the ASTH1 region was determined by comparison by BLAST of cDNA sequences to the genomic sequence of the region.
  • the genomic sequence of the ASHT1 region 5' to and overlapping ASTH1 J, is provided in SEQ ID NO:1.
  • Genomic structure of the ASTH1 I and ASTH1J genes is shown in Figure 1; the intron/exon junction sequences are in Table 2.
  • the protein encoded by ASTH1J (SEQ ID NO:5) is 300 amino acids in length.
  • a BLASTP search of the protein sequence against the public nonredundant sequence database (NCBI) revealed similarity to one protein domain of transcription factors of the ets family.
  • the ets family named for the E26 oncoprotein which originally defined this type of transcription factor, is a group of transcription factors which activate genes involved in a variety of immunological and other processes, or implicated in cancer.
  • the family members most similar to ASTH11 and ASTH1 J are: ETS1 , ESX, ETS2, ELF, ELK1 , TEL, NET, SAP-1 , NERF and FLI.
  • ASTH1 I and ASTH1J are predicted to contain two conserved modules, the N- terminal protein interaction domain (SAM-domain) and the C-terminal DNA-binding domain (ETS-domain).
  • SAM-domain N- terminal protein interaction domain
  • ETS-domain C-terminal DNA-binding domain
  • the ASTH1 I altl (SEQ ID NO:7), alt2 (SEQ ID NO:9) and alt3 (SEQ ID NO:11) forms are 265, 255 and 164 amino acids in length, respectively, and differ at their 5' ends.
  • the ASTH1 I and ASTH1J proteins show similarity to each other in the ets domain and between ASTH1J exon c and ASTH1 I exon e. They are more related to each other than to other proteins. Over the ets domain they are 66% similar (ie. have amino acids with similar properties in the same positions) and 46% identical to each other. All three forms of ASTH11 have the helix turn helix motif located near the carboxy terminal end of the protein.
  • the alternate forms of the ASTH1 I protein may differ in function in critical ways.
  • the activity of ets transcription factors can be affected by the presence of independently folding protein structural motifs which interact with the ets protein binding domain (helix loop helix).
  • the differing 5' ends of the ASTH1 I proteins may help modulate activity of the proteins in a tissue-specific manner.
  • Affected and unaffected individuals from the Toronto cohort were used to determine sequence variants, as were approximately 25 controls derived from populations not selected for asthma.
  • Affected and unaffected individuals from the Tristan da Cunha population were also chosen; the set to be assayed was also selected to represent all the major haplotypes for the ASTH1 region in that
  • Polymorphism analysis was accomplished by three techniques: comparative (heterozygote detection) sequencing, radioactive SSCP and fluorescent SSCP. Polymorphisms found by SSCP were sequenced to determine the exact sequence change involved.
  • PCR and sequencing primers were designed from genomic sequence flanking each exon of the coding region and 5' UTRs of ASTH1 I and ASTH1J.
  • the forward and reverse PCR primers were labeled with different dyes to allow visualization of both strands of the PCR product.
  • a variant seen in one strand of the product was also apparent in the other strand.
  • heterozygotes were also detected in sequences from both DNA strands.
  • Polymorphisms associated with the ASTH11 locus are listed in Table 3. The sequence flanking each variant is shown. Polymorphisms were also deduced from comparison of sequences from multiple independent cDNA clones spanning the same region of the transcripts, and comparison with genomic DNA sequence. The polymorphisms in the long 3' UTR regions of these genes were found by this method. One polymorphism in each gene is associated with an amino acid change in the protein sequence. An alanine/valine difference in exon c of ASTH1 J is a conservative amino acid change. A serine/cysteine variant in exon g of ASTH1 I is not a conservative change, but would be found only in the alt3 form of the protein.
  • the polymorphisms in the ASTH1 I and J transcribed regions were genotyped in the whole Tristan da Cunha and Toronto populations, as well as in a larger sample of non-asthma selected controls, by high throughput methods such as OLA (oligonucleotide ligation assay; Tobe et al. (1996) Nucl. Acids Res. 24:3728) or Taqman (Holland et al. (1992) Clin. Chem. 38: 462), or by PCR and restriction enzyme digestion.
  • OLA oligonucleotide ligation assay
  • Taqman Holland et al. (1992) Clin. Chem. 38: 462
  • the population-wide data were used in a statistical analysis for significant differences in the frequencies of ASTH1 I or ASTH1J alleles between asthmatics and non-asthmatics.
  • WIJ __Ia 05 +1103 AGACCCGATARGAGCTCCTTC 1 14499..
  • WIJ __Ia 06 +794 AAAAGTGGATMCTCTGCAAAC 1 15544.
  • WIJ__Ia 06 1535 AAAGGGTTAGYTTGTCCCCTT 1 15599.
  • Cross-species sequence conservation can reveal the presence of functionally important areas of sequence within a larger region. Approximately 90 kb of sequence lie between ASTH1 I and ASTH1 J, which are transcribed in opposite directions ( Figure 1). The transcriptional orientation of these genes may allow coordinate regulation of their expression. The expression patterns of these genes are similar but not identical. Sequences found 5' to genes are critical for expression. To search for regulatory or other important regions, the genomic sequence between ASTH1 I and ASTH1J, was examined and plasmid clones derived from genomic sequencing experiments chosen for cross-species hybridization experiments. The criterion for probe choice was a lack of repeat elements such as Alu or LINEs. Inserts from these clones were used as probes on Southern blots of EcoRI-digested human, mouse and pig or cow genomic DNA. Probes that produced discrete bands in more than one species were considered conserved.
  • conserved probes clustered in four locations. One region was located 5' to ASTH11 and spanned exon j of this gene. A second conserved region was located 5' to ASTH1 IJ, spanning approximately 10 kb and beginning 6 kb 5' to ASTH1J exon a (and is within SEQ ID NO:1). Two other clusters of conserved probes were noted in the region between ASTH1 I and J. They are approximately 10 and 6 kb in length. Promoters, enhancers and other important control regions are generally found near the 5' ends of genes or within introns.
  • Methods of identifying and characterizing such regions include: luciferase assays, chloramphenicol acetyl transferase (CAT) assays, gel shift assays, DNAsel protection assays (footprinting), methylation interference assays, DNAsel hypersensitivity assays to detect functionally relevant chromatin-ree regions, other types of chemical protection assays, transgenic mice with putative promoter regions linked to a reporter gene such as ⁇ -galactosidase, etc.
  • CAT chloramphenicol acetyl transferase
  • gel shift assays gel shift assays
  • DNAsel protection assays footprinting
  • methylation interference assays DNAsel hypersensitivity assays to detect functionally relevant chromatin-ree regions
  • transgenic mice with putative promoter regions linked to a reporter gene such as ⁇ -galactosidase, etc.
  • the ASTH1 locus is associated with asthma and bronchial hyperreactivity.
  • ASTH1 I and ASTH1J are transcription factors expressed in trachea, lung and several other tissues. The main site of their effect upon asthma may therefore be in trachea and lung tissues. Since ets family genes are transcription factors, a function for ASTH1 I and ASTH1J is activation of transcription of particular sets of genes within cells of the trachea and lung. Cytokines are extracellular signalling proteins important in inflammation, a common feature of asthma. Several ets family transcription factors activate expression of cytokines or cytokine receptors in response to their own activation by upstream signals. ELF, for example, activates IL-2, IL-3, IL-2 receptor ⁇ and GM-CSF, factors involved in signaling between cell types important in asthma. NET activates transcription of the IL-1 receptor antagonist gene. ETS1 activates the T cell receptor ⁇ gene, which has been linked to atopic asthma in some families (Moffatt et al. (1994) supra.)
  • Cytokines are produced by structural cells within the airway, including epithelial cells, endothelial cells and fibroblasts, bringing about recruitment of inflammatory cells into the airway.
  • a model for the role of ASTH11 and ASTH1 J in asthma that is consistent with the phenotype linked to ASTH1 , the expression pattern of these genes, the nature of the ASTH1 l/J genes, and the known function of similar genes is that aberrant function of ASTH1 I and/or ASTH1J in trachea or lung leads to altered expression of factors involved in the inflammatory process, leading to chronic inflammation and asthma.
  • Primer extension analyses performed using total RNA isolated from both bronchial and prostate epithelial cells have revealed one major and five minor transcription start sites for ASTH1 J.
  • the major site accounts for more than 90% of ASTH1 J gene transcriptional initiation. None of these sites are found when the primer extension analysis is performed using mRNA isolated from human lung fibroblasts that do not express ASTH1 J.
  • TATAAAAA putative TATA box
  • TATAAAA consensus sequence for TATA box protein binding as compared with 389 TATA elements
  • the nuclear factor-1 (NF-1) family of transcription factors comprises a large group of eukaryotic DNA binding proteins. Diversity within this gene family is contributed by multiple genes (including: NF-1 A, NF-1 B, NF-1C and NF-1X), differential splicing and heterodimerization.
  • C/EBP Transcription factor C/EBP (CCAAT-enhancer binding protein) is a heat stable, sequence-specific DNA binding protein first purified from rat liver nuclei.
  • C/EBP binds DNA through a bipartite structural motif and appears to function exclusively in terminally differentiated, growth arrested cells.
  • C/EBP ⁇ was originally described as NF-IL-6; it is induced by IL-6 in liver, where it is the major C/EBP binding component.
  • CRP 1 , C/EBP ⁇ and C/EBP ⁇ Three more recently described members of this gene family, designated CRP 1 , C/EBP ⁇ and C/EBP ⁇ , exhibit similar DNA binding specificities and affinities to C/EBP ⁇ .
  • C/EBP ⁇ and C/EBP ⁇ readily form heterodimers with each other as well as with C/EBP ⁇ .
  • DNA array is made by spotting DNA fragments onto glass microscope slides which are pretreated with poly-L-lysine. Spotting onto the array is accomplished by a robotic arrayer. The DNA is cross-linked to the glass by ultraviolet irradiation, and the free poly-L-lysine groups are blocked by treatment with 0.05% succinic anhydride, 50% 1-methyl-2-pyrrolidinone and 50% borate buffer.
  • the spots on the array are oligonucleotides synthesized on an ABI automated synthesizer. Each spot is one of the alternative polymorphic sequences indicated in Tables 3 to 8. For each pair of polymorphisms, both forms are included. Subsets include (1) the ASTH1J polymorphisms of Table 3, (2) the
  • Genomic DNA from patient samples is isolated, amplified and subsequently labeled with fluorescent nucleotides as follows: isolated DNA is added to a standard PCR reaction containing primers (100 pmoles each), 250uM nucleotides, and 5 Units of Taq polymerase (Perkin Elmer). In addition, fluorescent nucleotides (Cy3-dUTP (green fluorescence) or Cy5-dUTP (red fluorescence), sold by Amersham) are added to a final concentration of 60 uM. The reaction is carried out in a Perkin Elmer thermocycler (PE9600) for 30 cycles using the following cycle profile: 92°C for 30 seconds, 58°C for 30 seconds, and 72°C for 2 minutes. Unincorporated fluorescent nucleotides are removed by size exclusion chromatography (Microcon-30 concentration devices, sold by Amicon).
  • the sample is reduced to 5 ⁇ l and supplemented with 1.4 ⁇ l 20X SSC and 5 ⁇ g yeast tRNA. Particles are removed from this mixture by filtration through a pre-wetted 0.45 ⁇ microspin filter (Ultrafree-MC, Millipore, Bedford, Ma.). SDS is added to a 0.28% final concentration.
  • the fluorescently-labeled cDNA mixture is then heated to 98°C for 2 min., quickly cooled and applied to the DNA array on a microscope slide. Hybridization proceeds under a coverslip, and the slide assembly is kept in a humidified chamber at 65°C for 15 hours.
  • the slide is washed briefly in 1X SSC and 0.03% SDS, followed by a wash in 0.06% SSC.
  • the slide is kept in a humidified chamber until fluorescence scanning was done.
  • Fluorescence scanning and data acquisition Fluorescence scanning is set for 20 microns/pixel and two readings are taken per pixel. Data for channel 1 is set to collect fluorescence from Cy3 with excitation at 520 nm and emission at 550- 600 nm. Channel 2 collects signals excited at 647 nm and emitted at 660-705 nm, appropriate for Cy5. No neutral density filters are applied to the signal from either channel, and the photomultiplier tube gain is set to 5. Fine adjustments are then
  • Phage MW1-J was isolated by screening a mouse 129Sv genomic phage library (Stratagene) with the 443bp BamHI-Smal fragment from the 5' region of the human asth1-J cDNA clone PA1001A as probe. The 23kb insert in MW1-J was sequenced.
  • a 2.65kb Sad fragment (bp7115-bp9765) from MW1-J was isolated, cloned into the Sacl site of pUC19, isolated from the resultant plasmid as an EcoRI-Xbal fragment, inserted into the EcoRI-Xbal sites of pBluescriptll KS+ (Stratagene), and the 2.5kb Xhol-Mlul fragment isolated.
  • a 5.4kb Hindlll fragment (bp11515-bp16909) was isolated from MW1-J, inserted into the Hindlll site of pBluescriptll KS+, reisolated as a Xhol-Notl fragment, inserted into the Xhol-Notl sites of pPNT, and the 9.5kb Xhol-Mlul fragment isolated.
  • the two Xhol-Mlul fragments were ligated together to produce the final targeting construct plasmid, asthlexb.
  • Asthlexb was linearized by digestion with Notl and purified by CsCI banding.
  • RW4 ES cells Approximately 10 million RW4 ES cells (Genome Systems) were electroporated with 20 ⁇ g of linearized asthlexb and grown on mitomycin C inactivated MEFs (Mouse Embryo Fibroblasts) in ES cell medium (DMEM + 15% fetal bovine serum+1000U/ml LIF (Life Technologies)) and 400 ⁇ g/ml G418. After 24-48hrs, the cells were refed with ES cell medium. After 7-10 days in selection culture approximately 200 colonies were picked, trypsinized, grown in 96 well microtiter plates, and expanded in duplicate 24 well microtiter plates.
  • mice heterozygous for the Asthl-J targeted allele are interbred to obtain mice homozygous for the asth1-J targeted allele. Homozygotes are identified by Ndel Southern blot screening described above. The germline offspring of the chimeric founders are 50% A/J or C57BL6 and 50% 129SvJ in genetic background. Subsequent generations of backcrossing with wild type A/J or C57BL/6 mates will result in halving of the 129SvJ contribution to the background. The percentage A/J or C57BL/6 background is calculated for each homozygous mouse from its breeding history.
  • RNA and protein Various tissues of homozygotes, heterozygotes and wild type littermates at various stages of development from embryonic stages to mature adults are isolated and processed to obtain RNA and protein. Northern and western expression analyses as well as in situ hybridizations and immunohistochemical analyses are
  • C57BL/6 backgrounds at varying stages of development are assessed for gross pathology and overt behavioral phenotypic differences such as weight, breeding performance, alertness and activity level, etc.
  • a 3.4kb Hindlll fragment (bp17217-bp20622) was isolated from MW1-J, inserted into the Hindlll site of pBluescriptll KS+, reisolated as a Xhol-Notl fragment, inserted into the Xhol-Notl sites of pPNT, and the 9.5kb Rsrll-Mlul fragment isolated.
  • the two Rsrll-Mlul fragments were ligated together to produce the final targeting construct plasmid, Asthlexc. Asthlexc was linearized by digestion with Notl and purified by CsCI banding.
  • RW4 ES cells Approximately 10 million RW4 ES cells (Genome Systems) were electroporated with 20 ⁇ g of linearized asthlexc and grown on mitomycin C inactivated MEFs (Mouse Embryo Fibroblasts) in ES cell medium (DMEM + 15% fetal bovine serum+1000U/ml LIF (Life Technologies)) and 400 ⁇ g/ml G418. After 24-48hrs, the cells were refed with ES cell medium. After 7-10 days in selection culture approximately 200 colonies were picked, trypsinized, grown in 96 well
  • Targeted clones are injected into blastocysts and high percentage chimeras bred to A/J and C57BL/6 mates analogously to that done for asthl-Jexb knockout mice.
  • Heterozygote, homozygote and wild type littermates are obtained and analyzed analogously to that done for asthl-Jexb knockout mice.
  • ASTH11 and ASTH1 J are novel human genes linked to a history of clinical asthma and bronchial hyperreactivity in two asthma cohorts, the population of Tristan da Cunha and a set of Canadian asthma families.
  • a TDT curve in the ASTH1 region indicates that ASTH1 I and ASTH1 J are located in the region most highly associated with disease.
  • the genes have been characterized and their genetic structure determined. Full length cDNA sequence for three isoforms of ASTH11 and three isoforms of ASTH1 J are reported.
  • the genes are novel members of the ets family of transcription factors, which have been implicated in the activation of a variety of genes including the TCR ⁇ gene and cytokine genes known to be important in the aetiology of asthma.
  • Polymorphisms in the ASTH1 I and ASTH1J genes are described. These polymorphisms are useful in the presymptomatic diagnosis of asthma susceptibility, and in the confirmation of diagnosis of asthma and of asthma subtypes.
  • MOLECULE TYPE Genomic DNA
  • SEQUENCE DESCRIPTION SEQ ID NO : 1 : GCACTTTTTG GGGAAGGTGG AAGAATAAAA GTAAGGGAGG TGTGCTGAGA CTTCAATTTT 60
  • ATGTCTACTT TCAAGGTGCT CACAGGTCAG ATCTAGGATT ATTGCTACTA ACTGATATTT 540
  • TTCATTCATT CAAGATGGAA TTAGTGCCCC AGACACAGAG GCAGGGGATA AATAGCAAAC 2700
  • CAGAGTGGAT CTGGACATTC TGCATGAGCC CAGGGATCCT GAGAATGGAT TGGCTGAGCA 3360
  • CTGAGTCTAA CTGGAAGCCA GAGGGCAAAG GAGGTACCCT TTCCAGCTCT GCAATTCTCT 3840
  • CTCCTCTCAC CATCTGGTGG TCCCCGTGCC CACGCACCAG CTCGTTGGAT GGACATTTTG 7260
  • AAAAAAAAAA AGAACCACAG GAGGGAGAGA TCATATATGA CCCCGTATGT GTGAAAAGTC 11700
  • ATCAGTTCTA TACTTAATTA TAAATACTCT TGGGAATAAA ACATACTTAT CTAATAAGCA 15240
  • AAAAAGAGCA AAAGGGAAAA AAAACCCCAA GCAGATGAAA AGTAAAGAAG GCAATGGTTA 17820

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Abstract

A genetic locus associated with asthma is identified. The genes within the locus, ASTH1I and ASTH1J, and the regulatory sequences of the locus are characterized. The genes are used to produce the encoded proteins; in screening for compositions that modulate the expression or function of ASTH1 proteins; and in studying associated physiological pathways. The DNA is further used as a diagnostic for genetic predisposition to asthma.

Description

ASTHMA RELATED GENES
INTRODUCTION Asthma is a disease of reversible bronchial obstruction, characterized by airway inflammation, epithelial damage, airway smooth muscle hypertrophy and bronchial hyperreactivity. Many asthma symptoms can be controlled by medical intervention, but incidence of asthma-related death and severe illness continue to rise in the United States. The approximately 4,800 deaths in 1989 marked a 46 percent increase since 1980. As many as 12 million people in the United States have asthma, up 66 percent since 1980, and annually, the disease's medical and indirect costs are estimated at over $6 billion.
Two common subdivisions of asthma are atopic (allergic, or extrinsic) asthma and non-atopic (intrinsic) asthma. Atopy is characterized by a predisposition to raise an IgE antibody response to common environmental antigens. In atopic asthma, asthma symptoms and evidence of allergy, such as a positive skin test to common allergens, are both present. Non-atopic asthma may be defined as reversible airflow limitation in the absence of allergies.
The smooth muscle surrounding the bronchi are able to rapidly alter airway diameter in response to stimuli. When the response is excessive, it is termed bronchial hyperreactivity, a characteristic of asthma thought to have a heritable component. Studies have demonstrated a genetic predisposition to asthma by showing, for example, a greater concordance for this trait among monozygotic twins than among dizygotic twins. The genetics of asthma is complex, however, and shows no simple pattern of inheritance. Environment also plays a role in asthma development, for example, children of smokers are more likely to develop asthma than are children of non-smokers.
In recent years thousands of human genes have been cloned. In many cases, gene discovery has been based on prior knowledge about the corresponding protein, such as amino acid sequence, immunological reactivity, etc. This approach has been very successful, but is limited in some important ways. One limitation is that genes in these cases are identified based on knowledge of molecular level protein properties. For a large number of important human genes, however, there
-1- is little or no biochemical data concerning the encoded product. For example, genes that predispose to human diseases, such as cystic fibrosis, Huntington's disease, etc. are of interest because of their phenotypic effect. Biochemical characterization of such genes may be secondary to genetic characterization. A solution to this impasse has been found in combining classical genetic mapping with the ability to identify genes and, if necessary, to sequence large regions of chromosomes. Population and family studies enable genes associated with a trait of interest to be localized to a relatively small region of a chromosome. At this point, physical mapping can be used to identify candidate genes, and various molecular biology techniques used to pick out mutated genes in affected individuals. This "top-down" approach to gene discovery has been termed positional cloning, because genes are identified based on position in the genome. Positional cloning is now being applied to complex genetic diseases, which affect a greater fraction of humanity than do the more simple and usually rarer single gene disorders. Such studies must take into account the contribution of both environmental and genetic factors to the development of disease, and must allow for contributions to the genetic component by more than one, and potentially many, genes. The clinical importance of asthma makes it of considerable interest to characterize genes that underlie a genetic predisposition to this disease. Positional cloning provides an approach to this goal.
Relevant Literature
The symptoms and biology of asthma are reviewed in Chanez et al. (1994)
Odyssey 1 :24-33. A review of bronchial hyperreactivity may be found in Smith and McFadden (1995) Ann. Allerαv. Asthma and Immunol. 74:454. Moss (1989) Annals of Allergy 63:566 review the allergic etiology and immunology of asthma.
The genetic dissection of complex traits is discussed in Lander and Schork
(1994) Science 265:2037-2048. Genetic mapping of candidate genes for atopy and/or bronchial hyperreactivity is described in Postma et al. (1995) N.E.J.M. 333:894; Marsh et al. (1994) Science 264: 1152; and Meyers et al. (1994) Genomics
23:464.
-2- Lawrence et al. (1994) Ann. Hum. Genet. 58:359 discuss an approach to the genetic analysis of atopy and asthma. Genetic linkage between the alpha subunit of the T cell receptor and IgE reactions has been noted by Moffat et al. (1994) The
Lancet 343:1597. Caraballo and Hernandez (1990) Tissue Antigens 35:182 noted an association between HLA alleles and allergic asthma. Evidence of linkage of atopy to markers on chromosome 11q has been seen in some British asthma families (Cookson et al. (1989) Lancet M292-1295; Young et al. (1991) J. Med.
Genet. 29:236, but not in other British families (Lympany et al. (1992) Clin. Exp.
Allergy 22:1085-1092) or in families from Minnesota or Japan (Rich et al. (1992) Clin. Exp. Allerαv 22: 1070-1076; and Hizawa et al. (1992) Clin. Exp. Allergy
22:1065).
The association of a polymorphism for the FcεRI-β gene and risk of atopy is described in Hill et al. (1995) B.M.J. 311 :776; Hill and Cookson (1996) Human Mol.
Genet. 5:959; and Shirakawa et al. (1994) Nature Genetics 7:125; an association of FcεRI-β with bronchial hyperreactivity is described in van Herwerden (1995) The
Lancet 346:1262.
Collections of polymorphic markers from throughout the human genome have been tested for linkage to asthma, described in Meyers et al. (1996) Am. J.
Hum. Genet. 59:A228 and Daniels et al. (1996) Nature 383:247-250. No linkage to human chromosome 11 p was detected in these studies.
SUMMARY OF THE INVENTION Human genes associated with a genetic predisposition to asthma are provided. The genes, herein termed ASTH1I and ASTH1J, are located close to each other on human chromosome 11 p, have similar patterns of expression, and common sequence motifs. The nucleic acid compositions are used to produce the encoded proteins, which may be employed for functional studies, as a therapeutic, and in studying associated physiological pathways. The nucleic acid compositions and antibodies specific for the protein are useful as diagnostics to identify a hereditary predisposition to asthma.
-3- BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 : Genomic organization of the ΛS7H11 and ASTHl J genes. The sizes of the exons are not to scale. Alternative exons are hatched. The direction of transcription is indicated below each gene.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS The provided ASTH1 genes and fragments thereof, encoded protein, ASTH1 genomic regulatory regions, and anti-/AS7H1 antibodies are useful in the identification of individuals predisposed to development of asthma, and for the modulation of gene activity in vivo for prophylactic and therapeutic purposes. The encoded ASTl^l protein is useful as an immunogen to raise specific antibodies, in drug screening for compositions that mimic or modulate ASTH^ activity or expression, including altered forms of ASTH protein, and as a therapeutic.
Asthma, as defined herein, is reversible airflow limitation in a patient over a period of time. The disease is characterized by increased airway responsiveness to a variety of stimuli, and airway inflammation. A patient diagnosed as asthmatic will generally have multiple indications over time, including wheezing, asthmatic attacks, and a positive response to methacholine challenge, i.e. a PC20 on methacholine challenge of less than about 4 mg/ml. Guidelines for diagnosis may be found in the National Asthma Education Program Expert Panel. Guidelines for diagnosis and management of asthma. National Institutes of Health, 1991 ; Pub. #91-3042. Atopy, respiratory infection and environmental predisposing factors may also be present, but are not necessary elements of an asthma diagnosis. Asthma conditions strictly related to atopy are referred to as atopic asthma. The human ASTH1I and ASTH1J gene sequences are provided, as are the genomic sequences 5' to ASTH1J. The major sequences of interest provided in the sequence listing are as follows:
ASTH1J 5' Genomic Region DNA (SEQ ID NO:1)
ASTH1J altl cDNA (SEQ ID NO:2)
ASTH1J alt2 cDNA (SEQ ID NO:3) ASTH1J alt3 cDNA (SEQ ID NO:4)
-4- ASTH1J protein protein (SEQ ID NO:5)
ASTH1I altl cDNA (SEQ ID NO:6)
ASTH1 l alt1 protein protein (SEQ ID NO:7)
ASTH1I alt2 cDNA (SEQ ID NO:8)
ASTH1 l alt2 protein protein (SEQ ID NO:9)
ASTH1I alt3 cDNA (SEQ ID NO 10)
ASTH1 l alt3 protein protein (SEQ ID NO 11)
CAAT box "A" form DNA (SEQ ID NO 12)
CAAT box "G" form DNA (SEQ ID NO 13)
ASTH1J 5' promoter region DNA (SEQ ID NO 14)
Mouse asthlj cDNA (SEQ ID NO:338)
Mouse asthlj protein (SEQ ID NO:339)
Polymorphisms DNA (SEQ ID NO:16-159)
Microsatellite flanking sequences DNA (SEQ ID NO:160-281)
Microsatellite repeats DNA (SEQ ID NO:282-292) Intron-Exon boundaries DNA (SEQ ID NO:293-335)
The ASTH1 locus has been mapped to human chromosome 11p. The traits for a positive response to methacholine challenge and a clinical history of asthma were shown to be genetically linked in a genome scan of the population of Tristan da Cunha, a single large extended family with a high incidence of asthma (discussed in Zamel et al. (1996) Am. J. Respir. Crit. Care Med. 153:1902-1906). The linkage finding was replicated in a set of Canadian asthmatic families. The region of strongest linkage was the marker D11S907 on the short arm of chromosome 11. Additional markers were identified from the four megabase region surrounding D11S907 from public databases and by original cloning of new polymorphic microsatellite markers. Refinement of the region of interest was obtained by genotyping new markers in the studied populations, and applying the transmission disequilibrium test (TDT), which reflects the level of association between marker alleles and disease status. TDT curves were superimposed on the physical map. Molecular genetic techniques for gene identification were applied to the region of interest. A one megabase genomic region was sequenced to high accuracy, and the resulting data used for the sequence-based prediction of genes and determination of the intron/exon structure of genes in the region. Nucleic Acid Compositions
ASTH1 I produces a 2.8 kb mRNA expressed at high levels in trachea and prostate, and at lower levels in lung and kidney and possibly other tissues. ASTH11 cDNA clones have also been identified in prostate, testis and lung libraries. Sequence polymorphisms are shown in Table 3. ASTH11 has at least three alternate forms denoted as altl , alt2, and alt3. The alternative splicing and start codons give the three forms of ASTH1 I proteins different amino termini. The ASTH1 I proteins, altl , alt2 and alt3 are 265, 255 and 164 amino acids in length, respectively.
A domain of the ASTH1 I and ASTH1 J proteins is similar in sequence to transcription factors of the ets family. The ets family is a group of transcription factors that activate genes involved in a variety of immunological and other processes. The family members most similar to ASTH11 and ASTH1 J are: ETS1 , ETS2, ESX, ELF, ELK1 , TEL, NET, SAP-1 , NERF and FLI. The ASTH1 I and ASTH1 J proteins show similarity to each other. Over the ets domain they are 66% similar (ie. have amino acids with similar properties in the same positions) and 46% identical to each other. All forms of ASTH1 I and ASTH1J have a helix turn helix motif, characteristic of some transcription factors, located near the carboxy terminal end of the protein.
ASTH1J produces an approximately 6 kb mRNA expressed at high levels in the trachea, prostate and pancreas and at lower levels in colon, small intestine, lung and stomach. ASTH1J has at least three forms, consisting of the altl , alt2 and alt3 forms. The open reading frame is identical for the three forms, which differ only in the 5' UTR. The protein encoded by ASTH1 J is 300 amino acids in length.
Mouse coding region sequence of asthlj is provided in SEQ ID NO:326, and the amino acid sequence is provided in SEQ ID NO:327. The mouse and human proteins have 88.4% identity throughout their length. The match in the ets domain is 100%. The mouse cDNA was identified by hybridization of a full-length human cDNA to a mouse lung cDNA library (Stratagene).
The term "ASTH1 genes" is herein used generically to designate ASTH1I and ASTH1J genes and their alternate forms. The two genes lie in opposite orientations on a native chromosome, with the 5' regulatory sequences between them. Part of the genomic sequence between the two coding regions is provided as SEQ ID NO:1. The term "ASTH1 locus" is used herein to refer to the two genes in all alternate forms and the genomic sequence that lies between the two genes. Alternate forms include splicing variants, and polymorphisms in the sequence. Specific polymorphic sequences are provided in SEQ ID NOs:16-159. For some purposes the previously known EST sequences described herein may be excluded from the sequences defined as the ASTH1 locus.
The DNA sequence encoding ASTH1 may be cDNA or genomic DNA or a fragment thereof. The term "ASTH1 gene" shall be intended to mean the open reading frame encoding specific ASTH1 polypeptides, introns, as well as adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression, up to about 1 kb beyond the coding region, but possibly further in either direction. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host. The term "cDNA" as used herein is intended to include all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3' and 5' non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns removed by nuclear RNA splicing, to create a continuous open reading frame encoding the ASTH1 protein.
The genomic ASTH1 sequence has non-contiguous open reading frames, where introns interrupt the protein coding regions. A genomic sequence of interest comprises the nucleic acid present between the initiation codon and the stop codon, as defined in the listed sequences, including all of the introns that are normally present in a native chromosome. It may further include the 3' and 5' untranslated regions found in the mature mRNA. It may further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc.,
-7- including about 1 kb, but possibly more, of flanking genomic DNA at either the 5' or 3' end of the transcribed region. The genomic DNA may be isolated as a fragment of 100 kbp or smaller; and substantially free of flanking chromosomal sequence. Genomic regions of interest include the non-transcribed sequences 5' to ASTH1J, as provided in SEQ ID NO:1. This region of DNA contains the native promoter elements that direct expression of the linked ASTH1J gene. Usually a promoter region will have at least about 140 nt of sequence located 5' to the ASTH1 gene and further comprising a TATA box and CAAT box motif sequence (SEQ ID NO: 14, nt. 597-736). The promoter region may further comprise a consensus ets binding motif, (C/A)GGA(A/T) (SEQ ID NO:14, nt 1-5). A region of particular interest, containing the ets binding motif, TATA box and CAAT box motifs 5' to the ASTH1J gene, is provided in SEQ ID NO:14. The position of SEQ ID NO:14 within the larger sequence is SEQ ID NO:1 , nt 60359-61095. The promoter sequence may comprise polymorphisms within the CAAT box region, for example those shown in SEQ ID NO: 12 and SEQ ID NO: 13, which have been shown to affect the function of the promoter. The promoter region of interest may extend 5' to SEQ ID NO:14 within the larger sequence, e.g. SEQ ID NO:1 , nt 59000-61095; SEQ ID NO:1 , nt 5700-61095, etc.
The sequence of this 5' region, and further 5' upstream sequences and 3' downstream sequences, may be utilized for promoter elements, including enhancer binding sites, that provide for expression in tissues where ASTH1J is expressed. The tissue specific expression is useful for determining the pattern of expression, and for providing promoters that mimic the native pattern of expression. Naturally occurring polymorphisms in the promoter region are useful for determining natural variations in expression, particularly those that may be associated with disease. See, for example, SEQ ID NO: 12 and 13. Alternatively, mutations may be introduced into the promoter region to determine the effect of altering expression in experimentally defined systems. Methods for the identification of specific DNA motifs involved in the binding of transcriptional factors are known in the art, e.g. sequence similarity to known binding motifs, gel retardation studies, etc. For examples, see Blackwell et al. (1995) Mol Med 1: 194-205; Mortlock et al. (1996)
-8- Genome Res. 6: 327-33; and Joulin and Richard-Foy (1995) Eur J Biochem 232: 620-626.
The regulatory sequences may be used to identify cis acting sequences required for transcriptional or translational regulation of ASTH1 expression, especially in different tissues or stages of development, and to identify cis acting sequences and trans acting factors that regulate or mediate ASTH1 expression. Such transcription or translational control regions may be operably linked to a ASTH1 gene in order to promote expression of wild type or altered ASTH1 or other proteins of interest in cultured cells, or in embryonic, fetal or adult tissues, and for gene therapy.
The nucleic acid compositions of the subject invention may encode all or a part of the subject polypeptides. Fragments may be obtained of the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc. For the most part, DNA fragments will be of at least 15 nt, usually at least 18 nt, more usually at least about 50 nt. Such small DNA fragments are useful as primers for PCR, hybridization screening, etc. Larger DNA fragments, i.e. greater than 100 nt are useful for production of the encoded polypeptide. For use in amplification reactions, such as PCR, a pair of primers will be used. The exact composition of the primer sequences is not critical to the invention, but for most applications the primers will hybridize to the subject sequence under stringent conditions, as known in the art. It is preferable to choose a pair of primers that will generate an amplification product of at least about 50 nt, preferably at least about 100 nt. Algorithms for the selection of primer sequences are generally known, and are available in commercial software packages. Amplification primers hybridize to complementary strands of DNA, and will prime towards each other.
The ASTH1 genes are isolated and obtained in substantial purity, generally as other than an intact mammalian chromosome. Usually, the DNA will be obtained substantially free of other nucleic acid sequences that do not include an ASTH1 sequence or fragment thereof, generally being at least about 50%, usually at least about 90% pure and are typically "recombinant", i.e. flanked by one or more
-9- nucleotides with which it is not normally associated on a naturally occurring chromosome.
The DNA sequences are used in a variety of ways. They may be used as probes for identifying ASTH1 related genes. Mammalian homologs have substantial sequence similarity to the subject sequences, i.e. at least 75%, usually at least 90%, more usually at least 95% sequence identity with the nucleotide sequence of the subject DNA sequence. Sequence similarity is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al. (1990) J Mol Biol 215:403-10.
Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50°C and 10XSSC (0.9 M saline/0.09 M sodium citrate) and remain bound when subjected to washing at 55°C in 1XSSC. Sequence identity may be determined by hybridization under stringent conditions, for example, at 50°C or higher and 0.1XSSC (9 mM saline/0.9 mM sodium citrate). By using probes, particularly labeled probes of DNA sequences, one can isolate homologous or related genes. The source of homologous genes may be any species, e.g. primate species, particularly human; rodents, such as rats and mice, canines, felines, bovines, ovines, equines, yeast, Drosophila, Caenhorabditis, etc. The DNA may also be used to identify expression of the gene in a biological specimen. The manner in which one probes cells for the presence of particular nucleotide sequences, as genomic DNA or RNA, is well established in the literature and does not require elaboration here. mRNA is isolated from a cell sample. mRNA may be amplified by RT-PCR, using reverse transcriptase to form a complementary DNA strand, followed by polymerase chain reaction amplification using primers specific for the subject DNA sequences. Alternatively, mRNA sample is separated by gel electrophoresis, transferred to a suitable support, e.g. nitrocellulose, nylon, etc., and then probed with a fragment of the subject DNA as a probe. Other techniques, such as oligonucleotide ligation assays, in situ
-10- hybridizations, and hybridization to DNA probes arrayed on a solid chip may also find use. Detection of mRNA hybridizing to the subject sequence is indicative of ASTH1 gene expression in the sample.
The subject nucleic acid sequences may be modified for a number of purposes, particularly where they will be used intracellularly, for example, by being joined to a nucleic acid cleaving agent, e.g. a chelated metal ion, such as iron or chromium for cleavage of the gene; or the like.
The sequence of the ASTH1 locus, including flanking promoter regions and coding regions, may be mutated in various ways known in the art to generate targeted changes in promoter strength, sequence of the encoded protein, etc. The DNA sequence or product of such a mutation will be substantially similar to the sequences provided herein, i.e. will differ by at least one nucleotide or amino acid, respectively, and may differ by at least two but not more than about ten nucleotides or amino acids. The sequence changes may be substitutions, insertions or deletions. Deletions may further include larger changes, such as deletions of a domain or exon. Other modifications of interest include epitope tagging, e.g. with the FLAG system, HA, etc. For studies of subcellular localization, fusion proteins with green fluorescent proteins (GFP) may be used. Such mutated genes may be used to study structure-function relationships of ASTH1 polypeptides, or to alter properties of the protein that affect its function or regulation. For example, constitutively active transcription factors, or a dominant negatively active protein that binds to the ASTH1 DNA target site without activating transcription, may be created in this manner.
Techniques for in vitro mutagenesis of cloned genes are known. Examples of protocols for scanning mutations may be found in Gustin et al., Biotechniques 14:22 (1993); Barany, Gene 37:111-23 (1985); Colicelli et al., Mol Gen Genet 199:537-9 (1985); and Prentki et al. , Gene 29:303-13 (1984). Methods for site specific mutagenesis can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp. 15.3-15.108; Werner et al., Gene 126:35- 41 (1993); Sayers et al., Biotechniques 13:592-6 (1992); Jones and Winistorfer, Biotechniques 12:528-30 (1992); Barton et al., Nucleic Acids Res 18:7349-55
-11- (1990); Marotti and Tomich, Gene Anal Tech 6:67-70 (1989); and Zhu Anal Biochem 177:120-4 (1989).
Synthesis of ASTH1 Proteins The subject gene may be employed for synthesis of a complete ASTH1 protein, or polypeptide fragments thereof, particularly fragments corresponding to functional domains; binding sites; etc.; and including fusions of the subject polypeptides to other proteins or parts thereof. For expression, an expression cassette may be employed, providing for a transcriptional and translational initiation region, which may be inducible or constitutive, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region. Various transcriptional initiation regions may be employed that are functional in the expression host.
The polypeptides may be expressed in prokaryotes or eukaryotes in accordance with conventional ways, depending upon the purpose for expression. For large scale production of the protein, a unicellular organism, such as E. coli, B. subtilis, S. cerevisiae, or cells of a higher organism such as vertebrates, particularly mammals, e.g. COS 7 cells, may be used as the expression host cells. In many situations, it may be desirable to express the ASTH1 gene in mammalian cells, where the ASTH1 gene will benefit from native folding and post-translational modifications. Small peptides can also be synthesized in the laboratory.
With the availability of the polypeptides in large amounts, by employing an expression host, the polypeptides may be isolated and purified in accordance with conventional ways. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. The purified polypeptide will generally be at least about 80% pure, preferably at least about 90% pure, and may be up to and including 100% pure. Pure is intended to mean free of other proteins, as well as cellular debris.
The polypeptide is used for the production of antibodies, where short fragments provide for antibodies specific for the particular polypeptide, and larger fragments or the entire protein allow for the production of antibodies over the surface of the polypeptide. Antibodies may be raised to the wild-type or variant
-12- forms of ASTH Antibodies may be raised to isolated peptides corresponding to these domains, or to the native protein, e.g. by immunization with cells expressing ASTH1 , immunization with liposomes having ASTH1 inserted in the membrane, etc. Antibodies are prepared in accordance with conventional ways, where the expressed polypeptide or protein is used as an immunogen, by itself or conjugated to known immunogenic carriers, e.g. KLH, pre-S HBsAg, other viral or eukaryotic proteins, or the like. Various adjuvants may be employed, with a series of injections, as appropriate. For monoclonal antibodies, after one or more booster injections, the spleen is isolated, the lymphocytes immortalized by cell fusion, and then screened for high affinity antibody binding. The immortalized cells, i.e. hybridomas, producing the desired antibodies may then be expanded. For further description, see Monoclonal Antibodies: A Laboratory Manual. Harlow and Lane eds., Cold Spring Harbor Laboratories, Cold Spring Harbor, New York, 1988. If desired, the mRNA encoding the heavy and light chains may be isolated and mutagenized by cloning in E. coli, and the heavy and light chains mixed to further enhance the affinity of the antibody. Alternatives to in vivo immunization as a method of raising antibodies include binding to phage "display" libraries, usually in conjunction with in vitro affinity maturation.
Detection of ASTH1 Associated Asthma Diagnosis of ASTH1 associated asthma is performed by protein, DNA or
RNA sequence and/or hybridization analysis of any convenient sample from a patient, e.g. biopsy material, blood sample, scrapings from cheek, etc. A nucleic acid sample from a patient having asthma that may be associated with ASTH1, is analyzed for the presence of a predisposing polymorphism in ASTHL A typical patient genotype will have at least one predisposing mutation on at least one chromosome. The presence of a polymorphic ASTH1 sequence that affects the activity or expression of the gene product, and confers an increased susceptibility to asthma is considered a predisposing polymorphism. Individuals are screened by analyzing their DNA or mRNA for the presence of a predisposing polymorphism, as compared to an asthma neutral sequence. Specific sequences of interest include any polymorphism that leads to clinical bronchial hyperreactivity or is otherwise associated with asthma, including, but not limited to, insertions, substitutions and
-13- deletions in the coding region sequence, intron sequences that affect splicing, or promoter or enhancer sequences that affect the activity and expression of the protein. Examples of specific ASTH1 polymorphisms in asthma patients are listed in Tables 3-8. The CAAT box polymorphism of SEQ ID NO: 12 and 13 (which is located within SEQ ID NO:14) is of particular interest. The "G" form, SEQ ID NO:13, can be associated with a propensity to develop bronchial hyperreactivity or asthma. Other polymorphisms in the surrounding region affect this association. It has been found that substitution of "G" for "A" results in decreased binding of nuclear proteins to the DNA motif.
The effect of an ASTH1 predisposing polymorphism may be modulated by the patient genotype in other genes related to asthma and atopy, including, but not limited to, the Fcε receptor, Class I and Class II HLA antigens, T cell receptor and immunoglobulin genes, cytokines and cytokine receptors, and the like. Screening may also be based on the functional or antigenic characteristics of the protein. Immunoassays designed to detect predisposing polymorphisms in ASTH1 proteins may be used in screening. Where many diverse mutations lead to a particular disease phenotype, functional protein assays have proven to be effective screening tools. Biochemical studies may be performed to determine whether a candidate sequence polymorphism in the ASTH1 coding region or control regions is associated with disease. For example, a change in the promoter or enhancer sequence that affects expression of ASTH1 may result in predisposition to asthma. Expression levels of a candidate variant allele are compared to expression levels of the normal allele by various methods known in the art. Methods for determining promoter or enhancer strength include quantitation of the expressed natural protein; insertion of the variant control element into a vector with a reporter gene such as β-galactosidase, luciferase, chloramphenicol acetyltransferase, etc. that provides for convenient quantitation; and the like. The activity of the encoded ASTH1 protein may be determined by comparison with the wild-type protein.
-14- A number of methods are available for analyzing nucleic acids for the presence of a specific sequence. Where large amounts of DNA are available, genomic DNA is used directly. Alternatively, the region of interest is cloned into a suitable vector and grown in sufficient quantity for analysis. Cells that express ASTH1 genes, such as trachea cells, may be used as a source of mRNA, which may be assayed directly or reverse transcribed into cDNA for analysis. The nucleic acid may be amplified by conventional techniques, such as the polymerase chain reaction (PCR), to provide sufficient amounts for analysis. The use of the polymerase chain reaction is described in Saiki, et al. (1985) Science 239:487, and a review of current techniques may be found in Sambrook, et al. Molecular Cloning: A Laboratory Manual. CSH Press 1989, pp.14.2-14.33. Amplification may also be used to determine whether a polymorphism is present, by using a primer that is specific for the polymorphism. Alternatively, various methods are known in the art that utilize oligonucleotide ligation as a means of detecting polymorphisms, for examples see Riley et al. (1990) N.A.R. 18:2887-2890; and Delahunty et al. (1996) Am. J. Hum. Genet. 58: 1239-1246.
A detectable label may be included in an amplification reaction. Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive labels, e.g. 32P, 35S, 3H; etc. The label may be a two stage system, where the amplified DNA is conjugated to biotin, haptens, etc. having a high affinity binding partner, e.g. avidin, specific antibodies, etc., where the binding partner is conjugated to a detectable label. The label may be conjugated to one or both of the primers. Alternatively, the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product.
The sample nucleic acid, e.g. amplified or cloned fragment, is analyzed by one of a number of methods known in the art. The nucleic acid may be sequenced by dideoxy or other methods, and the sequence of bases compared to a neutral ASTH1 sequence. Hybridization with the variant sequence may also be used to
-15- determine its presence, by Southern blots, dot blots, etc. The hybridization pattern of a control and variant sequence to an array of oligonucleotide probes immobilised on a solid support, as described in US 5,445,934, or in WO95/35505, may also be used as a means of detecting the presence of variant sequences. Single strand conformational polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), mismatch cleavage detection, and heteroduplex analysis in gel matrices are used to detect conformational changes created by DNA sequence variation as alterations in electrophoretic mobility. Alternatively, where a polymorphism creates or destroys a recognition site for a restriction endonuclease (restriction fragment length polymorphism, RFLP), the sample is digested with that endonuclease, and the products size fractionated to determine whether the fragment was digested. Fractionation is performed by gel or capillary electrophoresis, particularly acrylamide or agarose gels.
The hybridization pattern of a control and variant sequence to an array of oligonucleotide probes immobilised on a solid support, as described in US
5,445,934, or in WO95/35505, may be used as a means of detecting the presence of variant sequences. In one embodiment of the invention, an array of oligonucleotides are provided, where discrete positions on the array are complementary to at least a portion of mRNA or genomic DNA of the ASTH1 locus. Such an array may comprise a series of oligonucleotides, each of which can specifically hybridize to a nucleic acid, e.g. mRNA, cDNA, genomic DNA, etc. from the ASTH1 locus.
An array may include all or a subset of the polymorphisms listed in Table 3 (SEQ ID NOs:16-126). One or both polymorphic forms may be present in the array, for example the polymorphism of SEQ ID NO: 12 and 13 may be represented by either, or both, of the listed sequences. Usually such an array will include at least 2 different polymorphic sequences, i.e. polymorphisms located at unique positions within the locus, usually at least about 5, more usually at least about 10, and may include as many as 50 to 100 different polymorphisms. The oligonucleotide sequence on the array will usually be at least about 12 nt in length, may be the length of the provided polymorphic sequences, or may extend into the flanking regions to generate fragments of 100 to 200 nt in length. For examples of arrays,
-16- see Hacia et al. (1996) Nature Genetics 14:441-447; Lockhart et al. (1996) Nature Biotechnol. 14:1675-1680; and De Risi et al. (1996) Nature Genetics 14:457-460.
Antibodies specific for ASTH1 polymorphisms may be used in screening immunoassays. A reduction or increase in neutral ASTH1 and/or presence of asthma associated polymorphisms is indicative that asthma is ASTH1 -associated. A sample is taken from a patient suspected of having ASTH1 -associated asthma. Samples, as used herein, include biological fluids such as tracheal lavage, blood, cerebrospinal fluid, tears, saliva, lymph, dialysis fluid and the like; organ or tissue culture derived fluids; and fluids extracted from physiological tissues. Also included in the term are derivatives and fractions of such fluids. Biopsy samples are of particular interest, e.g. trachea scrapings, etc. The number of cells in a sample will generally be at least about 103, usually at least 10 more usually at least about 105. The cells may be dissociated, in the case of solid tissues, or tissue sections may be analyzed. Alternatively a lysate of the cells may be prepared. Diagnosis may be performed by a number of methods. The different methods all determine the absence or presence or altered amounts of normal or abnormal ASTH1 in patient cells suspected of having a predisposing polymorphism in ASTHL For example, detection may utilize staining of cells or histological sections, performed in accordance with conventional methods. The antibodies of interest are added to the cell sample, and incubated for a period of time sufficient to allow binding to the epitope, usually at least about 10 minutes. The antibody may be labeled with radioisotopes, enzymes, fluorescers, chemiluminescers, or other labels for direct detection. Alternatively, a second stage antibody or reagent is used to amplify the signal. Such reagents are well known in the art. For example, the primary antibody may be conjugated to biotin, with horseradish peroxidase- conjugated avidin added as a second stage reagent. Final detection uses a substrate that undergoes a color change in the presence of the peroxidase. The absence or presence of antibody binding may be determined by various methods, including flow cytometry of dissociated cells, microscopy, radiography, scintillation counting, etc.
An alternative method for diagnosis depends on the in vitro detection of binding between antibodies and ASTH1 in a lysate. Measuring the concentration of
-17- ASTH1 binding in a sample or fraction thereof may be accomplished by a variety of specific assays. A conventional sandwich type assay may be used. For example, a sandwich assay may first attach ASTH1 -specific antibodies to an insoluble surface or support. The particular manner of binding is not crucial so long as it is compatible with the reagents and overall methods of the invention. They may be bound to the plates covalently or non-covalently, preferably non-covalently.
The insoluble supports may be any compositions to which polypeptides can be bound, which is readily separated from soluble material, and which is otherwise compatible with the overall method. The surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports to which the receptor is bound include beads, e.g. magnetic beads, membranes and microtiter plates. These are typically made of glass, plastic (e.g. polystyrene), polysaccharides, nylon or nitrocellulose. Microtiter plates are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples.
Patient sample lysates are then added to separately assayable supports (for example, separate wells of a microtiter plate) containing antibodies. Preferably, a series of standards, containing known concentrations of normal and/or abnormal ASTH1 is assayed in parallel with the samples or aliquots thereof to serve as controls. Preferably, each sample and standard will be added to multiple wells so that mean values can be obtained for each. The incubation time should be sufficient for binding, generally, from about 0.1 to 3 hr is sufficient. After incubation, the insoluble support is generally washed of non-bound components. Generally, a dilute non-ionic detergent medium at an appropriate pH, generally 7-8, is used as a wash medium. From one to six washes may be employed, with sufficient volume to thoroughly wash non-specifically bound proteins present in the sample.
After washing, a solution containing a second antibody is applied. The antibody will bind ASTH1 with sufficient specificity such that it can be distinguished from other components present. The second antibodies may be labeled to facilitate direct, or indirect quantification of binding. Examples of labels that permit direct measurement of second receptor binding include radiolabels, such as 3H or 125l, fluorescers, dyes, beads, chemilumninescers, colloidal particles, and the like.
-18- Examples of labels which permit indirect measurement of binding include enzymes where the substrate may provide for a colored or fluorescent product. In a preferred embodiment, the antibodies are labeled with a covalently bound enzyme capable of providing a detectable product signal after addition of suitable substrate. Examples of suitable enzymes for use in conjugates include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase and the like. Where not commercially available, such antibody-enzyme conjugates are readily produced by techniques known to those skilled in the art. The incubation time should be sufficient for the labeled ligand to bind available molecules. Generally, from about 0.1 to 3 hr is sufficient, usually 1 hr sufficing.
After the second binding step, the insoluble support is again washed free of non-specifically bound material. The signal produced by the bound conjugate is detected by conventional means. Where an enzyme conjugate is used, an appropriate enzyme substrate is provided so a detectable product is formed. Other immunoassays are known in the art and may find use as diagnostics.
Ouchterlony plates provide a simple determination of antibody binding. Western blots may be performed on protein gels or protein spots on filters, using a detection system specific for ASTH1 as desired, conveniently using a labeling method as described for the sandwich assay. Other diagnostic assays of interest are based on the functional properties of
ASTH1 proteins. Such assays are particularly useful where a large number of different sequence changes lead to a common phenotype, i.e. altered protein function leading to bronchial hyperreactivity. For example, a functional assay may be based on the transcriptional changes mediated by ASTH1 gene products. Other assays may, for example, detect conformational changes, size changes resulting from insertions, deletions or truncations, or changes in the subcellular localization of ASTH1 proteins.
In a protein truncation test, PCR fragments amplified from the ASTH1 gene or its transcript are used as templates for in vivo transcription/translation reactions to generate protein products. Separation by gel electrophoresis is performed to determine whether the polymorphic gene encodes a truncated protein, where truncations may be associated with a loss of function.
-19- Diagnostic screening may also be performed for polymorphisms that are genetically linked to a predisposition for bronchial hyperreactivity, particularly through the use of microsatellite markers or single nucleotide polymorphisms. Frequently the microsatellite polymorphism itself is not phenotypically expressed, but is linked to sequences that result in a disease predisposition. However, in some cases the microsatellite sequence itself may affect gene expression. Microsatellite linkage analysis may be performed alone, or in combination with direct detection of polymorphisms, as described above. The use of microsatellite markers for genotyping is well documented. For examples, see Mansfield et al. (1994) Genomics 24:225-233; Ziegle et al. (1992) Genomics 14:1026-1031 ; Dib et al., supra.
Microsatellite loci that are useful in the subject methods have the general formula:
U (R)n U', where U and U' are non-repetitive flanking sequences that uniquely identify the particular locus, R is a repeat motif, and n is the number of repeats. The repeat motif is at least 2 nucleotides in length, up to 7, usually 2-4 nucleotides in length. Repeats can be simple or complex. The flanking sequences U and U' uniquely identify the microsatellite locus within the human genome. U and U' are at least about 18 nucleotides in length, and may extend several hundred bases up to about 1 kb on either side of the repeat. Within U and U', sequences are selected for amplification primers. The exact composition of the primer sequences are not critical to the invention, but they must hybridize to the flanking sequences U and U', respectively, under stringent conditions. Criteria for selection of amplification primers are as previously discussed. To maximize the resolution of size differences at the locus, it is preferable to chose a primer sequence that is close to the repeat sequence, such that the total amplification product is between 100-500 nucleotides in length.
The number of repeats at a specific locus, n, is polymorphic in a population, thereby generating individual differences in the length of DNA that lies between the amplification primers. The number will vary from at least 1 repeat to as many as about 100 repeats or more.
-20- The primers are used to amplify the region of genomic DNA that contains the repeats. Conveniently, a detectable label will be included in the amplification reaction, as previously described. Multiplex amplification may be performed in which several sets of primers are combined in the same reaction tube. This is particularly advantageous when limited amounts of sample DNA are available for analysis. Conveniently, each of the sets of primers is labeled with a different fluorochrome.
After amplification, the products are size fractionated. Fractionation may be performed by gel electrophoresis, particularly denaturing acrylamide or agarose gels. A convenient system uses denaturing polyacrylamide gels in combination with an automated DNA sequencer, see Hunkapillar et al. (1991) Science 254:59-74. The automated sequencer is particularly useful with multiplex amplification or pooled products of separate PCR reactions. Capillary electrophoresis may also be used for fractionation. A review of capillary electrophoresis may be found in Landers, et al. (1993) BioTechniques 14:98-111. The size of the amplification product is proportional to the number of repeats (n) that are present at the locus specified by the primers. The size will be polymorphic in the population, and is therefore an allelic marker for that locus.
A number of markers in the region of the ASTH1 locus have been identified, and are listed in Table 1 in the Experimental section (SEQ ID NOs:160-273). Of particular interest for diagnostic purposes is the marker D11S2008, in which individuals having alleles C or F at this locus, particularly in combination with the CAAT box polymorphism and other polymorphisms, are predisposed to develop bronchial hyperreactivity or asthma. The association of D11S2008 alleles is as follows: llele Association with asthma Number of TATC repeats relative to allele C (SEQ ID NO:15)
A no -2
B no -1
C yes equivalent
D no +1
E no +2
F yes +3
G no +4 H no +5
-21- A DNA sequence of interest for diagnosis comprises the D11S2008 primer sequences shown in Table 1 (SEQ ID NO:242 and 243), flanking one or three repeats of SEQ ID NO: 15.
Other microsatellite markers of interest for diagnostic purposes are CA39_2; 774F; 774J; 7740; L19PENTA1 ; 65P14TE1 ; AFM205YG5; D11S907; D11S4200; 774N; CA11-11 ; 774L; AFM283WH9; ASMI14 and D11S1900 (primer sequences are provided in Table 1 , the repeats are provided in Table 1 B).
Regulation of ASTH1 Expression The ASTH1 genes are useful for analysis of ASTH1 expression, e.g. in determining developmental and tissue specific patterns of expression, and for modulating expression in vitro and in vivo. The regulatory region of SEQ ID NO:1 may also be used to investigate analysis of ASTH1 expression. Vectors useful for introduction of the gene include plasmids and viral vectors. Of particular interest are retroviral-based vectors, e.g. Moloney murine leukemia virus and modified human immunodeficiency virus; adenovirus vectors, etc. that are maintained transiently or stably in mammalian cells. A wide variety of vectors can be employed for transfection and/or integration of the gene into the genome of the cells. Alternatively, micro-injection may be employed, fusion, or the like for introduction of genes into a suitable host cell. See, for example, Dhawan et al. (1991) Science 254:1509-1512 and Smith et al. (1990) Molecular and Cellular Biology 3268-3271. Administration of vectors to the lungs is of particular interest. Frequently such methods utilize liposomal formulations, as described in Eastman et al. (1997) Hum Gene Ther 8:765-773: Oudrhiri et al. (1997) P.N.A.S. 94:1651-1656: McDonald et al. (1997) Hum Gene Ther 8:411-422. The expression vector will have a transcriptional initiation region oriented to produce functional mRNA. The native transcriptional initiation region, e.g. SEQ ID NO: 14, or an exogenous transcriptional initiation region may be employed. The promoter may be introduced by recombinant methods in vitro, or as the result of homologous integration of the sequence into a chromosome. Many strong promoters are known in the art, including the β-actin promoter, SV40 early and late promoters, human cytomegalovirus promoter, retroviral LTRs, methallothionein responsive element (MRE), tetracycline-inducible promoter constructs, etc.
-22- Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences. Transcription cassettes may be prepared comprising a transcription initiation region, the target gene or fragment thereof, and a transcriptional termination region. The transcription cassettes may be introduced into a variety of vectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like, where the vectors are able to transiently or stably be maintained in the cells, usually for a period of at least about one day, more usually for a period of at least about several days to several weeks. Antisense molecules are used to down-regulate expression of ASTH1 in cells. The anti-sense reagent may be antisense oligonucleotides (ODN), particularly synthetic ODN having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA. The antisense sequence is complementary to the mRNA of the targeted gene, and inhibits expression of the targeted gene products. Antisense molecules inhibit gene expression through various mechanisms, e.g. by reducing the amount of mRNA available for translation, through activation of RNAse H, or steric hindrance. One or a combination of antisense molecules may be administered, where a combination may comprise multiple different sequences.
Antisense molecules may be produced by expression of all or a part of the target gene sequence in an appropriate vector, where the transcriptional initiation is oriented such that an antisense strand is produced as an RNA molecule. Alternatively, the antisense molecule is a synthetic oligonucleotide. Antisense oligonucleotides will generally be at least about 7, usually at least about 12, more usually at least about 20 nucleotides in length, and not more than about 500, usually not more than about 50, more usually not more than about 35 nucleotides in length, where the length is governed by efficiency of inhibition, specificity, including absence of cross-reactivity, and the like. It has been found that short oligonucleotides, of from 7 to 8 bases in length, can be strong and selective inhibitors of gene expression (see Wagner et al. (1996) Nature Biotechnology 14:840-844).
A specific region or regions of the endogenous sense strand mRNA sequence is chosen to be complemented by the antisense sequence. Selection of
-23- a specific sequence for the oligonucleotide may use an empirical method, where several candidate sequences are assayed for inhibition of expression of the target gene in an in vitro or animal model. A combination of sequences may also be used, where several regions of the mRNA sequence are selected for antisense complementation.
Antisense oligonucleotides may be chemically synthesized by methods known in the art (see Wagner et al. (1993) supra, and Milligan et al., supra.) Preferred oligonucleotides are chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases.
Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3'-O'-5'-S-phosphorothioate, 3'-S-5'-O- phosphorothioate, 3'-CH2-5'-O-phosphonate and 3'-NH-5'-O-phosphoroamidate. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage. Sugar modifications are also used to enhance stability and affinity. The α-anomer of deoxyribose may be used, where the base is inverted with respect to the natural β-anomer. The 2'-OH of the ribose sugar may be altered to form 2'- O-methyl or 2'-O-allyl sugars, which provides resistance to degradation without comprising affinity. Modification of the heterocyclic bases must maintain proper base pairing. Some useful substitutions include deoxyuridine for deoxythymidine; 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine. 5- propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and deoxycytidine, respectively.
As an alternative to anti-sense inhibitors, catalytic nucleic acid compounds, e.g. ribozymes, anti-sense conjugates, etc. may be used to inhibit gene expression. Ribozymes may be synthesized in vitro and administered to the patient, or may be encoded on an expression vector, from which the ribozyme is synthesized in the
-24- targeted cell (for example, see International patent application WO 9523225, and Beigelman et al. (1995) Nucl. Acids Res 23:4434-42). Examples of oligonucleotides with catalytic activity are described in WO 9506764. Conjugates of anti-sense ODN with a metal complex, e.g. terpyridylCu(ll), capable of mediating mRNA hydrolysis are described in Bashkin et al. (1995) Appl Biochem Biotechnol 54:43-56.
Therapeutic Use of ASTH1 Protein A host may be treated with intact ASTH1 protein, or an active fragment thereof to modulate or reduce bronchial hypereactivity. Desirably, the peptides will not induce an immune response, particularly an antibody response. Xenogeneic analogs may be screened for their ability to provide a therapeutic effect without raising an immune response. The protein or peptides may also be administered to in vitro cell cultures.
Various methods for administration may be employed. The polypeptide formulation may be given orally, or may be injected intravascularly, subcutaneously, peritoneally, etc. Methods of administration by inhalation are well-known in the art. The dosage of the therapeutic formulation will vary widely, depending upon the nature of the disease, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. The initial dose may be larger, followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, etc. to maintain an effective dosage level. In many cases, oral administration will require a higher dose than if administered intravenously. The amide bonds, as well as the amino and carboxy termini, may be modified for greater stability on oral administration. The subject peptides may be prepared as formulations at a pharmacologically effective dose in pharmaceutically acceptable media, for example normal saline, PBS, etc. The additives may include bactericidal agents, stabilizers, buffers, or the like. In order to enhance the half-life of the subject peptide or subject peptide conjugates, the peptides may be encapsulated, introduced into the lumen of liposomes, prepared as a colloid, or another conventional technique may be employed that provides for an extended lifetime of the peptides.
-25- The peptides may be administered as a combination therapy with other pharmacologically active agents. The additional drugs may be administered separately or in conjunction with the peptide compositions, and may be included in the same formulation. Models for Asthma
The subject nucleic acids can be used to generate genetically modified non-human animals or site specific gene modifications in cell lines. The term "transgenic" is intended to encompass genetically modified animals having a deletion or other knock-out of ASTH1 gene activity, having an exogenous ASTH1 gene that is stably transmitted in the host cells, or having an exogenous ASTH1 promoter operably linked to a reporter gene. Transgenic animals may be made through homologous recombination, where the ASTH1 locus is altered. Alternatively, a nucleic acid construct is randomly integrated into the genome. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like. Of interest are transgenic mammals, e.g. cows, pigs, goats, horses, etc., and particularly rodents, e.g. rats, mice, etc.
A "knock-out" animal is genetically manipulated to substantially reduce, or eliminate endogenous ASTH1 function. Different approaches may be used to achieve the "knock-out". A chromosomal deletion of all or part of the native ASTH1 homolog may be induced. Deletions of the non-coding regions, particularly the promoter region, 3' regulatory sequences, enhancers, or deletions of gene that activate expression of ASTH1 genes. A functional knock-out may also be achieved by the introduction of an anti-sense construct that blocks expression of the native ASTH1 genes (for example, see Li and Cohen (1996) Cell 85:319-329). Transgenic animals may be made having exogenous ASTH1 genes. The exogenous gene is usually either from a different species than the animal host, or is otherwise altered in its coding or non-coding sequence. The introduced gene may be a wild-type gene, naturally occurring polymorphism, or a genetically manipulated sequence, for example those previously described with deletions, substitutions or insertions in the coding or non-coding regions. The introduced sequence may encode an ASTH1 polypeptide, or may utilize the ASTH1 promoter operably linked to a reporter gene. Where the introduced gene is a coding sequence, it usually
-26- operably linked to a promoter, which may be constitutive or inducible, and other regulatory sequences required for expression in the host animal.
Specific constructs of interest, but are not limited to, include anti-sense ASTH1, which will block ASTH1 expression, expression of dominant negative ASTH1 mutations, and over-expression of a ASTH1 gene. A detectable marker, such as lac Z may be introduced into the ASTH1 locus, where upregulation of ASTH1 expression will result in an easily detected change in phenotype. Constructs utilizing the ASTH1 promoter region, e.g. SEQ ID NO:1 ; SEQ ID NO:14, in combination with a reporter gene or with the coding region of ASTH1J or ASTH1I are also of interest.
The modified cells or animals are useful in the study of ASTH1 function and regulation. Animals may be used in functional studies, drug screening, efα, e.g. to determine the effect of a candidate drug on asthma. A series of small deletions and/or substitutions may be made in the ASTH1 gene to determine the role of different exons in DNA binding, transcriptional regulation, etc. By providing expression of ASTH1 protein in cells in which it is otherwise not normally produced, one can induce changes in cell behavior. These animals are also useful for exploring models of inheritance of asthma, e.g. dominant v. recessive; relative effects of different alleles and synergistic effects between ASTH1I and ASTH1J and other asthma genes elsewhere in the genome.
DNA constructs for homologous recombination will comprise at least a portion of the ASTH1 gene with the desired genetic modification, and will include regions of homology to the target locus. DNA constructs for random integration need not include regions of homology to mediate recombination. Conveniently, markers for positive and negative selection are included. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For various techniques for transfecting mammalian cells, see Keown et al. (1990) Methods in Enzymology 185:527-537.
For embryonic stem (ES) cells, an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, e.g. mouse, rat, guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder layer or grown in the presence of appropriate growth factors, such as leukemia inhibiting factor (LIF).
-27- When ES cells have been transformed, they may be used to produce transgenic animals. After transformation, the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be detected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct. Those colonies that are positive may then be used for embryo manipulation and blastocyst injection. Blastocysts are obtained from 4 to 6 week old superovulated females. The ES cells are trypsinized, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting litters screened for mutant cells having the construct. By providing for a different phenotype of the blastocyst and the ES cells, chimeric progeny can be readily detected.
The chimeric animals are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allogeneic or congenic grafts or transplants, or in in vitro culture.
Investigation of genetic function may utilize non-mammalian models, particularly using those organisms that are biologically and genetically well-characterized, such as C. elegans, D. melanogaster and S. cerevisiae. For example, transposon (Tc1) insertions in the nematode homolog of an ASTH1 gene or promoter region may be made. The subject gene sequences may be used to knock-out or to complement defined genetic lesions in order to determine the physiological and biochemical pathways involved in ASTH1 function. A number of human genes have been shown to complement mutations in lower eukaryotes. Drug screening may be performed in combination with the subject animal models. Many mammalian genes have homologs in yeast and lower animals. The study of such homologs' physiological role and interactions with other proteins can facilitate understanding of biological function. In addition to model systems based on genetic complementation, yeast has been shown to be a powerful tool for studying protein-protein interactions through the two hybrid system described in
-28- Chien et al. (1991) P.N.A.S. 88:9578-9582. Two-hybrid system analysis is of particular interest for exploring transcriptional activation by ASTH1 proteins.
Drug Screening Assays By providing for the production of large amounts of ASTH1 protein, one can identify ligands or substrates that bind to, modulate or mimic the action of ASTHL Areas of investigation are the development of asthma treatments. Drug screening identifies agents that provide a replacement or enhancement for ASTH1 function in affected cells. Conversely, agents that reverse or inhibit ASTH1 function may stimulate bronchial reactivity. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, protein-DNA binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, and the like. The purified protein may also be used for determination of three-dimensional crystal structure, which can be used for modeling intermolecular interactions, transcriptional regulation, etc.
The term "agent" as used herein describes any molecule, e.g. protein or pharmaceutical, with the capability of altering or mimicking the physiological function of ASTHL Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.
Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids,
-29- steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic Compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
Where the screening assay is a binding assay, one or more of the molecules may be joined to a label, where the label can directly or indirectly provide a detectable signal. Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e.g. magnetic particles, and the like. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc. For the specific binding members, the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures.
A variety of other reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used. The mixture of components are added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4 and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0.1 and 1 hours will be sufficient.
-30- Other assays of interest detect agents that mimic ASTH1 function. For example, candidate agents are added to a cell that lacks functional ASTH1 , and screened for the ability to reproduce ASTH1 in a functional assay.
The compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host for treatment of asthma attributable to a defect in ASTH1 function. The compounds may also be used to enhance ASTH1 function. The therapeutic agents may be administered in a variety of ways, orally, topically, parenterally e.g. subcutaneously, intraperitoneally, by viral infection, intravascularly, etc. Inhaled treatments are of particular interest. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt.%.
The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds. Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.
Pharmacogenetics Pharmacogenetics is the linkage between an individual's genotype and that individual's ability to metabolize or react to a therapeutic agent. Differences in metabolism or target sensitivity can lead to severe toxicity or therapeutic failure by altering the relation between bioactive dose and blood concentration of the drug. In the past few years, numerous studies have established good relationships between polymorphisms in metabolic enzymes or drug targets, and both response and toxicity. These relationships can be used to individualize therapeutic dose administration.
Genotyping of polymorphic alleles is used to evaluate whether an individual will respond well to a particular therapeutic regimen. The polymorphic sequences
-31- are also used in drug screening assays, to determine the dose and specificity of a candidate therapeutic agent. A candidate ASTH1 polymorphism is screened with a target therapy to determine whether there is an influence on the effectiveness in treating asthma. Drug screening assays are performed as described above. Typically two or more different sequence polymorphisms are tested for response to a therapy.
Drugs currently used to treat asthma include beta 2-agonists, glucocorticoids, theophylline, cromones, and anticholinergic agents. For acute, severe asthma, the inhaled beta 2-agonists are the most effective bronchodilators. Short-acting forms give rapid relief; long-acting agents provide sustained relief and help nocturnal asthma. First-line therapy for chronic asthma is inhaled glucocorticoids, the only currently available agents that reduce airway inflammation. Theophylline is a bronchodilator that is useful for severe and nocturnal asthma, but recent studies suggest that it may also have an immunomodulatory effect. Cromones work best for patients who have mild asthma: they have few adverse effects, but their activity is brief, so they must be given frequently. Cysteinil leukotrienes are important mediators of asthma, and inhibition of their effects may represent a potential breakthrough in the therapy of allergic rhinitis and asthma. Where a particular sequence polymorphism correlates with differential drug effectiveness, diagnostic screening may be performed. Diagnostic methods have been described in detail in a preceding section. The presence of a particular polymorphism is detected, and used to develop an effective therapeutic strategy for the affected individual.
EXPERIMENTAL
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are
-32- parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.
MATERIALS AND METHODS Asthma families for genetic mapping studies
Asthma phenotype measurements and blood samples were obtained from the inhabitants of Tristan da Cunha, an isolated island in the South Atlantic, and from asthma families in Toronto, Canada (see Zamel et al., (1996) supra.) The 282 inhabitants of Tristan da Cunha form a single large extended family descended from 28 original founders. Settlement of Tristan da Cunha occurred beginning in 1817 with soldiers who remained behind when a British garrison was withdrawn from the island, followed by the survivors of several shipwrecks. In 1827 five women from St. Helena, one with children, emigrated to Tristan da Cunha and married island men. One of these women is said to have been asthmatic, and could be the origin of a genetic founder effect for asthma in this population. Inbreeding has resulted in kinship resemblances of at least first cousin levels for all individuals.
The Tristan da Cunha family pedigrees were ascertained through review of baptismal, marriage and medical records, as well as reliably accurate historical records of the early inhabitants (Zamel (1995) Can. Respir. J. 2:18). The prevalence of asthma on Tristan da Cunha is high; 23% had a definitive diagnosis of asthma.
The Toronto cohort included 59 small families having at least one affected individual. These were ascertained based on the following criteria: (i) an affected proband; (ii) availability of at least one sibling of the proband, either affected or unaffected; (iii) at least one living parent from whom DNA could be obtained. A set of 156 "triad" families consisting of an affected proband and his or her parents were also collected. Signed consent forms were obtained from each individual prior to commencement of phenotyping and blood sample collection. The Toronto patients were mainly of mixed European ancestry.
-33- Clinical characterization
A standardized questionnaire based on that of the American Thoracic Society (American Lung Association recommended respiratory diseases questionnaire for use with adults and children in epidemiology research. 1978. American Review of Respiratory Disease 118(2):7-53) was used to record the presence of respiratory symptoms such as cough, sputum and wheezing; the presence of other chest disorders including recent upper respiratory tract infection, allergic history; asthmatic attacks including onset, offset, confirmation by a physician, prevalence, severity and precipitating factors; other illnesses and smoking history; and all medications used within the previous 3 months. A physician-confirmed asthmatic attack was the principal criterion for a diagnosis of asthma.
Skin atopy was determined by skin prick tests to common allergens: A. fumigatus, Cladospohum, Alternaria, egg, milk, wheat, tree, dog, grass, horse, house dust, cat, feathers, house dust mite D. farinae, and house dust mite
D. pteronyssinus. Atopy testing of Toronto subjects omitted D. pteronyssinus and added cockroach and ragweed allergens. Saline and histamine controls were also performed (Bencard Laboratories, Mississauga, Ontario). Antihistamines were withdrawn for at least 48 hours prior to testing. Wheal diameters were corrected by subtraction of the saline control wheal diameter, and a corrected wheal size of >3 mm recorded 10 min after application was considered a positive response.
Airway responsiveness was assessed by a methacholine challenge test in those subjects with a baseline FEV1 (forced exhalation volume in one second) > 70% of predicted (Crapo et al. (1981) Am. Rev. Respir. Pis. 123:659). Methacholine challenge response was determined using the tidal breathing method (Cockcroft et al. (1977) Clin. Allergy 7:235). Doubling doses of methacholine from 0.03 to 16 mg/ml were administered using a Wright nebulizer at 4-min intervals to measure the provocative concentration of methacholine producing a 20% fall in FEV1 (PC20). If FEV1 was <70% of predicted, a bronchodilator response to 400 mg salbutamol aerosol was used to determine airway responsiveness. Both methacholine challenges and bronchodilator responses were measured using a computerized bronchial challenge system (S&M Instrument Co. Inc., Doyleston, PA)
-34- consisting of a software package and interface board installed in a Toshiba T1850C laptop computer and connected to a flow sensor (RS232FS). The power source for instruments used on Tristan da Cunha has been described (Zamel et al. (1996) supra.) Increased airway responsiveness was defined as a PC20 < 4.0 mg/ml or a > 15% improvement in FEV1 15 min postbronchodilator. Participants were asked to withhold bronchodilators at least 8 h before testing; inhaled or systemic steroids were maintained at the usual dosage. Subjects with a history of an upper respiratory tract infection within a month of testing were rechallenged at a later date.
Genotyping
PCR primer pairs were synthesized using Applied Biosystems 394 automated oligo synthesizer. The forward primer of each pair was labeled with either FAM, HEX, or TET phosphoramidites (Applied Biosystems). No oligo purification step was performed. Genomic DNA was extracted from whole blood. PCR was performed using
PTC100 thermocyclers (MJ Research). Reactions contained 10 mM Tris-HCl, pH 8.3; 1.5-3.0 mM MgCI2; 50 mM KCI; 0.01 % gelatin; 250 μM each dGTP, dATP, dTTP, dCTP; 20 μM each PCR primer; 20 ng genomic DNA; and 0.75 U Taq Polymerase (Perkin Elmer Cetus) in a final volume of 20 μl. Reactions were performed in 96 well polypropylene microtiter plates (Robbins Scientific) with an initial 94°C, 3 min. denaturation followed by 35 cycles of 30 sec. at 94°C, 30 sec. at the annealing temp., and 30 sec. at 72°C, with a final 2 min. extension at 72°C following the last cycle. Dye label, annealing temperature, and final magnesium concentration were specific to the individual marker. Dye label intensity and quantity of PCR product (as assessed on agarose gels) were used to determine the amount to be pooled for each marker locus. The pooled products were precipitated and the product pellets mixed with 0.4 μl Genescan 500 Tamra size standard, 2 μl formamide, and 1 μl ABI loading dye. Plates of PCR product pools were heated to 80°C for 5 minutes and immediately placed on ice prior to gel loading.
-35- PCR products were electrophoresed on denaturing 6% polyacrylamide gels at a constant 1000 volts using ABI 373a instruments. Peak detection, sizing, and stutter band filtering were achieved using Genescan 1.2 and Genotyper 1.1 software (Applied Biosystems). Genotype data were subsequently submitted to quality control and consistency checks (Hall et al. (1996) Genome Res. 6:781). Genotyping of 'saturation' markers in the ASTH1 region was done by the method described above with several exceptions. In most cases, the unlabeled primer of each pair was modified with the sequence GTTTCTT at the 5' end (Smith et al. 1995 Genome Res. 5:312). Amplitaq Gold (Perkin Elmer Cetus) and buffer D (2.5 mM MgCI2, 33.5 mM Tris-HCl pH 8.0, 8.3 mM (NH4)2SO4, 25 mM KCI, 85 μg/ml BSA) were used in the PCR. A 'touchdown' amplification profile was employed in which the annealing temperature began at 66°C and decreased one degree per cycle to a final 20 cycles at 56°C. Products were run on 4.25% polyacrylamide gels using ABI 377 instruments. The data was processed with Genescan 2.1 and Genotyper 1.1 software.
The Genome Scan
A genome scan was performed in the population of Tristan da Cunha using 274 polymorphic microsatellite markers chosen from among those developed at Oxford (Reed et al. (1994) Nature Genetics 7:390), Genethon (Dib et al. (1996) Nature 380:152) and the Cooperative Human Linkage Center (CHLC, Murray et al. (1994) Science 265:2049). Markers with heterozygosity values of 0.75 or greater were selected to cover all the human chromosomes, as well as for ease of genotyping and size of PCR product for multiplexing of markers on gels. Fifteen multiplexed sets were used to provide a ladder of PCR products in each of three dyes when separated by size. Published distances were used initially to estimate map resolution. More accurate genetic distances were calculated using the study population as the data was generated. The 274 markers gave an average 14 cM interval for the genome scan.
-36- Linkage analysis
Parametric linkage analyses of marker data were conducted using the methods of Haseman and Elston (1972) Behav. Genet. 2:3, and FASTLINK (Schaffer et al. (1996) Hum. Hered. 46:226), assuming a dominant mode of transmission with incomplete penetrance. Linkage to three primary phenotypes including asthma diagnosis (history), airway responsiveness (PC20 < 4 mg/ml for methacholine challenge) and atopy (one or more skin-prick test which yielded a wheal diameter > 3 mm) and combinations of these, were tested.
Small scale yeast artificial chromosome (YAC) DNA preparation
Small scale isolation of YAC DNA for STS mapping was done by a procedure which uses glass beads and physical shearing to damage the yeast cell wall (Scherer and Tsui (1991) Cloning and analysis of large DNA molecules. In Advanced Techniques in Chromosome Research. (K.W. Adolph, ed.) pp. 33-72. Marcel Dekker, Inc. New York, Basel, Hong Kong.)
YAC block prep and pulsed field gel electrophoresis (PFGE)
A 50 ml culture of each YAC was grown in 2 x AHC at 30°C. The cells were pelleted by centrifugation and washed twice in sterile water. After resuspension of the cells in 4 ml of SCEM (1 M sorbitol, 0.1 M sodium citrate (pH 5.8), 10 mM
EDTA, 30 mM β-mercaptoethanol), 5 ml of 1.2% low melting temperature agarose in SCEM was added, mixed, pipetted into 100 ml plug molds and allowed to solidify.
Plugs were incubated overnight in 50 ml of SCEM containing 30 U/ml lyticase
(Sigma). Plugs were rinsed 3 times in TE (10 mM Tris pH 8.0, 1 mM EDTA) and incubated twice for 12 hours each at 50°C in lysis solution (0.5 M EDTA, pH 8.0;
1% w/v sodium lauryl sarcosine; 0.5 mg/ml proteinase K). They were washed 5 times with TE and stored in 0.5 M EDTA (pH 8.0) at 4°C.
YACs and yeast chromosomes were separated on pulsed field gels using a
CHEF Mapper (BIO-RAD) and according to methods supplied by the manufacturer, then transferred to nitrocellulose. YACs which comigrated with yeast chromosomes were visualized by hybridization of the blot with radiolabelled YAC vector sequences (Scherer and Tsui (1991) supra.)
-37- Hybridization of YAC DNA to bacterial artificial chromosome (BAC) and cosmid grids
Size-purified YAC DNA was prepared by pulsed field gel electrophoresis on a low melting temperature Seaplaque GTG agarose (FMC) gel, purified by GeneClean (BIO101) and radiolabeled for 30 mins with 32P-dCTP using the Prime-It II kit (Sfratagene). 50 μl of water was added and unincorporated nucleotide was removed by Quick Spin Column (Boehringer Mannheim). 23 μl of 11.2 mg/ml human placental DNA (Sigma) and 36 μl of 0.5 M Na2HPO4, pH 6.0 were added to the approximately 150 μl of eluant. The probe was boiled for 5 mins and incubated at 65°C for exactly 3 hours, then added to the prehybridized gridded BAC (Shizuya et al. (1992) Proc. Natl. Acad. Sci. 89:8794; purchased from Research Genetics) or chromosome 11 cosmid [Resource Center/ Primary Database of the German Human Genome Project, Berlin; Lehrach et al. (1990), In Davies, K.E. and Tilghman, S.M. (eds.). Genome Analysis Volume 1 : Genetic and Physical Mapping. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 39-81] filters in dextran sulfate hybridization mix (10% dextran sulfate, 1 % SDS, 1 M NaCl). Hybridizations were at 65°C for 12 - 48 hours, followed by 2 washes at room temperature in 2x SSC for 10 mins each, and 3 washes at 65°C in 0.2X SSC, 0.2% SDS for 20 mins each.
Metaphase fluorescence in situ hybridization (FISH) and direct visual in situ hybridisation (DIRVISH)
Metaphase FISH was carried out by standard methods (Heng and Tsui (1994) FISH detection on DAPI banded chromosomes. In Methods of Molecular Bioloαv: In Situ Hybridisation Protocols (K.H.A. Choo, ed.) pp. 35-49. Human Press, Clifton, N.J.). High resolution FISH, or DIRVISH, was used to map the relative positions of two or more clones on genomic DNA. The protocol used was as described by Parra and Windle (1993) Nature Genet. 5:17. Briefly, slides containing stretched DNA were prepared by adding 2 μl of a suspension of normal human lymphoblast cells at one end of a glass slide and allowing to dry. 8 μl lysis buffer (0.5% SDS, 50 mM EDTA, 200 mM Tris-HCL, pH 7.4) was added and the
-38- slide incubated at room temperature for 5 minutes. The slide was tilted so that the DNA ran down the slide, then dried. The DNA was fixed by adding 400 μl 3:1 methanol/acetic acid. Probes were labeled either with biotin or with digoxygenin by standard nick translation (Rigby et al. (1977) J. Mol. Biol. 113:237). Hybridization and detections were carried out using standard fluorescence in situ hybridization techniques (Heng and Tsui (1994) supra.). Results were visualised using a Mikrophot SA microscope (Nikon) equipped with a CCD camera (Photometries). Images were recorded using Smartcapture software (Vysis).
Gap filling
Clones flanking gaps in the map were end cloned by digestion with enzymes that do not cut the respective vector sequences (Nsil for BAC clones and Xbal for PAC clones), followed by religation and transformation into competent DH5α. Clones which produced two end fragments and plasmid vector upon digestion with Notl and Nsil or Xbal were sequenced. Gaps in the tiling path were filled by screening a gridded BAC library with the end clone probes or by screening DNA pools of a human genomic PAC library (loannou et al. (1994) Nature Genetics 6:84; licensed from Health Research, Inc.) by PCR using primers designed from end clone sequences.
Direct cDNA selection
Direct cDNA selection (Lovett et al., (1991) Proc. Natl. Acad. Sci. 88:9628) was carried out using cDNA derived from both adult whole lung tissue and fetal whole lung tissue (Clontech). 5 μg of Poly(A)+ RNA was converted to double stranded cDNA using the Superscript Choice System for cDNA synthesis and the supplied protocol (Gibco BRL). First strand priming was achieved by both oligo(dT) and random hexamers. The resulting cDNA was split into 2 equal aliquots and digested with either Mbol or Taql prior to the addition of specific linker primers. Linker primers for Mbol-digested DNA were as described by Morgan et al. (1992) Nucleic Acid Res. 20:5173. Linker primers for Taql-digested DNA were a modification of these:
-39- (SEQ ID NO:336 ) Taqla: 5'-CGAGAATTCACTCGAGCATCAGG; (SEQ ID NO:337 ) Taqlb: 5'-CCTGATGCTCGAGTGAATTCT. The modified cDNA was ethanol precipitated and resuspended in 200 μl of H2O. 1 μl of cDNA was amplified with the linker primer Mbolb in a 100 μl PCR reaction. The resulting cDNA products, approximately 1 μg, were blocked with 1 μg of COT1 DNA (Gibco BRL) for 4 hours at 60°C in 120 mM NaPO4 buffer, pH 7.0.
Approximately 1 μg of the appropriate genomic clones was biotinylated using the BioNick Labeling System (Gibco BRL). Unincorporated biotin was removed by spin column chromatography. Approximately 100 ng of biotinylated genomic DNA was denatured and allowed to hybridize to 1 μg of blocked cDNA in a total volume of 20 μl in 120 mM NaPO4 for 60 hours at 60°C under mineral oil. After hybridization, the biotinylated DNA was captured on streptavidin-coated magnetic beads (Dynal) in 100 μl of binding buffer (1 M NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA) for 20 minutes at room temperature with constant rotation. Two 15 minute washes at room temperature with 500 μl of 1X SSC/0.1% SDS were followed by four washes for 20 minutes at 65°C with 500 ul of 0.1 X SSC/0.1% SDS with constant rotation. After each wash, the beads were collected on the side of the tube using magnet separation and the supernatant was removed with a pipette. Following the last wash, the beads were briefly rinsed once with wash solution prior to eluting the bound cDNA with 50 μl of 0.1 M NaOH for 10 minutes at room temperature. The supernatant was removed and neutralized with 50 μl 1 M Tris pH 7.4. The primary selected cDNA was desalted using a Sephadex G-50 column (Boehringer Mannheim). PCR was performed on 1 , 2, 5, and 10 μl of eluate with Mbolb primers. Amplified products were analyzed on a 1.4% agarose gel. The reaction with the cleanest bands and least background was scaled up to produce approximately 1 μg of primary selected cDNA. This amplified primary selected cDNA was blocked with 1 μg of COT1 at 60°C for 1 hour followed by a second round of hybridization to 100 ng of the appropriate genomic DNA under the same conditions as the first round of selection. Washing of the bound cDNA, elution, and PCR of the selected cDNA was identical to the first round. 1 μl of PCR amplified secondary selected cDNA was cloned using the TA cloning system according to the
-40- manufacturers protocol (Invifrogen). Colonies were picked into 96-well microtiter plates and grown overnight prior to sequencing.
Exon Trapping Exon trapping was performed by the method of Buckler et al. (1991 , Proc.
Natl. Acad. Sci. USA 88:4005) with modifications described in Church et al., (1994) Nature Genetics 6:98. Each BAC clone of the minimal set of clones required to the cover the ASTH1 region (/'.e. the tiling path) was subject to exon trapping separately. Briefly, restriction fragments (Pstl or BamHI/Bglll) of each cosmid were shotgun subcloned into Pstl- or BamHI-digested and phosphatase-treated pSPL3B which had been modified as in Burns et al. (1995) Gene 161 :183 (GIBCO BRL). Ligations were electroporated into ElectroMax HB101 cells (Gibco BRL) and plated on 20 cm diameter LB ampicillin plates. DNA was prepared from plates with > 2000 colonies by collection of the bacteria in LB ampicillin liquid and plasmid DNA purification by a standard alkaline lysis protocol (Sambrook et al. (1989) supra.) 5 μg of DNA from each plasmid pool preparation were electroporated into Cos 7 cells (ATCC) and RNA harvested using TRIZOL (Gibco BRL) after 48 hours of growth. RT-PCR products were digested with BstXI prior to a second PCR amplification. Products were cloned into pAMP10 (Gibco BRL) and transformed into DH5 cells (Gibco BRL). 96 colonies per BAC were picked and analyzed for insert size by PCR.
Northern blot hybridisation
Northern hybridisation was performed using Multiple Tissue Northern (MTN) blots (Clontech). DNA probes were radioactively labeled by random priming
[Feinberg and Vogelstein (1984) Anal. Biochem. 137:266] using the Prime-It II kit (Sfratagene). Hybridizations were performed in ExpressHyb hybridisation solution (Clontech) according to the manufacturer's recommendations. Filters were exposed to autoradiographic film overnight or for 3 days.
-41- cDNA library screening
Phage cDNA libraries were plated and screened with radiolabeled probes (exon trapping or cDNA selection products amplified by PCR from plasmids containing these sequences) by standard methods (Sambrook et al. (1989) supra.)
Rapid amplification of cDNA ends (RACE)
RACE libraries were constructed using polyA+ RNA and the Marathon cDNA amplification kit (Clontech). Nested RACE primer sets were designed for each cDNA or potential gene fragment (trapped exon, predicted exon, conserved fragment, etc). The RACE libraries were tested by PCR using one primer pair for each potential gene fragment; the two strongly positive libraries were chosen for RACE experiments.
Genomic sequencing DNA from cosmid, PAC, and BAC clones was prepared using Qiagen DNA prep kits and further purified by CsCI gradient. DNA was sonicated and DNA fragments were repaired using nuclease BAL-31 and T4 DNA polymerase. DNA fragments of 0.8-2.2 kb were size-fractionated by agarose gel electrophoresis and ligated into pUC9 vector. Inserts of the plasmid clones were amplified by PCR and sequenced using standard ABI dye-primer chemistry.
ABI sample file data was reanalyzed using Phred (Phil Green, University of Washington) for base calling and quality analysis. Sequence assembly of reanalyzed sequence data was accomplished using Phrap (Phil Green, University of Washington). Physical gaps between assembled contigs and unjoined but overlapping contigs were identified by inspection of the assembled data using GFP (licensed from Baylor College of Medicine) and Consed (Phil Green, University of Washington). Material for sequence data generation across gaps was obtained by PCR amplification. Low coverage regions were resequenced using dye-primer and dye-terminator chemistries (ABI). Final base-perfect editing (to > 99% accuracy) was accomplished using Consed.
-42- Single stranded conformational polymorphism (SSCP) analysis
PCR primers flanking each exon of the ASTH1 I and ASTH1J genes, or more than one primer pair for large exons, were designed from genomic sequence generated using Primer (publicly available from the Whitehead Institute for Biomedical Research) or Oligo 4.0 (licensed from National Biosciences).
Radioactive SSCP was performed by the method of Orita et al. (1989, Proc. Natl. Acad. Sci. 86:2766). Briefly, radioactively labeled PCR products between 150 and 300 bp and spanning exons of the ASTH1 I and ASTH1 J genes were generated from a set of asthma patient and control genomic template DNAs, by incorporating α-32P dCTP in the PCR. PCR reactions (20 μl) included 1x reaction buffer, 100 μM dNTPs, 1 μM each forward and reverse primer, and 1 unit Taq DNA polymerase (Perkin-Elmer) and 1 μCi α-32P dCTP. A brief denaturation at 94°C was followed by 30-32 cycles of: 94°C for 30 sec, 30 sec at the annealling temperature, and 72°C for 30 sec; followed by 5 mins at 72°. Radiolabeled PCR products were diluted 1 :20 in water, mixed with an equal volume of denaturing loading dye (95% formamide,
0.25% bromophenol blue), and denatured for 10 minutes at 80°C immediately prior to electrophoresis. 0.5x MDE (FMC) gels with and without 8% glycerol in 1x TBE were run at 8-12 Watts for 16-20 hours at room temperature. Dried gels were exposed to autoradiographic film (Kodak XAR) for 1-2 days at -80°C. PCR products from individuals carrying SSCP variants were subcloned into the PCR2.1 or pZeroBlunt plasmid vector (Invitrogen). Inserts of the plasmid clones were amplified by PCR and sequenced using standard ABI dye-primer chemistry to determine the nature of the sequence variant responsible for the conformational changes detected by SSCP. Fluorescent SSCP was carried out according to the recommended ABI protocol (ABI User Bulletin entitled 'Multi Color Fluorescent SSCP'). Unlabeled PCR primers were used to amplify genomic DNA segments containing different exons of the ASTH11 or ASTH1 J genes, in patient or control DNA. Nested fluorescently labeled (TET, FAM or HEX) primers were then used to amplify smaller products, 150 to 300 bp containing the exon or region of interest. Amplification was done using a "touchdown' PCR protocol, in which the annealing temperature
-43- decreased from 57°C to 42°C, and Amplitaq Gold polymerase (Perkin Elmer, Cetus). In most cases the fluorescently labeled primers were identical in sequence to those used for conventional radioactive SSCP. The fluorescent PCR products were diluted and mixed with denaturing agents, GeneScan size standard (Genescan 500 labelled with Tamra) and Blue dextran dye. Samples were heated at 90°C and quick chilled on ice prior to loading on 6.5% standard or 0.5 X MDE (manufacturer) polyacrylamide gels containing 2.5% glycerol and run using externally temperature controlled modified ABI 377 instruments. Gels were run at 1240V and 20 °C for 7-9 hrs and analyzed using GeneScan software (ABI).
Comparative (heterozygote detection) sequencing
Unlabeled PCR primers were used to amplify genomic DNA segments containing different exons of the ASTH1 I or ASTH1J genes, from patient or control DNAs. A set of nested PCR primers was then used to reamplify the fragment. Unincorporated primers were removed from the PCR product by Centricon-100 column (Amicon), or by Centricon-30 column for products less than 130 bp. The nested primers and dye terminator sequencing chemistry (ABI PRISM dye terminator cycle sequencing ready reaction kit) were then used to cycle sequence the exon and flanking region. Volumes were scaled down to 5 μl and 10% DMSO added to increase peak height uniformity. Sequences were compared between samples and heterozygous positions detected by visual inspection of chromatograms and using Sequence Navigator (licensed from ABI).
For some exons, PCR products were also compared by subcloning and sequencing, and comparison of sequences for ten or more clones.
RESULTS
Genome scanning and linkage analysis
A genome scan was performed using polymorphic microsatellite markers from throughout the human genome, and DNA isolated from blood samples drawn from the inhabitants of Tristan da Cunha. Linkage analysis, an established statistical method used to map the locations of genes and markers relative to other markers, was applied to verify the marker orders and relative distances between
-44- markers on all human chromosomes, in the Tristan da Cunha population. Linkage analysis can detect cosegregation of a marker with disease, and was used as a means to detect genes influencing the development of asthma in this population. The most highly significant linkage in the genome scan (p = 0.0001 for history of asthma and p = 0.0009 for methacholine challenge) was obtained at D11S907, a marker on the short arm of chromosome 11. This significant linkage result indicated that a gene influencing predisposition to asthma in the Tristan da Cunha population was located near D11S907.
Replication of this finding was obtained in a collection of asthma families from Toronto, in which D11S907 and several nearby markers were tested for linkage. The significant linkage seen (p = 0.001 for history of asthma and p = 0.05 for methacholine challenge) supported the mapping of an asthma gene near D11S907 and indicated that the gene was likely to be relevant in the more diverse outbred Toronto group as well as in the inbred population of Tristan da Cunha. The approximate genetic location of the ASTH1 gene in the Tristan da
Cunha population was confirmed by genotyping and analyzing data from several markers near D11S907, spaced at intervals no greater than 5 cM across a possible linked region of about 30 cM. Sib-pair and affected pedigree member linkage analyses of these markers yielded confirmatory evidence for linkage and refined the genetic interval.
Physical mapping atASTHI: YAC contig construction
Yeast artificial chromosome (YAC) clones were derived from the CEPH megaYAC library (Cohen et al. 1993 Nature 366:698). Individual YAC addresses were obtained from a public physical map of CEPH megaYAC STS (sequence tagged site; Olson et al. (1989) Science 245:1434) mapping data maintained by the Whitehead Institute and accessible through the world wide web (Cohen et al. 1993. supra.; http://www-genome.wi.mit.edu/cgi-bin/contig/phys_map). YAC clones spanning or overlapping other YACs containing D11S907 were chosen for map construction; STSs mapping to these YACs were used for map and clone verification. Some YACs annotated in the public database as being chimeric were excluded from the analyses. Multiple colonies of each YAC, obtained from a freshly
-45- streaked plate inoculated from the CEPH megaYAC library masterplate, were scored using STS markers from the ASTH1 region. These markers included polymorphic microsatellite repeats, expressed sequence tags (ESTs) and STSs. Comparison of STS mapping data for each clone with the public map allowed choice of the individual clone which retained the greatest number of ASTH1 region STSs, and was therefore least likely to be deleted. YAC addresses for which clones differed in STS content were interpreted to be prone to deletion; those for which a subset of clones contained no ASTH1 region STSs were presumed to be contaminated with yeast cells containing a YAC from another region of the genome. Chimerism of the chosen clones was assessed by metaphase fluorescent in situ hybridization (FISH). Their sizes were determined by pulsed field gel electrophoresis (PFGE), Southern blotting and hybridization with a YAC vector probe. The PFGE analyses also showed that no YAC clone chosen contained more than one yeast artificial chromosome. An STS map based on assuming the least number of deletions in the YAC clones was generated. The STS marker order was in agreement with that of the Whitehead map. The STS retention pattern of individual YACs, however, was slightly different from that of the public data. In general, the chosen clones were positive for a greater number ASTH1 region markers, showing that the data set was likely to have fewer false negatives than the public map. Non-chimeric YAC clones spanning the region of greatest interest were chosen for use as hybridization probes for the identification of smaller BAC, PAC, P1 or cosmid clones from the region.
Conversion to a plasmid-based clone map
The YAC map at ASTH1 provided continuous coverage of a 4 Mb region, the central 1 Mb of which was of greatest interest. YAC clones comprising a minimal tiling path of this region were chosen, and the size purified artificial chromosomes were used as hybridization probes to identify BAC and cosmid clones. Gridded filters of a 3x human genomic BAC library and of a human chromosome 11 -specific cosmid library were hybridized with radiolabeled purified YAC. Clones corresponding to the grid coordinates of the positives were streaked to colony
-46- purity, and filters gridded with four clones of each BAC or cosmid. These secondary filters were hybridized with size-purified YAC DNAs. A proportion of both the BACs and cosmids were found to be non-clonal by these analyses. A positively hybridizing clone of each was chosen for further analysis. The BAC and cosmid clones were STS mapped to establish overlaps between the clones. The BACs were further localized by DIRVISH. BACs which did not contain an STS marker were mapped in pairwise fashion by simultaneous two-color DIRVISH with another BAC. The map produced had three gaps which were subsequently filled by end cloning and hybridization of the end clones to a human genomic PAC library. Genetic refinement of the ASTH1 region had occurred concurrently with mapping, rendering it unnecessary to extend the BAC- contigged region. Mapping data was recorded in ACeDB (Eeckman and Durbin (1995) Methods Cell Biol. 48:583).
Genomic sequencing and gene prediction
A minimal tiling path of BAC and cosmid clones was chosen for genomic sequencing. Over 1 Mb of genomic sequence was generated at ASTHL On average, sequencing was done to 12x coverage (12 times redundancy in sequences). Marker order was verified relative to the STS map. BLAST searches (Altschul et al. (1990) supra.) were performed to identify sequences in public databases that were related to those in the ASTH1 region. Sequence-based gene prediction was done with the GRAIL [Roberts (1991) Science 254:805] and Geneparser [Snyder and Stormo (1993) Nucleic Acids Res. 21 : 607] programs. Genomic sequence and feature data was stored in ACeBD.
Development of new microsatellite markers for genetic refinement of the ASTH1 region
Additional informative polymorphic markers were important for the genetic refinement of the ASTH1 region. 'Saturation' cloning of every microsatellite in the 1 Mb region surrounding D11S907 was performed. Plasmid libraries were constructed from PFGE purified DNA from each YAC, prescreened with a primer from each known microsatellite marker, then screened with radiolabeled (CA)15 or
-47- a pool of trinucleotide and tetranucleotide repeat oligonucleotides. The plasmid inserts were sequenced, the set of sequences compared with those of the known microsatellite markers in the region, using Power assembler (ABI) or Sequencher (Alsbyte). Primer pairs flanking each novel microsatellite repeat were designed, and the heterozygosity of each new marker was tested by Batched Analysis of Genotypes (BAGs; LeDuc et al., 1995. PCR Methods and Applications 4:331). Additional microsatellites were found by analysis of the genomic sequence in AceDB. Table 1 lists all the microsatellite markers used for genotyping in the ASTH1 region and their repeat type, source and primers. Table 1 B lists some repeat sequences.
TABLE 1 Polymorphic microsatellite markers in the ASTHl region
SEQ ID MARKER PRIMER 1
160. 110O5GT1 CTGCTGTGGACGAATAGG 1 16611.. TCAATATAATCTTGCTTAACTTGG
162. 139C7GT1 GACCTGTTTGGGTTGATTTCAG
163. GTTTCTTACAGTGTCTTGCTATCACATCACC
164. 171L24AT1 GAGGACTGGCAGTACCAAGTAAAC
165. GTTTCTTTGGTTCATTCTAAGATGGCTGG 1 16666.. 2 25533EE66GGTT11 GCTGAGGCAGGAGAAAAGACAAG
167. GTTTCTTCATGCAAAGGTCAGGAGGTAGG
168. 253E6TE1 GTTGCTTCCAGACGAGGTACATG
169. GTTTCTTCAATGGCTCCACAAACATCTCTG
170. 253E6TR1 AGGTTTAGGGGACAGGGTTTGG 1 17711.. GTTTCTTTCCTGGCTAACACGGTGAAATC
172. 65P14 GTTTCTTATTGCCTCCTCCCAAAATTC
173. AGAGGCCACTGGAAGACGAA
174. 65P14GT1 AACTGGAGTCAGGCAAAACGTG
175. GTTTCTTTGGCTGGTAAGGAAAGAAACCAC 1 17766.. 6 655PP1144TTEE11 GGCTAGGTTCATAAACTCTGTGCTG
177. GTTTCTTGATTGTTTGAGATCCTTGACCCAG
178. 65P14TE2 GCCGAAATCACAACACTGCATC 179. GTTTCTTGATTCTGCTCTTACTCTTGCCCC
-48- 180. 65P14TR1 GTAATAGAACCAAAGGGCTGAGAC
181. GTTTCTTCGGAGTCAGACCTTACATTGTTGAG
182. 774F ATCTCCCTGCTACCCACCTT
183. GTTTCTTGTTTTCAGTGAGTTTCTGTTGGG
184. 774J GTGTGCCAAACAACATTTGC
185. GTTTCTTCAAGCCATCAAGCTAGAGTGG
186. 774L GGGCTTTTAAACCCTTATTTAACC
187. GTTTCTTAGGTGATCTCAGAGCCACTCA
188. 774N AGGGCAGGTGGGAACTTACT
189. GTTTCTTTGGAGTCAGTTGAGCTTTCTACC
190. 7740 TGAACTTGCCTACCTCCCAG
191. GTTTCTTAGCATATATCCTTACACAAGCACA
192. 774T CATGGTTCCAAAGGCAAGTT
193. GTTTCTTTTGAGGCTGAATGAGCTGTG
194. 86J5AT2 ACAGGTGGGAAGACTGAATGTC
195. GTTTCTTGCAGTACACATCACATGACCTTG
196. 86J5CA1 GAAATAGGCGGAAACTGGTTC
197. GTTTCTTCGTTGTGGTTGTTCAGAAAGG
198. 86J5GT1 GGTCAAGTGTTCAGAACGCATC
199. GTTTCTTGCAGGGATTATGCTAGGTCTGTAG
200. 86 5GT2 AGCACTTCTGAGGAAGGGACAC
201. GTTTCTTAGGGCAGGCAGACATACAAAC
202. 86J5TE1 GCCAATGTGTTCCTAGAGCGAC
203. GTTTCTTTTAAAGGGGGTAGGGTGTCACC
204. 8E.PENTA1 GGAAGGGAAAAGGACAAGGTTTTG
205. GTTTCTTAGCAAGAGCACTGGTGTAGGAGTC
206. 8EP04D05 GCTTTTCAAGCACTTGTCTC
207. TGGGATTGTGACTTACCATG
208. 8016GT1 ACTTGGTGTCTTATAGAAAGGTG
209. GTTTCTTAGCTGTGTTTGCTGCATC
210. 8016GT2 AGATGTGTGATGAGATGCAG
211. GTTTCTTCAAATAGTGCAACAAACCC 212. AFM198YB1 TGTCATTCTGAAAGTGCTTCC
-49- 213. GTTTCTTCTGTAACTAACGATCTGTAGTGGTG
214. AFM205YG5 (G) TATCAAGGTAATATAGTAGCCACGG
215. AGGTCTTTCATGCAGAGTGG
216. AFM206XB2 (G) ATTGCCAAAACTTGGAAGC
217. AGGTGACATATCAAGACCCTG
218. AFM283 H9 (G) TTGTCAACGAAGCCCAC
219. GTTTCTTGCAAGATTGTGTGTATGGATG
220. AFM324YH5 (G) GCTCTCTATGTGTTTGGGTG
221. AAGAGTACGCTAGTGGATGG
222. AFMA154ZD1 (G) TCCATTAGACCCAGAAAGG
223. GTTTCTTCACCAGGCTGAGATGTTACT
224. ASMI14 AATCGTTCCTTATCAGGTAATTTGG
225. GTTTCTTCAAAGAAAGCAATTCCATCATAACA
226. ASMI14T GCATTTGTTGAAGCAAGCGG
227. CTTTGTTCCTTGGCTGATGG
228. CA11_11 AATAGTACCAGACACACGTG
229. CAATGGTTCACAGCCCTTTT
230. CA39_2 AGCCTGGGAGACAGAGTGAG
231. GTTTCTTGCACTTTTTGGGGAAGGTG
232. CD59 (L) GTTCCTCCCTTCCCTCTCC
233. GTTTCTTTCAGGGACTGGATTGTAG
234. D11S130KU) GTGTTCTTTATGTGTAGTTC
235. GTTTCTTGGCAACAGAGTGAGACTCA
236. D11S1751 (G) GTGACATCCAGTGTTGGGAG
237. GTTTCTTCCTAAGCAAGCAAGCAATCA
238. D11S1776 (G) AAAGGCAATTGGTGGACA
239. GTTTCTTTTCAATCCTTGATGCAAAGT
240. D11S1900(U) GGTGACAGAGCAAGATTTCG
241. GTTTCTTGTAGAGTTGAGGGAGCAGC
242. D11S2008/D11S1392 CATCCATCTCATCCCATCAT
(C)
243. GTTTCTTTTCACCCTACTGCCAACTTC 244. D11S2014 (C) CCGCCATTTTAGAGAGCATA
-50- 245 . GTTTCTTTTCTGGGACAATTGGTAGGA
246 . D11S4200 ( G) TTTGTGTTATTATTTCAGGTGC
247 . GTTTCTTGTTTTTTGTTTCA GTTTAGGAAC
248 . D11S907 (G) CATACCCAAATCGTTCTCTTCCTC
249 . GTTTCTTGGAAAAGCAAAG GCATCGTAGAG
250 . D11S935 (G) TACTAACCAAAAGAGTTGGGG
251 . CTATCATTCAGAAAATGTTGGC
252 . GATA- P18492 ( C ) GTATGGCAGTAGAGGGCATG
253 . AAGGTTACATTTCAAGAAATAAAGT
254 . GATA- P6915 ( C) CTGTTCAGGCCTCAATATATACC
255 . AAGAGGATAGGTGGGGTTTG
256 . L19CA3 CCTCCCACCTAGACACAAT
257 . ATATGATCTTTGCATCCCTG
258 . L19PENTA1 AAGAAAGACCTGGAAGGAAT
259 . AAACAGCAAAACCTCATCTC
260 L19TETRA5 CCACCACTTATTACCTGCAT
261 TGAATGAATGAATGAACGAA
262 LMP2 AACTGTGATTGTGCCACTGCACTC
263 GTTTCTTCACCGCCTTTATCCCTCAAATG
264 LMP3 GATGGGTGGAGGGCAGTTAAAG
265 GTCAAGCAACTTGTCCAAGGCTAC
266 LMP4 CAGGCTATCAGTTTCCTTTGGAG
267 GGCAGGTAATACTGGAGAATTAGG
268 LMP7 GACGGATCTCAGAGCCACTC
269 GTTTCTTAAAAGATAAGGGCTTTTAAACC
270 T18 5 AGTTTCACAGCTTGTTATGG
271 GGTTGATGAAGTGAGACTTT
272 T29 9 ATGGTGGATGCATCCTGTG
273 GTTTCTTGTATTGACTCCTCCTCTGC
274 774L CAGTAAACAT
275 TGTTGAGTGG
276 774N TCTCCTCAATGTGCATGT
-51- 277. ATTCTACATA 278. ASMI14 GTGTTTGCAT 279, ACAAGTTGGC 280, CA11 11 TAGTACCAGA 281 TACATCCAAGAAAA
The source of marker was Sequana Therapeutics, Inc. unless a letter in parenthesis is indicated after the name, where G = Genethon; L = Nothen and Dewald (1995) Clin. Genet. 47:165; U = the Utah genome center, see: The Utah Marker Development Group (1995) Am. J. Hum. Genet. 57:619; c= the cooperative Human Lineage Center.
Table IB
SEQ Marker Repeat and flanking sequence
282. CA39_2 GAGACTCTGA (CA) nAATATATATA
283. 774F TGTTGATCGC (CA) nAACCAAAATC
284. 774J AATGCATGTA (TG) 2TATA (TG) nGTGTGGTATG (TG) 3TACATATG CG
285. 7740 CCTCCCAGAA(CA)n ATCATGATAA
286. LI9PENT AGACAGTCTCAAAAAAT (ATTTT) nAAAGAAAAAGCTGGATAAAT
Al
287. 65P14TE AACTAGCTTTAAGAAAATAAGAAGAAAAAGAAAGAAG (AAAG) 2TAA
1 G (AAAG) nAGAAAGAAAAG (AAAG) nAAAAG (AAAG) nAGGAATGAT TGAC
288. 65P14 CGCGCACATA ( CA) nCCCTTTCTCT
289. 774L CAGTAAACAT (CA) n TGTTGAGTGG
290. 774N TCTCCTCAATGTGCATGT (GTGC) 2 ATGA (GTGC) 2 (AC) n ATTCTACATA
291. ASMI14 GTGTTTGCAT (GT)n T (GT) 3 ACAAGTTGGC 292. CA11 11 TAGTACCAGA (CA)2 CG(TG)2 (CA) 2 GGCAAGCG (CA)n C (CA)3 TACATCCAAGAAAA
Genetic refinement of the ASTH1 region
The microsatellite markers isolated from YACs from the ASTH1 region were genotyped in both the Tristan da Cunha and Toronto cohorts. Genetic refinement of the ASTH1 region was accomplished by applying the transmission/disequilibrium test (TDT; Spielman et al. (1993) Am. J. Hum. Genet. 52:506) to genetic data from the Tristan and Toronto populations, at markers throughout the ASTH1 region. The TDT statistic reflects the level of association between a marker allele and disease
-52- status. A multipoint version of the TDT test controls for variability in heterozygosities between loci, and results in a smoother regional TDT curve than would a plot of single locus TDT data. Significance of a TDT value is determined by means of the χ2 test; A χ2 value of 3.84 or greater is considered statistically significant at a probability level of 0.05. Figure 1 shows graphs of χ2 values for key ASTH1 region markers for both history of asthma with positive methacholine challenge, for the Toronto triad families, χ2 is plotted vs. genomic location of the marker on the physical map.
The Toronto TDT peak is located at marker D11S2008 (χ2= 11.6, p < .0001). The marker allele in disequilibrium is fairly rare (freq = 6%), representing the fourth most common allele at this marker. The relative risk of affection vs. normal for this allele is 5.25. This is also the peak marker for linkage and linkage disequilibrium in Tristan da Cunha, indicating that the ASTH1 gene is very close to this marker. The markers defining the limits of linkage disequilibrium were D11 S907 and 65P14TE1. The physical size of the refined region is approximately 100 kb.
A significant TDT test reflects the tendency of alleles of markers located near a disease locus (also said to be in "linkage disequilibrium" with the disease) to segregate with the disease locus, while alleles of markers located further from the disease locus segregate independently of affection status. An expectation that derives from this is that a population for which a disease gene (ie a disease predisposing polymorphism) was recently introduced would show statistically significant TDT over a larger region surrounding the gene than would a population in which the mutant gene had been segregating for a greater length of time. In the latter case, time would have allowed more opportunity for markers in the vicinity of the disease gene to recombine with it. This expectation is fulfilled in our populations. The Tristan da Cunha population, founded only 10 generations ago, shows a broader TDT curve than does the set of Toronto families, which are mixed European in derivation and thus represent an older and more diverse, less recently established population.
-53- Gene isolation and characterization
The tiling path of BACs, cosmids and PAC clones was subjected to exon trapping and cDNA selection to isolate sequences derived from ASTH1 region genes. Exon trap clones were isolated on the basis of size and ability to cross- hybridize. Approximately 300 putatively non-identical clones were sequenced. cDNA selection was performed with adult and fetal lung RNA using pools of tiling path clones. The cDNA selection clones were sequenced and the sequences assembled with those of the exon trap clones. Representative exon trapping clones spanning each assembly were chosen, and arranged as "masterplates" (96- well microtitre dishes) of clones. Exon trap masterplate clones and cDNA selection clones were subjected to expression studies.
Human multi-tissue Northern blots were probed with PCR products of masterplate clones. In some cases, exon trapping clones did not detect RNA species, either because they did not represent expressed sequences, or represented genes with very restricted patterns of expression, or due to small size of the exon probe.
Masterplate clones detecting discrete RNA species on Northern blots were used to screen lambda phage based cDNA libraries chosen on the basis of the expression pattern of the clone. The sequences of the cDNAs were determined by end sequencing and sequence walking. cDNAs were also isolated, or extended, by 5' and 3' rapid amplification of cDNA ends (RACE). In most cases, 5' RACE was necessary to obtain the 5' end of the cDNA.
ASTH1 I and ASTH1J were detected by exon trapping. ASTH1 I exons detected a 2.8 kb mRNA expressed at high levels in trachea and prostate, and at lower levels in lung and kidney. ASTH1 I exons were used as probes to screen prostate, lung and testis cDNA libraries; positive clones were obtained from each of these libraries. Isolation of a ASTH11 cDNA clone from testis demonstrates that this gene is expressed in this tissue, and possibly others, at a level not detectable by Northern blot analysis. ASTH1 J exons detected a 6.0 kb mRNA expressed at high levels in the trachea, prostate and pancreas and at lower levels in colon, small intestine, lung and stomach. Pancreas and prostate libraries were screened with exon clones
-54- from ASTH1 J. cDNA clone end sequences were assembled using Sequencher (Alsbyte) with the sequences of the exon trapped clones, producing sequence contigs used to design sequence walking and RACE primers. The additional sequences produced by these methods were assembled with the original sequences to produce longer contigs of cDNA sequences. It was evident from the sequence assemblies that both ASTH1 I and ASTH1J are alternatively spliced and/or have alternative transcription start sites at their 5' ends, since not all clones of either gene contained the same 5' sequence.
ASTH1 J has three splice forms consisting of the altl form, found in prostate and lung cDNA clones, and in which the exons (illustrated in Figure 1) are found in the order: 5' a, b, c, d, e, f, g, h, i 3'. A second form, alt2, in which the exon order is: 5' a2, b, c, d, e, f, g, h, i 3' was seen in a pancreas cDNA clone. A third form, alt3, contains an alternate exon, a3, between exons a2 and b. The start codon is within exon b, so that the open reading frame is identical for the three forms, which differ only in the 5' UTR. The ASTH1J cDNAs shown as SEQ ID NO:2 (form altl); SEQ ID NO:3 (form alt2); SEQ ID NO:4 (form alt3) are 5427, 5510 and 5667 bp in length, respectively. The sequence of the entire protein coding region and alternate 5' UTRs are provided. The 3' terminus, where the polyA tail is added, varies by 7 bp between clones. The provided sequences are the longest of these variants. The encoded protein product is provided as SEQ ID NO:5.
ASTH1 I was seen in three isoforms denoted as altl , alt2, and alt3. The exons of ASTH1 I and ASTH1J were given letter designations before the directionality of the cDNA was known, the order is different for the two genes. In the altl form of ASTH1 I, exons are in the following order: 5' i, f, e, d, c, b, a 3'. In the alt2 form of ASTH11, an alternative 5' exon, j, substitutes for exon i, with the following exon arrangement: 5' j, f, e, d, c, b, a 3'. The alt3 form of the gene has the exon order: 5' f, k, h, g, e, d, c, b, a 3'. The alternative splicing and start codons in each of exons i, f and e give the three forms of ASTH11 protein different amino termini. The common stop codon is located in exon a, which also contains a long 3' UTR. Two polyadenylation signals are present in the 3' UTR; some cDNA clones end with a polyA tract just after the first polyA signal and for others the polyA tract is at the end of the sequence shown. Since the sequences shown for the altl ,
-55- alt2, and alt3 forms of ASTH1 I (2428 bp; 2280 bp and 2498 bp; respectively) are close to the estimated Northern blot transcript size of 2.8 kb, these sequences are essentially full length.
EST matches
The nucleotide sequences of the altl , alt2 and alt3 forms of ASTH1J and the altl , alt2 and alt3 forms of ASTH1 I were used in BLAST searches against dbEST in order to identify EST sequences representing these genes. Perfect or near perfect matches were taken to represent sequence identity rather than relatedness. Accession numbers T65960, T64537, AA055924 and AA055327 represent the forward and reverse sequences of two clones which together span the last 546 bp (excluding the polyA tail) of the 3' UTR of ASTH1 I. No ESTs spanned any part of the coding region of this gene. One colon cDNA clone (accession number AA149006) spanned 402 bp including the last 21 bp of the ASTH1 J coding region and part of the 3' UTR.
Intronlexon structure determination
The genomic organization of genes in the ASTH1 region was determined by comparison by BLAST of cDNA sequences to the genomic sequence of the region. The genomic sequence of the ASHT1 region 5' to and overlapping ASTH1 J, is provided in SEQ ID NO:1. Genomic structure of the ASTH1 I and ASTH1J genes is shown in Figure 1; the intron/exon junction sequences are in Table 2.
TABLE 2: Genomic organization of the ASTHl I and ASTHIJ genes. *Exonic sequences are upper case, flanking sequences lower case.
SEQ NO Exon Size of Sequences at the ends of and exon flanking the exons of ASTHlI and (bp) ASTHIJ*
ASTHlI
293. i >214 ggaggctgagCAGGGGTGCC...
294. ...ACTCCCACAGgtacctgcag 295. j >66 ...CTGCCCTCACgtaagcgcct
-56- 296. f 125 gctgttgcagGGTAATGTTG...
297. ...CATCAGACAGgtgcgtaca
298. k 226 ggctggtgagGAGGGGCTGA... 299. ... CGCTCTGTGGgtgagcttca 300. h 93 tgtggaatagCCCAATTACA...
301. ...AGGGTGCTGAgtgagtagta
302. g 79 ttcttttcagGCCCTCGTGT...
303. ...TGCTGACCCGgtatggtggt
304. e 232 tttggtgcagCCTGTGACTC... 305. ...CGCACACAAGgtcagtgttc
306. d 51 tctttcccagGTTACTCCTT...
307. ...ATCAAAGACTgtaagtaacc
308. c 69 tctatttcagATGCTGATTC...
309. ...AGTAGAACAAgtaagtgcag 310. b 196 ttttcaaaagGCCTCCAAAG...
311. ...GAGCCCTGAGgtaagttaat
312. a 1522 gctttttcagATACTACTAT...
313. ...TAACATGTTCaactgtctgt
314. a 146 tgttatatgcATTTATCTTC... 315. ...GGTAAATGAGgtaagtcctg
316. a2 229 tcttgttaagATCGCTCTCT...
317. ...CCTTGCCCAGgttctcttaa
318. a3 157 gcaatcgcacCTGCACACCC...
319. ...ACTGCCCATTtctggtaaag 320. b 100 cccctaacagATCATGATTC...
321. ...ACGTGCAATGgtaagagggc
322. c 246 tgttttgcagTTTCCAGTGG...
323. ...AAGTGGAACGgtgactctct
324. d 63 tccttcacagGCCAGTGCAG...
-57- 325. ... GAACAAACTGgtg agtagta
326. e 69 ttttttgtagAGCCTTCCAT...
327. ...AGCACAGTAGgtaactaact
328. f 69 atggccacagATTTGTTGGA... 329. ...CTTCCTGTTGgtaagctgtc
330. g 63 ttctccttagCAGAGTCACC...
331. ...AAAAAGCACAgtaagttggc
332. h 196 ttttcatcagACCCGAGAGG...
333. ...GAGCTATGAGgtgaggagtt 334. i 4457 tttgttacagATATTACTAC ...
335. ...AGCCTGGAAAtgcgtgtttc
The deduced ASTH1I and ASTH1J proteins
The protein encoded by ASTH1J (SEQ ID NO:5) is 300 amino acids in length. A BLASTP search of the protein sequence against the public nonredundant sequence database (NCBI) revealed similarity to one protein domain of transcription factors of the ets family. The ets family, named for the E26 oncoprotein which originally defined this type of transcription factor, is a group of transcription factors which activate genes involved in a variety of immunological and other processes, or implicated in cancer. The family members most similar to ASTH11 and ASTH1 J are: ETS1 , ESX, ETS2, ELF, ELK1 , TEL, NET, SAP-1 , NERF and FLI. Secondary structure analysis and comparison of the protein sequence to the crystal structure of the human ETS1-DNA complex (Werner et al. (1995) Cell 83:761) confirmed that it has a winged helix turn helix motif characteristic of some DNA binding proteins which are transcription factors.
Multiple sequence alignment of ASTH1 I, ASTH1J, and other ETS-domain proteins detected a second, N-terminal domain shared by ASTH11, ASTH1 J and some, but not all, ETS-domain proteins. Conservation of this motif have been observed (Tei et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6856-6860), and its involvement in protein self-association have been documented for TEL, an ETS- domain protein, upon its fusion with platelet-derived growth factor β receptor (Carrol
-58- et al. (1996) Proc. Natl. Acad. Sci. USA 93:14845-14850). Alignment of the N- terminal conserved domain in the ETS proteins was converted into a generalized sequence profile to scan the protein databases using the Smith-Waterman algorithm. This search revealed that the N-terminal domain in ASTH1 I, ASTH1J and other ETS-domain proteins belongs to the SAM-domain family (Schultz et al. (1997) Protein Science 6:249-253). SAM domains are found in diverse developmental proteins where they are thought to mediate protein-protein interactions. Thus, both ASTH1 I and ASTH1J are predicted to contain two conserved modules, the N- terminal protein interaction domain (SAM-domain) and the C-terminal DNA-binding domain (ETS-domain). The sequence segments between these two domains is predicted to have elongated, non-globular structure and may be hinges between the two functional domains in ASTH1 I and ASTH1J.
The ASTH1 I altl (SEQ ID NO:7), alt2 (SEQ ID NO:9) and alt3 (SEQ ID NO:11) forms are 265, 255 and 164 amino acids in length, respectively, and differ at their 5' ends. The ASTH1 I and ASTH1J proteins show similarity to each other in the ets domain and between ASTH1J exon c and ASTH1 I exon e. They are more related to each other than to other proteins. Over the ets domain they are 66% similar (ie. have amino acids with similar properties in the same positions) and 46% identical to each other. All three forms of ASTH11 have the helix turn helix motif located near the carboxy terminal end of the protein.
The alternate forms of the ASTH1 I protein may differ in function in critical ways. The activity of ets transcription factors can be affected by the presence of independently folding protein structural motifs which interact with the ets protein binding domain (helix loop helix). The differing 5' ends of the ASTH1 I proteins may help modulate activity of the proteins in a tissue-specific manner.
Polymorphism analysis of ASTH1I and ASTH1J
Affected and unaffected individuals from the Toronto cohort were used to determine sequence variants, as were approximately 25 controls derived from populations not selected for asthma. Affected and unaffected individuals from the Tristan da Cunha population were also chosen; the set to be assayed was also selected to represent all the major haplotypes for the ASTH1 region in that
-59- population. This ensured that all chromosome types for Tristan were included in the analysis.
Polymorphism analysis was accomplished by three techniques: comparative (heterozygote detection) sequencing, radioactive SSCP and fluorescent SSCP. Polymorphisms found by SSCP were sequenced to determine the exact sequence change involved.
PCR and sequencing primers were designed from genomic sequence flanking each exon of the coding region and 5' UTRs of ASTH1 I and ASTH1J. For fluorescent SSCP, the forward and reverse PCR primers were labeled with different dyes to allow visualization of both strands of the PCR product. In general, a variant seen in one strand of the product was also apparent in the other strand. For comparative sequencing, heterozygotes were also detected in sequences from both DNA strands.
Polymorphisms associated with the ASTH11 locus are listed in Table 3. The sequence flanking each variant is shown. Polymorphisms were also deduced from comparison of sequences from multiple independent cDNA clones spanning the same region of the transcripts, and comparison with genomic DNA sequence. The polymorphisms in the long 3' UTR regions of these genes were found by this method. One polymorphism in each gene is associated with an amino acid change in the protein sequence. An alanine/valine difference in exon c of ASTH1 J is a conservative amino acid change. A serine/cysteine variant in exon g of ASTH1 I is not a conservative change, but would be found only in the alt3 form of the protein.
The polymorphisms in the ASTH1 I and J transcribed regions were genotyped in the whole Tristan da Cunha and Toronto populations, as well as in a larger sample of non-asthma selected controls, by high throughput methods such as OLA (oligonucleotide ligation assay; Tobe et al. (1996) Nucl. Acids Res. 24:3728) or Taqman (Holland et al. (1992) Clin. Chem. 38: 462), or by PCR and restriction enzyme digestion. The population-wide data were used in a statistical analysis for significant differences in the frequencies of ASTH1 I or ASTH1J alleles between asthmatics and non-asthmatics.
-60- TABLE 3: POLYMORPHISMS IN THE ASTHII AND ASTHIJ GENES.
Polymorphism Location Sequence
SEQ ASTHII Transcribed region
16. EXON B (+) 170 ACAGAATGACRTATGAAAAGT 17. INTRON D (+) 15 GTAACCAAGCKCAAGCCACCC
18. INTRON F (+) 24 AAGGAGCCCAYCTGAGTGCAG
19. EXON G ( +)62 ser→-cys CGTTCCATCTSTGCTCTGTGC
20. EXON H (+) 77 AGCGCCTCGGYTGGCTGAGGG
21. EXON A 3' UTR (+) 1176 TGTATTCAAGYGCTATAACAC 22. EXON I (+) 76 CACTGAGAAGCCC nACAGGCCTGT
23. EXON I (+) 86 CCCACAGGCCWGTCCCTCCAA
24. INTRON J (+) 93 CGTCCATCTCYAGCTCCAGGG
ASTHIJ Transcribed region
25. EXON A 5' UTR (+)38 GACTTGATAAYGCCCGTGGTG 26. EXON A 5' UTR (+)39 ACTTGATAACRCCCGTGGTGC
27. EXON A 5' UTR (+) 99 CTCCCCTCCAWGAGCCACAGC
28. INTRON A (+) 224/225 ATTTCCTGCATT/^GTCTGGACTT
29. INTRON A (+)48 ATCCAAACACYTGAGTGGAAA
30. EXON A3 (+)28 AGTTTCCTCARTGCGGGAGCT 31. EXON C (+) 158 GCGAGCACCTYTGCAGCATGA
32. EXON C ( +)190 ala→-val TTCACCCGGGYGGCAGGGACG
33. INTRON D (-) 36/37 CTGGGGAAAA(GA) /TGATCGCTGAC
34. INTRON F (-) 22 GTCAATTAAAYGGCTCTCATT
35. INTRON G (-)27 TAGATCATTCRTAACCTGCCT 36. EXON I (3' UTR) (+)22 AAAGAGAAATWCTGGAGCGTG
37. EXON I (3' UTR) (+)220 ATGAGGGGAAMAAGAAACTAC
38. EXON I (3' UTR) (+)475 TTTTGTATGTKACATGATTTA
39. EXON I (3' UTR) (+) 871 AGCTTGGTTCYTTTTTGCTCC
40. EXON I (3' UTR) (+) 1084 TTGACACCAGRAACCCCCCAG 5' to ASTHIJ
41. CAAT box -165 AAATGAGCCARTGTTTGTAAT
-61- 42. 5PWlJ_P01+399 ATCCATTTTGYATTCCTCATT
43. 5PW1J_P01+1604 CTGGAGCTCARACCAGACAGC
44. 5PW1J_P02+1382 GCCAGTGCAGSCATCATTACC
45. 5P 1J_P03+128 AGTTCAAATCRTAATTTTTAT
46. 5PWlJ_P03+556 TCATCAGAATYTAAATCTCCC
47. 5P 1J_P03+712 GGAGATTCAGA/^TGAAGCAAGA
48. 5PW1J_P03+781 TTTTTCCACAYCCAGCCTGGC
49. 5P 1J_P03+791 CCCAGCCTGGYGAACCCTGGC
50. 5P lJ_P03+820 CTCTTCATCAYGGTCAAATAC
51. 5P 1J_P03+1530 CAACTTGCTGYCAAAGTGCTG
52. 5P 1J_P03+1605 TACTATGTGCYAGATACTAAG
53. 5P 1J_P04+542/543 ATGCCACTTTRRACAACTTGAG
54. 5P lJ_P04+973 CGCATGCCTGKAAAGAAGAGA
55. 5P 1J_P04+1079 GGATAAGCACMAGTGAGCCTG
56. 5PW1J_P04+1153 AAAGCCAGACRGCAACTTGTG
57. 5PW1J_P04+1430 TCTCAAAAAGRGTGATAGGAG
58. 5PWlJ_P05+334 TCTGAATCCTSTCTCCTCCTT
59. 5PWlJ_P05+749 TAGAACCAGGWTGTGGGACCA
60. 5PW1J_P05+915 TTCTTGTGTCRGGCGCAAAAC
61. 5PWlJ_P06+529 AACCAACATGRAGAAACCCCA
62. 5PW1J_P06+1290 AATAAACTATRGTTCACCTAG
63. 5PW1J_P06+1573 ACATATTTGTRTCTCATATGA
64. 5PW1J_P06+1661 CAAAGCAGTTYCTAATAATCC
65. 5PWlJ_P07+335 AGATCCTAACYGGGGCCTCCT
66. 5PW1J_P07+731 CTCTTTCTCTYTGCTTCCTCC
67. 5P 1J_P07+1024 TTAGGAATCCWCAAATATGTA
68. 5P 1J_P07+1610 GTCTGACTCCRCCTCCCTCAT
69. 5PWlJ_P08+398 GAATCACATCRTGAGAAATGT
70. 5PWU_P08+439 AATTCAATCCYTCACAGACTT
71. 5P lJ_P08+580 GTGTAGCCAGRGTTGCTAATT
72. 5P lJ_P08+762 CCTAGAAATASCCAAGGGCAC
73. 5PWlJ_P08+952 AAATTCTCATRCCTCACCCTC
74. 5PW1J_P08+1172 TCCCACCCCTRTCACCTTCAT
75. 5PW1J_P08+1393 CCTCATTCTCRGAAGCCAACA
76. 5PW1J_P08+1433 GAAGAGCCGTYCAGTCCCTTT
77. 5PW1J_P08+1670 TCCATAGGCTYTTTATTTGGC
78. 5P 1J_P08+1730 TCGTTTAGTAYACAGGCTTTG
79. 5P lJ_P09+59 GCCTCAGTTGYCCCAGCTATA
80. 5P 1J_P09+145 AGCAAAATGCWCTATGCACTG
81. 5P lJ_P09+892 GTGTCCTGAC (TTGCACTCCAC) /-
ACACTGCCTG
82. 5P 1J_P10+1070 ATCAGATAACRCCTACACTTA
83. 5P 1J_P10+1511 TCTCTCTTCTSCCTGCCCTGT 84. 5PW1J P09+1132 TGGACACAGGKAGGGGAATAT
-62- 85. 5PW1J_P09+1688 TGTCACTTGCRCATACAAGGC
86. 5P 1J_P09+1900 ATCATCAGATYAGCCCAGAAT
87. 5PW1J W1R1-1060 TCAACAGAGARAGTTAATGGT
88. 5P 1J 1R1-1831 AGCAATAATGYTTCCCTTTTC 89. 5PW1J W1R1-2355 TCTAGCTTTTYTGTGTTTTTT
90. 5P 1J W1R1-3160 GATTCCTTAAYGCTTGATACT
91. 5PW1J 1R1-3787 CCTCCTCCAGYACCAAAGTGG
92. WlJ_CD+24 ATGGCCACAGRTCAAATCCTG 93. WlJ_CA+564 ACTGAGTGTTYATGCCAATTT 5' to ASTHII
94. WI_CL+94 GACAAGCCCTETCTGACACAC
95. WI_CN+134 TGAAAAGCCTYCTTGCTGCCT
96. I_CQ-28 TCCTGGAGTTYCTTTGCTCCC
97. WI_CQ+39 GATTCCAAAT AACTAAAGAT 98. P14-16+191662 GACCTCAAGTCRTCCACCCGCC
99. P14-16+192592 AACAAATACTMCCCCGCAACCC
100. P14-16+192762 ATTTTTTTTTT/ -AAGGAAAATA
101. P14-16+195066 AAATTTCCCCMAAACAAGCAG
102. P14-16+196590 GAGAAAGGGTRTGTGTGTGTG 103. P14-16+196617 GTGTGTGTGTGT- /GTOTATGTGCGCGTG
104. P14-16+196902 ATCGGGAACCYCATACCCCAA
105. P14-16+198040 TTTGTTTCGCMATGAGGTACG
106. P14-16+198240 TGAGGGTGTTSTGGGCTGGAC
107. P14-16+198840 TCTTCATTGGYATCTGAATGT 108. P14-16+200120 GCGAGCACCTYTGCAGCATGA
109. P14-16+200617 AACCCCCCCCMCACACACACA
110. J5-16+4454 TCAGTGCTCTSTAATCAGTCA
111. J5-16+4825 TCTTTGTGAAA-/ (GAlAATTAGTCTG*
112. J5-16+5426 GCTGCCCTGASAGCTGGGCCA 113. J5-16+5623 CCTTCTGATCYTTGTTTGCTG
114. J5-16+7386 GGAACACTGAKTCTTGATTAG
115. J5-16+7904 TAGGCTTCTCYTGATAATTGA
116. J5-16+8055 TCTTAAAATAMTTGGCTTGTA
117. J5-16+10595 TAGATCATTARTAACCTGCCT 118. J5-16+11140 ATGAGGGGAAMAAGAAACTAC
119. J5-16+12004 TTGACACCAGRAACCCCCCAG
120. J5-16+12219 TGTTTTAAATRTTAGGGACAA
121. J5-16+12303 GTAAGCATAGYAATGTAGCAG
122. J5-16+13504 GGCTCTTTCTKCAACCTTTCC 123. J5-16+14120 GACCCAGGTTRTGAGTTTTCC
124. ASTHII, exon B +169 GACAGAATGAYATATGAAAAG
125. ASTHII, exon I +69 TGTGTGACACYGAGAAGCCCA
-63- 126. ASTHIJ, exon C +56 AGTACTGGACMAAGTACCAGG
127. 5' ASTHIJ, WI_Cg -9 CCTGGGAGCARGTATTGCATT
ASTHIJ Intron A
128. WIJ_Ia01 +39 AGATTTGAGGYCTCAGGTCCC
129. WIJ_Ia01 +140 TGTCAATGTCRCATGATAAGC
130. WIJ_Ia01 +678 TTGCCCCAGTKTTCTCCGGGC
131. WIJ_Ia01 +855 TATGAGCAGCRTAGGGAGTGG
132. WIJ_Ia01 +929 AGTTGACTGA (AAAA) /-TAAATAAGAC
133. WIJ__Ia 03 +362 ATTCAAATAGSCTCTAGAAAC 1 13344.. W WIIJJ__IIaa 0 033 + +991188 CCCAGAATTTMATATCCATTC
135. WIJ_Ia 03 +943 TGACCCAACARAAACTCACTG
136. WIJ_Ia 03 +1569 CCAGAATATAWCATCAGCCCT
137. WIJ_Ia 03 +1580 CATCAGCCCTWCTGAGGAGAT
138. WIJ __Ia 02 +435 CCAGAACAGAYTTTATTCTGT 1 13399.. W WIIJJ_lIaa 0 022 + +558833 TTCAGCCATCYTTCCAGTTGT
140. WIJ_Ia 02 +643 TCACTAACTCWAAAACGACAT
141. WIJ_Ia 02 +648 AACTCAAAAAYGACATCCTCC
142. WIJ_Ia 02 +1048 GAACTGCACARGTTGCACACT
143. WIJ __Ia 02 +1061 TTGTTCCATGSACTACCTCCT 1 14444.. W WIIJJ_lIaa 0 022 + +11114422 ACAGCAGGCAYTCAACAAATT
145. WIJ_Ia 04 +410 TTATTTTTGGOTTTGTTTTAA
146. WIJ_Ia 04 +1056 TAGGCTGTTCYCTGCCATCAC
147. WIJ_Ia 05 +1484 GTGCTCTGGGMCACACAGCTC
148. WIJ __Ia 05 +1103 AGACCCGATARGAGCTCCTTC 1 14499.. W WIIJJ_lIaa 0 055 + +11882233 CATCTTGCGCRGTCATGTAAG
150. WIJ_Ia 05 +1852 CAGCACAGCTRTTCCCTCAAA
151. WIJ_Ia 05 +1906 TTTGGAAACAYGGTGAAGTAT
152. WIJ_Ia 05 +1913 ACACGGTGAARTATTGTCTCC
153. WIJ __Ia 06 +794 AAAAGTGGATMCTCTGCAAAC 1 15544.. W WIIJJ_lIaa 0 066 + +881144 CTTCAAATGCRGCTATTAAAG
155. WIJ_Ia 06 +1197 CCTGGGAGCAYGGTAAATCAG
156. WIJ_Ia 06 +1231 TGAAAATGTCRCTTTCTCACCT
157. WIJ_Ia 06 +1256 CCTGATATTTRCCAACAAGAA
158. WIJ__Ia 06 +1535 AAAGGGTTAGYTTGTCCCCTT 1 15599.. W WII_CCaaaa + +116633 TGAAAATAAAASACAATTTTTT
The sequences are listed with the variant residues represented by the appropriate single letter designation, i.e. A or G is shown by "R". The variant residues are underlined. Where the polymorphism is a deletion, the underlined residues are underlined, and the alternative form shown as a "-". aWhere intron 'a' is the intron 3' to exon 'a', etc. bPosition numbers correspond to the position within the intron or exon, with nucleotide +1 being the 5'-most base of the exon or the intron. Alternatively, negative numbers denote the number of bases from the 3' end of an intron. cPosition in cDNA = position # for the exon a form of ASTH1 J or the exon i form of ASTH11.
-64- dExonic sequences are uppercase, intronic sequences lower case. UTR = untranslated region. N/A = not applicable.
Cross-species sequence conservation Cross-species sequence conservation can reveal the presence of functionally important areas of sequence within a larger region. Approximately 90 kb of sequence lie between ASTH1 I and ASTH1 J, which are transcribed in opposite directions (Figure 1). The transcriptional orientation of these genes may allow coordinate regulation of their expression. The expression patterns of these genes are similar but not identical. Sequences found 5' to genes are critical for expression. To search for regulatory or other important regions, the genomic sequence between ASTH1 I and ASTH1J, was examined and plasmid clones derived from genomic sequencing experiments chosen for cross-species hybridization experiments. The criterion for probe choice was a lack of repeat elements such as Alu or LINEs. Inserts from these clones were used as probes on Southern blots of EcoRI-digested human, mouse and pig or cow genomic DNA. Probes that produced discrete bands in more than one species were considered conserved.
Conserved probes clustered in four locations. One region was located 5' to ASTH11 and spanned exon j of this gene. A second conserved region was located 5' to ASTH1 IJ, spanning approximately 10 kb and beginning 6 kb 5' to ASTH1J exon a (and is within SEQ ID NO:1). Two other clusters of conserved probes were noted in the region between ASTH1 I and J. They are approximately 10 and 6 kb in length. Promoters, enhancers and other important control regions are generally found near the 5' ends of genes or within introns. Methods of identifying and characterizing such regions include: luciferase assays, chloramphenicol acetyl transferase (CAT) assays, gel shift assays, DNAsel protection assays (footprinting), methylation interference assays, DNAsel hypersensitivity assays to detect functionally relevant chromatin-ree regions, other types of chemical protection assays, transgenic mice with putative promoter regions linked to a reporter gene such as β-galactosidase, etc. Such studies define the promoters and other critical
-65- control regions of ASTH1 I and ASTH1J and establish the functional significance of the evolutionarily conserved sequences between these genes.
Discussion The ASTH1 locus is associated with asthma and bronchial hyperreactivity.
ASTH1 I and ASTH1J are transcription factors expressed in trachea, lung and several other tissues. The main site of their effect upon asthma may therefore be in trachea and lung tissues. Since ets family genes are transcription factors, a function for ASTH1 I and ASTH1J is activation of transcription of particular sets of genes within cells of the trachea and lung. Cytokines are extracellular signalling proteins important in inflammation, a common feature of asthma. Several ets family transcription factors activate expression of cytokines or cytokine receptors in response to their own activation by upstream signals. ELF, for example, activates IL-2, IL-3, IL-2 receptor α and GM-CSF, factors involved in signaling between cell types important in asthma. NET activates transcription of the IL-1 receptor antagonist gene. ETS1 activates the T cell receptor α gene, which has been linked to atopic asthma in some families (Moffatt et al. (1994) supra.)
Activation of genes involved in inflammation by other members of the ets family suggest that the effect of these ASTH1 genes on development of asthma is exerted through influencing cytokine or receptor expression in trachea and/or lung. Cytokines are produced by structural cells within the airway, including epithelial cells, endothelial cells and fibroblasts, bringing about recruitment of inflammatory cells into the airway.
A model for the role of ASTH11 and ASTH1 J in asthma that is consistent with the phenotype linked to ASTH1 , the expression pattern of these genes, the nature of the ASTH1 l/J genes, and the known function of similar genes is that aberrant function of ASTH1 I and/or ASTH1J in trachea or lung leads to altered expression of factors involved in the inflammatory process, leading to chronic inflammation and asthma.
-66- Functional analysis of a ASTH1 J promoter sequence variant and location of the ASTH1J promoter
Primer extension analyses performed using total RNA isolated from both bronchial and prostate epithelial cells have revealed one major and five minor transcription start sites for ASTH1 J. The major site accounts for more than 90% of ASTH1 J gene transcriptional initiation. None of these sites are found when the primer extension analysis is performed using mRNA isolated from human lung fibroblasts that do not express ASTH1 J.
Identification of the ASTH1 J transcriptional start site has allowed the localization of a putative TATA box (TTTAAAA) between positions -24 and -30 (24 to 30 bp 5' to the transcription start site). Although the sequence is not that of a typical TATA box, it conforms to the consensus sequence (TATAAAA) for TATA box protein binding as compared with 389 TATA elements (Transfac database: http://transfac.gbf-braunschweig.de/, ID: V$TATA_01).
Analysis of the CAAT box "G" polymorphism bv αel shift assay
Binding of nuclear proteins to a polymorphism in the GCCAAT motif (GCCAAT or GCCAGT) found at position -140 (140 bp 5' to the transcription start of ASTH1 J as defined by primer extension experiments, previously referred to as "-165 bp"), has been assessed using electrophoretic mobility shift assays. These experiments clearly showed a remarkable difference when binding of nuclear proteins to radioactively-labelled double stranded oligonucleotides containing the normal "A" vs the mutant "G" nucleotide was examined. A specific set of nuclear proteins was able to bind to the normal oligonucleotide, but did not bind to the "G" oligonucleotide. The specificity of the DNA binding complexes was further addressed by competition with either normal or mutant unlabeled oligonucleotides. Addition of increasing amounts of normal unlabeled oligonucleotide effectively competed binding of nuclear proteins to the labeled normal oligonucleotide, while the addition of increasing amounts of unlabelled "G" oligonucleotide did not. The GCCAAT cis-element is found in many promoters at various locations relative to genes, as well as in distal enhancer elements. There is no known correlation between location of these elements and activity. Both positive and
-67- negative regulatory trans-acting factors are known to bind this class of cis element. These factors can be grouped into the NF-1 and C/EBP families.
The nuclear factor-1 (NF-1) family of transcription factors comprises a large group of eukaryotic DNA binding proteins. Diversity within this gene family is contributed by multiple genes (including: NF-1 A, NF-1 B, NF-1C and NF-1X), differential splicing and heterodimerization.
Transcription factor C/EBP (CCAAT-enhancer binding protein) is a heat stable, sequence-specific DNA binding protein first purified from rat liver nuclei. C/EBP binds DNA through a bipartite structural motif and appears to function exclusively in terminally differentiated, growth arrested cells. C/EBPα was originally described as NF-IL-6; it is induced by IL-6 in liver, where it is the major C/EBP binding component. Three more recently described members of this gene family, designated CRP 1 , C/EBP β and C/EBP δ, exhibit similar DNA binding specificities and affinities to C/EBP α. Furthermore, C/EBP β and C/EBP δ readily form heterodimers with each other as well as with C/EBP α.
Members of the C/EBP family of transcription factors, but not members of the NF-1 family, bind to the ASTH1 J promoter region, as determined by the use of commercially available antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA) that recognize all NF-1 and C/EBP family members known to date, in electrophoretic mobility shift assays.
Fabricating a DNA array of polymorphic sequences
DNA array: is made by spotting DNA fragments onto glass microscope slides which are pretreated with poly-L-lysine. Spotting onto the array is accomplished by a robotic arrayer. The DNA is cross-linked to the glass by ultraviolet irradiation, and the free poly-L-lysine groups are blocked by treatment with 0.05% succinic anhydride, 50% 1-methyl-2-pyrrolidinone and 50% borate buffer.
The spots on the array are oligonucleotides synthesized on an ABI automated synthesizer. Each spot is one of the alternative polymorphic sequences indicated in Tables 3 to 8. For each pair of polymorphisms, both forms are included. Subsets include (1) the ASTH1J polymorphisms of Table 3, (2) the
-68- ASTH1I polymorphisms of Table 3; and (3) the polymorphisms of Table 4. Some internal standards and negative control spots including non-polymorphic coding region sequences and bacterial controls are included.
Genomic DNA from patient samples is isolated, amplified and subsequently labeled with fluorescent nucleotides as follows: isolated DNA is added to a standard PCR reaction containing primers (100 pmoles each), 250uM nucleotides, and 5 Units of Taq polymerase (Perkin Elmer). In addition, fluorescent nucleotides (Cy3-dUTP (green fluorescence) or Cy5-dUTP (red fluorescence), sold by Amersham) are added to a final concentration of 60 uM. The reaction is carried out in a Perkin Elmer thermocycler (PE9600) for 30 cycles using the following cycle profile: 92°C for 30 seconds, 58°C for 30 seconds, and 72°C for 2 minutes. Unincorporated fluorescent nucleotides are removed by size exclusion chromatography (Microcon-30 concentration devices, sold by Amicon).
Buffer replacement, removal of small nucleotides and primers and sample concentration is accomplished by ultrafiltration over an Amicon microconcentrator- 30 (mwco = 30,000 Da) with three changes of 0.45 ml TE. The sample is reduced to 5 μl and supplemented with 1.4 μl 20X SSC and 5 μg yeast tRNA. Particles are removed from this mixture by filtration through a pre-wetted 0.45μ microspin filter (Ultrafree-MC, Millipore, Bedford, Ma.). SDS is added to a 0.28% final concentration. The fluorescently-labeled cDNA mixture is then heated to 98°C for 2 min., quickly cooled and applied to the DNA array on a microscope slide. Hybridization proceeds under a coverslip, and the slide assembly is kept in a humidified chamber at 65°C for 15 hours.
The slide is washed briefly in 1X SSC and 0.03% SDS, followed by a wash in 0.06% SSC. The slide is kept in a humidified chamber until fluorescence scanning was done.
Fluorescence scanning and data acquisition. Fluorescence scanning is set for 20 microns/pixel and two readings are taken per pixel. Data for channel 1 is set to collect fluorescence from Cy3 with excitation at 520 nm and emission at 550- 600 nm. Channel 2 collects signals excited at 647 nm and emitted at 660-705 nm, appropriate for Cy5. No neutral density filters are applied to the signal from either channel, and the photomultiplier tube gain is set to 5. Fine adjustments are then
-69- made to the photomultiplier gain so that signals collected from the two spots are equivalent.
Construction of an asthU Transgenic Mouse Isolation of mouse asth1-J genomic fragment:
Phage MW1-J was isolated by screening a mouse 129Sv genomic phage library (Stratagene) with the 443bp BamHI-Smal fragment from the 5' region of the human asth1-J cDNA clone PA1001A as probe. The 23kb insert in MW1-J was sequenced.
Assembly of asthl-Jexb targeting construct:
A 2.65kb Sad fragment (bp7115-bp9765) from MW1-J was isolated, cloned into the Sacl site of pUC19, isolated from the resultant plasmid as an EcoRI-Xbal fragment, inserted into the EcoRI-Xbal sites of pBluescriptll KS+ (Stratagene), and the 2.5kb Xhol-Mlul fragment isolated. A 5.4kb Hindlll fragment (bp11515-bp16909) was isolated from MW1-J, inserted into the Hindlll site of pBluescriptll KS+, reisolated as a Xhol-Notl fragment, inserted into the Xhol-Notl sites of pPNT, and the 9.5kb Xhol-Mlul fragment isolated. The two Xhol-Mlul fragments were ligated together to produce the final targeting construct plasmid, asthlexb. Asthlexb was linearized by digestion with Notl and purified by CsCI banding.
Identification of targeted ES clones:
Approximately 10 million RW4 ES cells (Genome Systems) were electroporated with 20 μg of linearized asthlexb and grown on mitomycin C inactivated MEFs (Mouse Embryo Fibroblasts) in ES cell medium (DMEM + 15% fetal bovine serum+1000U/ml LIF (Life Technologies)) and 400 μg/ml G418. After 24-48hrs, the cells were refed with ES cell medium. After 7-10 days in selection culture approximately 200 colonies were picked, trypsinized, grown in 96 well microtiter plates, and expanded in duplicate 24 well microtiter plates. Cells from one set of plates were trypsinized, resuspended in freezing medium (Joyner, A., ed., Gene Targeting, A Practical Approach. 1993. Oxford University Press), and stored at -85C. Genomic DNA was isolated from the other set of plates by standard
-70- methods (Joyner, supra.) Approximately 10 μg of genomic DNA per clone were digested with Ndel and screened by southern blotting using a 100 bp fragment (bp6164-bp6260) as probe. A banding pattern consistent with targeted replacement by homologous recombination at the asthl-J locus was detected in 10 of 113 clones screened.
Production of asth1-J knockout mice:
Two of the targeted clones, cl# 17 and cl#58, were expanded and injected into C57BL/6 blastocysts according to standard methods (Joyner, supra). High percentage male chimeric founder mice (as ascertained by extent of agouti coat color contribution) were bred to A/J and C57BL/6 female mice. Germline transmission was ascertained by chinchilla or albino coat color offspring from A J outcrosses and by agouti coat color offsprint from C57BL/6 outcrosses. The Ndel southern blot assay employed for ES cell screening was used to identify germline offspring carrying the targeted allele of Asthl-J. Germline offspring from both A/J and C57BL/6 outcrosses were identified and bred with A/J or C57BL/6 mates respectively.
Mice heterozygous for the Asthl-J targeted allele are interbred to obtain mice homozygous for the asth1-J targeted allele. Homozygotes are identified by Ndel Southern blot screening described above. The germline offspring of the chimeric founders are 50% A/J or C57BL6 and 50% 129SvJ in genetic background. Subsequent generations of backcrossing with wild type A/J or C57BL/6 mates will result in halving of the 129SvJ contribution to the background. The percentage A/J or C57BL/6 background is calculated for each homozygous mouse from its breeding history.
Molecular and cellular analysis of homozygous mice:
Various tissues of homozygotes, heterozygotes and wild type littermates at various stages of development from embryonic stages to mature adults are isolated and processed to obtain RNA and protein. Northern and western expression analyses as well as in situ hybridizations and immunohistochemical analyses are
-71- performed using cDNA probes and polyclonal and/or monoclonal antibodies specific for asthl-J protein.
Phenotypic analysis of homozygous mice: A/J, C57BL/6, wild type, heterozygous and homozygous mice in both A/J and
C57BL/6 backgrounds at varying stages of development are assessed for gross pathology and overt behavioral phenotypic differences such as weight, breeding performance, alertness and activity level, etc.
Metacholine challenge tests are performed according to published protocols (De Sanctis et al. (1995). Quantitative Locus Analysis of Airway
Hyperresponsiveness in A/J and C57BL/6J mice. Nat. Genet. 11 :150-154.).
Targeting at asth1-J exon C: Assembly of exon C targeting construct: A 3.2kb Hindlll-Xbal fragment (bp11515-bp14752) from MW1-J was isolated, cloned into the Hindlll-Xbal site of pUC19, isolated from the resultant plasmid as a Kpnl-Xbal fragment, inserted into the Kpnl-Xbal sites of pBluescriptll KS+ (Stratagene), and the 4.5kb Rsrll-Mlul fragment isolated. A 3.4kb Hindlll fragment (bp17217-bp20622) was isolated from MW1-J, inserted into the Hindlll site of pBluescriptll KS+, reisolated as a Xhol-Notl fragment, inserted into the Xhol-Notl sites of pPNT, and the 9.5kb Rsrll-Mlul fragment isolated. The two Rsrll-Mlul fragments were ligated together to produce the final targeting construct plasmid, Asthlexc. Asthlexc was linearized by digestion with Notl and purified by CsCI banding.
Identification of targeted ES clones:
Approximately 10 million RW4 ES cells (Genome Systems) were electroporated with 20μg of linearized asthlexc and grown on mitomycin C inactivated MEFs (Mouse Embryo Fibroblasts) in ES cell medium (DMEM + 15% fetal bovine serum+1000U/ml LIF (Life Technologies)) and 400 μg/ml G418. After 24-48hrs, the cells were refed with ES cell medium. After 7-10 days in selection culture approximately 200 colonies were picked, trypsinized, grown in 96 well
-72- microtiter plates, and expanded in duplicate 24 well microtiter plates. Cells from one set of plates were trypsinized, resuspended in freezing medium (Joyner, supra), and stored at -85C. Genomic DNA was isolated from the other set of plates by standard methods (Joyner, supra). Approximately 10 μg of genomic DNA per clone were digested with Ncol and screened by southern blotting using a 518bp fragment (bp8043-bp8560) as probe. A banding pattern consistent with targeted replacement by homologous recombination at the Asthl-J locus was detected in 3 of 46 clones screened.
Targeted clones are injected into blastocysts and high percentage chimeras bred to A/J and C57BL/6 mates analogously to that done for asthl-Jexb knockout mice. Heterozygote, homozygote and wild type littermates are obtained and analyzed analogously to that done for asthl-Jexb knockout mice.
The data presented above demonstrate that ASTH11 and ASTH1 J are novel human genes linked to a history of clinical asthma and bronchial hyperreactivity in two asthma cohorts, the population of Tristan da Cunha and a set of Canadian asthma families. A TDT curve in the ASTH1 region indicates that ASTH1 I and ASTH1 J are located in the region most highly associated with disease. The genes have been characterized and their genetic structure determined. Full length cDNA sequence for three isoforms of ASTH11 and three isoforms of ASTH1 J are reported. The genes are novel members of the ets family of transcription factors, which have been implicated in the activation of a variety of genes including the TCRα gene and cytokine genes known to be important in the aetiology of asthma. Polymorphisms in the ASTH1 I and ASTH1J genes are described. These polymorphisms are useful in the presymptomatic diagnosis of asthma susceptibility, and in the confirmation of diagnosis of asthma and of asthma subtypes.
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
-73- Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
-74- SEQUENCE LISTING (1) GENERAL INFORMATION (i) APPLICANT: AxyS Pharmaceuticals, Inc. (ii) TITLE OF THE INVENTION: Asthma Related Genes (iii) NUMBER OF SEQUENCES: 339 (iv) CORRESPONDENCE ADDRESS:
(A ADDRESSEE: Bozicevic & Reed, LLP (B STREET: 285 Hamilton Ave, Suite 200 (C CITY: Palo Alto (D STATE : CA (E COUNTRY: USA (F ZIP: 94301
(v) COMPUTER READABLE FORM:
(A MEDIUM TYPE: Diskette (B COMPUTER: IBM Compatible (C OPERATING SYSTEM: DOS (D SOFTWARE: FastSEQ for Windows Version 2.0
(vi) CURRENT APPLICATION DATA: (A APPLICATION NUMBER: (B FILING DATE: 21-JAN-1998 (C CLASSIFICATION:
(vii PRIOR APPLICATION DATA: (A APPLICATION NUMBER: (B FILING DATE:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sherwood, Pamela J
(B) REGISTRATION NUMBER: 36,677
(C) REFERENCE/DOCKET NUMBER: SEQ-4P
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 650-327-3231
(B) TELEFAX: 650-327-3231
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 72928 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : GCACTTTTTG GGGAAGGTGG AAGAATAAAA GTAAGGGAGG TGTGCTGAGA CTTCAATTTT 60
-75- AATATCTTAT TTCTTAGGTT GAGTGTTACA CAGGCATTTG TAATCATATA TACTTTTGTA 120
CACTTGAAAT ATATATATTT GTGTGTGTGT GTGTGTGTGT GTCAGAGTCT CACTCTGTCT 180
CCCAGGCTGG AGTGCAGTGG TGTGATCTTG GCTCATTGCA ACCTCCACCT CCCAGGTTCA 240
AGAGCTTTTT GTGCCTCCAT CTCCTGAGTA GCTGAGACTA CAGGCAAGCA CCACCACACC 300
GGCTAATTTT TGTATTTTTA GTAGAGATGG GGTTTCACCA TGTTGCCCAG GCTGGTCTCA 360
AATTCCTGGC CTCAAGTGAT CCAGTCACCT TGGCCTCCCA AAGTGCTGGA ATTACAGGCG 420
TGAGCCACCA TGCCCGGTCT GAAATATTTC AAAATGTAAA AAAGCTAAAC CCAAATCCAG 480
ATGTCTACTT TCAAGGTGCT CACAGGTCAG ATCTAGGATT ATTGCTACTA ACTGATATTT 540
ATTATCCCAG CACCAGCATG TTTGGCTGTG TGTCATGGGT AAGTTACTCA CCTTCTCTGC 600
GACAGTGTCA TCATTGTAAA ATAGGGATAA AAGAGTTTAG ACCTTGCAGA GTCCTTCAGA 660
TTAAAGGAGA TAATCAGTAC GTGGCACTGA GTACCTGCAA TATATTAAGT GGTGTGTGCT 720
CAGAGATATG ATCACATACA GTATCTTGGA TCTGCCCAGC AACTCTATGA AGATGAGGAA 780
ACAGACTCAG GCAGGTCAGA GCCAGAACAT AATGTTTCTG GAATTTGAAC GTAAACGTTC 840
CCCTTTCTCT TATCCAGGCT GAGTGCTAAA GGAATTGTAA AAATGGAATT TGCCTGTTGC 900
CTGCATCTCC CTCTCTTTTT CTTCCTCTGT GTCCTCTGAA TATCTAGCAC CAGTGGGACT 960
TTACAGTGTT GGCCTCAATG CTGTAGGGTG CTGTGTGCAC ACTTGTCTTC AGCTCCCTGA 1020
GTTAGCAGAG CATTGCCCCA ACTCTGCCCT CTGGCCAGCT CATGTGCCTT ACAACTTTCT 1080
GTTGCCAGAA GAGAGCCCTG CTCATTCTCT AGACTCAACC AACAAAAGCT GCCTACCATT 1140
TTCAGAATGC CAGTGGGCAG TGAGAAGTGC AGAGCTTGTG TCCTGAGCTT GGCAGCCATC 1200
TTGCTTGGTG TTAACAAAGA GTAATTAAGT GATCTCATAA AACTCAGTGG TGGAGGTTGT 1260
GGTTCAGAGC AAGCTGGGTC AATGCCAAGG CTACTTTGGC TTCATCTGGT CCATAGCCCC 1320
ACATTTCTCT TCTGATGGTT CAGTTCCGGG AATGAGAACC AGTCTGAGTG TAAGAAGACT 1380
TGGGTTTGAA TCTGTCTCCT CCAATCACTA GCTGACCTTA GAAAAGTGAC TTAACCTCCC 1440
GAGCTGCTAT TTCCTCATCT TAAATGGTGA TAGTAATCTT TCCTTACCTT AAGGTTGTTG 1500
AGCAGCTTAA ATAATATAAT GAGTTGAAAG CTTTTTGTAT GATCTGTTAT TAGGAGTCCA 1560
GATAGTGTTT TATAAACAAG AGGATAAAAA AAAAAAAAAA AAAAAAAACA GGATTCTGAA 1620
GGCTGGACTC ATTGCATTCC TTGCAAACTA CCCACTGAGC CCCAACTCTT CCGTCAGCTC 1680
AAAGTCACTT CTCAGAGCAA ACCAGATTGT CCTGAACCCA GCACTTGCCA ACATCTCCTC 1740
CTCTTCCCTG ATGAAAACTC TGGGCTGGAG TTGTGGTGGG TGAGGGGAAG GCAGGATAAA 1800
TCAAAAATTG ATGTTTTAAG AAAACTATGG TATTCTTGGA TGCAAAGGCA TGAGAATGAT 1860
ACCTTAGACT TTGGGGCTTG GGGAAAAGGG TGGGGGGTGG CGAGGGATAA AAGACTACAC 1920
ATTGGGTTTA GTGGACACTG CTCGGGTTAT GGGTGCACCA AAATCTCAGA AATCACCACT 1980
AAAGAACTGA TTCAGGTAAC CAAACACCAC CTGTTCCCCA AAAACCTATT GAAATAAAAA 2040
CAGAAAATTA AAAAAAAGAA AACCTATGGT ATTCTTGGAA GAAGCACAGT GGTGAAGTGG 2100
AGTAGACACA GATGTGGAAG TGATGTGAAC TTTGGTAAGT TGCTGAGCCT CTGAGGATGA 2160
TTTCCCTCAT CTGTCAATCA GGGAACAAAA TCCCTTACTT GTACAATGAG TATTATAAAG 2220
ATCAATTCAG ATGACGCATG TAAAGATGCA ATGTGGGACT GGTAGGTAGT AAGCATCCCA 2280
TAAATGGCAG CTATTAATAA GTAATAATCA CCGAGTGGTG GGCTGCCTTT CATGAAAACA 2340
TTCCCAGCAA GCTGCTCTTC TGTCGGCTCA AAGTCACTTC TCAGAGTAAA TGAGATTGGC 2400
CAGTTCTTTC TTTCCAAGGC TTTTCTGGAT ATTCATTTGT CCCAGATTTC TCCTGTATAC 2460
AAAGCTCAGG AGTGAGGACC CCCACAGTGG GGCTTGCACA AGGATAGCCT TGGGGGGCTT 2520
TTTCTAAGAG CTATGACTTT GAATGCTCTC TTCATCGATG CTGACAGATG AGGGCTGATG 2580
GAAGTGGTCA TGTTTTAAAA TGTCTGATGT CCAGAAACAC AGAGATGTGT ACGCAAAACA 2640
TTCATTCATT CAAGATGGAA TTAGTGCCCC AGACACAGAG GCAGGGGATA AATAGCAAAC 2700
AAGGCTTGAT TCCTGCCTTC ATAGAGCTTA CTGTCTTGTA GGGGAAACAT GAGTAAATTC 2760
AGCAGAGTAA GGGCTCTAAT TGGGTAAATG GGGGCTAGGC TGCCTGTGTC CTTGGGGTGG 2820
TGGGAAGGCT GCTGATCTGG GGTGCCAGAA GACCTGAGTT TTGATGCAGG CTCTGTGACT 2880
TTGAGCAGGT CGTTTCCAAC TTCTGAGCTT CCATTTCCCT AGCTGAAAAT GGGGGCTTGC 2940
CATACTCGAT GCTGTACTCT ATGAGTCTTT GCAGCTCTGT CATCTTTTTT TCTTTTGGTC 3000
ACTCAGAGAC TCCAGGATTG GGAGAACAAC CTGCATTCTG ATTTAAAGTG TGAATCTAAT 3060
AATTTCAAAA AGAAAGGGAC TAAAAGGGAC AAACTTGTTT CTGTTTATTT TCCATCCTTC 3120
TTTGGGGAAG TGTAACATTT GAAATCAAAT TCTCATTGGC TTAGCCAATG TGTAGACTTC 3180
GAGGGGAAAT TCTCACTGCC CAGAGAAGTG ACTAAAAATG ACCATTACAG CCAAAAAGAG 3240
AAGTTTTTTT TTTTTTAAAA TCTGTGCTCT ACAGATGGAT GAAGTGCTGC TGCACATGGA 3300
CAGAGTGGAT CTGGACATTC TGCATGAGCC CAGGGATCCT GAGAATGGAT TGGCTGAGCA 3360
TAGACAGGGT GACCTATCGA TGTTCACTGT GGTCCTGATC TATGTGGCCT CTTCCTAAGG 3420
-76- GAAGATTTTT CTTAAGGTTG TTTCCTTTCT CAGCAGATAT TTGTGAAGAA ΑCTGTATCTG 3480
TAGTCTCATT TTGTCCTTAT AATGACCCTG ATGGATGGGA GGTAGAGGGA TGATGATCAG 3540
TAAGAGCTGG GAAAGCACCA GGAACTAGCA AGAGCAGGAC ACCTTTTCCA CCACTAGGTA 3600
AATGGACCTA GTGACTGCTG GCACCGTGGG TGAGGGGACT GCCTGGCAGG AGCTGTGGCC 3660
GTAGCTAGGG GATTACAGCT ACGGCCACAA CTCTGGCCCT GTACGGAGGG AGTGGGGGAA 3720
ATAAAGAGTT CATATCACTC CCCTCTTTCC CTGGAGTCTC CTGCTGGTAC CTTGCATTGG 3780
CTGAGTCTAA CTGGAAGCCA GAGGGCAAAG GAGGTACCCT TTCCAGCTCT GCAATTCTCT 3840
TCAGACAGGG CTGGGATTTC TGGAGAGAAT TTGCAGAATC AGAAAGCAGA GCTTTCCAAT 3900
CAATGCCAAG CAAGAGACTC TGCAGACTCT CATAGCCTTG GGACCTGAGA AACCAGGTAT 3960
CCAGTGAGCA GTCACTTAAG CCTGTTCACC TGGCCCTCTC TTACTTTCTC TCCTATAGCA 4020
GCAGCAAAGG AGCGATGGGC CGAAGGGACT TGCTGGGTAG AAGTGGACCC ACATTCTAAA 4080
AAGGAATGGA AGAGAAACCT GATTTCTTTG ACTCGCCCTG TCCCTGAAGA TGAGGGGCAG 4140
GCACAGACCA GCCCTCTCCA GAAAGACAAA TATATTCTTC CATTCATGGG AGGGGTAGTA 4200
GAGACTAACA TTTGTTAAGT ATCTATTACA TGGGGGGTAT GGAGGTAGGC CCTTTGTGTG 4260
TGTTGCCTCT TTTAATCCTT TGGTGATCAA CTCATGAAAA TAAACAGCTC CAGAGCCAGC 4320
TGTCTTTGGA GGGTGTAGGC AGGCCCGGCT CTGGGAAACC TGGTGACACT GACCTAGTTT 4380
GACTTCCAAA TCTTCTCTCT TCTTCGATTC TGGTGAGCCC CACTCTAGCC CCATAGTATG 4440
TATGGCCAAG CACCCAGATA CTGCTTCCAT CAGGAGGAAA TAACATACCT GATGAATTTC 4500
TTCACTCAAG GTGTTAGGAG CTTAATGTGT TTCCCCCGCC CCCCGCACCA AGAGAATTTG 4560
TGTTTTCCAA GACAGTCAGA GAGTGGGTGG TGCTGAACTC AAAGGAGTGA ATCACTAATA 4620
GTGGAATCCC AGGCATTCAG GGAGGTCCTA TTTCTGGGGT GGGTTCCTTC CTGACACTTC 4680
ATTTTCTACA AAGGTGGCAG CCACCTATTG TCTCCAGAAA GGAGGCTGTC CCTGTGGGTG 4740
TGGTGACGGT GGGAAAGGAG AGGCACCTGC AGGCTGAAGC CAAGATCACC TGATTTTCAA 4800
AACCAAATCT GTCCCTACAA AGGAGAAGTG GCTTAAAAAT CCACACAGCC TCCCGAGTGG 4860
AGGGAAGAAT TCCCTCTCCT CTCTGGAACA GGGTTCCCTT CACCCAGAAC ACGGTGCTGT 4920
TGTTATGCAA TGTCCCTGTT GGCAAAGATA TTTGAGCCCC TTGTTTTCAG GTCTGTGTCA 4980
TTTCCAAGAA AGAGCTGTGG CCTTTGAGTA GGACTGGGCT CCTGAATAGG GTCCCTGGTG 5040
CCAAATGAGG GAGCCAAGAA AAGGCAGAGA AGAGGAAAGT CCTGACTTTT ACATGAAGAT 5100
GAGACAGCCA GCCCTGTGGC AGCCAGATGG CAGTCCTGTT GCTCTGTAGT GGCCTTGGGG 5160
TCAGACTAGG GGCAGAGCTG GGCTGAAGGC AGGAAGGCCA GGACAAGACA GGTGAGAAGG 5220
GCAAAGTCTC CTGTAACCTG GTGAGAAAAT GTGGGCTAAG CCATTCTCAT CTGGAGCTGA 5280
AGGCTTGGTG GAGAATGGCC CTCAACATTC AAGTTCACAC CCATGGATTT ATAAAAGGCA 5340
GGGCTGGGGG GAAAGGTTTT TCCCATTATA CTTAATAACA TTATCAACAA CAATAATCAC 5400
TACTATCATT TATTGAGCAT TGACTCAAAA GACAGTCCTT TTATGAAAAT TATTTACTTA 5460
AATCCTTACA AAGCTTCTAT TCATTCACCC AACACATATT TATTGAGTTC CTACTATGAG 5520
CCAGGCATTA TTCTAGGTGC TTAATTTAGA TCAAGGGACA AGACAGACAA AATCCCTGTT 5580
CTGGTGGCAG GGCTACTACA TGCAATTAAC AGCACACAAC TCTAGGGGGA GCCACATACA 5640
TGGGCCACCT TATGAATGGT GTGCCCTGAG GTTAAGCATC CTGGCAGCCC CTTTCTGTGA 5700
CATTTGCATT CTAGTGAAGG GAGTCTAATA CCAATGAAGT AGATGTCATT ATCCCCTGAC 5760
TACAGTTTAG GAAACAGAGA CACATAGGAA TTAAGTAACT TGCTGAGTTT TTCAGCCAAA 5820
AATGACTGAC CCATGATTTA TACTGAAGTC AGTCCTTGCA ATTCACCTGT GCCACGTACT 5880
TGCCTTTCTC TCCCTGGTGG GCACAGGGAA GAGGGAGTAG CCAGGCTGGC CAGATGAGTG 5940
CTGGGCTGGC TGGCCCAGTA GAGGCACCAT GTCCTGACTG GGTGGACAAA GACTGGGTAG 6000
GAGGTAACAG AGAATCCCTT GGTGAGTCTA ACTTAGCTAT AAGAAGGCTT GCTGAGAGCA 6060
GCTGCCTCCA TGCAGAGGGT GGGGTGACCG GCCTTTAATC CTTCCCAGCT GAGGATTTAG 6120
TCAAAGAAGC TTGTCTCTGG GGATAGCCTA TGGTCTTGAA GGGCCTGAGT TAGCTATTAG 6180
TTCACCCATT TATTTAACAT TCATTCATTA TTTTTAAAAA ATTTCCTAGC TATGTTTGGG 6240
GGCAGAGAAG TGGGTCCAGA GACCTAGAGG TTTGCAAGGG TAGCTTCTAA ACTCCTTTGG 6300
TTCAGAACAG AATAGAAAGT GTCCTCGGGT GACCTTGGGT CTGCTTCCCA AGCAAATTGA 6360
GCATACGCAG CCAGAACAAA GACTGCACTC TACTCTAGTG AGCTCAGCCT GCTAGGCTTG 6420
GATCTAGATT TTATAGCAAT AAGCTTGGAG TCTCACCTTT GGGTCAGACA GAGTACTACC 6480
CCAGACATGA GGTAGGGAGA GCCTAGTCTA TATTCCTCTG CCTTTGTCCA AGCCTGCTTT 6540
GTCCTTCCTC TTGACGAGGA ATAAAGATGG CTTCTGGGTG TGCATCCCCT TCCTTCTTCC 6600
ACCTGCAGAT GTACCTGTTT GTGTGCAGTG GGCTTCTGAG TCCTGGGCAG GGATGCCAGA 6660
GACCGCAAGC CAGATGCTTG GGATGCCAAT CCTTGGGACT TTGAGGAGAA AGAGAGGTTC 6720
TGAGGGGCAT CTGTCTATGG CACAGAGTCA AATGGAACAC ATGGAAGTCC CTTAGAAGGC 6780
-77- TGGTATCTAA GTGTTGGCCA CACAATGTCC GTTCTTCCTC CATTATTTGA ATTTCTCCTT 6840
CTCTATCCTT CTATCTTTCT TGGCACCTTG AGCCAGGTCT GGGGTGAGAG AAGGGATGGT 6900
GTAGGTGAAT TAGTGGTAGT TATTGGAGGA AGGCAATAAA CCCAGAAAAA GTGTCACGTG 6960
ACTTCTTTCT TGGGCCCAGT GTGACGCTTC TAGTTAGGCT AACGTGGGTC TTGGGACTGT 7020
TCCTGAGATT TTGTGGAAAA CTCTTTGTAT TTGTGCTGGT AACAGAAGGA AACCAGAGTT 7080
AGGGCTGGTG GGATGAAGCA GTGGGAACAC TGATTTCTCC TTTTTTTCAG ATTCAGGGAT 7140
TTCTGTCAGA GACATCCGTG GGGGAGGGAT GGGATTGGGA GTGAGGAGAA TCCCTTTCCT 7200
CTCCTCTCAC CATCTGGTGG TCCCCGTGCC CACGCACCAG CTCGTTGGAT GGACATTTTG 7260
ATTCCCTTAA GATGTACATT CTTCAAATCA TTGTTTGTCA TTAGCTCCCT GGAGAAAATG 7320
GAGGGGCTGA GATATTAGTG AGAAAACATA AAGTTAATTG GGTGATGGAG ACTGGGAGAA 7380
GGGGAATGTT AGAAGAAAGT GAGCGAGGTC TGCTAAAAGT GAACTTTATC TTCTTCTCAA 7440
TTTTGCCTAA GACTCGTGTT GCCTGGGCAG TCTCTTTTTG GAAGAGAAAT TTTCATGACA 7500
GTTTGGGCCA GAGATGGCAA ATAAATGCCT GACATGGTTG CTGCCAGCCC CTGTCTCCCG 7560
ACACGTTCAC AAGGGTGCAC ACCACTTCTC CTCTCTGTGA CCATAGACTC AGACCCATTG 7620
CAATCCAGCA TCCTGCATGG CCCCATTGGT CAGAGTTGAC ATTTGCAATG AAGCTGCTTC 7680
CCTATGCCTG GTTAGGCCTT TTGCTATGAA TTCTCTGGAG TTAACTATTT CCAAGGGGCT 7740
CCAACTTATT CTTGTGATTT CCACGGGATT TGGAGCCCCA GAAGACAATC CCATGTGGAT 7800
TCACAAAATG CCCTCTAAAT TTGATGGCTG TCAGTGCATA CTAAGTATGA CTGACTCACT 7860
GGTATCTGTT TCCTCCGCTG ACACAGCTGG TTCTTAGGCT CGGCAGGAGT TTGGGCTGAG 7920
ACCTCTCATT GCTCTATATT CCCTCTGTTA CTAATGAGGT GTTGTTCCTT AATTACTAGG 7980
TGCTGGATAC TAGAATTGCT TTTCTTTGTT TCAGGGGATT TAGCAAAGGG CTTATAAATA 8040
TTTCTTGTGT CTGGCATGAA CTACCTGATT TTTTTATTCT TCAGGTCACT GAGCTGGCAA 8100
TAAAGGCAAC TCAAAGTTAG CTGGGAATCA GAATGAAGGG GGACTAGGAA AAGTGATGCC 8160
TAGAACACCA ACAGGTGTGG GATCATCTTC ATTGTACCTT TCAGAGCCTA AGATATAAGT 8220
CCTCTGGATA CTCTCTGCTT GTTTATTTAA AGGAAAAAAT AATCAGAATG TGGGAGAAAT 8280
GGGTGCTTTG GGTAATTTCA TATTCTAATT GATGAACGTG TATGAAATTA TAATATTAAA 8340
CCACTACTAG CCCTTGCCGT AAAAAACTAT TCCAAAATAG CTGAGTCTAA GTTTCCTGCC 8400
TCAGTGTGTC CCACCTCTTG CGCTTGAGTC CTTAATGATC CAGAGTTTCA AGTCCCCAGT 8460
GCCCTAATCT TGAAAAGCAG AAACTTTAGA AGTTTGCTGA AGTTTATTAG TTGGCTATAC 8520
GATCCATCAA GAAATTGACT TTTTTGGATT AAATTCAAGA TAGTTTTTAA AAAATCAGAA 8580
GTTTCTTTAT CATGAAAGCT AAAAAAATAA TTGAAGGTAG AGGCTAGTTG GAATCCCAGT 8640
TAATAGATGG ATTTCTTCCT TCTTGAAGAA ACTTGTGTCC AAGGGCAAAC TGAATCCTGG 8700
TGGTCTATGC TGGCCACATT CAGCAAAAAA TGGCCCGAGG TTTTGATGGT TATCATTCTC 8760
AAAACTGTTC CTGCCAACAC ACTCTGATCC CAGGAGGTTA CCTGACCTTT ATAAGGCTCA 8820
GTTTCCTCCC CTGTAAAATG GGCAGGGTAA TCAAGCTAGG CAAAATATTT AACCTAAGTG 8880
AGGAAATTGT GCTATTAGTG CCCTGAAAAA CATGTAGAAA GACATTAGAC ATTATTTTAT 8940
TTAATATCAT GTTGAACTTA GTTTTTAAAA AGAAGACCTA TTGGATTTTC CAAGAACAAC 9000
TAAACTGATT CCTTGTAGAC AGTTTAGAGA ATACAGAAAA TTAGAAATAG GAAAAAAGCA 9060
AAACAAAACA AAAACCATCA AACAAAGTCT ACGCAAATAC AGTTTCTCTT AACTTTTGGT 9120
TTATTTCCTT CTAGTCATTT TTTAGGTGCA TTTTTAAATT GTGGTAAAAT ATATGTAATG 9180
TAGAATTTAC CATTGTAGCC ATTTTTAAGT GTAGAGTTCA GTGGCATTAA GTACATTTAT 9240
ATTGCCGTGC AACCATCACC ACCATCTATC TCCAGATTTT ATAACCCCAG ACTGAAACTC 9300
CATATCCATT AAATGATAAC TCCCCATTCC CCTCTCCCTA CCCTGGTGAC CACCATTTTA 9360
CTTTCTGTTT TTATGAATTT GACTTTCTTG GCGCCTCTTA TAAGTGGGAT CATTTTTAGT 9420
TGTTTTTATA ATCGGTTTCC TTCCTTTAAA AATATGAATG GAGCCTAATG AATATTGAAT 9480
TTAGTGTACT GGTTTCTTTG AACATTTCAG CATCATAAAC ATGTTTTTGT ATTCTACATT 9540
CTTCTTGTAT TGCTATATTC TCTATAGGAA TTTTTTTTTT TTTTTTGACA GAGTCTCACT 9600
CTGTTGCCCA GGCTGGAGTG CAGTGGCACA ATTTCAGCTC ACTGCAACCT CCGCCTACTG 9660
GGTTCAAATG ATTCTCCTGC CTCAGCCTCC CAAGTAGCTG GGACCAGAGG TGCATGCCAC 9720
CATGCCTGGC TAATTTTTGC ATTTTTAGTA GAGATGGGGT TTCATCATGT TGGCCAGGCT 9780
GGTCTTGAAC TCCTGACCTC AGGTGATCCG CCCACCTTGG CCTCCCAAAG TGCTGGAATT 9840
ACAGGTGTGA GCCATTGGCC CCAGCCTTGA ACATCATTTT TAATGGCTGA AGATTATAGA 9900
ATCCAGTGGG TGTGCCATCC ATTATTAGTA TTCTGTTGTT TCCAAATATT TGCTGTTTTA 9960
AACAGTGTTG TGAAAACATA TTTTTGTGTT GAACTTTTAT CATATTGAGA GGCACTTCCT 10020
CTGTGCAGAA TCAAGAAATT AATTACCGGT TTATAAGGAA TGTGAACCTT TCAGGCTCAT 10080
AATCTGTATT ACCAAATGGT TAGGAAAAAA ATGTTCAGAA GGTGCCATTC ACAGATGGAG 10140
-78- TGGGCTTCCA CCAGGGGCTG TGAAGCTCTA ATCTCAAAGG ATGTTGACTA CTGGTAGGGC 10200
TGATTCAAGT ATTAGATATC TAGGAAGGGT GGGAAGGGCA GAGAAGCTTC CAAAATTCCT 10260
ATGTAGGAGA GGCATAGGGG TGCTGATCTC TTCATAAGGG GTGACGGGAA TTTTCCTTGA 10320
AACAGCATGT GCAGATCAAG CACTGTTCTT TCCTTTAGAG TGTGTGTTTA TTTGGGGCGA 10380
CTTGGAGGGT TGCTAATTGA GATTATGGGG AATCTAAAGC CACACCCCAA ACCGCCCCTT 10440
GGTTCCCCTA CCTGGGGGAG AGTTGACACT AGTCAAACCT CTCCCATCTC TGAGATTTTG 10500
TGAATCTAGG ACTCTTGCCA CTGCACAGAC TCCAGCTGGA CCCAGGGACT CCAGCTTCTC 10560
ACATCACCCT GGCTCATCCA TAACTCTCTT TTGTTTCATC TCAAACATCA CTGAGAGATG 10620
GCTGCCTCTT CTCCCTTCCT AGGAAAGCCC ATGTCACAAT AAGCGCGCCT GTGCTTCTCA 10680
TCAGTGCTTT CCTGGTAGCA CCACCTGACA AACACTGCTC GCGGCTGCCT TCAGCTGCTC 10740
TCCAAGAAGA CGTCATAACC ACAAGAGATC TGAATCAGCC CATTTTTTCC CCTGTGGCAC 10800
TGTGTGCTTT GGCTGCCTGG CCAGAAAGCT GGGACTGTAT TTACCTATCA TTTTGATACT 10860
ATCTTGGGGT GTAATTGGAA TTGAGCTCTT AGTGTGGAAA TTCTTACTCA GAACACAAAG 10920
GATTGAAGAG TGCTTGGAGG CTGAACTCTG GAAGGACTCT TCCCTGAGGC CTCTTGGCAT 10980
CTGGCTCTTG TTTCTTGGAG CGGTGGTATG GCCCACAGGT GGGTGTTTCC TTTGGGAGCA 11040
ATTTCTTGCT TTTTCAGTAG CTCTGGGCTG TCATCGAGCC CACTGTTCCT TGTCTTCTCT 11100
GCACTGTTTA GTGATGATGT AGGTGAATTG CTCCACAGTT TAATTCCAGT GGTAGAGCAG 11160
TCACCATTTG TTGGTTTCTT TTTCTTATGG GAACTCTGGT CTGCATCTCA CTGTGTTTCC 11220
CTTGAACGTG TCTGGGGTCC TCCAAACAGC TTCGTGTCCC TCTGAGTGCG GACACTCAGA 11280
TTCTAACTCA GATTCTAAGT CAATGGTCTC AGCCTTTAGA ACCGCAGGAG GCCAGGCGCG 11340
GTGACTCACG CCTGTAATCC CAGGACTTTG GGAGGCCTAC GCGGGTGGAT CACCTAAGGT 11400
CAGGAGTTCG AGACCAGCCT GGCCAACACA GTGAAACCCC ATCTCTACTA AAAATACAAA 11460
AATTAGCCAG ATGTGGTGGC ATGTGCCTGT AATCCCGGCT ACTCAGGAGG CTGAGGCAGA 11520
GGCAGGAGAA TCGCTTGAAC ACGGGAGGTG GAGGTTGCAG TGAGCCGAGA TTGTGAGATT 11580
GTGCCATTGC ACTCTAGCCT GGGCAACAGA GTGAGACTCC ATCTCAAAAA AAAAAAAAAA 11640
AAAAAAAAAA AGAACCACAG GAGGGAGAGA TCATATATGA CCCCGTATGT GTGAAAAGTC 11700
CTATCATTGC TACCCACACC AACAATATTA GTGGAAAAAT GTCTTCAAAG GACATTCGAT 11760
TCAATGATAC ATGAGATTTG CTTCCTTCCT TAATTTTTCC CTGTACAGCT ATATAATGAT 11820
TTTTTCAATC AGATCCTCTT TTCCCCCTAT TAATTGTATT TATAGGATGA GATTGATTCT 11880
AACACAATAG CAAATGATGT ATGCACATTT AACACATTTC GTGAAGGCAG GAAAGGGCAC 11940
ACTATAAATT CTGTGAAATC CACATTAGAT CATGCCTCTC CTTTCTCAGT TGGGAGGTGG 12000
GCTCTGACAG TGCTCAAGAG AAAAAAAAAT CAAGTTGTGA CAGTTTAAAA AATATTTTAA 12060
A ATTAAACT ATTTATTATG GAACTTAAAA CATACACAGA AGTTGGCAGA ATAACATCAT 12120
GTACCCTAAA TATCTATCTC CAAACCTCAA CAGTGATCAA CCTGTGGTCA GTTCTGCCTC 12180
TTCTGGTTCC CATCTGCTCT CTGACTTCAG TTTATTTTGA AGCATGTCTC AGACATCTTG 12240
TGACTTCAGT ATTGCACGAT GTATGTCCTA AACGTAAGCA TTCCCTTTAA AACATGTATC 12300
TACTTTTTAA ATGAAGAACA ATTAGGTGCA TTTTCATAAG GGTTTTAGAA AGGGAAGAAA 12360
CTGTATTTCT TTAATTTAAA AATGTATCAG ACAACTAATC CATGTTTACT GTTTCTAACA 12420
CGGATACCAT AATAATAGGA TCATTCTATT ATACATAGAC TAGTGAGATC AATTTGTCAG 12480
ATAAACTTAG AAGGGCCATT AAGAAAGTTA TGTCATAATT TTTGTCACTT GCTGAAACCA 12540
AGACTTTAAT TCTGCAGAAC ATCATACCAG GATTCACAAT TGTATACACT GATTGTGTTT 12600
GTCCAGAGGT AATCTCAGAT CCACTGTATA TAATTTTCCA TTTGCCTAGC TATGGGGTTG 12660
GACACGTCAG TTTTTTCCAG ACCAAGGGTC TCCTAGCTTT TTTTTTATTT TTATTTTTAT 12720
TTTTTGAGAC AGAGTCTCTG TTGCCTGTGC TGGAGTACAC TGGTGCGATC TCGGCTCACT 12780
GCACCCTCCA CCTCTCAGAT TCAAGTGATT CTTGTGTCTC AGCCTCCTGA GTTGTAGGTG 12840
GGACTACAGG CACCTGCCAC CATGCCTGGA TTTTTTTTTT GTTTTTTTGT ATCTTTAGTA 12900
GAGATGGAGT TTTGCCATGT TGGCCAGGCT GGTCTTGACC TCTTGATCTT AGAAGATCTG 12960
CCCACCTTGG CCTCCCAAAG CTGGGATTAC AGGCATGAGC CACTGTGCCC AGCCTCCTAG 13020
CTGTTTTGGC TGCACACTTC TATCCGTAGA TAATTAAGCA TGTACCCTTA CTATTTTCCG 13080
CAATATAAAT TATTTACTTA TAAATTACAT TATGTACTCT ATCACACTGG TAAATTAAGT 13140
ATATTATAAA ACAGAAACTA AAAGTATGAA GTGAGAATTA AAAATGAATA GCAATTCTAA 13200
TATCTTCATC TTCCCCTCAG TGGATCCTCC TGTACATACT CCAATTTGCA GACCACTGGA 13260
GGAGGCTGTA GGAGGCAATA TTATATCCCA GTGAGGTGTG TGGGTTGTAA AGCCGAACAG 13320
CCTGAGTCCA CATCCCAGCT CCACCACTCC TTAGTTCTGT GACTTGGAAA CATCACTTAA 13380
CCTCTCTGAA TCTATCTTCT CACCTGTAAT ATGAGGGCAT TAACCCCTTA CAGGTTATTG 13440
TAAGGTTTCT TACACTGTGC CTGTGGTAAG CATCAATACA TTTTAGCCAA TAATAACAGT 13500
-79- AATGATAATA ACACATTCCT AGAGGGCTGG GATGGATCTA GATTTTTCTT CCCCTTTTAG 13560
TGGAAGACCA CAGCATGATG CATGAATTTA CATTTCCTCA GACATTCTGG TGCTGATGAA 13620
GGTAAAGATG GTGAGGCTGC GATGATGGTT TCAGGGATGG GTGTGTTGGG CGTGATGAAT 13680
AGCATGATGC ATATTGTCAC TCATTTAGTT TATCTGCACT GATGATGATG CTGATTATAT 13740
GATGACTGTT ACAGGGATGG TCACATTGTG GGTGATGAAT ATGACCAGAA AGGGAAGACT 13800
TTCACAGTTC CTACCCGAAC TACAACATCG ATATTTTCAT TTGTCTTTCC TAGGAACTCT 13860
TACCTTAATC ACCTGACCAA TATGCTGACG ACTAACATGT TGCGCCCTGC CTTTCTTCCG 13920
GGCCTCTCTG CCTTGCTGAT CTGTTTTGCT GGTGTGCCCT CCACTGTGCT CTTGGGTCTT 13980
TGTCTCTCGG TAAAGCCTAG TACTGTGGTT GCTGTACACA AAACCTGTAG ATGATTAAGA 14040
TCTCTGTTCA CTGCAGGGCC ATTCATCTCC CAGCAACTAT TTTATCCTTA AGTCAAGAGA 14100
CTTGCCTCTC AGCCCCTGGG GACCATGGAA AGAGTGCTAG AAACCTACAG AGTATGACCC 14160
TTTGTAGCCT TATGCAAGAA GTGACCTGTG TCTTTCCTGT CATGAGAGAG GACAGACATT 14220
GCAGGAATCA AACGCATAAC ACTAGTGCAA AACTGGGGAT AATGCCCAAA CCTGGTTAGG 14280
CAGGGGCGCC TGGAACATGC TTGTCCAGGA AATCTTCCAC TCAGTTCTGC TGCCTCCATG 14340
TCCCAGATGA TCACAGAAGC CTCCTGAGAA GGGTTGAATC CCCCGTCGCC TGGGGATCCC 14400
AAGAAAGCTG CAGAGGAAAG ACTTTCTCTT CCAAGATCAG AACAAAGGAC GGTTAGCATT 14460
GTGCCCAGTA GTGCCAAAAG GTAAGGTTGG GTTAAAATAA GAATTTGCCT TAAGCTCTTT 14520
TCCCGGGGGC TTGTTTTTTT CATTAACCTT GTTGGCTGGA CTTTAGGGAA GTATGCACCA 14580
TCTTCTCCAG AAGTGCTTCA GATTTTATAT TTTTAAGAAA TTCAAGAGTC TGAGTTAGGC 14640
ACTTTAATGT AACCTCCCCA AAGCTTTTGT TCCAGGAATT GACTTGGGGA TTAATCTGTT 14700
TAGCAAATTC TGACACAGAG GCATCTCATA ACCTTTTATT TTTTCTACAG ACCACATTGT 14760
ATCTACCTGG GATGTTTTGA AAATGAACAG TGACACCTAA GAATGTATAC TTATCTCTTC 14820
ATGCCAATTC TCCAAACTGG ATGTTGCCCA TGTCTCAAAA TTACTTGCCT CCAATTTTAG 14880
GGCATAAAGT GTGAGATTCT GTAGCATGAG ATCATATGCT CTTAAAATAC TAAGTATATA 14940
TAAATTATCC CTTAGCATCT TTAACATGCA ττττττττττ QTAGAGACAG TATCTCTACA 15000
AAAAAATCTC TCTGTATTGC TCAGGCTGGT CTTGAAATCC TGGGCTCAAG AGATCTTCCC 15060
ATCTCGGCTT CCCAAAATGC TAGAATTACA GGCATGAGTC TCCACACCTG GCCTAACATG 15120
AAATATTCTT TAACAGTATT CTTTAGGATA ATATATTATT CTATAGATTT GAAATAATTT 15180
ATCAGTTCTA TACTTAATTA TAAATACTCT TGGGAATAAA ACATACTTAT CTAATAAGCA 15240
AACAGTCGTG CTATTCCAAA CAATTTGGGA TTGCCTTTCC AAGCATTTTT TGGGGGTTTC 15300
TTCAACTGAT TGAGAGACCC CCGGCCGGGG AAGAGAAAGA GAATTTGATT TGTGACACTG 15360
ATGGAATGGA CTACAACCTT TTGGTGGTGA CTCTACTGGG GACTTGTCAC AGAGCTTATT 15420
TTCTAAACAG ATGTGAAAAA TGAAAGTCAG GCTGCTGTCT GGTTGGTAAG ATAAAGCTTT 15480
CATTAATACT TGGCAGCATT ATTTTAGCTA AAGTGTCAGA TCAAACGCCC ACATTATCAC 15540
CTCCCCTTCC TGATTCCAAC CGCCCATGAT AGAAAAGAAA TAAAAGACTA GGAATAGGTC 15600
CATCAACTGG TGAATGGCTA AACAAAATGA GGTATATACA TACAATAGAT GGTTATTGAA 15660
TCACAGTAGG GAATGAAGTA CTGATACATG CTACAATATA GATGATCGTC ATAAACATCA 15720
TGCTACGTGA AAGAGGCCAG ATGCAAAAAT GTCACATATT ATATGATTCT ACTTATTTGA 15780
AAAACTCAAA GTAGGCAAAT CCATAGAGAC AGAAAGCAGA CTGGTAGTTT CCCAGTGCTG 15840
GGGAGAAGGC AGACAGGGAA GTGACTGCTT AATGAGTATG AAGTTTCCTT TTGGGATGAT 15900
GAAAATGTCT TGGGACTTAG ATAGAAGTGA TGGTTGCACA ACACTGTGAA TGTAGTAATT 15960
GCCATGGAGA TGTACACCTC AAAATGGCTA AAATGAATTC TATGTTATGT GAATTTTACC 16020
TGAATTTAAA GAAGAGTAGA AACAAACACC AAGAAAAAGG GAGGAAAGGA GGCATTATTG 16080
AACAAGACAT TTCAACAAGT TTTGGAATAT GGAAAATATA CGGAGAAGTG GCAACTGACT 16140
TACCAGAGTG GCAGAAGAAA TAGTCTATGT GAGTGTGGGG AATGGGGTGG ATGTGGAACC 16200
AGTGAGAAAT AAGCCGCTTT ACTGGGAAGA ACTACAGAAA GACTGAGGCT TGGACGCAGC 16260
TTGTGCTACT ACAGGTAGCA GTAAACAGGG GGATTTGTTG AACTTCAGAA TATAGAGAAT 16320
TTTGATGTAA GAGGTTTTTT TTTTCTCGTC TCAAACCAGG AGACTTTTTT TGTTCTCTAG 16380
GTGAGGGAGA TCTAGAGACA GCCAAGTACA GGGTGCAGTA TCATCTAGAA AATAAAGAAG 16440
AGGTTTGAGT CTGCAGGTGA GACTCCTGCT CTCTTCCTGG AATGCTGGCA GCCAGGCTTA 16500
GATCAGCCTC TCTGCCCTGC TCCAGGCAGA AGATGGAAAG ATCCCTTTCT GGAGAAACTG 16560
ACTCATCCAA GAGATAACAG CTCATATTCT TACTTTTTAG AGCTCTCCAG TAAAATGCAG 16620
CTCAACACTT GATCAGTTTC CAGCGATGAC CCCTGATCAG GCCCTCACTA CGAACCTCTG 16680
GGTTTTAATT GGTTATTTAG TATCTCAATT TTAAAGATCA AAGACAGGAT CGCTTTTGAG 16740
GAAACTTCCA ACTTTAATGA AAGAATTTAA AAAAAAAAAG GAAAAAAAAC CTGATAGTGT 16800
AAAGAGCAGA GAAATGGCAG GGAAATGAAA ATTAAGTTAA AAAACAGAAA CTTTTATATA 16860
-80- ATTCTAATCC TTTGCAGAGA TAAAAAAATA CATTGCATAC CTAAAACAAG TACAAGTTGC 16920
CATGGAAACA GATTCATTAG TGAAGAGGAA AGAGATCTTG GAAATTAAAG ACATAAAAGA 16980
CAAAATAAAA ATTAAAAAAA TTAAACAGAA TTTAGAACAT AATGTTGAAA TGAGAGAACT 17040
TTAGATCTCA AAAACAACAG AGAATCAACC CAGGAGATTG TGTGTGACTA AAGAAGTCTC 17100
AGAAAGAGAA TAGAGGAAAG GAAGGAATAT TATAAGAAAA GTTTCAAGAA TAAAAGGTCA 17160
TGGGCCTCCA GACTGATAAA AATCCATCTT GTACCCAGAA AAAATTGACT TTTCAAGAAC 17220
TGAATCAGAA CCTATCCTGT GAAATGTTAG GACAAGTAGA TCCTAAAATC TTCCAGAGGG 17280
AATCCATTCA AAGGCCTTGA ATGGCATTAG ACTTCTCCAT ATCAATACTG GATGGTGAAA 17340
GAAAAAGAGC AATACCTTAA ACTTGCTAAA AGAAAATGAT TTTTAACTAG AATTCAATTT 17400
CCATCTCAAT TAAAAAACCC ACTGTAAAGA AAAAATTCAA ATCTTCTCAG GCATATAATA 17460
ACTCTAAAAT TCTACCTCCT GTGCACCTAA TTTTGGCAAG TATCTCAGGA AGATACACTT 17520
TGCTAGAACA AGGACATAGT TTAAGAAAGT GGAAGAAATC AGATCTGGGA ATCAGGGGAT 17580
CACATGATAC AGAGGCACAG CCAGAGGGAT CCCAGGGAGA GCATGTCCAG TGTGACAAGG 17640
AGTGGACAGC TTCAGAAGGG ACAGCACCAG GGGAAAAAAC AAAATGAATA TCTGATTGGC 17700
ATAAACATTT GGAAAGTAGT ATTAAAAATG TGTGTAACAG GTGTGTTGTT ACATTTGCCA 17760
AAAAAGAGCA AAAGGGAAAA AAAACCCCAA GCAGATGAAA AGTAAAGAAG GCAATGGTTA 17820
ACTACTGGAA AAACAAAAAA CAATATTCAA GAAAGGAAAC GAAATCATGG TATACTTCTT 17880
GACTAATGGG TGAAAAATGA AGATGTACAT AGTTATTAAA ATGCAAACAT TGATTATTGA 17940
GTTAACCCAA AGTTGTGACA TTTGGAAGCA CGGGTAGGCA CAGTGGGGTG TAAGAGACCT 18000
AAATCCTCAC TTACCGTAAT GTTTAAAAAA TTGCCATGTC AAAGAATAGC AGCATATCAT 18060
ATTATTTAGA AATATGGATG CAAATGCCAG AAGAAAAATT AAAGGAAGTG AAAAATGTTT 18120
TCCTCTAGGA ATAGGACAGG GGACGTAATA GGGAACAGAT ATTCTGCATT ATCTCAATTA 18180
ATTCTCACAA CTGTGACTGA AGCTCTTTTG CTCTCCTTGT TTTGCAGATG AGCAAACTCA 18240
CAGAGGGATG CAACTTGCCT AGGATCGTAT AGCCAGCAGC TCATGAGTGT GGAATGGGGA 18300
TTCAAATAAG GTCTAGGAGA CTCCAAAATC CATGTGCTTA ACCATGAAGT TTTACTACCC 18360
CTTCTCTGCT TCTTCATTAA GTATTTTTAG TGCCTAATTG CCCATGCTCT CTGCCAGGTG 18420
CAGTAAAGGA GGATTACACA GGTGCAATAT GAGCCATGAC TCTTGTTGAA ATCAGCACGT 18480
CAAAAATAAG GCTAATGAGC ACGTGAAAAG ATGCTCAACA TCACTAATCA TTAGGGAAAT 18540
GCAAAACTGC ATTAAAATAT CACCTCATAT ACATTAGGAT GGCTACTATG AAAAAAACCA 18600
GAAAATAACA AATATTGGCA AGGATGTGGA ATAACTGGAA CACTCATGCA CTGTTGGTGG 18660
GAATGTAAAA TGGTGCAGCT GCTGTGGAAA ACAGTATGAT GGCTCTTAAA AAAATTTTAA 18720
AAAAATAGAT TTCTCATATA ATTCTGCAAT TCCATTCCTG GATATATACC CCAAAGAATG 18780
GAGAAAACAG GATCCTGGAG AGATGTTTGT ATACCCATGT TCATAGCAGC ATTATTCACA 18840
ATAGCTAACA TCTGGCAGAA CCCAATGAAT GAGTGGATAA ACAAAATGTA GTATATACAC 18900
ACAATGGGAT ATTAGTCTTA AAAAGGAAGG AAATTCTGAC ACATGCCACA ACATGGAGGT 18960
GCCTTGAGGA CATTATGCTA AGTGAAATAA AGCCAGTCAC AAAAGGACAA ATATTATATG 19020
ATTCCATTTA TATAAGCTAC TTAGAGTGGT CAAATTCATA GAGACAGAAA GTAGAATGGT 19080
GGTTGCCGGG GATGTAAAGG TGGGCATTTC TCAAAAAACT GAGAAATACA GAAAAATAAA 19140
AATCACTCAC TGTTTGCCAC ACTTCTACCC TGGTTCTTTT TAAATCTATT TTTCTTACTC 19200
AAAGAAATAC ATGTTTATAG TTTAAACATT CAAATAGTAC TACAGGTTCG TAATAAACAA 19260
GAGCGGTCCA ACTCCCCTCC TCCTAGCCCT GTGCTCCAGT CCTTTCAGAT GTTGTTTCTG 19320
GTCTTTGTAT TTCTCAATAA CATGCCTAAA TGTATTTTCT GGCTCCTTGT ATTGTTTATT 19380
TATTATTTGT TGAGTTTATT GCTATGAAAA ATAGAGATTA GATCACTTAC AGGGTCTTCC 19440
TGACACCGTG CTCACCTTCC CCACCTATAT GTACAATTCA CCTTCCCTGT CCTCATGGAA 19500
ATAATATTAC TCTTTTAGTT AAGTCACAGG TCAGTATTTA TGTTATGATT ATGTAAATAT 19560
TGTTTATGTA ATGTGCTAGG GCTACTTTTT TTTTCTTTAA TTCCTTATCC TCCTTCACCC 19620
TCACCACCCA ACCCCAATCT CATCCTGGAG TTCACAGTTA TCTCATTTTT CCTTTGCTTG 19680
GTTTTCTAAA ATCTATCTCC TGGCTCTTTC TCCAACTCTT CTCTCAGTAA GATAGTTTCT 19740
CAGCTCTACC TTTTCCCCTT GTTGACATTG CTCCAGAGCC CTTCAACCTG CTCAGGTGGC 19800
TATTCTGCTT GGTCACTCAC TTGTCCTCCT AGGTTTTCTT ATCTCCATCA TCTTGGGGAT 19860
TCTGGTCTCC AATTTCCTGT GTTAGACCAA CTGTGTCCTG GATCCCATAT CTTTCTGTCT 19920
CTTAGTTTAT TTCTTTGCTT TGATTGAACA TACTACCTAT GACATTTCTG AGAAACAATG 19980
AAAGAGAAAT GATTTTTTGA GTTGTGGGAT GAATATTAAA GTCACTACCC GGGAAGGATC 20040
ATTGTGCCTC TATCTGTATG AGGGATTCCC CTTGCACTTC TCAACCATAG ACAGCTCTGT 20100
TCTGTCTCTT GAGCTCTTGG TGAACCCATC CCCCAGGACA ACATTTCTAT GTGTCTTGGT 20160
CTGGCACAAG GTGACTACCT ATTCCCAGCA AATGCCAATC AACACCTGTC TTAATAATAC 20220
-81- CTTAGCTTCA ACACCCAAGG TTTAAGTTGC ATTAATCACT TAATAAAGAA ACCTTCACAA 20280
ATGCTAATTA CTAACCTAGT CCTTAAACCA TACTCATTTA AAGAGGTGGC ATCTTAGAAG 20340
TTACAGTGTT TATAGTCATT CAACAAACAT TTATTGTCAG CCATATAGAA GACACCATGC 20400
AAGGGCTTTA CATGGGTTAT CCAATGTAGT CCTCATGAAG GTCCTGTGAA GTGGGAATTA 20460
TTGCCATTTG TGAATGAGTT TCAGAGAGAT AAAACTTCTC CAGCCATTCA TTCAACACAT 20520
TTACTGAGTA TCTACTATGT GCTAGAAAAT GAGGATACCG CAGGGGGCAG AGGCACATGT 20580
CCCTGACCTC TTGGAGTTTC TAGTCTAGCC TAGTCTGTTT CCAAGGGTAA CAGATATTAA 20640
ATAAATAATT TCACAAATAG TCTATTAAAT ACATTTGAGA CAAGTGTCAT GAAAAAGAAG 20700
TACAAGATGC TATGGGAATG TATAAAGGCC ATAAGCTGTC CTAGTCTGGG GCTCAGAGGT 20760
GGTTTTTCTG AAGCAGTGCA TTAAGTCTGC AGGATAAGGA AGAGTCAGCC AGATGAAATG 20820
AAGTCTAAGG TTGGAGAGAG GGAGGGAACA GCATGAGCAA AGGCTCAGGG GCAGGAAGGG 20880
GCTTTGCATA TACGAAGAAC TGAAAGGCCA ATGCGGCTGG AACAAAGAAT GGAATGGTGT 20940
GGCATAAAGT GCAGCAGGGA CCGGGTCAGG GAGAAGACCA TAAAGCATTT GTGCACGCTG 21000
TTAAAGAATC TGTATGCAAC CTTGGTGGAC GTGGGAGACA TGACTGCTGA ACTTGAAGCG 21060
CATCCCTGGA GATGGGGATA AATGGAGGGA TGCGGGATGT GTGAAGCAAG AGGCTTGTTC 21120
ATGGTCAGAA CCGGCATCTG AACCCAGCTC TCATGACAAG TCTGCTGCTC TTTTTGGTAC 21180
ACAAAACCCG TTTCTTTCTC TGTGTGAGAA TGAACAAGGT GCCTGCACAT TTTTCTGTCC 21240
CAGTGCAGTG TTTGAGGATG CTAAGTTACA CCCCAACAGC TGTGCAAAAT CTGTTTCTCT 21300
CTTGTGTAGT GATGGAGGCT ATACATTGTG TTGTGAAAGG TGTCACTCAT TTGGGAAATT 21360
AGAACAAAAC ATAGTCATTG CCTTTAACAG CACACAGCCT AATAGAGGCA ATAGGAATGT 21420
AAACAGGGTC CCAAGCCAAA ACTTAACATG AGCAAGTTAT AGAATCATAT ACAATTCTTA 21480
GGGTCATAAT TCTAGGGCTA CATGTTTTGA CTGTTTGACC ACACTATATG CAGCAGTATC 21540
GTTAATGGTC CTGGATCTAG GCAGCATTTT CCGAAGTAGA CTTAAAATAA CATCACTCTT 21600
AGACTGGTCT GATTCTCTGT TTTGGCTAGA AATTGTGTTC CTCAAGAATA ATAACACATT 21660
TAAAATCATC CTTATTTTTT AAGTTCAGAT ATTCTGCTAA ATCATTGATC TCCATGAATT 21720
CATTGGTCAA TGTTTTAAAA CTTTCTCACA AACGGGCTTA TTGGAAATGG AGGCAGAAAA 21780
TAAGGTGTTC AATAATATGA CCACATGGTC TAAATTTCCT ACAATACGCT TAGTTTACAT 21840
GTGCAACACC TTTGTCAGAC ATATACCCAA TTTTGGTTTG AAAATAGCAT TTACTTCCCA 21900
GGAGTGGTGT GTAGGAACTT AAGGGTCCTA GTATGTATGT CTCTAGTGGA AACTTTGGGG 21960
TTCAGTTTGA AAAGGCAGTG TATCTCATGT GGATCCCTGT GATTCTCAGG GATTCTATAC 22020
TAGGCAGTCC CTTGTGGATG CCTGGGGAAG TCGGGCTGTG ATCCTTACAG ACCTTCTCTG 22080
AGCTGCCATA CAGATGGGGC AGAGGGTGAA TGATGGAAAA AGAACAAATG TTGCTGATGG 22140
TCCATGATTC GTCCGCAAAT ATTGTAAAAC CCTGTACTAC CTGGCTGATG CTTTAACAAA 22200
ATAGCTTCAG GGACATTAAA AAAGTAGTGT TTCCTGGTGT GCTGGTAAAT ATTTATTGAT 22260
ACAAAGATTG TGTAATCACA ATTTAAAATA TACAGTACTC TTGATTGTAA ATTCCTTATA 22320
ACCAATTGAT CCCCACAGAA TGCTCTTGTT GACTTTTGTT TGAGGCTCTT GTATCTATAG 22380
TGTATCCAAT CTATTATTGC AATTGATGGA CAAGTGCCAT TCTGATAAGA ATGTGGGCTG 22440
AGATTTCCCT TTATGTTAAT GAGTAAGAAG AAAGGGAAAC AGCAGAGCTA GACACTGGGC 22500
CTTCAATCGT TTGTTAACAA CACGAGCAAC CTTTTTGTTG AACTGGATAA TAGTTTTTGA 22560
ATACTGGAAG AATATTTCCT CAGTCTTTTT CTGTTATTCA CCATGCATTG GCTACAGTCA 22620
CATTTTAGAA TTTAACCTGC ATTATTAGCA TTTCTCCATC ACTTTTTATA AGTCTAGACT 22680
GGGGATTATT AAACTGTGGT CTAGGGGCCA TATCTGGTCC CCTGACCTGT TTTCGTACAT 22740
AAAGTTTTCT GGAACACAAC CATGTCCACT AGTTTTATAT ATTGTATATG GCTGCTTTTG 22800
TATTACAATA GCAGAAGCAG AGTCGAGTAG GTTGGACAGA GATTTAATGG ACGCAAAGTC 22860
AAAATTATTT AATATCTGGC CTTTTGCAAA ATAAGATTTA CCAAGCCTTG GTCTGGGTGG 22920
TCAACAAAAC AATAAATCAA GCCTTGATCT GTAGTGTCTG CCAATTTCCA TGGTGTAAAT 22980
ACTCCCATCA TGGCCAATTT CTATCTACCA ACATGACACA GCAAAACATA GAGTTGGGAA 23040
GAGATGTGTA AAGTACACCG TTATAGAGTA TTCTCACTCT ATAGCTACAG TGGCTATAAA 23100
TAACTTCCAG AGCATAGACA ATAGTAAAAT GTAGTCATAA TTAAGAACTG GTAAGTTTTG 23160
AGTGTTTATT ACCTTTGTTT CTAAATACAA TTTATTTAAT TTTAAGTTTA TATTTTAATT 23220
TCGAATAATG GCTGGGTTTA ACAAGTGGTT TGCAAAATCT CTGAGAACTT AACAATCAGT 23280
TATCATGAGT TGGCACTATT GCTTTCCTTT GGTGCCCAGC TGTCTTCTTT TTTCAGCCAT 23340
TTCCCTGTCT CCAGGAGATA ATCCTTTTTT TTCTTCTCAG CCTGTCTGCT TCCCAAAGTA 23400
TCCTTTGTTC TTTTCATGGC CCTCTGGCTA CGCAGGGACC CCACTTTTTG CCAAACTAAT 23460
CTTTTAAAAC ATATGTCCCA CAGAGTACCA TTCCCTTTCA TCTGCTTCCC ATCAATACTC 23520
TTATTTCTAC AATAGGGTTG ATACCAAATG GCCAGCAACA ATTTGTAATA AGCTGTAAAT 23580
-82- GATTAATGGC CTGGAAACAC TTGCATTTTA AAAAAAGGAG TCTTGTTGAC CCAAAGGTTA 23640
TAGGGTTTGA ATGTCTGGCA ACATTGCAGG TGTGAGGAAC GTCTTTGGAA TTCCTAGTTC 23700
CCCCCAAAAG GTTACTGTCT TCTTCAGTGA CAAACAACCA ACCCAAGCGT GTACCCTGAT 23760
GCTCCTCATT ACCCCTCAAA ACTTTTTCCT TTTCAATCTT TTTAGTTTTA GCTCTTTATT 23820
TCCCCTCCAC TTTCATTCCT TATTTAAACC TCTCAATTGT AACTGAAGCA GATGTTATAT 23880
GGACTTGGGG AAAGGGATCA AGAAATCATT CAGTTGTTTG TGCTTATCTA GAACTGTCAG 23940
CCCCTGAATT GTGTGGTCTT GGCTGGCATC TGAGCACACC TGGTGCATCA GCAGAATCAG 24000
TGTTCTCTCA GTTCCTGGTT GGCTCTACTG TCTGGCACCA TTCGGCTGTT TGTACTTATC 24060
TGGAACTGCC AATGGGAAGA TCACATGGTC ATTGAGAAAC CGCACCCTGA AGAGATGGCT 24120
AAAAGCCTGG AGGGCATGCC CATCACAGCC TTGCCGGGAG TGTGAAAGGT GGTGTGAAGA 24180
CCCTGGGGCT CACAGGACTC CCTCACCATG GGGCACAGTG TAAGAAGGTC CACGGTGAAA 24240
ATGCAGTAGG AGGCAGTTAC ATCAGGCTCT GGATCGATGA TATCAAGGAA CAACCCAGGC 24300
TGAAGGAAAA GGCGTTTGTG TTTCAGGAAA GATGTATTGA GCCTCATCCA TGCTCCAGAC 24360
TTTGTTTAGG CCCTGGGTTA CAGCATGGAA TGGAATGAAA CCCCTGTTCT TTAGTTTCTT 24420
ACATGTTGAG TGGGTGAGAC AGAAAGCAGC AATATGGTAA AGAGGGGGGA ACAGGGGAAG 24480
AATGGTAGGA GATCAAGTTA GAGAGGGGAA TGGGCTAGAT CATGGAGCAA CCGGGGCAAG 24540
ATGTCAAGCC CTTGGAAGGT TTTGAGCAAG AGAGTGTTAT GTTCTGACTT ACGTCTTGAA 24600
ACACTCTAGT TGCTGTACAA GGAGACCAGG TCAGAGGCTA TTGCAGTTGT CCAGGTGAAG 24660
GTGGCCAGGT AGCGATGGAG GATGAGAAGT AGAAAATTCT GTGAAGGCAG AGCTGACAGG 24720
ATTTACAGAT GGATTGGCTC ATGAGAGGAA AAGAGGGACT CACGGATGAT GCCAAAGTTT 24780
TTGACCTGAG AAACTGGAAG AATGGAATTT CCACTTACTA TGATGGGAGA GGTTGTGAAA 24840
GGATGACTTA GGGGTTGGAG AAAACCAGGA GTTTGGATAT GGGCCTTAGA TATTGCCATG 24900
CAGATGTTGA GTAGACAGCT GCACATATGA GTTGGGAGTG CAGAGGGAGA GGCTGGGGTT 24960
CTGGGTATCA GTATATGAAT CATCTGTGTC CACATGGCAT TTAAAGGCAT GAGACCAGGT 25020
GACCCCCCTT ATAGAAAGAT TAGATCCAAA AGAGTAGTGG TCTGAGGACT GGGCTTTAGG 25080
CCCTGATGCT CAGAGGTGAG GACCCAGGAA AGGAGACACA GAGAATCCTC TTTGTCAGAG 25140
CATTACAAAA GGGCTATTTG GAAATAGTTC AGGTGGTGAC TGGGTGAAAA GCCCTTCGAA 25200
CAGCCTCAAG GACCCAGGCT GGTGGACTGC TGGCTGAGTC CTGTTGTGCC TCAGAGGATA 25260
TTGTAATATT TGGAAAAATT TCTCCAAGTC AAATTTAAAT TAACATGAAT GTCATATGGC 25320
TTTTTGGTAC GTCCTACAGT CAAGCAAATA ACAATTGGAT AGGGTAGCTG CAGGAAGACT 25380
GGGTGTCTCT ACAGTGGTCA AGTTGGAAGA ACAAAGAATG AGTGATTGAT CTTTTGCTAC 25440
TCCCCAAGGG GAGAAGCCAC TGATAGCTTC CTTGGAAGCA CTTTGTACCT CACCTGCCCC 25500
AGAGTAGATT AAATATTAAG TTTCCTCCCT TCTTTCAAGT CCTAGTGCTG CCATTGATAG 25560
TGCTGTGACT TCAGGAAAGT TGCTTAACTT TTCCAAACCT CTATTTCCTC ATTACTAATG 25620
AGTAATAATT CCCACCATAG GGTGTTTATA AAGATTAAAT AATTTTAAAT ATGTTGAAGC 25680
ATGTAGTGAA CTGCAAAGCA ATATGCAAAT ATAAGAGGTG GAAATGACTA TGCCTATAAT 25740
TACGTGGCTC AATTTACACA ATAATAGATT TTCACACTTT GCATAAATAA TGAGGGTTTT 25800
TATACTCAAG TCACTGAACT TACTATCTTC AGGATCCAAA ATCCCCAAAC AGAAGGCATC 25860
CCCTACTGTT AGCTCAAATA GCTCTTGCTG GTTTAGAGAG TTAATGCAAG CCCCACTGCC 25920
TCCTGAGCTG GAAACATGAA ACAGAAGTTT CAGTTCCCTA ATCAATCCAT TCTTTCTTCC 25980
TCTGGCTTCT GATAGGCCTC CTCCTTATCT TTGTAAACCC TGTAGCTGGT TGCTAGTTGA 26040
AAGTGCCTCT GATCTCCCTC TTCTGCCTCC CATGATGTTG ATAAAAAGCA CGAGGGCACA 26100
TGCAGGATGA AAACGATCGT GGTCCTGCCA GCCTGAATTA TTAAAGCATT TCAGTCCTAA 26160
GTATGAGGTG TGTATATGTT GGGGTGTGGA GTGAGTTGTG GAGATGAGAG ACAGCTGAAT 26220
TACATAAAGT TGAGAAGATC TGAGTTCTAG TCTTGAAATT CACAAGCCAT CTCTATACAA 26280
TAGTTCCGTT ACTCAGTAAA GTAAAAGCAT TGGATCTAAG CTTTAAGGAC CCTTCTAGTT 26340
CTTTCTGATT GGAATTCTGT GACTTCATCT TTTGTGGGTT AGAAACTCAT CACTCTGTCC 26400
AGTTATTTCT ATATTATGCC ACCAGATGGC AATGTTTCCT TAACCCCAAA GAAAGTTTTC 26460
ATTCTGGTAA AAAGTCAAGT TTTGTTGCCA ACTTTTCCCC CTCTGAACGT GCAAAAGAAT 26520
GATTTTCCGA AGCTGTGGAG GAAAGAAAGA ACTCTCCTTC TGAACATCTC AGGTGGTTTA 26580
TGCTGGAAAC AGACAGGACC CTGTTTAGAG AAGATCTCTC TTTTCTTCGT GGACTGGGAA 26640
CTCCAGTTGG AATGATGTCT CCTGTGATTG CGTATGGTGG GAGGTGGGAG ATGTTGGAAT 26700
TGGCGTGTCC TCAGGAGGCT TGGGGGTGGG GGAGATGTGC CCTAGCTGGT GGGCCTGCAT 26760
GAGCCCTGCA AAACTCTGAC TTATAGAGGG GCATCAGATG CCAAGTTTTA CCAGACCATG 26820
CAGAACTAGG AATTGCCAGA TGCACTCATA GGGCAGCTAA AATGGTCCTG GCAGAATCAG 26880
ACTCTTTCGC TCATAAAGGT CAGAGACGCA AGAAAGTGAC ATAAAGTCCA GCCCTTTTCT 26940
-83- TGTGCAGATG GGGAAATTGA GGCCTAGAGC AGGTCAGCTG TCCTGATTCT ATCTCCTTGC 27000
CAAGTTACTT TGTATTTAAA CATTTCAAGT AGACTTTTCA ATCATCTCAT CTTGCTGTGT 27060
TCAGCTAGCG CACCTTGTTA AGCCTGTTGG CCTCCGGGCC TGCCAAGCCC CTGCATCTAT 27120
ACACACCAGG GCATGCTGCA TGCGCTCAGT GAGACTTCAA CAGCTGACTG ATTCGTTCAA 27180
ACCTATCAAA CAGCAGACTT AGCTAGTTGG GGAGAAAAGT CATTTAAAGT AATTGCTTAT 27240
TAATCTGCAA AACAAGTCTC ATAGCAGGTT TTTATTTTAT TTTATTTTAT TTTTTTGCTT 27300
TTAACAACGA TATAATAACA ACAAACATTT GTTTAGTGTT TCCTGTGGAC CAGGCTCTGT 27360
GTTAAGCACT TAACATCACT ATATCATGCA CTTTTGCTAA TAAAGCTGTG AAATAGTTAT 27420
TACTATTTCT GCTTTACAGC TGCAACAGAG ACTCAGAGAG GTTAGGTAAC TTGCCCCAGG 27480
TCACAGAGCT GGAAGGAGCA GAGCCAATAT TCACACCCTG ATTTGCGTAA TTCCAGATTT 27540
GATCTTCTAG CTTCTATGCT GTGCTGCCTC TTCATGACAG TTTTTCTCAT GTACAGGATC 27600
TGATGCAGAA ACTTATCGGA GTTTCTTACC GGAGCACCAG TCACCTCTCA TCATTTTCCT 27660
GTTTTGACGT GAAGGCTCAG TGATAGTGAG CAGGCTCAGG GTCTACAGAG TTGGTGATAT 27720
CAGCATCACA CAGGACATTC AGAATGTTGA CTCCAGGGAT GTTGAGAGAT ACTCCTGCAC 27780
AAAGCTGCCA GCACCCGTGT CCAAGAAACA CTCAGAATCT AGGTCTCCTT GTATATTTTC 27840
CCCACTACCT GCAAAGGTAA AGAGGAACAG GCAGTGCTGG GACCGAGGGA GCGACAGTCC 27900
TAATGGAAGC TAGTGTGTTG AGAGTCTCCT CTGTGTCATG CTCTGAGCGA CATGTTTTAT 27960
ATGCACGATC TCATTTAGAC CTTGTGACAG CATGTTGTAG CAAGGACCCC ATCATCACAG 28020
GGGGCAAATG TCTGCAGTGC AGAAAGTCGT CCTGAAGAAA TGGATGTCAG ATAAAAACAG 28080
TCTTCATAAA TCAATGATCC TGTTTTACCT CAAAAGTGCA TGAAATGGAA ATGGAAATAT 28140
CTTGTGAAGA TGTAGACAAA TGACGGTCAT TGCCCAGAGC AGTAGTTACT GTCAGAAAAA 28200
GAGATAAGGA TTTCCAGTCT GACAGACTGG ATTCCTGGCT CAACACCACC CCCTTCTAAC 28260
CATGTGACCT TGGGCAAATT ACCTAACCTT TTCTGAGTCT CAATTTCCTC ATCTTCCAAA 28320
AGGGGATAAT ATCATATATG TTCCAAGATT GCTGTGAGTA TTAAATGAGA TGATGTATGT 28380
AAAGTACCTG GCCAGCAGTT TCTGGCACAT AGTAAGTATT CAATAAAGAC TAATGGTGGA 28440
GATGAGTATA GGGGCTACTA ATGCCCATCC TTACTCCAGA GACTTCTTTC TGACCATCAT 28500
GAGGCACTTT TGAATATCTA AACCCATTTA AAGCCCACTT TTCTCTATGG CTGGCCATTT 28560
CTGCCTATTG ACAGCTAATT TGCCTCATCC TACAGGACAC CTTCCATGTT TCCCCAGACT 28620
CCAGAAATCA GGTATTAAAT TATCAGGGCT TCAGGAGCCA TGGTCTATGA TGAGTTTACT 28680
ACCTGTGCCC AATAAATGTT TAAGAAATAA ATAAGAGCCA ATATAACTAT AAAGACCAAG 28740
AGCCAAAATA AGTCTCTTTG CTTGCGCTTT AGATCTTAAG AGTCCTTTAT ATTCAAGCTG 28800
CTCAGAGTCA AACGTGTGCC TAATAAACAT TCTACAAAGG TCCTGGCGTG GTGTGACCAA 28860
AGGAAGAGAG AGGGCTCCAG TGTCTGTCAC TGGGAGACCA GATGGACAGC CACGTGGGGC 28920
AGGGCCACTG GTGCCACATG TCCAGGTCTG TTAAGCCCTA TGAAAGACAC TTGAGTCAAA 28980
ATGTATTTCT ATCTAAGAAA GAAGACTATA AATGGAAAAG GGAGAGGGGA GAAGACCTCT 29040
CAAGGGCATC TCCCTCTAGA AGTAGAGATT GTGAATCTGC AGCAGAAAGG TTTTAAACAA 29100
GGGATAGCAG AATGCCTGGA TGGTGTTCTA GTGCCTGAAT GGAAAAAGGC CACAATGACC 29160
AACAAATCCC ACCTACATCC GCCTTCCTCG CTGCCTGAAA TCCCACCATT AGGATTTTTT 29220
TCCTTTTGGG TTAGCAACCA AGAAAGAGTA AAGTCTGGAA GACTCTTATT CCACATCTTC 29280
ACTTTGCAGC GCCTCTTTTT TTTTTTTTTT TTGAGATAGA GTCTTGCTCT GTCACCCAGG 29340
CTGGAGTGCA GTGATGCGAT CTCAGCTCAC TGCAAGCTCT GCTTCCTGGG TTCACATCAT 29400
TCTCGTGCTT CAGCCTCCCG AGTAGCTGGG ACTACAGGCA CCTGCCACCA CTCCCAGCTA 29460
TTTTTTTTTG TATTTTTAGT GGAGACAGGG TTTCACCGTG TTAGCCAGGA TGGTCTTGAC 29520
CTCATGATCC GTCCGCCTTG GCCTCCCAAA GTGCTGGGAT TACCACCTCT TCTTAATTAC 29580
AAACATAAAC AAAAACTAAC AACTTTCTAG TTTTTTCTTT TTCTTTTTTT TTAAATTACA 29640
AAAGAGATCC ATATTCGTCA GAGAATAATT GGAAAAAAGA GATAAGCAAA ATCAGAAAAA 29700
TAAATTCAGC CTGTAATCAC CCAGAGATAA CAATTATTAA AATTTAGGTA TTCACTTTGT 29760
TATTTCCTTT TATAACAAAA CTTTTTTTTC TTGTGAAATT TAATAGAATA CAATTGAACT 29820
ATTTTTTCCT TTATGGTTAA TGATTCTTGT TTCTTATTTA GGAAATCATT TCCTGAGTCA 29880
TAAAGAATTC TCTCATATTT TCTTCTAAAG CTTTATACAG TTTTGCCCTT CAAATAAGGT 29940
TAATAACCCA CCTAGAATTG ATTTCTGTGT ATGGCATGCA GTAGAGACAA GTTCTACTTT 30000
TTTCTCTCAA ATGAATATTC AGTTGGACCA AGGCTGTCCT TTCTCCACTA CTTTGCAGTT 30060
TCACTTTTTG TTGAAAAATC AATTGTTCAT ACATGGGTAG ATCTCTTTCT GGGCACTCTT 30120
GGAGTCTATT GGTCTGTCTA TAAGTTGAAC AGGATCAGAC AGGCTGTGCT TTGTTTCAGG 30180
TAACAAAGAA CCCCAACATC TCACTGATGA ATACACTAAA GTCATTTTTG TTTTCCATTG 30240
GCAGTTCACT TCTGATGCAG GAGATGCATC AGGGCAATCG CCCTTTGCAA GGTGAAGTGT 30300
-84- CTGCACCATT GGAAGTACTC TCCATCCAGG GGAGAGAGAC TGGAAATGGT CCATGAGGTT 30360
TTCATCGACC CAGAATGAAA GCATCACCCA TCATTCCCTT CTTGTTACAG CCTATTTGTC 30420
AGAACCAGTC AGAGTTCCAC CCACCTGCAA AAGGTTGTGA CGTGCTGTTT GCGATTTGCC 30480
TGGAAGGAGG GAATACCCAG ATACAGGAAA ATGCTAGTGA CGTGCACTTC CATCTAACTA 30540
TCTTTGAATG AAAATGACAG TCTTAATTAC TGCAGTAAGA TAAGCAGACT CTATACCTGG 30600
TAGAGCAAGT CCTCTTACCC CATTTCTTCT TCAAGAAGGT CTTGGCTAGT TTGGAACCTT 30660
GGCAATCCCA TATAAACTTT AGAAAATGCT AGTTAAGTTC TTTAAAAATC CTGCTGAGAC 30720
TTTTATTGAA TCCATAGCTT CATTTAGAGA GAGCTGACAT TTAAATTAGG GAGTGCTCCA 30780
AGCCACTAAC ATAGAATTTC TCTCTTTTAC TCCAGGTCTT CTTTAATTTC TCTCGAGTGT 30840
TTTGTAATGT TTTGCGCAAA GTTCTTGCAC ATCTTTTGAT AGATTTCCCC CTAGGTTTTG 30900
GATATTTTTA AGATGCTAGT GTAAATGTTA TTGCTATATA TTTTTCATTT TACAAATATA 30960
TGTGTTTAGT ATATAGAAAT TTAATTCATG TTTCTGTATT GACTTTATTG AGTAACCTTA 31020
TGAAACTTTC TTAAATTCTA AAAATTATCC ACAGCTTCCC ATAGATTTTC TATGTAGGTA 31080
ATAACATAAT CCACAAAAAT GACACTTCAA TTTTTTCCTT TCTGTTTCTT ATGTCTTTAT 31140
TTCTCTTTCT TGCATTTCCC ATGTGGGGTC CCTAGACACT GTTGAATAGA TGTCGTGATA 31200
GTGAGCATCC CTGTTCTGTA CACAGCCTCG AAAGGAAAAT TTTCAGAGTT TTGTTTTAAA 31260
CAATCTGGTT GTTATAGGTT TTATTGTAGC AGCTCTTCAC CAGATTACCT GCATGTTTTC 31320
TTTTTTCTAG TTTCTAAGAC TTTTAATCCA TTAATGAGTG GATGTTGAAT TTTAACAAAT 31380
GCTTGTCTCT GCATGTATTG AAATGACTAT ATGACTTTTC CCCAATTGAT CTGTTAAGTT 31440
GGTAAATTAC ACTGATATTC CAAAGTTAAA GCAATTTTTA CACTGGCACC CTCAAGTAAG 31500
CCAAATTTGG ACATGATGTA TTTTTAAATA TATATTGCTG GTGTTGGCCT GTTAATATTT 31560
TATTTAGAAT TGTTGAGCCT ATGTTCAAGA ATAAAATTGG CTTGTGATTT TCCTTCACAT 31620
ACTGTTCATA TTGGGTTTTG GTATCAAGAT TACTCAAGCC TCACAAAATA ACATAGGGAG 31680
TCTCATTTTT TCTATTTTCT GGAAGAGTTT GCATAAGTGT GGCATTATAT CTTCTTTATC 31740
TCATAAAATT TGCTTGAGCC ATCAAATCTT AACATTTTAT GACAGGTTGA TTTTTTATTA 31800
AATCAATGAT TTTAATAGTT ATAGGATTAT TAGGATTTTT TATTTCTTCT TTTGTTAATT 31860
TTAGTAAGTA GTGTTTTCCT AGGAATTTGT CTATTTTATC AAAATTTATA AATTAATTCA 31920
CAGAGTTGTT TATAATATCT TCTAATTATC TTTCTAATGT CTGCAACACA TGTAATAATG 31980
TTATTTTTGC TTATAAATTG ACAATTTATA ATTGCGTATA CTTATGGGGC ACAAAACAAT 32040
GTTATGATTT ATGAAAGCAA TGTGGAATAA TTAAATCTAG CAAATTAATA TATCCATCAC 32100
CTTAAATACT CATCATTTTT TGTGGTGAAA ACATTTGAAA TTCACTTTTT TTCACAATTT 32160
AAAAATGCAC AGTACACTAT TATTATCTAC AGGTGGTTCC TGACTTCTTA TGATGATTTG 32220
AATTATCACT TTTCAACTTT ACAATAATGT GAAAGGAATA TGCATTCAGT ATGCTCTATG 32280
ACTTATGTTG GGATTATGTC TGGATAAACC CATAGTAAGT TGAAAATATC AATGGGCTCA 32340
TCCAGATATA ACTCCATCAT AATTTGAGAA GCAGCTGTAT ATTTATCATG GTGTGCAATA 32400
AATCTCAAAA AAAGACTTAT TCCTCCCGTC TGAGATTTTG TACCCTTTGG CCATCACTCC 32460
TTCATTCCCC TCACCCACAG CCCCTGTAAC TACCATTCTA CTCTCTGCTT CTATGGATTT 32520
GATTGCTTGA GATTCCACAT GTAAGTGAGA ACATGTGGTG TTTGTCTTTC TGTGTCTGGC 32580
TTATTTTACT TAGCATGATG TTCTCCAGTT TCAGTGATGT TGTTGCAAAT GATAGAATTT 32640
CCTTCTGTTT AAAGGCTGAA TTATCCCATT GCATGTATAT ACTACATTTT ATTTATCCAT 32700
TCATCCATTG ATAGACACTT AGGTTGATTC CATAACTTGG CTAGTGTAAA TAGTGCTGCA 32760
GTGAACATGG GAGTAAGGAC ATGTCTTAGA CAATCTGATT TCAATATTTG GATAAACACC 32820
CAGAAGTGGA GTTACTTGGT CATATGATAA TCTAGTTTTA GTTTTTAAAG TAACTTTCAA 32880
ATAGTTTTTC ATGATGGCAG TACTAACATA CACTCCCAAC AGTGTACAAG GGTTCTCCTT 32940
TCTCCACAGA TGTTCTCTTT TTCATTACTG ACATGAGTTA TCTGTGCCTT TCCCATTTTT 33000
TGTCTTCATC TGTCTCAGCA GAGGTTTATC AATTTTATCA TTTAAAAGGT AAAAATTGTT 33060
ACCTTTTAAA TCTTGTCTAT TGTATTTTTT TGTTTCATTA ATTTTTGCTC TGATTTTTGT 33120
ACTTCCTTTT TTCCATATTT TTAGGAGATG ACTTTGCTGT TCTTCTAACT TCTCTTTCTA 33180
GGACTCCTAG AAATATGTTA AGTCTGCTCA TTGTATTTTT CTCACCTTTA TATTTTCCAT 33240
TGTTTTATCT CTTTCTTATT CATTCTGGGT AGTTTCTTCT AATCTACCTT CCAGTTCATT 33300
AATTATCTCT TTACCTGTGT TGAATTTGCT ATTAAACCTA TCTGAATGAC TTTTTCATTT 33360
TTTATTGGGT TTTTAAATGT TAAAATTCTC ATTCCTATTT GGTTCTTCCT CAAATTTGCA 33420
ATGATTTTGT TTCAGCTGAT TGCCAAAACG TTTTTAGTTC AAGTTCATCT CTTTGAGCAT 33480
AGTGAGCACT GTTGTTTTAC AGTCTTTATG TAAATACCTT CTCTTTTATT AATCTTTCCA 33540
CGTTTCTGGT GGAGGGACTG GCTATGAGAG ACAAAAACTT TCTTTCAGGT GCTTTTAGGA 33600
CTTACCCATA TTTCTTTCAT GGTGTCTATT ATTTTATTAT CTCATTATTT AGATACTTTT 33660
-85- CTCCTCTACT AAACTAATGG TTCAAGGCTT ATCAAAGATA AATCCTCTGT CTTGTTCATC 33720
TCTGTGTCTC TCATGGTATC TAGCAGACTT CCACCCAAGA TATAAAGACA CTATGACTAA 33780
GTGAATGATT TTAGTCTTAC CTACCTGCCT GTTAACTTAC CTACTTGCAT CTCACTTATA 33840
CTTCAACTTT TGGCTTCTTC CTCAACCTCA ACTACCCCAT TCTTCCCATG GCTCACTGTG 33900
CTCACTGGCC TCCATACTGT CCCTTAAATA AGGAAAGCTG CCCTAGCCTC AGGGCCTTTG 33960
CACCTGCTCT GCCTGCTGTT TGGAATGCTC TTCTTCCCAT ATACCCATCT GTTTTAATCC 34020
CTCATCTTTT ATTCCCTCAT CCCATCTCTT CAAATGTGAT TTCTACAGAG GGTTCTCTGA 34080
CCACCTTATC CAATAACCAG CATTCCGTCT CCCCTCTGCC ATTCTCCATC ATCTCACCAT 34140
GCTTTATATC ACATATCACT AAGTGACAGT ATACTATAAA CGTACCCATT TGTTTACTGT 34200
CTGCCTCCCT AACTAATGTA TAAGCTCTCT GAGGGCAGGG ACTCTGTTTT ATTTGTACAC 34260
CACAATTATC TCCAGTGCCT TGAATAGTGT CTGGCATGTA GAAGGAATTC AAGAAATACT 34320
TGTCAAGCTA GGTGCTGTGA TAACTACTTT ATATGAAATT AAGTATTTCT CCTCCAGCAG 34380
CTCTAAAAGT TTAGTATGTT ATTATTGTCT CTGTTTTACT GATGAGTGAA CTGAGGTTCA 34440
GAGAGGTTAT TTAGCATACG TATGAAGACA GAATTAGTGA GTGATTGACC TGAGATTTGA 34500
ACTCAACCTG TGCTGTCTAA AGCTAGCCAG GCAGCCTCAC ATACATGGCA AATGCCTACT 34560
GAGACATGAA CATGCAGGTT GGGATCCCAA ACTGTTGGGA AGCATAAAAG AAAAACACTA 34620
AAGATGTGGG GAGTGTAGGA CTTTTTTTTT TAATAGGCCA GTGGCCCTCT CTGCAACCCT 34680
TTGAATGATC AGCTTGATCA GAGAATCCCC TACCCCTACC CCTGCCTCAG CCAGTTTCTA 34740
TCTGGCTGTG TCATCAGCTG GCTGATCCAA ACAGCAATGT CAACAAAAGA ATGGTGATCA 34800
GGCACGTAAA GCAATGTGTC AGAAAGAAAG AAAAGGCAGC TCAGATGATG CAAGATCATC 34860
CAGATGTCAA GCACTGTGTG GTGGCACACT TGCCCGTTCA TGTTGTTGAT TTTTTAAACA 34920
TTTGTGATAA GAACAAAAAC TTAGTTGCTT CCCTCAGGTC CTCCCTGTAT GGATTAGTGC 34980
AGACATCTGC CGCTTCAGGC TTTCTGATTG GTTCCCACTG GTTTGGGGCA AAACCGGAAA 35040
CTTCTGAGCC AAGTGCAGGG GCAGAAGAGC TCCCAAGAGC TCCTGGGAAA ACTAGGAAGG 35100
ACAATCAAGA AACCACCGGC AGCTCCATTT GCAGGATCTC ATCCCATCAG GGGCTGTCTC 35160
AGGAGGGGGA ATTGGAATAC CATTCACCTG TCCCCTTTGC AGATACACCA ATGTCTCGTT 35220
CAAGAACAAG CAGAAAGGAA ACACCAGATT GCCCAGAGCA CAGGATTAGG ACACACCACA 35280
CAGAGCCAAC TCAGCGTATC ATTGTTTGCA TTGATCATCT GGGGATGAAG CAGGCTCCGT 35340
TCTGGAAGGG GCAACCTGAA TAGAGAAGAG TCTGACATTG GAGTCAAGCA GAACTTGGTT 35400
GGAATTTGGC TCATTGCTGG GTGATCCAGA GACAGTTATT TAATCTGAGA ATCAGATATC 35460
TTGTCTGTTA AATGGAAATT ATAGTAGCCA CTTCACAGGA TTGCTGTAAA GAGTACATAA 35520
AACCAGGTAC CTGCAATGTA TAGTGCTAAG CCTGACACGT AGCAGGGTGT TAGTAAGTGG 35580
TACCTCTGAC TGGGGATGGA AGCCAGAGGA GCTGGACCTT TATTTGACTG GCCAGAAGCC 35640
AGCTCTCTAG TCACCTTCCT GATCCTTCCT TCTTCTGTGT GTACACGGAC AATGTTTTTC 35700
TACATAATGG AACAGTGGCC CTCAAAACTT GTTTTCATAA GAATTATCCA GGTTGCTAGT 35760
TATTAATACT AGTTATCCAG GTTGCTAGTT ATTAATACTA GTTATCTGTG TTGCTAGCTA 35820
AAAATACACT CAGTTCCCAT CCCCAGATTT TTCTATTTCA GTAGGTGGTA GTGGGTTCAG 35880
GAAATCTGTG TTTTTACCAA AGTATCCCCT ACTATAGAAT TAATTTTTGT GTTCCCCCCT 35940
CATTCATATG TTGACATTTA AACCTCCACT GTGATGATAC CAGGTGGCTT TGGGAGGTGA 36000
TTAGGTGATA ACGATGAAGC CCTCATAAAT GTGATTACTG ACCTAATAAA AGAGACCCCA 36060
GAATGCCCCC TTGTCCCTTC TGCCATGTGA GGTCACGGTG AGAAGATGGC ATCTATGAAC 36120
TAGGAAGTGG GCCCTCACCA GACGCTGAAT CTGCTGGTGC CTTGCTCTTG GACTTCCCAG 36180
CCTCTAGAAT TCTGAATAAT AAATTTCCGT TGCTTGTAGC CTAGTCTATG ACATTCTTTT 36240
GTGGCAGCGT AAATGGACTA AGATGTGCAC CCTCATGCCC TTTAGGGAAT TGTGACTTTG 36300
AGAAATGCTG CCCTAGGATT TACAGAATGC TGACAAAGCT TTGTTGACTC AAATGCAAAA 36360
TATTCTTATA AAGACCAAAA TAGAAATGAA TACTCCCTTG AACTCCTTTG GATGTGCACT 36420
TTGCGTAGTT ATAGCACCTT TTCATCATGT GCAAATGAGA CGCAAATGAA TCCTTAGTTT 36480
GACCCAGAAA GAATGTCTTT GCTGGTAGGG ACTACGGGAG AGAGAGAAGA GCCAGAATAC 36540
TGTAGGAAAA TTAACACCGG CCACGAGACA ACTGGTTGCT AGCTCGGTAG CTGTGCAACA 36600
TTGGCATGTT ACTTGAACTT CTAGAAATCT GTTCTTTCTT CTGTAAAATG AATATGGTCT 36660
GGAAAGTAAA GACCAGTCAC CTCCTCTATC AGTTGGAGTC TAATCAGGAA GAAACCTAAG 36720
TGTCTTCAAC AGAGGGAATT TAATGCAGGG AATGGGTCAC ACCAGTGTTA GAAAAGCTGC 36780
AATGCCAAAG AGGGGATAAA GAGATAGCTC AAAGGTTAAT AAGAGCAGAA AGTCACTAGT 36840
ATTCATAGGC TGAAAAGAGA AAGGGAGGAG ATAGTGTTCC CGGAATCCCT GATGGGCTTG 36900
TCTGGAGGGC GCTGGGGCCA TGGAGGAAAT GTAGTAGCTG CTGGAGGCAT GCTCAGGGCA 36960
GAGAGGGAGC AGAGAAATAC CCTGGCTTCT CATTTTCTTT CTCCAGTCCT TGCAGGCACC 37020
-86- TCACTGGCTG AACTCAGGGG AGCATTTCTC CTCTACAGAA CAGAGTCTCC TTGCATACAA 37080
CAAGAGGGTC AAACAGAGGA TGGCTTAATT TTTCCTTCCA TTTCTCACTT CTATGATTCT 37140
CTCCCTTCAG GTTAAGTAAG TGAGGGTAAG TAAGCTGCCC AGTAAGTGAA CAGTTTTCCA 37200
AACAAGCCCA CAGCACCACC TCTATATACA GCAACTCTCT GTTTATCAGC ACTGCATTAA 37260
CCAGGACTCT CTATTAACTG GGACTTCCAG TTCCTTAAAT TTCTTCATGG TTCCTGTGTA 37320
CTCCCAAAGC ATCTTCATCA AACAAACATT AAGTTACGCT TAGAGACCAT TTCTCAATTG 37380
AATATAGATA AAAGATTCTA AGGCCTTGAA AAAAATTAAT ACATGCATAT TAGATATAGC 37440
TATAAAAGCC AGACTATCTG ATTAATTATG TGACTGGTGT TAAACTGTTT GGACAAAGGT 37500
TGGCTAAATT CCCTATGAAT ACTTACTTCC CTACTTCTGT GGACAAGGAA AAATAGACCA 37560
AAGGTTCAGA TAAAAGCTTG ATTCAATGTC ATCTCTTTTC TCACGAATCT TGGTCATGTG 37620
TGGGAAGTGA CCCAGATCTA GAACCTTAGC CTTTGGGACT TAAAAAAAAA ACAAAAAACT 37680
GTTGAGTTGA ATCATTAAGT GTTACTGAGG GACAGGAGAG AGGAGGGTAG CTTTCTTAGT 37740
TCCAAGACAA ATTTTGTTAA CAAAGATCTG TGGGTAGACT TGTGTCTGGG CAAAAGATCA 37800
GAAGATGTGC TGTTCTAGGC CTCTTTGCCC TCAGACCCAT TCCCTATCCT TTCCCCTTCA 37860
CTGTACCCCC TTATCTCCTC TTCTGCTGTC TTCCTCTGGG CCTGATGCTT GAGGATCCAG 37920
AAGTTTCTCA GGCTCCCATG TTCCAGCAAT CCAGGCCTCC TTCCCAGTAA GGGATGAGTA 37980
CAGGGGCCAC ACATAGCCCT GCAAGTTTTG TAATCCAACT TGAAATCCAA TGGCAGAATG 38040
AATGGTTATA TATGGTGTGA CCCAGGACCA CATGCAGTTG TATCACATGC ACTTACAAAA 38100
GAGCCCCATT TCTTGGACTC ATTCCCAGAC TCAATCTCTC TGAGGGTAGG ACCAGGAATT 38160
CGGCCCTTTT CACAATCTTC CCAGGTGATT CTCTACATAG TATAATAACA CAAACTCATG 38220
GAAATATATT TAATGAAAAA TGAATAAAAG AATAAATGAA ATAACAAATG GTGATGGCTG 38280
GCACAATGTG TGTATCCATT CTCCTACTGA GGTGCACTTA CTTTGCTTCC AAATGTTCAT 38340
TTGACAAGTA GTGATGCATT GAATATCCTT GTACATGTGA GCATGCAGTA AAGTTTCCAT 38400
GGGCTTATAT TTGCTGGATT ATGGGCACGT GCATCTTCCT CTTTTCTAGA TATTAACAAA 38460
TCACTCTCCA AAGTATTTAT AACAATCAAC ACTCCTGAAC AAGCAGTGGG TTGGAATTCC 38520
TTCCTCATCA CATCCTGGCC AACAATTATT ATCATCAGAT TTTTTAATTT TGCCAATTTG 38580
AAGGAAATGC AGTGGCTTCT CATGTGTTAG TGTTTCTGAT GATCAGTGAG GTTGAGTGTC 38640
ATTTTTTTTT ττττττττττ TTTTTTTTGA GATGGAGTTT TGCTCTTGTT GCCCAGGCTG 38700
GAGTGCAATG GTGCTATCTT GGCTCACTGC AACCTCCGCC TCCCGGGTTC AAGTGATTCT 38760
CCTGCTTCAG CCTCCCAAGT AGATGGGATT ACAGGCATGC ACCACCATGC CTGGCTAAGT 38820
TTTATATTTT TAGTAGAGAC AGGGTTTCAC CATGTTGGTC AGGCTGGTCT CAAACTCCTG 38880
ACCTCAAGTG ATCTGCCTGC CTCGGCCTCC CAAAGTGCTG GGATTACAGG CACGAGCCAC 38940
TGCACCTGGC CGATTGAGCA TCTTTTTATG TGTTTAATGA TGCTCATTTT TTATTGACTT 39000
CCTTCTGTGC TTTCTTTTTT TTAGCAGTGA ATTTGAGTTG TAAGAATATG TATTTCTTTC 39060
ACTCTGGGAT TCACCTACAT AAAGTAATTT TCACTTGAAT GAAAAAGAAA TCAGTTGTAT 39120
AAACATCTGT TTTTTCTGAA TTTTACTGGT GTAAAAATGG CCACTCAGCC CTGGAAGAAA 39180
CAAAGGCACT TTGCCAACTG AAGTTGCAGA TGGGAAATTT TTAGAAAGGT CCTGTTCAAC 39240
CTCTGGAAGG GGAAGATCAT ATCTGAAAGT CAGGGTAATC CACCCAACCC . AAATGTTTCT 39300
TCTACTATGG GTTCTGAGGA TTCGTCCATG TGCTTCTTCT GCATTGCTGC CATCTGATTT 39360
CCTTTGCTAG GCTCCTCTTG CAACTTGGGC TACAAAGAGG TGCTTCATAG TCCACAGTCT 39420
TTGCCTCACC TTCAGTCTTG AGGTGGTCCC CTAGGAGTTA TTGGTAGTTG CCGCTGGAAG 39480
CCATTCTAAC AAACCTGGCG AAGGCACAAA AGGATAGAAA GCCTTTAGCC AATATGGTGC 39540
CATCAAAAAC AAACAGAGCA CGCTGCCCAG TCCTCTTCTG GTTGCCTTTA CTAATGCATC 39600
AGTCATACTT CTTCTGCACT CGATCTTAGC CAAGAGGTCG AGAAGCCATA GTCATAATTC 39660
TTCTGAAATT AATCTCTTCC TGCCCCACCT CCCCATCATC TGTCTTTGAA TTCCCAGGGC 39720
TAGTACTCAT AAGATTATCT CTTTCTTCTC CTTTATGAGG AGACCCATTC TTTTTCACAA 39780
ACCAGCCACA AAAGCAAGTG TCATTACCCC CTACCGGAAA TACCAGACAG AGAGTTCATC 39840
TGGGGTTAGT TTCTAATCAA GCCTCCTGCC CGGGTTTTTC CTGCTCCTGT CTTGAAGCGA 39900
CCACAGGGGG AGAGCAGTTT CCAAATATGA TCCCTCCTTT CCACTGTCAC TTGTCCAACC 39960
CCGACCACTA TCATTCTTTT ATTTGCTTCT CCCCTGAGCC AGCCAAGAGC CTAGGTCAGT 40020
GACAGGGCAG GCAGAAGAGA GAGGGGCTTC CAGGAAGGAG AGGGAGCAAC CCACAGAAGA 40080
GGCAGCAAGA CAGGAAGGCG GGCAGGGGCT GAAAATCCAA TACATATCTA AGTACATTTT 40140
TCTAGGATGG GCTTCTACAC TCAGCCAAAA CATATATTGC ATATTGTTTG TATTTTTTAG 40200
AGGTTTACAG GTCTCCCTGA AAGTCCCTCT GTGGAATTAT AAACCTCTAA TAAAAAATCC 40260
CAGGGTTAAA GAAAGGAAAA GATGAAGGAG AGGCCCACAC TCTGAAAGGA AAGGGTTCAG 40320
CGACTCCTGG AAGGTTCTGG ATGGTGCTTC CTTGACCAAG TCAGCTGCTT CTTCTACCTG 40380
-87- GTCTCCTTTG TGGTTCAGCT GGGGTGGGGC TTACTAGAAA AAGCTGTGGG AGGTGGTTGC 40440
TCCAACGTAT GGGGGCTGTC TGTAAGTGTA GGTGTTATCT GATGAAAGCT GCCCCGGGTG 40500
AGGGTTTGTA CAGAAAGCTC CTGGTGGTGG GGAGATAATG TCAAGCTTCT CTCTCTCTCC 40560
CAGATCCTGG TTGTATCCTC TGTCCCTCTC CACCCCCACC CACTCACCCA CAGACTTCCA 40620
AGGAACCGGC GCCTGCAGAC ATGCCTCTCT GATGCCCTCC CAGTAACCCC TGGCAGGCAG 40680
CACAGCGCCA AACCTCTTGG CCTTACCCCA CTGGGCCCAT GACCCAGTGG CTGTGCCTCT 40740
GGGTCCTCCC TGTCCTGCAA AGAGAACTGG GCCCTCAGTC AGGTTCTTCT GCTCCAACCC 40800
AGTGGCCACC TGTGCTCTTG GGGAGCTCGG GGGAGGCTGG GAAACTTTCA AAGAGCAGTT 40860
AATCACTAAC TAGCTGGAGA TAAGAGAGAG AGAATGAAAC AATTGAGAAA ATGCCCAACC 40920
CAGAGGTTAG TGCTTCCCTG CCTGCACACG CCAGAACCTG GCCCGCCCAG AGAAACTGGC 40980
GATCAAACTG AGTTTGTTCA CTGGAGAGAG CTGACATACA GTCTCTAAGG GGCTGCAGTA 41040
TCCCAGGCTG AGGTCCAGTG GCAGCCGCTG CCCCTTTCCT CCTAGGGCCC TTTCCTTCAG 41100
CCATGCCTCA GCCCTGAAGA CAAACAGGAG CAGTTTTCAA GGAGCCCTTC CCTTATCTCT 41160
AAGGTCTGGG CCTGGAATTC AGCTTGGCCC ATTTACTATG CCAGCTCTGT GCAGGGTGCA 41220
GAGATCCAAG ATAAATCAGA CAGGGTCTCT GCTGTCAGTG TGCTCAAGGA AAGAGGCTTT 41280
TAGGGGAAAC AAATCTAAAC GACTGCCAGC TGGAACTTCA ACTCTGTAAA GCAGCACCCT 41340
GCCACATCTG CCTGCTGGAA CATTTTCATC TGCTGGGCTC ACGTAGCTGT GCAACAGCTG 41400
GGGCTGGGGT CACATTCTGG GCTAATCTGA TGATTATTTT GGCTAGAGTG AGCTCATCCT 41460
TTTTGTTTTC AGGAGCTGTT CAAGGGTGGT CTGATGGTTT GGATCAAGAC TAGCTGTATC 41520
CCGGAGAAGA ATACGTTGAC TTTTCTGGGG TGGGGTCTGG GGCAGAAAGC AAGAAGGCTG 41580
CCTTACTTCA AGGAAGGCTC TCCTTCCACC TTCTGCCCTC TGAGTGCCTT GTATGCGCAA 41640
GTGACACTAG ACAAAGTGCT TAACACTTAT TACCTGACTT GAATCTCCCA ATGGCCCTGT 41700
AAAGCAGGTA CTCCATTATC ATCACCACCC TTCTTTTTAC AGGCAAGAAA ACCAAGGCAC 41760
AGTCAGTTTA AATAACTGGC TCAAGGCTGC ACGGCCGATA AGTAGCAAAT TTGGACTTCG 41820
AATCTGGGCG CTCTGGCTTC AAAGTGTGCT GTCCATTGTT CAGGTTCTGG TCTGGTACTG 41880
GCAATGTCAG CCACACCTGG AAGCTTGCTA GGACTATAGA ATCCCCAGCT GACCCCAAAC 41940
TCCCCAAATT AGCACCATGA TTTTAACAAG ATCTCAGGTG ATTGGTGTAC ACATTACAGT 42000
TAGAGAAACA CTGCCCTTTT CACATTATAT GGCTCTGTGC TCAGTACAGA TTTAATTTTC 42060 ττττττττττ TTTTATTATA CTTTGAGTTC TGGGGTACAT GCGCAGAACA TGCAGGTTTG 42120
TTACATAGGT ATATATGTGC CATGGTGGCT TGCTGCACCC ATCAACAGGC CCCGGTGTGT 42180
GATATTCCCC TCCCTGTGTC CATGTGTTCT CATTGTTCAA CTCCCACTTA TGAGTGAGAA 42240
TGCGGTGTTT GGTTTTCTGT TCTTGTGTTA GTTTCACAAT CATTCTCAGA TTTAGCTTTC 42300
AAACTATTCA TTCCACCTGC CAACAATTAG CGAGCTCCAG ACATTGTGCC AGGTGAATGA 42360
TGGAGGTGAA GAGACAAATT TCCTTATAGA ACTTGGCCAT GCCCTTCATG CAGGCAGTGT 42420
GTGGAGTGCA AGTCAGGACA CTTGGATCTA AATCCAGTGC TACCACCTGC CGGCTGCGAG 42480
ACTGTGGCTG AGTCATTTCA CCTTCTTGGG TCCCAGGTTC CTAATCGGTA AAACCGGGAG 42540
GCAAGCCAGA GATGTCCGGC CCCAGCAGCA TATTCTATGT GAACAGGATG AGGTGCCCAG 42600
CAGGCAATCA GTGGGGATCT GCTGAATGAG GGAACCAGTA AATGAGTGAG TGAACCGATC 42660
ATCCACCACA AGGAAAGAGC CCTCCATTTC CAAATGAAGA AAAGAAGTAT GCTAGTGGAG 42720
GGGAGACGGG ATTATCTGCT GTGTGTCAGG GAAGAGTAGG GCCTTCCCAA GCTCCCTTAA 42780
TACTAACATT ACACAGGGGT CCTCGCTTGC CCTTCTCAAT GGTCCACTCA GATGATTTCT 42840
CTTGGCGAAT GTCTGCCCCA CATCTGTGTG TCACTCAGCA ACTTTGGCCA CCTATCCAGT 42900
GTGAGATCTC TAGATCACAA GGTGGGGAAA GGGGTGAGGA ATGACCTAGA ATCCTGGCCT 42960
CTGGCCTTAG AGCCTCACTT GTTAAAGGGA AAGGGGCAAA TAAGATCTGA ACATCAAAAA 43020
TTATTTCAGC TTGCCTTCCC TCTCACTTTT CTCTGTCCCC TTCTCCTCTT GTCTTCCCTG 43080
CAAACCACTT TGAGTCTCCT TTGGTTACCA AGATAAAACC AATCCACATT AACTATGGCT 43140
GGTATTTTTT TCGCTTTTAC TCCAAGCCAG TGCATAGTGC ATTTTGCTCA CATTAGATTA 43200
TGGAATCCTT CAAACAACCT GATGATGAGT GGGTGCCATT GATACCCCCA TTTTATAGCT 43260
GGGACAACTG AGGCACAGGG TTGTTAAGCA GCTAACCTGA GGCCACTCGG TCACTTCCTT 43320
GTGGTGGACC CAGGATTTGA ATCCAGGTTT GCTCAACTCC AAAGCCTGTG TACTAAACGA 43380
CACTTCCTGC CTTGATAAGA TAATTGTGGT TGTTACTTGG CCAAATAAAA AGCCTATGGA 43440
GAAGTTGTTT CCAATGAAGC ATATCAGCTT CTAAATCTGG CTGAACATTG GACTCTCCAA 43500
AGGGGCACAA AATACAGCTT TCCGGGCACC ATCTTGAAAT GACTGATTCA GCAAATTGGT 43560
CGTAGGCAGC GAGGCACCTG TAGTTTGGTA AAGCTCCCAG GTGATTCTGA TAATGAGCTT 43620
GTGCAGAACC CATTTACCTA AGGAGAACGC GGGTTCAAAG GGACTGGACG GCTCTTCCTT 43680
ATTTAGAGTA GGAGGCTGTT GGCTTCTGAG AATGAGGGCT AATTAACTTT GGGGAGCTTC 43740
-88- CTGCAGTGAC CTTTGCCTTC GGGGAAAGTG TGGGGATTGA GATAAGAGAG AGAAATCCTT 43800
GGCGGCTAGG AGGAAGGGTA GGGTGTTTGC TGTCAGGCTC CAGGCTTAGC CCTCGTGGTG 43860
TCCCTCCTGG AGATGGTGTG CACTGAGTGC AGTGGCTGCT GGAGAGTGGG TGGAGAGATG 43920
AAGGTGATAG GGGTGGGATT AATTAAAATA TCAGGCAGTG TGGCTGGGCG CAGTGGTTCA 43980
CACCTGTAAT CCCAGCGCTT TAGGAGGCCA AGGCAGGTGG ATCACCTGAG ATTGGGAATT 44040
CGAGTTTAGC GTGGCCAACA TGACGAAACC CTGTCTCTAC TAAAAAAATG TAAAAATTAG 44100
CTGGGTGTGG TGGTGCACTG CAATCCCAGC TACTCGGGAG GGTGAGGTAT GAGAATTTCT 44160
CAAACCCAGG AGGCAGAGGC TGCAAGTGAG CGGAGATCAC ACCACTGCAC TCCGGCTGGG 44220
ACAACAGAGA GAGACTCTGT CTCAAAGAAA AGAAGCAGTG AACCTTTAGA TTATCCCACT 44280
CTAAAAGTGA GGCAACCTTA GTTTTTCTGG GTCTTTAGAA GCAGAAGTGC CCTTGGGTAT 44340
TTCTAGGCTG AGGGCCCCAC CTAGTTCAAG CCTTCTAAAC ATCCAGTGTT TTGCTATATT 44400
CATTTACCAC TTGTCCTATT AGACTCTTAG GTCTTTTTTT TTAATGACTC ACTTATTAAA 44460
GAATGTGCAT TTATTTACAA GGCAATAATA TCACTACCTT TAATGGAAAA TTAGCAACCC 44520
TGGCTACACC TAGAAGGTAA CTGTTAATAA ATAGGATGAA ACCCAAGGCT GGAATTAACT 44580
TCTCATTGGA TCCTGCAGCC TATGCTCCTT TCACTGAAGG GTGATATCAG CCAACTGAGA 44640
CCTCCTCTAA AGTCTGTGAA GGATTGAATT AAGAGAATTG GAAAGGGCAC ACATTTCTCA 44700
TGATGTGATT CAATATTGAT TAATTCCAGG TTCACCTATT ATCTAAAACC ATGTTACTGA 44760
AAGTGGCTTA TAAATACCGC AGCACCAGAA TGTAAACTCC ACAAGGGCAG AGTTTTTGGT 44820
TTTGTTTTGT CTTTTAAAAA TCTGTTCATT GCTTTATTCC TAGACTCTGG AACAGTACTT 44880
GGAATATAGT AGGTGTTCAC ATATTTATTG ACTACGTGGA CTCTTTTTAG ACTGAGAAGC 44940
GGAATATAAA GTCAGAGGGT CCGACTGGTG ATCGAATGCC TTCGTTCTGT ACTCAAGCCC 45000
ACTCACCCAC TTAGTTTTGA GAACTCTGGT GACCCAACCT ACAGCCTGTC CCACCTTCAA 45060
CTTATTCCCA TTCCTTGGGT GCACGTGTTG CTGTGAGGAT CAGATGAGGT CATGGATGGG 45120
CAGGACTCTG AACTGCGTGC CCTCTGCACA GGGAAACAGC TGGGCCGATT ATAAATTGCA 45180
AAGGGGATGC CTGATGGTGG CCCCATGACT TTTCATATGC TTTGGGCTGT TGTGAGAGAG 45240
AGTGCCCAAA GCCTGATTCT GGAACATTTT CTTTGCTGTC TTCTAAATGA GAACCTGCTT 45300
GCTTCAATTC TCCCACTGAG CAATCATGCT GACATGAGGG AGGCGGAGTC AGACCTTACA 45360
TTGTTGAGAC CAGATTCTGT GTTCTACGAG TATTGGGAAG GGTGATGCAG GCAGGCACCC 45420
ACCATGTTCC CTGTGAGTGC TTATTTTTAA TAAAAACCTT GGTATACTGC TATTAATGAA 45480
AATAATAATA ATAATAATAA TTACTCCTGC TAATAATATA AGGAAACACC CACTGGTCTG 45540
TGACTGAGCC AGCCTTGCCT GAAGGCAGGG GAATGAATTC AATGACCTCT TGACACTGGT 45600
CTCAGCCCTT TGGTTCTATT ACCACCTTGT AAACCTGAGG TTGTTCTGTT TTTATCCCTA 45660
GGGAGTTGTG GTTAGAACCT GCCAGAAATT TCTCACTATG AATCAATCTT CCATTGGTCA 45720
CTGCCCTTTT CAACATGCCT GTCATTCAAG ACTTACGATT TCCTAGGCAT TGACAGAGAG 45780
AAACTGGCCA TGTGGACCAA GGCAGTGGGA TTTACGTGAC ACCCGCCAAG CCGGTGGGGC 45840
TAAGTTCCAT TGCTGAAGTC TGATACCTGT CATCTGCTGT GGGGTGACAT CCACACCATG 45900
TCATTCTCCA TTCGTTCAAT ACATATTTGT GGATTCCTAA AATGCCCCTG CTGCTGTGAT 45960
AGTCCAGCTC AAGAGAGAGG AAGTACATGA GATGTTACCA CACAGTGTGG TATGTGCTGG 46020
AGAGGTGAAG ACTCTGGAGC AGAGAGGCAA CAACTCAGGT GGGGACTGAA TGGTGGCGGG 46080
GTGAGCTCAT CAGGAAAGGC CCCCCCAGGG AAGCTGTGTT TGGGCTGGGG TCTAAGGATG 46140
AGCAGCAGTT AGCCAGGGAA GACAAGGAGT AAATGTACCT AGGCATGTGG GGCAGTCTAT 46200
GCAATAATGT GGGGAGGAAG CAAAGAGAAA GAGAATGGGA GAATGGCCTG CCTGTTTGGG 46260
GAAATGAAAG GAGCCAGTAT GTAAAAATCA GGTGAGAGAC AGCTGGAGAT GAGGCTGCAG 46320
AAATAGGTAG GTGCCAGGTC ACAGAGGGCC TTGTGAATAG TATCATGGAC GCTGGACTTT 46380
ACTCCAAAGG GCATGGGAGC CATCAAAGGG TGTTGAACAA GGAGATGCAC ATTATAGAAA 46440
GGCCAGGAAG GCCTCTGGGA TCTCCTCTTC TCCAAACTGT GGCTCTGGGG ACAGCTCCCT 46500
ATAGTGGTCT TGGGCAGCAC CAAACTGGTG TTTAGGCTCA GCTCACATGC AGCTCACAGC 46560
AAGATGGTGA CAAATGACTC ATCCTCAAAC AACAGAGCAG GCATAGGAAG GAGGCCCCAG 46620
TTAGGATCTT GCTTACCTGG TTTGCTGGTG GCCTATGCAT TTAATTGTAG AACAGAATGC 46680
CAAGCCACTT TTTAACCTTT CTTCTACACC ATGCCCTGCA CCTCCCCTTC TCTCTCTGCT 46740
CTTCTCCCTT CCACCCTCAA ATTTCTAAGC CATGTCCAGG TCTCGTTTTC ACCTGTGCCA 46800
GAGAAGATCT ATCTGACTTT GGCCATGGAA GAGGTATAGC AGGTATCAGT TGGAGAGGGC 46860
TGGAAAAGCT CCCTGGTGCT AGATATGGAC GACCTGAGCT TCCAGTCCTG GCTCTTGCAG 46920
CCACCAGGCA TTTGACATGG GCAGAAGCAC TTTTCCTCAC TGAGCCTCTG TTTCCTCATC 46980
TGTAAAATGG GAATCATGGT GATGGTGTGA TATTTGAACA AGTTTTTTTT TTTTTTTCAA 47040
AATTGCTTTG TAAACTGCAA AGCTCTGAAT AAGTGTTTAT TTGGGATTAT TAGGAACTGC 47100
-89- TTTGCTGGAA CAGTCTACCA GAGGGATGGA AGGAGAGGAA CTGAGAAATC GATTCTTTGA 47160
AATATTTTTA TCATATGAGA TACAAATATG TATCTATATA AATATAGATA TAAATATGAA 47220
CAAATATATC TGTCATAAAA TTTAAAAAAG GATGAACCTT GCCCCCAATC TCACCCCTAG 47280
CAGCAACTAT TAATTTTTTG TTGTATATCT GCCCAGACAC ATATAAAATA TATATTCAAA 47340
CAAAAAATAT AATCATATTA TAAACTTTGT TTTTTAGCTT GTTTATTCAC ATTACATGGA 47400
AATCTTTCAG CATCATGGCA TATAGATCTG TCTTTTTAAT ATTACTTCAT GGTCTAGGTG 47460
AACCATAGTT TATTTAGCAT TTTCCTTTTG GTAAACATTA AAGTTAGTTG CAATTTTTCA 47520
TCATATATTT TTTCTGGTCT TTTGTACATA TATCTATGAG AGAAATTCCT AGAAATAGGG 47580
TTGCTGACTC AAAGGATACC AGCATTTTAA ATTTTGGTAG GTACTACCAA ATTGCTCTTC 47640
ATAAAGAGTG TACAAATACA CCCTCCCACA AACAGAGTGC CTGCCTTCCA TGCCTGGACC 47700
AACCACAGGC ATTACCACCT CTGCTGAAGC TTTTTCATGA GACAAGGTCT TGCTCTGTTG 47760
CCCAGGCTGG AGTGCAGTGG CGTGATCTCT GCTCACTTCA ACCTCTGCCT CCCAGGTTCA 47820
AGTGACTGTC ATGTCTCAGC CTCTGGAGTA GCTGGGACTA CAGGTGCGTG CCACCAAACC 47880
TGGCTAATTT TTGTATTTTT GGTAGAGATG GGGTTTTGCC ATGTTGGCCA GGCTGGTCTC 47940
GAACTCCTGG CCTCAAGTGA TTTGACTGCC TTGGCCTCCC AAAGTGCTGG AATTACAGGC 48000
GTGAGCCACC ATGTCTGGAC TGCTGAGGTT TTTTTTTTTT TTTTGAGACC AAGTTTCACT 48060
CTTGTAGCCC AGGCTGCAGT GCAATGGCAT GATCTTGGCT CACTGCAACC TCCGCCTCCC 48120
AGGTTCAAGG GATTCTCCTG CCTCAGCCTT CCAAGTAGCT GGGATTATAG GCATGTGCCA 48180
CCATGCCCAG CTAATTTTGT ATTTTTAGTA GAGATGGGGT TTCTTCATGT TGGTTAGGCT 48240
GGTCTCGAAC TCCCAACCTC AGGTGATCTG CCCGCCTTGG CCTCTCAAAG TGCTGGGATT 48300
ACAGGCATGA ACCACTGCGC CCAGCCTTGC TGAGGCTTTT AAAACCATGA AACGCTCCTC 48360
CTCCCTCAAA TGGTCATGTG GCCACTGCCT GCTTCATCAC ACTGCTCCTC TGTCTGACAA 48420
GCCTGTTCTT ATATAACACC AGTAGGTAGG GCCATCCGAG ACATGGTTAT CCAATAAAAT 48480
GGTAAGAACC AGCCCTAGGG TATTTGGGAA ACTGGCTGTG AGGGTTCAAT GGAATATTCA 48540
CATTTCCAAA CATAAAATCT AGCAGCAATG GAGAAACGTA CTTTAAGCAG AGAGTTTTGC 48600
GCCTGACACA AGAAATTATT ATTATTGTTG TTATTGAAAG TTCTGACACA CAGATCTCGG 48660
TTGTGTTTGG AAGGAGGATA GTCAGAGAGA GGAGGAAGGT ATGAAGAGGT CGAGGTGTTA 48720
GTTTTAAAAA GTGTGTCTTT GTCATTGTCG AGCTGTGGCT GGTCCCACAA CCTGGTTCTA 48780
TCAGGCCTTT GGTGTTACAA AATGCAAAAC ACCAGGCAAC CAAATAGCGT TTCCATGGAA 48840
GTATCCCATG ACCTCTGGTG CTGTGTACAG GTGAGACAGT GAGCACTCAG AAAGGGATGG 48900
CCTGGGTGGG GAGGGCGAAA GGGGCCTCTC CAGCCTCTGC AACATAAAAC AAGGGGCCAA 48960
TGGAAAGTTC TGGAACTGGA TCACTAAGAA GACAGGCCCC ACTGCTGGCA TGAGTGGGAT 49020
GACCAAAGAA TTAGGAAACT GAGATTGGAG TTGGTCACCA ATTCAACTGG CCCATTTAAA 49080
AATTTTCATA AGCAGGGACA GAGGATCAAG CCAAGAGCAC TAGGGAGATG GTGATGAATG 49140
GAAATTGTGT AAGGTAGATG GCTATGTGCC GGGGAAGGAG GAGAGAGGAT TCAGAATTAT 49200
AGGAATAATA CATGAAATGA CTGACAAAAG TAGCCTTTTA TGTGTGTTAT GTAATTTAAT 49260
CCTCTTAACC TTATAGAGTT AGCACTGTCA GGATCCACAT TAAAAAAAAA AAAGACGAAG 49320
CAGAAGCTCG GAGAAGTCAA ATTACTTGGC CAAGGTCAAG GTCACACAGC CACATGTGGC 49380
AAATCTGGAA TACAAACTTA GGTCTATCTG ACTTTAAACC AAAATGCTGC ATATAGCTTC 49440
GATTTCAGCA CAGCAGGGTT CAACTTGGAG ATAGAGGGTG GTGTTATAGA TTACCAGATA 49500
CGATAGTGGT AGGTTTTCTT CTGTCTTGAT GAAAGATGAG CTATTTTTAT CCTGTTGCAG 49560
GACAACGCGA AGGATCATGA CTTCCATTTT TGAACTGACA TTGTAGATTT GTGTATATTT 49620
GACAGCTCTA CCACATTCCC AACCCTATGC CCTCCTATCA CTCTTTTTGA GAATACTGGG 49680
CTAGTTGGGG GCAGTGTTGG GGGACTTGGG CCTGGGCGTA TGCTGGGAGG AAAGGCAAGG 49740
AGATTATGCA GTGTGGTAGT AGGGACTGGG GGAAGTTTTT TTGTTTTTTG TTTTTGTTTT 49800
TAAAATCCTA GTTGGTCCCC AGTGGAGCCT CCAGACCTCC TCAAAGTCTT TGAGGTTGTG 49860
ATTAATTACC ATATAAACTA GACAGTCCTT GGCCTTGGTG TTGCCATTCC AGCCTGTAAT 49920
TATCTTCATC ACAAGTTGCT GTCTGGCTTT GTTCTGTAGG TAGAGGCTCT TTCGTAGGTC 49980
CCTGCATGTC CCTGAGTCAC TAGCAGGCTC ACTTGTGCTT ATCCAAACTG GTGAATCATT 50040
AGCTGTCACC CTGGAGAGCA GTGCAGTTTG GGAAGGCGTG GGTGCGCCCA TGGAGAGGGT 50100
GATCCCCTCT CTCTTCTTTC CAGGCATGCG TAAGGAGCAG TGGCAGAGAA TTACGGAACA 50160
GAGGATGCTA TCATAGGTGA CCTATGAGCC AGGCACGTAC ATACGTGTCA TCTCAATGAA 50220
AGCTTTACAG CACAGGTTAT ACAAGTAGTA CACAGGGATA AACAGCAAGG TTCTTAGGTG 50280
GGTTTCAGAC CTGGCTCTGT CATTTATCTA GAGGTATGAC CTTGGCCCAA CCTTCCTAAC 50340
TTGTCTATGC CTTGATTTCC TCAACTATAA AATAGAGATA AAAATGGTAA CTGCATCCAA 50400
GAGCTTTTGA GAGGAATTGA TGCAAAGATG CAAGTACAGT GCCTAGCAAA CTGAAGCACT 50460
-90- CCATGAGGAG TGGTGATGCG GATGCTAATG CTGATGCTGG GACAAACTTA CACCCACTTT 50520
ACAGATGGGA GAACTGAGCC TCAAGTTGTT TAAAGTGGCA TAGCTAGTAA GTGGTAGACT 50580
TGGGATGAAA ACCCCAGTCT GTTTCCAAGT CAGGAACCCT TTCCTCCATA ATGCCGTCTG 50640
CATAAATTAG ACTGTTGGAC TGAAAAACAA TCCGTTCAAA CCACAAGGGT ACATTGGCCC 50700
AGGTTGCTTC TATGTTTTAT CCTCAATCTG AAGCAATATA ATGAGCAATG TAATGAGATT 50760
ATGTTAATAT TTACTCAGGG TTCTGGGAAA CCCAGAAGGG TTTCAGGGTA AACCATCTCC 50820
CAGCAAGCAA GGGCTCGCCC GCTAATTCCC CTTTCTTCCA AGACTGATCA GATTGCCCAG 50880
TGCCTAGTAA AATGCCAGTT TCCTTCTATG TGGAAGGGAG CAAAGCTGTC AGCTCCTGCT 50940
GGGGCACAGG GAGAGGATGT TTCTTGTGGA TAGGTAGGTG GTGCTTAGGG GTAGAGGCTC 51000
TGAGATCAGG CAGACATGGT TTCTATCTGT CCTCCCAGCA GTGTGTCCTT GGGTAAGTTA 51060
CTTAATGTTT CTCAGCTTCA ATGTCCTCAT CTTAAGATGA GGGATTATCA TGCTACTTTG 51120
TGGGGCCTTT GTGAGGATTA AATGAGATCT TAGTATCTGG CACATAGTAA GTGCTTAATA 51180
AAAATAATAA GGCAGAGCTG GGTAGATTGA GGGTTTGGTT TACAGCACTT TGACAGCAAG 51240
TTGCTTGTTT CCTGCCATTC AGAGACCCTG GCCAAACTAT GTCCATTGTG GCCACAAGAC 51300
CATTGGCATG TCAGCCTCCA AAAGAGAGAT GACTGCTCAG CAGGCATTAA CCAGATCAGA 51360
GGTTCTTTGA TTCAGCACAG TGCTCTCTTT TTGCACTGCT CTCAGTCTAC CAACAGTATC 51420
AATCACAGCA ACCATTCATG GTGCAAGGTG ATCTCCCTAA ACTTACATTA TATCTTTAAT 51480
CCTCACAGCA GCCTTGGGGG ATGGTATTAT TTCCATCTGT AGATGAGACA ATAGGGGCTC 515 0
AGAGATGGTA GGTAATTGCC CAAGGACACA TAGCTGTTGG AGAAAGTAGT ATTGGAGCAA 51600
AATCTATGTG TGTGCATCTA GATTGACCAA CCTTCCTGGT TTGCCTGGGA ATATGGGGTT 51660
TTCTAGGATG TGGGGCATTC AGTGCTAAAA TCAGGAAAGT CTAAGATGAG TTGGTTACTC 51720
TATATGCGGC CTCTCCGTGG AGGGTTGGTT GGTGGGCCTG GAAAAGGGAT AGGGATAAGA 51780
GAGAGAAGAG GAGGACGCAG AGAGAATGGC AGAAGCAACT CTGCACTGTT TCTTTCTGCA 51840
AAGATGTCTT TTCAATTCAA CCTGCTTGTT CAGTTCAACA AGCAGGTTTG AATGCCCTCG 51900
TCCTTGGAGG GAGTCACGTC AGGACTTTCC GGGTATTTGA CCGTGATGAA GAGCGCTGTC 51960
TGCCAGGGTT CGCCAGGCTG GGTGTGGAAA AATGGTGCCC CAAACCAGCC CCACATGGCA 52020
GAATAGGAAA CATGCTGTCA TCTTGCTTCA TCTGAATCTC CATTCCATGA GGGCAGGAAT 52080
TGTTTTCTTT TTTACTTCTA TAGCTGAAGC CCCAGTGCCC AGAATATGGC AGAAACTCCA 52140
GAAACATTGG TGGAATGTAG ACTATTGAAT AATTCCAAGT ACAAACCAAT GGTCCAGGGA 52200
GATTTAGATT CTGATGAAGG CAATCTGGGG AAGACTGAAT GGAGAAATAG CATTGGAAAC 52260
GGTTTGGATA CCACGTGTTG GGATCAGGAA GCAGAGGAGC ACAGAATGCT TGTGCAGAAG 52320
TGACATGGGC CCACTGCACC TGGGGTGGAC CCTGTGAGGT AGAGTTGGAG ACCAAGGGCC 52380
TGAGGACTGG ACATGTCGGT GGAGACCAGG TGGTGGAGGA TGGAGAATGC CATGCCCTCA 52440
GGGAGTTTGG ACTGCCTGTC GTTAAGCCAT TTTTTTCTCC AAATTTCAAT CCCCCTCATT 52500
CCATTGTCAC CATATTTGCC ATGTCTGTGT ACCTACCTAT ATTACTTATT TAACACTTTT 52560
CCTTCAAGTG ACTTACTTTT TAACTTTACA TTTGTTTTCA TATCAAACAC ACATGGCTGT 52620
TAAAATAAAA ATTACGATTT GAACTTAGAA TCATCTTGCC TACCACATGA GGTAGGTGTA 52680
CTTCCCTCTG AGGACCACAG CTCCAGCAAC TGGGGAACCG ACAAAGATTT TTGAAAGAAG 52740
AAATGATTCA GTTGCTTTTT GGGAAGACTA CACACGTGAG GAAGTACTGA GTGGAAGATA 52800
TGTGCATAAA ACATTGGCGC AATTGTGACT AACATGGTAA GAAATATT T CAACGCAAGT 52860
TTGGGGGGCA TTTCAAAGTC TCTCAATGGT CATCCGGATG AAATATGCAA GAACTGCTCT 52920
CTCTCTCTCT CTCTCTGTCT TTTCTCTTCT TGGTCTCACT TTGCCCTCTT TCCCAGCAGC 52980
TCTGCCTTCT CCCCCATGCT TGCTGCCAAC AGCTCTGAGG AATGGGAGGG ATTGCAGTTC 53040
AAAGAGTAAA CAGGTCTACT CTGAGTAAGG CTGTGGGCTG TGCAGTGACC CCCAGTGGGT 53100
CTGGGTGCCT GGTAATGATG CCTGCACTGG CATGATGCTG TGGCTTTCCA GGCTTGTTTT 53160
ACCTGGTTGT GCAAAGAATG TTACCCCCAG CCAAGGCTCA AGTTCACAGA CCATTGGCCC 53220
ATCCCCTAAT AAGCATATTA TTCCCAGCTG GGCATTGAAC TTCCAAGTTA AGGTGACCTG 53280
CCAAACTGGA AAGAAAATGG ATTTGCAAAA ATCAGATGTT TGCCAACAGC ACCATCCCCC 53340
ACCACAACCA TAGACAATTG TGAGATCTAA AGTTGGACTC CCTGAGGTTT TCTGCCCTGG 53400
TGGTTCTGGC AACTCCTGGA GAGCCACAGA CTGATGAATT TGAGGATCAT AAACCTTAAG 53460
AAGACTTTAA AGTATTTTTG GCATTAATTG ACAAAGTCCA CAGCAAGCCA GGCATGCTCT 53520
TCTCTCCCAC TCCCCTTGTC AGAGATGTCT CTTTCCCCTT GCTCTTCTTA CCCCATTCTT 53580
TCCAGCATAA CCAAGCTTAA TAGCTTCCAT GTTTCCACTG TAAGGAAGTG AGCCGAGTGT 53640
GGTTGGTCTG TTTCACAGCA GGGCTATCCT CACACGAAAA GTTTTCAGAT GCATTGACTA 53700
TGCAGATTTT TGGCTCAGTT TGCAGAAGAC TTCCTTATTT CAGTTTTACT GTACACCCAC 53760
CTACATAATA CTTTTTGGTT CTTAGAATTT CAGAGCTATT AACCTCTAAA CTTAAATCAA 53820
-91- AATTCTCATC AAACTTTCCT AGGGCCTTGT CATAAAAGAA ACTAAGTCTC AAAATAGGAC 53880
TTTTTGGCCT AATTTCCTTG TCCAGGAAGA CAGATTGACT AATTCCAAAC CCTGGACTCA 53940
CATGTGATTG CTAAGAATAG GGGTGGGGGA GAGAGAGGGG ACAAAAGTCA TAGACATGCC 54000
ATGACACATA TTGGGAGATT TCATCTGAAT TTCCCCTGAG TATGAAATTA TTCAGAAATA 54060
ATTCCAAGGG CTTCTTTTCT GACATTCCAC CAGTGTGCAG GTGCATATGT TTTGAATGAA 54120
CTGAATGGAT AATTTATTTA TACAAATGAG TCTTTTTGAA TAGTTGCAAT GGATGTGCTG 54180
TCAACTCTCC AATATCACTT CCAGGGGGTT TGTAGATGCA TTCTTTCCAT GGGCATCAGC 54240
AGGTCTGGAT CTCCTGCTTT CTATCTGAAA GGCACTGGTC TGGATCTCCT GCTTTCTATC 54300
TGAAAGGCAT TATGGGCAGC AGTCTGGTTG ATTTTATACT ACTTTGATAC ACTCTCAATT 54360
GCATACTAAG AATGAGGATG GAGAAACTGA TAGTGACCCT CACCCCAATT AGGTTTCACT 54420
ACTGCCCTTG ACCTTCATAT TTAATGCCTT TGTTATCACA GCAACTCTTT GCTCTATTTC 54480
TGGATCCAAA TGCCTAAGGA TCTCCCTGGG GTGTTAAGCT TGCTCAGTGC TATTAAACCT 54540
GGTGGGTGGC AGAGTGACCT TTGTATCACA AGAGCCTCAT GACTTCCCAG GCAAGACCAA 54600
GTCACAACTT TTCCAATGGA TTTCCCCTCG ATTCTTATTC TGAGCATTTA GCTTTTTAAA 54660
TATTTGGCTC TGAAGGGCAG GGGCTAAACA TTGTTCTGTA AGATCCAAAC CTGCTTGTAT 54720
ATTTTATACT TTTGTTTTTT CATTTCAACT TTCCGATCTC GCTCTTCTGA GAAACATTCA 54780
CATTTCCAAT TGCATTCCAG AACTGAGCTT GACTTTCCAT GTCCATGTAA GATCTTGTAA 54840
TTCAAATTTC AGCCAGCTGC TAAGCTTCTC TTTTCTGGAG GGGATTGTGG TTAAGAGATC 54900
TTGTTTTGCA ATGACGCTGT CTGGTCTGAG CTCCAGCTAC TTGTCTTATT TACTGTGCAA 54960
CCTTGGCCAT GTTAACTTAT CAGGCTCATG AGGCTCGGTT TTCTCATCTA TAAAGTGAGA 55020
AAATGAATAG TACCTATCTG ATGGAGTTTT TCTAAGGCTT AAATGAAGTA ATGCAAATTA 55080
AATTCTTAGT CTAGTCACTG GGAAAAGATG AAAACTTAAC GAATATGAAT AGTCACTATT 55140
CTGTTTCTTT TTTTCTATGC CATTCCGGCT TCACCTCCTT CTCTTACTTT TTCCCTTTCT 55200
TTTTTCATTT GTTTTCTTTT TTTTTTTTTT CTTTTTTGAG ATGGAGTTTC GCTCTTGTTG 55260
CCCAGACTGG AGTACAATGG CATGATCTTG GCTCGCTGCA ACCTCCACCT CCCAGGTTCA 55320
AGCGATTCTC CTGCCTCAGC CTCTCGAGTA CCTGGGATTA CAGGTGCCCA CCACCATGCC 55380
TGGCTAATTT TTGTATTTTT AGTAGAGATG GGGTTTCACC ATGTTGGCCA GGCTGGTCTT 55440
GAACTTCTGA CCTCAGGTGA TCCACCCACC TCAGCCTCCC AAAGTGCTGG GATAACAGGA 55500
TGCCGCACCA CTGTGCCTGG CCTCTTCTAC TTTTTCTTAG AAACATGGAG GGTTAGTTCT 55560
CTGGCCACTC ATATGAAACT TCATTCCCTG CTAAGGTGGA AGTATTGGAG TTCAAGCTCT 55620
ACACTTAGTG GAGGGAGTAA ATAAGCATTT CCAGAGAGCC CACCAAGTGC CATGCAATCT 55680
CCTAATGCTT TGTACTATTT CTCATTTAAC CCCCCAAACA GCTCACTGAG TATGTTAATA 55740
TCCCCAATAA ACAGATAGGG AAACTGAGAC CTAAAGTTTG AGCAAATATG GCAAAGTTTT 55800
CCTAGGCTGT CTGGCTTTAA AAACAATGTC CTTTCACCGC ATCAGGCTGC TTCTGAGGAG 55860
CAGAGCCACC TTGCTTTTGT AAGTCTGTTG GAATAGGCTC TGAGATGCCA CACGTTATCC 55920
CAAATAATTA GGCATCTGGA TGGAGATTTT ATACATTTTC TACTTGGACC TGAGTTTGCT 55980
GTCTCTCATG GTTCCTGGGT GAAAGAGGCC AGGCCCTGAG ACCTTTACCC AAGGTTGGCT 56040
CTACCAAAAT ATCTTCTTGA GTGAGTTCTC TGGTTGATCA TCTGTGGAAC AATGTGGGAG 56100
CCTACTAAAT ATGAATGGAA AATGAGGAAT GCAAAATGGA TGGTTTTCTC CACTATCACC 56160
TCACCCTTGG AGGTGTTTGC TGATTTGGTA GATGTGTGGA GGAACTCAGG AGTCTGAATT 56220
TGTAAAGGTA ATTTGGATGC TTCATTAGCT TAGAAAGGAC ACAGCAGGGA GAACTATATA 56280
GCAGAGAAGG CTGGATGCCT ATGAGGGTAG GGAAGGGAAA ACAAGGGGGT GGGGCTGTAG 56340
CTGCCCTACC TCCGGTCCAT ATATGGCTGC ATTTCTTTAA TCTCTTTTAC TTTTGGGATT 56400
CCATGGTAGT AAACAAAGAG TTCTTATGTT AAAACAATTG CTATCTAATT GTACAGCATG 56460
GTGAATATAG TCAATAACAA TGTATCATGT ATTTGCAAAT TGCTAAGAGA GTAGATTGTG 56520
TTTTCACCAC ACACAAAAAT GGCAAGTATG TGAGGTAATG CCCATGTTAA TTAGCTCAAT 56580
TTAGCCACTC CACAATGTGT GTGTGTGTGT GTGTGTGTGT GTATATATAT ATATATGTTT 56640
ATGTATATAT ACACACACAC ATATATATGA CATGTCAGAA TGTCATGTTT TATTCCATAA 56700
ATATATACAA ATTTTATTTG TCAATATAAA AAGAATAATA CCTGGAAAAA CAAAAAAAAA 56760
ATCCTAAGTG CTATACTTAT AAAGAAATCT TCCTCATACA AAAAAGAAGA AATTCTGGCC 56820
ACAGGAAGGT TGCCTGAAAA TGGCCACCTT TTTCATGATT TTCCCTCCCT TTCTGAGACT 56880
GAGAAATGAG CCTTCTTGAA GACCCTGATG GAAATACTGT GAAGAAACTA AGACAGTTGG 56940
ATTCAAGAAC CAAAATGCTT ATCGTAGCAG TGAGGTTGGC TTGAAGTCAG GGAACAGTGT 57000
AAAGCTATTT GTGGGGAAAG ATAAGGCCAG AAAGAGATTG ATAAAATACA GGCGAGACCA 57060
AAGGAACAGG GCAGGGGCAA ATTAGTTTAG GCAAGAATAG AGGCGTCTTG ATATTAATTA 57120
AAATATGGAG GAGGAGTCCA GAAAATTCAT CCTTGGTGCT TGGGTAAGTT TAGCAACATG 57180
-92- TTCAGATGCC TGAGTTTTGT GTGTGTATGT GTGTGGGCAT GCACGTGTGT GTGTACACAG 57240
TGGGTCATTC TTCTCAGGAA GAGTGAGCCA CTCTCCCCTC CTCCAGCACC AAAGTGGCCC 57300
CCACCTTGGC ACGCCAGTGG CACATGCCAT TGGGCCAGGA TTTGCTCAGA ATGCAGGCAC 57360
ACAGACATAA TGTCAGGAGG CATTGCTGGT GTGTGTCACA TCAACCTGTT AGAACAACTG 57420
TCAACGTGTG ACCTCCCAAA CAGAACTCAG GTGCCCCCTT CAGAGACCGT AAAGCTTGTC 57480
CTTAGAGGAT AATGAAGATC CCCAGGAACC TCATCTAATC CAAAACCAAA AGATTTGGGA 57540
AATGTGACCT TTAGAGGGGA GTAGCATTAA GAAGCAAAAT GATACTTATT AATTCTGTTG 57600
CTTATTTGAC TGTAACCAGT ATAATAAATG ATCATATTCT GCTCGATTTA ATTCCCCCTC 57660
CCCATAAGTT TCACAAGACC AGAAGGAGTT TCTTCTTCCC ATTGGTCTTA CATTAATATT 57720
CTTGTACGGC TTTCACTAAA TAGATGCCGT GTTCTGCCCT GGAGGTAACA CCACGTCATT 57780
AGGAGGAGAT GATAGACAGA AATATATACA AACACACACT TGCTTTCAAA AATAAATATA 57840
GGCCCTCTAG TTAAAAGGTA TTGTGTAAAG TGTGTGAGCA TCCTCTTTCT TGCAAAGCAA 57900
GCACACAGCT TCCATTAATC TTGTAGCCAC AGCCTGTGTT GGTGTTAAGA CTCAGATTCC 57960
TTAACGCTTG ATACTTGGCT TAAAGAGATT CTTTGTCCTG GCCTTGATTT GGGAATTAAG 58020
ATCCCTAGGG TTTTTGGTTT TACAGTATGG ATCTTCTAGG AGACAACCCG ACTGACCTCC 58080
GGGTCTCCAG GCCACCACAC ACAACCTGGT TTGCTTTGCT CTGTTCCCCT TTTCCTCTGT 58140
GGGGACCAGC ACAGGACTCA ACTCAAGGGC TCTGTGTCTG TGCACAGGTT GGAGAGGGTG 58200
ATAGGGCCTT GACCTGTAGG GACAACCAGG AAGATTTCTA TGCAGAGTAA TTGGGTTTCT 58260
AGAGTTTGTT TCAGTTGATT TGAGGGCAAG CTGCTTGGCC TCTCTCTCTT GATTCTTCCC 58320
ATCCACAGAA TAAAGACAAT CAGCTTTGTT TATCACTCTG TTCATTTTGC TATGTCTTTA 58380
TCAGCCCCCC AGAGAATTCA GGAGCACAGA ACAAGTGCTG GAGGTCTCTC TTGCCAGAGT 58440
CCTCCTTGAG AACTTACAAT GTGTCCATAT TAAGGATCTG CTGTGTTTGA TGATTTTGTG 58500
ATTACACTTT AAACTTCTTA TCCATAAAGG ACATACTTGA TATATCTGAG ACTTGTAGTA 58560
GAAGGCCTTG AGACATCCAT CTCATCCCAT CATTATCTAT CTATCATCTA TCTATCTATC 58620
TATCTATCTA TCTATCTATC TATCTATCTA TCTATCATCT ATCTATCTAT CGCCAGTACT 58680
GTCTTGTTGA AGTTGGCAGT AGGGTGAAAG ACCTCAAACT CCAAAGGACT TTCCGTATGG 58740
ATGCAATATA CCTGCAATTC TAGCTTTTTT GTGTTTTTTT TTTTAGGTTG GGGGTGAGGG 58800
GTATTGTTTT CATTTTTGTT TTTCTTCTGG AAGGTTCAAC TAAGACCCAA GTAAAAAGAA 58860
GAATCAATAC TTAATAAGTA CCCAGCAAGT AGCAGGCACA CTTTTAGGTA CTTTATTTAC 58920
AAAAAAACCT CCACAAATAA AGTGGCTTGT GAGTATGAGG TGACATCTTT CCCTCCCCTC 58980
CCACCATCAC TACCCCAATA TGACTCGTCT CAATAGCCCT CCAATCTAAA ATGGACTAAA 59040
TACAAGTGGA TAAAGAAATG GAGATTTAAC CAGAATTCTT CAGCTATAAA TTACAGGGCC 59100
TATAATTAAA GGTGATTGGG ACTGGGTCAG AGAGCCACAT CACTTTTGTG GTTGCATTTG 59160
AAGTTCACTA TCTCTTGACC ACACAACCCT AGCCCTTCTA CTCCCACCCT GCTGTCTCAG 59220
GTTAATCTCA GGCAATGGTG TAAAGAAGGC CAAGTTTGTT TCCCTGGAGT CCCACGGGCT 59280
CTAGCAATAA TGCTTCCCTT TTCTCATGAG TGCCCCGCCA CCCACCCCCC TTCACCATCA 59340
CTACACACAA ATGCCCTGCA GTGGGTGGAA TGTAGTTACT TCAGGTTGTG CCTGATTTGT 59400
CTCTCAAGCA AAACTCCAGC AGGCCATTCC CTCAGGGCCC TGCTCTCAGA TCTGGAACTG 59460
ATAGACTAAT TGGGGCTAAT GTGATAATGG GAAATAATGA AATTTGTTGT TTTTATCAGT 59520
GTGTATATGG GGCGGGGTTT ACATTTGCAT TTTCACAGGG CCCTTGGCAA GTTCACAGGG 59580
TTGAACAGTT GGGAAGGGTG GGAATGTCTG GGGCAGGTTA GGGAGGCAGA GGGATTTATT 59640
AGAACTCCCC TAAACTGCAC TGACCAAAGC CTCAAGCCCT TCTTCAAGAC CTGCCCAGCT 59700
TCCAAGACCT TCCCAAGTCC ACCCTTGTTT TCCCACTGAG TCTTTTACAC TTTCAGAAAC 59760
CTCTGAATTT GTGTAGAAAC TAGAAAAAAT AAGTAAGAAA AGACTAATAC TACTGCACAC 59820
TCACTGTTCC CCCTTAATAT AATAACCAGT TTTTATTCTA TTCAGTCAGC CTTTGACCAT 59880
AAGCAGACCT TTTTTTTTTC TTTTTAACAC AAGTAACTTC TTGGTTTTGA TCACAAAATC 59940
TTTATCTCTG CCAAATCTCA ACTTCCCTTC CCTCTCCCAC AAAAGGGAGG CCCGTTGAGT 60000
CAAAGAAATC TGCTTAGACA CTTTGCTCAT GCCAGGCCAG- TGTCCTGGAA GGTTCAACAG 60060
AGAGAGTTAA TGGTTGGGGG ATGGTATTTT TCTTTGCTAG GAGCAGTCAT TCACCCGTAT 60120
GGGAGAAGGT ACATTTGTGA CCCAGTGAAG CAGGTACAGG TAACTCCCCA TATGTCCCTT 60180
GGCCCAAGGG AATAGAGGTT GCCTGGGTAT TTGAATCCGT AGATCCTCCC TAATATTCCA 60240
CCTTCTTCTT GTCCAAACTG TGCTTTTTTA TTTCCAGTTT CAGCATTTTG GTCTTCTCAT 60300
CTCTAACTCT TATAGGGAGT GTCAATAAAC CTTTTAAAAA AGATCATGTA AGTGTCAAGA 60360
GGAAGTGAAG AACCTAGATA ATCCACCAAC CGGATAATCA GCTCTTGCAT ATTTGAGAGT 60420
TGACTGCTTG ACCTAAGCAT CTCCTCATAA GGTACCCTCC CTCCCAGGAC CTTCCCTTTC 60480
AAACCTCTCA AGGCTCTTAC CTGGGGCCAG GGGAGATAGG CTTTTCAAAG TCCATTGAAT 60540
-93- TGCCAAGAGT CTCTGTCAAG AAGGCAGTCA TGGTGCCTGG AGAGGGAACT TGCTGGGAGC 60600
CCCTTCAGAG CCTGGTACTT ATAGAGCTAG GGAAAAGATC TTGATGCCAA AGCAGGGTGG 60660
ACTAAATACA GACTAATAAA TGAGACAGGT GCTCAAGAGG GCCCCTCCAT ACCATCATCT 60720
CCTCCAGATT TGGACTTCTA CTCACTTTGC TTTTACATTC CCTCTTCCCG ATGGTGTCTT 60780
TGGTGAGCAG GGTGCTTTTC ACCTGAAACA GCCTCTGAGC TGAAAAGAAC AGTCACCACC 60840
AAATCAATTC CTCATCCATT AACAGGTTGT CTCTCTGTTC TTGAGACACA GGCATTACCT 60900
GGTTAGACCT GTTTTGTTTG AACACTAACG TGTGAGTTGG CCAAATGCAA ATGAGCCAAT 60960
GTTTGTAATC CTTTATTTTA TTTTTTTAAA GGGCTGGGTA GCCAATCAGA AGAGGGGGAA 61020
GTGACTTAGG GAATTCCCGG TTGGTGGCTT ATTGCTTAAC ATCCTACAAA ATGATTTAAA 61080
ATTATTGTTA TATGCATTTA TCTTCACTCT GATGAGGGCT CAGACTTGAT AACGCCCGTG 61140
GTGCCCCATC CCTATAGGAG CTGGTGAGAT TGCAGCCTGC TGCCTCCCCT CCATCAGCCA 61200
CAGCTATTGG ATTTCCCACC CAGAATCTTT AGGTAAATGA GGTAAGTCCT GATTTTTAAA 61260
ACTTCTTTTG AATCTGGAAT CCAAACACTT GAGTGGAAAG AGAAGCCTGC TTTAAACTGG 61320
ACAGATGAAA CTAGAACAGA CTCTTGGAGA CGGCTGGCAG GAAGTGAAGC TCACCTTACC 61380
TGGGCTTACC TCACTGGGTC AAATCAGAAT TTTATTTTGG AGGGCAGGTT GGCTACTTTG 61440
GATATTATCT GTGAATTTCC TGCATTGTCT GGACTTCTAA TCTCTGTGAA TTTAAAAGCC 61500
CCCTCGTTTC CCTATGCCTG GGTGGCAAAA CCATTCCCCT GGGTTGAATT CTTCTGGAAC 61560
AAATAGGCAG CTAGAGATAG GTGGCTCTGA TATAGCTCAG AGAAGAAGTG GTTGGCTAAG 61620
TAGCTGTTAG GGCTCAGAGT ACACGGTCTC GCTTTCTAGA GATGTCTTCT GCTGGTAATT 61680
TTTCTGACTT ATGAGCTACA TGGAAAGGCC AATTTGTTTT TAATATGTTC CAGGACTGGA 61740
AAATGGCTAG AAATAGGCAA GAACATACAC AATCACACTG GAAAAAGTGG CCAGGCAGCC 61800
AAGGCAGGCA GAGGTATTGG GGAGAGCTGA ATATCTACAA AAACAAAAAT TCAGAAAAAA 61860
CAAAAATCAA TTTTGGCAAA GGGCTTCACT GTATAACAAG GGGACAAACT AACCCTTTGT 61920
TTACAAACTA ACCCTTTGTT TACTCCATTT TGTCCAGAAA ATACAACAAT CAGTTTTGGC 61980
AAAGGGCTTC ACTGTGTAAC AAGGGGACAA ACTAACCCTT TGTTTACTCC ATTTTGGGAG 62040
ACTATGATCA GACAGGCAGT TGTGACTCAG CAGCAACAAA TGCCTTCTGA GACAGGGATT 62100
CTTTTGATTT TGCTTGGACA TTGTGGAGAA GTGTTAGCCC CAATGTGGAC TGATCTGGGA 62160
ACAGTGGGAA ATTAACTTCT TGTTGGCAAA TATCAGGCTG AGGTGAGAAA GCGACATTTT 62220
CACCGTCCAT CTTTGCTGAT TTACCGTGCT CCCAGGATGG TGGGAGTGTG TGTTTTTAAG 62280
ATGGAGAGTG TATGCTTCTG GGTTCAAGTT CACAGGTGTC TCTGCTGGTT ATCTGCACTC 62340
ACCTTGGTAA CAGGGAGAAA GTGAGTGAAT GGATTCCAAG AACTTACTGA TGGAAGTCTA 62400
ATTCAGGAGT TGGTTCTGCA GCCATGGAGG TAAAGATGTG TTGATAGTCT TTCAATGTGT 62460
AAAAGGGCAA TTAGAGATTC TGTGTGACTG TGTGTTAATT CCACTGGGGT CAGGGGAAAA 62520
ATTTATTTCT AACAGAAAAG AAGAAGATAC GTTATTAGGA AGAATTTCAT GGCTAGGAGA 62580
TACTATCAGA AAAGGCTCTT AAGAGATTTT AAGGATGACT TTAATAGCCG CATTTGAAGT 62640
TTGCAGAGGA TCCACTTTTC CTCTTTTTGT GACCTAAAAT TCTGGGATGA TGAAATAACT 62700
CACCAATTCC ATCTTCTTAT AATATGGAGT CATGTAGACA ACACCATTTT CACACAAATG 62760
GCTAATGGTA TTTAAAAACC ATGATGGAAT GTGAATTGGG AGTCATTTGG AGGTCTGTAG 62820
TTGAACTTGA AAAAATAATA AATGTAATGG AGACAATACT TCACCGTGTT TCCAAAATAT 62880
TTTACAGAGG CATTTTAAAT GAAAGTCACT TTGAGGGAAC AGCTGTGCTG TAAGTTCTCT 62940
TACATGACTG CGCAAGATGG TAGCCTTCAT CAAGACCTCT CAAGGTAGTG TGGGTAGGGT 63000
GACGTGTTTG ATTCAGGCCT CGTTTGTTAT GAAAAGGCTC AAATTCAATT GTATTTGTTA 63060
TTTTTTTGGT TAAAAAGCAC CTATTTGTTC AATTCAAACA ATCCTTTTTG GTTTTTTTTT 63120
GAGATGAAGT CTCCGTCGCC CAGCCTGGAG TGCAGTGGCA TGATCTTGGC TGACTGCAAC 63180
CTCCGCCTCC CAGGTTCAAG TGATTCTCCC AACTCAGCCC CCCGAGTAGC TGGGATTACA 63240
TGTGCTCGCC ACTATGCCCA GTTAAGTTTT GTATTTTTAG TAGAGACGGG GTTTTGCCAT 63300
GTCAGCCAGG CTGGTTTTGA ACTCCTGACC TCAGGTGATC CACCTGCCTC AGCCTCCCAA 63360
AGTGCTGGGA TTATAGGCTT CAGCCACCGT GCCCAGCCAT ATTGTTTTCA TTTTTAATCT 63420
ATTAGTCTAT CGTGATCTCC CAGTGGAAGT ATCTTTGGCC TTTGTGGACG TCAGGAAAGC 63480
CCTACATTCC CACTCGCGAT TCCATGTTTA TGGGTACCCT AAATGCTCCC ATTAATTGAC 63540
CAACTTTACC CTGATCTTCT TTCAATATCT TTCTGACTCC TTGAAGGTAT GAGACAAAAT 63600
GGAAACTGAG AGGTTAAAAG GTTTACTAGG TTGCATTCAA TTAGCGAATT GGAAACTGGA 63660
AGGAGCTCCT ATCGGGTCTC AGGTCAGAAC GTGAGTGCTT TTGGCCAAAG TTCACTTCTG 63720
AGGAAGTAGA ATTTCGCTTT CTGGAATCTT GCGATATTTT ATTTCCTCTA TATCTTTCCC 63780
ATGCCCCCGA CCCACCCAAT CTCCACAAAT TTGGGGATTT GAGCACTGGG TTGTGATCGT 63840
TAGACCATCT TGCTTTTCTG AAAGCCCAGG GCAAGACCCC TGCTTCATGT CACAGTATCA 63900
-94- AACACAGACA TAGAAGCTTG TACAAATTAT TGAGAAGTTA TTGTCTTTTC TCCCTTCCTC 63960
CATATGGAGT CATCTCTATG CCCTTTCATA CAGATGTGAT TTACGAAGAC CTCTGGGTTA 64020
GGGGTGGGGT GGTGAGCAAG AATCCCGTGG CAGAATCTGC TAACACACTT GAGAAGCAAT 64080
GTTGTGGTTT TAAGGAACTC AATCTAAAGC TTGAACCTGA TTTTCAGGGA TACCATTTTG 64140
CTGCCGTTTC AGCCCATTTC TCTTGTTAAG ATCGCTCTCT GGTAGAGTTG ACGTGACACT 64200
CATTTCTGTT GTGGGTGGGG CCCTGGTTGG GAGGCATTGG CTCCACTGCA GCCTGGGTGT 64260
CTAGAGACCA CATTCTCACC CTGCCTTTGT TACTGGGAAA CCGAACGCGG CGCTGTGGCT 64320
TTCAGCTTGG GTAAGCCGGG TCTGCGGCGG GGATTGCCAT CTGAAGACAG AGGCAGGAGG 64380
GCAGCCACAC CTTGCCCAGG TTCTCTTAAA TCTCTTGCTC TATAACTGAA AGGAGGGCAT 64440
AGATAATTAA CTTTATTTGA CATTTTTC T ATCTAATTTT TAAGAATATG ATTTTAAAAT 64500
AATAGATTTG TTCTAAAGAG CAAACAATCT TGCTGTTATT AAAAACGTGT TTACTTAAAT 64560
TGAACGGGGT TTCAAAGGGC CAAGCTACTA AGCTGTGCAG GAAACAAACA GTGCAGTGAG 64620
GAGAATGGCT CCTCACCACA GCTATTCTTA GGGTGGGACA TAGTTTCAAG CCAAATGACA 64680
TTGATGTCCG GAAACCAGGA TGTGCTGAAG TAGAAATTTC CAGGGATCCC TCAGAGTTAT 64740
TTGCTAAAAT GTTTATTATT CTTCAGAGGG GGGTGGAAAT ATTTCTTTAA GAGTCTTCCT 64800
TGAAGAATTT TGAACTCCAG CTTTGGAGTG ATGGGAGCAC AGTGCAGGGA AGGCGGGATG 64860
TGAGGTGGTG TGCTGGACGG CAGTCTAGGG ACCTGGTCTA GCACTGGCAG AGCTGTGTGT 64920
CCCAGAGCAC ACATTCCCCT TTGCCAGGCT TTAGTTTCCT CCTCTAGGCA AAAGGGTTTG 64980
AACCTGACCA TCTTTAAGAT CCATTTTAAC CCTCAGATTC TGTGGCTGTG GTGATTGGGG 65040
GTGGTGGGAG TACCTGGGGG TCAGCAGGAT AAGCACGAAT CTGTGAGAGC TGAGAACAGG 65100
TGGGAGAAGC CTTCTAAGGA TGAGGCAGGA AAGATTAGCA AGAGCCCTTA AATGGATTCT 65160
TTAGGGCCTT CAGAATTTTG GCTAAAGGCT ATACTAGTGG AGGTACTAAG ACCTGACACC 65220
TGGAGCCTTT ATTAAGGATG TTAGAATCCA CTCCCATGAC AACATCCCAG CTTTGCCAAT 65280
TTGCCTCATG TGTCTCAAGC TGGTGGGAAT GTAGAAGTGG ATGAAACAGA CTGTTTTGTG 65340
ATGGCAGGGA ACAGCCTATG CACAGGGGCA GGTGCTCTAC TGGTGTCTTC TATAAAACGC 65400
CAAAGCAGCC CGCCAGAAAA TGGACATTTA GGCACTCGTG GTGTCTACTG AGTTTGTATG 65460
GTACTGATGA GCTTGCTTGA CTGATTATCC ATGACTTACT GAGTAGATCG AACGTATGTG 65520
GACTCACTTC TCCTAGAGGA AGACCCTGTG GCTGCCCCAG CCACTGAGCA GCCTAACCTG 65580
GAGACCCTGA TGTGCCCAGA AAGCGTCAAC CTTGTATCTG GAGAAACCAG AACTTGCAAC 65640
AGGGCCAAGC AGGGTGGCCC ATTTAAAGAG GCTCCTAGGG TTTTAATTGA CCTTGTTTTA 65700
AAAGAGACAC CCTGTAAAAT ACTCCTATGA AAACTTATTT CACAAGCACC TAACCGCATT 65760
CTGTCTTTGG TTTGTTTTAC GGGGCCGGGC CCCTTGTTCT GGTCAATTGG TCTGCATTAT 65820
CTCTCCTCCT CCAATCTCAC CACACACCCT GGCCTCTGGG AGGCTTCCTC CCTTCTTTTT 65880
TTTTGTTTGT TTTGTTTTTT TAGCATCTTA GTTGTTACTA GGGGTACTTG CCTACTTATT 65940
TAAAATATGG CCAGTATAGG TGCATACAAA ATGTGCTTTC TGATTAAAAC AAAGCCAAAA 66000
ATAAAAAGAA ACCAAAATGC CTATTATAGT AGTTGGATTT TTAGACTAAC AGACCACCTC 66060
ATTAACCCTG TCATTTTACC ATAACAACTT ATTTTTATCT TTGTATGACC TTGTCTCAAT 66120
GTCCTTTTTC TTTGATGTTG TTGCAATTAT GAACATCAAA TTTCATAGCT GCTTTTCCAC 66180
CCCACTTTCT ATCACAGAAG CACAATAAAT AATCTTGGGG GCTGGGCTCT TGTTGGCCCA 66240
ACTGTGGCTT CAAAACATTT CAGTTGCCTG TCCAGCCCTT TCTTAGCCTG ATACAACATC 66300
CCCCAAAAGT CTGTTGAGCT TTTCCTGGAA TAAGAAGAGG GTCTTCTACT TTTTGAATAG 66360
AGCAATGGAG ATTGGAGAAT ATGGTCATCT TGTGGAGGTT ATTCCAGGCT TCTTCTTAGG 66420
AACCTTAAAA AAAATCTCCT CAGTAGGGCT GATGATATAT TCTGGACAAT AAGGTGAGCA 66480
GAGTCTGAAA GATGAGAGCA ATTTTCAATC TTGTCATGAT TTCATCTAGT CAGCCTCATT 66540
TCATCTAGTC ATGAGGCTGA CTAATGATAA GACTTGCTTT GTCTTTGCAG TGTACTCTAG 66600
ATTTGACTCT AAATTCAGCC TCTGTCTTGA TCATGCCCAC TTAGAAAATT AGAGTGCAGC 66660
TAGCTCACCT TTTAGTCATC TTAATTCCAC TAGGCAGAAG GCTGTGGGTC AAGGAATGTT 66720
GATGGAGTAA AATTTGACTG CATGTGTATC TGAAGGGGTA GGAGGCTAAG AGATTTTATG 66780
GCTTGGAAGC TGCTGAGATG TGGTGTAAAG AACACTGGAC TTAGAGTCCA GACACCTGAG 66840
TTTAAGCTGG ACTCTACCAC TGGGTAGTTG AATGACTTTG AGTGAGTTAT ATAAGCTCTA 66900
GCATCTAAGT TTTCTCATCT GGAAAATGGA GTTAATAACA TCTACTGCAT TGGGCTGTTG 66960
TAAAGATTAA ATTAACAAAG AATGTGAAAG CACCTGAACA AAAGCTTGTG AGTAAATAAT 67020
TAGTAATTTG TGGAATGAAC ATCAAGGGAA GTCTTCAATT TGGGTGTTTT CAGTGAGTTT 67080
CTGTTGGGTC AGAGTGAATG GATATTAAAT TCTGGGATTT TGGTTTGTGT GTGTGTGTGT 67140
GTGTGTGTGT GTGTGTGTGT GGCGATCAAC ATTGGTTCTT CACTGTGACC TTAGGAAAGA 67200
AATGCAATAG GGTTTTTATT GGGAAGGTGG GTAGCAGGGA GATGCATGAA CCATATTAAG 67260
-95- GGGGGACCTC CAAATTGAAC CTTGTTTTGA GTCAACTGCA AACCACAACC AAGGAGGTCC 67320
TGGGAGACCT GGGGTGACTT GGGGTGATTG GGTATGCAGC ACATTCCTGT TCTTGTGTCC 67380
TGATGCCTGG CAAGTAGGGA CCTGCAGAAA ATACTGATTC TCCTCCAGGC AGTTCACATG 67440
ACTAGCTTTT AGGAGTGAGT ATACCGTTGC CCACCCCTAA AATTCTTGAT CATGTCTCCA 67500
GATGTCTACT GACCACTGAT GCTGAGGTCA TGAATCTTGG GCATTCTAGA GGCTTTGGGA 67560
AAAAAAATTC TACTTACTTC TTTTGCCCAG ACACTCTGGG GTCTACCTCT TGGTAAATTA 67620
TTCAAATGAG GTTTCTGGTC ATGCAAATGT GGTTTCTAGA GCCTATTTGA ATTGAACAAG 67680
TAGTTCTTAT TATTAGTAAA ACAGCAAGGA TCCCTAACTT GGGGTCCAAG GGTAAATTCA 67740
GGGTTTCTGT GAACTTGGAT GTAAAAAAAA ATTGTGTTTA TTTTCAATAA TCTCTAACTA 67800
GAATTTAACA TTTTCTTTCA ATATGAATGT AGGCAAAACT CCATGGTAGT ATTAGCTGCA 67860
ATTGTGACTA TCACCAGGAT AAATCACATT TTCATGTCTT ATTACACCTA TTACATATAT 67920
CACAAAAAGT GGGTATTTGA TATCAAGTTA GATCTGCACT AGGTAGATAT TCTTATTTAA 67980
TGTATTAACA AGGAAGCACA TATATTGTTA TCAGGTTGGT GCAAAAGTAA TTGTGGTTCT 68040
TGCCATTAAA AATAATTACA AAAACAGCCA GTCTGGCCAA CATGGCGAAA CCCCATCTCT 68100
ACTAAAAATA CAGGTGTGGT AGCACACACC TGTAATCCCA GCTACTTGGG AGGCTGAGGC 68160
AGGAGAATCA TTTGAACCTG GGAAGCAGAG GCTGCAGTGA GCCAAGATCA CACCACTGCA 68220
CTCTAGCCTG AGCAACAGAG TGAGACTCTG TCTCAAAAAA ATTAAAAAAT AAAAAAAAAC 68280
TCTGTAATTA CTTTTGCACC AACATAATAT GATATCACAC ATTTATTTTA AAAAGTATTT 68340
TGACATTGTC TTTTAATATA AATTTTTTTA AATCTTATAA TATTTTAATT TGTCATGTAA 68400
AAATATTATT TTGAGAAGAG GCCTGTAGGC CTCACTAGAT TACAAAACAG ATCCATCGTA 68460
CAGATGAAAG GTTAAGAACA CCTCATTTAC AGCATTCTCT CACACACGAC TAACGAAATG 68520
ACTTCTGAAC AGCGCCAGTT GATAGATGTT CTCTGCCAAA AGGGGAATAT GATCTTCCCA 68580
TATGTTCCTG CCTATGGGTA GCCTTGGAGT TGTGAAGGGA CTTTGGCATA ATGAAGATGA 68640
TAATAAGAAT GATAATGGTA ATTTGTTGAG TGCCTGCTGT AAGCCAGGTG GTTACAGTCC 68700
TGTTCAATGT CATGTTTAGT TTAATCCTCC CAATGACCTC AGGAGGTAGT GCATGGAACA 68760
AAGACAGAAG AGATCCCCTG CCCACCCACG GTAATGAAAC ATGGGTACAG GTGAAGGCAA 68820
AAGTGGGGAC TGACCCTTTG GAGATGGCTG ATGTCACGAG TGTGCAACCT GTGCAGTTCC 68880
ACGGGGCCCC ATGCTTAGAA AGGTTCCATG TTTGGTTTAA GGCTCTGCTG TTGCCATCTT 68940
AAAATTCTTC GTAAGTTTTG AACAAAGGGC CCTGCATGTT CCTTTTACAC TGAGCTCTGC 69000
AAATGATGTA GCTGGTCCTG CCTCTGGTTA TGGTGAAATG GAATGTATGA CAACTCCTGA 69060
GACTGGGAGT CTGGGAAGCT GCTGCGGAGA GCCCTCTCCT CATTTTCATC AGGCTCAGCT 69120
ACGCAACCTC TGGTGGAAAG CTATGGCCTG TTGAGGAGGG AGGATGTCGT TTTTGAGTTA 69180
GTGAGTTTTC CAGTTTTGTT TGAGCTCCAA AGCTTTCCTC CAAACAACTG GAAAGATGGC 69240
TGAATAATTG GCTGAAAGGG ATTTAATCCC TTGAAAAACC TTTCTGGTAG GGAGTTGCTG 69300
GCAATACTGG TGGGTTTTTC ATGATTTTAT TTTACAGAGG GCTTGCTACG TAAACCAGTG 69360
AGCCAGGAGA AACAGAATAA AGTCTGTTCT GGAAGGAAAA ATGAGACCTG GTGTGCCACG 69420
AGTCTAGTGT TCTCATAGGA AGGCTCTAAA AACAAACTCA GCTTTCCTGC TATTGAATGA 69480
TTATCTCTAT AAAAGGAAAC TTTACTTCTT CTAAAGGAGA GGTCGTCTAA TTTGTGAGAA 69540
AATTCAGATG TTATTTGCTT CTTAAGCTGC AAGGATGCTA ATGAAATAAT TCTCATGAAG 69600
TTCTGTTGGT GTTTTAGGGC TAAGTTTTTA TAGACTGTTC CAAAATTCAA AACAGGGATG 69660
TGGACGTAGT GATGGTGGAA GAGGGGAAGA CTTTTCCTCG ATTTCTTTGC CTGAGGGATG 69720
GAATTCAGGC TCCCCCAATA ACATATTCAT GGTCTTTCTC TGGTCAGTCA GTGATGTTCA 69780
TAACACAAGC AAGCCTGTCA TCAGGACCAA TCTGTGATGG CTGAGACATC AGGTGCTCTT 69840
CCAAAAGAGC CATAATTCAC CCTTCATTTC CCAAGGTTTT TTTTTTCTTG CTGTTATTAC 69900
TGCTCTTTTA TCATGGTTAA TAAGTCTGAG GTGGCTTCAG ACAGCCAGTC CTAACCCCTG 69960
AGTCAATCTG GGGCCTCTAA CAGGAAGCCA GACTGAAGTT CTGATAGATG GGTTTGAGTG 70020
GCTGTGAACT GTGTTTCTGT AGCATCCAGA CTGATTTGCA CTGAAAGGGA GCTTCCATAT 70080
TAGGGTACAA GGATGATCAA TATGTCTCCT GTTTATATTT GGTGGAAAAA GTTGTGGGAA 70140
TCGTGCTTAA AGGATCTCAA CTTTGAAATT AAAAGTATAA CGTCCTAACA GACATCCTCC 70200
TTCTCTTTAG AAACACAAGG ATCCATTTTC AAGTAATTTC AAAAGAACTA TGTTGCTTTC 70260
CCCACCCCTT CCCAAGTACA CTTATTATAA TATATCCAGT CCATTTGCTA GCTTTGTGTC 70320
TTTAGAAAAG TTGCTTAACC TCTCTCTGTA AAATGGTGCT TATATTAGTA CTAACATTCA 70380
GGGTTATTGT GAGGATTAAA TGAGGTAATT CATGTAATGA CTAGTTCTAT TTCTAGCACA 70440
ATTTAAACCC TCAACAAATA TGAACTATTA TCACTGTCAT AGTTTTTGTT GTTGTTTTCT 70500
AATTATATAA TCTTCAAGAT TCTGAGATGG GGGCTGTTGC TCTTTCCTTG ACTTGAACAT 70560
CTTGGTCTTT TCCTAGGAGG AAACTTGACT CTTGAAATGG TCAAATCCAT TGTCCTAGTT 70620
-96- CATCCTGACC CCTCCCTGGC TCCAATCCCC ACCCCTTACC GTCCTCCACC CTTCCTACAT 70680
TCCTGCACAG TTGGTCTTAT TTATTTTTCA GTCAACTAAG GGTGTTGTTA AATCTTTTAT 70740
TTTTCTGCTG CCCGATTTGG TTCTAAGCAC TCCACTCCCT ACGCTGCTCA TAACAAGAAT 70800
GCCTGGGAAC GCTCAGTCAG CCATATCCCT CCCCTGTCGG AACACCCAGT TCTTAATGCT 70860
CCTGGAGAGG CAACATTTCT GAGGCCCCAC TGCCATAAGC CCCCCTCCCC CATGAAGCCA 70920
GTGGTCTGGT AGTAATGAAC CCCCAACGGC CCGGAGAAAA CTGGGGCAAG GTGTTTGTCT 70980
GGGGAAATGT TGCATGTTGC CTTGACTGTG CTTTCTTCTA CAAAGCTTAA AAAGAGATAT 71040
TATATTATTT TATTTTTATT TTTATTTTTG AGATGGAGTC TTACTTGGTT GCCCAGGCTG 71100
GGTGTGCAGT GGCACAGTCA TGGCTCACTG CAACCTCCAC CTCCTGGGTT CAAGTGATTC 71160
TCCTGCCTCA GCCTCCCAAG TAGCTGGGAC TACAGGCACA TGCCACCATG CCTGGCTAAT 71220
TTGTATATTT TTAGTAGAGA CGGGGTTTCA CCATATTAAC TACATTGGTC TTGAACTCCT 71280
GACCTCAAGT GATATGCCCG CCTCGGACTC CCAAAGTGCT GGGATTAAAA GCATGAGCCA 71340
CTGCGCCCGG CCAAAAAGAG ATATTCAAAA GCTCCCTCTG ACTGTGTGTG CTGAAGGCTG 71400
AGTGCTGATG CCATTGCTTA ATTAATGTTG TTCATGATCT CCATTTGGGC GATTTGTTTA 71460
GCTCCTTGTG GCCCTTTTTG GACTTAGCTT ATCATGTGAC ATTGACAAAT TAATGAGAAG 71520
TGAGCATGTG ATGATGCTTG GATTAGGACA GAAATCACAT CTAGGACATC TCAGGCCCTT 71580
TCCACCTGGG ACCTGAGACC TCAAATCTCT TGGCAGGAGA TGAGTGGGTC TACACAGCCC 71640
GATTTTGAGG TAGGTGTGGC TAGCCTCATT TATGCGATGG GAAAACTGTG GTCCGGGAAC 71700
CAGGGGTTTT CAAATTATGC TTTTTGCCCA GGGCTGGATG TAGGATGTCT GGGGGAGAGG 71760
CTTGACTGAG ATCTGGGTAC ACTGAGCCTC CACTTTAGGA GGTAACCTAG AGACTACACC 71820
TACTCCCTAA ACTGTATTGA CTTTTGGAAG TCAACCATTT AGAAGAGTGT GGTTTTGGTT 71880
TCGATCGTAT CCCAGCAGTC TTTTCTCTGC CCTTGTTAAT CTGATTCATG ATCTGAACCT 71940
GGGCTGGCTG GAGGCTGGCC ATGTCACTTT GCAGACCATG GACACCCCTG AGTGCCCTCA 72000
CAGAACCAGC CAATGGAAAA GTACAACGTC TTCTGGCTTC TCAGCCTTGC CATCTCCCTC 72060
TGGCCTATTT GATACCCCCT TTTATATTGA GGGAGTGAAA ATGTAGCATC CAAACTGAAA 72120
ACGCAGGTTT TTCTTTGGTT TTTATAGGAA AAACAAATTG GCATGAACAC TCAGTCAAAC 72180
CAGCTCAGGC TGTTTGGGCA GATGCCTTTC TTTGCTTTTT TCTGTTTATT TTCCTACAAA 72240
TCAATGCTTA ACTGCGTTGT TATCGGAGCA GAGCAACAGG TGCAAAAAAA TAACTCTGCT 72300
GCCAACTCAA ATGAAAAGGT AGGGCTTATA CCCTCTGGGA GGTATTCAGA AGATAACAGA 72360
AGCCCCTGCC AGCAACTGAA TTAACAGCTC TGTTTACGGT GGGTTTTATG TTAACAACCT 72420
GCTCCTGACC CTCCTACACA TAAACACACC ATTGTCTCAG AGAGAGACAT TCAGCCATCC 72480
AGACAACCCA CTGCTTTATT CTGCCCTGAG TGGAGATTGG TTTTGGCTCA GGCTGCTTTG 72540
TGAAACTCAG AAGCATTATC CTCTCTGCCA ACTCCACGTC CTAGTCAGAG TTTTCTGTGA 72600
AGGCAAGGGC ATGGGGTTGC CGGAGAGAAG AGGATTGGTC CTGCTTTTAA GCCTAGCTGA 72660
AATTCTTTTC AAGGTTGGTC ATTCTCAAAT GCCAGAGAGG GTTGCCCGGC TCTCTCTGCT 72720
CTTGCCCCAT TCCATTCACA ACAGGAGGTG GGGAATGAGC TCAGATGACT TTGGAAGGAG 72780
CCACTATTAT TTTGGAAGCC GTGTCCTTGT GAATAGTCCA TCAGGGTAGG GCAGCGTCTA 72840
TGTTTTGTTA ACTATTGTAT CGCCAGCACC TAGCAAAGTG CCCAGCATCT AGTAGACACT 72900
TGGTAAATAT GTATGAATTA CAGAGGGT 72928
(2) INFORMATION FOR SEQ ID NO : 2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5427 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :
ATTTATCTTC ACTCTGATGA GGGCTCAGAC TTGATAACGC CCGTGGTGCC CCATCCCTAT 60
AGGAGCTGGT GAGATTGCAG CCTGCTGCCT CCCCTCCATC AGCCACAGCT ATTGGATTTC 120
CCACCCAGAA TCTTTAGGTA AATGAGATCA TGATTCTGGA AGGAGGTGGT GTAATGAATC 180
TCAACCCCGG CAACAACCTC CTTCACCAGC CGCCAGCCTG GACAGACAGC TACTCCACGT 240
-97- GCAATGTTTC CAGTGGGTTT TTTGGAGGCC AGTGGCATGA AATTCATCCT CAGTACTGGA 300
CCAAGTACCA GGTGTGGGAG TGGCTCCAGC ACCTCCTGGA CACCAACCAG CTGGATGCCA 360
ATTGTATCCC TTTCCAAGAG TTCGACATCA ACGGCGAGCA CCTCTGCAGC ATGAGTTTGC 420
AGGAGTTCAC CCGGGCGGCA GGGACGGCGG GGCAGCTCCT CTACAGCAAC TTGCAGCATC 480
TGAAGTGGAA CGGCCAGTGC AGTAGTGACC TGTTCCAGTC CACACACAAT GTCATTGTCA 540
AGACTGAACA AACTGAGCCT TCCATCATGA ACACCTGGAA AGACGAGAAC TATTTATATG 600
ACACCAACTA TGGTAGCACA GTAGATTTGT TGGACAGCAA AACTTTCTGC CGGGCTCAGA 660
TCTCCATGAC AACCACCAGT CACCTTCCTG TTGCAGAGTC ACCTGATATG AAAAAGGAGC 720
AAGACCCCCC TGCCAAGTGC CACACCAAAA AGCACAACCC GAGAGGGACT CACTTATGGG 780
AATTCATCCG CGACATCCTC TTGAACCCAG ACAAGAACCC AGGATTAATA AAATGGGAAG 840
ACCGATCTGA GGGCGTCTTC AGGTTCTTGA AATCAGAGGC AGTGGCTCAG CTATGGGGTA 900
AAAAGAAGAA CAACAGCAGC ATGACCTATG AAAAGCTCAG CCGAGCTATG AGATATTACT 960
ACAAAAGAGA AATACTGGAG CGTGTGGATG GACGAAGACT GGTATATAAA TTTGGGAAGA 1020
ATGCCCGAGG ATGGAGAGAA AATGAAAACT GAAGCTGCCA ATACTTTGGA CACAAACCAA 1080
AACACACACC AAATAATCAG AAACAAAGAA CTCCTGGACG TAAATATTTC AAAGACTACT 1140
TTTCTCTGAT ATTTATGTAC CATGAGGGGA AAAAGAAACT ACTTCTAACG GGAAGAAGAA 1200
ACACTACAGT CGATTAAAAA AATTATTTTG TTACTTCGAA GTATGTCCTA TATGGGGAAA 1260
AAACGTACAC AGTTTTCTGT GAAATATGAT GCTGTATGTG GTTGTGATTT TTTTTCACCT 1320
CTATTGTGAA TTCTTTTTCA CTGCAAGAGT AACAGGATTT GTAGCCTTGT GCTTCTTGCT 1380
AAGAGAAAGA AAAACAAAAT CAGAGGGCAT TAAATGTTTT GTATGTGACA TGATTTAGAA 1440
AAAGGTGATG CATCCTCCTC ACATAAGCAT CCATATGGCT TCGTCAAGGG AGGTGAACAT 1500
TGTTGCTGAG TTAAATTCCA GGGTCTCAGA TGGTTAGGAC AAAGTGGATG GATGCCGGGA 1560
AGTTTAACCT GAGCCTTAGG ATCCAATGAG TGGAGAATGG GGACTTCCAA AACCCAAGGT 1620
TGGCTATAAT CTCTGCATAA CCACATGACT TGGAATGCTT AAATCAGCAA GAAGAATAAT 1680
GGTGGGGTCT TTATACTCAT TCAGGAATGG TTTATCTGAT GCCAGGGCTG TCTTCCTTTC 1740
TCCCCTTTGG ATGGTTGGTG AAATACTTTA ATTGCCCTGT CTGCTCACTT CTAGCTATTT 1800
AAGAGAGAAC CCAGCTTGGT TCTTTTTTGC TCCAAGTGCT TAAAAATAAG TTGGAAAAAG 1860
GAGACGGTGG TGTGGAAATG GCTGAAGAGT TTGCTCTTGT ATCCCTATAG TCCAAGGTTT 1920
CTCAATCTGC ACAATTGACA TTTTTGGCCG GAGTGTTCTT TGTGGTGAGG GCTTTCCTGT 1980
GCATTGTAAG ATGTTCAGCA GTATCCACTC ATGGTCTCTA ACCACTTGAC ACCAGAAACC 2040
CCCCAGCTGT GATAACGCAA AATGTCTCTA GACATCACCA AATGTTCCCT GGGGGTGGCA 2100
AATTTGCCCT TGATTGAGAA CCACCAGTTT AGCTAGTCAA TATGAGGATG GTGGTTTATT 2160
CTCAGAAGAA AAAGATATGT AAGGTCTTTT AGCTCCTTAG AGTGAAGCAA AAGCAAGACT 2220
TCAACCTCAA CCTATCTTTA TGTTTTAAAT ATTAGGGACA ATAAGTTGAA ATAGCTAGAG 2280
GAGCTTCTTT TCAGAACCCC AGATGAGAGC CAATGTCAGA TAAAGTAAGC ATAGCAATGT 2340
AGCAGGAACT ACAATAGAAG ACATTTTCAC TGGAATTACA AAGCAGAATT AAAATTATAT 2400
TGTAGAAGGA AACACCAAGA AAAGAATTTC CAGGGAAAAT CCTCTTTGCA GGTATTAATT 2460
CTTATAATTT TTTGTCTTTT GGATTATCTG TTTACTGTCT CATCTGAACT GATCCCAGGT 2520
GAACGGTTTA TTGCCTAGAT TTGTACTCAG AGGAATTTTT TTTGTTTTGT TTTGTCTTTT 2580
AAGAAAGGAA AGAAAGGATG AAAAAAATAA ACAGAAAACT CAGCTCAGGC ACAATTGTCA 2640
CCAAGGAGTT AAAAGCTTCT TCTTCAATAG AGGAATTGTT CTGGGGGTCC TGGAGACTTA 2700
CCATTGAGCC ATGCAATCTG GGAAGCACAG GAATAAGTAG ACACTTTGAA AATGGATTTG 2760
AATGTTCTCA TCCCTTTTGC AGCTTTTCTT TTTGGCTCTC TCATGTCCTT GGCTTGCTCC 2820
TCTATTCTAC CTCTCTTTCT CCAGCAATAA TATGCAAATG AAGACATGTA TCCATAAGAA 2880
GGAGTGCTCT TCATCAACTA ATAGAGCACC TACCACAGTG TCATACCTGG TAGAGGTGAG 2940
CAATTCATAT TCAAAGGTTG CAAAGTGTTT GTAATATATT CATGAGGCTG GAAGTAAGAA 3000
GAATTAAAAA TTTGTCCTAA TTACAATGAG AACCATTCTA GGTAGTGATC TTGGAGCACA 3060
CATGAATAAC TTTCTGAAGG TGCAACCAAA TCCATTTTTA TTTCTGCCTG GCTTGGTCAC 3120
CTCTGTAAAG GTTTAACTTA GTGTTGTCAA GTAACAGTTA CTGAAAGAGC TGAGAAAAAG 3180
AACAATGAAC AGCAACGATC TTGACTGTGC AACTCAGACA TTCCTGCAGA AAAGACATAT 3240
GTTGCTTTAC AAGAAGGCCA AAGAACTATG GGGCCTTCCC AGCATTTGAC TGTTCATTGC 3300
ATAGAATGAA TTAAATATCC AGTTACTTGA ATGGGTATAA CGCATGAATA TTTGTGTGTC 3360
TGTGTGTGTG TCTGAGTTGT GTGATTTTAT TAGGGGCATC TGCCAATTCT CTCACTGTGG 3420
TTCCTTCTCT GACTTTGCCT GTTCATCATC TAAGGAGGCT AGATCCTTCG CTGACTTCAC 3480
CATTCCTCAA ACCTGTAAGT TTCTCACTTC TTCCAAATTG GCTTTGGCTC TTTCTTCAAC 3540
CTTTCCATTC. AAGAGCAATC TTTGCTAAGG AGTAAGTGAA TGTGAAGAGT ACCAACTACA 3600
-98- ACAATTCTAC AGATAATTAG TGGATTGTGT TGTTTGTTGA GAGTGAAGGT TTCTTGGCAT 3660
CTGGTGCCTG ATTAAGGCTT GAGTATTAAG TTCTCAGCAT ATCTCTCTAT TGTCTTGACT 3720
TGAGTTTGCT GCATTTTCTA TGTGCTGTTC GTGACTTGGA GAACTTAAAG TAATCGAGCT 3780
ATGCCAACTT GGGGTGGTAA CAGAGTACTT CCCACCACAG TGTTGAAAGG GAGAGCAAAG 3840
TCTTATGGAT AAACCCTCCT TTCTTTTGGG GACACATGGC TCTCACTTGA GAAGCTCACC 3900
TGTGCTGAAT GTCCACATGG TCACTAAACA TGTTATCCTT AAACCCCCCG TATGCCTGAG 3960
TTGAAAGGGC TCTCTCTTAT TAGGTTTTCA TGGGAACATG AGGCAGCAAA TCTATTGCTA 4020
AGACTTTACC AGGCTCAAAT CATCTGAGGC TGATAGATAT TTGACTTGGT AAGACTTAAG 4080
TAAGGCTCTG GCTCCCAGGG GCATAAGCAA CAGTTTCTTG AATGTGCCAT CTGAGAAGGG 4140
AGACCCAGGT TATGAGTTTT CCTTTGAACA CATTGGTCTT TTCTCAAAGT TCCTGCCTTG 4200
CTAGACTGTT AGCTCTTTGA GGACAGGGAC TATGTCTTAT CAATCACTAT TATTTTCCTG 4260
TTACCTAGCA TGGGACAAGT ACACAACACA TATTTGTTCA ATGAATGAAT GAATGTCTTC 4320
TAAAAGACTC CTCTGATTGG GAGACCATAT CTATAATTGG GATGTGAATC ATTTCTTCAG 4380
TGGAATAAGA GCACAACGGC ACAACCTTCA AGGACATATT ATCTACTATG AACATTTTAC 4440
TGTGAGACTC TTTATTTTGC CTTCTACTTG CGCTGAAATG AAACCAAAAC AGGCCGTTGG 4500
GTTCCACAAG TCAATATATG TTGGATGAGG ATTCTGTTGC CTTATTGGGA ACTGTGAGAC 4560
TTATCTGGTA TGAGAAGCCA GTAATAAACC TTTGACCTGT TTTAACCAAT GAAGATTATG 4620
AATATGTTAA TATGATGTAA ATTGCTATTT AAGTGTAAAG CAGTTCTAAG TTTTAGTATT 4680
TGGGGGATTG GTTTTTATTA TTTTTTTCCT TTTTGAAAAA TACTGAGGGA TCTTTTGATA 4740
AAGTTAGTAA TGCATGTTAG ATTTTAGTTT TGCAAGCATG TTGTTTTTCA AATATATCAA 4800
GTATAGAAAA AGGTAAAACA GTTAAGAAGG AAGGCAATTA TATTATTCTT CTGTAGTTAA 4860
GCAAACACTT GTTGAGTGCC TGCTATGTGC ACGGCATGGG CCCATATGTG TGAGGAGCTT 4920
GTCTAATTAT GTAGGAAGCA ATAGATCTCG GTAGTTACGT ATTGGGCAGA TACTTACTGT 4980
ATGAATGAAA GAACATCACA GTAATCACAA TATCAGAGCT GAATTATCCT CAGTGTAGCT 5040
TCTTGGAATT CAGTTTCTGG AACTAGAGAT AGAGCATTTA TTAAAAAAAA CTCCTGTTGA 5100
GACTGTGTCT TATGAACCTC TGAAACGTAC AAGCCTTCAC AAGTTTAACT AAATTGGGAT 5160
TAATCTTTCT GTAGTTATCT GCATAATTCT TGTTTTTCTT TCCATCTGGC TCCTGGGTTG 5220
ACAATTTGTG GAAACAACTC TATTGCTACT ATTTAAAAAA AATCAGAAAT CTTTCCCTTT 5280
AAGCTATGTT AAATTCAAAC TATTCCTGCT ATTCCTGTTT TGTCAAAGAA TTATATTTTT 5340
CAAAATATGT TTATTTGTTT GATGGGTCCC AGGAAACACT AATAAAAACC ACAGAGACCA 5400
GCCTGGAAAA AAAAAAAAAA AAAAAAA 5427
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5510 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
ATCGCTCTCT GGTAGAGTTG ACGTGACACT CATTTCTGTT GTGGGTGGGG CCCTGGTTGG 60
GAGGCATTGG CTCCACTGCA GCCTGGGTGT CTAGAGACCA CATTCTCACC CTGCCTTTGT 120
TACTGGGAAA CCGAACGCGG CGCTGTGGCT TTCAGCTTGG GTAAGCCGGG TCTGCGGCGG 180
GGATTGCCAT CTGAAGACAG AGGCAGGAGG GCAGCCACAC CTTGCCCAGA TCATGATTCT 240
GGAAGGAGGT GGTGTAATGA ATCTCAACCC CGGCAACAAC CTCCTTCACC AGCCGCCAGC 300
CTGGACAGAC AGCTACTCCA CGTGCAATGT TTCCAGTGGG TTTTTTGGAG GCCAGTGGCA 360
TGAAATTCAT CCTCAGTACT GGACCAAGTA CCAGGTGTGG GAGTGGCTCC AGCACCTCCT 420
GGACACCAAC CAGCTGGATG CCAATTGTAT CCCTTTCCAA GAGTTCGACA TCAACGGCGA 480
GCACCTCTGC AGCATGAGTT TGCAGGAGTT CACCCGGGCG GCAGGGACGG CGGGGCAGCT 540
CCTCTACAGC AACTTGCAGC ATCTGAAGTG GAACGGCCAG TGCAGTAGTG ACCTGTTCCA 600
GTCCACACAC AATGTCATTG TCAAGACTGA ACAAACTGAG CCTTCCATCA TGAACACCTG 660
GAAAGACGAG AACTATTTAT ATGACACCAA CTATGGTAGC ACAGTAGATT TGTTGGACAG 720
-99- CAAAACTTTC TGCCGGGCTC AGATCTCCAT GACAACCACC AGTCACCTTC CTGTTGCAGA 780
GTCACCTGAT ATGAAAAAGG AGCAAGACCC CCCTGCCAAG TGCCACACCA AAAAGCACAA 840
CCCGAGAGGG ACTCACTTAT GGGAATTCAT CCGCGACATC CTCTTGAACC CAGACAAGAA 900
CCCAGGATTA ATAAAATGGG AAGACCGATC TGAGGGCGTC TTCAGGTTCT TGAAATCAGA 960
GGCAGTGGCT CAGCTATGGG GTAAAAAGAA GAACAACAGC AGCATGACCT ATGAAAAGCT 1020
CAGCCGAGCT ATGAGATATT ACTACAAAAG AGAAATACTG GAGCGTGTGG ATGGACGAAG 1080
ACTGGTATAT AAATTTGGGA AGAATGCCCG AGGATGGAGA GAAAATGAAA ACTGAAGCTG 1140
CCAATACTTT GGACACAAAC CAAAACACAC ACCAAATAAT CAGAAACAAA GAACTCCTGG 1200
ACGTAAATAT TTCAAAGACT ACTTTTCTCT GATATTTATG TACCATGAGG GGAAAAAGAA 1260
ACTACTTCTA ACGGGAAGAA GAAACACTAC AGTCGATTAA AAAAATTATT TTGTTACTTC 1320
GAAGTATGTC CTATATGGGG AAAAAACGTA CACAGTTTTC TGTGAAATAT GATGCTGTAT 1380
GTGGTTGTGA TTTTTTTTCA CCTCTATTGT GAATTCTTTT TCACTGCAAG AGTAACAGGA 1440
TTTGTAGCCT TGTGCTTCTT GCTAAGAGAA AGAAAAACAA AATCAGAGGG CATTAAATGT 1500
TTTGTATGTG ACATGATTTA GAAAAAGGTG ATGCATCCTC CTCACATAAG CATCCATATG 1560
GCTTCGTCAA GGGAGGTGAA CATTGTTGCT GAGTTAAATT CCAGGGTCTC AGATGGTTAG 1620
GACAAAGTGG ATGGATGCCG GGAAGTTTAA CCTGAGCCTT AGGATCCAAT GAGTGGAGAA 1680
TGGGGACTTC CAAAACCCAA GGTTGGCTAT AATCTCTGCA TAACCACATG ACTTGGAATG 1740
CTTAAATCAG CAAGAAGAAT AATGGTGGGG TCTTTATACT CATTCAGGAA TGGTTTATCT 1800
GATGCCAGGG CTGTCTTCCT TTCTCCCCTT TGGATGGTTG GTGAAATACT TTAATTGCCC 1860
TGTCTGCTCA CTTCTAGCTA TTTAAGAGAG AACCCAGCTT GGTTCTTTTT TGCTCCAAGT 1920
GCTTAAAAAT AAGTTGGAAA AAGGAGACGG TGGTGTGGAA ATGGCTGAAG AGTTTGCTCT 1980
TGTATCCCTA TAGTCCAAGG TTTCTCAATC TGCACAATTG ACATTTTTGG CCGGAGTGTT 2040
CTTTGTGGTG AGGGCTTTCC TGTGCATTGT AAGATGTTCA GCAGTATCCA CTCATGGTCT 2100
CTAACCACTT GACACCAGAA ACCCCCCAGC TGTGATAACG CAAAATGTCT CTAGACATCA 2160
CCAAATGTTC CCTGGGGGTG GCAAATTTGC CCTTGATTGA GAACCACCAG TTTAGCTAGT 2220
CAATATGAGG ATGGTGGTTT ATTCTCAGAA GAAAAAGATA TGTAAGGTCT TTTAGCTCCT 2280
TAGAGTGAAG CAAAAGCAAG ACTTCAACCT CAACCTATCT TTATGTTTTA AATATTAGGG 2340
ACAATAAGTT GAAATAGCTA GAGGAGCTTC TTTTCAGAAC CCCAGATGAG AGCCAATGTC 2400
AGATAAAGTA AGCATAGCAA TGTAGCAGGA ACTACAATAG AAGACATTTT CACTGGAATT 2460
ACAAAGCAGA ATTAAAATTA TATTGTAGAA GGAAACACCA AGAAAAGAAT TTCCAGGGAA 2520
AATCCTCTTT GCAGGTATTA ATTCTTATAA TTTTTTGTCT TTTGGATTAT CTGTTTACTG 2580
TCTCATCTGA ACTGATCCCA GGTGAACGGT TTATTGCCTA GATTTGTACT CAGAGGAATT 2640
TTTTTTGTTT TGTTTTGTCT TTTAAGAAAG GAAAGAAAGG ATGAAAAAAA TAAACAGAAA 2700
ACTCAGCTCA GGCACAATTG TCACCAAGGA GTTAAAAGCT TCTTCTTCAA TAGAGGAATT 2760
GTTCTGGGGG TCCTGGAGAC TTACCATTGA GCCATGCAAT CTGGGAAGCA CAGGAATAAG 2820
TAGACACTTT GAAAATGGAT TTGAATGTTC TCATCCCTTT TGCAGCTTTT CTTTTTGGCT 2880
CTCTCATGTC CTTGGCTTGC TCCTCTATTC TACCTCTCTT TCTCCAGCAA TAATATGCAA 2940
ATGAAGACAT GTATCCATAA GAAGGAGTGC TCTTCATCAA CTAATAGAGC ACCTACCACA 3000
GTGTCATACC TGGTAGAGGT GAGCAATTCA TATTCAAAGG TTGCAAAGTG TTTGTAATAT 3060
ATTCATGAGG CTGGAAGTAA GAAGAATTAA AAATTTGTCC TAATTACAAT GAGAACCATT 3120
CTAGGTAGTG ATCTTGGAGC ACACATGAAT AACTTTCTGA AGGTGCAACC AAATCCATTT 3180
TTATTTCTGC CTGGCTTGGT CACCTCTGTA AAGGTTTAAC TTAGTGTTGT CAAGTAACAG 3240
TTACTGAAAG AGCTGAGAAA AAGAACAATG AACAGCAACG ATCTTGACTG TGCAACTCAG 3300
ACATTCCTGC AGAAAAGACA TATGTTGCTT TACAAGAAGG CCAAAGAACT ATGGGGCCTT 3360
CCCAGCATTT GACTGTTCAT TGCATAGAAT GAATTAAATA TCCAGTTACT TGAATGGGTA 3420
TAACGCATGA ATATTTGTGT GTCTGTGTGT GTGTCTGAGT TGTGTGATTT TATTAGGGGC 3480
ATCTGCCAAT TCTCTCACTG TGGTTCCTTC TCTGACTTTG CCTGTTCATC ATCTAAGGAG 3540
GCTAGATCCT TCGCTGACTT CACCATTCCT CAAACCTGTA AGTTTCTCAC TTCTTCCAAA 3600
TTGGCTTTGG CTCTTTCTTC AACCTTTCCA TTCAAGAGCA ATCTTTGCTA AGGAGTAAGT 3660
GAATGTGAAG AGTACCAACT ACAACAATTC TACAGATAAT TAGTGGATTG TGTTGTTTGT 3720
TGAGAGTGAA GGTTTCTTGG CATCTGGTGC CTGATTAAGG CTTGAGTATT AAGTTCTCAG 3780
CATATCTCTC TATTGTCTTG ACTTGAGTTT GCTGCATTTT CTATGTGCTG TTCGTGACTT 3840
GGAGAACTTA AAGTAATCGA GCTATGCCAA CTTGGGGTGG TAACAGAGTA CTTCCCACCA 3900
CAGTGTTGAA AGGGAGAGCA AAGTCTTATG GATAAACCCT CCTTTCTTTT GGGGACACAT 3960
GGCTCTCACT TGAGAAGCTC ACCTGTGCTG AATGTCCACA TGGTCACTAA ACATGTTATC 4020
CTTAAACCCC CCGTATGCCT GAGTTGAAAG GGCTCTCTCT TATTAGGTTT TCATGGGAAC 4080
-100- ATGAGGCAGC AAATCTATTG CTAAGACTTT ACCAGGCTCA AATCATCTGA GGCTGATAGA 4140
TATTTGACTT GGTAAGACTT AAGTAAGGCT CTGGCTCCCA GGGGCATAAG CAACAGTTTC 4200
TTGAATGTGC CATCTGAGAA GGGAGACCCA GGTTATGAGT TTTCCTTTGA ACACATTGGT 4260
CTTTTCTCAA AGTTCCTGCC TTGCTAGACT GTTAGCTCTT TGAGGACAGG GACTATGTCT 4320
TATCAATCAC TATTATTTTC CTGTTACCTA GCATGGGACA AGTACACAAC ACATATTTGT 4380
TCAATGAATG AATGAATGTC TTCTAAAAGA CTCCTCTGAT TGGGAGACCA TATCTATAAT 4440
TGGGATGTGA ATCATTTCTT CAGTGGAATA AGAGCACAAC GGCACAACCT TCAAGGACAT 4500
ATTATCTACT ATGAACATTT TACTGTGAGA CTCTTTATTT TGCCTTCTAC TTGCGCTGAA 4560
ATGAAACCAA AACAGGCCGT TGGGTTCCAC AAGTCAATAT ATGTTGGATG AGGATTCTGT 4620
TGCCTTATTG GGAACTGTGA GACTTATCTG GTATGAGAAG CCAGTAATAA ACCTTTGACC 4680
TGTTTTAACC AATGAAGATT ATGAATATGT TAATATGATG TAAATTGCTA TTTAAGTGTA 4740
AAGCAGTTCT AAGTTTTAGT ATTTGGGGGA TTGGTTTTTA TTATTTTTTT CCTTTTTGAA 4800
AAATACTGAG GGATCTTTTG ATAAAGTTAG TAATGCATGT TAGATTTTAG TTTTGCAAGC 4860
ATGTTGTTTT TCAAATATAT CAAGTATAGA AAAAGGTAAA ACAGTTAAGA AGGAAGGCAA 4920
TTATATTATT CTTCTGTAGT TAAGCAAACA CTTGTTGAGT GCCTGCTATG TGCACGGCAT 4980
GGGCCCATAT GTGTGAGGAG CTTGTCTAAT TATGTAGGAA GCAATAGATC TCGGTAGTTA 5040
CGTATTGGGC AGATACTTAC TGTATGAATG AAAGAACATC ACAGTAATCA CAATATCAGA 5100
GCTGAATTAT CCTCAGTGTA GCTTCTTGGA ATTCAGTTTC TGGAACTAGA GATAGAGCAT 5160
TTATTAAAAA AAACTCCTGT TGAGACTGTG TCTTATGAAC CTCTGAAACG TACAAGCCTT 5220
CACAAGTTTA ACTAAATTGG GATTAATCTT TCTGTAGTTA TCTGCATAAT TCTTGTTTTT 5280
CTTTCCATCT GGCTCCTGGG TTGACAATTT GTGGAAACAA CTCTATTGCT ACTATTTAAA 5340
AAAAATCAGA AATCTTTCCC TTTAAGCTAT GTTAAATTCA AACTATTCCT GCTATTCCTG 5400
TTTTGTCAAA GAATTATATT TTTCAAAATA TGTTTATTTG TTTGATGGGT CCCAGGAAAC 5460
ACTAATAAAA ACCACAGAGA CCAGCCTGGA AAAAAAAAAA AAAAAAAAAA 5510
(2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5667 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
ATCGCTCTCT GGTAGAGTTG ACGTGACACT CATTTCTGTT GTGGGTGGGG CCCTGGTTGG 60
GAGGCATTGG CTCCACTGCA GCCTGGGTGT CTAGAGACCA CATTCTCACC CTGCCTTTGT 120
TACTGGGAAA CCGAACGCGG CGCTGTGGCT TTCAGCTTGG GTAAGCCGGG TCTGCGGCGG 180
GGATTGCCAT CTGAAGACAG AGGCAGGAGG GCAGCCACAC CTTGCCCAGC TGCACACCCA 240
GTAACAAGTT TCCTCAGTGC GGGTATCTGC CACAGGCTGG GCTGGTCATC AAAGGGCCTC 300
AGTCATATTT TAATAGAGCT CTTCAAGTAT CTGGCTTTGT GATAATATCA GGAATCAGTT 360
GGTTTCTCTG ACAGACACTG CCCATTATCA TGATTCTGGA AGGAGGTGGT GTAATGAATC 420
TCAACCCCGG CAACAACCTC CTTCACCAGC CGCCAGCCTG GACAGACAGC TACTCCACGT 480
GCAATGTTTC CAGTGGGTTT TTTGGAGGCC AGTGGCATGA AATTCATCCT CAGTACTGGA 540
CCAAGTACCA GGTGTGGGAG TGGCTCCAGC ACCTCCTGGA CACCAACCAG CTGGATGCCA 600
ATTGTATCCC TTTCCAAGAG TTCGACATCA ACGGCGAGCA CCTCTGCAGC ATGAGTTTGC 660
AGGAGTTCAC CCGGGCGGCA GGGACGGCGG GGCAGCTCCT CTACAGCAAC TTGCAGCATC 720
TGAAGTGGAA CGGCCAGTGC AGTAGTGACC TGTTCCAGTC CACACACAAT GTCATTGTCA 780
AGACTGAACA AACTGAGCCT TCCATCATGA ACACCTGGAA AGACGAGAAC TATTTATATG 840
ACACCAACTA TGGTAGCACA GTAGATTTGT TGGACAGCAA AACTTTCTGC CGGGCTCAGA 900
TCTCCATGAC AACCACCAGT CACCTTCCTG TTGCAGAGTC ACCTGATATG AAAAAGGAGC 960
AAGACCCCCC TGCCAAGTGC CACACCAAAA AGCACAACCC GAGAGGGACT CACTTATGGG 1020
AATTCATCCG CGACATCCTC TTGAACCCAG ACAAGAACCC AGGATTAATA AAATGGGAAG 1080
ACCGATCTGA GGGCGTCTTC AGGTTCTTGA AATCAGAGGC AGTGGCTCAG CTATGGGGTA 1140
-101- AAAAGAAGAA CAACAGCAGC ATGACCTATG AAAAGCTCAG CCGAGCTATG AGATATTACT 1200
ACAAAAGAGA AATACTGGAG CGTGTGGATG GACGAAGACT GGTATATAAA TTTGGGAAGA 1260
ATGCCCGAGG ATGGAGAGAA AATGAAAACT GAAGCTGCCA ATACTTTGGA CACAAACCAA 1320
AACACACACC AAATAATCAG AAACAAAGAA CTCCTGGACG TAAATATTTC AAAGACTACT 1380
TTTCTCTGAT ATTTATGTAC CATGAGGGGA AAAAGAAACT ACTTCTAACG GGAAGAAGAA 1440
ACACTACAGT CGATTAAAAA AATTATTTTG TTACTTCGAA GTATGTCCTA TATGGGGAAA 1500
AAACGTACAC AGTTTTCTGT GAAATATGAT GCTGTATGTG GTTGTGATTT TTTTTCACCT 1560
CTATTGTGAA TTCTTTTTCA CTGCAAGAGT AACAGGATTT GTAGCCTTGT GCTTCTTGCT 1620
AAGAGAAAGA AAAACAAAAT CAGAGGGCAT TAAATGTTTT GTATGTGACA TGATTTAGAA 1680
AAAGGTGATG CATCCTCCTC ACATAAGCAT CCATATGGCT TCGTCAAGGG AGGTGAACAT 1740
TGTTGCTGAG TTAAATTCCA GGGTCTCAGA TGGTTAGGAC AAAGTGGATG GATGCCGGGA 1800
AGTTTAACCT GAGCCTTAGG ATCCAATGAG TGGAGAATGG GGACTTCCAA AACCCAAGGT 1860
TGGCTATAAT CTCTGCATAA CCACATGACT TGGAATGCTT AAATCAGCAA GAAGAATAAT 1920
GGTGGGGTCT TTATACTCAT TCAGGAATGG TTTATCTGAT GCCAGGGCTG TCTTCCTTTC 1980
TCCCCTTTGG ATGGTTGGTG AAATACTTTA ATTGCCCTGT CTGCTCACTT CTAGCTATTT 2040
AAGAGAGAAC CCAGCTTGGT TCTTTTTTGC TCCAAGTGCT TAAAAATAAG TTGGAAAAAG 2100
GAGACGGTGG TGTGGAAATG GCTGAAGAGT TTGCTCTTGT ATCCCTATAG TCCAAGGTTT 2160
CTCAATCTGC ACAATTGACA TTTTTGGCCG GAGTGTTCTT TGTGGTGAGG GCTTTCCTGT 2220
GCATTGTAAG ATGTTCAGCA GTATCCACTC ATGGTCTCTA ACCACTTGAC ACCAGAAACC 2280
CCCCAGCTGT GATAACGCAA AATGTCTCTA GACATCACCA AATGTTCCCT GGGGGTGGCA 2340
AATTTGCCCT TGATTGAGAA CCACCAGTTT AGCTAGTCAA TATGAGGATG GTGGTTTATT 2400
CTCAGAAGAA AAAGATATGT AAGGTCTTTT AGCTCCTTAG AGTGAAGCAA AAGCAAGACT 2460
TCAACCTCAA CCTATCTTTA TGTTTTAAAT ATTAGGGACA ATAAGTTGAA ATAGCTAGAG 2520
GAGCTTCTTT TCAGAACCCC AGATGAGAGC CAATGTCAGA TAAAGTAAGC ATAGCAATGT 2580
AGCAGGAACT ACAATAGAAG ACATTTTCAC TGGAATTACA AAGCAGAATT AAAATTATAT 2640
TGTAGAAGGA AACACCAAGA AAAGAATTTC CAGGGAAAAT CCTCTTTGCA GGTATTAATT 2700
CTTATAATTT TTTGTCTTTT GGATTATCTG TTTACTGTCT CATCTGAACT GATCCCAGGT 2760
GAACGGTTTA TTGCCTAGAT TTGTACTCAG AGGAATTTTT TTTGTTTTGT TTTGTCTTTT 2820
AAGAAAGGAA AGAAAGGATG AAAAAAATAA ACAGAAAACT CAGCTCAGGC ACAATTGTCA 2880
CCAAGGAGTT AAAAGCTTCT TCTTCAATAG AGGAATTGTT CTGGGGGTCC TGGAGACTTA 2940
CCATTGAGCC ATGCAATCTG GGAAGCACAG GAATAAGTAG ACACTTTGAA AATGGATTTG 3000
AATGTTCTCA TCCCTTTTGC AGCTTTTCTT TTTGGCTCTC TCATGTCCTT GGCTTGCTCC 3060
TCTATTCTAC CTCTCTTTCT CCAGCAATAA TATGCAAATG AAGACATGTA TCCATAAGAA 3120
GGAGTGCTCT TCATCAACTA ATAGAGCACC TACCACAGTG TCATACCTGG TAGAGGTGAG 3180
CAATTCATAT TCAAAGGTTG CAAAGTGTTT GTAATATATT CATGAGGCTG GAAGTAAGAA 3240
GAATTAAAAA TTTGTCCTAA TTACAATGAG AACCATTCTA GGTAGTGATC TTGGAGCACA 3300
CATGAATAAC TTTCTGAAGG TGCAACCAAA TCCATTTTTA TTTCTGCCTG GCTTGGTCAC 3360
CTCTGTAAAG GTTTAACTTA GTGTTGTCAA GTAACAGTTA CTGAAAGAGC TGAGAAAAAG 3420
AACAATGAAC AGCAACGATC TTGACTGTGC AACTCAGACA TTCCTGCAGA AAAGACATAT 3480
GTTGCTTTAC AAGAAGGCCA AAGAACTATG GGGCCTTCCC AGCATTTGAC TGTTCATTGC 3540
ATAGAATGAA TTAAATATCC AGTTACTTGA ATGGGTATAA CGCATGAATA TTTGTGTGTC 3600
TGTGTGTGTG TCTGAGTTGT GTGATTTTAT TAGGGGCATC TGCCAATTCT CTCACTGTGG 3660
TTCCTTCTCT GACTTTGCCT GTTCATCATC TAAGGAGGCT AGATCCTTCG CTGACTTCAC 3720
CATTCCTCAA ACCTGTAAGT TTCTCACTTC TTCCAAATTG GCTTTGGCTC TTTCTTCAAC 3780
CTTTCCATTC AAGAGCAATC TTTGCTAAGG AGTAAGTGAA TGTGAAGAGT ACCAACTACA 3840
ACAATTCTAC AGATAATTAG TGGATTGTGT TGTTTGTTGA GAGTGAAGGT TTCTTGGCAT 3900
CTGGTGCCTG ATTAAGGCTT GAGTATTAAG TTCTCAGCAT ATCTCTCTAT TGTCTTGACT 3960
TGAGTTTGCT GCATTTTCTA TGTGCTGTTC GTGACTTGGA GAACTTAAAG TAATCGAGCT 4020
ATGCCAACTT GGGGTGGTAA CAGAGTACTT CCCACCACAG TGTTGAAAGG GAGAGCAAAG 4080
TCTTATGGAT AAACCCTCCT TTCTTTTGGG GACACATGGC TCTCACTTGA GAAGCTCACC 4140
TGTGCTGAAT GTCCACATGG TCACTAAACA TGTTATCCTT AAACCCCCCG TATGCCTGAG 4200
TTGAAAGGGC TCTCTCTTAT TAGGTTTTCA TGGGAACATG AGGCAGCAAA TCTATTGCTA 4260
AGACTTTACC AGGCTCAAAT CATCTGAGGC TGATAGATAT TTGACTTGGT AAGACTTAAG 4320
TAAGGCTCTG GCTCCCAGGG GCATAAGCAA CAGTTTCTTG AATGTGCCAT CTGAGAAGGG 4380
AGACCCAGGT TATGAGTTTT CCTTTGAACA CATTGGTCTT TTCTCAAAGT TCCTGCCTTG 4440
CTAGACTGTT AGCTCTTTGA GGACAGGGAC TATGTCTTAT CAATCACTAT TATTTTCCTG 4500
-102- TTACCTAGCA TGGGACAAGT ACACAACACA TATTTGTTCA ATGAATGAAT GAATGTCTTC 4560
TAAAAGACTC CTCTGATTGG GAGACCATAT CTATAATTGG GATGTGAATC ATTTCTTCAG 4620
TGGAATAAGA GCACAACGGC ACAACCTTCA AGGACATATT ATCTACTATG AACATTTTAC 4680
TGTGAGACTC TTTATTTTGC CTTCTACTTG CGCTGAAATG AAACCAAAAC AGGCCGTTGG 4740
GTTCCACAAG TCAATATATG TTGGATGAGG ATTCTGTTGC CTTATTGGGA ACTGTGAGAC 4800
TTATCTGGTA TGAGAAGCCA GTAATAAACC TTTGACCTGT TTTAACCAAT GAAGATTATG 4860
AATATGTTAA TATGATGTAA ATTGCTATTT AAGTGTAAAG CAGTTCTAAG TTTTAGTATT 4920
TGGGGGATTG GTTTTTATTA TTTTTTTCCT TTTTGAAAAA TACTGAGGGA TCTTTTGATA 4980
AAGTTAGTAA TGCATGTTAG ATTTTAGTTT TGCAAGCATG TTGTTTTTCA AATATATCAA 5040
GTATAGAAAA AGGTAAAACA GTTAAGAAGG AAGGCAATTA TATTATTCTT CTGTAGTTAA 5100
GCAAACACTT GTTGAGTGCC TGCTATGTGC ACGGCATGGG CCCATATGTG TGAGGAGCTT 5160
GTCTAATTAT GTAGGAAGCA ATAGATCTCG GTAGTTACGT ATTGGGCAGA TACTTACTGT 5220
ATGAATGAAA GAACATCACA GTAATCACAA TATCAGAGCT GAATTATCCT CAGTGTAGCT 5280
TCTTGGAATT CAGTTTCTGG AACTAGAGAT AGAGCATTTA TTAAAAAAAA CTCCTGTTGA 5340
GACTGTGTCT TATGAACCTC TGAAACGTAC AAGCCTTCAC AAGTTTAACT AAATTGGGAT 5400
TAATCTTTCT GTAGTTATCT GCATAATTCT TGTTTTTCTT TCCATCTGGC TCCTGGGTTG 5460
ACAATTTGTG GAAACAACTC TATTGCTACT ATTTAAAAAA AATCAGAAAT CTTTCCCTTT 5520
AAGCTATGTT AAATTCAAAC TATTCCTGCT ATTCCTGTTT TGTCAAAGAA TTATATTTTT 5580
CAAAATATGT TTATTTGTTT GATGGGTCCC AGGAAACACT AATAAAAACC ACAGAGACCA 5640
GCCTGGAAAA AAAAAAAAAA AAAAAAA 5667
(2) INFORMATION FOR SEQ ID NO : 5 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 300 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Met lie Leu Glu Gly Gly Gly Val Met Asn Leu Asn Pro Gly Asn Asn
1 5 10 15
Leu Leu His Gin Pro Pro Ala Trp Thr Asp Ser Tyr Ser Thr Cys Asn
20 25 30
Val Ser Ser Gly Phe Phe Gly Gly Gin Trp His Glu lie His Pro Gin
35 40 45
Tyr Trp Thr Lys Tyr Gin Val Trp Glu Trp Leu Gin His Leu Leu Asp
50 55 60
Thr Asn Gin Leu Asp Ala Asn Cys lie Pro Phe Gin Glu Phe Asp lie 65 70 75 80
Asn Gly Glu His Leu Cys Ser Met Ser Leu Gin Glu Phe Thr Arg Ala
85 90 95
Ala Gly Thr Ala Gly Gin Leu Leu Tyr Ser Asn Leu Gin His Leu Lys
100 105 110
Trp Asn Gly Gin Cys Ser Ser Asp Leu Phe Gin Ser Thr His Asn Val
115 120 125 lie Val Lys Thr Glu Gin Thr Glu Pro Ser lie Met Asn Thr Trp Lys
130 135 140
Asp Glu Asn Tyr Leu Tyr Asp Thr Asn Tyr Gly Ser Thr Val Asp Leu 145 150 155 160
Leu Asp Ser Lys Thr Phe Cys Arg Ala Gin lie Ser Met Thr Thr Thr
165 170 175
Ser His Leu Pro Val Ala Glu Ser Pro Asp Met Lys Lys Glu Gin Asp
-103- 180 185 190-
Pro Pro Ala Lys Cys His Thr Lys Lys His Asn Pro Arg Gly Thr His
195 200 205
Leu Trp Glu Phe lie Arg Asp lie Leu Leu Asn Pro Asp Lys Asn Pro
210 215 220
Gly Leu lie Lys Trp Glu Asp Arg Ser Glu Gly Val Phe Arg Phe Leu 225 230 235 240
Lys Ser Glu Ala Val Ala Gin Leu Trp Gly Lys Lys Lys Asn Asn Ser
245 250 255
Ser Met Thr Tyr Glu Lys Leu Ser Arg Ala Met Arg Tyr Tyr Tyr Lys
260 265 270
Arg Glu lie Leu Glu Arg Val Asp Gly Arg Arg Leu Val Tyr Lys Phe
275 280 285
Gly Lys Asn Ala Arg Gly Trp Arg Glu Asn Glu Asn 290 295 300
(2) INFORMATION FOR SEQ ID NO : 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2428 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :
CAGGGGTGCC GGGTTGCTCA GGCCATGGGA GCCACACCTG TTATTGCTGC CTCTGATTTG 60
TGTGACACTG AGAAGCCCAC AGGCCTGTCC CTCCAACTCG GTGGACCCTC TCTGTGTGCA 120
TTTGGTGTGT GAGCCAGCTC TGAGAAGGGT TCAGAAGCCA CTGGAGGCAT CTGGGGACCT 180
CAGCTTCCAT GCCATCTCTG CCTCACTCCC ACAGGGTAAT GTTGGACTCG GTGACACACA 240
GCACCTTCCT GCCTAATGCA TCCTTCTGCG ATCCCCTGAT GTCGTGGACT GATCTGTTCA 300
GCAATGAAGA GTACTACCCT GCCTTTGAGC ATCAGACAGC CTGTGACTCA TACTGGACAT 360
CAGTCCACCC TGAATACTGG ACTAAGCGCC ATGTGTGGGA GTGGCTCCAG TTCTGCTGCG 420
ACCAGTACAA GTTGGACACC AATTGCATCT CCTTCTGCAA CTTCAACATC AGTGGCCTGC 480
AGCTGTGCAG CATGACACAG GAGGAGTTCG TCGAGGCAGC TGGCCTCTGC GGCGAGTACC 540
TGTACTTCAT CCTCCAGAAC ATCCGCACAC AAGGTTACTC CTTTTTTAAT GACGCTGAAG 600
AAAGCAAGGC CACCATCAAA GACTATGCTG ATTCCAACTG CTTGAAAACA AGTGGCATCA 660
AAAGTCAAGA CTGTCACAGT CATAGTAGAA CAAGCCTCCA AAGTTCTCAT CTATGGGAAT 720
TTGTACGAGA CCTGCTTCTA TCTCCTGAAG AAAACTGTGG CATTCTGGAA TGGGAAGATA 780
GGGAACAAGG AATTTTTCGG GTGGTTAAAT CGGAAGCCCT GGCAAAGATG TGGGGACAAA 840
GGAAGAAAAA TGACAGAATG ACGTATGAAA AGTTGAGCAG AGCCCTGAGA TACTACTATA 900
AAACAGGAAT TTTGGAGCGG GTTGACCGAA GGTTAGTGTA CAAATTTGGA AAAAATGCAC 960
ACGGGTGGCA GGAAGACAAG CTATGATCTG CTCCAGGCAT CAAGCTCATT TTATGGATTT 1020
CTGTCTTTTA AAACAATCAG ATTGCAATAG ACATTCGAAA GGCTTCATTT TCTTCTCTTT 1080
TTTTTTAACC TGCAAACATG CTGATAAAAT TTCTCCACAT CTCAGCTTAC ATTTGGATTC 1140
AGAGTTGTTG TCTACGGAGG GTGAGAGCAG AAACTCTTAA GAAATCCTTT CTTCTCCCTA 1200
AGGGGATGAG GGGATGATCT TTTGTGGTGT CTTGATCAAA CTTTATTTTC CTAGAGTTGT 1260
GGAATGACAA CAGCCCATGC CATTGATGCT GATCAGAGAA AAACTATTCA ATTCTGCCAT 1320
TAGAGACACA TCCAATGCTC CCATCCCAAA GGTTCAAAAG TTTTCAAATA ACTGTGGCAG 1380
CTCACCAAAG GTGGGGGAAA GCATGATTAG TTTGCAGGTT ATGGTAGGAG AGGGTGAGAT 1440
ATAAGACATA CATACTTTAG ATTTTAAATT ATTAAAGTCA AAAATCCATA GAAAAGTATC 1500
CCTTTTTTTT TTTTTTGAGA CGGGTTCTCA CTATGTTGCC CAGGGCTGGT CTTGAACTCC 1560
TATGCTCAAG TGATCCTCCC ACCTCGGCCT CCCAAAGTAC TGTGATTACA AGCGTGAGCC 1620
ACGGCACCTG GGCAGAAAAG TATCTTAATT AATGAAAGAG CTAAGCCATC AAGCTGGGAC 1680
-104- TTAATTGGAT TTAACATAGG TTCACAGAAA GTTTCCTAAC CAGAGCATCT TTTTGACCAC 1740
TCAGCAAAAC TTCCACAGAC ATCCTTCTGG ACTTAAACAC TTAACATTAA CCACATTATT 1800
AATTGTTGCT GAGTTTATTC CCCCTTCTAA CTGATGGCTG GCATCTGATA TGCAGAGTTA 1860
GTCAACAGAC ACTGGCATCA ATTACAAAAT CACTGCTGTT TCTGTGATTC AAGCTGTCAA 1920
CACAATAAAA TCGAAATTCA TTGATTCCAT CTCTGGTCCA GATGTTAAAC GTTTATAAAA 1980
CCGGAAATGT CCTAACAACT CTGTAATGGC AAATTAAATT GTGTGTCTTT TTTGTTTTGT 2040
CTTTCTACCT GATGTGTATT CAAGCGCTAT AACACGTATT TCCTTGACAA AAATAGTGAC 2100
AGTGAATTCA CACTAATAAA TGTTCATAGG TTAAAGTCTG CACTGACATT TTCTCATCAA 2160
TCACTGGTAT GTAAGTTATC AGTGACTGAC AGCTAGGTGG ACTGCCCCTA GGACTTCTGT 2220
TTCACCAGAG CAGGAATCAA GTGGTGAGGC ACTGAATCGC TGTACAGGCT GAAGACCTCC 2280
TTATTAGAGT TGAACTTCAA AGTAACTTGT TTTAAAAAAT GTGAATTACT GTAAAATAAT 2340
CTATTTTGGA TTCATGTGTT TTCCAGGTGG ATATAGTTTG TAAACAATGT GAATAAAGTA 2400
TTTAACATGT TCAAAAAAAA AAAAAAAA 2428
(2) INFORMATION FOR SEQ ID NO : 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 265 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :
Met Pro Ser Leu Pro His Ser His Arg Val Met Leu Asp Ser Val Thr
1 5 10 15
His Ser Thr Phe Leu Pro Asn Ala Ser Phe Cys Asp Pro Leu Met Ser
20 25 30
Trp Thr Asp Leu Phe Ser Asn Glu Glu Tyr Tyr Pro Ala Phe Glu His
35 40 45
Gin Thr Ala Cys Asp Ser Tyr Trp Thr Ser Val His Pro Glu Tyr Trp
50 55 60
Thr Lys Arg His Val Trp Glu Trp Leu Gin Phe Cys Cys Asp Gin Tyr 65 70 75 80
Lys Leu Asp Thr Asn Cys lie Ser Phe Cys Asn Phe Asn lie Ser Gly
85 90 95
Leu Gin Leu Cys Ser Met Thr Gin Glu Glu Phe Val Glu Ala Ala Gly
100 105 110
Leu Cys Gly Glu Tyr Leu Tyr Phe lie Leu Gin Asn lie Arg Thr Gin
115 120 125
Gly Tyr Ser Phe Phe Asn Asp Ala Glu Glu Ser Lys Ala Thr lie Lys
130 135 140
Asp Tyr Ala Asp Ser Asn Cys Leu Lys Thr Ser Gly lie Lys Ser Gin 145 150 155 160
Asp Cys His Ser His Ser Arg Thr Ser Leu Gin Ser Ser His Leu Trp
165 170 175
Glu Phe Val Arg Asp Leu Leu Leu Ser Pro Glu Glu Asn Cys Gly lie
180 185 190
Leu Glu Trp Glu Asp Arg Glu Gin Gly lie Phe Arg Val Val Lys Ser
195 200 205
Glu Ala Leu Ala Lys Met Trp Gly Gin Arg Lys Lys Asn Asp Arg Met
210 215 220
Thr Tyr Glu Lys Leu Ser Arg Ala Leu Arg Tyr Tyr Tyr Lys Thr Gly 225 230 235 240
-105- lie Leu Glu Arg Val Asp Arg Arg Leu Val Tyr Lys Phe Gly-Lys Asn
245 250 255
Ala His Gly Trp Gin Glu Asp Lys Leu 260 265
(2) INFORMATION FOR SEQ ID NO : 8 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2280 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
CTGGGAGCGC CTGCCTTCTC TTGCCTTGAA AGCCTCCTCT TTGGACCTAG CCACCGCTGC 60
CCTCACGGTA ATGTTGGACT CGGTGACACA CAGCACCTTC CTGCCTAATG CATCCTTCTG 120
CGATCCCCTG ATGTCGTGGA CTGATCTGTT CAGCAATGAA GAGTACTACC CTGCCTTTGA 180
GCATCAGACA GCCTGTGACT CATACTGGAC ATCAGTCCAC CCTGAATACT GGACTAAGCG 240
CCATGTGTGG GAGTGGCTCC AGTTCTGCTG CGACCAGTAC AAGTTGGACA CCAATTGCAT 300
CTCCTTCTGC AACTTCAACA TCAGTGGCCT GCAGCTGTGC AGCATGACAC AGGAGGAGTT 360
CGTCGAGGCA GCTGGCCTCT GCGGCGAGTA CCTGTACTTC ATCCTCCAGA ACATCCGCAC 420
ACAAGGTTAC TCCTTTTTTA ATGACGCTGA AGAAAGCAAG GCCACCATCA AAGACTATGC 480
TGATTCCAAC TGCTTGAAAA CAAGTGGCAT CAAAAGTCAA GACTGTCACA GTCATAGTAG 540
AACAAGCCTC CAAAGTTCTC ATCTATGGGA ATTTGTACGA GACCTGCTTC TATCTCCTGA 600
AGAAAACTGT GGCATTCTGG AATGGGAAGA TAGGGAACAA GGAATTTTTC GGGTGGTTAA 660
ATCGGAAGCC CTGGCAAAGA TGTGGGGACA AAGGAAGAAA AATGACAGAA TGACGTATGA 720
AAAGTTGAGC AGAGCCCTGA GATACTACTA TAAAACAGGA ATTTTGGAGC GGGTTGACCG 780
AAGGTTAGTG TACAAATTTG GAAAAAATGC ACACGGGTGG CAGGAAGACA AGCTATGATC 840
TGCTCCAGGC ATCAAGCTCA TTTTATGGAT TTCTGTCTTT TAAAACAATC AGATTGCAAT 900
AGACATTCGA AAGGCTTCAT TTTCTTCTCT TTTTTTTTAA CCTGCAAACA TGCTGATAAA 960
ATTTCTCCAC ATCTCAGCTT ACATTTGGAT TCAGAGTTGT TGTCTACGGA GGGTGAGAGC 1020
AGAAACTCTT AAGAAATCCT TTCTTCTCCC TAAGGGGATG AGGGGATGAT CTTTTGTGGT 1080
GTCTTGATCA AACTTTATTT TCCTAGAGTT GTGGAATGAC AACAGCCCAT GCCATTGATG 1140
CTGATCAGAG AAAAACTATT CAATTCTGCC ATTAGAGACA CATCCAATGC TCCCATCCCA 1200
AAGGTTCAAA AGTTTTCAAA TAACTGTGGC AGCTCACCAA AGGTGGGGGA AAGCATGATT 1260
AGTTTGCAGG TTATGGTAGG AGAGGGTGAG ATATAAGACA TACATACTTT AGATTTTAAA 1320
TTATTAAAGT CAAAAATCCA TAGAAAAGTA TCCCTTTTTT TTTTTTTTGA GACGGGTTCT 1380
CACTATGTTG CCCAGGGCTG GTCTTGAACT CCTATGCTCA AGTGATCCTC CCACCTCGGC 1440
CTCCCAAAGT ACTGTGATTA CAAGCGTGAG CCACGGCACC TGGGCAGAAA AGTATCTTAA 1500
TTAATGAAAG AGCTAAGCCA TCAAGCTGGG ACTTAATTGG ATTTAACATA GGTTCACAGA 1560
AAGTTTCCTA ACCAGAGCAT CTTTTTGACC ACTCAGCAAA ACTTCCACAG ACATCCTTCT 1620
GGACTTAAAC ACTTAACATT AACCACATTA TTAATTGTTG CTGAGTTTAT TCCCCCTTCT 1680
AACTGATGGC TGGCATCTGA TATGCAGAGT TAGTCAACAG ACACTGGCAT CAATTACAAA 1740
ATCACTGCTG TTTCTGTGAT TCAAGCTGTC AACACAATAA AATCGAAATT CATTGATTCC 1800
ATCTCTGGTC CAGATGTTAA ACGTTTATAA AACCGGAAAT GTCCTAACAA CTCTGTAATG 1860
GCAAATTAAA TTGTGTGTCT TTTTTGTTTT GTCTTTCTAC CTGATGTGTA TTCAAGCGCT 1920
ATAACACGTA TTTCCTTGAC AAAAATAGTG ACAGTGAATT CACACTAATA AATGTTCATA 1980
GGTTAAAGTC TGCACTGACA TTTTCTCATC AATCACTGGT ATGTAAGTTA TCAGTGACTG 2040
ACAGCTAGGT GGACTGCCCC TAGGACTTCT GTTTCACCAG AGCAGGAATC AAGTGGTGAG 2100
GCACTGAATC GCTGTACAGG CTGAAGACCT CCTTATTAGA GTTGAACTTC AAAGTAACTT 2160
GTTTTAAAAA ATGTGAATTA CTGTAAAATA ATCTATTTTG GATTCATGTG TTTTCCAGGT 2220
GGATATAGTT TGTAAACAAT GTGAATAAAG TATTTAACAT GTTCAAAAAA AAAAAAAAAA 2280
-106- (2) INFORMATION FOR SEQ ID NO : 9 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 255 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :
Met Leu Asp Ser Val Thr His Ser Thr Phe Leu Pro Asn Ala Ser Phe
1 5 10 15
Cys Asp Pro Leu Met Ser Trp Thr Asp Leu Phe Ser Asn Glu Glu Tyr
20 25 30
Tyr Pro Ala Phe Glu His Gin Thr Ala Cys Asp Ser Tyr Trp Thr Ser
35 40 45
Val His Pro Glu Tyr Trp Thr Lys Arg His Val Trp Glu Trp Leu Gin
50 55 60
Phe Cys Cys Asp Gin Tyr Lys Leu Asp Thr Asn Cys lie Ser Phe Cys 65 70 75 80
Asn Phe Asn lie Ser Gly Leu Gin Leu Cys Ser Met Thr Gin Glu Glu
85 90 95
Phe Val Glu Ala Ala Gly Leu Cys Gly Glu Tyr Leu Tyr Phe lie Leu
100 105 110
Gin Asn lie Arg Thr Gin Gly Tyr Ser Phe Phe Asn Asp Ala Glu Glu
115 120 ' 125
Ser Lys Ala Thr lie Lys Asp Tyr Ala Asp Ser Asn Cys Leu Lys Thr
130 135 140
Ser Gly lie Lys Ser Gin Asp Cys His Ser His Ser Arg Thr Ser Leu 145 150 155 160
Gin Ser Ser His Leu Trp Glu Phe Val Arg Asp Leu Leu Leu Ser Pro
165 170 175
Glu Glu Asn Cys Gly lie Leu Glu Trp Glu Asp Arg Glu Gin Gly lie
180 185 190
Phe Arg Val Val Lys Ser Glu Ala Leu Ala Lys Met Trp Gly Gin Arg
195 200 205
Lys Lys Asn Asp Arg Met Thr Tyr Glu Lys Leu Ser Arg Ala Leu Arg
210 215 220
Tyr Tyr Tyr Lys Thr Gly lie Leu Glu Arg Val Asp Arg Arg Leu Val 225 230 235 240
Tyr Lys Phe Gly Lys Asn Ala His Gly Trp Gin Glu Asp Lys Leu 245 250 255
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2498 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
-107- GAGGGGCTGA CAGCGGCGTC CCTCGTCTGG GCAGCCTCCG CTCTGCCACT CTCCTCCCGT 60
CCTGAGGATG GGACCCCCGG AAAAGCGGCC TCTGGAGGCC TGCCATGGCA CCCAGAGCAG 120
CCATTTTCCT CCCAGTTCTG GGGCTTTGGA AGGAGCTTGC GGATGAGGAG AGGGAGCCTC 180
CGCAGGGCTC TGGCTCCCCT CCAGGGGCCG AGGCCGCACA CAAAGCCGCT CTGTGGCCCA 240
ATTACACCTA CTGGATAGGA TTGTTGAGGG GACCTGAGAA ACTTGAGACG ACAAGAACGC 300
GTAGCGCCTC GGCTGGCTGA GGGTGCTGAG CCCTCGTGTT GTGTTCTCTC CAGCTTTCCC 360
CGTGCCTCAG CCACTCTTCA CGTTCCATCT GTGCTCTGTG CTGACCCGCC TGTGACTCAT 420
ACTGGACATC AGTCCACCCT GAATACTGGA CTAAGCGCCA TGTGTGGGAG TGGCTCCAGT 480
TCTGCTGCGA CCAGTACAAG TTGGACACCA ATTGCATCTC CTTCTGCAAC TTCAACATCA 540
GTGGCCTGCA GCTGTGCAGC ATGACACAGG AGGAGTTCGT CGAGGCAGCT GGCCTCTGCG 600
GCGAGTACCT GTACTTCATC CTCCAGAACA TCCGCACACA AGGTTACTCC TTTTTTAATG 660
ACGCTGAAGA AAGCAAGGCC ACCATCAAAG ACTATGCTGA TTCCAACTGC TTGAAAACAA 720
GTGGCATCAA AAGTCAAGAC TGTCACAGTC ATAGTAGAAC AAGCCTCCAA AGTTCTCATC 780
TATGGGAATT TGTACGAGAC CTGCTTCTAT CTCCTGAAGA AAACTGTGGC ATTCTGGAAT 840
GGGAAGATAG GGAACAAGGA ATTTTTCGGG TGGTTAAATC GGAAGCCCTG GCAAAGATGT 900
GGGGACAAAG GAAGAAAAAT GACAGAATGA CGTATGAAAA GTTGAGCAGA GCCCTGAGAT 960
ACTACTATAA AACAGGAATT TTGGAGCGGG TTGACCGAAG GTTAGTGTAC AAATTTGGAA 1020
AAAATGCACA CGGGTGGCAG GAAGACAAGC TATGATCTGC TCCAGGCATC AAGCTCATTT 1080
TATGGATTTC TGTCTTTTAA AACAATCAGA TTGCAATAGA CATTCGAAAG GCTTCATTTT 1140
CTTCTCTTTT TTTTTAACCT GCAAACATGC TGATAAAATT TCTCCACATC TCAGCTTACA 1200
TTTGGATTCA GAGTTGTTGT CTACGGAGGG TGAGAGCAGA AACTCTTAAG AAATCCTTTC 1260
TTCTCCCTAA GGGGATGAGG GGATGATCTT TTGTGGTGTC TTGATCAAAC TTTATTTTCC 1320
TAGAGTTGTG GAATGACAAC AGCCCATGCC ATTGATGCTG ATCAGAGAAA AACTATTCAA 1380
TTCTGCCATT AGAGACACAT CCAATGCTCC CATCCCAAAG GTTCAAAAGT TTTCAAATAA 1440
CTGTGGCAGC TCACCAAAGG TGGGGGAAAG CATGATTAGT TTGCAGGTTA TGGTAGGAGA 1500
GGGTGAGATA TAAGACATAC ATACTTTAGA TTTTAAATTA TTAAAGTCAA AAATCCATAG 1560
AAAAGTATCC CTTTTTTTTT TTTTTGAGAC GGGTTCTCAC TATGTTGCCC AGGGCTGGTC 1620
TTGAACTCCT ATGCTCAAGT GATCCTCCCA CCTCGGCCTC CCAAAGTACT GTGATTACAA 1680
GCGTGAGCCA CGGCACCTGG GCAGAAAAGT ATCTTAATTA ATGAAAGAGC TAAGCCATCA 1740
AGCTGGGACT TAATTGGATT TAACATAGGT TCACAGAAAG TTTCCTAACC AGAGCATCTT 1800
TTTGACCACT CAGCAAAACT TCCACAGACA TCCTTCTGGA CTTAAACACT TAACATTAAC 1860
CACATTATTA ATTGTTGCTG AGTTTATTCC CCCTTCTAAC TGATGGCTGG CATCTGATAT 1920
GCAGAGTTAG TCAACAGACA CTGGCATCAA TTACAAAATC ACTGCTGTTT CTGTGATTCA 1980
AGCTGTCAAC ACAATAAAAT CGAAATTCAT TGATTCCATC TCTGGTCCCA GATGTTAAAC 2040
GTTTATAAAA CCGGAAATGT CCTAACAACT CTGTAATGGC AAATTAAATT GTGTGTCTTT 2100
TTTGTTTTGT CTTTCTACCT GATGTGTATT CAAGCGCTAT AACACGTATT TCCTTGACAA 2160
AAATAGTGAC AGTGAATTCA CACTAATAAA TGTTCATAGG TTAAAGTCTG CACTGACATT 2220
TTCTCATCAA TCACTGGTAT GTAAGTTATC AGTGACTGAC AGCTAGGTGG ACTGCCCCTA 2280
GGACTTCTGT TTCACCAGAG CAGGAATCAA GTGGTGAGGC ACTGAATCGC TGTACAGGCT 2340
GAAGACCTCC TTATTAGAGT TGAACTTCAA AGTAACTTGT TTTAAAAAAT GTGAATTACT 2400
GTAAAATAAT CTATTTTGGA TTCATGTGTT TTCCAGGTGG ATATAGTTTG TAAACAATGT 2460
GAATAAAGTA TTTAACATGT TCAAAAAAAA AAAAAAAA 2498
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 164 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Met Thr Gin Glu Glu Phe Val Glu Ala Ala Gly Leu Cys Gly Glu Tyr
-108- 1 5 10 _15
Leu Tyr Phe lie Leu Gin Asn lie Arg Thr Gin Gly Tyr Ser Phe Phe
20 25 30
Asn Asp Ala Glu Glu Ser Lys Ala Thr lie Lys Asp Tyr Ala Asp Ser
35 40 45
Asn Cys Leu Lys Thr Ser Gly lie Lys Ser Gin Asp Cys His Ser His
50 55 60
Ser Arg Thr Ser Leu Gin Ser Ser His Leu Trp Glu Phe Val Arg Asp 65 70 75 80
Leu Leu Leu Ser Pro Glu Glu Asn Cys Gly lie Leu Glu Trp Glu Asp
85 90 95
Arg Glu Gin Gly lie Phe Arg Val Val Lys Ser Glu Ala Leu Ala Lys
100 105 110
Met Trp Gly Gin Arg Lys Lys Asn Asp Arg Met Thr Tyr Glu Lys Leu
115 120 125
Ser Arg Ala Leu Arg Tyr Tyr Tyr Lys Thr Gly lie Leu Glu Arg Val
130 135 140
Asp Arg Arg Leu Val Tyr Lys Phe Gly Lys Asn Ala His Gly Trp Gin 145 150 155 160
Glu Asp Lys Leu
(2) INFORMATION FOR SEQ ID NO : 12 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: AAATGAGCCA ATGTTTGTAA T 21
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: AAATGAGCCA GTGTTTGTAA T 21
(2) INFORMATION FOR SEQ ID NO : 14 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 736 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
-109- (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 14 :
AGGAAGTGAA GAACCTAGAT AATCCACCAA CCGGATAATC AGCTCTTGCA TATTTGAGAG 60
TTGACTGCTT GACCTAAGCA TCTCCTCATA AGGTACCCTC CCTCCCAGGA CCTTCCCTTT 120
CAAACCTCTC AAGGCTCTTA CCTGGGGCCA GGGGAGATAG GCTTTTCAAA GTCCATTGAA 180
TTGCCAAGAG TCTCTGTCAA GAAGGCAGTC ATGGTGCCTG GAGAGGGAAC TTGCTGGGAG 240
CCCCTTCAGA GCCTGGTACT TATAGAGCTA GGGAAAAGAT CTTGATGCCA AAGCAGGGTG 300
GACTAAATAC AGACTAATAA ATGAGACAGG TGCTCAAGAG GGCCCCTCCA TACCATCATC 360
TCCTCCAGAT TTGGACTTCT ACTCACTTTG CTTTTACATT CCCTCTTCCC GATGGTGTCT 420
TTGGTGAGCA GGGTGCTTTT CACCTGAAAC AGCCTCTGAG CTGAAAAGAA CAGTCACCAC 480
CAAATCAATT CCTCATCCAT TAACAGGTTG TCTCTCTGTT CTTGAGACAC AGGCATTACC 540
TGGTTAGACC TGTTTTGTTT GAACACTAAC GTGTGAGTTG GCCAAATGCA AATGAGCCAA 600
TGTTTGTAAT CCTTTATTTT ATTTTTTTAA AGGGCTGGGT AGCCAATCAG AAGAGGGGGA 660
AGTGACTTAG GGAATTCCCG GTTGGTGGCT TATTGCTTAA CATCCTACAA AATGATTTAA 720
AATTATTGTT ATATGC 736
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 333 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GCCAGAGTCC TCCTTGAGAA CTTACAATGT GTCCATATTA AGGATCTGCT GTGTTTGATG 60
ATTTTGTGAT TACACTTTAA ACTTCTTATC CATAAAGGAC ATACTTGATA TATCTGAGAC 120
TTGTAGTAGA AGGCCTTGAG ACATCCATCT CATCCCATCA TTATCTATCT ATCATCTATC 180
TATCTATCTA TCTATCTATC TATCTATCTA TCTATCATCT ATCTATCTAT CGCCAGTACT 240
GTCTTGTTGA AGTTGGCAGT AGGGTGAAAG ACCTCAAACT CCAAAGGACT TTCCGTATGG 300
ATGCAATATA CCTGCAATTC TAGCTTTTCT GTG 333
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
ACAGAATGAC RTATGAAAAG T 21
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
-110- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: GTAACCAAGC KCAAGCCACC C 21
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: AAGGAGCCCA YCTGAGTGCA G 21
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: CGTTCCATCT STGCTCTGTG C 21
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: AGCGCCTCGG YTGGCTGAGG G 21
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-111- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
TGTATTCAAG YGCTATAACA C 21
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CACTGAGAAG CCNACAGGCC TGT 23
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CCCACAGGCC WGTCCCTCCA A 21
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: CGTCCATCTC YAGCTCCAGG G 21
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: GACTTGATAA YGCCCGTGGT G 21
-112- (2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: ACTTGATAAC RCCCGTGGTG C 21
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: CTCCCCTCCA WGAGCCACAG C 21
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: ATTTCCTGCA TNGTCTGGAC TT 22
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
ATCCAAACAC YTGAGTGGAA A 21
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
-113- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: AGTTTCCTCA RTGCGGGAGC T 21
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: GCGAGCACCT YTGCAGCATG A 21
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: TTCACCCGGG YGGCAGGGAC G 21
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: CTGGGGAAAA NNGATCGCTG AC 22
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-114- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
GTCAATTAAA YGGCTCTCAT T 21
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: TAGATCATTC RTAACCTGCC T 21
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: AAAGAGAAAT WCTGGAGCGT G 21
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: ATGAGGGGAA MAAGAAACTA C 21
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: TTTTGTATGT KACATGATTT A 21
-115- (2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: AGCTTGGTTC YTTTTTGCTC C 21
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: TTGACACCAG RAACCCCCCA G 21
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: AAATGAGCCA RTGTTTGTAA T 21
(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
ATCCATTTTG YATTCCTCAT T 21
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
-116- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: CTGGAGCTCA RACCAGACAG C 21
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: GCCAGTGCAG SCATCATTAC C 21
(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45: AGTTCAAATC RTAATTTTTA T 21
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: TCATCAGAAT YTAAATCTCC C 21
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-117- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
GGAGATTCAG NTGAAGCAAG A 21
(2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48: TTTTTCCACA YCCAGCCTGG C 21
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: CCCAGCCTGG YGAACCCTGG C 21
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50: CTCTTCATCA YGGTCAAATA C 21
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: CAACTTGCTG YCAAAGTGCT G 21
-118- (2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: TACTATGTGC YAGATACTAA G 21
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: ATGCCACTTT RRGACAACTT GAG 23
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: CGCATGCCTG KAAAGAAGAG A 21
(2) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
GGATAAGCAC MAGTGAGCCT G 21
(2) INFORMATION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
-119- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56: AAAGCCAGAC RGCAACTTGT G 21
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57: TCTCAAAAAG RGTGATAGGA G 21
(2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: TCTGAATCCT STCTCCTCCT T 21
(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: TAGAACCAGG WTGTGGGACC A 21
(2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-120- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
TTCTTGTGTC RGGCGCAAAA C 21
(2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: AACCAACATG RAGAAACCCC A 21
(2) INFORMATION FOR SEQ ID NO:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62: AATAAACTAT RGTTCACCTA G 21
(2) INFORMATION FOR SEQ ID NO: 63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: ACATATTTGT RTCTCATATG A 21
(2) INFORMATION FOR SEQ ID NO: 64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: CAAAGCAGTT YCTAATAATC C 21
-121- (2) INFORMATION FOR SEQ ID NO: 65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65: AGATCCTAAC YGGGGCCTCC T 21
(2) INFORMATION FOR SEQ ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: CTCTTTCTCT YTGCTTCCTC C 21
(2) INFORMATION FOR SEQ ID NO: 67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: TTAGGAATCC WCAAATATGT A 21
(2) INFORMATION FOR SEQ ID NO: 68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:
GTCTGACTCC RCCTCCCTCA T 21
(2) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
-122- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69: GAATCACATC RTGAGAAATG T 21
(2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70: AATTCAATCC YTCACAGACT T 21
(2) INFORMATION FOR SEQ ID NO: 71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: GTGTAGCCAG RGTTGCTAAT T 21
(2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72: CCTAGAAATA SCCAAGGGCA C 21
(2) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-123- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
AAATTCTCAT RCCTCACCCT C 21
(2) INFORMATION FOR SEQ ID NO: 74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: TCCCACCCCT RTCACCTTCA T 21
(2) INFORMATION FOR SEQ ID NO: 75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75: CCTCATTCTC RGAAGCCAAC A 21
(2) INFORMATION FOR SEQ ID NO: 76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76: GAAGAGCCGT YCAGTCCCTT T 21
(2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: TCCATAGGCT YTTTATTTGG C 21
-124- (2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78: TCGTTTAGTA YACAGGCTTT G 21
(2) INFORMATION FOR SEQ ID NO: 79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79: GCCTCAGTTG YCCCAGCTAT A 21
(2) INFORMATION FOR SEQ ID NO: 80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80: AGCAAAATGC WCTATGCACT G 21
(2) INFORMATION FOR SEQ ID NO: 81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:
GTGTCCTGAC NNNNNNNNNN NACACTGCCT G 31
(2) INFORMATION FOR SEQ ID NO: 82:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
-125- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: ATCAGATAAC RCCTACACTT A 21
(2) INFORMATION FOR SEQ ID NO: 83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: TCTCTCTTCT SCCTGCCCTG T 21
(2) INFORMATION FOR SEQ ID NO: 84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84: TGGACACAGG KAGGGGAATA T 21
(2) INFORMATION FOR SEQ ID NO: 85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85: TGTCACTTGC RCATACAAGG C 21
(2) INFORMATION FOR SEQ ID NO: 86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-126- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86:
ATCATCAGAT YAGCCCAGAA T 21
(2) INFORMATION FOR SEQ ID NO: 87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87: TCAACAGAGA RAGTTAATGG T 21
(2) INFORMATION FOR SEQ ID NO: 88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88: AGCAATAATG YTTCCCTTTT C 21
(2) INFORMATION FOR SEQ ID NO: 89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89: TCTAGCTTTT YTGTGTTTTT T 21
(2) INFORMATION FOR SEQ ID NO: 90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90: GATTCCTTAA YGCTTGATAC T 21
-127- (2) INFORMATION FOR SEQ ID NO: 91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91: CCTCCTCCAG YACCAAAGTG G 21
(2) INFORMATION FOR SEQ ID NO : 92 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92: ATGGCCACAG RTCAAATCCT G 21
(2) INFORMATION FOR SEQ ID NO: 93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93: ACTGAGTGTT YATGCCAATT T 21
(2) INFORMATION FOR SEQ ID NO : 94 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:
GACAAGCCCT RTCTGACACA C 21
(2) INFORMATION FOR SEQ ID NO: 95:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
-128- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95: TGAAAAGCCT YCTTGCTGCC T 21
(2) INFORMATION FOR SEQ ID NO: 96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96: TCCTGGAGTT YCTTTGCTCC C 21
(2) INFORMATION FOR SEQ ID NO: 97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97: GATTCCAAAT WAACTAAAGA T 21
(2) INFORMATION FOR SEQ ID NO: 98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:98: GACCTCAAGT CRTCCACCCG CC 22
(2) INFORMATION FOR SEQ ID NO: 99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-129- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99:
AACAAATACT MCCCCGCAAC CC 22
(2) INFORMATION FOR SEQ ID NO: 100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100: ATTTTTTTTT NAAGGAAAAT A 21
(2) INFORMATION FOR SEQ ID NO: 101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101: AAATTTCCCC MAAACAAGCA G 21
(2) INFORMATION FOR SEQ ID NO: 102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:102: GAGAAAGGGT RTGTGTGTGT G 21
(2) INFORMATION FOR SEQ ID NO: 103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:103: GTGTGTGTGT NNNNGTATGT GCGCGTG 27
-130- (2) INFORMATION FOR SEQ ID NO: 104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104: ATCGGGAACC YCATACCCCA A 21
(2) INFORMATION FOR SEQ ID NO: 105:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105: TTTGTTTCGC MATGAGGTAC G 21
(2) INFORMATION FOR SEQ ID NO:106:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106: TGAGGGTGTT STGGGCTGGA C 21
(2) INFORMATION FOR SEQ ID NO: 107:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107:
TCTTCATTGG YATCTGAATG T 21
(2) INFORMATION FOR SEQ ID NO: 108:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
-131- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:108: GCGAGCACCT YTGCAGCATG A 21
(2) INFORMATION FOR SEQ ID NO: 109:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:109: AACCCCCCCC MCACACACAC A 21
(2) INFORMATION FOR SEQ ID NO: 110:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110: TCAGTGCTCT STAATCAGTC A 21
(2) INFORMATION FOR SEQ ID NO: 111:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111: TCTTTGTGAA ANNAATTAGT CTG 23
(2) INFORMATION FOR SEQ ID NO: 112:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-132- (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 112 :
GCTGCCCTGA SAGCTGGGCC A 21
(2) INFORMATION FOR SEQ ID NO: 113:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113: CCTTCTGATC YTTGTTTGCT G 21
(2) INFORMATION FOR SEQ ID NO: 114:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114: GGAACACTGA KTCTTGATTA G 21
(2) INFORMATION FOR SEQ ID NO: 115:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115: TAGGCTTCTC YTGATAATTG A 21
(2) INFORMATION FOR SEQ ID NO: 116:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116: TCTTAAAATA MTTGGCTTGT A 21
-133- (2) INFORMATION FOR SEQ ID NO: 117:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117: TAGATCATTA RTAACCTGCC T 21
(2) INFORMATION FOR SEQ ID NO: 118:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118: ATGAGGGGAA MAAGAAACTA C 21
(2) INFORMATION FOR SEQ ID NO: 119:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119: TTGACACCAG RAACCCCCCA G 21
(2) INFORMATION FOR SEQ ID NO: 120:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120:
TGTTTTAAAT RTTAGGGACA A 21
(2) INFORMATION FOR SEQ ID NO: 121:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
-134- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121: GTAAGCATAG YAATGTAGCA G 21
(2) INFORMATION FOR SEQ ID NO: 122:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 122: GGCTCTTTCT KCAACCTTTC C 21
(2) INFORMATION FOR SEQ ID NO: 123:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:123: GACCCAGGTT RTGAGTTTTC C 21
(2) INFORMATION FOR SEQ ID NO: 124:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:124: GACAGAATGA YATATGAAAA G 21
(2) INFORMATION FOR SEQ ID NO: 125:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-135- (ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 125: TGTGTGACAC YGAGAAGCCC A 21
(2) INFORMATION FOR SEQ ID NO: 126:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 126: AGTACTGGAC MAAGTACCAG G 21
(2) INFORMATION FOR SEQ ID NO: 127:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 127: CCTGGGAGCA RGTATTGCAT T 21
(2) INFORMATION FOR SEQ ID NO: 128:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
_ (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128: AGATTTGAGG YCTCAGGTCC C 21
(2) INFORMATION FOR SEQ ID NO: 129:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
-136- (ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 129: TGTCAATGTC RCATGATAAG C 21
(2) INFORMATION FOR SEQ ID NO: 130:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 130: TTGCCCCAGT KTTCTCCGGG C 21
(2) INFORMATION FOR SEQ ID NO: 131:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 131: TATGAGCAGC RTAGGGAGTG G 21
(2) INFORMATION FOR SEQ ID NO: 132:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:132: AGTTGACTGA AAAANTAAAT AAGAC 25
(2) INFORMATION FOR SEQ ID NO:133:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
-137- (ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:133: ATTCAAATAG SCTCTAGAAA C 21
(2) INFORMATION FOR SEQ ID NO: 134:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 134 : CCCAGAATTT MATATCCATT C 21
(2) INFORMATION FOR SEQ ID NO: 135:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 135: TGACCCAACA RAAACTCACT G 21
(2) INFORMATION FOR SEQ ID NO: 136:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:136: CCAGAATATA WCATCAGCCC T 21
(2) INFORMATION FOR SEQ ID NO: 137:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
-138- (ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 137: CATCAGCCCT WCTGAGGAGA T 21
(2) INFORMATION FOR SEQ ID NO: 138:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 138: CCAGAACAGA YTTTATTCTG T 21
(2) INFORMATION FOR SEQ ID NO: 139:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 139: TTCAGCCATC YTTCCAGTTG T 21
(2) INFORMATION FOR SEQ ID NO: 140:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 140: TCACTAACTC WAAAACGACA T 21
(2) INFORMATION FOR SEQ ID NO: 141:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
-139- (ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 141: AACTCAAAAA YGACATCCTC C 21
(2) INFORMATION FOR SEQ ID NO: 142:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 142: GAACTGCACA RGTTGCACAC T 21
(2) INFORMATION FOR SEQ ID NO:143:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 143: TTGTTCCATG SACTACCTCC T 21
(2) INFORMATION FOR SEQ ID NO: 144:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 144: ACAGCAGGCA YTCAACAAAT T 21
(2) INFORMATION FOR SEQ ID NO: 145:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
-140- (ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 145: TTATTTTTGG STTTGTTTTA A 21
(2) INFORMATION FOR SEQ ID NO:146:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 146: TAGGCTGTTC YCTGCCATCA C 21
(2) INFORMATION FOR SEQ ID NO: 147:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 147: GTGCTCTGGG MCACACAGCT C 21
(2) INFORMATION FOR SEQ ID NO: 148:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 148: AGACCCGATA RGAGCTCCTT C 21
(2) INFORMATION FOR SEQ ID NO: 149:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
-141- (ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 149: CATCTTGCGC RGTCATGTAA G 21
(2) INFORMATION FOR SEQ ID NO: 150:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 150: CAGCACAGCT RTTCCCTCAA A 21
(2) INFORMATION FOR SEQ ID NO: 151:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 151: TTTGGAAACA YGGTGAAGTA T 21
(2) INFORMATION FOR SEQ ID NO:152:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:152: ACACGGTGAA RTATTGTCTC C 21
(2) INFORMATION FOR SEQ ID NO: 153:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
-142- (ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:153: AAAAGTGGAT MCTCTGCAAA C 21
(2) INFORMATION FOR SEQ ID NO: 154:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 154: CTTCAAATGC RGCTATTAAA G 21
(2) INFORMATION FOR SEQ ID NO: 155:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 155: CCTGGGAGCA YGGTAAATCA G 21
(2) INFORMATION FOR SEQ ID NO: 156:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 156: TGAAAATGTC RCTTTCTCAC CT 22
(2) INFORMATION FOR SEQ ID NO: 157:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
-143- (ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:157: CCTGATATTT RCCAACAAGA A 21
(2) INFORMATION FOR SEQ ID NO:158:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 158: AAAGGGTTAG YTTGTCCCCT T 21
(2) INFORMATION FOR SEQ ID NO: 159:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 159: TGAAAATAAA ASACAATTTT TT 22
(2) INFORMATION FOR SEQ ID NO: 160:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 160: CTGCTGTGGA CGAATAGG 18
(2) INFORMATION FOR SEQ ID NO: 161:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-144- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 161:
TCAATATAAT CTTGCTTAAC TTGG 24
(2) INFORMATION FOR SEQ ID NO: 162:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 162: GACCTGTTTG GGTTGATTTC AG 22
(2) INFORMATION FOR SEQ ID NO:163:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 163: GTTTCTTACA GTGTCTTGCT ATCACATCAC C 31
(2) INFORMATION FOR SEQ ID NO: 164:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 164: GAGGACTGGC AGTACCAAGT AAAC 24
(2) INFORMATION FOR SEQ ID NO:165:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 165: GTTTCTTTGG TTCATTCTAA GATGGCTGG 29
-145- (2) INFORMATION FOR SEQ ID NO: 166:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:166: GCTGAGGCAG GAGAAAAGAC AAG 23
(2) INFORMATION FOR SEQ ID NO: 167:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 167: GTTTCTTCAT GCAAAGGTCA GGAGGTAGG 29
(2) INFORMATION FOR SEQ ID NO: 168:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:168: GTTGCTTCCA GACGAGGTAC ATG 23
(2) INFORMATION FOR SEQ ID NO: 169:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:169:
GTTTCTTCAA TGGCTCCACA AACATCTCTG 30
(2) INFORMATION FOR SEQ ID NO: 170:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs
-146- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 170: AGGTTTAGGG GACAGGGTTT GG 22
(2) INFORMATION FOR SEQ ID NO: 171:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 171: GTTTCTTTCC TGGCTAACAC GGTGAAATC 29
(2) INFORMATION FOR SEQ ID NO:172:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 172: GTTTCTTATT GCCTCCTCCC AAAATTC 27
(2) INFORMATION FOR SEQ ID NO:173:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 173: AGAGGCCACT GGAAGACGAA 20
(2) INFORMATION FOR SEQ ID NO: 174:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-147- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:174:
AACTGGAGTC AGGCAAAACG TG 22
(2) INFORMATION FOR SEQ ID NO: 175:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:175: GTTTCTTTGG CTGGTAAGGA AAGAAACCAC 30
(2) INFORMATION FOR SEQ ID NO:176:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:176: GGCTAGGTTC ATAAACTCTG TGCTG 25
(2) INFORMATION FOR SEQ ID NO: 177:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 177: GTTTCTTGAT TGTTTGAGAT CCTTGACCCA G 31
(2) INFORMATION FOR SEQ ID NO: 178:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:178: GCCGAAATCA CAACACTGCA TC 22
-148- (2) INFORMATION FOR SEQ ID NO: 179:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 179: GTTTCTTGAT TCTGCTCTTA CTCTTGCCCC 30
(2) INFORMATION FOR SEQ ID NO: 180:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:180: GTAATAGAAC CAAAGGGCTG AGAC 24
(2) INFORMATION FOR SEQ ID NO:181:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 181: GTTTCTTCGG AGTCAGACCT TACATTGTTG AG 32
(2) INFORMATION FOR SEQ ID NO: 182:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 182:
ATCTCCCTGC TACCCACCTT 20
(2) INFORMATION FOR SEQ ID NO:183:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs
-149- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:183: GTTTCTTGTT TTCAGTGAGT TTCTGTTGGG 30
(2) INFORMATION FOR SEQ ID NO: 184:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 184: GTGTGCCAAA CAACATTTGC 20
(2) INFORMATION FOR SEQ ID NO: 185:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 185: GTTTCTTCAA GCCATCAAGC TAGAGTGG 28
(2) INFORMATION FOR SEQ ID NO:186:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:186: GGGCTTTTAA ACCCTTATTT AACC 24
(2) INFORMATION FOR SEQ ID NO: 187:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-150- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 187:
GTTTCTTAGG TGATCTCAGA GCCACTCA 28
(2) INFORMATION FOR SEQ ID NO: 188:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:188: AGGGCAGGTG GGAACTTACT 20
(2) INFORMATION FOR SEQ ID NO: 189:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 189: GTTTCTTTGG AGTCAGTTGA GCTTTCTACC 30
(2) INFORMATION FOR SEQ ID NO: 190:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 190: TGAACTTGCC TACCTCCCAG 20
(2) INFORMATION FOR SEQ ID NO: 191:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 191: GTTTCTTAGC ATATATCCTT ACACAAGCAC A 31
-151- (2) INFORMATION FOR SEQ ID NO: 192:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 192: CATGGTTCCA AAGGCAAGTT 20
(2) INFORMATION FOR SEQ ID NO: 193:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 193: GTTTCTTTTG AGGCTGAATG AGCTGTG 27
(2) INFORMATION FOR SEQ ID NO: 194:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:194: ACAGGTGGGA AGACTGAATG TC 22
(2) INFORMATION FOR SEQ ID NO: 195:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 195:
GTTTCTTGCA GTACACATCA CATGACCTTG 30
(2) INFORMATION FOR SEQ ID NO: 196:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs
-152- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:196: GAAATAGGCG GAAACTGGTT C 21
(2) INFORMATION FOR SEQ ID NO: 197:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 197: GTTTCTTCGT TGTGGTTGTT CAGAAAGG 28
(2) INFORMATION FOR SEQ ID NO: 198:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 198: GGTCAAGTGT TCAGAACGCA TC 22
(2) INFORMATION FOR SEQ ID NO: 199:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 199: GTTTCTTGCA GGGATTATGC TAGGTCTGTA G 31
(2) INFORMATION FOR SEQ ID NO: 200:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-153- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 200:
AGCACTTCTG AGGAAGGGAC AC 22
(2) INFORMATION FOR SEQ ID NO: 201:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:201: GTTTCTTAGG GCAGGCAGAC ATACAAAC 28
(2) INFORMATION FOR SEQ ID NO:202:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 202: GCCAATGTGT TCCTAGAGCG AC 22
(2) INFORMATION FOR SEQ ID NO: 203:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:203: GTTTCTTTTA AAGGGGGTAG GGTGTCACC 29
(2) INFORMATION FOR SEQ ID NO:204:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 204: GGAAGGGAAA AGGACAAGGT TTTG 24
-154- (2) INFORMATION FOR SEQ ID NO: 205:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:205: GTTTCTTAGC AAGAGCACTG GTGTAGGAGT C 31
(2) INFORMATION FOR SEQ ID NO: 206:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:206: GCTTTTCAAG CACTTGTCTC 20
(2) INFORMATION FOR SEQ ID NO: 207:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 207: TGGGATTGTG ACTTACCATG 20
(2) INFORMATION FOR SEQ ID NO: 208:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 208:
ACTTGGTGTC TTATAGAAAG GTG 23
(2) INFORMATION FOR SEQ ID NO: 209:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs
-155- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 209: GTTTCTTAGC TGTGTTTGCT GCATC 25
(2) INFORMATION FOR SEQ ID NO: 210:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 210: AGATGTGTGA TGAGATGCAG 20
(2) INFORMATION FOR SEQ ID NO: 211:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 211: GTTTCTTCAA ATAGTGCAAC AAACCC 26
(2) INFORMATION FOR SEQ ID NO:212:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 212: TGTCATTCTG AAAGTGCTTC C 21
(2) INFORMATION FOR SEQ ID NO:213:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-156- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:213:
GTTTCTTCTG TAACTAACGA TCTGTAGTGG TG 32
(2) INFORMATION FOR SEQ ID NO: 214:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 214: TATCAAGGTA ATATAGTAGC CACGG 25
(2) INFORMATION FOR SEQ ID NO: 215:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 215: AGGTCTTTCA TGCAGAGTGG 20
(2) INFORMATION FOR SEQ ID NO: 216:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:216: ATTGCCAAAA CTTGGAAGC 19
(2) INFORMATION FOR SEQ ID NO: 217:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 217: AGGTGACATA TCAAGACCCT G 21
-157- (2) INFORMATION FOR SEQ ID NO: 218:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 218: TTGTCAACGA AGCCCAC 17
(2) INFORMATION FOR SEQ ID NO: 219:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:219: GTTTCTTGCA AGATTGTGTG TATGGATG 28
(2) INFORMATION FOR SEQ ID NO: 220:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 220: GCTCTCTATG TGTTTGGGTG 20
(2) INFORMATION FOR SEQ ID NO: 221:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 221:
AAGAGTACGC TAGTGGATGG 20
(2) INFORMATION FOR SEQ ID NO: 222:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs
-158- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 222: TCCATTAGAC CCAGAAAGG 19
(2) INFORMATION FOR SEQ ID NO:223:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:223: GTTTCTTCAC CAGGCTGAGA TGTTACT 27
(2) INFORMATION FOR SEQ ID NO: 224:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 224: AATCGTTCCT TATCAGGTAA TTTGG 25
(2) INFORMATION FOR SEQ ID NO: 225:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 225: GTTTCTTCAA AGAAAGCAAT TCCATCATAA CA 32
(2) INFORMATION FOR SEQ ID NO:226:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-159- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 226:
GCATTTGTTG AAGCAAGCGG 20
(2) INFORMATION FOR SEQ ID NO: 227:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 227: CTTTGTTCCT TGGCTGATGG 20
(2) INFORMATION FOR SEQ ID NO:228:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 228: AATAGTACCA GACACACGTG 20
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 229: CAATGGTTCA CAGCCCTTTT 20
(2) INFORMATION FOR SEQ ID NO: 230:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 230: AGCCTGGGAG ACAGAGTGAG 20
-160- (2) INFORMATION FOR SEQ ID NO: 231:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 231: GTTTCTTGCA CTTTTTGGGG AAGGTG 26
(2) INFORMATION FOR SEQ ID NO: 232:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:232: GTTCCTCCCT TCCCTCTCC 19
(2) INFORMATION FOR SEQ ID NO:233:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:233: GTTTCTTTCA GGGACTGGAT TGTAG 25
(2) INFORMATION FOR SEQ ID NO:234:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:234:
GTGTTCTTTA TGTGTAGTTC 20
(2) INFORMATION FOR SEQ ID NO:235:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs
-161- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 235: GTTTCTTGGC AACAGAGTGA GACTCA 26
(2) INFORMATION FOR SEQ ID NO: 236:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:236: GTGACATCCA GTGTTGGGAG 20
(2) INFORMATION FOR SEQ ID NO: 237:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 237: GTTTCTTCCT AAGCAAGCAA GCAATCA 27
(2) INFORMATION FOR SEQ ID NO: 238:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 238: AAAGGCAATT GGTGGACA 18
(2) INFORMATION FOR SEQ ID NO: 239:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-162- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:239:
GTTTCTTTTC AATCCTTGAT GCAAAGT 27
(2) INFORMATION FOR SEQ ID NO: 240:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 240: GGTGACAGAG CAAGATTTCG 20
(2) INFORMATION FOR SEQ ID NO: 241:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 241: GTTTCTTGTA GAGTTGAGGG AGCAGC 26
(2) INFORMATION FOR SEQ ID NO: 242:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 242: CATCCATCTC ATCCCATCAT 20
(2) INFORMATION FOR SEQ ID NO: 243:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:243: GTTTCTTTTC ACCCTACTGC CAACTTC 27
-163- (2) INFORMATION FOR SEQ ID NO: 244:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 244: CCGCCATTTT AGAGAGCATA 20
(2) INFORMATION FOR SEQ ID NO: 245:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:245: GTTTCTTTTC TGGGACAATT GGTAGGA 27
(2) INFORMATION FOR SEQ ID NO: 246:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:246: TTTGTGTTAT TATTTCAGGT GC 22
(2) INFORMATION FOR SEQ ID NO: 247:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 247:
GTTTCTTGTT TTTTGTTTCA GTTTAGGAAC 30
(2) INFORMATION FOR SEQ ID NO:248:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs
-164- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 248: CATACCCAAA TCGTTCTCTT CCTC 24
(2) INFORMATION FOR SEQ ID NO: 249:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 249: GTTTCTTGGA AAAGCAAAGG CATCGTAGAG 30
(2) INFORMATION FOR SEQ ID NO: 250:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:250: TACTAACCAA AAGAGTTGGG G 21
(2) INFORMATION FOR SEQ ID NO: 251:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 251: CTATCATTCA GAAAATGTTG GC 22
(2) INFORMATION FOR SEQ ID NO: 252:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-165- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:252:
GTATGGCAGT AGAGGGCATG 20
(2) INFORMATION FOR SEQ ID NO:253:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:253: AAGGTTACAT TTCAAGAAAT AAAGT 25
(2) INFORMATION FOR SEQ ID NO: 254:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 254: CTGTTCAGGC CTCAATATAT ACC 23
(2) INFORMATION FOR SEQ ID NO:255:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 255: AAGAGGATAG GTGGGGTTTG 20
(2) INFORMATION FOR SEQ ID NO: 256:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 256: CCTCCCACCT AGACACAAT 19
-166- (2) INFORMATION FOR SEQ ID NO: 257:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:257: ATATGATCTT TGCATCCCTG 20
(2) INFORMATION FOR SEQ ID NO: 258:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:258: AAGAAAGACC TGGAAGGAAT 20
(2) INFORMATION FOR SEQ ID NO: 259:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:259: AAACAGCAAA ACCTCATCTC 20
(2) INFORMATION FOR SEQ ID NO: 260:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 260:
CCACCACTTA TTACCTGCAT 20
(2) INFORMATION FOR SEQ ID NO: 261:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs
-167- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 261: TGAATGAATG AATGAACGAA 20
(2) INFORMATION FOR SEQ ID NO:262:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:262: AACTGTGATT GTGCCACTGC ACTC 24
(2) INFORMATION FOR SEQ ID NO: 263:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 263: GTTTCTTCAC CGCCTTTATC CCTCAAATG 29
(2) INFORMATION FOR SEQ ID NO: 264:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 264: GATGGGTGGA GGGCAGTTAA AG 22
(2) INFORMATION FOR SEQ ID NO: 265:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-168- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 265:
GTCAAGCAAC TTGTCCAAGG CTAC 24
(2) INFORMATION FOR SEQ ID NO-.266:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:266: CAGGCTATCA GTTTCCTTTG GAG 23
(2) INFORMATION FOR SEQ ID NO: 267:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:267: GGCAGGTAAT ACTGGAGAAT TAGG 24
(2) INFORMATION FOR SEQ ID NO: 268:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:268: GACGGATCTC AGAGCCACTC 20
(2) INFORMATION FOR SEQ ID NO: 269:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 269: GTTTCTTAAA AGATAAGGGC TTTTAAACC 29
-169- (2) INFORMATION FOR SEQ ID NO: 270:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 270: AGTTTCACAG CTTGTTATGG 20
(2) INFORMATION FOR SEQ ID NO: 271:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 271: GGTTGATGAA GTGAGACTTT 20
(2) INFORMATION FOR SEQ ID NO: 272:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 272: ATGGTGGATG CATCCTGTG 19
(2) INFORMATION FOR SEQ ID NO: 273:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:273:
GTTTCTTGTA TTGACTCCTC CTCTGC 26
(2) INFORMATION FOR SEQ ID NO: 274:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs
-170- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:274: CAGTAAACAT 10
(2) INFORMATION FOR SEQ ID NO: 275:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:275: TGTTGAGTGG 10
(2) INFORMATION FOR SEQ ID NO: 276:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:276: TCTCCTCAAT GTGCATGT 18
(2) INFORMATION FOR SEQ ID NO: 277:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 277:
ATTCTACATA 10
(2) INFORMATION FOR SEQ ID NO: 278:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs
-171- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:278: GTGTTTGCAT 10
(2) INFORMATION FOR SEQ ID NO: 279:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 279: ACAAGTTGGC 10
(2) INFORMATION FOR SEQ ID NO: 280:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 280: TAGTACCAGA 10
(2) INFORMATION FOR SEQ ID NO: 281:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:281:
TACATCCAAG AAAA 14
(2) INFORMATION FOR SEQ ID NO: 282:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs
-172- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 282: GAGACTCTGA CAAATATATA TA 22
(2) INFORMATION FOR SEQ ID NO: 283:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:283: TGTTGATCGC CAAACCAAAA TC 22
(2) INFORMATION FOR SEQ ID NO: 284:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 284: AATGCATGTA TGTATATGGT GTGGTATGTG TACATATG 38
(2) INFORMATION FOR SEQ ID NO: 285:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 285: CCTCCCAGAA CAATCATGAT AA 22
(2) INFORMATION FOR SEQ ID NO: 286:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 86 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-173- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:286:
AGACAGTCTC AAAAAATATT TTAAAGAAAA AGCTGGATAA ATAACTAGCT TTAAGAAAAT 60 AAGAAGAAAA AGAAAGAAGA AAGTAA 86
(2) INFORMATION FOR SEQ ID NO: 287:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 86 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 287:
AACTAGCTTT AAGAAAATAA GAAGAAAAAG AAAGAAGAAA GTAAGAAAGA GAAAGAAAAG 60 AAAGAAAAGA AAGAGGAATG ATTGAC 86
(2) INFORMATION FOR SEQ ID NO: 288:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 288: CGCGCACATA CACCCTTTCT CT 22
(2) INFORMATION FOR SEQ ID NO: 289:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 289: CAGTAAACAT CATGTTGAGT GG 22
(2) INFORMATION FOR SEQ ID NO: 290:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
-174- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:290:
TCTCCTCAAT GTGCATGTGT GCATGAGTGC ACATTCTACA TA 42
(2) INFORMATION FOR SEQ ID NO: 291:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 291: GTGTTTGCAT GTTGTACAAG TTGGC 25
(2) INFORMATION FOR SEQ ID NO: 292:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:292:
TAGTACCAGA CACGTGCAGG CAAGCGCACC ATACATCCAA GAAAA 45
(2) INFORMATION FOR SEQ ID NO:293:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 293: GGAGGCTGAG CAGGGGTGCC 20
(2) INFORMATION FOR SEQ ID NO:294:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 294:
-175- ACTCCCACAG GTACCTGCAG _ 20
(2) INFORMATION FOR SEQ ID NO: 295:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:295: CTGCCCTCAC GTAAGCGCCT 20
(2) INFORMATION FOR SEQ ID NO:296:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:296: GCTGTTGCAG GGTAATGTTG 20
(2) INFORMATION FOR SEQ ID NO: 297:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 297: CATCAGACAG GTGCGTACA 19
(2) INFORMATION FOR SEQ ID NO: 298:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 298: GGCTGGTGAG GAGGGGCTGA 20
(2) INFORMATION FOR SEQ ID NO: 299:
-176- (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:299: CGCTCTGTGG GTGAGCTTCA 20
(2) INFORMATION FOR SEQ ID NO: 300:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 300: TGTGGAATAG CCCAATTACA 20
(2) INFORMATION FOR SEQ ID NO: 301:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 301: AGGGTGCTGA GTGAGTAGTA 20
(2) INFORMATION FOR SEQ ID NO: 302:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 302: TTCTTTTCAG GCCCTCGTGT 20
(2) INFORMATION FOR SEQ ID NO: 303:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
-177- (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 303: TGCTGACCCG GTATGGTGGT 20
(2) INFORMATION FOR SEQ ID NO: 304:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:304: TTTGGTGCAG CCTGTGACTC 20
(2) INFORMATION FOR SEQ ID NO: 305:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 305: CGCACACAAG GTCAGTGTTC 20
(2) INFORMATION FOR SEQ ID NO: 306:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 306: TCTTTCCCAG GTTACTCCTT 20
(2) INFORMATION FOR SEQ ID NO: 307:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 307:
-178- ATCAAAGACT GTAAGTAACC - 20
(2) INFORMATION FOR SEQ ID NO:308:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:308: TCTATTTCAG ATGCTGATTC 20
(2) INFORMATION FOR SEQ ID NO: 309:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 309: AGTAGAACAA GTAAGTGCAG 20
(2) INFORMATION FOR SEQ ID NO: 310:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:310: TTTTCAAAAG GCCTCCAAAG 20
(2) INFORMATION FOR SEQ ID NO: 311:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 311: GAGCCCTGAG GTAAGTTAAT 20
(2) INFORMATION FOR SEQ ID NO:312:
-179- (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:312: GCTTTTTCAG ATACTACTAT 20
(2) INFORMATION FOR SEQ ID NO: 313:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 313: TAACATGTTC AACTGTCTGT 20
(2) INFORMATION FOR SEQ ID NO:314:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 314: TGTTATATGC ATTTATCTTC 20
(2) INFORMATION FOR SEQ ID NO: 315:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:315: GGTAAATGAG GTAAGTCCTG 20
(2) INFORMATION FOR SEQ ID NO: 316:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
-180- (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:316: TCTTGTTAAG ATCGCTCTCT 20
(2) INFORMATION FOR SEQ ID NO: 317:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:317: CCTTGCCCAG GTTCTCTTAA 20
(2) INFORMATION FOR SEQ ID NO: 318:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:318: GCAATCGCAC CTGCACACCC 20
(2) INFORMATION FOR SEQ ID NO: 319:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:319: ACTGCCCATT TCTGGTAAAG 20
(2) INFORMATION FOR SEQ ID NO: 320:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 320:
-181- CCCCTAACAG ATCATGATTC 20
(2) INFORMATION FOR SEQ ID NO: 321:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 321: ACGTGCAATG GTAAGAGGGC 20
(2) INFORMATION FOR SEQ ID NO:322:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:322: TGTTTTGCAG TTTCCAGTGG 20
(2) INFORMATION FOR SEQ ID NO: 323:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 323: AAGTGGAACG GTGACTCTCT 20
(2) INFORMATION FOR SEQ ID NO: 324:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 324: TCCTTCACAG GCCAGTGCAG 20
(2) INFORMATION FOR SEQ ID NO: 325:
-182- (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 325: GAACAAACTG GTGAGTAGTA 20
(2) INFORMATION FOR SEQ ID NO: 326:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:326: TTTTTTGTAG AGCCTTCCAT 20
(2) INFORMATION FOR SEQ ID NO: 327:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 327: AGCACAGTAG GTAACTAACT 20
(2) INFORMATION FOR SEQ ID NO: 328:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 328: ATGGCCACAG ATTTGTTGGA 20
(2) INFORMATION FOR SEQ ID NO: 329:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
-183- (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 329: CTTCCTGTTG GTAAGCTGTC 20
(2) INFORMATION FOR SEQ ID NO: 330:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 330: TTCTCCTTAG CAGAGTCACC 20
(2) INFORMATION FOR SEQ ID NO: 331:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 331: AAAAAGCACA GTAAGTTGGC 20
(2) INFORMATION FOR SEQ ID NO: 332:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:332: TTTTCATCAG ACCCGAGAGG 20
(2) INFORMATION FOR SEQ ID NO: 333:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 333:
-184- GAGCTATGAG GTGAGGAGTT . 20
(2) INFORMATION FOR SEQ ID NO: 334:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 334: TTTGTTACAG ATATTACTAC 20
(2) INFORMATION FOR SEQ ID NO: 335:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 335: AGCCTGGAAA TGCGTGTTTC 20
(2) INFORMATION FOR SEQ ID NO: 336:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:336: CGAGAATTCA CTCGAGCATC AGG 23
(2) INFORMATION FOR SEQ ID NO: 337:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 337: CCTGATGCTC GAGTGAATTC T 21
-185- (2) INFORMATION FOR SEQ ID NO: 338:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 848 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 1...848 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 338:
ATG ATT CTG GAA GGA AGT GGT GTA ATG AAT CTC AAC CCA GCC AAC AAC 48 Met lie Leu Glu Gly Ser Gly Val Met Asn Leu Asn Pro Ala Asn Asn 1 5 10 15
CTC CTT CAC CAG CAA CCA GCC TGG CCG GAC AGC TAC CCC ACA TGC AAT 96 Leu Leu His Gin Gin Pro Ala Trp Pro Asp Ser Tyr Pro Thr Cys Asn 20 25 30
GTT TCC AGC GGT TTT TTT GGA AGC CAG TGG CAT GAA ATC CAC CCT CAG 144 Val Ser Ser Gly Phe Phe Gly Ser Gin Trp His Glu lie His Pro Gin 35 40 45
TAC TGG ACC AAA TAC CAG GTG TGG GAA TGG CTG CAG CAC CTC CTG GAC 192 Tyr Trp Thr Lys Tyr Gin Val Trp Glu Trp Leu Gin His Leu Leu Asp 50 55 60
ACC AAC CAG CTA GAC GCT AGC TGC ATC CCT TTC CAG GAG TTC GAC ATT 240 Thr Asn Gin Leu Asp Ala Ser Cys lie Pro Phe Gin Glu Phe Asp lie 65 70 75 80
AGC GGA GAA CAC CTG TGC AGC ATG AGT CTG CAG GAG TTC ACG AGG GCA 288 Ser Gly Glu His Leu Cys Ser Met Ser Leu Gin Glu Phe Thr Arg Ala 85 90 95
GCA GGC TCA GCT GGG CAG CTG CTC TAC AGC AAC CTA CAG CAT CTC AAG 336 Ala Gly Ser Ala Gly Gin Leu Leu Tyr Ser Asn Leu Gin His Leu Lys 100 105 110
TGG AAC GGC CAA TGC AGC AGT GAC CTT TTC CAG TCC GCA CAC AAT GTC 384 Trp Asn Gly Gin Cys Ser Ser Asp Leu Phe Gin Ser Ala His Asn Val 115 120 125
ATT GTC AAG ACT GAA CAA ACC GAT CCT TCC ATC ATG AAC ACA TGG AAA 432 lie Val Lys Thr Glu Gin Thr Asp Pro Ser lie Met Asn Thr Trp Lys 130 135 140
GAA GAA AAC TAT CTC TAT GAT CCC AGC TAT GGT AGC ACA GTA GAT CTG 480 Glu Glu Asn Tyr Leu Tyr Asp Pro Ser Tyr Gly Ser Thr Val Asp Leu 145 150 155 160
-186- TTG GAC AGT AAG ACT TTC TGC CGG GCT CAG ATC TCC ATG ACA_ ACC TCC 528
Leu Asp Ser Lys Thr Phe Cys Arg Ala Gin lie Ser Met Thr Thr Ser
165 170 175
AGT CAC CTT CCA GTT GCA GAG TCA CCT GAT ATG AAA AAG GAG CAA GAC 576
Ser His Leu Pro Val Ala Glu Ser Pro Asp Met Lys Lys Glu Gin Asp
180 185 190
CAC CCT GTA AAG TCC CAC ACC AAA AAG CAC AAC CCA AGA GGC ACT CAC 624
His Pro Val Lys Ser His Thr Lys Lys His Asn Pro Arg Gly Thr His 195 200 205
TTA TGG GAG TTC ATC CGA GAC ATT CTC TTG AGC CCA GAC AAG AAC CCA 672
Leu Trp Glu Phe lie Arg Asp lie Leu Leu Ser Pro Asp Lys Asn Pro 210 215 220
GGG CTG ATC AAA TGG GAA GAC CGT TCG GAA GGC ATC TTC AGG TTC CTG 720
Gly Leu lie Lys Trp Glu Asp Arg Ser Glu Gly lie Phe Arg Phe Leu 225 230 235 240
AAG TCA GAA GCT GTG GCT CAG CTG TGG GGG AAA AAG AAA AAT AAC AGT 768
Lys Ser Glu Ala Val Ala Gin Leu Trp Gly Lys Lys Lys Asn Asn Ser
245 250 255
AGC ATG ACA TAC GAG AAG CTC AGC CGG GCT ATG AGA TAT TAC TAC AAA 816
Ser Met Thr Tyr Glu Lys Leu Ser Arg Ala Met Arg Tyr Tyr Tyr Lys
260 265 270
CGA GAA ATC CTG GAA CGT GTG GAT GGA CGA CG 848
Arg Glu lie Leu Glu Arg Val Asp Gly Arg Arg 275 280
(2) INFORMATION FOR SEQ ID NO: 339:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 283 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 339:
Met lie Leu Glu Gly Ser Gly Val Met Asn Leu Asn Pro Ala Asn Asn
1 5 10 15
Leu Leu His Gin Gin Pro Ala Trp Pro Asp Ser Tyr Pro Thr Cys Asn
20 25 30
Val Ser Ser Gly Phe Phe Gly Ser Gin Trp His Glu lie His Pro Gin
35 40 45
Tyr Trp Thr Lys Tyr Gin Val Trp Glu Trp Leu Gin His Leu Leu Asp
50 55 60
Thr Asn Gin Leu Asp Ala Ser Cys lie Pro Phe Gin Glu Phe Asp lie 65 70 75 80
Ser Gly Glu His Leu Cys Ser Met Ser Leu Gin Glu Phe Thr Arg Ala
-187- 85 90 95
Ala Gly Ser Ala Gly Gin Leu Leu Tyr Ser Asn Leu Gin His Leu Lys
100 105 110
Trp Asn Gly Gin Cys Ser Ser Asp Leu Phe Gin Ser Ala His Asn Val
115 120 125 lie Val Lys Thr Glu Gin Thr Asp Pro Ser lie Met Asn Thr Trp Lys
130 135 140
Glu Glu Asn Tyr Leu Tyr Asp Pro Ser Tyr Gly Ser Thr Val Asp Leu 145 150 155 160
Leu Asp Ser Lys Thr Phe Cys Arg Ala Gin lie Ser Met Thr Thr Ser
165 170 175
Ser His Leu Pro Val Ala Glu Ser Pro Asp Met Lys Lys Glu Gin Asp
180 185 190
His Pro Val Lys Ser His Thr Lys Lys His Asn Pro Arg Gly Thr His
195 200 205
Leu Trp Glu Phe lie Arg Asp lie Leu Leu Ser Pro Asp Lys Asn Pro
210 215 220
Gly Leu lie Lys Trp Glu Asp Arg Ser Glu Gly lie Phe Arg Phe Leu 225 230 235 240
Lys Ser Glu Ala Val Ala Gin Leu Trp Gly Lys Lys Lys Asn Asn Ser
245 250 255
Ser Met Thr Tyr Glu Lys Leu Ser Arg Ala Met Arg Tyr Tyr Tyr Lys
260 265 270
Arg Glu lie Leu Glu Arg Val Asp Gly Arg Arg 275 280
-188-

Claims

WHAT IS CLAIMED IS:
1. An isolated nucleic acid molecule comprising a sequence within a mammalian ASTH1 locus, or a polymorphic variant thereof.
2. An isolated nucleic acid molecule according to Claim 1 , wherein said nucleic acid molecule encodes an ASTH1 polypeptide.
3. An isolated nucleic acid molecule according to Claim 1 wherein said nucleic acid comprises a promoter or regulatory region.
4. An isolated nucleic acid molecule according to Claim 1 comprising a probe for detection of an ASTH1 locus polymorphism.
5. An array of oligonucleotides comprising: two or more probes according to Claim 4.
6. An isolated nucleic acid comprising a microsatellite repeat associated with a predisposition to asthma.
7. A nucleic acid according to any of claim 1 to 5, wherein said ASTH1 locus is human.
8. A cell comprising a nucleic acid composition according to any of claims 1 to 4.
9. A purified polypeptide composition comprising at least 50 weight % of the protein present as the product of the nucleic acid of Claim 1.
10. A method for detecting a predisposition to asthma in an individual, the method comprising: analyzing the genomic DNA or mRNA of said individual for the presence of at least one predisposing ASTH1 locus polymorphism or a sequence linked to a
-189- predisposing polymorphism; wherein the presence of said predisposing polymorphism is indicative of an increased susceptibility to asthma.
11. A method according to Claim 10, wherein said analyzing step comprises detection of specific binding between the genomic DNA or mRNA of said individual with a probe or probes according to either of Claims 4 or 5.
12. A method according to Claim 10, wherein said analyzing step comprises detection of specific binding between the genomic DNA or mRNA of said individual with a microsatellite marker listed in Table 1.
13. A non-human transgenic animal model for ASTH1 gene function comprising one of:
(a) a knockout of an ASTH1 gene; (b) an exogenous and stably transmitted mammalian ASTH1 gene sequence; or
(c) an ASTH1 promoter sequence operably linked to a reporter gene.
14. A method of screening for biologically active agents that modulate ASTH1 function, the method comprising: combining a candidate biologically active agent with any one of:
(a) a mammalian ASTH1 polypeptide;
(b) a cell comprising a nucleic acid encoding a mammalian ASTH1 polypeptide; or (c) a non-human transgenic animal model for ASTH1 gene function comprising one of: (i) a knockout of an ASTH1 gene; (ii) an exogenous and stably transmitted mammalian ASTH1 gene sequence; or (iii) an ASTH1 promoter sequence operably linked to a reporter gene; and determining the effect of said agent on ASTH1 function.
-190-
15. An isolated nucleic acid that hybridizes under stringent conditions to any one of: SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO.10, or SEQ ID NO:328.
16. An isolated nucleic acid that encodes a polypeptide or fragment thereof having an amino acid sequence substantially identical to the sequence as set forth within any one of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 , or SEQ ID NO:339.
-191-
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US7205146B1 (en) 2000-06-14 2007-04-17 Oscient Pharmaceuticals Corporation Nucleotide and amino acid sequences relating to respiratory diseases and obesity
EP1182255A1 (en) * 2000-08-16 2002-02-27 Universiteit Utrecht Genes involved in immune related responses observed with asthma
JP2002153299A (en) * 2000-11-22 2002-05-28 Sumikin Bioscience Kk GENOPOLYMORPHISM OF CONSTITUENT MASP-1 OF HUMAN COMPLEMENT-ACTIVATED LECTIN RaRF
IL159722A0 (en) * 2001-07-10 2004-06-20 Oligos Etc Inc Pharmaceutical compositions containing oligonucleotides
EP1498493A4 (en) * 2002-04-03 2006-01-04 Genox Research Inc Method of examining allergic disease
WO2008039941A2 (en) 2006-09-27 2008-04-03 The Government Of The Usa As Represented By The Secretary Of The Dpt. Of Health And Human Services Scgb3a2 as a growth factor and anti-apoptotic agent

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CA2314677A1 (en) 1999-07-29

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