Polymorphic markers of prostate carcinoma tumor antigen-1(PCTA-1)

Abstract

The invention concerns the geNo.mic sequence and cDNA sequences of the PCTA-1 gene. The invention also concerns biallelic markers of the PCTA-1 gene and the association established between these markers and prostate cancer. The invention provides means to determine the predisposition of individuals to prostate cancer as well as means for the diagNo.sis of prostate cancer and for the progNo.sis/detection of an eventual treatment response to agents acting against prostate cancer.

Description

FIELD OF THE INVENTION

The invention concerns the genomic and cDNA sequences of the PCTA-1 gene, biallelic markers of the PCTA-1 gene and the association established between these markers and prostate cancer. The invention provides means to determine the predisposition of individuals to prostate cancer as well as means for the diagnosis of this cancer and for the prognosis/detection of an eventual treatment response to therapeutic agents acting against prostate cancer.

BACKGROUND OF THE INVENTION

Prostate Cancer

The incidence of prostate cancer has dramatically increased over the last decades. It averages 30-50/100,000 males in Western European countries as well as within the US White male population. In these countries, it has recently become the most commonly diagnosed malignancy, being one of every four cancers diagnosed in American males. Prostate cancer's incidence is very much population specific, since it varies from 2/100,000 in China, to over 80/100,000 among African-American males.

In France, the incidence of prostate cancer is 35/100,000 males and it is increasing by 10/100,000 per decade. Mortality due to prostate cancer is also growing accordingly. It is the second cause of cancer death among French males, and the first one among French males aged over 70. This makes prostate cancer a serious burden in terms of public health.

Prostate cancer is a latent disease. Many men carry prostate cancer cells without overt signs of disease. Autopsies of individuals dying of other causes show prostate cancer cells in 30% of men at age 50 and in 60% of men at age 80. Furthermore, prostate cancer can take up to 10 years to kill a patient after the initial diagnosis.

The progression of the disease usually goes from a well-defined mass within the prostate to a breakdown and invasion of the lateral margins of the prostate, followed by metastasis to regional lymph nodes, and metastasis to the bone marrow. Cancer metastasis to bone is common and often associated with uncontrollable pain.

Unfortunately, in 80% of cases, diagnosis of prostate cancer is established when the disease has already metastasized to the bones. Of special interest is the observation that prostate cancers frequently grow more rapidly in sites of metastasis than within the prostate itself.

Early-stage diagnosis of prostate cancer mainly relies today on Prostate Specific Antigen (PSA) dosage, and allows the detection of prostate cancer seven years before clinical symptoms become apparent. The effectiveness of PSA dosage diagnosis is however limited, due to its inability to discriminate between malignant and non-malignant affections of the organ and because not all prostate cancers give rise to an elevated serum PSA concentration. Furthermore, PSA dosage and other currently available approaches such as physical examination, tissue biopsy and bone scans are of limited value in predicting disease progression.

Therefore, there is a strong need for a reliable diagnostic procedure which would enable a more systematic early-stage prostate cancer prognosis.

Although an early-stage prostate cancer prognosis is important, the possibility of measuring the period of time during which treatment can be deferred is also interesting as currently available medicaments are expensive and generate important adverse effects. However, the aggressiveness of prostate tumors varies widely. Some tumors are relatively aggressive, doubling every six months whereas others are slow-growing, doubling once every five years. In fact, the majority of prostate cancers grow relatively slowly and never becomes clinically manifest. Very often, affected patients are among the elderly and die from another disease before prostate cancer actually develops. Thus, a significant question in treating prostate carcinoma is how to discriminate between tumors that will progress and those that will not progress during the expected lifetime of the patient.

Hence, there is also a strong need for detection means which may be used to evaluate the aggressiveness or the development potential of prostate cancer tumors once diagnosed.

Furthermore, at the present time, there is no means to predict prostate cancer susceptibility. It would also be very beneficial to detect individual susceptibility to prostate cancer. This could allow preventive treatment and a careful follow up of the development of the tumor.

A further consequence of the slow growth rate of prostate cancer is that few cancer cells are actively dividing at any one time, rendering prostate cancer generally resistant to radiation and chemotherapy. Surgery is the mainstay of treatment but it is largely ineffective and removes the ejaculatory ducts, resulting in impotence. Oral oestrogens and luteinizing releasing hormone analogs are also used for treatment of prostate cancer. These hormonal treatments provide marked improvement for many patients, but they only provide temporary relief. Indeed, most of these cancers soon relapse with the development of hormone-resistant tumor cells and the oestrogen treatment can lead to serious cardiovascular complications. Consequently, there is a strong need for preventive and curative treatment of prostate cancer.

Efficacy/tolerance prognosis could be precious in prostate cancer therapy. Indeed, hormonal therapy, the main treatment currently available, presents important side effects. The use of chemotherapy is limited because of the small number of patients with chemosensitive tumors. Furthermore the age profile of the prostate cancer patient and intolerance to chemotherapy make the systematic use of this treatment very difficult.

Therefore, a valuable assessment of the eventual efficacy of a medicament to be administered to a prostate cancer patient as well as the patient's eventual tolerance to it may allow the benefit/risk ratio of prostate cancer treatment to be enhanced.

Prostate Carcinoma Tumor Antigen-1 (PCTA-1)

WO 96/21671 describes a new protein, named PCTA-1. The document describes the cloning and sequencing of a cDNA encoding PCTA-1 (GenBank L78132). This cDNA has 3.85 kb in length and presents about 80% sequence homology with rat galectin-8.

WO 96/21671 mentions that the PCTA-1 protein retains a number of conserved structural motifs that are found in most members of the galectin gene family. On the basis of its predicted amino acid sequence, PCTA-1 is said to appear to be a human homologue of rat galectin-8. The galectins display wide tissue distribution, clear developmental regulation, and differential levels in specific tissues, supporting the hypothesis that they contribute to many physiologically important processes in mammalian cells. Of direct relevance to cancer is the finding that the galectins can mediate both cell-cell and cell-matrix interactions.

SUMMARY OF THE INVENTION

The inventors have characterized the genomic sequence of the PCTA-1 gene, including its regulatory regions, and, through an association study, have shown that alleles of some biallelic markers of PCTA-1 are associated with prostate cancer.

Therefore, the present invention concerns the identification and characterization of the genomic sequence of the PCTA-1 gene, of new cDNA sequences and the proteins encoded by these cDNAs. The invention also concerns biallelic markers located in such sequences, as well as the selection of significant polymorphisms associated with prostate cancer.

Oligonucleotide probes and primers hybridizing specifically with a genomic sequence of PCTA-1 are also part of the invention. A further object of the invention consists of recombinant vectors comprising any of the nucleic acid sequences described in the present invention, and in particular of recombinant vectors comprising the regulatory region of PCTA-1 or a sequence encoding a PCTA-1 protein, as well as cell hosts comprising said nucleic acid sequences or recombinant vectors.

The selected polymorphisms are used in the design of assays for the reliable detection of genetic susceptibility to prostate cancer, of an early onset of prostate cancer, of the aggressiveness of prostate cancer tumors, of a modified or forthcoming expression of the PCTA-1 gene, of a modified or forthcoming production of the PCTA-1 protein, or of the production of a modified PCTA-1 protein. They can be used for diagnosis, staging, prognosis, and monitoring of such a disease, which processes can be further included within treatment approaches. The selected polymorphisms can also be used in the design of drug screening protocols to provide an accurate and efficient evaluation of the therapeutic and side-effect potential of new or already existing medicaments.

The invention also encompasses methods of screening of molecules which modulate or inhibit the expression of the PCTA-1 gene and more preferably of agent acting against prostate cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a diagram of the PCTA-1 gene with an indication of the relative position of the biallelic markers of the present invention. The upper line refers to the genomic sequence of PCTA-1. The middle line refers to the alternative cDNA comprising the exon 6bis with the biallelic markers localization. The lower line refers the PCTA-1 protein with the polymorphic amino acids due to the biallelic markers. {circle around (☆)} refers to frequent SNP (detected on pool of hundred DNA).

FIG. 1B is a diagram of the 3 alternative cDNAs of PCTA-1.

FIG. 2 is a graph demonstrating the association between some of the biallelic markers of the invention and prostate cancer with the absolute value of the logarithm (base 10) of the p-value of the chi-square values for each marker shown on the y-axis and a rough estimate of the position of each marker with respect to the PCTA-1 gene elements on the x-axis.

FIG. 3 is a block diagram of an exemplary computer system.

FIG. 4 is a flow diagram illustrating one embodiment of a process 200 for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database.

FIG. 5 is a flow diagram illustrating one embodiment of a process 250 in a computer for determining whether two sequences are homologous.

FIG. 6 is a flow diagram illustrating one embodiment of an identifier process 300 for detecting the presence of a feature in a sequence.

FIGS. 7A-D is an alignment of the mouse and human PCTA-1 proteins. The amino acid sequences provided in the alignment are presented as SEQ ID NOs: 13 (leg2), 14 (leg1), 15 (PCTA), 16 (PCTA.var), 17 (PCTA.mus), 18 (gal9-1), 19 (gal), 20 (leg7), 21 (gal4), and (consenus sequence).

BRIEF DESCRIPTION OF THE SEQUENCES PROVIDED IN THE SEQUENCE LISTING

SEQ ID No 1 contains a genomic sequence of PCTA-1 comprising the 5′ regulatory region (upstream untranscribed region), the exons (0, 1, 2, 3, 4, 5, 6, 6bis, 7, 8, 9, 9bis, and 9ter) and introns, and the 3′ regulatory region (downstream untranscribed region).

SEQ ID No 2 contains a cDNA sequence of PCTA-1 comprising the exons 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9.

SEQ ID No 3 contains a cDNA sequence of PCTA-1 comprising the exons 0, 1, 2, 3, 4, 5, 6, 6bis, 7, 8, and 9.

SEQ ID No 4 contains a cDNA sequence of PCTA-1 comprising the exons 0, 1, 2, 3, 4, 5, 6, 7, 8, 9bis and 9ter.

SEQ ID No 5 contains the amino acid sequence encoded by the cDNA of SEQ ID No 2.

SEQ ID No 6 contains the amino acid sequence encoded by the cDNA of SEQ ID No 3.

SEQ ID No 7 contains the amino acid sequence encoded by the cDNA of SEQ ID No 4.

SEQ ID No 8 contains a murine cDNA sequence of PCTA-1.

SEQ ID No 9 contains the amino acid sequence encoded by the cDNA of SEQ ID No 8.

SEQ ID No 10 contains a primer containing the additional PU 5′ sequence described further in Example 2.

SEQ ID No 11 contains a primer containing the additional RP 5′ sequence described further in Example 2.

SEQ ID NO: 12 is a version of the polynucleotide sequence of the PCTA-1 gene of SEQ ID NO: 1 that provides symbols appropriate for indicated allelic substitutions at nucleotide positions 402, 67092, 68525, 82234, and 82393. SEQ ID NO: 12.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Before describing the invention in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used to describe the invention herein.

The term “PCTA-1 gene” is intended to define an entity which can comprise some or all the following elements: exons, introns, promoter, regulatory regions, 5′UTR, 3′UTR and regions never transcribed and located either upstream or downstream of the coding sequence of PCTA-1 . The term “PCTA-1 gene”, when used herein, encompasses genomic, mRNA and cDNA sequences encoding a PCTA-1 protein.

The term “heterologous protein”, when used herein, is intended to designate any protein or polypeptide other than the PCTA-1 protein. More particularly, the heterologous protein is a compound which can be used as a marker in further experiments with a PCTA-1 regulatory region or as a toxin to certain cells in which it is intended to be produced, preferably a toxin to prostate cancer cells.

As used herein, the term “toxin gene” refers to a polynucleotide sequence which encodes a polypeptide that, when expressed in a eukaryotic cell, typically a mammalian cell, kills or disables the cell or causes the cell to exhibit apoptosis, cytostasis or senescence.

The term “isolated” requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.

The term “purified” does not require absolute purity; rather, it is intended as a relative definition. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. As an example, purification from 0.1% concentration to 10% concentration is two orders of magnitude. The term “purified” is used herein to describe a polynucleotide or polynucleotide vector of the invention which has been separated from other compounds including, but not limited to other nucleic acids, carbohydrates, lipids and proteins (such as the enzymes used in the synthesis of the polynucleotide), or the separation of covalently closed polynucleotides from linear polynucleotides. A polynucleotide is substantially pure when at least about 50%, preferably 60 to 75% of a sample exhibits a single polynucleotide sequence and conformation (linear versus covalently closed). A substantially pure polynucleotide typically comprises about 50%, preferably 60 to 90% weight/weight of a nucleic acid sample, more usually about 95%, and preferably is over about 99% pure. Polynucleotide purity or homogeneity is indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polynucleotide band upon staining the gel. For certain purposes higher resolution can be provided by using HPLC or other means well known in the art.

As used interchangeably herein, the terms “nucleic acids”, “oligonucleotides”, and “Polynucleotides” include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form. The term “nucleotide” as used herein as an adjective to describe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form. The term “nucleotide” is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a molecule, or individual unit in a larger nucleic acid molecule, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide. Although the term “nucleotide” is also used herein to encompass “modified nucleotides” which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, for examples of analogous linking groups, purine, pyrimidines, and sugars see for example PCT publication No. WO 95/04064. This may be especially oligonucleotides with α or β anomers, oligonucleotides with inter-nucleotide linkage of the phosphorothioate or methyl phosphonate type, or alternatively oligothionucleotide. The polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.

Throughout the present specification, the expression “nucleotide sequence” may be employed to designate indifferently a polynucleotide or a nucleic acid. More precisely, the expression “nucleotide sequence” encompasses the nucleic material itself and is thus not restricted to the sequence information (i.e. the succession of letters chosen among the four base letters) that biochemically characterizes a specific DNA or RNA molecule.

A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell required to initiate the specific transcription of a gene.

A sequence which is “overably linked” to a regulatory sequence such as a promoter means that said regulatory element is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the nucleic acid of interest. As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. More precisely, two DNA molecules (such as a polynucleotide containing a promoter region and a polynucleotide encoding a desired polypeptide or polynucleotide) are said to be “operably linked” if the nature of the linkage between the two polynucleotides does not (1) result in the introduction of a frame-shift mutation or (2) interfere with the ability of the polynucleotide containing the promoter to direct the transcription of the coding polynucleotide.

The term “primer” denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence. A primer serves as an initiation point for nucleotide polymerization catalyzed by either DNA polymerase, RNA polymerase or reverse transcriptase.

The term “probe” denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., polynucleotide as defined herein) which can be used to identify a specific polynucleotide sequence present in samples, said nucleic acid segment comprising a nucleotide sequence complementary of the specific polynucleotide sequence to be identified.

The terms “base paired” and “Watson & Crick base paired” are used interchangeably herein to refer to nucleotides which can be hydrogen bonded to one another by virtue of their sequence identities in a manner like that found in double-helical DNA with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds (See Stryer, L., Biochemistry , 4 th edition, 1995).

The terms “complementary” or “complement thereof” are used herein to refer to the sequences of polynucleotides which are capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region. For the purpose of the present invention, a first polynucleotide is deemed to be complementary to a second polynucleotide when each base in the first polynucleotide is paired with its complementary base. Complementary bases are, generally, A and T (or A and U), or C and G. “Complement” is used herein as a synonym of “complementary polynucleotide”, “complementary nucleic acid” and “complementary nucleotide sequence”. These terms are applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.

The term “Volypeptide” refers to a polymer of amino acids without regard to the length of the polymer, thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not specify or exclude post-expression modifications of polypeptides, for example, polypeptides which include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Also included within the definition are polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

The term “recombinant polypeptide” is used herein to refer to polypeptides that have been artificially designed and which comprise at least two polypeptide sequences that are not found as contiguous polypeptide sequences in their initial natural environment, or to refer to polypeptides which have been expressed from a recombinant polynucleotide.

The term “purified” is used herein to describe a polypeptide of the invention which has been separated from other compounds including, but not limited to nucleic acids, lipids, carbohydrates and other proteins. A polypeptide is substantially pure when at least about 50%, preferably 60 to 75% of a sample exhibits a single polypeptide sequence. A substantially pure polypeptide typically comprises about 50%, preferably 60 to 90% weight/weight of a protein sample, more usually about 95%, and preferably is over about 99% pure. Polypeptide purity or homogeneity is indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polypeptide band upon staining the gel. For certain purposes higher resolution can be provided by using HPLC or other means well known in the art.

As used herein, the term “non-human animal” refers to any non-human vertebrate, birds and more usually mammals, preferably primates, farm animals such as swine, goats, sheep, donkeys, and horses, rabbits or rodents, more preferably rats or mice. As used herein, the term “animal” is used to refer to any vertebrate, preferable a mammal. Both the terms “animal” and “mammal” expressly embrace human subjects unless preceded with the term “non-human”.

As used herein, the term “antibody” refers to a polypeptide or group of polypeptides which are comprised of at least one binding domain, where an antibody binding domain is formed from the folding of variable domains of an antibody molecule to form three-dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an antigenic determinant of an antigen, which allows an immunological reaction with the antigen. Antibodies include recombinant proteins comprising the binding domains, as wells as fragments, including Fab, Fab′, F(ab) 2 , and F(ab′) 2 fragments.

As used herein, an “antigenic determinant” is the portion of an antigen molecule, in this case a PCTA-1 polypeptide, that determines the specificity of the antigen-antibody reaction. An “epitope” refers to an antigenic determinant of a polypeptide. An epitope can comprise as few as 3 amino acids in a spatial conformation which is unique to the epitope. Generally an epitope consists of at least 6 such amino acids, and more usually at least 8-10 such amino acids. Methods for determining the amino acids which make up an epitope include x-ray crystallography, 2-dimensional nuclear magnetic resonance, and epitope mapping e.g. the Pepscan method described by Geysen et al. 1984; PCT Publication No. WO 84/03564; and PCT Publication No. WO 84/03506, the disclosures of which are incorporated herein by reference in their entireties.

The term “allele” is used herein to refer to variants of a nucleotide sequence. A biallelic polymorphism has two forms. Diploid organisms may be homozygous or heterozygous for an allelic form.

The term “heterozyposity rate” is used herein to refer to the incidence of individuals in a population which are heterozygous at a particular allele. In a biallelic system, the heterozygosity rate is on average equal to 2P a (1-P a ), where P a is the frequency of the least common allele. In order to be useful in genetic studies, a genetic marker should have an adequate level of heterozygosity to allow a reasonable probability that a randomly selected person will be heterozygous.

The term “genotype” as used herein refers the identity of the alleles present in an individual or a sample. In the context of the present invention, a genotype preferably refers to the description of the biallelic marker alleles present in an individual or a sample. The term “genotyping” a sample or an individual for a biallelic marker consists of determining the specific allele or the specific nucleotide carried by an individual at a biallelic marker.

The term “mutation” as used herein refers to a difference in DNA sequence between or among different genomes or individuals which has a frequency below 1%.

The term “haplotype” refers to a combination of alleles present in an individual or a sample. In the context of the present invention, a haplotype preferably refers to a combination of biallelic marker alleles found in a given individual and which may be associated with a phenotype.

The term “polymorphism” as used herein refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. “Polymorphic” refers to the condition in which two or more variants of a specific genomic sequence can be found in a population. A “polymorphic site” is the locus at which the variation occurs. A single nucleotide polymorphism is a single base pair change. Typically a single nucleotide polymorphism is the replacement of one nucleotide by another nucleotide at the polymorphic site. Deletion of a single nucleotide or insertion of a single nucleotide, also give rise to single nucleotide polymorphisms. In the context of the present invention “single nucleotide polymorphism” preferably refers to a single nucleotide substitution. However, the polymorphism can also involve an insertion or a deletion of at least one nucleotide, preferably between 1 and 5 nucleotides. The nucleotide modification can also involve the presence of several adjacent single base polymorphisms. This type of nucleotide modification is usually called a “variable motif”. Generally, a “variable motif” involves the presence of 2 to 10 adjacent single base polymorphisms. In some instances, series of two or more single base polymorphisms can be interrupted by single bases which are not polymorphic. This is also globally considered to be a “variable motif”. Typically, between different genomes or between different individuals, the polymorphic site may be occupied by two different nucleotides.

The term “biallelic polymorphism” and “biallelic marker” are used interchangeably herein to refer to a polymorphism, usually a single nucleotide, having two alleles at a fairly high frequency in the population. A “biallelic marker allele” refers to the nucleotide variants present at a biallelic marker site. Typically, the frequency of the less common allele of the biallelic markers of the present invention has been validated to be greater than 1%, preferably the frequency is greater than 10%, more preferably the frequency is at least 20% (i.e. heterozygosity rate of at least 0.32), even more preferably the frequency is at least 30% (i.e. heterozygosity rate of at least 0.42). A biallelic marker wherein the frequency of the less common allele is 30% or more is termed a “high quality biallelic marker”.

As used herein the terminology “defining a biallelic marker” means that a sequence includes a polymorphic base from a biallelic marker. The sequences defining a biallelic marker may be of any length consistent with their intended use, provided that they contain a polymorphic base from a biallelic marker. The sequence has between 2 and 100, preferably between 20, 30, or 40 and 60, and more preferably about 47 nucleotides in length. Likewise, the term “marker” or “biallelic marker” requires that the sequence is of sufficient length to practically (although not necessarily unambiguously) identify the polymorphic allele, which usually implies a length of at least 4, 5, 6, 10, 15, 20, 25, or 40 nucleotides.

As used herein the term “PCTA-1-related biallelic marker” or “biallelic marker of the PCTA-1 gene” relates to a set of biallelic markers in linkage disequilibrium with the PCTA-1 gene. The term PCTA-1-related biallelic marker encompasses all of the biallelic markers A1 to A125 disclosed in Table 2.

The location of nucleotides in a polynucleotide with respect to the center of the polynucleotide are described herein in the following manner. When a polynucleotide has an odd number of nucleotides, the nucleotide at an equal distance from the 3′ and 5′ ends of the polynucleotide is considered to be “at the center” of the polynucleotide, and any nucleotide immediately adjacent to the nucleotide at the center, or the nucleotide at the center itself is considered to be “within 1 nucleotide of the center.” With an odd number of nucleotides in a polynucleotide any of the five nucleotides positions in the middle of the polynucleotide would be considered to be within 2 nucleotides of the center, and so on. When a polynucleotide has an even number of nucleotides, there would be a bond and not a nucleotide at the center of the polynucleotide. Thus, either of the two central nucleotides would be considered to be “within 1 nucleotide of the center” and any of the four nucleotides in the middle of the polynucleotide would be considered to be “within 2 nucleotides of the center”, and so on. For polymorphisms which involve the substitution, insertion or deletion of 1 or more nucleotides, the polymorphism, allele or biallelic marker is “at the center” of a polynucleotide if the difference between the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 3′ end of the polynucleotide, and the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 5′ end of the polynucleotide is zero or one nucleotide. If this difference is 0 to 3, then the polymorphism is considered to be “within 1 nucleotide of the center.” If the difference is 0 to 5, the polymorphism is considered to be “within 2 nucleotides of the center.” If the difference is 0 to 7, the polymorphism is considered to be “within 3 nucleotides of the center,” and so on.

The terms “trait” and “phenotype” are used interchangeably herein and refer to any visible, detectable or otherwise measurable property of an organism such as symptoms of, or susceptibility to a disease for example. Preferably, the term “trait” or “phenotype”, when used herein, encompasses, but is not limited to, prostate cancer, an early onset of prostate cancer, a beneficial response to or side effects related to treatment or a vaccination against prostate cancer, a susceptibility to prostate cancer, the level of aggressiveness of prostate cancer tumors, a modified or forthcoming expression of the PCTA-1 gene, a modified or forthcoming production of the PCTA-1 protein, or the production of a modified PCTA-1 protein. However, the term “trait” or “phenotype” can refer to other types of cancer.

The term “susceptibility to prostate cancer” is used herein to designate a strong likelihood for an individual to develop in his lifetime a form of prostate cancer, particularly a form of prostate cancer in which a PCTA-1 protein is expressed. This likelihood is strongly related to the association established between the biallelic markers of the present invention and prostate cancer or other more specific characteristics which can lead to the development of the prostate cancer such as the modified expression of the PCTA-1 gene, the modified production of the PCTA-1 protein or the production of a modified PCTA-1 protein.

The term “aggressiveness” of prostate cancer tumors refers to the metastatic potential of these tumors.

The term “treatment of prostate cancer” when used herein is intended to designate the administration of substances either for prophylactic or curative purposes. When administered for prophylactic purposes, the treatment is provided in advance of the appearance of biologically or clinically significant cancer symptoms. When administered for curative purposes, the treatment is provided to attenuate the pathological symptoms of prostate cancer, to decrease the size or growth of cancer tumors or metastases or to remove them.

The terms “an agent acting against prostate cancer” refers to any drug or compound that is capable of reducing the growth rate, rate of metastasis, or viability of tumor cells in a mammal, is capable of reducing the size or eliminating tumors in a mammal, or is capable of increasing the average life span of a mammal or human with cancer. Agents acting against prostate cancer also include compounds which are able to reduce the risk of cancer developing in a population, particularly a high risk population. Examples of agents acting against prostate cancer include hormonal therapeutic agents (for example, medroxyprogesterone acetate, estramustine phosphate, gonadotrophin releasing hormone (GnRH) agonists, anti-androgens such as flutamide, nilutamide, groserelin, and cyprosterone acetate, anti-gonadotropic agents such as stilboestrol and other oestrogenic agents, progestogens such as megestrol acetate) or chemotherapeutic agents (for example, carboplatin, cisplatin, methotrexate, mitomycin, epirubicin, vinblastine, 5-fluorouracyl, mitozantrone, cyclophosphamide, interferon, N-(4-hydroxyphenyl) retinamide (4HPR)). These agents can be used in combination.

The term “side effects to an agent acting against prostate cancer” refers to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors. These side effects include, but are not limited to, adverse reactions such as dermatological, hematological or hepatological toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial En asthma to laryngeal edema and bronchoconstriction, hypotension, sexual dysfunction, and shock. More particularly, the side effects can be nausea/vomiting, cardiovascular side effects such as deep vein thrombosis and fluid retention, and gynaecomastia.

The term “response to an agent acting against prostate cancer” refers to drug efficacy, including but not limited to ability to metabolize a compound, to the ability to convert a pro-drug to an active drug, and to the pharmacokinetics (absorption, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.

In the context of the present invention, a “positive response” to a medicament can be defined as comprising a reduction of the symptoms related to the disease, an increase of survival time or condition to be treated.

In the context of the present invention, a “negative response” to a medicament can be defined as comprising either a lack of positive response to the medicament which does not lead to a symptom reduction or an increase of survival time, or which leads to a side-effect observed following administration of the medicament.

Variants and Fragments

1-Polynucleotides

The invention also relates to variants and fragments of the polynucleotides described herein, particularly of a PCTA-1 gene containing one or more biallelic markers according to the invention.

Variants of polynucleotides, as the term is used herein, are polynucleotides that differ from a reference polynucleotide. A variant of a polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms. Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical.

Variants of polynucleotides according to the invention include, without being limited to, nucleotide sequences which are at least 95% identical to a polynucleotide selected from the group consisting of the nucleotide sequences of SEQ ID Nos 1, 2, 3, 4, 8 or to any polynucleotide fragment of at least 8 consecutive nucleotides of a polynucleotide selected from the group consisting of the nucleotide sequences of SEQ ID Nos 1, 2, 3, 4, 8, and preferably at least 99% identical, more particularly at least 99.5% identical, and most preferably at least 99.8% identical to a polynucleotide selected from the group consisting of the nucleotide sequences of SEQ ID Nos 1, 2, 3, 4, 8 or to any polynucleotide fragment of at least 8 consecutive nucleotides of a polynucleotide selected from the group consisting of the nucleotide sequences of SEQ ID Nos 1, 2, 3, 4, 8.

Nucleotide changes present in a variant polynucleotide may be silent, which means that they do not alter the amino acids encoded by the polynucleotide. However, nucleotide changes may also result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.

In the context of the present invention, particularly preferred embodiments are those in which the polynucleotides encode polypeptides which retain substantially the same biological function or activity as the mature PCTA-1 protein, or those in which the polynucleotides encode polypeptides which maintain or increase a particular biological activity, while reducing a second biological activity.

A polynucleotide fragment is a polynucleotide having a sequence that entirely is the same as part but not all of a given nucleotide sequence, preferably the nucleotide sequence of a PCTA-1 gene, and variants thereof. The fragment can be a portion of an exon or of an intron of a PCTA-1 gene. It can also be a portion of the regulatory sequences of the PCTA-1 gene, preferably of the promoter. Preferably, such fragments comprise at least one of the biallelic markers A1 to A125, and the complements thereof, or a biallelic marker in linkage disequilibrium therewith.

Such fragments may be “free-standing”, i.e. not part of or fused to other polynucleotides, or they may be comprised within a single larger polynucleotide of which they form a part or region. However, several fragments may be comprised within a single larger polynucleotide.

As representative examples of polynucleotide fragments of the invention, there may be mentioned those which have from about 4, 6, 8, 15, 20, 25, 40, 10 to 30, 30 to 55, 50 to 100, 75 to 100 or 100 to 200 nucleotides in length. Preferred are those fragments having about 47 nucleotides in length, such as those of P1 to P125 and the complementary sequences thereto, and containing at least one of the biallelic markers of the PCTA-1 gene which are described herein. It will of course be understood that the polynucleotides P1 to P125 and the complementary sequences thereto can be shorter or longer, although it is preferred that they at least contain the biallelic marker of the primer which can be located at one end of the fragment.

2-Polypeptides

The invention also relates to variants, fragments, analogs and derivatives of the polypeptides described herein, including mutated PCTA-1 proteins.

The variant may be 1) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code, or 2) one in which one or more of the amino acid residues includes a substituent group, or 3) one in which the PCTA-1 protein is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or 4) one in which the additional amino acids are fused to the PCTA-1 protein, such as a leader or secretory sequence or a sequence which is employed for purification of the PCTA-1 protein or a preprotein sequence. Such variants are deemed to be within the scope of those skilled in the art

A polypeptide fragment is a polypeptide having a sequence that entirely is the same as part but not all of a given polypeptide sequence, preferably a polypeptide encoded by a PCTA-1 gene and variants thereof. Preferred fragments include those of the active region of the PCTA-1 protein that may play a role in prostate cancer and those regions possessing antigenic properties and which can be used to raise antibodies against the PCTA-1 protein.

In the case of an amino acid substitution in the amino acid sequence of a polypeptide according to the invention, one or several amino acids can be replaced by “equivalent” amino acids. The expression “equivalent” amino acid is used herein to designate any amino acid that may be substituted for one of the amino acids having similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Generally, the following groups of amino acids represent equivalent changes: (1) Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, Ile, Leu, Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, Trp, His.

A specific embodiment of a modified PCTA-1 peptide molecule of interest according to the present invention, includes, but is not limited to, a peptide molecule which is resistant to proteolysis, is a peptide in which the —CONH— peptide bond is modified and replaced by a (CH 2 NH) reduced bond, a (NHCO) retro inverso bond, a (CH 2 —O) methylene-oxy bond, a (CH 2 —S) thiomethylene bond, a (CH2CH 2 ) carba bond, a (CO—CH 2 ) cetomethylene bond, a (CHOH—CH 2 ) hydroxyethylene bond), a (N—N) bound, a E-alcene bond or also a —CH═CH— bond. The invention also encompasses a human PCTA-1 polypeptide or a fragment or a variant thereof in which at least one peptide bound has been modified as described above.

Such fragments may be “free-standing”, i.e. not part of or fused to other polypeptides, or they may be comprised within a single larger polypeptide of which they form a part or region. However, several fragments may be comprised within a single larger polypeptide.

As representative examples of polypeptide fragments of the invention, there may be mentioned those which have from about 5, 6, 7, 8, 9 or 10 to 15, 10 to 20, 15 to 40, or 30 to 55 amino acids long. Preferred are those fragments containing at least one amino acid mutation in the PCTA-1 protein.

Identity between Nucleic Acids or Polypeptides

The terms “percentage of sequence identity” and “percentage homology” are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Homology is evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988; Altschul et al., 1990; Thompson et al., 1994; Higgins et al., 1996; Altschul et al., 1993). In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool (“BLASr”) which is well known in the art (see, e.g., Karlin and Altschul, 1990; Altschul et al., 1990, 1993, 1997). In particular, five specific BLAST programs are used to perform the following task:

(1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database;

(2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database;

(3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database;

(4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and

(5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as “high-scoring segment pairs,” between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., 1992; Henikoff and Henikoff, 1993). Less preferably, the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978). The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user-specified percent homology. Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (see, e.g., Karlin and Altschul, 1990).

Stringent Hybridization Conditions

By way of example and not limitation, procedures using conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C., the preferred hybridization temperature, in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5−20×10 6 cpm of 32 P-labeled probe.

Alternatively, the hybridization step can be performed at 65° C. in the presence of SSC buffer, 1×SSC corresponding to 0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes can be done at 37° C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1×SSC at 50° C. for 45 min. Alternatively, filter washes can be performed in a solution containing 2×SSC and 0.1% SDS, or 0.5×SSC and 0.1% SDS, or 0.1×SSC and 0.1% SDS at 68° C. for 15 minute intervals. Following the wash steps, the hybridized probes are detectable by autoradiography. Other conditions of high stringency which may be used are well known in the art and as cited in Sambrook et al., 1989; and Ausubel et al., 1989. These hybridization conditions are suitable for a nucleic acid molecule of about 20 nucleotides in length. There is no need to say that the hybridization conditions described above are to be adapted according to the length of the desired nucleic acid, following techniques well known to the one skilled in the art. The suitable hybridization conditions may for example be adapted according to the teachings disclosed in the book of Hames and Higgins (1985) or in Sambrook et al.(1989).

Genomic Sequence of the PCTA-1 Gene

The present invention relates to a purified and/or isolated nucleic acid corresponding to the genomic sequence of the PCTA-1 gene. Preferably, this genomic PCTA-1 sequence comprises the nucleotide sequence of SEQ ID No 1, a sequence complementary thereto, a fragment or a variant thereof.

The present invention encompasses the genomic sequence of PCTA-1. The PTCA-1 gene sequence comprises a coding sequence including 13 exons included in SEQ ID No 1, namely exon 0, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 6bis, exon 7, exon 9, exon 9bis and exon 9ter, the intronic regions, the promoter, the 5′UTR, the 3′UTR, and regulatory regions located upstream and downstream of the coding region.

The localization of the exons and introns of the PCTA-1 gene is detailed in Table A and is described as feature in SEQ ID No 1.

TABLE A
	Position Range		Position range
	in SEQ ID No 1		in SEQ ID No 1
Exon	Beginning	End	Intron	Beginning	End
0	68648	68741	0	68742	70646
1	70647	70794	1	70795	82207
2	82208	82296	2	82297	83612
3	83613	83823	3	83824	85297
4	85298	85417	4	85418	86388
5	86389	86445	5	86446	87495
6	87496	87522	6	87523	87649
6bis	87650	87775	6bis	87776	88294
7	88295	88383	7	88384	89483
8	89484	89649	8	89650	92748
9	92749	97155	9bis	92884	95820
9bis	92749	92883
9ter	95821	97155

Intron 0 refers to the nucleotide sequence located between Exon 0 and Exon 1, and so on. The intron 6 refers to the nucleotide sequence located between Exon 6 and Exon 6bis. The intron 6bis refers to the nucleotide sequence located between Exon 6bis and Exon 7. The intron 8 refers to the nucleotide sequence located between Exon 8 and Exon 9 or 9bis. The intron 9bis refers to the nucleotide sequence located between Exon 9bis and Exon 9ter.

The invention also encompasses a purified, isolated, or recombinant polynucleotide comprising a mucleotide sequence having at least 70, 75, 80, 85, 90, or 95% nucleotide identity with a mucleotide sequence of SEQ ID No 1 or a complementary sequence thereto or a fragment therof. The nucleotide differences as regards to the nucleotide sequence of SEQ ID No 1 may be generally randomly distributed throughout the entire nucleic acid. Nevertheless, preferred nucleic acids are those wherein the nucleotide differences as regards to the nucleotide sequence of SEQ ID No 1 are predominantly located outside the coding sequences contained in the exons. These nucleic acids, as well as their fragments and variants, may be used as oligonucleotide primers or probes in order to detect the presence of a copy of the PCTA-1 gene in a test sample, or alternatively in order to amplify a target nucleotide sequence within the PCTA-1 sequences.

Another object of the invention consists of a purified, isolated, or recombinant nucleic acid that hybridizes with the nucleotide sequence of SEQ ID No 1 or a complementary sequence therto or a variant thereof, under the stringent hybridization conditions as defined above.

Particularly preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements therof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1: 1-70715, 70795-82207, 82297-83612, 83824-85297, 85418-86388, 86446-87495, 87523-88294, 88384-89483, 89650-92748, 97156-98309, 98476-99329, 99491-100026, 100212-100281, 100396-100538, 100682-100833, 100995-101920, 102087-102970, 103264-103724, and 103753-106746. Additional preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide G at positions 70728, 87860, 88297, 94432, and 95340 of SEQ ID No 1; a nucleotide A at positions 82218, 83644, 83808, 87787, 87806, 94218, and 97144 of SEQ ID No 1; a nucleotide C at positions 87902, 88215, 88283, 92760, 93726, and 94422 of SEQ ID No 1; and a nucleotide T at positions 93903, and 94170 of SEQ ID No 1. Other preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide G at positions 86435, 93592, 93680, 93681, 93682,93728, 93761, and 95445 of SEQ ID No 1; a nucleotide A at positions 86434, 88355, 93240, 93471, and 93747of SEQ ID No 1; a nucleotide C at positions 93683, 95126, and 95444 of SEQ ID No 1; and a nucleotide T at positions 94154, and 94430 of SEQ ID No 1. Other preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 1: 92975-92977, 93711-93715, 94151-94153, 94240-94243, 94770-94773, 94804-94808, 95121-95122, 95129-95135, 95148-95153, 95154-95159, 95173-95178, 95367-95374, 95410-95413, 95418-95420, 95430-95436, 95533-95535, and 95677-95677. It should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section.

A preferred aspect of the present invention is a purified and/or isolated and/or recombined PCTA-1 gene or a fragment thereof comprising at least one of the biallelic polymorphisms described below, a sequence complementary thereto, a fragment or a variant thereof. In some embodiments, the PCTA-1 gene or a fragment thereof may comprise at least one of the nucleotide sequences of P1 to P125, a sequence complementary thereto, a fragment or a variant thereof. In a preferred embodiment, the PCTA-1 gene or a fragment thereof comprises a biallelic marker selected from the group consisting of A1 to A125 and the complements thereof.

While this section is entitled “Genomic Sequences of The PCTA-1 Gene”, it should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section, flanking the genomic sequences of PCTA-1 on either side or between two or more such genomic sequences.

PCTA-1 cDNA Sequences

The invention also concerns a purified and/or isolated cDNA encoding a PCTA-1 protein; Preferably, the cDNA comprises a nucleotide sequence selected from the group consisting of SEQ ID Nos 2, 3, 4, sequences complementary thereto and functional fragments and variants thereof. Moreover, preferred polynucleotides of the invention include purified, isolated, or recombinant PCTA-1 cDNAs consisting of, consisting essentially of, or comprising a sequence selected from the group consisting of SEQ ID Nos 2, 3, 4 and the complementary sequence thereto.

The invention also pertains to a purified or isolated nucleic acid comprising a polynucleotide having at least 95% nucleotide identity with a polynucleotide selected from the group consisting of SEQ ID Nos 2, 3, 4, advantageously 99% nucleotide identity, preferably 0 99.5% nucleotide identity and most preferably 99.8% nucleotide identity with a polynucleotide selected from the group consisting of SEQ ID Nos 2, 3, 4, or a sequence complementary thereto or a biologically active fragment thereof.

Another object of the invention consists of purified, isolated or recombinant nucleic acids comprising a polynucleotide that hybridizes, under the stringent hybridization conditions defined herein, with a polynucleotide selected from the group consisting of the nucleotide sequences of SEQ ID Nos 2, 3, 4, or a sequence complementary thereto or a variant thereof or a biologically active fragment thereof.

The 5′UTR and 3′UTR regions of a gene are of particular importance in that they often comprise regulatory elements which can play a role in providing appropriate expression levels, particularly through the control of mRNA stability. The inventors have cloned a complete PCTA-1 cDNA (SEQ ID No 2) in which the 5′UTR is carried by exon 0 and a portion of exon 1 and the 3′UTR is carried by a portion of exon 9. Moreover, they have characterized a 5′EST, which is located as a feature in SEQ ID No 1, comprising the exons 0 and 1, and partially exon 2. Since an ATG codon is located at the beginning of the partial exon 1 disclosed in WO 96/21671, one could assume that the promoter of the PCTA-1 gene would be located immediately upstream of this codon. However, the inventors unexpectedly found that the PCTA-1 genomic DNA contains further exonic sequences upstream of the partial exon 1 disclosed in WO 96/21671. Without the knowledge of such sequences, the identification by the skilled person of the PCTA-1 promoter was extremely unlikely. Only the full genomic sequence of PCTA-1 and access by the inventors to a proprietary 5′EST database rendered possible the identification of a full cDNA sequence and of the PCTA-1 promoter. The invention concern the nucleotide sequence of 5′ EST consisting of the position range 1-266 in the SEQ ID No 2.

The main characteristics of the PCTA-1 cDNA comprising exons 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9 are detailed in Table B. The invention concerns the purified and/or isolated sequence of the 5′UTR and 3′UTR as described in Table B or a complementary sequence thereto or an allelic variant thereof set forth in SEQ ID No 2. Particularly preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60,70, 80, 90, 100, 150,200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 1-162 of SEQ ID No 2. Further preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527, 2471, and 5397 of SEQ ID No 2; a nucleotide C at positions 1013, 1979, and 2675 of SEQ ID No 2; a nucleotide G at positions 176, 749, 2685, 3593 of SEQ ID No 2; and a nucleotide T at positions 2156, and 2423 of SEQ ID No 2. Additional preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708, 807, 1493, 1724, and 2000; a nucleotide C at positions 1936, 3379, and 3697; a nucleotide G at positions 709, 1845, 1933, 1934, 1935, 1981, 2014, and 3698; and a nucleotide T at positions 2407, and 2683 of SEQ ID No 2. Other preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 2: 1229-1231, 1964-1968, 2404-2406, 2493-2496, 3023-3026, 3057-3061, 3374-3375, 3382-3388, 3401-3406, 3407-3412, 3426-3431, 3620-3627, 3663-3666, 3671-3673, 3683-3689, 3786-3788 and 3930-3932. It should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section.

The majority of interrupted genes are transcribed into an RNA that gives rise to a single type of spliced mRNA. But the RNAs of some genes follow patterns of alternative splicing, wherein a single gene gives rise to more than one mRNA species. In some cases, the ultimate pattern of expression is dictated by the primary transcript, because the use of different startpoints or termination sequences alters the splicing pattern. In other cases, a single primary transcript is spliced in more than one way, and internal exons are substituted, added or deleted. In some cases, the multiple products all are made in the same cell, but in others, the process is regulated so that particular splicing patterns occur only under particular conditions.

At least three PCTA-1 cDNAs are produced by alternative splicing. The inventors have identified a minor species of PCTA-1 cDNA, disclosed in SEQ ID No 3, and comprising an additional exon 6bis which encodes 42 additional amino acids. In a further embodiment, the present invention concerns the additional exon of the PCTA-1 gene located between exon 6 and exon 7, namely exon 6bis, detailed as a feature in SEQ ID No 1 and in Table A, a sequence complementary thereto, and a fragment or variant thereof. The present invention embodies a PCTA-1 cDNA comprising the exon 6bis disclosed in SEQ ID No 1.

The main characteristics of this second PCTA-1 cDNA comprising exons 0, 1, 2, 3, 4, 5, 6, 6bis, 7, 8, and 9 are detailed in Table B. The amino acid sequence of this new PCTA-1 protein is disclosed in SEQ ID No 6. Particularly preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 3: 1-162 and 747-872. Further preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527, 2597, and 5523 of SEQ ID No 3; a nucleotide C at positions 1139, 2105, and 2801 of SEQ ID No 3; a nucleotide G at positions 176, 875, 2811, 3719 of SEQ ID No 3; and a nucleotide T at positions 2282, and 2549 of SEQ ID No 3. Additional preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708, 807, 1619, 1850, and 2126; a nucleotide C at positions 2062, 3505, and 3823; a nucleotide G at positions 709, 1971, 2059, 2060, 2061, 2107, 2140, and 3824; and a nucleotide T at positions 2533, and 2809 of SEQ ID No 3. Other preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 3: 1355-1357, 1892-1894,2090-2094, 2530-2532, 2619-2622, 3149-3152, 3183-3187, 3500-3501, 3508-3514, 3527-3532, 3533-3538, 3552-3557, 3746-3749, 3789-3792, 3797-3799, 3809-3815, 3912-3914 and 4056-4058. It should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section.

The inventors have also identified a species of PCTA-1 cDNA comprising alternative exons to exon 9 which are called exons 9bis and 9ter. Its sequence is disclosed in SEQ ID No 4. The exon 9bis and 9ter correspond respectively to the beginning and the ends of the exon 9. The polynucleotide of the exon 9 located between exons 9bis and 9ter is spliced or deleted. The combination of exons 9bis and 9ter extends the ORF of the PCTA-1 gene.

The main characteristics of this second PCTA-1 cDNA comprising exons 0, 1, 2, 3, 4, 5, 6, 7, 8, 9bis and 9ter are detailed in Table B. The amino acid sequence of the new PCTA-1 protein encoded by this cDNA is disclosed in SEQ ID No 7. Particularly preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 1-162 of SEQ ID No 4. Further preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527 and 2460 of SEQ ID No 4; a nucleotide c at position 1013 of SEQ ID No 4 and a nucleotide G at positions 176, and 749 of SEQ ID No 4. Additionally preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708 and 807 and a nucleotide G at position 709 of SEQ No 4. Other preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises the pairs of nucleotide positions 1136-1137 of SEQ ID No 4. It should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section.

The invention further embodies purified, isolated, or recombinant polynucleotides comprising a nucleotide sequence selected from the group consisting of the 13 exons of the PCTA-1 gene, or a sequence complementary thereto. The invention also deals with purified, isolated, or recombinant nucleic acids comprising a combination of at least two exons of the PCTA-1 gene, wherein the polynucleotides are arranged within the nucleic acid, from the 5′-end to the 3 ′-end of said nucleic acid, in the same order as in SEQ ID No 1. In this specific embodiment of a purified or isolated nucleic acid according to the invention, said nucleic acid preferably comprises the exon 0 at its 5′ end and the exon 9 or 9ter at its 3′ end.

The 3′UTR sequence of PCTA-1 appears to include several polyadenylation sites. These polyadenylation sites could have an influence on the stability of the mRNA resulting from the transcription of the PCTA-1 genomic DNA.

The invention also concerns a purified and/or isolated cDNA sequence encoding a mouse PCTA-1 protein, particularly a cDNA comprising the nucleotide sequence of SEQ ID No 8, a sequence complementary thereto or a fragment and variant thereof. The main characteristics of the murine cDNA are detailed in Table B. Moreover, preferred polynucleotides of the invention include purified, isolated, or recombinant PCTA-1 cDNAs consisting of, consisting essentially of, or comprising the sequence of SEQ ID No 8 and the complementary sequence thereto.

The invention also pertains to a purified or isolated nucleic acid comprising a polynucleotide having at least 95% nucleotide identity with a polynucleotide of SEQ ID No 8, advantageously 99% nucleotide identity, preferably 99.5% nucleotide identity and most preferably 99.8% nucleotide identity with a polynucleotide of SEQ ID No 8, or a sequence complementary thereto or a biologically active fragment thereof.

Another object of the invention consists of purified, isolated or recombinant nucleic acids comprising a polynucleotide that hybridizes, under the stringent hybridization conditions defined herein, with a polynucleotide of SEQ ID No 8, or a sequence complementary thereto or a variant thereof or a biologically active fragment thereof.

Particularly preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 8 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 8: 1-500, 501-1000, 1001-1500, and 1501-1738.

	TABLE B
	Position range	Position range of ORF	Position range	Position range of
cDNA	of 5′UTR	ATG	STOP	of 3′UTR	polydenylation sites
SEQ ID No 2	1-200	201-203	1149-1151	1152-5408	1773-1778, 3624-3629,
					3828-3833, 5119-5124,
					5381-5386, 5386-5391
SEQ ID No 3	1-200	201-203	1275-1277	1278-5534	1899-1904, 3750-3755,
					3954-3959, 5245-5250,
					5507-5512, 5512-5517
SEQ ID No 4	1-200	201-203	1305-1307	1308-2471	2182-2187, 2444-2449,
					2449-2454
SEQ ID No 8	1-120	121-123	1068-1070	1071-1738

While this section is entitled “PCTA-1 cDNA Sequences,” it should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section, flanking the genomic sequences of PCTA-1 on either side or between two or more such genomic sequences.

Coding Regions

The invention also concerns a nucleotide sequence encoding the human PCTA-1 protein selected from the group consisting of SEQ ID No 5, 6, 7, sequences complementary thereto and fragments and variants thereof. The present invention embodies isolated, purified, and recombinant polynucleotides which encode polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 or 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 5, wherein said contiguous span includes:

a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 5; and/or

at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 5.

The present invention also embodies isolated, purified, and recombinant polynucleotides which encode polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 or 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 6, wherein said contiguous span includes:

a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 245 in SEQ ID No 6; and/or

at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 225 in SEQ ID No 6; and/or

at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exon 6bis, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 183-224 of the SEQ ID No 6.

The present invention further embodies isolated, purified, and recombinant polynucleotides which encode polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 or 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 7, wherein said contiguous span includes:

a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 7; and/or

at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 7; and/or

at least 1, 2, 3, 5 or 1 0 of the amino acid encoded by the exons 9bis and 9ter, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 313-368 of the SEQ ID No 7.

The invention also concerns a nucleotide sequence encoding the murine PCTA-1 protein of SEQ ID No 9, sequences complementary thereto and fragments and variants thereof More particularly, the present invention embodies isolated, purified, and recombinant polynucleotides which encode polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 or 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 9, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following amino acid positions of SEQ ID No 9: 1-50, 51-100, 101-150, 151-200, 201-250, and 251-316.

The above disclosed polynucleotide that contains the coding sequence of the PCTA-1 gene may be expressed in a desired host cell or a desired host organism, when this polynucleotide is placed under the control of suitable expression signals. The expression signals may be either the expression signals contained in the regulatory regions in the PCTA-1 gene of the invention or in contrast the signals may be exogenous regulatory nucleic sequences. Such a polynucleotide, when placed under the suitable expression signals, may also be inserted in a vector for its expression and/or amplification.

Regulator Sequences of the PCTA-1 Gene

The present invention also concerns the purified and/or isolated sequences of the upstream regulatory region (5′ regulatory region) of the PCTA-1 gene, sequences complementary thereto, and fragments or variants thereof, particularly the nucleotide sequence located between positions 1 and 68647 of SEQ ID No 1, as well as any sequence of 8 to 3000 consecutive nucleotides, preferably of 8 to 500 consecutive nucleotides, included therein. More particularly, the invention further includes specific elements within this regulatory region. These elements include a promoter region. The promoter region appears to be located in the 10 kb region, preferably in the 5 kb region, more preferably in the 2 kb region, still more preferably in the 1 kb region, and more particularly in the 500 bp, upstream of the first exon of the PCTA-1 gene. Preferably, the promoter region has a nucleotide sequence located between positions 66647 and 68647 of SEQ ID No 1 as well as any functional sequence of at least 8 consecutive nucleotide, preferably 8 to 400 consecutive nucleotides, more preferably of 8 to 300 nucleotides included therein, sequences complementary thereto and fragments and variants thereof. Further comments are provided below on this region which is of a particular importance in the present invention.

Also included in the invention are regulatory sequences downstream of the PCTA-1 coding sequence (3′ regulatory region) such as those included in the nucleotide sequence located between positions 97156 and 106746 of SEQ ID No 1, sequences complementary thereto and fragments and variants thereof.

In order to identify the relevant biologically active polynucleotide fragments or variants of the 5′ or 3′ regulatory region, the one skilled in the art will refer to the book of Sambrook et al. (Sambrook et al., 1989) which describes the use of a recombinant vector carrying a marker gene (i.e. beta galactosidase, chloramphenicol acetyl transferase, etc.) the expression of which will be detected when placed under the control of a biologically active polynucleotide fragments or variants of the 5′ or 3′ regulatory region. Genomic sequences located upstream of the first exon of the PCTA-1 gene are cloned into a suitable promoter reporter vector, such as the pSEAP-Basic, pSEAP-Enhancer, pβgal-Basic, pβgal-Enhancer, or pEGFP-1 Promoter Reporter vectors available from Clontech, or pGL2-basic or pGL3-basic promoterless luciferase reporter gene vector from Promega. Briefly, each of these promoter reporter vectors include multiple cloning sites positioned upstream of a reporter gene encoding a readily assayable protein such as secreted alkaline phosphatase, luciferase, beta galactosidase, or green fluorescent protein. The sequences upstream the PCTA-1 coding region are inserted into the cloning sites upstream of the reporter gene in both orientations and introduced into an appropriate host cell. The level of reporter protein is assayed and compared to the level obtained from a vector which lacks an insert in the cloning site. The presence of an elevated expression level in the vector containing the insert with respect to the control vector indicates the presence of a promoter in the insert. If necessary, the upstream sequences can be cloned into vectors which contain an enhancer for increasing transcription levels from weak promoter sequences. A significant level of expression above that observed with the vector lacking an insert indicates that a promoter sequence is present in the inserted upstream sequence.

Promoter sequences within the upstream genomic DNA may be further defined by constructing nested 5′ and/or 3′ deletions in the upstream DNA using conventional techniques such as Exonuclease III or appropriate restriction endonuclease digestion. The resulting deletion fragments can be inserted into the promoter reporter vector to determine whether the deletion has reduced or obliterated promoter activity. In this way, the boundaries of the promoters may be defined. If desired, potential individual regulatory sites within the promoter may be identified using site directed mutagenesis or linker scanning to obliterate potential transcription factor binding sites within the promoter individually or in combination. The effects of these mutations on transcription levels may be determined by inserting the mutations into cloning sites in promoter reporter vectors. This type of assay is well-known to those skilled in the art and is described in WO 97/17359, U.S. Pat. No. 5,374,544, EP 582,796, U.S. Pat. No. 5,698,389, U.S. Pat. No. 5,643,746, U.S. Pat. No. 5,502,176, and U.S. Pat. No. 5,266,488, the disclosures of which are incorporated herein by reference in their entireties.

The strength and the specificity of the promoter of the PCTA-1 gene can be assessed through the expression levels of a detectable polynucleotide operably linked to the PCTA-1 promoter in different types of cells and tissues. The detectable polynucleotide may be either a polynucleotide that specifically hybridizes with a predefined oligonucleotide probe, or a polynucleotide encoding a detectable protein, including a PCTA-1 polypeptide or a fragment or a variant thereof. This type of assay is well-known to those skilled in the art and is described in U.S. Pat. No. 5,502,176, and U.S. Pat. No. 5,266,488, the disclosures of which are incorporated herein by reference in their entireties. In one embodiment, the efficacy of the promoter of the PCTA-1 gene is assessed in normal and cancer cells. In a preferred embodiment, the efficacy of the promoter of the PCTA-1 gene is assessed in normal cells and in cancer cells which can present different degrees of malignancy, more preferably cells from prostate tissue. Some of the methods are discussed in more detail below.

Polynucleotides carrying the regulatory elements located at the 5′ end and at the 3′ end of the PCTA-1 coding region may be advantageously used to control the transcriptional and translational activity of an heterologous polynucleotide of interest.

Thus, the present invention also concerns a purified or isolated nucleic acid comprising a polynucleotide which is selected from the group consisting of the 5′ and 3′ regulatory regions, or a sequence complementary thereto or a biologically active fragment or variant thereof. “5′ regulatory region” refers to the nucleotide sequence located between positions 1 and 68647 of SEQ ID No 1. “3′ regulatory region” refers to the nucleotide sequence located between positions 97156 and 106746 of SEQ ID No 1.

The invention also pertains to a purified or isolated nucleic acid comprising a polynucleotide having at least 95% nucleotide identity with a polynucleotide selected from the group consisting of the 5′ and 3′ regulatory regions, advantageously 99% nucleotide identity, preferably 99.5% nucleotide identity and most preferably 99.8% nucleotide identity with a polynucleotide selected from the group consisting of the 5′ and 3′ regulatory regions, or a sequence complementary thereto or a biologically active fragment thereof.

Another object of the invention consists of purified, isolated or recombinant nucleic acids comprising a polynucleotide that hybridizes, under the stringent hybridization conditions defined herein, with a polynucleotide selected from the group consisting of the nucleotide sequences of the 5′- and 3′ regulatory regions, or a sequence complementary thereto or a variant thereof or a biologically active fragment thereof.

Preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1: 1-4000, 4001-8000, 8001-12000, 12001-16000, 16001-20000, 20001-24000, 24001-28000, 28001-32000, 32001-36000, 36001-40000, 40001-44000, 4400-8000, 48001-52000, 52001-56000, 56001-60000, 60001-64000, 64001-68647. Particularly preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1: 66647-68647.

“Biologically active” polynucleotide derivatives of SEQ ID No 1 are polynucleotides comprising or alternatively consisting of a fragment of said polynucleotide which is functional as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide in a recombinant cell host. It could act either as an enhancer or as a repressor.

For the purpose of the invention, a nucleic acid or polynucleotide is “functional” as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide if said regulatory polynucleotide contains nucleotide sequences which contain transcriptional and translational regulatory information, and such sequences are “operably linked” to nucleotide sequences which encode the desired polypeptide or the desired polynucleotide.

The regulatory polynucleotides of the invention may be prepared from the nucleotide sequence of SEQ ID No 1 by cleavage using suitable restriction enzymes, as described for example in the book of Sambrook et al.(1989). The regulatory polynucleotides may also be prepared by digestion of SEQ ID No 1 by an exonuclease enzyme, such as Bal3 1 (Wabiko et al., 1986). These regulatory polynucleotides can also be prepared by nucleic acid chemical synthesis, as described elsewhere in the specification.

A preferred 5′-regulatory polynucleotide of the invention includes the 5′-untranslated region (5′-UTR) of the PCTA-1 cDNA, or a biologically active fragment or variant thereof. A preferred 3′-regulatory,polynucleotide of the invention includes the 3′-untranslated region (3′-UTR) of the PCTA-1 cDNA, or a biologically active fragment or variant thereof.

A further object of the invention consists of a purified or isolated nucleic acid comprising:

a) a nucleic acid comprising a regulatory nucleotide sequence selected from the group consisting of:

(i) a nucleotide sequence comprising a polynucleotide of the 5′ regulatory region or a complementary sequence thereto;

(ii) a nucleotide sequence comprising a polynucleotide having at least 95% of nucleotide identity with the nucleotide sequence of the 5′ regulatory region or a complementary sequence thereto;

(iii) a nucleotide sequence comprising a polynucleotide that hybridizes under stringent hybridization conditions with the nucleotide sequence of the 5′ regulatory region or a complementary sequence thereto; and

(iv) a biologically active fragment or variant of the polynucleotides in (i), (ii) and (iii);

b) a polynucleotide encoding a desired polypeptide or a nucleic acid of interest, operably linked to the nucleic acid defined in (a) above; and

c) Optionally, a nucleic acid comprising a 3′-regulatory polynucleotide, preferably a 3′-regulatory polynucleotide of the PCTA-1 gene.

In a specific embodiment of the nucleic acid defined above, said nucleic acid includes the 5′-untranslated region (5′-UTR) of the PCTA-1 cDNA, or a biologically active fragment or variant thereof. In a second specific embodiment of the nucleic acid defined above, said nucleic acid includes the 3′-untranslated region (3′-UTR) of the PCTA-1 cDNA, or a biologically active fragment or variant thereof.

The regulatory polynucleotide of the 5′ regulatory region, or its biologically active fragments or variants, is operably linked at the 5′-end of the polynucleotide encoding the desired polypeptide or polynucleotide.

The regulatory polynucleotide of the 3′ regulatory region, or its biologically active fragments or variants, is advantageously operably linked at the 3′-end of the polynucleotide encoding the desired polypeptide or polynucleotide.

The desired polypeptide encoded by the above-described nucleic acid may be of various nature or origin, encompassing proteins of prokaryotic or eukaryotic origin. Among the polypeptides expressed under the control of a PCTA-1 regulatory region include bacterial, fungal or viral antigens. Also encompassed are eukaryotic proteins such as intracellular proteins, like “house keeping” proteins, membrane-bound proteins, like receptors, and secreted proteins like endogenous mediators such as cytokines. The desired polypeptide may be the PCTA-1 protein, especially the protein of a amino acid sequence selected from the group consisting of SEQ ID Nos 5, 6, 7, 9, or a fragment or a variant thereof.

The desired nucleic acids encoded by the above-described polynucleotide, usually an RNA molecule, may be complementary to a desired coding polynucleotide, for example to a PCTA-1 coding sequence, and thus useful as an antisense polynucleotide.

Such a polynucleotide may be included in a recombinant expression vector in order to express the desired polypeptide or the desired nucleic acid in host cell or in a host organism. Suitable recombinant vectors that contain a polynucleotide such as described herein are disclosed elsewhere in the specification.

Polynucleotide Constructs

The terms “polynucleotide construct” and “recombinant polynucleotide” are used interchangeably herein to refer to linear or circular, purified or isolated polynucleotides that have been artificially designed and which comprise at least two nucleotide sequences that are not found as contiguous nucleotide sequences in their initial natural environment.

DNA Construct that Enables Directing Temporal and Spatial PCTA-1 Gene Expression in Recombinant Cell Hosts and in Transgenic Animals.

In order to study the physiological and phenotypic consequences of a lack of synthesis of the PCTA-1 protein, both at the cell level and at the multi cellular organism level, the invention also encompasses DNA constructs and recombinant vectors enabling a conditional expression of a specific allele of the PCTA-1 genomic sequence or cDNA and also of a copy of this genomic sequence or cDNA harboring substitutions, deletions, or additions of one or more bases as regards to the PCTA-1 nucleotide sequence of SEQ ID Nos 1, 2, 3, 4, 8, or a fragment thereof, these base substitutions, deletions or additions being located either in an exon, an intron or a regulatory sequence, but preferably in the 5′-regulatory sequence or in an exon of the PCTA-1 genomic sequence or within a PCTA-1 cDNA of SEQ ID Nos 2, 3, 4, or 8. In a preferred embodiment, the PCTA-1 sequence comprises a biallelic marker of the present invention. In a preferred embodiment, the PCTA-1 sequence comprises a biallelic marker of the present invention, preferably one of the biallelic markers A1 to A125 and the complements thereof.

The present invention embodies recombinant vectors comprising any one of the polynucleotides described in the present invention. More particularly, the polynucleotide constructs according to the present invention can comprise any of the polynucleotides described in the “PCTA-1 cDNA Sequences” section, the “Coding Regions” section, and the “Oligonucleotide Probes And Primers” section.

A first preferred DNA construct is based on the tetracycline resistance operon tet from E. coli transposon Tn 10 for controlling the PCTA-1 gene expression, such as described by Gossen et al.(1992, 1995) and Furth et al.(1994). Such a DNA construct contains seven let operator sequences from Tn 10 (tetop) that are fused to either a minimal promoter or a 5′-regulatory sequence of the PCTA-1 gene, said minimal promoter or said PCTA-1 regulatory sequence being operably linked to a polynucleotide of interest that codes either for a sense or an antisense oligonucleotide or for a polypeptide, including a PCTA-1 polypeptide or a peptide fragment thereof. This DNA construct is functional as a conditional expression system for the nucleotide sequence of interest when the same cell also comprises a nucleotide sequence coding for either the wild type (tTA) or the mutant (rTA) repressor fused to the activating domain of viral protein VP16 of herpes simplex virus, placed under the control of a promoter, such as the HCMVIE1 enhancer/promoter or the MMTV-LTR. Indeed, a preferred DNA construct of the invention comprise both the polynucleotide containing the let operator sequences and the polynucleotide containing a sequence coding for the tTA or the rTA repressor.

In a specific embodiment, the conditional expression DNA construct contains the sequence encoding the mutant tetracycline repressor rTA, the expression of the polynucleotide of interest is silent in the absence of tetracycline and induced in its presence.

DNA Constructs Allowing Homologous Recombination: Replacement Vectors

A second preferred DNA construct will comprise, from 5′-end to 3′-end: (a) a first nucleotide sequence that is comprised in the PCTA-1 genomic sequence; (b) a nucleotide sequence comprising a positive selection marker, such as the marker for neomycine resistance (neo); and (c) a second nucleotide sequence that is comprised in the PCTA-1 genomic sequence, and is located on the genome downstream the first PCTA-1 nucleotide sequence (a).

In a preferred embodiment, this DNA construct also comprises a negative selection marker located upstream the nucleotide sequence (a) or downstream the nucleotide sequence (c). Preferably, the negative selection marker consists of the thymidine kinase (tk) gene (Thomas et al., 1986), the hygromycine beta gene (Te Riele et al., 1990), the hprt gene (Van der Lugt et al., 1991; Reid et al., 1990) or the Diphteria toxin A fragment (Dt-A) gene (Nada et al., 1993; Yagi et al. 1990). Preferably, the positive selection marker is located within a PCTA-1 exon sequence so as to interrupt the sequence encoding a PCTA-1 protein. These replacement vectors are described, for example, by Thomas et al.(1986; 1987), Mansour et al.(1988) and Koller et al.(1992).

The first and second nucleotide sequences (a) and (c) may be indifferently located within a PCTA-1 regulatory sequence, an intronic sequence, an exon sequence or a sequence containing both regulatory and/or intronic and/or exon sequences. The size of the nucleotide sequences (a) and (c) ranges from 1 to 50 kb, preferably from 1 to 10 kb, more preferably from 2 to 6 kb and most preferably from 2 to 4 kb.

DNA Constructs Allowing Homologous Recombination: Cre-LoxP System.

These new DNA constructs make use of the site specific recombination system of the P1 phage. The P1 phage possesses a recombinase called Cre which interacts specifically with a 34 base pairs loxP site. The loxP site is composed of two palindromic sequences of 13 bp separated by a 8 bp conserved sequence (Hoess et al., 1986). The recombination by the Cre enzyme between two loxP sites having an identical orientation leads to the deletion of the DNA fragment.

The Cre-loxP system used in combination with a homologous recombination technique has been first described by Gu et al.(1993, 1994). Briefly, a nucleotide sequence of interest to be inserted in a targeted location of the genome harbors at least two loxP sites in the same orientation and located at the respective ends of a nucleotide sequence to be excised from the recombinant genome. The excision event requires the presence of the recombinase (Cre) enzyme within the nucleus of the recombinant cell host. The recombinase enzyme may be brought at the desired time either by (a) incubating the recombinant cell hosts in a culture medium containing this enzyme, by injecting the Cre enzyme directly into the desired cell, such as described by Araki et al.(1995), or by lipofection of the enzyme into the cells, such as described by Baubonis et al.(1993); (b) transfecting the cell host with a vector comprising the Cre coding sequence operably linked to a promoter functional in the recombinant cell host, which promoter being optionally inducible, said vector being introduced in the recombinant cell host, such as described by Gu et al.(1993) and Sauer et al.(1988); (c) introducing in the genome of the cell host a polynucleotide comprising the Cre coding sequence operably linked to a promoter functional in the recombinant cell host, which promoter is optionally inducible, and said polynucleotide being inserted in the genome of the cell host either by a random insertion event or an homologous recombination event, such as described by Gu et al.(1994).

In a specific embodiment, the vector containing the sequence to be inserted in the PCTA-1 gene by homologous recombination is constructed in such a way that selectable markers are flanked by loxP sites of the same orientation, it is possible, by treatment by the Cre enzyme, to eliminate the selectable markers while leaving the PCTA-1 sequences of interest that have been inserted by an homologous recombination event. Again, two selectable markers are needed: a positive selection marker to select for the recombination event and a negative selection marker to select for the homologous recombination event. Vectors and methods using the Cre-toxP system are described by Zou et al.(1994).

Thus, a third preferred DNA construct of the invention comprises, from 5′-end to 3′-end: (a) a first nucleotide sequence that is comprised in the PCTA-1 genomic sequence; (b) a nucleotide sequence comprising a polynucleotide encoding a positive selection marker, said nucleotide sequence comprising additionally two sequences defining a site recognized by a recombinase, such as a loxP site, the two sites being placed in the same orientation; and (c) a second nucleotide sequence that is comprised in the PCTA-1 genomic sequence, and is located on the genome downstream of the first PCTA-1 nucleotide sequence (a).

The sequences defining a site recognized by a recombinase, such as a loxP site, are preferably located within the nucleotide sequence (b) at suitable locations bordering the nucleotide sequence for which the conditional excision is sought. In one specific embodiment, two loxP sites are located at each side of the positive selection marker sequence, in order to allow its excision at a desired time after the occurrence of the homologous recombination event.

In a preferred embodiment of a method using the third DNA construct described above, the excision of the polynucleotide fragment bordered by the two sites recognized by a recombinase, preferably two loxP sites, is performed at a desired time, due to the presence within the genome of the recombinant host cell of a sequence encoding the Cre enzyme operably linked to a promoter sequence, preferably an inducible promoter, more preferably a tissue-specific promoter sequence and most preferably a promoter sequence which is both inducible and tissue-specific, such as described by Gu et al.(1994).

The presence of the Cre enzyme within the genome of the recombinant cell host may result of the breeding of two transgenic animals, the first transgenic animal bearing the PCTA-1-derived sequence of interest containing the loxP sites as described above and the second transgenic animal bearing the Cre coding sequence operably linked to a suitable promoter sequence, such as described by Gu et al.(1994).

Spatio-temporal control of the Cre enzyme expression may also be achieved with an adenovirus based vector that contains the Cre gene thus allowing infection of cells, or in vivo infection of organs, for delivery of the Cre enzyme, such as described by Anton and Graham (1995) and Kanegae et al.(1995).

The DNA constructs described above may be used to introduce a desired nucleotide sequence of the invention, preferably a PCTA-1 genomic sequence or a PCTA-1 cDNA sequence, and most preferably an altered copy of a PCTA-1 genomic or cDNA sequence, within a predetermined location of the targeted genome, leading either to the generation of an altered copy of a targeted gene (knock-out homologous recombination) or to the replacement of a copy of the targeted gene by another copy sufficiently homologous to allow an homologous recombination event to occur (knock-in homologous recombination). In a specific embodiment, the DNA constructs described above may be used to introduce a PCTA-1 genomic sequence or a PCTA-1 cDNA sequence comprising at least one biallelic marker of the present invention, preferably at least one biallelic marker selected from the group consisting of A1 to A125 and the complements thereof.

Oligonucleotide Probes and Primers

Polynucleotides derived from the PCTA-1 gene are useful in order to detect the presence of at least a copy of a nucleotide sequence of SEQ ID No 1, or a fragment, complement, or variant thereof in a test sample.

Particularly preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1: 1-70715, 70795-82207, 82297-83612, 83824-85297, 85418-86388, 86446-87495, 87523-88294, 88384-89483, 89650-92748, 97156-98309, 98476-99329, 99491-100026, 100212-100281, 100396-100538, 100682-100833, 100995-101920, 102087-102970, 103264-103724, and 103753-106746. Additional preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide G at positions 70728, 87860, 88297, 94432, and 95340 of SEQ ID No 1; a nucleotide A at positions 82218, 83644, 83808, 87787, 87806, 94218, and 97144 of SEQ ID No 1; a nucleotide C at positions 87902, 88215, 88283, 92760, 93726, and 94422 of SEQ ID No 1; and a nucleotide T at positions 93903, and 94170 of SEQ ID No 1. Other preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide G at positions 86435, 93592, 93680, 93681, 93682, 93728, 93761, and 95445 of SEQ ID No 1; a nucleotide A at positions 86434, 88355, 93240, 93471, and 93747of SEQ ID No l; a nucleotide C at positions 93683, 95126, and 95444 of SEQ ID No 1; and a nucleotide T at positions 94154, and 94430 of SEQ ID No 1. Other preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 1: 92975-92977, 93711-93715, 94151-94153, 94240-94243, 94770-94773, 94804-94808, 95121-95122, 95129-95135, 95148-95153, 95154-95159, 95173-95178, 95367-95374, 95410-95413, 95418-95420, 95430-95436, 95533-95535, and 95677-95677.

Another object of the invention is a purified, isolated, or recombinant polynucleotide comprising the nucleotide sequence of SEQ ID No 2, complementary sequences thereto, as well as allelic variants, and fragments thereof. Particularly preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 1-162 of SEQ ID No 2. Additional preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527, 2471, and 5397 of SEQ ID No 2; a nucleotide C at positions 1013, 1979, and 2675 of SEQ ID No 2; a nucleotide G at positions 176, 749, 2685, 3593 of SEQ ID No 2; and a nucleotide T at positions 2156, and 2423 of SEQ ID No 2. Particularly preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708, 807, 1493, 1724, and 2000; a nucleotide C at positions 1936, 3379, and 3697; a nucleotide G at positions 709, 1845, 1933, 1934, 1935, 1981, 2014, and 3698; and a nucleotide T at positions 2407, and 2683 of SEQ ID No 2. Other preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 2: 1229-1231, 1964-1968, 24042406, 2493-2496, 3023-3026, 3057-3061, 3374-3375, 3382-3388, 3401-3406, 3407-3412, 3426-3431, 3620-3627, 3663-3666, 3671-3673, 3683-3689, 3786-3788 and 3930-3932.

A further object of the invention is a purified, isolated, or recombinant polynucleotide comprising the nucleotide sequence of SEQ ID No 3, complementary sequences thereto, as well as allelic variants, and fragments thereof. Particularly preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 3: 1-162 and 747-872. Additional preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527, 2597, and 5523 of SEQ ID No 3; a nucleotide C at positions 1139, 2105, and 2801 of SEQ ID No 3; a nucleotide G at positions 176, 875, 2811, 3719 of SEQ ID No 3; and a nucleotide T at positions 2282, and 2549 of SEQ ID No 3. Additional preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708, 807, 1619, 1850, and 2126; a nucleotide C at positions 2062, 3505, and 3823; a nucleotide G at positions 709, 1971, 2059, 2060, 2061, 2107, 2140, and 3824; and a nucleotide Tat positions 2533, and 2809 of SEQ ID No 3. Other preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 3: 1355-1357, 1892-1894, 2090-2094, 2530-2532, 2619-2622, 3149-3152, 3183-3187, 3500-3501, 3508-3514, 3527-3532, 3533-3538, 3552-3557, 3746-3749, 3789-3792, 3797-3799, 3809-3815, 3912-3914 and 4056-4058.

An additional object of the invention is a purified, isolated, or recombinant polynucleotide comprising the nucleotide sequence of SEQ ID No 4, complementary sequences thereto, as well as allelic variants, and fragments thereof. Particularly preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 1-162 of SEQ ID No 4. Additional preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527 and 2460 of SEQ ID No 4; a nucleotide C at position 1013 of SEQ ID No 4; and a nucleotide G at positions 176 and 749 of SEQ ID No 4. Additionally preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708 and 807 and a nucleotide G at position 709 of SEQ ID No 4. Other preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises the pairs of nucleotide positions 1136-1137 of SEQ ID No 4.

One more object of the invention is a purified, isolated, or recombinant polynucleotide comprising the nucleotide sequence of SEQ ID No 8, complementary sequences thereto, as well as allelic variants, and fragments thereof. Particularly preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 8 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 8: 1-500, 501-1000, 1001-1500, and 1501-1738.

Thus, the invention also relates to nucleic acid probes characterized in that they hybridize specifically, under the stringent hybridization conditions defined above, with a nucleic acid selected from the group consisting of the nucleotide sequences:

a) 1-70715, 70795-82207, 82297-83612, 83824-85297, 85418-86388, 86446-87495, 87523-88294, 88384-89483, 89650-92748, 97156-98309, 98476-99329, 99491-100026, 100212-100281, 100396-100538, 100682-100833, 100995-101920, 102087-102970, 103264-103724, and 103753-106746 of SEQ ID No 1 or a variant thereof or a sequence complementary thereto;

b) 1-162 of SEQ ID No 2 or a variant thereof or a sequence complementary thereto;

c) 1-162 and 747-872 of SEQ ID No 3 or a variant thereof or a sequence complementary thereto;

d) 1-162 of SEQ ID No 4 or a variant thereof or a sequence complementary thereto;

e) SEQ ID No 8 or a variant thereof or a sequence complementary thereto.

In a preferred embodiment, the oligonucleotides of the invention can hybridize with at least a portion of an intron or of the regulatory sequences of the PCTA-1 gene. Particularly preferred oligonucleotides of the invention hybridize with a sequence comprised in an intron or in the regulatory sequences of the PCTA-1 gene. In an other preferred embodiment, the oligonucleotides of the invention can hybridize with at least a portion of an exon selected from the group of exons 0, 1, 6bis, 9, and 9ter.

The present invention also concerns oligonucleotides and groups of oligonucleotides for the detection of alleles of biallelic markers of the PCTA-1 gene, preferably those associated with cancer, preferably with prostate cancer, with an early onset of prostate cancer, with a susceptibility to prostate cancer, with the level of aggressiveness of prostate cancer tumors, with a modified or forthcoming expression of the PCTA-1 gene, with a modified or forthcoming production of the PCTA-1 protein, or with the production of a modified PCTA-1 protein. These oligonucleotides are characterized in that they can hybridize with a PCTA-1 gene, preferably with a polymorphic PCTA-1 gene and more preferably with a region of a PCTA-1 gene comprising a polymorphic site containing a specific allele associated with prostate cancer, with the level of aggressiveness of prostate cancer tumors or with modifications in the regulation of expression of the PCTA-1 gene. These oligonucleotides are useful either as primers for use in various processes such as DNA amplification and microsequencing or as probes for DNA recognition in hybridization analyses.

Therefore, another preferred embodiment of a probe according to the invention consists of a nucleic acid comprising a biallelic marker selected from the group consisting of A1 to A125 or the complements thereof, for which the respective locations in the sequence listing are provided in Table 2. In some embodiments, the oligonucleotides comprise the polymorphic base of a sequence selected from P1 to P125, and the complementary sequences thereto. In other embodiments, the oligonucleotides have a 3′ terminus immediately adjacent to a polymorphic base in the PCTA-1 gene, such as a polymorphic base comprised in one of the sequences P1 to P125, and the complementary sequence thereto. In other embodiments, the oligonucleotide is capable of discriminating between different alleles of a biallelic marker in the PCTA-1 gene, including the biallelic markers A1 to A125 and the complements thereof.

In one embodiment the invention encompasses isolated, purified, and recombinant polynucleotides consisting of, or consisting essentially of a contiguous span of 8 to 50 nucleotides of any one of SEQ ID Nos 1, 2, 3, 4 and the complement thereof, wherein said span includes a PCTA-1-related biallelic marker in said sequence; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, and A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94 to A101, A108, A111 to A113, A115 to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said contiguous span is 18 to 47 nucleotides in length and said biallelic marker is within 4 nucleotides of the center of said polynucleotide; optionally, wherein said polynucleotide consists of said contiguous span and said contiguous span is 25 nucleotides in length and said biallelic marker is at the center of said polynucleotide; optionally, wherein said polynucleotide consists of said contiguous span and said contiguous span is 47 nucleotides in length and said biallelic marker is at the center of said polynucleotide; optionally, wherein the 3′ end of said contiguous span is present at the 3′ end of said polynucleotide; and optionally, wherein the 3′ end of said contiguous span is located at the 3′ end of said polynucleotide and said biallelic marker is present at the 3′ end of said polynucleotide. In a preferred embodiment, said probes comprises, consists of, or consists essentially of a sequence selected from the following sequences: P1 to P125 and the complementary sequences thereto.

In another embodiment the invention encompasses isolated, purified and recombinant polynucleotides comprising, consisting of, or consisting essentially of a contiguous span of 8 to 50 nucleotides of SEQ ID Nos 1, 2, 3, 4, or the complements thereof, wherein the 3′ end of said contiguous span is located at the 3′ end of said polynucleotide, and wherein the 3′ end of said polynucleotide is located within 20 nucleotides upstream of a PCTA-1-related biallelic marker in said sequence; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, and A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, and A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92A94to A101, A108, A111 to A113, A115to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein the 3′ end of said polynucleotide is located 1 nucleotide upstream of said PCTA-1-related biallelic marker in said sequence; and optionally, wherein said polynucleotide consists essentially of a sequence selected from the following sequences: D1 to D125 and E1 to E125.

In a further embodiment, the invention encompasses isolated, purified, or recombinant polynucleotides comprising, consisting of, or consisting essentially of a sequence selected from the following sequences: B1 to B47 and C1 to C47.

In an additional embodiment, the invention encompasses polynucleotides for use in hybridization assays, sequencing assays, and enzyme-based mismatch detection assays for determining the identity of the nucleotide at a PCTA-1-related biallelic marker in SEQ ID Nos 1, 2, 3, 4, or the complements thereof, as well as polynucleotides for use in amplifying segments of nucleotides comprising a PCTA-1-related biallelic marker in SEQ ID Nos 1, 2, 3, 4, or the complements thereof; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, Al107, A118, and A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, and A122, and complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55A56A59A92A94to A101, A108A111 to A113A115 to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith.

The formation of stable hybrids depends on the melting temperature (Tm) of the DNA. The Tm depends on the length of the primer or probe, the ionic strength of the solution and the G+C content. The higher the G+C content of the primer or probe, the higher is the melting temperature because G:C pairs are held by three H bonds whereas A:T pairs have only two. The GC content in the probes of the invention usually ranges between 10 and 75%, preferably between 35 and 60%, and more preferably between 40 and 55%.

A probe or a primer according to the invention has between 8 and 1000 nucleotides in length, or is specified to be at least 12, 15, 18, 20, 25, 35, 40, 50, 60, 70, 80, 100, 250, 500 or 1000 nucleotides in length. More particularly, the length of these probes and primers can range from 8, 10, 15, 20, or 30 to 100 nucleotides, preferably from 10 to 50, more preferably from 15 to 30 nucleotides. Shorter probes and primers tend to lack specificity for a target nucleic acid sequence and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer probes and primers are expensive to produce and can sometimes self-hybridize to form hairpin structures. The appropriate length for primers and probes under a particular set of assay conditions may be empirically determined by one of skill in the art. A preferred probe or primer consists of a nucleic acid comprising a polynucleotide selected from the group of the nucleotide sequences of P1 to P125 and the complementary sequence thereto, B1 to B47, C1 to C47, D1 to D125, E1 to E125, for which the respective locations in the sequence listing are provided in Tables 1, 2, 3 and 4.

The primers and probes can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphodiester method of Narang et al.(1979), the phosphodiester method of Brown et al.(1979), the diethylphosphoramidite method of Beaucage et al.(1981) and the solid support method described in EP0 707 592, the disclosure of which is incorporated herein by reference in its entirety.

Detection probes are generally nucleic acid sequences or uncharged nucleic acid analogs such as, for example peptide nucleic acids which are disclosed in International Patent Application WO 92/20702, morpholino analogs which are described in U.S. Pat. Nos. 5,185,444; 5,034,506and 5,142,047, the disclosures of which are incorporated herein by reference in their entireties. The probe may have to be rendered “non-extendable” in that additional dNTPs cannot be added to the probe. In and of themselves analogs usually are non-extendable and nucleic acid probes can be rendered non-extendable by modifying the 3′ end of the probe such that the hydroxyl group is no longer capable of participating in elongation. For example, the 3′ end of the probe can be functionalized with the capture or detection label to thereby consume or otherwise block the hydroxyl group. Alternatively, the 3′ hydroxyl group simply can be cleaved, replaced or modified, U.S. patent application Ser. No. 07/049,061 filed Apr. 19, 1993 describes modifications, which can be used to render a probe non-extendable.

Any of the polynucleotides of the present invention can be labeled, if desired, by incorporating any label known in the art to be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive substances (including, 32 p, 35 S, 3 H, 125 I), fluorescent dyes (including, 5-bromodesoxyuridin, fluorescein, acetylaminofluorene, digoxigenin) or biotin. Preferably, polynucleotides are labeled at their 3′ and 5′ ends. Examples of non-radioactive labeling of nucleic acid fragments are described in the French patent No. FR-7810975 or by Urdea et al (1988) or Sanchez-Pescador et al (1988), the disclosures of which are incorporated herein by reference in their entireties. In addition, the probes according to the present invention may have structural characteristics such that they allow the signal amplification, such structural characteristics being, for example, branched DNA probes as those described by Urdea et al. in 1991 or in the European patent No. EP0 225 807 (Chiron), the disclosures of which are incorporated herein by reference in their entireties.

A label can also be used to capture the primer, so as to facilitate the immobilization of either the primer or a primer extension product, such as amplified DNA, on a solid support. A capture label is attached to the primers or probes and can be a specific binding member which forms a binding pair with the solid's phase reagent's specific binding member (e.g. biotin and streptavidin). Therefore depending upon the type of label carried by a polynucleotide or a probe, it may be employed to capture or to detect the target DNA. Further, it will be understood that the polynucleotides, primers or probes provided herein, may, themselves, serve as the capture label. For example, in the case where a solid phase reagent's binding member is a nucleic acid sequence, it may be selected such that it binds a complementary portion of a primer or probe to thereby immobilize the primer or probe to the solid phase. In cases where a polynucleotide probe itself serves as the binding member, those skilled in the art will recognize that the probe will contain a sequence or “tail” that is not complementary to the target. In the case where a polynucleotide primer itself serves as the capture label, at least a portion of the primer will be free to hybridize with a nucleic acid on a solid phase. DNA Labeling techniques are well known to the skilled technician.

The probes of the present invention are useful for a number of purposes. They can be notably used in Southern hybridization to genomic DNA. The probes can also be used to detect PCR amplification products. They may also be used to detect mismatches in the PCTA-1 gene or mRNA using other techniques.

Any of the polynucleotides, primers and probes of the present invention can be conveniently immobilized on a solid support. Solid supports are known to those skilled in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes and others. The solid support is not critical and can be selected by one skilled in the art. Thus, latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and duracytes are all suitable examples. Suitable methods for immobilizing nucleic acids on solid phases include ionic, hydrophobic, covalent interactions and the like. A solid support, as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid support can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent. The additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent. As yet another alternative, the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid support and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a solid support material before the performance of the assay or during the performance of the assay. The solid phase thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, duracytes® and other configurations known to those of ordinary skill in the art. The polynucleotides of the invention can be attached to or immobilized on a solid support individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the invention to a single solid support. In addition, polynucleotides other than those of the invention may be attached to the same solid support as one or more polynucleotides of the invention.

Consequently, the invention also deals with a method for detecting the presence of a nucleic acid comprising a nucleotide sequence selected from a group consisting of SEQ ID Nos 1, 2, 3, 4, 8, a fragment or a variant thereof and a complementary sequence thereto in a sample, said method comprising the following steps of:

a) bringing into contact a nucleic acid probe or a plurality of nucleic acid probes which can hybridize with a nucleotide sequence included in a nucleic acid selected form the group consisting of the nucleotide sequences of SEQ ID Nos 1, 2, 3, 4, 8, a fragment or a variant thereof and a complementary sequence thereto and the sample to be assayed; and

b) detecting the hybrid complex formed between the probe and a nucleic acid in the sample.

The invention further concerns a kit for detecting the presence of a nucleic acid comprising a nucleotide sequence selected from a group consisting of SEQ ID Nos 1, 2, 3, 4, 8, a fragment or a variant thereof and a complementary sequence thereto in a sample, said kit comprising:

a) a nucleic acid probe or a plurality of nucleic acid probes which can hybridize with a nucleotide sequence included in a nucleic acid selected form the group consisting of the nucleotide sequences of SEQ ID Nos 1, 2, 3, 4, 8, a fragment or a variant thereof and a complementary sequence thereto; and

b) optionally, the reagents necessary for performing the hybridization reaction.

In a first preferred embodiment of this detection method and kit, said nucleic acid probe or the plurality of nucleic acid probes are labeled with a detectable molecule. In a second preferred embodiment of said method and kit, said nucleic acid probe or the plurality of nucleic acid probes has been immobilized on a substrate. In a third preferred embodiment, the nucleic acid probe or the plurality of nucleic acid probes comprise either a sequence which is selected from the group consisting of the nucleotide sequences of P1 to P125 and the complementary sequence thereto, B1 to B47, C1 to C47, D1 to D125, E1 to E125 or a biallelic marker selected from the group consisting of A1 to A125 and the complements thereto.

Oligonucleotide Arrays

A substrate comprising a plurality of oligonucleotide primers or probes of the invention may be used either for detecting or amplifying targeted sequences in the PCTA-1 gene and may also be used for detecting mutations in the coding or in the non-coding sequences of the PCTA-1 gene.

Any polynucleotide provided herein may be attached in overlapping areas or at random locations on the solid support. Alternatively the polynucleotides of the invention may be attached in an ordered array wherein each polynucleotide is attached to a distinct region of the solid support which does not overlap with the attachment site of any other polynucleotide. Preferably, such an ordered array of polynucleotides is designed to be “addressable” where the distinct locations are recorded and can be accessed as part of an assay procedure. Addressable polynucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. The knowledge of the precise location of each polynucleotides location makes these “addressable” arrays particularly useful in hybridization assays. Any addressable array technology known in the art can be employed with the polynucleotides of the invention. One particular embodiment of these polynucleotide arrays is known as the Genechips™, and has been generally described in U.S. Pat. No. 5,143,854; PCT publications WO 90/15070 and 92/10092, the disclosures of which are incorporated herein by reference in their entireties. These arrays may generally be produced using mechanical synthesis methods or light directed synthesis methods which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis (Fodor et al., 1991). The immobilization of arrays of oligonucleotides on solid supports has been rendered possible by the development of a technology generally identified as “Very Large Scale Immobilized Polymer Synthesis” (VLSIPS™) in which, typically, probes are immobilized in a high density array on a solid surface of a chip. Examples of VLSIPSυ technologies are provided in U.S. Pat. Nos. 5,143,854; and 5,412,087 and in PCT Publications WO 90/15070, WO 92/10092 and WO 95/11995, the disclosures of which are incorporated herein by reference in their entireties, which describe methods for forming oligonucleotide arrays through techniques such as light-directed synthesis techniques. In designing strategies aimed at providing arrays of nucleotides immobilized on solid supports, further presentation strategies were developed to order and display the oligonucleotide arrays on the chips in an attempt to maximize hybridization patterns and sequence information. Examples of such presentation strategies are disclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 and WO 97/31256.

In another embodiment of the oligonucleotide arrays of the invention, an oligonucleotide probe matrix may advantageously be used to detect mutations occurring in the PCTA-1 gene and preferably in its regulatory region. For this particular purpose, probes are specifically designed to have a nucleotide sequence allowing their hybridization to the genes that carry known mutations (either by deletion, insertion or substitution of one or several nucleotides). By known mutations, it is meant, mutations on the PCTA-1 gene that have been identified according, for example to the technique used by Huang et al.(1996) or Samson et al.(1996).

Another technique that is used to detect mutations in the PCTA-1 gene is the use of a high-density DNA array. Each oligonucleotide probe constituting a unit element of the high density DNA array is designed to match a specific subsequence of the PCTA-1 genomic DNA or cDNA. Thus, an array consisting of oligonucleotides complementary to subsequences of the target gene sequence is used to determine the identity of the target sequence with the wild gene sequence, measure its amount, and detect differences between the target sequence and the reference wild gene sequence of the PCTA-1 gene. In one such design, termed 4L tiled array, is implemented a set of four probes (A, C, G, T), preferably 15-nucleotide oligomers. In each set of four probes, the perfect complement will hybridize more strongly than mismatched probes. Consequently, a nucleic acid target of length L is scanned for mutations with a tiled array containing 4L probes, the whole probe set containing all the possible mutations in the known wild reference sequence. The hybridization signals of the 15-mer probe set tiled array are perturbed by a single base change in the target sequence. As a consequence, there is a characteristic loss of signal or a “footprint” for the probes flanking a mutation position. This technique was described by Chee et al. in 1996.

Consequently, the invention concerns an array of nucleic acid molecules comprising at least one polynucleotide described above as probes and primers. Preferably, the invention concerns an array of nucleic acid comprising at least two polynucleotides described above as probes and primers.

A further object of the invention consists of an array of nucleic acid sequences comprising either at least one of the sequences selected from the group consisting of P1 to P125, B1 to B47, C1 to C47, D1 to D 125, E1 to E125, the sequences complementary thereto, a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 30, or 40 consecutive nucleotides thereof, and at least one sequence comprising a biallelic marker selected from the group consisting of A1 to A125 and the complements thereto.

The invention also pertains to an array of nucleic acid sequences comprising either at least two of the sequences selected from the group consisting of P1 to P125, B1 to B47, C1 to C47, D1 to D 125, E1 to E125, the sequences complementary thereto, a fragment thereof of at least 8 consecutive nucleotides thereof, and at least two sequences comprising a biallelic marker selected from the group consisting of A1 to A125 and the complements thereof.

PCTA-1 Proteins and Polypeptide Fragments thereof

The term “PCTA-1 polypeptides” is used herein to embrace all of the proteins and polypeptides of the present invention. Also forming part of the invention are polypeptides encoded by the polynucleotides of the invention, as well as fusion polypeptides comprising such polypeptides.

The invention embodies PCTA-1 proteins from humans, including isolated or purified PCTA-1 proteins consisting, consisting essentially, or comprising the sequence of SEQ ID No 5. It should be noted the PCTA-1 proteins of the invention are based on the naturally-occurring variant of the amino acid sequence of human PCTA-1, wherein the valine residue of amino acid position 170 has been replaced with a serine residue and the glutamine residue of amino acid position 203 has been replaced with a lysine residue. This variant protein and the fragments thereof which contain a serine at the amino acid position 170 and a lysine at the amino acid position 203 of SEQ ID No 5 are collectively referred to herein as “170-Ser 203-Lys variants.” In another embodiment, the present invention concerns a purified and/or isolated nucleic acid encoding the PCTA-1 protein of SEQ ID No 5 or variant or fragment thereof.

The invention also concerns a purified and/or isolated PCTA-1 protein comprising a sequence selected from the group consisting of SEQ ID Nos 6, 7 and variants and functional fragments thereof. In another embodiment, the present invention concerns a purified and/or isolated nucleic acid encoding the PCTA-1 protein comprising a sequence selected from the group consisting of SEQ ID Nos 6, 7 or a variant or a fragment thereof.

The invention also encompasses the amino acid sequence of a murine PCTA-1 protein, such as that of SEQ ID No 9, fragments and variants thereof. The invention also concerns a nucleotide sequence encoding the murine PCTA-1 protein of SEQ ID No 9, sequences complementary thereto and fragments and variants thereof.

The invention also relates to modified human and mouse PCTA-1 proteins and to fragments and variants thereof. The term “modified PCTA-1 protein” is intended to designate a PCTA-1 protein which, when compared to a native PCTA-1 protein of SEQ ID No 5, 6, or 7, bears at least one amino acid substitution, deletion or addition. More particularly, preferred modified PCTA-1 proteins include the proteins bearing at least one of the following amino acid substitutions:

a substitution from F to Y at position 18, a substitution from R to C at position 35, a substitution from V to M at position 55 and a substitution from S to R at position 183 in SEQ ID No 5;

a substitution from F to Y at position 18, a substitution from R to C at position 35, a substitution from V to M at position 55, a substitution from D to Y at position 204 and a substitution from S to R at position 225 in SEQ ID No 6; and

a substitution from F to Y at position 18, a substitution from R to C at position 35, a substitution from V to M at position 55 and a substitution from S to R at position 183 in SEQ ID No 7.

Modified proteins bearing two or more of such substitutions also fall within the scope of the present invention. Other preferred embodiments include regions of the modified PCTA-1 proteins of the invention, and particularly those regions bearing at least one of the substitutions described above. Particularly preferred regions are those possessing antigenic properties and which can be used in vaccine agents or to raise antibodies against the PCTA-1 protein, and which most preferably comprise at least one of the particular substitutions referred to above.

The term “modified PCTA-1 protein” also designates a truncated PCTA-1 protein consisting of the amino acid sequence 1-211 of SEQ ID No 7.

The present invention embodies isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 5, wherein said contiguous span includes:

a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 5; and/or

at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 5.

The present invention embodies isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 6, wherein said contiguous span includes:

a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 245 in SEQ ID No 6; and/or

at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 225 in SEQ ID No 6; and/or

at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exon 6bis, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 183-224 of the SEQ ID No 6.

The present invention embodies isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 7, wherein said contiguous span includes:

a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 7; and/or

at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 7; and/or

at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exons 9bis and 9ter, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 313-368 of the SEQ ID No 7.

The invention also concerns the truncated PCTA-1 protein consisting essentially of or consisting of the amino acid positions 1-211 of SEQ ID No 7.

In other preferred embodiments the contiguous stretch of amino acids from SEQ ID Nos 5, 6, 7 comprises the site of a mutation or functional mutation, including a deletion, addition, swap or truncation of the amino acids in the PCTA-1 protein sequence.

The present invention embodies isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 9.

PCTA-1 proteins are preferably isolated from human or mammalian tissue samples or expressed from human or mammalian genes. The PCTA-1 polypeptides of the invention can be made using routine expression methods known in the art. The polynucleotide encoding the desired polypeptide, is ligated into an expression vector suitable for any convenient host. Both eukaryotic and prokaryotic host systems is used in forming recombinant polypeptides, and a summary of some of the more common systems. The polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use. Purification is by any technique known in the art, for example, differential extraction, salt fractionation, chromatography, centrifugation, and the like. See, for example, Methods in Enzymology for a variety of methods for purifying proteins.

In addition, shorter protein fragments is produced by chemical synthesis. Alternatively the proteins of the invention is extracted from cells or tissues of humans or non-human animals. Methods for purifying proteins are known in the art, and include the use of detergents or chaotropic agents to disrupt particles followed by differential extraction and separation of the polypeptides by ion exchange chromatography, affinity chromatography, sedimentation according to density, and gel electrophoresis.

Any PCTA-1 cDNA, including SEQ ID Nos 2, 3, 4, 8, is used to express PCTA-1 proteins and polypeptides. The nucleic acid encoding a PCTA-1 protein or polypeptide to be expressed is operably linked to a promoter in an expression vector using conventional cloning technology. The PCTA-1 insert in the expression vector may comprise the full coding sequence for a PCTA-1 protein or a fragment thereof. For example, the PCTA-1 derived insert may encode a polypeptide as described above.

The expression vector is any of the mammalian, yeast, insect or bacterial expression systems known in the art. Commercially available vectors and expression systems are available from a variety of suppliers including Genetics Institute (Cambridge, Mass.), Stratagene (La Jolla, Calif.), Promega (Madison, Wis.), and Invitrogen (San Diego, Calif.). If desired, to enhance expression and facilitate proper protein folding, the codon context and codon pairing of the sequence is optimized for the particular expression organism in which the expression vector is introduced, as explained by Hatfield, et al., U.S. Pat. No. 5,082,767, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the entire coding sequence of a PCTA-1 cDNA through the poly A signal of the cDNA are operably linked to a promoter in the expression vector. Alternatively, if the nucleic acid encoding a fragment of the PCTA-1 protein lacks a methionine to serve as the initiation site, an initiating methionine can be introduced next to the first codon of the nucleic acid using conventional techniques. Similarly, if the insert from a PCTA-1 cDNA lacks a poly A signal, this sequence can be added to the construct by, for example, splicing out the Poly A signal from pSG5 (Stratagene) using BglI and SalI restriction endonuclease enzymes and incorporating it into the mammalian expression vector pXT1 (Stratagene). pXT1 contains the LTRs and a portion of the gag gene from Moloney Murine Leukemia Virus. The position of the LTRs in the construct allow efficient stable transfection. The vector includes the Herpes Simplex Thymidine Kinase promoter and the selectable neomycin gene. The nucleic acid encoding a PCTA-1 protein or a fragment thereof is obtained by PCR from a bacterial vector containing a PCTA-1 cDNA selected from the group consisting of SEQ ID Nos 2, 3, 4, and 8 using oligonucleotide primers complementary to the PCTA-1 cDNA or fragment thereof and containing restriction endonuclease sequences for Pst I incorporated into the 5′ primer and BglII at the 5′ end of the corresponding cDNA3′ primer, taking care to ensure that the sequence encoding the PCTA-1 protein or a fragment thereof is positioned properly with respect to the poly A signal. The purified fragment obtained from the resulting PCR reaction is digested with PstI, blunt ended with an exonuclease, digested with BglII, purified and ligated to pXT1, now containing a poly A signal and digested with BglII.

The ligated product is transfected into mouse NIH 3T3 cells using Lipofectin (Life Technologies, Inc., Grand Island, N.Y.) under conditions outlined in the product specification. Positive transfectants are selected after growing the transfected cells in 600 ug/ml G418 (Sigma, St. Louis, Mo.).

Alternatively, the nucleic acids encoding the PCTA-1 protein or a fragment thereof is cloned into pED6dpc2 (Genetics Institute, Cambridge, Mass.). The resulting pED6dpc2 constructs is transfected into a suitable host cell, such as COS 1 cells. Methotrexate resistant cells are selected and expanded.

The above procedures may also be used to express a mutant PCTA-1 protein responsible for a detectable phenotype or a fragment thereof.

The expressed proteins is purified using conventional purification techniques such as ammonium sulfate precipitation or chromatographic separation based on size or charge. The protein encoded by the nucleic acid insert may also be purified using standard immunochromatography techniques. In such procedures, a solution containing the expressed PCTA-1 protein or fragment thereof, such as a cell extract, is applied to a column having antibodies against the PCTA-1 protein or fragment thereof is attached to the chromatography matrix. The expressed protein is allowed to bind the immunochromatography column. Thereafter, the column is washed to remove non-specifically bound proteins. The specifically bound expressed protein is then released from the column and recovered using standard techniques.

To confirm expression of a PCTA-1 protein or a fragment thereof, the proteins expressed from host cells containing an expression vector containing an insert encoding a PCTA-1 protein or a fragment thereof can be compared to the proteins expressed in host cells containing the expression vector without an insert. The presence of a band in samples from cells containing the expression vector with an insert which is absent in samples from cells containing the expression vector without an insert indicates that the PCTA-1 protein or a fragment thereof is being expressed. Generally,the bandwill have the mobility expected for the PCTA-1 protein or fragment thereof. However, the band may have a mobility different than that expected as a result of modifications such as glycosylation, ubiquitination, or enzymatic cleavage.

Antibodies capable of specifically recognizing the expressed PCTA-1 protein or a fragment thereof are described below.

If antibody production is not possible, the nucleic acids encoding the PCTA-1 protein or a fragment thereof is incorporated into expression vectors designed for use in purification schemes employing chimeric polypeptides. In such strategies the nucleic acid encoding the PCTA-1 protein or a fragment thereof is inserted in frame with the gene encoding the other half of the chimera. The other half of the chimera is β-globin or a nickel binding polypeptide encoding sequence. A chromatography matrix having antibody to β-globin or nickel attached thereto is then used to purify the chimeric protein. Protease cleavage sites is engineered between the β-globin gene or the nickel binding polypeptide and the PCTA-1 protein or fragment thereof. Thus, the two polypeptides of the chimera is separated from one another by protease digestion.

One useful expression vector for generating β-globin chimeric proteins is pSG5 (Stratagene), which encodes rabbit β-globin. Intron II of the rabbit β-globin gene facilitates splicing of the expressed transcript, and the polyadenylation signal incorporated into the construct increases the level of expression. These techniques are well known to those skilled in the art of molecular biology. Standard methods are published in methods texts such as Davis et al., (1986) and many of the methods are available from Stratagene, Life Technologies, Inc., or Promega. Polypeptide may additionally be produced from the construct using in vitro translation systems such as the In vitro Express™ Translation Kit (Stratagene).

Antibodies that Bind PCTA-1 Polypeptides of the Invention

Any PCTA-1 polypeptide or whole protein may be used to generate antibodies capable of specifically binding to an expressed PCTA-1 protein or fragments thereof as described.

One antibody composition of the invention is capable of specifically binding or specifically bind to the 170-Ser 203-Lys variant of the PCTA-1 protein of SEQ ID No 5. An other antibody composition of the invention is capable of specifically binding or specifically bind to the PCTA-1 protein selected from the group consisting of amino acid sequences of SEQ ID Nos 6, 7, 9. For an antibody composition to specifically bind to a first variant of PCTA-1, it must demonstrate at least a 5%, 10%, 15%, 20%, 25%, 50%, or 100% greater binding affinity for a full length first variant of the PCTA-1 protein than for a full length second variant of the PCTA-1 protein in an ELISA, RIA, or other antibody-based binding assay.

In a preferred embodiment, the invention concerns antibody compositions, either polyclonal or monoclonal, capable of selectively binding, or selectively bind to an epitope-containing any one of the following polypeptides:

a) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 5, wherein said epitope comprises:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 5; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 5;

b) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 6, wherein said epitope comprises:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 245 in SEQ ID No 6; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 225 in SEQ ID No 6; and/or

iii) at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exon 6bis, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 183-224 of the SEQ ID No6;

c) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 7, wherein said epitope comprises:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 7; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 7; and/or

iii) at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exons 9bis and 9ter, more particularly at least 1, 2, 3, 5or 10 of the amino acid positions 313-368 of the SEQ ID No 7; and

d) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 9.

The invention also concerns a purified or isolated antibody capable of specifically binding to a mutated PCTA-1 protein or to a fragment or variant thereof comprising an epitope of the mutated PCTA-1 protein. In another preferred embodiment, the present invention concerns an antibody capable of binding to a polypeptide comprising at least 10 consecutive amino acids of a PCTA-1 protein and including at least one of the amino acids which can be encoded by the trait causing mutations.

In a preferred embodiment, the invention concerns the use of any one of the following polypeptides in the manufacture of antibodies:

a) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 5, wherein said contigous span comprises:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 5; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 5;

b) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 6, wherein said contigous span comprises:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 245 in SEQ ID No 6; and/or

i) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 225 in SEQ ID No 6; and/or

ii) at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exon 6bis, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 183-224 of the SEQ ID No 6;

c) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 7, wherein said contigous span comprises:

) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 7; and/or

i) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 7; and/or

ii) at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exons 9bis and 9ter, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 313-368 of the SEQ ID No 7; and

d) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 9.

Non-human animals or mammals, whether wild-type or transgenic, which express a different species of PCTA-1 than the one to which antibody binding is desired, and animals which do not express PCTA-1 (i.e. a PCTA-1 knock out animal as described in herein) are particularly useful for preparing antibodies. PCTA-1 knock out animals will recognize all or most of the exposed regions of a PCTA-1 protein as foreign antigens, and therefore produce antibodies with a wider array of PCTA-1 epitopes. Moreover, smaller polypeptides with only 10 to 30 amino acids may be useful in obtaining specific binding to any one of the PCTA-1 proteins. In addition, the humoral immune system of animals which produce a species of PCTA-1 that resembles the antigenic sequence will preferentially recognize the differences between the animal's native PCTA-1 species and the antigen sequence, and produce antibodies to these unique sites in the antigen sequence. Such a technique will be particularly useful in obtaining antibodies that specifically bind to any one of the PCTA-1 proteins.

Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample. The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.

The antibodies of the invention may be labeled by any one of the radioactive, fluorescent or enzymatic labels known in the art.

Consequently, the invention is also directed to a method for detecting specifically the presence of a PCTA-1 polypeptide according to the invention in a biological sample, said method comprising the following steps:

a) bringing into contact the biological sample with a polyclonal or monoclonal antibody that specifically binds a PCTA-1 polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID Nos 5, 6, 7, 9, or to a peptide fragment or variant thereof; and

b) detecting the antigen-antibody complex formed.

The invention also concerns a diagnostic kit for detecting in vitro the presence of a PCTA-1 polypeptide according to the present invention in a biological sample, wherein said kit comprises:

a) a polyclonal or monoclonal antibody that specifically binds a PCTA-1 polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID Nos 5, 6, 7, 9, or to a peptide fragment or variant thereof, optionally labeled;

b) a reagent allowing the detection of the antigen-antibody complexes formed, said reagent carrying optionally a label, or being able to be recognized itself by a labeled reagent, more particularly in the case when the above-mentioned monoclonal or polyclonal antibody is not labeled by itself.

PCTA-1-Related Biallelic Markers

Advantages of the Biallelic Markers of the Present Invention

The PCTA-1-related biallelic markers of the present invention offer a number of important advantages over other genetic markers such as RFLP (Restriction fragment length polymorphism) and VNTR (Variable Number of Tandem Repeats) markers.

The first generation of markers, were RFLPs, which are variations that modify the length of a restriction fragment. But methods used to identify and to type RFLPs are relatively wasteful of materials, effort, and time. The second generation of genetic markers were VNTRs, which can be categorized as either minisatellites or microsatellites. Minisatellites are tandemly repeated DNA sequences present in units of 5-50 repeats which are distributed along regions of the human chromosomes ranging from 0.1 to 20 kilobases in length. Since they present many possible alleles, their informative content is very high. Minisatellites are scored by performing Southern blots to identify the number of tandem repeats present in a nucleic acid sample from the individual being tested. However, there are only 10 4 potential VNTRs that can be typed by Southern blotting. Moreover, both RFLP and VNTR markers are costly and time-consuming to develop and assay in large numbers.

Single nucleotide polymorphism or biallelic markers can be used in the same manner as RFLPs and VNTRs but offer several advantages. SNP are densely spaced in the human genome and represent the most frequent type of variation. An estimated number of more than 10 7 sites are scattered along the 3×10 9 base pairs of the human genome. Therefore, SNP occur at a greater frequency and with greater uniformity than RFLP or VNTR markers which means that there is a greater probability that such a marker will be found in close proximity to a genetic locus of interest. SNP are less variable than VNTR markers but are mutationally more stable.

Also, the different forms of a characterized single nucleotide polymorphism, such as the biallelic markers of the present invention, are often easier to distinguish and can therefore be typed easily on a routine basis. Biallelic markers have single nucleotide based alleles and they have only two common alleles, which allows highly parallel detection and automated scoring. The biallelic markers of the present invention offer the possibility of rapid, high throughput genotyping of a large number of individuals.

Biallelic markers are densely spaced in the genome, sufficiently informative and can be assayed in large numbers. The combined effects of these advantages make biallelic markers extremely valuable in genetic studies. Biallelic markers can be used in linkage studies in families, in allele sharing methods, in linkage disequilibrium studies in populations, in association studies of case-control populations or of trait positive and trait negative populations. An important aspect of the present invention is that biallelic markers allow association studies to be performed to identify genes involved in complex traits. Association studies examine the frequency of marker alleles in unrelated case- and control-populations and are generally employed in the detection of polygenic or sporadic traits. Association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families (linkage studies). Biallelic markers in different genes can be screened in parallel for direct association with disease or response to a treatment. This multiple gene approach is a powerful tool for a variety of human genetic studies as it provides the necessary statistical power to examine the synergistic effect of multiple genetic factors on a particular phenotype, drug response, sporadic trait, or disease state with a complex genetic etiology.

PCTA-1-Related Biallelic Markers and Polynucleotides Related Thereto

The invention also concerns a purified and/or isolated PCTA-1-related biallelic marker located in the sequence of the PCTA-1 gene, preferably a bialielic marker comprising an allele associated with prostate cancer, with an early onset of prostate cancer, with a response to a prophylactic or therapeutic agent administered for cancer treatment, particularly prostate cancer, with the level of aggressiveness of prostate cancer tumors, with a modified or forthcoming expression of the PCTA-1 gene, with a modified or forthcoming production of the PCTA-1 protein, or with the production of a modified PCTA-1 protein. The term PCTA-1-related biallelic marker includes the biallelic markers designated A1 to A125. The invention also concerns sets of these biallelic markers.

125 biallelic markers were identified. They include 3 deletions, 6 insertions and 2 variable motifs. 40 biallelic markers, namely A45, A54 to A56, A59 to A61, A75, A76, A85, A93 to A122, were located in exonic region. 39 biallelic markers, namely A44, A46 to A53, A57 to A58, A62 to A74, A77 to A84, A86 to A92, were localized in intronic region of the PCTA-1 gene. 3 biallelic markers A123, A124 and A125 were in the 3′ regulatory region. 43 biallelic markers, namely A1 to A43, were located in the 5′ regulatory region. More particularly, 16 of them, namely A28 to A43, were in the promoter of the PCTA-1 gene.

Among the exonic biallelic markers, 6 of them change the amino acid sequence of a PCTA-1 protein. First, the biallelic marker A54 encodes either a residue tyrosine or phenylalanine. The biallelic marker A56 encodes either a residue cysteine or arginine. The marker A60 encodes either a residue valine or methionine. The marker A75 encodes either a residue aspartic acid or tyrosine. The marker A76 encodes either a leucine residue or a STOP. Finally, the biallelic marker A85 encodes either a residue serine or arginine.

The invention also relates to a purified and/or isolated nucleotide sequence comprising a polymorphic base of a PCTA-1-related biallelic marker, preferably of a biallelic marker selected from the group consisting of A1 to A125, and the complements thereof. The sequence has between 8 and 1000 nucleotides in length, and preferably comprises at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 60, 70, 80, 100, 250, 500 or 1000 contiguous nucleotides, to the extent that such lengths are consistent with the specific sequence, of a nucleotide sequence selected from the group consisting of SEQ ID Nos 1, 2, 3, 4, or a variant thereof or a complementary sequence thereto. These nucleotide sequences comprise the polymorphic base of either allele 1 or allele 2 of the considered biallelic marker. Optionally, said biallelic marker may be within 6, 5, 4, 3, 2, or 1 nucleotides of the center of said polynucleotide or at the center of said polynucleotide. Optionally, the 3′ end of said contiguous span may be present at the 3′ end of said polynucleotide. Optionally, biallelic marker may be present at the 3′ end of said polynucleotide. Optionally, the 3′ end of said polynucleotide may be located within or at least 2, 4, 6, 8, 10, 12, 15, 18, 20, 25, 50, 100, 250, 500, or 1000 nucleotides upstream of a PCTA-1-related biallelic marker in said sequence. Optionally, the 3′ end of said polynucleotide may be located 1 nucleotide upstream of a PCTA-1-related biallelic marker in said sequence. Optionally, said polynucleotide may further comprise a label. Optionally, said polynucleotide can be attached to solid support. In a further embodiment, the polynucleotides defined above can be used alone or in any combination.

In a preferred embodiment, the sequences comprising a polymorphic base of one of the biallelic markers listed in Table 2 are selected from the group consisting of the nucleotide sequences that have a contiguous span of, that consist of, that are comprised in, or that comprises a polynucleotide having one of the sequences set forth as the amplicons listed in Table 1 or a variant thereof or a complementary sequence thereto.

The invention further concerns a nucleic acid encoding a PCTA-1 protein, wherein said nucleic acid comprises a polymorphic base of a biallelic marker selected from the group consisting of A1 to A125 and the complements thereof.

The invention also relates to a purified and/or isolated nucleotide sequence comprising a sequence defining a biallelic marker located in the sequence of the PCTA-1 gene. Preferably, the sequences defining a biallelic marker include the polymorphic base of one of the sequences P1 to P125 or the complementary sequence thereto. In some embodiments, the sequences defining a biallelic marker comprise one of the sequences selected from the group consisting of P1to P125, or a fragment or variant thereof or a complementary sequence thereto, said fragment comprising the polymorphic base.

The invention also concerns a set of the purified and/or isolated nucleotide sequences defined above. More particularly, the set of purified and/or isolated nucleotide sequences comprises a group of sequences defining a combination of biallelic markers located in the sequence of the PCTA-1 gene, preferably wherein alleles of said biallelic markers or the combinations thereof are associated with prostate cancer, with the level of aggressiveness of prostate cancer tumors, or with a level of expression of the PCTA-1 gene.

In a preferred embodiment, the invention relates to a set of purified and/or isolated nucleotide sequences, each sequence comprising a sequence defining a biallelic marker located in the sequence of the PCTA-1 gene, wherein the set is characterized in that between about 30 and 100%, preferably between about 40 and 60%, more preferably between 50 and 60%, of the sequences defining a biallelic marker are selected from the group consisting of P1 to P125, or a fragment or variant thereof or a complementary sequence thereto, said fragment comprising the polymorphic base.

More particularly, the invention concerns a set of purified and/or isolated nucleotide sequences, each sequence comprising a sequence defining a different bialielic marker located in the sequence of the PCTA-1 gene, said biallelic marker being either included in one of the nucleotide sequences of P1 to P125 or a complementary sequence thereto, or a biallelic marker preferably located in the sequence of the PCTA-1 gene, more preferably biallelic markers A1 to A125 and the complements thereof, and/or in linkage disequilibrium with one of the markers A1 to A125.

The invention also relates to a set of at least two, preferably four, five, six, seven, eight or more nucleotide sequences selected from the group consisting of P1 to P125, or a fragment or variant thereof or a complementary sequence thereto, said fragment comprising the polymorphic base.

The invention further concerns a nucleotide sequence selected from the group consisting of P1 to P125 or a fragment or a variant thereof or a complementary sequence thereto, said fragment comprising the polymorphic base.

The invention also encompasses the use of any polynucleotide for, or any polynucleotide for use in, determining the identity of one or more nucleotides at a PCTA-1-related biallelic marker. In addition, the polynucleotides of the invention for use in determining the identity of one or more nucleotides at a PCTA-1-related biallelic marker encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination. Optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, and A123 complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94 to A101, A108, A111 to 113, A115, A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, said polynucleotide may comprise a sequence disclosed in the present specification; Optionally, said polynucleotide may consist of, or consist essentially of any polynucleotide described in the present specification; Optionally, said determining may be performed in a hybridization assay, a sequencing assay, a microsequencing assay, or an enzyme-based mismatch detection assay; A preferred polynucleotide may be used in a hybridization assay for determining the identity of the nucleotide at a PCTA-1-related biallelic marker. Another preferred polynucleotide may be used in a sequencing or microsequencing assay for determining the identity of the nucleotide at a PCTA-1-related biallelic marker. A third preferred polynucleotide may be used in an enzyme-based mismatch detection assay for determining the identity of the nucleotide at a PCTA-1-related biallelic marker. A fourth preferred polynucleotide may be used in amplifying a segment of polynucleotides comprising a PCTA-1-related biallelic marker. Optionally, any of the polynucleotides described above may be attached to a solid support, array, or addressable array; Optionally, said polynucleotide may be labeled.

Additionally, the invention encompasses the use of any polynucleotide for, or any polynucleotide for use in, amplifying a segment of nucleotides comprising a PCTA-1-related biallelic marker. In addition, the polynucleotides of the invention for use in amplifying a segment of nucleotides comprising a PCTA-1-related biallelic marker encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, and A123 complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94 to A101, A108, A111 to A113, A115 to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, said polynucleotide may comprise a sequence disclosed in the present specification; Optionally, said polynucleotide may consist of, or consist essentially of any polynucleotide described in the present specification; Optionally, said amplifying may be performed by a PCR or LCR. Optionally, said polynucleotide may be attached to a solid support, array, or addressable array. Optionally, said polynucleotide may be labeled.

The primers for amplification or sequencing reaction of a polynucleotide comprising a biallelic marker of the invention may be designed from the disclosed sequences for any method known in the art. A preferred set of primers are fashioned such that the 3′ end of the contiguous span of identity with a sequence selected from the group consisting of SEQ ID Nos 1, 2, 3, 4 or a sequence complementary thereto or a variant thereof is present at the 3′ end of the primer. Such a configuration allows the 3′ end of the primer to hybridize to a selected nucleic acid sequence and dramatically increases the efficiency of the primer for amplification or sequencing reactions. Allele specific primers may be designed such that a polymorphic base of a biallelic marker is at the 3′ end of the contiguous span and the contiguous span is present at the 3′ end of the primer. Such allele specific primers tend to selectively prime an amplification or sequencing reaction so long as they are used with a nucleic acid sample that contains one of the two alleles present at a biallelic marker. The 3′ end of the primer of the invention may be located within or at least 2, 4, 6, 8, 10, 12, 15, 18, 20, 25, 50, 100, 250, 500, or 1000 nucleotides upstream of a PCTA-1-related biallelic marker in said sequence or at any other location which is appropriate for their intended use in sequencing, amplification or the location of novel sequences or markers. Thus, another set of preferred amplification primers comprise an isolated polynucleotide consisting essentially of a contiguous span of 8 to 50 nucleotides in a sequence selected from the group consisting of SEQ ID Nos 1, 2, 3, 4 or a sequence complementary thereto or a variant thereof, wherein the 3′ end of said contiguous span is located at the 3′ end of said polynucleotide, and wherein the 3′ end of said polynucleotide is located upstream of a PCTA-1-related biallelic marker in said sequence. Preferably, those amplification primers comprise a sequence selected from the group consisting of the sequences B1 to B47 and C1 to C47. Primers with their 3′ ends located 1 nucleotide upstream of a biallelic marker of PCTA-1 have a special utility as microsequencing assays. Preferred microsequencing primers arc described in Table 4. Optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A8 1, A82, A86 to A91, A107, A118, and A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, and A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94to A101, A108, A111 to A113, A115 to A117 and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, microsequencing primers are selected from the group consisting of the nucleotide sequences D1 to D125 and E1 to E125. More preferred microsequencing primers are Q selected from the group consisting of the nucleotides sequences D15, D24, D30, D34, D36, D38, D41, D44, D50, D53, D54, D56, D57, D59, D76, D85, D93, D108, D111, D115, D124, E11, E14, E22, E25, E26, E35, E42, E52, E53, E55, E56, E60, E61, E64, E73, E75, E93, E96.

The probes of the present invention may be designed from the disclosed sequences for any method known in the art, particularly methods which allow for testing if a marker disclosed herein is present. A preferred set of probes may be designed for use in the hybridization assays of the invention in any manner known in the art such that they selectively bind to one allele of a biallelic marker, but not the other under any particular set of assay conditions. Preferred hybridization probes comprise the polymorphic base of either allele I or allele 2 of the considered biallelic marker. Optionally, said biallelic marker may be within 6, 5, 4, 3, 2, or 1 nucleotides of the center of the hybridization probe or at the center of said probe. In a preferred embodiment, the probes are selected from the group consisting of the sequences P1 to P125 and the complementary sequence thereto.

It should be noted that the polynucleotides of the present invention are not limited to having the exact flanking sequences surrounding the polymorphic bases which are enumerated in Sequence Listing. Rather, it will be appreciated that the flanking sequences surrounding the biallelic markers may be lengthened or shortened to any extent compatible with their intended use and the present invention specifically contemplates such sequences. The flanking regions outside of the contiguous span need not be homologous to native flanking sequences which actually occur in human subjects. The addition of any nucleotide sequence which is compatible with the nucleotides intended use is specifically contemplated.

Primers and probes may be labeled or immobilized on a solid support as described in“Oligonucleotide probes and primers”.

The polynucleotides of the invention which are attached to a solid support encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, said polynucleotides may be specified as attached individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the invention to a single solid support. Optionally, polynucleotides other than those of the invention may attached to the same solid support as polynucleotides of the invention. Optionally, when multiple polynucleotides are attached to a solid support they may be attached at random locations, or in an ordered array. Optionally, said ordered array may be addressable.

The present invention also encompasses diagnostic kits comprising one or more polynucleotides of the invention with a portion or all of the necessary reagents and instructions for genotyping a test subject by determining the identity of a nucleotide at a PCTA-1-related biallelic marker. The polynucleotides of a kit may optionally be attached to a solid support, or be part of an array or addressable array of polynucleotides. The kit may provide for the determination of the identity of the nucleotide at a marker position by any method known in the art including, but not limited to, a sequencing assay method, a microsequencing assay method, a hybridization assay method, or an enzyme-based mismatch detection assay method.

Methods for De Novo Identification of Biallelic Markers

Any of a variety of methods can be used to screen a genomic fragment for single nucleotide polymorphisms such as differential hybridization with oligonucleotide probes, detection of changes in the mobility measured by gel electrophoresis or direct sequencing of the amplified nucleic acid. A preferred method for identifying biallelic markers involves comparative sequencing of genomic DNA fragments from an appropriate number of unrelated individuals.

In a first embodiment, DNA samples from unrelated individuals are pooled together, following which the genomic DNA of interest is amplified and sequenced. The nucleotide sequences thus obtained are then analyzed to identify significant polymorphisms. One of the major advantages of this method resides in the fact that the pooling of the DNA samples substantially reduces the number of DNA amplification reactions and sequencing reactions, which must be carried out. Moreover, this method is sufficiently sensitive so that a biallelic marker obtained thereby usually demonstrates a sufficient frequency of its less common allele to be useful in conducting association studies.

In a second embodiment, the DNA samples are not pooled and are therefore amplified and sequenced individually. This method is usually preferred when biallelic markers need to be identified in order to perform association studies within candidate genes. Preferably, highly relevant gene regions such as promoter regions or exon regions may be screened for biallelic markers. A biallelic marker obtained using this method may show a lower degree of informativeness for conducting association studies, e.g. if the frequency of its less frequent allele may be less than about 10%. Such a biallelic marker will, however, be sufficiently informative to conduct association studies and it will further be appreciated that including less informative biallelic markers in the genetic analysis studies of the present invention, may allow in some cases the direct identification of causal mutations, which may, depending on their penetrance, be rare mutations.

The following is a description of the various parameters of a preferred method used by the inventors for the identification of the biallelic markers of the present invention.

Genomic DNA Samples

The genomic DNA samples from which the biallelic markers of the present invention are generated are preferably obtained from unrelated individuals corresponding to a heterogeneous population of known ethnic background. The number of individuals from whom DNA samples are obtained can vary substantially, preferably from about 10 to about 1000, preferably from about 50 to about 200 individuals. It is usually preferred to collect DNA samples from at least about 100 individuals in order to have sufficient polymorphic diversity in a given population to identify as many markers as possible and to generate statistically significant results.

As for the source of the genomic DNA to be subjected to analysis, any test sample can be foreseen without any particular limitation. These test samples include biological samples, which can be tested by the methods of the present invention described herein, and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supernatants; fixed tissue specimens including tumor and non-tumor tissue and lymph node tissues; bone marrow aspirates and fixed cell specimens. The preferred source of genomic DNA used in the present invention is from peripheral venous blood of each donor. Techniques to prepare genomic DNA from biological samples are well known to the skilled technician. Details of a preferred embodiment are provided in Example 1. The person skilled in the art can choose to amplify pooled or unpooled DNA samples.

DNA Amplification

The identification of biallelic markers in a sample of genomic DNA may be facilitated through the use of DNA amplification methods. DNA samples can be pooled or unpooled for the amplification step. DNA amplification techniques are well known to those skilled in the art.

Amplification techniques that can be used in the context of the present invention include, but are not limited to, the ligase chain reaction (LCR) described in EP-A-320 308, WO 9320227 and EP-A439 182, the disclosures of which are incorporated herein by reference in their entireties, the polymerase chain reaction (PCR, RT-PCR) and techniques such as the nucleic acid sequence based amplification (NASBA) described in Guatelli J. C., et al.(1990) and in Compton J.(1991), Q-beta amplification as described in European Patent Application No 4544610, the disclosures of which are incorporated herein by reference in their entireties, strand displacement amplification as described in Walkeret al.(1996) and EP A684 315, the disclosures of which are incorporated herein by reference in their entireties, and, target mediated amplification as described in PCT Publication WO 9322461.

LCR and Gap LCR are exponential amplification techniques, both depend on DNA ligase to join adjacent primers annealed to a DNA molecule. In Ligase Chain Reaction (LCR), probe pairs are used which include two primary (first and second) and two secondary (third and fourth) probes, all of which are employed in molar excess to target. The first probe hybridizes to a first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5′ phosphate-3′ hydroxyl relationship, and so that a ligase can covalently fuse or ligate the two probes into a fused product. In addition, a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar abutting fashion. Of course, if the target is initially double stranded, the secondary probes also will hybridize to the target complement in the first instance. Once the ligated strand of primary probes is separated from the target strand, it will hybridize with the third and fourth probes, which can be ligated to form a complementary, secondary ligated product. It is important to realize that the ligated products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. A method for multiplex LCR has also been described (WO 9320227, the disclosure of which is incorporated herein by reference in its entirety). Gap LCR (GLCR) is a version of LCR where the probes are not adjacent but are separated by 2 to 3 bases.

For amplification of mRNAs, it is within the scope of the present invention to reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Pat. No. 5,322,770, the disclosure of which is incorporated herein by reference in its entirety, or, to use Asymmetric Gap LCR (RT-AGLCR) as described by Marshall et al.(1994). AGLCR is a modification of GLCR that allows the amplification of RNA.

The PCR technology is the preferred amplification technique used in the present invention. A variety of PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see White (1997) and the publication entitled “PCR Methods and Applications” (1991, Cold Spring Harbor Laboratory Press). In each of these PCR procedures, PCR primers on either side of the nucleic acid sequences to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase. The nucleic acid in the sample is denatured and the PCR primers are specifically hybridized to complementary nucleic acid sequences in the sample. The hybridized primers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites. PCR has further been described in several patents including U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188, the disclosures of which are incorporated herein by reference in their entireties.

The PCR technology is the preferred amplification technique used to identify new biallelic markers. A typical example of a PCR reaction suitable for the purposes of the present invention is provided in Example 2.

One of the aspects of the present invention is a method for the amplification of the human PCTA-1 gene, particularly of a fragment of the genomic sequence of SEQ ID No 1 or of the cDNA sequences of SEQ ID Nos 2, 3, 4, 8, or a fragment or a variant thereof in a test sample, preferably using the PCR technology. This method comprises the steps of:

a) contacting a test sample with amplification reaction reagents comprising a pair of amplification primers as described above and located on either side of the polynucleotide region to be amplified, and

b) optionally, detecting the amplification products.

The invention also concerns a kit for the amplification of a PCTA-1 gene sequence, particularly of a portion of the genomic sequence of SEQ ID No 1 or of the cDNA sequences of SEQ ID Nos 2, 3 4, 9, or a variant thereof in a test sample, wherein said kit comprises:

a) a pair of oligonucleotide primers located on either side of the PCTA-1 region to be amplified;

b) optionally, the reagents necessary for performing the amplification reaction.

In one embodiment of the above amplification method and kit, the amplification product is detected by hybridization with a labeled probe having a sequence which is complementary to the amplified region. In another embodiment of the above amplification method and kit, primers comprise a sequence which is selected from the group consisting of the nucleotide sequences of B1 to B47, C1 to C47, D1 to D125, and E1 to E125.

In a first embodiment of the present invention, biallelic markers are identified using genomic sequence information generated by the inventors. Sequenced genomic DNA fragments are used to design primers for the amplification of 500 bp fragments. These 500 bp fragments are amplified from genomic DNA and are scanned for biallelic markers. Primers may be designed using the OSP software (Hillier L. and Green P., 1991). All primers may contain, upstream of the specific target bases, a common oligonucleotide tail that serves as a sequencing primer. Those skilled in the art are familiar with primer extensions, which can be used for these purposes.

Preferred primers, useful for the amplification of genomic sequences encoding the candidate genes, focus on promoters, exons and splice sites of the genes. A biallelic marker presents a higher probability to be an eventual causal mutation if it is located in these functional regions of the gene. Preferred amplification primers of the invention include the nucleotide sequences B1 to B47 and C1 to C47, detailed further in Example 2, Table 1.

Sequencing of Amplified Genomic DNA and Identification of Single Nucleotide Polymorphisms

The amplification products generated as described above, are then sequenced using any method known and available to the skilled technician. Methods for sequencing DNA using either the dideoxy-mediated method (Sanger method) or the Maxam-Gilbert method are widely known to those of ordinary skill in the art. Such methods are for example disclosed in Sambrook et al.(1989). Alternative approaches include hybridization to high-density DNA probe arrays as described in Chee et al.(1996).

Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol. The products of the sequencing reactions are run on sequencing gels and the sequences are determined using gel image analysis. The polymorphism search is based on the presence of superimposed peaks in the electrophoresis pattern resulting from different bases occurring at the same position. Because each dideoxy terminator is labeled with a different fluorescent molecule, the two peaks corresponding to a biallelic site present distinct colors corresponding to two different nucleotides at the same position on the sequence. However, the presence of two peaks can be an artifact due to background noise. To exclude such an artifact, the two DNA strands are sequenced and a comparison between the peaks is carried out. In order to be registered as a polymorphic sequence, the polymorphism has to be detected on both strands.

The above procedure permits those amplification products, which contain biallelic markers to be identified. The detection limit for the frequency of biallelic polymorphisms detected by sequencing pools of 100 individuals is approximately 0.1 for the minor allele, as verified by sequencing pools of known allelic frequencies. However, more than 90% of the w biallelic polymorphisms detected by the pooling method have a frequency for the minor allele higher than 0.25. Therefore, the biallelic markers selected by this method have a frequency of at least 0.1 for the minor allele and less than 0.9 for the major allele. Preferably at least 0.2 for the minor allele and less than 0.8 for the major allele, more preferably at least 0.3 for the minor allele and less than 0.7 for the major allele, thus a heterozygosity rate higher than 0.18, preferably higher than 0.32, more preferably higher than 0.42.

In another embodiment, biallelic markers are detected by sequencing individual DNA samples, the frequency of the minor allele of such a biallelic marker may be less than 0. 1.

Validation of the Biallelic Markers of the Present Invention

The polymorphisms are evaluated for their usefulness as genetic markers by validating that both alleles are present in a population. Validation of the biallelic markers is accomplished by genotyping a group of individuals by a method of the invention and demonstrating that both alleles are present. Microsequencing is a preferred method of genotyping alleles. The validation by genotyping step may be performed on individual samples derived from each individual in the group or by genotyping a pooled sample derived from more than one individual. The group can be as small as one individual if that individual is heterozygous for the allele in question. Preferably the group contains at least three individuals, more preferably the group contains five or six individuals, so that a single validation test will be more likely to result in the validation of more of the biallelic markers that are being tested. It should be noted, however, that when the validation test is performed on a small group it may result in a false negative result if as a result of sampling error none of the individuals tested carries one of the two alleles. Thus, the validation process is less useful in demonstrating that a particular initial result is an artifact, than it is at demonstrating that there is a bonafide biallelic marker at a particular position in a sequence. All of the genotyping, haplotyping, association, and interaction study methods of the invention may optionally be performed solely with validated biallelic markers.

Evaluation of the Frequency of the Biallelic Markers of the Present Invention

The validated biallelic markers are further evaluated for their usefulness as genetic markers by determining the frequency of the least common allele at the biallelic marker site. The higher the frequency of the less common allele the greater the usefulness of the biallelic marker is association and interaction studies. The determination of the least common allele is accomplished by genotyping a group of individuals by a method of the invention and demonstrating that both alleles are present. This determination of frequency by genotyping step may be performed on individual samples derived from each individual in the group or by genotyping a pooled sample derived from more than one individual. The group must be large enough to be representative of the population as a whole. Preferably the group contains at least 20 individuals, more preferably the group contains at least 50 individuals, most preferably the group contains at least 100 individuals. Of course the larger the group the greater the accuracy of the frequency determination because of reduced sampling error. For an indication of the frequency for the less common allele of a particular biallelic marker of the invention see Table 2. A biallelic marker wherein the frequency of the less common allele is 30% or more is termed a “high quality biallelic marker.” All of the genotyping, haplotyping, association, and interaction study methods of the invention may optionally be performed solely with high quality biallelic markers.

Methods for Genotyping an Individual for Biallelic Markers

Methods are provided to genotype a biological sample for one or more biallelic markers of the present invention, all of which may be performed in vitro. Such methods of genotyping comprise determining the identity of a nucleotide at a PCTA-1 biallelic marker site by any method known in the art. These methods find use in genotyping case-control populations in association studies as well as individuals in the context of detection of alleles of biallelic markers which are known to be associated with a given trait, in which case both copies of the biallelic marker present in individual's genome are determined so that an individual may be classified as homozygous or heterozygous for a particular allele.

These genotyping methods can be performed on nucleic acid samples derived from a single individual or pooled DNA samples.

Genotyping can be performed using similar methods as those described above for the identification of the biallelic markers, or using other genotyping methods such as those further described below. In preferred embodiments, the comparison of sequences of amplified genomic fragments from different individuals is used to identify new biallelic markers whereas microsequencing is used for genotyping known biallelic markers in diagnostic and association study applications.

In one embodiment the invention encompasses methods of genotyping comprising determining the identity of a nucleotide at a PCTA-1-related biallelic marker or the complement thereof in a biological sample; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, and A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94 to A01, A108, A111 to A113, A115 to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said biological sample is derived from a single subject; optionally, wherein the identity of the nucleotides at said biallelic marker is determined for both copies of said biallelic marker present in said individual's genome; optionally, wherein said biological sample is derived from multiple subjects; Optionally, the genotyping methods of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination; Optionally, said method is performed in vitro; optionally, further comprising amplifying a portion of said sequence comprising the biallelic marker prior to said determining step; Optionally, wherein said amplifying is performed by PCR, LCR, or replication of a recombinant vector comprising an origin of replication and said fragment in a host cell; optionally, wherein said determining is performed by a hybridization assay, a sequencing assay, a microsequencing assay, or an enzyme-based mismatch detection assay.

Source of Nucleic Acids for Genotyping

Any source of nucleic acids, in purified or non-purified form, can be utilized as the starting nucleic acid, provided it contains or is suspected of containing the specific nucleic acid sequence desired. DNA or RNA may be extracted from cells, tissues, body fluids and the like as described above. While nucleic acids for use in the genotyping methods of the invention can be derived from any mammalian source, the test subjects and individuals from which nucleic acid samples are taken are generally understood to be human.

Amplification of DNA Fragments Comprising Biallelic Markers

Methods and polynucleotides are provided to amplify a segment of nucleotides comprising one or more biallelic marker of the present invention. It will be appreciated that amplification of DNA fragments comprising biallelic markers may be used in various methods and for various purposes and is not restricted to genotyping. Nevertheless, many genotyping methods, although not all, require the previous amplification of the DNA region carrying the biallelic marker of interest. Such methods specifically increase the concentration or total number of sequences that span the biallelic marker or include that site and sequences located either distal or proximal to it. Diagnostic assays may also rely on amplification of DNA segments carrying a biallelic marker of the present invention. Amplification of DNA may be achieved by any method known in the art. Amplification techniques are described above in the section entitled, “DNA amplification.”

The invention also concerns a method for the amplification of a PCTA-1 gene region, preferably containing at least one of the polymorphic bases identified in the context of the present invention, or a fragment or variant thereof, in a test sample. The method comprises the step of contacting a test sample suspected of containing the targeted PCTA-1 gene sequence or a fragment thereof with amplification reaction reagents comprising a pair of amplification primers, preferably located on either side of the polymorphic base. Preferred amplification primers consist of B1 to B47 and C1 to C47. The method may further comprise the step of detecting the amplification product. For example, the amplification product may be detected using a detection probe that can hybridize with an internal region of the amplicon sequences. In some embodiments, the polymorphic base is included in one of the sequences of P1 to P125, and the complementary sequences thereof.

Some of these amplification methods are particularly suited for the detection of single nucleotide polymorphisms and allow the simultaneous amplification of a target sequence and the identification of the polymorphic nucleotide as it is further described below.

The identification of biallelic markers as described above allows the design of appropriate oligonucleotides, which can be used as primers to amplify DNA fragments comprising the biallelic markers of the present invention. Amplification can be performed using the primers initially used to discover new biallelic markers which are described herein or any set of primers allowing the amplification of a DNA fragment comprising a biallelic marker of the present invention.

In some embodiments the present invention provides primers for amplifying a DNA fragment containing one or more biallelic markers of the present invention. Preferred amplification primers are listed in Example 2. It will be appreciated that the primers listed are merely exemplary and that any other set of primers which produce amplification products containing one or more biallelic markers of the present invention are also of use.

The spacing of the primers determines the length of the segment to be amplified. In the context of the present invention, amplified segments carrying biallelic markers can range in size from at least about 25 bp to 35 kbp. Amplification fragments from 25-3000 bp are typical, fragments from 50-1000 bp are preferred and fragments from 100-600 bp are highly preferred. It will be appreciated that amplification primers for the biallelic markers may be any sequence which allow the specific amplification of any DNA fragment carrying the markers. Amplification primers may be labeled or immobilized on a solid support as described in “Oligonucleotide probes and primers”.

Methods of Genotyping DNA Samples for Biallelic Markers

Any method known in the art can be used to identify the nucleotide present at a biallelic marker site. Since the biallelic marker allele to be detected has been identified and specified in the present invention, detection will prove simple for one of ordinary skill in the art by employing any of a number of techniques. Many genotyping methods require the previous amplification of the DNA region carrying the biallelic marker of interest. While the amplification of target or signal is often preferred at present, ultrasensitive detection methods which do not require amplification are also encompassed by the present genotyping methods. Methods well-known to those skilled in the art that can be used to detect biallelic polymorphisms include methods such as, conventional dot blot analyzes, single strand conformational polymorphism analysis (SSCP) described by Orita et al.(1989), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and other conventional techniques as described in Sheffield et al.(1991), White et al.(1992), Grompe et al.(1989 and 1993). Another method for determining the identity of the nucleotide present at a particular polymorphic site employs a specialized exonuclease-resistant nucleotide derivative as described in U.S. Pat. No. 4,656,127.

Preferred methods involve directly determining the identity of the nucleotide present at a biallelic marker site by sequencing assay, enzyme-based mismatch detection assay, or hybridization assay. The following is a description of some preferred methods. A highly preferred method is the microsequencing technique. The term “sequencing” is generally used herein to refer to polymerase extension of duplex primer/template complexes and includes both traditional sequencing and microsequencing.

1) Sequencing Assays

The nucleotide present at a polymorphic site can be determined by sequencing methods. In a preferred embodiment, DNA samples are subjected to PCR amplification before sequencing as described above. DNA sequencing methods are described in “Sequencing Of Amplified Genomic DNA And Identification Of Single Nucleotide Polymorphisms”.

Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol. Sequence analysis allows the identification of the base present at the biallelic marker site.

2) Microsequencing Assays

In microsequencing methods, the nucleotide at a polymorphic site in a target DNA is detected by a single nucleotide primer extension reaction. This method involves appropriate microsequencing primers which, hybridize just upstream of the polymorphic base of interest in the target nucleic acid. A polymerase is used to specifically extend the 3′ end of the primer with one single ddNTP (chain terminator) complementary to the nucleotide at the polymorphic site. Next the identity of the incorporated nucleotide is determined in any suitable way.

Typically, microsequencing reactions are carried out using fluorescent ddNTPs and the extended microsequencing primers are analyzed by electrophoresis on ABI 377 sequencing machines to determine the identity of the incorporated nucleotide as described in EP412 883, the disclosure of which is incorporated herein by reference in its entirety. Alternatively capillary electrophoresis can be used in order to process a higher number of assays simultaneously. An example of a typical microsequencing procedure that can be used in the context of the present invention is provided in Example 4.

Different approaches can be used for the labeling and detection of ddNTPs. A homogeneous phase detection method based on fluorescence resonance energy transfer has been described by Chen and Kwok (1997) and Chen et al.(1997). In this method, amplified genomic DNA fragments containing polymorphic sites are incubated with a 5′-fluorescein-labeled primer in the presence of allelic dye-labeled dideoxyribonucleoside triphosphates and a modified Taq polymerase. The dye-labeled primer is extended one base by the dye-terminator specific for the allele present on the template. At the end of the genotyping reaction, the fluorescence intensities of the two dyes in the reaction mixture are analyzed directly without separation or purification. All these steps can be performed in the same tube and the fluorescence changes can be monitored in real time. Alternatively, the extended primer may be analyzed by MALDI-TOF Mass Spectrometry. The base at the polymorphic site is identified by the mass added onto the microsequencing primer (see Haff and Smirnov, 1997).

Microsequencing may be achieved by the established microsequencing method or by developments or derivatives thereof. Alternative methods include several solid-phase microsequencing techniques. The basic microsequencing protocol is the same as described previously, except that the method is conducted as a heterogeneous phase assay, in which the primer or the target molecule is immobilized or captured onto a solid support. To simplify the primer separation and the terminal nucleotide addition analysis, oligonucleotides are attached to solid supports or are modified in such ways that permit affinity separation as well as polymerase extension. The 5′ ends and internal nucleotides of synthetic oligonucleotides can be modified in a number of different ways to permit different affinity separation approaches, e.g., biotinylation. If a single affinity group is used on the oligonucleotides, the oligonucleotides can be separated from the incorporated terminator regent. This eliminates the need of physical or size separation. More than one oligonucleotide can be separated from the terminator reagent and analyzed simultaneously if more than one affinity group is used. This permits the analysis of several nucleic acid species or more nucleic acid sequence information per extension reaction. The affinity group need not be on the priming oligonucleotide but could alternatively be present on the template. For example, immobilization can be carried out via an interaction between biotinylated DNA and streptavidin-coated microtitration wells or avidin-coated polystyrene particles. In the same manner, oligonucleotides or templates may be attached to a solid support in a high-density format. In such solid phase microsequencing reactions, incorporated ddNTPs can be radiolabeled (Syvänen, 1994) or linked to fluorescein (Livak and Hainer, 1994). The detection of radiolabeled ddNTPs can be achieved through scintillation-based techniques. The detection of fluorescein-linked ddNTPs can be based on the binding of antifluorescein antibody conjugated with alkaline phosphatase, followed by incubation with a chromogenic substrate (such asp-nitrophenyl phosphate). Other possible reporter-detection pairs include: ddNTP linked to dinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (Harju et al., 1993) or biotinylated ddNTP and horseradish peroxidase-conjugated streptavidin with o-phenylenediamine as a substrate (WO 92/15712, the disclosure of which is incorporated herein by reference in its entirety). As yet another alternative solid-phase microsequencing procedure, Nyren et al.(1993) described a method relying on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA).

Pastinen et al.(1997) describe a method for multiplex detection of single nucleotide polymorphism in which the solid phase minisequencing principle is applied to an oligonucleotide array format. High-density arrays of DNA probes attached to a solid support (DNA chips) are further described below.

In one aspect the present invention provides polynucleotides and methods to genotype one or more biallelic markers of the present invention by performing a microsequencing assay. Preferred microsequencing primers include the nucleotide sequences D1 to D125 and E1 to E125. More preferred microsequencing primers are selected from the group consisting of the nucleotide sequences D15, D24, D30, D34, D36, D38, D41, D44, D50, D53, D54, D56, D57, D59, D76, D85, D93, D108, D111, D115, D124, Eli, E14, E22, E25, E26, E35, E42, E52, E53, E55, E56, E60, E61, E64, E73, E75, E93, E96. It will be appreciated that the microsequencing primers listed in Example 4 are merely exemplary and that, any primer having a 3′ end immediately adjacent to the polymorphic nucleotide may be used. Similarly, it will be appreciated that microsequencing analysis may be performed for any biallelic marker or any combination of biallelic markers of the present invention. One aspect of the present invention is a solid support which includes one or more microsequencing primers listed in Example 4, or fragments comprising at least 8, 12, 15, 20, 25, 30, 40, or 50 consecutive nucleotides thereof, to the extent that such lengths are consistent with the primer described, and having a 3′ terminus immediately upstream of the corresponding biallelic marker, for determining the identity of a nucleotide at a biallelic marker site.

3) Mismatch Detection Assays Based on Polymerases and Ligases

In one aspect the present invention provides polynucleotides and methods to determine the allele of one or more biallelic markers of the present invention in a biological sample, by mismatch detection assays based on polymerases and/or ligases. These assays are based on the specificity of polymerases and ligases. Polymerization reactions places particularly stringent requirements on correct base pairing of the 3′ end of the amplification primer and the joining of two oligonucleotides hybridized to a target DNA sequence is quite sensitive to mismatches close to the ligation site, especially at the 3′ end. Methods, primers and various parameters to amplify DNA fragments comprising biallelic markers of the present invention are further described above in “Amplification Of DNA Fragments Comprising Biallelic Markers”.

Allele Specific Amplification Primers

Discrimination between the two alleles of a biallelic marker can also be achieved by allele specific amplification, a selective strategy, whereby one of the alleles is amplified without amplification of the other allele. For allele specific amplification, at least one member of the pair of primers is sufficiently complementary with a region of a PCTA-1 gene comprising the polymorphic base of a biallelic marker of the present invention to hybridize therewith and to initiate the amplification. Such primers are able to discriminate between the two alleles of a biallelic marker.

This is accomplished by placing the polymorphic base at the 3′ end of one of the amplification primers. Because the extension forms from the 3′ end of the primer, a mismatch at or near this position has an inhibitory effect on amplification. Therefore, under appropriate amplification conditions, these primers only direct amplification on their complementary allele. Determining the precise location of the mismatch and the corresponding assay conditions are well within the ordinary skill in the art.

Ligation/Amplification Based Methods

The “Oligonucleotide Ligation Assay” (OLA) uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target molecules. One of the oligonucleotides is biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate that can be captured and detected. OLA is capable of detecting single nucleotide polymorphisms and may be advantageously combined with PCR as described by Nickerson et al.(1990). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.

Other amplification methods which are particularly suited for the detection of single nucleotide polymorphism include LCR (ligase chain reaction), Gap LCR (GLCR) which are described above in “DNA Amplification”. LCR uses two pairs of probes to exponentially amplify a specific target. The sequences of each pair of oligonucleotides, is selected to permit the pair to hybridize to abutting sequences of the same strand of the target. Such hybridization forms a substrate for a template-dependant ligase. In accordance with the present invention, LCR can be performed with oligonucleotides having the proximal and distal sequences of the same strand of a biallelic marker site. In one embodiment, either oligonucleotide will be designed to include the biallelic marker site. In such an embodiment, the reaction conditions are selected such that the oligonucleotides can be ligated together only if the target molecule either contains or lacks the specific nucleotide that is complementary to the biallelic marker on the oligonucleotide. In an alternative embodiment, the oligonucleotides will not include the biallelic marker, such that when they hybridize to the target molecule, a “gap” is created as described in WO 90/01069, the disclosure of which is incorporated herein by reference in its entirety. This gap is then “filled” with complementary dNTPs (as mediated by DNA polymerase), or by an additional pair of oligonucleotides. Thus at the end of each cycle, each single strand has a complement capable of serving as a target during the next cycle and exponential allele-specific amplification of the desired sequence is obtained.

Ligase/Polymerase-mediated Genetic Bit Analysis™ is another method for determining the identity of a nucleotide at a preselected site in a nucleic acid molecule (WO 95/21271, the disclosure of which is incorporated herein by reference in its entirety). This method involves the incorporation of a nucleoside triphosphate that is complementary to the nucleotide present at the preselected site onto the terminus of a primer molecule, and their subsequent ligation to a second oligonucleotide. The reaction is monitored by detecting a specific label attached to the reaction's solid phase or by detection in solution.

4) Hybridization Assay Methods

A preferred method of determining the identity of the nucleotide present at a biallelic marker site involves nucleic acid hybridization. The hybridization probes, which can be conveniently used in such reactions, preferably include the probes defined herein. Any hybridization assay may be used including Southern hybridization, Northern hybridization, dot blot hybridization and solid-phase hybridization (see Sambrook et al., 1989).

Hybridization refers to the formation of a duplex structure by two single stranded nucleic acids due to complementary base pairing. Hybridization can occur between exactly complementary nucleic acid strands or between nucleic acid strands that contain minor regions of mismatch. Specific probes can be designed that hybridize to one form of a biallelic marker and not to the other and therefore are able to discriminate between different allelic forms. Allele-specific probes are often used in pairs, one member of a pair showing perfect match to a target sequence containing the original allele and the other showing a perfect match to the target sequence containing the alternative allele. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles. Stringent, sequence specific hybridization conditions, under which a probe will hybridize only to the exactly complementary target sequence are well known in the art (Sambrook et al., 1989). Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Although such hybridization can be performed in solution, it is preferred to employ a solid-phase hybridization assay. The target DNA comprising a biallelic marker of the present invention may be amplified prior to the hybridization reaction. The presence of a specific allele in the sample is determined by detecting the presence or the absence of stable hybrid duplexes formed between the probe and the target DNA. The detection of hybrid duplexes can be carried out by a number of methods. Various detection assay formats are well known which utilize detectable labels bound to either the target or the probe to enable detection of the hybrid duplexes. Typically, hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected. Those skilled in the art will recognize that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the primers and probes.

Two recently developed assays allow hybridization-based allele discrimination with no need for separations or washes (see Landegren U. et al., 1998). The TaqMan assay takes advantage of the 5′ nuclease activity of Taq DNA polymerase to digest a DNA probe annealed specifically to the accumulating amplification product. TaqMan probes are labeled with a donor-acceptor dye pair that interacts via fluorescence energy transfer. Cleavage of the TaqMan probe by the advancing polymerase during amplification dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence. All reagents necessary to detect two allelic variants can be assembled at the beginning of the reaction and the results are monitored in real time (see Livak et al., 1995). In an alternative homogeneous hybridization based procedure, molecular beacons are used for allele discriminations. Molecular beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogeneous solutions. When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore (Tyagi et al., 1998).

The polynucleotides provided herein can be used to produce probes which can be used in hybridization assays for the detection of biallelic marker alleles in biological samples. These probes are characterized in that they preferably comprise between 8 and 50 nucleotides, and in that they are sufficiently complementary to a sequence comprising a biallelic marker of the present invention to hybridize thereto and preferably sufficiently specific to be able to discriminate the targeted sequence for only one nucleotide variation. A particularly preferred probe is 25 nucleotides in length. Another particularly preferred probe is 47 nucleotides in length. Preferably the biallelic marker is within 4 nucleotides of the center of the polynucleotide probe. In particularly preferred probes, the biallelic marker is at the center of said polynucleotide. Preferred probes comprise a nucleotide sequence selected from the group consisting of amplicons listed in Table 1 and the sequences complementary thereto, or a fragment thereof, said fragment comprising at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides and containing a polymorphic base. Preferred probes comprise a nucleotide sequence selected from the group consisting of P1 to P125 and the sequences complementary thereto. In preferred embodiments the polymorphic base(s) are within 5, 4, 3, 2, 1, nucleotides of the center of the said polynucleotide, more preferably at the center of said polynucleotide.

Preferably the probes of the present invention are labeled or immobilized on a solid support. Labels and solid supports are further described in “Oligonucleotide Probes and Primers”. The probes can be non-extendable as described in “Oligonucleotide Probes and Primers”.

By assaying the hybridization to an allele specific probe, one can detect the presence or absence of a biallelic marker allele in a given sample. High-Throughput parallel hybridization in array format is specifically encompassed within “hybridization assays” and are described below.

5) Hybridization to Addressable Arrays of Oligonucleotides

Hybridization assays based on oligonucleotide arrays rely on the differences in hybridization stability of short oligonucleotides to perfectly matched and mismatched target sequence variants. Efficient access to polymorphism information is obtained through a basic structure comprising high-density arrays of oligonucleotide probes attached to a solid support (e.g., the chip) at selected positions. Each DNA chip can contain thousands to millions of individual synthetic DNA probes arranged in a grid-like pattern and miniaturized to the size of a dime.

The chip technology has already been applied with success in numerous cases. For example, the screening of mutations has been undertaken in the BRCA1 gene, in S. cerevisiae mutant strains, and in the protease gene of HIV-1 virus (Hacia et al., 1996; Shoemaker et al., 1996; Kozal et al., 1996). Chips of various formats for use in detecting biallelic polymorphisms can be produced on a customized basis by Affymetrix (GeneChip™), Hyseq (HyChip and HyGnostics), and Protogene Laboratories.

In general, these methods employ arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual which, target sequences include a polymorphic marker. EP785280, the disclosure of which is incorporated herein by reference in its entirety, describes a tiling strategy for the detection of single nucleotide polymorphisms. Briefly, arrays may generally be “tiled” for a large number of specific polymorphisms. By “tiling” is generally meant the synthesis of a defined set of oligonucleotide probes which is made up of a sequence complementary to the target sequence of interest, as well as preselected variations of that sequence, e.g., substitution of one or more given positions with one or more members of the basis set of nucleotides. Tiling strategies are further described in PCT application No. WO 95/11995, the disclosure of which is incorporated herein by reference in its entirety. In a particular aspect, arrays are tiled for a number of specific, identified biallelic marker sequences. In particular, the array is tiled to include a number of detection blocks, each detection block being specific for a specific biallelic marker or a set of biallelic markers. For example, a detection block may be tiled to include a number of probes, which span the sequence segment that includes a specific polymorphism. To ensure probes that are complementary to each allele, the probes are synthesized in pairs differing at the biallelic marker. In addition to the probes differing at the polymorphic base, monosubstituted probes are also generally tiled within the detection block. These monosubstituted probes have bases at and up to a certain number of bases in either direction from the polymorphism, substituted with the remaining nucleotides (selected from A, T, G, C and U). Typically the probes in a tiled detection block will include substitutions of the sequence positions up to and including those that are 5 bases away from the biallelic marker. The monosubstituted probes provide internal controls for the tiled array, to distinguish actual hybridization from artefactual cross-hybridization. Upon completion of hybridization with the target sequence and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes. The hybridization data from the scanned array is then analyzed to identify which allele or alleles of the biallelic marker are present in the sample. Hybridization and scanning may be carried out as described in PCT application No. WO 92/10092 and WO 95/11995 and U.S. Pat. No. 5,424,186, the disclosures of which are incorporated herein by reference in their entireties.

Thus, in some embodiments, the chips may comprise an array of nucleic acid sequences of fragments of about IS nucleotides in length. In further embodiments, the chip may comprise an array including at least one of the sequences selected from the group consisting of amplicons listed in table I and the sequences complementary thereto, or a fragment thereof, said fragment comprising at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides and containing a polymorphic base. In preferred embodiments the polymorphic base is within 5, 4, 3, 2, 1, nucleotides of the center of the said polynucleotide, more preferably at the center of said polynucleotide. In some embodiments, the chip may comprise an array of at least 2, 3, 4, 5, 6, 7, 8 or more of these polynucleotides of the invention. Solid supports and polynucleotides of the present invention attached to solid supports are further described in “Oligonucleotide Probes And Primers”.

6) Integrated Systems

Another technique, which may be used to analyze polymorphisms, includes multicomponent integrated systems, which miniaturize and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device. An example of such technique is disclosed in US patent 5,589,136, the disclosure of which is incorporated herein by reference in its entirety, which describes the integration of PCR amplification and capillary electrophoresis in chips.

Integrated systems can be envisaged mainly when microfluidic systems are used. These systems comprise a pattern of microchannels designed onto a glass, silicon, quartz, or plastic wafer included on a microchip. The movements of the samples are controlled by electric, electroosmotic or hydrostatic forces applied across different areas of the microchip to create functional microscopic valves and pumps with no moving parts.

For genotyping biallelic markers, the microfluidic system may integrate nucleic acid amplification, microsequencing, capillary electrophoresis and a detection method such as laser-induced fluorescence detection.

Methods of Genetic Analysis using the Biallelic Markers of the Present Invention

Different methods are available for the genetic analysis of complex traits (see Lander and Schork, 1994). The search for disease-susceptibility genes is conducted using two main methods: the linkage approach in which evidence is sought for cosegregation between a locus and a putative trait locus using family studies, and the association approach in which evidence is sought for a statistically significant association between an allele or a trait causing allele and a trait (Khoury et al., 1993). In general, the biallelic markers of the present invention find use in any method known in the art to demonstrate a statistically significant correlation between a genotype and a phenotype. The biallelic markers may be used in parametric and non-parametric linkage analysis methods. Preferably, the biallelic markers of the present invention are used to identify genes associated with detectable traits using association studies, an approach which does not require the use of affected families and which permits the identification of genes associated with complex and sporadic traits.

The genetic analysis using the biallelic markers of the present invention may be conducted on any scale. The whole set of biallelic markers of the present invention or any subset of biallelic markers of the present invention corresponding to the candidate gene may be used. Further, any set of genetic markers including a biallelic marker of the present invention may be used. A set of biallelic polymorphisms that could be used as genetic markers in combination with the biallelic markers of the present invention has been described in WO 98120165, the disclosure of which is incorporated herein by reference in its entirety. As mentioned above, it should be noted that the biallelic markers of the present invention may be included in any complete or partial genetic map of the human genome. These different uses are specifically contemplated in the present invention and claims.

Linkage Analysis

Linkage analysis is based upon establishing a correlation between the transmission of genetic markers and that of a specific trait throughout generations within a family. Thus, the aim of linkage analysis is to detect marker loci that show cosegregation with a trait of interest in pedigrees.

Parametric Methods

When data are available from successive generations there is the opportunity to study the degree of linkage between pairs of loci. Estimates of the recombination fraction enable loci to be ordered and placed onto a genetic map. With loci that are genetic markers, a genetic map can be established, and then the strength of linkage between markers and traits can be calculated and used to indicate the relative positions of markers and genes affecting those traits (Weir, 1996). The classical method for linkage analysis is the logarithm of odds (lod) score method (see Morton, 1955; Ott, 1991). Calculation of lod scores requires specification of the mode of inheritance for the disease (parametric method). Generally, the length of the candidate region identified using linkage analysis is between 2 and 20 Mb. Once a candidate region is identified as described above, analysis of recombinant individuals using additional markers allows further delineation of the candidate region. Linkage analysis studies have generally relied on the use of a maximum of 5,000 microsatellite markers, thus limiting the maximum theoretical attainable resolution of linkage analysis to about 600 kb on average.

Linkage analysis has been successfully applied to map simple genetic traits that show clear Mendelian inheritance patterns and which have a high penetrance (i.e., the ratio between the number of trait positive carriers of allele a and the total number of a carriers in the population). However, parametric linkage analysis suffers from a variety of drawbacks. First, it is limited by its reliance on the choice of a genetic model suitable for each studied trait. Furthermore, as already mentioned, the resolution attainable using linkage analysis is limited, and complementary studies are required to refine the analysis of the typical 2 Mb to 20 Mb regions initially identified through linkage analysis. In addition, parametric linkage analysis approaches have proven difficult when applied to complex genetic traits, such as those due to the combined action of multiple genes and/or environmental factors. It is very difficult to model these factors adequately in a lod score analysis. In such cases, too large an effort and cost are needed to recruit the adequate number of affected families required for applying linkage analysis to these situations, as recently discussed by Risch, N. and Merikangas, K. (1 996).

Non-Parametric Methods

The advantage of the so-called non-parametric methods for linkage analysis is that they do not require specification of the mode of inheritance for the disease, they tend to be more useful for the analysis of complex traits. In non-parametric methods, one tries to prove that the inheritance pattern of a chromosomal region is not consistent with random Mendelian segregation by showing that affected relatives inherit identical copies of the region more often than expected by chance. Affected relatives should show excess “allele sharing” even in the presence of incomplete penetrance and polygenic inheritance. In non-parametric linkage analysis the degree of agreement at a marker locus in two individuals can be measured either by the number of alleles identical by state (IBS) or by the number of alleles identical by descent (IBD). Affected sib pair analysis is a well-known special case and is the simplest form of these methods.

The biallelic markers of the present invention may be used in both parametric and non-parametric linkage analysis. Preferably biallelic markers may be used in non-parametric methods which allow the mapping of genes involved in complex traits. The biallelic markers of the present invention may be used in both IBD- and IBS-methods to map genes affecting a complex trait. In such studies, taking advantage of the high density of biallelic markers, several adjacent biallelic marker loci may be pooled to achieve the efficiency attained by multi-allelic markers (Zhao et al., 1998).

Population Association Studies

The present invention comprises methods for identifying if the PCTA-1 gene is associated with a detectable trait using the biallelic markers of the present invention. In one embodiment the present invention comprises methods to detect an association between a biallelic marker allele or a biallelic marker haplotype and a trait. Further, the invention comprises methods to identify a trait causing allele in linkage disequilibrium with any biallelic marker allele of the present invention.

Alternative approaches can be employed to perform association studies: genome-wide association studies, candidate region association studies and candidate gene association studies. In a preferred embodiment, the biallelic markers of the present invention are used to perform candidate gene association studies. The candidate gene analysis clearly provides a short-cut approach to the identification of genes and gene polymorphisms related to a particular trait when some information concerning the biology of the trait is available. Further, the biallelic markers of the present invention may be incorporated in any map of genetic markers of the human genome in order to perform genome-wide association studies. Methods to generate a high-density map of biallelic markers has been described in U.S. Provisional Patent application serial No. 60/082,614. The biallelic markers of the present invention may further be incorporated in any map of a specific candidate region of the genome (a specific chromosome or a specific chromosomal segment for example).

As mentioned above, association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families. Association studies are extremely valuable as they permit the analysis of sporadic or multifactor traits. Moreover, association studies represent a powerful method for fine-scale mapping enabling much finer mapping of trait causing alleles than linkage studies. Studies based on pedigrees often only narrow the location of the trait causing allele. Association studies using the biallelic markers of the present invention can therefore be used to refine the location of a trait causing allele in a candidate region identified by Linkage Analysis methods. Moreover, once a chromosome segment of interest has been identified, the presence of a candidate gene such as a candidate gene of the present invention, in the region of interest can provide a shortcut to the identification of the trait causing allele. Biallelic markers of the present invention can be used to demonstrate that a candidate gene is associated with a trait. Such uses are specifically contemplated in the present invention.

Determining the Frequency of a Biallelic Marker Allele or of a Biallelic Marker Haplotype in a Population

Association studies explore the relationships among frequencies for sets of alleles between loci.

Determining the Frequency of an Allele in a Population

Allelic frequencies of the biallelic markers in a populations can be determined using one of the methods described above under the heading “Methods For Genotyping An Individual For Biallelic Markers”, or any genotyping procedure suitable for this intended purpose. Genotyping pooled samples or individual samples can determine the frequency of a biallelic marker allele in a population. One way to reduce the number of genotypings required is to use pooled samples. A major obstacle in using pooled samples is in terms of accuracy and reproducibility for determining accurate DNA concentrations in setting up the pools. Genotyping individual samples provides higher sensitivity, reproducibility and accuracy and; is the preferred method used in the present invention. Preferably, each individual is genotyped separately and simple gene counting is applied to determine the frequency of an allele of a biallelic marker or of a genotype in a given population.

The invention also relates to methods of estimating the frequency of a PCTA-1-related biallelic marker allele in a population comprising: a) genotyping individuals from said population for said biallelic marker according to the method of the present invention; and b) determining the proportional representation of said biallelic marker in said population. In addition, the methods of estimating the frequency of an allele in a population of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, and A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, and A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94 to A101, A108, A111 to A113, A115 to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, determining the frequency of a biallelic marker allele in a population may be accomplished by determining the identity of the nucleotides for both copies of said biallelic marker present in the genome of each individual in said population and calculating the proportional representation of said nucleotide at said PCTA-1-related biallelic marker for the population; Optionally, determining the proportional representation may be accomplished by performing a genotyping method of the invention on a pooled biological sample derived from a representative number of individuals, or each individual, in said population, and calculating the proportional amount of said nucleotide compared with the total.

Determining the Frequency of a Haplotype in a Population

The gametic phase of haplotypes is unknown when diploid individuals are heterozygous at more than one locus. Using genealogical information in families gametic phase can sometimes be inferred (Perlin et al., 1994). When no genealogical information is available different strategies may be used. One possibility is that the multiple-site heterozygous diploids can be eliminated from the analysis, keeping only the homozygotes and the single-site heterozygote individuals, but this approach might lead to a possible bias in the sample composition and the underestimation of low-frequency haplotypes. Another possibility is that single chromosomes can be studied independently, for example, by asymmetric PCR amplification (see Newton et al, 1989; Wu et al., 1989) or by isolation of single chromosome by limit dilution followed by PCR amplification (see Ruano et al., 1990). Further, a sample may be haplotyped for sufficiently close biallelic markers by double PCR amplification of specific alleles (Sarkar, G. and Sommer S. S., 1991). These approaches are not entirely satisfying either because of their technical complexity, the additional cost they entail, their lack of generalization at a large scale, or the possible biases they introduce. To overcome these difficulties, an algorithm to infer the phase of PCR-amplified DNA genotypes introduced by Clark, A. G.(1990) may be used. Briefly, the principle is to start filling a preliminary list of haplotypes present in the sample by examining unambiguous individuals, that is, the complete homozygotes and the single-site heterozygotes. Then other individuals in the same sample are screened for the possible occurrence of previously recognized haplotypes. For each positive identification, the complementary haplotype is added to the list of recognized haplotypes, until the phase information for all individuals is either resolved or identified as unresolved. This method assigns a single haplotype to each multiheterozygous individual, whereas several haplotypes are possible when there are more than one heterozygous site. Alternatively, one can use methods estimating haplotype frequencies in a population without assigning haplotypes to each individual. Preferably, a method based on an expectation-maximization (EM) algorithm (Dempster et al., 1977) leading to maximum-likelihood estimates of haplotype frequencies under the assumption of Hardy-Weinberg proportions (random mating) is used (see Excoffier L. and Slatkin M., 1995). The EM algorithm is a generalized iterative maximum-likelihood approach to estimation that is useful when data are ambiguous and/or incomplete. The EM algorithm is used to resolve heterozygotes into haplotypes. Haplotype estimations are further described below under the heading “Statistical Methods.” Any other method known in the art to determine or to estimate the frequency of a haplotype in a population may be used.

The invention also encompasses methods of estimating the frequency of a haplotype for a set of biallelic markers in a population, comprising the steps of: a) genotyping at least one PCTA-1-related biallelic marker according to a method of the invention for each individual in said population; b) genotyping a second biallelic marker by determining the identity of the nucleotides at said second biallelic marker for both copies of said second biallelic marker present in the genome of each individual in said population; and c) applying a haplotype determination method to the identities of the nucleotides determined in steps a) and b) to obtain an estimate of said frequency. In addition, the methods of estimating the frequency of a haplotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, and A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, and A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94 to A101, A108, A111 to A113, A115 to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, said haplotype determination method is performed by asymmetric PCR amplification, double PCR amplification of specific alleles, the Clark algorithm, or an expectation-maximization algorithm.

Linkage Disequilibrium Analysis

Linkage disequilibrium is the non-random association of alleles at two or more loci and represents a powerful tool for mapping genes involved in disease traits (see Ajioka R. S. et al., 1997). Biallelic markers, because they are densely spaced in the human genome and can be genotyped in greater numbers than other types of genetic markers (such as RFLP or VNTR markers), are particularly useful in genetic analysis based on linkage disequilibrium.

When a disease mutation is first introduced into a population (by a new mutation or the immigration of a mutation carrier), it necessarily resides on a single chromosome and thus on a single “background” or “ancestral” haplotype of linked markers. Consequently, there is complete disequilibrium between these markers and the disease mutation: one finds the disease mutation only in the presence of a specific set of marker alleles. Through subsequent generations recombination events occur between the disease mutation and these marker polymorphisms, and the disequilibrium gradually dissipates. The pace of this dissipation is a function of the recombination frequency, so the markers closest to the disease gene will manifest higher levels of disequilibrium than those that are further away. When not broken up by recombination, “ancestral” haplotypes and linkage disequilibrium between marker alleles at different loci can be tracked not only through pedigrees but also through populations. Linkage disequilibrium is usually seen as an association between one specific allele at one locus and another specific allele at a second locus.

The pattern or curve of disequilibrium between disease and marker loci is expected to exhibit a maximum that occurs at the disease locus. Consequently, the amount of linkage disequilibrium between a disease allele and closely linked genetic markers may yield valuable information regarding the location of the disease gene. For fine-scale mapping of a disease locus, it is useful to have some knowledge of the patterns of linkage disequilibrium that exist between markers in the studied region. As mentioned above the mapping resolution achieved through the analysis of linkage disequilibrium is much higher than that of linkage studies. The high density of biallelic markers combined with linkage disequilibrium analysis provides powerful tools for fine-scale mapping. Different methods to calculate linkage disequilibrium are described below under the heading “Statistical Methods”.

Population-Based Case-Control Studies of Trait-Marker Associations

As mentioned above, the occurrence of pairs of specific alleles at different loci on the same chromosome is not random and the deviation from random is called linkage disequilibrium. Association studies focus on population frequencies and rely on the phenomenon of linkage disequilibrium. If a specific allele in a given gene is directly involved in causing a particular trait, its frequency will be statistically increased in an affected (trait positive) population, when compared to the frequency in a trait negative population or in a random control population. As a consequence of the existence of linkage disequilibrium, the frequency of all other alleles present in the haplotype carrying the trait-causing allele will also be increased in trait positive individuals compared to trait negative individuals or random controls. Therefore, association between the trait and any allele (specifically a biallelic marker allele) in linkage disequilibrium with the trait-causing allele will suffice to suggest the presence of a trait-related gene in that particular region. Case-control populations can be genotyped for biallelic markers to identify associations that narrowly locate a trait causing allele. As any marker in linkage disequilibrium with one given marker associated with a trait will be associated with the trait. Linkage disequilibrium allows the relative frequencies in case-control populations of a limited number of genetic polymorphisms (specifically biallelic markers) to be analyzed as an alternative to screening all possible functional polymorphisms in order to find trait-causing alleles. Association studies compare the frequency of marker alleles in unrelated case-control populations, and represent powerful tools for the dissection of complex traits.

Case-Control Populations (Inclusion Criteria)

Population-based association studies do not concern familial inheritance but compare the prevalence of a particular genetic marker, or a set of markers, in case-control populations. They are case-control studies based on comparison of unrelated case (affected or trait positive) individuals and unrelated control (unaffected, trait negative or random) individuals. Preferably the control group is composed of unaffected or trait negative individuals. Further, the control group is ethnically matched to the case population. Moreover, the control group is preferably matched to the case-population for the main known confusion factor for the trait under study (for example age-matched for an age-dependent trait). Ideally, individuals in the two samples are paired in such a way that they are expected to differ only in their disease status. The terms “trait positive population”, “case population” and “affected population” are used interchangeably herein.

An important step in the dissection of complex traits using association studies is the choice of case-control populations (see Lander and Schork, 1994). A major step in the choice of case-control populations is the clinical definition of a given trait or phenotype. Any genetic trait may be analyzed by the association method proposed here by carefully selecting the individuals to be included in the trait positive and trait negative phenotypic groups. Four criteria are often useful; clinical phenotype, age at onset, family history and severity. The selection procedure for continuous or quantitative traits (such as blood pressure for example) involves selecting individuals at opposite ends of the phenotype distribution of the trait under study, so as to include in these trait positive and trait negative populations individuals with overlapping phenotypes. Preferably, case-control populations consist of phenotypically homogeneous populations. Trait positive and trait negative populations consist of phenotypically uniform populations of individuals representing each between 1 and 98%, preferably between 1 and 80%, more preferably between 1 and 50%, and more preferably between 1 and 30%, most preferably between 1 and 20% of the total population under study, and preferably selected among individuals exhibiting non-overlapping phenotypes. The clearer the difference between the two trait phenotypes, the greater the probability of detecting an association with biallelic markers. The selection of those drastically different but relatively uniform phenotypes enables efficient comparisons in association studies and the possible detection of marked differences at the genetic level, provided that the sample sizes of the populations under study are significant enough.

In preferred embodiments, a first group of between 50 and 300 trait positive individuals, preferably about 100 individuals, are recruited according to their phenotypes. A similar number of control individuals are included in such studies.

In the present invention, typical examples of inclusion criteria include, but are not restricted to, prostate cancer or aggressiveness of prostate cancer tumors. In one preferred embodiment of the present invention, association studies are carried out on the basis of a presence (trait positive) or absence (trait negative) of prostate cancer.

Associations studies can be carried out by the skilled technician using the biallelic markers of the invention defined above, with different trait positive and trait negative populations. Suitable further examples of association studies using biallelic markers of the PCTA-1 gene, including the biallelic markers A1 to A125, involve studies on the following populations:

a trait positive population suffering from a cancer and a healthy unaffected population, or

a trait positive population suffering from prostate cancer treated with agents acting against prostate cancer and suffering from side-effects resulting from this treatment and an trait negative population suffering from prostate cancer treated with same agents without any substantial side-effects, or

a trait positive population suffering from prostate cancer treated with agents acting against prostate cancer showing a beneficial response and a trait negative population suffering from prostate cancer treated with same agents without any beneficial response, or

a trait positive population suffering from prostate cancer presenting highly aggressive prostate cancer tumors and a trait negative population suffering from prostate cancer with prostate cancer tumors devoid of aggressiveness.

Association Analysis

The general strategy to perform association studies using biallelic markers derived from a region carrying a candidate gene is to scan two groups of individuals (case-control populations) in order to measure and statistically compare the allele frequencies of the biallelic markers of the present invention in both groups.

If a statistically significant association with a trait is identified for at least one or more of the analyzed biallelic markers, one can assume that: either the associated allele is directly responsible for causing the trait (i.e. the associated allele is the trait causing allele), or more likely the associated allele is in linkage disequilibrium with the trait causing allele. The specific characteristics of the associated allele with respect to the candidate gene function usually give further insight into the relationship between the associated allele and the trait (causal or in linkage disequilibrium). If the evidence indicates that the associated allele within the candidate gene is most probably not the trait causing allele but is in linkage disequilibrium with the real trait causing allele, then the trait causing allele can be found by sequencing the vicinity of the associated marker, and performing further association studies with the polymorphisms that are revealed in an iterative manner.

Association studies are usually run in two successive steps. In a first phase, the frequencies of a reduced number of biallelic markers from the candidate gene are determined in the trait positive and control populations. In a second phase of the analysis, the position of the genetic loci responsible for the given trait is further refined using a higher density of markers from the relevant region. However, if the candidate gene under study is relatively small in length, as is the case for PCTA-1, a single phase may be sufficient to establish significant associations.

It is another object of the present invention to provide a method for the identification and characterization of an association between an allele of one or more biallelic markers of a PCTA-1 gene and a trait. The method comprises the steps of:

genotyping a marker or a group of biallelic markers according to the invention in trait positive and control individuals; and

establishing a statistically significant association between one allele of at least one marker and the trait.

The control individuals can be random or trait negative populations. Preferably, the trait positive and trait negative individuals are selected from non-overlapping phenotypes relating trait under study. In some embodiments, the biallelic marker is comprised in one or more of the sequences of P1 to P125, and the complementary sequences thereof.

The invention also comprises methods of detecting an association between a genotype and a phenotype, comprising the steps of a) determining the frequency of at least one PCTA-1-related biallelic marker in a trait positive population according to a genotyping method of the invention; b) determining the frequency of said PCTA-1-related biallelic marker in a control population according to a genotyping method of the invention; and c) determining whether a statistically significant association exists between said genotype and said phenotype. In addition, the methods of detecting an association between a genotype and a phenotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: Optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, and A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, and A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94 to A101, A108, A111 to A113, A115 to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, said control population may be a trait negative population, or a random population; Optionally, each of said genotyping steps a) and b) may be performed on a pooled biological sample derived from each of said populations; Optionally, each of said genotyping of steps a) and b) is performed separately on biological samples derived from each individual in said population or a subsample thereof; Optionally, the identity of the nucleotides at the biallelic markers of the PCTA-1 gene is determined in steps a) and b). Optionally, said phenotype is symptoms of, or susceptibility to cancer, preferably prostate cancer, the level of aggressiveness of prostate cancer tumors, an early onset of prostate cancer, a beneficial response to or side effects related to treatment against prostate cancer.

If the trait is a beneficial response or inversely a side effect to a treatment of prostate cancer, the method of the invention referred to above further comprises some or all of the following steps:

selecting a population or cohort of subjects diagnosed as suffering from prostate cancer;

administering a specified treatment of prostate cancer to said cohort of subjects;

monitoring the outcome of drug administration and identifying those individuals that are trait positive or trait negative relative to the treatment;

taking from said cohort biological samples containing DNA and testing this DNA for the presence of a specific allele or of a set of alleles of biallelic markers of the PCTA-1 gene;

analyzing the distribution of alleles of biallelic markers between trait positive and trait negative individuals; and

performing a statistical analysis to determine a statistically significant association between the presence or absence of alleles of biallelic markers of the PCTA-1 gene and the treatment related trait.

Haplotype Analysis

As described above, when a chromosome carrying a disease allele first appears in a population as a result of either mutation or migration, the mutant allele necessarily resides on a chromosome having a set of linked markers: the ancestral haplotype. This haplotype can be tracked through populations and its statistical association with a given trait can be analyzed. Complementing single point (allelic) association studies with multi-point association studies also called haplotype studies increases the statistical power of association studies. Thus, a haplotype association study allows one to define the frequency and the type of the ancestral carrier haplotype. A haplotype analysis is important in that it increases the statistical power of an analysis involving individual markers.

In a first stage of a haplotype frequency analysis, the frequency of the possible haplotypes based on various combinations of the identified biallelic markers of the invention is determined. The haplotype frequency is then compared for distinct populations of trait positive and control individuals. The number of trait positive individuals, which should be, subjected to this analysis to obtain statistically significant results usually ranges between 30 and 300, with a preferred number of individuals ranging between 50 and 150. The same considerations apply to the number of unaffected individuals (or random control) used in the study. The results of this first analysis provide haplotype frequencies in case-control populations, for each evaluated haplotype frequency a p-value and an odd ratio are calculated. If a statistically significant association is found the relative risk for an individual carrying the given haplotype of being affected with the trait under study can be approximated.

The present invention also provides a method for the identification and characterization of an association between a haplotype comprising alleles of several biallelic markers of the genomic sequence of the PCTA-1 gene and a trait. The method comprises the steps of:

genotyping a group of biallelic markers according to the invention in trait positive and control individuals; and

establishing a statistically significant association between a haplotype and the trait.

Preferably, the control individuals can be random or trait negative populations. In some embodiments, the haplotype comprises two or more biallelic markers comprised in the sequences of P1 to P125, and the complementary sequences thereof.

An additional embodiment of the present invention encompasses methods of detecting an association between a haplotype and a phenotype, comprising the steps of: a) estimating the frequency of at least one haplotype in a trait positive population, according to a method of the invention for estimating the frequency of a haplotype; b) estimating the frequency of said haplotype in a control population, according to a method of the invention for estimating the frequency of a haplotype; and c) determining whether a statistically significant association exists between said haplotype and said phenotype. In addition, the methods of detecting an association between a haplotype and a phenotype of the invention encompass methods with any further limitation described in this disclosure, or those following: Optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A1 to A44, A46 to A53, A57, A58, A62 to A76, A81, A82, A86 to A91, A107, A118, and A123 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A45, A54, A60, A61, A77 to A80, A83 to A85, A93, A102 to A106, A109, A110, A114, and A122, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said PCTA-1-related biallelic marker is selected from the group consisting of A55, A56, A59, A92, A94 to A101, A108, A111 to A113, A115 to A117, and A119 to A121, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; Optionally, said control population is a trait negative population, or a random population. Optionally, said phenotype is symptoms of, or susceptibility to cancer, preferably prostate cancer, the level of aggressiveness of prostate cancer tumors, an early onset of prostate cancer, a beneficial response to or side effects related to treatment against prostate cancer; Optionally, said method comprises the additional steps of determining the phenotype in said trait positive and said control populations prior to step c).

Interaction Analysis

The biallelic markers of the present invention may also be used to identify patterns of biallelic markers associated with detectable traits resulting from polygenic interactions. The analysis of genetic interaction between alleles at unlinked loci requires individual genotyping using the techniques described herein. The analysis of allelic interaction among a selected set of biallelic markers with appropriate level of statistical significance can be considered as a haplotype analysis. Interaction analysis consists of stratifying the case-control populations with respect to a given haplotype for the first loci and performing a haplotype analysis with the second loci with each subpopulation.

Statistical methods used in association studies are further described below.

Testing for Linkage in the Presence of Association

The biallelic markers of the present invention may further be used in TDT (transmission/disequilibrium test). TDT tests for both linkage and association and is not affected by population stratification. TDT requires data for affected individuals and their parents or data from unaffected sibs instead of from parents (see Spielmann S. et al., 1993; Schaid D. J. et al., 1996, Spielmann S. and Ewens W. J., 1998). Such combined tests generally reduce the false—positive errors produced by separate analyses.

Association of Biallelic Markers of the Invention with Prostate Cancer

Trait Positive and Control Populations

Two groups of independent individuals were used: the overall trait positive and the trait negative populations included 491 individuals suffering from prostate cancer and 313 individuals without any sign of prostate cancer. A specific protocol for the collection of DNA samples from trait positive and trait negative individuals is described in Example 5. The 491 individuals suffering from prostate cancer can be subdivided into a population of individuals who developed prostate cancer under 65 years-old and a population of individuals who developed prostate cancer after the age of 65. The population of individuals who are less than 65 years-old was used to determine an association with an early onset of prostate cancer. The affected individuals can also be subdivided in familial cases and sporadic cases.

In order to have as much certainty as possible on the absence of prostate cancer in trait negative individuals, it is preferred to conduct a PSA dosage analysis on this population. Several commercial assays can be used (WO 96/21042, the disclosure of which is incorporated herein by reference in its entirety). In one preferred embodiment, a Hybritech assay is used and trait negative individuals must have a level of PSA less than 2.8 ng/ml of serum in order to be selected as such. In a preferred embodiment, the Yang assay is used and trait negative individuals must have a level of PSA of less than 4 ng/ml of serum in order to be included in the population under study.

Association Analysis

In one preferred embodiment of the invention in which a correlation was found between biallelic markers of the PCTA-1 gene and prostate cancer, results of the association study, further details of which are provided in example 5, seem to indicate that prostate cancer, preferably familial prostate cancer, more preferably early onset familial prostate cancer, is associated most strongly with the biallelic markers A30 (99-1572/440) and A41 (5-171/204) which present a particular interest. These association results constitute new elements for studying the genetic susceptibility of individuals to prostate cancer, preferably to familial prostate cancer, more preferably familial early onset prostate cancer. Further details concerning this association study are provided below.

The biallelic markers most strongly associated with prostate cancer, namely A30 and A41, are located in the regulatory region of the PCTA-1 gene, more particularly in the promoter region. The consequences of the presence of these markers in these regions are discussed below.

Furthermore, the biallelic marker A2 (99-1601/402) was found to be also associated with prostate cancer, more particularly with sporadic prostate cancer. This biallelic marker is localized in the 5′ regulatory region of the PCTA-1 gene.

Similar association studies can also be carried out with other biallelic markers within the scope of the invention, preferably with biallelic markers in linkage disequilibrium with the markers associated with prostate cancer as described above, including the biallelic markers A1 to A125.

Analysis of Biallelic Marker Associations

Even though polymorphisms associated with prostate cancer have been identified in the coding region of the PCTA-1 gene, these polymorphisms do not appear to be as significant as those found in the upstream regulatory region of the PCTA-1 gene. The results further suggest that a trait-causing mutation is likely to be located within the 5′ regulatory region of the PCTA-1 gene. The extent to which the markers found within the coding region of PCTA-1 are significant in relation to cancer can be determined using haplotype analyses involving at least two of the biallelic markers of the present invention.

Six of the biallelic markers of the present invention result in a change in the amino acid sequence of a PCTA-1 protein. These are biallelic markers A54, A56, A60, A75, A76 and A85. These mutations may change the function and/or the stability of the PCTA-1 protein. An amino acid change in a PCTA-1 protein can lead to alterations in PCTA-1 biological activity. Either a modified function or an increased stability can be involved in prostate cancer appearance.

Furthermore, as the expression of the PCTA-1 gene has mainly been reported in prostate cancer cells, one can assume that its expression is closely linked to the development of cancer, particularly prostate cancer. Generally, a major control of gene expression proceeds at the level of the initiation of the transcription. This initiation involves the promoter which can be considered as a concentration of transcription factor binding sites. The initiation of the transcription also involves enhancers which modulate the efficiency of the initiation and consist of DNA binding sites which are located in regulatory regions of the considered gene which may be at a certain distance in 3′ or 5′ of the gene.

Most of the biallelic polymorphisms of the PCTA-1 gene associated with prostate cancer according to the present invention are located in the regulatory region upstream of the transcription start site of the PCTA-1 gene and particularly in the promoter. Biallelic marker A41, which is located about 120 bp upstream of the beginning of the first exon (exon 0), may be comprised in the proximal promoter of the PCTA-1 gene. This biallelic marker could be a trait causing mutation of prostate cancer. Biallelic marker A30, which is located about 1.5 kb upstream the beginning of the first exon (exon 0), may be comprised in the distal promoter of the PCTA-1 gene. Biallelic marker A2 is located in the 5′ regulatory region of the PCTA-1 gene.

As the expression of the PCTA-1 gene has mainly been reported in prostate cancer cells, the expression of PCTA-1 gene is modified during the carcinogenesis. The exact mechanism through which PCTA expression is modified is not understood. However, it is possible that the polymorphisms A41, A30, and A2 modulate PCTA-1 expression by modulating PCTA-1 transcription through DNA binding proteins, which will be explained in further detail below.

The regulation of PCTA-1 expression is a key factor in the onset and for development of cancer and particularly prostate cancer. In this regard, the polymorphisms located in the 5′ regulatory region of the PCTA-1 gene appear to play the most significant role in the association of PCTA-1 with cancer. It appears clear that the polymorphisms found in the promoter region adjacent to the transcription initiation site, and particularly those located in the proximal PCTA-1 promoter, are more strongly associated with prostate cancer than polymorphisms of the other promoter elements located further upstream of this site. Furthermore, some polymorphisms, such as the biallelic marker A41, are clearly associated with early onset prostate cancer. The polymorphisms found in the proximal 2000 to 3000 bp of the 5′ regulatory region are associated with early onset prostate cancer. The inventors have also shown an association between some of the biallelic markers of the present invention located at the 3′ end of the PCTA-1 genomic DNA and prostate cancer.

The involvement of the associated polymorphisms in the modification of the PCTA-1 expression in prostate cancer cells can be confirmed through the assays described below.

The expression levels of a PCTA-1 gene, preferably a gene comprising at least one biallelic marker according to the invention, in different tissues, can be determined by analyses of tissue samples from individuals typed for the presence or absence of a specific polymorphism. Any convenient method can be used such as Northern, or Dot blot or other hybridization analyses, and quantitative RT-PCR for mRNA quantitation, Western blot ELISA, R1A for protein quantitation. The tissue specific expression can then be correlated with the genotype. More details on some of these methods are provided below under the heading “Method For Screening”.

The effects of modifications in the regulatory regions of the PCTA-1 gene, and particularly in the sequence of its promoter, can be studied through the determination of expression levels by expression assays for the particular promoter sequence. The assays are performed with the PCTA-1 coding sequence or with a detectable marker sequence using a reporter gene. To determine tissue specificity, the assay is performed in cells from different sources. Preferably the assay is performed on normal tissue cells and cancerous cells of the same tissue type (e.g. prostate cells and on prostate cancer cells). More preferably, the assay is performed on a large range of cell lines with an increasing level of malignancy. Some methods are discussed in more detail below under the heading “Method For Screening”.

An assay to determine the effect of a sequence polymorphism on PCTA-1 expression may be performed in cell-free extracts, or in cellculture assays, such as transient or stable transfection assays. This assay is also within the scope of the present invention. Alterations in expression may be correlated to decreases or increases in the basic amounts of PCTA-1 mRNA and/or protein that are expressed in one or more cell types. Expression levels of different alleles are compared using various methods known in the art. Methods for determining whether the level of expression triggered by promoter or enhancer sequences is increased or decreased depending on the studied allele of said sequence include the insertion into a vector of said sequence upstream a reporter gene such as β-galactosidase, luciferase, green fluorescent protein or chloramphenicol acetyltransferase. Expression levels are assessed by quantitation of expressed reporter proteins that provides for convenient quantitation.

The changes in PCTA-1 expression can be the result of modifications in the modulation of PCTA-1 transcription by DNA binding proteins, which are able to activate or inhibit the initiation of the transcription of the PCTA-1 gene. The term “DNA binding protein” is intended to encompass more particularly transcriptional factors. The binding of these proteins on the sites located in the promoter is critical for a correct binding of polymerases and consequently for the initiation of transcription. The binding of these proteins on the sites located in the 5′ upstream regulatory regions modulates transcription.

The binding sites of DNA binding proteins, preferably transcription factors, are generally 6-20 nucleotides in length. A polymorphic site located in a transcription factor binding site may result in a difference of binding affinity of the said transcription factor between the two allele of the polymorphism. This difference of affinity could explain the changes of expression of the PCTA-1 gene.

When one or more alleles of the biallelic markers of the PCTA-1 gene associated with cancer are present in the genome of an individual since conception, there would be an event which provokes a drastic increase in the expression of PCTA-1. There are at least two possible hypotheses that can be formulated to explain this event. Firstly, as cancer is the result of a succession of mutations, one mutation could lead to either the expression of a new DNA binding activity, or the overexpression of a DNA binding factor which binds to the site containing the polymorphism and which is involved in the transcription of the PCTA-1 gene. Secondly the DNA binding factor readily binds to the site containing the polymorphism in normal cells where it is either unable to activate the transcription of PCTA-1 or repressor of the PCTA-1 transcription initiation. A mutation in the transcription factor can make the transcription factor either functional in the case of an activator or unfunctional in the case of a repressor. Likewise, a mutation in an additional protein can induce the binding of this protein which is needed by the DNA binding factor for activating the transcription of the PCTA-1 gene.

In order to confirm the capacity of transcription factors to bind sites containing the biallelic markers of the present invention, so as to assess the difference in affinity between the two alleles of the considered biallelic marker and to discriminate between these hypotheses, a gel retardation assay or DNA mobility shift assay can be carried out. This type of assay is well-known to those skilled in the art and is described in U.S. Pat. No. 5,698,389, U.S. Pat. No. 5,502, 176, Fried and Crothers (1981), Garner and Revzin (1981) and Dent and Latchman (1993).

This type of method relies on the principle that a fragment of DNA to which a protein has bound will move more slowly in gel electrophoresis than the same DNA fragment without the bound protein. The DNA mobility shift assay is carried out, therefore, by first labeling the specific DNA segment whose protein-binding properties are being investigated. The labeled DNA is then incubated with a nuclear (Dignam et al., 1983; Schreiber et al., 1989; Mulleret al., 1989; Mizokami et al., 1994) or whole cell (Manley et al., 1980) extract of cells prepared in such a way as to contain DNA-binding proteins. DNA-protein complexes are then allowed to form. The complexes are then electrophoresed on a non-denaturing polyacrylamide gel and the position of the labeled DNA is visualized by suitable techniques. Various types of suitable labels can be selected by the person skilled in the art. Notably, the radioactive labeling is appropriate. If no protein has bound to the DNA, all the label is free to migrate quickly, whereas labeled protein-DNA complexes migrate more slowly and hence give a different signal from that of the unbound DNA near the top of the gel. The interaction specificity can be estimated by carrying out a gel retardation assay with increasing amount of unlabeled DNA segment which can compete with the labeled one. A positive control can be realized with an oligonucleotide containing the androgene responsive element.

The investigated DNA segment preferably comprises the sequence of a potential binding site containing an allele of a polymorphism of the present invention, more preferably a sequence comprising a sequence selected from P1 to P125 and the complementary sequences thereto, still more preferably a sequence comprising a sequence selected from P1 to P43 and the complementary sequences thereto. In an embodiment, the polymorphism site is located in the middle of the DNA fragment. In an other embodiment, the polymorphism site can be located close to an end of the DNA fragment, for example at 6 nucleotides away from the end. The DNA fragment has a sufficient length to hybridize with the complementary strand and to form a stable double strand. For example, the DNA fragment comprises at least 8 nucleotides, preferably at least 20 nucleotides, more preferable 30 nucleotides. In a specific embodiment, the DNA fragment comprises the sequence of interest at the middle of the fragment and some poly G, poly C, or poly GC at its 5′ and/or 3′ ends.

In a preferred embodiment, the DNA segment consists of an oligonucleotide selected from the group consisting of Oligo1 to Oligo60 which are described in Table C and detailed as feature in SEQ ID No 1. For each polymorphic site, 4 oligonucleotides are generated and correspond to the two complementary strands of the DNA for each of the two alleles of the considered polymorphism. The DNA segments are designed such as the polymorphic base is surrounded with 14 nucleotides on each side.

TABLE C
					Complementary
			Position range of the		position range of the
		Oligo-	oligonucleotide	Oligo-	oligonucleotide in SEQ
Biallelic		nucleotide	in SEQ ID No 1	nucleotide	ID No 1
marker	All	name	Beginning	End	name	Beginning	End
5-169-208	A	Oligo1	67820	67848	Oligo31	67820	67850
5-169-208	G	Oligo2	67820	67848	Oligo32	67820	67850
5-169-331	C	Oligo3	67940	67969	Oligo33	67941	67969
5-169-331	T	Oligo4	67940	67969	Oligo34	67941	67969
5-169-97	C	Oligo5	67707	67737	Oligo35	67709	67738
5-169-97	G	Oligo6	67707	67737	Oligo36	67709	67738
5-170-238	A	Oligo7	68198	68227	Oligo37	68199	68228
5-170-238	G	Oligo8	68198	68227	Oligo38	68199	68228
5-170-288	A	Oligo9	68247	68277	Oligo39	68249	68277
5-170-288	C	Oligo10	68247	68277	Oligo40	68249	68277
5-171-156	G	Oligo11	68463	68491	Oligo41	68463	68492
5-171-156	T	Oligo12	68463	68491	Oligo42	68463	68492
5-171-204	C	Oligo13	68511	68539	Oligo43	68511	68539
5-171-204	T	Oligo14	68511	68539	Oligo44	68511	68539
5-171-273	A	Oligo15	68580	68608	Oligo45	68580	68608
5-171-273	G	Oligo16	68580	68608	Oligo46	68580	68608
5-171-289	C	Oligo17	68596	68624	Oligo47	68596	68626
5-171-289	T	Oligo18	68596	68624	Oligo48	68596	68626
5-171-54	C	Oligo19	68360	68389	Oligo49	68361	68389
5-171-54	G	Oligo20	68360	68389	Oligo50	68361	68389
99-1572-315	C	Oligo21	66951	66981	Oligo51	66953	66983
99-1572-315	T	Oligo22	66951	66981	Oligo52	66953	66983
99-1572-335	A	Oligo23	66973	67001	Oligo53	66973	67002
99-1572-335	G	Oligo24	66973	67001	Oligo54	66973	67002
99-1572-440	C	Oligo25	67078	67106	Oligo55	67078	67106
99-1572-440	T	Oligo26	67078	67106	Oligo56	67078	67106
99-1572-477	A	Oligo27	67113	67143	Oligo57	67115	67144
99-1572-477	T	Oligo28	67113	67143	Oligo58	67115	67144
99-1572-578	C	Oligo29	67212	67243	Oligo59	67215	67247
99-1572-578	T	Oligo30	67212	67243	Oligo60	67215	67247

Each oligonucleotide selected from Oligo1 to Oligo60 comprises 4 additional bases, namely GATC, at its 5′ end.

In a preferred embodiment, either the nuclear or whole cell extracts are provided from normal and cancer cells, particularly from normal prostate cells and prostate cancer cells. For example, suitable cell extracts can be provided from PZ-HPV-7 (ATCC: CRL-222 1), CA-HPV-10 (ATCC CRL-2220), PC-3 (ATCC: CRL-1435), DU 145 (ATCC: HTB-81), LNCaP-FGC (ATCC: CRL-10995 and CRL-1740), or NCI-H660 (ATCC: CRL-5813) cells. In a more preferred embodiment, the cell extracts are provided form PNT1A, PNT2, LNCaP-JMV, DU145 (ATCC Nr: HTB-81) or PC3 (ATCC Nr: CRL-1435) cells.

In case a new transcription factor is specifically expressed in cancer cells, a gel retardation assay will show a retarded or shifted band only when the DNA was incubated with cell extracts from prostate cancer cells. If the DNA binding activity already exists in normal cells, the gel retardation assay will show a shifted band with cell extracts from normal prostate cells and prostate cancer cells. Gel retardation assays will also allow to show a significant difference in affinity between a DNA binding factor and binding sites containing the two alleles of the considered polymorphism.

The interaction of the DNA segment described above with transcription factors can also be studied with an optical biosensor such as BIACORE. This technology is well-known to those skilled in the art and is described in Szabo et al. (1995) and Edwards et al. (1997). The main advantage of this method is that it allows the determination of the association rate between the DNA fragment which is investigated and the DNA binding protein. Typically, a DNA segment such as those defined above is biotinylated at its 5′ or 3′ ends and is immobilized on a streptavidin-coated sensor chip. Then, a whole or a nuclear extract of cells is placed in contact with the DNA segment. The binding of DNA binding proteins to the DNA fragment causes a change in the refractive index and/or thickness. This change is detected by the Biosensor provided it occurs in the evanescent field. The affinity of the DNA binding protein to the DNA fragment can then be measured.

In order to precisely localize the binding site of the transcription factors, DNAse I footprinting or DMS protection footprinting assays can also be carried out with DNA fragments which contain the sequence of a potential binding site containing an allele of a polymorphism of the present invention, preferably a sequence comprising a sequence selected from P1 to P125 and the complementary sequences thereto, more preferably a sequence comprising a sequence selected from P1 to P43 and the complementary sequences thereto. This type of assay is well-known to those skilled in the art and is described in Galas and Schmitz (1978), and Dynan and Tjian (1 983). Briefly, in the DNAse I footprinting assay, end-labeled DNA is incubated with protein extract and then partially digested with DNAse I. Specific binding of proteins to DNA will modify nuclease digestion at the site of interaction relative to free DNA, leaving an “imprint” which can be visualized after extraction of the labeled DNA and electrophoresis in a sequence gel.

The interaction with transcription factors can also be studied with the methylation interference assay which is well-known to those skilled in the art and is described in Siebenlist and Gilbert (1980) and Maxam and Gilbert (1980). Briefly, this method relies on the ability of DMS to methylate G residues, which can be cleaved with piperidine. The target DNA is partially methylated so that, on average, only one G residue per DNA molecule is methylated. These partially methylated molecules is used in a DNA mobility shift experiment with an appropriate cell extract containing transcription factors. After electrophoresis, the band produced by the DNA which has bound protein and that produced by the unbound DNA are excised from the gel and treated with piperidine to cleave the DNA at the methylated G residues and not at unmethylated G residues. If methylation of a particular G residue prevents transcription factors binding, then cleavage at this methylated G residue will be observed only in the DNA that failed to bind the protein.

In order to confirm the implication of a particular PCTA-1 derived sequence containing the biallelic marker as a binding site for a transcription regulator of PCTA-1 in cancer cells, a transient expression assay can be carried out in which a vector comprising the considered binding site upstream of the HSV1 thymidine kinase promoter operably linked to a reporter gene such as chloramphenicol acetyltransferase is transfected in appropriate cell lines. This assay is well-known to those skilled in the art and is described in Doucas et al. (1991). This assay can also be realized by cloning the considered binding site upstream the SV40 promoter into the pGL3-promoter luciferase vector (Promega) as described in Coles et al.(1998). Both normal and cancer cells, more particularly normal and cancer cells from prostate, are transfected with said vector. The effect of the binding site and more particularly of the alleles comprised in the binding site can be assessed through the expression level of the reporter gene.

The inventors believe that these polymorphisms, particularly the polymorphisms located on or close to polyadenylation sites have a direct although somewhat milder effect on prostate cancer development.

Haplotype Analysis

In the context of the present invention, a haplotype can be defined as a combination of biallelic markers found in a given individual and which may be associated more or less significantly, as a result of appropriate statistical analyses, with the expression of a given trait.

A two-marker haplotype including markers A30 and A41 (TT alleles respectively) was shown to be significantly associated with prostate cancer, preferably with a familial prostate cancer, more preferably with a familial early onset prostate cancer. As shown in Table 8, the “TT” haplotype present a p-value of 2.5×10 −6 for the familial early onset prostate cancer (see Example 5).

A three-marker haplotype including markers A2, A30, and A41 (ATT alleles respectively) was shown to be significantly associated with prostate cancer, preferably with a familial prostate cancer, more preferably with a familial early onset prostate cancer. As shown in table 8, the “ATT” haplotype present a p-value of 2.5×10 −7 for the familial early onset prostate cancer (see Example 5).

A first two-marker haplotype including markers A2 and A57 (99-1605/112) (TA alleles, respectively) was shown to be significantly associated with prostate cancer, preferably with a sporadic prostate cancer. As shown in table 8, the “TA” haplotype present a p-value of 3.4×10 −5 for the sporadic informative prostate cancer (see Example 5). A second two-marker haplotype including markers A2 and A55 (5-2/178) (TT alleles, respectively) was shown to be significantly associated with prostate cancer, preferably with a sporadic prostate cancer. As shown in table 8, the “TT” haplotype present a p-value of 1×10 −5 for the sporadic informative prostate cancer (see Example 5).

Therefore, one preferred haplotype of the present invention associated with a familial prostate cancer comprises a biallelic marker selected from the group consisting of A30 (allele T), A41 (allele T), A2 (allele A), A55 (allele C) and A57 (allele G). One more preferred haplotype of the present invention associated with a familial prostate cancer comprises a biallelic marker selected from the group consisting of A30 (allele T), A41 (allele T), and A2 (allele A). One still more haplotype of the present invention associated with a familial prostate cancer comprises a biallelic marker selected from the group consisting of A30 (allele T), and A41 (allele T).

Furthermore, one preferred haplotype of the present invention associated with a sporadic prostate cancer comprises a biallelic marker selected from the group consisting of A2 (allele T), A55 (allele T), A57 (allele A), A30 (allele T) and A41 (allele T). One more preferred haplotype of the present invention associated with a sporadic prostate cancer comprises a biallelic marker selected from the group consisting of A2 (allele T), A41 (allele T), A55 (allele T), A57 (allele A).

The permutation tests clearly validated the statistical significance of the association between these haplotypes and the prostate cancer (see Example 5). All these haplotypes can be used in diagnostic of prostate cancer, more particularly either familial prostate cancer or sporadic prostate cancer.

One can observe that the haplotypes associated to familial cases of prostate cancer are not associated with the sporadic cases of prostate cancer and that the haplotypes associated to the sporadic cases are not associated with the familial cases (see Table 7 of Example 5). Moreover, except the biallelic markers A2, the familial and sporadic cases haplotypes do not present any common biallelic marker. Therefore, the ancestral haplotypes would be different and the causing trait allele would not be the same.

This information is extremely valuable. The knowledge of a potential genetic predisposition to prostate cancer, even if this predisposition is not absolute, might contribute in a very significant manner to treatment efficacy of prostate cancer and to the development of new therapeutic and diagnostic tools.

Statistical Methods

In general, any method known in the art to test whether a trait and a genotype show a statistically significant correlation may be used.

1) Methods in Linkage Analysis

Statistical methods and computer programs useful for linkage analysis are well-known to those skilled in the art (see Terwilliger J. D. and Ott J., 1994; Ott J., 1991).

2) Methods to Estimate Haplotype Frequencies in a Population

As described above, when genotypes are scored, it is often not possible to distinguish heterozygotes so that haplotype frequencies cannot be easily inferred. When the gametic phase is not known, haplotype frequencies can be estimated from the multilocus genotypic data. Any method known to person skilled in the art can be used to estimate haplotype frequencies (see Lange K., 1997; Weir, B. S., 1996) Preferably, maximum-likelihood haplotype frequencies are computed using an Expectation- Maximization (EM) algorithm (see Dempster et al., 1977; Excoffier L. and Slatkin M., 1995). This procedure is an iterative process aiming at obtaining maximum-likelihood estimates of haplotype frequencies from multi-locus genotype data when the gametic phase is unknown. Haplotype estimations are usually performed by applying the EM algorithm using for example the EM-HAPLO program (Hawley M. E. et al., 1994) or the Arlequin program (Schneider et al., 1997). The EM algorithm is a generalized iterative maximum likelihood approach to estimation and is briefly described below.

Please note that in the present section, “Methods To Estimate Haplotype Frequencies In A Population,” of this text, phenotypes will refer to multi-locus genotypes with unknown phase. Genotypes will refer to known-phase multi-locus genotypes.

A sample of N unrelated individuals is typed for K markers. The data observed are the unknown-phase K-locus phenotypes that can categorized in F different phenotypes. Suppose that we have H underlying possible haplotypes (in case of K biallelic markers, H=2 K ).

For phenotype j, suppose that c j genotypes are possible. We thus have the following equation $$ P j = ∑ i = 1 c j ⁢ pr ⁡ ( genotype i ) = ∑ i = 1 c j ⁢ pr ⁡ ( h k , h l ) Equation ⁢   ⁢ 1

where P j is the probability of the phenotype j, h k and h l are the two haplotypes constituent the genotype i. Under the Hardy-Weinberg equilibrium, pr(h k , h l ) becomes:

pr ( h k , h l )= pr ( h k ) 2 if h k =h l , pr ( h k , h l )=2 pr ( h k ). pr ( h l ) if h k ≠h l .  Equation 2

The successive steps of the E-M algorithm can be described as follows:

Starting with initial values of the of haplotypes frequencies, noted p 1 (0) , p 2 (0) , . . . p H (0) , these initial values serve to estimate the genotype frequencies (Expectation step) and then estimate another set of haplotype frequencies (Maximization step), noted p 1 (1) , p 2 (1) , . . . p H (1) , these two steps are iterated until changes in the sets of haplotypes frequency are very small.

A stop criterion can be that the maximum difference between haplotype frequencies between two iterations is less than 10 −7 . These values can be adjusted according to the desired precision of estimations.

At a given iteration s, the Expectation step consists of calculating the genotypes frequencies by the following equation: $$ &AutoLeftMatch; pr ⁡ ( genotype i ) ( s ) =   ⁢ pr ⁡ ( phenotype j ) ·   ⁢ pr ⁢ ( genotype i &RightBracketingBar; ⁢ phenotype j ) ( s ) =   ⁢ n j N · pr ⁡ ( h k , h l ) ( s ) P j ( s ) Equation ⁢   ⁢ 3

where genotype i occurs in phenotype j, and where h k and h l constitute genotype i. Each probability is derived according to eq. 1, and eq. 2 described above.

Then the Maximization step simply estimates another set of haplotype frequencies given the genotypes frequencies. This approach is also known as the gene-counting method (Smith, 1957). $$ p t ( s + 1 ) = 1 2 ⁢ ∑ j = 1 F ⁢ ∑ i = 1 c j ⁢ δ it · pr ⁡ ( genotype i ) ( s ) Equation ⁢   ⁢ 4

Where δ it is an indicator variable which count the number of time haplotype t in genotype i. It takes the values of 0, 1 or 2.

To ensure that the estimation finally obtained is the maximum-likelihood estimation several values of departures are required. The estimations obtained are compared and if they are different the estimations leading to the best likelihood are kept.

3) Methods to Calculate Linkage Disequilibrium Between Markers

A number of methods can be used to calculate linkage disequilibrium between any two genetic positions, in practice linkage disequilibrium is measured by applying a statistical association test to haplotype data taken from a population.

Linkage disequilibrium between any pair of biallelic markers comprising at least one of the biallelic markers of the present invention (M i , M j ) having alleles (a i /b i ) at marker M i and alleles (a j /b j ) at marker M j can be calculated for every allele combination (a i ,a j , a i ,b j : b i ,a j and b i ,b j ), according to the Piazza formula:

Δ aiaj =θ4−(θ4+θ3) (θ4+θ2), where:

θ4=−−=frequency of genotypes not having allele a i at M i and not having allele a j at M j

θ3=−+=frequency of genotypes not having allele a i at M i and having allele a j at M j

θ2=+−=frequency of genotypes having allele a i at M i and not having allele a j at M j

Linkage disequilibrium (LD) between pairs of biallelic markers (M i ; M j ) can also be calculated for every allele combination (ai,aj; ai,bj; b i ,a j and b i ,b j ), according to the maximum-likelihood estimate (MLE) for delta (the composite genotypic disequilibrium coefficient), as described by Weir (Weir B. S., 1996). The MLE for the composite linkage disequilibrium is:

D aiaj (2 n 1 +n 2 +n 3 +n 4 /2)/ N −2( pr ( a i ). pr ( a j ))

Where n 1 =Σ phenotype (a i /a i ,a j /a j ), n 2 =Σ phenotype (a i /a i , a j /b j ), n 3 =Σ phenotype (a i ,/b i , a j /a j ), n 4 =Σ phenotype (a i /b i , a j /b j ) and N is the number of individuals in the sample.

This formula allows linkage disequilibrium between alleles to be estimated when only genotype, and not haplotype, data are available.

Another means of calculating the linkage disequilibrium between markers is as follows. For a couple of biallelic markers, M i (a i /b i ) and M j (a j /b j ), fitting the Hardy-Weinberg equilibrium, one can estimate the four possible haplotype frequencies in a given population according to the approach described above.

The estimation of gametic disequilibrium between ai and aj is simply:

D aiaj =pr (haplotype( a i ,a j ))− pr ( a i ). pr ( a j ).

Where pr(a i ) is the probability of allele a i and pr(a i ) is the probability of allele a j and where pr(haplotype (a i , a j )) is estimated as in Equation 3 above.

For a couple of biallelic marker only one measure of disequilibrium is necessary to describe the association between M i and M j .

Then a normalized value of the above is calculated as follows:

D ′ aiaj =D aiaj /max (− pr ( a i ). pr (a j ), − pr ( b i ). pr ( b j )) with D aiaj <0

D ′ aiaj =D aiaj /max ( pr ( b i ). pr (a j ), pr ( a i ). pr ( b j )) with D aiaj >0

The skilled person will readily appreciate that other linkage disequilibrium calculation methods can be used.

Linkage disequilibrium among a set of biallelic markers having an adequate heterozygosity rate can be determined by genotyping between 50 and 1000 unrelated individuals, preferably between 75 and 200, more preferably around 100.

4) Testing for Association

Methods for determining the statistical significance of a correlation between a phenotype and a genotype, in this case an allele at a biallelic marker or a haplotype made up of such alleles, may be determined by any statistical test known in the art and with any accepted threshold of statistical significance being required. The application of particular methods and thresholds of significance are well with in the skill of the ordinary practitioner of the art.

Testing for association is performed by determining the frequency of a biallelic marker allele in case and control populations and comparing these frequencies with a statistical test to determine if their is a statistically significant difference in frequency which would indicate a correlation between the trait and the biallelic marker allele under study. Similarly, a haplotype analysis is performed by estimating the frequencies of all possible haplotypes for a given set of biallelic markers in case and control populations, and comparing these frequencies with a statistical test to determine if their is a statistically significant correlation between the haplotype and the phenotype (trait) under study. Any statistical tool useful to test for a statistically significant association between a genotype and a phenotype may be used. Preferably the statistical test employed is a chi-square test with one degree of freedom. A P-value is calculated (the P-value is the probability that a statistic as large or larger than the observed one would occur by chance).

Statistical Significance

In preferred embodiments, significance for diagnosis purposes, either as a positive basis for further diagnostic tests or as a preliminary starting point for early preventive therapy, the p value related to a biallelic marker association is preferably about 1×10 −2 or less, more preferably about 1×10 −4 or less, for a single biallelic marker analysis and about 1×10 −3 or less, still more preferably 1×10 −6 or less and most preferably of about 1×10 −8 or less, for a haplotype analysis involving two or more markers. These values are believed to be applicable to any association studies involving single or multiple marker combinations.

The skilled person can use the range of values set forth above as a starting point in order to carry out association studies with biallelic markers of the present invention. In doing so, significant associations between the biallelic markers of the present invention and prostate cancer, the level of aggressiveness of prostate cancer tumors, an early onset of prostate cancer, or a beneficial response to or side effects related to treatment against prostate cancer can be revealed and used for diagnosis and drug screening purposes.

Phenotypic Permutation

In order to confirm the statistical significance of the first stage haplotype analysis described above, it might be suitable to perform further analyses in which genotyping data from case-control individuals are pooled and randomized with respect to the trait phenotype. Each individual genotyping data is randomly allocated to two groups, which contain the same number of individuals as the case-control populations used to compile the data obtained in the first stage. A second stage haplotype analysis is preferably run on these artificial groups, preferably for the markers included in the haplotype of the first stage analysis showing the highest relative risk coefficient. This experiment is reiterated preferably at least between 100 and 10000 times. The repeated iterations allow the determination of the probability to obtain by chance the tested haplotype.

Assessment of Statistical Association

To address the problem of false positives similar analysis may be performed with the same case-control populations in random genomic regions. Results in random regions and the candidate region are compared as described in a co-pending US Provisional Patent Application entitled “Methods, Software And Apparati For Identifying Genomic Regions Harboring A Gene Associated With A Detectable Trait,” U.S. Ser. No. 60/107,986, filed Nov. 10, 1998.

5) Evaluation of Risk Factors

The association between a risk factor (in genetic epidemiology the risk factor is the presence or the absence of a certain allele or haplotype at marker loci) and a disease is measured by the odds ratio (OR) and by the relative risk (RR). If P(R + ) is the probability of developing the disease for individuals with R and P(R − ) is the probability for individuals without the risk factor, then the relative risk is simply the ratio of the two probabilities, that is:

RR=P ( R + )/ P ( R − )

In case-control studies, direct measures of the relative risk cannot be obtained because of the sampling design. However, the odds ratio allows a good approximation of the relative risk for low-incidence diseases and can be calculated:

OR =( F + /(1 −F + ))/( F − /(1 −F − ))

F + is the frequency of the exposure to the risk factor in cases and F − is the frequency of the exposure to the risk factor in controls. F + and F − are calculated using the allelic or haplotype frequencies of the study and further depend on the underlying genetic model (dominant, recessive, additive . . . ).

One can further estimate the attributable risk (AR) which describes the proportion of individuals in a population exhibiting a trait due to a given risk factor. This measure is important in quantifying the role of a specific factor in disease etiology and in terms of the public health impact of a risk factor. The public health relevance of this measure lies in estimating the proportion of cases of disease in the population that could be prevented if the exposure of interest were absent. AR is determined as follows:

AR=P E ( RR −1)/( P E ( RR− 1 )+1)

AR is the risk attributable to a biallelic marker allele or a biallelic marker haplotype. P E is the frequency of exposure to an allele or a haplotype within the population at large; and RR is the relative risk which, is approximated with the odds ratio when the trait under study has a relatively low incidence in the general population.

Identification of Biallelic Markers in Linkage Disequilibrium with the PCTA-1-Related Biallelic Markers

Once an association has been demonstrated between a given biallelic marker and a trait, the discovery of additional biallelic markers associated to trait and in linkage disequilibrium with one of the biallelic markers disclosed herein can easily be carried out by the skilled person.

The present invention then also concerns biallelic markers in linkage disequilibrium with the specific biallelic markers described above, more particularly with biallelic markers Al to A125, and which are expected to present similar characteristics in terms of their respective association with a given trait.

Hence, once linkage disequilibrium has been demonstrated between a trait and a given biallelic marker, all the biallelic markers shown to be in linkage disequilibrium with the given biallelic marker are expected to present similar characteristics in terms of their respective association with a given trait. The discovery of additional biallelic markers associated with this trait is of great interest in order to increase the density of biallelic markers in this particular region because the causal mutation will be found in the vicinity of the marker or set of markers showing the highest correlation with the trait. These additional markers which can be identified and sequenced by the skilled person using the teachings of the present application also fall within the scope of the present invention.

The invention also concerns a method for the identification and characterization of a biallelic marker in linkage disequilibrium with a biallelic marker of the PCTA-1 gene, preferably a biallelic marker of the PCTA-1 gene of which one allele is associated with a trait In one embodiment, the biallelic marker of the PCTA-1 gene is outside of the PCTA-1 gene itself. In another embodiment, the biallelic marker in linkage disequilibrium with a biallelic marker of the PCTA-1 gene is itself located within the PCTA-1 gene. The method comprises the following steps: (a) amplifying a genomic fragment, preferably comprising a first biallelic marker, from a plurality of individuals; (b) identifying second biallelic markers in said amplified portion; (c) conducting a linkage disequilibrium analysis between said first biallelic marker and second biallelic markers; and, (d) identifying second biallelic markers in linkage disequilibrium with said first marker. Subcombinations comprising steps (b) and (c) are also contemplated. Optionally, the first biallelic marker is selected from the group consisting of A1 to A125 and the complements thereof. Preferably, the first biallelic marker is selected from the group consisting of A2, A30, A41, A55, A57 and the complements thereof.

Methods to identify biallelic markers and to conduct linkage disequilibrium analysis are described herein and can be carried out by the skilled person without undue experimentation.

Once identified, the sequences in linkage disequilibrium with a biallelic marker of the PCTA-1 gene may be used in any of the methods described herein, including methods for determining an association between a biallelic marker and a trait, methods for identifying individuals having a predisposition for a trait, methods of administration of prophylactic or therapeutic agents disease treatment, methods of identifying individuals likely to respond positively or negatively to said agents, and methods of using drugs and vaccines.

An example of identification of additional biallelic markers associated to a trait based on the previous knowledge of the localization of a first marker associated to a given trait is given below.

Biallelic Markers in Linkage Disequilibrium with a Particular Marker:Apo E4

The following example relating to the identification of markers in linkage disequilibrium with the apoE4 allele is representative of the procedures of the present invention in which markers in LD with a target gene are identified. 3 major isoforms of human apolipoprotein E (apoE2, -E3, and -E4) have been identified by isoelectric focusing and are coded for by 3 alleles (ε2, 3, and 4) of the Apo E gene. As originally reported by Strittrnatter et al. and by Saunders et al. in 1993, the Apo E ε4 allele is strongly associated with both late-onset familial and sporadic Alzheimer's Disease (AD).

Biallelic markers in linkage disequilibrium with the Apo E ε4 allele were identified. This example is illustrative of the general principle that the generation of biallelic markers associated with a trait leads to markers in linkage disequilibrium with any biallelic marker already known to be associated with the trait.

An Apo E marker was used to screen the human genomic BAC library. A BAC, which gave a unique hybridization signal on chromosomal region 19q13.2.3 by FISH, was selected for finding biallelic markers.

This BAC contained an insert of 205 kb that was subcloned. Fifty BAC subclones were randomly selected and sequenced. Twenty-five subclone sequences were selected and used to design twenty-five couples of PCR primers that allowed amplicons of approximately 500 bp to be generated. These PCR primers were then used to amplify the corresponding genomic sequences in a pool of DNA from 100 individuals (French origin, blood donors) as already described. Amplification products from pooled DNA were sequenced and analyzed for the presence of biallelic polymorphisms using the software described herein. Five amplicons were shown to contain a polymorphic base in the pool of 100 individuals, and therefore these polymorphisms (99-366/274; 99-344/439; 99-365/344; 99-359/308; 99-355/219) were selected as the random biallelic markers in the vicinity of the Apo E gene.

An additional couple of primers was designed that allowed amplification of the genomic fragment carrying the already known polymorphism of Apo E, (99-2452/54 C/T).

An association study was then performed. As expected, there was a clear association between Alzheimer disease (AD) and the known Apo E4 polymorphism (biallelic marker 99-2452/54), the C allele frequency being increased in 26% in the AD case population studied compared to the AD control population analyzed (pvalue of this difference=2×10 −21 ).

In addition, the association study with the random markers generated in the vicinity of the Apo E gene showed that the biallelic marker 99-365/344 C/T is also associated to AD, the T allele frequency being increased of 17% in the AD case population respect to the AD control population under study (pvalue of this allele frequency difference =7×10 −10 ). Thus individuals who possess a T allele at the biallelic marker 99-365/344 are at risk of developing AD.

Among the biallelic markers generated in the Apo E region, 99-365/344 is in LD with the previously known Apo E4 marker 99-2452/54. The linkage disequilibrium is detected in a control population (LD value=0.08) and is clearly increased in the AD case population (LD=0.21). Hence the generated biallelic marker which are associated with Alzheimer's disease, namely the biallelic marker 99-365, is in linkage disequilibrium with the biallelic marker 99-2452 already known to be associated with this disease.

Identification of a Trait Causing Mutation in the PCTA-1 Gene

If a statistically significant association with a trait is identified for at least one or more of the analyzed PCTA-1-related biallelic markers, one can assume that: either the associated allele is directly responsible for causing the trait, or more likely the associated allele is in linkage disequilibrium with the trait causing allele. More probably, the trait causing mutation would be found near to the associated biallelic markers.

Mutations in the PCTA-1 gene which are responsible for a detectable phenotype may be identified by comparing the sequences of the PCTA-1 gene from trait positive and trait negative individuals. Preferably, trait positive individuals to be sequenced carry a single marker allele or a haplotype shown to be associated to the trait and trait negative individuals to be sequenced do not carry such allele or haplotype associated to the trait. The detectable phenotype may comprise cancer, preferably prostate cancer, a response to or side effects related to a prophylactic or curative agent acting against prostate cancer, the aggressiveness of prostate cancer tumors, expression of the PCTA-1 gene, a modified or forthcoming production of the PCTA-1 protein, or the production of a modified PCTA-1 protein. The mutations may comprise point mutations, deletions, or insertions in the PCTA-1 gene. These mutations are called trait causing mutations and are at least partly responsible for a particular detectable phenotype in an individual. The mutations may lie within the coding sequence for the PCTA-1 protein or within intronic and/or within regulatory regions in the PCTA-1 gene, including splice sites, 5′ UTRs, 3′ UTRs and promoter sequences, including one or more transcription factor binding sites.

A further embodiment of the invention is a method to identify a trait causing mutation in the PCTA-1 gene pursuant to the detection of an association between alleles of one or several of the biallelic markers of the present invention and a particular trait.

This method comprises the following steps:

amplifying a region of the PCTA-1 gene comprising a biallelic marker or a group of biallelic markers associated to the considered trait from DNA samples of trait positive and trait negative individuals;

sequencing the amplified region;

comparing DNA sequences from trait positive and trait negative individuals; and

determining mutations specific to trait positive patients.

In some embodiments, the amplified region is a region located close to a biallelic marker of PCTA-1 gene. In further embodiments, the amplified region is located close to one or more of the biallelic markers A1 to A125 and the complements thereof. In a preferred embodiment, the amplified region is located close to one or more of the biallelic markers A2, A30, A41, AS5, A57 and the complements thereof.

Oligonucleotide primers are constructed as described previously to amplify the sequences of each of the exons, introns, the promoter region and the regulatory regions of the PCTA-1 gene. Amplification is carried out on genomic DNA samples from trait positive patients and trait negative controls, preferably using the PCR conditions described in the examples. Amplification products from the genomic PCRs are then subjected to sequencing, preferably through automated dideoxy terminator sequencing reactions and electrophoresed, preferably on ABI 377 sequencers. Following gel image analysis and DNA sequence extraction, ABI sequence data are automatically analyzed to detect the presence of sequence variations among trait positive and trait negative individuals. Sequences are verified by determining the sequences of both DNA strands for each individual.

Candidate polymorphisms suspected of being responsible for the detectable phenotype, are then verified by screening a larger population of trait positive and trait negative individuals using polymorphism analysis techniques such as the techniques described above. Polymorphisms which exhibit a statistically significant correlation with the detectable phenotype are deemed responsible for the detectable phenotype.

The invention also concerns a mutated PCTA-1 gene comprising a trait causing mutation, and particularly the mutated genes obtained by the process described above.

A mutated PCTA-1 gene can be defined as a gene encoding either a modified or native PCTA-1 protein through a nucleotide sequence which is different from the nucleotide sequence of the PCTA-1 gene found in a majority of trait negative individuals.

The region of the PCTA-1 gene containing the mutation responsible for the detectable phenotype may be used in diagnostic techniques such as those described below. For example, microsequencing oligonucleotides, or oligonucleotides containing the mutation responsible for the detectable phenotype for amplification, or hybridization based diagnostics, such as those described herein, may be used for detecting individuals suffering from the detectable phenotype or individuals at risk of developing the detectable phenotype at a subsequent time. In addition, the PCTA-1 allele responsible for the detectable phenotype may be used in gene therapy. The PCTA-1 allele responsible for the detectable phenotype may also be cloned into an expression vector to express the mutant PCTA-1 protein as described herein.

Biallelic Markers of the Invention in Methods of Genetic Diagnostics

The biallelic markers of the present invention can also be used to develop diagnostics tests capable of identifying individuals who express a detectable trait as the result of a specific genotype or individuals whose genotype places them at risk of developing a detectable trait at a subsequent time. The trait analyzed using the present diagnostics may be any detectable trait, including susceptibility to cancer, preferably prostate cancer, the level of aggressiveness of prostate cancer tumors, an early onset of prostate cancer, a beneficial response to or side effects related to treatment against prostate cancer.

Information resulting from single marker association and for haplotype analyses is extremely valuable as it can, in certain circumstances, be used to initiate preventive treatments or to allow an individual carrying a significant haplotype to foresee warning signs such as minor symptoms. In diseases such as prostate cancer, in which metastasis can be fatal if not stopped in time, the knowledge of a potential predisposition, might contribute in a very significant manner to treatment efficacy. Similarly, a diagnosed predisposition to a potential side-effect could immediately direct the physician toward a treatment for which such side-effects have not been observed during clinical trials.

The invention concerns a method for the detection in an individual of alleles of PCTA-1-related biallelic markers associated with a trait preferably selected from prostate cancer, an early onset of prostate cancer, a susceptibility to prostate cancer, the level of aggressiveness of prostate cancer tumors, or the level of expression of the PCTA-1 gene. The information obtained using this method is useful in the diagnosis, staging, monitoring, prognosis and/or prophylactic or curative therapy of prostate cancer. The method also concerns the detection of specific alleles present within the PCTA-1 gene expressing a modified level of PCTA-1 mRNA or an altered PCTA-1 mRNA, coding for an altered PCTA-1 protein. The identities of the polymorphic bases may be determined using any of the genotyping procedures described above in “Method For Genotyping An Individual For Biallelic Markers”. More particularly, the invention concerns the detection of a PCTA-1 nucleic acid comprising at least one of the nucleotide sequences of P1 to P125 and the complementary sequence thereof. This method comprises the following steps;

obtaining a nucleic acid sample from the individual to be tested; and

determining the presence in the sample of an allele of a biallelic marker or of a group of biallelic markers of the PCTA-1 gene which, when taken alone or in combination with another/other biallelic marker/s of the PCTA-1 gene, is indicative of prostate cancer, of an early onset of prostate cancer, of the level of aggressiveness of prostate cancer tumors, of a modified or forthcoming expression of the PCTA-1 gene, of a modified or forthcoming production of the PCTA-1 protein, or of the production of a modified PCTA-1 protein.

In some embodiments, the biallelic marker comprises at least one of the biallelic markers defined by the sequences P1 to P125, and the complementary sequences thereto, more preferably at least one biallelic marker selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. In a preferred embodiment, the biallelic marker comprises at least one of the biallelic markers defined by the sequences of P2, P30, P41, P55, P57, and the complementary sequence thereto, more particularly at least one biallelic marker selected from the group consisting of A2, A30, A41, A55, A57 and the complement thereof. In a preferred embodiment, the detection method comprises an additional step of amplifying a nucleotide sequence of the PCTA-1 gene comprising biallelic markers. Optionally, the amplification primers can be selected from the group consisting of B1 to B47 and C1 to C47.

In preferred embodiments of the detection method described above, the presence of alleles of one or more biallelic markers of the PCTA-1 gene is determined through microsequencing reactions. Optionally, the microsequencing primers are selected from the group consisting of D1 to D125 and E1 to E125. Optionally, the microsequencing primers can be bound to a solid support, preferably in the form of arrays of primers attached to appropriate chips or be used in microfluidic devices. Such arrays are described in further detail in the “Oligonucleotide arrays” section. Optionally, the microsequencing primers can be labeled.

In additional preferred embodiments of the detection method, the presence of alleles of one or more biallelic markers of the PCTA-1 gene is determined through an allele specific amplification assay or an enzyme based mismatch detection assay. Optionally, the allele specific amplification assay comprises a step of detecting the presence of the amplification product.

In further preferred embodiments of the detection method, the presence of alleles of one or more biallelic markers of the PCTA-1 gene is determined through a hybridization assay. The probes used in the hybridization assay may include a probe selected from the group consisting of P1 to P125, a complementary sequence thereto or a fragment thereof, said fragment comprising the polymorphic base. Preferably, the probe is labeled.

A diagnostic method according to the present invention can also consist on the detection of an allele of the PCTA-1 gene comprising a trait causing mutation.

The invention also specifically relates to a method of determining whether an individual suffering from prostate cancer or susceptible of developing prostate cancer is likely to respond positively to treatment with a selected medicament acting against prostate cancer.

The method comprises the following steps:

obtaining a DNA sample from the individual to be tested; and

analyzing said DNA sample to determine whether it comprises alleles of one or more biallelic markers associated with a positive response to treatment with the medicament and/or alleles of one or more biallelic markers associated with a negative response to treatment with the medicament.

In a preferred embodiment, the biallelic marker is selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith.

The detection methods of the present invention can be applied to, for example, the preliminary screening of patient populations suffering from prostate cancer. This preliminary screening is useful to initiate adequate treatment when needed or to determine and select appropriate patient populations for clinical trials on new compounds in order to avoid the potential occurrence of specific side effects or to enhance the probability of beneficial patient response. By establishing in advance a homogeneous genotype selection for the population to be tested, the assessment of drug efficacy and/or toxicity can be more readily achieved and less hampered by divergences in population response. This approach can yield better therapeutic approaches based on patient population targeting resulting from pharmacogenomics studies.

The invention also relates to diagnostic kits useful for determining the presence in a DNA sample of alleles associated with the trait, preferably with prostate cancer, with an early onset of prostate cancer, with the level of aggressiveness of prostate cancer tumors, with a modified or forthcoming expression of the PCTA-1 gene, with a modified or forthcoming production of the PCTA-1 protein, or with the production of a modified PCTA-1 protein. Diagnostic kits can comprise any of the polynucleotides of the present invention.

In a first embodiment, the kit comprises primers such as those described above, preferably forward and reverse primers which are used to amplify the PCTA-1 gene or a fragment thereof. In some embodiments, at least one of the primers is complementary to a nucleotide sequence of the PCTA-1 gene comprising a biallelic marker associated with prostate cancer, with an early onset of prostate cancer, with the level of aggressiveness of prostate cancer tumors, with a modified or forthcoming expression of the PCTA-1 gene, with a modified or forthcoming production of the PCTA-1 protein, or with the production of a modified PCTA-1 protein. In one embodiment, the biallelic marker is comprised in one of the sequences P1 to P125 and the complementary sequences thereto. Optionally, the kit comprises an amplification primer which includes a polymorphic base of at least one biallelic marker selected from the group consisting of A1 to A125 and the complements thereof. In a preferred embodiment, the kit comprises one or more of the sequences B1 to B47 and C1 to C47. In a more preferred 1 5 embodiment, the kit comprises one or more of the sequences B1, B16, B20, B23, B24 and C1, C16, C20, C23, C24.

In a second embodiment, the kit comprises microsequencing primers, wherein at least one of said primers is an oligonucleotide capable of hybridizing, either with the coding or with the non-coding strand, immediately upstream of the polymorphic base of a biallelic marker selected from the group consisting of A1 to A125 and the complements thereof, preferably those of D1 to D125 and E1 to E125, more preferably those of D2, D30, D41, D55, D57 and E2, E30, E41, E55, E57.

In a third embodiment, the kit comprises a hybridization DNA probe, that is or eventually becomes immobilized on a solid support, which is capable of hybridizing with the PCTA-1 gene or fragment thereof, preferably which is capable of hybridizing with a region of the PCTA-1 gene which comprises an allele of a biallelic marker of the present invention, more preferably an allele associated with prostate cancer, with an early onset of prostate cancer, with a susceptibility to prostate cancer, with the level of aggressiveness of prostate cancer tumors, with a modified or forthcoming expression of the PCTA-1 gene, with a modified or forthcoming production of the PCTA-1 protein, or with the production of a modified PCTA-1 protein. In a preferred embodiment, the probe is selected from the group consisting of P1 to P125 and the complementary sequences thereto, or a fragment thereof, said fragment comprising the polymorphic base. In a more preferred embodiment, the probe is selected from the group consisting of P2, P30, P41, P55, P57 and the complementary sequences thereto, or a fragment thereof, said fragment comprising the polymorphic base.

The kits of the present invention can also comprise optional elements including appropriate amplification reagents such as DNA polymerases when the kit comprises primers, reagents useful in hybridization reactions and reagents useful to reveal the presence of a hybridization reaction between a labeled hybridization probe and the PCTA-1 gene containing at least one biallelic marker.

Treatment of Cancer or Prostate Cancer

The invention also concerns methods for the treatment of prostate cancer using an allele of a biallelic marker or of a group of biallelic markers, preferably markers of the PCTA-1 gene, associated with a susceptibility to prostate cancer, with an aggressive form of prostate cancer or with a positive or negative response to treatment with an effective amount of a medicament acting against prostate cancer.

As the metastasis of prostate cancer can be fatal, it is important to detect prostate cancer susceptibility of individuals. Consequently, the invention also concerns a method for the treatment of prostate cancer comprising the following steps:

selecting an individual whose DNA comprises alleles of a biallelic marker or of a group of biallelic markers, preferably markers of the PCTA-1 gene, associated with prostate cancer;

following up said individual for the appearance (and optionally the development) of tumors in prostate; and

administering an effective amount of a medicament acting against prostate cancer to said individual at an appropriate stage of the prostate cancer.

In some embodiments, the biallelic marker is comprised in one of the sequences P1 to P125 and the complementary sequences thereto. Preferably the biallelic marker is at least one biallelic marker selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. In particular embodiments, the individual is selected by genotyping one or more biallelic markers of the present invention.

The prophylactic administration of a treatment serves to prevent, attenuate or inhibit the growth of cancer cells.

Therefore, another embodiment of the present invention consists of a method for the treatment of prostate cancer comprising the following steps:

selecting an individual whose DNA comprises alleles of a biallelic marker or of a group of biallelic markers, preferably markers of the PCTA-1 gene, associated with prostate cancer; and

administering to said individual, preferably as a preventive treatment of prostate cancer, an effective amount of a medicament acting against prostate cancer such as 4HPR or of a vaccine composition capable of conferring immunity against PCTA-1 related prostate cancer.

In some embodiments, the biallelic marker is comprised in one of the sequences P1 to P125 and the complementary sequences thereto. Preferably the biallelic marker is at least one biallelic marker selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. More preferably the biallelic marker is at least one biallelic marker selected from the group consisting of A2, A30, A41, A55, A57, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. In particular embodiments, the individual is selected by genotyping one or more biallelic markers of the present invention.

In a further embodiment, the present invention concerns a method for the treatment of prostate cancer comprising the following steps:

selecting an individual whose DNA comprises alleles of a biallelic marker or of a group of biallelic markers, preferably markers of the PCTA-1 gene, associated with a susceptibility prostate cancer;

administering to said individual, as a preventive treatment of prostate cancer, an effective amount of a medicament acting against prostate cancer such as 4HPR or of a vaccine composition capable of conferring immunity against PCTA-1-related prostate cancer;

following up said individual for the appearance and the development of tumors in prostate; and optionally

administering an effective amount of a medicament acting against prostate cancer to said individual at the appropriate stage of the prostate cancer.

In some embodiments, the biallelic marker is comprised in one of the sequences P1 to P125 and the complementary sequences thereto. Preferably the biallelic marker is at least one biallelic marker selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. More preferably the biallelic marker is at least one biallelic marker selected from the group consisting of A2, A30, A41, A55, A57, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. In particular embodiments, the individual is selected by genotyping one or more biallelic markers of the present invention.

To enlighten the choice of the appropriate beginning of the treatment of prostate cancer, the present invention also concerns a method for the treatment of prostate cancer comprising the following steps:

selecting an individual suffering from a prostate cancer whose DNA comprises alleles of a biallelic marker or of a group of biallelic markers, preferably markers of the PCTA-1 gene, associated with the aggressiveness of prostate cancer tumors; and

administering an effective amount of a medicament acting against prostate cancer to said individual.

In some embodiments, the biallelic marker is comprised in one of the sequences P1 to P125 and the complementary sequences thereto. Preferably the biallelic marker is at least one biallelic marker selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. More preferably the biallelic marker is at least one biallelic marker selected from the group consisting of A2, A30, A41, A55, A57, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. In particular embodiments, the individual is selected by genotyping one or more biallelic markers of the present invention.

The invention concerns a method of determining whether a subject is likely to respond positively to treatment with a selected medicament acting against prostate cancer.

The invention also concerns a method for the treatment of prostate cancer in a selected population of individuals. The method comprises:

selecting an individual suffering from prostate cancer and

whose DNA comprises alleles of a biallelic marker or of a group of biallelic markers, preferably markers of the PCTA-1 gene, associated with a positive response to treatment with an effective amount of a medicament acting against prostate cancer,

and/or whose DNA does not comprise alleles of a biallelic marker or of a group of biallelic markers, preferably markers of the PCTA-1 gene, associated with a negative response to treatment with said medicament; and

administering at suitable intervals an effective amount of said medicament to said selected individual.

In some embodiments, the biallelic marker is comprised in one of the sequences P1 to P125 and the complementary sequences thereto. Preferably the biallelic marker is at least one biallelic marker selected from the group consisting of A1 to A125, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. In particular embodiments, the individual is selected by genotyping one or more biallelic markers of the present invention.

Another aspect of the invention is a method of using a medicament acting against prostate cancer. The method comprises obtaining a DNA sample from a subject, determining whether the DNA sample contains one or more biallelic markers associated with a positive response to the medicament and/or whether the DNA sample contains one or more biallelic markers associated with a negative response to the medicament, and administering the medicament to the subject if the DNA sample contains one or more biallelic markers associated with a positive response to the medicament and/or if the DNA sample lacks one or more biallelic markers associated with a negative response to the medicament.

The invention also concerns a method for the clinical testing of a medicament, preferably a medicament acting against prostate cancer.

In some embodiments, the medicament may be administered to the subject in a clinical trial if the DNA sample contains alleles of one or more biallelic markers associated with a positive response to treatment with the medicament and/or if the DNA sample lacks alleles of one or more biallelic markers associated with a negative response to treatment with the medicament. In preferred embodiments, the medicament is a drug acting against prostate cancer. In other embodiments, the biallelic marker is selected from the group consisting of A1 to A125 and the complements thereof or optionally the biallelic markers in linkage disequilibrium therewith.

Using the method of the present invention, the evaluation of drug efficacy may be conducted in a population of individuals likely to respond favorably to the medicament.

The invention also concerns a method for the clinical testing of a medicament, preferably a medicament acting against prostate cancer. The method comprises the following steps:

administering a medicament, preferably a medicament susceptible of acting against prostate cancer to a heterogeneous population of individuals;

identifying a first population of individuals who respond positively to said medicament and a second population of individuals who respond negatively to said medicament;

identifying biallelic markers in said first population which are associated with a positive response to said medicament;

selecting individuals whose DNA comprises biallelic markers associated with a positive response to said medicament; and

administering said medicament to said individuals.

Such methods are deemed to be extremely useful to increase the benefit/risk ratio resulting from the administration of medicaments which may cause undesirable side effects and/or be inefficacious to a portion of the patient population to which it is normally administered.

Once an individual has been diagnosed as suffering from a prostate cancer, selection tests are carried out to determine whether the DNA of this individual comprises alleles of a biallelic marker or of a group of biallelic markers associated with a positive response to treatment or with a negative response to treatment which may include either side effects or unresponsiveness.

The selection of the patient to be treated using the method of the present invention can be carried out through the detection methods described above. The individuals which are to be selected are preferably those whose DNA does not comprise alleles of a biallelic marker or of a group of biallelic markers associated with a negative response to treatment. The knowledge of an individual's genetic predisposition to unresponsiveness or side effects to particular medicaments allows the clinician to direct treatment toward appropriate drugs against prostate cancer.

Once the patient's genetic predispositions have been determined, the clinician can select appropriate treatment for which negative response, particularly side effects, has not been reported or has been reported only marginally for the patient.

Recombinant Vectors

The term “vector” is used herein to designate either a circular or a linear DNA or RNA molecule, which is either double-stranded or single-stranded, and which comprise at least one polynucleotide of interest that is sought to be transferred in a cell host or in a unicellular or multicellular host organism.

Another embodiment of the present invention is a recombinant vector. This recombinant vector comprises a nucleotide sequence encoding a regulatory region of the PCTA-1 gene, the promoter region of the PCTA-1 gene, an intron of the PCTA-1 gene, exon 0 and/or exon 1 of the PCTA-1 gene, exon 6bis of the PCTA-1 gene, exon 9bis of the PCTA-1 gene, the genomic sequence of the PCTA-1 gene, a cDNA sequence of the PCTA-1 gene, or combinations of such sequences, or complementary sequences thereto or fragments or variants thereof. Preferred nucleotide sequences included in such an expression vector include at least one nucleotide sequence selected from the group consisting of SEQ ID Nos 1, 2, 3, 4, 8 or fragments or variants thereof or a complementary sequence thereto.

Generally, a recombinant vector of the invention may comprise any of the polynucleotides described herein, including regulatory sequences and coding sequences, as well as any PCTA-1 primer or probe as defined above. More particularly, the recombinant vectors of the present invention can comprise any of the polynucleotides described in the “PCTA-1 cDNA Sequences” section, the “Coding Regions” section, and the “Oligonucleotide Probes And Primers” section.

In another embodiment, the vector includes a PCTA-1 gene or cDNA or a fragment thereof comprising at least one of the biallelic markers described herein, and more preferably a mutated PCTA-1 gene or cDNA comprising a trait causing mutation, particularly a mutation determined using the method described above. Preferably, the biallelic marker is selected from the group consisting of A1 to A125 and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith.

One embodiment of the invention is the production of a PCTA-1 protein under the control of its own promoter or of an exogenous promoter. The present invention also relates to expression vectors which include nucleic acids encoding a native or mutated PCTA-1 protein under the control of either a native PCTA-1 regulatory region, preferably a native PCTA-1 promoter which comprises at least one of the biallelic markers of the present invention, more particularly at least one among the A1 to A43 and the complements thereof, or an exogenous promoter.

More particularly, the present invention relates to expression vectors which include nucleic acids encoding a PCTA-1 protein, preferably a PCTA-1 protein comprising a amino acid sequence selected from the group consisting of SEQ ID Nos 5, 6, 7, 9 or variants or fragments thereof, under the control of a regulatory sequence selected among the PCTA-1 regulatory polynucleotides, or alternatively under the control of an exogenous regulatory sequence.

The present invention also concerns an expression vector comprising a PCTA-1 regulatory region or any sequence thereof of 10 to 3000 nucleotides capable of regulating the expression of a nucleotide sequence encoding a protein and operably linked to the regulatory region. A further preferred regulatory region is the promoter sequence. In this regard, it is to be noted that a portion of the promoter can be used in the expression vector as long as it can influence the transcription of the coding sequence operably linked thereto.

Any nucleotide sequence encoding a polypeptide of interest can be included in an expression vector comprising a PCTA-1 regulatory region and operably linked thereto. Preferred polypeptides are therapeutic proteins which are described in further detail later on.

In some embodiments, expression vectors are employed to express a PCTA-1 polypeptide which can be then purified and, for example be used in ligand screening assays or as an immunogen in order to raise specific antibodies directed against a PCTA-1 protein. In other embodiments, the expression vectors are used for constructing transgenic animals and also for gene therapy.

Some of the elements which can be found in the vectors of the present invention are described in further detail in the following sections.

1. General Features of the Expression Vectors of the Invention

A recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid, a plasmid or even a linear DNA molecule which may consist of a chromosomal, non-chromosomal, semi-synthetic or synthetic DNA. Such a recombinant vector can comprise a transcriptional unit comprising an assembly of:

(1) a genetic element or elements having a regulatory role in gene expression, for example promoters or enhancers. Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp in length that act on the promoter to increase the transcription.

(2) a structural or coding sequence which is transcribed into mRNA and eventually translated into a polypeptide, said structural or coding sequence being operably linked to the regulatory elements described in (1); and

(3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, when a recombinant protein is expressed without a leader or transport sequence, it may include a N-terminal residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.

Generally, recombinant expression vectors will include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or the extracellular medium. In a specific embodiment wherein the vector is adapted for transfecting and expressing desired sequences in mammalian host cells, preferred vectors will comprise an origin of replication in the desired host, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′-flanking non-transcribed sequences. DNA sequences derived from the SV40 viral genome, for example SV40 origin, early promoter, enhancer, splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.

The in vivo expression of a PCTA-1 polypeptide may be useful in order to correct a genetic defect related to the expression of the native gene in a host organism or to the production of a biologically inactive PCTA-1 protein.

Consequently, the present invention also deals with recombinant expression vectors mainly designed for the in vivo production of a PCTA-1 polypeptide of SEQ ID Nos 5, 6, 7, 9 or fragments or variants thereof by the introduction of the appropriate genetic material in the organism of the patient to be treated. This genetic material may be introduced in vitro in a cell that has been previously extracted from the organism, the modified cell being subsequently reintroduced in the said organism, directly in vivo into the appropriate tissue.

2. Regulatory Elements

Promoters

The suitable promoter regions used in the expression vectors according to the present invention are chosen taking into account the cell host in which the heterologous gene has to be I,X;l expressed. The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell, such as, for example, a human or a viral promoter.

A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed. Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.

Promoter regions can be selected from any desired gene using, for example, CAT (chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7 vectors.

Preferred bacterial promoters are the LacI, LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL and trp promoters (EP 0036776, the disclosure of which is incorporated herein by reference in its entirety), the polyhedrin promoter, or the p10 protein promoter from baculovirus (Kit Novagen) (Smith et al., 1983; O'Reilly et al., 1992), the lambda PR promoter or also the trc promoter.

Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionine-L. Selection of a convenient vector and promoter is well within the level of ordinary skill in the art.

The choice of a promoter is well within the ability of a person skilled in the field of genetic engineering. For example, one may refer to the book of Sambrook et al.(1989) or also to the procedures described by Fuller et al.(1996).

Other Regulatory Elements

Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

The vector containing the appropriate DNA sequence as described above, more preferably PCTA-1 gene regulatory polynucleotide, a polynucleotide encoding a PCTA-1 polypeptide selected from the group consisting of SEQ ID No 1 or a fragment or a variant thereof and SEQ ID Nos 2, 3,4, 8, or both of them, can be utilized to transform an appropriate host to allow the expression of the desired polypeptide or polynucleotide.

3. Selectable Markers

Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct. The selectable marker genes for selection of transformed host cells are preferably dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, TRPI for S. cerevisiae or tetracycline, rifampicin or ampicillin resistance in E. coli , or levan saccharase for mycobacteria, this latter marker being a negative selection marker.

4. Preferred Vectors.

Bacterial Vectors

As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and a bacterial origin of replication derived from commercially available plasmids comprising genetic elements of pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and GEMI (Promega Biotec, Madison, Wis., USA).

Large numbers of other suitable vectors are known to those of skill in the art, and commercially available, such as the following bacterial vectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress).

Bacteriophage Vectors

The P1 bacteriophage vector may contain large inserts ranging from about 80 to about 100 kb.

The construction of P1 bacteriophage vectors such as p158 or p158/neo8 are notably described by Sternberg (1994). Recombinant P1 clones comprising PCTA-1 nucleotide sequences may be designed for inserting large polynucleotides of more than 40 kb (Linton et al., 1993). To generate P1 DNA for transgenic experiments, a preferred protocol is the protocol described by McCormick et al.(1994). Briefly, E. coli (preferably strain NS3529) harboring the P1 plasmid are grown overnight in a suitable broth medium containing 25 μg/ml of kanamycin. The P1 DNA is prepared from the E. coli by alkaline lysis using the Qiagen Plasmid Maxi kit (Qiagen, Chatsworth, Calif., USA), according to the manufacturer's instructions. The P1 DNA is purified from the bacterial lysate on two Qiagen-tip 500 columns, using the washing and elution buffers contained in the kit. A phenol/chloroform extraction is then performed before precipitating the DNA with 70% ethanol. After solubilizing the DNA in TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA), the concentration of the DNA is assessed by spectrophotometry.

When the goal is to express a P1 clone comprising PCTA-1 nucleotide sequences in a transgenic animal, typically in transgenic mice, it is desirable to remove vector sequences from the P1 DNA fragment, for example by cleaving the P1 DNA at rare-cutting sites within the P1 polylinker (SfiI, NotI or SalI). The P1 insert is then purified from vector sequences on a pulsed-field agarose gel, using methods similar using methods similar to those originally reported for the isolation of DNA from YACs (Schedl et al., 1993a; Peterson et al., 1993). At this stage, the resulting purified insert DNA can be concentrated, if necessary, on a Millipore Ultrafree-MC Filter Unit (Millipore, Bedford, Mass., USA-30,000 molecular weight limit) and then dialyzed against microinjection buffer(10 mM Tris-HCl, pH 7.4; 250 μM EDTA) containing 100 mM NaCl, 30 μM spermine, 70 μM spermidine on a microdyalisis membrane (type VS, 0.025 μM from Millipore). The intactness of the purified P1 DNA insert is assessed by electrophoresis on 1% agarose (Sea Kem GTG; FMC Bio-products) pulse-field gel and staining with ethidium bromide.

Baculovirus Vectors

A suitable vector for the expression of a PCTA-1 polypeptide of SEQ ID Nos 5, 6, 7, 9 or fragments or variants thereof is a baculovirus vector that can be propagated in insect cells and in insect cell lines. A specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC N o CRL 1711) which is derived from Spodoptera frugiperda.

Other suitable vectors for the expression of a PCTA-1 polypeptide of SEQ ID Nos 5, 6, 7, 9 or fragments or variants thereof in a baculovirus expression system include those described by Chai et al.(l993), Vlasak et al.(l983) and Lenhard et al.(1996).

Viral Vectors

In one specific embodiment, the vector is derived from an adenovirus. Preferred adenovirus vectors according to the invention are those described by Feldman and Steg (1996) or Ohno et al.(l994). Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal origin (French patent application N o FR-93.05954).

Retrovirus vectors and adeno-associatcd virus vectors are generally understood to be the recombinant gene delivery systems of choice for the transfer of exogenous polynucleotides in vivo, particularly to mammals, including humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal. DNA of the host.

Particularly preferred retroviruses for the preparation or construction of retroviral in vitro or in vitro gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma virus. Particularly preferred Murine Leukemia Viruses include the 4070A and the 1504A viruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCC No VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus (ATCC No VR-190; PCT Application No WO 94/24298). Particularly preferred Rous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657, VR-726, VR-659 and VR-728). Other preferred retroviral vectors are those described in Roth et al.(1996), PCT Application No WO 93/25234 (the disclosure of which is incorporated herein by reference in its entirety), PCT Application No WO 94/ 06920 (the disclosure of which is incorporated herein by reference in its entirety), Roux et al., 1989, Julan et al., 1992 and Neda et al., 1991.

Yet another viral vector system that is contemplated by the invention consists of the adeno-associated virus (AAV). The adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al., 1992). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (Flotte et al., 1992; Samulski et al., 1989; McLaughlin et al., 1989). One advantageous feature of AAV derives from its reduced efficacy for transducing primary cells relative to transformed cells.

BAC Vectors

The bacterial artificial chromosome (BAC) cloning system (Shizuya et al , 1992) has been developed to stably maintain large fragments of genomic DNA (100-300 kb) in E. coli . A preferred BAC vector consists of pBeloBAC 11 vector that has been described by Kim et al.(1996). BAC libraries are prepared with this vector using size-selected genomic DNA that has been partially digested using enzymes that permit ligation into either the Bam HI or HindIII sites in the vector. Flanking these cloning sites are T7 and SP6 RNA polymerase transcription initiation sites that can be used to generate end probes by either RNA transcription or PCR methods. After the construction of a BAC library in E. coli , BAC DNA is purified from the host cell as a supercoiled circle. Converting these circular molecules into a linear form precedes both size determination and introduction of the BACs into recipient cells. The cloning site is flanked by two Not I sites, permitting cloned segments to be excised from the vector by Not I digestion. Alternatively, the DNA insert contained in the pBeloBAC11 vector may be linearized by treatment of the BAC vector with the commercially available enzyme lambda terminase that leads to the cleavage at the unique cosN site, but this cleavage method results in a full length BAC clone containing both the insert DNA and the BAC sequences.

5. Delivery of the Recombinant Vectors

In order to effect expression of the polynucleotides and polynucleotide constructs of the invention, these constructs must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cell lines, or in vivo or ex vivo, as in the treatment of certain diseases states.

One mechanism is viral infection where the expression construct is encapsulated in an infectious viral particle.

Several non-viral methods for the transfer of polynucleotides into cultured mammalian cells are also contemplated by the present invention, and include, without being limited to, calcium phosphate precipitation (Graham et al., 1973; Chen et al., 1987;), DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), direct microinjection (Harland et al., 1985), DNA-loaded liposomes (Nicolau et al., 1982; Fraley et al., 1979), and receptor-mediate transfection (Wu and Wu, 1987; 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.

Once the expression polynucleotide has been delivered into the cell, it may be stably integrated into the genome of the recipient cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle.

One specific embodiment for a method for delivering a protein or peptide to the interior of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a physiologically acceptable carrier and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a physiological effect. This is particularly applicable for transfer in vitro but it may be applied to in vivo as well.

Compositions for use in vitro and in vivo comprising a “naked” polynucleotide are described in PCT application N o WO 90/11092 (Vical Inc.) and also in PCT application No. WO 95/11307 (Institut Pasteur, INSERM, Universitéd'Ottawa) as well as in the articles of Tacson et al.(l996) and of Huygen et al.(1996).

In still another embodiment of the invention, the transfer of a naked polynucleotide of the invention, including a polynucleotide construct of the invention, into cells may be proceeded with a particle bombardment (biolistic), said particles being DNA-coated microprojectiles accelerated to a high velocity allowing them to pierce cell membranes and enter cells without killing them, such as described by Klein et al.(1987).

In a further embodiment, the polynucleotide of the invention may be entrapped in a liposome (Ghosh and Bacchawat, 1991; Wong et al., 1980; Nicolau et al., 1987)

In a specific embodiment, the invention provides a composition for the in vivo production of a PCTA-1 protein or polypeptide described herein. It comprises a naked polynucleotide operatively coding for this polypeptide, in solution in a physiologically acceptable carrier, and suitable for introduction into a tissue to cause cells of the tissue to express the said protein or polypeptide.

The amount of vector to be injected to the desired host organism varies according to the site of injection. As an indicative dose, it will be injected between 0,1 and 100 μg of the vector in an animal body, preferably a mammal body, for example a mouse body.

In another embodiment of the vector according to the invention, it may be introduced in vitro in a host cell, preferably in a host cell previously harvested from the animal to be treated and more preferably a somatic cell such as a muscle cell. In a subsequent step, the cell that has been transformed with the vector coding for the desired PCTA-1 polypeptide or the desired fragment thereof is reintroduced into the animal body in order to deliver the recombinant protein within the body either locally or systemically.

Cell Hosts

The invention also concerns host cells transformed by one of the vectors described above that produce either a heterologous protein, a PCTA-1 protein or fragments thereof encoded by the PCTA-1 gene, preferably comprising at least one of the biallelic polymorphisms described herein, and more preferably a mutated PCTA-1 gene comprising the trait causing mutation determined using the above-noted method.

Another object of the invention consists of a host cell that has been transformed or transfected with one of the polynucleotides described herein, and in particular a polynucleotide either comprising a PCTA-1 regulatory polynucleotide or the coding sequence of a PCTA-1 polypeptide selected from the group consisting of SEQ ID No 1 2, 3, 4, 8 or a fragment or a variant thereof. Also included are host cells that are transformed (prokaryotic cells) or that are transfected (eukaryotic cells) with a recombinant vector such as one of those described above. More particularly, the cell hosts of the present invention can comprise any of the polynucleotides described in the “PCTA-1 cDNA Sequences” section, the “Coding Regions” section, and the “Oligonucleotide Probes And Primers” section.

A further recombinant cell host according to the invention comprises a polynucleotide containing a biallelic marker selected from the group consisting of A1 to A125, and the complements thereof.

Generally, a recombinant host cell of the invention comprises any one of the polynucleotides or the recombinant vectors described herein.

Preferred host cells used as recipients for the expression vectors of the invention are the following:

a) Prokaryotic host cells: Escherichia coli strains (I.E.DH5-α strain), Bacillus subtilis, Salmonella typhimurium , and strains from species like Pseudomonas, Streptomyces and Staphylococcus.

b) Eukaryotic host cells: HeLa cells (ATCC N o CCL2; N o CCL2. 1; N o CCL2.2), Cv 1 cells (ATCC N o CCL70), COS cells (ATCC N o CRL1650; N o CRL1651), Sf-9 cells (ATCC N o CRL1711), C127 cells (ATCC N o CRL-1804),3T3 (ATCC N o CRL-6361), CHO (ATCC N o CCL-61), human kidney 293. (ATCC N o 45504; N o CRL-1573) and BHK (ECACC N o 84100501; N o 84111301).

c) Other mammalian host cells.

The PCTA-1 gene expression in mammalian, and typically human, cells may be rendered defective, or alternatively it may be proceeded with the insertion of a PCTA-1 genomic or cDNA sequence with the replacement of the PCTA-1 gene counterpart in the genome of an animal cell by a PCTA-1 polynucleotide according to the invention. These genetic alterations may be generated by homologous recombination events using specific DNA constructs that have been previously described.

One kind of cell hosts that may be used are mammal zygotes, such as murine zygotes. For example, murine zygotes may undergo microinjection with a purified DNA molecule of interest, for example a purified DNA molecule that has previously been adjusted to a concentration range from 1 ng/ml -for BAC inserts- 3 ng/μl -for P1 bacteriophage inserts- in 10 mM Tris-HCl, pH 7.4, 250 μM EDTA containing 100 mM NaCl, 30 μM spermine, and70 μM spermidine. When the DNA to be microinjected has a large size, polyamines and high salt concentrations can be used in order to avoid mechanical breakage of this DNA, as described by Schedl et al (1993b).

Anyone of the polynucleotides of the invention, including the DNA constructs described herein, may be introduced in an embryonic stem (ES) cell line, preferably a mouse ES cell line. ES cell lines are derived from pluripotent, uncommitted cells of the inner cell mass of pre-implantation blastocysts. Preferred ES cell lines are the following: ES-E 14TG2a (ATCC n o CRL-1821), ES-D3 (ATCC n o CRL1934 and n o CRL-11632), YS001 (ATCC n o CRL-11776),36.5 (ATCC n o CRL-11116). To maintain ES cells in an uncommitted state, they are cultured in the presence of growth inhibited feeder cells which provide the appropriate signals to preserve this embryonic phenotype and serve as a matrix for ES cell adherence. Preferred feeder cells consist of primary embryonic fibroblasts that are established from tissue of day 13-day 14 embryos of virtually any mouse strain, that are maintained in culture, such as described by Abbondanzo et al.(1993) and are inhibited in growth by irradiation, such as described by Robertson (1987), or by the presence of an inhibitory concentration of LIF, such as described by Pease and Williams (1990).

The constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.

Following transformation of a suitable host and growth of the host to an appropriate cell density, the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in the expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known by the skill artisan.

Transgenic Animals

The invention also relates to transgenic animals having an exogenous PCTA-1 regulatory region or a PCTA-1 gene, preferably comprising at least one of the biallelic polymorphisms described herein, and more preferably to a mutated PCTA-1 gene comprising the trait causing mutation determined using the above-noted method. Preferably, the biallelic marker is selected from the group consisting of A1 to A125 and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith. In another embodiment, the invention concerns animals, preferably a mouse, having the mouse PCTA-1 gene which is modified or knocked out. These animals could be used to screen compounds of interest.

The terms “transgenic animals” or “host animals” are used herein to designate animals that have their genome genetically and artificially manipulated so as to include one of the nucleic acids according to the invention. Preferred animals are non-human mammals and include those belonging to a genus selected from Mus (e.g. mice), Rattus (e.g. rats) and Oryctogalus (e.g. rabbits) which have their genome artificially and genetically altered by the insertion of a nucleic acid according to the invention.

In one embodiment, the invention encompasses non-human host mammals and animals comprising a recombinant vector of the invention, a polynucleotide construct according to the invention, or a PCTA-1 gene disrupted by homologous recombination with a knock out vector. Generally, a transgenic animal according the present invention comprises any one of the polynucleotides, the recombinant vectors and the cell hosts described in the present invention. More particularly, the transgenic animals according to the present invention can comprise any of the polynucleotides described in the “PCTA-1 cDNA Sequences” section, the “Coding Regions” section, and the “Oligonucleotide Probes And Primers” section.

The transgenic animals of the invention all include within a plurality of their cells a cloned recombinant or synthetic DNA sequence, more specifically one of the purified or isolated nucleic acids comprising a PCTA-1 coding sequence, a PCTA-1 regulatory polynucleotide or a DNA sequence encoding an antisense polynucleotide such as described in the present specification, and still more preferably a nucleotide comprising an allele of at least one biallelic marker of the PCTA-1 gene.

In a first preferred embodiment, these transgenic animals may be good experimental models in order to study cancer, preferably prostate cancer, in particular concerning the transgenic animals within the genome of which has been inserted one or several copies of a polynucleotide encoding a native PCTA-1 protein, or alternatively a mutant PCTA-1 protein.

In a second preferred embodiment, these transgenic animals may express a desired polypeptide of interest under the control of the regulatory polynucleotides of the PCTA-1 gene, leading to good yields in the synthesis of this protein of interest, and eventually a tissue specific expression of this protein of interest.

The design of the transgenic animals of the invention may be made according to the conventional techniques well known from the one skilled in the art. For more details regarding the production of transgenic animals, and specifically transgenic mice, it may be referred to U.S. Pat. Nos. 4,873,191, issued Oct. 10, 1989; U.S. Pat. No. 5,464,764 issued Nov. 7, 1995; and U.S. Pat. No. 5,789,215, issued Aug. 4, 1998, the disclosures of which are incorporated herein by reference in their entireties.

Transgenic animals of the present invention are produced by the application of procedures which result in an animal with a genome that incorporates exogenous genetic material which is integrated into the genome. The procedure involves obtaining the genetic material, or a portion thereof, which encodes either a PCTA-1 coding sequence, a PCTA-1 regulatory polynucleotide or a DNA sequence encoding an antisense polynucleotide such as described in the present specification.

A recombinant polynucleotide of the invention is inserted into an embryonic or ES stem cell line. The insertion is made using electroporation. The cells subjected to electroporation are screened (e.g. Southern blot analysis) to find positive cells which have integrated the exogenous recombinant polynucleotide into their genome. An illustrative positive-negative selection procedure that may be used according to the invention is described by Mansour et al. (1988).

Then, the positive cells are isolated, cloned and injected into 3.5 days old blastocysts from mice. The blastocysts are then inserted into a female host animal and allowed to grow to term.

Alternatively, the positive ES cells are brought into contact with embryos at the 2.5 days old 8-16 cell stage (morulae) such as described by Wood et al.(1993) or by Nagy et al.(1993), the ES cells being intemalized to colonize extensively the blastocyst including the cells which will give rise to the germ line.

The offsprings of the female host are tested to determine which animals are transgenic e.g. include the inserted exogenous DNA sequence and which are wild-type.

Thus, the present invention also concerns a transgenic animal containing a nucleic acid, a recombinant expression vector or a recombinant host cell according to the invention.

Recombinant Cell Lines Derived from the Transgenic Animals of the Invention.

A further object of the invention consists of recombinant host cells obtained from a transgenic animal described herein. In one embodiment the invention encompasses cells derived from non-human host mammals and animals comprising a recombinant vector of the invention or a PCTA-1 gene disrupted by homologous recombination with a knock out vector.

Recombinant cell lines may be established in vitro from cells obtained from any tissue of a transgenic animal according to the invention, for example by transfection of primary cell cultures with vectors expressing one-genes such as SV40 large T antigen, as described by Chou (1989) and Shay et al.(l991).

Screening of Agents Acting Against Prostate Cancer

In a further embodiment, the present invention also concerns a method for the screening of new agents, or candidate substances, acting against cancer, preferably against prostate cancer and which may be suitable for the treatment of a patient whose DNA comprises an allele of the PCTA-1 gene associated with cancer, preferably with prostate cancer, with an early onset of prostate cancer, or with the aggressiveness of prostate cancer tumors, or more generally with a modified or forthcoming expression of the PCTA-1 gene, with a modified or forthcoming production of the PCTA-1 protein, or with the production of a modified PCTA-1 protein.

In a preferred embodiment, the invention relates to a method for the screening of candidate substances for cancer treatment, preferably prostate cancer treatment. The method comprises the following steps:

providing a cell line, an organ, or a mammal expressing a PCTA-1 gene or a fragment thereof, preferably the regulatory region or the promoter region of the PCTA-1 gene;

obtaining a candidate substance, preferably a candidate substance capable of inhibiting the binding of a transcription factor to the PCTA-1 regulatory region; and

testing the ability of the candidate substance to decrease the symptoms of cancer, preferably of prostate cancer and/or to modulate the expression levels of PCTA-1.

In some embodiments, the cell line, organ or mammal expresses a heterologous protein, the coding sequence of which is operably linked to the PCTA-1 regulatory or promoter sequence. In other embodiments, they express a PCTA-1 gene comprising alleles of one or more biallelic markers associated with cancer, preferably with prostate cancer, an early onset of prostate cancer, or the aggressiveness of prostate cancer tumors, or a mutated PCTA-1 gene comprising a trait causing mutation determined using the above-noted method. Optionally, the biallelic marker is selected from the group consisting of A1 to A125 and the complements thereof. Preferably, the biallelic marker is selected from the group consisting of A2, A30, A41, A55, A57 and the complements thereof. In a further embodiment, a mice expressing a PCTA-1 protein, preferably a mouse PCTA-1 protein encoded by a nucleic acid sequence of SEQ ID No 9 or a variant or a fragment thereof can be used to screen agents acting against cancer, preferably prostate cancer.

A candidate substance is a substance which can interact with or modulate, by binding or other intermolecular interactions, expression, stability, and function of PCTA-1. Such substances may be potentially interesting for patients who are not responsive to existing drugs or develop side effects to them. Screening may be effected using either in vitro methods or in vivo methods.

Such methods can be carried out in numerous ways such as on transformed cells which express the considered alleles of the PCTA-1 gene, on tumors induced by said transformed cells, for example in mice, or on PCTA-1 protein encoded by the considered allelic variant of PCTA-1. This method preferably includes preparing transformed cells with different forms of PCTA-1 sequences containing particular alleles of one or more biallelic markers and/or trait causing mutations described above. Optionally, the biallelic marker is selected from the group consisting of A1 to A125 and the complements thereof.

Screening assays of the present invention generally involve determining the ability of a candidate substance to present a cytotoxic effect, to change the characteristics of transformed cells such as proliferative and invasive capacity, to affect the tumor growth, or to modify the expression level of PTCA-1.

Typical examples of such drug screening assays are provided below. It is to be understood that the parameters set forth in these examples can be modified by the skilled person without undue experimentation.

Screening Substances Interacting with the Regulatory Sequences of the PCTA-1 Gene.

The present invention also concerns a method for screening substances or molecules that are able to interact with the regulatory sequences of the PCTA-1 gene, such as for example promoter or enhancer sequences.

Nucleic acids encoding proteins which are able to interact with the regulatory sequences of the PCTA-1 gene, more particularly a nucleotide sequence selected from the group consisting of the polynucleotides of the 5′ and 3′ regulatory region or a fragment or variant thereof, and preferably a variant comprising one of the biallelic markers of the invention, may be identified by using a one-hybrid system, such as that described in the booklet enclosed in the Matchmaker One-Hybrid System kit from Clontech (Catalog Ref. n o K1603-1). Briefly, the target nucleotide sequence is cloned upstream of a selectable reporter sequence and the resulting DNA construct is integrated in the yeast genome ( Saccharomyces cerevisiae ). The yeast cells containing the reporter sequence in their genome are then transformed with a library consisting of fusion molecules between cDNAs encoding candidate proteins for binding onto the regulatory sequences of the PCTA-1 gene and sequences encoding the activator domain of a yeast transcription factor such as GAL4. The recombinant yeast cells are plated in a culture broth for selecting cells expressing the reporter sequence. The recombinant yeast cells thus selected contain a fusion protein that is able to bind onto the target regulatory sequence of the PCTA-1 gene. Then, the cDNAs encoding the fusion proteins are sequenced and may be cloned into expression or transcription vectors in vitro. The binding of the encoded polypeptides to the target regulatory sequences of the PCTA-1 gene may be confirmed by techniques familiar to the one skilled in the art, such as gel retardation assays or DNAse protection assays. Such assays are detailed in the section “Analysis Of Biallelic Markers Of The Invention With Prostate Cancer”.

Screening for Expression Modifiers

The PCTA-1 gene appears to be involved in a series of events which most likely include a modification of at least one step of its transcription process. In fact, and as mentioned previously, there is a strong possibility that this modification is directly related to the binding efficiency of DNA binding factors to sites of the PCTA-1 regulatory region.

Screening programs can be used to test potentially therapeutic compounds, either by competitively binding to the sites of the PCTA-1 promoter which would normally bind the DNA transcription factor, or directly binding to the DNA binding factor itself. These compounds could reduce the speed at which the cascade of events leading to the development of PCTA-1 related cancers takes place. In fact, even though it seems clear that a combination of several DNA binding sites may be involved in the development of a PCTA-1 related prostate cancer, binding inhibition of only a few such sites is likely to be sufficient to significantly impact on PCTA-1 production and hence the proliferation of cancer.

The screening of expression modifiers is important as it can be used for detecting modifiers specific to one allele or a group of alleles of the PCTA-1 gene. The alteration of PCTA-1 expression in response to a modifier can be determined by administering or combining the candidate modifier with an expression system such as animals, cells, and in vitro transcription assay.

The term “expression modifier” is intended to encompass but is not limited to chemical agents and polypeptides that modulate the action of PCTA-1 through modulation of the PCTA-1 gene expression.

The effect of the modifier on PCTA-1 transcription and/or steady state mRNA levels can be also determined. As with the basic expression levels, tissue specific interactions are of interest. Correlations are made between the ability of an expression modifier to affect PCTA-1 activity, and the presence of the targeted polymorphisms. A panel of different modifiers may be screened in order to determine the effect under a number of different conditions.

Another subject of the present invention is a method for screening molecules that modulate the expression of the PCTA-1 protein. Such a screening method comprises the steps of:

a) cultivating a prokaryotic or an eukaryotic cell that has been transfected with a nucleotide sequence encoding the PCTA-1 protein or a variant or a fragment thereof, placed under the control of its own promoter;

b) bringing into contact the cultivated cell with a molecule to be tested; and

c) quantifying the expression of the PCTA-1 protein or a variant or a fragment thereof.

In an embodiment, the nucleotide sequence encoding the PCTA-1 protein or a variant or a fragment thereof comprises an allele of at least one of the biallelic markers A1 to A125, preferably A2, A30, A41, A55, A57, and the complements thereof.

Using DNA recombination techniques well known by the one skilled in the art, the PCTA-1 protein encoding DNA sequence is inserted into an expression vector, downstream from its promoter sequence.

The quantification of the expression of the PCTA-1 protein may be realized either at the mRNA level or at the protein level. In the latter case, polyclonal or monoclonal antibodies may be used to quantify the amounts of the PCTA-1 protein that have been produced, for example in an ELISA or a RIA assay.

In a preferred embodiment, the quantification of the PCTA-1 mRNA is realized by a quantitative PCR amplification of the cDNA obtained by a reverse transcription of the total mRNA of the cultivated PCTA-1-transfected host cell, using a pair of primers specific for PCTA-1.

Thus, is also part of the present invention a method for screening of a candidate substance or molecule that modulated the expression of the PCTA-1 gene, this method comprises the following steps:

providing a recombinant cell host containing a nucleic acid, wherein said nucleic acid comprises a nucleotide sequence of the 5′ regulatory region or a biologically active fragment or variant thereof located upstream a polynucleotide encoding a detectable protein;

obtaining a candidate substance; and

determining the ability of the candidate substance to modulate the expression levels of the polynucleotide encoding the detectable protein.

In a further embodiment, the nucleic acid comprising the nucleotide sequence of the 5′ regulatory region or a biologically active fragment or variant thereof also includes a 5′UTR region of the PCTA-1 cDNAs, or one of its biologically active fragments or variants thereof.

Among the preferred polynucleotides encoding a detectable protein, there may be cited polynucleotides encoding luciferase, beta galactosidase, green fluorescent protein (GFP) and chloramphenicol acetyl transferase (CAT).

In another embodiment of a method for the screening of a candidate substance or molecule that modulates the expression of the PCTA-1 gene, wherein said method comprises the following steps:

a) providing a recombinant host cell containing a nucleic acid, wherein said nucleic acid comprises the 5′UTR sequence of a PCTA-1 cDNA, or one of its biologically active fragments or variants, the 5′UTR sequence or its biologically active fragment or variant being operably linked to a polynucleotide encoding a detectable protein;

b) obtaining a candidate substance; and

c) determining the ability of the candidate substance to modulate the expression levels of the polynucleotide encoding the detectable protein.

In one particular embodiment of the above screening method, the nucleic acid that comprises a nucleotide sequence selected from the group consisting of the 5′UTR sequence of a PCTA-1 cDNA or one of its biologically active fragments or variants, includes a promoter sequence which is exogenous with respect to the PCTA-1 5′UTR sequence defined therein. In a further preferred embodiment, the nucleic acid comprising the 5′-UTR sequence of a PCTA-1 cDNA or the biologically active fragments thereof includes a biallelic marker selected from the group consisting of A1 to A125, preferably A2, A30, A41, A55, A57, or the complements thereof.

The invention also pertains to kits useful for performing the herein described screening method. Preferably, such kits comprise a recombinant vector that allows the expression of a nucleotide sequence of the 5′ regulatory region or a biologically active fragment or variant thereof located upstream and operably linked to a polynucleotide encoding a detectable protein or a PCTA-1 protein or a fragment or a variant thereof. Moreover, the kit can comprise a recombinant vector that comprises a nucleic acid including a 5′UTR sequence of a PCTA-1 cDNA, or one of their biologically active fragments or variants, said nucleic acid being operably linked to a polynucleotide encoding a detectable protein or a PCTA-1 protein or a fragment or a variant thereof.

For the design of suitable recombinant vectors useful for performing the screening methods described above, it will be referred to the section of the present specification wherein the preferred recombinant vectors of the invention are detailed.

Expression levels and patterns of PCTA-1 may be analyzed by solution hybridization with long probes as described in International Patent Application No. WO 97/05277. Briefly, a PCTA-1 cDNA or the PCTA-1 genomic DNA described above, or fragments thereof, is inserted at a cloning site immediately downstream of a bacteriophage (T3, T7 or SP6) RNA polymerase promoter to produce antisense RNA. Preferably, the PCTA-1 insert comprises at least 100 or more consecutive nucleotides of the genomic DNA sequence or a cDNA sequence, particularly those comprising at least one of biallelic markers according the present invention, preferably at least one of the biallelic markers A1 to A125 and the complements thereof or those comprising the trait causing mutation. The plasmid is linearized and transcribed in the presence of ribonucleotides comprising modified ribonucleotides (i.e. biotin-UTP and DIG-UTP). An excess of this doubly labeled RNA is hybridized in solution with mRNA isolated from cells or tissues of interest. The hybridizations are performed under standard stringent conditions (40-50° C. for 16 hours in an 80% formamide, 0.4 M NaCl buffer, pH 7-8). The unhybridized probe is removed by digestion with ribonucleases specific for single-stranded RNA (i.e. RNases CL3, T1, Phy M, U2 or A). The presence of the biotin-UTP modification enables capture of the hybrid on a microtitration plate coated with streptavidin. The presence of the DIG modification enables the hybrid to be detected and quantified by ELISA using an anti-DIG antibody coupled to alkaline phosphatase.

Quantitative analysis of the PCTA-1 gene expression may also be performed using arrays. As used herein, the term array means a one dimensional, two dimensional, or multidimensional arrangement of a plurality of nucleic acids of sufficient length to permit specific detection of expression of mRNAs capable of hybridizing thereto. For example, the arrays may contain a plurality of nucleic acids derived from genes whose expression levels are to be assessed. The arrays may include the PCTA-1 genomic DNA, a PCTA-1 cDNA sequence or the sequences complementary thereto or fragments thereof, particularly those comprising at least one of the biallelic markers according the present invention, preferably at least one of the biallelic markers A1 to A125 and the complements thereof or those comprising a trait causing mutation. Preferably, the fragments are at least 15 nucleotides in length. In other embodiments, the fragments are at least 25 nucleotides in length. In some embodiments, the fragments are at least 50 nucleotides in length. More preferably, the fragments are at least 100 nucleotides in length. In another preferred embodiment, the fragments are more than 100 nucleotides in length. In some embodiments the fragments may be more than 500 nucleotides in length.

For example, quantitative analysis of PCTA-1 gene expression may be performed with a complementary DNA microarray as described by Schena et al. (1995 and 1996). Full length PCTA-1 cDNAs or fragments thereof are amplified by PCR and arrayed from a 96-well microtiter plate onto silylated microscope slides using high-speed robotics. Printed arrays are incubated in a humid chamber to allow rehydration of the array elements and rinsed, once in 0.2% SDS for 1 min, twice in water for 1 min and once for 5 min in sodium borohydride solution. The arrays are submerged in water for 2 min at 95° C., transferred into 0.2% SDS for 1 min, rinsed twice with water, air dried and stored in the dark at 25° C.

Cell or tissue mRNA is isolated or commercially obtained and probes are prepared by a single round of reverse transcription. Probes are hybridized to 1 cm 2 microarrays under a 14×14 mm glass coverslip for 6-12 hours at 60° C. Arrays are washed for 5 min at 25° C. in low stringency wash buffer (1×SSC/0.2% SDS), then for 10 min at room temperature in high stringency wash buffer (0.1×SSC/0.2% SDS). Arrays are scanned in 0.1×SSC using a fluorescence laser scanning device fitted with a custom filter set. Accurate differential expression measurements are obtained by taking the average of the ratios of two independent hybridizations.

Quantitative analysis of PCTA-1 gene expression may also be performed with full length PCTA-1 cDNAs or fragments thereof in complementary DNA arrays as described by Pietu et al. (1996). The full length PCTA-1 cDNA or fragments thereof is PCR amplified and spotted on membranes. Then, mRNAs originating from various tissues or cells are labeled with radioactive nucleotides. After hybridization and washing in controlled conditions, the hybridized mRNAs arc detected by phospho-imaging or autoradiography. Duplicate experiments are performed and a quantitative analysis of differentially expressed mRNAs is then performed.

Alternatively, expression analysis using the PCTA-1 genomic DNA, a PCTA-1 cDNA, or fragments thereof can be done through high density nucleotide arrays as described by Lockhart et al. (1996) and Sosnowsky et al. (1997). Oligonucleotides of 15-50 nucleotides from the sequence of the PCTA-1 genomic DNA, a PCTA-1 cDNA sequence, particularly a sequence comprising at least one of biallelic markers according the present invention, preferably at least one of the biallelic markers A1 to A125 and the complements thereof or comprising the trait causing mutation, or a sequence complementary thereto, are synthesized directly on the chip (Lockhart et al., supra) or synthesized and then addressed to the chip (Sosnowski et al., supra). Preferably, the oligonucleotides are about 20 nucleotides in length.

PCTA-1 cDNA probes labeled with an appropriate compound, such as biotin, digoxigenin or fluorescent dye, are synthesized from the appropriate mRNA population and then randomly fragmented to an average size of 50 to 100 nucleotides. The said probes are then hybridized to the chip. After washing as described in Lockhart et al., supra and application of different electric fields (Sosnowsky et al., 1997)., the dyes or labeling compounds are detected and quantified. Duplicate hybridizations are performed. Comparative analysis of the intensity of the signal originating from cDNA probes on the same target oligonucleotide in different cDNA samples indicates a differential expression of PCTA-1 mRNA.

Screening for Molecules Interacting with a PCTA-1 Protein

The PCTA-1 proteins or fragments thereof described above may be used in drug screening procedures to identify molecules which are agonists, antagonists, or inhibitors of PCTA-1 activity. In a preferred embodiment, the PCTA-1 proteins or fragments thereof comprise at least one mutation provided either by biallelic markers of the present invention, preferably at least one mutation encoding by the biallelic markers A54, A56, A60, A75, A76, A85, or by a trait causing mutation according to the present invention. The PCTA-1 proteins or fragments thereof used in such analyses may be free in solution or linked to a solid support Alternatively, the PCTA-1 proteins or fragments thereof can be expressed on a cell surface. The cell may naturally express a PCTA-1 protein or a fragment thereof or, alternatively, the cell may express a PCTA-1 protein or a fragment thereof from an expression vector such as those described above.

In one method of drug screening, eucaryotic or procaryotic host cells which are stably transformed with recombinant polynucleotides in order to express a PCTA-1 protein or a fragment thereof are used in conventional competitive binding assays or standard direct binding assays.

To study the interaction of a PCTA-1 protein or a fragment thereof with drugs or small molecules, such as molecules generated through combinatorial chemistry approaches, the microdialysis coupled to HPLC method described by Wang et al. (1997) or the affinity capillary electrophoresis method described by Bush et al. (I 997) can be used.

In further methods, molecules which interact with a PCTA-1 protein or a fragment thereof may be identified using assays such as the following. The molecule to be tested for binding is labeled with a detectable label, such as a fluorescent, radioactive, or enzymatic tag and placed in contact with an immobilized PCTA-1 protein or a fragment thereof under conditions which permit specific binding to occur. After removal of non-specifically bound molecules, bound molecules are detected using appropriate means.

Another object of the present invention consists of methods and kits for the screening of candidate substances that interact with a PCTA-1 polypeptide.

A method for the screening of a candidate substance comprises the following steps: a) providing a polypeptide consisting of a PCTA-1 protein or a fragment thereof; b) obtaining a candidate substance; c) bringing into contact said polypeptide with said candidate substance; and d) detecting the complexes formed between said polypeptide and said candidate substance. Optionally, said PCTA-1 protein or fragment thereof is selected from the group consisting of polypeptides of SEQ ID Nos 5, 6, 7, 9 and fragments thereof.

The invention also pertains to kits useful for performing the hereinbefore described screening method. Preferably, such kits comprise a PCTA-1 polypeptide or a fragment thereof, and optionally means useful to detect the complex formed between a PCTA-1 polypeptide or a fragment thereof and the candidate substance. In a preferred embodiment the detection means consist in monoclonal or polyclonal antibodies directed against the corresponding PCTA-1 polypeptide or a fragment thereof.

Various candidate substances or molecules can be assayed for interaction with a PCTA-1 protein or a fragment thereof. These substances or molecules include, without being limited to, natural or synthetic organic compounds or molecules of biological origin such as polypeptides, antibodies, fatty acids and lipoproteins. When the candidate substance or molecule consists of a polypeptide, this polypeptide may be the resulting expression product of a phage clone belonging to a phage-based random peptide library, or alternatively the polypeptide may be the resulting expression product of a cDNA library cloned in a vector suitable for performing a two-hybrid screening assay.

For the purpose of the present invention, a ligand means a molecule, such as a protein, a peptide, an antibody, a fatty acid, a lipoprotein, or any synthetic chemical compound capable of binding to a PCTA-1 protein or a fragment thereof.

A. Candidate Ligands Obtained from Random Peptide Libraries

In a particular embodiment of the screening method, the putative ligand is the expression product of a DNA insert contained in a phage vector (Parm ley and Smith, 1988). Specifically, random peptide phages libraries are used. The random DNA inserts encode for peptides of 8 to 20 amino acids in length (Oldenburg K. R. et al., 1992; Valadon P., et al., 1996; Lucas A. H., 1994; Westerink M. A. J., 1995; Felici F. et al., 1991). According to this particular embodiment, the recombinant phages expressing a protein that binds to the immobilized PCTA-1 protein or a fragment thereof is retained and the complex formed between the PCTA-1 polypeptide and the recombinant phage may be subsequently immunoprecipitated by a polyclonal or a monoclonal antibody directed against the PCTA-1 polypeptide.

Once the ligand library in recombinant phages has been constructed, the phage population is brought into contact with the immobilized PCTA-1 protein or a fragment thereof. Then the preparation of complexes is washed in order to remove the non-specifically bound recombinant phages. The phages that bind specifically to the PCTA-1 protein or a fragment thereof are then eluted by a buffer (acid pH) or immunoprecipitated by the monoclonal antibody produced by the hybridoma anti-PCTA-1, and this phage population is subsequently amplified by an over-infection of bacteria (for example E. coli ). The selection step may be repeated several times, preferably 2-4 times, in order to select the more specific recombinant phage clones. The last step consists of characterizing the peptide produced by the selected recombinant phage clones either by expression in infected bacteria and isolation, expressing the phage insert in another host-vector system, or sequencing the insert contained in the selected recombinant phages.

B. Candidate Ligands Obtained by Competition Experiments.

Alternatively, peptides, drugs or small molecules which bind to the PCTA-1 protein, or a fragment thereof may be identified in competition experiments. In such assays, the PCTA-1 protein or a fragment thereof is immobilized to a surface, such as a plastic plate. Increasing amounts of the peptides, drugs or small molecules are placed in contact with the immobilized PCTA-1 protein or a fragment thereof in the presence of a detectable labeled known PCTA-1 protein ligand. For example, the PCTA-1 ligand may be detectably labeled with a fluorescent, radioactive, or enzymatic tag. The ability of the test molecule to bind the PCTA-1 protein or a fragment thereof is determined by measuring the amount of detectably labeled known ligand bound in the presence of the test molecule. A decrease in the amount of known ligand bound to the PCTA-1 protein or a fragment thereof when the test molecule is present indicated that the test molecule is able to bind to the PCTA-1 protein or a fragment thereof.

C. Candidate Ligands Obtained by Affinity Chromatography.

Proteins or other molecules interacting with the PCTA-1 protein or a fragment thereof can also be found using affinity columns which contain the PCTA-1 protein or a fragment thereof. The PCTA-1 protein or a fragment thereof may be attached to the column using conventional techniques including chemical coupling to a suitable column matrix such as agarose, Affi Gel®, or other matrices familiar to those of skill in art. In some embodiments of this method, the affinity column contains chimeric proteins in which the PCTA-1 protein or a fragment thereof is fused to glutathion S transferase (GST). A mixture of cellular proteins or pool of expressed proteins as described above is applied to the affinity column. Proteins or other molecules interacting with the PCTA-1 protein or a fragment thereof attached to the column can then be isolated and analyzed on 2-D electrophoresis gel as described in Ramunsen et al. (1997). Alternatively, the proteins retained on the affinity column can be purified by electrophoresis based methods and sequenced. The same method can be used to isolate antibodies, to screen phage display products, or to screen phage display human antibodies.

D. Candidate Ligands Obtained by Optical Biosensor Methods

Proteins interacting with the PCTA-1 protein or a fragment thereof can also be screened by using an Optical Biosensor as described in Edwards and Leatherbarrow (1997) and also in Szabo et al. (1995). This technique permits the detection of interactions between molecules in real time, without the need of labeled molecules. This technique is based on the surface plasmon resonance (ISPR) phenomenon. Briefly, the candidate ligand molecule to be tested is attached to a surface (such as a carboxymethyi dextran matrix). A light beam is directed towards the side of the surface that does not contain the sample to be tested and is reflected by said surface. The SPR phenomenon causes a decrease in the intensity of the reflected light with a specific association of angle and wavelength. The binding of candidate ligand molecules cause a change in the refraction index on the surface, which change is detected as a change in the SPR signal. For screening of candidate ligand molecules or substances that are able to interact with the PCTA-1 protein or a fragment thereof, the PCTA-1 polypeptide is immobilized onto a surface. This surface consists of one side of a cell through which flows the candidate molecule to be assayed. The binding of the candidate molecule on the PCTA-1 protein or a fragment thereof is detected as a change of the SPR signal. The candidate molecules tested may be proteins, peptides, carbohydrates, lipids, or small molecules generated by combinatorial chemistry. This technique may also be performed by immobilizing eukaryotic or prokaryotic cells or lipid vesicles exhibiting an endogenous or a recombinantly expressed PCTA-1 protein at their surface.

The main advantage of the method is that it allows the determination of the association rate between the PCTA-1 protein and molecules interacting with the PCTA-1 protein. It is thus possible to select specifically ligand molecules interacting with the PCTA-1 protein, or a fragment thereof, through strong or conversely weak association constants.

E. Candidate Ligands Obtained through a Two-Hybrid Screening Assay.

The yeast two-hybrid system is designed to study protein-protein interactions in vivo (Fields and Song, 1989), and relies upon the fusion of a bait protein to the DNA binding domain of the yeast Gal4 protein. This technique is also described in the U.S. Patent N o U.S. Pat. No. 5,667,973 and the U.S. Pat. N o 5,283,173 (Fields et al.), the disclosures of which are incorporated herein by reference in their entireties.

The general procedure of library screening by the two-hybrid assay may be performed as described by Harperet al. (1993) or as described by Cho et al. (1998) or also Fromont-Racine et al. (1997).

The bait protein or polypeptide consists of a PCTA-1 polypeptide or a fragment thereof.

More precisely, the nucleotide sequence encoding the PCTA-1 polypeptide or a fragment thereof is fused to a polynucleotide encoding the DNA binding domain of the GAL4 protein, the fused nucleotide sequence being inserted in a suitable expression vector, for example pAS2 or pM3.

Then, a human cDNA library is constructed in a specially designed vector, such that the human cDNA insert is fused to a nucleotide sequence in the vector that encodes the transcriptional domain of the GAL4 protein. Preferably, the vector used is the pACT vector. The polypeptides encoded by the nucleotide inserts of the human cDNA library are termed “pray” polypeptides.

A third vector contains a detectable marker gene, such as beta galactosidase gene or CAT gene that is placed under the control of a regulation sequence that is responsive to the binding of a complete Gal4 protein containing both the transcriptional activation domain and the DNA binding domain. For example, the vector pG5EC may be used.

Two different yeast strains are also used. As an illustrative but non limiting example the two different yeast strains may be the followings:

Y190, the phenotype of which is (MATa, Leu2-3, 112 ura3-12, trp1-901, his3-D200, ade2-101, gal4Dga1180D URA3 GAL-LacZ, LYS GAL-HIS3, cyh');

Y187, the phenotype of which is (MaTa gal4 ga180 his3 trp1-901 ade2-101 ura3-52 leu2-3, -112 URA3 GAL-lacZmet′), which is the opposite mating type of Y190.

Briefly, 20 μg of pAS2/PCTA-1 and 20 μg of pACT-cDNA library are co-transformed into yeast strain Y190. The transformants are selected for growth on minimal media lacking histidine, leucine and tryptophan, but containing the histidine synthesis inhibitor 3-AT (50 mM). Positive colonies are screened for beta galactosidase by filter lift assay. The double positive colonies (His + , beta-gal + ) are then grown on plates lacking histidine, leucine, but containing tryptophan and cycloheximide (10 mg/ml) to select for loss of pAS2/PCTA-1 plasmids bu retention of pACT-cDNA library plasmids. The resulting Y190 strains are mated with Y187 strains expressing PCTA-1 or non-related control proteins; such as cyclophilin B, lamin, or SNF1, as Gal4 fusions as described by Harper et al. (1 993) and by Bram et al. (1993), and screened for beta galactosidase by filter lift assay. Yeast clones that are beta gal- after mating with the control Gal4 fusions are considered false positives.

In another embodiment of the two-hybrid method according to the invention, interaction between the PCTA-1 or a fragment thereof with cellular proteins may be assessed using the Matchmaker Two Hybrid System 2 (Catalog No. K1604-1, Clontech). As described in the manual accompanying the Matchmaker Two Hybrid System 2 (Catalog No. K1604-1, Clontech), nucleic acids encoding the PCTA-1 protein or a fragment thereof, are inserted into an expression vector such that they are in frame with DNA encoding the DNA binding domain of the yeast transcriptional activator GAL4. A desired cDNA, preferably human cDNA, is inserted into a second expression vector such that they are in frame with DNA encoding the activation domain of GAL4. The two expression plasmids are transformed into yeast and the yeast are plated on selection medium which selects for expression of selectable markers on each of the expression vectors as well as GALA dependent expression of the HIS3 gene. Transformants capable of growing on medium lacking histidine are screened for GAL4 dependent lacZ expression. Those cells which are positive in both the histidine selection and the lacZ assay contain interaction between PCTA-1 and the protein or peptide encoded by the initially selected cDNA insert.

Screening through Spontaneous Metastatic Assay

Screening of new compounds can be carried out through a spontaneous metastatic assay as described in Nihei et al. (1995). Hence, it can be possible to assess the decrease of metastatic potential of transformed cells related to treatment of said compounds. Indeed, according to the present invention, the metastatic potential of cells constitutes the major criteria of the aggressiveness of prostate cancer tumors.

To evaluate the metastatic ability, about 5×10 5 cells expressing a PCTA-1 gene comprising alleles for one or more biallelic markers associated with cancer, preferably with prostate cancer, or with the aggressiveness of prostate cancer tumors, are injected subcutaneously in the flank of male athymic nude mice. The mice are treated with the screened compounds. Tumor volume and tumor volume doubling time are used as the index of the tumor growth rate and are determined as described in Isaacs & Hukku, 1988). The tumor-bearing animals are scored for lung metastases at spontaneous death or when killed at day 35 post-inoculation.

Gene Therapy

Gene therapy involves the alteration of the phenotypic expression of a targeted cell, usually a cancer cell through the alteration of the cell's genotypic content. The desired effect of gene therapy is a reduction or interruption of tumor growth or, ideally, the destruction of the cell itself. An appropriate gene for gene therapy must be capable of altering the biological behavior of the cancer cell in order to slow growth, reduce local invasive potential, or induce apoptosis. The PCTA-1 gene, or certain portions thereof, is a good candidate for gene therapy.

The present invention also comprises the use of the genomic PCTA-1 DNA described above or a fragment thereof, in gene therapy strategies, such as antisense and triple helix strategies, and in the introduction of a therapeutic gene. Preferred nucleotide sequences useful in gene therapy include the sequences of SEQ ID Nos 1, 2, 3, 4, 8, complementary sequences thereto, and fragments thereof. More particularly, preferred nucleotide sequences comprise any of the polynucleotides described in the “PCTA-1 cDNA Sequences” section, the “Coding Regions” section, and the “Oligonucleotide Probes And Primers” section. Preferred PCTA-1 DNA fragments used in such approaches are those comprising a nucleotide sequence comprising a PCTA-1 regulatory region or a fragment thereof. More particularly, the regulatory regions comprise at least one of the biallelic markers according to the invention, more particularly those comprising a biallelic marker selected from the group consisting of A1 to A125, preferably A2, A30, A41, A55, A57, or a trait causing mutation, or complementary sequences thereof or variants or fragments thereof.

In a first embodiment, the invention therefore concerns a method for the treatment of prostate cancer. The method comprises: (a) selecting an individual whose DNA comprises an allele of biallelic marker or of a group of biallelic markers, preferably markers of the PCTA-1 gene, associated with a susceptibility to prostate cancer; and (b) administering to the individual an effective amount of a molecule capable of modifying the expression of the PCTA-1 gene:

In one embodiment, the molecule is an antisense nucleotide sequence, capable of competitively binding to the mRNA sequence resulting from the transcription of the PCTA-1 genomic DNA so as to prevent the translation of said mRNA. In preferred embodiments of this method, the antisense nucleotide sequence is characterized in that it hybridizes with exons of the PCTA-1 gene, preferably with a region of such exons comprising a least an allele of one of the biallelic markers of the present invention. Optionally, the antisense nucleotide sequence hybridizes with exons 0, 1, 6bis, 9 or 9ter of the PCTA-1 gene.

In an other embodiment, the molecule is a nucleotide sequence comprising a homopurine or homopyridine, preferably a 10-mer to 20-mer homopurine or homopyridine, which is complementary to a homopurine or homopyridine sequence of the PCTA-1 genomic DNA so as to prevent transcription of said genomic DNA into mRNA.

In a further embodiment, the molecule is a nucleotide sequence comprising a DNA sequence encoding a protein capable, when expressed, of exerting a therapeutic effect on prostate cancer cells, operably linked to the promoter of PCTA-1 gene, so as to kill or disable said prostate cancer cells.

The invention also concerns a method for the treatment of prostate cancer comprising:

administering to an individual an effective amount of a nucleotide sequence comprising a DNA sequence encoding a protein capable, when expressed, of exerting a therapeutic effect on prostate cancer cells, operably linked to the promoter of PCTA-1 gene.

The gene encoding a protein capable of exerting a therapeutic effect on prostate cancer cells is called the therapeutic gene in the present application. In some embodiments, the therapeutic gene is a toxin gene encoding a cytotoxic or cytostatic gene product. In another embodiment, the therapeutic gene is a gene encoding an immunogenic antigen which is highly visible to the immune system. In further embodiment, the therapeutic gene is a gene encoding a lymphokine which activates an anti-tumor immune response. In additional embodiments, the therapeutic gene encodes an antisense sequence having as a target the coding region of an essential gene for the proliferation or viability of the cell.

Antisense Approach

In antisense approaches, nucleic acid sequences complementary to a targeted mRNA are hybridized to the mRNA intracellularly, thereby blocking the expression of the protein encoded by the mRNA. The antisense sequences can prevent gene expression through a variety of mechanisms. For example, the antisense sequences may inhibit the ability of ribosomes to translate the mRNA. Alternatively, the antisense sequences may block transport of the mRNA from the nucleus to the cytoplasm, thereby limiting the amount of mRNA available for translation. Another mechanism through which antisense sequences may inhibit gene expression is by interfering with mRNA splicing. In yet another strategy, the antisense nucleic acid may be incorporated in a ribozyme capable of specifically cleaving the target mRNA.

The antisense nucleic acid molecules to be used in gene therapy may be either DNA or RNA sequences. They comprise a nucleotide sequence complementary to the targeted sequence of the PCTA-1 genomic DNA or a PCTA-1 cDNA. The targeted DNA or RNA sequence preferably comprises at least one of the biallelic markers according to the present invention, particularly a biallelic marker selected from the group consisting of A1 to A125 and the complements thereof, or comprises a trait causing mutation. In a preferred embodiment, the antisense oligonucleotide are able to hybridize with at least one of the splicing sites of the targeted PCTA-1 gene, with the 3′UTR or the 5′UTR, with exon 0, 1, 6bis, 9 or 9ter, or with an exonic region comprising at least one of the biallelic markers of the present invention or comprising a trait causing mutation.

Preferred methods using antisense polynucleotide according to the present invention are the procedures described by Sczakiel et al.(1995).

Preferably, the antisense tools are chosen among the polynucleotides (15-200 bp long) that are complementary to the 5′ end of the PCTA-1 mRNA. In another embodiment, a combination of different antisense polynucleotides complementary to different parts of the desired targeted gene are used.

Preferred antisense polynucleotides according to the present invention are complementary to a sequence of the mRNAs of PCTA-1 that contains either the translation initiation codon ATG or a splicing donor or acceptor site.

The antisense nucleic acids should have a length and melting temperature sufficient to permit formation of an intracellular duplex having sufficient stability to inhibit the expression of the PCTA-1 mRNA in the duplex. Strategies for designing antisense nucleic acids suitable for use in gene therapy are disclosed in Green et al., (1986) and Izant and Weintraub, (1984).

In some strategies, antisense molecules are obtained by reversing the orientation of the PCTA-1 coding region with respect to a promoter so as to transcribe the opposite strand from that which is normally transcribed in the cell. The antisense molecules may be transcribed using in vitro transcription systems such as those which employ T7 or SP6 polymerase to generate the transcript. Another approach involves transcription of PCTA-1 antisense nucleic acids in vivo by operably linking DNA containing the antisense sequence to a promoter in a suitable expression vector.

Alternatively,suitable antisense strategies are those described by Rossi et al.(1991), in the International Applications Nos. WO 94/23026, WO 95/04141, WO 92/18522, WO 96/31523 and in the European Patent Application No. EP 0 572 287 A2, the disclosures of which are incorporated herein by reference in their entireties.

An alternative to the antisense technology that is used according to the present invention consists of using ribozymes that will bind to a target sequence via their complementary polynucleotide tail and that will cleave the corresponding RNA by hydrolyzing its target site (namely “hammerhead ribozymes”). Briefly, the simplified cycle of a hammerhead ribozyme consists of (1) sequence specific binding to the target RNA via complementary antisense sequences; (2) site-specific hydrolysis of the cleavable motif of the target strand; and (3) release of cleavage products, which gives rise to another catalytic cycle. Indeed, the use of long-chain antisense polynucleotide (at least 30 bases long) or ribozymes with long antisense arms are advantageous. A preferred delivery system for antisense ribozyme is achieved by covalently linking these antisense ribozymes to lipophilic groups or to use liposomes as a convenient vector. Preferred antisense ribozymes according to the present invention are prepared as described by Sczakiel et al.(1995).

Triple Helix Approach

The PCTA-1 genomic DNA, preferably comprising at least one of the biallelic markers according to the invention, more preferably at least one biallelic marker selected from the group consisting of A1 to A125, or comprising a trait causing mutation, or complementary sequences, variants or fragments thereof, may also be used in gene therapy approaches based on intracellular triple helix formation.

Triple helix oligonucleotides are used to inhibit transcription from a genome. They are particularly useful for studying alterations in cell activity when it is associated with a particular gene. Fragments of the PCTA-1 genomic DNA can be used to inhibit gene expression in individuals suffering from prostate cancer or from another detectable phenotype, or in individuals at risk of developing prostate cancer or another detectable phenotype at a later date as a result of their PCTA-1 genotype.

Similarly, a portion of the PCTA-1 genomic DNA can be used to study the effect of inhibiting PCTA-1 transcription within a cell. Traditionally, homopurine sequences were considered the most useful for triple helix strategies. However, homopyrimidine sequences can also inhibit gene expression. Such homopyrimidine oligonucleotides bind to the major groove at homopurine:homopyrimidine sequences. Thus, both types of sequences from the PCTA-1 genomic DNA, preferably comprising at least one of the biallelic markers according to the invention, more preferably at least one of the biallelic markers A1 to A125, or comprising the trait causing mutation, or complementary sequences thereof, variants thereof, are contemplated within the scope of this invention.

To carry out gene therapy strategies using the triple helix approach, the sequences of the PCTA-1 genomic DNA, preferably comprising at least one of the biallelic markers according to the invention, or comprising the trait causing mutation, or complementary sequences thereof, or variants thereof, are first scanned to identify 10-mer to 20-mer homopyrimidine or homopurine stretches which could be used in triple-helix based strategies for inhibiting PCTA-1 expression. Following identification of candidate homopyrimidine or homopurine stretches, their efficiency in inhibiting PCTA-1 expression is assessed by introducing varying amounts of oligonucleotides containing the candidate sequences into tissue culture cells which express the PCTA-1 gene.

The oligonucleotides can be introduced into the cells using a variety of methods known to those skilled in the art, including but not limited to calcium phosphate precipitation, DEAE-Dextran, electroporation, liposome-mediated transfection or native uptake.

Treated cells are monitored for altered cell function or reduced PCTA-1 expression using techniques such as Northern blotting, RNase protection assays, or PCR based strategies to monitor the transcription levels of the PCTA-1 gene in cells which have been treated with the oligonucleotide.

The oligonucleotides which are effective in inhibiting gene expression in tissue culture cells may then be introduced in vivo using the techniques described above in the antisense approach at a dosage calculated based on the in vitro results, as described in antisense approach.

In some embodiments, the natural (beta) anomers of the oligonucleotide units can be replaced with alpha anomers to render the oligonucleotide more resistant to nucleases. Further, an intercalating agent such as ethidium bromide, or the like, can be attached to the 3′ end of the alpha oligonucleotide to stabilize the triple helix. For information on the generation of oligonucleotides suitable for triple helix formation see Griffin et al. (1989).

Introduction of a Therapeutic Gene

One important aspect of the present invention concerns a promoter specifically expressed in prostate cancer cells. More particularly, the present invention relates to the regulatory sequences, and particularly the promoter of the PCTA-1 gene. The expression of PCTA-1 appears to be specific to prostate cancer cells.

The term “specific”, when used herein with reference to a promoter, is intended to designate a promoter which is specifically expressed in prostate cancer cells, at a level which is sufficient to have a significant impact on the metabolism of such cells. In other words, the promoter is specific in activity, effect or function. However, the term does not necessarily designate a promoter which is expressed solely in prostate cancer cells. Indeed, it is possible that the PCTA-1 gene is expressed, under the control of its promoter, in other cells at levels which are sufficiently low to be undetectable by current detection techniques such as those involving antibodies, hybridization with a probe or even PCR. The promoter of the PCTA-1 gene can be advantageously used to introduce a therapeutic gene which will be expressed specifically in prostate cancer cells.

The invention therefore also concerns an expression vector comprising a DNA sequence encoding a functional protein, particularly a functional protein capable of exerting a therapeutic effect on prostate cancer cells, operably linked to the promoter of the PCTA-1 gene which is specifically expressed in prostate cancer cells.

Furthermore, the PCTA-1 promoter preferably comprises biallelic markers according to the invention, more particularly those described previously. Some alleles of the biallelic markers of the invention show an association with prostate cancer and may be involved in a modified or forthcoming expression of the PCTA-1 gene in prostate cancer cells. It may therefore advantageous to use the PCTA-1 promoter comprising such an allele to introduce a therapeutic gene for enhancing its expression in prostate cancer cells.

The term “therapeutic gene” is intended to designate DNA encoding an amino acid sequence corresponding to a functional peptide or protein capable of exerting a therapeutic effect on prostate cancer cells preferably by killing or disabling such cells, or having a regulatory effect on the expression of an important function in prostate cells.

In one embodiment, a single enhancer element or multiple enhancer elements which amplify the expression of the therapeutic gene without compromising tissue specificity can also be combined with the promoter of the PCTA-1 gene. In a preferred embodiment, the enhancer element may be a portion of the cytomegalovirus LTR, SV40 enhancer sequences, or MMTV LTR. Preferably, the enhancer element is positioned upstream of the PCTA-1 promoter.

The term “enhancer element” is intended to designate a nucleotide sequence that increases the rate of transcription of therapeutic genes or genes of interest but does not have promoter activity. An enhancer can be moved upstream, downstream, and to the other side of the PCTA-1 promoter without significant loss of activity.

In a preferred embodiment, a vector is constructed by inserting the therapeutic gene downstream of the PCTA-1 promoter. The therapeutic gene is inserted so as to be operably linked to the promoter.

Examples of therapeutic genes include suicide genes. These are gene sequences, the expression of which produces a protein or agent that inhibits prostate tumor cell growth or induces prostate tumor cell death. Genes of interest include genes encoding enzymes, oncogenes, tumor suppressor genes, genes encoding toxins, genes encoding cytokines, or a gene encoding oncostatin. The purpose of the therapeutic genes is to inhibit the growth of or kill prostate cancer cells or to produce cytokines or other cytotoxic agents which directly or indirectly inhibit the growth of or kill prostate cancer cell.

Suitable enzymes include thymidine kinase, xanthine-guanine phosphoribosyltransferase, cytosine deaminase, and hypoxanthine phosphoribosyl transferase. Suitable oncogenes and tumor suppressor genes include neu, EGF, ras, p53, retinoblastoma tumor suppressor gene (Rb), Wilm's tumor gene product, phosphotyrosine phosphatase, and nm23. Suitable toxins include Pseudomonas exotoxin A and S, diphteria toxin, E. coli LT toxins, Shiga toxin, Shiga-like toxins, ricin, abrin, supporin, and gelonin. Suitable cytokines include interferons, GM-CSF interleukins, tumor necrosis factor.

Other gene therapy strategies include antisense sequences as mentioned above of at least about 30 bp, preferably 50 pb, having as target the coding sequence of an essential gene for the proliferation or viability of the cell. Numerous proteins associated with transcription, translation, metabolic pathways, cytostructural genes can be used as target, preferably those which are essential, present at relatively low levels, and particularly associated with cancer cells.

The three presently available methodologies for DNA delivery are well-known by the skilled artisan: transfection with a viral vector, fusion with a lipid; and cationic supported DNA introduction. A suitable DNA delivery method should meet the following criteria: 1) capable of directing the therapeutic polynucleotides into specific target cell types, 2) highly efficient in mediating uptake of the therapeutic polynucleotide into the target cells, and 3) suited for use in vivo for therapeutic application.

The preferred method relies on replication-defective viral vectors harboring the therapeutic polynucleotide sequence as part of retroviral genome. Preferred vectors for use in the present invention are viral including adenoviruses, retroviral vectors, and adeno-associated viral vectors. Retroviral vectors and adenoviruses offer an efficient, useful, and presently the best-characterized means of introducing and expressing foreign genes efficiently in mammalian cells. These vectors have very broad host and cell type range and express genes stably and efficiently.

Other virus vectors that may be used for gene transfer into cells include retroviruses such as Moloney murine lekemia virus, papovaviruses such as JC, SV40, polyoma, and adenoviruses, Epstein-Barr virus, papilloma viruses such as bovine papilloma virus type I, vaccinia, and poliovirus.

Another gene transfer method is physical transfer of plasmid DNA comprising the therapeutic polynucleotide in liposomes directly into prostate, preferably into tumors cells in situ. Immunoliposomes may improve cell type specificity as compared to liposomes by virtue of the inclusion of specific antibodies which presumably bind to surface antigens specific of prostate cells. In one embodiment, antibodies are directed against PCTA-1 protein which is specific to prostate cancer cells.

Direct physical application of naked DNA comprising the therapeutic polynucleotide to the target cells is believed to be preferred in many cases.

Vaccine Composition

The invention concerns a vaccine composition comprising a vaccination agent including one of the following polypeptide:

a) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15,20, 25,30,40, 50, or 100 amino acids of SEQ ID No 5, wherein said contigous span comprises:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 5; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 5;

b) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25,30,40, 50, or 100 amino acids of SEQ ID No 6, wherein said contigous span comprises:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 245 in SEQ ID No 6; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 225 in SEQ ID No 6; and/or

iii) at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exon 6bis, more CF particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 183-224 of the SEQ ID No 6;

c) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20,25,30, 40, 50, or 100 amino acids of SEQ ID No 7, wherein said contigous span comprises:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 7; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 7; and/or

iii) at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exons 9bis and 9ter, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 313-368 of the SEQ ID No 7; and

d) a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25,30, 40, 50, or 100 amino acids of SEQ ID No 9.

“Vaccine agent or vaccination agent” is intended to designate a substance which has the ability, when administered to a patient in suitable amounts, to generate an immunogenic reaction which can confer either immunity to the patent against prostate cancer or kill or disable prostate cancer cells bearing on their surface the PCTA-1 protein or a fragment thereof.

The vaccine compositions of the present invention are intended to be administered to patients in an amount sufficient to inhibit the growth of cancer cells expressing the PCTA-1 protein. More particularly the vaccine composition is intended to decrease the growth rate, rate of division, or viability of the prostate cancer cells.

The administration of a vaccine composition of the invention may be for either a “prophylactic” or “therapeutic” purposes. When provided prophylactically, the vaccine agent are provided in advance of symptoms indicative of prostate cancer. The prophylactic administration of vaccine agent serves to prevent, attenuate, or inhibit of the growth of prostate cancer cells. The therapeutic administration of the vaccine agent serves to attenuate the pathological symptoms of prostate cancer, to decrease the size or growth of cancer tumors and or metastasis or to remove them.

The term “inhibition of growth” refers in the present application to the decrease of the rate of growth, rate of division, or viability of the cells in question.

Indeed, as the PCTA-1 gene is specifically expressed in prostate cancer cells, these vaccine agents can initiate the production of PCTA-1 specific cytotoxic T lymphocytes which lyse cells bearing, preferably on their surface, PCTA-1, a fragment of PCTA-1, or one or more PCTA-1 epitope peptides thereof and which lead to an inhibition of the growth of cancer also bearing the PCTA-1 protein.

Vaccine preparations which contain protein or peptide sequences as active substances are generally well known in the art, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, the disclosures of which are incorporated herein by reference in their entireties.

A vaccine according to the present invention may further contain auxiliary vaccine constituents, such as carriers, buffers, stabilizers, solubilizers, adjuvants and preservatives.

In order to enhance the immunogenic character of the polypeptides taken from the mutated PCTA-1 protein, the polypeptides may be prepared as homopolymers (a multitude of identical polypeptides coupled to one another) or heteropolymers (a multitude of at least two different polypeptides coupled to one another).

The vaccine agents of the present invention can be used in native form or can be modified to form a chemical derivative. As used herein, a molecule is said to be a “chemical derivative” of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, et. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980).

The vaccine agents of the present invention may be administered in a convenient manner such as by oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, or intradermal routes. The vaccine agents of the present invention are administered in an amount which is effective for treatment and/or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 μg/kg body weight per day and in most cases they are administered in an amount not in excess of about 8 mg/kg body weight per day. In most cases, the dosage is from about 10 μg/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.

When administering the vaccine agent of the present invention to a patient, the dosage of the administered vaccine agent varies depending upon such factors as the patient's age, weight, sex, general medical condition, previous medical history. In general, it is desirable to provide the recipient with a dosage of vaccine agent which is in the range of from about 1 pg/kg to 10 mg/kg body weight, although a lower or higher dosage may be administered. The therapeutically effective dose can be lowered by using combinations of the vaccine agents of the present invention or other agents.

It is normally necessary to have multiple administrations of the vaccine agents, usually not exceeding six vaccinations, more usually not exceeding four vaccinations, preferably one or more vaccinations, more preferably about three vaccinations. The vaccinations will be normally be at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years will be desirable to maintain levels of protective immunity.

The vaccine agents of the present invention are intended to be provided to recipient subjects in an amount sufficient to inhibit the growth (as defined above) of cancer cells bearing PCTA-1 protein.

The effect of the vaccine agents of the present invention can be assessed through the measurement of released IFN-γ from memory T-lymphocytes. The stronger of the immune response, the more IFN-γ will be released. Accordingly, a vaccine according to the invention comprises a polypeptide capable of releasing from the memory T-lymphocytes at least 1500 pg/ml, preferably 200 pg/ml, and more preferably 300 pg/ml of IFN-γ. Practically, the levels of IFN-γ from the primed lymphocytes are measured with in vitro proliferation assays of peripheral blood lymphocytes co-cultured with a vaccine agents to be tested. These techniques are well known and may be found in a wide variety of patents, such as U.S. Pat. No. 3,791,932; 4,174,384; and 3,949,064, the disclosures of which are incorporated herein by reference in their entireties, as illustrative of these types of assays.

The administration of the vaccine agent of the invention may be for either a “prophylactic” or “therapeutic” purposes. When provided prophylactically, the vaccine agent are administered in advance of any symptoms indicative of prostate cancer. The prophylactic administration of the vaccine agent serves to prevent, attenuate, or inhibit of the growth of prostate cancer cells. The therapeutic administration of the vaccine agent serves to attenuate the pathological symptoms of prostate cancer and to decrease the size of prostate cancer tumors or to remove them.

Typically, such vaccine agents are prepared as injectable either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may be emulsified. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the vaccine agent. Suitable excipients are, for example, water, saline, dextrose, ethanol, or the like, and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.

PCTA-1 protein and peptides, preferably mutated, may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts which are formed between the free amino groups of the peptide, and inorganic acids, such as hydrochloric or phosphoric acids, or organic acids, such as acetic oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxydes, or from organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

Some of the polypeptides of the vaccine agents of the invention are sufficiently immunogenic in a vaccine, but the immune response can be enhanced if the vaccine further comprises an adjuvant substance.

Various methods of achieving adjuvant effects for vaccines include the use of agents such as aluminim hydroxide or phosphate, commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperature ranging between 70° C. and 101 C for 30 second to 2 minute periods, respectively. Aggregation by reacting with pepsin treated antibodies (Fab) to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide monoleatc (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed. According to the invention, dimethyldioctadecylammonium bromide is an interesting candidate for an adjuvant, but also Freund's complete and incomplete adjuvants as well as QuilA and RIBI are interesting possibilities. Other possibilities involve the use of immune modulating substances such as lymphokines (e.g. IFN-γ, IL-2 and IL-12) or synthetic IFN-γ inducers such as poly I:C in combination with the above-mentioned adjuvants.

The vaccine agent of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby immunogenic peptides, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, such as human serum albumin, are described Remington's Pharmaceutical Sciences (1980). In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of one or more of the vaccine agents of the present invention, together with a suitable amount of a carrier vehicle.

Additional pharmaceutical methods may be employed to control the duration of action. Control release preparations may be achieved through the use of polymers to complex or absorb one or more of the vaccine agents of the present invention. The controlled delivery may be exercised by selecting appropriate macromolecule (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, protamine, or sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate vaccine agents of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these vaccine agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, bu coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine-microcapsules and poly(methylmethacylate) microcapsules, respectively, orin colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980).

The invention further provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the vaccine compositions of the invention.

Computer-Related Embodiments

As used herein the term “nucleic acid codes of the invention” encompass the nucleotide sequences comprising, consisting essentially of, or consisting of any one of the following: a) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1: 1-70715, 70795-82207, 82297-83612, 83824-85297, 85418-86388, 86446-87495, 87523-88294, 88384-89483, 89650-92748, 97156-98309, 98476-99329, 99491-100026, 100212-100281, 100396-100538, 100682-100833, 100995-101920, 102087-102970, 103264-103724, and 103753-106746; b) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200,500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide G at positions 70728, 87860, 88297, 94432, and 95340 of SEQ ID No 1; a nucleotide A at positions 82218, 83644, 83808, 87787, 87806, 94218, and 97144 of SEQ ID No 1; a nucleotide C at positions 87902, 88215, 88283, 92760,93726, and 94422 of SEQ ID No 1; and a nucleotide T at positions 93903, and 94170 of SEQ ID No 1; c)a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide G at positions 86435, 93592, 93680, 93681, 93682, 93728, 93761, and 95445 of SEQ ID No 1; a nucleotide A at positions 86434, 88355, 93240, 93471, and 93747of SEQ ID No 1; a nucleotide C at positions 93683, 95126, and 95444 of SEQ ID No 1; and a nucleotide T at positions 94154, and 94430 of SEQ ID No 1; d) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 1: 92975-92977, 93711-93715,94151-94153, 94240-94243, 94770-94773, 94804-94808, 95121-95122, 95129-95135,95148-95153, 95154-95159, 95173-95178, 95367-95374, 95410-95413, 95418-95420, 95430-95436, 95533-95535, and 95677-95677; e) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 1-162 of SEQ ID No 2; f) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527, 2471, and 5397 of SEQ ID No 2; a nucleotide C at positions 1013, 1979, and 2675 of SEQ ID No 2; a nucleotide G at positions 176, 749, 2685, 3593 of SEQ ID No 2; and a nucleotide T at positions 2156, and 2423 of SEQ ID No 2; g) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708, 807, 1493, 1724, and 2000; a nucleotide C at positions 1936, 3379, and 3697; a nucleotide G at positions 709, 1845, 1933, 1934, 1935, 1981, 2014, and3698; and a nucleotide T at positions 2407, and 2683 of SEQ ID No 2; h) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 2: 1229-1231, 1964-1968, 2404-2406, 2493-2496, 3023-3026, 3057-3061, 3374-3375, 3382-3388, 3401-3406, 3407-3412, 3426-3431, 3620-3627, 3663-3666, 3671-3673, 3683-3689, 3786-3788 and 3930-3932; i) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3,5, or 10 of the following nucleotide positions of SEQ ID No 3: 1-162 and 747-872; j) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527, 2597, and 5523 of SEQ ID No 3; a nucleotide C at positions 1139, 2105, and 2801 of SEQ ID No 3; a nucleotide G at positions 176, 875, 2811, 3719 of SEQ ID No 3; and a nucleotide T at positions 2282, and 2549 of SEQ ID No 3; k) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708, 807, 1619, 1850, and 2126; a nucleotide C at positions 2062, 3505, and 3823; a nucleotide G at positions 709, 1971, 2059, 2060, 2061, 2107, 2140, and 3824; and a nucleotide T at positions 2533, and 2809 of SEQ ID No 3; 1) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises nucleotide positions selected from the group consisting of the nucleotide positions of SEQ ID No 3: 1355-1357, 1892-1894, 2090-2094, 2530-2532, 2619-2622, 3149-3152, 3183-3187, 3500-3501, 3508-3514, 3527-3532, 3533-3538, 3552-3557, 3746-3749, 3789-3792, 3797-3799, 3809-3815, 3912-3914 and 4056-4058; m) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 1-162 of SEQ ID No 4; n) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 253, 363, 527 and 2460 of SEQ ID No 4; a nucleotide C at position 1013 of SEQ ID No 4 and a nucleotide G at positions 176, and 749 of SEQ ID No 4; o) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least one nucleotide selected from the group consisting of a nucleotide A at positions 708 and 807 and a nucleotide G at position 709 of SEQ ID No 4; p) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises the pairs of nucleotide positions 1136-1137 of SEQ ID No 4; q) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 8 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 8: 1-500, 501-1000, 1001-1500, and 1501-1738; and, r) a nucleotide sequence complementary to any one of the preceding nucleotide sequences.

The “nucleic acid codes of the invention” further encompass nucleotide sequences homologous to: a) a contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1: 1-70715, 70795-82207, 82297-83612, 83824-85297, 85418-86388, 86446-87495, 87523-88294, 88384-89483, 89650-92748, 97156-98309, 98476-99329, 99491-100026, 100212-100281, 100396-100538, 100682-100833, 100995-101920, 102087-102970, 103264-103724, and 103753-106746; b) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 1-162 of SEQ ID No 2; c) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 3: 1-162 and 747-872; d) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 1-162 of SEQ ID No 4; e) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 8 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 8: 1-500, 501-1000, 1001-1500, and 1501-1738; and f) sequences complementary to all of the preceding sequences. Homologous sequences refer to a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% homology to these contiguous spans. Homology may be determined using any method described herein, including BLAST2N with the default parameters or with any modified parameters. Homologous sequences also may include RNA sequences in which uridines replace the thymines in the nucleic acid codes of the invention. It will be appreciated that the nucleic acid codes of the invention can be represented in the traditional single character format (See the inside back cover of Stryer, Lubert. Biochemistry, 3 rd edition. W. H Freeman & Co., New York.) or in any other format or code which records the identity of the nucleotides in a sequence.

As used herein the term “polypeptide codes of the invention” encompass the polypeptide sequences comprising:

a) a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 5, wherein said contiguous span includes:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 5; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 5;

b) a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 6, wherein said contiguous span includes:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 245 in SEQ ID No 6; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 225 in SEQ ID No 6; and/or

iii) at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exon 6bis, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 183-224 of the SEQ ID No 6;

c) a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 7, wherein said contiguous span includes:

i) a serine residue at amino acid position 170 and/or a lysine residue at amino acid position 203 in SEQ ID No 7; and/or

ii) at least one residue selected from the group consisting of a tyrosine residue at amino acid position 18, a cysteine residue at amino acid position 35, a methionine residue at amino acid position 55 and an arginine residue at amino acid position 183 in SEQ ID No 7; and/or

iii) at least 1, 2, 3, 5 or 10 of the amino acid encoded by the exons 9bis and 9ter, more particularly at least 1, 2, 3, 5 or 10 of the amino acid positions 313-368 of the SEQ ID No 7; and,

d) a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 9.

It will be appreciated that the polypeptide codes of the invention can be represented in the traditional single character format or three letter format (See the inside back cover of Stryer, Lubert. Biochemistry, 3 rd edition. W. H Freeman & Co., New York.) or in any other format or code which records the identity of the polypeptides in a sequence.

It will be appreciated by those skilled in the art that the nucleic acid codes of the invention and polypeptide codes of the invention can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer. As used herein, the words “recorded” and “stored” refer to a process for storing information on a computer medium. A skilled artisan can readily adopt any of the presently known methods for recording information on a computer readable medium to generate manufactures comprising one or more of the nucleic acid codes of the invention, or one or more of the polypeptide codes of the invention. Another aspect of the present invention is a computer readable medium having recorded thereon at least 2, 5, 10, 15, 20, 25, 30, or 50 nucleic acid codes of the invention. Another aspect of the present invention is a computer readable medium having recorded thereon at least 2, 5, 10, 15, 20, 25, 30, or 50 polypeptide codes of the invention.

Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media. For example, the computer readable media may be a hard disk, a floppy disk, a magnetic tape, CAROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) as well as other types of other media known to those skilled in the art.

Embodiments of the present invention include systems, particularly computer systems which store and manipulate the sequence information described herein. One example of a computer system 100 is illustrated in block diagram form in FIG. 3 . As used herein, “a computer system” refers to the hardware components, software components, and data storage components used to analyze the nucleotide sequences of the nucleic acid codes of the invention or the amino acid sequences of the polypeptide codes of the invention. In one embodiment, the computer system 100 is a Sun Enterprise 1000 server (Sun Microsystems, Palo Alto, Calif.). The computer system 100 preferably includes a processor for processing, accessing and manipulating the sequence data. The processor 105 can be any well-known type of central processing unit, such as the Pentium III from Intel Corporation, or similar processor from Sun, Motorola, Compaq or International Business Machines.

Preferably, the computer system 100 is a general purpose system that comprises the processor 105 and one or more internal data storage components 110 for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components. A skilled artisan can readily appreciate that any one of the currently available computer systems are suitable.

In one particular embodiment, the computer system 100 includes a processor 105 connected to a bus which is connected to a main memory 115 (preferably implemented as RAM) and one or more internal data storage devices 110 , such as a hard drive and/or other computer readable media having data recorded thereon. In some embodiments, the computer system 100 further includes one or more data retrieving device 118 for reading the data stored on the internal data storage devices 110 .

The data retrieving device 118 may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, etc. In some embodiments, the internal data storage device 110 is a removable computer readable medium such as a floppy disk, a compact disk, a magnetic tape, etc. containing control logic and/or data recorded thereon. The computer system 100 may advantageously include or be programmed by appropriate software for reading the control logic and/or the data from the data storage component once inserted in the data retrieving device.

The computer system 100 includes a display 120 which is used to display output to a computer user. It should also be noted that the computer system 100 can be linked to other computer systems 125 a-c in a network or wide area network to provide centralized access to the computer system 100 .

Software for accessing and processing the nucleotide sequences of the nucleic acid codes of the invention or the amino acid sequences of the polypeptide codes of the invention (such as search tools, compare tools, and modeling tools etc.) may reside in main memory 115 during execution.

In some embodiments, the computer system 100 may further comprise a sequence comparer for comparing the above-described nucleic acid codes of the invention or the polypeptide codes of the invention stored on a computer readable medium to reference nucleotide or polypeptide sequences stored on a computer readable medium. A “sequence comparer” refers to one or more programs which are implemented on the computer system 100 to compare a nucleotide or polypeptide sequence with other nucleotide or polypeptide sequences and/or compounds including but not limited to peptides, peptidomimetics, and chemicals stored within the data storage means. For example, the sequence comparer may compare the nucleotide sequences of nucleic acid codes of the invention or the amino acid sequences of the polypeptide codes of the invention stored on a computer readable medium to reference sequences stored on a computer readable medium to identify homologies, motifs implicated in biological function, or structural motifs. The various sequence comparer programs identified elsewhere in this patent specification are particularly contemplated for use in this aspect of the invention.

FIG. 4 is a flow diagram illustrating one embodiment of a process 200 for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database. The database of sequences can be a private database stored within the computer system 100 , or a public database such as GENBANK, PIR OR SWISSPROT that is available through the Internet.

The process 200 begins at a start state 201 and then moves to a state 202 wherein the new sequence to be compared is stored to a memory in a computer system 100 . As discussed above, the memory could be any type of memory, including RAM or an internal storage device.

The process 200 then moves to a state 204 wherein a database of sequences is opened for analysis and comparison. The process 200 then moves to a state 206 wherein the first sequence stored in the database is read into a memory on the computer. A comparison is then performed at a state 210 to determine if the first sequence is the same as the second sequence. It is important to note that this step is not limited to performing an exact comparison between the new sequence and the first sequence in the database. Well-known methods are known to those of skill in the art for comparing two nucleotide or protein sequences, even if they are not identical. For example, gaps can be introduced into one sequence in order to raise the homology level between the two tested sequences. The parameters that control whether gaps or other features are introduced into a sequence during comparison are normally entered by the user of the computer system.

Once a comparison of the two sequences has been performed at the state 210 , a determination is made at a decision state 210 whether the two sequences are the same. Of course, the term “same” is not limited to sequences that are absolutely identical. Sequences that are within the homology parameters entered by the user will be marked as “same” in the process 200 .

If a determination is made that the two sequences are the same, the process 200 moves to a state 214 wherein the name of the sequence from the database is displayed to the user. This state notifies the user that the sequence with the displayed name fulfills the homology constraints that were entered. Once the name of the stored sequence is displayed to the user, the process 200 moves to a decision state 218 wherein a determination is made whether more sequences exist in the database. If no more sequences exist in the database, then the process 200 terminates at an end state 220 . However, if more sequences do exist in the database, then the process 200 moves to a state 224 wherein a pointer is moved to the next sequence in the database so that it can be compared to the new sequence. In this manner, the new sequence is aligned and compared with every sequence in the database.

It should be noted that if a determination had been made at the decision state 212 that the sequences were not homologous, then the process 200 would move immediately to the decision state 218 in order to determine if any other sequences were available in the database for comparison.

Accordingly, one aspect of the present invention is a computer system comprising a processor, a data storage device having stored thereon a nucleic acid code of the invention or a By polypeptide code of the invention, a data storage device having retrievably stored thereon reference nucleotide sequences or polypeptide sequences to be compared to the nucleic acid code of the invention or polypeptide code of the invention and a sequence comparer for conducting the comparison. The sequence comparer may indicate a homology level between the sequences compared or identify structural motifs in the nucleic acid code of the invention and polypeptide codes of the invention or it may identify structural motifs in sequences which are compared to these nucleic acid codes and polypeptide codes. In some embodiments, the data storage device may have stored thereon the sequences of at least 2, 5, 10, 15, 20, 25, 30, or 50 of the nucleic acid codes of the invention or polypeptide codes of the invention.

Another aspect of the present invention is a method for determining the level of homology between a nucleic acid code of the invention and a reference nucleotide sequence, comprising the steps of reading the nucleic acid code and the reference nucleotide sequence through the use of a computer program which determines homology levels and determining homology between the nucleic acid code and the reference nucleotide sequence with the computer program. The computer program may be any of a number of computer programs for determining homology levels, including those specifically enumerated herein, including BLAST2N with the default parameters or with any modified parameters. The method may be implemented using the computer systems described above. The method may also be performed by reading 2, 5, 10, 15, 20, 25, 30, or 50 of the above described nucleic acid codes of the invention through the use of the computer program and determining homology between the nucleic acid codes and reference nucleotide sequences.

FIG. 5 is a flow diagram illustrating one embodiment of a process 250 in a computer for determining whether two sequences are homologous. The process 250 begins at a start state 252 and then moves to a state 254 wherein a first sequence to be compared is stored to a memory. The second sequence to be compared is then stored to a memory at a state 256 . The process 250 then moves to a state 260 wherein the first character in the first sequence is read and then to a state 262 wherein the first character of the second sequence is read. It should be understood that if the sequence is a nucleotide sequence, then the character would normally be either A, T, C, G or U. If the sequence is a protein sequence, then it should be in the single letter amino acid code so that the first and sequence sequences can be easily compared.

A determination is then made at a decision state 264 whether the two characters are the same. If they are the same, then the process 250 moves to a state 268 wherein the next characters in the first and second sequences are read. A determination is then made whether the next characters are the same. If they are, then the process 250 continues this loop until two characters are not the same. If a determination is made that the next two characters are not the same, the process 250 moves to a decision state 274 to determine whether there are any more characters either sequence to read.

If there aren't any more characters to read, then the process 250 moves to a state 276 wherein the level of homology between the first and second sequences is displayed to the user. The level of homology is determined by calculating the proportion of characters between the sequences that were the same out of the total number of sequences in the first sequence. Thus, if every character in a first 100 nucleotide sequence aligned with a every character in a second sequence, the homology level would be 100%.

Alternatively, the computer program may be a computer program which compares the nucleotide sequences of the nucleic acid codes of the present invention, to reference nucleotide sequences in order to determine whether the nucleic acid code of the invention differs from a reference nucleic acid sequence at one or more positions. Optionally such a program records the length and identity of inserted, deleted or substituted nucleotides with respect to the sequence of either the reference polynucleotide or the nucleic acid code of the invention. In one embodiment, the computer program may be a program which determines whether the nucleotide sequences of the nucleic acid codes of the invention contain one or more single nucleotide polymorphisms (SNP) with respect to a reference nucleotide sequence. These single nucleotide polymorphisms may each comprise a single base substitution, insertion, or deletion.

Another aspect of the present invention is a method for determining the level of homology between a polypeptide code of the invention and a reference polypeptide sequence, comprising the steps of reading the polypeptide code of the invention and the reference polypeptide sequence through use of a computer program which determines homology levels and determining homology between the polypeptide code and the reference polypeptide sequence using the computer program.

Accordingly, another aspect of the present invention is a method for determining whether a nucleic acid code of the invention differs at one or more nucleotides from a reference nucleotide sequence comprising the steps of reading the nucleic acid code and the reference nucleotide sequence through use of a computer program which identifies differences between nucleic acid sequences and identifying differences between the nucleic acid code and the reference nucleotide sequence with the computer program. In some embodiments, the computer program is a program which identifies single nucleotide polymorphisms The method may be implemented by the computer systems described above and the method illustrated in FIG. 5 . The method may also be performed by reading at least 2, 5, 10, 15, 20, 25, 30, or 50 of the nucleic acid codes of the invention and the reference nucleotide sequences through the use of the computer program and identifying differences between the nucleic acid codes and the reference nucleotide sequences with the computer program.

In other embodiments the computer based system may further comprise an identifier for identifying features within the nucleotide sequences of the nucleic acid codes of the invention or the amino acid sequences of the polypeptide codes of the invention.

An “identifier” refers to one or more programs which identifies certain features within the above-described nucleotide sequences of the nucleic acid codes of the invention or the amino acid sequences of the polypeptide codes of the invention. In one embodiment, the identifier may comprise a program which identifies an open reading frame in the cDNAs codes of the invention.

FIG. 6 is a flow diagram illustrating one embodiment of an identifier process 300 for detecting the presence of a feature in a sequence. The process 300 begins at a start state 302 and then moves to a state 304 wherein a first sequence that is to be checked for features is stored to a memory 115 in the computer system 100 . The process 300 then moves to a state 306 wherein a database of sequence features is opened. Such a database would include a list of each feature's attributes along with the name of the feature. For example, a feature name could be “Initiation Codon” and the attribute would be “ATG”. Another example would be the feature name “TTATAA Box” and the feature attribute would be “TATAA”. An example of such a database is produced by the University of Wisconsin Genetics Computer Group (Worldwide Web address: gcg.com).

Once the database of features is opened at the state 306 , the process 300 moves to a state 308 wherein the first feature is read from the database. A comparison of the attribute of the first feature with the first sequence is then made at a state 310 . A determination is then made at a decision state 316 whether the attribute of the feature was found in the first sequence. If the attribute was found, then the process 300 moves to a state 318 wherein the name of the found feature is displayed to the user.

The process 300 then moves to a decision state 320 wherein a determination is made whether move features exist in the database. If no more features do exist, then the process 300 terminates at an end state 324 . However, if more features do exist in the database, then the process 300 reads the next sequence feature at a state 326 and loops back to the state 310 wherein the attribute of the next feature is compared against the first sequence.

It should be noted, that if the feature attribute is not found in the first sequence at the decision state 316 , the process 300 moves directly to the decision state 320 in order to determine if any more features exist in the database.

In another embodiment, the identifier may comprise a molecular modeling program which determines the 3-dimensional structure of the polypeptides codes of the invention. In some embodiments, the molecular modeling program identifies target sequences that are most compatible with profiles representing the structural environments of the residues in known three-dimensional protein structures. (See, e.g., Eisenberg et al., U.S. Pat. No. 5,436,850 issued Jul. 25, 1995, the disclosure of which is incorporated herein by reference in its entirety). In another technique, the known three-dimensional structures of proteins in a given family are superimposed to define the structurally conserved regions in that family. This protein modeling technique also uses the known three-dimensional structure of a homologous protein to approximate the structure of the polypeptide codes of the invention. (See e.g., Srinivasan, et al., U.S. Pat. No. 5,557,535 issued Sep. 17, 1996, the disclosure of which is incorporated herein by reference in its entirety). Conventional homology modeling techniques have been used routinely to build models of proteases and antibodies. (Sowdhamini et al., 1997). Comparative approaches can also be used to develop three-dimensional protein models when the protein of interest has poor sequence identity to template proteins. In some cases, proteins fold into similar three-dimensional structures despite having very weak sequence identities. For example, the three-dimensional structures of a number of helical cytokines fold in similar three-dimensional topology in spite of weak sequence homology.

The recent development of threading methods now enables the identification of likely folding patterns in a number of situations where the structural relatedness between target and template(s) is not detectable at the sequence level. Hybrid methods, in which fold recognition is performed using Multiple Sequence Threading (MST), structural equivalencies are deduced from the threading output using a distance geometry program DRAGON to construct a low resolution model, and a full-atom representation is constructed using a molecular modeling package such as QUANTA.

According to this 3-step approach, candidate templates are first identified by using the novel fold recognition algorithm MST, which is capable of performing simultaneous threading of multiple aligned sequences onto one or more 3-D structures. In a second step, the structural equivalencies obtained from the MST output are converted into interresidue distance restraints and fed into the distance geometry program DRAGON, together with auxiliary information obtained from secondary structure predictions. The program combines the restraints in an unbiased manner and rapidly generates a large number of low resolution model confirmations. In a third step, these low resolution model confirmations are converted into full-atom models and subjected to energy minimization using the molecular modeling package QUANTA. (See e.g., Aszodi et al., 1997).

The results of the molecular modeling analysis may then be used in rational drug design techniques to identify agents which modulate the activity of the polypeptide codes of the invention.

Accordingly, another aspect of the present invention is a method of identifying a feature within the nucleic acid codes of the invention or the polypeptide codes of the invention comprising reading the nucleic acid code(s) or the polypeptide code(s) through the use of a computer program which identifies features therein and identifying features within the nucleic acid code(s) or polypeptide code(s) with the computer program. In one embodiment, computer program comprises a computer program which identifies open reading frames. In a further embodiment, the computer program identifies structural motifs in a polypeptide sequence. In another embodiment, the computer program comprises a molecular modeling program. The method may be performed by reading a single sequence or at least 2, 5, 10, 15, 20, 25, 30, or 50 of the nucleic acid codes of the invention or the polypeptide codes of the invention through the use of the computer program and identifying features within the nucleic acid codes or polypeptide codes with the computer program.

The nucleic acid codes of the invention or the polypeptide codes of the invention may be stored and manipulated in a variety of data processor programs in a variety of formats. For example, they may be stored as text in a word processing file, such as MicrosoftWORD or WORDPERFECT or as an ASCII file in a variety of database programs familiar to those of skill in the art, such as DB2, SYBASE, or ORACLE. In addition, many computer programs and databases may be used as sequence comparers, identifiers, or sources of reference nucleotide or polypeptide sequences to be compared to the nucleic acid codes of the invention or the polypeptide codes of the invention. The following list is intended not to limit the invention but to provide guidance to programs and databases which are useful with the nucleic acid codes of the invention or the polypeptide codes of the invention. The programs and databases which may be used include, but are not limited to: MacPattern (EMBL), DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Applications Group), Look (Molecular Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, 1990), FASTA (Pearson and Lipman, 1988), FASTDB (Brutlag et al., 1990), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (Molecular Simulations Inc.), Cerius 2 .DBAccess (Molecular Simulations Inc.), HypoGen (Molecular Simulations Inc.), Insight II, (Molecular Simulations Inc.), Discover (Molecular Simulations Inc.), CHARMm (Molecular Simulations Inc.), Felix (Molecular Simulations Inc.), DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.), Homology (Molecular Simulations Inc.), Modeler (Molecular Simulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/Protein Design (Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.), WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer (Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), the EMBL/Swissprotein database, the MDL Available Chemicals Directory database, the MDL Drug Data Report data base, the Comprehensive Medicinal Chemistry database, Derwents's World Drug Index database, the BioByteMasterFile database, the Genbank database, and the Genseqn database. Many other programs and data bases would be apparent to one of skill in the art given the present disclosure.

Motifs which may be detected using the above programs include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides which direct the secretion of the encoded proteins, sequences implicated in transcription regulation such as homeoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.

Throughout this application, various publications, patents and published patent applications are cited. The disclosures of these publications, patents and published patent specification referenced in this application are hereby incorporated by reference into the present disclosure to more fully describe the sate of the art to which this invention pertains.

EXAMPLES

Example 1

Detection of PCTA-1 Biallelic Markers

DNA Extraction

Blood donors were from French Caucasian origin. They presented a sufficient diversity for being representative of a French heterogeneous population. The DNA from 100 unrelated and healthy individuals was extracted, pooled and tested for the detection of biallelic markers. The pool was constituted by mixing equivalent quantities of DNA from each individual.

30 ml of peripheral venous blood were taken from each donor in the presence of EDTA. Cells (pellet) were collected after centrifugation for 10 minutes at 2000 rpm. Red cells were lysed by a lysis solution (50 ml final volume: 10 mM Tris pH7.6; 5 mM MgCl 2 ; 10 mM NaCl). The solution was centrifuged (10 minutes, 2000 rpm) as many times as necessary to eliminate the residual red cells present in the supernatant, after resuspension of the pellet in the lysis solution.

The pellet of white cells was lysed overnight at 42° C. with 3.7 ml of lysis solution composed of:

3 ml TE 10-2 (Tris-HCl 10 mM, EDTA 2 mM)/NaCl 0.4 M

200 μl SDS 10%

500 μl K-proteinase (2 mg K-proteinase in TE 10-2/NaCl 0.4 M).

For the extraction of proteins, 1 ml saturated NaCl (6M) (1/3.5 v/v) was added. After vigorous agitation, the solution was centrifuged for 20 minutes at 10000 rpm.

For the precipitation of DNA, 2 to 3 volumes of 100% ethanol were added to the previous supernatant, and the solution was centrifuged for 30 minutes at 2000 rpm. The DNA solution was rinsed three times with 70% ethanol to eliminate salts, and centrifuged for 20 minutes at 2000 rpm. The pellet was dried at 37° C., and resuspended in 1 ml TE 10- 1 or 1 ml water. The DNA concentration was evaluated by measuring the OD at 260 nm (1 unit OD=50 μg/ml DNA).

To determine the presence of proteins in the DNA solution, the OD 260/OD 280 ratio was determined. Only DNA preparations having a OD 260/OD 280 ratio between 1.8 and 2 were used in the subsequent examples described below.

Example 2

Detection of the Biallelic Markers

Amplification of Genomic DNA by PCR

The amplification of specific genomic sequences of the DNA samples of example 1 was carried out on the pool of DNA obtained previously. In addition, 10 individual samples were similarly amplified.

Final volume                     25 μl
DNA                              2 ng/μl
MgCl                                                                        2 2 mM
dNTP (each)                      200 μM
primer (each)                    2.9 ng/μl
Ampli Taq Gold DNA polymerase    0.05 unit/μl
PCR buffer (10× = 0.1M TrisHCl pH 8.3 0.5M KCl 1×

Each pair of primers was designed using the sequence information of our total genomic sequence (SEQ ID No 1) and the OSP software (Hillier& Green, 1991). These primers had about 20 nucleotides in length and their respective sequences are disclosed in Table 1 and had the sequences disclosed in Table 1 in the columns labeled “Position range of amplification primer in SEQ ID No 1” and “Complementary position range of amplification primer in SEQ ID No 1”.

The primers contained a common oligonucleotide tail upstream of the specific bases targeted for amplification which was useful for sequencing.

Primers from the columns labeled “Position range of amplification primer in SEQ ID No 1,” contain the following additional PU 5′ sequence: TGTAAAACGACGGCCAGT; and primers from the columns labeled “Complementary position range of amplification primer in SEQ ID No 1,” contain the following RP 5′ sequence: CAGGAAACAGCTATGACC. The primer containing the additional PU 5′ sequence is listed in SEQ ID No 10. The primer containing the additional RP 5′ sequence is listed in SEQ ID No 11.

The synthesis of these primers was performed following the phosphoramidite method, on a GENSET UFPS 24.1 synthesizer.

DNA amplification was performed on a Genius II thermocycler. After heating at 94° C. for 10 min, 40 cycles were performed. Each cycle comprised: 30 sec at 94° C., 55° C. for 1 min, and 30 sec at 72° C. For final elongation, 7 min at 72° C. end the amplification. The quantities of the amplification products obtained were determined on 96-well microtiter plates, using a fluorometer and Picogreen as intercalant agent (Molecular Probes).

TABLE 1
					Complementary
	Position range		Position range of		position range of
	of the amplicon	Primer	amplification primer	primer	amplification primer
Amplicon	in SEQ ID 1	name	in SEQ ID No 1	name	in SEQ ID No 1
99-1601	1	506	B1	1	18	C1	486	506
99-13801	2607	3054	B2	2607	2627	C2	3035	3054
99-13806	11883	12331	B3	11883	11902	C3	12313	12331
99-13799	12379	12909	B4	12379	12399	C4	12889	12909
99-13798	17442	17887	B5	17442	17462	C5	17868	17887
99-1602	21881	22506	B6	21881	21899	C6	22487	22506
99-13794	28669	29149	B7	28669	28689	C7	29131	29149
99-13812	30941	31457	B8	30941	30961	C8	31437	31457
99-13805	31560	32075	B9	31560	31579	C9	32057	32075
99-1587	34515	34909	B10	34515	34535	C10	34890	34909
99-1582	45325	46018	B11	45325	45343	C11	46000	46018
99-1585	49765	50310	B12	49765	49784	C12	50291	50310
99-1607	54726	55325	B13	54726	54746	C13	55307	55325
99-1577	64135	64536	B14	64135	64153	C14	64518	64536
99-1591	65202	65834	B15	65202	65219	C15	65815	65834
99-1572	66653	67295	B16	66653	66671	C16	67275	67295
5-169	67627	68043	B17	67627	67646	C17	68024	68043
5-264	67246	67696	B18	67246	67263	C18	67678	67696
5-170	67977	68424	B19	67977	67994	C19	68406	68424
5-171	68322	68742	B20	68322	68340	C20	68725	68742
5-1	70507	70928	B21	70507	70524	C21	70909	70928
99-1578	79940	80575	B22	79940	79957	C22	80557	80575
99-1605	82057	82504	B23	82057	82077	C23	82484	82504
5-2	82058	82492	B24	82058	82077	C24	82473	82492
5-3	83561	83982	B25	83561	83578	C25	83965	83982
5-4	83597	84017	B26	83597	83616	C26	83999	84017
5-260	83793	84167	B27	83793	83812	C27	84148	84167
5-9	85153	85576	B28	85153	85170	C28	85559	85576
5-5	86239	86539	B29	86239	86257	C29	86519	86539
5-202	87619	88050	B30	87619	87638	C30	88033	88050
5-7	88104	88536	B31	88104	88122	C31	88519	88536
5-181	89338	89758	B32	89338	89357	C32	89739	89758
5-10	92722	93142	B33	92722	92741	C33	93124	93142
5-11	93090	93509	B34	93090	93108	C34	93490	93509
5-12	93460	93881	B35	93460	93478	C35	93862	93881
5-13	93759	94192	B36	93759	93776	C36	94175	94192
5-14	94127	94554	B37	94127	94144	C37	94535	94554
5-15	94504	94921	B38	94504	94521	C38	94904	94921
5-16	94833	95251	B39	94833	94850	C39	95232	95251
5-17	95124	95561	B40	95124	95142	C40	95542	95561
5-18	95290	95708	B41	95290	95308	C41	95689	95708
5-300	95533	95952	B42	95533	95551	C42	95934	95952
5-262	96097	96591	B43	96097	96115	C43	96574	96591
5-263	96548	97001	B44	96548	96565	C44	96982	97001
5-265	96901	97309	B45	96901	96918	C45	97292	97309
99-7183	102156	102604	B46	102156	102176	C46	102584	102604
99-7207	105570	106074	B47	105570	105588	C47	106056	106074

Example 3

Detection of the Biallelic Markers

Sequencing of Amplified Genomic DNA and Identification of Polymorphisms

The sequencing of the amplified DNA obtained in example 2 was carried out on ABI 377 sequencers. The sequences of the amplification products were determined using automated dideoxy terminator sequencing reactions with a dye terminator cycle sequencing protocol. The products of the sequencing reactions were run on sequencing gels and the sequences were determined using gel image analysis (ABI Prism DNA Sequencing Analysis software (2.1.2 version)).

The sequence data were further evaluated using the above mentioned polymorphism analysis software designed to detect the presence of biallelic markers among the pooled amplified fragments. The polymorphism search was based on the presence of superimposed peaks in the electrophoresis pattern resulting from different bases occurring at the same position as described previously.

47 fragments of amplification were analyzed. In these segments, 125 markers were detected. The localization of the biallelic markers was as shown in Table 2. Table 3 comprises the polynucleotides defining the PCTA-1-related biallelic markers. They could be used as probes and their sequence are disclosed in Table 3 in “Position range of probes in SEQ ID No 1”.

	TABLE 2
		Localization	Polymorphism
	Marker	in PCTA-1	(frequency %)	BM position in SEQ ID
Amplicon	BM	Name	gene	all1	all2	No 1	No 2	No 3	No 4
99-1601	A1	99-1601-278	5′regulatory	A	C	278
99-1601	A2	99-1601-402	5′regulatory	A (66)	T	402
99-1601	A3	99-1601-472	5′regulatory	A	T	472
99-13801	A4	99-13801-100	5′regulatory	T	C	2955
99-13806	A5	99-13806-166	5′regulatory	G	A	12167
99-13799	A6	99-13799-376	5′regulatory	T	G	12536
99-13798	A7	99-13798-297	5′regulatory	T	C	17593
99-13798	A8	99-13798-284	5′regulatory	T	C	17606
99-1602	A9	99-1602-200	5′regulatory	C	G	22079
99-13794	A10	99-13794-186	5′regulatory	T	C	28964
99-13794	A11	99-13794-147	5′regulatory	C	G	29003
99-13812	A12	99-13812-384	5′regulatory	T	C	31077
99-13805	A13	99-13805-313	5′regulatory	T	C	31766
99-1587	A14	99-1587-281	5′regulatory	A	G	34791
99-1582	A15	99-1582-430	5′regulatory	C	T	45751
99-1585	A16	99-1585-465	5′regulatory	T	C	49847
99-1585	A17	99-1585-457	5′regulatory	T	C	49855
99-1585	A18	99-1585-426	5′regulatory	G	A	49886
99-1585	A19	99-1585-412	5′regulatory	G	A	49900
99-1585	A20	99-1585-406	5′regulatory	C	A	49906
99-1585	A21	99-1585-391	5′regulatory	C	A	49921
99-1585	A22	99-1585-373	5′regulatory	G	A	49939
99-1585	A23	99-1585-55	5′regulatory	C	A	50256
99-1607	A24	99-1607-373	5′regulatory	T	C	54955
99-1577	A25	99-1577-105	5′regulatory	A (54)	G	64239
99-1591	A26	99-1591-235	5′regulatory	A	G	65436
99-1591	A27	99-1591-295	5′regulatory	G	T	65496
99-1572	A28	99-1572-315	Promoter	C	T	66967
99-1572	A29	99-1572-335	Promoter	A	G	66987
99-1572	A30	99-1572-440	Promoter	C (32)	T	67092
99-1572	A31	99-1572-477	Promoter	A	T	67129
99-1572	A32	99-1572-578	Promoter	C	T	67229
5-264	A33	5-264-188	Promoter	A	G	67433
5-169	A34	5-169-97	Promoter	G (18)	C	67723
5-169	A35	5-169-208	Promoter	A (<1)	G	67834
5-169	A36	5-169-331	Promoter	C (99)	T	67955
5-170	A37	5-170-238	Promoter	A	G	68213
5-170	A38	5-170-288	Promoter	A (1)	C	68263
5-170	A39	5-170-400	Promoter	G	C	68375
5-171	A40	5-171-156	Promoter	G	T	68477
5-171	A41	5-171-204	Promoter	C (30)	T	68525
5-171	A42	5-171-273	Promoter	A	G	68594
5-171	A43	5-171-289	Promoter	C	T	68610
5-1	A44	5-1-60	Intron 0	C (1)	T	70566
5-1	A45	5-1-222	Exon 1	A	G	70728	176	176	176
99-1578	A46	99-1578-99	Intron 1	G	T	80038
99-1578	A47	99-1578-179	Intron 1	A	T	80118
99-1578	A48	99-1578-231	Intron 1	Ins		80170
				AC
99-1578	A49	99-1578-245	Intron 1	del AT		80183
99-1578	A50	99-1578-496	Intron 1	C	T	80435
5-2	A51	5-2-30	Intron 1	Ins		82090
				CAG
5-2	A52	5-2-109	Intron 1	G	T	82165
5-2	A53	5-2-113	Intron 1	Del		82169
				GTTT
5-2	A54	5-2-162	Exon 2	A (67)	T	82218	253	253	253
5-2	A55	5-2-178	Exon 2	C (67)	T	82234	269	269	269
5-2	A56	5-2-213	Exon 2	C (33)	T	82268	303	303	303
99-1605	A57	99-1605-112	Intron 2	T (67)	C	82393
5-3	A58	5-3-27	Intron 2	A	G	83587
5-3	A59	5-3-83	Exon 3	C (39)	T	83643	362	362	362
5-3	A60	5-3-84	Exon 3	A (29)	G	83644	363	363	363
5-3	A61	5-3-248	Exon 3	A	G	83808	527	527	527
5-3	A62	5-3-321	Intron 3	G	T	83881
5-3	A63	5-3-324	Intron 3	C	T	83884
5-4	A64	5-4-313	Intron 3	A	G	83909
5-3	A65	5-3-377	Intron 3	ins		83937
				TTTG
5-4	A66	5-4-351	Intron 3	C	T	83947
5-4	A67	5-4-386	Intron 3	A	G	83982
5-4	A68	5-4-392	Intron 3	GGG	TA	83988
5-260	A69	5-260-255	Intron 3	C	T	84047
5-260	A70	5-260-300	Intron 3	C	T	84092
5-260	A71	5-260-353	Intron 3	C	T	84145
5-9	A72	5-9-50	Intron 3	C	T	85202
5-5	A73	5-5-21	Intron 4	A	G	86259
5-5	A74	5-5-85	Intron 4	TATA	ACAG	86323
				AAAT	GTTA
				ATT	TATA
5-202	A75	5-202-95	Exon 6bis	G	T (<1)	87713		810
5-202	A76	5-202-117	Exon 6bis	A (<1)	T	87735		832
5-202	A77	5-202-169	Intron 6bis	A	C	87787
5-202	A78	5-202-188	Intron 6bis	A	G	87806
5-202	A79	5-202-242	Intron 6bis	A	G	87860
5-202	A80	5-202-284	Intron 6bis	C	T	87902
5-202	A81	5-202-362	Intron 6bis	del CC		87980
5-202	A82	5-202-394	Intron 6bis	C	T	88012
5-7	A83	5-7-113	Intron 6bis	C	T	88215
5-7	A84	5-7-181	Intron 6bis	G	C	88283
5-7	A85	5-7-195	Exon 7	G (25)	C	88297	749	875	749
5-7	A86	5-7-340	Intron 7	C	T	88442
5-7	A87	5-7-369	Intron 7	A	T	88471
5-7	A88	5-7-378	Intron 7	C	T	88480
5-181	A89	5-181-57	Intron 7	A	G	89394
5-181	A90	5-181-127	Intron 7	C	T	89464
5-181	A91	5-181-134	Intron 7	C	T	89471
5-181	A92	5-181-321	Intron 8	A	C	89658
5-10	A93	5-10-39	Exon 9	C	T	92760	1013	1139	1013
			exon 9bis
5-10	A94	5-10-302	Exon 9	A	G	93023	1276	1402
			Intron 9bis
5-10	A95	5-10-334	Exon 9	A	C	93055	1308	1434
			Intron 9bis
5-11	A96	5-11-158	Exon 9	A (22)	G	93247	1500	1626
			Intron 9bis
5-11	A97	5-11-230	Exon 9	G	T	93319	1572	1698
			Intron 9bis
5-11	A98	5-11-234	Exon9	C	T	93323	1576	1702
			Intron 9bis
5-11	A99	5-11-299	Exon9	A	T	93388	1641	1767
			Intron 9bis
5-11	A100	5-11-304	Exon9	A	C	93393	1646	1772
			Intron 9bis
5-11	A101	5-11-329	Exon 9	C	T	93418	1671	1797
			Intron 9bis
5-12	A102	5-12-56	Exon 9	ins		93515	1768	1894
			Intron 9bis	CTTT
5-12	A103	5-12-267	Exon 9	A	C	93726	1979	2105
			Intron 9bis
5-13	A104	5-13-145	Exon 9	C	T	93903	2156	2282
			Intron 9bis
5-14	A105	5-14-44	Exon 9	C	T	94170	2423	2549
			Intron 9bis
5-14	A106	5-14-93	Exon 9	A	T	94218	2471	2597
			Intron 9bis
5-14	A107	5-14-144	Exon 9	ins T		94269	2522	2648
			Intron 9bis
5-14	A108	5-14-165	Exon 9	C	T	94290	2543	2669
			Intron 9bis
5-14	A109	5-14-297	Exon 9	A	C	94422	2675	2801
			Intron 9bis
5-14	A110	5-14-307	Exon 9	G	T	94432	2685	2811
			Intron 9bis
5-15	A111	5-15-219	Exon 9	A	T	94720	2973	3099
			Intron 9bis
5-16	A112	5-16-157	Exon 9	A	G	94989	3242	3368
			Intron 9bis
5-17	A113	5-17-140	Exon 9	A	G	95261	3514	3640
			Intron 9bis
5-18	A114	5-18-51	Exon 9	G	T	95340	3593	3719
			Intron 9bis
5-18	A115	5-18-208	Exon 9	A	C	95497	3750	3876
			Intron 9bis
5-300	A116	5-300-238	Exon 9	C	T	95770	4023	4149
			Intron 9bis
5-300	A117	5-300-287	Exon 9	A	G	95819	4072	4198
			Intron 9bis
5-262	A118	5-262-49	Exon 9	ins C		96145	4398	4524	1461
			Exon 9ter
5-262	A119	5-262-85	Exon 9	C	T	96181	4434	4560	1497
			Exon 9ter
5-262	A120	5-262-254	Exon 9	C	T	96350	4603	4729	1666
			Exon 9ter
5-263	A121	5-263-404	Exon 9	C	T	96951	5204	5330	2267
			Exon 9ter
5-265	A122	5-265-244	Exon 9	A	G	97144	5397	5523	2460
			Exon 9ter
5-265	A123	5-265-376	3′regulatory	A	G	97276
99-7183	A124	99-7183-338	3′regulatory	C	T	102267
99-7207	A125	99-7207-138	3′regulatory	A	G	105937

BM refers to “biallelic marker”. A111 and a112 refer respectively to allele 1 and allele 2 of the biallelic marker. “Frequency%” refers to the frequency of the allele in percentage in control population. Frequencies corresponded to a population of random blood donors of French Caucasian origin.

	TABLE 3
			Position range of
			probes in SEQ
	BM	Marker Name	ID No 1	Probes
	A1	99-1601-278	255	301	P1
	A2	99-1601-402	379	425	P2
	A3	99-1601-472	449	495	P3
	A4	99-13801-100	2932	2978	P4
	A5	99-13806-166	12144	12190	P5
	A6	99-13799-376	12513	12559	P6
	A7	99-13798-297	17570	17616	P7
	A8	99-13798-284	17583	17629	P8
	A9	99-1602-200	22056	22102	P9
	A10	99-13794-186	28941	28987	P10
	A11	99-13794-147	28980	29026	P11
	A12	99-13812-384	31054	31100	P12
	A13	99-13805-313	31743	31789	P13
	A14	99-1587-281	34768	34814	P14
	A15	99-1582-430	45728	45774	P15
	A16	99-1585-465	49824	49870	P16
	A17	99-1585-457	49832	49878	P17
	A18	99-1585-426	49863	49909	P18
	A19	99-1585-412	49877	49923	P19
	A20	99-1585-406	49883	49929	P20
	A21	99-1585-391	49898	49944	P21
	A22	99-1585-373	49916	49962	P22
	A23	99-1585-55	50233	50279	P23
	A24	99-1607-373	54932	54978	P24
	A25	99-1577-105	64216	64262	P25
	A26	99-1591-235	65413	65459	P26
	A27	99-1591-295	65473	65519	P27
	A28	99-1572-315	66944	66990	P28
	A29	99-1572-335	66964	67010	P29
	A30	99-1572-440	67069	67115	P30
	A31	99-1572-477	67106	67152	P31
	A32	99-1572-578	67206	67252	P32
	A33	5-264-188	67410	67456	P33
	A34	5-169-97	67700	67746	P34
	A35	5-169-208	67811	67857	P35
	A36	5-169-331	67932	67978	P36
	A37	5-170-238	68190	68236	P37
	A38	5-170-288	68240	68286	P38
	A39	5-170-400	68352	68398	P39
	A40	5-171-156	68454	68500	P40
	A41	5-171-204	68502	68548	P41
	A42	5-171-273	68571	68617	P42
	A43	5-171-289	68587	68633	P43
	A44	5-1-60	70543	70589	P44
	A45	5-1-222	70705	70751	P45
	A46	99-1578-99	80015	80061	P46
	A47	99-1578-179	80095	80141	P47
	A48	99-1578-231	80147	80193	P48
	A49	99-1578-245	80160	80206	P49
	A50	99-1578-496	80412	80458	P50
	A51	5-2-30	82067	82113	P51
	A52	5-2-109	82142	82188	P52
	A53	5-2-113	82146	82192	P53
	A54	5-2-162	82195	82241	P54
	A55	5-2-178	82211	82257	P55
	A56	5-2-213	82245	82291	P56
	A57	99-1605-112	82370	82416	P57
	A58	5-3-27	83564	83610	P58
	A59	5-3-83	83620	83666	P59
	A60	5-3-84	83621	83667	P60
	A61	5-3-248	83785	83831	P61
	A62	5-3-321	83858	83904	P62
	A63	5-3-324	83861	83907	P63
	A64	5-4-313	83886	83932	P64
	A65	5-3-377	83914	83960	P65
	A66	5-4-351	83924	83970	P66
	A67	5-4-386	83959	84005	P67
	A68	5-4-392	83965	84011	P68
	A69	5-260-255	84024	84070	P69
	A70	5-260-300	84069	84115	P70
	A71	5-260-353	84122	84168	P71
	A72	5-9-50	85179	85225	P72
	A73	5-5-21	86236	86282	P73
	A74	5-5-85	86300	86346	P74
	A75	5-202-95	87690	87736	P75
	A76	5-202-117	87712	87758	P76
	A77	5-202-169	87764	87810	P77
	A78	5-202-188	87783	87829	P78
	A79	5-202-242	87837	87883	P79
	A80	5-202-284	87879	87925	P80
	A81	5-202-362	87957	88003	P81
	A82	5-202-394	87989	88035	P82
	A83	5-7-113	88192	88238	P83
	A84	5-7-181	88260	88306	P84
	A85	5-7-195	88274	88320	P85
	A86	5-7-340	88419	88465	P86
	A87	5-7-369	88448	88494	P87
	A88	5-7-378	88457	88503	P88
	A89	5-181-57	89371	89417	P89
	A90	5-181-127	89441	89487	P90
	A91	5-181-134	89448	89494	P91
	A92	5-181-321	89635	89681	P92
	A93	5-10-39	92737	92783	P93
	A94	5-10-302	93000	93046	P94
	A95	5-10-334	93032	93078	P95
	A96	5-11-158	93224	93270	P96
	A97	5-11-230	93296	93342	P97
	A98	5-11-234	93300	93346	P98
	A99	5-11-299	93365	93411	P99
	A100	5-11-304	93370	93416	P100
	A101	5-11-329	93395	93441	P101
	A102	5-12-56	93492	93538	P102
	A103	5-12-267	93703	93749	P103
	A104	5-13-145	93880	93926	P104
	A105	5-14-44	94147	94193	P105
	A106	5-14-93	94195	94241	P106
	A107	5-14-144	94246	94292	P107
	A108	5-14-165	94267	94313	P108
	A109	5-14-297	94399	94445	P109
	A110	5-14-307	94409	94455	P110
	A111	5-15-219	94697	94743	P111
	A112	5-16-157	94966	95012	P112
	A113	5-17-140	95238	95284	P113
	A114	5-18-51	95317	95363	P114
	A115	5-18-208	95474	95520	P115
	A116	5-300-238	95747	95793	P116
	A117	5-300-287	95796	95842	P117
	A118	5-262-49	96122	96168	P118
	A119	5-262-85	96158	96204	P119
	A120	5-262-254	96327	96373	P120
	A121	5-263-404	96928	96974	P121
	A122	5-265-244	97121	97167	P122
	A123	5-265-376	97253	97299	P123
	A124	99-7183-338	102244	102290	P124
	A125	99-7207-138	105914	105960	P125

Example 4

Validation of the Polymorphisms through Microseguencing

The biallelic markers identified in example 3 were further confirmed and their respective frequencies were determined through microsequencing. Microsequencing was carried out for each individual DNA sample described in Example 1.

Amplification from genomic DNA of individuals was performed by PCR as described above for the detection of the biallelic markers with the same set of PCR primers (Table 1).

The preferred primers used in microsequencing had about 19 nucleotides in length and hybridized just upstream of the considered polymorphic base. Their sequences are disclosed in Table 4 in columns labeled “Position range of microsequencing primer mis. 1 in SEQ ID No 1” and “Complementary position range of microsequencing primer mis. 2 in SEQ ID No 1”.

Mis 1 and Mis 2 respectively refer to microsequencing primers which hybridized with the non-coding strand of the PCTA-1 gene or with the coding strand of the PCTA-1 gene.

The microsequencing reaction was performed as follows:

10 μl of PCR products were added to 20 μl of microsequencing reaction mixture containing: 10 pmol microsequencing oligonucleotide (crude synthesis, 5 OD), 1 U Thermosequenase (Amersham E79000G), 1.25 μl Thermosequenase buffer (260 mM Tris HCl pH 9.5, 65 mM MgCl 2 ), and the appropriate fluorescent ddNTPs complementary to the nucleotides at the polymorphic site corresponding to the polymorphic bases (11.25 nM TAMRA-ddTTP; 16.25 nM ROX-ddCTP; 1.675 nM REG-ddATP; 1.25 nM RHO-ddGTP; Perkin Elmer, Dye Terminator Set 401095). After 4 minutes at 94° C., 20 PCR cycles of 15 sec at 55° C., 5 sec at 72° C., and 10 sec at 94° C. were carried out in a thermocycler. After amplification, the unincorporated dye terminators were removed by ethanol precipitation. After discarding the supernatants, the microplate was evaporated to dryness under reduced pressure (Speed Vac); samples were resuspended in 2.5 μl formamide EDTA loading buffer and heated for 2 min at 95° C. 0.8 μl microsequencing reaction were loaded on a 10% (19:1) polyacrylamide sequencing gel. The data were collected by an ABI PRISM 377 DNA sequencer and processed using the GENESCAN software (Perkin Elmer).

Following gel analysis, data were automatically processed with software that allows the determination of the alleles of biallelic markers present in each amplified fragment.

The software evaluates such factors as whether the intensities of the signals resulting from the above microsequencing procedures are weak, normal, or saturated, or whether the signals are ambiguous. In addition, the software identifies significant peaks (according to shape and height criteria). Among the significant peaks, peaks corresponding to the targeted site are identified based on their position. When two significant peaks are detected for the same position, each sample is categorized classification as homozygous or heterozygous type based on the height ratio.

	TABLE 4
			Complementary
	Position range of		position range of
	microsequencing		microsequencing
	primer mis. 1 in		primer mis. 2 in
Marker Name	BM	Mis. 1	SEQ ID No 1	Mis. 2	SEQ ID No 1
99-1601-278	A1	D1	258	277	E1	279	298
99-1601-402	A2	D2	382	401	E2	403	422
99-1601-472	A3	D3	452	471	E3	473	492
99-13801-100	A4	D4	2935	2954	E4	2956	2975
99-13806-166	A5	D5	12147	12166	E5	12168	12187
99-13799-376	A6	D6	12516	12535	E6	12537	12556
99-13798-297	A7	D7	17573	17592	E7	17594	17613
99-13798-284	A8	D8	17586	17605	E8	17607	17626
99-1602-200	A9	D9	22059	22078	E9	22080	22099
99-13794-186	A10	D10	28944	28963	E10	28965	28984
99-13794-147	A11	D11	28983	29002	E11	29004	29023
99-13812-384	A12	D12	31057	31076	E12	31078	31097
99-13805-313	A13	D13	31746	31765	E13	31767	31786
99-1587-281	A14	D14	34771	34790	E14	34792	34811
99-1582-430	A15	D15	45731	45750	E15	45752	45771
99-1585-465	A16	D16	49827	49846	E16	49848	49867
99-1585-457	A17	D17	49835	49854	E17	49856	49875
99-1585-426	A18	D18	49866	49885	E18	49887	49906
99-1585-412	A19	D19	49880	49899	E19	49901	49920
99-1585-406	A20	D20	49886	49905	E20	49907	49926
99-1585-391	A21	D21	49901	49920	E21	49922	49941
99-1585-373	A22	D22	49919	49938	E22	49940	49959
99-1585-55	A23	D23	50236	50255	E23	50257	50276
99-1607-373	A24	D24	54935	54954	E24	54956	54975
99-1577-105	A25	D25	64219	64238	E25	64240	64259
99-1591-235	A26	D26	65416	65435	E26	65437	65456
99-1591-295	A27	D27	65476	65495	E27	65497	65516
99-1572-315	A28	D28	66947	66966	E28	66968	66987
99-1572-335	A29	D29	66967	66986	E29	66988	67007
99-1572-440	A30	D30	67072	67091	E30	67093	67112
99-1572-477	A31	D31	67109	67128	E31	67130	67149
99-1572-578	A32	D32	67209	67228	E32	67230	67249
5-264-188	A33	D33	67413	67432	E33	67434	67453
5-169-97	A34	D34	67703	67722	E34	67724	67743
5-169-208	A35	D35	67814	67833	E35	67835	67854
5-169-331	A36	D36	67935	67954	E36	67956	67975
5-170-238	A37	D37	68193	68212	E37	68214	68233
5-170-288	A38	D38	68243	68262	E38	68264	68283
5-170-400	A39	D39	68355	68374	E39	68376	68395
5-171-156	A40	D40	68457	68476	E40	68478	68497
5-171-204	A41	D41	68505	68524	E41	68526	68545
5-171-273	A42	D42	68574	68593	E42	68595	68614
5-171-289	A43	D43	68590	68609	E43	68611	68630
5-1-60	A44	D44	70546	70565	E44	70567	70586
5-1-222	A45	D45	70708	70727	E45	70729	70748
99-1578-99	A46	D46	80018	80037	E46	80039	80058
99-1578-179	A47	D47	80098	80117	E47	80119	80138
99-1578-231	A48	D48	80150	80169	E48	80171	80190
99-1578-245	A49	D49	80163	80182	E49	80184	80203
99-1578-496	A50	D50	80415	80434	E50	80436	80455
5-2-30	A51	D51	82070	82089	E51	82091	82110
5-2-109	A52	D52	82145	82164	E52	82166	82185
5-2-113	A53	D53	82149	82168	E53	82170	82189
5-2-162	A54	D54	82198	82217	E54	82219	82238
5-2-178	A55	D55	82214	82233	E55	82235	82254
5-2-213	A56	D56	82248	82267	E56	82269	82288
99-1605-112	A57	D57	82373	82392	E57	82394	82413
5-3-27	A58	D58	83567	83586	E58	83588	83607
5-3-83	A59	D59	83623	83642	E59	83644	83663
5-3-84	A60	D60	83624	83643	E60	83645	83664
5-3-248	A61	D61	83788	83807	E61	83809	83828
5-3-321	A62	D62	83861	83880	E62	83882	83901
5-3-324	A63	D63	83864	83883	E63	83885	83904
5-4-313	A64	D64	83889	83908	E64	83910	83929
5-3-377	A65	D65	83917	83936	E65	83938	83957
5-4-351	A66	D66	83927	83946	E66	83948	83967
5-4-386	A67	D67	83962	83981	E67	83983	84002
5-4-392	A68	D68	83968	83987	E68	83989	84008
5-260-255	A69	D69	84027	84046	E69	84048	84067
5-260-300	A70	D70	84072	84091	E70	84093	84112
5-260-353	A71	D71	84125	84144	E71	84146	84165
5-9-50	A72	D72	85182	85201	E72	85203	85222
5-5-21	A73	D73	86239	86258	E73	86260	86279
5-5-85	A74	D74	86303	86322	E74	86324	86343
5-202-95	A75	D75	87693	87712	E75	87714	87733
5-202-117	A76	D76	87715	87734	E76	87736	87755
5-202-169	A77	D77	87767	87786	E77	87788	87807
5-202-188	A78	D78	87786	87805	E78	87807	87826
5-202-242	A79	D79	87840	87859	E79	87861	87880
5-202-284	A80	D80	87882	87901	E80	87903	87922
5-202-362	A81	D81	87960	87979	E81	87981	88000
5-202-394	A82	D82	87992	88011	E82	88013	88032
5-7-113	A83	D83	88195	88214	E83	88216	88235
5-7-181	A84	D84	88263	88282	E84	88284	88303
5-7-195	A85	D85	88277	88296	E85	88298	88317
5-7-340	A86	D86	88422	88441	E86	88443	88462
5-7-369	A87	D87	88451	88470	E87	88472	88491
5-7-378	A88	D88	88460	88479	E88	88481	88500
5-181-57	A89	D89	89374	89393	E89	89395	89414
5-181-127	A90	D90	89444	89463	E90	89465	89484
5-181-134	A91	D91	89451	89470	E91	89472	89491
5-181-321	A92	D92	89638	89657	E92	89659	89678
5-10-39	A93	D93	92740	92759	E93	92761	92780
5-10-302	A94	D94	93003	93022	E94	93024	93043
5-10-334	A95	D95	93035	93054	E95	93056	93075
5-11-158	A96	D96	93227	93246	E96	93248	93267
5-11-230	A97	D97	93299	93318	E97	93320	93339
5-11-234	A98	D98	93303	93322	E98	93324	93343
5-11-299	A99	D99	93368	93387	E99	93389	93408
5-11-304	A100	D100	93373	93392	E100	93394	93413
5-11-329	A101	D101	93398	93417	E101	93419	93438
5-12-56	A102	D102	93495	93514	E102	93516	93535
5-12-267	A103	D103	93706	93725	E103	93727	93746
5-13-145	A104	D104	93883	93902	E104	93904	93923
5-14-44	A105	D105	94150	94169	E105	94171	94190
5-14-93	A106	D106	94198	94217	E106	94219	94238
5-14-144	A107	D107	94249	94268	E107	94270	94289
5-14-165	A108	D108	94270	94289	E108	94291	94310
5-14-297	A109	D109	94402	94421	E109	94423	94442
5-14-307	A110	D110	94412	94431	E110	94433	94452
5-15-219	A111	D111	94700	94719	E111	94721	94740
5-16-157	A112	D112	94969	94988	E112	94990	95009
5-17-140	A113	D113	95241	95260	E113	95262	95281
5-18-51	A114	D114	95320	95339	E114	95341	95360
5-18-208	A115	D115	95477	95496	E115	95498	95517
5-300-238	A116	D116	95750	95769	E116	95771	95790
5-300-287	A117	D117	95799	95818	E117	95820	95839
5-262-49	A118	D118	96125	96144	E118	96146	96165
5-262-85	A119	D119	96161	96180	E119	96182	96201
5-262-254	A120	D120	96330	96349	E120	96351	96370
5-263-404	A121	D121	96931	96950	E121	96952	96971
5-265-244	A122	D122	97124	97143	E122	97145	97164
5-265-376	A123	D123	97256	97275	E123	97277	97296
99-7183-338	A124	D124	102247	102266	E124	102268	102287
99-7207-138	A125	D125	105917	105936	E125	105938	105957

Example 5

Association Study between Prostate Cancer and the Biallelic Markers of the PCTA-1 Gene

Collection of DNA Samples from Affected and Non-Affected Individuals

Affected Population:

The positive trait followed in this association study was prostate cancer. Prostate cancer patients were recruited according to a combination of clinical, histological and biological inclusion criteria. Clinical criteria can include rectal examination and prostate biopsies. Biological criteria can include PSA assays. The affected individuals were recorded as familial forms when at least two persons affected by prostate cancer have been diagnosed in the family. Remaining cases were classified as non-familial informative cases (at least two sibs of the case both aged over 50 years old are unaffected), or non-familial uniformative cases (no information about sibs over 50 years old is available). All affected individuals included in the statistical analysis of this patent were unrelated. Cases were also separated following the criteria of diagnosis age: early onset prostate cancer (under 65 years old) and late onset prostate cancer (65 years old or more).

Unaffected Population:

Control individuals included in this study were checked for both the absence of all clinical and biological criteria defining the presence or the risk of prostate cancer (PSA<4) (WO 96/21042), and for their age (aged 65 years old or more). All unaffected individuals included in the statistical analysis of this patent were unrelated.

The affected group was composed by 491 unrelated individuals, comprising:

197 familial cases among which 91 individuals were under 65 years old and 106 individuals were 65 years old or more; and

294 sporadic cases.

The unaffected group contained 313 individuals which were 65 years or older.

As used herein, the term “early onset cancer” refers to a cancer in which the individuals are under 65 years old.

Genotyping of Affected and Control Individuals

The general strategy to perform the association studies was to individually scan the DNA samples from all individuals in each of the populations described above in order to establish the allele frequencies of the above described biallelic markers in each of these populations. More particularly, the biallelic markers used in the present association study are A2, A9, A15, A22, A24, A25, A26, A30, A34, A35, A36, A38, A41, A42, A44, A51, A52, A54, A55, A56, A57, A59, A60, A64, A73, A75, A76, A85, A93, A96, A108, A111, A115.

Allelic frequencies of the above-described biallelic markers in each population were determined by performing microsequencing reactions on amplified fragments obtained by genomic PCR performed on the DNA samples from each individual. Genomic PCR and microsequencing were performed as detailed above in examples 2 and 4 using the described PCR and microsequencing primers.

Association Study between Prostate Cancer and the Biallelic Markers of the PCTA-1 Gene

The alleles of two biallelic markers, namely (T) A30 and (T) A41, have been shown to be significantly associated to familial prostate cancer, more particularly early onset familial prostate cancer. Indeed, the allele T of the biallelic marker A30 showed a p-value of 1.08×10 −2 for the early onset familial prostate cancer and of 3.39×10 −2 for the familial prostate cancer. The allele T of the biallelic marker A41 presented a p-value of 4.04×10 −2 for the early onset familial prostate cancer. These two markers could be then used in diagnostics.

Some other biallelic markers, namely A54, A55, A56, A57, A59, A60, A61, A85, A96, A108, A115, showed a moderate association. These biallelic markers are localized in the exons and introns of the PCTA-1 gene.

The inventors observed that all the PCTA-1-related biallelic markers were in linkage disequilibrium with each other in the controls individuals. In the familial cases of prostate cancer, the biallelic markers localized in the promoter did not show a linkage disequilibrium with those localized in exonic and intronic region of the PCTA-1 gene and were not in linkage disequilibrium with each other. This lack of linkage disequilibrium for the promoter biallelic markers suggests that this region comprises a trait causing mutation and could explain the cases haplotypes.

Haplotype Frequency Analysis

One way of increasing the statistical power of individual markers, is by performing haplotype association analysis.

Haplotype analysis for association of PCTA-1 markers and prostate cancer was performed by estimating the frequencies of all possible haplotypes comprising biallelic markers selected from the group consisting of A2, A9, Al5, A22, A24, A25, A26, A30, A34, A35, A36, A38, A41, A42, A44, A51, A52, A54, A55, A56, A57, A59, A60, A64, A73, A75, A76, A85, A93, A96, A108, A111, A115 in the cases and control populations described in Example 5, and comparing these frequencies by means of a chi square statistical test (one degree of freedom). Haplotype estimations were performed by applying the Expectation-Maximization (EM) algorithm (Excofflier L & Slatkin M, 1995), using the EM-HAPLO program (Hawley M E, Pakstis A J & Kidd K K, 1994).

Haplotype Frequency Analysis for Familial Cases of Prostate Cancer

The most significant haplotypes obtained with the familial cases of prostate cancer are shown in Table 5. These haplotypes comprise the biallelic markers A2, A30, A41, A55, A57, and 5-202/95.

The preferred two-markers haplotypes are described in Table 5 as H1 to H7 of PT2. The more preferred haplotype is the haplotype H1/PT2 and comprises the biallelic markers A30 (99-1572/440 allele T) and A41 (allele T). This haplotype presented a pvalue of 1.1×10 −4 and an odd-ratio of 1.67. Estimated haplotype frequencies were 57.2% in the cases and 44.4% in the controls.

The preferred three-markers haplotypes are described in Table 5 as H1, H2, H3, H7, H8, H9, H10, H11, and H12 of PT3. The more preferred haplotype is the haplotype H1/PT3 and comprises the biallelic markers A2 (allele A), A30 (99-1572/440 allele T) and A41 (allele T). This haplotype presented a p-value of 1.1×10 −5 and an odd-ratio of 1.84. Estimated haplotype frequencies were 42.9% in the cases and 29% in the controls.

The preferred four-markers haplotypes are described in Table 5 as H1, H2, H4, H5, H7, H9, H16, H17, H18 and H19 of PT4.

In conclusion, most preferred haplotypes for the familial cases of prostate cancer comprise the biallelic markers A30 (99-1572/440 allele T) and/or A41 (allele T). Some other preferred haplotypes for the familial cases of prostate cancer comprise the biallelic marker A2 (allele A). Optionally, preferred haplotypes for the familial cases of prostate cancer comprise the biallelic markers A55 (allele C) and/or A57 (allele G). These haplotypes can be used in diagnostic of prostate cancer susceptibility.

Haplotype Frequency Analysis for Sporadic Cases of Prostate Cancer

The most significant haplotypes obtained with the sporadic cases of prostate cancer are shown in Table 6. These haplotypes comprise the biallelic markers A2, A30, A41, A55, A57, and 5-202/95.

The preferred two-markers haplotypes are described in Table 6 as H1 to H4, H6, and H7 of PT2. The first more preferred haplotype is the haplotype H1/PT2 and comprises the biallelic markers A2 (allele T) and A55 (allele T). This haplotype presented a p-value of 2.4×10 −4 and an odd-ratio of 1.94. Estimated haplotype frequencies were 16.2% in the cases and 9% in the controls. The second more preferred haplotype is the haplotype H2/PT2 and comprises the biallelic markers A2 (allele T) and A57 (allele A). This haplotype presented a p-value of 5.3×10 −4 and an odd-ratio of 1.84. Estimated haplotype frequencies were 16.3% in the cases and 9.5% in the controls.

The preferred three-markers haplotypes are described in Table 6 as H1, H2, H3, H4, H6, H7, and H8 of PT3. The more preferred haplotype is the haplotype H2/PT3 and comprises the biallelic markers A2 (allele T), A55 (allele T), and A57 (allele A). This haplotype presented a p-value of 2.3×10 −3 and an odd-ratio of 1.75. Estimated haplotype frequencies were 15% in the cases and 9.2% in the controls.

The preferred four-markers haplotypes are described in Table 6 as H1, H2, H3, H4, and H6 of PT4.

In conclusion, most preferred haplotypes for the sporadic cases of prostate cancer comprise a biallelic marker selected from the group consisting of A2 (allele T), A55 (allele T), and A57 (allele A). Optionally, preferred haplotypes for the familial cases of prostate cancer comprise the biallelic markers A30 (allele T) and/or A41 (allele T). These haplotypes can be used in diagnostic of prostate cancer.

Summary of Haplotype Frequency Analysis

The most preferred two- and three-biallelic markers haplotypes for the familial and sporadic prostate cancer are summarized in Table 7. These haplotypes can be used in diagnostic of prostate cancer susceptibility.

The statistical significance of the results obtained for the haplotype analysis was evaluated by a phenotypic permutation test reiterated 1000 or 10,000 times on a computer. For this computer simulation, data from the cases and control individuals were pooled and randomly allocated to two groups which contained the same number of individuals as the case-control populations used to produce the haplotype frequency analysis data. A haplotype analysis was then run on these artificial groups for the five haplotypes of the Table 7 which presented a strong association with prostate cancer. This experiment was reiterated 1000 times and the results are shown in Table 8.

The haplotypes 1 and 2 of the Table 7 are clearly associated with familial prostate cancer and more particularly with familial cases which were under 65 years and with>3caP familial cases. The permutation test clearly validate the statistical significance of the association between these haplotypes and familial prostate cancer since, among 1000 iterations, none of the obtained haplotypes had a p-value comparable to the one obtained for the haplotypes 1 and 2 of Table 7 for the familial cases, the familial cases under 65 years and the>3caP familial cases.

The haplotypes 3, 4, and 5 of the Table 7 are clearly associated with the sporadic prostate cancer. The permutation test clearly validate the statistical significance of the association between these haplotypes and sporadic prostate cancer since, among 1000 iterations, less than 6 of the obtained haplotypes had a p-value comparable to the one obtained for the haplotypes 3, 4 and 5 of Table 7 for the sporadic cases. Moreover, among 1000 iterations, none of the obtained haplotypes had a p-value comparable to the one obtained for the haplotypes 3, 4 and 5 of Table 7 for the informative sporadic cases.

Attributable Risk

The attributable risk has been calculated as described in the “Evaluation of risk factors” of the part entitled “Statistic method”. The results are disclosed in Table 9.

These results show that the preferred haplotypes disclosed in the present invention are highly significant for the prostate cancer. Indeed, 16.92% of the sporadic prostate cancer cases carried the haplotype 4 of the Table 7 considering a dominant model which is the more relevant model for prostate cancer. Moreover, 60.77% of the familial early onset prostate cancer cases carried the haplotype 1 of the Table 7 considering a dominant model.

TABLE 5

Haplotype frequency analysis for the familial cases of prostate cancer

A2

A30

A41

A55

A57

A75

67/67

72/66

75/71

72/68

72/69

95/95

frequency %

(A)

(T)

(T)

(C)

(G)

(G)

haplotype

abs diff freq all

0,1

6,4

4,2

3,8

3,4

0

frequencies

Odds

Pvalue

pvalue

7,5e-01

3,3e-02

1,4e-01

2,0e-01

2,5e-01

7,5e-01

cases

Controls

ratio

(1df)

Cases/controls ↓

H1

PT2

183/298

T

T

0.572

0.444

1.67

1.1e-04

H2

188/298

T

G

0.540

0.431

1.55

8.6e-04

H3

183/296

T

C

0.536

0.428

1.54

1.1e-03

H4

183/299

A

T

0.543

0.460

1.40

1.1e-02

H5

192/299

A

T

0.517

0.440

1.36

1.8e-02

H6

184/300

T

T

0.046

0.022

2.15

3.4e-02

H7

183/298

A

C

0.518

0.451

1.31

4.3e-02

H1

PT3

181/294

A

T

T

0.429

0.290

1.84

1.1e-05

H2

186/295

A

T

G

0.406

0.274

1.82

1.8e-05

H3

181/292

A

T

C

0.405

0.274

1.80

3.2e-05

H7

180/294

T

T

C

0.506

0.396

1.56

9.1e-04

H8

179/295

T

G

G

0.547

0.436

1.56

9.1e-04

H9

181/293

T

C

G

0.542

0.432

1.55

1.0e-03

H10

181/295

T

T

G

0.534

0.426

1.54

1.2e-03

H11

179/291

T

C

G

0.540

0.433

1.54

1.4e-03

H12

178/293

T

T

G

0.510

0.404

1.54

1.5e-03

H1

PT4

177/288

A

T

C

G

0.413

0.276

1.85

1.5e-05

H2

177/292

A

T

G

G

0.415

0.278

1.84

1.5e-05

H4

179/289

A

T

C

G

0.409

0.279

1.79

3.4e-05

H5

176/290

A

T

T

G

0.389

0.260

1.81

3.7e-05

H7

178/290

A

T

T

C

0.383

0.260

1.77

6.7e-05

H9

179/291

A

T

T

G

0.395

0.280

1.67

2.7e-04

H16

177/288

T

C

G

G

0.545

0.438

1.54

1.5e-03

H17

178/291

T

T

C

G

0.506

0.400

1.53

1.5e-03

H18

176/289

T

T

C

G

0.508

0.405

1.52

2.1e-03

H19

176/290

T

T

G

G

0.510

0.408

1.51

2.2e-03

1df refers to one degree of freedom.

TABLE 6

Haplotype frequency analysis of the sporadic cases of prostate cancer

haplotype

frequencies

Pvalue

A2

A30

A41

A55

A57

A75

cases

Controls

Odds ratio

(1 df)

frequency %

60/67

64/66

73/71

64/68

65/69

94/95

(A)

(T)

(T)

(C)

(G)

(G)

abs diff freq. all

−7,4

−2,0

2,7

−3,7

−3,9

−1

pvalue

7,7e-03

4,3e-01

2,9e-01

1,6e-01

1,4e-01

3,4e-01

Cases/controls ↓

H1

PT2

281/298

T

T

0.162

0.090

1.94

2.4e-04

H2

282/301

T

A

0.163

0.095

1.85

5.3e-04

H3

282/301

T

T

0.140

0.083

1.79

2.1e-03

H4

283/298

T

A

0.136

0.083

1.74

3.6e-03

H6

283/299

T

T

0.317

0.246

1.42

7.3e-03

H7

279/300

T

T

0.045

0.022

2.08

3.0e-02

H1

PT3

278/295

T

T

T

0.083

0.037

2.33

1.1e-03

H2

277/294

T

T

A

0.150

0.092

1.75

2.3e-03

H3

279/295

T

T

A

0.081

0.040

2.12

3.4e-03

H4

278/294

T

T

A

0.134

0.082

1.75

3.8e-03

H6

277/295

T

T

G

0.126

0.076

1.76

4.7e-03

H7

277/293

T

T

A

0.091

0.048

1.96

4.7e-03

H8

275/294

T

T

T

0.093

0.051

1.91

5.5e-03

H1

PT4

273/290

T

T

T

A

0.046

0.010

4.76

2.0e-04

H2

271/290

T

T

T

T

0.044

0.010

4.54

3.9e-04

H3

274/291

T

T

T

A

0.078

0.038

2.15

3.6e-03

H4

274/292

T

T

T

G

0.053

0.021

2.57

4.4e-03

H6

272/289

T

T

T

A

0.090

0.048

1.95

5.5e-03

TABLE 7
Haplotype frequency analysis of the preferred haplotypes
	Pvalue haplo. Frequency %
	(cases vs controls)
	HAPLOTYPE	familial cases	sporadic cases
	MARKERS	A2	A30	A41	A55	A57	vs controls	vs controls
FAMILIAL	PT2	haplotype 1		T	T			1e-04 (57/44)	6e-01 (45/44)
CASES	PT3	haplotype 2	A	T	T			1e-05 (43/29)	2e-01 (26/29)
HAPLOTYPES
SPORADICS	PT2	haplotype 3	T				A	4e-01 (11/10)	5e-04 (16/10)
CASES		haplotype 4	T			T		3e-01 (11/9)	2e-04 (16/9)
HAPLOTYPES	PT3	haplotype 5	T			T	A	3e-01 (11/9)	2e-03 (15/9)

TABLE 8
Haplotype frequency analysis with permutation test results
	number	haplotype			PERMUTATIONS
	cases/	frequency		Pvalue	TEST
SAMPLES	controls	cases	controls	Odds ratio	(1 df)	Iter/nb of Iter.
HAPLOTYPE 1 of Table 7
cases vs controls	463/298	0.501	0.444	1.26	2.8e-02	30/1000
cases (<=65 years) vs controls	176/298	0.546	0.444	1.51	2.3e-03	3/1000
cases (>65 years) vs controls	283/298	0.467	0.444	1.10	4.0e-01	273/1000
sporadic cases vs controls	280/298	0.455	0.444	1.04	6.5e-01	572/1000
sporadic cases (<=65 years) vs controls	89/298	0.454	0.444	1.04	7.5e-01	696/1000
sporadic cases (>65 years) vs controls	187/298	0.450	0.444	1.02	7.5e-01	771/1000
sporadic informatif vs controls	67/298	0.434	0.444	0.96	7.5e-01	699/1000
familial cases vs controls	183/298	0.572	0.444	1.67	1.1e-04	0/1000
familial cases (<=65 years) vs controls	87/298	0.646	0.444	2.28	2.7e-06	0/1000
familial cases (>65 years) vs controls	96/298	0.501	0.444	1.25	1.7e-01	103/1000
familial cases (>=3 caP) vs controls	82/298	0.588	0.444	1.79	1.1e-03	0/1000
HAPLOTYPE 2 of Table 7
cases vs controls	457/294	0.325	0.290	1.18	1.4e-01	127/1000
cases (<=65 years) vs controls	174/294	0.362	0.290	1.39	2.1e-02	24/1000
cases (>65 years) vs controls	279/294	0.297	0.290	1.03	7.5e-01	770/1000
sporadic cases vs controls	276/294	0.257	0.290	0.85	2.1e-01	176/1000
sporadic cases (<=65 years) vs controls	88/294	0.229	0.290	0.73	1.1e-01	99/1000
sporadic cases (>65 years) vs controls	184/294	0.265	0.290	0.88	4.0e-01	351/1000
sporadic informatif vs controls	67/294	0.176	0.290	0.52	6.9e-03	9/1000
familial cases vs controls	181/294	0.429	0.290	1.84	1.1e-05	0/1000
familial cases (<=65 years) vs controls	86/294	0.501	0.290	2.46	2.5e-07	0/1000
familial cases (>65 years) vs controls	95/294	0.365	0.290	1.41	4.8e-02	48/1000
familial cases (>=3 caP) vs controls	82/294	0.467	0.290	2.14	2.0e-05	0/1000
HAPLOTYPE 3 of Table 7
cases vs controls	470/301	0.143	0.095	1.58	5.8e-03	15/1000
cases (<=65 years) vs controls	175/301	0.132	0.095	1.44	7.8e-02	96/1000
cases (>65 years) vs controls	292/301	0.145	0.095	1.61	8.2e-03	14/1000
sporadic cases vs controls	282/301	0.163	0.095	1.85	5.3e-04	4/1000
sporadic cases (<=65 years) vs controls	90/301	0.163	0.095	1.85	1.1e-02	11/1000
sporadic cases (>65 years) vs controls	189/301	0.158	0.095	1.77	3.4e-03	10/1000
sporadic informatif vs controls	70/301	0.221	0.095	2.69	3.4e-05	0/1000
familial cases vs controls	188/301	0.110	0.095	1.17	4.4e-01	487/1000
familial cases (<=65 years) vs controls	85/301	0.096	0.095	1.00	7.5e-01	991/1000
familial cases (>65 years) vs controls	103/301	0.121	0.095	1.31	2.7e-01	317/1000
familial cases (>=3 caP) vs controls	83/301	0.074	0.095	0.76	3.7e-01	462/1000
HAPLOTYPE 4 of Table 7
cases vs controls	464/298	0.143	0.090	1.68	2.2e-03	7/1000
cases (<=65 years) vs controls	174/298	0.135	0.090	1.57	3.0e-02	47/1000
cases (>65 years) vs controls	286/298	0.145	0.090	1.70	3.8e-03	9/1000
sporadic cases vs controls	281/298	0.162	0.090	1.94	2.4e-04	2/1000
sporadic cases (<=65 years) vs controls	88/298	0.165	0.090	2.00	4.7e-03	17/1000
sporadic cases (>65 years) vs controls	189/298	0.156	0.090	1.87	1.7e-03	4/1000
sporadic informatif vs controls	69/298	0.223	0.090	2.89	1.1e-05	0/1000
familial cases vs controls	183/298	0.110	0.090	1.25	2.9e-01	318/1000
familial cases (<=65 years) vs controls	86/298	0.100	0.090	1.12	6.5e-01	726/1000
familial cases (>65 years) vs controls	97/298	0.120	0.090	1.37	2.2e-01	271/1000
familial cases (>=3 caP) vs controls	81/298	0.084	0.090	0.93	7.5e-01	839/1000
HAPLOTYPE 5 of Table 7
cases vs controls	456/294	0.136	0.092	1.56	9.1e-03	14/1000
cases (<=65 years) vs controls	171/294	0.131	0.092	1.48	6.5e-02	80/1000
cases (>65 years) vs controls	282/294	0.136	0.092	1.55	1.8e-02	30/1000
sporadic cases vs controls	277/294	0.150	0.092	1.75	2.3e-03	6/1000
sporadic cases (<=65 years) vs controls	88/294	0.155	0.092	1.81	1.7e-02	27/1000
sporadic cases (>65 years) vs controls	186/294	0.142	0.092	1.64	1.5e-02	34/1000
sporadic informatif vs controls	69/294	0.226	0.092	2.89	1.0e-05	0/1000
familial cases vs controls	179/294	0.112	0.092	1.24	3.2e-01	354/1000
familial cases (<=65 years) vs controls	83/294	0.102	0.092	1.12	6.5e-01	733/1000
familial cases (>65 years) vs controls	96/294	0.121	0.092	1.36	2.4e-01	262/1000
familial cases (>=3 caP) vs controls	79/294	0.082	0.092	0.88	6.5e-01	749/1000

Familial forms in which at least three persons are affected by prostate cancer in the family are described in the present application as>3CaP. Sporadic cases were classified as informative sporadic cases when at least two sibs of the case both aged over 50 years old are unaffected.

TABLE 9
Attributable risk for prostate cancer
	Estimating of haplotype	Dominant Model	Recessif Model
	frequency	Carriers		Carriers
				Random	frequency		frequency
	Sample sizes		Controls	controls	(cases vs	Attributable	(cases vs	Attributable
	cases vs controls	cases	(unaffected)	(French)	controls)	Risk %	controls)	Risk %
Haplotype 4	281 vs 298	16.2%	9%	10.3%	30% vs 17%	16,92	3% vs 1%	2,38
of Table 7
Haplotype 1	87 vs 298	64.6%	44%	48%	88% vs 69%	60,77	42% vs 20%	30,63
of Table 7
CARRIER: Individual carrying the haplotype
ODD RATIO of Carrier (OR): Carrier of cases * (1-Carrier of controls)/Carrier of controls * (1-Carrier of cases)
ATTRIBUTABLE RISK (RR): Carrier of Randoms controls * (OR − 1)/(Carrier of Randoms controls * (OR − 1) + 1)
(ref: Epidémiologie - Principes et méthodes quantitatives - J. Bouyer, D. Hémon, S. Cordier 1995)

Example 6

Mouse PCTA-1 Protein

The inventors have cloned a cDNA molecule encoding a mouse homologue of the PCTA-1 protein (SEQ ID No 8). The deduce amino acid sequence is provided in SEQ ID No 9. FIGS. 7A-D show alignments between the human and mouse PCTA-1 protein sequences of the inventions, as well as that of GenBank L78132. It shows an 80% homology between the human and mouse homologues.

Further comparisons between these mouse and human cDNA and protein sequences, taking into consideration the position of significant polymorphisms in relation with potentially conserved motifs, should allow the person skilled in the art to identify regions of specific physiological interest, in the design of suitable vaccine or therapeutic candidates. Two galactoside binding sites shown in FIGS. 7A-D are of special interest. These sites are conserved among the PCTA-1 proteins and the galectins, and seem to be involved in the cell-cell and cell-matrix interactions which are of high relevance to cancer. Two other sites, HFNPRF and VVCN, are also highly conserved among all these proteins.

Example 7

Preparation of Antibody Compositions to a PCTA-1 Protein

Substantially pure protein or polypeptide is isolated from transfected or transformed cells containing an expression vector encoding a PCTA-1 protein or a portion thereof. The concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms/ml. Monoclonal or polyclonal antibody to the protein can then be prepared as follows:

A. Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes in a PCTA-1 protein or a portion thereof can be prepared from murine hybridomas according to the classical method of Kohler et al. (1975) or derivative methods thereof. Also see Harlow et al. 1988.

Briefly, a mouse is repetitively inoculated with a few micrograms of a PCTA-1 protein or a portion thereof over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall, (1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis et al.

B. Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogeneous epitopes in a PCTA-protein or a portion thereof can be prepared by immunizing suitable non-human animal with this PCTA-1 protein or a portion thereof, which can be unmodified or modified to enhance immunogenicity. A suitable non-human animal is preferably a non-human mammal is selected, usually a mouse, rat, rabbit, goat, or horse. Alternatively, a crude preparation which has been enriched for the PCTA-1 concentration can be used to generate antibodies. Such proteins, fragments or preparations are introduced into the non-human mammal in the presence of an appropriate adjuvant (e.g. aluminum hydroxide, RIBI, etc.) which is known in the art. In addition the protein, fragment or preparation can be pretreated with an agent which will increase antigenicity, such agents are known in the art and include, for example, methylated bovine serum albumin (mBSA), bovine serum albumin (BSA), Hepatitis B surface antigen, and keyhole limpet hemocyanin (KLH). Serum from the immunized animal is collected, treated and tested according to known procedures. If the serum contains polyclonal antibodies to undesired epitopes, the polyclonal antibodies can be purified by immunoaffinity chromatography.

Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. Also, host animals vary in response to site of inoculations and b dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable. Techniques for producing and processing polyclonal antisera are known in the art, see for example, Mayer and Walker (1987). An effective immunization protocol for rabbits can be found in Vaitukaitis, et al. (1971).

Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony, O. et al., (1973). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 μM). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, (1980).

Antibody preparations prepared according to either the monoclonal or the polyclonal protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they arc also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample. The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.

While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein by the one skilled in the art without departing from the spirit and scope of the invention. Accordingly, the scope of this invention is intended to be defined only by reference to the appended claims.

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22 1 106746 DNA Homo sapiens misc_feature 1..68647 5′regulation region 1ttggcttggc agggcaacca gctcaccaga ctctctgcag acccgaagtc attacataca 60gtatgataac agggaatgga cccgaccagc atttgctgga gatgatatct ggtgtcagcc 120cgacaggccc ctacctgctt ctcttgatat gcaggaatcc cttcaagctc caacaagatc 180tgtttaatag actggagagt cctttagttc cttcctctaa gggaaaatca gatcgttctg 240gtttgcttgg taactcctta cttcatccct gatgggaagt ttatagaatg aggaaccagg 300gctattacat gaaactataa aactgcctag agcacatact tggtattttt aacattgttg 360agagggactc acttaattca gccttgcagc tattgcattc ctgtccaaac caacggcagg 420ttctcaaaac aagcggtgaa agggttcctg ttgcagagct gtctggacat ttaaagaagg 480gagaggaaat ctcaaggggt cggttgcact ggaatagaaa tcgcctgttc tttttttttg 540agacggagtc tcgctctgtc acccaggctg gagagcagtt gcgcgatctt tgctcactgc 600aacctctgcc tcccgggttc acgccattct cctgcctcag cctcctgaat agctgggact 660acaggcgccc gccaccacgt ctggctcatt ttttgtattt ttagtagaga tggagtttca 720ccattttagc caggatggtc tcgatctgct gaccttgtaa tccacccgcc tcggcctccc 780aaagtacagg gattataggc gtgagccacc gcgcccaggt gcctgttcct tttttaagag 840tctcactctg tcgcccaggc tggcgtgcag tggcgcgatc tctgcttact gcagtctccg 900tctcctgagt tcaaatcaag cgagaaatca cttgttctct tctgtgaacg gaagcatcgc 960agatctctct tggcctcaca ctcctccatc tccctgattc ctctgttctt catttaccta 1020ccttcccagc agtctgcaga gctggccgct cactcacctc tagtaagggg atggagggtc 1080ctgtgttgga ataactcact gaccgctaga aagttaaaaa taaatgggta atgccaggag 1140aacttggctg gtgccttaaa agccatagaa cttctctttc catctgtaga taactgtaga 1200caattttgtc caaaacagat aatgatctga ttctacctcc cattggtatt tcccttcctc 1260ggcctgtgac atctcacttt ctctagactg aactttatcc cagactgtga ccttgccatg 1320accttcctcc tgcgtgtgcc tctgccacca caggaatggc cacgcctcag atcatgtcac 1380cgctgggaac aaaccctcta cctgcgactc tgaagttccc tctctgaccc tcttttcttt 1440cttcccctcc cccctcccct cactccctct gcacctgtgt ttcgctgtca cgctcccaac 1500tcatccctgt agagctggtg aagagatgct gatgtagttc ttgaccttga accccagccc 1560tgcagccgtc ctgtggcctc actgacccag cgtcatgccc tggtcaagca ttttggtgat 1620gctcttggtg attttcaatg ggacctgcct tgccaagccc tgggcttagg tgaaccagga 1680ccacctgcat tctatgtttt tgattgctgg aaaaaaatca tgaaatgtca actgttgttc 1740tcatttttcc cactgccagt tcctgctacc caacctccgc cctcatttca aggccttgag 1800tacttttttt ctatagtgaa gtctcccaaa aatgatattt ttttaaaaaa gaaaagccat 1860agtactctga tttgatgtgg tctgttaata cctatgggct ttgacttgtt tctgctttta 1920gacctagaca aaataaaata tctgtggtaa aacatattca agtttaccgg gcacgggggc 1980tcacgcctgt aatcccagca ctttgggagg ctggggcagg cagatcactt gagcccagga 2040gtttgagacc agcctgggca acagggtgaa acaacatctc tacaaaaata caaaaaatac 2100ctgggcattg tggtgcatgc ctgtagtcgc agctactcgg gagactgagg tgggaggatg 2160gcttgagctc tggaggcgga ggtcatagtg agccaagatc gtgccactga actccagcct 2220gggcaacaga ggcagattct ttctctctaa aaaacataaa ataaaaaaag gccaggcgca 2280gtggctcaca cctgtaatcc cagcactttg ggaggctgag gggggcggac gaagaggtca 2340ggagatagag accatcctgg ccaacatggt gaaaccctgc ctctactaaa aatacaaaaa 2400ttagccgggt gtagtggtgc atgcctgtaa tctcaactac tcaggaggct gaggcaggaa 2460aatcgcttga acccaggagg cggaggctgc agtgagccaa gatcgcacca ctgcactcca 2520gcctgggtga cagagcaaga ctctgtcccc caccaaaaaa aataaataaa taaatcaggc 2580caaagggcaa aaatgcttgc tttttagcac ttagtagtta tttccccaag aagagcggga 2640gagaagttta ttaataatga aactggacag ttctttatca gctctaattg tttgactcaa 2700tggcttctct tctcattacc atgcagtgct ctgctggctg caatgccttt gaacttcaca 2760agaaggttag aatttcactg agacattcgg atggtgtggg tgtcagggtg cagctctcac 2820acatagttga gagtgtaaat tgatacaact ttatggaaaa ttaattggga gtacccattc 2880acactcctgt ctagcaatct cactttaagg acttgatcct acagaactca ttacatggtg 2940caaggttcac agtgtggcat tcaaaataga gaagagctgc gggtaactcc catgcccgtt 3000ggcaggaact ggttgaataa attatggtgc atcagtgctg tggggtatca ttaaaccatt 3060aaaaagaaga gagagtcctg gccttaaaaa aaacttatct gatgtattgt taaacagata 3120aagcaagttg tagatcaatg tgatttgggg ctaaaaaaat atttctatat aggtgtgaac 3180atggccatga ctaaggaatc aggaaggaag tacctagatt gtaaccagta acatgtcggg 3240agtgagatgg gattgagaga cgtaataata gattgagaga aaaagatttt cccatctctt 3300tttgattttt taagaaaaca gcatgatttt cagtaatttt tacttttgtg tgtttttggt 3360attttttctt tttctttttt tttttttttt ttttgagacg gagtttcact cttgttgccc 3420aggctggaat gcgatggccc agtcccagct cactgcaacc ttcacttccc aggttcaaga 3480gattctcctg tctcagcctc ccgagtagct gggattacag gcccctgcca ttacgcccag 3540ctactttttg tatttttagt agagatgggt ttcaccgtgt tggttaggct ggtttgaact 3600cctgagctca ggcgatctgc ctacttcagc ctcccaaaat gctgggatta cagccgtgag 3660ccaccgcccc cagccggtat tttttcaaat caaagaaaaa ataatagagt aaatcatcca 3720aaactttaga tggtatttag actcagtaaa cttttcatat atgacagatg aagccaaatg 3780gtctttctgt gcagtcagct agcacacaat tgtgcacccg aggaaaatta gagactgaac 3840cggggtgtct gtggatgcat ttcctcagca ttcagccttc cttttgcccg tgttctagca 3900ttacttctgt cctacagcct gggatttgtg aatgaaatag acaggtgcaa aaactccctg 3960cctgtctgta atatccatag ccccgtgctc tacttgtatt tgcatgtaca aaccataatc 4020tcctgtaaaa tactctgtga tatttctgaa taataataaa ctctacatcc tacacaaagg 4080caaaacccct gtatctttca tctttgaaac catagcaaag gtatgaaatt acacctgagc 4140atgcctggcc tcaaagtcct ggaacggtta tgtctttgac cctcacttca actcaactcc 4200agaagaagca ggtcttcctt gtaattggat agaaaactca ttgtagagaa gaaagatcta 4260caggtcaaga aacccacagg tttgctgtaa tccgagcaaa gcactgtagc atttatttta 4320tattttcact cttcttattt agctcttttt tttttttttt ttttgagatg gagtttcatt 4380cttgtcaccc agcctggagc aatggtgcca tctcggctca ctgcaacctc tgcctcccag 4440gttcaagtga ttctcctgcc tcagcctcct gagtagctgg ggttacaggc tcccaccgcc 4500acacccagcc aattttttgt atttttagta gagacggggt ttcaccatgt tagccagact 4560ggtctcaaac tcctggcctt aggtgatcca cccgcctcag catcccaaag tgctgggatt 4620acaggcgcac cggccttagc tcttttatcc ttaatgaaat gctcctcatt ccctgaggtc 4680tcacttgaat tcttgcccac ctctgggttg ccttcctctt ctgtctgtgc tttgtaacac 4740gtggttcctt atgatgtcaa tatttatgca tatgtcttca ttccattact ggattataat 4800cttgaagcaa cagatttttg tctctatatc ccagagccta gaatggattc ttacactggg 4860cagtaagtac ttaataaatg tatcccaaat caaataaata catttcttct ttttcttttc 4920tttttttttt ttttttgaga cagggttcca ctctgtcacc caggctggag tgtaatgaca 4980tgatctcagc ttactacagc ctcaatctcc tgggcttaag caatcctccc acctcagcct 5040cccacatagc taggactaca ggcgctcacc acaacacctc atttttgtat attttttgta 5100gagattgggg gatctcacta cgttgccccg gctggttttg aacttctggg ctcagacaat 5160ccacccacct tggcctccca aactgttgag attacaggaa tgagccacca ttccctggcc 5220aaatacattt ctaaaagcca gtttctggag tatactgtca aataatagat atatgtccac 5280atttttatac ggacttatat tgtaagaaaa agtaaaaata agtgtgaagt tattacagta 5340atagtaatta ttttgcagaa aaagaactga gtttaaacag gctttttaga aaaacccaac 5400aggagattca cagtctggta ctaacgttta gacatggatc atcagtaaat gtgttccaaa 5460gagttacaca gataccagct ttgtcttggg aattcttacc cctgaaaatt gattgactat 5520cactgactgt gtgacatgag aaagttttgt ggggtttttt ttttgtattt ttttgagacg 5580tatcttgctc tgtcacccaa gctggagtcc actggcgcga tcttggctca ctacaacctc 5640tgccgcctgg ttcaagcgat tctcctgcct cagcctccag aatagctgcg attacaggca 5700cctgccacca tgcccggcta atttttgtat ttttagtaaa gacggggttt catcgtgttg 5760gccaggatgg tcttgaactc ctgacctcag gtgatctgcc cacctcagcc tcccaaagtg 5820ctgggattac aggcatgagc caccgtgccc agcctgaaaa agttttgaac ggtctaaatc 5880catatgctgt gaatcctatt accatcacac acttaggcat ttaaaatcat attttcaagg 5940ccaggtactg aaatattttc tgcaagcaga gagatcaaac tttagcattg ttattcttgt 6000agtagtttca tagtttgagg tcttagattt aagtcttcca ttgattttga tttgattttt 6060gtatatggag ataggggtct agtttcattc ttttgcatat ggatatccag ttttcccagc 6120accatttatt gaagagactg tctttttcac cagtgtatgc tcttggcacc ttcgtcaaaa 6180atgagttccc tgtaggtgtg tgggtttgct tctggttctc tattctgtcc cattggtcta 6240agtgtttggt tttatgctac taccatgctg gttggtatag ctctgcagta taacttgaaa 6300gcaggtaatg tgattccaaa gaagctagtt aagtaattga gctaaactgg aacctcaggt 6360gtagaagtca taagcgtggg gagcgtttct tctcaggttc tctgcctata atttagtttg 6420ccacaccaga tgaacagtga caacttggtc ttggtgttcg tggtggtttc caaccaaact 6480ttggtcataa caggtgaacc agcctggggc atgctttccc attcggttat cctccccata 6540gtttgcaaag tagcaaagat gaactcttca tgagttggct aagcatagac atttcaagac 6600caaactaaac gtcctgaaga gcatgtttca cagaaaacta gcccctaagg gaccagtggg 6660ggctgtcaga gaacaaggtt tcaacgtact gagttttaaa gatctaattg gcttttaata 6720acaattcatg aaccaggcac catagtctac aaaatagaca gggtttctgc tgggcactgc 6780aggacagttg gtttttggaa ggtggcttga gcaggaacaa ggaaaaagca ccgtgccaag 6840agtggattgg ttaacatcag gggacttcgg gtgactttcc ttctatgggt taaagcaaag 6900gggacttccc tagcatgtca gctcaggttg actgggcccc tttggattgg ttgctgtgaa 6960tctcctagtt ttttttgttt tggtttggtt tgggtctttt ggggaaaacg ggccagtttg 7020gagattcagc tattatttct ctctcctgat atcagaagat cagatcttat gagtacacag 7080ctgaggtttt gggttggtga tgtggaaccc tggtgtgagt gactccattt tgggttggtc 7140tattggggtc tcggtgcagg agctcagtcc aaatcagtgg cctctcctca tttttatttg 7200acttctccat caatctatcc gtgtctcccg tcacatcagt ccattccccc gtgggctgca 7260cattcagctc ggagctgaga gcttttccca gggtgtgccc tggggtttct gctgcttgca 7320gcctgatatt aaatctcagg tgtaaatctt cagaggcaac tgttccttag tacccagagc 7380tttcagctcc ctgagcagaa atgggacttg actgtcagtt tataaactaa ccaaggtgtg 7440aaattcatgc aacttagccg actttctgtt caaagaattc ttggcagcag ttaatacatt 7500ttgcccaaat ataagataat tcccttgtac tcacaatgag aaagttttac aaaatggggg 7560ttttctttag tttacttgaa tataaaacat aggtgttcca ctctgcagta ccttaacagt 7620tcttaaggag atgtttgaaa caacccatgt ccaggcctca cacctcgcca attaaataaa 7680tgagaagttc ttcccagcca gtgttaagaa aaattaacat caagttttag gaaggtagac 7740agattatgca aatgcatacc tatatgattt aagttattac attaatttac acacacatat 7800ttaaaatcat agattaatct aatttagaga tgctgcattt tttccatctc tcctgtttca 7860taaatgttat tcacacggca tttctctgct atcctcggaa tagtgtttgt atcgtgtcac 7920tctggcacgg ggctctacag aacatgtcga gcgtgttgcc ttccctactg cccacatcgt 7980ttgagagaac acattttaaa cattttttta ttgtggtaaa atacacataa cataaaagtt 8040acgattttaa ccttttttaa ctctgtcatc caggctggag tgcagtggcg agatcttggt 8100tcactgcaac ctccgcctcc taggtccaag tgattctcct gcctcagcct tccgagtagc 8160tgggattaca ggtgcacacc accacgnccg gctaattttg tatttttagt agatgcgggg 8220tttcaccatg ttagccaggt tggtctcgaa ctcccgacct caggtgatca gcccgcctcg 8280cctccccagt gctgggatta caggcgtgcg ccactgtgcc gggcccattt taaccacttt 8340taagtgcaca gttcagtggc attaagtata ttcgcggtgt tgtgcgaccg tcaccaccat 8400tcacctccag aacttctctg tcttcccaaa ctgaaattct gtacccattg aacggtaact 8460ccccattccc catttctgct tcctaggccc tgacatggag gctgggccaa cggatatctc 8520acctcccttc aggcttctcc agatttgccc ccgtttttct ccctctttgt cccatctcca 8580aagaaatggt gtcttttcat catcaaggtc catcccttgc tccttgaata cactccaggc 8640ccagtggaac aggcatcctg tggggtgcac ggacagggtg cctggggaac acccagggca 8700cagaacccag accgggggtt tggagaaggt gtcctagcag aagtgatgtc taagctgagg 8760ccctacagat aagagaaagt aagcagatga aagggctggg gagggtggca tttcaggcct 8820acacaaccac acgcgtgttc ttcagccatc tccatggcct cactgcccac ctggtatcag 8880ccggccacca cccggctaga acggctttca aaatcgctgc tcgtctactc ctcaccaaat 8940cttgtcttca cttggtgctc aagcccatca cctttctgca agtattattt tttttttttt 9000ggagatggag tctcgctctg tcacccgggc tggagtgcag tggttcaatg atagctcact 9060gcaaccttga actcctgggc tcaagatcct cttgccacag cctcccaaag tgctgagatt 9120acaggcacaa gccaccatgc gtggtccttg ctgcaacttt tttttttttt tttttttttt 9180ttttgagaca gaatctcgct ctgtcgctca ggctggagtg cagtggtgtg atctcggctc 9240aatgcaacct ccgcctcccg ggttcaggtg attctcctgc ctcaccctcc tgagtagcta 9300ggaacacagg cgctcaccac cacatccagc taatttttgt gtttttagta gagccggggt 9360tttgccatgt tggccaggct tctctcaaac tcctggacct cgggcgattg gcccgcctcg 9420gcctcccaaa atgctggaat tacaggcatg agccaccgtg cctggccatt tgctgcaact 9480tttgacactg ctccccctgc ttttcttccc ctctctgacc tcctttctct gctgtccttt 9540cgttccttcc tctgccactg aagtgtcctt ctcaggtcct tctcaaggtt gtgaccttac 9600agctgtctct tcacttccag tcatttcttt cataatcact ttgacatcct tattttcatc 9660tcctgccctg gcctctccca gggaccagga ccatgcattc agctcctggg ggcatctcaa 9720gcttgttgtg tgtgagcctg cccttgttgt cttctccgtc acctcttcac agcttgctct 9780gcatttcacc tcctttcctg ttttccccag tgatcgcatc tctacagcgg ctctcacttc 9840atccccttct ctcctagagg agtgatgcgg agtctcatta atccttgctt atgtcattct 9900tcccccttct ctgtccatca cctccacatg tcctgttccc ccatgcgtcc tacactgtag 9960ccaggtgggt atttcctgtg ctggtcttag acaccccctg aggataccct gcttcaggcg 10020agagccctca gtgactccct gttgtccgga atgacgtcca gctccttgga cagtccccag 10080tgtattcacc tgtctcatct ccttcttttc gttttgtttg tttttcttaa cttccagccc 10140gatttctgaa tcatctccct cttgcccctc ccattgcctt tgcttaagac taaatgctcc 10200ttcctcccaa gtccccactg cccagatttc agcagggtcc atctcaaaca tgtctgtctc 10260caagaaactg cctctgattt ttttcataag aagacacctg tcctctctga cttcatctgt 10320acccctctct tggaagtcac tatcttgtgc cttgcatttt cgttgtttaa gtggtctcca 10380tttcccagca tatcttgagg tcaagggttc aggtcatttt atctttgtct atgcattgca 10440atatgggggt ttttacatat tagctgctca ataaatcggt gttgaataaa ggcatgtgta 10500tgctttcatt aagactatga aacccacaaa aatcagtggt tttcctattt cacccttaga 10560aaacaaaccc acaacatagc acaacctgat attcagagct aagaacaaag gtcatgcata 10620ttaatctaaa ttctatcttt atcaactttc acaagtaatt cgtatttccc tgtctgcatc 10680acggggatga ttctggccag acattgacct tggtaaaatt tcctccagat tatgagaaat 10740caagtcaaat atgccaagta acatagtttc tacttagagt caggttcatg ttttagcagg 10800aacctcaaat accacaaaat ctgtcaagtt ctaacatttg tatctctcga cagtacctga 10860agttcctgtt tctgtttcct cagcccaggt ttccaattca gtgagcagaa cggtgactgt 10920gttggtaaaa gagcccacat acctgcccga tcctgcagga gtgttgcaga tgcaaacagg 10980cgggtctcca catgacctgc ggagtaatga ctagtgtccc taaagtcatg gggcttctgg 11040ggttagcctt gaaaaaagct aaaggttgca tagagagaga tttctatccg ttcagagact 11100cactataatt ctctctttct gtctctgtcc ttcatctgtt tctctctttc tctctcactc 11160tctctctctg atacacacac acacacacac acacacacac actcacactc acacactcct 11220gagtaaggga aatgtgagaa gaaggtaaaa cttcaactaa atgaaaagaa attgtatgaa 11280ttatggtaag caggttggtt tttagttcca gtaaagatag aaatatttag attacttagg 11340agaaaagtct agctggtaac acatgggaat gtgcctgtgt gaaaacaaaa caaaacaaaa 11400aatctaggct tgtggttagg tgaaggtatg tacactgctg agacatggcg atgggtgagc 11460ttgggatgag gagaaaggct tctctgagaa gattaagaga gaaagattgt ttaaaaatgt 11520ttaaacatgc tgggcactgt ggctcacacc tgtaatccca acactttggg aggccaaggt 11580gggcggatca tgaggtcagg agttcgagac catcccggcc aacatggtga aaccctgtct 11640ctgctaaaaa tacaaaaatt agccaggcgt ggtggcgggt ggctgtagtc ccagctactt 11700gggaggctga ggcaggagaa tggcgtgaac ccaggaggcg gagatgcagt gagccgagat 11760tgtgccactg cactccagcc tgggcgacag agcaagactc cgtctcaaaa aaaacaaaaa 11820aacaaaaaaa aaacacacat tgacaccagg acggagttag cacatcttta caggtgagac 11880tctcagaccc gagaaaatag aggcacttta gagctgagct aatcccacag ccacctcaac 11940acacaaacgg ggaatctgag acccgcattg gcaccgtgcc tgaggttcta aagcccaggg 12000cttctgactc gcctcttgtg cttcttcagt actgtgggtg ggggtggggt ggggggtgac 12060attagctgat gagaaagatt ttggttttag aaagatggag ttaacataaa cgaaggtgta 12120ctgggactgg tctcctctgc tgacttcatg ggaagcacac acacgcacac acacacacac 12180acacacacac acacacacac atacacacac ctgtccaaga tcagaaaaaa tccctcacat 12240ccctgtagca tgatcctgat tgtaaaaatg gagccctaat cagaagggca gaagcatgat 12300tgcctctcaa gagatttgga cgccactttt tcatagttgg ttttagctgc tttgcgatat 12360atactgaaat aaatagaaaa gggaaagaat tgtaacctgg attgacagac aacaagccct 12420gacagacaaa aagcagataa gaaataaaat aaggaagata acccataatg taaaataaaa 12480atagcacatt gttgcatgca ttgataccct tttttttttt tctttgagat cttgctctgt 12540ctttcaggcc gaagtacagt gtctcaatca tagctcactg cagcctccag cttctgggct 12600caagcaatct tcccatctca gccacccaag tagctggggc tgcaggcacg aactatggtg 12660cccagctgat aatttttaaa aatagggaca ttagtgcatt tagcaaattt gagtgtctgc 12720tgtgtatcaa gcactgttct gggcactggg acagcacagg gagcaaataa acaaaagccc 12780ctgcgctcaa ggtgctcgta ttctagaggg agatgctgag ttcacctccc attaaaatgc 12840cattctcaag atccagtccc tccacccacc ccagccccca gggttttggt ggaaatttaa 12900ctaagttgga agattgataa tatctccatt cacatttgga tatgatttta atgaaggttg 12960ctttttggtt tttagggaga agaaaatggc tttccagata gcactggaga tcctcttcca 13020ggtaaatgat tgattctaaa gctatctggg ctaatagcta gtgtggctga ataaaagata 13080atttgaggcc agggtcggtg actcatgcct gtaattccag cactttggga ggccaaggtg 13140ggcggatcac ctgaggtcag gagttcaaga ccagcctggc caacatggta aaaccccgtc 13200tctaccaaaa atacaaaaat tagctggttg tggtgggcgc ctgtaatccc agctactcgg 13260aggctgaggc aggagaatcg cttgaacccg ggaggcggag gttgcagtga gccaagatca 13320caccactgca ctccagcctg gacaacagag cgaaactcca tctcaaaaaa ttaaattaaa 13380taaaataaat aatttgagac tatgtttatc attaacttta aaatctgtac tgcagaatag 13440agcaactttc tacctgcggt gcactgcagg gaaagccgta tcttacaaga cttcacaaaa 13500gccttcaaag agtattttct ctgcactaac cttcctttgc atgtgagggg cacggcaggg 13560ttctgaatgg ggcaggttta ggatcaggcc agtcgggact gagtggattc ttcttccctc 13620tgagttctaa gagccatagc attggtggag aacatgctgt ttgttgcttg gtggaaggga 13680ccagaagcca gctgggtcat ctctctgttt gtgccttggc cacttaggta gccaaaggag 13740ccctcctgac attaggtcag gtgttagtcc ctctcctttt ctgcttttag tgtgtttaag 13800caaataaaca ttaaagttca tttctccccg ctcccctttt ttaatcataa gacagacatg 13860tttgcaatgt ttaaatttct cattaatcag aagggatagg gagtgaggga gtaagcatta 13920aaataagcta gcaaatggcc aggtgtggtg gctcacacct gtaatcccag gactttggga 13980ggccaaggtg ggcagatcac ttgaggccag gagttcaaga ccagcatggc caacatggca 14040aaactccatc tctactaaaa atacaaaaat tagccaggcg tggtgatggg cacctataat 14100ctgagctact cgggaggctg aggcagagaa ttgcttgaac ccgggaggca aagattgcag 14160tgagctgaga ctgcaccact gcattccagc ctgggtgaca gagcaagact ccatctcaaa 14220aaaatgctag caaaataata ataataataa taataaaaca tacctcacca acattttcta 14280catcttgtaa agcatacatt gactgactga agtcaccaga gttttgtttc tttctttctt 14340aagcagggtg gggaacccgt agagccctca ggggcagcta tcatcagccc aggtaaccaa 14400gctgaaaaac cagaaggtgc agtgcgtact caactttttc cccttagaaa cacgatatta 14460gaaaatacac caataccaac atgtgagcaa cagttctctc tggaaggtgc agttctgggt 14520gatttttttt tcattccata gatttttttt ttcttgagac ggagtttcgc actcttgttg 14580cctaggctgg agtgcaatgg tgcgccacca cgcccggcta atttttgtat ttttagtaga 14640gacggggttt caccatgttg gccaggctgg tctcgaactc ctgacctcag gtgatccacc 14700tgcttcggcc tcctaaagtg ctgggatgac aggtgtctca ctatgttgcc taagcttttc 14760tcgaacccct gagctcaagc ctcctcccac ctcagccatc caaagtgctg ggattacagg 14820catgagccac cacgcctggt gagtttttat tttctttcca ctatccntat atttctaaaa 14880tttctaacat gagctggtat cagaactgcc cctccgcatt taatctgtgt atacaaatgt 14940atatataaca aatgatcaca tgttggtaan gtataccttg ctgcatggtg aaataaccaa 15000ggaaacttct aaaaggttaa ctgtggttgg cctgggtaat gggagcatta attttttcca 15060tatgctcatc tgaattttca gatttgctat gacaagcaca tatttatttt ctaattttaa 15120aaatctatat ttaaactctt taaagactaa caccctacac actaatgtgg cacgttagct 15180aaaataaaaa taaatacaga aatttgttta gaaatatttg taaacccttc aaggactctt 15240ctgaatgata gtcattatta attagcaggt taattttaat caggcttctg gtcatcttca 15300aacatttttt acttgtgtca aaatgaacca ccagagtgtg ggtttttttg ttattttttt 15360tgtttttttg agacagagtt tcactcttgt tgcccaggct ggagtgcaat ggcgagatct 15420cggctcactg caacctctgc ctcctgggtt caagcagctc tcctgcctca gcctcctcct 15480gagtagctgg gattacaggc gcccaccacc acacccagct aatttttgta tttttagtag 15540agatgggttt tgccatgttg gccaggttgg tcttgaactc ctcacctcag acgatccacc 15600cacctcagcc tcccaaagtg ctgggactac agatgcacac caccacaccc ggttaatttt 15660tgtattttta gtaaggacgg gggttcccca tgttggccag gctggtctca aactcctgac 15720ctcaagtgat tcacctgcct tggcctccca aagtgctggc attacaggcc tccgccaccg 15780cacccagccc aacctgggtc cttttgtatg tgagagtttg cttgtttttt tcacgtgctt 15840tctctactcc agttttattc tatgacaaaa ttgaggccca acatgattta cttgcctgga 15900tccacccaac ctgtcagtta cttcccagtg ctgctgccaa cttaatgtct ccttaaaagg 15960atgctttaga gaaaacgaaa tcatgttgtt tttccccttt ggttaagaga tcaaacgccc 16020accaaaagcc cttgggtcag tttcttagta gataaaaata attcttcgtc actttctgaa 16080agcggctaac atataaccct tatgatgaat aatgtggtgt gtgtgtgtgt gcgcgcccca 16140aattccaatg agttatcaaa gccagaaact tatattttaa atatgtttat ttcccaacca 16200cactggaaac cacacacaga aaaaaaaaaa agcatgatta taccccctta ataaccgtta 16260ctgcagaagg atgtgactct ccttcaacac ttgttggtat tttacagcct ccaaatctga 16320ccatgtataa ccacctggga tagagttatt ttatttcaga accataatac ttagctatct 16380cggaagttgc caatataaaa tgtttactct ctaatggttt tgaactaact caagacctgg 16440ttatcccggg gagcatcctt acaaatgatc tgagagctaa cagtcctctt gcagcagtgg 16500agggaaacac tcccgtggca atcactctcc aaaagccaga atgtgcaaga taaaagggca 16560ccttccctgc agggaggcac attaagtcag tctgtgatct gctgccaaca tcctgactgg 16620agccgtttct acgcctaact aatcatgacg tttgtgaatt gtgaagcttg ttgcaattca 16680caattaactg ttaattgacc catattttat aacccgccag ccatgaactt acaagttaga 16740tacagacact accagacatt cactattttt ttttacaatt gttttaaatg acattaatga 16800gcatgcttga ttcctgaact cttctttaca gtataatttt aaaatatttg agtgggatac 16860gatggagagg agggaggtgg gggaagaaat gccccatgga aaacccactc atcaggttga 16920gagtgtggag aagccctgtg tatctgagaa ctcttaatca tccacagaca tggtatctct 16980caaagagaag tgggtgtaat tccaaaatct aattttggca ggcgctcctg actaaatact 17040taatctggag atgtcttcaa ggcaggcgga ggttttcagt cctggctgca cattagaagt 17100cccaggggag ctttaaaaaa ttcccacgtc ctccctgcat cccagactaa ttaatcggga 17160tctccgaggg tgggaccaca catcagggtt ttgtaaattt ccctgggggt ttggtggggt 17220tgggggtgga ggcgtctatc ctatggccaa ggttgagaac cactgctttt taaaagactg 17280tttgcttgtt tttgagatgg ggtctcgctc tgtcacccag gctggagtgc agtggcgcaa 17340tctcagctca ctgcaacctc tgcctcctgg gctcaagcaa ttctcctgaa aaaggctgtt 17400ggttattaat gcttccccac agctattcta ttcattgttg catgcttctt acgtgtgcta 17460ggatgggagc tttaaaggat tacctcattt aatcctcaca accaccttgt gagagaggtg 17520tcattatccc tgtttggaga gtgagacagg ggcttagcaa gctcagtaac ctgtccaagt 17580cacacatctg catggggtta gctgctgcta aagctcatgc cgttaatctc catggtacac 17640ggtgtcctct ccatagcaat cttgcggctg ccttgttaac accaaaaaaa cttgcatcag 17700ctggtttgac aatttctaga taaagagctc ttttcgggct gctaagaagc ctaatttttc 17760atttgatttt cttcttgaac tgtgtcacac tcctcattca tttgatatat tcatcaaata 17820cttattgagc acctgctgtg tgcctggtgt gcagcagtga caccagacat ccaaagtcct 17880tttcctctta gagcttattc tatctgggag agacagataa taaacacaaa atcagtaagt 17940cattttatat ggtggtaggt gccttgagga agatgagcca ggttaatggg attaagcctg 18000gtagggggag ggtgccactt tagctcggaa agggtagcga gacccaaaca atgcaaagga 18060cccggcccgt ggagatctaa gacaggagga tgccagggac aggaagttgc tggggcaaag 18120cccctgaggc tggactgagc tcagtgttct aggacgggcg tgggcagtga ggagcagcag 18180aggaggtgag ctgggagata gcctggggac tctttcttct gcctccttca aaaaataaaa 18240ctagccaggt gtggtggctc acacctgtaa tcccaacaat ttgggaagct gatgtaggtg 18300gattgcttga gtccaggagt tcgagaccag cctgggcaac atagtgagac ccctcccccc 18360atttctacca aaaaatcaaa aaattagctg ggcccggtgg cgtgcgcctg tggtcccagc 18420tactcaggag gctgaggtgg gagcattgtt tgaacccggg aggtggaggc tgcagtgagg 18480cgtgattgtg ccactgtact ctagcctggg tgacagagtg agactctgtc tctaaataaa 18540taagtaaatc tagaacctaa catcttggag tgcagtggca ccaccatggc tcactgcagc 18600ctcaatctcc tgagctaatc gagcctcccc ttcagcctcc tgagtagctg ggactatagg 18660cgtgcaccac catacctgaa taatcaaaac ctaacatctt taaagaacat tggcataaga 18720cttggcaaaa atggcatctt gtccctcatc tcatttagtc caagcgatac aggaaatgct 18780gccacctcca ttttatagat gaggagtctg acgttcctag aggttcaatg ccctgaaacg 18840tcaagccttg aggaagttgg agcactggga ttcgaagagc accatccaat acagacccag 18900aatcaggatg atttgggatt atgcttgtca aggactcagg gcagggctac catacattag 18960gcacaagaat tttgatagtg ataattactg tgttcattgt cacttcatca tgacagttac 19020cgtgatgata agaaacctgg cccttcttca cctgacaaag gctttcttcg tttgagccac 19080tgctcaaacg agactgacca agaataaatc ctcggggctt tggcctttaa aataggaagt 19140catcataaat gacttgatgt ggtgtgtttc attcttgctt tgcaccagtg gaaaatatac 19200aggtcaagca tcaaaacatg gcaaatgggg accccaatta ttagagaatc taagttaatt 19260tttatgtata attaattatt caacaaccct ctcctctcca aaccaataat taatccatct 19320tttgtatttt aagaccaatt ctgtagtatt ttccatcaat atctatttac tgctagcaga 19380tatcagctac attctttctc ctttaataga agttccctct ttaggtatta agattcatta 19440aacaacaata acaaatctac cttgcctccc agggacaatg cacagttctc attcatttgt 19500tcatttagca gataattttt gaatttccac tgtacagcag ccctgtgctt gtggttggcc 19560tgttatttga gaagcatcaa ataataatct cattttttgg ctgggtgtga tagctcacgc 19620ctgtagtccc agcactttgg gaggctgagg cgggtggatc acttgaggat gggcgttgga 19680gaccagcctg gctaacatgg tgaaacctcg tctctattaa aaatacaaaa attagccagg 19740tgtggtggca gacacctgta atcccagcta ctcgggaggg tgaggcagga gaatcgcttg 19800aacctgggag gcagaggttg cagtgagccg agatcgcccc attgcactcc agcctgggca 19860acaagagcga gactccgtct caaaaaacaa aacaaaacaa gacaaaaaaa aacccaacaa 19920ataaaataaa taatcccatt tttctccatt tttgagaaag atttctttgg tctgaagtct 19980ttctctcccc tctccgaggc attacccagt ttaacctttc atgtataata tatatgatag 20040ttatttaaag tatagcagga caaaatgtat ttgataggag aaaaccttgt ttgctctgtg 20100ttaagtcctc cagagagcta attagagttt gtgattctaa aaggcaacta tagattcact 20160tatattagca gttcatgtag attccagtta aggaaatggt ttgtcacttg tgttattgaa 20220aacacacaca gggcgagcac tgtggcccat gctggtaatc ccagcgtttt gggaggctga 20280ggtgggcaga tcacggggtc aggagtttga gatcagcctg gccaacatgg tgaaaacccg 20340tctctactat aaatacaaaa aattagctgg cagtagtggc aggcgcctct aatctcagct 20400actcgggagg ctgaggtagg agaatcgctt gaacccagga gtcggaggtt gcagtgagtc 20460gagatcgcac cattgcactc cagcttgggc aacaagggca agactccgtc tcaaaaaaaa 20520agaaagaaaa cacacacaca aaaaaacttt agtagatctt tcggcatatt attttttaaa 20580ataaactgat aatggttgat atgattgttc aaagaaataa gagcttttca taaactcagt 20640ttaaagaaac tttacaggcc gggcgcggtg gctcatgccc gtaatcctag cactttggga 20700ggccaaggcg ggtggatcac ctgaggtcaa gagttcgaga ccagcctggc caacatggta 20760aaagcctgtc tctattaaaa aatacaaaaa ttagccaggt gtgttggctg gcgcctgtaa 20820tctcagcaac tcaggaggct gaagcaggag aatcgctgga acctggtagg cagaggttgc 20880agtgagacaa aatcgtgcca ttgcactcca gccccagctg acaacagcga gactccatct 20940caaataaata aataaataaa taaataaata aataaataaa ggagctttac agaaaccttc 21000tgatgttttt ttcttcttga cgataacatt gccaacactg aatcttacaa agataagaca 21060agaaagggac cttcagacac cattacatgt aattctggac ttagtggttt aaatccttat 21120ttttctatga cattaaaaaa atgtatattt taggccaggc acagggctca cacctgtaat 21180cccagcactt cgggaggccg aggcaggtgg attgcttcag cccaggagtt caagagcagc 21240ctggggaaca tagtgagacc cctgtcccta cagatttttt ttttttgttt gagatggagt 21300tttgctcatg ttgcctaggc tggagtgcag tggcacgatc tcggttcact gcaacctctg 21360cctcctgggt tcaagcaatt ctcctgcctc agcctcccaa gtagctggga ttacaggcat 21420gtgccaccac acccggctaa ttttgtattt ttggcagaga ctgggtttct ccatgttggt 21480caggctggtc ttgaactccc aacctcaggt gatctgcctc cctcagcctc ccaaagtact 21540gggattacag gcgtgagcca ccttgcccag cctacaaaaa gttttaaaaa attaaaaaat 21600tagttgggca tggaggtgca tgccagctac tcgggaggct gaggcaggag gattgcttga 21660gcccatgaag tggaggctgc agtgagccat aattgcagca ctgcactcca gcctgggcca 21720tagagcaaga ccctgtctca aaaatatata tagtatccaa ataaacacaa taattacaga 21780aaattgaaaa gtgcccataa gcaaaaaaaa aaaaaagaaa aaattaatca cctgcgttct 21840catcacccag aattaaccat tgttaatatt tttgttatag atccttccaa acttttctcc 21900atgcttgtga ttgtatttat tatacatgat ttacagggat ataaacgact gtattattag 21960tcattagaag aactggatta tggccgggca cggtggctca cacctgtaat ctcagtactc 22020tgggaggctg aagtgagcag atcatgaggt caggaaatcg agaccatcct ggctaacaga 22080gtgaaacccc gtctctacta aaaatacaaa aaattacctg ggcgtggtgg caggcgcctg 22140tagtcccagc tactcgggag gctcaggcag gagcagagat acctatctgt tctcaggatt 22200ttaaggtgtt gcgcggaaat aagaaaaccg tacagtgttt ctcactacaa agcagggtca 22260ggagatgcaa acaaactgat gtgggggttc caagtgaggt ggaattccag acaggggccg 22320ggaagacttc gtggaaaggg agaatctgag gtgggttttc taggatgggt aaagttcatt 22380agaggaagag aagtgcaaca gaggaagttc ggtgagaggt agagggaagg cgttctgatc 22440atgaaggaaa cactagaaaa ggtatggaga tagaaaaaga taaggcctga ttttttaacc 22500taccacttaa aaaaaatcct tgaaaagaga tttttaaaac gaatacttgg tgctgacaaa 22560ggtgaaatga ccgggcgcgg tggctcacac ctgtaatctc agcacattgg gaggctgagg 22620cgggcagatc acttgagctc aggagtttga gaccagcgtg gccaacatgg caaaactcca 22680tctctactaa aaatataaaa attagacggg tgtgatggtg ggtgcctgta gtcccaacta 22740ctcaggaggc tgaggcagga gaattgcttg aacccgagag gcggaggttg ctgtgagctg 22800agattgtgcc actgcactcc agcctggata gcaggatgag actgtctcaa aaaaagaaag 22860aaaaggaaag aaaaaaaaat ccgtactgta aactggtaaa ggctttcttt ctggagagca 22920atttggggca catgcaccag tagccttaga aggctcatgc ttttgaccta attatcctat 22980tagtggtgag atgattaaag atgtggcccc aatttatgtg aaaggtatgc atcacatctt 23040cactcataat caggagagtt ggggaaaacc ctagctgtta atagtttatc caaaatccat 23100atatatatgt gtgtgtgtgt gtgtgtgtgt gtgtgtatgg atttatatat atatataaat 23160ggatatatat atatatctgg atggatatat aaatatgata tatatatgtg tgtgtgtgta 23220tatatatatg tgtatatatg tatatatata tgatggaata ctatttagcc ataaaaagga 23280atgaattaat ggcattcgca gtaacctgga tggacttgga gaccattatt attttatttt 23340atttatttat ttttgagacg gagtctcgct ctgtcaccca ggctggagtg cagtggctcg 23400agctcagctc actgcaagct ccacctcccg agttgacgcc attctcctgc ctcagcctcc 23460tgagtagctg ggactgcagg cgcccgccat cacgcccaga taactttttg tatttttagt 23520agagactggg tttcaccgtg ttagccggga tggtctccat ctgctgacct catgatccac 23580ccgcctcggc ctcccaaagt gctgggatta caggcgtgag ccaccgcgcc cagcgagact 23640gttattctaa gtgaagtaac tcaggaatgg aaaaccaaac atcgtatgtt ctcactcata 23700agtgggagtt atgctatgag gacgcaaagg cataagaatg atacgataga ctttggggac 23760tcagggaaaa ggtgggaagg gggtgaagga taaaagatac aaattgggtg cagtgtatac 23820tgctcgggtg atgggtgcac caaaatctca taaatcacca ctaatgaact tactcatgta 23880accaaatacc acctgttcct caataaacca tggaaattaa aaaagaaaaa agaaaaagta 23940ccctggaaaa aaaatttctc cctggccagt cacggtggct catacctgta atcccagcaa 24000ttcgagaggc tgaggcagga ggatcacttg agcccagtag ttcaaaacca gccagtgcaa 24060catagtggga ccctgtctca aataaaatct aaaaattagc caggtgtgtt ggtgcatgtc 24120tgtggtccca gctactcagg aggctgaggt gagagtattg cttgagccta ggaggttaag 24180gcggcagtga gccgtgattg tgccactgcc atccaacctg ggcaacaaag caagaccctg 24240tctcaaaaaa aaagaaaaaa aaaacctctc tattcgcctt ttaagaatac ctgggcttct 24300ctgtgtacac ttaagcttca ttggagtctt tagacttttt ttttgctgta tctgtccagt 24360taccaagtcc cagcttctac tccatgctcc ccatgctctc ttcctatttt attttccatg 24420actgcctcgg tataacttgt gctcaaccaa actggactac tcaattccct gcattttctt 24480ttttaaagtt taatcaaaaa aaaaaagaaa actggctggg cacagtgggc ttctgcccac 24540aatctcggtg ctttgggaaa ctgaggcagg aggattgctt aaggccaaga gttcaagacc 24600agcctgggta acatagcaag acctccatct ccacaaaaaa atttaaaaat tgactgagtg 24660tgatggtgtg cacctagtcc cagctgcttg ggaggctgag gcaggagaat tgcttgagcc 24720caggagttcc aggttatgat gagctatgac tgtgccaccg cactccagcc agggtaacag 24780agtgggactg tctcaaaaaa caaaacaaaa tccctaatat aatctcagtg tgccttttaa 24840gtatgccata tatatatata tatatatata tatatatata tatatatatc acattttctt 24900tatccactca ttgattttca tgtagttcta atcgtagaat tcatacattc tttctatctt 24960ccatctttca cataacatca caaacatttt ctaggttgcc atattgtctt catagttact 25020taaataatat tccatcaagt agcacaatca tttatttcac tagtcctcta actgtagaca 25080ttttggttgt ttttgaaact taataatgta aataacaccg tgataacaat gtttatgtaa 25140attcatattt tggattatct ccttagggtg gattcccaga agtcacatta gtaggtcaaa 25200gagtatgagc ctattttcaa ggctcttgtt ttattacctt ttaatttcca cttgcctcaa 25260tattgctggt ttgctccctt atgatcacca gagttactcc gtcggtccaa attctttacc 25320ttccgaaact gggaaggcca tgactcaatg ttatatatat agtaaaggct actataacct 25380tccccagaat tttccaagcc agtggtctct aaagtgacct ttggctgtta aaatctgaat 25440tcagagggtt catgagactc agtgttgttg tagaatttaa gctccttaat ttgccacgtt 25500gtttagacac cacttaatac tttattgcaa atgacttgtc aacgcctctc acctacaaac 25560ttcatcctcc tacaaatata cctcctgcta atcaaatgag gctacagttg agtctttaag 25620tttcagtaga aagatggccc ttcctctggg gtaggcgcat gctcttcatg ctgaagctca 25680gctgaaaagc ctcctgctga gttttctgcc tctttccctc ccactgcaca caccccaggg 25740tgttggcgcc acttcaaagg gagcctgtgg atgaagaaaa cacaggtaaa ggcagagggc 25800tcataagggg gccataaatt taaaaagtta agattcctgg cactatcaac tctcacttgt 25860tttcaaatat gcatatggag tggatattcc agttttcatg tctgtgttgt tgtttttaaa 25920aaaagacctt tcaaagaact gtgcattttt tacaggctga caggctgtgt ttggtgttaa 25980actgtcaggg ctgactggtc acttggaaag ggcaagggct gaggtgcatg caagtgtcgg 26040ctggttactc acagacacag cagccccttt taccccggag agagttctgt ttgctggagc 26100ccttattctg gccagcagtg tcacaaatgc acactgtaag acatagacag tcttggaaag 26160aaagggaaac tggctttaaa aattcttact ccttctagca aagcaattca tctttggcta 26220taaagaataa cacagccagg tgcggtggct catgcttgta atcccagcac tttgggaggt 26280caaggtgggc agatcacttg agtctaggag ttcaagacca gcctgggaaa catggtgaaa 26340ccccacctct accaaaaaaa aaaaaaagaa agaaagaaaa gattagccag gtttggtggt 26400acgtgcctgt agtcccaggt actcgggaag ctgaggtggg aggatcgctt gagcctggag 26460ggcggaggtt gcagtgagcc gagatcatgc cactgcactc cagcctgggc aacagagtga 26520caccctgtat caaaaaaaaa aaaaaaaaag aacagtaaca cattattaga aatgagcatt 26580ctgaggccag gcacggtggc tcatgcctat aatcgcagca ctttgggagg ccgaggcggg 26640tggatcacaa ggtcaggaga tcgagaccat cctggctaac acggtgaaac cccgtcttta 26700ctaaaaacac aaaaaattag ccgggtgcag tggcgggtgc ctatagtccc agctactcag 26760gaggctgagg caggagaatg gcgtgaaccc ctgggaggcg gagcttgcag tgagccgaga 26820tagtgccact gcactccagc ctgggcgaaa gagcgagact ccatctcaaa aaaaaaaaaa 26880aaaagaaaga aacgagcatt ctgaaatagt cttccatatg atgcttttga caattcagca 26940ggaaaataaa ggatgtaaga aatgaatgca tatgttaggc ctcttgttga cctgtggact 27000aaattgtttc tccctgcaga gatcagcaag gacaactcct gcaaagaaaa ctgtacttgt 27060tcctcctgct tgctccgggc ccccaccata agtgacttgc tcaatgatca ggacttacta 27120gacgtgatca ggataaagct ggatccgtgt cacccaacgg tgaaaaactg gaggaatttt 27180gcaagcaaat gggggatgtc ctatgacgaa ttgtgcttcc tggagcagag gccacagagc 27240cccaccttgg agttcttgct ccggaacagt cagaggacgg tgggccagct gatggagctc 27300tgcaggctct accacagggc cgacgtggag aaggttctgc gcaggtgggt ggacgaggag 27360tggcccaagc gggagcgtgg agacccctcc aggcacttct agagctcttc ttcttccttc 27420attggcctct ccggatgttg aaacaaccac aggtcaagaa ggaatgtgaa tctgttgttt 27480tataagagtt taggacaagg acgtggaaca gtggacactg gttttcccca aagctggcag 27540ttttgtggag gggtagcttg tttcggtggt ggatctctgt ttatttttgc acatctgtta 27600taatttaata ttcaaatctg gaattaagaa aacatatttt ctagtatcct ctaagggcca 27660aagtcctaca atcggaatgg attcatgcca cgttgaagat aaaattatcc tctctctgaa 27720atacggtaaa gatttaaata ggtcctgaga ctgttgatag ccccagacat acccacagca 27780ttatatgtaa catctctcct gatcagtgcc attcccacgg tttcaaagaa aacagctaca 27840aggaatgctt acctgagtgt ctgcagcacc ctccacttct ctcctaggca atgagaccca 27900gtggctagaa attcaccatg tctattctca agatccatgc cagggagctc tttgactctc 27960gtgggaatcc cactgttgag gttgatctct tcacctcaga aggtctcttc agagctgctg 28020tgcccagtgg tgcttcaact ggtatctatg aggtcctaga gctccaggac aatgataaga 28080ctcgctatat ggggaagggt gtctcaaagc ctgttgagcc catcaataaa actattgcac 28140ctgtcctggt tagcaagaaa ctgaacgtca cagaacaaga gaagattgac aaacttatga 28200tagagatgga tggaacagaa aataaatcta aatttggtgc aaatgccatt ctgggagtgt 28260ccctcgctgc ctgcaaagct agtgctgttg agaagggggt tcccctgtac caccacatcg 28320ccgacttgtc tggcaactcc aaagtcatct tgccagtccc ggtgttcaat gtcatcaatg 28380gcagttctca tgctgtcacc aagctggcca tgcaggagtt catggtcctc ccagtcggtg 28440cagcaaactt cagggaagcc atgcccattg gagcggaggt ttaccacagc ctgaagaatg 28500tcatcaagga gaaatatggg aaagatgcca ccggtgtggg ggatggaggc gcgtttgctc 28560ccaacatcct ggagaataaa gaaggcctgg agctgctgaa gactgcgatt gggaaagctg 28620gctacactga taaggtgatc gtcagcatgg acgtagaggc ctccgagttc ttcaggtctg 28680gaaagtatga cctggaattc aagtttctcg acgaccccac caggtacatc tcacctgact 28740gtctggctga cctgtacaag tccttcatca aaaactaccc agtggtgtct actgaagatc 28800cctttgacca ggatgactgg ggagcttggc agaagttcac ggccagtgca ggaatccagg 28860tagtggagga tgatctcaga gtgaccaacc caaagaggac agcctcggcc gtgaatgaga 28920agaagtgcaa ctgcctcctg ctcaaagtga accagattcg ctctgtgact gagtcccttc 28980aggcgtgcaa gctggcccag gccaatggtt ggtgtgtcat ggtgcctcat cattctgggg 29040agactgaaaa taccttcatc actgacctgg tggtggggct gtgacctggg cagctcaaga 29100ctggtgcccc ttgctgatct gagcgcttgg ccaagtacaa ccagctcctc agaattgaag 29160aggagctggg cagcaaggct aagtttgccg gcaggaactt cagnaacccc ccagccaagt 29220aagctgtggg caggcaagcc cttcagtcac ctggtggcta attagacccc tccccttgtg 29280tcaactccgg cagctcaaga cccccgagca acatttgtag gggccgctgc tagttagcta 29340cccttgccca ccgccgtgga gttcgcacct cttccttaga acttctacag aagcaggttg 29400cagtgagccg agattgcgcc actgcacacc agtttggaga cagagtgaga gtccgtccca 29460gaaaaaaaaa aaaaaaaaaa gaacttntac agaagccaag ctccctggag ccctgttggc 29520agctctagcc ttgcagtcat gtaattggcc caaatcaccg gagccacgtg accctccagt 29580gtcatctccg gggtggccac aggcaagatc cccagtgatt ttgtgctcaa aataaaaagc 29640ctcattgacc catgagaaaa aagaaaacag caatgagaag tgaccctgtc ttgttggttt 29700attacttttt ttgttataaa gtactttggt gaattaacag gatgctagta ttacatggtg 29760atactcttca gaacacctgc cccatctttt ttatgcaagt atgtttacaa tcagtggact 29820atcagtaatg tcatttgctc aaatattttt taaagaccta cagaaactga tggttattgg 29880gaaaacagtc aggaagtagt gaggtaatca aggccatggg aatagtgttt gacaaagaga 29940gtactccaaa tcccttttgg ttacccagga ctttaaaaaa gagagtactc catcacacct 30000gtaatcccag cactttggga ggccgaggcg ggtggatcac gaggtcagga gatcgagacc 30060atcatagcta acatggtgaa accccgtctc tactaaaaat acaaaacatt agccgggtgt 30120ggtggcgggc gcctgtagtc ccatctactc aggaggctga ggcaggagga tggcttgaac 30180ccaggaggcg gacttgcagt gagccgagat agcaccactg cactccagcc tgggcgacag 30240agcaagactg tgtctcaaaa aaaaaaaaaa aagagtgctc caaatctcct ttggttaccc 30300gggactttaa aaaatttaat gtgatagtta ggccgggtgt ggttctcacg cctgtaatcc 30360tagcactttt ggaagctgag gcgggtggat catttgaggt caggagttgg agaccagcct 30420ggccaacatg gcgaaacccc gtctctacta aaaatacaaa aattagccag gcgtggtggt 30480gggcgcctgt aatcccagct cctcgggaaa ttgaggcact agaattgctt gaacccagga 30540ggtggaggtt gcagtgagcc gagattgcgc cactgcactc cagcctaggc aacagagcga 30600ggttccatct caaaaaaaaa aattgtaata ataataataa caatgtaata tttacttttt 30660catcctttat ataaggctga gtgcttcacc cctgagatga agctcagtta agaaataaat 30720gaaaatcccg taacctattg gtgaaaggta accaccccca gctcctacta gcccaactta 30780aaacaggacc ccatcacact acacagcagt ttagccaaga aaagggggtc tttatgtgga 30840cactgggagg gaagggattc cttcaaatcc aaactttaaa ggattttaaa caaatgaaac 30900atttggttca aagaatagct gatgttttta tttgatgatt ttggagaaag gaaagtgtgg 30960ggcataatgg ggtttgttat tggaaagatc agattttcta ggtaatttgg gtggagaaag 31020acaaaaggca aagctttgac tgacaattcc atgaaagtgc tatttggttt tggttatggg 31080cttagaaaat taagacactt agttcaattt ggaaggattc tgtataagtc cctgattaaa 31140ataagcaaaa atgatgaata acactgattc agtgcaaccg aaagattagg attaactcaa 31200aagaaagtta ttttctaaac caccgtgatt ttttccactg acaattacag cggttttcat 31260taggttgctg acacatgaag tcagcctcac catcagttgc aaactctaaa ctagcaaaat 31320ctattacaga gacatactta tcacttctga tttagtgcta atctcaccca gctcatcttc 31380tcttgtcaga tttatgagat aaatgtcaga tttatcacca gatatattga aagtaacagc 31440cagtaataaa atgtgagatt ttaaaaaata gattctttgg caaattggtg ttcagtgagg 31500caattattaa acatttttgt cagccaggtt ccaggcactg tacagaagct gttaggagtt 31560ctcaccatct acgaatttga tttgatgtat tgtattctca ttaagctatg tgtgacacat 31620tgtcatttat tagcccagaa tttaaaaagc tgtggttgtt tagtgttggt ggtagcagac 31680cccagcagtc tgatggtctg cactccttcc atcctgccac cccctgggga tgcaaagact 31740ggatctcagg gtgacaatct tcttgcgcac gactgcctgg ccaagtgcct ccagaaagcc 31800ccttccttcc cccatttcca cccaggccca cttgtcacct cagcctaaca ccagcctgca 31860cagtctacgg ccaccatcca ggcagtggga gagggaaagg ggaggagggt ggaagggaaa 31920acccctttct atacctctcc tcagcctgct ctttcctcct cccacctctg agcctccgcc 31980tcccccagac agagacagaa aagatggaag aacaggtggg acctccaccc ccaccccaag 32040ccttcatccc ggtggagggg gatgggaaga tttctctcat ttcaagagac tcctccacct 32100cagactgaca aaaggcagag gcctggcaag aagaaagggc accctgggga agaagggcat 32160tgaaatagca cctgccgggc cgggcacggt ggctcacgcc tgtaatccca acactttggg 32220aggccgaggc gcgtggatca cggggtcagg agttcaagac cagcctggcc aagatggtga 32280aaccccgtct ctactaaaaa tacaaaaagt agccaggcgt ggtagcgggt gcctgtagtc 32340ccagctactc cggaggctga gacagggaac tgcttgaact gggaaggtgg aggttgcagt 32400gagccaagat cgtgccatgc actccagcct gggcgacaga gtgagactcc atctcaaaaa 32460accaaaacag aaatagcacc tgcccccacc ccctgcccgc cctccttccc gcccccgtcc 32520tttcctagac ttcactcaag tcctctgctc agaggaagcc ctgctctact gaaagccaca 32580aggccattct cggtggcctg ggacagcagc ccaagacgtg ggcttctaac tgcctccgaa 32640ggggccacag cagcaaacat aaataaaaat agtaaaatgt tcttaaatta taaatttaaa 32700atttggaaaa tttagtgagc acagcttcta gggggcatgt ttccaaaatt ccaaccacaa 32760aagtgcagtc tcaaaactga ctgtaaaccg aacataccat ctcatctcag acacagctat 32820tgttcacgag tgtcagtgga actttcctcc cttgagatgg accaaaaacg tcaagcaaga 32880tgacatttgc tgatttgcag gcttcaggca gataagatac gggcagagtt gagtgtgcgc 32940ctttaccctt aaattcagga atagcaggaa cagcaggaaa aacgtaggac cacagcgtac 33000gtcccacttg tctttcattt tgatatcatt atttccagag tcctgattgc tagtcatgtc 33060taacactgga tttattatca tctcattgct agcatggcta ggaaagcttt gaacatcctt 33120atcattctat tttaattcct attataattg catggggaag ttccagggtg gaaaaatttc 33180cttttctttc tttttttttt tttaatagga gagttggctg ggcacggtgg ctcacgcctg 33240tgatcccaac actttgggag gccaaggcgg gtggatcacc tgaggtcagg agttcgagac 33300caacctggcc aacatagtga aaacctatct ctactaaaaa tacaaagtta gctgggtatg 33360gtggtgcaca cctatagtcc cagctactgg ggaggctgag acaggagaat cacttgaata 33420cgggaggcag cagtgagcca agatcatgcc actgcactcc aacctgggag agacagagtg 33480ccatctcaaa aaaaacaaac aaaagagttg atataaattt gctgttataa tttgactgta 33540ctgtttcttg cacatgttga catctgtaat gactggagtt tatgaaaatt tttgatgagt 33600aggagcatac cattaacaga gagaaattta atcaaaagat ttttaaagtt ccttcagagt 33660ccagactttg actaagtgta gtatgattta tatctatgtt gcatacaaaa atatcaaaca 33720gtaattccca actgaaatac aagtatcaat caattgtgta acaatgcaaa atcatttaat 33780ttaaagttaa tttatagcaa atgagtactg taatagcata agcatgccga tactttacaa 33840aggagagagt ggaaaggtag gatattataa ctaattgatc aaatcattgt taaaatttaa 33900gtttattaat acttttactt ctgtccgtag ggatccatgt taaattgggt atattataaa 33960cttaactgct aatgatgagg tccttttgct attagaaatc tattttttat ttttctttat 34020tattttttga ggcagggtct tgctctgttg cccaggctgg agtgcagtgg tgaaattata 34080gctcactgca gcctcaacct cctgggctca aggaatcctc ctgcctcagc ctcccaagta 34140atggaaactg cagtcgtata caagcacacc cagcaatttt tttttttttt tttggtaaga 34200tggggtttag ctatgctgtc caagctggtc tcaaactcct ggcctcaagt gatcctccca 34260ccttancctc caaagtgctg ggattacagg cgggagccac cattcccagc ctagaatgaa 34320atatctttag ctaaattaca gggctggatg tggtggctca tgcctgtaat cccagcactt 34380tgggagactg agggcgggag ggtcacttga gatcaggagt ttgagaccac cctgggcaac 34440acagtgagaa ttctgtctct attttaaaaa gagaaaaatc tagggtatat tctcttaaac 34500aaaactttca tctataatgg tagttgatga ggtcctatgt aatatgcatt tccttggttg 34560caatagcaaa ttactacaca cacagaaagg aaagccacac tccccgacac atctacacac 34620aggaggactc acacaggagg gagactcaaa gaaggcacgt gacttttaca ttgttagggc 34680ttacatggtc ctgggatttc ccaccagtac tcaaaagatc aattgtatga acaagtcacc 34740tatttttacg gcactaaata attattattc aacaacatgg aaaatatgtg gtagcagacc 34800tggattttcc ttaagagtta tttttatgtg gtactgcccc ctgctggaat ataacatcta 34860tacacatcct ttctggctgg gctgacatcc taaaaccagc ccaggaccag ccttttatta 34920atattaattc ttggccaggc gcggtggctc gcctgtaatc ccagtacttt gggagtccag 34980ggcgggcgga tcacgaggtc aggagttcaa gaccagcctg gccaacatgg tgaaactccg 35040gctctactaa aaatacaaaa cttagctggg catagtggca cattcctgta atcccagtta 35100ctcgagaggc tgaggcagga gaattgcctg aaccgggacc cgggaggtgg aggttgcggt 35160gagccgaaat cgtgccactg cactccagcc tgggctacag agcgagactc cgtctcaaaa 35220ataaataaat aaaaattaaa attaaaaaat aattcttggt tgtatgctaa aagccttgca 35280agtagcccca ctggaagata ggaagagtgg ggctgtttta caaatgagca catataagca 35340gaacgaggcc gccataattg aaatgaaggt ccccgtcccg tggatgtgtt catcgctact 35400tcaccctgtc attcggatcc aatgtgtgac cagccagctc caataacagt tccatactct 35460gggaattatt tttaacactc ggcaggatgc tttcttcctg tagttttagg cttagccctt 35520tgtgcacttt tggtctcttt ccctttcaat ttagcatcca aggaagcggc tgtgaccaaa 35580ggtagctgtc atgttaaagg acaaagttca tagttacagc aaatattgac ccagagcact 35640atccttgccc cttcctctat aatgtgcaat gcaaaaatat gttcttttaa gtacaatatt 35700aataagtaag gtctaggaga ttttcttccc ccttcctttc tcttttagat gagtaaatgt 35760tttatctagt tttgaggaga ctatccttct tatcacatct ctttccactt ctgctctcct 35820tgttttataa ttttcctctc ctttgggtcc gtgtcattat ttcgtgtcgc ttgttttcga 35880gccatgcact catttatcaa atcagatttc ctccgtatgc cgacggcctt cctctccctg 35940ccacgggctt cctttttccc tgactatgca gaagcaattt gttcgcttgt gtttcttttt 36000ttttttgaga cagagtctcg ctctgtcacc caggctggag tgcagtggcg acatctcggc 36060tcactgcaac ctccgcctgt caggttcaag caattctcat gcgtcagcct ccagagtagc 36120tgggattaca ggtgtttgcc accaaggctg gctaattttt agtagagacg gggtttcacc 36180atgttggcca ggcttgtctc gaactcccaa catcagttga tccacccatc tcggccttcc 36240aaaatgctgg gattataggc atgaaccacc gcatctggcc ttgtctttca tccttaatga 36300cactttagtc ctaataatgc taaaatcatt ttctactctt tgaattgaaa cacagcttat 36360ctacatgagc ccaaggcagt agcaacattc acctccattt cttctctgat ctctaccttc 36420tgaaccctgt ggacttggtt gtaaatggat gagggcaagt cttgcttcct tcccctgtgt 36480ttacagagga tcgtggctga gatgctgggc cacactctgg gcctgctggc acccctgggc 36540cggtggctgc tgcccctcag ggtgctcacc acctagacca gaagaaccaa ggtgagggag 36600agcctgtttt ctttcttcct gtggctgcgg gggctgtgag gcatgggtct agtggctgtg 36660tttagctggg gatgcctcct agaaatcagc tccaccgttg aagagatcaa agcaatgcac 36720agtgccactt gaaatgaaac gattgagctt atcagcgctt ttgcaaatgt acaagagggt 36780agctcccccg gacatcctga actgagccat gctcttctat tttgtgtaac agcccagtga 36840cccctgaatc ttcccctgag gcaggtcccc gaagcttcat ggaggatgtt cctcagctga 36900ccaaggtgag gctcttgagc tcctaaatct ttgtgatact gtttatacat ctttgtgctg 36960tactttttaa gctgacttcg tgttatcacc tgtatgattt tatgttttgc ttctaaataa 37020gtacagatta ttttaaactc taataatggg tgctacaaaa ttaaagatta tgtcaatcac 37080tgtctctgat gagttatttt atgtagattt caacacaatc attgattcat gtgtactctt 37140ggtcagtcat cagtcatctg agtacctagt gggtttccaa aatgggtcct ggatgctggg 37200gatgcaaaga taagcaacac atttctatcc tcaacagcct gtagatgagg gagaatcact 37260gcggacaatc agggaagtta ccggagagag cagtgcacat gtggtctaga aactggtgga 37320acaaagttga gaatcactga actaggagga aagacaggtc actgacaatc caaggcacag 37380tgactcacac tctaatctca gcactttggg aggccaaggc aggaagatcc actgagctca 37440ggagataaag accagcctgg ctgacctatg gagacccttc tctaccaaaa aaaaaaaaaa 37500aaaaaaaaac attagcctgg catggtgggg tgtgcctgtg gtaccagcta ttcaggaggc 37560tgaagtggga ggatcgcttg agcccgggag gtcaagactg cagtgaatca tgatcacacc 37620attgcactcc agcctaggga agagagcaag aaagaccctg tctcaaaaac agaaaaaaat 37680ccagtaaaat gtttcagatg ttgttaaagg tgatttcact gttacttttc acctctcctc 37740attttacatc tctgacctat gcttgtcctc tgacttgcca gacattccta gctatggact 37800tgatgtctcg acatggaggc tcacaggcac cccaaactca gcctgcccta agctgaaccc 37860atgatctttc cttccaaact tgtttctcac cagagttccc atcttatcat ccacctagtt 37920gttcaagtca tccttaagac ctccctctcc ttcactgtct attctacctc cctaatatct 37980cttaaatcct tccctcctct cccacctcac agccaccatc ctaacctaag cagccaccct 38040ttctcaccct ataatgacct cctggctgtt ctctatagag ttggtgaatc ctttcgtctt 38100cagcctgaac cccctttcga gggattctta tatatataca tagatataca caaatatata 38160tgtacatatg tacatatgtg tgtatatatg tacatatgtg tatggatata catatgtaca 38220tatgtggatg tacatatgta catgtgtatg ggtgtacata tgtacatgtg tatgggtgta 38280catatgtaca tatgtgtatg ggtgtacata tgtacatatg tatatgggtg tacatatgta 38340catgtgtgta tgggtgtaca tatgtacatg tgtgtatggg tgtacatatg tacatgtgtg 38400tatatatgta catgtgtgta tgtacgcatg tacatatgtg catgtatgtg catgtgtatg 38460tgtgtgtatg tacacgtgtg catatgtgtg tatatgtgta cacgtgtacg tgtgtatata 38520tatatatata tatactggct ggagtgcagt gggaaagttt tggctcacca caaactccac 38580ctcccaggtt caagtgattc tcctgcctca gtctcctgag tagctgggat tacaggcgtg 38640caccaccatg cccagctaat ttttgtattt ttagtagaga cggggtttca ccatgttggc 38700caggctggtc ttgaactcct gacctcaggt gatccaccca cctcggcctc ccaaagtgct 38760gggattacag gcgtgagcca ccgtgcctgg ccggattcct atcttgaaga cgaagcccca 38820gaccatcgac acggcctcaa ggccctgcat gacgcctcct gccccaacac ctcgtgtcat 38880cttgctctcc tctcccgcag ctcctgaggc tttagccacc ctggaattcc aagtccccat 38940ggtcattttt ttttcctgct caagatatca ccatgtgctg tcccctctgc ccttgtctac 39000acccacgtgt ccttctcccg ccccggccac actcatgggg cacactgtcc ttccctggct 39060aatcctccca cactcgatac cactttctct gggatattgc acccgatcct cagccgcagt 39120tgtcttccta tgacccactc ccacactctc gccacaatgg taattgtttg attcctactt 39180gttgtccctg tgagactgca aaccccagag gacaggggcc ctgggttctc cttcgcctct 39240ggatcatcag cactaactga atacctggcc tagaagagat gctaacgatg ctgaatgaat 39300aaataagtgg aaagactctc agtaaagcaa aacctttctt taccatttta tggccgtcaa 39360ggaggaaaac acattatcag tggaaaacgc aaaatgaggg gatttgctta gcaaacgatg 39420aattcctctg gcaccctggc agccttggtt tcttttgatg aggtccaccc ccttccatcc 39480atcttctggg cttaagagat caaagcaaaa catgctgtgg aattcgatac tggtgcaggt 39540tgcacaacat tgtgactgaa ctaaaagcca ctggatggta caactgaaaa tggtgaattg 39600cgtgttgcat gaattataac ctaactgggg aaaaaaaggc ttaaaaagag acaaagcttc 39660ccccacaatg gaaaggaagg tataatagaa acagcagctt tcaaaccttg gcaggataat 39720gaaaccccgt ttctattttt aaaaattagc tgggtacagt ggcacgtgcc tgtagaccca 39780gctactcggg aggctgaggc tggaagatcg cttgagccca ggagttcaag gctgtggtaa 39840actataatca cactactgca ctccagcctg ggtgacagag aaagaccctg tctcaaaaaa 39900ggaaggaaag aaggaaggaa ggaaggaagg agggagggag ggagggaagg agggaaggaa 39960ggaaggaagg aaggaaatag cagctctgag cttagaaata ggagtctatt tctaagtggg 40020agatggggag aaggagggaa ctggggaggt gaggaagaag caggtattgt caccagtgag 40080gactgtgctg ttgtgagccc agctaggcaa ctggcaattc cattctgtta gtgacagcta 40140caataaccca aagccctctg gagccctgct ttcctctgct ctcttcgtgg cttgactagg 40200agctgaagat cctgtccctc ttagagcatt ggggcggccc accccacctc caccctcctc 40260cacctgctgc ctcgaggccc ctcccactcc cggggtagac aaaacagttt agaggctgaa 40320gtcaccgggg ctgtaactgt tggatttgca catgtcatag aaaatcatca tatgttttgt 40380gtggactcca tgcataacaa caagagaacc aaccagaccc catagacaga agggagtgtg 40440aattggagac aaaatttaaa ttatgagttg ccttctattc agatttctcc catttttaac 40500aaaaaggagc ccaaattcct aaatgttatg gttgtttgca gcaacttatc atctttctcc 40560tttccttcat agccaaggtt tttgaaagag ctatctgagg ccgggaatgg tgactcacgc 40620ctgtaatcct agcacagagg ctgaggtgag tggatcacct gaggtcagga gttcaagacc 40680accctggcca acatggcgaa atcccgtctc tactaaaaat acaaaaatta gccgggcatg 40740gtggcgtgtg cctgtaatcc cagctactca ggaggctgag gcaggagaat cacttgaacc 40800caggaggtgg aggttgcagt gagccaagat ctcaccactg cactccagcc tgggcaacag 40860agtgagactc catctaaaac aaaaaaaaaa gtagctgtct gttctttctt ctcgaactct 40920ttttcccgct ggagtctgtg acctgctgcc gtctgcctca agtgagaggg actagcagat 40980ctggtgaatt accttctaat gcccgtaccc tgcccatacc agcttcaatc tgtatgtaga 41040agcttagctt gctccatgca tggcctccag catccactgg tcacaaaata acacaaaata 41100gcatgagaga gaatggtcgc atggagcgga ggagctgctg agactgaacc caagccaggg 41160ctactgctgg gtggaactgg acatgcccag cccatgggaa agtcttccca cagaagtcat 41220atttgcaggg gtctcccagg agacagcaca ttctgagcaa aggagtgagg cagagataac 41280tattcaggaa ccaagagact cgctggaaag aagcagagat tttcagccca gcgtagtgga 41340tgtttcttga atcttcccct gtggatgccc caaaccttga gatccttcca acaaatagca 41400cactactaac aaactgtgac tcaaagagag ggaaacatgg tcccctgctc tgtcacaaat 41460cactgtgaag ctttggcacc ctgactgctc aggtggccac caacacagaa ggaccacgaa 41520tggctgagtc aggaagtcac agccgtgtgg ctggaagagg ctctgccttg ctctgggaga 41580aatgcctatc cccaaggaag ccttagtatc catgggagag aaacactgta gcaatggccc 41640ccaggactct cgggaagcca cttctggtgg gaggggactc aaagggtgct gggggacctg 41700tgtctgcatc tggaagtgag gagccaggaa aattttcttt cagtttcttt cttttttctt 41760ttcttttttt tttttttttt ttgagaaagg gccttgccct gtcgctcagg ctgaaacata 41820gtggtgcgat ctcggctcac tgcaacctcc acctcccagg ttcgagtgat tctcctgcct 41880cagcctcccg agtagctggg actacaggca tgcaccccca cccacgccca gctaattttt 41940gtatttttgg tagagatgtg gtttcgccat gttggccagg ctggtctcga actcctggcc 42000tcaagtgatc ctcccgatgt gctgggatta caggtgtgag ccaccacgcc cggcctcttt 42060ctgcttcatt taacattaat ggtcatccca cagcatggtg ctgtgcacct gtagtcccag 42120ctactcaggt ggctgaggtg ggagaatcac ttgcgttcca gctgtagtga gccttgattg 42180tgtctgtgaa taaatgccac ttctctccag cttgagcaac atagggagac tgtctcttaa 42240aaaacaaaac aaaacaggct gggctcggtg gcccacgcct acaatcccag cactttggga 42300ggccaaggca agaggattgc ttgagcccag gaggtcaaga gcagcctggg caaaataggg 42360agaccccatc tctacaaaaa gataaaaaat aaaaaaatta actgggcatg gtgatacacc 42420tgtagtccca gctactctgg aggctgagat aggagtattg cttgagcctg ggaggtcgag 42480gctgcagcga gccatgatca tgccactaca ctccagtcca ggcagcagag tgagatcccg 42540cctcaaaaaa ataaaacaaa acaaaactca tctctccctt ggctcctgag actacaatcc 42600ctcacggttc ttttctactt ctctgttttt ctcttcttgt ctcccttttt ttctggtctc 42660tctgtcaccc aggctggagt gcagtggtgt gatcatagct cactgcaacc ttgacctcct 42720gggttcaaga gatcctccca cctcagcctc tcgagtagct aggactacag gctcacacca 42780ccatgcctag ctaatatttg tagattttgt agagatgggg tcttgctatg ctgtccaggc 42840tggtctcaag ctcctggcct caagtgatcc acccacctca gccacccaaa gttctgggat 42900tacaggggtg agccaccgcg cccagccgat aattgttgaa aaatcatttt cagttaaggt 42960atccagtcaa ggtcagaaaa tgagaaaatg ttaaaaaaaa aaaagctata agtaaaacag 43020attcagtcgg gacatgatgg ttcacgcctg caatcccagc actttgggag gttgaggtag 43080gataatcact tgagcccagg agttcgagac cagcctgggc aacatagcga gaccttatcc 43140atacaaaaaa atttaaaaaa tacccaggca tggtggcata ctcctgcatt ccctgctaat 43200tggatgggtg aaggggagga tcccttgaaa taggagtaga ggtgcaggaa atatgattgt 43260gccgtgtaat ccagcctggt tgacagagca agatgttccc cacccccctg aaaaaaaaaa 43320aaaaacctaa atccaaattt taaaagtttc cttgactctt caacttgctc accctccacc 43380aaataaaata actacgaagg aggcttattt tttactattt ccagggatac gatatatgtt 43440tgtcctgaaa atatacatca tggctttact caagccacag tgatgaggcc tcattgtcac 43500tgtagcctaa ttacgatttt ataactccat ttaaaattca atttaaacac agtttaaaaa 43560ttcagtccaa gtcaaacatg ctctcagtag ctagaagcaa aactctgttc aggtccttga 43620tggatctatt tgtactttct ttcatgaaaa cagaaagtcc ttttttacac accatgcaac 43680aggaaaattc ataacggaca ttgttttacc tgttcttggc aaagacaagt gagctcttaa 43740caagcaaggt aactatggag atgatgtttt gctccaagtt aacacttaca tatttaatta 43800gaaagatttc aaaggtgggc agattcactg gaaagtttcc aaaagcttca cttgttcaac 43860aaataatgtt agagagggag caccgtgccc tcgggcccct aggaattagt tccacatggt 43920ccggtcctct gtccagtgtg cccagcatcc acttgggaga acagcatggc cttctgtcca 43980gggcagccca cgccagcact gcctgccctt tcaggcccat ggctcccatt aagtgccatt 44040tcgagcatac ttagccaagt ttccctacca tggccaacaa agaggttgtt caaaaatgct 44100tgtcaggtcg ggcatggtgg ctcacgcctg tagtcccggc actttgggag gctgaggcgg 44160gtggatcacc tgaggtcagg aattcaagac cagcctggcc gacatggtga aaccccgtct 44220ccacaaaaat acaaacatta gttgggcatg atggcgggtg cctgtaatcc cagctgctca 44280ggaggctgag acaggagaat tgcttgaacc cgggaggtga aggttgcact gagctgagat 44340cacaccattg cactccagcc tgggcgacag agtgagaatc catctcgaaa aaaaaaaaag 44400tttgtcaacg gtttcactga atccagaata cttttctaaa atgtcaaccc tatagaatac 44460attttataaa attatgaagg cctggtctgg tgtagtggct cacgcttgta atcccagcac 44520tttgggcagc caaggcaggt ggatcgcttg aggctgggag tttgagacta gcctggccaa 44580caaggcaaaa ccctgactct actaaaaaat acaaaaatta actgggcgtg gtggtgcaca 44640cctgtaatcc cagctactca ggaggttgag acaggagaat cacttgaacc caggaggtgg 44700aggttgcagt gagtggagat tgcgccattg cactctagcc tgggtgacag agcaagactc 44760tatcttcaaa aaatagataa ataaataaaa attaaaacaa aataaaattc tgaaggcctt 44820aggtcagaga attaccgagg gaatattcaa agttatacct ccaagtatct acaatgaaga 44880tactttcatc agaaaaaagg agtttacggc caggccctgt ggttcatgcc tataatctca 44940gcactttggg aagccaaggc tgaggcagga ggatcacttg aggccaggag ttcgagacca 45000gcctgagcaa aaacgtgaga tcccatttct accaaaaata aaaatgtaag gtaggcatgc 45060aactgtagtc ccagctactc gagaggctga ggcaagagga tcgcttaaac ccaggactcc 45120agcctgagca acagagcgag accctgttta taaaaaaaaa agaaaaaaaa aaagaagaag 45180aagaaggaga agaaaggaaa taaaatttaa gaaaaaaaaa aggacttaat aaggttgaat 45240gaaggcaaga atattcttag ctctgtttaa gtcaagacct gagtagtagc tctacgtagc 45300tgtatgtcga taatgttttt gagacagcac tactgataaa ttgttacata ataaactgtt 45360atggctggat gcagtggctc atgcccataa tcccagcacc ttgggaggcc gaagtgagtg 45420gatcacctga ggtcaggagt tcgagactag cctgatcaat atggtgaaat cccatttcta 45480ctaaaaaaat aaaaattagc tgggcatggt ggcgcacctg taatcccagc tactcaggag 45540gctggggcag gaggattgct tgaacccagg agacagaggt tgcagtgagc cgagattgcg 45600ccattgcact ccagcctaga agacagagcg agactccatc tcaaataaat aaactgttaa 45660attaagttta gcctaaagct acccccttac atattttaag ttcagtctaa aggtttccct 45720gcacatagtg aactgtaacc taactggatg cgtaaacaga ctataaccta ctcttgggcc 45780agtcactgag ttttggtcaa tcaaaggcag ccaactgttc aaaccaggtt aaaataaggc 45840agatgctgag ctctaaccag tccagccatt tctgtacctt gcttccattt tctgtccatc 45900actttccctt ttctgtccat aaatcttcca ccacgtggct gtgctggagc cactgtgaaa 45960ctattctgtt tcaggggctg cccaattcat gaatcattcc ttgctcaatt aaactctgtt 46020catttaattt gtctaatatt tttcttttaa tcaaagtaat ttggccgggc acagtggctc 46080acgcctgtaa tcccaacact tcgggaggcc gaggtaggtg gatcacctga ggtcaagggt 46140tcaagactag cctggccaac atggtgaaac cccgtctcta ctaaaagtac aaaaattagc 46200cgggtgtggt ggcgggcgcc tgtaatccca gctactcggg aggctgaggg aggagaatcg 46260cttgaacccg ggaggtggaa gttgcagtga gctgagattg tgccattgca ctccagcttg 46320ggcgacaggg caagactctg tctcaaaaaa aaaaaaaaaa ttaattcaga gacctactca 46380tgtgaagttg tattttttta ttctccatat tacaaaacag aacaattggc acagggatga 46440agaaatactt tgcaaaacat ctagagaggt taaatgccat gagtctttaa aatgtaagac 46500tgctttcacc tgagcaatct agtgtccatt tctagagcta gcttaaatgt ccgtgtaaat 46560ccccgtaatt ggttgggata acaattacct atgttgtata acttgagtca aaaactacgt 46620ttccactgcc tgccacccct atggatggtt ttctcttaag gtatcaaatt ttactgggaa 46680agacctagat aaaatacagc gaaaatgagg cggggcgtcc tggcacatgc ctgtaatccc 46740agcgctttgg gaggctgagt cagaaagatc tttgaattca ggagttcaag accagcctgg 46800gcaatatagt gaaatcctgt ctttacaaaa aattaaaaat tagccaggca tgggggcatg 46860ggcctgtagt cccagctact tgggttgggt gactgatgtg ggaggatcac ttgagcccag 46920gaggttgagg ctgcagtgag ctctgaccat gcccctgcac tccagcctgg gtgacagagc 46980aagacccagt ctcaaaaaga aaagaaaaag agtaatgtta ggtcaaggta gaacctacct 47040tgactttctg ttactatgga agatattctg gggtatctct gagatccaag tattatggca 47100cttaagtaat tcctatctat tgttctactt ggttcctcgg gagtaaaagt catattcaaa 47160ccaaaaaggc tgtgggattt ccagaatttt aaaagcaata atagttaatg ttctcccatg 47220ggagttactc cacattttta catatgttcc atatgttaac tcatttagac cttaccttta 47280tgaggtaagt cctcttctta tccccacttt agaggtggga aaactgaggc acagaaagag 47340taagttgctt gcctaaggcc ctgttactag caggtggtga aaccagcatt ccaacccggg 47400agtctggcaa atgtgtgtga agagcacacg tttggaaatg acagtcatga ggacactgta 47460agacttctgg aatgtttata atttcacctt tgcttgttat ttttcctgtc tgtttcccta 47520gagtgagctg agtgaaaaaa gaaagaagaa agaaagaaga aagagaaaga gaaagaaagg 47580agagaaaaag aaaaaagaaa agaaaaacag aaaaagggaa agaaagaaga aatgaaagaa 47640agaaaagaaa agaagaaaga aaagaaagag agaaaggaag gaaggaaggt gggaggggag 47700gaaaggaaga agaaagaaag aagggggaca gagggaggga atgaaggagg gagagaggga 47760gggaaggagg aagaaataaa aagatgagga tctgtatgct tgaggggtgg aggtgggggg 47820cttgggtggg agtgtgggat gggcagaaag ctggagggag ccctggaccg actgcattcc 47880acagaggatt gtgggtgcaa cgtaggtggc agattgagaa aagcaaacaa acaagctcag 47940cctttggagc ttcggggaag aaaaaaagct gagcagtgaa tgctggcttc ccacggagaa 48000ggcaggctgc ttcgccagct cacatccttc cgcgcaccca cttcctcttt ccggaggtca 48060ctttagattg ctttatggca ggatctccag gtcacaggaa tgttatgttt cgactggggt 48120ttccccctcc cctgggatgc ctgggccagc tccccaaggg ctagtctctg tcccaggccc 48180cacactccca tagcactcag caaaagccta gagagagcac cgcaaaatgc caaacgcaac 48240aggaccgcgt aggaagaaga cgcttggaat gacagggaca ctagaactgc ccatggtcgt 48300ggtctcaaat ttttgttcca tggtctgaaa tactaaaagt tcttaaacag ctacttgatt 48360tcatactatt gttttgaaga aaacagtgtt tgtttgttgt tttgtttgtt tgtttgtttg 48420agacagagtt ttgctcttgt tgccgagttt ggtccatgtt ggtcaggctg gtctcgaact 48480cctgacatca ggtaatccac ccacctctgc ctcccaaagt gctgggatta caggaaaaca 48540gttgtttctt taaaacaatt atataggctg ggcacggtag ctcatgcctg taatcccagc 48600actttgggag gctgaggtgg gtgaattacc tgaggtcagc agttcgagac cagcctggcc 48660aacatggtga acctccgtct ctactaaaaa tgcaaaaaat tagccgggcg tggtggtgca 48720ttcctgtaat accaggtact caggaggctg aggcaggaga atcacttgaa cccaggaggt 48780ggaggttgca gtgagctgag atggcaccac tgcactccag cctgggcaac aagagcaaaa 48840ctccatctca caatctcaaa aaaataaaat aaaataaaat aaaataaatg gttatataag 48900ctaccttatt gatgcagtta caaatgagcc gctgaaacat ataaatttta aagaacaagc 48960cacatatctt tcatcaccca cagcttcacc aactaaaggt gtatgtagta cttttgtgga 49020aggcatttcc acatgctttg agggaccttg aaatactgct atgattacat gatttttcta 49080aaaccagact actcctacat tacaagaatt gaaaagttca gagtaaatat ttgtaagacc 49140tagaaaagat gatgttcttt aaaaaaaacg atgcccatct ttgtagcgaa aagaaagaga 49200gatcagactg ttactgtgtc tatgtagaaa cagaagacat aagagactcc attttgaaaa 49260agacctgtac tttaaacaat tgctttgctg agatgttgtt aatttgtagc tttgccccag 49320ccactttgac ccaactactt tgacccaacc tggagctcac aaaaatatat gttgtatgaa 49380atcaaggttt aagggatcta gggctgtgca ggacgtgcct tgttaacaaa atgtttgcaa 49440gcagtatact tggtaaaagt catcgccatt ctctagtctc aataaaccag gggcacaagg 49500cactgtggaa agccgcaggg acctctgccc tggaaagcgg ggtgttgtcc aaggtttctc 49560cccatgtggt agtctgaaat atggcctcgt gggatgagaa agacctgacc atcccccagc 49620ccaacacctg taaagggtct gtgccgaggt ggattagtca aagaggaaag cctcttgcag 49680ttgagataga ggaaggccac tgtctcctgc ctgcccctgg gaactgaatg tcttggtata 49740aaacccgatt gtacatttgt tcaattctga gataggagaa aaaccgccct atggcgggag 49800gcgagacatg tttgcagcaa tgctgccttg ttattcttta ctccaccgag atgtttgggt 49860ggagagaaac ataaatctgg cttacgtgca cgtccagtca tagtaacttc ccttgaactt 49920aattatgacg tagattctgt tgctcacatg ttcgttgctg accttctcct tattatcacc 49980ctgctctcct actacattcc tttttgctga aataacgaag ataataatca ataaaaactg 50040agggaactca gagatggtgc cggtgcaggt ccttggtatg ctgagcgccg gttccctggg 50100cccactgttg tttctctata ctttgtctct gtgttttatt tattttctca gtctctcgtc 50160ccacctgact agaaatatcc acaggtgtgg aggggcaggc caccccttca catcttgtct 50220ccacttcctt gattaaaaaa aagaaaagaa aaaaaaattt gccgaagttg gattcattca 50280cagaattcta cacattaaaa atgttgcagg tcgggtgtgg tggcagctcc caaagctgcc 50340tataatccca gcgctttggg aggcttgagc ccaggaggtc aaggctgcag tgaactgaga 50400tcgcaccact gcactccagc ctgggcgaca gagcaagacc ctgtctcaaa gaaaaaaaaa 50460aaaacagaaa aaaataacgt tacagaaaaa gtacaatatt tttaatatat atatatatat 50520tttttttttc tgagacagag tgttgctctg tcacccaggc cggagagcta tggctcgatc 50580tcagctcact gcaacctcca cctcccgggt tcaagcgatt ctcctgcctc agcctcccga 50640gtagctggga ttacaggcac ccaccaccac gcctggctaa tttttgtatt tttagtagag 50700acggggtttc cccatgttgg ccaggctggc ctcgaactcc tgactttatg atccgcctgc 50760cttggcctcc caaagtgttg ggattacagg tgtgagccac catgcccagc caaaagtaca 50820atatttttaa tgacatataa agatgttcat tctttgtggt tgccctgggt gagagggact 50880attgatactc aatagtgttt cttttgtttc tacattgttt ctatagtgaa aatacgcatt 50940ggctttgtat taaaaaatgt atagtaaaaa tggttttatt aaaaatagca aataactaca 51000aaaactccat tgcaatggaa agcagccctt ggattttcta gttgaatgaa acgagtaatt 51060tatccaatgt tagaaatgtc taaaggctcg ctcaggtttc atgagcagaa caggaattgt 51120atatccaatt aaatgtgaaa ttgcaatgcc tggtgcggtg gcttatgcct gtaatcccag 51180cactttggga agccgaggca ggggatcgct tgagcccagg agttcgagac caccatgggt 51240aacatgggga ggccccatct ctacaaaaaa taaaaatcgt tagccgggca ggttggtgca 51300tgcttgtgtt cccagctact tgggaggctg aggtggaagg atcctctgag cccaggagga 51360tgaggctgca gtgagacatg atcgatgcac tccagcctgg atgacagagt gagaccctgt 51420ctcaaaaaaa aaaaaaaaga aaagaaagta caatcgcaat taaatgtctt tgcgttggtg 51480gctcctgacc aaattcccta agcaagcagt atgttaatga gcagaggggc cacagctcac 51540cttgctcaat taaaggcagg agcaggccgg gcgtggtggc tcacgcctgt aatcccagca 51600ctttgggagg ccaaggtggg cggatcacga ggtcaagaga tcgagaccat cctggccaac 51660atggtgaaac cctgtctgta ctaaaaatac gaaaattaac tgggcatgtg gcatgagcct 51720gtaatcccag ctactcggga ggctgaggca gaagaattgc ttgaacccgg gaggtggagg 51780ttgcagtgag ccgagattgc accactgccc tccagcctgg tgacagagcg agacttcatc 51840ttaaaaaaaa aaaaaaaaaa ggcaggagca agtatgggcc agacagaaat caaggtgtaa 51900attgggcaga tcctcaggcc cagtgctgaa ttttggtttg atgaaataaa acattacatt 51960tcaaggttgg cagagaggaa tgaaggtgga agaggaatct agggccattt agggaagcca 52020tgaagcctcc tgcccacact agtgggtaga gtggagccag gcgttttgct agggcttgct 52080atatctcttg gcagggtgct ctgctgccaa agccaagaat tctaaattag attaaatagc 52140cagaaagaat gttaaacatt tggacatgat atcctccctc acagattagc tagagtgtag 52200ttctgctgtg ctagatactt aaataaatac ctccctagct gtgaagcctg cttatcacag 52260tactatattt taggatgagg tcattatttt cctatgcata cacatgcatt gtataatctt 52320gccaatgtag gtcagcccaa aagaagtgac aaatgtgtag aacacacatt ggactagctt 52380gggacaaaat tagtatacct aaagatgaca gatttcttaa ctaattttat gagccatgca 52440gctttgtatt ctagcagaga cagacattag gaatcttata aaatcaaaaa ttttaatttt 52500tgcctgaata gctccaaagg gctaagatct caagcaaatg cgtgtaggtt ttgtttttgt 52560ggttgttgtt gtttttagag acagggtctt gctctgtcac ccatgctgaa gtgcagcggt 52620gcagtcctag ctcactgcag ccttgacctc tcaggcttaa gtgatcctcc tgccttagcc 52680tcccgagtag ctgggactac aggcgcatgc caccaccccg agtaattttt tatttttatt 52740tttacttttg tagagacagg ggtctcaata tgttgctcag gctagtatct tttttctttt 52800tgagacagtc tcgctcaatt gcccaggctg gagtgcagtg gtgccatctc ggctcactgc 52860aagctccgcc tcccgggttc acgccattct cctgcctcag cctcccgagt agctgggact 52920acaggcgccc gccaccatgc ccagctaatt ttttttgtat ttttagtaga gacggggttt 52980caccgtgtta gccaggatgg tctcgatctt ctgacctcgt gatccacccg cctcagcctc 53040ccaaagtgct gagattacag gcgtgagccc tcgcgcccgg cccagtcttg taacttaact 53100ttaaagctac ttattcccaa atgaagatgg gatggtacac agattttaag tattagctgg 53160tttggagctt ctgtctttta aagcaacatt ttactttgcc acagggtggt ggggcggggg 53220ccatcctaga aagaagagtg tgagtttcat gggatagggt ctggggaggt ggctggagga 53280gtttaggttc ttttgatatc tgtggctaca cagacagata accaaggaaa atgtccaaac 53340agtgaaatta agtgctcact gcactaacac agagaaggac cctgatgtct ggccgcaggc 53400ctttgttctc attggcttca aagaacttct tgatgtctac cttaatttca ttattattta 53460cccaggagtc attcaggagc aggttgttca attgccatgt agttatgtgg ttttgagtga 53520gtttcttaat actgagttct aatttgattg tgctgtggtc tgagacactg tttcgatttc 53580agttcttttg catttgctga ggaatgtttc atttccaatt atgtggtcga ttttagagta 53640agtgccacgt gacgctgaga agaatacata ttctgttgat ttcggggggg agagttctgt 53700agatatctat taggtccact tgatccagag ctgagctcaa gtcttgaata tccttattca 53760ttttctgtct cgttaatctg tctaatattg acagtggggt attcaagtct cccactatta 53820ttgtgtggaa gtttaagtct ctgtgtaggt ctctaaaaac ttgttttatg aatctgggtg 53880ctcctgtatt gggtgcatat gtatttagga gagttagctc ttcttgttga attgctccct 53940ttaccattat gtaatgccca tctttgtctt ttttgatctt tgttgggata aaattacatt 54000ttatgtcccc cttcctatag tttgtcactg agggttggca gaagttgaaa ggaagaagac 54060atttgggtgt ttggtttggg gttatattag gttataaggt tcattgcctc cacctctttc 54120aaaacattta gtttctaaat gaatccagct ttaaatgact gcaggagtgc ccatgcacaa 54180ttttgtttct caaatctttg ggatttttcc ttgaagaata ttcacaggga atggggctgt 54240cttgcttcat agttactctt ttgtatacat gatctcaaga atcgcctgat cactgctaga 54300gttaaaccaa tacactaact gcctgaagtg ctgaaaagtc aaatgggggc ttagaacctc 54360actccagatc ctacacaagc tgatggttct gttcccagaa acaacccagc ttcctcatca 54420tctatggcca gtgccttgta gcggagctgg agatcaccct ttagtgggct cttcagctgg 54480atctagaaat caaattgaca ccaggcagat taacaagaga aaagtataca gattttattg 54540cttttatatg tacttgggaa tctgcacaag ggcaaagtcc gaagaggtgg ccaaagcaag 54600gtgcttttat acatttttag aaaaagagcc aaaaaattgg agaagaaatg ataggacaaa 54660gaaaatctag ccaggcagta aattttctag gagaatcact aggacatata tgaggaaggg 54720tgtgtaaaac aggtgaaaga taagggctag ttcattaaac atgtttactc tggtccattg 54780tagcctctac gataaggagt attttctcgc tctggtgtgg acagggcacg cctcccagag 54840caacctttat cacttactgc atgcaggaag agacaggtca gcccgccctt cctgaaacta 54900caatttcttc agtgttttca actcaaaata atcaataccc cccatctggc atatctgggg 54960atggcacgtc ctttactcct tcaggctctt ctccctgaag gtcctttgca tagttgggaa 55020tctccaccag gaggggtagc tctttggtct aaacccatgg tggcagagtt tcgacaatat 55080tcccaactta aatgtttctg attctgagtg gtggttagat ccctttgtac acccctgtcc 55140ccagtgccta cagaatgggc atgttaataa gtgttggctg aacattcaat gatggataag 55200gaagaatagg aggcaagaga gacggtggtc tccagtgcca agccccagtg ctaactgggg 55260tgattttttt tcatgactca ttttcctaaa atcaccctca agggtcctac aaaactcttc 55320ccaacagcta aatcacagac taatctggcc catcgacgtc ttccctgatt atactaattt 55380ttttgtgttt ttttttttga gatggagtct tgctctgtca cccaggttgg agtgcaatgg 55440cactatctca gctcacagca acttccacct cctgggttca agcgattctc ctgcctcagc 55500ctcctgagta gctgggactg ccagcatgcg ccaccatgcc cggctaattt tttttttttt 55560ttttttagta gagatgagat ggggtttcac catgttggcc agggtggtct tgaactcctg 55620accgcaagtg atccgcttgc ctcggcctcc caaagtgctg ggattacagg tgtgagccac 55680tgcgcccgac catatattaa tggtttttga tgaatttgtt ccatagatta aaatcttgtg 55740ccccatcgcg tgtggggctc catcgcatgt ggggcacagg gttcctgagt gtttgtggct 55800gtcaaaccaa gatgatttct tgcttaatca agcagatttg aaagttcatc tctgctacca 55860ggaagcactt gctcaactca gaagacaatg tcctatcagt ctttcactat cacgcatctg 55920ttcttcaaga tccgtcaaat tagctccagt gaaacggagg ctaaagtgaa actttttctc 55980ttatatagat ttttattcat aactagggaa aaattaggca cccacagaaa aataataacc 56040taaaaaaatt aggctgaacg taagaaaaat ttgtgatgaa ataaacattt caatcaacag 56100aaaatatttt tctgactttt tatgtgccac cattagttac atcattgaga aaacaatatt 56160tgtattaaaa aaagagctgg tgaaaatctg gcaattggtc gggcatagtg gctcgtgcct 56220gtaatcccag cactttggaa ggccgaggca ggcggatcac ttgaggtcag gagtttgagg 56280ccagcctgac ccacgtggtg caaccccctc tcaactaaaa atacaaaaat tagctgggcg 56340tggtggcagg cgcatgtaat cccagctact agggaggtta aggcaggaga attgcttgaa 56400tctgggagat agaggttgca gtgagccgag actgagccac tgccttccag cctggtgaca 56460gagcaagact tcatctctct ctcttttact ttttttaaag acttcttctc aaaaataaaa 56520agaaagaaag aaaatctggc aatccagtaa aaactggcca ctatggcatg catgtgctat 56580gcataaacgt aaattgatgc ataaacttaa ttttagaact ggaaggaaat ctggagttct 56640ttaggagcca ggttttacac atgcagaaac ctaacagctt cagtttcgat tcgataaaat 56700ttgactaact aaacttaaga taagcatagt tacgcattag agtattaact ctcaaacttt 56760taaaaaagaa ttcttccttt gcttgttaat tttctttctt tctttttttt ttttttgaga 56820tagggtcttg ctgtcgtcca ggccgaagtg cagtgacgtc atcatagttc actgcagcct 56880ctacctcccc ggctcaagta atcctcctgc ctcagccttc tgagtatctg ggactacagg 56940catgagccac catgcccagc cttttctttt cttttctttc ttttctttct ctctctgtct 57000ctctttcttt cttttttcct ccctttcttc tttttttttg atggagtctt gcactatcgc 57060caggctggaa tgcagtggtg cgatcttggc tcactgcaac ctccgcctcc gggttcaagc 57120aattctcctg cctcagcctc ctgagtagct gagactacag gtgtgtgcca ccacgccagc 57180taattttttt atttttctag agacggggtt ttaccatgtt ggccaggatg gtcttgatct 57240cctgacctca ttctccacct gcctaggcct cccgaagtgc tgggattaca ggcatgagcc 57300accgtacctg gccctttctt tctttttatc aagacaacaa catgtcttta tagtgctccc 57360aaggctaaag tataccttac gtctatgtaa acactcaacc tgagctttgc aatggcccat 57420gttggcagta gtgcaaacaa aaacaattat gaaacccatt ttcctttgac aaagagaaat 57480aagtggcaag aattggttct ttctcttagt atgggtctct gaaaagaacc agatcagtca 57540aaaggggaat atttttctga agggataggt ttggcctagt ggcttctacc tcttttagat 57600gactgctgtt tctcgtttta atgttaaata gacactaata ggagaaatca cattaattca 57660gtcaacaaac atttactgag cacttcctgt agtcaggccc tctgttaact tctgggaata 57720caatgacaac tctgacaatc ccaacccaag gagccaacaa gtccgggaat agagacagac 57780aagaaaacag acaattacaa ctctaccgtt agaataaagg tacattgaga acttgcaaca 57840aatattcctc atcccttatc ttaattattc ataacatgtt taccaccaat aagaatagca 57900ataacaataa atgcccaact cagacagcaa tgtccattta ccctgtgttt acacagcata 57960atacaagcaa gctgtggaca gagattctct tgtttagtcc tcacaactct gcaaggtggg 58020ttttattact ctccatttct agataaagga tctcacctaa tattacatgg gccagtggtc 58080ttccagttgg ggtatgcaca accctagggg taggtgagga ccctgcctgg ggtcttcagg 58140tggggaccat caacctccat ttgtactctt ttctgaacat tggtctgaga cagaaagtcc 58200ctgcaattaa ggcattaagc tggctctttt tctatttctc atttcataat tgcccttctc 58260ctgctttacc aaaatctttc accccccatc atatatatat atccccatac atattctata 58320tatacatacc ctacatatgc atgcacacac atcatatata tgtatgcata tatgatatat 58380acatatatgc tatgtaaaca tatatagtgt gtatatacat gtgtgtatgt gtatatgtgt 58440gtatatgtgt atatatacac gtgtgtgtgt acatatatac atcatatatg tgtgtctata 58500tatgtatata tgggtgtgtc tatatatgtg tatatgtacg caaatacgta tatgtgatgt 58560atatatataa gatgtgtgtg tgtatatata tgtgtttgca tgtgtgtgtg tatatatata 58620tagtatatac atattttttg agacagcatc tcactctgtc gcccaggctg aagttcggtg 58680gctgatgaca gctcactgca cctcccggct caagtgattc ttccacctca gcttcctgaa 58740tggctgggac tagaggcgtg tgtcaccaca cccagttagt tattttattt ttcgtagaga 58800tggtggtctc actgtattgc ccaggctggt ctcgaactcc tgggctcaag cgcttttcca 58860cctcgacttc ccaaattgct gggattacag gtgtgagcca ctgcaccggc ccatccttta 58920ttttaatatt atgcagtgcc ctgagacata taaaaaaccc accttcccaa gtaaaggaaa 58980ttcaagctga tgcctgcaga gccttcttta acaaaggctc tgaaataccc tctctcatta 59040aaatgatact ttccaataaa attttgttta acaatgattt acaaaatgat aaaatttatt 59100tattttgatt gtgtatggat catggtaaca ataaaaagac ttgtaaaaat aactaaattg 59160aaagaatctt gaacatttag agccttaaga ctgtaggaat tgaagaccac agaattatta 59220atttatatta atatttttgt tgcagagaca taatgaatga tcaacgaaag gcttttaagc 59280gttaaaaata tattacacta gataaaatta tttgcgggaa tgggatggaa atacattttc 59340aagagagaaa ggagcaatgt aaaatgaaga tgtaaaatcc ttctgctggt tgtccttggg 59400gttttctttt aaagaaaagc ttggcagtgt ttttcttttt ttccattgga tgatggtgaa 59460tatcaaatca ctttggtgct aatatttcat ttaatacatt aattttaaaa ttttctgtag 59520aggtgggatc tcactatgtt gtccaggctg gtttcaaact cctggcctca agcaatctcc 59580ctgcctcagc ctcccacagt gctgggatta caggtgtgag ccactgcatc cggccccatt 59640taatacattt aaaagagtgg tgtaacaatt tttatttaaa atgtcatatt tacaatattc 59700tagaatgtat atcttttcaa ctcattaaac ctaaacatcc ttgtaaaaag tgtgaaaagt 59760tatatagttt ttcaaaattc gattagcagt tacataagca taaatgttta aagtatgtat 59820ggtacagcca ggcttcagtt ccctgtctta aacacaaaga tccatatcaa ttccagatac 59880tgcaatggtt tgctgttttt cctgcttccc ccatctccaa ataaactaaa gcatcaacat 59940gcctcacctc acataaccct aagttttcag cagttggcag ttacacctgg aaaccatttt 60000tctaaaataa acaacaactg tttgcttacg gatcaaaatg caaaggacca taacatttag 60060cctcaccttc ctactacaga tcgagtttaa aagtgccatg gtatagctaa attatgaaga 60120aagatatgaa tataactgca aaagtggaag gagatttggg ataattcttg cccattttgt 60180taggccaaat gcatctttgt gcaaattaga aaaaggtggt cttcatccct tcactcctat 60240ccttttgggg gtggaggggc agtggctaaa gtacagacta ggtttcagct accacatcct 60300ccttcagtta gctgccctcg gcgtgacaga aacatgtgca aacagccctg tgcctttgtc 60360ttatgttcca gccagccaag aaaaatagtt gtaaaagagc agctgctgtt tggggtaatg 60420accttggacc ctccccaatt tgttccaagc ctgtttttgt attcattttt cccacattta 60480tgttcctgga tggaagcttc catatctgct cttggcccta tttgaaattc cccagatttc 60540cttcctggct cctggccttt ggtttttcat gtggctcctg atcccacacg ctccctgaat 60600ttggattctc ctgtcatttc aggtgcgagg tttcccacta cagcctcttg ggcctcacct 60660ccaatacctc tttcccatca gaacagcccg gaccttcccc tatggtagag cagagacaga 60720atttaaatga attctcaaga agtgcttgga ctcatatcta gcaaaattac atggcattta 60780acctttgaca caaaaaatgc agcttctagg aatctatcta aagatacact gtggcaaata 60840tacaaaaaga agcattattt atcaagcact atttcctaat aaaataattc ttaggtcagg 60900cgcaatggct caggcctgta atcccagcac cctgggaggc tgaggaaggc agatggcttg 60960agctctggag ttcaggacca gcgtgggtaa catgacaaaa ccccatctct aacaaaaata 61020caaaaattag ccgggcatgg tggcatgcac ctgtagtccc agctactcga gaggctgagg 61080tgggaggatc gcttgagcct gggaggcaca ggttgcagtg agccaagatc gcaccactgc 61140actccagcct gggtgacaga gtgagaccct gtcaaagaaa gagagagaga gagagaaagg 61200aaagaaagga agaaaggaag aaagagaaag gaaagagaaa gaaagagaaa aagaaagaaa 61260gaaggaaaga aagaaagaaa aaagaaagaa aaagaaagag aaaggagaaa aagaattctt 61320actaataaat gcaggagaaa tgatagaatt gaaatatcac cattttcaat tcctaatgaa 61380ataacgtatc taggcaatga ccatcaatag ctagatgcta aaatcatctg atcaaacact 61440gatgggaact tcgtaacaga tggatcaggc taacaacatc tgaaaccact aactggtttt 61500gatgtcataa aaagaaaaac aaccagatat tttctgtctc ctgatgagtt gcaattggag 61560ctacatatca cctgtaaagt cttctggcca aaaaattaag cccagccgga ccttattaaa 61620cctttaaatc taacaattag ttttgaagct tttacagatt aaatgaagtc tgagatttgc 61680ttcaaaatga accagtggtg gggaggaagt gggtgaggtg taggtgaaac aagattggcc 61740acgtcgataa ttgctggagc tgggcgatga aagcacaggt atttatcaca ccatctctct 61800acttttgtgt gtttttttgt ttgtttttgg ttttggtttt aaggagcaga gagtctaata 61860ggcaagaaag aaaagagaag gctgaaggaa gacgctcccc cgtacagaga cagagggagg 61920gggctccaaa gccgaaagag gaggtcctct tgtgtatgtt ttaaaatact cccagataaa 61980atatttttgg aagagtactt ggttggattc aacagctttt ttttaattta aaaaaatcac 62040ctcaattttt ttgcttgctc taacgtgcca tagaaattcc tgaggtttta cttgttgctt 62100tacaatgaac tgtgtaaaca caagctggaa gagatcagct atgcgctgga agggttggtt 62160aaatattgag actgccttgc tgagggaagc cttttaatga atctcagtaa ttttgcaaga 62220gaaaagataa caatgaacac tacattaaac atcattcttt tgcactttgc taaatatgtg 62280tatgtaaatt actgtctgac tgttactgga tatatacagc atatacatat gcactttttt 62340tactgttttt tttttttttt tttttttttt tttttttaca gagcttgctc tgtcacctag 62400gctggagggc agtggcgcag tctcagctca ctgcaatctc cgcctcccag gttcaagcga 62460ttctcctgtc tcagcctcca aagtagctgg gactacaggc gcctgccacc gctcctggct 62520aatttttgta tttttagtag agacagggtt tcactatgtt gtccaggctg gtctcgaact 62580cctgacctcg tgatccatct gcctcggcct cccaaagtgc tgggattaca ggcataagcc 62640accgcgcccg gcccatgtat actatttata catttttagt atcattttgt ctttacattt 62700tacataattt cagatacatt ttcctcatat caaataattc agcatttttt agtactaaca 62760tcatagtctg taagccattc aaaaaatgta tttcacaaaa taggctatct catcctttga 62820gctattgaga tgaattaatt tatactcctc ctaagatccc tctcgtcact aagattcttt 62880tattttatga caaaaccata gttctagaag cttgtttctc ccacctgaaa agactggatt 62940tgggacatga tcctgtagaa cttcggaggt aagcctggtg aatcagatca tagggggtct 63000ggagggtgaa aaaaaagggt ttggtgctca tggatggggc tagtattggg gtgtagggga 63060gattaggtca aagcaagagg attcaaagga gaaatgaatt cctttagatt ggggaagata 63120atcggaagag gtaaaagaca ccgtccatga cacttcctgg ggaagcagat gtatgtataa 63180ggatgtgagt attgtggttt tgtaaagaat gcattcctga agatgttgca taatttaaaa 63240cctacatatt ttgattaatt ttctcatgag aatagcaggg tatgtgttct cggcgctcac 63300aaatgtataa tccattgtgg caaatttttg ctttcacata ttttttttta tcattattgt 63360cacaggttct gtgacggagt tctggtttct aaattcacag cataccaagg cagttcttta 63420aagttcttga tactctttta tcatatctaa cttgtattcc aaaattattg agttggagca 63480cattttccca gcacttagca ccgctatttc atggatggtt ggagaggggg tccaaaaatt 63540ttacaattat gttaacaaaa gtaacacagc aacaaaacaa taagcaaaat cactgccaga 63600gtattcctta gcttgaaaca atacggttca catcgataga atatggcatc tatttctgtt 63660taatcagtta accctgctaa gtagcaagag cttacaattc atgtctaaaa tcatgatttt 63720tttactagtt ttttaaaaaa tgtgggctct atatatataa tttaacattt tgcttgtaag 63780acttaatttt gcctgggtat ggtggctcat gcctgtaatc ctagcacttc aggaggttga 63840ggcaggagga ctgcttgaac ccaggagttc aagaccagcc tgggcaacac agtgggaccc 63900catcactacc aaaaaaaaaa aaaattagtc aggcatggtg gtgtgcactt gtagtcccag 63960ctacttggga ggctgaggtg ggaggatcac ttgagcccag gaggtcactg ctgcagtgag 64020ccattattgt accacaacac tccagcctgg gtaatagagt gagactctgt ctcaaaaaaa 64080aaaaaagact atttctaaat gtgtggctat attataccat ataaatgtgg cttcttgggc 64140aaggaaagag gacaatatag atgaaaaaga aattgatcct accagaagtg atccttttat 64200ctgcataact ctcaggcagt tgtggcaaat aattggcaat atctattgtt ctgaaactgg 64260ttttcgcaac ttttattggg aacaccatcc cctctcctgc atgatcagtt tctcctctcc 64320acggatcatt cacatgagta aagtcagtag cgtgctggta aatgtttaag atcttgttct 64380ttgggggaaa aagttcctaa gttctagcag ttgccctgga taacttcaag gtatcaacat 64440ggaagttatg tacaaaaatg gctgtcacaa gccagtatga gctaacacca acatactacc 64500cagtgttctt caaacttcag ctcacagccc attagtgggg cttgcaaaca ttttagtgga 64560ctataagcag cattttttta aaatgaaaaa gtagattgtt ttacacataa caggagtatt 64620gttttgtaca attttttttt tttttttttt tttttagaca cagtttcact ctgttgccca 64680ggctggagta cagtggtaca atctcagctc actgcaacct cggccttctg ggttcaagcg 64740attctcatgc ctcagcctcc cgagtagctg aaattacagg catgcgccac aaggcccagc 64800taattattgt atttttagta gaaacagggt ttcaccatat tggccaggct ggtctcaaac 64860tcctgacctc aagtgatcca cctgcctcgg cttcccaaag tgctgggatt acaggagtga 64920gccaacgtgc ctggtcaaaa tttttgtttt cgtaatttta agtatgtgtg tagtaagtct 64980caatggaaat gtaattctta tggcaggtca cttgaaaaaa aagaagtcta aaagtcacca 65040atgtagtatc ttctctttaa aaaaaaaaaa aaacaacagg agaaaacctg aatctgccct 65100ttgctccact ccttcctcag ctataatgct gcttctccat tcctcctcac agcaaacctt 65160tctgaaatct ttatagtcat ggtttccacc agttcttcac ctcccatttc tcaacacact 65220tcagagtcag agtcagcaat gacatccatg ttcctaagcc cattgcttac ttccgtcctc 65280cttggcctct cagcacactt ggcacacagg ctgtttctct ttctttggca tctgtgacac 65340cactctcagc aaattcccct ggttgtccct tctcagtctc atttattggc gttgtcttat 65400ctccccaggg ctgtccgagg tgattttctc ccactactct cctaggtggt gccatccaat 65460ctcatgatgt catatcattc ttccctcatg cttcagccat actggttggt ggcctttgtt 65520tcctgaacac atttaatgca ttctcaagac cctcagggac tttgcagcag ctgctcgcta 65580aggctggaat gctcttcccc accatcttca tatggctgtt tcctttcttt cactcaccag 65640cagcttaaac tttgactcct ctgagagact ttccttgtca cccaactaag gttgccactc 65700aggtgctccc aatttaatct tctctaaaac acatcactgt atgtgtcctc aactagagta 65760taagcttcct cgaaacaaga acaatcaaaa ctccttgccc tcatggagtt tatagtctta 65820tgatgggtga agtaacataa aataaaaagg caccttatat agtatattag catgacaaat 65880gttagccaga aataaagcaa ggaagagttg ctagggagtg tgtatgagtg tgttttggga 65940gagtgtttgc aattttaaat attggtggtc aggaagggcc ccactgagaa ctgacatttg 66000agtagacttg aaaagggaaa aggaaatatt gagtaaagat tttaggatgg gagtgtgaca 66060ggcctgctag gagaatagca aagtcgctgt ggctgctgca gaaaaagtga gaaggaaagt 66120agtaggagat gaaatcacag tgtgtgagga ttcgggcaga tcaggaagtg ctcgtgtaag 66180aactggatct ttactcaaag aatgagcaaa aattagtaga cggttggccg gatgcagtgg 66240ctcacacctg caatcccagg ttacaggagg ccgaggcggg cggatcactt gaggtcagga 66300gttcaagact agcctgggca acatggtgaa acctcatctc tactaaaaat acaaaactta 66360gctgggcatg gtggcgcgca cctgtagtcc cagctactca ggaggctgag gcacatgaat 66420cacttgaaca cggtaggcag aggctgcagt gagctgagaa tgtaacactg tactccactc 66480caacctgggc aacagagtga gactgcctca aaaaaaaaaa aaaagtaggt tttagtaagg 66540gattaacatg atctgaatta tgttttgtca tgacttctct ggctgttgtg ttgagactac 66600attgcagagg ggcaagggca aatataggga gaccgattag gatactgcag taataatgta 66660agagatgtgg gactctatct agaagggccc atgaggtcct ttgcatgcta gtattcttta 66720ctgctgtgcc tggccatgat aggcattcag tgaatatttg cttatttaaa ataacacact 66780gggctaattg aacaacagtg ccaaatgagg gagatatttc taggaataag ttcttaggat 66840ttatgaacat tttaatccag attttctttg ttaactctgc tctctggccc tttcactcag 66900ccccgtttgc acctaaatat gacttacaaa agaaacacag catttatgtg tacttatttc 66960aacttacttt agctttgtaa agaagtacaa ggttgactca gggcccagct tggtgtctca 67020tgcctgtaat ttcagcattg tgggaggcca aggcaggaag atattgtgag cccaggagtt 67080tgagaccagc ccgggcaaca cagtggaccc tgtctctaca aaaaaaaatt tttaaattag 67140ctgggcatag tggtgtgcgt ctatagtccc agctactcct ggggctgagg tgggaggatc 67200acttgaggga aaccctgtct caaagtggcg gggctggggg gagactcagg cagaattgtg 67260aagatattca attgctcctg actttatcaa taatctaaca tttcaaccta acattgatat 67320ctattttatg caaagcatta cactatgcac tggagactgt ataagacaag ttccttttct 67380caaactacag tcgagttgga tagataaaac acacaacaca taccaaaaga cagctataaa 67440tccaaggcag tgtatgtcaa gggtaaattc acctattcag attggatctt gagaagtgca 67500tcaggcttgg aaaatgggta aggaggagag aaaagcaaca gtgaatcaga acatgagttc 67560ccagttatgg gacttgtaat gaattcctca attaaaacaa aaaataatga aaacaaaagc 67620cagggaggag aaagcccacg ttaatgacac taaaatatat ctttccaaac aaatgtggat 67680aaaagccaag tagagaagat gagaactttg aggtccctaa cacaaaataa acagtaagca 67740gccagccatt ccaagtggct gacatgactt tgtttaactt tatttgtatt tctggctggt 67800gtgtttacag ccaataggtc aaactatcag tcagtgtagg gccctgagaa gtcgggtatt 67860taagagcatc taataggcac agaattgtgc tccatactgc ttaaactgtt ccctaagtgt 67920ccaatttgga gaaaacaccc acacgcagga taaccggcga gtgacgcgga gtggctgcga 67980gtccaagtta tcactaacgg atggggagct tgggctgggc acagtccagc gtactgaacc 68040cttcccccac cgtttcacct gcatacagag gtgtgtactg tcaaaaagca gcgcctccaa 68100gtctcttctg gcactgtctg gacttggatc cgaggcagac gaggaagctg agaaaaccct 68160ggcgttgacc ccgtggacct gggcgccccg ggaaggccag cgcttggtcc aggcaggcgg 68220ggcctgtgcg gtgaccaccc tggtcctgaa aagtcccagc cccgagcgcc ctccctccta 68280gacctggagg cctggaacag ccaggtggac gtcggcccac ctttcttttc tttccttccc 68340attttcctac cacctcccac cccactccgc cttccgggca aaggcagcca gatccaccca 68400ggacacattc tttgtcctta tccctctgtg ctcgtcccac agcaagccag tcgcggtcca 68460aggctccaga ggctgtgcag gaggccgagc tgggtggcga tcagcggcgg gtccctgtcc 68520aaaatccagc agagccgcca gggacgcccc agacacagaa ggcggggcgc ggggagggtg 68580gggagaccac agcagtgagg cgcgcgagcc gggaagtgaa cgaggactga ctcctgtcgc 68640ttcccgtagc cgcccacgga cgccagagcc gggaaccctg acggcactta gctgctgaca 68700aacaacctgc tccgtggagc gcctgaaaca ccagtctttg ggtgagtcgc gcgacccccg 68760gcctcgggtg gcggggcagt cgctagaggc gtggctgctc tgagggtctc gccagtggag 68820gatggcattc ggatgtcacg gctcctaaat caccatttga tgggtgggac agtgtccagt 68880ccaccccgac ccgccggtcc tcaccgcggc agagccgggg ctgggtggcg gggacgctgc 68940ctctgcaggc gaggcgctcc ggggcataag ggattatcag gagtcgcggc ctttcttgga 69000catccctggc tggggtcagg ctgtttgccc tggggtgtct cctcgctgca aacccacccc 69060acctgggctg ctttctcacc tgttccctcc tagcctgagg ccgagcgcca cctccaagtg 69120gaggaatctg gggaagtttc cttcccggaa tttgtagtga cagtggagtg acctccattg 69180cgttccctgc ctctaacacg ctctttagga tgccgagtca tttgactgca gtgttaaaca 69240ttgcaaagcg caagtcatgt gacttccttt gaccgtacgt gaaacttaag tgatggctgc 69300ttgtgatgca tacgaagtgt tcatgctggc gggacctgtc cctggggata cttcgggggt 69360tgcgtgattt aatgcaagca gatggcttaa attgggtcac tggcttgtta ttatacatgt 69420gtatggcaac tcggcatcca ttctttttgc tcttgttctt acttcctgaa ttgagtcacg 69480gagccagagt tttgaggttt tgactaacga attaagttaa tgacatgggg ctatatttag 69540gtggtaaacc aagagggata cagttttttt tcttaataaa gaaaaagtga tagatttgat 69600cggtgtgtat tgttggtgtg cagtataatg acagaattgc tggaagtaaa atacaggaag 69660ctctggtttc atttcccctt tagttctgct taaagtcgag tttttcctgg agctattaaa 69720tgtagtgtag tgtccatgag tgcttttatc ttaaaaaatg tggctgatgc tttccaacac 69780tcccctgccc tgtgattatt atttttttaa gcaacagaga aaactgtatc ttaatagtat 69840taaaagtatt ggatttttcc ctactttgat ttgtttaaat tggaggagga agagcaattc 69900tttctattca caataataat agctaacata gcgcttactc tttcgctgtt ttattaactc 69960aatcctcaga acaaaccaat gatgtgaata ctgtaattct cattttatgg aaatgaaaat 70020ttaaatgaat acctctgata attgtacggg actgtttgat tagtatttac cattaattaa 70080ttaaattttt tttttttttg agatggagtc tcgttctgtt gcccaggctg gagtgcagtg 70140gcacaatctc ggctcacttc aacctccgtc tcccaggttc aagcaattgt cctgtctcag 70200cctcctgagt agctgggact acaggtgcat gccactacgt ctggctaatt tttgtatttt 70260tagtagggat gggatttcac catattggtc aggctggtct ccaactcctg aacctcaggt 70320gatccacccg ccttggcctc ccaaagtgct gggattacag gtgtgagcca ccatgcctgg 70380cctattttag tatttttaat aataaattcc atgttagaaa ttttctactg atgtattttt 70440taagtcaata tttcctacac tcacaatcca aaattattta gtatatgagc acactggtaa 70500gaatgggagg cagatcgttg attgtaataa tattctatta tttggtaaat atcagtaaca 70560taatatataa tttaaatttt aaaataggat atgaagaaaa atgctacatg cttacttttc 70620ttttcctcta tttttacttt acacagggcc agtgcctcag tttcaatcca ggtaaccttt 70680aaatgaaact tgcctaaaat cttaggtcat acacagaaga gactccaatc gacaagaagc 70740tggaaaagaa tgatgttgtc cttaaacaac ctacagaata tcatctataa cccggtaact 70800gatttctata agataacttt ttacctatgc caggacagat ccaatagaat attaattatc 70860cattgggaga cagggcaaga ataaaagcca gtgaacatat ttaaagcacc tactatgtaa 70920tagagatggt ggtgggtgct gattacgaaa cagctcttgt cctctagtgg aggaagaagt 70980cacaatgata atatgacgtg atgaaacagt gttatgaaca gggaacgtct gggtagagtg 71040gagggaatgc caacttttgg tgatgggagg aggctcagct aatcataaat tgtagttttt 71100aaaggaaaat ggatttctta ctctacaagt ttttcatttt cttttttaat tagagctgtc 71160catgagaagt taatgtctcg atctttccct cagcctttca aatactgctt ggcccttgag 71220cagggaaaat gtcaaaagcc aatggggaga tggagagtgt gaagtagtaa gggtctcgtg 71280cagttcaggc aggtcctaga atccctgaat gactgtaatt gctggaaatt gccctgtaat 71340cctgagcagt aaagagcttg ttttagtttt atgtggtggt gagaatcttt aggaatgtct 71400agtttccacg tatctgaagc tgaatcctga atcgaggtct gaaaaaggac agccactttt 71460ttagtaaacc gcctagaaga ttcttgggca aaaggaaggg tgagaatcct taaaatgagg 71520ccctaaacca gttttgttag tgtgtgtggg ttcaagtttt tgtcatttac tttatagctg 71580tatttccttt ttccctaagt tttaatgtca ttgtgtaaga atgaggtatc gctgctgtat 71640caagcaaagt cagttttagg agaaatagcc tttcagtggt agtaagttta aaaaagatga 71700cttcctgaag cggaagcttg tgagacattt aagatgactt tgcgcatgtt agagttaaaa 71760acatcccaag gttgtaaact gatttcctgc aaagatctta acaacaacaa caacaacaac 71820aaactaggct gcctgccacg ggtgtctgaa gtatcatctt ggctcaagct gggagaatgg 71880ataaaggtta cactgttcat ttctgccctt cacacagaaa agaagataat tttataggta 71940aaattcgtgc atatcttgat tctagcatac tgctgattcc tgtagtttct ggggtcagta 72000ctctcaacta ttgaggtgga acaaaaataa gtagacttca tttcttgagg aaggggatct 72060ggagaagtag ttctgcgcta gagcagaaaa tgccttcagt cttgtggcat gggctggatg 72120ctgttctgag gataatgcat ttccaaggga gatatttttg gcaaatagct tttttttctt 72180tcttttcaaa attctctgtt ttattatcag ttctcacaaa agagtcggaa aggttagagg 72240tagactgaac tgaatggcaa aaacattttg cgctctcttt acgtttcact gctgtaaaat 72300atttatagta taaagggcct gtattgcact gaatttctct catttgtagc tagttgccct 72360ttcaatgttc caaaaaaaag gctgtaaata acttatttta tttattcaat taattttttt 72420ttttaaattt tttgagatat agtttccctc tggtcaccca ggctggagtg aaatgatgca 72480atctcggctt actgcaattt ctgcctcccg ggttcaagca attctagtgc ctcagcctcc 72540tgagtagctg ggactgcagg cacgtgctac catgcccggc taatttttgt gtttttagta 72600gatatggggt ttcacagtgt tggccagcct agtctcaaac tcctgacctc aggtgatgtg 72660cccaccttga cctccaaaag tgctgggatt acaggcgtga ggcaccatgc ctggccaact 72720tagttattta aagataatca attagtatat tttataagct agacttagga aaactgtttt 72780cagctgggca tggtggctca cacctgtaat cccagcactt tgggaggccg aggcaggtgg 72840atcacgaggt caggagttca agaccagcct ggccaagatg gcgaaactcc gtctctactt 72900aaaaatacaa aacttagcca ggcgtgatgg cagcctcctg taatcccagc tactcgggag 72960gctgaggcaa gagaatcact tgaacctggg aggcggaggt tgcagtgagc cgagatggtg 73020ccactgcact ccagcctggg tgacagagcg agactccatc tcaaaaaaaa aaaaaccccc 73080cccacacaca aaacctgttt tcttgaatca tggttgtttt gttactgata ggttcaataa 73140gtaaatatat ttattgtctg ttgtattctt tattaggcat tataaacaca ccgccacttt 73200ttaattttta tttcattaat gtttccaatt tttttttttt tttttttttt tttaagacag 73260aggctcgctc tgtcatccag gctggagtgg agtggtgcag tcttacccca ctgcaacctc 73320cacctcctgg gctcagcctt gtaaatagct gggactacag gcatgcacca ccatgcctgg 73380ctaatttttg tatttttttt ggtaaagaca gagttttgcc atgtttctca gtctggtcaa 73440gcactcctcc cgcctcggcc tcccaaagtg ttgggattac aggcatgagc caccatgcct 73500ggcctatttc taatattttg gtccacattg gtgttagacc aactgtccac attaagtttt 73560cttggaaaag atgaagtaaa tattgcaact ggcctatgta tttttttccc tatttagtat 73620atttctttga ctagttcaac tgatagaatt ccaagactta aaaaagtcag gctctaaggc 73680tgggtccaga ggctcatgcc tgtaattcca gcactgtggg aggccaaggc tagtggatca 73740cttgagccca ggagttcaag accagcttga gcaacatagt gagaccttgt ctctctataa 73800aaatacaaaa attaactggg gattgtggcg catgtctgta gtcccagcta tgaggaagag 73860tgaggtggga ggattgcttg agcccaggag gttgaggctg cagtgagctg tgagtttgac 73920actgtgcttc attctgggtg acagagcaag aaccatgttc aaaaataaaa ataaaaagtc 73980agagtccggg tgctgcggct catgcctgta atcccagcac tttgggaagc cgaggcgaga 74040ggatcacttg aggtcaggag ttcgagacca gcctgactaa cacagtgaaa ccccgtctct 74100actaaaaata caaaaattag ccgggcatgg tggcggtggc ctgtaatccc agctacatgg 74160gaggttgagg caggagaatc acttgaaccc gggaggtgga ggttgtaatg agccaagatt 74220gcacaactgc actgcatcct gggcgacaga gtgatacttc atctcaaaaa aaaaaaaaga 74280aaaaaaaagt taggcttcct tttctgtttt ttttttcttt tttcttctct tttttttttt 74340ttttttaaga gatggaggct tgctctattg cccatgctgg agtgcagtgg tgcaatctcg 74400gctcactgcc acctttgcct cctgggttct agcaattctc ctgcctcagc ctcccgagta 74460gctgggacta caggcgcaca ccgccacgcc ccgctaattg ttcttttgta ttttagtaga 74520gacggggttt caccatgttg gccagcctgg tctcgaactc ctgagctcag gcaatccgcc 74580cgcctcggcc tcccaaagtg ctaggattat aggcgtgaac caccgtggct ggccacttac 74640ttttctttct attgaatttg aatgaataat ttggaagaca gtatctttac ttcataccag 74700gaatgctgcc agtgaaattt cttgtttggc agttcattat ctacctatat atttaatttt 74760gctattgttt atagagttct taagatatga ttaaatgcta gctggttaag aaatcattta 74820gaaatgaaac agaattggtt gttactccaa gttaataagt tgcttgtcaa cataaatcct 74880acctggtacc cagttttctt aggaaccttg cttccatgtt tatccttttc tgcttagtat 74940tctaagtact ccttttttac cttacaattt agtcttaaaa cacaacacag tcaagtcttt 75000cttttgtaac ctgtgaggta ccttctagcc tttgtgctgt ttttcttctt tttttgctgc 75060ctgccttcct gactgagagt ggatttcctc actaaggctc tgccctctga tttttcactc 75120tcttttcttt tttggtttta ctagtgaaat tttgtcttta atgtctcttt cttttatgtc 75180tttaccgatc actcataaat ttttttttcc atatgtatcc agttccaacc tttcacctaa 75240tgtgaacccc caactctcag ttgctcagcc agcccttcaa gactaggagt tcaaaaccaa 75300acttgcatct tccttcccaa accagctttc ctcttgcagt tttctgcagc aggatccttc 75360tgctgtttaa cttttgcctc ctcccttgtt tcctagcacc caatagttgg aagatagtct 75420gtcttcaaaa ttttaaacta catttatgtc caaaccagtg gcttttcctt ttaaaaaaat 75480ttaaagataa tatgtgcaaa tcattttttt aaaattcaaa cagtatttaa gagtttcagt 75540gaaacatgca ttttccttct accctggtac ttagttttac tccccaaggg caatcacttt 75600ttactggttt ttagaaatat atccttcctg agatacttat gaatatccaa aagtgtgtgt 75660gttgtgtata tcacctttta tatatcctgt ctctttacgt gcatgcattt taccgtataa 75720actgttttct accctgcttt ttctatttga cctattttgg aaatgtcatt ttatttagaa 75780cttcctcatt tattttaaca gctgcataat tagcagtaaa acttatgtaa gcagtccctt 75840gtgaagggct gtgtcttttt gcgattatat ccggtgctat agtgtacatc cttgtgtgtg 75900catcttggtg tgcctgtgct acgtatttct gtaggataaa tctgtaaaag tggaatcact 75960aggtcagagg gtatggtcca ttttctttac ttatttattt tatttattta ttcatttatt 76020tttgagacag agtcttgctc tgtcgcccag gctggagtgc agtggcatga tcttggctta 76080ctgcaagctc cgcctcccgg gttcacacca ttctcctgcc tcagcctccg gagtagctgg 76140gactacaggc gcccgccacc cacgcctggc taattttttt gtatttttag tagagacggg 76200gtttcaccat gttagccagg atggtctcga tctcctgacc tcgtgatcca cccatctcag 76260cctcccaaag tgctgggatt acaggtgtga gccaccgcgc ccggccccat tttattatct 76320ttatttgctt ggatccttct tagcttcttc aatgttaaag atattgacag ttttcctctt 76380actgaaattt ataaatccat tgactccctt gatattattg ccctggcctg actgattctt 76440ctctctcctt tctcttctca ccccatgttg aggtccccaa ggtcacaccc agttttgatg 76500actcaccagc atagagttgt acttgtgcct atgatttatt gcggtgaaag gatatagagc 76560aaaattgcaa acggaaaggc acctggggtg aattccaggg gaaatccagt gcaagttcca 76620aggtcgcctc ccagtggagt cacataggat gtgcttacat cctccagcaa ggagttgtga 76680caacacttgt gaaatgtgga ctgccaggga agctcatcag agcctcagtg cctagggttt 76740ttactggagg ctggtcacat aagcaccctc acacatatca aaaaattctg gtcccccaga 76800aggaaagcag gtgtttagca taaccatatt atttgcatga acagttcagg tacaggaaat 76860ccccgttacc agttaggttg gtgggtgccc ttctcaaatc ccaagttccc agacaccagc 76920caggggcctg cctcgtaagg aggcctttcc aggacagcag tcaggcctgc caatgttaat 76980tcttttctgc atacctccta attttagaaa ccaccgagcc tttgctgcct gacctgtcct 77040gcttttcgat ttctttatct actttgatat ctttacaaat gatctttacc ctgactttta 77100aatgtgtgct ctggccattc acctagcgtg tggttctgag tctccaagtc ttagcagatt 77160tgctctcaga tgctctgcca acgcttcaca ccaagtatta caaactaaac tcgtcatctt 77220cctcctgaaa cctgtctccc aggccaggcg cggtggctca cacttgtaat cccagtactt 77280tgggaggccg aggtgggtgg atcacctgag gtcaggagtt cgagaccagc ctggccaact 77340tggtaaaacc ccatctctac gaaaaataca aaaaaattag ccaggcgtgg tggcaggcac 77400ctgtaatccc agctattcag gaggctgagg caggagaatc gcttgaaccc gggaggcgga 77460gattgcagta agccgagatc acgccattgc actccagcct gggcaacaaa agtgaaactc 77520catctcaaga aaaacaaaaa acaaaaaaca aaaaacctgt tttctcccca gctttgtcat 77580gtatttagtg gccttatgta gacagtttcc tttgaaacat ctcttggact tctctgctct 77640tccagggcca ttgccaccga cctggaatgt gtccttatcg tttcacgcca ggcttatggc 77700agcagtcagt cacccagatg acctcctgac ctctggctta tttcaccccc actggactgt 77760tgttcctaaa cacttctttc gtatgtcact ctaaaatctg accctggctg tacctttctt 77820taactactcc ctgactgcgt gctgagagaa gatgggtctt gtcttttcct gcctctctgc 77880ttttgtaaac tgccatttct acctgaagtg gcaactgaaa tcatatcttc ttcataaact 77940gtctttggct acctcagtta gaattcctta tcccattttc ctgaagcatt tctttgactc 78000ttctttactg ctcccccacc cttttttttt tctttgagac tgaattttgc ttgttgccca 78060ggctggagtg caatggcccg atctcggctc attgcaacct ccgcctcctg ggttcaagtg 78120attctcctgc ctcagcctcc tgagtagctg ggattacagt catgtgccac catgcccggc 78180taattttgta tttttagtag agatggggtt tctccacgtt ggtcaggctg gtcttcaact 78240cccaacctca agtgatctgc ccaccttcgc ctcccaaaat gctgggatta caggtgttag 78300ccactgcgcc tgacccccat tttttttttt tttaaagatg ttgaattggt cagggtttgt 78360agttacaagc aacagaagcc aactctttaa gcagaaaagg aatttgctaa atgatagtgc 78420agagttctca gaatctctag caggatgaag aaccaggctt ggagaatagg tagccacaga 78480tacacaagca tactgtagga cggttcccat gaagaggcat ctgttgtcac cactggacac 78540agatggtact gtgtctctgc tactctacca atgccactgc tgtctctgac cccagatgta 78600gctccctctg accctggatg cagctccctc tgaccctgga tgcagctccc tctgaccctc 78660gatatagctg cccctgaccc cggatgtagc tgcctttgac cccagatgta gctttctcca 78720aacccagata tagcggctgc ccccttgcca gagtgaatac tgcgtcattg tggcttcttc 78780ttgtcactgg ttcttactta aaagctgagc tggaagttct aatgggcagt tttgtcacct 78840gctcttacct tgttgcagtc tagatgaggt ctaatgttca taagctaggg gattttcaga 78900tatggaaagg gataccaatt ttcagcagcc aaatagagta tcacattttc actccatgtt 78960tcctgggtgt ctgttatgtt tcctgggtgt ctgactctta ggcttctttc aagctgcagt 79020ctgcctaata gagagccttg catttaatca tcaaaaaggc aaagcaatat gaatcagcaa 79080gggtgttttg gcaaataaca gcaaacctga ctgtggcgta agcttgtggt attgtctcca 79140gtgtgatcag atctgtattt taatttttta aatgtaaatt aataatgatc tgtgaatcac 79200caaagtagct tggagtagcc tagaaaacaa tgtatgtcct ccgttttcac agaagccaca 79260tagtcgtggg ttaaatgagt cagcggcagg gcactgtgtc tcatagttaa aaaaaaaaaa 79320aagtattact gaagtaatgc aggatctttt ctgaagtaga aggcatgatg aacccagaaa 79380actaaagcag caagtggcca ccgttcttag catagttgtt tctcaaactg gaacaaccta 79440taaacagttg tgaacaaggt attagaagtg atgggggccg ggtgcggtag cttctcccaa 79500agctcattac ctcccaaagc aaccccagta ctttgggagg atcactttga gcccaggagt 79560tccagaccag cccggccaac atggcaaaac cccatctctc taaaactaca aaaaattagc 79620tgggcatggt ggcacatgcc tgtagtccca gctacttgga tggctgaggc aggagaatcg 79680cttgaaccgg gaggcagagg ttgcagtgag ctgagatcac gccactgcac tctagcctgg 79740gggacagagt gagtctctgt ctcaaaaaaa aaaaaaaaaa aaaaaagtga tgggaataga 79800ttgttttgtc tcaaaaagct ctttccaaca ctaaaatgaa acatataatt aaaaatattt 79860ttctggctat aaaaatatcg atgcttatta tagacatctg caaagtatga aaatatatga 79920agaaaaaaat taaaatgcca tcatccccca tgaaaactat tgttatcatt tttgtctgat 79980ttctttagtg tttctctttt tcttttttta atttttaatt tttttgagta tgtagtatgt 80040atatctattt atggggtata tggcatattt tgatacagga tacagtgtgt attagcaagg 80100ttttcttttt aatgtttata tttatttagt tgagatcata ctatatatgg ctctatagat 80160tactttctct tatattacta acatttgtgt tattaaatat tctgcataaa gataatttta 80220agatgaaatt tgatgttata aaaacttctc attttattaa gagattaacg ctatgaaacc 80280tgctgctata tattcttgga accagctgtg acccaaaaga tcaatgtagg gatgtaggtc 80340cttccccatt ctctacacac aaaatcagat actctgatgt gcagctgtag ccccagtcta 80400cactgtctgt tgtatttttt gttttctggt gtcacgtgcc tcccaccctg ctcctagcaa 80460ttgccatgac aacaaataga taattggctt ccgtaatttc tcatcttatt gcctaaggca 80520acagagagct tgtgggctca gcttgcggtt cagcagctgc tttgttgcct ctcctctgta 80580tgtgtgaggc ctgccagagc ccactttcca gacaggtgag agttcattca ttcaccatgc 80640agttaccgat cgtctcttga cctgtgtcct ggggaggtaa aggtgacgag ccagttctgc 80700cccatgcagc tcacagtcta ggcaaagcta catgcaaaca aacagaatcc aaagtgctat 80760catgaaccct ctgagagggg ctgactcagc agcccaggga gcttgaagaa ggctccacag 80820aggaggctgt gcctcaaggc gatttcggtt taggagccac caatttataa ccacttttct 80880gtggcccgtc ttattttatt tcttatttct tgacaatcag aagtaccttg ggtaggtttt 80940accatgcaca tcgtaatttg agtgagctta gtgtgaggct taacggtgtg tgggctgtac 81000atcctggtca gatgctccag atggaggcag atggttgtga tgcaggagag gcagccacat 81060agcacaggtc cccagccagt ggactgggaa gacagtgtag tcatctctgg ggaaggggaa 81120tgacaagatc tggcagtgtg gcaggtccca gaaaaaaagg gctgggttct gggcagtgag 81180ggtgcaggtt gagacctgaa tactgggtgg agccagctgt cagagtccac gcctgcagac 81240tggactggtc cacggcaggt ggatgccatg tcttgaagac ccacaggcac ccactcatcc 81300tcatgatcat gcagttctct ggtttctaac agtgcagtct gggttgcagt ctgggagtcc 81360agcagagaag agcaggccct ggaatcccag gtgtgggggc gtggcttaac gtggagtttc 81420cttcagaggc agtgagtgct tgtcattgtc tccgtcagca ttggctttgg gcctagtgtg 81480gcctcgaacc ttctgttggg atcagcagtg gaacagtagg aaaaggaatg agtagacatg 81540gcattgcaac aagtcttttt ttttttttct gttagaatta tcatattaag cagaagtttt 81600gcttcacaaa ctctcagcca aatacaaaat actatgaata gtatttacct tgtgtctctt 81660tccaaagaac tcatagtggt ttgcagctat tgcagatatc ctggccatgc ggtatgcggt 81720tccttttttt tgtttttttt ttttttttga gacggagcct tgctctgtcg cccaggctgg 81780agcatagtgg cgcgatctcg gctcactgca agctccgcct cccgggttcg tgccattctc 81840ctgcctcagc ctcccgagta gctgggacta caggcgcccg ccaccacacc cggctaattt 81900ttgtattttt agtagagacg gggtttcacc gtgttagcca ggatggtctc gatctcctga 81960cctcgtgatc ctcctgcctc ggcctcccaa agtgctggga ttacaggcgt gagccaccgc 82020gcccggccgc agttcctttt tatagctgtt tgaataggaa agatgacttg gaaaatgctg 82080gattctgaga tttatgtgca gccttaaaaa gtgtagtttt tctctatcaa taatgagtgt 82140gggttgtaat tgcttagtaa gtaattttgt ttatgtaaac gtacatttgt taaatttttt 82200ttcttaggta atcccgtatg ttggcaccat tcccgatcag ctggatcctg gaactttgat 82260tgtgatatgt gggcatgttc ctagtgacgc agacaggtaa aatcactgtg ctaaaggaag 82320gagcatgaat aggctgtctt tttgtgattg tggaatgata acagagtaag gcgggagaga 82380ccatttgata ctctgaggcc caattagctt tcatcagcag ccctggccaa ggtgctgagg 82440agattggaat gaatgactaa ataaaggtta ttgggattta tttcattgct gtaagtctga 82500tttcagtata aaaaaattag aactatcagc tggatgtggt gacttacaca tacttttcca 82560gcactttggg aggccaaggc gggaggattg tttgaggcca ggagttcgag accagcctgg 82620gcaacatagt gagacccccc ccatctgtta aaaaaaaaaa aaaaattaaa aattaactgg 82680gcttggtggt gtgcgcctgt agttgtagct actcaggggg ctgaggtagg aggatccctt 82740gagcccagga gtttgaggtt gcagtgagct gtgatggagc cactgcacta tagcctgggt 82800gacaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaagaaaga aaaagaatta ggtatgtcat 82860taaagaaagg aattgtggtc agatgacagg gagagtctag ttttagtctg acattcccac 82920agcatcacag atctagttca gatggtttta ctgaatactt gctttggata caagctgtgg 82980tatcattagt gttgggctca gctctgtgta cctaacacct gaagagcagt ggtttaagat 83040gtgaaaatta agtctcaagg agacagccca ggccttttca gttaactcct tcaagtcgtt 83100agagaagtag actccttcca gcttaccact ctgctatctt gagggtgagg tgaggtcccc 83160tttcccatta tccttggcag ctagatttcc agccctcact tctgtgcttt gggtagctgg 83220atgggtgcat gtggtgtttg cggggaaaca gagctggaca aaaggcaagt gcttgctgac 83280ttttaaggca gtttctagta gccttccctg agcacttcac ttccatctta tcagcagagc 83340tttagctgca caggcaggcc tagctgcgag ggaggctggg aaaggtaggt ttttattctg 83400ggcagattca gacccagttc aaactcaggg gctattttac tgaggaagac agaaaagatt 83460agacagtcag ctctttaggc ctcatagtga atgaatgagg agggattggt cagtcccttg 83520tcactgggcc tggagtgtag tgcctgctgg gcctttactg gtggctttcc tttctgagca 83580ctcatgaggc ccctgtgtct tccctcatat agattccagg tggatctgca gaatggcagc 83640agtgtgaaac ctcgagccga tgtggccttt catttcaatc ctcgtttcaa aagggccggc 83700tgcattgttt gcaatacttt gataaatgaa aaatggggac gggaagagat cacctatgac 83760acgcctttca aaagagaaaa gtcttttgag atcgtgatta tggtgctaaa ggacaaattc 83820caggtaggtt ttggagaggg acaggttgag tcctcattag tgagcaggag tgcacagggg 83880ggcctttcac atttgtgagc ccagccttgt atttcctaca cctgagatat agtttggctt 83940tgtagtcttt ctccataaaa ggaccaggaa ggcacctaaa tatgaggggg tggcaccact 84000actctccagc cagttgttgc catgcagaaa tatggtccac tgtgactaga tctttttatt 84060agatcctatt tctcctagca gggctgagtt ctgaattgac acagtattat gttcatgatg 84120ggagggtaag ttataatata accgtcacca cctgaagaac taacaagggc aatcccagca 84180tagaaatcag aagggttttg taaattcaag tcttgccaca agacagttct gtaggatcat 84240gagattttta gacccagagg acatcctaga aatccttgat gtcagttcca tctctggctt 84300catggagtgt cttataccta gcgcgcgtgt gtatggttga atttggtccc agaagctctt 84360acacctgctg gccctctggc ctgtggagct ttcccacagt agaggtttgt accaacgtga 84420gagaagactc acatgcctct ggcacagatc ctttctgatc ttcgggatac tgctcctgcc 84480cgaaagtctt tctgaatctc ccaaactcca ttcacctctc ccttctctgg ccttttgagc 84540ccgtgtctgt atcattcttt ttcacagttt ttaacagttg tgctttggct ttatgtgttt 84600attttgcctc cacaatggga tttaaagctc cttgagtcag agactatatt gtatgctgct 84660cgcgttttct gcctataacc taacgtggta cctggcattt gagagggagg gagggaggag 84720gctcgtagcg tgccgaggac ctgcagaagc tactttctcg tcatcttact gtagtctgtt 84780gaggtagaga ttgttcctac ttcagaataa gaaaaccgaa ttcaaatatg ttgggtaact 84840tgtccatatt aatttattta gcaaatacaa cagattttga gtgtctgcca catgggtggt 84900ctccagggac agtgttgtgg ggagctcgca ggcagatctt taacctgggt tcacaatctc 84960cagggcacct gtgcctgggc ttccaggcga ccttcgaacc cagatgtctc acatgtatgc 85020agaggcgcac acaagcacac gcacatatac ttatgactgc ctgtttgtct ggggagagac 85080agttcctggt gcttaatcaa atcaggaact caaaagaagt tcggaagcac tgctggtgtt 85140ttgggtgctt tcggttacca tttggtcacg tgtgtggaga cctgtgggaa caggtataaa 85200attggacgca aggaaacatt taaatttgga taataagtta atttattaac tgtttttttt 85260tggtggcggg ggggctctgt cttctgtatc tctctaggtg gctgtaaatg gaaaacatac 85320tctgctctat ggccacagga tcggcccaga gaaaatagac actctgggca tttatggcaa 85380agtgaatatt cactcaattg gttttagctt cagctcggtg agtgaccttc cacagcttgg 85440ggtcttttat gaggatggtt tctgatgaga tggtagaaaa aatcttcaaa taacacttct 85500attgacataa aaaggacgta tctccctgac tgtagtatta attttttgga agtgaactgt 85560tcacactagc agaaggctgt ttatcagcca gggcttcatt gtctgtagga tctcaaacct 85620agtgtggttt taataaaaca cacacagttt ttagctgggt agcagctatt tcctttgcat 85680gggcataaaa tggagtattt ctgtaagaca ggttcctagg ctgggagtgt ctgagtcaaa 85740gagcacagtc atgtgttgca taaggacagt tcagtcaaag atgaaccgca tatacaaccg 85800tggtcccata agattgtcat atactgtatt tttaccatac cttttctatg tttaggtaag 85860tttatatgca caaatactta ccatcctgct ctggttgcct acagtatttg gtacagtgcc 85920tgctgtacag attcactggc caggagctat aggccacacc ctacagccta ggtgtgtagt 85980tggcagtacc atctaaggtt gttaagtaat attctgtgat gtttgcacga tgacaaaagt 86040catgtaagga cacatttctc agaacatacc cccttcgtta agcaacacat gactgtcttt 86100gcattgaaaa ttttgataga tactaactcg cccttcacaa gggtaaaaac agtttgcact 86160ctcaacagcc atgctcccac cttcttgttg acattacatc ttattctctg taatgtttgc 86220caatctgatg gggggcggaa aggaccccag tgtcaagtat atttttggat catagttttc 86280aagcatattt ttagtaccat ttataatttt tttatatgtc aaacaggtta tatatagaaa 86340atattttcta ctggatgtta caaattaatc tttattatct tttctcagga cttacaaagt 86400acccaagcat ctagtctgga actgacagag ataagtagag aaaatgtaaa tattaaatct 86460tttaatgagc cactggttta aaaatgttgt tttagctgcc atgttaatga aatggcaaga 86520aggctgggtt tttgaaaatt atgcttttag aacgcaagta atcacttgaa aattgagata 86580catacttgtg gtgccaggca cgcagtaagt ttttgctgat gattcacctg tcagtttctg 86640taactgccac tcactgttct tatgtaaaaa gcactctctc actcttaact gctgaatagt 86700actgttctgg ggtatttcca aatattgaac atcagccagt gcactggcaa atgaacttcc 86760atgtgtatct tcaacccctg ggagaataac tgcaatttaa aaatgcgctg ttattaatgg 86820agaaagtgag gtcttaccga ctggcacgtt cacacctcac agacagaata gaatcttagc 86880attctggggg caccctggaa aggacaacta agacacgttt gaagttcatg tagtgctggg 86940tgaaggtggt ggctcaggcc tgtagtccca gcgctttggc tgaggtgggg ggattgcttg 87000agcctaggag tttgagatca gcctgggcca catagggaga accccatctc tacaaaaaat 87060taaaaaatta tctgggcatg gtggcgcatg gctgtgatcc cagctttggg tggctgaagt 87120aggcggatga cttgagccca ggaggttgag gctgcagtga gccatgattg agccactgca 87180tcccagtgtg gatgacagag taagaccctg tctcttaaaa aaatttcata tagttctatg 87240aaaaattatt aatttatggt ggaggataaa ggactcagat gaacagggat atcagactct 87300cttctcaacc cgtgtagccc ttcacaacac cataccattc cgtcataaag caccagctgc 87360ctggaggtca caccagagtg gagcaggaac atcccaggct ccggccaggc tcagctcagc 87420acaaccaaga cttcagatta taaactataa ttcttcccct tctaacattg ttgtgttttg 87480tttcttttcc aataggttcc aaagtctggc acgccccagc ttgtgagtat ttttgcctgg 87540gttatttcat gtggaatatt ttataaagtt gcatagaaaa tgaacagttt aaaccgtgga 87600gggcagcttc attcattcca ttccttactg tagaactgtt tccctacagc ctagtaatag 87660aggaggagac atttctaaaa tcgcacccag aactgtctac accaagagca aagattcgac 87720tgtcaatcac actttgactt gcaccaaaat accacctatg aactatgtgt caaaggtttg 87780aagagcccca aattttctta actctgtata aaaattaagt tgtaatgagc tgttacgagt 87840aacctgtatc cacaatagaa gcccaaagca gccccctctg catttgtgtg ccgtccctgg 87900atggattcga gagtcaacca ggcctgcctc tgagccattc ctgtgtattt cctcagcacc 87960tccctgcttg gctgcttccc cttcaggcag aacacagtac tgcctcagac cccaggcaca 88020gggggccttc ctggcgtgtt tcactcatac agagggcatc gggtcccacc ctgtcactca 88080tttcatcgtc taaaatgtaa tcatgagtgt ttgcttcgag ccagggacag tgctgctgca 88140ggggacccag ctgggaccaa ggcagactgt ctctcccctc ctgggattta cagggtcatg 88200gctctgaaac attctgtagt gttctttgga cacgagtttt ccctggagat cgctttctgc 88260aggcctcttg gtcctgactg tggcttcttt tcagagcctg ccattcgctg caaggttgaa 88320cacccccatg ggccctggac gaactgtcgt cgttaaagga gaagtgaatg caaatgccaa 88380aaggtcagta tccttcggta ccagtcacag tgcagatact tccgtgcctg ttaccgcctt 88440ctacccgtga acggtcctgt gagctggaag tagggctagt gtcagaatct tcatttccaa 88500agtgagatga ttcaagcagg aggtggttag attgtgaaca gccagtgggc agcagagccg 88560actaaggccg tgttctgacc tcggcttttt ctggccagac aagagagtag catttttgtc 88620cacgaggcct atccttgcct tgtagaactc cagagcagcc ccgtaagatc aggcaacatc 88680ttttcttttt ttttttgaga tggagcctca ctgtgtcacc caggctggag tgcagtgtca 88740caatcccagc acaccacaac ctccgcttcc tgggtgttca agccattctc ctacctcagc 88800ctctggagta gctgggatta caggcgcacc accacgccca gctacttttt gaatttttgt 88860atttttagta gagacagggg ttcaccatgt tggccaggct ggtctggaac tcctgacctc 88920aagtgatccg cccacttcgc cctcccagtg ctgggattac aggtgtgagt cgccacgccc 88980agcccaggca acatttttta gggcccctct tgtcatgtga tttagaaaat ttctgcttta 89040acaacttttt ccacagacgt ccagccttct gaaagcttga aattagagct atttcctaga 89100aagtggcata ctttcaagaa ggaaggaaca cgggtagatg atgaaaagag aatacctgct 89160tgagaggatc ccaggctcct gcagcctgaa gtagtcattc agtttagcgt taaaccttcc 89220atttctgtcc aaccacatct cagcctcaat gctgatttta aaggggtttt ttttttcgta 89280tttttatttt gcaagtaacg aattagtgga atgctgactg ggtttaaaat ttcaacttca 89340cctgcattcc catgtccatg tggatacgtg tgtttcatag agttagaatc atagttcaag 89400tctggtcact aacattgctg aaattgccac tactctgtcc tacttggtta attaaggttt 89460tttttttctt tctttctcaa aagctttaat gttgacctac tagcaggaaa atcaaaggat 89520attgctctac acttgaaccc acgcctgaat attaaagcat ttgtaagaaa ttcttttctt 89580caggagtcct ggggagaaga agagagaaat attacctctt tcccatttag tcctgggatg 89640tactttgagg tgaggttcca gtttttgaaa atgggacagc aataagaatc ctgggagcag 89700gggtgggata agtggtccat ttaaatcaag tcctaactca gtatgtggag gttgtgtatg 89760ttttttgttt acttggagat tgtaatttgc cccttccttt ttataacgtg ggcaatcagt 89820ataaatggca aagccagtag agtgtcaaat tatgcacatt ggaattgaca tttgtcatca 89880tattaaaatt cctgtgtagc cccatattga taggaattta accaggaagc ttgtctcagg 89940actggagtca cacatttaat catataagca gacttgagga ctggagaccc taaaactgct 90000tgcttgcact ggccatcatc tcccatcagg gtaggtggca gtcctttctc ctaaggagtt 90060agtcttgttt atatgtattc aaggaaaaat acatcagtcc cttggaacta aaaggcatgc 90120agtcctgagt ccccagatag gtgaatattg taacacatac ctttcccgaa atatgtttct 90180gggatgctga gcagagaata gtctccttgt gatgtggatg ccgggtgttt ggccagcctc 90240aatcaccagc tcaggtgcca ctgcctcaca cagtcactta gggtcattgg tttaggttat 90300cattctacag cattttaaac tgacacattg tctggaccat gtgggttctt gaggactcat 90360caaaacccgt tactaaaagc atgaatatca ggcgaaatag atagcaatgt gacattcgta 90420tttatcccta agttccagtc taatgcagtg ccctggtatg tggagtgtag acagatgtgg 90480gctaatcatg gaaggttccc tggaagttgt ggatattggt ttcgaattca gaaagctggg 90540aaggatgtgg aaggctgaag gttggctttt ctagatttag ggcatgattt gaacaagtcc 90600ttagaggtgg gaagggcagc acagggttgt tggcttggca agagtcaagg tgcaaagggt 90660gacttggggt tcactggagg gaaacagaga tgagtgctct agaaggaagt tgagccttgt 90720ggtgggtgac aggaaaccaa tgatgtaact tgtttttgac ctatctgggc cccaagtttg 90780gatctgctat attaatataa aaaaggataa taatgataca ttcaaataat gctgaaaaat 90840actaagatga aaatacctcc aacttcgtaa ttcaaaccat accattagga ttaggtgaac 90900cacattccag gcgttttttt gcagagacag tgaaagggat ggctggctga aggaatgaat 90960agatgaatgt tatatgcttt tgaacaatcg tcttttccat ttaattttct aattcaggag 91020cagtaattat ccttgtgttg atcactgctg acgattttct atactgatag gtcctttccg 91080ggggcttcca tctcttgcct tttaaatatg cttgcattga gattatctca ggtctttcca 91140ttatgccatt actttcattt taaatcttct tgctctttca aatacacttt agttgtatct 91200acagtgtttt aaaaacaatc tcattcagtg ttgtaatttc atctgtgggc tcttcctctg 91260gatgaaatcc gtgttcctcc cagctgttcg gcagcatcag atggttgtga gggattctgt 91320tgttctgttt tcttctaggc aaaggatgtg ccttcttttc atttgcagta gtctgctcac 91380ccggaagcat gtcatttctt tgccacttgc ttgtaattca ctggctttgc acttgctctg 91440atacagtaca ggtaactaat tgactccctc tgctgccaac ttggttttcc ttctgagcta 91500tagcatcagg ctgtgtgttt tgtgttttct tgagattttg ttaaatatat ctggggtccc 91560ttctacctgg ttggaactgg gattcccacc attcttgtgg ggatagaatc tcaggttaca 91620cctatttccc caatcctctg tagccacaga agcttcatct tggccagctc tgttatcaga 91680gtgcaggact tgggctgaaa tttcctcccc ttcctgattt tccttgacag tcctttccac 91740tgctcctatc aatcaaaaga atgaaaaccc tcaacttgct gctttgcaga ttcaggtttt 91800gtgcttcttt ctggcctctc ggggtggggc cgggttagca gcaaggctga gctgcccctc 91860tttcttctga agccttcatg ggggcgagga gcacagggag agctcagtgc agggcctccc 91920agtggccttc tcagagtggg tggaaaccca gcctggcact ggcagcgtgg caccagaagt 91980atgaagtgta ggtgtaaagg tgatgtaaaa ggctagtagg ttttttggtt tttcattgtt 92040tgagttttgg gcatagatga ctgtgaaggg cgaacactgc cgatggatct gaatgaattt 92100gtagtatgtg caccacttcc aacttacggg atacccagct ttgacggctt tggacaaaca 92160cactgaggcc aagatgtgct gagcttatca ggatcaggat caccaagcag ctgtaaaaac 92220cctagcaagt gccttaagct gctgaaattt catattaatt gtctggtttg ttcatggtcc 92280tagagtttga ggcagaaaag tcaggatcca agtcccttgg ttccaggcta cagctggaaa 92340cagcatctcg gtgaactaaa gcaaccatat taggagtttt cctgctttag gagagtcccc 92400agcatcggcg aggagggggc agcactctgg ctttccagga gcaaggggca ggatgcggcc 92460gagggagagg ggctgtgttg aggaaaggag ggccgcaggc cctggggatg gtgtgaggct 92520ccaaacatgt ccgagtcact tccctgggtg ggatgaggca gacagtgcca ccaccaggga 92580cactttagtt agattagggt cttggaagtc acagaaggaa gtcagcagca gcaggctgga 92640acttttctat gtataatcaa atggtttact ctgacaccgt tagcatgtaa caaacacaaa 92700attttaaact aaggggaacc actaatggca tgtttccttt cctttcagat gataatttac 92760tgtgatgtta gagaattcaa ggttgcagta aatggcgtac acagcctgga gtacaaacac 92820agatttaaag agctcagcag tattgacacg ctggaaatta atggagacat ccacttactg 92880gaagtaagga gctggtagcc tacctacaca gctgctacaa aaaccaaaat acagaatggc 92940ttctgtgata ctggccttgc tgaaacgcat ctcactgtca ttctattgtt tatattgtta 93000aaatgagctt gtgcaccatt agatcctgct gggtgttctc agtccttgcc atgaagtatg 93060gtggtgtcta gcactgaatg gggaaactgg gggcagcaac acttatagcc agttaaagcc 93120actctgccct ctctcctact ttggctgact cttcaagaat gccattcaac aagtatttat 93180ggagtaccta ctataataca gtagctaaca tgtattgagc acagattttt tttggtaaaa 93240ctgtgaggag ctaggatata tacttggtga aacaaaccag tatgttccct gttctcttga 93300gcttcgactc ttctgtgctc tattgctgcg cactgctttt tctacaggca ttacatcaac 93360tcctaagggg tcctctggga ttagttaagc agctattaaa tcacccgaag acactaattt 93420acagaagaca caactccttc cccagtgatc actgtcataa ccagtgctct accgtatccc 93480atcactgagg actgatgttg actgacatca ttttatcgta ataaacatgt ggctctatta 93540gctgcaagct ttaccaagta attggcatga catctgagca cagaaattaa ggcaaaaaac 93600caaagcaaaa caaatacatg gtgctgaaat taacttgatg ccaagcccaa ggcagctgat 93660ttctgtgtat ttgaacttag ggcaaatcag agtctacaca gacgcctaca gaaagtttca 93720ggaagaggca agatgcattc aatttgaaag atatttatgg gcaacaaagt aaggtcagga 93780ttagacttca ggcattcata aggcaggcac tatcagaaag tgtacgccaa ctaagggacc 93840cacaaagcag gcagaggtaa tgcagaaatc tgttttgttc ccatgaaatc accaatcaag 93900gcctccgttc ttctaaagat tagtccatca tcattagcaa ctgagatcaa agcactcttc 93960cactttacgt gattaaaatc aaacctgtat cagcaagtta aatggttcca tttctgtgat 94020ttttctatta tttgagggga gttggcagaa gttccatgta tatgggatct ttacaggtca 94080gatcttgtta caggaaattt caaaggtttg ggagtgggga gggaaaaaag ctcagtcagt 94140gaggatcatt ttatcacatt agactggggc agaactctgc caggatttag gaatattttc 94200agaacagatt ttagatatta tttctatcca tatattgaaa agaataccat tgtcaatctt 94260atttttttaa aagtactcag tgtagaaatt gctagccctt aattcttttc cagcttttca 94320tattaatgta tgcagagtct caccaagctc aaagacactg gttgggggtg gagggtgcca 94380cagggaaagc tgtagaaggc aagaagactc gagaatcccc cagagttatt tttctccata 94440aagaccatca gagtgcttaa ctgagctgtt ggagactgtg aggcatttag gaaaaaaata 94500gcccactcac atcattcctt gtaagtctta agttcatttt cattttacgt ggaggaaaaa 94560aatttaaaaa gctattagta tttattaatg aattttactg agacatttct tagaaatatg 94620cacttctata ctagcaagct ctgtctctaa aatgcaagtt ggccttttgc ttgccacatt 94680tctgcattaa acttctatat tagcttcaaa ggcttttaaa ctcaatgcga acattctacg 94740ggatgttctt agatgccttt aaaaaggggg cagatctaat tttatttgaa ccctcacttt 94800ccaacttcac catgacccag tactagagat tagggcactt caaagcattg aaaaaaatct 94860actgatactt actttcttag acaagtagtt cttagttaac caccaatgga actgggttca 94920ttctgaatcc tggaggagct tcctcgtgcc acccagtgtt tctgggccct ctgtgtgagc 94980agccaggtat gagctgtttt agaagcagcg tgttgccttc atctctcccg tttcccaaaa 95040gaacaaagga taaaggtgac agtcacactc ctgggttaaa aaaagcattc cagaaccact 95100tctctttatg ggcacaacaa agaaacgaag gctgaagttc gcctacccaa aatgaaaagt 95160aggctttaca gtcaaaagta cttctgttga ttgctaaata acttcatttt cttgaaatag 95220agcaactttg agtgaaatct gcaacatgga taccatgtat ataagatact gctgtacaga 95280agagttaagg cttacagtgc aaatgaggcg tcagctttgg gtgctaaaat taacaagtct 95340aatattatta ccatcaatca ggaagagaat aataaatgtt taaacaaaca cagcagtctg 95400tataaaaata ccgtgtatca tttactcttt ctgcagctct atacgatagg caggagaggc 95460ttatgtggca gcacaagcca ggtggggatt ttgtaacgaa gtgataaaac atttgtaagt 95520aatccaagta ggtgtattaa ggcaccaaaa gtaacatggc acccaacacc caaaaataaa 95580aatatgaaat atgagtgtga actctgagta gagtatgaaa caccacagaa agtcttagaa 95640atagctctgg agtggctctc ccaggacagt ttccagttgc tgaatagtct tttggcactg 95700atgttctact tcttcacatt catctaaaaa aaaaaaaaaa aaaaatcaaa attaaaatct 95760gagtcagtct gcctgcctcg gttctcatta gtttaattct taatgccttg cactttccag 95820caatcattca atcaaaagag tgaaatgaag cacattaaca aagcaggagg cgccacggac 95880cgcctccctc cacaccgctc cttccgcctt cattccttgc ccacaggctt gcactggaag 95940ctgaataaga atccccaaaa ctcaaacttc ctagggatgc caccccttta gtagctcaca 96000cctcccccct ccaagagcta agaaacaaag gagaatgtac ttttgtagct tagataagca 96060atgaatcagt aaaggactga tctacttgct ccaccacccc tcccttaata ataacattta 96120ctgttatttc ctgggcctaa gacttatgtt ccagaactgt cacagctccc catgtcacac 96180ccactagctt gtgatctttg tcaaataact gaaatctttt aagcctctag tttcttcctt 96240tgtaaaacag agataaaatg ttgtggtttt taagtgagat aatccaagta aagcacctaa 96300catggagtag tgaatgaaca tcggttgcta ctaaaagtgg acatcctacc gcatccttaa 96360tgccactagg catttccata caatctgggg accaaaactt caatcatata aatgtatgag 96420gttaattaaa aacactactg taatctgctt gtatgatcac aaaccaccac aaaagaaaag 96480atcgtgaaga ttacactgta aacggactct caaatgatca ggaggtggtc acttcgcaac 96540ttgctccctc cacccaactc aaaacaggag ctcgagcctg cctgtatttg agactggagc 96600tgcctgtatg aggactggat caactgctag tcacgttata tccaaatctg cattatcatt 96660gggcacattt tcacagaatt ttactgaatt attccttaat tgtttaatgg ttgggaatag 96720tttgggaatt accttccatc aactctgcta agaaaggaat ggattctggt agcaagacaa 96780tataattctc ctttagtttt tcagccagtg ctaacacagt aatcaaagca gcaaatcgaa 96840cctgaaaggg ataaaagagc aaagaaataa aaagtagtgt tactgtattt attatcttaa 96900gagctgtact gacttgagac aagctctaac tttttaaaca ttagttcaca cgcgtttatt 96960cacttcatta tgttcattaa gctttcatct tagaatacca gtttcaccat ttgggagctg 97020tttgtaatat gtgcaacctt ataaatagtg ttttccaaac tgtgtcccag gactgcaaat 97080ctttaatgtg aaatgtcttt ttataatctc ttcctttaaa aaaaaccaat aaaataaaat 97140gccacatgca aactcaagtg tgtcaccaga ttttacttca ttggcgctcg ccagcccgcc 97200aggctggcaa taaagtgcct ccagccacct ctggcaggtc tcctcaccca cagcccctga 97260ctggtcacca ctatagttgt atgaggggcc aggacaatcg cttgggataa actcccatct 97320cagcactgaa taaaaaacat tctgtgtcac aatatcctag ttttggggct ttaaaaacgt 97380ctaggtgttc ctcacatgcc ttgtctataa taaggaaagc aagcagtagt tgggtattgt 97440tagcttttga aacaaaagcc ctactggtct tctaattttg gatattttaa ttaaagaata 97500tctggacagt acaaagtgaa ttattaaaaa accatttgta actacctaga ttcaatcagg 97560atttccttga tttgtgcaaa gtaaaatatt acaataaatt tgatactgct acttgtataa 97620aaacctatgg tttaaaatgt gggggttcat cataatagtc tcattgttag catatcctaa 97680taaagaattt gaactaataa atcctattaa taaaattctg ctttggtctg ttatagccag 97740taaagttcta atacaatcat tagtttgaga aatggtgact cattgctaaa acagtttgaa 97800atttgtaaca cttgggtgtc aaattttgac ttccactcaa cctacccatg ttttatttcc 97860actgccacca cttactcaac aagatcataa gcctagtatc tataaacaac agaatgtatt 97920gctctaactc aaaagactat agtgtggata aattcaatgc atttctctct ggagcacaat 97980gacatttcaa tagcacttaa aaaagaagga attacttcaa atctttgtta tttaaaagta 98040tttagaaagt attttagtac ttctgcccaa cgcaccattg gggtggggat agggcattgc 98100tattctttac aaatagccta taagtaaaaa acaaaatttt cttaggcaca aatttctgcc 98160taatacaaaa gaccagacct ctagtactgg atgacaaata gcaatgttct tccctgccag 98220tttactaggg ggcctacatc tgtgaccacc tgcaggctgt ttaggctatg cagtgaaaag 98280atgcagtttc agtacttgtc acgcagttcc taaccttagg cgaggagtct ctcgtcttta 98340gcagaatctg gtagttcagt ggtttccaaa gagagtcatc cgccatggcc actgaaaact 98400gtgcgatgca tggtatcagg tgctttgtca cccgttcctg gaatttctct tctcccccaa 98460gcctgttttc cagctaggaa gagtaagaca aagactttga acaacaagtc tcatttcttt 98520cttctgtttg aaaaaatgtc caacatacaa atattttact atctttcatg atattagcag 98580gttcaaaaac caggcattat tctaatactc tctagggcaa atgtattgcc ttctagaact 98640caaatggaat ctcataccct ttatcatcgc ccctttctct ccagcagaac atctcagagg 98700agctctttgc tccagaggac agccatgctc tgacacgttc tcagtgaggc ccagttaaaa 98760caaatgaata cattaaccat gacagcttat atcatgtctg tcttttgagc agtttaaaaa 98820ataaaaaata aaaaataact cagggccagg catggtggct cacgcctgta atcccagcag 98880tttgggaggc caaggtgggt ggatcacttg aggtcaggag ttcgagacca gcctggccaa 98940catggcaaaa cctcatccct actaaaaata caaaaattag ccaggtgtgg aggcgggcgc 99000ctgtgatccc agctattcgg gaggctgagg cacaagaatt gcttgaaccc gggaggtgga 99060ggttgcagcg agccgagatt gcaccactgc actccagcct gggtgacaga gcaagaccct 99120gtctcaaaac aacaaaacaa aactcaaatt ccacaatgaa gttatatctt tgaaaaaaca 99180attttcaaat aaaacatttc attaaaaaga ccagaaaaaa caaccttaca aagaaaaatc 99240ctagcaagct gtcatttgag cagatctaaa acctgccaag ctcgaacagt gatggcttcc 99300tcagcaacga aagatgattc tgtttggtta cctgatccac cagaggcatc atcaaggctc 99360ctgctctctc tttacttata aaatgctggg tatcaaaaag gaagattttg tataaacagt 99420tcaaaataaa ctgcaacagc aagcagcact tttcagggtc attttcagag tcaaaaaatg 99480cttcatctgt agacgtggga agagtaaaaa tgaaaaaaca ctgaacttaa ccatttaatc 99540tccaatgttt acattgaaat cactattaaa ataactaaat cagaagagtc taaaatgatc 99600tagaaatcat aatcaggacg aaggcagaac acaatggatg gtctctcgaa gaatgattcc 99660ttcttttaga gttaagattc taacactcac tctggcaagt taaattccct caactgtcaa 99720gtgggtcacg tattagcatt agagaataaa ctaatcttaa tttttgcgtt ttaaagttac 99780ttccagtaac tgacagtaac ggccatttac tttattcttt ctcccaagtg aggtgactta 99840taacattcgc tcatcatgct aaaacaacac ttcactgtct gacaacaatg aagtaaaaaa 99900ttcaccctcc ttagcttagg acttaagaac ctctaaaatc ttgcttccaa gcactagctt 99960gtgtcttact ggtaccttgt ataaggcaca caggacaagg gtgacagctg aactgaagcg 100020accacccacc tgttttggag atgttcacct ggtccaaggt gtcagcaaaa ggcttcacta 100080agtggccggc aaacagagta aaaagccctt tcagcttttc agcaatgcaa tctgccaagt 100140tgtaaaatgt caacaacctg tcctttgggg catcttctgt tttagcccaa tcaaacagct 100200gaaaggataa gacagtatta gtttcttcga catcttgtca cttaaatctg agcacaaaag 100260agaggaagag gaagaaagcg tcaccttgaa gaacaggggc ctgaatgtga cctcggaaag 100320tttgacaacc atggctacta gacagtcaat gatacaattt tccgtttttc caacttcctc 100380cagatcgttc tgaaaacaga agagcccatt tattagagtg ctgatacctg actgtaaatt 100440attttggcaa gtaccactgt tacacggcta gattgttctc ggactcttca ataggtggat 100500aacagcttta ggatttggag gagtgaacct gagcttacct cagagtgctg ggctcggaag 100560tccagggcct ccaggaaaaa ggcggttagc tgagactgat gggaggtgag ctcttccttc 100620ttcatcgccc caatatgctc ttgcaagatg ctcataaacg gacccatgtg attctaccaa 100680taacacagga aaaagatgtg ccattttcaa atgattccta gagttcagcg gtgtgtattt 100740ttaaaaacta aatcttcttc tttaagtcaa agtttacaca ttgcagtacc acctctccct 100800tctccaaagt cttaataccc aataagatct aaccttccag ttcttctcaa tctgcttgta 100860agtttttttg atggcgggca acaggactcg gggtgcaagt gtggtagcca gtgtcttttt 100920aagagatgtg agacggatat tagcctgtga cgcagaaccc atttcactag tgattttctc 100980cagatgaatc acctacagga atataaaaaa agtgatcagg gccactgcag atcttcgctg 101040acaaacacac acttacagag aggcttcatg atgaggtact agtgtttgga aaatgcttag 101100cactttttaa ctacacacag agttcctttt aaagtcagcc ctaaacgtca gtggataaaa 101160ctgggcagac acctcttgcc caacttgcga tcagggacga aggccgatgg tagacgcaga 101220cgcacacaca gcacccagac agatgatttt cttagaggac aggaatgcaa gggaccacgg 101280caagagtcaa gttgctaaaa aactgagaaa gctcctcaga gcacaggccc ctttctctga 101340gaaggctact tttaaaccct ggctgtggtg taagtgaagc ggtttaatca tttgccccat 101400ggtaatgaag gctcctaacc ttgtaaatgg caaatgatca acacaatgga acagccaggt 101460ctcaacactc ttgagcatct tcaatcataa ataccactgg cccctagcgt gttgacagga 101520aaccgctgac gtgcaataca aaaattctgc tttgcaagat gccttaggat taaacctctc 101580acagtagaaa cagggcccat caatttccac aagtaataaa aggcggctct accagcccaa 101640ctccaaagat ctcacagaag aaaaaaaagc cagaatacat tccgcacaat taaagaagag 101700aagcatctcg ctaaaaagtg acccccatat caatttcaag attaagtggc aaggatgatg 101760gaagagaaaa agtacacatt taataaaagc aagcacatct cttcagaaat aagactcctt 101820tctgtcaaac ggaaactaac ccttaaagaa aaaacaaaat cactacattt gtgatctttt 101880accttcccca gccaccctgc gtagcatgtc gtggctatcg tggctcacct gggagagaat 101940gccttccaga taggggctga tgaagtgcgg gagagtctcc acaaccttct gcagagcagc 102000caaggcactg agcaggtaga cctcgctgga gaccagctcg ctggtgttct tcattgttgt 102060cagcaacgat ggcatcaggc tagaaacaaa gtaagagctt tagaagaact tgaagcagaa 102120acagaggcta gggaatggag tagagggcat tatgaaaaaa accagcaaac tgtgcctatt 102180acatcgctat ctgcctcata gcctaaaaag cagtgtctat acattttatg tggctaagca 102240caagaaatct cccagtgcta acagtatgga cacaacagta atttaaaaaa taacaatgtc 102300tttcattaac tgaacactta ctatgtgtca ggcactatgc aaaactcctt gcaagcactg 102360ccctacagaa atcctatgag gtagatactg tctctgtttt atagacagca aagctctaac 102420aggttaagga acatactggc tgtacagtaa ggaactacca cagccaggag cttctaactt 102480ccaaatttgg cagcagaagg cagctttggc cttgcctaac tgggtgggcc cctctgccaa 102540gaaccttcac ccactgcttt ttgactatac tagacaaaag gaaggaagaa tggaggacga 102600ttaacactgc aaagcagtgc atctgaagat aaacgggaag gctgcatctt tctgtttgaa 102660gattaattat ttttattatt atttctttaa gagacagggt ctcactctgt tgcccaggct 102720acagtgcagt ggtgcagtca tagctcactg cagcctcaaa ctcctgggct caaatgatct 102780ccctgccttg gcctcccaaa gtgctgggat cacagccgtg agccaccaca ccctgcaaga 102840tcaattcttt aacaaattcc aattttatgc aacgtctact cagaggaaaa aaaaaaaaag 102900tcaccaaagt gttatttttc aatgtgtgcc aggcggtaac agctcctgtt ccaagtctcc 102960ggccgcatac ctgggaagct gggggatggc cagcgcctcc agggtggagg tcacctctgc 103020tatgcacagc agcgcgcttc ccaagacatt cttctcctcc tttctctctg gagcaatcag 103080tttcacagca gtgctcagca ctgggacaaa aggatctgga ttttctgcac caaaattctt 103140gcataaaagc tttaaggtat acaacgctgt ctgtctgttg attgcttgtt cttcttcccc 103200ttcctttttc ttacgctgca caatggccaa aaggtctgga accagtttta ggaaacgggt 103260aacctgaagg ggacagccag aatccccaaa tcattaaagc tgcaaaaaat gtttgtccat 103320tttcccattg tcacagcttg agattgtcta aatggaaatc agactcgggg gtcctgagtc 103380acacagtcat gctaagcgat gtgcatgttc tagccagtgt ttcacttata caaagcaccc 103440actgatctgg agtaaaaggg acttagaact atgctaaggc taaggccacg taagctctgt 103500agtaagcaag aattccacta ggctgaaatt ccattctaag agctcttaca acacacatat 103560attcccgtta gaattaacgt cacattttaa aacatgtcat ggtattatat tcagataata 103620atatacttca atttgaaatt gtaccactag agaaattgaa gggagttaaa tgcagctctt 103680tgataaagca aagtacagta aatgggtgtg tcctgggtct tcactcacta ttgtcttctt 103740ccaggatata ttttgctgca gcttgttatt caaaaggtcc agcgctttgc ggcgaacaga 103800tggcagggga ttgcccacca gccctctgat cacaggaatg aatgtctctg tgggcagcaa 103860ggcattgacc taaagagaaa ttttatattt aacatgaaaa gaaaaacaaa ttaaaaaaaa 103920aatcaacttc aattaagaca gactgctgtc cactgcacac ctccaggcac caggcacttc 103980cacacacatt ttcttattta attcttaaaa taacctttca ggtaggcatt accaaccaca 104040cattatcgaa caaaacaaaa gcctgatgtc aggaggaagt gccaaaggca tgcagctaaa 104100tgactgagct agatttgaat cagcaatcct aacttcgagg ccagtgatat gtatgtaata 104160tacttcatac ttttatttta ttccacttga ataaagtaga acagtatata ttatatgact 104220taattattaa aatatacgag gtacatgttc tcataactgg taaggaaaca attttttcca 104280gacaaatcta tttctagtca tcaagagatt gttttctaag aaaaatctga gcttcattat 104340attcataaaa ggaattgcta agtttattct taaaaacttt acataatttc acaataattt 104400aaaaaacagc aacaaaacag taattccagg gagaaatgaa cacctacctt atctaacagg 104460tcgtaagctt tactaaggag cgcgcgccag aacttcacgg tgagtttgtc tgcgttcctt 104520tccatggact gtgcaactgc actgatatag ccgagaacgg tctccagcaa cctgaaacac 104580agaggctcgc tcagcaaacg gcagctgaag aaactcagag aacttgttca tgtctacctt 104640atgctaaatg tttcaagtag aaagacgagt taaataattc tgtactaaat tatttcaaaa 104700actactcgga aagaaaggaa atgagggatt attgccatag acagagatca tcaagaagta 104760actaggcgct tctgtgcaga agcatcgacc tcgctcagac tctgtgaggt gctgaataag 104820caacagatgc tgaaagcgtt taaggaactc actcatatct agctcatgct cagtggatct 104880cactgggctg tccaagtggg gtgttcaggg agttatggcc ctaggttaat ggcaggtgtg 104940tgcgtgcaca cacacacagg cacacacacg cacacataca catgcacaca caccatacac 105000catttatata aagagaaata ttaatagaaa tgaacatata acccacttct ttcacattat 105060taggagacaa aaaaaaagac tacaaacttc aaataacttg taattagaaa agcacacacc 105120aaattccaac acagctgcca ctggagatcc ccccactgct gccagcctga ggggggagct 105180agagggaaga gtggagacag aagttgacac cgcacagcag aggaggggag aagggggcgc 105240agacaaaatc agctccaaaa acgaaagtcc tacgcatagc gctacaagtc agcccacagg 105300actggaactc agcagctcac attcctggct gcagggcagg cactttccag tggaaggggc 105360aggacagtgg ccctgggaat gccatgcatc tgaaaaggag gtacacagca aggccaggag 105420gcaaaccccg aggacatggg agagaaagga aaattcctgc acccaaatat ataatggcag 105480catatggatt agaatccacg gaataaagaa ttcatgagcc catagaaatc agggccagat 105540tgagacacta aacagatact gcaactcaat acaatacaca gacttgacat ggatcatgat 105600gcagaaacac atgcggtgta aaggacagtg ttgggataat tagggagact ggagtatgaa 105660ctgtagatta catcactgga ttggatcaat gttaaatttt ctgaatttga tcaatgtact 105720gtggttttat aagaacatct cttattctta gagacataat gtatatgatt tactttcaaa 105780tggctcagag aaaaaaccct acatagggag aacgctaagg caaatgtggc agaaagtatt 105840atcaaatggt gaacctggtt gtaaagagta tatgaatttt ctgtactgtt tttccaggtt 105900ttctataagt ttgaagtcat ttccaaataa aaagtaaaaa aagaaaagga aacatacctc 105960tcttcaaggc cttttaaaat ctcaggacca ccactctcaa ctacctaatt tttaaagaag 106020acgtcattag aacggtatgg aagtcaataa taaaagtcat ttcaagtcag ttcaatgaaa 106080ctcggaccat tcactgaaac cttccacagc aactgttttc tgacattaca atttaatcag 106140gttcatagca tcttcattat actgtagtaa ctctatttct cttaatttat tttaattata 106200ttctactggt agtatctaaa aagtactaca atggttcaga aaaatacagc aatcaacact 106260caattagcac taccgaattc tatgacatgc tgatctggtg agctcacata tcctttgttg 106320agaagttaaa cattacagat tcagctggaa tcccccaagt actgctcctt ggtcctattc 106380tccctctacc ccaagcccca caaacaaaac catcatccca aatctgcttc caaatgtttc 106440aaacactaca tatcacggaa caacatgttt ttctggaaac atatttttga gatctatgca 106500tggtgactta tgttctagtt ccttcatttt aactgcatat gatattcctc tataaatacc 106560acttatctat ccatttgcct ctgttgttag atgtttagtt tatgtccatt ttttcccctt 106620ttactaataa tgctagagaa gaacattttt atgtcccttt gatcatcttg ggaagttttt 106680acagcatata tacctaagga agggaatgac cagatcacag gaattactgg aactttcaac 106740ctcatg 106746 2 5408 DNA Homo sapiens CDS 201..1151 polyA_signal 1773..1778 AATAAA 2agccgcccac ggacgccaga gccgggaacc ctgacggcac ttagctgctg acaaacaacc 60tgctccgtgg agcgcctgaa acaccagtct ttggggccag tgcctcagtt tcaatccagg 120taacctttaa atgaaacttg cctaaaatct taggtcatac acagaagaga ctccaatcga 180caagaagctg gaaaagaatg atg ttg tcc tta aac aac cta cag aat atc atc 233 Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile 1 5 10tat aac ccg gta atc ccg tat gtt ggc acc att ccc gat cag ctg gat 281Tyr Asn Pro Val Ile Pro Tyr Val Gly Thr Ile Pro Asp Gln Leu Asp 15 20 25cct gga act ttg att gtg ata tgt ggg cat gtt cct agt gac gca gac 329Pro Gly Thr Leu Ile Val Ile Cys Gly His Val Pro Ser Asp Ala Asp 30 35 40aga ttc cag gtg gat ctg cag aat ggc agc agt gtg aaa cct cga gcc 377Arg Phe Gln Val Asp Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala 45 50 55gat gtg gcc ttt cat ttc aat cct cgt ttc aaa agg gcc ggc tgc att 425Asp Val Ala Phe His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile60 65 70 75gtt tgc aat act ttg ata aat gaa aaa tgg gga cgg gaa gag atc acc 473Val Cys Asn Thr Leu Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr 80 85 90tat gac acg cct ttc aaa aga gaa aag tct ttt gag atc gtg att atg 521Tyr Asp Thr Pro Phe Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met 95 100 105gtg cta aag gac aaa ttc cag gtg gct gta aat gga aaa cat act ctg 569Val Leu Lys Asp Lys Phe Gln Val Ala Val Asn Gly Lys His Thr Leu 110 115 120ctc tat ggc cac agg atc ggc cca gag aaa ata gac act ctg ggc att 617Leu Tyr Gly His Arg Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile 125 130 135tat ggc aaa gtg aat att cac tca att ggt ttt agc ttc agc tcg gac 665Tyr Gly Lys Val Asn Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp140 145 150 155tta caa agt acc caa gca tct agt ctg gaa ctg aca gag ata agt aga 713Leu Gln Ser Thr Gln Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg 160 165 170gaa aat gtt cca aag tct ggc acg ccc cag ctt agc ctg cca ttc gct 761Glu Asn Val Pro Lys Ser Gly Thr Pro Gln Leu Ser Leu Pro Phe Ala 175 180 185gca agg ttg aac acc ccc atg ggc cct gga cga act gtc gtc gtt aaa 809Ala Arg Leu Asn Thr Pro Met Gly Pro Gly Arg Thr Val Val Val Lys 190 195 200gga gaa gtg aat gca aat gcc aaa agc ttt aat gtt gac cta cta gca 857Gly Glu Val Asn Ala Asn Ala Lys Ser Phe Asn Val Asp Leu Leu Ala 205 210 215gga aaa tca aag gat att gct cta cac ttg aac cca cgc ctg aat att 905Gly Lys Ser Lys Asp Ile Ala Leu His Leu Asn Pro Arg Leu Asn Ile220 225 230 235aaa gca ttt gta aga aat tct ttt ctt cag gag tcc tgg gga gaa gaa 953Lys Ala Phe Val Arg Asn Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu 240 245 250gag aga aat att acc tct ttc cca ttt agt cct ggg atg tac ttt gag 1001Glu Arg Asn Ile Thr Ser Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu 255 260 265atg ata att tac tgt gat gtt aga gaa ttc aag gtt gca gta aat ggc 1049Met Ile Ile Tyr Cys Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly 270 275 280gta cac agc ctg gag tac aaa cac aga ttt aaa gag ctc agc agt att 1097Val His Ser Leu Glu Tyr Lys His Arg Phe Lys Glu Leu Ser Ser Ile 285 290 295gac acg ctg gaa att aat gga gac atc cac tta ctg gaa gta agg agc 1145Asp Thr Leu Glu Ile Asn Gly Asp Ile His Leu Leu Glu Val Arg Ser300 305 310 315tgg tag cctacctaca cagctgctac aaaaaccaaa atacagaatg gcttctgtga 1201Trp *tactggcctt gctgaaacgc atctcactgt cattctattg tttatattgt taaaatgagc 1261ttgtgcacca ttagatcctg ctgggtgttc tcagtccttg ccatgaagta tggtggtgtc 1321tagcactgaa tggggaaact gggggcagca acacttatag ccagttaaag ccactctgcc 1381ctctctccta ctttggctga ctcttcaaga atgccattca acaagtattt atggagtacc 1441tactataata cagtagctaa catgtattga gcacagattt tttttggtaa aactgtgagg 1501agctaggata tatacttggt gaaacaaacc agtatgttcc ctgttctctt gagcttcgac 1561tcttctgtgc tctattgctg cgcactgctt tttctacagg cattacatca actcctaagg 1621ggtcctctgg gattagttaa gcagctatta aatcacccga agacactaat ttacagaaga 1681cacaactcct tccccagtga tcactgtcat aaccagtgct ctaccgtatc ccatcactga 1741ggactgatgt tgactgacat cattttatcg taataaacat gtggctctat tagctgcaag 1801ctttaccaag taattggcat gacatctgag cacagaaatt aaggcaaaaa accaaagcaa 1861aacaaataca tggtgctgaa attaacttga tgccaagccc aaggcagctg atttctgtgt 1921atttgaactt agggcaaatc agagtctaca cagacgccta cagaaagttt caggaagagg 1981caagatgcat tcaatttgaa agatatttat gggcaacaaa gtaaggtcag gattagactt 2041caggcattca taaggcaggc actatcagaa agtgtacgcc aactaaggga cccacaaagc 2101aggcagaggt aatgcagaaa tctgttttgt tcccatgaaa tcaccaatca aggcctccgt 2161tcttctaaag attagtccat catcattagc aactgagatc aaagcactct tccactttac 2221gtgattaaaa tcaaacctgt atcagcaagt taaatggttc catttctgtg atttttctat 2281tatttgaggg gagttggcag aagttccatg tatatgggat ctttacaggt cagatcttgt 2341tacaggaaat ttcaaaggtt tgggagtggg gagggaaaaa agctcagtca gtgaggatca 2401ttttatcaca ttagactggg gcagaactct gccaggattt aggaatattt tcagaacaga 2461ttttagatat tatttctatc catatattga aaagaatacc attgtcaatc ttattttttt 2521aaaagtactc agtgtagaaa ttgctagccc ttaattcttt tccagctttt catattaatg 2581tatgcagagt ctcaccaagc tcaaagacac tggttggggg tggagggtgc cacagggaaa 2641gctgtagaag gcaagaagac tcgagaatcc cccagagtta tttttctcca taaagaccat 2701cagagtgctt aactgagctg ttggagactg tgaggcattt aggaaaaaaa tagcccactc 2761acatcattcc ttgtaagtct taagttcatt ttcattttac gtggaggaaa aaaatttaaa 2821aagctattag tatttattaa tgaattttac tgagacattt cttagaaata tgcacttcta 2881tactagcaag ctctgtctct aaaatgcaag ttggcctttt gcttgccaca tttctgcatt 2941aaacttctat attagcttca aaggctttta aactcaatgc gaacattcta cgggatgttc 3001ttagatgcct ttaaaaaggg ggcagatcta attttatttg aaccctcact ttccaacttc 3061accatgaccc agtactagag attagggcac ttcaaagcat tgaaaaaaat ctactgatac 3121ttactttctt agacaagtag ttcttagtta accaccaatg gaactgggtt cattctgaat 3181cctggaggag cttcctcgtg ccacccagtg tttctgggcc ctctgtgtga gcagccaggt 3241atgagctgtt ttagaagcag cgtgttgcct tcatctctcc cgtttcccaa aagaacaaag 3301gataaaggtg acagtcacac tcctgggtta aaaaaagcat tccagaacca cttctcttta 3361tgggcacaac aaagaaacga aggctgaagt tcgcctaccc aaaatgaaaa gtaggcttta 3421cagtcaaaag tacttctgtt gattgctaaa taacttcatt ttcttgaaat agagcaactt 3481tgagtgaaat ctgcaacatg gataccatgt atataagata ctgctgtaca gaagagttaa 3541ggcttacagt gcaaatgagg cgtcagcttt gggtgctaaa attaacaagt ctaatattat 3601taccatcaat caggaagaga ataataaatg tttaaacaaa cacagcagtc tgtataaaaa 3661taccgtgtat catttactct ttctgcagct ctatacgata ggcaggagag gcttatgtgg 3721cagcacaagc caggtgggga ttttgtaacg aagtgataaa acatttgtaa gtaatccaag 3781taggtgtatt aaggcaccaa aagtaacatg gcacccaaca cccaaaaata aaaatatgaa 3841atatgagtgt gaactctgag tagagtatga aacaccacag aaagtcttag aaatagctct 3901ggagtggctc tcccaggaca gtttccagtt gctgaatagt cttttggcac tgatgttcta 3961cttcttcaca ttcatctaaa aaaaaaaaaa aaaaaaatca aaattaaaat ctgagtcagt 4021ctgcctgcct cggttctcat tagtttaatt cttaatgcct tgcactttcc agcaatcatt 4081caatcaaaag agtgaaatga agcacattaa caaagcagga ggcgccacgg accgcctccc 4141tccacaccgc tccttccgcc ttcattcctt gcccacaggc ttgcactgga agctgaataa 4201gaatccccaa aactcaaact tcctagggat gccacccctt tagtagctca cacctccccc 4261ctccaagagc taagaaacaa aggagaatgt acttttgtag cttagataag caatgaatca 4321gtaaaggact gatctacttg ctccaccacc cctcccttaa taataacatt tactgttatt 4381tcctgggcct aagacttatg ttccagaact gtcacagctc cccatgtcac acccactagc 4441ttgtgatctt tgtcaaataa ctgaaatctt ttaagcctct agtttcttcc tttgtaaaac 4501agagataaaa tgttgtggtt tttaagtgag ataatccaag taaagcacct aacatggagt 4561agtgaatgaa catcggttgc tactaaaagt ggacatccta ccgcatcctt aatgccacta 4621ggcatttcca tacaatctgg ggaccaaaac ttcaatcata taaatgtatg aggttaatta 4681aaaacactac tgtaatctgc ttgtatgatc acaaaccacc acaaaagaaa agatcgtgaa 4741gattacactg taaacggact ctcaaatgat caggaggtgg tcacttcgca acttgctccc 4801tccacccaac tcaaaacagg agctcgagcc tgcctgtatt tgagactgga gctgcctgta 4861tgaggactgg atcaactgct agtcacgtta tatccaaatc tgcattatca ttgggcacat 4921tttcacagaa ttttactgaa ttattcctta attgtttaat ggttgggaat agtttgggaa 4981ttaccttcca tcaactctgc taagaaagga atggattctg gtagcaagac aatataattc 5041tcctttagtt tttcagccag tgctaacaca gtaatcaaag cagcaaatcg aacctgaaag 5101ggataaaaga gcaaagaaat aaaaagtagt gttactgtat ttattatctt aagagctgta 5161ctgacttgag acaagctcta actttttaaa cattagttca cacgcgttta ttcacttcat 5221tatgttcatt aagctttcat cttagaatac cagtttcacc atttgggagc tgtttgtaat 5281atgtgcaacc ttataaatag tgttttccaa actgtgtccc aggactgcaa atctttaatg 5341tgaaatgtct ttttataatc tcttccttta aaaaaaacca ataaaataaa atgccacatg 5401caaactc 5408 3 5534 DNA Homo sapiens misc_feature 201..203 ATG 3agccgcccac ggacgccaga gccgggaacc ctgacggcac ttagctgctg acaaacaacc 60tgctccgtgg agcgcctgaa acaccagtct ttggggccag tgcctcagtt tcaatccagg 120taacctttaa atgaaacttg cctaaaatct taggtcatac acagaagaga ctccaatcga 180caagaagctg gaaaagaatg atg ttg tcc tta aac aac cta cag aat atc atc 233 Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile 1 5 10tat aac ccg gta atc ccg tat gtt ggc acc att ccc gat cag ctg gat 281Tyr Asn Pro Val Ile Pro Tyr Val Gly Thr Ile Pro Asp Gln Leu Asp 15 20 25cct gga act ttg att gtg ata tgt ggg cat gtt cct agt gac gca gac 329Pro Gly Thr Leu Ile Val Ile Cys Gly His Val Pro Ser Asp Ala Asp 30 35 40aga ttc cag gtg gat ctg cag aat ggc agc agt gtg aaa cct cga gcc 377Arg Phe Gln Val Asp Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala 45 50 55gat gtg gcc ttt cat ttc aat cct cgt ttc aaa agg gcc ggc tgc att 425Asp Val Ala Phe His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile60 65 70 75gtt tgc aat act ttg ata aat gaa aaa tgg gga cgg gaa gag atc acc 473Val Cys Asn Thr Leu Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr 80 85 90tat gac acg cct ttc aaa aga gaa aag tct ttt gag atc gtg att atg 521Tyr Asp Thr Pro Phe Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met 95 100 105gtg cta aag gac aaa ttc cag gtg gct gta aat gga aaa cat act ctg 569Val Leu Lys Asp Lys Phe Gln Val Ala Val Asn Gly Lys His Thr Leu 110 115 120ctc tat ggc cac agg atc ggc cca gag aaa ata gac act ctg ggc att 617Leu Tyr Gly His Arg Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile 125 130 135tat ggc aaa gtg aat att cac tca att ggt ttt agc ttc agc tcg gac 665Tyr Gly Lys Val Asn Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp140 145 150 155tta caa agt acc caa gca tct agt ctg gaa ctg aca gag ata agt aga 713Leu Gln Ser Thr Gln Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg 160 165 170gaa aat gtt cca aag tct ggc acg ccc cag ctt cct agt aat aga gga 761Glu Asn Val Pro Lys Ser Gly Thr Pro Gln Leu Pro Ser Asn Arg Gly 175 180 185gga gac att tct aaa atc gca ccc aga act gtc tac acc aag agc aaa 809Gly Asp Ile Ser Lys Ile Ala Pro Arg Thr Val Tyr Thr Lys Ser Lys 190 195 200gat tcg act gtc aat cac act ttg act tgc acc aaa ata cca cct atg 857Asp Ser Thr Val Asn His Thr Leu Thr Cys Thr Lys Ile Pro Pro Met 205 210 215aac tat gtg tca aag agc ctg cca ttc gct gca agg ttg aac acc ccc 905Asn Tyr Val Ser Lys Ser Leu Pro Phe Ala Ala Arg Leu Asn Thr Pro220 225 230 235atg ggc cct gga cga act gtc gtc gtt aaa gga gaa gtg aat gca aat 953Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn Ala Asn 240 245 250gcc aaa agc ttt aat gtt gac cta cta gca gga aaa tca aag gat att 1001Ala Lys Ser Phe Asn Val Asp Leu Leu Ala Gly Lys Ser Lys Asp Ile 255 260 265gct cta cac ttg aac cca cgc ctg aat att aaa gca ttt gta aga aat 1049Ala Leu His Leu Asn Pro Arg Leu Asn Ile Lys Ala Phe Val Arg Asn 270 275 280tct ttt ctt cag gag tcc tgg gga gaa gaa gag aga aat att acc tct 1097Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu Glu Arg Asn Ile Thr Ser 285 290 295ttc cca ttt agt cct ggg atg tac ttt gag atg ata att tac tgt gat 1145Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys Asp300 305 310 315gtt aga gaa ttc aag gtt gca gta aat ggc gta cac agc ctg gag tac 1193Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu Glu Tyr 320 325 330aaa cac aga ttt aaa gag ctc agc agt att gac acg ctg gaa att aat 1241Lys His Arg Phe Lys Glu Leu Ser Ser Ile Asp Thr Leu Glu Ile Asn 335 340 345gga gac atc cac tta ctg gaa gta agg agc tgg tag cctacctaca 1287Gly Asp Ile His Leu Leu Glu Val Arg Ser Trp * 350 355cagctgctac aaaaaccaaa atacagaatg gcttctgtga tactggcctt gctgaaacgc 1347atctcactgt cattctattg tttatattgt taaaatgagc ttgtgcacca ttagatcctg 1407ctgggtgttc tcagtccttg ccatgaagta tggtggtgtc tagcactgaa tggggaaact 1467gggggcagca acacttatag ccagttaaag ccactctgcc ctctctccta ctttggctga 1527ctcttcaaga atgccattca acaagtattt atggagtacc tactataata cagtagctaa 1587catgtattga gcacagattt tttttggtaa aactgtgagg agctaggata tatacttggt 1647gaaacaaacc agtatgttcc ctgttctctt gagcttcgac tcttctgtgc tctattgctg 1707cgcactgctt tttctacagg cattacatca actcctaagg ggtcctctgg gattagttaa 1767gcagctatta aatcacccga agacactaat ttacagaaga cacaactcct tccccagtga 1827tcactgtcat aaccagtgct ctaccgtatc ccatcactga ggactgatgt tgactgacat 1887cattttatcg taataaacat gtggctctat tagctgcaag ctttaccaag taattggcat 1947gacatctgag cacagaaatt aaggcaaaaa accaaagcaa aacaaataca tggtgctgaa 2007attaacttga tgccaagccc aaggcagctg atttctgtgt atttgaactt agggcaaatc 2067agagtctaca cagacgccta cagaaagttt caggaagagg caagatgcat tcaatttgaa 2127agatatttat gggcaacaaa gtaaggtcag gattagactt caggcattca taaggcaggc 2187actatcagaa agtgtacgcc aactaaggga cccacaaagc aggcagaggt aatgcagaaa 2247tctgttttgt tcccatgaaa tcaccaatca aggcctccgt tcttctaaag attagtccat 2307catcattagc aactgagatc aaagcactct tccactttac gtgattaaaa tcaaacctgt 2367atcagcaagt taaatggttc catttctgtg atttttctat tatttgaggg gagttggcag 2427aagttccatg tatatgggat ctttacaggt cagatcttgt tacaggaaat ttcaaaggtt 2487tgggagtggg gagggaaaaa agctcagtca gtgaggatca ttttatcaca ttagactggg 2547gcagaactct gccaggattt aggaatattt tcagaacaga ttttagatat tatttctatc 2607catatattga aaagaatacc attgtcaatc ttattttttt aaaagtactc agtgtagaaa 2667ttgctagccc ttaattcttt tccagctttt catattaatg tatgcagagt ctcaccaagc 2727tcaaagacac tggttggggg tggagggtgc cacagggaaa gctgtagaag gcaagaagac 2787tcgagaatcc cccagagtta tttttctcca taaagaccat cagagtgctt aactgagctg 2847ttggagactg tgaggcattt aggaaaaaaa tagcccactc acatcattcc ttgtaagtct 2907taagttcatt ttcattttac gtggaggaaa aaaatttaaa aagctattag tatttattaa 2967tgaattttac tgagacattt cttagaaata tgcacttcta tactagcaag ctctgtctct 3027aaaatgcaag ttggcctttt gcttgccaca tttctgcatt aaacttctat attagcttca 3087aaggctttta aactcaatgc gaacattcta cgggatgttc ttagatgcct ttaaaaaggg 3147ggcagatcta attttatttg aaccctcact ttccaacttc accatgaccc agtactagag 3207attagggcac ttcaaagcat tgaaaaaaat ctactgatac ttactttctt agacaagtag 3267ttcttagtta accaccaatg gaactgggtt cattctgaat cctggaggag cttcctcgtg 3327ccacccagtg tttctgggcc ctctgtgtga gcagccaggt atgagctgtt ttagaagcag 3387cgtgttgcct tcatctctcc cgtttcccaa aagaacaaag gataaaggtg acagtcacac 3447tcctgggtta aaaaaagcat tccagaacca cttctcttta tgggcacaac aaagaaacga 3507aggctgaagt tcgcctaccc aaaatgaaaa gtaggcttta cagtcaaaag tacttctgtt 3567gattgctaaa taacttcatt ttcttgaaat agagcaactt tgagtgaaat ctgcaacatg 3627gataccatgt atataagata ctgctgtaca gaagagttaa ggcttacagt gcaaatgagg 3687cgtcagcttt gggtgctaaa attaacaagt ctaatattat taccatcaat caggaagaga 3747ataataaatg tttaaacaaa cacagcagtc tgtataaaaa taccgtgtat catttactct 3807ttctgcagct ctatacgata ggcaggagag gcttatgtgg cagcacaagc caggtgggga 3867ttttgtaacg aagtgataaa acatttgtaa gtaatccaag taggtgtatt aaggcaccaa 3927aagtaacatg gcacccaaca cccaaaaata aaaatatgaa atatgagtgt gaactctgag 3987tagagtatga aacaccacag aaagtcttag aaatagctct ggagtggctc tcccaggaca 4047gtttccagtt gctgaatagt cttttggcac tgatgttcta cttcttcaca ttcatctaaa 4107aaaaaaaaaa aaaaaaatca aaattaaaat ctgagtcagt ctgcctgcct cggttctcat 4167tagtttaatt cttaatgcct tgcactttcc agcaatcatt caatcaaaag agtgaaatga 4227agcacattaa caaagcagga ggcgccacgg accgcctccc tccacaccgc tccttccgcc 4287ttcattcctt gcccacaggc ttgcactgga agctgaataa gaatccccaa aactcaaact 4347tcctagggat gccacccctt tagtagctca cacctccccc ctccaagagc taagaaacaa 4407aggagaatgt acttttgtag cttagataag caatgaatca gtaaaggact gatctacttg 4467ctccaccacc cctcccttaa taataacatt tactgttatt tcctgggcct aagacttatg 4527ttccagaact gtcacagctc cccatgtcac acccactagc ttgtgatctt tgtcaaataa 4587ctgaaatctt ttaagcctct agtttcttcc tttgtaaaac agagataaaa tgttgtggtt 4647tttaagtgag ataatccaag taaagcacct aacatggagt agtgaatgaa catcggttgc 4707tactaaaagt ggacatccta ccgcatcctt aatgccacta ggcatttcca tacaatctgg 4767ggaccaaaac ttcaatcata taaatgtatg aggttaatta aaaacactac tgtaatctgc 4827ttgtatgatc acaaaccacc acaaaagaaa agatcgtgaa gattacactg taaacggact 4887ctcaaatgat caggaggtgg tcacttcgca acttgctccc tccacccaac tcaaaacagg 4947agctcgagcc tgcctgtatt tgagactgga gctgcctgta tgaggactgg atcaactgct 5007agtcacgtta tatccaaatc tgcattatca ttgggcacat tttcacagaa ttttactgaa 5067ttattcctta attgtttaat ggttgggaat agtttgggaa ttaccttcca tcaactctgc 5127taagaaagga atggattctg gtagcaagac aatataattc tcctttagtt tttcagccag 5187tgctaacaca gtaatcaaag cagcaaatcg aacctgaaag ggataaaaga gcaaagaaat 5247aaaaagtagt gttactgtat ttattatctt aagagctgta ctgacttgag acaagctcta 5307actttttaaa cattagttca cacgcgttta ttcacttcat tatgttcatt aagctttcat 5367cttagaatac cagtttcacc atttgggagc tgtttgtaat atgtgcaacc ttataaatag 5427tgttttccaa actgtgtccc aggactgcaa atctttaatg tgaaatgtct ttttataatc 5487tcttccttta aaaaaaacca ataaaataaa atgccacatg caaactc 5534 4 2471 DNA Homo sapiens misc_feature 201..203 ATG 4agccgcccac ggacgccaga gccgggaacc ctgacggcac ttagctgctg acaaacaacc 60tgctccgtgg agcgcctgaa acaccagtct ttggggccag tgcctcagtt tcaatccagg 120taacctttaa atgaaacttg cctaaaatct taggtcatac acagaagaga ctccaatcga 180caagaagctg gaaaagaatg atg ttg tcc tta aac aac cta cag aat atc atc 233 Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile 1 5 10tat aac ccg gta atc ccg tat gtt ggc acc att ccc gat cag ctg gat 281Tyr Asn Pro Val Ile Pro Tyr Val Gly Thr Ile Pro Asp Gln Leu Asp 15 20 25cct gga act ttg att gtg ata tgt ggg cat gtt cct agt gac gca gac 329Pro Gly Thr Leu Ile Val Ile Cys Gly His Val Pro Ser Asp Ala Asp 30 35 40aga ttc cag gtg gat ctg cag aat ggc agc agt gtg aaa cct cga gcc 377Arg Phe Gln Val Asp Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala 45 50 55gat gtg gcc ttt cat ttc aat cct cgt ttc aaa agg gcc ggc tgc att 425Asp Val Ala Phe His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile60 65 70 75gtt tgc aat act ttg ata aat gaa aaa tgg gga cgg gaa gag atc acc 473Val Cys Asn Thr Leu Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr 80 85 90tat gac acg cct ttc aaa aga gaa aag tct ttt gag atc gtg att atg 521Tyr Asp Thr Pro Phe Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met 95 100 105gtg cta aag gac aaa ttc cag gtg gct gta aat gga aaa cat act ctg 569Val Leu Lys Asp Lys Phe Gln Val Ala Val Asn Gly Lys His Thr Leu 110 115 120ctc tat ggc cac agg atc ggc cca gag aaa ata gac act ctg ggc att 617Leu Tyr Gly His Arg Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile 125 130 135tat ggc aaa gtg aat att cac tca att ggt ttt agc ttc agc tcg gac 665Tyr Gly Lys Val Asn Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp140 145 150 155tta caa agt acc caa gca tct agt ctg gaa ctg aca gag ata agt aga 713Leu Gln Ser Thr Gln Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg 160 165 170gaa aat gtt cca aag tct ggc acg ccc cag ctt agc ctg cca ttc gct 761Glu Asn Val Pro Lys Ser Gly Thr Pro Gln Leu Ser Leu Pro Phe Ala 175 180 185gca agg ttg aac acc ccc atg ggc cct gga cga act gtc gtc gtt aaa 809Ala Arg Leu Asn Thr Pro Met Gly Pro Gly Arg Thr Val Val Val Lys 190 195 200gga gaa gtg aat gca aat gcc aaa agc ttt aat gtt gac cta cta gca 857Gly Glu Val Asn Ala Asn Ala Lys Ser Phe Asn Val Asp Leu Leu Ala 205 210 215gga aaa tca aag gat att gct cta cac ttg aac cca cgc ctg aat att 905Gly Lys Ser Lys Asp Ile Ala Leu His Leu Asn Pro Arg Leu Asn Ile220 225 230 235aaa gca ttt gta aga aat tct ttt ctt cag gag tcc tgg gga gaa gaa 953Lys Ala Phe Val Arg Asn Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu 240 245 250gag aga aat att acc tct ttc cca ttt agt cct ggg atg tac ttt gag 1001Glu Arg Asn Ile Thr Ser Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu 255 260 265atg ata att tac tgt gat gtt aga gaa ttc aag gtt gca gta aat ggc 1049Met Ile Ile Tyr Cys Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly 270 275 280gta cac agc ctg gag tac aaa cac aga ttt aaa gag ctc agc agt att 1097Val His Ser Leu Glu Tyr Lys His Arg Phe Lys Glu Leu Ser Ser Ile 285 290 295gac acg ctg gaa att aat gga gac atc cac tta ctg gaa caa tca ttc 1145Asp Thr Leu Glu Ile Asn Gly Asp Ile His Leu Leu Glu Gln Ser Phe300 305 310 315aat caa aag agt gaa atg aag cac att aac aaa gca gga ggc gcc acg 1193Asn Gln Lys Ser Glu Met Lys His Ile Asn Lys Ala Gly Gly Ala Thr 320 325 330gac cgc ctc cct cca cac cgc tcc ttc cgc ctt cat tcc ttg ccc aca 1241Asp Arg Leu Pro Pro His Arg Ser Phe Arg Leu His Ser Leu Pro Thr 335 340 345ggc ttg cac tgg aag ctg aat aag aat ccc caa aac tca aac ttc cta 1289Gly Leu His Trp Lys Leu Asn Lys Asn Pro Gln Asn Ser Asn Phe Leu 350 355 360ggg atg cca ccc ctt tag tagctcacac ctcccccctc caagagctaa 1337Gly Met Pro Pro Leu * 365gaaacaaagg agaatgtact tttgtagctt agataagcaa tgaatcagta aaggactgat 1397ctacttgctc caccacccct cccttaataa taacatttac tgttatttcc tgggcctaag 1457acttatgttc cagaactgtc acagctcccc atgtcacacc cactagcttg tgatctttgt 1517caaataactg aaatctttta agcctctagt ttcttccttt gtaaaacaga gataaaatgt 1577tgtggttttt aagtgagata atccaagtaa agcacctaac atggagtagt gaatgaacat 1637cggttgctac taaaagtgga catcctaccg catccttaat gccactaggc atttccatac 1697aatctgggga ccaaaacttc aatcatataa atgtatgagg ttaattaaaa acactactgt 1757aatctgcttg tatgatcaca aaccaccaca aaagaaaaga tcgtgaagat tacactgtaa 1817acggactctc aaatgatcag gaggtggtca cttcgcaact tgctccctcc acccaactca 1877aaacaggagc tcgagcctgc ctgtatttga gactggagct gcctgtatga ggactggatc 1937aactgctagt cacgttatat ccaaatctgc attatcattg ggcacatttt cacagaattt 1997tactgaatta ttccttaatt gtttaatggt tgggaatagt ttgggaatta ccttccatca 2057actctgctaa gaaaggaatg gattctggta gcaagacaat ataattctcc tttagttttt 2117cagccagtgc taacacagta atcaaagcag caaatcgaac ctgaaaggga taaaagagca 2177aagaaataaa aagtagtgtt actgtattta ttatcttaag agctgtactg acttgagaca 2237agctctaact ttttaaacat tagttcacac gcgtttattc acttcattat gttcattaag 2297ctttcatctt agaataccag tttcaccatt tgggagctgt ttgtaatatg tgcaacctta 2357taaatagtgt tttccaaact gtgtcccagg actgcaaatc tttaatgtga aatgtctttt 2417tataatctct tcctttaaaa aaaaccaata aaataaaatg ccacatgcaa actc 2471 5 316 PRT Homo sapiens VARIANT 18 5-2-162 polymorphic amino acid Tyr or Phe 5Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val Ile1 5 10 15Pro Tyr Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu Ile 20 25 30Val Ile Cys Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val Asp 35 40 45Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr Leu65 70 75 80Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro Phe 85 90 95Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asp Lys 100 105 110Phe Gln Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His Arg 115 120 125Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys Val Asn 130 135 140Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser Thr Gln145 150 155 160Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg Glu Asn Val Pro Lys 165 170 175Ser Gly Thr Pro Gln Leu Ser Leu Pro Phe Ala Ala Arg Leu Asn Thr 180 185 190Pro Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn Ala 195 200 205Asn Ala Lys Ser Phe Asn Val Asp Leu Leu Ala Gly Lys Ser Lys Asp 210 215 220Ile Ala Leu His Leu Asn Pro Arg Leu Asn Ile Lys Ala Phe Val Arg225 230 235 240Asn Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu Glu Arg Asn Ile Thr 245 250 255Ser Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys 260 265 270Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu Glu 275 280 285Tyr Lys His Arg Phe Lys Glu Leu Ser Ser Ile Asp Thr Leu Glu Ile 290 295 300Asn Gly Asp Ile His Leu Leu Glu Val Arg Ser Trp305 310 315 6 358 PRT Homo sapiens VARIANT 18 5-2-162 polymorphic amino acid Tyr or Phe 6Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val Ile1 5 10 15Pro Tyr Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu Ile 20 25 30Val Ile Cys Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val Asp 35 40 45Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr Leu65 70 75 80Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro Phe 85 90 95Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asp Lys 100 105 110Phe Gln Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His Arg 115 120 125Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys Val Asn 130 135 140Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser Thr Gln145 150 155 160Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg Glu Asn Val Pro Lys 165 170 175Ser Gly Thr Pro Gln Leu Pro Ser Asn Arg Gly Gly Asp Ile Ser Lys 180 185 190Ile Ala Pro Arg Thr Val Tyr Thr Lys Ser Lys Asp Ser Thr Val Asn 195 200 205His Thr Leu Thr Cys Thr Lys Ile Pro Pro Met Asn Tyr Val Ser Lys 210 215 220Ser Leu Pro Phe Ala Ala Arg Leu Asn Thr Pro Met Gly Pro Gly Arg225 230 235 240Thr Val Val Val Lys Gly Glu Val Asn Ala Asn Ala Lys Ser Phe Asn 245 250 255Val Asp Leu Leu Ala Gly Lys Ser Lys Asp Ile Ala Leu His Leu Asn 260 265 270Pro Arg Leu Asn Ile Lys Ala Phe Val Arg Asn Ser Phe Leu Gln Glu 275 280 285Ser Trp Gly Glu Glu Glu Arg Asn Ile Thr Ser Phe Pro Phe Ser Pro 290 295 300Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys Asp Val Arg Glu Phe Lys305 310 315 320Val Ala Val Asn Gly Val His Ser Leu Glu Tyr Lys His Arg Phe Lys 325 330 335Glu Leu Ser Ser Ile Asp Thr Leu Glu Ile Asn Gly Asp Ile His Leu 340 345 350Leu Glu Val Arg Ser Trp 355 7 368 PRT Homo sapiens VARIANT 18 5-2-162 polymorphic amino acid Tyr or Phe 7Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val Ile1 5 10 15Pro Tyr Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu Ile 20 25 30Val Ile Cys Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val Asp 35 40 45Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr Leu65 70 75 80Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro Phe 85 90 95Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asp Lys 100 105 110Phe Gln Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His Arg 115 120 125Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys Val Asn 130 135 140Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser Thr Gln145 150 155 160Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg Glu Asn Val Pro Lys 165 170 175Ser Gly Thr Pro Gln Leu Ser Leu Pro Phe Ala Ala Arg Leu Asn Thr 180 185 190Pro Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn Ala 195 200 205Asn Ala Lys Ser Phe Asn Val Asp Leu Leu Ala Gly Lys Ser Lys Asp 210 215 220Ile Ala Leu His Leu Asn Pro Arg Leu Asn Ile Lys Ala Phe Val Arg225 230 235 240Asn Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu Glu Arg Asn Ile Thr 245 250 255Ser Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys 260 265 270Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu Glu 275 280 285Tyr Lys His Arg Phe Lys Glu Leu Ser Ser Ile Asp Thr Leu Glu Ile 290 295 300Asn Gly Asp Ile His Leu Leu Glu Gln Ser Phe Asn Gln Lys Ser Glu305 310 315 320Met Lys His Ile Asn Lys Ala Gly Gly Ala Thr Asp Arg Leu Pro Pro 325 330 335His Arg Ser Phe Arg Leu His Ser Leu Pro Thr Gly Leu His Trp Lys 340 345 350Leu Asn Lys Asn Pro Gln Asn Ser Asn Phe Leu Gly Met Pro Pro Leu 355 360 365 8 1738 DNA Mus musculus 8gagtgttact accaccgggg acaagttttt actttgagta atccttaaat gaagagtggg 60taaagtgtgt atacggaaga gagactccaa tcaacaatat caataagttg aaaaagaaaa 120atg ttg tcc tta aat aac cta caa aat atc atc tat aac ccg ata atc 168Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Ile Ile1 5 10 15ccc tat gtt ggc acc att act gag caa ttg aag cct ggc tct ctg att 216Pro Tyr Val Gly Thr Ile Thr Glu Gln Leu Lys Pro Gly Ser Leu Ile 20 25 30gta atc cgt ggg cat gtc cct aaa gat tca gaa aga ttc cag gtt gac 264Val Ile Arg Gly His Val Pro Lys Asp Ser Glu Arg Phe Gln Val Asp 35 40 45ttt cag ctg ggc aac agc ctg aag cca aga gca gac gtg gcc ttc cac 312Phe Gln Leu Gly Asn Ser Leu Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60ttt aac cct cgg ttc aaa agg tct agc tgc att gtt tgt aac aca ctg 360Phe Asn Pro Arg Phe Lys Arg Ser Ser Cys Ile Val Cys Asn Thr Leu65 70 75 80aca cag gag aag tgg ggc tgg gag gag atc acc tac gac atg ccc ttc 408Thr Gln Glu Lys Trp Gly Trp Glu Glu Ile Thr Tyr Asp Met Pro Phe 85 90 95aga aaa gaa aag tcc ttt gag atc gtg ttc atg gtg ctc aag aac aaa 456Arg Lys Glu Lys Ser Phe Glu Ile Val Phe Met Val Leu Lys Asn Lys 100 105 110ttc cag gtg gct gtg aac gga agg cat gtt ctg ctg tac gcc cac agg 504Phe Gln Val Ala Val Asn Gly Arg His Val Leu Leu Tyr Ala His Arg 115 120 125atc agc ccg gag cag atc gac aca gtg ggc atc tac ggc aaa gtg aac 552Ile Ser Pro Glu Gln Ile Asp Thr Val Gly Ile Tyr Gly Lys Val Asn 130 135 140atc cac tcc atc ggg ttc aga ttc agc tcg gat tta cag agt atg gaa 600Ile His Ser Ile Gly Phe Arg Phe Ser Ser Asp Leu Gln Ser Met Glu145 150 155 160aca tct gct ctg gga ctg aca cag ata aac aga gag aat ata caa aag 648Thr Ser Ala Leu Gly Leu Thr Gln Ile Asn Arg Glu Asn Ile Gln Lys 165 170 175cca ggc aag ctc cag ctg agc ctg cca ttt gaa gca agg ttg aat gcc 696Pro Gly Lys Leu Gln Leu Ser Leu Pro Phe Glu Ala Arg Leu Asn Ala 180 185 190tcc atg ggt cct gga cga acc gtt gtc att aaa ggg gaa gtg aac acc 744Ser Met Gly Pro Gly Arg Thr Val Val Ile Lys Gly Glu Val Asn Thr 195 200 205aat gcc cga agc ttt aat gtt gac cta gtg gca gga aaa aca agg gat 792Asn Ala Arg Ser Phe Asn Val Asp Leu Val Ala Gly Lys Thr Arg Asp 210 215 220atc gct ctg cac ttg aac cca cgc ctc aat gtg aaa gca ttt gta aga 840Ile Ala Leu His Leu Asn Pro Arg Leu Asn Val Lys Ala Phe Val Arg225 230 235 240aat tcc ttt ctt cag gat gcc tgg gga gaa gag gag aga aat att acc 888Asn Ser Phe Leu Gln Asp Ala Trp Gly Glu Glu Glu Arg Asn Ile Thr 245 250 255tgc ttc cca ttt agt tct ggg atg tac ttt gag atg ata atc tac tgt 936Cys Phe Pro Phe Ser Ser Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys 260 265 270gat gtc cgg gaa ttc aag gtt gct ata aat ggt gtg cac agc ctg gag 984Asp Val Arg Glu Phe Lys Val Ala Ile Asn Gly Val His Ser Leu Glu 275 280 285tac aaa cac aga ttt aaa gac cta agc agt att gat aca cta tca gtc 1032Tyr Lys His Arg Phe Lys Asp Leu Ser Ser Ile Asp Thr Leu Ser Val 290 295 300gat ggt gat atc cgt ttg ctg gat gta agg agc tgg tag ctaccatgac 1081Asp Gly Asp Ile Arg Leu Leu Asp Val Arg Ser Trp *305 310 315tgccaaaacc cccgaaatac aaaatggctt atccggtact ggccatgtca aatgcatctc 1141gctttcacca tattgtttat attgctaagt tgagctcctc caacatcaag tcctactggt 1201gttgtcaggt ctggccatgc agtacattca gaggaacaga gccggggcaa tcacagctca 1261ctgccagaga ggctctgcac actgggtccc tcttataaac cacactcagc aaatatttaa 1321gtgcctaata tactacatat actagctaat agggatggca agcatacttc ctttgtatat 1381tctctgagcc gggcacagac atggcagggc ccagaacttg tgtggtccat gttttctagc 1441acttcgtacc agtttctggc ctcctaatgt agggtcttct tgctggcatt gcattaaccc 1501cactaggggc ctttgcagtt aaggtcagaa aaatatacta atggatggca aacactactt 1561ccccagcaac ccttttcata atcagcattc tatcatatct cataattgaa gactgcatag 1621catttactta gctctcaccg ctttaaactt tataaaatgt atgatgctga acacagcaga 1681aaaactgagg ccaaaaccct gaattatgac aaaacaagtg ttctgctcca agcagat 1738 9 316 PRT Mus musculus 9Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Ile Ile1 5 10 15Pro Tyr Val Gly Thr Ile Thr Glu Gln Leu Lys Pro Gly Ser Leu Ile 20 25 30Val Ile Arg Gly His Val Pro Lys Asp Ser Glu Arg Phe Gln Val Asp 35 40 45Phe Gln Leu Gly Asn Ser Leu Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60Phe Asn Pro Arg Phe Lys Arg Ser Ser Cys Ile Val Cys Asn Thr Leu65 70 75 80Thr Gln Glu Lys Trp Gly Trp Glu Glu Ile Thr Tyr Asp Met Pro Phe 85 90 95Arg Lys Glu Lys Ser Phe Glu Ile Val Phe Met Val Leu Lys Asn Lys 100 105 110Phe Gln Val Ala Val Asn Gly Arg His Val Leu Leu Tyr Ala His Arg 115 120 125Ile Ser Pro Glu Gln Ile Asp Thr Val Gly Ile Tyr Gly Lys Val Asn 130 135 140Ile His Ser Ile Gly Phe Arg Phe Ser Ser Asp Leu Gln Ser Met Glu145 150 155 160Thr Ser Ala Leu Gly Leu Thr Gln Ile Asn Arg Glu Asn Ile Gln Lys 165 170 175Pro Gly Lys Leu Gln Leu Ser Leu Pro Phe Glu Ala Arg Leu Asn Ala 180 185 190Ser Met Gly Pro Gly Arg Thr Val Val Ile Lys Gly Glu Val Asn Thr 195 200 205Asn Ala Arg Ser Phe Asn Val Asp Leu Val Ala Gly Lys Thr Arg Asp 210 215 220Ile Ala Leu His Leu Asn Pro Arg Leu Asn Val Lys Ala Phe Val Arg225 230 235 240Asn Ser Phe Leu Gln Asp Ala Trp Gly Glu Glu Glu Arg Asn Ile Thr 245 250 255Cys Phe Pro Phe Ser Ser Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys 260 265 270Asp Val Arg Glu Phe Lys Val Ala Ile Asn Gly Val His Ser Leu Glu 275 280 285Tyr Lys His Arg Phe Lys Asp Leu Ser Ser Ile Asp Thr Leu Ser Val 290 295 300Asp Gly Asp Ile Arg Leu Leu Asp Val Arg Ser Trp305 310 315 10 18 DNA Artificial Sequence sequencing oligonucleotide PrimerPU 10tgtaaaacga cggccagt 18 11 18 DNA Artificial Sequence sequencing oligonucleotide PrimerRP 11caggaaacag ctatgacc 18 12 106746 DNA Homo sapiens misc_feature 1..68647 5′regulation region 12ttggcttggc agggcaacca gctcaccaga ctctctgcag acccgaagtc attacataca 60gtatgataac agggaatgga cccgaccagc atttgctgga gatgatatct ggtgtcagcc 120cgacaggccc ctacctgctt ctcttgatat gcaggaatcc cttcaagctc caacaagatc 180tgtttaatag actggagagt cctttagttc cttcctctaa gggaaaatca gatcgttctg 240gtttgcttgg taactcctta cttcatccct gatgggaagt ttatagaatg aggaaccagg 300gctattacat gaaactataa aactgcctag agcacatact tggtattttt aacattgttg 360agagggactc acttaattca gccttgcagc tattgcattc cwgtccaaac caacggcagg 420ttctcaaaac aagcggtgaa agggttcctg ttgcagagct gtctggacat ttaaagaagg 480gagaggaaat ctcaaggggt cggttgcact ggaatagaaa tcgcctgttc tttttttttg 540agacggagtc tcgctctgtc acccaggctg gagagcagtt gcgcgatctt tgctcactgc 600aacctctgcc tcccgggttc acgccattct cctgcctcag cctcctgaat agctgggact 660acaggcgccc gccaccacgt ctggctcatt ttttgtattt ttagtagaga tggagtttca 720ccattttagc caggatggtc tcgatctgct gaccttgtaa tccacccgcc tcggcctccc 780aaagtacagg gattataggc gtgagccacc gcgcccaggt gcctgttcct tttttaagag 840tctcactctg tcgcccaggc tggcgtgcag tggcgcgatc tctgcttact gcagtctccg 900tctcctgagt tcaaatcaag cgagaaatca cttgttctct tctgtgaacg gaagcatcgc 960agatctctct tggcctcaca ctcctccatc tccctgattc ctctgttctt catttaccta 1020ccttcccagc agtctgcaga gctggccgct cactcacctc tagtaagggg atggagggtc 1080ctgtgttgga ataactcact gaccgctaga aagttaaaaa taaatgggta atgccaggag 1140aacttggctg gtgccttaaa agccatagaa cttctctttc catctgtaga taactgtaga 1200caattttgtc caaaacagat aatgatctga ttctacctcc cattggtatt tcccttcctc 1260ggcctgtgac atctcacttt ctctagactg aactttatcc cagactgtga ccttgccatg 1320accttcctcc tgcgtgtgcc tctgccacca caggaatggc cacgcctcag atcatgtcac 1380cgctgggaac aaaccctcta cctgcgactc tgaagttccc tctctgaccc tcttttcttt 1440cttcccctcc cccctcccct cactccctct gcacctgtgt ttcgctgtca cgctcccaac 1500tcatccctgt agagctggtg aagagatgct gatgtagttc ttgaccttga accccagccc 1560tgcagccgtc ctgtggcctc actgacccag cgtcatgccc tggtcaagca ttttggtgat 1620gctcttggtg attttcaatg ggacctgcct tgccaagccc tgggcttagg tgaaccagga 1680ccacctgcat tctatgtttt tgattgctgg aaaaaaatca tgaaatgtca actgttgttc 1740tcatttttcc cactgccagt tcctgctacc caacctccgc cctcatttca aggccttgag 1800tacttttttt ctatagtgaa gtctcccaaa aatgatattt ttttaaaaaa gaaaagccat 1860agtactctga tttgatgtgg tctgttaata cctatgggct ttgacttgtt tctgctttta 1920gacctagaca aaataaaata tctgtggtaa aacatattca agtttaccgg gcacgggggc 1980tcacgcctgt aatcccagca ctttgggagg ctggggcagg cagatcactt gagcccagga 2040gtttgagacc agcctgggca acagggtgaa acaacatctc tacaaaaata caaaaaatac 2100ctgggcattg tggtgcatgc ctgtagtcgc agctactcgg gagactgagg tgggaggatg 2160gcttgagctc tggaggcgga ggtcatagtg agccaagatc gtgccactga actccagcct 2220gggcaacaga ggcagattct ttctctctaa aaaacataaa ataaaaaaag gccaggcgca 2280gtggctcaca cctgtaatcc cagcactttg ggaggctgag gggggcggac gaagaggtca 2340ggagatagag accatcctgg ccaacatggt gaaaccctgc ctctactaaa aatacaaaaa 2400ttagccgggt gtagtggtgc atgcctgtaa tctcaactac tcaggaggct gaggcaggaa 2460aatcgcttga acccaggagg cggaggctgc agtgagccaa gatcgcacca ctgcactcca 2520gcctgggtga cagagcaaga ctctgtcccc caccaaaaaa aataaataaa taaatcaggc 2580caaagggcaa aaatgcttgc tttttagcac ttagtagtta tttccccaag aagagcggga 2640gagaagttta ttaataatga aactggacag ttctttatca gctctaattg tttgactcaa 2700tggcttctct tctcattacc atgcagtgct ctgctggctg caatgccttt gaacttcaca 2760agaaggttag aatttcactg agacattcgg atggtgtggg tgtcagggtg cagctctcac 2820acatagttga gagtgtaaat tgatacaact ttatggaaaa ttaattggga gtacccattc 2880acactcctgt ctagcaatct cactttaagg acttgatcct acagaactca ttacatggtg 2940caaggttcac agtgtggcat tcaaaataga gaagagctgc gggtaactcc catgcccgtt 3000ggcaggaact ggttgaataa attatggtgc atcagtgctg tggggtatca ttaaaccatt 3060aaaaagaaga gagagtcctg gccttaaaaa aaacttatct gatgtattgt taaacagata 3120aagcaagttg tagatcaatg tgatttgggg ctaaaaaaat atttctatat aggtgtgaac 3180atggccatga ctaaggaatc aggaaggaag tacctagatt gtaaccagta acatgtcggg 3240agtgagatgg gattgagaga cgtaataata gattgagaga aaaagatttt cccatctctt 3300tttgattttt taagaaaaca gcatgatttt cagtaatttt tacttttgtg tgtttttggt 3360attttttctt tttctttttt tttttttttt ttttgagacg gagtttcact cttgttgccc 3420aggctggaat gcgatggccc agtcccagct cactgcaacc ttcacttccc aggttcaaga 3480gattctcctg tctcagcctc ccgagtagct gggattacag gcccctgcca ttacgcccag 3540ctactttttg tatttttagt agagatgggt ttcaccgtgt tggttaggct ggtttgaact 3600cctgagctca ggcgatctgc ctacttcagc ctcccaaaat gctgggatta cagccgtgag 3660ccaccgcccc cagccggtat tttttcaaat caaagaaaaa ataatagagt aaatcatcca 3720aaactttaga tggtatttag actcagtaaa cttttcatat atgacagatg aagccaaatg 3780gtctttctgt gcagtcagct agcacacaat tgtgcacccg aggaaaatta gagactgaac 3840cggggtgtct gtggatgcat ttcctcagca ttcagccttc cttttgcccg tgttctagca 3900ttacttctgt cctacagcct gggatttgtg aatgaaatag acaggtgcaa aaactccctg 3960cctgtctgta atatccatag ccccgtgctc tacttgtatt tgcatgtaca aaccataatc 4020tcctgtaaaa tactctgtga tatttctgaa taataataaa ctctacatcc tacacaaagg 4080caaaacccct gtatctttca tctttgaaac catagcaaag gtatgaaatt acacctgagc 4140atgcctggcc tcaaagtcct ggaacggtta tgtctttgac cctcacttca actcaactcc 4200agaagaagca ggtcttcctt gtaattggat agaaaactca ttgtagagaa gaaagatcta 4260caggtcaaga aacccacagg tttgctgtaa tccgagcaaa gcactgtagc atttatttta 4320tattttcact cttcttattt agctcttttt tttttttttt ttttgagatg gagtttcatt 4380cttgtcaccc agcctggagc aatggtgcca tctcggctca ctgcaacctc tgcctcccag 4440gttcaagtga ttctcctgcc tcagcctcct gagtagctgg ggttacaggc tcccaccgcc 4500acacccagcc aattttttgt atttttagta gagacggggt ttcaccatgt tagccagact 4560ggtctcaaac tcctggcctt aggtgatcca cccgcctcag catcccaaag tgctgggatt 4620acaggcgcac cggccttagc tcttttatcc ttaatgaaat gctcctcatt ccctgaggtc 4680tcacttgaat tcttgcccac ctctgggttg ccttcctctt ctgtctgtgc tttgtaacac 4740gtggttcctt atgatgtcaa tatttatgca tatgtcttca ttccattact ggattataat 4800cttgaagcaa cagatttttg tctctatatc ccagagccta gaatggattc ttacactggg 4860cagtaagtac ttaataaatg tatcccaaat caaataaata catttcttct ttttcttttc 4920tttttttttt ttttttgaga cagggttcca ctctgtcacc caggctggag tgtaatgaca 4980tgatctcagc ttactacagc ctcaatctcc tgggcttaag caatcctccc acctcagcct 5040cccacatagc taggactaca ggcgctcacc acaacacctc atttttgtat attttttgta 5100gagattgggg gatctcacta cgttgccccg gctggttttg aacttctggg ctcagacaat 5160ccacccacct tggcctccca aactgttgag attacaggaa tgagccacca ttccctggcc 5220aaatacattt ctaaaagcca gtttctggag tatactgtca aataatagat atatgtccac 5280atttttatac ggacttatat tgtaagaaaa agtaaaaata agtgtgaagt tattacagta 5340atagtaatta ttttgcagaa aaagaactga gtttaaacag gctttttaga aaaacccaac 5400aggagattca cagtctggta ctaacgttta gacatggatc atcagtaaat gtgttccaaa 5460gagttacaca gataccagct ttgtcttggg aattcttacc cctgaaaatt gattgactat 5520cactgactgt gtgacatgag aaagttttgt ggggtttttt ttttgtattt ttttgagacg 5580tatcttgctc tgtcacccaa gctggagtcc actggcgcga tcttggctca ctacaacctc 5640tgccgcctgg ttcaagcgat tctcctgcct cagcctccag aatagctgcg attacaggca 5700cctgccacca tgcccggcta atttttgtat ttttagtaaa gacggggttt catcgtgttg 5760gccaggatgg tcttgaactc ctgacctcag gtgatctgcc cacctcagcc tcccaaagtg 5820ctgggattac aggcatgagc caccgtgccc agcctgaaaa agttttgaac ggtctaaatc 5880catatgctgt gaatcctatt accatcacac acttaggcat ttaaaatcat attttcaagg 5940ccaggtactg aaatattttc tgcaagcaga gagatcaaac tttagcattg ttattcttgt 6000agtagtttca tagtttgagg tcttagattt aagtcttcca ttgattttga tttgattttt 6060gtatatggag ataggggtct agtttcattc ttttgcatat ggatatccag ttttcccagc 6120accatttatt gaagagactg tctttttcac cagtgtatgc tcttggcacc ttcgtcaaaa 6180atgagttccc tgtaggtgtg tgggtttgct tctggttctc tattctgtcc cattggtcta 6240agtgtttggt tttatgctac taccatgctg gttggtatag ctctgcagta taacttgaaa 6300gcaggtaatg tgattccaaa gaagctagtt aagtaattga gctaaactgg aacctcaggt 6360gtagaagtca taagcgtggg gagcgtttct tctcaggttc tctgcctata atttagtttg 6420ccacaccaga tgaacagtga caacttggtc ttggtgttcg tggtggtttc caaccaaact 6480ttggtcataa caggtgaacc agcctggggc atgctttccc attcggttat cctccccata 6540gtttgcaaag tagcaaagat gaactcttca tgagttggct aagcatagac atttcaagac 6600caaactaaac gtcctgaaga gcatgtttca cagaaaacta gcccctaagg gaccagtggg 6660ggctgtcaga gaacaaggtt tcaacgtact gagttttaaa gatctaattg gcttttaata 6720acaattcatg aaccaggcac catagtctac aaaatagaca gggtttctgc tgggcactgc 6780aggacagttg gtttttggaa ggtggcttga gcaggaacaa ggaaaaagca ccgtgccaag 6840agtggattgg ttaacatcag gggacttcgg gtgactttcc ttctatgggt taaagcaaag 6900gggacttccc tagcatgtca gctcaggttg actgggcccc tttggattgg ttgctgtgaa 6960tctcctagtt ttttttgttt tggtttggtt tgggtctttt ggggaaaacg ggccagtttg 7020gagattcagc tattatttct ctctcctgat atcagaagat cagatcttat gagtacacag 7080ctgaggtttt gggttggtga tgtggaaccc tggtgtgagt gactccattt tgggttggtc 7140tattggggtc tcggtgcagg agctcagtcc aaatcagtgg cctctcctca tttttatttg 7200acttctccat caatctatcc gtgtctcccg tcacatcagt ccattccccc gtgggctgca 7260cattcagctc ggagctgaga gcttttccca gggtgtgccc tggggtttct gctgcttgca 7320gcctgatatt aaatctcagg tgtaaatctt cagaggcaac tgttccttag tacccagagc 7380tttcagctcc ctgagcagaa atgggacttg actgtcagtt tataaactaa ccaaggtgtg 7440aaattcatgc aacttagccg actttctgtt caaagaattc ttggcagcag ttaatacatt 7500ttgcccaaat ataagataat tcccttgtac tcacaatgag aaagttttac aaaatggggg 7560ttttctttag tttacttgaa tataaaacat aggtgttcca ctctgcagta ccttaacagt 7620tcttaaggag atgtttgaaa caacccatgt ccaggcctca cacctcgcca attaaataaa 7680tgagaagttc ttcccagcca gtgttaagaa aaattaacat caagttttag gaaggtagac 7740agattatgca aatgcatacc tatatgattt aagttattac attaatttac acacacatat 7800ttaaaatcat agattaatct aatttagaga tgctgcattt tttccatctc tcctgtttca 7860taaatgttat tcacacggca tttctctgct atcctcggaa tagtgtttgt atcgtgtcac 7920tctggcacgg ggctctacag aacatgtcga gcgtgttgcc ttccctactg cccacatcgt 7980ttgagagaac acattttaaa cattttttta ttgtggtaaa atacacataa cataaaagtt 8040acgattttaa ccttttttaa ctctgtcatc caggctggag tgcagtggcg agatcttggt 8100tcactgcaac ctccgcctcc taggtccaag tgattctcct gcctcagcct tccgagtagc 8160tgggattaca ggtgcacacc accacgnccg gctaattttg tatttttagt agatgcgggg 8220tttcaccatg ttagccaggt tggtctcgaa ctcccgacct caggtgatca gcccgcctcg 8280cctccccagt gctgggatta caggcgtgcg ccactgtgcc gggcccattt taaccacttt 8340taagtgcaca gttcagtggc attaagtata ttcgcggtgt tgtgcgaccg tcaccaccat 8400tcacctccag aacttctctg tcttcccaaa ctgaaattct gtacccattg aacggtaact 8460ccccattccc catttctgct tcctaggccc tgacatggag gctgggccaa cggatatctc 8520acctcccttc aggcttctcc agatttgccc ccgtttttct ccctctttgt cccatctcca 8580aagaaatggt gtcttttcat catcaaggtc catcccttgc tccttgaata cactccaggc 8640ccagtggaac aggcatcctg tggggtgcac ggacagggtg cctggggaac acccagggca 8700cagaacccag accgggggtt tggagaaggt gtcctagcag aagtgatgtc taagctgagg 8760ccctacagat aagagaaagt aagcagatga aagggctggg gagggtggca tttcaggcct 8820acacaaccac acgcgtgttc ttcagccatc tccatggcct cactgcccac ctggtatcag 8880ccggccacca cccggctaga acggctttca aaatcgctgc tcgtctactc ctcaccaaat 8940cttgtcttca cttggtgctc aagcccatca cctttctgca agtattattt tttttttttt 9000ggagatggag tctcgctctg tcacccgggc tggagtgcag tggttcaatg atagctcact 9060gcaaccttga actcctgggc tcaagatcct cttgccacag cctcccaaag tgctgagatt 9120acaggcacaa gccaccatgc gtggtccttg ctgcaacttt tttttttttt tttttttttt 9180ttttgagaca gaatctcgct ctgtcgctca ggctggagtg cagtggtgtg atctcggctc 9240aatgcaacct ccgcctcccg ggttcaggtg attctcctgc ctcaccctcc tgagtagcta 9300ggaacacagg cgctcaccac cacatccagc taatttttgt gtttttagta gagccggggt 9360tttgccatgt tggccaggct tctctcaaac tcctggacct cgggcgattg gcccgcctcg 9420gcctcccaaa atgctggaat tacaggcatg agccaccgtg cctggccatt tgctgcaact 9480tttgacactg ctccccctgc ttttcttccc ctctctgacc tcctttctct gctgtccttt 9540cgttccttcc tctgccactg aagtgtcctt ctcaggtcct tctcaaggtt gtgaccttac 9600agctgtctct tcacttccag tcatttcttt cataatcact ttgacatcct tattttcatc 9660tcctgccctg gcctctccca gggaccagga ccatgcattc agctcctggg ggcatctcaa 9720gcttgttgtg tgtgagcctg cccttgttgt cttctccgtc acctcttcac agcttgctct 9780gcatttcacc tcctttcctg ttttccccag tgatcgcatc tctacagcgg ctctcacttc 9840atccccttct ctcctagagg agtgatgcgg agtctcatta atccttgctt atgtcattct 9900tcccccttct ctgtccatca cctccacatg tcctgttccc ccatgcgtcc tacactgtag 9960ccaggtgggt atttcctgtg ctggtcttag acaccccctg aggataccct gcttcaggcg 10020agagccctca gtgactccct gttgtccgga atgacgtcca gctccttgga cagtccccag 10080tgtattcacc tgtctcatct ccttcttttc gttttgtttg tttttcttaa cttccagccc 10140gatttctgaa tcatctccct cttgcccctc ccattgcctt tgcttaagac taaatgctcc 10200ttcctcccaa gtccccactg cccagatttc agcagggtcc atctcaaaca tgtctgtctc 10260caagaaactg cctctgattt ttttcataag aagacacctg tcctctctga cttcatctgt 10320acccctctct tggaagtcac tatcttgtgc cttgcatttt cgttgtttaa gtggtctcca 10380tttcccagca tatcttgagg tcaagggttc aggtcatttt atctttgtct atgcattgca 10440atatgggggt ttttacatat tagctgctca ataaatcggt gttgaataaa ggcatgtgta 10500tgctttcatt aagactatga aacccacaaa aatcagtggt tttcctattt cacccttaga 10560aaacaaaccc acaacatagc acaacctgat attcagagct aagaacaaag gtcatgcata 10620ttaatctaaa ttctatcttt atcaactttc acaagtaatt cgtatttccc tgtctgcatc 10680acggggatga ttctggccag acattgacct tggtaaaatt tcctccagat tatgagaaat 10740caagtcaaat atgccaagta acatagtttc tacttagagt caggttcatg ttttagcagg 10800aacctcaaat accacaaaat ctgtcaagtt ctaacatttg tatctctcga cagtacctga 10860agttcctgtt tctgtttcct cagcccaggt ttccaattca gtgagcagaa cggtgactgt 10920gttggtaaaa gagcccacat acctgcccga tcctgcagga gtgttgcaga tgcaaacagg 10980cgggtctcca catgacctgc ggagtaatga ctagtgtccc taaagtcatg gggcttctgg 11040ggttagcctt gaaaaaagct aaaggttgca tagagagaga tttctatccg ttcagagact 11100cactataatt ctctctttct gtctctgtcc ttcatctgtt tctctctttc tctctcactc 11160tctctctctg atacacacac acacacacac acacacacac actcacactc acacactcct 11220gagtaaggga aatgtgagaa gaaggtaaaa cttcaactaa atgaaaagaa attgtatgaa 11280ttatggtaag caggttggtt tttagttcca gtaaagatag aaatatttag attacttagg 11340agaaaagtct agctggtaac acatgggaat gtgcctgtgt gaaaacaaaa caaaacaaaa 11400aatctaggct tgtggttagg tgaaggtatg tacactgctg agacatggcg atgggtgagc 11460ttgggatgag gagaaaggct tctctgagaa gattaagaga gaaagattgt ttaaaaatgt 11520ttaaacatgc tgggcactgt ggctcacacc tgtaatccca acactttggg aggccaaggt 11580gggcggatca tgaggtcagg agttcgagac catcccggcc aacatggtga aaccctgtct 11640ctgctaaaaa tacaaaaatt agccaggcgt ggtggcgggt ggctgtagtc ccagctactt 11700gggaggctga ggcaggagaa tggcgtgaac ccaggaggcg gagatgcagt gagccgagat 11760tgtgccactg cactccagcc tgggcgacag agcaagactc cgtctcaaaa aaaacaaaaa 11820aacaaaaaaa aaacacacat tgacaccagg acggagttag cacatcttta caggtgagac 11880tctcagaccc gagaaaatag aggcacttta gagctgagct aatcccacag ccacctcaac 11940acacaaacgg ggaatctgag acccgcattg gcaccgtgcc tgaggttcta aagcccaggg 12000cttctgactc gcctcttgtg cttcttcagt actgtgggtg ggggtggggt ggggggtgac 12060attagctgat gagaaagatt ttggttttag aaagatggag ttaacataaa cgaaggtgta 12120ctgggactgg tctcctctgc tgacttcatg ggaagcacac acacgcacac acacacacac 12180acacacacac acacacacac atacacacac ctgtccaaga tcagaaaaaa tccctcacat 12240ccctgtagca tgatcctgat tgtaaaaatg gagccctaat cagaagggca gaagcatgat 12300tgcctctcaa gagatttgga cgccactttt tcatagttgg ttttagctgc tttgcgatat 12360atactgaaat aaatagaaaa gggaaagaat tgtaacctgg attgacagac aacaagccct 12420gacagacaaa aagcagataa gaaataaaat aaggaagata acccataatg taaaataaaa 12480atagcacatt gttgcatgca ttgataccct tttttttttt tctttgagat cttgctctgt 12540ctttcaggcc gaagtacagt gtctcaatca tagctcactg cagcctccag cttctgggct 12600caagcaatct tcccatctca gccacccaag tagctggggc tgcaggcacg aactatggtg 12660cccagctgat aatttttaaa aatagggaca ttagtgcatt tagcaaattt gagtgtctgc 12720tgtgtatcaa gcactgttct gggcactggg acagcacagg gagcaaataa acaaaagccc 12780ctgcgctcaa ggtgctcgta ttctagaggg agatgctgag ttcacctccc attaaaatgc 12840cattctcaag atccagtccc tccacccacc ccagccccca gggttttggt ggaaatttaa 12900ctaagttgga agattgataa tatctccatt cacatttgga tatgatttta atgaaggttg 12960ctttttggtt tttagggaga agaaaatggc tttccagata gcactggaga tcctcttcca 13020ggtaaatgat tgattctaaa gctatctggg ctaatagcta gtgtggctga ataaaagata 13080atttgaggcc agggtcggtg actcatgcct gtaattccag cactttggga ggccaaggtg 13140ggcggatcac ctgaggtcag gagttcaaga ccagcctggc caacatggta aaaccccgtc 13200tctaccaaaa atacaaaaat tagctggttg tggtgggcgc ctgtaatccc agctactcgg 13260aggctgaggc aggagaatcg cttgaacccg ggaggcggag gttgcagtga gccaagatca 13320caccactgca ctccagcctg gacaacagag cgaaactcca tctcaaaaaa ttaaattaaa 13380taaaataaat aatttgagac tatgtttatc attaacttta aaatctgtac tgcagaatag 13440agcaactttc tacctgcggt gcactgcagg gaaagccgta tcttacaaga cttcacaaaa 13500gccttcaaag agtattttct ctgcactaac cttcctttgc atgtgagggg cacggcaggg 13560ttctgaatgg ggcaggttta ggatcaggcc agtcgggact gagtggattc ttcttccctc 13620tgagttctaa gagccatagc attggtggag aacatgctgt ttgttgcttg gtggaaggga 13680ccagaagcca gctgggtcat ctctctgttt gtgccttggc cacttaggta gccaaaggag 13740ccctcctgac attaggtcag gtgttagtcc ctctcctttt ctgcttttag tgtgtttaag 13800caaataaaca ttaaagttca tttctccccg ctcccctttt ttaatcataa gacagacatg 13860tttgcaatgt ttaaatttct cattaatcag aagggatagg gagtgaggga gtaagcatta 13920aaataagcta gcaaatggcc aggtgtggtg gctcacacct gtaatcccag gactttggga 13980ggccaaggtg ggcagatcac ttgaggccag gagttcaaga ccagcatggc caacatggca 14040aaactccatc tctactaaaa atacaaaaat tagccaggcg tggtgatggg cacctataat 14100ctgagctact cgggaggctg aggcagagaa ttgcttgaac ccgggaggca aagattgcag 14160tgagctgaga ctgcaccact gcattccagc ctgggtgaca gagcaagact ccatctcaaa 14220aaaatgctag caaaataata ataataataa taataaaaca tacctcacca acattttcta 14280catcttgtaa agcatacatt gactgactga agtcaccaga gttttgtttc tttctttctt 14340aagcagggtg gggaacccgt agagccctca ggggcagcta tcatcagccc aggtaaccaa 14400gctgaaaaac cagaaggtgc agtgcgtact caactttttc cccttagaaa cacgatatta 14460gaaaatacac caataccaac atgtgagcaa cagttctctc tggaaggtgc agttctgggt 14520gatttttttt tcattccata gatttttttt ttcttgagac ggagtttcgc actcttgttg 14580cctaggctgg agtgcaatgg tgcgccacca cgcccggcta atttttgtat ttttagtaga 14640gacggggttt caccatgttg gccaggctgg tctcgaactc ctgacctcag gtgatccacc 14700tgcttcggcc tcctaaagtg ctgggatgac aggtgtctca ctatgttgcc taagcttttc 14760tcgaacccct gagctcaagc ctcctcccac ctcagccatc caaagtgctg ggattacagg 14820catgagccac cacgcctggt gagtttttat tttctttcca ctatccntat atttctaaaa 14880tttctaacat gagctggtat cagaactgcc cctccgcatt taatctgtgt atacaaatgt 14940atatataaca aatgatcaca tgttggtaan gtataccttg ctgcatggtg aaataaccaa 15000ggaaacttct aaaaggttaa ctgtggttgg cctgggtaat gggagcatta attttttcca 15060tatgctcatc tgaattttca gatttgctat gacaagcaca tatttatttt ctaattttaa 15120aaatctatat ttaaactctt taaagactaa caccctacac actaatgtgg cacgttagct 15180aaaataaaaa taaatacaga aatttgttta gaaatatttg taaacccttc aaggactctt 15240ctgaatgata gtcattatta attagcaggt taattttaat caggcttctg gtcatcttca 15300aacatttttt acttgtgtca aaatgaacca ccagagtgtg ggtttttttg ttattttttt 15360tgtttttttg agacagagtt tcactcttgt tgcccaggct ggagtgcaat ggcgagatct 15420cggctcactg caacctctgc ctcctgggtt caagcagctc tcctgcctca gcctcctcct 15480gagtagctgg gattacaggc gcccaccacc acacccagct aatttttgta tttttagtag 15540agatgggttt tgccatgttg gccaggttgg tcttgaactc ctcacctcag acgatccacc 15600cacctcagcc tcccaaagtg ctgggactac agatgcacac caccacaccc ggttaatttt 15660tgtattttta gtaaggacgg gggttcccca tgttggccag gctggtctca aactcctgac 15720ctcaagtgat tcacctgcct tggcctccca aagtgctggc attacaggcc tccgccaccg 15780cacccagccc aacctgggtc cttttgtatg tgagagtttg cttgtttttt tcacgtgctt 15840tctctactcc agttttattc tatgacaaaa ttgaggccca acatgattta cttgcctgga 15900tccacccaac ctgtcagtta cttcccagtg ctgctgccaa cttaatgtct ccttaaaagg 15960atgctttaga gaaaacgaaa tcatgttgtt tttccccttt ggttaagaga tcaaacgccc 16020accaaaagcc cttgggtcag tttcttagta gataaaaata attcttcgtc actttctgaa 16080agcggctaac atataaccct tatgatgaat aatgtggtgt gtgtgtgtgt gcgcgcccca 16140aattccaatg agttatcaaa gccagaaact tatattttaa atatgtttat ttcccaacca 16200cactggaaac cacacacaga aaaaaaaaaa agcatgatta taccccctta ataaccgtta 16260ctgcagaagg atgtgactct ccttcaacac ttgttggtat tttacagcct ccaaatctga 16320ccatgtataa ccacctggga tagagttatt ttatttcaga accataatac ttagctatct 16380cggaagttgc caatataaaa tgtttactct ctaatggttt tgaactaact caagacctgg 16440ttatcccggg gagcatcctt acaaatgatc tgagagctaa cagtcctctt gcagcagtgg 16500agggaaacac tcccgtggca atcactctcc aaaagccaga atgtgcaaga taaaagggca 16560ccttccctgc agggaggcac attaagtcag tctgtgatct gctgccaaca tcctgactgg 16620agccgtttct acgcctaact aatcatgacg tttgtgaatt gtgaagcttg ttgcaattca 16680caattaactg ttaattgacc catattttat aacccgccag ccatgaactt acaagttaga 16740tacagacact accagacatt cactattttt ttttacaatt gttttaaatg acattaatga 16800gcatgcttga ttcctgaact cttctttaca gtataatttt aaaatatttg agtgggatac 16860gatggagagg agggaggtgg gggaagaaat gccccatgga aaacccactc atcaggttga 16920gagtgtggag aagccctgtg tatctgagaa ctcttaatca tccacagaca tggtatctct 16980caaagagaag tgggtgtaat tccaaaatct aattttggca ggcgctcctg actaaatact 17040taatctggag atgtcttcaa ggcaggcgga ggttttcagt cctggctgca cattagaagt 17100cccaggggag ctttaaaaaa ttcccacgtc ctccctgcat cccagactaa ttaatcggga 17160tctccgaggg tgggaccaca catcagggtt ttgtaaattt ccctgggggt ttggtggggt 17220tgggggtgga ggcgtctatc ctatggccaa ggttgagaac cactgctttt taaaagactg 17280tttgcttgtt tttgagatgg ggtctcgctc tgtcacccag gctggagtgc agtggcgcaa 17340tctcagctca ctgcaacctc tgcctcctgg gctcaagcaa ttctcctgaa aaaggctgtt 17400ggttattaat gcttccccac agctattcta ttcattgttg catgcttctt acgtgtgcta 17460ggatgggagc tttaaaggat tacctcattt aatcctcaca accaccttgt gagagaggtg 17520tcattatccc tgtttggaga gtgagacagg ggcttagcaa gctcagtaac ctgtccaagt 17580cacacatctg catggggtta gctgctgcta aagctcatgc cgttaatctc catggtacac 17640ggtgtcctct ccatagcaat cttgcggctg ccttgttaac accaaaaaaa cttgcatcag 17700ctggtttgac aatttctaga taaagagctc ttttcgggct gctaagaagc ctaatttttc 17760atttgatttt cttcttgaac tgtgtcacac tcctcattca tttgatatat tcatcaaata 17820cttattgagc acctgctgtg tgcctggtgt gcagcagtga caccagacat ccaaagtcct 17880tttcctctta gagcttattc tatctgggag agacagataa taaacacaaa atcagtaagt 17940cattttatat ggtggtaggt gccttgagga agatgagcca ggttaatggg attaagcctg 18000gtagggggag ggtgccactt tagctcggaa agggtagcga gacccaaaca atgcaaagga 18060cccggcccgt ggagatctaa gacaggagga tgccagggac aggaagttgc tggggcaaag 18120cccctgaggc tggactgagc tcagtgttct aggacgggcg tgggcagtga ggagcagcag 18180aggaggtgag ctgggagata gcctggggac tctttcttct gcctccttca aaaaataaaa 18240ctagccaggt gtggtggctc acacctgtaa tcccaacaat ttgggaagct gatgtaggtg 18300gattgcttga gtccaggagt tcgagaccag cctgggcaac atagtgagac ccctcccccc 18360atttctacca aaaaatcaaa aaattagctg ggcccggtgg cgtgcgcctg tggtcccagc 18420tactcaggag gctgaggtgg gagcattgtt tgaacccggg aggtggaggc tgcagtgagg 18480cgtgattgtg ccactgtact ctagcctggg tgacagagtg agactctgtc tctaaataaa 18540taagtaaatc tagaacctaa catcttggag tgcagtggca ccaccatggc tcactgcagc 18600ctcaatctcc tgagctaatc gagcctcccc ttcagcctcc tgagtagctg ggactatagg 18660cgtgcaccac catacctgaa taatcaaaac ctaacatctt taaagaacat tggcataaga 18720cttggcaaaa atggcatctt gtccctcatc tcatttagtc caagcgatac aggaaatgct 18780gccacctcca ttttatagat gaggagtctg acgttcctag aggttcaatg ccctgaaacg 18840tcaagccttg aggaagttgg agcactggga ttcgaagagc accatccaat acagacccag 18900aatcaggatg atttgggatt atgcttgtca aggactcagg gcagggctac catacattag 18960gcacaagaat tttgatagtg ataattactg tgttcattgt cacttcatca tgacagttac 19020cgtgatgata agaaacctgg cccttcttca cctgacaaag gctttcttcg tttgagccac 19080tgctcaaacg agactgacca agaataaatc ctcggggctt tggcctttaa aataggaagt 19140catcataaat gacttgatgt ggtgtgtttc attcttgctt tgcaccagtg gaaaatatac 19200aggtcaagca tcaaaacatg gcaaatgggg accccaatta ttagagaatc taagttaatt 19260tttatgtata attaattatt caacaaccct ctcctctcca aaccaataat taatccatct 19320tttgtatttt aagaccaatt ctgtagtatt ttccatcaat atctatttac tgctagcaga 19380tatcagctac attctttctc ctttaataga agttccctct ttaggtatta agattcatta 19440aacaacaata acaaatctac cttgcctccc agggacaatg cacagttctc attcatttgt 19500tcatttagca gataattttt gaatttccac tgtacagcag ccctgtgctt gtggttggcc 19560tgttatttga gaagcatcaa ataataatct cattttttgg ctgggtgtga tagctcacgc 19620ctgtagtccc agcactttgg gaggctgagg cgggtggatc acttgaggat gggcgttgga 19680gaccagcctg gctaacatgg tgaaacctcg tctctattaa aaatacaaaa attagccagg 19740tgtggtggca gacacctgta atcccagcta ctcgggaggg tgaggcagga gaatcgcttg 19800aacctgggag gcagaggttg cagtgagccg agatcgcccc attgcactcc agcctgggca 19860acaagagcga gactccgtct caaaaaacaa aacaaaacaa gacaaaaaaa aacccaacaa 19920ataaaataaa taatcccatt tttctccatt tttgagaaag atttctttgg tctgaagtct 19980ttctctcccc tctccgaggc attacccagt ttaacctttc atgtataata tatatgatag 20040ttatttaaag tatagcagga caaaatgtat ttgataggag aaaaccttgt ttgctctgtg 20100ttaagtcctc cagagagcta attagagttt gtgattctaa aaggcaacta tagattcact 20160tatattagca gttcatgtag attccagtta aggaaatggt ttgtcacttg tgttattgaa 20220aacacacaca gggcgagcac tgtggcccat gctggtaatc ccagcgtttt gggaggctga 20280ggtgggcaga tcacggggtc aggagtttga gatcagcctg gccaacatgg tgaaaacccg 20340tctctactat aaatacaaaa aattagctgg cagtagtggc aggcgcctct aatctcagct 20400actcgggagg ctgaggtagg agaatcgctt gaacccagga gtcggaggtt gcagtgagtc 20460gagatcgcac cattgcactc cagcttgggc aacaagggca agactccgtc tcaaaaaaaa 20520agaaagaaaa cacacacaca aaaaaacttt agtagatctt tcggcatatt attttttaaa 20580ataaactgat aatggttgat atgattgttc aaagaaataa gagcttttca taaactcagt 20640ttaaagaaac tttacaggcc gggcgcggtg gctcatgccc gtaatcctag cactttggga 20700ggccaaggcg ggtggatcac ctgaggtcaa gagttcgaga ccagcctggc caacatggta 20760aaagcctgtc tctattaaaa aatacaaaaa ttagccaggt gtgttggctg gcgcctgtaa 20820tctcagcaac tcaggaggct gaagcaggag aatcgctgga acctggtagg cagaggttgc 20880agtgagacaa aatcgtgcca ttgcactcca gccccagctg acaacagcga gactccatct 20940caaataaata aataaataaa taaataaata aataaataaa ggagctttac agaaaccttc 21000tgatgttttt ttcttcttga cgataacatt gccaacactg aatcttacaa agataagaca 21060agaaagggac cttcagacac cattacatgt aattctggac ttagtggttt aaatccttat 21120ttttctatga cattaaaaaa atgtatattt taggccaggc acagggctca cacctgtaat 21180cccagcactt cgggaggccg aggcaggtgg attgcttcag cccaggagtt caagagcagc 21240ctggggaaca tagtgagacc cctgtcccta cagatttttt ttttttgttt gagatggagt 21300tttgctcatg ttgcctaggc tggagtgcag tggcacgatc tcggttcact gcaacctctg 21360cctcctgggt tcaagcaatt ctcctgcctc agcctcccaa gtagctggga ttacaggcat 21420gtgccaccac acccggctaa ttttgtattt ttggcagaga ctgggtttct ccatgttggt 21480caggctggtc ttgaactccc aacctcaggt gatctgcctc cctcagcctc ccaaagtact 21540gggattacag gcgtgagcca ccttgcccag cctacaaaaa gttttaaaaa attaaaaaat 21600tagttgggca tggaggtgca tgccagctac tcgggaggct gaggcaggag gattgcttga 21660gcccatgaag tggaggctgc agtgagccat aattgcagca ctgcactcca gcctgggcca 21720tagagcaaga ccctgtctca aaaatatata tagtatccaa ataaacacaa taattacaga 21780aaattgaaaa gtgcccataa gcaaaaaaaa aaaaaagaaa aaattaatca cctgcgttct 21840catcacccag aattaaccat tgttaatatt tttgttatag atccttccaa acttttctcc 21900atgcttgtga ttgtatttat tatacatgat ttacagggat ataaacgact gtattattag 21960tcattagaag aactggatta tggccgggca cggtggctca cacctgtaat ctcagtactc 22020tgggaggctg aagtgagcag atcatgaggt caggaaatcg agaccatcct ggctaacaga 22080gtgaaacccc gtctctacta aaaatacaaa aaattacctg ggcgtggtgg caggcgcctg 22140tagtcccagc tactcgggag gctcaggcag gagcagagat acctatctgt tctcaggatt 22200ttaaggtgtt gcgcggaaat aagaaaaccg tacagtgttt ctcactacaa agcagggtca 22260ggagatgcaa acaaactgat gtgggggttc caagtgaggt ggaattccag acaggggccg 22320ggaagacttc gtggaaaggg agaatctgag gtgggttttc taggatgggt aaagttcatt 22380agaggaagag aagtgcaaca gaggaagttc ggtgagaggt agagggaagg cgttctgatc 22440atgaaggaaa cactagaaaa ggtatggaga tagaaaaaga taaggcctga ttttttaacc 22500taccacttaa aaaaaatcct tgaaaagaga tttttaaaac gaatacttgg tgctgacaaa 22560ggtgaaatga ccgggcgcgg tggctcacac ctgtaatctc agcacattgg gaggctgagg 22620cgggcagatc acttgagctc aggagtttga gaccagcgtg gccaacatgg caaaactcca 22680tctctactaa aaatataaaa attagacggg tgtgatggtg ggtgcctgta gtcccaacta 22740ctcaggaggc tgaggcagga gaattgcttg aacccgagag gcggaggttg ctgtgagctg 22800agattgtgcc actgcactcc agcctggata gcaggatgag actgtctcaa aaaaagaaag 22860aaaaggaaag aaaaaaaaat ccgtactgta aactggtaaa ggctttcttt ctggagagca 22920atttggggca catgcaccag tagccttaga aggctcatgc ttttgaccta attatcctat 22980tagtggtgag atgattaaag atgtggcccc aatttatgtg aaaggtatgc atcacatctt 23040cactcataat caggagagtt ggggaaaacc ctagctgtta atagtttatc caaaatccat 23100atatatatgt gtgtgtgtgt gtgtgtgtgt gtgtgtatgg atttatatat atatataaat 23160ggatatatat atatatctgg atggatatat aaatatgata tatatatgtg tgtgtgtgta 23220tatatatatg tgtatatatg tatatatata tgatggaata ctatttagcc ataaaaagga 23280atgaattaat ggcattcgca gtaacctgga tggacttgga gaccattatt attttatttt 23340atttatttat ttttgagacg gagtctcgct ctgtcaccca ggctggagtg cagtggctcg 23400agctcagctc actgcaagct ccacctcccg agttgacgcc attctcctgc ctcagcctcc 23460tgagtagctg ggactgcagg cgcccgccat cacgcccaga taactttttg tatttttagt 23520agagactggg tttcaccgtg ttagccggga tggtctccat ctgctgacct catgatccac 23580ccgcctcggc ctcccaaagt gctgggatta caggcgtgag ccaccgcgcc cagcgagact 23640gttattctaa gtgaagtaac tcaggaatgg aaaaccaaac atcgtatgtt ctcactcata 23700agtgggagtt atgctatgag gacgcaaagg cataagaatg atacgataga ctttggggac 23760tcagggaaaa ggtgggaagg gggtgaagga taaaagatac aaattgggtg cagtgtatac 23820tgctcgggtg atgggtgcac caaaatctca taaatcacca ctaatgaact tactcatgta 23880accaaatacc acctgttcct caataaacca tggaaattaa aaaagaaaaa agaaaaagta 23940ccctggaaaa aaaatttctc cctggccagt cacggtggct catacctgta atcccagcaa 24000ttcgagaggc tgaggcagga ggatcacttg agcccagtag ttcaaaacca gccagtgcaa 24060catagtggga ccctgtctca aataaaatct aaaaattagc caggtgtgtt ggtgcatgtc 24120tgtggtccca gctactcagg aggctgaggt gagagtattg cttgagccta ggaggttaag 24180gcggcagtga gccgtgattg tgccactgcc atccaacctg ggcaacaaag caagaccctg 24240tctcaaaaaa aaagaaaaaa aaaacctctc tattcgcctt ttaagaatac ctgggcttct 24300ctgtgtacac ttaagcttca ttggagtctt tagacttttt ttttgctgta tctgtccagt 24360taccaagtcc cagcttctac tccatgctcc ccatgctctc ttcctatttt attttccatg 24420actgcctcgg tataacttgt gctcaaccaa actggactac tcaattccct gcattttctt 24480ttttaaagtt taatcaaaaa aaaaaagaaa actggctggg cacagtgggc ttctgcccac 24540aatctcggtg ctttgggaaa ctgaggcagg aggattgctt aaggccaaga gttcaagacc 24600agcctgggta acatagcaag acctccatct ccacaaaaaa atttaaaaat tgactgagtg 24660tgatggtgtg cacctagtcc cagctgcttg ggaggctgag gcaggagaat tgcttgagcc 24720caggagttcc aggttatgat gagctatgac tgtgccaccg cactccagcc agggtaacag 24780agtgggactg tctcaaaaaa caaaacaaaa tccctaatat aatctcagtg tgccttttaa 24840gtatgccata tatatatata tatatatata tatatatata tatatatatc acattttctt 24900tatccactca ttgattttca tgtagttcta atcgtagaat tcatacattc tttctatctt 24960ccatctttca cataacatca caaacatttt ctaggttgcc atattgtctt catagttact 25020taaataatat tccatcaagt agcacaatca tttatttcac tagtcctcta actgtagaca 25080ttttggttgt ttttgaaact taataatgta aataacaccg tgataacaat gtttatgtaa 25140attcatattt tggattatct ccttagggtg gattcccaga agtcacatta gtaggtcaaa 25200gagtatgagc ctattttcaa ggctcttgtt ttattacctt ttaatttcca cttgcctcaa 25260tattgctggt ttgctccctt atgatcacca gagttactcc gtcggtccaa attctttacc 25320ttccgaaact gggaaggcca tgactcaatg ttatatatat agtaaaggct actataacct 25380tccccagaat tttccaagcc agtggtctct aaagtgacct ttggctgtta aaatctgaat 25440tcagagggtt catgagactc agtgttgttg tagaatttaa gctccttaat ttgccacgtt 25500gtttagacac cacttaatac tttattgcaa atgacttgtc aacgcctctc acctacaaac 25560ttcatcctcc tacaaatata cctcctgcta atcaaatgag gctacagttg agtctttaag 25620tttcagtaga aagatggccc ttcctctggg gtaggcgcat gctcttcatg ctgaagctca 25680gctgaaaagc ctcctgctga gttttctgcc tctttccctc ccactgcaca caccccaggg 25740tgttggcgcc acttcaaagg gagcctgtgg atgaagaaaa cacaggtaaa ggcagagggc 25800tcataagggg gccataaatt taaaaagtta agattcctgg cactatcaac tctcacttgt 25860tttcaaatat gcatatggag tggatattcc agttttcatg tctgtgttgt tgtttttaaa 25920aaaagacctt tcaaagaact gtgcattttt tacaggctga caggctgtgt ttggtgttaa 25980actgtcaggg ctgactggtc acttggaaag ggcaagggct gaggtgcatg caagtgtcgg 26040ctggttactc acagacacag cagccccttt taccccggag agagttctgt ttgctggagc 26100ccttattctg gccagcagtg tcacaaatgc acactgtaag acatagacag tcttggaaag 26160aaagggaaac tggctttaaa aattcttact ccttctagca aagcaattca tctttggcta 26220taaagaataa cacagccagg tgcggtggct catgcttgta atcccagcac tttgggaggt 26280caaggtgggc agatcacttg agtctaggag ttcaagacca gcctgggaaa catggtgaaa 26340ccccacctct accaaaaaaa aaaaaaagaa agaaagaaaa gattagccag gtttggtggt 26400acgtgcctgt agtcccaggt actcgggaag ctgaggtggg aggatcgctt gagcctggag 26460ggcggaggtt gcagtgagcc gagatcatgc cactgcactc cagcctgggc aacagagtga 26520caccctgtat caaaaaaaaa aaaaaaaaag aacagtaaca cattattaga aatgagcatt 26580ctgaggccag gcacggtggc tcatgcctat aatcgcagca ctttgggagg ccgaggcggg 26640tggatcacaa ggtcaggaga tcgagaccat cctggctaac acggtgaaac cccgtcttta 26700ctaaaaacac aaaaaattag ccgggtgcag tggcgggtgc ctatagtccc agctactcag 26760gaggctgagg caggagaatg gcgtgaaccc ctgggaggcg gagcttgcag tgagccgaga 26820tagtgccact gcactccagc ctgggcgaaa gagcgagact ccatctcaaa aaaaaaaaaa 26880aaaagaaaga aacgagcatt ctgaaatagt cttccatatg atgcttttga caattcagca 26940ggaaaataaa ggatgtaaga aatgaatgca tatgttaggc ctcttgttga cctgtggact 27000aaattgtttc tccctgcaga gatcagcaag gacaactcct gcaaagaaaa ctgtacttgt 27060tcctcctgct tgctccgggc ccccaccata agtgacttgc tcaatgatca ggacttacta 27120gacgtgatca ggataaagct ggatccgtgt cacccaacgg tgaaaaactg gaggaatttt 27180gcaagcaaat gggggatgtc ctatgacgaa ttgtgcttcc tggagcagag gccacagagc 27240cccaccttgg agttcttgct ccggaacagt cagaggacgg tgggccagct gatggagctc 27300tgcaggctct accacagggc cgacgtggag aaggttctgc gcaggtgggt ggacgaggag 27360tggcccaagc gggagcgtgg agacccctcc aggcacttct agagctcttc ttcttccttc 27420attggcctct ccggatgttg aaacaaccac aggtcaagaa ggaatgtgaa tctgttgttt 27480tataagagtt taggacaagg acgtggaaca gtggacactg gttttcccca aagctggcag 27540ttttgtggag gggtagcttg tttcggtggt ggatctctgt ttatttttgc acatctgtta 27600taatttaata ttcaaatctg gaattaagaa aacatatttt ctagtatcct ctaagggcca 27660aagtcctaca atcggaatgg attcatgcca cgttgaagat aaaattatcc tctctctgaa 27720atacggtaaa gatttaaata ggtcctgaga ctgttgatag ccccagacat acccacagca 27780ttatatgtaa catctctcct gatcagtgcc attcccacgg tttcaaagaa aacagctaca 27840aggaatgctt acctgagtgt ctgcagcacc ctccacttct ctcctaggca atgagaccca 27900gtggctagaa attcaccatg tctattctca agatccatgc cagggagctc tttgactctc 27960gtgggaatcc cactgttgag gttgatctct tcacctcaga aggtctcttc agagctgctg 28020tgcccagtgg tgcttcaact ggtatctatg aggtcctaga gctccaggac aatgataaga 28080ctcgctatat ggggaagggt gtctcaaagc ctgttgagcc catcaataaa actattgcac 28140ctgtcctggt tagcaagaaa ctgaacgtca cagaacaaga gaagattgac aaacttatga 28200tagagatgga tggaacagaa aataaatcta aatttggtgc aaatgccatt ctgggagtgt 28260ccctcgctgc ctgcaaagct agtgctgttg agaagggggt tcccctgtac caccacatcg 28320ccgacttgtc tggcaactcc aaagtcatct tgccagtccc ggtgttcaat gtcatcaatg 28380gcagttctca tgctgtcacc aagctggcca tgcaggagtt catggtcctc ccagtcggtg 28440cagcaaactt cagggaagcc atgcccattg gagcggaggt ttaccacagc ctgaagaatg 28500tcatcaagga gaaatatggg aaagatgcca ccggtgtggg ggatggaggc gcgtttgctc 28560ccaacatcct ggagaataaa gaaggcctgg agctgctgaa gactgcgatt gggaaagctg 28620gctacactga taaggtgatc gtcagcatgg acgtagaggc ctccgagttc ttcaggtctg 28680gaaagtatga cctggaattc aagtttctcg acgaccccac caggtacatc tcacctgact 28740gtctggctga cctgtacaag tccttcatca aaaactaccc agtggtgtct actgaagatc 28800cctttgacca ggatgactgg ggagcttggc agaagttcac ggccagtgca ggaatccagg 28860tagtggagga tgatctcaga gtgaccaacc caaagaggac agcctcggcc gtgaatgaga 28920agaagtgcaa ctgcctcctg ctcaaagtga accagattcg ctctgtgact gagtcccttc 28980aggcgtgcaa gctggcccag gccaatggtt ggtgtgtcat ggtgcctcat cattctgggg 29040agactgaaaa taccttcatc actgacctgg tggtggggct gtgacctggg cagctcaaga 29100ctggtgcccc ttgctgatct gagcgcttgg ccaagtacaa ccagctcctc agaattgaag 29160aggagctggg cagcaaggct aagtttgccg gcaggaactt cagnaacccc ccagccaagt 29220aagctgtggg caggcaagcc cttcagtcac ctggtggcta attagacccc tccccttgtg 29280tcaactccgg cagctcaaga cccccgagca acatttgtag gggccgctgc tagttagcta 29340cccttgccca ccgccgtgga gttcgcacct cttccttaga acttctacag aagcaggttg 29400cagtgagccg agattgcgcc actgcacacc agtttggaga cagagtgaga gtccgtccca 29460gaaaaaaaaa aaaaaaaaaa gaacttntac agaagccaag ctccctggag ccctgttggc 29520agctctagcc ttgcagtcat gtaattggcc caaatcaccg gagccacgtg accctccagt 29580gtcatctccg gggtggccac aggcaagatc cccagtgatt ttgtgctcaa aataaaaagc 29640ctcattgacc catgagaaaa aagaaaacag caatgagaag tgaccctgtc ttgttggttt 29700attacttttt ttgttataaa gtactttggt gaattaacag gatgctagta ttacatggtg 29760atactcttca gaacacctgc cccatctttt ttatgcaagt atgtttacaa tcagtggact 29820atcagtaatg tcatttgctc aaatattttt taaagaccta cagaaactga tggttattgg 29880gaaaacagtc aggaagtagt gaggtaatca aggccatggg aatagtgttt gacaaagaga 29940gtactccaaa tcccttttgg ttacccagga ctttaaaaaa gagagtactc catcacacct 30000gtaatcccag cactttggga ggccgaggcg ggtggatcac gaggtcagga gatcgagacc 30060atcatagcta acatggtgaa accccgtctc tactaaaaat acaaaacatt agccgggtgt 30120ggtggcgggc gcctgtagtc ccatctactc aggaggctga ggcaggagga tggcttgaac 30180ccaggaggcg gacttgcagt gagccgagat agcaccactg cactccagcc tgggcgacag 30240agcaagactg tgtctcaaaa aaaaaaaaaa aagagtgctc caaatctcct ttggttaccc 30300gggactttaa aaaatttaat gtgatagtta ggccgggtgt ggttctcacg cctgtaatcc 30360tagcactttt ggaagctgag gcgggtggat catttgaggt caggagttgg agaccagcct 30420ggccaacatg gcgaaacccc gtctctacta aaaatacaaa aattagccag gcgtggtggt 30480gggcgcctgt aatcccagct cctcgggaaa ttgaggcact agaattgctt gaacccagga 30540ggtggaggtt gcagtgagcc gagattgcgc cactgcactc cagcctaggc aacagagcga 30600ggttccatct caaaaaaaaa aattgtaata ataataataa caatgtaata tttacttttt 30660catcctttat ataaggctga gtgcttcacc cctgagatga agctcagtta agaaataaat 30720gaaaatcccg taacctattg gtgaaaggta accaccccca gctcctacta gcccaactta 30780aaacaggacc ccatcacact acacagcagt ttagccaaga aaagggggtc tttatgtgga 30840cactgggagg gaagggattc cttcaaatcc aaactttaaa ggattttaaa caaatgaaac 30900atttggttca aagaatagct gatgttttta tttgatgatt ttggagaaag gaaagtgtgg 30960ggcataatgg ggtttgttat tggaaagatc agattttcta ggtaatttgg gtggagaaag 31020acaaaaggca aagctttgac tgacaattcc atgaaagtgc tatttggttt tggttatggg 31080cttagaaaat taagacactt agttcaattt ggaaggattc tgtataagtc cctgattaaa 31140ataagcaaaa atgatgaata acactgattc agtgcaaccg aaagattagg attaactcaa 31200aagaaagtta ttttctaaac caccgtgatt ttttccactg acaattacag cggttttcat 31260taggttgctg acacatgaag tcagcctcac catcagttgc aaactctaaa ctagcaaaat 31320ctattacaga gacatactta tcacttctga tttagtgcta atctcaccca gctcatcttc 31380tcttgtcaga tttatgagat aaatgtcaga tttatcacca gatatattga aagtaacagc 31440cagtaataaa atgtgagatt ttaaaaaata gattctttgg caaattggtg ttcagtgagg 31500caattattaa acatttttgt cagccaggtt ccaggcactg tacagaagct gttaggagtt 31560ctcaccatct acgaatttga tttgatgtat tgtattctca ttaagctatg tgtgacacat 31620tgtcatttat tagcccagaa tttaaaaagc tgtggttgtt tagtgttggt ggtagcagac 31680cccagcagtc tgatggtctg cactccttcc atcctgccac cccctgggga tgcaaagact 31740ggatctcagg gtgacaatct tcttgcgcac gactgcctgg ccaagtgcct ccagaaagcc 31800ccttccttcc cccatttcca cccaggccca cttgtcacct cagcctaaca ccagcctgca 31860cagtctacgg ccaccatcca ggcagtggga gagggaaagg ggaggagggt ggaagggaaa 31920acccctttct atacctctcc tcagcctgct ctttcctcct cccacctctg agcctccgcc 31980tcccccagac agagacagaa aagatggaag aacaggtggg acctccaccc ccaccccaag 32040ccttcatccc ggtggagggg gatgggaaga tttctctcat ttcaagagac tcctccacct 32100cagactgaca aaaggcagag gcctggcaag aagaaagggc accctgggga agaagggcat 32160tgaaatagca cctgccgggc cgggcacggt ggctcacgcc tgtaatccca acactttggg 32220aggccgaggc gcgtggatca cggggtcagg agttcaagac cagcctggcc aagatggtga 32280aaccccgtct ctactaaaaa tacaaaaagt agccaggcgt ggtagcgggt gcctgtagtc 32340ccagctactc cggaggctga gacagggaac tgcttgaact gggaaggtgg aggttgcagt 32400gagccaagat cgtgccatgc actccagcct gggcgacaga gtgagactcc atctcaaaaa 32460accaaaacag aaatagcacc tgcccccacc ccctgcccgc cctccttccc gcccccgtcc 32520tttcctagac ttcactcaag tcctctgctc agaggaagcc ctgctctact gaaagccaca 32580aggccattct cggtggcctg ggacagcagc ccaagacgtg ggcttctaac tgcctccgaa 32640ggggccacag cagcaaacat aaataaaaat agtaaaatgt tcttaaatta taaatttaaa 32700atttggaaaa tttagtgagc acagcttcta gggggcatgt ttccaaaatt ccaaccacaa 32760aagtgcagtc tcaaaactga ctgtaaaccg aacataccat ctcatctcag acacagctat 32820tgttcacgag tgtcagtgga actttcctcc cttgagatgg accaaaaacg tcaagcaaga 32880tgacatttgc tgatttgcag gcttcaggca gataagatac gggcagagtt gagtgtgcgc 32940ctttaccctt aaattcagga atagcaggaa cagcaggaaa aacgtaggac cacagcgtac 33000gtcccacttg tctttcattt tgatatcatt atttccagag tcctgattgc tagtcatgtc 33060taacactgga tttattatca tctcattgct agcatggcta ggaaagcttt gaacatcctt 33120atcattctat tttaattcct attataattg catggggaag ttccagggtg gaaaaatttc 33180cttttctttc tttttttttt tttaatagga gagttggctg ggcacggtgg ctcacgcctg 33240tgatcccaac actttgggag gccaaggcgg gtggatcacc tgaggtcagg agttcgagac 33300caacctggcc aacatagtga aaacctatct ctactaaaaa tacaaagtta gctgggtatg 33360gtggtgcaca cctatagtcc cagctactgg ggaggctgag acaggagaat cacttgaata 33420cgggaggcag cagtgagcca agatcatgcc actgcactcc aacctgggag agacagagtg 33480ccatctcaaa aaaaacaaac aaaagagttg atataaattt gctgttataa tttgactgta 33540ctgtttcttg cacatgttga catctgtaat gactggagtt tatgaaaatt tttgatgagt 33600aggagcatac cattaacaga gagaaattta atcaaaagat ttttaaagtt ccttcagagt 33660ccagactttg actaagtgta gtatgattta tatctatgtt gcatacaaaa atatcaaaca 33720gtaattccca actgaaatac aagtatcaat caattgtgta acaatgcaaa atcatttaat 33780ttaaagttaa tttatagcaa atgagtactg taatagcata agcatgccga tactttacaa 33840aggagagagt ggaaaggtag gatattataa ctaattgatc aaatcattgt taaaatttaa 33900gtttattaat acttttactt ctgtccgtag ggatccatgt taaattgggt atattataaa 33960cttaactgct aatgatgagg tccttttgct attagaaatc tattttttat ttttctttat 34020tattttttga ggcagggtct tgctctgttg cccaggctgg agtgcagtgg tgaaattata 34080gctcactgca gcctcaacct cctgggctca aggaatcctc ctgcctcagc ctcccaagta 34140atggaaactg cagtcgtata caagcacacc cagcaatttt tttttttttt tttggtaaga 34200tggggtttag ctatgctgtc caagctggtc tcaaactcct ggcctcaagt gatcctccca 34260ccttancctc caaagtgctg ggattacagg cgggagccac cattcccagc ctagaatgaa 34320atatctttag ctaaattaca gggctggatg tggtggctca tgcctgtaat cccagcactt 34380tgggagactg agggcgggag ggtcacttga gatcaggagt ttgagaccac cctgggcaac 34440acagtgagaa ttctgtctct attttaaaaa gagaaaaatc tagggtatat tctcttaaac 34500aaaactttca tctataatgg tagttgatga ggtcctatgt aatatgcatt tccttggttg 34560caatagcaaa ttactacaca cacagaaagg aaagccacac tccccgacac atctacacac 34620aggaggactc acacaggagg gagactcaaa gaaggcacgt gacttttaca ttgttagggc 34680ttacatggtc ctgggatttc ccaccagtac tcaaaagatc aattgtatga acaagtcacc 34740tatttttacg gcactaaata attattattc aacaacatgg aaaatatgtg gtagcagacc 34800tggattttcc ttaagagtta tttttatgtg gtactgcccc ctgctggaat ataacatcta 34860tacacatcct ttctggctgg gctgacatcc taaaaccagc ccaggaccag ccttttatta 34920atattaattc ttggccaggc gcggtggctc gcctgtaatc ccagtacttt gggagtccag 34980ggcgggcgga tcacgaggtc aggagttcaa gaccagcctg gccaacatgg tgaaactccg 35040gctctactaa aaatacaaaa cttagctggg catagtggca cattcctgta atcccagtta 35100ctcgagaggc tgaggcagga gaattgcctg aaccgggacc cgggaggtgg aggttgcggt 35160gagccgaaat cgtgccactg cactccagcc tgggctacag agcgagactc cgtctcaaaa 35220ataaataaat aaaaattaaa attaaaaaat aattcttggt tgtatgctaa aagccttgca 35280agtagcccca ctggaagata ggaagagtgg ggctgtttta caaatgagca catataagca 35340gaacgaggcc gccataattg aaatgaaggt ccccgtcccg tggatgtgtt catcgctact 35400tcaccctgtc attcggatcc aatgtgtgac cagccagctc caataacagt tccatactct 35460gggaattatt tttaacactc ggcaggatgc tttcttcctg tagttttagg cttagccctt 35520tgtgcacttt tggtctcttt ccctttcaat ttagcatcca aggaagcggc tgtgaccaaa 35580ggtagctgtc atgttaaagg acaaagttca tagttacagc aaatattgac ccagagcact 35640atccttgccc cttcctctat aatgtgcaat gcaaaaatat gttcttttaa gtacaatatt 35700aataagtaag gtctaggaga ttttcttccc ccttcctttc tcttttagat gagtaaatgt 35760tttatctagt tttgaggaga ctatccttct tatcacatct ctttccactt ctgctctcct 35820tgttttataa ttttcctctc ctttgggtcc gtgtcattat ttcgtgtcgc ttgttttcga 35880gccatgcact catttatcaa atcagatttc ctccgtatgc cgacggcctt cctctccctg 35940ccacgggctt cctttttccc tgactatgca gaagcaattt gttcgcttgt gtttcttttt 36000ttttttgaga cagagtctcg ctctgtcacc caggctggag tgcagtggcg acatctcggc 36060tcactgcaac ctccgcctgt caggttcaag caattctcat gcgtcagcct ccagagtagc 36120tgggattaca ggtgtttgcc accaaggctg gctaattttt agtagagacg gggtttcacc 36180atgttggcca ggcttgtctc gaactcccaa catcagttga tccacccatc tcggccttcc 36240aaaatgctgg gattataggc atgaaccacc gcatctggcc ttgtctttca tccttaatga 36300cactttagtc ctaataatgc taaaatcatt ttctactctt tgaattgaaa cacagcttat 36360ctacatgagc ccaaggcagt agcaacattc acctccattt cttctctgat ctctaccttc 36420tgaaccctgt ggacttggtt gtaaatggat gagggcaagt cttgcttcct tcccctgtgt 36480ttacagagga tcgtggctga gatgctgggc cacactctgg gcctgctggc acccctgggc 36540cggtggctgc tgcccctcag ggtgctcacc acctagacca gaagaaccaa ggtgagggag 36600agcctgtttt ctttcttcct gtggctgcgg gggctgtgag gcatgggtct agtggctgtg 36660tttagctggg gatgcctcct agaaatcagc tccaccgttg aagagatcaa agcaatgcac 36720agtgccactt gaaatgaaac gattgagctt atcagcgctt ttgcaaatgt acaagagggt 36780agctcccccg gacatcctga actgagccat gctcttctat tttgtgtaac agcccagtga 36840cccctgaatc ttcccctgag gcaggtcccc gaagcttcat ggaggatgtt cctcagctga 36900ccaaggtgag gctcttgagc tcctaaatct ttgtgatact gtttatacat ctttgtgctg 36960tactttttaa gctgacttcg tgttatcacc tgtatgattt tatgttttgc ttctaaataa 37020gtacagatta ttttaaactc taataatggg tgctacaaaa ttaaagatta tgtcaatcac 37080tgtctctgat gagttatttt atgtagattt caacacaatc attgattcat gtgtactctt 37140ggtcagtcat cagtcatctg agtacctagt gggtttccaa aatgggtcct ggatgctggg 37200gatgcaaaga taagcaacac atttctatcc tcaacagcct gtagatgagg gagaatcact 37260gcggacaatc agggaagtta ccggagagag cagtgcacat gtggtctaga aactggtgga 37320acaaagttga gaatcactga actaggagga aagacaggtc actgacaatc caaggcacag 37380tgactcacac tctaatctca gcactttggg aggccaaggc aggaagatcc actgagctca 37440ggagataaag accagcctgg ctgacctatg gagacccttc tctaccaaaa aaaaaaaaaa 37500aaaaaaaaac attagcctgg catggtgggg tgtgcctgtg gtaccagcta ttcaggaggc 37560tgaagtggga ggatcgcttg agcccgggag gtcaagactg cagtgaatca tgatcacacc 37620attgcactcc agcctaggga agagagcaag aaagaccctg tctcaaaaac agaaaaaaat 37680ccagtaaaat gtttcagatg ttgttaaagg tgatttcact gttacttttc acctctcctc 37740attttacatc tctgacctat gcttgtcctc tgacttgcca gacattccta gctatggact 37800tgatgtctcg acatggaggc tcacaggcac cccaaactca gcctgcccta agctgaaccc 37860atgatctttc cttccaaact tgtttctcac cagagttccc atcttatcat ccacctagtt 37920gttcaagtca tccttaagac ctccctctcc ttcactgtct attctacctc cctaatatct 37980cttaaatcct tccctcctct cccacctcac agccaccatc ctaacctaag cagccaccct 38040ttctcaccct ataatgacct cctggctgtt ctctatagag ttggtgaatc ctttcgtctt 38100cagcctgaac cccctttcga gggattctta tatatataca tagatataca caaatatata 38160tgtacatatg tacatatgtg tgtatatatg tacatatgtg tatggatata catatgtaca 38220tatgtggatg tacatatgta catgtgtatg ggtgtacata tgtacatgtg tatgggtgta 38280catatgtaca tatgtgtatg ggtgtacata tgtacatatg tatatgggtg tacatatgta 38340catgtgtgta tgggtgtaca tatgtacatg tgtgtatggg tgtacatatg tacatgtgtg 38400tatatatgta catgtgtgta tgtacgcatg tacatatgtg catgtatgtg catgtgtatg 38460tgtgtgtatg tacacgtgtg catatgtgtg tatatgtgta cacgtgtacg tgtgtatata 38520tatatatata tatactggct ggagtgcagt gggaaagttt tggctcacca caaactccac 38580ctcccaggtt caagtgattc tcctgcctca gtctcctgag tagctgggat tacaggcgtg 38640caccaccatg cccagctaat ttttgtattt ttagtagaga cggggtttca ccatgttggc 38700caggctggtc ttgaactcct gacctcaggt gatccaccca cctcggcctc ccaaagtgct 38760gggattacag gcgtgagcca ccgtgcctgg ccggattcct atcttgaaga cgaagcccca 38820gaccatcgac acggcctcaa ggccctgcat gacgcctcct gccccaacac ctcgtgtcat 38880cttgctctcc tctcccgcag ctcctgaggc tttagccacc ctggaattcc aagtccccat 38940ggtcattttt ttttcctgct caagatatca ccatgtgctg tcccctctgc ccttgtctac 39000acccacgtgt ccttctcccg ccccggccac actcatgggg cacactgtcc ttccctggct 39060aatcctccca cactcgatac cactttctct gggatattgc acccgatcct cagccgcagt 39120tgtcttccta tgacccactc ccacactctc gccacaatgg taattgtttg attcctactt 39180gttgtccctg tgagactgca aaccccagag gacaggggcc ctgggttctc cttcgcctct 39240ggatcatcag cactaactga atacctggcc tagaagagat gctaacgatg ctgaatgaat 39300aaataagtgg aaagactctc agtaaagcaa aacctttctt taccatttta tggccgtcaa 39360ggaggaaaac acattatcag tggaaaacgc aaaatgaggg gatttgctta gcaaacgatg 39420aattcctctg gcaccctggc agccttggtt tcttttgatg aggtccaccc ccttccatcc 39480atcttctggg cttaagagat caaagcaaaa catgctgtgg aattcgatac tggtgcaggt 39540tgcacaacat tgtgactgaa ctaaaagcca ctggatggta caactgaaaa tggtgaattg 39600cgtgttgcat gaattataac ctaactgggg aaaaaaaggc ttaaaaagag acaaagcttc 39660ccccacaatg gaaaggaagg tataatagaa acagcagctt tcaaaccttg gcaggataat 39720gaaaccccgt ttctattttt aaaaattagc tgggtacagt ggcacgtgcc tgtagaccca 39780gctactcggg aggctgaggc tggaagatcg cttgagccca ggagttcaag gctgtggtaa 39840actataatca cactactgca ctccagcctg ggtgacagag aaagaccctg tctcaaaaaa 39900ggaaggaaag aaggaaggaa ggaaggaagg agggagggag ggagggaagg agggaaggaa 39960ggaaggaagg aaggaaatag cagctctgag cttagaaata ggagtctatt tctaagtggg 40020agatggggag aaggagggaa ctggggaggt gaggaagaag caggtattgt caccagtgag 40080gactgtgctg ttgtgagccc agctaggcaa ctggcaattc cattctgtta gtgacagcta 40140caataaccca aagccctctg gagccctgct ttcctctgct ctcttcgtgg cttgactagg 40200agctgaagat cctgtccctc ttagagcatt ggggcggccc accccacctc caccctcctc 40260cacctgctgc ctcgaggccc ctcccactcc cggggtagac aaaacagttt agaggctgaa 40320gtcaccgggg ctgtaactgt tggatttgca catgtcatag aaaatcatca tatgttttgt 40380gtggactcca tgcataacaa caagagaacc aaccagaccc catagacaga agggagtgtg 40440aattggagac aaaatttaaa ttatgagttg ccttctattc agatttctcc catttttaac 40500aaaaaggagc ccaaattcct aaatgttatg gttgtttgca gcaacttatc atctttctcc 40560tttccttcat agccaaggtt tttgaaagag ctatctgagg ccgggaatgg tgactcacgc 40620ctgtaatcct agcacagagg ctgaggtgag tggatcacct gaggtcagga gttcaagacc 40680accctggcca acatggcgaa atcccgtctc tactaaaaat acaaaaatta gccgggcatg 40740gtggcgtgtg cctgtaatcc cagctactca ggaggctgag gcaggagaat cacttgaacc 40800caggaggtgg aggttgcagt gagccaagat ctcaccactg cactccagcc tgggcaacag 40860agtgagactc catctaaaac aaaaaaaaaa gtagctgtct gttctttctt ctcgaactct 40920ttttcccgct ggagtctgtg acctgctgcc gtctgcctca agtgagaggg actagcagat 40980ctggtgaatt accttctaat gcccgtaccc tgcccatacc agcttcaatc tgtatgtaga 41040agcttagctt gctccatgca tggcctccag catccactgg tcacaaaata acacaaaata 41100gcatgagaga gaatggtcgc atggagcgga ggagctgctg agactgaacc caagccaggg 41160ctactgctgg gtggaactgg acatgcccag cccatgggaa agtcttccca cagaagtcat 41220atttgcaggg gtctcccagg agacagcaca ttctgagcaa aggagtgagg cagagataac 41280tattcaggaa ccaagagact cgctggaaag aagcagagat tttcagccca gcgtagtgga 41340tgtttcttga atcttcccct gtggatgccc caaaccttga gatccttcca acaaatagca 41400cactactaac aaactgtgac tcaaagagag ggaaacatgg tcccctgctc tgtcacaaat 41460cactgtgaag ctttggcacc ctgactgctc aggtggccac caacacagaa ggaccacgaa 41520tggctgagtc aggaagtcac agccgtgtgg ctggaagagg ctctgccttg ctctgggaga 41580aatgcctatc cccaaggaag ccttagtatc catgggagag aaacactgta gcaatggccc 41640ccaggactct cgggaagcca cttctggtgg gaggggactc aaagggtgct gggggacctg 41700tgtctgcatc tggaagtgag gagccaggaa aattttcttt cagtttcttt cttttttctt 41760ttcttttttt tttttttttt ttgagaaagg gccttgccct gtcgctcagg ctgaaacata 41820gtggtgcgat ctcggctcac tgcaacctcc acctcccagg ttcgagtgat tctcctgcct 41880cagcctcccg agtagctggg actacaggca tgcaccccca cccacgccca gctaattttt 41940gtatttttgg tagagatgtg gtttcgccat gttggccagg ctggtctcga actcctggcc 42000tcaagtgatc ctcccgatgt gctgggatta caggtgtgag ccaccacgcc cggcctcttt 42060ctgcttcatt taacattaat ggtcatccca cagcatggtg ctgtgcacct gtagtcccag 42120ctactcaggt ggctgaggtg ggagaatcac ttgcgttcca gctgtagtga gccttgattg 42180tgtctgtgaa taaatgccac ttctctccag cttgagcaac atagggagac tgtctcttaa 42240aaaacaaaac aaaacaggct gggctcggtg gcccacgcct acaatcccag cactttggga 42300ggccaaggca agaggattgc ttgagcccag gaggtcaaga gcagcctggg caaaataggg 42360agaccccatc tctacaaaaa gataaaaaat aaaaaaatta actgggcatg gtgatacacc 42420tgtagtccca gctactctgg aggctgagat aggagtattg cttgagcctg ggaggtcgag 42480gctgcagcga gccatgatca tgccactaca ctccagtcca ggcagcagag tgagatcccg 42540cctcaaaaaa ataaaacaaa acaaaactca tctctccctt ggctcctgag actacaatcc 42600ctcacggttc ttttctactt ctctgttttt ctcttcttgt ctcccttttt ttctggtctc 42660tctgtcaccc aggctggagt gcagtggtgt gatcatagct cactgcaacc ttgacctcct 42720gggttcaaga gatcctccca cctcagcctc tcgagtagct aggactacag gctcacacca 42780ccatgcctag ctaatatttg tagattttgt agagatgggg tcttgctatg ctgtccaggc 42840tggtctcaag ctcctggcct caagtgatcc acccacctca gccacccaaa gttctgggat 42900tacaggggtg agccaccgcg cccagccgat aattgttgaa aaatcatttt cagttaaggt 42960atccagtcaa ggtcagaaaa tgagaaaatg ttaaaaaaaa aaaagctata agtaaaacag 43020attcagtcgg gacatgatgg ttcacgcctg caatcccagc actttgggag gttgaggtag 43080gataatcact tgagcccagg agttcgagac cagcctgggc aacatagcga gaccttatcc 43140atacaaaaaa atttaaaaaa tacccaggca tggtggcata ctcctgcatt ccctgctaat 43200tggatgggtg aaggggagga tcccttgaaa taggagtaga ggtgcaggaa atatgattgt 43260gccgtgtaat ccagcctggt tgacagagca agatgttccc cacccccctg aaaaaaaaaa 43320aaaaacctaa atccaaattt taaaagtttc cttgactctt caacttgctc accctccacc 43380aaataaaata actacgaagg aggcttattt tttactattt ccagggatac gatatatgtt 43440tgtcctgaaa atatacatca tggctttact caagccacag tgatgaggcc tcattgtcac 43500tgtagcctaa ttacgatttt ataactccat ttaaaattca atttaaacac agtttaaaaa 43560ttcagtccaa gtcaaacatg ctctcagtag ctagaagcaa aactctgttc aggtccttga 43620tggatctatt tgtactttct ttcatgaaaa cagaaagtcc ttttttacac accatgcaac 43680aggaaaattc ataacggaca ttgttttacc tgttcttggc aaagacaagt gagctcttaa 43740caagcaaggt aactatggag atgatgtttt gctccaagtt aacacttaca tatttaatta 43800gaaagatttc aaaggtgggc agattcactg gaaagtttcc aaaagcttca cttgttcaac 43860aaataatgtt agagagggag caccgtgccc tcgggcccct aggaattagt tccacatggt 43920ccggtcctct gtccagtgtg cccagcatcc acttgggaga acagcatggc cttctgtcca 43980gggcagccca cgccagcact gcctgccctt tcaggcccat ggctcccatt aagtgccatt 44040tcgagcatac ttagccaagt ttccctacca tggccaacaa agaggttgtt caaaaatgct 44100tgtcaggtcg ggcatggtgg ctcacgcctg tagtcccggc actttgggag gctgaggcgg 44160gtggatcacc tgaggtcagg aattcaagac cagcctggcc gacatggtga aaccccgtct 44220ccacaaaaat acaaacatta gttgggcatg atggcgggtg cctgtaatcc cagctgctca 44280ggaggctgag acaggagaat tgcttgaacc cgggaggtga aggttgcact gagctgagat 44340cacaccattg cactccagcc tgggcgacag agtgagaatc catctcgaaa aaaaaaaaag 44400tttgtcaacg gtttcactga atccagaata cttttctaaa atgtcaaccc tatagaatac 44460attttataaa attatgaagg cctggtctgg tgtagtggct cacgcttgta atcccagcac 44520tttgggcagc caaggcaggt ggatcgcttg aggctgggag tttgagacta gcctggccaa 44580caaggcaaaa ccctgactct actaaaaaat acaaaaatta actgggcgtg gtggtgcaca 44640cctgtaatcc cagctactca ggaggttgag acaggagaat cacttgaacc caggaggtgg 44700aggttgcagt gagtggagat tgcgccattg cactctagcc tgggtgacag agcaagactc 44760tatcttcaaa aaatagataa ataaataaaa attaaaacaa aataaaattc tgaaggcctt 44820aggtcagaga attaccgagg gaatattcaa agttatacct ccaagtatct acaatgaaga 44880tactttcatc agaaaaaagg agtttacggc caggccctgt ggttcatgcc tataatctca 44940gcactttggg aagccaaggc tgaggcagga ggatcacttg aggccaggag ttcgagacca 45000gcctgagcaa aaacgtgaga tcccatttct accaaaaata aaaatgtaag gtaggcatgc 45060aactgtagtc ccagctactc gagaggctga ggcaagagga tcgcttaaac ccaggactcc 45120agcctgagca acagagcgag accctgttta taaaaaaaaa agaaaaaaaa aaagaagaag 45180aagaaggaga agaaaggaaa taaaatttaa gaaaaaaaaa aggacttaat aaggttgaat 45240gaaggcaaga atattcttag ctctgtttaa gtcaagacct gagtagtagc tctacgtagc 45300tgtatgtcga taatgttttt gagacagcac tactgataaa ttgttacata ataaactgtt 45360atggctggat gcagtggctc atgcccataa tcccagcacc ttgggaggcc gaagtgagtg 45420gatcacctga ggtcaggagt tcgagactag cctgatcaat atggtgaaat cccatttcta 45480ctaaaaaaat aaaaattagc tgggcatggt ggcgcacctg taatcccagc tactcaggag 45540gctggggcag gaggattgct tgaacccagg agacagaggt tgcagtgagc cgagattgcg 45600ccattgcact ccagcctaga agacagagcg agactccatc tcaaataaat aaactgttaa 45660attaagttta gcctaaagct acccccttac atattttaag ttcagtctaa aggtttccct 45720gcacatagtg aactgtaacc taactggatg cgtaaacaga ctataaccta ctcttgggcc 45780agtcactgag ttttggtcaa tcaaaggcag ccaactgttc aaaccaggtt aaaataaggc 45840agatgctgag ctctaaccag tccagccatt tctgtacctt gcttccattt tctgtccatc 45900actttccctt ttctgtccat aaatcttcca ccacgtggct gtgctggagc cactgtgaaa 45960ctattctgtt tcaggggctg cccaattcat gaatcattcc ttgctcaatt aaactctgtt 46020catttaattt gtctaatatt tttcttttaa tcaaagtaat ttggccgggc acagtggctc 46080acgcctgtaa tcccaacact tcgggaggcc gaggtaggtg gatcacctga ggtcaagggt 46140tcaagactag cctggccaac atggtgaaac cccgtctcta ctaaaagtac aaaaattagc 46200cgggtgtggt ggcgggcgcc tgtaatccca gctactcggg aggctgaggg aggagaatcg 46260cttgaacccg ggaggtggaa gttgcagtga gctgagattg tgccattgca ctccagcttg 46320ggcgacaggg caagactctg tctcaaaaaa aaaaaaaaaa ttaattcaga gacctactca 46380tgtgaagttg tattttttta ttctccatat tacaaaacag aacaattggc acagggatga 46440agaaatactt tgcaaaacat ctagagaggt taaatgccat gagtctttaa aatgtaagac 46500tgctttcacc tgagcaatct agtgtccatt tctagagcta gcttaaatgt ccgtgtaaat 46560ccccgtaatt ggttgggata acaattacct atgttgtata acttgagtca aaaactacgt 46620ttccactgcc tgccacccct atggatggtt ttctcttaag gtatcaaatt ttactgggaa 46680agacctagat aaaatacagc gaaaatgagg cggggcgtcc tggcacatgc ctgtaatccc 46740agcgctttgg gaggctgagt cagaaagatc tttgaattca ggagttcaag accagcctgg 46800gcaatatagt gaaatcctgt ctttacaaaa aattaaaaat tagccaggca tgggggcatg 46860ggcctgtagt cccagctact tgggttgggt gactgatgtg ggaggatcac ttgagcccag 46920gaggttgagg ctgcagtgag ctctgaccat gcccctgcac tccagcctgg gtgacagagc 46980aagacccagt ctcaaaaaga aaagaaaaag agtaatgtta ggtcaaggta gaacctacct 47040tgactttctg ttactatgga agatattctg gggtatctct gagatccaag tattatggca 47100cttaagtaat tcctatctat tgttctactt ggttcctcgg gagtaaaagt catattcaaa 47160ccaaaaaggc tgtgggattt ccagaatttt aaaagcaata atagttaatg ttctcccatg 47220ggagttactc cacattttta catatgttcc atatgttaac tcatttagac cttaccttta 47280tgaggtaagt cctcttctta tccccacttt agaggtggga aaactgaggc acagaaagag 47340taagttgctt gcctaaggcc ctgttactag caggtggtga aaccagcatt ccaacccggg 47400agtctggcaa atgtgtgtga agagcacacg tttggaaatg acagtcatga ggacactgta 47460agacttctgg aatgtttata atttcacctt tgcttgttat ttttcctgtc tgtttcccta 47520gagtgagctg agtgaaaaaa gaaagaagaa agaaagaaga aagagaaaga gaaagaaagg 47580agagaaaaag aaaaaagaaa agaaaaacag aaaaagggaa agaaagaaga aatgaaagaa 47640agaaaagaaa agaagaaaga aaagaaagag agaaaggaag gaaggaaggt gggaggggag 47700gaaaggaaga agaaagaaag aagggggaca gagggaggga atgaaggagg gagagaggga 47760gggaaggagg aagaaataaa aagatgagga tctgtatgct tgaggggtgg aggtgggggg 47820cttgggtggg agtgtgggat gggcagaaag ctggagggag ccctggaccg actgcattcc 47880acagaggatt gtgggtgcaa cgtaggtggc agattgagaa aagcaaacaa acaagctcag 47940cctttggagc ttcggggaag aaaaaaagct gagcagtgaa tgctggcttc ccacggagaa 48000ggcaggctgc ttcgccagct cacatccttc cgcgcaccca cttcctcttt ccggaggtca 48060ctttagattg ctttatggca ggatctccag gtcacaggaa tgttatgttt cgactggggt 48120ttccccctcc cctgggatgc ctgggccagc tccccaaggg ctagtctctg tcccaggccc 48180cacactccca tagcactcag caaaagccta gagagagcac cgcaaaatgc caaacgcaac 48240aggaccgcgt aggaagaaga cgcttggaat gacagggaca ctagaactgc ccatggtcgt 48300ggtctcaaat ttttgttcca tggtctgaaa tactaaaagt tcttaaacag ctacttgatt 48360tcatactatt gttttgaaga aaacagtgtt tgtttgttgt tttgtttgtt tgtttgtttg 48420agacagagtt ttgctcttgt tgccgagttt ggtccatgtt ggtcaggctg gtctcgaact 48480cctgacatca ggtaatccac ccacctctgc ctcccaaagt gctgggatta caggaaaaca 48540gttgtttctt taaaacaatt atataggctg ggcacggtag ctcatgcctg taatcccagc 48600actttgggag gctgaggtgg gtgaattacc tgaggtcagc agttcgagac cagcctggcc 48660aacatggtga acctccgtct ctactaaaaa tgcaaaaaat tagccgggcg tggtggtgca 48720ttcctgtaat accaggtact caggaggctg aggcaggaga atcacttgaa cccaggaggt 48780ggaggttgca gtgagctgag atggcaccac tgcactccag cctgggcaac aagagcaaaa 48840ctccatctca caatctcaaa aaaataaaat aaaataaaat aaaataaatg gttatataag 48900ctaccttatt gatgcagtta caaatgagcc gctgaaacat ataaatttta aagaacaagc 48960cacatatctt tcatcaccca cagcttcacc aactaaaggt gtatgtagta cttttgtgga 49020aggcatttcc acatgctttg agggaccttg aaatactgct atgattacat gatttttcta 49080aaaccagact actcctacat tacaagaatt gaaaagttca gagtaaatat ttgtaagacc 49140tagaaaagat gatgttcttt aaaaaaaacg atgcccatct ttgtagcgaa aagaaagaga 49200gatcagactg ttactgtgtc tatgtagaaa cagaagacat aagagactcc attttgaaaa 49260agacctgtac tttaaacaat tgctttgctg agatgttgtt aatttgtagc tttgccccag 49320ccactttgac ccaactactt tgacccaacc tggagctcac aaaaatatat gttgtatgaa 49380atcaaggttt aagggatcta gggctgtgca ggacgtgcct tgttaacaaa atgtttgcaa 49440gcagtatact tggtaaaagt catcgccatt ctctagtctc aataaaccag gggcacaagg 49500cactgtggaa agccgcaggg acctctgccc tggaaagcgg ggtgttgtcc aaggtttctc 49560cccatgtggt agtctgaaat atggcctcgt gggatgagaa agacctgacc atcccccagc 49620ccaacacctg taaagggtct gtgccgaggt ggattagtca aagaggaaag cctcttgcag 49680ttgagataga ggaaggccac tgtctcctgc ctgcccctgg gaactgaatg tcttggtata 49740aaacccgatt gtacatttgt tcaattctga gataggagaa aaaccgccct atggcgggag 49800gcgagacatg tttgcagcaa tgctgccttg ttattcttta ctccaccgag atgtttgggt 49860ggagagaaac ataaatctgg cttacgtgca cgtccagtca tagtaacttc ccttgaactt 49920aattatgacg tagattctgt tgctcacatg ttcgttgctg accttctcct tattatcacc 49980ctgctctcct actacattcc tttttgctga aataacgaag ataataatca ataaaaactg 50040agggaactca gagatggtgc cggtgcaggt ccttggtatg ctgagcgccg gttccctggg 50100cccactgttg tttctctata ctttgtctct gtgttttatt tattttctca gtctctcgtc 50160ccacctgact agaaatatcc acaggtgtgg aggggcaggc caccccttca catcttgtct 50220ccacttcctt gattaaaaaa aagaaaagaa aaaaaaattt gccgaagttg gattcattca 50280cagaattcta cacattaaaa atgttgcagg tcgggtgtgg tggcagctcc caaagctgcc 50340tataatccca gcgctttggg aggcttgagc ccaggaggtc aaggctgcag tgaactgaga 50400tcgcaccact gcactccagc ctgggcgaca gagcaagacc ctgtctcaaa gaaaaaaaaa 50460aaaacagaaa aaaataacgt tacagaaaaa gtacaatatt tttaatatat atatatatat 50520tttttttttc tgagacagag tgttgctctg tcacccaggc cggagagcta tggctcgatc 50580tcagctcact gcaacctcca cctcccgggt tcaagcgatt ctcctgcctc agcctcccga 50640gtagctggga ttacaggcac ccaccaccac gcctggctaa tttttgtatt tttagtagag 50700acggggtttc cccatgttgg ccaggctggc ctcgaactcc tgactttatg atccgcctgc 50760cttggcctcc caaagtgttg ggattacagg tgtgagccac catgcccagc caaaagtaca 50820atatttttaa tgacatataa agatgttcat tctttgtggt tgccctgggt gagagggact 50880attgatactc aatagtgttt cttttgtttc tacattgttt ctatagtgaa aatacgcatt 50940ggctttgtat taaaaaatgt atagtaaaaa tggttttatt aaaaatagca aataactaca 51000aaaactccat tgcaatggaa agcagccctt ggattttcta gttgaatgaa acgagtaatt 51060tatccaatgt tagaaatgtc taaaggctcg ctcaggtttc atgagcagaa caggaattgt 51120atatccaatt aaatgtgaaa ttgcaatgcc tggtgcggtg gcttatgcct gtaatcccag 51180cactttggga agccgaggca ggggatcgct tgagcccagg agttcgagac caccatgggt 51240aacatgggga ggccccatct ctacaaaaaa taaaaatcgt tagccgggca ggttggtgca 51300tgcttgtgtt cccagctact tgggaggctg aggtggaagg atcctctgag cccaggagga 51360tgaggctgca gtgagacatg atcgatgcac tccagcctgg atgacagagt gagaccctgt 51420ctcaaaaaaa aaaaaaaaga aaagaaagta caatcgcaat taaatgtctt tgcgttggtg 51480gctcctgacc aaattcccta agcaagcagt atgttaatga gcagaggggc cacagctcac 51540cttgctcaat taaaggcagg agcaggccgg gcgtggtggc tcacgcctgt aatcccagca 51600ctttgggagg ccaaggtggg cggatcacga ggtcaagaga tcgagaccat cctggccaac 51660atggtgaaac cctgtctgta ctaaaaatac gaaaattaac tgggcatgtg gcatgagcct 51720gtaatcccag ctactcggga ggctgaggca gaagaattgc ttgaacccgg gaggtggagg 51780ttgcagtgag ccgagattgc accactgccc tccagcctgg tgacagagcg agacttcatc 51840ttaaaaaaaa aaaaaaaaaa ggcaggagca agtatgggcc agacagaaat caaggtgtaa 51900attgggcaga tcctcaggcc cagtgctgaa ttttggtttg atgaaataaa acattacatt 51960tcaaggttgg cagagaggaa tgaaggtgga agaggaatct agggccattt agggaagcca 52020tgaagcctcc tgcccacact agtgggtaga gtggagccag gcgttttgct agggcttgct 52080atatctcttg gcagggtgct ctgctgccaa agccaagaat tctaaattag attaaatagc 52140cagaaagaat gttaaacatt tggacatgat atcctccctc acagattagc tagagtgtag 52200ttctgctgtg ctagatactt aaataaatac ctccctagct gtgaagcctg cttatcacag 52260tactatattt taggatgagg tcattatttt cctatgcata cacatgcatt gtataatctt 52320gccaatgtag gtcagcccaa aagaagtgac aaatgtgtag aacacacatt ggactagctt 52380gggacaaaat tagtatacct aaagatgaca gatttcttaa ctaattttat gagccatgca 52440gctttgtatt ctagcagaga cagacattag gaatcttata aaatcaaaaa ttttaatttt 52500tgcctgaata gctccaaagg gctaagatct caagcaaatg cgtgtaggtt ttgtttttgt 52560ggttgttgtt gtttttagag acagggtctt gctctgtcac ccatgctgaa gtgcagcggt 52620gcagtcctag ctcactgcag ccttgacctc tcaggcttaa gtgatcctcc tgccttagcc 52680tcccgagtag ctgggactac aggcgcatgc caccaccccg agtaattttt tatttttatt 52740tttacttttg tagagacagg ggtctcaata tgttgctcag gctagtatct tttttctttt 52800tgagacagtc tcgctcaatt gcccaggctg gagtgcagtg gtgccatctc ggctcactgc 52860aagctccgcc tcccgggttc acgccattct cctgcctcag cctcccgagt agctgggact 52920acaggcgccc gccaccatgc ccagctaatt ttttttgtat ttttagtaga gacggggttt 52980caccgtgtta gccaggatgg tctcgatctt ctgacctcgt gatccacccg cctcagcctc 53040ccaaagtgct gagattacag gcgtgagccc tcgcgcccgg cccagtcttg taacttaact 53100ttaaagctac ttattcccaa atgaagatgg gatggtacac agattttaag tattagctgg 53160tttggagctt ctgtctttta aagcaacatt ttactttgcc acagggtggt ggggcggggg 53220ccatcctaga aagaagagtg tgagtttcat gggatagggt ctggggaggt ggctggagga 53280gtttaggttc ttttgatatc tgtggctaca cagacagata accaaggaaa atgtccaaac 53340agtgaaatta agtgctcact gcactaacac agagaaggac cctgatgtct ggccgcaggc 53400ctttgttctc attggcttca aagaacttct tgatgtctac cttaatttca ttattattta 53460cccaggagtc attcaggagc aggttgttca attgccatgt agttatgtgg ttttgagtga 53520gtttcttaat actgagttct aatttgattg tgctgtggtc tgagacactg tttcgatttc 53580agttcttttg catttgctga ggaatgtttc atttccaatt atgtggtcga ttttagagta 53640agtgccacgt gacgctgaga agaatacata ttctgttgat ttcggggggg agagttctgt 53700agatatctat taggtccact tgatccagag ctgagctcaa gtcttgaata tccttattca 53760ttttctgtct cgttaatctg tctaatattg acagtggggt attcaagtct cccactatta 53820ttgtgtggaa gtttaagtct ctgtgtaggt ctctaaaaac ttgttttatg aatctgggtg 53880ctcctgtatt gggtgcatat gtatttagga gagttagctc ttcttgttga attgctccct 53940ttaccattat gtaatgccca tctttgtctt ttttgatctt tgttgggata aaattacatt 54000ttatgtcccc cttcctatag tttgtcactg agggttggca gaagttgaaa ggaagaagac 54060atttgggtgt ttggtttggg gttatattag gttataaggt tcattgcctc cacctctttc 54120aaaacattta gtttctaaat gaatccagct ttaaatgact gcaggagtgc ccatgcacaa 54180ttttgtttct caaatctttg ggatttttcc ttgaagaata ttcacaggga atggggctgt 54240cttgcttcat agttactctt ttgtatacat gatctcaaga atcgcctgat cactgctaga 54300gttaaaccaa tacactaact gcctgaagtg ctgaaaagtc aaatgggggc ttagaacctc 54360actccagatc ctacacaagc tgatggttct gttcccagaa acaacccagc ttcctcatca 54420tctatggcca gtgccttgta gcggagctgg agatcaccct ttagtgggct cttcagctgg 54480atctagaaat caaattgaca ccaggcagat taacaagaga aaagtataca gattttattg 54540cttttatatg tacttgggaa tctgcacaag ggcaaagtcc gaagaggtgg ccaaagcaag 54600gtgcttttat acatttttag aaaaagagcc aaaaaattgg agaagaaatg ataggacaaa 54660gaaaatctag ccaggcagta aattttctag gagaatcact aggacatata tgaggaaggg 54720tgtgtaaaac aggtgaaaga taagggctag ttcattaaac atgtttactc tggtccattg 54780tagcctctac gataaggagt attttctcgc tctggtgtgg acagggcacg cctcccagag 54840caacctttat cacttactgc atgcaggaag agacaggtca gcccgccctt cctgaaacta 54900caatttcttc agtgttttca actcaaaata atcaataccc cccatctggc atatctgggg 54960atggcacgtc ctttactcct tcaggctctt ctccctgaag gtcctttgca tagttgggaa 55020tctccaccag gaggggtagc tctttggtct aaacccatgg tggcagagtt tcgacaatat 55080tcccaactta aatgtttctg attctgagtg gtggttagat ccctttgtac acccctgtcc 55140ccagtgccta cagaatgggc atgttaataa gtgttggctg aacattcaat gatggataag 55200gaagaatagg aggcaagaga gacggtggtc tccagtgcca agccccagtg ctaactgggg 55260tgattttttt tcatgactca ttttcctaaa atcaccctca agggtcctac aaaactcttc 55320ccaacagcta aatcacagac taatctggcc catcgacgtc ttccctgatt atactaattt 55380ttttgtgttt ttttttttga gatggagtct tgctctgtca cccaggttgg agtgcaatgg 55440cactatctca gctcacagca acttccacct cctgggttca agcgattctc ctgcctcagc 55500ctcctgagta gctgggactg ccagcatgcg ccaccatgcc cggctaattt tttttttttt 55560ttttttagta gagatgagat ggggtttcac catgttggcc agggtggtct tgaactcctg 55620accgcaagtg atccgcttgc ctcggcctcc caaagtgctg ggattacagg tgtgagccac 55680tgcgcccgac catatattaa tggtttttga tgaatttgtt ccatagatta aaatcttgtg 55740ccccatcgcg tgtggggctc catcgcatgt ggggcacagg gttcctgagt gtttgtggct 55800gtcaaaccaa gatgatttct tgcttaatca agcagatttg aaagttcatc tctgctacca 55860ggaagcactt gctcaactca gaagacaatg tcctatcagt ctttcactat cacgcatctg 55920ttcttcaaga tccgtcaaat tagctccagt gaaacggagg ctaaagtgaa actttttctc 55980ttatatagat ttttattcat aactagggaa aaattaggca cccacagaaa aataataacc 56040taaaaaaatt aggctgaacg taagaaaaat ttgtgatgaa ataaacattt caatcaacag 56100aaaatatttt tctgactttt tatgtgccac cattagttac atcattgaga aaacaatatt 56160tgtattaaaa aaagagctgg tgaaaatctg gcaattggtc gggcatagtg gctcgtgcct 56220gtaatcccag cactttggaa ggccgaggca ggcggatcac ttgaggtcag gagtttgagg 56280ccagcctgac ccacgtggtg caaccccctc tcaactaaaa atacaaaaat tagctgggcg 56340tggtggcagg cgcatgtaat cccagctact agggaggtta aggcaggaga attgcttgaa 56400tctgggagat agaggttgca gtgagccgag actgagccac tgccttccag cctggtgaca 56460gagcaagact tcatctctct ctcttttact ttttttaaag acttcttctc aaaaataaaa 56520agaaagaaag aaaatctggc aatccagtaa aaactggcca ctatggcatg catgtgctat 56580gcataaacgt aaattgatgc ataaacttaa ttttagaact ggaaggaaat ctggagttct 56640ttaggagcca ggttttacac atgcagaaac ctaacagctt cagtttcgat tcgataaaat 56700ttgactaact aaacttaaga taagcatagt tacgcattag agtattaact ctcaaacttt 56760taaaaaagaa ttcttccttt gcttgttaat tttctttctt tctttttttt ttttttgaga 56820tagggtcttg ctgtcgtcca ggccgaagtg cagtgacgtc atcatagttc actgcagcct 56880ctacctcccc ggctcaagta atcctcctgc ctcagccttc tgagtatctg ggactacagg 56940catgagccac catgcccagc cttttctttt cttttctttc ttttctttct ctctctgtct 57000ctctttcttt cttttttcct ccctttcttc tttttttttg atggagtctt gcactatcgc 57060caggctggaa tgcagtggtg cgatcttggc tcactgcaac ctccgcctcc gggttcaagc 57120aattctcctg cctcagcctc ctgagtagct gagactacag gtgtgtgcca ccacgccagc 57180taattttttt atttttctag agacggggtt ttaccatgtt ggccaggatg gtcttgatct 57240cctgacctca ttctccacct gcctaggcct cccgaagtgc tgggattaca ggcatgagcc 57300accgtacctg gccctttctt tctttttatc aagacaacaa catgtcttta tagtgctccc 57360aaggctaaag tataccttac gtctatgtaa acactcaacc tgagctttgc aatggcccat 57420gttggcagta gtgcaaacaa aaacaattat gaaacccatt ttcctttgac aaagagaaat 57480aagtggcaag aattggttct ttctcttagt atgggtctct gaaaagaacc agatcagtca 57540aaaggggaat atttttctga agggataggt ttggcctagt ggcttctacc tcttttagat 57600gactgctgtt tctcgtttta atgttaaata gacactaata ggagaaatca cattaattca 57660gtcaacaaac atttactgag cacttcctgt agtcaggccc tctgttaact tctgggaata 57720caatgacaac tctgacaatc ccaacccaag gagccaacaa gtccgggaat agagacagac 57780aagaaaacag acaattacaa ctctaccgtt agaataaagg tacattgaga acttgcaaca 57840aatattcctc atcccttatc ttaattattc ataacatgtt taccaccaat aagaatagca 57900ataacaataa atgcccaact cagacagcaa tgtccattta ccctgtgttt acacagcata 57960atacaagcaa gctgtggaca gagattctct tgtttagtcc tcacaactct gcaaggtggg 58020ttttattact ctccatttct agataaagga tctcacctaa tattacatgg gccagtggtc 58080ttccagttgg ggtatgcaca accctagggg taggtgagga ccctgcctgg ggtcttcagg 58140tggggaccat caacctccat ttgtactctt ttctgaacat tggtctgaga cagaaagtcc 58200ctgcaattaa ggcattaagc tggctctttt tctatttctc atttcataat tgcccttctc 58260ctgctttacc aaaatctttc accccccatc atatatatat atccccatac atattctata 58320tatacatacc ctacatatgc atgcacacac atcatatata tgtatgcata tatgatatat 58380acatatatgc tatgtaaaca tatatagtgt gtatatacat gtgtgtatgt gtatatgtgt 58440gtatatgtgt atatatacac gtgtgtgtgt acatatatac atcatatatg tgtgtctata 58500tatgtatata tgggtgtgtc tatatatgtg tatatgtacg caaatacgta tatgtgatgt 58560atatatataa gatgtgtgtg tgtatatata tgtgtttgca tgtgtgtgtg tatatatata 58620tagtatatac atattttttg agacagcatc tcactctgtc gcccaggctg aagttcggtg 58680gctgatgaca gctcactgca cctcccggct caagtgattc ttccacctca gcttcctgaa 58740tggctgggac tagaggcgtg tgtcaccaca cccagttagt tattttattt ttcgtagaga 58800tggtggtctc actgtattgc ccaggctggt ctcgaactcc tgggctcaag cgcttttcca 58860cctcgacttc ccaaattgct gggattacag gtgtgagcca ctgcaccggc ccatccttta 58920ttttaatatt atgcagtgcc ctgagacata taaaaaaccc accttcccaa gtaaaggaaa 58980ttcaagctga tgcctgcaga gccttcttta acaaaggctc tgaaataccc tctctcatta 59040aaatgatact ttccaataaa attttgttta acaatgattt acaaaatgat aaaatttatt 59100tattttgatt gtgtatggat catggtaaca ataaaaagac ttgtaaaaat aactaaattg 59160aaagaatctt gaacatttag agccttaaga ctgtaggaat tgaagaccac agaattatta 59220atttatatta atatttttgt tgcagagaca taatgaatga tcaacgaaag gcttttaagc 59280gttaaaaata tattacacta gataaaatta tttgcgggaa tgggatggaa atacattttc 59340aagagagaaa ggagcaatgt aaaatgaaga tgtaaaatcc ttctgctggt tgtccttggg 59400gttttctttt aaagaaaagc ttggcagtgt ttttcttttt ttccattgga tgatggtgaa 59460tatcaaatca ctttggtgct aatatttcat ttaatacatt aattttaaaa ttttctgtag 59520aggtgggatc tcactatgtt gtccaggctg gtttcaaact cctggcctca agcaatctcc 59580ctgcctcagc ctcccacagt gctgggatta caggtgtgag ccactgcatc cggccccatt 59640taatacattt aaaagagtgg tgtaacaatt tttatttaaa atgtcatatt tacaatattc 59700tagaatgtat atcttttcaa ctcattaaac ctaaacatcc ttgtaaaaag tgtgaaaagt 59760tatatagttt ttcaaaattc gattagcagt tacataagca taaatgttta aagtatgtat 59820ggtacagcca ggcttcagtt ccctgtctta aacacaaaga tccatatcaa ttccagatac 59880tgcaatggtt tgctgttttt cctgcttccc ccatctccaa ataaactaaa gcatcaacat 59940gcctcacctc acataaccct aagttttcag cagttggcag ttacacctgg aaaccatttt 60000tctaaaataa acaacaactg tttgcttacg gatcaaaatg caaaggacca taacatttag 60060cctcaccttc ctactacaga tcgagtttaa aagtgccatg gtatagctaa attatgaaga 60120aagatatgaa tataactgca aaagtggaag gagatttggg ataattcttg cccattttgt 60180taggccaaat gcatctttgt gcaaattaga aaaaggtggt cttcatccct tcactcctat 60240ccttttgggg gtggaggggc agtggctaaa gtacagacta ggtttcagct accacatcct 60300ccttcagtta gctgccctcg gcgtgacaga aacatgtgca aacagccctg tgcctttgtc 60360ttatgttcca gccagccaag aaaaatagtt gtaaaagagc agctgctgtt tggggtaatg 60420accttggacc ctccccaatt tgttccaagc ctgtttttgt attcattttt cccacattta 60480tgttcctgga tggaagcttc catatctgct cttggcccta tttgaaattc cccagatttc 60540cttcctggct cctggccttt ggtttttcat gtggctcctg atcccacacg ctccctgaat 60600ttggattctc ctgtcatttc aggtgcgagg tttcccacta cagcctcttg ggcctcacct 60660ccaatacctc tttcccatca gaacagcccg gaccttcccc tatggtagag cagagacaga 60720atttaaatga attctcaaga agtgcttgga ctcatatcta gcaaaattac atggcattta 60780acctttgaca caaaaaatgc agcttctagg aatctatcta aagatacact gtggcaaata 60840tacaaaaaga agcattattt atcaagcact atttcctaat aaaataattc ttaggtcagg 60900cgcaatggct caggcctgta atcccagcac cctgggaggc tgaggaaggc agatggcttg 60960agctctggag ttcaggacca gcgtgggtaa catgacaaaa ccccatctct aacaaaaata 61020caaaaattag ccgggcatgg tggcatgcac ctgtagtccc agctactcga gaggctgagg 61080tgggaggatc gcttgagcct gggaggcaca ggttgcagtg agccaagatc gcaccactgc 61140actccagcct gggtgacaga gtgagaccct gtcaaagaaa gagagagaga gagagaaagg 61200aaagaaagga agaaaggaag aaagagaaag gaaagagaaa gaaagagaaa aagaaagaaa 61260gaaggaaaga aagaaagaaa aaagaaagaa aaagaaagag aaaggagaaa aagaattctt 61320actaataaat gcaggagaaa tgatagaatt gaaatatcac cattttcaat tcctaatgaa 61380ataacgtatc taggcaatga ccatcaatag ctagatgcta aaatcatctg atcaaacact 61440gatgggaact tcgtaacaga tggatcaggc taacaacatc tgaaaccact aactggtttt 61500gatgtcataa aaagaaaaac aaccagatat tttctgtctc ctgatgagtt gcaattggag 61560ctacatatca cctgtaaagt cttctggcca aaaaattaag cccagccgga ccttattaaa 61620cctttaaatc taacaattag ttttgaagct tttacagatt aaatgaagtc tgagatttgc 61680ttcaaaatga accagtggtg gggaggaagt gggtgaggtg taggtgaaac aagattggcc 61740acgtcgataa ttgctggagc tgggcgatga aagcacaggt atttatcaca ccatctctct 61800acttttgtgt gtttttttgt ttgtttttgg ttttggtttt aaggagcaga gagtctaata 61860ggcaagaaag aaaagagaag gctgaaggaa gacgctcccc cgtacagaga cagagggagg 61920gggctccaaa gccgaaagag gaggtcctct tgtgtatgtt ttaaaatact cccagataaa 61980atatttttgg aagagtactt ggttggattc aacagctttt ttttaattta aaaaaatcac 62040ctcaattttt ttgcttgctc taacgtgcca tagaaattcc tgaggtttta cttgttgctt 62100tacaatgaac tgtgtaaaca caagctggaa gagatcagct atgcgctgga agggttggtt 62160aaatattgag actgccttgc tgagggaagc cttttaatga atctcagtaa ttttgcaaga 62220gaaaagataa caatgaacac tacattaaac atcattcttt tgcactttgc taaatatgtg 62280tatgtaaatt actgtctgac tgttactgga tatatacagc atatacatat gcactttttt 62340tactgttttt tttttttttt tttttttttt tttttttaca gagcttgctc tgtcacctag 62400gctggagggc agtggcgcag tctcagctca ctgcaatctc cgcctcccag gttcaagcga 62460ttctcctgtc tcagcctcca aagtagctgg gactacaggc gcctgccacc gctcctggct 62520aatttttgta tttttagtag agacagggtt tcactatgtt gtccaggctg gtctcgaact 62580cctgacctcg tgatccatct gcctcggcct cccaaagtgc tgggattaca ggcataagcc 62640accgcgcccg gcccatgtat actatttata catttttagt atcattttgt ctttacattt 62700tacataattt cagatacatt ttcctcatat caaataattc agcatttttt agtactaaca 62760tcatagtctg taagccattc aaaaaatgta tttcacaaaa taggctatct catcctttga 62820gctattgaga tgaattaatt tatactcctc ctaagatccc tctcgtcact aagattcttt 62880tattttatga caaaaccata gttctagaag cttgtttctc ccacctgaaa agactggatt 62940tgggacatga tcctgtagaa cttcggaggt aagcctggtg aatcagatca tagggggtct 63000ggagggtgaa aaaaaagggt ttggtgctca tggatggggc tagtattggg gtgtagggga 63060gattaggtca aagcaagagg attcaaagga gaaatgaatt cctttagatt ggggaagata 63120atcggaagag gtaaaagaca ccgtccatga cacttcctgg ggaagcagat gtatgtataa 63180ggatgtgagt attgtggttt tgtaaagaat gcattcctga agatgttgca taatttaaaa 63240cctacatatt ttgattaatt ttctcatgag aatagcaggg tatgtgttct cggcgctcac 63300aaatgtataa tccattgtgg caaatttttg ctttcacata ttttttttta tcattattgt 63360cacaggttct gtgacggagt tctggtttct aaattcacag cataccaagg cagttcttta 63420aagttcttga tactctttta tcatatctaa cttgtattcc aaaattattg agttggagca 63480cattttccca gcacttagca ccgctatttc atggatggtt ggagaggggg tccaaaaatt 63540ttacaattat gttaacaaaa gtaacacagc aacaaaacaa taagcaaaat cactgccaga 63600gtattcctta gcttgaaaca atacggttca catcgataga atatggcatc tatttctgtt 63660taatcagtta accctgctaa gtagcaagag cttacaattc atgtctaaaa tcatgatttt 63720tttactagtt ttttaaaaaa tgtgggctct atatatataa tttaacattt tgcttgtaag 63780acttaatttt gcctgggtat ggtggctcat gcctgtaatc ctagcacttc aggaggttga 63840ggcaggagga ctgcttgaac ccaggagttc aagaccagcc tgggcaacac agtgggaccc 63900catcactacc aaaaaaaaaa aaaattagtc aggcatggtg gtgtgcactt gtagtcccag 63960ctacttggga ggctgaggtg ggaggatcac ttgagcccag gaggtcactg ctgcagtgag 64020ccattattgt accacaacac tccagcctgg gtaatagagt gagactctgt ctcaaaaaaa 64080aaaaaagact atttctaaat gtgtggctat attataccat ataaatgtgg cttcttgggc 64140aaggaaagag gacaatatag atgaaaaaga aattgatcct accagaagtg atccttttat 64200ctgcataact ctcaggcagt tgtggcaaat aattggcaat atctattgtt ctgaaactgg 64260ttttcgcaac ttttattggg aacaccatcc cctctcctgc atgatcagtt tctcctctcc 64320acggatcatt cacatgagta aagtcagtag cgtgctggta aatgtttaag atcttgttct 64380ttgggggaaa aagttcctaa gttctagcag ttgccctgga taacttcaag gtatcaacat 64440ggaagttatg tacaaaaatg gctgtcacaa gccagtatga gctaacacca acatactacc 64500cagtgttctt caaacttcag ctcacagccc attagtgggg cttgcaaaca ttttagtgga 64560ctataagcag cattttttta aaatgaaaaa gtagattgtt ttacacataa caggagtatt 64620gttttgtaca attttttttt tttttttttt tttttagaca cagtttcact ctgttgccca 64680ggctggagta cagtggtaca atctcagctc actgcaacct cggccttctg ggttcaagcg 64740attctcatgc ctcagcctcc cgagtagctg aaattacagg catgcgccac aaggcccagc 64800taattattgt atttttagta gaaacagggt ttcaccatat tggccaggct ggtctcaaac 64860tcctgacctc aagtgatcca cctgcctcgg cttcccaaag tgctgggatt acaggagtga 64920gccaacgtgc ctggtcaaaa tttttgtttt cgtaatttta agtatgtgtg tagtaagtct 64980caatggaaat gtaattctta tggcaggtca cttgaaaaaa aagaagtcta aaagtcacca 65040atgtagtatc ttctctttaa aaaaaaaaaa aaacaacagg agaaaacctg aatctgccct 65100ttgctccact ccttcctcag ctataatgct gcttctccat tcctcctcac agcaaacctt 65160tctgaaatct ttatagtcat ggtttccacc agttcttcac ctcccatttc tcaacacact 65220tcagagtcag agtcagcaat gacatccatg ttcctaagcc cattgcttac ttccgtcctc 65280cttggcctct cagcacactt ggcacacagg ctgtttctct ttctttggca tctgtgacac 65340cactctcagc aaattcccct ggttgtccct tctcagtctc atttattggc gttgtcttat 65400ctccccaggg ctgtccgagg tgattttctc ccactactct cctaggtggt gccatccaat 65460ctcatgatgt catatcattc ttccctcatg cttcagccat actggttggt ggcctttgtt 65520tcctgaacac atttaatgca ttctcaagac cctcagggac tttgcagcag ctgctcgcta 65580aggctggaat gctcttcccc accatcttca tatggctgtt tcctttcttt cactcaccag 65640cagcttaaac tttgactcct ctgagagact ttccttgtca cccaactaag gttgccactc 65700aggtgctccc aatttaatct tctctaaaac acatcactgt atgtgtcctc aactagagta 65760taagcttcct cgaaacaaga acaatcaaaa ctccttgccc tcatggagtt tatagtctta 65820tgatgggtga agtaacataa aataaaaagg caccttatat agtatattag catgacaaat 65880gttagccaga aataaagcaa ggaagagttg ctagggagtg tgtatgagtg tgttttggga 65940gagtgtttgc aattttaaat attggtggtc aggaagggcc ccactgagaa ctgacatttg 66000agtagacttg aaaagggaaa aggaaatatt gagtaaagat tttaggatgg gagtgtgaca 66060ggcctgctag gagaatagca aagtcgctgt ggctgctgca gaaaaagtga gaaggaaagt 66120agtaggagat gaaatcacag tgtgtgagga ttcgggcaga tcaggaagtg ctcgtgtaag 66180aactggatct ttactcaaag aatgagcaaa aattagtaga cggttggccg gatgcagtgg 66240ctcacacctg caatcccagg ttacaggagg ccgaggcggg cggatcactt gaggtcagga 66300gttcaagact agcctgggca acatggtgaa acctcatctc tactaaaaat acaaaactta 66360gctgggcatg gtggcgcgca cctgtagtcc cagctactca ggaggctgag gcacatgaat 66420cacttgaaca cggtaggcag aggctgcagt gagctgagaa tgtaacactg tactccactc 66480caacctgggc aacagagtga gactgcctca aaaaaaaaaa aaaagtaggt tttagtaagg 66540gattaacatg atctgaatta tgttttgtca tgacttctct ggctgttgtg ttgagactac 66600attgcagagg ggcaagggca aatataggga gaccgattag gatactgcag taataatgta 66660agagatgtgg gactctatct agaagggccc atgaggtcct ttgcatgcta gtattcttta 66720ctgctgtgcc tggccatgat aggcattcag tgaatatttg cttatttaaa ataacacact 66780gggctaattg aacaacagtg ccaaatgagg gagatatttc taggaataag ttcttaggat 66840ttatgaacat tttaatccag attttctttg ttaactctgc tctctggccc tttcactcag 66900ccccgtttgc acctaaatat gacttacaaa agaaacacag catttatgtg tacttatttc 66960aacttacttt agctttgtaa agaagtacaa ggttgactca gggcccagct tggtgtctca 67020tgcctgtaat ttcagcattg tgggaggcca aggcaggaag atattgtgag cccaggagtt 67080tgagaccagc cygggcaaca cagtggaccc tgtctctaca aaaaaaaatt tttaaattag 67140ctgggcatag tggtgtgcgt ctatagtccc agctactcct ggggctgagg tgggaggatc 67200acttgaggga aaccctgtct caaagtggcg gggctggggg gagactcagg cagaattgtg 67260aagatattca attgctcctg actttatcaa taatctaaca tttcaaccta acattgatat 67320ctattttatg caaagcatta cactatgcac tggagactgt ataagacaag ttccttttct 67380caaactacag tcgagttgga tagataaaac acacaacaca taccaaaaga cagctataaa 67440tccaaggcag tgtatgtcaa gggtaaattc acctattcag attggatctt gagaagtgca 67500tcaggcttgg aaaatgggta aggaggagag aaaagcaaca gtgaatcaga acatgagttc 67560ccagttatgg gacttgtaat gaattcctca attaaaacaa aaaataatga aaacaaaagc 67620cagggaggag aaagcccacg ttaatgacac taaaatatat ctttccaaac aaatgtggat 67680aaaagccaag tagagaagat gagaactttg aggtccctaa cacaaaataa acagtaagca 67740gccagccatt ccaagtggct gacatgactt tgtttaactt tatttgtatt tctggctggt 67800gtgtttacag ccaataggtc aaactatcag tcagtgtagg gccctgagaa gtcgggtatt 67860taagagcatc taataggcac agaattgtgc tccatactgc ttaaactgtt ccctaagtgt 67920ccaatttgga gaaaacaccc acacgcagga taaccggcga gtgacgcgga gtggctgcga 67980gtccaagtta tcactaacgg atggggagct tgggctgggc acagtccagc gtactgaacc 68040cttcccccac cgtttcacct gcatacagag gtgtgtactg tcaaaaagca gcgcctccaa 68100gtctcttctg gcactgtctg gacttggatc cgaggcagac gaggaagctg agaaaaccct 68160ggcgttgacc ccgtggacct gggcgccccg ggaaggccag cgcttggtcc aggcaggcgg 68220ggcctgtgcg gtgaccaccc tggtcctgaa aagtcccagc cccgagcgcc ctccctccta 68280gacctggagg cctggaacag ccaggtggac gtcggcccac ctttcttttc tttccttccc 68340attttcctac cacctcccac cccactccgc cttccgggca aaggcagcca gatccaccca 68400ggacacattc tttgtcctta tccctctgtg ctcgtcccac agcaagccag tcgcggtcca 68460aggctccaga ggctgtgcag gaggccgagc tgggtggcga tcagcggcgg gtccctgtcc 68520aaaayccagc agagccgcca gggacgcccc agacacagaa ggcggggcgc ggggagggtg 68580gggagaccac agcagtgagg cgcgcgagcc gggaagtgaa cgaggactga ctcctgtcgc 68640ttcccgtagc cgcccacgga cgccagagcc gggaaccctg acggcactta gctgctgaca 68700aacaacctgc tccgtggagc gcctgaaaca ccagtctttg ggtgagtcgc gcgacccccg 68760gcctcgggtg gcggggcagt cgctagaggc gtggctgctc tgagggtctc gccagtggag 68820gatggcattc ggatgtcacg gctcctaaat caccatttga tgggtgggac agtgtccagt 68880ccaccccgac ccgccggtcc tcaccgcggc agagccgggg ctgggtggcg gggacgctgc 68940ctctgcaggc gaggcgctcc ggggcataag ggattatcag gagtcgcggc ctttcttgga 69000catccctggc tggggtcagg ctgtttgccc tggggtgtct cctcgctgca aacccacccc 69060acctgggctg ctttctcacc tgttccctcc tagcctgagg ccgagcgcca cctccaagtg 69120gaggaatctg gggaagtttc cttcccggaa tttgtagtga cagtggagtg acctccattg 69180cgttccctgc ctctaacacg ctctttagga tgccgagtca tttgactgca gtgttaaaca 69240ttgcaaagcg caagtcatgt gacttccttt gaccgtacgt gaaacttaag tgatggctgc 69300ttgtgatgca tacgaagtgt tcatgctggc gggacctgtc cctggggata cttcgggggt 69360tgcgtgattt aatgcaagca gatggcttaa attgggtcac tggcttgtta ttatacatgt 69420gtatggcaac tcggcatcca ttctttttgc tcttgttctt acttcctgaa ttgagtcacg 69480gagccagagt tttgaggttt tgactaacga attaagttaa tgacatgggg ctatatttag 69540gtggtaaacc aagagggata cagttttttt tcttaataaa gaaaaagtga tagatttgat 69600cggtgtgtat tgttggtgtg cagtataatg acagaattgc tggaagtaaa atacaggaag 69660ctctggtttc atttcccctt tagttctgct taaagtcgag tttttcctgg agctattaaa 69720tgtagtgtag tgtccatgag tgcttttatc ttaaaaaatg tggctgatgc tttccaacac 69780tcccctgccc tgtgattatt atttttttaa gcaacagaga aaactgtatc ttaatagtat 69840taaaagtatt ggatttttcc ctactttgat ttgtttaaat tggaggagga agagcaattc 69900tttctattca caataataat agctaacata gcgcttactc tttcgctgtt ttattaactc 69960aatcctcaga acaaaccaat gatgtgaata ctgtaattct cattttatgg aaatgaaaat 70020ttaaatgaat acctctgata attgtacggg actgtttgat tagtatttac cattaattaa 70080ttaaattttt tttttttttg agatggagtc tcgttctgtt gcccaggctg gagtgcagtg 70140gcacaatctc ggctcacttc aacctccgtc tcccaggttc aagcaattgt cctgtctcag 70200cctcctgagt agctgggact acaggtgcat gccactacgt ctggctaatt tttgtatttt 70260tagtagggat gggatttcac catattggtc aggctggtct ccaactcctg aacctcaggt 70320gatccacccg ccttggcctc ccaaagtgct gggattacag gtgtgagcca ccatgcctgg 70380cctattttag tatttttaat aataaattcc atgttagaaa ttttctactg atgtattttt 70440taagtcaata tttcctacac tcacaatcca aaattattta gtatatgagc acactggtaa 70500gaatgggagg cagatcgttg attgtaataa tattctatta tttggtaaat atcagtaaca 70560taatatataa tttaaatttt aaaataggat atgaagaaaa atgctacatg cttacttttc 70620ttttcctcta tttttacttt acacagggcc agtgcctcag tttcaatcca ggtaaccttt 70680aaatgaaact tgcctaaaat cttaggtcat acacagaaga gactccaatc gacaagaagc 70740tggaaaagaa tgatgttgtc cttaaacaac ctacagaata tcatctataa cccggtaact 70800gatttctata agataacttt ttacctatgc caggacagat ccaatagaat attaattatc 70860cattgggaga cagggcaaga ataaaagcca gtgaacatat ttaaagcacc tactatgtaa 70920tagagatggt ggtgggtgct gattacgaaa cagctcttgt cctctagtgg aggaagaagt 70980cacaatgata atatgacgtg atgaaacagt gttatgaaca gggaacgtct gggtagagtg 71040gagggaatgc caacttttgg tgatgggagg aggctcagct aatcataaat tgtagttttt 71100aaaggaaaat ggatttctta ctctacaagt ttttcatttt cttttttaat tagagctgtc 71160catgagaagt taatgtctcg atctttccct cagcctttca aatactgctt ggcccttgag 71220cagggaaaat gtcaaaagcc aatggggaga tggagagtgt gaagtagtaa gggtctcgtg 71280cagttcaggc aggtcctaga atccctgaat gactgtaatt gctggaaatt gccctgtaat 71340cctgagcagt aaagagcttg ttttagtttt atgtggtggt gagaatcttt aggaatgtct 71400agtttccacg tatctgaagc tgaatcctga atcgaggtct gaaaaaggac agccactttt 71460ttagtaaacc gcctagaaga ttcttgggca aaaggaaggg tgagaatcct taaaatgagg 71520ccctaaacca gttttgttag tgtgtgtggg ttcaagtttt tgtcatttac tttatagctg 71580tatttccttt ttccctaagt tttaatgtca ttgtgtaaga atgaggtatc gctgctgtat 71640caagcaaagt cagttttagg agaaatagcc tttcagtggt agtaagttta aaaaagatga 71700cttcctgaag cggaagcttg tgagacattt aagatgactt tgcgcatgtt agagttaaaa 71760acatcccaag gttgtaaact gatttcctgc aaagatctta acaacaacaa caacaacaac 71820aaactaggct gcctgccacg ggtgtctgaa gtatcatctt ggctcaagct gggagaatgg 71880ataaaggtta cactgttcat ttctgccctt cacacagaaa agaagataat tttataggta 71940aaattcgtgc atatcttgat tctagcatac tgctgattcc tgtagtttct ggggtcagta 72000ctctcaacta ttgaggtgga acaaaaataa gtagacttca tttcttgagg aaggggatct 72060ggagaagtag ttctgcgcta gagcagaaaa tgccttcagt cttgtggcat gggctggatg 72120ctgttctgag gataatgcat ttccaaggga gatatttttg gcaaatagct tttttttctt 72180tcttttcaaa attctctgtt ttattatcag ttctcacaaa agagtcggaa aggttagagg 72240tagactgaac tgaatggcaa aaacattttg cgctctcttt acgtttcact gctgtaaaat 72300atttatagta taaagggcct gtattgcact gaatttctct catttgtagc tagttgccct 72360ttcaatgttc caaaaaaaag gctgtaaata acttatttta tttattcaat taattttttt 72420ttttaaattt tttgagatat agtttccctc tggtcaccca ggctggagtg aaatgatgca 72480atctcggctt actgcaattt ctgcctcccg ggttcaagca attctagtgc ctcagcctcc 72540tgagtagctg ggactgcagg cacgtgctac catgcccggc taatttttgt gtttttagta 72600gatatggggt ttcacagtgt tggccagcct agtctcaaac tcctgacctc aggtgatgtg 72660cccaccttga cctccaaaag tgctgggatt acaggcgtga ggcaccatgc ctggccaact 72720tagttattta aagataatca attagtatat tttataagct agacttagga aaactgtttt 72780cagctgggca tggtggctca cacctgtaat cccagcactt tgggaggccg aggcaggtgg 72840atcacgaggt caggagttca agaccagcct ggccaagatg gcgaaactcc gtctctactt 72900aaaaatacaa aacttagcca ggcgtgatgg cagcctcctg taatcccagc tactcgggag 72960gctgaggcaa gagaatcact tgaacctggg aggcggaggt tgcagtgagc cgagatggtg 73020ccactgcact ccagcctggg tgacagagcg agactccatc tcaaaaaaaa aaaaaccccc 73080cccacacaca aaacctgttt tcttgaatca tggttgtttt gttactgata ggttcaataa 73140gtaaatatat ttattgtctg ttgtattctt tattaggcat tataaacaca ccgccacttt 73200ttaattttta tttcattaat gtttccaatt tttttttttt tttttttttt tttaagacag 73260aggctcgctc tgtcatccag gctggagtgg agtggtgcag tcttacccca ctgcaacctc 73320cacctcctgg gctcagcctt gtaaatagct gggactacag gcatgcacca ccatgcctgg 73380ctaatttttg tatttttttt ggtaaagaca gagttttgcc atgtttctca gtctggtcaa 73440gcactcctcc cgcctcggcc tcccaaagtg ttgggattac aggcatgagc caccatgcct 73500ggcctatttc taatattttg gtccacattg gtgttagacc aactgtccac attaagtttt 73560cttggaaaag atgaagtaaa tattgcaact ggcctatgta tttttttccc tatttagtat 73620atttctttga ctagttcaac tgatagaatt ccaagactta aaaaagtcag gctctaaggc 73680tgggtccaga ggctcatgcc tgtaattcca gcactgtggg aggccaaggc tagtggatca 73740cttgagccca ggagttcaag accagcttga gcaacatagt gagaccttgt ctctctataa 73800aaatacaaaa attaactggg gattgtggcg catgtctgta gtcccagcta tgaggaagag 73860tgaggtggga ggattgcttg agcccaggag gttgaggctg cagtgagctg tgagtttgac 73920actgtgcttc attctgggtg acagagcaag aaccatgttc aaaaataaaa ataaaaagtc 73980agagtccggg tgctgcggct catgcctgta atcccagcac tttgggaagc cgaggcgaga 74040ggatcacttg aggtcaggag ttcgagacca gcctgactaa cacagtgaaa ccccgtctct 74100actaaaaata caaaaattag ccgggcatgg tggcggtggc ctgtaatccc agctacatgg 74160gaggttgagg caggagaatc acttgaaccc gggaggtgga ggttgtaatg agccaagatt 74220gcacaactgc actgcatcct gggcgacaga gtgatacttc atctcaaaaa aaaaaaaaga 74280aaaaaaaagt taggcttcct tttctgtttt ttttttcttt tttcttctct tttttttttt 74340ttttttaaga gatggaggct tgctctattg cccatgctgg agtgcagtgg tgcaatctcg 74400gctcactgcc acctttgcct cctgggttct agcaattctc ctgcctcagc ctcccgagta 74460gctgggacta caggcgcaca ccgccacgcc ccgctaattg ttcttttgta ttttagtaga 74520gacggggttt caccatgttg gccagcctgg tctcgaactc ctgagctcag gcaatccgcc 74580cgcctcggcc tcccaaagtg ctaggattat aggcgtgaac caccgtggct ggccacttac 74640ttttctttct attgaatttg aatgaataat ttggaagaca gtatctttac ttcataccag 74700gaatgctgcc agtgaaattt cttgtttggc agttcattat ctacctatat atttaatttt 74760gctattgttt atagagttct taagatatga ttaaatgcta gctggttaag aaatcattta 74820gaaatgaaac agaattggtt gttactccaa gttaataagt tgcttgtcaa cataaatcct 74880acctggtacc cagttttctt aggaaccttg cttccatgtt tatccttttc tgcttagtat 74940tctaagtact ccttttttac cttacaattt agtcttaaaa cacaacacag tcaagtcttt 75000cttttgtaac ctgtgaggta ccttctagcc tttgtgctgt ttttcttctt tttttgctgc 75060ctgccttcct gactgagagt ggatttcctc actaaggctc tgccctctga tttttcactc 75120tcttttcttt tttggtttta ctagtgaaat tttgtcttta atgtctcttt cttttatgtc 75180tttaccgatc actcataaat ttttttttcc atatgtatcc agttccaacc tttcacctaa 75240tgtgaacccc caactctcag ttgctcagcc agcccttcaa gactaggagt tcaaaaccaa 75300acttgcatct tccttcccaa accagctttc ctcttgcagt tttctgcagc aggatccttc 75360tgctgtttaa cttttgcctc ctcccttgtt tcctagcacc caatagttgg aagatagtct 75420gtcttcaaaa ttttaaacta catttatgtc caaaccagtg gcttttcctt ttaaaaaaat 75480ttaaagataa tatgtgcaaa tcattttttt aaaattcaaa cagtatttaa gagtttcagt 75540gaaacatgca ttttccttct accctggtac ttagttttac tccccaaggg caatcacttt 75600ttactggttt ttagaaatat atccttcctg agatacttat gaatatccaa aagtgtgtgt 75660gttgtgtata tcacctttta tatatcctgt ctctttacgt gcatgcattt taccgtataa 75720actgttttct accctgcttt ttctatttga cctattttgg aaatgtcatt ttatttagaa 75780cttcctcatt tattttaaca gctgcataat tagcagtaaa acttatgtaa gcagtccctt 75840gtgaagggct gtgtcttttt gcgattatat ccggtgctat agtgtacatc cttgtgtgtg 75900catcttggtg tgcctgtgct acgtatttct gtaggataaa tctgtaaaag tggaatcact 75960aggtcagagg gtatggtcca ttttctttac ttatttattt tatttattta ttcatttatt 76020tttgagacag agtcttgctc tgtcgcccag gctggagtgc agtggcatga tcttggctta 76080ctgcaagctc cgcctcccgg gttcacacca ttctcctgcc tcagcctccg gagtagctgg 76140gactacaggc gcccgccacc cacgcctggc taattttttt gtatttttag tagagacggg 76200gtttcaccat gttagccagg atggtctcga tctcctgacc tcgtgatcca cccatctcag 76260cctcccaaag tgctgggatt acaggtgtga gccaccgcgc ccggccccat tttattatct 76320ttatttgctt ggatccttct tagcttcttc aatgttaaag atattgacag ttttcctctt 76380actgaaattt ataaatccat tgactccctt gatattattg ccctggcctg actgattctt 76440ctctctcctt tctcttctca ccccatgttg aggtccccaa ggtcacaccc agttttgatg 76500actcaccagc atagagttgt acttgtgcct atgatttatt gcggtgaaag gatatagagc 76560aaaattgcaa acggaaaggc acctggggtg aattccaggg gaaatccagt gcaagttcca 76620aggtcgcctc ccagtggagt cacataggat gtgcttacat cctccagcaa ggagttgtga 76680caacacttgt gaaatgtgga ctgccaggga agctcatcag agcctcagtg cctagggttt 76740ttactggagg ctggtcacat aagcaccctc acacatatca aaaaattctg gtcccccaga 76800aggaaagcag gtgtttagca taaccatatt atttgcatga acagttcagg tacaggaaat 76860ccccgttacc agttaggttg gtgggtgccc ttctcaaatc ccaagttccc agacaccagc 76920caggggcctg cctcgtaagg aggcctttcc aggacagcag tcaggcctgc caatgttaat 76980tcttttctgc atacctccta attttagaaa ccaccgagcc tttgctgcct gacctgtcct 77040gcttttcgat ttctttatct actttgatat ctttacaaat gatctttacc ctgactttta 77100aatgtgtgct ctggccattc acctagcgtg tggttctgag tctccaagtc ttagcagatt 77160tgctctcaga tgctctgcca acgcttcaca ccaagtatta caaactaaac tcgtcatctt 77220cctcctgaaa cctgtctccc aggccaggcg cggtggctca cacttgtaat cccagtactt 77280tgggaggccg aggtgggtgg atcacctgag gtcaggagtt cgagaccagc ctggccaact 77340tggtaaaacc ccatctctac gaaaaataca aaaaaattag ccaggcgtgg tggcaggcac 77400ctgtaatccc agctattcag gaggctgagg caggagaatc gcttgaaccc gggaggcgga 77460gattgcagta agccgagatc acgccattgc actccagcct gggcaacaaa agtgaaactc 77520catctcaaga aaaacaaaaa acaaaaaaca aaaaacctgt tttctcccca gctttgtcat 77580gtatttagtg gccttatgta gacagtttcc tttgaaacat ctcttggact tctctgctct 77640tccagggcca ttgccaccga cctggaatgt gtccttatcg tttcacgcca ggcttatggc 77700agcagtcagt cacccagatg acctcctgac ctctggctta tttcaccccc actggactgt 77760tgttcctaaa cacttctttc gtatgtcact ctaaaatctg accctggctg tacctttctt 77820taactactcc ctgactgcgt gctgagagaa gatgggtctt gtcttttcct gcctctctgc 77880ttttgtaaac tgccatttct acctgaagtg gcaactgaaa tcatatcttc ttcataaact 77940gtctttggct acctcagtta gaattcctta tcccattttc ctgaagcatt tctttgactc 78000ttctttactg ctcccccacc cttttttttt tctttgagac tgaattttgc ttgttgccca 78060ggctggagtg caatggcccg atctcggctc attgcaacct ccgcctcctg ggttcaagtg 78120attctcctgc ctcagcctcc tgagtagctg ggattacagt catgtgccac catgcccggc 78180taattttgta tttttagtag agatggggtt tctccacgtt ggtcaggctg gtcttcaact 78240cccaacctca agtgatctgc ccaccttcgc ctcccaaaat gctgggatta caggtgttag 78300ccactgcgcc tgacccccat tttttttttt tttaaagatg ttgaattggt cagggtttgt 78360agttacaagc aacagaagcc aactctttaa gcagaaaagg aatttgctaa atgatagtgc 78420agagttctca gaatctctag caggatgaag aaccaggctt ggagaatagg tagccacaga 78480tacacaagca tactgtagga cggttcccat gaagaggcat ctgttgtcac cactggacac 78540agatggtact gtgtctctgc tactctacca atgccactgc tgtctctgac cccagatgta 78600gctccctctg accctggatg cagctccctc tgaccctgga tgcagctccc tctgaccctc 78660gatatagctg cccctgaccc cggatgtagc tgcctttgac cccagatgta gctttctcca 78720aacccagata tagcggctgc ccccttgcca gagtgaatac tgcgtcattg tggcttcttc 78780ttgtcactgg ttcttactta aaagctgagc tggaagttct aatgggcagt tttgtcacct 78840gctcttacct tgttgcagtc tagatgaggt ctaatgttca taagctaggg gattttcaga 78900tatggaaagg gataccaatt ttcagcagcc aaatagagta tcacattttc actccatgtt 78960tcctgggtgt ctgttatgtt tcctgggtgt ctgactctta ggcttctttc aagctgcagt 79020ctgcctaata gagagccttg catttaatca tcaaaaaggc aaagcaatat gaatcagcaa 79080gggtgttttg gcaaataaca gcaaacctga ctgtggcgta agcttgtggt attgtctcca 79140gtgtgatcag atctgtattt taatttttta aatgtaaatt aataatgatc tgtgaatcac 79200caaagtagct tggagtagcc tagaaaacaa tgtatgtcct ccgttttcac agaagccaca 79260tagtcgtggg ttaaatgagt cagcggcagg gcactgtgtc tcatagttaa aaaaaaaaaa 79320aagtattact gaagtaatgc aggatctttt ctgaagtaga aggcatgatg aacccagaaa 79380actaaagcag caagtggcca ccgttcttag catagttgtt tctcaaactg gaacaaccta 79440taaacagttg tgaacaaggt attagaagtg atgggggccg ggtgcggtag cttctcccaa 79500agctcattac ctcccaaagc aaccccagta ctttgggagg atcactttga gcccaggagt 79560tccagaccag cccggccaac atggcaaaac cccatctctc taaaactaca aaaaattagc 79620tgggcatggt ggcacatgcc tgtagtccca gctacttgga tggctgaggc aggagaatcg 79680cttgaaccgg gaggcagagg ttgcagtgag ctgagatcac gccactgcac tctagcctgg 79740gggacagagt gagtctctgt ctcaaaaaaa aaaaaaaaaa aaaaaagtga tgggaataga 79800ttgttttgtc tcaaaaagct ctttccaaca ctaaaatgaa acatataatt aaaaatattt 79860ttctggctat aaaaatatcg atgcttatta tagacatctg caaagtatga aaatatatga 79920agaaaaaaat taaaatgcca tcatccccca tgaaaactat tgttatcatt tttgtctgat 79980ttctttagtg tttctctttt tcttttttta atttttaatt tttttgagta tgtagtatgt 80040atatctattt atggggtata tggcatattt tgatacagga tacagtgtgt attagcaagg 80100ttttcttttt aatgtttata tttatttagt tgagatcata ctatatatgg ctctatagat 80160tactttctct tatattacta acatttgtgt tattaaatat tctgcataaa gataatttta 80220agatgaaatt tgatgttata aaaacttctc attttattaa gagattaacg ctatgaaacc 80280tgctgctata tattcttgga accagctgtg acccaaaaga tcaatgtagg gatgtaggtc 80340cttccccatt ctctacacac aaaatcagat actctgatgt gcagctgtag ccccagtcta 80400cactgtctgt tgtatttttt gttttctggt gtcacgtgcc tcccaccctg ctcctagcaa 80460ttgccatgac aacaaataga taattggctt ccgtaatttc tcatcttatt gcctaaggca 80520acagagagct tgtgggctca gcttgcggtt cagcagctgc tttgttgcct ctcctctgta 80580tgtgtgaggc ctgccagagc ccactttcca gacaggtgag agttcattca ttcaccatgc 80640agttaccgat cgtctcttga cctgtgtcct ggggaggtaa aggtgacgag ccagttctgc 80700cccatgcagc tcacagtcta ggcaaagcta catgcaaaca aacagaatcc aaagtgctat 80760catgaaccct ctgagagggg ctgactcagc agcccaggga gcttgaagaa ggctccacag 80820aggaggctgt gcctcaaggc gatttcggtt taggagccac caatttataa ccacttttct 80880gtggcccgtc ttattttatt tcttatttct tgacaatcag aagtaccttg ggtaggtttt 80940accatgcaca tcgtaatttg agtgagctta gtgtgaggct taacggtgtg tgggctgtac 81000atcctggtca gatgctccag atggaggcag atggttgtga tgcaggagag gcagccacat 81060agcacaggtc cccagccagt ggactgggaa gacagtgtag tcatctctgg ggaaggggaa 81120tgacaagatc tggcagtgtg gcaggtccca gaaaaaaagg gctgggttct gggcagtgag 81180ggtgcaggtt gagacctgaa tactgggtgg agccagctgt cagagtccac gcctgcagac 81240tggactggtc cacggcaggt ggatgccatg tcttgaagac ccacaggcac ccactcatcc 81300tcatgatcat gcagttctct ggtttctaac agtgcagtct gggttgcagt ctgggagtcc 81360agcagagaag agcaggccct ggaatcccag gtgtgggggc gtggcttaac gtggagtttc 81420cttcagaggc agtgagtgct tgtcattgtc tccgtcagca ttggctttgg gcctagtgtg 81480gcctcgaacc ttctgttggg atcagcagtg gaacagtagg aaaaggaatg agtagacatg 81540gcattgcaac aagtcttttt ttttttttct gttagaatta tcatattaag cagaagtttt 81600gcttcacaaa ctctcagcca aatacaaaat actatgaata gtatttacct tgtgtctctt 81660tccaaagaac tcatagtggt ttgcagctat tgcagatatc ctggccatgc ggtatgcggt 81720tccttttttt tgtttttttt ttttttttga gacggagcct tgctctgtcg cccaggctgg 81780agcatagtgg cgcgatctcg gctcactgca agctccgcct cccgggttcg tgccattctc 81840ctgcctcagc ctcccgagta gctgggacta caggcgcccg ccaccacacc cggctaattt 81900ttgtattttt agtagagacg gggtttcacc gtgttagcca ggatggtctc gatctcctga 81960cctcgtgatc ctcctgcctc ggcctcccaa agtgctggga ttacaggcgt gagccaccgc 82020gcccggccgc agttcctttt tatagctgtt tgaataggaa agatgacttg gaaaatgctg 82080gattctgaga tttatgtgca gccttaaaaa gtgtagtttt tctctatcaa taatgagtgt 82140gggttgtaat tgcttagtaa gtaattttgt ttatgtaaac gtacatttgt taaatttttt 82200ttcttaggta atcccgtatg ttggcaccat tccygatcag ctggatcctg gaactttgat 82260tgtgatatgt gggcatgttc ctagtgacgc agacaggtaa aatcactgtg ctaaaggaag 82320gagcatgaat aggctgtctt tttgtgattg tggaatgata acagagtaag gcgggagaga 82380ccatttgata ctytgaggcc caattagctt tcatcagcag ccctggccaa ggtgctgagg 82440agattggaat gaatgactaa ataaaggtta ttgggattta tttcattgct gtaagtctga 82500tttcagtata aaaaaattag aactatcagc tggatgtggt gacttacaca tacttttcca 82560gcactttggg aggccaaggc gggaggattg tttgaggcca ggagttcgag accagcctgg 82620gcaacatagt gagacccccc ccatctgtta aaaaaaaaaa aaaaattaaa aattaactgg 82680gcttggtggt gtgcgcctgt agttgtagct actcaggggg ctgaggtagg aggatccctt 82740gagcccagga gtttgaggtt gcagtgagct gtgatggagc cactgcacta tagcctgggt 82800gacaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaagaaaga aaaagaatta ggtatgtcat 82860taaagaaagg aattgtggtc agatgacagg gagagtctag ttttagtctg acattcccac 82920agcatcacag atctagttca gatggtttta ctgaatactt gctttggata caagctgtgg 82980tatcattagt gttgggctca gctctgtgta cctaacacct gaagagcagt ggtttaagat 83040gtgaaaatta agtctcaagg agacagccca ggccttttca gttaactcct tcaagtcgtt 83100agagaagtag actccttcca gcttaccact ctgctatctt gagggtgagg tgaggtcccc 83160tttcccatta tccttggcag ctagatttcc agccctcact tctgtgcttt gggtagctgg 83220atgggtgcat gtggtgtttg cggggaaaca gagctggaca aaaggcaagt gcttgctgac 83280ttttaaggca gtttctagta gccttccctg agcacttcac ttccatctta tcagcagagc 83340tttagctgca caggcaggcc tagctgcgag ggaggctggg aaaggtaggt ttttattctg 83400ggcagattca gacccagttc aaactcaggg gctattttac tgaggaagac agaaaagatt 83460agacagtcag ctctttaggc ctcatagtga atgaatgagg agggattggt cagtcccttg 83520tcactgggcc tggagtgtag tgcctgctgg gcctttactg gtggctttcc tttctgagca 83580ctcatgaggc ccctgtgtct tccctcatat agattccagg tggatctgca gaatggcagc 83640agtgtgaaac ctcgagccga tgtggccttt catttcaatc ctcgtttcaa aagggccggc 83700tgcattgttt gcaatacttt gataaatgaa aaatggggac gggaagagat cacctatgac 83760acgcctttca aaagagaaaa gtcttttgag atcgtgatta tggtgctaaa ggacaaattc 83820caggtaggtt ttggagaggg acaggttgag tcctcattag tgagcaggag tgcacagggg 83880ggcctttcac atttgtgagc ccagccttgt atttcctaca cctgagatat agtttggctt 83940tgtagtcttt ctccataaaa ggaccaggaa ggcacctaaa tatgaggggg tggcaccact 84000actctccagc cagttgttgc catgcagaaa tatggtccac tgtgactaga tctttttatt 84060agatcctatt tctcctagca gggctgagtt ctgaattgac acagtattat gttcatgatg 84120ggagggtaag ttataatata accgtcacca cctgaagaac taacaagggc aatcccagca 84180tagaaatcag aagggttttg taaattcaag tcttgccaca agacagttct gtaggatcat 84240gagattttta gacccagagg acatcctaga aatccttgat gtcagttcca tctctggctt 84300catggagtgt cttataccta gcgcgcgtgt gtatggttga atttggtccc agaagctctt 84360acacctgctg gccctctggc ctgtggagct ttcccacagt agaggtttgt accaacgtga 84420gagaagactc acatgcctct ggcacagatc ctttctgatc ttcgggatac tgctcctgcc 84480cgaaagtctt tctgaatctc ccaaactcca ttcacctctc ccttctctgg ccttttgagc 84540ccgtgtctgt atcattcttt ttcacagttt ttaacagttg tgctttggct ttatgtgttt 84600attttgcctc cacaatggga tttaaagctc cttgagtcag agactatatt gtatgctgct 84660cgcgttttct gcctataacc taacgtggta cctggcattt gagagggagg gagggaggag 84720gctcgtagcg tgccgaggac ctgcagaagc tactttctcg tcatcttact gtagtctgtt 84780gaggtagaga ttgttcctac ttcagaataa gaaaaccgaa ttcaaatatg ttgggtaact 84840tgtccatatt aatttattta gcaaatacaa cagattttga gtgtctgcca catgggtggt 84900ctccagggac agtgttgtgg ggagctcgca ggcagatctt taacctgggt tcacaatctc 84960cagggcacct gtgcctgggc ttccaggcga ccttcgaacc cagatgtctc acatgtatgc 85020agaggcgcac acaagcacac gcacatatac ttatgactgc ctgtttgtct ggggagagac 85080agttcctggt gcttaatcaa atcaggaact caaaagaagt tcggaagcac tgctggtgtt 85140ttgggtgctt tcggttacca tttggtcacg tgtgtggaga cctgtgggaa caggtataaa 85200attggacgca aggaaacatt taaatttgga taataagtta atttattaac tgtttttttt 85260tggtggcggg ggggctctgt cttctgtatc tctctaggtg gctgtaaatg gaaaacatac 85320tctgctctat ggccacagga tcggcccaga gaaaatagac actctgggca tttatggcaa 85380agtgaatatt cactcaattg gttttagctt cagctcggtg agtgaccttc cacagcttgg 85440ggtcttttat gaggatggtt tctgatgaga tggtagaaaa aatcttcaaa taacacttct 85500attgacataa aaaggacgta tctccctgac tgtagtatta attttttgga agtgaactgt 85560tcacactagc agaaggctgt ttatcagcca gggcttcatt gtctgtagga tctcaaacct 85620agtgtggttt taataaaaca cacacagttt ttagctgggt agcagctatt tcctttgcat 85680gggcataaaa tggagtattt ctgtaagaca ggttcctagg ctgggagtgt ctgagtcaaa 85740gagcacagtc atgtgttgca taaggacagt tcagtcaaag atgaaccgca tatacaaccg 85800tggtcccata agattgtcat atactgtatt tttaccatac cttttctatg tttaggtaag 85860tttatatgca caaatactta ccatcctgct ctggttgcct acagtatttg gtacagtgcc 85920tgctgtacag attcactggc caggagctat aggccacacc ctacagccta ggtgtgtagt 85980tggcagtacc atctaaggtt gttaagtaat attctgtgat gtttgcacga tgacaaaagt 86040catgtaagga cacatttctc agaacatacc cccttcgtta agcaacacat gactgtcttt 86100gcattgaaaa ttttgataga tactaactcg cccttcacaa gggtaaaaac agtttgcact 86160ctcaacagcc atgctcccac cttcttgttg acattacatc ttattctctg taatgtttgc 86220caatctgatg gggggcggaa aggaccccag tgtcaagtat atttttggat catagttttc 86280aagcatattt ttagtaccat ttataatttt tttatatgtc aaacaggtta tatatagaaa 86340atattttcta ctggatgtta caaattaatc tttattatct tttctcagga cttacaaagt 86400acccaagcat ctagtctgga actgacagag ataagtagag aaaatgtaaa tattaaatct 86460tttaatgagc cactggttta aaaatgttgt tttagctgcc atgttaatga aatggcaaga 86520aggctgggtt tttgaaaatt atgcttttag aacgcaagta atcacttgaa aattgagata 86580catacttgtg gtgccaggca cgcagtaagt ttttgctgat gattcacctg tcagtttctg 86640taactgccac tcactgttct tatgtaaaaa gcactctctc actcttaact gctgaatagt 86700actgttctgg ggtatttcca aatattgaac atcagccagt gcactggcaa atgaacttcc 86760atgtgtatct tcaacccctg ggagaataac tgcaatttaa aaatgcgctg ttattaatgg 86820agaaagtgag gtcttaccga ctggcacgtt cacacctcac agacagaata gaatcttagc 86880attctggggg caccctggaa aggacaacta agacacgttt gaagttcatg tagtgctggg 86940tgaaggtggt ggctcaggcc tgtagtccca gcgctttggc tgaggtgggg ggattgcttg 87000agcctaggag tttgagatca gcctgggcca catagggaga accccatctc tacaaaaaat 87060taaaaaatta tctgggcatg gtggcgcatg gctgtgatcc cagctttggg tggctgaagt 87120aggcggatga cttgagccca ggaggttgag gctgcagtga gccatgattg agccactgca 87180tcccagtgtg gatgacagag taagaccctg tctcttaaaa aaatttcata tagttctatg 87240aaaaattatt aatttatggt ggaggataaa ggactcagat gaacagggat atcagactct 87300cttctcaacc cgtgtagccc ttcacaacac cataccattc cgtcataaag caccagctgc 87360ctggaggtca caccagagtg gagcaggaac atcccaggct ccggccaggc tcagctcagc 87420acaaccaaga cttcagatta taaactataa ttcttcccct tctaacattg ttgtgttttg 87480tttcttttcc aataggttcc aaagtctggc acgccccagc ttgtgagtat ttttgcctgg 87540gttatttcat gtggaatatt ttataaagtt gcatagaaaa tgaacagttt aaaccgtgga 87600gggcagcttc attcattcca ttccttactg tagaactgtt tccctacagc ctagtaatag 87660aggaggagac atttctaaaa tcgcacccag aactgtctac accaagagca aagattcgac 87720tgtcaatcac actttgactt gcaccaaaat accacctatg aactatgtgt caaaggtttg 87780aagagcccca aattttctta actctgtata aaaattaagt tgtaatgagc tgttacgagt 87840aacctgtatc cacaatagaa gcccaaagca gccccctctg catttgtgtg ccgtccctgg 87900atggattcga gagtcaacca ggcctgcctc tgagccattc ctgtgtattt cctcagcacc 87960tccctgcttg gctgcttccc cttcaggcag aacacagtac tgcctcagac cccaggcaca 88020gggggccttc ctggcgtgtt tcactcatac agagggcatc gggtcccacc ctgtcactca 88080tttcatcgtc taaaatgtaa tcatgagtgt ttgcttcgag ccagggacag tgctgctgca 88140ggggacccag ctgggaccaa ggcagactgt ctctcccctc ctgggattta cagggtcatg 88200gctctgaaac attctgtagt gttctttgga cacgagtttt ccctggagat cgctttctgc 88260aggcctcttg gtcctgactg tggcttcttt tcagagcctg ccattcgctg caaggttgaa 88320cacccccatg ggccctggac gaactgtcgt cgttaaagga gaagtgaatg caaatgccaa 88380aaggtcagta tccttcggta ccagtcacag tgcagatact tccgtgcctg ttaccgcctt 88440ctacccgtga acggtcctgt gagctggaag tagggctagt gtcagaatct tcatttccaa 88500agtgagatga ttcaagcagg aggtggttag attgtgaaca gccagtgggc agcagagccg 88560actaaggccg tgttctgacc tcggcttttt ctggccagac aagagagtag catttttgtc 88620cacgaggcct atccttgcct tgtagaactc cagagcagcc ccgtaagatc aggcaacatc 88680ttttcttttt ttttttgaga tggagcctca ctgtgtcacc caggctggag tgcagtgtca 88740caatcccagc acaccacaac ctccgcttcc tgggtgttca agccattctc ctacctcagc 88800ctctggagta gctgggatta caggcgcacc accacgccca gctacttttt gaatttttgt 88860atttttagta gagacagggg ttcaccatgt tggccaggct ggtctggaac tcctgacctc 88920aagtgatccg cccacttcgc cctcccagtg ctgggattac aggtgtgagt cgccacgccc 88980agcccaggca acatttttta gggcccctct tgtcatgtga tttagaaaat ttctgcttta 89040acaacttttt ccacagacgt ccagccttct gaaagcttga aattagagct atttcctaga 89100aagtggcata ctttcaagaa ggaaggaaca cgggtagatg atgaaaagag aatacctgct 89160tgagaggatc ccaggctcct gcagcctgaa gtagtcattc agtttagcgt taaaccttcc 89220atttctgtcc aaccacatct cagcctcaat gctgatttta aaggggtttt ttttttcgta 89280tttttatttt gcaagtaacg aattagtgga atgctgactg ggtttaaaat ttcaacttca 89340cctgcattcc catgtccatg tggatacgtg tgtttcatag agttagaatc atagttcaag 89400tctggtcact aacattgctg aaattgccac tactctgtcc tacttggtta attaaggttt 89460tttttttctt tctttctcaa aagctttaat gttgacctac tagcaggaaa atcaaaggat 89520attgctctac acttgaaccc acgcctgaat attaaagcat ttgtaagaaa ttcttttctt 89580caggagtcct ggggagaaga agagagaaat attacctctt tcccatttag tcctgggatg 89640tactttgagg tgaggttcca gtttttgaaa atgggacagc aataagaatc ctgggagcag 89700gggtgggata agtggtccat ttaaatcaag tcctaactca gtatgtggag gttgtgtatg 89760ttttttgttt acttggagat tgtaatttgc cccttccttt ttataacgtg ggcaatcagt 89820ataaatggca aagccagtag agtgtcaaat tatgcacatt ggaattgaca tttgtcatca 89880tattaaaatt cctgtgtagc cccatattga taggaattta accaggaagc ttgtctcagg 89940actggagtca cacatttaat catataagca gacttgagga ctggagaccc taaaactgct 90000tgcttgcact ggccatcatc tcccatcagg gtaggtggca gtcctttctc ctaaggagtt 90060agtcttgttt atatgtattc aaggaaaaat acatcagtcc cttggaacta aaaggcatgc 90120agtcctgagt ccccagatag gtgaatattg taacacatac ctttcccgaa atatgtttct 90180gggatgctga gcagagaata gtctccttgt gatgtggatg ccgggtgttt ggccagcctc 90240aatcaccagc tcaggtgcca ctgcctcaca cagtcactta gggtcattgg tttaggttat 90300cattctacag cattttaaac tgacacattg tctggaccat gtgggttctt gaggactcat 90360caaaacccgt tactaaaagc atgaatatca ggcgaaatag atagcaatgt gacattcgta 90420tttatcccta agttccagtc taatgcagtg ccctggtatg tggagtgtag acagatgtgg 90480gctaatcatg gaaggttccc tggaagttgt ggatattggt ttcgaattca gaaagctggg 90540aaggatgtgg aaggctgaag gttggctttt ctagatttag ggcatgattt gaacaagtcc 90600ttagaggtgg gaagggcagc acagggttgt tggcttggca agagtcaagg tgcaaagggt 90660gacttggggt tcactggagg gaaacagaga tgagtgctct agaaggaagt tgagccttgt 90720ggtgggtgac aggaaaccaa tgatgtaact tgtttttgac ctatctgggc cccaagtttg 90780gatctgctat attaatataa aaaaggataa taatgataca ttcaaataat gctgaaaaat 90840actaagatga aaatacctcc aacttcgtaa ttcaaaccat accattagga ttaggtgaac 90900cacattccag gcgttttttt gcagagacag tgaaagggat ggctggctga aggaatgaat 90960agatgaatgt tatatgcttt tgaacaatcg tcttttccat ttaattttct aattcaggag 91020cagtaattat ccttgtgttg atcactgctg acgattttct atactgatag gtcctttccg 91080ggggcttcca tctcttgcct tttaaatatg cttgcattga gattatctca ggtctttcca 91140ttatgccatt actttcattt taaatcttct tgctctttca aatacacttt agttgtatct 91200acagtgtttt aaaaacaatc tcattcagtg ttgtaatttc atctgtgggc tcttcctctg 91260gatgaaatcc gtgttcctcc cagctgttcg gcagcatcag atggttgtga gggattctgt 91320tgttctgttt tcttctaggc aaaggatgtg ccttcttttc atttgcagta gtctgctcac 91380ccggaagcat gtcatttctt tgccacttgc ttgtaattca ctggctttgc acttgctctg 91440atacagtaca ggtaactaat tgactccctc tgctgccaac ttggttttcc ttctgagcta 91500tagcatcagg ctgtgtgttt tgtgttttct tgagattttg ttaaatatat ctggggtccc 91560ttctacctgg ttggaactgg gattcccacc attcttgtgg ggatagaatc tcaggttaca 91620cctatttccc caatcctctg tagccacaga agcttcatct tggccagctc tgttatcaga 91680gtgcaggact tgggctgaaa tttcctcccc ttcctgattt tccttgacag tcctttccac 91740tgctcctatc aatcaaaaga atgaaaaccc tcaacttgct gctttgcaga ttcaggtttt 91800gtgcttcttt ctggcctctc ggggtggggc cgggttagca gcaaggctga gctgcccctc 91860tttcttctga agccttcatg ggggcgagga gcacagggag agctcagtgc agggcctccc 91920agtggccttc tcagagtggg tggaaaccca gcctggcact ggcagcgtgg caccagaagt 91980atgaagtgta ggtgtaaagg tgatgtaaaa ggctagtagg ttttttggtt tttcattgtt 92040tgagttttgg gcatagatga ctgtgaaggg cgaacactgc cgatggatct gaatgaattt 92100gtagtatgtg caccacttcc aacttacggg atacccagct ttgacggctt tggacaaaca 92160cactgaggcc aagatgtgct gagcttatca ggatcaggat caccaagcag ctgtaaaaac 92220cctagcaagt gccttaagct gctgaaattt catattaatt gtctggtttg ttcatggtcc 92280tagagtttga ggcagaaaag tcaggatcca agtcccttgg ttccaggcta cagctggaaa 92340cagcatctcg gtgaactaaa gcaaccatat taggagtttt cctgctttag gagagtcccc 92400agcatcggcg aggagggggc agcactctgg ctttccagga gcaaggggca ggatgcggcc 92460gagggagagg ggctgtgttg aggaaaggag ggccgcaggc cctggggatg gtgtgaggct 92520ccaaacatgt ccgagtcact tccctgggtg ggatgaggca gacagtgcca ccaccaggga 92580cactttagtt agattagggt cttggaagtc acagaaggaa gtcagcagca gcaggctgga 92640acttttctat gtataatcaa atggtttact ctgacaccgt tagcatgtaa caaacacaaa 92700attttaaact aaggggaacc actaatggca tgtttccttt cctttcagat gataatttac 92760tgtgatgtta gagaattcaa ggttgcagta aatggcgtac acagcctgga gtacaaacac 92820agatttaaag agctcagcag tattgacacg ctggaaatta atggagacat ccacttactg 92880gaagtaagga gctggtagcc tacctacaca gctgctacaa aaaccaaaat acagaatggc 92940ttctgtgata ctggccttgc tgaaacgcat ctcactgtca ttctattgtt tatattgtta 93000aaatgagctt gtgcaccatt agatcctgct gggtgttctc agtccttgcc atgaagtatg 93060gtggtgtcta gcactgaatg gggaaactgg gggcagcaac acttatagcc agttaaagcc 93120actctgccct ctctcctact ttggctgact cttcaagaat gccattcaac aagtatttat 93180ggagtaccta ctataataca gtagctaaca tgtattgagc acagattttt tttggtaaaa 93240ctgtgaggag ctaggatata tacttggtga aacaaaccag tatgttccct gttctcttga 93300gcttcgactc ttctgtgctc tattgctgcg cactgctttt tctacaggca ttacatcaac 93360tcctaagggg tcctctggga ttagttaagc agctattaaa tcacccgaag acactaattt 93420acagaagaca caactccttc cccagtgatc actgtcataa ccagtgctct accgtatccc 93480atcactgagg actgatgttg actgacatca ttttatcgta ataaacatgt ggctctatta 93540gctgcaagct ttaccaagta attggcatga catctgagca cagaaattaa ggcaaaaaac 93600caaagcaaaa caaatacatg gtgctgaaat taacttgatg ccaagcccaa ggcagctgat 93660ttctgtgtat ttgaacttag ggcaaatcag agtctacaca gacgcctaca gaaagtttca 93720ggaagaggca agatgcattc aatttgaaag atatttatgg gcaacaaagt aaggtcagga 93780ttagacttca ggcattcata aggcaggcac tatcagaaag tgtacgccaa ctaagggacc 93840cacaaagcag gcagaggtaa tgcagaaatc tgttttgttc ccatgaaatc accaatcaag 93900gcctccgttc ttctaaagat tagtccatca tcattagcaa ctgagatcaa agcactcttc 93960cactttacgt gattaaaatc aaacctgtat cagcaagtta aatggttcca tttctgtgat 94020ttttctatta tttgagggga gttggcagaa gttccatgta tatgggatct ttacaggtca 94080gatcttgtta caggaaattt caaaggtttg ggagtgggga gggaaaaaag ctcagtcagt 94140gaggatcatt ttatcacatt agactggggc agaactctgc caggatttag gaatattttc 94200agaacagatt ttagatatta tttctatcca tatattgaaa agaataccat tgtcaatctt 94260atttttttaa aagtactcag tgtagaaatt gctagccctt aattcttttc cagcttttca 94320tattaatgta tgcagagtct caccaagctc aaagacactg gttgggggtg gagggtgcca 94380cagggaaagc tgtagaaggc aagaagactc gagaatcccc cagagttatt tttctccata 94440aagaccatca gagtgcttaa ctgagctgtt ggagactgtg aggcatttag gaaaaaaata 94500gcccactcac atcattcctt gtaagtctta agttcatttt cattttacgt ggaggaaaaa 94560aatttaaaaa gctattagta tttattaatg aattttactg agacatttct tagaaatatg 94620cacttctata ctagcaagct ctgtctctaa aatgcaagtt ggccttttgc ttgccacatt 94680tctgcattaa acttctatat tagcttcaaa ggcttttaaa ctcaatgcga acattctacg 94740ggatgttctt agatgccttt aaaaaggggg cagatctaat tttatttgaa ccctcacttt 94800ccaacttcac catgacccag tactagagat tagggcactt caaagcattg aaaaaaatct 94860actgatactt actttcttag acaagtagtt cttagttaac caccaatgga actgggttca 94920ttctgaatcc tggaggagct tcctcgtgcc acccagtgtt tctgggccct ctgtgtgagc 94980agccaggtat gagctgtttt agaagcagcg tgttgccttc atctctcccg tttcccaaaa 95040gaacaaagga taaaggtgac agtcacactc ctgggttaaa aaaagcattc cagaaccact 95100tctctttatg ggcacaacaa agaaacgaag gctgaagttc gcctacccaa aatgaaaagt 95160aggctttaca gtcaaaagta cttctgttga ttgctaaata acttcatttt cttgaaatag 95220agcaactttg agtgaaatct gcaacatgga taccatgtat ataagatact gctgtacaga 95280agagttaagg cttacagtgc aaatgaggcg tcagctttgg gtgctaaaat taacaagtct 95340aatattatta ccatcaatca ggaagagaat aataaatgtt taaacaaaca cagcagtctg 95400tataaaaata ccgtgtatca tttactcttt ctgcagctct atacgatagg caggagaggc 95460ttatgtggca gcacaagcca ggtggggatt ttgtaacgaa gtgataaaac atttgtaagt 95520aatccaagta ggtgtattaa ggcaccaaaa gtaacatggc acccaacacc caaaaataaa 95580aatatgaaat atgagtgtga actctgagta gagtatgaaa caccacagaa agtcttagaa 95640atagctctgg agtggctctc ccaggacagt ttccagttgc tgaatagtct tttggcactg 95700atgttctact tcttcacatt catctaaaaa aaaaaaaaaa aaaaatcaaa attaaaatct 95760gagtcagtct gcctgcctcg gttctcatta gtttaattct taatgccttg cactttccag 95820caatcattca atcaaaagag tgaaatgaag cacattaaca aagcaggagg cgccacggac 95880cgcctccctc cacaccgctc cttccgcctt cattccttgc ccacaggctt gcactggaag 95940ctgaataaga atccccaaaa ctcaaacttc ctagggatgc caccccttta gtagctcaca 96000cctcccccct ccaagagcta agaaacaaag gagaatgtac ttttgtagct tagataagca 96060atgaatcagt aaaggactga tctacttgct ccaccacccc tcccttaata ataacattta 96120ctgttatttc ctgggcctaa gacttatgtt ccagaactgt cacagctccc catgtcacac 96180ccactagctt gtgatctttg tcaaataact gaaatctttt aagcctctag tttcttcctt 96240tgtaaaacag agataaaatg ttgtggtttt taagtgagat aatccaagta aagcacctaa 96300catggagtag tgaatgaaca tcggttgcta ctaaaagtgg acatcctacc gcatccttaa 96360tgccactagg catttccata caatctgggg accaaaactt caatcatata aatgtatgag 96420gttaattaaa aacactactg taatctgctt gtatgatcac aaaccaccac aaaagaaaag 96480atcgtgaaga ttacactgta aacggactct caaatgatca ggaggtggtc acttcgcaac 96540ttgctccctc cacccaactc aaaacaggag ctcgagcctg cctgtatttg agactggagc 96600tgcctgtatg aggactggat caactgctag tcacgttata tccaaatctg cattatcatt 96660gggcacattt tcacagaatt ttactgaatt attccttaat tgtttaatgg ttgggaatag 96720tttgggaatt accttccatc aactctgcta agaaaggaat ggattctggt agcaagacaa 96780tataattctc ctttagtttt tcagccagtg ctaacacagt aatcaaagca gcaaatcgaa 96840cctgaaaggg ataaaagagc aaagaaataa aaagtagtgt tactgtattt attatcttaa 96900gagctgtact gacttgagac aagctctaac tttttaaaca ttagttcaca cgcgtttatt 96960cacttcatta tgttcattaa gctttcatct tagaatacca gtttcaccat ttgggagctg 97020tttgtaatat gtgcaacctt ataaatagtg ttttccaaac tgtgtcccag gactgcaaat 97080ctttaatgtg aaatgtcttt ttataatctc ttcctttaaa aaaaaccaat aaaataaaat 97140gccacatgca aactcaagtg tgtcaccaga ttttacttca ttggcgctcg ccagcccgcc 97200aggctggcaa taaagtgcct ccagccacct ctggcaggtc tcctcaccca cagcccctga 97260ctggtcacca ctatagttgt atgaggggcc aggacaatcg cttgggataa actcccatct 97320cagcactgaa taaaaaacat tctgtgtcac aatatcctag ttttggggct ttaaaaacgt 97380ctaggtgttc ctcacatgcc ttgtctataa taaggaaagc aagcagtagt tgggtattgt 97440tagcttttga aacaaaagcc ctactggtct tctaattttg gatattttaa ttaaagaata 97500tctggacagt acaaagtgaa ttattaaaaa accatttgta actacctaga ttcaatcagg 97560atttccttga tttgtgcaaa gtaaaatatt acaataaatt tgatactgct acttgtataa 97620aaacctatgg tttaaaatgt gggggttcat cataatagtc tcattgttag catatcctaa 97680taaagaattt gaactaataa atcctattaa taaaattctg ctttggtctg ttatagccag 97740taaagttcta atacaatcat tagtttgaga aatggtgact cattgctaaa acagtttgaa 97800atttgtaaca cttgggtgtc aaattttgac ttccactcaa cctacccatg ttttatttcc 97860actgccacca cttactcaac aagatcataa gcctagtatc tataaacaac agaatgtatt 97920gctctaactc aaaagactat agtgtggata aattcaatgc atttctctct ggagcacaat 97980gacatttcaa tagcacttaa aaaagaagga attacttcaa atctttgtta tttaaaagta 98040tttagaaagt attttagtac ttctgcccaa cgcaccattg gggtggggat agggcattgc 98100tattctttac aaatagccta taagtaaaaa acaaaatttt cttaggcaca aatttctgcc 98160taatacaaaa gaccagacct ctagtactgg atgacaaata gcaatgttct tccctgccag 98220tttactaggg ggcctacatc tgtgaccacc tgcaggctgt ttaggctatg cagtgaaaag 98280atgcagtttc agtacttgtc acgcagttcc taaccttagg cgaggagtct ctcgtcttta 98340gcagaatctg gtagttcagt ggtttccaaa gagagtcatc cgccatggcc actgaaaact 98400gtgcgatgca tggtatcagg tgctttgtca cccgttcctg gaatttctct tctcccccaa 98460gcctgttttc cagctaggaa gagtaagaca aagactttga acaacaagtc tcatttcttt 98520cttctgtttg aaaaaatgtc caacatacaa atattttact atctttcatg atattagcag 98580gttcaaaaac caggcattat tctaatactc tctagggcaa atgtattgcc ttctagaact 98640caaatggaat ctcataccct ttatcatcgc ccctttctct ccagcagaac atctcagagg 98700agctctttgc tccagaggac agccatgctc tgacacgttc tcagtgaggc ccagttaaaa 98760caaatgaata cattaaccat gacagcttat atcatgtctg tcttttgagc agtttaaaaa 98820ataaaaaata aaaaataact cagggccagg catggtggct cacgcctgta atcccagcag 98880tttgggaggc caaggtgggt ggatcacttg aggtcaggag ttcgagacca gcctggccaa 98940catggcaaaa cctcatccct actaaaaata caaaaattag ccaggtgtgg aggcgggcgc 99000ctgtgatccc agctattcgg gaggctgagg cacaagaatt gcttgaaccc gggaggtgga 99060ggttgcagcg agccgagatt gcaccactgc actccagcct gggtgacaga gcaagaccct 99120gtctcaaaac aacaaaacaa aactcaaatt ccacaatgaa gttatatctt tgaaaaaaca 99180attttcaaat aaaacatttc attaaaaaga ccagaaaaaa caaccttaca aagaaaaatc 99240ctagcaagct gtcatttgag cagatctaaa acctgccaag ctcgaacagt gatggcttcc 99300tcagcaacga aagatgattc tgtttggtta cctgatccac cagaggcatc atcaaggctc 99360ctgctctctc tttacttata aaatgctggg tatcaaaaag gaagattttg tataaacagt 99420tcaaaataaa ctgcaacagc aagcagcact tttcagggtc attttcagag tcaaaaaatg 99480cttcatctgt agacgtggga agagtaaaaa tgaaaaaaca ctgaacttaa ccatttaatc 99540tccaatgttt acattgaaat cactattaaa ataactaaat cagaagagtc taaaatgatc 99600tagaaatcat aatcaggacg aaggcagaac acaatggatg gtctctcgaa gaatgattcc 99660ttcttttaga gttaagattc taacactcac tctggcaagt taaattccct caactgtcaa 99720gtgggtcacg tattagcatt agagaataaa ctaatcttaa tttttgcgtt ttaaagttac 99780ttccagtaac tgacagtaac ggccatttac tttattcttt ctcccaagtg aggtgactta 99840taacattcgc tcatcatgct aaaacaacac ttcactgtct gacaacaatg aagtaaaaaa 99900ttcaccctcc ttagcttagg acttaagaac ctctaaaatc ttgcttccaa gcactagctt 99960gtgtcttact ggtaccttgt ataaggcaca caggacaagg gtgacagctg aactgaagcg 100020accacccacc tgttttggag atgttcacct ggtccaaggt gtcagcaaaa ggcttcacta 100080agtggccggc aaacagagta aaaagccctt tcagcttttc agcaatgcaa tctgccaagt 100140tgtaaaatgt caacaacctg tcctttgggg catcttctgt tttagcccaa tcaaacagct 100200gaaaggataa gacagtatta gtttcttcga catcttgtca cttaaatctg agcacaaaag 100260agaggaagag gaagaaagcg tcaccttgaa gaacaggggc ctgaatgtga cctcggaaag 100320tttgacaacc atggctacta gacagtcaat gatacaattt tccgtttttc caacttcctc 100380cagatcgttc tgaaaacaga agagcccatt tattagagtg ctgatacctg actgtaaatt 100440attttggcaa gtaccactgt tacacggcta gattgttctc ggactcttca ataggtggat 100500aacagcttta ggatttggag gagtgaacct gagcttacct cagagtgctg ggctcggaag 100560tccagggcct ccaggaaaaa ggcggttagc tgagactgat gggaggtgag ctcttccttc 100620ttcatcgccc caatatgctc ttgcaagatg ctcataaacg gacccatgtg attctaccaa 100680taacacagga aaaagatgtg ccattttcaa atgattccta gagttcagcg gtgtgtattt 100740ttaaaaacta aatcttcttc tttaagtcaa agtttacaca ttgcagtacc acctctccct 100800tctccaaagt cttaataccc aataagatct aaccttccag ttcttctcaa tctgcttgta 100860agtttttttg atggcgggca acaggactcg gggtgcaagt gtggtagcca gtgtcttttt 100920aagagatgtg agacggatat tagcctgtga cgcagaaccc atttcactag tgattttctc 100980cagatgaatc acctacagga atataaaaaa agtgatcagg gccactgcag atcttcgctg 101040acaaacacac acttacagag aggcttcatg atgaggtact agtgtttgga aaatgcttag 101100cactttttaa ctacacacag agttcctttt aaagtcagcc ctaaacgtca gtggataaaa 101160ctgggcagac acctcttgcc caacttgcga tcagggacga aggccgatgg tagacgcaga 101220cgcacacaca gcacccagac agatgatttt cttagaggac aggaatgcaa gggaccacgg 101280caagagtcaa gttgctaaaa aactgagaaa gctcctcaga gcacaggccc ctttctctga 101340gaaggctact tttaaaccct ggctgtggtg taagtgaagc ggtttaatca tttgccccat 101400ggtaatgaag gctcctaacc ttgtaaatgg caaatgatca acacaatgga acagccaggt 101460ctcaacactc ttgagcatct tcaatcataa ataccactgg cccctagcgt gttgacagga 101520aaccgctgac gtgcaataca aaaattctgc tttgcaagat gccttaggat taaacctctc 101580acagtagaaa cagggcccat caatttccac aagtaataaa aggcggctct accagcccaa 101640ctccaaagat ctcacagaag aaaaaaaagc cagaatacat tccgcacaat taaagaagag 101700aagcatctcg ctaaaaagtg acccccatat caatttcaag attaagtggc aaggatgatg 101760gaagagaaaa agtacacatt taataaaagc aagcacatct cttcagaaat aagactcctt 101820tctgtcaaac ggaaactaac ccttaaagaa aaaacaaaat cactacattt gtgatctttt 101880accttcccca gccaccctgc gtagcatgtc gtggctatcg tggctcacct gggagagaat 101940gccttccaga taggggctga tgaagtgcgg gagagtctcc acaaccttct gcagagcagc 102000caaggcactg agcaggtaga cctcgctgga gaccagctcg ctggtgttct tcattgttgt 102060cagcaacgat ggcatcaggc tagaaacaaa gtaagagctt tagaagaact tgaagcagaa 102120acagaggcta gggaatggag tagagggcat tatgaaaaaa accagcaaac tgtgcctatt 102180acatcgctat ctgcctcata gcctaaaaag cagtgtctat acattttatg tggctaagca 102240caagaaatct cccagtgcta acagtatgga cacaacagta atttaaaaaa taacaatgtc 102300tttcattaac tgaacactta ctatgtgtca ggcactatgc aaaactcctt gcaagcactg 102360ccctacagaa atcctatgag gtagatactg tctctgtttt atagacagca aagctctaac 102420aggttaagga acatactggc tgtacagtaa ggaactacca cagccaggag cttctaactt 102480ccaaatttgg cagcagaagg cagctttggc cttgcctaac tgggtgggcc cctctgccaa 102540gaaccttcac ccactgcttt ttgactatac tagacaaaag gaaggaagaa tggaggacga 102600ttaacactgc aaagcagtgc atctgaagat aaacgggaag gctgcatctt tctgtttgaa 102660gattaattat ttttattatt atttctttaa gagacagggt ctcactctgt tgcccaggct 102720acagtgcagt ggtgcagtca tagctcactg cagcctcaaa ctcctgggct caaatgatct 102780ccctgccttg gcctcccaaa gtgctgggat cacagccgtg agccaccaca ccctgcaaga 102840tcaattcttt aacaaattcc aattttatgc aacgtctact cagaggaaaa aaaaaaaaag 102900tcaccaaagt gttatttttc aatgtgtgcc aggcggtaac agctcctgtt ccaagtctcc 102960ggccgcatac ctgggaagct gggggatggc cagcgcctcc agggtggagg tcacctctgc 103020tatgcacagc agcgcgcttc ccaagacatt cttctcctcc tttctctctg gagcaatcag 103080tttcacagca gtgctcagca ctgggacaaa aggatctgga ttttctgcac caaaattctt 103140gcataaaagc tttaaggtat acaacgctgt ctgtctgttg attgcttgtt cttcttcccc 103200ttcctttttc ttacgctgca caatggccaa aaggtctgga accagtttta ggaaacgggt 103260aacctgaagg ggacagccag aatccccaaa tcattaaagc tgcaaaaaat gtttgtccat 103320tttcccattg tcacagcttg agattgtcta aatggaaatc agactcgggg gtcctgagtc 103380acacagtcat gctaagcgat gtgcatgttc tagccagtgt ttcacttata caaagcaccc 103440actgatctgg agtaaaaggg acttagaact atgctaaggc taaggccacg taagctctgt 103500agtaagcaag aattccacta ggctgaaatt ccattctaag agctcttaca acacacatat 103560attcccgtta gaattaacgt cacattttaa aacatgtcat ggtattatat tcagataata 103620atatacttca atttgaaatt gtaccactag agaaattgaa gggagttaaa tgcagctctt 103680tgataaagca aagtacagta aatgggtgtg tcctgggtct tcactcacta ttgtcttctt 103740ccaggatata ttttgctgca gcttgttatt caaaaggtcc agcgctttgc ggcgaacaga 103800tggcagggga ttgcccacca gccctctgat cacaggaatg aatgtctctg tgggcagcaa 103860ggcattgacc taaagagaaa ttttatattt aacatgaaaa gaaaaacaaa ttaaaaaaaa 103920aatcaacttc aattaagaca gactgctgtc cactgcacac ctccaggcac caggcacttc 103980cacacacatt ttcttattta attcttaaaa taacctttca ggtaggcatt accaaccaca 104040cattatcgaa caaaacaaaa gcctgatgtc aggaggaagt gccaaaggca tgcagctaaa 104100tgactgagct agatttgaat cagcaatcct aacttcgagg ccagtgatat gtatgtaata 104160tacttcatac ttttatttta ttccacttga ataaagtaga acagtatata ttatatgact 104220taattattaa aatatacgag gtacatgttc tcataactgg taaggaaaca attttttcca 104280gacaaatcta tttctagtca tcaagagatt gttttctaag aaaaatctga gcttcattat 104340attcataaaa ggaattgcta agtttattct taaaaacttt acataatttc acaataattt 104400aaaaaacagc aacaaaacag taattccagg gagaaatgaa cacctacctt atctaacagg 104460tcgtaagctt tactaaggag cgcgcgccag aacttcacgg tgagtttgtc tgcgttcctt 104520tccatggact gtgcaactgc actgatatag ccgagaacgg tctccagcaa cctgaaacac 104580agaggctcgc tcagcaaacg gcagctgaag aaactcagag aacttgttca tgtctacctt 104640atgctaaatg tttcaagtag aaagacgagt taaataattc tgtactaaat tatttcaaaa 104700actactcgga aagaaaggaa atgagggatt attgccatag acagagatca tcaagaagta 104760actaggcgct tctgtgcaga agcatcgacc tcgctcagac tctgtgaggt gctgaataag 104820caacagatgc tgaaagcgtt taaggaactc actcatatct agctcatgct cagtggatct 104880cactgggctg tccaagtggg gtgttcaggg agttatggcc ctaggttaat ggcaggtgtg 104940tgcgtgcaca cacacacagg cacacacacg cacacataca catgcacaca caccatacac 105000catttatata aagagaaata ttaatagaaa tgaacatata acccacttct ttcacattat 105060taggagacaa aaaaaaagac tacaaacttc aaataacttg taattagaaa agcacacacc 105120aaattccaac acagctgcca ctggagatcc ccccactgct gccagcctga ggggggagct 105180agagggaaga gtggagacag aagttgacac cgcacagcag aggaggggag aagggggcgc 105240agacaaaatc agctccaaaa acgaaagtcc tacgcatagc gctacaagtc agcccacagg 105300actggaactc agcagctcac attcctggct gcagggcagg cactttccag tggaaggggc 105360aggacagtgg ccctgggaat gccatgcatc tgaaaaggag gtacacagca aggccaggag 105420gcaaaccccg aggacatggg agagaaagga aaattcctgc acccaaatat ataatggcag 105480catatggatt agaatccacg gaataaagaa ttcatgagcc catagaaatc agggccagat 105540tgagacacta aacagatact gcaactcaat acaatacaca gacttgacat ggatcatgat 105600gcagaaacac atgcggtgta aaggacagtg ttgggataat tagggagact ggagtatgaa 105660ctgtagatta catcactgga ttggatcaat gttaaatttt ctgaatttga tcaatgtact 105720gtggttttat aagaacatct cttattctta gagacataat gtatatgatt tactttcaaa 105780tggctcagag aaaaaaccct acatagggag aacgctaagg caaatgtggc agaaagtatt 105840atcaaatggt gaacctggtt gtaaagagta tatgaatttt ctgtactgtt tttccaggtt 105900ttctataagt ttgaagtcat ttccaaataa aaagtaaaaa aagaaaagga aacatacctc 105960tcttcaaggc cttttaaaat ctcaggacca ccactctcaa ctacctaatt tttaaagaag 106020acgtcattag aacggtatgg aagtcaataa taaaagtcat ttcaagtcag ttcaatgaaa 106080ctcggaccat tcactgaaac cttccacagc aactgttttc tgacattaca atttaatcag 106140gttcatagca tcttcattat actgtagtaa ctctatttct cttaatttat tttaattata 106200ttctactggt agtatctaaa aagtactaca atggttcaga aaaatacagc aatcaacact 106260caattagcac taccgaattc tatgacatgc tgatctggtg agctcacata tcctttgttg 106320agaagttaaa cattacagat tcagctggaa tcccccaagt actgctcctt ggtcctattc 106380tccctctacc ccaagcccca caaacaaaac catcatccca aatctgcttc caaatgtttc 106440aaacactaca tatcacggaa caacatgttt ttctggaaac atatttttga gatctatgca 106500tggtgactta tgttctagtt ccttcatttt aactgcatat gatattcctc tataaatacc 106560acttatctat ccatttgcct ctgttgttag atgtttagtt tatgtccatt ttttcccctt 106620ttactaataa tgctagagaa gaacattttt atgtcccttt gatcatcttg ggaagttttt 106680acagcatata tacctaagga agggaatgac cagatcacag gaattactgg aactttcaac 106740ctcatg 106746 13 132 PRT Homo sapiens MISC_FEATURE (1)..(132) amino acid sequence of leg2 13Met Thr Gly Glu Leu Glu Val Lys Asn Met Asp Met Lys Pro Gly Ser1 5 10 15Thr Leu Lys Ile Thr Gly Ser Ile Ala Asp Gly Thr Asp Gly Phe Val 20 25 30Ile Asn Leu Gly Gln Gly Thr Asp Lys Leu Asn Leu His Phe Asn Pro 35 40 45Arg Phe Ser Glu Ser Thr Ile Val Cys Asn Ser Leu Asp Gly Ser Asn 50 55 60Trp Gly Gln Glu Gln Arg Glu Asp His Leu Cys Phe Ser Pro Gly Ser65 70 75 80Glu Val Lys Phe Thr Val Thr Phe Glu Ser Asp Lys Phe Lys Val Lys 85 90 95Leu Pro Asp Gly His Glu Leu Thr Phe Pro Asn Arg Leu Gly His Ser 100 105 110His Leu Ser Tyr Leu Ser Val Arg Gly Gly Phe Asn Met Ser Ser Phe 115 120 125Lys Leu Lys Glu 130 14 134 PRT Homo sapiens MISC_FEATURE (1)..(134) amino acid sequence of leg1 14Ala Cys Gly Leu Val Ala Ser Asn Leu Asn Leu Lys Pro Gly Glu Cys1 5 10 15Leu Arg Val Arg Gly Glu Val Ala Pro Asp Ala Lys Ser Phe Val Leu 20 25 30Asn Leu Gly Lys Asp Ser Asn Asn Leu Cys Leu His Phe Asn Pro Arg 35 40 45Phe Asn Ala His Gly Asp Ala Asn Thr Ile Val Cys Asn Ser Lys Asp 50 55 60Gly Gly Ala Trp Gly Thr Glu Gln Arg Glu Ala Val Phe Pro Phe Gln65 70 75 80Pro Gly Ser Val Ala Glu Val Cys Ile Thr Phe Asp Gln Ala Asn Leu 85 90 95Thr Val Lys Leu Pro Asp Gly Tyr Glu Phe Lys Phe Pro Asn Arg Leu 100 105 110Asn Leu Glu Ala Ile Asn Tyr Met Ala Ala Asp Gly Asp Phe Lys Ile 115 120 125Lys Cys Val Ala Phe Asp 130 15 316 PRT Homo sapiens MISC_FEATURE (1)..(316) amino acid sequence of PCTA 15Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val Ile1 5 10 15Pro Tyr Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu Ile 20 25 30Val Ile Cys Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val Asp 35 40 45Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr Leu65 70 75 80Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro Phe 85 90 95Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asp Lys 100 105 110Phe Gln Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His Arg 115 120 125Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys Val Asn 130 135 140Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser Thr Gln145 150 155 160Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg Glu Asn Val Pro Lys 165 170 175Ser Gly Thr Pro Gln Leu Ser Leu Pro Phe Ala Ala Arg Leu Asn Thr 180 185 190Pro Met Gly Pro Gly Arg Thr Val Val Val Lys Gly Glu Val Asn Ala 195 200 205Asn Ala Lys Ser Phe Asn Val Asp Leu Leu Ala Gly Lys Ser Lys Asp 210 215 220Ile Ala Leu His Leu Asn Pro Arg Leu Asn Ile Lys Ala Phe Val Arg225 230 235 240Asn Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu Glu Arg Asn Ile Thr 245 250 255Ser Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys 260 265 270Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His Ser Leu Glu 275 280 285Tyr Lys His Arg Phe Lys Glu Leu Ser Ser Ile Asp Thr Leu Glu Ile 290 295 300Asn Gly Asp Ile His Leu Leu Glu Val Arg Ser Trp305 310 315 16 358 PRT Homo sapiens MISC_FEATURE (1)..(358) amino acid sequence of PCTA.var 16Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val Ile1 5 10 15Pro Tyr Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr Leu Ile 20 25 30Val Ile Cys Gly His Val Pro Ser Asp Ala Asp Arg Phe Gln Val Asp 35 40 45Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile Val Cys Asn Thr Leu65 70 75 80Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile Thr Tyr Asp Thr Pro Phe 85 90 95Lys Arg Glu Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys Asp Lys 100 105 110Phe Gln Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly His Arg 115 120 125Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys Val Asn 130 135 140Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser Thr Gln145 150 155 160Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg Glu Asn Val Pro Lys 165 170 175Ser Gly Thr Pro Gln Leu Pro Ser Asn Arg Gly Gly Asp Ile Ser Lys 180 185 190Ile Ala Pro Arg Thr Val Tyr Thr Lys Ser Lys Asp Ser Thr Val Asn 195 200 205His Thr Leu Thr Cys Thr Lys Ile Pro Pro Met Asn Tyr Val Ser Lys 210 215 220Ser Leu Pro Phe Ala Ala Arg Leu Asn Thr Pro Met Gly Pro Gly Arg225 230 235 240Thr Val Val Val Lys Gly Glu Val Asn Ala Asn Ala Lys Ser Phe Asn 245 250 255Val Asp Leu Leu Ala Gly Lys Ser Lys Asp Ile Ala Leu His Leu Asn 260 265 270Pro Arg Leu Asn Ile Lys Ala Phe Val Arg Asn Ser Phe Leu Gln Glu 275 280 285Ser Trp Gly Glu Glu Glu Arg Asn Ile Thr Ser Phe Pro Phe Ser Pro 290 295 300Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys Asp Val Arg Glu Phe Lys305 310 315 320Val Ala Val Asn Gly Val His Ser Leu Glu Tyr Lys His Arg Phe Lys 325 330 335Glu Leu Ser Ser Ile Asp Thr Leu Glu Ile Asn Gly Asp Ile His Leu 340 345 350Leu Glu Val Arg Ser Trp 355 17 315 PRT Mus musculus MISC_FEATURE (1)..(315) amino acid sequence of PCTA.mus 17Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Ile Ile1 5 10 15Pro Tyr Val Gly Thr Ile Thr Glu Gln Leu Lys Pro Gly Ser Leu Ile 20 25 30Val Ile Arg Gly His Val Pro Lys Asp Ser Glu Arg Phe Gln Val Asp 35 40 45Phe Gln Leu Gly Asn Ser Leu Lys Pro Arg Ala Asp Val Ala Phe His 50 55 60Phe Asn Pro Arg Phe Lys Arg Ser Ser Cys Ile Val Cys Asn Thr Leu65 70 75 80Thr Gln Glu Lys Trp Gly Trp Glu Glu Ile Thr Tyr Asp Met Pro Phe 85 90 95Arg Lys Glu Lys Ser Phe Glu Ile Val Phe Met Val Leu Lys Asn Lys 100 105 110Phe Gln Val Ala Val Asn Gly Arg His Val Leu Leu Tyr Ala His Arg 115 120 125Ile Ser Pro Glu Gln Ile Asp Thr Val Gly Ile Tyr Gly Lys Val Asn 130 135 140Ile His Ser Ile Gly Phe Arg Phe Ser Ser Asp Leu Gln Ser Met Glu145 150 155 160Thr Ser Ala Leu Gly Leu Thr Gln Ile Asn Arg Glu Asn Ile Gln Lys 165 170 175Pro Gly Lys Leu Gln Leu Ser Leu Pro Phe Glu Ala Arg Leu Asn Ala 180 185 190Ser Met Gly Pro Gly Arg Thr Val Val Ile Lys Gly Glu Val Asn Thr 195 200 205Asn Ala Arg Ser Phe Asn Val Asp Leu Val Ala Gly Lys Thr Arg Asp 210 215 220Ile Ala Leu His Leu Asn Pro Arg Leu Asn Lys Ala Phe Val Arg Asn225 230 235 240Ser Phe Leu Gln Asp Ala Trp Gly Glu Glu Glu Arg Asn Ile Thr Cys 245 250 255Phe Pro Phe Ser Ser Gly Met Tyr Phe Glu Met Ile Ile Tyr Cys Asp 260 265 270Val Arg Glu Phe Lys Val Ala Ile Asn Gly Val His Ser Leu Glu Tyr 275 280 285Lys His Arg Phe Lys Asp Leu Ser Ser Ile Asp Thr Leu Ser Val Asp 290 295 300Gly Asp Ile Arg Leu Leu Asp Val Arg Ser Trp305 310 315 18 355 PRT Homo sapiens MISC_FEATURE (1)..(355) amino acid sequence of gal9-1 18Met Ala Phe Ser Gly Ser Gln Ala Pro Tyr Leu Ser Pro Ala Val Pro1 5 10 15Phe Ser Gly Thr Ile Gln Gly Gly Leu Gln Asp Gly Leu Gln Ile Thr 20 25 30Val Asn Gly Thr Val Leu Ser Ser Ser Gly Thr Arg Phe Ala Val Asn 35 40 45Phe Gln Thr Gly Phe Ser Gly Asn Asp Ile Ala Phe His Phe Asn Pro 50 55 60Arg Phe Glu Asp Gly Gly Tyr Val Val Cys Asn Thr Arg Gln Asn Gly65 70 75 80Ser Trp Gly Pro Glu Glu Arg Lys Thr His Met Pro Phe Gln Lys Gly 85 90 95Met Pro Phe Asp Leu Cys Phe Leu Val Gln Ser Ser Asp Phe Lys Val 100 105 110Met Val Asn Gly Ile Leu Phe Val Gln Tyr Phe His Arg Val Pro Phe 115 120 125His Arg Val Asp Thr Ile Ser Val Asn Gly Ser Val Gln Leu Ser Tyr 130 135 140Ile Ser Phe Gln Asn Pro Arg Thr Val Pro Val Gln Pro Ala Phe Ser145 150 155 160Thr Val Pro Phe Ser Gln Pro Val Cys Phe Pro Pro Arg Pro Arg Gly 165 170 175Arg Arg Gln Lys Pro Pro Gly Val Trp Pro Ala Asn Pro Ala Pro Ile 180 185 190Thr Gln Thr Val Ile His Thr Val Gln Ser Ala Pro Gly Gln Met Phe 195 200 205Ser Thr Pro Ala Ile Pro Pro Met Met Tyr Pro His Pro Ala Tyr Pro 210 215 220Met Pro Phe Ile Thr Thr Ile Leu Gly Gly Leu Tyr Pro Ser Lys Ser225 230 235 240Ile Leu Leu Ser Gly Thr Val Leu Pro Ser Ala Gln Arg Phe His Ile 245 250 255Asn Leu Cys Ser Gly Asn His Ile Ala Phe His Leu Asn Pro Arg Phe 260 265 270Asp Glu Asn Ala Val Val Arg Asn Thr Gln Ile Asp Asn Ser Trp Gly 275 280 285Ser Glu Glu Arg Ser Leu Pro Arg Lys Met Pro Phe Val Arg Gly Gln 290 295 300Ser Phe Ser Val Trp Ile Leu Cys Glu Ala His Cys Leu Lys Val Ala305 310 315 320Val Asp Gly Gln His Leu Phe Glu Tyr Tyr His Arg Leu Arg Asn Leu 325 330 335Pro Thr Ile Asn Arg Leu Glu Val Gly Gly Asp Ile Gln Leu Thr His 340 345 350Val Gln Thr 355 19 323 PRT Homo sapiens MISC_FEATURE (1)..(323) amino acid sequence of gal 19Met Ala Phe Ser Gly Ser Gln Ala Pro Tyr Leu Ser Pro Ala Val Pro1 5 10 15Phe Ser Gly Thr Ile Gln Gly Gly Leu Gln Asp Gly Leu Gln Ile Thr 20 25 30Val Asn Gly Thr Val Leu Ser Ser Ser Gly Thr Arg Phe Ala Val Asn 35 40 45Phe Gln Thr Gly Phe Ser Gly Asn Asp Ile Ala Phe His Phe Asn Pro 50 55 60Arg Phe Glu Asp Gly Gly Tyr Val Val Cys Asn Thr Arg Gln Asn Gly65 70 75 80Ser Trp Gly Pro Glu Glu Arg Arg Thr His Met Pro Phe Gln Lys Gly 85 90 95Met Pro Phe Asp Leu Cys Phe Leu Val Gln Ser Ser Asp Phe Lys Val 100 105 110Met Val Asn Gly Ile Leu Phe Val Gln Tyr Phe His Arg Val Pro Phe 115 120 125His Arg Val Asp Thr Ile Phe Val Asn Gly Ser Val Gln Leu Ser Tyr 130 135 140Ile Ser Phe Gln Pro Pro Gly Val Trp Pro Ala Asn Pro Ala Pro Ile145 150 155 160Thr Gln Thr Val Ile His Thr Val Gln Ser Ala Pro Gly Gln Met Phe 165 170 175Ser Thr Pro Ala Ile Pro Pro Met Met Tyr Pro His Pro Ala Tyr Pro 180 185 190Met Pro Phe Ile Thr Thr Ile Leu Gly Gly Leu Tyr Pro Ser Lys Ser 195 200 205Ile Leu Leu Ser Gly Thr Val Leu Pro Ser Ala Gln Arg Phe His Ile 210 215 220Asn Leu Cys Ser Gly Asn His Ile Ala Phe His Leu Asn Leu Arg Phe225 230 235 240Asp Glu Asn Ala Val Val Arg Asn Thr Gln Ile Asp Asn Ser Trp Gly 245 250 255Ser Glu Glu Arg Ser Leu Pro Arg Lys Met Pro Phe Val Arg Gly Gln 260 265 270Ser Phe Ser Val Trp Ile Leu Cys Gly Ala His Cys Leu Lys Val Ala 275 280 285Val Asp Gly Gln His Leu Phe Glu Tyr Tyr His Arg Leu Arg Asn Leu 290 295 300Pro Thr Ile Asn Arg Leu Glu Val Gly Gly Asp Ile Gln Leu Thr His305 310 315 320Val Gln Thr 20 135 PRT Homo sapiens MISC_FEATURE (1)..(135) amino acid sequence of leg7 20Ser Asn Val Pro His Lys Ser Ser Leu Pro Glu Gly Ile Arg Pro Gly1 5 10 15Thr Val Leu Arg Ile Arg Gly Leu Val Pro Pro Asn Ala Ser Arg Phe 20 25 30His Val Asn Leu Leu Cys Gly Glu Glu Gln Gly Ser Asp Ala Ala Leu 35 40 45His Phe Asn Pro Arg Leu Asp Thr Ser Glu Val Val Phe Asn Ser Lys 50 55 60Glu Gln Gly Ser Trp Gly Arg Glu Glu Arg Gly Pro Gly Val Pro Phe65 70 75 80Gln Arg Gly Gln Pro Phe Glu Val Leu Ile Ile Ala Ser Asp Asp Gly 85 90 95Phe Lys Ala Val Val Gly Asp Ala Gln Tyr His His Phe Arg His Arg 100 105 110Leu Pro Leu Ala Arg Val Arg Leu Val Glu Val Gly Gly Asp Val Gln 115 120 125Leu Asp Ser Val Arg Ile Phe 130 135 21 323 PRT Homo sapiens MISC_FEATURE (1)..(323) amino acid sequence of gal4 21Met Ala Tyr Val Pro Ala Pro Gly Tyr Gln Pro Thr Tyr Asn Pro Thr1 5 10 15Leu Pro Tyr Tyr Gln Pro Ile Pro Gly Gly Leu Asn Val Gly Met Ser 20 25 30Val Tyr Ile Gln Gly Val Ala Ser Glu His Met Lys Arg Phe Phe Val 35 40 45Asn Phe Val Val Gly Gln Asp Pro Gly Ser Asp Val Ala Phe His Phe 50 55 60Asn Pro Arg Phe Asp Gly Trp Asp Lys Val Val Phe Asn Thr Leu Gln65 70 75 80Gly Gly Lys Trp Gly Ser Glu Glu Arg Lys Arg Ser Met Pro Phe Lys 85 90 95Lys Gly Ala Ala Phe Glu Leu Val Phe Ile Val Leu Ala Glu His Tyr 100 105 110Lys Val Val Val Asn Gly Asn Pro Phe Tyr Glu Tyr Gly His Arg Leu 115 120 125Pro Leu Gln Met Val Thr His Leu Gln Val Asp Gly Asp Leu Gln Leu 130 135 140Gln Ser Ile Asn Phe Ile Gly Gly Gln Pro Leu Arg Pro Gln Gly Pro145 150 155 160Pro Met Met Pro Pro Tyr Pro Gly Pro Gly His Cys His Gln Gln Leu 165 170 175Asn Ser Leu Pro Thr Met Glu Gly Pro Pro Thr Phe Asn Pro Pro Val 180 185 190Pro Tyr Phe Gly Arg Leu Gln Gly Gly Leu Thr Ala Arg Arg Thr Ile 195 200 205Ile Ile Lys Gly Tyr Val Pro Pro Thr Gly Lys Ser Phe Ala Ile Asn 210 215 220Phe Lys Val Gly Ser Ser Gly Asp Ile Ala Leu His Ile Asn Pro Arg225 230 235 240Met Gly Asn Gly Thr Val Val Arg Asn Ser Leu Leu Asn Gly Ser Trp 245 250 255Gly Ser Glu Glu Lys Lys Ile Thr His Asn Pro Phe Gly Pro Gly Gln 260 265 270Phe Phe Asp Leu Ser Ile Arg Cys Gly Leu Asp Arg Phe Lys Val Tyr 275 280 285Ala Asn Gly Gln His Leu Phe Asp Phe Ala His Arg Leu Ser Ala Phe 290 295 300Gln Arg Val Asp Thr Leu Glu Ile Gln Gly Asp Val Thr Leu Ser Tyr305 310 315 320Val Gln Ile 22 466 PRT Artificial Sequence conserved sequence among SEQ ID NOs. 13-21 22Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110Xaa Pro Xaa Xaa Pro Xaa Xaa Xaa Xaa Ile Pro Xaa Gly Leu Xaa Pro 115 120 125Gly Xaa Xaa Xaa Xaa Ile Xaa Gly Xaa Val Xaa Pro Xaa Xaa Ala Xaa 130 135 140Arg Phe Xaa Val Asn Leu Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Gly Xaa145 150 155 160Asp Xaa Ala Phe His Phe Asn Pro Arg Phe Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175Xaa Xaa Val Val Cys Asn Thr Xaa Xaa Xaa Gly Xaa Trp Gly Xaa Glu 180 185 190Glu Arg Xaa Xaa Xaa Xaa Pro Phe Xaa Xaa Gly Xaa Xaa Phe Glu Xaa 195 200 205Xaa Xaa Xaa Val Xaa Xaa Asp Xaa Phe Lys Val Xaa Val Asn Gly Xaa 210 215 220Xaa Xaa Xaa Xaa Tyr Xaa His Arg Leu Xaa Pro Leu Xaa Xaa Val Xaa225 230 235 240Xaa Xaa Xaa Val Xaa Gly Asp Val Gln Leu Xaa Ser Ile Xaa Phe Xaa 245 250 255Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 260 265 270Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 275 280 285Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 290 295 300Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa305 310 315 320Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa 325 330 335Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 340 345 350Gly Xaa Val Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa Xaa 355 360 365Xaa Xaa Xaa Xaa Xaa Ile Ala Xaa His Xaa Asn Xaa Arg Xaa Xaa Xaa 370 375 380Xaa Xaa Xaa Val Arg Asn Xaa Xaa Xaa Xaa Xaa Xaa Trp Gly Xaa Glu385 390 395 400Glu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Phe Xaa Xaa Gly Xaa Xaa Phe 405 410 415Xaa Xaa Xaa Ile Xaa Cys Xaa Xaa Xaa Xaa Xaa Lys Val Xaa Xaa Xaa 420 425 430Gly Xaa His Xaa Xaa Xaa Xaa Xaa His Arg Xaa Xaa Xaa Xaa Xaa Xaa 435 440 445Xaa Xaa Xaa Leu Xaa Xaa Xaa Gly Asp Xaa Xaa Leu Xaa Xaa Val Xaa 450 455 460Xaa Xaa465

Claims

1. A method of genotyping comprising determining the identity of a nucleotide at a biallelic marker of SEQ ID NO: 12, or the complement of said nucleotide, in a biological sample, wherein said biallelic marker is selected from the group consisting of biallelic markers of SEQ ID No:1 located at positions 402 (A2), 67092 (A30), 68525 (A41), 82234 (A55), 82393 (A57), and 87713 (A75).

2. The method according to claim 1, wherein said biological sample is derived from a single subject.

3. The method according to claim 2, wherein the identity of the nucleotides at said biallelic marker is determined for both copies of said biallelic marker present in said individual's genome.

4. A method of estimating the frequency of a haplotype for a set of biallelic markers of SEQ ID NO: 12 in a population, comprising:a) genotyping at least one biallelic marker of SEQ ID NO: 12 according to claim 2 for each individual in said population; b) genotyping a second biallelic marker of SEQ ID NO: 12 by determining the identity of the nucleotide at said second biallelic marker for both copies of said second biallelic marker present in the genome of each individual in said population; and c) applying a haplotype determination method to the identities of the nucleotides determined in steps a) and b) to obtain an estimate of said frequency.

5. The method according to claim 4, wherein said haplotype determination method is selected from the group consisting of asymmetric PCR amplification, double PCR amplification of specific alleles, the Clark algorithm, or an expectation-maximization algorithm.

6. A method of detecting an association between a haplotype and a trait, comprising the steps of:a) estimating the frequency of at least one haplotype in a trait positive population according to the method of claim 4; b) estimating the frequency of said haplotype in a control population according to the method of claim 4; and c) determining whether a statistically significant association exists between said haplotype and said trait, wherein said trait is familial or sporadic prostate cancer.

7. The method according to claim 6, wherein said control population is a trait negative population.

8. The method according to claim 6, wherein said control population is a random population.

9. The method according to claim 1, wherein said biological sample is derived from multiple subjects.

10. The method according to claim 1, further comprising amplifying a portion of said sequence comprising the biallelic marker prior to said determining step.

11. The method according to claim 10, wherein said amplifying is performed by PCR.

12. The method according to claim 1, wherein said determining is performed by a hybridization assay.

13. The method according to claim 1, wherein said determining is performed by a sequencing assay.

14. The method according to claim 1, wherein said determining is performed by a microsequencing assay.

15. The method according to claim 1, wherein said determining is performed by an enzyme-based mismatch detection assay.

16. A method of estimating the frequency of an allele of a biallelic marker of SEQ ID NO: 12 in a population comprising:a) genotyping individuals from said population for said biallelic marker according to the method of claim 1; and b) determining the proportional representation of said biallelic marker in said population.

17. A method of detecting an association between a genotype and a trait, comprising the steps of:a) performing the method of claim 16 to determine the frequency of at least one biallelic marker of SEQ ID NO: 12 in trait positive population; b) performing the method of claim 16 to determine the frequency of at least one biallelic marker of SEQ ID NO: 12 in a control population; and c) determining whether a statistically significant association exists between said genotype and said trait, wherein said trait is familial or sporadic prostate cancer.

18. The method according to claim 17, wherein said genotyping steps a) and b) are performed on a single pooled biological sample derived from each of said populations.

19. The method according to claim 17, wherein said genotyping steps a) and b) performed separately on biological samples derived from each individual in said populations.

20. The method according to claim 17, wherein said control population is a trait negative population.

21. The method according to claim 17, wherein said control population is a random population.

22. A method of determining whether an individual is at risk of developing familial or sporadic prostate cancer, comprising:a) genotyping at least one biallelic marker of SEQ ID NO: 12 according to the method of claim 3; b) determining if the individual has a biallelic marker or combination of biallelic markers that is associated with familial or sporadic prostate cancer; and c) correlating the result of step b) with a biallelic marker or one or more combinations of biallelic markers that are associated with a risk of developing familial or sporadic prostate cancer; wherein: for familial cases of prostate cancer said biallelic marker or combinations of biallelic markers are selected from the group consisting of: 1) A30 and A41; 2) A30 and A57; 3); A30 and A55; 4) A2 and A41; 5) A2 and A30; 6) A30 and A75; 7) A2 and A55; 8) A2, A30, and A41; 9) A2, A30, and A57; 10) A2, A30, and A55; 11) A30, A41, and A55; 12) A30, A57, and A75; 13) A30, A55, and A75; 14) A30, A41, and A75; 15) A30, A55, and A57; 16) A30, A41, and A57; 17) A2, A30, A55, and A57; 18) A2, A30, A57, and A75; 19) A2, A30, A55, and A75; 20) A2, A30, A41, and A57; 21) A2, A30, A41, and A55; 22) A2, A30, A41, and A75; 23) A30, A55, A57, and A75; 24) A30, A41, A55, and A75; 25) A30, A41, A55, and A57; 26) A30, A41, A57, and A75 and A30; and 27) A30; and for sporadic cases of prostate cancer said combinations of biallelic markers are selected from the group consisting of: 1) A2 and A55; 2) A2 and A57; 3) A41 and A55; 4) A41 and A57; 5) A2 and A41; 6) A30 and A75; 7) A2, A41, and A55; 8) A2, A55, and A57; 9) A2, A41, and A57; 10) A41, A55, and A57; 11) A2, A55, and A75; 12) A30, A41, and A57; 13) A30, A41, and A55; 14) A2, A30, A41, and A57; 15) A2, A30, A41, and A55; 16) A2, A41, A55, and A57; 17) A2, A41, A55, and A75; and 18) A30, A41, A55, and A57.