WO2006022633A1

WO2006022633A1 - Methods for identifying a risk of type ii diabetes and treatments thereof

Info

Publication number: WO2006022633A1
Application number: PCT/US2004/023903
Authority: WO
Inventors: Maria L. Langdown; Matthew Roberts Nelson; Rikard Henry Reneland; Stefan M. Kammerer; Andreas Braun; Mikhail F. Denissenko; Kenneth W. Henry; Carolyn R. Hoyal-Wrightson
Original assignee: Sequenom, Inc.
Priority date: 2004-07-22
Filing date: 2004-07-22
Publication date: 2006-03-02

Abstract

Provided herein are methods for identifying a risk of type II diabetes in a subject, reagents and kits for carrying out the methods, methods for identifying candidate therapeutics for treating type II diabetes, and therapeutic and preventative methods applicable to type II diabetes. These embodiments are based upon an analysis of polymorphic variations in nucleotide sequences within the human genome.

Description

METHODS FOR IDENTIFYING RISK OF TYPE II DIABETES AND TREATMENTS THEREOF

Field of the Invention

[0001] The invention relates to genetic methods for identifying predisposition to type II diabetes, also known as non-insulin dependent diabetes, and treatments that specifically target the disease.

Background

[0002] Diabetes is among the most common of all metabolic disorders, affecting up to 11% of the population by age 70. Type I diabetes (insulin-dependent diabetes) represents about 5 to 10% of this group and is the result of progressive autoimmune destruction of the pancreatic beta-cells with subsequent insulin deficiency.

[0003] Type II diabetes (non-insulin dependent diabetes) represents 90-95% of the affected population, more than 100 million people worldwide. Approximately 17 million Americans suffer from type II diabetes, although 6 million do not even know they have the disease. The prevalence of the disease has jumped 33% in the last decade and is expected to rise further as the baby boomer generation gets older and more overweight. The global figure of people with diabetes is set to rise to an estimated 150 to 220 million in 2010, and 300 million in 2025. The widespread problem of diabetes has crept up on an unsuspecting health care community and has already imposed a huge burden on health-care systems (Zimmet et α/.(2001) Nature 414: 782-787).

[0004] Often, the onset of type II diabetes can be insidious, or even clinically unapparent, making diagnosis difficult. Even when the disease is properly diagnosed, many of those treated do not have adequate control over their diabetes, resulting in elevated sugar levels in the bloodstream that slowly destroys the kidneys, eyes, blood vessels and nerves. This late damage is an important factor contributing to mortality in diabetics.

[0005] Type II diabetes is associated with peripheral insulin resistance, elevated hepatic glucose production, and inappropriate insulin secretion (DeFronzo, R. A. (1988) Diabetes 37:667-687), although the primary pathogenic lesion on type II diabetes remains elusive. Many have suggested that primary insulin resistance of the peripheral tissues is the initial event. Genetic epidemiological studies have supported this view. Similarly, insulin secretion abnormalities have been argued as the primary defect in type II diabetes. It is likely that both phenomena are important in the development of type II diabetes, and genetic defects predisposing to both are likely to be important contributors to the disease process (Rimoin, D.L., et α/.(1996) Emery and Rimoin's Principles and Practice of Medical Genetics 3rd Ed. 1: 1401-1402). [0006] Evidence from familial aggregation and twins studies point to a genetic component in the etiology of diabetes (Newman et α/.(1987) Diabetologia 30:763-768; Kobberling, J. (1971) Diabetologia 7:46-49; Cook, J. T. E. (1994) Diabetologia 37:1231-1240), however, there is little agreement as to the nature of the genetic factors involved. This confusion can largely be attributed to the genetic heterogeneity known to exist in diabetes.

Summary

[0007] It has been discovered that certain polymorphic variations in human genomic DNA are associated with the occurrence of type II diabetes, also known as non-insulin dependent diabetes. In particular, polymorphic variants in a locus containing PP2Ce gene (also known as PPMlL and hereafter referred to as "PP2Ce") and B3GALT3 gene in human genomic DNA have been associated with risk of type II diabetes.

[0008] Thus, featured herein are methods for identifying a subject at risk of type II diabetes and/or a risk of type II diabetes in a subject, which comprise detecting the presence or absence of one or more polymorphic variations associated with type II diabetes in and around the locus described herein in a human nucleic acid sample. In an embodiment, two or more polymorphic variations are detected and in some embodiments, 3 or more, or 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more polymorphic variants are detected.

[0009] Also featured are nucleic acids that include one or more polymorphic variations associated with occurrence of type II diabetes, as well as polypeptides encoded by these nucleic acids. In addition, provided are methods for identifying candidate therapeutic molecules for treating type II diabetes and other insulin-related disorders, as well as methods for treating type II diabetes in a subject by identifying a subject at risk of type II diabetes and treating the subject with a suitable prophylactic, treatment or therapeutic molecule.

[0010] Also provided are compositions comprising a cell from a subject having type II diabetes or at risk of type II diabetes and/or a PP2Ce nucleic acid, with a nucleic acid having a nucleotide sequence complementary to a PP2Ce nucleotide sequence, or a RNAi, siRNA, antisense DNA or RNA, or ribozyme nucleic acid designed from a PP2Ce nucleotide sequence. In an embodiment, the RNAi, siRNA, antisense DNA or RNA, or ribozyme nucleic acid is designed from a PP2Ce nucleotide sequence that includes one or more type II diabetes associated polymorphic variations, and in some instances, specifically interacts with such a nucleotide sequence. Further, provided are arrays of nucleic acids bound to a solid surface, in which one or more nucleic acid molecules of the array have a PP2Ce nucleotide sequence, or a fragment or substantially identical nucleic acid thereof, or a complementary nucleic acid of the foregoing. Featured also are compositions comprising a cell from a subject having type II diabetes or at risk of type II diabetes and/or a PP2Ce polypeptide, with an antibody that specifically binds to the polypeptide. In an embodiment, the antibody specifically binds to an epitope in the polypeptide that includes a non-synonymous amino acid modification associated with type II diabetes (e.g., results in an amino acid substitution in the encoded polypeptide associated with type II diabetes).

[0011] Also provided are compositions comprising a cell from a subject having type II diabetes or at risk of type II diabetes and/or a B3GALT3 nucleic acid, with a nucleic acid having a nucleotide sequence complementary to a B3GALT3 nucleotide sequence, or a RNAi, siRNA, antisense DNA or RNA, or ribozyme nucleic acid designed from a B3GALT3 nucleotide sequence. In an embodiment, the RNAi, siRNA, antisense DNA or RNA, or ribozyme nucleic acid is designed from a B3GALT3 nucleotide sequence that includes one or more type II diabetes associated polymorphic variations, and in some instances, specifically interacts with such a nucleotide sequence. Further, provided are arrays of nucleic acids bound to a solid surface, in which one or more nucleic acid molecules of the array have a B3GALT3 nucleotide sequence, or a fragment or substantially identical nucleic acid thereof, or a complementary nucleic acid of the foregoing. Featured also are compositions comprising a cell from a subject having type II diabetes or at risk of type II diabetes and/or a B3GALT3 polypeptide, with an antibody that specifically binds to the polypeptide. In an embodiment, the antibody specifically binds to an epitope in the polypeptide that includes a non- synonymous amino acid modification associated with type II diabetes (e.g., results in an amino acid substitution in the encoded polypeptide associated with type II diabetes). In an embodiment, the antibody specifically binds to an epitope comprising an asparagine at position 126, or an aspartic acid at position 126, in a B3GALT3 polypeptide (SEQ ID NO: 11).

Brief Description of the Drawings

[0012] Figures 1A-1C show proximal SNP p-values (based on allelotyping results in the discovery cohort) in the PP2Ce/B3GALT3 region for females, males, and males and females combined, respectively. Figures 1D-1F show proximal SNP p-values (based on allelotyping results in the replication cohort) in the PP2Ce/B3GALT3 region for females, males, and males and females combined, respectively. Positions of each SNP on the chromosome are shown on the x-axis and the y-axis provides the negative logarithm of the p-value comparing the estimated allele to that of the control group. Also shown are exons and introns of genes in approximate chromosomal positions.

[0013] Figure 2 shows meta-analysis results for PP2Ce/B3GALT3.

Detailed Description

[0014] It has been discovered that polymorphic variants in a PP2Ce/B3GALT3 locus in human genomic DNA are associated with occurrence of type II diabetes in subjects. Thus, detecting genetic determinants in and around this locus associated with an increased risk of type II diabetes occurrence can lead to early identification of a risk of type II diabetes and early application of preventative and treatment measures. Associating the polymorphic variants with type II diabetes also has provided new targets for diagnosing type II diabetes, and methods for screening molecules useful in diabetes treatments and diabetes preventatives.

[0015] PP2Ce is a protein phosphatase believed to be involved in the IL-I -induced regulation of TAKl (Li et al . J Biol Chem. 2003 Apr 4;278(14):12013-21). Protein phosphatase 2C (PP2C) is one of the four major classes of mammalian serine/threonine specific protein phosphatases. PP2C is a monomeric enzyme of about 42 Kd which shows broad substrate specificity and is dependent on divalent cations (mainly manganese and magnesium) for its activity. Three isozymes are currently known in mammals: PP2C-alpha, beta and -gamma.

[0016] PP2C does not appear to be evolutionary related to the main family of serine/threonine phosphatases: PPl, PP2A and PP2B. However, it is significantly similar to the catalytic subunit of pyruvate dehydrogenase phosphatase (PDPC), which catalyzes dephosphorylation and concomitant reactivation of the alpha subunit of the El component of the pyruvate dehydrogenase complex. PDPC is a mitochondrial enzyme and, like PP2C, is magnesium-dependent.

[0017] The B3GALT3 gene is a member of the beta-l,3-galactosyltransferase gene family. This family encodes type II membrane-bound glycoproteins with diverse enzymatic functions using different donor substrates (UDP-galactose and UDP-N-acetylglucosamine) and different acceptor sugars (N-acetylglucosamine, galactose, N-acetylgalactosamine). Four transcript variants have been described which differ in both the 5' and 3' UTR sequences. All transcript variants encode an identical protein.

Type II Diabetes and Sample Selection

[0018] The term "type II diabetes" as used herein refers to non-insulin-dependent diabetes. Type II diabetes refers to an insulin-related disorder in which there is a relative disparity between endogenous insulin production and insulin requirements, leading to elevated hepatic glucose production, elevated blood glucose levels, inappropriate insulin secretion, and peripheral insulin resistance. Type II diabetes has been regarded as a relatively distinct disease entity, but type II diabetes is often a manifestation of a much broader underlying disorder (Zimmet et α/.(2001) Nature 414: 782-787), which may include metabolic syndrome (syndrome X), diabetes (e.g., type I diabetes, type II diabetes, gestational diabetes, autoimmune diabetes), hyperinsulinemia, hyperglycemia, impaired glucose tolerance (IGT), hypoglycemia, B-cell failure, insulin resistance, dyslipidemias, atheroma, insulinoma, hypertension, hypercoagulability, microalbuminuria, and obesity and obesity-related disorders such as visceral obesity, central fat, obesity-related type II diabetes, obesity-related atherosclerosis, heart disease, obesity-related insulin resistance, obesity- related hypertension, microangiopathic lesions resulting from obesity-related type II diabetes, ocular lesions caused by microangiopathy in obese individuals with obesity-related type II diabetes, and renal lesions caused by microangiopathy in obese individuals with obesity-related type II diabetes.

[0019] Some of the more common adult onset diabetes symptoms include fatigue, excessive thirst, frequent urination, blurred vision, a high rate of infections, wounds that heal slowly, mood changes and sexual problems. Despite these known symptoms, the onset of type II diabetes is often not discovered by health care professionals until the disease is well developed. Once identified, type II diabetes can be recognized in a patient by measuring fasting plasma glucose levels and/or casual plasma glucose levels, measuring fasting plasma insulin levels and/or casual plasma insulin levels, or administering oral glucose tolerance tests or hyperinsulinemic euglycemic clamp tests.

[0020] Based in part upon selection criteria set forth above, individuals having type II diabetes can be selected for genetic studies. Also, individuals having no history of metabolic disorders, particularly type II diabetes, often are selected for genetic studies as controls. The individuals selected for each pool of case and controls, were chosen following strict selection criteria in order to make the pools as homogenous as possible. Selection criteria for the study described herein included patient age, ethnicity, BMI, GAD (Glutamic Acid Decarboxylase) antibody concentration, and HbAIc (glycosylated hemoglobin AIc) concentration. GAD antibody is present in association with islet cell destruction, and therefore can be utilized to differentiate insulin dependent diabetes (type I diabetes) from non-insulin dependent diabetes (type II diabetes). HbAIc levels will reveal the average blood glucose over a period of 2-3 months or more specifically, over the life span of a red blood cell, by recording the number of glucose molecules attached to hemoglobin.

Polymorphic Variants Associated with Type II Diabetes

[0021] A genetic analysis provided herein linked type II diabetes with polymorphic variant nucleic acid sequences in the human genome. As used herein, the term "polymorphic site" refers to a region in a nucleic acid at which two or more alternative nucleotide sequences are observed in a significant number of nucleic acid samples from a population of individuals. A polymorphic site may be a nucleotide sequence of two or more nucleotides, an inserted nucleotide or nucleotide sequence, a deleted nucleotide or nucleotide sequence, or a microsatellite, for example. A polymorphic site that is two or more nucleotides in length may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, 20 or more, 30 or more, 50 or more, 75 or more, 100 or more, 500 or more, or about 1000 nucleotides in length, where all or some of the nucleotide sequences differ within the region. A polymorphic site is often one nucleotide in length, which is referred to herein as a "single nucleotide polymorphism" or a "SNP."

[0022] Where there are two, three, or four alternative nucleotide sequences at a polymorphic site, each nucleotide sequence is referred to as a "polymorphic variant" or "nucleic acid variant." Where two polymorphic variants exist, for example, the polymorphic variant represented in a minority of samples from a population is sometimes referred to as a "minor allele" and the polymorphic variant that is more prevalently represented is sometimes referred to as a "major allele." Many organisms possess a copy of each chromosome (e.g., humans), and those individuals who possess two major alleles or two minor alleles are often referred to as being "homozygous" with respect to the polymorphism, and those individuals who possess one major allele and one minor allele are normally referred to as being "heterozygous" with respect to the polymorphism. Individuals who are homozygous with respect to one allele are sometimes predisposed to a different phenotype as compared to individuals who are heterozygous or homozygous with respect to another allele.

[0023] In genetic analysis that associate polymorphic variants with type II diabetes, samples from individuals having type II diabetes and individuals not having type II diabetes often are allelotyped and/or genotyped. The term "allelotype" as used herein refers to a process for determining the allele frequency for a polymorphic variant in pooled DNA samples from cases and controls. By pooling DNA from each group, an allele frequency for each SNP in each group is calculated. These allele frequencies are then compared to one another. The term "genotyped" as used herein refers to a process for determining a genotype of one or more individuals, where a "genotype" is a representation of one or more polymorphic variants in a population.

[0024] A genotype or polymorphic variant may be expressed in terms of a "haplotype," which as used herein refers to two or more polymorphic variants occurring within genomic DNA in a group of individuals within a population. For example, two SNPs may exist within a gene where each SNP position includes a cytosine variation and an adenine variation. Certain individuals in a population may carry one allele (heterozygous) or two alleles (homozygous) having the gene with a cytosine at each SNP position. As the two cytosines corresponding to each SNP in the gene travel together on one or both alleles in these individuals, the individuals can be characterized as having a cytosine/cytosine haplotype with respect to the two SNPs in the gene.

[0025] As used herein, the term "phenotype" refers to a trait which can be compared between individuals, such as presence or absence of a condition, a visually observable difference in appearance between individuals, metabolic variations, physiological variations, variations in the function of biological molecules, and the like. An example of a phenotype is occurrence of type II diabetes.

[0026] Researchers sometimes report a polymorphic variant in a database without determining whether the variant is represented in a significant fraction of a population. Because a subset of these reported polymorphic variants are not represented in a statistically significant portion of the population, some of them are sequencing errors and/or not biologically relevant. Thus, it is often not known whether a reported polymorphic variant is statistically significant or biologically relevant until the presence of the variant is detected in a population of individuals and the frequency of the variant is determined. Methods for detecting a polymorphic variant in a population are described herein, specifically in Example 2. A polymorphic variant is statistically significant and often biologically relevant if it is represented in 5% or more of a population, sometimes 10% or more, 15% or more, or 20% or more of a population, and often 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more of a population.

[0027] A polymorphic variant may be detected on either or both strands of a double-stranded nucleic acid. Also, a polymorphic variant may be located within an intron or exon of a gene or within a portion of a regulatory region such as a promoter, a 5' untranslated region (UTR), a 3' UTR, and in DNA (e.g., genomic DNA (gDNA) and complementary DNA (cDNA)), RNA (e.g., mRNA, tRNA, and rRNA), or a polypeptide. Polymorphic variations may or may not result in detectable differences in gene expression, polypeptide structure, or polypeptide function.

[0028] It was determined that polymorphic variations associated with an increased risk of type II diabetes exist in a PP2Ce/B3GALT3 locus . An incident polymorphic variant described in Table IA was associated with type II diabetes.

TABLE IA

[0029] Public information pertaining to the polymorphism and the genomic sequence that includes the polymorphism are indicated. The genomic sequence identified in Table IA may be accessed at the http address www.ncbi.nih.gov/entrez/query.fcgi, for example, by using the publicly available SNP reference number (e.g., rs 1947686). The chromosome position refers to the position of the SNP within NCBFs Genome build 34, which may be accessed at the following http address: www.ncbi.nlm.nih.gov/mapview/map_search.cgi?chr=hum_chr.inf&query=. The "Contig Position" provided in Table IA corresponds to a nucleotide position set forth in the contig sequence, and designates the polymorphic site corresponding to the SNP reference number. The sequence containing the polymorphisms also may be referenced by the "Sequence Identification" set forth in Table IA. The "Sequence Identification" corresponds to cDNA sequence that encodes associated polypeptides (e.g., PP2Ce or B3GALT3) of the invention. The position of the SNP within the cDNA sequence is provided in the "Sequence Position" column of Table IA. All nucleotide sequences referenced and accessed by the parameters set forth in Table IA are incorporated herein by reference. [0030] The polymorphic variant in Table IA and others proximal to it were associated with type II diabetes. In the PP2Ce/B3GALT3 region, polymorphic variants at positions selected from the group consisting of rsl947686, 1580253, rsl580254, rs980975, rs980976, rs898681, rs898682, rs881453, rslO37888, rs898685, rs898684, rs898683, rs2306061, rs2279108, rs2279107, rs2279106, rs2290915, rslO45448, rsl3256, rs2290914, rsl599384, rs2231257, rs2231256, rs2231255, rs2231254, rs2231253, rs2231252, «2231251, rsl610158, rs 1947685, rs 1903423, rs731363 and rsl599386 were tested for association with type II diabetes. Polymorphic variants at positions rsl947686, rs898685, rs898684, rs898683, rs2306061, rs2279106, rslO45448, rsl599384, rs2231252, rs731363 and rsl599386 were associated with an increased risk of type II diabetes. At these positions, an adenine at position 45062, a thymine at position 15913, a cytosine at position 16079, a guanine at position 16109, a guanine at position 20565, a cytosine at position 22192, a cytosine at position 29417, a thymine at position 33065, an adenine at position 38415, a cytosine at position 82157, and a thymine at position 89377 were associated with risk of type II diabetes.

[0031] Based in part upon analyses summarized in Figures 1 A-IC, regions with significant association have been identified in PP2Ce/B3GALT3 locus associated with type II diabetes. Any polymorphic variants associated with type II diabetes in a region of significant association can be utilized for embodiments described herein. The following reports such regions, where "begin" and "end" designate the boundaries of the region according to chromosome positions within NCBI's Genome build 34. The chromosome on which the PP2Ce/B3GALT3 locus resides and an incident polymorphism in the locus also are noted.

COMBINED

Incident chr begin end size rs 1947686 3 162103763 162177227 73464

FEMALES Incident chr begin end size rs 1947686 3 162092323 162163165 70842

MALES

Incident chr Begin End size rsl947686 3 162103763 162117267 13504 For example, polymorphic variants in a region spanning chromosome positions 162103763 to 162177227 in the PP2Ce/B3GALT3 locus have significant association based upon a combined analysis of genetic information from males and females.

Additional Polymorphic Variants Associated with Type II Diabetes

[0032] Also provided is a method for identifying polymorphic variants proximal to an incident, founder polymorphic variant associated with type II diabetes. Thus, featured herein are methods for identifying a polymorphic variation associated with type II diabetes that is proximal to an incident polymorphic variation associated with type II diabetes, which comprises identifying a polymorphic variant proximal to the incident polymorphic variant associated with type II diabetes, where the incident polymorphic variant is in a PP2Ce/B3GALT3 nucleotide sequence. The nucleotide sequence often comprises a polynucleotide sequence selected from the group consisting of (a) a polynucleotide sequence of SEQ ID NO: 1 ; (b) a polynucleotide sequence that encodes a polypeptide having an amino acid sequence encoded by a polynucleotide sequence of SEQ ID NO: 1; and (c) a polynucleotide sequence that encodes a polypeptide having an amino acid sequence that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1 or a polynucleotide sequence 90% or more identical to the polynucleotide sequence of SEQ ID NO: 1. The presence or absence of an association of the proximal polymorphic variant with type II diabetes then is determined using a known association method, such as a method described in the Examples hereafter. In an embodiment, the incident polymorphic variant is a polymorphic variant associated with type II diabetes described herein. In another embodiment, the proximal polymorphic variant identified sometimes is a publicly disclosed polymorphic variant, which for example, sometimes is published in a publicly available database. In other embodiments, the polymorphic variant identified is not publicly disclosed and is discovered using a known method, including, but not limited to, sequencing a region surrounding the incident polymorphic variant in a group of nucleic samples. Thus, multiple polymorphic variants proximal to an incident polymorphic variant are associated with type II diabetes using this method.

[0033] The proximal polymorphic variant often is identified in a region surrounding the incident polymorphic variant. In certain embodiments, this surrounding region is about 50 kb flanking the first polymorphic variant (e.g. about 50 kb 5' of the first polymorphic variant and about 50 kb 3' of the first polymorphic variant), and the region sometimes is composed of shorter flanking sequences, such as flanking sequences of about 40 kb, about 30 kb, about 25 kb, about 20 kb, about 15 kb, about 10 kb, about 7 kb, about 5 kb, or about 2 kb 5' and 3' of the incident polymorphic variant. In other embodiments, the region is composed of longer flanking sequences, such as flanking sequences of about 55 kb, about 60 kb, about 65 kb, about 70 kb, about 75 kb, about 80 kb, about 85 kb, about 90 kb, about 95 kb, or about 100 kb 5' and 3' of the incident polymorphic variant. [0034] In certain embodiments, polymorphic variants associated with type II diabetes are identified iteratively. For example, a first proximal polymorphic variant is associated with type II diabetes using the methods described above and then another polymorphic variant proximal to the first proximal polymorphic variant is identified {e.g., publicly disclosed or discovered) and the presence or absence of an association of one or more other polymorphic variants proximal to the first proximal polymorphic variant with type II diabetes is determined.

[0035] The methods described herein are useful for identifying or discovering additional polymorphic variants that may be used to further characterize a gene, region or loci associated with a condition, a disease (e.g., type II diabetes), or a disorder. For example, allelotyping or genotyping data from the additional polymorphic variants may be used to identify a functional mutation or a region of linkage disequilibrium. In certain embodiments, polymorphic variants identified or discovered within a region comprising the first polymorphic variant associated with type II diabetes are genotyped using the genetic methods and sample selection techniques described herein, and it can be determined whether those polymorphic variants are in linkage disequilibrium with the first polymorphic variant. The size of the region in linkage disequilibrium with the first polymorphic variant also can be assessed using these genotyping methods. Thus, provided herein are methods for determining whether a polymorphic variant is in linkage disequilibrium with a first polymorphic variant associated with type II diabetes, and such information can be used in prognosis/diagnosis methods described herein.

Isolated Nucleic Acids

[0036] Featured herein are isolated PP2Ce nucleic acid variants depicted in SEQ ID NO: 1-5, isolated B3GALT3 nucleic acid variants depicted in SEQ ID NO: 10, and substantially identical , nucleic acids thereof. A nucleic acid variant may be represented on one or both strands in a double- stranded nucleic acid or on one chromosomal complement (heterozygous) or both chromosomal complements (homozygous).

[0037] As used herein, the term "nucleic acid" includes DNA molecules (e.g., a complementary DNA (cDNA) and genomic DNA (gDNA)) and RNA molecules (e.g., mRNA, rRNA, siRNA and tRNA) and analogs of DNA or RNA, for example, by use of nucleotide analogs. The nucleic acid molecule can be single-stranded and it is often double-stranded. The term "isolated or purified nucleic acid" refers to nucleic acids that are separated from other nucleic acids present in the natural source of the nucleic acid. For example, with regard to genomic DNA, the term "isolated" includes nucleic acids which are separated from the chromosome with which the genomic DNA is naturally associated. An "isolated" nucleic acid is often free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and/or 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5 ' and/or 3 ' nucleotide sequences which flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. As used herein, the term "gene" refers to a nucleotide sequence that encodes a polypeptide.

[0038] The nucleic acid often comprises a part of or all of a nucleotide sequence in SEQ ID NO: 1, 2, 3, 4 and/or 5, or a substantially identical sequence thereof. Such a nucleotide sequence sometimes is a 5' and/or 3' sequence flanking a polymorphic variant described above that is 5-10000 nucleotides in length, or in some embodiments 5-5000, 5-1000, 5-500, 5-100, 5-75, 5-50, 5-45, 5- 40, 5-35, 5-30, 5-25 or 5-20 nucleotides in length.

[0039] Also included herein are nucleic acid fragments. These fragments often are a nucleotide sequence identical to a nucleotide sequence of SEQ ID NO: 1-5 or 10, a nucleotide sequence substantially identical to a nucleotide sequence of SEQ ID NO: 1-5 or 10, or a nucleotide sequence that is complementary to the foregoing. The nucleic acid fragment may be identical, substantially identical or homologous to a nucleotide sequence in an exon or an intron in a nucleotide sequence of SEQ ID NO: 1, and may encode a domain or part of a domain of a polypeptide. Sometimes, the fragment will comprises one or more of the polymorphic variations described herein as being associated with type II diabetes. The nucleic acid fragment is often 50, 100, or 200 or fewer base pairs in length, and is sometimes about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 3000, 4000, 5000, 10000, 15000, or 20000 base pairs in length. A nucleic acid fragment that is complementary to a nucleotide sequence identical or substantially identical to a nucleotide sequence in SEQ ID NO: 1-5 or 10 and hybridizes to such a nucleotide sequence under stringent conditions is often referred to as a "probe." Nucleic acid fragments often include one or more polymorphic sites, or sometimes have an end that is adjacent to a polymorphic site as described hereafter.

[0040] An example of a nucleic acid fragment is an oligonucleotide. As used herein, the term "oligonucleotide" refers to a nucleic acid comprising about 8 to about 50 covalently linked nucleotides, often comprising from about 8 to about 35 nucleotides, and more often from about 10 to about 25 nucleotides. The backbone and nucleotides within an oligonucleotide may be the same as those of naturally occurring nucleic acids, or analogs or derivatives of naturally occurring nucleic acids, provided that oligonucleotides having such analogs or derivatives retain the ability to hybridize specifically to a nucleic acid comprising a targeted polymorphism. Oligonucleotides described herein may be used as hybridization probes or as components of prognostic or diagnostic assays, for example, as described herein. [0041] Oligonucleotides are typically synthesized using standard methods and equipment, such as the ABF^M3900 High Throughput DNA Synthesizer and the EXPEDITE™ 8909 Nucleic Acid Synthesizer, both of which are available from Applied Biosystems (Foster City, CA). Analogs and derivatives are exemplified in U.S. Pat. Nos. 4,469,863; 5,536,821; 5,541,306; 5,637,683; 5,637,684; 5,700,922; 5,717,083; 5,719,262; 5,739,308; 5,773,601; 5,886,165; 5,929,226; 5,977,296; 6,140,482; WO 00/56746; WO 01/14398, and related publications. Methods for synthesizing oligonucleotides comprising such analogs or derivatives are disclosed, for example, in the patent publications cited above and in U.S. Pat. Nos. 5,614,622; 5,739,314; 5,955,599; 5,962,674; 6,117,992; in WO 00/75372; and in related publications.

[0042] Oligonucleotides may also be linked to a second moiety. The second moiety may be an additional nucleotide sequence such as a tail sequence (e.g., a polyadenosine tail), an adapter sequence (e.g., phage M 13 universal tail sequence), and others. Alternatively, the second moiety may be a non-nucleotide moiety such as a moiety which facilitates linkage to a solid support or a label to facilitate detection of the oligonucleotide. Such labels include, without limitation, a radioactive label, a fluorescent label, a chemiluminescent label, a paramagnetic label, and the like. The second moiety may be attached to any position of the oligonucleotide, provided the oligonucleotide can hybridize to the nucleic acid comprising the polymorphism.

Uses for Nucleic Acid Sequence

[0043] Nucleic acid coding sequences (e.g., SEQ ID NO: 2-5, 10) may be used for diagnostic purposes for detection and control of polypeptide expression. Also, included herein are oligonucleotide sequences such as antisense RNA, small-interfering RNA (siRNA) and DNA molecules and ribozymes that function to inhibit translation of a polypeptide. Antisense techniques and RNA interference techniques are known in the art and are described herein.

[0044] Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. For example, hammerhead motif ribozyme molecules may be engineered that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences corresponding to or complementary to PP2Ce nucleotide sequences. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between fifteen (15) and twenty (20) ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.

[0045] Antisense RNA and DNA molecules, siRNA and ribozymes may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

[0046] DNA encoding a polypeptide also may have a number of uses for the diagnosis of diseases, including type II diabetes, resulting from aberrant expression of a target gene described herein. For example, the nucleic acid sequence may be used in hybridization assays of biopsies or autopsies to diagnose abnormalities of expression or function (e.g., Southern or Northern blot analysis, in situ hybridization assays).

[0047] In addition, the expression of a polypeptide during embryonic development may also be determined using nucleic acid encoding the polypeptide. As addressed, infra, production of functionally impaired polypeptide is the cause of various disease states, such as type II diabetes. In situ hybridizations using polypeptide as a probe may be employed to predict problems related to type II diabetes. Further, as indicated, infra, administration of human active polypeptide, recombinantly produced as described herein, may be used to treat disease states related to functionally impaired polypeptide. Alternatively, gene therapy approaches may be employed to remedy deficiencies of functional polypeptide or to replace or compete with dysfunctional polypeptide.

Expression Vectors, Host Cells, and Genetically Engineered Cells

[0048] Provided herein are nucleic acid vectors, often expression vectors, which contain a PP2Ce or B3GALT3 nucleotide sequence or a substantially identical sequence thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid, or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors may include replication defective retroviruses, adenoviruses and adeno-associated viruses for example. [0049] A vector can include a PP2Ce or B3GALT3 nucleotide sequence in a form suitable for expression of an encoded target polypeptide or target nucleic acid in a host cell. A "target polypeptide" is a polypeptide encoded by a PP2Ce or B3GALT3 nucleotide sequence or a substantially identical nucleotide sequence thereof. The recombinant expression vector typically includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, and the like. Expression vectors can be introduced into host cells to produce target polypeptides, including fusion polypeptides.

[0050] Recombinant expression vectors can be designed for expression of target polypeptides in prokaryotic or eukaryotic cells. For example, target polypeptides can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0051] Expression of polypeptides in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant polypeptide; 2) to increase the solubility of the recombinant polypeptide; and 3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith & Johnson, Gene 67: 31-40 (1988)), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.

[0052] Purified fusion polypeptides can be used in screening assays and to generate antibodies specific for target polypeptides. In a therapeutic embodiment, fusion polypeptide expressed in a retroviral expression vector is used to infect bone marrow cells that are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks). [0053] Expressing the polypeptide in host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide is often used to maximize recombinant polypeptide expression (Gottesman, S., Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, California 185: 119-128 (1990)). Another strategy is to alter the nucleotide sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et ai, Nucleic Acids Res. 20: 2111-2118 (1992)). Such alteration of nucleotide sequences can be carried out by standard DNA synthesis techniques.

[0054] When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. Recombinant mammalian expression vectors are often capable of directing expression of the nucleic acid in a particular cell type {e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include an albumin promoter (liver-specific; Pinkert et ai, Genes Dev. 1: 268-277 (1987)), lymphoid-specific promoters (Calame & Eaton, Adv. Immunol. 43: 235-275 (1988)), promoters of T cell receptors (Winoto & Baltimore, EMBOJ. 8: 729-733 (1989)) promoters of immunoglobulins (Banerji et al.Cell 33: 729-740 (1983); Queen & Baltimore, Cell 33: 741-748 (1983)), neuron-specific promoters (e.g., the neurofilament promoter; Byrne & Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477 (1989)), pancreas-specific promoters (Edlund et al., Science 230: 912-916 (1985)), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are sometimes utilized, for example, the murine hox promoters (Kessel & Gruss, Science 249: T>1A-?>19 (1990)) and the alpha-fetopolypeptide promoter (Campes & Tilghman, Genes Dev. 3: 537-546 (1989)).

[0055] A PP2Ce or B3GALT3 nucleic acid may also be cloned into an expression vector in an antisense orientation. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a PP2Ce or B3GALT3 nucleic acid cloned in the antisense orientation can be chosen for directing constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types. Antisense expression vectors can be in the form of a recombinant plasmid, phagemid or attenuated virus. For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub et al., Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) (1986).

[0056] Also provided herein are host cells that include a PP2Ce or B3GALT3 nucleotide sequence within a recombinant expression vector or a fragment of such a nucleotide sequence which facilitate homologous recombination into a specific site of the host cell genome. The terms

"host cell" and "recombinant host cell" are used interchangeably herein. Such terms refer not only to the particular subject cell but rather also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic or eukaryotic cell. For example, a target polypeptide can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

[0057] Vectors can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, transduction/infection, DEAE-dextran-mediated transfection, lipofection, or electroporation.

[0058] A host cell provided herein can be used to produce (i.e., express) a target polypeptide or a substantially identical polypeptide thereof. Accordingly, further provided are methods for producing a target polypeptide using host cells described herein. In one embodiment, the method includes culturing host cells into which a recombinant expression vector encoding a target polypeptide has been introduced in a suitable medium such that a target polypeptide is produced. In another embodiment, the method further includes isolating a target polypeptide from the medium or the host cell.

[0059] Also provided are cells or purified preparations of cells which include a PP2Ce or B3GALT3 transgene, or which otherwise misexpress target polypeptide. Cell preparations can consist of human or non-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a PP2Ce or B3GALT3 transgene (e.g., a heterologous form of a PP2Ce or B3GALT3 gene, such as a human gene expressed in non-human cells). The transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene which misexpress an endogenous target polypeptide (e.g., expression of a gene is disrupted, also known as a knockout). Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed alleles or for use in drug screening. Also provided are human cells (e.g., a hematopoietic stem cells) transformed with a PP2Ce or B3GALT3 nucleic acid.

[0060] Also provided are cells or a purified preparation thereof (e.g., human cells) in which an endogenous PP2Ce or B3GALT3 nucleic acid is under the control of a regulatory sequence that does not normally control the expression of the endogenous gene corresponding to a PP2Ce or B3GALT3 nucleotide sequence. The expression characteristics of an endogenous gene within a cell (e.g., a cell line or microorganism) can be modified by inserting a heterologous DNA regulatory element into the genome of the cell such that the inserted regulatory element is operably linked to the corresponding endogenous gene. For example, an endogenous corresponding gene (e.g. , a gene which is "transcriptionally silent," not normally expressed, or expressed only at very low levels) may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell. Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g., Chappel, US 5,272,071; WO 91/06667, published on May 16, 1991.

Transgenic Animals

[0061] Non-human transgenic animals that express a heterologous target polypeptide {e.g., expressed from a PP2Ce or B3GALT3 nucleic acid or substantially identical sequence thereof) can be generated. Such animals are useful for studying the function and/or activity of a target polypeptide and for identifying and/or evaluating modulators of the activity of PP2Ce or B3GALT3 nucleic acids and encoded polypeptides. As used herein, a "transgenic animal" is a non-human animal such as a mammal (e.g., a non-human primate such as chimpanzee, baboon, or macaque; an ungulate such as an equine, bovine, or caprine; or a rodent such as a rat, a mouse, or an Israeli sand rat), a bird (e.g., a chicken or a turkey), an amphibian (e.g., a frog, salamander, or newt), or an insect (e.g., Drosophila melanogaster), in which one or more of the cells of the animal includes a transgene. A transgene is exogenous DNA or a rearrangement (e.g., a deletion of endogenous chromosomal DNA) that is often integrated into or occurs in the genome of cells in a transgenic animal. A transgene can direct expression of an encoded gene product in one or more cell types or tissues of the transgenic animal, and other transgenes can reduce expression (e.g., a knockout). Thus, a transgenic animal can be one in which an endogenous nucleic acid homologous to a PP2Ce or B3GALT3 nucleic acid has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal (e.g., an embryonic cell of the animal) prior to development of the animal.

[0062] Intronic sequences and polyadenylation signals can also be included in the transgene to increase expression efficiency of the transgene. One or more tissue-specific regulatory sequences can be operably linked to a PP2Ce or B3GALT3 nucleotide sequence to direct expression of an encoded polypeptide to particular cells. A transgenic founder animal can be identified based upon the presence of a PP2Ce or B3GALT3 nucleotide sequence in its genome and/or expression of encoded mRNA in tissues or cells of the animals. A transgenic founder animal can then be used'to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a PP2Ce or B3GALT3 nucleotide sequence can further be bred to other transgenic animals carrying other transgenes.

[0063] Target polypeptides can be expressed in transgenic animals or plants by introducing, for example, a PP2Ce or B3GALT3 nucleic acid into the genome of an animal that encodes the target polypeptide. In preferred embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Also included is a population of cells from a transgenic animal.

Target Polypeptides

[0064] Also featured herein are isolated target polypeptides, which are encoded by a PP2Ce nucleotide sequence (e.g., SEQ ID NO: 1-5 or 10) or a substantially identical nucleotide sequence thereof, such as the polypeptides having amino acid sequences in SEQ ID NO: 6-9 or 11. The term "polypeptide" as used herein includes proteins and peptides. An "isolated" or "purified" polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. In one embodiment, the language "substantially free" means preparation of a target polypeptide having less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-target polypeptide (also referred to herein as a "contaminating protein"), or of chemical precursors or non-target chemicals. When the target polypeptide or a biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, specifically, where culture medium represents less than about 20%, sometimes less than about 10%, and often less than about 5% of the volume of the polypeptide preparation. Isolated or purified target polypeptide preparations are sometimes 0.01 milligrams or more or 0.1 milligrams or more, and often 1.0 milligrams or more and 10 milligrams or more in dry weight.

[0065] Further included herein are target polypeptide fragments. The polypeptide fragment may be a domain or part of a domain of a target polypeptide. For example, PP2Ce domains are indicated in Table IB below and the BSGALTS domains consist of the galactosy transferases domain from amino acids 33-329 of SEQ ID NO: 11. The polypeptide fragment may have increased, decreased or unexpected biological activity. The polypeptide fragment is often 50 or fewer, 100 or fewer, or 200 or fewer amino acids in length, and is sometimes 300, 400, 500, 600, 700, or 900 or fewer amino acids in length.

TABLE IB

[0066] Substantially identical target polypeptides may depart from the amino acid sequences of target polypeptides in different manners. For example, conservative amino acid modifications may be introduced at one or more positions in the amino acid sequences of target polypeptides. A "conservative amino acid substitution" is one in which the amino acid is replaced by another amino acid having a similar structure and/or chemical function. Families of amino acid residues having similar structures and functions are well known. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Also, essential and non-essential amino acids may be replaced. A "non-essential" amino acid is one that can be altered without abolishing or substantially altering the biological function of a target polypeptide, whereas altering an "essential" amino acid abolishes or substantially alters the biological function of a target polypeptide. Amino acids that are conserved among target polypeptides are typically essential amino acids.

[0067] Also, target polypeptides may exist as chimeric or fusion polypeptides. As used herein, a target "chimeric polypeptide" or target "fusion polypeptide" includes a target polypeptide linked to a non-target polypeptide. A "non-target polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a polypeptide which is not substantially identical to the target polypeptide, which includes, for example, a polypeptide that is different from the target polypeptide and derived from the same or a different organism. The target polypeptide in the fusion polypeptide can correspond to an entire or nearly entire target polypeptide or a fragment thereof. The non-target polypeptide can be fused to the N-terminus or C-terminus of the target polypeptide.

[0068] Fusion polypeptides can include a moiety having high affinity for a ligand. For example, the fusion polypeptide can be a GST-target fusion polypeptide in which the target sequences are fused to the C-terminus of the GST sequences, or a polyhistidine-target fusion polypeptide in which the target polypeptide is fused at the N- or C-terminus to a string of histidine residues. Such fusion polypeptides can facilitate purification of recombinant target polypeptide. Expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide), and a nucleotide sequence in SEQ ID NO: 1-5 OR 10, or a substantially identical nucleotide sequence thereof, can be cloned into an expression vector such that the fusion moiety is linked in-frame to the target polypeptide. Further, the fusion polypeptide can be a target polypeptide containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression, secretion, cellular internalization, and cellular localization of a target polypeptide can be increased through use of a heterologous signal sequence. Fusion polypeptides can also include all or a part of a serum polypeptide (e.g., an IgG constant region or human serum albumin).

[0069] Target polypeptides can be incorporated into pharmaceutical compositions and administered to a subject in vivo. Administration of these target polypeptides can be used to affect the bioavailability of a substrate of the target polypeptide and may effectively increase target polypeptide biological activity in a cell. Target fusion polypeptides may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a target polypeptide; (ii) mis-regulation of the gene encoding the target polypeptide; and (iii) aberrant post-translational modification of a target polypeptide. Also, target polypeptides can be used as immunogens to produce anti-target antibodies in a subject, to purify target polypeptide ligands or binding partners, and in screening assays to identify molecules which inhibit or enhance the interaction of a target polypeptide with a substrate.

[0070] In addition, polypeptides can be chemically synthesized using techniques known in the art (See, e.g., Creighton, 1983 Proteins. New York, N. Y.: W. H. Freeman and Company; and Hunkapiller et α/.,(1984) Nature July 12 -18;310(5973):105-l 1). For example, a relative short fragment can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the fragment sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3- amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b- alanine, fluoroamino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

[0071] Polypeptides and polypeptide fragments sometimes are differentially modified during or after translation, e.g. , by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; and the like. Additional post-translational modifications include, for example, N-linked or O-linked carbohydrate chains, processing of N- terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The polypeptide fragments may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the polypeptide.

[0072] Also provided are chemically modified derivatives of polypeptides that can provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see e.g., U.S. Pat. No: 4,179,337. The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

[0073] The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile {e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).

[0074] The polymers should be attached to the polypeptide with consideration of effects on functional or antigenic domains of the polypeptide. There are a number of attachment methods available to those skilled in the art (e.g., EP 0 401 384 (coupling PEG to G-CSF) and Malik et al.(\992) Exp Hematol. September 20(8): 1028-35 (pegylation of GM-CSF using tresyl chloride)). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. For therapeutic purposes, the attachment sometimes is at an amino group, such as attachment at the N-terminus or lysine group.

[0075] Proteins can be chemically modified at the N-terminus. Using polyethylene glycol as an illustration of such a composition, one may select from a variety of polyethylene glycol molecules

(by molecular weight, branching, and the like), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N- terminus may be accomplished by reductive alkylation, which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

Substantially Identical Nucleic Acids and Polypeptides

[0076] Nucleotide sequences and polypeptide sequences that are substantially identical to a PP2Ce nucleotide sequence and the target polypeptide sequences encoded by those nucleotide sequences, respectively, are included herein. The term "substantially identical" as used herein refers to two or more nucleic acids or polypeptides sharing one or more identical nucleotide sequences or polypeptide sequences, respectively. Included are nucleotide sequences or polypeptide sequences that are 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more (each often within a 1%, 2%, 3% or 4% variability) identical to a PP2Ce nucleotide^equence or the encoded target polypeptide amino acid sequences. One test for determining whether two nucleic acids are substantially identical is to determine the percent of identical nucleotide sequences or polypeptide sequences shared between the nucleic acids or polypeptides.

[0077] Calculations of sequence identity are often performed as follows. Sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is sometimes 30% or more, 40% or more, 50% or more, often 60% or more, and more often 70% or more, 80% or more, 90% or more, or 100% of the length of the reference sequence. The nucleotides or amino acids at corresponding nucleotide or polypeptide positions, respectively, are then compared among the two sequences. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, the nucleotides or amino acids are deemed to be identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, introduced for optimal alignment of the two sequences. [0078] Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. Percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers & Miller, CABIOS 4: 11-17 (1989), which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Also, percent identity between two amino acid sequences can be determined using the Needleman & Wunsch, J. MoI. Biol. 48: 444-453 (1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at the http address www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http address www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A set of parameters often used is a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0079] Another manner for determining if two nucleic acids are substantially identical is to assess whether a polynucleotide homologous to one nucleic acid will hybridize to the other nucleic acid under stringent conditions. As use herein, the term "stringent conditions" refers to conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. , 6.3.1-6.3.6 (1989). Aqueous and non-aqueous methods are described in that reference and either can be used. An example of stringent hybridization conditions is hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45⁰C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50⁰C. Another example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 55⁰C. A further example of stringent hybridization conditions is hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45⁰C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 6O⁰C. Often, stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65⁰C. More often, stringency conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C.

[0080] An example of a substantially identical nucleotide sequence to a nucleotide sequence in SEQ ID NO: 1-5 or 10 is one that has a different nucleotide sequence but still encodes the same polypeptide sequence encoded by the nucleotide sequence in SEQ ID NO: 1-5 or 10. Another example is a nucleotide sequence that encodes a polypeptide having a polypeptide sequence that is more than 70% or more identical to, sometimes more than 75% or more, 80% or more, or 85% or more identical to, and often more than 90% or more and 95% or more identical to a polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO: 1-5 or 10. As used herein, "SEQ ID NO: 1-5 or 10" typically refers to one or more sequences in SEQ ID NO: 1, 2, 3, 4, 5 and/or 10. Many of the embodiments described herein are applicable to (a) a nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5 and/or 10; (b) a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1, 2, 3, 4 and/or 5; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5 and/or 10, or a nucleotide sequence about 90% or more identical to a nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5 and/or 10; (d) a fragment of a nucleotide sequence of (a), (b), or (c); and/or a nucleotide sequence complementary to the nucleotide sequences of (a), (b), (c) and/or (d), where nucleotide sequences of (b) and (c), fragments of (b) and (c) and nucleotide sequences complementary to (b) and (c) are examples of substantially identical nucleotide sequences. Examples of substantially identical nucleotide sequences include nucleotide sequences from subjects that differ by naturally occurring genetic variance, which sometimes is referred to as background genetic variance (e.g., nucleotide sequences differing by natural genetic variance sometimes are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one another).

[0081] Nucleotide sequences in SEQ ID NO: 1-5 or 10 and amino acid sequences of encoded polypeptides can be used as "query sequences" to perform a search against public databases to identify other family members or related sequences, for example. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al, J. MoI. Biol. 215: 403- 10 (1990). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleotide sequences in SEQ ID NO: 1-5 or 10. BLAST polypeptide searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to polypeptides encoded by the nucleotide sequences of SEQ ID NO: 1-5 or 10. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et ah, Nucleic Acids Res. 25(17): 3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see the http address www.ncbi.nlm.nih.gov).

[0082) A nucleic acid that is substantially identical to a nucleotide sequence in SEQ ID NO: 1-5 or 10 may include polymorphic sites at positions equivalent to those described herein when the sequences are aligned. For example, using the alignment procedures described herein, SNPs in a sequence substantially identical to a sequence in SEQ ID NO: 1-5 or 10 can be identified at nucleotide positions that match or correspond (e.g., align) with nucleotides at SNP positions in each nucleotide sequence in SEQ ID NO: 1-5 or 10. Also, where a polymorphic variation results in an insertion or deletion, insertion or deletion of a nucleotide sequence from a reference sequence can change the relative positions of other polymorphic sites in the nucleotide sequence.

[0083] Substantially identical nucleotide and polypeptide sequences include those that are naturally occurring, such as allelic variants (same locus), splice variants, homologs (different locus), and orthologs (different organism) or can be non-naturally occurring. Non-naturally occurring variants can be generated by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product). Orthologs, homologs, allelic variants, and splice variants can be identified using methods known in the art. These variants normally comprise a nucleotide sequence encoding a polypeptide that is 50% or more, about 55% or more, often about 70-75% or more or about 80-85% or more, and sometimes about 90-95% or more identical to the amino acid sequences of target polypeptides or a fragment thereof. Such nucleic acid molecules can readily be identified as being able to hybridize under stringent conditions to a nucleotide sequence in SEQ ID NO: 1-5 or 10 or a fragment of this sequence. Nucleic acid molecules corresponding to orthologs, homologs, and allelic variants of a nucleotide sequence in SEQ ID NO: 1-5 or 10 can further be identified by mapping the sequence to the same chromosome or locus as the nucleotide sequence in SEQ ID NO: 1-5 or 10.

[0084] Also, substantially identical nucleotide sequences may include codons that are altered with respect to the naturally occurring sequence for enhancing expression of a target polypeptide in a particular expression system. For example, the nucleic acid can be one in which one or more codons are altered, and often 10% or more or 20% or more of the codons are altered for optimized expression in bacteria (e.g., E. coli.), yeast (e.g., S. cervesiae), human (e.g., 293 cells), insect, or , rodent (e.g., hamster) cells.

Methods for Identifying Subjects at Risk of Diabetes and Risk of Diabetes in a Subject [0085] Methods for prognosing and diagnosing type II diabetes and its related disorders (e.g., metabolic disorders, syndrome X, obesity, insulin resistance, hyperglycemia) are included herein. These methods include detecting the presence or absence of one or more polymorphic variations in a nucleotide sequence associated with type II diabetes, such as variants in or around the locus set forth herein, or a substantially identical sequence thereof, in a sample from a subject, where the presence of a polymorphic variant described herein is indicative of a risk of type II diabetes or one or more type II diabetes related disorders (e.g., metabolic disorders, syndrome X, obesity, insulin resistance, hyperglycemia). Determining a risk of type II diabetes refers to determining whether an individual is at an increased risk of type II diabetes (e.g., intermediate risk or higher risk). [0086] Thus, featured herein is a method for identifying a subject who is at risk of type II diabetes, which comprises detecting a type II diabetes-associated aberration in a nucleic acid sample from the subject. An embodiment is a method for detecting a risk of type II diabetes in a subject, which comprises detecting the presence or absence of a polymorphic variation associated with type II diabetes at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject, where the nucleotide sequence comprises a polynucleotide sequence selected from the group consisting of: (a) a nucleotide sequence of SEQ ID NO: 1-5 or 10; (b) a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-5 or 10; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-5 or 10, or a nucleotide sequence about 90% or more identical to a nucleotide sequence of SEQ ID NO: 1-5 or 10; and (d) a fragment of a nucleotide sequence of (a), (b), or (c) comprising the polymorphic site; whereby the presence of the polymorphic variation is indicative of a predisposition to type II diabetes in the subject. In certain embodiments, polymorphic variants at the positions described herein are detected for determining a risk of type II diabetes, and polymorphic variants at positions in linkage disequilibrium with these positions are detected for determining a risk of type II diabetes.

[0087] Results from prognostic tests may be combined with other test results to diagnose type II diabetes related disorders, including metabolic disorders, syndrome X, obesity, insulin resistance, hyperglycemia. For example, prognostic results may be gathered, a patient sample may be ordered based on a determined predisposition to type II diabetes, the patient sample is analyzed, and the results of the analysis may be utilized to diagnose the type II diabetes related condition (e.g., metabolic disorders, syndrome X, obesity, insulin resistance, hyperglycemia). Also type II diabetes diagnostic methods can be developed from studies used to generate prognostic methods in which populations are stratified into subpopulations having different progressions of a type II diabetes related disorder or condition. In another embodiment, prognostic results may be gathered, a patient's risk factors for developing type II diabetes (e.g., age, weight, race, diet) analyzed, and a patient sample may be ordered based on a determined predisposition to type II diabetes.

[0088] Risk of type II diabetes sometimes is expressed as a probability, such as an odds ratio, percentage, or risk factor. The risk sometimes is expressed as a relative risk with respect to a population average risk of type II diabetes, and sometimes is expressed as a relative risk with respect to the lowest risk group. Such relative risk assessments often are based upon penetrance values determined by statistical methods and are particularly useful to clinicians and insurance companies for assessing risk of type II diabetes (e.g., a clinician can target appropriate detection, prevention and therapeutic regimens to a patient after determining the patient's risk of type II diabetes, and an insurance company can fine tune actuarial tables based upon population genotype assessments of type II diabetes risk). Risk of type II diabetes sometimes is expressed as an odds ratio, which is the odds of a particular person having a genotype has or will develop type II diabetes with respect to another genotype group (e.g., the most disease protective genotype or population average). The risk often is based upon the presence or absence of one or more polymorphic variants described herein, and also may be based in part upon phenotypic traits of the individual being tested. In an embodiment, two or more polymorphic variations are detected in a PP2Ce/B3GALT3 locus. In certain embodiments, 3 or more, or 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more polymorphic variants are detected in the sample. Methods for calculating risk based upon patient data are well known (see, e.g., Agresti, Categorical Data Analysis, 2nd Ed. 2002. Wiley). Allelotyping and genotyping analyses may be carried out in populations other than those exemplified herein to enhance the predictive power of the prognostic method.

[0089] The nucleic acid sample typically is isolated from a biological sample obtained from a subject. For example, nucleic acid can be isolated from blood, saliva, sputum, urine, cell scrapings, and biopsy tissue. The nucleic acid sample can be isolated from a biological sample using standard techniques, such as the technique described in Example 2. As used herein, the term "subject" refers primarily to humans but also refers to other mammals such as dogs, cats, and ungulates (e.g., cattle, sheep, and swine). Subjects also include avians (e.g., chickens and turkeys), reptiles, and fish (e.g., salmon), as embodiments described herein can be adapted to nucleic acid samples isolated from any of these organisms. The nucleic acid sample may be isolated from the subject and then directly utilized in a method for determining the presence of a polymorphic variant, or alternatively, the sample may be isolated and then stored (e.g., frozen) for a period of time before being subjected to analysis.

[0090] The presence or absence of a polymorphic variant is determined using one or both chromosomal complements represented in the nucleic acid sample. Determining the presence or absence of a polymorphic variant in both chromosomal complements represented in a nucleic acid sample from a subject having a copy of each chromosome is useful for determining the zygosity of an individual for the polymorphic variant (i.e., whether the individual is homozygous or heterozygous for the polymorphic variant). Any oligonucleotide-based diagnostic may be utilized to determine whether a sample includes the presence or absence of a polymorphic variant in a sample. For example, primer extension methods, ligase sequence determination methods (e.g., U.S. Pat. Nos. 5,679,524 and 5,952,174, and WO 01/27326), mismatch sequence determination methods (e.g., U.S. Pat. Nos. 5,851,770; 5,958,692; 6,110,684; and 6,183,958), microarray sequence determination methods, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism detection (SSCP) (e.g., U.S. Pat. Nos. 5,891,625 and 6,013,499), PCR- based assays (e.g., TAQMAN^® PCR System (Applied Biosystems)), and nucleotide sequencing methods may be used.

[0091] Oligonucleotide extension methods typically involve providing a pair of oligonucleotide primers in a polymerase chain reaction (PCR) or in other nucleic acid amplification methods for the purpose of amplifying a region from the nucleic acid sample that comprises the polymorphic variation. One oligonucleotide primer is complementary to a region 3' of the polymorphism and the other is complementary to a region 5' of the polymorphism. A PCR primer pair may be used in methods disclosed in U.S. Pat. Nos. 4,683,195; 4,683,202, 4,965,188; 5,656,493; 5,998,143; 6,140,054; WO 01/27327; and WO 01/27329 for example. PCR primer pairs may also be used in any commercially available machines that perform PCR, such as any of the GENEAMP^® Systems available from Applied Biosystems. Also, those of ordinary skill in the art will be able to design oligonucleotide primers based upon a PP2Ce or B3GALT3nυc\eotide sequence using knowledge available in the art.

[0092] Also provided is an extension oligonucleotide that hybridizes to the amplified fragment adjacent to the polymorphic variation. As used herein, the term "adjacent" refers to the 3' end of the extension oligonucleotide being often 1 nucleotide from the 5' end of the polymorphic site, and sometimes 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5' end of the polymorphic site, in the nucleic acid when the extension oligonucleotide is hybridized to the nucleic acid. The extension oligonucleotide then is extended by one or more nucleotides, and the number and/or type of nucleotides that are added to the extension oligonucleotide determine whether the polymorphic variant is present. Oligonucleotide extension methods are disclosed, for example, in U.S. Pat. Nos. 4,656,127; 4,851,331; 5,679,524; 5,834,189; 5,876,934; 5,908,755; 5,912,118; 5,976,802; 5,981,186; 6,004,744; 6,013,431; 6,017,702; 6,046,005; 6,087,095; 6,210,891; and WO 01/20039. Oligonucleotide extension methods using mass spectrometry are described, for example, in U.S. Pat. Nos. 5,547,835; 5,605,798; 5,691,141; 5,849,542; 5,869,242; 5,928,906; 6,043,031; and 6,194,144, and a method often utilized is described herein in Example 2.

[0093] A microarray can be utilized for determining whether a polymorphic variant is present or absent in a nucleic acid sample. A microarray may include any oligonucleotides described herein, and methods for making and using oligonucleotide microarrays suitable for diagnostic use are disclosed in U.S. Pat. Nos. 5,492,806; 5,525,464; 5,589,330; 5,695,940; 5,849,483; 6,018,041; 6,045,996; 6,136,541; 6,142,681; 6,156,501; 6,197,506; 6,223,127; 6,225,625; 6,229,911; 6,239,273; WO 00/52625; WO 01/25485; and WO 01/29259. The microarray typically comprises a solid support and the oligonucleotides may be linked to this solid support by covalent bonds or by non-covalent interactions. The oligonucleotides may also be linked to the solid support directly or by a spacer molecule. A microarray may comprise one or more oligonucleotides complementary to a polymorphic site set forth herein. [0094] A kit also may be utilized for determining whether a polymorphic variant is present or absent in a nucleic acid sample. A kit often comprises one or more pairs of oligonucleotide primers useful for amplifying a fragment of a nucleotide sequence of SEQ ID NO: 1-5 or 10 or a substantially identical sequence thereof, where the fragment includes a polymorphic site. The kit sometimes comprises a polymerizing agent, for example, a thermostable nucleic acid polymerase such as one disclosed in U.S. Pat. Nos. 4,889,818 or 6,077,664. Also, the kit often comprises an elongation oligonucleotide that hybridizes to a PP2Ce or B3GALT3 nucleotide sequence in a nucleic acid sample adjacent to the polymorphic site. Where the kit includes an elongation oligonucleotide, it also often comprises chain elongating nucleotides, such as dATP, dTTP, dGTP, dCTP, and dITP, including analogs of dATP, dTTP, dGTP, dCTP and dITP, provided that such analogs are substrates for a thermostable nucleic acid polymerase and can be incorporated into a nucleic acid chain elongated from the extension oligonucleotide. Along with chain elongating nucleotides would be one or more chain terminating nucleotides such as ddATP, ddTTP, ddGTP, ddCTP, and the like. In an embodiment, the kit comprises one or more oligonucleotide primer pairs, a polymerizing agent, chain elongating nucleotides, at least one elongation oligonucleotide, and one or more chain terminating nucleotides. Kits optionally include buffers, vials, microtiter plates, and instructions for use.

[0095] An individual identified as being at risk of type II diabetes may be heterozygous or homozygous with respect to the allele associated with a higher risk of type II diabetes. A subject homozygous for an allele associated with an increased risk of type II diabetes is at a comparatively high risk of type II diabetes, a subject heterozygous for an allele associated with an increased risk of type II diabetes is at a comparatively intermediate risk of type II diabetes, and a subject homozygous for an allele associated with a decreased risk of type II diabetes is at a comparatively low risk of type II diabetes. A genotype may be assessed for a complementary strand, such that the complementary nucleotide at a particular position is detected.

[0096] Also featured are methods for determining risk of type II diabetes and/or identifying a subject at risk of type II diabetes by contacting a polypeptide or protein encoded by a PP2Ce or BSGAL ^nucleotide sequence from a subject with an antibody that specifically binds to an epitope associated with increased risk of type II diabetes in the polypeptide.

Applications of Prognostic and Diagnostic Results to Pharmacogenomic Methods [0097] Pharmacogenomics is a discipline that involves tailoring a treatment for a subject according to the subject's genotype as a particular treatment regimen may exert a differential effect depending upon the subject's genotype. For example, based upon the outcome of a prognostic test described herein, a clinician or physician may target pertinent information and preventative or therapeutic treatments to a subject who would be benefited by the information or treatment and avoid directing such information and treatments to a subject who would not be benefited (e.g., the treatment has no therapeutic effect and/or the subject experiences adverse side effects).

[0098] The following is an example of a pharmacogenomic embodiment. A particular treatment regimen can exert a differential effect depending upon the subject's genotype. Where a candidate therapeutic exhibits a significant interaction with a major allele and a comparatively weak interaction with a minor allele (e.g., an order of magnitude or greater difference in the interaction), such a therapeutic typically would not be administered to a subject genotyped as being homozygous for the minor allele, and sometimes not administered to a subject genotyped as being heterozygous for the minor allele. In another example, where a candidate therapeutic is not significantly toxic when administered to subjects who are homozygous for a major allele but is comparatively toxic when administered to subjects heterozygous or homozygous for a minor allele, the candidate therapeutic is not typically administered to subjects who are genotyped as being heterozygous or homozygous with respect to the minor allele.

[0099] The methods described herein are applicable to pharmacogenomic methods for preventing, alleviating or treating type II diabetes conditions such as metabolic disorders, syndrome X, obesity, insulin resistance, hyperglycemia. For example, a nucleic acid sample from an individual may be subjected to a prognostic test described herein. Where one or more polymorphic variations associated with increased risk of type II diabetes are identified in a subject, information for preventing or treating type II diabetes and/or one or more type II diabetes treatment regimens then may be prescribed to that subject.

[0100] In certain embodiments, a treatment or preventative regimen is specifically prescribed and/or administered to individuals who will most benefit from it based upon their risk of developing type II diabetes assessed by the methods described herein. Thus, provided are methods for identifying a subject predisposed to type II diabetes and then prescribing a therapeutic or preventative regimen to individuals identified as having a predisposition. Thus, certain embodiments are directed to a method for reducing type II diabetes in a subject, which comprises: detecting the presence or absence of a polymorphic variant associated with type II diabetes in a nucleotide sequence in a nucleic acid sample from a subject, where the nucleotide sequence comprises a polynucleotide sequence selected from the group consisting of: (a) a nucleotide sequence of SEQ ID NO: 1-5 or 10; (b) a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-5 or 10; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-5 or 10, or a nucleotide sequence about 90% or more identical to a nucleotide sequence of SEQ ID NO: 1-5 or 10; and (d) a fragment of a polynucleotide sequence of (a), (b), or (c); and prescribing or administering a treatment regimen to a subject from whom the sample originated where the presence of a polymorphic variation associated with type II diabetes is detected in the nucleotide sequence. In these methods, predisposition results may be utilized in combination with other test results to diagnose type II diabetes associated conditions, such as metabolic disorders, syndrome X, obesity, insulin resistance, hyperglycemia.

[0101] Certain preventative treatments often are prescribed to subjects having a predisposition to type II diabetes and where the subject is diagnosed with type II diabetes or is diagnosed as having symptoms indicative of early stage type II diabetes, (e.g., impaired glucose tolerance, or IGT). For example, recent studies have highlighted the potential for intervention in IGT subjects to reduce progression to type II diabetes. One such study showed that over three years lifestyle intervention (targeting diet and exercise) reduced the risk of progressing from IGT to diabetes by 58% (The Diabetes Prevention Program. (1999) Diabetes Care 22:623-634). In a similar Finnish study, the cumulative incidence of diabetes after four years was 11% in the intervention group and 23% in the control group. During the trial, the risk of diabetes was reduced by 58% in the intervention group (Tuomilehto et al.(200\) N. Eng. J Med. 344:1343-1350). Clearly there is great benefit in the early diagnosis and subsequent preventative treatment of type II diabetes.

[0102] The treatment sometimes is preventative (e.g., is prescribed or administered to reduce the probability that a type II diabetes associated condition arises or progresses), sometimes is therapeutic, and sometimes delays, alleviates or halts the progression of a type II diabetes associated condition. Any known preventative or therapeutic treatment for alleviating or preventing the occurrence of a type II diabetes associated disorder is prescribed and/or administered. For example, the treatment sometimes includes changes in diet, increased exercise, and the administration of therapeutics such as sulphonylureas (and related insulin secretagogues), which increase insulin release from pancreatic islets; metformin, (Glucophage™), which acts to reduce hepatic glucose production; peroxisome proliferator-activated receptor-gamma (PPAR) agonists (thiozolidinediones such as Avandia® and Actos®), which enhance insulin action; alpha- glucosidase inhibitors (e.g., Precose®, Voglibose®, and Miglitol®), which interfere with gut glucose absorption; and insulin itself, which suppresses glucose production and augments glucose utilization (MoMer Nature 414, 821-827 (2001)).

[0103] As therapeutic approaches for type II diabetes continue to evolve and improve, the goal of treatments for type II diabetes related disorders is to intervene even before clinical signs (e.g., impaired glucose tolerance, or IGT) first manifest. Thus, genetic markers associated with susceptibility to type II diabetes prove useful for early diagnosis, prevention and treatment of type II diabetes.

[0104] As type II diabetes preventative and treatment information can be specifically targeted to subjects in need thereof (e.g., those at risk of developing type II diabetes or those that have early stages of type II diabetes), provided herein is a method for preventing or reducing the risk of developing type II diabetes in a subject, which comprises: (a) detecting the presence or absence of a polymorphic variation associated with type II diabetes at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) identifying a subject with a predisposition to type II diabetes, whereby the presence of the polymorphic variation is indicative of a predisposition to type II diabetes in the subject; and (c) if such a predisposition is identified, providing the subject with information about methods or products to prevent or reduce type II diabetes or to delay the onset of type II diabetes. Also provided is a method of targeting information or advertising to a subpopulation of a human population based on the subpopulation being genetically predisposed to a disease or condition, which comprises: (a) detecting the presence or absence of a polymorphic variation associated with type II diabetes at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) identifying the subpopulation of subjects in which the polymorphic variation is associated with type II diabetes; and (c) providing information only to the subpopulation of subjects about a particular product which may be obtained and consumed or applied by the subject to help prevent or delay onset of the disease or condition.

[0105] Pharmacogenomics methods also may be used to analyze and predict a response to a type II diabetes treatment or a drug. For example, if pharmacogenomics analysis indicates a likelihood that an individual will respond positively to a type II diabetes treatment with a particular drug, the drug may be administered to the individual. Conversely, if the analysis indicates that an individual is likely to respond negatively to treatment with a particular drug, an alternative course of treatment may be prescribed. A negative response may be defined as either the absence of an efficacious response or the presence of toxic side effects. The response to a therapeutic treatment can be predicted in a background study in which subjects in any of the following populations are genotyped: a population that responds favorably to a treatment regimen, a population that does not respond significantly to a treatment regimen, and a population that responds adversely to a treatment regiment (e.g., exhibits one or more side effects). These populations are provided as examples and other populations and subpopulations may be analyzed. Based upon the results of these analyses, a subject is genotyped to predict whether he or she will respond favorably to a treatment regimen, not respond significantly to a treatment regimen, or respond adversely to a treatment regimen.

[0106] The tests described herein also are applicable to clinical drug trials. One or more polymorphic variants indicative of response to an agent for treating type II diabetes or to side effects to an agent for treating type II diabetes may be identified using the methods described herein. Thereafter, potential participants in clinical trials of such an agent may be screened to identify those individuals most likely to respond favorably to the drug and exclude those likely to experience side effects. In that way, the effectiveness of drug treatment may be measured in individuals who respond positively to the drug, without lowering the measurement as a result of the inclusion of individuals who are unlikely to respond positively in the study and without risking undesirable safety problems.

[0107] Thus, another embodiment is a method of selecting an individual for inclusion in a clinical trial of a treatment or drug comprising the steps of: (a) obtaining a nucleic acid sample from an individual; (b) determining the identity of a polymorphic variation which is associated with a positive response to the treatment or the drug, or at least one polymorphic variation which is associated with a negative response to the treatment or the drug in the nucleic acid sample, and (c) including the individual in the clinical trial if the nucleic acid sample contains said polymorphic variation associated with a positive response to the treatment or the drug or if the nucleic acid sample lacks said polymorphic variation associated with a negative response to the treatment or the drug. In addition, the methods described herein for selecting an individual for inclusion in a clinical trial of a treatment or drug encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination. The polymorphic variation may be in a sequence selected individually or in any combination from the group consisting of (i) a nucleotide sequence of SEQ ID NO: 1-5 or 10; (ii) a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-5 or 10; (iii) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-5 or 10, or a nucleotide sequence about 90% or more identical to a nucleotide sequence of SEQ ID NO: 1-5 or 10; and (iv) a fragment of a polynucleotide sequence of (i), (ii), or (iii) comprising the polymorphic site. The including step (c) optionally comprises administering the drug or the treatment to the individual if the nucleic acid sample contains the polymorphic variation associated with a positive response to the treatment or the drug and the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug.

[0108] Also provided herein is a method of partnering between a diagnostic/prognostic testing provider and a provider of a consumable product, which comprises: (a) the diagnostic/prognostic testing provider detects the presence or absence of a polymorphic variation associated with type II diabetes at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) the diagnostic/prognostic testing provider identifies the subpopulation of subjects in which the polymorphic variation is associated with type II diabetes; (c) the diagnostic/prognostic testing provider forwards information to the subpopulation of subjects about a particular product which may be obtained and consumed or applied by the subject to help prevent or delay onset of the disease or condition; and (d) the provider of a consumable product forwards to the diagnostic test provider a fee every time the diagnostic/prognostic test provider forwards information to the subject as set forth in step (c) above. Compositions Comprising Diabetes-Directed Molecules

[0109] Featured herein is a composition comprising a cell from a subject having type II diabetes or at risk of type II diabetes and one or more molecules specifically directed and targeted to a nucleic acid comprising a PP2Ce or B3GALT3nucleotide sequence or amino acid sequence. Such directed molecules include, but are not limited to, a compound that binds to a PP2Ce or B3GALT3 nucleotide sequence or amino acid sequence referenced herein; a nucleic acid comprising or consisting of a nucleotide sequence that hybridizes under stringent conditions to a PP2Ce or B3GALT3 nucleotide sequence; a RNAi or siRNA molecule having a strand complementary to a PP2Ce or B3GALT3 nucleotide sequence; an antisense nucleic acid complementary to an RNA encoded by a PP2Ce or B3GALT3 nucleotide sequence; a ribozyme that hybridizes to a PP2Ce or B3GALT3 nucleotide sequence; a nucleic acid aptamer that specifically binds a polypeptide encoded by PP2Ce or B3GALT3 nucleotide sequence; and an antibody that specifically binds to a polypeptide encoded by PP2Ce or B3GALT3 nucleotide sequence or binds to a nucleic acid having such a nucleotide sequence. In specific embodiments, the diabetes directed molecule interacts with a nucleic acid or polypeptide variant associated with diabetes, such as variants referenced herein. In other embodiments, the diabetes directed molecule interacts with a polypeptide involved in a signal pathway of a polypeptide encoded by a PP2Ce or B3GALT3 nucleotide sequence, or a nucleic acid comprising such a nucleotide sequence.

[0110] Compositions sometimes include an adjuvant known to stimulate an immune response, and in certain embodiments, an adjuvant that stimulates a T-cell lymphocyte response. Adjuvants are known, including but not limited to an aluminum adjuvant (e.g., aluminum hydroxide); a cytokine adjuvant or adjuvant that stimulates a cytokine response (e.g., interleukin (IL)-12 and/or γ- interferon cytokines); a Freund-type mineral oil adjuvant emulsion (e.g., Freund's complete or incomplete adjuvant); a synthetic lipoid compound; a copolymer adjuvant (e.g., TitreMax); a saponin; Quil A; a liposome; an oil-in-water emulsion (e.g., an emulsion stabilized by Tween 80 and pluronic polyoxyethlene/polyoxypropylene block copolymer (Syntex Adjuvant Formulation); TitreMax; detoxified endotoxin (MPL) and mycobacterial cell wall components (TDW, CWS) in 2% squalene (Ribi Adjuvant System)); a muramyl dipeptide; an immune-stimulating complex (ISCOM, e.g., an Ag-modified saponin/cholesterol micelle that forms stable cage-like structure); an aqueous phase adjuvant that does not have a depot effect (e.g., Gerbu adjuvant); a carbohydrate polymer (e.g., AdjuPrime); L-tyrosine; a manide-oleate compound (e.g., Montanide); an ethylene- vinyl acetate copolymer (e.g., Elvax 40Wl, 2); or lipid A, for example. Such compositions are useful for generating an immune response against a diabetes directed molecule (e.g., an HLA- binding subsequence within a polypeptide encoded by a PP2Ce or B3GALT3 nucleotide sequence). In such methods, a peptide having an amino acid subsequence of a polypeptide encoded by a

PP2Ce or B3GALT3 nucleotide sequence is delivered to a subject, where the subsequence binds to an HLA molecule and induces a CTL lymphocyte response. The peptide sometimes is delivered to the subject as an isolated peptide or as a minigene in a plasmid that encodes the peptide. Methods for identifying HLA-binding subsequences in such polypeptides are known (see e.g., publication WO02/20616 and PCT application US98/01373 for methods of identifying such sequences).

[0111] The cell may be in a group of cells cultured in vitro or in a tissue maintained in vitro or present in an animal in vivo (e.g., a rat, mouse, ape or human). In certain embodiments, a composition comprises a component from a cell such as a nucleic acid molecule (e.g., genomic DNA), a protein mixture or isolated protein, for example. The aforementioned compositions have utility in diagnostic, prognostic and pharmacogenomic methods described previously and in diabetes therapeutics described hereafter. Certain diabetes directed molecules are described in greater detail below.

Compounds

[0112] Compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive (see, e.g., Zuckermann et al, J. Med. Chem.37: 2678-85 (1994)); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; "one-bead one-compound" library methods; and synthetic library methods using affinity chromatography selection. Biological library and peptoid library approaches are typically limited to peptide libraries, while the other approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, (1997)). Examples of methods for synthesizing molecular libraries are described, for example, in DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90: 6909 (1993); Erb et al, Proc. Natl. Acad. Sci. USA 91: 11422 (1994); Zuckermann et al, J. Med. Chem. 37: 2678 (1994); Cho ef α/.,Science 261: 1303 (1993); Carrell et al, Angew. Chem. Int. Ed. Engl. 33: 2059 (1994); Carell et al, Angew. Chem. Int. Ed. Engl. 33: 2061 (1994); and in Gallop et al, J. Med. Chem. 37: 1233 (1994).

[0113] Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13: 412-421 (1992)), or on beads (Lam, Nature 354: 82-84 (1991)), chips (Fodor, Nature 364: 555-556 (1993)), bacteria or spores (Ladner, United States Patent No. 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. USA 89: 1865-1869 (1992)) or on phage (Scott and Smith, Science 249: 386-390 (1990); Devlin, Science 249: 404-406 (1990); Cwirla et al, Proc. Natl. Acad. Sci. 87: 6378-6382 (1990); Felici, J. MoI. Biol. 222: 301-310 (1991); Ladner supra.).

[0114] A compound sometimes alters expression and sometimes alters activity of a polypeptide target and may be a small molecule. Small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

Antisense Nucleic Acid Molecules, Ribozymes, RNAi, siRNA and Modified Nucleic Acid Molecules

[0115] An "antisense" nucleic acid refers to a nucleotide sequence complementary to a "sense" nucleic acid encoding a polypeptide, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire coding strand (e.g., SEQ ID NO: 2-5), or to a portion thereof or a substantially identical sequence thereof. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence (e.g., 5' and 3' untranslated regions in SEQ ID NO: 1).

[0116] An antisense nucleic acid can be designed such that it is complementary to the entire coding region of an mRNA encoded by a nucleotide sequence (e.g., SEQ ID NO: 1-5 or 10), and often the antisense nucleic acid is an oligonucleotide antisense to only a portion of a coding or noncoding region of the mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length. The antisense nucleic acids, which include the ribozymes described hereafter, can be designed to target a PP2Ce or B3GALT3 nucleotide sequence, often a variant associated with diabetes, or a substantially identical sequence thereof. Among the variants, minor alleles and major alleles can be targeted, and those associated with a higher risk of diabetes are often designed, tested, and administered to subjects.

[0117] An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using standard procedures. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[0118] When utilized as therapeutics, antisense nucleic acids typically are administered to a subject (e.g., by direct injection at a tissue site) or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a polypeptide and thereby inhibit expression of the polypeptide, for example, by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then are administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, for example, by linking antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. Sufficient intracellular concentrations of antisense molecules are achieved by incorporating a strong promoter, such as a pol II or pol III promoter, in the vector construct.

[0119] Antisense nucleic acid molecules sometimes are alpha-anomeric nucleic acid molecules. An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual beta-units, the strands run parallel to each other (Gaultier et al, Nucleic Acids. Res. 15: 6625-6641 (1987)). Antisense nucleic acid molecules can also comprise a 2'-o-methylribonucleotide (Inoue et al, Nucleic Acids Res. 15: 6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al, FEBS Lett. 215: 327-330 (1987)). Antisense nucleic acids sometimes are composed of DNA or PNA or any other nucleic acid derivatives described previously.

[0120] In another embodiment, an antisense nucleic acid is a ribozyme. A ribozyme having specificity for a PP2Ce or B3GALT3 nucleotide sequence can include one or more sequences complementary to such a nucleotide sequence, and a sequence having a known catalytic region responsible for mRNA cleavage (see e.g., U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach, Nature 334: 585-591 (1988)). For example, a derivative of a Tetrahymena L-19 IVS RNA is sometimes utilized in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a mRNA (see e.g., Cech et al. U.S. Patent No. 4,987,071; and Cech et al U.S. Patent No. 5,116,742). Also, target mRNA sequences can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see e.g., Bartel & Szostak, Science 261 : 1411-1418 (1993)).

[0121] Diabetes directed molecules include in certain embodiments nucleic acids that can form triple helix structures with a PP2Ce or B3GALT3 nucleotide sequence or a substantially identical sequence thereof, especially one that includes a regulatory region that controls expression of a polypeptide. Gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a nucleotide sequence referenced herein or a substantially identical sequence (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of a gene in target cells (see e.g., Helene, Anticancer Drug Des. 6(6): 569-84 (1991); Helene et al, Ann. N.Y. Acad. Sci. 660: 27-36 (1992); and Maher, Bioassays 14(12): 807-15 (1992). Potential sequences that can be targeted for triple helix formation can be increased by creating a so-called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5 '-3', 3 '-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

[0122] Diabetes directed molecules include RNAi and siRNA nucleic acids. Gene expression may be inhibited by the introduction of double-stranded RNA (dsRNA), which induces potent and specific gene silencing, a phenomenon called RNA interference or RNAi. See, e.g., Fire et al., US Patent Number 6,506,559; Tuschl et al. PCT International Publication No. WO 01/75164; Kay et al. PCT International Publication No. WO 03/010180A1; or Bosher JM, Labouesse, Nat Cell Biol

2000 Feb;2(2):E31-6. This process has been improved by decreasing the size of the double- stranded RNA to 20-24 base pairs (to create small-interfering RNAs or siRNAs) that "switched off' genes in mammalian cells without initiating an acute phase response, i.e., a host defense mechanism that often results in cell death (see, e.g., Caplen et al. Proc Natl Acad Sci U S A. 2001 Aug 14;98(17):9742-7 and Elbashir et al. Methods 2002 Feb;26(2): 199-213). There is increasing evidence of post-transcriptional gene silencing by RNA interference (RNAi) for inhibiting targeted expression in mammalian cells at the mRNA level, in human cells. There is additional evidence of effective methods for inhibiting the proliferation and migration of tumor cells in human patients, and for inhibiting metastatic cancer development (see, e.g., U.S. Patent Application No.

US2001000993183; Caplen et al. Proc Natl Acad Sci U S A; and Abderrahmani et alMol Cell Biol

2001 Nov21(21):7256-67).

[0123] An "siRNA" or "RNAi" refers to a nucleic acid that forms a double stranded RNA and has the ability to reduce or inhibit expression of a gene or target gene when the siRNA is delivered to or expressed in the same cell as the gene or target gene. "siRNA" refers to short double-stranded RNA formed by the complementary strands. Complementary portions of the siRNA that hybridize to form the double stranded molecule often have substantial or complete identity to the target molecule sequence. In one embodiment, an siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA.

[0124] When designing the siRNA molecules, the targeted region often is selected from a given DNA sequence beginning 50 to 100 nucleotides downstream of the start codon. See, e.g., Elbashir et al,. Methods 26:199-213 (2002). Initially, 5' or 3' UTRs and regions nearby the start codon were avoided assuming that UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP or RISC endonuc lease complex. Sometimes regions of the target 23 nucleotides in length conforming to the sequence motif AA(Nl 9)TT (N, an nucleotide), and regions with approximately 30% to 70% G/C-content (often about 50% G/C-content) often are selected. If no suitable sequences are found, the search often is extended using the motif NA(N21). The sequence of the sense siRNA sometimes corresponds to (N19) TT or N21 (position 3 to 23 of the 23-nt motif), respectively. In the latter case, the 3' end of the sense siRNA often is converted to TT. The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3' overhangs. The antisense siRNA is synthesized as the complement to position 1 to 21 of the 23-nt motif. Because position 1 of the 23- nt motif is not recognized sequence-specifically by the antisense siRNA, the 3 '-most nucleotide residue of the antisense siRNA can be chosen deliberately. However, the penultimate nucleotide of the antisense siRNA (complementary to position 2 of the 23-nt motif) often is complementary to the targeted sequence. For simplifying chemical synthesis, TT often is utilized. siRNAs corresponding to the target motif NAR(N 17) YNN, where R is purine (A,G) and Y is pyrimidine (C,U), often are selected. Respective 21 nucleotide sense and antisense siRNAs often begin with a purine nucleotide and can also be expressed from pol III expression vectors without a change in targeting site. Expression of RNAs from pol III promoters often is efficient when the first transcribed nucleotide is a purine.

[0125] The sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof. Often, the siRNA is about 15 to about 50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, sometimes about 20-30 nucleotides in length or about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The siRNA sometimes is about 21 nucleotides in length. Methods of using siRNA are well known in the art, and specific siRNA molecules may be purchased from a number of companies including Dharmacon Research, Inc. An siRNA molecule sometimes is composed of a different chemical composition as compared to native RNA that imparts increased stability in cells (e.g., decreased susceptibility to degradation), and sometimes includes one or more modifications in siSTABLE RNA described at the http address www.dharmacon.com.

[0126] Antisense, ribozyme, RNAi and siRNA nucleic acids can be altered to form modified nucleic acid molecules. The nucleic acids can be altered at base moieties, sugar moieties or phosphate backbone moieties to improve stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup et al, Bioorganic & Medicinal Chemistry 4 (1): 5-23

(1996)). As used herein, the terms "peptide nucleic acid" or "PNA" refers to a nucleic acid mimic such as a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. Synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described, for example, in Hyrup et «/.,(1996) supra and Perry-O'Keefe et al, Proc. Natl. Acad. Sci. 93: 14670-675 (1996).

[0127] PNA nucleic acids can be used in prognostic, diagnostic, and therapeutic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNA nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, {e.g., by PNA-directed PCR clamping); as "artificial restriction enzymes" when used in combination with other enzymes, (e.g., Sl nucleases (Hyrup (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup et α/.,(1996) supra; Perry-O'Keefe supra).

[0128] In other embodiments, oligonucleotides may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across cell membranes (see e.g., Letsinger et al, Proc. Natl. Acad. Sci. USA 86: 6553-6556 (1989); Lemaitre et al, Proc. Natl. Acad. Sci. USA 84: 648-652 (1987); PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al, Bio-Techniques 6: 958-976 (1988)) or intercalating agents. (See, e.g., Zon, Pharm. Res. 5: 539-549 (1988) ). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

[0129] Also included herein are molecular beacon oligonucleotide primer and probe molecules having one or more regions complementary to a PP2Ce or B3GALT3 nucleotide sequence or a substantially identical sequence thereof, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantifying the presence of the nucleic acid in a sample. Molecular beacon nucleic acids are described, for example, in Lizardi et al, U.S. Patent No. 5,854,033; Nazarenko et al, U.S. Patent No. 5,866,336, and Livak et al, U.S. Patent No. 5,876,930.

Antibodies

[0130] The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')₂ fragments which can be generated by treating the antibody with an enzyme such as pepsin. An antibody sometimes is a polyclonal, monoclonal, recombinant (e.g. , a chimeric or humanized), fully human, non-human (e.g., murine), or a single chain antibody. An antibody may have effector function and can fix complement, and is sometimes coupled to a toxin or imaging agent.

[0131] A full-length polypeptide or antigenic peptide fragment encoded by a nucleotide sequence referenced herein can be used as an immunogen or can be used to identify antibodies made with other immunogens, e.g., cells, membrane preparations, and the like. An antigenic peptide often includes at least 8 amino acid residues of the amino acid sequences encoded by a nucleotide sequence referenced herein, or substantially identical sequence thereof, and encompasses an epitope. Antigenic peptides sometimes include 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, or 30 or more amino acids. Hydrophilic and hydrophobic fragments of polypeptides sometimes are used as immunogens.

[0132] Epitopes encompassed by the antigenic peptide are regions located on the surface of the polypeptide (e.g., hydrophilic regions) as well as regions with high antigenicity. For example, an Emini surface probability analysis of the human polypeptide sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface of the polypeptide and are thus likely to constitute surface residues useful for targeting antibody production. The antibody may bind an epitope on any domain or region on polypeptides described herein.

[0133] Also, chimeric, humanized, and completely human antibodies are useful for applications which include repeated administration to subjects. Chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, can be made using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et α/.,Science 240: 1041-1043 (1988); Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443 (1987); Liu et al., J. Immunol. 139: 3521-3526 (1987); Sun et al, Proc. Natl. Acad. Sci. USA 84: 214-218 (1987); Nishimura et al, Cane. Res. 47: 999-1005 (1987); Wood et al., Nature 314: 446-449 (1985); and Shaw et al, J. Natl. Cancer Inst. 80: 1553-1559 (1988); Morrison, S. L., Science 229: 1202-1207 (1985); Oi et al, BioTechniques 4: 214 (1986); Winter U.S. Patent 5,225,539; Jones et al, Nature 321 : 552-525 (1986); Verhoeyan ef α/.,Science 239: 1534; and Beidler et al, J. Immunol. 141: 4053-4060 (1988).

[0134] Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. See, for example, Lonberg and Huszar, Int. Rev. Immunol. 13: 65-93 (1995); and U.S. Patent Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806. In addition, companies such as Abgenix, Inc. (Fremont, CA) and Medarex, Inc. (Princeton, NJ), can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above. Completely human antibodies that recognize a selected epitope also can be generated using a technique referred to as "guided selection." In this approach a selected non- human monoclonal antibody (e.g., a murine antibody) is used to guide the selection of a completely human antibody recognizing the same epitope. This technology is described for example by Jespers et ai, Bio/Technology 12: 899-903 (1994).

[0135] An antibody can be a single chain antibody. A single chain antibody (scFV) can be engineered (see, e.g., Colcher et ai, Ann. N Y Acad. Sci. 880: 263-80 (1999); and Reiter, Clin. Cancer Res. 2: 245-52 (1996)). Single chain antibodies can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target polypeptide.

[0136] Antibodies also may be selected or modified so that they exhibit reduced or no ability to bind an Fc receptor. For example, an antibody may be an isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor {e.g., it has a mutagenized or deleted Fc receptor binding region).

[0137] Also, an antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g. , methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g. , mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti¬ mitotic agents (e.g., vincristine and vinblastine).

[0138] Antibody conjugates can be used for modifying a given biological response. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a polypeptide such as tumor necrosis factor, gamma-interferon, alpha-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-I"), interleukin-2 ("IL- 2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors. Also, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, for example.

[0139] An antibody (e.g., monoclonal antibody) can be used to isolate target polypeptides by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an antibody can be used to detect a target polypeptide (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide. Antibodies can be used diagnostically to monitor polypeptide levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labeling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵1, ¹³¹1, ³⁵S or ³H. Also, an antibody can be utilized as a test molecule for determining whether it can treat diabetes, and as a therapeutic for administration to a subject for treating diabetes.

[0140] An antibody can be made by immunizing with a purified antigen, or a fragment thereof, e.g., a fragment described herein, a membrane associated antigen, tissues, e.g., crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions.

[0141] Included herein are antibodies which bind only a native polypeptide, only denatured or otherwise non-native polypeptide, or which bind both, as well as those having linear or conformational epitopes. Conformational epitopes sometimes can be identified by selecting antibodies that bind to native but not denatured polypeptide. Also featured are antibodies that specifically bind to a polypeptide variant associated with diabetes.

Methods for Identifying Candidate Therapeutics for Treating Type II Diabetes [0142] Current therapies for the treatment of type II diabetes have limited efficacy, limited tolerability and significant mechanism-based side effects, including weight gain and hypoglycemia. Few of the available therapies adequately address underlying defects such as obesity and insulin resistance (Moller D. Nature. 414:821-827 (2001)). Current therapeutic approaches were largely developed in the absence of defined molecular targets or even a solid understanding of disease pathogenesis. Therefore, provided are methods of identifying candidate therapeutics that target biochemical pathways related to the development of diabetes.

[0143] Thus, featured herein are methods for identifying a candidate therapeutic for treating type II diabetes. The methods comprise contacting a test molecule with a target molecule in a system. A "target molecule" as used herein refers to a PP2Ce or B3GALT3 nucleic acid, a substantially identical nucleic acid thereof, or a fragment thereof, and an encoded polypeptide of the foregoing. The methods also comprise determining the presence or absence of an interaction between the test molecule and the target molecule, where the presence of an interaction between the test molecule and the nucleic acid or polypeptide identifies the test molecule as a candidate type II diabetes therapeutic. The interaction between the test molecule and the target molecule may be quantified.

[0144] Test molecules and candidate therapeutics include, but are not limited to, compounds, antisense nucleic acids, siRNA molecules, ribozymes, polypeptides or proteins encoded by a PP2Ce or B3GALT3 nucleotide sequence, or a substantially identical sequence or fragment thereof, and immunotherapeutics (e.g., antibodies and HLA-presented polypeptide fragments). A test molecule or candidate therapeutic may act as a modulator of target molecule concentration or target molecule function in a system. A "modulator" may agonize (i.e., up-regulates) or antagonize (i.e., down-regulates) a target molecule concentration partially or completely in a system by affecting such cellular functions as DNA replication and/or DNA processing (e.g., DNA methylation or DNA repair), RNA transcription and/or RNA processing (e.g., removal of intronic sequences and/or translocation of spliced mRNA from the nucleus), polypeptide production (e.g., translation of the polypeptide from mRNA), and/or polypeptide post-translational modification (e.g., glycosylation, phosphorylation, and proteolysis of pro-polypeptides). A modulator may also agonize or antagonize a biological function of a target molecule partially or completely, where the function may include adopting a certain structural conformation, interacting with one or more binding partners, ligand binding, catalysis (e.g., phosphorylation, dephosphorylation, hydrolysis, methylation, and isomerization), and an effect upon a cellular event (e.g., effecting progression of type II diabetes). In certain embodiments, a candidate therapeutic increases glucose uptake in cells of a subject (e.g., in certain cells of the pancreas).

[0145] As used herein, the term "system" refers to a cell free in vitro environment and a cell- based environment such as a collection of cells, a tissue, an organ, or an organism. A system is "contacted" with a test molecule in a variety of manners, including adding molecules in solution and allowing them to interact with one another by diffusion, cell injection, and any administration routes in an animal. As used herein, the term "interaction" refers to an effect of a test molecule on test molecule, where the effect sometimes is binding between the test molecule and the target molecule, and sometimes is an observable change in cells, tissue, or organism.

[0146] There are many standard methods for detecting the presence or absence of interaction between a test molecule and a target molecule. For example, titrametric, acidimetric, radiometric, NMR, monolayer, polarographic, spectrophotometric, fluorescent, and ESR assays probative of a target molecule interaction may be utilized.

[0147] Test molecule/target molecule interactions can be detected and/or quantified using assays known in the art. For example, an interaction can be determined by labeling the test molecule and/or the target molecule, where the label is covalently or non-covalently attached to the test molecule or target molecule. The label is sometimes a radioactive molecule such as ¹²⁵I, ¹³¹1, ³⁵S or ³H, which can be detected by direct counting of radioemission or by scintillation counting. Also, enzymatic labels such as horseradish peroxidase, alkaline phosphatase, or luciferase may be utilized where the enzymatic label can be detected by determining conversion of an appropriate substrate to product. In addition, presence or absence of an interaction can be determined without labeling. For example, a microphysiometer (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indication of an interaction between a test molecule and target molecule (McConnell, H. M. et al.,Science 257: 1906-1912 (1992)).

[0148] In cell-based systems, cells typically include a PP2Ce or B3GALT3 nucleic acid, an encoded polypeptide, or substantially identical nucleic acid or polypeptide thereof, and are often of mammalian origin, although the cell can be of any origin. Whole cells, cell homogenates, and cell fractions (e.g., cell membrane fractions) can be subjected to analysis. Where interactions between a test molecule with a target polypeptide are monitored, soluble and/or membrane bound forms of the polypeptide may be utilized. Where membrane-bound forms of the polypeptide are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N- methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_n, 3-[(3-cholamidopropyl)dimethylamminio]-l -propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-l -propane sulfonate (CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-l -propane sulfonate.

[0149] An interaction between a test molecule and target molecule also can be detected by monitoring fluorescence energy transfer (FET) (see, e.g., Lakowicz et ai, U.S. Patent No. 5,631,169; Stavrianopoulos et al. U.S. Patent No. 4,868,103). A fluorophore label on a first, "donor" molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, "acceptor" molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the "donor" polypeptide molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the "acceptor" molecule label may be differentiated from that of the "donor". Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the "acceptor" molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

[0150] In another embodiment, determining the presence or absence of an interaction between a test molecule and a target molecule can be effected by monitoring surface plasmon resonance (see, e.g., Sjolander & Urbaniczk, Anal. Chem. 63: 2338-2345 (1991) and Szabo et ai, Curr. Opin. Struct. Biol. 5: 699-705 (1995)). "Surface plasmon resonance" or "biomolecular interaction analysis (BIA)" can be utilized to detect biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

[0151] In another embodiment, the target molecule or test molecules are anchored to a solid phase, facilitating the detection of target molecule/test molecule complexes and separation of the complexes from free, uncomplexed molecules. The target molecule or test molecule is immobilized to the solid support. In an embodiment, the target molecule is anchored to a solid surface, and the test molecule, which is not anchored, can be labeled, either directly or indirectly, with detectable labels discussed herein.

[0152] It may be desirable to immobilize a target molecule, an anti-target molecule antibody, and/or test molecules to facilitate separation of target molecule/test molecule complexes from uncomplexed forms, as well as to accommodate automation of the assay. The attachment between a test molecule and/or target molecule and the solid support may be covalent or non-covalent (see, e.g., U.S. Patent No. 6,022,688 for non-covalent attachments). The solid support may be one or more surfaces of the system, such as one or more surfaces in each well of a microtiter plate, a surface of a silicon wafer, a surface of a bead (see, e.g., Lam, Nature 354: 82-84 (1991)) that is optionally linked to another solid support, or a channel in a microfluidic device, for example. Types of solid supports, linker molecules for covalent and non-covalent attachments to solid supports, and methods for immobilizing nucleic acids and other molecules to solid supports are well known (see, e.g., U.S. Patent Nos. 6,261,776; 5,900,481 ; 6,133,436; and 6,022,688; and WIPO publication WO 01/18234). [0153] In an embodiment, target molecule may be immobilized to surfaces via biotin and streptavidin. For example, biotinylated target polypeptide can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques known in the art {e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). In another embodiment, a target polypeptide can be prepared as a fusion polypeptide. For example, glutathione-S-transferase/target polypeptide fusion can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivitized microtiter plates, which are then combined with a test molecule under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, or the matrix is immobilized in the case of beads, and complex formation is determined directly or indirectly as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of target molecule binding or activity is determined using standard techniques.

[0154] In an embodiment, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that a significant percentage of complexes formed will remain immobilized to the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of manners. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface, e.g., by adding a labeled antibody specific for the immobilized component, where the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody.

[0155] In another embodiment, an assay is performed utilizing antibodies that specifically bind target molecule or test molecule but do not interfere with binding of the target molecule to the test molecule. Such antibodies can be derivitized to a solid support, and unbound target molecule may be immobilized by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

[0156] Cell free assays also can be conducted in a liquid phase. In such an assay, reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation {see, e.g., Rivas, G., and Minton, Trends Biochem Sci Aug;18(8): 284-7 (1993)); chromatography (gel filtration chromatography, ion- exchange chromatography); electrophoresis {see, e.g., Ausubel et ai, eds. Current Protocols in

Molecular Biology , J. Wiley: New York (1999)); and immunoprecipitation (see, e.g., Ausubel et

Al al., eds., supra). Media and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, JMoI. Recognit. Winter; 11(1-6): 141-8 (1998); Hage & Tweed, J. Chromatogr. B Biomed. Sci. Appl. Oct 10; 699 (1-2): 499-525 (1997)). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

[0157] In another embodiment, modulators of target molecule expression are identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of target mRNA or target polypeptide is evaluated relative to the level of expression of target mRNA or target polypeptide in the absence of the candidate compound. When expression of target mRNA or target polypeptide is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as an agonist of target mRNA or target polypeptide expression. Alternatively, when expression of target mRNA or target polypeptide is less (e.g., less with statistical significance) in the presence of the candidate compound than in its absence, the candidate compound is identified as an antagonist or inhibitor of target mRNA or target polypeptide expression. The level of target mRNA or target polypeptide expression can be determined by methods described herein.

[0158] In another embodiment, binding partners that interact with a target molecule are detected. The target molecules can interact with one or more cellular or extracellular macromolecules, such as polypeptides in vivo, and these interacting molecules are referred to herein as "binding partners." Binding partners can agonize or antagonize target molecule biological activity. Also, test molecules that agonize or antagonize interactions between target molecules and binding partners can be useful as therapeutic molecules as they can up-regulate or down-regulated target molecule activity in vivo and thereby treat type II diabetes.

[0159] Binding partners of target molecules can be identified by methods known in the art. For example, binding partners may be identified by lysing cells and analyzing cell lysates by electrophoretic techniques. Alternatively, a two-hybrid assay or three-hybrid assay can be utilized (see, e.g., U.S. Patent No. 5,283,317; Zervos et ai.Cell 72:223-232 (1993); Madura et al, J. Biol. Chem. 268: 12046-12054 (1993); Bartel et al., Biotechniques 14: 920-924 (1993); Iwabuchi et al., Oncogene 8: 1693-1696 (1993); and Brent WO94/10300). A two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. The assay often utilizes two different DNA constructs. In one construct, a PP2Ce or B3GALT3 nucleic acid (sometimes referred to as the "bait") is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In another construct, a DNA sequence from a library of DNA sequences that encodes a potential binding partner (sometimes referred to as the "prey") is fused to a gene that encodes an activation domain of the known transcription factor. Sometimes, a PP2Ce or B3GALT3 nucleic acid can be fused to the activation domain. If the "bait" and the "prey" molecules interact in vivo, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to identify the potential binding partner.

[0160] In an embodiment for identifying test molecules that antagonize or agonize complex formation between target molecules and binding partners, a reaction mixture containing the target molecule and the binding partner is prepared, under conditions and for a time sufficient to allow complex formation. The reaction mixture often is provided in the presence or absence of the test molecule. The test molecule can be included initially in the reaction mixture, or can be added at a time subsequent to the addition of the target molecule and its binding partner. Control reaction mixtures are incubated without the test molecule or with a placebo. Formation of any complexes between the target molecule and the binding partner then is detected. Decreased formation of a complex in the reaction mixture containing test molecule as compared to in a control reaction mixture indicates that the molecule antagonizes target molecule/binding partner complex formation. Alternatively, increased formation of a complex in the reaction mixture containing test molecule as compared to in a control reaction mixture indicates that the molecule agonizes target molecule/binding partner complex formation. In another embodiment, complex formation of target molecule/binding partner can be compared to complex formation of mutant target molecule/binding partner (e.g., amino acid modifications in a target polypeptide). Such a comparison can be important in those cases where it is desirable to identify test molecules that modulate interactions of mutant but not non-mutated target gene products.

[0161] The assays can be conducted in a heterogeneous or homogeneous format. In heterogeneous assays, target molecule and/or the binding partner are immobilized to a solid phase, and complexes are detected on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the molecules being tested. For example, test compounds that agonize target molecule/binding partner interactions can be identified by conducting the reaction in the presence of the test molecule in a competition format. Alternatively, test molecules that agonize preformed complexes, e.g., molecules with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed.

[0162] In a heterogeneous assay embodiment, the target molecule or the binding partner is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored molecule can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the molecule to be anchored can be used to anchor the molecule to the solid surface. The partner of the immobilized species is exposed to the coated surface with or without the test molecule. After the reaction is complete, unreacted components are removed {e.g., by washing) such that a significant portion of any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface is indicative of complex. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored to the surface; e.g., by using a labeled antibody specific for the initially non- immobilized species. Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

[0163] In another embodiment, the reaction can be conducted in a liquid phase in the presence or absence of test molecule, where the reaction products are separated from unreacted components, and the complexes are detected (e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes). Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified.

[0164] In an alternate embodiment, a homogeneous assay can be utilized. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared. One or both of the target molecule or binding partner is labeled, and the signal generated by the label(s) is quenched upon complex formation {e.g., U.S. Patent No. 4,109,496 that utilizes this approach for immunoassays). Addition of a test molecule that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target molecule/binding partner complexes can be identified.

[0165] Candidate therapeutics for treating type II diabetes are identified from a group of test molecules that interact with a target molecule. Test molecules are normally ranked according to the degree with which they modulate {e.g., agonize or antagonize) a function associated with the target molecule {e.g., DNA replication and/or processing, RNA transcription and/or processing, polypeptide production and/or processing, and/or biological function/activity), and then top ranking modulators are selected. Also, pharmacogenomic information described herein can determine the rank of a modulator. The top 10% of ranked test molecules often are selected for further testing as candidate therapeutics, and sometimes the top 15%, 20%, or 25% of ranked test molecules are selected for further testing as candidate therapeutics. Candidate therapeutics typically are formulated for administration to a subject. Therapeutic Formulations

[0166] Formulations and pharmaceutical compositions typically include in combination with a pharmaceutically acceptable carrier one or more target molecule modulators. The modulator often is a test molecule identified as having an interaction with a target molecule by a screening method described above. The modulator may be a compound, an antisense nucleic acid, a ribozyme, an antibody, or a binding partner. Also, formulations may comprise a target polypeptide or fragment thereof in combination with a pharmaceutically acceptable carrier.

[0167] As used herein, the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. Pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0168] A pharmaceutical composition typically is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0169] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. [0170] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as. lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0171] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0172] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0173] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. Molecules can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0174] In one embodiment, active molecules are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0175] It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0176] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀ZED₅₀. Molecules which exhibit high therapeutic indices are preferred. While molecules that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0177] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such molecules lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any molecules used in the methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (j e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. [0178] As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, sometimes about 0.01 to 25 mg/kg body weight, often about 0.1 to 20 mg/kg body weight, and more often about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, sometimes between 2 to 8 weeks, often between about 3 to 7 weeks, and more often for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

[0179] With regard to polypeptide formulations, featured herein is a method for treating type II diabetes in a subject, which comprises contacting one or more cells in the subject with a first polypeptide, where the subject comprises a second polypeptide having one or more polymorphic variations associated with cancer, and where the first polypeptide comprises fewer polymorphic variations associated with cancer than the second polypeptide. The first and second polypeptides are encoded by a nucleic acid which comprises a nucleotide sequence in SEQ ID NO: 1-5 or 10; a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence referenced in SEQ ID NO: 1-5 or 10; a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-5 or 10 and a nucleotide sequence 90% or more identical to a nucleotide sequence in SEQ ID NO: 1-5 or 10. The subject often is a human.

[0180] For antibodies, a dosage of 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg) is often utilized. If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is often appropriate. Generally, partially human antibodies and fully human antibodies have a longer half- life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et ai, J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193 (1997).

[0181] Antibody conjugates can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a polypeptide such as tumor necrosis factor, .alpha.-interferon, .beta.-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL- 1"), interleukin-2 ("IL-2"), interIeukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors. Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.

[0182] For compounds, exemplary doses include milligram or microgram amounts of the compound per kilogram of subject or sample weight, for example, about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid described herein, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[0183] With regard to nucleic acid formulations, gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et α/.,(1994) Proc. Natl. Acad. Sci. USA 97:3054-3057). Pharmaceutical preparations of gene therapy vectors can include a gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells (e.g., retroviral vectors) the pharmaceutical preparation can include one or more cells which produce the gene delivery system. Examples of gene delivery vectors are described herein.

Therapeutic Methods

[0184] A therapeutic formulation described above can be administered to a subject in need of a therapeutic for inducing a desired biological response. Therapeutic formulations can be administered by any of the paths described herein. With regard to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from pharmacogenomic analyses described herein. [0185] As used herein, the term "treatment" is defined as the application or administration of a therapeutic formulation to a subject, or application or administration of a therapeutic agent to an isolated tissue or cell line from a subject with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect type II diabetes, symptoms of type II diabetes or a predisposition towards type II diabetes. A therapeutic formulation includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides. Administration of a therapeutic formulation can occur prior to the manifestation of symptoms characteristic of type II diabetes, such that type II diabetes is prevented or delayed in its progression. The appropriate therapeutic composition can be determined based on screening assays described herein.

[0186] In related aspects, embodiments include methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising a polypeptide described herein, or a fragment thereof, or a therapeutic formulation described herein, wherein said biological response is selected from the ^' group consisting of: (a) modulating circulating (either blood, serum or plasma) levels (concentration) of glucose, wherein said modulating is preferably lowering; (b) increasing cell or tissue sensitivity to insulin, particularly muscle, adipose, liver or brain; (c) inhibiting the progression from impaired glucose tolerance to insulin resistance; (d) increasing glucose uptake in skeletal muscle cells; (e) increasing glucose uptake in adipose cells; (f) increasing glucose uptake in neuronal cells; (g) increasing glucose uptake in red blood cells; (h) increasing glucose uptake in the brain; and (i) significantly reducing the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal.

[0187] In other embodiments, a pharmaceutical or physiologically acceptable composition can be utilized as an insulinvsensitizer, or can be used in: a method to improve insulin sensitivity in some persons with type II diabetes in combination with insulin therapy; a method to improve insulin sensitivity in some persons with type II diabetes without insulin therapy; or a method of treating individuals with gestational diabetes. Gestational diabetes refers to the development of diabetes in an individual during pregnancy, usually during the second or third trimester of pregnancy. In further embodiments, the pharmaceutical or physiologically acceptable composition can be used in a method of treating individuals with impaired fasting glucose (IFG). Impaired fasting glucose (IFG) is a condition in which fasting plasma glucose levels in an individual are elevated but not diagnostic of overt diabetes (i.e. plasma glucose levels of less than 126 mg/dl and greater than or equal to 110 mg/dl).

[0188] In other embodiments, the pharmaceutical or physiologically acceptable composition can be used in a method of treating and preventing impaired glucose tolerance (IGT) in an individual. By providing therapeutics and methods for reducing or preventing IGT (i.e., for normalizing insulin resistance) the progression to type II diabetes can be delayed or prevented. Furthermore, by providing therapeutics and methods for reducing or preventing insulin resistance, provided are methods for reducing and/or preventing the appearance of Insulin-Resistance Syndrome (ERS). In further embodiments, the pharmaceutical or physiologically acceptable composition can be used in a method of treating a subject having polycystic ovary syndrome (PCOS). PCOS is among the most common disorders of premenopausal women, affecting 5-10% of this population. Insulin- sensitizing agents (e.g., troglitazone) have been shown to be effective in PCOS and that, in particular, the defects in insulin action, insulin secretion, ovarian steroidogenesis and fibrinolysis are improved (Ehrman et α/.(1997) J Clin Invest 100:1230), such as in insulin-resistant humans. Accordingly, provided are methods for reducing insulin resistance, normalizing blood glucose thus treating and/or preventing PCOS.

[0189] In certain embodiments, the pharmaceutical or physiologically acceptable composition can be used in a method of treating a subject having insulin resistance, where a subject having insulin resistance is treated to reduce or cure the insulin resistance. As insulin resistance is also often associated with infections and cancer, preventing or reducing insulin resistance may prevent or reduce infections and cancer.

[0190] In other embodiments, the pharmaceutical compositions and methods described herein are useful for: preventing the development of insulin resistance in a subject, e.g., those known to have an increased risk of developing insulin resistance; controlling blood glucose in some persons with type II diabetes in combination with insulin therapy; increasing cell or tissue sensitivity to insulin, particularly muscle, adipose, liver or brain; inhibiting or preventing the progression from impaired glucose tolerance to insulin resistance; improving glucose control of type II diabetes patients alone, without an insulin secretagogue or an insulin sensitizing agent; and administering a complementary therapy to type II diabetes patients to improve their glucose control in combination with an insulin secretagogue (preferably oral form) or an insulin sensitizing (preferably oral form) agent. In the latter embodiment, the oral insulin secretagogue sometimes is l,l-dimethyl-2-(2- morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide. The insulin sensitizing agent sometimes is selected from metformin, ciglitazone, troglitazone and pioglitazone.

[0191] Further embodiments include methods of administering a pharmaceutical or physiologically acceptable composition concomitantly or concurrently, with an insulin secretagogue or insulin sensitizing agent, for example, in the form of separate dosage units to be used simultaneously, separately or sequentially (e.g., before or after the secretagogue or before or after the sensitizing agent). Accordingly, provided is a pharmaceutical or physiologically acceptable composition and an insulin secretagogue or insulin sensitizing agent as a combined preparation for simultaneous, separate or sequential use for the improvement of glucose control in type II diabetes patients. [0192] Thus, any test known in the art or a method described herein can be used to determine that a subject is insulin resistant, and an insulin resistant patient can then be treated according to the methods described herein to reduce or cure the insulin resistance. Alternatively, the methods described herein also can be used to prevent the development of insulin resistance in a subject, e.g., those known to have an increased risk of developing insulin-resistance.

[0193] As discussed, successful treatment of type II diabetes can be brought about by techniques that serve to agonize target molecule expression or function, or alternatively, antagonize target molecule expression or function. These techniques include administration of modulators that include, but are not limited to, small organic or inorganic molecules; antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab')₂ and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof); and peptides, phosphopeptides, or polypeptides.

[0194] Further, antisense and ribozyme molecules that inhibit expression of the target gene can also be used to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above. It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Alternatively, in instances in that the target gene encodes an extracellular polypeptide, it can be preferable to co¬ administer normal target gene polypeptide into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.

[0195] Another method by which nucleic acid molecules may be utilized in treating or preventing type II diabetes is use of aptamer molecules specific for target molecules. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to ligands (see, e.g., Osborne, et al, Curr. Opin. Chem. Biol.l(l): 5-9 (1997); and Patel, D. J., Curr. Opin. Chem. Biol. Jun;l(l): 32-46 (1997)).

[0196] Yet another method of utilizing nucleic acid molecules for type II diabetes treatment is gene therapy, which can also be referred to as allele therapy. Provided herein is a gene therapy method for treating type II diabetes in a subject, which comprises contacting one or more cells in the subject or from the subject with a nucleic acid having a first nucleotide sequence. Genomic DNA in the subject comprises a second nucleotide sequence having one or more polymorphic variations associated with type II diabetes (e.g., the second nucleic acid has a nucleotide sequence in SEQ ID NO: 1-5 or 10). The first and second nucleotide sequences typically are substantially identical to one another, and the first nucleotide sequence comprises fewer polymorphic variations associated with type II diabetes than the second nucleotide sequence. The first nucleotide sequence may comprise a gene sequence that encodes a full-length polypeptide or a fragment thereof. The subject is often a human. Allele therapy methods often are utilized in conjunction with a method of first determining whether a subject has genomic DNA that includes polymorphic variants associated with type II diabetes.

[0197] In another allele therapy embodiment, provided herein is a method which comprises contacting one or more cells in the subject or from the subject with a polypeptide encoded by a nucleic acid having a first nucleotide sequence. Genomic DNA in the subject comprises a second nucleotide sequence having one or more polymorphic variations associated with type II diabetes (e.g., the second nucleic acid has a nucleotide sequence in SEQ ID NO: 1-5 or 10). The first and second nucleotide sequences typically are substantially identical to one another, and the first nucleotide sequence comprises fewer polymorphic variations associated with type II diabetes than the second nucleotide sequence. The first nucleotide sequence may comprise a gene sequence that encodes a full-length polypeptide or a fragment thereof. The subject is often a human.

[0198] For antibody-based therapies, antibodies can be generated that are both specific for target molecules and that reduce target molecule activity. Such antibodies may be administered in instances where antagonizing a target molecule function is appropriate for the treatment of type II diabetes.

[0199] In circumstances where stimulating antibody production in an animal or a human subject by injection with a target molecule is harmful to the subject, it is possible to generate an immune response against the target molecule by use of anti-idiotypic antibodies {see, e.g., Herlyn, Ann. Med.;31(l): 66-78 (1999); and Bhattacharya-Chatterjee & Foon, Cancer Treat. Res.; 94: 51-68 (1998)). Introducing an anti-idiotypic antibody to a mammal or human subject often stimulates production of anti-anti-idiotypic antibodies, which typically are specific to the target molecule. Vaccines directed to type II diabetes also may be generated in this fashion.

[0200] In instances where the target molecule is intracellular and whole antibodies are used, internalizing antibodies may be preferred. Lipofectin or liposomes can be used to deliver the antibody or a fragment of the Fab region that binds to the target antigen into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target antigen is preferred. For example, peptides having an amino acid sequence corresponding to the Fv region of the antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see, e.g., Marasco et al., Proc. Natl. Acad. ScL USA 90: 7889- 7893 (1993)).

[0201] Modulators can be administered to a patient at therapeutically effective doses to treat type II diabetes. A therapeutically effective dose refers to an amount of the modulator sufficient to result in amelioration of symptoms of type II diabetes. Toxicity and therapeutic efficacy of modulators can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀ZED₅₀. Modulators that exhibit large therapeutic indices are preferred. While modulators that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such molecules to the site of affected tissue in order to minimize potential damage to uninfected cells, thereby reducing side effects.

[0202] Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0203] Another example of effective dose determination for an individual is the ability to directly assay levels of "free" and "bound" compound in the serum of the test subject. Such assays may utilize antibody mimics and/or "biosensors" that have been created through molecular imprinting techniques. Molecules that modulate target molecule activity are used as a template, or "imprinting molecule", to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix which contains a repeated "negative image" of the compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell et al., Current Opinion in Biotechnology 7: 89-94 (1996) and in Shea, Trends in Polymer Science 2: 166-173 (1994). Such "imprinted" affinity matrixes are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example of the use of such matrixes in this way can be seen in Vlatakis, et al., Nature 361: 645-647 (1993). Through the use of isotope-labeling, the "free" concentration of compound which modulates target molecule expression or activity readily can be monitored and used in calculations of IC₅₀. Such "imprinted" affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes readily can be assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC₅₀. An example of such a "biosensor" is discussed in Kriz et al, Analytical Chemistry 6-7: 2142-2144 (1995).

[0204] The examples set forth below are intended to illustrate but not limit the invention.

Examples

[0205] In the following studies a group of subjects was selected according to specific parameters pertaining to type II diabetes. Nucleic acid samples obtained from individuals in the study group were subjected to genetic analyses that identified associations between type II diabetes and certain polymorphic variants in human genomic DNA. This procedure was repeated in a second and third group of subjects that served as replication cohorts. See Examples 3-4. Polymorphic variants proximal to the incident SNP were identified and analyzed in cases and controls. See Example 5. Methods are described for producing PP2Ce or BSGALTS polypeptides encoded by the nucleic acids of SEQ ID NO: 1-5 or 10 in vitro or in vivo, which can be utilized in methods that screen test molecules for those that interact with PP2Ce or BSGALTS polypeptides. Test molecules identified as being interactors with PP2Ce or BSGALTS polypeptides can be screened further as type II diabetes therapeutics.

Example 1 Samples and Pooling Strategies

Sample Selection

[0206] Blood samples were collected from individuals diagnosed with type II diabetes, which were referred to as case samples. Also, blood samples were collected from individuals not diagnosed with type II diabetes or a history of type II diabetes; these samples served as gender and age-matched controls. A database was created that listed all phenotypic trait information gathered from individuals for each case and control sample. Genomic DNA was extracted from each of the blood samples for genetic analyses.

DNA Extraction from Blood Samples

[0207] Six to ten milliliters of whole blood was transferred to a 50 ml tube containing 27 ml of red cell lysis solution (RCL). The tube was inverted until the contents were mixed. Each tube was incubated for 10 minutes at room temperature and inverted once during the incubation. The tubes were then centrifuged for 20 minutes at 3000 x g and the supernatant was carefully poured off. 100-200 μl of residual liquid was left in the tube and was pipetted repeatedly to resuspend the pellet in the residual supernatant. White cell lysis solution (WCL) was added to the tube and pipetted repeatedly until completely mixed. While no incubation was normally required, the solution was incubated at 37°C or room temperature if cell clumps were visible after mixing until the solution was homogeneous. 2 ml of protein precipitation was added to the cell lysate. The mixtures were vortexed vigorously at high speed for 20 sec to mix the protein precipitation solution uniformly with the cell lysate, and then centrifuged for 10 minutes at 3000 x g. The supernatant containing the DNA was then poured into a clean 15 ml tube, which contained 7 ml of 100% isopropanol. The samples were mixed by inverting the tubes gently until white threads of DNA were visible. Samples were centrifuged for 3 minutes at 2000 x g and the DNA was visible as a small white pellet. The supernatant was decanted and 5 ml of 70% ethanol was added to each tube. Each tube was inverted several times to wash the DNA pellet, and then centrifuged for 1 minute at 2000 x g. The ethanol was decanted and each tube was drained on clean absorbent paper. The DNA was dried in the tube by inversion for 10 minutes, and then 1000 μl of IX TE was added. The size of each sample was estimated, and less TE buffer was added during the following DNA hydration step if the sample was smaller. The DNA was allowed to rehydrate overnight at room temperature, and DNA samples were stored at 2-8⁰C.

[0208] DNA was quantified by placing samples on a hematology mixer for at least 1 hour. DNA was serially diluted (typically 1:80, 1 :160, 1:320, and 1 :640 dilutions) so that it would be within the measurable range of standards. 125 μl of diluted DNA was transferred to a clear U- bottom microtitre plate, and 125 μl of IX TE buffer was transferred into each well using a multichannel pipette. The DNA and IX TE were mixed by repeated pipetting at least 15 times, and then the plates were sealed. 50 μl of diluted DNA was added to wells A5-H12 of a black flat bottom microtitre plate. Standards were inverted six times to mix them, and then 50 μl of IX TE buffer was pipetted into well Al, 1000 ng/ml of standard was pipetted into well A2, 500 ng/ml of standard was pipetted into well A3, and 250 ng/ml of standard was pipetted into well A4. PicoGreen (Molecular Probes, Eugene, Oregon) was thawed and freshly diluted 1:200 according to the number of plates that were being measured. PicoGreen was vortexed and then 50μl was pipetted into all wells of the black plate with the diluted DNA. DNA and PicoGreen were mixed by pipetting repeatedly at least 10 times with the multichannel pipette. The plate was placed into a Fluoroskan Ascent Machine (microplate fluorometer produced by Labsystems) and the samples were allowed to incubate for 3 minutes before the machine was run using filter pairs 485 nm excitation and 538 nm emission wavelengths. Samples having measured DNA concentrations of greater than 450 ng/μl were re-measured for conformation. Samples having measured DNA concentrations of 20 ng/μl or less were re-measured for confirmation.

Pooling Strategies

[0209] Samples were placed into one of four groups based on disease status. The four groups were female case samples, female control samples, male case samples and male control samples. A select set of samples from each group were utilized to generate pools, and one pool was created for each group. Each individual sample in a pool was represented by an equal amount of genomic DNA. For example, where 25 ng of genomic DNA was utilized in each PCR reaction and there were 200 individuals in each pool, each individual would provide 125 pg of genomic DNA. Inclusion or exclusion of samples for a pool was based upon the following criteria and detailed in the tables below: patient ethnicity, diagnosis with type II diabetes, GAD antibody concentration, HbAIc concentration, body mass (BMI), patient age, date of primary diagnosis, and age of individual as of primary diagnosis. (See Table 2 below). Cases with elevated GAD antibody titers and low age of diagnosis were excluded to increase the homogeneity of the diabetes sample in terms of underlying pathogenesis. Controls with elevated HbAIc were excluded to remove any potentially undiagnosed diabetics. Control samples were derived from non-diabetic individuals with no family history of type II diabetes. Secondary phenotypes were also measured in the diabetic cases, including HDL levels, LDL levels, triglyceride levels, insulin levels, C-peptide levels, nephropathy status, and neuropathy status, to name a few. The phenotype data collected may be used to perform secondary analysis of the cases in order to elucidate the potential pathway of a disease gene.

TABLE 2

[0210] The selection process yielded the pools described in Table 3, which were used in the studies described herein.

TABLE 3

Example 2 Association of Polymorphic Variants with type II diabetes

[0211] A whole-genome screen was performed to identify particular SNPs associated with occurrence of type II diabetes. As described in Example 1, two sets of samples were utilized: female individuals having type II diabetes (female cases) and samples from female individuals not having type II diabetes or any history of type II diabetes (female controls), and male individuals having type II diabetes (male cases) and samples from male individuals not having type II diabetes or any history of type II diabetes (male controls). The initial screen of each pool was performed in an allelotyping study, in which certain samples in each group were pooled. By pooling DNA from each group, an allele frequency for each SNP in each group was calculated. These allele frequencies were then compared to one another. Particular SNPs were considered as being associated with type II diabetes when allele frequency differences calculated between case and control pools were statistically significant. SNP disease association results obtained from the allelotyping study were then validated by genotyping each associated SNP across all samples from each pool. The results of the genotyping were then analyzed, allele frequencies for each group were calculated from the individual genotyping results, and a p-value was calculated to determine whether the case and control groups had statistically significantly differences in allele frequencies for a particular SNP. When the genotyping results agreed with the original allelotyping results, the SNP disease association was considered validated at the genetic level. SNP Panel Used for Genetic Analyses- First Pass

[0212] The pools studied in the screen are described in Example 1. The SNPs analyzed in this study were part of a set of 25,488 SNPs confirmed as being statistically polymorphic as each is characterized as having a minor allele frequency of greater than 10%. The SNPs in the set reside in genes or in close proximity to genes, and many reside in gene exons. Specifically, SNPs in the set are located in exons, introns, and within 5,000 base-pairs upstream of a transcription start site of a gene. In addition, SNPs were selected according to the following criteria: they are located in ESTs; they are located in Locuslink or Ensembl genes; and they are located in Genomatix promoter predictions. An additional 3088 SNPs were included with these 25,488 SNPs and these additional SNPs had been chosen on the basis of gene location, with preference to non-synonymous coding SNPs located in disease candidate genes. SNPs in the set were also selected on the basis of even spacing across the genome, as depicted in Table 4.

TABLE 4

General Statistics Spacing Statistics

Total # of SNPs 25,488 Median 37,058 bp

# of Exonic SNPs >4,335 (17%) Minimum* 1,000 bp

# SNPs with refSNP ID 20,776 (81%) Maximum* 3,000,000 bp

Gene Coverage > 10,000 Mean 122,412 bp

Chromosome Coverage All Std Deviation 373,325 bp

^Excludes outliers

Genotyping Results

[0213] The genetic studies summarized above and described in more detail below identified allelic variants associated with type II diabetes. The allelic variant identified from the SNP panel described in Table 4 is summarized in Table IA.

Assay for Verifying. Allelotvping. and Genotvping SNPs

[0214] A MassARRAY™ system (Sequenom, Inc.) was utilized to perform SNP genotyping in a high-throughput fashion. This genotyping platform was complemented by a homogeneous, single- tube assay method (hME™ or homogeneous MassEXTEND™ (Sequenom, Inc.)) in which two genotyping primers anneal to and amplify a genomic target surrounding a polymorphic site of interest. A third primer (the MassEXTEND™ primer), which is complementary to the amplified target up to but not including the polymorphism, was then enzymatically extended one or a few bases through the polymorphic site and then terminated.

[0215] For each polymorphism, SpectroDESIGNER™ software (Sequenom, Inc.) was used to generate a set of PCR primers and a MassEXTEND™ primer which where used to genotype the polymorphism. Other primer design software could be used or one of ordinary skill in the art could manually design primers based on his or her knowledge of the relevant factors and considerations in designing such primers. Table 5 shows PCR primers and Table 6 shows extension probes used for analyzing the polymorphism set forth in Table 1. The initial PCR amplification reaction was performed in a 5 μl total volume containing IX PCR buffer with 1.5 mM MgC^ (Qiagen), 200 μM each of dATP, dGTP, dCTP, dTTP (Gibco-BRL), 2.5 ng of genomic DNA, 0.1 units of HotStar DNA polymerase (Qiagen), and 200 nM each of forward and reverse PCR primers specific for the polymorphic region of interest.

TABLE 5: PCR Primers

[0216] Samples were incubated at 95°C for 15 minutes, followed by 45 cycles of 95°C for 20 seconds, 56⁰C for 30 seconds, and 72°C for 1 minute, finishing with a 3 minute final extension at 72°C. Following amplification, shrimp alkaline phosphatase (SAP) (0.3 units in a 2 μl volume) (Amersham Pharmacia) was added to each reaction (total reaction volume was 7 μl) to remove any residual dNTPs that were not consumed in the PCR step. Samples were incubated for 20 minutes at 37°C, followed by 5 minutes at 85°C to denature the SAP.

[0217] Once the SAP reaction was complete, a primer extension reaction was initiated by adding a polymorphism-specific MassEXTEND™ primer cocktail to each sample. Each MassEXTEND™ cocktail included a specific combination of dideoxynucleotides (ddNTPs) and deoxy nucleotides (dNTPs) used to distinguish polymorphic alleles from one another. Methods for verifying, allelotyping and genotyping SNPs are disclosed, for example, in U.S. Pat. No. 6,258,538, the content of which is hereby incorporated by reference. In Table 6, ddNTPs are shown and the fourth nucleotide not shown is the dNTP.

TABLE 6: Extension Primers

[0218] The MassEXTEND™ reaction was performed in a total volume of 9 μl, with the addition of IX Thermo Sequenase buffer, 0.576 units of ThermoSequenase (Amersham Pharmacia), 600 nM MassEXTEND™ primer, 2 mM of ddATP and/or ddCTP and/or ddGTP and/or ddTTP, and 2 mM of dATP or dCTP or dGTP or dTTP. The deoxy nucleotide-(dNTP) used in the assay normally was complementary to the nucleotide at the polymorphic site in the amplicon. Samples were incubated at 94°C for 2 minutes, followed by 55 cycles of 5 seconds at 94°C, 5 seconds at 52°C, and 5 seconds at 72°C.

[0219] Following incubation, samples were desalted by adding 16 μl of water (total reaction volume was 25 μl), 3 mg of SpectroCLEAN™ sample cleaning beads (Sequenom, Inc.) and allowed to incubate for 3 minutes with rotation. Samples were then robotically dispensed using a piezoelectric dispensing device (SpectroJET™ (Sequenom, Inc.)) onto either 96-spot or 384-spot silicon chips containing a matrix that crystallized each sample (SpectroCHIP™ (Sequenom, Inc.)). Subsequently, MALDI-TOF mass spectrometry (Biflex and Autoflex MALDI-TOF mass spectrometers (Bruker Daltonics) can be used) and SpectroTYPER RT™ software (Sequenom, Inc.) were used to analyze and interpret the SNP genotype for each sample.

Genetic Analysis

[0220] The minor allelic frequency for the polymorphism set forth in Table 1 was verified as being 10% or greater using the extension assay described above in a group of samples isolated from 92 individuals originating from the state of Utah in the United States, Venezuela and France (Coriell cell repositories).

[0221] Genotyping results for the allelic variant set forth in Table 1 is shown for female pools in Table 7 and for male pools in Table 8. In Tables 7, "F case" and "F control" refer to female case and female control group allele frequencies, and in Table 8, "M case" and "M control" refer to male case and male control group allele frequencies.

TABLE 7: Female Genotyping Results

[0222] The single marker alleles set forth in Table 1 were considered validated, since the genotyping data for the females, males or both pools were significantly associated with type II diabetes, and because the genotyping results agreed with the original allelotyping results. Particularly significant associations with type II diabetes are indicated by a calculated p-value of less than 0.05 for genotype results, which are set forth in bold text. [0223] Odds ratio results are shown in Tables 7 and 8. An odds ratio is an unbiased estimate of relative risk which can be obtained from most case-control studies. Relative risk (RR) is an estimate of the likelihood of disease in the exposed group (susceptibility allele or genotype carriers) compared to the unexposed group (not carriers). It can be calculated by the following equation:

RR = /A//a

/A is the incidence of disease in the A carriers and /a is the incidence of disease in the non- carriers.

RR > 1 indicates the A allele increases disease susceptibility.

RR < 1 indicates the a allele increases disease susceptibility.

For example, RR = 1.5 indicates that carriers of the A allele have 1.5 times the risk of disease than non-carriers, i.e., 50% more likely to get the disease.

[0224] Case-control studies do not allow the direct estimation of /A and /a, therefore relative risk cannot be directly estimated. However, the odds ratio (OR) can be calculated using the following equation:

OR = (nDAnda)/(nd AnDa) = /?DA(l - pdA)//?dA(l - pDA), or

OR = ((case f) / (1- case f)) / ((control f) / (1 -control f)), where f = susceptibility allele frequency.

[0225] An odds ratio can be interpreted in the same way a relative risk is interpreted and can be directly estimated using the data from case-control studies, i.e., case and control allele frequencies. The higher the odds ratio value, the larger the effect that particular allele has on the development of breast cancer. Possessing an allele associated with a relatively high odds ratio translates to having a higher risk of developing or having type II diabetes.

Example 3 Samples and Pooling Strategies for the Replication Cohort

[0226] The single marker polymorphism set forth in Table 1 was genotyped again in two replication cohorts to further validate its association with type II diabetes. Like the original study population described in Examples 1 and 2, the replication cohorts consisted of type II diabetics (cases) and non-diabetics (controls). The case and control samples were selected and genotyped as described below.

Sample Selection and Pooling Strategies - Newfoundland

[0227] Blood samples were collected from individuals diagnosed with type II diabetes, which were referred to as case samples. Also, blood samples were collected from individuals not diagnosed with type II diabetes or a history of type II diabetes; these samples served as gender and age-matched controls. All of the samples were collected from individuals residing in Newfoundland, Canada. Residents of Newfoundland represent a preferred population for genetic studies because of their relatively small founder population and resulting homogeneity.

[0228] Genetic linkage studies from Newfoundland have proved particularly useful for mapping disease genes for both monogenic and complex diseases as evidenced in studies of autosomal dominant polycystic kidney disease, von Hippel-Lindau disease, ankylosing spondylitis, major depression, Grave's eye disease; retinitis pigmentosa, hereditary nonopolyposis colorectal cancer, Kallman syndrome, ocular albinism type I, late infantile type 2 neuronal ceroid lipofuscinosis, Bardet-Biedl syndrome, adenine phosphoriboysl-transferase deficiency, and arthropathy- camptodactyly syndrome, Familial multiple endocrine neoplasia type 1 (MENl). Thus Newfoundland's genetically enriched population provides a unique setting to rapidly identify disease-related genes in selected complex diseases.

[0229] Phenotypic trait information was gathered from individuals for each case and control sample, and genomic DNA was extracted from each of the blood samples for genetic analyses.

[0230] Samples were placed into one of four groups based on disease status. The four groups were female case samples, female control samples, male case samples, and male control samples. A select set of samples from each group were utilized to generate pools, and one pool was created for each group.

[0231] Patients were included in the case pools if a) they were diagnosed with type II diabetes as documented in their medical record, b) they were treated with either insulin or oral hypoglycemic agents, and c) they were of Caucasian ethnicity. Patients were excluded in the case pools if a) they were diabetic or had a history of diabetes, b) they suffered from diet controlled glucose intolerance, or c) they (or any their relatives) were diagnosed with MODY or gestational diabetes.

[0232] Phenotype information included, among others, patient ethnicity, country or origin of mother and father, diagnosis with type II diabetes (date of primary diagnosis, age of individual as of primary diagnosis), body weight, onset of obesity, retinopathy, glaucoma, cataracts, nephropathy, heart disease, hypertension, myocardial infarction, ulcers, required treatment (onset of insulin treatment, oral hypoglycemic agent), blood glucose levels, and MODY.

[0233] In total, the final selection consisted of 199 female cases, 241 Female Controls, 140 Male Case, and 62 Male Controls as set forth in Table 9. TABLE 9

Sample Selection and Pooling Strategies - Denmark

[0234] The polymorphism described in Table 5 was genotyped again in a second replication cohort, consisting of individuals of Danish ancestry, to further validate its association with type II diabetes. Blood samples were collected from individuals diagnosed with type II diabetes, which were referred to case samples. Also, blood samples were collected from individuals not diagnosed with type II diabetes or a history of type II diabetes; these samples served as gender and age- matched controls.

[0235] Phenotypic trait information was gathered from individuals for each case and control sample, and genomic DNA was extracted from each of the blood samples for genetic analyses.

[0236] Samples were placed into one of four groups based on disease status. The four groups were female case samples, female control samples, male case samples, and male control samples. A select set of samples from each group were utilized to generate pools, and one pool was created for each group.

[0237] In total, the final selection consisted of 197 female cases (average age 63) and 277 male cases (average age 60) as set forth in Table 10. All cases had been diagnosed with type II diabetes in their mid 50's, and were of Danish ancestry. Members selected for the cohort were recruited through the outpatient clinic at Steno Diabetes Center, Copenhagen. Diabetes was diagnosed according to the 1985 World Health Organization criteria. For the controls, 152 females (average age 50), and 136 males (average age 55) were selected. All control subjects underwent a 2-hour oral glucose tolerance test (OGTT) and were deemed to be glucose tolerant, and all were of Danish ancestry. In addition, all control subjects were living in the same area of Copenhagen as the type II diabetic patients.

[0238] Additional phenotype were measured in both the case and control group. Phenotype information included, among others, e.g. body mass index , waist/hip ratio, blood pressure, serum insulin, glucose, C-peptide, cholesterol, hdl, triglyceride, Hb_Aic, urine, creatinine, free fatty acids (mmol/1), GAD antibodies. TABLE 10

DNA Extraction from Blood Samples

[0239] Blood samples for DNA preparation were taken in 5 EDTA tubes. If it was not possible to get a blood sample from a patient, a sample from the cheek mucosa was taken. Red blood cells were lysed to facilitate their separation from the white blood cells. The white cells were pelleted and lysed to release the DNA. Lysis was done in the presence of a DNA preservative using an anionic detergent to solubilize the cellular components. Contaminating RNA was removed by treatment with an RNA digesting enzyme. Cytoplasmic and nuclear proteins were removed by salt precipitation.

[0240] Genomic DNA was then isolated by precipitation with alcohol (2-propanol and then ethanol) and rehydrated in water. The DNA was transferred to 2-ml tubes and stored at 4°C for short-term storage and at -7O⁰C for long-term storage.

Example 4 Association of Polymorphic Variants with Type II Diabetes in the Replication Cohort

[0241] The associated SNP from the initial scan was re-validated by genotyping the associated SNP across the replication cohort described in Example 3. The results of the genotyping were then analyzed, allele frequencies for each group were calculated from the individual genotyping results, and a p-value was calculated to determine whether the case and control groups had statistically significantly differences in allele frequencies for a particular SNP. The replication genotyping results were considered significant with a calculated p-value of less than 0.05 for genotype results, which are set forth in bold text. See Table 11 below.

Assay for Verifying. AUelotyping, and Genotyping SNPs

[0242] Genotyping of the replication cohort was performed using the same methods used for the original genotyping, as described herein. A MassARRAY™ system (Sequenom, Inc.) was utilized to perform SNP genotyping in a high-throughput fashion. This genotyping platform was complemented by a homogeneous, single-tube assay method (hME™ or homogeneous MassEXTEND™ (Sequenom, Inc.)) in which two genotyping primers anneal to and amplify a genomic target surrounding a polymorphic site of interest. A third primer (the MassEXTEND™ primer), which is complementary to the amplified target up to but not including the polymorphism, was then enzymatically extended one or a few bases through the polymorphic site and then terminated.

[0243] For each polymorphism, SpectroDESIGNER™ software (Sequenom, Inc.) was used to generate a set of PCR primers and a MassEXTEND™ primer which where used to genotype the polymorphism. Other primer design software could be used or one of ordinary skill in the art could manually design primers based on his or her knowledge of the relevant factors and considerations in designing such primers. Table 5 shows PCR primers and Table 6 shows extension probes used for analyzing {e.g., genotyping) polymorphisms. The initial PCR amplification reaction was performed in a 5 μl total volume containing IX PCR buffer with 1.5 mM MgCIj (Qiagen), 200 μM each of dATP, dGTP, dCTP, dTTP (Gibco-BRL), 2.5 ng of genomic DNA, 0.1 units of HotStar DNA polymerase (Qiagen), and 200 nM each of forward and reverse PCR primers specific for the polymorphic region of interest.

[0244] Samples were incubated at 95⁰C for 15 minutes, followed by 45 cycles of 95°C for 20 seconds, 56⁰C for 30 seconds, and 72°C for 1 minute, finishing with a 3 minute final extension at 72⁰C. Following amplification, shrimp alkaline phosphatase (SAP) (0.3 units in a 2 μl volume) (Amersham Pharmacia) was added to each reaction (total reaction volume was 7 μl) to remove any residual dNTPs that were not consumed in the PCR step. Samples were incubated for 20 minutes at 37⁰C, followed by 5 minutes at 85°C to denature the SAP.

[0245] Once the SAP reaction was complete, a primer extension reaction was initiated by adding a polymorphism-specific MassEXTEND™ primer cocktail to each sample. Each MassEXTEND™ cocktail included a specific combination of dideoxynucleotides (ddNTPs) and deoxynucleotides (dNTPs) used to distinguish polymorphic alleles from one another. Methods for verifying, allelotyping and genotyping SNPs are disclosed, for example, in U.S. Pat. No. 6,258,538, the content of which is hereby incorporated by reference. In Table 6, ddNTPs are shown and the fourth nucleotide not shown is the dNTP.

[0246] The MassEXTEND™ reaction was performed in a total volume of 9 μl, with the addition of IX ThermoSequenase buffer, 0.576 units of ThermoSequenase (Amersham Pharmacia), 600 nM MassEXTEND™ primer, 2 mM of ddATP and/or ddCTP and/or ddGTP and/or ddTTP, and 2 mM of dATP or dCTP or dGTP or dTTP. The deoxy nucleotide (dNTP) used in the assay normally was complementary to the nucleotide at the polymorphic site in the amplicon. Samples were incubated at 94°C for 2 minutes, followed by 55 cycles of 5 seconds at 94°C, 5 seconds at 52°C, and 5 seconds at 72⁰C. [0247] Following incubation, samples were desalted by adding 16 μl of water (total reaction volume was 25 μl), 3 mg of SpectroCLEAN™ sample cleaning beads (Sequenom, Inc.) and allowed to incubate for 3 minutes with rotation. Samples were then robotically dispensed using a piezoelectric dispensing device (SpectroJET™ (Sequenom, Inc.)) onto either 96-spot or 384-spot silicon chips containing a matrix that crystallized each sample (SpectroCHIP™ (Sequenom, Inc.)). Subsequently, MALDI-TOF mass spectrometry (Biflex and Autoflex MALDI-TOF mass spectrometers (Bruker Daltonics) can be used) and SpectroTYPER RT™ software (Sequenom, Inc.) were used to analyze and interpret the SNP genotype for each sample.

Genetic Analysis

[0248] The minor allelic frequency for the polymorphism set forth in Table 1 was verified as being 10% or greater using the extension assay described above in a group of samples isolated from 92 individuals originating from the state of Utah in the United States, Venezuela and France (Coriell cell repositories).

[0249] Replication genotyping results are shown for female and male pools in Table 11.

TABLE 11: Replication Genotyping Results

[0250] Meta-analysis was performed on rs 1947686 based on genotype results provided in Tables 7, 8 and 11. Figure 2 depicts the combined meta analysis odds ratio for rsl947686, across the discovery samples and replication samples (see Examples 1-4). The boxes are centered over the odds ratio for each sample, with the size of the box correlated to the contribution of each sample to the combined meta analysis odds ratio. The lines extending from each box are the 95% confidence interval values. The diamond is centered over the combined meta analysis odds ratio with the ends of the diamond depicting the 95% confidence interval values. The meta-analysis further illustrates the strong association the incident SNP has with type II diabetes across multiple case and control samples.

[0251] The subjects available for discovery from Germany included 498 cases and 498 controls. The subjects available for replication from Newfoundland included 350 type 2 diabetes cases and 300 controls. The subjects available for replication from Denmark included 474 type 2 diabetes cases and 287 controls. Meta analyses, combining only the results of both the Canadian and Danish replication sample, were carried out using a random effects (DerSimonian-Laird) procedure. The combined p-values and odd ratios are P < 0.096 and 1.15, respectively.

[0252] The absence of a statistically significant association in the replication cohort for males should not be interpreted as minimizing the value of the original finding. There are many reasons why a biologically derived association identified in a sample from one population would not replicate in a sample from another population. The most important reason is differences in population history. Due to bottlenecks and founder effects, there may be common disease predisposing alleles present in one population that are relatively rare in another, leading to a lack of association in the candidate region. Also, because common diseases such as diabetes are the result of susceptibilities in many genes and many environmental risk factors, differences in population- specific genetic and environmental backgrounds could mask the effects of a biologically relevant allele. For these and other reasons, statistically strong results in the original, discovery sample that did not replicate in the Newfoundland replication sample or the Denmark replication sample may be further evaluated in additional replication cohorts and experimental systems.

Example 5 PP2Ce/B3GALT3 Proximal SNPs

[0253] It has been discovered that polymorphic variations spanning a region that includes PP2Ce, a novel member of the protein phosphatase 2C family, and B3GALT3, a member of the beta-l,3-galactosyltransferase (beta3GalT) gene family, are associated with the occurrence of type II diabetes (see Examples 1-4). Thirty-two additional allelic variants proximal to rsl947686 were identified and subsequently allelotyped in diabetes case and control sample sets as described in Examples 1 and 2. The polymorphic variants are set forth in Table 12. The chromosome position provided in column four of Table 12 is based on Genome "Build 34" of NCBI's GenBank.

TABLE 12

Assay for Verifying and Allelotvping SNPs

[0254] The methods used to verify and allelotype the 32 proximal SNPs of Table 12 are the same methods described in Examples 1 and 2 herein. The primers and probes used in these assays are provided in Table 13 and Table 14, respectively.

TABLE 13

TABLE 14

16

Genetic Analysis

[0255] Allelotyping results are shown for female (F), male (M) and combined cases and controls in Table 15, 16 and 17, respectively. The allele frequency for the A2 allele is noted in the fifth and sixth columns for diabetes case pools and control pools, respectively, where "AF" is allele frequency. The allele frequency for the Al allele can be easily calculated by subtracting the A2 allele frequency from 1 (Al AF = 1-A2 AF). For example, the SNP rs 1580253 has the following case and control allele frequencies: case Al (T) = 0.697; case A2 (C) = 0.303; control Al (T) = 0.700; and control A2 (C) = 0.300, where the nucleotide is provided in paranthesis. Some SNPs are labeled "untyped" because of failed assays. Also, the SNP rs2231257 codes for an amino acid change D126N in B3GALT3, where the asparagine is associated with an increased risk of type II diabetes.

TABLE 15: Female Allelotyping Results

TABLE 16: Male Allelotyping Results

[0256] Allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold. The allelotyping p-values were plotted in Figures 1 A-C for females, males and combined, respectively. The position of each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group. The minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in Figures IA-C can be determined by consulting Tables 15 and 16. For example, the left-most X on the left graph is at position 162088063. By proceeding down the Table from top to bottom and across the graphs from left to right the allele frequency associated with each symbol shown can be determined.

[0257] To aid the interpretation, multiple lines have been added to the graph. The broken horizontal lines are drawn at two common significance levels, 0.05 and 0.01. The vertical broken lines are drawn every 20kb to assist in the interpretation of distances between SNPs. Two other lines are drawn to expose linear trends in the association of SNPs to the disease. The light gray line (or generally bottom-most curve) is a nonlinear smoother through the data points on the graph using a local polynomial regression method (W. S. Cleveland, E. Grosse and W.M. Shyu (1992) Local regression models. Chapter 8 of Statistical Models in S eds J.M. Chambers and T.J. Hastie, Wadsworth & Brooks/Cole.). The black line (or generally top-most curve, e.g., see peak in left¬ most graph just to the left of position 92150000) provides a local test for excess statistical significance to identify regions of association. This was created by use of a 10kb sliding window with lkb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than 10^'8 were truncated at that value.

[0258] Finally, the exons and introns of the genes in the covered region are plotted below each graph at the appropriate chromosomal positions. The gene boundary is indicated by the broken horizontal line. The exon positions are shown as thick, unbroken bars. An arrow is place at the 3' end of each gene to show the direction of transcription. Proximal SNP Replication

[0259] The proximal SNPs disclosed above were also allelotyped in the Newfoundland replication cohort described in Examples 3 and 4. Allelotyping results are shown for female (F), male (M), and combined cases and controls in Table 18, 19 and 20 respectively. The allele frequency for the A2 allele is noted in the fifth and sixth columns for diabetes case pools and control pools, respectively, where "AF" is allele frequency. The allele frequency for the Al allele can be easily calculated by subtracting the A2 allele frequency from 1 (Al AF = 1-A2 AF). Some SNPs may be labeled "untyped" because of failed assays.

TABLE 18: Female Replication Allelotyping Results

[0260] Allelotyping results were considered particularly significant with a calculated p-value of less than or equal to 0.05 for allelotype results. These values are indicated in bold. The allelotyping p-values were plotted in Figures 1 D-F for females, males and combined, respectively. The position of each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group. The minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in Figures ID-F can be determined by consulting Tables 18, 19 and 20 For example, the left-most X on the left graph is at position 162088063. By proceeding down the Table from top to bottom and across the graphs from left to right the allele frequency associated with each symbol shown can be determined. Genotyping

[0261] Three proximal SNPs set forth in Table 12 were also genotyped in diabetic case and control samples as described in Examples 1 and 2. These three proximal SNPs (rs898685, rs898683 and 2279106) were genotyped using the same PCR primers, extend primer and termination mix used to allelotype the SNPs (see Tables 13 and 14). The combined genotyping results for female (F) and male (M) cases and controls are shown in Table 21. Allele frequency is noted in the fourth and fifth columns for type II diabetics and controls.

TABLE 21: Male Genotyping Results

[0262] Each SNP is significantly associated with disease status (Table 21), and marker genotype frequencies approximate Hardy- Weinberg equilibrium (HWE) (Table 22).

TABLE 22: Hardy- Weinberg equilibrium

Haplotype Analysis

[0263] Haplotype frequencies, linkage disequilibrium statistics, and haplotype association tests are presented for each set of markers. Pairwise LD statistics estimated for rs898683, rs898685, and rs2279106 are highly significant (Table 23), indicating very strong LD between each pair of loci.

TABLE 23: Pairwise LD Statistics (r^Λ2)

[0264] Haplotypes are also significantly associated with diabetes when males and females are analyzed together (Tables 24 and 25). The general distribution of haplotypes among cases and controls is approximately the same in each subgroup, with the "GTC" haplotype occurring at the highest frequency among cases.

TABLE 24: Test of Association between Haplotype and type II diabetes

TABLE 25: Test of Association between Haplotype and type II diabetes (most frequent haplotypes)

Pearson X" (w/Yates') = 6.565, df = 1, p-value = 0.0104

[0265] The two most frequent haplotypes (AAT and GTC) are both significantly associated with type II diabetes as set forth in Table 25 (p-value of 0.0104). Thus, for the haplotype defined by positions 15913, 16109 and 22192 in SEQ ID NO: 1, the AAT haplotype is protective against type II diabetes and individuals having the GTC haplotype are at risk of type II diabetes.

Example 6 Isolation of a cDNA that Codes for the Human Homolog of the Mouse PP2Ce Gene

[0266] The mouse PP2Ce gene was previously characterized as an open reading frame (ORF) coding for a 360 amino acid protein. Two relevant putative cDNA sequences were discovered that represent human homologs. The first putative transcript contained an ORF that coded for an amino acid sequence that aligned, with very high homology, to the first 191 amino acids of the mouse protein (see SEQ ID NO: 9 alignment hereafter). After this homologous coding region, the DNA sequence diverges significantly. The second putative transcript contained an ORF that coded for an amino acid sequence that aligned, with very high homology, to the last 227 amino acids of the mouse protein. This homologous coding region is preceded by significantly divergent DNA sequence. These two reading frames code for a perfectly overlapping 58 amino acids sequence. The high homology at the DNA level through this overlapping coding region suggested that these two transcripts are mRNA splice variants of each other.

[0267] To demonstrate that a third mRNA splice variant, that codes for a human homolog of the entire mouse PP2Ce gene, exists, PCR primers were designed from the two previously described transcripts: 5'Primer; 5'GTCGCTGGTGGTGGTTS'; 3'Primer,

5'TGTTGAAAGTCCTTTAGTCTAAa'. AS a template source for PCR amplification, a mixture of cDNAs was used created from 56 established cell lines. Gel electrophoresis of the PCR reaction demonstrated the amplification of a single DNA band of the expected size (see SEQ ID NO: 2). Amplification of this band required the presence of both primers in the PCR reaction. Isolation and DNA sequencing of this band confirmed it as the predicted DNA sequence coding for a full length human homolog of the mouse PP2Ce protein. The human ORF codes for a 360 amino acid protein, with four alternate amino acids relative to the mouse ORF. This makes the predicted human gene product 99% identical to the corresponding mouse gene product.

[0268] Analysis of the in vitro phosphatase activity of the mouse PP2Ce protein demonstrates that the integrity of at least four conserved motifs of the PP2C family of phosphatases must be maintained for phosphatase function (Li, M.G., et. al., J. Bio. Chem., 278(14):12013-21, 2003). Because of the high degree of conserved identity between the mouse and human gene product, it is believed that the same conserved motifs are required for human PP2Ce function. Because neither of the previously described putative human PP2Ce transcripts code for all four of these conserved motifs, it is expected that they are nonfunctional. In addition, it is believed that the expression of truncated peptides might affect the final overall efficiency of the full length human PP2Ce gene product by competing for substrates or cofactors in a dominantly negative fashion.

Example 7 In Vitro Tests of Metabolic-Related Activity

[0269] In vitro assays described hereafter are useful for identifying therapeutics for treating human diabetes. As used in Examples hereafter directed to in vitro assays, rodent models and studies in humans, the term "test molecule" refers to a molecule that is added to a system, where an agonist effect, antagonist effect, or lack of an effect of the molecule on PP2Ce or B3GALT3 function or a related physiological function in the system is assessed. An example of a test molecule is a test compound, such as a test compound described in the section "Compositions Comprising Diabetes-Directed Molecules" above. Another example of a test molecule is a test peptide, which includes, for example, a PP2Ce or B3GALT3-related test peptide such as a soluble, extracellular form of PP2Ce or B3GALT3, a biologically active fragment of PP2Ce or B3GALT3, a PP2Ce or B3GALT3 binding partner or ligand, or a functional fragment of the foregoing. A concentration range or amount of test molecule utilized in the assays and models is selected from a variety of available ranges and amounts. For example, a test molecule sometimes is introduced to an assay system in a concentration range between 1 nanomolar and 100 micromolar or a concentration range between 1 nanograms/mL and 100 micrograms/mL. An effect of a test molecule on PP2Ce or B3GALT3 function or a related physiological function often is determined by comparing an effect in a system administered the test molecule against an effect in system not admininstered the test molecule. Described directly hereafter are examples of in vitro assays.

Glucose Uptake Assay

[0270] One of the many responses of adipocytes and muscle cells after exposure to insulin is the transport of glucose intracellularly. This transport is mediated by GLUT4, an insulin-regulatable glucose transporter. Insulin binding to insulin receptors on the cell surface results in autophosphorylation and activation of the intrinsic tyrosine kinase activity of the insulin receptor. Phosphorylated tyrosine residues on the insulin receptor and its endogenous targets activate several intracellular signaling pathways that eventually lead to the translocation of GLUT4 from intracellular stores to the extracellular membrane.

Methods

[0271] Cells are plated in 6-well dishes, and grown to confluency. Cells are then differentiated with DMEM plus 10% fetal calf serum (FCS), 10 ug/mL insulin, 390 ng/mL dexamethasone and 112 ug/mL isobutylmethylxanthine for 2 days. After 2 days of differentiation, media is changed to maintenance media DMEM plus 10% FCS and 5 ug/mL insulin. Media is changed every 2 days thereafter. Cells are assayed for insulin-mediated glucose uptake 10 days after differentiation. On the day of the assay, cells are washed once with PBS, and serum starved by adding 2 mL of DMEM plus 2mg/mL BSA for 3 hours. During serum starvation, recombinant rat PP2Ce or B3GALT3/Fc chimeric ligand is preclustered. In a solution of PBS plus 2 mg/mL BSA, recombinant rat PP2Ce or B3GALT3/Fc chimeria is added to a concentration of 1.75 ug/mL, and anti-human IgG, Fc γ fragment specific antibody to a final concentration of 17.5 ug/mL. After 3 hours of serum starvation, media is replaced with 2 mL of preclustered PP2Ce or B3GALT3, and incubated for 10, 40 and 90 min at 37 deg. After 10 min, porcine insulin is added to a final concentration of 100 nM for 10 min at 37 deg. For every 2 mL of media, 100 uL of PBS-2-DOG label is added to give a final concentration of 2 uCi. Cells are immediately placed on ice, washed three times with ice cold PBS, and lysed with 0.7 mL of 0.2 N NaOH. Lysates are read in a Wallac 1450 Microbeta Liquid Scintillation and Luminescence Counter.

Example 8 Triacylglycerol (TG) Assay

[0272] A direct metabolic consequence of glucose transport intracellularly is its incorporation into the fatty acid and glycerol moieties of triacylglycerol (TG). TGs are highly concentrated stores of metabolic energy, and are the major energy reservoir of cells. In mammals, the major site of accumulation of triacylglycerols is the cytoplasm of adipose cells. Adipocytes are specialized for the synthesis, and storage of TG, and for their mobilization into fuel molecules that are transported to other tissues through the bloodstream. It is likely that changes in the transport of glucose intracellularly can affect cytoplasmic stores of triacylglycerols.

Methods

[0273J Cells are plated in 6-well dishes, and grown to confluency. When cells reached confluency, cells are differentiated with DMEM plus 10% fetal calf serum (FCS), 10 ug/mL insulin, 390 ng/mL dexamethasone and 112 ug/mL isobutylmethylxanthine for 2 days. After 2 days of differentiation, media is changed to maintenance media DMEM plus 10% FCS and 5 ug/mL insulin. On the day of the assay (day 9 post-differentiation), cells are washed once with PBS, and serum starved by adding 2 mL of DMEM plus 2 mg/mL BSA for 3 hours. During serum starvation, recombinant rat PP2Ce or B3GALT3/Fc chimeric ligand is preclustered. In a solution of PBS plus 2 mg/mL BSA recombinant rat PP2Ce or B3GALT3/Fc chimeria is added to a concentration of 1.75 ug/mL, and anti-hiiman IgG, Fc γ fragment specific antibody to a final concentration of 17.5 ug/mL. After 3 hours of serum starvation, media is replaced with pre-clustered PP2Ce or B3GALT3 solution, and incubated for 10 minutes at 37 degrees. Cells are then treated with 100 nM porcine insulin for 2 hours at 37 degrees. Cells are immediately placed on ice, and washed twice with ice cold PBS. Cells are lysed with 1% SDS, 1.2 mM Tris, pH 7.0 and heat treated at 95 degrees for 5 minutes. Samples are assayed using INFINITY Tryglyceride reagent. In a 96-well, flat bottom, transparent microliter plate, 3 uL of sample are added to 300 uL of INFINITY Triglyceride Reagent. Samples are incubated at room temperature for 10 minutes. The assay is read at 500-550 nm.

Example 9 Quantitative Assessment of mResistin Levels

[0274] Resistin is a secreted factor specifically expressed in white adipocyte. It was initially discovered in a screen for genes downregulated in adipocytes by PPAR gamma, and expression was found to be attenuated by insulin. Elevated levels of resistin have been measured in genetically obese, and high fat fed obese mice. It is therefore thought that resistin contributes to peripheral tissue insulin unresponsiveness, one of the pathological hallmarks of diabetes.

Methods

[0275] 3T3-L1 cells are differentiated for 3 days as previously described and maintained for three days prior to splitting. At day 5 post-differentiation, differentiated cells are plated in 10 cm dish at a cell density of 3X10⁶ cells. Cells are then serum starved on day 7 after initiation of differentiation. On day 8, cells are treated with pre-clustered recombinant rat PP2Ce or B3GALT3/Fc chimera as described above for 10 min and treated with 10 nM insulin for 2 hours. Cells are harvested, mRNA extracted using magnetic DYNAL beads and reverse transcribed to cDNA using Superscript First-Strand Synthesis as described by the manufacturer. Appropriate primers are designed and used in 15 uL PCR reaction using 55 deg annealing temperature and 30 cycles of amplification.

Example 10 Effect on Muscle Differentiation

[0276] C2C12 cells (murine skeletal muscle cell line; ATCC CRL 1772, Rockville, MD) are seeded sparsely (about 15-20%) in complete DMEM (w/glutamine, pen/strep, etc) + 10% FCS. Two days later they become 80-90% confluent. At this time, the media is changed to DMEM+2% horse serum to allow differentiation. The media is changed daily. Abundant myotube formation occurs after 3-4 days of being in 2% horse serum, although the exact time course of C2C12 differentiation depends on how long they have been passaged and how they have been maintained, among other factors.

[0277] To test the effect of the presence of test molecules on muscle differentiation, test molecules (e.g., test peptides added in a range of 1 to 2.5 μg/mL) are added the day after seeding when the cells are still in DMEM with 10% FCS. Two days after plating the cells (one day after the test molecule was first added), at about 80-90% confluency, the media is changed to DMEM+2% horse serum plus the test molecule.

Effect on Muscle Cell Fatty Acid Oxidation

[0278] C2C12 cells are differentiated in the presence or absence of 2 μg/mL test molecules for 4 days. On day 4, oleate oxidation rates are determined by measuring conversion of l-¹⁴C-oleate (0.2 mM) to ¹⁴Cθ₂ for 90 min. This experiment can be used to screen for active polypeptides and peptides as well as agonists and antagonists or activators and inhibitors of PP2Ce or B3GALT3 polypeptides or binding partners.

[0279] The effect of test molecules on the rate of oleate oxidation can be compared in differentiated C2C12 cells (murine skeletal muscle cells; ATCC, Manassas, VA CRL-1772) and in a hepatocyte cell line (Hepal-6; ATCC, Manassas, VA CRL-1830). Cultured cells are maintained according to manufacturer's instructions. The oleate oxidation assay is performed as previously described (Muoio et al (1999) Biochem J 338;783-791). Briefly, nearly confluent myocytes are kept in low serum differentiation media (DMEM, 2.5% Horse serum) for 4 days, at which time formation of myotubes becomes maximal. Hepatocytes are kept in the same DMEM medium supplemented with 10% FCS for 2 days. One hour prior to the experiment the media is removed and 1 mL of preincubation media (MEM, 2.5% Horse serum, 3 mM glucose, 4 mM Glutamine,

25 mM Hepes, 1% FFA free BSA, 0.25 mM Oleate, 5 μg/mL gentamycin) is added. At the start of the oxidation experiment ¹⁴C-Oleic acid (lμCi/mL, American Radiolabeled Chemical Inc., St. Louis, MO) is added and cells are incubated for 90 min at 37⁰C in the absence/presence of test molecule (e.g., 2.5 μg/mL of PP2Ce or B3GALT3-τelated test peptide). After the incubation period 0.75 mL of the media is removed and assayed for ¹⁴C-oxidation products as described below for the muscle FFA oxidation experiment.

Triglyceride and Protein Analysis following Oleate Oxidation in Cultured Cells [0280] Following transfer of media for oleate oxidation assay, cells are placed on ice. To determine triglyceride and protein content, cells are washed with 1 mL of Ix PBS to remove residual media. To each well 300 μL of cell dissociation solution (Sigma) is added and incubated at 37⁰C for 10 min. Plates are tapped to loosen cells, and 0.5 mL of Ix PBS is added. The cell suspension is transferred to an Eppendorf tube, each well is rinsed with an additional 0.5 mL of Ix PBS, and is transferred to the appropriate Eppendorf tube. Samples are centrifuged at 1000 rpm for 10 minutes at room temperature. Each supernatant is discarded and 750 μL of Ix PBS/2% CHAPS is added to cell pellet. The cell suspension is vortexed and placed on ice for 1 hour. Samples are then centrifuged at 13000 rpm for 20 min at 4⁰C. Each supernatant is transferred to a new tube and frozen at -2O⁰C until analyzed. Quantitative measure of triglyceride level in each sample is determined using Sigma Diagnostics GPO-TRINDER enzymatic kit. The procedure outlined in the manual is followed, with the following exceptions: the assay is performed in 48 well plate, 350 μL of sample volume is assayed, a control blank consists of 350 μL PBS/2% CHAPS, and a standard contains 10 μL standard provide in the kit with 690 μL PBS/2% CHAPS. Analysis of samples is carried out on a Packard Spectra Count at a wavelength of 550 nm. Protein analysis is carried out on 25 μL of each supernatant sample using the BCA protein assay (Pierce) following manufacturer's instructions. Analysis of samples is carried out on a Packard Spectra Count at a wavelength of 550 nm.

Stimulation of insulin secretion in HIT-Tl 5 cells

[0281] HIT-T 15 (ATCC CRL#1777) is an immortalized hamster insulin-producing cell line. It is known that stimulation of cAMP in HIT-Tl 5 cells causes an increase in insulin secretion when the glucose concentration in the culture media is changed from 3mM to 15 mM. Thus, test molecules also are tested for their ability to stimulate glucose-dependent insulin secretion (GSIS) in HIT-Tl 5 cells. In this assay, 30,000 cells/well in a 12-well plate are incubated in culture media containing 3 mM glucose and no serum for 2 hours. The media is then changed, wells receive media containing either 3 mM or 15 mM glucose, and in both cases the media contains either vehicle (DMSO) or test molecule at a concentration of interest. Some wells receive media containing 1 micromolar forskolin as a positive control. All conditions are tested in triplicate. Cells are incubated for 30 minutes, and the amount of insulin secreted into the media is determined by ELISA, using a kit from either Peninsula Laboratories (Cat # ELIS-7536) or Crystal Chem Inc. (Cat # 90060).

Stimulation of insulin secretion in isolated rat islets

[0282] As with HIT-Tl 5 cells, it is known that stimulation of cAMP in isolated rat islets causes an increase in insulin secretion when the glucose concentration in the culture media is changed from 60 mg/dl to 300 mg/dl. Ligands are tested for their ability to stimulate GSIS in rat islet cultures. This assay is performed as follows:

1. Select 75-150 islet equivalents (IEQ) for each assay condition using a dissecting microscope. Incubate overnight in low-glucose culture medium. (Optional.)

2. Divide the islets evenly into triplicate samples of 25-40 islet equivalents per sample. Transfer to 40 μm mesh sterile cell strainers in wells of a 6-well plate with 5 ml of low (60 mg/dl) glucose Krebs-Ringers Buffer (KRB) assay medium.

3. Incubate 30 minutes (1 hour if overnight step skipped) at 37° C and 5% CO₂. Save the supernatants if a positive control for the RIA is desired.

4. Move strainers with islets to new wells with 5ml/well low glucose KRB. This is the second pre-incubation and serves to remove residual or carryover insulin from the culture medium. Incubate 30 minutes.

5. Move strainers to next wells (Low 1) with 4 or 5 ml low glucose KRB. Incubate at 37° C for 30 minutes. Collect supernatants into low-binding polypropylene tubes pre- labelled for identification and keep cold.

6. Move strainers to high glucose wells (300mg/dl, which is equivalent to 16.7mM). Incubate and collect supernatants as before. Rinse islets in their strainers in low- glucose to remove residual insulin. If the rinse if to be collected for analysis, use one rinse well for each condition (i.e. set of triplicates.)

7. Move strainers to final wells with low-glucose assay medium (Low 2). Incubate and collect supernatants as before.

8. Maintaining a cold temperature, centrifuge supernatants at 1800rpm for 5 minutes at 4- 8°C to remove small islets/islet pieces that escape the 40mm mesh. Remove all but lower 0.5 - 1 ml and distribute in duplicate to pre-labelled low-binding tubes. Freeze and store at <-20° C until insulin concentrations can be determined. 9. Insulin determinations are performed as above, or by Linco Labs as a custom service, using a rat insulin RIA (Cat. # RI- 13K).

Example 11 Effect of PP2Ce or B3GALT3-Related Test Peptides on Mice Fed a High-Fat Diet

[0283] Following is a representative rodent model for identifying thereapeutics for treating human diabetes. Experiments are performed using approximately 6 week old C57B1/6 mice (8 per group). All mice are housed individually. The mice are maintained on a high fat diet throughout each experiment. The high fat diet (cafeteria diet; D12331 from Research Diets, Inc.) has the following composition: protein kcal% 16, sucrose kcal% 26, and fat kcal% 58. The fat is primarily composed of coconut oil, hydrogenated.

[0284] After the mice are fed a high fat diet for 6 days, micro-osmotic pumps are inserted using isoflurane anesthesia, and are used to provide test molecule, saline, and a control molecule (e.g., an irrelevant peptide) to the mice subcutaneously (s.c.) for 18 days. For example, PP2Ce or B3GALT3-τelated test peptides are provided at doses of 100, 50, 25, and 2.5 μg/day and an irrelevant peptide is provided at 10 μg/day. Body weight is measured on the first, third and fifth day of the high fat diet, and then daily after the start of treatment. Final blood samples are taken by cardiac puncture and are used to determine triglyceride (TG), total cholesterol (TC), glucose, leptin, and insulin levels. The amount of food consumed per day is also determined for each group.

Example 12 In vivo Effects of Test Molecules on Glucose Homeostasis in Mice

[0285] Following are representative rodent models for identifying thereapeutics for treating human diabetes.

Oral Glucose tolerance test (oGTT)

[0286] Male C57bl/6N mice at age of 8 weeks are fasted for 18 hours and randomly grouped (n=l 1) to receive an PP2Ce or B3GALT3-related test peptide, a test molecule at indicated doses, or with control extendin-4 (ex-4, 1 mg/kg), a GLP-I peptide analog known to stimulate glucose- dependent insulin secretion. Thirty minutes after administration of PP2Ce or B 3GALT3 -related test peptides, test compound and control ex-4, mice are administered orally with dextrose at 5 g/kg dose. Test molecule is delivered orally via a gavage needle (p.o. volume at 100 ml). Control Ex-4 is delivered intraperitoneally. Levels of blood glucose are determined at regular time points using Glucometer Elite XL (Bayer). Acute response of db mice to test molecule

[0287] Male db mice (C57BL/KsOlahsd-Leprdb, diabetic, Harlan) at age of 10 weeks are randomly grouped (n=6) to receive vehicle (oral gavage), PP2Ce or B3GALT3-related test peptides (at concentration of interest), test molecule (e.g., 60 mg/kg, or another concentration of interest, oral gavage), or Ex-4 (1 mg/kg, intraperitoneally). After peptide and/or compound administration, food is removed and blood glucose levels are determined at regular time intervals. Reduction in blood glucose at each time point may be expressed as percentage of original glucose levels, averaged from the number of animals for each group. Results show the effect PP2Ce or B3GALT3-related test peptides and test molecules for improving glucose homeostasis in diabetic animals.

Example 13 Effect of Test Molecules on Plasma Free Fatty Acid in C57 BL/6 Mice

[0288] Following is a representative rodent model for identifying thereapeutics for treating human diabetes. The effect of test molecules on postprandial lipemia (PPL) in normal C57BL6/J mice is tested.

[0289] The mice used in this experiment are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken. All blood samples are taken from the tail using EDTA coated capillary tubes (50 μL each time point). At time 0 (8:30 AM), a standard high fat meal (6g butter, 6 g sunflower oil, 1O g nonfat dry milk, 1O g sucrose, 12 mL distilled water prepared fresh following Nb#6, JF, pg.1) is given by gavage (vol.=l% of body weight) to all animals.

[0290] Immediately following the high fat meal, a test molecule is injected i.p. in 100 μL saline (e.g., 25μg of test peptide). The same dose (25μg/mL in lOOμL) is again injected at 45 min and at 1 hr 45 min. Control animals are injected with saline (3xl00μL). Untreated and treated animals are handled in an alternating mode.

[0291] Blood samples are taken in hourly intervals, and are immediately put on ice. Plasma is prepared by centrifugation following each time point. Plasma is kept at -20⁰C and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako). Due to the limited amount of plasma available, glucose is determined in duplicate using pooled samples. For each time point, equal volumes of plasma from all 8 animals per treatment group are pooled.

Example 14 Effect of Test Molecules on Plasma FFA. TG and Glucose in C57 BL/6 Mice

[0292] Following is a representative rodent model for identifying thereapeutics for treating human diabetes. The experimental procedure is similar to that described in Example 13. Briefly, 14 mice are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken.

All blood samples are taken from the tail using EDTA coated capillary tubes (50 μL each time point). At time 0 (9:00AM), a standard high fat meal (see Example 4) is given by gavage (vol.=l% of body weight) to all animals. Immediately following the high fat meal, 4 mice are injected with a test molecule i.p. in lOOμL saline (e.g., 25 μg of test peptide). The same dose is again injected at 45 min and at 1 hr 45 min. A second treatment group receives 3 times a higher amount of the test molecule (e.g., 50 μg of test peptide) at the same intervals. Control animals are injected with saline (e.g., 3xl00μL). Untreated and treated animals are handled in an alternating mode.

[0293] Blood samples are immediately put on ice. Plasma is prepared by centrifugation following each time point. Plasma is kept at -20 ⁰C and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako).

Example 15 IEffect of Test Molecules on FFA following Epinephrine Injection

[0294] Following is a representative rodent model for identifying thereapeutics for treating human diabetes. In mice, plasnia free fatty acids increase after intragastric administration of a high fat/sucrose test meal. These free fatty acids are mostly produced by the activity of lipolytic enzymes i.e. lipoprotein lipase (LPL) and hepatic lipase (HL). In this species, these enzymes are found in significant amounts both bound to endothelium and freely circulating in plasma. Another source of plasma free fatty acids is hormone sensitive lipase (HSL) that releases free fatty acids from adipose tissue after β-adrenergic stimulation. To test whether test molecules also regulate the metabolism of free fatty acid released by HSL, mice are injected with epinephrine.

[0295] Two groups of mice are given epinephrine (5μg) by intraperitoneal injection. A treated group is injected with a test molecule (e.g., 25μg of test peptide) one hour before and again together with epinephrine, while control animals receive saline. Plasma is isolated and free fatty acids and glucose are measured as described above.

Example 16 Effect of Test Molecules on Muscle FFA Oxidation

[0296] Following is a representative rodent model for identifying thereapeutics for treating human diabetes. To investigate the effect of test molecules on muscle free fatty acid oxidation, intact hind limb muscles from C57BL/6J mice are isolated and FFA oxidation is measured using oleate as substrate (Clee, S. M. et al. Plasma and vessel wall lipoprotein lipase have different roles in atherosclerosis. J Lipid Res 41, 521-531 (2000); Muoio, D. M., Dohm, G. L., Tapscott, E. B. & Coleman, R. A. Leptin opposes insulin's effects on fatty acid partitioning in muscles isolated from obese ob/ob mice. Am J Physiol 276, E913-921 (1999)) Oleate oxidation in isolated muscle is measured as previously described (Cuendet et al (1976) J Clin Invest 58:1078-1088; Le Marchand- Brustel, Y., Jeanrenaud, B. & Freychet, P. Insulin binding and effects in isolated soleus muscle of lean and obese mice. Am J Physiol 234, E348-E358 (1978). Briefly, mice are sacrificed by cervical dislocation and soleus and EDL muscles are rapidly isolated from the hind limbs. The distal tendon of each muscle is tied to a piece of suture to facilitate transfer among different media. All incubations are carried out at 3O⁰C in 1.5 mL of Krebs-Henseleit bicarbonate buffer (118.6 mM NaCl, 4.76 mM KCl, 1.19 mM KH₂PO₄, 1.19 mM MgSO₄, 2.54 mM CaCl₂, 25mM NaHCO₃, 10 mM Hepes, pH 7.4) supplemented with 4% FFA free bovine serum albumin (fraction V, RIA grade, Sigma) and 5 mM glucose (Sigma). The total concentration of oleate (Sigma) throughout the experiment is 0.25 mM. All media are oxygenated (95% O₂; 5% CO₂) prior to incubation. The gas mixture is hydrated throughout the experiment by bubbling through a gas washer (Kontes Inc., Vineland, NJ).

[0297] Muscles are rinsed for 30 min in incubation media with oxygenation. The muscles are then transferred to fresh media (1.5 mL) and incubated at 30⁰C in the presence of lμCi/mL [1-¹⁴C] oleic acid (American Radiolabeled Chemicals). The incubation vials containing this media are sealed with a rubber septum from which a center well carrying a piece of Whatman paper (1.5 cm x 11.5 cm) is suspended.

[0298] After an initial incubation period of lOmin with constant oxygenation, gas circulation is removed to close the system to the outside environment and the muscles are incubated for 90 min at 3O⁰C. At the end of this period, 0.45 mL of Solvable (Packard Instruments, Meriden, CT) is injected onto the Whatman paper in the center well and oleate oxidation by the muscle is stopped by transferring the vial onto ice.

[0299] After 5 min, the muscle is removed from the medium, and an aliquot of 0.5 mL medium is also removed. The vials are closed again and 1 mL of 35% perchloric acid is injected with a syringe into the media by piercing through the rubber septum. The CO₂ released from the acidified media is collected by a Solvable in the center well. After a 90 min collection period at 30⁰C, the Whatman paper is removed from the center well and placed in scintillation vials containing 15 mL of scintillation fluid (HionicFlour, Packard Instruments, Meriden, CT). The amount of ¹⁴C radioactivity is quantitated by liquid scintillation counting. The rate of oleate oxidation is expressed as nmol oleate produced in 90min/g muscle.

[0300] To test the effect of test molecules on oleate oxidation, the each test molecule is added to the media (e.g., a final concentration of 2.5 μg/mL of test peptide) and maintained in the media throughout the procedure.

Example 17 Effect of Test Molecules on FFA following Intralipid Injection

[0301] Following is a representative rodent model for identifying thereapeutics for treating human diabetes. Two groups of mice are intravenously (tail vein) injected with 30 μL bolus of Intralipid-20% (Clintec) to generate a sudden rise in plasma FFAs, thus by-passing intestinal absorption. (Intralipid is an intravenous fat emulsion used in nutritional therapy). A treated group (treated with test molecule) is injected with a test molecule (e.g., 25μg of a test peptide) at 30 and 60 minutes before Intralipid is given, while control animals receive saline. Plasma is isolated and FFAs are measured as described previously. The effect of a test molecule on the decay in plasma FFAs following the peak induced by Intralipid injection is then monitored.

Example 18 In Vivo Tests for Metabolic-related Activity in Rodent Diabetes Models

[0302] Following are representative rodent models for identifying thereapeutics for treating human diabetes. As metabolic profiles differ among various animal models of obesity and diabetes, analysis of multiple models is undertaken to separate the effects of test molecules on hyperglycemia, hyperinsulinemia, hyperlipidemia and obesity. Mutations within colonies of laboratory animals and different sensitivities to dietary regimens have made the development of animal models with non-insulin dependent diabetes associated with obesity and insulin resistance possible. Genetic models such as db/db and ob/ob (See Diabetes, (1982) 31(1): 1-6) in mice and fa/fa in zucker rats have been developed by the various laboratories for understanding the pathophysiology of disease and testing the efficacy of new antidiabetic compounds (Diabetes, (1983) 32: 830-838; Annu Rep Sankyo Res Lab (1994) 46: 1-57). The homozygous animals, C57 BL/KsJ-db/db mice developed by Jackson Laboratory, US, are obese, hyperglycemic, hyperinsulinemic and insulin resistant (J Clin Invest, (1990) 85: 962-967), whereas heterozygous animals are lean and normoglycemic. The db/db mice progressively develop insulinopenia with age, a feature commonly observed in late stages of human type II diabetes when blood sugar levels are insufficiently controlled. The state of the pancreas and its course vary according to the models. Since this is a model of type II diabetes mellitus, test molecules are tested for blood sugar and triglycerides lowering activities. Zucker (fa/fa) rats are severely obese, hyperinsulinemic, and insulin resistant (Coleman, Diabetes 31 :1, 1982; E. Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte, Jr. Eds. (Elsevier Science Publishing Co., Inc., New York, ed. 4, 1990), pp. 299-340), and the fa/fa mutation may be the rat equivalent of the murine db mutation (Friedman et al., Cell 69:217- 220, 1992; Truett et al., Proc. Natl. Acad. Sci. USA 88:7806, 1991). Tubby (tub/tub) mice are characterized by obesity, moderate insulin resistance and hyperinsulinemia without significant hyperglycemia (Coleman et al., J. Heredity 81 :424, 1990).

[0303] Previously, leptin is reported to reverse insulin resistance and diabetes mellitus in mice with congenital lipodystrophy (Shimomura et al. Nature 401: 73-76 (1999). Leptin is found to be less effective in a different lipodystrophic rodent model of lipoatrophic diabetes (Gavrilova et al Nature 403: 850 (2000); hereby incorporated herein in its entirety including any drawings, figures, or tables). [0304] The streptozotocin (STZ) model for chemically-induced diabetes is tested to examine the effects of hyperglycemia in the absence of obesity. STZ-treated animals are deficient in insulin and severely hyperglycemic (Coleman, Diabetes 31:1, 1982; E. Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte, Jr. Eds. (Elsevier Science Publishing Co., Inc., New York, ed. 4, 1990), pp. 299-340). The monosodium glutamate (MSG) model for chemically-induced obesity (Olney, Science 164:719, 1969; Cameron et al., Clin Exp Pharmacol Physiol 5:41, 1978), in which obesity is less severe than in the genetic models and develops without hyperphagia, hyperinsulinemia and insulin resistance, is also examined. Also, a non-chemical, non-genetic model for induction of obesity includes feeding rodents a high fat/high carbohydrate (cafeteria diet) diet ad libitum.

[0305] Test molecules are tested for reducing hyperglycemia in any or all of the above rodent diabetes models or in humans with type II diabetes or other metabolic diseases described previously or models based on other mammals. In some assays, the test molecule sometimes is combined with another compatible pharmacologically active antidiabetic agent such as insulin, leptin (US provisional application No 60/155,506), or troglitazone, either alone or in combination.

[0306] Tests described in Gavrilova et al. ((2000) Diabetes 49: 1910-6; (2000) Nature 403:850) using A-ZIP/F-1 mice sometimes are utilized, test molecules are administered intraperitoneally, subcutaneous Iy, intramuscularly or intravenously. Glucose and insulin levels of the mice are tested, food intake and liver weight monitored, and other factors, such as leptin, FFA, and TG levels, often are measured in these tests.

In Vivo Assay for Anti-hyperglvcemic Activity of Test Molecules

[0307] Genetically altered obese diabetic mice (db/db) (male, 7-9 weeks old) are housed (7-9 mice/cage) under standard laboratory conditions at 22° C and 50% relative humidity, and maintained on a diet of Purina rodent chow and water ad libitum. Prior to treatment, blood is collected from the tail vein of each animal and blood glucose concentrations are determined using One Touch Basic Glucose Monitor System (Lifescan). Mice that have plasma glucose levels between 250 to 500 mg/dl are used. Each treatment group consists of seven mice that are distributed so that the mean glucose levels are equivalent in each group at the start of the study, db/db mice are dosed by micro-osmotic pumps, inserted using isoflurane anesthesia, to provide test molecules, saline, and an irrelevant peptide to the mice subcutaneous Iy (s.c). Blood is sampled from the tail vein hourly for 4 hours and at 24, 30 h post-dosing and analyzed for blood glucose concentrations. Food is withdrawn from 0-4 h post dosing and reintroduced thereafter. Individual body weights and mean food consumption (each cage) are also measured after 24 h. Significant differences between groups (comparing test molecule treated to saline-treated) are evaluated using a Student t-test. Example 19 Tests of Metabolic-Related Activity in Humans

[0308] Tests of the efficacy of test molecules in humans are performed in accordance with a physician's recommendations and with established guidelines. The parameters tested in mice are also tested in humans (e.g. food intake, weight, TG, TC, glucose, insulin, leptin, FFA). It is expected that the physiological factors are modified over the short term. Changes in weight gain sometimes require a longer period of time. In addition, diet often is carefully monitored. Test molecules often are administered in daily doses (e.g., about 6 mg test peptide per 70 kg person or about 10 mg per day). Other doses are tested, for instance 1 mg or 5 mg per day up to 20 mg, 50 mg, or 100 mg per day.

Example 20 PP2Ce Expression Profile

[0309] cDNA profiling was completed in a panel of nine normal tissues (using the following primers: forward- CTATGATGAAGCAGGCACAA and reverse-

CAGGTCAAAGGTCAGGATGT), and expression was detected in adipose tissue, liver tissue, pancreas tissue and in skeletal muscle tissue. In addition, PP2Ce expression was detected in a mouse, adipocyte cell line (3T3-L1) using the following primers: forward- CGCACCCGTCCATCTT and reverse- TGCTTCATCATAGGATACAG.

Example 21 In vitro Production of PP2Ce polypeptides

[0310] PP2Ce polypeptides encoded by the polynucleotides provided in SEQ ID NO: 1-5 may be produced by the methods described herein.

[0311] cDNA is cloned into a pFVEX 2.3-MCS vector (Roche Biochem) using a directional cloning method. A cDNA insert is prepared using PCR with forward and reverse primers having 5' restriction site tags (in frame) and 5-6 additional nucleotides in addition to 3' gene-specific portions, the latter of which is typically about twenty to about twenty-five base pairs in length. A Sal I restriction site is introduced by the forward primer and a Sma I restriction site is introduced by the reverse primer. The ends of PCR products are cut with the corresponding restriction enzymes (i.e., Sal I and Sma I) and the products are gel-purified. The pFVEX 2.3-MCS vector is linearized using the same restriction enzymes, and the fragment with the correct sized fragment is isolated by gel-purification. Purified PCR product is ligated into the linearized pFVEX 2.3-MCS vector and E. coli cells transformed for plasmid amplification. The newly constructed expression vector is verified by restriction mapping and used for protein production. [0312] E. coli lysate is reconstituted with 0.25 ml of Reconstitution Buffer, the Reaction Mix is reconstituted with 0.8 ml of Reconstitution Buffer; the Feeding Mix is reconstituted with 10.5 ml of Reconstitution Buffer; and the Energy Mix is reconstituted with 0.6 ml of Reconstitution Buffer. 0.5 ml of the Energy Mix was added to the Feeding Mix to obtain the Feeding Solution. 0.75 ml of Reaction Mix, 50 μl of Energy Mix, and 10 μg of the template DNA is added to the E. coli lysate.

[0313] Using the reaction device (Roche Biochem), 1 ml of the Reaction Solution is loaded into the reaction compartment. The reaction device is turned upside-down and 10 ml of the Feeding Solution is loaded into the feeding compartment. All lids are closed and the reaction device is loaded into the RTS500 instrument. The instrument is run at 3O⁰C for 24 hours with a stir bar speed of 150 rpm. The pIVEX 2.3 MCS vector includes a nucleotide sequence that encodes six consecutive histidine amino acids on the C-terminal end of the PP2Ce polypeptide for the purpose of protein purification. PP2Ce polypeptide is purified by contacting the contents of reaction device with resin modified with Ni²⁺ ions. PP2Ce polypeptide is eluted from the resin with a solution containing free Ni²⁺ ions.

Example 22 Cellular Production of PP2Ce and B3GALT3 polypeptides

[0314] Nucleic acids are cloned into DNA plasmids having phage recombination cites and PP2Ce or β5GΛLr3polypeptides are expressed therefrom in a variety of host cells. Alpha phage genomic DNA contains short sequences known as attP sites, and E. coli genomic DNA contains unique, short sequences known as attB sites. These regions share homology, allowing for integration of phage DNA into E. coli via directional, site-specific recombination using the phage protein Int and the E. coli protein IHF. Integration produces two new art sites, L and R, which flank the inserted prophage DNA. Phage excision from E. coli genomic DNA can also be accomplished using these two proteins with the addition of a second phage protein, Xis. DNA vectors have been produced where the integration/excision process is modified to allow for the directional integration or excision of a target DNA fragment into a backbone vector in a rapid in vitro reaction (Gateway™ Technology (Invitrogen, Inc.)).

[0315] A first step is to transfer the nucleic acid insert into a shuttle vector that contains attL sites surrounding the negative selection gene, ccdB (e.g. pENTER vector, Invitrogen, Inc.). This transfer process is accomplished by digesting the nucleic acid from a DNA vector used for sequencing, and to ligate it into the multicloning site of the shuttle vector, which will place it between the two attL sites while removing the negative selection gene ccdB. A second method is to amplify the nucleic acid by the polymerase chain reaction (PCR) with primers containing attB sites. The amplified fragment then is integrated into the shuttle vector using Int and IHF. A third method is to utilize a topoisomerase-mediated process, in which the nucleic acid is amplified via PCR using gene-specific primers with the 5' upstream primer containing an additional CACC sequence (e.g.,

TOPO^® expression kit (Invitrogen, Inc.)). In conjunction with Topoisomerase I, the PCR amplified fragment can be cloned into the shuttle vector via the attL sites in the correct orientation.

[0316] Once the nucleic acid is transferred into the shuttle vector, it can be cloned into an expression vector having attR sites. Several vectors containing attR sites for expression of PP2Ce or B3GALT3po\ypeptide as a native polypeptide, N-fusion polypeptide, and C-fusion polypeptides are commercially available (e.g., pDEST (Invitrogen, Inc.)), and any vector can be converted into an expression vector for receiving a nucleic acid from the shuttle vector by introducing an insert having an attR site flanked by an antibiotic resistant gene for selection using the standard methods described above. Transfer of the nucleic acid from the shuttle vector is accomplished by directional recombination using Int, IHF, and Xis (LR clonase). Then the desired sequence can be transferred to an expression vector by carrying out a one hour incubation at room temperature with Int, IHF, and Xis, a ten minute incubation at 37⁰C with proteinase K, transforming bacteria and allowing expression for one hour, and then plating on selective media. Generally, 90% cloning efficiency is achieved by this method. Examples of expression vectors are pDEST 14 bacterial expression vector with att7 promoter, pDEST 15 bacterial expression vector with a T7 promoter and a N-terminal GST tag, pDEST 17 bacterial vector with a T7 promoter and a N-terminal polyhistidine affinity tag, and pDEST 12.2 mammalian expression vector with a CMV promoter and neo resistance gene. These expression vectors or others like them are transformed or transfected into cells for expression of the PP2Ce or 53GΛZ, rjpolypeptide or polypeptide variants. These expression vectors are often transfected, for example, into murine-transformed cell lines (e.g., adipocyte cell line 3T3-L1, (ATCC), human embryonic kidney cell line 293, and rat cardiomyocyte cell line H9C2).

[0317] Provided hereafter is a PP2Ce/B3GALT3 genomic nucleotide sequence (SEQ ID NO: 1). Polymorphic variants are designated in IUPAC format. The following nucleotide representations are used throughout the specification and figures: "A" or "a" is adenosine, adenine, or adenylic acid; "C" or "c" is cytidine, cytosine, or cytidylic acid; "G" or "g" is guanosine, guanine, or guanylic acid; "T" or "t" is thymidine, thymine, or thymidylic acid; and "I" or "i" is inosine, hypoxanthine, or inosinic acid. SNPs are designated by the following convention: "R" represents A or G, "M" represents A or C; "W" represents A or T; "Y" represents C or T; "S" represents C or G; "K" represents G or T; "V" represents A, C or G; "H" represents A, C, or T; "D" represents A, G, or T; "B" represents C, G, or T; and "N" represents A, G, C, or T.

>3:162087851-162177450

1 tacctctaat cccagctacc tgggaagctg gggtgggagg atcacttgag gtgaggagat

61 cgaggctgca atgagccagg attgtgccac tgcgctctag cctgggcagc aaagtgagat

121 cctgtctcaa aaaaaaaacc aaaccaaaac aaaacaaaac aaaaaaaccc taaaacaaaa

181 acaagaaact ttctgagtac caacatgatg ctYaaaggaa atgctcattc gagcattttg 241 gattttggat ttttggattt gggatgccca acctgcataa actttaaaga atccttaaat

301 aaagttatat gctgctaaaa ctaactgcct ccttgccaca Ytcctaataa ggttctacat

361 cttgcagaaa tttttaaaag cctgatttag ggtgtcagac ctgggttcaa aatcttcgtc

421 ttctactcaa aagcaatctg atattggtga gcttcttatt ccatatctgt agaatgggat

481 aggggatggt aataataatc ccatgtacct tgcaggattt ttgtgaaact ggataaggtc

541 aggtatgtgt gaacagcaaa tggagtgtct ggcccattgt agctgtttag ttaataacat

601 ttcctgttgt gacttgatat ctggtttgag attctggcat cctgtaactc ctcctcctca

661 ccagccctgc cctccatcag cctggtcccc tgtacagtgt gacacctact cagtaattgc

721 ccatagcaga aacagactca ccagctgttc tagagctggc tctccctggt caaagcttcc

781 catgcagcaa gtgtttcata atcagcctgt tcctgctgcc gtctgccact tttcccccga

841 ccttcctggc catcccttgt cccttcctgg gcaaaagcat ctaaccatgt ggtatggtgg

901 tgtcacctga gtgtggtgtc agcaaaccca gagctttcca agcctcccat gatgagtcta

961 tgacctggcg ttccctgttc cttctgatac tgttttttct ttctctggaa ctacattggc

1021 caaaacagaa aacagggagt cagtaaggtt tatcaatatt tcacttgcat agccgcagtc

1081 ttaagttact gaaatgacag gttgatccag tgaggaaaat caaacaacta tcaaaaagac

1141 cagtcaagtc attgtagacc atgacaaaag accctcagta ggaaaggaaa aaatgcctaa

1201 caccaagaaa catgaaagta aaaagctctg aatttggtaa aagtgaagta atttctataa

1261 atgagtcata ttgtatctag gaaattagga aaatttcaga aaagtttttt taaaaattca

1321 aatcaacttt aaaagctgat atcctttttt cttctaatat ctttggcata tatttgtata

1381 cacatttcaa gtatttaaat gaatgaaatt ggaattttta tatatagttc ttttcaagta

1441 aaaatgctaa tgtaatattt atttttaaat tttataagaa gtgttttatt ttttacttaa

1501 aatatgagac tttctcatga caatatatat tcttcagaaa cactataatt aatgtccttg

1561 taatgctttc ttgtttggat agttaataat gcattttgtc atactatagc tactcatttt

1621 ggctgtcttc attgttttgc tactttaagt aattctatga ggtcaatcct tgtacataaa

1681 gctcagttca aatctctgat tatttcctta ggataaaatc caagaattgg cattattagg

1741 tcaaagagca taaacaagta tttttttcta ttctttgttt tgaaagtgaa ttacaaagaa

1801 atcctctctt aaaagttgct cctttcattt ggttttattc tacagattgc ttctttacct

1861 ccttctttgt gtcaagaacc atattaaact atgtgaattc ctctctgttg catctttaag

1921 agagagttga ggtctcactt cctcccagct atcctctcca ccacccgcca tctcctcaga

1981 ctgagacaag ttcccttctc tgctatcacc cacacccata catctgggct ctcatagtac

2041 tgtcatagca tttatcatgc tgaattctaa gtgtctgttt ggttttattt tggcattaga

2101 cagtaagtgc cttgcgggta aggactatat tttattcatc tttatttctc cagaaaacac

2161 cgagtacttt gtacatagta ggcactgaat gagggaacaa ggaacacaga accatttagt

2221 cactacacac acacacacac acacacacac acacaaccat cctgtcctat attattaaat

2281 acaggagata gaagtgtgag ggagaaagag ggtcatcttc attcagtaat atgcttgtat

2341 tttctcagat atagatgtat ggatgagata tcactttttg atatgtgtcc agataggcca

2401 gatataacta atatcaggat tttatcaggt cattgggacc acagccttcc tggaaacata

2461 gctttagagt cagttttata gtctgctgag cccaaaacaa gctagataat taataatatt

2521 attggatcaa aatggcccaa aatctgatta ggggttattt atcctacatg tacatgatag

2581 catagtcttt attttgtccc tccaagtgag tttataagtg actgtgttct gtttggatgg

2641 aaaatatcat ctggaaataa tttatgagta actgcagaac ctgccaatgg catgctttac

2701 aagggcttat ttcattcact tccacgtata tcccataatt tcctgatttt ctcaggttct

2761 tttgtgtaaa taccaaattg accttatgtt tttgtccgcc ttttccccag gggcgatcta

2821 agtgtcagga tggtgttatt gacctggcat ctgcgggggc gacccagagt ttccatgacc

2881 tgtggttgga aggcccactc attggggcac tgaagctcct gagagttact gttctcactc

2941 aatgggaggc cagcggttct cagcagtttt ctctgcttca caccagactg tccagttagc

3001 ttcttattca gccattgcct ttccatactg gagcaaaggt ttatgattta ttaggctatt

3061 cccaggaaag caagttctat gtcttctgtg ctttgagctc cacataattc ttggagatcc

3121 tcacatcaaa gactccctct tggttttcct ctttgtctcc cttgcatctt cctctactca

3181 cacccctgct gtctggccag ccttacttat cagggagagc aggaagagag gattcatggg

3241 caggaaaact cactgtttgc tccatcttcg tagtcctggg gtgtggccca aaaccttggt

3301 tgatagattt gtaaaaagac ctttatcttg agaatattgt tgctttgtga atttccacag

3361 tctccttgaa attcaaaagc tgagcataac tatttctttt tctttgcatt tcagccatag

3421 caatcctaat agcccctagg acaatgagtg tctcaggctg aatggaagag ggtcatatac

3481 atgcaagggg atgggaattt aatatctcta aatatcatcc taccaacaca tgtatatgta

3541 aatagattta taactatatg gtcatttgta taaggataac ctaataatat caaaggacag

3601 gttcatttta aattaaaact atagggaaga gtgttgattc tacaccaata acaaaaaggt

3661 taaagaaagt gtaattctct tttaagtacc agatgcttca gactgtgcct ctcattttca

3721 gccttcttct ccccttttcc tgtctctgcc tttctcctct ctgcctttta ttgaaaggca

3781 aatgcccctt gtaataaggt tcaatttcag aagttcctgt tataccccac tcccatagca

3841 ttcataaaaa aagcagagac cgtagttata atttgctggg aaccctaaaa gagaagtgag

3901 acaactcact gaagaatcct caaataacac gaagacgtga aaaaatatag ctggtgcttc

3961 tctggcttat gtaggaagaa tcaaagcaac catgaaatta attttattta ccattaaaag

4021 atactggact tagaattcag gttgtgatta tgtaattata aattaatttc agtagcattg

4081 tataatctga aatgccagca atagttatga gaagtatgtt aatgaggatc agaaaagctt

4141 gtctgcttga atgtacctca aaatagtcat gcccagagag tatatatgaa gcctcctgca 4201 gcaagtggac ccactgtggt ttcctaagac tctccaatgc atgtgatcag tttactagga

4261 agcagctctc tttaatatgt tcatacttaa cagaaaagga aggaccagaa ggttgcccat

4321 aggctgcacc caagtgtgaa ataaacatta ttattacagc agttgaaatg tgaagttgaa

4381 gaacatttta atttcaaaat agtattcatg agaatcagat gacacatcta tactttaact

4441 ctggtgaagt gtgaaaatta aatgaacact ccRtcagatt ctgaaagttg aattatgacc

4501 cccacctccc tcactaaaga gatatttttt catatttagg gtggcatacc tcatttgtgg

4561 aaaaggtctt tctggcactc atcccctgtt tctcaattta ttgtagccat cttattaatc

4621 actgtgtttg ctggggaaga gccaaagtat acctaaatct agaattaaac tatatagcac

4681 tagggtttaa aataatagaa tttgtcatca agttgactgc ctcatattca Kccagtgtca

4741 aactcaatag ttgatatgtt tgcttatgca aaaatatcag agaggaaaag aaaattttaa

4801 tttgtaatgg atctatttct cataaaatgt cattcctgcc gctctgctct ttagcacatg

4861 atgaggggaa gctgaggcag cttctattgt aaaggaagac ccaaggaggt gccattttag

4921 gggaccttca gtctccaccc agttcagggg attcacatga gcctccctgc atatatgggc

4981 gtgtcccatc actgcttctt ctgcttggaa gcccacaaag atggtttgct atcctagtac

5041 ctcaggtctt gatggaagaa ggccttggtg tgttcttaat tatatccttt gcctcagatg

5101 ttttgttgtc agtgtttcac tcttcccagt caggttttag ggctgctacc agtctctcca

5161 tgaaatagaa accccctaat gtgctgttct gctgaacttc ctcattgact gaccttgtac

5221 tatgatgtct ttcttcctgg ggtcagtggg tacgggataa agcctctaag cagggttgcc

5281 catatggttg tgtatgtttg gattacccca cagctctgga cggcaccatt cacactgtgg

5341 tcattgtcga tttaaatatg tattatggcc attttatgga gatggtagta aagggtcttg

5401 atgaagacat acctttttct ggtttgcaca caaaaaatta aacacaaggt agcaatggcc

5461 tgccctgatg ttgaaagcaa gactctggca acaggctagg tgggttctac cccttactag

5521 ctgtgtgacc ttgggctagt cacataatct cagttttatc atctgtaaga tgggaataat

5581 aatagtacat cataggctat taggattaaa gaaattatgt atgccaagga ctaagtacag

5641 agcctgacgc atggtgaatg ctcagtatat ggcagctgtc acaatgtggc ccatttgtgt

5701 ccttccatat agatagatca ttttccaatt tccaattact ttctacacat cctctcttaa

5761 cataaaatga ctttcaagcc cattactaca agacttatta caggggacct atggacttgt

5821 gatttgaatc gtaattagta ttaatttaat agtctcttca cagagttaga ctaatatgtc

5881 aggtgacaca gtgtcttagc ccagccacac acattgatat gactctgatg atggcctgcc

5941 tctcagtgaa gtcagagcag ggggtacaag gactcaggca atagacaaga gagaaaagaa

6001 agattaaagc ttactgttta tagtgttttc cacacaaggt atcatccctg acaagtcttt

6061 ctatgccatg gcattgatta ttagaagaaa gactcgtaaa aacctctagg caggtcagat

6121 tttggcagac ttaaagatta tgcagacaag agtaggggtc Rgggagtctt atctgagagc

6181 tgccgggtac tttatStgtg tttcaggaca gggctgacca cagtgttggc tgtggaaggt

6241 gggggctgga aacctctgct gacagtgatc ttatctttcc catgtgatag tttcattaat

6301 gactgccttc acctgctctt tttccagtat gttgcactcc aatgtaattt ctgtccgtta

6361 tcaggccttg atgtcatcta tgccctgaaa ttgtgttatt gaactaacac tcattgtcaa

6421 acctttacga gataaaccta ttttatttct cctcatccta aaaatgtgtt gattctcctg

6481 tgctgtgtat aatgaatggc cctttggctg ttgtaacacc ttcactgaaa tccttgaagt

6541 gtgtgtgtgc ctatctctct atgtctcgcc ccctttcgcc tcctcccctt cctcacccct

6601 tgccacccct tccttaccct ccccaatctc tctctctctt tcctaaagaa caaaaatggc

6661 agtttccctt attctcatag aaaagacttt atgtcacaga agggagctgc tattagcata

6721 agcagtctgg cattgtgtgg gtgagtgtcc atgtgcatta tcaRgcaagg cttataaacc

6781 atgttaattc cacacaactt tctctactcc atccagcttc caatccgtac cccacaacat

6841 aaaaagtgac atcactcact taagaaaatg ttgcagtaag gtggattttc ataaaataag

6901 gattctcagg actccaattt ttaaatcata cctgtgtgcc tacagaaagg ttggaggggg

6961 gcaagggagt gaggatctta attggcaccc aaggcagaat gtttgctctg aggagaagga

7021 ttgagtggca ctacttaaga gatgcagcaa ggcagtgcag ggagagccta aaagcagctg

7081 aggctctcaa tgtcacccag acactgatgg acagtcattg atatttgaat ctgaatctga

7141 atgtgtacta tcaaaaaggc tgctgtcatc attgtcacca ctattcctct gagtcctttt

7201 ccatctctac catcttactc tctgtaacgg gattgtaagt tctatatttt gttttatatc

7261 ttgtatactt tgcttgatgc agtaaatatg ctactgaaac actgtgaata agtaaaactt

7321 cacctgactg caacatttaa ataacattaa attcatctga atacagaaaa atataaagaa

7381 gaaaacaaaa aggtctaaaa tcctatcacc aagatagccc tgttaatatt tgttaaaaat

7441 ataagtatgt gtgtggacat gattagagtg tctatatttg catagtataa ggggtaaatt

7501 atactagtac aattaatagg tataaattga aaagcaccta catttctttc gctcctaccc

7561 caatccttag cccctccctc agcattatgt ctgtgcagtg tacaaagcac tcttataaga

7621 atacagcaat gggccggcac gttggctcac ccctgtaatc ctagtacttt gggaggccga

7681 ggtgggtaga tcacctgtgg tcaggagttc aagaccaacc tggacaatat ggtgaaaccc

7741 cgtctctact aaaaatacaa aaattagcct gggcatggtg gcgcacgcct gtaatcccag

7801 ctacttggga ggctgaggca ggagaatcac ttgaacctag gaggcggagg ttgcagtgag

7861 ccgagatcgc gccactgcac tccagcccgg gcaacaagag tgaactccat ctcaaaaaaa

7921 taaaaaagaa tacagcaatg actaaggcat ctccctggcc ctaaaaggga gtttcagcct

7981 aatgggtcaa agaatcataa ataaggcata taaatagcat gatatgtgat gtacagtaag

8041 ttggcagctg gcagccatga gatgtgatgc caacattacc ctcagctgct acaggtctct

8101 gttgtcatcc cttccctcct ccgcctcacc taactagcca catgacctta aagaaatacc 8161 tgaactgtct ttacccattt tatgcattta taaggtagat gtgataattc ctccctgcct

8221 gatctgatga gctttgagaa tcgagtgcca gaatgggtaa gaaaacactt cacgactcat

8281 aaaacagacc agaagtgtaa ggtttcattg tcaWctttat tgttatacaa aggggtacta

8341 tgaagaatca tagtaatctt gggcgttaat ttaatagaaa gcccgggagc acagggtcac

8401 caacagccca gatttgacca acagctgcct ttcgcttggc ttgtactgct ttttaaaaga

8461 aaagttgcta acgtttaaaa atcaggagag ttcatacaaa aaagacttcc cttgaaaaat

8521 cggaggaaac attcacagtg gacctgcatt cctacgcggc agccattttc gttgctgagg

8581 aactgctttc ccctgagcac gtgttctccc atttgtcaca agcccccccg actccctttt

8641 gtagccccaa cactgaaacc tgatgtcagt ttcacttgtc atcatgcttg cccttcctta

8701 gagccagaaa atgttttaga acccaggttc ccatcaaaag tagcagggta aatagctact

8761 ccaagaaggc caagattttt cttatacctt acctgaaagc attggagttt gcaatttcaa

8821 tttaagatct gcagaatatt caagctgaaa ggggcctgga ggttaattaa tccaaacgtc

8881 ttgtttttga gttccagaga ggttaagtat ataataaaat gtttagcatt ataataaaat

8941 actttagtag ggtccatcat tttctttcct tttttttttt tttttttttt tttgacgcag

9001 agtctcactc tgtcagccag gctggagtgc agtggcacga tcttggctca ctgcaacctc

9061 tgtctcgcgg gctcaagcaa ttctcctgcc tcagcctccg gagtaactgg gattacaggc

9121 atgtgccact acacctggct aatttttgta tttttagtag agatggggtt tcaccatgtt

9181 ggccaggctg gtctcgaact cctaacctca ggtaatctgc ctgcctcagc ctccgaaagt

9241 gctgggatta caggcatgag ccaccacacc cagcccatca ttttcattat tactaaagta

9301 acaattttgg attcccaggt tttttagtca tttagtcatt cagtacatat gcattgagca

9361 atgtaatcag ggataagaca cttggggttt catggaaaag aaacttttta tcatagtgct

9421 ttgtatacta tacaaactca taatgctggg ggaggggctg aggattgggg tgggcgtgaa

9481 ggataagata tttgaggttt ctgagtcccg ttttcctcat ctgcttagag ggagaatacc

9541 tgccctcccc actctgaatg tttgaaaatc aattgcaagt atgtttgtga gccacttagt

9601 agagtttata gtactgaaca aatcgttatt acaactttgt tatgaattta actgagaaag

9661 ttttaaaagt atttataact aggaatctaa aagatgtctg agatacccca agttacataa

9721 ccctcattag gatttacagt tttctcaagc tctacttgtc aaatgaatta attgtctaat

9781 attatctaag caaaatactt agttaagctt agaataactt ttttgtatac tgttactttg

9841 tctgcattcc agaaattaaa tcagagggga gaatgttggt ttacaggaat agactgatac

9901 ataatttgca tttgtgtgtc ttccctaagg aaaacaataa ttattagtcc aacacaaaaa

9961 catgcccatt ttgctgaagt acatattgag ttcagaagtg gacacagatt gtcctcataa

10021 tgaccactgt aatgtacctt tattgcttca caaggtgcca tggctttgga tggtgtggtg

10081 aggatggaag ccctacccat atgttatttg cactggacca gaatcttgga tggaggaaag

10141 aactttggac atatctgggc cagaagttct ctccacctac caggagtgag catgggagca

10201 tgagctatac atgtacagga gggtcatcgt gttttcatca gggtgtaaag tgtattttgc 10261 tacatctgat agctcttttt atggcaatag ctggacaatg gtgttgtcca ttgtgtactg 10321 aactgggctg acctgtcaga gcaagacttt agaagcttag cattatgtca ggtcaggaca 10381 ttatgtgtga acgcttattc attcatctat taatttgctt atttagccta tgttgggtgc 10441 ttttaatatt ctagacatca gcattgtagt gggaaaatcc aaaacttctg atacggtttg 10501 gatctgtgtc ccttcccaaa tctcatgtca aattgtaatc cccattgttg gaggtggggc 10561 ctggtgggag gcaactggat cctgggggcg gtttctcatg aatgatttgt cactgtcctt 10621 cttggtgctg ttcttgtgat agtgagttct tgtgagatct ggttgtttaa aagtgtattg 10681 cacctccccc cacccacttc cttctgctcc agctatgtga agtgcctcat gcctcctttg 10741 cctcctgcca tgattggaag cttcttgagg cctccccaga agcagaagcc actatgcttc 10801 cagtacagcc tccagaatca tgagccaatt aaacctcttt tctgtataaa ttacccagtc 10861 tcaggtattt ctttatagca gtgtaagaat ggactaatat atcttgtgaa gtttatattc 10921 taaagggaaa gactgacact aaataaataa atagtctaaa taaataagca agatatctac 10981 ataattgtgt gttgtaaggg caataaacaa agaggaaata caatccctgt cagaaaaggc 11041 ctctctgaag atgtcacatt tgagctacaa catcttcata tgggtcagtc aaccaatgtc 11101 aaggacagga gaacaagcca ggcagagaga acagcgagta caaaagccca aagatgacaa 11161 aaaggttggc atgtatgggc aacaggaggg aggccttgag caaggaaggg agtggtgtga 11221 catgatatta gacaaattag ctaggggcca ggtcatgcag gacttgccac cattgtttct 11281 cacctggact actgaaatag cccgggggct aaaatgacag tggttgggac tggagtcaat 11341 gctgtgagaa caaactggaa gggtttaaga gatattttag aggtaagagt aacagaactt 11401 aatgatggac tgaatgtcag gaaagaggaa aagagaaaaa ttaaggatga ttcttaggtt 11461 ctggcttgag caatggagtg gattatggtg ccattttctg aggcttaaaa gtcaaataac 11521 ttgccaaacc tcacttagct aggacattga agtctggtcc atgtgaattc gctttttttt 11581 tttttttaag ctttttgggt ttttttttaa acccatgaga taaggttatt aggttaaatt 11641 tccaagatct ctttcagatt tgcaattaag ttctcatcta gattaaattg ttaatatgga 11701 aatatcctga ggttcagaat taagtaacat tgtctggccc tggtattttt gataaatata 11761 gtgctatgat ggaataaata ttttatctca gaactaatag ccagaatttt gcaagtgttt 11821 tgtctcaagc ataaaaagaa aatgattttt tcattttctc ttgggcataa tgtcttttct 11881 ggagtttccc agcaacaagc agtttgttct gtcaagtaag atgagattgt aaactatctg 11941 ttataatcct gagctaaaac tgcaaagccc aaacagtaag ttttttttgg tgtcctggtt 12001 tgtttcagat cattcttaag cctcccaccc cagtgctcag cattgctttg cacgtattat 12061 ttctccagaa ccacacaaac ctcacccagg ataggggcag gtttcctctg tagagacagg 12121 aatggagaca tttcagtaat tcagaatacc acctataact cataaccctg ccatctgggg

12181 ctcacccatt ttggccaggt ctaagattct gttgctaaca taggcaggat agtcctgaaa

12241 acttcaccat gaggtagatc ttaatttacc catagaaaca gtgggatttg tttgtgacaa

12301 aggcgggtca tcgatcttaa gtatgttcaa tagcatgtga gtacagttgg ctcttcatgt

12361 ccataggttg tacatttgtg gattcaacca accacagatc aaaaatattt ttaaagcaat

12421 aaaaaacaat ataacatcat acaaattaaa aaacaatata acaactattt acgtagcatt

12481 tacattatag attataagta atctagagat gacttaaagt atacaagagg atgtacatag

12541 gttatatgca aatactaagc cattttatat aagggacttg gccatccgtg gattttgcta

12601 tctatagggg gtctagaacc aatcaccagc agatcccaag ggacaactgt gtagctaaac

12661 agccacactc taaatctctc atcaaattca gatattacag ggaacaacaa caacaaaaaa

12721 ttcaactcaa aaagtctcaa acaagaaagg catttattgt ctcattgaac aaggacaggg

12781 cagctctagg ctggctcagc agttccctga tattctccag gactcaggct ctactgttta

12841 cctctgtgct ctaccatcct gcacttgttg ccatgaccct aagggatatc ccttcatagc

12901 tgcaagatgg ctgccacact tctgggaaac acataggcta gcactgggcc aaataaacag

12961 tcattgtctg ttctgtgtct cttttagtgg ggggggaaaa aaaaaagcct ttctcagaag

13021 cccccagcag acttttttga gtcctcgtcc atggaatgcc cagtttctcc catctagata

13081 tacgggtatt atcctaaact tctcccctca tcctttattc tcctcagttt ctatgagtga

13141 tccattctgg tttttaaaaa cctgtcacag gagctgggca cagtggctca cacctgtaat

13201 cccagcactt taaaaggcca gcaggcggat cacttgaggc caggagttca aaaccagtct

13261 aggcaatata gtgagaaccc atctctacaa aataaaaaat aagaacctat cacctaccct

13321 ctatccctat atttattaca ttaattcaga tcctcattaa ttccacctgt tctcactgct

13381 cccaatttgt ctatcttctg cattgcccct ggaatcatct tttaaaatat gaatctcatt

13441 gtgtcactat cctgcttaaa acccttcagt gattcttaat caaatatggg actaaatttt

13501 attttatttt tctagctgca agaatgcctc cttctccctt ttttaaaaag aaatcttgcg

13561 tggcagctga cttgttagct g-ttaacacgt ggaagcagag ctgtcctagt tgaaatgcgg

13621 ctggggaagg ggcctttaac tcagccagct ggattcctcc ccttccctct catggaggcc

13681 ctaaagcctt tccttggatc ccctggagta caggcaaatg gaaacgccta ctaggcagtt

13741 ggtgtagacc tcaggagtgt caaccagcct aactgcatag acttagaaag tgtggaagcc

13801 aaggtcggcg acatgctcac ctagaaagtg cagagtgaga agagaagaga gtcctataga

13861 gagaggactc caagagtgat gctgaagtaa tgcctacaag ccatgtaata tcccatgaag

13921 caatctttct attttacctt tcaaactcta agactgtgcc ttataggaga atttatgcta

13981 tgattgtgta ggataattta attcaatcag atgtttgggg acaaagtgag gatatgttga

14041 ataccaaaat ttcagtttcc aatataaaaa aaaaatcaaa aactggtaac aacaatttct

14101 aattgtgtgg attgtgcttt tgcttaattt tcattcttat agctttttaa aaattgtttt

14161 gataatccat gaaattagtc ttttttttcc cccaaggcct tttgtattag aaatcctgtg

14221 ttaaccaaca tttcatgtgt ttccatatct gatcaaggca tttttagtta aataagctta

14281 agaatggaat ttcaagcaga actataataa ctgattatat tatgatagtt attttaattc

14341 tctctgctct tgatatcaac ctgtgtttat tctttatgtc tatttaagaa ttcagtgttg

14401 ttgttttttt ttctgttggt gggagtctaa attagttcaa ccattgtgga agacagtgtg

14461 gcaattcctc aaggatctag aaccagaaat accatttgac tcagcaatcc cattactggg

14521 tatataccca aaggattata aaccattcta ctataaagac acatacatac atatgtttat

14581 tgcagcacta ttcacaatag caaagacttg gaacccatcc aaatgtccat caatgataga

14641 ctggataaag aaaatgtggc acatatacac cacggaatac tatgcagcca taaaaaagga

14701 tgaattcatg tcctttgcag ggacgtggat gaagctggaa gccatcattc tcagcaaagt

14761 tacactggaa cagaaaacca aacaccacat gttctcactc gtaagtggga gttgaacaat

14821 gagaacatat gggcacaggg aggggaacat cacacaccag ggtctgtcag ggggtgtggg

14881 gcaaggggag ggagagcatt aggacaaata cctaatgcat gtagggctta aaacttagat

14941 gacaggttga tgggtacagc aaaccaccat ggcacatgta tacctatgta acaagcctgc

15001 acattctgca cgtgtcccag aacttaaagt aaaattaaaa aaaaaaaaaa agaattcagt

15061 gttttttttc aagccagcac catggcatgc acctgtagtc ccagctactc cagaggctga

15121 ggtgggggaa ttgcttgagc cgaggaattt gaggtttcag tgagttatga tcacaacact

15181 gctccctagc ctagatgaca gagcaagacc ctatctctta aaaaaagaaa aaaaaaatcg

15241 gtcttttttg ttgttccatg aaaactaaag agccatttag atacagttga cattcttgtt

15301 atagaatatg accttaatga ggaaggaaac gttaatgaag actaattagt gcataatttt

15361 aacgagggag atatgtttga gtcaagagct gaatgggttg tgcatttcag caggtatttg

15421 catattgagt gacactgtca aaaaggaaga ggtgaacaga aaggagctgg acagggcagg

15481 ctccaactag accagaccgt tcaactctgt ttgcttttat ccattcccat gtcaaaaagg

15541 atttgagcag agcttgtcct ttgtgtcttg tgtgggcttc aggcaagcac gtcttcaatc

15601 ttgctttctt ctgactcgaa ccaccctctc cctaggcctc agttctgtca acaccagagc

15661 tttctctgct tctgtcttta tggtctgcct ccatcacacc ctggagtgga cctgtttctg

15721 gccaccagtc tttgctgtca gactttgtat aggcaagctc aacctgtcct gacatcagca

15781 gcttcctcat gctccacatg attcagctgt tggcatttct aggccctcct agaaatgagg

15841 agcccaccgc acacccccat ccttcccact tccaactggg acttcacaga aactcagcac

15901 caagcagaag ccWaaagtgt gtatcagttg actgaaactt caaacgattt tgtttctctc

15961 ctaatccttt taaatgtgat atgtgtttaa gagtagcaga gacaaagtga tttaggaaaa

16021 ttataatgtt atcctagcct attttagtga cactctaaca tggttaagaa aattgactKt 16081 cagatttgat cattgttgtc gaagtgaaYa gccatcattc tcagagtggc tggagtcaaa

16141 ttctcaccca gcagaagcaa ttttaatgac tgaagaatcc agttaccaca agaagcaatg

16201 gaacctgaat gcactgctga cctcccgcgt tctctctctc ttctattttt caggcacaac

16261 gtgtttgatt gctctgctat cagataaaga cctcactgtg gccaacgtgg gtgactcgcg

16321 cggggtcctg tgtgacaaag atgggaacgc tattcctttg tctcatgatc acaagcctta

16381 ccagttgaag gaaagaaaga ggataaagag agcaggtgag cttgtgaaca cctccaagaa

16441 atgtctgctg tcttgctggt gaacaaggcg gcttgcttgg agagctcctg gataaaatgt

16501 ccagttatgc agtattagag aggctgctac tgaggtaact gagattgact ttgtaacatt

16561 acctatcaaa taaatggcca ctaaatctgt cttccttcat agccaaagat tccaaaagaa

16621 ctaaaaagcc catttccagt ttaaaggcta ataagatctt tgggccccgg gttctcactt

16681 ttctctagcc tgttgctata tgctgcttcc ctcggagcca caactaacta ctaatcattc

16741 ctaaaaagac cttgtttctg atacctctgt gcctttgcaa gtgctgttct gggtcacaat

16801 tacttgtccc ataaaactca gtttaaggtg ttcttttgga agctttccat gtcacactgt

16861 ggcaagggcc ttcccattct ggggtggatg cccctctgcc tttccccagc atcctatgtg

16921 gacgtcttgg gtcacctttt acaatacatg gacatacctg gctctcctat ttgccttgcc

16981 ttcctgtcac ctctttgaag acaggaactt ggttttgttc atctctatgc cttgagcatt

17041 tggcttagtg cctggtataa gtaaatattt cagagcaagg ttgggggtag aggggtccct

17101 tattcattca ttcattggtt tattcaatat tttaggagct ggggataaaa caaaacccca

17161 ttctcaagct ttcatgaaac ttacattctg aaattggcat gggggtgagg aatggagtaa

17221 atagacatta acaaaaaaaa atagtaaaag taagtcattc atatagtata ttagaagttg

17281 ataactgcta tggaaagaca tgaagcaggt aaggggagtg tcagggagtt gcagttttaa

17341 gtagtgtggc ctgtgtgggc cttgctagag tgatgtttgg acaaagactc ggaggataat

17401 tatcagttga ttattacatt ttcataattc gatttcagag aataccttta tacctatgta

17461 agggaagaat tctgtaactt ttcagactga gagctgcagg atgtatgctc ttcatgatgt

17521 gccaggcagc aggccagctg tcagagggga gcaagcgggg tgctcatgta ccctagtatc

17581 tgaccaggat gggcccttgc tgtctgggtt aatctgcaca ttgactctcg atctctacag

17641 gtcagcttct gcccagtgaa tgaactgaat gaacctaatc aacattttag ttttcacagt

17701 agggaaccat actgttaagt caaaatgtgt tgaattttat tacaatctcc tgcttcagtg

17761 actgcttgag ttgcaaaata gtttttaatg aaagcattgg tcaggttttc tgttgatggg

17821 atgaaacgaa tgatatttta ggagcatcta tcaagctttg atgacctcca ggcagtagga

17881 gcaaagttct ctggcttcct acttgtaaaa caccatctgc ctgtaggagt ctgctgtgaa

17941 attaagacac agttagctca gtcttgcatc agcatcaggc aaggatgtgt acacaataca

18001 cctgcaaatg tcatcttacc attcagtgct ctcttttgaa atatcatggc cattttatgc

18061 ttcctcttct tccctgtcat acccactacc cacgcctgca ccctatgttg attctttgta

18121 gaagcaaatt tgcacaccgt ctccaaggag gttaatctaa cctctctgtt cttttacata

18181 atttgatcaa ttgcatattt tacacaaggc ttaagaaaca gtgattggtt ttaccatctt

18241 catgtgtgtt ttctctggcc tcagcctctt ttcaggacat ctttccccct cctgcttagc

18301 atgacagtaa ggtccagcca gtctgggcca aatacatgtc tttctgtcgc cactccttat

18361 actgtttcca acaacaacat tagctgtcat ttattgtgtg cttatcctgt gctggcagca

18421 aagcctagca ctttacacat acactttctc tttgcctgtc ttaagaggtt gataatatta

18481 ttctcatttt gtagccaaca aactggaact tcacagaggc ttatgtgccc aagcacacac

18541 agctagtgaa tggtgagacc acagctgaaa cccaggactg ggttttatgc tcttgcctac

18601 tagaccatgc ttcctggaat gtcctcctac atatcctgcc catagatgtt tcacccatcg

18661 ctcaaagccc acctttaatg ttcattctac cctcatgatc agaactggaa atcatccctc

18721 ccttctccat ctgcctacag catttagcag acactggtct cgccctgaag aataccactt

18781 tgtgcttatt gtatttaata ctttttgtgg cttaattctc ctgccaggct gtctactttt

18841 ttaaagaaag gtctggatct tgtataactt catatcctgc acattgcctt ccatggagta

18901 ggtatttagt aaatatctga caaatatgtg gatgaagaaa aaaataatag atacaattgt

18961 taagtgcata ttatgttcag ggactgtgcc aaatgactta tgtggaataa gaccttcatt

19021 tctcaacaac ccaataaaat agatactgtc tctattttgt agccagggag actgaggatc

19081 agagaaagag tttctcatcc aaagtgagac atggttagaa gagacagagc caggaatcac

19141 attcaagtag tctgacctga gcctttaatg actatactag tgtgtgcttt ttcttaatct

19201 tgatcctatt tggggggaca gagctgtttt gagacctata taggcacact tatgagtggc

19261 ctcaggccaa atgatatata cggtgcctgt tcacctgcct ttcagcattc ctttgccact

19321 ctcctcaaat ccctaccctc atgctctcta ctgtcaatca ctgagccacc acggtgttaa

19381 gttaaaaacc aatgccctcc cccatatgta tccgccacct tcagtactct tattgatata

19441 ttattgaaat ccttcaagat gccaggatga gggtatgagg atcaactggt agagtataaa

19501 accccaggcc atgcagtggg tcccaggagt gatggggtta aggggagtgc ccacagccca

19561 gttcacatga agtaggtcac cctgttgcgc acgtacctag actatcccag gtaagtgaga

19621 tatcagctgg tcaaactaat gggctcatcc tgtctttcta ggtggtttca tcagtttcaa

19681 tggctcctgg agggtccagg gaatcctggc catgtctcgg tccctggggg attatccgct

19741 gaaaaatctc aacgtggtca tcccagaccc agacatcctg acctttgacc tggacaagct

19801 tcagcctgag ttcatgatct tggcatcaga tggtctctgg gatgctttca gcaatgaaga

19861 agcagttcga ttcatcaagg agcgcttgga tgaacctcac tttggggcca agagcatagt

19921 tttacagtca ttttacagag gctgccctga caatataaca gtcatggtgg tgaagttcag

19981 aaatagcagc aaaacagaag agcagtgaac ccttcagggg tctcagctgc cttagactaa 20041 aggactttca acacactggt ctcttttaat ttagtgaaaa gtgtgggagt tgtaattagg

20101 atcatccacc ccagacatgg aatcccccct ccctggtggt cttaggtcta taatcagtga

20161 cgaacagagg gtgcccttgg ccaatgtagt taagaaactg gaaaatggtt tcttcatgtt

20221 ttcccaactc tttcatccag tgtccaaaat atataagtaa atagctgtag agtcacatat

20281 atgaagtgaa tagcatatgt gtcatttagt ctccctgaag attcttttca agatcctgtt

20341 cagggtcctc caggcatcag ctgttgtgtc ctctctttgt aacagtggac aggacagacc

20401 acccagtgct gcaggagaca ggccactgcg tcacctgtga gtggtcaggg gctgatgtgg

20461 caacaccctc tgccaagaga cagagctgtc ctgagaatgc tttgtccttc tgagcccatg

20521 ttttctgctc agtagcagct tggaagcaga tttggaatgg tttaYtattt tggctgctct

20581 tggggactgc gagaagcaga gagaatgaga gaccagtggc aactgcctgc acagcagaga

20641 taaccctctt cccttgcttc ctttaatagt taaatagact ttgtatacca cctgaccagc

20701 ctttgtgcat ttatcctaat catgcatgac cgttaacctt ttgcttagtc cttaccatat

20761 gtaataggca gctgttaaat tcaccaacag ataccctgat ttttcatctt acgtgaccaa

20821 gaaaccacgt taggggaaat gaaaaaagca agccacaata ccatgattcc ttccattttc

20881 aacagtagat gaaggaaatg atactgaatg agtcacagtg ttccctggca agtaagctgt

20941 ttgcattgag aaaggagtga gctggtgagg ttaccaccct gaattgagct ccagctgcca

21001 gtttttgtgt ttttccttgc ccctttccaa gtggttttca agtgtcaggc agtgttctga

21061 gaagcagcag cctataactg tatgtgtgtt ccttgaagcc aggtgcagag ttcccagcta

21121 ctgcagcttg ggatttggtg ggaaactact gggataagct tctccttgac aatggaaagg

21181 cagcagtctt caacatttgg ttgcaaatct ccatccacat cagggagctt tccccaggca

21241 aatacaaacc gccccgtggc ctgcaggcct gcaggggagg cagcaaaggg acctggcagt

21301 tgcaacacag taagtcagga attcaagcgt gaacccatat tgataagtgg gccagaatta

21361 tttaggggcc tggcttgctg ctctccattt cctgtatcgc taatttatca tttttccaac

21421 cttggtattt tatattattt agagaaaatg ctgggtcact ctctttgcct aaggtgactc

21481 aaacagccaa acgaaacttc cgttgcctcc cagccctctg tatccacctg gttttgttct

21541 ttccctgtga gcatttgtgg ttatagcttc ccatatgcca cattcaccgg ccacctcctc

21601 Rccgtggaac cctgccctct cccctcctgc tgctgtagag ttttcccaaa gcagtgtcca

21661 ggagagaatt tgagagagtg aagaaattgc cttgtagatg tggagggctg caatctgtct

21721 tgatttctcc aagcacttaa ctttctttga ctgcactgaK aattgctaat gatttcccat

21781 gagatttgct tacttttgta tactgtattt tccagcatta cagaaccttg gttattgttt

21841 tttagccata gaatcttcta gtaaaaaata tctgccacca ttttagattt aagcatttgc

21901 ctatggggag acactgaata tgtggatgtg tgtattaata tttggggtgg ggacagggaa

21961 gggaatgtgg aaaacaaatg ctggctgtga gcagtgctga gatggccagg ccaggcggct

22021 gagtttgctt ggaaattcag gacattctga ctcctaagag ttgcccccac ccaccatcaa

22081 actgaaatca gcaccaatgg tgtcagcact ttacagccca tagccaactt tctttatttt

22141 taacgtagca caaaaatgta taatagcaag gaaaagacat ttttaaattc cRgttatttt

22201 tattgtctaa aatgaaagca acagtgtttt gataaagatg aaaaagaaaa gctactaaat

22261 tagtaaatca gtggttacgt gccctgcaga atttcttaac agatggtgct gagtgcacga

22321 gttacataac tttctctcta attgaggttc acaaggcgtc ttctaaattt tgctttgtac

22381 aattaattca tttctgatgt taaccaaata gagtgtatat atcctactcc cattactgcc

22441 tctttccccc ctactatggc ttgtagattt tcaaaagata gaagttctag gcaaaactgt

22501 agcagtttca ttaatagttg ttaggatagt tatatctaaa atcagagtat tttgttccct

22561 tccttctgga gctctctctc cctagccctt ttccagctgt tggggatgtg cagacatcac

22621 tcctgggggt gccctatggt acactggctg ctccccaggg acctgggaat gctgaatcta

22681 gccattaggg aaattgattt gagatgtttg catgtgaagc ttccctaagc agcactgcca

22741 aagctttgga attgtcaccc aaggcttcct agactcccta gctagctcga gttcgaattg

22801 ccaatagtcc ccatggtgcc aaattttgςg atggttttac agtcttttag aaatgaagac

22861 aggaatggcc atgtgcttct gagaccccag cacgatgcca ggcccaggtc tccattaatg

22921 cagtgctctg ctgctgctcc actggcaatt tgtactctta acataggttg ggggagggac

22981 agctagggaa atttttttta attaattgta tctaaaattc tttcatcata ggaataaaca

23041 acacatatag aaagctccaa agctgttccc aagacctgta ttatttattt tatatgacca

23101 acttgcccca aggcaattaa ctctgaattg ctgttctaat gtgacatatc tgtgtacata

23161 tgtacatagt actggtaacc agtggctgca ttttattcct tgctgtggac tgaggtattg

23221 gcttcttgaa tcttgtatat gtcataagaa tattatacag agagtgttat agtgaaggat

23281 gtaagctgga taatttaaaa cagaccttat ttcatacaag tgtaattatt acatcatttt

23341 gggtaaatgg caaacaggat tgcatgagag tgatggcatc atcatttcaa ttgaatctga

23401 agtgattcta ggaaattgat tccactgttg gattctagga aattgagtcc actgttttcc

23461 tttgcgttta atttcctttg tgtttaattt gaccacagaa acttttttga gtagcaccta

23521 gtaacattgc aaggaacttg tgtatcaagg tgtatcttga ttatcctgat tttttttgtt

23581 ctctaagttg ttgccagatc tctcttgcaa ctgtgtcaat gaaaggtctg tgtttagaaa

23641 aggcagcata tcattgtata tttgaaacta taggaatata tactttgtat aaaacttttc

23701 caatgatttc agaaattctt ttttcctcct tttgacccac aactagactg ttcataccct

23761 aatagttccc caaaattgcc ttagcatgtc acaccagcat ttgtcatcca gctctggaga

23821 aatgacatga ctgttagata ctatgcctgt ttaattgcct ctggattaag tcattgatag

23881 ctagactttt gagctagtta gctgtagaaa taatagaatc cagtgtttcc caaagtgtgt

23941 tccaaagaac acaagttcca aagatgctct tctaaatata ggattctagg atgaaataag 24001 tttggaaaac acataccatc ttagaaagtt agtgaacatg agcacatcaa aggctctaat

24061 ttaaatgtcc tgtgaggaga gaaaaacaca acaaatatct actgtcagtt agcccagtgt

24121 tttccagatt aatttgacca cagaaacttt tttgagtggc acctagtaac actgcaagga

24181 acttgtgttc taaagaacac cctttgggaa attctggtat attgaaatat tgctgtgttt

24241 tcattcacga tgactcttag tagcagtaca atttgcaact tagaagcatg agcctttcat

24301 atatgaagct gacttgttaa taaaagcagt gttaagaagt agtatgactt aatatgacca

24361 acagcagcct catattgata gcagaacaat cctacttaaa gctctaaaca tcatcccccc

24421 cttttttttt ttaacggaat ctcgctctgt cacccaggct ggaatgccgt ggcgcaatgt

24481 caggtcactg caagctccgc ctcccaggtt cacaccattc tcctgcctca gcctcctgag

24541 cagctgggac tacaggcggc ctccaccaca cctggctaat tttttgtatt tttagtagag

24601 acggggtttc accatgttag ccaggatggt cttgatatcc tgaccttgta atccaccagc

24661 cttggcctcc caaagtgctg ggattacagg tgtgagccac tgcacccggc catccctttc

24721 ttttatatga ataatgagca agagccctgc catagctaaa tatgtctcaa attcattcca

24781 agatctttcc attgcttttt tgcagttacc tttgcttttt gggttgaatc aaaatgaata

24841 tttttatatt tcattactat atgatattta aaaatataca gaaaatcaca gaaaaggaaa

24901 aacagaaatt tgattttaat tcacttttga aattttaacg atttttaaaa ctaatgatct

24961 gttatataat aactgaaatg taaactatta acagttattt actttctttc ctttattgtc

25021 ctctctggat tcagtgaaat aatttgaaat caattagagc tcataccttc taaaactcct

25081 gtcccatatc atcccacctt tcatgtttta tgtacatagt taagtttggc atgttatctc

25141 ttgcctaaaa tgtgaggcct tcctgtagtt ccacagtagt tactcttggc tgcagaaata

25201 ggtttgggaa gctatgagag attaccccaa agtcatggat gaaacaaatc tagtcgaaaa

25261 catggtacag agtgaattaa ggcaaaagtc attaacttaa aattacatcc aatatttagg

25321 aatagctcat cctctgcaca ttatccaaaa tattttaaaa aactaaaagt aagatctctt

25381 aggctaggtg gcggattgca aagggcaggg ggcagttgat accatgaagc ttccaataga

25441 tgcatttcta aacatttttt ctaatatgta aatttgctga tccttagtaa tgttaatgct

25501 ttgtctattt gttattttta ccactgttga acagcaaact gttgaggcta gggttgatta

25561 ttgataattt ttatgtgttt agtaataacc acagcatcat acgtacaact gctttaataa

25621 atccaagtta aggactttga ggagagcatg tagcaagtat tcattttaac aaatcatcac

25681 agacagtttc cctgttcaaa gctccaccaa aaagcaagca tgcagcaaag gcatagactg

25741 tatttagaga tgtggtttca tttattcttg aagtcctata accttgcatt atttaaacac

25801 aaaatcccac ctaacatttg ccaaatccaa gttaatctca aaaacctccg aggacattga

25861 gcacaagcag attactgtaa gggcactggt ggcagattgt ggtggagaga gggctgagtg

25921 acccagagca aagcctgccc gggttactcg ctgccacttc aggagaggga ttgtatcagc

25981 tctcattacc ttttgtcatg aggcagttca taaattaaat atgatatcat gcattatctc

26041 aaaatatttc tatattaatt agataattca cgatgtaaaa cgtgttcatg cagaagcaaa

26101 ggcggatgta atagtcaagt atcagggaaa aaccccttta ggatacccag acattaaaaa

26161 tgtaaaaagt agcttataag aaatgtggat tatcagattt ttactatata taggttgtgt

26221 ttaatagtaa tttctgacac ctgcacatag atgaagaaaa ggcagttgtt accacttttc

26281 accattcaaa aaatgggctt gtctcttaga gtattggtta atgctttgct tatttgttaa

26341 tgaagacagc attttaattt taaagcttcc ttttgtagga ctgatggcac gggcagaatc

26401 acatttaaga aaaatggttt ggaacacttt ttaataaaaa attgtaaaat cagtttccag

26461 tgtggtcaaa attagaatgt aaaggaaagc atttttaaga aaatacagaa gcccaaatca

26521 agggagagta atataaaagg aaatatttta aattaagaaa aggaggtgtt atagtttatt

26581 acatcatgaa ttatgctttc tgtacttacg ctcttagtga aggagataac attgaagtga

26641 gaaaatgaac attctcagtt atttgcgaag ttagtaaaat tgcagtccct attccatttt

26701 tttctgatga aaatggatac atgagatctc atttattgct ctcctcaaca cctttactgt

26761 cccagattcc attcacgttc caaatcgtgg acagtaaaac tgaagtcaga taaacacagt

26821 cacaggtaca actgggagcg agttttaaca tagtggtaat tttgcaaatg ccaacatgaa

26881 tactagtgag tactcaattt acgacagaca atttcacaaa aaccaaccac agagtgattt

26941 gatattcttg cttcttagag agttttctaa tccatataat gcaatgggtc aggttgctca

27001 tatgattaaa aaaattaatc ctgtagaaat gatgtattag acatctctaa cccaagaaaa

27061^" agatgtcttt tgtataactt ttaaattccc atttgtctat cttaggacaa atgatgactt

27121 cttcgtgtag taatgaaaac gtctaaatcc tgctctgtgg agaaaagcta gaaccatggg

27181 acattaagca agaatactcc tctgatttag gctcccagac tacaactaac agtctttatg

27241 tatttatcat cttggttatt gttttatctc aagcatatac agtgtgcaga aagtagaata

27301 atgaataaaa tatgaccatg gagaaaaccc ataacttttg cttctattat agaaagcagt

27361 gtttgttctt tttaatttat tacatgttca cattgaaaca ttaacatcta caatattgtc

27421 tttcagttta ctcttcaatt actaaagcaa atgattttga tttatagcac ttacacaact

27481 gcagttcagg tcacagaatt gaaatctatt tgataatgtc tcagcattct catgggcagc

27541 caggaaaaaa aaaaagagag agaaagacaa atagaaattt tagtgggttc agataaatga

27601 ttattctgac aaacctggaa aatagtaatt aaacagttca ttccaaaaga ccccaatgta

27661 aactctgctc acatacataa tttgtataag gaaaatgtat gagttttttt cttaaaaata

27721 aatagaagca tcattgcatt tataacagat tgatcaatct tatgaacaga ttacattggg

27781 aaagtcacta ttattgctgt gtgtttgtat attttattct ttcctttttt gtgtgagcat

27841 ctactatgtg ccaggcagtg actgtgtctg agtgaatgta tccatggtga gcactcaggc

27901 gccatcactg ccttcttggg gcttacaccc tattggggtg tcagagatat tataacactc 27961 aatattgacc caatgggatg aaaattgtta tcttcatgat ttcttccaat gggcttagat 28021 ttgttgggac agggcagaat tagaataatt atttctcaca tataagagta agtgatatga 28081 gattgggaat ttcttgccaa cactcaagca aactgcagaa gataattact ccattttact 28141 agctcaccta gtttctgaag gatggggtgg ggagtgagac atggagcctc tggggacaag 28201 gatagggggg tgggactgtc ccagtggagg gtgccactgg aagcctccac ccaagtgggc 28261 cagcagctgt gcaagggtgc agcagtaggc tggtgaaagc caggttgtcc agatctttct 28321 aggggaagag aaggattata gaaaaggaga aaagggagga gcaagaacat ctctccatgg 28381 cctattttta tttttgttcc caagagacta gtgtaataac cttatgcccc caaagacagc 28441 ccagaactga gtatcttcag tgttcctgag tgtaaatcag tgaatctgtt atatagaaag 28501 ttaacatttc aggagcacaa ttatttcctg atttcaaata tgaacagaag actaaatgtc 28561 atttttttaa atcataaatt tggtatccat agttacttta cacattccag agtctaagca 28621 tggaatttaa gttttgtatt ttgaacaaaa atagcaacat ctgttttctg tatggcaaag 28681 aaaaactaaa tcccagactt ttatttttaa ccacagaaac catcacttct ttctaaatta 28741 tttgcttgcc tgctcatttc ccctaaacat ctctgtttat gccaatggct ttttaaatcc 28801 aaataatact agttattgcc aaattgcatt agaatcctat tgaattagaa taattcatat 28861 acttcatata ctaggcagta aatggttaga gacggtaaag atctgattgt gatcaaaagt 28921 agggtttttg ttttgttttt aatcatgagc acatataaaa ggaaattcaa atgaaaccaa 28981 gccaatttct gatttagtaa gattaaaacc atagatggag ctcagttatt aaggatggaa 29041 aggaggaaag cacagagtga ggagagaaaa aggggaagga aaaaaagtag tgtttttttg 29101 gggaacagta taatgtctca gaagttcttc tctcttatcc tgctgatgtt gagacagtgg 29161 ctttgtataa gcttttattt atacctattt caacatcatg aaaatttttc catcagcatt 29221 ttcttgcaac tttaaaaatc acaagggatt atttgtttat aaagagatac aggatctata 29281 atttattggc aatactttat tttaggaatc ttcaaattgt acttaataaa taaatcacag 29341 gtcaaaatga ctgtaaaatc aatcaggctt tctcRtaagt tatttcctga aaaccatcca 29401 tgataaatac aacaagSctt ggcgaaacct cattactctt ggaagtcttc agaggttatt 29461 atacctactg atctgtattt gtctctaaga gacatttgaa acacagttac aatgtcaatt 29521 tggtgtaaca ttacctgtag tagagctcag agtatatggc agtgcacRgt cactcaagat 29581 gcatggcgtg tcatttgggt cgctttgatg ggtgctcggg tagaattagc cttgtagaga 29641 ctaatgtgtt catgctatct ctgatcctgt ggtgttgtat aaaaatgaac agctaaaaat 29701 ttacccatga caagattaaa gcaaaaataa acacaaagtt gttagacttt aaacatgaga 29761 ctgctggtat tcttttaagt taaactcatt ttccattcta acatctgttc tWgaatttta 29821 taatttctct atgcttttga gttggagtag cttgagctag ggttctatgt ggaaagcaaa 29881 gaaacaattc atgggtagag gataagacca aagcctaaca gaagcaagag gcaggaggtt 29941 ttgagacttt ggtgctttca tttggaatgg gaataagcct ggctctggtt caaaacaagg 30001 caatttataa acatatatac aatgtatgca acaaaaaatg tttattgtaa acctaacaca 30061 acttttaccc agctttatag taacacatcg acaaagttta tatagtaatt actataaggt 30121 aacaagggtt acattataca cagatgtagg ctccagcaac acagcatgat gcacctctga 30181 gaaaagtgtt cagaacagaa tatccatctg caaaggtttt cactcaatgc aggaagaaat 30241 actgtaaaat ttggagacag aatgaaatag aggtaaactc tctccaggat tctagactct 30301 gaagagtggg tagtactcat ggcttaggcc atattgtagt aacacggtga cttggactaa 30361 ccatgtagat ggagttcgtg gattagacaa ttctcagaat tttctagaaa aagttggggc 30421 cagacatagt ggctctcacc tgtaatacct atactttggg aggccaaagc agaaggatca 30481 cttgagctca ggagtttgag accagcctgg tcaacacagt gagaccccat ctcttaaaaa 30541 gaataatttt ttttaaaaaa aaaggggaaa gaaaaggagt ttgttacgca gacgaaacaa 30601 ttgcctactc tacctgcttt caaattcaag tctgataaat ataaaagttc atttctacct 30661 ataaaatgct tttttaaaaa attatcttgt ttatatgcta aggttcacta aaggagaaaa 30721 aaaaggaagt gcaaaagttt taaaggtaca tataaaaagg gattcactac aaaagtgaag 30781 ttaaatttct ctcctaatcc tactattcat tgagctatga gatagagtga actatcactt 30841 gatttgtgaa caccaaatta acctctacct atagtttttc aactgttctt ctcctgaaac 30901 cgtgaaagac tttttaaaat aaatcaatca cagcagcctt gagggatggg agaactgcaa 30961 gatctgtgtc cagagaaaca gtcattgcaa tgtgaattta ccagaagtca gaaattttcc 31021 accttttcat ttgtgaagag ggcagagtga atgttgttaa catcaggccc agaacctact 31081 ttgtgccagg ttcataaagt aaaccttccc tgatgctgtt cagtgagtaa gcagtaaaag 31141 gcaagcaaat gtgaccctgt cctctctcct cgttcaaatg ggaagacctg gctccatatt 31201 gtgatggctt cattagtgag agatgtgtac actcttcagg tttgtaacca ggatgctgtg 31261 cccctttccg tgctcccagc agataggaat tcagactctg ctgcctgaaa tggaaggtaa 31321 tcacctattg aggagcaagg aagagaggaa cagaaggcat tttttaattt ccgctatccc .31381 caatccatca gtgtgggctg ctcactcagg ctgggatcca ttctccaaca agatgcatcc 31441 cagagcatgg agctcctgtg tcacggacaa gctgctttct atctgcctca taggaaattg 31501 ggtttatctc ttcacttcgg ccccgctcat cattggcgct gaccctcagg aagaggaaaa 31561 ctgtttcgta tgttcctaaa ctttggatgc cctcagctgt aacagaccac tgcacccacc 31621 atggtggtga actcacatat gcaggggcaa gaagtctgtg gtcacagggt tggttcagtg 31681 gctcaatgat gccatcaaga gccagggtct ccacctcagc cactgccctg gcctttaggc 31741 tgttctcatg ggacccacac ggttgcttca gatactatca ttgcagacac aacatctggc 31801 caaagaaaag aatttctcct ttgtctttat tagaaaggaa tacccccaga agcccaccag 31861 cagacatcgc ctggggtccc actggctggg attggttcac atgtcaaagc atgaggggca 31921 acaaaggctg gtaaagggca gtggcagcct gagcaacata gtaagcccca tctctacaaa

31981 gtttttgaat aattagccag gtgtggtggc acacgcctgt agtcccagat acttgggggg

32041 tgctgaggtg gatcacttgg acccaaaagc tcgaggctgt agtaaagcta tgatcgtgcc

32101 actgtattcc agcctgagcg acagagcgag accgtcttaa acaacaaaag tgactgagga

32161 tgggaggcag cccctgcctg cacagaagaa agcgggatgg tgggcacgtg aggctggggg

32221 cttaccggca aacacagcat ttggcctgca ccgcagcagc ctttgctcat aacattttga

32281 tgccactttt tacctttacc taaatttgtt aatgtatttg ttaaaacatc tgtgttataa

32341 aaatgattta tcctcattgt tgagaatgta ataaaaggta cttttaaagt aggaagaaat

32401 cttttgagag gcattttctg ttcaaatttt gttatatttt catcctgttt ctgtagtttg

32461 tacatatgtt gcaagagagg gaagagtggc atttagaacc atcacagcag ctgctgcaac

32521 cttttaaaag aatgacaaaa tttatcttta gagaagttga tttcctttta tctgggaaat

32581 gccttgaatg agtttttttg gggggagtgt ggggggggag aaatgttaag cagcattcac

32641 ccctgccttg cttcccacat ggtctgaaag gagaaagtga aacagaaaga gggctaagtg

32701 gaaaggagtt ttggaaatta ctcgttgata acctcaagca ggggtaaggc ccggcatgta

32761 tagaggagtg gccagatgtg aggagagaag tcttggcccc ggggccacag acaatgggaa

32821 atttccttgg tgagaaatgt tttaattatc gtaagaaagc atccccaaag ttggcagcat

32881 taggcaaaaa ttatttctta catttctggt ttacagggcc taaattgggg tctgttgcag

32941 agttgtctgg cattgtctga aggctccctc tccctttcag acactgagtg tggcagctta

33001 cagtacgact gcactcagtt tttcattccc cattctgtgg aacaatacat tcctgcatgc

33061 tgcaRcatca cttgcagtta cttcctgcaa gagaaatata cttccccacc tcagtgacac

33121 tgggcttggc aagggattca tctcagccag tggatgtatt aatatatgcc actgtgcttc

33181 caccagcttt ttaatttcct tctataagaa cagcatgttc caaacgggct gcccatcagc

33241 ctggatcctg gaatgaaaaa gacatagagc agggttgcag ccaaccactg ccatgcaact

33301 gagtgaaaac attggttgtc atcaagtctg agatttgggc gttactgtag catttgtgac

33361 ccttagcaaa gctaactaat- gtgctaaaag tcacaaactc aacacctgca gacaaggaaa

33421 gcaagtgagg cagagggtgt gtgaaactat ggggatggca gggctaaggc agatgggacc

33481 aaatcagccc aaggataagc tccagtatta aacgttgtta tatgagttaa aatttttaaa

33541 gctgctgatt tggttaaaca ccaggtcatg attttaataa cacagctaat atttacttcc

33601 tgaaagctta atatagttga aaacaaaaaa tataatgacc caatctctta atgttttcct

33661 aatcatttaa ctttgtaggt tcttttaggc accaatcttc agttcaaaag aaacaatctt

33721 ctgaaacttg tttgtttttt ttttgttttt tggtcttttc taaaagtgaa agggaattat

33781 tttttatgtt aaaagttctc tctagaaatg ttttgagtta gcttcccaaa catcgatata

33841 cagaaatgtg gcacttagca atggagatac attctgagaa ataggtgatt ttgtcgttgt

33901 gcaaacatta gagtgtactt acacaaacgt agatggcata gcctactata cacctaggct

33961 acatggtaga gcctatcgct tctaggctat aaacctgtac agaatgttac tgtactgaat

34021 acttactgtt ggaaactgtc acacaatggt actggtgtat ctaaaataga aaaagcacag

34081 taaaaaaaac caaacagtat aaaagataaa aaatagtaca cctgtatagg gagggcactt

34141 accattaata gagcttgcag gggtggaagt tgctctgtgt gagtcactaa gtgagtggtg

34201 ggtgaatgtg aaggcctcaa acattactgt gcactactgt agactttaca aacatggcac

34261 atttaaagta ggccacacta aattcattta acaattttct ttcttcagta attaacctta

34321 actcaccgta acttaacttt acaaactttc aaactttgac tcttttgtca aagttttttg

34381 acacttagct taaatttaaa acacaaacat cttgtaaaac tgcacaaaaa tatctttata

34441 tccttattct gtaagttttt tcctatttta aactttattt ttctactttt taaactttgt

34501 taaaaactta agacacaaac acacacatta gcctaggccc acagggtcag gatcatcagt

34561 atcactgtct tctatcacca catcttgtct cactggaagg tcttcaggag aaatagcatg

34621 aatggagctg tcatctccta tgataacaat gctttcttct ggaatacctc ctgaaggacc

34681 tgcccaaggc tctattaaac atttttttaa taagtaggaa tagaacactc taattataaa

34741 aagtaaatac acaaaccggt gtcatttatt gtcattatca gtattacata ctgtacataa

34801 ctgaatgtgc tgtactttta tacaactggc agtgtagtag gtttgtttac accagcatct

34861 ccacagacac gtgagtaatg cactaggcta tgacattaaa acggctaaaa catcatgagg

34921 caatagattt tctgctccat tataatttta tgggaccact attgtatatg tcattatgca

34981 gtacataact gtaatcttct gcaaaaccag tcaaatctaa ccatgccaca aaaataacta

35041 ccaccatctg tcttaaagaa cgtgactcct atatggatgg agattaacaa gggcatagat

35101 cgattctata gaatatttca tttaattgat tttattataa ctggattagg tctgagccct

35161 gggaaacaga catcaccttg ttatacagaa tcacacatta tagttttaac tgaagttgtt

35221 tttcatatag gacaatcata aatatgagta ttacaagagc tgactacata ctttcaagtt

35281 tatatttcct gacaaaggat atgacctttt tccatcctgt aattcagaga aaaagaacca

35341 cttgccctat ataaactcta aaaagaaaac tgcataaacc caggaacagg gggcaaggag

35401 tgctcccctt tgacctacta ttagcaggtc caacaaatga tatatcacca cgtctatgaa

35461 gaaatttctt tctataaaag cacaaaattt aaatcggatt tgactttaca tattagggct

35521 ttcatcttga tgagaaactc ataacagcag cagctcaaac tgattgttaa aaggaaaaaa

35581 cacagggcag attaataatg caacttggat cttcactctt ctctttccaa ttcaaatgct

35641 ggaatatgac actcttttta taatattctg cccatgaggt tctctccctt cttttctgcc

35701 ttcagagtcc tttccaaaag gaaattactg ccttgcattc tacattattc ctaatttcca

35761 ggtggaaaaa tgagacaaat taagtgactt ctaaaaacac taggaactgt attttttaaa

35821 aacaagacaa tacttcagat tttcttgctt taattcttct ctatattacc acagtaaaat 35881 atttaacaaa gtccaagaga ttactgatat gcaataatga cctatgactt tacattaatg

35941 gagtgatgta tcaataataa actgatcagt taagtaactg gaaaatgttt gcatgtaaag

36001 aatgattcac tatccttttt atcttgtatt gaaatcgtca aaacatttaa aaacacaaag

36061 ttgaagtaat tttaaataat aataactgtg aaatactgca acatcttgaa gtactttata

36121 aatgaccaaa aacaggtaaa attttgttca gtataacttc agtgaagaag ttttttgaca

36181 cagaactaca tatattttta aattggtaat ccacataaga tatacacaaa accttcaagt

36241 gactacattg ttcaataaat aaaactctac attgttttgt ttttacagcc tagtgagctt

36301 ataactcagt aacacccttt taagaaattc tagtctacag aatgacatgt ccaaattgtt

36361 tggtcctatt aatttcttta gcaaaaacct ccaattcctt tatatttaat tcctccacat

36421 atcatctttg aagggcctga ctaataaatc acaagtgtaa ccctccagtc tccagtctgg

36481 gtttttcatg agtttcagtt cagtgtaagc cagcacactg acctccccat gaattttcca

36541 cagtacctac tttatttaac actttccaca aagtatcctg tccttctagg ctttttgtag

36601 aatgtgaagt taataatggc atgtggtgtt ccttagcatg acctgccaaa aagtgatgat

36661 ctccttggaa gaaaagccat gggctgcaat cacacgtctc agttgacaga catccaaatg

36721 gattctatat agaaagaaaa gatttgtgtc ttctggaata tgaatgttca cttttaataa

36781 attcaaacag atcccgacat aaacatcttc aaacttgatg ggttttacgt gacccatcat

36841 .ttcatagatc cttggcacca aatctctgga cattatataa cccaacccac tgcagtatgg

36901 agggaacacc ttgaaaggat actcctggta agaaatatgg gttttttggt aaaatcctct

36961 ataggaataa ttatcaatta gaggataacc tgtgaaaaac ttctctgagt ggtttaggtt

37021 taaaagatac ttcactaaat tgccagtatt gatgaaaaca tcagtgtctg tcttcattac

37081 gtacttggca ttggggcaaa actcagttac ccacctgaat gccataatgg ttttcaaggt

37141 caggttatta tatgtgtcta aaaaatcttg tcggattatg tcaccataaa gaaggtgttc

37201 atcctctaag gacaatgcca acattttgtY ttccttttca gcctcttggc ctaataagaa

37261 aaatgtaaga acctcatatc cccaccaaga ctttttttca ccccaagtaa ctctaatggc

37321 ctgcctggct ttcacatctg aagggtggga ggtcaccaga atgaccagaa atggattttg

37381 atgagagcag tttgaatgct ctcgaagtgt gaagtgaaag tcttgtctgt aaatcggctc

37441 atactcatag aagtacatcc agttcacgcg ttctatcaca ttgtagtggg gaaggctgag

37501 gtaccacatc acaaagaaac tcaggagtga cagcagcagg aggctccatt tgagggatct

37561 cagtgacatc ctactcggaa ggacagtcca gagagccgag gccatccaca gcagctcaga

37621 agcacgcgag cYgaaggttc tggaagagtt atcaaagatt gggttaatat tccacaccga

37681 aaacacattt tcaaaagaca tctgactatc tactcaagga gaaagagtag gagaacagaa

37741 tctccagagt aaaacaaaaa atYctgactt gaatctactt catttaactg taagcaaata

37801 gaaaccaata accaaaaatc tattttagct tcttacatca agaccaaggt taaaacatat

37861 cctttgatta ttttgtagta gttatcctac tctacctaaa ttaacagttt actgttaaat

37921 ataattctat Sctaggtgca gatatgatag ggacagaaRg agatgggtaa tgatgggtta

37981 aaaaataaat ctaccataaa aacaaataaa taaaatgtat ctcctcttta taactataaa

38041 aaaagaagga tttaattcaa ggggatgtta ctagccacat gcccaaataa acgaagggtc

38101 tttctcagct gtgtgttctg atacggattc tagcaaatct ggataagaaa aaccctatag

38161 tga^'tcattat tctattctta gtgactgctg atgtgggata aggcagagat tcttcacctg

38221 aaaggcaagt cctccagaca caaataggat catctctctt gagttttcaa acactgacta

38281 tgaaacagct tttacctgcc ccaaacacca cctctcccct ccttttacct cattggttaa

38341 actcaatgct cagtctgcat agcaactgtt taaatcaaag gcccgtcctt agtattaagt

38401 tttcacattt cccaYtccaa cctcttcaga aatcacacac ccaatcagaa tcatgggatg

38461 gaaRctccta aaacacttgc ttttacacag aagctccagc tgagacaaga ggaggcacag

38521 aacattcaga ttgtgaagtg acagctctgt tcctgatctc aaggcagagg gtggtttata

38581 attcgggatt ccatagagct ttccttgcac catgagcata gacttcagtc taagggcatt

38641 catgtatgta tatttactgg acatctgctt ccaaagacca ggcactaagg agaccaaagg

38701 aagtaggaga cgggagaaag aagaaatccc aagacaacgt ataaacatgg cagcacagtg

38761 cagtacacac tgtttacatc aaatgctaag gaactttagc ataagggaca gatgaacaac

38821 atgaggaagg gtgggctgag gaggctgctg gcaggtgccc ttttacccac aattatatgg

38881 gggagggttc ttggaaagct ggaattaact atggaggaca gacattaatg tccttatggg

38941 aaggctagtc taccaagggt tctaccttgg gtcaacttta gggtctctgg acatacagct

39001 cctctgtctc ttaatacata tgaaaataac aggatttcaa ggctccttgt cctcttcatc

39061 tggtcatctc ttacctgccc tgattacgtc tcaccacaat tatcttttct atatgtaatg

39121 ctttccatat tttaacccac acaacgcaat gtaatgtttt agcaaactct ctccagtttg

39181 gttccacgta tatgtgtctt gcctctccac ttacattcta tgaccttgtt ggcaaaaaca

39241 atttttctct caggagtcaa agtgcaagac aaagggaggc ctacagcagg agtgcaatgg

39301 acactgaggc ttgatatgct atctccattt gtaaaaggat aaaatataga tctgtccacc

39361 cacctcctat cctcccctat cccctttact tgccatacac tgccaatccc agaaaaaaac

39421 ctgaaatgga atagtttgag agcaagaatt atgaaagtaa ttaacactgc ctttatacgt

39481 tttctcctca cctggctact gggcactacg caacccttga gggaaccatt tacaactcag

39541 ccactaacga cttgtacatt tatgacaaca aatatctcat tctaacaaat cagcatattt

39601 caacagattt tggaaaaaca gaaggcaagt gttgtaagag gtgccattta taattgcata

39661 tggatgatgg gggccctact actcttcaat aatgggaaga acttaaaatg gcaaatctta

39721 ttataaaagt tattttttgc acattctata agccatagtt tctaaatgtg agaatgtgac

39781 tatggttatg atgtctatca tttatggacc catgaataaa ataaagcagc agcaaaaaga 39841 ggagaaacta tctagtccaa aagcaaggaa cggactctaa agaactggcc agaacagact

39901 aggtcaccta caacacaggt ggctccaaag ccagccacca aagtccactc tccagctctg

39961 tcctttacca accaggatga atgggtatgt tatgtatctc ttgaagcctc agtttcctgt

40021 ctctatattg aagatattaa tatagtgcaa attagtgata atatatgcaa aacttctggc

40081 ctgtgttagg tattcaacaa acagaagctg ttattatcag ctgtggttct acccttggca

40141 tcatttccat tctatttgtg ttctctaaaa tgattcttac tgtcaaaata gattatcctg

40201 aaaaggaggg aggggcaaaa aattttttca tgtggttaaa aaatgtaact aaaatactag

40261 gtaaaaatga catgatggct acaaaaagat gtttcatagg aaaagtaaaa tatgctaaca

40321 ttcttagtat attttaacaa agagatatga ataaaccttc acattttgct aaaaacttca

40381 tttaagaatg tacattaacc tttacattga tcactgaaag aacaaatata gaacactatc

40441 ttccaaattg attgagaaat agaagacaca aattaagtta tctgtctatt caatataagg

40501 taggaaaacg ggaaataaaa agaaaaaatg catgcaaaca caaaatgcaa aagatgatgg

40561 caggaataaa tataaatgtg ctgttaatta aggtaaatgt aaatgtaaac ttatctacta

40621 aaaaaaaaag actctcaggc tgggcatggt ggctcatgcc tgtaattcca gaactttggg

40681 aggccaaggc aggcggatca cctgatgtca ggagttcagg accaacctgg ccaacatggc

40741 aaaaccccat ctctactaaa aacacaaaaa ttagccaggc atggtggtgc gcacctgtag

40801 tcccacctac ttgggaggct gaggcaggag aactgcttaa acctgagaga cggagattgc

40861 agtgagctga gatcctgcca ctgtactcca gcctgggcaa cagagcagca agactgtctc

40921 aaaataaata aacaaacatc attaataagg gacaaaggag atgttattac taacagaaga

40981 aaccatccaa aaagatactt tatcataaag ctctgtaaga agaaaccatc caaaaagata

41041 atttaacaat catgaagctc tataaccata aaaacacagt atcaaaatat ataaagcaaa

41101 acctgaagag tatgggtata aatgggaaaa cttataatcc taatgggaga tttttaatac

41161 atctaagtaa ctgatacatc aagcagataa aaaaggatat gaatgtttca aataatatta

41221 ttaatcttga tcaaatgaaa catttagtac ctttcatcca ataaaaagaa tagaaacatt

41281 catttttaag acaacataac acatttaaaa agaaaactaa taatattagg taatacaagc

41341 cttctcaaca attttaaaaa caataataca ggctacattc tctaaccaca gcataattat

41401 aaatcaattg caaaaaatat ggaaaacact taaaaaattt aaaaaccata aaatttctta

41461 aaaatttgtt tagaagccca aggtgggtgg atcacgaggt caggagatta agaccatcct

41521 ggctaacacg gtgaaacccc atctctactt taaaaaaaaa aaaaattgtt tagaagctca

41581 aaatccttat atacacaatt tgtttcatcc ataaatttaa agagaacacc atatctgcac

41641 tgcacatgag caatgtaaaa tgtttcaact cggtagctac tataccaatc tcttacttga

41701 aaattactgg gatgatggag gaaagacagc attttttatt tcctgcttta aagaatcaat

41761 catctatagt tcagaggaag caaatagctc taattgaaga gaaaaagctc ttctttatag

41821 aaaaatgcca cttataggct gggtgtcatg gctcacacct gtaatcctaa tgctttggga

41881 ggctaaggtg ggaggatcac ttgagaccag gagttcaaaa ccagcctggg caacatagtg

41941 agaccctgtc tctacaaaag aataataaaa ttagccaggc atggtggcat gagcctgtgg

42001 tcccagctac ccgggaggct gaggcagaag gatggtttgg gtgtgggagg tccgggctgc

42061 agtgagctgt gattgtgcca ctgcactgta gcctgggtga caaagcgtga ccgtttcaaa

42121 aataaataaa taaataattt aaaaattttt aaaagaaaaa tgccaattag aaatgtctag

42181 aaattacccc tgcagtttcc tggcccatag tttcagttat ccacagtcaa ctacagtctg

42241 gaaatattaa gtgaaaaatt ccagaaataa acaattcata agctttaaat cgcgcactgt

42301 tctgagtaat atgacaaaat ctcgcaccat ccttctctgt ctcaggtaga acatgaatcc

42361 tccctcttcc cagcgtatcc acactatata tgctacccac ctgttagtca cataggagcc

42421 ctcagttacc agattcactg tcatggtatc tcagtacttg tgtgcaagtc acccttattt

42481 tacctaataa tggccccaag gcagaaaagt aggatgctga cacttcagat aggtcaaaga

42541 gaagctcaaa gtgcttcctt taagtgaaaa ggtgaccttg gcattctttg ggtagggtgt

42601 ggtactatca tggtttcagg catgtttggg ggacaggctg ttgaacacag cccccatgga

42661 taagcagaga ctactataga tggaatgaca gaataagaaa actgtcattt tgcaatcttt

42721 catgaaatta ctgatgatga ttatacatca gagttttgat aataaataaa aggttgatgg

42781 acaattggct atctgtatga tgccaaagta acaccacgaa gattactttt tagttgcaag

42841 aactgaaaaa atataaactt tacattggag gatcagggtg tcaccattct aacccaataa

42901 ttaatcgtaa catcataaat gatggaacaa acacctatta tgtatctcct aatgagatac

42961 aatgtaatgt ttaactggaa tctaatcaaa ttttacaaga aatacagagg atagaaagaa

43021 aagctaagga acatcacaag aaagcaatca aatccataaa tatatgtcca taagataata

43081 aataaaagtt ggtatgtctt ccaatggaat cctatatgat agctaaatga attggatttg

43141 tatacagaac atcaatttaa ctctcagaaa taaaacgttg agtataaaca agttacataa

43201 tgatatatgt ataaattatc atttataaaa attctaaaac tacacacaag aagtgatgaa

43261 cctttagaag caactacςta taaataatga tttttttcta atgggactac aggaaaacat

43321 accacaccta tcagcaggaa gaatgaaagg aacatgtggt taaaggggga aaaaaacact

43381 ggtcataaat actgtactgt aattaacagc tgctaattca gggcgatgga aatataggtg

43441 ttctctaata tttacagttt tctttgtata ttaagtttat taaaacaaac aggaaaaaag

43501 cataataaaa atgctttgga tatcagagcc aatccaccct aaatcttgac tgcaacaatg

43561 actagttgta tgaccttagg catgttagtt accttctcaa aagtttcatt tttctttttt

43621 tttttttttt tttttgagat ggagtctcgc tctgtcgccc aggctggagt gcagtggtgc

43681 aatctcggct cactgcgcct cccgggttca caccattctc ctgcctcagc cgcctgagta

43741 gctgggacta caggtgctcc accacgcccg gctaattttt tgtattttta gtagacatgg 43801 ggtttcacca tgttagccag gatggtctcg atctcctgac cttgtgatcc acccgcctcg

43861 gcctcccaaa gtgctgggat tacagacgtg agccaccgca ccaggccaaa agtttcattt

43921 ttcagctata aatcaggcat cacaaaacta attattgtga ggtttaatgg aaagaacttg

43981 gattaaccag tcagcacagg gcctggtcca ggaccaagag cacttaatgg tttctgaata

44041 ttcccaacat aaagaaatga taaatttttg aggttataga tatcctgaat ttctgatttg

44101 accattacac atttatgcat gtatcaaaat atcacatgta ctttataaat atgtacactt

44161 atcaatcaat aaaaatattt ttaaaacgat actcaggcat gtagataaat aagggctcaa

44221 ttgaagtggc ctctgtgttc atagcatgtg tgtatcatag aaatcaaact ggccagagat

44281 ctcggattgg ctcatttcct gatagtgaga gcacagggca gctggcctgc ctccttagga

44341 cagctgagca gtgggctaca aggaccccga cactgttcta aatcccatgt cccaggggag

44401 ggacggagag aagaatcaac aatattgacg aatagaaaac aatatgaaaa gccccaacag

44461 ggaagtacac tattatttta ttactgactt cttaaatgcc acacatcact gtcaagaaag

44521 aaattataac ttaaaaataa tttccatttt ctaaaagata aacttgtggc caggcactgt

44581 ggttcacacc tataatccca ggactttagg aggctaaggc gggaggatcg ctggaggcca

44641 ggagttcaag accaacctga gggaacagtg agaccccata gataaaaaaa aaaaatttta

44701 agataaactt gtgcttatat taactacata tgaatttgcc aactataagc aatttccatt

44761 aagcaaacag tatcttatct atcccaccaa cttagtgcaa tgctgggtga acagcaagcc

44821 accagtaatt acttgttgat ttaggggttg atctgcagca gctgaaaaaa catctcaaat

44881 actagaaaag ctcagaaaac ctccaaagat aaaaaaggtt atcagtttcc agtgttgtaa

44941 gtggtagaga caaactcaaa caagactcca gatctcacat tcttggctgc tagatcccag

45001 ggagtgtgct gcctgctcac tatgcttcct atgctaagga gctcttctga ccagagctgc

45061 tYtgagtttt aatttcttgc aaccattgaa ctttgatata aacagtatgg cagcatcaca

45121 gccttatcat tttcacaagg aagactcaaa attcctaccc ttaaaacctg aaatacaaat

45181 ttttaacaag aacagtaact aattgttcat gcagatatac acctttttat ttaacaccaa

45241 aaacatactg gacgtcacta gaaaataaat ggaggtatca gtgtttaaat gcaaagattc

45301 caggaaaaca gacaaacaga gatactacaa tggcacaatt atttaagcca aaataacctg

45361 aaactctttc ctggggtgca aaaatgttgg ctatttcctg caaaaagaaa tcaaatgtaa

45421 ttatttaaaa agtaattatt ctaatttcaa atgtaatata tgttcattag agaaaattta

45481 gggaatgcca gaagagcaca gagacaataa aagcttaaaa ataacaatat taacattaaa

45541 aaaaaattta gagatgtgtc ttgctatgtt tcccaggctg gacagcagtg gccattcaca

45601 ggcacaatta ttttgcactg cagactcaaa ctcctggcct caagtgatcc accccagtag

45661 ccaggaccac aggcatttgc cacaatgccg ggcaacaacg ttcacatttt ttatgaagat

45721 ctgtcaagtc ttctggctcc atgggtttat ataagcccct ctttcatctg ctacaactac

45781 tccctcccaa tcctatttca accacaatgg cttccgttct tttttattct tttaatcttt

45841 taattaaaaa atatataaat atatagagat gggggtcttg ctatgttacc caagctggtc

45901 tcgaactcct ggactcaagc aatcctccca ccgcagcctc ccaaagtgct ggcattatag

45961 atgtgggcca ccaaacccgg cctgactccc attcttatcc ctgaatatgt caactatttt

46021 cctctcttgc agagcctgca cttactctgc ccctccaacc aaaaagctgg ttgttggaaa

46081 ggctggctcc ttctcatgcc tcacatctca gcacagtgtc acttaggaga gtccttccct

46141 gaccccttat ctcaagtagt tcccccagca cttattttct atcacatccc tcattattcc

46201 cttccataaa gtatatgaat gtgctttggt taaggaatag gctaaggcga atatctgggc

46261 caaagtgact tagcaagttt agggcacagg tgcatacttc acttgttaga taacctgtct

46321 ttgtaagttt atacttggct ttgagttgct attgtttgta aaaggtataa ctgccctgct

46381 gatgctgccc atggggtttg ggttggcttg acatggcttg gaggtgcact aatgcccaga

46441 gagagtaatg ctactgaccc ctgtaaggaa gagtggattg ccttgaaggc gggaagtggg

46501 gacttatgcc cagagggaaa aaattaagct gctaaccctg gtagtatagg ccagggagtg

46561 cagctgcaag gctgggggca gcaggagcag gctcataaga ggagccacaa agctgaagca

46621 aacagccaag ataaagaaac tgtgagaaac agttagtgtg aggaagctgc tgattaaaaa

46681 gctgctgaat aaaactacat ttcacctgcc taaagccccc cgagtgttct ttcagcaatt

46741 cgcccatcca cccactcccc tcggacctca actgggactc aaacctaaca ccttgttgga

46801 cctcagctgg gacttaaacc taacaccttc tcagcattta accctgtcta gacttttaaa

46861 aatctatttc tatttgttta ttgtctattc ccttcaaaag aatataagat tggagagagg

46921 gaaaacctga ccccacttat tcactgctat atgtccagtg tctaacacat agtaggctct

46981 ctgtaatatt tgcatacttt gcaaatgatt tggtaatgta cttttatcca cttagtaaat

47041 attcctaaga ataagctgaa tccaaaagta acctgaattt gttgattatg tttttcttca

47101 gctgtcttct aataacaaaa ccttattgta ataggctatg ctgattctga tcatgtgcat

47161 tttaaaaggt ccaaaaaaac aagaaacaaa tttcagttaa attttaggtc caagcacgct

47221 agagtcagga tcatccatat gctaactatt atagataatt accaaactac tgtcagtttt

47281 gcaacattct aacttttctt ctacataaaa tactgcaaag ggagtggaaa tcttaattgc

47341 ctaaaaaaag gtcctgtgca tattaagttt aattcaaatc atcattacac caatcatgta

47401 tattccttat aaagggaaaa taactatcaa ttcacatctt tgagggaaat tcccccacta

47461 ccctttcaaa ctaaacacat taactcataa agcaatataa aactctacca ttcaattaga

47521 cttgtcaatt aaacatctaa ttattttcac aactcactgc tgctttgtgt ttgaggccct

47581 cccctgatta tattagaaat aaacacaacc agtgccatct gaccactact ttttcttccc

47641 ctcttcttta acccagtggt tctcactttg cggcacatta taatctcctg gagaacttga

47701 aaaatactgg aagccaagcg ggatcccagg ccaactaaat cagagtttca gagggtggca 47761 gggctcaggt ggcagtaatt ttagtatacg tgctgccaaa gcgagcacca ggtggcagta 47821 tttttaaagc tcccaggtga tttgaatgtg atgcctagac cctcaccact caaagtgcat 47881 gggcaccacc taggagtgta ttaggaatac aaagccccag gccccaccct agacctagtg 47941 aatcagaaac atttttaaca ggtggcttgt ggacatactg aagtttgaga agcaatgaaa 48001 tccacttgag caaattactc aaccttttta agcttcagtt accttatctg taaaatggga 48061 gcaataatac cttctttgcg ggtttattgc cagagctaca tgaaataata cgYgctaaac 48121 atctaggatg gggctggcac acagtaaggg ctaagtagat ggtattgctg ttattattag 48181 agtccacact atatgcttcc tctacatata ttatttgtct acatttcctt tacaaacctt 48241 gctcctaact cccaaatgtg tcttacatgg catgacatgc tctgtaatac aattaaacat 48301 gcaaataaat cttagtggat ccaccttctc cttccaaatc agtaagtctt ctcctagagt 48361 ccaaggtttc tgttttccta gaatctccca gtgcctgatg ttctgctatc ctgcaaatac 48421 gagccccaaa gaactactgg tagaggtcca caaagccaaa cgcacatttc tgggttccgc 48481 ctctgcacat gtgctcccct ctcctgggat ccttctttct gccccatggt ctttctacaa 48541 ctgaacatca ttttttctgg gaaccactca cactgtcaaa agtctcacca taggcacttg 48601 tagcagctgg aggagagctg caaaatacaa gaaatttttc cagctgtagt gcgtgtgata 48661 ttcaagtggt gtcaggctac tttgaggatt gtgcatattt gatatggttt acatttttct 48721 ccttactttg gagcatacag ctggacatat aataggaagt gaaattttat ttactgaatg 48781 aatgaatagc aattggtata tctgcttaat ttgataacac ttaaaaatga aatatgcttt 48841 actctgtcag ggtaaatagg ctcacattaa aaagaacctg ctttctactc tatccaattt 48901 cttcatactc ccctctctcc aaaattttag caatagtcct tgcttggtcc tggcactgta 48961 gtaagtatgt tttatatttc cattcgtttg atgactgccg caatcttatg aagtaggcac 49021 caatattata acatttatct ttcagttaac cagcttgaaa tttggacacc tgttcaggag 49081 tcagggtgtc agagcctgca aagcagcact aaaaactcag gtatattcac caccccaagt 49141 ggagtctgta gccactgcac tacaaaagct tccctcctgc cttgttttat cctattggcc 49201 tggtatcttc atactttcac agtgcagcac ctctgttctt cttcagaggt gaagagttgt 49261 cagagctcaa cgagaaactt ttggccgggc atggtggctc atgcctgtaa tcccagcact 49321 ttgggaggcc aaggctggca gactgcttga ggtcaggagt tcaagagcag cctggcaaca 49381 tggtgaaacc tcatctctac taaaaacaca aaaattagcc aggcatactg gtgcatgcct 49441 gtaatgccag ctattcgagt ggctaaacca ggaactttaa agaaagggaa gtttagtcca 49501 atttccatgc aaatcatgct cacccagttt tcaacaagtc ctctccaaca agttcacttt 49561 ttggcaatgg agaaattcct gaagggacta atgttaatgc atctgtattc caaataaccc 49621 tgaataagtt gctgagactc tgtaaacatt agtgaacact tatgacacaa acacaatgac 49681 catcaggagc tagaagagat tcactgagaa tgctctgttt gactatccta agccccattt 49741 tcaatagggt tcttcaatag aagatcaagg taactcgcag gtaacaccta agaatcaagc 49801 ttctctgtaa agagagctgg acacttaaag tccttctgaa caaaatggga aaatgtgagt 49861 aataatagta gctagatggc ttaaaaattg gctgatccat catagccagt gtatttttca 49921 tgcagtcatt tgttcaagaa acatttcttg ggcttgtgct ggactctggg aatagtgttc 49981 cacaaaagtg ccagagactt gtcctcctgc tgatgttcta aggttgactg gagcatcttt 50041 gtcaaactgg ggaaaaaaat cacaggccta tctttagcct tttctcattt catatttcta 50101 attaaagaac gtgggcttat tcaattttgt aggcaggaaa aaaaatgaca aattcattaa 50161 aagatgtaat tacaatccaa aaggttttaa caggtgagaa aaagctaaag acagcaagat 50221 gaaatttgat gggaacacat ttattgtcct atgtcagagt tcgaaaaacc agctgcacaa 50281 gggcaggatg gggggccatt ttaacagcag cctaaacaga ccttacagtt tagctgagta 50341 tgagtccaat atgataaaag gtggtgaagt aaaggaataa ctgggcagtc aggaaacaag 50401 gattctagtt ctggcaggga ggccaagtca ttgaatttga ggagttattt ctcaacagaa 50461 ttttaggaca ttgggctagg atacctctca ggcaccatct ggccctacca gcagagggat 50521 gatgttcatt cttgcacact gtgtccccag cacctagcat agtgcctggc acataggaag 50581 cactcccaag tacttgtgaa tgaatggcag tgctaacaga aactctacaa gtgaatacta 50641 gattgtgtcc cccttctcta acacacacac acacacacac acccctaata cactcctagg 50701 ttcaagaaac tggaaaagca tctgggcaaa gggggtcata attccatagc cccatatgag 50761 ttccattttg aaagggtcac taacaagtta gaagagggta actcatttgg tgaagggttt 50821 atcatctccc ataaggattg catgacatgt ggctgaagga tctggggatg ttctgttctg 50881 gagacaagaa aagcttagaa gtgccatggt agatactttc aaattatttt gaataataaa 50941 atMtgtaagt atcactccag aaggcaaatg agaaaccaaa gatagtgtta ttctaacaaa 51001 cgtcgaaggt cccttctaac tacaaggttc cacaactttg ttattgatgt ttaaaatgta 51061 gttcatagaa aaggtttcat tttggcatat tttaaattgt tcaattttta agaatgaatt 51121 tgaagtattt actgagttta agaaaaaagc ccacctcgag caccactctc tgtagttgtc 51181 acacagtttt aggtagacag tgagtataaa tacctatact cattaccatt tcccaattct 51241 gcccttgaaa ctctgctggt tgtagcacca ccaccctcag gtatcttcta agatgtatgg 51301 gagaatgtct ttcattaaag cttcattaaa agggaagcct gctgcttcat tcagtgtgct 51361 gaggtataag acatctgagt ttttaaagac acatgtaagt aagacatatt atatatactg 51421 ttatacaaac tactttcttg taggcaaaac aatccctgta tatgggaatc tctgatagac 51481 ttctgggttg tggcatgcag aatcttttag ggatttctgg gtttgggctg agaaaaatga 51541 ggagcaaaca aaagcagcag gactttagaa ggaaaattta gccgcaattt tctttttgct 51601 tctgccctat ctgcatattc caataagaac ataaaaatca ctcttaataa tttgcaatac 51661 atgttaggat ttttagcagg gctgtttttt tttttaaagg catagtgaac ttctgggcac 51721 tatacaagtt tatagtgctt ctaactacaa attacaaagc acatttcatg acactgtaaa

51781 atctgtgtta acatcaatcc cattttccag ggaacagtgc agcccaggta gaggaagaca

51841 tggaatccaa actcaagccc ttcaactcca aatgccaagc tccttctgcc agggcacagt

51901 gccccaggag agaccaacct cacatctaat ctcacagcct cctgagattc ctgggcaggg

51961 agcaaagaat gcagcagaag cagacacacc tttacactcg gggtgagtag tcggagagca

52021 ccacgtgata gagcagccag cgggaagagc caacaggtca accgggtcca gggagctaag

52081 aaaactggag cagagcaaag atcacaaagt caggcagaac agcagcaaga ctgccctgaa

52141 agctgggctt tctgtgtctg ttttgttaca aagagcttta tccactggag gaagagtatc

52201 ggggagctgc tgcagttaac atttcttccc attactaccc taaattgaga atattacagc

52261 tctttactat tcttcttagt taagccatca attacctttg ctttagtcat ctgagggtat

52321 ttcattcctt cattcagtgt tctggacctg tgttagaggt tggggcagca gtggggaagc

52381 agacagacaa gccctgtcct caagttgccg acactcaatg cagagcgtgg tggaagactc

52441 acaacgacac atttcacacc aaacatcatg agtgaggccc acctagagaa catgggggag

52501 gctttccagc tcaaatgttt ctaaactagt gaccaaaatc cacagcaggg aactgagttt

52561 gcactctcag aatgtgttag tgcccaatag aggcataaat ttagagcctg ttatcagtat

52621 ggagaaggga actatttaat actaagagat aaaactggac ggtaattttt tatcttagaa

52681 agtgcgtaac tccattctca acgacaaagt ctgtaaccaa aatagcagcc aaatacaatt

52741 ccagatatga gatatggttt gagccctatg acatgctgca ggaataggtg gctgcaggct

52801 tgtgaaatgt tttaacagga ggacttgtgt attctaactt ttatgagact attcaaaatg

52861 cacagcttga gtttagcaga aatccatgtt ctatcccaga agcgggcaaa cttttttccc

52921 cataaaaggc agatagtaaa tatcttcttt gagggcccta gggtctgtct cagctactcc

52981 attctgcttt tgcatagaaa gtggtaactc cattcttgaa aacaaactct ggaaacaaaa

53041 tagctaaata tagttccaga taacagacac agaaagagaa gcaacagaca atgagatcct

53101 acctacctac ctgtgttcag agtcagaaat acatggggtg ggaaaccgat aacctactct

53161 agctttcttt tagttcctta atgtctcaga gattcaagga gtgcaggtta tctccccagc

53221 caaatctccc aaccttccag cacttggggc cctaccctac agatgttctg tggcttcatc

53281 ttccttcccc atgctgatgc gccccctaca aactgccgta tttctagctc atcctctcat

53341 cttcccctag cctctaattc acaatatgcc aaaaggactg gtaacaaaag caaatcagta

53401 tttataatat acaatttttt attagtataa aagcattgtc catagtccca agagagatgt

53461 ttgccaaaca gcctgctgtt aaatgaactg taacttttga atgtatctgt tgtgcaaggt

53521 cattaagagt atcctgtttg atcccacgtg cttgacccca tccatgtggc tatctaccac

53581 acagtgggag tttaccccat agcgagcaac catgatccat cctgttaaaa gcttctttgg

53641 tctggacatg atatagttgt gaaagggaca aagacatttc ccaaaagagg gttctacctg

53701 catcatccaa tggtggatgg aacaaagctg gaggctgggc acaagaagaa gagacatcat

53761 ccatccaaag gggaaagaga aaaagtactc aatggagcat tttcttaggc ataaagggaa

53821 ttgagtcaga acttgaagtt gaatatgacc caccaaatta actttttata gttattgggt

53881 tttcctgaag aaccatggta ttttaataat tcaatttttt agaaaatata tatattatta

53941 gagccgggtg aggtggcaca cacctgaagt cctcgctact ctgcaggctt aggtaggagg

54001 atcatttgag cccaggggtt ccagtccagc ctgggaaaca taaagtggcc ttgtctctat

54061 ttttcaagca agaaaaaaga aaaaaatata ttatccaaaa tggcttagta gagcttcact

54121 cttgaatttc ttcaaatcct ctgatgaaag aatcgtacta gaatatacaa caaacttcgt

54181 ggctgcatca acgatactta aaaccttgta aagggcatta ttatgggcat ttttaaccaa

54241 ttaggataat ttataagtta aaagtagatg aacttacctg tggaattcca gatgaaatta

54301 aagcttaagt tttttgttgt tttctgtatt ccatgcttaa ttaaaacact cagatctgtt

54361 gtgaactact gaacagaaga acagaaaaaa atatatatga gtcataacat taggaattat

54421 gacatctaaa atcaatgttg tatacttagc aagttgtttt tggagccatt tattacatat

54481 caacactttt ttcatttttg gaaaagcctt gatcctctct tctcgctttc aaatcacctc

54541 atgctctaat ctgaaattat acttggacac tgtttttttg ttttttgttt gttttggaga

54601 tggagtctca ctctgttgcc caggctggag tgcaatggca caatctcggc tcactgcaac

54661 ctctgcctcc cgggttcgag caattctcct gcctcagccc cccgaggaac tgggatttca

54721 ggtgtgtgcc accacaccca gctaattttg tatatttagt agagacgggg tttcaccacg

54781 ttggccaagc cgatctcgaa ctcccgacct gaggtgatct gcccgcctcg gcctcccaaa

54841 gtgctggaat tactggcgtg agccaccgcg cccggcctgg atgctgtttt gacaacagtg

54901 tttattttca ggtctggagt tatccaatgg ccattcattt gaacgaacat tttagtctat

54961 gaggggaaaa aagtcatagt ctcttatctg ttagtaataa acgattctgt tctccagact

55021 gatacgcgtt cagtaatttg tgcttttatc aggcatcgac acatgcatac taagagcaca

55081 aattcagcgc ccttcttttg cttaagcctc ctgggagaag ctaaaaaact tgttcccagt

55141 tgaacactgc aaacccaaat tttccttacc tctgaaaaca gaaaaaaaga catggtttgg

55201 caatttcttc cttgatagct tttcccacaa cgcagccacc tcctaaacgc aggcaatctg

55261 tgcaaaacgc agaggaaaca aaatcatgaa agccctgcaa atctccctcc taaatccaac

55321 attaagaagg cgaacggata ctgaattcaa agactacccg tacttgagga tggcatcatt

55381 ccttactgct tggaaaagtg agactccgtt gcacctctag gaatcttttt aaaacgccct

55441 gtatgttcct taacactgcc tgtaggaatc ctgtagccct agccccgtgg gatttccttc

55501 tctctccgcc cgttctccac attccccact tagtttcaca ttccttccca atccctccga

55561 catccctgcg ccggggccct tccctagcgc tccgggaaag cgttcgccgc aggcctgtta

55621 ggggcccacc aataccgtaa caccgttccc gtcctcttcc tcggcaccgc tgcaaacgcc 55681 ccgggtcgct acccggattt gacggtggct ggagcggcac gggcgacgca aggcaggtat

55741 tgcaccctgg ggcaaggcaa ggtggacaac cagcggatgc ggcgcggccg cggcctgggc

55801 ctgggccctc cgcgtcgccc actgccccac tgggcccctt ctcccgcgct caccggaggc

55861 gggcagctgc tcgtcaccga gacccgagac ccacggtgcg gcggctcaac ctgggccacg

55921 ctccgctcta gaggagtggg cggtgcctcc cggcctagct gccggggtct aggcgtttcc

55981 gaggacggcg cgttccggga aaggcgggtc cggtggctat accctccacc cagtggcagg

56041 actcaaccgc ctcggagacc ctgcagggcc gcccttctca ctcacctcct gtgctcggcg

56101 cgcacccagc tcggaagcgg gaaggtggcc ggcgttctct ccctgcgcac acacgcagcc

56161 tccccgcatc cgcacgcacg gccgggcgcg cgcagaccac gcgccagggc cgcggccgga

56221 agcccctggc tgcacgcgcg ggcccaggcg gcccccagcc ctgtgagctc gcatggcgcg

56281 ccctctgggt cctcgcggtc accttgcatt ccgagcacgg ccgtgttatg tatgccttta

56341 gttgatgctg gccaaaatta gacaacttag tggcagacgt cgagctagaa tttttgtttg

56401 agatcctttt tccctggtcg tctttccaaa ctgcgtgagt gcatggcctc gaggcggccc

56461 tggagggatt ttcttgttaa acttcataca gaactaatta ggaaagagga aatcttagga

56521 ggagttcttt gctgtctcag aagaaatgcc agttcttctt ccggaatcga tggggaatgg

56581 aatgggggta agagagacac gtcaggtttc aatggcctgt gtatggatgg gagtcccagg

56641 gagttgcttt tgtacacctg agcatagctg acctaaactc ccagatccat ctcctcaaac

56701 tccttttacc aagatgtgca gtattgattg tactggtcct ttcctcaaaa tcaagtgggt

56761 ctggatcatg tatttaacca ctaccaagct cagccatgag caccacccct gattgtaacc

56821 cacacatttg catcaagact aaacgtggcc caccatttct ttttgtttac tgactctttg

56881 gcctgactgc ttcttcctta tagccttttt ctgcaaaatt acttaggata gtttgaaggt

56941 tctctgaaaa taataggggt gaatatagag atcaaaatgg atatacaaca aaagtaagtt

57001 tagatagtag cagtgtaagt caaggactta gttgacactg cataaataat cagatctaaa

57061 ttataataat cagatctaaa ttatggttga gattgttttc ctggttccag ccttggcagg

57121 tagctgtgtc ctttgcagag ggtaaagtga ttgctgccct gctgcacgcc tttatcacag

57181 ccattcttca ccctacctgg gctgacagtt cacctcacta agtcaaggag gtccattaat

57241 aacaaaacca gacaggtttc actgcagata gattaatatg ctgaaaaaat agacctctta

57301 tttggaagaa tgctttaggg cattaatact atattttaca aattccgttt cttaaaattt

57361 tagaaaagtt gtatttcaat actcattttt aaaatgtttt gaatgtcctt tgagtaacca

57421 gcaggtatat gtgctcatag ctgccaaaac ttccctggaa tgcaaagcag agccatggac

57481 aacagattgg ggtaagaaat tctcctaaat gaagtaagtg agacaaaaag gcccaaaacc

57541 tttatgagat tatttccttt gaagcagtct cctttttctt tgtgtagcat ttatcagctt

57601 gtgattgagt catctattgc tgcagatcga actctcccaa aatatagttg cttgcaaaaa

57661 tgtcaattta atatttctca taattctgaa ggttggttat gtggttcctc tctggttttg

57721 atttggctca ctcgtataga tacattcagc tgtagcaggg gttggccaag ttttctgtaa

57781 aacaggatca gatagtaaat attttagact ttgtctctgt cacatattct tttttattta

57841 atttttataa actattaaaa acccaaagct attcatagct caagggccat acagaagcag

57901 gctatggcct acaggctgta gtttgctgat ccctgaactg gagaatcagc taagctgaaa

57961 gtccaaggtg gcctcactca tgtgtctgct gttggctcca tgtggcctct tatcctgcag

58021 taggctatgt ttttggtctg tgggcaacat ccagaagaga gcaggcagaa gctgtaaaat

58081 ttcttaaggc ctagacttgg gagtcacaca agatcatata tgccaaattc tacaaatcaa

58141 agcaaattgt gaggcctctc caaatccaag gggcaggcaa atagtctctg cctcttgatg

58201 gaaggagctg cagataatgt ggccagaact agctaaacta agtggcagga accttattct

58261 tgaaagtaaa tcatcaaact tcaaatttat gatatctgat ggcagctatt gttcttactc

58321 acttctctcc ccaaactacc caaacctgtt tataattctg tgagcactca cttgcctcag

58381 aacttgttta cagatgtctc tgccatgaga ttatcagcaa cctgagggaa gagacaggcc

58441 ctaccgcact gccttgctgt aatatatctt caatagatat atatggaatt tagctgaata

58501 gccttactga ggtttaaagt caggttattt gcttacttca caaaagcaaa ataaatttaa

58561 gtatcctgca ctatctacaa attccagaag aaaagcacac atcaaagagt tatatgtcac

58621 actcacatac catctgacca ataagatgga aaccttccta gcaattttcc gaacatataa

58681 tcaagagtta ggaagccata tgcctaatga taagtttgaa ctagcagtca cctttaaggc

58741 tatattacat tttaacctta aatttgtctg cagatgaaaa ttgaaatgta acaacagttc

58801 tttcgtaata tgtgctatat atgtttcaag gcagatgtgg ttctgggcag ggtttctaat

58861 ctacaggaaa tgcttctact aattgttcag ttgaattttt caatatgtgc cttctccaaa

58921 atctgactgt gactgtcttc ttccacttga tggaaagctt tgacaattta ttatgcttag

58981 aagcagctgg ggtgtatttc taattgatga tttgttatat ttcttacttg actatccaca

59041 tctttcattc tcttcagcat tttcctccag cacttcctgt cttactgaat ttgatatatg

59101. acactgcctt tgtgcaaatg tagacaatat cttcagcctt ctgcctctgt ccttcagatt

59161 acaaatacat gcgagagtca tgctagtaac acgtatgtga cacttaaaca ttaatacagt

59221 atattcacat acactttatc agttgattat taatacacca atactgtgga ggtggatact

59281 gtcattacta tcatttggca ggtagggaaa ctggcgaaca aatgtctcag agatacctca

59341 aacttaacat gtctaggcca acctcagtac cttcccataa aatgtgctta ttctcctgga

59401 ttcctgattt gtataaagca gctccattta tcactcatcc aagttcaaat ctcacttcat

59461 ccttctttct attccacatg tccagtcagt caccaagtcc ctgtggattc tctctctttc

59521 tccctctcct ctctgagatg cctgcagcca agaaagcctc attcttcatt taggtttgta

59581 tcagagcttt ctgtctgctc ccttgacttc caagttgccc atcaggtcct tcagattacc 59641 actaggctgt cttcctcaaa cccagatctt gtcaggcatt cctattttaa attggtcagt 59701 gacttcccac agggcacaat ccaaagggct acagaacctt ttgtaatcta cctcagccta 59761 ccagctcctg ccatgcatgc attttcatgc actgtcattg tgcccactgt tacctcagag 59821 tacagtttta ttctctgagt ccctccatcc cagaaccaca cccatctgac tcatccaagt 59881 cccacaacca ctatagtaat gcttctcaaa cctaaatgtg cataggaatc actggagaat 59941 cttattaaaa gcagattctg attcattagg tcctggatag ggctggagat tctgcctatt 60001 aacaaaccaa ctgtgcaggg cacggtggct catgcctgta atctcagcac tttgggaggc 60061 tgagatgggc agatagcttg agctcctgag ttcaagacca acctgggcaa catagcaaaa 60121 ccctgcctct actaaaaata caaaaattag cctggcatgg gggcgtgcat ctgtagtccc 60181 agctactcag gaggctgagc tgggaggatt gcttgagctt gggaggtcaa ggctgcagtg 60241 agctgtgatt gcaccactgc actccagcct gggtgacaga gcaagacctt gtctcaagac 60301 aaaaacaaaa acagacaacc caccaattgg taataggata ctgctggttg ttaggctgca 60361 atttgagtag caagagtcta gagctgcact gttcaataga actttctgca atgataaaaa 60421 tgctcttagg tgttttctaa tattgtagaa actaatgaca tgtagctatt gaatactcga 60481 aatgtagcta atgagactga ggaactgaat tttaaatttt acttagttta agtttcagta 60541 gctacatgtg agtaagtggc caccatgttg aacaatgcag atgtatagga atccctttca 60601 tccttcaaaa ctcattcaga cttctggttt tcagtccagc atgtaagacc ttagaagtca 60661 ccattccatc ctaataacaa gtaaaaagct aaacaaactt aaaaatactt aactctttgt 60721 atattttttg gaaaagtgag gttatggggc aaactgcatt tcccaaactg gagagacaga 60781 caggtagata cagagaatta cagaattaaa agaaaagaaa aaaaatgaca ttttgagctg 60841 gacaaataaa aacaaacaac aaaacaaaca gaaaatacag agaattataa cttatcagag 60901 cagaaaacca tgagctgaaa tctctacagg aaccagtgct ggggtaggaa aacctaaact 60961 gttaaatgat gaattgctag aagttctgtg ttggcaagtc tgagagttaa aaacttcagg 61021 gggacctagt catggtgggt ggaacccata cttttagtgt gttttaactc ctggagttct 61081 accaggttca cacagtgaat atcagagaaa aaccccccag tgcttccggc ggagggagga 61141 gaaaagaaac cattttgaaa tgtgccagaa cattctgttc ttaacaagag ctgccctcag 61201 aagaaagtag ctaagtggaa cctagcctgc tggggtttta tcagagccta actgacctgg 61261 aggaagggaa atactgaact ccattccctc tagacttcca catatgagaa gggaaatacc 61321 caaatccagc ccattctagc tatcctatcc tacctaacat gttaggggtg agtatagaag 61381 taaagaactg agaggcactt gtgaagttca tgatccagag gcacaggctc actaaaagac 61441 tgagacctaa tcataggact atagaatgct cccccaccta tacatacacc ttaccatcac 61501 attactaaag gaacatcaca gcaatttctt taacccagta cattatgtcc agttatcaag 61561 aaaaaattac gaggcatact aaaaggaaaa acacagtttg aagagacagt acaagcatca 61621 gaaccaaatt caagatatgg tagagatgtt ggtattagac cactaattta aaataactat 61681 gattaattta ctaagagctt taacacataa agtagatagc atgaaaagac caataggtaa 61741 tgtaagcaga gagatagata atttaataag gtaccaaaaa agatggtaga gatcaaaagc 61801 actgtagcag aaatgaagaa tgcctttgat ggtcttagta gactggacac agctgaagga 61861 aaaaatctct gaacatgaag ccatctcaat agaaactgcc aaaactgaaa aacagaaaaa 61921 agactgaaaa aaaagaaaac ctacccagaa cagaatatac aagagctgtg gctcaactac 61981 aaaaggcgca acatgtgtaa tgggaataac caagagaaga aaaagagaaa gaaacaaaag 62041 aaatatttga aacaataatg acaaaatttc ccctaaatta atgtcagaca gcaaaccaca 62101 agtccagaaa gctgagagaa caccaagcag aataaatgta aaaaacaccc acacctaagc 62161 ataatgtatt caaactacaa aaaaatcaaa gataaaagaa aattctgaaa gaagtcagtg 62221 aaataaaacc caccttatct atagaggagc aaggataaga attacacctg gtttttcctc 62281 ggaaaccatg caagcaaaaa aagagtagag tgaaatattt aaagtgttga gagaagcaaa 62341 caaactggaa ttctgcaaca tgtaaaatta tccttcaaaa gtaaaaaaaa aaaaaaaaaa 62401 aaaaaaaaaa ttgagggaat ttgtttccag tggactgctt tggaagaaat gttaaaaaaa 62461 aaaaagtcct ttagagagaa ggaaaataat agaggtcaga aatttggacc tgtgtaaaga 62521 aaagaatagc aatggagaag gagtaaatga agataaaaat aataacattc tgacatggtt 62581 tgaatctgtg ttcctaccaa aatctcatgt tgaaatgtta tccccaatgc tggaggtgag 62641 gcctggtggg agacaactgg atcatgggag tggtttctca tagcttaaca tcattgcccc 62701 cttggtgctg tcattgcaat gagctctcat gaaatctggt catttaatag tgtgtggcac 62761 ctcactcccc aacgccccca cacctgccaa cacttcctcc tgctctggcc atgtgaagag 62821 ttggcttccc gttcctcttc caccatgatt gtaagattcc tgaggcctcc ccagaaactg 62881 agtagatgct gccttgcttc ctgtacagcc tgtggaacca tgagccaatt aaacctcttt 62941 gctttatata ttacccagtt ttaggtattt ctttatggca gtgtgataac caactaatat 63001 actttttaaa attcttaatt gatctgacag ataacagttt gttcaagata gtaataacgt 63061. atttgattat gtatgtttgt gtatggctat atataactga aatgaatgaa agcaatgaaa 63121 caaggatagg atggagaaat gaggattatt ttgttattac aaggtactca cactacccat 63181 gaagtggtat agtgttattt gaaagatgac ttgaacttgt tttaaatgta tattgcaaac 63241 tctagggcac ccactaaaaa aagttttaaa aaataaatat aactgccggg tgcggtggct 63301 cacgcctgta atcccagaac tttgagaggc tgaggtcacg aggtcaggag tttgagaaca 63361 gcctgaccaa catggtgaaa ccccgtgtct actaaaaata tgaaaattag ccaggcatgg .63421 tggcacacgc ctgtaatctc agctactcag gaggctgagg caggagaatc acttgaaccc 63481 aggaggcaga ggttgcagtg agctgagttc gcaccactgc actccagcct gggcgacagc 63541 gagactccat ctcaaaaata aataaataaa taaatataac tgatatgcta agaaaggaaa 63601 gaagatggag tcatataaaa tgctcaatta aaaattacaa agggcagaaa atgtggaggg 63661 caaaaggtag gaacaaagaa caagggtgat gatatagttt ggatattcat ttccttcaaa 63721 tgtcatgttg aaatttgatt cccagtgttg gaggtggggc cttgtgggag gtatttgggt 63781 catggaggca gatcacttat aaatggcttg gtgccattcc tacaataata agtgaattct 63841 cagctctgtt cattcatatg agagctgatt gtttaagaga acatgacatc cctctcctct 63901 ctcacttgct ccctctcttg ccatataatg ccagcttcca cttcccattg ccatgattgg 63961 cagcttcctg aagccctagt cggaagcaga tattggcacg atgcttcttg tacagcctgc 64021 agaattgtga gccaaataaa cctattttct ttataaatta cccagcctca ggtattgttt 64081 tatagcaata caaatgacta agaggtgaca agtagaaaac tgtaacaaaa atgagctggg 64141 agcagtggct tacacctgta atcccagcac tttggaaggc tgaagccaga gggtcacttg 64201 aagacaggag tttgagacca gcctggccaa catggtaaaa ccccatctct actaaaaata 64261 tgaaaattag gtgtggtggt acacacctgt aatcctagct acttgggtga ggcaggagaa 64321 tcacttgaac ccaggaagcg aaggctgcag tgagccaaga ccacactact gtgctccagc 64381 ttggatgaca gcaaaactcc atctcaaaaa aatgaaaaag cactaatgaa tatggaagat 64441 attaatccaa ctatatgaat aatcactttg aatgtccatg gtctaaatgc accaattaaa 64501 agacagacat tgtccaagtg aatcaaaaga taagacccaa ttatttttta caaaaaatcc 64561 attttaaata taaatatata aattaaaagt aggtggatag ggaaagatat accatggtaa 64621 cactaatttt tgaaaggtag ggtagctaca ataacttcag atggagcaga cttcagagca 64681 aggaaggata tcaaggataa acaatgacat tacataatga tatagggata aattctccaa 64741 caagacataa caattctttt tttttttttt tttttttgag acggagtctt gcactgttgc 64801 ccaggctgga gtgcagtggc agaatctcgg ctcactgcaa gctccaactc ccagatacac 64861 gccattctcc tgcctcagcc tcccgagtat ctgggactac agttgcctgc caccactcct 64921 ggctaatttt tgtattttta gtagagacgg ggtttcacca tgttaaccag gctggtctcg 64981 aactcctgac cttgtgatcc acccgccttg gcctcccaaa gtgctgggaa ttcaagcatg 65041 agtcaccaca cccggccaag acataacaat tcataatgtg aatacactta aaaacagagc 65101 atcaaaatat gggagacaaa aactgatagg atgacaacaa gaaatagatg aatctaccat 65161 tatagctgga gacgtccaca tttctttaac agatgtggac acattccgca ggcaaaaaat 65221 cagactatag tggaactcaa caacaccatc aatcaactgg atataatggg catctataga 65281 ctatctcatc cagtaacagc agaatacaca tttttcttaa tctcacatag aacattctgc 65341 aagataggtc acattcttgg ccataaaaca caccataacc catttaaaag aataaaaagc 65401 atacagtgtc tgccctcaga ctacagtgga attaaactag aaatcaatac cagaaagaca 65461 gctggagaat ccccaaatac ttggagatta aacaacatgc ttcgaaatga aacatgggtc 65521 aaaaagtaac tctcaagata aattttaaaa tattttcaac taaatgaaaa caaaaacaca 65581 agttatcaaa cttcatggga tgcaatgaaa gtgcttaaag ggaaatttat agcattgaat 65641 acttaaagaa aaaagatcca aaaatcaatc atataagctt tcattttagg aaacaagaaa 65701 aagaaggcca aattaaaatc agagaaagat aaatattata gcaaaaatta atgaattgaa 65761 aatatgaaat cagtagaaaa accaatgaag ctaaaagctg gttctttgaa aagatcaata 65821 aaattgataa ttatctaacc ctgctaactg tattaggcca ttcttgcatt actacaaata 65881 attagctgag actaggtaat ttataaagaa aagaggttta attggctcat ggttctgcag 65941 gttgtacaac catgtcactg gcatctgctt ggcttctgcg gaggccttag tgagctttta 66001 gtcatggcag aaggcaaaac gatagctggc acatcacaag gcaaaagcag gagcaagaga 66061 ggggtggagg aggtgccaca cacttttaaa ttaccagatc tcatgagaac tcactcactg 66121 ccttgtggac agcaccaagc tgtgaggaat ccaccctcat gacccacaca cctcccatca ~66181 ggccccacct ccaacactgg ggattacatc tcaacacgaa atttggatga ggacaaatat 66241 tcaacctgtg tcactaagaa aaaagaggag acaaaatgct aatatcaaaa atgaaaaagg 66301 ggacactaaa gagcccagag acattgaaag ggtaatcaag aaatattctg aacaattcta 66361 ggtccacaaa tttgataacc tagatgaaat ggaagaacac tttgaaagac aatatttgcc 66421 aaaactcaaa caaggagaaa tagacaatct gaaccagcct atatctatca aaaatattga 66481 gtcaataatt aatgaccttg caaaatagaa agaagcaggc ttagatgtgt tcactggtga 66541 attctaccaa aaattaaagg aaacttcaga agatagaagc aagagcttct ggctagctga 66601 ccacatgagg gttcctggag agggggcata cccagggaag acagagaaat gccatgcacc 66661 ctcccccata cgttacccca tgtatcactt cttctgtatc ctttgtaata aacttgaaaa 66721 tttaaaaaag aagaagatga aaacagagag aatacttctt aacttattct atgaggccag 66781 cattactcta ataccaaaac caggcaaagg cattacaaga aaagaaaact acagagatat 66841 cttacataaa cataaatgca aatgtcctca acaaaatatt atcaaatcaa atccaacaat 66901 gttaaaaact attatacacc acaagtaagt gagatttatt gtaagtctgg ttcagcattc 66961 aaaaaaagca gtgaatataa tctgtcacat caaaagacta aagaggaaaa atcacatgat 67021 catatcaata gatgcagaaa aagcattcga caaaatcctg catccattaa tggataaaat 67081 aacaataaag gctcagtaaa ctgaaatagt gcaacttcct caacttgatt tttaaaaacc 67141 tattaacaaa atcctgtgaa aataccaatg aaattcttca cggaaataga gaaaacaatt 67201 ctaaaattta tatgaaacat aaaagactca aaatagccaa agctattgta agtaaaaaga 67261 aaaaactgga ggaatcacct tacctgatgt caaattatgc tacagtaaaa cagtatggta 67321 ctagcataaa aacagacacc taggccaatg gaacaaaact gagaacccag aaacaaaccc 67381 acacgcctac catgaactcg tttttaacaa agttgccaag aacatacgct ggggaaaaga 67441 caatgtcttc aataaatggt gctgggaaac tggatatcca tatgcagaag aatgaaacta 67501 gacccctgtc tcttgccata tattaaaata aaaatggatt gaaaacttaa atataagacc 67561 tcaaattatg aaactactac aagaaaacat tggagaagct ctccaggaca ttggtctggg

67621 caaaaatttc ttgagcaata ccccacaagc acaagcaacc aaagcaaaaa tggacaaatg

67681 ggatcacatc aagttaaaaa gcttctgcac agcaaaggat acaatcaaca aagtgaagac

67741 agccaacagg atgggaaaaa atatttgcaa actactcatc aggcaaggga ttaataacca

67801 ggatatataa ggagcttaca caagtctata ggaaaaaaat ctagtaatcc agaaaaatat

67861 gggcagtaga tttgaataga catttttcaa aagaagacat acaaatggaa aacaggtgta

67921 tgaaaatgtg cccaacatca ctgatcatca gagaaatgca aatcaaaact acaatgagat

67981 atcatctcac cccagttaaa ataacttata tccaaaaggc aggcaataac aaatgctgac

68041 aaggatgtag agaaaaggaa acccccatac actgttggtg ggaatgtaaa ttagtacaac

68101 cactatggag aacagttgga gattcctaaa aataaataaa aattgagtca ccgtatgatc

68161 cagcaatctc actgctggat atatactcaa aaggaaagag atatctgcac tcctatgttt

68221 gttgcagcac tgtctaaaat agtaagattt ggaagcaacc taagtgtcca tcaacggatg

68281 aatggataaa gaaaatgtgg tacatataca cgatggatta ccattcagtc catgagggcc

68341 aggcgcggtg gctcttgcct gtaatcccag cactttggga ggccgaggcg ggcagatcac

68401 taggtcaaga gatagaaacc atcctgccta acatggtgaa accccatctc tactaaaaat

68461 gcagaattat ctggacctgg tgtcatgtgc ctgtagtctc agctacttgg gaggctgagg

68521 caggggaatc gcttgaaccc aggaggtgga ggttgcagtg agccaagatc ctgccactgc

68581 actccagcct gggagacaga gcaagactcc gttaaaaaca aaacaaaaca aaacaaaaaa

68641 ggtcctttca ttttgtaaca atatgaatgg aactggaagt caggaagtca ttatgttaag

68701 tgagataagc cagccacaga aagacaaaca ttgggtgttc tcacttgtcg gattttaaaa

68761 gtgaaaacaa ttgaatacat ggacatagag ggtagaagga tggttaccag aagctgggaa

68821 gggtagtggg ggtctggggg gtaggtgggg atagttaatg ggtacaaaag aaaatagaaa

68881 gaataaataa ggcctactat ttgatagtac aatggggtga ctatcatcaa taatacctta

68941 attgtatact ttagagtaac ttagagaatg taattggatt gtttgtaact caaaggataa

69001 atgcttgagg ggatggaaaa taatctataa aaa'aaaccct cctcatcaaa atgccaatga

69061 aattctttga agaaatggaa aaaaaatccc caaatttata tggaaccaca aaagaccctg

69121 aataggcaaa gcaactctga ccaaaatgga caaactggaa ggatcacact accaacttca

69181 aaatatacta caaagctata gtaaccaagt cagcatggga ctggcataaa aatagacaca

69241 tagaccaatg taacaaaata gagaacccag atacaaatcc atggatttac tgctattaat

69301 aacattttga cagttaccaa taacatacaa tggggaaagg acagtcacat caatgtgaaa

69361 attctcaaca aaatactggc aaacagaatc caacagcaca tcaaaaagct taaccgtcac

69421 gatcaagtca acttcatccc tgggatgcaa gactggttca acatatgcaa atcaataaac

69481 ataatccatc acgtaaacaa aaccaatgac aaaaaccaca tgattatctc aatagatgca

69541 gaaaaggcct tcgataaaac tcaataccct gtcatgccaa aaccctcaat aaagtaggta

69601 ttgatggtac atgtctcaaa aagagctatt tatgacaaat ccacagccaa tatactgaat

69661 gggtaaaagc tggaagcatt ccctttgaaa actggcgcaa gacaaggatg ccctctctca

69721 ccactcctat tcaacatagt attgaaagtt ctggccaggg caatcaggca agagaaagaa

69781 agaaagggta ttcaattagg aaaagaggaa gtcaaattgt ctctgtttgc agatgacatg

69841 attgtcaact caagccattg cttcagtggg tgcaagcccc aagctttggt ggctactatt

69901 tggtgttggg cctgtgggtg cgcaaaagac aacagttgag gtttgggaac ctccacctag

69961 atgtcacagg atgtacggaa actcctggat gtccaggcag aagtctattg caggggtgga

70021 gccctcatgg agaacctcta ctagggcagt acagagggga aatgtgggat tggagcctcc

70081 acacagagtc cccaccaggg cactgcatag tggacctgtg agaagagggc cattatcctc

70141 cagaccctag aatggtagat ccaccaacat cttgcactat gcacttggaa aagctgtaga

70201 cactcaaggc cagcccatga aagcagctgg aggggctgta ccctgcagag ccagaggggt

70261 ggagctgcct aaggccttgg gagcccacct cttgcatcag catgccctgt atatgagaca

70321 tgaaatcaaa ggagatcatt tcggagcttt aagatttaat ggctaccctg ctgggttttg

70381 gacttgcatg gggcctgtaa cccctttgtt ttggccaatt tctccaattt ggattgggaa

70441 catttactca atgtctgtac caccattgta tcttggaagt aactaacttg ggaacattta

70501 ctcaatgtct gtaccaccac tgtatcttag aagtaactaa cttgtttttg attttacagg

70561 cttatagcca gaagaaactt gccttgtctc agatgagact ttggacttgg acttttgggt

70621 taatgctgga atgagttaag actttgggag actgttggaa atgcatgaat gtaaaaagga

70681 catgagattt gggaggagcc acaggcaaaa tgatatgatt tggccctgtg tccccaccca

70741 aatcttatcc tgaatggtaa tccccatgtg ttgagggagg gacctggtgg gaggtgattg

70801 gatcatgggt ttggtttccc ctatgctgtt ctcatgatag tgagtaagtt ctcaagagat

70861 ctgatggttt aaaagtgtga cacttccccg ccattctctt tctctcctgc caccctgtaa

70921 gatgtgcctt gctttccctt tgccttctgc catgactgta agtttcctga ggcttcccca

70981 gcca.tgtgta actgtgagtc aagtaaactt cttttcttaa taaattacac agtctcaggt

71041 agttctttat agcagtgtga aaacagacta atacattcac tattggacgt atatccagag

71101 gaaaggaaat atattgaaga gatatctcca ctcccatgtt tattgcagta ttattcacaa

71161 aagccaaaat atggaatcat cctaaatgcc atgaatggag gaatggaaaa agaaaatggt

71221 atatatacaa tgaagtaaaa agaatgaaat tctgttattt gcagcaacat gggtgaaact

71281 gaaggtcatt atgttaagta aaatgagcca agctgagaaa gataaatatc atgtgttctt

71341 acatatgtgg gagctaaaaa agtggatttc atgaagatgg agagaagatt ggtgattacc

71401 agagaccagg agaggtagga gggagggaaa tgaagagagg ttgattaatg ggtacaatta

71461 gatagaaaaa ataacactta gcactcagta gatcagaagg ggattatagt taatattaat 71521 ctatggtaca tttcaaaata gctagaacag aataacgtag atgttcttag catagaagta

71581 atggatgtct caataacctt gatttttaca ctttatatga atgtatcaaa taatcatatt

71641 taccctacaa atatgttcat ctgttattta tcaataaaag ccaaatccta tactatgttg

71701 aataggagtg gtgatagagg acatccttgt cttgtgccag ttttcaaagg gaatgcttct

71761 agcttttgcc cattcagtat gatattggct gtgggtttgt cataaatagc tcttattatt

71821 ttcagataca ttccatcaac acctagttta ctgagtgttt ttagcataaa ggtgtattga

71881 attttatcga aggccttttc tgcatctatt gagataatca tgtgtttttt gtcagtggtt

71941 ctgtttacgt gatggattat gtttattgat ttgtgtatgt tgaaccagac ttgcatccca

72001 gggaagaagc tgacttgatc atggtcattt taatgtgctg ctgagtttgg tttgccagta

72061 ttttattaag gattttcaca taaatgttaa tcaggaatat tggcctgaaa ttttcttctt

72121^' ttgttatgtc tctgccaggt tttggtatca agatgatgct gacctcataa aatgagttag

72181 ggagaggtcc ctctttttct attgtttgga atagtttcag aaggaatggt accagctcct

72241 ctttgtacct ctggtagaat tcggctgtga atctgtctag tcctgggctt ttttggttgg

72301 taggctatta attactgtct caatttcaga ggcagtaatt aactggtcta ttcaaggatt

72361 ccacttcttc ctggcttagt cttggaagtg tttatgtgtc caggaattta tccatttatt

72421 cttagatttt tcaagtttat ttgcagagag gtgtttatag tattctctaa tggtagtttg

72481 tatttctgtg ggattagtgg tgatatcctc tatcattttt attgtgtcta tttgattctt

72541 ctctcttttc ttttttatta gtctggctag caatctatct attttgttaa tcttttgaaa

72601 aaactagctc ctggaagttc aggctagggt aatcagacaa gagaaagaaa taaagggtat

72661 tcgaatagga agagaggaag taaaattatc tctgtttgca gatgacatga ttttatattt

72721 agaaaacccc atcatctcag gccaaaaact ccttaagctg ataagcaact tcacaaagtc

72781 tcaggatact taatcaatct tataatcata cttataagaa tgcttgtgca aaaagcacaa

72841 acattcttac acaccaataa tagacaagca gccaaatcat gagtgaactc ccattcacaa

72901 gtgctacaaa gagaataaag tacctaggaa tacaacttac aaaggatgtg aaggacctct

72961 tcaaggagaa ctacaaacca ctgctcaagg aaataagaga gaacacaaac aaatggaaaa

73021 acattccatg ctcatggata ggaagaatca atatcatgaa aatggccata ctgcctaaag

73081 caatttatag attcaatgct attcccatca tgctaccatt gactttcttt acagaattag

73141 aaaaaactac cttaaatttc atgtggaacc aaaaaagggc ccatatagcc aagacaatcc

73201 taagcaaaaa gaacaaagct ggaggtatca cgctatctga cttcaagcta tactacaagg

73261 ctacagtaac caaaagagca tggtactggt accaaaacag atatatagat taatggaaca

73321 gaacagagcc ctcagaaata acaccacaca tctacaacca tctgatcttt gacaaacctg

73381 acaaaaaagc aatggggaaa ggattcccta tttaataaat ggggttggga aaactagcta

73441 gccagatgca gaaaactgaa agtggacccc ttctttatgc cttataaaaa atgactcaag

73501 atggattaaa gacttaaaca taagaactaa aagcataaaa accctagaag aaaacctcgg

73561 caataccatt caggacatag gcatgggcaa agacttcatg actaaaacac caaaagcaat

73621 ggcaacaaaa gccaaaattg acaatggggt ctaattaaac taaagagctt ctgcacagca

73681 aatgaaacta tcatcagagt gaacaggcag cctgcagaat gggagaaaat tttttgcaat

73741 ctatccatct gacaaagggg ctaatatcca gattctacaa ggaacttaaa acaaatttac

73801 aagaaaaaaa caaccccacc aaaaagtggg cgaaggatat gaacagacac ttatcaaaag

73861 aagacattta tgcagccaac aaagatgaga aaaatctcat catcactggt cattagaaaa

73921 atgcaaatca aaaccacaat gagataccat ctcatgtcag ttagaatggc agtcattaaa

73981 aagtcaggaa acaacagatg ctggagagga tgtggagaaa taggaatgct tttacactgt

74041 tggtgggagt gtaaattagt tcaactgttg tggaagacag tgtggtgatt cctcattgtg

74101 gaaaacagtg tggtgattct atatctagaa ctggaaatac cacttgaccc agcaatccca

74161 ttactgggta tatattcaaa ggattataaa tcattctact ataaagacag attctactat

74221 aaagacacat gcacacatat gtttattgca gcactcttta caatagcaaa gacttggaac

74281 caacccaaat gcccatgaat gatagactgg ataaagaaaa tgtggcacat atatgccatg

74341 gaatactata cagccataaa aatgaatgag ttcatttcct ttgcagggat gtggatgaag

74401 ctggaaaccg tcattctcag caaactaaca caggaacaga aaaccaaaca ctgcatgttc

74461 tcactcataa gtgggagttg aacaatgaaa acatatggac atagggaggc aaacagcaca

74521 caccagggcc tgtcagggtg ggaggcaagg aaagggatag cattaggaga aatacctaat

74581 atagatgaca ggtttatggg tgcagcaaac caccatggca catatatacc tacgtaacaa

74641 acctgcacat tctgcacatg tatcttagaa cttaaagtat aatttaaata aaaaaacaac

74701 agaaaaacac aaatcctagg gcacaaaatt ttaaaaaggt tgtcctaatg ggccgggtgt

74761 gctggctcac gcctgtaatc ccaacacttt gggaggccaa ggcaggcgga ttacctgagg

74821 tcaggagttc aagaccagct ggccaacatg atgaaaccct gtccctactg aaaatacaaa

74881 aattagctgg gtgtcatggc agacgcctgt aatccagcta ctcgggaggc tgaggcaggg

74941 .gaatcagtta aacctaggag ttggtgttta cagtgagctg atatcgcacc gctgcactcc

75001 agcctgggaa acatagtgag actctgtctc aaaaaaaaaa aaaaattgac ttggtggtgg

75061 ttaactagaa gctctcctgt aagatcaagg agcaaggcaa ggatatgccc tctcaccact

75121 gctttttaac atcatactga aagtgctaga taatgcaata agacaagaaa agaaaataaa

75181 ggcatacaga ttgaaaagaa ggaactagaa ctgactttgt tcacagataa catgatcgtc

75241 tacatagaaa acccagaaga attagcaaaa aaaggaaaac tctccaggaa ctaataagtg

75301 attataagaa tgtcRcagga ttcaaagtta atatacaaaa ggcaaatgtt tttctatata

75361 ccagcaaaaa gcagatggca tttaaaatta aaacacaata ttatttacat cagcactccc

75421 aaatggacac attagcagga tctatataag gaaaaccaca aaaatctcat aagagaaata 75481 aaagaattaa atagggaaat attccatgtt tatagataga aaacttaata ttgtcaagat

75541 gccagttctt cccgtttata gattcaatgc aatgccagcc aaaattacag caagtgattt

75601 tatgtatgtt aacaaaataa ttctaaagtt tatgtagagt gtcaaaagac ttagggtagt

75661 caatacaata ttaaaggagt tataaagttg aaggactgac acttacatga cttcaagact

75721 tattataaga ctacagtaat caaattatgt ggtattaggg aaagaaaaga caaataaatt

75781 agtgtaatga aatagaaaac ctagaaatag atccacaaaa aaataatcaa ctgaccttta

75841 aagattcata ttgcaaagac aatacaaaaa aaaatcttta aacaaatggt gctggaatga

75901 ctggatatcc tcctgccaat aaaaagaaat gaatctacac acagacctta cacccttctt

75961 aaaaatcaat ttgaaatgga tcacagatct aaatgtaaaa tgctatgaat ctcctagaag

76021 ataatgtaga aaaatattta tataagctta gtttggtgat gactccttag aggcaacact

76081 gaaggcacaa tacatgagag aaagaactga taagctggac gtcatgaaaa tttgttttct

76141 gtgaaagaca ctgtcaagtg aataaaaaga caagccaaag actgggagaa aatatttgca

76201 aaacatatat ctcataaaga tctgttattc aaaatataca cagaaccctt aaaactcaaa

76261 aataagaaaa caaacaacct gactaaatac tgggccaaag accttaataa tgcaagtact

76321 agaaagcatg aatgactttt tttttataat aggggagagg gaaaacttta caagctatca

76381 ctcaaaatta agctctgagc actttgggag gtcagggagg gaggattgct tgagaccagg

76441 agttcaaaac cagccagggc aacatagcaa gacccatctc tacaagattt tttaaaaaaa

76501 ttatctgggc atgatggcat gcacctatag tcctagctac ttgggaggct gaagtgagag

76561 gattgcttga acccaggagt tcaaggctgc agtgagccat gatcacacca ctgcactcca

76621 gcctaggcaa cagagtgaga ctctgtttaa aaaagaaaaa agaatcaata aaaaacataa

76681 atgatgtgtc tatatataaa ctttaaaaca tttttatgag gaaaaacaac acagtaaaaa

76741 atgacaactt ggaagaaata tttataatta tttttataga tttataatta gatataattt

76801 ataattagat ataattagat atataattat aattataatt gttttataat tatattatta

76861 gatgactaat atttttccca atatttcatt atgaacattt tgaaacataa agttttatag

76921 gcaacaccca aaatccatca catacattct cccattaata ttttactcta cttgtttaat

76981 tatattatat agaatttatc cattttttat gcatttcaaa gtaaattgca cacatcaata

77041 tacttctttc taagtatttc tgcatgtata tcattaacca tttctgcatg tatatcatta

77101 aatacttccc tctaagtatt tctgcatgta tatataacca tgtatagctc agtatttgtt

77161 tacaggtttt tctcccaatg taaaatataa atacaatgaa attcaaaaat cttaagtgtg

77221 cattactgat ttttgacaaa tgcatacatc tatcaaaccc ctatccagag ggatgatttt

77281 tttcagtggt ttatacaaga tccaagttta tctctttttt acataaattg ggtcatccag

77341 gtgtgtttga ttatgtcagg ggcttaaggg cctttctatt ttattgttcc tccattttca

77401 gcaacacagc taaaacctta aaccatcata tctgtattcc agctactagg aagaaaaaaa

77461 gtgcagagaa ggacctacct cctcctttca aggtcatttc tagaagttgc atgtgctgct

77521 ctggttatat cccagaacat aatcatatag taaacaaagt tgcaaaggaa gctggaaaat

77581 gttgacttta ttctggtcgc caaatttcca gctaaaaata ggtgattcta ttaataagga

77641 agtaggagtg actggctttt gggaaacaag tagtagtctg tatcataggc atttttattt

77701 agttttttcc cttcatgttc cctatatcca aagggccgat attaataaaa tatagagagt

77761 tccaaaaatg ttgagaagaa aaaataccaa cagtgcaata gaaaaattgg taaaacagct

77821 actcaggggg ctgaggcagg agaattgctt gaacctggga ggctgaggtt gcagtgagcc

77881 aagatcatgc cactgcactc cagcctgggt gacagagcaa gactctgtct caaaaaaaaa

77941 aaaatttaat taaaaaaata aagaaaaatg agtaaaagat atgaatggac agttccaaag

78001 aaaacgaaat ggaccctaaa catataaaaa gatgtcagac catgcagagt ggctcatgcc

78061 tgtaatccca gcactttggg aggctgagct gggaggatta cttgaaccca ggagtttgag

78121 actagtctgg gcaacataga gagacctcat ctctacaaaa aattttaaaa aattagctgg

78181 gtgtggtggt acatgcctgt agtctcagct acttgggagg cagaggtggg aggatcactt

78241 gggcccagaa ggtcgaggct acagcgagct atgattacac cactgcactc cagcctgggc

78301 aacagagtga gaccctgtct taaaaaaaaa agaaagatgc ccaacctgct tataataaaa

78361 aaataatgct taatagtgac caacccaaaa ggaaattaag aaaatcccat ttacaacagc

78421 atcaagtaga ataaaatagt ttgcctccat ggaggcaaaa gacttgtgca actaaaaatg

78481 acaaaacatt gctgaaaaaa atgaaagaaa acataaataa atggaaagat atcccatgtt

78541 catgaatgaa agacttaata ttgttaagat aagttaacta cagattcaat gcaatccctc

78601 tcagaatcaa actatagttt ttgcataaat agaaatatcc attctaaaat ttatatggaa

78661 tctcaaggga ccctgaagag agccaaacca ataaaaataa cagatttgga ggtctcacac

78721 ttattacaat tctaaaagta atcaaaacag tgtggtattg tcataaagac agacatacgg

78781 accaacgaaa cagtataggg agcccagtaa ataatcccta acatatatgg tcaaataatt

78841 ttcaaaaaag taccaagatt attcgtgggg agagtagaat cttttcaaca aattatactg

78901 ggaaaactgg atatccacat gcaaaagaat gaagttggac acttaacacc atatacaaaa

78961 attagctcaa aatggatcaa aggcctatat ataaccacta aaactgtaaa acccagaaaa

79021 aaaaatatgg gggaaaatca tgacataagt ttgacaatga tttcttaggt atgaccaatg

79081 gtacaggcaa caaaaaaaaa atggaaaaac tggacttcat caatattaaa aaatttttgg

79141 gcgtcaaagg acactatgaa tggagtaaaa agataaacca cagaaaagga gaagatattt

79201 gcaaatcata tatcagataa ggaattaata tccagaatat acacacaact cctaaaacta

79261 aatatcaaca gcaacaacac aattcaattt aaaaagtaga caaagtctct ggttgtctag

79321 tgtctaggaa acaaacaagt ggacaaagga ctttaattga gatttcctca aaaaagatac

79381 acaaatggcc aaacaggaca tgaaaagtta ttcaacatca caaattatta gagaaatgca 79441 gatcaaaacc acaataaaat atcacttcag atccattaaa atggttacta taaaaagacc

79501 caaaaactgt tggtgcagat gtgtagaaac tgaaagcctt gtgcaatgct agtgggaatg

79561 taaaattatg cagccactaa ggaaaacagt atggtggttc ctcaaaaaat taaacagaat

79621 taccatataa tccagcaatt ttacttctgg gtatacacac aaaataattc aaagcaggaa

79681 tttςaacaaa tatttgtaca ttaatgctca taacagcatt attcacaata gccaaaaggt

79741 ggaaacaact caaatgtcca tcaacagata aatggagaaa caaaatgtgg tatatacata

79801 caatgaaata ttagccttaa aagaaattac attctaatat atgacacaat gtggatgaac

79861 cttgaagacg taatggcaag tgagataagc caaacacaaa aggatacata ttttatgatt

79921 ccgcttatat gaggtaccta gaatactcaa atgcatggag acagaaagta aaacagtggt

79981 tacccagaac taaggggaga agaaatgtga gttattgttt aatgggtttg gactttcagt

80041 ttggaatgat gaaaaagttc tggatgtgag ctgtagtgat ggttacacaa caatgtgaat

80101 gtacttcatg ccactgaagt gtacacttaa aagtggttaa aatgctaagt ctcaggtata

80161 ttttgccaca ccaatttttt tttaaataaa ttttaaaaag aaggccaggt gcagtggctt

80221 gtgcctgtaa tcccaacact ttgggaggcc aagtcaggaa aacaggttga atctaggaat

80281 ttgagaccag cctgggcaac aaagtgacac cctgtctcta caaaaaaaaa aaaaaaaaaa

80341 agaaaagaaa aacaaattag ctgggcgcag tggctcatac ctatagtccc ctgatgcagt

80401 gacaactctt gcacctatag tcccagctac ttaggaggct gaggtgggag gatttcatga

80461 gcccagaagc tcaaggcttt ggtaagctat gatcatgcca ctgcagtgca gcttgggtga

80521 cagagcaaga caccatctgt taaaaaaagt ttgtttttag actggacaca gtggctcatg

80581 cttgtaatcc tagtactttg ggaggccgag gcaggaggat tccttgaggc caggagttca

80641 agaccagcct aggcaacata atgagatctt catctctaca aaaaaagagg gagaaatact

80701 aactaaaagt atattgtggc tgggcgcggt ggctcacacc tgtagtccta gcactttggg

80761 aggctgaggc aggtggatcg cctgagctca ggagttcaag accagactgg ccaacatagt

80821 gaaaccctgt ctctactaaa aatacaaaaa ttagccaagc gtgctggcag ccttctgtaa

80881 tcccagctac tcgggaggct gaggcatgag aatcgcttga acctgggagg cagtggttgc

80941 agtgagccga gattacacca ctgcattcca acctgggcga taaagcaaga ctttgtctca

81001 aaaaaaaaaa aaaaaaaagt atattgagat gtcatttttc agccatcaga ttgacaagaa

81061 tacaaaagct tggcaatgca aggctgtggg gaaacaggcc tctctcattg ctttgggaag

81121 ttaaaaatgt tatactcact atgatgggga atttggtaat gtctaagaac attaggtgag

81181 catttatcct ttcatccaac aatcccattt ctaggacatc tcccaaagat acacctgcaa

81241 aaaaataaaa taaaattata ggcacaaagt tatttatggt gaccttattt gctatagtag

81301 aaaaccagaa acaacctaaa cgtccagctg tagggactgg tttaataaac tgtggtacat

81361 ttaagcaata gagaattata caactgtaaa aacaatgagg aacacttcta gatgctggta

81421 tggaacaatt gccaggatat attattaagt gaaataaagc aagatgcaga acaatgttta

81481 tggtgtgcta ccttttgtgt gaaggaaaca aatatataaa catatttgtt tgttttcaaa

81541 aaagaaacaa tgaaatgaca tactaacaaa tggacaaaaa tggtctcctc catttggaga

81601 gagggtgcag gatgaagaag caagtacaga aggaaaggtt ctatgggtgc atctttttat

81661 gtagttttgg ctttggaacc atataaacga attatgtatt tatacaatgg gaaagacaaa

81721 cccctgaaat cgaaaacaaa agaaacagaa acaaatgatt ctaactatac atcattttgg

81781 cactgaaact gtgtagagaa agtaattctt tcattttaaa tttcagtaca cctctagtag

81841 gatatattct actaggaaaa agtaccacaa aaaaacctac actgcattca gttgtcttat

81901 tgtaatacta ataataacat gatacagaaa aagaaaagca aatatttgtt ttaattccat

81961 taggaactga gattttcaca gtaagaaaaa agagatccaa gcataaaatc aaagaagtta

82021 tgtacaaatt gtaaagaata atgactgcga tggattaaaa tatcaactat atacaaattt

82081 gtgagctcat tgtgatactt caaaaaatgc attggtttcc atgggaggtt gctagatcac

82141 taagtgacta ctctgcKcat tgataaagtc ggggagggat aaagaattct tcctttcctg

82201 tataaataaa ataattaatg aaggaaagtt tagtttttgt tttattttat ttatttattt

82261 attttggaga cagggtcttg ctttgtctgc tctgtcaccc aggctggagt agctggatca

82321 tggctcactg cagccttgaa ctcctgggct caagtggtct tcccacctca atctccacct

82381 cctgaacagc tgggacttca ggctcacgcc ataaccccta cattgaccag cctggtcttg

82441 agctcctggc ctcaagcgat cctcccacct tggccttcca aagtgcttga ctttcaggtg

82501 tgagccactg tgccagcccc atcctctttt ttgtagatga attacagtca atcaaaaaag

82561 aagcagtaat aaaatagccc actaccattt gcatattaaa gttacccacc aacgttttac

82621 ccatgccatt agaagttgca aatgacagat tctaatgaca tgggtaaaac attggtgggt

82681 aactttaaaa tggaggaaac tagctgacat caccagaacc cagtgatcaa ctttgcaaca

82741 ataaaactag gcaaccagta acttgtatct cctggtgtga gtaatttgag gtatgcaaca

82801 tcatcagtga agtactcttg aatgaaaatc tgaccctgca tctaatcaga cttctaaatc

82861 tcacttccat tttacaggaa atatagatga tagaggaaca agtaaagcag ggataagagg

82921 aaaatcaata gccttaacta attttaccaa actggaaaaa taaaaataaa tgaacaaaat

82981 gtttgattcc aaatctagaa acataaacca aaagaaggca ggagggagaa aataataaat

83041 attaaaggac agattaatga ttagaacaac tacaagaact aataaatctt tggaaaaaaa

83101 taaatacttc tttgggaaaa acataggtaa accaccagct aacctaatta agaacaaaaa

83161 aggaagaaat acataagaaa tgataagaga gacataagca ttgaaacatt ttttttaagt

83221 ttaagagact acttgcacaa ctttatgcaa atacctttga aaacctagtt gaaataaata

83281 attccctggg aaaatacatt taaaaaaaaa ttatccccaa aagagaggaa aatttgaaca

83341 aatcaatttt catatgagaa atagagaaca acctaaatac tcatctattt aaaactagtt 83401 aaataaacaa tgatgcatgc aaactataaa gtatgtacta tgtagcaaaa atctctatga

83461 actgatatgg aatgtttcca aggatttttt tttttttttt acagagtctt gctctgtccc

83521 ccaggctgga gtgcagtggc accatcttgg cccattgcaa cctctgcctc tgaggttcaa

83581 gcaacccgcc cacctcagcc tcccaagtag ctgggactac aggcatgtac tgctaatcct

83641 ggctaatttt tgtatttttg tgtgtgtgtg tatatatata tatataattt ttttttgttt

83701 tttttttttt ggtagagacg ggatttcacc atattgccca ggctggtctc aaacttctgg

83761 tctcaagcga tcctcctacc ttagcctccc aaagtgctgg gattacaggt gtgagccacc

83821 acgcctggcc tcaaggaaat tttgttaaaa gaaatatgtg aaagagtata tataacatga

83881 acactttgta agaaaaagga gaaaatatga ctccatatat atttgcttgt tcttaaaaaa

83941 gaaatatagg aagaataagc cagaaacaaa tgaaaaagca taaaatgaag gtgtgcaact

84001 tctctgaatt tacctcttta atatagtttt tcttttgaac catataaacg ttttgcatat

84061 tag^'atcaaca aaaatttaaa agcaaaacct aatgtagaac acaaacagcc acaatttaag

84121 ctaactaaag aaactctcaa gttggtacca taacaattca gaaatagaat taattcaagt

84181 aacttttgaa cacagcactc ccctaacaga acgtattttc agtgcaaaaa gaattgcaaa

84241 aaaatattga actttctcta ctaggttttt aaaaaattat tgttgttttt aattgataca

84301 taattgtaca tatttatgga gtatagtgtg gtatttcaat acatgtatac aatgtgtaat

84361 gctcaaatca gggtaattag catatctatc acgacaaaca tttatcattt ctttgtgttg

84421 ggaacaattc aaaatctgct cttctagctt tttgaaaata tacaataaat tgttgtttat

84481 tatagtcact ctgtagggct gtggaacact ggaacttatt tctcctaaca agctgtactc

84541 ttgcatgcgt taaccaacag ctgactatcc ccttgacctc ttccaaaccc tttcccagcc

84601 tctagtaacc actattctac tatctacttc tttttttatt tgtttgtttt gagatggagt

84661 tttgctcttg ttgcccaggc tggagtgcaa tggcgtcacc atggctcact gcaacctctg

84721 cctcctgggt tcaagcgatt ctcctccctc agcctcctga gtagctggga ttgcaggcgc

84781 ccgccagcat gcccagctaa tttttgcatt tagtagaggt ggggtttcac catattggca

84841 aggctggtct cgaactcctg acttcatgtg atccacccac ctcggcctca caaagtgcta

84901 ggataacagg cgtgagccac cacacctggt cacggtattt atctttctat gcctagctta

84961 cttcacttat cagaatgtcc ttcaaggtca tccatattgc catgaatgac agaattttat

85021 tattttaaat tgctaactag tattccatga tgtatatata ccacatttct tttatcattc

85081 atctgttgat ggacatgtag gttgatcaca tatcttggct attatgaaaa atgcttcaat

85141 aaacacgagg gcagatatct atttgacata ctgatttcct ttcctttggc taaataccca

85201 gtagtgggat tgctggatca tagggtagtt ctatttgtag ttttttgaag aacctccata

85261 cttttctcca taatggttaa atttgccttc ccaccaacat tgtgtaaaag ctcccatttc

85321 tctccatccg tgccagcatt tgttattttt tgcctttttg atagtagtca ttttaactgg

85381 agtgaggtga tatctcattg tggttttgat ttgcatttcc gtgatgttta gtgatttttt

85441 ttaataataa tatcaatgaa gaaattctga agttattttg aatgtattat agatagagaa

85501 aattgtggga aaccagggtt ctttcctcac tgtaggagaa gggagtaaga aaaaaatata

85561 tagctcagag cagtctgagt tatgtgagga atacaaaatt tatcaggccc agagagacat

85621 aagtatggca tttcagccat gcttccttcc tacgttcatg cctggggcag ttctttaaag

85681 gcattttgtt tctaatttgc tgcttcaccc acctgttatg ttcatattcc tgggaggtgt

85741 gatgcaagga acaatgatag ttaataaatt atgttatttt aatgcaaatt cttggttaaa

85801 aaaattagaa actgattctt cttttttacg ttaaaaactc attagttact gctgctaatt

85861 ggagtatgta tacagggcaa cttaaatcta tgctcctgag ttgcagtcct caaacttggc

85921 ccaaataaac tctctacttt gcctcagctt cttcttgtag gttgacattg gaattagagg

85981 tatcagtatt aactcatgat tctttaaaat tttggatttc tagctttgtc tcccaaaaaa

86041 agcatagaag caataacatt ccaatagcta atttcaagtc tgggaaaggg gaggttcaca

86101 gtaagctgga aacatcttac gcacaaaagt aagaaagtgc tcaaaattga tgggaacaca

86161 agagttgact tgaagctgtt cccactggct aaatttgtga caagctgaac atcaaaaaga

86221 ataataatgg tgatggatta tattaccttg aaataagaaa agaacccatg aatccatagt

86281 gacactcaaa ataaaggtgg aagtggaagt gagctctctt ctttcaaaga ctgcagccta

86341 caaaatacag aagaaattat agaattagaa aaatcaccat ttttcaacca ccattggaat

86401 aattcaggca agatcacctg tagatgctaa acttattgag tgaaaggttt ttgaggatta

86461 ttgggcagta tgatattcac agtattgtat ctcacacaca aattacttat ttttatttaa

86521 ttattttttg agacagggtc tcacactgtc acccaggcta gagtgcagtg gcacaatcat

86581 ggctcaccac agctgcaacc tcccaggctc aggtgatcct cccacctcag cctcctgagt

86641 agctaggact ataggtgcct gccatcatgc ctggctaatt tttgtatttt ttgtagagat

86701 ggggttttgc catattactc aggcttgtct tgaactcctg tgctcaagaa atccaccctc

86761 ctgggcctcc caaaatgctg ggattatagg tgtgagccac cgcgcctggc ccacagatta

86821 cttattaatt acataaacca aatagtatta tttgaggcag gtctcaatca atttagagat

86881 tttgccaagg ttaaggacat gaccagtgac acagcctaag aagcttctga gaacatgtgc

86941 ccgaggtgct tgggttacag tttggcttta catattttag ggagacagaa gttacaagca

87001 aaggcataaa tcaatacatg gaaggtatat atttgttcag cctggaaagg caggacatct

87061 tgaagctgag ggaggagtct tctaagtcat aggtggattc aaatattttg ttattggcaa

87121 ttggctgaaa gagttaagct ctgcttgttg agttgaagtc agaataaaga aatgcttgag

87181 ttagattggg gagttgtgga agccaaggtt cttgtatgaa gatgaagcct ccaggcttca

87241 gagaaaatag attataaaca tctcttctct ggagacctta ctctccagaa aagacctagt

87301 agcaaaggaa ggagattctc tacagaatac acattttctt tctaatgact tgtgatgtag 87361 aaaaaagaaa aatgcaaatt tcccctacaa ggacctcttt gcaaggccat gccacaatat

87421 gtcaaagaaa tatatttagg gccagttaca gtgactcttg cctgtaatcc tagcactttg

87481 ggaggctgag gcaggagaat ggcttgaggc caggagtttg agaccaaccc gagcaacata

87541 atgagacccc tatttctaca aaaatttttt aaaaattaat tgagcatggt ggcatatgcc

87601 gtccaactac tcaggaggct gaggtgggag gatctcttaa gcccaggact ttgagggttc

87661 agtgtgctat gatcatgcca ctgcatttca gcctgggtga caagagcagg atactgtctc

87721 taaaaaataa ataaataaat gaaaaataaa gtaacaaagg aagaaagaaa ttaaaacata

87781 tatttagggc ctgttatctg tcatgtgatg ctataccagt atcaagttgg agttcagtat

87841 cttattgcta caaagagttt gtcttattca tcttatgatc tctatttttg tgttaatgct

87901 ggtcagttgt gcctaagctc caaggggagc catgtactac cccccattcc catcgtaggc

87961 tgaaatagtt tttcagcttt ctctgggatc cccttggcca agagtggatc cattcagtca

88021 gctgtcgggc ttagaatttt attttccatg tacaattaca atatagtaca cactcacatg

88081 catgtgcata tgcacacatg cacaaaatat gttgagatag tatgctgatc tttatgaaca

88141 tgtaatagcc cagaaatggg agaagcgctt tttaaagaga tttttcaacc acaaatcttc

88201 actgaaagaa gagacttctg tctatcttgg atatatgtct tttgggacac tcctacctct

88261 agagaccaaa aacaattgtt cctgagagaa agtgaagtca cccgagtaaa ggtgcttagg

88321 ttgagaagct aagccaagtg ctttattttg taatgtaaat ccatgtctgt cagcagcaaa

88381 gccaagtccc tgcagctttg tatcttccac aggagaaagg attattttct atatttggat

88441 ttttattgtt ttgggttggt tttttattgc tggagatgtg tatatgttct ttatgtagta

88501 aatcacagag aatcaggagc agaagcccta aagctactgt agagataggc atgcctgatc

88561 atcctatggg gctttaaaaa agtgacctgg acttagtcca gtttagcaca gcatgggttg

88621 tggttatgcc cactaagatt ggataactca caatgttgtt attgatttgg cggccgttat

88681 ttgtaattgt cctgagaggt ggagctcagg gtaaccataa cactgagggt tagggtggtg

88741 catcttttaa aaggagacat aattccacaa tcatccacct gagtagtagt tataaattac

88801 tggtgtgcaa aaattgagat ggaggaagag cagaaaccag agggaaagga gacccttttt

88861 cttgtttagg tccttattga gagtctggca agaaccttga ggcatgaaga tagatgagag

88921 tcaaataggg aggcaatcga ggttggtctc aaatacagaa ctgggggtgg tgcatcggag

88981 cgaagtcatt tttttttttt tttttttttt ttttgagaca gagtttcgct cttgttgtcc

89041 aggctggagt gcaatggcgc gatctcggcc cctggcaacc tccgcctccc gtgttcaagc

89101 gattctcctg cctcagcctt cctgagtagc tgggattaca cgcatgcacc accacgccca

89161 gctaattttg tatttttagc agaggtggag tttctccatg ttggtcaggc tggtctcgaa

89221 ctcccgacct cagatgatcc gcctgcctcg gcctcccaaa gtgctgggat tacaggcatg

89281 agccaccgca cccggccctg agcaaagtca tttttaacag caattggaag taactcattg

89341 tgctaagctt cctgccagcc agtctcagat atgcctRgga aattaagaaa tgggcctgcc

89401 cgtgcttggg gaacaatggt aaagggcaaa acactgcctc cccctgaggc caaaggggag

89461 aaagcaggta tgtccccagt tatcaaggat acaaaaatag tttccaaagg gtagaggtca

89521 tcaggcataa attctctata agtcgattca ttaacaaagt atgtagtcag tcctcacttg

89581 tctccttata attggttcac

[0318] Following is afirstPP2CecomplementaryDNAsequence(cDNA; SEQIDNO: 2).

TranscriptVariant 1: 1083 bp

ATGATAGAGGATACAATGACTTTGCTGTCTCTGCTGGGTCGCATCATGCGCTACTTCTTGCTGAGACCCGAGA

CTTAAACAGCATCTTCAGGACTACGAGAAAGACAAAGAAAATAGTGTATTATCTTACCAGACCATCCTTGAAC AGCAGATTTTGTCAATTGACCGAGAAATGCTAGAAAAATTGACTGTATCCTATGATGAAGCAGGCACAACGTG TTTGATTGCTCTGCTATCAGATAAAGACCTCACTGTGGCCAACGTGGGTGACTCGCGCGGGGTCCTGTGTGAC AAAGATGGGAACGCTATTCCTTTGTCTCATGATCACAAGCCTTACCAGTTGAAGGAAAGAAAGAGGATAAAGA GAGCAGGTGGTTTCATCAGTTTCAATGGCTCCTGGAGGGTCCAGGGAATCCTGGCCATGTCTCGGTCCCTGGG GGATTATCCGCTGAAAAATCTCAACGTGGTCATCCCAGACCCAGACATCCTGACCTTTGACCTGGACAAGCTT

TGACAATATAACAGTCATGGTGGTGAAGTTCAGAAATAGCAGCAAAACAGAAGAGCAGTGA [0319] FollowingisasecondPP2CecDNAsequence(SEQIDNO: 3).

TranscriptVariant2: 912 bp

ATGGTGAAGGGCAAGGTAGCCGAGATCATGCAGAACGATCGACTCGGGGGGCTTGATGTGCTCGAGGCCGAGT TTTCCAAGACCTGGGAGTTCAAGAACCACAACGTGGCGGTGTACTCCATCCAGGGCCGGAGAGACCACATGGA GGACCGCTTCGAAGTTCTCACGGATCTGGCCAACAAGACGCACCCGTCCATCTTCGGGATCTTCGACGGGCAC

AGAAAGACAAAGAAAATAGTGTATTATCTTACCAGACCATCCTTGAACAGCAGATTTTGTCAATTGACCGAGA AATGCTAGAAAAATTGACTGTATCCTATGATGAAGCAGGCACAACGTGTTTGATTGCTCTGCTATCAGATAAA

TGGCTCCTGGAGGGTCCAGGGAATCCTGGCCATGTCTCGGTCCCTGGGGGATTATCCGCTGAAAAATCTCAAC GTGGTCATCCCAGACCCAGACATCCTGACCTTTGACCTGGACAAGCTTCAGCCTGAGTTCATGATCTTGGCAT

CTTTGGGGCCAAGAGCATAGTTTTACAGTCATTTTACAGAGGCTGCCCTGACAATATAACAGTCATGGTGGTG AAGTTCAGAAATAGCAGCAAAACAGAAGAGCAGTGA

[0320] Following isathirdPP2CecDNAsequence(SEQIDNO: 4).

TranscriptVariant3: 546 bp

ATGCTAGAAAAATTGACTGTATCCTATGATGAAGCAGGCACAACGTGTTTGATTGCTCT

GCTATCAGATAAAGACCTCACTGTGGCCAACGTGGGTGACTCGCGCGGGGTCCTGTGT

GACAAAGATGGGAACGCTATTCCTTTGTCTCATGATCACAAGCCTTACCAGTTGAAGG

AAAGAAAGAGGATAAAGAGAGCAGGTGGTTTCATCAGTTTCAATGGCTCCTGGAGGGT

CCAGGGAATCCTGGCCATGTCTCGGTCCCTGGGGGATTATCCGCTGAAAAATCTCAAC

GTGGTCATCCCAGACCCAGACATCCTGACCTTTGACCTGGACAAGCTTCAGCCTGAGTT

CATGATCTTGGCATCAGATGGTCTCTGGGATGCTTTCAGCAATGAAGAAGCAGTTCGAT

TCATCAAGGAGCGCTTGGATGAACCTCACTTTGGGGCCAAGAGCATAGTTTTACAGTC

ATTTTACAGAGGCTGCCCTGACAATATAACAGTCATGGTGGTGAAGTTCAGAAATAGC

AGCAAAACAGAAGAGCAGTGA

[0321] FollowingisafourthPP2CecDNAsequence(SEQIDNO: 5).

TranscriptVariant4: 573 bp

CGCTTTTCCTGCTGTGCATCAGCTTGGCTCTATGGAGTTACTTCTTCCACACCGACGAGGTGAAGACCATCGT GGGCTTGATGTGCTCGAGGCCGAGTTTTCCAAGACCTGGGAGTTCAAGAACCACAACGTGGCGGTGTACTCCA CATCTTCGGGATCTTCGACGGGCACGGGGGAGAGACTGCAGCTGAATATGTAAAATCTCGACTCCCAGAGGCC AGCAGATTTTGTCAATTGACCGAGAAATGCTAGAAAAATTGACTGTATCCTATGATGAAGCA [0322] FollowingisafirstPP2Ceaminoacidsequence(SEQIDNO: 6).

Isoform 1:360aminoacids

MiEDTMTLLSLLGRiMRYFLLRPETLFLLCisLAL WSYFFHTDEVKTΓVKSSRDAVKMVKGK

VAEIMQNDRLGGLDVLEAEFSKTWEFKNHNVAVYSIQGRRDHMEDRFEVLTDLANKTHP

SIFGIFDGHGGETAAEYVKSRLPEALKQHLQDYEKDKENSVLSYQTILEQQILSDDREMLEK

LTVSYDEAGTTCLIALLSDKDLTV ANVGDSRGVLCDKDGNAIPLSHDHKPYQLKERKRIKR AGGFISFNGSWRVQGILAMSRSLGDYPLKNLNVVIPDPDILTFDLDKLQPEFMILASDGLWD AFSNEEAVRFIKERLDEPHFGAKSIVLQSFYRGCPDNITVMWKFRNSSKTEEQ

[0323] Following is a second PP2Ce amino acid sequence (SEQ ID NO: 7).

MVKGKV AEIMQNDRLGGLDVLEAEFSKTWEFKNHNV AVYSIQGRRDHMEDRFEVLTDLA NKTHPSIFGIFDGHGGETAAEYVKSRLPEALKQHLQDYEKDKENSVLSYQTILEQQILSIDR EMLEKLTVSYDEAGTTCLIALLSDKDLTV ANVGDSRGVLCDKDGNAIPLSHDHKPYQLKE RKRIKRAGGFISFNGSWRVQGILAMSRSLGDYPLKNLNVVIPDPDILTFDLDKLQPEFMILA SDGL WD AFSNEEA VRFIKERLDEPHFGAKSΓVLQSFYRGCPDNITVMVVKFRNSSKTEEQ

[0324] Following is a third PP2Ce amino acid sequence (SEQ ID NO: 8).

Isoform 3: 181 amino acids

MLEKLTVSYDEAGTTCLIALLSDKDLTVANVGDSRGVLCDKDGNAIPLSHDHKPYQLKER KRIKRAGGFISFNGSWRVQGILAMSRSLGDYPLKNLNVVIPDPDILTFDLDKLQPEFMILAS DGL WD AFSNEEA VRFIKERLDEPHFGAKSΓVLQSFYRGCPDNITVMWKFRNSSKTEEQ

[0325] Following is a fourth PP2Ce amino acid sequence (SEQ ID NO: 9).

Isoform 4: 191 amino acids

MIEDTMTLLSLLGRIMRYFLLRPETLFLLCISLALWSYFFHTDEVKTIVKSSRDAVKMVKGK VAEIMQNDRLGGLDVLEAEFSKTWEFKNHNVAVYSIQGRRDHMEDRFEVLTDLANKTHP SIFGIFDGHGGETAAEYVKSRLPEALKQHLQDYEKDKENSVLSYQTILEQQILSIDREMLEK LTVSYDEA

[0326] FollowingisaB3GALT3cDNAsequence(SEQIDNO: 10).

B3GALT3 Sequence

gtgcggatgcggggaggctgcgtgtgtgcgcagggagagaacgccggccaccttcccgcttccgagctgggtg cgcgccgagcacaggagattgcctgcgtttaggaggtggctgcgttgtgggaaaagctatcaaggaagaaatt gccaaaccatgtctttttttctgtttcagagtagttcacaacagatctgagtgttttaattaagcatggaata cagaaaacaacaaaaaacttaagctttaatttcatctggaattccacagttttcttagctccctggacccggt tgacctgttggtctcttcccgctggctgctctatcacgtggtgctctccgactactcaccccgagtgtaaaga accttcggctcgcgtgcttctgagctgctgtggatggcctcggctctctggactgtccttccgagtaggatgt cactgagatccctcaaatggagcctcctgctgctgtcactcctgagtttctttgtgatgtggtacctcagcct tccccactacaatgtgatagaacgcgtgaactggatgtacttctatgagtatgagccgatttacagacaagac tttcacttcacacttcgagagcattcaaactgctctcatcaaaatccatttctggtcattctggtgacctccc acccttcagatgtgaaagccaggcaggccattagagttacttggggtgaaaaaaagtcttggtggggatatga ggttcttacatttttcttattaggccaagaggctgaaaaggaagacaaaatgttggcattgtccttagaggat gaacaccttctttatggtgacataatccgacaagattttttagacacatataataacctgaccttgaaaacca ttatggcattcaggtgggtaactgagttttgccccaatgccaagtacgtaatgaagacagacactgatgtttt catcaatactggcaatttagtgaagtatcttttaaacctaaaccactcagagaagtttttcacaggttatcct ctaattgataattattcctatagaggattttaccaaaaaacccatatttcttaccaggagtatcctttcaagg tgttccctccatactgcagtgggttgggttatataatgtccagagatttggtgccaaggatctatgaaatgat gggtcacgtaaaacccatcaagtttgaagatgtttatgtcgggatctgtttgaatttattaaaagtgaacatt catattccagaagacacaaatcttttctttctatatagaatccatttggatgtctgtcaactgagacgtgtga ttgcagcccatggcttttcttccaaggagatcatcactttttggcaggtcatgctaaggaacaccacatgcca ttattaacttcacattctacaaaaagcctagaaggacaggatactttgtggaaagtgttaaataaagtaggta ctgtggaaaattcatggggaggtcagtgtgctggcttacactgaactgaaactcatgaaaaacccagactgga gactggagggttacacttgtgatttattagtcaggcccttcaaagatgatatgtggaggaattaaatataaag gaattggaggtttttgctaaagaaattaataggaccaaacaatttggacatgtcattctgtagactagaattt cttaaaagggtgttactgagttataagctcactaggctgtaaaaacaaaacaatgtagagttttatttattga acaatgtagtcacttgaaggttttgtgtatatcttatgtggattaccaatttaaaaatatatgtagttctgtg tcaaaaaacttcttcactgaagttatactgaacaaaatattatttaaaattacttcaactttgtgtttttaaa tgttttgacgatttcaatacaagataaaaaggatagtgaatcattctttacatgcaaacattttccagttact taactgatcagtttattattgatacatcactccattaatgtaaagtcataggtcattattgcatatcagtaat ctcttggactttgttaaatattttactgtggtaatatagagaagaattaaagcaagaaaatctgaagtattgt cttgtttttaaaaaatacagttcctagtgtttatagaagtcacttaatttgtctcatttttccacctggaaat taggaataatgtagaatgcaaggcagtaatttccttttggaaaggactctgaaggcagaaaagaagggagaga acctcatgggcagaatattataaaaagagtgtcatattccagcatttgaattggaaagagaagagtgaagatc caagttgcattattaatctgccctgtgttttttccttttaacaatcagtttgagctgctgctgttatgagttt ctcatcaagatgaaagccctaatatgtaaagtcaaatccgatttaaattttgtgcttttatagaaagaaattt cttcatagacgtggtgatatatcatttcttggacctgctaatagtaggtcaaaggggagcactccttgccccc tgttcctgggtttatgcagttttctttttagagtttatatagggcaagtggttctttttctctgaattacagg atggaaaaaggtcatatcctttgtcaggaaatataaacttgaaagtatgtagtcagctcttgtaatactcata tttatgattgtcctatatgaaaaacaacttcagttaaaactataatgtgtgattctgtataacaaggtgatgt ctgtttcccagggctcagacctaatccagttataataaaatcaattaaatgaaatattctatagaatcgatct atgcccttgttaatctccatccatataggagtcacgttctttaagacagatggtggtagttatttttgtggca tggttagatttgactggttttgcagaagattacagttatgtactgcataatgacatatacaatagtggtccca taaaattataatggagcagaaaatctattgcctcatgatgttttagccgttttaatgtcatagcctagtgcat tactcacgtgtctgtggagatgctggtgtaaacaaacctactacactgccagttgtataaaagtacagcacat tcagttatgtacagtatgtaatactgataatgacaataaatgacaccggtttgtgtatttactttttataa

[0327] FollowingisaB3GALT3aminoacidsequence(SEQIDNO: 11).

B3GALT3Sequence

MASALWTVLPSRMSLRSLKWSLLLLSLLSFFVMWYLSLPHYNVIERVNWMYFYEYEPIYR QDFHFTLREHSNCSHQNPFLVILVTSHPSDVKARQAIRVTWGEKKSWWGYEVLTFFLLGQ EAEKEDKMLALSLEDEHLLYGDIIRQDFLDTYNNLTLKTIMAFRWVTEFCPNAKYVMKTD TDVFINTGNLVKYLLNLNHSEKFFTGYPLIDNYSYRGFYQKTHISYQEYPFKVFPPYCSGLG YIMSRDLVPRIYEMMGHVKPIKFEDVYVGICLNLLKVNIHIPEDTNLFFLYRIHLDVCQLRR VIAAHGFSSKEIITFWQVMLRNTTCHY

[0328] Following is a predicted PP2Ce amino acid sequence alignment between human and mouse counterparts.

Predicted Human vs Mouse Amino Acid Alignment

OriGene MIEDTMTLLSLLGRIMRYFLLRPETLFLLCISLALWSYFFHTDEVKTIVK

NCBI

Mouse MIEDIMTLLSLLGRIMRYFLLRPETLFLLCISLALWSYFFHTDEVKTIVK

OriGene SSRDAVKMVKGKVAEIMQNDRLGGLDVLEAEFSKTWEFKNHNVAVYSIQG

NCBI

Mouse SSRDAVKMVKGKVAEIMQNDRLGGLDVLEAEFSKTWEFESHNVAVYSIQG

OriGene RRDHMEDRFEVLTDLANKTHPSIFGIFDGHGGETAAEYVKSRLPEALKQH

NCBI TAAEYVKSRLPEALKQH

Mouse RRDHMEDRFEVLTDLANKTHPSIFGIFDGHGGETAAEYVKSRLPEALKOH

OriGene LQDYEKDKENSVLrfYQTILEQQILSIDREMLEKLTVSYDEA

NCBI LQDYEKDKENSVLSYQTILEQQILSIDREMLEKLTVSYDEAGTTCLIALL

Mouse LQDYEKDKENSVLTYQTILEQQILSIDREMLEKLTVSYDEAGTTCLIALL

OriGene

NCBI SDKDLTVANVGDSRGVLCDKDGNAIPLSHDHKPYQLKERKRIKRAGGFIS

Mouse SDKDLTVANVGDSRGVLCDKDGNAIPLSHDHKPYOLKERKRIKRAGGFIS

OriGene

NCBI FNGSWRVQGILAMSRSLGDYPLKNLNVVIPDPDILTFDLDKLQPEFMILA

Mouse FNGSWRVQGILAMSRSLGDYPLKNLNVVIPDPDILTFDLDKLOPEFMILA

OriGene

NCBI SDGLWDAFSNEEAVRFIKERLDEPHFGAKSIVLQSFYRGCPDNITVMWK

Mouse SDGLWDAFSNEEAVRFIKERLDEPHFGAKSIVLOSFYRGCPDNITVMWK

OriGene

NCBI FRNSSKTEEC*

Mouse FRNSSKTEEh*

[0329] Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the invention, as set forth in the claims which follow. Also, citation of the above publications or documents is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Each patent, patent application and other publication and document referenced are incorporated herein by reference in its entirety, including drawings, tables and cited documents.

Claims

What is claimed is:

1. A method for identifying a subject at risk of type II diabetes, which comprises detecting the presence or absence of one or more polymorphic variations associated with type II diabetes in a nucleic acid sample from a subject, wherein the one or more polymorphic variations are detected in a nucleotide sequence in SEQ ED NO: 1, a substantially identical sequence thereof or a fragment of the foregoing, whereby the presence of the polymorphic variation is indicative of the subject being at risk of type II diabetes.

2. The method of claim 1, which further comprises obtaining the nucleic acid sample from the subject.

3. The method of claim 1, wherein the one or more polymorphic variations are detected at one or more positions selected from the group consisting of rsl947686, 1580253, rsl580254, rs980975, rs980976, rs898681, rs898682, rs881453, rslO37888, rs898685, rs898684, rs898683, rs2306061, rs2279108, rs2279107, rs2279106, rs2290915, rslO45448, rsl3256, rs2290914, rsl599384, rs2231257, rs2231256, rs2231255, rs2231254, rs2231253, rs2231252, rs2231251, rsl610158, rsl947685, rsl903423, rs731363 and rsl599386.

4. The method of claim 1, wherein the one or more polymorphic variations are detected at positions selected from the group consisting of rs 1947686, rs898685, rs898684, rs898683, rs2306061, rs2279106, rslO45448, rsl599384, rs2231252, rs731363 and rsl599386.

5. The method of claim 1, wherein the one or more polymorphic variations are detected at one or more positions selected from the group consisting of rsl3256, rs2231252 and rs 1903423.

6. The method of claim 1, wherein the one or more polymorphic variations are detected in a region spanning positions 15913 to 89377 in SEQ ID NO: 1.

7. The method of claim 1, wherein the one or more polymorphic variations are detected at one or more positions in linkage disequilibrium with one or more positions in claim 3.

8. The method of claim 1, wherein detecting the presence or absence of the one or more polymorphic variations comprises: hybridizing an oligonucleotide to the nucleic acid sample, wherein the oligonucleotide is complementary to a nucleotide sequence in the nucleic acid and hybridizes to a region adjacent to the polymorphic variation; extending the oligonucleotide in the presence of one or more nucleotides, yielding extension products; and detecting the presence or absence of a polymorphic variation in the extension products.

9. The method of claim 1, wherein the subject is a human.

10. A method for identifying a polymorphic variation associated with type II diabetes proximal to an incident polymorphic variation associated with type II diabetes, which comprises: identifying a polymorphic variation proximal to the incident polymorphic variation associated with type II diabetes, wherein the polymorphic variation is detected in a nucleotide sequence in SEQ ID NO: 1, a substantially identical sequence thereof or a fragment of the foregoing; and determining the presence or absence of an association of the proximal polymorphic variant with type II diabetes.

11. The method of claim 10, wherein the incident polymorphic variation is at one or more positions in claim 3.

12. The method of claim 10, wherein the proximal polymorphic variation is within a region between about 5 kb 5' of the incident polymorphic variation and about 5 kb 3' of the incident polymorphic variation.

13. The method of claim 10, which further comprises determining whether the proximal polymorphic variation is in linkage disequilibrium with the incident polymorphic variation.

14. The method of claim 10, which further comprises identifying a second polymorphic variation proximal to the identified proximal polymorphic variation associated with type II diabetes and determining if the second proximal polymorphic variation is associated with type II diabetes.

15. The method of claim 14, wherein the second proximal polymorphic variant is within a region between about 5 kb 5' of the incident polymorphic variation and about 5 kb 3' of the proximal polymorphic variation associated with type II diabetes.

16. An isolated nucleic acid which comprises an adenine at a position corresponding to position 37230 in SEQ ID NO: 1.

17. An oligonucleotide comprising a nucleotide sequence complementary to a portion of the nucleic acid of claim 16, wherein the 3' end of the oligonucleotide is adjacent to position 37230.

18. A microarray comprising an isolated nucleic acid of claim 16 linked to a solid support.

19. An isolated polypeptide encoded by the isolated nucleic acid sequence of claim 16.

20. A method for identifying a candidate molecule that increases glucose uptake in a cell, which comprises:

(a) introducing a test molecule to a system which comprises a nucleic acid comprising a nucleotide sequence in SEQ ID NO: 1, a substantially identical sequence thereof, or a fragment of the foregoing; or introducing a test molecule to a system which comprises a protein encoded by a nucleotide sequence in SEQ ID NO: 1, a substantially identical sequence thereof, or a fragment of the foregoing; and

(b) determining the presence or absence of an interaction between the test molecule and the nucleic acid or protein, whereby the presence of an interaction between the test molecule and the nucleic acid or protein identifies the test molecule as a candidate molecule that modulates cell proliferation.

21. The method of claim 20, wherein the system is an animal.

22. The method of claim 20, wherein the system is a cell.

23. The method of claim 20, wherein the nucleotide sequence comprises one or more polymorphic variations associated with type II diabetes.

24. The method of claim 23, wherein the one or more polymorphic variations associated with type II diabetes are at one or more positions in claim 3.

25. A method for treating type II diabetes in a subject, which comprises administering a candidate molecule identified by the method of claim 20 to a subject in need thereof, whereby the candidate molecule treats type II diabetes in the subject.

26. A method for identifying a candidate therapeutic for treating type II diabetes, which comprises:

(b) determining the presence or absence of an interaction between the test molecule and the nucleic acid or protein, whereby the presence of an interaction between the test molecule and the nucleic acid or protein identifies the test molecule as a candidate therapeutic for treating type II diabetes.

27. A method for treating type II diabetes in a subject, which comprises contacting one or more cells of a subject in need thereof with a nucleic acid, wherein the nucleic acid comprises a nucleotide sequence in SEQ ID NO: 1, a substantially identical sequence thereof, a fragment of the foregoing, or complementary nucleotide sequence of the foregoing; whereby contacting the one or more cells of the subject with the nucleic acid treats type II diabetes in the subject.

28. The method of claim 26, wherein the nucleic acid is RNA or PNA.

29. The method of claim 28, wherein the nucleic acid is duplex RNA.

30. A method for treating type II diabetes in a subject, which comprises contacting one or more cells of a subject in need thereof with a protein, wherein the protein is encoded by a nucleotide sequence which comprises a polynucleotide sequence in SEQ ID NO: 1, a substantially identical sequence thereof or a fragment of the foregoing; whereby contacting the one or more cells of the subject with the protein treats type II diabetes in the subject.

31. A method for treating type II diabetes in a subject, which comprises: detecting the presence or absence of one or more polymorphic variations associated with type II diabetes in a nucleic acid sample from a subject, wherein the one or more polymorphic variation are detected in a nucleotide sequence in SEQ ID NO: 1, a substantially identical sequence thereof or a fragment of the foregoing; and administering a type II diabetes treatment to a subject in need thereof based upon the presence or absence of the one or more polymorphic variations in the nucleic acid sample.

32. The method of claim 31, wherein the one or more polymorphic variations are detected at one or more positions in claim 3.

33. The method of claim 31, which further comprises determining blood glucose levels in the subject.

34. The method of claim 31, wherein the^" treatment is selected from the group consisting of administering insulin, a hypoglycemic, a starch blocker, a liver glucose regulating agent, an insulin sensitizer, a glucose level monitoring regimen, dietary counseling, a dietary regimen for managing blood glucose levels, and combinations of the foregoing.

35. A method for detecting or preventing type II diabetes in a subject, which comprises: detecting the presence or absence of one or more polymorphic variations associated with type II diabetes in a nucleic acid sample from a subject, wherein the polymorphic variation is detected in a nucleotide sequence in SEQ ID NO: 1, a substantially identical sequence thereof or a fragment of the foregoing; and administering a type II diabetes treatment or detection procedure to a subject in need thereof based upon the presence or absence of the one or more polymorphic variations in the nucleic acid sample.

36. The method of claim 35, wherein the one or more polymorphic variations are detected at one or more positions in claim 3.

37. The method of claim 35, wherein the type II diabetes treatment is selected from the group consisting of administering insulin, a hypoglycemic, a starch blocker, a liver glucose regulating agent, an insulin sensitizer, a glucose level monitoring regimen, dietary counseling, a dietary regimen for managing blood glucose levels, and combinations of the foregoing.

38. A method of targeting information for preventing or treating type II diabetes to a subject in need thereof, which comprises: detecting the presence or absence of one or more polymorphic variations associated with type II diabetes in a nucleic acid sample from a subject, wherein the polymorphic variation is detected in a nucleotide sequence in SEQ ID NO: 1, a substantially identical sequence thereof or a fragment of the foregoing; and directing information for preventing or treating type II diabetes to a subject in need thereof based upon the presence or absence of the one or more polymorphic variations in the nucleic acid sample.

39. The method of claim 38, wherein the one or more polymorphic variations are detected at one or more positions in claim 3.

40. The method of claim 38, wherein the information comprises a description of a type II diabetes detection procedure or treatment.

41. The method of claim 40, wherein the treatment is selected from the group consisting of administering insulin, a hypoglycemic, a starch blocker, a liver glucose regulating agent, an insulin sensitizer, a glucose level monitoring regimen, dietary counseling, a dietary regimen for managing blood glucose levels, and combinations of the foregoing

42. A composition comprising a cell from a subject having type II diabetes or at risk of type II diabetes and an antibody that specifically binds to a protein, polypeptide or peptide encoded by a nucleotide sequence identical to or 90% or more identical to a nucleotide sequence in SEQ ID NO: 1-5 or 10.

43. A composition comprising a cell from a subject having type II diabetes or at risk of type II diabetes and a RNA, DNA, PNA or ribozyme molecule comprising a nucleotide sequence identical to or 90% or more identical to a portion of a nucleotide sequence in SEQ ID NO: 1-5 or 10, or complementary sequence to the foregoing.

44. The composition of claim 43, wherein the RNA molecule is a short inhibitory RNA molecule.

45. An isolated nucleic acid, which comprises a nucleotide sequence that encodes a human PP2Ce comprising the amino acid sequence of SEQ ID NO: 6, 7 or 9.

46. The nucleic acid molecule of claim 45, wherein the nucleotide sequence comprises a polynucleotide sequence in SEQ ID NO: 2, 3 or 5.

47. A nucleic acid vector comprising the isolated nucleic acid molecule of claim 45, wherein the vector is selected from the group consisting of a plasmid or phage.

48. A recombinant host cell transfected with the isolated nucleic acid molecule of claim 45.

49. An isolated nucleic acid, which comprises a nucleotide sequence that encodes a polypeptide comprising amino acids 192-350 in SEQ ID NO: 6, amino acids 135-293 in SEQ ID NO: 7 or amino acids 13-171 in SEQ ID NO: 8.

50. An isolated nucleic acid, which comprises a nucleotide sequence that encodes all or part of a protein fragment consisting of amino acids 192-350 in SEQ ID NO: 6, amino acids 135- 293 in SEQ ID NO: 7 or amino acids 13-171 in SEQ ID NO: 8.

51. An antibody that specifically binds to a polypeptide comprising amino acids 192- 350 in SEQ ID NO: 6, amino acids 135-293 in SEQ ID NO: 7 or amino acids 13-171 in SEQ ID NO: 8, or that specifically binds to a polypeptide consisting of all or part of the amino acid sequence in SEQ ID NO: 6, 7 or 9.